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WO2025017207A1 - Inhibitors of smndc1 and their therapeutic use - Google Patents

Inhibitors of smndc1 and their therapeutic use Download PDF

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Publication number
WO2025017207A1
WO2025017207A1 PCT/EP2024/070646 EP2024070646W WO2025017207A1 WO 2025017207 A1 WO2025017207 A1 WO 2025017207A1 EP 2024070646 W EP2024070646 W EP 2024070646W WO 2025017207 A1 WO2025017207 A1 WO 2025017207A1
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alkyl
alkylene
haloalkyl
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coo
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Lennart ENDERS
Marton SIKLOS
Stefan Kubicek
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CEMM Forschungszentrum fuer Molekulare Medizin GmbH
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CEMM Forschungszentrum fuer Molekulare Medizin GmbH
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D277/38—Nitrogen atoms
    • C07D277/44—Acylated amino or imino radicals
    • C07D277/46—Acylated amino or imino radicals by carboxylic acids, or sulfur or nitrogen analogues thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/10—Spiro-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0821—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp
    • C07K5/0823—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp and Pro-amino acid; Derivatives thereof

Definitions

  • the present invention provides novel SMNDC1 inhibitors, specifically the compounds of formula (I), as well as pharmaceutical compositions containing these compounds.
  • the SMNDC1 inhibitors provided herein exhibit particularly beneficial properties in terms of potency and selectivity, which renders these compounds highly advantageous for use in therapy, including, e.g., in the treatment or prevention of cancer or diabetes.
  • SNDC1 Survival motor neuron domain-containing protein 1
  • SPF30 Survival of motor neuron-related-splicing factor 30
  • SPF30 is an essential splicing factor required for the formation of the spliceosome
  • SPF30 Is an Essential Human Splicing Factor Required for Assembly of the U4/U5/U6 Tri-small Nuclear Ribonucleoprotein into the Spliceosome. J. Biol.
  • SMNDC1 binds to methylated arginines on Sm-proteins using its6.1 domain (Rappsilber, J. et al. loc. cit; Cote, J. & Richard, S. Mathematics Domains Bind Symmetrical Dimethylated Arginines*. Journal of Biological Chemistry 280, 28476-28483 (2005)), similar to its better- studied paralog survival of motor neuron (SMN) protein (Cheng, D., Cote, J., Shaaban, S. & Bedford, M. T.
  • SSN motor neuron
  • the Arginine Methyltransferase CARM1 Regulates the Coupling of Transcription and mRNA Processing. Molecular Cell 25, 71-83 (2007); Pellizzoni, L, Kataoka, N., Charroux, B. & Dreyfuss, G. A Novel Function for SMN, the Spinal Muscular Atrophy Disease Gene Product, in Pre-mRNA Splicing. Cell 95, 615-624 (1998); Fischer, U., Liu, Q. & Dreyfuss, G. The SMN-SIP1 Complex Has an Essential Role in Spliceosomal snRNP Biogenesis. Cell 90, 1023— 1029 (1997)).
  • SMNDC1 Glucose metabolism and pancreatic defects in spinal muscular atrophy. Annals of Neurology 72, 256-268 (2012)). In contrast, for SMNDC1 it was recently shown that its knockdown causes the upregulation of insulin in a-cells through splicing changes in key chromatin remodelers and induction of the beta cell transcription factor PDX1 (Casteels, T. et al. SMNDC1 links chromatin remodeling and splicing to regulate pancreatic hormone expression. Cell Reports 40, (2022)). SMNDC1 further is essential for cell proliferation in different contexts, and a recent study reported worse survival in hepatocellular carcinoma patients with high SMNDC1 (Zhu, R., Wang, X., Yu, Q., Guo, W.
  • the SMN6.1 domain is sufficient for formation of a phase-separated compartment dependent on the dimethylarginine (DMA) modification of binding proteins (Courchaine, E. M. et al. DMA-tudor interaction modules control the specificity of in vivo condensates.
  • DMA dimethylarginine
  • T udor-SN interacts with and co-localizes with G3BP in stress granules under stress conditions.
  • SMNDC1 has a speckled localization within the nucleus that - based on co-localization - was attributed to the sub- nuclear structures Cajal bodies and nuclear speckles (Rappsilber, J. et al. loc. cit), which were later defined as prime examples of membraneless organelles (Zhu, L. & Brangwynne, C. P. Nuclear bodies: the emerging biophysics of nucleoplasmic phases. Curr. Opin. Cell Biol. 34, 23-30 (2015)), i.e. biomolecular condensates formed by liquid-liquid phase separation (LLPS).
  • LLPS liquid-liquid phase separation
  • RNA-binding proteins including SMNDC1 , were found to phase separate together with RNA, but also with chromatin (Shao, W. et al. Phase separation of RNA-binding protein promotes polymerase binding and transcription. Nat Chem Biol (2021) doi: 10.1038/s41589-021 -00904-5). Amongst other factors phase separation behavior can be initiated by RNA (Garcia-Jove Navarro, M. et al.
  • RNA is a critical element for the sizing and the composition of phase-separated RNA-protein condensates. Nat Commun 10, 3230 (2019)) and regulated by the secondary structure of RNAs and the ratio of RNA to RBPs (Banerjee, P. R., Milin, A. N., Moosa, M. M., Onuchic, P. L. & Deniz, A. A. Reentrant Phase Transition Drives Dynamic Substructure Formation in Ribonucleoprotein Droplets. Angewandte Chemie International Edition 56, 11354-11359 (2017); Langdon, E. M. etal. mRNA structure determines specificity of a polyQ-driven phase separation.
  • RNA polymerase II Cho, W.-K. et al. Mediator and RNA polymerase II clusters associate in transcription-dependent condensates. Science 361, 412-415 (2018)) to RNA processing and (alternative) splicing (Gueroussov, S. et al. Regulatory Expansion in Mammals of Multivalent hnRNP Assemblies that Globally Control Alternative Splicing. Ce// 170, 324-339. e23 (2017)).
  • SMNDC1 Small molecules that specifically inhibit SMNDC1
  • a further object of the invention is to provide potent SMNDC1 Vietnamese domain inhibitors that are selective for SMNDC1 over other Six domain-containing proteins, particularly over SMN.
  • the present invention addresses these needs and solves the problem of providing novel and therapeutically advantageous SMNDC1 inhibitors.
  • the phase-separating behavior of SMNDC1 was studied both in vitro and within cells (see Example B below), and specific inhibitors against the Tale domain of SMNDC1 , influencing the sub-cellular localization and phase separation of their target, were developed. It was surprisingly found that the compounds of formula (I) as provided herein are inhibitors of SMNDC1 exhibiting particularly favorable properties in terms of potency and selectivity, which makes these compounds highly advantageous for use in therapy.
  • the present invention provides a compound of the following formula (I) or a pharmaceutically acceptable salt or solvate thereof: X 1 -X 2 S.UQ / N R 1
  • one of the ring atoms X 1 and X 2 is S, and the other one is C(-R x ).
  • the group R x is selected from hydrogen, C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-O(Ci-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-NH2, -(C0-5 alkylene)-NH(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci
  • R 1 is -L 1 -L 2 -R 11 .
  • L 2 is selected from a covalent bond, C1-10 alkylene, C2-10 alkenylene, C2-10 alkynylene, -(C0-5 alkylene)-carbocyclyl- (C0-5 alkylene)-, and -(C0-5 alkylene)-heterocyclyl-(Co-5 alkylene)-, wherein said C1-10 alkylene, said C2-10 alkenylene, said C2-10 alkynylene, the two C0-5 alkylene groups in said -(C0-5 alkylene)-carbocyclyl-(Co-5 alkylene)-, and the two C0-5 alkylene groups in said -(C0-5 alkylene)-heterocyclyl-(Co-5 alkylene)- are each optionally substituted with one or more groups R Alk , wherein one or more -CH2- units comprised in said C1-10 alkylene, said C2-10 alkenylene, said C2-10 alkynylene, any of
  • R 11 is selected from hydrogen, -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(Ci-5 alkyl), -S(Ci-5 alkylene)-SH, -S(Ci- 5 alkylene)-S(Ci. 5 alkyl), -NH 2 , -NH(CI- 5 alkyl), -N(CI_ 5 alkyl)(Ci. 5 alkyl), -NH- OH, -N(CI-5 alkyl)-OH, -NH-O(CI.
  • each alkyl or alkylene comprised in any of the aforementioned groups is optionally substituted with one or more groups R Alk , wherein one or more -CH 2 - units comprised in the Co-15 alkylene in said -(Co-15 alkylene)-carbocyclyl or in the Co-15 alkylene in said -(Co-15 alkylene)-heterocyclyl are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2- , -CO-, -NH-, and -N(CI-5 alkyl)-, and wherein the carbocyclyl in said -(Co-15 alkylene)-carbocyclyl and the heterocyclyl in said -(Co-15 alkylene)-heter
  • R 2 is selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, -(Co-10 alkylene)-OH, -(Co-10 alkylene)-O(Ci-5 alkyl), -(Co-10 alkylene)-SH, -(Co-10 alkylene)-S(Ci-5 alkyl), -(C1-10 alkylene)-NH2, -(C1-10 alkylene)-NH(Ci-5 alkyl), -(C1-10 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(CMO alkylene)-halogen, C1-10 haloalkyl, -(Co-10 alkylene)-O-(Ci-5 haloalkyl), -(Co-10 alkylene)-CN, -(Co-10 alkylene)-NO2, -(Co-10 alkylene)-CHO, -(Co-10 alkylene)-CO-(Ci-io
  • R 3 is -(C0-5 alky lene)-ary I or -(C0-5 alkylene)-heteroaryl, wherein the aryl in said -(C0-5 alky lene)-aryl and the heteroaryl in said -(C0-5 alkylene)-heteroaryl are each optionally substituted with one or more groups R 31 , and wherein one or more -CH 2 - units comprised in the C0-5 alkylene in said -(C0-5 alkylene)-aryl or in the C0-5 alkylene in said -(C0-5 alky lene)-heteroary I are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO 2 -, -CO- , -NH-, and -N(CI- 5 alkyl)-.
  • Each R 31 is independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-O(Ci-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-NH2, -(C0-5 alkylene)-NH(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alky
  • Each R Alk is independently selected from -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(Ci. 5 alkyl), -S(Ci. 5 alkylene)-SH, -S(Ci. 5 alkylene)-S(Ci. 5 alkyl), -NH 2 , -NH(CI. 5 alkyl), -N(CI. 5 alkyl)(Ci. 5 alkyl), -NH-OH, -N(CI. 5 alkyl)-OH, -NH-O(CI. 5 alkyl), -N(CI.
  • Each R Cyc is independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-O(Ci-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-NH2, -(C0-5 alkylene)-NH(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 al
  • Each L z is independently selected from a covalent bond, C1-35 alkylene, C2-35 alkenylene, and C2-35 alkynylene, wherein said alkylene, said alkenylene and said alkynylene are each optionally substituted with one or more groups independently selected from halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -CN, -OH, -O(Ci-5 alkyl), -SH, -S(Ci-5 alkyl), -NH2, -NH(CI-5 alkyl), and -N(CI-5 alkyl)(Ci-5 alkyl), wherein one or more CH units comprised in said alkylene, said alkenylene or said alkynylene are each optionally replaced by a nitrogen atom, and further wherein one or more -CH2- units comprised in said alkylene, said alkenylene or said alkynylene are each optionally replaced by a group independently selected from -O
  • Each R z is independently selected from -OH, -0(Ci-5 alkyl), -0(Ci-5 alkylene)-OH, -0(Ci-5 alkylene)-0(Ci-5 alkyl), -SH, -S(Ci. 5 alkyl), -S(Ci. 5 alkylene)-SH, -S(Ci. 5 alkylene)-S(Ci. 5 alkyl), -NH 2 , -NH(Ci. 5 alkyl), -N(Ci. 5 alkyl)(Ci. 5 alkyl), -NH-OH, -N(Ci. 5 alkyl)-OH, -NH-O(Ci.
  • the present invention also relates to a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, in combination with a pharmaceutically acceptable excipient. Accordingly, the invention relates to a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof (or a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable excipient) for use as a medicament.
  • the compounds of formula (I) have been found to be highly effective as SMNDC1 inhibitors, particularly as SMNDC1 Vietnamese domain inhibitors (see Examples B and C below). Accordingly, the present invention provides potent SMNDC1 inhibitors. Moreover, the invention also provides SMNDC1 inhibitors that are advantageously selective for SMNDC1 over SMN (see also Examples B and C).
  • SMNDC1 is a Vietnamese domain protein that recognizes di-methylated arginines and controls gene expression as an essential splicing factor.
  • the specific contributions of the SMNDC1 Vietnamese domain to protein-protein interactions, subcellular localization, and molecular function were studied.
  • the inventors developed small molecule inhibitors, particularly the compounds of formula (I), targeting the dimethyl arginine binding pocket of the SMNDC1 Vietnamese domain.
  • SMNDC1 localizes to phase-separated membraneless organelles that partially overlap with nuclear speckles.
  • SMNDC1 This condensation behavior is driven by the unstructured C-terminal region of SMNDC1 , depends on RNA interaction and can be recapitulated in vitro. Inhibitors of the protein's Vietnamese domain drastically alter protein-protein interactions and subcellular localization, causing splicing changes for SMNDCI-dependent genes.
  • the SMNDC1 inhibitors provided herein can advantageously be used in therapy, particularly for the treatment or prevention of SMNDCI-related (or SMNDCI-mediated) diseases/disorders, including cancer or diabetes.
  • inhibitors of SMNDC1 are expected to induce the production of insulin in a-cells and, consequently, to be suitable for the treatment of diabetes or diabetes-related diseases/disorders (see, e.g., Casteels, T. et al. SMNDC1 links chromatin remodeling and splicing to regulate pancreatic hormone expression. Cell Reports 40, (2022)). Moreover, SMNDC1 has been reported to be essential for cell proliferation, particularly in the context of cancer, and inhibitors of SMNDC1 are expected to be effective in reducing or inhibiting cancer cell proliferation and/or migration (see, e.g., Zhu, R. et al.
  • the present invention thus further relates to a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising any of the aforementioned entities and a pharmaceutically acceptable excipient, for use in the treatment or prevention of an SMNDCI-related disease or disorder (or an SMNDCI-mediated disease or disorder).
  • the invention also provides a pharmaceutical composition comprising, as an active ingredient, a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, together with a pharmaceutically acceptable excipient, for use in the treatment or prevention of an SMNDCI-related disease/disorder (or an SMNDCI-mediated disease/disorder).
  • the present invention relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for the treatment or prevention of an SMNDC1 -related disease or disorder (or an SMNDC1 -mediated disease or disorder).
  • the invention likewise relates to a method of treating or preventing an SMNDC1 -related disease or disorder (or an SMNDCI-mediated disease or disorder), the method comprising administering a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising any of the aforementioned entities in combination with a pharmaceutically acceptable excipient, to a subject (preferably a human) in need thereof.
  • a pharmaceutically acceptable amount of the compound of formula (I) or the pharmaceutically acceptable salt thereof (or of the pharmaceutical composition) is to be administered in accordance with this method.
  • the disease or disorder to be treated or prevented with a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof (or a corresponding pharmaceutical composition) in accordance with the present invention includes any SMNDC1 -related (or SMNDCI-mediated) disease or disorder. It is preferred that the disease/disorder to be treated or prevented in accordance with the invention is cancer or diabetes.
  • the diabetes may be, e.g., type 1 diabetes or type 2 diabetes.
  • the cancer may be, e.g., a solid cancer or a hematological cancer.
  • the cancer may be selected from lung cancer (e.g., small cell lung cancer or non-small cell lung cancer), renal cancer (or kidney cancer; e.g., renal carcinoma), gastrointestinal cancer, stomach cancer, colorectal cancer (e.g., colorectal carcinoma), colon cancer, anal cancer, genitourinary cancer, bladder cancer, liver cancer (e.g., hepatocellular carcinoma), pancreatic cancer (e.g., pancreatic adenocarcinoma or pancreatic ductal adenocarcinoma), cervical cancer, endometrial cancer, vaginal cancer, vulvar cancer, ovarian cancer (e.g., ovarian carcinoma), uterine cancer, prostate cancer (e.g., hormone-refractory prostate cancer), testicular cancer, biliary tract cancer, hepatobiliary cancer, neuroblastoma, brain cancer (e.g., glioblastoma), breast cancer (e.g., triple-negative breast cancer, including in particular
  • the present invention particularly relates to a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising any of the aforementioned entities and a pharmaceutically acceptable excipient, for use in the treatment or prevention of cancer (e.g., any one of the specific types of cancer mentioned herein above) or diabetes (e.g., type 1 diabetes or type 2 diabetes).
  • cancer e.g., any one of the specific types of cancer mentioned herein above
  • diabetes e.g., type 1 diabetes or type 2 diabetes.
  • the present invention furthermore relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof as an inhibitor of survival motor neuron domain-containing protein 1 (SMNDC1) in research, particularly as a research tool compound for inhibiting SMNDC1.
  • the invention refers to the in vitro use of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof as an SMNDC1 inhibitor and, in particular, to the in vitro use of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof as a research tool compound acting as an SMNDC1 inhibitor.
  • the invention likewise relates to a method, particularly an in vitro method, of inhibiting SMNDC1 , the method comprising the application of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof.
  • the invention further relates to a method of inhibiting SMNDC1 , the method comprising applying a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof to a test sample (e.g., a biological sample) or a test animal (i.e., a non-human test animal).
  • the invention also refers to a method, particularly an in vitro method, of inhibiting SMNDC1 in a sample (e.g., a biological sample), the method comprising applying a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof to said sample.
  • a sample e.g., a biological sample
  • the present invention further provides a method of inhibiting SMNDC1 , the method comprising contacting a test sample (e.g., a biological sample) or a test animal (i.e., a non-human test animal) with a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof.
  • sample includes, without being limited thereto: a cell, a cell culture or a cellular or subcel lular extract; biopsied material obtained from an animal (e.g., a human), or an extract thereof; or blood, serum, plasma, saliva, urine, feces, or any other body fluid, or an extract thereof.
  • in vitro is used in this specific context in the sense of "outside a living human or animal body”, which includes, in particular, experiments performed with cells, cellular or subcellular extracts, and/or biological molecules in an artificial environment such as an aqueous solution or a culture medium which may be provided, e.g., in a flask, a test tube, a Petri dish, a microtiter plate, etc.
  • one of the ring atoms X 1 and X 2 is S, and the other one is C(-R x ).
  • X 1 is S and X 2 is C(-R x ), or alternatively, X 1 is C(-R x ) and X 2 is S.
  • the 5-membered ring containing the ring atoms X 1 and X 2 is aromatic, as also reflected by the inner circle drawn within this ring in formula (I).
  • the compound of formula (I) may have one of the following structures:
  • X 1 is S
  • X 2 is C(-R x )
  • X 1 is C(-R x )
  • X 2 is S
  • X 1 is S
  • X 2 is C(-R x ). Accordingly, it is preferred that the compound of formula (I) has the following structure:
  • the group R x is selected from hydrogen, C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-O(Ci-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-NH2, -(C0-5 alkylene)-NH(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci
  • R x is selected from hydrogen, C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(Ci-5 alkyl), -S(Ci-5 alkylene)-SH, -S(Ci-5 alkylene)-S(Ci-5 alkyl), -NH 2 , -NH(CI. 5 alkyl), -N(CI. 5 alkyl)(Ci. 5 alkyl), -NH-OH, -N(CI.
  • R x is selected from hydrogen, C1-5 alkyl, -OH, -O(Ci-5 alkyl), -SH, -S(Ci-5 alkyl), -NH2, -NH(CI-5 alkyl), -N(CI-5 alkyl)(Ci-5 alkyl), halogen (e.g., -F or -Cl), C1-5 haloalkyl (e.g., -CF3), -O-(Ci-5 haloalkyl) (e.g., -OCF3), and -ON.
  • R x is selected from hydrogen, C1-5 alkyl, halogen (e.g., -F, -Cl or -Br), C1-5 haloalkyl (e.g., -CF3), -O-(Ci-5 haloalkyl) (e.g., -OCF3), and -CN.
  • halogen e.g., -F, -Cl or -Br
  • C1-5 haloalkyl e.g., -CF3
  • -O-(Ci-5 haloalkyl) e.g., -OCF3
  • -CN e.g., -CN
  • R x is hydrogen or methyl.
  • R x may be methyl. It is especially preferred that R x is hydrogen.
  • R 1 is -L 1 -L 2 -R 11 .
  • L 1 is selected from a covalent bond, -CO-, and -SO2-.
  • L 1 is a covalent bond or -CO-.
  • L 2 is selected from a covalent bond, C1-10 alkylene, C2-10 alkenylene, C2-10 alkynylene, -(C0-5 alkylene)-carbocyclyl- (C0-5 alkylene)-, and -(C0-5 alkylene)-heterocyclyl-(Co-5 alkylene)-, wherein said C1-10 alkylene, said C2-10 alkenylene, said C2-10 alkynylene, the two C0-5 alkylene groups in said -(C0-5 alkylene)-carbocyclyl-(Co-5 alkylene)-, and the two C0-5 alkylene groups in said -(C0-5 alkylene)-heterocyclyl-(Co-5 alkylene)- are each optionally substituted with one or more (e.g., one, two or three) groups R Alk , wherein one or more (e.g., one, two or three) -CH2- units comprised in said C1-10 al
  • L 2 is selected from a covalent bond, C1-10 alkylene, C2-10 alkenylene, -(C0-5 alkylene)-cycloalkyl-(Co-5 alkylene)-, -(C0-5 alkylene)-heterocycloalkyl-(Co-5 alkylene)-, -(C0-5 alkylene)-aryl-(Co-5 alkylene)-, and -(C0-5 alky lene)-heteroary l-(Co-5 alkylene)-, wherein said C1-10 alkylene, said C2-10 alkenylene, or any C0-5 alkylene comprised in any of the aforementioned groups are each optionally substituted with one or more groups R Alk , wherein one or more -CH2- units comprised in said C1-10 alkylene, said C2-10 alkenylene, or any C0-5 alkylene comprised in any of the aforementioned groups are each optionally replaced by a group independently selected from -O-,
  • L 2 is selected from a covalent bond, C1-5 alkylene, -(C0-5 alkylene)-aryl-(Co-5 alkylene)-, and -(C0-5 alkylene)-heteroaryl-(Co-5 alkylene)-, wherein said C1-5 alkylene, the two C0-5 alkylene groups in said -(C0-5 alkylene)-aryl-(Co-5 alkylene)-, and the two C0-5 alkylene groups in said -(C0-5 alkylene)-heteroaryl-(Co-5 alkylene)- are each optionally substituted with one or more groups R Alk , wherein one or more -CH2- units comprised in said C1-5 alkylene or any of the C0-5 alkylene groups comprised in said -(C0-5 alkylene)-aryl-(Co-5 alkylene)- or said -(C0-5 alkylene)-heteroaryl-(Co-5 alkylene)- are each
  • L 2 is selected from a covalent bond, C1-5 alkylene, -(C0-5 alkylene)-aryl-(Co-5 alkylene)-, and -(C0-5 alkylene)-heteroaryl-(Co-5 alkylene)-, wherein the aryl in said -(C0-5 alkylene)-aryl-(Co-5 alkylene)- and the heteroaryl in said -(C0-5 alkylene)-heteroaryl-(Co-5 alkylene)- are each optionally substituted with one or more groups RCy Ci
  • L 2 is selected from a covalent bond, aryl, and heteroaryl, wherein said aryl and said heteroaryl are each optionally substituted with one or more groups R Cyc .
  • R 11 is selected from hydrogen, -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(Ci-5 alkyl), -S(Ci.5 alkylene)-SH, -S(Ci. 5 alkylene)-S(Ci. 5 alkyl), -NH 2 , -NH(CI. 5 alkyl), -N(CI. 5 alkyl)(Ci_ 5 alkyl), -NH- OH, -N(CI.5 alkyl)-OH, -NH-O(CI. 5 alkyl), -N(CI.
  • each alkyl or alkylene comprised in any of the aforementioned groups is optionally substituted with one or more (e.g., one, two, three, or four) groups R Alk , wherein one or more (e.g., one, two, three, of four) -CH2- units comprised in the Co-15 alkylene in said -(Co-15 alkylene)-carbocyclyl or in the Co-15 alkylene in said -(Co-15 alkylene)-heterocyclyl are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -NH-, and -N(0I-5 alkyl)-, and wherein the carb
  • R 11 is selected from hydrogen, -OH, -O(Ci-5 alkyl), -SH, -S(Ci-5 alkyl), -NH2, -NH(CI-5 alkyl), -N(0I-5 alkyl)(Ci.5 alkyl), -NH-OH, -N(CI. 5 alkyl)-OH, -NH-O(CI. 5 alkyl), -N(CI. 5 alkyl)-O(Ci_ 5 alkyl), halogen, C1.5 haloalkyl, -O-(Ci. 5 haloalkyl), -CN, -NO 2 , -CHO, -CO-(Ci.
  • each C1-5 alkyl or C0-5 alkylene comprised in any of the aforementioned groups is optionally substituted with one or more groups R Alk , wherein one or more (e.g., one or two) -CH2- units comprised in the C0-5 alkylene in said -(C0-5 alkylene)-cycloalkyl, in the C0-5 alkylene in said -(C0-5 alkylene)-heterocycloalkyl, in the C0-5 alkylene in said -
  • R 1 examples include, in particular, any of the specific groups R 1 comprised in the exemplary compounds of formula (I) described in the present specification, including any of the exemplary compounds of formula (I) described in Example A, B or 0 below.
  • R 1 examples include, in particular, any of the specific groups R 1 comprised in the exemplary compounds of formula (I) described in the present specification, including any of the exemplary compounds of formula (I) described in Example A, B or 0 below.
  • any of these exemplary compounds of formula (I) has hydrogen as one of the two groups R 1 and R 2 , it will be assumed that said hydrogen is R 2 and that the other one of the said two groups is R 1 .
  • R 2 is selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, -(Co-10 alkylene)-OH, -(Co-10 alkylene)-0(Ci-5 alkyl), -(Co-10 alkylene)-SH, -(Co-10 alkylene)-S(Ci-5 alkyl), -(C1-10 alkylene)-NH2, -(C1-10 alkylene)-N H(CI-5 alkyl), -(C1-10 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C1-10 alkylene)-halogen, C1-10 haloalkyl, -(Co-10 alkylene)-0-(Ci-5 haloalkyl), -(Co-10 alkylene)-CN, -(Co-10 alkylene)-N02, -(Co-10 alkylene)-CHO, -(Co-10 alkylene)-CO-(Ci-i
  • R 2 is selected from hydrogen, C1.5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-0(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C1.5 alkylene)-NH2, -(C1.5 alkylene)-NH(Ci-5 alkyl), -(C1-5 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C1.5 alkylene)-halogen, C1.5 haloalkyl, -(C0-5 alkylene)-0-(Ci-5 haloalkyl), -(C0-5 alkylene)-CN, -(C0-5 alkylene)-N02, -(C0-5 alkylene)-CHO, -(C0-5 alkylene)-(C0
  • R 2 is selected from hydrogen, C1-5 alkyl, C2-5 alkenyl, and C1-5 haloalkyl.
  • R 2 is hydrogen or C1-5 alkyl.
  • R 2 is hydrogen
  • R 3 is -(C0-5 alky lene)-ary I or -(C0-5 alkylene)-heteroaryl, wherein the aryl in said -(C0-5 alky lene)-aryl and the heteroaryl in said -(C0-5 alkylene)-heteroaryl are each optionally substituted with one or more (e.g., one, two or three) groups R 31 , and wherein one or more (e.g., one, two or three) -CH2- units comprised in the C0-5 alkylene in said -(C0-5 alkylene)-aryl or in the C0-5 alkylene in said -(C0-5 alkylene)-heteroaryl are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -NH-, and -N(CI-5 alkyl)-.
  • R 3 examples include, in particular, any of the specific groups R 3 comprised in the exemplary compounds of formula (I) described in the present specification, including any of the exemplary compounds of formula (I) described in Example A, B or C below.
  • R 3 is -(C0-5 alkylene)-aryl or -(C0-5 alkylene)-heteroaryl, wherein the aryl in said -(C0-5 alkylene)-aryl and the heteroaryl in said -(C0-5 alkylene)-heteroaryl are each optionally substituted with one or more groups R 31 .
  • R 3 is aryl, -Chfe-aryl, heteroaryl, or -Chh-heteroaryl, wherein said aryl, the aryl in said -Chfe-aryl, said heteroaryl, and the heteroaryl in said -CH2-heteroaryl are each optionally substituted with one or more groups R 31 .
  • R 3 is aryl or heteroaryl, wherein said aryl and said heteroaryl are each optionally substituted with one or more groups R 31 .
  • the aryl (including, e.g., the aryl comprised in the above-mentioned group -(C0-5 alkylene)-aryl) is phenyl, naphthyl or tetralinyl (e.g., tetralin- 6-yl), more preferably phenyl.
  • the heteroaryl (including, e.g., the heteroaryl comprised in the above-mentioned group -(C0-5 alkylene)-heteroaryl) is a monocyclic 5- or 6-membered heteroaryl or a bicyclic 9- or 10-membered heteroaryl, more preferably a monocyclic 5- or 6-membered heteroaryl.
  • a corresponding bicyclic 9- or 10-membered heteroaryl may, e.g., have 1 , 2, 3, 4 or 5 ring heteroatoms selected independently from nitrogen, oxygen and sulfur, while all remaining ring atoms are carbon atoms.
  • a corresponding monocyclic 5- or 6-membered heteroaryl may, e.g., have 1, 2 or 3 ring heteroatoms selected independently from nitrogen, oxygen and sulfur, while all remaining ring atoms are carbon atoms.
  • Examples of a corresponding heteroaryl include pyridinyl (e.g., pyridin-2-yl), pyrrolyl (e.g., 1 H-pyrrol-2-yl), 1 ,3-benzodioxolyl (e.g., 1 ,3-benzodioxol-5-yl), furanyl (e.g., furan-2-yl), or thiophenyl (e.g., thiophen-2-yl).
  • a corresponding heteroaryl include pyridinyl (e.g., pyridin-2-yl or pyridin-4-yl) or pyrrolyl (e.g., 1 H-pyrrol-2-yl), even more preferably pyridin-2-yl or 1 H-pyrrol-2-yl.
  • An especially preferred example of a corresponding heteroaryl is pyridin-2-yl. It is furthermore preferred that the heteroaryl (in R 3 ) is not pyridin-3-yl, more preferably it is not pyridin-3- yl, pyridin-4-yl or thiophen-2-yl.
  • R 3 is phenyl or a monocyclic 5- or 6-membered heteroaryl, wherein said aryl and said heteroaryl are each optionally substituted with one or more groups R 31 .
  • R 3 is selected from phenyl, pyridin-2-yl, and 1 H-pyrrol-2-yl, wherein said phenyl, said pyridin-2-yl, and said 1 H-pyrrol-2-yl are each optionally substituted with one or more groups R 31 .
  • R 3 is pyridin-2-yl which is optionally substituted with one or more groups R 31 .
  • Each R 31 is independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-O(Ci-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-NH2, -(C0-5 alkylene)-NH(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alky
  • R 31 examples include, in particular, any of the specific groups R 31 comprised in the exemplary compounds of formula (I) described in the present specification, including any of the exemplary compounds of formula (I) described in Example A, B or C below.
  • each R 31 is independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(0i-5 alkyl), -S(0i-5 alkylene)-SH, -S(0i-5 alkylene)-S(Ci-5 alkyl), -NH 2 , -NH(CI. 5 alkyl), -N(CI. 5 alkyl)(Ci. 5 alkyl), -NH-OH, -N(CI.
  • each R 31 is independently selected from C1-5 alkyl, C2-5 alkenyl, -OH, -O(Ci-5 alkyl), -SH, -S(Ci-5 alkyl), -NH2, -NH(CI-5 alkyl), -N(CI-5 alkyl)(Ci-5 alkyl), halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), and -ON.
  • Each R Alk is independently selected from -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(Ci. 5 alkyl), -S(Ci. 5 alkylene)-SH, -S(Ci. 5 alkylene)-S(Ci. 5 alkyl), -NH 2 , -NH(Ci. 5 alkyl), -N(Ci. 5 alkyl)(Ci. 5 alkyl), -NH-OH, -N(Ci. 5 alkyl)-OH, -NH-O(CI. 5 alkyl), -N(CI.
  • Each R Cyc is independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-O(Ci-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-NH2, -(C0-5 alkylene)-NH(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 al
  • each R 0 '' 0 is independently selected from C1.5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(Ci-5 alkyl), -S(Ci-5 alkylene)-SH, -S(Ci-5 alkylene)-S(Ci-5 alkyl), -NH 2 , -NH(CI. 5 alkyl), -N(CI. 5 alkyl)(Ci. 5 alkyl), -NH-OH, -N(Ci.
  • Each L z is independently selected from a covalent bond, C1-35 alkylene, C2-35 alkenylene, and C2-35 alkynylene, wherein said alkylene, said alkenylene and said alkynylene are each optionally substituted with one or more (e.g., one, two, or three) groups independently selected from halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -CN, -OH, -O(Ci-5 alkyl), -SH, -S(Ci-5 alkyl), -NH2, -NH(CI-5 alkyl), and -N(CI-5 alkyl)(Ci-5 alkyl), wherein one or more (e.g., one, two or three) CH units comprised in said alkylene, said alkenylene or said alkynylene are each optionally replaced by a nitrogen atom (e.g., and further wherein one or more (e.g., one, two or
  • each L z is independently selected from a covalent bond, C1-5 alkylene, C2-5 alkenylene, and C2-5 alkynylene, wherein said alkylene, said alkenylene and said alkynylene are each optionally substituted with one or more (e.g., one, two, or three) groups independently selected from halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -CN, -OH, -O(Ci. 5 alkyl), -SH, -S(Ci. 5 alkyl), -NH 2 , -NH(Ci. 5 alkyl), and -N(Ci. 5 alkyl)(Ci.
  • one or more -CH2- units comprised in said alkylene, said alkenylene or said alkynylene are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -NH-, and -N(Ci-5 alkyl)-.
  • Each R z is independently selected from -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(Ci. 5 alkyl), -S(Ci. 5 alkylene)-SH, -S(Ci. 5 alkylene)-S(Ci. 5 alkyl), -NH 2 , -NH(Ci. 5 alkyl), -N(Ci. 5 alkyl)(Ci. 5 alkyl), -NH-OH, -N(Ci. 5 alkyl)-OH, -NH-O(Ci.
  • one or more groups independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -CN, -NO2, -OH,
  • each R z is independently selected from -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(Ci_ 5 alkyl), -S(Ci_ 5 alkylene)-SH, -S(Ci. 5 alkylene)-S(Ci_ 5 alkyl), -NH 2 , -NH(CI_ 5 alkyl), -N(CI_ 5 alkyl)(Ci. 5 alkyl), -NH-OH, -N(CI_ 5 alkyl)-OH, -NH-O(CI. 5 alkyl), -N(CI.
  • the compound of formula (I) is any one of the specific compounds of formula (I) described in the examples section of this specification, including any one of the specific compounds of formula (I) described in Example A, B or C herein below, either in non-salt form or as a pharmaceutically acceptable salt of the respective compound.
  • Example A, B or C While several exemplary compounds of formula (I) have been described in Example A, B or C as compounds having a specific stereochemical configuration, the present invention also relates to each of these compounds without specific configuration.
  • the invention further relates specifically and individually to all other possible stereoisomers of such compounds that have been described with a specific configuration, including an enantiomer or a diastereoisomer of each respective compound, as well as mixtures thereof, including racemic mixtures (racemates).
  • the compound of formula (I) is selected from any one of the following compounds: or a pharmaceutically acceptable salt or solvate of any one of the above-depicted compounds.
  • the present invention also specifically relates to each one of the above-depicted compounds in non-salt form.
  • the compounds of Examples 1 to 19, 63 to 87 and 110 are excluded from formula (I). Accordingly, it is preferred that the compound of formula (I) is not a compound of any one of Examples 1 to 19, 63 to 87 or 110 (or a pharmaceutically acceptable salt or solvate thereof).
  • the compound of formula (I) is a compound of the following formula (la) or pharmaceutically acceptable salt or solvate thereof, wherein: o one of the ring atoms X 1 and X 2 is S, and the other one is C(-R x ); o R x is selected from hydrogen, C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-O(Ci-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -(C0-5 alkylene)-SH,
  • each alkyl or alkylene comprised in any of the aforementioned groups is optionally substituted with one or more groups R Alk , wherein one or more -CH2- units comprised in the C1-15 alkylene in said -(C1-15 alkylene)-carbocyclyl or in the C1-15 alkylene in said -(C1-15 alkylene)-heterocyclyl are each replaced by a group independently selected from -O-, -S-, -SO-,
  • each R Cyc is independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-O(Ci-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S
  • haloalkyl -ON, -NO 2 , -OH, -O(Ci. 5 alkyl), -SH, -S(Ci. 5 alkyl), -NH 2 , -NH(CI. 5 alkyl), -N(CI. 5 alkyl)(Ci.5 alkyl), -OHO, -CO-(Ci. 5 alkyl), -COOH, -CO-O-(Ci. 5 alkyl), -O-CO-(Ci. 5 alkyl), -CO-NH 2 , -CO-NH(CI. 5 alkyl), -CO-N(CI.5 alkyl)(Ci. 5 alkyl), -NH-CO-(CI.
  • each R z is independently selected from -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(Ci. 5 alkyl), -S(Ci. 5 alkylene)-SH, -S(Ci. 5 alkylene)-S(Ci. 5 alkyl), -NH 2 , -NH(CI. 5 alkyl), -N(CI.
  • the compound of formula (I) is a compound of the following formula (lb) or pharmaceutically acceptable salt or solvate thereof, wherein: o one of the ring atoms X 1 and X 2 is S, and the other one is C(-R x ); o R x is selected from hydrogen, C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-O(Ci-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -(C0-5-5
  • each alkyl or alkylene comprised in any of the aforementioned groups is optionally substituted with one or more groups R Alk , wherein one or more -CH 2 - units comprised in the Co-15 alkylene in said -(Co-15 alkylene)-carbocyclyl or in the Co-15 alkylene in said -(Co-15 alkylene)-heterocyclyl are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO 2 - , -CO-, -NH-, and -N(CI-5 alkyl)-, wherein the carbocyclyl in said -(Co-15 alkylene)-carbocyclyl and the heterocyclyl in said -(Co-15 alkylene)-heterocyclyl are each optionally substituted with
  • each R Cyc is independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-O(Ci-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -
  • haloalkyl -CN, -NO 2 , -OH, -O(Ci. 5 alkyl), -SH, -S(Ci. 5 alkyl), -NH 2 , -NH(CI. 5 alkyl), -N(CI. 5 alkyl)(Ci.5 alkyl), -OHO, -CO-(Ci. 5 alkyl), -COCH, -CO-O-(Ci. 5 alkyl), -O-CO-(Ci. 5 alkyl), -CO-NH 2 , -CO-NH(CI. 5 alkyl), -CO-N(Ci-5 alkyl)(Ci.
  • each R z is independently selected from -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(Ci. 5 alkyl), -S(Ci. 5 alkylene)-SH, -S(Ci. 5 alkylene)-S(Ci. 5 alkyl), -NH 2 , -NH(Ci. 5 alkyl), -N(Ci.
  • hydrocarbon group refers to a group consisting of carbon atoms and hydrogen atoms.
  • alicyclic is used in connection with cyclic groups and denotes that the corresponding cyclic group is non-aromatic.
  • alkyl refers to a monovalent saturated acyclic (i.e., non-cyclic) hydrocarbon group which may be linear or branched. Accordingly, an “alkyl” group does not comprise any carbon-to-carbon double bond or any carbon-to-carbon triple bond.
  • a “C1-5 alkyl” denotes an alkyl group having 1 to 5 carbon atoms. Preferred exemplary alkyl groups are methyl, ethyl, propyl (e.g., n-propyl or isopropyl), or butyl (e.g., n-butyl, isobutyl, sec-butyl, or tert- butyl).
  • alkyl preferably refers to C1-4 alkyl, more preferably to methyl or ethyl, and even more preferably to methyl.
  • alkenyl refers to a monovalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon double bonds while it does not comprise any carbon-to-carbon triple bond.
  • C2-5 alkenyl denotes an alkenyl group having 2 to 5 carbon atoms.
  • Preferred exemplary alkenyl groups are ethenyl, propenyl (e.g., prop-1 -en-1-yl, prop-1-en-2-yl, or prop-2-en-1-yl), butenyl, butadienyl (e.g., buta-1,3-dien-1-yl or buta-1 ,3-dien-2-yl), pentenyl, or pentadienyl (e.g., isoprenyl).
  • alkenyl preferably refers to C2-4 alkenyl.
  • alky ny I refers to a monovalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon triple bonds and optionally one or more (e.g., one or two) carbon-to-carbon double bonds.
  • C2-5 alkynyl denotes an alkynyl group having 2 to 5 carbon atoms.
  • Preferred exemplary alkynyl groups are ethynyl, propynyl (e.g., propargyl), or butynyl.
  • alkynyl preferably refers to C2-4 alkynyl.
  • alkylene refers to an alkanediyl group, i.e. a divalent saturated acyclic hydrocarbon group which may be linear or branched.
  • a “C1-5 alkylene” denotes an alkylene group having 1 to 5 carbon atoms, and the term “C0-5 alkylene” indicates that a covalent bond (corresponding to the option "Co alkylene”) or a C1-5 alkylene is present.
  • Preferred exemplary alkylene groups are methylene (-CH2-), ethylene (e.g., -CH2-CH2- or -CH(-CH3)-), propylene (e.g., -CH2-CH2-CH2-, -CH(-CH 2 -CH 3 )-, -CH 2 -CH(-CH 3 )-, or -CH(-CH 3 )-CH 2 -), or butylene (e.g., -CH2-CH2- CH2-CH2-).
  • alkylene preferably refers to C1-4 alkylene (including, in particular, linear C1-4 alkylene), more preferably to methylene or ethylene, and even more preferably to methylene.
  • alkenylene refers to an alkenediyl group, i.e. a divalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon double bonds while it does not comprise any carbon-to-carbon triple bond.
  • a "C2-5 alkenylene” denotes an alkenylene group having 2 to 5 carbon atoms.
  • alkenylene preferably refers to C2-4 alkenylene (including, in particular, linear C2-4 alkenylene).
  • alkynylene refers to an alkynediyl group, i.e. a divalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon triple bonds and optionally one or more (e.g., one or two) carbon-to-carbon double bonds.
  • a "C2-5 alkynylene” denotes an alkynylene group having 2 to 5 carbon atoms.
  • alkynylene preferably refers to C2-4 alkynylene (including, in particular, linear C2-4 alkynylene).
  • carbocyclyl refers to a hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings), wherein said ring group may be saturated, partially unsaturated (i.e., unsaturated but not aromatic) or aromatic.
  • “carbocyclyl” preferably refers to aryl, cycloalkyl or cycloalkenyl.
  • the term “carbocyclylene” refers to a carbocyclyl group, as defined herein above, but having two points of attachment, i.e.
  • a divalent hydrocarbon ring group including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings), wherein said ring group may be saturated, partially unsaturated (i.e., unsaturated but not aromatic) or aromatic.
  • “carbocyclylene” preferably refers to arylene, cycloalkylene or cycloalkenylene.
  • heterocycly I refers to a ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings), wherein said ring group comprises one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group), and further wherein said ring group may be saturated, partially unsaturated (i.e., unsaturated but not aromatic) or aromatic.
  • each heteroatom-containing ring comprised in said ring group may contain one or two O atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring.
  • heterocyclyl preferably refers to heteroaryl, heterocycloalkyl or heterocycloalkenyl.
  • heterocyclylene refers to a heterocyclyl group, as defined herein above, but having two points of attachment, i.e. a divalent ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings), wherein said ring group comprises one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from O, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group), and further wherein said ring group may be saturated, partially unsaturated (i.e., unsaturated but not aromatic)
  • each heteroatom-containing ring comprised in said ring group may contain one or two 0 atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring.
  • heterocyclylene preferably refers to heteroarylene, heterocycloalkylene or heterocycloalkenylene.
  • aryl refers to an aromatic hydrocarbon ring group, including monocyclic aromatic rings as well as bridged ring and/or fused ring systems containing at least one aromatic ring (e.g., ring systems composed of two or three fused rings, wherein at least one of these fused rings is aromatic; or bridged ring systems composed of two or three rings, wherein at least one of these bridged rings is aromatic).
  • aryl is a bridged and/or fused ring system which contains, besides one or more aromatic rings, at least one non-aromatic ring (e.g., a saturated ring or an unsaturated alicyclic ring), then one or more carbon ring atoms in each non-aromatic ring may optionally be oxidized (i.e., to form an oxo group).
  • non-aromatic ring e.g., a saturated ring or an unsaturated alicyclic ring
  • carbon ring atoms in each non-aromatic ring may optionally be oxidized (i.e., to form an oxo group).
  • Aryl may, e.g., refer to phenyl, naphthyl, dialinyl (i.e., 1,2-dihydronaphthyl), tetralinyl (i.e., 1 ,2,3,4-tetrahydronaphthyl), indanyl, indenyl (e.g., 1 H-indenyl), anthracenyl, phenanthrenyl, 9H- fluorenyl, or azulenyl.
  • an "aryl” preferably has 6 to 14 ring atoms, more preferably 6 to 10 ring atoms, even more preferably refers to phenyl or naphthyl, and most preferably refers to phenyl.
  • arylene refers to an aryl group, as defined herein above, but having two points of attachment, i.e. a divalent aromatic hydrocarbon ring group, including monocyclic aromatic rings as well as bridged ring and/or fused ring systems containing at least one aromatic ring (e.g., ring systems composed of two or three fused rings, wherein at least one of these fused rings is aromatic; or bridged ring systems composed of two or three rings, wherein at least one of these bridged rings is aromatic).
  • the arylene is a bridged and/or fused ring system which contains, besides one or more aromatic rings, at least one non-aromatic ring (e.g., a saturated ring or an unsaturated alicyclic ring), then one or more carbon ring atoms in each non-aromatic ring may optionally be oxidized (i.e., to form an oxo group).
  • at least one non-aromatic ring e.g., a saturated ring or an unsaturated alicyclic ring
  • one or more carbon ring atoms in each non-aromatic ring may optionally be oxidized (i.e., to form an oxo group).
  • Arylene may, e.g., refer to phenylene (e.g., phen-1, 2-diyl, phen-1 ,3-diyl, or phen-1, 4- diyl), naphthylene (e.g., naphthalen-1 ,2-diyl, naphthalen-1, 3-diyl, naphthalen-1, 4-diyl, naphthalen-1, 5-diyl, naphthalen-1, 6-diyl, naphthalen-1, 7-diyl, naphthalen-2, 3-diyl, naphthalen-2, 5-diyl, naphthalen-2, 6-diyl, naphthalen- 2,7-diyl, or naphthalen-2, 8-diyl), 1 ,2-dihydronaphthylene, 1 ,2,3,4-tetrahydronaphthylene, indanylene, ind
  • an "arylene” preferably has 6 to 14 ring atoms, more preferably 6 to 10 ring atoms, even more preferably refers to phenylene or naphthylene, and most preferably refers to phenylene (particularly phen-1, 4-diyl).
  • heteroaryl refers to an aromatic ring group, including monocyclic aromatic rings as well as bridged ring and/or fused ring systems containing at least one aromatic ring (e.g., ring systems composed of two or three fused rings, wherein at least one of these fused rings is aromatic; or bridged ring systems composed of two or three rings, wherein at least one of these bridged rings is aromatic), wherein said aromatic ring group comprises one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, and further wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group).
  • aromatic ring group comprises one or more (such as, e.g., one, two, three
  • each heteroatom-containing ring comprised in said aromatic ring group may contain one or two 0 atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring.
  • Heteroaryl may, e.g., refer to thienyl (i.e., thiophenyl), benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl (i.e., furanyl), benzofuranyl, isobenzofuranyl, chromanyl, chromenyl (e.g., 2H-1-benzopyranyl or 4H-1-benzopyranyl), isochromenyl (e.g., 1 H-2-benzopyranyl), chromonyl, xanthenyl, phenoxathiinyl, pyrrolyl (e.g., 1 H-pyrrolyl), imidazolyl, pyrazolyl, pyridyl (i.e., pyridinyl; e.g., 2-pyridyl, 3-pyridyl, or 4-pyridyl), pyrazin
  • heteroaryl preferably refers to a 5 to 14 membered (more preferably 5 to 10 membered) monocyclic ring or fused ring system comprising one or more (e.g., one, two, three or four) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized; even more preferably, a “heteroaryl” refers to a 5 or 6 membered monocyclic ring comprising one or more (e.g., one, two or three) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized.
  • heteroaryl examples include pyridinyl (e.g., 2-pyridyl, 3-pyridyl, or 4-pyridyl), imidazolyl, thiazolyl, 1 H-tetrazolyl, 2H-tetrazolyl, thienyl (i.e., thiophenyl), or pyrimidinyl.
  • heteroarylene refers to a heteroaryl group, as defined herein above, but having two points of attachment, i.e. a divalent aromatic ring group, including monocyclic aromatic rings as well as bridged ring and/or fused ring systems containing at least one aromatic ring (e.g., ring systems composed of two or three fused rings, wherein at least one of these fused rings is aromatic; or bridged ring systems composed of two or three rings, wherein at least one of these bridged rings is aromatic), wherein said aromatic ring group comprises one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, and further wherein one or more carbon ring atoms may optionally be oxidized (i).
  • each heteroatom-containing ring comprised in said aromatic ring group may contain one or two 0 atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three, or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatomcontaining ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring.
  • Heteroarylene may, e.g., refer to thienylene (i.e., thiophenylene; e.g., thien-2,3-diyl, thien-2,4-diyl, or thien-2,5-diyl), benzo[b]thienylene, naphtho[2,3-b]thienylene, thianthrenylene, furylene (i.e., furanylene; e.g., furan-2,3-diyl, furan-2,4-diyl, or furan-2,5-diyl), benzofuranylene, isobenzofuranylene, chromanylene, chromenylene, isochromenylene, chromonylene, xanthenylene, phenoxathiinylene, pyrrolylene, imidazolylene, pyrazolylene, pyridylene (i.e., pyridinylene),
  • heteroarylene preferably refers to a divalent 5 to 14 membered (more preferably 5 to 10 membered) monocyclic ring or fused ring system comprising one or more (e.g., one, two, three or four) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized; even more preferably, a "heteroarylene” refers to a divalent 5 or 6 membered monocyclic ring comprising one or more (e.g., one, two or three) ring heteroatoms independently selected from 0, S, and N, wherein one or more S ring atoms (if present) and/or one
  • heteroarylene including any of the specific heteroarylene groups described herein, may be attached through two carbon ring atoms, particularly through those two carbon ring atoms that have the greatest distance from one another (in terms of the number of ring atoms separating them by the shortest possible connection) within one single ring or within the entire ring system of the corresponding heteroarylene.
  • particularly preferred examples of a “heteroarylene” include pyridinylene, imidazolylene, thiazolylene, 1 H-tetrazolylene, 2H-tetrazolylene, thienylene (i.e., thiophenylene), or pyrimidinylene.
  • cycloalky I refers to a saturated hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings).
  • Cycloalkyl may, e.g., refer to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, decalinyl (i.e., decahydronaphthyl), or adamantyl.
  • cycloalkyl preferably refers to a C3-11 cycloalkyl, and more preferably refers to a C3-7 cycloalkyl.
  • a particularly preferred "cycloalkyl” is a monocyclic saturated hydrocarbon ring having 3 to 7 ring members.
  • particularly preferred examples of a “cycloalkyl” include cyclohexyl or cyclopropyl, particularly cyclohexyl.
  • cycloalkylene refers to a cycloalkyl group, as defined herein above, but having two points of attachment, i.e. a divalent saturated hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings).
  • Cycloalkylene may, e.g., refer to cyclopropylene (e.g., cyclopropan-1,1-diyl or cyclopropan-1 ,2-diyl), cyclobutylene (e.g., cyclobutan-1 ,1-diyl, cyclobutan-1,2-diyl, or cyclobutan-1 ,3-diyl), cyclopentylene (e.g., cyclopentan-1,1 -diyl, cyclopentan-1 , 2-diyl, or cyclopentan- 1,3-diyl), cyclohexylene (e.g., cyclohexan-1 , 1-diyl, cyclohexan-1 ,2-diyl, cyclohexan-1 ,3-diyl, or cyclohexan-1 ,4-diyl), cycloheptylene, de
  • cycloalkylene preferably refers to a C3-11 cycloalkylene, and more preferably refers to a C3-7 cycloalkylene.
  • a particularly preferred "cycloalkylene” is a divalent monocyclic saturated hydrocarbon ring having 3 to 7 ring members.
  • particularly preferred examples of a “cycloalkylene” include cyclohexylene or cyclopropylene, particularly cyclohexylene.
  • heterocycloalkyl refers to a saturated ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said ring group contains one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, and further wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group).
  • each heteroatom-containing ring comprised in said saturated ring group may contain one or two 0 atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatomcontaining ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring.
  • Heterocycloalkyl may, e.g., refer to aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, piperazinonyl (e.g., piperazin-2-on-1-yl or piperazin-3-on-1-yl), azepanyl, diazepanyl (e.g., 1,4-diazepanyl), oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, morpholinyl (e.g., morpholin-4-yl), thiomorpholinyl (e.g., thiomorpholin-4-yl), oxazepanyl, oxiranyl, oxetanyl, tetrahydrofuranyl,
  • heterocycloalkyl preferably refers to a 3 to 11 membered saturated ring group, which is a monocyclic ring or a fused ring system (e.g., a fused ring system composed of two fused rings), wherein said ring group contains one or more (e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized; more preferably, "heterocycloalkyl” refers to a 5 to 7 membered saturated monocyclic ring group containing one or more (e.g., one, two, or three) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms
  • heterocycloalkyl examples include tetrahydropyranyl, piperidinyl, piperazinyl, piperazinonyl, morpholinyl, pyrrolidinyl, or tetrahydrofuranyl.
  • heterocycloalkylene refers to a heterocycloalkyl group, as defined herein above, but having two points of attachment, i.e. a divalent saturated ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said ring group contains one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, and further wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo
  • each heteroatom-containing ring comprised in said saturated ring group may contain one or two 0 atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring.
  • Heterocycloalkylene may, e.g., refer to aziridinylene, azetidinylene, pyrrolidinylene, imidazolidinylene, pyrazolidinylene, piperidinylene, piperazinylene, azepanylene, diazepanylene (e.g., 1 ,4-diazepanylene), oxazolidinylene, isoxazolidinylene, thiazolidinylene, isothiazolidinylene, morpholinylene, thiomorpholinylene, oxazepanylene, oxiranylene, oxetanylene, tetrahydrofuranylene, 1 ,3-dioxolanylene, tetrahydropyranylene, 1,4-dioxanylene, oxepanylene, thiiranylene, thietanylene, tetrahydrothiophenylene (i.
  • heterocycloalkylene preferably refers to a divalent 3 to 11 membered saturated ring group, which is a monocyclic ring or a fused ring system (e.g., a fused ring system composed of two fused rings), wherein said ring group contains one or more (e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized; more preferably, "heterocycloalkylene” refers to a divalent 5 to 7 membered saturated monocyclic ring group containing one or more (e.g., one, two, or three) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N
  • heterocycloalkylene examples include tetrahydropyranylene, piperidinylene, piperazinylene, morpholinylene, pyrrolidinylene, or tetrahydrofuranylene.
  • cycloalkenyl refers to an unsaturated alicyclic (non-aromatic) hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said hydrocarbon ring group comprises one or more (e.g., one or two) carbon-to-carbon double bonds and does not comprise any carbon-to-carbon triple bond.
  • Cycloalkenyl may, e.g., refer to cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, or cycloheptadienyl.
  • cycloalkenyl preferably refers to a C3-11 cycloalkenyl, and more preferably refers to a C3-7 cycloalkenyl.
  • a particularly preferred "cycloalkenyl” is a monocyclic unsaturated alicyclic hydrocarbon ring having 3 to 7 ring members and containing one or more (e.g., one or two; preferably one) carbon-to-carbon double bonds.
  • cycloalkenylene refers to a cycloalkenyl group, as defined herein above, but having two points of attachment, i.e. a divalent unsaturated alicyclic (i.e., non-aromatic) hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said hydrocarbon ring group comprises one or more (e.g., one or two) carbon-to-carbon double bonds and does not comprise any carbon-to-carbon triple bond.
  • a divalent unsaturated alicyclic (i.e., non-aromatic) hydrocarbon ring group including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings
  • Cycloalkenylene may, e.g., refer to cyclopropenylene, cyclobutenylene, cyclopentenylene, cyclohexenylene, cyclohexadienylene, cycloheptenylene, or cycloheptadienylene.
  • cycloalkenylene preferably refers to a C3-11 cycloalkenylene, and more preferably refers to a C3-7 cycloalkenylene.
  • a particularly preferred "cycloalkenylene” is a divalent monocyclic unsaturated alicyclic hydrocarbon ring having 3 to 7 ring members and containing one or more (e.g., one or two; preferably one) carbon-to-carbon double bonds.
  • heterocycloalkenyl refers to an unsaturated alicyclic (non-aromatic) ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said ring group contains one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, wherein one or more carbon ring atoms may optionally be oxidized (i.e.
  • each heteroatom-containing ring comprised in said unsaturated alicyclic ring group may contain one or two 0 atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatomcontaining ring.
  • Heterocycloalkenyl may, e.g., refer to imidazolinyl (e.g., 2-imidazolinyl (i.e., 4,5-dihydro-1 H- imidazolyl), 3-imidazolinyl, or 4-imidazolinyl), tetrahydropyridinyl (e.g., 1,2,3,6-tetrahydropyridinyl), dihydropyridinyl (e.g., 1 ,2-dihydropyridinyl or 2,3-dihydropyridinyl), pyranyl (e.g., 2H-pyranyl or 4H-pyranyl), thiopyranyl (e.g., 2H-thiopyranyl or 4H-thiopyranyl), dihydropyranyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrazinyl, dihydroisoindolyl, o
  • heterocycloalkenyl preferably refers to a 3 to 11 membered unsaturated alicyclic ring group, which is a monocyclic ring or a fused ring system (e.g., a fused ring system composed of two fused rings), wherein said ring group contains one or more (e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, wherein one or more carbon ring atoms are optionally oxidized, and wherein said ring group comprises at least one double bond between adjacent ring atoms and does not comprise any triple bond between adjacent ring atoms; more preferably, "heterocycloalkenyl” refers to a 5 to 7 membered monocyclic unsaturated non-aromatic ring group containing one or more (e.g
  • heterocycloalkenylene refers to a heterocycloalkenyl group, as defined herein above, but having two points of attachment, i.e. a divalent unsaturated alicyclic (i.e., non-aromatic) ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said ring group contains one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, wherein one or more carbon ring atoms may optionally be oxidized, wherein one or
  • each heteroatom-containing ring comprised in said unsaturated alicyclic ring group may contain one or two 0 atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatomcontaining ring.
  • Heterocycloalkenylene may, e.g., refer to imidazolinylene, tetrahydropyridinylene, dihydropyridinylene, pyranylene, thiopyranylene, dihydropyranylene, dihydrofuranylene, dihydropyrazolylene, dihydropyrazinylene, dihydroisoindolylene, octahydroquinolinylene, or octahydroisoquinolinylene.
  • heterocycloalkenylene preferably refers to a divalent 3 to 11 membered unsaturated alicyclic ring group, which is a monocyclic ring or a fused ring system (e.g., a fused ring system composed of two fused rings), wherein said ring group contains one or more (e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, wherein one or more carbon ring atoms are optionally oxidized, and wherein said ring group comprises at least one double bond between adjacent ring atoms and does not comprise any triple bond between adjacent ring atoms; more preferably, "heterocycloalkenylene” refers to a divalent 5 to 7 membered monocyclic unsaturated nonaromatic ring group containing one or more (
  • halogen refers to fluoro (-F), chloro (-CI), bromo (-Br), or iodo (-I).
  • haloalkyl refers to an alkyl group substituted with one or more (preferably 1 to 6, more preferably 1 to 3) halogen atoms which are selected independently from fluoro, chloro, bromo and iodo, and are preferably all fluoro atoms. It will be understood that the maximum number of halogen atoms is limited by the number of available attachment sites and, thus, depends on the number of carbon atoms comprised in the alkyl moiety of the haloalkyl group.
  • Haloalkyl may, e.g., refer to -CF 3 , -CHF 2 , -CH 2 F, -CF 2 -CH 3 , -CH 2 -CF 3 , -CH 2 -CHF 2 , -CH 2 -CF 2 -CH 3 , -CH 2 -CF 2 -CF 3 , or -CH(CF 3 ) 2 .
  • a preferred "haloalkyl” group is fluoroalkyl.
  • a particularly preferred "haloalkyl” group is -CF 3 .
  • fluoroalky I refers to an alkyl group substituted with one or more (preferably 1 to 6, more preferably 1 to 3) fluoro atoms (-F). It will be understood that the maximum number of fluoro atoms is limited by the number of available attachment sites and, thus, depends on the number of carbon atoms comprised in the alkyl moiety of the fluoroalkyl group.
  • “Fluoroalkyl” may, e.g., refer to -CF 3 , -CHF 2 , -CH 2 F, -CF 2 -CH 3 , -CH 2 -CF 3 , -CH 2 -CHF 2 , -CH 2 -CF 2 -CH 3 , -CH 2 -CF 2 -CF 3 , or -CH(CF 3 ) 2 .
  • a particularly preferred “fl uoroalky I” group is -CF 3 .
  • the terms "bond” and "covalent bond” are used herein synonymously, unless explicitly indicated otherwise or contradicted by context.
  • the terms “optional”, “optionally” and “may” denote that the indicated feature may be present but can also be absent.
  • the present invention specifically relates to both possibilities, i.e., that the corresponding feature is present or, alternatively, that the corresponding feature is absent.
  • the expression “X is optionally substituted with Y” (or “X may be substituted with Y”) means that X is either substituted with Y or is unsubstituted.
  • a component of a composition is indicated to be “optional”, the invention specifically relates to both possibilities, i.e., that the corresponding component is present (contained in the composition) or that the corresponding component is absent from the composition.
  • substituents such as, e.g., one, two, three or four substituents. It will be understood that the maximum number of substituents is limited by the number of attachment sites available on the substituted moiety.
  • the "optionally substituted” groups referred to in this specification carry preferably not more than two substituents and may, in particular, carry only one substituent.
  • the optional substituents are absent, i.e. that the corresponding groups are unsubstituted.
  • substituent groups comprised in the compounds of the present invention may be attached to the remainder of the respective compound via a number of different positions of the corresponding specific substituent group. Unless defined otherwise, the preferred attachment positions for the various specific substituent groups are as illustrated in the examples.
  • compositions comprising “a” compound of formula (I) can be interpreted as referring to a composition comprising "one or more” compounds of formula (I).
  • the term "about” preferably refers to ⁇ 10% of the indicated numerical value, more preferably to ⁇ 5% of the indicated numerical value, and in particular to the exact numerical value indicated. If the term “about” is used in connection with the endpoints of a range, it preferably refers to the range from the lower endpoint -10% of its indicated numerical value to the upper endpoint +10% of its indicated numerical value, more preferably to the range from of the lower endpoint -5% to the upper endpoint +5%, and even more preferably to the range defined by the exact numerical values of the lower endpoint and the upper endpoint.
  • the term “comprising” (or “comprise”, “comprises”, “contain”, “contains”, or “containing”), unless explicitly indicated otherwise or contradicted by context, has the meaning of “containing, inter alia”, i.e., “containing, among further optional elements, .. In addition thereto, this term also includes the narrower meanings of “consisting essentially of' and “consisting of'.
  • a comprising B and C has the meaning of "A containing, inter alia, B and C”, wherein A may contain further optional elements (e.g., "A containing B, C and D” would also be encompassed), but this term also includes the meaning of "A consisting essentially of B and C” and the meaning of "A consisting of B and C” (i.e., no other components than B and C are comprised in A).
  • the scope of the invention embraces all pharmaceutically acceptable salt forms of the compounds of formula (I) which may be formed, e.g., by protonation of an atom carrying an electron lone pair which is susceptible to protonation, such as an amino group, with an inorganic or organic acid, or as a salt of an acid group (such as a carboxylic acid group) with a physiologically acceptable cation.
  • Exemplary base addition salts comprise, for example: alkali metal salts such as sodium or potassium salts; alkaline earth metal salts such as calcium or magnesium salts; zinc salts; ammonium salts; aliphatic amine salts such as trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, procaine salts, meglumine salts, ethylenediamine salts, or choline salts; aralkyl amine salts such as N, N-dibenzylethylenediamine salts, benzathine salts, benethamine salts; heterocyclic aromatic amine salts such as pyridine salts, picoline salts, quinoline salts or isoquinoline salts; quaternary ammonium salts such as tetramethylammonium salts, tetraethylammonium salts, benzyltrimethylammonium salts, benzyltriethylam
  • Exemplary acid addition salts comprise, for example: mineral acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate salts (such as, e.g., sulfate or hydrogensulfate salts), nitrate salts, phosphate salts (such as, e.g., phosphate, hydrogenphosphate, or dihydrogenphosphate salts), carbonate salts, hydrogencarbonate salts, perchlorate salts, borate salts, or thiocyanate salts; organic acid salts such as acetate, propionate, butyrate, pentanoate, hexanoate, heptanoate, octanoate, cyclopentanepropionate, decanoate, undecanoate, oleate, stearate, lactate, maleate, oxalate, fumarate, tartrate, malate, citrate, succinate, adipate, gluconate, glycolate, nic
  • Preferred pharmaceutically acceptable salts of the compounds of formula (I) include a hydrochloride salt, a hydrobromide salt, a mesylate salt, a sulfate salt, a tartrate salt, a fumarate salt, an acetate salt, a citrate salt, and a phosphate salt.
  • a particularly preferred pharmaceutically acceptable salt of the compound of formula (I) is a hydrochloride salt.
  • the compound of formula (I), including any one of the specific compounds of formula (I) described herein is in the form of a hydrochloride salt, a hydrobromide salt, a mesylate salt, a sulfate salt, a tartrate salt, a fumarate salt, an acetate salt, a citrate salt, or a phosphate salt, and it is particularly preferred that the compound of formula (I) is in the form of a hydrochloride salt.
  • the present invention also specifically relates to the compound of formula (I), including any one of the specific compounds of formula (I) described herein, in non-salt form.
  • the scope of the invention embraces the compounds of formula (I) in any solvated form, including, e.g., solvates with water (i.e., as a hydrate) or solvates with organic solvents such as, e.g., methanol, ethanol, isopropanol, acetic acid, ethyl acetate, ethanolamine, DMSO, or acetonitrile. All physical forms, including any amorphous or crystalline forms (i.e., polymorphs), of the compounds of formula (I) are also encompassed within the scope of the invention. It is to be understood that such solvates and physical forms of pharmaceutically acceptable salts of the compounds of the formula (I) are likewise embraced by the invention.
  • the invention also specifically relates to the compounds of formula (I) in unsolvated form, i.e., not in the form of a solvate.
  • the compounds of formula (I) may exist in the form of different stereoisomers (including, e.g., geometric isomers (or cis/trans isomers), enantiomers and diastereomers) or tautomers (including, in particular, prototropic tautomers, such as keto/enol tautomers or thione/thiol tautomers). All such isomers of the compounds of formula (I) are contemplated as being part of the present invention, either in admixture or in pure or substantially pure form.
  • stereoisomers the invention embraces the isolated optical isomers of the compounds according to the invention as well as any mixtures thereof (including, in particular, racemic mixtures/racemates).
  • the racemates can be resolved by physical methods, such as, e.g., fractional crystallization, separation or crystallization of diastereomeric derivatives, or separation by chiral column chromatography.
  • the individual optical isomers can also be obtained from the racemates via salt formation with an optically active acid followed by crystallization.
  • the present invention further encompasses any tautomers of the compounds of formula (I). It will be understood that some compounds may exhibit tautomerism. In such cases, the formulae provided herein expressly depict only one of the possible tautomeric forms.
  • the formulae and chemical names as provided herein are intended to encompass any tautomeric form of the corresponding compound and not to be limited merely to the specific tautomeric form depicted by the drawing or identified by the name of the compound.
  • the scope of the invention also embraces compounds of formula (I), in which one or more atoms are replaced by a specific isotope of the corresponding atom.
  • the invention encompasses compounds of formula (I), in which one or more hydrogen atoms (or, e.g., all hydrogen atoms) are replaced by deuterium atoms (i.e., 2 H; also referred to as “D”).
  • deuterium atoms i.e., 2 H; also referred to as “D”.
  • the invention also embraces compounds of formula (I) which are enriched in deuterium.
  • Naturally occurring hydrogen is an isotopic mixture comprising about 99.98 mol-% hydrogen-1 ( 1 H) and about 0.0156 mol-% deuterium ( 2 H or D).
  • the content of deuterium in one or more hydrogen positions in the compounds of formula (I) can be increased using deuteration techniques known in the art.
  • a compound of formula (I) or a reactant or precursor to be used in the synthesis of the compound of formula (I) can be subjected to an H/D exchange reaction using, e.g., heavy water (D2O).
  • D2O heavy water
  • deuteration techniques are described in: Atzrodt J et al., Bioorg Med Chem, 20(18), 5658-5667, 2012; William JS et al., Journal of Labelled Compounds and Radiopharmaceuticals, 53(11-12), 635-644, 2010; Modvig A et al., J Org Chem, 79, 5861-5868, 2014.
  • the content of deuterium can be determined, e.g., using mass spectrometry or NMR spectroscopy.
  • it is preferred that the compound of formula (I) is not enriched in deuterium. Accordingly, the presence of naturally occurring hydrogen atoms or 1 H hydrogen atoms in the compounds of formula (I) is preferred.
  • the present invention also embraces compounds of formula (I), in which one or more atoms are replaced by a positron-emitting isotope of the corresponding atom, such as, e.g., 18 F, 11 C, 13 N, 15 0, 76 Br, 77 Br, 120 l and/or 124 l.
  • a positron-emitting isotope of the corresponding atom such as, e.g., 18 F, 11 C, 13 N, 15 0, 76 Br, 77 Br, 120 l and/or 124 l.
  • Such compounds can be used as tracers, trackers or imaging probes in positron emission tomography (PET).
  • the invention thus includes (I) compounds of formula (I), in which one or more fluorine atoms (or, e.g., all fluorine atoms) are replaced by 18 F atoms, (ii) compounds of formula (I), in which one or more carbon atoms (or, e.g., all carbon atoms) are replaced by 11 C atoms, (ill) compounds of formula (I), in which one or more nitrogen atoms (or, e.g., all nitrogen atoms) are replaced by 13 N atoms, (iv) compounds of formula (I), in which one or more oxygen atoms (or, e.g., all oxygen atoms) are replaced by 15 O atoms, (v) compounds of formula (I), in which one or more bromine atoms (or, e.g., all bromine atoms) are replaced by 76 Br atoms, (vi) compounds of formula (I), in which one or more bromine atoms (or, e.g., all bromine
  • the compounds provided herein may be administered as compounds perse or may be formulated as medicaments.
  • the medicaments/pharmaceutical compositions may optionally comprise one or more pharmaceutically acceptable excipients, such as carriers, diluents, fillers, disintegrants, lubricating agents, binders, colorants, pigments, stabilizers, preservatives, antioxidants, and/or solubility enhancers.
  • the pharmaceutical compositions may comprise one or more solubility enhancers, such as, e.g., polyethylene glycol), including polyethylene glycol) having a molecular weight in the range of about 200 to about 5,000 Da (e.g., PEG 200, PEG 300, PEG 400, or PEG 600), ethylene glycol, propylene glycol, glycerol, a non-ionic surfactant, tyloxapol, polysorbate 80, macrogol-15-hydroxystearate (e.g., Kolliphor® HS 15, CAS 70142-34-6), a phospholipid, lecithin, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, a cyclodextrin, o-cyclodextrin, p-cyclodextrin, y-cyclodextrin, hydroxyethyl-p-cyclodextrin,
  • the pharmaceutical compositions may also comprise one or more preservatives, particularly one or more antimicrobial preservatives, such as, e.g., benzyl alcohol, chlorobutanol, 2-ethoxyethanol, m-cresol, chlorocresol (e.g., 2-chloro-3-methyl-phenol or 4-chloro-3-methyl-phenol), benzalkonium chloride, benzethonium chloride, benzoic acid (or a pharmaceutically acceptable salt thereof), sorbic acid (or a pharmaceutically acceptable salt thereof), chlorhexidine, thimerosal, or any combination thereof.
  • preservatives particularly one or more antimicrobial preservatives, such as, e.g., benzyl alcohol, chlorobutanol, 2-ethoxyethanol, m-cresol, chlorocresol (e.g., 2-chloro-3-methyl-phenol or 4-chloro-3-methyl-phenol), benzalkonium chloride, benzethonium chloride, benzoic
  • compositions can be formulated by techniques known to the person skilled in the art, such as the techniques published in "Remington: The Science and Practice of Pharmacy”, Pharmaceutical Press, 22 nd edition.
  • the pharmaceutical compositions can be formulated as dosage forms for oral, parenteral, such as intramuscular, intravenous, subcutaneous, intradermal, intraarterial, intracardial, rectal, nasal, topical, aerosol or vaginal administration.
  • Dosage forms for oral administration include coated and uncoated tablets, soft gelatin capsules, hard gelatin capsules, lozenges, troches, solutions, emulsions, suspensions, syrups, elixirs, powders and granules for reconstitution, dispersible powders and granules, medicated gums, chewing tablets and effervescent tablets.
  • Dosage forms for parenteral administration include solutions, emulsions, suspensions, dispersions and powders and granules for reconstitution. Emulsions are a preferred dosage form for parenteral administration.
  • Dosage forms for rectal and vaginal administration include suppositories and ovula.
  • Dosage forms for nasal administration can be administered via inhalation and insufflation, for example by a metered inhaler.
  • Dosage forms for topical administration include creams, gels, ointments, salves, patches and transdermal delivery systems.
  • the compounds of formula (I) or the pharmaceutically acceptable salts or solvates thereof, or the above described pharmaceutical compositions comprising any of the aforementioned entities may be administered to a subject by any convenient route of administration, whether systemically/peri pheral ly or at the site of desired action, including but not limited to one or more of: oral (e.g., as a tablet, capsule, or as an ingestible solution), topical (e.g., transdermal, intranasal, ocular, buccal, and sublingual), parenteral (e.g., using injection techniques or infusion techniques, and including, for example, by injection, e.g., subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, or intrasternal by, e.g., implant of a depot, for
  • examples of such administration include one or more of: intravenously, intraarterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternally, intracardially, intracranially, intramuscularly or subcutaneously administering the compounds or pharmaceutical compositions, and/or by using infusion techniques.
  • parenteral administration the compounds are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
  • the aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary.
  • the preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.
  • Said compounds or pharmaceutical compositions can also be administered orally in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavoring or coloring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications.
  • the tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycolate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included. Solid compositions of a similar type may also be employed as fillers in gelatin capsules.
  • excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine
  • disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glyco
  • Preferred excipients in this regard include lactose, starch, a cellulose, or high molecular weight polyethylene glycols.
  • the agent may be combined with various sweetening or flavoring agents, coloring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
  • the compounds or pharmaceutical compositions are preferably administered by oral ingestion, particularly by swallowing.
  • the compounds or pharmaceutical compositions can thus be administered to pass through the mouth into the gastrointestinal tract, which can also be referred to as "oral-gastrointestinal” administration.
  • said compounds or pharmaceutical compositions can be administered in the form of a suppository or pessary, or may be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment or dusting powder.
  • the compounds of the present invention may also be dermally or transdermally administered, for example, by the use of a skin patch.
  • sustained-release compositions include semi-permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules.
  • Sustained-release matrices include, e.g., polylactides, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, poly(2-hydroxyethyl methacrylate), ethylene vinyl acetate, or poly-D-(— )-3- hydroxybutyric acid.
  • Sustained-release pharmaceutical compositions also include liposomally entrapped compounds. The present invention thus also relates to liposomes containing a compound of the invention.
  • Said compounds or pharmaceutical compositions may also be administered by the pulmonary route, rectal routes, or the ocular route.
  • they can be formulated as micronized suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzalkonium chloride.
  • they may be formulated in an ointment such as petrolatum.
  • dry powder formulations of the compounds of formula (I) for pulmonary administration may be prepared by spray drying under conditions which result in a substantially amorphous glassy or a substantially crystalline bioactive powder. Accordingly, dry powders of the compounds of the present invention can be made according to an emulsification/spray drying process.
  • said compounds or pharmaceutical compositions can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, emulsifying wax and water.
  • they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, 2-octyldodecanol, benzyl alcohol and water.
  • the present invention thus relates to the compounds or the pharmaceutical compositions provided herein, wherein the corresponding compound or pharmaceutical composition is to be administered by any one of: an oral route; topical route, including by transdermal, intranasal, ocular, buccal, or sublingual route; parenteral route using injection techniques or infusion techniques, including by subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, intrasternal, intraventricular, intraurethral, or intracranial route; pulmonary route, including by inhalation or insufflation therapy; gastrointestinal route; intrauterine route; intraocular route; subcutaneous route; ophthalmic route, including by intravitreal, or intracameral route; rectal route; or vaginal route.
  • Preferred routes of administration are oral administration or parenteral administration.
  • a physician will determine the actual dosage which will be most suitable for an individual subject.
  • the specific dose level and frequency of dosage for any particular individual subject may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual subject undergoing therapy.
  • the precise dose and also the route of administration will ultimately be at the discretion of the attendant physician or veterinarian.
  • the compound of formula (I) or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition comprising any of the aforementioned entities can be administered in monotherapy (e.g., without concomitantly administering any further therapeutic agents, or without concomitantly administering any further therapeutic agents against the same disease that is to be treated or prevented with the compound of formula (I)).
  • the compound of formula (I) or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition comprising any of the aforementioned entities can also be administered in combination with one or more further therapeutic agents. If the compound of formula (I) is used in combination with a second therapeutic agent active against the same disease or condition, the dose of each compound may differ from that when the corresponding compound is used alone, in particular, a lower dose of each compound may be used.
  • the combination of the compound of formula (I) with one or more further therapeutic agents may comprise the simultaneous/concomitant administration of the compound of formula (I) and the further therapeutic agent(s) (either in a single pharmaceutical formulation or in separate pharmaceutical formulations), or the sequential/separate administration of the compound of formula (I) and the further therapeutic agent(s). If administration is sequential, either the compound of formula (I) according to the invention or the one or more further therapeutic agents may be administered first. If administration is simultaneous, the one or more further therapeutic agents may be included in the same pharmaceutical formulation as the compound of formula (I), or they may be administered in two or more different (separate) pharmaceutical formulations.
  • the one or more further therapeutic agents to be administered in combination with a compound of the present invention are preferably anticancer drugs.
  • the anticancer drug(s) to be administered in combination with a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof may, e.g., be selected from: a tumor angiogenesis inhibitor (e.g., a protease inhibitor, an epidermal growth factor receptor kinase inhibitor, or a vascular endothelial growth factor receptor kinase inhibitor); a cytotoxic drug (e.g., an antimetabolite, such as purine and pyrimidine analog antimetabolites); an antimitotic agent (e.g., a microtubule stabilizing drug or an antimitotic alkaloid); a platinum coordination complex; an anti-tumor antibiotic; an alkylating agent (e.g., a nitrogen mustard or a nitrosourea); an endocrine agent (e.g., an adre
  • An alkylating agent which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, a nitrogen mustard (such as cyclophosphamide, mechlorethamine (chlormethine), uramustine, melphalan, chlorambucil, ifosfamide, bendamustine, or trofosfamide), a nitrosourea (such as carmustine, streptozocin, fotemustine, lomustine, nimustine, prednimustine, ranimustine, or semustine), an alkyl sulfonate (such as busulfan, mannosulfan, or treosulfan), an aziridine (such as hexamethylmelamine (altretamine), triethylenemelamine, ThioTEPA (N.N'N'-triethylenethiophosphoramide), carboquone, or triaziquone), a hydrazine (such as procarbazine),
  • a platinum coordination complex which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, or triplatin tetranitrate.
  • a cytotoxic drug which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, an antimetabolite, including folic acid analogue antimetabolites (such as aminopterin, methotrexate, pemetrexed, or raltitrexed), purine analogue antimetabolites (such as cladribine, clofarabine, fludarabine, 6-mercaptopurine (including its prodrug form azathioprine), pentostatin, or 6-thioguanine), and pyrimidine analogue antimetabolites (such as cytarabine, decitabine, 5-fluorouracil (including its prodrug forms capecitabine and tegafur), floxuridine, gemcitabine, enocitabine, or sapacitabine).
  • folic acid analogue antimetabolites such as aminopterin, methotrexate, pemetrexed, or raltitrexed
  • An antimitotic agent which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, a taxane (such as docetaxel, larotaxel, ortataxel, paclitaxel/taxol, tesetaxel, or nab-paclitaxel (e.g., Abraxane®)), a Vinca alkaloid (such as vinblastine, vincristine, vinflunine, vindesine, or vinorelbine), an epothilone (such as epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, or epothilone F) or an epothilone B analogue (such as ixabepilone/azaepothilone B).
  • a taxane such as docetaxel, larotaxel, ortataxel, paclitaxel/taxol
  • An anti-tumor antibiotic which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, an anthracycline (such as aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, amrubicin, pirarubicin, valrubicin, or zorubicin), an anthracenedione (such as mitoxantrone, or pixantrone) or an anti-tumor antibiotic isolated from Streptomyces (such as actinomycin (including actinomycin D), bleomycin, mitomycin (including mitomycin C), or plicamycin).
  • an anthracycline such as aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, amrubicin, pirarubicin, valrubicin, or zorubicin
  • a tyrosine kinase inhibitor which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, axitinib, bosutinib, cediranib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, lestaurtinib, nilotinib, semaxanib, sorafenib, sunitinib, axitinib, nintedanib, ponatinib, vandetanib, or vemurafenib.
  • a topoisomerase inhibitor which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, a topoisomerase I inhibitor (such as irinotecan, topotecan, camptothecin, belotecan, rubitecan, or lamellarin D) or a topoisomerase II inhibitor (such as amsacrine, etoposide, etoposide phosphate, teniposide, or doxorubicin).
  • a topoisomerase I inhibitor such as irinotecan, topotecan, camptothecin, belotecan, rubitecan, or lamellarin D
  • a topoisomerase II inhibitor such as amsacrine, etoposide, etoposide phosphate, teniposide, or doxorubicin.
  • a PARP inhibitor which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, niraparib, olaparib, rucaparib, talazoparib, veliparib, pamiparib (BGB-290), BMN-673, CEP 9722, MK 4827, E7016, or 3-aminobenzamide.
  • An EGFR inhibitor/antagonist which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, gefitinib, erlotinib, lapatinib, afatinib, neratinib, osimertinib, brigatinib, dacomitinib, vandetanib, pelitinib, canertinib, icotinib, poziotinib, ABT-414, AV-412, PD 153035, PKI-166, BMS-690514, CUDC- 101 , AP26113, XL647, cetuximab, panitumumab, zalutumumab, nimotuzumab, or matuzumab.
  • anticancer drugs may also be used in combination with a compound of the present invention.
  • the anticancer drugs may comprise biological or chemical molecules, like TNF-related apoptosis-inducing ligand (TRAIL), tamoxifen, amsacrine, bexarotene, estramustine, irofulven, trabectedin, cetuximab, panitumumab, tositumomab, alemtuzumab, bevacizumab, edrecolomab, gemtuzumab, alvocidib, seliciclib, aminolevulinic acid, methyl aminolevulinate, efaproxiral, porfimer sodium, talaporfin, temoporfin, verteporfin, alitretinoin, tretinoin, anagrelide, arsenic trioxide, atrasentan, bortezomib, carmofur,
  • biological drugs like antibodies, antibody fragments, antibody constructs (for example, single-chain constructs), and/or modified antibodies (like CDR-grafted antibodies, humanized antibodies, "fully human” antibodies, etc.) directed against cancer or tumor markers/factors/cytokines involved in proliferative diseases can be employed in cotherapy approaches with the compounds of the invention.
  • biological molecules are anti-HER2 antibodies (e.g. trastuzumab, Herceptin®), anti-CD20 antibodies (e.g. Rituximab, Rituxan®, MabThera®, Reditux®), anti-CD19/CD3 constructs, and anti-TNF antibodies (see, e.g., Taylor PC, Curr Opin Pharmacol, 2003, 3(3):323-328).
  • An anticancer drug which can be used in combination with a compound of the present invention may be, in particular, an immunooncology therapeutic (such as an antibody (e.g., a monoclonal antibody or a polyclonal antibody), an antibody fragment, an antibody construct (e.g., a single-chain construct), or a modified antibody (e.g., a CDR-grafted antibody, a humanized antibody, or a "fully human” antibody) or a small molecule) targeting any one of CTLA-4, PD-1 , PD-L1 , TIGIT, TIM3, LAG3, 0X40, CSF1 R, IDO, or CD40.
  • an immunooncology therapeutic such as an antibody (e.g., a monoclonal antibody or a polyclonal antibody), an antibody fragment, an antibody construct (e.g., a single-chain construct), or a modified antibody (e.g., a CDR-grafted antibody, a humanized antibody, or a "fully human”
  • Such immunooncology therapeutics include, e.g., an anti- CTLA-4 antibody (e.g., ipilimumab or tremelimumab), an anti-PD-1 antibody (e.g., nivolumab (BMS-936558), pembrolizumab (MK-3475), pidilizumab (CT-011), cemiplimab, dostarlimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, zimberelimab, AMP-224, AMP-514 (or MEDI0680), JTX-4014, INCMGA00012 (or MGA012), or APE02058), an anti-PD-L1 antibody (e.g., atezolizumab, avelumab, durvalumab, KN035, CK-301 , BMS- 936559, MEDI4736,
  • a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising any of the aforementioned entities may be administered in combination with an immune checkpoint inhibitor, preferably an antibody (or an antigen-binding fragment thereof, or an antibody construct) directed against CTLA-4, PD-1, PD-L1, TIGIT, or LAG3.
  • an immune checkpoint inhibitor preferably an antibody (or an antigen-binding fragment thereof, or an antibody construct) directed against CTLA-4, PD-1, PD-L1, TIGIT, or LAG3.
  • Corresponding preferred examples include, but are not limited to, any one of the anti-CTLA-4 antibodies ipilimumab or tremelimumab, any one of the anti-PD-1 antibodies nivolumab, pembrolizumab, pidilizumab, cemiplimab, dostarlimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, zimberelimab, AMP-224, AMP-514, JTX-4014, INCMGA00012, or APE02058, any one of the anti-PD-L1 antibodies atezolizumab, avelumab, durvalumab, KN035, CK-301 , BMS-936559, MEDI4736, MPDL3280A, MDX- 1105, MEDI6469 or bintrafusp alfa, any one of the anti-TIGIT antibodies tiragolumab
  • the present invention thus relates to a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising any of the aforementioned entities optionally in combination with a pharmaceutically acceptable excipient, for use in the treatment or prevention of cancer, wherein the compound or the pharmaceutical composition is to be administered in combination with one or more immune checkpoint inhibitors, wherein said one or more immune checkpoint inhibitors are preferably selected from anti-CTLA-4 antibodies, anti- PD-1 antibodies, anti-PD-L1 antibodies, anti-TIGIT antibodies, and/or anti-LAG3 antibodies (for example, said one or more immune checkpoint inhibitors may be selected from anti-CTLA-4 antibodies, anti-PD-1 antibodies and/or anti- PD-L1 antibodies, such as, e.g., ipilimumab, tremelimumab, nivolumab, pembrolizumab, cemiplimab, spartalizumab, camrelizumab, sintilimab
  • the present invention thus particularly relates to a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising any of the aforementioned entities optionally in combination with a pharmaceutically acceptable excipient, for use in the treatment or prevention of cancer, wherein the compound or the pharmaceutical composition is to be administered in combination with one or more anticancer drugs (including any one or more of the specific anticancer drugs described herein above).
  • the combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formulation.
  • the individual components of such combinations may be administered either sequentially or simultaneously/concomitantly in separate or combined pharmaceutical formulations by any convenient route.
  • administration is sequential, either the compound of the present invention (i.e., the compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof) or the further therapeutic agent(s) may be administered first.
  • administration is simultaneous, the combination may be administered either in the same pharmaceutical composition or in different pharmaceutical compositions.
  • the two or more compounds must be stable and compatible with each other and the other components of the formulation.
  • they may be provided in any convenient formulation and may be administered by any convenient route.
  • the individual components of such combinations are provided in separate pharmaceutical formulations.
  • the subject or patient to be treated in accordance with the present invention may be an animal (e.g., a non-human animal).
  • the subject/patient is a mammal.
  • the subject/patient is a human (e.g., a male human or a female human) or a non-human mammal (such as, e.g., a guinea pig, a hamster, a rat, a mouse, a rabbit, a dog, a cat, a horse, a monkey, an ape, a marmoset, a baboon, a gorilla, a chimpanzee, an orangutan, a gibbon, a sheep, cattle, or a pig).
  • the subject/patient to be treated in accordance with the invention is a human.
  • Treatment of a disorder or disease, as used herein, is well known in the art.
  • Treatment of a disorder or disease implies that a disorder or disease is suspected or has been diagnosed in a patient/subject.
  • a patient/subject suspected of suffering from a disorder or disease typically shows specific clinical and/or pathological symptoms which a skilled person can easily attribute to a specific pathological condition (i.e., diagnose a disorder or disease).
  • the "treatment” of a disorder or disease may, for example, lead to a halt in the progression of the disorder or disease (e.g., no deterioration of symptoms) or a delay in the progression of the disorder or disease (in case the halt in progression is of a transient nature only).
  • the "treatment” of a disorder or disease may also lead to a partial response (e.g., amelioration of symptoms) or complete response (e.g., disappearance of symptoms) of the subject/patient suffering from the disorder or disease.
  • the "treatment” of a disorder or disease may also refer to an amelioration of the disorder or disease, which may, e.g., lead to a halt in the progression of the disorder or disease or a delay in the progression of the disorder or disease.
  • Such a partial or complete response may be followed by a relapse.
  • a subject/patient may experience a broad range of responses to a treatment (such as the exemplary responses as described herein above).
  • the treatment of a disorder or disease may, inter alia, comprise curative treatment (preferably leading to a complete response and eventually to healing of the disorder or disease) and palliative treatment (including symptomatic relief).
  • curative treatment preferably leading to a complete response and eventually to healing of the disorder or disease
  • palliative treatment including symptomatic relief.
  • prevention of a disorder or disease is also well known in the art. For example, a pati en t/su bject suspected of being prone to suffer from a disorder or disease may particularly benefit from a prevention of the disorder or disease.
  • the subject/patient may have a susceptibility or predisposition for a disorder or disease, including but not limited to hereditary predisposition.
  • a predisposition can be determined by standard methods or assays, using, e.g., genetic markers or phenotypic indicators.
  • a disorder or disease to be prevented in accordance with the present invention has not been diagnosed or cannot be diagnosed in the patient/subject (for example, the patient/subject does not show any clinical or pathological symptoms).
  • prevention comprises the use of a compound of the present invention before any clinical and/or pathological symptoms are diagnosed or determined or can be diagnosed or determined by the attending physician.
  • the present invention specifically relates to each and every combination of features and embodiments described herein, including any combination of general and/or preferred features/embodiments.
  • the invention specifically relates to each combination of meanings (including general and/or preferred meanings) for the various groups and variables comprised in formula (I).
  • Figure 1 SMNDC1 co-localizes with nuclear speckle markers
  • a Overview of SMNDCTs structure with numbered truncations (for Fig. 2f), intrinsic disorder prediction plot (MetaDisorder; Kozlowski, L. P. & Bujnicki, J. M. MetaDisorder: a meta-server for the prediction of intrinsic disorder in proteins. BMC Bioinformatics 13, 111 (2012)), AlphaFold structure prediction with Vietnamese domain and positions of GFP intron-tags marked, b, Live images of clonal cell lines (aTC1 and HAP1) with the endogenous GFP-tag.
  • c Immunoblots showing expression of WT SMNDC1 and SMNDC1-GFP fusion proteins in clonal cell lines with GFP-tag in different introns
  • d Live (SMNDC1-GFP intron 2-3, aTC1) and immunofluorescence images (aTC1 WT) with nuclear staining (DRAQ5TM I DAPI).
  • e Live imaging (SMNDC1-GFP intron 2-3, aTC1) with DRAQ5TM nuclear staining showing a cell during M-phase.
  • f Immunofluorescence images (aTC1 WT) with SMNDC1 and SC35 antibody, and overlap.
  • SMNDC1 shows biomolecular condensation in vitro and in cellular systems
  • a In vitro droplet formation assay with 10 piM GFP or SMNDC1-GFP fusion protein +/- 10% PEG-8000
  • b In vitro droplet formation assay of SMNDC1-GFP over time with droplet fusion event, marked by arrows
  • c In vitro droplet formation assay of SMNDC1-GFP with quantified number of droplets with different protein and NaCI concentrations, +/- 10 ng/pil RNA.
  • d In vitro droplet formation assay of SMNDC1-GFP with the addition of 10 ng/pil total cellular RNA and RNase
  • e In vitro droplet formation assay of SMNDC1-GFP with 100 ng/pil Cy5-labeled RNA, and overlap
  • f In vitro droplet formation assay of different truncations of SMNDC1-GFP + 10 ng/pil total cellular RNA.
  • g Live imaging (SMNDC1-GFP intron 2-3, aTC1), cells were treated with 2.5% or 5 % 1 ,6-hexanediol. Quantifications of GFP intensity and GFP spots/nucleus in 4 different clonal cell lines.
  • Figure 3 Characterization of SMNDCTs interactome by proximity labeling, a, Scheme of proximity labeling by APEX2 fusion proteins followed by mass spectrometry-based proteomics, b, Immunofluorescence images (aTC1 WT) with staining against SMNDC1, Biotin via Streptavidin and nuclear staining DAPI. c, Volcano plot showing Iog2 abundance against -Iog10 adjusted p-value (one-way ANOVA, Benjamini-Hochberg correction for multiple comparisons) of APEX2-SMNDC1 FL versus APEX2-SMNDC1 TD biotinylated and enriched proteins.
  • d Enrichr analysis of APEX2-SMNDC1 FL enriched proteins, GO Biological Process 2021 terms plotted with their odds ratio and their adjusted p-value (Benjamini- Hochberg method for correction for multiple hypotheses testing).
  • Figure 4 Identification of an inhibitor against SMNDCTs Vietnamese domain, a, Scheme of AlphaScreen set-up with the NMR-structure of SMNDCTs Very domain (PDB: 4A4H).
  • b Screening strategy starting with -90,000 compound library, c, Overview of AlphaScreen of full -90,000 compound library with DMSO, positive control (quencher), and compound hits, d, AlphaScreen percentage of DMSO control with SMNDC1/ sDMA-peptide vs crosslinking peptide. Remaining hits marked, e-k, Chemical structure and AlphaScreen 9-point compound titration with SMNDC1/ sDMA- peptide vs.
  • Figure 5 Structure-activity relationships of the 2-aryl-4-aminothiazole Although Domain inhibitors. Grey scale-coded chemical structures illustrating structure activity relationships for compound 1. Modifications to the 2-aryl moiety, replacements of the thiazole group, linker modifications and arylamide analogs are shown in different grey scales. Underneath are IC50 values (piM) for SMNDC1 in black (on the left) and SMN in grey (on the right).
  • Figure 6 Inhibitor binds at the aromatic cage of the Vietnamese domain, a, Chemical shift perturbations (CSP) of Tale domains SMNDC1 and SMN in presence of 0.8 mM compound 13. Residues forming the aromatic cage are highlighted in bold. Left inlet shows cartoon representation of SMNDC1 in complex with compound 13 (grey sticks) calculated using semi-rigid body docking of compound 13 based on 23 intermolecular NOE restraints (see d and Table 3). CSP per residue in presence of 8 mM compound 13 are displayed in grey shades. Right inlet shows compound 13 with numbers indicating the assignment used in d and Table 3. b, Zoomed view of the binding site with residues forming the aromatic cage shown as sticks.
  • CSP Chemical shift perturbations
  • d Live imaging of cells (SRRM2-RFP intron 9-10, aTC1) treated with DMSO, 50 piM compound 1, or 50 piM compound 9.
  • e Quantification of SRRM2-RFP spots/ nucleus in live imaging data of SRRM2-RFP cell line (aTC1) treated with DMSO, 50 piM compound 1, or 50 piM compound 9.
  • RNA-sequencing data plotted are all overlapping alternative splicing events between compound 1 over DMSO treatment (x-axis) and SMNDC1 knockdown (KD) over empty vector (EV) (y-axis) with their respective dPSI-values. Results from a simple linear regression analysis. Events confirmed via PCR in light grey, not confirmed events in dark grey. I, DNA-bands on agarose gel after reverse transcription and PCR amplification of RNA to confirm alternative splicing events. RNA was isolated from aTC1 cells transfected with empty vector or SMNDC1 knock-down (KD) plasmid and treated with DMSO, 2 pi M compound 1 for 5 days, or 50 piM compound 1 for 16 h.
  • d Live imaging of SMNDC1-GFP intron 2-3 cell line with DRAQ5TM nuclear staining, control (no overexpression), overexpressing CLK1 or DYRK3.
  • e Co-localization analyses of IF images of aTC1 WT with SMNDCI-antibody, SC35-antibody, and DAPI nuclear staining. Pearson correlation between different channels of maximum intensity projections of z-stack images. Data shown as scatter plot + median, analyzed by unpaired t-test.
  • f Comparison of Pearson correlation values in co-localization analyses of mitotic and interphase cells, live imaging corresponding to Fig. 1g+h. Data shown as scatter plot + median.
  • Figure 9 Characterization of SMNDCTs interactome by proximity labeling
  • a Depiction of APEX2-fusion constructs APEX2-SMNDC1 FL and APEX2-SMNDC1 TD .
  • b Ponceau S staining of all proteins and western blot with Streptavidin- HRP showing all biotinylated proteins
  • c Volcano plot showing Iog2 abundance against -Iog10 adjusted p-value of APEX2-SMNDC1 FL versus APEX2-SMNDC1 TD biotinylated and enriched proteins.
  • Nuclear proteins highlighted, d Venn diagrams showing the overlap of all proteins with a known sDMA modification or aDMA modification (light grey; circles on the left), and APEX2-SMNDC1 FL enriched proteins or all proteins identified with SMNDC1 proximity labeling (dark grey; circles on the right).
  • Figure 11 SMNDCI/inhibitor complex, a, Inter-molecular NOEs (dashed line) used in the restraint-driven docking simulation. NOEs derived from different protons of the inhibitor are colored individually. Phe or Tyr protons HE and HD were not specifically assigned, and a dashed line is only shown for the proton with the closest distance to the NOE contact. The inlet shows the relative orientation of the structure compared to Figure 6.
  • d Quantification of nuclear SRRM2-RFP in live imaging data of different SRRM2-RFP cell lines (aTC1) treated with DMSO, or 50 pM compound 1. Data analyzed by ratio-paired t- test.
  • KD SMNDC1 knock-down
  • Top panel Live imaging of cells (SMN-RFP intron 5-6, aTC1) treated with DMSO or 50 pM compound 1. Quantification of whole cell SMN-RFP and SMN-RFP spots/ cytoplasm in live imaging data of different SMN-RFP cell lines (aTC1).
  • Figure 13 Effects of SMNDC1 Sixfold domain inhibition on interactome and splicing
  • a Volcano plot showing Iog2 protein abundance against -Iog10 adjusted p-value of compound 1 treated cells over DMSO control after APEX2- SMNDC1 TD proximity labeling and biotin enrichment.
  • b Volcano plot showing Iog2 protein abundance against -Iog10 adjusted p-value of compound 1 treated cells over DMSO control after APEX2-SMNDC1 FL proximity labeling and biotin enrichment.
  • Nuclear speckle proteins marked, c Volcano plot showing Iog2 protein abundance against -Iog10 adjusted p-value of compound 1 treated cells over DMSO control after APEX2-SMNDC1 FL proximity labeling and biotin enrichment.
  • Proteins identified by SRSF7-APEX2 proximity labeling marked, d Western blot with Streptavidin-HRP showing all biotinylated proteins, antibodies against SFPQ, APEX2, and SMNDC1.
  • Cells overexpressing APEX2-SMNDC1 FL or APEX2-SMNDC1 TD were treated with DMSO or compound 1, and proximity-labeled.
  • e Fluorescence recovery after photobleaching (FRAP) experiment in SMNDC1-GFP intron 2-3, SRRM2-RFP intron 9-10, aTC1-cells, treated with DMSO (filled symbols) or 50 piM compound 1 (empty symbols). Relative intensity of Hoechst SMNDC1-GFP, and SRRM2-RFP only in reference region over time (bleach region: see Fig. 7i). Data plotted as mean with standard error of the mean, n > 11. f, DNA- bands on agarose gel after reverse transcription and PCR amplification of RNA to confirm alternative splicing events.
  • FRAP Fluorescence recovery after photobleaching
  • RNAbindRplus of SMNDC1 RNAbindRplus score of amino acids (AA) over length of SMNDC1 protein. Areas over threshold of 0.5 marked in dark grey.
  • the compounds described in this section are defined by their chemical formulae and their corresponding chemical names.
  • the present invention relates to both the compound defined by the chemical formula and the compound defined by the chemical name, and particularly relates to the compound defined by the chemical formula.
  • Solvents and inorganic reagents were obtained from Lactan, Carl Roth, Sigma-Aldrich or TCI and were used as received. Reagents were obtained from abcr, Enamine or Sigma-Aldrich and were used as received; detailed sources are given further below.
  • the carboxylic acid (1.0 mmol) was dissolved in anhydrous DMF (2 ml), and diisopropylethylamine (3 mmol). With stirring, PyBOP (1.2 mmol) was added, and the activation mixture was stirred for 20 min at room temperature (rt). A solution of the amine substrate (1.0 mmol) in anhydrous DMF was added and the reaction was stirred at ambient temperature for 20 h. The reaction was then diluted with water and extracted with ethyl acetate (3 x 50 mL).
  • the pomalidomide analogs used as reagents were synthesized as described in J. Med. Chem. 2018, 61, 462-481.
  • Examples 1-19 sources and catalog numbers of commercial compounds (structures depicted in Example C below)
  • Example 1 8016-5462, ChemDiv
  • Example 4 F0463-0182, Life Chemicals Example 5: F0737-0296, Life Chemicals Example 6: F0789-0042, Life Chemicals Example 7: F0866-0370, Life Chemicals Example 8: F1166-0142, Life Chemicals Example 9: F2450-0086, Life Chemicals Example 10: F2536-1240, Life Chemicals Example 11 : F5127-0237, Life Chemicals Example 12: G786-1089, ChemDiv Example 13: G786-1145, ChemDiv Example 14: G786-1153 ChemDiv Example 15: G786-1206, ChemDiv Example 16: G786-1438, ChemDiv Example 17: F0700-0008
  • Examples 63-87 sources and catalog numbers of commercial compounds (structures depicted in Example C below)
  • Example 20 tert-butyl 4-(2-(3-(4-methylpiperazine-1-carbonyl)-5-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)phenoxy)ethyl)piperazine-1-carboxylate
  • Example 20B 3-(4-methylpiperazine-1-carbonyl)-5-(2-(piperazin-1-yl)ethoxy)-N-(4-(pyridin-2-yl)thiazol-2- yl)benzamide
  • Example 20 (68 mg) was dissolved in 4M HOI I dioxane (1 mL) and the resulting solution was stirred for 18 h. The reaction mixture was evaporated, and the resulting red film was used without purification.
  • Example 21 3-(2-(3-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)-N-(3-(3-(but-3-yn-1-yl)-3H-diazirin-3- yl)propanoyl)propanamido)ethoxy)-5-(4-methylpiperazine-1-carbonyl)-N-(4-(pyridin-2-yl)thiazol-2- yl)benzamide
  • Example 23 3-(2-(4-(3-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)propanoyl)piperazin-1-yl)ethoxy)-5-(4- methylpiperazine-1-carbonyl)-N-(4-(pyridin-2-yl)thiazol-2-yl)benzamide
  • the title compound was prepared according to general procedure A from 3-(3-(but-3-yn-1-yl)-3H-diazirin-3- yl)propanoic acid (13.9 mg, 84 pimol), HATU (42.2 mg, 111 pimol), iP ⁇ NEt (80 piL), Example 20B (38.8 mg, 60 pimol) in anhydrous DMF (1.0 mL). The reaction mixture was stirred under exclusion of light for 18 h. The clear amber solution was then diluted with brine and extracted with EtOAc (3 x 20 mL). The organic solutions were washed with H2O and brine, dried on Na2SO4 and evaporated. The crude product was subjected to flash chromatography (SiO2, CH2CI2 1 MeOH (0-20%). The title compound was obtained as a brownish film (7.8 mg, 11.4 pimol, 19%).
  • the title compound was prepared according to general procedure A from 3-(2-aminoethoxy)-5-(4-methylpiperazine- 1-carbonyl)-N-(4-(pyridin-2-yl)thiazol-2-yl)benzamide (HCI salt, 25 mg, 46 pimol), 3-(3-(but-3-yn-1-yl)-3H-diazirin-3- yl)propanoic acid (8.4 mg, 50 pimol), HATU (24 mg, 63 pimol), iP ⁇ NEt (50 piL), anhydrous DMF (0.4 mL). The reaction mixture was stirred for 18 h under light exclusion. The solution was diluted with brine and extracted with EtOAc (3 x 20 mL).
  • Example 25 3-hydroxy-5-(4-methylpiperazine-1-carbonyl)-N-(4-(pyridin-2-yl)thiazol-2-yl)benzamide 5-Hydroxyisophthalic acid (29.30 g, 0.161 mol) was dissolved in DMF (150 mL) and IP ⁇ NEt (87 mL, 0.499 mol) and treated with PyBOP (84.6 g, 0.163 mol) and N-methylpiperazine (16.15 g, 0.161 mol).
  • Example 26 tert-butyl 2-(3-(4-methylpiperazine-1-carbonyl)-5-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)phenoxy)acetate
  • Example 27 3-(2-((3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)propyl)amino)-2-oxoethoxy)- 5-(4-methylpiperazine-1-carbonyl)-N-(4-(pyridin-2-yl)thiazol-2-yl)benzamide
  • the title compound was prepared according to the general procedure A from 2-(3-(4-methylpiperazine-1-carbonyl)- 5-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)phenoxy)acetic acid (24.8 mg, 51.5 pimol), HATU (31.2 mg, 82 pimol) in anhydrous DMF (1.5 mL), IP ⁇ NEt (100 piL), 4-((3-aminopropyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3- dione TFA salt (29.2 mg, 52.3 pimol). Chromatographic purification of the crude product (SiO2, C ⁇ Ch/MeOH (0-20%) afforded the title compound as a yellow film (15.3 mg, 19.3 pimol, 37%).
  • the title compound was prepared according to the general procedure A, from 2-(3-(4-methylpiperazine-1 -carbonyl)- 5-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)phenoxy)acetic acid (22.3 mg, 46.3 pimol), HATU (24.5 mg, 64.4 pimol) in anhydrous DMF (1.5 mL), IP ⁇ NEt (100 piL), 4-((6-aminohexyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1 , 3-dione (TFA salt, 24.7 mg, 41 pimol). Chromatographic purification of the crude product (SiO2, CF C /MeOH (0-20%) afforded the title compound as a tan solid glass (11.6 mg, 13.9 pimol, 30%).
  • Example 29 3-(2-((2-(2-((2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethyl)amino)-2- oxoethoxy)-5-(4-methylpiperazine-1-carbonyl)-N-(4-(pyridin-2-yl)thiazol-2-yl)benzamide
  • the title compound was prepared according to the general procedure A, from 2-(3-(4-methylpiperazine-1 -carbonyl)- 5-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)phenoxy)acetic acid (25.7 mg, 53.4 pimol), HATU (28.6 mg, 75.2 pimol) in anhydrous DMF (1.5 mL), IPr2NEt (100 piL), 4-((2-(2-aminoethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline- 1 ,3-dione TFA salt (33.5 mg, 58.5 pimol). Chromatographic purification of the crude product (SiO2, C ⁇ C /MeOH (0- 20%) afforded the title compound as a yellow film (14.5 mg, 17.6 pimol, 33%).
  • Example 30 3-((20-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-2,14-dioxo-6,9,12-trioxa-3,15- diazaicosyl)oxy)-5-(4-methylpiperazine-1-carbonyl)-N-(4-(pyridin-2-yl)thiazol-2-yl)benzamide
  • the title compound was prepared according to the general procedure A, from 2-(3-(4-methylpiperazine-1 -carbonyl)- 5-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)phenoxy)acetic acid (23.9 mg, 49.6 pimol), HATU (25.1 mg, 66 pimol) in anhydrous DMF (1.5 mL), IP ⁇ NEt (100 piL), 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-N-(5-((2-(2,6-dioxopiperidin-3- yl)-1 ,3-dioxoisoindolin-4-yl)amino)pentyl)acetamide TFA salt (32.2 mg, 41.5 pimol).
  • Neat tert-butyl 2-(3-(4-methylpiperazine-1-carbonyl)-5-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)phenoxy)acetate (0.75 g, 1.395 mmol) was cooled to 0°C and treated slowly with TFA (3.0 mL) while stirring. The solution was stirred at 0°C for 1 h, then it was allowed to warm to ambient temperature and was stirred for a further 1.5 h. The solvent was evaporated below 40°C and traces of TFA were removed by repeated evaporation with CH2CI2. The hygroscopic residue was dried in vacuo and used without purification.
  • Example 36 (2S,4R)-1-((S)-2-(tert-butyl)-14-(3-(4-methylpiperazine-1-carbonyl)-5-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)phenoxy)-4,13-dioxo-6,9-dioxa-3,12-diazatetradecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5- yl)benzyl)pyrrolidine-2-carboxamide
  • the title compound was prepared according to the general procedure A, from 2-(3-(4-methylpiperazine-1 -carbonyl)- 5-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)phenoxy)acetic acid (27.7 mg, 57.5 pimol), HATU (24.0 mg, 63.1 pimol) in anhydrous DMF (1.5 mL), IP ⁇ NEt (100 piL), (2S,4R)-1-((S)-2-(2-(2-(2-aminoethoxy)ethoxy)acetamido)-3,3- dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide HCI (VHL ligand 1-PEG2- NH2 HCI, 31.6 mg, 51.6 pimol). Chromatographic purification of the crude product (SiO2, C ⁇ C /MeOH (0-25%). The title compound was obtained as a
  • the title compound was prepared according to the general procedure A, from 2-(3-(4-methylpiperazine-1 -carbonyl)- 5-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)phenoxy)acetic acid (25.6 mg, 53.2 pimol), HATU (26.1 mg, 68.6 pimol) in anhydrous DMF (2.0 mL), IP ⁇ NEt (100 piL), VHL ligand I-C8-NH2 HCI (29.9 mg, 48 pimol). Chromatographic purification of the crude product (SiO2, C ⁇ Ch/MeOH (0-25%). The title compound was obtained as a tan film (19.1 mg, 17.8 pimol, 34%).
  • the title compound was prepared according to the general procedure A, from 2-(3-(4-methylpiperazine-1 -carbonyl)- 5-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)phenoxy)acetic acid (30.0 mg, 62.3 pimol), HATU (27.4 mg, 72.1 pimol) in anhydrous DMF (1.5 mL), iP ⁇ NEt (100 piL), VHL ligand 1-PEG4-NH2 HCI, 36.7 mg, 52.5 pimol). Chromatographic purification of the crude product (SiO2, CH2Cl2/MeOH (0-25%). The title compound was obtained as a tan gum (28.9 mg, 25.6 pimol, 41 %).
  • the title compound was prepared according to the general procedure A, from 2-(3-(4-methylpiperazine-1 -carbonyl)- 5-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)phenoxy)acetic acid (28.4 mg, 59 pimol), HATU (29.8 mg, 78.4 pimol) in anhydrous DMF (2.0 mL), iP ⁇ NEt (120 piL), (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4- methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (27 mg, 57.8 pimol). After chromatographic purification of the crude product (SiO2, C ⁇ Ch/MeOH (0-25%), the title compound was obtained as a light tan solid glass (17.9 mg, 20.0 pimol, 34%).
  • Example 40 3-(2-((10-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)decyl)amino)-2- oxoethoxy)-5-(4-methylpiperazine-1-carbonyl)-N-(4-(pyridin-2-yl)thiazol-2-yl)benzamide
  • the title compound was prepared according to the general procedure A, from 2-(3-(4-methylpiperazine-1 -carbonyl)- 5-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)phenoxy)acetic acid (26.8 mg, 55.7 pimol), HATU (34.5 mg, 90.7 pimol) in anhydrous DMF (1.5 mL), iP ⁇ NEt (100 piL), 4-((10-aminodecyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3- dione TFA salt (29.6 mg, 45.1 pimol). Chromatographic purification of the crude product (SiO2, C ⁇ Ch/MeOH (0-20%) afforded the title compound as a yellow film (21 .8 mg, 24.4 pimol, 44%).
  • the title compound was prepared according to the general procedure A, from 4-(dimethylsulfamoyl)benzoic acid (71.4 mg, 0.312 mmol), HATU (160 mg, 0.421 mmol), 5-ethyl-4-(pyridin-2-yl)thiazol-2-amine (67.9 mg, 0.325 mmol), iP ⁇ NEt (250 pi L) in DMF (3.5 mL). After chromatographic purification (SiO2, C ⁇ C /MeOH (0-25%)) the title product was obtained as yellow solid (64.3 mg, 0.155 mmol, 50%).
  • the title compound was prepared according to the general procedure A, from 4-(dimethylsulfamoyl)benzoic acid (74.2 mg, 0.324 mmol), HATU (163 mg, 0.429 mmol), 5-bromo-4-(pyridin-2-yl)thiazol-2-amine (89 mg, 0.264 mmol), iP ⁇ NEt (250 piL) in DMF (1.5 mL). After chromatographic purification (SiO2, Cf ⁇ C /MeOH (0-25%)) the title product was obtained as yellow solid weighing 102 mg, 0.218 mmol, 67%.
  • Example 45 4-(Pyridin-2-yl)thiazol-2-amine (Example 45) (0.100 g, 0.564 mmol) was dissolved in acetone (2 mL) and treated with a solution of 85% H 3 PO 4 (40 piL) in acetone. The precipitated monobasic phosphate of the compound was filtered and washed with acetone, then air dried to afford the salt in quantitative yield.
  • Step 1 methyl 4-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)benzoate
  • Step 2 4-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)benzoic acid
  • Methyl 4-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)benzoate was ground with 10 mL MeOH until a homogenous suspension had been obtained, then 10 M NaOH (5 mL) was added slowly with vigorous stirring. The reaction mixture thickened and was stirred overnight. This suspension of the Na salt of the product was filtered and washed with MeOH. The salt was suspended in H2O (8 mL) and acidified with cc. HOI. The obtained white solid was filtered and washed with H2O. After drying, the title compound was obtained as an off-white solid, which could be recrystallized from boiling MeOH to increase purity. 1 .85 g of a white solid was obtained.
  • the title compound was prepared according to general procedure B from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (20.3 mg, 62.6 pimol), PyBOP (47.9 mg, 92.1 pimol), iP ⁇ NEt (100 piL), and 4-((6- aminohexyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1 , 3-dione (TFA salt, 34.6 mg, 57.6 pimol) in anhydrous DMF (1.5 mL).
  • the crude reaction product was subjected to flash chromatography (SiO2, CH2CI2 1 MeOH (0-15%)).
  • the desired compound was obtained as a yellow film (4.3 mg, 6.3 pimol, 11 %).
  • the title compound was prepared according to general procedure B from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (25.5 mg, 79 pimol), PyBOP (53.4 mg, 103 pimol), IP ⁇ NEt (200 piL), and 4-((5- aminopentyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1, 3-dione (TFA salt, 42.3 mg, 76.5 pimol) in anhydrous DMF (2.0 mL).
  • the crude reaction product was subjected to flash chromatography (SiO2, CH2CI2 1 MeOH (0-15%)). The desired compound was obtained as a yellow solid (17.6 mg, 26.4 pimol, 33%).
  • the title compound was prepared according to general procedure A from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (32.8 mg, 101 pimol), HATU (45.7 mg, 120 pimol), IP ⁇ NEt (200 piL), and 4-((2-(2- aminoethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1, 3-dione (TFA salt, 61.8 mg, 105 pimol) in anhydrous DMF (2.5 mL).
  • the crude reaction product was subjected to flash chromatography (SIO2, CH2CI21 MeOH
  • the title compound was prepared according to general procedure A from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (28.6 mg, 88 pimol), HATU (39.3 mg, 103 pimol), IP ⁇ NEt (200 piL), and 4-((2-(2-(2- aminoethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1, 3-dione (TFA salt, 55.3 mg, 87.4 pimol) in anhydrous DMF (1 .5 mL).
  • the crude reaction product was subjected to flash chromatography (SIO2, CH2CI21 MeOH (0-15%)). Yellow solid glass (12.6 mg, 17.7 mol, 20%).
  • the title compound was prepared according to general procedure A from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (28.9 mg, 89 pimol), HATU (45.0 mg, 118 pimol), IP ⁇ NEt (100 piL), and VHL ligand 1- PEGI-NH2 (HCI salt, 39.8 mg, 70 pimol) in anhydrous DMF (2.0 mL).
  • the crude reaction product was subjected to flash chromatography (SIO2, CH2CI2 1 MeOH (0-15%)). A tan amorphous solid was obtained (25.9 mg, 30.9 pimol, 35%).
  • Example 54 N1-(7-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)- 3,3-dimethyl-1-oxobutan-2-yl)amino)-7-oxoheptyl)-N4-(4-(pyridin-2-yl)thiazol-2-yl)terephthalamide
  • Example 55 N1-((S)-16-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1- carbonyl)-17,17-dimethyl-14-oxo-3,6,9,12-tetraoxa-15-azaoctadecyl)-N4-(4-(pyridin-2-yl)thiazol-2- yl)terephthalamide
  • Example 56 N1-(3-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)- 3,3-dimethyl-1-oxobutan-2-yl)amino)-3-oxopropyl)-N4-(4-(pyridin-2-yl)thiazol-2-yl)terephthalamide
  • the title compound was prepared according to general procedure A from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (30.0 mg, 92.6 pimol), HATU (47.9 mg, 126 pimol), IP ⁇ NEt (250 piL), and (2S,4R)-1-((S)- 2-(3-aminopropanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2- carboxamide (34.1 mg, 63.4 pimol) in anhydrous DMF (2.0 mL).
  • Example 57 N1-(2-(2-(2-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1- yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)ethoxy)ethyl)-N4-(4-(pyridin-2-yl)thiazol-2- yl)terephthalamide
  • the title compound was prepared according to general procedure A from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (24.4 mg, 75 pimol), HATU (48.4 mg, 127.3 pimol), IP ⁇ NEt (200 piL), and (2S,4R)-1-((S)- 2-(2-(2-(2-aminoethoxy)ethoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5- yl)benzyl)pyrrolidine-2-carboxamide HCI (38.2 mg, 62 pimol) in anhydrous DMF (2.0 mL). The crude reaction product was subjected to flash chromatography (SiO2, CH2CI2 1 MeOH (0-25%)). A light tan gum was obtained (31 mg, 35 pimol, 47%).
  • Example 58 N1-((S)-13-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1- carbonyl)-14,14-dimethyl-11-oxo-3,6,9-trioxa-12-azapentadecyl)-N4-(4-(pyridin-2-yl)thiazol-2- yl)terephthalamide
  • the title compound was prepared according to general procedure A from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (29.3 mg, 90 pimol), HATU (46.7 mg, 122.8 pimol), IP ⁇ NEt (250 piL), and (2S,4R)-1-((S)- 14-amino-2-(tert-butyl)-4-oxo-6,9,12-trioxa-3-azatetradecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5- yl)benzyl)pyrrolidine-2-carboxamide (41.2 mg, 63 pimol) in anhydrous DMF (2.0 mL). The crude reaction product was subjected to flash chromatography (SIO2, CH2CI2 1 MeOH (0-25%)). A yellowish glass was obtained (7.9 mg, 8.5 pimol, 13.5%).
  • the title compound was prepared according to general procedure A from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (17.3 mg, 53 pimol), HATU (31.7 mg, 83.4 pimol), IP ⁇ NEt (200 piL), and 4-((8- aminooctyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1 , 3-dione (TFA salt, 26.9 mg, 40.2 pimol) in anhydrous DMF (2.0 mL).
  • the crude reaction product was subjected to flash chromatography (SIO2, CH2CI2 1 MeOH (0-25%)). Yellow solid glass (22 mg, 31 pimol, 77%).
  • Example 60 N1-(9-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)- 3,3-dimethyl-1-oxobutan-2-yl)amino)-9-oxononyl)-N4-(4-(pyridin-2-yl)thiazol-2-yl)terephthalamide
  • the title compound was prepared according to general procedure A from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (29.5 mg, 91 pimol), HATU (41.4 mg, 108 pimol), IP ⁇ NEt (200 piL), and (2S,4R)-1-((S)-2- (9-aminononanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (43.1 mg, 69 pimol) in anhydrous DMF (2.0 mL).
  • the crude reaction product was subjected to flash chromatography (SiO2, EtOAc / MeOH (0-30%)). A yellowish glass was obtained (13.5 mg, 15 pimol, 22%).
  • Example 61 N1-(2-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4- yl)amino)ethoxy)ethoxy)ethyl)-N4-(4-(pyridin-2-yl)thiazol-2-yl)terephthalamide
  • the title compound was prepared according to general procedure A from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (26.8 mg, 82.7 pimol), HATU (45.1 mg, 118.6 pimol), IP ⁇ NEt (200 piL), and 4-((2-(2-(2-(2- aminoethoxy)ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1 , 3-dione TFA salt (50.5 mg, 74.7 pimol) in anhydrous DMF (2.0 mL).
  • the crude reaction product was subjected to flash chromatography (SIO2, CH2CI2 I MeOH (0-25%)). Yellow solid glass (22 mg, 29 pmol, 39%).
  • the title compound was prepared according to the general procedure B from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (0.716 g, 2.137 mmol), PyBOP (1.973 g, 3.79 mmol), iP ⁇ NEt (2.0 mL), N-Boc-1 ,2- ethylenediamine (0.375 g, 2.34 mmol) in anhydrous DMF (10 mL). The crude product was taken up in acetone and spontaneously crystallized. The desired compound was obtained as a white solid (0.665 g, 1 .42 mmol, 67%).
  • Example 88 tert-butyl 4-(4-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)benzoyl)piperazine-1-carboxylate
  • the title compound was prepared according to the general procedure B from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (0.132 g, 0.405 mmol), PyBOP (0.4028 g, 0.636 mmol), iP ⁇ NEt (0.25 mL), Boc-piperazine (0.106 g, 0.569 mmol) in anhydrous DMF (2.5 mL).
  • the crude reaction product was subjected to column chromatography Si O2, CH2CI21 MeOH (0-20%).
  • the desired compound was obtained as a white solid (0. 108 g, 0.219 mmol, 55%).
  • Example 88B tert-butyl (3-(4-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)benzamido)propyl)carbamate
  • the title compound was prepared according to general procedure B from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (0.119 g, 0.365 mmol), PyBOP (0.331 g, 0.636 mmol), IP ⁇ NEt (0.25 mL), tert-butyl-3- aminopropyl carbamate (0.143 g, 0.821 mmol) in anhydrous DMF (3 mL). Column chromatography of the crude product (SIO2, hexanes/EtOAc (0-100%)) afforded the title compound as white solid (0.048 g, 102 pimol, 28%).
  • the title compound was prepared according to the general procedure B for amide couplings from 4-((4-(pyridin-2- yl)thiazol-2-yl)carbamoyl)benzoic acid (0.1088 g, 0.334 mmol), PyBOP (0.310 g, 0.596 mmol), IP ⁇ NEt (0.37 mL), t- butyl 4-aminobutyl carbamate (0.102 g, 0.54 mmol) in anhydrous DMF (3 mL). The crude reaction product was subjected to column chromatography (SIO2, CH2CI21 MeOH (0-5%)). The desired compound was obtained as white solid (0.0256 g, 0.052 mmol, 16%).
  • the title compound was prepared according to the general procedure A for HATU couplings from 4-((4-(py ridin-2- yl)thiazol-2-yl)carbamoyl)benzoic acid (0.095 g, 0.291 mmol), HATU (0.158 g, 0.416 mmol), IP ⁇ NEt (0.25 mL), t-butyl 5-aminopentyl carbamate (0.074 g, 0.37 mmol) in anhydrous DMF (2.0 mL). The crude reaction product was washed with acetone to obtain the pure product as an off-white powder (71 mg, 0.139 mmol, 48%).
  • the title compound was prepared according to the general procedure A for HATU couplings from 4-((4-(py ridin-2- yl)thiazol-2-yl)carbamoyl)benzoic acid (0.095 g, 0.292 mmol), HATU (0.143 g, 0.376 mmol), iP ⁇ NEt (0.25 mL), Boc- PEG1 -diamine (0.084 g, 0.411 mmol) in anhydrous DMF (2.5 mL).
  • the crude reaction product was subjected to column chromatography (SiO2, CH2CI2 1 MeOH (0-5%) twice to obtain the title compound as a yellowish oil that set up to a solid (23.7 mg).
  • the title compound was prepared according to the general procedure A for HATU couplings from 4-((4-(py ridin-2- yl)thiazol-2-yl)carbamoyl)benzoic acid (0.095 g, 0.292 mmol), HATU (0.145 g, 0.381 mmol), iP ⁇ NEt (0.20 mL), glycine t-butyl ester HCI (0.0696 g, 0.415 mmol) in anhydrous DMF (2.5 mL). The reaction mixture was stirred at ambient temperature overnight, diluted with brine and extracted with EtOAc (3 x 15 mL).
  • the title compound was prepared according to the general procedure A for HATU coupling from 6-methyl-2,3-dihydro- 1-benzothiophene-2-carboxylic acid (31.7 mg, 0.163 mmol), HATU (86.2 mg, 0.227 mmol), iP ⁇ NEt (0.150 mL), and 4-(pyridin-2-yl)thiazol-2-amine (HBr salt, 37.2 mg, 0.144 mmol).
  • the reaction mixture was stirred overnight at ambient temperature and was then subjected to EtOAc I H2O workup.
  • the crude product was purified by column chromatography (hexane - EtOAc gradient 0-60%) affording the title compound as a light tan solid (48.3 mg, 0.137 mmol, 95%).
  • Example 95 Methyl 2-amino-4-(pyridin-2-yl)thiazole-5-carboxylate Methyl 3-oxo-3-(pyridine-2-yl) propanoate (0.57 g, 3.18 mmol), thiourea (0.541 g, 7.12 mmol), and I2 (0.847 g, 3.34 mmol) were refluxed in EtOH (5 mL) for 18 h. After cooling, the yellow precipitate of product was filtered and washed with cold EtOH. The compound was obtained as 0.709 g of yellow solid, 95%.
  • Example 96 The Boc protected compound of Example 96 (0.50 g) was dissolved in EtOAc (10 mL) and MeOH (3 mL) and treated with 4M HOI in dioxane (3 mL). The reaction mixture was stirred at ambient temperature for 24 h and evaporated to dryness. The residual white solid was used without further purification.
  • Example 98 Methyl 2-(4-(N,N-dimethylsulfamoyl)benzamido)-4-(pyridin-2-yl)thiazole-5-carboxylate
  • the title compound was prepared according to the general procedure B for PyBOP coupling from 4- dimethylsulfamoylbenzoic acid (58.7 mg, 0.256 mmol), PyBOP (0.1667 g, 0.320 mmol), and Example 95 (45.4 mg, 0.193 mmol) in a mixture of DMF (2.5 mL) and iP ⁇ NEt (0.35 mL).
  • Example 100 tert-butyl (2-(4-((5-ethyl-4-(pyridin-2-yl)thiazol-2 yl)carbamoyl)benzamido)ethyl)carbamate
  • the title compound was prepared according to the general procedure B for PyBOP coupling from 4-((2-((tert- butoxycarbonyl)amino)ethyl)carbamoyl)benzoic acid (77 mg, 0.250 mmol), 5-ethyl-4-(pyridin-2-yl)thiazol-2-amine (49 mg, 0.239 mmol) and PyBOP (0.2462 g, 0.473 mmol) in a mixture of iP ⁇ NEt (0.50 mL) and anhydrous DMF (2.5 mL). The reaction mixture was stirred for 24 h at ambient temperature, then it was partitioned between EtOAc and brine.
  • Example 101 5-pivalamido-N-(4-(pyridin-2-yl)thiazol-2-yl)picolinamide
  • Example 104 N1-(adamantan-1-yl)-N4-(4-(pyridin-2-yl)thiazol-2-yl)terephthalamide
  • the title compound was prepared according to the general procedure A for HATU coupling from 4-((4-(pyridin-2- yl)thiazol-2-yl)carbamoyl)benzoic acid (87.2 mg, 0.268 mmol), HATU (148 mg, 0.389 mmol) and adamantaneamine HCI (60 mg, 0.320 mmol) in anhydrous DMF (2.0 mL) and iP ⁇ NEt (0.25 mL).
  • the title compound was prepared according to the general procedure A for HATU coupling from (4-((4-(pyridin-2- yl)thiazol-2-yl)carbamoyl)benzoyl)glycine (93 mg, 0.243 mmol), HATU (0.200 g, 0.526 mmol), iP ⁇ NEt (0.20 mL) and adamantaneamine HCI (80 mg, 0.426 mmol) in anhydrous DMF (2.0 mL). Purification of the crude product by trituration with MeOH I acetone afforded the title compound as a yellowish solid (6.0 mg, 11 .6 pi mol, 5%).
  • Example 106 3-(2-(adamantan-1-ylamino)-2-oxoethoxy)-5-(4-methylpiperazine-1-carbonyl)-N-(4-(pyridin-2- yl)thiazol-2-yl)benzamide
  • Example 107 tert-butyl 4-(4-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)phenyl)piperazine-1-carboxylate 4-(4-(Tert-butoxycarbonyl)piperazin-1 -yl)benzoic acid (1.765 g, 5.76 mmol) was dissolved in anhydrous DMF (20 mL) and IP ⁇ NEt (1.0 mL), then it was treated with COMU (2.794 g, 6.52 mmol) as a solid. The solution soon turned orange-red and was stirred for 10 min.
  • Example 109 (tert-butyl 3-(4-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)benzamido)propanoate) was deprotected by stirring in 4M HOI I dioxane in EtOAc. After completion of the reaction, the white suspension was evaporated to dryness and the product was dried under high vacuum affording a white solid.
  • the title compound was prepared according to the general procedure A for HATU coupling from 4-((4-(pyridin-2- yl)thiazol-2-yl)carbamoyl)benzoic acid (226.8 mg, 0.697 mmol), HATU (436.9 mg, 1.15 mmol) and p-alanine-tert-butyl ester HOI (127.9 mg, 0.704 mmol) in a mixture of IP ⁇ NEt (0.7 mL) and anhydrous DMF (5 mL). The reaction was carried out over 18 h at ambient temperature under N2. EtOAc I H2O workup was used to isolate the crude product which crystallized. The substance could be recrystallized from MeOH - acetone (190 mg nearly white solid, 0.386 mmol, 55%).
  • Example 110 Z45411712, Enamine
  • Example B Pharmacological perturbation of the phase-separating protein SMNDC1
  • the murine oTC1 cell line was obtained from ATCC (Cat#CRL-2934, RRID:CVCL_B036). Cells were grown in low- glucose DMEM medium (Biowest L0066) supplemented with 10% FBS, 50 U/mL penicillin and 50 pg/mL streptomycin. HAP1 cells (Horizon discovery) were grown in IMDM medium (Sigma I6529) supplemented with 10% FBS, 50 U/mL penicillin and 50 pg/mL streptomycin. The Lenti-XTM 293 T cell line was purchased from Takara Bio (632180).
  • Cells were grown in high-glucose DMEM medium (Sigma D5796) supplemented with 10% FBS, 1 mM sodium pyruvate, 50 U/mL penicillin and 50 pg/mL streptomycin. Intron tagging and live imaging of cells
  • Clones were validated for the correct integration of the intron-tag via comparison of live cell images to publicly available or in-house IF images, genomic DNA PCR amplification of the respective loci, and western blots with antibodies against the target protein and/or the fluorescent tag.
  • Cells were imaged on a PerkinElmer Opera Phenix automated microscope with 500 ms exposure time in either GFP or RFP channel, or on a Zeiss LSM 980 microscope.
  • condition-independent identification nuclear markers such as Hoechst or DRAQ5TM were used.
  • Cells were fixed in the 96-well imaging plates they were growing in before by adding 37% formaldehyde solution 1 :10 to the culture medium for a final concentration of 3.7%. Cells were incubated with this for 15 min at room temperature (RT). Next, cells were washed once with PBS, followed by a 30 min permeabilization step with PBST (0.2% Tween). Afterwards, cells were blocked with a 3% BSA in PBST solution for 1 h.
  • nuclei were identified in the channel of the nuclear marker (DAPI/ Hoechst/ DRAQ5TM) (with Method C, Common Threshold 0.75, Area > 10 m 2 ). After the identification of nuclei, their corresponding cytoplasm was also identified using the respective nucleic acid marker (with Method A, Individual Threshold 0.15). Even though the highest staining of these nuclear markers is obviously detected in the nucleus they still produce a significant staining of the cytoplasm above background. After defining the respective cell areas, mean intensity in the different channels was measured. Finally, spots were identified with the according "Spots” algorithm (with Method A, Relative Spot Intensity > 0.053, Splitting Sensitivity: 1.0).
  • BL21 (DE3) competent E. coli cells were transformed with the respective plasmids and liquid stocks frozen at -80°C. Volumes described here are for 450 ml total volume bacterial culture but were adjusted according to protein amounts needed. From frozen liquid stocks, 200 ml LB Kanamycin cultures were grown at 30°C overnight, diluted with 250 ml fresh LB and grown until ODeoo reached 0.8-1. Protein expression was induced with 1 mM IPTG and bacteria grown for another 24h at 20°C. Bacteria were harvested by centrifugation at 4000xg for 15min at 4°C. Pellets were washed in 35ml PBS and spun down again at 6000xg for 10 min at 4°C. After removal of supernatant PBS, pellets can be stored at -80°C.
  • pellets were resuspended in 13 ml Lysis buffer (50 mM TRIS pH 7.7, 500 mM NaCI, 1 % Igepal, 2.5 mg/ml Lysozyme, 0.1 mg/ml DNase I), incubated for at least 15min and sonicated to ensure cell lysis. Afterwards, lysates were spun down again for 20min at 8500xg and 4°C to remove debris pellet. In parallel, 1 ml of Ni-NTA resin (Qiagen) were added to a 15ml tube and centrifuged at 700xg for 2min.
  • Lysis buffer 50 mM TRIS pH 7.7, 500 mM NaCI, 1 % Igepal, 2.5 mg/ml Lysozyme, 0.1 mg/ml DNase I
  • Proteins for droplet assays were then purified further by size exclusion chromatography (SEC) on a Superdex increase 200 10/300 GL column with 50mM Tris pH 7.5, 125mM NaCI, 10% glycerol and 1 mM DTT running buffer.
  • SEC size exclusion chromatography
  • FRAP experiments cells harboring intron-tags in SMNDC1 and SRRM2 were seeded 24h before imaging on a Zeiss LSM 980 microscope. 15 min before imaging, medium was changed to medium without phenol red containing DRAQ5TM 1 : 1000 to reduce autofluorescence and to mark nuclei. If cells were treated with compounds, these were added in the same step. After identifying a suitable cell, bleach and reference regions were defined. After taking one reference image, the bleach region was bleached 15 times for 5 milliseconds with 100% laser power at 488 nm for GFP and with 20% laser power at 546 nm for RFP. After bleaching, a new image was taken approximately every 3 seconds until 150 seconds after bleaching. Fluorescence intensities were quantified in the bleach and reference regions for every image and normalized to the intensity before bleaching.
  • Proximity labeling with APEX2 was done following the described protocol for imaging and proteomic analysis (Hung, V. et al. Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2. Nat Protoc 11, 456-475 (2016)).
  • Cell lines with a stable expression of APEX2-fusion proteins were generated using lentiviral transduction of plasmids generated with Gateway cloning of the respective fusion protein into pLEX305.
  • pLEX_305 was a gift from David Root (Addgene plasmid # 41390; http://n2t.net/addgene:41390; RRID:Addgene_41390).
  • the APEX2 sequence was amplified from APEX2-csGBP which was a gift from Rob Parton (Addgene plasmid # 108874; http://n2t.net/addgene: 108874; RRID:Addgene_108874). Briefly, cells were incubated with 0.5mM biotin-phenol for 30min, after which 1 mM H2O2 was added for exactly 1 min. Afterwards the labeling reaction was quenched by 3 quick washes with Quenching solution (10mM sodium ascorbate, 10mM sodium azide, 5mM Trolox in PBS). Cells were then fixed for IF analysis or detached from the plates with a cell scraper for WB or MS analysis.
  • streptavidin agarose beads (PierceTM Streptavidin Agarose, Thermo Scientific, 20353) per sample were taken to 5 ml tubes in batches of 400 pil. To settle down the beads, tubes were centrifuged for 30sec in a table-top spin centrifuge and settled further on ice for 3min before taking off the supernatant. Beads were washed twice in 4ml PBS. After the last washing, beads were resuspended in PBS and combined to a final volume of 100 pil/ sample. After distribution of 100 pil/ sample, 1.35 ml of PBS were added, and beads stored at 4°C.
  • BioRad Minispin columns were equilibrated on vacuum manifold with 1 ml Wash buffer 1 (0.2% SDS in PBS). Beads with enriched proteins were transferred from tubes to columns by resuspending in 2x 0.5ml Wash buffer 1 . Afterwards, beads were washed 10x in 0.5ml Wash buffer 2 (8M Urea in PBS) and 4x in 0.5ml PBS. After closing of columns, beads were resuspended in 2x 0.5ml digestion buffer (H2O (HPLC grade), 50mM Ammonium bicarbonate, 0.2M Guanidine hydrochloride, 1 mM Calcium chloride) and transferred into fresh 1.5ml lo-bind tubes.
  • H2O HPLC grade
  • tubes were centrifuged for 30sec in a table-top spin centrifuge and settled further on ice for 3min before taking off the supernatant.
  • 250pil Digestion buffer were added to the beads, and beads were stored at 4°C before the overnight digest.
  • 10pil trypsin 0.1 pig/pil, total 1 g were added to each tube at the end of the day, incubation at 37°C rotating inside the incubator overnight ( ⁇ 14h).
  • stage tips were prepared as follows. 32x 1 mm in diameter C18 material was punched out from Empore C18 disk using blunt syringe needle and plunged into filter-less P200 pipette tip, pushing towards narrow end of the tip. The metal piston was pressed down to fix the C18. 24pil oligo R3 solution (15mg/ml in 100% acetonitrile (ACN)) were applied to the C18 tip, centrifuged at 1 ,000xg for 1 min inside of a collection tube. C18 was activated by washing 2x with lOOpil 100% ACN, centrifugation at 1,000xg for 1 min.
  • ACN acetonitrile
  • beads were separated via centrifugation at 1 ,000xg for 30sec, and complete supernatants transferred into fresh 1.5ml lo-bind tubes. Beads were washed with 200pil H2O for HPLC using wide pipette tips, centrifuged again for 30sec at 1,000xg and supernatant combined with digest. The peptide samples were then acidified with 16pil 30% TFA ( ⁇ 1 % final) and loaded to the C18 columns in fractions of max. 250 l, and centrifuged at 1 ,000xg for 3min each. After loading the full volume, columns were washed with 200 l 0.1 % TFA, and centrifuged at 1 ,000xg for 3min.
  • Reaction was stopped by adding 1 .5pl of 5% hydroxylamine solution in H2O for HPLC (prepared fresh from 50% hydroxylamine stock solution), vortexing, spinning down in spin-centrifuge and incubation at 25°C and 300rpm for 15min. Full volumes of respective TMTpro channels were then pooled into fresh 1.5ml lo-bind tube.
  • samples were fractionated by on-tip high pH fractionation.
  • Fresh ammonium formate (AF) buffer was prepared right before using, as it is volatile: 100mM ammonium formate in 2ml tube (6.3mg into 1 ml H2O for HPLC) mixed into 4ml H2O for HPLC in 15ml tube, pH 10 adjusted with two drops of 25% ammonia solution ( ⁇ 35pil, final concentration 20mM).
  • 1 ml of 20mM freshly prepared AF was added to 320pil of pooled sample.
  • C18 columns were prepared as described above. The eluate was loaded in fractions (max. capacity 200pil at once), centrifuged at 1 ,000xg for 3min each.
  • Fraction 1 Elution with 50pl 16% ACN (24pl ACN +126pl 20mM AF), centrifuged at 1 ,000xg for 2min, washed with 20pl of same buffer, collected together in tube #1 , centrifuged at 1 ,000xg for 1 min.
  • Fraction 2 Elution with 50pl 20% ACN (30pl ACN +120pl 20mM AF), centrifuged at 1,000xg for 2min, washed with 20pl of same buffer, collected together in tube #2, centrifuged at 1 ,000xg for 1 min.
  • Fraction 3 Elution with 50pl 24% ACN (36pl ACN 114pl 20mM AF), centrifuged at 1,000xg for 2min, washed with 20pl of same buffer, collected together in tube #3, centrifuged at 1 ,000xg for 1 min.
  • Fraction 4 Elution with 50pl 28% ACN (42pl ACN +108pl 20mM AF), centrifuged at 1,000xg for 2min, washed with 20pl of same buffer, collected together in tube #4, centrifuged at 1 ,000xg for 1 min.
  • Fraction 5 Elution with 50pl 80% ACN (120pl ACN +30pl 20mM AF), centrifuged at 1,000xg for 2min, washed with 20pl of same buffer, collected together in tube #5, centrifuged at 1 ,000xg for 1 min.
  • proteins were separated through application of an electric field (120V for 15 min, 160V for 90 min).
  • an electric field 120V for 15 min, 160V for 90 min.
  • gels were stained with Coomassie Blue. To do so, the gel was fixed in fixing solution (50% methanol, 10% glacial acetic acid) for 1 h with gentle agitation. The gel was then stained in staining solution (0.1 % Coomassie Brilliant Blue R-250, 50% methanol and 10% glacial acetic acid) for 20 min, followed by several rounds of destaining with destaining solution (40% methanol, 10% glacial acetic acid).
  • a nitrocellulose membrane For visualization of individual proteins, they were transferred to a nitrocellulose membrane (GE Healthcare Life Science) by electrophoresis. The membrane was blocked by 5% Milk solution in TBST for at least 1 h at RT, followed by incubation in primary antibody solution (SMNDC1 : Novus Biologicals Cat#NBP1-47302; RRID:AB_10010256; SFPQ: Atlas Antibodies Cat#HPA047513; RRID:AB_2680073; APEX2 Innovagen PA-APX2-100; for all dilution 1 : 1000 in 5% Milk TBST) at 4°C o/n.
  • SNDC1 Novus Biologicals Cat#NBP1-47302
  • RRID AB_10010256
  • SFPQ Atlas Antibodies Cat#HPA047513
  • RRID AB_2680073
  • PA-APX2-100 for all dilution 1 : 1000 in 5% Milk TBST
  • Membranes were then washed 3 times in TBST, followed by incubation with HRP-coupled secondary antibody solution (Peroxidase AffiniPure Donkey Anti-Mouse IgG Jackson ImmunoResearch Cat#715-035-151 ; RRID:AB_2340771; Peroxidase AffiniPure Donkey Anti-Rabbit IgG Jackson ImmunoResearch Cat#711-035-152; RRID:AB_10015282; Goat Anti-Chicken IgY H&L (HRP) Abeam ab97135; RRID:AB_10680105; for all dilution 1 :20000 in 5% Milk TBST) for at least 1 h at RT. After 3 more washing steps, signal was detected by application of Clarity ECL Western Blotting Substrate (Bio-Rad) to the membrane with a ChemiDoc MP Imaging System (Bio-Rad) with Image Lab Touch Software Version 2.3.0.07.
  • Mass spectrometry analysis was performed on an Orbitrap Fusion Lumos Tribrid mass spectrometer (ThermoFisher Scientific, San Jose, CA) coupled to a Dionex Ultimate 3000 RSLCnano system (ThermoFisher Scientific, San Jose, CA) via a Nanospray Flex Ion Source (ThermoFisher Scientific, San Jose, CA) interface. Peptides were loaded onto a trap column (PepMap 100 C18, 5 pm, 5 x 0.3 mm, ThermoFisher Scientific, San Jose, CA) at a flow rate of 10 pL/min using 0.1 % TFA as loading buffer.
  • Separation was achieved by applying a four-step gradient over 151 min at the flow rate of 230 nL/min (initial gradient increase from 6% to 9% solvent B within 1 min, 9% to 30% solvent B within 146 min, 30% to 65% solvent B within 8 min and, 65% to 100% solvent B within 1 min, 100% solvent B for 6 min before equilibrating at 6% solvent B for 23 min prior to next injection).
  • electrospray ionization was enabled by applying a voltage of 1.8 kV directly to the liquid to be sprayed, and non-coated silica emitters were used.
  • the mass spectrometer was operated in a data-dependent acquisition mode (DDA) and used a synchronous precursor selection (SPS) approach, which enables more accurate multiplexed quantification of peptides and proteins at the MS3 level.
  • DDA data-dependent acquisition mode
  • SPS synchronous precursor selection
  • For both MS2 and MS3 level a survey scan of 400-1600 m/z in the Orbitrap was collected at a resolution of 120000 (FTMS1), an AGO target was set to 'standard' and a maximum injection time (IT) of 50 ms was applied.
  • Precursor ions were filtered according to charge state (2-6), dynamic exclusion (60 s with a ⁇ 10 ppm window), and monoisotopic precursor selection.
  • Precursor ions for data-dependent MS n (ddMS n ) analysis were selected using 10 dependent scans (TopN approach). Charge state filter was used to select precursors for data- dependent scans.
  • IMS2 dual-pressure linear ion trap
  • Quadrupole isolation window was set to 0.7 Da and collision induced dissociation (CID) fragmentation technique was used at a normalized collision energy of 35%.
  • Normalized AGC target value was set to 200% with a maximum IT of 35 ms.
  • HCD high-energy collision induced dissociation
  • FTMS3 Orbitrap analysis
  • Results were filtered to include peptide spectrum matches with Sequest HT cross-correlation factor (Xcorr) scores of >1 and high peptide confidence assigned by Percolator.
  • MS3 signal-to-noise values (S/N) values of TMTpro reporter ions were used to calculate peptide/protein abundance values.
  • Peptide spectrum matches with precursor isolation interference values of >70%, SPS mass matches ⁇ 65% and average TMTpro reporter ion S/N ⁇ 10 were excluded from quantitation. Both unique and razor peptides were used for TMT quantitation. Isotopic impurity correction was applied. Data were normalized on total peptide amount for correction of experimental bias and scaled ‘on all average'.
  • Protein ratios are directly calculated from the grouped protein abundances using a one-way ANOVA hypothesis test, followed by Benjamini-Hochberg correction for multiple comparisons.
  • the mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD037092 and 10.6019/PXD037092.
  • Isotope-enriched SMN84-147 and SMNDC165-128 were expressed and purified as described in Tripsianes et al., 2011 (Tripsianes, K. et al. Structural basis for dimethylarginine recognition by the6.1 domains of human SMN and SPF30 proteins. Nature Structural & Molecular Biology 18, 1414-1420 (2011)).
  • NMR experiments were performed on Bruker Avance III spectrometers operating at 600 MHz or 800 MHz 1 H frequencies using H/N/C triple-resonance cryogenic probes. All NMR acquisition was performed in 3 mm tubes at 25°C. Spectra were processed using Topspin 3.5 (Bruker) and analyzed with Cara 1.9.17 (Keller, R.
  • the SMNDC1/sDMA structure (PDB: 4A4H) (Tripsianes, K. et al. Structural basis for dimethylarginine recognition by the6.1 domains of human SMN and SPF30 proteins. Nature Structural & Molecular Biology 18, 1414-1420 (2011)) was used as a protein model with the sDMA removed beforehand. Instead of defining active/passive residues, intermolecular NOE contacts were introduced as ambiguous restraints. Visible and assigned NOE crosspeaks were defined as distance restraints with a lower limit of 0.5 A and upper limit of 5 A. Peak intensities of crosspeaks were measured and normalized to the strongest peak.
  • SMNDC1 knock-down was performed as described in Casteels et al. (Casteels, T. et al. SMNDC1 links chromatin remodeling and splicing to regulate pancreatic hormone expression. Cell Reports 40, (2022)). Briefly, Smndcl shRNA from the TRC shRNA library (https://portals.broadinstitute.org/gpp/public/) (TRCN0000123795) was cloned into pLKO.1 (Addgene plasmid #10878).
  • This plasmid was packaged into lentivirus in Lenti-XTM 293 T cells (BOSC-23, RRID:CVCL_4401, TakaraBio Cat#632180) with LipofectamineTM 3000 (Thermo Fisher Scientific L3000008) and packaging plasmids psPAX2 (Addgene plasmid #12260) and pMD2.G (Addgene plasmid #12259).
  • Target cells were transduced with viral supernatant after filtering and addition of 8 pig/ml Polybrene® (Santa Cruz Biotechnology sc- 134220) 48h after transfection. Medium was changed 24h later.
  • PCR products were run on a 1 % Agarose gel for 30 min at 100 Volt.
  • RNA sequencing libraries were prepared from low-input samples using the Smart-seq2 protocol (Picelli, S. et al. Full- length RNA-seq from single cells using Smart-seq2. Nat Protoc 9, 171-181 (2014)).
  • the subsequent library preparation from the amplified cDNA was performed using the Nextera XT DNA library prep kit (Illumina, San Diego, CA, USA). Library concentrations were quantified with the Qubit 2.0 Fluorometric Quantitation system (Life Technologies, Carlsbad, CA, USA) and the size distribution was assessed using the Experion Automated Electrophoresis System (Bio-Rad, Hercules, CA, USA).
  • samples were diluted and pooled into NGS libraries in equimolar amounts.
  • NGS reads were mapped to the Genome Reference Consortium GRCm38 assembly via "Spliced Transcripts Alignment to a Reference” (STAR) (Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15- 21 (2013)) utilising the "basic” Ensembl transcript annotation from version e100 (April 2020) as reference transcriptome.
  • the mm10 assembly of the UCSC Genome Browser was used for downstream data processing, and the Ensembl transcript annotations were adjusted to UCSC Genome Browser sequence region names. STAR was run with options recommended by the ENCODE project.
  • NGS read alignments overlapping Ensembl transcript features were counted with the Bioconductor (3.
  • SMNDC1 co-localizes with nuclear speckle markers
  • the inventors analyzed the protein sequence by comparing predictions for disordered regions by MetaDisorder (Kozlowski, L. P. & Bujnicki, J. M. MetaDisorder: a meta-server for the prediction of intrinsic disorder in proteins. BMC Bioinformatics 13, 111 (2012)) and for the full- length structure by AlphaFold (Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583-589 (2021); Varadi, M. et al. AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models.
  • the inventors employed an endogenous tagging system that targets introns and introduces a GFP-tag as an artificial exon (Serebrenik, Y. V., Sansbury, S. E., Kumar, S. S., Henao-Mejia, J. & Shalem, O. Efficient and flexible tagging of endogenous genes by homology-independent intron targeting. Genome Res 29, 1322-1328 (2019)) to characterize SMNDCTs cellular functions. To rule out disrupting effects of the tag on protein localization, the inventors targeted all of SMNDCTs introns in murine alphaTCI cells, and then isolated clonal sublines.
  • the targeted introns result in GFP integrations covering all regions of the protein, including one at the N-terminus (before residue 1), the N-terminal region (residue 40), the Vietnamese domain (residue 88), and a long stretch in the C-terminal region (residue 142, residue 193) which is predicted to be disordered (Kozlowski, L. P. & Bujnicki, J. M. MetaDisorder: a meta-server for the prediction of intrinsic disorder in proteins. BMC Bioinformatics 13, 111 (2012)) (Fig. 1 a, b). Furthermore, the inventors also tagged intron 2-3 in human HAP1 cells.
  • SMNDC1 also reacted to the overexpression of the cell-cycle dependent kinases DYRK3 and CLK1 , which is known to dissolve nuclear speckles (Rai, A. K., Chen, J.-X., Selbach, M. & Pelkmans, L. Kinase-controlled phase transition of membraneless organelles in mitosis. Nature 559, 211-216 (2018); Sacco-Bubulya, P. & Spector, D. L. Disassembly of interchromatin granule clusters alters the coordination of transcription and pre-mRNA splicing. Journal of Cell Biology 156, 425-436 (2002)), with a loss of its focal nuclear localization (Fig. 8d).
  • SMNDCTs To further characterize SMNDCTs localization in the nucleus, the inventors co-stained cells with antibodies against SMNDC1 and SC35, a marker for nuclear speckles. Both signals overlap to a large degree and avoid chromatin- dense regions, whereby SMNDC1 shows a wider less focal distribution (Fig. 1f, co-localization analysis Fig. 8e). To be able to visualize nuclear speckles in live cells the inventors RFP-tagged SRRM2 in the SMNDC1 -GFP-tagged cells (Fig. 1g). SRRM2 is the target of the SC35 antibody (llik, i. A. et al. SON and SRRM2 are essential for nuclear speckle formation.
  • SMNDC1-GFP and SRRM2-RFP co-localized to a large degree, both in interphase and during mitosis (Fig. 1 h). Even though co-localization was maintained in the mitotic interchromatin granules, there SMNDC1 showed a higher degree of diffuse localization, leading to a lower average Pearson correlation score compared to interphase cells (Fig. 8f). Overall, the inventors find that SMNDC1 shows behavior and localization typical for proteins in nuclear speckles, which have been described as membraneless organelles in the nucleus formed by LLPS. SMNDC1 undergoes biomolecular condensation in vitro and in cellular systems
  • RNA RNA to the PEG-8000 containing buffer enhanced SMNDCTs droplet formation while high NaCI concentrations prevented droplet formation. Digestion of RNA by RNase led to the dissolution of droplets, even after their formation (Fig. 2d). RNA also physically localized to the protein droplets (Fig. 2e).
  • SMNDC1-GFP exhibited the expected phenotype in live cells treated with 1,6-hexanediol by losing its focal localization within the nucleus (Fig. 2g).
  • SMNDC1 Full-length SMNDC1 interacts with nuclear speckle proteins
  • the inventors set out to characterize SMNDCTs interactome using proximity labeling by overexpressing an SMNDC1- APEX2 fusion protein (Fig. 3a).
  • SMNDCT1- APEX2 fusion protein Fig. 3a
  • Co-IP co-immunoprecipitation
  • this recently developed method Hung, V. et al. Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2. Nat Protoc 11, 456-475 (2016)
  • SMNDC1 FL full-length SMNDC1
  • the inventors also performed proximity labeling with a fusion protein of APEX2 with a truncated SMNDC1 consisting of only the Vietnamese domain and therefore lacking N-terminal and C-terminal regions, and the nuclear localization signal (NLS) (APEX2-SMNDC1 TD ) (Fig. 9a).
  • the inventors performed proximity labeling followed by an immunofluorescence (IF) staining against SMNDC1 and biotin.
  • IF immunofluorescence
  • APEX2-SMNDC1 FL caused biotinylation in the areas where SMNDC1 is localized: nuclear while avoiding chromatin-dense regions (Fig. 3b).
  • APEX2-SMNDC1 FL Analyzing the biotinylated and enriched proteins by mass spectrometry (MS), the inventors identified and quantified a large number of proteins (-3200) in the proximity of APEX2-SMNDC1 FL and APEX2-SMNDC1 TD . Compared to the proximity interactome of APEX2-SMNDC1 TD , APEX2-SMNDC1 FL showed overall less interactions (Fig. 3c). The inventors attribute this to the higher specificity of interactions happening with the correctly localized full form of SMNDC1.
  • the inventors then filtered for proteins enriched in APEX2-SMNDC1 FL over APEX2-SMNDC1 TD (adjusted p-value ⁇ 0.1 , abundance ratio >1.1) which reduced the number of proteins they considered specific interactors of SMNDC1 FL to 750. As expected, they found an enrichment of proteins associated with mRNA processing, and more specifically splicing, but also an enrichment of proteins associated with ribosome biogenesis and rRNA processing amongst these (Fig. 3d). When comparing these interactors to an SMNDC1 Co-IP dataset generated in their lab (Casteels, T. et al. SMNDC1 links chromatin remodeling and splicing to regulate pancreatic hormone expression.
  • APEX2-SMNDC1 TD should bind these proteins, too. Consequently, only a small subset was enriched in APEX2-SMNDC1 FL over APEX2-SMNDC1 TD .
  • the inventors therefore also compared the sDMA-modified proteins to all proteins identified in their SMNDC1-APEX2 experiments and found most of them (67 out of 87 known sDMA-modified proteins). There was also an enrichment, although to a lesser degree, of proteins with asymmetrical di-methylations. These protein sets partially overlap, as the same arginine sites can often alternatively be symmetrically or asymmetrically di-methylated.
  • SMNDCTs Small nuclear ribonucleoprotein Sm D3 containing four sDMAs
  • Table 1 Small molecule screening data.
  • Raw signal was normalized pl ate-specif ical ly by correcting row and column-specific mean signals to the mean signal of the entire plate, each after removing the highest and lowest 25% of values.
  • Raw signal was then converted to percent of control signal, but linear regression to plate-specific mean signal of DMSO wells (set to 100 percent of control) and positive control wells (set to 0% of control), after outlier removal using a Grubbs test.
  • Hit rate Primary screen 511 small molecules (including unspecific quenchers), Secondary screen (with crosslinking peptide): 40 small molecules, Tertiary screen (titration): 14 small molecules.
  • the inventors then selected the 4-arylthiazole-2-amine series for thorough exploration of structure-activity relationships, also due to the synthetic ease of access (Fig. 5). They found the 2-pyridyl substitution to be advantageous for binding affinity, as its replacement with certain other aryl groups led to drastic loss of potency (e.g., compounds 3-7). A 2-substituted pyrrole could be used with some loss of potency in compound 8. Omission of the aromatic group by replacement with ethoxycarbonyl resulted in complete loss of activity (compound 9).
  • the five-membered heterocycle in the core scaffold could be replaced with the isomeric scaffold 2-(pyridin-2- yl)thiazol-4-amine in compound 14.
  • the third possible isomer, compound 15, had significant loss of activity and preferentially inhibited SMN over SMNDC1.
  • the thiazole was replaced with an analogous oxazole in the compound 16, there was a 40-fold drop in potency.
  • Replacement of the thiazole with 1 ,2,4-thiadiazoles resulted in inactive compounds.
  • Substitution of the 5-position of the thiazole of 1 with a methyl group was tolerated without loss of potency (compound 10) but an ethyl group or a bromine atom decreased the IC50 threefold (compounds 11 and 12).
  • the pyridine of compound 13 forms tight TT- TT stacking contacts with the aromatic rings of F83 and Y111 with distances of 3.7 A to each, which underlines the importance of an aromatic substituent at the thiazole 4-position.
  • the aromatic moieties of Y90 and F108 stand perpendicular to the pyridine ring while the sidechain of N113 is enclosing it from the opposite site.
  • the structure shows high similarity to SMNDC1/sDMA (PDB: 4A4H) (Fig. 11c) and is fully consistent with the predicted binding mode.
  • Table 3 Assigned intermolecular NOEs between SMNDC1 and inhibitor 13 and associated restraint upper limits.
  • Table 4 SMNDC1/ compound 13 molecular-docking results of structure ensemble generated using the HADDOCK webserver. Statistics generated over all 200 analyzed structures. a Restraints listed in Table 3. b The violated distance involves proton 13-H2 and Y90-HE. SMNDC1 Six Domain inhibitors impact protein localization and splicing
  • the inventors then went on to analyze the effects of the identified small molecule binders on SMNDCTs phase separation. Using the endogenously tagged cell lines, they observed strong effects on the levels and distribution of SMNDC1. Treating the cells with 50 piM of compound 1 for 12-16h leads to a loss of SMNDC1 within the nucleus (Fig. 7a, quantification Fig. 7b,). Additionally, the subnuclear distribution changed and less spots were detected within the nucleus (quantification Fig. 7c). These effects were not observed with the negative control compound 9 which lacks the 2-pyridy I crucial for the binding to SMNDC1 .
  • the inventors tested the SMNDCI-selective compound 28 for its effects on SMNDC1 and SRRM2 localization and could confirm the effects observed for the non-selective compound 1 (Fig. 12h), even at lower concentrations (Fig. 121).
  • SMN1 was endogenously tagged with RFP. Treating these cells with 50 piM of compound 1 for 16h showed effects on SMN. Overall intensity of SMN decreased while number of spots (supposedly stress granules) in the cytoplasm increased (Fig. 12j).
  • the inventors analyzed the effect of compound 1 on the proximity interactome of SMNDC1. Overall, they observed that upon inhibitor treatment, more proteins showed a reduced interaction (volcano plot skewed towards down-regulated side, many more significantly down-regulated than up-regulated proteins, Fig. 7g). This indicates that the inhibitor blocks SMNDCTs function to bind to its interaction partners. Compared to APEX2-SMNDC1 FL , the inhibitor effects in APEX2-SMNDC1 TD were diminished, presumably due to less specific interactions in the truncated form at baseline (Fig. 13a). 126 proteins were significantly depleted in APEX2-SMNDC1 FL treated with inhibitor vs.
  • the inventors can confirm the loss of interactions to specific proteins, e.g., the sDMA-modified splicing factor SFPQ or the loss of trans-interactions to SMNDC1 itself. Interactions to SMNDC1 itself are lost both to the endogenous protein (30 kDa band with antibody against SMNDC1) and APEX2-Fusion protein (60 kDa band with antibodies against SMNDC1 and APEX2).
  • specific proteins e.g., the sDMA-modified splicing factor SFPQ or the loss of trans-interactions to SMNDC1 itself.
  • Interactions to SMNDC1 itself are lost both to the endogenous protein (30 kDa band with antibody against SMNDC1) and APEX2-Fusion protein (60 kDa band with antibodies against SMNDC1 and APEX2).
  • the inventors did not observe instantaneous effects of the inhibitor on the architecture of nuclear speckles by quantifying intensity or spots per nucleus, they tested whether the inhibitor immediately influenced mobility of proteins within nuclear speckles. To this end, they applied the inhibitor 1 to live cells at a concentration of 50 piM and measured FRAP within a timeframe of 15- 45 min (Fig. 7i). Indeed, they detected a lower recovery after photobleaching for both SMNDC1 and SRRM2 when cells were treated with compound 1 while reference regions were not affected (Fig. 13e).
  • VAST-TOOLS Vertebrate Alternative Splicing and Transcription Tools
  • Fig. 7j Directly comparing the differential percentage spliced-in (dPSI) values for alternative splicing events the inventors found a significant correlation between knock-down and small-molecule inhibition of SMNDC1 (Fig. 7k). They went on to test a panel of individual events with the biggest dPSI values in both knock-down and compound 1 treatment or known to be differentially spliced upon knock-down of SMNDC1 (Casteels, T. et al. loc. cit.) (3 selected events Fig. 7I and the full panel Fig 13f).
  • dPSI differential percentage spliced-in
  • the inventors demonstrated specific effects of the inhibitor on the splicing function and the localization of SMNDC1 to nuclear speckles and its proximity to interaction partners, and to the architecture of nuclear speckles in general.
  • RNAbindRplus Walia, R. R. et al. RNABindRPIus: A Predictor that Combines Machine Learning and Sequence Homology-Based Methods to Improve the Reliability of Predicted RNA-Binding Residues in Proteins. PLOS ONE 9, e97725 (2014)
  • RNAbindRplus Walia, R. R. et al. RNABindRPIus: A Predictor that Combines Machine Learning and Sequence Homology-Based Methods to Improve the Reliability of Predicted RNA-Binding Residues in Proteins. PLOS ONE 9, e97725 (2014)
  • RNA-terminal IDR is binding to RNAs which in turn recruit further proteins.
  • This model is consistent with their earlier observation (Casteels, T. et al. SMNDC1 links chromatin remodeling and splicing to regulate pancreatic hormone expression. Cell Reports 40, (2022)) that the majority of SMNDC1 protein interactions are lost when RNA is hydrolyzed.
  • RNA-mediated interactors together with arginine methyl interactions mediated by the SMNDC1 constitute the multivalent binding platform that is a typical prerequisite in the formation of biomolecular condensates.
  • the inventors focused on the protein's Vietnamese domain.
  • the Very Domain domain exhibits a well-defined structure with a characteristic aromatic cage that mediates specific recognition of dimethylarginine ligands. This feature potentially enables small molecule binding often referred to as druggability.
  • the inventors thus set out to identify small molecule inhibitors of SMNDCTs Vietnamese domain using an AlphaScreen set-up.
  • the inventors have identified the first specific inhibitors of SMNDCTs Six domain influencing SMNDCTs phaseseparation behavior and splicing and architecture of nuclear speckles. These compounds are chemically distinct from inhibitors previously described for other organised domain proteins (Arrowsmith, C. H. & Schapira, M. Targeting non- bromodomain chromatin readers. Nat Struct Mol Biol 26, 863-869 (2019); Zhu, H., Wei, T., Cai, Y. & Jin, J. Small Molecules Targeting the Specific Domains of Histone-Mark Readers in Cancer Therapy. Molecules 25, 578 (2020)), namely TP53B1 (Sun, Y. et al.
  • Example C Evaluation of compounds according to the invention for their inhibitory activity on SMNDC and SMN1
  • Example B In addition to the compounds of formula (I) tested in Example B, a number of further exemplary compounds of formula (I) as well as several reference compounds were tested for their inhibitory activity on SMNDC and SMN1 , respectively, following the same procedure as described in Example B. The thus determined IC50 values of these compounds for SMNDC and for SMN1 are summarized in the following table:
  • Example D Inhibition of cell viability in pancreatic cancer and ovarian cancer cells
  • the pancreatic cancer cell line PANC1 was cultivated in standard tissue culture conditions in DMEM containing 10% fetal bovine serum.
  • the ovarian cancer cell line OV90 was cultivated in standard conditions using a 1 :1 mixture of MCDB 105 medium containing a final concentration of 1.5 g/L sodium bicarbonate and Medium 199 containing a final concentration of 2.2 g/L sodium bicarbonate supplemented with 15% fetal bovine serum.
  • 50 pl cell suspension was seeded on top of 60 nl compound in DMSO in 384-well plates (Corning 3701). After 72 h cell viability was measured using the Cell Titer Gio Luminescent Cell Viability Assay (Promega) on an Envision Plate Reader (Perkin Elmer).

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Abstract

The present invention provides novel SMNDC1 inhibitors, specifically the compounds of formula (I), (I) as well as pharmaceutical compositions containing these compounds. The SMNDC1 inhibitors provided herein exhibit particularly beneficial properties in terms of potency and selectivity, which renders these compounds highly advantageous for use in therapy, including, e.g., in the treatment or prevention of cancer or diabetes.

Description

Inhibitors of SMNDC1 and their therapeutic use
The present application claims the benefit of priority of European patent application EP23186842.3 filed on July 20, 2023, which is incorporated herein by reference in its entirety.
The present invention provides novel SMNDC1 inhibitors, specifically the compounds of formula (I), as well as pharmaceutical compositions containing these compounds. The SMNDC1 inhibitors provided herein exhibit particularly beneficial properties in terms of potency and selectivity, which renders these compounds highly advantageous for use in therapy, including, e.g., in the treatment or prevention of cancer or diabetes.
Survival motor neuron domain-containing protein 1 (SMNDC1), also called Survival of motor neuron-related-splicing factor 30 (SPF30), is an essential splicing factor required for the formation of the spliceosome (Meister, G. et al. SMNrp is an essential pre-mRNA splicing factor required for the formation of the mature spliceosome. The EMBO Journal 20, 2304—2314 (2001); Rappsilber, J., Ajuh, P., Lamond, A. I. & Mann, M. SPF30 Is an Essential Human Splicing Factor Required for Assembly of the U4/U5/U6 Tri-small Nuclear Ribonucleoprotein into the Spliceosome. J. Biol. Chem. 276, 31142-31150 (2001)). To promote spliceosome assembly SMNDC1 binds to methylated arginines on Sm-proteins using its Tudor domain (Rappsilber, J. et al. loc. cit; Cote, J. & Richard, S. Tudor Domains Bind Symmetrical Dimethylated Arginines*. Journal of Biological Chemistry 280, 28476-28483 (2005)), similar to its better- studied paralog survival of motor neuron (SMN) protein (Cheng, D., Cote, J., Shaaban, S. & Bedford, M. T. The Arginine Methyltransferase CARM1 Regulates the Coupling of Transcription and mRNA Processing. Molecular Cell 25, 71-83 (2007); Pellizzoni, L, Kataoka, N., Charroux, B. & Dreyfuss, G. A Novel Function for SMN, the Spinal Muscular Atrophy Disease Gene Product, in Pre-mRNA Splicing. Cell 95, 615-624 (1998); Fischer, U., Liu, Q. & Dreyfuss, G. The SMN-SIP1 Complex Has an Essential Role in Spliceosomal snRNP Biogenesis. Cell 90, 1023— 1029 (1997)). The Tudor domain structures of both proteins are highly conserved, revealing binding of their substrate symmetric dimethylated arginine (sDMA) in an aromatic cage through cation-rt interactions (Tripsianes, K. et al. Structural basis for dimethylarginine recognition by the Tudor domains of human SMN and SPF30 proteins. Nature Structural & Molecular Biology 18, 1414-1420 (2011)). Functionally, both proteins play essential and apparently opposite roles in the regulation of gene expression and cell identity in the endocrine pancreas. Patients and animal models with SMN mutations experience increased numbers of glucagon producing alpha cells and a reduction of insulin producing beta cells (Bowerman, M. et al. Glucose metabolism and pancreatic defects in spinal muscular atrophy. Annals of Neurology 72, 256-268 (2012)). In contrast, for SMNDC1 it was recently shown that its knockdown causes the upregulation of insulin in a-cells through splicing changes in key chromatin remodelers and induction of the beta cell transcription factor PDX1 (Casteels, T. et al. SMNDC1 links chromatin remodeling and splicing to regulate pancreatic hormone expression. Cell Reports 40, (2022)). SMNDC1 further is essential for cell proliferation in different contexts, and a recent study reported worse survival in hepatocellular carcinoma patients with high SMNDC1 (Zhu, R., Wang, X., Yu, Q., Guo, W. & Zhu, L. A systems biology-based approach to screen key splicing factors in hepatocellular carcinoma. Molecular Carcinogenesis n/a, (2023)). SMNDC1 knock-down led to decreased proliferation and migration of hepatocellular carcinoma cells, establishing SMNDC1 as a new therapeutic target. Both SMN and SMNDC1 show distinct and focal subcellular localization patterns. The SMN Tudor domain is sufficient for formation of a phase-separated compartment dependent on the dimethylarginine (DMA) modification of binding proteins (Courchaine, E. M. et al. DMA-tudor interaction modules control the specificity of in vivo condensates. Cell 184, 3612-3625.e17 (2021)) and was shown to be required for the regulation of the phase-separated stress granules via symmetric dimethylarginine (sDMA) (Chitiprolu, M. et al. A complex of C9ORF72 and p62 uses arginine methylation to eliminate stress granules by autophagy. Nature Communications 9, 2794 (2018)). Arginine methylation in RGG/RG motifs recognized by Tudor domains can affect phase separation of Fused in sarcoma (FUS) (Hofweber, M. et al. Phase Separation of FUS Is Suppressed by Its Nuclear Import Receptor and Arginine Methylation. Ce// 173, 706-719. e13 (2018); Qamar, S. etal. FUS Phase Separation Is Modulated by a Molecular Chaperone and Methylation of Arginine Cation-n Interactions. Ce// 173, 720-734.e15 (2018)) and other proteins (Chong, P. A., Vernon, R. M. & Forman-Kay, J. D. RGG/RG Motif Regions in RNA Binding and Phase Separation. Journal of Molecular Biology 430, 4650-4665 (2018)), and further Tudor domain containing proteins themselves have been shown to be involved in phase separation (Gao, X. et al. T udor-SN interacts with and co-localizes with G3BP in stress granules under stress conditions. FEBS Lett. 584, 3525-3532 (2010); Su, C. et al. Phosphorylation of Tudor-SN, a novel substrate of JNK, is involved in the efficient recruitment of Tudor-SN into stress granules. Biochim Biophys Acta Mol Cell Res 1864, 562-571 (2017)).
SMNDC1 has a speckled localization within the nucleus that - based on co-localization - was attributed to the sub- nuclear structures Cajal bodies and nuclear speckles (Rappsilber, J. et al. loc. cit), which were later defined as prime examples of membraneless organelles (Zhu, L. & Brangwynne, C. P. Nuclear bodies: the emerging biophysics of nucleoplasmic phases. Curr. Opin. Cell Biol. 34, 23-30 (2015)), i.e. biomolecular condensates formed by liquid-liquid phase separation (LLPS). These assemblies can consist of proteins, nucleic acids, and other molecules and are found both in the cytoplasm and the nucleus (Boeynaems, S. et al. Protein Phase Separation: A New Phase in Cell Biology. Trends in Cell Biology 28, 420-435 (2018); Strom, A. R. & Brangwynne, C. P. The liquid nucleome - phase transitions in the nucleus at a glance. J. Cell. Sci. 132, (2019)). An important feature present in many proteins that were found to undergo LLPS are intrinsically disordered regions (IDRs), which do not adopt a well-defined globular structure. IDRs can enable multiple and multivalent interactions that mediate binding to other proteins (Wright, P. E. & Dyson, H. J. Intrinsically disordered proteins in cellular signalling and regulation. Nat Rev Mol Cell Biol 16, 18-29 (2015)). Many RNA-binding proteins (RBPs), including SMNDC1 , were found to phase separate together with RNA, but also with chromatin (Shao, W. et al. Phase separation of RNA-binding protein promotes polymerase binding and transcription. Nat Chem Biol (2021) doi: 10.1038/s41589-021 -00904-5). Amongst other factors phase separation behavior can be initiated by RNA (Garcia-Jove Navarro, M. et al. RNA is a critical element for the sizing and the composition of phase-separated RNA-protein condensates. Nat Commun 10, 3230 (2019)) and regulated by the secondary structure of RNAs and the ratio of RNA to RBPs (Banerjee, P. R., Milin, A. N., Moosa, M. M., Onuchic, P. L. & Deniz, A. A. Reentrant Phase Transition Drives Dynamic Substructure Formation in Ribonucleoprotein Droplets. Angewandte Chemie International Edition 56, 11354-11359 (2017); Langdon, E. M. etal. mRNA structure determines specificity of a polyQ-driven phase separation. Science 360, 922-927 (2018); Maharana, S. et al. RNA buffers the phase separation behavior of prion-like RNA binding proteins. Science 360, 918-921 (2018)). Given the fact that the nucleus and its sub-compartments are enriched in IDR-containing proteins (IDPs) (Frege, T. & Uversky, V. N. Intrinsically disordered proteins in the nucleus of human cells. Biochem Biophys Rep , 33-51 (2015)) and the obvious abundance of negatively charged nucleic acids (both DNA and RNA) the nucleus is primed for LLPS (Aumiller, W. M. & Keating, C. D. Phosphorylation-mediated RNA/peptide complex coacervation as a model for intracellular liquid organelles. Nat Chem 8, 129-137 (2016)). Functionally, these LLPS events control gene expression within the different nuclear compartments (Bhat, P., Honson, D. & Guttman, M. Nuclear compartmentalization as a mechanism of quantitative control of gene expression. Nat Rev Mol Cell Biol 22, 653-670 (2021)) from the formation of heterochromatin (Strom, A. R. et al. Phase separation drives heterochromatin domain formation. Nature 547, 241— 245 (2017); Larson, A. G. et al. Liquid droplet formation by HP1o suggests a role for phase separation in heterochromatin. Nature 547, 236-240 (2017)) over transcription by RNA polymerase II (Cho, W.-K. et al. Mediator and RNA polymerase II clusters associate in transcription-dependent condensates. Science 361, 412-415 (2018)) to RNA processing and (alternative) splicing (Gueroussov, S. et al. Regulatory Expansion in Mammals of Multivalent hnRNP Assemblies that Globally Control Alternative Splicing. Ce// 170, 324-339. e23 (2017)).
Tudor domains have not been targeted extensively by small-molecule inhibitors. Only recently, a study disclosed a fragment unspecifically binding to both SMN and SMNDC1 in isothermal titration calorimetry (ITC), and with cellular specificity for SMN (Liu, Y. et al. A small molecule antagonist of SMN disrupts the interaction between SMN and RNAP I I. Nat Commun 13, 5453 (2022)). Similarly, specific agents perturbing biomolecular condensation events are lacking, and pharmacological approaches often rely on unspecific agents like 1,6-hexanediol (Kroschwald, S., Maharana, S. & Simon, A. Hexanediol: a chemical probe to investigate the material properties of membrane-less compartments. Matters 3, e201702000010 (2017)) at concentrations of several hundred millimolar.
In view of the lack of small molecules that specifically inhibit SMNDC1 , it would be highly desirable to provide such SMNDC1 inhibitors. Accordingly, it is an object of the present invention to provide novel and/or improved SMNDC1 Tudor domain inhibitors. A further object of the invention is to provide potent SMNDC1 Tudor domain inhibitors that are selective for SMNDC1 over other Tudor domain-containing proteins, particularly over SMN.
The present invention addresses these needs and solves the problem of providing novel and therapeutically advantageous SMNDC1 inhibitors. In the context of the invention, the phase-separating behavior of SMNDC1 was studied both in vitro and within cells (see Example B below), and specific inhibitors against the Tudor domain of SMNDC1 , influencing the sub-cellular localization and phase separation of their target, were developed. It was surprisingly found that the compounds of formula (I) as provided herein are inhibitors of SMNDC1 exhibiting particularly favorable properties in terms of potency and selectivity, which makes these compounds highly advantageous for use in therapy.
Accordingly, the present invention provides a compound of the following formula (I) or a pharmaceutically acceptable salt or solvate thereof: X1-X2 S.UQ / N R1
(I)
In formula (I), one of the ring atoms X1 and X2 is S, and the other one is C(-Rx).
The group Rx is selected from hydrogen, C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-O(Ci-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-NH2, -(C0-5 alkylene)-NH(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-OH, -(C0-5 alkylene)-N(Ci-5 alkyl)-OH, -(C0-5 alkylene)-NH-O(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-O(Ci-5 alkyl), -(C0-5 alkylene)-halogen, -(C0-5 alkylene)-(Ci-5 haloalkyl), -(C0-5 alkylene)-O-(Ci-5 haloalkyl), -(C0-5 alkylene)-CN, -(C0-5 alkylene)-NO2, -(C0-5 alkylene)-CHO, -(C0-5 alkylene)-CO-(Ci-5 alkyl), -(C0-5 alkylene)-CO-(Ci-5 haloalkyl), -(C0-5 alkylene)-COOH, -(C0-5 alkylene)-CO-O-(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-(Ci-5 alkyl), -(C0-5 alkylene)-CO-NH2, -(C0-5 alkylene)-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-CO-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-CO-(Ci-5 alkyl), -(C0-5 alkylene)-NH-COO(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-COO(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-SO2-NH2, -(C0-5 alkylene)-SO2-NH(Ci-5 alkyl), -(C0-5 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-SO-(Ci-5 alkyl), -(C0-5 alkylene)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-carbocyclyl, -(C0-5 alkylene)-heterocyclyl, and -Lz-Rz, wherein the carbocyclyl in said -(C0-5 alkylene)-carbocyclyl and the heterocyclyl in said -(C0-5 alkylene)-heterocyclyl are each optionally substituted with one or more groups RCyc.
R1 is -L1-L2-R11.
L1 is selected from a covalent bond, -CO-, -SO-, -SO2-, -CO-N(RN)-, -SO-N(RN)-, -SO2-N(RN)-, and -C(=N-RN)-N(RN)- , wherein each RN is independently hydrogen or C1-5 alkyl.
L2 is selected from a covalent bond, C1-10 alkylene, C2-10 alkenylene, C2-10 alkynylene, -(C0-5 alkylene)-carbocyclyl- (C0-5 alkylene)-, and -(C0-5 alkylene)-heterocyclyl-(Co-5 alkylene)-, wherein said C1-10 alkylene, said C2-10 alkenylene, said C2-10 alkynylene, the two C0-5 alkylene groups in said -(C0-5 alkylene)-carbocyclyl-(Co-5 alkylene)-, and the two C0-5 alkylene groups in said -(C0-5 alkylene)-heterocyclyl-(Co-5 alkylene)- are each optionally substituted with one or more groups RAlk, wherein one or more -CH2- units comprised in said C1-10 alkylene, said C2-10 alkenylene, said C2-10 alkynylene, any of the C0-5 alkylene groups in said -(C0-5 alkylene)-carbocyclyl-(Co-5 alkylene)-, or any of the C0-5 alkylene groups in said -(C0-5 alkylene)-heterocyclyl-(Co-5 alkylene)- are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -N(RL2)-, and -C(RL2)=C(RL2)-, wherein each RL2 is independently hydrogen or C1-5 alkyl, and further wherein the carbocyclyl group in said -(C0-5 alkylene)-carbocyclyl- (Co-5 alkylene)- and the heterocyclyl group in said -(C0-5 alkylene)-heterocyclyl are each optionally substituted with one or more groups F .
R11 is selected from hydrogen, -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(Ci-5 alkyl), -S(Ci-5 alkylene)-SH, -S(Ci-5 alkylene)-S(Ci.5 alkyl), -NH2, -NH(CI-5 alkyl), -N(CI_5 alkyl)(Ci.5 alkyl), -NH- OH, -N(CI-5 alkyl)-OH, -NH-O(CI.5 alkyl), -N(CI_5 alkyl)-O(Ci.5 alkyl), halogen, C1.5 haloalkyl, -O-(Ci-5 haloalkyl), -ON, -NO2, -CHO, -C0-(Ci.5 alkyl), -COOH, -CO-O-(Ci.5 alkyl), -O-CO-(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(CI.5 alkyl)(Ci.5 alkyl), -NH-CO-(CI.5 alkyl), -N(CI_5 alkyl)-CO-(Ci.5 alkyl), -NH-COO(CI.5 alkyl), -N(CI_5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(CI-5 alkyl), -O-CO-N(CI.5 alkyl)(Ci_5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci_5 alkyl), -NH-SO2-(CI.5 alkyl), -N(CI_5 alkyl)-SO2-(Ci-5 alkyl), -SO-(Ci.5 alkyl), -SO2-(Ci.5 alkyl), -(Co-15 alkylene)-carbocyclyl, -(Co-15 alkylene)-heterocyclyl, and -Lz-Rz, wherein each alkyl or alkylene comprised in any of the aforementioned groups is optionally substituted with one or more groups RAlk, wherein one or more -CH2- units comprised in the Co-15 alkylene in said -(Co-15 alkylene)-carbocyclyl or in the Co-15 alkylene in said -(Co-15 alkylene)-heterocyclyl are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2- , -CO-, -NH-, and -N(CI-5 alkyl)-, and wherein the carbocyclyl in said -(Co-15 alkylene)-carbocyclyl and the heterocyclyl in said -(Co-15 alkylene)-heterocyclyl are each optionally substituted with one or more groups RCyc.
R2 is selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, -(Co-10 alkylene)-OH, -(Co-10 alkylene)-O(Ci-5 alkyl), -(Co-10 alkylene)-SH, -(Co-10 alkylene)-S(Ci-5 alkyl), -(C1-10 alkylene)-NH2, -(C1-10 alkylene)-NH(Ci-5 alkyl), -(C1-10 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(CMO alkylene)-halogen, C1-10 haloalkyl, -(Co-10 alkylene)-O-(Ci-5 haloalkyl), -(Co-10 alkylene)-CN, -(Co-10 alkylene)-NO2, -(Co-10 alkylene)-CHO, -(Co-10 alkylene)-CO-(Ci-io alkyl), -(Co-10 alkylene)-COOH, -(Co-10 alkylene)-CO-O-(Ci-5 alkyl), -(Co-10 alkylene)-O-CO-(Ci-5 alkyl), -(Co-10 alkylene)-CO-NH2, -(Co-10 alkylene)-CO-NH(Ci-5 alkyl), -(Co-10 alkylene)-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C1-10 alkylene)-NH-CO-(Ci-5 alkyl), -(C1-10 alkylene)-N(Ci-5 alkyl)-CO-(Ci-5 alkyl), -(C1-10 alkylene)-NH-COO(Ci-5 alkyl), -(C1-10 alkylene)-N(Ci-5 alkyl)-COO(Ci-5 alkyl), -(Co-10 alkylene)-O-CO-NH(Ci-5 alkyl), -(Co-10 alkylene)-O-CO- N(CI-5 alkyl)(Ci-5 alkyl), -(Co-10 alkylene)-SO2-NH2, -(Co-10 alkylene)-SO2-NH(Ci-5 alkyl), -(Co-10 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(CMO alkylene)-NH-SO2-(Ci-5 alkyl), -(CMO alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(Co-10 alkylene)-SO-(Ci-5 alkyl), -(Co-10 alkylene)-SO2-(Ci-5 alkyl), -(Co-10 alkylene)-carbocyclyl, and -(Co-10 alkylene)-heterocyclyl, wherein one or more -CH2- units comprised in said C1-10 alkyl, said C2-10 alkenyl, said C2-10 alkynyl, or in any of the aforementioned Co-10 alkylene or C1-10 alkylene groups are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -NH-, and -N(CI-5 alkyl)-, wherein the carbocyclyl in said -(Co-10 alkylene)-carbocyclyl and the heterocyclyl in said -(Co-10 alkylene)-heterocyclyl are each optionally substituted with one or more groups RCyc.
R3 is -(C0-5 alky lene)-ary I or -(C0-5 alkylene)-heteroaryl, wherein the aryl in said -(C0-5 alky lene)-aryl and the heteroaryl in said -(C0-5 alkylene)-heteroaryl are each optionally substituted with one or more groups R31, and wherein one or more -CH2- units comprised in the C0-5 alkylene in said -(C0-5 alkylene)-aryl or in the C0-5 alkylene in said -(C0-5 alky lene)-heteroary I are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO- , -NH-, and -N(CI-5 alkyl)-. Each R31 is independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-O(Ci-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-NH2, -(C0-5 alkylene)-NH(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-OH, -(C0-5 alkylene)-N(Ci-5 alkyl)-OH, -(C0-5 alkylene)-NH-O(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-O(Ci-5 alkyl), -(C0-5 alkylene)-halogen, -(C0-5 alkylene)-(Ci-5 haloalkyl), -(C0-5 alkylene)-O-(Ci-5 haloalkyl), -(C0-5 alkylene)-CN, -(C0-5 alkylene)-NO2, -(C0-5 alkylene)-CHO, -(C0-5 alkylene)-CO-(Ci-5 alkyl), -(C0-5 alkylene)-COOH, -(C0-5 alkylene)-CO-O-(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-(Ci-5 alkyl), -(C0-5 alkylene)-CO-NH2, -(C0-5 alkylene)-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-CO-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-CO-(Ci-5 alkyl), -(C0-5 alkylene)-NH-COO(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-COO(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-SO2-NH2, -(C0-5 alkylene)-SO2-NH(Ci-5 alkyl), -(C0-5 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-SO-(Ci-5 alkyl), -(C0-5 alkylene)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-carbocyclyl, -(C0-5 alkylene)-heterocyclyl, and -Lz-Rz, wherein the carbocyclyl in said -(C0-5 alkylene)-carbocyclyl and the heterocyclyl in said -(C0-5 alkylene)-heterocyclyl are each optionally substituted with one or more groups R°yc, and wherein one or more -CH2- units comprised in the C0-5 alkylene in said -(C0-5 alkylene)-carbocyclyl or in the C0-5 alkylene in said -(C0-5 alkylene)-heterocyclyl are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -NH-, and -N(CI-5 alkyl)-.
Each RAlk is independently selected from -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(Ci.5 alkyl), -S(Ci.5 alkylene)-SH, -S(Ci.5 alkylene)-S(Ci.5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI.5 alkyl)(Ci.5 alkyl), -NH-OH, -N(CI.5 alkyl)-OH, -NH-O(CI.5 alkyl), -N(CI.5 alkyl)-O(Ci.5 alkyl), halogen, C1.5 haloalkyl, -O-(Ci.5 haloalkyl), -ON, -NO2, -OHO, -CO(Ci.5 alkyl), -COOH, -COO(Ci.5 alkyl), -O-CO(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(CI-5 alkyl)(Ci.5 alkyl), -NH-CO(CI.5 alkyl), -N(CI.5 alkyl)-CO(Ci_5 alkyl), -NH-COO(CI.5 alkyl), -N(CI.5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(CI.5 alkyl), -O-CO-N(CI.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci.5 alkyl), -NH-SO2-(CI.5 alkyl), -N(CI.5 alkyl)-SO2-(Ci.5 alkyl), -SO2-(Ci.5 alkyl), -SO-(Ci.5 alkyl), -(C0-5 alkylene)-cycloalkyl, -(C0-5 alkylene)-heterocycloalkyl, and -Lz-Rz.
Each RCyc is independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-O(Ci-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-NH2, -(C0-5 alkylene)-NH(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-OH, -(C0-5 alkylene)-N(Ci-5 alkyl)-OH, -(C0-5 alkylene)-NH-O(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-O(Ci-5 alkyl), -(C0-5 alkylene)-halogen, -(C0-5 alkylene)-Ci-5 haloalkyl, -(C0-5 alkylene)-O-(Ci-5 haloalkyl), -(C0-5 alkylene)-CN, -(C0-5 alkylene)-NO2, -(C0-5 alkylene)-CHO, -(C0-5 alkylene)-CO(Ci-5 alkyl), -(C0-5 alkylene)-COOH, -(C0-5 alkylene)-COO(Ci-5 alkyl), -(C0-5 alkylene)-O-CO(Ci-5 alkyl), -(C0-5 alkylene)-CO-NH2, -(C0-5 alkylene)-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-CO(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-CO(Ci-5 alkyl), -(C0-5 alkylene)-NH-COO(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-COO(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-SO2-NH2, -(C0-5 alkylene)-SO2-NH(Ci-5 alkyl), -(C0-5 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-S0-(Ci-5 alkyl), -(C0-5 alkylene)-carbocyclyl, -(C0-5 alkylene)-heterocyclyl, and -Lz-Rz.
Each Lz is independently selected from a covalent bond, C1-35 alkylene, C2-35 alkenylene, and C2-35 alkynylene, wherein said alkylene, said alkenylene and said alkynylene are each optionally substituted with one or more groups independently selected from halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -CN, -OH, -O(Ci-5 alkyl), -SH, -S(Ci-5 alkyl), -NH2, -NH(CI-5 alkyl), and -N(CI-5 alkyl)(Ci-5 alkyl), wherein one or more CH units comprised in said alkylene, said alkenylene or said alkynylene are each optionally replaced by a nitrogen atom, and further wherein one or more -CH2- units comprised in said alkylene, said alkenylene or said alkynylene are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -NH-, -N(CI-5 alkyl)-, carbocyclylene, and heterocyclylene, wherein said carbocyclylene and said heterocyclylene are each optionally substituted with one or more groups independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -CN, -NO2, -OH, -O(Ci.5 alkyl), -SH, -S(Ci.5 alkyl), -NH2, -NH(Ci.5 alkyl), -N(CI.5 alkyl)(Ci.5 alkyl), -OHO, -CO-(Ci.5 alkyl), -COCH, -CO-O-(Ci.5 alkyl), -O-CO-(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(CI.5 alkyl)(Ci.5 alkyl), -NH-CO-(Ci-5 alkyl), -N(Ci.5 alkyl)-CO-(Ci.5 alkyl), -NH-COO(Ci.5 alkyl), -N(Ci.5 alkyl)-COO(Ci.5 alkyl), -O-CO- NH(0I-5 alkyl), -O-CO-N(Ci.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(Ci.5 alkyl), -SO2-N(Ci.5 alkyl)(Ci.5 alkyl), -NH-SO2-(CI.5 alkyl), -N(Ci.5 alkyl)-SO2-(Ci.5 alkyl), -SO-(Ci.5 alkyl), and -SO2-(Ci.5 alkyl).
Each Rz is independently selected from -OH, -0(Ci-5 alkyl), -0(Ci-5 alkylene)-OH, -0(Ci-5 alkylene)-0(Ci-5 alkyl), -SH, -S(Ci.5 alkyl), -S(Ci.5 alkylene)-SH, -S(Ci.5 alkylene)-S(Ci.5 alkyl), -NH2, -NH(Ci.5 alkyl), -N(Ci.5 alkyl)(Ci.5 alkyl), -NH-OH, -N(Ci.5 alkyl)-OH, -NH-O(Ci.5 alkyl), -N(Ci.5 alkyl)-O(Ci.5 alkyl), halogen, C1.5 haloalkyl, -O-(Ci.5 haloalkyl), -CN, -N02, -OHO, -C0(Ci.5 alkyl), -COCH, -C00(Ci.5 alkyl), -0-C0(Ci.5 alkyl), -CO-NH2, -CO-NH(Ci.5 alkyl), -C0-N(Ci-5 alkyl)(Ci.5 alkyl), -NH-CO(Ci.5 alkyl), -N(Ci.5 alkyl)-CO(Ci.5 alkyl), -NH-COO(Ci.5 alkyl), -N(Ci.5 alkyl)-C00(Ci.5 alkyl), -O-CO-NH(Ci.5 alkyl), -O-CO-N(Ci.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(Ci.5 alkyl), -SO2-N(CI.5 alkyl)(Ci.5 alkyl), -NH-SO2-(Ci.5 alkyl), -N(Ci.5 alkyl)-SO2-(Ci.5 alkyl), -SO2-(Ci.5 alkyl), -SO-(Ci.5 alkyl), aryl, heteroaryl, cycloalkyl, and heterocycloalkyl, wherein said aryl, said heteroaryl, said cycloalkyl, and said heterocycloalkyl are each optionally substituted with one or more groups independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, halogen, C1-5 haloalkyl, -0-(Ci-5 haloalkyl), -CN, -NO2, -OH, -0(Ci-5 alkyl), -SH, -S(Ci-5 alkyl), -NH2, -NH(CI.5 alkyl), -N(Ci.5 alkyl)(Ci.5 alkyl), -OHO, -C0-(Ci.5 alkyl), -COCH, -C0-0-(Ci.5 alkyl), -0-C0-(Ci.5 alkyl), -CO-NH2, -C0-NH(Ci-5 alkyl), -CO-N(Ci.5 alkyl)(Ci.5 alkyl), -NH-CO-(Ci.5 alkyl), -N(Ci.5 alkyl)-CO-(Ci.5 alkyl), -NH-C00(Ci-5 alkyl), -N(Ci.5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(Ci.5 alkyl), -O-CO-N(Ci.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(Ci-5 alkyl)(Ci.5 alkyl), -NH-SO2-(Ci-5 alkyl), -N(Ci.5 alkyl)-SO2-(Ci-5 alkyl), -S0-(Ci-5 alkyl), -SO2-(Ci-5 alkyl), carbocyclyl, and heterocyclyl, wherein said carbocyclyl and said heterocyclyl are each optionally substituted with one or more groups independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, halogen, C1.5 haloalkyl, -O-(Ci.5 haloalkyl), -CN, -N02, -OH, -O(Ci.5 alkyl), -SH, -S(Ci.5 alkyl), -NH2, -NH(Ci.5 alkyl), -N(0I-5 alkyl)(Ci.5 alkyl), -OHO, -C0-(Ci.5 alkyl), -COCH, -C0-0-(Ci.5 alkyl), -0-C0-(Ci.5 alkyl), -CO-NH2, -C0-NH(Ci-5 alkyl), -CO-N(Ci.5 alkyl)(Ci.5 alkyl), -NH-CO-(Ci.5 alkyl), -N(Ci.5 alkyl)-CO-(Ci.5 alkyl), -NH-C00(Ci-5 alkyl), -N(Ci.5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(Ci.5 alkyl), -O-CO-N(Ci.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(Ci-5 alkyl)(Ci.5 alkyl), -NH-SO2-(Ci-5 alkyl), -N(Ci.5 alkyl)-SO2-(Ci-5 alkyl), -S0-(Ci-5 alkyl), and -SO2-(Ci-5 alkyl). The present invention also relates to a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, in combination with a pharmaceutically acceptable excipient. Accordingly, the invention relates to a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof (or a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable excipient) for use as a medicament.
The compounds of formula (I) have been found to be highly effective as SMNDC1 inhibitors, particularly as SMNDC1 Tudor domain inhibitors (see Examples B and C below). Accordingly, the present invention provides potent SMNDC1 inhibitors. Moreover, the invention also provides SMNDC1 inhibitors that are advantageously selective for SMNDC1 over SMN (see also Examples B and C).
As explained above, SMNDC1 is a Tudor domain protein that recognizes di-methylated arginines and controls gene expression as an essential splicing factor. In the context of the present invention, the specific contributions of the SMNDC1 Tudor domain to protein-protein interactions, subcellular localization, and molecular function were studied. To perturb the protein function in cells, the inventors developed small molecule inhibitors, particularly the compounds of formula (I), targeting the dimethyl arginine binding pocket of the SMNDC1 Tudor domain. As detailed in Example B, it was found that SMNDC1 localizes to phase-separated membraneless organelles that partially overlap with nuclear speckles. This condensation behavior is driven by the unstructured C-terminal region of SMNDC1 , depends on RNA interaction and can be recapitulated in vitro. Inhibitors of the protein's Tudor domain drastically alter protein-protein interactions and subcellular localization, causing splicing changes for SMNDCI-dependent genes. The SMNDC1 inhibitors provided herein can advantageously be used in therapy, particularly for the treatment or prevention of SMNDCI-related (or SMNDCI-mediated) diseases/disorders, including cancer or diabetes.
Without being bound by theory, inhibitors of SMNDC1 are expected to induce the production of insulin in a-cells and, consequently, to be suitable for the treatment of diabetes or diabetes-related diseases/disorders (see, e.g., Casteels, T. et al. SMNDC1 links chromatin remodeling and splicing to regulate pancreatic hormone expression. Cell Reports 40, (2022)). Moreover, SMNDC1 has been reported to be essential for cell proliferation, particularly in the context of cancer, and inhibitors of SMNDC1 are expected to be effective in reducing or inhibiting cancer cell proliferation and/or migration (see, e.g., Zhu, R. et al. A systems biology-based approach to screen key splicing factors in hepatocellular carcinoma. Molecular Carcinogenesis, doi: 10.1002/mc.23549 (2023); Zhang, Y. et al. Activation of ERK by altered RNA splicing in cancer. bioRxiv, doi: 10.1101/2022.08.31.505957 (2022)).
The present invention thus further relates to a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising any of the aforementioned entities and a pharmaceutically acceptable excipient, for use in the treatment or prevention of an SMNDCI-related disease or disorder (or an SMNDCI-mediated disease or disorder). Accordingly, the invention also provides a pharmaceutical composition comprising, as an active ingredient, a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, together with a pharmaceutically acceptable excipient, for use in the treatment or prevention of an SMNDCI-related disease/disorder (or an SMNDCI-mediated disease/disorder). Moreover, the present invention relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for the treatment or prevention of an SMNDC1 -related disease or disorder (or an SMNDC1 -mediated disease or disorder).
The invention likewise relates to a method of treating or preventing an SMNDC1 -related disease or disorder (or an SMNDCI-mediated disease or disorder), the method comprising administering a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising any of the aforementioned entities in combination with a pharmaceutically acceptable excipient, to a subject (preferably a human) in need thereof. It will be understood that a therapeutically effective amount of the compound of formula (I) or the pharmaceutically acceptable salt thereof (or of the pharmaceutical composition) is to be administered in accordance with this method.
As explained above, the disease or disorder to be treated or prevented with a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof (or a corresponding pharmaceutical composition) in accordance with the present invention includes any SMNDC1 -related (or SMNDCI-mediated) disease or disorder. It is preferred that the disease/disorder to be treated or prevented in accordance with the invention is cancer or diabetes. The diabetes may be, e.g., type 1 diabetes or type 2 diabetes. The cancer may be, e.g., a solid cancer or a hematological cancer. Preferably, the cancer may be selected from lung cancer (e.g., small cell lung cancer or non-small cell lung cancer), renal cancer (or kidney cancer; e.g., renal carcinoma), gastrointestinal cancer, stomach cancer, colorectal cancer (e.g., colorectal carcinoma), colon cancer, anal cancer, genitourinary cancer, bladder cancer, liver cancer (e.g., hepatocellular carcinoma), pancreatic cancer (e.g., pancreatic adenocarcinoma or pancreatic ductal adenocarcinoma), cervical cancer, endometrial cancer, vaginal cancer, vulvar cancer, ovarian cancer (e.g., ovarian carcinoma), uterine cancer, prostate cancer (e.g., hormone-refractory prostate cancer), testicular cancer, biliary tract cancer, hepatobiliary cancer, neuroblastoma, brain cancer (e.g., glioblastoma), breast cancer (e.g., triple-negative breast cancer, including in particular COX-2 expressing triple-negative breast cancer, or breast cancer having a BRCA1 and/or BRCA2 gene mutation), head and/or neck cancer (e.g., head and neck squamous cell carcinoma), skin cancer, melanoma, Merkel-cell cancer (e.g., Merkel-cell carcinoma), epidermoid cancer, squamous cell cancer (or squamous cell carcinoma; including, e.g., oral squamous cell carcinoma/squamous-cell mouth carcinoma, squamous-cell skin cancer, squamous-cell lung carcinoma, squamous-cell thyroid carcinoma, squamous-cell esophageal carcinoma, or squamous-cell vaginal carcinoma), bone cancer (e.g., osteosarcoma or osteogenic sarcoma), fibrosarcoma, Ewing's sarcoma, malignant mesothelioma, esophageal cancer, laryngeal cancer, mouth cancer, thymoma, neuroendocrine cancer (e.g., neuroendocrine carcinoma), goblet cell cancer (e.g., goblet cell carcinoid), hematological cancer, leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, or chronic myeloid leukemia), lymphoma (e.g., Hodgkin lymphoma or non-Hodgkin lymphoma, such as, e.g., follicular lymphoma or diffuse large B-cell lymphoma), and multiple myeloma. Moreover, the cancer (including any one of the aforementioned specific types of cancer) may also be a metastatic cancer.
Accordingly, the present invention particularly relates to a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising any of the aforementioned entities and a pharmaceutically acceptable excipient, for use in the treatment or prevention of cancer (e.g., any one of the specific types of cancer mentioned herein above) or diabetes (e.g., type 1 diabetes or type 2 diabetes).
The present invention furthermore relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof as an inhibitor of survival motor neuron domain-containing protein 1 (SMNDC1) in research, particularly as a research tool compound for inhibiting SMNDC1. Accordingly, the invention refers to the in vitro use of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof as an SMNDC1 inhibitor and, in particular, to the in vitro use of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof as a research tool compound acting as an SMNDC1 inhibitor. The invention likewise relates to a method, particularly an in vitro method, of inhibiting SMNDC1 , the method comprising the application of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof. The invention further relates to a method of inhibiting SMNDC1 , the method comprising applying a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof to a test sample (e.g., a biological sample) or a test animal (i.e., a non-human test animal). The invention also refers to a method, particularly an in vitro method, of inhibiting SMNDC1 in a sample (e.g., a biological sample), the method comprising applying a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof to said sample. The present invention further provides a method of inhibiting SMNDC1 , the method comprising contacting a test sample (e.g., a biological sample) or a test animal (i.e., a non-human test animal) with a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof. The terms "sample”, "test sample” and "biological sample” include, without being limited thereto: a cell, a cell culture or a cellular or subcel lular extract; biopsied material obtained from an animal (e.g., a human), or an extract thereof; or blood, serum, plasma, saliva, urine, feces, or any other body fluid, or an extract thereof. It is to be understood that the term “in vitro" is used in this specific context in the sense of "outside a living human or animal body”, which includes, in particular, experiments performed with cells, cellular or subcellular extracts, and/or biological molecules in an artificial environment such as an aqueous solution or a culture medium which may be provided, e.g., in a flask, a test tube, a Petri dish, a microtiter plate, etc.
The compounds of formula (I) as well as the pharmaceutically acceptable salts and solvates thereof will be described in more detail in the following.
Figure imgf000011_0001
In formula (I), one of the ring atoms X1 and X2 is S, and the other one is C(-Rx).
Accordingly, X1 is S and X2 is C(-Rx), or alternatively, X1 is C(-Rx) and X2 is S. It will be understood that the 5-membered ring containing the ring atoms X1 and X2 is aromatic, as also reflected by the inner circle drawn within this ring in formula (I). Thus, depending on the meanings of X1 and X2, the compound of formula (I) may have one of the following structures:
Figure imgf000012_0001
X1 is S, and X2 is C(-Rx) X1 is C(-Rx), and X2 is S
Preferably, X1 is S, and X2 is C(-Rx). Accordingly, it is preferred that the compound of formula (I) has the following structure:
Figure imgf000012_0002
The group Rx is selected from hydrogen, C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-O(Ci-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-NH2, -(C0-5 alkylene)-NH(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-OH, -(C0-5 alkylene)-N(Ci-5 alkyl)-OH, -(C0-5 alkylene)-NH-O(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-O(Ci-5 alkyl), -(C0-5 alkylene)-halogen, -(C0-5 alkylene)-(Ci-5 haloalkyl), -(C0-5 alkylene)-O-(Ci-5 haloalkyl), -(C0-5 alkylene)-CN, -(C0-5 alkylene)-NO2, -(C0-5 alkylene)-CHO, -(C0-5 alkylene)-CO-(Ci-5 alkyl), -(C0-5 alkylene)-CO-(Ci-5 haloalkyl), -(C0-5 alkylene)-COOH, -(C0-5 alkylene)-CO-O-(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-(Ci-5 alkyl), -(C0-5 alkylene)-CO-NH2, -(C0-5 alkylene)-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-CO-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-CO-(Ci-5 alkyl), -(C0-5 alkylene)-NH-COO(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-COO(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-SO2-NH2, -(C0-5 alkylene)-SO2-NH(Ci-5 alkyl), -(C0-5 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-SO-(Ci-5 alkyl), -(C0-5 alkylene)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-carbocyclyl, -(C0-5 alkylene)-heterocyclyl, and -Lz-Rz, wherein the carbocyclyl in said -(C0-5 alkylene)-carbocyclyl and the heterocyclyl in said -(C0-5 alkylene)-heterocyclyl are each optionally substituted with one or more (e.g., one, two or three) groups RCyc.
Preferably, Rx is selected from hydrogen, C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(Ci-5 alkyl), -S(Ci-5 alkylene)-SH, -S(Ci-5 alkylene)-S(Ci-5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI.5 alkyl)(Ci.5 alkyl), -NH-OH, -N(CI.5 alkyl)-OH, -NH-O(CI.5 alkyl), -N(CI.5 alkyl)-O(Ci-5 alkyl), halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -CN, -CHO, -CO-(Ci-5 alkyl), -CO-(Ci-5 haloalkyl), -COCH, -CO-O-(Ci.5 alkyl), -O-CO-(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(CI.5 alkyl)(Ci.5 alkyl), -NH-CO-(CI-5 alkyl), -N(CI.5 alkyl)-CO-(Ci.5 alkyl), -NH-COO(CI.5 alkyl), -N(CI.5 alkyl)-COO(Ci.5 alkyl), -O-CO- NH(CI.5 alkyl), -O-CO-N(CI.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(CI-5 alkyl), -SO2-N(CI-5 alkyl)(Ci.5 alkyl), -NH-SO2-(CI.5 alkyl), -N(CI.5 alkyl)-SO2-(Ci-5 alkyl), -SO-(Ci.5 alkyl), -SO2-(Ci-5 alkyl), -(C0-5 alkylene)-carbocyclyl, -(C0-5 alkylene)-heterocyclyl, and -Lz-Rz, wherein the carbocyclyl in said -(C0-5 alky lene)-carbocycly I and the heterocyclyl in said -(C0-5 alky lene)-heterocy cly I are each optionally substituted with one or more groups RCyc.
More preferably, Rx is selected from hydrogen, C1-5 alkyl, -OH, -O(Ci-5 alkyl), -SH, -S(Ci-5 alkyl), -NH2, -NH(CI-5 alkyl), -N(CI-5 alkyl)(Ci-5 alkyl), halogen (e.g., -F or -Cl), C1-5 haloalkyl (e.g., -CF3), -O-(Ci-5 haloalkyl) (e.g., -OCF3), and -ON.
Even more preferably, Rx is selected from hydrogen, C1-5 alkyl, halogen (e.g., -F, -Cl or -Br), C1-5 haloalkyl (e.g., -CF3), -O-(Ci-5 haloalkyl) (e.g., -OCF3), and -CN.
Yet even more preferably, Rx is hydrogen or methyl. For example, Rx may be methyl. It is especially preferred that Rx is hydrogen.
R1 is -L1-L2-R11.
L1 is selected from a covalent bond, -CO-, -SO-, -SO2-, -CO-N(RN)-, -SO-N(RN)-, -SO2-N(RN)-, and -C(=N-RN)-N(RN)- , wherein each RN is independently hydrogen or C1-5 alkyl.
Preferably, L1 is selected from a covalent bond, -CO-, and -SO2-.
More preferably, L1 is a covalent bond or -CO-.
L2 is selected from a covalent bond, C1-10 alkylene, C2-10 alkenylene, C2-10 alkynylene, -(C0-5 alkylene)-carbocyclyl- (C0-5 alkylene)-, and -(C0-5 alkylene)-heterocyclyl-(Co-5 alkylene)-, wherein said C1-10 alkylene, said C2-10 alkenylene, said C2-10 alkynylene, the two C0-5 alkylene groups in said -(C0-5 alkylene)-carbocyclyl-(Co-5 alkylene)-, and the two C0-5 alkylene groups in said -(C0-5 alkylene)-heterocyclyl-(Co-5 alkylene)- are each optionally substituted with one or more (e.g., one, two or three) groups RAlk, wherein one or more (e.g., one, two or three) -CH2- units comprised in said C1-10 alkylene, said C2-10 alkenylene, said C2-10 alkynylene, any of the C0-5 alkylene groups in said -(C0-5 alkylene)-carbocyclyl-(Co-5 alkylene)-, or any of the C0-5 alkylene groups in said -(C0-5 alkylene)-heterocyclyl-(Co-5 alkylene)- are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -N(RL2)-, and -C(RL2)=C(RL2)-, wherein each RL2 is independently hydrogen or C1-5 alkyl, and further wherein the carbocyclyl group in said -(C0-5 alkylene)-carbocyclyl-(Co-5 alkylene)- and the heterocyclyl group in said -(C0-5 alkylene)-heterocyclyl are each optionally substituted with one or more (e.g., one, two or three) groups RCyc.
Preferably, L2 is selected from a covalent bond, C1-10 alkylene, C2-10 alkenylene, -(C0-5 alkylene)-cycloalkyl-(Co-5 alkylene)-, -(C0-5 alkylene)-heterocycloalkyl-(Co-5 alkylene)-, -(C0-5 alkylene)-aryl-(Co-5 alkylene)-, and -(C0-5 alky lene)-heteroary l-(Co-5 alkylene)-, wherein said C1-10 alkylene, said C2-10 alkenylene, or any C0-5 alkylene comprised in any of the aforementioned groups are each optionally substituted with one or more groups RAlk, wherein one or more -CH2- units comprised in said C1-10 alkylene, said C2-10 alkenylene, or any C0-5 alkylene comprised in any of the aforementioned groups are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2- , -CO-, -N(RL2)-, and -C(RL2)=C(RL2)-, wherein each RL2 is independently hydrogen or C1-5 alkyl, and further wherein the cycloalkyl in said -(C0-5 alkylene)-cycloalkyl-(Co-5 alkylene)-, the heterocycloalkyl in said -(C0-5 alkylene)-heterocycloalkyl-(Co-5 alkylene)-, the aryl in said -(C0-5 alkylene)-aryl-(Co-5 alkylene)-, and the heteroaryl in said -(C0-5 alkylene)-heteroaryl-(Co-5 alkylene)- are each optionally substituted with one or more groups F .
More preferably, L2 is selected from a covalent bond, C1-5 alkylene, -(C0-5 alkylene)-aryl-(Co-5 alkylene)-, and -(C0-5 alkylene)-heteroaryl-(Co-5 alkylene)-, wherein said C1-5 alkylene, the two C0-5 alkylene groups in said -(C0-5 alkylene)-aryl-(Co-5 alkylene)-, and the two C0-5 alkylene groups in said -(C0-5 alkylene)-heteroaryl-(Co-5 alkylene)- are each optionally substituted with one or more groups RAlk, wherein one or more -CH2- units comprised in said C1-5 alkylene or any of the C0-5 alkylene groups comprised in said -(C0-5 alkylene)-aryl-(Co-5 alkylene)- or said -(C0-5 alkylene)-heteroaryl-(Co-5 alkylene)- are each optionally replaced by a group independently selected from -O-, -SO2- , -CO-, -NH-, and -N(CI-5 alkyl)-, and further wherein the aryl in said -(C0-5 alkylene)-aryl-(Co-5 alkylene)- and the heteroaryl in said -(C0-5 alkylene)-heteroaryl-(Co-5 alkylene)- are each optionally substituted with one or more groups RCyCi
Even more preferably, L2 is selected from a covalent bond, C1-5 alkylene, -(C0-5 alkylene)-aryl-(Co-5 alkylene)-, and -(C0-5 alkylene)-heteroaryl-(Co-5 alkylene)-, wherein the aryl in said -(C0-5 alkylene)-aryl-(Co-5 alkylene)- and the heteroaryl in said -(C0-5 alkylene)-heteroaryl-(Co-5 alkylene)- are each optionally substituted with one or more groups RCyCi
Yet even more preferably, L2 is selected from a covalent bond, aryl, and heteroaryl, wherein said aryl and said heteroaryl are each optionally substituted with one or more groups RCyc.
R11 is selected from hydrogen, -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(Ci-5 alkyl), -S(Ci.5 alkylene)-SH, -S(Ci.5 alkylene)-S(Ci.5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI.5 alkyl)(Ci_5 alkyl), -NH- OH, -N(CI.5 alkyl)-OH, -NH-O(CI.5 alkyl), -N(CI.5 alkyl)-O(Ci.5 alkyl), halogen, C1.5 haloalkyl, -O-(Ci.5 haloalkyl), -CN, -NO2, -CHO, -C0-(Ci.5 alkyl), -COCH, -CO-O-(Ci.5 alkyl), -O-CO-(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(CI.5 alkyl)(Ci.5 alkyl), -NH-CO-(CI.5 alkyl), -N(CI.5 alkyl)-CO-(Ci-5 alkyl), -NH-COO(CI.5 alkyl), -N(CI.5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(CI-5 alkyl), -O-CO-N(CI.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(CI-5 alkyl), -SO2-N(CI-5 alkyl)(Ci.5 alkyl), -NH-SO2-(CI.5 alkyl), -N(CI.5 alkyl)-SO2-(Ci-5 alkyl), -SO-(Ci.5 alkyl), -SO2-(Ci-5 alkyl), -(Co-15 alkylene)-carbocyclyl, -(Co-15 alkylene)-heterocyclyl, and -Lz-Rz, wherein each alkyl or alkylene comprised in any of the aforementioned groups is optionally substituted with one or more (e.g., one, two, three, or four) groups RAlk, wherein one or more (e.g., one, two, three, of four) -CH2- units comprised in the Co-15 alkylene in said -(Co-15 alkylene)-carbocyclyl or in the Co-15 alkylene in said -(Co-15 alkylene)-heterocyclyl are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -NH-, and -N(0I-5 alkyl)-, and wherein the carbocyclyl in said -(Co-15 alkylene)-carbocyclyl and the heterocyclyl in said -(Co-15 alkylene)-heterocyclyl are each optionally substituted with one or more (e.g., one, two or three) groups RCyc.
Preferably, R11 is selected from hydrogen, -OH, -O(Ci-5 alkyl), -SH, -S(Ci-5 alkyl), -NH2, -NH(CI-5 alkyl), -N(0I-5 alkyl)(Ci.5 alkyl), -NH-OH, -N(CI.5 alkyl)-OH, -NH-O(CI.5 alkyl), -N(CI.5 alkyl)-O(Ci_5 alkyl), halogen, C1.5 haloalkyl, -O-(Ci.5 haloalkyl), -CN, -NO2, -CHO, -CO-(Ci.5 alkyl), -COOH, -CO-O-(Ci.5 alkyl), -O-CO-(Ci.5 alkyl), -CO-NH2, -CO-NH(CI-5 alkyl), -CO-N(CI.5 alkyl)(Ci-5 alkyl), -NH-CO-(CI.5 alkyl), -N(CI.5 alkyl)-CO-(Ci_5 alkyl), -NH-COO(CI-5 alkyl), -N(CI.5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(CI.5 alkyl), -O-CO-N(CI.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI-5 alkyl)(Ci-5 alkyl), -NH-SO2-(CI-5 alkyl), -N(CI.5 alkyl)-SO2-(Ci-5 alkyl), -SO-(Ci-5 alkyl), -SO2-(Ci-5 alkyl), -(C0-5 alkylene)-cycloalkyl, -(C0-5 alkylene)-heterocycloalkyl, -(C0-5 alkylene)-aryl, -(C0-5 alkylene)-heteroaryl, and -Lz-Rz, wherein each C1-5 alkyl or C0-5 alkylene comprised in any of the aforementioned groups is optionally substituted with one or more groups RAlk, wherein one or more (e.g., one or two) -CH2- units comprised in the C0-5 alkylene in said -(C0-5 alkylene)-cycloalkyl, in the C0-5 alkylene in said -(C0-5 alkylene)-heterocycloalkyl, in the C0-5 alkylene in said -(C0-5 alkylene)-aryl, or in the C0-5 alkylene in said -(C0-5 alky lene)-heteroary I are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO- , -NH-, and -N(CI-5 alkyl)-, and wherein the cycloalkyl in said -(C0-5 alkylene)-cycloalkyl, the heterocycloalkyl in said -(C0-5 alkylene)-heterocycloalkyl, the aryl in said -(C0-5 alkylene)-aryl, and the heteroaryl in said -(C0-5 alky lene)-heteroary I are each optionally substituted with one or more groups RCyc.
Examples of R1 include, in particular, any of the specific groups R1 comprised in the exemplary compounds of formula (I) described in the present specification, including any of the exemplary compounds of formula (I) described in Example A, B or 0 below. For this purpose, if any of these exemplary compounds of formula (I) has hydrogen as one of the two groups R1 and R2, it will be assumed that said hydrogen is R2 and that the other one of the said two groups is R1.
R2 is selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, -(Co-10 alkylene)-OH, -(Co-10 alkylene)-0(Ci-5 alkyl), -(Co-10 alkylene)-SH, -(Co-10 alkylene)-S(Ci-5 alkyl), -(C1-10 alkylene)-NH2, -(C1-10 alkylene)-N H(CI-5 alkyl), -(C1-10 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C1-10 alkylene)-halogen, C1-10 haloalkyl, -(Co-10 alkylene)-0-(Ci-5 haloalkyl), -(Co-10 alkylene)-CN, -(Co-10 alkylene)-N02, -(Co-10 alkylene)-CHO, -(Co-10 alkylene)-CO-(Ci-io alkyl), -(Co-10 alkylene)-COOH, -(Co-10 alkylene)-C0-0-(Ci-5 alkyl), -(Co-10 alkylene)-0-C0-(Ci-5 alkyl), -(Co-10 alkylene)- CO-NH2, -(Co-10 alkylene)-C0-NH(Ci-5 alkyl), -(Co-10 alkylene)-C0-N(Ci-5 alkyl)(Ci-5 alkyl), -(C1-10 alkylene)- NH-C0-(CI-5 alkyl), -(C1-10 alkylene)-N(Ci-5 alkyl)-C0-(Ci-5 alkyl), -(C1-10 alkylene)-NH-C00(Ci-5 alkyl), -(C1-10 alkylene)-N(Ci-5 alkyl)-C00(Ci-5 alkyl), -(Co-10 alkylene)-0-C0-NH(Ci-5 alkyl), -(Co-10 alkylene)-0-C0-N(Ci-5 alkyl)(Ci-5 alkyl), -(Co-10 alkylene)-SO2-NH2, -(Co-10 alkylene)-SO2-NH(Ci-5 alkyl), -(Co-10 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C1-10 alkylene)-NH-SO2-(Ci-5 alkyl), -(C1-10 alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(Co-10 alkylene)-S0-(Ci-5 alkyl), -(Co-10 alkylene)-SO2-(Ci-5 alkyl), -(Co-10 alkylene)-carbocyclyl, and -(Co-10 alkylene)-heterocyclyl, wherein one or more (e.g., one, two or three) -CH2- units comprised in said C1-10 alkyl, said C2-10 alkenyl, said C2-10 alkynyl, or in any of the aforementioned Co-10 alkylene or C1-10 alkylene groups are each optionally replaced by a group independently selected from -0-, -S-, -SO-, -SO2-, -CO-, -NH-, and -N(CI-5 alkyl)-, wherein the carbocyclyl in said -(Co-10 alkylene)-carbocyclyl and the heterocyclyl in said -(Co-10 alkylene)-heterocyclyl are each optionally substituted with one or more (e.g., one, two or three) groups RCyc.
Preferably, R2 is selected from hydrogen, C1.5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-0(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C1.5 alkylene)-NH2, -(C1.5 alkylene)-NH(Ci-5 alkyl), -(C1-5 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C1.5 alkylene)-halogen, C1.5 haloalkyl, -(C0-5 alkylene)-0-(Ci-5 haloalkyl), -(C0-5 alkylene)-CN, -(C0-5 alkylene)-N02, -(C0-5 alkylene)-CHO, -(C0-5 alkylene)-C0-(Ci-5 alkyl), -(C0-5 alkylene)-COOH, -(C0-5 alkylene)-C0-0-(Ci-5 alkyl), -(C0-5 alkylene)-0-C0-(Ci-5 alkyl), -(C0-5 alkylene)-C0-NH2, -(C0-5 alkylene)-C0-NH(Ci-5 alkyl), -(C0-5 alkylene)-C0-N(Ci-5 alkyl)(Ci-5 alkyl), -(C1.5 alkylene)-NH-C0-(Ci-5 alkyl), -(C1.5 alkylene)-N(Ci-5 alkyl)-C0-(Ci-5 alkyl), -(C1.5 alkylene)-NH-C00(Ci-5 alkyl), -(C1.5 alkylene)-N(Ci-5 alkyl)-C00(Ci-5 alkyl), -(C0-5 alkylene)-0-C0-NH(Ci-5 alkyl), -(C0-5 alkylene)-0-C0-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-SC>2-NH2, -(C0-5 alkylene)-SO2-NH(Ci-5 alkyl), -(C0-5 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C1-5 alkylene)-NH-SO2-(Ci-5 alkyl), -(C1-5 alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-S0-(Ci-5 alkyl), -(C0-5 alkylene)-SC>2-(Ci-5 alkyl), -(C0-5 alkylene)-carbocyclyl, and -(C0-5 alkylene)-heterocyclyl, wherein one or more (e.g., one, two, or three) -CH2- units comprised in said C1-5 alkyl, said C2-5 alkenyl, said C2-5 alkynyl, or in any of the aforementioned C0-5 alkylene or C1-5 alkylene groups are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -NH-, and -N(CI-5 alkyl)-, wherein the carbocyclyl in said -(C0-5 alkylene)-carbocyclyl and the heterocyclyl in said -(C0-5 alkylene)-heterocyclyl are each optionally substituted with one or more (e.g., one, two, or three) groups RCyc.
More preferably, R2 is selected from hydrogen, C1-5 alkyl, C2-5 alkenyl, and C1-5 haloalkyl.
Even more preferably, R2 is hydrogen or C1-5 alkyl.
Yet even more preferably, R2 is hydrogen.
R3 is -(C0-5 alky lene)-ary I or -(C0-5 alkylene)-heteroaryl, wherein the aryl in said -(C0-5 alky lene)-aryl and the heteroaryl in said -(C0-5 alkylene)-heteroaryl are each optionally substituted with one or more (e.g., one, two or three) groups R31, and wherein one or more (e.g., one, two or three) -CH2- units comprised in the C0-5 alkylene in said -(C0-5 alkylene)-aryl or in the C0-5 alkylene in said -(C0-5 alkylene)-heteroaryl are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -NH-, and -N(CI-5 alkyl)-.
Examples of R3 include, in particular, any of the specific groups R3 comprised in the exemplary compounds of formula (I) described in the present specification, including any of the exemplary compounds of formula (I) described in Example A, B or C below.
Preferably, R3 is -(C0-5 alkylene)-aryl or -(C0-5 alkylene)-heteroaryl, wherein the aryl in said -(C0-5 alkylene)-aryl and the heteroaryl in said -(C0-5 alkylene)-heteroaryl are each optionally substituted with one or more groups R31. More preferably, R3 is aryl, -Chfe-aryl, heteroaryl, or -Chh-heteroaryl, wherein said aryl, the aryl in said -Chfe-aryl, said heteroaryl, and the heteroaryl in said -CH2-heteroaryl are each optionally substituted with one or more groups R31. Even more preferably, R3 is aryl or heteroaryl, wherein said aryl and said heteroaryl are each optionally substituted with one or more groups R31.
In the above-described general and preferred definitions of R3, it is furthermore preferred that the aryl (including, e.g., the aryl comprised in the above-mentioned group -(C0-5 alkylene)-aryl) is phenyl, naphthyl or tetralinyl (e.g., tetralin- 6-yl), more preferably phenyl. Moreover, in these general and preferred definitions of R3, it is preferred that the heteroaryl (including, e.g., the heteroaryl comprised in the above-mentioned group -(C0-5 alkylene)-heteroaryl) is a monocyclic 5- or 6-membered heteroaryl or a bicyclic 9- or 10-membered heteroaryl, more preferably a monocyclic 5- or 6-membered heteroaryl. A corresponding bicyclic 9- or 10-membered heteroaryl may, e.g., have 1 , 2, 3, 4 or 5 ring heteroatoms selected independently from nitrogen, oxygen and sulfur, while all remaining ring atoms are carbon atoms. A corresponding monocyclic 5- or 6-membered heteroaryl may, e.g., have 1, 2 or 3 ring heteroatoms selected independently from nitrogen, oxygen and sulfur, while all remaining ring atoms are carbon atoms. Examples of a corresponding heteroaryl include pyridinyl (e.g., pyridin-2-yl), pyrrolyl (e.g., 1 H-pyrrol-2-yl), 1 ,3-benzodioxolyl (e.g., 1 ,3-benzodioxol-5-yl), furanyl (e.g., furan-2-yl), or thiophenyl (e.g., thiophen-2-yl). Particularly preferred examples of a corresponding heteroaryl include pyridinyl (e.g., pyridin-2-yl or pyridin-4-yl) or pyrrolyl (e.g., 1 H-pyrrol-2-yl), even more preferably pyridin-2-yl or 1 H-pyrrol-2-yl. An especially preferred example of a corresponding heteroaryl is pyridin-2-yl. It is furthermore preferred that the heteroaryl (in R3) is not pyridin-3-yl, more preferably it is not pyridin-3- yl, pyridin-4-yl or thiophen-2-yl.
Thus, even more preferably, R3 is phenyl or a monocyclic 5- or 6-membered heteroaryl, wherein said aryl and said heteroaryl are each optionally substituted with one or more groups R31. Yet even more preferably, R3 is selected from phenyl, pyridin-2-yl, and 1 H-pyrrol-2-yl, wherein said phenyl, said pyridin-2-yl, and said 1 H-pyrrol-2-yl are each optionally substituted with one or more groups R31. Still more preferably, R3 is pyridin-2-yl which is optionally substituted with one or more groups R31.
Each R31 is independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-O(Ci-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-NH2, -(C0-5 alkylene)-NH(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-OH, -(C0-5 alkylene)-N(0i-5 alkyl)-OH, -(C0-5 alkylene)-NH-O(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-O(Ci-5 alkyl), -(C0-5 alkylene)-halogen, -(C0-5 alkylene)-(Ci-5 haloalkyl), -(C0-5 alkylene)-O-(Ci-5 haloalkyl), -(C0-5 alkylene)-CN, -(C0-5 alkylene)-NO2, -(C0-5 alkylene)-CHO, -(C0-5 alkylene)-CO-(Ci-5 alkyl), -(C0-5 alkylene)-COOH, -(C0-5 alkylene)-CO-O-(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-(Ci-5 alkyl), -(C0-5 alkylene)-CO-NH2, -(C0-5 alkylene)-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-CO-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-CO-(Ci-5 alkyl), -(C0-5 alkylene)-NH-COO(Ci-5 alkyl), -(C0-5 alkylene)-N(0i-5 alkyl)-COO(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-SO2-NH2, -(C0-5 alkylene)-SO2-NH(Ci-5 alkyl), -(C0-5 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-SO-(Ci-5 alkyl), -(C0-5 alkylene)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-carbocyclyl, -(C0-5 alkylene)-heterocyclyl, and -Lz-Rz, wherein the carbocyclyl in said -(C0-5 alkylene)-carbocyclyl and the heterocyclyl in said -(C0-5 alkylene)-heterocyclyl are each optionally substituted with one or more (e.g., one, two or three) groups R0^, and wherein one or more (e.g., one or two) -CH2- units comprised in the C0-5 alkylene in said -(C0-5 alkylene)-carbocyclyl or in the C0-5 alkylene in said -(C0-5 alkylene)-heterocyclyl are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2- , -CO-, -NH-, and -N(CI.5 alkyl)-.
Examples of R31 include, in particular, any of the specific groups R31 comprised in the exemplary compounds of formula (I) described in the present specification, including any of the exemplary compounds of formula (I) described in Example A, B or C below.
Preferably, each R31 is independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(0i-5 alkyl), -S(0i-5 alkylene)-SH, -S(0i-5 alkylene)-S(Ci-5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI.5 alkyl)(Ci.5 alkyl), -NH-OH, -N(CI.5 alkyl)-OH, -NH-O(CI.5 alkyl), -N(CI.5 alkyl)-O(Ci-5 alkyl), halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -ON, -NO2, -OHO, -C0-(Ci-5 alkyl), -COOH, -CO-O-(Ci.5 alkyl), -O-CO-(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(CI.5 alkyl)(Ci.5 alkyl), -NH-CO-(CI-5 alkyl), -N(Ci.5 alkyl)-CO-(Ci_5 alkyl), -NH-COO(CI.5 alkyl), -N(CI.5 alkyl)-COO(Ci_5 alkyl), -0-00- NH(CI.5 alkyl), -O-CO-N(CI.5 alkyl)(Ci_5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci_5 alkyl), -NH-SO2-(CI.5 alkyl), -N(Ci.5 alkyl)-SO2-(Ci.5 alkyl), -SO-(Ci.5 alkyl), -SO2-(Ci.5 alkyl), -(C0-5 alkylene)-carbocyclyl, -(C0-5 alkylene)-heterocyclyl, and -Lz-Rz, wherein the carbocyclyl in said -(C0-5 alky lene)-carbocycly I and the heterocyclyl in said -(C0-5 alky lene)-heterocy cly I are each optionally substituted with one or more groups RCyc.
More preferably, each R31 is independently selected from C1-5 alkyl, C2-5 alkenyl, -OH, -O(Ci-5 alkyl), -SH, -S(Ci-5 alkyl), -NH2, -NH(CI-5 alkyl), -N(CI-5 alkyl)(Ci-5 alkyl), halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), and -ON.
Each RAlk is independently selected from -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(Ci.5 alkyl), -S(Ci.5 alkylene)-SH, -S(Ci.5 alkylene)-S(Ci.5 alkyl), -NH2, -NH(Ci.5 alkyl), -N(Ci.5 alkyl)(Ci.5 alkyl), -NH-OH, -N(Ci.5 alkyl)-OH, -NH-O(CI.5 alkyl), -N(CI.5 alkyl)-O(Ci.5 alkyl), halogen, C1.5 haloalkyl, -O-(Ci.5 haloalkyl), -ON, -NO2, -OHO, -CO(Ci.5 alkyl), -COOH, -COO(Ci.5 alkyl), -O-CO(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(CI.5 alkyl)(Ci.5 alkyl), -NH-CO(CI.5 alkyl), -N(Ci.5 alkyl)-CO(Ci.5 alkyl), -NH-COO(CI.5 alkyl), -N(Ci.5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(CI.5 alkyl), -O-CO-N(CI.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci.5 alkyl), -NH-SO2-(CI.5 alkyl), -N(CI.5 alkyl)-SO2-(Ci.5 alkyl), -SO2-(Ci.5 alkyl), -SO-(Ci.5 alkyl), -(C0-5 alkylene)-cycloalkyl, -(C0-5 alkylene)-heterocycloalkyl, and -Lz-Rz.
Each RCyc is independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-O(Ci-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-NH2, -(C0-5 alkylene)-NH(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-OH, -(C0-5 alkylene)-N(Ci-5 alkyl)-OH, -(C0-5 alkylene)-NH-O(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-O(Ci-5 alkyl), -(C0-5 alkylene)-halogen, -(C0-5 alkylene)-Ci-5 haloalkyl, -(C0-5 alkylene)-O-(Ci-5 haloalkyl), -(C0-5 alkylene)-CN, -(C0-5 alkylene)-NO2, -(C0-5 alkylene)-CHO, -(C0-5 alkylene)-CO(Ci-5 alkyl), -(C0-5 alkylene)-COOH, -(C0-5 alkylene)-COO(Ci-5 alkyl), -(C0-5 alkylene)-O-CO(Ci-5 alkyl), -(C0-5 alkylene)-CO-NH2, -(C0-5 alkylene)-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-CO(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-CO(Ci-5 alkyl), -(C0-5 alkylene)-NH-COO(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-COO(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-SO2-NH2, -(C0-5 alkylene)-SO2-NH(Ci-5 alkyl), -(C0-5 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-SO-(Ci-5 alkyl), -(C0-5 alkylene)-carbocyclyl, -(C0-5 alkylene)-heterocyclyl, and -Lz-Rz.
Preferably, each R0''0 is independently selected from C1.5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(Ci-5 alkyl), -S(Ci-5 alkylene)-SH, -S(Ci-5 alkylene)-S(Ci-5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI.5 alkyl)(Ci.5 alkyl), -NH-OH, -N(Ci.5 alkyl)-OH, -NH-O(Ci.5 alkyl), -N(Ci.5 alkyl)-O(Ci.5 alkyl), halogen, C1.5 haloalkyl, -O-(Ci.5 haloalkyl), -ON, -NO2, -OHO, -CO(Ci.5 alkyl), -COOH, -COO(Ci.5 alkyl), -O-CO(Ci-5 alkyl), -CO-NH2, -CO-NH(Ci.5 alkyl), -CO-N(Ci.5 alkyl)(Ci.5 alkyl), -NH-CO(Ci.5 alkyl), -N(Ci.5 alkyl)-CO(Ci.5 alkyl), -NH-COO(Ci.5 alkyl), -N(Ci.5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(Ci.5 alkyl), -O-CO-N(Ci.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(Ci-5 alkyl), -SO2-N(Ci-5 alkyl)(Ci.5 alkyl), -NH-SO2-(Ci-5 alkyl), -N(Ci.5 alkyl)-SO2-(Ci-5 alkyl), -SO2-(Ci-5 alkyl), -S0-(Ci-5 alkyl), -(C0-3 alkylene)-cycloalkyl, -(C0-3 alkylene)-heterocycloalkyl, and -Lz-Rz.
Each Lz is independently selected from a covalent bond, C1-35 alkylene, C2-35 alkenylene, and C2-35 alkynylene, wherein said alkylene, said alkenylene and said alkynylene are each optionally substituted with one or more (e.g., one, two, or three) groups independently selected from halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -CN, -OH, -O(Ci-5 alkyl), -SH, -S(Ci-5 alkyl), -NH2, -NH(CI-5 alkyl), and -N(CI-5 alkyl)(Ci-5 alkyl), wherein one or more (e.g., one, two or three) CH units
Figure imgf000019_0001
comprised in said alkylene, said alkenylene or said alkynylene are each optionally replaced by a nitrogen atom (e.g.,
Figure imgf000019_0002
and further wherein one or more (e.g., one, two or three) -CH2- units comprised in said alkylene, said alkenylene or said alkynylene are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -NH-, -N(CI-5 alkyl)-, carbocyclylene, and heterocyclylene, wherein said carbocyclylene and said heterocyclylene are each optionally substituted with one or more (e.g., one, two or three) groups independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -CN, -NO2, -OH, -O(Ci.5 alkyl), -SH, -S(Ci.5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI.5 alkyl)(Ci.5 alkyl), -OHO, -CO-(Ci.5 alkyl), -COCH, -CO-O-(Ci.5 alkyl), -O-CO-(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(Ci-5 alkyl)(Ci.5 alkyl), -NH-CO-(Ci.5 alkyl), -N(Ci.5 alkyl)-CO-(Ci.5 alkyl), -NH-COO(Ci.5 alkyl), -N(Ci.5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(Ci.5 alkyl), -O-CO-N(Ci.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(Ci-5 alkyl), -SO2-N(CI.5 alkyl)(Ci.5 alkyl), -NH-SO2-(Ci-5 alkyl), -N(Ci.5 alkyl)-SO2-(Ci-5 alkyl), -SO-(Ci.5 alkyl), and -SO2-(Ci-5 alkyl).
Preferably, each Lz is independently selected from a covalent bond, C1-5 alkylene, C2-5 alkenylene, and C2-5 alkynylene, wherein said alkylene, said alkenylene and said alkynylene are each optionally substituted with one or more (e.g., one, two, or three) groups independently selected from halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -CN, -OH, -O(Ci.5 alkyl), -SH, -S(Ci.5 alkyl), -NH2, -NH(Ci.5 alkyl), and -N(Ci.5 alkyl)(Ci.5 alkyl), and further wherein one or more -CH2- units comprised in said alkylene, said alkenylene or said alkynylene are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -NH-, and -N(Ci-5 alkyl)-.
Each Rz is independently selected from -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(Ci.5 alkyl), -S(Ci.5 alkylene)-SH, -S(Ci.5 alkylene)-S(Ci.5 alkyl), -NH2, -NH(Ci.5 alkyl), -N(Ci.5 alkyl)(Ci.5 alkyl), -NH-OH, -N(Ci.5 alkyl)-OH, -NH-O(Ci.5 alkyl), -N(Ci.5 alkyl)-O(Ci.5 alkyl), halogen, C1.5 haloalkyl, -O-(Ci.5 haloalkyl), -CN, -NO2, -OHO, -CO(Ci.5 alkyl), -COCH, -COO(Ci.5 alkyl), -O-CO(Ci.5 alkyl), -CO-NH2, -CO-NH(Ci.5 alkyl), -CO-N(Ci-5 alkyl)(Ci.5 alkyl), -NH-CO(Ci.5 alkyl), -N(Ci.5 alkyl)-CO(Ci.5 alkyl), -NH-COO(Ci.5 alkyl), -N(Ci.5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(Ci.5 alkyl), -O-CO-N(Ci.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(Ci-5 alkyl), -SO2-N(CI.5 alkyl)(Ci.5 alkyl), -NH-SO2-(Ci-5 alkyl), -N(Ci.5 alkyl)-SO2-(Ci-5 alkyl), -SO2-(Ci-5 alkyl), -SO-(Ci.5 alkyl), aryl, heteroaryl, cycloalkyl, and heterocycloalkyl, wherein said aryl, said heteroaryl, said cycloalkyl, and said heterocycloalkyl are each optionally substituted with one or more (e.g., one, two or three) groups independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -CN, -NO2, -OH, -O(Ci-5 alkyl), -SH, -S(Ci.5 alkyl), -NH2, -NH(Ci.5 alkyl), -N(Ci.5 alkyl)(Ci.5 alkyl), -OHO, -CO-(Ci.5 alkyl), -COCH, -CO-O-(Ci.5 alkyl), -O-CO-(Ci.5 alkyl), -CO-NH2, -CO-NH(Ci.5 alkyl), -CO-N(Ci.5 alkyl)(Ci.5 alkyl), -NH-CO-(Ci.5 alkyl), -N(Ci.5 alkyl)-CO-(Ci-5 alkyl), -NH-COO(CI.5 alkyl), -N(CI_5 alkyl)-COO(Ci_5 alkyl), -O-CO-NH(CI.5 alkyl), -O-CO-N(CI.5 alkyl)(Ci-5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci_5 alkyl), -NH-SO2-(CI.5 alkyl), -N(CI.5 alkyl)-SC>2-(Ci-5 alkyl), -S0-(Ci-5 alkyl), -SO2-(Ci-5 alkyl), carbocyclyl, and heterocyclyl, wherein said carbocyclyl and said heterocyclyl are each optionally substituted with one or more (e.g., one, two or three) groups independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -CN, -NO2, -OH, -O(Ci-5 alkyl), -SH, -S(Ci.5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI_5 alkyl)(Ci.5 alkyl), -CHO, -CO-(Ci.5 alkyl), -COOH, -CO-O-(Ci.5 alkyl), -0-C0-(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(CI.5 alkyl)(Ci_5 alkyl), -NH-CO-(CI.5 alkyl), -N(CI_5 alkyl)-CO-(Ci.5 alkyl), -NH-COO(CI.5 alkyl), -N(CI_5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(CI.5 alkyl), -O-CO-N(CI.5 alkyl)(Ci-5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci_5 alkyl), -NH-SO2-(CI.5 alkyl), -N(CI.5 alkyl)-SO2-(Ci-5 alkyl), -SO-(Ci-5 alkyl), and -SO2-(Ci-5 alkyl).
Preferably, each Rz is independently selected from -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(Ci_5 alkyl), -S(Ci_5 alkylene)-SH, -S(Ci.5 alkylene)-S(Ci_5 alkyl), -NH2, -NH(CI_5 alkyl), -N(CI_5 alkyl)(Ci.5 alkyl), -NH-OH, -N(CI_5 alkyl)-OH, -NH-O(CI.5 alkyl), -N(CI.5 alkyl)-O(Ci.5 alkyl), halogen, C1.5 haloalkyl, -O-(Ci.5 haloalkyl), -CN, -NO2, -CHO, -CO(Ci.5 alkyl), -COOH, -COO(Ci.5 alkyl), -O-CO(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(CI-5 alkyl)(Ci_5 alkyl), -NH-CO(CI.5 alkyl), -N(CI_5 alkyl)-CO(Ci_5 alkyl), -NH-COO(CI.5 alkyl), -N(CI_5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(CI.5 alkyl), -O-CO-N(CI.5 alkyl)(Ci_5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci_5 alkyl), -NH-SO2-(CI.5 alkyl), -N(CI.5 alkyl)-SO2-(Ci.5 alkyl), -SO2-(Ci.5 alkyl), -SO-(Ci.5 alkyl), aryl, heteroaryl, cycloalkyl, and heterocycloalkyl, wherein said aryl, said heteroaryl, said cycloalkyl, and said heterocycloalkyl are each optionally substituted with one or more groups independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -CN, -OH, -O(Ci-5 alkyl), -SH, -S(Ci-5 alkyl), -NH2, -NH(CI.5 alkyl), and -N(CI_5 alkyl)(Ci_5 alkyl).
It is preferred that the compound of formula (I) is any one of the specific compounds of formula (I) described in the examples section of this specification, including any one of the specific compounds of formula (I) described in Example A, B or C herein below, either in non-salt form or as a pharmaceutically acceptable salt of the respective compound.
Moreover, while several exemplary compounds of formula (I) have been described in Example A, B or C as compounds having a specific stereochemical configuration, the present invention also relates to each of these compounds without specific configuration. The invention further relates specifically and individually to all other possible stereoisomers of such compounds that have been described with a specific configuration, including an enantiomer or a diastereoisomer of each respective compound, as well as mixtures thereof, including racemic mixtures (racemates).
It is particularly preferred that the compound of formula (I) is selected from any one of the following compounds:
Figure imgf000021_0001
Figure imgf000022_0001
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
or a pharmaceutically acceptable salt or solvate of any one of the above-depicted compounds. The present invention also specifically relates to each one of the above-depicted compounds in non-salt form.
Insofar as the present invention relates to novel compounds, it is preferred that the compounds of Examples 1 to 19, 63 to 87 and 110 are excluded from formula (I). Accordingly, it is preferred that the compound of formula (I) is not a compound of any one of Examples 1 to 19, 63 to 87 or 110 (or a pharmaceutically acceptable salt or solvate thereof).
In a first specific embodiment, the compound of formula (I) is a compound of the following formula (la)
Figure imgf000030_0002
or pharmaceutically acceptable salt or solvate thereof, wherein: o one of the ring atoms X1 and X2 is S, and the other one is C(-Rx); o Rx is selected from hydrogen, C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-O(Ci-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-NH2, -(C0-5 alkylene)-NH(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-OH, -(C0-5 alkylene)-N(Ci-5 alkyl)-OH, -(C0-5 alkylene)-NH-O(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-O(Ci-5 alkyl), -(C0-5 alkylene)-halogen, -(C0-5 alkylene)-(Ci-5 haloalkyl), -(C0-5 alkylene)-O-(Ci-5 haloalkyl), -(C0-5 alkylene)-CN, -(C0-5 alkylene)-NO2, -(C0-5 alkylene)-CHO, -(C0-5 alkylene)-CO-(Ci-5 alkyl), -(C0-5 alkylene)-CO-(Ci-5 haloalkyl), -(C0-5 alkylene)-COOH, -(C0-5 alkylene)-CO-O-(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-(Ci-5 alkyl), -(C0-5 alkylene)-CO-NH2, -(C0-5 alkylene)-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-CO-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-CO-(Ci-5 alkyl), -(C0-5 alkylene)-NH-COO(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-COO(Ci-5 alkyl), -(C0-5 alkylene)-O-CO- NH(CI-5 alkyl), -(C0-5 alkylene)-O-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-SO2-NH2, -(C0-5 alkylene)-SO2-NH(Ci-5 alkyl), -(C0-5 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-SC>2-(Ci-5 alkyl), -(C0-5 alkylene)-SO-(Ci-5 alkyl), -(C0-5 alkylene)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-carbocyclyl, -(C0-5 alkylene)-heterocyclyl, and -Lz-Rz, wherein the carbocyclyl in said -(C0-5 alky lene)-carbocycly I and the heterocyclyl in said -(C0-5 alky lene)-heterocycly I are each optionally substituted with one or more groups RCyc; R1 is -L1-L2-R11; L1 is selected from a covalent bond, -CO-, and -SO-; preferably, L1 is -CO-; L2 is selected from -(C0-5 alkylene)-aryl-(Co-5 alkylene)- and -(C0-5 alkylene)-heteroaryl-(Co-5 alkylene)-, wherein the two C0-5 alkylene groups in said -(C0-5 alkylene)-aryl-(Co-5 alkylene)- and the two C0-5 alkylene groups in said -(C0-5 alkylene)-heteroaryl-(Co-5 alkylene)- are each optionally substituted with one or more groups RAlk, wherein one or more -CH2- units comprised in any of the C0-5 alkylene groups in said -(C0-5 alky lene)-ary l-(Co-5 alkylene)- or any of the C0-5 alkylene groups in said -(C0-5 al ky lene)-heteroary l-(Co-5 alkylene)- are each optionally replaced by a group independently selected from -O-, -SO2-, -CO-, -NH-, and -N(CI-5 alkyl)-, and further wherein the aryl group in said -(C0-5 alkylene)-aryl-(Co-5 alkylene)- and the heteroaryl group in said -(C0-5 alky lene)-heteroary l-(Co-5 alkylene)- are each optionally substituted with one or more groups RCyc; preferably, L2 is selected from aryl and heteroaryl, wherein said aryl and said heteroaryl are each optionally substituted with one or more groups RCyc; R11 is selected from -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci.5 alkyl), -NH-SO2-(CI.5 alkyl), -N(CI.5 alkyl)-SO2-(Ci-5 alkyl), -SO-(Ci-5 alkyl), -SO2-(Ci-5 alkyl), -(C1-15 alkylene)-carbocyclyl, and -(C1-15 alkylene)-heterocyclyl, wherein each alkyl or alkylene comprised in any of the aforementioned groups is optionally substituted with one or more groups RAlk, wherein one or more -CH2- units comprised in the C1-15 alkylene in said -(C1-15 alkylene)-carbocyclyl or in the C1-15 alkylene in said -(C1-15 alkylene)-heterocyclyl are each replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -NH-, and -N(CI-5 alkyl)-, and wherein the carbocyclyl in said -(C1-15 alky lene)-carbocycly I and the heterocyclyl in said -(C1-15 alky lene)-heterocycly I are each optionally substituted with one or more groups RCyc; preferably, R11 is selected from -SO2-NH2, -SO2-NH(CI-5 alkyl), -SO2-N(CI-5 alkyl)(Ci-5 alkyl), -(C1-15 alkylene)-carbocyclyl, and -(C1-15 alkylene)-heterocyclyl, wherein one or more -CH2- units comprised in the C1-15 alkylene in said -(C1-15 alky lene)-carbocycly I or in the C1-15 alkylene in said -(C1-15 alky lene)-heterocycly I are each replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -NH-, and -N(CI-5 alkyl)-, and wherein the carbocyclyl in said -(C1-15 alkylene)-carbocyclyl and the heterocyclyl in said -(C1-15 alky lene)-heterocycly I are each optionally substituted with one or more groups RCyc; for example, R11 may be -SO2-heterocyclyl, wherein the heterocyclyl in said -SO2-heterocyclyl is optionally substituted with one or more groups RCyc (and wherein the heterocyclyl in said -SO2-heterocycly I may be attached via a ring nitrogen atom or a ring carbon atom, preferably via a ring nitrogen atom, to the SO2 group in said -SO2-heterocyclyl); R2 is selected from hydrogen, C1-5 alkyl, C2-5 alkenyl, and C1-5 haloalkyl; R3 is pyridinyl which is optionally substituted with one or more groups R31; preferably, R3 is pyridin-2-yl which is optionally substituted with one or more groups R31; more preferably, R3 is pyridin-2-yl; each R31 is independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-O(Ci-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-NH2, -(C0-5 alkylene)-NH(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-OH, -(C0-5 alkylene)-N(Ci-5 alkyl)-OH, -(C0-5 alkylene)-NH-O(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-O(Ci-5 alkyl), -(C0-5 alkylene)-halogen, -(C0-5 alkylene)-(Ci-5 haloalkyl), -(C0-5 alkylene)-O-(Ci-5 haloalkyl), -(C0-5 alkylene)-CN, -(C0-5 alkylene)-NO2, -(C0-5 alkylene)-CHO, -(C0-5 alkylene)-CO-(Ci-5 alkyl), -(C0-5 alkylene)-COOH, -(C0-5 alkylene)-CO-O-(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-(Ci-5 alkyl), -(C0-5 alkylene)-CO-NH2, -(C0-5 alkylene)-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-CO-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-CO-(Ci-5 alkyl), -(C0-5 alkylene)-NH-COO(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-COO(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-SO2-NH2, -(C0-5 alkylene)-SO2-NH(Ci-5 alkyl), -(C0-5 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-SO-(Ci-5 alkyl), -(C0-5 alkylene)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-carbocyclyl, -(C0-5 alkylene)-heterocyclyl, and -Lz-Rz, wherein the carbocyclyl in said -(C0-5 alky lene)-carbocycly I and the heterocyclyl in said -(C0-5 alky lene)-heterocycly I are each optionally substituted with one or more groups R°yc, and wherein one or more -CH2- units comprised in the C0-5 alkylene in said -(C0-5 alkylene)-carbocyclyl or in the C0-5 alkylene in said -(C0-5 alkylene)-heterocyclyl are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -NH-, and -N(CI-5 alkyl)-; each RAlk is independently selected from -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(Ci.5 alkyl), -S(Ci.5 alkylene)-SH, -S(Ci.5 alkylene)-S(Ci_5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI.5 alkyl)(Ci.5 alkyl), -NH-OH, -N(CI.5 alkyl)-OH, -NH-O(CI.5 alkyl), -N(CI.5 alkyl)-O(Ci_5 alkyl), halogen, C1.5 haloalkyl, -O-(Ci.5 haloalkyl), -ON, -NO2, -OHO, -CO(Ci-5 alkyl), -COOH, -COO(Ci.5 alkyl), -O-CO(Ci.5 alkyl), -CO-NH2, -CO-NH(CI-5 alkyl), -CO-N(CI.5 alkyl)(Ci.5 alkyl), -NH-CO(CI.5 alkyl), -N(CI.5 alkyl)-CO(Ci_5 alkyl), -NH-COO(CI-5 alkyl), -N(CI.5 alkyl)-COO(Ci_5 alkyl), -O-CO-NH(CI.5 alkyl), -O-CO-N(CI.5 alkyl)(Ci_5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI-5 alkyl)(Ci_5 alkyl), -NH-SO2-(CI-5 alkyl), -N(CI.5 alkyl)-SO2-(Ci-5 alkyl), -SO2-(Ci-5 alkyl), -SO-(Ci-5 alkyl), -(C0-5 alkylene)-cycloalkyl, -(C0-5 alkylene)-heterocycloalkyl, and -Lz-Rz; each RCyc is independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-O(Ci-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-NH2, -(C0-5 alkylene)-NH(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-OH, -(C0-5 alkylene)-N(Ci-5 alkyl)-OH, -(C0-5 alkylene)-NH-O(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-O(Ci-5 alkyl), -(C0-5 alkylene)-halogen, -(C0-5 alkylene)-Ci-5 haloalkyl, -(C0-5 alkylene)-O- (C1-5 haloalkyl), -(C0-5 alkylene)-CN, -(C0-5 alkylene)-NO2, -(C0-5 alkylene)-CHO, -(C0-5 alkylene)-CO(Ci-5 alkyl), -(C0-5 alkylene)-COOH, -(C0-5 alkylene)-COO(Ci-5 alkyl), -(C0-5 alkylene)-O-CO(Ci-5 alkyl), -(C0-5 alkylene)-CO-NH2, -(C0-5 alkylene)-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-CO(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-CO(Ci-5 alkyl), -(C0-5 alkylene)-NH-COO(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-COO(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-O-CO- N(CI-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-SO2-NH2, -(C0-5 alkylene)-SO2-NH(Ci-5 alkyl), -(C0-5 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-SO-(Ci-5 alkyl), -(C0-5 alkylene)-carbocyclyl, -(C0-5 alkylene)-heterocyclyl, and -Lz-Rz; each Lz is independently selected from a covalent bond, C1-35 alkylene, C2-35 alkenylene, and C2-35 alkynylene, wherein said alkylene, said alkenylene and said alkynylene are each optionally substituted with one or more groups independently selected from halogen, C1.5 haloalkyl, -O-(Ci-5 haloalkyl), -CN, -OH, -O(Ci-5 alkyl), -SH, -S(Ci-5 alkyl), -NH2, -NH(CI-5 alkyl), and -N(CI-5 alkyl)(Ci-5 alkyl), wherein one or more CH units comprised in said alkylene, said alkenylene or said alkynylene are each optionally replaced by a nitrogen atom, and further wherein one or more -CH2- units comprised in said alkylene, said alkenylene or said alkynylene are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -NH-, -N(CI-5 alkyl)- , carbocyclylene, and heterocyclylene, wherein said carbocyclylene and said heterocyclylene are each optionally substituted with one or more groups independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, halogen, C1.5 haloalkyl, -O-(Ci.5 haloalkyl), -ON, -NO2, -OH, -O(Ci.5 alkyl), -SH, -S(Ci.5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI.5 alkyl)(Ci.5 alkyl), -OHO, -CO-(Ci.5 alkyl), -COOH, -CO-O-(Ci.5 alkyl), -O-CO-(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(CI.5 alkyl)(Ci.5 alkyl), -NH-CO-(CI.5 alkyl), -N(CI.5 alkyl)-CO-(Ci.5 alkyl), -NH-COO(CI.5 alkyl), -N(CI-5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(CI.5 alkyl), -O-CO-N(CI.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci.5 alkyl), -NH-SO2-(CI.5 alkyl), -N(CI.5 alkyl)-SO2-(Ci-5 alkyl), -SO-(Ci-5 alkyl), and -SO2-(Ci-5 alkyl); and o each Rz is independently selected from -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(Ci.5 alkyl), -S(Ci.5 alkylene)-SH, -S(Ci.5 alkylene)-S(Ci.5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI.5 alkyl)(Ci.5 alkyl), -NH-OH, -N(CI.5 alkyl)-OH, -NH-O(CI.5 alkyl), -N(CI.5 alkyl)-O(Ci.5 alkyl), halogen, C1.5 haloalkyl, -O-(Ci.5 haloalkyl), -ON, -NO2, -OHO, -CO(Ci.5 alkyl), -COOH, -COO(Ci.5 alkyl), -O-CO(Ci.5 alkyl), -CO-NH2, -CO-NH(CI-5 alkyl), -CO-N(CI.5 alkyl)(Ci.5 alkyl), -NH-CO(CI.5 alkyl), -N(CI.5 alkyl)-CO(Ci.5 alkyl), -NH-COO(CI-5 alkyl), -N(CI.5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(CI.5 alkyl), -O-CO-N(CI.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci.5 alkyl), -NH-SO2-(CI.5 alkyl), -N(CI.5 alkyl)-SO2-(Ci.5 alkyl), -SO2-(Ci-5 alkyl), -SO-(Ci-5 alkyl), aryl, heteroaryl, cycloalkyl, and heterocycloalkyl, wherein said aryl, said heteroaryl, said cycloalkyl, and said heterocycloalkyl are each optionally substituted with one or more groups independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -ON, -NO2, -OH, -O(Ci.5 alkyl), -SH, -S(Ci.5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI.5 alkyl)(Ci.5 alkyl), -OHO, -CO-(Ci.5 alkyl), -COOH, -CO-O-(Ci.5 alkyl), -O-CO-(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(CI.5 alkyl)(Ci.5 alkyl), -NH-CO-(CI-5 alkyl), -N(CI.5 alkyl)-CO-(Ci.5 alkyl), -NH-COO(CI.5 alkyl), -N(CI.5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(CI-5 alkyl), -O-CO-N(CI.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci.5 alkyl), -NH-SO2-(CI.5 alkyl), -N(CI.5 alkyl)-SO2-(Ci.5 alkyl), -SO-(Ci.5 alkyl), -SO2-(Ci.5 alkyl), carbocyclyl, and heterocyclyl, wherein said carbocyclyl and said heterocyclyl are each optionally substituted with one or more groups independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, halogen, C1-5 haloalkyl, -0- (C1.5 haloalkyl), -ON, -NO2, -OH, -O(Ci.5 alkyl), -SH, -S(Ci.5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI.5 alkyl)(Ci.5 alkyl), -OHO, -CO-(Ci.5 alkyl), -COOH, -CO-O-(Ci.5 alkyl), -O-CO-(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(CI-5 alkyl)(Ci.5 alkyl), -NH-CO-(CI.5 alkyl), -N(CI.5 alkyl)-CO-(Ci.5 alkyl), -NH-COO(CI.5 alkyl), -N(CI.5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(CI.5 alkyl), -O-CO-N(CI.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci.5 alkyl), -NH-SO2-(CI.5 alkyl), -N(CI.5 alkyl)-SO2-(Ci.5 alkyl), -SO-(Ci-5 alkyl), and -SO2-(Ci-5 alkyl).
The exemplary and preferred meanings described herein above for each group/variable in formula (I) likewise apply to the corresponding group/variable in formula (la), insofar as such exemplary or preferred meanings are consistent with the above definition of the compound of formula (la). In a second specific embodiment, the compound of formula (I) is a compound of the following formula (lb)
Figure imgf000034_0001
or pharmaceutically acceptable salt or solvate thereof, wherein: o one of the ring atoms X1 and X2 is S, and the other one is C(-Rx); o Rx is selected from hydrogen, C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-O(Ci-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-NH2, -(C0-5 alkylene)-NH(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-OH, -(C0-5 alkylene)-N(Ci-5 alkyl)-OH, -(C0-5 alkylene)-NH-O(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-O(Ci-5 alkyl), -(C0-5 alkylene)-halogen, -(C0-5 alkylene)-(Ci-5 haloalkyl), -(C0-5 alkylene)-O-(Ci-5 haloalkyl), -(C0-5 alkylene)-CN, -(C0-5 alkylene)-NO2, -(C0-5 alkylene)-CHO, -(C0-5 alkylene)-CO-(Ci-5 alkyl), -(C0-5 alkylene)-CO-(Ci-5 haloalkyl), -(C0-5 alkylene)-COOH, -(C0-5 alkylene)-CO-O-(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-(Ci-5 alkyl), -(C0-5 alkylene)-CO-NH2, -(C0-5 alkylene)-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-CO-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-CO-(Ci-5 alkyl), -(C0-5 alkylene)-NH-COO(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-COO(Ci-5 alkyl), -(C0-5 alkylene)-O-CO- NH(CI-5 alkyl), -(C0-5 alkylene)-O-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-SO2-NH2, -(C0-5 alkylene)-SO2-NH(Ci-5 alkyl), -(C0-5 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-SO-(Ci-5 alkyl), -(C0-5 alkylene)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-carbocyclyl, -(C0-5 alkylene)-heterocyclyl, and -Lz-Rz, wherein the carbocyclyl in said -(C0-5 alky lene)-carbocycly I and the heterocyclyl in said -(C0-5 alky lene)-heterocycly I are each optionally substituted with one or more groups RCyc; o R1 is -L1-L2-R11; o L1 is selected from a covalent bond, -CO-, -SO-, -CO-N(RN)-, -SO-N(RN)-, -SO2-N(RN)-, and -C(=N-RN)-N(RN)-, wherein each RN is independently hydrogen or C1-5 alkyl; o L2 is selected from a covalent bond, C1-10 alkylene, C2-10 alkenylene, C2-10 alkynylene, -(C0-5 alky lene)-carbocycly I- (C0-5 alkylene)-, and -(C0-5 alkylene)-heterocyclyl-(Co-5 alkylene)-, wherein said C1-10 alkylene, said C2-10 alkenylene, said C2-10 alkynylene, the two C0-5 alkylene groups in said -(C0-5 alky lene)-carbocyclyl-(Co-5 alkylene)- , and the two C0-5 alkylene groups in said -(C0-5 alkylene)-heterocyclyl-(Co-5 alkylene)- are each optionally substituted with one or more groups RAlk, wherein one or more -CH2- units comprised in said C1-10 alkylene, said C2-10 alkenylene, said C2-10 alkynylene, any of the C0-5 alkylene groups in said -(C0-5 alky lene)-carbocycly l-(Co-5 alkylene)-, or any of the C0-5 alkylene groups in said -(C0-5 alkylene)-heterocyclyl-(Co-5 alkylene)- are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -CO-, -N(RL2)-, and -C(RL2)=C(RL2)-, wherein each RL2 is independently hydrogen or C1-5 alkyl, and further wherein the carbocyclyl group in said -(C0-5 alkylene)-carbocyclyl-(Co-5 alkylene)- and the heterocyclyl group in said -(C0-5 alkylene)-heterocyclyl are each optionally substituted with one or more groups RCyc; R11 is selected from -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(Ci-5 alkyl), -S(Ci-5 alkylene)-SH, -S(Ci-5 alkylene)-S(Ci_5 alkyl), -NH2, -NH(Ci-5 alkyl), -N(Ci-5 alkyl)(Ci_5 alkyl), -NH- OH, -N(CI-5 alkyl)-OH, -NH-O(CI.5 alkyl), -N(Ci_5 alkyl)-O(Ci_5 alkyl), halogen, -O-(Ci-5 haloalkyl), -ON, -NO2, -CHO, -COOH, -CO-O-(CI-5 alkyl), -O-CO-(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(CI.5 alkyl)(Ci_5 alkyl), -NH-CO-(CI.5 alkyl), -N(Ci_5 alkyl)-CO-(Ci_5 alkyl), -NH-COO(CI.5 alkyl), -N(Ci_5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(CI.5 alkyl), -O-CO-N(CI.5 alkyl)(Ci_5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci-5 alkyl), -NH-SO2-(CI.5 alkyl), -N(Ci_5 alkyl)-SO2-(Ci.5 alkyl), -SO-(Ci.5 alkyl), -SO2-(Ci.5 alkyl), -(Co-15 alkylene)-carbocyclyl, and -(Co-15 alkylene)-heterocyclyl, wherein each alkyl or alkylene comprised in any of the aforementioned groups is optionally substituted with one or more groups RAlk, wherein one or more -CH2- units comprised in the Co-15 alkylene in said -(Co-15 alkylene)-carbocyclyl or in the Co-15 alkylene in said -(Co-15 alkylene)-heterocyclyl are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2- , -CO-, -NH-, and -N(CI-5 alkyl)-, wherein the carbocyclyl in said -(Co-15 alkylene)-carbocyclyl and the heterocyclyl in said -(Co-15 alkylene)-heterocyclyl are each optionally substituted with one or more groups R°yc, and wherein the heterocyclyl in said -(Co-15 al ky lene)-heterocycly I is selected from heteroaryl and heterocycloalkyl; R2 is selected from hydrogen, C1-10 alkyl, C2- alkenyl, C2-io alkynyl, -(Co-10 alkylene)-OH, -(Co-10 alkylene)-O(Ci-5 alkyl), -(Co-10 alkylene)-SH, -(Co-10 alkylene)-S(Ci-5 alkyl), -(C1-10 alkylene)-NH2, -(C1-10 alkylene)-NH(Ci-5 alkyl), -(C1-10 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C1-10 alkylene)-halogen, C1-10 haloalkyl, -(Co-10 alkylene)-O-(Ci-5 haloalkyl), -(Co-10 alkylene)-CN, -(Co-10 alkylene)-NO2, -(Co-10 alkylene)-CHO, -(Co-10 alkylene)-CO-(Ci-io alkyl), -(Co-10 alkylene)-COOH, -(Co-10 alkylene)-CO-O-(Ci-5 alkyl), -(Co-10 alkylene)-O-CO-(Ci-5 alkyl), -(Co-10 alkylene)-CO-NH2, -(Co-10 alkylene)-CO-NH(Ci-5 alkyl), -(Co-10 alkylene)-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C1-10 alkylene)-NH-CO-(Ci-5 alkyl), -(C1-10 alkylene)-N(Ci-5 alkyl)-CO-(Ci-5 alkyl), -(C1-10 alkylene)-NH-COO(Ci-5 alkyl), -(C1-10 alkylene)-N(Ci-5 alkyl)-COO(Ci-5 alkyl), -(Co-10 alkylene)-O-CO-NH(Ci-5 alkyl), -(Co-10 alkylene)-O-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(Co-10 alkylene)-SO2-NH2, -(Co-10 alkylene)-SO2-NH(Ci-5 alkyl), -(Co-10 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C1-10 alkylene)-NH-SO2-(Ci-5 alkyl), -(C1-10 alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(Co-10 alkylene)-SO-(Ci-5 alkyl), -(Co-10 alkylene)-SO2-(Ci-5 alkyl), -(Co-10 alkylene)-carbocyclyl, and -(Co-10 alkylene)-heterocyclyl, wherein one or more -CH2- units comprised in said C1-10 alkyl, said C2-10 alkenyl, said C2-10 alkynyl, or in any of the aforementioned Co-10 alkylene or C1-10 alkylene groups are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -NH-, and -N(CI-5 alkyl)-, and further wherein the carbocyclyl in said -(Co-10 alky lene)-carbocycly I and the heterocyclyl in said -(Co-10 alkylene)-heterocyclyl are each optionally substituted with one or more groups RCyc; R3 is -(C0-5 alkylene)-aryl or -(C0-5 alkylene)-heteroaryl, wherein the aryl in said -(C0-5 alkylene)-aryl is selected from phenyl, naphthyl and tetralinyl, wherein the heteroaryl in said -(C0-5 alkylene)-heteroaryl is selected from pyrrolyl, 1 ,3-benzodioxolyl, furanyl and thiophenyl, wherein the aryl in said -(C0-5 alkylene)-aryl and the heteroaryl in said -(C0-5 alkylene)-heteroaryl are each optionally substituted with one or more groups R31, and wherein one or more -CH2- units comprised in the C0-5 alkylene in said -(C0-5 alkylene)-aryl or in the C0-5 alkylene in said -(C0-5 alkylene)-heteroaryl are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2- , -CO-, -NH-, and -N(CI_5 alkyl)-; preferably, R3 is -(C0-5 alkylene)-heteroaryl, wherein the heteroaryl in said -(C0-5 alkylene)-heteroaryl is selected from pyrrolyl, 1 ,3-benzodioxolyl, furanyl and thiophenyl, wherein the heteroaryl in said -(C0-5 alkylene)-heteroaryl is optionally substituted with one or more groups R31, and wherein one or more -CH2- units comprised in the C0-5 alkylene in said -(C0-5 alkylene)-heteroaryl are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -NH-, and -N(Ci_5 alkyl)-; each R31 is independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-O(Ci-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-NH2, -(C0-5 alkylene)-NH(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-OH, -(C0-5 alkylene)-N(Ci-5 alkyl)-OH, -(C0-5 alkylene)-NH-O(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-O(Ci-5 alkyl), -(C0-5 alkylene)-halogen, -(C0-5 alkylene)-(Ci-5 haloalkyl), -(C0-5 alkylene)-O-(Ci-5 haloalkyl), -(C0-5 alkylene)-CN, -(C0-5 alkylene)-NO2, -(C0-5 alkylene)-CHO, -(C0-5 alkylene)-CO-(Ci-5 alkyl), -(C0-5 alkylene)-COOH, -(C0-5 alkylene)-CO-O-(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-(Ci-5 alkyl), -(C0-5 alkylene)-CO-NH2, -(C0-5 alkylene)-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-CO-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-CO-(Ci-5 alkyl), -(C0-5 alkylene)-NH-COO(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-COO(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-SO2-NH2, -(C0-5 alkylene)-SO2-NH(Ci-5 alkyl), -(C0-5 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-SO-(Ci-5 alkyl), -(C0-5 alkylene)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-carbocyclyl, -(C0-5 alkylene)-heterocyclyl, and -Lz-Rz, wherein the carbocyclyl in said -(C0-5 alky lene)-carbocycly I and the heterocyclyl in said -(C0-5 alky lene)-heterocycly I are each optionally substituted with one or more groups R°yc, and wherein one or more -CH2- units comprised in the C0-5 alkylene in said -(C0-5 alkylene)-carbocyclyl or in the C0-5 alkylene in said -(C0-5 alkylene)-heterocyclyl are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -NH-, and -N(CI-5 alkyl)-; each RAlk is independently selected from -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(Ci.5 alkyl), -S(Ci.5 alkylene)-SH, -S(Ci.5 alkylene)-S(Ci.5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI.5 alkyl)(Ci.5 alkyl), -NH-OH, -N(CI.5 alkyl)-OH, -NH-O(Ci.5 alkyl), -N(CI.5 alkyl)-O(Ci.5 alkyl), halogen, C1.5 haloalkyl, -O-(Ci.5 haloalkyl), -CN, -NO2, -CHO, -CO(Ci.5 alkyl), -COOH, -COO(Ci.5 alkyl), -O-CO(Ci.5 alkyl), -CO-NH2, -C0-NH(CI.5 alkyl), -CO-N(Ci.5 alkyl)(Ci.5 alkyl), -NH-CO(Ci.5 alkyl), -N(CI.5 alkyl)-CO(Ci.5 alkyl), -NH-C00(CI.5 alkyl), -N(CI.5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(Ci.5 alkyl), -O-CO-N(Ci.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -S02-NH(CI.5 alkyl), -SO2-N(Ci.5 alkyl)(Ci.5 alkyl), -NH-SO2-(Ci.5 alkyl), -N(CI.5 alkyl)-SO2-(Ci.5 alkyl), -SO2-(Ci-5 alkyl), -SO-(Ci-5 alkyl), -(C0-5 alkylene)-cycloalkyl, -(C0-5 alkylene)-heterocycloalkyl, and -Lz-Rz; each RCyc is independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-O(Ci-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-NH2, -(C0-5 alkylene)-NH(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-OH, -(C0-5 alkylene)-N(Ci-5 alkyl)-OH, -(C0-5 alkylene)-NH-O(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-O(Ci-5 alkyl), -(C0-5 alkylene)-halogen, -(C0-5 alkylene)-Ci-5 haloalkyl, -(C0-5 alkylene)-O- (C1-5 haloalkyl), -(C0-5 alkylene)-CN, -(C0-5 alkylene)-NO2, -(C0-5 alkylene)-CHO, -(C0-5 alkylene)-CO(Ci-5 alkyl), -(C0-5 alkylene)-COOH, -(C0-5 alkylene)-COO(Ci-5 alkyl), -(C0-5 alkylene)-O-CO(Ci-5 alkyl), -(C0-5 alkylene)-CO-NH2, -(C0-5 alkylene)-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-CO(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-CO(Ci-5 alkyl), -(C0-5 alkylene)-NH-COO(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-COO(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-O-CO- N(CI-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-SO2-NH2, -(C0-5 alkylene)-SO2-NH(Ci-5 alkyl), -(C0-5 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-SC>2-(Ci-5 alkyl), -(C0-5 alkylene)-SO-(Ci-5 alkyl), -(C0-5 alkylene)-carbocyclyl, and -(C0-5 alkylene)-heterocyclyl; each Lz is independently selected from a covalent bond, C1.35 alkylene, C2-35 alkenylene, and C2-35 alkynylene, wherein said alkylene, said alkenylene and said alkynylene are each optionally substituted with one or more groups independently selected from halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -CN, -OH, -O(Ci-5 alkyl), -SH, -S(Ci-5 alkyl), -NH2, -NH(CI-5 alkyl), and -N(CI-5 alkyl)(Ci-5 alkyl), wherein one or more CH units comprised in said alkylene, said alkenylene or said alkynylene are each optionally replaced by a nitrogen atom, and further wherein one or more -CH2- units comprised in said alkylene, said alkenylene or said alkynylene are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -NH-, -N(CI-5 alkyl)- , carbocyclylene, and heterocyclylene, wherein said carbocyclylene and said heterocyclylene are each optionally substituted with one or more groups independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, halogen, C1.5 haloalkyl, -O-(Ci.5 haloalkyl), -CN, -NO2, -OH, -O(Ci.5 alkyl), -SH, -S(Ci.5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI.5 alkyl)(Ci.5 alkyl), -OHO, -CO-(Ci.5 alkyl), -COCH, -CO-O-(Ci.5 alkyl), -O-CO-(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(Ci-5 alkyl)(Ci.5 alkyl), -NH-CO-(Ci.5 alkyl), -N(Ci.5 alkyl)-CO-(Ci.5 alkyl), -NH-COO(Ci.5 alkyl), -N(OI-5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(Ci.5 alkyl), -O-CO-N(Ci.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(Ci-5 alkyl)(Ci.5 alkyl), -NH-SO2-(Ci-5 alkyl), -N(CI.5 alkyl)-SO2-(Ci-5 alkyl), -SO-(Ci-5 alkyl), and -SO2-(Ci-5 alkyl); and each Rz is independently selected from -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(Ci.5 alkyl), -S(Ci.5 alkylene)-SH, -S(Ci.5 alkylene)-S(Ci.5 alkyl), -NH2, -NH(Ci.5 alkyl), -N(Ci.5 alkyl)(Ci.5 alkyl), -NH-OH, -N(Ci.5 alkyl)-OH, -NH-O(Ci.5 alkyl), -N(CI.5 alkyl)-O(Ci.5 alkyl), halogen, C1.5 haloalkyl, -O-(Ci.5 haloalkyl), -CN, -NO2, -OHO, -CO(Ci.5 alkyl), -COCH, -COO(Ci.5 alkyl), -O-CO(Ci.5 alkyl), -CO-NH2, -CO-NH(Ci-5 alkyl), -CO-N(Ci.5 alkyl)(Ci.5 alkyl), -NH-CO(Ci.5 alkyl), -N(CI.5 alkyl)-CO(Ci.5 alkyl), -NH-COO(Ci-5 alkyl), -N(CI.5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(Ci.5 alkyl), -O-CO-N(Ci.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(Ci-5 alkyl)(Ci.5 alkyl), -NH-SO2-(Ci-5 alkyl), -N(CI.5 alkyl)-SO2-(Ci-5 alkyl), -SO2-(Ci-5 alkyl), -SO-(Ci-5 alkyl), aryl, heteroaryl, cycloalkyl, and heterocycloalkyl, wherein said aryl, said heteroaryl, said cycloalkyl, and said heterocycloalkyl are each optionally substituted with one or more groups independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -CN, -NO2, -OH, -O(Ci.5 alkyl), -SH, -S(Ci.5 alkyl), -NH2, -NH(Ci.5 alkyl), -N(CI.5 alkyl)(Ci.5 alkyl), -OHO, -CO-(Ci.5 alkyl), -COCH, -CO-O-(Ci.5 alkyl), -O-CO-(Ci.5 alkyl), -CO-NH2, -CO-NH(Ci.5 alkyl), -CO-N(Ci.5 alkyl)(Ci.5 alkyl), -NH-CO-(Ci-5 alkyl), -N(CI.5 alkyl)-CO-(Ci.5 alkyl), -NH-COO(Ci.5 alkyl), -N(CI.5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(Ci-5 alkyl), -O-CO-N(Ci.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(Ci-5 alkyl), -SO2-N(Ci-5 alkyl)(Ci.5 alkyl), -NH-SO2-(CI-5 alkyl), -N(CI.5 alkyl)-SO2-(Ci-5 alkyl), -SO-(Ci.5 alkyl), -SO2-(Ci-5 alkyl), carbocyclyl, and heterocyclyl, wherein said carbocyclyl and said heterocyclyl are each optionally substituted with one or more groups independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, halogen, C1-5 haloalkyl, -0- (C1.5 haloalkyl), -CN, -NO2, -OH, -O(Ci.5 alkyl), -SH, -S(Ci.5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI.5 alkyl)(Ci.5 alkyl), -OHO, -CO-(Ci.5 alkyl), -COCH, -CO-O-(Ci.5 alkyl), -O-CO-(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(CI.5 alkyl)(Ci.5 alkyl), -NH-CO-(CI.5 alkyl), -N(CI.5 alkyl)-CO-(Ci.5 alkyl), -NH-COO(CI.5 alkyl), -N(CI.5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(CI.5 alkyl), -O-CO-N(CI.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci_5 alkyl), -NH-SO2-(CI.5 alkyl), -N(Ci_5 alkyl)-SO2-(Ci.5 alkyl), -S0-(Ci-5 alkyl), and -SO2-(Ci-5 alkyl).
The exemplary and preferred meanings described herein above for each group/variable in formula (I) likewise apply to the corresponding group/variable in formula (lb), insofar as such exemplary or preferred meanings are consistent with the above definition of the compound of formula (lb).
For a person skilled in the field of synthetic chemistry, various ways for the preparation of the compounds of formula (I) and their pharmaceutically acceptable salts and solvates will be readily apparent. For example, the compounds of formula (I) can be synthesized in accordance with the methods described in any of the following general/exemplary schemes, or in accordance with (or in analogy to) the synthetic procedures described in Example A herein below:
Figure imgf000038_0001
Figure imgf000039_0001
The following definitions apply throughout the present specification and the claims, unless specifically indicated otherwise.
The term "hydrocarbon group” refers to a group consisting of carbon atoms and hydrogen atoms.
The term "alicyclic” is used in connection with cyclic groups and denotes that the corresponding cyclic group is non-aromatic.
As used herein, the term "alkyl” refers to a monovalent saturated acyclic (i.e., non-cyclic) hydrocarbon group which may be linear or branched. Accordingly, an "alkyl” group does not comprise any carbon-to-carbon double bond or any carbon-to-carbon triple bond. A “C1-5 alkyl” denotes an alkyl group having 1 to 5 carbon atoms. Preferred exemplary alkyl groups are methyl, ethyl, propyl (e.g., n-propyl or isopropyl), or butyl (e.g., n-butyl, isobutyl, sec-butyl, or tert- butyl). Unless defined otherwise, the term "alkyl” preferably refers to C1-4 alkyl, more preferably to methyl or ethyl, and even more preferably to methyl.
As used herein, the term "alkenyl” refers to a monovalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon double bonds while it does not comprise any carbon-to-carbon triple bond. The term "C2-5 alkenyl” denotes an alkenyl group having 2 to 5 carbon atoms. Preferred exemplary alkenyl groups are ethenyl, propenyl (e.g., prop-1 -en-1-yl, prop-1-en-2-yl, or prop-2-en-1-yl), butenyl, butadienyl (e.g., buta-1,3-dien-1-yl or buta-1 ,3-dien-2-yl), pentenyl, or pentadienyl (e.g., isoprenyl). Unless defined otherwise, the term "alkenyl” preferably refers to C2-4 alkenyl.
As used herein, the term “alky ny I” refers to a monovalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon triple bonds and optionally one or more (e.g., one or two) carbon-to-carbon double bonds. The term "C2-5 alkynyl” denotes an alkynyl group having 2 to 5 carbon atoms. Preferred exemplary alkynyl groups are ethynyl, propynyl (e.g., propargyl), or butynyl. Unless defined otherwise, the term "alkynyl” preferably refers to C2-4 alkynyl.
As used herein, the term "alkylene” refers to an alkanediyl group, i.e. a divalent saturated acyclic hydrocarbon group which may be linear or branched. A “C1-5 alkylene” denotes an alkylene group having 1 to 5 carbon atoms, and the term "C0-5 alkylene” indicates that a covalent bond (corresponding to the option "Co alkylene”) or a C1-5 alkylene is present. Preferred exemplary alkylene groups are methylene (-CH2-), ethylene (e.g., -CH2-CH2- or -CH(-CH3)-), propylene (e.g., -CH2-CH2-CH2-, -CH(-CH2-CH3)-, -CH2-CH(-CH3)-, or -CH(-CH3)-CH2-), or butylene (e.g., -CH2-CH2- CH2-CH2-). Unless defined otherwise, the term "alkylene” preferably refers to C1-4 alkylene (including, in particular, linear C1-4 alkylene), more preferably to methylene or ethylene, and even more preferably to methylene.
As used herein, the term "alkenylene” refers to an alkenediyl group, i.e. a divalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon double bonds while it does not comprise any carbon-to-carbon triple bond. A "C2-5 alkenylene” denotes an alkenylene group having 2 to 5 carbon atoms. Unless defined otherwise, the term "alkenylene” preferably refers to C2-4 alkenylene (including, in particular, linear C2-4 alkenylene).
As used herein, the term "alkynylene” refers to an alkynediyl group, i.e. a divalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon triple bonds and optionally one or more (e.g., one or two) carbon-to-carbon double bonds. A "C2-5 alkynylene” denotes an alkynylene group having 2 to 5 carbon atoms. Unless defined otherwise, the term "alkynylene” preferably refers to C2-4 alkynylene (including, in particular, linear C2-4 alkynylene).
As used herein, the term "carbocyclyl” refers to a hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings), wherein said ring group may be saturated, partially unsaturated (i.e., unsaturated but not aromatic) or aromatic. Unless defined otherwise, "carbocyclyl” preferably refers to aryl, cycloalkyl or cycloalkenyl. As used herein, the term "carbocyclylene” refers to a carbocyclyl group, as defined herein above, but having two points of attachment, i.e. a divalent hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings), wherein said ring group may be saturated, partially unsaturated (i.e., unsaturated but not aromatic) or aromatic. Unless defined otherwise, "carbocyclylene” preferably refers to arylene, cycloalkylene or cycloalkenylene.
As used herein, the term “heterocycly I” refers to a ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings), wherein said ring group comprises one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group), and further wherein said ring group may be saturated, partially unsaturated (i.e., unsaturated but not aromatic) or aromatic. For example, each heteroatom-containing ring comprised in said ring group may contain one or two O atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring. Unless defined otherwise, "heterocyclyl” preferably refers to heteroaryl, heterocycloalkyl or heterocycloalkenyl.
As used herein, the term "heterocyclylene” refers to a heterocyclyl group, as defined herein above, but having two points of attachment, i.e. a divalent ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings), wherein said ring group comprises one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from O, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group), and further wherein said ring group may be saturated, partially unsaturated (i.e., unsaturated but not aromatic) or aromatic. For example, each heteroatom-containing ring comprised in said ring group may contain one or two 0 atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring. Unless defined otherwise, "heterocyclylene” preferably refers to heteroarylene, heterocycloalkylene or heterocycloalkenylene.
As used herein, the term "aryl” refers to an aromatic hydrocarbon ring group, including monocyclic aromatic rings as well as bridged ring and/or fused ring systems containing at least one aromatic ring (e.g., ring systems composed of two or three fused rings, wherein at least one of these fused rings is aromatic; or bridged ring systems composed of two or three rings, wherein at least one of these bridged rings is aromatic). If the aryl is a bridged and/or fused ring system which contains, besides one or more aromatic rings, at least one non-aromatic ring (e.g., a saturated ring or an unsaturated alicyclic ring), then one or more carbon ring atoms in each non-aromatic ring may optionally be oxidized (i.e., to form an oxo group). "Aryl” may, e.g., refer to phenyl, naphthyl, dialinyl (i.e., 1,2-dihydronaphthyl), tetralinyl (i.e., 1 ,2,3,4-tetrahydronaphthyl), indanyl, indenyl (e.g., 1 H-indenyl), anthracenyl, phenanthrenyl, 9H- fluorenyl, or azulenyl. Unless defined otherwise, an "aryl” preferably has 6 to 14 ring atoms, more preferably 6 to 10 ring atoms, even more preferably refers to phenyl or naphthyl, and most preferably refers to phenyl.
As used herein, the term "arylene” refers to an aryl group, as defined herein above, but having two points of attachment, i.e. a divalent aromatic hydrocarbon ring group, including monocyclic aromatic rings as well as bridged ring and/or fused ring systems containing at least one aromatic ring (e.g., ring systems composed of two or three fused rings, wherein at least one of these fused rings is aromatic; or bridged ring systems composed of two or three rings, wherein at least one of these bridged rings is aromatic). If the arylene is a bridged and/or fused ring system which contains, besides one or more aromatic rings, at least one non-aromatic ring (e.g., a saturated ring or an unsaturated alicyclic ring), then one or more carbon ring atoms in each non-aromatic ring may optionally be oxidized (i.e., to form an oxo group). "Arylene” may, e.g., refer to phenylene (e.g., phen-1, 2-diyl, phen-1 ,3-diyl, or phen-1, 4- diyl), naphthylene (e.g., naphthalen-1 ,2-diyl, naphthalen-1, 3-diyl, naphthalen-1, 4-diyl, naphthalen-1, 5-diyl, naphthalen-1, 6-diyl, naphthalen-1, 7-diyl, naphthalen-2, 3-diyl, naphthalen-2, 5-diyl, naphthalen-2, 6-diyl, naphthalen- 2,7-diyl, or naphthalen-2, 8-diyl), 1 ,2-dihydronaphthylene, 1 ,2,3,4-tetrahydronaphthylene, indanylene, indenylene, anthracenylene, phenanthrenylene, 9H-fluorenylene, or azulenylene. Unless defined otherwise, an "arylene” preferably has 6 to 14 ring atoms, more preferably 6 to 10 ring atoms, even more preferably refers to phenylene or naphthylene, and most preferably refers to phenylene (particularly phen-1, 4-diyl).
As used herein, the term "heteroaryl” refers to an aromatic ring group, including monocyclic aromatic rings as well as bridged ring and/or fused ring systems containing at least one aromatic ring (e.g., ring systems composed of two or three fused rings, wherein at least one of these fused rings is aromatic; or bridged ring systems composed of two or three rings, wherein at least one of these bridged rings is aromatic), wherein said aromatic ring group comprises one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, and further wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group). For example, each heteroatom-containing ring comprised in said aromatic ring group may contain one or two 0 atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring. "Heteroaryl” may, e.g., refer to thienyl (i.e., thiophenyl), benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl (i.e., furanyl), benzofuranyl, isobenzofuranyl, chromanyl, chromenyl (e.g., 2H-1-benzopyranyl or 4H-1-benzopyranyl), isochromenyl (e.g., 1 H-2-benzopyranyl), chromonyl, xanthenyl, phenoxathiinyl, pyrrolyl (e.g., 1 H-pyrrolyl), imidazolyl, pyrazolyl, pyridyl (i.e., pyridinyl; e.g., 2-pyridyl, 3-pyridyl, or 4-pyridyl), pyrazinyl, pyrimidinyl, pyridazinyl, indolyl (e.g., 1 H-indolyl), isoindolyl, indazolyl, indolizinyl, purinyl, quinolyl, isoquinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, cinnolinyl, pteridinyl, carbazolyl, p-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl (e.g., [1,10]phenanthrolinyl, [1,7]phenanthrolinyl, or [4,7]phenanthrolinyl), phenazinyl, thiazolyl, isothiazolyl, phenothiazinyl, oxazolyl, isoxazolyl, oxadiazolyl (e.g., 1 ,2,4-oxadiazolyl, 1,2,5-oxadiazolyl (i.e., furazanyl), or 1,3,4-oxadiazolyl), thiadiazolyl (e.g., 1,2,4-thiadiazolyl, 1,2,5- thiadiazolyl, or 1,3,4-thiadiazolyl), phenoxazinyl, pyrazolo[1 ,5-a]pyrimidinyl (e.g., pyrazolo[1 ,5-a]pyrimidin-3-yl), 1 ,2-benzoisoxazol-3-yl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzo[b]thiophenyl (i.e., benzothienyl), triazolyl (e.g., 1 H-1,2,3-triazolyl, 2H-1 ,2,3-triazolyl, 1 H-1,2,4-triazolyl, or 4H- 1 ,2,4-triazolyl), benzotriazolyl, 1 H-tetrazolyl, 2H-tetrazolyl, triazinyl (e.g., 1,2,3-triazinyl, 1 ,2,4-triazinyl, or 1,3,5- triazinyl), furo[2,3-c]pyridinyl, dihydrofuropyridinyl (e.g., 2,3-dihydrofuro[2,3-c]pyridinyl or 1 ,3-dihydrofuro[3,4- c]pyridinyl), imidazopyridinyl (e.g., imidazo[1 ,2-a]pyridinyl or imidazo[3,2-a]pyridinyl), quinazolinyl, thienopyridinyl, tetrahydrothienopyridinyl (e.g., 4,5,6,7-tetrahydrothieno[3,2-c]pyridinyl), dibenzofuranyl, 1 ,3-benzodioxolyl, benzodioxanyl (e.g., 1,3-benzodioxanyl or 1 ,4-benzodioxanyl), or coumarinyl. Unless defined otherwise, the term "heteroaryl” preferably refers to a 5 to 14 membered (more preferably 5 to 10 membered) monocyclic ring or fused ring system comprising one or more (e.g., one, two, three or four) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized; even more preferably, a "heteroaryl” refers to a 5 or 6 membered monocyclic ring comprising one or more (e.g., one, two or three) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized. Moreover, unless defined otherwise, particularly preferred examples of a "heteroaryl” include pyridinyl (e.g., 2-pyridyl, 3-pyridyl, or 4-pyridyl), imidazolyl, thiazolyl, 1 H-tetrazolyl, 2H-tetrazolyl, thienyl (i.e., thiophenyl), or pyrimidinyl.
As used herein, the term "heteroarylene” refers to a heteroaryl group, as defined herein above, but having two points of attachment, i.e. a divalent aromatic ring group, including monocyclic aromatic rings as well as bridged ring and/or fused ring systems containing at least one aromatic ring (e.g., ring systems composed of two or three fused rings, wherein at least one of these fused rings is aromatic; or bridged ring systems composed of two or three rings, wherein at least one of these bridged rings is aromatic), wherein said aromatic ring group comprises one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, and further wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group). For example, each heteroatom-containing ring comprised in said aromatic ring group may contain one or two 0 atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three, or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatomcontaining ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring. "Heteroarylene” may, e.g., refer to thienylene (i.e., thiophenylene; e.g., thien-2,3-diyl, thien-2,4-diyl, or thien-2,5-diyl), benzo[b]thienylene, naphtho[2,3-b]thienylene, thianthrenylene, furylene (i.e., furanylene; e.g., furan-2,3-diyl, furan-2,4-diyl, or furan-2,5-diyl), benzofuranylene, isobenzofuranylene, chromanylene, chromenylene, isochromenylene, chromonylene, xanthenylene, phenoxathiinylene, pyrrolylene, imidazolylene, pyrazolylene, pyridylene (i.e., pyridinylene), pyrazinylene, pyrimidinylene, pyridazinylene, indolylene, isoindolylene, indazolylene, indolizinylene, purinylene, quinolylene, isoquinolylene, phthalazinylene, naphthyridinylene, quinoxalinylene, cinnolinylene, pteridinylene, carbazolylene, p-carbolinylene, phenanthridinylene, acridinylene, perimidinylene, phenanthrolinylene, phenazinylene, thiazolylene (e.g., thiazol-2,4-diyl, thiazol-2,5-diyl, or thiazol-4,5-diyl), isothiazolylene (e.g., isothiazol-3,4-diyl, isothiazol-3,5-diyl, or isothiazol-4,5-diyl), phenothiazinylene, oxazolylene (e.g., oxazol-2,4-diyl, oxazol-2,5-diyl, or oxazol-4,5-diyl), isoxazolylene (e.g., isoxazol-3,4-diyl, isoxazol-3,5-diyl, or isoxazol-4,5-diyl), oxadiazolylene (e.g., 1 ,2,4-oxadiazol-3,5-diyl, 1 ,2,5- oxadiazol-3,4-diyl, or 1 ,3,4-oxadiazol-2,5-diyl), thiadiazolylene (e.g., 1,2,4-thiadiazol-3,5-diyl, 1 ,2,5-thiadiazol-3,4- diyl, or 1 ,3,4-thiadiazol-2,5-diyl), phenoxazinylene, pyrazolo[1,5-a]pyrimidinylene, 1 ,2-benzoisoxazolylene, benzothiazolylene, benzothiadiazolylene, benzoxazolylene, benzisoxazolylene, benzimidazolylene, benzo[b]thiophenylene (i.e., benzothienylene), triazolylene (e.g., 1 H-1,2,3-triazolylene, 2H-1,2,3-triazolylene, 1 H- 1 ,2,4-triazolylene, or 4H-1,2,4-triazolylene), benzotriazolylene, 1 H-tetrazolylene, 2H-tetrazolylene, triazinylene (e.g.,
1.2.3-triazinylene, 1 ,2,4-triazinylene, or 1,3,5-triazinylene), furo[2,3-c]pyridinylene, dihydrofuropyridinylene (e.g., 2,3- dihydrofuro[2,3-c]pyridinylene or 1 ,3-dihydrofuro[3,4-c]pyridinylene), imidazopyridinylene (e.g., imidazo[1 ,2- a]py ridinylene or imidazo[3,2-a]pyridinylene), quinazolinylene, thienopyridinylene, tetrahydrothienopyridinylene (e.g., 4,5,6,7-tetrahydrothieno[3,2-c]pyridinylene), dibenzofuranylene, 1,3-benzodioxolylene, benzodioxanylene (e.g.,
1.3-benzodioxanylene or 1 ,4-benzodioxanylene), or coumarinylene. Unless defined otherwise, the term "heteroarylene” preferably refers to a divalent 5 to 14 membered (more preferably 5 to 10 membered) monocyclic ring or fused ring system comprising one or more (e.g., one, two, three or four) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized; even more preferably, a "heteroarylene” refers to a divalent 5 or 6 membered monocyclic ring comprising one or more (e.g., one, two or three) ring heteroatoms independently selected from 0, S, and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized. A "heteroarylene”, including any of the specific heteroarylene groups described herein, may be attached through two carbon ring atoms, particularly through those two carbon ring atoms that have the greatest distance from one another (in terms of the number of ring atoms separating them by the shortest possible connection) within one single ring or within the entire ring system of the corresponding heteroarylene. Moreover, unless defined otherwise, particularly preferred examples of a "heteroarylene” include pyridinylene, imidazolylene, thiazolylene, 1 H-tetrazolylene, 2H-tetrazolylene, thienylene (i.e., thiophenylene), or pyrimidinylene.
As used herein, the term “cycloalky I” refers to a saturated hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings). "Cycloalkyl” may, e.g., refer to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, decalinyl (i.e., decahydronaphthyl), or adamantyl. Unless defined otherwise, "cycloalkyl” preferably refers to a C3-11 cycloalkyl, and more preferably refers to a C3-7 cycloalkyl. A particularly preferred "cycloalkyl” is a monocyclic saturated hydrocarbon ring having 3 to 7 ring members. Moreover, unless defined otherwise, particularly preferred examples of a "cycloalkyl” include cyclohexyl or cyclopropyl, particularly cyclohexyl.
As used herein, the term "cycloalkylene” refers to a cycloalkyl group, as defined herein above, but having two points of attachment, i.e. a divalent saturated hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings). "Cycloalkylene” may, e.g., refer to cyclopropylene (e.g., cyclopropan-1,1-diyl or cyclopropan-1 ,2-diyl), cyclobutylene (e.g., cyclobutan-1 ,1-diyl, cyclobutan-1,2-diyl, or cyclobutan-1 ,3-diyl), cyclopentylene (e.g., cyclopentan-1,1 -diyl, cyclopentan-1 , 2-diyl, or cyclopentan- 1,3-diyl), cyclohexylene (e.g., cyclohexan-1 , 1-diyl, cyclohexan-1 ,2-diyl, cyclohexan-1 ,3-diyl, or cyclohexan-1 ,4-diyl), cycloheptylene, decalinylene (i.e., decahydronaphthylene), or adamantylene. Unless defined otherwise, "cycloalkylene” preferably refers to a C3-11 cycloalkylene, and more preferably refers to a C3-7 cycloalkylene. A particularly preferred "cycloalkylene” is a divalent monocyclic saturated hydrocarbon ring having 3 to 7 ring members. Moreover, unless defined otherwise, particularly preferred examples of a "cycloalkylene” include cyclohexylene or cyclopropylene, particularly cyclohexylene.
As used herein, the term “heterocycloalkyl” refers to a saturated ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said ring group contains one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, and further wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group). For example, each heteroatom-containing ring comprised in said saturated ring group may contain one or two 0 atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatomcontaining ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring. "Heterocycloalkyl” may, e.g., refer to aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, piperazinonyl (e.g., piperazin-2-on-1-yl or piperazin-3-on-1-yl), azepanyl, diazepanyl (e.g., 1,4-diazepanyl), oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, morpholinyl (e.g., morpholin-4-yl), thiomorpholinyl (e.g., thiomorpholin-4-yl), oxazepanyl, oxiranyl, oxetanyl, tetrahydrofuranyl,
1.3-dioxolanyl, tetrahydropyranyl, 1,4-dioxanyl, oxepanyl, thiiranyl, thietanyl, tetrahydrothiophenyl (i.e., thiolanyl),
1.3-dithiolanyl, thianyl, thiepanyl, decahydroquinolinyl, decahydroisoquinolinyl, or 2-oxa-5-aza-bicyclo[2.2.1]hept-5- yl. Unless defined otherwise, "heterocycloalkyl” preferably refers to a 3 to 11 membered saturated ring group, which is a monocyclic ring or a fused ring system (e.g., a fused ring system composed of two fused rings), wherein said ring group contains one or more (e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized; more preferably, "heterocycloalkyl” refers to a 5 to 7 membered saturated monocyclic ring group containing one or more (e.g., one, two, or three) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized. Moreover, unless defined otherwise, particularly preferred examples of a "heterocycloalkyl” include tetrahydropyranyl, piperidinyl, piperazinyl, piperazinonyl, morpholinyl, pyrrolidinyl, or tetrahydrofuranyl.
As used herein, the term "heterocycloalkylene” refers to a heterocycloalkyl group, as defined herein above, but having two points of attachment, i.e. a divalent saturated ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said ring group contains one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, and further wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group). For example, each heteroatom-containing ring comprised in said saturated ring group may contain one or two 0 atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring. "Heterocycloalkylene” may, e.g., refer to aziridinylene, azetidinylene, pyrrolidinylene, imidazolidinylene, pyrazolidinylene, piperidinylene, piperazinylene, azepanylene, diazepanylene (e.g., 1 ,4-diazepanylene), oxazolidinylene, isoxazolidinylene, thiazolidinylene, isothiazolidinylene, morpholinylene, thiomorpholinylene, oxazepanylene, oxiranylene, oxetanylene, tetrahydrofuranylene, 1 ,3-dioxolanylene, tetrahydropyranylene, 1,4-dioxanylene, oxepanylene, thiiranylene, thietanylene, tetrahydrothiophenylene (i.e., thiolanylene), 1,3-dithiolanylene, thianylene, thiepanylene, decahydroquinolinylene, decahydroisoquinolinylene, or 2- oxa-5-aza-bicyclo[2.2.1]hept-5-ylene. Unless defined otherwise, "heterocycloalkylene” preferably refers to a divalent 3 to 11 membered saturated ring group, which is a monocyclic ring or a fused ring system (e.g., a fused ring system composed of two fused rings), wherein said ring group contains one or more (e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized; more preferably, "heterocycloalkylene” refers to a divalent 5 to 7 membered saturated monocyclic ring group containing one or more (e.g., one, two, or three) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized. Moreover, unless defined otherwise, particularly preferred examples of a "heterocycloalkylene” include tetrahydropyranylene, piperidinylene, piperazinylene, morpholinylene, pyrrolidinylene, or tetrahydrofuranylene.
As used herein, the term “cycloalkenyl” refers to an unsaturated alicyclic (non-aromatic) hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said hydrocarbon ring group comprises one or more (e.g., one or two) carbon-to-carbon double bonds and does not comprise any carbon-to-carbon triple bond. “Cycloalkenyl” may, e.g., refer to cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, or cycloheptadienyl. Unless defined otherwise, "cycloalkenyl” preferably refers to a C3-11 cycloalkenyl, and more preferably refers to a C3-7 cycloalkenyl. A particularly preferred "cycloalkenyl” is a monocyclic unsaturated alicyclic hydrocarbon ring having 3 to 7 ring members and containing one or more (e.g., one or two; preferably one) carbon-to-carbon double bonds.
As used herein, the term "cycloalkenylene” refers to a cycloalkenyl group, as defined herein above, but having two points of attachment, i.e. a divalent unsaturated alicyclic (i.e., non-aromatic) hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said hydrocarbon ring group comprises one or more (e.g., one or two) carbon-to-carbon double bonds and does not comprise any carbon-to-carbon triple bond. "Cycloalkenylene” may, e.g., refer to cyclopropenylene, cyclobutenylene, cyclopentenylene, cyclohexenylene, cyclohexadienylene, cycloheptenylene, or cycloheptadienylene. Unless defined otherwise, "cycloalkenylene” preferably refers to a C3-11 cycloalkenylene, and more preferably refers to a C3-7 cycloalkenylene. A particularly preferred "cycloalkenylene” is a divalent monocyclic unsaturated alicyclic hydrocarbon ring having 3 to 7 ring members and containing one or more (e.g., one or two; preferably one) carbon-to-carbon double bonds.
As used herein, the term "heterocycloalkenyl” refers to an unsaturated alicyclic (non-aromatic) ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said ring group contains one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, wherein one or more carbon ring atoms may optionally be oxidized (i.e. , to form an oxo group), and further wherein said ring group comprises at least one double bond between adjacent ring atoms and does not comprise any triple bond between adjacent ring atoms. For example, each heteroatom-containing ring comprised in said unsaturated alicyclic ring group may contain one or two 0 atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatomcontaining ring. “Heterocycloalkenyl” may, e.g., refer to imidazolinyl (e.g., 2-imidazolinyl (i.e., 4,5-dihydro-1 H- imidazolyl), 3-imidazolinyl, or 4-imidazolinyl), tetrahydropyridinyl (e.g., 1,2,3,6-tetrahydropyridinyl), dihydropyridinyl (e.g., 1 ,2-dihydropyridinyl or 2,3-dihydropyridinyl), pyranyl (e.g., 2H-pyranyl or 4H-pyranyl), thiopyranyl (e.g., 2H-thiopyranyl or 4H-thiopyranyl), dihydropyranyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrazinyl, dihydroisoindolyl, octahydroquinolinyl (e.g., 1,2,3,4,4a,5,6,7-octahydroquinolinyl), or octahydroisoquinolinyl (e.g., 1 ,2,3,4,5,6,7,8-octahydroisoquinolinyl). Unless defined otherwise, "heterocycloalkenyl” preferably refers to a 3 to 11 membered unsaturated alicyclic ring group, which is a monocyclic ring or a fused ring system (e.g., a fused ring system composed of two fused rings), wherein said ring group contains one or more (e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, wherein one or more carbon ring atoms are optionally oxidized, and wherein said ring group comprises at least one double bond between adjacent ring atoms and does not comprise any triple bond between adjacent ring atoms; more preferably, "heterocycloalkenyl” refers to a 5 to 7 membered monocyclic unsaturated non-aromatic ring group containing one or more (e.g., one, two, or three) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, wherein one or more carbon ring atoms are optionally oxidized, and wherein said ring group comprises at least one double bond between adjacent ring atoms and does not comprise any triple bond between adjacent ring atoms.
As used herein, the term "heterocycloalkenylene” refers to a heterocycloalkenyl group, as defined herein above, but having two points of attachment, i.e. a divalent unsaturated alicyclic (i.e., non-aromatic) ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said ring group contains one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, wherein one or more carbon ring atoms may optionally be oxidized (i.e. , to form an oxo group), and further wherein said ring group comprises at least one double bond between adjacent ring atoms and does not comprise any triple bond between adjacent ring atoms. For example, each heteroatom-containing ring comprised in said unsaturated alicyclic ring group may contain one or two 0 atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatomcontaining ring. "Heterocycloalkenylene” may, e.g., refer to imidazolinylene, tetrahydropyridinylene, dihydropyridinylene, pyranylene, thiopyranylene, dihydropyranylene, dihydrofuranylene, dihydropyrazolylene, dihydropyrazinylene, dihydroisoindolylene, octahydroquinolinylene, or octahydroisoquinolinylene. Unless defined otherwise, "heterocycloalkenylene” preferably refers to a divalent 3 to 11 membered unsaturated alicyclic ring group, which is a monocyclic ring or a fused ring system (e.g., a fused ring system composed of two fused rings), wherein said ring group contains one or more (e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, wherein one or more carbon ring atoms are optionally oxidized, and wherein said ring group comprises at least one double bond between adjacent ring atoms and does not comprise any triple bond between adjacent ring atoms; more preferably, "heterocycloalkenylene” refers to a divalent 5 to 7 membered monocyclic unsaturated nonaromatic ring group containing one or more (e.g., one, two, or three) ring heteroatoms independently selected from
O, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, wherein one or more carbon ring atoms are optionally oxidized, and wherein said ring group comprises at least one double bond between adjacent ring atoms and does not comprise any triple bond between adjacent ring atoms.
As used herein, the term "halogen” refers to fluoro (-F), chloro (-CI), bromo (-Br), or iodo (-I).
As used herein, the term “haloalkyl” refers to an alkyl group substituted with one or more (preferably 1 to 6, more preferably 1 to 3) halogen atoms which are selected independently from fluoro, chloro, bromo and iodo, and are preferably all fluoro atoms. It will be understood that the maximum number of halogen atoms is limited by the number of available attachment sites and, thus, depends on the number of carbon atoms comprised in the alkyl moiety of the haloalkyl group. "Haloalkyl” may, e.g., refer to -CF3, -CHF2, -CH2F, -CF2-CH3, -CH2-CF3, -CH2-CHF2, -CH2-CF2-CH3, -CH2-CF2-CF3, or -CH(CF3)2. A preferred "haloalkyl” group is fluoroalkyl. A particularly preferred "haloalkyl” group is -CF3.
As used herein, the term "fluoroalky I” refers to an alkyl group substituted with one or more (preferably 1 to 6, more preferably 1 to 3) fluoro atoms (-F). It will be understood that the maximum number of fluoro atoms is limited by the number of available attachment sites and, thus, depends on the number of carbon atoms comprised in the alkyl moiety of the fluoroalkyl group. “Fluoroalkyl” may, e.g., refer to -CF3, -CHF2, -CH2F, -CF2-CH3, -CH2-CF3, -CH2-CHF2, -CH2-CF2-CH3, -CH2-CF2-CF3, or -CH(CF3)2. A particularly preferred “fl uoroalky I” group is -CF3. The terms "bond” and "covalent bond” are used herein synonymously, unless explicitly indicated otherwise or contradicted by context.
As used herein, the terms "optional”, "optionally” and "may” denote that the indicated feature may be present but can also be absent. Whenever the term "optional”, "optionally” or "may” is used, the present invention specifically relates to both possibilities, i.e., that the corresponding feature is present or, alternatively, that the corresponding feature is absent. For example, the expression "X is optionally substituted with Y” (or "X may be substituted with Y”) means that X is either substituted with Y or is unsubstituted. Likewise, if a component of a composition is indicated to be "optional”, the invention specifically relates to both possibilities, i.e., that the corresponding component is present (contained in the composition) or that the corresponding component is absent from the composition.
Various groups are referred to as being "optionally substituted” in this specification. Generally, these groups may carry one or more substituents, such as, e.g., one, two, three or four substituents. It will be understood that the maximum number of substituents is limited by the number of attachment sites available on the substituted moiety. Unless defined otherwise, the "optionally substituted” groups referred to in this specification carry preferably not more than two substituents and may, in particular, carry only one substituent. Moreover, unless defined otherwise, it is preferred that the optional substituents are absent, i.e. that the corresponding groups are unsubstituted.
A skilled person will appreciate that the substituent groups comprised in the compounds of the present invention may be attached to the remainder of the respective compound via a number of different positions of the corresponding specific substituent group. Unless defined otherwise, the preferred attachment positions for the various specific substituent groups are as illustrated in the examples.
As used herein, unless explicitly indicated otherwise or contradicted by context, the terms "a”, "an” and "the” are used interchangeably with "one or more” and "at least one”. Thus, for example, a composition comprising "a” compound of formula (I) can be interpreted as referring to a composition comprising "one or more” compounds of formula (I).
It is to be understood that wherever numerical ranges are provided/disclosed herein, all values and subranges encompassed by the respective numerical range are meant to be encompassed within the scope of the invention. Accordingly, the present invention specifically and individually relates to each value that falls within a numerical range disclosed herein, as well as each subrange encompassed by a numerical range disclosed herein.
As used herein, the term "about” preferably refers to ±10% of the indicated numerical value, more preferably to ±5% of the indicated numerical value, and in particular to the exact numerical value indicated. If the term "about” is used in connection with the endpoints of a range, it preferably refers to the range from the lower endpoint -10% of its indicated numerical value to the upper endpoint +10% of its indicated numerical value, more preferably to the range from of the lower endpoint -5% to the upper endpoint +5%, and even more preferably to the range defined by the exact numerical values of the lower endpoint and the upper endpoint. As used herein, the term "comprising” (or "comprise”, "comprises”, "contain”, "contains”, or "containing”), unless explicitly indicated otherwise or contradicted by context, has the meaning of "containing, inter alia”, i.e., "containing, among further optional elements, .. In addition thereto, this term also includes the narrower meanings of "consisting essentially of' and "consisting of'. For example, the term "A comprising B and C” has the meaning of "A containing, inter alia, B and C”, wherein A may contain further optional elements (e.g., "A containing B, C and D” would also be encompassed), but this term also includes the meaning of "A consisting essentially of B and C” and the meaning of "A consisting of B and C” (i.e., no other components than B and C are comprised in A).
The scope of the invention embraces all pharmaceutically acceptable salt forms of the compounds of formula (I) which may be formed, e.g., by protonation of an atom carrying an electron lone pair which is susceptible to protonation, such as an amino group, with an inorganic or organic acid, or as a salt of an acid group (such as a carboxylic acid group) with a physiologically acceptable cation. Exemplary base addition salts comprise, for example: alkali metal salts such as sodium or potassium salts; alkaline earth metal salts such as calcium or magnesium salts; zinc salts; ammonium salts; aliphatic amine salts such as trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, procaine salts, meglumine salts, ethylenediamine salts, or choline salts; aralkyl amine salts such as N, N-dibenzylethylenediamine salts, benzathine salts, benethamine salts; heterocyclic aromatic amine salts such as pyridine salts, picoline salts, quinoline salts or isoquinoline salts; quaternary ammonium salts such as tetramethylammonium salts, tetraethylammonium salts, benzyltrimethylammonium salts, benzyltriethylammonium salts, benzyltributylammonium salts, methyltrioctylammonium salts or tetrabutylammonium salts; and basic amino acid salts such as arginine salts, lysine salts, or histidine salts. Exemplary acid addition salts comprise, for example: mineral acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate salts (such as, e.g., sulfate or hydrogensulfate salts), nitrate salts, phosphate salts (such as, e.g., phosphate, hydrogenphosphate, or dihydrogenphosphate salts), carbonate salts, hydrogencarbonate salts, perchlorate salts, borate salts, or thiocyanate salts; organic acid salts such as acetate, propionate, butyrate, pentanoate, hexanoate, heptanoate, octanoate, cyclopentanepropionate, decanoate, undecanoate, oleate, stearate, lactate, maleate, oxalate, fumarate, tartrate, malate, citrate, succinate, adipate, gluconate, glycolate, nicotinate, benzoate, salicylate, ascorbate, pamoate (embonate), camphorate, glucoheptanoate, or pivalate salts; sulfonate salts such as methanesulfonate (mesylate), ethanesulfonate (esylate), 2-hydroxyethanesulfonate (isethionate), benzenesulfonate (besylate), p-toluenesulfonate (tosylate), 2-naphthalenesulfonate (napsylate), 3-phenylsulfonate, or camphorsulfonate salts; glycerophosphate salts; and acidic amino acid salts such as aspartate or glutamate salts. Further pharmaceutically acceptable salts are described in the literature, e.g., in Stahl PH & Wermuth CG (eds.), "Handbook of Pharmaceutical Salts: Properties, Selection, and Use”, Wiley-VCH, 2002 and in the references cited therein. Preferred pharmaceutically acceptable salts of the compounds of formula (I) include a hydrochloride salt, a hydrobromide salt, a mesylate salt, a sulfate salt, a tartrate salt, a fumarate salt, an acetate salt, a citrate salt, and a phosphate salt. A particularly preferred pharmaceutically acceptable salt of the compound of formula (I) is a hydrochloride salt. Accordingly, it is preferred that the compound of formula (I), including any one of the specific compounds of formula (I) described herein, is in the form of a hydrochloride salt, a hydrobromide salt, a mesylate salt, a sulfate salt, a tartrate salt, a fumarate salt, an acetate salt, a citrate salt, or a phosphate salt, and it is particularly preferred that the compound of formula (I) is in the form of a hydrochloride salt. The present invention also specifically relates to the compound of formula (I), including any one of the specific compounds of formula (I) described herein, in non-salt form.
Moreover, the scope of the invention embraces the compounds of formula (I) in any solvated form, including, e.g., solvates with water (i.e., as a hydrate) or solvates with organic solvents such as, e.g., methanol, ethanol, isopropanol, acetic acid, ethyl acetate, ethanolamine, DMSO, or acetonitrile. All physical forms, including any amorphous or crystalline forms (i.e., polymorphs), of the compounds of formula (I) are also encompassed within the scope of the invention. It is to be understood that such solvates and physical forms of pharmaceutically acceptable salts of the compounds of the formula (I) are likewise embraced by the invention. The invention also specifically relates to the compounds of formula (I) in unsolvated form, i.e., not in the form of a solvate.
Furthermore, the compounds of formula (I) may exist in the form of different stereoisomers (including, e.g., geometric isomers (or cis/trans isomers), enantiomers and diastereomers) or tautomers (including, in particular, prototropic tautomers, such as keto/enol tautomers or thione/thiol tautomers). All such isomers of the compounds of formula (I) are contemplated as being part of the present invention, either in admixture or in pure or substantially pure form. As for stereoisomers, the invention embraces the isolated optical isomers of the compounds according to the invention as well as any mixtures thereof (including, in particular, racemic mixtures/racemates). The racemates can be resolved by physical methods, such as, e.g., fractional crystallization, separation or crystallization of diastereomeric derivatives, or separation by chiral column chromatography. The individual optical isomers can also be obtained from the racemates via salt formation with an optically active acid followed by crystallization. The present invention further encompasses any tautomers of the compounds of formula (I). It will be understood that some compounds may exhibit tautomerism. In such cases, the formulae provided herein expressly depict only one of the possible tautomeric forms. The formulae and chemical names as provided herein are intended to encompass any tautomeric form of the corresponding compound and not to be limited merely to the specific tautomeric form depicted by the drawing or identified by the name of the compound.
The scope of the invention also embraces compounds of formula (I), in which one or more atoms are replaced by a specific isotope of the corresponding atom. For example, the invention encompasses compounds of formula (I), in which one or more hydrogen atoms (or, e.g., all hydrogen atoms) are replaced by deuterium atoms (i.e., 2H; also referred to as “D”). Accordingly, the invention also embraces compounds of formula (I) which are enriched in deuterium. Naturally occurring hydrogen is an isotopic mixture comprising about 99.98 mol-% hydrogen-1 (1H) and about 0.0156 mol-% deuterium (2H or D). The content of deuterium in one or more hydrogen positions in the compounds of formula (I) can be increased using deuteration techniques known in the art. For example, a compound of formula (I) or a reactant or precursor to be used in the synthesis of the compound of formula (I) can be subjected to an H/D exchange reaction using, e.g., heavy water (D2O). Further suitable deuteration techniques are described in: Atzrodt J et al., Bioorg Med Chem, 20(18), 5658-5667, 2012; William JS et al., Journal of Labelled Compounds and Radiopharmaceuticals, 53(11-12), 635-644, 2010; Modvig A et al., J Org Chem, 79, 5861-5868, 2014. The content of deuterium can be determined, e.g., using mass spectrometry or NMR spectroscopy. Unless specifically indicated otherwise, it is preferred that the compound of formula (I) is not enriched in deuterium. Accordingly, the presence of naturally occurring hydrogen atoms or 1H hydrogen atoms in the compounds of formula (I) is preferred. The present invention also embraces compounds of formula (I), in which one or more atoms are replaced by a positron-emitting isotope of the corresponding atom, such as, e.g., 18F, 11C, 13N, 150, 76Br, 77Br, 120l and/or 124l. Such compounds can be used as tracers, trackers or imaging probes in positron emission tomography (PET). The invention thus includes (I) compounds of formula (I), in which one or more fluorine atoms (or, e.g., all fluorine atoms) are replaced by 18F atoms, (ii) compounds of formula (I), in which one or more carbon atoms (or, e.g., all carbon atoms) are replaced by 11C atoms, (ill) compounds of formula (I), in which one or more nitrogen atoms (or, e.g., all nitrogen atoms) are replaced by 13N atoms, (iv) compounds of formula (I), in which one or more oxygen atoms (or, e.g., all oxygen atoms) are replaced by 15O atoms, (v) compounds of formula (I), in which one or more bromine atoms (or, e.g., all bromine atoms) are replaced by 76Br atoms, (vi) compounds of formula (I), in which one or more bromine atoms (or, e.g., all bromine atoms) are replaced by 77Br atoms, (vii) compounds of formula (I), in which one or more iodine atoms (or, e.g., all iodine atoms) are replaced by 120l atoms, and (viii) compounds of formula (I), in which one or more iodine atoms (or, e.g., all iodine atoms) are replaced by 124l atoms. In general, it is preferred that none of the atoms in the compounds of formula (I) are replaced by specific isotopes.
The compounds provided herein may be administered as compounds perse or may be formulated as medicaments. The medicaments/pharmaceutical compositions may optionally comprise one or more pharmaceutically acceptable excipients, such as carriers, diluents, fillers, disintegrants, lubricating agents, binders, colorants, pigments, stabilizers, preservatives, antioxidants, and/or solubility enhancers.
The pharmaceutical compositions may comprise one or more solubility enhancers, such as, e.g., polyethylene glycol), including polyethylene glycol) having a molecular weight in the range of about 200 to about 5,000 Da (e.g., PEG 200, PEG 300, PEG 400, or PEG 600), ethylene glycol, propylene glycol, glycerol, a non-ionic surfactant, tyloxapol, polysorbate 80, macrogol-15-hydroxystearate (e.g., Kolliphor® HS 15, CAS 70142-34-6), a phospholipid, lecithin, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, a cyclodextrin, o-cyclodextrin, p-cyclodextrin, y-cyclodextrin, hydroxyethyl-p-cyclodextrin, hydroxypropyl-p- cyclodextrin, hydroxyethyl-y-cyclodextrin, hydroxypropyl-y-cyclodextrin, dihydroxypropyl-p-cyclodextrin, sulfobutylether-p-cyclodextrin, sulfobutylether-y-cyclodextrin, glucosyl-a-cyclodextrin, glucosyl-p-cyclodextrin, diglucosyl-p-cyclodextrin, maltosyl-a-cyclodextrin, maltosyl-p-cyclodextrin, maltosyl-y-cyclodextrin, maltotriosyl-p- cyclodextrin, maltotriosyl-y-cyclodextrin, dimaltosyl-p-cyclodextrin, methyl-p-cyclodextrin, a carboxyalkyl thioether, hydroxypropyl methylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, a vinyl acetate copolymer, vinyl pyrrolidone, sodium lauryl sulfate, dioctyl sodium sulfosuccinate, or any combination thereof.
The pharmaceutical compositions may also comprise one or more preservatives, particularly one or more antimicrobial preservatives, such as, e.g., benzyl alcohol, chlorobutanol, 2-ethoxyethanol, m-cresol, chlorocresol (e.g., 2-chloro-3-methyl-phenol or 4-chloro-3-methyl-phenol), benzalkonium chloride, benzethonium chloride, benzoic acid (or a pharmaceutically acceptable salt thereof), sorbic acid (or a pharmaceutically acceptable salt thereof), chlorhexidine, thimerosal, or any combination thereof. The pharmaceutical compositions can be formulated by techniques known to the person skilled in the art, such as the techniques published in "Remington: The Science and Practice of Pharmacy”, Pharmaceutical Press, 22nd edition. The pharmaceutical compositions can be formulated as dosage forms for oral, parenteral, such as intramuscular, intravenous, subcutaneous, intradermal, intraarterial, intracardial, rectal, nasal, topical, aerosol or vaginal administration. Dosage forms for oral administration include coated and uncoated tablets, soft gelatin capsules, hard gelatin capsules, lozenges, troches, solutions, emulsions, suspensions, syrups, elixirs, powders and granules for reconstitution, dispersible powders and granules, medicated gums, chewing tablets and effervescent tablets. Dosage forms for parenteral administration include solutions, emulsions, suspensions, dispersions and powders and granules for reconstitution. Emulsions are a preferred dosage form for parenteral administration. Dosage forms for rectal and vaginal administration include suppositories and ovula. Dosage forms for nasal administration can be administered via inhalation and insufflation, for example by a metered inhaler. Dosage forms for topical administration include creams, gels, ointments, salves, patches and transdermal delivery systems.
The compounds of formula (I) or the pharmaceutically acceptable salts or solvates thereof, or the above described pharmaceutical compositions comprising any of the aforementioned entities, may be administered to a subject by any convenient route of administration, whether systemically/peri pheral ly or at the site of desired action, including but not limited to one or more of: oral (e.g., as a tablet, capsule, or as an ingestible solution), topical (e.g., transdermal, intranasal, ocular, buccal, and sublingual), parenteral (e.g., using injection techniques or infusion techniques, and including, for example, by injection, e.g., subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, or intrasternal by, e.g., implant of a depot, for example, subcutaneously or intramuscularly), pulmonary (e.g., by inhalation or insufflation therapy using, e.g., an aerosol, e.g., through mouth or nose), gastrointestinal, intrauterine, intraocular, subcutaneous, ophthalmic (including intravitreal or intracameral), rectal, or vaginal administration.
If said compounds or pharmaceutical compositions are administered parenterally, then examples of such administration include one or more of: intravenously, intraarterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternally, intracardially, intracranially, intramuscularly or subcutaneously administering the compounds or pharmaceutical compositions, and/or by using infusion techniques. For parenteral administration, the compounds are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.
Said compounds or pharmaceutical compositions can also be administered orally in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavoring or coloring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications.
The tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycolate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included. Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the agent may be combined with various sweetening or flavoring agents, coloring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
For oral administration, the compounds or pharmaceutical compositions are preferably administered by oral ingestion, particularly by swallowing. The compounds or pharmaceutical compositions can thus be administered to pass through the mouth into the gastrointestinal tract, which can also be referred to as "oral-gastrointestinal” administration.
Alternatively, said compounds or pharmaceutical compositions can be administered in the form of a suppository or pessary, or may be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment or dusting powder. The compounds of the present invention may also be dermally or transdermally administered, for example, by the use of a skin patch.
Said compounds or pharmaceutical compositions may also be administered by sustained release systems. Suitable examples of sustained-release compositions include semi-permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained-release matrices include, e.g., polylactides, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, poly(2-hydroxyethyl methacrylate), ethylene vinyl acetate, or poly-D-(— )-3- hydroxybutyric acid. Sustained-release pharmaceutical compositions also include liposomally entrapped compounds. The present invention thus also relates to liposomes containing a compound of the invention.
Said compounds or pharmaceutical compositions may also be administered by the pulmonary route, rectal routes, or the ocular route. For ophthalmic use, they can be formulated as micronized suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.
It is also envisaged to prepare dry powder formulations of the compounds of formula (I) for pulmonary administration, particularly inhalation. Such dry powders may be prepared by spray drying under conditions which result in a substantially amorphous glassy or a substantially crystalline bioactive powder. Accordingly, dry powders of the compounds of the present invention can be made according to an emulsification/spray drying process.
For topical application to the skin, said compounds or pharmaceutical compositions can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, 2-octyldodecanol, benzyl alcohol and water.
The present invention thus relates to the compounds or the pharmaceutical compositions provided herein, wherein the corresponding compound or pharmaceutical composition is to be administered by any one of: an oral route; topical route, including by transdermal, intranasal, ocular, buccal, or sublingual route; parenteral route using injection techniques or infusion techniques, including by subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, intrasternal, intraventricular, intraurethral, or intracranial route; pulmonary route, including by inhalation or insufflation therapy; gastrointestinal route; intrauterine route; intraocular route; subcutaneous route; ophthalmic route, including by intravitreal, or intracameral route; rectal route; or vaginal route. Preferred routes of administration are oral administration or parenteral administration. For each of the compounds or pharmaceutical compositions provided herein, it is particularly preferred that the respective compound or pharmaceutical composition is to be administered orally (particularly by oral ingestion).
Typically, a physician will determine the actual dosage which will be most suitable for an individual subject. The specific dose level and frequency of dosage for any particular individual subject may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual subject undergoing therapy. The precise dose and also the route of administration will ultimately be at the discretion of the attendant physician or veterinarian.
The compound of formula (I) or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition comprising any of the aforementioned entities, can be administered in monotherapy (e.g., without concomitantly administering any further therapeutic agents, or without concomitantly administering any further therapeutic agents against the same disease that is to be treated or prevented with the compound of formula (I)). However, the compound of formula (I) or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition comprising any of the aforementioned entities, can also be administered in combination with one or more further therapeutic agents. If the compound of formula (I) is used in combination with a second therapeutic agent active against the same disease or condition, the dose of each compound may differ from that when the corresponding compound is used alone, in particular, a lower dose of each compound may be used. The combination of the compound of formula (I) with one or more further therapeutic agents may comprise the simultaneous/concomitant administration of the compound of formula (I) and the further therapeutic agent(s) (either in a single pharmaceutical formulation or in separate pharmaceutical formulations), or the sequential/separate administration of the compound of formula (I) and the further therapeutic agent(s). If administration is sequential, either the compound of formula (I) according to the invention or the one or more further therapeutic agents may be administered first. If administration is simultaneous, the one or more further therapeutic agents may be included in the same pharmaceutical formulation as the compound of formula (I), or they may be administered in two or more different (separate) pharmaceutical formulations. For the treatment or prevention of cancer, the one or more further therapeutic agents to be administered in combination with a compound of the present invention are preferably anticancer drugs. The anticancer drug(s) to be administered in combination with a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof may, e.g., be selected from: a tumor angiogenesis inhibitor (e.g., a protease inhibitor, an epidermal growth factor receptor kinase inhibitor, or a vascular endothelial growth factor receptor kinase inhibitor); a cytotoxic drug (e.g., an antimetabolite, such as purine and pyrimidine analog antimetabolites); an antimitotic agent (e.g., a microtubule stabilizing drug or an antimitotic alkaloid); a platinum coordination complex; an anti-tumor antibiotic; an alkylating agent (e.g., a nitrogen mustard or a nitrosourea); an endocrine agent (e.g., an adrenocorticosteroid, an androgen, an anti-androgen, an estrogen, an anti-estrogen, an aromatase inhibitor, a gonadotropin-releasing hormone agonist, or a somatostatin analog); a compound that targets an enzyme or receptor that is overexpressed and/or otherwise involved in a specific metabolic pathway that is deregulated (or misregulated) in the tumor cell (e.g., ATP and GTP phosphodiesterase inhibitors, histone deacetylase inhibitors, protein kinase inhibitors (such as serine, threonine and tyrosine kinase inhibitors, e.g., Abelson protein tyrosine kinase inhibitors) and the various growth factors, their receptors and corresponding kinase inhibitors (such as epidermal growth factor receptor kinase inhibitors, vascular endothelial growth factor receptor kinase inhibitors, fibroblast growth factor inhibitors, insulin-like growth factor receptor inhibitors and platelet-derived growth factor receptor kinase inhibitors)); methionine; an aminopeptidase inhibitor; a proteasome inhibitor; a cyclooxygenase inhibitor (e.g., a cyclooxygenase-1 inhibitor or a cyclooxygenase- 2 inhibitor); a topoisomerase inhibitor (e.g., a topoisomerase I inhibitor or a topoisomerase II inhibitor); a poly ADP ribose polymerase inhibitor (PARP inhibitor); and an epidermal growth factor receptor (EGFR) inhibitor/antagonist. An alkylating agent which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, a nitrogen mustard (such as cyclophosphamide, mechlorethamine (chlormethine), uramustine, melphalan, chlorambucil, ifosfamide, bendamustine, or trofosfamide), a nitrosourea (such as carmustine, streptozocin, fotemustine, lomustine, nimustine, prednimustine, ranimustine, or semustine), an alkyl sulfonate (such as busulfan, mannosulfan, or treosulfan), an aziridine (such as hexamethylmelamine (altretamine), triethylenemelamine, ThioTEPA (N.N'N'-triethylenethiophosphoramide), carboquone, or triaziquone), a hydrazine (such as procarbazine), a triazene (such as dacarbazine), or an imidazotetrazine (such as temozolomide).
A platinum coordination complex which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, or triplatin tetranitrate.
A cytotoxic drug which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, an antimetabolite, including folic acid analogue antimetabolites (such as aminopterin, methotrexate, pemetrexed, or raltitrexed), purine analogue antimetabolites (such as cladribine, clofarabine, fludarabine, 6-mercaptopurine (including its prodrug form azathioprine), pentostatin, or 6-thioguanine), and pyrimidine analogue antimetabolites (such as cytarabine, decitabine, 5-fluorouracil (including its prodrug forms capecitabine and tegafur), floxuridine, gemcitabine, enocitabine, or sapacitabine).
An antimitotic agent which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, a taxane (such as docetaxel, larotaxel, ortataxel, paclitaxel/taxol, tesetaxel, or nab-paclitaxel (e.g., Abraxane®)), a Vinca alkaloid (such as vinblastine, vincristine, vinflunine, vindesine, or vinorelbine), an epothilone (such as epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, or epothilone F) or an epothilone B analogue (such as ixabepilone/azaepothilone B). An anti-tumor antibiotic which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, an anthracycline (such as aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, amrubicin, pirarubicin, valrubicin, or zorubicin), an anthracenedione (such as mitoxantrone, or pixantrone) or an anti-tumor antibiotic isolated from Streptomyces (such as actinomycin (including actinomycin D), bleomycin, mitomycin (including mitomycin C), or plicamycin).
A tyrosine kinase inhibitor which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, axitinib, bosutinib, cediranib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, lestaurtinib, nilotinib, semaxanib, sorafenib, sunitinib, axitinib, nintedanib, ponatinib, vandetanib, or vemurafenib.
A topoisomerase inhibitor which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, a topoisomerase I inhibitor (such as irinotecan, topotecan, camptothecin, belotecan, rubitecan, or lamellarin D) or a topoisomerase II inhibitor (such as amsacrine, etoposide, etoposide phosphate, teniposide, or doxorubicin).
A PARP inhibitor which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, niraparib, olaparib, rucaparib, talazoparib, veliparib, pamiparib (BGB-290), BMN-673, CEP 9722, MK 4827, E7016, or 3-aminobenzamide.
An EGFR inhibitor/antagonist which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, gefitinib, erlotinib, lapatinib, afatinib, neratinib, osimertinib, brigatinib, dacomitinib, vandetanib, pelitinib, canertinib, icotinib, poziotinib, ABT-414, AV-412, PD 153035, PKI-166, BMS-690514, CUDC- 101 , AP26113, XL647, cetuximab, panitumumab, zalutumumab, nimotuzumab, or matuzumab.
Further anticancer drugs may also be used in combination with a compound of the present invention. The anticancer drugs may comprise biological or chemical molecules, like TNF-related apoptosis-inducing ligand (TRAIL), tamoxifen, amsacrine, bexarotene, estramustine, irofulven, trabectedin, cetuximab, panitumumab, tositumomab, alemtuzumab, bevacizumab, edrecolomab, gemtuzumab, alvocidib, seliciclib, aminolevulinic acid, methyl aminolevulinate, efaproxiral, porfimer sodium, talaporfin, temoporfin, verteporfin, alitretinoin, tretinoin, anagrelide, arsenic trioxide, atrasentan, bortezomib, carmofur, celecoxib, demecolcine, elesclomol, elsamitrucin, etoglucid, lonidamine, lucanthone, masoprocol, mitobronitol, mitoguazone, mitotane, oblimersen, omacetaxine, sitimagene, ceradenovec, tegafur, testolactone, tiazofurine, tipifarnib, vorinostat, iniparib, or copanlisib.
Also biological drugs, like antibodies, antibody fragments, antibody constructs (for example, single-chain constructs), and/or modified antibodies (like CDR-grafted antibodies, humanized antibodies, "fully human” antibodies, etc.) directed against cancer or tumor markers/factors/cytokines involved in proliferative diseases can be employed in cotherapy approaches with the compounds of the invention. Examples of such biological molecules are anti-HER2 antibodies (e.g. trastuzumab, Herceptin®), anti-CD20 antibodies (e.g. Rituximab, Rituxan®, MabThera®, Reditux®), anti-CD19/CD3 constructs, and anti-TNF antibodies (see, e.g., Taylor PC, Curr Opin Pharmacol, 2003, 3(3):323-328). An anticancer drug which can be used in combination with a compound of the present invention may be, in particular, an immunooncology therapeutic (such as an antibody (e.g., a monoclonal antibody or a polyclonal antibody), an antibody fragment, an antibody construct (e.g., a single-chain construct), or a modified antibody (e.g., a CDR-grafted antibody, a humanized antibody, or a "fully human” antibody) or a small molecule) targeting any one of CTLA-4, PD-1 , PD-L1 , TIGIT, TIM3, LAG3, 0X40, CSF1 R, IDO, or CD40. Such immunooncology therapeutics include, e.g., an anti- CTLA-4 antibody (e.g., ipilimumab or tremelimumab), an anti-PD-1 antibody (e.g., nivolumab (BMS-936558), pembrolizumab (MK-3475), pidilizumab (CT-011), cemiplimab, dostarlimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, zimberelimab, AMP-224, AMP-514 (or MEDI0680), JTX-4014, INCMGA00012 (or MGA012), or APE02058), an anti-PD-L1 antibody (e.g., atezolizumab, avelumab, durvalumab, KN035, CK-301 , BMS- 936559, MEDI4736, MPDL3280A (RG7446), MDX-1105, MEDI6469, or bintrafusp alfa), an anti-TIGIT antibody (e.g., tiragolumab, vibostolimab, domvanalimab, etigilimab, BMS-986207, EOS-448, COM902, ASP8374, SEA-TGT, BGB- A1217, IBI-939, or M6223), an anti-TIM3 antibody, an anti-LAG3 antibody (e.g., relatlimab (or BMS-986016), ieramilimab (or LAG525), encelimab (or TSR-033), tebotelimab (or MGD013), REGN3767 (or R3767), FS118, IMP701 , or IMP731 ), an anti-OX40 antibody (e.g., MEDI0562), an anti-CSF1 R antibody (e.g., IMC-CS4 or RG7155), an anti-IDO antibody, or an anti-CD40 antibody (e.g., CP-870,893 or Chi Lob 7/4). Further immunooncology therapeutics are known in the art and are described, e.g., in: Kyi C et al., FEBS Lett, 2014, 588(2):368-76; Intlekofer AM et al., J Leukoc Biol, 2013, 94(1):25-39; Callahan MK et al., J Leukoc Biol, 2013, 94(1):41-53; Ngiow SF et al., Cancer Res, 2011, 71 (21):6567-71; and Blattman JN et al., Science, 2004, 305(5681 ):200-5.
In particular, a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising any of the aforementioned entities, may be administered in combination with an immune checkpoint inhibitor, preferably an antibody (or an antigen-binding fragment thereof, or an antibody construct) directed against CTLA-4, PD-1, PD-L1, TIGIT, or LAG3. Corresponding preferred examples include, but are not limited to, any one of the anti-CTLA-4 antibodies ipilimumab or tremelimumab, any one of the anti-PD-1 antibodies nivolumab, pembrolizumab, pidilizumab, cemiplimab, dostarlimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, zimberelimab, AMP-224, AMP-514, JTX-4014, INCMGA00012, or APE02058, any one of the anti-PD-L1 antibodies atezolizumab, avelumab, durvalumab, KN035, CK-301 , BMS-936559, MEDI4736, MPDL3280A, MDX- 1105, MEDI6469 or bintrafusp alfa, any one of the anti-TIGIT antibodies tiragolumab, vibostolimab, domvanalimab, etigilimab, BMS-986207, EOS-448, CCM902, ASP8374, SEA-TGT, BGB-A1217, I BI-939 or M6223, and/or any one of the anti-LAG3 antibodies relatlimab, ieramilimab, encelimab, tebotelimab, REGN3767, FS118, IMP701 , or IMP731. The present invention thus relates to a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising any of the aforementioned entities optionally in combination with a pharmaceutically acceptable excipient, for use in the treatment or prevention of cancer, wherein the compound or the pharmaceutical composition is to be administered in combination with one or more immune checkpoint inhibitors, wherein said one or more immune checkpoint inhibitors are preferably selected from anti-CTLA-4 antibodies, anti- PD-1 antibodies, anti-PD-L1 antibodies, anti-TIGIT antibodies, and/or anti-LAG3 antibodies (for example, said one or more immune checkpoint inhibitors may be selected from anti-CTLA-4 antibodies, anti-PD-1 antibodies and/or anti- PD-L1 antibodies, such as, e.g., ipilimumab, tremelimumab, nivolumab, pembrolizumab, cemiplimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, AMP-224, AMP-514, atezolizumab, avelumab, durvalumab, KN035, or CK- 301); more preferably, said one or more immune checkpoint inhibitors are selected from ipilimumab, tremelimumab, nivolumab, pembrolizumab, pidilizumab, cemiplimab, dostarlimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, zimberelimab, AMP-224, AMP-514, JTX-4014, INCMGA00012, APE02058, atezolizumab, avelumab, durvalumab, KN035, CK-301 , BMS-936559, MEDI4736, MPDL3280A, MDX-1105, MEDI6469, bintrafusp alfa, tiragolumab, vibostolimab, domvanalimab, etigilimab, BMS-986207, EOS-448, CCM902, ASP8374, SEA-TGT, BGB-A1217, IBI-939, M6223, relatlimab, ieramilimab, encelimab, tebotelimab, REGN3767, FS118, IMP701 , and IMP731.
The present invention thus particularly relates to a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising any of the aforementioned entities optionally in combination with a pharmaceutically acceptable excipient, for use in the treatment or prevention of cancer, wherein the compound or the pharmaceutical composition is to be administered in combination with one or more anticancer drugs (including any one or more of the specific anticancer drugs described herein above).
The combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formulation. The individual components of such combinations may be administered either sequentially or simultaneously/concomitantly in separate or combined pharmaceutical formulations by any convenient route. When administration is sequential, either the compound of the present invention (i.e., the compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof) or the further therapeutic agent(s) may be administered first. When administration is simultaneous, the combination may be administered either in the same pharmaceutical composition or in different pharmaceutical compositions. When combined in the same formulation, it will be appreciated that the two or more compounds must be stable and compatible with each other and the other components of the formulation. When formulated separately, they may be provided in any convenient formulation and may be administered by any convenient route. For the combinations described above, it is preferred that the individual components of such combinations are provided in separate pharmaceutical formulations.
The subject or patient to be treated in accordance with the present invention may be an animal (e.g., a non-human animal). Preferably, the subject/patient is a mammal. More preferably, the subject/patient is a human (e.g., a male human or a female human) or a non-human mammal (such as, e.g., a guinea pig, a hamster, a rat, a mouse, a rabbit, a dog, a cat, a horse, a monkey, an ape, a marmoset, a baboon, a gorilla, a chimpanzee, an orangutan, a gibbon, a sheep, cattle, or a pig). Most preferably, the subject/patient to be treated in accordance with the invention is a human.
The term "treatment” of a disorder or disease, as used herein, is well known in the art. "Treatment” of a disorder or disease implies that a disorder or disease is suspected or has been diagnosed in a patient/subject. A patient/subject suspected of suffering from a disorder or disease typically shows specific clinical and/or pathological symptoms which a skilled person can easily attribute to a specific pathological condition (i.e., diagnose a disorder or disease).
The "treatment” of a disorder or disease may, for example, lead to a halt in the progression of the disorder or disease (e.g., no deterioration of symptoms) or a delay in the progression of the disorder or disease (in case the halt in progression is of a transient nature only). The "treatment” of a disorder or disease may also lead to a partial response (e.g., amelioration of symptoms) or complete response (e.g., disappearance of symptoms) of the subject/patient suffering from the disorder or disease. Accordingly, the "treatment” of a disorder or disease may also refer to an amelioration of the disorder or disease, which may, e.g., lead to a halt in the progression of the disorder or disease or a delay in the progression of the disorder or disease. Such a partial or complete response may be followed by a relapse. It is to be understood that a subject/patient may experience a broad range of responses to a treatment (such as the exemplary responses as described herein above). The treatment of a disorder or disease may, inter alia, comprise curative treatment (preferably leading to a complete response and eventually to healing of the disorder or disease) and palliative treatment (including symptomatic relief). The term "prevention” of a disorder or disease, as used herein, is also well known in the art. For example, a pati en t/su bject suspected of being prone to suffer from a disorder or disease may particularly benefit from a prevention of the disorder or disease. The subject/patient may have a susceptibility or predisposition for a disorder or disease, including but not limited to hereditary predisposition. Such a predisposition can be determined by standard methods or assays, using, e.g., genetic markers or phenotypic indicators. It is to be understood that a disorder or disease to be prevented in accordance with the present invention has not been diagnosed or cannot be diagnosed in the patient/subject (for example, the patient/subject does not show any clinical or pathological symptoms). Thus, the term "prevention” comprises the use of a compound of the present invention before any clinical and/or pathological symptoms are diagnosed or determined or can be diagnosed or determined by the attending physician.
It is to be understood that the present invention specifically relates to each and every combination of features and embodiments described herein, including any combination of general and/or preferred features/embodiments. In particular, the invention specifically relates to each combination of meanings (including general and/or preferred meanings) for the various groups and variables comprised in formula (I).
In this specification, a number of documents including patent applications and scientific literature are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.
The reference in this specification to any prior publication (or information derived therefrom) is not and should not be taken as an acknowledgment or admission or any form of suggestion that the corresponding prior publication (or the information derived therefrom) forms part of the common general knowledge in the technical field to which the present specification relates.
The invention is also described by the following illustrative figures:
Figure 1 : SMNDC1 co-localizes with nuclear speckle markers, a, Overview of SMNDCTs structure with numbered truncations (for Fig. 2f), intrinsic disorder prediction plot (MetaDisorder; Kozlowski, L. P. & Bujnicki, J. M. MetaDisorder: a meta-server for the prediction of intrinsic disorder in proteins. BMC Bioinformatics 13, 111 (2012)), AlphaFold structure prediction with Tudor domain and positions of GFP intron-tags marked, b, Live images of clonal cell lines (aTC1 and HAP1) with the endogenous GFP-tag. c, Immunoblots showing expression of WT SMNDC1 and SMNDC1-GFP fusion proteins in clonal cell lines with GFP-tag in different introns, d, Live (SMNDC1-GFP intron 2-3, aTC1) and immunofluorescence images (aTC1 WT) with nuclear staining (DRAQ5™ I DAPI). e, Live imaging (SMNDC1-GFP intron 2-3, aTC1) with DRAQ5™ nuclear staining showing a cell during M-phase. f, Immunofluorescence images (aTC1 WT) with SMNDC1 and SC35 antibody, and overlap. DAPI nuclear staining, g, Live imaging (SMNDC1-GFP intron 2-3, SRRM2-RFP intron 9-10, aTC1) with DRAQ5™ nuclear staining showing a cell during telophase, h, Co-localization analyses of interphase (n=114) and mitotic (n=13) cells, Pearson correlation between different channels of maximum intensity projections of z-stack images. Data shown as scatter plot + median line, analyzed by unpaired t-test.
Figure 2: SMNDC1 shows biomolecular condensation in vitro and in cellular systems, a, In vitro droplet formation assay with 10 piM GFP or SMNDC1-GFP fusion protein +/- 10% PEG-8000, b, In vitro droplet formation assay of SMNDC1-GFP over time with droplet fusion event, marked by arrows, c, In vitro droplet formation assay of SMNDC1-GFP with quantified number of droplets with different protein and NaCI concentrations, +/- 10 ng/pil RNA. d, In vitro droplet formation assay of SMNDC1-GFP with the addition of 10 ng/pil total cellular RNA and RNase, e, In vitro droplet formation assay of SMNDC1-GFP with 100 ng/pil Cy5-labeled RNA, and overlap, f, In vitro droplet formation assay of different truncations of SMNDC1-GFP + 10 ng/pil total cellular RNA. g, Live imaging (SMNDC1-GFP intron 2-3, aTC1), cells were treated with 2.5% or 5 % 1 ,6-hexanediol. Quantifications of GFP intensity and GFP spots/nucleus in 4 different clonal cell lines. Data presented as scatter plot with mean line (n=4), analyzed by two-tailed, multiple paired t-tests with False Discovery Rate q calculated by Two-stage step-up (Benjamin!, Y., Krieger, A. M. & Yekutieli, D. Adaptive linear step-up procedures that control the false discovery rate. Biometrika 93, 491-507 (2006)). h, Fluorescence recovery after photobleaching (FRAP) experiment in SMNDC1- GFP intron 2-3, SRRM2-RFP intron 9-10, aTC1-cells. Top: Relative intensity of Hoechst, SMNDC1-GFP, and SRRM2-RFP in reference (filled symbols) and bleach region (empty symbols) over time. Data plotted as mean with standard deviation, n=3. Bottom: representative images of nucleus with marked reference and bleach region at 3 different time points, 0 sec (before bleaching), 3 sec (directly after bleaching), and 120 sec (after recovery).
Figure 3: Characterization of SMNDCTs interactome by proximity labeling, a, Scheme of proximity labeling by APEX2 fusion proteins followed by mass spectrometry-based proteomics, b, Immunofluorescence images (aTC1 WT) with staining against SMNDC1, Biotin via Streptavidin and nuclear staining DAPI. c, Volcano plot showing Iog2 abundance against -Iog10 adjusted p-value (one-way ANOVA, Benjamini-Hochberg correction for multiple comparisons) of APEX2-SMNDC1FL versus APEX2-SMNDC1TD biotinylated and enriched proteins. Highlighted dots indicate 750 enriched proteins (adjusted p-value <0.1, abundance ratio >1.1). d, Enrichr analysis of APEX2-SMNDC1FL enriched proteins, GO Biological Process 2021 terms plotted with their odds ratio and their adjusted p-value (Benjamini- Hochberg method for correction for multiple hypotheses testing). Terms with a -Iog10 (adjusted p-value) >60 named, e, Venn diagram showing the overlap of proteins identified by SMNDC1-ColP, and APEX2-SMNDC1FL enriched, f, Venn diagram showing the overlap of proteins identified by SRSF7-APEX2, and APEX2-SMNDC1FL enriched.
Figure 4: Identification of an inhibitor against SMNDCTs Tudor domain, a, Scheme of AlphaScreen set-up with the NMR-structure of SMNDCTs Tudor domain (PDB: 4A4H). b, Screening strategy starting with -90,000 compound library, c, Overview of AlphaScreen of full -90,000 compound library with DMSO, positive control (quencher), and compound hits, d, AlphaScreen percentage of DMSO control with SMNDC1/ sDMA-peptide vs crosslinking peptide. Remaining hits marked, e-k, Chemical structure and AlphaScreen 9-point compound titration with SMNDC1/ sDMA- peptide vs. SMN/ sDMA-peptide. Data presented as mean +/- SD (n=2). Figure 5: Structure-activity relationships of the 2-aryl-4-aminothiazole Tudor domain inhibitors. Grey scale-coded chemical structures illustrating structure activity relationships for compound 1. Modifications to the 2-aryl moiety, replacements of the thiazole group, linker modifications and arylamide analogs are shown in different grey scales. Underneath are IC50 values (piM) for SMNDC1 in black (on the left) and SMN in grey (on the right).
Figure 6: Inhibitor binds at the aromatic cage of the Tudor domain, a, Chemical shift perturbations (CSP) of Tudor domains SMNDC1 and SMN in presence of 0.8 mM compound 13. Residues forming the aromatic cage are highlighted in bold. Left inlet shows cartoon representation of SMNDC1 in complex with compound 13 (grey sticks) calculated using semi-rigid body docking of compound 13 based on 23 intermolecular NOE restraints (see d and Table 3). CSP per residue in presence of 8 mM compound 13 are displayed in grey shades. Right inlet shows compound 13 with numbers indicating the assignment used in d and Table 3. b, Zoomed view of the binding site with residues forming the aromatic cage shown as sticks. Stacking contacts between the thiazole moiety of compound 13 and tryptophane 83 or tyrosine 111 indicated with dashed lines, c, Overlay of 1H,15N-HSQC spectra of SMNDC1 in the presence of 0, 0.05, 0.15, 0.25, 0.5, 0.8, and 8 mM compound 13. Zoomed view shows residues in and near the aromatic cages, d, Section of an in co i-13C-filtered NOESY spectrum shows crosspeaks between inhibitor protons (grey arrows) and aromatic cage protons (black) arrows. The crossing points of the dashed lines indicate the locations of the intermolecular NOEs.
Figure 7: Cellular effects of SMNDC1 Tudor domain inhibition, a-c, Live imaging and quantifications of different SMNDC1-GFP cell lines (aTC1) treated with DMSO, 50 piM compound 1, or 50 piM compound 9. Data shown as mean + standard deviation, n=3, analyzed by ratio-paired t-test. d, Live imaging of cells (SRRM2-RFP intron 9-10, aTC1) treated with DMSO, 50 piM compound 1, or 50 piM compound 9. e, Quantification of SRRM2-RFP spots/ nucleus in live imaging data of SRRM2-RFP cell line (aTC1) treated with DMSO, 50 piM compound 1, or 50 piM compound 9. Data shown as mean + standard deviation, n=3, analyzed by unpaired t-test, n=3. f, Quantification of nuclear SMNDC1-GFP, SMNDC1-GFP spots/ nucleus, nuclear SRRM2-RFP, and SRRM2-RFP spots/ nucleus in live imaging data of double-tagged SMNDC1-GFP (intron 2-3) SRRM2-RFP (intron 9-10) cell line (aTC1) treated with DMSO or 50 piM compound 1 and transduced with Empty Vector or SMNDC1 knock-down (KD) plasmids. Data shown as mean + standard deviation, n=3. g, Volcano plot showing Iog2 protein abundance against -Iog10 adjusted p-value of compound 1 treated cells over DMSO control after APEX2-SMNDC1 FL proximity labeling and biotin enrichment. 126 proteins significantly depleted vs. 6 proteins significantly enriched, adjusted p-value <0.05, |log2FC| > 2. h, Same as in g. Proteins with known sDMA modification marked and named, i, Fluorescence recovery after photobleaching (FRAP) experiment in SMNDC1-GFP intron 2-3, SRRM2-RFP intron 9-10, aTC1-cells, treated with DMSO (filled symbols) or 50 piM compound 1 (empty symbols). Relative intensity of Hoechst SMNDC1-GFP, and SRRM2-RFP in bleach region over time (Reference region: see Fig. 13e). Data plotted as mean with standard error of the mean, n > 11 . Last time point analyzed by unpaired t-test. j, Splicing analysis of RNA-sequencing data, plotted are all alternative splicing events and their density with their differential percentage spliced-in (dPSI) value of compound 1 over DMSO treatment. Exon events are colored dark grey, while intron events are colored light grey, k, Splicing analysis of RNA-sequencing data, plotted are all overlapping alternative splicing events between compound 1 over DMSO treatment (x-axis) and SMNDC1 knockdown (KD) over empty vector (EV) (y-axis) with their respective dPSI-values. Results from a simple linear regression analysis. Events confirmed via PCR in light grey, not confirmed events in dark grey. I, DNA-bands on agarose gel after reverse transcription and PCR amplification of RNA to confirm alternative splicing events. RNA was isolated from aTC1 cells transfected with empty vector or SMNDC1 knock-down (KD) plasmid and treated with DMSO, 2 pi M compound 1 for 5 days, or 50 piM compound 1 for 16 h.
Figure 8: SMNDC1 co-localizes with nuclear speckle markers, a, AlphaFold structure predictions of SMNDC1 with GFP integrated in different introns, b, Quantification of SMNDC1-GFP band divided by endogenous SMNDC1 band in different clonal intron-tag cell lines (aTC1), on a Iog2 scale. Data shown as mean, n=4. c, Immunoblots showing expression of WT SMNDC1 and SMNDC1-GFP fusion proteins in clonal cell lines with GFP-tag in different introns, 4 replicates. Ponceau staining to show comparable loading, d, Live imaging of SMNDC1-GFP intron 2-3 cell line with DRAQ5™ nuclear staining, control (no overexpression), overexpressing CLK1 or DYRK3. e, Co-localization analyses of IF images of aTC1 WT with SMNDCI-antibody, SC35-antibody, and DAPI nuclear staining. Pearson correlation between different channels of maximum intensity projections of z-stack images. Data shown as scatter plot + median, analyzed by unpaired t-test. f, Comparison of Pearson correlation values in co-localization analyses of mitotic and interphase cells, live imaging corresponding to Fig. 1g+h. Data shown as scatter plot + median.
Figure 9: Characterization of SMNDCTs interactome by proximity labeling, a, Depiction of APEX2-fusion constructs APEX2-SMNDC1 FL and APEX2-SMNDC1TD. b, Ponceau S staining of all proteins and western blot with Streptavidin- HRP showing all biotinylated proteins, c, Volcano plot showing Iog2 abundance against -Iog10 adjusted p-value of APEX2-SMNDC1 FL versus APEX2-SMNDC1TD biotinylated and enriched proteins. Nuclear proteins highlighted, d, Venn diagrams showing the overlap of all proteins with a known sDMA modification or aDMA modification (light grey; circles on the left), and APEX2-SMNDC1FL enriched proteins or all proteins identified with SMNDC1 proximity labeling (dark grey; circles on the right).
Figure 10: Establishment of AlphaScreen, a, Coomassie staining of all proteins in different samples along the protein purification process. SMNDC1-Tudor domain marked by arrow, b, Cross-titration of different protein and peptide concentrations in AlphaScreen, c, Titration of AlphaScreen acceptor and donor beads with 50 nM peptide, 75 nM protein. Data shown as mean + standard deviation, n=3.
Figure 11 : SMNDCI/inhibitor complex, a, Inter-molecular NOEs (dashed line) used in the restraint-driven docking simulation. NOEs derived from different protons of the inhibitor are colored individually. Phe or Tyr protons HE and HD were not specifically assigned, and a dashed line is only shown for the proton with the closest distance to the NOE contact. The inlet shows the relative orientation of the structure compared to Figure 6. b, Ramachandran plot of the four lowest-energy structures, 88.9% and 11.1 % of the backbone torsion angles are found in the favored regions and additional allowed regions, respectively, c, Overlay of SMNDC1/ compound 13 complex (four lowest-energy structures, shades of green; compound 13, light grey) and SMNDC1/sDMA (dark grey/grey, PDB: 4A4H) shows high structural similarity (backbone rmsd 0.295-0.375 A). Figure 12: Effects of SMNDC1 Tudor domain inhibition on localization, a, Live imaging of cells (SMNDC1-GFP with different introns tagged, aTC1) treated with DMSO, 50 M compound 1, or 50 M compound 9. Nuclei stained with Hoechst, corresponding to images in Fig. 7a. b, Quantification of AnnexinV+and PI + cells (aTC1), treated with DMSO or 50 pM compound 1. Data shown as mean + standard deviation, n=3. c, Quantification of nuclear SRRM2-RFP in live imaging data of SRRM2-RFP cell line (aTC1) treated with DMSO, 50 pM compound 1, or 50 pM compound 9. Data shown as mean + standard deviation, n=3. d, Quantification of nuclear SRRM2-RFP in live imaging data of different SRRM2-RFP cell lines (aTC1) treated with DMSO, or 50 pM compound 1. Data analyzed by ratio-paired t- test. e, Quantification of SRRM2-RFP spots/ nucleus in live imaging data of different SRRM2-RFP cell lines (aTC1) treated with DMSO, or 50 pM compound 1. Data analyzed by ratio-paired t-test. f, Upper panel: Immunofluorescence images of cells (aTC1 WT) stained with antibodies against SMNDC1 , SC25, and nuclear marker DAPI, treated with DMSO or 50 pM compound 1. Lower panel: Quantification of SMNDC1-AB intensity and SC35-AB intensity in the nucleus in immunofluorescence imaging data (aTC1), cells treated with DMSO or 50 pM compound 1. Data analyzed by ratio-paired t-test. g, Representative images of quantifications shown in Fig. 7f. Live imaging data of double-tagged SMNDC1-GFP (intron 2-3) SRRM2-RFP (intron 9-10) cell line (aTC1) treated with DMSO or 50 pM compound 1 and transduced with Empty Vector or SMNDC1 knock-down (KD) plasmids, h, Live imaging data of double-tagged SMNDC1-GFP (intron 2-3) SRRM2-RFP (intron 9-10) cell line (aTC1) and multiple SMNDC1-GFP clonal cell lines single-tagged in different introns, treated with DMSO, 50 pM compound 1, or 50 pM compound 28. Quantification of nuclear SMNDC1-GFP and SMNDC1-GFP spots/ nucleus. Data shown as mean + standard deviation, n=3, analyzed by ratio-paired t-test. i, Quantification of live imaging data of double-tagged SMNDC1-GFP (intron 2-3) SRRM2-RFP (intron 9-10) cell line (aTC1) treated with DMSO, 50 pM compound 1, or 50/ 25/ 12.5/ 6.25/ 3.125 pM compound 28. Nuclear SMNDC1-GFP, SMNDC1-GFP spots/ nucleus, nuclear SRRM2-RFP, and SRRM2-RFP spots/ nucleus. Data shown as mean + standard deviation, n=3. j, Top panel: Live imaging of cells (SMN-RFP intron 5-6, aTC1) treated with DMSO or 50 pM compound 1. Quantification of whole cell SMN-RFP and SMN-RFP spots/ cytoplasm in live imaging data of different SMN-RFP cell lines (aTC1).
Figure 13: Effects of SMNDC1 Tudor domain inhibition on interactome and splicing, a, Volcano plot showing Iog2 protein abundance against -Iog10 adjusted p-value of compound 1 treated cells over DMSO control after APEX2- SMNDC1TD proximity labeling and biotin enrichment. Significantly enriched proteins in light gray and significantly depleted proteins in dark grey. 0 proteins significantly depleted vs. 5 proteins significantly enriched, adjusted p-value <0.05, |log2FC| > 2. b, Volcano plot showing Iog2 protein abundance against -Iog10 adjusted p-value of compound 1 treated cells over DMSO control after APEX2-SMNDC1 FL proximity labeling and biotin enrichment. Nuclear speckle proteins marked, c, Volcano plot showing Iog2 protein abundance against -Iog10 adjusted p-value of compound 1 treated cells over DMSO control after APEX2-SMNDC1 FL proximity labeling and biotin enrichment. Proteins identified by SRSF7-APEX2 proximity labeling marked, d, Western blot with Streptavidin-HRP showing all biotinylated proteins, antibodies against SFPQ, APEX2, and SMNDC1. Cells overexpressing APEX2-SMNDC1 FL or APEX2-SMNDC1TD were treated with DMSO or compound 1, and proximity-labeled. Biotinylated proteins were enriched and separated in an SDS-PAGE. Representative images of n=2. e, Fluorescence recovery after photobleaching (FRAP) experiment in SMNDC1-GFP intron 2-3, SRRM2-RFP intron 9-10, aTC1-cells, treated with DMSO (filled symbols) or 50 piM compound 1 (empty symbols). Relative intensity of Hoechst SMNDC1-GFP, and SRRM2-RFP only in reference region over time (bleach region: see Fig. 7i). Data plotted as mean with standard error of the mean, n > 11. f, DNA- bands on agarose gel after reverse transcription and PCR amplification of RNA to confirm alternative splicing events. RNA was isolated from aTC1 cells transfected with empty vector or SMNDC1 knock-down (KD) plasmid and treated with DMSO, 2 piM compound 1 for 5 days, or 50 piM compound 1 for 16 h. Boxes show relevant bands for confirmed events.
Figure 14: RNAbindRplus of SMNDC1. RNAbindRplus score of amino acids (AA) over length of SMNDC1 protein. Areas over threshold of 0.5 marked in dark grey.
The invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of the scope of the present invention.
EXAMPLES
The compounds described in this section are defined by their chemical formulae and their corresponding chemical names. In case of conflict between any chemical formula and the corresponding chemical name indicated herein, the present invention relates to both the compound defined by the chemical formula and the compound defined by the chemical name, and particularly relates to the compound defined by the chemical formula.
Example A: Synthesis of compounds of formula (I)
The following examples illustrate the synthesis of representative compounds of the present invention. These examples are not intended, nor are they to be construed, as limiting the scope of the present invention and the embodiments provided herein.
Solvents and inorganic reagents were obtained from Lactan, Carl Roth, Sigma-Aldrich or TCI and were used as received. Reagents were obtained from abcr, Enamine or Sigma-Aldrich and were used as received; detailed sources are given further below.
Automated preparative flash chromatography was performed using KP-SIL Biotage cartridges on a Biotage Isolera Prime.
1H NMR and 13C NMR spectra were recorded on a Bruker Avance III 600 MHz NMR spectrometer. Chemical shifts are given in parts per million (ppm), coupling constants J are given in Hertz, and spin multiplicities are given as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), or b (broad signal). HRMS was recorded on a TSQ Vantage mass spectrometer (Thermo Scientific) with ESI-source.
General procedure A. Amide coupling using HATU (WO 2010/001169)
A 50 mL flask was charged with a magnetic stir bar, the carboxylic acid (1 mmol), anhydrous DMF (2 ml), and diisopropylethylamine (5 mmol). With stirring, HATU (1.5 mmol) was added, followed by a solution of the amine substrate (1 mmol) in DMF and the reaction was stirred at ambient temperature for 20 h. The reaction was then diluted with water and extracted with ethyl acetate (3 x 50 mL). The combined organic extract was dried with Na2SO4, filtered, and concentrated in vacuo to yield the crude product which was purified via silica gel chromatography using ethyl acetate/hexanes gradient or ethyl acetate/methanol (0-20%) gradient as eluent to provide the title compound.
General procedure B. Amide coupling using PyBOP
The carboxylic acid (1.0 mmol) was dissolved in anhydrous DMF (2 ml), and diisopropylethylamine (3 mmol). With stirring, PyBOP (1.2 mmol) was added, and the activation mixture was stirred for 20 min at room temperature (rt). A solution of the amine substrate (1.0 mmol) in anhydrous DMF was added and the reaction was stirred at ambient temperature for 20 h. The reaction was then diluted with water and extracted with ethyl acetate (3 x 50 mL). The combined organic extract was dried with Na2SO4, filtered, and concentrated in vacuo to yield the crude product which was purified via silica gel chromatography using ethyl acetate/hexanes gradient or ethyl acetate/methanol (0-20%) gradient as eluent to provide the title compound.
The pomalidomide analogs used as reagents were synthesized as described in J. Med. Chem. 2018, 61, 462-481.
Examples 1-19: sources and catalog numbers of commercial compounds (structures depicted in Example C below) Example 1 : 8016-5462, ChemDiv
Example 2: F0098-0393, Life Chemicals
Example 3: F0147-0092, Life Chemicals
Example 4: F0463-0182, Life Chemicals Example 5: F0737-0296, Life Chemicals Example 6: F0789-0042, Life Chemicals Example 7: F0866-0370, Life Chemicals Example 8: F1166-0142, Life Chemicals Example 9: F2450-0086, Life Chemicals Example 10: F2536-1240, Life Chemicals Example 11 : F5127-0237, Life Chemicals Example 12: G786-1089, ChemDiv Example 13: G786-1145, ChemDiv Example 14: G786-1153 ChemDiv Example 15: G786-1206, ChemDiv Example 16: G786-1438, ChemDiv Example 17: F0700-0008
Example 18-19: custom synthesis, Enamine
Examples 63-87: sources and catalog numbers of commercial compounds (structures depicted in Example C below) Example 63: Y042-5014, ChemDiv
Example 64: Y042-5014, ChemDiv
Example 65: Y042-5708, ChemDiv
Example 66: Z1259485437, Enamine
Example 67: Z1559503155, Enamine Example 68: Z1917788786, Enamine
Example 69: Z238189326, Enamine
Example 70: Z28306748, Enamine
Example 71 : Z28307393, Enamine
Example 72: Z29466325, Enamine
Example 73: Z29466475, Enamine
Example 74: Z29466475, Enamine
Example 75: Z29467591, Enamine
Example 76: Z29467644, Enamine
Example 77: Z29467754, Enamine
Example 78: Z29467852, Enamine
Example 79: G786-1153, ChemDiv
Example 80: Z2468078, Enamine
Example 81 : Z3297024656, Enamine
Example 82: Z48849973, Enamine
Example 83: Z51932376, Enamine
Example 84: Z56789173, Enamine
Example 85: Z56794310, Enamine
Example 86: Z99481352, Enamine
Example 87: Z99482088, Enamine
Example 20: tert-butyl 4-(2-(3-(4-methylpiperazine-1-carbonyl)-5-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)phenoxy)ethyl)piperazine-1-carboxylate
Figure imgf000067_0001
To a suspension of 3-hydroxy-5-(4-methylpiperazine-1 -carbonyl)-N-(4-(pyridin-2-yl)thiazol-2-yl)benzamide (0.426 g, 1.03 mmol), K2CO3 (0.958 g, 6.93 mmol), and KI (37 mg) in anhydrous DMF (3.0 mL), 1-Boc-4-(2- chloroethyl)piperazine (0.274 g, 1.10 mmol) was added. The mixture was heated to 70°C for 5 h, then it was stirred at ambient temperature for 60 h. The dark suspension was dissolved in H2O (15 mL) and extracted with EtOAc (3 x 10 mL). The combined extracts were washed with H2O and brine, then dried on Na2SO4 and evaporated in vacuo. The resulting oil was subjected to flash chromatography (SIO2, CH2CI2 + 1% EtsN /MeOH (0-20%)). The title compound was obtained as an amber gum (118 mg, 0.186 mmol, 19%).
HRMS (ESI): m/z calcd for C32H41N7O5S (M + H)+, 636.2963, found 636.2967.
Example 20B: 3-(4-methylpiperazine-1-carbonyl)-5-(2-(piperazin-1-yl)ethoxy)-N-(4-(pyridin-2-yl)thiazol-2- yl)benzamide
Figure imgf000068_0001
Example 20 (68 mg) was dissolved in 4M HOI I dioxane (1 mL) and the resulting solution was stirred for 18 h. The reaction mixture was evaporated, and the resulting red film was used without purification.
Example 21 : 3-(2-(3-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)-N-(3-(3-(but-3-yn-1-yl)-3H-diazirin-3- yl)propanoyl)propanamido)ethoxy)-5-(4-methylpiperazine-1-carbonyl)-N-(4-(pyridin-2-yl)thiazol-2- yl)benzamide
Figure imgf000068_0002
General procedure A was followed under light exclusion for 18 h. The reaction mixture was subjected to EtOAc/H2O workup, then the crude product was purified by flash chromatography (SiO2, hexanes I EtOAc (0-100%)).
HRMS (ESI): m/z calcd for C31H34N8O4S (M + H)+, 615.2496, found 615.2495.
Example 22: Tert-butyl (2-(3-(4-methylpiperazine-1-carbonyl)-5-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)phenoxy)ethyl)carbamate
Figure imgf000068_0003
3-Hydroxy-5-(4-methylpiperazine-1-carbonyl)-N-(4-(pyridin-2-yl)thiazol-2-yl)benzamide (0.355 g, 0.86 mmol) was dissolved in anhydrous DMF (5 mL) and tert-butyl (2-bromoethyl)carbamate (0.287 g, 1.29 mmol), K2CO3 (0.977 g, 7.07 mmol), KI (43 mg, 0.26 mmol) were added. The suspension was heated to 65°C for 2 days. Saturated brine was added and the pH was adjusted to 7. The solution was extracted several times with EtOAc, then the pH was set to 8- 9 and the extraction was repeated. The aqueous solution was evaporated to dryness and was triturated with hot MeOH. All the organic phases were combined and evaporated, then the material was subjected to chromatographic purification (SiO2, C^C /MeOH (0-25%)). The fraction containing the product was purified again by reverse phase chromatography (C18-SiO2, H2O/ACN (0-100%)) affording the product as a gummy solid (0.174 g, 0.307 mmol, 36%). HRMS (ESI): m/z calcd for C28H34N6O5S (M + H)+, 567.2384, found 567.2383. Example 23: 3-(2-(4-(3-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)propanoyl)piperazin-1-yl)ethoxy)-5-(4- methylpiperazine-1-carbonyl)-N-(4-(pyridin-2-yl)thiazol-2-yl)benzamide
Figure imgf000069_0001
The title compound was prepared according to general procedure A from 3-(3-(but-3-yn-1-yl)-3H-diazirin-3- yl)propanoic acid (13.9 mg, 84 pimol), HATU (42.2 mg, 111 pimol), iP^NEt (80 piL), Example 20B (38.8 mg, 60 pimol) in anhydrous DMF (1.0 mL). The reaction mixture was stirred under exclusion of light for 18 h. The clear amber solution was then diluted with brine and extracted with EtOAc (3 x 20 mL). The organic solutions were washed with H2O and brine, dried on Na2SO4 and evaporated. The crude product was subjected to flash chromatography (SiO2, CH2CI2 1 MeOH (0-20%). The title compound was obtained as a brownish film (7.8 mg, 11.4 pimol, 19%).
HRMS (ESI): m/z calcd for C35H41N9O4S (M + H)+, 684.3075, found 684.3073.
Example 24: 3-(2-(3-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)propanamido)ethoxy)-5-(4-methylpiperazine-1- carbonyl)-N-(4-(pyridin-2-yl)thiazol-2-yl)benzamide
Figure imgf000069_0002
The title compound was prepared according to general procedure A from 3-(2-aminoethoxy)-5-(4-methylpiperazine- 1-carbonyl)-N-(4-(pyridin-2-yl)thiazol-2-yl)benzamide (HCI salt, 25 mg, 46 pimol), 3-(3-(but-3-yn-1-yl)-3H-diazirin-3- yl)propanoic acid (8.4 mg, 50 pimol), HATU (24 mg, 63 pimol), iP^NEt (50 piL), anhydrous DMF (0.4 mL). The reaction mixture was stirred for 18 h under light exclusion. The solution was diluted with brine and extracted with EtOAc (3 x 20 mL). The organic solutions were washed with water and brine, dried on Na2SO4, and evaporated. Purification of the crude compound by flash chromatography (SiO2, C^Ch/MeOH (0-20%)) afforded the title compound as a brownish gum (7.2 mg, 11.7 pimol, 25%).
HRMS (ESI): m/z calcd for C31H34N8O4S (M + H)+, 615.2496, found 615.2495.
Example 25: 3-hydroxy-5-(4-methylpiperazine-1-carbonyl)-N-(4-(pyridin-2-yl)thiazol-2-yl)benzamide
Figure imgf000069_0003
5-Hydroxyisophthalic acid (29.30 g, 0.161 mol) was dissolved in DMF (150 mL) and IP^NEt (87 mL, 0.499 mol) and treated with PyBOP (84.6 g, 0.163 mol) and N-methylpiperazine (16.15 g, 0.161 mol). The reaction mixture was stirred overnight and HATU (61.2 g, 0.161 mol), IPr2NEt (27.9 mL, 0.16 mol) followed by the HBr salt of 5-(pyridin-2-yl)thiazol- 2-amine (37.0 g, 0.1434 mol) were added. The dark reaction mixture was stirred for 24 h and the solvents were removed in vacuo. The remaining syrupy liquid was treated with saturated brine and 5N HCI until a grayish brown slush was obtained. This was extracted with 3 portions of EtOAc, and the combined organic solutions were extracted with 3 portions of 0.5 N HCI. The original aq. phase and the dilute HCI solutions were combined and the pH was set to 8-9 with 10M NaOH, then the mixture was extracted with EtOAc (4 x 150 mL). The pooled organic phases were washed with brine, dried on anhydrous Na2SO4, and evaporated. The residue was purified by chromatography (SIO2, EtOAc-MeOH (0-5%) then MeOH; the most polar fractions were combined and treated with 4N HCI/dioxane and acetone. The separated hygroscopic solid was filtered and washed with acetone then dried in vacuo to afford 9.7 g of the title compound as HCI salt, brownish solid.
HRMS (ESI): m/z calcd for C21H21N5O3S (M + H)+, 424.1438, found 424.1439.
Example 26: tert-butyl 2-(3-(4-methylpiperazine-1-carbonyl)-5-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)phenoxy)acetate
Figure imgf000070_0001
The phenol 3-hydroxy-5-(4-methylpiperazine-1 -carbonyl)-N-(4-(pyridin-2-yl)thiazol-2-yl)benzamide (HCI salt, 2.34 g, 5.09 mmol) was dissolved in anhydrous DMF (7 mL) and a solution of KOtBu (1 M in THF, 15 mL, 15 mmol) was added with stirring. The orange-brown solution was stirred for 30 min. t-Butyl bromoacetate (0.75 mL, 5.08 mmol) was then slowly added and the reaction mixture was stirred for 22 h. The reaction was then quenched with saturated brine and extracted with EtOAc (4 x 50 mL). The combined extracts were washed with water and brine, dried on anhydrous Na2SO4, evaporated, and subjected to flash chromatography (SIO2, C^Ch/MeOH (0-20%) affording the title compound as brownish solid (0.753 g, 1.40 mmol, 27.5%).
Example 27: 3-(2-((3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)propyl)amino)-2-oxoethoxy)- 5-(4-methylpiperazine-1-carbonyl)-N-(4-(pyridin-2-yl)thiazol-2-yl)benzamide
Figure imgf000071_0001
The title compound was prepared according to the general procedure A from 2-(3-(4-methylpiperazine-1-carbonyl)- 5-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)phenoxy)acetic acid (24.8 mg, 51.5 pimol), HATU (31.2 mg, 82 pimol) in anhydrous DMF (1.5 mL), IP^NEt (100 piL), 4-((3-aminopropyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3- dione TFA salt (29.2 mg, 52.3 pimol). Chromatographic purification of the crude product (SiO2, C^Ch/MeOH (0-20%) afforded the title compound as a yellow film (15.3 mg, 19.3 pimol, 37%).
HRMS (ESI): m/z calcd for C39H39N9O8S (M + H)+, 794.2715, found 794.2699.
Example 28: 3-(2-((6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)hexyl)amino)-2-oxoethoxy)-5-
(4-methylpiperazine-1-carbonyl)-N-(4-(pyridin-2-yl)thiazol-2-yl)benzamide
Figure imgf000071_0002
The title compound was prepared according to the general procedure A, from 2-(3-(4-methylpiperazine-1 -carbonyl)- 5-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)phenoxy)acetic acid (22.3 mg, 46.3 pimol), HATU (24.5 mg, 64.4 pimol) in anhydrous DMF (1.5 mL), IP^NEt (100 piL), 4-((6-aminohexyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1 , 3-dione (TFA salt, 24.7 mg, 41 pimol). Chromatographic purification of the crude product (SiO2, CF C /MeOH (0-20%) afforded the title compound as a tan solid glass (11.6 mg, 13.9 pimol, 30%).
HRMS (ESI): m/z calcd for C42H44N8O9S (M + H)+, 837.3025, found 837.3026.
Example 29: 3-(2-((2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethyl)amino)-2- oxoethoxy)-5-(4-methylpiperazine-1-carbonyl)-N-(4-(pyridin-2-yl)thiazol-2-yl)benzamide
Figure imgf000072_0001
The title compound was prepared according to the general procedure A, from 2-(3-(4-methylpiperazine-1 -carbonyl)- 5-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)phenoxy)acetic acid (25.7 mg, 53.4 pimol), HATU (28.6 mg, 75.2 pimol) in anhydrous DMF (1.5 mL), IPr2NEt (100 piL), 4-((2-(2-aminoethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline- 1 ,3-dione TFA salt (33.5 mg, 58.5 pimol). Chromatographic purification of the crude product (SiO2, C^C /MeOH (0- 20%) afforded the title compound as a yellow film (14.5 mg, 17.6 pimol, 33%).
HRMS (ESI): m/z calcd for C40H41N9O9S (M + H)+, 824.2821 , found 824.2815.
Example 30: 3-((20-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-2,14-dioxo-6,9,12-trioxa-3,15- diazaicosyl)oxy)-5-(4-methylpiperazine-1-carbonyl)-N-(4-(pyridin-2-yl)thiazol-2-yl)benzamide
Figure imgf000072_0002
The title compound was prepared according to the general procedure A, from 2-(3-(4-methylpiperazine-1 -carbonyl)- 5-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)phenoxy)acetic acid (23.9 mg, 49.6 pimol), HATU (25.1 mg, 66 pimol) in anhydrous DMF (1.5 mL), IP^NEt (100 piL), 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-N-(5-((2-(2,6-dioxopiperidin-3- yl)-1 ,3-dioxoisoindolin-4-yl)amino)pentyl)acetamide TFA salt (32.2 mg, 41.5 pimol). Chromatographic purification of the crude product (SiO2, CF C /MeOH (0-20%) afforded the title compound as a yellow film (8.5 mg, 8.4 pimol, 17%). HRMS (ESI): m/z calcd for C49H58N10O12S (M + H)+, 1011.4029, found 1011.4024.
Example 31 : (2S,4R)-1-((S)-3,3-dimethyl-2-(5-(2-(3-(4-methylpiperazine-1-carbonyl)-5-((4-(pyridin-2-yl)thiazol- 2-yl)carbamoyl)phenoxy)acetamido)pentanamido)butanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5- yl)benzyl)pyrrolidine-2-carboxamide
Figure imgf000073_0001
The title compound was prepared according to the general procedure A, from 2-(3-(4-methylpiperazine-1 -carbonyl)- 5-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)phenoxy)acetic acid (24.6 mg, 51.1 pimol), HATU (26.8 mg, 70.5 pimol) in anhydrous DMF (1.5 mL), IP^NEt (100 piL), VHL-C4-NH2 HCI (25.5 mg, 45.1 pimol). Chromatographic purification of the crude product (SiO2, C^Ch/MeOH (0-20%) afforded the title compound as a tan film (13.0 mg, 13.1 pimol, 26%). HRMS (ESI): m/z calcd for C50H60N10O8S2 (M + H)+, 993.4110, found 993.4116.
Example 32: (2S,4R)-1-((S)-3,3-dimethyl-2-(7-(2-(3-(4-methylpiperazine-1-carbonyl)-5-((4-(pyridin-2-yl)thiazol- 2-yl)carbamoyl)phenoxy)acetamido)heptanamido)butanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5- yl)benzyl)pyrrolidine-2-carboxamide
Figure imgf000073_0002
The title compound was prepared according to the general procedure A, from 2-(3-(4-methylpiperazine-1 -carbonyl)- 5-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)phenoxy)acetic acid (24.3 mg, 50.5 pimol), HATU (27.0 mg, 71.0 pimol) in anhydrous DMF (1.5 mL), IP^NEt (100 piL), VHL-C6-NH2 HCI (30.2 mg, 50.3 pimol). Chromatographic purification of the crude product (SiO2, CH2Cl2/MeOH (0-20%) afforded the title compound as a tan film (13.0 mg, 13.1 pimol, 26%). HRMS (ESI): m/z calcd for C52H64N10O8S2 (M + H)+, 1021.4423, found 1021.4418.
Example 33: 2-(3-(4-methylpiperazine-1-carbonyl)-5-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)phenoxy)acetic acid )
Figure imgf000073_0003
Neat tert-butyl 2-(3-(4-methylpiperazine-1-carbonyl)-5-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)phenoxy)acetate (0.75 g, 1.395 mmol) was cooled to 0°C and treated slowly with TFA (3.0 mL) while stirring. The solution was stirred at 0°C for 1 h, then it was allowed to warm to ambient temperature and was stirred for a further 1.5 h. The solvent was evaporated below 40°C and traces of TFA were removed by repeated evaporation with CH2CI2. The hygroscopic residue was dried in vacuo and used without purification.
MS (ESI): m/z calcd for C23H23N5O5S (M + H)+, 482.15, found 482.17.
Example 34: (2S,4R)-1-((S)-3,3-dimethyl-2-(2-(2-(2-(3-(4-methylpiperazine-1-carbonyl)-5-((4-(pyridin-2- yl)thiazol-2-yl)carbamoyl)phenoxy)acetamido)ethoxy)acetamido)butanoyl)-4-hydroxy-N-(4-(4-methylthiazol- 5-yl)benzyl)pyrrolidine-2-carboxamide
Figure imgf000074_0001
2-(3-(4-Methylpiperazine-1-carbonyl)-5-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)phenoxy)acetic acid (28.3 mg, 59 pimol) was dissolved in anhydrous DMF (1.5 mL) and iP^NEt (80 piL) and activated with HATU (26.5 mg, 70 pimol) for 15 min at rt. VHL ligand I-PEGI-NH2 (HCI salt, 33.1 mg, 58 pimol) was added and the reaction mixture was stirred for 18 h. The solvents were removed in vacuo and the residue was purified by flash chromatography (SiO2, CF C /MeOH (0-20%). The title compound was obtained as a tan solid (20.4 mg, 20.5 pimol, 35%).
HRMS (ESI): m/z calcd for C49H58O9N10S2 (M + H)+, 995.3891, found 995.3902.
Example 35: (2S,4R)-1-((S)-3,3-dimethyl-2-(3-(2-(3-(4-methylpiperazine-1-carbonyl)-5-((4-(pyridin-2-yl)thiazol- 2-yl)carbamoyl)phenoxy)acetamido)propanamido)butanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5- yl)benzyl)pyrrolidine-2-carboxamide
Figure imgf000074_0002
The title compound was prepared according to the general procedure A, from 2-(3-(4-methylpiperazine-1 -carbonyl)- 5-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)phenoxy)acetic acid (24.5 mg, 50.8 pimol), HATU (21.3 mg, 56 pimol) in anhydrous DMF (1.5 mL), iP^NEt (100 piL), VHL ligand 1-C2-NH2 HCI (24.3 mg, 45.2 pimol). Chromatographic purification of the crude product (SiO2, C^Ch/MeOH (0-20%) afforded the title compound as a tan solid (20.6 mg, 20.5 pimol, 35%). HRMS (ESI): m/z calcd for C48H56NIO08S2 (M + H)+, 965.3797, found 965.3795.
Example 36: (2S,4R)-1-((S)-2-(tert-butyl)-14-(3-(4-methylpiperazine-1-carbonyl)-5-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)phenoxy)-4,13-dioxo-6,9-dioxa-3,12-diazatetradecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5- yl)benzyl)pyrrolidine-2-carboxamide
Figure imgf000075_0001
The title compound was prepared according to the general procedure A, from 2-(3-(4-methylpiperazine-1 -carbonyl)- 5-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)phenoxy)acetic acid (27.7 mg, 57.5 pimol), HATU (24.0 mg, 63.1 pimol) in anhydrous DMF (1.5 mL), IP^NEt (100 piL), (2S,4R)-1-((S)-2-(2-(2-(2-aminoethoxy)ethoxy)acetamido)-3,3- dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide HCI (VHL ligand 1-PEG2- NH2 HCI, 31.6 mg, 51.6 pimol). Chromatographic purification of the crude product (SiO2, C^C /MeOH (0-25%). The title compound was obtained as a light beige solid (17.7 mg, 17.0 pimol, 30%).
HRMS (ESI): m/z calcd for C51 H62N10O10S2 (M + H)+, 1039.4165, found 1039.4159.
Example 37: (2S,4R)-1-((S)-3,3-dimethyl-2-(9-(2-(3-(4-methylpiperazine-1-carbonyl)-5-((4-(pyridin-2-yl)thiazol- 2-yl)carbamoyl)phenoxy)acetamido)nonanamido)butanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5- yl)benzyl)pyrrolidine-2-carboxamide
Figure imgf000075_0002
The title compound was prepared according to the general procedure A, from 2-(3-(4-methylpiperazine-1 -carbonyl)- 5-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)phenoxy)acetic acid (25.6 mg, 53.2 pimol), HATU (26.1 mg, 68.6 pimol) in anhydrous DMF (2.0 mL), IP^NEt (100 piL), VHL ligand I-C8-NH2 HCI (29.9 mg, 48 pimol). Chromatographic purification of the crude product (SiO2, C^Ch/MeOH (0-25%). The title compound was obtained as a tan film (19.1 mg, 17.8 pimol, 34%).
HRMS (ESI): m/z calcd for C54H68N10O8S2 (M + H)+, 1071.4736, found 1071.4746. Example 38: (2S,4R)-1-((S)-2-(tert-butyl)-20-(3-(4-methylpiperazine-1-carbonyl)-5-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)phenoxy)-4,19-dioxo-6,9,12,15-tetraoxa-3,18-diazaicosanoyl)-4-hydroxy-N-(4-(4-methylthiazol- 5-yl)benzyl)pyrrolidine-2-carboxamide
Figure imgf000076_0001
The title compound was prepared according to the general procedure A, from 2-(3-(4-methylpiperazine-1 -carbonyl)- 5-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)phenoxy)acetic acid (30.0 mg, 62.3 pimol), HATU (27.4 mg, 72.1 pimol) in anhydrous DMF (1.5 mL), iP^NEt (100 piL), VHL ligand 1-PEG4-NH2 HCI, 36.7 mg, 52.5 pimol). Chromatographic purification of the crude product (SiO2, CH2Cl2/MeOH (0-25%). The title compound was obtained as a tan gum (28.9 mg, 25.6 pimol, 41 %).
HRMS (ESI): m/z calcd for C55H70N10O12S2 (M + H)+, 1127.4689, found 1127.4710.
Example 39: (2S,4R)-1-((S)-3,3-dimethyl-2-(2-(3-(4-methylpiperazine-1-carbonyl)-5-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)phenoxy)acetamido)butanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2- carboxamide
Figure imgf000076_0002
The title compound was prepared according to the general procedure A, from 2-(3-(4-methylpiperazine-1 -carbonyl)- 5-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)phenoxy)acetic acid (28.4 mg, 59 pimol), HATU (29.8 mg, 78.4 pimol) in anhydrous DMF (2.0 mL), iP^NEt (120 piL), (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4- methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (27 mg, 57.8 pimol). After chromatographic purification of the crude product (SiO2, C^Ch/MeOH (0-25%), the title compound was obtained as a light tan solid glass (17.9 mg, 20.0 pimol, 34%).
HRMS (ESI): m/z calcd for C45H51N9O7S2 (M + H)+, 894.3426, found 894.3438.
Example 40: 3-(2-((10-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)decyl)amino)-2- oxoethoxy)-5-(4-methylpiperazine-1-carbonyl)-N-(4-(pyridin-2-yl)thiazol-2-yl)benzamide
Figure imgf000077_0001
The title compound was prepared according to the general procedure A, from 2-(3-(4-methylpiperazine-1 -carbonyl)- 5-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)phenoxy)acetic acid (26.8 mg, 55.7 pimol), HATU (34.5 mg, 90.7 pimol) in anhydrous DMF (1.5 mL), iP^NEt (100 piL), 4-((10-aminodecyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3- dione TFA salt (29.6 mg, 45.1 pimol). Chromatographic purification of the crude product (SiO2, C^Ch/MeOH (0-20%) afforded the title compound as a yellow film (21 .8 mg, 24.4 pimol, 44%).
Example 41 : 5-ethyl-4-(pyridin-2-yl)thiazol-2-amine
Figure imgf000077_0002
The title compound was synthesized according to the procedure described in WO 2016/183173 A1.
1H NMR (600 MHz, DMSO) 5 8.55 (ddd, J = 4.8, 1.9, 1.0 Hz, 1 H), 7.84 (dt, J = 8.0, 1.1 Hz, 1 H), 7.77 (td, J = 7.7, 1.9 Hz, 1 H), 7.20 (ddd, J = 7.4, 4.8, 1.3 Hz, 1 H), 6.82 (s, 2H), 3.19 (q, J = 7.4 Hz, 2H), 1.17 (t, J = 7.4 Hz, 3H).
Example 42: 4-(N,N-dimethylsulfamoyl)-N-(5-ethyl-4-(pyridin-2-yl)thiazol-2-yl)benzamide
Figure imgf000077_0003
The title compound was prepared according to the general procedure A, from 4-(dimethylsulfamoyl)benzoic acid (71.4 mg, 0.312 mmol), HATU (160 mg, 0.421 mmol), 5-ethyl-4-(pyridin-2-yl)thiazol-2-amine (67.9 mg, 0.325 mmol), iP^NEt (250 pi L) in DMF (3.5 mL). After chromatographic purification (SiO2, C^C /MeOH (0-25%)) the title product was obtained as yellow solid (64.3 mg, 0.155 mmol, 50%).
HRMS (ESI): m/z calcd for C19H20N4O3S2 (M + H)+, 417.1050, found 417.1053.
1H NMR (600 MHz, DMSO) 5 12.89 (s, 1 H), 8.64 (ddd, J = 4.8, 1.9, 1.0 Hz, 1 H), 8.40 - 8.28 (m, 2H), 8.01 (dt, J = 8.0, 1 .1 Hz, 1 H), 7.98 - 7.86 (m, 3H), 7.37 - 7.29 (m, 1 H), 2.74 - 2.59 (m, 6H), 1 .29 (t, J = 7.5 Hz, 3H).
13C NMR (151 MHz, DMSO) 5 164.03, 154.15, 148.93, 138.12, 136.90, 129.93, 129.21 , 129.11 , 128.52, 128.40, 128.25, 127.65, 122.24, 121.99, 37.53, 20.05, 16.14.
Example 43: 5-bromo-4-(pyridin-2-yl)thiazol-2-amine
Figure imgf000078_0001
The title compound was synthesized according to the procedure described in US 2014/249154 A1.
Example 44: N-(5-bromo-4-(pyridin-2-yl)thiazol-2-yl)-4-(N,N-dimethylsulfamoyl)benzamide
Figure imgf000078_0002
The title compound was prepared according to the general procedure A, from 4-(dimethylsulfamoyl)benzoic acid (74.2 mg, 0.324 mmol), HATU (163 mg, 0.429 mmol), 5-bromo-4-(pyridin-2-yl)thiazol-2-amine (89 mg, 0.264 mmol), iP^NEt (250 piL) in DMF (1.5 mL). After chromatographic purification (SiO2, Cf^C /MeOH (0-25%)) the title product was obtained as yellow solid weighing 102 mg, 0.218 mmol, 67%.
HRMS (ESI): m/z calcd for Ci7Hi5BrN4O3S2 (M + H)+, 466.9842, found 466.9834.
Example 45: 4-(pyridin-2-yl)thiazol-2-amine
Figure imgf000078_0003
The title compound was synthesized according to the procedure described in J. Het. Chem. 7 (5), 1137-41 (1970).
Example 46: 4-(pyridin-2-yl)thiazol-2-amine HBr salt
The title compound was synthesized according to the procedure described in J. Het. Chem. 7 (5), 1137-41 (1970).
Example 47: 4-(pyridin-2-yl)thiazol-2-amine phosphate salt
4-(Pyridin-2-yl)thiazol-2-amine (Example 45) (0.100 g, 0.564 mmol) was dissolved in acetone (2 mL) and treated with a solution of 85% H3PO4 (40 piL) in acetone. The precipitated monobasic phosphate of the compound was filtered and washed with acetone, then air dried to afford the salt in quantitative yield.
Example 48: 4-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)benzoic acid
Figure imgf000078_0004
Step 1 : methyl 4-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)benzoate
4-(Pyridin-2-yl)thiazol-2-amine (HBr salt, 5.6 g, 21.69 mmol) and DMAP (31 mg) were suspended in CH2CI2 (80 mL) and Et3N (10.5 mL). To this solution, a solution of methyl 4-(chlorocarbonyl)benzoate (4.40 g, 22.16 mmol) in 20 mL CH2CI2 was added slowly at 0°C. A clear amber solution was obtained, which was stirred for a further 14 h. The reaction mixture was washed with H2O (2 x 80 mL), dried on Na2SO4, and the organic layer was evaporated to a solid. This crude product was suspended in acetone and filtered, then washed with cold acetone to yield the title compound as a white solid (4.336 g, 12.78 mmol, 59%).
Step 2: 4-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)benzoic acid
Methyl 4-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)benzoate was ground with 10 mL MeOH until a homogenous suspension had been obtained, then 10 M NaOH (5 mL) was added slowly with vigorous stirring. The reaction mixture thickened and was stirred overnight. This suspension of the Na salt of the product was filtered and washed with MeOH. The salt was suspended in H2O (8 mL) and acidified with cc. HOI. The obtained white solid was filtered and washed with H2O. After drying, the title compound was obtained as an off-white solid, which could be recrystallized from boiling MeOH to increase purity. 1 .85 g of a white solid was obtained.
HRMS (ESI): m/z calcd for CI6HI 1 N3O3S (M + H)+, 326.0594, found 326.0595.
1H NMR (600 MHz, DMSO) 5 13.32 - 12.91 (m, 1 H), 8.75 (d, J = 5.4 Hz, 1 H), 8.53 (s, 1 H), 8.37 (t, J = 7.8 Hz, 1 H), 8.30 (d, J = 8.0 Hz, 1 H), 8.21 (d, J = 7.9 Hz, 2H), 8.08 (d, J = 8.0 Hz, 2H), 7.73 (t, J = 6.5 Hz, 1 H), 3.16 (s, 1 H). 13C NMR (151 MHz, DMSO) 5 166.55, 165.05, 159.35, 144.93, 135.38, 134.31 , 129.44, 129.38, 128.73, 128.58, 128.53, 124.52, 122.55, 116.83.
Example 49: N1-(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexyl)-N4-(4-(pyridin-2- yl)thiazol-2-yl)terephthalamide
Figure imgf000079_0001
The title compound was prepared according to general procedure B from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (20.3 mg, 62.6 pimol), PyBOP (47.9 mg, 92.1 pimol), iP^NEt (100 piL), and 4-((6- aminohexyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1 , 3-dione (TFA salt, 34.6 mg, 57.6 pimol) in anhydrous DMF (1.5 mL). The crude reaction product was subjected to flash chromatography (SiO2, CH2CI2 1 MeOH (0-15%)). The desired compound was obtained as a yellow film (4.3 mg, 6.3 pimol, 11 %).
HRMS (ESI): m/z calcd for C35H33N7O6S (M + H)+, 680.2286, found 680.2276.
Example 50: N1-(5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)pentyl)-N4-(4-(pyridin-2- yl)thiazol-2-yl)terephthalamide
Figure imgf000080_0001
The title compound was prepared according to general procedure B from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (25.5 mg, 79 pimol), PyBOP (53.4 mg, 103 pimol), IP^NEt (200 piL), and 4-((5- aminopentyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1, 3-dione (TFA salt, 42.3 mg, 76.5 pimol) in anhydrous DMF (2.0 mL). The crude reaction product was subjected to flash chromatography (SiO2, CH2CI2 1 MeOH (0-15%)). The desired compound was obtained as a yellow solid (17.6 mg, 26.4 pimol, 33%).
HRMS (ESI): m/z calcd for C34H31N7O6S (M + H)+ 666.2129, found 666.2130.
Example 51: N1-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethyl)-N4-(4-
(pyridin-2-yl)thiazol-2-yl)terephthalamide
Figure imgf000080_0002
The title compound was prepared according to general procedure A from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (32.8 mg, 101 pimol), HATU (45.7 mg, 120 pimol), IP^NEt (200 piL), and 4-((2-(2- aminoethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1, 3-dione (TFA salt, 61.8 mg, 105 pimol) in anhydrous DMF (2.5 mL). The crude reaction product was subjected to flash chromatography (SIO2, CH2CI21 MeOH
(0-15%)). The desired compound was obtained as a yellow solid glass (12.4 mg, 18.6 pimol, 18%).
HRMS (ESI): m/z calcd for C33H29N7O7S (M + H)+ 668.1922, found 668.1899.
Example 52: N1-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethyl)-N4-
(4-(pyridin-2-yl)thiazol-2-yl)terephthalamide
Figure imgf000080_0003
The title compound was prepared according to general procedure A from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (28.6 mg, 88 pimol), HATU (39.3 mg, 103 pimol), IP^NEt (200 piL), and 4-((2-(2-(2- aminoethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1, 3-dione (TFA salt, 55.3 mg, 87.4 pimol) in anhydrous DMF (1 .5 mL). The crude reaction product was subjected to flash chromatography (SIO2, CH2CI21 MeOH (0-15%)). Yellow solid glass (12.6 mg, 17.7 mol, 20%).
HRMS (ESI): m/z calcd for C35H33N7O8S (M + H)+, 712.2184, found 712.2175.
Example 53: N1-(2-(2-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)- 3,3-dimethyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)ethyl)-N4-(4-(pyridin-2-yl)thiazol-2-yl)terephthalamide
Figure imgf000081_0001
The title compound was prepared according to general procedure A from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (28.9 mg, 89 pimol), HATU (45.0 mg, 118 pimol), IP^NEt (100 piL), and VHL ligand 1- PEGI-NH2 (HCI salt, 39.8 mg, 70 pimol) in anhydrous DMF (2.0 mL).The crude reaction product was subjected to flash chromatography (SIO2, CH2CI2 1 MeOH (0-15%)). A tan amorphous solid was obtained (25.9 mg, 30.9 pimol, 35%).
HRMS (ESI): m/z calcd for C42H46N8O7S2 (M + H)+, 839.3004, found 839.2990.
Example 54: N1-(7-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)- 3,3-dimethyl-1-oxobutan-2-yl)amino)-7-oxoheptyl)-N4-(4-(pyridin-2-yl)thiazol-2-yl)terephthalamide
Figure imgf000081_0002
The title compound was prepared according to general procedure A from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (29.7 mg, 91.7 pimol), HATU (46.2 mg, 122 pimol), IP^NEt (200 piL), and VHL ligand 1- C6-NH2 (HCI salt, 42.6 mg, 71.7 pimol) in anhydrous DMF (2.0 mL). The crude reaction product was subjected to flash chromatography (SIO2, CH2CI21 MeOH (0-20%)). A yellowish solid foam was obtained (22 mg, 25.4 pimol, 28%). HRMS (ESI): m/z calcd for C45H52N8O6S2 (M + H)+, calc 865.3524, found 865.3521.
Example 55: N1-((S)-16-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1- carbonyl)-17,17-dimethyl-14-oxo-3,6,9,12-tetraoxa-15-azaoctadecyl)-N4-(4-(pyridin-2-yl)thiazol-2- yl)terephthalamide
Figure imgf000082_0001
The title compound was prepared according to general procedure A from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (28.2 mg, 88 pimol), HATU (47.4 mg, 124.6 pimol), IP^NEt (200 piL), and VHL ligand 1- PEG4-NH2 HCI (44.1 mg, 72 pimol) in anhydrous DMF (2.0 mL). The crude reaction product was subjected to flash chromatography (SIO2, EtOAc / MeOH (0-25%)). A light tan glass was obtained (15.0 mg, 15.4 pimol, 17.5%). HRMS (ESI): m/z calcd for C48H58N8O10S2 (M + H)+, 971.3790, found 971.3790.
Example 56: N1-(3-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)- 3,3-dimethyl-1-oxobutan-2-yl)amino)-3-oxopropyl)-N4-(4-(pyridin-2-yl)thiazol-2-yl)terephthalamide
Figure imgf000082_0002
The title compound was prepared according to general procedure A from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (30.0 mg, 92.6 pimol), HATU (47.9 mg, 126 pimol), IP^NEt (250 piL), and (2S,4R)-1-((S)- 2-(3-aminopropanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2- carboxamide (34.1 mg, 63.4 pimol) in anhydrous DMF (2.0 mL). The crude reaction product was subjected to flash chromatography (SiO2, CH2CI21 MeOH (0-25%)). A yellowish solid glass was obtained (16.2 mg, 20 pimol, 22%). HRMS (ESI): m/z calcd for C41H44N8O6S2 (M + H)+, 809.2898, found 809.2888.
Example 57: N1-(2-(2-(2-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1- yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)ethoxy)ethyl)-N4-(4-(pyridin-2-yl)thiazol-2- yl)terephthalamide
Figure imgf000083_0001
The title compound was prepared according to general procedure A from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (24.4 mg, 75 pimol), HATU (48.4 mg, 127.3 pimol), IP^NEt (200 piL), and (2S,4R)-1-((S)- 2-(2-(2-(2-aminoethoxy)ethoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5- yl)benzyl)pyrrolidine-2-carboxamide HCI (38.2 mg, 62 pimol) in anhydrous DMF (2.0 mL). The crude reaction product was subjected to flash chromatography (SiO2, CH2CI2 1 MeOH (0-25%)). A light tan gum was obtained (31 mg, 35 pimol, 47%).
HRMS (ESI): m/z calcd for C44H50N8O8S2 (M + H)+, 883.3266, found 883.3260.
Example 58: N1-((S)-13-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1- carbonyl)-14,14-dimethyl-11-oxo-3,6,9-trioxa-12-azapentadecyl)-N4-(4-(pyridin-2-yl)thiazol-2- yl)terephthalamide
Figure imgf000083_0002
The title compound was prepared according to general procedure A from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (29.3 mg, 90 pimol), HATU (46.7 mg, 122.8 pimol), IP^NEt (250 piL), and (2S,4R)-1-((S)- 14-amino-2-(tert-butyl)-4-oxo-6,9,12-trioxa-3-azatetradecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5- yl)benzyl)pyrrolidine-2-carboxamide (41.2 mg, 63 pimol) in anhydrous DMF (2.0 mL). The crude reaction product was subjected to flash chromatography (SIO2, CH2CI2 1 MeOH (0-25%)). A yellowish glass was obtained (7.9 mg, 8.5 pimol, 13.5%).
Example 59: N1-(8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)octyl)-N4-(4-(pyridin-2- yl)thiazol-2-yl)terephthalamide
Figure imgf000084_0001
The title compound was prepared according to general procedure A from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (17.3 mg, 53 pimol), HATU (31.7 mg, 83.4 pimol), IP^NEt (200 piL), and 4-((8- aminooctyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1 , 3-dione (TFA salt, 26.9 mg, 40.2 pimol) in anhydrous DMF (2.0 mL). The crude reaction product was subjected to flash chromatography (SIO2, CH2CI2 1 MeOH (0-25%)). Yellow solid glass (22 mg, 31 pimol, 77%).
HRMS (ESI): m/z calcd for C37H37N7O6S (M + H)+, 708.2599, found 708.2612.
Example 60: N1-(9-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)- 3,3-dimethyl-1-oxobutan-2-yl)amino)-9-oxononyl)-N4-(4-(pyridin-2-yl)thiazol-2-yl)terephthalamide
Figure imgf000084_0002
The title compound was prepared according to general procedure A from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (29.5 mg, 91 pimol), HATU (41.4 mg, 108 pimol), IP^NEt (200 piL), and (2S,4R)-1-((S)-2- (9-aminononanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (43.1 mg, 69 pimol) in anhydrous DMF (2.0 mL). The crude reaction product was subjected to flash chromatography (SiO2, EtOAc / MeOH (0-30%)). A yellowish glass was obtained (13.5 mg, 15 pimol, 22%).
HRMS (ESI): m/z calcd for C47H56N8O6S2 (M + H)+, 893.3837, found 893.3862.
Example 61 : N1-(2-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4- yl)amino)ethoxy)ethoxy)ethoxy)ethyl)-N4-(4-(pyridin-2-yl)thiazol-2-yl)terephthalamide
Figure imgf000084_0003
The title compound was prepared according to general procedure A from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (26.8 mg, 82.7 pimol), HATU (45.1 mg, 118.6 pimol), IP^NEt (200 piL), and 4-((2-(2-(2-(2- aminoethoxy)ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1 , 3-dione TFA salt (50.5 mg, 74.7 pimol) in anhydrous DMF (2.0 mL). The crude reaction product was subjected to flash chromatography (SIO2, CH2CI2 I MeOH (0-25%)). Yellow solid glass (22 mg, 29 pmol, 39%).
HRMS (ESI): m/z calcd for C37H37N7O9S (M + H)+, 756.2446, found 756.2441.
Example 62: tert-butyl (2-(4-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)benzamido)ethyl)carbamate
Figure imgf000085_0001
The title compound was prepared according to the general procedure B from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (0.716 g, 2.137 mmol), PyBOP (1.973 g, 3.79 mmol), iP^NEt (2.0 mL), N-Boc-1 ,2- ethylenediamine (0.375 g, 2.34 mmol) in anhydrous DMF (10 mL). The crude product was taken up in acetone and spontaneously crystallized. The desired compound was obtained as a white solid (0.665 g, 1 .42 mmol, 67%).
1H NMR (600 MHz, DMSO) 5 12.94 (s, 1 H), 8.85 - 8.43 (m, 2H), 8.21 (d, J = 7.9 Hz, 2H), 8.15 - 7.78 (m, 5H), 7.35 (dd, J = 7.4, 4.8 Hz, 1 H), 6.94 (t, J = 5.8 Hz, 1 H), 3.31 (d, J = 6.4 Hz, 2H), 3.14 (q, J = 6.4 Hz, 2H), 1.38 (s, 9H).
13C NMR (151 MHz, DMSO) 5 165.55, 164.73, 158.65, 155.74, 152.03, 149.55, 149.42, 137.99, 137.30, 134.02, 128.17, 127.39, 122.89, 120.07, 112.30, 77.68, 39.70, 28.22, 28.08.
Examples 63 to 87: commercially available compounds (see above)
Example 88: tert-butyl 4-(4-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)benzoyl)piperazine-1-carboxylate
Figure imgf000085_0002
The title compound was prepared according to the general procedure B from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (0.132 g, 0.405 mmol), PyBOP (0.4028 g, 0.636 mmol), iP^NEt (0.25 mL), Boc-piperazine (0.106 g, 0.569 mmol) in anhydrous DMF (2.5 mL). The crude reaction product was subjected to column chromatography Si O2, CH2CI21 MeOH (0-20%). The desired compound was obtained as a white solid (0. 108 g, 0.219 mmol, 55%).
HRMS (ESI): m/z calcd for C25H27N5O4S (M + H)+, 494.1857, found 494.1859.
1H NMR (600 MHz, DMSO) 5 12.91 (s, 1 H), 8.71 - 8.51 (m, 1 H), 8.19 (d, J = 8.0 Hz, 2H), 8.03 (d, J = 7.9 Hz, 1 H), 7.95 - 7.86 (m, 2H), 7.58 (d, J = 8.0 Hz, 2H), 7.39 - 7.31 (m, 1 H), 3.62 (s, 2H), 3.43 (s, 2H), 3.33 (d, J = 32.1 Hz, 4H), 1.41 (s, 9H).
Example 88B: tert-butyl (3-(4-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)benzamido)propyl)carbamate
Figure imgf000086_0001
The title compound was prepared according to general procedure B from 4-((4-(pyridin-2-yl)thiazol-2- yl)carbamoyl)benzoic acid (0.119 g, 0.365 mmol), PyBOP (0.331 g, 0.636 mmol), IP^NEt (0.25 mL), tert-butyl-3- aminopropyl carbamate (0.143 g, 0.821 mmol) in anhydrous DMF (3 mL). Column chromatography of the crude product (SIO2, hexanes/EtOAc (0-100%)) afforded the title compound as white solid (0.048 g, 102 pimol, 28%).
Example 89: tert-butyl (4-(4-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)benzamido)butyl)carbamate
Figure imgf000086_0002
The title compound was prepared according to the general procedure B for amide couplings from 4-((4-(pyridin-2- yl)thiazol-2-yl)carbamoyl)benzoic acid (0.1088 g, 0.334 mmol), PyBOP (0.310 g, 0.596 mmol), IP^NEt (0.37 mL), t- butyl 4-aminobutyl carbamate (0.102 g, 0.54 mmol) in anhydrous DMF (3 mL).The crude reaction product was subjected to column chromatography (SIO2, CH2CI21 MeOH (0-5%)). The desired compound was obtained as white solid (0.0256 g, 0.052 mmol, 16%).
HRMS (ESI): m/z calcd for C25H29N5O4S (M + H)+, 496.2013, found 496.2016.
Example 90: tert-butyl (5-(4-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)benzamido)pentyl)carbamate
Figure imgf000086_0003
The title compound was prepared according to the general procedure A for HATU couplings from 4-((4-(py ridin-2- yl)thiazol-2-yl)carbamoyl)benzoic acid (0.095 g, 0.291 mmol), HATU (0.158 g, 0.416 mmol), IP^NEt (0.25 mL), t-butyl 5-aminopentyl carbamate (0.074 g, 0.37 mmol) in anhydrous DMF (2.0 mL).The crude reaction product was washed with acetone to obtain the pure product as an off-white powder (71 mg, 0.139 mmol, 48%).
HRMS (ESI): m/z calcd for C26H31N5O4S (M + H)+, 510.2170, found 510.2173.
1H NMR (700 MHz, DMSO) 5 12.93 (s, 1 H), 8.65 - 8.60 (m, 2H), 8.22 - 8.19 (m, 2H), 8.03 (dt, J = 7.9, 1 . 1 Hz, 1 H), 8.01 - 7.97 (m, 2H), 7.93 - 7.88 (m, 2H), 7.35 (ddd, J = 7.4, 4.7, 1.2 Hz, 1 H), 6.77 (t, J = 5.6 Hz, 1 H), 3.27 (q, J = 6.6 Hz, 2H), 2.92 (q, J = 6.7 Hz, 2H), 1.54 (p, J = 7.3 Hz, 2H), 1.41 (p, J = 7.3 Hz, 2H), 1.36 (s, 9H), 1.29 (m, 2H). 13C NMR (176 MHz, DMSO) 5 206.45, 165.24, 164.74, 158.65, 155.57, 152.04, 149.55, 149.42, 138.12, 137.30, 133.94, 128.19, 127.32, 122.88, 120.07, 112.29, 77.28, 29.20, 28.71, 28.25, 23.77.
Example 91 : tert-butyl (2-(2-(4-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)benzamido)ethoxy)ethyl)carbamate
Figure imgf000087_0001
The title compound was prepared according to the general procedure A for HATU couplings from 4-((4-(py ridin-2- yl)thiazol-2-yl)carbamoyl)benzoic acid (0.095 g, 0.292 mmol), HATU (0.143 g, 0.376 mmol), iP^NEt (0.25 mL), Boc- PEG1 -diamine (0.084 g, 0.411 mmol) in anhydrous DMF (2.5 mL).The crude reaction product was subjected to column chromatography (SiO2, CH2CI2 1 MeOH (0-5%) twice to obtain the title compound as a yellowish oil that set up to a solid (23.7 mg).
HRMS (ESI): m/z calcd for C25H29N5O5S (M + H)+, 512.1962, found 512.1966.
Example 92: tert-butyl (4-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)benzoyl)glycinate
Figure imgf000087_0002
The title compound was prepared according to the general procedure A for HATU couplings from 4-((4-(py ridin-2- yl)thiazol-2-yl)carbamoyl)benzoic acid (0.095 g, 0.292 mmol), HATU (0.145 g, 0.381 mmol), iP^NEt (0.20 mL), glycine t-butyl ester HCI (0.0696 g, 0.415 mmol) in anhydrous DMF (2.5 mL). The reaction mixture was stirred at ambient temperature overnight, diluted with brine and extracted with EtOAc (3 x 15 mL). The combined organic solutions were washed with water and brine, dried on Na2SO4 and evaporated; the crude material crystallized and was suspended in acetone and filtered to afford the title compound as 68.2 mg off-white solid (0.155 mmol, 53%). HRMS (ESI): m/z calcd for C22H22N4O4S (M + H)+, 439.1435, found 439.1437.
1H NMR (600 MHz, DMSO) 5 12.93 (s, 1 H), 9.06 (t, J = 5.9 Hz, 1 H), 8.62 (d, J = 4.8 Hz, 1 H), 8.22 (d, J = 8.0 Hz, 2H), 8.02 (t, J = 8.4 Hz, 3H), 7.97 - 7.86 (m, 2H), 7.35 (dd, J = 7.4, 4.7 Hz, 1 H), 3.94 (d, J = 5.9 Hz, 2H), 1.43 (s, 9H).
13C NMR (151 MHz, DMSO) 5 168.84, 165.79, 164.77, 158.66, 152.03, 149.55, 149.42, 137.32, 137.18, 134.43, 128.35, 127.41 , 122.90, 120.08, 112.30, 80.75, 45.96, 41.95, 38.23, 35.77, 30.76, 27.73.
Example 93: 4-(piperazine-1-carbonyl)-N-(4-(pyridin-2-yl)thiazol-2-yl)benzamide
Figure imgf000088_0001
Tert-butyl 4-(4-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)benzoyl)piperazine-1-carboxylate (53 mg) was suspended in EtOAc (4 mL) and treated with 4M HCI in dioxane (1.5 mL). Stirring was continued for 16 h, at which point all the material had been converted according to TLC. The reaction mixture was evaporated to dryness to afford the HCI salt of the title compound.
HRMS (ESI): m/z calcd for C20H19N5O2S (M + H)+, 394.1329 found 394.1335.
1H NMR (600 MHz, DMSO) 5 12.97 (s, 1 H), 9.11 (s, 2H), 8.71 - 8.59 (m, 1 H), 8.27 - 8.17 (m, 2H), 8.07 (d, J = 7.9 Hz, 1 H), 7.97 (d, J = 6.2 Hz, 2H), 7.65 (d, J = 8.2 Hz, 2H), 7.40 (dd, J = 7.4, 4.8 Hz, 1 H), 3.84 (s, 4H), 3.56 (s, 4H). 13C NMR (151 MHz, DMSO) 5 168.75, 165.18, 159.24, 151.93, 149.44, 149.17, 139.22, 138.50, 133.45, 128.91, 127.75, 123.57, 120.85, 113.27, 66.78, 42.99.
Example 94: 6-methyl-N-(4-(pyridin-2-yl)thiazol-2-yl)-2,3-dihydrobenzo[b]thiophene-2-carboxamide
Figure imgf000088_0002
The title compound was prepared according to the general procedure A for HATU coupling from 6-methyl-2,3-dihydro- 1-benzothiophene-2-carboxylic acid (31.7 mg, 0.163 mmol), HATU (86.2 mg, 0.227 mmol), iP^NEt (0.150 mL), and 4-(pyridin-2-yl)thiazol-2-amine (HBr salt, 37.2 mg, 0.144 mmol). The reaction mixture was stirred overnight at ambient temperature and was then subjected to EtOAc I H2O workup. The crude product was purified by column chromatography (hexane - EtOAc gradient 0-60%) affording the title compound as a light tan solid (48.3 mg, 0.137 mmol, 95%).
MS (ESI): m/z calcd for C18H15N3OS2 (M + H)+, 354.07, found 354.08.
1H NMR (700 MHz, DMSO) 5 12.52 (s, 1 H), 8.60 (ddd, J = 4.8, 1.9, 1.0 Hz, 1 H), 7.94 (dt, J = 7.9, 1.2 Hz, 1 H), 7.89 (td, J = 7.7, 1 .8 Hz, 1 H), 7.84 (s, 1 H), 7.33 (ddd, J = 7.5, 4.7, 1 .3 Hz, 1 H), 7.14 (d, J = 7.7 Hz, 1 H), 7.01 (s, 1 H), 6.88 - 6.84 (m, 1 H), 4.69 (dd, J = 8.6, 3.5 Hz, 1 H), 3.62 (dd, J = 16.1 , 3.5 Hz, 1 H), 3.42 (dd, J = 16.0, 8.6 Hz, 1 H), 2.23 (s, 3H).
13C NMR (176 MHz, DMSO) 5 170.24, 158.11, 151.92, 149.53, 149.16, 138.63, 137.31 , 136.74, 136.34, 125.41, 124.24, 122.88, 121.75, 119.94, 112.02, 48.28, 36.57, 20.63.
Example 95: Methyl 2-amino-4-(pyridin-2-yl)thiazole-5-carboxylate
Figure imgf000088_0003
Methyl 3-oxo-3-(pyridine-2-yl) propanoate (0.57 g, 3.18 mmol), thiourea (0.541 g, 7.12 mmol), and I2 (0.847 g, 3.34 mmol) were refluxed in EtOH (5 mL) for 18 h. After cooling, the yellow precipitate of product was filtered and washed with cold EtOH. The compound was obtained as 0.709 g of yellow solid, 95%.
Example 96: tert-butyl (6-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)pyridin-3-yl)carbamate
Figure imgf000089_0001
5-((Tert-butoxycarbonyl)amino)picolinic acid (1.081 g, 4.54 mmol) was dissolved in anhydrous DMF (20 mL), and treated with iP^NEt (4.0 mL) and PyBOP (2.895 g, 5.56 mmol). The mixture was stirred for 20 min to achieve activation of the carboxylic acid. Then 4-(pyridin-2-yl)thiazol-2-amine was added (1.172 g, 4.54 mmol, as the HBr salt) and the reaction mixture was stirred overnight at ambient temperature. The solution was then diluted with brine and extracted with EtOAc 4x. The extracts were washed with H2O and brine then dried on Na2SO4. The organic solution was evaporated and the solid residue was washed with acetone affording the title compound as a white solid. The filtrate was evaporated and the residue was subjected to column chromatography affording an additional quantity of product. The total yield was 0.74 g, 1.86 mmol, 41 %.
HRMS (ESI): m/z calcd for C19H19N3O5S (M + H)+, 398.1281 , found 398.1291.
1H NMR (600 MHz, DMSO) 5 11.85 (s, 1 H), 10.08 (s, 1 H), 8.81 - 8.77 (m, 1 H), 8.60 (ddd, J = 4.7, 1.8, 0.9 Hz, 1 H), 8.17 - 8.10 (m, 2H), 8.02 (dt, J = 7.9, 1.1 Hz, 1 H), 7.93 - 7.86 (m, 2H), 7.33 (ddd, J = 7.5, 4.7, 1.2 Hz, 1 H), 1.51 (s, 9H).
13C NMR (151 MHz, DMSO) 5 162.47, 157.61 , 152.50, 151.92, 149.47, 149.41 , 140.93, 139.86, 138.51 , 137.25, 125.06, 123.66, 122.89, 120.21 , 112.26, 80.42, 45.83, 45.80, 27.94.
Example 97: 5-amino-N-(4-(pyridin-2-yl)thiazol-2-yl)picolinamide
Figure imgf000089_0002
The Boc protected compound of Example 96 (0.50 g) was dissolved in EtOAc (10 mL) and MeOH (3 mL) and treated with 4M HOI in dioxane (3 mL). The reaction mixture was stirred at ambient temperature for 24 h and evaporated to dryness. The residual white solid was used without further purification.
HRMS (ESI): m/z calcd for C14H11 N5O3S (M + H)+, 298.0757, found 298.0758.
Example 98: Methyl 2-(4-(N,N-dimethylsulfamoyl)benzamido)-4-(pyridin-2-yl)thiazole-5-carboxylate
Figure imgf000089_0003
The title compound was prepared according to the general procedure B for PyBOP coupling from 4- dimethylsulfamoylbenzoic acid (58.7 mg, 0.256 mmol), PyBOP (0.1667 g, 0.320 mmol), and Example 95 (45.4 mg, 0.193 mmol) in a mixture of DMF (2.5 mL) and iP^NEt (0.35 mL). After a reaction time of 12 h, the crude product was extracted with EtOAc and subjected to chromatographic purification (hexanes - EtOAc 0-100%) affording the desired product as a white solid (24.8 mg, 55.5 pimol, 29%).
HRMS (ESI): m/z calcd for C19H18N4O5S2 (M + H)+, found 447.0793, calc 447.0791.
Example 99 3-hydroxy-N-(4-(pyridin-2-yl)thiazol-2-yl)benzamide
Figure imgf000090_0001
The general procedure B for PyBOP coupling was followed using 3-hydroxybenzoic acid (0.4905 g, 3.55 mmol), PyBOP (2.301 g, 4.42 mmol), iP^NEt (5.0 mL), 4-(pyridin-2-yl)thiazol-2-amine (as HBr salt, 1.042 g, 4.04 mmol) in anhydrous DMF (20 mL). The reaction mixture was stirred overnight, then it was quenched with brine and extracted with EtOAc (3 x 70 mL). The combined extracts were washed with H2O and brine, dried on Na2SO4 and evaporated. The obtained red oil was purified by column chromatography (hexane - EtOAc 0-80%) to afford the title compound as a dull yellow solid (0.42 g, 1.41 mmol, 40%).
HRMS (ESI): m/z calcd for C15H11 N3O2S (M + Na)+, 320.0464, found 320.0463.
Example 100: tert-butyl (2-(4-((5-ethyl-4-(pyridin-2-yl)thiazol-2 yl)carbamoyl)benzamido)ethyl)carbamate
Figure imgf000090_0002
The title compound was prepared according to the general procedure B for PyBOP coupling from 4-((2-((tert- butoxycarbonyl)amino)ethyl)carbamoyl)benzoic acid (77 mg, 0.250 mmol), 5-ethyl-4-(pyridin-2-yl)thiazol-2-amine (49 mg, 0.239 mmol) and PyBOP (0.2462 g, 0.473 mmol) in a mixture of iP^NEt (0.50 mL) and anhydrous DMF (2.5 mL). The reaction mixture was stirred for 24 h at ambient temperature, then it was partitioned between EtOAc and brine. The aqueous solution was extracted with two additional portions of EtOAc and the combined organic extracts were washed with H2O and brine, dried on Na2SO4 and evaporated. Purification by column chromatography (SiO2, hexanes - EtOAc 0-100%) afforded the title compound as a reddish solid (14.1 mg, 0.028 mmol, 12%).
HRMS (ESI): m/z calcd for C25H29N5O4S (M + H)+, 496.2013, found 496.2013.
Example 101 : 5-pivalamido-N-(4-(pyridin-2-yl)thiazol-2-yl)picolinamide
Figure imgf000090_0003
To a solution of 5-amino-N-(4-(pyridin-2-yl)thiazol-2-yl)picolinamide HCI (101.2 mg, 0.273 mmol) in CH2CI2 (2.0 mL) and IP^NEt (0.3 mL) held at 0°C pivaloyl chloride (39 mg, 0.325 mmol) in CH2CI2 (0.5 mL) was added slowly and the reaction mixture was stirred for 3 h, allowing the temperature to return to ambient temperature after 0.5 h. The solution was then evaporated and the crude product was purified by column chromatography (SIO2, hexanes-EtOAc 0-70%) to 34 mg of white solid.
HRMS (ESI): m/z calcd for C19H19N5O2S (M + H)+, 382.1332, found 382.1326.
Example 102: 5-(3,3-dimethylbutanamido)-N-(4-(pyridin-2-yl)thiazol-2-yl)picolinamide
Figure imgf000091_0001
5-Ami no- N- (4-(py r idin-2-y l)th I azol-2-y I) picolin amide HCI (29 mg, 78 pimol) was suspended in a mixture of CH2CI2 (1.0 mL) and EtsN (0.1 mL), then it was cooled to 0°C under Ar and treated slowly with a solution of 2,2-dimethylbutanoyl chloride (17.5 mg) in CH2CI2 (0.5 mL). The reaction mixture was stirred at 0°C for 30 min then at ambient temperature for 4 h. The clear solution was then washed with water, evaporated and the residue was subjected to chromatographic purification (hexanes-EtOAc 0-100%), affording the product as an off-white solid (11.5 mg, 25 pimol, 32%).
HRMS (ESI): m/z calcd for C26H26N4O2S (M + H)+, 459.1849, found 459.1848.
Example 103: N-(4-(pyridin-2-yl)thiazol-2-yl)-5-(2,2,2-trifluoroacetamido)picolinamide
Figure imgf000091_0002
5-Amino-N-(4-(pyridin-2-yl)thiazol-2-yl)picolinamide HCI (0.0353 g, 0.095 mmol) was suspended in CH2CI2 (1.5 mL) and EtaN (0.10 mL). Trifluoroacetic anhydride was added (0.030 g, 0.142 mmol) and the solution was stirred at ambient temperature for 7 h. It was then diluted with an additional 5 mL of CH2CI2, washed with H2O (2 x 3 mL), dried on Na2SO4 and evaporated. Purification of the crude material by column chromatography (SIO2, hexane - EtOAc gradient 0-90%) afforded an off-white solid (12 mg, 30.5 pimol, 32%).
HRMS (ESI): m/z calcd for C16H10F3N5O2S (M + H)+, 394.0577, found 394.0574.
Example 104: N1-(adamantan-1-yl)-N4-(4-(pyridin-2-yl)thiazol-2-yl)terephthalamide
Figure imgf000091_0003
The title compound was prepared according to the general procedure A for HATU coupling from 4-((4-(pyridin-2- yl)thiazol-2-yl)carbamoyl)benzoic acid (87.2 mg, 0.268 mmol), HATU (148 mg, 0.389 mmol) and adamantaneamine HCI (60 mg, 0.320 mmol) in anhydrous DMF (2.0 mL) and iP^NEt (0.25 mL). The crude product was isolated by EtOAc I H2O workup and purification by column chromatography (SiO2, hexane-EtOAc 0-100%). White solid, 45 mg. HRMS (ESI): m/z calcd for C26H26N4O2S (M + H)+, 459.1849, found 459.1848.
Example 105: N1-(2-(adamantan-1-ylamino)-2-oxoethyl)-N4-(4-(pyridin-2-yl)thiazol-2-yl)terephthalamide
Figure imgf000092_0001
The title compound was prepared according to the general procedure A for HATU coupling from (4-((4-(pyridin-2- yl)thiazol-2-yl)carbamoyl)benzoyl)glycine (93 mg, 0.243 mmol), HATU (0.200 g, 0.526 mmol), iP^NEt (0.20 mL) and adamantaneamine HCI (80 mg, 0.426 mmol) in anhydrous DMF (2.0 mL). Purification of the crude product by trituration with MeOH I acetone afforded the title compound as a yellowish solid (6.0 mg, 11 .6 pi mol, 5%).
HRMS (ESI): m/z calcd for C28H29N5O3S (M + H)+, 516.2064, found 516.2067.
Example 106: 3-(2-(adamantan-1-ylamino)-2-oxoethoxy)-5-(4-methylpiperazine-1-carbonyl)-N-(4-(pyridin-2- yl)thiazol-2-yl)benzamide
Figure imgf000092_0002
2-(3-(4-Methylpiperazine-1-carbonyl)-5-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)phenoxy)acetic acid (88.9 mg, 0.185 mmol) was dissolved in anhydrous DMF (1.5 mL) and iP^NEt (250 piL) then treated with HATU as a solid (102.5 mg, 0.270 mmol) and stirred for 20 min at ambient temperature. Adamantaneamine HCI (35.3 mg, 0.188 mmol) was then added in a single portion as a solid. The reaction mixture was stirred overnight. Brine (10 mL) was then added to the solution and the product was extracted with EtOAc (3 x 10 mL). The pooled extracts were washed with H2O and brine, dried on Na2SO4, evaporated and the crude product was purified by automated column chromatography (SiO2, CH2Cl2/MeOH (0-20%). The product was obtained as a brownish gum (15 mg, 0.024 mmol, 13%).
HRMS (ESI): m/z calcd for C33H38N6O4S (M + H)+, 615.2748, found 615.2741.
Example 107: tert-butyl 4-(4-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)phenyl)piperazine-1-carboxylate
Figure imgf000092_0003
4-(4-(Tert-butoxycarbonyl)piperazin-1 -yl)benzoic acid (1.765 g, 5.76 mmol) was dissolved in anhydrous DMF (20 mL) and IP^NEt (1.0 mL), then it was treated with COMU (2.794 g, 6.52 mmol) as a solid. The solution soon turned orange-red and was stirred for 10 min. 4-(pyridin-2-yl)thiazol-2-amine HBr salt (1.590 g, 6.16 mmol) was added and the reaction mixture was stirred at ambient temperature for 24 h. The reaction mixture was then diluted with brine and extracted with EtOAc. The pooled extracts were washed with brine and evaporated. The crude material was recrystallized from MeOH, and the filtrate was evaporated then purified by flash chromatography (SIO2, hexanes - EtOAc 0-100%). The fractions containing product by MS (amber oil, 115 mg) were eluted again (SIO2, hexanes - EtOAc gradient) to afford 63 mg of a gum (13.5 pimol, 0.2%).
HRMS (ESI): m/z calcd for C24H27N5O3S (M + Na)+, 488.17, found 488.22.
Example 108: 3-(4-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)benzamido)propanoic acid
Figure imgf000093_0001
Example 109 (tert-butyl 3-(4-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)benzamido)propanoate) was deprotected by stirring in 4M HOI I dioxane in EtOAc. After completion of the reaction, the white suspension was evaporated to dryness and the product was dried under high vacuum affording a white solid.
MS (ESI): m/z calcd for C19H16N4O4S (M + H)+, 395.08, found 394.98.
Example 109: tert-butyl 3-(4-((4-(pyridin-2-yl)thiazol-2-yl)carbamoyl)benzamido)propanoate
Figure imgf000093_0002
The title compound was prepared according to the general procedure A for HATU coupling from 4-((4-(pyridin-2- yl)thiazol-2-yl)carbamoyl)benzoic acid (226.8 mg, 0.697 mmol), HATU (436.9 mg, 1.15 mmol) and p-alanine-tert-butyl ester HOI (127.9 mg, 0.704 mmol) in a mixture of IP^NEt (0.7 mL) and anhydrous DMF (5 mL). The reaction was carried out over 18 h at ambient temperature under N2. EtOAc I H2O workup was used to isolate the crude product which crystallized. The substance could be recrystallized from MeOH - acetone (190 mg nearly white solid, 0.386 mmol, 55%).
MS (ESI): m/z calcd for C23H24N4O4S (M + H)+, 453.16, found 453.16.
1H NMR (600 MHz, DMSO) 5 12.94 (s, 1 H), 8.74 (t, J = 5.5 Hz, 1 H), 8.63 (ddd, J = 4.8, 1.9, 0.9 Hz, 1 H), 8.25 - 8.18 (m, 2H), 8.04 (dt, J = 7.9, 1 . 1 Hz, 1 H), 8.00 - 7.95 (m, 2H), 7.95 - 7.89 (m, 2H), 7.36 (ddd, J = 7.5, 4.7, 1.2 Hz, 1 H), 3.50 (td, J = 7.0, 5.4 Hz, 2H), 2.52 (m, 2H), 1.41 (s, 9H).
13C NMR (151 MHz, DMSO) 5 170.56, 170.54, 165.39, 164.74, 158.64, 152.03, 149.56, 149.42, 137.80, 137.32, 134.12, 128.25, 127.30, 122.90, 120.06, 112.30, 35.71 , 34.88, 27.72.
Example 110: Z45411712, Enamine Example B: Pharmacological perturbation of the phase-separating protein SMNDC1
Methods
Nomenclature
To reduce confusion due to the difference between gene and protein name, the inventors have decided to only use SMNDC1 for both.
AlphaFold
AlphaFold (Jumper, J. etal. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583-589 (2021)) predictions were run via Col ab Fold (Mirdita, M. et al. Col ab Fold : making protein folding accessible to all. Nat Methods 19, 679-682 (2022)) (v1.2.0) with the AlphaFold2 algorithm and the following parameters: msa_method=mmseqs2 homooligomer=1 pair_mode=unpaired cov=0 qid=0 max_msa=512:1024 subsample_msa=T rue num_reiax=0 use_turbo=True use_ptm=True rank_by=pLDDT num_models=5 num_samples=1 num_ensemble=1 max_recycles=3 tol=0 is_training=False use_templates=False
Cell culture
The murine oTC1 cell line was obtained from ATCC (Cat#CRL-2934, RRID:CVCL_B036). Cells were grown in low- glucose DMEM medium (Biowest L0066) supplemented with 10% FBS, 50 U/mL penicillin and 50 pg/mL streptomycin. HAP1 cells (Horizon discovery) were grown in IMDM medium (Sigma I6529) supplemented with 10% FBS, 50 U/mL penicillin and 50 pg/mL streptomycin. The Lenti-X™ 293 T cell line was purchased from Takara Bio (632180). Cells were grown in high-glucose DMEM medium (Sigma D5796) supplemented with 10% FBS, 1 mM sodium pyruvate, 50 U/mL penicillin and 50 pg/mL streptomycin. Intron tagging and live imaging of cells
Cell lines with fluorescent tags in the endogenous intron loci of different genes were generated as described in Serebrenik et al., and Reicher et al. (Serebrenik, Y. V., Sansbury, S. E., Kumar, S. S., Henao-Mejia, J. & Shalem, 0. Efficient and flexible tagging of endogenous genes by homology-independent intron targeting. Genome Res 29, 1322-1328 (2019); Reicher, A., Koren, A. & Kubicek, S. Pooled protein tagging, cellular imaging, and in situ sequencing for monitoring drug action in real time. Genome Res. (2020) doi: 10.1101/gr.261503.120). Cells were transiently transfected using Avalanche - Everyday Transfection Reagent with three plasmids in parallel: (1) the donor plasmid containing the artificial intron with splice acceptor and splice donor site, the fluorescent tag GFP or RFP, and a possible correction for the frame of the targeted intron, (2) a pX330 backbone containing Cas9 and the gRNA against the donor plasmid, and (3) a plasmid expressing the gRNA against the target intron (see table below). After 3-5 days, GFP- and/or RFP-positive cells were sorted on a SONY SH800 Cell Sorter to get fluorescent single cell clones. Clones were validated for the correct integration of the intron-tag via comparison of live cell images to publicly available or in-house IF images, genomic DNA PCR amplification of the respective loci, and western blots with antibodies against the target protein and/or the fluorescent tag.
Cells were imaged on a PerkinElmer Opera Phenix automated microscope with 500 ms exposure time in either GFP or RFP channel, or on a Zeiss LSM 980 microscope. For condition-independent identification nuclear markers such as Hoechst or DRAQ5™ were used.
Figure imgf000095_0001
Immunofluorescence
Cells were fixed in the 96-well imaging plates they were growing in before by adding 37% formaldehyde solution 1 :10 to the culture medium for a final concentration of 3.7%. Cells were incubated with this for 15 min at room temperature (RT). Next, cells were washed once with PBS, followed by a 30 min permeabilization step with PBST (0.2% Tween). Afterwards, cells were blocked with a 3% BSA in PBST solution for 1 h. Primary antibodies (SMNDC1 : Thermo Fisher Scientific Cat#PA5-31148; RRID:AB_2548622, 1 :500; SC35: GeneTex Cat#GTX11826; RRID:AB_372954, 1 :500) in 1 .5% BSA in PBST were added in their individual concentrations and incubated overnight (o/n) at 4°C. On the next day, wells were washed 3x with PBST, before incubation with secondary antibodies (Goat anti-Rabbit IgG Alexa Fluor 546 Thermo Fisher Scientific Cat#A-11010; RRID:AB_2534077, 1 :500; Goat anti-Mouse IgG Alexa Fluor 488 Thermo Fisher Scientific Cat#A-11001; RRID:AB_2534069, 1 :500) and DAPI (5 mg/ml, 1 :2000) for 1-2h. After 3 washing steps with PBST, cells were ready to be imaged. Cells can be stored at 4°C before imaging on the PerkinElmer Opera Phenix automated microscope or on a Zeiss LSM 980 microscope.
Imaging quantifications
Images were analyzed using the high-content image acquisition and analysis software Harmony® 4.9 developed by PerkinElmer. First, nuclei were identified in the channel of the nuclear marker (DAPI/ Hoechst/ DRAQ5™) (with Method C, Common Threshold 0.75, Area > 10 m2). After the identification of nuclei, their corresponding cytoplasm was also identified using the respective nucleic acid marker (with Method A, Individual Threshold 0.15). Even though the highest staining of these nuclear markers is obviously detected in the nucleus they still produce a significant staining of the cytoplasm above background. After defining the respective cell areas, mean intensity in the different channels was measured. Finally, spots were identified with the according "Spots” algorithm (with Method A, Relative Spot Intensity > 0.053, Splitting Sensitivity: 1.0).
Colocalization analysis
Images were preprocessed in Python version 3.7.9. Z-stacks in czi format were loaded with czifile library, version 2019.7.2, and reduced using maximum intensity Z projection. Segmentation of nuclei was carried out with Cellpose (Stringer, C., Wang, T., Michaelos, M. & Pachitariu, M. Cellpose: a generalist algorithm for cellular segmentation. Nat Methods 18, 100-106 (2021)) (version 0.6.1) based on the DAPI channel. Additional segmentation masks (mitotic nuclei only) were created manually. Preprocessed images and segmentation masks were saved in PNG format. CellProfiler (Stirling, D. R. et al. CellProfiler 4: improvements in speed, utility and usability. BMC Bioinformatics 22, 433 (2021)) (4.0.7) was used to extract fluorescence intensity measurements for non-mitotic and mitotic nuclei separately.
All preprocessing code and the CellProfiler pipeline are available at https://github.com/reinisj/colocalization_analysis and under the DOI 10.5281/zenodo.8091256.
In-vitro protein expression
Expression plasmids for SMNDCTs and SMNTs Tudor domain as used in Tripsianes et al., 2011 (Tripsianes, K. et al. Structural basis for dimethylarginine recognition by the Tudor domains of human SMN and SPF30 proteins. Nature Structural & Molecular Biology 18, 1414-1420 (2011)) were a kind gift from Michael Sattler. Protein expression plasmids with a GFP-fusion for droplet assays were generated by ligation independent cloning (Aslanidis, C. & de Jong, P. J. Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res 18, 6069-6074 (1990); Stols, L. et al. A new vector for high-throughput, ligation-independent cloning encoding a tobacco etch virus protease cleavage site. Protein Expr Purif 25, 8-15 (2002)) using pET His6 GFP TEV LIC cloning vector (1 GFP) which was a gift from Scott Gradia (Addgene plasmid # 29663; http://n2t.net/addgene:29663; RRID:Addgene_29663) and amplification of the respective sequences from cDNA.
BL21 (DE3) competent E. coli cells were transformed with the respective plasmids and liquid stocks frozen at -80°C. Volumes described here are for 450 ml total volume bacterial culture but were adjusted according to protein amounts needed. From frozen liquid stocks, 200 ml LB Kanamycin cultures were grown at 30°C overnight, diluted with 250 ml fresh LB and grown until ODeoo reached 0.8-1. Protein expression was induced with 1 mM IPTG and bacteria grown for another 24h at 20°C. Bacteria were harvested by centrifugation at 4000xg for 15min at 4°C. Pellets were washed in 35ml PBS and spun down again at 6000xg for 10 min at 4°C. After removal of supernatant PBS, pellets can be stored at -80°C.
For protein purification, pellets were resuspended in 13 ml Lysis buffer (50 mM TRIS pH 7.7, 500 mM NaCI, 1 % Igepal, 2.5 mg/ml Lysozyme, 0.1 mg/ml DNase I), incubated for at least 15min and sonicated to ensure cell lysis. Afterwards, lysates were spun down again for 20min at 8500xg and 4°C to remove debris pellet. In parallel, 1 ml of Ni-NTA resin (Qiagen) were added to a 15ml tube and centrifuged at 700xg for 2min. Supernatant was removed, and resin washed once by RIPA w/o (= without) EDTA (50 mM TRIS pH 7.7, 500 mM NaCI, 1 % Igepal). Lysate supernatant was then added to equilibrated resin and rotated at 4°C for 3h. Beads were then spun down again at 700xg for 2min, and washed rotating for 10min twice by adding 14 ml RIPA w/o EDTA. Eventually, bound protein was eluted 5x with 1 ml elution buffer (250 mM Imidazole in RIPA w/o EDTA) by rotating at room temperature for 20 min, 25 min, 30 min, 45 min, o/n (= overnight).
Proteins for droplet assays were then purified further by size exclusion chromatography (SEC) on a Superdex increase 200 10/300 GL column with 50mM Tris pH 7.5, 125mM NaCI, 10% glycerol and 1 mM DTT running buffer.
In-vitro droplet assays
In vitro droplet assays were performed as described in Klein, Boija et al. (Klein, I. A. et al. Partitioning of cancer therapeutics in nuclear condensates. Science 368, 1386-1392 (2020)). Recombinant GFP-fusion protein purified by SEC in 50mM Tris pH 7.5, 125mM NaCI, 10% glycerol and 1 mM DTT running buffer was diluted to 10 piM with a concentrated PEG-8000 solution in the same buffer (and additional buffer according to protein concentration) to a final PEG-8000 concentration of 15%. In some of the experiments, total RNA isolated from aTC1 cells (10 ng/pil, Fig. 11, k) or in wfro-transcribed RNA (100 ng/pil, Fig. 1j) was added. 10 l of this solution were loaded onto PerkinElmer PhenoPlate™ 384-well microplates (formerly named CellCarrier Ultra microplates) and imaged immediately on the PerkinElmer Opera Phenix automated microscope with a 63x objective at the bottom of the well.
Fluorescence recovery after photobleachinq (FRAP)
For FRAP experiments, cells harboring intron-tags in SMNDC1 and SRRM2 were seeded 24h before imaging on a Zeiss LSM 980 microscope. 15 min before imaging, medium was changed to medium without phenol red containing DRAQ5™ 1 : 1000 to reduce autofluorescence and to mark nuclei. If cells were treated with compounds, these were added in the same step. After identifying a suitable cell, bleach and reference regions were defined. After taking one reference image, the bleach region was bleached 15 times for 5 milliseconds with 100% laser power at 488 nm for GFP and with 20% laser power at 546 nm for RFP. After bleaching, a new image was taken approximately every 3 seconds until 150 seconds after bleaching. Fluorescence intensities were quantified in the bleach and reference regions for every image and normalized to the intensity before bleaching.
AlphaScreen
Compounds and controls were transferred on PerkinElmer OptiPlate-384 plates using an acoustic liquid handler (Echo, Labcyte). The AlphaScreen was conducted in 20 mM sodium phosphate pH 6.5, 50 mM NaCI, Tween 0.01 %, BSA 0.1 %. Protein concentration was optimized for each batch of purified protein. Optimal concentration was chosen at the lowest concentration with -80% of maximum signal. The biotinylated binding peptide (Sequence: AAR*GR*GR*GMGR*GNIFQKRR, R*=sDMA) (Tripsianes, K. et al. Structural basis for dimethylarginine recognition by the Tudor domains of human SMN and SPF30 proteins. Nature Structural & Molecular Biology 18, 1414-1420 (2011)) was used at 50 nM, and donor and acceptor beads at 5 pig/ml final concentration. In the first step, 10 pil of the protein solution containing either SMNDCTs or SMN's Tudor domain coupled to a 6xHis-Tag were distributed to 384-well plates pre-spotted with compounds and controls, shaken and incubated for 30 min at RT. Afterwards, 10 pl of peptide solution was added to each well and incubated for 1 h at RT. Finally, 5 pl of a solution containing both Streptavidin Donor and nickel chelate (Ni-NTA) Acceptor beads was added and again incubated for 1 h at RT. AlphaScreen signal was read out on a 2104 EnVision Multilabel Plate Reader with AlphaScreen settings, excitation time 180 ms, total measurement time 550 ms.
Proximity labeling with APEX2
Proximity labeling with APEX2 was done following the described protocol for imaging and proteomic analysis (Hung, V. et al. Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2. Nat Protoc 11, 456-475 (2016)). Cell lines with a stable expression of APEX2-fusion proteins were generated using lentiviral transduction of plasmids generated with Gateway cloning of the respective fusion protein into pLEX305. pLEX_305 was a gift from David Root (Addgene plasmid # 41390; http://n2t.net/addgene:41390; RRID:Addgene_41390). The APEX2 sequence was amplified from APEX2-csGBP which was a gift from Rob Parton (Addgene plasmid # 108874; http://n2t.net/addgene: 108874; RRID:Addgene_108874). Briefly, cells were incubated with 0.5mM biotin-phenol for 30min, after which 1 mM H2O2 was added for exactly 1 min. Afterwards the labeling reaction was quenched by 3 quick washes with Quenching solution (10mM sodium ascorbate, 10mM sodium azide, 5mM Trolox in PBS). Cells were then fixed for IF analysis or detached from the plates with a cell scraper for WB or MS analysis.
Biotin enrichment after proximity labeling
After proximity labeling, cells (approx. 10 Mio. cells, 15 cm dish) were harvested, washed 2x in PBS, snap frozen and stored at -80°C. Cell pellets were resuspended in 200 piL freshly prepared lysis buffer (1x PBS, 1 % SDS, 2mM MgCh, Protease inhibitors, Benzonase), vortexed and incubated at 37°C for 30min. Samples were then centrifuged for 30min at 18,000xg and +4°C, supernatants transferred into fresh 1.5ml lo-bind tubes on ice. After quantification of protein amounts by Pierce™ 660nm Protein Assay, samples were normalized to 500 pig total protein input in a final volume of 300 pil lysis buffer. For reduction, 30 pil of 50 mM TCEP were added for a final concentration of 4.5 mM, vortexed and incubated on a shaking thermoblock at 56°C for 1 h. After adjustment of pH by addition of 80pil 1 M HEPES pH 7.5, 45pil of freshly prepared 200mM iodoacetamide were added for alkylation. Samples were vortexed and incubated on a shaking thermoblock at 25°C for 30min with light protection.
During reduction and alkylation 100 pil of streptavidin agarose beads (Pierce™ Streptavidin Agarose, Thermo Scientific, 20353) per sample were taken to 5 ml tubes in batches of 400 pil. To settle down the beads, tubes were centrifuged for 30sec in a table-top spin centrifuge and settled further on ice for 3min before taking off the supernatant. Beads were washed twice in 4ml PBS. After the last washing, beads were resuspended in PBS and combined to a final volume of 100 pil/ sample. After distribution of 100 pil/ sample, 1.35 ml of PBS were added, and beads stored at 4°C. For enrichment, reduced and alkylated samples were then added to the prepared beads and rotated at 25°C for 1 h. To settle down the beads, tubes were centrifuged for 30sec in a table-top spin centrifuge and settled further at RT before taking off the supernatant.
BioRad Minispin columns were equilibrated on vacuum manifold with 1 ml Wash buffer 1 (0.2% SDS in PBS). Beads with enriched proteins were transferred from tubes to columns by resuspending in 2x 0.5ml Wash buffer 1 . Afterwards, beads were washed 10x in 0.5ml Wash buffer 2 (8M Urea in PBS) and 4x in 0.5ml PBS. After closing of columns, beads were resuspended in 2x 0.5ml digestion buffer (H2O (HPLC grade), 50mM Ammonium bicarbonate, 0.2M Guanidine hydrochloride, 1 mM Calcium chloride) and transferred into fresh 1.5ml lo-bind tubes. To settle down the beads, tubes were centrifuged for 30sec in a table-top spin centrifuge and settled further on ice for 3min before taking off the supernatant. 250pil Digestion buffer were added to the beads, and beads were stored at 4°C before the overnight digest. 10pil trypsin (0.1 pig/pil, total 1 g) were added to each tube at the end of the day, incubation at 37°C rotating inside the incubator overnight (~14h).
For solid phase extraction (SPE) stage tips were prepared as follows. 32x 1 mm in diameter C18 material was punched out from Empore C18 disk using blunt syringe needle and plunged into filter-less P200 pipette tip, pushing towards narrow end of the tip. The metal piston was pressed down to fix the C18. 24pil oligo R3 solution (15mg/ml in 100% acetonitrile (ACN)) were applied to the C18 tip, centrifuged at 1 ,000xg for 1 min inside of a collection tube. C18 was activated by washing 2x with lOOpil 100% ACN, centrifugation at 1,000xg for 1 min. Columns were equilibrated with 200pil 0.1 % TFA, centrifuged at 1 ,000xg for 30sec, wrapped in parafilm and stored at 4°C overnight. Right before using them for clean-up of digests the next day, C18 columns were centrifuged at 1 ,000xg for 2min, equilibrated again with 200pil 0.1 % TFA, and centrifuged at 1 ,000xg for 3min.
After overnight digest, beads were separated via centrifugation at 1 ,000xg for 30sec, and complete supernatants transferred into fresh 1.5ml lo-bind tubes. Beads were washed with 200pil H2O for HPLC using wide pipette tips, centrifuged again for 30sec at 1,000xg and supernatant combined with digest. The peptide samples were then acidified with 16pil 30% TFA (~1 % final) and loaded to the C18 columns in fractions of max. 250 l, and centrifuged at 1 ,000xg for 3min each. After loading the full volume, columns were washed with 200 l 0.1 % TFA, and centrifuged at 1 ,000xg for 3min. Samples were eluted with 2x 50 l elution buffer (90% ACN, 10% of 0.1 % Trifluoroacetic acid (final 0.01 %)) by centrifugation at 1 ,000xg for 3min. Eluates were dried in vacuum centrifuge at V-AQ, 45°C for 1 ,5h and stored at -20°C until TMT-labeling.
Dried pellets after SPE were reconstituted in 15pl of 100mM HEPES pH 8.5 in H2O for HPLC (diluted from 1 M HEPES pharmaceutical standard stock solution, pH adjusted using NaOH for HPLC). Aliquots of frozen TMT labels (Lot no. WA314599) were equilibrated at RT for 5min, spun down in spin-centrifuge, vortexed and spun down again. 4pl of respective TMTpro label were added, vortexed, spun down in spin-centrifuge and incubated at 25°C and 300rpm for 1 h. Reaction was stopped by adding 1 .5pl of 5% hydroxylamine solution in H2O for HPLC (prepared fresh from 50% hydroxylamine stock solution), vortexing, spinning down in spin-centrifuge and incubation at 25°C and 300rpm for 15min. Full volumes of respective TMTpro channels were then pooled into fresh 1.5ml lo-bind tube.
For a 2D analysis, samples were fractionated by on-tip high pH fractionation. Fresh ammonium formate (AF) buffer was prepared right before using, as it is volatile: 100mM ammonium formate in 2ml tube (6.3mg into 1 ml H2O for HPLC) mixed into 4ml H2O for HPLC in 15ml tube, pH 10 adjusted with two drops of 25% ammonia solution (~35pil, final concentration 20mM). For 2D analysis, 1 ml of 20mM freshly prepared AF was added to 320pil of pooled sample. C18 columns were prepared as described above. The eluate was loaded in fractions (max. capacity 200pil at once), centrifuged at 1 ,000xg for 3min each. The column was washed with 200JJI 20mM AF, and centrifuged at 1 ,000xg for 3min. Each fraction was eluted in a fresh 1 ,5ml lo-bind tube. All fractionation buffers (100% ACN and 20mM AF mixed at different ratios) were prepared fresh:
Fraction 1 : Elution with 50pl 16% ACN (24pl ACN +126pl 20mM AF), centrifuged at 1 ,000xg for 2min, washed with 20pl of same buffer, collected together in tube #1 , centrifuged at 1 ,000xg for 1 min.
Fraction 2: Elution with 50pl 20% ACN (30pl ACN +120pl 20mM AF), centrifuged at 1,000xg for 2min, washed with 20pl of same buffer, collected together in tube #2, centrifuged at 1 ,000xg for 1 min.
Fraction 3: Elution with 50pl 24% ACN (36pl ACN 114pl 20mM AF), centrifuged at 1,000xg for 2min, washed with 20pl of same buffer, collected together in tube #3, centrifuged at 1 ,000xg for 1 min.
Fraction 4: Elution with 50pl 28% ACN (42pl ACN +108pl 20mM AF), centrifuged at 1,000xg for 2min, washed with 20pl of same buffer, collected together in tube #4, centrifuged at 1 ,000xg for 1 min.
Fraction 5: Elution with 50pl 80% ACN (120pl ACN +30pl 20mM AF), centrifuged at 1,000xg for 2min, washed with 20pl of same buffer, collected together in tube #5, centrifuged at 1 ,000xg for 1 min.
All 5 eluates were dried in vacuum centrifuge at 45°C, V-AQ for at least 2h (until dry) and frozen at -20°C until analysis.
For a WB analysis instead of the described preparation of samples for MS, samples were not reduced and alkylated, but instead loaded on to streptavidin beads directly after lysis, quantification, and normalization. Instead of digesting proteins on the beads after enrichment, beads were transferred to lo-bind tubes with 2x 0.5mL PBS. After removal of supernatant, proteins were eluted from the beads in 3 rounds. First, 50 pl 4x LB was added, beads incubated at 95°C for 10 mins, spun down and supernatant transferred to a new tube. Second, 50 l 1x LB was used and combined. Last, 50 pl PBS was used and combined. Typically, 30 pl of sample were loaded on an SDS-PAGE gel.
SDS-PAGE followed by Coomassie staining or western blotting
To separate proteins according to size, cell lysates/ protein solutions were loaded onto SDS-polyacrylamide gels (12%) with 4x Laemmli loading buffer (LB):
17.6ml 0.5M Tris pH 6.8
17.6ml Glycerol
8.8ml 20% SDS
2ml 1 % bromophenol blue
2ml beta-mercaptoethanol
Afterwards, proteins were separated through application of an electric field (120V for 15 min, 160V for 90 min). For visualization of total protein, gels were stained with Coomassie Blue. To do so, the gel was fixed in fixing solution (50% methanol, 10% glacial acetic acid) for 1 h with gentle agitation. The gel was then stained in staining solution (0.1 % Coomassie Brilliant Blue R-250, 50% methanol and 10% glacial acetic acid) for 20 min, followed by several rounds of destaining with destaining solution (40% methanol, 10% glacial acetic acid).
For visualization of individual proteins, they were transferred to a nitrocellulose membrane (GE Healthcare Life Science) by electrophoresis. The membrane was blocked by 5% Milk solution in TBST for at least 1 h at RT, followed by incubation in primary antibody solution (SMNDC1 : Novus Biologicals Cat#NBP1-47302; RRID:AB_10010256; SFPQ: Atlas Antibodies Cat#HPA047513; RRID:AB_2680073; APEX2 Innovagen PA-APX2-100; for all dilution 1 : 1000 in 5% Milk TBST) at 4°C o/n. Membranes were then washed 3 times in TBST, followed by incubation with HRP-coupled secondary antibody solution (Peroxidase AffiniPure Donkey Anti-Mouse IgG Jackson ImmunoResearch Cat#715-035-151 ; RRID:AB_2340771; Peroxidase AffiniPure Donkey Anti-Rabbit IgG Jackson ImmunoResearch Cat#711-035-152; RRID:AB_10015282; Goat Anti-Chicken IgY H&L (HRP) Abeam ab97135; RRID:AB_10680105; for all dilution 1 :20000 in 5% Milk TBST) for at least 1 h at RT. After 3 more washing steps, signal was detected by application of Clarity ECL Western Blotting Substrate (Bio-Rad) to the membrane with a ChemiDoc MP Imaging System (Bio-Rad) with Image Lab Touch Software Version 2.3.0.07.
2D-RP/RP Liquid Chromatography - Tandem Mass Spectrometry analysis
Mass spectrometry analysis was performed on an Orbitrap Fusion Lumos Tribrid mass spectrometer (ThermoFisher Scientific, San Jose, CA) coupled to a Dionex Ultimate 3000 RSLCnano system (ThermoFisher Scientific, San Jose, CA) via a Nanospray Flex Ion Source (ThermoFisher Scientific, San Jose, CA) interface. Peptides were loaded onto a trap column (PepMap 100 C18, 5 pm, 5 x 0.3 mm, ThermoFisher Scientific, San Jose, CA) at a flow rate of 10 pL/min using 0.1 % TFA as loading buffer. After loading, the trap column was switched in-line with an Acclaim PepMap nanoHPLC C18 analytical column (2.0 pirn particle size, 75pim IDx500mm, catalog number 164942, ThermoFisher Scientific, San Jose, CA). Column temperature was maintained at 50 °C. Mobile-phase A consisted of 0.4% formic acid in water and mobile-phase B of 0.4% formic acid in a mix of 90% acetonitrile and 10% water. Separation was achieved by applying a four-step gradient over 151 min at the flow rate of 230 nL/min (initial gradient increase from 6% to 9% solvent B within 1 min, 9% to 30% solvent B within 146 min, 30% to 65% solvent B within 8 min and, 65% to 100% solvent B within 1 min, 100% solvent B for 6 min before equilibrating at 6% solvent B for 23 min prior to next injection). In a liquid-junction set-up, electrospray ionization was enabled by applying a voltage of 1.8 kV directly to the liquid to be sprayed, and non-coated silica emitters were used.
The mass spectrometer was operated in a data-dependent acquisition mode (DDA) and used a synchronous precursor selection (SPS) approach, which enables more accurate multiplexed quantification of peptides and proteins at the MS3 level. For both MS2 and MS3 level a survey scan of 400-1600 m/z in the Orbitrap was collected at a resolution of 120000 (FTMS1), an AGO target was set to 'standard' and a maximum injection time (IT) of 50 ms was applied. Precursor ions were filtered according to charge state (2-6), dynamic exclusion (60 s with a ±10 ppm window), and monoisotopic precursor selection. Precursor ions for data-dependent MSn (ddMSn) analysis were selected using 10 dependent scans (TopN approach). Charge state filter was used to select precursors for data- dependent scans. In ddMS2 analysis, spectra were acquired using a single charge state per branch (from z=2 to z=5) in a dual-pressure linear ion trap (ITMS2). Quadrupole isolation window was set to 0.7 Da and collision induced dissociation (CID) fragmentation technique was used at a normalized collision energy of 35%. Normalized AGC target value was set to 200% with a maximum IT of 35 ms. During the ddMS3 analyses, precursors were isolated using SPS waveform and different MS1 isolation windows (1.3 m/z for z=2, 1.2 m/z for z=3, 0.8 m/z for z=4 and 0.7 m/z for z=5). Target MS2 fragment ions were further fragmented by high-energy collision induced dissociation (HCD) followed by Orbitrap analysis (FTMS3). The HCD normalized collision energy was set to 45% and normalized AGC target was set to 300% with a maximum IT of 100 ms. The resolution was set to 50 000 with defined scan range from 100 to 500 m/z. Xcalibur version 4.3.73.11 and Tune 3.4.3072.18 were used to operate the instrument. Data processing and data analysis
Following data acquisition, acquired raw data files were processed using the Proteome Discoverer v.2.4.1.15 platform, choosing a TMT16plex quantification method. In the processing step the inventors used Sequest HT database search engine and Percolator validation software node to remove false positives with a false discovery rate (FDR) of 1 % on peptide and protein level under strict conditions. Searches were performed with full tryptic digestion against the mouse SwissProt database v.2017.10.25 (SwissProt TaxlD=10090, 25 097 sequences and appended known contaminants and streptavidin) with a maximum of two allowed miscleavage sites. Oxidation (+15.994 Da) of methionine and acetylation of protein N termini (+42.011 Da), as well as methionine loss (-131.040 Da) and acetylation of protein N termini with methionine loss (-89.030Da) were set as variable modification, while carbamidomethylation (+57.021 Da) of cysteine residues and tandem mass tag (TMT) 16-plex labeling of peptide N termini and lysine residues (+304.207Da) were set as fixed modifications. Data was searched with mass tolerances of ±10 ppm and ±0.6 Da on the precursor and fragment ions, respectively. Results were filtered to include peptide spectrum matches with Sequest HT cross-correlation factor (Xcorr) scores of >1 and high peptide confidence assigned by Percolator. MS3 signal-to-noise values (S/N) values of TMTpro reporter ions were used to calculate peptide/protein abundance values. Peptide spectrum matches with precursor isolation interference values of >70%, SPS mass matches <65% and average TMTpro reporter ion S/N<10 were excluded from quantitation. Both unique and razor peptides were used for TMT quantitation. Isotopic impurity correction was applied. Data were normalized on total peptide amount for correction of experimental bias and scaled ‘on all average'. Protein ratios are directly calculated from the grouped protein abundances using a one-way ANOVA hypothesis test, followed by Benjamini-Hochberg correction for multiple comparisons. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD037092 and 10.6019/PXD037092.
Enrichr analysis
Gene symbols of proteins identified by mass-spectrometry or subsets thereof were analyzed for enrichment of gene ontology (GO) Biological Process 2021 terms with the online tool "Enrichr” (https://maayanlab.doud/Enrichr/) (Chen, E. Y. et al. Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinformatics 14, 128 (2013)). For each term that is associated with at least one identified gene symbol an odds ratio was calculated based on the size of the input dataset and the size of the group of gene symbols associated with that term. Furthermore, a p-value, and an adjusted p-value (Benjamini-Hochberg method for correction for multiple hypotheses testing) was determined.
NMR experiments
Isotope-enriched SMN84-147 and SMNDC165-128 were expressed and purified as described in Tripsianes et al., 2011 (Tripsianes, K. et al. Structural basis for dimethylarginine recognition by the Tudor domains of human SMN and SPF30 proteins. Nature Structural & Molecular Biology 18, 1414-1420 (2011)). NMR experiments were performed on Bruker Avance III spectrometers operating at 600 MHz or 800 MHz 1H frequencies using H/N/C triple-resonance cryogenic probes. All NMR acquisition was performed in 3 mm tubes at 25°C. Spectra were processed using Topspin 3.5 (Bruker) and analyzed with Cara 1.9.17 (Keller, R. L. J. Optimizing the process of nuclear magnetic resonance spectrum analysis and computer aided resonance assignment. (ETH Zurich, 2005). doi: 10.3929/ethz-a-005068942) or NMRglue (Helmus, J. J. & Jaroniec, C. P. Nmrglue: an open source Python package for the analysis of multidimensional NMR data. J Biomol NMR 55, 355-367 (2013))-based Python scripts. Chemical shift assignments were transferred from Selenko et al. (Selenko, P. et al. SMN Tudor domain structure and its interaction with the Sm proteins. Nat Struct Mol Biol 8, 27-31 (2001)) and Tripsianes et al., 2011 (loc. cit.) (BMRB: 4899 for SMN, 18006 for SMNDC1). All titration measurements were performed in 20 mM sodium phosphate buffer pH 6.5, 50 mM NaCI, 4 mM dithiothreitol and 10% (v/v) D2O as deuterium lock. Aqueous, buffered inhibitor stock solutions of maximal 20 mM concentration were prepared and carefully adjusted to pH 6.5. Inhibitor concentration was measured by addition of 100 piM DSS, peak integration and calculating with C = 1 Ndss Cdss where C, I, N and CDSS, IDSS, NDSS is the IDSS’N concentration, peak intensity, and number of protons of inhibitor and DSS, respectively. Titration experiments were performed with 50 piM 15N-labeled SMN84-147 and SMNDC165-128 and addition of 0, 0.05, 0.1 , 0.25, 0.5 and 0.8 mM compound 13. Since the titration did not reach saturation, an additional point was measured for SMNDC1 with 8 mM compound 13. An apparent dissociation constant KD of around 1 mM was calculated from CSP data using CSP = CSP -c
— with c being the concentration of compound 13 and CSPmax the CSP at saturation. However, this value is c+Kp obstructed by solubility issues of 13 and therefore not comparable with the IC50 values from the AlphaScreen assay, where lower concentrations were used. To record intermolecular NOE data, a 1 mM sample of 13C,15N-labeled SMNDCi65-i28 was prepared, lyophilized, and resolved in D2O containing 20 mM buffered compound 13. To confirm saturation of binding, a 1H,15N-HSQC spectrum was acquired and compared with the titration data. coi-13C-filtered and a>2-13C-filtered two-dimensional NOESY and a>2-13C-filtered, coi-13C-edited three-dimensional NOESY experiments (Sattler, M., Schleucher, J. & Griesinger, C. Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients. Progress in Nuclear Magnetic Resonance Spectroscopy 34, 93-158 (1999)) were recorded with 150 ms mixing times. Chemical shift assignments were transferred from Tripsianes et al., 2011 (loc. cit.).
Docking calculation
Docking calculations were performed using the HADDOCK webserver (van Zundert, G. C. P. etal. The HADDOCK2.2 Web Server: User-Friendly Integrative Modeling of Biomolecular Complexes. Journal of Molecular Biology 428, 720— 725 (2016); Honorato, R. V. et al. Structural Biology in the Clouds: The WeNMR-EOSC Ecosystem. Frontiers in Molecular Biosciences 8, (2021)). Structure and topology files for compound 13 were generated by prodrg2 (Schuttelkopf, A. W. & van Aalten, D. M. F. PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Cryst D 60, 1355-1363 (2004)). The SMNDC1/sDMA structure (PDB: 4A4H) (Tripsianes, K. et al. Structural basis for dimethylarginine recognition by the Tudor domains of human SMN and SPF30 proteins. Nature Structural & Molecular Biology 18, 1414-1420 (2011)) was used as a protein model with the sDMA removed beforehand. Instead of defining active/passive residues, intermolecular NOE contacts were introduced as ambiguous restraints. Visible and assigned NOE crosspeaks were defined as distance restraints with a lower limit of 0.5 A and upper limit of 5 A. Peak intensities of crosspeaks were measured and normalized to the strongest peak. According to their relative intensities, upper distance limits were gradually lowered to 3.5 A for non-overlapping crosspeaks. 1000, 400 and 400 structures were calculated for the different stages of rigid body docking, semi-flexible refinement, and final refinement, respectively. Parameters were chosen as suggested by HADDOCK for small molecule docking. 200 structures were analyzed and clustered by RMSD with a cutoff of 1.5 A resulting in one cluster, which contains all analyzed structures. SMNDC1 knock-down
SMNDC1 knock-down was performed as described in Casteels et al. (Casteels, T. et al. SMNDC1 links chromatin remodeling and splicing to regulate pancreatic hormone expression. Cell Reports 40, (2022)). Briefly, Smndcl shRNA from the TRC shRNA library (https://portals.broadinstitute.org/gpp/public/) (TRCN0000123795) was cloned into pLKO.1 (Addgene plasmid #10878). This plasmid was packaged into lentivirus in Lenti-X™ 293 T cells (BOSC-23, RRID:CVCL_4401, TakaraBio Cat#632180) with Lipofectamine™ 3000 (Thermo Fisher Scientific L3000008) and packaging plasmids psPAX2 (Addgene plasmid #12260) and pMD2.G (Addgene plasmid #12259). Target cells were transduced with viral supernatant after filtering and addition of 8 pig/ml Polybrene® (Santa Cruz Biotechnology sc- 134220) 48h after transfection. Medium was changed 24h later.
Splicing PCRs
To perform splicing PCRs, RNA was isolated from pelleted cells using the RNeasy Mini Kit (Qiagen, #74106). RNA was then reverse transcribed with LunaScript RT SuperMix Kit (NEB #E3010). cDNA was PCR-amplified with OneTaq® Quick-Load® 2X Master Mix (NEB # M0486) for 35 cycles with the following primers as suggested on vastdb.crg.eu:
Figure imgf000104_0001
Figure imgf000105_0001
The PCR products were run on a 1 % Agarose gel for 30 min at 100 Volt.
RNA sequencing and transcriptome analysis
RNA sequencing libraries were prepared from low-input samples using the Smart-seq2 protocol (Picelli, S. et al. Full- length RNA-seq from single cells using Smart-seq2. Nat Protoc 9, 171-181 (2014)). The subsequent library preparation from the amplified cDNA was performed using the Nextera XT DNA library prep kit (Illumina, San Diego, CA, USA). Library concentrations were quantified with the Qubit 2.0 Fluorometric Quantitation system (Life Technologies, Carlsbad, CA, USA) and the size distribution was assessed using the Experion Automated Electrophoresis System (Bio-Rad, Hercules, CA, USA). For sequencing, samples were diluted and pooled into NGS libraries in equimolar amounts.
Expression profiling libraries were sequenced on NovaSeq 6000 instrument (Illumina, San Diego, CA, USA) with a 100-base-pair, paired-end setup. Raw data acquisition and base calling was performed on-instrument. Subsequent raw data processing off the instruments involved two custom programs (https://github.com/epigen/picard/) based on Picard tools (2.19.2) (https://broadinstitute.github.io/picard/). In a first step, base calls were converted into lanespecific, multiplexed, unaligned BAM files suitable for long-term archival (IHuminaBasecallsToMultiplexSam, 2.19.2- CeMM). In a second step, archive BAM files were demultiplexed into sample-specific, unaligned BAM files (llluminaSamDemux, 2.19.2-CeMM).
NGS reads were mapped to the Genome Reference Consortium GRCm38 assembly via "Spliced Transcripts Alignment to a Reference” (STAR) (Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15- 21 (2013)) utilising the "basic” Ensembl transcript annotation from version e100 (April 2020) as reference transcriptome. The mm10 assembly of the UCSC Genome Browser was used for downstream data processing, and the Ensembl transcript annotations were adjusted to UCSC Genome Browser sequence region names. STAR was run with options recommended by the ENCODE project. NGS read alignments overlapping Ensembl transcript features were counted with the Bioconductor (3. 11) GenomicAlignments (1 .24.0) package via the summarizeOverlaps function in Union mode, ignoring secondary alignments and alignments not passing vendor quality filtering. Since the Smart-seq2 protocol is not strand specific, all alignments irrespective of the gene or transcript orientation were counted. Transcript-level counts were aggregated to gene-level counts and the Bioconductor DESeq2 (Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology 15, 550 (2014)) (1.28.1) package was used to test for differential expression based on a model using the negative binomial distribution.
Splicing analysis
Alternative splicing events were characterised and quantified using VAST-TOOLS (Tapial, J. et al. An atlas of alternative splicing profiles and functional associations reveals new regulatory programs and genes that simultaneously express multiple major isoforms. Genome Res. 27, 1759-1768 (2017)) (2.5.1) in conjunction with the Mus musculus database (vastdb. mm2.23.06.20), based on the Genome Reference Consortium assembly GR0m38.p5 and Ensembl transcript annotation 88 (March 2017). Briefly, NGS reads were aligned for each read group independently, read groups were merged into samples and samples were combined into a summary table. The differential splicing events were called via the VAST-TOOLS "compare” algorithm (min_dPSI > 15, min_range > 5) and further filtered for genes showing statistical significance (adjusted P-value <= 0.1) and a sizable effect (absolute Iog2-fold change >= 1.0) in the differential expression analysis.
Results
SMNDC1 co-localizes with nuclear speckle markers
To identify features associated with subcellular SMNDC1 localization, the inventors analyzed the protein sequence by comparing predictions for disordered regions by MetaDisorder (Kozlowski, L. P. & Bujnicki, J. M. MetaDisorder: a meta-server for the prediction of intrinsic disorder in proteins. BMC Bioinformatics 13, 111 (2012)) and for the full- length structure by AlphaFold (Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583-589 (2021); Varadi, M. et al. AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Research 50, D439-D444 (2022)) (Fig. 1 a). The experimentally solved Tudor domain structure (Tripsianes, K. et al. Structural basis for dimethylarginine recognition by the Tudor domains of human SMN and SPF30 proteins. Nature Structural & Molecular Biology 18, 1414-1420 (2011)) (residues 64-128) and two interacting N-terminal alpha-helices (residues 2-25, and 30-52) are visible both in the AlphaFold prediction and in the disorder tendency plot as ordered regions. AlphaFold in addition predicts a long C-terminal alpha-helix, for which however currently no other experimental evidence exists.
The inventors employed an endogenous tagging system that targets introns and introduces a GFP-tag as an artificial exon (Serebrenik, Y. V., Sansbury, S. E., Kumar, S. S., Henao-Mejia, J. & Shalem, O. Efficient and flexible tagging of endogenous genes by homology-independent intron targeting. Genome Res 29, 1322-1328 (2019)) to characterize SMNDCTs cellular functions. To rule out disrupting effects of the tag on protein localization, the inventors targeted all of SMNDCTs introns in murine alphaTCI cells, and then isolated clonal sublines. The targeted introns result in GFP integrations covering all regions of the protein, including one at the N-terminus (before residue 1), the N-terminal region (residue 40), the Tudor domain (residue 88), and a long stretch in the C-terminal region (residue 142, residue 193) which is predicted to be disordered (Kozlowski, L. P. & Bujnicki, J. M. MetaDisorder: a meta-server for the prediction of intrinsic disorder in proteins. BMC Bioinformatics 13, 111 (2012)) (Fig. 1 a, b). Furthermore, the inventors also tagged intron 2-3 in human HAP1 cells. Typically, these monoallelic tagging events resulted in cells expressing both un-tagged and GFP-tagged SMNDC1 at comparable levels as shown by western blot (WB) (Fig. 1c, quantifications and full membranes Fig. 8b, c). The GFP-tag within the Tudor domain (intron 3-4) showed the lowest relative expression levels, indicating possible interference with folding efficiency.
AlphaFold structure predictions (Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583-589 (2021); Varadi, M. et al. AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Research 50, D439-D444 (2022)) for SMNDC1 with and without GFP in the different introns revealed that the GFP-tag does not seem to disrupt the overall structure of the protein (Fig. 8a). All structural elements such as the N- and C-terminal a-helices and the Tudor domain are predicted to form normally, even when the GFP-tag interrupts the Tudor domain (intron 3-4). Accordingly, all of the different intron tagged clones, including the intra-Tudor GFP integration showed consistent subcellular localization pattern (Fig. 1 b). These GFP fusions showed the same speckled nuclear localization avoiding DNA-dense regions as observed for the endogenous protein by antibody-based immunofluorescence (IF) (Fig. 1d). During M- phase of the cell cycle SMNDC1 dissipated to the whole cell and formed distinct droplets called mitotic interchromatin granules (Ferreira, J. A., Carmo-Fonseca, M. & Lamond, A. I. Differential interaction of splicing snRNPs with coiled bodies and interchromatin granules during mitosis and assembly of daughter cell nuclei. Journal of Cell Biology 126, 11-23 (1994); Tripathi, K. & Parnaik, V. K. Differential dynamics of splicing factor SC35 during the cell cycle. J Biosci 33, 345-354 (2008)) (Fig. 1e), a behavior which is typical for nuclear speckle proteins (Av§ar Ihk, i. &Akta§, T. Nuclear speckles: dynamic hubs of gene expression regulation. FEBS J (2021) doi:10.1111/febs.16117; Rai, A. K., Chen, J.- X., Selbach, M. & Pelkmans, L. Kinase-controlled phase transition of membraneless organelles in mitosis. Nature 559, 211-216 (2018)). SMNDC1 also reacted to the overexpression of the cell-cycle dependent kinases DYRK3 and CLK1 , which is known to dissolve nuclear speckles (Rai, A. K., Chen, J.-X., Selbach, M. & Pelkmans, L. Kinase- controlled phase transition of membraneless organelles in mitosis. Nature 559, 211-216 (2018); Sacco-Bubulya, P. & Spector, D. L. Disassembly of interchromatin granule clusters alters the coordination of transcription and pre-mRNA splicing. Journal of Cell Biology 156, 425-436 (2002)), with a loss of its focal nuclear localization (Fig. 8d).
To further characterize SMNDCTs localization in the nucleus, the inventors co-stained cells with antibodies against SMNDC1 and SC35, a marker for nuclear speckles. Both signals overlap to a large degree and avoid chromatin- dense regions, whereby SMNDC1 shows a wider less focal distribution (Fig. 1f, co-localization analysis Fig. 8e). To be able to visualize nuclear speckles in live cells the inventors RFP-tagged SRRM2 in the SMNDC1 -GFP-tagged cells (Fig. 1g). SRRM2 is the target of the SC35 antibody (llik, i. A. et al. SON and SRRM2 are essential for nuclear speckle formation. eLife 9, e60579 (2020)) and scaffolding protein of nuclear speckles (Xu, S. et al. SRRM2 organizes splicing condensates to regulate alternative splicing. Nucleic Acids Research gkac669 (2022) doi: 10.1093/nar/gkac669).
Endogenously tagged SMNDC1-GFP and SRRM2-RFP co-localized to a large degree, both in interphase and during mitosis (Fig. 1 h). Even though co-localization was maintained in the mitotic interchromatin granules, there SMNDC1 showed a higher degree of diffuse localization, leading to a lower average Pearson correlation score compared to interphase cells (Fig. 8f). Overall, the inventors find that SMNDC1 shows behavior and localization typical for proteins in nuclear speckles, which have been described as membraneless organelles in the nucleus formed by LLPS. SMNDC1 undergoes biomolecular condensation in vitro and in cellular systems
A common way to prove phase-separating behavior of a protein is to show its ability to form droplets in a purified form in vitro. To do so, the inventors expressed and purified full SMNDC1 with an N-terminal GFP-tag and mixed it with PEG-8000 as a surrogate for the crowded environment of a cell. They observed droplet formation (Fig. 2a) and fusion of droplets (Fig. 2b). Subsequently the inventors tested the influence of other biomolecules and salt concentration on droplet formation (Fig. 2c). Addition of RNAto the PEG-8000 containing buffer enhanced SMNDCTs droplet formation while high NaCI concentrations prevented droplet formation. Digestion of RNA by RNase led to the dissolution of droplets, even after their formation (Fig. 2d). RNA also physically localized to the protein droplets (Fig. 2e).
To further understand which part of the protein is responsible for the formation of droplets, the inventors fused different SMNDC1 truncations (Fig. 1 a) to GFP and subjected them to the same treatment in buffer containing RNA and PEG- 8000. These experiments clearly displayed that the C-terminal region after the Tudor domain (constructs 5 and 6), which is predicted to be intrinsically disordered (Kozlowski, L. P. & Bujnicki, J. M. MetaDisorder: a meta-server for the prediction of intrinsic disorder in proteins. BMC Bioinformatics 13, 111 (2012)), was sufficient to induce droplet formation with RNA (Fig. 2f, see Fig. 1a for a scheme of the truncated forms), which fit the predicted IDR scores (Kozlowski, L. P. & Bujnicki, J. M. loc. cit.) (Fig. 1 a). The inventors also confirmed that the Tudor domain alone (construct s) cannot form droplets, consistent with previous literature (Courchaine, E. M. et al. DMA-tudor interaction modules control the specificity of in vivo condensates. Cell 184, 3612-3625.e17 (2021)).
To show the reversibility of phase separation in vivo, the aliphatic alcohol 1 ,6-hexanediol which interferes with weak hydrophobic interactions is often used to dissolve protein condensates (Kroschwald, S., Maharana, S. & Simon, A. Hexanediol: a chemical probe to investigate the material properties of membrane-less compartments. Matters 3, e201702000010 (2017)). SMNDC1-GFP exhibited the expected phenotype in live cells treated with 1,6-hexanediol by losing its focal localization within the nucleus (Fig. 2g). Another way to characterize the molecular dynamics and mobility of phase-separating proteins in cells is to analyze the diffusion of a fluorescently labeled protein by fluorescence recovery after photobleaching (FRAP). When bleaching SMNDC1-GFP and SRRM2-RFP, fluorescence recovered within 30 seconds (Fig. 2h), consistent with liquid-like behavior rather than protein aggregation. These data provide evidence that SMNDC1 undergoes phase separation, both in vitro and in membraneless organelles within the nucleus, presumably nuclear speckles.
Full-length SMNDC1 interacts with nuclear speckle proteins
The inventors set out to characterize SMNDCTs interactome using proximity labeling by overexpressing an SMNDC1- APEX2 fusion protein (Fig. 3a). Compared to classical co-immunoprecipitation (Co-IP), this recently developed method (Hung, V. et al. Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2. Nat Protoc 11, 456-475 (2016)) is better suited to capture weak and transient interactions as they are expected in phase-separated compartments like nuclear speckles. In addition to full-length SMNDC1 (APEX2-SMNDC1 FL), the inventors also performed proximity labeling with a fusion protein of APEX2 with a truncated SMNDC1 consisting of only the Tudor domain and therefore lacking N-terminal and C-terminal regions, and the nuclear localization signal (NLS) (APEX2-SMNDC1TD) (Fig. 9a). To verify their approach, the inventors performed proximity labeling followed by an immunofluorescence (IF) staining against SMNDC1 and biotin. APEX2-SMNDC1 FL caused biotinylation in the areas where SMNDC1 is localized: nuclear while avoiding chromatin-dense regions (Fig. 3b). Much less biotinylation was observed when omitting H2O2. The control overexpression of APEX2-SMNDC1TD on the other hand showed a uniform localization throughout the cell and a corresponding biotinylation pattern. On a western blot, a ladder of biotin- labelled proteins was visible, but absent when leaving out the H2O2 during the labeling. More proteins appear to be labelled by the ubiquitously localized APEX2-SMNDC1TD fusion (Fig. 9b).
Analyzing the biotinylated and enriched proteins by mass spectrometry (MS), the inventors identified and quantified a large number of proteins (-3200) in the proximity of APEX2-SMNDC1 FL and APEX2-SMNDC1TD. Compared to the proximity interactome of APEX2-SMNDC1TD, APEX2-SMNDC1 FL showed overall less interactions (Fig. 3c). The inventors attribute this to the higher specificity of interactions happening with the correctly localized full form of SMNDC1. The fact that SMNDC1 itself was enriched in APEX2-SMNDC1 FL over APEX2-SMNDC1TD suggests that labeling in trans works better if SMNDC1 is correctly localized and concentrated in its phase-separated compartment leading to more SMNDC1 protein in its proximity. Similarly, proteins known to be localized to the nucleus were not depleted in APEX2-SMNDC1 FL over APEX2-SMNDC1TD, reflecting the loss of correct localization when the NLS is missing (Fig. 9c).
The inventors then filtered for proteins enriched in APEX2-SMNDC1 FL over APEX2-SMNDC1TD (adjusted p-value <0.1 , abundance ratio >1.1) which reduced the number of proteins they considered specific interactors of SMNDC1 FL to 750. As expected, they found an enrichment of proteins associated with mRNA processing, and more specifically splicing, but also an enrichment of proteins associated with ribosome biogenesis and rRNA processing amongst these (Fig. 3d). When comparing these interactors to an SMNDC1 Co-IP dataset generated in their lab (Casteels, T. et al. SMNDC1 links chromatin remodeling and splicing to regulate pancreatic hormone expression. Cell Reports 40, (2022)) the inventors found a significant overlap but confirm that proximity labeling can detect more and different interactions compared to a Co-IP (Fig. 3e). A majority of APEX2-SMNDC1 FL interactors was also identified by SRSF7- APEX2 proximity labeling48 (Fig. 3f), suggesting that APEX2-SMNDC1 FL proximity labeling did enrich for proteins localized to nuclear speckles. Furthermore, the inventors compared the interactors to proteins identified as symmetrically di-methylated on arginine residues in a deep protein methylation profiling study (Hartel, N. G., Chew, B., Qin, J., Xu, J. & Graham, N. A. Deep Protein Methylation Profiling by Combined Chemical and Immunoaffinity Approaches Reveals Novel PRMT 1 Targets*. Molecular & Cellular Proteomics
Figure imgf000109_0001
2149-2164 (2019)) (Fig. 9d). Since these interactions are expected to be mediated through the Tudor domain APEX2-SMNDC1TD should bind these proteins, too. Consequently, only a small subset was enriched in APEX2-SMNDC1 FL over APEX2-SMNDC1TD. The inventors therefore also compared the sDMA-modified proteins to all proteins identified in their SMNDC1-APEX2 experiments and found most of them (67 out of 87 known sDMA-modified proteins). There was also an enrichment, although to a lesser degree, of proteins with asymmetrical di-methylations. These protein sets partially overlap, as the same arginine sites can often alternatively be symmetrically or asymmetrically di-methylated.
Overall, the inventors found a large interactome of SMNDC1 enriched for proteins interacting with RNA, localized to nuclear speckles, and with known sDMA modifications. They therefore suspected that the Tudor domain is responsible for a subset of SMNDCTs specific interactions.
A screen for small molecule SMNDC1 Tudor domain inhibitors
To pharmacologically perturb SMNDC1 function, the inventors set out to identify small molecule inhibitors of SMNDCTs Tudor domain based on perturbing its interaction with a dimethyl-arginine peptide. To establish an AlphaScreen (Bosse, R., Illy, C., Elands, J. & Chelsky, D. Miniaturizing screening: how low can we go today? Drug Discovery Today 5, 42-47 (2000)), they coupled donor beads to purified SMNDCTs (or SMN's) Tudor domain via a His-Tag and acceptor beads to a biotinylated peptide corresponding to the C-terminal region of the Small nuclear ribonucleoprotein Sm D3 containing four sDMAs (Fig. 4a). These interaction partners had previously been used in the structural study of SMNDC1 and SMN (Tripsianes, K. et al. Structural basis for dimethylarginine recognition by the Tudor domains of human SMN and SPF30 proteins. Nature Structural & Molecular Biology 18, 1414-1420 (2011)). Protein domains were purified employing their His-Tag (Fig. 10a). To identify ideal concentrations for screening, the inventors performed a cross-titration of Tudor domains and binding peptides (Fig. 10b). Since the AlphaScreen signal was sufficient for screening, they reduced the concentration of acceptor and donor beads to 5 pig/ml (Fig. 10c).
Using this set-up with their in-house library of -90,000 compounds (overview over screening strategy Fig. 4b, Table 1), the inventors identified 511 hits with signal <50 % of control (POC) (Fig. 4c). Since the AlphaScreen is susceptible to unspecific quenching of the singlet oxygen energy transfer they then performed a counter-screen using a crosslinking peptide which combines both affinity tags and therefore always brings donor and acceptor beads in close proximity (Fig. 4d). This led to a reduction to 40 hits that selectively inhibited the interaction between SMNDC1 , and its arginine methylated binding partner. These compounds were next tested in dose response with the Tudor domains of both SMNDC1 and SMN. Several chemical scaffolds (Fig. 4e-k) of inhibitors were discovered by this screen, with IC50 values of 0.2 to 2 pM and different degrees of selectivity between the different Tudor domains. The molecules with the best physicochemical and structural properties were then used for the design of further analogs, aiming at improving potency and selectivity (Fig. 5).
Table 1: Small molecule screening data.
Category Parameter Description
Assay Type of assay In vitro AlphaScreen / luminescence proximity
Target Tudor domain of SMNDC1
Primary measurement Decrease in luminescence signal reflecting disruption of heterodimeric complex between SMNDC1-Tudor domain and a peptide corresponding to the C-terminus of Small nuclear ribonucleoprotein Sm D3 containing 4 sDMAs (Sequence:
AAR*GR*GR*GMGR*GNIFQKRR, R*=sDMA)
Key reagents AlphaScreen no-wash assay kit containing
Streptavidin Donor beads and nickel chelate (Ni-NTA) AlphaScreen Acceptor beads (PerkinElmer Part Number 6760619).
Assay protocol Described in Methods section
Additional comments
Library Library size 89,355 small molecules
Library composition Structural diversity, NIH clinical collection, natural products, approved drugs, known bioactives (e.g., kinase, epigenetic modifiers, ...), natural products, drug-like molecules
Source Cayman chemical, Enamine Ltd, LC Labs,
MedChem Express, Selleck Chemicals, Sigma Aldrich, Tocris, Toronto Research Chemicals, Chemietek, Merck Millipore, Specs, ChemDiv, Zelinsky
Additional comments
Screen Format PerkinElmer OptiPlate-384 well plate
Concentration(s) tested Typically 10 pM (0.1% DMSO)
Plate controls 32 positive control wells (Mitoxantrone, quencher), 32 negative control wells (DMSO)
Reagent/ compound dispensing Echo 520 Liquid Handler Multidrop™ Combi system Reagent Dispenser
Detection instrument and software 2104 EnVision Multilabel Plate Reader Assay validation/QC Average Z'-score= 0.821
Correction factors Normalization Raw signal was normalized pl ate-specif ical ly by correcting row and column-specific mean signals to the mean signal of the entire plate, each after removing the highest and lowest 25% of values. Raw signal was then converted to percent of control signal, but linear regression to plate-specific mean signal of DMSO wells (set to 100 percent of control) and positive control wells (set to 0% of control), after outlier removal using a Grubbs test.
Additional comments
Post-HTS Hit criteria Percent of control < 50 % analysis
Hit rate Primary screen: 511 small molecules (including unspecific quenchers), Secondary screen (with crosslinking peptide): 40 small molecules, Tertiary screen (titration): 14 small molecules.
Additional assay(s) Secondary screen with crosslinking peptide,
Tertiary screen as 4-point titration, also with related Tudor domain of SMN
Confirmation of hit purity and structure Re-ordered and tested selected hits (Fig. 3 f-l) Additional comments
4-arylthiazole-2-amines show clear structure-activity relationships as SMNDC1 Tudor domain inhibitors
Among the most potent hit compounds were 2-amino-4-arylthiazoles and benzoxazepines. Of these classes, benzoxazepines had undesirable physicochemical properties including very low polarity (Fig. 4f, clogP = 6.33) along with poor solubility. Therefore, the inventors abandoned this series after testing a limited set of analogs (Table 2).
The inventors then selected the 4-arylthiazole-2-amine series for thorough exploration of structure-activity relationships, also due to the synthetic ease of access (Fig. 5). They found the 2-pyridyl substitution to be advantageous for binding affinity, as its replacement with certain other aryl groups led to drastic loss of potency (e.g., compounds 3-7). A 2-substituted pyrrole could be used with some loss of potency in compound 8. Omission of the aromatic group by replacement with ethoxycarbonyl resulted in complete loss of activity (compound 9).
In contrast, a wide variety of substituents were tolerated in the 2-position of the thiazole. Even the unmodified aminothiazole 13 showed a submicromolar IC50. This compound also served as the synthetic starting point for this series and related chemical probes. The amide linkage between the thiazole and the aryl group is dispensable for activity as demonstrated by the alkylamine 17 and the sulfonamide 18. Replacement of the aromatic amine by guanidine 19 decreased the IC50. The aryl amide could be substituted or replaced with a wide selection of groups, both aromatic and aliphatic rings with minor effects on potency (compounds 21-24). Among the most potent compounds were compound 1 and its morpholinosulfamoyl analog 2. The arylsulfonamide could be replaced with other groups with minimal loss of potency (compounds 25-28), whereby larger substituents as in compounds 25 and 28 increased selectivity for SMNDC1 over SMN.
The five-membered heterocycle in the core scaffold could be replaced with the isomeric scaffold 2-(pyridin-2- yl)thiazol-4-amine in compound 14. The third possible isomer, compound 15, had significant loss of activity and preferentially inhibited SMN over SMNDC1. When the thiazole was replaced with an analogous oxazole in the compound 16, there was a 40-fold drop in potency. Replacement of the thiazole with 1 ,2,4-thiadiazoles resulted in inactive compounds. Substitution of the 5-position of the thiazole of 1 with a methyl group was tolerated without loss of potency (compound 10) but an ethyl group or a bromine atom decreased the IC50 threefold (compounds 11 and 12).
These extensive structure-activity relationships revealed features that are absolutely essential for the binding of this scaffold to Tudor domains and indicate for substructures required for achieving selectivity between SMNDC1 and SMN. In the following biological characterization, the inventors focus on compound 1 as a potent Tudor domain inhibitor, validate findings with the SMNDC1 -specific compound 28 and use the inactive compound 9 as a negative control.
Table 2: Overview of tested small molecules.
Figure imgf000113_0001
Figure imgf000114_0001
Figure imgf000115_0001
Figure imgf000116_0001
2-amino-4-arylthiazoles bind the methyl-arginine pocket of the SMNDC1 Tudor domain
To prove specific binding of SMNDC1 inhibitors to the aromatic cage of SMNDC1 and to obtain structural information on the binding modes, the inventors applied nuclear magnetic resonance spectroscopy (NMR). The Tudor domains of SMN (residues 84-147) and SMNDC1 (residues 65-128) were expressed in isotope-enriched medium and purified as described elsewhere (Tripsianes, K. et al. Structural basis for dimethylarginine recognition by the Tudor domains of human SMN and SPF30 proteins. Nature Structural & Molecular Biology 18, 1414-1420 (2011)). Since organic solvents such as dimethyl sulfoxide showed non-specific binding to the Tudor domains and led to interference in the NMR experiments, the inventors attempted to use an aqueous, buffered solution of compound 1, which however exhibited insufficient solubility for NMR experiments. They therefore prepared an aqueous, buffered solution of the monobasic phosphate salt of compound 13 and assessed its concentration by comparing signal intensities to a DSS standard. NMR titrations of compound 13 with SMN84-W and SMNDC165-128 showed significant chemical shift perturbations (CSP) with binding kinetics reflecting fast-exchange (gradual change of chemical shift with increasing ligand concentration (Gobi, C., Madl, T., Simon, B. & Sattler, M. NMR approaches for structural analysis of multidomain proteins and complexes in solution. Progress in Nuclear Magnetic Resonance Spectroscopy 80, 26-63 (2014))) for both proteins (Fig. 6a, c). CSP are highly sensitive to changes of the local chemical environment of the observed nuclear spin and therefore excellent reporters to map binding sites of a ligand and (potentially associated) conformational changes. The largest CSP are observed for the amino acids forming the aromatic cage (W83, Y90, F108, Y111) and the surrounding residues. Additionally, some parts of the P2-strand show significant chemical shifts with increasing concentration of compound 13. The affected residues and CSP match very well the ones published for sDMA binding (Tripsianes, K. et al. loc. cit), with exception of residues W83 and S84, suggesting a different interaction with the aromatic cage's tryptophan, as well as N113.
In order to obtain higher resolution structural information of the recognition of compound 13 by the Tudor domain the inventors recorded 13C-filtered NOESY experiments using a 1 mM 15N,13C-labeled SMNDC1 Tudor domain with a 20- fold excess of compound 13. They could observe a number of contacts between the Tudor domain and the ligand by intermolecular nuclear Overhauser effects (NOE), most prominently with aromatic protons of the Tudor binding site identified by the CSP (Fig. 6b, d, Table 3). Using the intermolecular NOEs the inventors calculated a rigid model docking calculation using HADDOCK (van Zundert, G. C. P. et al. The HADDOCK2.2 Web Server: User-Friendly Integrative Modeling of Biomolecular Complexes. Journal of Molecular Biology 428, 720-725 (2016); Honorato, R. V. et al. Structural Biology in the Clouds: The WeNMR-EOSC Ecosystem. Frontiers in Molecular Biosciences 8, (2021)), which yielded one cluster with low structural deviation (Table 4). The structure indicates that the ring nitrogens of compound 13 are in a cis conformation in the complex. The pyridine moiety stacks inside the aromatic cage and its aromatic protons (H2-H5) are showing multiple contacts with the protein's aromatic residues, while the thiazole proton has considerably less contacts to the protein (Fig. 6d, Table 3, Fig. 11 a). The pyridine of compound 13 forms tight TT- TT stacking contacts with the aromatic rings of F83 and Y111 with distances of 3.7 A to each, which underlines the importance of an aromatic substituent at the thiazole 4-position. The aromatic moieties of Y90 and F108 stand perpendicular to the pyridine ring while the sidechain of N113 is enclosing it from the opposite site. Overall, the structure shows high similarity to SMNDC1/sDMA (PDB: 4A4H) (Fig. 11c) and is fully consistent with the predicted binding mode.
Table 3: Assigned intermolecular NOEs between SMNDC1 and inhibitor 13 and associated restraint upper limits.
Figure imgf000117_0001
Table 4: SMNDC1/ compound 13 molecular-docking results of structure ensemble generated using the HADDOCK webserver. Statistics generated over all 200 analyzed structures.
Figure imgf000117_0002
a Restraints listed in Table 3. b The violated distance involves proton 13-H2 and Y90-HE. SMNDC1 Tudor domain inhibitors impact protein localization and splicing
The inventors then went on to analyze the effects of the identified small molecule binders on SMNDCTs phase separation. Using the endogenously tagged cell lines, they observed strong effects on the levels and distribution of SMNDC1. Treating the cells with 50 piM of compound 1 for 12-16h leads to a loss of SMNDC1 within the nucleus (Fig. 7a, quantification Fig. 7b,). Additionally, the subnuclear distribution changed and less spots were detected within the nucleus (quantification Fig. 7c). These effects were not observed with the negative control compound 9 which lacks the 2-pyridy I crucial for the binding to SMNDC1 . Co-staining nuclei with Hoechst showed that nuclear structure was not affected and that these cells were in interphase (Fig. 12a). Longer treatment with compound 1 resulted in cell death. While the percentage of AnnexinV and Propidium Iodide (PI) positive cells was elevated, the majority of cells was not apoptotic or undergoing other forms of cell death at this timepoint (Fig. 12b), and cell death was only observed at later timepoints.
Using the cell-line in which SMNDC1 and SRRM2 are both tagged the inventors examined the effects of compound 1 on nuclear speckles. Upon treatment with inhibitor 1, but not compound 9, SRRM2 and therefore general organization of nuclear speckles was also affected. The overall SRRM2 intensity upon treatment was slightly reduced, and spots appeared to dissolve into the nucleoplasm (Fig. 7d, quantifications Fig. 7e and Fig. 12c). Treating several independent SRRM2-RFP clones replicated the results for inhibitor 1 (Fig 12f). These results could also be confirmed using antibodies against SMNDC1 and SC35 in IF (Fig. 12f).
To check whether inhibition of SMNDC1 is indeed responsible for the effects on nuclear speckles, the inventors silenced SMNDC1 with and without the inhibitor (Fig. 7f, images Fig. 12g). The knock-down of SMNDC1 also led to a reduction of SRRM2 intensity and even more pronounced to a reduction of SRRM2 spots in the nucleus, confirming the importance of SMNDC1 for the integrity of nuclear speckles. Treatment with the inhibitor could not further increase these effects, hinting that it is not unspecific effects of the inhibitor that cause the disruption of nuclear speckles. Furthermore, the inventors tested the SMNDCI-selective compound 28 for its effects on SMNDC1 and SRRM2 localization and could confirm the effects observed for the non-selective compound 1 (Fig. 12h), even at lower concentrations (Fig. 121).
To directly test the effect on SMN with its similar Tudor domain, the inventors created cell lines in which SMN1 was endogenously tagged with RFP. Treating these cells with 50 piM of compound 1 for 16h showed effects on SMN. Overall intensity of SMN decreased while number of spots (supposedly stress granules) in the cytoplasm increased (Fig. 12j).
Next, the inventors analyzed the effect of compound 1 on the proximity interactome of SMNDC1. Overall, they observed that upon inhibitor treatment, more proteins showed a reduced interaction (volcano plot skewed towards down-regulated side, many more significantly down-regulated than up-regulated proteins, Fig. 7g). This indicates that the inhibitor blocks SMNDCTs function to bind to its interaction partners. Compared to APEX2-SMNDC1 FL, the inhibitor effects in APEX2-SMNDC1TD were diminished, presumably due to less specific interactions in the truncated form at baseline (Fig. 13a). 126 proteins were significantly depleted in APEX2-SMNDC1 FL treated with inhibitor vs. none in APEX2-SMNDC1TD (adjusted p-value <0.05, Iog2 fold-change <-2). Proteins with known sDMA modifications identified in SMNDCTs interactome were among the most depleted upon inhibitor treatment (Fig. 7h). The same is true for proteins with assigned localization to nuclear speckles identified in the dataset, including SRRM2 and the other main nuclear speckle organizer SON (llik, i. A. et al. SON and SRRM2 are essential for nuclear speckle formation. eLife 9, e60579 (2020)) (Fig. 13b), and for proteins identified by SRSF7-APEX2 (Barutcu, A. R. et al. Systematic mapping of nuclear domain-associated transcripts reveals speckles and lamina as hubs of functionally distinct retained introns. Mol Cell 82, 1035-1052. e9 (2022)) (Fig. 13c). To confirm the observed effects of the inhibitor on the interactome the inventors performed a western blot analysis of the biotin-labeled proteins after pull-down (Fig. 13d). Also using this orthogonal technique, they see a general loss of labeled interactors upon treatment with the inhibitor, only with APEX2-SMNDC1 FL but not with APEX2-SMNDC1TD. Furthermore, the inventors can confirm the loss of interactions to specific proteins, e.g., the sDMA-modified splicing factor SFPQ or the loss of trans-interactions to SMNDC1 itself. Interactions to SMNDC1 itself are lost both to the endogenous protein (30 kDa band with antibody against SMNDC1) and APEX2-Fusion protein (60 kDa band with antibodies against SMNDC1 and APEX2).
As the inventors did not observe instantaneous effects of the inhibitor on the architecture of nuclear speckles by quantifying intensity or spots per nucleus, they tested whether the inhibitor immediately influenced mobility of proteins within nuclear speckles. To this end, they applied the inhibitor 1 to live cells at a concentration of 50 piM and measured FRAP within a timeframe of 15- 45 min (Fig. 7i). Indeed, they detected a lower recovery after photobleaching for both SMNDC1 and SRRM2 when cells were treated with compound 1 while reference regions were not affected (Fig. 13e). To test specific cellular effects of the SMNDC1 inhibitor 1 the inventors performed paired-end RNA sequencing and analyzed alternative splicing events using Vertebrate Alternative Splicing and Transcription Tools (VAST-TOOLS) (Tapial, J. et al. An atlas of alternative splicing profiles and functional associations reveals new regulatory programs and genes that simultaneously express multiple major isoforms. Genome Res. 27, 1759-1768 (2017)). They observed that inhibition of SMNDC1 with compound 1 led to an increased retention of introns and skipping of exons, very similar to the effects of SMNDC1 knock-down (Casteels, T. et al. SMNDC1 links chromatin remodeling and splicing to regulate pancreatic hormone expression. Cell Reports 40, (2022)) (Fig. 7j). Directly comparing the differential percentage spliced-in (dPSI) values for alternative splicing events the inventors found a significant correlation between knock-down and small-molecule inhibition of SMNDC1 (Fig. 7k). They went on to test a panel of individual events with the biggest dPSI values in both knock-down and compound 1 treatment or known to be differentially spliced upon knock-down of SMNDC1 (Casteels, T. et al. loc. cit.) (3 selected events Fig. 7I and the full panel Fig 13f). For the majority of events, the inventors confirmed the expected effect of the inhibition of SMNDC1 leading to the appearance of alternative spliced isoforms, comparable to SMNDC1 knock-down. Interestingly, they could also see the effect with a long-term, low-dose treatment (2 piM over 5 days) which is more comparable to the 5 days knock-down. Combining knock-down and inhibitor treatments again did not show synergistic effects.
Overall, the inventors demonstrated specific effects of the inhibitor on the splicing function and the localization of SMNDC1 to nuclear speckles and its proximity to interaction partners, and to the architecture of nuclear speckles in general.
Discussion
Previous work has shown that the interaction of the Tudor domain of SMNDCTs paralog SMN with dimethylarginine causes biomolecular condensation (Courchaine, E. M. et al. DMA-tudor interaction modules control the specificity of in vivo condensates. Ce// 184, 3612-3625.e17 (2021)). Here, using endogenous fluorescent tags and in vitro assays, the inventors show that also SMNDC1 undergoes phase separation. They find that SMNDC1 localizes to phase- separated membraneless organelles within the nucleus, partially overlapping with nuclear speckles. Consistent with previous findings (Courchaine, E. M. et al. loc. cit.) and in contrast to SMN, the SMNDC1 Tudor domain alone is not driving this condensation behavior. Rather, the protein's C-terminal I DR is sufficient for droplet formation in vitro. An RNA-binding prediction algorithm, RNAbindRplus (Walia, R. R. et al. RNABindRPIus: A Predictor that Combines Machine Learning and Sequence Homology-Based Methods to Improve the Reliability of Predicted RNA-Binding Residues in Proteins. PLOS ONE 9, e97725 (2014)), suggests that the C-terminus, especially residues 177-201 , interacts with RNA (Fig. 14). The inventors therefore hypothesize that the C-terminal IDR is binding to RNAs which in turn recruit further proteins. This model is consistent with their earlier observation (Casteels, T. et al. SMNDC1 links chromatin remodeling and splicing to regulate pancreatic hormone expression. Cell Reports 40, (2022)) that the majority of SMNDC1 protein interactions are lost when RNA is hydrolyzed. Likely these RNA-mediated interactors together with arginine methyl interactions mediated by the SMNDC1 Tudor domain constitute the multivalent binding platform that is a typical prerequisite in the formation of biomolecular condensates. Only the combination of the C- terminal IDR and the Tudor domain (and the nuclear localization signal) is sufficient for the correct localization of SMNDC1 and the full spectrum of interactions to both proteins and RNA. Picking apart the individual contributions of the different parts of the proteins is challenging, as the C-terminal region also harbors the NLS responsible for correct organelle localization, with the regions flanking the NLS particularly disordered (Fig. 1 a). From the present data it is not certain whether SMNDC1 can form nuclear droplets on its own in cellulo as a scaffold or which proteins are required for SMNDC1 to localize to pre-formed membraneless organelles as a client. However, the inventors do observe the dissolution of nuclear speckles upon SMNDC1 inhibition or knock-down. While other factors like SON and SRRM2 are known to be important for the formation of nuclear speckles (llik, i. A. et al. SON and SRRM2 are essential for nuclear speckle formation. eLife 9, e60579 (2020)), these data hint at an important structural role of SMNDC1 in these membraneless organelles.
To generate chemical tools for the dose- and time-dependent study of SMNDC1 function, the inventors focused on the protein's Tudor domain. In contrast to the C-terminal region, the Tudor domain exhibits a well-defined structure with a characteristic aromatic cage that mediates specific recognition of dimethylarginine ligands. This feature potentially enables small molecule binding often referred to as druggability. The inventors thus set out to identify small molecule inhibitors of SMNDCTs Tudor domain using an AlphaScreen set-up. Some of the hit structures from their 90,000 compound library showed selectivity in only binding the SMNDC1 but not the SMN Tudor domain and follow up studies allowed them to derive structure-activity relationships for these compounds. Interestingly, these compounds are also active in cellular assays, although relatively high concentrations are needed. Then, the most promising inhibitor led to a loss of SMNDC1 from the nucleus and nuclear speckles and diminished SMNDCTs interaction with its partners. The kinetics the inventors observe provide a first hint how inhibiting SMNDCTs Tudor domain might influence the architecture of nuclear speckles. While it does not immediately disrupt existing nuclear speckles, mobility and potentially inclusion of new proteins into the phase-separated compartment might be affected, leading to disruption over time. These data suggest that in cells the inhibition of the Tudor domain mediated interactions with its dimethylarginine binding partners drastically affects the protein function even with an intact C- terminal region. This perturbation then results in global splicing changes, consistent with the canonical function of the protein.
The inventors have identified the first specific inhibitors of SMNDCTs Tudor domain influencing SMNDCTs phaseseparation behavior and splicing and architecture of nuclear speckles. These compounds are chemically distinct from inhibitors previously described for other Tudor domain proteins (Arrowsmith, C. H. & Schapira, M. Targeting non- bromodomain chromatin readers. Nat Struct Mol Biol 26, 863-869 (2019); Zhu, H., Wei, T., Cai, Y. & Jin, J. Small Molecules Targeting the Specific Domains of Histone-Mark Readers in Cancer Therapy. Molecules 25, 578 (2020)), namely TP53B1 (Sun, Y. et al. Discovery of a novel 53BP1 inhibitor through AlphaScreen-based high-throughput screening. Bioorganic & Medicinal Chemistry 34, 116054 (2021); Perfetti, M. T. et al. Identification of a fragment-like small molecule ligand for the methyl-lysine binding protein, 53BP1. ACS C/?em. Biol. 10, 1072-1081 (2015)), Spindlin- 1 (Wagner, T. et al. Identification of a small-molecule ligand of the epigenetic reader protein Spindlin 1 via a versatile screening platform. Nucleic Acids Res. 44, e88 (2016); Bae, N. et al. Developing Spindlinl Small Molecule Inhibitors Using Protein Microarrays. Nat Chem Biol 13, 750-756 (2017); Fagan, V. et al. A Chemical Probe for Tudor Domain Protein Spindlinl to Investigate Chromatin Function. J. Med. Chem. 62, 9008-9025 (2019); Xiong, Y. et al. Discovery of a Potent and Selective Fragment-like Inhibitor of Methyllysine Reader Protein Spindlin 1 (SPIN1). J. Med. Chem. (2019) doi:10.1021/acs.jmedchem.9b00522), UHRF1 (Senisterra, G. et al. Discovery of Small-Molecule Antagonists of the H3K9me3 Binding to UHRF1 Tandem Tudor Domain. SLAS DISCOVERY: Advancing the Science of Drug Discovery 23, 930-940 (2018); Chang, L. et al. Discovery of small molecules targeting the tandem tudor domain of the epigenetic factor UHRF1 using fragment-based ligand discovery. Sci Rep 11, (2021); Kori, S. et al. Structurebased screening combined with computational and biochemical analyses identified the inhibitor targeting the binding of DNA Ligase 1 to UHRF1. Bioorganic & Medicinal Chemistry 52, 116500 (2021)), TDRD3 (Liu, J. et al. Structural plasticity of the TDRD3 Tudor domain probed by a fragment screening hit. FEBS J. 285, 2091-2103 (2018)), KDM4A (Upadhyay, A. K. et al. Targeting lysine specific demethylase 4A (KDM4A) tandem TUDOR domain - A fragment based approach. Bioorg. Med. Chem. Lett. 28, 1708-1713 (2018)), SETDB1 (Mader, P. et al. Identification and characterization of the first fragment hits for SETDB1 Tudor domain. Bioorganic & Medicinal Chemistry 27, 3866- 3878 (2019)), PHF1 (Engelberg, I. A. etal. Discovery of an H3K36me3-Derived Peptidomimetic Ligand with Enhanced Affinity for Plant Homeodomain Finger Protein 1 (PHF1). J Med Chem (2021) doi:10.1021/acs.jmedchem.1c00430), and SMN (Liu, Y. et al. A small molecule antagonist of SMN disrupts the interaction between SMN and RNAP II. Nat Commun 13, 5453 (2022)). The inventors' structure-activity relationships indicate that it is feasible to develop these compounds further to achieve specificity for SMNDC1 compared to its closest paralog SMN. Since the other ~34 human Tudor domain proteins in humans (Blanc, R. S. & Richard, S. Arginine Methylation: The Coming of Age. Mol. Cell 65, 8-24 (2017); Gayatri, S. & Bedford, M. T. Readers of histone methylarginine marks. Biochimica etBiophysica Acta (BBA) - Gene Regulatory Mechanisms 1839, 702-710 (2014)) are less conserved, the inventors expect even lower affinities, but it will be important to conduct unbiased analyses of potential off-targets and their contribution to cellular phenotypes.
Example C: Evaluation of compounds according to the invention for their inhibitory activity on SMNDC and SMN1
In addition to the compounds of formula (I) tested in Example B, a number of further exemplary compounds of formula (I) as well as several reference compounds were tested for their inhibitory activity on SMNDC and SMN1 , respectively, following the same procedure as described in Example B. The thus determined IC50 values of these compounds for SMNDC and for SMN1 are summarized in the following table:
Figure imgf000122_0001
Figure imgf000123_0001
Figure imgf000124_0001
Figure imgf000125_0001
Figure imgf000126_0001
Figure imgf000127_0001
Figure imgf000128_0001
Figure imgf000129_0001
Figure imgf000130_0001
Figure imgf000131_0001
Figure imgf000132_0001
Figure imgf000133_0001
Figure imgf000134_0001
These results demonstrate that the compounds of formula (I) are potent inhibitors of SMNDC1. Moreover, as also reflected by these results, the present invention provides inhibitors that are selective for SMNDC1 over SMN. These properties render the compounds provided in accordance with the present invention highly advantageous for therapeutic use. Example D: Inhibition of cell viability in pancreatic cancer and ovarian cancer cells
Various compounds provided herein were tested for their cytotoxic effect on pancreatic cancer cells and ovarian cancer cells using a cell viability assay.
The pancreatic cancer cell line PANC1 was cultivated in standard tissue culture conditions in DMEM containing 10% fetal bovine serum. The ovarian cancer cell line OV90 was cultivated in standard conditions using a 1 :1 mixture of MCDB 105 medium containing a final concentration of 1.5 g/L sodium bicarbonate and Medium 199 containing a final concentration of 2.2 g/L sodium bicarbonate supplemented with 15% fetal bovine serum. To test compound effects, 50 pl cell suspension was seeded on top of 60 nl compound in DMSO in 384-well plates (Corning 3701). After 72 h cell viability was measured using the Cell Titer Gio Luminescent Cell Viability Assay (Promega) on an Envision Plate Reader (Perkin Elmer).
The I C50 values thus determined for the tested compounds are shown in the following table:
Figure imgf000135_0001
Figure imgf000136_0001
These results further demonstrate that the compounds according to the present invention are therapeutically effective in the treatment of cancer, including pancreatic cancer and ovarian cancer.

Claims

1 . A compound of the following formula (I)
Figure imgf000137_0001
wherein: one of the ring atoms X1 and X2 is S, and the other one is C(-Rx);
Rx is selected from hydrogen, C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alky lene)-0(Ci-5 alkyl), -(C0-5 alky lene)-0(Ci-5 alky lene)-OH, -(C0-5 alky lene)-0(Ci-5 alky lene)-0(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-NH2, -(C0-5 alkylene)-NH(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-OH, -(C0-5 alkylene)-N(Ci-5 alkyl)-OH, -(C0-5 alkylene)-NH-O(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-O(Ci-5 alkyl), -(C0-5 alkylene)-halogen, -(C0-5 alkylene)-(Ci-5 haloalkyl), -(C0-5 alkylene)-O-(Ci-5 haloalkyl), -(C0-5 alkylene)-CN, -(C0-5 alkylene)-NO2, -(C0-5 alkylene)-CHO, -(C0-5 alkylene)-CO-(Ci-5 alkyl), -(C0-5 alkylene)-CO-(Ci-5 haloalkyl), -(C0-5 alkylene)-COOH, -(C0-5 alkylene)-CO-O-(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-(Ci-5 alkyl), -(C0-5 alkylene)-CO-NH2, -(C0-5 alkylene)-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-CO-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-CO-(Ci-5 alkyl), -(C0-5 alkylene)-NH-COO(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-COO(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-SO2-NH2, -(C0-5 alkylene)-SO2-NH(Ci-5 alkyl), -(C0-5 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-SO-(Ci-5 alkyl), -(C0-5 alkylene)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-carbocyclyl, -(C0-5 alkylene)-heterocyclyl, and -Lz-Rz, wherein the carbocyclyl in said -(C0-5 alkylene)-carbocyclyl and the heterocyclyl in said -(C0-5 alky lene)-heterocycly I are each optionally substituted with one or more groups RCyc;
R1 is -L1-L2-R11;
L1 is selected from a covalent bond, -CO-, -SO-, -SO2-, -CO-N(RN)-, -SO-N(RN)-, -SO2-N(RN)-, and -C(=N-RN)-N(RN)-, wherein each RN is independently hydrogen or C1.5 alkyl; L2 is selected from a covalent bond, CMO alkylene, C2-10 alkenylene, C2-10 alkynylene, -(C0-5 alkylene)-carbocyclyl-(Co-5 alkylene)-, and -(C0-5 alkylene)-heterocyclyl-(Co-5 alkylene)-, wherein said C1-10 alkylene, said C2-10 alkenylene, said C2-10 alkynylene, the two C0-5 alkylene groups in said -(C0-5 alkylene)-carbocyclyl-(Co-5 alkylene)-, and the two C0-5 alkylene groups in said -(C0-5 alky lene)-heterocycly l-(Co-5 alkylene)- are each optionally substituted with one or more groups RAlk, wherein one or more -CH2- units comprised in said C1-10 alkylene, said C2-10 alkenylene, said C2-10 alkynylene, any of the C0-5 alkylene groups in said -(C0-5 alky lene)-carbocyclyl-(Co-5 alkylene)-, or any of the C0-5 alkylene groups in said -(C0-5 alkylene)-heterocyclyl-(Co-5 alkylene)- are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -N(RL2)-, and -C(RL2)=C(RL2)-, wherein each RL2 is independently hydrogen or C1-5 alkyl, and further wherein the carbocyclyl group in said -(C0-5 alky lene)-carbocycly l-(Co-5 alkylene)- and the heterocyclyl group in said -(C0-5 alkylene)-heterocyclyl are each optionally substituted with one or more groups RCyc;
R11 is selected from hydrogen, -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(Ci_5 alkyl), -S(Ci_5 alkylene)-SH, -S(Ci-5 alkylene)-S(Ci_5 alkyl), -NH2, -NH(CI-5 alkyl), -N(CI-5 alkyl)(Ci_5 alkyl), -NH-OH, -N(CI.5 alkyl)-OH, -NH-O(CI.5 alkyl), -N(CI.5 alkyl)-O(Ci_5 alkyl), halogen, C1.5 haloalkyl, -O-(Ci-5 haloalkyl), -ON, -NO2, -OHO, -C0-(Ci-5 alkyl), -COCH, -CO-O-(Ci.5 alkyl), -O-CO-(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(CI.5 alkyl)(Ci.5 alkyl), -NH-CO-(Ci.5 alkyl), -N(CI-5 alkyl)-CO-(Ci_5 alkyl), -NH-COO(Ci.5 alkyl), -N(CI-5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(Ci.5 alkyl), -O-CO-N(Ci.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(OI.5 alkyl), -SO2-N(Ci-5 alkyl)(Ci.5 alkyl), -NH-SO2-(Ci-5 alkyl), -N(CI-5 alkyl)-SO2-(Ci-5 alkyl), -SO-(Ci-5 alkyl), -SO2-(Ci-5 alkyl), -(Co-15 alkylene)-carbocyclyl, -(Co-15 alkylene)-heterocyclyl, and -Lz-Rz, wherein each alkyl or alkylene comprised in any of the aforementioned groups is optionally substituted with one or more groups RAlk, wherein one or more -CH2- units comprised in the Co-15 alkylene in said -(Co-15 alkylene)-carbocyclyl or in the Co-15 alkylene in said -(Co-15 alky lene)-heterocycly I are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -NH-, and -N(CI-5 alkyl)-, and wherein the carbocyclyl in said -(Co-15 alky lene)-carbocycly I and the heterocyclyl in said -(Co-15 alky lene)-heterocycly I are each optionally substituted with one or more groups RCyc;
R2 is selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, -(Co-10 alkylene)-OH, -(Co-10 alkylene)-O(Ci-5 alkyl), -(Co-10 alkylene)-SH, -(Co-10 alkylene)-S(Ci-5 alkyl), -(C1-10 alkylene)-NH2, -(C1-10 alkylene)-NH(Ci-5 alkyl), -(C1-10 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C1-10 alkylene)-halogen, C1-10 haloalkyl, -(Co-10 alkylene)-O-(Ci-5 haloalkyl), -(Co-10 alkylene)-CN, -(Co-10 alkylene)-NO2, -(Co-10 alkylene)-CHO, -(Co-10 alkylene)-CO-(Ci-io alkyl), -(Co-10 alkylene)-COOH, -(Co-10 alkylene)-CO-O-(Ci-5 alkyl), -(Co-10 alkylene)-O-CO-(Ci-5 alkyl), -(Co-10 alkylene)-CO-NH2, -(Co-10 alkylene)-CO-NH(Ci-5 alkyl), -(Co-10 alkylene)-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C1-10 alkylene)-NH-CO-(Ci-5 alkyl), -(C1-10 alkylene)-N(Ci-5 alkyl)-CO-(Ci-5 alkyl), -(C1-10 alkylene)-NH-COO(Ci-5 alkyl), -(C1-10 alkylene)-N(Ci-5 alkyl)-COO(Ci-5 alkyl), -(Co-10 alkylene)-O-CO-NH(Ci-5 alkyl), -(Co-10 alkylene)-O-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(Co-10 alkylene)-SC>2-NH2, -(Co-10 alkylene)-SO2-NH(Ci-5 alkyl), -(Co-10 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C1-10 alkylene)-NH-SO2-(Ci-5 alkyl), -(C1-10 alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(Co-10 alkylene)-S0-(Ci-5 alkyl), -(Co-10 alkylene)-SO2-(Ci-5 alkyl), -(Co-10 alkylene)-carbocyclyl, and -(Co-10 alkylene)-heterocyclyl, wherein one or more -CH2- units comprised in said C1-10 alkyl, said C2-10 alkenyl, said C2-10 alkynyl, or in any of the aforementioned Co-10 alkylene or C1-10 alkylene groups are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO- , -NH-, and -N(CI-5 alkyl)-, and further wherein the carbocyclyl in said -(Co-10 alkylene)-carbocyclyl and the heterocyclyl in said -(Co-10 alkylene)-heterocyclyl are each optionally substituted with one or more groups RCyc;
R3 is -(C0-5 alkylene)-aryl or -(C0-5 alkylene)-heteroaryl, wherein the aryl in said -(C0-5 alkylene)-aryl and the heteroaryl in said -(C0-5 alkylene)-heteroaryl are each optionally substituted with one or more groups R31, and wherein one or more -CH2- units comprised in the C0-5 alkylene in said -(C0-5 alkylene)-aryl or in the C0-5 alkylene in said -(C0-5 alkylene)-heteroaryl are each optionally replaced by a group independently selected from -0-, -S-, -SO-, -SO2-, -CO-, -NH-, and -N(Ci-5 alkyl)-; each R31 is independently selected from C1.5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-0(Ci-5 alkyl), -(C0-5 alkylene)-0(Ci-5 alkylene)-OH, -(C0-5 alkylene)-0(Ci-5 alkylene)-0(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-NH2, -(C0-5 alkylene)-NH(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-OH, -(C0-5 alkylene)-N(Ci-5 alkyl)-OH, -(C0-5 alkylene)-NH-0(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-0(Ci-5 alkyl), -(C0-5 alkylene)-halogen, -(C0-5 alkylene)-(Ci-5 haloalkyl), -(C0-5 alkylene)-0-(Ci-5 haloalkyl), -(C0-5 alkylene)-CN, -(C0-5 alkylene)-N02, -(C0-5 alkylene)-CHO, -(C0-5 alkylene)-C0-(Ci-5 alkyl), -(C0-5 alkylene)-COOH, -(C0-5 alkylene)-C0-0-(Ci-5 alkyl), -(C0-5 alkylene)-0-C0-(Ci-5 alkyl), -(C0-5 alkylene)-C0-NH2, -(C0-5 alkylene)-C0-NH(Ci-5 alkyl), -(C0-5 alkylene)-C0-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-C0-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-C0-(Ci-5 alkyl), -(C0-5 alkylene)-NH-C00(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-C00(Ci-5 alkyl), -(C0-5 alkylene)-0-C0-NH(Ci-5 alkyl), -(C0-5 alkylene)-0-C0-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-SO2-NH2, -(C0-5 alkylene)-SO2-NH(Ci-5 alkyl), -(C0-5 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-S0-(Ci-5 alkyl), -(C0-5 alkylene)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-carbocyclyl, -(C0-5 alkylene)-heterocyclyl, and -Lz-Rz, wherein the carbocyclyl in said -(C0-5 alkylene)-carbocyclyl and the heterocyclyl in said -(C0-5 alky lene)-heterocycly I are each optionally substituted with one or more groups R°yc, and wherein one or more -CH2- units comprised in the C0-5 alkylene in said -(C0-5 alkylene)-carbocyclyl or in the C0-5 alkylene in said -(C0-5 alkylene)-heterocyclyl are each optionally replaced by a group independently selected from -0-, -S-, -SO-, -SO2-, -CO-, -NH-, and -N(CI-5 alkyl)-; each RAlk is independently selected from -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(Ci-5 alkyl), -S(Ci-5 alkylene)-SH, -S(Ci-5 alkylene)-S(Ci-5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI.5 alkyl)(Ci_5 alkyl), -NH-OH, -N(CI.5 alkyl)-OH, -NH-O(CI.5 alkyl), -N(CI-5 alkyl)-O(Ci-5 alkyl), halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -ON, -NO2, -CHO, -CO(Ci.5 alkyl), -COOH, -COO(Ci.5 alkyl), -O-CO(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(CI.5 alkyl)(Ci_5 alkyl), -NH-CO(CI.5 alkyl), -N(CI.5 alkyl)-CO(Ci.5 alkyl), -NH-COO(CI.5 alkyl), -N(CI-5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(CI.5 alkyl), -O-CO-N(CI.5 alkyl)(Ci_5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci_5 alkyl), -NH-SO2-(CI.5 alkyl), -N(CI.5 alkyl)-SO2-(Ci-5 alkyl), -SO2-(Ci-5 alkyl), -SO-(Ci-5 alkyl), -(C0-5 alkylene)-cycloalkyl, -(C0-5 alkylene)-heterocycloalkyl, and -Lz-Rz; each RCyc is independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-O(Ci-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-NH2, -(C0-5 alkylene)-NH(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-OH, -(C0-5 alkylene)-N(Ci-5 alkyl)-OH, -(C0-5 alkylene)-NH-O(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-O(Ci-5 alkyl), -(C0-5 alkylene)-halogen, -(C0-5 alkylene)-Ci-5 haloalkyl, -(C0-5 alkylene)-O-(Ci-5 haloalkyl), -(C0-5 alkylene)-CN, -(C0-5 alkylene)-NO2, -(C0-5 alkylene)-CHO, -(C0-5 alkylene)-CO(Ci-5 alkyl), -(C0-5 alkylene)-COOH, -(C0-5 alkylene)-COO(Ci-5 alkyl), -(C0-5 alkylene)-O- CO(Ci-5 alkyl), -(C0-5 alkylene)-CO-NH2, -(C0-5 alkylene)-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-CO(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-CO(Ci-5 alkyl), -(C0-5 alkylene)-NH-COO(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-COO(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-SO2-NH2, -(C0-5 alkylene)-SO2-NH(Ci-5 alkyl), -(C0-5 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-SO-(Ci-5 alkyl), -(C0-5 alkylene)-carbocyclyl, -(C0-5 alkylene)-heterocyclyl, and -Lz-Rz; each Lz is independently selected from a covalent bond, C1-35 alkylene, C2-35 alkenylene, and C2-35 alkynylene, wherein said alkylene, said alkenylene and said alkynylene are each optionally substituted with one or more groups independently selected from halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -CN, -OH, -O(Ci.5 alkyl), -SH, -S(Ci.5 alkyl), -NH2, -NH(CI.5 alkyl), and -N(CI.5 alkyl)(Ci.5 alkyl), wherein one or more CH units comprised in said alkylene, said alkenylene or said alkynylene are each optionally replaced by a nitrogen atom, and further wherein one or more -CH2- units comprised in said alkylene, said alkenylene or said alkynylene are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -NH-, -N(CI-5 alkyl)-, carbocyclylene, and heterocyclylene, wherein said carbocyclylene and said heterocyclylene are each optionally substituted with one or more groups independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -CN, -NO2, -OH, -O(Ci-5 alkyl), -SH, -S(Ci-5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI.5 alkyl)(Ci.5 alkyl), -CHO, -CO-(Ci.5 alkyl), -COOH, -CO-O-(Ci.5 alkyl), -O-CO-(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(CI.5 alkyl)(Ci_5 alkyl), -NH-CO-(CI.5 alkyl), -N(CI.5 alkyl)-CO-(Ci.5 alkyl), -NH-COO(CI.5 alkyl), -N(CI.5 alkyl)-COO(Ci_5 alkyl), -O-CO- NH(CI.5 alkyl), -O-CO-N(CI.5 alkyl)(Ci_5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci.5 alkyl), -NH-SO2-(CI.5 alkyl), -N(CI.5 alkyl)-SO2-(Ci.5 alkyl), -SO-(Ci.5 alkyl), and -SO2-(Ci.5 alkyl); and each Rz is independently selected from -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(Ci-5 alkyl), -S(Ci-5 alkylene)-SH, -S(Ci-5 alkylene)-S(Ci-5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI.5 alkyl)(Ci_5 alkyl), -NH-OH, -N(CI.5 alkyl)-OH, -NH-O(CI.5 alkyl), -N(CI-5 alkyl)-O(Ci-5 alkyl), halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -ON, -NO2, -CHO, -CO(Ci.5 alkyl), -COOH, -COO(Ci.5 alkyl), -O-CO(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(CI-5 alkyl)(Ci.5 alkyl), -NH-CO(CI.5 alkyl), -N(CI.5 alkyl)-CO(Ci.5 alkyl), -NH-COO(CI.5 alkyl), -N(CI.5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(CI.5 alkyl), -O-CO-N(CI.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci.5 alkyl), -NH-SO2-(CI.5 alkyl), -N(CI.5 alkyl)-SO2-(Ci-5 alkyl), -SO2-(Ci-5 alkyl), -SO-(Ci-5 alkyl), aryl, heteroaryl, cycloalkyl, and heterocycloalkyl, wherein said aryl, said heteroaryl, said cycloalkyl, and said heterocycloalkyl are each optionally substituted with one or more groups independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -ON, -NO2, -OH, -O(Ci-5 alkyl), -SH, -S(Ci.5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI.5 alkyl)(Ci_5 alkyl), -CHO, -CO-(Ci.5 alkyl), -COOH, -CO-O-(Ci.5 alkyl), -O-CO-(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(CI.5 alkyl)(Ci.5 alkyl), -NH-CO-(CI.5 alkyl), -N(CI.5 alkyl)-CO-(Ci.5 alkyl), -NH-COO(CI.5 alkyl), -N(CI.5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(CI.5 alkyl), -O-CO-N(CI.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci.5 alkyl), -NH-SO2-(CI.5 alkyl), -N(CI.5 alkyl)-SO2-(Ci-5 alkyl), -SO-(Ci-5 alkyl), -SO2-(Ci-5 alkyl), carbocyclyl, and heterocyclyl, wherein said carbocyclyl and said heterocyclyl are each optionally substituted with one or more groups independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -ON, -NO2, -OH, -O(Ci.5 alkyl), -SH, -S(Ci.5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI.5 alkyl)(Ci.5 alkyl), -CHO, -CO-(Ci.5 alkyl), -COOH, -CO-O-(Ci.5 alkyl), -O-CO-(Ci.5 alkyl), -CO-NH2, -CO-NH(CI-5 alkyl), -CO-N(CI.5 alkyl)(Ci_5 alkyl), -NH-CO-(CI.5 alkyl), -N(CI.5 alkyl)-CO-(Ci.5 alkyl), -NH-COO(CI.5 alkyl), -N(CI.5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(CI.5 alkyl), -O-CO-N(CI-5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci.5 alkyl), -NH-SO2-(CI.5 alkyl), -N(CI.5 alkyl)-SO2-(Ci.5 alkyl), -SO-(Ci.5 alkyl), and -SO2-(Ci.5 alkyl); or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment or prevention of cancer or diabetes.
2. The compound for use according to claim 1 , wherein X1 is S, and X2 is C(-Rx).
3. The compound for use according to claim 1 or 2, wherein Rx is selected from hydrogen, C1-5 alkyl, -OH, -O(Ci.5 alkyl), -SH, -S(Ci.5 alkyl), -NH2, -NH(CI.5 alkyl), -N(Ci_5 alkyl)(Ci_5 alkyl), halogen, C1.5 haloalkyl, -O-(Ci-5 haloalkyl), and -ON; preferably wherein Rx is hydrogen or methyl.
4. The compound for use according to any one of claims 1 to 3, wherein L1 is selected from a covalent bond, -CO-, and -SO2-.
5. The compound for use according to any one of claims 1 to 4, wherein L2 is selected from a covalent bond, C1-5 alkylene, -(C0-5 alkylene)-aryl-(Co-5 alkylene)-, and -(C0-5 alkylene)-heteroaryl-(Co-5 alkylene)-, wherein said C1-5 alkylene, the two C0-5 alkylene groups in said -(C0-5 alkylene)-aryl-(Co-5 alkylene)-, and the two C0-5 alkylene groups in said -(C0-5 alky lene)-heteroary l-(Co-5 alkylene)- are each optionally substituted with one or more groups RAlk, wherein one or more -CH2- units comprised in said C1-5 alkylene or any of the C0-5 alkylene groups comprised in said -(C0-5 alkylene)-aryl-(Co-5 alkylene)- or said -(C0-5 alkylene)-heteroaryl-(Co-5 alkylene)- are each optionally replaced by a group independently selected from -O-, -SO2-, -CO-, -NH-, and -N(0I-5 alkyl)-, and further wherein the aryl in said -(C0-5 alkylene)-aryl-(Co-5 alkylene)- and the heteroaryl in said -(C0-5 alky lene)-heteroary l-(Co-5 alkylene)- are each optionally substituted with one or more groups RCyc.
6. The compound for use according to any one of claims 1 to 5, wherein R2 is selected from hydrogen, C1-5 alkyl, C2-5 alkenyl, and C1-5 haloalkyl.
7. The compound for use according to any one of claims 1 to 6, wherein R3 is aryl or heteroaryl, wherein said aryl and said heteroaryl are each optionally substituted with one or more groups R31.
8. The compound for use according to any one of claims 1 to 7, wherein R3 is selected from phenyl, pyridin-2- yl, and 1 H-pyrrol-2-yl, wherein said phenyl, said pyridin-2-yl, and said 1 H-pyrrol-2-yl are each optionally substituted with one or more groups R31.
9. The compound for use according to claim 1 , wherein: o one of the ring atoms X1 and X2 is S, and the other one is C(-Rx); o Rx is selected from hydrogen, C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-0(Ci-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkylene)-0(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-NH2, -(C0-5 alkylene)-NH(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-OH, -(C0-5 alkylene)-N(Ci-5 alkyl)-OH, -(C0-5 alkylene)-NH-O(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-O(Ci-5 alkyl), -(C0-5 alkylene)-halogen, -(C0-5 alkylene)-(Ci-5 haloalkyl), -(C0-5 alkylene)-O-(Ci-5 haloalkyl), -(C0-5 alkylene)-CN, -(C0-5 alkylene)-NO2, -(C0-5 alkylene)-CHO, -(C0-5 alkylene)-CO-(Ci-5 alkyl), -(C0-5 alkylene)-CO-(Ci-5 haloalkyl), -(C0-5 alkylene)-COOH, -(C0-5 alkylene)-CO-O-(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-(Ci-5 alkyl), -(C0-5 alkylene)-CO-NH2, -(C0-5 alkylene)-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(Co-5 alkylene)-NH-C0-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-C0-(Ci-5 alkyl), -(C0-5 alkylene)-NH-C00(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-C00(Ci-5 alkyl), -(C0-5 alkylene)-O-CO- NH(CI-5 alkyl), -(C0-5 alkylene)-0-C0-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-SO2-NH2, -(C0-5 alkylene)-SO2-NH(Ci-5 alkyl), -(C0-5 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-S0-(Ci-5 alkyl), -(C0-5 alkylene)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-carbocyclyl, -(C0-5 alkylene)-heterocyclyl, and -Lz-Rz, wherein the carbocyclyl in said -(C0-5 alkylene)-carbocyclyl and the heterocyclyl in said -(C0-5 alky lene)-heterocycly I are each optionally substituted with one or more groups RCyc; R1 is -L1-L2-R11; L1 is selected from a covalent bond, -CO-, and -SO-; L2 is selected from -(C0-5 alkylene)-aryl-(Co-5 alkylene)- and -(C0-5 alkylene)-heteroaryl-(Co-5 alkylene)-, wherein the two C0-5 alkylene groups in said -(C0-5 alkylene)-aryl-(Co-5 alkylene)- and the two C0-5 alkylene groups in said -(C0-5 alkylene)-heteroaryl-(Co-5 alkylene)- are each optionally substituted with one or more groups RAlk, wherein one or more -CH2- units comprised in any of the C0-5 alkylene groups in said -(C0-5 alkylene)-aryl-(Co-5 alkylene)- or any of the C0-5 alkylene groups in said -(C0-5 alkylene)-heteroaryl-(Co-5 alkylene)- are each optionally replaced by a group independently selected from -O-, -SO2-, -CO-, -NH-, and -N(0I-5 alkyl)-, and further wherein the aryl group in said -(C0-5 alky lene)-ary l-(Co-5 alkylene)- and the heteroaryl group in said -(C0-5 alky lene)-heteroaryl-(Co-5 alkylene)- are each optionally substituted with one or more groups RCyc; R11 is selected from -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci-5 alkyl), -NH-SO2-(CI.5 alkyl), -N(0I-5 alkyl)-SO2-(Ci-5 alkyl), -SO-(Ci-5 alkyl), -SO2-(Ci-5 alkyl), -(C1-15 alkylene)-carbocyclyl, and -(C1-15 alkylene)-heterocyclyl, wherein each alkyl or alkylene comprised in any of the aforementioned groups is optionally substituted with one or more groups RAlk, wherein one or more -CH2- units comprised in the C1-15 alkylene in said -(C1-15 alkylene)-carbocyclyl or in the C1-15 alkylene in said -(C1-15 alky lene)-heterocycly I are each replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -NH-, and -N(0I-5 alkyl)-, and wherein the carbocyclyl in said -(C1-15 alkylene)-carbocyclyl and the heterocyclyl in said -(C1-15 alkylene)-heterocyclyl are each optionally substituted with one or more groups RCyc; R2 is selected from hydrogen, C1-5 alkyl, C2-5 alkenyl, and C1-5 haloalkyl; R3 is pyridin-2-yl which is optionally substituted with one or more groups R31; each R31 is independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-O(Ci-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-NH2, -(C0-5 alkylene)-NH(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-OH, -(C0-5 alkylene)-N(Ci-5 alkyl)-OH, -(C0-5 alkylene)-NH-O(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-O(Ci-5 alkyl), -(C0-5 alkylene)-halogen, -(C0-5 alkylene)-(Ci-5 haloalkyl), -(C0-5 alkylene)-O-(Ci-5 haloalkyl), -(C0-5 alkylene)-CN, -(C0-5 alkylene)-NO2, -(C0-5 alkylene)-CHO, -(C0-5 alkylene)-CO-(Ci-5 alkyl), -(C0-5 alkylene)-COOH, -(C0-5 alkylene)-CO-O-(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-(Ci-5 alkyl), -(C0-5 alkylene)-CO-NH2, -(C0-5 alkylene)-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-CO-(Ci-5 alkyl), -(Co-5 alkylene)-N(Ci-5 alkyl)-C0-(Ci-5 alkyl), -(C0-5 alkylene)-NH-C00(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-C00(Ci-5 alkyl), -(C0-5 alkylene)-0-C0-NH(Ci-5 alkyl), -(C0-5 alkylene)-O-CO- N(CI-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-SO2-NH2, -(C0-5 alkylene)-SO2-NH(Ci-5 alkyl), -(C0-5 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-S0-(Ci-5 alkyl), -(C0-5 alkylene)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-carbocyclyl, -(C0-5 alkylene)-heterocyclyl, and -Lz-Rz, wherein the carbocyclyl in said -(C0-5 alkylene)-carbocyclyl and the heterocyclyl in said -(C0-5 alkylene)-heterocyclyl are each optionally substituted with one or more groups R°yc, and wherein one or more -CH2- units comprised in the C0-5 alkylene in said -(C0-5 alkylene)-carbocyclyl or in the C0-5 alkylene in said -(C0-5 alkylene)-heterocyclyl are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -NH-, and -N(0I-5 alkyl)-; each RAlk is independently selected from -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(0i-5 alkyl), -S(0i-5 alkylene)-SH, -S(0i-5 alkylene)-S(Ci-5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI.5 alkyl)(Ci.5 alkyl), -NH-OH, -N(Ci_5 alkyl)-OH, -NH-O(CI.5 alkyl), -N(0I-5 alkyl)-O(Ci-5 alkyl), halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -ON, -NO2, -OHO, -C0(Ci-5 alkyl), -COOH, -COO(Ci.5 alkyl), -O-CO(Ci.5 alkyl), -CO-NH2, -CO-NH(Ci.5 alkyl), -CO-N(Ci.5 alkyl)(Ci.5 alkyl), -NH-CO(CI.5 alkyl), -N(Ci_5 alkyl)-CO(Ci.5 alkyl), -NH-COO(Ci.5 alkyl), -N(CI.5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(CI.5 alkyl), -O-CO-N(Ci.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(Ci.5 alkyl), -SO2-N(Ci.5 alkyl)(Ci.5 alkyl), -NH-SO2-(Ci.5 alkyl), -N(Ci_5 alkyl)-SO2-(Ci.5 alkyl), -SO2-(Ci.5 alkyl), -SO-(Ci.5 alkyl), -(C0-5 alkylene)-cycloalkyl, -(C0-5 alkylene)-heterocycloalkyl, and -Lz-Rz; each RCyc is independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-O(Ci-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-NH2, -(C0-5 alkylene)-NH(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-OH, -(C0-5 alkylene)-N(Ci-5 alkyl)-OH, -(C0-5 alkylene)-NH-O(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-O(Ci-5 alkyl), -(C0-5 alkylene)-halogen, -(C0-5 alkylene)-Ci-5 haloalkyl, -(C0-5 alkylene)-O-(Ci-5 haloalkyl), -(C0-5 alkylene)-CN, -(C0-5 alkylene)- NO2, -(C0-5 alkylene)-CHO, -(C0-5 alkylene)-CO(Ci-5 alkyl), -(C0-5 alkylene)-COOH, -(C0-5 alkylene)-COO(Ci-5 alkyl), -(C0-5 alkylene)-O-CO(Ci-5 alkyl), -(C0-5 alkylene)-CO-NH2, -(C0-5 alkylene)-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-CO(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-CO(Ci-5 alkyl), -(C0-5 alkylene)-NH-COO(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-COO(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-O-CO- N(CI-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-SO2-NH2, -(C0-5 alkylene)-SO2-NH(Ci-5 alkyl), -(C0-5 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-SO-(Ci-5 alkyl), -(C0-5 alkylene)-carbocyclyl, -(C0-5 alkylene)-heterocyclyl, and -Lz-Rz; each Lz is independently selected from a covalent bond, C1-35 alkylene, C2-35 alkenylene, and C2-35 alkynylene, wherein said alkylene, said alkenylene and said alkynylene are each optionally substituted with one or more groups independently selected from halogen, C1.5 haloalkyl, -O-(Ci-5 haloalkyl), -CN, -OH, -O(Ci.5 alkyl), -SH, -S(Ci.5 alkyl), -NH2, -NH(CI.5 alkyl), and -N(CI.5 alkyl)(Ci.5 alkyl), wherein one or more CH units comprised in said alkylene, said alkenylene or said alkyny lene are each optionally replaced by a nitrogen atom, and further wherein one or more -CH2- units comprised in said alkylene, said alkenylene or said alkyny lene are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -NH-, -N(CI-5 alkyl)-, carbocyclylene, and heterocyclylene, wherein said carbocyclylene and said heterocyclylene are each optionally substituted with one or more groups independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -ON, -NO2, -OH, -O(Ci.5 alkyl), -SH, -S(Ci.5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI.5 alkyl)(Ci.5 alkyl), -OHO, -CO-(Ci.5 alkyl), -COOH, -CO-O-(Ci.5 alkyl), -O-CO-(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(CI.5 alkyl)(Ci.5 alkyl), -NH-CO-(CI.5 alkyl), -N(CI.5 alkyl)-CO-(Ci.5 alkyl), -NH-COO(CI.5 alkyl), -N(CI-5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(CI.5 alkyl), -O-CO-N(CI.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci.5 alkyl), -NH-SO2-(CI.5 alkyl), -N(CI.5 alkyl)-SO2-(Ci-5 alkyl), -SO-(Ci-5 alkyl), and -SO2-(Ci-5 alkyl); and o each Rz is independently selected from -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(Ci-5 alkyl), -S(Ci-5 alkylene)-SH, -S(Ci-5 alkylene)-S(Ci-5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI.5 alkyl)(Ci.5 alkyl), -NH-OH, -N(CI.5 alkyl)-OH, -NH-O(CI.5 alkyl), -N(0I-5 alkyl)-O(Ci-5 alkyl), halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -ON, -NO2, -OHO, -C0(Ci-5 alkyl), -COOH, -COO(Ci.5 alkyl), -O-CO(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(CI.5 alkyl)(Ci.5 alkyl), -NH-CO(CI-5 alkyl), -N(CI.5 alkyl)-CO(Ci.5 alkyl), -NH-COO(CI.5 alkyl), -N(CI.5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(CI-5 alkyl), -O-CO-N(CI.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci.5 alkyl), -NH-SO2-(CI.5 alkyl), -N(CI.5 alkyl)-SO2-(Ci.5 alkyl), -SO2-(Ci.5 alkyl), -SO-(Ci.5 alkyl), aryl, heteroaryl, cycloalkyl, and heterocycloalkyl, wherein said aryl, said heteroaryl, said cycloalkyl, and said heterocycloalkyl are each optionally substituted with one or more groups independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -ON, -NO2, -OH, -O(Ci.5 alkyl), -SH, -S(Ci.5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI.5 alkyl)(Ci.5 alkyl), -OHO, -CO-(Ci.5 alkyl), -COOH, -CO-O-(Ci.5 alkyl), -O-CO-(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(CI-5 alkyl)(Ci.5 alkyl), -NH-CO-(CI.5 alkyl), -N(CI.5 alkyl)-CO-(Ci.5 alkyl), -NH-COO(CI.5 alkyl), -N(CI.5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(CI.5 alkyl), -O-CO-N(CI.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci.5 alkyl), -NH-SO2-(CI.5 alkyl), -N(CI.5 alkyl)-SO2-(Ci-5 alkyl), -SO-(Ci-5 alkyl), -SO2-(Ci-5 alkyl), carbocyclyl, and heterocyclyl, wherein said carbocyclyl and said heterocyclyl are each optionally substituted with one or more groups independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -ON, -NO2, -OH, -O(Ci.5 alkyl), -SH, -S(Ci.5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI.5 alkyl)(Ci.5 alkyl), -OHO, -CO-(Ci.5 alkyl), -COOH, -CO-O-(Ci.5 alkyl), -O-CO-(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(CI-5 alkyl)(Ci.5 alkyl), -NH-CO-(CI.5 alkyl), -N(CI.5 alkyl)-CO-(Ci.5 alkyl), -NH-COO(CI.5 alkyl), -N(CI.5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(CI.5 alkyl), -O-CO-N(CI.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci_5 alkyl), -NH-SO2-(CI.5 alkyl), -N(CI.5 alkyl)-SO2-(Ci-5 alkyl), -SO-(Ci-5 alkyl), and -SO2-(Ci-5 alkyl).
10. The compound for use according to claim 9, wherein L1 is -CO-.
11 . The compound for use according to claim 9 or 10, wherein L2 is selected from aryl and heteroaryl, wherein said aryl and said heteroaryl are each optionally substituted with one or more groups RCyc.
12. The compound for use according to any one of claims 9 to 11 , wherein R11 is selected from -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI-5 alkyl)(Ci-5 alkyl), -(C1-15 alkylene)-carbocyclyl, and -(C1-15 alkylene)-heterocyclyl, wherein one or more -CH2- units comprised in the C1-15 alkylene in said -(C1-15 alkylene)-carbocyclyl or in the C1-15 alkylene in said -(C1-15 alkylene)-heterocyclyl are each replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -NH-, and -N(CI-5 alkyl)-, and wherein the carbocyclyl in said -(C1-15 alky lene)-carbocycly I and the heterocyclyl in said -(C1-15 alky lene)-heterocycly I are each optionally substituted with one or more groups RCyc.
13. The compound for use according to claim 1 , wherein: o one of the ring atoms X1 and X2 is S, and the other one is C(-Rx); o Rx is selected from hydrogen, C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-O(Ci-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-NH2, -(C0-5 alkylene)-NH(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-OH, -(C0-5 alkylene)-N(Ci-5 alkyl)-OH, -(C0-5 alkylene)-NH-O(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-O(Ci-5 alkyl), -(C0-5 alkylene)-halogen, -(C0-5 alkylene)-(Ci-5 haloalkyl), -(C0-5 alkylene)-O-(Ci-5 haloalkyl), -(C0-5 alkylene)-CN, -(C0-5 alkylene)-NO2, -(C0-5 alkylene)-CHO, -(C0-5 alkylene)-CO-(Ci-5 alkyl), -(C0-5 alkylene)-CO-(Ci-5 haloalkyl), -(C0-5 alkylene)-COOH, -(C0-5 alkylene)-CO-O-(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-(Ci-5 alkyl), -(C0-5 alkylene)-CO-NH2, -(C0-5 alkylene)-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-CO-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-CO-(Ci-5 alkyl), -(C0-5 alkylene)-NH-COO(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-COO(Ci-5 alkyl), -(C0-5 alkylene)-O-CO- NH(CI-5 alkyl), -(C0-5 alkylene)-O-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-SO2-NH2, -(C0-5 alkylene)-SO2-NH(Ci-5 alkyl), -(C0-5 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-SO-(Ci-5 alkyl), -(C0-5 alkylene)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-carbocyclyl, -(C0-5 alkylene)-heterocyclyl, and -Lz-Rz, wherein the carbocyclyl in said -(C0-5 alkylene)-carbocyclyl and the heterocyclyl in said -(C0-5 alky lene)-heterocycly I are each optionally substituted with one or more groups RCyc;
O R1 is -L1-L2-R11; o L1 is selected from a covalent bond, -CO-, -SO-, -CO-N(RN)-, -SO-N(RN)-, -SO2-N(RN)-, and -C(=N-RN)- N(RN)-, wherein each RN is independently hydrogen or C1-5 alkyl; o L2 is selected from a covalent bond, C1-10 alkylene, C2-10 alkenylene, C2-10 alkynylene, -(C0-5 alkylene)-carbocyclyl-(Co-5 alkylene)-, and -(C0-5 alkylene)-heterocyclyl-(Co-5 alkylene)-, wherein said C1-10 alkylene, said C2-10 alkenylene, said C2-10 alkynylene, the two C0-5 alkylene groups in said -(C0-5 alkylene)-carbocyclyl-(Co-5 alkylene)-, and the two C0-5 alkylene groups in said -(C0-5 alkylene)-heterocyclyl-(Co-5 alkylene)- are each optionally substituted with one or more groups RAlk, wherein one or more -CH2- units comprised in said C1-10 alkylene, said C2-10 alkenylene, said C2-10 alkynylene, any of the C0-5 alkylene groups in said -(C0-5 alky lene)-carbocy cly l-(Co-5 alkylene)-, or any of the C0-5 alkylene groups in said -(C0-5 al ky lene)-heterocycly l-(Co-5 alkylene)- are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -CO-, -N(RL2)-, and -C(RL2)=C(RL2)-, wherein each RL2 is independently hydrogen or C1-5 alkyl, and further wherein the carbocyclyl group in said -(C0-5 alky lene)-carbocycly l-(Co-5 alkylene)- and the heterocyclyl group in said -(C0-5 alky lene)-heterocycly I are each optionally substituted with one or more groups RCyc; R11 is selected from -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-0(Ci-5 alkyl), -SH, -S(Ci_5 alkyl), -S(Ci_5 alkylene)-SH, -S(Ci-5 alkylene)-S(Ci.5 alkyl), -NH2, -NH(Ci.5 alkyl), -N(0I-5 alkyl)(Ci.5 alkyl), -NH-OH, -N(Ci_5 alkyl)-OH, -NH-O(CI.5 alkyl), -N(Ci_5 alkyl)-O(Ci.5 alkyl), halogen, -O-(Ci_5 haloalkyl), -ON, -NO2, -OHO, -COOH, -CO-O-(Ci.5 alkyl), -O-CO-(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(CI.5 alkyl)(Ci.5 alkyl), -NH-CO-(CI.5 alkyl), -N(Ci_5 alkyl)-CO-(Ci.5 alkyl), -NH-COO(CI.5 alkyl), -N(Ci_5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(CI.5 alkyl), -O-CO- N(CI-5 alkyl)(Ci-5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci.5 alkyl), -NH-SO2-(CI.5 alkyl), -N(0I-5 alkyl)-SO2-(Ci-5 alkyl), -S0-(Ci-5 alkyl), -SO2-(Ci-5 alkyl), -(Co-15 alkylene)-carbocyclyl, and -(Co-15 alkylene)-heterocyclyl, wherein each alkyl or alkylene comprised in any of the aforementioned groups is optionally substituted with one or more groups RAlk, wherein one or more - CH2- units comprised in the Co-15 alkylene in said -(Co-15 alkylene)-carbocyclyl or in the Co-15 alkylene in said -(Co-15 alkylene)-heterocyclyl are each optionally replaced by a group independently selected from -0-, -S-, -SO-, -S02-, -CO-, -NH-, and -N(CI-5 alkyl)-, wherein the carbocyclyl in said -(Co-15 alkylene)-carbocyclyl and the heterocyclyl in said -(Co-15 alkylene)-heterocyclyl are each optionally substituted with one or more groups R°yc, and wherein the heterocyclyl in said -(Co-15 alky lene)-heterocycly I is selected from heteroaryl and heterocycloalkyl; R2 is selected from hydrogen, C1-10 alkyl, C2.w alkenyl, C2.w alkynyl, -(Co-10 alkylene)-OH, -(Co-10 alkylene)-0(Ci-5 alkyl), -(Co-10 alkylene)-SH, -(Co-10 alkylene)-S(Ci-5 alkyl), -(C1-10 alkylene)-N H2, -(CMO alkylene)-NH(Ci-5 alkyl), -(C1-10 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C1-10 alkylene)-halogen, C1-10 haloalkyl, -(Co-10 alkylene)-0-(Ci-5 haloalkyl), -(Co-10 alkylene)-CN, -(Co-10 alkylene)-NO2, -(Co-10 alkylene)-CHO, -(Co-10 alkylene)-CO-(Ci-io alkyl), -(Co-10 alkylene)-COOH, -(Co-10 alkylene)-C0-0-(Ci-5 alkyl), -(Co-10 alkylene)-0-C0-(Ci-5 alkyl), -(Co-10 alkylene)-CO-NH2, -(Co-10 alkylene)-C0-NH(Ci-5 alkyl), -(Co-10 alkylene)-C0-N(Ci-5 alkyl)(Ci-5 alkyl), -(C1-10 alkylene)-NH-C0-(Ci-5 alkyl), -(C1-10 alkylene)-N(Ci-5 alkyl)-C0-(Ci-5 alkyl), -(C1-10 alkylene)-NH-C00(Ci-5 alkyl), -(C1-10 alkylene)-N(Ci-5 alkyl)-C00(Ci-5 alkyl), -(Co-10 alkylene)-0-C0-NH(Ci-5 alkyl), -(Co-10 alkylene)-0-C0-N(Ci-5 alkyl)(Ci-5 alkyl), -(Co-10 alkylene)-SO2-NH2, -(Co-10 alkylene)-SO2-NH(Ci-5 alkyl), -(Co-10 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C1-10 alkylene)-NH-SO2-(Ci-5 alkyl), -(C1-10 alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(Co-10 alkylene)-S0-(Ci-5 alkyl), -(Co-10 alkylene)-SO2-(Ci-5 alkyl), -(Co-10 alkylene)-carbocyclyl, and -(Co-10 alkylene)-heterocyclyl, wherein one or more -CH2- units comprised in said C1-10 alkyl, said C2-io alkenyl, said C2-10 alkynyl, or in any of the aforementioned Co-10 alkylene or C1-10 alkylene groups are each optionally replaced by a group independently selected from -0-, -S-, -SO-, -S02-, -CO-, -NH-, and -N(CI-5 alkyl)-, and further wherein the carbocyclyl in said -(Co-10 alkylene)-carbocyclyl and the heterocyclyl in said -(Co-10 alky lene)-heterocycly I are each optionally substituted with one or more groups RCyC; R3 is -(Co-5 alkylene)-aryl or -(C0-5 alkylene)-heteroaryl, wherein the aryl in said -(C0-5 alkylene)-aryl is selected from phenyl, naphthyl and tetralinyl, wherein the heteroaryl in said -(C0-5 alkylene)-heteroaryl is selected from pyrrolyl, 1,3-benzodioxolyl, furanyl and thiophenyl, wherein the aryl in said -(C0-5 alkylene)-aryl and the heteroaryl in said -(C0-5 alkylene)-heteroaryl are each optionally substituted with one or more groups R31, and wherein one or more -CH2- units comprised in the C0-5 alkylene in said -(C0-5 alky lene)-ary I or in the C0-5 alkylene in said -(C0-5 alky lene)-heteroary I are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -NH-, and -N(0I-5 alkyl)-; each R31 is independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-O(Ci-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-NH2, -(C0-5 alkylene)-NH(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-OH, -(C0-5 alkylene)-N(Ci-5 alkyl)-OH, -(C0-5 alkylene)-NH-O(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-O(Ci-5 alkyl), -(C0-5 alkylene)-halogen, -(C0-5 alkylene)-(Ci-5 haloalkyl), -(C0-5 alkylene)-O-(Ci-5 haloalkyl), -(C0-5 alkylene)-CN, -(C0-5 alkylene)-NO2, -(C0-5 alkylene)-CHO, -(C0-5 alkylene)-CO-(Ci-5 alkyl), -(C0-5 alkylene)-COOH, -(C0-5 alkylene)-CO-O-(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-(Ci-5 alkyl), -(C0-5 alkylene)-CO-NH2, -(C0-5 alkylene)-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-CO-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-CO-(Ci-5 alkyl), -(C0-5 alkylene)-NH-COO(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-COO(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-O-CO- N(CI-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-SO2-NH2, -(C0-5 alkylene)-SO2-NH(Ci-5 alkyl), -(C0-5 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-SO-(Ci-5 alkyl), -(C0-5 alkylene)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-carbocyclyl, -(C0-5 alkylene)-heterocyclyl, and -Lz-Rz, wherein the carbocyclyl in said -(C0-5 alkylene)-carbocyclyl and the heterocyclyl in said -(C0-5 alkylene)-heterocyclyl are each optionally substituted with one or more groups R0^, and wherein one or more -CH2- units comprised in the C0-5 alkylene in said -(C0-5 alkylene)-carbocyclyl or in the C0-5 alkylene in said -(C0-5 alkylene)-heterocyclyl are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -NH-, and -N(0I-5 alkyl)-; each RAlk is independently selected from -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(0i-5 alkyl), -S(0i-5 alkylene)-SH, -S(0i-5 alkylene)-S(0i-5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI.5 alkyl)(Ci.5 alkyl), -NH-OH, -N(CI.5 alkyl)-OH, -NH-O(CI.5 alkyl), -N(0I-5 alkyl)-O(Ci-5 alkyl), halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -ON, -NO2, -OHO, -C0(Ci-5 alkyl), -COOH, -COO(Ci.5 alkyl), -O-CO(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(CI.5 alkyl)(Ci.5 alkyl), -NH-CO(CI-5 alkyl), -N(CI.5 alkyl)-CO(Ci.5 alkyl), -NH-COO(CI.5 alkyl), -N(CI.5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(CI-5 alkyl), -O-CO-N(CI.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci.5 alkyl), -NH-SO2-(CI.5 alkyl), -N(CI.5 alkyl)-SO2-(Ci.5 alkyl), -SO2-(Ci.5 alkyl), -SO-(Ci.5 alkyl), -(C0-5 alkylene)-cycloalkyl, -(C0-5 alkylene)-heterocycloalkyl, and -Lz-Rz; each RCyc is independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-O(Ci-5 alkylene)-OH, -(C0-5 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-S(Ci-5 alkylene)-SH, -(C0-5 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-5 alkylene)-NH2, -(C0-5 alkylene)-NH(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-OH, -(C0-5 alkylene)-N(Ci-5 alkyl)-OH, -(C0-5 alkylene)-NH-0(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-0(Ci-5 alkyl), -(C0-5 alkylene)-halogen, -(C0-5 alkylene)-Ci-5 haloalkyl, -(C0-5 alkylene)-O-(Ci-5 haloalkyl), -(C0-5 alkylene)-CN, -(C0-5 alkylene)- NO2, -(C0-5 alkylene)-CHO, -(C0-5 alkylene)-CO(Ci-5 alkyl), -(C0-5 alkylene)-COOH, -(C0-5 alkylene)-COO(Ci-5 alkyl), -(C0-5 alkylene)-O-CO(Ci-5 alkyl), -(C0-5 alkylene)-CO-NH2, -(C0-5 alkylene)-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-CO(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-CO(Ci-5 alkyl), -(C0-5 alkylene)-NH-COO(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-COO(Ci-5 alkyl), -(C0-5 alkylene)-O-CO-NH(Ci-5 alkyl), -(C0-5 alkylene)-O-CO- N(CI-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-SO2-NH2, -(C0-5 alkylene)-SO2-NH(Ci-5 alkyl), -(C0-5 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-5 alkylene)-NH-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-SO2-(Ci-5 alkyl), -(C0-5 alkylene)-SO-(Ci-5 alkyl), -(C0-5 alkylene)-carbocyclyl, and -(C0-5 alkylene)-heterocyclyl; each Lz is independently selected from a covalent bond, C1-35 alkylene, C2-35 alkenylene, and C2-35 alkynylene, wherein said alkylene, said alkenylene and said alkynylene are each optionally substituted with one or more groups independently selected from halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -CN, -OH, -O(Ci-s alkyl), -SH, -S(Ci.5 alkyl), -NH2, -NH(CI.5 alkyl), and -N(CI.5 alkyl)(Ci.5 alkyl), wherein one or more CH units comprised in said alkylene, said alkenylene or said alkynylene are each optionally replaced by a nitrogen atom, and further wherein one or more -CH2- units comprised in said alkylene, said alkenylene or said alkynylene are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -NH-, -N(0I-5 alkyl)-, carbocyclylene, and heterocyclylene, wherein said carbocyclylene and said heterocyclylene are each optionally substituted with one or more groups independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -CN, -NO2, -OH, -O(Ci.5 alkyl), -SH, -S(Ci.5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI.5 alkyl)(Ci_5 alkyl), -OHO, -CO-(Ci.5 alkyl), -COOH, -CO-O-(Ci.5 alkyl), -O-CO-(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(CI-5 alkyl)(Ci_5 alkyl), -NH-CO-(Ci.5 alkyl), -N(CI.5 alkyl)-CO-(Ci_5 alkyl), -NH-COO(CI.5 alkyl), -N(CI-5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(CI.5 alkyl), -O-CO-N(CI.5 alkyl)(Ci_5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci.5 alkyl), -NH-SO2-(CI.5 alkyl), -N(CI.5 alkyl)-SO2-(Ci-5 alkyl), -SO-(Ci-5 alkyl), and -SO2-(Ci-5 alkyl); and each Rz is independently selected from -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(Ci-5 alkyl), -S(Ci-5 alkylene)-SH, -S(Ci-5 alkylene)-S(Ci-5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI.5 alkyl)(Ci.5 alkyl), -NH-OH, -N(CI.5 alkyl)-OH, -NH-O(CI.5 alkyl), -N(CI-5 alkyl)-O(Ci-5 alkyl), halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -CN, -NO2, -CHO, -C0(Ci-5 alkyl), -COOH, -COO(Ci.5 alkyl), -O-CO(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(CI.5 alkyl)(Ci.5 alkyl), -NH-CO(CI-5 alkyl), -N(CI.5 alkyl)-CO(Ci.5 alkyl), -NH-COO(CI.5 alkyl), -N(CI.5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(CI-5 alkyl), -O-CO-N(CI.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci.5 alkyl), -NH-SO2-(CI.5 alkyl), -N(CI.5 alkyl)-SO2-(Ci.5 alkyl), -SO2-(Ci.5 alkyl), -SO-(Ci.5 alkyl), aryl, heteroaryl, cycloalkyl, and heterocycloalkyl, wherein said aryl, said heteroaryl, said cycloalkyl, and said heterocycloalkyl are each optionally substituted with one or more groups independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -CN, -N02, -OH, -O(CI-5 alkyl), -SH, -S(Ci.5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI.5 alkyl)(Ci_5 alkyl), -CHO, -CO-(Ci.5 alkyl), -COOH, -CO-O-(Ci.5 alkyl), -O-CO-(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(CI.5 alkyl)(Ci.5 alkyl), -NH-CO-(CI.5 alkyl), -N(CI.5 alkyl)-CO-(Ci.5 alkyl), -NH-COO(CI.5 alkyl), -N(CI-5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(CI.5 alkyl), -O-CO-N(CI.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci_5 alkyl), -NH-SO2-(CI.5 alkyl), -N(CI.5 alkyl)-SO2-(Ci-5 alkyl), -SO-(Ci-5 alkyl), -SO2-(Ci-5 alkyl), carbocyclyl, and heterocyclyl, wherein said carbocyclyl and said heterocyclyl are each optionally substituted with one or more groups independently selected from C1-5 alkyl, C2.5 alkenyl, C2.5 alkynyl, halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -ON, -NO2, -OH, -O(CI-5 alkyl), -SH, -S(Ci.5 alkyl), -NH2, -NH(CI.5 alkyl), -N(CI.5 alkyl)(Ci.5 alkyl), -CHO, -CO-(Ci.5 alkyl), -COOH, -CO-O-(Ci.5 alkyl), -O-CO-(Ci.5 alkyl), -CO-NH2, -CO-NH(CI.5 alkyl), -CO-N(CI-5 alkyl)(Ci.5 alkyl), -NH-CO-(CI.5 alkyl), -N(CI.5 alkyl)-CO-(Ci.5 alkyl), -NH-COO(CI.5 alkyl), -N(CI.5 alkyl)-COO(Ci.5 alkyl), -O-CO-NH(CI.5 alkyl), -O-CO-N(CI.5 alkyl)(Ci.5 alkyl), -SO2-NH2, -SO2-NH(CI.5 alkyl), -SO2-N(CI.5 alkyl)(Ci_5 alkyl), -NH-SO2-(CI.5 alkyl), -N(CI.5 alkyl)-SO2-(Ci-5 alkyl), -SO-(Ci-5 alkyl), and -SO2-(Ci-5 alkyl).
14. The compound for use according to claim 13, wherein R3 is -(C0-5 alkylene)-heteroaryl, wherein the heteroaryl in said -(C0-5 alkylene)-heteroaryl is selected from pyrrolyl, 1 ,3-benzodioxolyl, furanyl and thiophenyl, wherein the heteroaryl in said -(C0-5 alkylene)-heteroaryl is optionally substituted with one or more groups R31, and wherein one or more -CH2- units comprised in the C0-5 alkylene in said -(C0-5 alky lene)-heteroary I are each optionally replaced by a group independently selected from -O-, -S-, -SO-, -SO2-, -CO-, -NH-, and -N(CI-5 alkyl)-.
The compound for use according to claim 1 , wherein said compound is selected from:
Figure imgf000150_0001
Figure imgf000151_0001
Figure imgf000152_0001
Figure imgf000153_0001
Figure imgf000154_0001
Figure imgf000155_0001
Figure imgf000156_0001
Figure imgf000157_0001
Figure imgf000158_0001
Figure imgf000159_0001
Figure imgf000160_0001
or a pharmaceutically acceptable salt or solvate thereof.
16. A pharmaceutical composition comprising a compound as defined in any one of claims 1 to 15 and a pharmaceutically acceptable excipient for use in the treatment or prevention of cancer or diabetes.
17. The compound for use according to any one of claims 1 to 15 or the pharmaceutical composition for use according to claim 16, wherein said compound or said pharmaceutical composition is for use in the treatment or prevention of diabetes.
18. The compound for use according to any one of claims 1 to 15 or the pharmaceutical composition for use according to claim 16, wherein said compound or said pharmaceutical composition is for use in the treatment or prevention of cancer.
19. The compound for use according to claim 18 or the pharmaceutical composition for use according to claim 18, wherein said cancer is selected from lung cancer, renal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, colon cancer, anal cancer, genitourinary cancer, bladder cancer, liver cancer, pancreatic cancer, cervical cancer, endometrial cancer, vaginal cancer, vulvar cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, biliary tract cancer, hepatobiliary cancer, neuroblastoma, brain cancer, breast cancer, head and/or neck cancer, skin cancer, melanoma, Merkel-cell cancer, epidermoid cancer, squamous cell cancer, bone cancer, fibrosarcoma, Ewing's sarcoma, malignant mesothelioma, esophageal cancer, laryngeal cancer, mouth cancer, thymoma, neuroendocrine cancer, goblet cell cancer, hematological cancer, leukemia, lymphoma, and multiple myeloma.
20. The compound for use according to claim 18 or 19 or the pharmaceutical composition for use according to claim 18 or 19, wherein said compound or said pharmaceutical composition is to be administered in combination with one or more anticancer drugs.
21. A compound as defined in any one of claims 1 to 15; with the proviso that the compound is not any one of the following compounds or a pharmaceutically acceptable salt or solvate thereof:
Figure imgf000161_0001
Figure imgf000162_0001
22. A compound which is selected from:
Figure imgf000162_0002
Figure imgf000163_0001
Figure imgf000164_0001
Figure imgf000165_0001
Figure imgf000166_0001
Figure imgf000167_0001
Figure imgf000168_0001
Figure imgf000169_0001
Figure imgf000170_0001
or a pharmaceutically acceptable salt or solvate thereof.
23. A pharmaceutical composition comprising the compound of claim 21 or 22 and a pharmaceutically acceptable excipient.
24. In vitro use of a compound as defined in any one of claims 1 to 15 as an SMNDC1 inhibitor.
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