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WO2025228947A1 - Bifunctional compounds capable of inducing degradation of polo-like kinase1 - Google Patents

Bifunctional compounds capable of inducing degradation of polo-like kinase1

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Publication number
WO2025228947A1
WO2025228947A1 PCT/EP2025/061648 EP2025061648W WO2025228947A1 WO 2025228947 A1 WO2025228947 A1 WO 2025228947A1 EP 2025061648 W EP2025061648 W EP 2025061648W WO 2025228947 A1 WO2025228947 A1 WO 2025228947A1
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Prior art keywords
group
cancer
alkyl
tlb
conjugate
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Pending
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PCT/EP2025/061648
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French (fr)
Inventor
Adrianus Petrus Antonius De Man
Rogier Christian Buijsman
Joost Cornelis Marinus UITDEHAAG
Erik ENSING
Jan Gerard STERRENBURG
Yvonne Gertruda Theodora Hendrika VAN MIL
Joeri Johannes Petrus DE WIT
Freek VAN CAUTER
Sander Petrus Wilhelmus VAN GEMERT
Milan Jurgen HOFFMANN
Martine Berendina Wilhemina PRINSEN
Winfried Robert Mulder
Michelle MULLER
Diep VU-PHAM
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Crossfire Oncology BV
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Crossfire Oncology BV
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Publication of WO2025228947A1 publication Critical patent/WO2025228947A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/545—Heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6869—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from a cell of the reproductive system: ovaria, uterus, testes, prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • Bifunctional compounds capable of inducing degradation of Polo-like kinasei
  • kinases are enzymes that transfer a phosphate group from ATP to a protein while phosphatases remove a phosphate group from protein. Together, these two enzymatic processes regulate cellular functions such as cell proliferation, subcellular translocation, apoptosis, inflammation and metabolism (Attwood et al. Trends in kinase drug discovery targets, indications and inhibitor design. Nat Rev Drug Discov 20, pages 839-861 (2021)).
  • the human kinome is composed of over 500 kinases.
  • Polo-like kinase 1 is a member of the PLK-family of kinases which plays a key role in cell-cycle progression through mitosis via its effects on chromosome segregation, spindle assembly, and cytokinesis.
  • PLK1 phosphorylates the substrate CDC25, thereby activating the cyclin B/CDC2 complex, which triggers cell proliferation.
  • PLK1 is activated in the cell by the kinase Aurora A and its co-factor Bora. Bora acts on PLK1 by altering its conformation, which in turn allows Aurora A to phosphorylate PLK1 on threonine residue 210, promoting mitotic entry.
  • PLK1 Once PLK1 has been activated, its expression accumulates during S phase, peaks during G2-M transition, and declines rapidly upon mitotic exit, (reviewed in Liu X. Targeting polo-like kinases: a promising therapeutic approach for cancer treatment. Transl Oncol 8, pages:185-95 (2015), M Chiappa et al, Present and Future Perspective on PLK1 Inhibition in Cancer Treatment, Frontiers in Oncology 12, 903016 (2022).
  • PLK1 is an important regulator of the DNA damage checkpoint.
  • cells suffer DNA damage, for instance genotoxic stress, in the G2 phase of the cell cycle, they do not progress into mitosis, but arrest in the G2/M DNA damage checkpoint where PLK1 is inhibited, and its degradation is induced.
  • Upstream activators of PLK1 are similarly affected by the DNA damage response, preventing activation of PLK1 at multiple levels.
  • active PLK1 mediates phosphorylation of checkpoint kinase 2 (CHK2), which inhibits checkpoint kinase 1 (CHK1) and CHK2 activation, thereby preventing DNA repair during mitosis, which would be harmful to the cell.
  • CHK2 checkpoint kinase 2
  • CHK1 checkpoint kinase 1
  • CHK1 checkpoint kinase 1
  • PLK1 is so intimately involved in cell-cycle regulation pathways and DNA damage repair, which are very important for tumor growth, maintenance and therapy resistance, it is not surprising that PLK1 has been proposed as a drug target in a variety of cancers.
  • CRISPR CRISPR
  • shRNA shRNA
  • PLK1 has significantly higher expression levels in several late stage cancers than in early stage. PLK1 expression is elevated in non-small-cell lung cancer, head and neck cancer, esophageal cancer, gastric cancer, melanomas, breast cancer, ovarian cancer, endometrial cancer, colorectal cancer, gliomas, and thyroid cancer.
  • PLK1 gene and protein expression has been proposed as a new prognostic marker for many types of malignancies, and PLK1 is a potential target for cancer therapy (reviewed in Z Liu et al., PLK1 , A potential target for cancer therapy, Translational Oncology 10, pages 22-32 (2017) and N Takai, Polo-like kinases (PLKs) and cancer. Oncogene 24, pages 287-291 (2005)).
  • PLK1 plays a role in inflammation, and PLK1 inhibitors show effects in inflammatory disease models.
  • targeting PLK1 could be useful in treating diseases caused or exacerbated by the immune system (Baldrighi et al., PLK1 inhibition dampens NLRP3 inflammasome-elicited response in inflammatory disease models, Journal of Clinical Investigation 133, page e162129 (2023).
  • PLK1 targeting modalities can also be used in transplantation, to treat or prevent graft versus host disease.
  • PLK1 is involved in neurological disorders such as Huntington’s disease and Alzheimer’s disease.
  • PLK1 p-amyloid
  • PLK1 Polo-like kinase 1
  • PLK1 inhibitors for cancer treatment.
  • Much studied inhibitors are BI2536, GSK461364, BI6727 (volasertib), CYC140 (plogosertib) and NMS-P937 (onvansertib), which all have been tested in clinical phase, particularly in patients with leukemias, lymphomas, lung cancer, pancreatic cancer, prostate cancer and in general advanced solid cancers.
  • PLK1 inhibitors do not sufficiently inhibit PLK1 activity at concentrations that are clinically safe (reviewed in M Chiappa et al, Frontiers in Oncology 12, 903016 (2022). If the cell cycle of cancer cells is only temporarily delayed by PLK1 inhibition, cancer cells will eventually restart the cell cycle, which negates any clinical effects.
  • PLK1 inhibitors Another reason for the clinical failure of PLK1 inhibitors is that many functional studies demonstrating the involvement of PLK1 in tumors used biological techniques involving knockingout or knocking-down PLK1 . To obtain the effective responses observed in these preclinical models by way of a PLK1 protein target, a therapeutic modality that inhibits or removes the entire PLK1 protein might be desired.
  • the ubiquitin-proteasome system is a highly conserved mechanism for degradation of both normal, mutated and misfolded proteins in eukaryotic cells, thus keeping intracellular protein homeostasis (J Bard et al. Structure and function of the 26S proteasome. Annu Rev Biochem, 87, pages 697-724 (2016), G Kleiger et al., Perilous journey: a tour of the ubiquitin- proteasome system. Trends Cell Biol. 24, pages 352-359 (2014); M Hipp et al. The proteostasis network and its decline in ageing. Nat Rev Mol Cell Biol. 20, pages 421-435 (2019)).
  • proteins to be degraded are covalently tagged with ubiquitin (Ub, a 76-amino acid protein), and this tagging process is catalyzed by three enzymes known as Ub-activating enzyme (E1), Ub- conjugating enzyme (E2) and Ub-ligase (E3): free Ub is activated by E1 and then attached to the cysteine residue (Cys) of E1 to form a thioester bond via an ATP-dependent reaction; the Ub- tagged E1 transfers its Ub to the Cys of E2 through a trans-thioesterification reaction; E3 recruits Ub-tagged E2 and E3 substrate to label the ubiquitin at the lysine residue (Lys) of the substrate.
  • E1 Ub-activating enzyme
  • E2 Ub- conjugating enzyme
  • E3 Ub-ligase
  • Such repeated ubiquitination processes generate a poly-Ub chain (mainly linked through Lys48 of Ub) on the target protein, which guides the substrate to 26S proteasome for degradation (D Komander D et al. The ubiquitin code. Annu Rev Biochem. 81 , pages 203-229 (2012); R Yau, M Rape. The increasing complexity of the ubiquitin code. Nat Cell Biol. 18, pages 579-586 (2016)).
  • the human proteome there are two E1s, about forty E2s and more than 600 E3s. Among them, the E3 ligases are responsible for specifically recognizing substrates.
  • HBCs Heterobifunctional compounds
  • PCI protein of interest
  • E3 ligand E3 ligase
  • HBC simultaneously recruits E3 ligase and PCI, forming the “E3 ligase-HBC-POl” ternary complex.
  • the HBC technology aims to completely eliminate the protein and all its functionality, rather than merely inhibiting their enzymatic activity. Therefore, the resistance to inhibition activity caused by kinase mutants, as observed with classical inhibitors, can be overcome by HBCs.
  • induced protein degradation offers several potential advantages over traditional target inhibition.
  • greater target selectivity can be achieved, even when starting with promiscuous binding ligands (Bondeson D. et al. Lessons in PROTAC design from selective degradation with promiscuous warheads. Cell. Chem. Biol, 25, pages 78-87 (2018)).
  • a further advantage of HBCs is their ability to overcome classical inhibitor resistance caused by feedback loops that upregulate the target.
  • HBCs have the ability to address scaffolding functions of target proteins that are not addressable with inhibitor molecules (K Samaransinghe and C Crews. Targeted protein degradation: a promise for undruggable targets. Cell Chem Biol 28, pages 934-951 (2021)).
  • EP3865152A1 discloses a conjugate of target molecule-linker-E3 ligase ligand as shown in the structure of A-L1-B (formula I), wherein the A is the monovalent group of the target molecule, the B is the monovalent group of the E3 ligase ligand, the L1 is the spacer linking A and B.
  • conjugates are described including an antibody attached to a flexible spacer between A and B and connected via an amine attachment point at a position between a PLK1- binding unit (A) and the E2 ligase binding unit (B).
  • PLK1 targeting HBCs which includes CN106543185, wherein tetrahydropteridin-based HBCs are disclosed that degrade PLK1 by hijacking the UPS system.
  • PLK1 degraders as therapeutic modalities, there is a clear need for improved PLK1 degradation inducing HBCs with potent PLK1 degrading activity and potent antiproliferative activity.
  • Still another object of embodiments of the present invention is to provide a therapeutic use of the compounds.
  • an aim of embodiments of the present invention is to provide bifunctional compounds providing improved pharmacological activity towards kinases, in particular towards Polo-like Kinase 1 .
  • Another aim of certain embodiments of this invention is to provide novel cancer treatments.
  • bifunctional compounds capable of inducing degradation of PLK1 kinases and conjugates based on bifunctional moieties derived from the bifunctional compounds according to the invention. Also provided herein are methods of using the bifunctional compounds and the conjugates according to the invention as medicament, and for treating disorders, including cancer, or in the prevention or treatment of disorders through modulation of Polo-like Kinase 1 (PLK1).
  • PLK1 Polo-like Kinase 1
  • TLB is a Targeting Ligase Binding group capable of binding to a E3 ligase
  • PB is a Protein Binding group capable of binding to Polo-like Kinase 1 (PLK1) and Sp is a spacer, which covalently links the Targeting Ligase Binding group (TLB) to the Protein Binding group (PB);
  • PB has the chemical structure BPLKI-X, wherein BPLKI has the formula: , wherein Ri is selected from the group consisting of H, methyl, ethyl or propyl; wherein R2 is selected from the group consisting of (1 -6C)cycloalkyl, (1 -6C)alkyl or benzyl; wherein any of said cycloalkyl, alkyl or benzyl is optionally and independently substituted with one or more substituents selected from halogen, hydroxy and cyano; wherein R3 is selected from the group consisting of OMe, OCHF2, OCF3, OCD3, (1-
  • bifunctional compounds of the invention are capable of providing improved kinase degradation, in particular of PLK1 kinase.
  • conjugates based on a bifunctional moiety derived from these bifunctional compounds are capable of providing improved kinase degradation, in particular of PLK1 kinase.
  • TLB is a Targeting Ligase Binding group capable of binding to a E3 ligase
  • PB is a Protein Binding group capable of binding to Polo-like Kinase 1 (PLK1)
  • Sp is a spacer, which covalently links the Targeting Ligase Binding group (TLB) to the Protein Binding group (PB);
  • PB has the chemical structure BPLKI-X, wherein BPLKI has the formula:
  • R1 is selected from the group consisting of H, methyl, ethyl or propyl
  • R2 is selected from the group consisting of (1 -6C)cycloalkyl, (1 -6C)alkyl or benzyl; wherein any of said cycloalkyl, alkyl or benzyl is optionally and independently substituted with one or more substituents selected from halogen, hydroxy and cyano
  • R3 is selected from the group consisting of OMe, OCHF2, OCF3, OCD3, (1- 3C)alky I, O(CH 2 ) 2 OH or O(CH 2 ) 3 OH
  • R4 is selected from the group consisting of H or methyl
  • the X group has a formula (X-1) to (X-28) selected from the group consisting of:
  • any of said formula X-1 to X-28 is optionally and independently substituted with one or more substituents selected from fluoro, hydroxy, (1 -3C)alkyl and (1 -3C)alkoxy; wherein n is 1 to 4; wherein —
  • a conjugate comprising a bifunctional compound according to the first aspect, having Formula (I), coupled to a moiety or agent.
  • D is a bifunctional moiety capable of inducing degradation of PLK1 ;
  • T comprises or consists of a targeting moiety capable of binding to a cell surface molecule
  • L is a linker, covalently bound to T and D; a is an integer from 1 to 8;
  • D is a bifunctional moiety having the chemical structure TLB-Sp-PB, wherein TLB, Sp and PB have the chemical structure according to the invention and wherein the X group of PB is covalently bound to the linker L, optionally by conversion of the -OH group of the X group to a bonding group containing -O-.
  • a bifunctional compound or the conjugate according to the invention for use in the treatment of disorders through modulation of Polo-like Kinase 1 (PLK1).
  • PKA1 Polo-like Kinase 1
  • a bifunctional compound or the conjugate according to the invention or a pharmaceutically acceptable salt thereof for use in the treatment of cancer.
  • a use of the bifunctional compound or the conjugate according to the invention or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament is provided.
  • composition which comprises the bifunctional compound or the conjugate according to the invention or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients.
  • a method for treating of cancer in a subject in need thereof comprising administering to the subject the bifunctional compound or the conjugate according to the invention or a pharmaceutically acceptable salt thereof in an amount effective to treat cancer.
  • a method for treating a subject suffering with a disorder comprising administering to the subject the bifunctional compound or the conjugate of the invention in an amount effective to treat the disorder through modulation of PLK1 .
  • a bifunctional compound or the conjugate according to the invention for use in the treatment of disorders or diseases are selected from the group consisting of cancer, benign tumor, a neurological disorder, an inflammatory disease, a disease characterized by inflammasome activation, an autoimmune disease, a hyperproliferative disease, a disorder that is caused by tissue rejection by the immune system, or for use in a therapy to prevent tissue rejection by the immune system.
  • Each of the sub-formulas 1 - 152 of the compounds of the invention is a preferred embodiment of the present application.
  • HBCs heterobifunctional compounds
  • the bifunctional compounds or bifunctional moieties according to the invention have crucial and novel characteristics. First they are able to induce degradation of PLK1 , enabling therapeutic applications that more closely mimic tumor targeting phenotypes in biological knockdown experiments.
  • the compounds according to the invention contain a unique functional moiety, in the form of a hydroxyalkyl group or side-chain, such as a hydroxymethylene or hydroxyethylene group, which has the unexpected characteristic that it simultaneously improves the capability of the bifunctional compounds for PLK1 degradation and their cytoxicity towards tumor cells.
  • the inventors have found and experimentally shown that having the hydroxyalkyl group or hydroxyalkyl side-chain of the Protein Binding group (PB), wherein PB has the chemical structure BPLK1-X, simultaneously improves the capability of the bifunctional compounds for PLK1 degradation and their cytoxicity towards tumor cells.
  • the Protein Binding group (PB) is the targeting group for targeting the PLK1 protein.
  • the inventors have found that the structural configuration of the X-group, including the configuration of the hydroxyalkyl group or hydroxyalkyl side-chain, in connection to the binding moiety (BPLKI) of the Protein Binding group (PB) cooperatively improve the capability of the bifunctional compounds for PLK1 degradation and their cytoxicity towards tumor cells.
  • the X-group is directly covalently connected to the amide (-C(O)- NH-) group of moiety BPLKI and by its direct connection cooperates with binding moiety BPLKI and positions the X-group, including its unique functional moiety of the X-group, close to the BPLKI group of the Protein Binding group (PB).
  • PB Protein Binding group
  • PB Protein Binding group
  • BPLKI binding moiety
  • Another advantage of certain embodiments of this invention is to provide cancer treatments based on the administration of the bifunctional compounds or conjugates of the invention.
  • composition as used herein has its conventional meaning and refers to a composition which is pharmaceutically acceptable.
  • the term “pharmaceutically acceptable” as used herein has its conventional meaning and refers to compounds, material, compositions and/or dosage forms, which are, within the scope of sound medical judgment suitable for contact with the tissues of mammals, especially humans, without excessive toxicity, irritation, allergic response and other problem complications commensurate with a reasonable benefit/risk ratio.
  • the term "effective amount as used herein, refers to an amount of the compound of the invention, and/or an additional therapeutic agent, or a composition thereof, that is effective in producing the desired therapeutic, ameliorative, inhibitory or preventative effect when administered to a subject suffering from a disease or disorder, such as a disease or disorder that can be treated by modulation of PLK1 .
  • as effective amount can refer to each individual agent or to the combination as a whole, wherein the amounts of all agents administered are together effective, but wherein the component agent of the combination may not be present individually in an effective amount.
  • a “subject” is a human or non-human mammal. In one embodiment, a subject is a human.
  • controlling is intended to refer to all processes wherein there may be a slowing, interrupting, arresting or stopping of the progression of the diseases and conditions affecting the mammal. However, “controlling” does not necessarily indicate a total elimination of all disease and condition symptoms and is intended to include prophylactic treatment.
  • excipient as used herein has its conventional meaning and refers to a pharmaceutically acceptable ingredient, which is commonly used in the pharmaceutical technology for preparing a granulate, solid or liquid oral dosage formulation.
  • salt as used herein has its conventional meaning and includes the acid addition and base salts of the compound of the invention.
  • solvate as used herein has its conventional meaning.
  • One or more compounds of the invention or the pharmaceutically acceptable salts thereof may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like.
  • “Solvate” means a physical association of a compound of this invention with one or more solvent molecules. This physical association Involves varying degrees of ionic and covalent bonding. Including hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid.
  • Solvate encompasses both solution-phase and isolatable solvates. Examples of suitable solvates include ethanolates, methanolates, and the like.
  • “Hydrate” is a solvate wherein the solvent molecule is H2O and includes any hydrate of the compound or the salt of said compound.
  • treatment has its conventional meaning and refers to curative, palliative and prophylactic treatment.
  • unit dosage form has its conventional meaning and refers to a dosage form which has the capacity of being administered to a subject, preferably a human, to be effective, and which can be readily handled and packaged, remaining as a physically and chemically stable unit dose comprising the therapeutic agent, i.e. the compound of the invention.
  • heterobifunctional compounds (HBCs) or bifunctional compounds or bifunctional moieties as used herein has its conventional meaning.
  • HBC consist of three covalently bound moieties: a ligand moiety to bind the POI (POI ligand), another ligand moiety to bind E3 ligase (E3 ligand) and a spacer to conjugate the two moieties.
  • HBC simultaneously recruits E3 ligase and POI, forming the “E3 ligase-HBC-POl” ternary complex. This complex potentiates the substrate recognition by E3 ligase and promotes the transfer of Ub to POI, accelerating the poly- ubiquitination and subsequent proteasome-mediated degradation of POI.
  • the bifunctional compounds according to the invention are heterobifunctional compounds (HBCs).
  • PLK1 degrader as used herein has its conventional meaning and refers to a degrading inducing compound for Polo-like Kinase 1 (PLK1).
  • a Polo-like Kinase 1 (PLK1) degrader may be a small molecule degrader. Degraders eliminate whole functions of the PLK1 protein, rather than merely inhibiting its enzymatic activity .
  • targeting moiety refers to a molecule which is capable of binding (selectively) to a target cell-surface molecule expressed on a target cell.
  • the targeting moiety is in preferred embodiments of the invention an antibody or an antigen-binding fragment thereof.
  • the targeting moiety is indicated in the chemical structures in the context of this invention with T.
  • Immunoglobulins are typically glycoproteins consisting of two pairs of polypeptide chains, a pair of heavy chains and a pair of light chains, all connected by disulfide bonds. Variant immunoglobulins only containing a single polypeptide chain also occur in nature, for example in camelids and cartilaginous fishes.
  • Heavy chains typically consist of a variable region (VH) and a constant region, which typically is comprised of three domains, CH1 , CH2, and CH3.
  • Light chains are typically comprised of a light chain variable region (VL) and a light chain constant region (CL).
  • the VH and VL regions contain regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved, termed framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • VH and VL regions typically comprise three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.
  • an "antigen-binding fragment" of an antibody is defined as a fragment that essentially maintains the antigen-binding properties of a parental antibody.
  • a fragment may be generated by any method known in the art, including recombinant production.
  • Antibodies and antigen-binding fragments thereof in accordance with the invention include, but are not limited to, F(ab')2 fragments, Fab' fragments, Fab fragments, scFv, dsFv, single-domain antibody (sdAb), monovalent IgG, scFv-Fc, reduced IgG (rlgG), minibody, diabodies, triabodies, tetrabodies, Fc fusion proteins, nanobodies, variable V domains such as VHH, Vh, and other types of antigen recognizing immunoglobulin fragments and domains, such as derived from camelids or cartilaginous fishes.
  • full-length antibody refers to an antibody comprising two pairs of heavy and light chains, each containing the heavy and light chain constant and variable domains that are normally found in a wild-type antibody of that isotype.
  • a full-length lgG1 antibody contains VH, CH1 , CH2, CH3, hinge, VL and CL domains.
  • Full-length antibodies may comprise mutations relative to the sequence of the wild-type antibody, such as introduced cysteines as described herein.
  • the antibodies (immunoglobulins) of the present invention may be bi- or multispecific.
  • a bispecific antibody has one arm having a specificity for one target antigen, while the other arm recognizes a different target antigen.
  • each arm of the bispecific antibody may have specificity for a different epitope of the same antigen of the target cell.
  • antibodies may be monovalent (e.g. comprise a single antigen-binding domain for a given target antigen) or multivalent, such as bivalent (comprising multiple antigen-binding domains for a given target antigen).
  • the antibodies (immunoglobulins) of the present invention may be, but are not limited to, polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, mouse antibodies, rat antibodies, rat/mouse hybrid antibodies, llama antibodies, llama heavy-chain only antibodies, heavy-chain only antibodies, and veterinary antibodies.
  • the antibody (immunoglobulin) of the present invention is a monoclonal antibody.
  • any other molecules than antibodies that bind to a cell receptor or antigen of a target cell can also be used as the cell surface molecule targeting moieties for the conjugates of the present invention.
  • These targeting moieties include, but are not limited to, proteins, polypeptides, peptides, RNA-based probes, DNA-based probes, small molecules or enzyme substrate mimics. Examples of these non-antibody targeting moieties are interferons (e.g.
  • IFN-a, IFN-p, and IFN-y transferrins, lectins, epidermal growth factors (EGF) and EGF-like domains, gastrin-releasing peptides (GRP), platelet-derived growth factors (PDGF), transforming growth factors (TGF), vaccinia growth factor (VGF), insulin and insulin-like growth factors (IGF, e.g. IGF-1 and IGF-2), other suitable hormones such as thyrotropin releasing hormones (TRH), melanocyte-stimulating hormones (MSH), steroid hormones (e.g. estrogen and androgen), somatostatin, lymphokines (e.g.
  • CSF colony-stimulating factors
  • M-CSF and GM-CSF colony-stimulating factors
  • aptamers e.g. AS-1411 , GBI-10, RNA aptamers against HIV glycoprotein
  • small molecules e.g. folate, anisamide phenylboronic acid, PSMA-617
  • vitamins e.g., vitamin D
  • carbohydrates e.g. hyaluronic acid, galactose
  • DAR as used herein has its conventional meaning and refers to the drug antibody ratio of the conjugate, which is the average number of degrader-linker complexes linked to each antibody. In certain aspects, the DAR of the conjugates described herein is from 1 to 10.
  • linker as used herein has its conventional meaning and refers to the chemical connector between the antibody and degrader payload; it may be non-cleavable or cleavable; cleavage can take place with proteases, hydrolysis or reduction mechanisms.
  • cleavable linker as used herein has its conventional meaning and refers to the linker that is susceptible to acid-induced cleavage, photo-induced cleavage, bioreductive cleavage, enzymatic cleavage, or the like, at conditions under which the bifunctional degrader D and/or targeting moiety T can remain active.
  • a non-exhaustive list of examples of cleavable linkers can be found in J Bargh et al. Cleavable linkers in antibody-drug conjugates. Chemical Society Reviews 48, pages 4361 -4374 (2019) and references therein.
  • non-cleavable linker 3 has its conventional meaning and refers to any chemical moiety that is capable of linking the targeting moiety T to the bifunctional degrader D in a stable, covalent manner.
  • a targeting moiety in the form of an antibody might get processed intracellularly, leading to release of the degrader still attached to the non-cleavable linker, which in turn is attached to a residual amino acid. In general, this metabolite then has the desired pharmacological activity.
  • a non-exhaustive list of examples of non-cleavable linkers can be found J Dugal-Tessier and N Jain. Non-cleavable Linkers: Permanently Linked, for Better or for Worse. Chapter 4 IN: Chemical Linkers in Antibody-Drug Conjugates (ADCs). Editor: Floris van Delft, ISBN 978-1-83916-515-3 (2021) and references therein.
  • proteavable as used herein has its conventional meaning and refers to a linker in which hydrolysis by the lysosomal proteases like Cathepsins, releases the bifunctional degrader D.
  • pyrophosphate or phosphate cleavable linker as used herein has its conventional meaning and refers to a linker in which hydrolysis by the lysosomal phosphatases releases the bifunctional degrader D.
  • beta-glucuronidase cleavable linker as used herein has its conventional meaning and refers to a linker in which hydrolysis by the lysosomal enzyme beta-glucuronidase releases the bifunctional degrader D.
  • click-to-release linker has its conventional meaning and refers to a linker where release of the degrader is chemically triggered by a tetrazine or related compound.
  • bioreducible linker as used herein is a linker that keep conjugates intact during systemic circulation, and are selectively cleaved by the high intracellular concentration of glutathione, releasing the active drugs (degraders) at the tumor sites from the non-toxic prodrugs.
  • EWG electron withdrawing group
  • Exemplary electron withdrawing groups include, but are not limited to -C(O), -CN, -NO2, -CX3, -X, -C(O)OR , - C(O)N(R)2, -C(O)R, -C(O)X, -S(O) 2 R, -S(O) 2 OR, -S(O) 2 NHR,-S(O) 2 N(R)2, -P(O)(OR)2, -P(O)(CH3)NHR, -NO, -N(R)3 + , wherein X is -F, -Br, -Cl, or -I, and R in some aspects is, at each occurrence, independently selected from the group consisting of hydrogen and (1 -6C)alkyl, and certain O-linked moieties such as acyloxy.
  • IC50 as used herein has its conventional meaning and refers to the concentration of a substance that results in a 50% effect on some measure of biochemical function or substance-target binding interaction.
  • DC50 as used herein has its conventional meaning and refers to the half- maximal degradation concentration that resulted in a 50% targeted protein degradation.
  • a bicyclic ring system refers to heterocyclic (heterocyclyl) groups, to cyclic groups having carbon groups only, i.e. without hetero atoms, within the cycle, and to combinations of a heterocyclic (heterocyclyl) group and a cyclic group having carbon groups only, i.e. without hetero atoms, within the cycle.
  • a bicyclic ring system includes 6-12 (e.g. 8-12, or 9-, 10-, or 11-) membered structures that form two rings, wherein the two rings have at least one atom in common (e.g. two atoms in common).
  • Bicyclic ring systems include bicycloaliphatics (e.g. bicycloalkyl or bicycloalkenyl), bicycloheteroaliphatics, bicyclic aryls, and bicyclic heteroaryls.
  • the rings can be different in size and nature, or identical in size and nature. Examples include spirobutane, spiropentane, spirohexane, spiroheptane, spirooctane, spirononane, or spirodecane.
  • One of both rings in a spirocycle can be fused to another ring carbocyclic, heterocyclic, aromatic, or heteroaromatic ring.
  • a (C3-Ci2)spirocycloalkyl is a spirocycle containing between 3 and 12 carbon atoms.
  • spiroheterocycloalkyl or “spiroheterocyclyl” means a spirocycle wherein at least one of the rings is a heterocycle wherein one or more of the carbon atoms can be substituted with a heteroatom (e.g., one or more of the carbon atoms can be substituted with a heteroatom in at least one of the rings).
  • One or both of the rings in a spiroheterocycle can be fused to another ring carbocyclic, heterocyclic, aromatic, or heteroaromatic ring.
  • a monocylic ring system refers both to a heterocyclic (heterocyclyl) group, and to a cyclic group having carbon groups only, i.e. without hetero atoms, within the cycle.
  • heterocyclic (heterocyclyl) group refers to both heteroaryl groups and heterocycloalkyl groups.
  • a heterobicyclic group refers to a bicyclic group having one or more heteroatoms, which is saturated, partially unsaturated or unsaturated.
  • aromatic groups include aromatic carbocyclic ring systems (e.g. phenyl) and fused polycyclic aromatic ring systems (e.g. naphthyl and 1 , 2,3,4- tetrahydronaphthyl).
  • heteroaryl refers to an aryl group having one or more hetero atoms.
  • alkyl refers to an aliphatic hydrocarbon group having one of its hydrogen atoms replaced with a bond having the specified number of carbon atoms.
  • an alkyl group contains, for example, from 1 to 6 carbon atoms (1 -6C)Alkyl or from 1 to 3 carbon atoms (1 -3C)Alkyl.
  • alkyl groups include methyl, ethyl, n- propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, neopentyl, isopentyl, n-hexyl, isohexyl and neohexyl.
  • an alkyl group is linear. In another embodiment, an alkyl group is branched.
  • alkyl includes both branched- and straight-chain saturated aliphatic hydrocarbon groups, including all isomers, having the specified number of carbon atoms; for example, “(1-6C)Alkyl” includes all of the hexyl alkyl and pentyl alkyl isomers as well as n-, iso- , sec- and t-butyl, n- and isopropyl, ethyl and methyl.
  • Alkylene refers to both branched- and straight-chain saturated aliphatic hydrocarbon groups, including all isomers, having the specified number of carbons, and having two terminal end chain attachments; for example, the term “A-C4 alkylene-B” represents, for example, A-CH2-CH2-CH2-CH2-B, A-CH2-CH2-CH(CH 3 )-CH 2 -B, A-CH2- CH(CH 2 CH 3 )-B, A-CH 2 -C(CH 3 )(CH 3 )-B, and the like.
  • alkylcarbonyl refers to an aliphatic hydrocarbon group having one of its hydrogen atoms replaced with a bond attached to a carbonyl group, wherein the aliphatic hydrocarbon group has the specified number of carbon atoms.
  • an alkyl group or aliphatic hydrocarbon group contains, for example, from 1 to 6 carbon atoms (1-6C)Alkyl or from 1 to 3 carbon atoms (1 -3C)Alkyl.
  • alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, neopentyl, isopentyl, n- hexyl, isohexyl and neohexyl.
  • an alkyl group is linear. In another embodiment, an alkyl group is branched.
  • Cycloalkyl means a cycloalkyl group having the recited number of carbon atoms, with the same meaning as previously defined, such as cyclopropyl, cyclobutyl, or cyclopentyl. “Cycloalkyl” refers to a cycloalkyl-group represented by an indicated number of carbon atoms; for example “(3- 6C)cycloalkyl” includes cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.
  • Heterocycloalkyl means a cycloalkyl group having the recited number of carbon atoms, and 1-3 heteroatoms selected from N, O and/or S, with the same meaning as previously defined.
  • Haloalkyl means a branched or unbranched alkyl group having the recited number of carbon atoms, in which one and up to all hydrogen atoms are replaced by a halogen; halogen is as defined herein.
  • branched or straight chained haloalkyl groups useful in the present invention include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl and n- butyl substituted independently with one or more halogens, e.g., fluoro, chloro, bromo and iodo.
  • a halo(1-3C)alkyl means a branched or unbranched alkyl group having 1 ,2, or 3 carbon atoms, in which at least one hydrogen atom is replaced by a halogen.
  • haloalkyl include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 1 -fluoroethyl, 2-fluoroethyl, 2,2-difluoroethy I, 2,2,2-trifluoroethy I, and perfluoro-n-propyl.
  • Alkoxy means an alkoxy group having the recited number of carbon atoms, the alkyl moiety having the same meaning as previously defined, e.g., “Alkoxy” refers to an alkyl-O-group represented by a linear or branched alkyl group of indicated number of carbon atoms attached through an oxygen bridge; for example “(1 -6C)Alkoxy” includes CH 3 -O-, CH2CH 3 -O-, CH(CH 3 )2-O- , (CH 2 ) 5 CH 3 -O-, and the like.
  • Cycloalkoxy means a cycloalkyl group having the recited number of carbon atoms, with the same meaning as previously defined, attached via a ring carbon atom to an exocyclic oxygen atom, such as cyclopropoxyl, cyclobutoxyl,or cyclopentoxyl.
  • Cycloalkoxy refers to a cycloalkyl- O-group represented by a cycloalkyl group of indicated number of carbon atoms attached through an oxygen bridge; for example “(3-6C)cycloalkoxy” includes cyclopropyl-O-, cyclobutyl-O-, cyclopentyl-O-, or cyclohexyl-O-.
  • Heterocycloalkoxy means a cycloalkyl group having the recited number of carbon atoms, and 1 -3 heteroatoms selected from N, O and/or S, with the same meaning as previously defined, attached via a ring carbon atom to an exocyclic oxygen atom.
  • alkyl groups are unsubstituted or substituted with 1 to 3 substituents on each carbon atom.
  • compositions comprising components A and B
  • the only enumerated components of the composition are A and B, and further the claim should be interpreted as including equivalents of those components.
  • indefinite article “a” or “an” does not exclude the possibility that more than one of the element or component are present, unless the context clearly requires that there is one and only one of the elements or components.
  • the indefinite article “a” or “an” thus usually means “at least one”.
  • Figure 1 Results for the A-549 PLK1-HiBiT high throughput degradation assay.
  • Compounds according to Example 1 and Example 2 show dose-dependent complete reduction of the HiBiT signal, demonstrating complete reduction of the levels of HiBiT tagged PLK1 in the cell line.
  • MG132 inhibits the proteasome. Adding MG132 leads to reduction of the potency of Example 1 and Example 2, indicating that their activity is dependent on proteasome activity.
  • FIG. 2 Western Blot of PLK1 degradation by Example 1 and Example 2 in the A549 PLK1 -HiBiT cell line. Co-incubation with the proteasome inhibitor MG132 abrogates PLK1 degradation, indicating PLK1 degradation by the Example compounds is proteasome-dependent. Onvansertib, BI2536 and CC885 are included as references. Cells were exposed for 24h to 10 pM compound.
  • Figure 3 Western Blot of dose dependent PLK1 degradation by Example 1 and Example 2 in the A549 PLK1 -HiBiT cell line. Cells were exposed to compound for 24h. Both Examples were analyzed on the same blot, dashed line was added for ease of viewing.
  • FIG. 4 Western Blot of dose dependent PLK1 degradation by Example 1 and Example 2 in the A549 PLK1 -HiBiT cell line, detect via an antibody to the HiBiT tag that is present on the N- terminus of PLK1 . Cells were exposed to compound for 24h. Both Examples were analyzed on the same blot, dashed line was added for ease of viewing.
  • Figure 5 Western Blot of dose dependent PLK1 degradation by Example 1 and Example 2 in the AU565 cell line. Cells were exposed to compound for 24h. Both Examples were analyzed on the same blot, dashed line was added for ease of viewing.
  • FIG. 6 Western Blot of PLK1 degradation by Example 1 (1 pM) in the AU565 cell line, and competition by the PLK1 inhibitor BI2536. AU565 cells were exposed to compounds simultaneously for 24h. All samples were analyzed on the same blot, solid lines indicate the removal of redundant samples. Addition of equimolar or higher concentrations of the PLK1 inhibitor BI2536 can abrogate PLK1 degradation by Example 1 , confirming that Example 1 acts through PLK1 binding.
  • Figure 7 Western Blot of PLK1 degradation by Example 1 (1 pM) in the AU565 cell line, and competition by the cereblon binding drug pomalidomide. AU565 cells were exposed to compounds simultaneously for 24h. All samples were analyzed on the same blot, solid lines indicate the removal of redundant samples. Addition of equimolar or higher concentrations of pomalidomide can abrogate PLK1 degradation by Example 1 , confirming that Example 1 acts through cereblon binding.
  • Figure 8 Incubation of Example 154m with papain in buffer leads to proteolytic cleavage of the linker and release of Example 2.
  • Figure 8A Chromatogram (LC) of Example 154m in absence of papain.
  • Figure 8B Chromatogram after 24h incubation with papain.
  • Figure 8C Mass spectrum (MS) of peak number 6 in Figure 8B, indicating presence of a compound with identical mass to Example 2.
  • Figure 9 Results of an SDS PAGE of various conjugates to the antibody rosopatamab and the parental rosopatamab batch.
  • the conjugates the light chain and heavy chain protein bands are shifted to higher molecular weight, indicating a covalent attachment of degrader-linker moieties and thus successful conjugation.
  • Figure 10 Results of a proliferation assay of the PSMA-positive cell line LNCaP.
  • Conjugates of ready-to-conjugate intermediate examples 157m, 158m and 159m to the PSMA binding antibody rosopatamab show dose-dependent cytotoxicity on this cell line, whereas the unconjugated antibody (rosopatamab) does not have any effect. This shows that the conjugation procedure causes the antibody to become cytotoxic.
  • Figure 11 Results of a proliferation assay of the PSMA-negative cell line PC3.
  • Conjugates of ready-to-conjugate intermediate examples 157m, 158m and 159m to the PSMA binding antibody rosopatamab show very limited toxicity on this cell line, compared to the cytotoxicity on LNCaP presented in Figure 10. This shows that the activity of the conjugates depends on their binding to PSMA and thus on functional antibody binding.
  • the present application addresses the provision of PLK1 targeting bifunctional compounds (HBCs) that are suitable as therapeutic compounds for targeting and inducing degradation of PLK1 kinases.
  • HBCs PLK1 targeting bifunctional compounds
  • the present application also addresses the provision of conjugates containing bifunctional moieties based on PLK1 targeting bifunctional compounds (HBCs) according to Formula (I) that are suitable as therapeutic compounds for targeting and inducing degradation of PLK1 kinases.
  • HBCs PLK1 targeting bifunctional compounds
  • the compounds according to the invention have a Targeting Ligase Binding group (TLB), which is a group that is capable of binding to a E3 ligase, a Protein Binding group (PB) capable of binding to Polo-like Kinase 1 (PLK1), wherein the Protein Binding group (PB) has the chemical structure BPLKI-X, and a spacer (Sp).
  • the spacer (Sp) covalently binds the Targeting Ligase Binding group (TLB) to the X group of the Protein Binding group (PB). As such the spacer covalently links the Protein Binding group (PB) to the Targeting Ligase Binding group (TLB).
  • the bifunctional compounds (HBCs) according to the invention have crucial and novel characteristics. First they are able to induce degradation of PLK1 , enabling therapeutic applications that more closely mimic tumor targeting phenotypes in biological knockdown experiments.
  • the compounds according to the invention in particular the Protein Binding Group of these compounds, have an X-group, which contains a unique functional moiety, in the form of a hydroxyalkyl group, such as a hydroxymethylene or hydroxyethylene side-group, which has the unexpected characteristic that it simultaneously improves the capability of the HBCs for binding PLK1 and for PLK1 degradation and their cytoxicity towards tumor cells.
  • the inventors have found that the bifunctional compounds (HBCs) due to the X group of the Protein Binding group (PB) having an unique functional moiety, wherein the X group, which comprises a hydroxyalkyl group improves the degradation effect of the bifunctional compounds (HBCs).
  • the X-group is covalently connected to the amide (-C(O)-NH-) group of BPLKI and positions the unique functional moiety of the X-group close to the BPLKI group of the Protein Binding group (PB).
  • the hydroxyalkyl functional group of the X group supports or improves the binding process of the Protein Binding group (PB) to the PLK1 and at the same time supports or improves the degradation process of the PLK1 by the bifunctional compound, when the PLK1 protein is (temporarily) bound by the Protein Binding group (PB) of the bifunctional compound.
  • PLK1 targeting bifunctional compounds (HBCs) or bifunctional moieties according to the invention are suitable for inducing PLK1 degradation.
  • Another advantage of certain embodiments of this invention is to provide cancer treatments based on the administration of the bifunctional compounds or conjugates of the invention.
  • bifunctional compounds according to the invention and in particular the X group of the bifunctional compounds, are suitable for forming conjugates comprising the bifunctional moieties.
  • the bifunctional compounds according to the invention can also be used as intermediate compounds for forming Degrader Antibody Conjugates (DAC), i.e. conjugates with an antibody group or conjugates with target functional fragments of antibodies.
  • DAC Degrader Antibody Conjugates
  • ready-to-conjugate intermediate compounds may be provided based on the bifunctional moieties according to the invention coupled to a ready-to-conjugate linker. These ready-to-conjugate intermediate compounds can easily be coupled to targeting moieties, preferably to antibodies or binding fragments thereof.
  • DAC Degrader Antibody Conjugates
  • Degrader Antibody Conjugates according to the invention are in preferred embodiments structured to reform the original bifunctional compounds according to the invention under (internal) cell conditions, wherein the Protein Binding group (PB) has the chemical structure BPLKI-X, including the original hydroxyalkyl functional group of the X group.
  • PB Protein Binding group
  • BPLKI-X the chemical structure of the degrader moiety as defined according to the invention is easily obtained from the Degrader Antibody Conjugates (DAC) according to the invention.
  • the conjugates have double selectivity: one from antibodies and the other from the PLK1 targeting bifunctional moieties.
  • the antibody in the conjugate the invention may bind a specific antigen on a cancer cell, and then the conjugate enters the cell via internalization.
  • the PLK1 targeting bifunctional moiety finds PLK1 and brings it to E3 ubiquitin ligase for ubiquitination.
  • the ubiquitinated PLK1 is marked for degradation by proteasomes. This happens only in cells expressing the specific antigen. Thus, for therapeutic effect, antigen expression and PLK1 expression is required.
  • a conjugate of the invention is highly selective and will have less adverse effects.
  • Bifunctional compounds of the invention are according to Formula (I):
  • TLB is a Targeting Ligase Binding group capable of binding to a E3 ligase
  • PB is a Protein Binding group capable of binding to Polo-like Kinase 1 (PLK1)
  • Sp is a spacer, which covalently links the Targeting Ligase Binding group (TLB) to the Protein Binding group (PB);
  • PB has the chemical structure BPLKI-X, wherein BPLKI has the formula:
  • Ri is selected from the group consisting of H, methyl, ethyl or propyl
  • R2 is selected from the group consisting of (1 -6C)cycloalkyl, (1 -6C)alkyl or benzyl; wherein any of said cycloalkyl, alkyl or benzyl is optionally and independently substituted with one or more substituents selected from halogen, hydroxy and cyano
  • R3 is selected from the group consisting of OMe, OCHF2, OCF3, OCD3, (1 -3C)alky I, O(CH 2 ) 2 OH or O(CH 2 ) 3 OH
  • R4 is selected from the group consisting of H or methyl; wherein denotes the point of attachment to the X group; wherein the X group has a formula (X-1) to (X-28) selected from the group consisting of:
  • a conjugate comprising a bifunctional compound according to Formula (I) coupled to a moiety or agent.
  • the moiety or agent is selected from the group consisting of a targeting moiety, a radioisotope, a protein, an enzyme, a dye, a drug, a pharmacologically active small molecule.
  • the moiety is a targeting moiety capable of binding to a cell surface molecule.
  • the conjugates according to the invention have the chemical structure: T - (L - D) a or a pharmaceutically acceptable salt thereof, wherein:
  • T comprises or consists of a targeting moiety capable of binding to a cell surface molecule
  • L is a linker, covalently bound to T and D; a is an integer from 1 to 8;
  • the bifunctional moiety D is based on the bifunctional compound according to the invention (according to the first aspect), which is also a ready to conjugate compound Dm, which has the chemical structure TLB-Sp-PB, wherein PB has the chemical structure BPLKI-X, and wherein PB is capable of binding to Polo-like Kinase 1 (PLK1) kinases.
  • Dm which has the chemical structure TLB-Sp-PB
  • PB has the chemical structure BPLKI-X
  • PB is capable of binding to Polo-like Kinase 1 (PLK1) kinases.
  • the X group of the bifunctional moiety D is covalently bound to the linker L by conversion of the -OH group of the X group to a bonding group containing -O-.
  • the bonding group provides a covalent bond to the linker L.
  • EP3865152A1 discloses a conjugate of target molecule-linker-E3 ligase ligand as shown in the structure of A-L1-B (formula I), wherein the A is the monovalent group of the target molecule, the B is the monovalent group of the E3 ligase ligand, the L1 is the spacer linking A and B.
  • conjugates are described including an antibody attached to a flexible spacer moiety between A and B and connected via an amine attachment point at a position between a PLK1-binding unit (A) and the E2 ligase binding unit (B).
  • the amine attachment point for connection via a linker to the antibody impairs the ternary complex formation.
  • Dragovitch et al. Chem. Soc. Rev. (2022) 51 , 3886 has reported that several reported chimeric degraders do not contain chemical groups that can be utilized for the covalent attachment of cleavable linkers (e.g., primary, secondary, and/or tertiary amines). In such instances, careful consideration must be given regarding whether to purposefully incorporate the necessary chemical groups into the degrader structure (and possibly alter its associated biological properties) or to exploit existing degrader functionality such as hydroxyl/phenol groups or add such a hydroxyl functionality for using ADC linking approaches.
  • cleavable linkers e.g., primary, secondary, and/or tertiary amines
  • the present compounds are provided containing the chemical structure BPLKI-X, wherein the alkylhydroxy group is provided as part of the targeting moiety (showing both inhibitor and degrader effects) to exploit improved degrader functionality and at the same time provide new ADC linking approaches.
  • the Protein Binding group PB of the conjugate has chemical structure
  • any of said formula Xc-1 to Xc-28 is optionally and independently substituted with one or more substituents selected from fluoro, hydroxy, (1 -3C)alkyl and (1-3C)alkoxy; wherein n is 1 to 4; wherein the —
  • the Xc groups according to any one of formula (Xc-1) to (Xc-28) are derived from the X groups of the bifunctional compounds according to any one of formula (X-1) to (X-28), respectively, by converting the -OH group of the X group to a bonding group containing -O-.
  • the Xc-group of D is selected from the group consisting of: , wherein the — i — * denotes the point of attachment of the X group to the amide (-
  • the Protein Binding group PB is capable of binding to Polo-like Kinase 1 (PLK1).
  • the Protein Binding group PB has the chemical structure BPLKI-X.
  • the Protein Binding group PB thus contains binding moiety BPLKI as shown below, and the X-group.
  • the Protein Binding group (PB) as a whole is the targeting group for targeting the PLK1 protein.
  • the inventors have found that the structural configuration of the X-group, including the configuration of the hydroxyalkyl group or hydroxyalkyl side-chain, in connection to the binding moiety (BPLKI) of the Protein Binding group (PB) cooperatively improve the capability of the bifunctional compounds for PLK1 degradation and their cytoxicity towards tumor cells.
  • the X-group is directly covalently connected to the amide (-C(O)- NH-) group of moiety BPLKI and by its direct connection cooperates with binding moiety BPLKI and positions the X-group, including its unique functional moiety of the X-group, close to the BPLKI group of the Protein Binding group (PB).
  • PB Protein Binding group
  • the Protein Binding Group has the Formula:
  • Ri is selected from the group consisting of H, methyl, ethyl or propyl
  • R2 is selected from the group consisting of (1 -6C)cycloalkyl, (1 -6C)alkyl or benzyl; wherein any of said cycloalkyl, alkyl or benzyl is optionally and independently substituted with one or more substituents selected from halogen, hydroxy and cyano
  • R3 is selected from the group consisting of OMe, OCHF2, OCF3, OCD3, (1-3C)alkyl, O(CH 2 ) 2 OH or O(CH 2 ) 3 OH
  • R4 is selected from the group consisting of H or methyl; wherein denotes the point of attachment to the X group.
  • R1 is H
  • the stereochemistry of R1 is not relevant as also the other subsituent is H.
  • R1 is ethyl or propyl
  • R2 is isopropyl, cyclohexyl, or cyclopentyl
  • R3 is OMe, OCD3 or OCF3, and wherein R4 is H or methyl
  • R1 is ethyl or propyl.
  • R2 is isopropyl, cyclohexyl, or cyclopentyl.
  • R3 is OMe
  • R4 is H or methyl.
  • the X-group of the Protein Binding group PB is further described below.
  • the X-group is directly covalently connected to the amide (-C(O)-NH-) group of BPLKI and positions the unique functional moiety of the X-group close to the rest of the BPLKI group of the Protein Binding group (PB).
  • the X group of the Protein Binding group PB is covalently bound to the spacer (Sp).
  • the X group has a formula (X-1) to (X-28) selected from the group consisting of:
  • any of said formula X-1 to X-28 is optionally and independently substituted with one or more substituents selected from fluoro, hydroxy, (1 -3C)alkyl and (1-3C)alkoxy; wherein n is 1 to 4; wherein —
  • the X-group contains a hydroxyalkyl group, such as a hydroxy-methylene or hydroxyethylene group, that is located near to the BPLKI . This improves the PLK1 binding effect of the Protein Binding group PB and improves the degradation effect of the bifunctional compounds (HBCs).
  • the X-group is directly covalently connected to the amide (-C(O)-NH-) group of BPLKI
  • the inventors have further found that an hydroxy group, which is directly attached to the (hetero)cyclic moiety of the X-group, instead of an alkylhydroxy substituent, is not suitable as substituent of the (hetero)cyclic moiety of the X-group.
  • X is selected from the group consisting of:
  • n 1 ,2 or 3; wherein ⁇
  • the Protein Binding group PB has the chemical structure BPLKI-X, which is selected from the group consisting of:
  • Ri is selected from the group consisting of H, methyl, ethyl or propyl; wherein R2 is selected from the group consisting of (1 -6C)cycloalkyl, (1 -6C)alkyl or benzyl; wherein R4 is selected from the group consisting of H or methyl; wherein "T* denotes the point of attachment to the spacer (Sp).
  • the spacer (Sp) covalently binds the Targeting Ligase Binding group (TLB) to the X group of the Protein Binding group PB.
  • the spacer (Sp) has the chemical structure -S 1 -S 2 -S 3 -S 4 -S 5 - , wherein S 1 is connected to the Targeting Ligase Binding group (TLB) and S 5 is connected to X, wherein:
  • 5 2 is independently selected from the group consisting of: a direct bond, -N(R S2 )-, -O-, (1- 4C)alkyl, -CH 2 -CH 2 -N(R S2 )-, -(CH 2 -CH 2 -O) m -, -(CH 2 -CH 2 -CH 2 -O) m -, -(O-CH 2 -CH 2 ) m -, (6- 10C)aryl, (3-12C)cycloalkyl, (3-12C)heterocycloalkyl, 7-12 membered spiro bicyclic heterocycloalkyl, 7-12 membered fused bicyclic heterocycloalkyl, 7-12 membered spiro bicyclic cycloalkyl and 7-12 membered fused bicyclic cycloalkyl; wherein any of said alkyl, cycloalkyl or heterocycloalkyl group is optionally and independently substituted with one
  • 5 3 is independently selected from the group consisting of: a direct bond, (1 -8C)alky I, -CEC-, N(R S3 )-, -O-, -N(R S3 )-C(O)-, -C(O)-N(R S3 )-, -C(O)-, -(O-CH 2 -CH 2 ) U -, -(CH 2 -CH 2 -O) U - and (3- 12C)heterocycloalkyl; wherein any of said heterocycloalkyl group is optionally and independently substituted with one or more substituents selected from halogen, oxo, hydroxy, (1 -3C)alkyl and (1-3C)alkoxy; each R S3 is independently -H or (1 -4C)alkyl;
  • 5 4 is independently selected from the group consisting of: a direct bond, (1 -4C)alky I, -N(R S4 ), - C(O)-, -(O-CH 2 -CH 2 ) P -, -(CH 2 -CH 2 -O) P -, (3-12C)cycloalkyl, (3-12C)heterocycloalkyl, 7-12 membered spiro bicyclic heterocycloalkyl and 7-12 membered fused bicyclic heterocycloalkyl; wherein any of said alkyl, cycloalkyl or heterocycloalkyl group is optionally and independently substituted with one or more substituents selected from halogen, oxo, hydroxy, (1 -3C)alkyl and (1-3C)alkoxy; each R S4 is independently -H or (1 -4C)alkyl;
  • 5 5 is independently selected from the group consisting of: a direct bond, -N(R S5 )-, N(R S5 )-C(O)-, -C(O)-, (1 -4C)alkyl, -(O-CH 2 -CH 2 ) q - and -(O-CH 2 -CH 2 -CH 2 ) q -; each R S5 is independently -H or (1 -4C)alkyl; each m, p, u and q is independently an integer from 1 to 3.
  • S 1 is selected from the group consisting of: a direct bond, - N(R S1 )-, -O-, -C(O)-N(R S1 )-, -N(R S1 )-C(O)-, -C(O)-, -CEC-, -OCH 2 C(O)-, (3-12C)heterocyclo- alkyl and (1 -4C)alkyl; wherein any said alkyl or heterocycloalkyl group is optionally and independently substituted with one or more substituents selected from fluoro and hydroxy; each R S1 is independently -H or methyl.
  • S 2 is selected from the group consisting of: a direct bond, (1 - 4C)alkyl, -CH 2 -CH 2 -N(R S2 )-, -(CH 2 -CH 2 -O)m-, -(CH2-CH2-CH 2 -O) m -, (6-10C)aryl, (3- 12C)cycloalkyl, (3-12C)heterocycloalkyl, 7-12 membered spiro bicyclic heterocycloalkyl, 7-12 membered fused bicyclic heterocycloalkyl, 7-12 membered spiro bicyclic cycloalkyl and 7-12 membered fused bicyclic cycloalkyl; wherein any of said alkyl, cycloalkyl or heterocycloalkyl group is optionally and independently substituted with one or more substituents selected from halogen, oxo, hydroxy, (1 -3C)alkyl and (1-3C)alkoxy
  • S 3 is selected from the group consisting of: a direct bond, (1 - 8C)alkyl, -N(R S3 )-, -O-, -N(R S3 )-C(O)-, -C(O)-N(R S3 )-, -C(O)-, -(O-CH 2 -CH 2 )u-, -(CH2-CH2- O) u - and (3-12C)heterocycloalkyl; wherein any of said or heterocycloalkyl group is optionally and independently substituted with one or more substituents selected from halogen, oxo, hydroxy, (1 -3C)alkyl and (1-3C)alkoxy; each R S3 is independently -H or methyl; u is an integer from 1 to 2.
  • S 4 is selected from the group consisting of: a direct bond, (1 - 4C)alkyl, -(O-CH 2 -CH 2 ) P -, -(CH 2 -CH 2 -O) P -, (3-12C)cycloalkyl and (3-12C)heterocycloalkyl; wherein any of said alkyl, cycloalkyl or heterocycloalkyl group is optionally and independently substituted with one or more substituents selected from halogen, oxo, hydroxy, (1 -3C)alkyl and (1-3C)alkoxy; p is an integer from 1 to 2.
  • S 5 is selected from the group consisting of: a direct bond, - N(R S5 )-, -N(R S5 )-C(O)-, -C(O)-, (1 -4C)alkyl, -(O-CH 2 -CH 2 ) q and -(O-CH 2 -CH2-CH 2 )q; each R S5 is independently -H or methyl; q is independently an integer from 1 to 2.
  • S 1 is selected from the group consisting of: a direct bond, -
  • any of said alkyl, cycloalkyl or heterocycloalkyl group is optionally and independently substituted with one or more substituents selected from halogen, oxo, hydroxy, (1 -3C)alkyl and (1-3C)alkoxy; m is an integer from 1 to 2.
  • S 3 is selected from the group consisting of: a direct bond, (1 -4C)alkyl, -N(R S3 )-, -O-, -N(R S3 )-C(O)-, -C(O)-N(R S3 )-, -C(O)-, -(O-CH 2 -CH 2 )u-, -(CH 2 -CH 2 - O) u - and (3-12C)heterocycloalkyl; wherein any of said alkyl or heterocycloalkyl group is optionally and independently substituted with one or more substituents selected from halogen, oxo, hydroxy, (1 -3C)alkyl and (1-3C)alkoxy; each R S3 is independently -H or methyl; u is an integer from 1 to 2.
  • S 4 is selected from the group consisting of: a direct bond, (1 -4C)alkyl, -N(R S4 ), -C(0)-, -(O-CH 2 -CH 2 ) P -, -(CH 2 -CH 2 -O) P - ,
  • any of said alkyl, cycloalkyl or heterocycloalkyl group is optionally and independently substituted with one or more substituents selected from halogen, oxo, hydroxy, (1 -3C)alkyl and (1-3C)alkoxy; each R S4 is independently hydrogen or methyl; p is an integer from 1 to 2.
  • S 5 is selected from the group consisting of: a direct bond, - N(R S5 )-, -N(R S5 )-C(O)-, -C(O)- and (1 -4C)alkyl; each R S5 is independently hydrogen or methyl.
  • TLB Targeting Ligase Binding group in case S 1 is a direct bond; and wherein the I marks the point of attachment to X; and wherein each R L is hydrogen or methyl; and wherein s is an integer from 0 to 5; and wherein t is an integer from 0 to 6.
  • the spacer (Sp) is selected from the group consisting of:
  • TLB Targeting Ligase Binding group
  • TLB Targeting Ligase Binding group
  • TLB Targeting Ligase Binding group
  • HBC heterobifunctional compounds
  • POI protein of interest
  • HBC consists of three covalently-bonded moieties: a ligand to bind POI (POI ligand), another ligand to recognize E3 ligase (E3 ligand) and a linker to conjugate the two ligands.
  • E3 ligand another ligand to recognize E3 ligase
  • HBC simultaneously recruits E3 ligase and POI, forming the “E3-HBC-POI” ternary complex. This complex potentiates the substrate recognition by E3 ligase and promotes the transfer of Ub to POI, accelerating the poly-ubiquitination and subsequent proteasome-mediated degradation of POI (Lai AC and Crews CM.
  • VHL Von-Hippel-Lindau
  • lAPs inhibitors of apoptosis proteins
  • CRBN Cereblon
  • TLB Targeting Ligase Binding group
  • each R TLB1 is independently selected from halo, cyano and (1 -4C)alkyl, wherein any of said alkyl group is optionally and independently substituted with one or more substituents selected from halogen, cyano, -COOH, COONH2, -NH2 and CF3;
  • each R TLB4 is independently selected from hydrogen, (1 -4C)alkyl and (1-3C)alkoxy;
  • each R TLB5 is independently selected from hydrogen, halo, cyano and (1 -3C)alkyl;
  • Z is -C(R TLB6 ) 2 or -C(O)-; each R TLB6 is independently selected from hydrogen and (1 -4C)alkyl;
  • Z’ is a bond, -C(O)- or -CH 2 C(O)-; Z” is -CH2- or -NH-; HAr is a (1-9C)heteroaryl, optionally substituted with one or more substituents selected from fluoro, methyl and methoxy; and heterocyclic ring G is selected from:
  • TLB Targeting Ligase Binding group
  • T ⁇ marks the point of attachment to the spacer (Sp) or S 1 of the spacer (Sp); wherein each R TLB5 is independently hydrogen, fluoro or cyano;
  • HAr is a (1-9C)heteroaryl optionally substituted with one or more substituents selected from fluoro, methyl and methoxy.
  • TLB Targeting Ligase Binding group
  • the Targeting Ligase Binding group is selected from the group consisting of: wherein the marks the point of attachment to the spacer (Sp) or S 1 of the spacer (Sp).
  • T comprises or consists of a targeting moiety capable of binding to a cell surface molecule.
  • targeting moiety refers to a molecule which is capable of binding to a target cellsurface molecule expressed on a target cell.
  • the targeting moiety is an antibody or an antigen-binding fragment thereof.
  • antibody as used herein is used in the broadest sense, which may refer to an immunoglobulin (Ig) defined as a protein belonging to the class IgG, IgM, IgE, IgA, or IgD (or any subclass thereof, for example IgG 1 , lgG2, lgG3 or lgG4), or a functional binding fragment or binding domain of an immunoglobulin.
  • An antibody is an immunoglobulin capable of specifically binding a target protein. Immunoglobulins are well-known proteins, described for example in Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)).
  • Immunoglobulins are typically glycoproteins consisting of two pairs of polypeptide chains, a pair of heavy chains and a pair of light chains, all connected by disulfide bonds. Variant immunoglobulins only containing a single polypeptide chain also occur in nature, for example in camelids.
  • Heavy chains typically consist of a variable region (VH) and a constant region, which typically is comprised of three domains, CH1 , CH2, and CH3.
  • Light chains are typically comprised of a light chain variable region (VL) and a light chain constant region (CL).
  • the VH and VL regions contain regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved, termed framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • VH and VL regions typically comprise three CDRs and four FRs, arranged from amino-terminus to carboxyterminus in the order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.
  • Any other molecules than antibodies that bind to a cell receptor or antigen of a target cell can also be used as the targeting moiety for the conjugates of the present invention.
  • the targeting moiety is selected from the group consisting of proteins, polypeptides, peptides, small molecules and enzyme substrate mimics.
  • non-antibody targeting moieties are interferons (e.g. IFN-a, IFN-p, and IFN-y), transferrins, lectins, epidermal growth factors (EGF) and EGF-like domains, gastrinreleasing peptides (GRP), platelet-derived growth factors (PDGF), transforming growth factors (TGF), vaccinia growth factor (VGF), insulin and insulin-like growth factors (IGF, e.g. IGF-1 and IGF-2), other suitable hormones such as thyrotropin releasing hormones (TRH), melanocyte-stimulating hormones (MSH), steroid hormones (e.g. estrogen and androgen), somatostatin, lymphokines (e.g.
  • interferons e.g. IFN-a, IFN-p, and IFN-y
  • transferrins e.g. IFN-a, IFN-p, and IFN-y
  • lectins e.g. IFN-
  • CSF colony-stimulating factors
  • M-CSF and GM-CSF colony-stimulating factors
  • aptamers e.g. AS-1411 , GBI-10, RNA aptamers against HIV glycoprotein
  • small molecules e.g. folate, anisamide phenylboronic acid, PSMA-617
  • vitamins e.g., vitamin D
  • carbohydrates e.g. hyaluronic acid, galactose
  • the conjugates according to the invention comprise a Linker (L), covalently bonding the T (targeting moiety) to D.
  • the linker L is a cleavable linker.
  • cleavable linker as used herein has its conventional meaning and refers to the linker that is susceptible to acid-induced cleavage, photo-induced cleavage, bioreductive cleavage, enzymatic cleavage, or the like, at conditions under which the bifunctional degrader D and/or targeting moiety T can remain active.
  • a non-exhaustive list of examples of cleavable linkers can be found in J Bargh et al. Cleavable linkers in antibody-drug conjugates. Chemical Society Reviews 48, pages 4361-4374 (2019) and references therein.
  • a linker (L) of the Degrader Antibody Conjugates (DAC) according to the invention is selected capable to reform the original bifunctional compounds according to the invention under (internal) cell conditions, wherein the Protein Binding group (PB) has the chemical structure BPLKI-X, including the original hydroxyalkyl functional group of the X group.
  • PB Protein Binding group
  • the degrader moiety D as defined according to the invention is easily obtained from the Degrader Antibody Conjugates (DAC) according to the invention in vivo under known cell conditions.
  • linker chemistry and Linker moieties based on common general knowledge on linker technology, which are able to reform the hydroxyalkyl functional group of the X group of the Protein Binding group (PB), which has the chemical structure BPLK1-X.
  • the cleavable linker is cleavable by a protease.
  • the linker L is selected from the group consisting of:
  • c is an integer from 2 to 10;
  • Z 1 , Z 2 , Z 3 , and Z 4 are each independently absent or a naturally occurring amino acid residue or the enantiomer thereof, provided that at least two of Z 1 , Z 2 , Z 3 , and Z 4 are amino acid residues;
  • the * denotes the point of attach me nt to T; and wherein the I denotes the point of attachment to Xc.
  • Z 1 , Z 2 , Z 3 , and Z 4 are independently absent or selected from the group consisting of L-valine, D-valine, L-citrulline, D-citrulline, L-alanine, D-alanine, L-glutamine, D- glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-asparagine, D- asparagine, L-phenylalanine, D-phenylalanine, L-lysine, D-lysine, L-proline, D-proline, and glycine, provided that at least two of Z 1 , Z 2 , Z 3 , and Z 4 are amino acid residues.
  • Z 1 is absent or selected from the group consisting of glycine, L-citru Hi ne or D-citru Hi ne ;
  • Z 2 is absent or selected from the group consisting of L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L aspartic acid, D-aspartic acid, L-alanine, D-alanine, L-valine, D-valine and glycine;
  • Z 3 is selected from the group consisting of L-valine, D-valine, L-alanine, D-alanine, L- phenylalanine, D-phenylalanine, L-proline, D-proline and glycine;
  • Z 4 is selected from the group consisting of L-alanine, D-alanine, L-citrulline, D-citrulline, L- asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine, and glycine.
  • L is selected from the group consisting of: wherein c is an integer from 2 to 10; wherein the TM * denotes the point of attachment to T; and wherein the denotes the point of attachment to Xc.
  • linker L is a pyrophosphate or phosphate cleavable linker.
  • linker L is selected from the group consisting of: wherein c is an integer from 2 to 10; wherein d is an integer 1 or 2; wherein the T ⁇ * denotes
  • linker L is a beta-glucuronidase cleavable linker.
  • linker L is selected from the group consisting of:
  • c is an integer from 2 to 10; wherein EWG is an electron withdrawing group; wherein the T ⁇ denotes the point of attachment to T; and wherein the denotes the point of attachment to Xc.
  • linker L is selected from the group consisting of: wherein c is an integer from 2 to 10; wherein the T ⁇ * denotes the point of attachment to T; and wherein the denotes the point of attachment to Xc.
  • linker L is a click-to-release linker
  • the Linker L is selected from the group consisting of: wherein c is an integer from 2 to 10; wherein the T ⁇ * denotes the point of attachment to T; denotes the point of attachment to Xc.
  • linker L is a bio-reducible linker
  • the linker L is selected from the group consisting of: wherein c is an integer from 2 to 10; R, R’, R”, and R’” are each independently selected from hydrogen, (1 -6C)alkoxy(1-6C)alkyl, (1-6C)2N(1-6C)alkyl, and (1 -6C)alkyl, or, two geminal R groups, together with the carbon atom to which they are attached, form a cyclobutyl or cyclopropyl ring; wherein the T ⁇ * denotes the point of attachment to T; and wherein the
  • I denotes the point of attachment to Xc.
  • linker L is a non-cleavable linker
  • non-cleavable linker 3 has its conventional meaning and refers to any chemical moiety that is capable of linking the targeting moiety T to the bifunctional degrader D in a stable, covalent manner.
  • a targeting moiety in the form of an antibody might get processed intracellularly, leading to release of the degrader still attached to the non-cleavable linker, which in turn is attached to a residual amino acid. In general, this metabolite then has the desired pharmacological activity.
  • a non-exhaustive list of examples of non-cleavable linkers can be found J Dugal-Tessier and N Jain. Non-cleavable Linkers: Permanently Linked, for Better or for Worse. Chapter 4 IN: Chemical Linkers in Antibody-Drug Conjugates (ADCs). Editor: Floris van Delft, ISBN 978-1-83916-515-3 (2021) and references therein.
  • linker L is selected from the group consisting of: wherein b is an integer from 1 to 10; wherein the * denotes the point of attachment to T ; and wherein the denotes the point of attachment to Xc.
  • linker L is:
  • the bifunctional compound has a sub-formula 1 - 152 selected from the group consisting of:
  • the invention further provides ready-to-conjugate intermediate compounds of Formula (II):
  • MC-Li-D Formula II wherein MC is a maleimide group; wherein the linker Li is a cleavable linker or a non-cleavable linker (preferably according to any one of the described embodiments for linker L), wherein D is a bifunctional moiety as defined according to any of the embodiments of the invention.
  • linker Li is a cleavable linker
  • said linker Li contains a cleavable bond at the point of attachment to the Xc-group of the protein binding group PB of the conjugates (see section Conjugates).
  • said linker Li contains a cleavable bond that subsequently releases or reforms the X-group of the protein binding group PB, which X-group is in embodiments according to any one of formula (X-1) to (X-28) as described in this application (and contains the hydroxyalkyl group).
  • MC-Li is selected from the group consisting of:
  • c is an integer from 2 to 10;
  • Z 1 , Z 2 , Z 3 , and Z 4 are each independently absent or a naturally occurring amino acid residue or enantiomer thereof, provided that at least two of Z 1 , Z 2 ,
  • Z 3 , and Z 4 are amino acid residues; wherein the I denotes the point of attachment to Xc.
  • Z 1 , Z 2 , Z 3 , and Z 4 are independently absent or selected from the group consisting of L-valine, D-valine, L-citrulline, D-citrulline, L-alanine, D-alanine, L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-asparagine, D- asparagine, L-phenylalanine, D-phenylalanine, L-lysine, D-lysine, L-proline, D-proline, and glycine, provided that at least two of Z 1 , Z 2 , Z 3 , and Z 4 are amino acid residues.
  • Z 1 is absent or selected from the group consisting of glycine, L-citrulline or D-citrulline;
  • Z 2 is absent or selected from the group consisting of L-glutamine, D-glutamine, L- glutamic acid, D-glutamic acid, L aspartic acid, D-aspartic acid, L-alanine, D-alanine, L-valine, D- valine and glycine;
  • Z 3 is selected from the group consisting of L-valine, D-valine, L-alanine, D-alanine, L- phenylalanine, D-phenylalanine, L-proline, D-proline and glycine;
  • Z 4 is selected from the group consisting of L-alanine, D-alanine, L-citrulline, D-citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine, and glycine.
  • MC-Li is selected from the group consisting of:
  • MC-Li is selected from the group consisting of:
  • MC-Li is selected from the group consisting of: wherein c is an integer from 2 to 10; wherein EWG is an electron withdrawing group; and wherein the denotes the point of attachment to Xc.
  • MC-Li is selected from the group consisting of:
  • c is an integer from 2 to 10; and wherein the denotes the point of attachment to Xc. More preferably, c is 5.
  • MC-Li is selected from the group consisting of:
  • MC-Li is selected from the group consisting of: wherein c is an integer from 2 to 10; R, R’, R”, and R’” are each independently selected from hydrogen, (1-6C)alkoxy(1-6C)alkyl, (1-6C)2N(1-6C)alkyl, and (1 -6C)alkyl, or, two geminal R groups, together with the carbon atom to which they are attached, form a cyclobutyl or cyclopropyl ring; and wherein the denotes the point of attachment to Xc.
  • MC-Li is selected from the group consisting of: wherein b is an integer from 1 to 10; and wherein the denotes the point of attachment to Xc.
  • MC-Li is:
  • b is 4 or 5.
  • a ready-to-conjugate intermediate compound has a sub-formula 154m - 162m selected from the group consisting of:
  • a conjugate has a sub-formula conjugate 154 - conjugate 162
  • conjugate 162 wherein a is an integer from 1 to 8.
  • compositions in accordance with the present invention comprise, as the active ingredient (‘API’), bifunctional compound of Formula (I) or a pharmaceutically acceptable salt, hydrate or solvate thereof.
  • a pharmaceutically acceptable salt includes any salt that retains the activity of the active agent(s) and is acceptable for pharmaceutical use.
  • a pharmaceutically acceptable salt also refers to any salt which may form in vivo as a result of administration of an acid, another salt, or a prodrug which is converted into an acid or salt.
  • the pharmaceutically acceptable salt is the HCI-salt of the compound of the invention.
  • the pharmaceutically acceptable salt of the disclosed compounds may be prepared by methods of pharmacy well known to those skilled in the art.
  • compositions can comprise compounds according to the invention in the form of a solvate, comprising a pharmaceutically acceptable solvent, such as water (‘hydrate’), ethanol, and the like.
  • a pharmaceutically acceptable solvent such as water (‘hydrate’), ethanol, and the like.
  • the solvated forms are considered equivalent to the unsolvated forms for the purposes of the present invention.
  • composition refers to a composition comprising a compound according to the invention or a salt or solvate thereof and, as the case may be, one or more additional, non-toxic ingredients, which composition is in a form suitable for administration to a (human) subject, through any route of administration, and which composition is physiologically tolerated upon such administration.
  • compositions of the invention may thus comprise one or more additional ingredients.
  • the composition comprises one or more carriers and/or excipients.
  • the appropriate choice of excipients is dependent on multiple factors, including the physicochemical properties of the API, the preferred pharmaceutical form, the preferred route of administration, the desired rate of release, etc.
  • the compositions of the invention can be formulated for a variety of routes of administration, oral administration being particularly preferred.
  • the composition is preferably provided in a unit dosage form.
  • unit dosage form refers to a physically discrete unit suitable as a unitary dosage for human subjects, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with any suitable pharmaceutical carrier(s) and/or excipient(s).
  • Exemplary, non-limiting unit dosage forms include a tablet (e.g., a chewable tablet), caplet, capsule (e.g., a hard capsule or a soft capsule), lozenge, film, strip, gelcap as well as any metered volume of a solution, suspension, syrup or elixir or the like, which may be contained, for instance in a vial, syringe, applicator device, sachet, spray, micropump etc.
  • the unit dosage form is a unit dosage form that is suitable for oral administration. Most preferably, it is a solid unit dosage form, such as a tablet.
  • pharmaceutically acceptable salts thereof may also be used.
  • Pharmaceutically acceptable salts of compounds of the invention include the acid addition and base salts thereof, such as preferably the calcium, potassium or sodium salts.
  • suitable salts reference is made “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).
  • salts of compounds according to the invention may be readily prepared by mixing together solutions of compounds according to the invention and the desired acid or base, as appropriate.
  • the salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent.
  • the degree of ionisation in the salt may vary from completely ionised to almost non-ionised.
  • the compounds, the conjugates and the pharmaceutical compositions of the present invention are useful as degradation-inducing compounds of Polo-like kinasei (PLK1).
  • compounds and conjugates of this invention are useful as degraders of Polo-like kinase 1 (PLK1), that is important in hyper-proliferative diseases, especially in cancer and inflammation.
  • the compounds, the conjugates and the pharmaceutical compositions of the present invention are useful for preventing, delaying or treating diseases through modulation of PLK1
  • the compounds, the conjugates and the pharmaceutical compositions of the invention is capable of fundamentally degrading the target protein, PLK1 in view of the mechanism of action, thereby achieving an excellent PLK1 inhibitory effect as compared to the conventional PLK1 small molecule inhibitor that inhibits the simple activity of PLK1 .
  • the pharmaceutical composition including the compound represented by Formula I of the invention or a pharmaceutically acceptable salt thereof may be effectively employed for selective degradation of PLK1 .
  • An embodiment of the present invention is a composition for preventing or treating diseases through modulation of PLK1 including the compound or the conjugates of the invention.
  • An another embodiment of the invention is a method for the prevention or treatment of diseases through modulation of PLK1 comprising administering the composition to a subject in need thereof.
  • the disease treated through modulation of PLK1 refers to any disease or condition capable of being treated, alleviated, delayed, inhibited or prevented by the induction of degradation or inhibition of activity of PLK1 .
  • the disease treated through modulation of PLK1 may be a cancer (malignant tumor), a benign tumor, or other genetic or non-genetic diseases caused by excessive cell division.
  • the cancer includes all cancers capable of exhibiting prophylactic or therapeutic efficacy due to inhibition or degradation of PLK1 activity, and may be solid cancer or blood cancer.
  • the compounds and conjugates of the present invention are also useful in the treatment of cancer, such as related indications defined by tissue origin such as prostate cancer, breast cancer, lymphoma, acute myelogenous leukemia, lung cancer, and pancreatic cancer.
  • bifunctional compounds according to the invention having Formula (I) and the conjugates based on bifunctional moieties derived from these bifunctional compounds having Formula (I) and pharmaceutical compositions thereof can be used to treat or prevent a variety of conditions, diseases or disorders through modulation of Polo-like kinase 1 (PLK1).
  • PLK1 Polo-like kinase 1
  • bifunctional compounds according to the invention having Formula (I) and the conjugates based on bifunctional moieties derived from these bifunctional compounds having Formula (I) and pharmaceutical compositions thereof can be used in the treatment of disorders or diseases selected from the group consisting of cancer, benign tumor, a neurological disorder, an inflammatory disease, a disease characterized by inflammasome activation, an autoimmune disease, a hyperproliferative disease, and other genetic or non-genetic diseases caused by excessive cell division, a disorder that is caused by tissue rejection by the immune system.
  • the bifunctional compounds according to the invention having Formula (I) and the conjugates based on bifunctional moieties derived from these bifunctional compounds having Formula (I) and pharmaceutical compositions thereof can be used in a therapy to prevent tissue rejection by the immune system.
  • therapies to prevent tissue rejection by the immune system include but are not limited to stem cell therapy graft versus host disease, and allogeneic organ transplants.
  • the neurological disorder is one or more selected from the group consisting of central nervous system disease, neurodegenerative disease, Alzheimer's disease, Parkinson's disease, multiple sclerosis, Huntington's disease, senile dementia, epilepsy, Lou Gehrig, stroke, and nerve damage and axonal degeneration-related disorders following brain or spinal cord injury.
  • the cancer or benign tumor is one or more selected from the group consisting of squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, skin cancer, skin or intraocular melanoma, rectal cancer, anal muscle cancer, esophageal cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia (including but not limited to acute myelogenous leukemia and complex karyotype acute myelogenous leukemia), lymphocytic lymphoma, double hit lymphoma, hepatocellular carcinoma, gastrointestinal cancer, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumor, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary
  • Suitable routes of administration may, for example, include oral, eyedrop, rectal, transmucosal, topical, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • compositions of the present invention may be manufactured in a mannerthat is itself known, e.g., by means of conventional mixing, dissolving, granulating, drageemaking, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • compositions for oral use can be obtained by combining the active compound with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the compounds can be formulated for parenteral administration by injection, e.g. bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g. in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form.
  • suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly or by intramuscular injection).
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • the compounds of the present invention can be prepared by methods well known in the art of organic chemistry. See, for example, J. March, ‘Advanced Organic Chemistry’ 4 th Edition, John Wiley and Sons. During synthetic sequences it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This is achieved by means of conventional protecting groups, such as those described in T.W. Greene and P.G.M. Wutts ‘Protective Groups in Organic Synthesis’ 3 rd Edition, John Wiley and Sons, 1999. The protective groups are optionally removed at a convenient subsequent stage using methods well known in the art.
  • the products of the reactions are optionally isolated and purified, if desired, using conventional techniques, including but not limited to, filtration, distillation, crystallization, chromatography and the like. Such materials are optionally characterized using conventional means, including physical constants and spectral data.
  • Scheme I show a general synthetic route in relation to an exemplary compound of Formula I.
  • Carboxylic acid containing pteridine IV can be prepared from commercially available substituted 2-chloro-7,8-dihydro-5/7-pteridin-6-ones II using appropriate aminobenzoic acids III in a solvent mixture such as ethanol/water at elevated temperatures.
  • Derivatives of Formula VI can be prepared via amide-coupling with amino-alcohols of Formula V and an appropriate couplings reagent such as HATU or EDCLHCI in a suitable solvent like DMF at appropriate temperature.
  • Finally deprotection of compounds of Formula VI can be accomplished using strong acids like HCI or TFA in the presence of water and a suitable cation scavenger like triisopropylsilane (TIS) at appropriate temperature.
  • TIS triisopropylsilane
  • Subsequent coupling of deprotected compounds of Formula VI to obtain compounds of Formula l-a can be accomplished by amide-coupling, alkylation or reductive amination using appropriate reagents such as HATU, EDCLHCI, NaBH(OAc)3 or NaBH(t-BuO)3. Subsequent purification using silicagel chromatography or preparative HLPC afforded pure compounds of Formula l-a.
  • the eluents used for this system are A (95/5 v/v% Milli-Q water/acetonitrile + 0.1 % formic acid) and B (acetonitrile + 0.1 % formic acid).
  • the eluents used for this system are A (95/5 v/v% Milli-Q water/acetonitrile + 0.1 % formic acid) and B (acetonitrile + 0.1 % formic acid).
  • the eluents used for this system are A (95/5 v/v% Milli-Q water/acetonitrile + 0.1 % formic acid) and B (acetonitrile + 0.1 % formic acid).
  • This compound was prepared in an analogues manner as described in Intermediate 2, starting from 1-A/-Cbz-3-pyrrolidinone and 3-ethoxy-3-oxo-propanoic acid to afford the title compound (28 mg, 28%).
  • This compound was prepared in an analogues manner as described in Intermediate 6, starting from tert-butyl 3, 3-difluoro-4-oxopiperidine-1 -carboxylate and malonic acid to afford the title compound (888 mg, 24%).
  • This compound was prepared in an analogues manner as described in Intermediate 2, starting from cis-fe/Y-butyl 5-oxohexahydrocyclopenta[c]pyrrole-2(1 /7)-carboxylate and 3-ethoxy-3- oxo-propanoic acid to afford the title compound (95 mg, 73.4%).
  • This compound was prepared in an analogues manner as described in Intermediate 2, starting from te/Y-butyl 3-oxo-1-oxa-8-azaspiro[4.5]decane-8-carboxylate and 3-ethoxy-3-oxo- propanoic acid to afford the title compound (130 mg, 99%).
  • This compound was prepared in an analogues manner as described in Intermediate 6, starting from tert-butyl 2-oxo-7-azaspiro[3.5]nonane-7-carboxylate, malonic acid and sodium borodeuteride for reduction in the second step, to afford the title compound (65 mg, 67.7%).
  • This compound was prepared in an analogues manner as described in Intermediate 2, starting from te/Y-butyl 7-oxo-5-oxa-2-azaspiro[3.4]octane-2-carboxylate and 3-ethoxy-3-oxo- propanoic acid to afford the title compound.
  • 3-Amino-4-methoxybenzoic acid (5 g, 29.9 mmol) was suspended in acrylic acid (8.05 mL, 117 mmol). The resulting suspension was stirred at 100 °C. After 10 min, a thick slurry/solid appeared, which was heated at 100 °C for another 3 h. After cooling, acetic acid (33 mL) was added and the suspension was heated to 100 °C and stirred for 10 min. Then, urea (11 g, 183 mmol) was added and the resulting mixture was stirred at 120 °C o/n. The resulting brown solution was then quenched into a ice-cold solution of water (150 mL) and concentrated HCI (10 mL).
  • Des-Martin periodinane (1.14 g, 2.69 mmol) was added to a solution of 2-(2,6-dioxo-3- piperidyl)-5-[(3/?)-3-(hydroxymethyl)pyrrolidin-1-yl]isoin-doline-1 ,3-dione in dichloromethane (480 mg, 1.34 mmol). The mixture was allowed to stir at room temperature for one hour. Dichloromethane and aqueous Na2SOs were subsequently added.
  • This compound was prepared in an analogous manner as described in Intermediate TLB- Sp5, starting from tert-butyl 2-[(3R)-pyrrolidin-3-yl]oxyacetate_(lntermediate Sp2) and 2-(2,6- dioxopiperidin-3-yl)-5-fluoroisoindoline-1 ,3-dione to afford 125 mg of the title compound.
  • A/,A/-diisopropylethylamine (12.5 mL, 75.63 mmol) was added to methyl 4-bromo-2- (bromomethyl)benzoate (7.32 g, 23.77 mmol) and 3-aminopiperidine-2, 6-dione hydrochloride (5.87 g, 35.66 mmol) in acetonitrile (100 mL). The resulting suspension was stirred at 80 °C for 48 hours. The reaction mixture was cooled to room temperature and filtered.
  • the reaction mixture was recharged with the same amounts of copper iodide (29 mg, 0.155 mmol), bis(triphenylphosphine)palladium chloride (108 mg, 0.155) and tert- butyl hept-6-ynoate (620 mg, 3.4 mmol) and again stirred at 80°C for 5 h.
  • the reaction mixture was added dropwise to ethyl acetate (100 mL)/10% citric acid (100 mL) under heavy stirring.
  • the dark black mixture was filtered over a CeliteTM and rinsed with ethyl acetate (60 mL) until the filtrate was colorless.
  • 2,2,2-Trichloroethyl chloroformate (4.79 mL, 34.8 mmol) was slowly added to a cooled solution of tert-butyl 3-[(3-amino-3-oxo-propyl)amino]azetidine-1-carboxylate (13.6 g, 34.8 mmol, theor.) and triethylamine (4.85 mL, 34.8 mmol) in dichloromethane (50 mL).
  • the reaction mixture was stirred for 4 h allowing the mixture to come to room temperature.
  • the organic layer was washed with water (3x50 mL) and filtered over a PE filter. The filtrate was concentrated in vacuo.
  • This compound was prepared in an analogous manner as described in Intermediate TLB- Sp16, starting from 6-[3-(2,4-dioxohexahydropyrimidin-1-yl)azetidin-1-yl]pyridine-3-carboxylic acid and tert-buty I 3-[2-(2-aminoethoxy)ethoxy]propanoate to afford 50.5 mg of the title compound (yield: 92%).
  • This compound was prepared in an analogous manner as described in Intermediate TLB- Sp16, starting from 6-[3-(2,4-dioxohexahydropyrimidin-1-yl)azetidin-1-yl]pyridine-3-carboxylic acid and fe/Y-butyl 3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]propanoate to afford 38.6 mg of the title compound (yield: 70%).
  • TLB1 starting from 2-[4-(2,6-dioxo-3-piperidyl)phenoxy]acetic acid (Intermediate TLB-Sp33) to afford the title compound (352 mg, 17%).
  • Intermediate TLB-Sp34 2-[4-(2,6-dioxo-3-piperidyl)phenoxy]acetic acid (Intermediate TLB-Sp33) to afford the title compound (352 mg, 17%).
  • 6-(2,4-Dioxo-1 ,3,8-triazaspiro[4.5]decan-8-yl)pyridine-3-carboxylic acid 52 mg, 0.1 mmol
  • tert-butyl 3-(2-aminoethoxy)propanoate (19.1 pL, 0.11 mmol) were dissolved in DMF (1.2 mL).
  • HATU 40 mg, 0.105 mmol
  • DiPEA (0.12 mL, 0.7 mmol) were added subsequently and the mixture stirred at room temperature for 1 h.
  • This compound was prepared from subsequent HATU-coupling and acid-deprotection reactions starting from commercially available (R)-1 ,2,3, 4-tetrahydronaphthalen-1 -amine, Boc-7- hydroxy-(S)-1 ,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid, Boc-Tle-OH and Boc-N-Me-Ala-OH to give 150 mg of the title compound.
  • Dess-Martin periodinane (283 mg, 0.67 mmol) was added to a suspesion of 3-[3-fluoro-4- [4-(hydroxymethyl)-1-piperidyl]phenyl]piperidine-2, 6-dione (144 mg, 0.45 mmol) in dichloromethane (3 mL). The mixture was allowed to stir at room temperature for five hours. Dichloromethane and aqueous Na2SOs were added.
  • This compound was prepared in an analogous manner as described in Intermediate TLB- Sp40, and starting from 4-bromo-2-fluoro-1 -iodobenzene and te/Y-butyl 3-piperazin-1- ylpropanoate dihydrochloride (Intermediate Sp1) to afford after deprotection with trifluoro acetic acid, the title compound (440 mg, 92%) as a TFA-salt.
  • This compound was prepared in an analogous manner as described in Intermediate TLB- Sp7, and starting from 2,3,4,5,6-Pentafluorophenyl) 3-(2,4-dioxohexahydropyrimidin-1-yl)-4- methyl-benzoate (Intermediate TLB2) and fe/Y-butyl 3-piperazin-1-ylpropanoate to afford after deprotection with trifluoro acetic acid, the title compound (354 mg, 100%) as a TFA-salt.
  • TLB-Sp54 This compound was prepared in an analogous manner as described in Intermediate TLB- Sp49, starting from 1 ,2-difluoro-4-nitro-benzene, fe/Y-butyl piperidine-4-carboxylate and 3- bromopiperidine-2, 6-dione to afford 360 mg of the title compound after tert-butyl ester deprotection with TFA/dichloromethane.
  • This compound was prepared in an analogous manner as described in Intermediate TLB- Sp40, and starting from 1-bromo-2-fluoro-4-iodo-benzene, fe/Y-butyl piperidine-4-carboxy-late and 2,6-dibenzyloxy-3-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)pyridine to afford 298 mg of the title compound.
  • Example 19 4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1piperidine-4-carbonyl1-6-(2-hvdroxyethyl)-2-azaspiro[3.31heptan-6-yl1-3- methoxy-benzamide

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Abstract

The present invention relates to bifunctional compounds capable of inducing degradation of Polo- like kinase 1 (PLK1) via ubiquitin proteasome pathway. More specifically, the invention relates to bifunctional compounds and conjugates derived thereof, along with processes to prepare the compounds or conjugates, uses thereof and methods for treating diseases through modulation of Polo-like kinase 1 (PLK1). Specifically, the invention relates to the use of the bifunctional compounds or conjugates in the treatment of cancer.

Description

Bifunctional compounds capable of inducing degradation of Polo-like kinasei
Field of the invention
The present invention relates to bifunctional compounds capable of inducing degradation of Polo-like kinase 1 (PLK1) via ubiquitin proteasome pathway. More specifically, the invention relates to bifunctional compounds and conjugates derived thereof, along with processes to prepare the compounds or conjugates, uses thereof and methods for treating diseases through modulation of Polo-like kinase 1 (PLK1). Specifically, the invention relates to the use of the bifunctional compounds or conjugates derived thereof in the treatment of cancer.
Background of the invention
Kinases are enzymes that transfer a phosphate group from ATP to a protein while phosphatases remove a phosphate group from protein. Together, these two enzymatic processes regulate cellular functions such as cell proliferation, subcellular translocation, apoptosis, inflammation and metabolism (Attwood et al. Trends in kinase drug discovery targets, indications and inhibitor design. Nat Rev Drug Discov 20, pages 839-861 (2021)). The human kinome is composed of over 500 kinases.
There is clinical evidence that supports the driver role of kinases in cancer owing to their aberrant activation by either translocations or activating mutations. Identification and characterization of these disease drivers has facilitated the design and approval of molecularly guided cancer therapies, beginning with the pioneering example of imatinib to treat CML driven by the BCR-ABL translocation, which results in a protein with elevated tyrosine kinase activity. Many small-molecule kinase inhibitors approved by the FDA that target kinases have oncology indications. Recent developments of kinase inhibitors, including developments of kinase inhibitors for oncology, have been reported, see: Attwood et al. Trends in kinase drug discovery targets, indications and inhibitor design. Nat Rev Drug Discov, pages 839-861 (2021).
Polo-like kinase 1 (PLK1) is a member of the PLK-family of kinases which plays a key role in cell-cycle progression through mitosis via its effects on chromosome segregation, spindle assembly, and cytokinesis. PLK1 phosphorylates the substrate CDC25, thereby activating the cyclin B/CDC2 complex, which triggers cell proliferation. During cell-cycle progression, PLK1 is activated in the cell by the kinase Aurora A and its co-factor Bora. Bora acts on PLK1 by altering its conformation, which in turn allows Aurora A to phosphorylate PLK1 on threonine residue 210, promoting mitotic entry. Once PLK1 has been activated, its expression accumulates during S phase, peaks during G2-M transition, and declines rapidly upon mitotic exit, (reviewed in Liu X. Targeting polo-like kinases: a promising therapeutic approach for cancer treatment. Transl Oncol 8, pages:185-95 (2015), M Chiappa et al, Present and Future Perspective on PLK1 Inhibition in Cancer Treatment, Frontiers in Oncology 12, 903016 (2022).
In addition to having an essential role in mitosis, PLK1 is an important regulator of the DNA damage checkpoint. When cells suffer DNA damage, for instance genotoxic stress, in the G2 phase of the cell cycle, they do not progress into mitosis, but arrest in the G2/M DNA damage checkpoint where PLK1 is inhibited, and its degradation is induced. Upstream activators of PLK1 are similarly affected by the DNA damage response, preventing activation of PLK1 at multiple levels. Vice versa, active PLK1 mediates phosphorylation of checkpoint kinase 2 (CHK2), which inhibits checkpoint kinase 1 (CHK1) and CHK2 activation, thereby preventing DNA repair during mitosis, which would be harmful to the cell. (T Takaki et al., Polo-like kinase 1 reaches beyond mitosis — cytokinesis, DNA damage response, and development., Curr Opin Cell Biol 20, pages 650-60 (2008)
Given that PLK1 is so intimately involved in cell-cycle regulation pathways and DNA damage repair, which are very important for tumor growth, maintenance and therapy resistance, it is not surprising that PLK1 has been proposed as a drug target in a variety of cancers. In a systematic CRISPR knockout study of all available cancer cell lines, nearly all cell lines are sensitive to PLK1 knockout by CRISPR, or knockdown by shRNA (www.depmap.org). Studies on patient materials show that PLK1 is highly expressed in malignant tumors but scarcely detectable in normal tissues. PLK1 was found to be significantly higher expressed, compared to normal tissue, in at least 18 different cancers, including lung, breast, prostate, colorectal and pancreatic cancer. Kaplan-Meier analysis showed that patients with higher expression levels of PLK1 have significantly worse overall survival prognoses than those with lower expression levels of PLK1 in at least 10 cancer types. Moreover, PLK1 has significantly higher expression levels in several late stage cancers than in early stage. PLK1 expression is elevated in non-small-cell lung cancer, head and neck cancer, esophageal cancer, gastric cancer, melanomas, breast cancer, ovarian cancer, endometrial cancer, colorectal cancer, gliomas, and thyroid cancer. PLK1 gene and protein expression has been proposed as a new prognostic marker for many types of malignancies, and PLK1 is a potential target for cancer therapy (reviewed in Z Liu et al., PLK1 , A potential target for cancer therapy, Translational Oncology 10, pages 22-32 (2017) and N Takai, Polo-like kinases (PLKs) and cancer. Oncogene 24, pages 287-291 (2005)).
Outside cancer, it has been observed that PLK1 plays a role in inflammation, and PLK1 inhibitors show effects in inflammatory disease models. Thus, targeting PLK1 could be useful in treating diseases caused or exacerbated by the immune system (Baldrighi et al., PLK1 inhibition dampens NLRP3 inflammasome-elicited response in inflammatory disease models, Journal of Clinical Investigation 133, page e162129 (2023). Because targeting PLK1 leads to depletion of immune cells, PLK1 targeting modalities can also be used in transplantation, to treat or prevent graft versus host disease. Additionally, PLK1 is involved in neurological disorders such as Huntington’s disease and Alzheimer’s disease. For instance, depletion of PLK1 reduces p-amyloid (Ap)-induced neuronal cell death (reviewed in lliaki et al, Polo-like kinase 1 (PLK1) signalling in cancer and beyond, Biochemical Pharmacology 193, 1 14747).
Because so many studies implicate PLK1 in tumor growth and maintenance, researchers have explored PLK1 inhibitors for cancer treatment. Much studied inhibitors are BI2536, GSK461364, BI6727 (volasertib), CYC140 (plogosertib) and NMS-P937 (onvansertib), which all have been tested in clinical phase, particularly in patients with leukemias, lymphomas, lung cancer, pancreatic cancer, prostate cancer and in general advanced solid cancers. Despite these efforts, most PLK1 inhibitors have failed at the clinical trial stage, and thus no commercially available PLK1 inhibitors have been approved by any authority for use as anticancer drugs, although some remain in clinical trials.
One of the probable reasons for the clinical failure of PLK1 inhibitors is that conventional PLK1 inhibitors do not sufficiently inhibit PLK1 activity at concentrations that are clinically safe (reviewed in M Chiappa et al, Frontiers in Oncology 12, 903016 (2022). If the cell cycle of cancer cells is only temporarily delayed by PLK1 inhibition, cancer cells will eventually restart the cell cycle, which negates any clinical effects.
Another reason for the clinical failure of PLK1 inhibitors is that many functional studies demonstrating the involvement of PLK1 in tumors used biological techniques involving knockingout or knocking-down PLK1 . To obtain the effective responses observed in these preclinical models by way of a PLK1 protein target, a therapeutic modality that inhibits or removes the entire PLK1 protein might be desired.
The ubiquitin-proteasome system (UPS) is a highly conserved mechanism for degradation of both normal, mutated and misfolded proteins in eukaryotic cells, thus keeping intracellular protein homeostasis (J Bard et al. Structure and function of the 26S proteasome. Annu Rev Biochem, 87, pages 697-724 (2018), G Kleiger et al., Perilous journey: a tour of the ubiquitin- proteasome system. Trends Cell Biol. 24, pages 352-359 (2014); M Hipp et al. The proteostasis network and its decline in ageing. Nat Rev Mol Cell Biol. 20, pages 421-435 (2019)). In UPS, proteins to be degraded are covalently tagged with ubiquitin (Ub, a 76-amino acid protein), and this tagging process is catalyzed by three enzymes known as Ub-activating enzyme (E1), Ub- conjugating enzyme (E2) and Ub-ligase (E3): free Ub is activated by E1 and then attached to the cysteine residue (Cys) of E1 to form a thioester bond via an ATP-dependent reaction; the Ub- tagged E1 transfers its Ub to the Cys of E2 through a trans-thioesterification reaction; E3 recruits Ub-tagged E2 and E3 substrate to label the ubiquitin at the lysine residue (Lys) of the substrate. Such repeated ubiquitination processes generate a poly-Ub chain (mainly linked through Lys48 of Ub) on the target protein, which guides the substrate to 26S proteasome for degradation (D Komander D et al. The ubiquitin code. Annu Rev Biochem. 81 , pages 203-229 (2012); R Yau, M Rape. The increasing complexity of the ubiquitin code. Nat Cell Biol. 18, pages 579-586 (2016)). In the human proteome, there are two E1s, about forty E2s and more than 600 E3s. Among them, the E3 ligases are responsible for specifically recognizing substrates.
Inspired by UPS, researchers designed Heterobifunctional compounds (HBCs) to hijack the UPS and degrade a protein of interest (PCI). HBCs consist of three covalently bound moieties: a ligand moiety to bind the PCI (PCI ligand), another ligand moiety to bind E3 ligase (E3 ligand) and a spacer to conjugate the two moieties. HBC simultaneously recruits E3 ligase and PCI, forming the “E3 ligase-HBC-POl” ternary complex. This complex potentiates the substrate recognition by E3 ligase and promotes the transfer of Ub to POI, accelerating the poly- ubiquitination and subsequent proteasome-mediated degradation of POI (A Lai and C Crews. Induced protein degradation: an emerging drug discovery paradigm. Nat Rev. Drug. Discov. 16, pages 101-114 (2017)). Current HBC developments have mainly been focused on three E3 ligases, i.e. Von-Hippel-Lindau (VHL), inhibitors of apoptosis proteins (lAPs) and cereblon (CRBN).
Unlike traditional inhibitors, the HBC technology aims to completely eliminate the protein and all its functionality, rather than merely inhibiting their enzymatic activity. Therefore, the resistance to inhibition activity caused by kinase mutants, as observed with classical inhibitors, can be overcome by HBCs.
The principle of induced degradation of protein targets as a potential therapeutic approach has been described by C Crews, Inducing protein degradation as a therapeutic strategy. J. Med. Med. 61 , pages 403-404 (2018) and references cited therein.
It has been clear that induced protein degradation offers several potential advantages over traditional target inhibition. First, only sub-stoichiometric drug concentrations are needed given the catalytic nature of HBCs, allowing one HBC molecule to degrade multiple POI molecules”. Furthermore, greater target selectivity can be achieved, even when starting with promiscuous binding ligands (Bondeson D. et al. Lessons in PROTAC design from selective degradation with promiscuous warheads. Cell. Chem. Biol, 25, pages 78-87 (2018)). A further advantage of HBCs is their ability to overcome classical inhibitor resistance caused by feedback loops that upregulate the target. Also, HBCs have the ability to address scaffolding functions of target proteins that are not addressable with inhibitor molecules (K Samaransinghe and C Crews. Targeted protein degradation: a promise for undruggable targets. Cell Chem Biol 28, pages 934-951 (2021)).
EP3865152A1 discloses a conjugate of target molecule-linker-E3 ligase ligand as shown in the structure of A-L1-B (formula I), wherein the A is the monovalent group of the target molecule, the B is the monovalent group of the E3 ligase ligand, the L1 is the spacer linking A and B. In particular examples, conjugates are described including an antibody attached to a flexible spacer between A and B and connected via an amine attachment point at a position between a PLK1- binding unit (A) and the E2 ligase binding unit (B).
Some examples of PLK1 targeting HBCs are known, which includes CN106543185, wherein tetrahydropteridin-based HBCs are disclosed that degrade PLK1 by hijacking the UPS system. To apply PLK1 degraders as therapeutic modalities, there is a clear need for improved PLK1 degradation inducing HBCs with potent PLK1 degrading activity and potent antiproliferative activity.
Therefore, there is an unsatisfied demand for a more effective PLK1 -inhibiting or PLK1 - removing (degrading) compounds, with no or minimal side effects.
An aim of embodiments of the present invention is to provide PLK1 degradation-inducing compounds with potent antiproliferative activity. Another aim of embodiments of the present invention is to provide a method for preparing the compounds.
Still another object of embodiments of the present invention is to provide a therapeutic use of the compounds.
Therefore, an aim of embodiments of the present invention is to provide bifunctional compounds providing improved pharmacological activity towards kinases, in particular towards Polo-like Kinase 1 .
Another aim of certain embodiments of this invention is to provide novel cancer treatments.
Summary of the invention
Provided herein are bifunctional compounds capable of inducing degradation of PLK1 kinases and conjugates based on bifunctional moieties derived from the bifunctional compounds according to the invention. Also provided herein are methods of using the bifunctional compounds and the conjugates according to the invention as medicament, and for treating disorders, including cancer, or in the prevention or treatment of disorders through modulation of Polo-like Kinase 1 (PLK1).
The inventors have found that a bifunctional compound of Formula (I):
TLB-Sp-PB
Formula (I), or a pharmaceutically acceptable salt, hydrate, and/or solvate thereof, wherein:
TLB is a Targeting Ligase Binding group capable of binding to a E3 ligase; PB is a Protein Binding group capable of binding to Polo-like Kinase 1 (PLK1) and Sp is a spacer, which covalently links the Targeting Ligase Binding group (TLB) to the Protein Binding group (PB); wherein PB has the chemical structure BPLKI-X, wherein BPLKI has the formula: , wherein Ri is selected from the group consisting of H, methyl, ethyl or propyl; wherein R2 is selected from the group consisting of (1 -6C)cycloalkyl, (1 -6C)alkyl or benzyl; wherein any of said cycloalkyl, alkyl or benzyl is optionally and independently substituted with one or more substituents selected from halogen, hydroxy and cyano; wherein R3 is selected from the group consisting of OMe, OCHF2, OCF3, OCD3, (1- 3C)alky I, O(CH2)2OH or O(CH2)3OH ; wherein R4 is selected from the group consisting of H or methyl; wherein "T* denotes the point of attachment to the X group; wherein the X group has a formula (X-1) to (X-28) as described below, and wherein said compound is in embodiments as further described below, is capable of providing or inducing improved PLK1 kinase degradation.
The inventors have found that these bifunctional compounds of the invention are capable of providing improved kinase degradation, in particular of PLK1 kinase.
The inventors have also found that conjugates based on a bifunctional moiety derived from these bifunctional compounds, are capable of providing improved kinase degradation, in particular of PLK1 kinase.
Aspects of the invention
In a first aspect of the invention is provided a bifunctional compound of Formula (I): TLB-Sp-PB Formula (I), or a pharmaceutically acceptable salt, hydrate, and/or solvate thereof, wherein:
TLB is a Targeting Ligase Binding group capable of binding to a E3 ligase; PB is a Protein Binding group capable of binding to Polo-like Kinase 1 (PLK1) and Sp is a spacer, which covalently links the Targeting Ligase Binding group (TLB) to the Protein Binding group (PB); wherein PB has the chemical structure BPLKI-X, wherein BPLKI has the formula:
(BPLKI) , wherein R1 is selected from the group consisting of H, methyl, ethyl or propyl; wherein R2 is selected from the group consisting of (1 -6C)cycloalkyl, (1 -6C)alkyl or benzyl; wherein any of said cycloalkyl, alkyl or benzyl is optionally and independently substituted with one or more substituents selected from halogen, hydroxy and cyano; wherein R3 is selected from the group consisting of OMe, OCHF2, OCF3, OCD3, (1- 3C)alky I, O(CH2)2OH or O(CH2)3OH ; wherein R4 is selected from the group consisting of H or methyl; wherein denotes the point of attachment to the X group; wherein the X group has a formula (X-1) to (X-28) selected from the group consisting of:
(X-21) (X-22) (X-23) (X-24) (X-25) wherein any of said formula X-1 to X-28 is optionally and independently substituted with one or more substituents selected from fluoro, hydroxy, (1 -3C)alkyl and (1 -3C)alkoxy; wherein n is 1 to 4; wherein — | — * denotes the point of attachment to the amide (-C(O)-NH-) group of BPLKI , and wherein denotes the point of attachment to the spacer (Sp).
In another aspect of the invention there is a provided a conjugate comprising a bifunctional compound according to the first aspect, having Formula (I), coupled to a moiety or agent.
In another aspect of the invention there is a provided a conjugate having the chemical structure:
T - (L - D)a or a pharmaceutically acceptable salt thereof, wherein:
D is a bifunctional moiety capable of inducing degradation of PLK1 ;
T comprises or consists of a targeting moiety capable of binding to a cell surface molecule;
L is a linker, covalently bound to T and D; a is an integer from 1 to 8;
Wherein D is a bifunctional moiety having the chemical structure TLB-Sp-PB, wherein TLB, Sp and PB have the chemical structure according to the invention and wherein the X group of PB is covalently bound to the linker L, optionally by conversion of the -OH group of the X group to a bonding group containing -O-.
In another aspect of the invention there is provided a bifunctional compound or the conjugate according to the invention for use as a medicament.
In another aspect of the invention there is provided a bifunctional compound or the conjugate according to the invention for use in therapy.
In another aspect of the invention there is provided a bifunctional compound or the conjugate according to the invention for use in the treatment of disorders through modulation of Polo-like Kinase 1 (PLK1).
In another aspect of the invention is provided a bifunctional compound or the conjugate according to the invention or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer. In another aspect of the invention is provided a use of the bifunctional compound or the conjugate according to the invention or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament.
In another aspect of the invention is provided a pharmaceutical composition which comprises the bifunctional compound or the conjugate according to the invention or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients.
In another aspect of the invention is provided a method for treating of cancer in a subject in need thereof comprising administering to the subject the bifunctional compound or the conjugate according to the invention or a pharmaceutically acceptable salt thereof in an amount effective to treat cancer.
In another aspect of the invention is provided a method for treating a subject suffering with a disorder comprising administering to the subject the bifunctional compound or the conjugate of the invention in an amount effective to treat the disorder through modulation of PLK1 .
In another aspect of the invention there is provided a bifunctional compound or the conjugate according to the invention for use in the treatment of disorders or diseases are selected from the group consisting of cancer, benign tumor, a neurological disorder, an inflammatory disease, a disease characterized by inflammasome activation, an autoimmune disease, a hyperproliferative disease, a disorder that is caused by tissue rejection by the immune system, or for use in a therapy to prevent tissue rejection by the immune system.
Each of the sub-formulas 1 - 152 of the compounds of the invention is a preferred embodiment of the present application.
The present application addresses the provision of PLK1 targeting heterobifunctional compounds (HBCs) that are suitable as therapeutic compounds capable of targeting and inducing degradation PLK1 or that are suitable as effector moieties for preparing therapeutic conjugates capable of targeting and inducing degradation of PLK1.
The bifunctional compounds or bifunctional moieties according to the invention have crucial and novel characteristics. First they are able to induce degradation of PLK1 , enabling therapeutic applications that more closely mimic tumor targeting phenotypes in biological knockdown experiments. The compounds according to the invention contain a unique functional moiety, in the form of a hydroxyalkyl group or side-chain, such as a hydroxymethylene or hydroxyethylene group, which has the unexpected characteristic that it simultaneously improves the capability of the bifunctional compounds for PLK1 degradation and their cytoxicity towards tumor cells.
The inventors have found and experimentally shown that having the hydroxyalkyl group or hydroxyalkyl side-chain of the Protein Binding group (PB), wherein PB has the chemical structure BPLK1-X, simultaneously improves the capability of the bifunctional compounds for PLK1 degradation and their cytoxicity towards tumor cells. The Protein Binding group (PB) is the targeting group for targeting the PLK1 protein. The inventors have found that the structural configuration of the X-group, including the configuration of the hydroxyalkyl group or hydroxyalkyl side-chain, in connection to the binding moiety (BPLKI) of the Protein Binding group (PB) cooperatively improve the capability of the bifunctional compounds for PLK1 degradation and their cytoxicity towards tumor cells. The X-group is directly covalently connected to the amide (-C(O)- NH-) group of moiety BPLKI and by its direct connection cooperates with binding moiety BPLKI and positions the X-group, including its unique functional moiety of the X-group, close to the BPLKI group of the Protein Binding group (PB).
The inventors have thus found that the functionality of the Protein Binding group (PB), which targets the protein, can be improved, i.e. by combining a binding moiety (BPLKI) and the X- group, as described.
This unexpected characteristic is exemplified by the enhanced activity of Example 1 when compared to Comparative Example 1 , where the latter has the same chemical structure, but lacks the hydroxy methylene group (Tables 1 a and Tables 2a). This unexpected characteristic is further exemplified by the enhanced activity of Example 2 when compared to Comparative Example 1 , where the latter has the same chemical structure, but lacks the hydroxyethylene group (Tables 1 a and Tables 2a). The surprising effects of the inventive structure of compounds of the invention are further demonstrated, for example, by the enhanced activity of Example 34 compared to Comparative example 2, by the enhanced activity of Example 33, compared to Comparative Example 3, by the enhanced activity of Example 144 compared to Comparative Example 4, and by the enhanced activity of the Example 36 compared to Comparative Example 5.
In particular, we have synthesized a series of bifunctional compounds described by Formula (I) which have surprisingly potent PLK1 degradation inducing activity and surprisingly potent antiproliferative activity.
There is a need for more effective PLK1 degrading compounds and the present application addresses the generation of bifunctional degradation inducing molecules that are capable of degrading PLK1 kinase as therapeutics
Another advantage of certain embodiments of this invention is to provide cancer treatments based on the administration of the bifunctional compounds or conjugates of the invention.
Definitions
The term “pharmaceutical composition” as used herein has its conventional meaning and refers to a composition which is pharmaceutically acceptable.
The term “pharmaceutically acceptable” as used herein has its conventional meaning and refers to compounds, material, compositions and/or dosage forms, which are, within the scope of sound medical judgment suitable for contact with the tissues of mammals, especially humans, without excessive toxicity, irritation, allergic response and other problem complications commensurate with a reasonable benefit/risk ratio. The term "effective amount as used herein, refers to an amount of the compound of the invention, and/or an additional therapeutic agent, or a composition thereof, that is effective in producing the desired therapeutic, ameliorative, inhibitory or preventative effect when administered to a subject suffering from a disease or disorder, such as a disease or disorder that can be treated by modulation of PLK1 . In the combination therapies of the present invention, as effective amount can refer to each individual agent or to the combination as a whole, wherein the amounts of all agents administered are together effective, but wherein the component agent of the combination may not be present individually in an effective amount.
A "subject" is a human or non-human mammal. In one embodiment, a subject is a human.
The term “controlling” is intended to refer to all processes wherein there may be a slowing, interrupting, arresting or stopping of the progression of the diseases and conditions affecting the mammal. However, “controlling” does not necessarily indicate a total elimination of all disease and condition symptoms and is intended to include prophylactic treatment.
The term “excipient” as used herein has its conventional meaning and refers to a pharmaceutically acceptable ingredient, which is commonly used in the pharmaceutical technology for preparing a granulate, solid or liquid oral dosage formulation.
The term “salt” as used herein has its conventional meaning and includes the acid addition and base salts of the compound of the invention.
The term “solvate” as used herein has its conventional meaning. One or more compounds of the invention or the pharmaceutically acceptable salts thereof may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. “Solvate” means a physical association of a compound of this invention with one or more solvent molecules. This physical association Involves varying degrees of ionic and covalent bonding. Including hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. “Solvate” encompasses both solution-phase and isolatable solvates. Examples of suitable solvates include ethanolates, methanolates, and the like. “Hydrate” is a solvate wherein the solvent molecule is H2O and includes any hydrate of the compound or the salt of said compound.
The term "treatment” as used herein has its conventional meaning and refers to curative, palliative and prophylactic treatment.
The term “unit dosage form” has its conventional meaning and refers to a dosage form which has the capacity of being administered to a subject, preferably a human, to be effective, and which can be readily handled and packaged, remaining as a physically and chemically stable unit dose comprising the therapeutic agent, i.e. the compound of the invention. The term heterobifunctional compounds (HBCs) or bifunctional compounds or bifunctional moieties as used herein has its conventional meaning. HBC consist of three covalently bound moieties: a ligand moiety to bind the POI (POI ligand), another ligand moiety to bind E3 ligase (E3 ligand) and a spacer to conjugate the two moieties. HBC simultaneously recruits E3 ligase and POI, forming the “E3 ligase-HBC-POl” ternary complex. This complex potentiates the substrate recognition by E3 ligase and promotes the transfer of Ub to POI, accelerating the poly- ubiquitination and subsequent proteasome-mediated degradation of POI. The bifunctional compounds according to the invention are heterobifunctional compounds (HBCs).
The term “PLK1” as used herein has its conventional meaning and refers to Polo-like Kinase 1. Polo-like Kinase 1 (PLK1) is a member of the Polo-like Kinase family of protein kinases which are a subset of kinases which play a central role in the regulation of a wide variety of cellular signaling processes.
The term ”PLK1 degrader” as used herein has its conventional meaning and refers to a degrading inducing compound for Polo-like Kinase 1 (PLK1). A Polo-like Kinase 1 (PLK1) degrader may be a small molecule degrader. Degraders eliminate whole functions of the PLK1 protein, rather than merely inhibiting its enzymatic activity .
The term "targeting moiety" refers to a molecule which is capable of binding (selectively) to a target cell-surface molecule expressed on a target cell. The targeting moiety is in preferred embodiments of the invention an antibody or an antigen-binding fragment thereof. The targeting moiety is indicated in the chemical structures in the context of this invention with T.
The term "antibody" as used herein is used in the broadest sense, which may refer to an immunoglobulin (Ig) defined as a protein belonging to the class IgG, IgM, IgE, IgA, or IgD (or any subclass thereof, for example lgG1 , lgG2, lgG3 or lgG4), or a functional binding fragment or binding domain of an immunoglobulin. An antibody is an immunoglobulin capable of specifically binding a target protein. Immunoglobulins are well-known proteins, described for example in Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Immunoglobulins are typically glycoproteins consisting of two pairs of polypeptide chains, a pair of heavy chains and a pair of light chains, all connected by disulfide bonds. Variant immunoglobulins only containing a single polypeptide chain also occur in nature, for example in camelids and cartilaginous fishes.
Heavy chains typically consist of a variable region (VH) and a constant region, which typically is comprised of three domains, CH1 , CH2, and CH3. Light chains are typically comprised of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions contain regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved, termed framework regions (FRs). VH and VL regions typically comprise three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. In the context of the present invention, an "antigen-binding fragment" of an antibody is defined as a fragment that essentially maintains the antigen-binding properties of a parental antibody. A fragment may be generated by any method known in the art, including recombinant production. "Antibodies and antigen-binding fragments thereof in accordance with the invention include, but are not limited to, F(ab')2 fragments, Fab' fragments, Fab fragments, scFv, dsFv, single-domain antibody (sdAb), monovalent IgG, scFv-Fc, reduced IgG (rlgG), minibody, diabodies, triabodies, tetrabodies, Fc fusion proteins, nanobodies, variable V domains such as VHH, Vh, and other types of antigen recognizing immunoglobulin fragments and domains, such as derived from camelids or cartilaginous fishes. The fragments and domains may be engineered, for example to minimize or completely remove the intermolecular disulphide interactions that occur between the CH1 and CL domains or to introduce additional cysteine residues which can be used as attachment points for conjugation. Also the CDRs of an antibody may be grafted onto a nonimmunoglobulin scaffold. Also included are non-natural sequences that are the result of optimization using algorithms or affinity maturation techniques, and which bear sequence homology to naturally occurring antibodies.
The term “full-length antibody” refers to an antibody comprising two pairs of heavy and light chains, each containing the heavy and light chain constant and variable domains that are normally found in a wild-type antibody of that isotype. A full-length lgG1 antibody contains VH, CH1 , CH2, CH3, hinge, VL and CL domains. Full-length antibodies may comprise mutations relative to the sequence of the wild-type antibody, such as introduced cysteines as described herein.
The antibodies (immunoglobulins) of the present invention may be bi- or multispecific. For example, a bispecific antibody has one arm having a specificity for one target antigen, while the other arm recognizes a different target antigen. Alternatively, each arm of the bispecific antibody may have specificity for a different epitope of the same antigen of the target cell. Furthermore, antibodies may be monovalent (e.g. comprise a single antigen-binding domain for a given target antigen) or multivalent, such as bivalent (comprising multiple antigen-binding domains for a given target antigen).
The antibodies (immunoglobulins) of the present invention may be, but are not limited to, polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, mouse antibodies, rat antibodies, rat/mouse hybrid antibodies, llama antibodies, llama heavy-chain only antibodies, heavy-chain only antibodies, and veterinary antibodies. Preferably, the antibody (immunoglobulin) of the present invention is a monoclonal antibody.
Any other molecules than antibodies that bind to a cell receptor or antigen of a target cell can also be used as the cell surface molecule targeting moieties for the conjugates of the present invention. These targeting moieties include, but are not limited to, proteins, polypeptides, peptides, RNA-based probes, DNA-based probes, small molecules or enzyme substrate mimics. Examples of these non-antibody targeting moieties are interferons (e.g. IFN-a, IFN-p, and IFN-y), transferrins, lectins, epidermal growth factors (EGF) and EGF-like domains, gastrin-releasing peptides (GRP), platelet-derived growth factors (PDGF), transforming growth factors (TGF), vaccinia growth factor (VGF), insulin and insulin-like growth factors (IGF, e.g. IGF-1 and IGF-2), other suitable hormones such as thyrotropin releasing hormones (TRH), melanocyte-stimulating hormones (MSH), steroid hormones (e.g. estrogen and androgen), somatostatin, lymphokines (e.g. IL-2, IL-3, IL-4, and IL- 6), colony-stimulating factors (CSF, e.g. G-CSF, M-CSF and GM-CSF), bombesin, gastrin, Arg- Gly-Asp or RGD, aptamers (e.g. AS-1411 , GBI-10, RNA aptamers against HIV glycoprotein), small molecules (e.g. folate, anisamide phenylboronic acid, PSMA-617), vitamins (e.g., vitamin D), carbohydrates (e.g. hyaluronic acid, galactose).
The term “DAR” as used herein has its conventional meaning and refers to the drug antibody ratio of the conjugate, which is the average number of degrader-linker complexes linked to each antibody. In certain aspects, the DAR of the conjugates described herein is from 1 to 10.
The term “linker” as used herein has its conventional meaning and refers to the chemical connector between the antibody and degrader payload; it may be non-cleavable or cleavable; cleavage can take place with proteases, hydrolysis or reduction mechanisms.
The term “cleavable linker” as used herein has its conventional meaning and refers to the linker that is susceptible to acid-induced cleavage, photo-induced cleavage, bioreductive cleavage, enzymatic cleavage, or the like, at conditions under which the bifunctional degrader D and/or targeting moiety T can remain active. A non-exhaustive list of examples of cleavable linkers can be found in J Bargh et al. Cleavable linkers in antibody-drug conjugates. Chemical Society Reviews 48, pages 4361 -4374 (2019) and references therein.
The term “non-cleavable linker3’ as used herein has its conventional meaning and refers to any chemical moiety that is capable of linking the targeting moiety T to the bifunctional degrader D in a stable, covalent manner. As is known in the field, a targeting moiety in the form of an antibody might get processed intracellularly, leading to release of the degrader still attached to the non-cleavable linker, which in turn is attached to a residual amino acid. In general, this metabolite then has the desired pharmacological activity. A non-exhaustive list of examples of non-cleavable linkers can be found J Dugal-Tessier and N Jain. Non-cleavable Linkers: Permanently Linked, for Better or for Worse. Chapter 4 IN: Chemical Linkers in Antibody-Drug Conjugates (ADCs). Editor: Floris van Delft, ISBN 978-1-83916-515-3 (2021) and references therein.
The term “protease cleavable” as used herein has its conventional meaning and refers to a linker in which hydrolysis by the lysosomal proteases like Cathepsins, releases the bifunctional degrader D. The term “pyrophosphate or phosphate cleavable linker” as used herein has its conventional meaning and refers to a linker in which hydrolysis by the lysosomal phosphatases releases the bifunctional degrader D.
The term “beta-glucuronidase cleavable linker” as used herein has its conventional meaning and refers to a linker in which hydrolysis by the lysosomal enzyme beta-glucuronidase releases the bifunctional degrader D.
The term “click-to-release linker” as used herein has its conventional meaning and refers to a linker where release of the degrader is chemically triggered by a tetrazine or related compound.
The term “bioreducible linker” as used herein is a linker that keep conjugates intact during systemic circulation, and are selectively cleaved by the high intracellular concentration of glutathione, releasing the active drugs (degraders) at the tumor sites from the non-toxic prodrugs.
The term “EWG (electron withdrawing group)” as used herein means a functional group or electronegative atom that draws electron density away from an atom to which it is bonded either inductively and/or through resonance, whichever is more dominant (i.e. , a functional group or atom may be electron withdrawing inductively but may overall be electron donating through resonance) and tends to stabilize anions or electron-rich moieties. The electron withdrawing effect is typically transmitted inductively, albeit in attenuated form, to other atoms attached to the bonded atom that has been made electron deficient by the electron withdrawing group (EWG), thus affecting the electrophilicity of a more remote reactive center. Exemplary electron withdrawing groups include, but are not limited to -C(O), -CN, -NO2, -CX3, -X, -C(O)OR , - C(O)N(R)2, -C(O)R, -C(O)X, -S(O)2R, -S(O)2OR, -S(O)2NHR,-S(O)2N(R)2, -P(O)(OR)2, -P(O)(CH3)NHR, -NO, -N(R)3+, wherein X is -F, -Br, -Cl, or -I, and R in some aspects is, at each occurrence, independently selected from the group consisting of hydrogen and (1 -6C)alkyl, and certain O-linked moieties such as acyloxy.
The term “IC50” as used herein has its conventional meaning and refers to the concentration of a substance that results in a 50% effect on some measure of biochemical function or substance-target binding interaction.
The term “DC50” as used herein has its conventional meaning and refers to the half- maximal degradation concentration that resulted in a 50% targeted protein degradation.
A bicyclic ring system, as used herein, refers to heterocyclic (heterocyclyl) groups, to cyclic groups having carbon groups only, i.e. without hetero atoms, within the cycle, and to combinations of a heterocyclic (heterocyclyl) group and a cyclic group having carbon groups only, i.e. without hetero atoms, within the cycle. A bicyclic ring system includes 6-12 (e.g. 8-12, or 9-, 10-, or 11-) membered structures that form two rings, wherein the two rings have at least one atom in common (e.g. two atoms in common). Bicyclic ring systems include bicycloaliphatics (e.g. bicycloalkyl or bicycloalkenyl), bicycloheteroaliphatics, bicyclic aryls, and bicyclic heteroaryls.
As used herein, “spirocycloalkyl” or spirocyclyl” means carbogenic bicyclic ring systems with both rings connected through a single atom. The rings can be different in size and nature, or identical in size and nature. Examples include spirobutane, spiropentane, spirohexane, spiroheptane, spirooctane, spirononane, or spirodecane. One of both rings in a spirocycle can be fused to another ring carbocyclic, heterocyclic, aromatic, or heteroaromatic ring. For example, a (C3-Ci2)spirocycloalkyl is a spirocycle containing between 3 and 12 carbon atoms.
As used herein, “spiroheterocycloalkyl” or “spiroheterocyclyl” means a spirocycle wherein at least one of the rings is a heterocycle wherein one or more of the carbon atoms can be substituted with a heteroatom (e.g., one or more of the carbon atoms can be substituted with a heteroatom in at least one of the rings). One or both of the rings in a spiroheterocycle can be fused to another ring carbocyclic, heterocyclic, aromatic, or heteroaromatic ring.
A monocylic ring system, as used herein, refers both to a heterocyclic (heterocyclyl) group, and to a cyclic group having carbon groups only, i.e. without hetero atoms, within the cycle.
A heterocyclic (heterocyclyl) group, as used herein, refers to both heteroaryl groups and heterocycloalkyl groups.
A heterobicyclic group, as used herein, refers to a bicyclic group having one or more heteroatoms, which is saturated, partially unsaturated or unsaturated.
As used herein, aromatic groups (or aryl groups) include aromatic carbocyclic ring systems (e.g. phenyl) and fused polycyclic aromatic ring systems (e.g. naphthyl and 1 , 2,3,4- tetrahydronaphthyl).
The term “heteroaryl”, as used herein, refers to an aryl group having one or more hetero atoms.
The term “alkyl,” as used herein, refers to an aliphatic hydrocarbon group having one of its hydrogen atoms replaced with a bond having the specified number of carbon atoms. In different embodiments, an alkyl group contains, for example, from 1 to 6 carbon atoms (1 -6C)Alkyl or from 1 to 3 carbon atoms (1 -3C)Alkyl. Non-limiting examples of alkyl groups include methyl, ethyl, n- propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, neopentyl, isopentyl, n-hexyl, isohexyl and neohexyl. In one embodiment, an alkyl group is linear. In another embodiment, an alkyl group is branched.
Unless specified otherwise, “alkyl” includes both branched- and straight-chain saturated aliphatic hydrocarbon groups, including all isomers, having the specified number of carbon atoms; for example, “(1-6C)Alkyl” includes all of the hexyl alkyl and pentyl alkyl isomers as well as n-, iso- , sec- and t-butyl, n- and isopropyl, ethyl and methyl. “Alkylene” refers to both branched- and straight-chain saturated aliphatic hydrocarbon groups, including all isomers, having the specified number of carbons, and having two terminal end chain attachments; for example, the term “A-C4 alkylene-B” represents, for example, A-CH2-CH2-CH2-CH2-B, A-CH2-CH2-CH(CH3)-CH2-B, A-CH2- CH(CH2CH3)-B, A-CH2-C(CH3)(CH3)-B, and the like.
The term "alkylcarbonyl," as used herein, refers to an aliphatic hydrocarbon group having one of its hydrogen atoms replaced with a bond attached to a carbonyl group, wherein the aliphatic hydrocarbon group has the specified number of carbon atoms. In different embodiments, an alkyl group or aliphatic hydrocarbon group contains, for example, from 1 to 6 carbon atoms (1-6C)Alkyl or from 1 to 3 carbon atoms (1 -3C)Alkyl. Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, neopentyl, isopentyl, n- hexyl, isohexyl and neohexyl. In one embodiment, an alkyl group is linear. In another embodiment, an alkyl group is branched.
Cycloalkyl means a cycloalkyl group having the recited number of carbon atoms, with the same meaning as previously defined, such as cyclopropyl, cyclobutyl, or cyclopentyl. “Cycloalkyl” refers to a cycloalkyl-group represented by an indicated number of carbon atoms; for example “(3- 6C)cycloalkyl” includes cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.
Heterocycloalkyl means a cycloalkyl group having the recited number of carbon atoms, and 1-3 heteroatoms selected from N, O and/or S, with the same meaning as previously defined.
Haloalkyl means a branched or unbranched alkyl group having the recited number of carbon atoms, in which one and up to all hydrogen atoms are replaced by a halogen; halogen is as defined herein. Examples of such branched or straight chained haloalkyl groups useful in the present invention include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl and n- butyl substituted independently with one or more halogens, e.g., fluoro, chloro, bromo and iodo.
For example, a halo(1-3C)alkyl means a branched or unbranched alkyl group having 1 ,2, or 3 carbon atoms, in which at least one hydrogen atom is replaced by a halogen. Examples of “haloalkyl” include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 1 -fluoroethyl, 2-fluoroethyl, 2,2-difluoroethy I, 2,2,2-trifluoroethy I, and perfluoro-n-propyl.
Alkoxy means an alkoxy group having the recited number of carbon atoms, the alkyl moiety having the same meaning as previously defined, e.g., “Alkoxy” refers to an alkyl-O-group represented by a linear or branched alkyl group of indicated number of carbon atoms attached through an oxygen bridge; for example “(1 -6C)Alkoxy” includes CH3-O-, CH2CH3-O-, CH(CH3)2-O- , (CH2)5CH3-O-, and the like.
Cycloalkoxy means a cycloalkyl group having the recited number of carbon atoms, with the same meaning as previously defined, attached via a ring carbon atom to an exocyclic oxygen atom, such as cyclopropoxyl, cyclobutoxyl,or cyclopentoxyl. “Cycloalkoxy” refers to a cycloalkyl- O-group represented by a cycloalkyl group of indicated number of carbon atoms attached through an oxygen bridge; for example “(3-6C)cycloalkoxy” includes cyclopropyl-O-, cyclobutyl-O-, cyclopentyl-O-, or cyclohexyl-O-.
Heterocycloalkoxy means a cycloalkyl group having the recited number of carbon atoms, and 1 -3 heteroatoms selected from N, O and/or S, with the same meaning as previously defined, attached via a ring carbon atom to an exocyclic oxygen atom.
Unless otherwise specifically noted as only “unsubstituted” or only “substituted”, alkyl groups are unsubstituted or substituted with 1 to 3 substituents on each carbon atom.
It should be noted that any carbon as well as heteroatom with unsatisfied valences in the text, schemes, examples and tables herein is assumed to have the sufficient number of hydrogen atom(s) to satisfy the valences.
The terms first, second, third and the like in the description and in the claims, are used for distinguishing between for example similar elements, compositions, constituents in a composition, or separate method steps, and not necessarily for describing a sequential or chronological order. The terms are interchangeable under appropriate circumstances and the embodiments of the invention can operate in other sequences than described or illustrated herein, unless specified otherwise.
Furthermore, the various embodiments, although referred to as “preferred” or “e.g.” or “for example” or “in particular” and the like are to be construed as exemplary manners in which the invention may be implemented rather than as limiting the scope of the invention.
The term “comprising”, used in the claims, should not be interpreted as being restricted to for example the elements or the method steps or the constituents of a compositions listed thereafter; it does not exclude other elements or method steps or constituents in a certain composition. It needs to be interpreted as specifying the presence of the stated features, integers, (method) steps or components as referred to, but does not preclude the presence or addition of one or more other features, integers, steps or components, or groups thereof. Thus, the scope of the expression “a method comprising steps A and B” should not be limited to a method consisting only of steps A and B, rather with respect to the present invention, the only enumerated steps of the method are A and B, and further the claim should be interpreted as including equivalents of those method steps. Thus, the scope of the expression “a composition comprising components A and B” should not be limited to a composition consisting only of components A and B, rather with respect to the present invention, the only enumerated components of the composition are A and B, and further the claim should be interpreted as including equivalents of those components.
In addition, reference to an element or a component by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element or component are present, unless the context clearly requires that there is one and only one of the elements or components. The indefinite article “a” or “an” thus usually means “at least one”. Legend to the figures
Figure 1 : Results for the A-549 PLK1-HiBiT high throughput degradation assay. Compounds according to Example 1 and Example 2 show dose-dependent complete reduction of the HiBiT signal, demonstrating complete reduction of the levels of HiBiT tagged PLK1 in the cell line. MG132 inhibits the proteasome. Adding MG132 leads to reduction of the potency of Example 1 and Example 2, indicating that their activity is dependent on proteasome activity.
Figure 2: Western Blot of PLK1 degradation by Example 1 and Example 2 in the A549 PLK1 -HiBiT cell line. Co-incubation with the proteasome inhibitor MG132 abrogates PLK1 degradation, indicating PLK1 degradation by the Example compounds is proteasome-dependent. Onvansertib, BI2536 and CC885 are included as references. Cells were exposed for 24h to 10 pM compound.
Figure 3: Western Blot of dose dependent PLK1 degradation by Example 1 and Example 2 in the A549 PLK1 -HiBiT cell line. Cells were exposed to compound for 24h. Both Examples were analyzed on the same blot, dashed line was added for ease of viewing.
Figure 4: Western Blot of dose dependent PLK1 degradation by Example 1 and Example 2 in the A549 PLK1 -HiBiT cell line, detect via an antibody to the HiBiT tag that is present on the N- terminus of PLK1 . Cells were exposed to compound for 24h. Both Examples were analyzed on the same blot, dashed line was added for ease of viewing.
Figure 5: Western Blot of dose dependent PLK1 degradation by Example 1 and Example 2 in the AU565 cell line. Cells were exposed to compound for 24h. Both Examples were analyzed on the same blot, dashed line was added for ease of viewing.
Figure 6: Western Blot of PLK1 degradation by Example 1 (1 pM) in the AU565 cell line, and competition by the PLK1 inhibitor BI2536. AU565 cells were exposed to compounds simultaneously for 24h. All samples were analyzed on the same blot, solid lines indicate the removal of redundant samples. Addition of equimolar or higher concentrations of the PLK1 inhibitor BI2536 can abrogate PLK1 degradation by Example 1 , confirming that Example 1 acts through PLK1 binding.
Figure 7: Western Blot of PLK1 degradation by Example 1 (1 pM) in the AU565 cell line, and competition by the cereblon binding drug pomalidomide. AU565 cells were exposed to compounds simultaneously for 24h. All samples were analyzed on the same blot, solid lines indicate the removal of redundant samples. Addition of equimolar or higher concentrations of pomalidomide can abrogate PLK1 degradation by Example 1 , confirming that Example 1 acts through cereblon binding.
Figure 8: Incubation of Example 154m with papain in buffer leads to proteolytic cleavage of the linker and release of Example 2. Figure 8A: Chromatogram (LC) of Example 154m in absence of papain. Figure 8B: Chromatogram after 24h incubation with papain. Figure 8C: Mass spectrum (MS) of peak number 6 in Figure 8B, indicating presence of a compound with identical mass to Example 2.
Figure 9: Results of an SDS PAGE of various conjugates to the antibody rosopatamab and the parental rosopatamab batch. In the conjugates, the light chain and heavy chain protein bands are shifted to higher molecular weight, indicating a covalent attachment of degrader-linker moieties and thus successful conjugation.
Figure 10: Results of a proliferation assay of the PSMA-positive cell line LNCaP.
Conjugates of ready-to-conjugate intermediate examples 157m, 158m and 159m to the PSMA binding antibody rosopatamab show dose-dependent cytotoxicity on this cell line, whereas the unconjugated antibody (rosopatamab) does not have any effect. This shows that the conjugation procedure causes the antibody to become cytotoxic.
Figure 11 : Results of a proliferation assay of the PSMA-negative cell line PC3.
Conjugates of ready-to-conjugate intermediate examples 157m, 158m and 159m to the PSMA binding antibody rosopatamab show very limited toxicity on this cell line, compared to the cytotoxicity on LNCaP presented in Figure 10. This shows that the activity of the conjugates depends on their binding to PSMA and thus on functional antibody binding.
The present invention will be illustrated further by means of the following non-limiting examples.
Detailed description of the invention
The present application addresses the provision of PLK1 targeting bifunctional compounds (HBCs) that are suitable as therapeutic compounds for targeting and inducing degradation of PLK1 kinases.
The present application also addresses the provision of conjugates containing bifunctional moieties based on PLK1 targeting bifunctional compounds (HBCs) according to Formula (I) that are suitable as therapeutic compounds for targeting and inducing degradation of PLK1 kinases.
The compounds according to the invention have a Targeting Ligase Binding group (TLB), which is a group that is capable of binding to a E3 ligase, a Protein Binding group (PB) capable of binding to Polo-like Kinase 1 (PLK1), wherein the Protein Binding group (PB) has the chemical structure BPLKI-X, and a spacer (Sp). The spacer (Sp) covalently binds the Targeting Ligase Binding group (TLB) to the X group of the Protein Binding group (PB). As such the spacer covalently links the Protein Binding group (PB) to the Targeting Ligase Binding group (TLB).
The bifunctional compounds (HBCs) according to the invention have crucial and novel characteristics. First they are able to induce degradation of PLK1 , enabling therapeutic applications that more closely mimic tumor targeting phenotypes in biological knockdown experiments. The compounds according to the invention, in particular the Protein Binding Group of these compounds, have an X-group, which contains a unique functional moiety, in the form of a hydroxyalkyl group, such as a hydroxymethylene or hydroxyethylene side-group, which has the unexpected characteristic that it simultaneously improves the capability of the HBCs for binding PLK1 and for PLK1 degradation and their cytoxicity towards tumor cells.
The inventors have found that the bifunctional compounds (HBCs) due to the X group of the Protein Binding group (PB) having an unique functional moiety, wherein the X group, which comprises a hydroxyalkyl group improves the degradation effect of the bifunctional compounds (HBCs). The X-group is covalently connected to the amide (-C(O)-NH-) group of BPLKI and positions the unique functional moiety of the X-group close to the BPLKI group of the Protein Binding group (PB).
It is shown in exemplary model tests, that the hydroxyalkyl functional group of the X group supports or improves the binding process of the Protein Binding group (PB) to the PLK1 and at the same time supports or improves the degradation process of the PLK1 by the bifunctional compound, when the PLK1 protein is (temporarily) bound by the Protein Binding group (PB) of the bifunctional compound.
Another advantage of certain embodiments of this invention is that PLK1 targeting bifunctional compounds (HBCs) or bifunctional moieties according to the invention are suitable for inducing PLK1 degradation.
Another advantage of certain embodiments of this invention is to provide cancer treatments based on the administration of the bifunctional compounds or conjugates of the invention.
Another advantage of the invention is that the bifunctional compounds according to the invention, and in particular the X group of the bifunctional compounds, are suitable for forming conjugates comprising the bifunctional moieties.
As such, the bifunctional compounds according to the invention can also be used as intermediate compounds for forming Degrader Antibody Conjugates (DAC), i.e. conjugates with an antibody group or conjugates with target functional fragments of antibodies. In advantageous embodiments ready-to-conjugate intermediate compounds may be provided based on the bifunctional moieties according to the invention coupled to a ready-to-conjugate linker. These ready-to-conjugate intermediate compounds can easily be coupled to targeting moieties, preferably to antibodies or binding fragments thereof.
It has been found that the Degrader Antibody Conjugates (DAC) according to the invention can be suitably and effectively used for targeting specific cells. The inventors have experimentally confirmed, see the experimental section described, that commonly known linker technologies, for example based on cleavable linker technologies, are suitable to provide Degrader Antibody Conjugates (DAC) capable to (re)form the bifunctional compounds according to the invention in its original end effective form (as also described throughout this application), when subjected to cleaving conditions. As such, Degrader Antibody Conjugates (DAC) according to the invention are in preferred embodiments structured to reform the original bifunctional compounds according to the invention under (internal) cell conditions, wherein the Protein Binding group (PB) has the chemical structure BPLKI-X, including the original hydroxyalkyl functional group of the X group. Thus, the degrader moiety as defined according to the invention is easily obtained from the Degrader Antibody Conjugates (DAC) according to the invention.
In addition, the conjugates have double selectivity: one from antibodies and the other from the PLK1 targeting bifunctional moieties. For example, the antibody in the conjugate the invention may bind a specific antigen on a cancer cell, and then the conjugate enters the cell via internalization. Once in the cell, the PLK1 targeting bifunctional moiety finds PLK1 and brings it to E3 ubiquitin ligase for ubiquitination. The ubiquitinated PLK1 is marked for degradation by proteasomes. This happens only in cells expressing the specific antigen. Thus, for therapeutic effect, antigen expression and PLK1 expression is required. Thus, a conjugate of the invention is highly selective and will have less adverse effects.
Embodiments
Bifunctional compounds of the invention
Bifunctional compounds of the invention are according to Formula (I):
TLB-Sp-PB
Formula (I), or a pharmaceutically acceptable salt, hydrate, and/or solvate thereof, wherein:
TLB is a Targeting Ligase Binding group capable of binding to a E3 ligase; PB is a Protein Binding group capable of binding to Polo-like Kinase 1 (PLK1) and Sp is a spacer, which covalently links the Targeting Ligase Binding group (TLB) to the Protein Binding group (PB); wherein PB has the chemical structure BPLKI-X, wherein BPLKI has the formula:
(BPLKI) , wherein Ri is selected from the group consisting of H, methyl, ethyl or propyl; wherein R2 is selected from the group consisting of (1 -6C)cycloalkyl, (1 -6C)alkyl or benzyl; wherein any of said cycloalkyl, alkyl or benzyl is optionally and independently substituted with one or more substituents selected from halogen, hydroxy and cyano; wherein R3 is selected from the group consisting of OMe, OCHF2, OCF3, OCD3, (1 -3C)alky I, O(CH2)2OH or O(CH2)3OH; wherein R4 is selected from the group consisting of H or methyl; wherein denotes the point of attachment to the X group; wherein the X group has a formula (X-1) to (X-28) selected from the group consisting of:
(X-16) (X-17) (X-18) (X-19) (X-20)
(X-26) (X-27) (X-28) wherein any of said formula X-1 to X-28 is optionally and independently substituted with one or more substituents selected from fluoro, hydroxy, (1 -3C)alkyl and (1 -3C)alkoxy; wherein n is 1 to 4; preferably n is 1 , 2 or 3, more preferably n is 1 or 2; wherein I denotes the point of attachment to the amide (-C(O)-NH-) group of BPLK-I, denotes the point of attachment to the spacer (Sp).
In one aspect of the invention there is provided a conjugate comprising a bifunctional compound according to Formula (I) coupled to a moiety or agent. In one embodiment of the conjugate, the moiety or agent is selected from the group consisting of a targeting moiety, a radioisotope, a protein, an enzyme, a dye, a drug, a pharmacologically active small molecule. In one embodiment, the moiety is a targeting moiety capable of binding to a cell surface molecule.
In preferred embodiments, the conjugates according to the invention have the chemical structure: T - (L - D)a or a pharmaceutically acceptable salt thereof, wherein:
D is a bifunctional moiety capable of inducing degradation of PLK1 ;
T comprises or consists of a targeting moiety capable of binding to a cell surface molecule;
L is a linker, covalently bound to T and D; a is an integer from 1 to 8;
Wherein D is a bifunctional moiety having the chemical structure TLB-Sp-PB, wherein TLB, Sp and PB have the chemical structure according to the invention and wherein the X group of PB is covalently bound to the linker L, optionally by conversion of the -OH group of the X group to a bonding group containing -O-. Techniques for conjugating therapeutic agents to a targeting moiety, such as proteins, and in particular to antibodies, are well-known. (See, e.g., Arnon et al, “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in Monoclonal Antibodies And Cancer Therapy (Reisfeld et al. eds., Alan R. Liss, Inc., 1985); Hellstrom et al, “Antibodies For Drug Delivery,” in Controlled Drug Delivery (Robinson et al. eds., Marcel Dekker, Inc., 2nd ed. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biological And Clinical Applications (Pinchera et al. eds., 1985); “Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy,”; Walsh S.J. et al. (2021) Chem Soc Rev, 50, 1305; “Site selective modification strategies in antibody-drug conjugates”; in Monoclonal Antibodies For Cancer Detection And Therapy (Baldwin et al. eds., Academic Press, 1985); and Thorpe et al, 1982, Immunol. Rev. 62:1 19-58. See also, e.g., PCT publication WO 89/12624.)
The bifunctional moiety D is based on the bifunctional compound according to the invention (according to the first aspect), which is also a ready to conjugate compound Dm, which has the chemical structure TLB-Sp-PB, wherein PB has the chemical structure BPLKI-X, and wherein PB is capable of binding to Polo-like Kinase 1 (PLK1) kinases.
In preferred embodiments, the X group of the bifunctional moiety D is covalently bound to the linker L by conversion of the -OH group of the X group to a bonding group containing -O-. The bonding group provides a covalent bond to the linker L.
In exemplary embodiments, the bonding group may be selected from -O- (ether group), -O-C(O)- NH- , -0-C(0)-0 and -O-C(O)-.
EP3865152A1 discloses a conjugate of target molecule-linker-E3 ligase ligand as shown in the structure of A-L1-B (formula I), wherein the A is the monovalent group of the target molecule, the B is the monovalent group of the E3 ligase ligand, the L1 is the spacer linking A and B. In particular examples, conjugates are described including an antibody attached to a flexible spacer moiety between A and B and connected via an amine attachment point at a position between a PLK1-binding unit (A) and the E2 ligase binding unit (B). However, the amine attachment point for connection via a linker to the antibody impairs the ternary complex formation.
Dragovitch et al. ( Chem. Soc. Rev. (2022) 51 , 3886) has reported that several reported chimeric degraders do not contain chemical groups that can be utilized for the covalent attachment of cleavable linkers (e.g., primary, secondary, and/or tertiary amines). In such instances, careful consideration must be given regarding whether to purposefully incorporate the necessary chemical groups into the degrader structure (and possibly alter its associated biological properties) or to exploit existing degrader functionality such as hydroxyl/phenol groups or add such a hydroxyl functionality for using ADC linking approaches. The present compounds are provided containing the chemical structure BPLKI-X, wherein the alkylhydroxy group is provided as part of the targeting moiety (showing both inhibitor and degrader effects) to exploit improved degrader functionality and at the same time provide new ADC linking approaches. In preferred embodiments, the Protein Binding group PB of the conjugate has chemical structure
BPLKI-XC, wherein the Xc-group has any one of the following structures according to formula (Xc-
1) to (Xc-28) selected from the group consisting of:
(Xc-16) (Xc-17) (Xc-18) (Xc-19)
wherein any of said formula Xc-1 to Xc-28 is optionally and independently substituted with one or more substituents selected from fluoro, hydroxy, (1 -3C)alkyl and (1-3C)alkoxy; wherein n is 1 to 4; wherein the — | — * denotes the point of attachment of the X group to the amide (-C(O)-NH-) group of Formula BPLK-I ; wherein I denotes the point of attachment to spacer Sp; wherein the
# denotes the point of attachment to linker L.
The Xc groups according to any one of formula (Xc-1) to (Xc-28) are derived from the X groups of the bifunctional compounds according to any one of formula (X-1) to (X-28), respectively, by converting the -OH group of the X group to a bonding group containing -O-. In more preferred embodiments, the Xc-group of D is selected from the group consisting of: , wherein the — i — * denotes the point of attachment of the X group to the amide (-
C(O)-NH-) group of BPLKI ; wherein WT,W denotes the point of attachment to Sp; wherein the denotes the point of attachment to linker L.
Protein Binding group PB
The Protein Binding group PB is capable of binding to Polo-like Kinase 1 (PLK1). The Protein Binding group PB has the chemical structure BPLKI-X. The Protein Binding group PB thus contains binding moiety BPLKI as shown below, and the X-group. The Protein Binding group (PB) as a whole is the targeting group for targeting the PLK1 protein. The inventors have found that the structural configuration of the X-group, including the configuration of the hydroxyalkyl group or hydroxyalkyl side-chain, in connection to the binding moiety (BPLKI) of the Protein Binding group (PB) cooperatively improve the capability of the bifunctional compounds for PLK1 degradation and their cytoxicity towards tumor cells. The X-group is directly covalently connected to the amide (-C(O)- NH-) group of moiety BPLKI and by its direct connection cooperates with binding moiety BPLKI and positions the X-group, including its unique functional moiety of the X-group, close to the BPLKI group of the Protein Binding group (PB).
The Protein Binding Group has the Formula:
, wherein Ri is selected from the group consisting of H, methyl, ethyl or propyl; wherein R2 is selected from the group consisting of (1 -6C)cycloalkyl, (1 -6C)alkyl or benzyl; wherein any of said cycloalkyl, alkyl or benzyl is optionally and independently substituted with one or more substituents selected from halogen, hydroxy and cyano; wherein R3 is selected from the group consisting of OMe, OCHF2, OCF3, OCD3, (1-3C)alkyl, O(CH2)2OH or O(CH2)3OH; wherein R4 is selected from the group consisting of H or methyl; wherein denotes the point of attachment to the X group.
Please note that when R1 is H, the stereochemistry of R1 is not relevant as also the other subsituent is H.
In preferred embodiments, R1 is ethyl or propyl, and wherein R2 is isopropyl, cyclohexyl, or cyclopentyl, and wherein R3 is OMe, OCD3 or OCF3, and wherein R4 is H or methyl.
In preferred embodiments, R1 is ethyl or propyl.
In preferred embodiments, R2 is isopropyl, cyclohexyl, or cyclopentyl.
In preferred embodiments, R3 is OMe.
In preferred embodiments, R4 is H or methyl.
The X-group of the Protein Binding group PB is further described below. X-group
The X-group is directly covalently connected to the amide (-C(O)-NH-) group of BPLKI and positions the unique functional moiety of the X-group close to the rest of the BPLKI group of the Protein Binding group (PB). The X group of the Protein Binding group PB is covalently bound to the spacer (Sp).
In preferred embodiments the X group has a formula (X-1) to (X-28) selected from the group consisting of:
(X-16) (X-17) (X-18) (X-19) (X-20) wherein any of said formula X-1 to X-28 is optionally and independently substituted with one or more substituents selected from fluoro, hydroxy, (1 -3C)alkyl and (1-3C)alkoxy; wherein n is 1 to 4; wherein — | — * denotes the point of attachment to the amide (C(O)-NH-) group of formula , denotes the point of attachment to the Spacer (Sp).
The X-group contains a hydroxyalkyl group, such as a hydroxy-methylene or hydroxyethylene group, that is located near to the BPLKI . This improves the PLK1 binding effect of the Protein Binding group PB and improves the degradation effect of the bifunctional compounds (HBCs). The X-group is directly covalently connected to the amide (-C(O)-NH-) group of BPLKI
The inventors have further found that an hydroxy group, which is directly attached to the (hetero)cyclic moiety of the X-group, instead of an alkylhydroxy substituent, is not suitable as substituent of the (hetero)cyclic moiety of the X-group.
In preferred embodiments, X is selected from the group consisting of:
(X-21) (X-22) (X-23) (X-24) (X-28) wherein n is 1 ,2 or 3; wherein~~| “denotes the point of attachment to the amide (-C(O)-NH-) group of formula BPLKI and wherein denotes the point of attachment to the Spacer (Sp).
Sub-structure BPLKI-X In preferred embodiments, the Protein Binding group PB has the chemical structure BPLKI-X, which is selected from the group consisting of:
(Bpi_Ki-X-2a) (BpLKi-X-2b1) (BPLKI -X-2c)
(Bpi_Ki-X-9a) (BpLKi-X-9b) (BPLKi-X-10b)
(BpLKi-X-2b3) wherein Ri is selected from the group consisting of H, methyl, ethyl or propyl; wherein R2 is selected from the group consisting of (1 -6C)cycloalkyl, (1 -6C)alkyl or benzyl; wherein R4 is selected from the group consisting of H or methyl; wherein "T* denotes the point of attachment to the spacer (Sp). Spacer (Sp)
The spacer (Sp) covalently binds the Targeting Ligase Binding group (TLB) to the X group of the Protein Binding group PB.
In preferred embodiments, the spacer (Sp) has the chemical structure -S1-S2-S3-S4-S5- , wherein S1 is connected to the Targeting Ligase Binding group (TLB) and S5 is connected to X, wherein:
51 is independently selected from the group consisting of: a direct bond, -N(RS1)-, -O-, -C(O)- N(RS1)-, -N(RS1)-C(O)-, -N(RS1)-C(O)-O-, -C(O)-, -CEC-, -C=C-, -OCH2C(O)-, (1 -4C)alkyl, (3- 12C)cycloalkyl and (3-12C)heterocycloalkyl; wherein any of said alkyl, cycloalkyl or heterocycloalkyl group is optionally and independently substituted with one or more substituents selected from halogen, hydroxy and (1 -3C)alkyl; each RS1 is independently -H or (1-4C)alkyl;
52 is independently selected from the group consisting of: a direct bond, -N(RS2)-, -O-, (1- 4C)alkyl, -CH2-CH2-N(RS2)-, -(CH2-CH2-O)m-, -(CH2-CH2-CH2-O)m-, -(O-CH2-CH2)m-, (6- 10C)aryl, (3-12C)cycloalkyl, (3-12C)heterocycloalkyl, 7-12 membered spiro bicyclic heterocycloalkyl, 7-12 membered fused bicyclic heterocycloalkyl, 7-12 membered spiro bicyclic cycloalkyl and 7-12 membered fused bicyclic cycloalkyl; wherein any of said alkyl, cycloalkyl or heterocycloalkyl group is optionally and independently substituted with one or more substituents selected from halogen, oxo, hydroxy, (1 -3C)alkyl and (1-3C)alkoxy; each RS2 is independently -H or (1 -4C)alkyl;
53 is independently selected from the group consisting of: a direct bond, (1 -8C)alky I, -CEC-, N(RS3)-, -O-, -N(RS3)-C(O)-, -C(O)-N(RS3)-, -C(O)-, -(O-CH2-CH2)U-, -(CH2-CH2-O)U- and (3- 12C)heterocycloalkyl; wherein any of said heterocycloalkyl group is optionally and independently substituted with one or more substituents selected from halogen, oxo, hydroxy, (1 -3C)alkyl and (1-3C)alkoxy; each RS3 is independently -H or (1 -4C)alkyl;
54 is independently selected from the group consisting of: a direct bond, (1 -4C)alky I, -N(RS4), - C(O)-, -(O-CH2-CH2)P-, -(CH2-CH2-O)P-, (3-12C)cycloalkyl, (3-12C)heterocycloalkyl, 7-12 membered spiro bicyclic heterocycloalkyl and 7-12 membered fused bicyclic heterocycloalkyl; wherein any of said alkyl, cycloalkyl or heterocycloalkyl group is optionally and independently substituted with one or more substituents selected from halogen, oxo, hydroxy, (1 -3C)alkyl and (1-3C)alkoxy; each RS4 is independently -H or (1 -4C)alkyl;
55 is independently selected from the group consisting of: a direct bond, -N(RS5)-, N(RS5)-C(O)-, -C(O)-, (1 -4C)alkyl, -(O-CH2-CH2)q- and -(O-CH2-CH2-CH2)q-; each RS5 is independently -H or (1 -4C)alkyl; each m, p, u and q is independently an integer from 1 to 3. In more preferred embodiments, S1 is selected from the group consisting of: a direct bond, - N(RS1)-, -O-, -C(O)-N(RS1)-, -N(RS1)-C(O)-, -C(O)-, -CEC-, -OCH2C(O)-, (3-12C)heterocyclo- alkyl and (1 -4C)alkyl; wherein any said alkyl or heterocycloalkyl group is optionally and independently substituted with one or more substituents selected from fluoro and hydroxy; each RS1 is independently -H or methyl.
In more preferred embodiments, S2 is selected from the group consisting of: a direct bond, (1 - 4C)alkyl, -CH2-CH2-N(RS2)-, -(CH2-CH2-O)m-, -(CH2-CH2-CH2-O)m-, (6-10C)aryl, (3- 12C)cycloalkyl, (3-12C)heterocycloalkyl, 7-12 membered spiro bicyclic heterocycloalkyl, 7-12 membered fused bicyclic heterocycloalkyl, 7-12 membered spiro bicyclic cycloalkyl and 7-12 membered fused bicyclic cycloalkyl; wherein any of said alkyl, cycloalkyl or heterocycloalkyl group is optionally and independently substituted with one or more substituents selected from halogen, oxo, hydroxy, (1 -3C)alkyl and (1-3C)alkoxy; each RS2 is independently -H or methyl; m is an integer from 1 to 2.
In more preferred embodiments, S3 is selected from the group consisting of: a direct bond, (1 - 8C)alkyl, -N(RS3)-, -O-, -N(RS3)-C(O)-, -C(O)-N(RS3)-, -C(O)-, -(O-CH2-CH2)u-, -(CH2-CH2- O)u- and (3-12C)heterocycloalkyl; wherein any of said or heterocycloalkyl group is optionally and independently substituted with one or more substituents selected from halogen, oxo, hydroxy, (1 -3C)alkyl and (1-3C)alkoxy; each RS3 is independently -H or methyl; u is an integer from 1 to 2.
In more preferred embodiments, S4 is selected from the group consisting of: a direct bond, (1 - 4C)alkyl, -(O-CH2-CH2)P-, -(CH2-CH2-O)P-, (3-12C)cycloalkyl and (3-12C)heterocycloalkyl; wherein any of said alkyl, cycloalkyl or heterocycloalkyl group is optionally and independently substituted with one or more substituents selected from halogen, oxo, hydroxy, (1 -3C)alkyl and (1-3C)alkoxy; p is an integer from 1 to 2.
In more preferred embodiments, S5 is selected from the group consisting of: a direct bond, - N(RS5)-, -N(RS5)-C(O)-, -C(O)-, (1 -4C)alkyl, -(O-CH2-CH2)q and -(O-CH2-CH2-CH2)q; each RS5 is independently -H or methyl; q is independently an integer from 1 to 2.
In even more preferred embodiments, S1 is selected from the group consisting of: a direct bond, -
wherein any of said alkyl, cycloalkyl or heterocycloalkyl group is optionally and independently substituted with one or more substituents selected from halogen, oxo, hydroxy, (1 -3C)alkyl and (1-3C)alkoxy; m is an integer from 1 to 2.
In even more preferred embodiments, S3 is selected from the group consisting of: a direct bond, (1 -4C)alkyl, -N(RS3)-, -O-, -N(RS3)-C(O)-, -C(O)-N(RS3)-, -C(O)-, -(O-CH2-CH2)u-, -(CH2-CH2- O)u- and (3-12C)heterocycloalkyl; wherein any of said alkyl or heterocycloalkyl group is optionally and independently substituted with one or more substituents selected from halogen, oxo, hydroxy, (1 -3C)alkyl and (1-3C)alkoxy; each RS3 is independently -H or methyl; u is an integer from 1 to 2.
In even more preferred embodiments, S4 is selected from the group consisting of: a direct bond, (1 -4C)alkyl, -N(RS4), -C(0)-, -(O-CH2-CH2)P-, -(CH2-CH2-O)P- ,
wherein any of said alkyl, cycloalkyl or heterocycloalkyl group is optionally and independently substituted with one or more substituents selected from halogen, oxo, hydroxy, (1 -3C)alkyl and (1-3C)alkoxy; each RS4 is independently hydrogen or methyl; p is an integer from 1 to 2.
In even more preferred embodiments, S5 is selected from the group consisting of: a direct bond, - N(RS5)-, -N(RS5)-C(O)-, -C(O)- and (1 -4C)alkyl; each RS5 is independently hydrogen or methyl.
In even more preferred embodiments, the spacer (Sp) has the chemical structure -S1-SpX-, wherein S1 is selected from the group consisting of: a direct bond, -NH-, -O-, -C(O)NH-, -NH- C(O)-, -C(O)-, -C=C-, -OCH2C(O)-, (1 -4C)alkyl; wherein S1 is connected to the Target Ligase Binding group (TLB) and SpX is connected to the X group, and wherein SpX is selected from the group consisting of:
wherein the marks the point of attachment to S1 or marks the point of attachment to the
Targeting Ligase Binding group (TLB) in case S1 is a direct bond; and wherein the I marks the point of attachment to X; and wherein each RL is hydrogen or methyl; and wherein s is an integer from 0 to 5; and wherein t is an integer from 0 to 6.
In particular preferred embodiments, the spacer (Sp) is selected from the group consisting of:
wherein the marks the point of attachment to the Targeting Ligase Binding group (TLB); I marks the point of attachment to X; and wherein s is an integer from 0 to 3.
Targeting Ligase Binding group (TLB)
The Targeting Ligase Binding group (TLB) is capable of binding to a E3 ligase.
Many groups are known to be capable of binding to a E3 ligase.
Inspired by UPS, researchers designed heterobifunctional compounds (HBC) to hijack the UPS and degrade a protein of interest (POI). HBC consists of three covalently-bonded moieties: a ligand to bind POI (POI ligand), another ligand to recognize E3 ligase (E3 ligand) and a linker to conjugate the two ligands. HBC simultaneously recruits E3 ligase and POI, forming the “E3-HBC-POI” ternary complex. This complex potentiates the substrate recognition by E3 ligase and promotes the transfer of Ub to POI, accelerating the poly-ubiquitination and subsequent proteasome-mediated degradation of POI (Lai AC and Crews CM. Induced protein degradation: an emerging drug discovery paradigm. Nat Rev. Drug. Discov. (2017) 16, 101 -1 14). Recent HBC developments have mainly been focused on three E3 ligases, i.e. Von-Hippel-Lindau (VHL), inhibitors of apoptosis proteins (lAPs) and Cereblon (CRBN).
In preferred embodiments, the Targeting Ligase Binding group (TLB) is any one of:

wherein the marks point of attachment to Sp or to S1 of Sp; wherein, t is the number of R(TLB1) substituents and is an integer from 0 to 2; each RTLB1 is independently selected from halo, cyano and (1 -4C)alkyl, wherein any of said alkyl group is optionally and independently substituted with one or more substituents selected from halogen, cyano, -COOH, COONH2, -NH2 and CF3; each RTLB4 is independently selected from hydrogen, (1 -4C)alkyl and (1-3C)alkoxy; each RTLB5 is independently selected from hydrogen, halo, cyano and (1 -3C)alkyl;
Z is -C(RTLB6)2 or -C(O)-; each RTLB6 is independently selected from hydrogen and (1 -4C)alkyl;
Z’ is a bond, -C(O)- or -CH2C(O)-; Z” is -CH2- or -NH-; HAr is a (1-9C)heteroaryl, optionally substituted with one or more substituents selected from fluoro, methyl and methoxy; and heterocyclic ring G is selected from:
In preferred embodiments, the Targeting Ligase Binding group (TLB) is selected from the group consisting of:
wherein the T~ marks the point of attachment to the spacer (Sp) or S1 of the spacer (Sp); wherein each RTLB5 is independently hydrogen, fluoro or cyano; and
HAr is a (1-9C)heteroaryl optionally substituted with one or more substituents selected from fluoro, methyl and methoxy.
In preferred embodiments, the Targeting Ligase Binding group (TLB) is selected from the group
wherein the marks the point of attachment to the spacer (Sp) or S1 of the spacer (Sp).
In further preferred embodiments, the Targeting Ligase Binding group (TLB) is selected from the group consisting of: wherein the marks the point of attachment to the spacer (Sp) or S1 of the spacer (Sp).
T (targeting moiety)
T comprises or consists of a targeting moiety capable of binding to a cell surface molecule.
The term "targeting moiety" refers to a molecule which is capable of binding to a target cellsurface molecule expressed on a target cell. On one embodiment, the targeting moiety is an antibody or an antigen-binding fragment thereof.
The term "antibody" as used herein is used in the broadest sense, which may refer to an immunoglobulin (Ig) defined as a protein belonging to the class IgG, IgM, IgE, IgA, or IgD (or any subclass thereof, for example IgG 1 , lgG2, lgG3 or lgG4), or a functional binding fragment or binding domain of an immunoglobulin. An antibody is an immunoglobulin capable of specifically binding a target protein. Immunoglobulins are well-known proteins, described for example in Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Immunoglobulins are typically glycoproteins consisting of two pairs of polypeptide chains, a pair of heavy chains and a pair of light chains, all connected by disulfide bonds. Variant immunoglobulins only containing a single polypeptide chain also occur in nature, for example in camelids.
Heavy chains typically consist of a variable region (VH) and a constant region, which typically is comprised of three domains, CH1 , CH2, and CH3. Light chains are typically comprised of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions contain regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved, termed framework regions (FRs). VH and VL regions typically comprise three CDRs and four FRs, arranged from amino-terminus to carboxyterminus in the order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.
Any other molecules than antibodies that bind to a cell receptor or antigen of a target cell can also be used as the targeting moiety for the conjugates of the present invention.
In embodiments, the targeting moiety is selected from the group consisting of proteins, polypeptides, peptides, small molecules and enzyme substrate mimics.
Examples of these non-antibody targeting moieties are interferons (e.g. IFN-a, IFN-p, and IFN-y), transferrins, lectins, epidermal growth factors (EGF) and EGF-like domains, gastrinreleasing peptides (GRP), platelet-derived growth factors (PDGF), transforming growth factors (TGF), vaccinia growth factor (VGF), insulin and insulin-like growth factors (IGF, e.g. IGF-1 and IGF-2), other suitable hormones such as thyrotropin releasing hormones (TRH), melanocyte-stimulating hormones (MSH), steroid hormones (e.g. estrogen and androgen), somatostatin, lymphokines (e.g. IL-2, IL-3, IL-4, and IL-6), colony-stimulating factors (CSF, e.g. G-CSF, M-CSF and GM-CSF), bombesin, gastrin, Arg-Gly-Asp or RGD, aptamers (e.g. AS-1411 , GBI-10, RNA aptamers against HIV glycoprotein), small molecules (e.g. folate, anisamide phenylboronic acid, PSMA-617), vitamins (e.g., vitamin D), carbohydrates (e.g. hyaluronic acid, galactose).
Linker (L)
The conjugates according to the invention comprise a Linker (L), covalently bonding the T (targeting moiety) to D. In preferred embodiments, the linker L is a cleavable linker.
The term “cleavable linker” as used herein has its conventional meaning and refers to the linker that is susceptible to acid-induced cleavage, photo-induced cleavage, bioreductive cleavage, enzymatic cleavage, or the like, at conditions under which the bifunctional degrader D and/or targeting moiety T can remain active. A non-exhaustive list of examples of cleavable linkers can be found in J Bargh et al. Cleavable linkers in antibody-drug conjugates. Chemical Society Reviews 48, pages 4361-4374 (2019) and references therein.
In preferred embodiments, a linker (L) of the Degrader Antibody Conjugates (DAC) according to the invention is selected capable to reform the original bifunctional compounds according to the invention under (internal) cell conditions, wherein the Protein Binding group (PB) has the chemical structure BPLKI-X, including the original hydroxyalkyl functional group of the X group. Thus, the degrader moiety D as defined according to the invention is easily obtained from the Degrader Antibody Conjugates (DAC) according to the invention in vivo under known cell conditions.
A skilled person can suitably select suitable linker chemistry and Linker moieties based on common general knowledge on linker technology, which are able to reform the hydroxyalkyl functional group of the X group of the Protein Binding group (PB), which has the chemical structure BPLK1-X.
For example see: Jain N. et al. (2015) Pharm Res, 32, 3526-3540, Su Z. et al. (2021) Acta Pharm Sinica, 11 , 3889-3907, Walsh S.J. et al. (2021) Chem. Soc. Rev., 50, 1305 and Sheyi R. et al. (2022) Pharmaceutics, 14, 396, for:
• Protease cleavable linker;
• pyrophosphate or phosphate cleavable linker;
• beta-glucuronidase cleavable linker;
• click-to-release linker;
• bio-reducible linker.
In embodiments, the cleavable linker is cleavable by a protease.
In embodiments, the linker L is selected from the group consisting of:
wherein: c is an integer from 2 to 10; Z1, Z2, Z3, and Z4 are each independently absent or a naturally occurring amino acid residue or the enantiomer thereof, provided that at least two of Z1, Z2, Z3, and Z4 are amino acid residues; wherein the * denotes the point of attach me nt to T; and wherein the I denotes the point of attachment to Xc. In embodiments, Z1, Z2, Z3, and Z4 are independently absent or selected from the group consisting of L-valine, D-valine, L-citrulline, D-citrulline, L-alanine, D-alanine, L-glutamine, D- glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-asparagine, D- asparagine, L-phenylalanine, D-phenylalanine, L-lysine, D-lysine, L-proline, D-proline, and glycine, provided that at least two of Z1, Z2, Z3, and Z4 are amino acid residues.
In embodiments, wherein:
Z1 is absent or selected from the group consisting of glycine, L-citru Hi ne or D-citru Hi ne ;
Z2 is absent or selected from the group consisting of L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L aspartic acid, D-aspartic acid, L-alanine, D-alanine, L-valine, D-valine and glycine;
Z3 is selected from the group consisting of L-valine, D-valine, L-alanine, D-alanine, L- phenylalanine, D-phenylalanine, L-proline, D-proline and glycine; and
Z4 is selected from the group consisting of L-alanine, D-alanine, L-citrulline, D-citrulline, L- asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine, and glycine.
In embodiments, wherein L is selected from the group consisting of: wherein c is an integer from 2 to 10; wherein the ™ * denotes the point of attachment to T; and wherein the denotes the point of attachment to Xc.
In embodiments, wherein c is 5. In embodiments, wherein the linker L is a pyrophosphate or phosphate cleavable linker.
In embodiments, wherein the linker L is selected from the group consisting of: wherein c is an integer from 2 to 10; wherein d is an integer 1 or 2; wherein the T~ * denotes
# the point of attachment to T; and wherein the I denotes the point of attachment to Xc.
In embodiments, wherein c is 5.
In embodiments, wherein the linker L is a beta-glucuronidase cleavable linker.
In embodiments, wherein the linker L is selected from the group consisting of:
wherein c is an integer from 2 to 10; wherein EWG is an electron withdrawing group; wherein the T~ denotes the point of attachment to T; and wherein the denotes the point of attachment to Xc.
In embodiments, wherein the linker L is selected from the group consisting of: wherein c is an integer from 2 to 10; wherein the T~ * denotes the point of attachment to T; and wherein the denotes the point of attachment to Xc.
In embodiments, wherein c is 5.
In embodiments, wherein the linker L is a click-to-release linker.
In embodiments, wherein the Linker L is selected from the group consisting of: wherein c is an integer from 2 to 10; wherein the T~ * denotes the point of attachment to T; denotes the point of attachment to Xc.
In embodiments, wherein the linker L is a bio-reducible linker.
In embodiments, wherein the linker L is selected from the group consisting of: wherein c is an integer from 2 to 10; R, R’, R”, and R’” are each independently selected from hydrogen, (1 -6C)alkoxy(1-6C)alkyl, (1-6C)2N(1-6C)alkyl, and (1 -6C)alkyl, or, two geminal R groups, together with the carbon atom to which they are attached, form a cyclobutyl or cyclopropyl ring; wherein the T~ * denotes the point of attachment to T; and wherein the
#
I denotes the point of attachment to Xc.
In embodiments, wherein the linker L is a non-cleavable linker.
The term “non-cleavable linker3’ as used herein has its conventional meaning and refers to any chemical moiety that is capable of linking the targeting moiety T to the bifunctional degrader D in a stable, covalent manner. As is known in the field, a targeting moiety in the form of an antibody might get processed intracellularly, leading to release of the degrader still attached to the non-cleavable linker, which in turn is attached to a residual amino acid. In general, this metabolite then has the desired pharmacological activity. A non-exhaustive list of examples of non-cleavable linkers can be found J Dugal-Tessier and N Jain. Non-cleavable Linkers: Permanently Linked, for Better or for Worse. Chapter 4 IN: Chemical Linkers in Antibody-Drug Conjugates (ADCs). Editor: Floris van Delft, ISBN 978-1-83916-515-3 (2021) and references therein.
In embodiments, wherein the linker L is selected from the group consisting of: wherein b is an integer from 1 to 10; wherein the * denotes the point of attachment to T ; and wherein the denotes the point of attachment to Xc.
In embodiments, wherein the linker L is:
In embodiments, wherein b is 4 or 5.
Compounds
In preferred embodiments, the bifunctional compound has a sub-formula 1 - 152 selected from the group consisting of:
3 4
17 18 19
5
44 45 46
80 81 82
001- 66 86 3
125 126 127
152
These are exemplary compounds shown to be suitable for inducing degradation of Polo-like Kinase 1 (PLK1). Ready- to-conjugate intermediate compounds
The invention further provides ready-to-conjugate intermediate compounds of Formula (II):
MC-Li-D Formula II; wherein MC is a maleimide group; wherein the linker Li is a cleavable linker or a non-cleavable linker (preferably according to any one of the described embodiments for linker L), wherein D is a bifunctional moiety as defined according to any of the embodiments of the invention.
In case the linker Li is a cleavable linker, preferably said linker Li contains a cleavable bond at the point of attachment to the Xc-group of the protein binding group PB of the conjugates (see section Conjugates). Preferably said linker Li contains a cleavable bond that subsequently releases or reforms the X-group of the protein binding group PB, which X-group is in embodiments according to any one of formula (X-1) to (X-28) as described in this application (and contains the hydroxyalkyl group).
In preferred embodiments, MC-Li is selected from the group consisting of:
wherein: c is an integer from 2 to 10; Z1, Z2, Z3, and Z4 are each independently absent or a naturally occurring amino acid residue or enantiomer thereof, provided that at least two of Z1, Z2,
Z3, and Z4 are amino acid residues; wherein the I denotes the point of attachment to Xc.
In preferred embodiments, Z1, Z2, Z3, and Z4 are independently absent or selected from the group consisting of L-valine, D-valine, L-citrulline, D-citrulline, L-alanine, D-alanine, L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-asparagine, D- asparagine, L-phenylalanine, D-phenylalanine, L-lysine, D-lysine, L-proline, D-proline, and glycine, provided that at least two of Z1, Z2, Z3, and Z4 are amino acid residues.
In preferred embodiments:
Z1 is absent or selected from the group consisting of glycine, L-citrulline or D-citrulline;
Z2 is absent or selected from the group consisting of L-glutamine, D-glutamine, L- glutamic acid, D-glutamic acid, L aspartic acid, D-aspartic acid, L-alanine, D-alanine, L-valine, D- valine and glycine;
Z3 is selected from the group consisting of L-valine, D-valine, L-alanine, D-alanine, L- phenylalanine, D-phenylalanine, L-proline, D-proline and glycine; and
Z4 is selected from the group consisting of L-alanine, D-alanine, L-citrulline, D-citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine, and glycine.
In preferred embodiments, MC-Li is selected from the group consisting of:
In preferred embodiments, MC-Li is selected from the group consisting of:
In preferred embodiments, MC-Li is selected from the group consisting of: wherein c is an integer from 2 to 10; wherein EWG is an electron withdrawing group; and wherein the denotes the point of attachment to Xc.
In preferred embodiments, MC-Li is selected from the group consisting of:
# wherein c is an integer from 2 to 10; and wherein the denotes the point of attachment to Xc. More preferably, c is 5.
In preferred embodiments, MC-Li is selected from the group consisting of:
# wherein c is an integer from 2 to 10; and wherein the denotes the point of attachment to Xc.
In preferred embodiments, MC-Li is selected from the group consisting of: wherein c is an integer from 2 to 10; R, R’, R”, and R’” are each independently selected from hydrogen, (1-6C)alkoxy(1-6C)alkyl, (1-6C)2N(1-6C)alkyl, and (1 -6C)alkyl, or, two geminal R groups, together with the carbon atom to which they are attached, form a cyclobutyl or cyclopropyl ring; and wherein the denotes the point of attachment to Xc.
In preferred embodiments, MC-Li is selected from the group consisting of: wherein b is an integer from 1 to 10; and wherein the denotes the point of attachment to Xc.
In preferred embodiments, MC-Li is:
More preferably, b is 4 or 5.
In a preferred embodiment, a ready-to-conjugate intermediate compound has a sub-formula 154m - 162m selected from the group consisting of:
154m 155m
160m 161m
In a preferred embodiment, a conjugate has a sub-formula conjugate 154 - conjugate 162
5 selected from the group consisting of: conjugate 156 conjugate 157
conjugate 162 wherein a is an integer from 1 to 8.
Pharmaceutical composition Pharmaceutical compositions in accordance with the present invention comprise, as the active ingredient (‘API’), bifunctional compound of Formula (I) or a pharmaceutically acceptable salt, hydrate or solvate thereof. As used herein, “a pharmaceutically acceptable salt” includes any salt that retains the activity of the active agent(s) and is acceptable for pharmaceutical use. A pharmaceutically acceptable salt also refers to any salt which may form in vivo as a result of administration of an acid, another salt, or a prodrug which is converted into an acid or salt. Preferably, the pharmaceutically acceptable salt is the HCI-salt of the compound of the invention. The pharmaceutically acceptable salt of the disclosed compounds may be prepared by methods of pharmacy well known to those skilled in the art.
Furthermore, the compositions can comprise compounds according to the invention in the form of a solvate, comprising a pharmaceutically acceptable solvent, such as water (‘hydrate’), ethanol, and the like. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the present invention.
As used herein, the term “pharmaceutical composition” refers to a composition comprising a compound according to the invention or a salt or solvate thereof and, as the case may be, one or more additional, non-toxic ingredients, which composition is in a form suitable for administration to a (human) subject, through any route of administration, and which composition is physiologically tolerated upon such administration.
The compositions of the invention may thus comprise one or more additional ingredients. In a preferred embodiment, the composition comprises one or more carriers and/or excipients. As is known by those of average skill in the art, the appropriate choice of excipients is dependent on multiple factors, including the physicochemical properties of the API, the preferred pharmaceutical form, the preferred route of administration, the desired rate of release, etc. The compositions of the invention can be formulated for a variety of routes of administration, oral administration being particularly preferred. It is within the purview of those of average skill in the art to conceive and develop suitable formulations, relying on the common general knowledge as reflected in text books such as Remington’s Pharmaceutical Sciences (Meade Publishing Co., Easton, Pa., 20.sup.th Ed., 2000), the entire disclosure of which is herein incorporated by reference, and routine development efforts.
In accordance with the various aspects of the invention, the composition is preferably provided in a unit dosage form. The term ‘unit dosage form’ refers to a physically discrete unit suitable as a unitary dosage for human subjects, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with any suitable pharmaceutical carrier(s) and/or excipient(s). Exemplary, non-limiting unit dosage forms include a tablet (e.g., a chewable tablet), caplet, capsule (e.g., a hard capsule or a soft capsule), lozenge, film, strip, gelcap as well as any metered volume of a solution, suspension, syrup or elixir or the like, which may be contained, for instance in a vial, syringe, applicator device, sachet, spray, micropump etc. In accordance with particularly preferred embodiments of the invention, the unit dosage form, is a unit dosage form that is suitable for oral administration. Most preferably, it is a solid unit dosage form, such as a tablet. Besides the compound according to the invention as such, pharmaceutically acceptable salts thereof may also be used. Pharmaceutically acceptable salts of compounds of the invention include the acid addition and base salts thereof, such as preferably the calcium, potassium or sodium salts. For a review on suitable salts, reference is made “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).
Pharmaceutically acceptable salts of compounds according to the invention may be readily prepared by mixing together solutions of compounds according to the invention and the desired acid or base, as appropriate. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionisation in the salt may vary from completely ionised to almost non-ionised.
Medical use
The compounds, the conjugates and the pharmaceutical compositions of the present invention are useful as degradation-inducing compounds of Polo-like kinasei (PLK1). In particular, compounds and conjugates of this invention are useful as degraders of Polo-like kinase 1 (PLK1), that is important in hyper-proliferative diseases, especially in cancer and inflammation.
The compounds, the conjugates and the pharmaceutical compositions of the present invention are useful for preventing, delaying or treating diseases through modulation of PLK1
The compounds, the conjugates and the pharmaceutical compositions of the invention is capable of fundamentally degrading the target protein, PLK1 in view of the mechanism of action, thereby achieving an excellent PLK1 inhibitory effect as compared to the conventional PLK1 small molecule inhibitor that inhibits the simple activity of PLK1 .
Accordingly, the pharmaceutical composition including the compound represented by Formula I of the invention or a pharmaceutically acceptable salt thereof may be effectively employed for selective degradation of PLK1 .
An embodiment of the present invention is a composition for preventing or treating diseases through modulation of PLK1 including the compound or the conjugates of the invention. An another embodiment of the invention is a method for the prevention or treatment of diseases through modulation of PLK1 comprising administering the composition to a subject in need thereof.
In the present invention, the disease treated through modulation of PLK1 refers to any disease or condition capable of being treated, alleviated, delayed, inhibited or prevented by the induction of degradation or inhibition of activity of PLK1 . In an embodiment, the disease treated through modulation of PLK1 may be a cancer (malignant tumor), a benign tumor, or other genetic or non-genetic diseases caused by excessive cell division.
The cancer includes all cancers capable of exhibiting prophylactic or therapeutic efficacy due to inhibition or degradation of PLK1 activity, and may be solid cancer or blood cancer. The compounds and conjugates of the present invention are also useful in the treatment of cancer, such as related indications defined by tissue origin such as prostate cancer, breast cancer, lymphoma, acute myelogenous leukemia, lung cancer, and pancreatic cancer.
The bifunctional compounds according to the invention having Formula (I) and the conjugates based on bifunctional moieties derived from these bifunctional compounds having Formula (I) and pharmaceutical compositions thereof can be used to treat or prevent a variety of conditions, diseases or disorders through modulation of Polo-like kinase 1 (PLK1).
The bifunctional compounds according to the invention having Formula (I) and the conjugates based on bifunctional moieties derived from these bifunctional compounds having Formula (I) and pharmaceutical compositions thereof can be used in the treatment of disorders or diseases selected from the group consisting of cancer, benign tumor, a neurological disorder, an inflammatory disease, a disease characterized by inflammasome activation, an autoimmune disease, a hyperproliferative disease, and other genetic or non-genetic diseases caused by excessive cell division, a disorder that is caused by tissue rejection by the immune system.
In further embodiments, the bifunctional compounds according to the invention having Formula (I) and the conjugates based on bifunctional moieties derived from these bifunctional compounds having Formula (I) and pharmaceutical compositions thereof can be used in a therapy to prevent tissue rejection by the immune system.
In exemplary embodiments of therapies to prevent tissue rejection by the immune system include but are not limited to stem cell therapy graft versus host disease, and allogeneic organ transplants.
In preferred embodiments, the neurological disorder is one or more selected from the group consisting of central nervous system disease, neurodegenerative disease, Alzheimer's disease, Parkinson's disease, multiple sclerosis, Huntington's disease, senile dementia, epilepsy, Lou Gehrig, stroke, and nerve damage and axonal degeneration-related disorders following brain or spinal cord injury.
In preferred embodiments, the cancer or benign tumor is one or more selected from the group consisting of squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, skin cancer, skin or intraocular melanoma, rectal cancer, anal muscle cancer, esophageal cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia (including but not limited to acute myelogenous leukemia and complex karyotype acute myelogenous leukemia), lymphocytic lymphoma, double hit lymphoma, hepatocellular carcinoma, gastrointestinal cancer, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumor, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, brain cancer, osteosarcoma, Barrett's esophagus, colon adenoma and polyp, breast fibroadenoma and cyst, monoclonal gammopathy of undetermined significance (MGUS), and monoclonal lymphocytosis.
Routes of administration
Suitable routes of administration may, for example, include oral, eyedrop, rectal, transmucosal, topical, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
Alternatively, one may administer the compound in a local rather than a systemic manner, for example, via injection of the compound directly into an edematous site, often in a depot or sustained release formulation.
Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with endothelial cell-specific antibody.
Composition/Formulation
The pharmaceutical compositions of the present invention may be manufactured in a mannerthat is itself known, e.g., by means of conventional mixing, dissolving, granulating, drageemaking, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
Pharmaceutical preparations for oral use can be obtained by combining the active compound with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The compounds can be formulated for parenteral administration by injection, e.g. bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g. in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly or by intramuscular injection). Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
Synthesis of the compounds
The compounds of the present invention can be prepared by methods well known in the art of organic chemistry. See, for example, J. March, ‘Advanced Organic Chemistry’ 4th Edition, John Wiley and Sons. During synthetic sequences it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This is achieved by means of conventional protecting groups, such as those described in T.W. Greene and P.G.M. Wutts ‘Protective Groups in Organic Synthesis’ 3rd Edition, John Wiley and Sons, 1999. The protective groups are optionally removed at a convenient subsequent stage using methods well known in the art.
The products of the reactions are optionally isolated and purified, if desired, using conventional techniques, including but not limited to, filtration, distillation, crystallization, chromatography and the like. Such materials are optionally characterized using conventional means, including physical constants and spectral data.
Compounds of any one of Formula (I), wherein the Protein Binding group PB, Formula BPKLI , X, R1 to R4, spacer (Sp) and Target Ligase Binding group (TLB) have the previously defined meanings, can be prepared by the general synthetic route shown in scheme I.
Scheme I show a general synthetic route in relation to an exemplary compound of Formula I.
Scheme I
Carboxylic acid containing pteridine IV can be prepared from commercially available substituted 2-chloro-7,8-dihydro-5/7-pteridin-6-ones II using appropriate aminobenzoic acids III in a solvent mixture such as ethanol/water at elevated temperatures. Derivatives of Formula VI can be prepared via amide-coupling with amino-alcohols of Formula V and an appropriate couplings reagent such as HATU or EDCLHCI in a suitable solvent like DMF at appropriate temperature. Finally deprotection of compounds of Formula VI can be accomplished using strong acids like HCI or TFA in the presence of water and a suitable cation scavenger like triisopropylsilane (TIS) at appropriate temperature. Subsequent coupling of deprotected compounds of Formula VI to obtain compounds of Formula l-a can be accomplished by amide-coupling, alkylation or reductive amination using appropriate reagents such as HATU, EDCLHCI, NaBH(OAc)3 or NaBH(t-BuO)3. Subsequent purification using silicagel chromatography or preparative HLPC afforded pure compounds of Formula l-a.
The invention is illustrated by the following examples.
Examples
The following examples are illustrative embodiments of the invention, not limiting the scope of the invention in any way. Reagents are either commercially available or are prepared according to procedures known in the literature. Method LCMS (A)
LC-MS system equipped with a Waters 2998 Photodiode Array Detector, Waters Acquity QDa Detector, Waters 2767 autosampler and Waters 2545 binary gradient module was used for sample analyses with a XTerra ® MS C18 column (2.5 pm, 4.6 x 50 mm) for 10 min measurements.
The eluents used for this system are A (95/5 v/v% Milli-Q water/acetonitrile + 0.1 % formic acid) and B (acetonitrile + 0.1 % formic acid).
Method LCMS (A): 95% A to 95% B in 7 min, then 95% A.
Method LCMS (B)
LC-MS system equipped with a Waters 2998 Photodiode Array Detector, Waters Acquity QDa Detector, Waters 2767 autosampler and Waters 2545 binary gradient module was used for sample analyses with a XTerra ® MS C18 column (2.5 pm, 4.6 x 50 mm) for 30 min measurements.
The eluents used for this system are A (95/5 v/v% Milli-Q water/acetonitrile + 0.1 % formic acid) and B (acetonitrile + 0.1 % formic acid).
Method LCMS (B): 95% A to 95% B in 22 min, then switched to 95% A.
Method Preparative HPLC
LC-MS system equipped with a Waters 2998 Photodiode Array Detector, Waters Acquity QDa Detector, Waters 2767 autosampler and Waters 2545 binary gradient module was used for Preparative reversed phase chromatography with a Luna ® 5 pm C18(2) 100 A (150 x 21 mm).
The eluents used for this system are A (95/5 v/v% Milli-Q water/acetonitrile + 0.1 % formic acid) and B (acetonitrile + 0.1 % formic acid).
The following abbreviations are used throughout the application with respect to chemical terminology:
TFA Trifluoracetic acid
HATU O-(7-Azabenzotriazol-1-yl)-1 ,1 ,3,3-tetramethyluroniumhexafluorophosphate
DMF A/,A/-Dimethylformamide
THF Tetra hydrofuran
DCM Dichloromethane
TMS-CI Chlorotrimethylsilane
DiPEA A/,A/-Diisopropylethylamine
HPLC High Performance Liquid Chromatography
LCMS Liquid Chromatography with Mass Spectrometry detection
4-DMAP 4-Dimethylamino pyridine
Boc te/Y-Butyloxycarbonyl
Cbz Benzyloxycarbonyl
□HMDS Lithium bis(trimethylsilyl)amide
DBU 1 ,8-Diazabicyclo[5.4.0]undec-7-ene
DEAD Diethyl azodicarboxylate o/n Overnight
Pd(dppf)Cl2 1 ,1 '-bis(diphenylphosphino)ferrocene palladium(ll) chloride
AIBN Azobisisobutyronitril
ZrCp2(H)CI zirconocene hydrochloride (Schwartz Reagent)
1H-NMR Proton nuclear magnetic resonance
BOC2O Di-tert-buty I decarbonate
DPPA Diphenylphosphoryl azide
T3P Propylphosphonic anhydride
NIS A/-lodosuccinimide
PyBOP benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate
TBTU 2-(1 H-Benzotriazole-1-yl)-1 ,1 ,3,3-tetramethylaminium tetrafluoroborate
EDCI 1 -Ethyl-3-(3-dimethylaminopropyl)carbodiimide
2MeTHF 2-M ethyltetra hydrofuran
Z-ONSu A/-(Benzyloxycarbonyloxy)succinimide o/w Over the weekend
TCEP T ris(2-carboxyethyl)phosphine
Tris 2-Amino-2-(hydroxymethyl)propane-1 ,3-diol
The names of the final products in the intermediates and examples are generated using Biovia Draw (version 16.1). In cases were Biovia Draw could not generate a name, molecular structures are given.
Intermediate 1 te/Y-Butyl 4-amino-4-(hvdroxymethyl)piperidine-1 -carboxylate
Under N2, a mixture of NaBFU (3.43 g, 90.7 mmol) in anhydrous THF (280 mL) was treated with 4-amino-1-terAbutoxycarbonyl-piperidine-4-carboxylic acid (9.23 g, 37.8 mmol) portion-wise and the reaction mixture was cooled to 0 °C. To this mixture was added a solution of I2 (9.58 g, 37.8 mmol) in anhydrous THF (80 mL) dropwise over 10 min. (gas evolution). After addition of I2 was complete and gas evolution ceased, the reaction mixture was heated to reflux overnight. The reaction mixture was cooled to room temperature and methanol (600 mL) was added cautiously until the mixture became clear. After stirring for 0.5 h, the solvent was removed to afford a white residue, which was taken up in 20% aq KOH (ca. 500 mL) and stirred for 4 h at rt. The reaction was extracted with CH2CI2 (3 x 250 mL) and the combined organic extracts were dried over Na2SO4, filtered and concentrated under reduced pressure to afford the crude title compound (7.92 g, 91 %) as a colorless oil, which was used without further purification.
Intermediate 2
Benzyl 4-amino-4-(2-hvdroxyethyl)piperidine-1 -carboxylate
(a) Benzyl 4-amino-4-(2-ethoxy-2-oxo-ethyl)piperidine-1 -carboxylate
To a solution of benzyl 4-oxopiperidine-1 -carboxylate (2.11 g, 8.89 mmol) in ethanol (18 mL) was added ammonium acetate (1.03 g, 13.34 mmol) and the reaction mixture was stirred at 80°C for 1 h. Subsequently a solution of 3-ethoxy-3-oxo-propanoic acid (1.17 g, 8.89 mmol) in ethanol (3.5 mL) was added drop-wise and the mixture was stirred at 85°C for 3 h, afterwhich the reaction mixture was allowed to cool to room temperature. The mixture was stirred overnight and then concentrated under reduced pressure and the residue was dissolved in ethyl acetate. The ethyl acetate layer was washed with 5% aq. NaHCOs-solution, and extracted with 0.5M HCI- solution. After addition of sat. aq. NaHCOs-solution until a basic solution was obtained, the water layer was extracted with ethyl acetate. The combined ethyl acetate layers were washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to give 1 .63 g. of the title compound (yield: 30%).
(b) Benzyl 4-amino-4-(2-hvdroxyethyl)piperidine-1 -carboxylate (Intermediate 2)
To a cold (4 °C) solution of benzyl 4-amino-4-(2-ethoxy-2-oxo-ethyl)piperidine-1- carboxylate (855 mg, 2.67 mmol) in THF (6.5 mL) was added LiBFL (87 mg, 4 mmol) and the reaction mixture was stirred for 30 at 4°C. The mixture was heated to 70 °C and stirred for an additional 2 h. After cooling the reaction, water (6.5 mL) was added carefully to quench the excess of LiBFk Ethyl acetate and water were added. The organic phase was separated, washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (dichloromethane/methanol = 95/5 to 8/2 v/v%) to give 364 mg of the title compound (yield: 49%). Intermediate 3
Benzyl 3-amino-3-(2-hvdroxyethyl)pyrrolidine-1 -carboxylate (Intermediate 3)
This compound was prepared in an analogues manner as described in Intermediate 2, starting from 1-A/-Cbz-3-pyrrolidinone and 3-ethoxy-3-oxo-propanoic acid to afford the title compound (28 mg, 28%).
Intermediate 4
Benzyl 3-amino-3-(2-hvdroxyethyl)azetidine-1 -carboxylate (Intermediate 4)
This compound was prepared in an analogues manner as described in Intermediate 2, starting from 1-A/-Cbz-3-azetidinone and 3-ethoxy-3-oxo-propanoic acid to afford the title compound (144 mg, 38%). Butyl 6-amino-6-(2-hvdroxyethyl)-2-azaspiro[3.31heptane-2-carboxylate (Intermediate 5)
(a) 2-(6-Amino-2-te/Y-butoxycarbonyl-2-azaspiro[3.31heptan-6-yl)acetic acid
To a stirred solution of fe/Y-butyl 6-oxo-2-azaspiro[3.3]heptane-2-carboxylate (5.0 g, 23.7 mmol) in n-butanol (47 mL) was subsequently added malonic acid (2.7 g, 26.0 mmol) and ammonium acetate (4.19 g, 54.4 mmol). The reaction mixture was stirred at reflux under nitrogen atmosphere for 3 h. After cooling of the reaction mixture, a precipitate formed. The solids were filtered, washed with cold ethyl acetate (10 mL) and dried under high vacuum to give 1.69 g of the title compound as a white fluffy solid (yield: 26%).
(b) tert-Butyl 6-amino-6-(2-hvdroxyethyl)-2-azaspiro[3.31heptane-2-carboxylate (Intermediate 5) This compound was prepared in an analogues manner as described in Intermediate 1 , starting from 2-(6-amino-2-te/Y-butoxycarbonyl-2-azaspiro[3.3]heptan-6-yl)acetic acid to afford the title compound (1 .55 g, 97%). te/Y-Butyl 2-amino-2-(2-hvdroxyethvD-7-azaspiro[3.51nonane-7-carboxylate (Intermediate 6)
(a) 2-(2-Amino-7-te/Y-butoxycarbonyl-7-azaspiro[3.51nonan-2-yl)acetic acid
To a stirred solution of fe/Y-butyl 2-oxo-7-azaspiro[3.5]nonane-7-carboxylate (2.5 g, 10.4 mmol) in n-butanol (21 mL) was subsequently added malonic acid (1.2 g, 1 1.5 mmol) and ammonium acetate (1.84 g, 23.9 mmol). The reaction mixture was stirred at reflux under nitrogen atmosphere for 3 h. After cooling of the reaction mixture, a precipitate formed. The solids were filtered, washed with cold ethyl acetate (5 mL) and dried under high vacuum to give 1.54 g of the title compound as a white powder (yield: 50%).
(b) tert-Butyl 2-amino-2-(2-hvdroxyethyl)-7-azaspiro[3.51nonane-7-carboxylate (Intermediate 6)
This compound was prepared in an analogues manner as described in Intermediate 1 , starting from 2-(2-amino-7-te/Y-butoxycarbonyl-7-azaspiro[3.5]nonan-2-yl)acetic acid to afford the title compound (516 mg, quantitative yield). Butyl 4-amino-3-fluoro-4-(2-hvdroxyethyl)piperidine-1 -carboxylate (Intermediate 7)
This compound was prepared in an analogues manner as described in Intermediate 6, starting from fe/Y-butyl 3-fluoro-4-oxo-piperidine-1 -carboxylate and malonic acid to afford the title compound (1.43 g, 35%). Intermediate 8 tert-Butyl 7-amino-7-(2-hvdroxyethyl)-2-azaspiro[3.51nonane-2-carboxylate (Intermediate 8)
This compound was prepared in an analogues manner as described in Intermediate 6, starting from tert-butyl 7-oxo-2-azaspiro[3.5]nonane-2-carboxylate and malonic acid to afford the title compound (1.1 g, quantitative).
Intermediate 9
2-[1-Amino-4-[[1-(4-bromo-2-fluoro-phenyl)-4-piperidyl1oxy1cvclohexyl1ethanol (Intermediate 9)
This compound was prepared in an analogues manner as described in Intermediate TLB- Sp55, Intermediate 6 and Intermediate 2 starting from 4-bromo-2-fluoro-1 -iodobenzene, 4-(1 ,4- dioxaspiro[4.5]decan-8-yloxy)piperidine and 3-ethoxy-3-oxo-propanoic acid to afford the title compound (815 mg). tert-Butyl 4-amino-3,3-difluoro-4-(2-hvdroxyethyl)piperidine-1 -carboxylate (Intermediate 10)
This compound was prepared in an analogues manner as described in Intermediate 6, starting from tert-butyl 3, 3-difluoro-4-oxopiperidine-1 -carboxylate and malonic acid to afford the title compound (888 mg, 24%).
tert-Butyl 9-amino-9-(2-hvdroxyethyl)-3-azaspiro[5.51undecane-3-carboxylate (Intermediate 11)
This compound was prepared in an analogues manner as described in Intermediate 6, starting from tert-butyl 9-oxo-3-azaspiro[5.5]undecane-3-carboxylate and malonic acid to afford the title compound (350 mg, quantitative).
Intermediate 12 tert-Butyl 6-amino-6-(2-hvdroxyethvD-2-azaspiro[3.41octane-2-carboxylate (Intermediate 12)
This compound was prepared in an analogues manner as described in Intermediate 2, starting from tert-butyl 6-oxo-2-azaspiro[3.4]octane-2-carboxylate and 3-ethoxy-3-oxo-propanoic acid to afford the title compound (396 mg, 82.6%). tert-butyl 4-amino-4-(2-hvdroxyethyl)azepane-1 -carboxylate (Intermediate 13)
This compound was prepared in an analogues manner as described in Intermediate 2, starting from tert-butyl 4-oxoazepane-1 -carboxylate and 3-ethoxy-3-oxo-propanoic acid to afford the title compound (495 mg, 98.7%). Intermediate 14 te/Y-Butyl (3aS, 6af?)-5-amino-5-(2-hvdroxyethvD-1 ,3,3a,4,6,6a-hexahydrocvclopenta[c1pyrrole-2- carboxylate (Intermediate 14)
This compound was prepared in an analogues manner as described in Intermediate 2, starting from cis-fe/Y-butyl 5-oxohexahydrocyclopenta[c]pyrrole-2(1 /7)-carboxylate and 3-ethoxy-3- oxo-propanoic acid to afford the title compound (95 mg, 73.4%).
Intermediate 15 te/Y-Butyl 3-amino-3-(2-hvdroxyethyl)-1-oxa-8-azaspiro[4.51decane-8-carboxylate (I ntermed iate 15)
This compound was prepared in an analogues manner as described in Intermediate 2, starting from te/Y-butyl 3-oxo-1-oxa-8-azaspiro[4.5]decane-8-carboxylate and 3-ethoxy-3-oxo- propanoic acid to afford the title compound (130 mg, 99%).
Intermediate 16 Butyl 8-amino-8-(2-hvdroxyethyl)-3-azabicvclo[3.2.11octane-3-carboxylate (Intermediate 16)
This compound was prepared in an analogues manner as described in Intermediate 6, starting from fe/Y-butyl 8-oxo-3-azabicyclo[3.2.1]octane-3-carboxylate and malonic acid to afford the title compound (939 mg, 99%). Intermediate 17 te/Y-Butyl 7-amino-7-(2-hydroxyethyl)-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (Intermediate 17)
This compound was prepared in an analogues manner as described in Intermediate 6, starting from te/Y-butyl 7-oxo-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate and malonic acid to afford the title compound (62 mg, 52.9%).
Intermediate 18 ediate 18)
This compound was prepared in an analogues manner as described in Intermediate 2, starting from fe/Y-butyl 3-methyl-4-oxo-piperidine-1 -carboxylate and 3-ethoxy-3-oxo-propanoic acid to afford the title compound (1 .67 g, 98.1%). tert-butyl 2-amino-2-i (Intermediate 19)
This compound was prepared in an analogues manner as described in Intermediate 2, starting from fert-butyl 2-oxo-6-azaspiro[3.4]octane-6-carboxylate and 3-ethoxy-3-oxo-propanoic acid to afford the title compound (567 mg, 99.1%). Intermediate 20 tert-Butyl 2-amino-2-(2,2-dideuterio-2-hvdroxy-ethvD-7-azaspiro[3.51nonane-7-carboxylate (Intermediate 20)
This compound was prepared in an analogues manner as described in Intermediate 6, starting from tert-butyl 2-oxo-7-azaspiro[3.5]nonane-7-carboxylate, malonic acid and sodium borodeuteride for reduction in the second step, to afford the title compound (65 mg, 67.7%).
Intermediate 21 tert-B uty I 4-amino-4-(3-hvdroxypropyl)piperidine-1 -carboxylate
(a) tert-Butyl 4-(3-ethoxy-3-oxo-propyl)-4-nitro-piperidine-1 -carboxylate tert-Butyl 4-nitropiperidine-1 -carboxylate (1 g, 4.34 mmol) and potassium carbonate (1.8 g, 13.0 mmol) were suspended in ethanol (12.4 mL) at room temperature. Methyl prop-2-enoate (450 pL, 4.99 mmol) was added to obtain a blue suspension. The reaction mixture was stirred for 3 h under a nitrogen atmosphere. The mixture was concentrated in vacuo and the residue was dissolved in EtOAc/ water (1 :1 v/v%) (50 mL). The bi-phasic mixture was separated, and the aqueous layer was extracted with EtOAc (2x25 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo. The resulting residue was purified by flash column chromatography (heptane/ethyl acetate = 10/0 to 55/45 v/v%) to give 1 .05 g of the title compound (yield: 73.2%).
(b) tert-B uty I 4-(3-hvdroxypropyl)-4-nitro-piperidine-1 -carboxylate
To a cold (4 °C) solution of tert-butyl 4-(3-ethoxy-3-oxo-propyl)-4-nitro-piperidine-1- carboxylate (885 mg, 2.68 mmol) in ethanol (9 mL) was added NaBEL (233 mg, 6.16 mmol) and the reaction mixture was stirred for 3 h at 50 °C. After cooling the reaction, water (6.5 mL) was added carefully to quench the excess of NaBEk Ethyl acetate and water were added. The organic phase was separated, washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (heptane/ethyl acetate = 8/2 to 1/9 v/v%) to give 583 mg of the title compound (yield: 75.5%). (c) tert-Butyl 4-amino-4-(3-hvdroxypropyl)piperidine-1 -carboxylate (Intermediate 21)
To a solution of tert-butyl 4-(3-hydroxypropyl)-4-nitro-piperidine-1-carboxylate (277 mg, 0.96 mmol) in ethanol/water (3/1 , v/v, 20 mL) was added ammonium chloride (540 mg, 10.1 mmol) and zinc (628 mg, 9.61 mmol) and the reaction mixture was stirred at 80°C o/n. After cooling the reaction, the mixture was filtered and concentrated under reduced pressure . Dichloromethane and 5% aq. NaHCOs-solution were added. The organic phase was separated over a PE-filter and concentrated under reduced pressure to give 195 mg of the title compound (yield: 78.6%).
Intermediate 22 rmediate 22)
This compound was prepared in an analogues manner as described in Intermediate 2, starting from te/Y-butyl 3-methoxy-4-oxo-piperidine-1 -carboxylate and 3-ethoxy-3-oxo-propanoic acid to afford the title compound (668 mg, 91 .9%). te/Y-Butyl 9-amino-9-(2-hydroxyethyl)-3-oxa-7-azabicyclo[3.3.1]nonane-7-carboxylate (Intermediate 23)
This compound was prepared in an analogues manner as described in Intermediate 6, starting from te/Y-butyl 9-oxo-3-oxa-7-azabicyclo[3.3.1]nonane-7-carboxylate and malonic acid to afford the title compound (193 mg, quantitative).
Intermediate 24 te/Y-Butyl 7-amino-7-(2-hvdroxyethyl)-5-oxa-2-azaspiro[3.41octane-2-carboxylate (I ntermed iate 24)
This compound was prepared in an analogues manner as described in Intermediate 2, starting from te/Y-butyl 7-oxo-5-oxa-2-azaspiro[3.4]octane-2-carboxylate and 3-ethoxy-3-oxo- propanoic acid to afford the title compound.
Intermediate A
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-3-methoxy-benzoic acid
Intermediate A was prepared according to the procedures described in WO 2006/018182 pp 58.
Intermediate C
4-[[(7/?)-7-ethyl-8-isopropyl-5-methyl-6-oxo-7H-pteridin-2-yl1amino1-3-methoxy-benzoic acid
Intermediate C was prepared according to the procedures described in WO 2006/018182 pp 58.
Intermediate D
4-[[(7/?)-7-ethyl-8-isopropyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-3-(trifluoromethoxy)benzoic acid Intermediate D was prepared according to the procedures described in WO 2006/018182 pp 58.
Intermediate B1
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[4-(hvdroxymethyl)-4- piperidyll-3-methoxy-benzamide (Intermediate B1)
(a) tert-Butyl 4-[[4-[[(7R)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-3-metho- xy-benzoyl1amino1-4-(hvdroxymethyl)piperidine-1 -carboxylate te/Y-Butyl 4-amino-4-(hydroxymethyl)piperidine-1 -carboxylate (456 mg, 1 .98 mmol) and 4- [[(7R)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl]amino]-3-methoxy-benzoic acid (Intermediate A, 768 mg, 1.80 mmol) were suspended in DMF (6.0 mL). HATU (685 mg, 1.8 mmol) and DiPEA (940 pL, 5.4 mmol) were added subsequently and the mixture stirred at room temperature o/n. Ethyl acetate was added and the mixture was washed with aq. 0.1 M HCI-solution. The organic layer was separated and washed with 5% NaHCOs-solution, water and brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (dichloromethane to methanol = 99/1 to 94/6 v/v%) to afford 967 mg of the title compound (yield: 84%).
(b) 4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[4-(hvdroxymethyl)-4- piperidyll-3-methoxy-benzamide (Intermediate B1)
To a cold (0 °C) solution of te/Y-butyl 4-[[4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7H- pteridin-2-yl]amino]-3-methoxy-ben-zoyl]amino]-4-(hydroxymethyl)piperidine-1 -carboxylate (961 .4 mg, 1.51 mmol) in DCM (10 mL) was added iodotrimethylsilane (691 pL, 4.83 mmol) and the reaction mixture was stirred for 3 minutes at 0 °C. The mixture was quenched with methanol (2.0 mL) and allowed come to room temperature. The mixture was diluted with DCM/MeOH (9:1 v/v%) (50 mL), and aq. 5% NaHCOs solution (8 mL) was added dropwise to the mixture. The mixture was separated and the organic layer filtered over a PE-filter and concentrated under reduced pressure to obtain 4-[[(7R)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7H-pteridin-2-yl]amino]-A/-[4- (hydroxymethyl)-4-piperidyl]-3-methoxy-benzamide (Intermediate B1) (834 mg, quantitative yield). Intermediate B2
4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yllaminol-/\/-[4-(2-hydroxyethyl)-4-
(Intermediate B2)
This compound was prepared in an analogous manner as described in Intermediate B1 , starting from te/Y-butyl 4-amino-4-(hydroxyethyl)piperidine-1 -carboxylate and 4-[[(7R)-8- cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl]amino]-3-methoxy-benzoic acid
(Intermediate A) to afford the title compound (81 mg, 92%).
4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yllaminol-A/-[3-(2- lidin-3-yll-3-i -benzamide (Intermediate B3)
This compound was prepared in an analogous manner as described in Intermediate B1 , starting from benzyl 3-amino-3-(2-hydroxyethyl)pyrrolidine-1 -carboxylate (Intermediate 3) and 4- [[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl]amino]-3-methoxy-benzoic acid (Intermediate A) to afford the title compound after catalytic hydrogenation of the Cbz-protected intermediate compound using 10% Pd/C and ammonium formate as described for Intermediate TLB-Sp23-d (47 mg, 99%). Intermediate B4
4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yllaminol-/\/-[3-(2-
:idin-3-yll-3-i -benzamide: (Intermediate B4)
This compound was prepared in an analogous manner as described in Intermediate B1 , starting from benzyl 3-amino-3-(2-hydroxyethyl)pyrrolidine-1 -carboxylate (Intermediate 3) and 4- [[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl]amino]-3-methoxy-benzoic acid (Intermediate A) to afford the title compound after catalytic hydrogenation of the Cbz-protected intermediate compound using 10% Pd/C, hydrogen and aq. HCI-solution (1 eq.) as described for Intermediate TLB-Sp1-b (91 mg, quantitative).
Intermediate B5
4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yllaminol-A/-[6-(2-hydroxyethyl)-2- i-6-yll-3-i -benzamide (Intermediate B5)
This compound was prepared in an analogous manner as described in Intermediate B1 , starting from fe/Y-butyl 6-amino-6-(2-hydroxyethyl)-2-azaspiro[3.3]heptane-2-carboxylate (Intermediate 5) and 4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl]amino]-3- methoxy-benzoic acid (Intermediate A) to afford the title compound (480 mg, quantitative). Intermediate B6
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-(2-hvdroxyethyl)-7- azaspiro[3.51nonan-2-yl1-3-methoxy-benzamide (Intermediate B6) This compound was prepared in an analogous manner as described in Intermediate B1 , starting from tert-butyl 2-amino-2-(2-hydroxyethyl)-7-azaspiro[3.5]nonane-7-carboxylate (Intermediate 6) and 4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl]amino]-3- methoxy-benzoic acid (Intermediate A) to afford the title compound (1 .03 g, quantitative yield). Intermediate B7
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[3-fluoro-4-(2- hvdroxyethyl)-4-piperidyl1-3-methoxy-benzamide (Intermediate B7)
This compound was prepared in an analogous manner as described in Intermediate B1 , starting from tert-butyl 4-amino-3-fluoro-4-(2-hydroxyethyl)piperidine-1 -carboxylate (Intermediate 7) and 4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl]amino]-3-methoxy- benzoic acid (Intermediate A) to afford the title compound (540 mg, 95%). Intermediate B8
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[7-(2-hvdroxyethyl)-2- azaspiro[3.51nonan-7-yl1-3-methoxy-benzamide (Intermediate B8)
This compound was prepared in an analogous manner as described in Intermediate B1 , starting from tert-butyl 7-amino-7-(2-hydroxyethyl)-2-azaspiro[3.5]nonane-2-carboxylate (Intermediate 8) and 4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl]amino]-3- methoxy-benzoic acid (Intermediate A) to afford the title compound (1 .72 g, 64.3%).
Intermediate B9
Cis- and trans-A/-[4-[[1-(4-bromo-2-fluoro-phenyl)-4-piperidyl1oxy1-1-(2-hvdroxyethyl)cvclohexyl1- 4-[[(7f?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7H-pteridin-2-yl1amino1-3-methoxy-benzamide (Intermediate B9a and B9b)
This compound was prepared in an analogous manner as described in Intermediate B1 , starting from 2-[1 -amino-4-[[1 -(4-bromo-2-fluoro-phenyl)-4-piperidyl]oxy]cyclohexyl]ethanol (Intermediate 9) and 4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl]amino]-3- methoxy-benzoic acid (Intermediate A) to afford the title compounds (Intermediate B9a, 302 mg, 21.8%) and (Intermediate B9b, 317 mg, 22.9%).
Intermediate B10
4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yllaminol-A/-[3,3-difluoro-4-(2-
-benzamide (Intermediate B10)
This compound was prepared in an analogous manner as described in Intermediate B1 , starting from tert-butyl 4-amino-3,3-difluoro-4-(2-hydroxyethyl)piperidine-1 -carboxylate (Intermediate 10) and 4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl]amino]-3- methoxy-benzoic acid (Intermediate A) to afford the title compound (90 mg, quantitative).
Intermediate B11
4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yllaminol-A/-[9-(2-hydroxyethyl)-3- i-9-yll-3-i -benzamide (Intermediate B11)
This compound was prepared in an analogous manner as described in Intermediate B1 , starting from tert-butyl 9-amino-9-(2-hydroxyethyl)-3-azaspiro[5.5]undecane-3-carboxylate (Intermediate 11) and 4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl]amino]-3- methoxy-benzoic acid (Intermediate A) to afford the title compound (680 mg). Intermediate B12
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[6-(2-hvdroxyethyl)-2- azaspiro[3.41octan-6-yl1-3-methoxy-benzamide (Intermediate B12) This compound was prepared in an analogous manner as described in Intermediate B1 , starting from tert-butyl 6-amino-6-(2-hydroxyethyl)-2-azaspiro[3.4]octane-2-carboxylate (Intermediate 12) and 4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl]amino]-3- methoxy-benzoic acid (Intermediate A) to afford the title compound (455 mg, 47.7%). Intermediate B13
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[4-(2- hvdroxyethyl)azepan-4-yl1-3-methoxy-benzamide (Intermediate B13)
This compound was prepared in an analogous manner as described in Intermediate B1 , starting from fert-butyl 6-amino-6-(2-hydroxyethyl)-2-azaspiro[3.4]octane-2-carboxylate (Intermediate 13) and 4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl]amino]-3- methoxy-benzoic acid (Intermediate A) to afford the title compound (180 mg, 95.4%). Intermediate B14
-2,3,3a,4,6,6a-hexahydro-1 H-< l-5-yll-4-[[(7/?)-8- oxo-7/7-pteridin-2-yllaminol-3-i -benzamide (Intermediate
This compound was prepared in an analogous manner as described in Intermediate B1 , starting from te/Y-butyl (3aS, 6a/?)-5-amino-5-(2-hydroxyethyl)-1 ,3, 3a, 4,6, 6a-hexahydro- cyclopenta[c]pyrrole-2-carboxylate (Intermediate 14) and 4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl- 6-oxo-7/7-pteridin-2-yl]amino]-3-methoxy-benzoic acid (Intermediate A) to afford the title compound (32 mg, 51.9%).
Intermediate B15
4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yllaminol-A/-[3-(2-l i-1- -benzamide (Intermediate B15)
This compound was prepared in an analogous manner as described in Intermediate B1 , starting from fe/Y-butyl 3-amino-3-(2-hydroxyethyl)-1-oxa-8-azaspiro[4.5]decane-8-carboxy-late (Intermediate 15) and 4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl]amino]-3- methoxy-benzoic acid (Intermediate A) to afford the title compound (180 mg, 73.0%). Intermediate B16
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[8-(2-hvdroxyethyl)-3- azabicyclo[3.2.11octan-8-yl1-3-methoxy-benzamide (Intermediate B16) This compound was prepared in an analogous manner as described in Intermediate B1 , starting from te/Y-butyl 8-amino-8-(2-hydroxyethyl)-3-azabicyclo[3.2.1]octane-3-carboxylate (Intermediate 16) and 4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl]amino]-3- methoxy-benzoic acid (Intermediate A) to afford the title compound (374 mg, 98.0%). Intermediate B17
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[7-(2-hvdroxyethyl)-3- oxa-9-azabicvclo[3.3.11nonan-7-yl1-3-methoxy-benzamide (Intermediate B17)
This compound was prepared in an analogous manner as described in Intermediate B1 , starting from fe/Y-butyl 7-amino-7-(2-hydroxyethyl)-3-oxa-9-azabicyclo[3.3.1]nonane-9- carboxylate (Intermediate 17) and 4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2- yl]amino]-3-methoxy-benzoic acid (Intermediate A) to afford the title compound (83 mg, 83.9%). Intermediate B18
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[4-(2-hvdroxyethyl)-3- methyl-4-piperidyl1-3-methoxy-benzamide (Intermediate B18) This compound was prepared in an analogous manner as described in Intermediate B1 , starting from te/Y-butyl 4-amino-4-(2-hydroxyethyl)-3-methyl-piperidine-1 -carboxylate (Intermediate 18) and 4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl]amino]-3- methoxy-benzoic acid (Intermediate A) to afford the title compound (248 mg, 83.4%). Intermediate B19
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-(2-hvdroxyethyl)-6- azaspiro[3.41octan-2-yl1-3-methoxy-benzamide (Intermediate B19)
This compound was prepared in an analogous manner as described in Intermediate B1 , starting from fe/Y-butyl 2-amino-2-(2-hydroxyethyl)-6-azaspiro[3.4]octane-6-carboxylate (Intermediate 19) and 4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl]amino]-3- methoxy-benzoic acid (Intermediate A) to afford the title compound (230 mg, quantitative). Intermediate B20
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-(2,2-dideuterio-2- hvdroxy-ethyl)-7-azaspiro[3.51nonan-2-yl1-3-methoxy-benzamide (Intermediate B20) This compound was prepared in an analogous manner as described in Intermediate B1 , starting from te/Y-butyl 2-amino-2-(2,2-dideuterio-2-hydroxy-ethyl)-7-azaspiro[3.5]nonane-7- carboxylate (Intermediate 20) and 4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2- yl]amino]-3-methoxy-benzoic acid (Intermediate A) to afford the title compound (103 mg, quantitative).
Intermediate B21
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[4-(3-hvdroxypropyl)-4- piperidyll-3-methoxy-benzamide (Intermediate B21) This compound was prepared in an analogous manner as described in Intermediate B1 , starting from fe/Y-butyl 4-amino-4-(3-hydroxypropyl)piperidine-1 -carboxylate (Intermediate 21) and 4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl]amino]-3-methoxy-benzoic acid (Intermediate A) to afford the title compound (33 mg, 62.6%). Intermediate B22
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[4-(2-hvdroxyethyl)-3- methoxy -4-piperidyl1-3-methoxy-benzamide (Intermediate B22) This compound was prepared in an analogous manner as described in Intermediate B1 , starting from tert-butyl 4-amino-4-(2-hydroxyethyl)-3-methoxy-piperidine-1 -carboxylate (Intermediate 22) and 4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl]amino]-3- methoxy-benzoic acid (Intermediate A) to afford the title compound (183 mg, 71.5%). Intermediate B23
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[9-(2-hvdroxyethyl)-3- oxa-7-azabicvclo[3.3.11nonan-9-yl1-3-methoxy-benzamide (Intermediate B23)
This compound was prepared in an analogous manner as described in Intermediate B1 , starting from tert-butyl 9-amino-9-(2-hydroxyethyl)-3-oxa-7-azabicyclo[3.3.1]nonane-7- carboxylate (Intermediate 23) and 4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2- yl]amino]-3-methoxy-benzoic acid (Intermediate A) to afford the title compound (124 mg, 65.0%). Intermediate B24
4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yllaminol-/\/-[7-(2-hydroxyethyl)-5- oxa-2-; I-7-VI1-3-I -benzamide (Intermediate B24)
This compound was prepared in an analogous manner as described in Intermediate B1 , starting from te/Y-butyl 7-amino-7-(2-hydroxyethyl)-5-oxa-2-azaspiro[3.4]octane-2-carboxylate (Intermediate 24) and 4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl]amino]-3- methoxy-benzoic acid (Intermediate A) to afford the title compound.
4-[[(7R)-7-ethyl-8-isopropyl-5-methyl-6-oxo-7H-pteridin-2-yllaminol-N-[4-(2-hydroxyethyl)-4-
-benzamide (Intermediate C1)
This compound was prepared in an analogous manner as described in Intermediate B1 , starting from benzyl 4-amino-4-(2-hydroxyethyl)piperidine-1 -carboxylate (Intermediate 2) and 4- [[(7R)-7-ethyl-8-isopropyl-5-methyl-6-oxo-7/7-pteridin-2-yl]amino]-3-methoxy-benzoic acid (Intermediate C) to afford the title compound after catalytic hydrogenation of the Cbz-protected intermediate compound using 10% Pd/C and ammonium formate as described for Intermediate TLB-Sp23-d (98 mg, 100%). Intermediate C2
4-[[(7/?)-7-ethyl-8-isopropyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[7-(2-hvdroxyethyl)-2- azaspiro[3.51nonan-7-yl1-3-methoxy-benzamide (Intermediate C2) This compound was prepared in an analogous manner as described in Intermediate B1 , starting from tert-butyl 2-amino-2-(2-hydroxyethyl)-7-azaspiro[3.5]nonane-7-carboxylate (Intermediate 8) and 4-[[(7/?)-7-ethyl-8-isopropyl-5-methyl-6-oxo-7H-pteridin-2-yl]amino]-3- methoxy-benzoic acid (Intermediate C) to afford the title compound (591 mg, quantitative). Intermediate C3
4-[[(7/?)-7-ethyl-8-isopropyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-(2-hvdroxyethyl)-7- azaspiro[3.51nonan-2-yl1-3-methoxy-benzamide (Intermediate C3)
This compound was prepared in an analogous manner as described in Intermediate B1 , starting from tert-butyl 7-amino-7-(2-hydroxyethyl)-2-azaspiro[3.5]nonane-2-carboxylate (Intermediate 6) and 4-[[(7/?)-7-ethyl-8-isopropyl-5-methyl-6-oxo-7H-pteridin-2-yl]amino]-3- methoxy-benzoic acid (Intermediate C) to afford the title compound (128 mg, quantitative). Intermediate D1
4-[[(7R)-7-ethyl-8-isopropyl-5-methyl-6-oxo-7H-pteridin-2-yl1amino1-N-[2-(2-hvdroxyethyl)-7- azaspiro[3.51nonan-2-yl1-3-(trifluoromethoxy') benzamide (Intermediate D1) This compound was prepared in an analogous manner as described in Intermediate B1 , starting from tert-butyl 7-amino-7-(2-hydroxyethyl)-2-azaspiro[3.5]nonane-2-carboxylate (Intermediate 6) and 4-[[(7R)-7-ethyl-8-isopropyl-5-methyl-6-oxo-7/7-pteridin-2-yl]amino]-3- (trifluoromethoxy)benzoic acid (Intermediate D) to afford the title compound (128 mg, quantitative).
Intermediate TLB1
(2,3,4,5,6-Pentafluorophenyl) 3-(2,4-dioxohexahvdropyrimidin-1-yl)-4-methoxy-benzoate (Intermediate TLB1)
(a) 3-(2,4-Dioxohexahvdropyrimidin-1 -yl)-4-methoxy-benzoic acid
3-Amino-4-methoxybenzoic acid (5 g, 29.9 mmol) was suspended in acrylic acid (8.05 mL, 117 mmol). The resulting suspension was stirred at 100 °C. After 10 min, a thick slurry/solid appeared, which was heated at 100 °C for another 3 h. After cooling, acetic acid (33 mL) was added and the suspension was heated to 100 °C and stirred for 10 min. Then, urea (11 g, 183 mmol) was added and the resulting mixture was stirred at 120 °C o/n. The resulting brown solution was then quenched into a ice-cold solution of water (150 mL) and concentrated HCI (10 mL). After stirring, the resulting beige suspension was stored o/n in the fridge at 5 °C, and then filtered. The residue was washed with water and dried to afford a brown solid. The solid was taken up in a 0.05M aq. HCI-solution and filtered. The residue was washed with TBME (3x25 mL) and dried at 40 °C under reduced pressure to afford 6.05 g of the title compound as a beige solid (Yield: 76.6%).
(b) (2,3,4,5,6-Pentafluorophenyl) 3-(2,4-dioxohexahvdropyrimidin-1-vD-4-methoxy-benzoate (Intermediate TLB1)
To a cold (0 °C) suspension of 3-(2,4-dioxohexahydropyrimidin-1-yl)-4-methoxy-benzoic acid (2 g, 7.57 mmol) and pentafluorophenyl 2,2,2-trifluoroacetate (2.54 g, 9.08 mmol) was added dropwise DI PEA (5 mL, 30.2 mmol) and the reaction mixture was stirred o/n allowing the temperature to come to room temperature. The mixture was diluted with water (50 mL). Then the mixture was extracted with ethyl acetate (2 x 50 mL). The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to give 2.93 g of the title compound (yield: 89.9%). Intermediate TLB2
(2,3,4,5,6-Pentafluorophenyl) 3-(2,4-dioxohexahvdropyrimidin-1-yl)-4-methyl-benzoate
(Intermediate TLB2)
This compound was prepared in an analogous manner as described in Intermediate TLB1 , starting from 3-amino-4-methyl-benzoic acid and acrylic acid to afford the title compound (5.09 g, 79%).
Intermediate DL1
H— Cl H-CI tert-Butyl 3-piperazin-1-ylpropanoate dihydrochloride (I ntermed iate Sp1 )
(a) Benzyl 4-(3-tert-butoxy-3-oxo-propyl)piperazine-1 -carboxylate
To a solution of benzyl piperazine-1 -carboxylate (5 mL, 25.92 mmol) in ethanolabs (15 mL) was added tert-butyl acrylate (5 mL, 45.84 mmol) and the reaction mixture was stirred at 100 °C for 5 h. Diethylether (35 mL) water (15 mL) and 1 M HCI-solution (35 mL) were added subsequently to the mixture and the water layer was separated. Ethyl acetate (150 mL) and 1 M NaOH-solution (35 mL) were added to the water layer, the organic phase was separated, dried over sodium sulfate, filtered and concentrated under reduced pressure to afford an oil. Yield: 8.25 g (91.3%, crude)
(b) tert-Butyl 3-piperazin-1-ylpropanoate dihydrochloride (Intermediate Sp1)
To a solution of benzyl 4-(3-tert-butoxy-3-oxo-propyl)piperazine-1-carboxylate (8.1 g, 23.25 mmol) in methanol (150 mL) was added 4M HCI-solution (11.62 mL, 46.5 mmol) and a suspension of 10% Pd on charcoal (800 mg) in ethanol (5 mL). Catalytic hydrogenation was perfomed for 2 h at room temperature. The palladium-catalyst was removed by filtration and the filtrate was concentrated under reduced pressure to give of the title compound in quantitative crude yield. Intermediate DL2 Butyl 2-[(3F?)-pyrrolidin-3-yl1oxyacetate (Intermed iate S p2)
(a) Benzyl (3F?)-3-(2-te/Y-butoxy-2-oxo-ethoxy)pyrrolidine-1 -carboxylate
To a cold (0 °C) solution of benzyl (3R)-3-hydroxypyrrolidine-1-carboxylate (221 mg, 1 mmol) in THF (9 mL) was added sodium hydride (60% dispersion in mineral oil, 60 mg, 1 .5 mmol). The mixture was stirred at 0 °C for 1 h, then a solution of fert-butyl bromoacetate (390 mg, 2 mmol) in THF (1 mL) and the reaction mixture is stirred for 3 h allowing the temperature to reach room temperature. Saturated ammoniumchloride solution and ethyl acetate were added. The ethyl acetate layers was separated, washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (heptane/ethyl acetate = 10/0 to 0/10 v/v%) to give 169 mg of the title compound (yield: 50%).
(b) tert-Butyl 2-[(3F?)-pyrrolidin-3-yl1oxyacetate (Intermediate Sp2)
To a solution of benzyl (3R)-3-(2-te/Y-butoxy-2-oxo-ethoxy)pyrrolidine-1-carboxylate (219 mg, 0.65 mmol) in ethanol (30 mL) was added 10% Pd on charcoal (35 mg). Catalytic hydrogenation was perfomed for 2 h at room temperature. The palladium-catalyst was removed by filtration and the filtrate was concentrated in vacuo to give of the title compound in quantitative crude yield.
Intermediate T
3-[2-[2-[2-[[2-(2,6-Dioxo-3-piperidyl)-1 ,3-dioxo-isoindolin-4- yllaminolethoxylethoxylethoxylpropanoic acid (Intermediate TLB-Sp1)
(a) tert-Butyl 3-[2-[2-[2-[[2-(2,6-dioxo-3-piperidyl)-1 ,3-dioxo-isoindolin-4-yl1amino1ethoxy1ethoxy1- ethoxylpropanoate
A solution of 2-(2,6-dioxo-piperidin-3-yl)-4-fluoroisoindoline-1 ,3-dione (200 mg, 0.72 mmol), fe/Y-butyl 12-amino-4,7,10-trioxadodecanoate (241 mg, 0.87 mmol) and DiPEA (480 pL, 2.90 mmol) in DMF (5 mL) was stirred at 80 °C o/n. The mixture was concentrated under vacuum at 60 °C. Purification was performed using preparative LCMS. Fractions containing the title compound were collected and lyophylised to afford 116.0 mg (30.0%) as a yellow oil. (b) 3-[2-[2-[2-[[2-(2,6-Dioxo-3-piperidyl)-1 ,3-dioxo-isoindolin-4- yllaminolethoxylethoxylethoxylpropanoic acid (Intermediate TLB-Sp1)
To a solution of te/Y-butyl 3-[2-[2-[2-[[2-(2,6-dioxo-3-piperidyl)-1 ,3-dioxo-isoindolin-4- yl]amino]-ethoxy]ethoxy]ethoxy]propanoate (116 mg, 0.22 mmol) in dichloromethane (2 mL) was added trifluoroacetic acid (2 mL). The resulting solution was stirred at room temperatur for 1 h and then concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (dichloromethane/methanol = 10/0 to 8/2 v/v%) to give 98.4 mg of the title compound (yield: 94.8%).
3-[2-[2-[[2-(2,6-Dioxo-3-piperidyl)-1 ,3-dioxo-isoindolin-4-yl1amino1ethoxy1ethoxy1propanoic acid (Intermediate TLB-Sp2)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp1 , starting from 2-(2,6-dioxo-piperidin-3-yl)-4-fluoroisoindoline-1 ,3-dione and H2N-PEG2-CO- OtBu to afford the title compound (86.7 mg, 92.3%).
Intermediate TLB-Sp3 yllaminolethoxylethoxylethoxylethoxyl-propanoic acid (Intermediate TLB-Sp3)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp1 , starting from 2-(2,6-dioxo-piperidin-3-yl)-4-fluoroisoindoline-1 ,3-dione and terf-buty 1 3-amino- (PEG4)propionate to afford the title compound (110.3 mg, 88.6%). Intermediate TLB-Sp4
3-[2-[2-[2-[2-[2-[2-[[2-(2,6-dioxo-3-piperidyl)-1 ,3-dioxo-isoindolin-4- yllaminolethoxylethoxylethoxyl-ethoxylethoxylethoxylpropanoic acid (Intermediate TLB-Sp4)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp1 , starting from 2-(2,6-dioxo-piperidin-3-yl)-4-fluoroisoindoline-1 ,3-dione and H2N-PEG6-CO- OtBu to afford the title compound (125.3 mg, quant.).
Intermediate TLB-Sp5
3-[4-[2-(2,6-Dioxo-3-piperidyl)-1 , 3-dioxo-isoindolin-5-yl1 piperazin-1 -yllpropanoic acid (Intermediate TLB-Sp5)
(a) te/Y-Butyl 3-[4-[2-(2,6-dioxo-3-piperidyl)-1 , 3-dioxo-isoindolin-5-yl1 piperazin-1 -yllpropanoate
A solution of 2-(2,6-dioxo-piperidin-3-yl)-5-fluoroisoindoline-1 ,3-dione (750 mg, 2.72 mmol), te/Y-butyl 3-piperazin-1-ylpropanoate dihydrochloride (764 mg, 2.66 mmol) and DiPEA (1 .35 mL, 8.15 mmol) in DMF (3 mL) was stirred at 80 °C o/n. The mixture was concentrated under reduced pressure at 60 °C. The residue was purified by column chromatography (dichloromethane/methanol = 99/1 to 97/3 v/v%) to afford tert-buty I 3-[4-[2-(2,6-dioxo-3-piperidyl)- 1 ,3-dioxo-isoindolin-5-yl]piperazin-1-yl]propanoate (601.9 mg, 48.1 %).
(b) 3-[4-[2-(2,6-Dioxo-3-piperidyl)-1 ,3-dioxo-isoindolin-5-yl1pi perazin-1 -yllpropanoic acid (Intermediate TLB-Sp5)
To a solution of terf-butyl 3-[4-[2-(2,6-dioxo-3-piperidyl)-1 ,3-dioxo-isoindolin-5- yl]piperazin-1-yl]propanoate (255.6 mg, 0.54 mmol) in dichloromethane (2 mL) was added trifluoroacetic acid (2 mL) and the mixture was stirred for 2 h at room temperature. The mixture was concentrated in vacuo. The residue was triturated with dichloromethane and dried in vacuo to give 246 mg of the title compound (Yield: quant). Intermediate TLB-Sp6 o-3-piperidyl)-1 ,3-dioxo-isoindolin-5-yl1pyrrolidine-3-carbaldehvde -Sp6) piperidyl)-5-[(3/?)-3-(hvdroxymethyl)pyrrolidin-1-yl1isoin-doline-1 ,3-dione
A solution of 2-(2,6-dioxo-piperidin-3-yl)-5-fluoroisoindoline-1 ,3-dione (750 mg, 2.72 mmol), (R)-pyrrolidin-3-ylmethanol (269 mg, 2.66 mmol) and DiPEA (1 .35 mL, 8.15 mmol) in DMF (3 mL) was stirred at 80 °C o/n. The mixture was concentrated under reduced pressure at 60 °C. The residue was purified by column chromatography (dichloromethane/methanol = 99/1 to 96/4 v/v%) to afford 2-(2,6-dioxo-3-piperidyl)-5-[(3/?)-3-(hydroxymethyl)pyrrolidin-1-yl]isoin-doline-1 ,3- dione (651.9 mg, 63.5%).
(b) (3/?)-1-[2-(2,6-Dioxo-3-piperidyl)-1 ,3-dioxo-isoindolin-5-yl1pyrrolidine-3-carbaldehvde (Intermediate TLB-Sp6
Des-Martin periodinane (1.14 g, 2.69 mmol) was added to a solution of 2-(2,6-dioxo-3- piperidyl)-5-[(3/?)-3-(hydroxymethyl)pyrrolidin-1-yl]isoin-doline-1 ,3-dione in dichloromethane (480 mg, 1.34 mmol). The mixture was allowed to stir at room temperature for one hour. Dichloromethane and aqueous Na2SOs were subsequently added. The organic layer was dried over sodium sulfate, filtered, concentrated, and purified by column chromatography (ethyl acetate) to afford (3R)-1-[2-(2,6-dioxo-3-piperidyl)-1 ,3-dioxo-isoindolin-5-yl]pyrrolidine-3-carbaldehyde
3-[4-[3-(2,4-Dioxohexahvdropyrimidin-1-yl)-4-methoxy-benzoyl1piperazin-1-yl1propanoic acid (Intermediate TLB-Sp7)
(a) tert-Butyl 3-[4-[3-(2,4-dioxohexahvdropyrimidin-1-yl)-4-methoxy-benzoyl1piperazin-1- yllpropanoate
To a solution of (2,3,4,5,6-pentafluorophenyl) 3-(2,4-dioxohexahydropyrimidin-1-yl)-4- methoxy-benzoate (Intermediate TLB1 , 250 mg, 0.58 mmol) in DMF (3 mL) was added subsequently A/,A/-diisopropylethylamine (288 pL, 1 .74 mmol) and te/Y-butyl 3-piperazin-1- ylpropanoate (131 mg, 0.61 mmol). The reaction mixture was stirred at room temperature o/n. The reaction mixture was concentrated in vacuo and the residue was purified by column chromatography using SiO2 and dichloromethane/methanol = 99/1 to 95/5 v/v%. All fractions containing the title compound were collected and concentrated in vacuo to give 242 mg of the title compound (Yield 90.5%).
(b) 3-[4-[3-(2,4-Dioxohexahvdropyrimidin-1-yl)-4-methoxy-benzoyl1piperazin-1-yl1propanoic acid (Intermediate TLB-Sp7)
To a solution of te/Y-butyl 3-[4-[3-(2,4-dioxohexahydropyrimidin-1-yl)-4-methoxy- benzoyl]piperazin-1-yl]propanoate (151 mg, 0.33 mmol) in dioxane (1 mL) was added 4M HCI/dioxane solution (3 mL) and the reaction mixture was stirred at room temperature for 3 h. The mixture was concentrated in vacuo. The residue was triturated with dichloromethane and dried in vacuo to give the title compound in quantitative crude yield.
Intermediate TLB-Sp8
3-[2-[[3-(2,4-Dioxohexahydropyrimidin-1-yl)-4-methoxy-benzoyl]amino]ethoxy]propanoic acid
(Intermediate TLB-Sp8)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp7, starting from (2,3,4,5,6-pentafluorophenyl) 3-(2,4-dioxohexahydropyrimidin-1-yl)-4-methoxy- benzoate (Intermediate TLB1) and te/Y-butyl 3-(2-aminoethoxy)propanoate to afford the title compound (107 mg, 79%).
Intermediate TLB-Sp9 iidin-1-yl)-4-methyl-benzoyllami noic acid
This compound was prepared in an analogous manner as described in Intermediate TLB-
Sp7, starting from (2,3,4,5,6-pentafluorophenyl) 3-(2,4-dioxohexahydropyrimidin-1-yl)-4-methyl- benzoate (Intermediate TLB2) and fert-butyl 3-(2-ammoethoxy)propanoate to afford the title compound (93 mg, 85%).
Intermediate TLB-Sp10
3-[2-[2-[[3-(2,4-Dioxohexahvdropyrimidin-1-yl)-4-methoxy- benzoyllaminolethoxylethoxylpropanoic acid (Intermediate TLB-Sp10)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp7, starting from (2,3,4,5,6-pentafluorophenyl) 3-(2,4-dioxohexahydropyrimidin-1-yl)-4-methoxy- benzoate (Intermediate TLB1) and fert-butyl 3-[2-(2-aminoethoxy)ethoxy]propanoate to afford the title compound (1 g, 70%).
Intermediate TLB-Sp11
3-[2-[2-[[3-(2,4-Dioxohexahvdropyrimidin-1-yl)-4-methyl-benzoyl1amino1ethoxy1ethoxy1propanoic acid (Intermediate TLB-Sp11)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp7, starting from (2,3,4,5,6-pentafluorophenyl) 3-(2,4-dioxohexahydropyrimidin-1-yl)-4-methyl- benzoate (Intermediate TLB2) and tert-butyl 3-[2-(2-aminoethoxy)ethoxy]propanoate to afford the title compound (258 mg, 63%).
Intermediate TLB-Sp12 2-[(3F?)-1-[2-(2,6-Dioxo-3-piperidyl)-1 ,3-dioxo-isoindolin-5-yl1pyrrolidin-3-yl1oxyacetic acid
(Intermediate TLB-Sp12)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp5, starting from tert-butyl 2-[(3R)-pyrrolidin-3-yl]oxyacetate_(lntermediate Sp2) and 2-(2,6- dioxopiperidin-3-yl)-5-fluoroisoindoline-1 ,3-dione to afford 125 mg of the title compound.
Intermediate TLB-Sp13
7-[2-(2,6-Dioxo-3-piperidyl)-1-oxo-isoindolin-5-yl1hept-6-ynoic acid (Intermediate TLB-Sp13)
(a) 3-(5-Bromo-1-oxo-isoindolin-2-yl) pi peridine-2, 6-dione
A/,A/-diisopropylethylamine (12.5 mL, 75.63 mmol) was added to methyl 4-bromo-2- (bromomethyl)benzoate (7.32 g, 23.77 mmol) and 3-aminopiperidine-2, 6-dione hydrochloride (5.87 g, 35.66 mmol) in acetonitrile (100 mL). The resulting suspension was stirred at 80 °C for 48 hours. The reaction mixture was cooled to room temperature and filtered. The solid was washed with acetonitrile (30 mL), acetonitri le:diethy I ether (50 mL [2:3]) and diethylether (2x50 mL) to afford 3-(5-bromo-1-oxo-isoindolin-2-yl)piperidine-2, 6-dione (7.38 g, 95.8%) as a dark purple solid.
(b) tert-Butyl hept-6-ynoate
To a cold (0 °C) solution of 6-heptynoic acid (3.03 g, 24 mmol) in THF (48 mL) was added trifluoroacetic acid anhydride (6.68 mL, 48 mmol) and the reaction mixture was stirred for 1 h at room temperature. tert-BuOH (24 mL) was added and the mixture was stirred overnight. The reaction mixture was quenched with 5% NaHCOs-solution (200 mL) and extracted with heptane:ethyl acetate=1 :1 (3x50 mL). The combined organic layers were washed with sat. NaHCOs-solution (50 mL), dried over sodium sulfate, filtered and concentrated in vacuo to give the title compound in quantitative crude yield.
(c) tert-Butyl 7-[2-(2,6-dioxo-3-piperidyl)-1-oxo-isoindolin-5-yl1hept-6-ynoate
A solution of 3-(5-bromo-1-oxo-isoindolin-2-yl)piperidine-2, 6-dione (1 g, 3.1 mmol) and triethylamine (2.6 mL) in DMF (10 mL) was purged with nitrogen for 5 minutes, followed by addition of copper iodide (29 mg, 0.155 mmol) and bis(triphenylphosphine)palladium chloride (108 mg, 0.155 mmol). Finally tert-butyl hept-6-ynoate (620 mg, 3.4 mmol) was added and the mixture was stirred at 80°C for 5 h. The reaction mixture was recharged with the same amounts of copper iodide (29 mg, 0.155 mmol), bis(triphenylphosphine)palladium chloride (108 mg, 0.155) and tert- butyl hept-6-ynoate (620 mg, 3.4 mmol) and again stirred at 80°C for 5 h. The reaction mixture was added dropwise to ethyl acetate (100 mL)/10% citric acid (100 mL) under heavy stirring. The dark black mixture was filtered over a Celite™ and rinsed with ethyl acetate (60 mL) until the filtrate was colorless. The dark black layers were seperated and the aqueous layer was extracted with ethyl acetate (2x50 mL). The combined organic layers were washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography using SiO2 and heptane/ethyl acetate = 9/1 to 1/1 v/v%. All fractions containing the title compound were collected and concentrated in vacuo to give 853 mg of the title compound (yield 65%).
(d) 7-[2-(2,6-Dioxo-3-piperidyl)-1-oxo-isoindolin-5-yl1hept-6-ynoic acid (Intermediate TLB-Sp13) To a solution of te/Y-butyl 7-[2-(2,6-dioxo-3-piperidyl)-1-oxo-isoindolin-5-yl]hept-6-ynoate (853 mg, 2.05 mmol) in dichloromethane (4 mL) was added trifluoroacetic acid (4 mL) and the mixture was stirred at room temperature o/n. The mixture added dropwise to diethyl ether (80 mL) and the precipitate formed was filtered. The precipitate was dried in vacuo to give 485 mg of the title compound (yield: 65%).
Intermediate TLB-Sp14
6-[2-(2,6-Dioxo-3-piperidyl)-1-oxo-isoindolin-5-yl1hex-5-ynoic acid (Intermediate TLB-Sp14)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp13, starting from 3-(5-bromo-1-oxo-isoindolin-2-yl)piperidine-2, 6-dione and tert-butyl hex-5- ynoate to afford 459 mg of the title compound (yield: 98%).
5-[2-(2,6-Dioxo-3-piperidyl)-1-oxo-isoindolin-5-yl1pent-4-ynoic acid (Intermediate TLB-Sp15)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp13, starting from 3-(5-bromo-1-oxo-isoindolin-2-yl)piperidine-2, 6-dione and tert-butyl pent-4- ynoate to afford 124 mg of the title compound (yield: 42%).
3-[[6-[3-(2,4-Dioxohexahvdropyrimidin-1-yl)azetidin-1-yl1pyridine-3-carbonyl1amino1propanoic acid (Intermediate TLB-Sp16)
(a) tert-Butyl 3-[(3-amino-3-oxo-propyl)amino1azetidine-1 -carboxylate
Acrylamide (7.43 g, 104.5 mmol) and triethylamine (9.71 mL, 69.7 mmol) were successively added to a solution of 3-amino-1-A/-Boc-azetidine (6 g, 34.8 mmol) in ethanol (40 mL). The reaction mixture was stirred at 65°C for 24 hrs. The reaction mixture was concentrated in vacuo to give 13.6 g of crude tert-butyl 3-[(3-amino-3-oxo-propyl)amino]azetidine-1-carboxylate which was used directly in the next step.
(b) tert-Butyl 3-[(3-amino-3-oxo-propyl)-(2,2,2-trichloroethoxycarbonyl)-amino1azetidine-1- carboxylate
2,2,2-Trichloroethyl chloroformate (4.79 mL, 34.8 mmol) was slowly added to a cooled solution of tert-butyl 3-[(3-amino-3-oxo-propyl)amino]azetidine-1-carboxylate (13.6 g, 34.8 mmol, theor.) and triethylamine (4.85 mL, 34.8 mmol) in dichloromethane (50 mL). The reaction mixture was stirred for 4 h allowing the mixture to come to room temperature. The organic layer was washed with water (3x50 mL) and filtered over a PE filter. The filtrate was concentrated in vacuo. The residue (thick oil) was again dissolved in dichloromethane, washed with 5% citric acid solution, brine and filtered over a PE filter. The filtrate was concentrated in vacuo, to give 12.75 g of the title compound as a white foam (yield: 87.5%).
(c) tert-Butyl 3-(2,4-dioxohexahvdropyrimidin-1 -yl)azetidine-1 -carboxylate Sodium methoxide (4.93 g, 91.35 mmol) was added to a solution of tert-butyl 3-[(3-amino- 3-oxo-propyl)-(2, 2, 2-trichloroethoxycarbonyl)amino]azetidine-1 -carboxylate (12.75 g, 30.45 mmol) in methanol (150 mL) under inert atmosphere. The reaction mixture was stirred at 65 °C for 1 h. After cooling, the reaction mixture was concentrated to a small volume. MTBE (300 mL) was added and after cooling, the mixture was acidified to pH 5-6 by adding 5% citric acid solution dropwise. The aqueous layer was separated and extracted with dichloromethane. De combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by column chromatography using SiO2 and dichloromethane/methanol = 99/1 to 95/5 v/v%. All fractions containing the title compound were collected and concentrated in vacuo to give 9.05 g of the title compound (Yield : 71 .0%) as a white solid.
(d) 1 -(Azetidin-3-yl)hexahvdropyrimidine-2, 4-dione 2,2,2-trifluoroacetic acid
A solution of tert-butyl 3-(2,4-dioxohexahydropyrimidin-1-yl)azetidine-1 -carboxylate (9.05 g, 30.45 mmol) in DCM/TFA = 1/1 v/v% (30 mL) was stirred for 1 h. at room temperature. The mixture was partially concentrated (10 mL) and carefully added dropwise to diethylether (400 mL) and stirred for 24 h at room temperature. The precipitate formed was filtered, washed with diethylether and dried under vacuum at 40 °C to give 6.4 g of the title compound as a white powder in a quantitative yield.
(e) tert-Butyl 6-[3-(2,4-dioxohexahvdropyrimidin-1-yl)azetidin-1-yl1pyridine-3-carboxylate
To a solution of 1 -(azetidin-3-yl)hexahydropyrimidine-2, 4-dione 2,2,2-trifluoroacetic acid (1 .2 g, 4.2 mmol) in acetonitrile (12 mL) was added subsequently triethylamine (1 .2 mL, 12.0 mmol) and 6-chloronicotinic acid tert-buty I ester (943 mg, 4.4 mmol) and the reaction mixture was stirred at 80 °C for o/n. After cooling, the mixture was concentrated under reduced pressure and the residue dissolved in dichloromethane/methanol = 9/1 v/v%, and washed with 5% citric acid solution, water and brine, dried over sodium sulfate and filtered. The filtrate was concentrated in vacuo to give 833 mg (57%) of the title product.
(f) 6-[3-(2,4-Dioxohexahvdropyrimidin-1-yl)azetidin-1-yl1pyridine-3-carboxylic acid
A solution of tert-butyl 6-[3-(2,4-dioxohexahydropyrimidin-1-yl)azetidin-1-yl]pyridine-3- carboxylate (803 mg, 2.3 mmol) in DCM/TFA = 1/1 v/v% (10 mL) was stirred for room temperature o/n. The mixture was added dropwise to diethylether (100 mL) and stirred for 24 h at room temperature. The precipitate was filtered off, washed with diethyl ether, and dried in vacuo to afford the title compound (761 mg, 81 %) as an off-white solid.
(g) tert-Butyl 3-[[6-[3-(2,4-dioxohexahvdropyrimidin-1-yl)azetidin-1-yl1pyridine-3- carbonyllaminolpropa-noate
6-[3-(2,4-Dioxohexahydropyrimidin-1-yl)azetidin-1-yl]pyridine-3-carboxylic acid (50 mg, 0.124 mmol) and tert-butyl 3-aminopropanoate (20.3 pL, 0.137 mmol) were suspended in DCM (1 mL). HATU (49.6 mg, 0.13 mmol) and DiPEA (60.5 pL, 0.868 mmol) were added subsequently and the mixture stirred at room temperature o/n. The mixture was washed with 5% NaHCOs- solution and 5% citric acid solution. The organic layer was separated over a PE-filter. The organic layer was concentrated under reduced pressure and the residue was purified by column chromatography (dichloromethane to methanol = 98/2 to 9/1 v/v%) to afford 34.3 mg of the title compound (yield: 66.9%).
(h) 3-[[6-[3-(2,4-Dioxohexahvdropyrimidin-1-yl)azetidin-1-yl1pyridine-3-carbonyl1amino1propanoic acid (Intermediate TLB-Sp16)
A solution of te/Y-butyl 3-[[6-[3-(2,4-dioxohexahydropyrimidin-1-yl)azetidin-1-yl]pyridine-3- carbonyl]amino]propanoate (34.3 mg, 0.08 mmol) in DCM/TFA = 1/1 v/v% (3 mL) was stirred at room temperature o/n. The mixture was subsequently added dropwise to diethylether (20 mL) and stirred for 24 h at room temperature. The supernatant was decanted and the residue dried in vacuo to afford the title compound (32.7 mg, 82.9%) as an off-white solid.
Intermediate TLB-Sp17
3-[2-[[6-[3-(2,4-Dioxohexahvdropyrimidin-1-yl)azetidin-1-yl1pyridine-3carbonyl1amino1ethoxy1 propanoic acid (Intermediate TLB-Sp17)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp16, starting from 6-[3-(2,4-dioxohexahydropyrimidin-1-yl)azetidin-1-yl]pyridine-3-carboxylic acid and te/Y-butyl 3-(2-aminoethoxy)propanoate to afford 48.5 mg of the title compound (yield:
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp16, starting from 6-[3-(2,4-dioxohexahydropyrimidin-1-yl)azetidin-1-yl]pyridine-3-carboxylic acid and tert-buty I 3-[2-(2-aminoethoxy)ethoxy]propanoate to afford 50.5 mg of the title compound (yield: 92%).
Intermediate TLB-Sp19 -1 -yl)azetidin-1 -yllpyridine-3- ic acid (Intermediate TLB-Sp19)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp16, starting from 6-[3-(2,4-dioxohexahydropyrimidin-1-yl)azetidin-1-yl]pyridine-3-carboxylic acid and tert-butyl 3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propanoate to afford 39.2 mg of the title compound (yield: 71%).
Intermediate TLB-Sp20
3-[2-[2-[2-[2-[[6-[3-(2,4-dioxohexahvdropyrimidin-1-yl)azetidin-1-yl1pyridine-3-carbonyl1amino1- ethoxyl-ethoxylethoxylethoxylpropanoic acid (Intermediate TLB-Sp20)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp16, starting from 6-[3-(2,4-dioxohexahydropyrimidin-1-yl)azetidin-1-yl]pyridine-3-carboxylic acid and fe/Y-butyl 3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]propanoate to afford 38.6 mg of the title compound (yield: 70%).
Intermediate TLB-Sp21
6-[6-[3-(2,4-dioxohexahvdropyrimidin-1-yl)azetidin-1-yl1-3-pyridyl1hex-5-vnoic acid (I ntermed iate TLB-Sp21)
To 1-(azetidin-3-yl)hexahydropyrimidine-2, 4-dione 2,2,2-trifluoroacetic acid (150 mg, 0.5 mmol) was added 6-(6-chloro-3-pyridyl)hex-5-ynoic acid (90.6 mg, 0.4 mmol) followed by CS2CO3 (727 mg, 2.2 mmol). Under an inert atmosphere, dry dioxane (6.0 mL) was added, followed by tBuXPhos Pd G3 (32.2 mg, 0.04 mmol). The mixture was degassed and stirred at 105 °C for 20 hours. The solvent was removed under a stream of nitrogen and the residue was dissolved in water. The pH was adjusted to 4 and the mixture was filtered through a C18 column. The fractions containing product were lyophilized and the residue was purified by HPLC to afford the title compound (6.0 mg, 4.1 %) as an orange solid. A second batch of the title compound was made on a one-third scale (5.3 mg, 1 1 %)
Intermediate TLB-Sp22
4-[[(1 S)-1-[(2S,4/?)-4-hvdroxy-2-[[4-(4-methylthiazol-5-yl)phenyl1methylcarbamoyl1pyrrolidine-1- carbonyl1-2,2-dimethyl-propyl1amino1-4-oxo-butanoic acid (Intermediate TLB-Sp22)
Succinic anhydride (46.5 mg, 0.46 mmol) was added to a stirred suspension of (2S,4R)- 1 -((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-A/-(4-(4-methylthiazol-5-yl) benzyl) pyrrolidine-2- carboxamide hydrochloride (100 mg, 0.21 mmol) in pyridine (0.5 mL) and the reaction mixture was stirred for 3 days at room temperature. The mixture was concentrated under reduced pressure. Purification was performed using preparative LCMS. Fractions containing the title compound were lyophilised to afford 84 mg (yield: 75%) of the title compound.
Intermediate TLB-Sp23
3-[4-[[1-[3-(2,4-dioxohexahvdropyrimidin-1-yl)-4-methoxy-benzoyl1-4-piperidyl1oxy1-1- piperidyllpropanoic acid (Intermediate TLB-Sp23)
(a) te/Y-Butyl 4-(4-pyridyloxy)piperidine-1 -carboxylate
To an ice-cold (4 °C) solution of te/Y-butyl 4-hydroxypiperidine-1-carboxylate (2.12 g, 10.5 mmol), pyridin-4-ol (1 g, 10.5 mmol) and triphenylphosphine (4.14 g, 15.8 mmol) in THF (20 mL) was added dropwise a solution of diisopropyl azodicarboxylate (3.1 mL, 15.8 mmol) in THF (10 mL). The mixture was stirred for 30 min at 4 °C and then stirred at room temperature o/n. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography using SiO2 and heptane/ethyl acetate = 4/1 to 0/10 v/v%. All fractions containing the title compound were collected and concentrated in vacuo to give 1 .41 g of tert-buty I 4-(4- pyridyloxy)piperidine-1 -carboxylate (yield: 48 %).
(b) tert-B uty I 4-(1 -benzylpyridin-1 -ium-4-yl)oxypiperidine-1 -carboxylate bromide
To a solution of te/Y-butyl 4-(4-pyridyloxy)piperidine-1-carboxylate (750 mg, 2.69 mmol) in DCM (8 mL) was added benzyl bromide (644 pL, 5.38 mmol). The reaction mixture was stirred at room temperature for 4 h. The mixture was concentrated to a small volume and diethyl ether (15 mL) was added. The precipitate formed was filtered, washed with diethyl ether and dried in vacuo to give 1.18 g of the title compound (yield: 98%). (c) te/Y-Butyl 4-[(1-benzyl-3,6-dihvdro-2H-pyridin-4-yl)oxy1piperidine-1 -carboxylate
To an ice-cold (4 °C) solution of te/Y-butyl 4-(1-benzylpyridin-1-ium-4-yl)oxypiperidine-1- carboxylate bromide (1.17 g, 2.60 mmol) in methanol (6 mL) was added sodium borohydride (197 mg, 5.20 mmol). The reaction mixture was stirred for 30 min allowing the temperature to come to room temperature. After quenching with acetone (2 mL), the mixture was stirred for 15 min. The mixture was concentrated and the residue dissolved in ethyl acetate, washed with 5% aq. HCO3- solution, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to give 970 mg of the title compound (quantitative yield).
(d) te/Y-Butyl 4-(4-piperidyloxy)piperidine-1 -carboxylate
To a stirred solution of te/Y-butyl 4-[(1-benzyl-3,6-dihydro-2/7-pyridin-4-yl)oxy]piperidine-1- carboxylate (870 mg, 2.34 mmol) in methanol (11 mL) was added a suspension of 10% Pd on charcoal (124 mg) in ethanol (4 mL). The reaction mixture was stirred at room temperature for 15 min under a nitrogen atmosphere. Then, ammonium formate (1.48 g, 23.4 mmol) was added and the reaction mixture was heated to reflux temperature for 15 min. The reaction mixture was cooled, filtered over Decalite® and concentrated in vacuo to give the title compound (744 mg, quantitative).
(e) Benzyl 4-[(1 -te/Y-butoxycarbonyl-4-piperidyl)oxy1piperidine-1 -carboxylate
To an ice-cold (4 °C) solution of te/Y-butyl 4-(4-piperidyloxy)piperidine-1-carboxylate (744 mg, 2.34 mmol) in THF (7.5 mL) was added triethylamine (652 pL, 4.68 mmol). To the mixture was added drop-wise a solution of A/-(benzyloxycarbonyloxy)succinimide (641 mg, 2.57 mmol) in THF (2.5 mL) and the resulting mixture was stirred at room temperature o/n. Ethyl acetate was added and the resulting mixture was washed with 5% citric acid solution, water and brine. The organic layer was separated, dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography using SiO2 and heptane/ethyl acetate = 10/0 to 0/10 v/v%. All fractions containing the title compound were collected and concentrated in vacuo to give 849 mg of benzyl 4-[(1-te/Y-butoxycarbonyl-4-piperidyl)oxy]piperidine-1-carboxylate (yield: 87%).
(f) Benzyl 4-(4-piperidyloxy)piperidine-1 -carboxylate
To a stirred solution of benzyl 4-[(1-te/Y-butoxycarbonyl-4-piperidyl)oxy]piperidine-1- carboxylate (849 mg, 2.02 mmol) in dichloromethane (6 mL) was added TFA (1 .5 mL). The reaction mixture was stirred at room temperature for 1 h. The mixture was concentrated under reduced pressure and the residue was purified by column chromatography using SiO2 and dichloromethane/7N NH3 in methanol = 9/1 v/v%. All fractions containing the title compound were collected and concentrated in vacuo to give 602 mg of the title compound (yield: 94%).
(g) Benzyl 4-[[1-(3-te/Y-butoxy-3-oxo-propyl)-4-piperidyl1oxy1piperidine-1 -carboxylate
To a solution of benzyl 4-(4-piperidyloxy)piperidine-1-carboxylate (600 mg, 1.88 mmol) and triethylamine (524 pL, 3.76 mmol) in ethanolabs (9.5 mL) was added tert-buty I acrylate (682 pL, 4.7 mmol) and the reaction mixture is stirred at room temperature o/n. The mixture was concentrated under reduced pressure and the residue was purified by column chromatography using SiO2 and dichloromethane/methanol = 9/1 to 85/15 v/v%. All fractions containing the title compound were collected and concentrated in vacuo to give 645 mg of the title compound (yield: 77%).
(h) te/Y-Butyl 3-[4-(4-piperidyloxy)-1-piperidyl1propanoate -1 -carboxylate
To a solution of benzyl 4-[[1-(3-te/Y-butoxy-3-oxo-propyl)-4-piperidyl]oxy]piperidine-1- carboxylate (645 mg, 1.44 mmol) in methanol (7 mL) was added 10% Pd on charcoal (76 mg). Catalytic hydrogenation was performed for 2 h at room temperature. The palladium-catalyst was filtered and the filtrate was evaporated to give of the title compound in quantitative crude yield.
(i) te/Y-Butyl 3-[4-[[1-[3-(2,4-dioxohexahvdropyrimidin-1-yl)-4-methoxy-benzoyl1-4-piperidyl1oxy1-
1 -piperidyllpropanoate
To a solution of (2,3,4,5,6-pentafluorophenyl) 3-(2,4-dioxohexahydropyrimidin-1-yl)-4- methoxy-benzoate (Intermediate TLB1 , 124 mg, 0.29 mmol) in DMC/DMF=3/1 v/v% (6 mL) was added subsequently A/,A/-diisopropylethylamine (151 pL, 0.86 mmol) and te/Y-butyl 3-[4-(4- piperidyloxy)-1-piperidyl]propanoate (90 mg, 0.29 mmol). The reaction mixture was stirred at room temperature o/n. The reaction mixture was concentrated in vacuo and the residue was purified by column chromatography using SiO2 and dichloromethane/methanol = 95/5 to 9/1 v/v%. All fractions containing the title compound were collected and concentrated in vacuo to give 120 mg of the title compound (Yield 74%).
(j) 3-[4-[[1-[3-(2,4-dioxohexahvdropyrimidin-1 -yl)-4-methoxy-benzoyl1-4-piperidyl1oxy1-1- piperidyllpro-panoic acid (Intermediate TLB-Sp23)
To a solution of te/Y-butyl 3-[4-[[1-[3-(2,4-dioxohexahydropyrimidin-1-yl)-4-methoxy- benzoyl]-4-piperidyl]oxy]-1-piperidyl]propanoate (120 mg, 0.21 mmol) in dioxane (1 mL) was added 4M HCI/dioxane solution (3 mL) and the reaction mixture was stirred at room temperature for 2 h. The mixture was concentrated in vacuo. The residue was triturated with dichloromethane and dried in vacuo to give the title compound in quantitative crude yield.
Intermediate TLB-Sp24
3-[4-[[1-[3-(2,4-Dioxohexahvdropyrimidin-1-yl)-4-methyl-benzoyl1-4-piperidyl1oxy1-1- piperidyllpropanoic acid (Intermediate TLB-Sp24) This compound was prepared in an analogous manner as described in Intermediate TLB- Sp24, starting using (2,3,4,5,6-pentafluorophenyl) 3-(2,4-dioxohexahydropyrimidin-1-yl)-4-methyl- benzoate (Intermediate TLB2) in the last sequences of steps to afford 136 mg of the title compound (yield: quantitative).
2-[1-[[6-[3-(2,4-Dioxohexahvdropyrimidin-1-yl)azetidin-1-yl1-3-pyridyl1methyl1-4-piperidyl1acetic acid (Intermediate TLB-Sp25)
(a) 6-[3-(2,4-Dioxohexahvdropyrimidin-1-yl)azetidin-1-yl1pyridine-3-carbaldehvde
To a flask charged with 1 -(azetidin-3-yl)hexahydropyrimidine-2, 4-dione 2,2,2- trifluoroacetic acid (1 .0 g, 3.6 mmol) in acetonitrile (35 mL) was added triethylamine (1 .1 mL, 10.6 mmol) followed by 6-chloropyridine-3-carbaldehyde (560 mg, 4.0 mmol). The mixture was stirred and heated to 95 °C overnight. After cooling to room temperature, the solvent was concentrated in vacuo and DCM/MeOH = 9/1 v/v% (80 mL) was added to the residue, followed by the addition of water (30 mL). The layers were separated and the aqueous layer was extracted with DCM/MeOH = 9/1 v/v% (3 x 25 mL). The combined organic layers were filtered over a PE filter and concentrated in vacuo. The residue was purified by column chromatography using SiO2 and dichloromethane/methanol = 99/1 to 9/1 v/v%. All fractions containing the title compound were collected and concentrated in vacuo to give 460 mg of the title compound (Yield 46%).
(b) te/Y-Butyl 3-[4-[[6-[3-(2,4-dioxohexahvdropyrimidin-1-yl)azetidin-1-yl1-3- pyridyllmethyll pi perazin-1 -yllpropanoate
To a tube charged with 6-[3-(2,4-dioxohexahydropyrimidin-1-yl)azetidin-1-yl]pyridine-3- carbaldehyde (40 mg, 0.15 mmol) in THF/DMF = 1/1 v/v% (2 mL) was added te/Y-butyl piperidine- 4-carboxylate (22.6 pL, 0.15 mmol) followed by acetic acid (8.6 pL, 0.15 mmol). The mixture was stirred for 15 minutes, after which NaBHsCN (13.2 mg, 0.22 mmol) was added. The mixture was stirred o/w. The mixture was recharged with AcOH, NaBHsCN, and amine as needed. The solvent was concentrated in vacuo and the residue was dissolved in DCM/MeOH = 9/1 v/v% (5 mL) and washed with sat. aq. NaHCOs-solution (2 x 2 mL). The combined aqueous phases were extracted with DCM/MeOH = 9/1 v/v% (2 mL). The combined organic phases were filtered through a PE filter and concentrated in vacuo. The residue was purified by column chromatography using SiO2 and dichloromethane/7N NH3 in methanol = 10/0 to 9/1 v/v% . All fractions containing the title compound were collected and concentrated in vacuo to give 29.2 mg of the title compound (Yield 43%).
(c) 2-[1 -[[6-[3-(2,4-Dioxohexahvdropyrimidin-1 -vDazetidine-1 -v 11-3-pyrid v II methyll-4- piperidyllacetic acid (Intermediate TLB-Sp25)
To a flask charged with te/Y-butyl 3-[4-[[6-[3-(2,4-dioxohexahydropyrimidin-1-yl)azetidin-1- yl]-3-pyridyl]methyl]piperazin-1-yl]propanoate (28 mg, 0.06 mmol) was added DCM/TFA = 1/1 v/v% (2 mL). The mixture was stirred for 1 hour. Diethyl ether (5 mL) was added and the mixture was concentrated in vacuo to afford 2-[1-[[6-[3-(2,4-dioxohexahydropyrimidin-1-yl)azetidine-1-yl]- 3-pyridyl]methyl]-4-piperidyl]acetic acid (Intermediate TLB-Sp25) (50 mg, quant) which was used directly for coupling.
Intermediate TLB-Sp26
A/-(2,6-dioxo-3-piperidyl)-5-(4-formyl-1-piperidyl)pyridine-2-carboxamide (Intermediate TLB- Sp26)
(a) Benzyl 4-(dimethoxymethyl)piperidine-1 -carboxylate
A solution of 4-formyl-A/-Cbz-piperidine (5 g, 20.22 mmol) in methanol:trimethylorthoformate (3:1 v/v%, 80 mL) containing a trace amount of p-toluenesulfonic acid was stirred at ambient temperature for 1 hour. The mixture was concentrated in vacuo to provide of the title compound in quantitative crude yield.
(b) 4-(Dimethoxymethyl)piperidine
To a solution of benzyl 4-(dimethoxymethyl)piperidine-1-carboxylate (6.23 g, 21.2 mmol) in methanol (100 mL) was added a slurry of 20% Pd(OH)2 on charcoal (500 mg) in methanol (5 mL). Catalytic hydrogenation was performed for 4 h at room temperature. The palladium-catalyst was filtered and the filtrate was evaporated to afford the title compound (3.09 g, 92%) as an oil. (c) Methyl 5-[4-(dimethoxymethyl)-1-piperidyl1pyridine-2-carboxylate
To a vial charged with methyl 5-fluoropicolinate (974 mg, 6.28 mmol) in DMSO (6.3 mL) was added 4-(dimethoxymethyl)piperidine (1.0 g, 6.28 mmol) followed by DIPEA (2.18 mL, 12.6 mmol). The mixture was heated to 100 °C and stirred overnight. The mixture was cooled to room temperature and water (8.0 mL) was added. The solids were filtered and the residue was dried in vacuo to afford the title compound (1 .31 g, 71 %) as a beige solid.
(d) Lithium 5-[4-(dimethoxymethyl)-1-piperidyl1pyridine-2-carboxylate
To a solution of methyl 5-[4-(dimethoxymethyl)-1-piperidyl]pyridine-2-carboxylate (1 .31 g, 4.45 mmol) in THF/water=4/1 v/v% (45 mL) was added lithium hydroxide (118 mg, 4.89 mmol) and the reaction mixture was stirred overnight. Water (30 mL) was added, followed by the removal of volatiles in vacuo. The residue was lyophilised to afford the title compound (1.34 g, 105%) as a beige solid.
(e) 5-[4-(Dimethoxymethyl)-1-piperidyl1-A/-(2,6-dioxo-3-piperidyl)pyridine-2-carboxamide
Lithium 5-[4-(dimethoxymethyl)-1-piperidyl]pyridine-2-carboxylate (1 .34 g, 4.68 mmol) and 3-aminopiperidine-2, 6-dione hydrochloride (770 mg, 4.68 mmol) were dissolved in DMF (25 mL). HATU (3.55 g, 9.36 mmol) and DiPEA (2.02 mL, 11.7 mmol) were added subsequently and the mixture stirred at room temperature for 1 h. To the mixture was added dichloromethane/methanol=1/1 v/v% (100 mL) and the mixture was washed with 5% NaHCOs- solution and 5% citric acid solution. The organic layer was separated over a PE-filter. The organic layer was concentrated under reduced pressure and the residue was purified by column chromatography (dichloromethane to methanol = 10/0 to 9/1 v/v%) to afford 1.2 g of the title compound (yield: 66%).
(f) A/-(2,6-dioxo-3-piperidyl)-5-(4-formyl-1-piperidyl)pyridine-2-carboxamide (Intermediate TLB- Sp26)
To a solution of 5-[4-(dimethoxymethyl)-1-piperidyl]-N-(2,6-dioxo-3-piperidyl)pyridine-2- carboxamide (200 mg, 0.52 mmol) in THF (4 mL) was added 2N aq. HCI (2 mL). The mixture was stirred for 2 hours. The mixture was neutralized with 5% aq. sodium bicarbonate and the aqueous phase was extracted with dichloromethane/methanol=9/1 v/v% (2 x 10 mL). The organic phase was concentrated in vacuo to afford the intermediate aldehyde (Intermediate TLB-Sp26) (140 mg, 77%) which was used directly for reductive amination reactions with the Intermediate B-x .
Intermediate TLB-Sp27
2-[1-[[1-[6-[(2,6-Dioxo-3-piperidyl)carbamoyl1-3-pyridyl1-4-piperidyl1methyl1-4-piperidyl1acetic acid (Intermediate TLB-Sp27)
(a) tert-Butyl 2-[1-[[1-[6-[(2,6-dioxo-3-piperidyl)carbamoyl1-3-pyridyl1-4-piperidyl1methyl1-4- piperidyllacetate
To a tube charged with A/-(2,6-dioxo-3-piperidyl)-5-(4-formyl-1-piperidyl)pyridine-2-carbox- amide (66 mg, 0.19 mmol) in DCM (2 mL) was added te/Y-butyl piperidine-4-carboxylate (49.6 pL, 0.23 mmol) followed by acetic acid (10.9 pL, 0.15 mmol). The mixture was stirred for 15 minutes at room temperature, after which sodium cyanoborohydride (7.2 mg, 0.116 mmol) was added and stirring was continued for 4 hours. The mixture was quenched by addition of methanol (0.5 mL). Dichloromethane (4.5 mL) was and added and the mixture was washed with saturated aqueous NaHCOs-solution (2 x 2 mL). The organic phase was filtered over a PE filter and concentrated in vacuo. The residue was purified by preparative HPLC. Fractions containing product were pooled and lyophilised to afford the title compound (32.6 mg) as a beige solid.
(b) 2-[1-[[1-[6-[(2,6-Dioxo-3-piperidyl)carbamoyl1-3-pyridyl1-4-piperidyl1methyl1-4-piperidyl1acetic acid (Intermediate TLB-Sp27)
To a flask charged with te/Y-butyl 2-[1-[[1-[6-[(2,6-dioxo-3-piperidyl)carbamoyl]-3-pyridyl]- 4-piperidyl]methyl]-4-piperidyl]acetate (32.6 mg) was added DCM/TFA = 1/1 v/v% (2 mL). The mixture was stirred for 1 hour. Diethyl ether (5 mL) was added and the mixture was concentrated in vacuo to afford 2-[1-[[1-[6-[(2,6-dioxo-3-piperidyl)carbamoyl]-3-pyridyl]-4-piperidyl]methyl]-4- piperidyl]acetic acid (Intermediate TLB-Sp27) quantitatively, which was used directly for coupling to the Intermediate B-x . te/Y-Butyl 2-[1-(4-piperidylmethyl)-4-piperidyl1acetate (I ntermed iate S p3)
(a) Benzyl 4-[[4-(2-te/Y-butoxy-2-oxo-ethyl)-1-piperidyl1methyl1piperidine-1 -carboxylate To a solution of tert-buty I 2-(4-piperidyl)acetate (0.5 g, 2.51 mmol) and 4-formyl-A/-Cbz- piperidine in THF (7.5 mL) was added acetic acid (180 pL) and the reaction mixture was stirred at room temperature o/n. After cooling to 0 °C sodium cyanoborohydride (0.189 g, 3.0 mmol) was added and the mixture was stirred for 1 h allowing the mixture to reach room temperature. 5% aq. NaHCOs-solution was added and the mixture was extracted with ethyl acetate. The ethyl acetate layers was separated, washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (heptane/ethyl acetate = 10/0 to 0/10 v/v%) to give 606 mg of the title compound (yield: 56%).
(b) tert-Butyl 2-[1-(4-piperidylmethyl)-4-piperidyl1acetate (Intermediate Sp3)
To a solution of benzyl 4-[[4-(2-te/Y-butoxy-2-oxo-ethyl)-1-piperidyl]methyl]piperidine-1- carboxylate (606 mg, 1.41 mmol) in ethanol (7.5 mL) was added 10% Pd on charcoal (60 mg). Catalytic hydrogenation was performed for 2 h at room temperature. The palladium catalyst was removed by filtration and the filtrate was concentrated in vacuo to give te/Y-butyl 2-[1-(4- piperidylmethyl)-4-piperidyl]acetate (Intermediate Sp3) in quantitative crude yield.
Intermediate TLB-Sp28
2-[1-[[1-[2-(2,6-Dioxo-3-piperidyl)-1 ,3-dioxo-isoindolin-5-yl1-4-piperidyl1methyl1-4-piperidyl1acetic acid (Intermediate TLB-Sp28)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp5, starting from 2-(2,6-dioxo-3-piperidyl)-5-fluoro-isoindoline-1 ,3-dione and te/Y-butyl 2-[1-(4- piperidyl-methyl)-4-piperidyl]acetate (Intermediate Sp3) to afford the title compound (22.4 mg, 3-[1-[[1-[6-[(2,6-dioxo-3-piperidyl)carbamoyl1-3-pyridyl1-4-piperidyl1methyl1-4-piperidyl1propanoic acid (Intermediate TLB-Sp29)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp27, starting from A/-(2,6-dioxo-3-piperidyl)-5-(4-formyl-1-piperidyl)pyridine-2-carbox-amide (Intermediate TLB-Sp26) and tert-buty I 3-(4-piperidyl)propanoate to afford 50.3 mg of the title compound (quant, yield).
Intermediate TLB-Sp30
2-[3-[3-(2,4-dioxohexahvdropyrimidin-1-yl)-4-methyl-benzoyl1-3,9-diazaspiro[5.51undecan-9- yllacetic acid (Intermediate TLB-Sp30)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp24, starting from (2,3,4,5,6-pentafluorophenyl) 3-(2,4-dioxohexahydropyrimidin-1-yl)-4-methyl- benzoate (Intermediate TLB2) and tert-buty I 3,9-diazaspiro[5.5]undecane-3-carboxylate to afford 318 mg of the title compound (yield: 92%).
Intermediate TLB-Sp31
3-[3-[3-(2,4-dioxohexahvdropyrimidin-1-yl)-4-methyl-benzoyl1-3,9-diazaspiro[5.51undecan-9- yllpropanoic acid (Intermediate TLB-Sp31)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp24, starting from (2,3,4,5,6-pentafluorophenyl) 3-(2,4-dioxohexahydropyrimidin-1-yl)-4-methyl- ben-zoate (Intermediate TLB2) and tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate to afford 330 mg of the title compound (yield: 89%).
Intermediate TLB-Sp32 yridyllmethyll-4- ic acid (Intermediate TLB-Sp32)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp25, starting from 6-[3-(2,4-dioxohexahydropyrimidin-1-yl)azetidin-1-yl]pyridine-3-carbaldehyde and tert-butyl 3-(4-piperidyl)propanoate to afford 83.2 mg of the title compound.
Intermediate TLB-Sp33 TLB-Sp33)
This compound was prepared in an analogous manner as described in Angew. Chem. Int.
Ed. 2021 , 60, 26663 to afford 1 .31 g of the title compound (yield: 88%).
Intermediate TLB3 (Intermediate TLB3)
This compound was prepared in an analogous manner as described in Intermediate
TLB1 , starting from 2-[4-(2,6-dioxo-3-piperidyl)phenoxy]acetic acid (Intermediate TLB-Sp33) to afford the title compound (352 mg, 17%). Intermediate TLB-Sp34
3-[4-[[1-[2-(2,6-dioxo-3-piperidyl)-1 ,3-dioxo-isoindolin-5-yl1-4-piperidyl1oxy1-1-piperidyl1propanoic acid (Intermediate TLB-Sp34)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp5, starting from 2-(2,6-dioxo-3-piperidyl)-5-fluoro-isoindoline-1 ,3-dione and te/Y-butyl 3-[4-(4- piperidyloxy)-1-piperidyl]propanoate (Intermediate TLB-Sp23-h) to afford the title compound (160 mg, quant.).
Intermediate TLB-Sp35
2-[1-[[1-[3-(2,4-dioxohexahvdropyrimidin-1-yl)-4-methyl-benzoyl1-4-piperidyl1methyl1-4- piperidyllacetic acid (Intermediate TLB-Sp35) This compound was prepared in an analogous manner as described in Intermediate TLB-
Sp24, starting from (2,3,4,5,6-pentafluorophenyl) 3-(2,4-dioxohexahydropyrimidin-1-yl)-4-methyl- ben-zoate (Intermediate TLB2) and fe/Y-butyl 2-[1-(4-piperidylmethyl)-4-piperidyl]acetate (Intermediate Sp3) to afford 108 mg of the title compound (yield: quantitative).
3-[2-[[6-(2,4-Dioxo-1 ,3,8-triazaspiro[4.51decan-8-yl)pyridine-3-carbonyl1amino1ethoxy1propanoic acid (Intermediate TLB-Sp36)
(a) tert-Butyl 6-(2,4-dioxo-1 ,3,8-triazaspiro[4.51decan-8-yl)pyridine-3-carboxylate To a solution of 1 ,3, 8-triazaspiro[4.5]decane-2, 4-dione 2,2,2-trifluoroacetic acid salt (283 mg, 1 mmol) and tert-butyl 6-chloropyridine-3-carboxylate (230 mg, 1.08 mmol) in DMF (5 mL) was added DiPEA (696 pl, 4 mmol) and the reaction mixture was stirred at 120 °C overnight. Water/brine=1/1 v/v% (75 mL) was added and the mixture was extracted with ethyl acetate. The ethyl acetate layers was separated, washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (dichloromethane/methanol = 98/2 to 9/1 v/v%) to give 198 mg of the title compound (yield: 57%).
(b) 6-(2,4-Dioxo-1 ,3,8-triazaspiro[4.51decan-8-yl)pyridine-3-carboxylic acid
To a stirred solution of tert-butyl 6-(2,4-dioxo-1 ,3,8-triazaspiro[4.5]decan-8-yl)pyridine-3- carboxylate (195 mg, 0.56 mmol) in dichloromethane (1 .5 mL) was added TFA (1 .5 mL). The reaction mixture was stirred at room temperature for 1 h. The mixture was concentrated under reduced pressure and traces of trifluoroacetic acid were removed by co-evaporation twice with dichloromethane. The title compound was isolated as the trifluoroacetic acid salt in quantitative crude yield (286 mg).
(c) tert-Butyl 3-[2-[[6-(2,4-dioxo-1 ,3,8-triazaspiro[4.51decan-8-yl)pyridine-3- carbonyllaminolethoxylpro-panoate
6-(2,4-Dioxo-1 ,3,8-triazaspiro[4.5]decan-8-yl)pyridine-3-carboxylic acid (52 mg, 0.1 mmol) and tert-butyl 3-(2-aminoethoxy)propanoate (19.1 pL, 0.11 mmol) were dissolved in DMF (1.2 mL). HATU (40 mg, 0.105 mmol) and DiPEA (0.12 mL, 0.7 mmol) were added subsequently and the mixture stirred at room temperature for 1 h. To the mixture was added to water/brine/ethyl acetate=1/1/1 v/v% (30 mL) and the mixture was stirred for 15 min. The ethyl acetate layer was separated, washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (dichloromethane to methanol = 10/0 to 9/1 v/v%) to afford 34 mg of the title compound (yield: 75%).
(d) 3-[2-[[6-(2,4-Dioxo-1 ,3,8-triazaspiro[4.51decan-8-yl)pyridine-3- carbonyllaminolethoxylpropanoic acid (Intermediate TLB-Sp36)
To a stirred solution of tert-butyl 3-[2-[[6-(2,4-dioxo-1 ,3,8-triazaspiro[4.5]decan-8- yl)pyridine-3-carbonyl]amino]ethoxy]propanoate (34 mg, 0.074 mmol) in dichloromethane (1 mL) was added TFA (1 mL). The reaction mixture was stirred at room temperature for 1 h. The mixture was concentrated under reduced pressure and traces of trifluoroacetic acid were removed by coevaporation twice with dichloromethane to give 37 mg of the title compound as the trifluoroacetic acid salt in quantitative crude yield. Intermediate Sp4 te/Y-Butyl 2-(azetidin-3-yloxy)acetate (Intermed iate S p4)
This compound was prepared in an analogous manner as described in Intermediate Sp2, starting from benzyl 3-hydroxyazetidine-1 -carboxylate and fe/Y-butyl bromoacetate to afford 160 mg of the title compound (yield: quantitative).
Intermediate TLB-Sp37
2-[1-[2-(2,6-Dioxo-3-piperidyl)-1 ,3-dioxo-isoindolin-5-yl1azetidin-3-yl1oxyacetic acid (Intermediate TLB-Sp37)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp5, starting from 2-(2,6-dioxo-3-piperidyl)-5-fluoro-isoindoline-1 ,3-dione and fe/Y-butyl 2- (azetidin-3-yloxy)acetate (Intermediate Sp4) to afford the title compound (50 mg, 20%).
3-[4-[6-(2,4-Dioxo-1 , 3, 8-triazaspiro[4.51decan-8-yl)pyridine-3-carbonyl1 pi perazin-1 -yllpropanoic acid (Intermediate TLB-Sp38)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp37, starting from 6-(2,4-dioxo-1 ,3,8-triazaspiro[4.5]decan-8-yl)pyridine-3-carboxylic acid (Intermediate TLB-Sp37-b) and fe/Y-butyl 3-piperazin-1-ylpropanoate dihydrochloride (Intermediate Sp1) to afford the title compound (53 mg, 97%). Intermediate TLB-Sp39
2-[2-[2-[[(3S)-2-[(2S)-2-[[(2S)-2-[te/Y-butoxycarbonyl(methyl)amino1propanoyl1amino1-3,3- dimethyl-butanoyl1-3-[[(1 F?)-tetral i n- 1 -yl1carbamoyl1-3,4-dihydro-1 /-/-isoquinolin-7- ylloxylethoxylethoxylacetic acid (Intermediate TLB-Sp39)
(a) tert-Butyl A/-[(1 S)-2-[[(1 S)-1 -[(3S)-7-hydroxy-3-[[(1 F?)-tetrali n-1 -yl1carbamoyl1-3,4-dihydro-1 H- isoquinoline-2-carbonyl1-2,2-dimethyl-propyl1amino1-1-methyl-2-oxo-ethyl1-A/-methyl-carbamate
This compound was prepared from subsequent HATU-coupling and acid-deprotection reactions starting from commercially available (R)-1 ,2,3, 4-tetrahydronaphthalen-1 -amine, Boc-7- hydroxy-(S)-1 ,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid, Boc-Tle-OH and Boc-N-Me-Ala-OH to give 150 mg of the title compound.
(b) Methyl 2-[2-[2-[[(3S)-2-[(2S)-2-[[(2S)-2-[te/Y-butoxycarbonyl(methyl)amino1propanoyl1amino1- 3,3-dimethyl-butanoyl1-3-[[(1 /?)-tetralin-1-yl1carbamoyl1-3,4-dihydro-1 /7-isoquinolin-7- ylloxylethoxylethoxylacetate
To a solution of fert-butyl A/-[(1 S)-2-[[(1 S)-1-[(3S)-7-hydroxy-3-[[(1R)-tetralin-1- yl]carbamoyl]-3,4-dihydro-1 /7-isoquinoline-2-carbonyl]-2,2-dimethyl-propyl]amino]-1-methyl-2- oxo-ethyl]-A/-methyl-carbamate (136 mg, 0.22 mmol) in DMF (3.9 mL) was added a solution of methyl 2-[2-(2-chloroethoxy)ethoxy]acetate (47 mg, 0.24 mmol) in DMF (0.5 mL) followed by potassium carbonate (91 mg, 0.66 mmol). The reaction mixture was stirred at 80 °C for 3 h. An additional portion of methyl 2-[2-(2-chloroethoxy)ethoxy]acetate (47 mg, 0.22 mol) was added and the mixture was stirred for 4 h at 80 °C. The mixture was added to water/brine/ethyl acetate=1/1/1 v/v% (140 mL) and the mixture was stirred for 15 min. The ethyl acetate layer was separated, washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (heptane to ethyl acetate = 1/4 to 0/10 v/v%) to afford 79 mg of the title compound (yield: 46%).
(c) 2-[2-[2-[[(3S)-2-[(2S)-2-[[(2S)-2-[te/Y-butoxycarbonyl(methyl)amino1propanoyl1amino1-3,3- dimethyl-butanoyl1-3-[[(1 /?)-tetralin-1-yl1carbamoyl1-3,4-dihydro-1 /7-isoquinolin-7- ylloxylethoxylethoxylacetic acid (Intermediate TLB-Sp39).
To a solution of methyl 2-[2-[2-[[(3S)-2-[(2S)-2-[[(2S)-2-[te/Y-butoxycarbonyl(methyl)- amino]-propanoyl]amino]-3,3-dimethyl-butanoyl]-3-[[(1 /?)-tetralin-1-yl]carbamoyl]-3,4-dihydro-1 /7- isoquinolin-7-yl]oxy]ethoxy]ethoxy]acetate (79 mg, 0.1 mmol) in dioxane/water=3/1 v/v% (1 .32 mL) was added lithium hydroxide (5.4 mg, 0.15 mmol) and the reaction mixture was stirred overnight. Water (5 mL) was added, followed by the removal of the volatiles in vacuo. The residue was lyophilised to afford the title compound (67 mg, 87%) as the lithium salt.
1-[4-(2,6-Dioxo-3-piperidyl)-2-fluoro-phenyl1piperidine-4-carbaldehyde (Intermediate TLB-Sp40)
(a) Ethyl 1 -(4-bromo-2-fluoro-phenyl)piperidine-4-carboxylate
To a solution of 4-bromo-2-fluoro-1 -iodobenzene (5.0 g, 16.61 mmol) and ethyl isonipecotate (3.12 g, 19.92 mmol) in toluene (100 mL) was added cesium carbonate (16.23 g, 49.83 mmol). After degassing with nitrogen, XantPhos Pd G4 (394 mg, 0.41 mmol) was added and the reaction mixture was stirred for 16 h at 100 °C. The reaction mixture was poured into toluene:water 1 :1 (200ml) and the bi-phasic mixture was stirred for 15 min. The mixture was filtered over Celite™ and the layers were separated. The organic layer was washed with 1 M aq. HCI- solution (50 mL), sat. aq. NaHCOs-solution (50 mL), 50% brine (50 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (heptane/ethyl acetate = 10/0 to 3/1 v/v%) to give 3.11 g of the title compound (yield: 57%).
(b) Ethyl 1 -[4-(2,6-dibenzyloxy-3-pyridyl)-2-fluoro-phenyl1piperidine-4-carboxylate
A mixture of ethyl 1-(4-bromo-2-fluoro-phenyl)piperidine-4-carboxylate (3.0 g, 9.08 mmol), 2,6-dibenzyloxy-3-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)pyridine (3.78 g, 9.08 mmol) and potassium phosphate tribasic (5.78 g, 27.24 mmol) in dioxane/water = 4/1 v/v% (63 mL) was degassed with nitrogen for 5 min at 30 °C. CataCXium® A Pd G3 (328 mg, 0.45 mmol) was added and the mixture was again degassed with nitrogen for 5 min at 30 °C. The reaction mixture was stirred for 16 h at 80 °C. After cooling ethyl acetate was added and the mixture was stirred for 5 min. The mixture was filtered over Decalite™ and the aqueous layer was extracted with heptane/ethyl acetate=4/1 v/v% (100 mL). The combined organic layers were washed with brine (100 mL), dried over sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by column chromatography using SiO2 and heptane/ethyl acetate = 10/0 to 3/1 v/v%. All fractions containing the title compound were collected and concentrated in vacuo to give 3.43 g of the title compound (Yield: 70%).
(c) [1-[4-(2,6-Dibenzyloxy-3-pyridyl)-2-fluoro-phenyl1-4-piperidyl1methanol
To a cold (5 °C) solution of ethyl 1 -[4-(2,6-dibenzyloxy-3-pyridyl)-2-fluoro- phenyl]piperidine-4-carboxylate (2.5 g, 4.62 mmol) in THF (25 mL) was added dropwise lithium aluminium hydride (1 M solution in THF, 5.08 mL) and the reaction mixture was stirred for 2 h allowing the temperature to come to room temperature. The mixture was cooled on an ice-bath and carefully quenched by addition of a sat. aq. solution of Rochelle salt (50 mL). Water (50 mL) and ethyl acetate/heptane=9/1 v/v% (100 mL) was added and the bi-phasic mixture was stirred for 1 h at rt. The layers were separated and the combined organic layers were washed with water/brine=1/1 v/v% (25 mL), dried over sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by column chromatography using SiO2 and dichloromethane/methanol = 95/5 v/v%. All fractions containing the title compound were collected and concentrated in vacuo to give 2.0 g of the title compound (Yield: 88%).
(d) 3-[3-Fluoro-4-[4-(hvdroxymethyl)-1-piperidyl1phenyl1piperidine-2, 6-dione
To a solution of [1-[4-(2,6-dibenzyloxy-3-pyridyl)-2-fluoro-phenyl]-4-piperidyl]methanol (1.7 g, 3.41 mmol) THF (17 mL) and ethanol (153 mL) was added. After addition of 10% Pd on charcoal (340 mg) catalytic hydrogenation was performed at room temperature o/n. The palladiumcatalyst was subsequently removed by filtration and the filtrate was concentrated in vacuo to give 990 mg of 3-[3-fluoro-4-[4-(hydroxymethyl)-1-piperidyl]phenyl]piperidine-2, 6-dione (yield: 90%).
(e) 1-[4-(2,6-Dioxo-3-piperidyl)-2-fluoro-phenyl1piperidine-4-carbaldehyde (Intermediate TLB- Sp40)
Dess-Martin periodinane (283 mg, 0.67 mmol) was added to a suspesion of 3-[3-fluoro-4- [4-(hydroxymethyl)-1-piperidyl]phenyl]piperidine-2, 6-dione (144 mg, 0.45 mmol) in dichloromethane (3 mL). The mixture was allowed to stir at room temperature for five hours. Dichloromethane and aqueous Na2SOs were added. The organic layer was dried over sodium sulfate, filtered, concentrated, and purified by column chromatography (ethyl acetate) to afford 1- [4-(2,6-dioxo-3-piperidyl)-2-fluoro-phenyl]piperidine-4-carbaldehyde (Intermediate TLB-Sp40) (120 mg, 89%). Intermediate TLB-Sp41
3-[4-[4-(2,6-Dioxo-3-piperidyl)-2-fluoro-phenyl1 piperazin-1 -yllpropanoic acid (Intermediate TLB- Sp41)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp40, and starting from 4-bromo-2-fluoro-1 -iodobenzene and te/Y-butyl 3-piperazin-1- ylpropanoate dihydrochloride (Intermediate Sp1) to afford after deprotection with trifluoro acetic acid, the title compound (440 mg, 92%) as a TFA-salt.
3-[4-[3-(2,4-dioxohexahvdropyrimidin-1-yl)-4-methyl-benzoyl1piperazin-1 -yllpropanoic acid (Intermediate TLB-Sp42)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp7, and starting from 2,3,4,5,6-Pentafluorophenyl) 3-(2,4-dioxohexahydropyrimidin-1-yl)-4- methyl-benzoate (Intermediate TLB2) and fe/Y-butyl 3-piperazin-1-ylpropanoate to afford after deprotection with trifluoro acetic acid, the title compound (354 mg, 100%) as a TFA-salt.
Intermediate TLB-Sp43
2-[4-[[1-[2-(2,6-dioxo-3-piperidyl)-1 ,3-dioxo-isoindolin-5-yl1-4-piperidyl1oxy1-1-piperidyl1acetic acid (Intermediate TLB-Sp43)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp5, starting from 2-(2,6-dioxo-3-piperidyl)-5-fluoro-isoindoline-1 ,3-dione and te/Y-butyl 2-[4-(4- piperidyloxy)-1-piperidyl]acetate to afford the title compound (253 mg, quant.).
1-[2-(2,6-Dioxo-3-piperidyl)-1 ,3-dioxo-isoindolin-5-yllazetidine-3-i (Intermediate
TLB-Sp44)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp6, starting from 2-(2,6-dioxo-piperidin-3-yl)-5-fluoroisoindoline-1 ,3-dione and azetidin-3-yl- methanol hydrochloride to afford the title compound (367 mg, 61 %.).
1-[4-(2,6-Dioxo-3-pi -2-fluoro-i idine-4-i lie acid (Intermediate TLB-
Sp45)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp40, and starting from 4-bromo-2-fluoro-1 -iodobenzene, 2,6-dibenzyloxy-3-(4,4,5,5-tetramethyl- 1 ,3,2-dioxaborolan-2-yl)pyridine and ethyl isonipecotate to afford the title compound (188 mg, 46%). Intermediate TLB-Sp46
3-[1-[2-(2,6-dioxo-3-piperidyl)-1 ,3-dioxo-isoindolin-5-yl1-4-piperidyl1propanoic acid (Intermediate TLB-Sp46)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp5, and starting from 2-(2,6-dioxo-piperidin-3-yl)-5-fluoroisoindoline-1 ,3-dione and tert-buty I 3- (4-piperidyl)propanoate to afford the title compound (474 mg, 49%).
Intermediate TLB-Sp47
(3S)-1-[2-(2,6-Dioxo-3-piperidyl)-1 ,3-dioxo-isoindolin-5-yl1pyrrolidine-3-carbaldehvde
(Intermediate TLB-Sp47)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp6, starting from 2-(2,6-dioxo-piperidin-3-yl)-5-fluoroisoindoline-1 ,3-dione and [(3S)-pyrrolidin-3- yl]methanol to afford the title compound (660 mg, 88%).
Intermediate TLB-Sp48
A/-[(3S)-2,6-dioxo-3-piperidyl1-5-(4-formyl-1 -piperidyl) pyridine-2-carboxamide (Intermediate TLB-
Sp48) This compound was prepared in an analogous manner as described in Intermediate TLB- Sp26, starting from 4-formyl-A/-Cbz-piperidine, 5-fluoropicolinate and (S)-3-amino-piperidine-2,6- dione hydrochloride to afford the title compound (206.2 mg, 87%).
1-[4-[(2,6-dioxo-3-piperidyl)amino1-2-fluoro-phenyl1piperidine-4-carbaldehyde (I nte rmed iate TLB-Sp49)
(a) 4-(Dimethoxymethyl)-1-(2-fluoro-4-nitro-phenyl) piperidine
To a solution of 4-(dimethoxymethyl)piperidine (3.32 g, 20.9 mmol) in DMSO (12 mL) was added 3,4-difluoronitrobenzene (3.5 g, 22 mmol). The reaction mixture was stirred for 16 h at 90 °C. Ethyl aceate (100 mL) and water (100 mLO were aded to the mixture and the bi-phasic mixture was stirred for 15 min. The organic layer was separated and washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by column chromatography using SiO2 and heptane/ethyl acetate = 10/0 to 35/65 v/v%. All fractions containing the title compound were collected and concentrated in vacuo to give 3.39 g of the title compound (Yield: 54%).
(b) 4-[4-(Dimethoxymethyl)-1-piperidyl1-3-fluoro-aniline
To a solution of 4-(dimethoxymethyl)-1-(2-fluoro-4-nitro-phenyl)piperidine (2.84 g, 9.5 mmol) in THF (95 mL) was added 10% Pd on charcoal (280 mg) and catalytic hydrogenation was performed at room temperature o/n. The palladium-catalyst was subsequently removed by filtration and the filtrate was concentrated in vacuo to give 2.69 g of 4-[4-(dimethoxymethyl)-1-piperidyl]-3- fluoro-aniline (crude yield: 105%).
(c) 3-[4-[4-(DimethoxymethvD-1 -pi peridyl1-3-fluoro-anilino1piperidine-2, 6-dione
4-[4-(Dimethoxymethyl)-1-piperidyl]-3-fluoro-aniline (2.66 g, 9.9 mmol) and 3- bromopiperidine-2, 6-dione (2.85 g, 14.9 mmol) were dissolved in DMF (25 mL) at room temperature. NaHCOs (2.50 g, 29.7 mmol) was added and the reaction mixture was stirred for 16 h at 65 °C. After cooling of the reaction mixture, ethyl acetate (250 mL) and water (220 mL) were added and the mixture was stirred for 15 min. The organic layer was separated, washed with water and brine, dreid over sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by column chromatography using SiO2 and heptane/ethyl acetate = 8/2 to 0/10 v/v%. All fractions containing the title compound were collected and concentrated in vacuo to give 2.95 g of the title compound (Yield: 79%).
(d) 1-[4-[(2,6-Dioxo-3-piperidyl)amino1-2-fluoro-phenyl1piperidine-4-carbaldehyde (Intermediate TLB-Sp49)
To a solution of 3-[4-[4-(dimethoxymethyl)-1-piperidyl]-3-fluoro-anilino]piperidine-2,6- dione (166 mg, 0.43 mmol) in THF (3.33 mL) was added 2N aq. HCI (1.66 mL). The mixture was stirred for 4 hours. The mixture was neutralized with 5% aq. sodium bicarbonate (20 mL) and the aqueous phase was extracted with dichloromethane/methanol=9/1 v/v% (2 x 10 mL). The organic phase was separated and concentrated in vacuo to afford the intermediate aldehyde (Intermediate TLB-Sp49) (153.5 mg, quantitative) which was used directly for reductive amination reactions with Intermediate B-x.
(3F?)-1-[4-(2,6-dioxo-3-piperidvD-3,5-difluoro-phenyl1pyrrolidine-3-carboxylic acid (Intermediate TLB-Sp50)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp40, and starting from 2-bromo-1 ,3-difluoro-5-iodobenzene and (R)-methyl pyrrolidine-3- carboxylate hydrochloride and 2,6-dibenzyloxy-3-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2- yl)pyridine to afford 319 mg of the title compound. Data: LCMS (A) Rt : 3.80 min; m/z 339.1 [M+H]+.
Intermediate TLB-Sp51
1-[4-(2,6-Diioxo-3-piperidyl)-2-fluoro-phenyl1-4-fluoro-piperidine-4-carbaldehyde (I ntermed iate
TLB-Sp51)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp40, and starting from 4-bromo-2-fluoro-1 -iodobenzene and ethyl 4-fluoropiperidine-4- carboxylate hydrochloride and 2,6-dibenzyloxy-3-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2- yl)pyridine to afford 160 mg of the crude title compound which was used directly in the reductive amination reactions. Data: LCMS (A) Rt : 2.61 min; m/z 355.2 [M+FW+HJT
(3/?)-1-[4-(2,6-dioxo-3-piperidyl)-3,5-difluoro-phenyl1pyrrolidine-3-carbaldehvde (Intermediate TLB-Sp52)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp40, and starting from 2-bromo-1 ,3-difluoro-5-iodobenzene and (R)-methyl pyrrolidine-3- carboxylate hydrochloride and 2,6-dibenzyloxy-3-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2- yl)pyridine to afford 133 mg of the title compound. Data: LCMS (A) Rt : 3.80 min; m/z 341.1 [M+H2O+H]+.
2-[1-[4-[(2,6-Dioxo-3-piperidyl)amino1phenyl1-4-hvdroxy-4-piperidyl1acetic acid (Intermediate TLB-Sp53)
This compound was prepared in an analogous manner as described in WO 2021/255212, starting from 1-(4-nitrophenyl)piperidin-4-one, te/Y-butyl acetate and 3-bromopiperidine-2, 6-dione to afford 1 .02 g of the title compound as the hydrochloride salt. Data: LCMS (A) Rt : 0.76 min; m/z 362.2 [M+H]+.
Intermediate TLB-Sp54
1-[4-[(2,6-Dioxo-3-piperidyl)amino1-2-fluoro-phenyl1piperidine-4-carboxylic acid (Intermediate
TLB-Sp54) This compound was prepared in an analogous manner as described in Intermediate TLB- Sp49, starting from 1 ,2-difluoro-4-nitro-benzene, fe/Y-butyl piperidine-4-carboxylate and 3- bromopiperidine-2, 6-dione to afford 360 mg of the title compound after tert-butyl ester deprotection with TFA/dichloromethane. Data: LCMS (A) Rt : 1.34 min; m/z 350.2 [M+H]+.
Intermediate TLB-Sp55
1-[4-(2,4-Dioxohexahvdropyrimidin-1-yl)-2-fluoro-phenyl1piperidine-4-carbaldehyde
(Intermediate TLB-Sp55)
(a) [1-(2-Fluoro-4-nitro-phenyl)-4-piperidyl1methanol
To a solution of 4-piperidylmethanol (4.0 g, 34.8 mmol) in methanol (12 mL) was added 1 ,2-difluoro-4-nitro-benzene (3.95 g, 24.8 mmol) and triethylamine (5.5 mL, 39.7 mmol). The reaction mixture was stirred for 16 h at 60 °C. Ethyl aceate (100 mL) and water (100 mL) were aded to the mixture and the bi-phasic mixture was stirred for 15 min. The organic layer was separated and washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo. to give 6.2 g of crude [1-(2-fluoro-4-nitro-phenyl)-4-piperidyl]methanol (yield: 98.2%).
(b) [1-(4-Amino-2-fluoro-phenyl)-4-piperidyl1methanol
To a solution of [1-(2-fluoro-4-nitro-phenyl)-4-piperidyl]methanol (6.2 g, 24.4 mmol) in ethanol (250 mL) was added 10% Pd on charcoal (620 mg) and catalytic hydrogenation was performed at room temperature o/n. The palladium-catalyst was subsequently removed by filtration and the filtrate was concentrated in vacuo to give 6.38 g of [1-(4-amino-2-fluoro-phenyl)-4- piperidyl]methanol (quantitative crude yield).
(c) 3-[3-Fluoro-4-[4-(hvdroxymethyl)-1-piperidyl1anilino1propanoic acid
To a solution of [1 -(4-amino-2-fluoro-phenyl)-4-piperidyl]methanol (615 mg, 2.74 mmol) in toluene (8 mL) was added acrylic acid (583 uL, 8.50 mmol) and the reaction mixture was stirred at 100 °C for 2.5 h under a nitrogen atmosphere. The reaction mixture was allowed to come to room temperature and was stirred for 16 h. The solvent was removed carefully and the residue was concentrated in vacuo to obtain crude 3-[3-fluoro-4-[4-(hydroxymethyl)-1- piperidyl]anilino]propanoic acid (1 .0 g) as a brown oil.
(d) [1 -[4-(2,4-Dioxohexahvdropyrimidin-1 -yl)-2-fluoro-phenyl1-4-piperidyl1methyl acetate
To a solution of 3-[3-fluoro-4-[4-(hydroxymethyl)-1-piperidyl]anilino]propanoic acid (1 .0 g, 3.39 mmol) in acetic acid (11 mL) was added urea (910 mg, 15.2 mmol) and the reaction mixture was stirred at 120 °C for 12 h under a nitrogen atmosphere. The reaction mixture was allowed to come to room temperature and concentrated in vacuo. The residue was partitioned over water (10 mL) and dichloromethane (10 mL). The organic layer was separated over a PE-filter and evaporated and concentrated in vacuo. The crude product was purified by column chromatography using SiO2 and heptane/ethyl acetate (8/2 to 0/10, v/v). All fractions containing the title compound were collected and concentrated in vacuo to give 321 mg of the title compound (yield: 26%).
(e) 1-[3-Fluoro-4-[4-(hvdroxymethyl)-1-piperidyl1phenyl1hexahvdropyrimidine-2, 4-dione
To a solution of 1-[4-(2,4-dioxohexahydropyrimidin-1-yl)-2-fluoro-phenyl]-4-piperidyl]- methyl acetate (291 mg, 0.81 mmol) in methanol (4 mL) was added acetyl chloride (63 pL, 0.881 mmol) and the reaction mixture was stirred at room temperature o/n. Water (4 mL) was added to the reaction mixture the pH was adjusted to pH = 7 with 5% aq. NaHCOs solution (2 mL). The solids formed were filtered and dried under high vacuum at 40 °C to obtain 1-[3-fluoro-4-[4- (hydroxymethyl)-1-piperidyl]phenyl]hexahydropyrimidine-2, 4-dione (160 mg, 62.2%) as a white solid.
(f) 1-[4-(2,4-dioxohexahvdropyrimidin-1-yl)-2-fluoro-phenyl1piperidine-4-carbaldehyde (Intermediate TLB-Sp55)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp40, and starting from 1-[3-fluoro-4-[4-(hydroxymethyl)-1-piperidyl]phenyl]hexahydro-pyrimidine- 2,4-dione to afford 50 mg of the crude title compound which was used directly in the reductive amination reactions. Data: LCMS (A) Rt : 2.74 min; m/z 338.1 [M+H2O+HP.
Intermediate TLB-Sp56
(3S)-1-[4-(2,6-dioxo-3-piperidyl)-3,5-difluoro-phenyl1pyrrolidine-3-carboxylic acid (I ntermed iate
TLB-Sp56)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp40, and starting from 2-bromo-1 ,3-difluoro-5-iodobenzene and (S)-methyl pyrrolidine-3- carboxylate hydrochloride and 2,6-dibenzyloxy-3-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2- yl)py ridine to afford 680 mg of the title compound. Data: LCMS (A) Rt : 3.83 min; m/z 339.1 [M+H]+.
Intermediate TLB-Sp57
(3S)-1-[4-(2,6-dioxo-3-piperidyl)-3,5-difluoro-phenyl1pyrrolidine-3-carbaldehvde (Intermediate TLB-Sp57)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp40, and starting from 2-bromo-1 ,3-difluoro-5-iodobenzene and (S)-methyl pyrrolidine-3- carboxylate hydrochloride and 2,6-dibenzyloxy-3-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2- yl)pyridine to afford 310 mg of the title compound. Data: LCMS (A) Rt : 3.80 min; m/z 341.1 [M+H2O+H]+.
Intermediate TLB-Sp58
1-[4-[(2,6-dioxo-3-piperidyl)-methyl-amino1-2-fluoro-phenyl1piperidine-4-carbaldehyde
(Intermediate TLB-Sp58)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp49, starting from 1 ,2-difluoro-4-nitro-benzene, fe/Y-butyl piperidine-4-carboxylate and 3- bromopiperidine-2, 6-dione to afford 980 mg of the title compound. Data: LCMS (A) Rt : 1.50 min; m/z 366.2 [M+H2O+H]+.
Intermediate TLB-Sp59
1-[4-[(2,6-Dioxo-3-piperidyl)amino1-2-fluoro-phenyl1-4-fluoro-piperidine-4-carbaldehyde (Intermediate TLB-Sp59)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp49, starting from 1 ,2-difluoro-4-nitro-benzene, ethyl 4-fluoropiperidine-4-carboxylate hydrochloride and 3-bromopiperidine-2, 6-dione to afford 1.8 g of the title compound. Data: LCMS
1-[4-(2,6-Dioxo-3-piperidyl)-3,5-difluoro-phenyl1piperidine-4-carboxylic acid (Intermediate TLB- Sp60)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp40, and starting from 2-bromo-1 ,3-difluoro-5-iodobenzene and fe/Y-butyl piperidine-4- carboxylate and 2,6-dibenzyloxy-3-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)pyridine to afford 115 mg of the title compound. Data: LCMS (A) Rt : 3.73 min; m/z 353.1 [M+H]+.
1-[4-(2,6-dioxo-3-piperidyl)-3,5-difluoro-phenyl1piperidine-4-carbaldehyde (Intermediate TLB- Sp61)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp40, and starting from 2-bromo-1 ,3-difluoro-5-iodobenzene and 4-(dimethoxymethyl)-piperidine and 2,6-dibenzyloxy-3-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)pyridine to afford 210 mg of the title compound. Data: LCMS (A) Rt : 3.98 min; m/z 355.2 [M+FW+HJT
1-[4-(2,6-Dioxo-3-piperidyl)-3,5-difluoro-phenyl1azetidine-3-carbaldehyde (Intermediate TLB-
Sp62)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp40, and starting from 2-bromo-1 ,3-difluoro-5-iodobenzene and azetidin-3-ylmethanol hydrochloride and 2,6-dibenzyloxy-3-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)pyridine to afford 250 mg of the title compound. Data: LCMS (A) Rt : 2.90 min; m/z 327.2 [M+FW+HJT
Intermediate TLB-Sp63
1-[4-(2,6-Dioxo-3-piperidyl)-2-fluoro-phenyl1azetidine-3-carbaldehyde (Intermediate TLB-Sp63)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp40, and starting from 4-bromo-2-fluoro-1 -iodobenzene and azetidin-3-ylmethanol hydrochloride and 2,6-dibenzyloxy-3-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)pyridine to afford 385 mg of the title compound. Data: LCMS (A) Rt : 2.58 min; m/z 309.1 [M+FW+HJT
Intermediate TLB-Sp64
1-[4-(2,6-Dioxo-3-piperidyl)-3,5-difluoro-phenyl1azetidine-3-carboxylic acid (Intermediate TLB- Sp64)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp40, and starting from 2-bromo-1 ,3-difluoro-5-iodobenzene and methyl azetidine-3-carboxylate hydrochloride and 2,6-dibenzyloxy-3-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)pyridine to afford 400 mg of the title compound. Data: LCMS (A) Rt : 3.46 min; m/z 325.1 [M+H]+.
(3/?)-1-[4-(2,6-dioxo-3-piperidyl)-3,5-difluoro-phenyl1piperidine-3-carboxylic acid (I ntermed iate TLB-Sp65)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp40, and starting from 2-bromo-1 ,3-difluoro-5-iodobenzene and methyl (3R)-piperidine-3- carboxylate hydrochloride and 2,6-dibenzyloxy-3-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2- yl)py ridine to afford 521 mg of the title compound. Data: LCMS (A) Rt : 3.92 min; m/z 353.2 [M+H]+. Intermediate TLB-Sp66
(3/?)-1-[4-(2,6-dioxo-3-piperidyl)-3,5-difluoro-phenyl1piperidine-3-carbaldehyde (Intermediate TLB-Sp66)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp40, and starting from 2-bromo-1 ,3-difluoro-5-iodobenzene and methyl (3R)-piperidine-3- carboxylate hydrochloride and 2,6-dibenzyloxy-3-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2- yl)pyridine to afford 201 mg of the title compound. Data: LCMS (A) Rt : 4.0 min; m/z 355.2 [M+H2O+H]+.
5-[3-(2,4-Dioxohexahvdropyrimidin-1-yl)-2-methyl-phenyl1pent-4-vnoic acid (Intermediate TLB-
Sp67)
This compound was prepared in an analogous manner as described in J. Med. Chem. (2023), 66, 4, 2904-2917 starting from 3-iodo-2-methyl-aniline and acrylic acid and 4-pentynoic acid to afford 19.3 mg of the title compound. Data: LCMS (A) Rt : 3.30 min; m/z 301 .1 [M+H]+.
Intermediate TLB-Sp68
6-[3-(2,4-Dioxohexahvdropyrimidin-1-yl)-2-methyl-phenyl1hex-5-vnoic acid (Intermediate TLB- Sp68)
This compound was prepared in an analogous manner as described in J. Med. Chem. (2023), 66, 4, 2904-2917 starting from 3-iodo-2-methyl-aniline and acrylic acid and 4-hexynoic acid to afford 158 mg of the title compound. Data: LCMS (A) Rt : 3.56 min; m/z 315.1 [M+H]+. Intermediate TLB-Sp69
(3S)-1-[4-(2,6-dioxo-3-piperidyl)-3,5-difluoro-phenyl1piperidine-3-carbaldehyde (I ntermed iate TLB-Sp69)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp40, and starting from 2-bromo-1 ,3-difluoro-5-iodobenzene and methyl (3S)-piperidine-3- carboxylate hydrochloride and 2,6-dibenzyloxy-3-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2- yl)pyridine to afford 236 mg of the title compound. Data: LCMS (A) Rt : 4.42 min; m/z 355.2 [M+H2O+H]+.
(3S)-1-[4-(2,6-dioxo-3-piperidyl)-3,5-difluoro-phenyl1piperidine-3-carboxylic acid (Intermediate TLB-Sp70)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp40, and starting from 2-bromo-1 ,3-difluoro-5-iodobenzene and methyl (3S)-piperidine-3- carboxylate hydrochloride and 2,6-dibenzyloxy-3-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2- yl)py ridine to afford 550 mg of the title compound. Data: LCMS (A) Rt : 3.96 min; m/z 353.2 [M+H]+.
1-[3-(2,4-Dioxohexahvdropyrimidin-1-yl)-4-methyl-benzoyl1piperidine-4-carbaldehyde (Intermediate TLB-Sp71)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp24 and Intermediate TLB-Sp49 starting from (2,3,4,5,6-pentafluorophenyl) 3-(2,4- dioxohexahydropyrimidin-1-yl)-4-methyl-benzoate (Intermediate TLB2) and 4- (dimethoxymethyl)piperidine to afford 109 mg of the title compound. Data: LCMS (A) Rt : 2.65 min; m/z 344.2 [M+H]+. Intermediate TLB-Sp72
1-[3-(2,4-Dioxohexahvdropyrimidin-1-yl)-4-methoxy-benzoyl1piperidine-4-carbaldehyde (Intermediate TLB-Sp72)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp24 and Intermediate TLB-Sp49 starting from (2,3,4,5,6-pentafluorophenyl) 3-(2,4- dioxohexahydropyrimidin-1-yl)-4-methoxy-benzoate (Intermediate TLB1) and 4- (dimethoxymethyl)piperidine to afford 141 mg of the title compound. Data: LCMS (A) Rt : 2.58 min; m/z 360.2 [M+H]+.
1-[4-(2,6-Dioxo-3-piperidyl)-3-fluoro-phenyl1piperidine-4-carboxylic acid (Intermediate TLB-
Sp73)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp40, and starting from 1-bromo-2-fluoro-4-iodo-benzene, fe/Y-butyl piperidine-4-carboxy-late and 2,6-dibenzyloxy-3-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)pyridine to afford 298 mg of the title compound.
1-[4-(2,6-Dioxo-3-piperidyl)-3-methoxy-phenyl1piperidine-4-carboxylic acid (Intermediate TLB- Sp74)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp40, and starting from 1-bromo-4-iodo-2-methoxy-benzene, fe/Y-butyl piperidine-4-carboxy-late and 2,6-dibenzyloxy-3-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)pyridine to afford 790 mg of the title compound. Data: LCMS (A) Rt : 2.22 min; m/z 347.2 [M+H]+. Intermediate TLB-Sp75
1-[6-(2,6-Dioxo-3-piperidyl)-5-fluoro-3-pyridyl1piperidine-4-carboxylic acid (Intermediate TLB- Sp75)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp40, and starting from 5-bromo-2-chloro-3-fluoro-pyridine, te/Y-butyl piperidine-4-carboxy-late and 2,6-dibenzyloxy-3-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)pyridine to afford 188 mg of the title compound. Data: LCMS (A) Rt : 3.02 min; m/z 336.2 [M+H]+.
3-[3-(2,4-Dioxohexahvdropyrimidin-1-yl)-4-methoxy-phenyl1prop-2-vnoic acid (I nte rmed iate
TLB-Sp76)
This compound was prepared in an analogous manner as described in J. Med. Chem. (2023) 66, 4, 2904-291 starting from 5-iodo-2-methoxy-aniline and propiolic acid to afford 104 mg of the title compound. Data: LCMS (A) Rt : 2.79 min; m/z 289.1 [M+H]+.
Intermediate TLB-Sp77
1 -[4-(2,4-Dioxohexahvdropyrimidin-1 -yl)-3-methoxy-phenyl1piperidine-4-carboxylic acid
(Intermediate TLB-Sp77)
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp55, and starting from 5-Fluoro-2-nitroanisole and fe/Y-butyl piperidine-4-carboxylate to afford 874 mg of the title compound. Data: LCMS (A) Rt : 2.25 min; m/z 348.2 [M+H]+.
General Procedure A: Reductive amination of E3-ligase-Spacer-COH (TLB-Sp-CHO) to amine containing PLK1 -ligands (Intermediates B-x) A tube was charged with E3-ligase-Spacer-CH0 (0.02 mol) and Intermediate B-x (0.02 mmol) in DCM (1 mL). After stirring at room temperature for 1 h, NaBH(OAc)3 (0.03 mmol) was subsequently added and the mixture was stirred at room temperature o/n. The mixture was quenched with methanol and concentrated under reduced pressure. Purification was performed using preparative LCMS. Fractions containing the title compound were lyophilised to afford the final compounds.
General Procedure B: HATU-coupling of E3-ligase-Spacer-COOH (TLB-Sp-COOH) to amine containing PLK1 -ligands (Intermediates B-x)
A tube was charged with Intermediate TLB-Spx-COOH (0.02 mol) and Intermediate B- x (0.02 mmol) in THF/DMF (1 mL). A/-Ethylmorpholine (0.06 mol) and HATU (0.02 mmol) were subsequently added and the mixture was stirred at room temperature for 4 h. The mixture was concentrated under reduced pressure. Purification was performed using preparative LCMS. Fractions containing the title compound was lyophilised to afford the final compounds.
Example 1
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1-4-piperidyl1methyl1-4-(hvdroxymethyl)-4-piperidyl1-3-methoxy- benzamide
This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B1 and Intermediate TLB-Sp40 to afford 45.5 mg of the title compound (yield: 49%). Data: LCMS (B) Rt : 6.57 min; m/z 840.6 [M+H]+.
Example 2
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1-4-piperidyl1methyl1-4-(2-hvdroxyethyl)-4-piperidyl1-3-methoxy- benzamide
This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B2 and Intermediate TLB-Sp40 to afford 19.9 mg of the title compound (yield: 50%). Data: LCMS (B) Rt : 6.84 min; m/z 854.5 [M+H]+.
Example 3
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1piperidine-4-carbonyl1-4-(2-hvdroxyethyl)-4-piperidyl1-3-methoxy- benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B2 and Intermediate TLB-Sp45 to afford 24.3 mg of the title compound (yield: 55%). Data: LCMS (B) Rt : 7.19 min; m/z 868.5 [M+H]+. Example 4
5-[4-[[4-[[4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-3-methoxy- benzoyl1amino1-4-(2-hvdroxyethyl)-1-piperidyl1methyl1-1-piperidyl1-A/-[(3S)-2,6-dioxo-3- piperidyllpyridine-2-carboxamide
This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B2 and Intermediate TLB-Sp48 to afford 28.0 mg of the title compound (yield: 60%). Data: LCMS (B) Rt : 5.64 min; m/z 880.5 [M+H]+. Example 5
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[[1-[4-[(2,6-dioxo-3- piperidyl)amino1-2-fluoro-phenyl1-4-piperidyl1methyl1-4-(2-hvdroxyethyl)-4-piperidyl1-3-methoxy- benzamide This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B2 and Intermediate TLB-Sp49 to afford 20.2 mg of the title compound (yield: 48%). Data: LCMS (B) Rt : 5.01 min; m/z 869.5 [M+H]+. Example 6
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[1-[[1-[4-[(2,6-dioxo-3- piperidyl)amino1-2-fluoro-phenyl1-4-piperidyl1methyl1-4-(hvdroxymethyl)-4-piperidyl1-3-methoxy- benzamide
This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B1 and Intermediate TLB-Sp49 to afford 15.5 mg of the title compound (yield: 41 %). Data: LCMS (B) Rt : 5.02 min; m/z 855.5 [M+H]+. Example 7
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[1-[[(3S)-1-[2-(2,6-dioxo-
3-piperidyl)-1 ,3-dioxo-isoindolin-5-yl1pyrrolidin-3-yl1methyl1-4-(2-hvdroxyethyl)-4-piperidyl1-3- methoxy-benzamide This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B2 and Intermediate TLB-Sp47 to afford 27.2 mg of the title compound (yield: 60.2%). Data: LCMS (B) Rt : 6.23 min; m/z 891 .5 [M+H]+. Example 8
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[[(3R)-1-[2-(2,6-dioxo-
3-piperidyl)-1 ,3-dioxo-isoindolin-5-yl1pyrrolidin-3-yl1methyl1-4-(2-hvdroxyethyl)-4-piperidyl1-3- methoxy-benzamide
This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B2 and Intermediate TLB-Sp6 to afford 19.3 mg of the title compound (yield: 41 %). Data: LCMS (B) Rt : 6.23 min; m/z 891.6 [M+H]+. Example 9
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[1-[3-[1-[2-(2,6-dioxo-3- piperidyl)-1 ,3-dioxo-isoindolin-5-yl1-4-piperidyl1propanoyl1-4-(hvdroxymethyl)-4-piperidyl1-3- methoxy-benzamide This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate B1 and Intermediate TLB-Sp46 to afford 22.8 mg of the title compound (yield: 46%). Data: LCMS (A) Rt : 3.74 min; m/z 933.5 [M+H]+. Example 10
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[3-[4-[2-(2,6-dioxo-3- piperidyl)-1 ,3-dioxo-isoindolin-5-yl1piperazin-1-yl1propanoyl1-4-(hvdroxymethyl)-4-piperidyl1-3- methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B1 and Intermediate TLB-Sp5 to afford 13.7 mg of the title compound (yield: 33%). Data: LCMS (A) Rt : 2.84 min; m/z 934.5 [M+H]+. Example 11
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1-4-piperidyl1methyl1-3-(2-hvdroxyethyl)pyrrolidin-3-yl1-3-methoxy- benzamide This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B3 and Intermediate TLB-Sp40 to afford 7.0 mg of the title compound (yield: 21 %). Data: LCMS (B) Rt : 6.44 min; m/z 840.6 [M+H]+. Example 12
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1piperidine-4-carbonyl1-3-(2-hvdroxyethyl)pyrrolidin-3-yl1-3-methoxy- benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B3 and Intermediate TLB-Sp45 to afford 13.0 mg of the title compound (yield: 45%). Data: LCMS (B) Rt : 7.11 min; m/z 854.5 [M+H]+. Example 13
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[(3R)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidine-3-carbonyl1-4-(hvdroxymethyl)-4-piperidyl1-3-methoxy- benzamide This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate B1 and Intermediate TLB-Sp50 to afford 7.5 mg of the title compound (yield: 22%). Data: LCMS (B) Rt : 8.31 min; m/z 858.5 [M+H]+. Example 14
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[(3R)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidine-3-carbonyl1-4-(2-hvdroxyethyl)-4-piperidyl1-3-methoxy- benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B2 and Intermediate TLB-Sp50 to afford 16.0 mg of the title compound (yield: 34%). Data: LCMS (B) Rt : 8.23 min; m/z 872.5 [M+H]+. Example 15
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1piperidine-4-carbonyl1-3-(2-hvdroxyethyl)azetidin-3-yl1-3-methoxy- benzamide This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate B4 and Intermediate TLB-Sp45 to afford 10.6 mg of the title compound (yield: 45%). Data: LCMS (B) Rt : 7.13 min; m/z 840.5 [M+H]+. Example 16
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1-4-piperidyl1methyl1-3-(2-hvdroxyethyl)azetidin-3-yl1-3-methoxy- benzamide
This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B4 and Intermediate TLB-Sp40 to afford 12.7 mg of the title compound (yield: 45%). Data: LCMS (B) Rt : 6.29 min; m/z 826.5 [M+H]+. Example 17
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-[[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1-4-piperidyl1methyl1-6-(2-hvdroxyethyl)-2-azaspiro[3.31heptan-6-yl1-3- methoxy-benzamide This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B5 and Intermediate TLB-Sp40 to afford 51.0 mg of the title compound (yield: 40%). Data: LCMS (B) Rt : 6.02 min; m/z 866.6 [M+H]+. Example 18
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[1-[[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1-4-fluoro-4-piperidyl1methyl1-4-(2-hvdroxyethyl)-4-piperidyl1-3-methoxy- benzamide This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B2 and Intermediate TLB-Sp51 to afford 22.0 mg of the title compound (yield: 17%). Data: LCMS (B) Rt : 6.42 min; m/z 872.6 [M+H]+.
Example 19 4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1piperidine-4-carbonyl1-6-(2-hvdroxyethyl)-2-azaspiro[3.31heptan-6-yl1-3- methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B5 and Intermediate TLB-Sp45 to afford 42.0 mg of the title compound (yield: 32%). Data: LCMS (B) Rt : 7.33 min; m/z 880.5 [M+H]+.
Example 20
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[7-[[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1-4-piperidyl1methyl1-2-(2-hvdroxyethyl)-7-azaspiro[3.51nonan-2-yl1-3- methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B6 and Intermediate TLB-Sp40 to afford 17.0 mg of the title compound (yield: 49%). Data: LCMS (B) Rt : 6.24 min; m/z 894.6 [M+H]+. Example 21
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[7-[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1piperidine-4-carbonyl1-2-(2-hvdroxyethyl)-7-azaspiro[3.51nonan-2-yl1-3- methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B6 and Intermediate TLB-Sp45 to afford 9.5 mg of the title compound (yield: 33%). Data: LCMS (B) Rt : 7.76 min; m/z 908.5 [M+H]+. Example 22
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[(3/?,4S)-1-[[1-[4-(2,6- dioxo-3-piperidyl)-2-fluoro-phenyl1-4-piperidyl1methyl1-3-fluoro-4-(2-hvdroxyethyl)-4-piperidyl1-3- methoxy-benzamide This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B7 and Intermediate TLB-Sp40 to afford 55 mg of the title compound (yield: 63%). Data: LCMS (B) Rt : 5.95 min; m/z 872.6 [M+H]+. Example 23
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[1-[[(3S)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidin-3-yl1methyl1-4-(2-hvdroxyethyl)-4-piperidyl1-3-methoxy- benzamide This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B2 and Intermediate TLB-Sp52 to afford 11 .6 mg of the title compound (yield: 28%). Data: LCMS (B) Rt : 6.67 min; m/z 858.8 [M+H]+.
Example 24 4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[7-[(3R)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidine-3-carbonyl1-2-(2-hvdroxyethyl)-7-azaspiro[3.51nonan-
2-yl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate B6 and Intermediate TLB-Sp50 to afford 6.1 mg of the title compound (yield: 18.8%). Data: LCMS (B) Rt : 8.82 min; m/z 912.5 [M+H]+.
Example 25
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[7-[[1-[4-[(2,6-dioxo-3- piperidyl)amino1-2-fluoro-phenyl1-4-piperidyl1methyl1-2-(2-hvdroxyethyl)-7-azaspiro[3.51nonan-2- yll-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B6 and Intermediate TLB-Sp49 to afford 25.8 mg of the title compound (yield: 59.2%). Data: LCMS (B) Rt : 5.12 min; m/z 909.6 [M+H]+. Example 26
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-[[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1-4-piperidyl1methyl1-7-(2-hvdroxyethyl)-2-azaspiro[3.51nonan-7-yl1-3- methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B8 and Intermediate TLB-Sp40 to afford 14.6 mg of the title compound (yield: 43.8%). Data: LCMS (B) Rt : 6.26 min; m/z 894.6 [M+H]+. Example 27
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[1-[2-[1-[4-[(2,6-dioxo-3- piperidyl)amino1phenyl1-4-hvdroxy-4-piperidyl1acetyl1-4-(2-hvdroxyethyl)-4-piperidyl1-3-methoxy- benzamide This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate B2 and Intermediate TLB-Sp53 to afford 16.0 mg of the title compound (yield: 12.9%). Data: LCMS (B) Rt : 5.46 min; m/z 895.6 [M+H]+. Example 28
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1piperidine-4-carbonyl1-7-(2-hvdroxyethyl)-2-azaspiro[3.51nonan-7-yl1-3- methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B8 and Intermediate TLB-Sp45 to afford 6.5 mg of the title compound (yield: 19.2%). Data: LCMS (B) Rt : 7.63 min; m/z 908.6 [M+H]+. Example 29
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-[[1-[4-[(2,6-dioxo-3- piperidyl)amino1-2-fluoro-phenyl1-4-piperidyl1methyl1-6-(2-hvdroxyethyl)-2-azaspiro[3.31heptan-6- yll-3-methoxy-benzamide This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B5 and Intermediate TLB-Sp49 to afford 17.0 mg of the title compound (yield: 47.2%). Data: LCMS (B) Rt : 5.01 min; m/z 881 .6 [M+H]+. Example 30
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-[(3R)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidine-3-carbonyl1-6-(2-hvdroxyethyl)-2-azaspiro[3.31heptan-
6-yl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate B5 and Intermediate TLB-Sp50 to afford 7.0 mg of the title compound (yield: 19.4%). Data: LCMS (B) Rt : 8.37 min; m/z 884.6 [M+H]+.
Example 31 4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[7-[1-[4-[(2,6-dioxo-3- piperidyl)amino1-2-fluoro-phenyl1piperidine-4-carbonyl1-2-(2-hvdroxyethyl)-7-azaspiro[3.51nonan- 2-yl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B6 and Intermediate TLB-Sp54 to afford 16.7 mg of the title compound (yield: 40.8%). Data: LCMS (B) Rt : 6.24 min; m/z 923.7 [M+H]+.
Example 32
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[1-[4-[(2,6-dioxo-3- piperidyl)amino1-2-fluoro-phenyl1piperidine-4-carbonyl1-4-(2-hvdroxyethyl)-4-piperidyl1-3-methoxy- benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B2 and Intermediate TLB-Sp54 to afford 13.4 mg of the title compound (yield: 44.6%). Data: LCMS (B) Rt : 5.76 min; m/z 883.6 [M+H]+. Example 33
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[2-[[(3S)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidin-3-yl1methyl1-7-(2-hvdroxyethyl)-2-azaspiro[3.51nonan-7- yll-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B8 and Intermediate TLB-Sp52 to afford 8.9 mg of the title compound (yield: 26.6%). Data: LCMS (B) Rt : 6.80 min; m/z 898.5 [M+H]+. Example 34
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-[(3R)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidine-3-carbonyl1-7-(2-hvdroxyethyl)-2-azaspiro[3.51nonan-
7-yl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate B8 and Intermediate TLB-Sp50 to afford 15.2 mg of the title compound (yield: 37.6%). Data: LCMS (B) Rt : 8.60 min; m/z 912.5 [M+H]+. Example 35
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-[1-[4-[(2,6-dioxo-3- piperidyl)amino1-2-fluoro-phenyl1piperidine-4-carbonyl1-7-(2-hvdroxyethyl)-2-azaspiro[3.51nonan-
7-yl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B8 and Intermediate TLB-Sp54 to afford 10.1 mg of the title compound (yield: 24.7%). Data: LCMS (B) Rt : 6.19 min; m/z 923.6 [M+H]+. Example 36
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[7-[[(3S)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidin-3-yl1methyl1-2-(2-hvdroxyethyl)-7-azaspiro[3.51nonan-2- yll-3-methoxy-benzamide This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B6 and Intermediate TLB-Sp52 to afford 12.0 mg of the title compound (yield: 31 .4%). Data: LCMS (B) Rt : 6.90 min; m/z 898.6 [M+H]+. Example 37
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[7-[[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1-4-fluoro-4-piperidyl1methyl1-2-(2-hvdroxyethyl)-7-azaspiro[3.51nonan-2- yll-3-methoxy-benzamide This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B6 and Intermediate TLB-Sp51 to afford 6.4 mg of the title compound (yield: 19.8%). Data: LCMS (B) Rt : 6.52 min; m/z 912.5 [M+H]+.
Example 38 4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[2-[[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1-4-fluoro-4-piperidyl1methyl1-7-(2-hvdroxyethyl)-2-azaspiro[3.51nonan-7- yll-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B8 and Intermediate TLB-Sp51 to afford 7.9 mg of the title compound (yield: 26.5%). Data: LCMS (B) Rt : 6.48 min; m/z 912.6 [M+H]+.
Example 39
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[[1-[4-(2,4- dioxohexahvdropyrimidin-1-yl)-2-fluoro-phenyl1-4-piperidyl1methyl1-4-(2-hvdroxyethyl)-4- piperidyll-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B2 and Intermediate TLB-Sp55 to afford 17.3 mg of the title compound (yield: 58.4%). Data: LCMS (B) Rt : 5.92 min; m/z 855.6 [M+H]+. Example 40
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-[[1-[4-(2,4- dioxohexahvdropyrimidin-1-yl)-2-fluoro-phenyl1-4-piperidyl1methyl1-7-(2-hvdroxyethyl)-2- azaspiro[3.51nonan-7-yl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B8 and Intermediate TLB-Sp55 to afford 16.4 mg of the title compound (yield: 55.7%). Data: LCMS (B) Rt : 5.95 min; m/z 895.6 [M+H]+. Example 41
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[7-[[1-[4-(2,4- dioxohexahvdropyrimidin-1-yl)-2-fluoro-phenyl1-4-piperidyl1methyl1-2-(2-hvdroxyethyl)-7- azaspiro[3.51nonan-2-yl1-3-methoxy-benzamide This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B6 and Intermediate TLB-Sp55 to afford 16.8 mg of the title compound (yield: 57.0%). Data: LCMS (B) Rt : 5.96 min; m/z 895.6 [M+H]+. Example 42 cis-4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[4-[[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1-4-piperidyl1oxy1-1-(2-hvdroxyethyl)cvclohexyl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp40b, starting from Intermediate B9b and 2,6-bis(benzyloxy)-3-(4,4,5,5-tetramethyl-1 ,3,2- dioxaborolan-2-yl)pyridine to afford, after catalytic hydrogenation and preparative LCMS purification 28.6 mg of the title compound (yield: 9.2%). Data: LCMS (B) Rt : 8.01 min; m/z 855.6 [M+H]+. Example 43 trans-4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[4-[[1 -[4-(2,6-dioxo-
3-piperidyl)-2-fluoro-phenyl1-4-piperidyl1oxy1-1-(2-hvdroxyethyl)cvclohexyl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in Intermediate TLB- Sp40b, starting from Intermediate B9a and 2,6-bis(benzyloxy)-3-(4,4,5,5-tetramethyl-1 ,3,2- dioxaborolan-2-yl)pyridine to afford, after catalytic hydrogenation and preparative LCMS purification 36.0 mg of the title compound (yield: 13%). Data: LCMS (B) Rt : 8.53 min; m/z 855.6 [M+H]+.
Example 44
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[7-[(3S)-1-[4-(2,6-dioxo- 3-piperidyl)-3,5-difluoro-phenyl1pyrrolidine-3-carbonyl1-2-(2-hvdroxyethyl)-7-azaspiro[3.51nonan-
2-yl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate B6 and Intermediate TLB-Sp56 to afford 9.7 mg of the title compound (yield: 30%). Data: LCMS (B) Rt : 8.81 min; m/z 912.6 [M+H]+. Example 45
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[(3S)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidine-3-carbonyl1-4-(2-hvdroxyethyl)-4-piperidyl1-3-methoxy- benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B2 and Intermediate TLB-Sp56 to afford 10.3 mg of the title compound (yield: 30%). Data: LCMS (B) Rt : 8.34 min; m/z 872.6 [M+H]+. Example 46
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-[(3S)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidine-3-carbonyl1-7-(2-hvdroxyethyl)-2-azaspiro[3.51nonan-
7-yl1-3-methoxy-benzamide This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate B8 and Intermediate TLB-Sp56 to afford 10.6 mg of the title compound (yield: 31.2%). Data: LCMS (B) Rt : 8.57 min; m/z 912.6 [M+H]+. Example 47
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[7-[[(3R)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidin-3-yl1methyl1-2-(2-hvdroxyethyl)-7-azaspiro[3.51nonan-2- yll-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B6 and Intermediate TLB-Sp57 to afford 2.9 mg of the title compound (yield: 10.7%). Data: LCMS (B) Rt : 6.83 min; m/z 898.6 [M+H]+. Example 48
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-[[(3R)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidin-3-yl1methyl1-7-(2-hvdroxyethyl)-2-azaspiro[3.51nonan-7- yll-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B8 and Intermediate TLB-Sp57 to afford 15.8 mg of the title compound (yield: 34.4%). Data: LCMS (B) Rt : 6.79 min; m/z 898.6 [M+H]+. Example 49
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[[(3R)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidin-3-yl1methyl1-4-(2-hvdroxyethyl)-4-piperidyl1-3-methoxy- benzamide
This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B2 and Intermediate TLB-Sp57 to afford 5.3 mg of the title compound (yield: 13.8%). Data: LCMS (B) Rt : 6.74 min; m/z 858.6 [M+H]+. Example 50
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[1-[[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1-4-piperidyl1methyl1-3,3-difluoro-4-(2-hvdroxyethyl)-4-piperidyl1-3- methoxy-benzamide This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B10 and Intermediate TLB-Sp45 to afford 5.0 mg of the title compound (yield: 11 .0%). Data: LCMS (B) Rt : 6.58 min; m/z 890.6 [M+H]+. Example 51
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[[1-[4-[(2,6-dioxo-3- piperidyl)-methyl-amino1-2-fluoro-phenyl1-4-piperidyl1methyl1-4-(2-hvdroxyethyl)-4-piperidyl1-3- methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B2 and Intermediate TLB-Sp58 to afford 18.2 mg of the title compound (yield: 57.6%). Data: LCMS (B) Rt : 5.60 min; m/z 883.6 [M+H]+. Example 52
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[7-[[1-[4-[(2,6-dioxo-3- piperidyl)-methyl-amino1-2-fluoro-phenyl1-4-piperidyl1methyl1-2-(2-hvdroxyethyl)-7- azaspiro[3.51nonan-2-yl1-3-methoxy-benzamide This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B6 and Intermediate TLB-Sp58 to afford 19.8 mg of the title compound (yield: 60.4%). Data: LCMS (B) Rt : 5.63 min; m/z 923.7 [M+H]+.
Example 53 4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[1-[[1-[4-[(2,6-dioxo-3- piperidyl)amino1-2-fluoro-phenyl1-4-fluoro-4-piperidyl1methyl1-4-(2-hvdroxyethyl)-4-piperidyl1-3- methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B2 and Intermediate TLB-Sp59 to afford 35.0 mg of the title compound (yield: 19.9%). Data: LCMS (B) Rt : 5.99 min; m/z 887.6 [M+H]+.
Example 54
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[2-[[(3S)-1-[4-(2,6-dioxo- 3-piperidyl)-3,5-difluoro-phenyl1pyrrolidin-3-yl1methyl1-6-(2-hvdroxyethyl)-2-azaspiro[3.31heptan- 6-yl1-3-methoxy-benzamide This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B5 and Intermediate TLB-Sp52 to afford 10.0 mg of the title compound (yield: 4.5%). Data: LCMS (A) Rt : 2.98 min; m/z 870.6 [M+H]+. Example 55
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[1-[4-(2,6-dioxo-3- piperidyl)-3,5-difluoro-phenyl1piperidine-4-carbonyl1-4-(2-hvdroxyethyl)-4-piperidyl1-3-methoxy- benzamide This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate B2 and Intermediate TLB-Sp60 to afford 5.2 mg of the title compound (yield: 9.1 %). Data: LCMS (B) Rt : 8.11 min; m/z 886.7 [M+H]+.
Example 56 4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[7-[[1-[4-(2,6-dioxo-3- piperidyl)-3,5-difluoro-phenyl1-4-piperidyl1methyl1-2-(2-hvdroxyethyl)-7-azaspiro[3.51nonan-2-yl1-
3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B6 and Intermediate TLB-Sp61 to afford 15.2 mg of the title compound (yield: 46.9%). Data: LCMS (B) Rt : 6.75 min; m/z 912.7 [M+H]+.
Example 57
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[[1-[4-(2,6-dioxo-3- piperidyl)-3,5-difluoro-phenyl1-4-piperidyl1methyl1-4-(2-hvdroxyethyl)-4-piperidyl1-3-methoxy- benzamide
This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B2 and Intermediate TLB-Sp61 to afford 17.4 mg of the title compound (yield: 55.7%). Data: LCMS (B) Rt : 6.77 min; m/z 872.6 [M+H]+. Example 58
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-[[1-[4-(2,6-dioxo-3- piperidyl)-3,5-difluoro-phenyl1-4-piperidyl1methyl1-7-(2-hvdroxyethyl)-2-azaspiro[3.51nonan-7-yl1-
3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B8 and Intermediate TLB-Sp61 to afford 16.4 mg of the title compound (yield: 50.6%). Data: LCMS (B) Rt : 6.81 min; m/z 912.6 [M+H]+. Example 59
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[1-[[1-[4-(2,6-dioxo-3- piperidyl)-3,5-difluoro-phenyl1azetidin-3-yl1methyl1-4-(2-hvdroxyethyl)-4-piperidyl1-3-methoxy- benzamide This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B2 and Intermediate TLB-Sp62 to afford 20.8 mg of the title compound (yield: 68.7%). Data: LCMS (B) Rt : 6.27 min; m/z 844.6 [M+H]+. Example 60
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[7-[[1-[4-(2,6-dioxo-3- piperidyl)-3,5-difluoro-phenyl1azetidin-3-yl1methyl1-2-(2-hvdroxyethyl)-7-azaspiro[3.51nonan-2-yl1-
3-methoxy-benzamide This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B6 and Intermediate TLB-Sp62 to afford 12.6 mg of the title compound (yield: 40.1 %). Data: LCMS (B) Rt : 6.44 min; m/z 884.6 [M+H]+.
Example 61
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-[[1-[4-(2,6-dioxo-3- piperidyl)-3,5-difluoro-phenyl1azetidin-3-yl1methyl1-7-(2-hvdroxyethyl)-2-azaspiro[3.51nonan-7-yl1-
3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B8 and Intermediate TLB-Sp62 to afford 15.2 mg of the title compound (yield: 48.4%). Data: LCMS (B) Rt : 6.41 min; m/z 884.6 [M+H]+. Example 62
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[1-[[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1azetidin-3-yl1methyl1-4-(2-hvdroxyethyl)-4-piperidyl1-3-methoxy- benzamide This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B2 and Intermediate TLB-Sp63 to afford 24.0 mg of the title compound (yield: 80.9%). Data: LCMS (B) Rt : 5.91 min; m/z 826.6 [M+H]+. Example 63
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[7-[[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1azetidin-3-yl1methyl1-2-(2-hvdroxyethyl)-7-azaspiro[3.51nonan-2-yl1-3- methoxy-benzamide This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B6 and Intermediate TLB-Sp63 to afford 19.1 mg of the title compound (yield: 62.0%). Data: LCMS (B) Rt : 6.07 min; m/z 866.6 [M+H]+.
Example 64 4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-[[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1azetidin-3-yl1methyl1-7-(2-hvdroxyethyl)-2-azaspiro[3.51nonan-7-yl1-3- methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B8 and Intermediate TLB-Sp63 to afford 14.6 mg of the title compound (yield: 47.4%). Data: LCMS (B) Rt : 6.101 min; m/z 866.6 [M+H]+.
Example 65
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[1-[4-(2,6-dioxo-3- piperidyl)-3,5-difluoro-phenyl1azetidine-3-carbonyl1-4-(2-hvdroxyethyl)-4-piperidyl1-3-methoxy- benzamide
This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate B2 and Intermediate TLB-Sp64 to afford 12.9 mg of the title compound (yield: 42.0%). Data: LCMS (B) Rt : 7.91 min; m/z 858.5 [M+H]+. Example 66
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[7-[(3R)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1piperidine-3-carbonyl1-2-(2-hvdroxyethyl)-7-azaspiro[3.51nonan-2- yll-3-methoxy-benzamide This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate B6 and Intermediate TLB-Sp65 to afford 11 .3 mg of the title compound (yield: 34.4%). Data: LCMS (B) Rt : 8.87 min; m/z 926.7 [M+H]+. Example 67
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[(3R)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1piperidine-3-carbonyl1-4-(2-hvdroxyethyl)-4-piperidyl1-3-methoxy- benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B2 and Intermediate TLB-Sp65 to afford 12.3 mg of the title compound (yield: 38.8%). Data: LCMS (B) Rt : 8.37 min; m/z 886.6 [M+H]+. Example 68
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-[(3R)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1piperidine-3-carbonyl1-7-(2-hvdroxyethyl)-2-azaspiro[3.51nonan-7- yll-3-methoxy-benzamide This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate B8 and Intermediate TLB-Sp65 to afford 10.1 mg of the title compound (yield: 30.7%). Data: LCMS (B) Rt : 8.55 min; m/z 926.7 [M+H]+. Example 69
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-[(3S)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidine-3-carbonyl1-6-(2-hvdroxyethyl)-2-azaspiro[3.31heptan-
6-yl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate B5 and Intermediate TLB-Sp56 to afford 12.5 mg of the title compound (yield: 37.9%). Data: LCMS (B) Rt : 8.30 min; m/z 884.6 [M+H]+. Example 70
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[7-[2-[4-(2,6-dioxo-3- piperidyl)phenoxy1acetyl1-2-(2-hvdroxyethyl)-7-azaspiro[3.51nonan-2-yl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B6 and Intermediate TLB-Sp33 to afford 10.6 mg of the title compound (yield: 35.5%). Data: LCMS (B) Rt : 7.34 min; m/z 837.6 [M+H]+. Example 71
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[7-[[(3S)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1-3-piperidyl1methyl1-2-(2-hvdroxyethyl)-7-azaspiro[3.51nonan-2- yll-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B6 and Intermediate TLB-Sp66 to afford 9.4 mg of the title compound (yield: 30.6%). Data: LCMS (B) Rt : 6.91 min; m/z 912.6 [M+H]+. Example 72
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[1-[[(3S)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1-3-piperidyl1methyl1-4-(2-hvdroxyethyl)-4-piperidyl1-3-methoxy- benzamide This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B2 and Intermediate TLB-Sp66 to afford 4.5 mg of the title compound (yield: 12.7%). Data: LCMS (B) Rt : 6.86 min; m/z 872.6 [M+H]+. Example 73
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[2-[[(3S)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1-3-piperidyl1methyl1-7-(2-hvdroxyethyl)-2-azaspiro[3.51nonan-7- yll-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B8 and Intermediate TLB-Sp66 to afford 4.8 mg of the title compound (yield: 16.9%). Data: LCMS (B) Rt : 6.90 min; m/z 912.7 [M+H]+. Example 74
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-[5-[3-(2,4- dioxohexahvdropyrimidin-1-yl)-2-methyl-phenyl1pent-4-vnoyl1-7-(2-hvdroxyethyl)-2- azaspiro[3.51nonan-7-yl1-3-methoxy-benzamide This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate B8 and Intermediate TLB-Sp67 to afford 5.8 mg of the title compound (yield: 18.7%). Data: LCMS (B) Rt : 7.96 min; m/z 874.6 [M+H]+. Example 75
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[7-[6-[3-(2,4- dioxohexahvdropyrimidin-1-yl)-2-methyl-phenyl1hex-5-vnoyl1-2-(2-hvdroxyethyl)-7- azaspiro[3.51nonan-2-yl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate B6 and Intermediate TLB-Sp68 to afford 6.5 mg of the title compound (yield: 20.6%). Data: LCMS (B) Rt : 8.27 min; m/z 888.7 [M+H]+. Example 76
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-[[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1azetidin-3-yl1methyl1-6-(2-hvdroxyethyl)-2-azaspiro[3.31heptan-6-yl1-3- methoxy-benzamide This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B5 and Intermediate TLB-Sp63 to afford 12.6 mg of the title compound (yield: 40.2%). Data: LCMS (B) Rt : 5.98 min; m/z 838.6 [M+H]+. Example 77
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-[[1-[4-(2,6-dioxo-3- piperidyl)-3,5-difluoro-phenyl1azetidin-3-yl1methyl1-6-(2-hvdroxyethyl)-2-azaspiro[3.31heptan-6-yl1-
3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B5 and Intermediate TLB-Sp62 to afford 12.9 mg of the title compound (yield: 40.3%). Data: LCMS (B) Rt : 6.36 min; m/z 856.6 [M+H]+. Example 78
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[2-[1-[4-(2,6-dioxo-3- piperidyl)-3,5-difluoro-phenyl1azetidine-3-carbonyl1-6-(2-hvdroxyethyl)-2-azaspiro[3.31heptan-6- yll-3-methoxy-benzamide This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B5 and Intermediate TLB-Sp64 to afford 14.6 mg of the title compound (yield: 44.9%). Data: LCMS (B) Rt : 6.63 min; m/z 870.4 [M+H]+. Example 79
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[[(3R)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1-3-piperidyl1methyl1-4-(2-hvdroxyethyl)-4-piperidyl1-3-methoxy- benzamide This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B2 and Intermediate TLB-Sp69 to afford 17.1 mg of the title compound (yield: 54.8%). Data: LCMS (B) Rt : 6.85 min; m/z 872.7 [M+H]+.
Example 80 4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-[[(3R)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1-3-piperidyl1methyl1-7-(2-hvdroxyethyl)-2-azaspiro[3.51nonan-7- yll-3-methoxy-benzamide This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B8 and Intermediate TLB-Sp69 to afford 6.5 mg of the title compound (yield: 20.1 %). Data: LCMS (B) Rt : 6.93 min; m/z 912.7 [M+H]+. Example 81
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-[[1-[4-(2,6-dioxo-3- piperidyl)-3,5-difluoro-phenyl1-4-piperidyl1methyl1-6-(2-hvdroxyethyl)-2-azaspiro[3.31heptan-6-yl1-
3-methoxy-benzamide This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B5 and Intermediate TLB-Sp61 to afford 16.1 mg of the title compound (yield: 48.8%). Data: LCMS (B) Rt : 6.71 min; m/z 884.6 [M+H]+.
Example 82 4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-[(3R)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1piperidine-3-carbonyl1-6-(2-hvdroxyethyl)-2-azaspiro[3.31heptan-
6-yl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B5 and Intermediate TLB-Sp65 to afford 12.5 mg of the title compound (yield: 37.3%). Data: LCMS (B) Rt : 8.20 min; m/z 898.6 [M+H]+.
Example 83
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[(3S)-1-[4-(2,6-dioxo- 3-piperidyl)-3,5-difluoro-phenyl1piperidine-3-carbonyl1-4-(2-hvdroxyethyl)-4-piperidyl1-3-methoxy- benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B2 and Intermediate TLB-Sp70 to afford 4.4 mg of the title compound (yield: 13.0%). Data: LCMS (B) Rt : 8.25 min; m/z 886.6 [M+H]+. Example 84
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[7-[(3S)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1piperidine-3-carbonyl1-2-(2-hvdroxyethyl)-7-azaspiro[3.51nonan-2- yll-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B6 and Intermediate TLB-Sp70 to afford 12.5 mg of the title compound (yield: 35.5%). Data: LCMS (B) Rt : 8.81 min; m/z 926.6 [M+H]+.
Example 85
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-[(3S)-1-[4-(2,6-dioxo- 3-piperidyl)-3,5-difluoro-phenyl1piperidine-3-carbonyl1-7-(2-hvdroxyethyl)-2-azaspiro[3.51nonan-7- yll-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B8 and Intermediate TLB-Sp70 to afford 11 .0 mg of the title compound (yield: 31 .2%). Data: LCMS (B) Rt : 8.49 min; m/z 926.6 [M+H]+. Example 86
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[2-[(3S)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1piperidine-3-carbonyl1-6-(2-hvdroxyethyl)-2-azaspiro[3.31heptan-
6-yl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate B5 and Intermediate TLB-Sp70 to afford 14.4 mg of the title compound (yield: 42.1 %). Data: LCMS (B) Rt : 8.19 min; m/z 898.6 [M+H]+.
Example 87
A/-[1-[(3S)-1-[4-(2,6-dioxo-3-piperidyl)-3,5-difluoro-phenyl1pyrrolidine-3-carbonyl1-4-(2- hvdroxyethyl)-4-piperidyl1-4-[[(7/?)-7-ethyl-8-isopropyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-3- methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate C1 and Intermediate TLB-Sp56 to afford 34 mg of the title compound (yield: 21 .6%). Data: LCMS (B) Rt : 7.90 min; m/z 846.6 [M+H]+. Example 88
A/-[2-[1-[4-(2,6-dioxo-3-piperidyl)-3,5-difluoro-phenyl1piperidine-4-carbonyl1-7-(2-hvdroxyethyl)-2- azaspiro[3.51nonan-7-yl1-4-[[(7/?)-7-ethyl-8-isopropyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-3- methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate C2 and Intermediate TLB-Sp60 to afford 20 mg of the title compound (yield: 45.6%). Data: LCMS (B) Rt : 7.96 min; m/z 900.6 [M+H]+.
Example 89
A/-[7-[1-[4-(2,6-dioxo-3-piperidyl)-3,5-difluoro-phenyl1piperidine-4-carbonyl1-2-(2-hvdroxyethyl)-7- azaspiro[3.51nonan-2-yl1-4-[[(7/?)-7-ethyl-8-isopropyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-3- methoxy-benzamide
This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate C3 and Intermediate TLB-Sp60 to afford 11 mg of the title compound (yield: 3.5%). Data: LCMS (B) Rt : 8.13 min; m/z 900.7 [M+H]+. Example 90
A/-[2-[[(3S)-1-[4-(2,6-dioxo-3-piperidyl)-3,5-difluoro-phenyl1pyrrolidin-3-yl1methyl1-7-(2- hvdroxyethyl)-2-azaspiro[3.51nonan-7-yl1-4-[[(7/?)-7-ethyl-8-isopropyl-5-methyl-6-oxo-7/7-pteridin-
2-yl1amino1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate C2 and Intermediate TLB-Sp52 to afford 10 mg of the title compound (yield: 28.5%). Data: LCMS (B) Rt : 6.44 min; m/z 872.6 [M+H]+. Example 91
A/-[1-[[(3S)-1-[4-(2,6-dioxo-3-piperidyl)-3,5-difluoro-phenyl1pyrrolidin-3-yl1methyl1-4-(2- hvdroxyethyl)-4-piperidyl1-4-[[(7/?)-7-ethyl-8-isopropyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-3- methoxy-benzamide This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate C1 and Intermediate TLB-Sp52 to afford 18.2 mg of the title compound (yield: 27.2%). Data: LCMS (B) Rt : 6.28 min; m/z 832.6 [M+H]+. Example 92
A/-[7-[[(3S)-1-[4-(2,6-dioxo-3-piperidyl)-3,5-difluoro-phenyl1pyrrolidin-3-yl1methyl1-2-(2- hvdroxyethyl)-7-azaspiro[3.51nonan-2-yl1-4-[[(7/?)-7-ethyl-8-isopropyl-5-methyl-6-oxo-7/7-pteridin-
2-yl1amino1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate C3 and Intermediate TLB-Sp52 to afford 22.2 mg of the title compound (yield: 32.6%). Data: LCMS (B) Rt : 6.37 min; m/z 872.6 [M+H]+. Example 93
A/-[2-[(3S)-1-[4-(2,6-dioxo-3-piperidyl)-3,5-difluoro-phenyl1pyrrolidine-3-carbonyl1-7-(2- hvdroxyethyl)-2-azaspiro[3.51nonan-7-yl1-4-[[(7/?)-7-ethyl-8-isopropyl-5-methyl-6-oxo-7/7-pteridin-
2-yl1amino1-3-methoxy-benzamide This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate C2 and Intermediate TLB-Sp56 to afford 13 mg of the title compound (yield: 30.7%). Data: LCMS (B) Rt : 8.20 min; m/z 886.5 [M+H]+. Example 94
A/-[1-[(3S)-1-[4-(2,6-dioxo-3-piperidyl)-3,5-difluoro-phenyl1pyrrolidine-3-carbonyl1-4-(2- hvdroxyethyl)-4-piperidyl1-4-[[(7/?)-7-ethyl-8-isopropyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-3- methoxy-benzamide
This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate C1 and Intermediate TLB-Sp56 to afford 21 .5 mg of the title compound (yield: 33.4%). Data: LCMS (B) Rt : 7.89 min; m/z 846.5 [M+H]+. Example 95
A/-[7-[(3S)-1-[4-(2,6-dioxo-3-piperidyl)-3,5-difluoro-phenyl1pyrrolidine-3-carbonyl1-2-(2- hvdroxyethyl)-7-azaspiro[3.51nonan-2-yl1-4-[[(7/?)-7-ethyl-8-isopropyl-5-methyl-6-oxo-7/7-pteridin-
2-yl1amino1-3-methoxy-benzamide This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate C3 and Intermediate TLB-Sp56 to afford 23 mg of the title compound (yield: 36.5%). Data: LCMS (B) Rt : 8.42 min; m/z 886.6 [M+H]+. Example 96
A/-[2-[[(3/?)-1-[4-(2,6-dioxo-3-piperidyl)-3,5-difluoro-phenyl1-3-piperidyl1methyl1-7-(2-hvdroxyethyl)-
2-azaspiro[3.51nonan-7-yl1-4-[[(7/?)-7-ethyl-8-isopropyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-3- methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate C2 and Intermediate TLB-Sp69 to afford 15.3 mg of the title compound (yield: 32.7%). Data: LCMS (B) Rt : 6.48 min; m/z 886.6 [M+H]+. Example 97
A/-[1-[[(3/?)-1-[4-(2,6-dioxo-3-piperidyl)-3,5-difluoro-phenyl1-3-piperidyl1methyl1-4-(2-hvdroxyethyl)-
4-piperidyl1-4-[[(7/?)-7-ethyl-8-isopropyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-3-methoxy- benzamide This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate C1 and Intermediate TLB-Sp69 to afford 21 .2 mg of the title compound (yield: 30.5%). Data: LCMS (B) Rt : 6.43 min; m/z 846.6 [M+H]+. Example 98
A/-[7-[[(3/?)-1-[4-(2,6-dioxo-3-piperidyl)-3,5-difluoro-phenyl1-3-piperidyl1methyl1-2-(2-hvdroxyethyl)-
7-azaspiro[3.51nonan-2-yl1-4-[[(7/?)-7-ethyl-8-isopropyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-3- methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate C3 and Intermediate TLB-Sp69 to afford 26.6 mg of the title compound (yield: 40.2%). Data: LCMS (B) Rt : 6.50 min; m/z 886.6 [M+H]+. Example 99
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[3-[(3S)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidine-3-carbonyl1-9-(2-hvdroxyethyl)-3- azaspiro[5.51undecan-9-yl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate B11 and Intermediate TLB-Sp56 to afford 12.4 mg of the title compound (yield: 39.0%). Data: LCMS (B) Rt : 9.23 min; m/z 940.7 [M+H]+. Example 100
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[3-[[(3S)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidin-3-yl1methyl1-9-(2-hvdroxyethyl)-3-azaspiro[5.51undecan-
9-yl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B11 and Intermediate TLB-Sp52 to afford 19.9 mg of the title compound (yield: 63.4%). Data: LCMS (B) Rt : 6.98 min; m/z 926.6 [M+H]+.
Example 101
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[3-[[(3/?)-1-[4-(2,6-dioxo- 3-piperidyl)-3,5-difluoro-phenyl1-3-piperidyl1methyl1-9-(2-hvdroxyethyl)-3-azaspiro[5.51undecan-9- yll-3-methoxy-benzamide This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B11 and Intermediate TLB-Sp69 to afford 16.7 mg of the title compound (yield: 52.5%). Data: LCMS (B) Rt : 7.06 min; m/z 940.7 [M+H]+.
Example 102
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-[(3R)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidine-3-carbonyl1-6-(2-hvdroxyethyl)-2-azaspiro[3.41octan-6- yll-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B12 and Intermediate TLB-Sp50 to afford 22.0 mg of the title compound (yield: 53.9%). Data: LCMS (B) Rt : 8.41 min; m/z 898.6 [M+H]+.
Example 103
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[2-[[(3S)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidin-3-yl1methyl1-6-(2-hvdroxyethyl)-2-azaspiro[3.41octan-6- yll-3-methoxy-benzamide This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B12 and Intermediate TLB-Sp52 to afford 12.5 mg of the title compound (yield: 31.1 %). Data: LCMS (B) Rt : 6.74 min; m/z 884.6 [M+H]+. Example 104
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[2-[[1-[4-(2,6-dioxo-3- piperidyl)-3,5-difluoro-phenyl1azetidin-3-yl1methyl1-6-(2-hvdroxyethyl)-2-azaspiro[3.41octan-6-yl1-
3-methoxy-benzamide This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B12 and Intermediate TLB-Sp62 to afford 20.5 mg of the title compound (yield: 51 .7%). Data: LCMS (B) Rt : 6.49 min; m/z 870.6 [M+H]+.
Example 105 4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[7-[[1-[3-(2,4- dioxohexahvdropyrimidin-1-yl)-4-methyl-benzoyl1-4-piperidyl1methyl1-2-(2-hvdroxyethyl)-7- azaspiro[3.51nonan-2-yl1-3-methoxy-benzamide This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B6 and Intermediate TLB-Sp71 to afford 16.8 mg of the title compound (yield: 41.2%). Data: LCMS (B) Rt : 5.63 min; m/z 919.7 [M+H]+. Example 106
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[2-[[1-[3-(2,4- dioxohexahvdropyrimidin-1-yl)-4-methyl-benzoyl1-4-piperidyl1methyl1-7-(2-hvdroxyethyl)-2- azaspiro[3.51nonan-7-yl1-3-methoxy-benzamide This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B8 and Intermediate TLB-Sp71 to afford 17.6 mg of the title compound (yield: 43.2%). Data: LCMS (B) Rt : 5.60 min; m/z 919.7 [M+H]+.
Example 107 4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[7-[[1-[3-(2,4- dioxohexahvdropyrimidin-1-yl)-4-methoxy-benzoyl1-4-piperidyl1methyl1-2-(2-hvdroxyethyl)-7- azaspiro[3.51nonan-2-yl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B6 and Intermediate TLB-Sp72 to afford 21 mg of the title compound (yield: 51 .3%). Data: LCMS (B) Rt : 5.54 min; m/z 935.7 [M+H]+.
Example 108
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[1-[4-(2,6-dioxo-3- piperidyl)-3,5-difluoro-phenyl1piperidine-4-carbonyl1-4-(2-hvdroxyethyl)azepan-4-yl1-3-methoxy- benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B13 and Intermediate TLB-Sp60 to afford 30 mg of the title compound (yield: 28.0%). Data: LCMS (B) Rt : 8.17 min; m/z 900.6 [M+H]+. Example 109
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[(3R)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1piperidine-3-carbonyl1-4-(2-hvdroxyethyl)azepan-4-yl1-3-methoxy- benzamide
This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate B13 and Intermediate TLB-Sp65 to afford 22 mg of the title compound (yield: 20.6%). Data: LCMS (B) Rt : 8.42 min; m/z 900.6 [M+H]+. Example 110
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[7-[(3S)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidine-3-carbonyl1-2-(2-hvdroxyethyl)-7-azaspiro[3.51nonan-
2-yl1-3-(trifluoromethoxy') benzamide This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate D1 and Intermediate TLB-Sp56 to afford 23 mg of the title compound (yield: 56.2%). Data: LCMS (B) Rt : 9.88 min; m/z 966.6 [M+H]+. Example 111
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[7-[[(3R)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidin-3-yl1methyl1-2-(2-hvdroxyethyl)-7-azaspiro[3.51nonan-2- yl1-3-(trifluoromethoxy') benzamide
This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate D1 and Intermediate TLB-Sp57 to afford 17 mg of the title compound (yield: 42.1 %). Data: LCMS (B) Rt : 7.71 min; m/z 952.6 [M+H]+. Example 112
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[7-[[1-[4-(2,6-dioxo-3- piperidyl)-3,5-difluoro-phenyl1-4-piperidyl1methyl1-2-(2-hvdroxyethyl)-7-azaspiro[3.51nonan-2-yl1-
3-(trifluoromethoxy') benzamide This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate D1 and Intermediate TLB-Sp61 to afford 18 mg of the title compound (yield: 44.0%). Data: LCMS (B) Rt : 7.74 min; m/z 966.6 [M+H]+. Example 113
A/-[(3aS,6a/?)-2-[1-[4-(2,6-dioxo-3-piperidyl)-3,5-difluoro-phenyl1piperidine-4-carbonyl1-5-(2- hydroxyethvD-1 ,3,3a,4,6,6a-hexahvdrocvclopenta[c1pyrrol-5-yl1-4-[[(7R)-8-cvclopentyl-7-ethyl-5- methyl-6-oxo-7H-pteridin-2-yl1amino1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate B14 and Intermediate TLB-Sp60 to afford 3.9 mg of the title compound (yield: 24.8%). Data: LCMS (B) Rt : 8.10 min; m/z 912.6 [M+H]+. Example 114
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[[(3R)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1-3-piperidyl1methyl1-4-(2-hvdroxyethyl)azepan-4-yl1-3-methoxy- benzamide This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B13 and Intermediate TLB-Sp69 to afford 37 mg of the title compound (yield: 32.5%). Data: LCMS (B) Rt : 7.00 min; m/z 886.5 [M+H]+. Example 115
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1-4-piperidyl1methyl1-4-(2-hvdroxyethyl)azepan-4-yl1-3-methoxy- benzamide
This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B13 and Intermediate TLB-Sp40 to afford 43 mg of the title compound (yield: 38.7%). Data: LCMS (B) Rt : 6.26 min; m/z 868.6 [M+H]+. Example 116
A/-[(3a/?,6aS)-2-[[(3S)-1-[4-(2,6-dioxo-3-piperidyl)-3,5-difluoro-phenyl1-3-piperidyl1methyl1-5-(2- hydroxyethvD-l ,3,3a,4,6,6a-hexahvdrocvclopenta[c1pyrrol-5-yl1-4-[[(7/?)-8-cvclopentyl-7-ethyl-5- methyl-6-oxo-7/7-pteridin-2-yl1amino1-3-methoxy-benzamide This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B14 and Intermediate TLB-Sp66 to afford 8.3 mg of the title compound (yield: 42.2%). Data: LCMS (B) Rt : 7.20 min; m/z 898.6 [M+H]+. Example 117
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[8-[(3S)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidine-3-carbonyl1-3-(2-hvdroxyethyl)-1-oxa-8- azaspiro[4.51decan-3-yl1-3-methoxy-benzamide This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate B15 and Intermediate TLB-Sp56 to afford 29 mg of the title compound (yield: 38.8%). Data: LCMS (B) Rt : 8.45 min; m/z 928.5 [M+H]+.
Example 118 4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[8-[[(3R)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidin-3-yl1methyl1-3-(2-hvdroxyethyl)-1-oxa-8- azaspiro[4.51decan-3-yl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B15 and Intermediate TLB-Sp57 to afford 29 mg of the title compound (yield: 40.8%). Data: LCMS (B) Rt : 6.75 min; m/z 914.6 [M+H]+.
Example 119
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[8-[[1-[4-(2,6-dioxo-3- piperidyl)-3,5-difluoro-phenyl1-4-piperidyl1methyl1-3-(2-hvdroxyethyl)-1-oxa-8-azaspiro[4.51decan-
3-yl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B15 and Intermediate TLB-Sp61 to afford 35 mg of the title compound (yield: 48.5%). Data: LCMS (B) Rt : 6.73 min; m/z 928.6 [M+H]+. Example 120
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[3-[1-[4-(2,6-dioxo-3- piperidyl)-3,5-difluoro-phenyl1piperidine-4-carbonyl1-8-(2-hvdroxyethyl)-3-azabicyclo[3.2.11octan-
8-yl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B16 and Intermediate TLB-Sp60 to afford 14 mg of the title compound (yield: 37.3%). Data: LCMS (B) Rt : 8.15 min; m/z 912.5 [M+H]+. Example 121
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[3-[[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1-4-piperidyl1methyl1-8-(2-hvdroxyethyl)-3-azabicyclo[3.2.11octan-8-yl1-3- methoxy-benzamide This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B16 and Intermediate TLB-Sp40 to afford 19 mg of the title compound (yield: 49.0%). Data: LCMS (B) Rt : 5.93 min; m/z 880.6 [M+H]+. Example 122
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-N-[3-[[(3S)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidin-3-yl1methyl1-8-(2-hvdroxyethyl)-3- azabicvclo[3.2.11octan-8-yl1-3-methoxy-benzamide This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B16 and Intermediate TLB-Sp66 to afford 20 mg of the title compound (yield: 54.0%). Data: LCMS (B) Rt : 6.33 min; m/z 884.6 [M+H]+.
Example 123 4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[3-[[(3R)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1-3-piperidyl1methyl1-8-(2-hvdroxyethyl)-3-azabicyclo[3.2.11octan-
8-yl1-3-methoxy-benzamide This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B16 and Intermediate TLB-Sp69 to afford 18 mg of the title compound (yield: 44.1 %). Data: LCMS (B) Rt : 6.73 min; m/z 898.5 [M+H]+. Example 124
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[9-[[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1-4-piperidyl1methyl1-7-(2-hvdroxyethyl)-3-oxa-9-azabicyclo[3.3.11nonan-
7-yl1-3-methoxy-benzamide This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B17 and Intermediate TLB-Sp40 to afford 18 mg of the title compound (yield: 45.7%). Data: LCMS (B) Rt : 6.01 min; m/z 896.6 [M+H]+.
Example 125 4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[9-[[(3S)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidin-3-yl1methyl1-7-(2-hvdroxyethyl)-3-oxa-9- azabicvclo[3.3.11nonan-7-yl1-3-methoxy-benzamide This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B17 and Intermediate TLB-Sp52 to afford 17 mg of the title compound (yield: 52.4%). Data: LCMS (B) Rt : 6.69 min; m/z 900.6 [M+H]+. Example 126
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[9-[[(3R)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1-3-piperidyl1methyl1-7-(2-hvdroxyethyl)-3-oxa-9- azabicvclo[3.3.11nonan-7-yl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B17 and Intermediate TLB-Sp69 to afford 15 mg of the title compound (yield: 45.6%). Data: LCMS (B) Rt : 6.78 min; m/z 914.7 [M+H]+.
Example 127 4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[1-[4-(2,6-dioxo-3- piperidyl)-3-fluoro-phenyl1piperidine-4-carbonyl1-4-(2-hvdroxyethyl)-4-piperidyl1-3-methoxy- benzamide This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B2 and Intermediate TLB-Sp73 to afford 25 mg of the title compound (yield: 59.9%). Data: LCMS (B) Rt : 7.39 min; m/z 868.7 [M+H]+. Example 128
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-[1-[4-(2,6-dioxo-3- piperidyl)-3-fluoro-phenyl1piperidine-4-carbonyl1-7-(2-hvdroxyethyl)-2-azaspiro[3.51nonan-7-yl1-3- methoxy-benzamide This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate B8 and Intermediate TLB-Sp73 to afford 12 mg of the title compound (yield: 27.8%). Data: LCMS (B) Rt : 7.67 min; m/z 908.6 [M+H]+.
Example 129 4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[3-[1-[4-(2,6-dioxo-3- piperidyl)-3-methoxy-phenyl1piperidine-4-carbonyl1-9-(2-hvdroxyethyl)-3-azaspiro[5.51undecan-9- yll-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B11 and Intermediate TLB-Sp74 to afford 11 mg of the title compound (yield: 28.8%). Data: LCMS (B) Rt : 6.99 min; m/z 948.7 [M+H]+.
Example 130
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[7-[1-[4-(2,6-dioxo-3- piperidyl)-3-methoxy-phenyl1piperidine-4-carbonyl1-2-(2-hvdroxyethyl)-7-azaspiro[3.51nonan-2-yl1- 3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B6 and Intermediate TLB-Sp74 to afford 10.4 mg of the title compound (yield: 28.0%). Data: LCMS (B) Rt : 6.62 min; m/z 920.7 [M+H]+. Example 131
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[1-[4-(2,6-dioxo-3- piperidyl)-3-methoxy-phenyl1piperidine-4-carbonyl1-4-(2-hvdroxyethyl)-4-piperidyl1-3-methoxy- benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B2 and Intermediate TLB-Sp74 to afford 14.2 mg of the title compound (yield: 40.0%). Data: LCMS (B) Rt : 6.25 min; m/z 880.7 [M+H]+. Example 132
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[(3R)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1piperidine-3-carbonyl1-4-(2-hvdroxyethyl)-3-methyl-4-piperidyl1-3- methoxy-benzamide This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate B18 and Intermediate TLB-Sp65 to afford 1 1.8 mg of the title compound (yield: 35.3%). Data: LCMS (B) Rt : 8.58 min; m/z 900.7 [M+H]+. Example 133
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1-4-piperidyl1methyl1-4-(2-hvdroxyethyl)-3-methyl-4-piperidyl1-3-methoxy- benzamide
This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B18 and Intermediate TLB-Sp40 to afford 15.9 mg of the title compound (yield: 49.2%). Data: LCMS (B) Rt : 6.38/6.67 min; m/z 868.5 [M+H]+ (mixture of isomers).
Example 134
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[1-[[(3S)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1-3-piperidyl1methyl1-4-(2-hvdroxyethyl)-3-methyl-4-piperidyl1-3- methoxy-benzamide
This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B18 and Intermediate TLB-Sp66 to afford 13.6 mg of the title compound (yield: 41.3%). Data: LCMS (B) Rt : 7.08/7.34 min; m/z 886.7 [M+H]+ (mixture of isomers).
Example 135
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[6-[[(3R)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidin-3-yl1methyl1-2-(2-hvdroxyethyl)-6-azaspiro[3.41octan-2- yll-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B19 and Intermediate TLB-Sp57 to afford 12.5 mg of the title compound (yield: 38.8%). Data: LCMS (A) Rt : 3.05 min; m/z 884.7 [M+H]+.
Example 136
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[6-[[1-[4-(2,6-dioxo-3- piperidyl)-3,5-difluoro-phenyl1-4-piperidyl1methyl1-2-(2-hvdroxyethyl)-6-azaspiro[3.41octan-2-yl1-3- methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure A, starting from Intermediate B19 and Intermediate TLB-Sp61 to afford 17.8 mg of the title compound (yield: 54.5%). Data: LCMS (A) Rt : 3.03 min; m/z 898.8 [M+H]+. Example 137
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[7-[1-[6-(2,6-dioxo-3- piperidyl)-5-fluoro-3-pyridyl1piperidine-4-carbonyl1-2-(2-hvdroxyethyl)-7-azaspiro[3.51nonan-2-yl1- 3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B6 and Intermediate TLB-Sp75 to afford 9.1 mg of the title compound (yield: 23.7%). Data: LCMS (B) Rt : 7.62 min; m/z 909.7 [M+H]+.
Example 138
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[2-[1-[6-(2,6-dioxo-3- piperidyl)-5-fluoro-3-pyridyl1piperidine-4-carbonyl1-7-(2-hvdroxyethyl)-2-azaspiro[3.51nonan-7-yl1- 3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate B8 and Intermediate TLB-Sp75 to afford 8 mg of the title compound (yield: 20.8%). Data: LCMS (A) Rt : 3.39 min; m/z 909.7 [M+H]+.
Example 140
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-(2,2-dideuterio-2- hvdroxy-ethyl)-7-[[(3/?)-1-[4-(2,6-dioxo-3-piperidyl)-3,5-difluoro-phenyl1pyrrolidin-3-yl1methyl1-7- azaspiro[3.51nonan-2-yl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B20 and Intermediate TLB-Sp57 to afford 23.4 mg of the title compound (yield: 61.9%). Data: LCMS (A) Rt : 3.10 min; m/z 900.7 [M+H]+. Example 141
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[6-[(3S)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidine-3-carbonyl1-2-(2-hvdroxyethyl)-6-azaspiro[3.41octan-2- yll-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B19 and Intermediate TLB-Sp56 to afford 3.4 mg of the title compound (yield: 10.4%). Data: LCMS (A) Rt : 3.86 min; m/z 898.6 [M+H]+. Example 142
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[1-[1-[4-(2,6-dioxo-3- piperidyl)-3,5-difluoro-phenyl1piperidine-4-carbonyl1-4-(3-hvdroxypropyl)-4-piperidyl1-3-methoxy- benzamide This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate B21 and Intermediate TLB-Sp60 to afford 18 mg of the title compound (yield: 64.8%). Data: LCMS (A) Rt : 3.72 min; m/z 900.5 [M+H]+. Example 143
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[7-[1-[4-(2,6-dioxo-3- piperidyl)-3,5-difluoro-phenyl1azetidine-3-carbonyl1-2-(2-hvdroxyethyl)-7-azaspiro[3.51nonan-2-yl1-
3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate B6 and Intermediate TLB-Sp64 to afford 17.9 mg of the title compound (yield: 56.1 %). Data: LCMS (B) Rt : 8.40 min; m/z 898.6 [M+H]+. Example 144
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[2-[1-[4-(2,6-dioxo-3- piperidyl)-3,5-difluoro-phenyl1azetidine-3-carbonyl1-7-(2-hvdroxyethyl)-2-azaspiro[3.51nonan-7-yl1-
3-methoxy-benzamide This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B8 and Intermediate TLB-Sp64 to afford 13 mg of the title compound (yield: 40.7%). Data: LCMS (B) Rt : 8.19 min; m/z 898.6 [M+H]+. Example 145
A/-[1-[1-[4-(2,6-dioxo-3-piperidyl)-3,5-difluoro-phenyl1piperidine-4-carbonyl1-4-(2-hvdroxyethyl)-4- piperidyl1-4-[[(7/?)-7-ethyl-8-isopropyl-5-methyl-6-oxo- pteridin-2-yl1amino1-3-methoxy- benzamide This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate C1 and Intermediate TLB-Sp60 to afford 30 mg of the title compound (yield: 38.2%). Data: LCMS (B) Rt : 7.63 min; m/z 860.6 [M+H]+.
Example 146 4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[8-[1-[4-(2,6-dioxo-3- piperidyl)-3,5-difluoro-phenyl1piperidine-4-carbonyl1-3-(2-hvdroxyethyl)-1-oxa-8- azaspiro[4.51decan-3-yl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B15 and Intermediate TLB-Sp60 to afford 26 mg of the title compound (yield: 44.1 %). Data: LCMS (B) Rt : 8.22 min; m/z 942.6 [M+H]+.
Example 147
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[7-[3-[3-(2,4- dioxohexahvdropyrimidin-1-yl)-4-methoxy-phenyl1prop-2-vnoyl1-2-(2-hvdroxyethyl)-7- azaspiro[3.51nonan-2-yl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B6 and Intermediate TLB-Sp76 to afford 3.5 mg of the title compound (yield: 9.6%). Data: LCMS (B) Rt : 7.60 min; m/z 862.6 [M+H]+. Example 148
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[7-[1-[4-(2,4- dioxohexahvdropyrimidin-1-yl)-3-methoxy-phenyl1piperidine-4-carbonyl1-2-(2-hvdroxyethyl)-7- azaspiro[3.51nonan-2-yl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from Intermediate B6 and Intermediate TLB-Sp77 to afford 16 mg of the title compound (yield: 41 .1 %). Data: LCMS (B) Rt : 6.62 min; m/z 921 .6 [M+H]+. Example 149
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[1-[1-[4-(2,6-dioxo-3- piperidyl)-3,5-difluoro-phenyl1piperidine-4-carbonyl1-4-(2-hvdroxyethyl)-3-methoxy-4-piperidyl1-3- methoxy-benzamide This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate B22 and Intermediate TLB-Sp60 to afford 19 mg of the title compound (yield: 54.6%). Data: LCMS (B) Rt : 8.04 min; m/z 916.6 [M+H]+. Example 150
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[7-[[(3S)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidin-3-yl1methyl1-9-(2-hvdroxyethyl)-3-oxa-7- azabicvclo[3.3.11nonan-9-yl1-3-methoxy-benzamide This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B23 and Intermediate TLB-Sp52 to afford 21 mg of the title compound (yield: 57.8%). Data: LCMS (B) Rt : 6.62 min; m/z 900.6 [M+H]+.
Example 151 4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[7-[1-[4-(2,6-dioxo-3- piperidyl)-3,5-difluoro-phenyl1azetidine-3-carbonyl1-9-(2-hvdroxyethyl)-3-oxa-7- azabicyclo[3.3.11nonan-9-yl1-3-methoxy-benzamide This compound was prepared in an analogous manner as described in General
Procedure B, starting from Intermediate B23 and Intermediate TLB-Sp64 to afford 16 mg of the title compound (yield: 46.1 %). Data: LCMS (B) Rt : 7.70 min; m/z 900.6 [M+H]+. Example 152
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[7-[[(3R)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1-3-piperidyl1methyl1-9-(2-hvdroxyethyl)-3-oxa-7- azabicvclo[3.3.11nonan-9-yl1-3-methoxy-benzamide This compound was prepared in an analogous manner as described in General
Procedure A, starting from Intermediate B23 and Intermediate TLB-Sp69 to afford 15 mg of the title compound (yield: 48.8%). Data: LCMS (B) Rt : 6.68 min; m/z 914.6 [M+H]+.
Comparative Example 1 4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1-4-piperidyl1methyl1-4-piperidyl1-3-methoxy-benzamide This compound was prepared in an analogous manner as described in General Procedure A, starting from 4-[[(7R)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl]amino]- 3-methoxy-A/-(4-piperidyl)benzamide hydrochloride and Intermediate TLB-Sp40 to afford 20 mg of the title compound (yield: 54%). Data: LCMS (B) Rt : 6.26 min; m/z 810.5 [M+H]+.
Comparative example 2
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-[(3R)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidine-3-carbonyl1-2-azaspiro[3.51nonan-7-yl1-3-methoxy- benzamide
This compound was prepared in an analogous manner as described in General
Procedure B, starting from A/-(2-azaspiro[3.5]nonan-7-yl)-4-[[(7R)-8-cyclopentyl-7-ethyl-5-methyl- 6-oxo-7/7-pteridin-2-yl]amino]-3-methoxy-benzamide and Intermediate TLB-Sp50 to afford 23 mg of the title compound (yield: 50%). Data: LCMS (B) Rt : 9.40 min; m/z 868.6 [M+H]+.
Comparative example 3
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[2-[[(3S)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidin-3-yl1methyl1-2-azaspiro[3.51nonan-7-yl1-3-methoxy- benzamide
This compound was prepared in an analogous manner as described in General
Procedure A, starting from A/-(2-azaspiro[3.5]nonan-7-yl)-4-[[(7R)-8-cyclopentyl-7-ethyl-5-methyl- 6-oxo-7/7-pteridin-2-yl]amino]-3-methoxy-benzamide and Intermediate TLB-Sp52 to afford 26 mg of the title compound (yield: 46.5%). Data: LCMS (B) Rt : 7.13 min; m/z 854.6 [M+H]+.
Comparative example 4
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[2-[1-[4-(2,6-dioxo-3- piperidyl)-3,5-difluoro-phenyl1azetidine-3-carbonyl1-2-azaspiro[3.51nonan-7-yl1-3-methoxy- benzamide This compound was prepared in an analogous manner as described in General
Procedure B, starting from A/-(2-azaspiro[3.5]nonan-7-yl)-4-[[(7R)-8-cyclopentyl-7-ethyl-5-methyl- 6-oxo-7/7-pteridin-2-yl]amino]-3-methoxy-benzamide and Intermediate TLB-Sp64 to afford 25 mg of the title compound (yield: 51.7%). Data: LCMS (B) Rt : 9.01 min; m/z 854.5 [M+H]+.
Comparative example 5
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[7-[[(3S)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidin-3-yl1methyl1-7-azaspiro[3.51nonan-2-yl1-3-methoxy- benzamide
This compound was prepared in an analogous manner as described in General
Procedure A, starting from A/-(7-azaspiro[3.5]nonan-2-yl)-4-[[(7R)-8-cyclopentyl-7-ethyl-5-methyl- 6-oxo-7H-pteridin-2-yl]amino]-3-methoxy-benzamide and Intermediate TLB-Sp52 to afford 20 mg of the title compound (yield: 45.1 %). Data: LCMS (B) Rt : 7.16 min; m/z 854.5 [M+H]+. Example 154m (ready-to-conjugate intermediate 1)
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1-4-piperidyl1methyl1-4-[2-[[[2-[[(2S)-2-[[2-[[2-[6-(2,5-dioxopyrrol-1 -yl)- hexanoylamino1acetyl1amino1acetyl1amino1-3-phenyl-propanoyl1amino1acetyl1amino1methoxy1- ethyl1-4-piperidyl1-3-methoxy-benzamide
(a) Benzyl 4-[[4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-3-methoxy- benzoyl1amino1-4-(2-hvdroxyethyl) pi peridine-1 -carboxylate
Benzyl 4-amino-4-(2-hydroxyethyl)piperidine-1 -carboxylate (Intermediate 2, 322 mg, 1 .16 mmol) and 4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl]amino]-3-methoxy- benzoic acid (Intermediate A, 488 mg, 1 .05 mmol) were suspended in DMF (5.5 mL). HATU (400 mg, 1 .05 mmol) and DiPEA (561 pL, 3.16 mmol) were added subsequently and the mixture stirred at room temperature o/n. Ethyl acetate was added and the mixture was washed with aq. 0.1 M HCI- solution. The organic layer was separated and washed with 5% NaHCOs-solution, water and brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (dichloromethane to methanol = 99/1 to 94/6 v/v%) to afford 516 mg of the title compound (yield: 72%).
(b) Benzyl 4-[[4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-3-methoxy- benzoyl1amino1-4-[2-[[[2-(9/7-fluoren-9-ylmethoxycarbonylamino)acetyl1amino1methoxy1ethyl1- piperidine-1 -carboxylate
9/7-Fluoren-9-ylmethyl A/-[2-(chloromethylamino)-2-oxo-ethyl]carbamate (0.51 mmol) was dissolved in DMF (4 ml) and benzyl 4-[[4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin- 2-yl]amino]-3-methoxy-benzoyl]amino]-4-(2-hydroxyethyl)piperidine-1-carboxylate (117 mg, 0.17 mmol) was added in one portion. The mixture was stirred at room temperature o/n. Ethyl acetate (100 mL), water (50 mL) and brine (50 mL) were added to the mixture and the bi-phasic mixture was stirred for 15 min. The organic layer was separated and washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by column chromatography using SiO2 and dichloromethane/methanol = 10/0 to 9/1 v/v%. All fractions containing the title compound were collected and concentrated in vacuo to give 60 mg of the title compound (Yield: 36%).
(c) 9/7-Fluoren-9-ylmethyl A/-[2-[2-[4-[[4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2- yl1amino1-3-methoxy-benzoyl1amino1-4-piperidyl1ethoxymethylamino1-2-oxo-ethyl1 carbamate
To a solution of benzyl 4-[[4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2- yl]amino]-3-methoxy-benzoyl]amino]-4-[2-[[[2-(9/7-fluoren-9-ylmethoxycarbonylamino)acetyl]- amino]methoxy]ethyl]-piperidine-1 -carboxylate (87 mg, 0.088 mmol) in methanol (7.5 mL) was added 2M HCI-solution (43.8 pL, 0.088 mmol) and a suspension of 10% Pd on charcoal (1 1 .4 mg) in ethanol (2.3 mL). Catalytic hydrogenation was performed at room temperature o/n. The palladium-catalyst was removed by filtration and the filtrate was concentrated under reduced pressure to give the crude title compound in quantitative yield.
(d) 9/7-Fluoren-9-ylmethyl A/-[2-[2-[4-[[4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin- 2-yl1amino1-3-methoxy-benzoyl1amino1-1-[[1-[4-(2,6-dioxo-3-piperidyl)-2-fluoro-phenyl1-4- piperidyl1methyl1-4-piperidyl1ethoxymethylamino1-2-oxo-ethyl1 carbamate
This compound was prepared in an analogous manner as described in General Procedure A, starting from 9/7-Fluoren-9-ylmethyl A/-[2-[2-[4-[[4-[[(7R)-8-cyclopentyl-7-ethyl-5- methyl-6-oxo-7/7-pteridin-2-yl]amino]-3-methoxy-benzoyl]amino]-4-piperidyl]ethoxymethylami- no]-2-oxo-ethyl]carbamate and Intermediate TLB-Sp40 to afford 57.3 mg of the title compound (yield: 46%).
(e) A/-[4-[2-[[(2-aminoacetyl)amino1methoxy1ethyl1-1 -[[1-[4-(2,6-dioxo-3-piperidyl)-2-fluoro-phe- nyl1-4-piperidyl1methyl1-4-piperidyl1-4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2- yllaminol-3-methoxy-benzamide
To a cold (0 °C) solution of 9/7-fluoren-9-ylmethyl A/-[2-[2-[4-[[4-[[(7R)-8-cyclopentyl-7- ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl]amino]-3-methoxy-benzoyl]amino]-1-[[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl]-4-piperidyl]methyl]-4-piperidyl]ethoxymethylamino]-2-oxo- ethylcarbamate (54.5 mg, 0.047 mmol) in DMF (545 pl) was added 25 eq. of morpholine and the mixture was stirred at 0 °C for 2 h. Purification was performed using preparative LCMS. Fractions containing the title compound was lyophilised to afford 29 mg of the final compound (yield: 66%).
(f) 4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[[1-[4-(2,6-dioxo-3- piperidvD-2-fluoro-phenyl1-4-piperidyl1methyl1-4-[2-[[[2-[[(2S)-2-[[2-[[2-[6-(2,5-dioxopyrrol-1-vD- hexanoylamino1acetyl1amino1acetyl1amino1-3-phenyl-propanoyl1amino1acetyl1amino1methoxy1- ethyl1-4-piperidyl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in General Procedure B, starting from A/-[4-[2-[[(2-aminoacetyl)amino]methoxy]ethyl]-1-[[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl]-4-piperidyl]methyl]-4-piperidyl]-4-[[(7/?)-8-cyclopentyl-7-ethyl-5-me- thyl-6-oxo-7/7-pteridin-2-yl]amino]-3-methoxy-benzamide and (2S)-2-[[2-[[2-[6-(2,5-dioxopyrrol-1- yl)hexanoylamino]acetyl]amino]acetyl]amino]-3-phenyl-propanoic acid to afford 28.0 mg of the title compound (yield: 70%). Data: LCMS (B) Rt : 7.50 min; m/z 698.1 [M+2H]2+. Example 155m (ready-to-conjugate intermediate 2)
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1-4-piperidyl1methyl1-4-[[[2-[[(2S)-2-[[2-[[2-[6-(2,5-dioxopyrrol-1-yl)he- xanoylamino1acetyl1amino1acetyl1amino1-3-phenyl-propanoyl1amino1acetyl1amino1methoxy- -methoxy-benzamide
This compound was prepared in an analogous manner as described in Example 154m (step b, e and f) starting from 4-[[(7R)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7H-pteridin-2- yl]amino]-N-[1-[[1-[4-(2,6-dioxo-3-piperidyl)-2-fluoro-phenyl]-4-piperidyl]methyl]-4-(hydroxyme- thyl)-4-piperidyl]-3-methoxy-benzamide (Example 1) to afford after preparative LCMS 22.0 mg of the title compound (yield: 30.5%). Data: LCMS (A) Rt : 3.08 min; m/z 691 .0 [M+2H]2+.
Example 156m (ready-to-conjugate intermediate 3)
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[1-[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl1piperidine-4-carbonyl1-4-[2-[[[2-[[(2S)-2-[[2-[[2-[6-(2,5-dioxopyrrol-1- yl)hexanoylamino1acetyl1amino1acetyl1amino1-3-phenyl- propanoyl1amino1acetyl1amino1methoxy1ethyl1-4-piperidyl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in Example 154m (step b, e and f) starting from 4-[[(7R)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2- yl]amino]-A/-[1-[[1-[4-(2,6-dioxo-3-piperidyl)-2-fluoro-phenyl]-4-piperidyl]methyl]-4-(hydroxyme- thyl)-4-piperidyl]-3-methoxy-benzamide (Example 3) to afford after preparative LCMS 12.7 mg of the title compound (yield: 24%). Data: LCMS (B) Rt : 8.39 min; m/z 705.2 [M+2H]2+. Example 157m (ready-to-conjugate intermediate 4)
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[1-[[1-[4-[(2,6-dioxo-3- piperidyl)amino1-2-fluoro-phenyl1-4-piperidyl1methyl1-4-[2-[[[2-[[(2S)-2-[[2-[[2-[6-(2,5-dioxopyrrol-1- yl)hexanoylamino1acetyl1amino1acetyl1amino1-3-phenyl- propanoyl1amino1acetyl1amino1methoxy1ethyl1-4-piperidyl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in Example 154m (step b, e and f) starting from 4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2- yl]amino]-A/-[1-[[1-[4-[(2,6-dioxo-3-piperidyl)amino]-2-fluoro-phenyl]-4-piperidyl]methyl]-4-(2- hydroxyethyl)-4-piperidyl]-3-methoxy-benzamide (Example 5) to afford after preparative LCMS 2.3 mg of the title compound (yield: 10.9%). Data: LCMS (B) Rt : 6.68 min; m/z 705.7 [M+2H]2+.
Example 158m (ready-to-conjugate intermediate 5)
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-A/-[2-[[(3S)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidin-3-yl1methyl1-7-[2-[[[2-[[(2S)-2-[[2-[[2-[6-(2,5-dioxopyrrol-
1-yl)hexanoylamino1acetyl1amino1acetyl1amino1-3-phenyl- propanoyl1amino1acetyl1amino1methoxy1ethyl1-2-azaspiro[3.51nonan-7-yl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in Example 154m (step b, e and f) starting from 4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2- yl]amino]-A/-[2-[[(3S)-1-[4-(2,6-dioxo-3-piperidyl)-3,5-difluoro-phenyl]pyrrolidin-3-yl]methyl]-7-(2- hydroxyethyl)-2-azaspiro[3.5]nonan-7-yl]-3-methoxy-benzamide (Example 33) to afford after preparative LCMS 2.25 mg of the title compound (yield: 28.1 %). Data: LCMS (B) Rt : 7.97 min; m/z 720.3 [M+2H]2+.
Example 159m (ready-to-conjugate intermediate 6)
4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-A/-[2-[(3R)-1-[4-(2,6-dioxo-
3-piperidyl)-3,5-difluoro-phenyl1pyrrolidine-3-carbonyl1-7-[2-[[[2-[[(2S)-2-[[2-[[2-[6-(2,5-dioxopyrrol-
1-yl)hexanoylamino1acetyl1amino1acetyl1amino1-3-phenyl- propanoyl1amino1acetyl1amino1methoxy1ethyl1-2-azaspiro[3.51nonan-7-yl1-3-methoxy-benzamide
This compound was prepared in an analogous manner as described in Example 154m (step b, e and f) starting from 4-[[(7R)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2- yl]amino]-A/-[2-[(3R)-1-[4-(2,6-dioxo-3-piperidyl)-3,5-difluoro-phenyl]pyrrolidine-3-carbonyl]-7-(2- hydroxyethyl)-2-azaspiro[3.5]nonan-7-yl]-3-methoxy-benzamide (Example 34) to afford after preparative LCMS 5.4 mg of the title compound (yield: 58%). Data: LCMS (A) Rt : 3.99 min; m/z 727.2 [M+2H]2+.
Example 160m (ready-to-conjugate intermediate 7)
[2-[4-[[4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-3-methoxy- benzoyl1amino1-1-[[1-[4-(2,6-dioxo-3-piperidyl)-2-fluoro-phenyl1-4-piperidyl1methyl1-4- piperidyllethoxy-hydroxy-phosphoryll 2-[6-(2,5-dioxopyrrol-1 -vDhexanoylaminolethyl hydrogen phosphate
(a) Benzyl 4-[[4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-3-methoxy- benzoyl1amino1-4-(2-di-te/Y-butoxyphosphoryloxyethyl) pi peridine-1 -carboxylate
To a solution of di-te/Y-butyl N,N-diethylphosphoramidite in anhydrous THF (10 mL) were added subsequently benzyl 4-[[4-[[(7R)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2- yl]amino]-3-methoxy-benzoyl]amino]-4-(2-hydroxyethyl)piperidine-1-carboxylate (1.5 g, 2.19 mmol) and tetrazole (0.45M solution in acetonitrile, 0.913 mL) and the reaction mixture was stirred for 45 min at room temperature. The reaction mixture was cooled to -40 °C and a solution of 3- chloroperbenzoic acid (1 .57 g, 6.56 mmol) in dichloromethane (5 mL) was added. The mixture was stirred for 15 min allowing the temperature to come to room temperature. The mixture was quenched by addition of 5% aq. sodium sulfite solution. The mixture was extracted with MTBE. The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo. The resulting residue was purified by flash column chromatography (heptane/ethyl acetate = 1/1 to 0/10, v/v) followed by elution with dichloromethane/methanol (95/5, v/v) to give 1.57 g of the title compound (yield: 81.8%).
(b) Di-te/Y-butyl 2-[4-[[4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-3- methoxy-benzoyllaminol-4-piperidyllethyl phosphate
To a solution of benzyl 4-[[4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2- yl]amino]-3-methoxy-benzoyl]amino]-4-(2-di-te/Y-butoxyphosphoryloxyethyl)piperidine-1- carboxylate (1.62 g, 1.85 mmol) in methanol (90 mL) was added 2M HCI-solution (0.92 mL, 1.85 mmol) and a suspension of 10% Pd on charcoal (162 mg) in ethanol (10 mL). Catalytic hydrogenation was performed at room temperature o/n. The palladium-catalyst was removed by filtration and the filtrate was concentrated under reduced pressure to give of the title compound in quantitative crude yield. (c) Di-te/Y-butyl 2-[4-[[4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-3- methoxy-benzoyl1amino1-1-[[1-[4-(2,6-dioxo-3-piperidyl)-2-fluoro-phenyl1-4-piperidyl1methyl1-4- piperidyllethyl phosphate
This compound was prepared in an analogous manner as described in General Procedure A, starting from di-te/Y-butyl 2-[4-[[4-[[(7R)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7- pteridin-2-yl]amino]-3-methoxy-benzoyl]amino]-4-piperidyl]ethyl phosphate and Intermediate TLB-Sp40 to afford 482 mg of the title compound (yield: 71.9%). Data: LCMS (B) Rt : 8.23 min; m/z 1046.7 [M+H]+.
(d) 2-[4-[[4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-3-methoxy- benzoyl1amino1-1-[[1-[4-(2,6-dioxo-3-piperidyl)-2-fluoro-phenyl1-4-piperidyl1methyl1-4- piperidyllethyl dihydroqen phosphate
To solution of di-te/Y-butyl 2-[1-[[1-[4-(2,6-dioxo-3-piperidyl)-2-fluoro-phenyl]-4- piperidyl]methyl]-4-[[3-methoxy-4-[[rac-(7R)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2- yl]amino]benzoyl]amino]-4-piperidyl]ethyl phosphate (339 mg, 0.32 mmol) in dichloromethane (5 mL) was added trifluoroacetic acid (0.48 mL, 6.48 mmol) and the reaction mixture was stirred at room temperature o/n. MTBE (10 mL) was added and the solids formed were filtered, washed with MTBE, dried under vacuum to give 326 mg of the title compound (96% crude yield).
(e) [2-Aminoethoxy(hvdroxy)phosphoryl1 2-[4-[[4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7- pteridin-2-yl1amino1-3-methoxy-benzoyl1amino1-1-[[1-[4-(2,6-dioxo-3-piperidyl)-2-fluoro-phenyl1-4- piperidyllmethvH-4-piperidyllethyl hydrogen phosphate
A tube was charged with te/Y-butyl A/-(2-phosphonooxyethyl)carbamate N,N- diethylethanamine (9.8 mg, 28.6 pmol) and DMF (250 pL) under nitrogen atmosphere. To the mixture was added triethylamine (1 .7 pL, 1 1 .9 pmol) and 1 ,1 '-carbonyldiimidazole (9.7 mg, 0.0596 mmol) . The reaction mixture was stirred for 30 min at room temperature and then treated with methanol (1 .9 pL, 47.7 pmol) . After 30 min zinc chloride (26 mg, 0.191 mmol) and 2-[4-[[4-[[(7R)- 8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl]amino]-3-methoxy-benzoyl]amino]-1-[[1-[4- (2,6-dioxo-3-piperidyl)-2-fluoro-phenyl]-4-piperidyl]methyl]-4-piperidyl]ethyl dihydrogen phosphate (25 mg, 23.9 pmol) were added, and the mixture was stirred at room temperature o/n. Water (1 .5 mL) was added to the mixture and the precipitate formed was separated by centrifugation. The crude was suspended in dichloromethane/trifluoroacetic acid = 3/2, v/v (2.5 mL) and stirred for 2 h at room temperature. The solvent was removed to afford after preparative LCMS 13 mg of the title compound (yield: 45.5%). Data: LCMS (A) Rt : 2.92 min; m/z 1057.2 [M+2H]2+.
(f) [2-[4-[[4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-3-methoxy- benzoyl1amino1-1-[[1-[4-(2,6-dioxo-3-piperidyl)-2-fluoro-phenyl1-4-piperidyl1methyl1-4- piperidyllethoxy-hydroxy-phosphoryll 2-[6-(2,5-dioxopyrrol-1 -vDhexanoylaminolethyl hydrogen phosphate (Example 160m)
This compound was prepared in an analogous manner as described in General Procedure B, starting from [2-aminoethoxy(hydroxy)phosphoryl] 2-[4-[[4-[[(7R)-8-cyclopentyl-7- ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl]amino]-3-methoxy-benzoyl]amino]-1-[[1-[4-(2,6-dioxo-3- piperidyl)-2-fluoro-phenyl]-4-piperidyl]methyl]-4-piperidyl]ethyl hydrogen phosphate and IV- succinimidyl 6-maleimidohexanoate to afford 11 mg of the title compound (yield: 72%). Data: LCMS (B) Rt : 7.39 min; m/z 626.1 [M+2H]2+.
Example 161m (ready-to-conjugate intermediate 8)
[2-[7-[[4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo- pteridin-2-yl1amino1-3-methoxy- benzoyl1amino1-2-[(3/?)-1-[4-(2,6-dioxo-3-piperidyl)-3,5-difluoro-phenyl1pyrrolidine-3-carbonyl1-2- azaspiro[3.51nonan-7-yl1ethoxy-hvdroxy-phosphoryl1 2-[6-(2,5-dioxopyrrol-1- vDhexanoylaminolethyl hydrogen phosphate
This compound was prepared in an analogous manner as described in Example 160m starting from 2-[7-[[4-[[(7/?)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl]amino]-3- methoxy-benzoyl]amino]-2-azaspiro[3.5]nonan-7-yl]ethyl dihydrogen phosphate and Intermediate TLB-Sp50 to afford after preparative LCMS 90 mg of the title compound (yield: 67.6%). Data: LCMS (B) Rt : 9.16 min; m/z 655.1 [M+2H]2+.
Example 162m (ready-to-conjugate intermediate 9)
[2-[7-[[4-[[(7/?)-8-cvclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl1amino1-3-methoxy- benzoyl1amino1-2-[(3/?)-1-[4-(2,6-dioxo-3-piperidyl)-3,5-difluoro-phenyl1pyrrolidine-3-carbonyl1-2- azaspiro[3.51nonan-7-yl1ethoxy-hvdroxy-phosphoryl1 2-[3-[[2-(2,5-dioxopyrrol-1- vDacetyllaminolpropanoylaminolethyl hydrogen phosphate This compound was prepared in an analogous manner as described in Example 160m starting from 2-[7-[[4-[[(7R)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-7/7-pteridin-2-yl]amino]-3- methoxy-benzoyl]amino]-2-azaspiro[3.5]nonan-7-yl]ethyl dihydrogen phosphate, Intermediate TLB-Sp50 and 3-[[2-(2,5-dioxopyrrol-1-yl)acetyl]amino]propanoic acid to afford after preparative LCMS 160 mg of the title compound (yield: 43.8%). Data: LCMS (B) Rt : 8.67 min; m/z 662.6 [M+2H]2+.
Example A
Biochemical kinase assay PLK1
To determine the inhibitory activity of compounds on Polo-like kinase 1 (PLK1), the Lanthanide Chelate Excite (LANCE)® Ultra TR-FRET assay (Revvity) was used. Compounds were serial diluted in dimethyl sulfoxide (DMSO) and subsequently in LANCE reaction buffer, consisting of 50 mM HEPES pH 7.5, 10 mM MgCI2, 1 mM EGTA, pH 8.0, 0.01 % Tween-20, and 2 mM DTT. Compound serial dilutions and a vehicle control (DMSO) were mixed with an equal volume of full- length PLK1 enzyme (Carna Biosciences, cat. no. 05-157) in LANCE reaction buffer. After preincubation of 1 hour in the dark at room temperature, L///ghf-p70S6K (Thr389) substrate (Revvity, cat. no. TRF0126) with ATP was added to start the reaction, leading to a final PLK1 enzyme concentration of 7.5 nM, a final substrate concentration of 50 nM and a final ATP concentration of 5 pM (KM, ATP). The final DMSO concentration in each well was 1 %. The reaction was allowed to proceed for 2 hours in the dark at room temperature. The reaction was stopped by adding 40 mM EDTA STOP solution, diluted in 1x LANCE® detection buffer (Revvity, cat. no. CR97-100). After 5 minutes of incubation, 4 nM Europium-anti-phospho-p70S6K (Thr389) (Revvity, cat. no. TRF0214) diluted in 1x LANCE® detection buffer was added. The mixture was incubated for 1 hour in the dark at room temperature. If PLK1 is able to phosphorylate its substrate, exciting the Europium label on the anti-phospho antibody will lead to energy transfer to the Ulight substrate and result in emitted light at 665 nm. The emitted light is proportional to the amount of substrate phosphorylated. The 665 nm signal was recorded on an Envision multilabel reader (Revvity). Half maximal inhibitory concentration (IC50) was calculated using IDBS XLfit ™5 software (ID Business Solution).
Results of the PLK1 binding affinity are shown in Table 1 below.
AU565 cells (ATCC, CRL-2351) were cultured in RPMI-1640 cell culture medium (ThermoScientific, cat. no. 61870036) supplemented with 10% (v/v) HyClone Bovine Calf Serum (BCS) (Cytiva, cat. no. SH30072.03) and 1 % (v/v) penicillin/streptomycin (ThermoScientific cat. no. 15140122).
OVCAR-3 cells (ATCC, HTB-161) were cultured in RPMI-1640 cell culture medium (ThermoScientific, cat. no. 61870036) supplemented with 10% (v/v) HyClone Bovine Calf Serum (BCS) (Cytiva, cat. no. SH30072.03) and 1 % (v/v) penicillin/streptomycin (ThermoScientific cat. no. 15140122).
SK-BR-3 cells (ATCC, HTB-30) were cultured in McCoy's 5A (Modified) Medium (ThermoScientific, cat. no. 36600088) supplemented with 10% (v/v) Avantor® Seradigm Fetal Bovine calf Serum (FBS) (VWR, cat. no. 97068-258) and 1 % (v/v) penicillin/streptomycin (ThermoScientific cat. no. 15140122).
Hs 578T cells (ATCC, HTB-126) were cultured in Dulbecco's Modified Eagle Medium (DMEM) (ThermoScientific, cat. no. 31966047) supplemented with 10% (v/v) HyClone Bovine Calf Serum (BCS) (Cytiva, cat. no. SH30072.03) and 1 % (v/v) penicillin/streptomycin (ThermoScientific cat. no. 15140122).
LNCaP cells (ATCC, CRL-1740) were cultured in RPMI-1640 cell culture medium (ThermoScientific, cat. no. 61870036) supplemented with 10% (v/v) HyClone Bovine Calf Serum (BCS, Cytiva, cat. no. SH30072.03) and 1 % (v/v) penicillin/streptomycin (ThermoScientific cat. no. 15140122).
PC3 cells (ATCC, CRL-1435) were cultured in Ham’s F-12K (Kaighn’s) Medium (ThermoScientific, cat. No. 21127022) supplemented with 10% (v/v) HyClone Bovine Calf Serum and 1 % (v/v) penicillin/streptomycin.
The identity of all cell lines was confirmed by short tandem repeat (STR) profiling. Cells were banked and used as indicated in the experiments described below.
A day before the assay, a total of 400 cells per well (in 45 pL) for Hs 578T or PC3, 800 cells per well for AU565 or LNCaP, 1600 cells per well for OVCAR-3, and 1000 cells per well for SK-BR-3 were seeded in white 384-well culture plates (Greiner Bio-One, cat. no. 781080) and allowed to attach to the surface for 16-24 hours at 37°C, 95% humidity, and 5% CO2. Subsequently, 5 pl compound solution (final DMSO concentration 0.3%) was added to the cells and incubation was continued for 120 hours, followed by the addition of 24pl ATPIite 1 Step™ (Revvity, cat. no. 6016736) solution to each well. Luminescence was recorded on an Envision multilabel reader (Revvity). Luminescence at 0 hours of compound incubation was recorded separately to distinguish between cell population growth and net cytotoxic effect. In addition, maximum growth was determined by incubation of cells in duplicate in the presence of 0.3% DMSO and no compound. Percentage viability, relative to the maximum growth signal, was used as the main y- axis signal. Half maximal inhibitory concentration (IC50) was fitted by non-linear regression using IDBS XLfit™5 (ID Business Solution) using a 4-parameter logistic curve, yielding a maximum signal, minimum signal, hill-parameter and IC50.
Results of the Hs 578T, AU-565, OVCAR-3 and SK-BR-3 proliferation are shown in Tables 1 a and 1 b below. Table 1a\ IC50 values for in vitro biochemical and cellular assays.
Table 1 a shows the unexpected characteristic that the hydroxyalkyl group or hydroxyalkyl side-chain, in the form of a hydroxymethylene or hydroxyethylene group, simultaneously improves the capability of the bifunctional compounds for PLK1 degradation and their cytoxicity towards tumor cells. This unexpected characteristic is exemplified by the enhanced activity of Example 1 when compared to Comparative 1 , where the latter has the same chemical structure, but lacks the hydroxymethylene group. This unexpected characteristic is further exemplified by the enhanced activity of Example 2 when compared to Comparative 1 , where the latter has the same chemical structure, but lacks the hydroxyethylene group.
Other exemplary results are shown in Table 1 b.
Table 1b: IC50 values for in vitro biochemical and cellular assays
Example C
In vitro cellular A-549 PLK1 -HiBiT assay
Generation of PLK1 -HiBiT expressing A-549 cells
A pool of A549 cells harboring the HiBiT tag at the N terminus of PLK1 (A549_PLK1-HiBiT) was purchased from Promega and cultured in Ham's F-12K (Kaighn's) Medium (ThermoScientific, cat. no. 21127022) supplemented with 10% (v/v) Avantor® Seradigm Fetal Bovine calf Serum (FBS) (VWR, cat. no. 97068-258) and 1 % (v/v) penicillin/streptomycin (ThermoScientific cat. no. 15140122). Cells were single cell cloned and the presence of the HiBiT tag was confirmed by adding the Nano-Gio® HiBiT Lytic Detection System (Promega, N3040) followed by luminescence readout on an Envision multilabel reader (Revvity). A-549 PLK1 -HiBiT cell proliferation assay
For the proliferation assay, 400 cells per well (in 45 pL) for A549_PLK1 -HiBiT were seeded in a white 384-well culture plate (Greiner Bio-One, cat. no. 781080) and allowed to attach to the surface for 16-24 hours at 37°C, 95% humidity, and 5% CO2. 5 pl compound solution was added to the cells and incubation was continued for 6, 24, or 120 hours, followed by the addition of 24pl ATPIite 1Step™ (Revvity, cat no. 6016736) solution to each well. Luminescence was recorded on an Envision multilabel reader (Revvity). Luminescence at 0 hours of compound incubation was recorded separately to distinguish between cell population growth and net cytotoxic effect. In addition, maximum growth was determined by incubation of cells in duplicate in the presence of vehicle control (0.3% DMSO). Percentage viability, relative to the maximum growth signal, was used as the main y-axis signal. Half maximal inhibitory concentration (IC50) was fitted by non-linear regression using IDBS XLfit™5 (ID Business Solution) using a 4-parameter logistic curve, yielding a maximum signal, minimum signal, hill-parameter and IC50.
Results of the A-549 PLK1 -HiBiT cell proliferation assay are shown in Tables 2a and 2b below.
A-549 PLK1-HiBiT high throughput degradation assay
A549_PLK1 -HiBiT cells were seeded at 400 cells/well (in 40 pL) in white 384-well culture plates (Greiner Bio-One, cat. no. 781080) and allowed to attach to the surface for 16-24 hours at 37°C, 95% humidity, and 5% CO2. Cells were incubated in the absence or presence of the proteasome inhibitor MG-132 (Sigma-Aldrich, cat. no. 474790, final concentration 10 pM). MG-132 is a well-described inhibitor of the proteasome and added to check the mechanism of the degradation effect. If a HBC works through the proteasome, its efficacy and potency should be negatively affected by presence of MG132.
All cells were preincubated for 2 hours at 37°C, 95% humidity, and 5% CO2, after which 5 pL compound solution in a range of concentrations was added to the cells. DMSO was used as a vehicle control and the final DMSO concentration was 0.3% in all wells. Incubation was continued for 6 or 24 hours followed by the addition of 24 pL complete Nano-Gio® HiBiT Lytic Detection System (Promega, N3040) solution. Luminescence was recorded on an Envision multilabel reader (Revvity) at room temperature. Percentage HiBiT signal was used as the main y-axis. Degradation concentration 50 (DC50) was fitted by non-linear regression using IDBS XLfit™5 (ID Business Solution) using a 4-parameter logistic curve, yielding a maximum signal, minimum signal, hillparameter and DC50. Curves for Example 1 and Example 2 are shown in Figure 1 . Results of the A-549 PLK1 -HiBiT degradation assay are shown in Tables 2a - 2c below.
Table 2a\ IC50 and DC50 values for in vitro A-549 PLK1 -HiBiT cellular assays. Percentage after the @ symbol indicates the maximum degradation level observed.
Table 2a shows the unexpected characteristic that the hydroxyalkyl group or hydroxyalkyl side-chain, in the form of a hydroxymethylene or hydroxyethylene group, simultaneously improves the capability of the bifunctional compounds for PLK1 degradation and their cytoxicity towards tumor cells. This unexpected characteristic is exemplified by the enhanced activity of Example 1 when compared to Comparative 1 , where the latter has the same chemical structure, but lacks the hydroxymethylene group. This unexpected characteristic is further exemplified by the enhanced activity of Example 2 when compared to Comparative 1 , where the latter has the same chemical structure, but lacks the hydroxyethylene group. Other exemplary results are shown in Table 2b.
Table 2b'. IC50 and DC50 values for in vitro A-549 PLK1-HiBiT cellular assays. Percentage after the
@ symbol indicates the maximum degradation level observed.
Other exemplary results are shown in Table 2c. Table 2c. IC50 values for in vitro biochemical and cellular assays.
Table 2c shows additional unexpected characteristic that the hydroxyalkyl group or hydroxyalkyl side-chain, in the form of a hydroxymethylene or hydroxyethylene group, simultaneously improves the capability of the bifunctional compounds for PLK1 degradation and their cytoxicity towards tumor cells. This unexpected characteristic is exemplified by the enhanced activity of Example 34 when compared to Comparative 2, where the latter has the same chemical structure, but lacks the hydroxyethylene group. This unexpected characteristic is further exemplified by the enhanced activity of Example 33 when compared to Comparative 3, Example 144 when compared to Comparative 4 and Example 36 when compared to Comparative 5 where the latter has the same chemical structure, but lacks the hydroxyethylene group.
Additional exemplary results are shown in Table 3a.
Results of the A-549 PLK1 -HiBit degradation are shown in Table 3a below.
DC50 A-549 PLK1 -HiBit: A means IC50 < 50 nM
B means IC50 between 50 nM and 250 nM
C means IC50 > 250 nM
Results of the A-549 PLK1 -HiBit + MG132 degradation are shown in Table 3a below.
DC50 A-549 PLK1 -HiBit + MG132: A means IC50 < 100 nM
B means IC50 between 100 nM and 316 nM
C means IC50 > 316 nM
Results of the A-549 PLK1 -HiBit proliferation are shown in Table 3a below.
IC50 A-549 PLK1 -HiBit: A means IC50 < 10 nM
B means IC50 between 10 nM and 25 nM
C means IC50 > 25 nM
Table 3a'. IC50 values for in vitro biochemical and cellular assays.
Additional exemplary results are shown in Table 3b-f.
Results of the AU-565 proliferation are shown in Table 3b below. I C50 AU-565 : A means IC50 < 25 nM
B means IC50 between 25 nM and 100 nM
C means IC50 > 100 nM Table 3b\ IC50 values for in vitro cellular AU-565 proliferation assay.
Results of the LNCaP proliferation are shown in Table 3c below.
IC50 LNCaP : A means IC50 < 5 nM
B means IC50 between 5 nM and 25 nM
C means IC50 > 25 nM
Table 3c. IC50 values for in vitro cellular LNCaP proliferation assay.
Results of the OVCAR-3 proliferation are shown in Table 3d below.
IC50 OVCAR-3 : A means IC50 < 25 nM
B means IC50 between 25 nM and 100 nM C means IC50 > 100 nM
Table 3d'. IC50 values for in vitro cellular OVCAR-3 proliferation assay. Results of the Hs578T proliferation are shown in Table 3e below.
IC50 Hs578T : A means IC50 < 25 nM
B means IC50 between 25 nM and 100 nM
C means IC50 > 100 nM
Table 3e: IC50 values for in vitro cellular Hs578T proliferation assay.
Results of the SKBR3 proliferation are shown in Table 3f below. IC50 SKBR3 : A means IC50 < 25 nM
B means IC50 between 25 nM and 100 nM
C means IC50 > 100 nM
Table 3f. IC50 values for in vitro cellular SKBR3 proliferation assay.
Example D
In vitro cellular PLK1 degradation assay
Western blot sample preparation - single dose, in vitro cellular PLK1 degradation
A549_PLK1-HiBiT cells were seeded in 24 well plates (Greiner Bio-One, cat. no. 662165) at a density of 200.000 cells/well in 800 pl culture medium and allowed to attach to the surface for 16-24 hours at 37°C, 95% humidity, and 5% CO2. Cells were incubated in the absence or presence of the proteasome inhibitor MG-132 (Sigma-Aldrich, cat. no. 474790, final concentration 10 pM). DMSO was used as a vehicle control. All cells were preincubated for 2 hours, after which 100 pL compound solution, to a final concentration of 10 pM was added. DMSO was used as a vehicle control and all wells contained 0.3% DMSO. After 24 hours incubation cells were lysed in 50 pL of RIPA lysis buffer (ThermoScientific, cat. no. 89900) supplemented with protease and phosphatase inhibitors (ThermoScientific, cat. no. 78429 and 78426, respectively). Protein concentration of the lysates was determined using the Pierce™ BOA Protein Assay kit (ThermoScientific, cat. no. 23227). Samples were used to perform western blot as described below. Results can be seen in Figure 2. SDS-PAGE and western blot
SDS samples were prepared by diluting lysates in NuPAGE™ LDS Sample Buffer (ThermoScientific, cat. no. NP0007) supplemented with NuPAGE™ Sample Reducing Agent (ThermoScientific, cat. no. NP0009), to a final concentration of 10 pg protein. Samples were denaturated for 5 minutes at 95°C. The samples were, together with a pre-stained protein ladder (ThermoScientific, cat. no. 26616 or 26619), separated on 4-12% Bis-Tris polyacrylamide gels (ThermoScientific, cat. no. NP0329BOX). The separated protein was subsequently transferred to a nitrocellulose membrane (ThermoScientific, cat. no. 88018). Blots were immunologically stained with PLK1 rabbit mAb (Cell Signaling, cat. no. 4513S) and beta-actin rabbit mAb (Cell Signaling, cat. no. 4967S) , followed by HRP conjugated anti-rabbit goat IgG (Cell Signaling, cat. no. 7074S). Alternatively, as indicated, blots were stained by anti-HiBiT Monoclonal Antibody (Promega, cat. no. N7200), followed by HRP conjugated anti-mouse IgG (Cell Signaling, cat. no. 7076S) to detect the HiBiT tag that is exclusively present on the N-terminus of PLK1 . Stained blots were incubated with Clarity™ Western ECL Substrate (BioRad cat. no. 170-5060) and analyzed on a Chemidoc™ imaging system (Biorad). Processing of western blot images was done using ImageLab software (BioRad) with limited contrast adjustment. Beta-actin levels were used as loading control.
Western blot sample preparation: Dose response curve, in vitro cellular PLK1 degradation
A549_PLK1 -HiBiT or AU565 cells were seeded in 24 well plates (Greiner Bio-One, cat. no. 662165) at a density of 200.000 cells/well in 900 pl culture medium and allowed to attach to the surface for 16-24 hours at 37°C, 95% humidity, and 5% CO2. Subsequently, 100 pL compound solution was added, leading to a final concentration range between 3.16 and 10 pM, or vehicle control. The final DMSO concentration in all wells was 0.3%. After 24 hours incubation, cells were lysed in 50 pL RIPA lysis buffer (ThermoScientific, cat. no. 89900) supplemented with protease and phosphatase inhibitors (ThermoScientific, cat. no. 78429 and 78426, respectively). Protein concentration of the lysates was determined using the Pierce™ BCA Protein Assay kit (ThermoScientific, cat. no. 23227). Western blot was performed as described above. The results of PLK1 staining on the A549_PLK1 -HiBiT cell samples can be seen in Figure 3. The results of HiBiT tag staining on A549_PLK1 -HiBiT cells samples can be seen in Figure 4. The results of PLK1 staining on the AU565 cells can be seen in Figure 5.
Western blot sample preparation: Competition assay, in vitro Cellular PLK1 degradation
AU565 cells were seeded in 135 pl at a density of 30.000 cells/well in a 96 well culture plate (Greiner Bio-One, cat. no. 655182) and allowed to attach to the surface for 16-24 hours at 37°C, 95% humidity, and 5% CO2. To determine if Example 1-induced PLK1 protein degradation is mediated through CRBN and PLK1 binding, pomalidomide (a CRBN binder) and BI-2536, (reference PLK1 inhibitor), were combined with Example 1 to investigate binding competition. Compounds were serially diluted in DMSO and subsequently in 20 mM HEPES pH 7.5. 15 pl compound solution was added to the cells. Final compound concentrations ranged from 10000 nM to 100 nM for each compound, in 10-fold dilution steps. DMSO was used as a vehicle control. Final DMSO concentration was 0.4% in all wells. After 24 hours incubation, cells were lysed in 30 pL RIPA lysis buffer (ThermoScientific, cat. no. 89900) supplemented with protease and phosphatase inhibitors (ThermoScientific, cat. no. 78429 and 78426, respectively). Protein concentration of the lysates was determined using the Pierce™ BCA Protein Assay kit (ThermoScientific, cat. no. 23227). Western blot was performed as described above. The results of the competition with Bl- 2536 can be seen in Figure 6. The results of the competition with pomalidomide can be seen in Figure 7.
Example E
Papain cleavage assay
Reaction buffer was freshly prepared (20 mM NaFhPCh, 1 mM EDTA, 20 mM L-cysteine HCI, pH 7.0) and used to wash 100 pl of immobilized papain (agarose resin, Thermo Fisher, Cat# 20341) three times. For the reaction, papain beads were resuspended in 95 pl reaction buffer and 5 pl 10mM ready to conjugate intermediate (Example 154m or Example 155m, final concentration in the assay: 500 pM). The mixture was incubated for 4 and 24 hours at 37°C. After incubation, 110 pl of pure acetonitrile was added, followed by centrifugation to remove precipitated protein and agarose resin. Of the supernatant, 20 pl was injected on a Waters LC-MS system consisting of a Xterra C18 column running a gradient from 5% to 95% of solvent B (acetonitrile + 0.1 % formic acid) mixed in A (95/5 v/v% Milli-Q water/acetonitrile + 0.1 % formic acid) followed by analysis with a Waters 2998 Photodiode Array Detector and Waters Acquity Qda Mass detector. In control samples that are incubated with reaction buffer in absence of papain, a product appears in which the maleimide group has reacted with cysteine in the reaction buffer. In the test samples containing papain, successful protease cleavage of the linker and subsequent self-immolation results in a product that is identical in retention time and mass to the heterobifunctional degrader. Results of the papain assay are shown in Table 4.
Table 4. Results of the papain cleavage assay
Figure 8A shows a chromatogram (LC) of Example 154m in absence of papain. Figure 8B shows a chromatogram after 24h incubation with papain. Figure 8C shows a mass spectrum (MS) of peak number 6 in Figure 8B, indicating presence of a compound with identical mass to Example 2. Example F
Ready to conjugate intermediates Examples 157m, 158m and 159m were coupled to rosopatamab, an antibody that binds human PSMA, according to the following conjugation and analysis procedures:
Conjugation procedure
The stock of the antibody rosopatamab was diluted to 1 mg/mL in 1 mL of phosphate buffered saline pH 7.4 (PBS, ThermoScientific, cat.no. 14190250) with 5 mM EDTA. Specific disulphide bridges in the antibody were reduced by adding 5 molar equivalents of tris(2- carboxyethyl)phosphine (TCEP) (cat. No. OR-5119) to the antibody, followed by incubation for 2 hours at 37°C. After the reduction, 12 molar equivalents of ready-to-conjugate intermediate Examples 157m, 158m or 159m were added to the mixture followed by incubation overnight at room temperature in the dark. Subsequently, the reaction mixture was applied on a pre-equilibrated NAP5 (Cytiva, cat. no., 45-000-151) column and eluted with 0.3 mL PBS with 0.02% Tween-20 added. This removed all excess TCEP and ready-to-conjugate intermediate from the antibody and therefore stopping the conjugation reaction. The protein concentration of the product of the conjugation reaction was determined by absorption at 280 nm and by the bicinchoninic acid (BCA) assay (ThermoScientific, cat. No., 23227).
SDS Page analysis
The products of the conjugation reaction were analyzed by SDS page. Samples were diluted in NuPAGE™ LDS Sample Buffer (ThermoScientific, cat. No. NP0007) supplemented with NuPAGE™ Sample Reducing Agent (ThermoScientific, cat. No. NP0009), to a final concentration of 1 pg protein. Samples were denaturated for 5 minutes at 70°C. The samples were, together with a pre-stained protein ladder (ThermoScientific, cat. No. 26619), separated on 4-12% Bis-Tris polyacrylamide gels (ThermoScientific, cat. No. NP0329BOX) in MES running buffer (ThermoScientific, cat. No. NP0002). Protein in the gel was stained by InstantBlue (Abeam, cat. No. ab1 19211). The results of the SDS-PAGE can be seen in Figure 9, showing covalent conjugation of the ready-to-conjugate intermediates to rosopatamab.
Cell proliferation assay
The PSMA expressing LNCaP cell line was obtained from the ATCC (cat. No. CRL-1740) and cultured in RPMI-1640 cell culture medium (ThermoScientific, cat. no. 61870036) supplemented with 10% (v/v) HyClone Bovine Calf Serum (BCS, Cytiva, cat. no. SH30072.03) and 1 % (v/v) penicillin/streptomycin (ThermoScientific cat. no. 15140122). The PSMA-negative cell line PC3 was obtained from the ATCC (cat. No. CRL-1435) and cultured in Ham’s F-12K (Kaighn’s) Medium (ThermoScientific, cat. No. 21127022) supplemented with 10% (v/v) HyClone Bovine Calf Serum and 1 % (v/v) penicillin/streptomycin. For the proliferation assays, 800 cells per well (in 45 pL) for LNCaP and 400 cells per well for PC3 were seeded in a white 384-well culture plate (Greiner Bio-One, cat. No. 781080) and allowed to attach to the surface for 16-24 hours at 37°C, 95% humidity, and 5% CO2. 5 pl solution of compound or antibody conjugate was added to the cells and incubation was continued for 72 or 120 hours, followed by the addition of 24 pl ATPIite 1 Step™ (Revvity, cat no. 6016736) solution to each well. Luminescence was recorded on an Envision multilabel reader (Revvity). Luminescence at 0 hours of compound incubation was recorded separately to distinguish between cell population growth and net cytotoxic effect. In addition, maximum growth was determined by incubation of cells in duplicate in the presence of vehicle control (0.3% DMSO). Percentage viability, relative to the maximum growth signal, was used as the main y-axis signal. Half maximal growth inhibitory concentration (IC50) was fitted by non-linear regression using IDBS Xlfit ™5 (ID Business Solution) using a 4-parameter logistic curve, yielding a maximum signal, minimum signal, hill-parameter and I C50.
The effects of the pay loads in the LNCaP and PC3 proliferation assays are shown in Table 5. The effects of the conjugates in the LNCaP proliferation assay can be seen in Figure 10. The effects of the conjugates in the PC3 proliferation assay can be seen in Figure 11. The data show that after conjugation the rosopatamab conjugates acquire potent cytotoxic activity. This activity is dependent on presence of the functional antibody, because conjugates are only active when PSMA is present on the cell surface.
Table 5 IC50 values in proliferation assays of the payloads released by Example DACs*.

Claims

1 . A bifunctional compound of Formula (I):
TLB-Sp-PB
Formula (I), or a pharmaceutically acceptable salt, hydrate, and/or solvate thereof, wherein:
TLB is a Targeting Ligase Binding group capable of binding to a E3 ligase; PB is a Protein Binding group capable of binding to Polo-like Kinase 1 (PLK1) and Sp is a spacer, which covalently links the Targeting Ligase Binding group (TLB) to the Protein Binding group (PB), wherein PB has the chemical structure BPLKI-X, wherein BPLKI has the formula:
(BPLKI) , wherein Ri is selected from the group consisting of H, methyl, ethyl or propyl; wherein R2 is selected from the group consisting of (1 -6C)cycloalkyl, (1 -6C)alkyl or benzyl; wherein any of said cycloalkyl, alkyl or benzyl is optionally and independently substituted with one or more substituents selected from halogen, hydroxy and cyano; wherein R3 is selected from the group consisting of OMe, OCHF2, OCF3, OCD3, (1- 3C)alkyl, O(CH2)2OH or O(CH2)3OH; wherein R4 is selected from the group consisting of H or methyl; wherein "T* denotes the point of attachment to the X group; wherein the X group has a formula (X-1) to (X-28) selected from the group consisting of:
(X-1) (X-2) (X-3) (X-4) (X-5)
wherein any of said formula X-1 to X-28 is optionally and independently substituted with one or more substituents selected from fluoro, hydroxy, (1 -3C)alkyl and (1-3C)alkoxy; wherein n is 1 to 4; wherein * denotes the point of attachment to the amide (-C(O)-NH-) group of BPLKI , and wherein denotes the point of attachment to the spacer (Sp).
2. The bifunctional compound of claim 1 , wherein the spacer (Sp) is -S1-S2-S3-S4-S5- , wherein S1 is connected to the Targeting Ligase Binding group (TLB) and S5 is connected to X, wherein:
51 is independently selected from the group consisting of: a direct bond, -N(RS1)-, -O-, -C(O)- N(RS1)-, -N(RS1)-C(O)-, -N(RS1)-C(O)-O-, -C(O)-, -CEC-, -C=C-, -OCH2C(O)-, (1 -4C)alkyl, (3-12C)cycloalkyl and (3-12C)heterocycloalkyl; wherein any of said alkyl, cycloalkyl or heterocycloalkyl group is optionally and independently substituted with with one or more substituents selected from halogen, hydroxy and (1 -3C)alkyl; each RS1 is independently -H or (1-4C)alkyl;
52 is independently selected from the group consisting of: a direct bond, -N(RS2)-, -O-, (1- 4C)alkyl, -CH2-CH2-N(RS2)-, -(CH2-CH2-O)m-, -(CH2-CH2-CH2-O)m-, -(O-CH2-CH2)m-, (6- 10C)aryl, (3-12C)cycloalkyl, (3-12C)heterocycloalkyl, 7-12 membered spiro bicyclic heterocycloalkyl, 7-12 membered fused bicyclic heterocycloalkyl, 7-12 membered spiro bicyclic cycloalkyl and 7-12 membered fused bicyclic cycloalkyl; wherein any of said alkyl, cycloalkyl or heterocycloalkyl group is optionally and independently substituted with with one or more substituents selected from halogen, oxo, hydroxy, (1 -3C)alkyl and (1- 3C)alkoxy; each RS2 is independently -H or (1 -4C)alkyl;
53 is independently selected from the group consisting of: a direct bond, (1 -8C)alkyl, -CEC-, -N(RS3)-, -O-, -N(RS3)-C(O)-, -C(O)-N(RS3)-, -C(O)-, -(O-CH2-CH2)U-, -(CH2-CH2-O)U- and (3-12C)heterocycloalkyl; wherein any of said heterocycloalkyl group is optionally and independently substituted with with one or more substituents selected from halogen, oxo, hydroxy, (1 -3C)alkyl and (1 -3C)alkoxy; each RS3 is independently -H or (1 -4C)alkyl;
S4 is independently selected from the group consisting of: a direct bond, (1 -4C)alkyl, -N(RS4), -C(O)-, -(O-CH2-CH2)P-, -(CH2-CH2-O)P-, (3-12C)cycloalkyl, (3-12C)heterocycloalkyl, 7-12 membered spiro bicyclic heterocycloalkyl and 7-12 membered fused bicyclic heterocycloalkyl; wherein any of said alkyl, cycloalkyl or heterocycloalkyl group is optionally and independently substituted with with one or more substituents selected from halogen, oxo, hydroxy, (1 -3C)alkyl and (1-3C)alkoxy; each RS4 is independently -H or (1- 4C)alkyl;
S5 is independently selected from the group consisting of: a direct bond, -N(RS5)-, -N(RS5)-C(O)-, -C(O)-, (1 -4C)alkyl, -(O-CH2-CH2)q- and -(O-CH2-CH2-CH2)q-; each RS5 is independently -H or (1 -4C)alkyl; each m, p, u and q is independently an integer from 1 to 3.
3. The bifunctional compound of any one of the preceding claims, wherein the Targeting Ligase Binding group (TLB) is any one of:
wherein the T marks point of attachment to Sp or to S1 of Sp; wherein, t is the number of R(TLB1) substituents and is an integer from 0 to 2; each RTLB1 is independently selected from halo, cyano and (1 -4C)alkyl, wherein any of said alkyl group is optionally and independently substituted with one or more substituents selected from halogen, cyano, -COOH, COONH2, -NH2 and CF3; each RTLB4 is independently selected from hydrogen, (1 -4C)alkyl and (1-3C)alkoxy; each RTLB5 is independently selected from hydrogen, halo, cyano and (1 -3C)alkyl;
Z is -C(RTLB6)2 or -C(O)-; each RTLB6 is independently selected from hydrogen and (1 - 4C)alkyl;
Z’ is a bond, -C(O)- or -CH2C(O)-;
Z” is -CH2- or -NH-;
HAr is a (1 -9C)heteroaryl, optionally substituted with one or more substituents selected from fluoro, methyl and methoxy; and heterocyclic ring G is selected from:
4. Bifunctional compound according to any of the preceding claims, wherein X is selected from
(X-8) (X-9) (X-18) (X-19) (X-20)
(X-21) (X-22) (X-23) (X-24) (X-28) wherein n is 1 ,2 or 3; wherein the * denotes the point of attachment to the amide (C(O)-NH-) group of denotes the point of attachment to the spacer (Sp).
5. Bifunctional compound according to any of the preceding claims, wherein R1 is ethyl or propyl, and wherein R2 is isopropyl, cyclohexyl, or cyclopentyl, and wherein R3 is OMe, OCD3 or OCF3, and wherein R4 is H or methyl .
6. Bifunctional compound according to any one of the preceding claims 2-5, wherein S1 is selected from the group consisting of: a direct bond, -N(RS1)-, -O-, -C(O)-N(RS1)-, -N(RS1)- C(O)-, -C(O)-, -C=C-, -OCH2C(O)-, (3-12C)heterocycloalkyl and (1 -4C)alkyl; wherein any said alkyl or heterocycloalkyl group is optionally and independently substituted with with one or more substituents selected from fluoro and hydroxy; each RS1 is independently -H or methyl.
7. Bifunctional compound according to any one of the preceding claims 2-6, wherein S2 is selected from the group consisting of: a direct bond, (1 -4C)alkyl, -CH2-CH2-N(RS2)-, -(CH2- CH2-O)m-, -(CH2-CH2-CH2-O)m-, (6-10C)aryl, (3-12C)cycloalkyl, (3-12C)heterocycloalkyl, 7-12 membered spiro bicyclic heterocycloalkyl, 7-12 membered fused bicyclic heterocycloalkyl, 7- 12 membered spiro bicyclic cycloalkyl and 7-12 membered fused bicyclic cycloalkyl; wherein any of said alkyl, cycloalkyl or heterocycloalkyl group is optionally and independently substituted with with one or more substituents selected from halogen, oxo, hydroxy, (1- 3C)alkyl and (1-3C)alkoxy; each RS2 is independently -H or methyl; m is an integer from 1 to 2.
8. Bifunctional compound according to any one of the preceding claims 1 -7, wherein S3 is selected from the group consisting of: a direct bond, (1 -8C)alkyl, -N(RS3)-, -O-, -N(RS3)-C(O)-, -C(O)-N(RS3)-, -C(O)-, -(O-CH2-CH2)U-, -(CH2-CH2-O)U- and (3-12C)heterocycloalkyl; wherein any of said or heterocycloalkyl group is optionally and independently substituted with with one or more substituents selected from halogen, oxo, hydroxy, (1 -3C)alkyl and (1-3C)alkoxy; each RS3 is independently -H or methyl; u is an integer from 1 to 2.
9. Bifunctional compound according to any one of the preceding claims 1 -8, wherein S4 is selected from the group consisting of: a direct bond, (1 -4C)alkyl, -(O-CH2-CH2)p-, -(CH2-CH2- O)P-, (3-12C)cycloalkyl and (3-12C)heterocycloalkyl; wherein any of said alkyl, cycloalkyl or heterocycloalkyl group is optionally and independently substituted with with one or more substituents selected from halogen, oxo, hydroxy, (1 -3C)alkyl and (1-3C)alkoxy; p is an integer from 1 to 2.
10. Bifunctional compound according to any one of the preceding claims 1 -9, wherein S5 is selected from the group consisting of: a direct bond, -N(RS5)-, -N(RS5)-C(O)-, -C(O)-, (1- 4C)alky I, -(O-CH2-CH2)q and -(O-CH2-CH2-CH2)q; each RS5 is independently -H or methyl; q is independently an integer from 1 to 2.
11. Bifunctional compound according to any one of the preceding claims 1 -10, wherein S1 is selected from the group consisting of: a direct bond, -NH-, -O-, -C(O)-NH-, -NH-C(O)-, C(O)-, -C=C-, -OCH2C(O)-, (1 -4C)alkyl.
12. Bifunctional compound according to any one of the preceding claims 1 -11 , wherein S2 is selected from the group consisting of: a direct bond, (1 -4C)alkyl, -CH2-CH2-NH-, -(CH2-CH2-
wherein any of said alkyl, cycloalkyl or heterocycloalkyl group is optionally and independently substituted with with one or more substituents selected from halogen, oxo, hydroxy, (1- 3C)alkyl and (1-3C)alkoxy; m is an integer from 1 to 2.
13. Bifunctional compound according to any one of the preceding claims 1 -12, wherein S3 is selected from the group consisting of: a direct bond, (1 -4C)alkyl, -N(RS3)-, -O-, -N(RS3)-C(O)-, -C(O)-N(RS3)-, -C(O)-, -(O-CH2-CH2)U-, -(CH2-CH2-O)U- and (3-12C)heterocycloalkyl; wherein any of said alkyl or heterocycloalkyl group is optionally and independently substituted with with one or more substituents selected from halogen, oxo, hydroxy, (1 -3C)alkyl and (1- 3C)alkoxy; each RS3 is independently -H or methyl; u is an integer from 1 to 2.
14. Bifunctional compound according to any one of the preceding claims 1 -13, wherein S4 is selected from the group consisting of: a direct bond, (1 -4C)alkyl, -N(RS4), -C(O)-, -(O-CH2-
wherein any of said alkyl, cycloalkyl or heterocycloalkyl group is optionally and independently substituted with one or more substituents selected from halogen, oxo, hydroxy, (1 -3C)alkyl and (1-3C)alkoxy; each RS4 is independently hydrogen or methyl; p is an integer from 1 to 2.
15. Bifunctional compound according to any one of the preceding claims 1 -14 wherein S5 is selected from the group consisting of: a direct bond, -N(RS5)-, -N(RS5)-C(O)-, -C(O)- and (1- 4C)alkyl; each RS5 is independently hydrogen or methyl.
16. Bifunctional compound according to any one of the preceding claims 1 -15, wherein the spacer (Sp) has the chemical structure -S1-SpX-, wherein S1 is selected from the group consisting of: a direct bond, -NH-, -O-, -C(O)NH-, -NH-C(O)-, -C(O)-, -CEC-, -OCH2C(O)-, (1- 4C)alkyl; wherein S1 is connected to the Target Ligase Binding group (TLB) and SpX is connected to the X group, and wherein SpX is selected from the group consisting of:
wherein the marks the point of attachment to S1 or marks the point of attachment to the Targeting Ligase Binding group (TLB) in case S1 is a direct bond; wherein the I marks the point of attachment to X; wherein each RL is hydrogen or methyl; wherein s is an integer from 0 to 5; and wherein t is an integer from 0 to 6.
17. Bifunctional compound according to any one of the preceding claims 1 -16, wherein spacer (Sp) is selected from the group consisting of: wherein the marks the point of attachment to the Targeting Ligase Binding group (TLB); and wherein the “T" marks the point of attachment to X.
18. Bifunctional compound according to any one of the preceding claims 1 -17, wherein the Targeting Ligase Binding group (TLB) is selected from the group consisting of:
wherein the T~ marks the point of attachment to the spacer (Sp) or S1 of the spacer (Sp); wherein each RTLB5 is independently hydrogen, fluoro or cyano; and HAr is a (1 -9C)heteroaryl optionally substituted with one or more substituents selected from fluoro, methyl and methoxy.
19. Bifunctional compound according to any one of the preceding claims 1 -18, wherein the Targeting Ligase Binding group (TLB) is selected from the group consisting of: wherein the marks the point of attachment to the spacer (Sp) or S1 of the spacer (Sp).
20. Bifunctional compound according to any one of the preceding claims 1 -19, wherein the
Targeting Ligase Binding group (TLB) is selected from the group consisting of: wherein the T~ marks the point of attachment to the spacer (Sp) or S1 of the spacer (Sp).
21. Bifunctional compound according to any one of the preceding claims 1-20, wherein the chemical structure BPLKI-X is selected from the group consisting of:
(BpLKi-X-7b) (Bpi_Ki-X-8a) (BpLKi-X-8b)
(BpLKi-X-24b) (BpLKi-X-28b) (BpLKi-X-2b2)
(BpLKi-X-2b3) wherein Ri is selected from the group consisting of H, methyl, ethyl or propyl; wherein R2 is selected from the group consisting of (1-6C)cycloalkyl, (1 -6C)alkyl or benzyl; wherein R4 is selected from the group consisting of H or methyl; wherein denotes the point of attachment to the spacer (Sp).
22. Bifunctional compound according to claim 1 , wherein the bifunctional compound has a sub- formula 1 - 152 selected from the group consisting of:
1 2
41 42 43
50 51 52
5
68 69 70
14
87 88
5
104 105 106
113 114 115
152
23. Bifunctional compound according to any one of the preceding claims, wherein said compound is suitable for inducing intracellular degradation of Polo-like Kinase 1 (PLK1), wherein preferably said bifunctional compound has a sub-formula as shown in claim 22 selected from the group consisting of 1 - 152.
24. Bifunctional compound according to any of the preceding claims or a pharmaceutically acceptable salt thereof, for use as a medicament.
25. Bifunctional compound according to any of the claims 1 - 23 or a pharmaceutically acceptable salt thereof, for use in therapy.
26. Bifunctional compound according to any of the claims 1 - 23 or a pharmaceutically acceptable salt thereof, for use in the prevention or treatment of disorders through modulation of Polo-like Kinase 1 (PLK1).
27. Bifunctional compound according to any of the claims 1 - 23 or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer.
28. Bifunctional compound according to any of the claims 1 - 23 or a pharmaceutically acceptable salt thereof, for use in the treatment of disorders or diseases selected from the group consisting of cancer, benign tumor, a neurological disorder, an inflammatory disease, a disease characterized by inflammasome activation, an autoimmune disease, a hyperproliferative disease, a disorder that is caused by tissue rejection by the immune system, or for use in a therapy to prevent tissue rejection by the immune system.
29. Bifunctional compound according to claim 28, where the neurological disorder is one or more selected from the group consisting of central nervous system disease, neurodegenerative disease, Alzheimer's disease, Parkinson's disease, multiple sclerosis, Huntington's disease, senile dementia, epilepsy, Lou Gehrig, stroke, and nerve damage and axonal degeneration- related disorders following brain or spinal cord injury.
30. Bifunctional compound according to claim 28, wherein the cancer or benign tumor is one or more selected from the group consisting of squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, skin cancer, skin or intraocular melanoma, rectal cancer, anal muscle cancer, esophageal cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, double hit lymphoma, hepatocellular carcinoma, gastrointestinal cancer, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumor, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, brain cancer, osteosarcoma, Barrett's esophagus, colon adenoma and polyp, breast fibroadenoma and cyst, monoclonal gammopathy of undetermined significance (MGUS), and monoclonal lymphocytosis.
31 . Bifunctional compound according to claim 28, wherein the cancer or benign tumor is caused by dysregulation of a biological pathway that is related to cell cycle regulation, DNA damage repair or hormone signaling.
32. A pharmaceutical formulation, which comprises a bifunctional compound according to any one of the claims 1 - 31 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.
33. A conjugate, having the chemical structure:
T - (L - D)a or a pharmaceutically acceptable salt thereof, wherein:
D is a bifunctional moiety capable of inducing degradation of PLK1 ;
T comprises or consists of a targeting moiety capable of binding to a cell surface molecule; L is a linker, covalently bound to T and D; a is an integer from 1 to 8;
Wherein D is a bifunctional moiety having the chemical structure TLB-Sp-PB, wherein TLB, Sp and PB have the chemical structure according to any one of the preceding claims 1 - 23 and wherein the X group of PB is covalently bound to the linker L, optionally by conversion of the -OH group of the X group to a bonding group containing -O-.
34. The conjugate according to claim 33, wherein PB of the conjugate has the chemical structure
BPLKI-XC and wherein the Xc-group has any one of the following structures according to formula (Xc-1) to (Xc-28) selected from the group consisting of:
(Xc-20) (Xc-21) (Xc-22) (Xc-23) (Xc-24)
wherein any of said formula Xc-1 to Xc-28 is optionally and independently substituted with one or more substituents selected from fluoro, hydroxy, (1 -3C)alkyl and (1-3C)alkoxy; wherein n is 1 to 4; wherein the * denotes the point of attachment to the amide (-C(O)-NH-) group of Formula BPLKI ; wherein l~~ denotes the point of attachment to spacer Sp; wherein the
# denotes the point of attachment to linker L.
35. The conjugate according to claim 33 or claim 34, wherein the Xc -group of D is selected from the group consisting of:
(Xc-21) (Xc-22) (Xc-23) (Xc-24) (Xc-28) wherein n is 1 , 2 or 3; wherein the -*q — * denotes the point of attachment to the amide (-C(O)-NH-) group # of BPLK-I ; wherein denotes the point of attachment to Sp; wherein the denotes the point of attachment to linker L.
36. Conjugate according to any of the claims 33 - 35, wherein the linker L is a cleavable linker.
37. Conjugate of claim 36 wherein the cleavable linker is cleavable by a protease.
wherein: c is an integer from 2 to 10; Z1, Z2, Z3, and Z4 are each independently absent or a naturally occurring amino acid residue or the enantiomer thereof, provided that at least two of Z1, Z2, Z3, and Z4 are amino acid residues; wherein the T~ * denotes the point of attachment to T ; and wherein the denotes the point of attachment to Xc.
39. Conjugate of claim 38, wherein Z1, Z2, Z3, and Z4 are independently absent or selected from the group consisting of L-valine, D-valine, L-citrulline, D-citrulline, L-alanine, D-alanine, L- glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L- asparagine, D-asparagine, L-phenylalanine, D-phenylalanine, L-lysine, D-lysine, L-proline, D- proline, and glycine, provided that at least two of Z1 , Z2, Z3, and Z4 are amino acid residues.
40. Conjugate of claim 38, wherein:
Z1 is absent or selected from the group consisting of glycine, L-citru Hi ne or D-citru Hi ne ;
Z2 is absent or selected from the group consisting of L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L aspartic acid, D-aspartic acid, L-alanine, D-alanine, L-valine, D-valine and glycine; Z3 is selected from the group consisting of L-valine, D-valine, L-alanine, D-alanine, L- phenylalanine, D-phenylalanine, L-proline, D-proline and glycine; and
Z4 is selected from the group consisting of L-alanine, D-alanine, L-citrulline, D-citrulline, L- asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine, and glycine.
41 . Conjugate of claim 36 - 40, wherein L is selected from the group consisting of: wherein c is an integer from 2 to 10; wherein the T~ * denotes the point of attachment to T; and wherein the denotes the point of attachment to Xc.
42. Conjugate of claim 41 , wherein c is 5.
43. Conjugate of claim 36, wherein the linker L is a pyrophosphate or phosphate cleavable linker.
44. Conjugate of claim 36 or 43, wherein the linker L is selected from the group consisting of:
wherein c is an integer from 2 to 10; wherein d is an integer 1 or 2; wherein the T~ * denotes the point of attachment to T ; and wherein the I denotes the point of attachment to Xc.
45. Conjugate of claim 44, wherein c is 5.
46. Conjugate of claim 36, wherein the linker L is a beta-glucuronidase cleavable linker.
47. Conjugate of claim 36 or 46, wherein the linker L is selected from the group consisting of:
wherein c is an integer from 2 to 10; wherein EWG is an electron withdrawing group; wherein the T~ * denotes the point of attachment to T; and wherein the I denotes the point of attachment to Xc.
48. Conjugate of claim 47, wherein the linker L is selected from the group consisting of:
wherein c is an integer from 2 to 10; wherein the T~ * denotes the point of attachment to T; and wherein the denotes the point of attachment to Xc.
49. Conjugate of claim 48 wherein c is 5.
50. Conjugate of claim 36, wherein the linker L is a click-to-release linker.
51 . Conjugate of claim 36 or 50, wherein the Linker L is selected from the group consisting of: wherein c is an integer from 2 to 10; wherein the T~ * denotes the point of attachment to T; and wherein the denotes the point of attachment to Xc.
52. Conjugate of claim 36, wherein the linker L is a bio-reducible linker.
53. Conjugate of claim 36 or 52, wherein the linker L is selected from the group consisting of: and wherein c is an integer from 2 to 10; R, R’, R”, and R’” are each independently selected from hydrogen, (1 -6C)alkoxy(1-6C)alkyl, (1 -6C)2N(1 -6C)alkyl, and (1 -6C)alkyl, or, two geminal R groups, together with the carbon atom to which they are attached, form a cyclobutyl or cyclopropyl ring; wherein the ™ * denotes the point of attachment to T; and wherein the # denotes the point of attachment to Xc.
54. Conjugate of any one of the preceding claims 33-35, wherein the linker L is a non-cleavable linker.
55. Conjugate of claim 33 or 54, wherein the linker L is selected from the group consisting of: wherein b is an integer from 1 to 10; wherein the T~ * denotes the point of attachment to T ; and wherein the denotes the point of attachment to Xc.
56. Conjugate of claim 55 wherein the linker L is:
57. Conjugate of claim 55 or 56, wherein b is 4 or 5.
58. Conjugate of any one of the preceding claims 33-57, wherein the conjugate has a subformula conjugate 154 - conjugate 162 selected from the group consisting of: conjugate 156 conjugate 157
conjugate 162 wherein a is an integer from 1 to 8.
59. A ready-to-conjugate intermediate compound wherein the compound has a sub-formula
154m - 162m selected from the group consisting of:
m 159m
162m
60. A pharmaceutical formulation, which comprises a conjugate according to any one of the claims 33 - 58 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.
61 . Conjugate according to any of the claims 33 - 58 or a pharmaceutically acceptable salt thereof or pharmaceutical formulation of claim 60, for use as a medicament.
62. Conjugate according to any of the claims 33 - 58 or a pharmaceutically acceptable salt thereof or pharmaceutical formulation of claim 60, for use in therapy.
63. Conjugate according to any of the claims 33 - 58 or a pharmaceutically acceptable salt thereof or pharmaceutical formulation of claim 60, for use in the prevention or treatment of disorders modulated through Polo-like Kinase 1 (PLK1).
64. Conjugate according to any of the claims 33 - 58 or a pharmaceutically acceptable salt thereof or pharmaceutical formulation of claim 60, for use in the treatment of cancer.
65. Conjugate according to any of the claims 33 - 58 or a pharmaceutically acceptable salt thereof or pharmaceutical formulation of claim 60, for use in the treatment of disorders or diseases selected from the group consisting of cancer, benign tumor, a neurological disorder, an inflammatory disease, a disease characterized by inflammasome activation, an autoimmune disease, a hyperproliferative disease, a disorder that is caused by tissue rejection by the immune system, or for use in a therapy to prevent tissue rejection by the immune system.
66. Conjugate according to claim 65, where the neurological disorder is one or more selected from the group consisting of central nervous system disease, neurodegenerative disease, Alzheimer's disease, Parkinson's disease, multiple sclerosis, Huntington's disease, senile dementia, epilepsy, Lou Gehrig, stroke, and nerve damage and axonal degeneration-related disorders following brain or spinal cord injury.
67. Conjugate according to claim 65, wherein the cancer or benign tumor is one or more selected from the group consisting of squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, skin cancer, skin or intraocular melanoma, rectal cancer, anal muscle cancer, esophageal cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia (including but not limited to acute myelogenous leukemia and complex karyotype acute myelogenous leukemia), lymphocytic lymphoma, double hit lymphoma, hepatocellular carcinoma, gastrointestinal cancer, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumor, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, brain cancer, osteosarcoma, Barrett's esophagus, colon adenoma and polyp, breast fibroadenoma and cyst, monoclonal gammopathy of undetermined significance (MGUS), and monoclonal lymphocytosis.
68. Conjugate according to claim 65, wherein the cancer or benign tumor is caused by dysregulation of a biological pathway that is related to cell cycle regulation, DNA damage repair or hormone signaling.
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