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WO2023089601A2 - Inhibiteurs d'usp9x - Google Patents

Inhibiteurs d'usp9x Download PDF

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Publication number
WO2023089601A2
WO2023089601A2 PCT/IB2023/050332 IB2023050332W WO2023089601A2 WO 2023089601 A2 WO2023089601 A2 WO 2023089601A2 IB 2023050332 W IB2023050332 W IB 2023050332W WO 2023089601 A2 WO2023089601 A2 WO 2023089601A2
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Prior art keywords
compound
substituted
usp9x
cancer
bcl
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WO2023089601A3 (fr
Inventor
Richard Martinelli
Julian F. Bond
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Prodeg LLC
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Prodeg LLC
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Priority claimed from PCT/US2022/050069 external-priority patent/WO2023091464A1/fr
Application filed by Prodeg LLC filed Critical Prodeg LLC
Publication of WO2023089601A2 publication Critical patent/WO2023089601A2/fr
Publication of WO2023089601A3 publication Critical patent/WO2023089601A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/81—Amides; Imides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D498/08—Bridged systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/07—Optical isomers

Definitions

  • Ubiquitination is a covalent post-translational modification of cellular proteins involving a complex enzymatic cascade. Emerging evidence suggests that many enzymes of the ubiquitination cascade are differentially expressed or activated in several diseases and may therefore be appropriate therapeutic targets.
  • Protein ubiquitination is a dynamic two-way process that can be reversed or regulated by deubiquitinating (deubiquitinase, DUB) enzymes.
  • DUB deubiquitinating
  • the human genome codes for nearly 100 proteins with putative DUB activity which can be broadly divided into two main sub- groups: ubiquitin C-terminal hydrolase (UCH) and the ubiquitin-specific proteases (USP).
  • UCH ubiquitin C-terminal hydrolase
  • USPs comprise the largest subclass of DUBs in humans, while only 4 known UCH DUBs have been described.
  • DUBs primarily serve to counterbalance ubiquitin-protein conjugation and also facilitate the cleavage of ubiquitin from its precursors and unanchored polyubiquitin chains.
  • DUBs regulate and maintain the homeostasis of free ubiquitin pools in the cell.
  • DUBs have been reported to regulate deubiquitination of histones, DNA damage repair, cellular proliferation (USP2) and cytokine signaling (DUB-A).
  • DUBs such as USP 14, Uch37 and RPN 11 have been shown to associate with the regulatory sub-unit of the proteasome ( 19S) and edit polyubiquitin chains on proteasome substrates.
  • the invention relates to the discovery of compounds that inhibit DUBs, particularly USP9X. Inhibition of DUBs, particularly USP9X, induces the targeted degradation of the substrates of the deubiquitylase. These substrates include Mcl-1, erbB2, beta-catenin and aldehyde dehydrogenase 1 A3.
  • the invention also includes methods of inhibiting DUBs, including USP9X.
  • the invention includes methods of treating, inhibiting or suppressing cancer, such as myeloma, lung cancer (e.g., non-small cell lung cancer), colon cancer, central nervous system (CNS) cancer, melanoma, ovarian cancer, renal cancer, prostate cancer, breast cancer (such as a triple negative breast cancer), pancreatic cancer, a virus-induced cancer, a Kaposi's sarcoma, a nasopharyngeal carcinoma (EBV), leukemia, a chronic myelogenous leukemia (CML), lymphoma, acute lymphocytic leukemia, a chronic lymphocytic leukemia, an acute myelogenous leukemia, a B-cell lymphoma, a mantle cell lymphoma, a multiple myeloma, a plasma cell dyscrasia, a myeloproliferative disorder, or a glioblastoma.
  • cancer such as myeloma, lung cancer (e
  • the invention also includes methods of inducing degradation of a USP9X substrate in a cell, such as Mcl-1 or erbB-2.
  • the invention includes methods of treating a condition in a subject wherein the condition is associated with a pathologic cell that expresses Mcl-1, comprising administrating to the subject an effective amount of the compound.
  • the condition is a cancer.
  • the degradation level of the USP9X substrate, such as Mcl-1 may be controlled, slowed down, or terminated by co- treating the cell with a proteasome inhibitor, such as Velcade.
  • Mcl-1 may serve as an anti-apoptotic factor that confers resistance, e.g., for a Bcl-2 family protein, to chemotherapy.
  • the invention also includes methods of enhancing potency of a Bcl-2 family inhibitor in a cell expressing Mcl- 1 and at least one other anti-apoptotic Bcl-2 family protein (such Bcl-2, Bcl-xL, or both), comprising co-treating the cell with the compound and the Bcl-2 family inhibitor.
  • the Bcl-2 family inhibitor is a BH3 mimetic such as Navitoclax or Venetoclax.
  • the invention also includes methods of treating a condition in a subject wherein the condition is associated with a pathologic cell that expresses Mcl-1 and at least one other anti-apoptotic Bcl-2 family protein, comprising co- administrating to the subject an effective amount of a pharmaceutical composition of the compound and an effective amount of a pharmaceutical composition of a Bcl-2 family inhibitor.
  • the condition is a cancer, more preferably, acute myeloid leukemia (AML) or breast cancer.
  • the compound can be incorporated into a PROTAC construct to target USP9X, leading to its ubiquitylation by an E3 ubiquitin ligase and consequently inducing the degradation of USP9X.
  • the invention also includes methods of treating inflammation, infection, such as a pathogenic infection, or a neurodegenerative disorder or symptoms of a neurodegenerative disorder, or a genetic disorder mediated to the DUB. Additionally, provided are methods of treating a condition arising from a pathogen infection comprising contacting the pathogen or a cell infected by the pathogen with the compound or composition as disclosed herein.
  • the condition can be gastroenteritis, encephalitis, a respiratory tract infection, SARS, virus- induced cancer, rabies, a hemorrhagic fever, Rift valley fever, listeriosis, or toxoplasmosis.
  • the condition is meningitis, myocarditis, hepatitis, bacteremia, or a skin infection.
  • the pathogen can be a virus, bacterium, fungus, or parasite.
  • the virus can be a calicivirus, a norovirus, a sapovirus, a picomavirus, a Togavirus, a Bunyavirus, a Rhabdovirus, a herpes virus, an adenovirus, an arterivirus, a coronavirus, a flavivirus, a paramyxovirus, a papillomavirus, a virus encoding for an ovarian tumor (OTU)-like protease, a baculovirus, or a nairovirus.
  • the virus can be a polyoma virus, a retrovirus or coronavirus.
  • the virus is selected from the group consisting of encephalomyocarditis virus (EMCV), Sindbis virus (SiNV), La Crosse virus (LaCV), Norwalk virus, Epstein-Barr (EBV), herpesvirus, Dengue virus, papillomavirus or coronavirus.
  • the virus can be cytomegalovirus, BK virus, hepatitis C virus, or HIV.
  • the bacterium can be Chlamydia, Escherichia, Salmonella, Yersinia, Burkholderia, Haemophilus, Listeria, or Mycobacterium. In some cases, the bacterium is Staphylococcus aureus.
  • the bacterium is methicillin- resistant Staph aureus (MRSA).
  • MRSA methicillin- resistant Staph aureus
  • the parasite or fungus can be Plasmodium falciparum, Toxoplasma gondii, Entamoeba histolytica, Giardia lamblia, Trypanosoma brucei, Trypanosoma cruzi, Cestoda, Clonorchis, Opisthorchis, Strongylocides, Candida, Aspergillus, or Cryptococcus.
  • the invention also includes methods of treating developmental disorders, such as intellectual disability, epilepsy, autism and developmental delay, and neurodegeneration, including Parkinson’s and Alzheimer’s disease, as well as autoimmune diseases and inflammation.
  • developmental disorders such as intellectual disability, epilepsy, autism and developmental delay, and neurodegeneration, including Parkinson’s and Alzheimer’s disease, as well as autoimmune diseases and inflammation.
  • R2, R3, R4, R6, and R7 are each independently selected from H, 2 H, halogen, substituted or unsubstituted alkyl, and substituted or unsubstituted alkoxy; and optionally, R3 and R4 together form a substituted or unsubstituted 5- or 6-membered heterocyclic ring;
  • R5 and R9 are each independently selected from H, 2 H, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, and one or more aryl substituents, such as halogen;
  • R8 is absent, or substituted or unsubstituted 1- to 4-carbon alkylene;
  • R11 is a hydrophobic alkyl selected from substituted or unsubstituted alkyl, and substituted or unsubstituted cycloalkyl;
  • Z1, Z2, and Z3 are each independently selected from CH and N;
  • X1 is selected from -O-, -S-, -NH-, -CH2-;
  • X2 is absent, or selected from -L-NRA-, -L-NRAC(O)-, -L-C(O)-, -L-OC(O)-, -L- C(O)O-, -L-CH(COORA)-, -L-C(O)NRA-L-, -L-C(O)NRAC(O)-, wherein RA is selected from H, 2 H, substituted or unsubstituted alkyl; and optionally, RA, R8, and X2 together with the aryl to which R8 is attached to form a substituted or unsubstituted 5- or 6-membered heterocyclic ring;
  • R1 and R6 are H; R2, R3, R4, and R5 are each independently selected from H, F, Cl, -CHc and -OCHa; R7 is H or methyl (more preferably methyl); R11 is selected from -CH2C(CH3)3, -CH2CH(CH3)2, -CH2-cyclo-CaHs, and -CH2- cyclo-C4H7; and X is -O- or -CH2-.
  • L is -CH2CH2CH2- or -CH2C(CH3)2CH2-. and more preferably, -CH2C(CH3)2CH2-.
  • X2 is -CH2C(CH3)2CH2C(O)N(CH3)-.
  • R8 is a) a substituted 2-carbon alkene, wherein the substituent is selected from halogen such as F or Cl, oxo, -COOH, -CONH2, -CONH-C2-Ci2-alkyl, - CONH-C2-C8-alkenyl, -CONH-C2-C8-alkynyl. -CONH-C3-C12-cycloalkyl, -CONH-aryl, - CONH-heteroaryl, -CONH-heterocycloalkyl, and -CONH-C2-C8-alkoxy: or b) -CH2CH2-.
  • R8 is selected from -CH(COOH)CH2-, -CH[C(O)NHCH2CH2OCH3]CH2-, and - CH2CH2-; and more preferably, is -CH2CH2-.
  • each of Z1, Z2, and Z3 is CH; one of Z1, Z2, and Z3 can be N; or two of Z1, Z2, and Z3 can be N.
  • Zi is N, Zi and Z2 are CH.
  • m 1, 2, or 3.
  • the compound can be of Formula (II) or (III): and pharmaceutically acceptable salts thereof, wherein R1, R2, R3, R4, R5, R6 , R7, and R8 are independently hydrogen or a substituted or unsubstituted alkyl,
  • R9, and Rio are independently hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted acyl, L is a linker group, such as a substituted or unsubstituted, saturated or unsaturated 1 to
  • Z1, Z2, and Z3 are independently selected from N or CH, Xi, X2, X3 are independently H or one or more aryl substituents, such as a halogen, e.g., fluorine, n and m are independently 0 or an integer, such as 1, 2, 3, 4, or 5.
  • a halogen e.g., fluorine
  • n and m are independently 0 or an integer, such as 1, 2, 3, 4, or 5.
  • Ri is hydrogen
  • R2 is hydrogen
  • R3 is hydrogen or methyl (more preferably methyl)
  • R4 is hydrogen or methyl (more preferably methyl)
  • R5 is hydrogen
  • R6 is hydrogen or methyl (more preferably hydrogen)
  • R7 is hydrogen or methyl (more preferably hydrogen).
  • R8 is preferably an alkyl group, and more preferably a branched alkyl, such as a 4, 5, or 6 carbon alkyl. -CH2C(CH3)3 is preferred. Where one or more of R2, R5, and R8 are a substituted or unsubstituted alkyl, a chiral center is formed.
  • the invention contemplates racemates and purified or isolated stereoisomers, enantiomers and diastereomers.
  • the linker L is -(CH2)2C(CH3)2(CH2)2-.
  • the linker can include one or more heteroatoms, such as an oxy, amine, amido or ester.
  • the linker can be - (CH 2 )2O(CH 2 )2-, -(CH 2 )2NH(CH 2 )2-, -(CH 2 ) 2 CONH (CH 2 )2-, or -(CH 2 )2CO2(CH 2 )2-.
  • each of Z1, Z2, and Z3 are CH.
  • one of Z1, Z2, and Z3 can be N.
  • Z1 and Z2 are CH and Z3 is CH or N.
  • compositions comprising a compound as described herein and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition can be formulated for oral, topical, intravenous, subcutaneous, intramuscular, intrathecal, ophthalmic, or inhalational route of administration.
  • Figure 1A is a graph showing a curve of fluorescence (RFU) vs. the concentration of Compound 1A.
  • the effect of Compound 1A was evaluated in an assay measuring the ability of USP9X to catalyze the hydrolysis of the amide bond between the C terminal carboxylate of ubiquitin and rhodamine 110 of the fluorogenic substrate ubiquitin-rhodamine 110.
  • Figure IB is a graph depicting relative response vs. time curves for Compound 2A binding to USP9X as determined by the BIACORETM assay at Compound 2A concentration ranging from 0.3* 10 -6 mol/1 to 20 x 10 -6 mol/1 described in Example 2.
  • Figure 1C is a graph depicting affinity determination of Compound 2A binding to USP9X as determined by the BIACORETM assay described in Example 2.
  • Figure ID is a graph depicting the effects of compounds Compound 2A, Comparator 1, Compound 3 A, Comparator 2, and Compound 4A on the enzyme activity of USP9X on the fluorogenic substate ubiquitin-rhodaminel lO (BPS-Biosciences).
  • the fluorescent signals generated by the USP9X mediated generation of the fluorescent product were monitored over time at the presence of these compounds at varying concentrations and at the presence of ubiquitin aldehyde as a positive control, respectively.
  • Figure IE illustrates the set of reactions that comprise the inhibition of enzyme activity.
  • the values of all the parameters may be determined by a series of steady state kinetic measurements.
  • the values determined for the parameters a and ⁇ determine the specific mode of inhibition.
  • Figure IF is a graph comparing the experimental data of Compound 2A (solid dot) with the partial inhibition dose-response curve based on the kinetic model of hyperbolic mixed inhibition.
  • Figure 2A is a graph depicting a cell viability dose-response curve of Compound 9A inhibiting Mia-Paca-2 cell proliferation after 72-hour treatment at 37 °C as assessed by the WST assay.
  • Figure 2B is a graph depicting a cell viability dose-response curve of Compound 9A inhibiting MDA-MB-231 cell proliferation after 72-hour treatment at 37 °C as assessed by the WST assay.
  • Figure 2C is a graph depicting dose-response curves of RKO colon cell viability for Compound 7A, Compound 6A, Compound 8A in an RKO colon cell line.
  • Figure 2D is a graph depicting dose-response curves of BT-549 Breast cancer cell viability for Compound 7A, Compound 6A, Compound 8A in a BT-549 Breast cancer cell line.
  • Figure 2E is a graph depicting dose-response curves of MIA Paca-2 Pancreatic cell viability for Compound 7A, Compound 6A, Compound 8A in a MIA Paca-2 Pancreatic cell line.
  • Figure 3C is a graph showing the erbB2 level changes overtime during 72 hours of Compound 7A treatment from western blot analysis, normalized by the housekeeping protein actin as a reference, compared with that during 72 hours of vehicle treatment only. Displayed at the upper right comer is a representative blot of erbB2 treated with 10 ⁇ M Compound 7A after 24, 48, and 72 hours compared with erbB2 treated with vehicle only after 24, 48, and 72 hours.
  • FIG. 4A is a western blot image showing Mcl-1 bands in U937 cell line at different stages of treatments.
  • U937cells were treated with either 10 pg/mL cycloheximide, 10 ⁇ M MG132, the combination of cycloheximide and MG132, or vehicle (DMSO) for one hour prior to the treatment with Compound 5A for 24 hours. Bands of tubulin undergoing the same treatments are shown as a reference.
  • Figure 4B left is a graph of capture ELISA results showing the percent decreases of Mcl-1 in MIA-PaCa-2 cells treated with Compound 7A at 25 ⁇ M, 12.5 ⁇ M, 6.25 ⁇ M, and 3.125 ⁇ M, respectively, for 24 hours.
  • Right is graph of capture ELISA results showing the percent increases of ubiquitylated Mcl-1 in MIA-PaCa-2 cells treated with Compound 7A at 25 ⁇ M, 12.5 ⁇ M, 6.25 ⁇ M, and 3.125 ⁇ M, respectively, for 24 hours.
  • Figure 5A is a graph depicting cell viability dose-response curves, as assessed by the WST assay, of ABT737 inhibiting U937 cells with and without the co-treatment of 0.5 ⁇ M Compound 7A, respectively.
  • the IC50 value of ABT737 decreases to 0.06 ⁇ M from 3.4 ⁇ M when treated with ABT737 alone.
  • Co-treatment of 0.5 ⁇ M Compound 7A results in 57-fold enhancement in ABT737’s potency for inhibiting U937 cells.
  • Figure 5B is a graph depicting cell viability dose-response curves of ABT737 inhibiting MDA-MB-231 cells with and without the co-treatment of 10 ⁇ M Compound 5 A, respectively.
  • the IC50 value of ABT737 decreases to 1.8 ⁇ M from 8.7 ⁇ M when treated with ABT737 alone.
  • Co-treatment of 10 ⁇ M Compound 5A results in 5-fold enhancement in ABT737’s potency for inhibiting MDA-MB-231 cells.
  • USP9X/FAM The ubiquitin-specific protease 9X
  • USP9X plays a significant role in cancer, both as an oncogene or tumor suppressor, developmental disorders, such as intellectual disability, epilepsy, autism and developmental delay, neurodegeneration, including Parkinson’s and Alzheimer’s disease, and inflammation such as autoimmune diseases.
  • a cell such as a cancer cell
  • the USP9X inhibitor and “the compound” as used herein are interchangeable.
  • cancer cell and “tumor cell” as used herein are interchangeable.
  • the compounds of the invention include a compound of Formula (I), or optionally a formula with a linear backbone structure obtained by breaking a carbon-carbon or carbon-nitrogen single bond on the cyclic backbone of Formula (I) at any site, and salts thereof:
  • Preferred alkyls include lower alkyls, such as methyl, ethyl, n-propyl, i-propyl, n- butyl, sec-butyl, i-butyl, tert-butyl, n-pentyl, s-pentyl, i-pentyl, or t-amyl.
  • the invention contemplates racemates and purified or isolated stereoisomers, enantiomers and diastereomers.
  • the compounds of the invention include a compound of Formula (II) or (III), and salts thereof:
  • alkyls include lower alkyls, such as methyl, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl, i-butyl, tert-butyl, n- pentyl, s-pentyl, i-pentyl, ort-amyl.
  • Preferred acyls include alkylcarbonyls, such as acetyl or t-boc.
  • the invention contemplates racemates and purified or isolated stereoisomers, enantiomers and diastereomers.
  • Table 1 provides additional representative examples of the invention.
  • DRBs Deubiquitinases
  • Deubiquitinating enzymes i.e., deubiquitinases or DUBs
  • DUBs deubiquitinases
  • UFP ubiquitin-specific proteases
  • UCH ubiquitin C-terminal hydrolases
  • DUBs include, for instance, USP5, USP6, USP4, USP5, USP13, USP2, USP11, USP14, USP7, USP9X, USP10, USP1, USP12, USP16, USP15, USP17, USP19, USP20, USP3, USP9Y, USP18, USP21, USP22, USP33, USP29, USP25, USP36, USP32, USP26, USP24, USP42, USP46, USP37, USP28, USP47, USP38, USP44, USP50, USP35, USP30, Memame-AA088peptidase, Memame-AA091 peptidase, USP45, USP51, USP34, USP48, USP40, USP31, Memame-AA129peptidase, USP49, USP17-like peptidase, USP54, USP53, USP39,
  • alkyl refers to saturated, unsaturated, straight- or branched-chain, or cyclic hydrocarbon radicals.
  • C1-C4 alkyl refers to alkyl groups containing from one to four, one to six, one to eight, one to twelve, 2 to 4 and 3 to 6 carbon atoms respectively.
  • Examples of C1-C8 alkyl radicals include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl. tert-butyl, neopentyl, n-hexyl, heptyl and octyl radicals.
  • alkenyl denotes a monovalent group derived from a hydrocarbon moiety having at least one carbon-carbon double bond by the removal of a single hydrogen atom.
  • C2-C8 alkenyl refers to alkenyl groups containing from two to eight, two to twelve, two to four, three to four or three to six carbon atoms respectively.
  • Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, 2-methyl-2-buten-2-yl, heptenyl, octenyl, and the like.
  • alkynyl denotes a monovalent group derived from a hydrocarbon moiety having at least one carbon-carbon triple bond by the removal of a single hydrogen atom.
  • C2-C8 alkynyl refers to alkynyl groups containing from two to eight, two to twelve, two to four, three to four or three to six carbon atoms respectively.
  • alkynyl groups include, but are not limited to, for example, ethynyl, 2-propynyl, 2-butynyl, heptynyl, octynyl, and the like.
  • Alkenyls and alkynyls are “unsaturated alkyls.”
  • cycloalkyl refers to a monocyclic or polycyclic saturated (or unsaturated) carbocyclic ring or a bi- or tri -cyclic group fused, bridged or spiro system, and the carbon atoms may be optionally oxo-substituted or optionally substituted with exocyclic olefinic double bond.
  • Preferred cycloalkyl groups include C3-C12 cycloalkyl, C3-C6 cycloalkyl, C3-C8 cycloalkyl and C4-C7 cycloalkyl.
  • C3-C12 cycloalkyl examples include, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentyl, cyclooctyl, 4-methylene-cyclohexyl, bicyclo[2.2.1]heptyl, bicyclo[3.1.0]hexyl, spiro[2.5]octyl, 3- methylenebicyclo[3.2.1]octyl, spiro[4.4]nonanyl, and the like.
  • cycloalkenyl refers to monocyclic or polycyclic carbocyclic ring or a bi- or tri -cyclic group fused, bridged or spiro system having at least one carbon-carbon double bond and the carbon atoms may be optionally oxo-substituted or optionally substituted with exocyclic olefinic double bond.
  • Preferred cycloalkenyl groups include C3-C12 cycloalkenyl, C3-C8 cycloalkenyl or C5-C7 cycloalkenyl groups.
  • C3-C12 cycloalkenyl examples include, but not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, bicyclo[2.2.1]hept-2-enyl, bicyclo[3.1.0]hex-2- enyl, spiro[2.5]oct-4-enyl, spiro[4.4]non-2-enyl, bicyclo[4.2.1]non-3-en- 12-yl, and the like.
  • aryl refers to a mono- or polycyclic carbocyclic ring system having one or two aromatic rings including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like.
  • heteroaryl refers to a mono- or polycyclic (e.g. bi-, or tri- cyclic or more) aromatic radical or ring having from five to ten ring atoms of which one or more ring atom is selected from, for example, S, O and N; zero, one or two ring atoms are additional heteroatoms independently selected from, for example, S, O and N; and the remaining ring atoms are carbon, wherein any N or S contained within the ring may be optionally oxidized.
  • Heteroaryl includes, but is not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and the like.
  • arylalkyl means a functional group wherein an alkylene chain is attached to an aryl group, e.g., -CH2CH2 -phenyl.
  • substituted arylalkyl means an arylalkyl functional group in which the aryl group is substituted.
  • heteroarylalkyl means a functional group wherein an alkylene chain is attached to a heteroaryl group.
  • substituted heteroarylalkyl means a heteroarylalkyl functional group in which the heteroaryl group is substituted.
  • alkoxy employed alone or in combination with other terms means, unless otherwise stated, an alkyl group having the designated number of carbon atoms connected to the rest of the molecule via an oxygen atom, such as, for example, methoxy, ethoxy, 2-propoxy, 2-propoxy (isopropoxy) and the higher homologs and isomers.
  • Preferred alkoxy are (C2-C3) alkoxy. It is understood that any alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic and cycloalkenyl moiety described herein can also be an aliphatic group or an alicyclic group.
  • An “aliphatic” group is a non-aromatic moiety comprised of any combination of carbon atoms, hydrogen atoms, halogen atoms, oxygen, nitrogen or other atoms, and optionally contains one or more units of unsaturation, e.g., double and/or triple bonds.
  • aliphatic groups are functional groups, such as alkyl, alkenyl, alkynyl, O, OH, NH, NH 2 , C(O), S(O)2, C(O)O, C(O)NH, OC(O)O, OC(O)NH, OC(O)NH 2 , S(O) 2 NH, S(O) 2 NH 2 , NHC(O)NH 2 , NHC(O)C(O)NH, NHS(O) 2 NH, NHS(O) 2 NH 2 , C(O)NHS(O) 2 , C(O)NHS(O) 2 NH or C(O)NHS(O) 2 NH 2 , and the like, groups comprising one or more functional groups, non-aromatic hydrocarbons (optionally substituted), and groups wherein one or more carbons of a non-aromatic hydrocarbon (optionally substituted) is replaced by a functional group.
  • groups comprising one or more functional groups, non-aro
  • Carbon atoms of an aliphatic group can be optionally oxo-substituted.
  • An aliphatic group may be straight chained, branched, cyclic, or a combination thereof and preferably contains between about 1 and about 24 carbon atoms, more typically between about 1 and about 12 carbon atoms.
  • aliphatic groups expressly include, for example, alkoxyalkyls, polyalkoxyalkyls, such as polyalkylene glycols, polyamines, and polyimines, for example. Aliphatic groups may be optionally substituted.
  • heterocyclic or “heterocycloalkyl” can be used interchangeably and referred to a non-aromatic ring or a bi- or tri-cyclic group fused, bridged or spiro system, where (i) each ring system contains at least one heteroatom independently selected from oxygen, sulfur and nitrogen, (ii) each ring system can be saturated or unsaturated (iii) the nitrogen and sulfur heteroatoms may optionally be oxidized, (iv) the nitrogen heteroatom may optionally be quatemized, (v) any of the above rings may be fused to an aromatic ring, and (vi) the remaining ring atoms are carbon atoms which may be optionally oxo-substituted or optionally substituted with exocyclic olefinic double bond.
  • heterocycloalkyl groups include, but are not limited to, 1,3-dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, 2-azabicyclo[2.2.1]-heptyl, 8-azabicyclo[3.2.1]octyl, 5 -azaspiro [2.5] octyl, 2- oxa-7-azaspiro[4.4]nonanyl, 7-oxooxepan-4-yl, and tetrahydrofuryl.
  • heterocyclic groups may be further substituted.
  • Heteroaryl or heterocyclic groups can be C-attached or N-attached (where possible). It is understood that any alkyl, alkenyl, alkynyl, alicyclic, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclic, aliphatic moiety or the like, described herein can also be a divalent or multivalent group when used as a linkage to connect two or more groups or substituents, which can be at the same or different atom(s).
  • One of skill in the art can readily determine the valence of any such group from the context in which it occurs.
  • substituted refers to substitution by independent replacement of one, two, or three or more of the hydrogen atoms on an alkyl group (substituted alkyl) or aryl group (substituted aryl) with substituents including, but not limited to, -F, -Cl, -Br, -I, -OH, C1-C12- alkyl; C 2 -Ci2-alkenyl, C2-C 12-alkynyl, -C3-C12-cycloalkyl, protected hydroxy, -NO2, -N3, - CN, -NH2, protected amino, oxo, thioxo, -NH-C2-Ci2-alkyl, -NH-C2-C8-alkenyl.
  • -NH-C2-C8- alkynyl -NH-C3-Ci2-cycloalkyl, -NH-aryl, -NH-heteroaryl, -NH-heterocycloalkyl, - dialkylamino, -diarylamino, -diheteroarylamino, -O-C2-Ci2-alkyl, -O-C2-C8-alkenyl. -O-C2- C8-alkynyl.
  • the substituents are independently selected from halo, preferably Cl and F; Ci- C4-alkyl, preferably methyl and ethyl; halo-Ci-C4-alkyl, such as fluoromethyl, difluoromethyl, and trifluoromethyl; C2-C4-alkenyl; halo-C2-C4-alkenyl; C3-C6-cycloalkyl.
  • Ci-C4-alkoxy such as methoxy and ethoxy
  • halo-Ci-C4 -alkoxy such as fluoromethoxy, difluoromethoxy, and trifluoromethoxy
  • -CN cyclopropyl
  • Ci-C4-alkoxy such as methoxy and ethoxy
  • halo-Ci-C4 -alkoxy such as fluoromethoxy, difluoromethoxy, and trifluoromethoxy
  • -CN -OH
  • NH2 Ci-C4-alkylamino
  • di(Ci-C4-alkyl)amino and NO2.
  • each substituent in a substituted moiety is additionally optionally substituted with one or more groups, each group being independently selected from Ci-C4-alkyl; -CF3, -OCH3, -OCF3, -F, -Cl, -Br, -I, -OH, -NO2, -CN, and -NH2.
  • a substituted alkyl group is substituted with one or more halogen atoms, more preferably one or more fluorine or chlorine atoms.
  • halo or “halogen” alone or as part of another substituent, as used herein, refers to a fluorine, chlorine, bromine, or iodine atom.
  • the term “optionally substituted”, as used herein, means that the referenced group may be substituted or unsubstituted. In one embodiment, the referenced group is optionally substituted with zero substituents, i.e., the referenced group is unsubstituted. In another embodiment, the referenced group is optionally substituted with one or more additional group(s) individually and independently selected from groups described herein.
  • hydroxogen includes hydrogen and deuterium ( 2 H or D).
  • deuterium 2 H or D
  • the recitation of an atom includes other isotopes of that atom so long as the resulting compound is pharmaceutically acceptable.
  • the salts, e.g., pharmaceutically acceptable salts, of the disclosed therapeutics or compounds may be prepared by reacting the appropriate base or acid with a stoichiometric equivalent of the therapeutic or compound.
  • Acids commonly employed to form pharmaceutically acceptable salts include inorganic acids such as hydrogen bisulfide, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid and phosphoric acid, as well as organic acids such as para-toluenesulfonic acid, salicylic acid, tartaric acid, bitartaric acid, ascorbic acid, maleic acid, besylic acid, fumaric acid, gluconic acid, glucuronic acid, formic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, lactic acid, oxalic acid, para-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid and acetic acid, as well as related inorganic and organic acids.
  • inorganic acids such as hydrogen bisulfide, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid and phosphoric acid
  • Such pharmaceutically acceptable salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne- 1,4-dioate, hexyne- 1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate, xylene sulfonate, phenylacetate, phenylpropionate
  • the terms “inhibit” as used herein with respect to the activity of the USP9X inhibitor refer to the ability to substantially antagonize, prohibit, prevent, restrain, slow, disrupt, alter, eliminate, stop, or reverse the progression or severity of, for example, the catalytic activity of a DUB such as USP9X, the survival or proliferation of a cancer cell, or a disease or condition associated with USP9X.
  • the USP9X inhibitor of the invention preferably inhibits the catalytic activity of USP9X by at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 100%, or a range defined by any two of the foregoing values.
  • the compounds as described can inhibit USP9X by binding to an active site of USP9X.
  • the compound binding to USP9X according to the present disclosure will, in at least some embodiments, have a KD value of 1.Ox 10 -6 mol/1 or lower at 25 °C, in one embodiment a KD value of 1.Ox 10 -6 mol/1 or lower at 25 °C, in another embodiment a KD value of 0.5 x 10 -6 mol/1 or lower at 25 °C.
  • the binding activity is determined with a standard binding assay, such as a Biacore surface plasmon resonance instrument. A method for determining the KD value of binding is described in the Examples Section.
  • Ubiquitin aldehyde can be used as a reference in analyzing the inhibition results determined via Biacore.
  • Ubiquitin aldehyde is a potent inhibitor of all ubiquitin deconjugating enzymes, including UCHs (ubiquitin C-terminal hydrolases), USPs (ubiquitin-specific proteases) and DUBs (deubiquitinylating enzymes), and binds covalently to the thiol group of the active site Cys of USP9X.
  • the compounds as disclosed can partially inhibit USP9X.
  • the compounds as disclosed can partially inhibit USP9X, as compared with the complete inhibition of ubiquitin aldehyde (for example, as shown in Figure IE; ubiquitin-aldehyde is an irreversible inhibitor of USP9X, forming a covalent bond with the thiol group of the active site Cys).
  • a partial USP9X inhibition mechanism of the compounds as disclosed is proposed in the present application, which in some embodiments can be evaluated and confirmed by comparing the experimental data of the compounds with the partial inhibition dose-response curve based on the kinetic model of hyperbolic mixed inhibition (for example, as shown in Figure IF).
  • the partial inhibition dose-response curve can be generated using the rate equation as shown below: wherein a, ⁇ , KI, Ks values can be determined from the experimental data for each compound (as described herein in Example 2).
  • IC50 or “the half maximal inhibitory concentration” is used as a measure of the potency of an inhibitor in inhibiting a specific biological or biochemical function. IC50 is a quantitative measure that indicates how much of a particular inhibitor is needed to inhibit, in vitro, a given biological process or biological component by 50%.
  • the biological component is a DUB, especially a USP9X. IC50 values are typically expressed as molar concentration.
  • IC50 and “potency” can be used interchangeably.
  • the compounds as disclosed have a IC50 value from 0.001 to 10 ⁇ M. In some embodiments, the compounds as disclosed have a IC50 value from 0.005 to 10 ⁇ M. In some embodiments, the compounds as disclosed have a IC50 value from 0.01 to 10 ⁇ M. In some embodiments, the compounds as disclosed have a IC50 value from 1 to 10 ⁇ M. In some embodiments, the compounds as disclosed have a IC50 value from 0.001 to 1 ⁇ M. In some embodiments, the compounds as disclosed have a IC50 value from 0.001 to 0.1 ⁇ M. In some embodiments, the compounds as disclosed have a IC50 value from 0.001 to 0.01 ⁇ M. In some embodiments, the compounds as disclosed have a IC50 value from 0.01 to 0. 1 ⁇ M. In some embodiments, the compounds as disclosed have a IC50 value from 0.1 to 1 ⁇ M.
  • the compounds as disclosed have lower IC50 values than other USP9X inhibitor in multiple cell lines, and such other USP9X inhibitor may be FT709, EOAI3402143, or WP1130 (also called Degrasyn).
  • the compounds as disclosed may be about 1- to about 10-fold more potent than other USP9X inhibitor in multiple cell lines.
  • the compounds as disclosed may be about 2-fold, 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, or about 9-fold more potent than other USP9X inhibitor in multiple cell lines.
  • the compounds as disclosed may be about 4-fold more potent than WP1130 in multiple cell lines.
  • the compounds as disclosed also exhibit a high binding specificity for USP9X.
  • specificity and “selectivity” are interchangeable and describe the selective inhibition of the compounds towards a particular enzyme and minimal or no detectable influence on other enzymes’ enzymatic activities when in contact over a sufficient period of time.
  • the compounds can inhibit one or more homologous enzymes’ enzymatic activities by less than 10%.
  • the compounds can inhibit one or more homologous enzymes’ enzymatic activities by less than 6%.
  • the compounds can inhibit one or more homologous enzymes’ enzymatic activities by less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1%. In some embodiments, rather than USP9X, the compounds have no detectable effect on one or more homologous enzymes’ enzymatic activities, i.e., the compounds inhibit one or more homologous enzymes’ enzymatic activities by 0%. Homologous enzymes include other DUBs rather than USP9X.
  • the compounds (such as Compound 5A as shown in Example 2) have no detectable effect on the enzymatic activities on DUBs including, but not limited to, CYLD, USP2, USP5, USP7, USP13, USP25, USP4, and UCH-L3.
  • the compounds (such as Compound 5A as shown in Example 2) have a minimal detectable effect on the enzymatic activities of other DUBs such as, but not limited to, inhibiting USP30’s enzymatic activity by less than about 5%, inhibiting USPlO’s enzymatic activity by less than about 1%, inhibiting USP15’s enzymatic activity by less than about 2%, inhibiting VCPIP’s enzymatic activity by less than about 3%, and inhibiting UCH-Ll’s enzymatic activity by less than about 2%.
  • the selectivity of the compound is determined with a standard screening assay, such as screening the compound on a DUB selectivity panel using ubiquitin- AMC.
  • the compounds exhibit higher selectivity against USP9X than other USP9X inhibitors, such as EOAI3402143 and WP1130.
  • USP9X EOAI3402143 also inhibits USP9X, USP24, and USP5; while WP1130 also inhibits USP5, USP14, UCH-1L, and UCH37.
  • Methods disclosed herein include methods of treating a disorder, such as a disorder associated with DUB activity or a disorder affected by modulation of DUB activity, or use of a compound disclosed herein in the preparation of a medicament to treat a disorder associated with DUB activity and/or affected by modulation of DUB activity. Further contemplated are methods of treatment wherein DUB catalytic activity is inhibited. In some cases, provided herein are methods that further include identifying a subject having a disorder affected by modulation of activity of a DUB and administering to the subject a compound as disclosed herein.
  • provided herein are methods of inhibiting proliferation of a cell comprising contacting the cell with an effective amount of a compound as disclosed herein to inhibit proliferation.
  • the cell is a cancer cell. Cancer cells contemplated are described elsewhere herein.
  • the compound inhibits a DUB endogenous to the cell and inhibits proliferation.
  • provided herein are methods of inhibiting Usp9x.
  • provided herein are methods of treating, inhibiting, or suppressing cancer in a subject, comprising administrating to the subject an effective amount of a compound as disclosed herein to inhibit cancer cell proliferation, thereby treating, inhibiting, or suppressing cancer. Cancer contemplated are described elsewhere herein.
  • the methods provided herein are prophylactic methods, and a compound or composition as disclosed herein is administered prior to onset of a disorder.
  • the method further comprises identifying a subject at risk of contracting a disorder associated with DUB activity and/or affected by DUB modulation (e.g., a virus, bacterium, and/or parasite as disclosed herein), and administering an effective amount of a compound as disclosed herein.
  • a disorder associated with DUB activity and/or affected by DUB modulation e.g., a virus, bacterium, and/or parasite as disclosed herein
  • neuropathic or inflammatory pain comprising contacting a cell with a compound disclosed herein in an amount sufficient to reduce or alleviate the pain, or to inhibit Usp5 in the cell.
  • the contacting comprises administering the compound to a subject suffering from neuropathic or inflammatory pain.
  • the methods disclosed herein further comprises administering a second therapeutic agent.
  • the second therapeutic agent can be administered at the same time as the compound as disclosed herein, or at a different time (e.g., separated by a time period of about 1 hour to about 12 hours).
  • the agents can be co-formulated, or formulated in separate formulations but given at the same time or within about 30 minutes of each other.
  • Contemplated second agents include, e.g., an antiviral, antiparasitic, antibacterial, anticancer agent, agent that treats one or more symptoms of a genetic disorder, and/or an agent that treats a neurodegenerative disorder.
  • Cancer is a disease of the genome characterized by a diverse mutational landscape and genomic alterations that give rise to mutations that lead to abnormal cell transduction cascades.
  • Signal transduction cascades relay growth signals from the cell membrane into the nucleus to initiate transcriptional responses or post-translational protein modifications. Dysregulation of signal transduction cascades in cancer ultimately results in increased cell survival and abnormal cell proliferation.
  • Signal transduction cascades can be regulated by phosphorylation that controls protein function, and ubiquitination that regulates protein turnover and degradation.
  • Phosphorylation or kinase signaling cascades and the proteasome are major targets in cancer therapy.
  • the anticancer activity of kinase and proteasome inhibitors arise from the disruption of multiple signaling pathways that support the growth, proliferation, and survival of malignant cells.
  • B cell cancers such as multiple myeloma (MM), mantle cell lymphoma (MCL) and chronic myeloid leukemia
  • proteasome inhibitors such as botezomib, carfilzomib
  • immunomodulatory drugs thalidomide, lenalidomide, pomalidomide
  • Btk inhibitors of kinase signal transduction cascades involved in B cell signaling
  • Ubiquitin/proteasome-mediated protein degradation is one of the major mechanisms used by cells for protein turnover or degradation. It involves two successive steps: 1) the attachment of ubiquitin 76 amino acid polypeptide, to a protein substrate mediated by the ubiquitin activating, conjugating and ligating enzymes El, E2, and E3, and 2) the degradation of the tagged or poly-ubiquitinylated protein by the 26s proteasome complex or lysosome. (Oncogene (2012) 31, 2373-2388 and Acta Pharmacol Sin 2007 September; 28 (9): 1325- 1330).
  • Deubiquitylation is a reversible process where ubiquitin can be removed from ubiquitinylated proteins by an enzymatic reaction catalyzed by deubiquitinases (DUB).
  • DUB deubiquitinases
  • Deubiquitinating enzymes are known to have important roles in the regulation of protein stability, proofreading of protein ubiquitination, recycling of ubiquitin and, maintaining free ubiquitin concentrations. DUBs can enhance protein stability by preventing protein degradation.
  • DUBs Consistent with the role of ubiquitination and DUBs in protein turnover and stability, dysregulation in the activity and expression of these enzymes have been linked to cancer development and progression. Due to their role in stabilizing the expression of oncogenic or tumor suppressor proteins, DUBs have been a focus of attention as drug targets or as diagnostic and prognostic biomarkers in oncology research. Several mutated DUBs have been found to act as oncogenes or tumor suppressors, and changes in the expression levels of DUBs were found in several hematologic and malignant solid tumors (lung, pancreas, prostate, colon, thyroid and breast). (Annu Rev Biochem. 2009; 78:363-97).
  • the DUB USP9X has recently received considerable attention as potential therapeutic target in several B cell malignancies (MM, MCL, chronic myeloid leukemia) based on the ability of USP9X to associate and stabilize the expression of the oncogenic protein Myeloid cell leukemia-1 (Mcl-1).
  • Mcl-1 protein is known to promote tumor growth and survival by inhibiting apoptotic or cell death pathways. Mcl-1 is overexpressed in MM, MCL and chronic myeloid leukemia.
  • the Mcl-1 gene was found to be amplified in 10.9% of cancers across multiple tissue types including breast, lung, skin (melanoma), neural tissue and sarcoma. (Nature. 2010 Feb. 18; 463(7283): 899-905).
  • Mcl-1 protein expression levels correlate with resistance to chemotherapy, disease relapse and poor survival.
  • high expression levels of USP9X were also found in MCL and MM which may be an underlying mechanism of increased Mcl-1 stability in these diseases.
  • the removal of ubiquitin from Mcl-1 by USP9X rescues it from proteasomal degradation and helps promote high levels of Mcl-1 inside of cells.
  • Higher levels of intracellular Mcl-1 helps confer resistance to apoptosis, which is a known hallmark of cancer cells.
  • any inducement of lower levels of intracellular Mcl-1 renders may render cell more susceptible to apoptosis, especially in response to standard chemotherapeutic agents.
  • knocking down USP9X expression in MM and MCL cells reduced Mcl-1 levels, reduced MM cell survival and blocked cell proliferation.
  • Mcl-1 via inhibition of USP9X can also be used in preparation for stem cell transplant for cancer patients.
  • patients are treated with a debilitating dose of chemotherapy and/or radiation to ablate hematopoietic cells. Since this stem cell population seems to uniquely dependent upon Mcl-1 for viability, USP9X inhibition may provide a milder and more specific protocol for achieving the same result.
  • One of the concerns about any therapeutic strategy that antagonizes Mcl-1 as a cancer treatment is the attendant toxicity for normal cells.
  • the compounds of the invention may be conjugated to an antibody directed against a surface antigen (e.g., CD34) specific for these stem cells.
  • a surface antigen e.g., CD34
  • USP9X was also found to be overexpressed in melanoma cells and in melanoma patients.
  • the use of compounds in melanoma cell lines resulted in the increased expression of the tumor suppressor p53, reduction in Mcl-1 protein, increased cell death, suppression of tumor cell invasiveness, and inhibition of cell proliferation.
  • the compound also enhanced and further increased the apoptotic and anti -cell proliferation effect of the kinase inhibitor vemurafenib that was recently used in about.60% of melanoma patients that harbor a mutation in BRAF, a component of kinase signaling cascade involved in cell proliferation and survival.
  • use of monotherapy reduced tumor growth and did not have any notable side effects in animal weight, behavior and mobility.
  • USP9X is implicated in regulating endocytosis of the breast cancer oncogene ERBB2.
  • the human protein erbB2 encoded by ERBB2 is also called HER2 (human epidermal growth factor receptor 2) or CD340 (cluster of differentiation 340).
  • HER2 human epidermal growth factor receptor 2
  • CD340 cluster of differentiation 340.
  • erbB2 co-immunoprecipitates with a complex containing c-Cbl and USP9X.
  • Reduction in USP9X levels increases bortezomib-induced downregulation of erbB2, suggesting that USP9X is associated with the internalisation and ubiquitylation status of erbB2.
  • erbB2 Overexpression of erbB2 is observed in breast and ovarian cancers (Slamon et al., Science, 1987, 235: 177-182; Slamon et al., Science, 1989, 244:707-712; and U.S. Pat. No. 4,968,603), and in other carcinomas including carcinomas of the stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas and bladder.
  • erbB2 may be overexpressed in prostate cancer (Gu et al. Cancer Lett., 1996, 99: 185-189; Ross et al. Hum. Pathol., 1997, 28:827-33; Ross et al. Cancer, 1997, 79:2162-70; and Sadasivan et al. J. Urol., 1993, 150: 126-31).
  • cancers contemplated include, but are not limited to, chronic myelogenous leukemia (CML), melanoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, B-cell lymphoma, mantle cell lymphoma, multiple myeloma, plasma cell dyscrasia, myeloproliferative disorders, glioblastoma, Kapsi's sarcoma, nasopharyngeal carcinoma (EBV), lung cancer, colon cancer, pancreatic cancer, breast cancer, prostate cancer, melanoma, and solid tumors.
  • CML chronic myelogenous leukemia
  • melanoma acute lymphocytic leukemia
  • chronic lymphocytic leukemia acute myelogenous leukemia
  • B-cell lymphoma mantle cell lymphoma
  • multiple myeloma multiple myeloma
  • plasma cell dyscrasia
  • the sensitivities of multiple cancer cells can be determined by screening the compounds on a cancer cell line panel, such as an ONCOLINESTM Profiler (Netherlands Translational Research Center) assayed with various tumor cell lines.
  • ONCOLINESTM Network of Co-LinesTM
  • the compounds inhibit the proliferation of a majority of the tumor cell lines being tested, such as at least about 80% of the tumor cell lines being tested or at least 50 of 66 tumor cell lines being tested.
  • Compound 7A inhibited the proliferation of 57 of the 66 cell lines being tested.
  • the compounds can have a IC50 value of no greater than 1 ⁇ M.
  • a tumor cell line sensitive to the compounds is referred to that the proliferation of a tumor cell line, when contacted with the compounds, is inhibited by at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.
  • cancer cell line completed may include the colon carcinoma cell line RKO (that expresses high levels of Bel -2 and very high levels of Mcl-1 but is resistant to the Bcl-2 and Bcl-xl inhibitor ABT737), the pancreatic cancer cell line MiaPaca-2 (that has mutations in both copies of Ras gene and over expresses erbB2 and Mcl- 1), the human breast cancer cell line SK-BR3 (that expresses erbB2 and Mcl-1 and is especially used in erbB2 targeting), the human breast ductal carcinoma cell line BT-549, the triple-negative breast cancer (TNBC) cell line MDA-MB-231, and the histiocytic lymphoma cell line U937.
  • RKO colon carcinoma cell line
  • MDA-MB-231 triple-negative breast cancer
  • the induced degradations of USP9X substrates (such as Mcl-1, erbB2, beta-catenin and aldehyde dehydrogenase 1A3) in multiple cancer cell lines by the compounds, can be investigated using a standard analysis method, such as western blot, flow cytometry, or ELISA.
  • the normalized signals from USP9X substrates decrease by at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, indicating that the compounds induce the substrate degradation by inhibiting USP9X.
  • the induced degradation leads to the inhibition of cancer cell proliferation, resulting in the decrease in cell viability.
  • even partial degradation of the substrates induced by the compounds can be sufficient to inhibit the proliferation of cancer cells completely, such as disclosed in Example 3 wherein the 50% decrease in Mcl-1 due to the 24-hour treatment of the compound led to the inhibition the U937 cell proliferation by 100% (i.e., cell viability decreases to 0%).
  • the mechanism of action for induced degradation of USP9X substrate can be further examined by comparing the combined effect of the compound and a proteasome inhibitor with the effect of the compound alone, on cells that express the USP9X substrate.
  • a proteasome inhibitor As illustrated in Example 4, when U937 cells treated with both MG132 and Compound 5A, decline in Mcl-1 was reduced compared to U937 cells treated with Compound 5A only.
  • These results confirmed intracellular inhibition of USP9X by the compounds to enhance proteasomal degradation of Mcl-1.
  • concentration ratios of the compound and proteasome inhibitor for treating cells it is possible to control the degradation of a USP9X substrate so the USP9X substrate can decline to a desirable level rather than degrade entirely.
  • the combined use of the compound and proteasome inhibitor provides a novel approach for inducing controlled degradation of a USP9X substrate wherein the USP9X substrate declines by about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more.
  • PD-L1 Programmed death-ligand 1
  • USP9X Programmed death-ligand 1
  • PD-L1 as a transmembrane protein is expressed on tumor cells and capable of blunting the immune response to tumors by interacting with PD1 on the surface of T cells that otherwise would attack tumor antigens on the surface of these cells and eradicate them.
  • the PDL1-PD1 interaction can be blunted by therapeutic antibodies and has shown clinical benefit in cancer patients.
  • the compounds as described herein may be capable of inducing PD-L1 degradation by inhibiting USP9X and thus enhancing the immune response of T cells against tumor cells. People skilled in the art will recognize that other USP9X substrates, have been described and are continually being discovered. All such USP9X substrates can be therapeutic, prognostic, or research targets of the compounds as described herein and encompassed by the present invention.
  • the methods and compounds disclosed herein are useful in treating pathogenic infections, e.g., preventing, inhibiting and/or ameliorating a pathogenic infection or symptom of a pathogenic infection. In some cases, the methods and compounds disclosed herein are useful in treating a condition due to a pathogenic infection.
  • Intentional contamination of the food and water supplies represents a major threat to the health and health-related services in the US population as a whole and to our armed forces serving throughout the world.
  • Many of the category B water- and food-borne pathogens have specific properties, e.g. low infectious dose, high stability, that make them attractive candidates for this type of bioterrorism.
  • methods or agents that provide protection or prophylaxis against these defined pathogens are urgently needed.
  • agents that provide protection against a wide spectrum of threats would be desirable.
  • the compounds disclosed herein have broad activity against multiple pathogens.
  • a potent inhibitor of diverse category A and B pathogens, and related family members e.g., murine norovirus, Tulane virus, Listeria monocytogenes, Toxoplasma gondii infection.
  • COVID- 19 replication requires deubiquitylase activity.
  • the compounds disclosed herein inhibit a deubiquitinase and this action results in accumulation of ubiquitinated proteins in the cytoplasmic and aggresomal compartment of the cell. This can establish an inhospitable environment for pathogen infection or replication within the target cell. Thus, these compounds are used as an antimicrobial inhibitor that can effectively suppress multiple pathogens.
  • the compounds disclosed herein block the infectivity of category A and/or B pathogens, and/or related family members.
  • pathogens that use a DUB in their infection mechanism.
  • the pathogen uses a DUB endogenous to the infected cell.
  • the pathogen uses a DUB endogenous to the pathogen.
  • Contemplated diseases or disorders due to a pathogenic infection include gastroenteritis, encephalitis, respiratory tract infections (e.g., SARS), virus-induced cancers, rabies, hemorrhagic fevers (e.g., Crimean-Congo, Dengue), Rift valley fever, listeriosis, or toxoplasmosis.
  • diseases or disorders due to a pathogenic infection include meningitis, myocarditis, hepatitis, bacteremia, and skin infections.
  • Contemplated pathogens include viral, bacterial, fungal, and parasitic pathogens.
  • Contemplated pathogenic viruses include a calicivirus (e.g., norovirus, sapovirus), a picomavirus, a Togavirus, a Bunyavirus, a Rhabdovirus, a herpes virus, an adenovirus, an arterivirus, a coronavirus, a flavivirus, a paramyxovirus, a papillomavirus, a virus encoding for an ovarian tumor (OTU)-like protease, a baculovirus, or a nairovirus.
  • Other contemplated pathogenic viruses include polyoma viruses and retroviruses.
  • viruses contemplated include encephalomyocarditis virus (EMCV), Sindbis virus (SiNV), La Crosse virus (LaCV), Norwalk virus, Tulane virus, rotavirus, Epstein-Barr (EBV), herpesvirus, Dengue virus, and papillomavirus. Further specific viruses contemplated include cytomegalovirus, BK virus, hepatitis C virus, and HIV.
  • Contemplated bacteria include Chlamydia, Escherichia, Salmonella, Yersinia, Burkholderia, Haemophilus, Listeria, and Mycobacterium.
  • Other bacteria contemplated include Staphylococcus aureus, or more specifically methicillin-resistant Staph aureus (MRSA).
  • Contemplated parasites or fungi include Plasmodium falciparum, Toxoplasma gondii, Entamoeba histolytica, Giardia lamblia, Trypanosoma brucei, Trypanosoma cruzi, Cestoda, Clonorchis, Opisthorchis, Strongylocides, Candida, Aspergillus, and Cryptococcus.
  • therapeutically effective amount and “prophylactically effective amount,” as used herein, refer to an amount of a compound sufficient to treat, ameliorate, or prevent the identified disease or condition, or to exhibit a detectable therapeutic, prophylactic, or inhibitory effect. The effect can be detected by, for example, an improvement in clinical condition, reduction in symptoms, or by any of the assays or clinical diagnostic tests described herein.
  • the precise effective amount for a subject will depend upon the subject's body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration. Therapeutically and prophylactically effective amounts for a given situation can be determined by routine experimentation that is within the skill and judgment of the clinician.
  • Dosages of the therapeutic can alternately be administered as a dose measured in mg/kg.
  • Contemplated mg/kg doses of the disclosed therapeutics include about 0.001 mg/kg to about 1000 mg/kg. Specific ranges of doses in mg/kg include about 0.1 mg/kg to about 500 mg/kg, about 0.5 mg/kg to about 200 mg/kg, about 1 mg/kg to about 100 mg/kg, about 2 mg/kg to about 50 mg/kg, and about 5 mg/kg to about 30 mg/kg.
  • the compounds described herein may be formulated in pharmaceutical compositions with a pharmaceutically acceptable excipient, carrier, or diluent.
  • the compound or composition comprising the compound is administered by any route that permits treatment of the disease or condition.
  • One route of administration is oral administration.
  • the compound or composition comprising the compound may be delivered to a patient using any standard route of administration, including parenterally, such as intravenously, intraperitoneally, intrapulmonary, subcutaneously or intramuscularly, intrathecally, topically, transdermally, rectally, orally, nasally or by inhalation.
  • Slow release formulations may also be prepared from the agents described herein in order to achieve a controlled release of the active agent in contact with the body fluids in the gastro intestinal tract, and to provide a substantial constant and effective level of the active agent in the blood plasma.
  • the crystal form may be embedded for this purpose in a polymer matrix of a biological degradable polymer, a water-soluble polymer or a mixture of both, and optionally suitable surfactants. Embedding can mean in this context the incorporation of micro-particles in a matrix of polymers. Controlled release formulations are also obtained through encapsulation of dispersed micro-particles or emulsified micro-droplets via known dispersion or emulsion coating technologies.
  • Administration may take the form of single dose administration, or a compound as disclosed herein can be administered over a period of time, either in divided doses or in a continuous-release formulation or administration method (e.g., a pump).
  • a compound as disclosed herein can be administered over a period of time, either in divided doses or in a continuous-release formulation or administration method (e.g., a pump).
  • the compounds of the embodiments are administered to the subject, the amounts of compound administered and the route of administration chosen should be selected to permit efficacious treatment of the disease condition.
  • the pharmaceutical compositions are formulated with one or more pharmaceutically acceptable excipient, such as carriers, solvents, stabilizers, adjuvants, diluents, etc., depending upon the particular mode of administration and dosage form.
  • the pharmaceutical compositions should generally be formulated to achieve a physiologically compatible pH, and may range from a pH of about 3 to a pH of about 11, preferably about pH 3 to about pH 7, depending on the formulation and route of administration.
  • the pH is adjusted to a range from about pH 5.0 to about pH 8.
  • the pharmaceutical compositions may comprise a therapeutically or prophylactically effective amount of at least one compound as described herein, together with one or more pharmaceutically acceptable excipients.
  • the pharmaceutical compositions may comprise a combination of the compounds described herein, or may include a second active ingredient useful in the treatment or prevention of bacterial infection (e.g., anti-bacterial or anti-microbial agents).
  • Formulations e.g., for parenteral or oral administration, are most typically solids, liquid solutions, emulsions or suspensions, while inhalable formulations for pulmonary administration are generally liquids or powders.
  • a pharmaceutical composition can also be formulated as a lyophilized solid that is reconstituted with a physiologically compatible solvent prior to administration.
  • Alternative pharmaceutical compositions may be formulated as syrups, creams, ointments, tablets, and the like.
  • pharmaceutically acceptable excipient refers to an excipient for administration of a pharmaceutical agent, such as the compounds described herein.
  • the term refers to any pharmaceutical excipient that may be administered without undue toxicity.
  • Pharmaceutically acceptable excipients are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there exists a wide variety of suitable formulations of pharmaceutical compositions (see, e.g., Remington's Pharmaceutical Sciences).
  • Suitable excipients may be carrier molecules that include large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles.
  • Other exemplary excipients include antioxidants (e.g., ascorbic acid), chelating agents (e.g., EDTA), carbohydrates (e.g., dextrin, hydroxyalkylcellulose, and/or hydroxyalkylmethylcellulose), stearic acid, liquids (e.g., oils, water, saline, glycerol and/or ethanol) wetting or emulsifying agents, pH buffering substances, and the like.
  • Liposomes are also included within the definition of pharmaceutically acceptable excipients.
  • compositions described herein are formulated in any form suitable for an intended method of administration.
  • tablets, troches, lozenges, aqueous or oil suspensions, non-aqueous solutions, dispersible powders or granules (including micronized particles or nanoparticles), emulsions, hard or soft capsules, syrups or elixirs may be prepared.
  • Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents including sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide a palatable preparation.
  • compositions particularly suitable for use in conjunction with tablets include, for example, inert diluents, such as celluloses, calcium or sodium carbonate, lactose, calcium or sodium phosphate; disintegrating agents, such as cross-linked povidone, maize starch, or alginic acid; binding agents, such as povidone, starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc.
  • inert diluents such as celluloses, calcium or sodium carbonate, lactose, calcium or sodium phosphate
  • disintegrating agents such as cross-linked povidone, maize starch, or alginic acid
  • binding agents such as povidone, starch, gelatin or acacia
  • lubricating agents such as magnesium stearate, stearic acid or talc.
  • Tablets may be uncoated or may be coated by known techniques including microencapsulation to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.
  • Formulations for oral use may be also presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example celluloses, lactose, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with non-aqueous or oil medium, such as glycerin, propylene glycol, polyethylene glycol, peanut oil, liquid paraffin or olive oil.
  • an inert solid diluent for example celluloses, lactose, calcium phosphate or kaolin
  • non-aqueous or oil medium such as glycerin, propylene glycol, polyethylene glycol, peanut oil, liquid paraffin or olive oil.
  • compositions may be formulated as suspensions comprising a compound of the embodiments in admixture with at least one pharmaceutically acceptable excipient suitable for the manufacture of a suspension.
  • compositions may be formulated as dispersible powders and granules suitable for preparation of a suspension by the addition of suitable excipients.
  • Excipients suitable for use in connection with suspensions include suspending agents (e.g., sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia); dispersing or wetting agents (e.g., a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycethanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan monooleate)); and thickening agents (e.g., carbomer, beeswax, hard paraffin or cetyl alcohol).
  • suspending agents
  • the suspensions may also contain one or more preservatives (e.g., acetic acid, methyl or n-propyl p-hydroxy-benzoate); one or more coloring agents; one or more flavoring agents; and one or more sweetening agents such as sucrose or saccharin.
  • preservatives e.g., acetic acid, methyl or n-propyl p-hydroxy-benzoate
  • coloring agents e.g., acetic acid, methyl or n-propyl p-hydroxy-benzoate
  • flavoring agents e.g., methyl or n-propyl p-hydroxy-benzoate
  • sweetening agents such as sucrose or saccharin.
  • the pharmaceutical compositions may also be in the form of oil-in water emulsions.
  • the oily phase may be a vegetable oil, such as olive oil or arachis oil, a mineral oil, such as liquid paraffin, or a mixture of these.
  • Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth; naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids; hexitol anhydrides, such as sorbitan monooleate; and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate.
  • the emulsion may also contain sweetening and flavoring agents.
  • Syrups and elixirs may be formulated with sweetening agents, such as glycerol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, a flavoring or a coloring agent.
  • sweetening agents such as glycerol, sorbitol or sucrose.
  • Such formulations may also contain a demulcent, a preservative, a flavoring or a coloring agent.
  • compositions may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous emulsion or oleaginous suspension.
  • a sterile injectable preparation such as a sterile injectable aqueous emulsion or oleaginous suspension.
  • This emulsion or suspension may be formulated by a person of ordinary skill in the art using those suitable dispersing or wetting agents and suspending agents, including those mentioned above.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,2-propane-diol.
  • the sterile injectable preparation may also be prepared as a lyophilized powder.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile fixed oils may be employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids e.g., oleic acid
  • a pharmaceutically acceptable salt of a compound described herein may be dissolved in an aqueous solution of an organic or inorganic acid, such as 0.3 M solution of succinic acid, or more preferably, citric acid. If a soluble salt form is not available, the compound may be dissolved in a suitable co-solvent or combination of co-solvents. Examples of suitable co- solvents include alcohol, propylene glycol, polyethylene glycol 300, polysorbate 80, glycerin and the like in concentrations ranging from about 0 to about 60% of the total volume. In one embodiment, the active compound is dissolved in DMSO and diluted with water.
  • the pharmaceutical composition may also be in the form of a solution of a salt form of the active ingredient in an appropriate aqueous vehicle, such as water or isotonic saline or dextrose solution.
  • an appropriate aqueous vehicle such as water or isotonic saline or dextrose solution.
  • compounds which have been modified by substitutions or additions of chemical or biochemical moieties which make them more suitable for delivery e.g., increase solubility, bioactivity, palatability, decrease adverse reactions, etc.
  • esterification glycosylation, PEGylation, etc.
  • the compounds described herein may be formulated for oral administration in a lipid-based formulation suitable for low solubility compounds.
  • Lipid- based formulations can generally enhance the oral bioavailability of such compounds.
  • compositions comprise a therapeutically or prophylactically effective amount of a compound described herein, together with at least one pharmaceutically acceptable excipient selected from the group consisting of medium chain fatty acids and propylene glycol esters thereof (e.g., propylene glycol esters of edible fatty acids, such as caprylic and capric fatty acids) and pharmaceutically acceptable surfactants, such as polyoxyl 40 hydrogenated castor oil.
  • pharmaceutically acceptable excipient selected from the group consisting of medium chain fatty acids and propylene glycol esters thereof (e.g., propylene glycol esters of edible fatty acids, such as caprylic and capric fatty acids) and pharmaceutically acceptable surfactants, such as polyoxyl 40 hydrogenated castor oil.
  • cyclodextrins may be added as aqueous solubility enhancers.
  • exemplary cyclodextrins include hydroxypropyl, hydroxyethyl, glucosyl, maltosyl and maltotriosyl derivatives of alpha-, beta-, and gamma-cyclodextrin.
  • a specific cyclodextrin solubility enhancer is hydroxypropyl-o-cyclodextrin (BPBC), which may be added to any of the above-described compositions to further improve the aqueous solubility characteristics of the compounds of the embodiments.
  • BPBC hydroxypropyl-o-cyclodextrin
  • the composition comprises about 0.1% to about 20% hydroxypropyl-o-cyclodextrin, more preferably about 1% to about 15% hydroxypropyl-o-cyclodextrin, and even more preferably from about 2.5% to about 10% hydroxypropyl-o-cyclodextrin.
  • solubility enhancer employed will depend on the amount of the compound of the invention in the composition.
  • the methods of the embodiments also include the use of a compound or compounds as described herein together with one or more additional therapeutic agents for the treatment of disease conditions.
  • the combination of active ingredients may be: (1) conjugated, (2) co-formulated and administered or delivered simultaneously in a combined formulation; (3) delivered by alternation or in parallel as separate formulations; or (4) by any other combination therapy regimen known in the art.
  • the methods described herein may comprise administering or delivering the active ingredients sequentially, e.g., in separate solution, emulsion, suspension, tablets, pills or capsules, or by different injections in separate syringes.
  • an effective dosage of each active ingredient is administered sequentially, i.e., serially
  • simultaneous therapy effective dosages of two or more active ingredients are administered together.
  • Various sequences of intermittent combination therapy may also be used.
  • a compound disclosed herein is administered and/or formulated with a second therapeutic—e.g., a chemotherapeutic.
  • Chemotherapeutic agents contemplated for use include, without limitation, alkylating agents including: nitrogen mustards, such as mechlor-ethamine, cyclophosphamide, ifosfamide, melphalan and chlorambucil; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU), and semustine (methyl-CCNU); ethylenimines/methylmelamine such as thriethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine); alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC); antimetabolites including folic acid analogs such as methotrexate and trimetrexate, pyrimidine analogs such as 5 -fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cy
  • the combination therapy can include aldesleukin (e.g., PROLEUKIN), alemtuzumab (e.g., CAMPATH), asparaginase Erwinia chrysanthemi (e.g., ERWINAZE), bevacizumab (e.g., AVASTIN), blinatumomab (e.g., BLINCYTO), brentuximab vedotin (e.g., ADCETRIS), cetuximab (e.g., ERBITUX), denosumab (e.g., PROLIA, XGEVA), Dinutuximab (e.g., UNITUXIN), ibritumomab tiuxctan (e.g., ZEVALIN), ipilimumab (e.g., Y), aldesleukin (e.g., PROLEUKIN), alemtuzumab (e.g., C
  • Conjugation can be achieved by covalently reacting a moiety on the conjugate or protein and a moiety substituted on the compound of formula (I).
  • the compound of formula (I) can be substituted by an attachment group, such as an electrophilic group or group that can react with a crosslinking agent.
  • conjugation moieties include thiols, hydroxy groups and amines.
  • maleimide groups, activated disulfides, active esters such as NHS esters and HOBt esters, haloformates, acid halides, alkyl and benzyl halides such as haloacetamides can be used.
  • Self-stabilizing maleimides and bridging disulfides can also be used in accordance with the disclosure.
  • targeting agents such as, peptides, proteins, small molecules, antibodies or ligands specific to cell receptors
  • targeting agents such as, peptides, proteins, small molecules, antibodies or ligands specific to cell receptors
  • the compounds as described can be incorporated into PROTAC constructs to target USP9X, leading to its ubiquitylation by an E3 ubiquitin ligase and consequently inducing the degradation of USP9X.
  • cell receptors on cancer cells can be targeted.
  • antibodies or ligands that bind one of the following antigens can be used: Aminopeptidase N (CD13), Annexin Al, B7-H3 (CD276, various cancers), CA125, CA15-3 (carcinomas), CA19-9 (carcinomas), L6 (carcinomas), Lewis Y (carcinomas), Lewis X (carcinomas), alpha fetoprotein (carcinomas), CA242, placental alkaline phosphatase (carcinomas), prostate specific antigen (prostate), prostatic acid phosphatase (prostate), epidermal growth factor (carcinomas), CD2 (Hodgkin's disease, NHL lymphoma, multiple myeloma), CD3 epsilon (T cell lymphoma, lung, breast, gastric, ovarian cancers, autoimmune diseases, malignant ascites), CD 19 (B cell malignancies), CD20 (non- Hodgkin's lymphoma), CD22 (leukemia, lymphot lymph
  • EGFR Epidermal Growth Factor Receptor
  • CTLA4 melanoma
  • CXCR4 CD 184, Heme-oncology, solid tumors
  • Endoglin CD 105, solid tumors
  • EPCAM epidermal cell adhesion molecule
  • ERBB2 Epidermal Growth Factor Receptor 2; lung, breast, prostate cancers
  • FCGR1 autoimmune diseases
  • FOLR farnesoleukin receptor
  • GD2 ganglioside cancers
  • G-28 a cell surface antigen glycolipid, melanoma
  • GD3 idiotype cancers
  • Heat shock proteins cancers
  • HER1 lung, stomach cancers
  • HER2 breast and ovarian cancers
  • HLA-DR10 HLA-DR10
  • HLA-DRB HLA-DRB
  • MAGE-1 (carcinomas), MAGE-2 (carcinomas), MAGE-3 (carcinomas), MAGE 4 (carcinomas), anti-transferrin receptor (carcinomas), p97 (melanoma)
  • MS4A1 (membrane-spanning 4-domains subfamily A member 1, Non-Hodgkin's B cell lymphoma, leukemia), MUC1 or MUC1-KLH (breast, ovarian, cervix, bronchus and gastrointestinal cancer), MUC16 (CA125) (Ovarian cancers), CEA (colorectal), gplOO (melanoma), MARTI (melanoma), MPG (melanoma), MS4A1 (membrane -spanning 4-domains subfamily A, small cell lung cancers, NHL),
  • CD4 Cluster of Differentiations
  • DLL4 -like -4
  • VEGFR-2 VEGFR-2 (CD309), CXCR4 9CD184
  • Targeting agents that can target cells implicated in autoimmune diseases include: anti -elastin antibody; Abys against epithelial cells antibody; Anti-Basement Membrane Collagen Type IV Protein antibody; Anti-Nuclear Antibody; Anti ds DNA; Anti ss DNA, Anti Cardiolipin Antibody IgM, IgG; anti-celiac antibody; Anti Phospholipid Antibody IgK, IgG; Anti SM Antibody; Anti Mitochondrial Antibody; Thyroid Antibody; Microsomal Antibody, T-cells antibody; Thyroglobulin Antibody, Anti SCL-70; Anti-Jo; Anti- U.sub.lRNP; Anti-La/SSB; Anti SSA; Anti SSB; Anti Perital Cells Antibody; Anti Histones; Anti RNP; C-ANCA; P-ANCA; Anti centromere; Anti-Fibrillarin, and Anti GBM Antibody, Anti -ganglioside antibody; Anti-Desmogein 3 antibody; Anti-p62 antibody; Anti
  • Targeting agents for infectious diseases include antibodies that bind pathogens, such as Poxyiridae, Herpesviridae, Adenoviridae, Papovaviridae, Enteroviridae, Picomaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza viruses, parainfluenza viruses, mumps, measles, respiratory syncytial virus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae, Non- A/Non-B Hepatitis virus, Rhinoviridae, Coronaviridae (including COVID-19 such as targeting-he so-called spike protein), Rotoviridae, Oncovirus [such as, HBV (Hepatocellular carcinoma), HPV (Cervical cancer, Anal cancer), Kaposi's sarcoma-associated herpesvirus (Kaposi's sarcoma), Epstein-Barr virus (Nasopharyngeal carcinoma, Burkitt
  • the ratio of the proteasome inhibitor and the compound may be selected from any values between 0.01 and 100, preferably, between 0.1 and 10.
  • the proteasome inhibitor used herein may be, as a nonlimiting example, MG132.
  • the USP9X substrate is Mcl-1.
  • the induced degradation can be controlled to reach a desirable level, at which, for example, the USP9X substrate declines by about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, or about 90% or more.
  • the proteasome inhibitor MG132 has been frequently used as a laboratory research tool to examine the consequences of blocking the ubiquitin-proteasome pathway.
  • MG 132 has been modified (by introducing a boronate group to the peptide backbone) to yield the proteasome inhibitor bortezomib which is marketed as Velcade.
  • a proteasome inhibitor drug such as Velcade may take antagonistic action against a drug of the compounds as described.
  • provided may also be methods of treating a subject with a condition by co-administering the compounds as described and subsequently a proteasome inhibitor drug (such as Velcade) to the subject, wherein a USP9X substrate (preferably Mcl- 1) in a cell of the subject is induced to degrade by the drug of the compounds as described and the induced degradation is controlled, slowed down, or terminated by the proteasome inhibitor drug.
  • a proteasome inhibitor drug such as Velcade
  • the USP9X substrate Mcl-1 may serve as an anti-apoptotic factor conferring resistance to chemotherapy, e.g., providing protection for other pathologic proteins against their inhibitors.
  • pathologic proteins may be an overexpressed anti-apoptotic protein such as, but are not limited to, Bcl-2 and Bcl-xL.
  • ABT737 is a prototype of new class of anti-cancer drugs known as BH3 mimetics, such as Navitoclax or Venetoclax. This class of drugs functions by antagonizing the action of anti-apoptotic proteins such as Bcl2, Bcl-xL, and Mcl-1 by blocking their ability to prevent the activation of pro-apoptotic proteins such as Bak and Bax.
  • ABT737 is a small molecule that binds to Bcl2 and Bcl-xL but not Mcl-1.
  • the binding site for ABT737 on Bcl2 and Bcl- xL is the same surface groove employed by Bcl2 and Bcl-xL to bind the BH3 domains of pro-apoptotic proteins culminating in the activation of Bak and Bax, the permeabilization of the outer membrane of the mitochondrion and the activation of caspases to dismantle the cell.
  • Treatment of cancer cells with ABT737 alone, may induce apoptosis but only if the intracellular level of Mcl-1 is low enough. This suggests that treatment of ABT737-resistant cells with USP9X inhibitors may augment the cell killing potential of ABT737 and similar compounds.
  • the at least one other anti-apoptotic Bcl-2 family protein may be Bcl-2, Bcl-xL, or both Bcl-2 and Bcl-xL.
  • the cancer cell may be selected from, but not limited to, acute myeloid leukemia (AML) cell and breast cancer cell.
  • the Bcl-2 family inhibitor is a Bcl- 2/Bcl-xL inhibitor, such as ABT737, Navitoclax or Venetoclax.
  • the cell is a cancer cell, such as acute myeloid leukemia (AML) cell or breast cancer cell.
  • the Bcl-2 family inhibitor is a Bcl-2/Bcl-xL inhibitor, such as ABT737, Navitoclax or Venetoclax.
  • the potency of the Bcl-2 family inhibitor may be enhanced by about 2 to about 100 folds. In some embodiments, the potency of the Bcl-2 family inhibitor may be enhanced by about 2, about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80 folds, or about 90 folds. In some embodiments, the potency of the Bcl-2 family inhibitor may be enhanced by about 50 folds.
  • the Bcl-2 family inhibitor is a Bcl-2/Bcl-xL inhibitor, such as ABT737, Navitoclax or Venetoclax.
  • the condition is associated with a pathologic cell that expresses Mcl- 1 and at least one other anti- apoptotic Bcl-2 family protein, by co-administrating to the subject an effective amount of a pharmaceutical composition of the compound as disclosed and an effective amount of a pharmaceutical composition of a Bcl-2 family inhibitor.
  • the condition is a cancer, such as acute myeloid leukemia (AML) or breast cancer.
  • the at least one other anti-apoptotic Bcl-2 family protein may be Bcl-2, Bcl-xL, or both Bcl-2 and Bcl-xL.
  • the Bcl-2 family inhibitor is a Bcl-2/Bcl-xL inhibitor, such as a clinically acceptable derivative of ABT737 (e.g., Venetoclax and Navitoclax).
  • reagents and solvents were used as purchased from commercial suppliers. Solvent removal was accomplished usually using a rotary evaporator at ⁇ 15 mm Hg pressure unless otherwise specified. Thin-layer chromatography (TLC) was performed using silica-gel 60 plates (F254, Merck) and visualized by UV light (254 nm). Column chromatography and flash column chromatography were carried out using silica gel (60-203 mesh and 40-60 mesh, respectively) unless otherwise specified.
  • Preferred compounds of the invention include:
  • Step 1 N-methyl-2-(4-nitrophenyl)ethan-l -amine hydrochloride (500 mg, 2.3 mmol) was dissolved in DCM (23 mL) before DIEA (2.0 mL, 11.5 mmol) and 4,4-dimethyldihydro-2H- pyran-2,6(3H)-dione (361 mg, 2.5 mmol) were added. The mixture was stirred at room temperature and monitored by LCMS. After 15 min, the reaction complete.
  • Step 2 3,3-dimethyl-5-(methyl(4-nitrophenethyl)amino)-5-oxopentanoic acid (0.616 g, 1.9 mmol) and 10% Pd/C (0.2 g, 0. 19 mmol) were stirred in EtOAc (19 ml) under a hydrogen atmosphere (1 atm). After the reaction was complete as determined by LCMS, the mixture was filtered and concentrated to give 5-((4-aminophenethyl)(methyl)amino)-3,3-dimethyl-5- oxopentanoic acid (0.373 g, 66.8 % yield) as a yellow oil, which was used as is.
  • Step 3 5-((4-aminophenethyl)(methyl)amino)-3,3-dimethyl-5-oxopentanoic acid (0.373 g, 1.3 mmol) and potassium carbonate (0.212 g, 1.5 mmol) were dissolved in Water (6 ml). The mixture was cooled to 0°C before a solution of (9H-fluoren-9-yl)methyl carbonochloridate (0.330 g, 1.3 mmol) in acetonitrile (6 ml) was added dropwise. The reaction was warmed to room temperature slowly and monitored by LCMS. After 30 min, the reaction was acidified with IM HC1 and extracted with ether.
  • Step 1 5-Cyano-2-fluorobenzoic acid (3.0 g, 18.3 mmol) and 10% palladium on carbon (0.3 g, 2.7 mmol) were shaken in methanol (200mL) under a hydrogen atmosphere (20 PSI). After 3h, the reaction was complete by LCMS. The mixture is filtered and concentrated to give 2. 1 g of the desired product (68%), which was used as is.
  • Step 2 5 -(aminomethyl)-2 -fluorobenzoic acid (2 g, 11.8 mmol) and (9H-fluoren-9-yl)methyl (2,5-dioxopyrrolidin-l-yl) carbonate (3.99 g, 11.8 mmol) were dissolved in THF (250mL). A saturated K2CO3 solution was added until the pH was non-acidic. The mixture was stirred at room temperature and monitored by LCMS. After 2h, the mixture was concentrated and the residue was washed with water and DCM (2x) and dried overnight to give the desired product in >75% yield.
  • Step 1 Methyl 4-hydroxybenzoate (0.250 g, 1.6 mmol) was dissolved in THF (16 ml) before tert-butyl (2-hydroxyethyl)(methyl)carbamate (0.333 ml, 2.0 mmol) and Ph3P (0.560 g, 2.1 mmol) were added. The reaction was cooled to 0°C and DIAD (0.383 ml, 2.0 mmol) was added dropwise and the reaction was monitored by LCMS. After 40 min, the reaction was concentrated the crude residue was purified by silica gel chromatography to give 0.440 g (87%) of desired product.
  • Step 2 4- Methyl 4-(2-((tert-butoxycarbonyl)(methyl)amino)ethoxy)benzoate (0.440 g, 1.4 mmol) and IM aqueous LiOH (7. 11 ml, 14.2 mmol) were stirred in THF (14 ml) at 60°C and the reaction was monitored by LCMS. After stirring overnight, the reaction was complete. The mixture was acidified (pH 2-3) with IM aqueous HC1 and extracted with DCM (3x).
  • Solid-phase Fmoc-deprotection-Resin (1.0 equiv) was suspended in DMF (4 mL x 5 min) and mixed with a stream of N2 every 30 seconds.
  • the Fmoc group was removed from the resin- supported building block by mixing the resin twice with a solution of 2% DBU, 2% piperidine in DMF (4 mL x 5 min) while agitating with a stream of N2 every 30 seconds.
  • the resin was washed six times with DMF (4 mL x 30 sec) and used in the next step as is.
  • Solid-phase amide formation The appropriate carboxylic acid (0.
  • Cyclization Dissolved the linear amino acid (1.0 equiv) in DMF (0.05 M) before N-ethyl-N- isopropylpropan-2 -amine (5.0 equiv) was added. The resulting solution was added to a pre- mixed solution of HATU (1.2 equiv) and HOAt (1.2 equiv) in DMF (0.011 M) at ⁇ 2 mL/min. The reaction was monitored by LCMS and additional HATU was added until complete conversion to the desired product was observed. The mixture was concentrated and purified by either preparative HPLC (small-scale) or reverse-phase silica gel chromatography to give the desired product.
  • Acid reagent (7, 314 mg, 1.3 mmol, 1.1 equiv) was dissolved in anhydrous DCM (3.5 mL) then DIPEA (301 mg, 2.33 mmol, 2.0 equiv), HOBt (190 mg, 1.4 mmol, 1.2 equiv) and EDCI (268 mg, 1.4 mmol, 1.2 equiv) were added. The resulting mixture was stirred at rt for 30 min then starting compound 6 (406 mg, 1.16 mmol, 1.0 equiv) was added and stirring was continued at rt for 20 h.
  • DIPEA 301 mg, 2.33 mmol, 2.0 equiv
  • HOBt 190 mg, 1.4 mmol, 1.2 equiv
  • EDCI 268 mg, 1.4 mmol, 1.2 equiv
  • DIPEA 100 mg, 0.77 mmol, 6 equiv
  • HOAt 52.6 mg, 0.39 mmol, 3.0 equiv
  • EDCI 74.1 mg, 0.39 mmol, 3 equiv
  • a solution of starting amino acid 18 100 mg, 0.13 mmol, 1.0 equiv
  • DIPEA 50 mg, 0.39 mmol, 3.0 equiv
  • the inhibition property of compound Compound 1A was evaluated in assays measuring the ability of USP9X to catalyze the hydrolysis of the amide bond between the C terminal carboxylate of ubiquitin and rhodamine 110 of the fluorogenic substrate ubiquitin- rhodamine 110.
  • Assay buffer 100 mM NaCl, 1 mM DTT, 0.1% bovine serum albumin, 0.05% Tween 20, 50 mM Tris, pH 7.5).
  • USP9X enzyme assays were set up in a 96 well plate to assess the abilities of Compound 3 A and Compound 4A to inhibit the activity of USP9X, respectively.
  • Ubiquitin- rhodaminel 10 was used as the substrate and ubiquitin aldehyde was included in the assay as a positive control.
  • Ubiquitin aldehyde binds covalently to the thiol group of the active site Cys of USP9X.
  • the activity of USP9X was completely abolished by incubation with ubiquitin aldehyde.
  • the activity of USP9X was reduced approximately 60% with the higher concentrations of Compound 2A, Compound 3 A, and Compound 4A (> 10 ⁇ M).
  • the partial inhibition of USP9X was not due to the lack of solubility of these compounds at high concentrations but instead was shown to have a mechanistic basis.
  • the IC50 for the inhibition of USP9X activity by Compound 2A was calculated from a four parameter, variable slope fit of the data in GraphPad Prism to be 0.39 ⁇ M. This value was similar to the values of the dissociation equilibrium constant determined for this compound from the surface plasmon resonance spectrometry.
  • the IC50 values of Compound 2A, Compound 5A, Compound 7A, and Compound 11A against USP9X were determined to be 0.39 ⁇ M, 0.03 ⁇ M, 0.011 ⁇ M, and 0.048 ⁇ M, respectively.
  • the values of all the parameters, Ks, Ki, kcat, a, and P could be determined from the steady state measurements.
  • the enzyme reactions were set up as described in the Inhibiting the catalytic activity of USP9X section.
  • the concentration of the substrate ubqiutin-rhodaminel 10 was varied.
  • the initial rates of the reactions were determined at several concentrations of the inhibitor Compound 2A.
  • the values of a and P could then be determined by fitting the initial rate data of the enzyme reaction at various concentrations of Compound 2A to a modified version of the Michaelis-Menten equation, designating both Vmax and Ks apparent to distinguish the apparent values of the parameters determined from these plots to the true values for these parameters determined from the steady state kinetics in the absence of Compound 2A.
  • the values of a and P could be determined from the variation of the slopes and intercepts of the series of double reciprocal plots of 1/v versus 1/[S] for each concentration of Compound 2A. The plots of 1/A slope vs. l/[ Compound 2A] or 1/A intercept vs.
  • the two values for Ki were determined by two different methods for determining this parameter from the kinetic data: 1 represented a value determined from the slope of the 1/A intercept vs 1/[I] plot; 2 represented a value determined from the y intercept of the 1/A slope vs 1/[I] plot.
  • 1 represented a value determined from the slope of the 1/A intercept vs 1/[I] plot
  • 2 represented a value determined from the y intercept of the 1/A slope vs 1/[I] plot.
  • These four compounds inhibited USP9X catalytic activity with the same mechanism - hyperbolic mixed inhibition. Only the values of the parameters varied for the compounds.
  • the potency of the inhibition of USP9X improved significantly for this series of compounds beginning with several hundred nM for Compound 2A and increasing by nearly two orders of magnitude for Compound 7A, whether comparing the IC50s from dose- response curves or the values for Ki determined from steady-state enzyme inhibition kinetics.
  • Screening for various cell lines Compounds can be screened for DUB inhibitory and apoptotic activity in a panel of CMU, myeloma and Mantle cell lymphoma cell lines. Selected compounds can also be tested for DUB inhibition in intact cells and in isolated DUB (USP9X-UCH domain) enzyme preparations.
  • Chemical structures can be screened for inhibitory activity using this assay. Fluorescent scans are used to assess inhibitory activity in this enzyme assay.
  • Mia-Paca-2 and MDA-MB-231 cells were seeded into the wells of a 96 well tissue culture plate in complete medium (10% fetal calf serum) and incubated overnight at 37 °C/5% CO2). The medium was exchanged for fresh medium containing varying concentrations of Compound 9 A and the plate was incubated for 72 hours at 37 °C/5% CO2. The viability of cells at the end of this incubation was assessed with the WST-1 reagent.
  • Compound 9A inhibited the proliferation of both types of cells, with an IC50 of 10 nM for Mi- aPaCa-2 cells and 2 ⁇ M for MDA-MB-231 cells.
  • the compounds were profiled on an ONCOLINESTM Profiler (Netherlands Translational Research Center) assayed with 66 cancer cell lines to determine if they were capable of inhibiting the proliferation of these cancer cells.
  • the results showed 57 of 66 cancer cell lines were sensitive to Compound 7A, wherein Compound 7A had IC50 values ⁇ 1 ⁇ M in 9 cancer cell lines.
  • Compound 7A had an IC50 value over 32 ⁇ M in the malignant melanoma cell line MeWo.
  • Compound 7A had an IC50 value of 2.4 ⁇ M in the ovarian cancer cell line OVCAR-3.
  • Compound 7A had an IC50 value of 0.88 ⁇ M in the colon cancer cell line RKO.
  • Compound 7A had an IC50 value of 0.79 ⁇ M in the pancreatic cancer cell line MIA PaCa-2. Compound 7A had an IC50 value of 0.2 ⁇ M in the triple negative breast cancer cell line BT-549. Table 3 provided IC50 values for cell lines wherein Compound 7A were active at IC50 ⁇ 5 ⁇ M. Table 3. IC50 of Compound 7A in Multiple Cell Lines Compound 7A had an IC50 value of 0.486 ⁇ M in the CML cell line K-562. According to BH3 profding (a functional assay to determine which of the pro-survival Bcl-2 proteins that a cell depends upon for survival), Mcl-1 was not essential for survival of K562. Inhibition of USP9X in K562 cells led to the K63 polyubiquitylation of Bcr-Abl (the oncogenic driver in K562) with consequent relocation to peri-nuclear aggresomes and thus loss of activity.
  • Compound 7A was about 4-fold more potent than WP1130.
  • Compound 7A appeared to be inactive and WP1130 was above 4-fold more potent than Compound 7A.
  • the 3 cell lines expressed high levels of the anti-apoptotic Bcl- xL, known to correlate with resistance to antagonism of Mcl-1.
  • WP1130 also inhibited the activity of other deubiquitylases, which might account for the observed higher potencies of WP1130 in certain cell lines. The results indicated that the compound was generally more potent than WP1130.
  • USP9X Another substrate of USP9X is the receptor tyrosine kinase erbB2.
  • the involvement of USP9X in the regulation of erbB2 suggested that its degradation may be induced by treatment with USP9X inhibitors.
  • the breast cancer cell line SK-BR3 expresses high levels of erbB2. This cell line was treated with Compound 7A and the effects on cell viability as well as expression levels of erbB2 were examined.
  • Compound 7A inhibited the viability of SK-BR3.
  • the levels of erbB2 were determined by Western blot and found to decrease in parallel with the decline in cell viability. After 72-hour treatment of 10 pm Compound 7A, the level of erbB2 declined to about zero. The loss of erbB2 from the plasma membrane was also confirmed by flow cytometry.
  • MIA-PaCa-2 cells were treated with Compound 7A at 25 ⁇ M, 12.5 ⁇ M, 6.25 ⁇ M, and 3.125 ⁇ M, respectively, for 24 hours.
  • Capture ELISA of Mcl-1 was performed using a dilution series of MIA-PaCa-2 cell lysates equalized for total protein in a 96-well plate coated with sheep anti-human Mcl-1 (R&D Systems, #AF8281). Detection of Mcl-1 was performed using biotinylated sheep anti -human Mcl-1 followed by streptavidin-horseradish peroxidase and TMB development.
  • Mcl-1 For detection of ubiquitylated Mcl-1 the same capture conditions were used but employing a biotinylated mouse anti-human ubiquitin antibody (R&D Systems, #MAB701), again detecting with streptavidin-horseradish peroxidase and TMB development. Lysate samples were run in triplicate and percentages were calculated based on the signal generated from cell lysates with no compound added (vehicle control samples). After treading cells with 25 ⁇ M Compound 7A for 24 hours, Mcl-1 declined by about 23.0% whereas ubiquitylated Mcl-1 increased by about 27.5%. With 12.5 ⁇ M Compound 7A, Mcl-1 declined by about 25.0% whereas ubiquitylated Mcl-1 increased by about 14.0%.
  • R&D Systems, #MAB701 biotinylated mouse anti-human ubiquitin antibody
  • Mcl-1 can serve as an anti-apoptotic factor conferring resistance to chemotherapy.
  • Western blot analysis was carried out to investigate the effect of the compounds on other inhibitors of which the targeted proteins could otherwise be protected by Mcl-1 and thus resistant to these inhibitors.
  • the TNBC cell line MDA-MB-231 express high levels of Mcl-1 and USP9X in addition to Bcl-2 and Bcl-xL.
  • MDA-MB-231 cells were treated with ABT737 for 72 hours at varying concentrations and underwent western blot analysis.
  • the IC50 value of ABT737 was determined to be 8.7 ⁇ M from the dose-response curve.
  • the IC50 value of ABT737 was determined to be 1.8 ⁇ M from the dose-response curve.
  • Co-treatment of 10 ⁇ M Compound 5A resulted in 5-fold enhancement in the sensitivity of MDA-MB-231 cells to ABT737.
  • PDL1 Programmed death-ligand 1
  • MIA PaCa-2 cells expressing detectable levels of PD-L1 are treated with Compound 7A at various concentrations for 24 hours, and the ensuing level of PD-L1 are determined by Western blot to decrease by about 50% after the treatment.
  • Example 7 In vivo animal model test
  • USP9X is highly expressed and activated in melanoma cells.
  • the effect of compounds on USP9X activity in a representative melanoma cell line, A375, and an A375 variant cell line that is resistant to the BRAF kinase inhibitor, vemurafenib, can be examined.
  • mice are treated intravenously (IV) or by oral gavage (PO).
  • IV intravenously
  • PO oral gavage
  • Compound is administered once to two mice per group at the indicated dosage level and route (IV or PO) and plasma is collected after administration.
  • Compound concentration in the plasma is measured by high performance liquid chromatography coupled with mass spectroscopy detection (LC/MS).
  • Tumor cells are injected into the dorsal region of twenty female NOD/SCID/gamma- 2 knockout mice (NSG) weighing about 20 grams each. After 3 weeks tumors become visible and measurable with calipers. Mice are separated into four groups of 5 mice each and IP injected with the compound of the invention dissolved in 55% dimethyl sulfoxide, 25% polyethylene glycol 300, 20% phosphate-buffered saline at dose levels of 0, 2.5, 5 and 10 mg/kg mouse body weight. Animals are injected once per day for 14 days and tumor growth (measured with calipers) and animal weight are monitored over the treatment interval.
  • NSG NOD/SCID/gamma- 2 knockout mice
  • Results show that tumor growth in mice treated with the compound at dose levels of 2.5, 5 and 10 mg/kg are inhibited at different levels, compared with that in the group of untreated mice; and the dose of 10 mg/kg of the compound lead to the maximum inhibition of tumor growth by about 80% among the three treated groups, increasing the life span by about 200% compared with untreated mice.

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Abstract

L'invention concerne des composés qui inhibent les DUB, en particulier USP9X. L'invention concerne également des procédés d'inhibition de DUB, y compris USP9X, et des procédés de traitement de cancers.
PCT/IB2023/050332 2021-11-16 2023-01-13 Inhibiteurs d'usp9x Ceased WO2023089601A2 (fr)

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PCT/US2022/050069 WO2023091464A1 (fr) 2021-11-16 2022-11-16 Inhibiteurs de l'usp9x
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CN119113118A (zh) * 2024-09-19 2024-12-13 中南大学 USP33抑制剂在制备抗SARS-CoV-2病毒药物中的应用、抗病毒药物及其制备方法

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US8030279B2 (en) * 2003-03-21 2011-10-04 The Trustees Of The University Of Pennsylvania Tamandarin analogs and fragments thereof and methods of making and using
MY201925A (en) * 2017-07-28 2024-03-23 Turning Point Therapeutics Inc Macrocyclic compounds and uses thereof
KR20210061400A (ko) * 2018-09-19 2021-05-27 포르마 세라퓨틱스 인크. 유비퀴틴 특이적 펩티다아제 9x의 억제
EP3902533A1 (fr) * 2018-12-26 2021-11-03 Forma Therapeutics, Inc. Inhibition de la peptidase 9x spécifique de l'ubiquitine

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CN119113118A (zh) * 2024-09-19 2024-12-13 中南大学 USP33抑制剂在制备抗SARS-CoV-2病毒药物中的应用、抗病毒药物及其制备方法

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