Compounds and uses thereof

COMPOUNDS AND THEIR USES

Background

The invention relates to compounds useful for modulating BRG1 or BRM-associated factor (BAF) complexes. In particular, the invention relates to compounds useful for the treatment of disorders associated with the function of the BAF complex.

Chromatin regulation is essential for gene expression, and ATP-dependent chromatin remodeling is one mechanism by which such gene expression occurs. The human Switch/Sucrose non-fermentable chromatin remodeling complex (SWI/SNF), also known as the BAF complex, has two SWI2-like ATPases known as BRG1 (Brahma-related gene 1) and BRM (Brahma). The transcriptional activator BRG1, also known as ATP-dependent chromatin remodeler SMARCA4, is encoded by the SMARCA4 gene on chromosome 19. BRG1 is overexpressed in some cancer tumors and is required for cancer cell proliferation. BRM, also known as SNF2L2 probable global transcription activator and/or ATP-dependent chromatin remodeler SMARCA2, is encoded by the SMARCA2 gene on chromosome 9 and has been shown to be essential for tumor cell growth in cells characterized by mutations. of loss of function BRG1. Inactivation of BRG and/or BRM results in downstream effects on cells, including cell cycle arrest and tumor suppression.

Compendium

The present invention features compounds useful for modulating a BAF complex. In some embodiments, the compounds are useful for the treatment of disorders associated with an alteration in a BAF complex, for example, a disorder associated with an alteration in one or both of the BRG1 and BRM proteins. The compounds of the invention, alone or in combination with other pharmaceutically active agents, can be used to treat such disorders.

In one aspect, the invention features a compound, N-(1-((4-(6-(2,6-dimethylmorpholino)pyridin-2yl)thiazol-2-yl)amino)-3-methoxy-1-oxopropan- 2-yl)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamide, or a pharmaceutically acceptable salt thereof, having the structure:

<img file="CO2022010547A2_D0001.tif" />

In some embodiments, the compound, or a pharmaceutically acceptable salt thereof, has the structure:

<img file="CO2022010547A2_D0002.tif" />

In some embodiments, the compound, or a pharmaceutically acceptable salt thereof, has the structure:

<img file="CO2022010547A2_D0003.tif" />

In some embodiments, the compound, or a pharmaceutically acceptable salt thereof, has the structure:

<img file="CO2022010547A2_D0004.tif" />

In another aspect, the invention features a compound, N-(1-((4-(6-(2,6-dimethylmorpholino)pyridin2-yl)thiazol-2-yl)amino)-3-(methoxy-d3)- 1-oxopropan-2-yl-3,3-d2)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamide, or a pharmaceutically acceptable salt thereof, having the structure:

<img file="CO2022010547A2_D0005.tif" />

In some embodiments, the compound, or a pharmaceutically acceptable salt thereof, has the structure:

<img file="CO2022010547A2_D0006.tif" />

In some embodiments, the compound, or a pharmaceutically acceptable salt thereof, has the structure:

<img file="CO2022010547A2_D0007.tif" />

In some embodiments, the compound, or a pharmaceutically acceptable salt thereof, has the structure:

<img file="CO2022010547A2_D0008.tif" />

In another aspect, the invention features a pharmaceutical composition that includes any of the above compounds and a pharmaceutically acceptable excipient.

In another aspect, the invention features a method for decreasing the activity of a BAF complex in a cell or subject. This method includes contacting the cell with, or administering to the subject, an effective amount of any of the foregoing compounds or pharmaceutical compositions.

In another aspect, the invention features a method for inhibiting BRM in a cell or subject. This method includes contacting the cell with, or administering to the subject, an effective amount of any of the foregoing compounds or pharmaceutical compositions.

In another aspect, the invention features a method for inhibiting BRG1 in a cell or subject. This method includes contacting the cell with, or administering to the subject, an effective amount of any of the foregoing compounds or pharmaceutical compositions.

In another aspect, the invention features a method of inhibiting BRM and BRG1 in a cell or subject. This method includes contacting the cell with, or administering to the subject, an effective amount of any of the foregoing compounds or pharmaceutical compositions.

In another aspect, the invention features a method for inducing apoptosis in a cell or subject. This method includes contacting the cell with, or administering to the subject, an effective amount of any of the foregoing compounds or pharmaceutical compositions.

In some embodiments of any of the above methods, the cell is a cancer cell and/or the subject has cancer.

In another aspect, the invention features a method of treating a BAF complex-related disorder in a subject in need thereof. This method includes administering to the subject an effective amount of any of the foregoing compounds or pharmaceutical compositions.

In another aspect, the invention features a method of treating a BRG1 loss-of-function mutation-related disorder in a subject in need thereof. This method includes administering to the subject an effective amount of any of the foregoing compounds or pharmaceutical compositions. In some embodiments, the subject is determined to have a BRG1 loss-of-function disorder (eg. eg, the disorder and/or subject have been determined to include cells with a loss-of-function mutation of BRG1).

In some embodiments of the above methods, the BAF complex-related disorder or BRG1 loss-of-function mutation-related disorder is cancer, viral infection, Coffin Siris, neurofibromatosis (for example, NF-1, NF- 2 or Schwannomatosis) or Multiple Meningioma.

In another aspect, the invention features a method for treating cancer in a subject in need thereof. This method includes administering to the subject an effective amount of any of the foregoing compounds or pharmaceutical compositions.

In another aspect, the invention features a method of reducing cancer tumor growth in a subject in need thereof. This method includes administering to the subject an effective amount of any of the foregoing compounds or pharmaceutical compositions.

In another aspect, the invention features a method for suppressing the metastatic progression of cancer in a subject in need thereof. This method includes administering an effective amount of any of the foregoing compounds or pharmaceutical compositions.

In another aspect, the invention features a method of suppressing metastatic colonization (eg, metastatic colonization in the lung and/or brain) of cancer in a subject in need thereof. This method includes administering an effective amount of any of the foregoing compounds or pharmaceutical compositions.

In another aspect, the invention features a method for reducing the level and/or activity of BRG1 and/or BRM in a cancer in a cell or subject in need thereof. This method includes contacting the cell with, or administering to the subject, an effective amount of any of the foregoing compounds or pharmaceutical compositions.

In some embodiments of any of the above methods, the cancer is non-small cell lung cancer, colorectal cancer, bladder cancer, cancer of unknown primary, glioma, breast cancer, melanoma, non-melanoma skin cancer, cancer of the endometrial cancer, esophagogastric cancer, esophageal cancer, pancreatic cancer, hepatobiliary cancer, soft tissue sarcoma, ovarian cancer, head and neck cancer, renal cell carcinoma, bone cancer, non-Hodgkin lymphoma, small cell lung cancer, prostate cancer, embryonal tumor, germ cell tumor, cervical cancer, thyroid cancer, salivary gland cancer, gastrointestinal neuroendocrine tumor, uterine sarcoma, gastrointestinal stromal tumor, cancer of the CNS, thymic tumor, adrenocortical carcinoma, appendiceal cancer, small intestine cancer, penile cancer, bone cancer or hematological cancer. In some forms of any of the above methods, the cancer is esophageal cancer.

In some embodiments of any of the above methods, the cancer is non-small cell lung cancer, colorectal cancer, bladder cancer, cancer of unknown primary, glioma, breast cancer, melanoma, non-melanoma skin cancer, cancer of the endometrial cancer, penile cancer, bone cancer, renal cell carcinoma, prostate cancer, or a blood cancer. In some embodiments of any of the above methods, the cancer is non-small cell lung cancer.

In some embodiments of any of the above methods, the cancer is melanoma, prostate cancer, breast cancer, bone cancer, renal cell carcinoma, or hematologic cancer.

In some forms, the cancer is melanoma (eg, uveal melanoma, mucosal melanoma, or cutaneous melanoma). In some embodiments, the cancer is prostate cancer. In some modalities, the cancer is a hematologic cancer (eg, multiple myeloma, large cell lymphoma, acute T-cell leukemia, acute myeloid leukemia, myelodysplastic syndrome, lambda immunoglobulin A myeloma, diffuse mixed histiocytic and lymphocytic lymphoma, B-cell lymphoma, acute lymphoblastic leukemia (eg. (e.g., T-cell acute lymphoblastic leukemia or B-cell acute lymphoblastic leukemia), diffuse large cell lymphoma, or non-Hodgkin lymphoma). In some embodiments, the cancer is breast cancer (eg, ER-positive breast cancer, ER-negative breast cancer, triple-positive breast cancer, or triple-negative breast cancer). In some modalities, the cancer is a bone cancer (eg, Ewing's sarcoma). In some forms, the cancer is a renal cell carcinoma (eg, microphthalmia transcription factor (MITF) familial translocation renal cell carcinoma (tRCC) ).

In some embodiments, the cancer expresses the BRG1 and/or BRM protein and/or the cell or subject has been identified as expressing BRG1 and/or BRM. In some embodiments, the cancer expresses the BRG1 protein and/or the cell or subject has been identified as expressing BRG1. In some embodiments, the cancer expresses the BRM protein and/or the cell or subject has been identified as expressing BRM. In some embodiments, the subject or the cancer has and/or has been identified as having a loss-of-function mutation of BRG1. In some embodiments, the subject or the cancer has and/or has been identified as having a BRM loss-of-function mutation.

In some embodiments of any of the above methods, the cancer has or has been determined to have one or more BRG1 mutations (eg, homozygous mutations). In some embodiments, the one or more BRG1 mutations include a mutation in the ATPase catalytic domain of the protein. In some embodiments, the one or more BRG1 mutations include a C-terminal deletion of BRG1.

In some embodiments of any of the above methods, the cancer does not have, or has been determined not to have, an epidermal growth factor receptor (EGFR) mutation. In some embodiments of any of the above methods, the cancer does not have, or has been determined not to have, an anaplastic lymphoma kinase (ALK) driver mutation. In some embodiments of any of the above methods, the cancer has, or has been determined to have, a KRAS mutation.

In some modalities, the cancer has, or has been determined to have, a mutation in

GNAQ. In some modalities, the cancer has, or has been determined to have, a mutation in

GNA11. In some modalities, the cancer has, or has been determined to have, a mutation in

PLCB4. In some modalities, the cancer has, or has been determined to have, a mutation in

CYSLTR2. In some embodiments, the cancer has, or has been determined to have, a mutation in BAP1. In some embodiments, the cancer has, or has been determined to have, a mutation in SF3B1. In some embodiments, the cancer has, or has been determined to have, a mutation in EIF1AX. In some embodiments, the cancer has, or has been determined to have, a TFE3 translocation. In some embodiments, the cancer has, or has been determined to have, a TFEB translocation. In some embodiments, the cancer has, or has been determined to have, a MITF translocation. In some embodiments, the cancer has, or has been determined to have, an EZH2 mutation. In some embodiments, the cancer has, or has been determined to have, a SUZ12 mutation. In some embodiments, the cancer has, or has been determined to have, an EED mutation.

In some modalities, the cancer is metastatic. For example, cancer includes cells that exhibit migration and/or migrating cell invasion and/or includes cells that exhibit endothelial recruitment and/or angiogenesis. Metastatic cancer can be spread by seeding on the surface of the peritoneal, pleural, pericardial, or subarachnoid spaces. Alternatively, metastatic cancer can spread through the lymphatic system or spread via the bloodstream. In some embodiments, the cancer is a cell migrating cancer (eg, a non-metastatic cell migrating cancer).

In some embodiments of any of the above methods, the cancer is drug resistant (eg, the cancer has been determined to be resistant, or is likely to be resistant, to chemotherapeutic or cytotoxic agents such as genetic markers, or is likely to be resistant to chemotherapeutic or cytotoxic agents (such as a cancer that has not responded to a chemotherapeutic or cytotoxic agent) and/or has not responded to prior therapy (eg. g., a chemotherapeutic or cytotoxic agent, immunotherapy, surgery, radiation therapy, thermotherapy, or photocoagulation, or a combination thereof).

In some modalities, the cancer is resistant and/or has not responded to vemurafenib, dacarbazine, a CTLA4 inhibitor, a PD1 inhibitor, interferon therapy, a BRAF inhibitor, a MEK inhibitor, radiation therapy, temozolimide, irinotecan, a CAR-T therapy, herceptin, perjeta, tamoxifen, xeloda, docetaxol, platinum agents such as carboplatin, taxanes such as paclitaxel and docetaxel, ALK inhibitors, MET inhibitors, alimta, abraxane, doxorubicin, gemcitabine, avastin, halaven, neratinib, a PARP inhibitor, brilanestrant, an mTOR inhibitor, topotecan, gemzar, a VEGFR2 inhibitor, a folate receptor antagonist, demcizumab, fosbretabulin, or a PDL1 inhibitor, or combinations thereof.

In some modalities of any of the above methods, the cancer is resistant and/or has not responded to dacarbazine, temozolomide, cisplatin, treosulfan, fotemustine, IMCgplOO, a CTLA-4 inhibitor (eg, ipilimumab), an inhibitor PD-1 inhibitor (eg, nivolumab or pembrolizumab), a PD-L1 inhibitor (eg, atezolizumab, avelumab, or durvalumab), a mitogen-activated protein kinase (MEK) inhibitor (eg. selumetinib, binimetinib, or tametinib) and/or a protein kinase C (PKC) inhibitor (eg, sotrastaurin or IDE196).

In some embodiments of any of the above methods, the cancer is resistant and/or unresponsive to a previously administered treatment used for the treatment of uveal melanoma, for example, a MEK inhibitor or a PKC inhibitor. For example, in some modalities, the cancer is resistant and/or unresponsive to a mitogen-activated protein kinase (MEK) inhibitor (eg, selumetinib, binimetinib, or tametinib) and/or a protein kinase inhibitor. C(PKC) (p. g., sotrastaurin or IDE196).

In some embodiments, the method further includes administering to the subject or contacting the cell with an anticancer therapy, eg, a chemotherapeutic or cytotoxic agent, immunotherapy, surgery, radiation therapy, thermotherapy, or photocoagulation, or combinations thereof. In some embodiments, the cancer therapy is a chemotherapeutic or cytotoxic agent, eg, an antimetabolite, antimitotic, antitumor antibiotic, asparagine-specific enzyme, bisphosphonate, antineoplastic, alkylating agent, DNA repair enzyme inhibitor, histone inhibitor deacetylase, corticosteroid, demethylating agent, immunomodulator, janus-associated kinase inhibitor, phosphinositide 3-kinase inhibitor, proteasome inhibitor or tyrosine kinase inhibitor, or combinations thereof.

In some embodiments, the compound of the invention is used in combination with another antineoplastic therapy used for the treatment of uveal melanoma such as surgery, a MEK inhibitor and/or a PKC inhibitor, or combinations thereof. For example, in some embodiments, the method further comprises performing surgery before, after, or at the same time as administration of the compound of the invention. In some embodiments, the method further comprises administration of a MEK inhibitor (eg, selumetinib, binimetinib, or tametinib) and/or a PKC inhibitor (eg, sotrastaurin or IDE196) before, after, or at the same time. time than the administration of the compound of the invention.

In some embodiments, the cancer therapy and the compound of the invention are administered within 28 days (eg, within 21 days, within 14 days, or within 7 days) of each other. and each in an amount that together are effective in treating the subject.

In another aspect, the disclosure provides a method for treating a viral infection in a subject in need thereof. This method includes administering to the subject an effective amount of any of the foregoing compounds or pharmaceutical compositions. In some embodiments, the viral infection is infection with a virus of the Retroviridae family such as lentiviruses (eg, human immunodeficiency virus (HIV)) and deltaretroviruses (eg. human T-cell leukemia virus I (HTLV-I), human T-cell leukemia virus II (HTLV-II), a virus of the family Hepadnaviridae (eg, hepatitis B (HBV)), a virus of the Flaviviridae family (eg, hepatitis C virus (HCV)), a virus of the Adenoviridae family (eg, human adenovirus), a virus of the family Herpesviridae (p. eg, human cytomegalovirus (HCMV), Epstein-Barr virus, herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), human herpes virus 6 (HHV-6), Herpesvitus K *, CMV, varicella-zoster virus), a virus of the Papillomaviridae family (eg, Human Papillomavirus (HPV, HPV E1)), a virus of the Parvoviridae family (eg, Parvovirus B19 ), a virus of the Polyomaviridae family (eg, JC virus and BK virus), a virus of the Paramyxoviridae family (eg. measles virus) or a virus from the Togaviridae family (eg rubella virus).

In some embodiments of any of the above methods, the effective amount of the compound reduces the level and/or activity of BRG1 by at least 5% (eg. g., at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, 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 95%, or at least 99%) compared to a reference.

In some embodiments of any of the above methods, the effective amount of the compound reduces the level and/or activity of BRG1 by at least 5% (eg. g., at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, 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 95%, or at least 99%) compared to a reference for at least 12 hours (for example, at least 14 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 22 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 48 hours, at least 72 hours, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 14 days, at least 21 days, at least 28 days or more).

In some embodiments of any of the above methods, the effective amount of the compound reduces the level and/or activity of BRM by at least 5% (eg. g., at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, 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 95%, or at least 99%) compared to a reference.

In some embodiments of any of the above methods, the effective amount of the compound reduces the level and/or activity of BRM by at least 5% (eg. g., at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, 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 95%, or at least 99%) compared to a reference for at least 12 hours (for example, at least 14 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 22 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 48 hours, at least 72 hours, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 14 days, at least 21 days, at least 28 days or more).

In some embodiments, the effective amount of the compound of the invention is an amount effective to inhibit metastatic colonization of cancer in the liver and/or brain.

chemical terms

The compounds of the invention may have one or more asymmetric carbon atoms and may exist as optically pure enantiomers, mixtures of enantiomers such as racemates, optically pure diastereoisomers, mixtures of diastereoisomers, diastereoisomeric racemates, or mixtures of diastereoisomeric racemates. The optically active forms can be obtained, for example, by resolution of the racemates, by asymmetric synthesis or by asymmetric chromatography (chromatography with a chiral adsorbent or eluent). That is, some of the described compounds can exist in various stereoisomeric forms. Stereoisomers are compounds that differ only in their spatial arrangement. Enantiomers are pairs of stereoisomers whose mirror images are not superimposable, most commonly because they contain an asymmetrically substituted carbon atom that acts as a chiral center. Enantiomer means one of a pair of molecules that are mirror images of each other and are not superimposable. Diastereomers are stereoisomers that are not related as mirror images, most commonly because they contain two or more asymmetrically substituted carbon atoms and represent the configuration of substituents around one or more chiral carbon atoms. Enantiomers of a compound can be prepared, for example, by separating an enantiomer from a racemate using one or more well-known techniques and methods, such as chiral chromatography and separation methods based thereon. The appropriate technique and/or method for separating an enantiomer of a compound described herein from a racemic mixture can be readily determined by those skilled in the art. Racemate or racemic mixture means a compound containing two enantiomers, where such mixtures do not exhibit optical activity; that is, they do not rotate the plane of polarized light. "Geometric isomer" means isomers that differ in the orientation of the substituent atoms relative to a carbon-carbon double bond, a cycloalkyl ring, or a bridged bicyclic system. The atoms (other than H) on each side of a carbon-carbon double bond can be in an E (substituents are on opposite sides of the carbon-carbon double bond) or Z (substituents are oriented on the same side) configuration. . R, S, S*, R*, E, Z, cis and trans indicate configurations relative to the core molecule. Some of the described compounds may exist in atropisomeric forms. Atropisomers are stereoisomers that result from hindered rotation over single bonds where the steric strain barrier to rotation is high enough to allow isolation of conformers. The compounds of the invention can be prepared as individual isomers by isomer-specific synthesis or they can be resolved from a mixture of isomers. Conventional resolution techniques include salting the free base of each isomer of an isomeric pair using an optically active acid (followed by fractional crystallization and regeneration of the free base), salting the acid form of each isomer of an isomeric pair using an optically active amine (followed by fractional crystallization and regeneration of the free acid), forming an ester or amide of each of the isomers of an isomeric pair using an acid, optically pure amine or alcohol (followed by chromatographic separation and removal of the chiral auxiliary), or resolving an isomeric mixture of a starting material or a final product using various well known chromatographic methods. When the stereochemistry of a described compound is named or represented by structure, the stereoisomer named or represented is at least 60%, 70%, 80%, 90%, 99%, or 99.9% by weight relative to the other stereoisomers. When a single enantiomer is named or represented by structure, the represented or named enantiomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% by weight optically pure. When a single diastereomer is named or represented by structure, the represented or named diastereomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% by weight pure. The percent optical purity is the ratio of the weight of the enantiomer or above the weight of the enantiomer plus the weight of its optical isomer. Diastereomeric purity by weight is the ratio of the weight of one diastereomer or above to the weight of all diastereomers. When the stereochemistry of a described compound is named or represented by structure, the stereoisomer named or represented has at least a purity of at least 60%, 70%, 80%, 90%, 99%, or 99.9% by mole fraction in relative to the other stereoisomers. When a single enantiomer is named or represented by structure, the represented or named enantiomer has a purity of at least 60%, 70%, 80%, 90%, 99%, or 99.9% by mole fraction. When a single diastereomer is named or represented by structure, the represented or named diastereomer has a purity of at least 60%, 70%, 80%, 90%, 99%, or 99.9% by mole fraction. The percent purity per mole fraction is the ratio of the moles of the enantiomer or above to the moles of the enantiomer plus the moles of its optical isomer. Similarly, percent purity per mole fraction is the ratio of the moles of the diastereomer or over the moles of the diastereomer plus the moles of its isomer. When a described compound is named or represented by its structure without indicating stereochemistry, and the compound has at least one chiral center, the name or structure should be understood to encompass any enantiomer of the free compound of the corresponding optical isomer, a racemic mixture of the compound, or mixtures enriched in an enantiomer with respect to its corresponding optical isomer. When a described compound is named or represented by its structure without indicating stereochemistry and has two or more chiral centers, the name or structure should be understood to encompass one diastereomer free from other diastereomers, several free diastereomers from other pairs of diastereomers, mixtures of diastereomers, mixtures of pairs of diastereomers, mixtures of diastereomers in which one diastereomer is enriched relative to the other diastereomers, or mixtures of diastereomers in which one or more diastereomers are enriched with respect to the other diastereomers. The invention encompasses all of these forms.

Unless otherwise indicated, the structures depicted herein also include compounds that differ only in the presence of one or more isotopically enriched atoms. Exemplary isotopes that can be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, and iodine, such as²h,³H, 11C,¹³c,¹⁴c,¹³No,¹⁵No,¹⁵EITHER,¹⁷EITHER,¹⁸EITHER,³²Q,³³Q,³⁵Yes,¹⁸F,³⁶Cl,¹²³I and¹²⁵I. Isotopically labeled compounds (eg, those labeled with³h and¹⁴C) may be useful in assays for the distribution of compounds or substrates in tissues. Tritiated isotopes (i.e.,³H) and carbon-14 (i.e.,¹⁴C) can be useful because of their ease of preparation and detection. Also, substitution with heavier isotopes such as deuterium (i.e.,²H) may provide certain therapeutic advantages resulting from increased metabolic stability (eg, increased in vivo half-life or reduced dosage requirements). In some embodiments, one or more hydrogen atoms are replaced by²H o³H, or one or more carbon atoms are replaced by carbon enriched with¹³C or¹⁴C. Positron-emitting isotopes such as¹⁵EITHER,¹³N, 11C and¹⁸F are useful for positron emission tomography (PET) studies to examine substrate receptor occupancy. Preparations of isotopically labeled compounds are known to those skilled in the art. For example, isotopically labeled compounds can generally be prepared by following procedures analogous to those described for the compounds of the present invention described herein, substituting a non-isotopically labeled reagent for an isotopically labeled reagent.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as that normally ascribed to them by a person skilled in the art to which this invention pertains. Methods and materials are described herein for use in the present disclosure; Other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, shall control.

Definitions

In this application, unless otherwise apparent from the context, (i) the term a can be understood to mean at least one; (ii) the term "or" can be understood in the sense of "and/or"; and (iii) the terms comprising and including may be understood to encompass detailed components or steps, whether presented alone or in conjunction with one or more additional components or steps.

As used herein, the terms "around" and "about" refer to a value that is within 10% above or below the value being described. For example, the term around 5 nM indicates a range from 4.5 to 5.5 nM.

As used herein, the term "administration" refers to the administration of a composition (eg, a compound or a preparation including a compound as described herein) to a subject or system. Administration to an animal subject (eg, a human) can be by any appropriate route. For example, in some modalities, administration may be bronchial (including by bronchial instillation), buccal, enteral, interdermal, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intratumoral, intravenous, intraventricular, mucosal, nasal , oral, rectal, subcutaneous, sublingual, topical, tracheal (including intratracheal instillation), transdermal, vaginal, and vitreous.

As used herein, the term "BAF complex" refers to the complex of factors associated with BRG1 or HBRM in a human cell.

As used herein, the term "BAF complex-related disorder" refers to a disorder caused or affected by the level of activity of a BAF complex.

As used herein, the term BRG1 loss-of-function mutation refers to a mutation in BRG1 that leads to the protein having decreased activity (eg, at least a 1% reduction in BRG1 activity). eg, 2%, 5%, 10%, 25%, 50%, or 100% reduction in BRG1 activity). Exemplary BRG1 loss-of-function mutations include, but are not limited to, a BRG1 homozygous mutation and a BRG1 C-terminal deletion.

As used herein, the term BRG1 loss-of-function disorder refers to a disorder (eg, cancer) that exhibits a reduction in BRG1 activity (eg, at least a reduction in 1% in BRG1 activity, eg, 2%, 5%, 10%, 25%, 50%, or 100% reduction in BRG1 activity).

The term "cancer" refers to a condition caused by the proliferation of malignant neoplastic cells, such as tumors, neoplasms, carcinomas, sarcomas, leukemias, and lymphomas.

As used herein, a combination therapy or administered in combination means that two (or more) different agents or treatments are administered to a subject as part of a defined treatment regimen for a particular disease or condition. The treatment regimen defines the doses and periodicity of administration of each agent so that the effects of the separate agents on the subject overlap. In some embodiments, the administration of the two or more agents is simultaneous or concurrent and the agents may be co-formulated. In some embodiments, the two or more agents are not co-formulated and are administered sequentially as part of a prescribed regimen. In some modalities, the administration of two or more agents or treatments in combination is such that the reduction in a symptom or other disorder-related parameter is greater than would be observed with one agent or treatment administered alone or in the absence of the other. The effect of the two treatments may be partially additive, fully additive, or greater than additive (eg, synergistic). Sequential or substantially simultaneous administration of each therapeutic agent can be effected by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues. The therapeutic agents can be administered by the same route or by different routes. For example, a first combination therapeutic agent can be administered by intravenous injection while a second combination therapeutic agent can be administered orally.

The term CTLA-4 inhibitor, as used herein, refers to a compound such as an antibody capable of inhibiting the activity of the protein that in humans is encoded by the CTLA4 gene. Known CTLA-4 inhibitors include ipilimumab.

By determining the level of a protein or RNA is meant the detection of a protein or RNA, by methods known in the art, either directly or indirectly. “Directly determine” means to perform a process (eg, perform an assay or test on a sample or “analyze a sample,” as that term is defined herein) to obtain the physical entity or value. “Indirectly determining” means receiving the physical entity or value from another party or source (for example, a third-party lab that directly acquired the physical entity or value). Methods for measuring protein level generally include, but are not limited to, Western blotting, immunoblotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, immunofluorescence, surface plasmon resonance, chemiluminescence, fluorescent polarization, phosphorescence, assay immunohistochemistry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), liquid chromatography (LC)-mass spectrometry, microcytometry, microscopy, fluorescence activated cell sorting (FACS), and flow cytometry, as well as assays based on a property of a protein including, but not limited to, enzyme activity or interaction with other associated proteins. Methods for measuring RNA levels are known in the art and include, but are not limited to, quantitative polymerase chain reaction (qPCR) and Northern blot analyses.

By a decreased level or an increased level of a protein or RNA is meant a decrease or increase, respectively, in a protein or RNA level, compared to a control (eg. e.g., a decrease or increase of approximately 5%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500% or more; a decrease or increase of more than 10%, about 15%, about 20%, about 50%, about 75%, about 100%, or about 200%, compared to a reference; a decrease or increase of less than about 0.01 times, about 0.02 times, about 0.1 times, about 0.3 times,

<td>about 0.5</td><td>times, about 0.8</td><td>times or less; or a raise</td><td>of</td><td>more of</td>

<td>about 1.2</td><td>times, approximately</td><td>1.4 times approximately</td><td> 1,5</td><td>times,</td>

<td>about 1.8</td><td>times, approximately</td><td>2.0 times approximately</td><td> 3,0</td><td>times,</td>

<td>about 3.5</td><td>times, approximately</td><td>4.5 times approximately</td><td> 5,0</td><td>times,</td>

<td>about 10</td><td>times, approximately</td><td>about 15 times</td><td> 20</td><td>times,</td>

<td>about 30</td><td>times, approximately</td><td>40 times, approximately</td><td> 50</td><td>times,</td>

<td colspan="5">about 100 times, about 1000 times or more). The level of a protein can</td>

<td colspan="5">expressed in mass/vol (eg, g/dL, mg/mL, μg/mL, ng/mL) or percent relative to total protein in</td>

a sample.

By decreasing the activity of a BAF complex is meant decreasing the level of an activity related to a BAF complex, or a related after effect. A non-limiting example of decreasing the activity of a BAF complex is the activation of Sox2. The activity level of a BAF complex can be measured using any method known in the art, for example, the methods described in Kadoch et al. Cell, 2013, 153, 71-85, whose methods are incorporated herein by reference.

As used herein, the term "derivative" refers to the natural, synthetic, and semi-synthetic analogs of a compound, peptide, protein, or other substance described herein. A derivative of a compound, peptide, protein, or other substance described herein may retain or enhance the biological activity of the parent material.

A cancer determined to be drug resistant, as used herein, refers to a cancer that is drug resistant, based on a lack of response or reduced response to a chemotherapeutic agent, or is predicted to be resistant to drugs based on a prognostic assay (eg, a gene expression assay).

By drug resistant is meant a cancer that does not respond to, or shows a decreased response to, one or more chemotherapeutic agents (eg, any agent described herein).

As used herein, the term "prior therapy unresponsive" or "prior therapy refractory" refers to cancer that has progressed despite therapy treatment.

As used herein, the term "BRM inhibition and/or BRG1 inhibition" refers to blocking or reducing the level or activity of the ATPase catalytic binding domain or bromodomain of the protein. Inhibition of BRM and/or BRG1 can be determined using methods known in the art, for example, a BRM and/or BRG1 ATPase assay, a Nano DSF assay, or a BRM cell and/or BRG1 Luciferase assay.

As used herein, the term LXS196, also known as IDE196, refers to the PKC inhibitor having the structure:

<img file="CO2022010547A2_D0009.tif" />

or a pharmaceutically acceptable salt thereof.

As used herein, "metastatic nodule" refers to an aggregation of tumor cells in the body at a site other than the original tumor site.

As used herein, metastatic cancer refers to a tumor or cancer in which the tumor-forming cancer cells have a high potential or have begun to metastasize or spread from one location to another location or locations within a subject. , through the lymphatic system or via hematogenous spread, for example, creating secondary tumors within the subject. Such metastatic behavior may be indicative of malignant tumors. In some cases, metastatic behavior may be associated with an increase in the cellular migration and/or invasion behavior of tumor cells.

Examples of cancers that can be defined as metastatic include, but are not limited to, lung cancer (eg. non-small cell lung cancer), breast cancer, ovarian cancer, colorectal cancer, biliary tract cancer, bladder cancer, brain cancer, including glioblastomas and medulablastomas, cervical cancer, choriocarcinoma, cancer of the endometrial cancer, esophageal cancer, gastric cancer, hematological malignancies, multiple myeloma, leukemia, intraepithelial neoplasias, liver cancer, lymphomas, neuroblastomas, oral cancer, pancreatic cancer, prostate cancer, sarcoma, skin cancer including melanoma, basal cell cancer, squamous cell cancer, testicular cancer, stromal tumors, germ cell tumors, thyroid cancer, and renal cancer.

Non-metastatic cell migrating cancer as used herein refers to cancers that do not migrate through the lymphatic system or through hematogenous spread.

The term PD-1 inhibitor, as used herein, refers to a compound such as an antibody capable of inhibiting the activity of the protein that in humans is encoded by the PDCD1 gene. Known PD-1 inhibitors include nivolumab, pembrolizumab, pidilizumab, BMS 936559, and atezolizumab.

The term PD-L1 inhibitor, as used herein, refers to a compound such as an antibody capable of inhibiting the activity of the protein that in humans is encoded by the CD274 gene. Known PD-L1 inhibitors include atezolizumab and durvalumab.

The term pharmaceutical composition, as used herein, means a composition containing a compound described herein formulated with a pharmaceutically acceptable excipient and suitable for administration to a mammal, eg, a human. Typically, a pharmaceutical composition is manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of disease in a mammal. The pharmaceutical compositions can be formulated, for example, for oral administration in unit dosage form (eg, a tablet, capsule, gelcap, or syrup); for topical administration (eg, as a cream, gel, lotion, or ointment); for intravenous administration (eg. g., as a sterile solution free of particle emboli and in a solvent system suitable for intravenous use); or in any other pharmaceutically acceptable formulation.

A pharmaceutically acceptable excipient, as used herein, refers to any ingredient other than the compounds described herein (for example, a carrier capable of suspending or dissolving the active compound) and having the properties of being substantially non-toxic and non-inflammatory in a patient. Excipients may include, for example: release agents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (thinners), film formers or coatings, flavors, fragrances, glidants (stain enhancers). flow), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners and waters of hydration.

As used herein, the term "pharmaceutically acceptable salt" means any pharmaceutically acceptable salt of a compound described herein. Pharmaceutically acceptable salts of any of the compounds described herein may include those that are within the scope of good medical judgment, suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. PH Stahl and CG Wermuth), Wiley -VCH, 2008. Salts can be prepared in situ during final isolation and purification of the compounds described herein or separately by reacting a free base group with a suitable organic acid.

The compounds of the invention may have ionizable groups so that they can be prepared as pharmaceutically acceptable salts. These salts can be, for example, acid addition salts involving inorganic or organic acids or the salts can, in the case of acid forms of the compounds of the invention, be prepared from inorganic or organic bases. Often, the compounds are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases and methods for the preparation of the appropriate salts are well known in the art. Salts can be prepared from non-toxic pharmaceutically acceptable acids and bases, including inorganic and organic acids and bases.

Progression-free survival, as used herein, refers to the period of time during and after medication or treatment during which the disease being treated (eg, cancer) does not worsen.

"Proliferation", as used in this application, implies the reproduction or multiplication of similar forms (cells) due to constituent (cellular) elements.

By reference is meant any useful reference used to compare protein or RNA levels. The reference can be any sample, standard, standard curve, or level that is used for comparative purposes. The reference can be a normal reference sample or a reference standard or level. A reference sample can be, for example, a control, eg a predetermined negative control value such as a normal control or a previous sample taken from the same subject; a sample from a normal healthy subject, such as a normal cell or normal tissue;

a sample (eg, a cell or tissue) from a subject who does not have a disease; a sample from a subject diagnosed with a disease, but not yet treated with a compound of the invention; a sample from a subject that has been treated with a compound of the invention; or a sample of a purified protein or RNA (eg, any of those described herein) at a known normal concentration. By "reference standard or level" is meant a value or number derived from a reference sample. A normal control value is a predetermined value indicative of a disease-free state, eg, a value expected in a healthy control subject. Typically, a normal control value is expressed as a range (between X and Y), a high threshold (no higher than X), or a low threshold (no lower than X). A subject having a measured value within the normal control value for a particular biomarker is generally referred to as within normal limits for that biomarker. A normal reference standard or level can be a value or number derived from a normal subject who does not have a disease or disorder (eg, cancer); a subject who has been treated with a compound of the invention. In preferred embodiments, the reference, standard, or level sample is compared to the sample subject's sample on at least one of the following criteria: age, weight, sex, stage of disease, and general health. A standard curve of levels of a purified protein or RNA, eg, any of those described herein, within the normal reference range, may also be used as a reference.

As used herein, slowing the spread of metastasis refers to reducing or stopping the formation of new loci; or reduce, arrest, or reverse tumor burden.

As used herein, the term "subject" refers to any organism to which a composition according to the invention may be administered, eg, for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include any animal (eg, mammals such as mice, rats, rabbits, non-human primates, and humans). A subject may seek or need treatment, require treatment, receive treatment, receive treatment in the future, or be a human or animal under the care of a trained professional for a particular disease or condition .

As used herein, the terms treat, treated, or treatment mean therapeutic treatment or any measure intended to slow down (lessen) an unwanted physiological condition, disorder, or disease, or to obtain beneficial or desired clinical results. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms; decreasing the extent of a condition, disorder or disease; stabilized (ie, not worsening) state of condition, disorder or disease; delay in onset or slowing of the disease, condition or disease progression; improvement of the condition, disorder or disease state or remission (whether partial or total); an improvement of at least one measurable physical parameter, not necessarily perceptible by the patient; or improvement or favoring of a condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival compared to the expected survival if no treatment is received. The compounds of the invention may also be used to prophylactically treat or prevent a disorder, eg, in a subject at increased risk of developing the disorder.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is normally ascribed to them by a person skilled in the art to which this invention pertains. Methods and materials are described herein for use in the present disclosure; Other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, shall control.

Details of one or more embodiments of the invention are set forth in the following description. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.

Brief description of the Figures

The FIG. 1 is a graph illustrating the inhibition of cell proliferation of various cancer cell lines by a BRG1/BRM inhibitor (Compound A).

The FIG. 2 is a graph illustrating the inhibition of cell proliferation of uveal melanoma cell line 92-1 by a BRG1/BRM inhibitor (Compound A), a MEK inhibitor (Selumetinib) and a PKC inhibitor (LXS196).

The FIG. 3 is a graph illustrating the inhibition of cell proliferation of the uveal melanoma cell line MP41 by a BRG1/BRM inhibitor (Compound A), a MEK inhibitor (Selumetinib) and a PKC inhibitor (LXS196).

The FIG. 4 is a graph illustrating the inhibition of cell proliferation of various cancer cell lines by a BRG1/BRM inhibitor (Compound B).

The FIG. 5 is a graph illustrating the area under the curves (AUC) calculated from dose-response curves for cancer cell lines treated with a BRG1/BRM inhibitor (Compound B).

The FIG. 6 is a graph illustrating the inhibition of cell proliferation of non-small cell lung cancer and uveal melanoma cell lines by a BRG1/BRM inhibitor (Compound B).

The FIG. 7 is a graph illustrating the inhibition of cell proliferation of uveal melanoma cell line 92-1 by a BRG1/BRM inhibitor (Compound B), a MEK inhibitor (Selumetinib) and a PKC inhibitor (LXS196).

The FIG. 8 is a graph illustrating the inhibition of cell proliferation of the uveal melanoma cell line MP41 by a BRG1/BRM inhibitor (Compound B), a MEK inhibitor (Selumetinib) and a PKC inhibitor (LXS196).

The FIG. 9 is a graph illustrating the inhibition of cell proliferation of parental and PKC inhibitor refractory uveal melanoma cell lines by a PKC inhibitor (LXS196).

The FIG. 10 is a graph illustrating the inhibition of cell proliferation of parental and PKC inhibitor refractory uveal melanoma cell lines by a BRG1/BRM inhibitor (Compound B).

The FIG. 11 is a graph illustrating the inhibition of tumor growth in mice engrafted with uveal melanoma cell lines by a BRG1/BRM inhibitor (Compound C).

The FIG. 12 is an illustration of the size of tumors from mice engrafted with uveal melanoma cell lines and dosed with a BRG1/BRM inhibitor (Compound C).

The FIG. 13 is a graph illustrating the change in body weight of mice engrafted with uveal melanoma cell lines and dosed with a BRG1/BRM inhibitor (Compound C).

The FIG. 14 is a graph illustrating the inhibition of cell proliferation of various uveal melanoma cell lines by N-((S)-1-((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl )thiazol-2-yl)amino)3-methoxy-1-oxopropan-2-yl)-1-(methylsulfonyl)-1H-pyrrolo-3-carboxamide.

The FIG. 15 is a graph illustrating the inhibition of tumor growth in mice grafted with uveal melanoma cell lines by N-((S)-1-((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2- yl)thiazol-2-yl)amino)-3-methoxy-1-oxopropan-2-yl)-1-(methylsulfonyl)-1H-pyrrolo-3-carboxamide.

The FIG. 16 is a graph illustrating the change in body weight of mice engrafted with uveal melanoma cell lines and dosed with N-((S)-1-((4-(6-(cis-2,6-dimethylmorpholino)pyridin2 -yl)thiazol-2-yl)amino)-3-methoxy-1-oxopropan-2-yl)-1-(methylsulfonyl)-1H-pyrrolo-3-carboxamide.

Detailed description

The present description presents useful compounds for the inhibition of BRG1 and/or BRM. These compounds can be used to modulate the activity of a BAF complex, for example, for the treatment of a BAF-related disorder, such as cancer. Exemplary compounds, or pharmaceutically acceptable salts thereof, described herein include 10 compounds having the structure:

<img file="CO2022010547A2_D0010.tif" />

<img file="CO2022010547A2_D0011.tif" />

<img file="CO2022010547A2_D0012.tif" />

<img file="CO2022010547A2_D0013.tif" />

<img file="CO2022010547A2_D0014.tif" />

Other embodiments, as well as exemplary methods for the synthesis of the production of these compounds, are described herein.

pharmaceutical uses

The compounds described herein are useful in the methods of the invention and, while not bound by theory, are believed to exert their ability to modulate the level, status, and/or activity of a BAF complex, i.e. , by inhibiting the activity of the BRG1 and/or BRM proteins within the BAF complex in a mammal. Disorders related to the BAF complex include, but are not limited to, disorders related to the BRG1 loss-of-function mutation.

One aspect of the present invention relates to methods of treating disorders related to BRG1 loss-of-function mutations such as cancer (eg, non-small cell lung cancer, colorectal cancer, bladder cancer, primary cancer). unknown, glioma, breast cancer, melanoma, non-melanoma skin cancer, endometrial cancer, or penile cancer) in a subject in need. In some embodiments, the present invention relates to methods of treating melanoma (eg, uveal melanoma), prostate cancer, breast cancer, bone cancer, renal cell carcinoma, or a hematologic cancer.

In some embodiments, the compound is administered in an amount and for a time effective to result in one or more (eg. g., two or more, three or more, four or more) of: (a) reduced tumor size, (b) reduced tumor growth rate, (c) increased tumor cell death (d) reduced progression of the tumor, (e) reduction in the number of metastases, (f) reduction in the metastasis rate, (g) decrease in tumor recurrence (h) increase in subject survival, (i) increase in disease-free survival subject progression.

Cancer treatment may result in a reduction in the size or volume of a tumor. For example, after treatment, the size of the tumor is reduced by 5% or more (eg, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) relative to their size before treatment. The size of a tumor can be measured by any reproducible measurement means. For example, the size of a tumor can be measured as the diameter of the tumor.

Cancer treatment may also result in a decrease in the number of tumors. For example, after treatment, the number of tumors is reduced by 5% or more (eg, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) relative to the number before treatment. The number of tumors can be measured by any reproducible means of measurement, for example, the number of tumors can be measured by counting tumors visible to the naked eye or at a specific magnification (for example, 2x, 3x, 4x, 5x, 10x or 50x).

Cancer treatment may result in a decrease in the number of metastatic nodules in other tissues or organs distant from the primary tumor site. For example, after treatment, the number of metastatic nodules is reduced by 5% or more (eg, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%). % or more) relative to the number before treatment. The number of metastatic nodules can be measured by any reproducible measurement means. For example, the number of metastatic nodules can be measured by counting metastatic nodules visible to the naked eye or under specific magnification (eg, 2x, 10x, or 50x).

Cancer treatment may result in an increased median survival time of a population of subjects treated in accordance with the present invention as compared to a population of untreated subjects. For example, the median survival time increases by more than 30 days (more than 60 days, 90 days, or 120 days). An increase in the median survival time of a population can be measured by any reproducible means. An increase in the median survival time of a population can be measured, for example, by calculating for a population the median survival time after initiation of treatment with the compound of the invention. An increase in the median survival time of a population can also be measured, for example, by calculating for a population the median survival time after completion of a first round of treatment with a pharmaceutically acceptable salt of the invention.

Cancer treatment may also result in a decreased mortality rate of a population of treated subjects compared to an untreated population. For example, the mortality rate is reduced by more than 2% (eg, more than 5%, 10%, or 25%). A decrease in the mortality rate of a population of treated subjects can be measured by any reproducible means, for example, by calculating for a population the mean number of disease-related deaths per unit time after initiation of treatment with a pharmaceutically acceptable salt. of the invention. A decrease in the mortality rate can also be measured by any means, for example, by calculating for a population the mean number of disease-related deaths per unit of time after completion of a first round of treatment with a pharmaceutically acceptable salt of the invention.

Examples of cancers that can be treated by the invention include, but are not limited to, non-small cell lung cancer, small cell lung cancer, colorectal cancer, bladder cancer, glioma, breast cancer, melanoma, lung cancer, non-melanoma skin, endometrial cancer, esophagogastric cancer, esophageal cancer, pancreatic cancer, hepatobiliary cancer, soft tissue sarcoma, ovarian cancer, head and neck cancer, renal cell carcinoma, bone cancer, non-Hodgkin lymphoma, prostate cancer, embryonal tumor, germ cell tumor, cervical cancer, thyroid cancer, salivary gland cancer, gastrointestinal neuroendocrine tumor, uterine sarcoma, gastrointestinal stromal tumor, CNS cancer, thymic tumor, carcinoma adrenocortical cancer, appendiceal cancer, small bowel cancer, hematologic cancer, and penile cancer.

Combined formulations and their uses

The compounds of the invention may be combined with one or more therapeutic agents. In particular, the therapeutic agent may be one that treats or prophylactically treats any cancer described herein.

Combination therapies

A compound of the invention may be used alone or in combination with an additional therapeutic agent, for example, other agents that treat cancer or the symptoms associated therewith, or in combination with other types of treatment to treat cancer. In combination treatments, the doses of one or more of the therapeutic compounds may be reduced from the standard doses when administered alone. For example, doses can be determined empirically from drug combinations and permutations or can be inferred by isobolographic analysis (eg, Black et al., Neurology 65:S3-S6, 2005). In this case, the doses of the compounds when combined should provide a therapeutic effect.

In some embodiments, the second therapeutic agent is a chemotherapeutic agent (eg, a cytotoxic agent or other chemical useful in the treatment of cancer). These include alkylating agents, antimetabolites, folic acid analogues, pyrimidine analogues, purine analogues and related inhibitors, vinca alkaloids, epipodopilotoxins, antibiotics, L-asparaginase, topoisomerase inhibitors, interferons, platinum coordination complexes, substituted urea. with anthracenedione, methylhydrazine derivatives, adrenocortical suppressant, adrenocorticosteroids, progestogens, estrogens, antiestrogens, androgens, antiandrogens and gonadotropin-releasing hormone analogues. Also included is 5-fluorouracil (5-FU), leucovorin (LV), irenotecan, oxaliplatin, capecitabine, paclitaxel, and doxetaxel. Non-limiting examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analog topotecan); bryostatin; calistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin, and bizelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictine; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, colophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembiquine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics such as the enediyne antibiotics (eg, calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, p. eg, Agnew, Chem. Intl. Ed. Engl. 33:183-186 (1994)); dynemycin, including dynemycin A; bisphosphonates, such as clodronate; a esperamycin; as well as neocarzinostatin chromophore and related antibiotic enediyne chromoprotein chromophores), aclacinomycins, actinomycin, autramycin, azaserin, bleomycins, cactinomycin, carabicin, cadomycin, carzinophyllin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L- norleucine, Adriamycin® (doxorubicin, including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxidoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, chelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisanthrene; edatraxate; defofamine; demecolcine; Diaziquone; elfomitin; elliptinium acetate; an epothilone; ethoglycide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; fenamet; pyrarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triazicuone; 2,2',2'' trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidin); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids, eg, Taxol® paclitaxel (Bristol-Myers

Squibb Oncology, Princeton, NJ), ABraxane®, albumin-modified nanoparticle formulation without paclitaxel cremophor (American Pharmaceutical Partners, Schaumberg, Ill.), and Taxotere® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine Gemzar®; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; Navelbine® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (eg, CPT-11); RFS 2000 topoisomerase inhibitor; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Two or more chemotherapeutic agents can be used in a cocktail to be administered in combination with the first therapeutic agent described herein. Suitable dosage regimens of combination chemotherapies are known in the art and are described, for example, in Saltz et al. (1999) Proc ASCO 18:233a and Douillard et al. (2000) Lancet 355:1041-7.

In some embodiments, the second therapeutic agent is a therapeutic agent that is a biological agent such as a cytokine (eg, interferon or an interleukin (eg, IL-2)) used in the treatment of cancer. In some embodiments, the biologic is an antiangiogenic agent, such as an anti-VEGF agent, eg, bevacizumab (Avastin®). In some embodiments, the biologic is an immunoglobulin-based biologic, eg, a monoclonal antibody (eg, a humanized antibody, fully human antibody, Fc fusion protein, or functional fragment thereof) that agonizes a target to stimulate an anticancer response, or antagonizes an antigen important to cancer. Such agents include Rituxan (Rituximab); Zenapax (Daclizumab); Simulect (Basiliximab); Synagis (Palivizumab); Remicade (Infliximab); Herceptin (Trastuzumab); Mylotarg (Gemtuzumab ozogamicin); Campath (Alemtuzumab); Zevalin (Ibritumomab tiuxetan); Humira (Adalimumab); Xolair (Omalizumab); Bexxar (Tositumomab-I131); Raptiva (Efalizumab); Erbitux (Cetuximab); Avastin (Bevacizumab); Tysabri (Natalizumab); Actemra (Tocilizumab); Vectibix (Panitumumab); Lucentis (Ranibizumab); Soliris (Eculizumab); Cimzia (Certolizumab pegol); Simponi (Golimumab); Ilaris (Canakinumab); Stelara (Ustekinumab); Arzerra (Ofatumumab); Prolia (Denosumab); Numax (Motavizumab); ABThrax (Raxibacumab); Benlysta (Belimumab); Yervoy (Ipilimumab); Adcetris (Brentuximab Vedotin); Perjeta (Pertuzumab); Kadcyla (Ado-trastuzumab emtansine); and Gazyva (Obinutuzumab). Antibody-drug conjugates are also included.

The second agent may be a therapeutic agent that is a non-drug treatment. For example, the second therapeutic agent is radiation therapy, cryotherapy, hyperthermia, and/or surgical excision of tumor tissue.

The second agent may be a checkpoint inhibitor. In one embodiment, the checkpoint inhibitor is an inhibitory antibody (eg, a monospecific antibody such as a monoclonal antibody). The antibody can be, for example, humanized or fully human. In some embodiments, the checkpoint inhibitor is a fusion protein, eg, an Fc receptor fusion protein. In some embodiments, the checkpoint inhibitor is an agent, such as an antibody, that interacts with a checkpoint protein. In some embodiments, the checkpoint inhibitor is an agent, such as an antibody, that interacts with the ligand of a checkpoint protein. In some embodiments, the checkpoint inhibitor is an inhibitor (eg, an inhibitory antibody or a small molecule inhibitor) of CTLA-4 (eg. g., an anti-CTLA4 antibody such as ipilimumab/Yervoy or tremelimumab). In some modalities, the checkpoint inhibitor is an inhibitor (eg, an inhibitory antibody or small molecule inhibitor) of PD-1 (eg, nivolumab/Opdivo®; pembrolizumab/Keytruda®; pidilizumab /CT-011). In some embodiments, the checkpoint inhibitor is an inhibitor (eg, an inhibitory antibody or a small molecule inhibitor) of PDL1 (eg. eg MPDL3280A/RG7446; MEDI4736; MSB0010718C; BMS 936559). In some embodiments, the checkpoint inhibitor is an inhibitor (eg, an inhibitory antibody or Fc fusion or small molecule inhibitor) of PDL2 (eg, an Ig/PDL2 fusion protein such as AMP 224 ). In some embodiments, the checkpoint inhibitor is an inhibitor (eg, an inhibitory antibody or a small molecule inhibitor) of B7-H3 (eg. MGA271), B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, B-7 family ligands, or a combination of the same.

In some embodiments, the compound of the invention is used in combination with another antineoplastic therapy used for the treatment of uveal melanoma such as surgery, a MEK inhibitor and/or a PKC inhibitor, or a combination thereof. For example, in some embodiments, the method further comprises performing surgery before, after, or at the same time as administration of the compound of the invention. In some embodiments, the method further comprises administration of a MEK inhibitor (eg, selumetinib, binimetinib, or tametinib) and/or a PKC inhibitor (eg, sotrastaurin or IDE196) before, after, or at the same time. time than the administration of the compound of the invention.

In any of the combination modalities described herein, the first and second therapeutic agents are administered simultaneously or sequentially, in any order. The first therapeutic agent may be administered immediately, up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to 8 hours, up to 9 hours, up to 10 hours, up to 11 hours, up to 12 hours, up to 13 hours, 14 hours, up to 16 hours, up to 17 hours, up to 18 hours, up to 19 hours, up to 20 hours, up to 21 hours, up to 22 hours, up to 23 hours, up to 24 hours or until 1-7, 1-14, 1-21 or 1-30 days before or after the second therapeutic agent.

pharmaceutical compositions

The compounds of the invention are preferably formulated into pharmaceutical compositions for administration to a mammal, preferably a human, in a biologically compatible form suitable for administration in vivo. Accordingly, in one aspect, the present invention provides a pharmaceutical composition comprising a compound of the invention in admixture with a suitable diluent, carrier or excipient.

The compounds of the invention can be used in the form of the free base, in the form of salts, solvates and as prodrugs. All forms are within the scope of the invention. In accordance with the methods of the invention, the disclosed compounds or salts, solvates or prodrugs thereof can be administered to a patient in a variety of forms depending on the route of administration selected, as will be understood by those skilled in the art. The compounds of the invention can be administered, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, patch, pump, or transdermal administration and the pharmaceutical compositions formulated accordingly. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal and topical administration forms. Parenteral administration may be by continuous infusion over a selected period of time.

A compound of the invention can be administered orally, for example, with an inert diluent or with an edible assimilable vehicle, or it can be enclosed in hard or soft-shell gelatin capsules, or it can be compressed into tablets, or it can be incorporate directly with the food of the diet. For oral therapeutic administration, a compound of the invention may be incorporated with an excipient and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, and wafers.

A compound of the invention can also be administered parenterally. Solutions of a compound of the invention can be prepared in water suitably mixed with a surfactant, such as hydroxypropyl cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2003, 20^to ed.) and in The United States Pharmacopeia: The National

Formulary (USP 24 NF19), published 1999. Dosage forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that it can be easily administered by syringe.

A compound described herein can be administered intratumorally, for example, as an intratumoral injection. Intratumoral injection is an injection directly into the tumor vasculature and is specifically contemplated for discrete, solid, and accessible tumors. Local, regional or systemic administration may also be appropriate. A compound described herein can be advantageously contacted by administering a single injection or multiple injections to the tumor, spaced, for example, at intervals of about

1 cm. In the case of surgical intervention, the present invention can be used preoperatively, such as to render an inoperable tumor subject to resection. Continuous administration can also be applied where appropriate, for example, by implanting a catheter into a tumor or into the tumor vasculature.

The compounds of the invention may be administered to an animal, eg, a human, alone or in combination with pharmaceutically acceptable carriers, as indicated herein, the ratio of which is determined by the solubility and chemical nature of the compound. , the route of administration chosen and the usual pharmaceutical practice.

Dose

The dosage of compounds of the invention and/or compositions comprising a compound of the invention may vary depending on many factors, such as the pharmacodynamic properties of the compound; the mode of administration; the age, health and weight of the beneficiary; the nature and extent of the symptoms; the frequency of treatment and the type of concurrent treatment, if any; and the rate of elimination of the compound in the animal to be treated. One skilled in the art can determine the appropriate dosage based on the above factors. The compounds of the invention may initially be administered in a suitable dosage which may be adjusted as required, depending on the clinical response. In general, satisfactory results can be obtained when the compounds of the invention are administered to a human in a daily dose of, for example, between 0.05 mg and 3000 mg (measured as solid form).

Alternatively, the dosage amount can be calculated using the patient's body weight. For example, the dose of a compound, or pharmaceutical composition thereof, administered to a patient can range from 0.1 to 50 mg/kg.

examples

The following abbreviations are used throughout the examples section.

<td>mouth</td><td>tert-butoxycarbonyl</td>

<td>DCM</td><td>dichloromethane</td>

<td>DIPEA or DIEA</td><td>NN-diisopropylethylamine</td>

<td>DMF</td><td>NN-dimethylformamide</td>

<td>DMSO</td><td>dimethyl sulfoxide</td>

<td>EDCI</td><td>N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride</td>

<td>EEDQ</td><td>2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline</td>

<td>EtOH</td><td>ethyl alcohol</td>

<td>ho hr</td><td>hour</td>

<td>HOBt or HOBT</td><td>1-hydroxybenzotriazole hydrate</td>

<td>MeOH</td><td>methyl alcohol</td>

<td>mscl</td><td>methanesulfonyl chloride</td>

<td>NaHMDS</td><td>sodium bis(trimethylsilyl)amide</td>

PdCl2(dtbpf) dichloro[1,1'-bis(di-t-butylphosphino)ferrocene]palladium(II)

THF tetra hid break or

TMSCHN2 (diazomethyl)trimethylsilane

Example 1. Preparation of N-((S)-1-((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)3-methoxy-1- oxopropan-2-yl)-1 -(methylsulfonyl)-1H-pyrrole-3-carboxamide

N-((S)-1-((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)-3-methoxy-1-oxopropan-25-yl )-1-(methylsulfonyl)-1H-pyrrolo-3-carboxamide was synthesized as shown in Scheme 1 below.

Scheme 1.

<img file="CO2022010547A2_D0015.tif" />

Step 1: Preparation of 6-fluoropyridine-2-carbonyl chloride (Intermediate B)

F

<img file="CO2022010547A2_D0016.tif" />

B.

To a cooled (0 °C) solution of 6-fluoropihd¡n-2-carboxylic acid (50.0 g, 354 mmol) in dichloromethane (500 mL) and Ν,Ν-dimethylformamide (0.26 mL, 3 0.54 mmol) oxalyl chloride (155 mL, 1.77 mol) was added. After the oxalyl chloride addition was complete, the reaction mixture was warmed to room temperature. After 0.5 hour, the mixture was concentrated in vacuo to give Intermediate

B (56.50 g) as a white solid, which was used in the next step without further purification.

Step 2: Preparation of 2-chloro-1-(6-fluoro-2-pyridyl)ethenone (Intermediate C)

<img file="CO2022010547A2_D0017.tif" />

To a cooled (0 °C) mixture of Intermediate B (56.0 g, 351 mmol) in 1,4-dioxane (800 mL) was added dropwise a solution of 2 M trimethylsilyldiazomethane in hexanes (351 mL, 702 mmol). The resulting reaction mixture was stirred at 25 °C for 10 h. The reaction mixture was subsequently quenched with a solution of 4M HCl in 1,4-dioxane (500 mL, 2.0 mol). After stirring for 2h, the reaction solution was concentrated in vacuo to give an oil. The residue was diluted with saturated aqueous NaHCO3 and extracted three times with ethyl acetate. The combined organic layers were washed twice with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give Intermediate C (35.5 g) as a white solid, which was used directly in the next step.

LCMS (ESI) m/z: [M+H]+ = 173.8.

Step 3: Preparation of 4-(6-fluoro-2-pyridyl)thiazol-2-amine (Intermediate E)

<img file="CO2022010547A2_D0018.tif" />

To a solution of Intermediate C (35.5 g, 205 mmol) and thiourea (14.0 g, 184 mmol) in a mixture of methanol (250 mL) and water (250 mL) at room temperature was added NaF (3 0.56 g, 84.8 mmol). After stirring for 0.5 h, the reaction mixture was partially concentrated in vacuo to remove MeOH and the resulting solution was acidified to pH ~3 with 2M aqueous HCl. After 15 minutes, the solution was extracted three times with ethyl acetate. The organic layers were discarded and the aqueous phase was made alkaline with saturated aqueous NaHCO3 and stirred for 30 minutes and extracted three times with ethyl acetate. The combined organic layers were washed three times with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was triturated with petroleum ether and stirred at 25°C for 10 minutes and filtered. The resulting solids were dried in vacuo to give Intermediate E (28.0 g, 143 mmol, 70.1% yield, 100% purity) as a white solid.

LCMS (ESI) m/z: [M+H]+ = 195.8.

¹H NMR (400 MHz, DMSO-de) δ 8.00 - 7.96 (m, 1H), 7.72 (d, J = 7.2 Hz, 1H), 7.24 (s, 1H), 7 .16 (s, 2H), 7.02 (d, J = 8.0 Hz, 1H).

Step 4: Preparation of 4-[6-[cis-2,6-dimethylmorpholin-4-yl]-2-pyridyl]thiazol-2-amine (Intermediate G)

<img file="CO2022010547A2_D0019.tif" />

Ten separate mixtures of Intermediate E (2.00 g, 10.3 mmol), cis-2,6-dimethylmorpholine (3.54 g, 30.7 mmol), and DIPEA (5.35 mL, 30.7 mmol) in dimethyl sulfoxide (10 mL) were stirred in parallel at 120 °C under N2 atmosphere. After 36h, the reaction mixtures were combined and added dropwise to the water. The resulting slurry was filtered and the filter cake washed three times with water and once with petroleum ether, then dried under reduced pressure to give Intermediate G (25.5 g, 87.8 mmol, 95.2% yield) as a yellow solid.

LCMS (ESI) m/z: [M+H]⁺= 291,2.

¹H NMR (400 MHz, DMSO-d6) δ 7.56 - 7.54 (m, 1H), 7.17 (s, 1H), 7.13 (d, J = 7.6 Hz, 1H), 7 .01 (s, 2H), 6.72 (d, J = 8.8 Hz, 1H), 4.26 - 4.15 (m, 2H), 3.67 - 3.55 (m, 2H), 2.38-2.34 (m, 2H), 1.17 (d, J=6.4Hz, 6H).

Step 5: Preparation of tert-Butyl N-[(1S)-2-[[4-[6-[cis-2,6-dimethylmorpholin-4-yl]-2-pyridyl]thiazol-2yl]amino]-1 -(methoxymethyl)-2-oxo-ethyl]carbamate (Intermediate I)

<img file="CO2022010547A2_D0020.tif" />

To a solution of Intermediate G (12.0 g, 41.3 mmol) and (2S)-2-(tert-butoxycarbonylamino)3-methoxypropanoic acid (10.9 g, 49.6 mmol) in dichloromethane (60 mL) was added EEDQ (12.3g, 49.6mmol). After stirring at room temperature for 16 h, the reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 2:1 to 3:2) to give Intermediate I (20.0 g, 40.7 mmol, 98.5% of performance) like a yellow rubber.

LCMS (ESI) m/z: [M+H]+ = 492.2.

¹H NMR (400 MHz, DMSO-de) δ 12.37 (s, 1H), 7.78 (s, 1H), 7.64-7.60 (m, 1H), 7.25 (d, J = 7.2 Hz, 1H), 7.16 (d, J = 7.2 Hz, 1H), 6.79 (d, J = 8.4 Hz, 1H), 4.50 - 4.48 (m, 1H), 4.25 (d, J = 11.6 Hz, 2H), 3.70 - 3.51 (m, 4H), 3.26 (s, 3H), 2.44 - 2.40 (m , 2H), 1.39 (s, 9H), 1.18 (d, J = 6.4 Hz, 6H).

Step 6: Preparation of (S)-4-(4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)-1-methoxy-3-oxobutan-2-aminium chloride (Intermediate J)

<img file="CO2022010547A2_D0021.tif" />

To a solution of 4 M HCl in 1,4-dioxane (200 mL, 800 mmol) was added a solution of Intermediate I (20.0 g, 40.7 mmol) in dichloromethane (50 mL). After stirring at room temperature for 2 h, the mixture was diluted with methyl tert-butyl ether resulting in a suspension. The solid was collected by filtration, washed twice with methyl tert-butyl ether and dried in vacuo to give Intermediate J (19.0 g) as a yellow solid, which was used in the next step without further purification.

LCMS (ESI) m/z: [M+H]⁺= 392,3.

¹H NMR (400 MHz, DMSO-de) δ 13.44 - 12.30 (m, 1H), 8.65 (d, J = 4.4 Hz, 3H), 7.87 (s, 1H), 7 .66 - 7.64 (m, 1Η), 7.25 (d, J = 7.2 Hz, 1H), 6.83 (d, J = 8.8 Hz, 1H), 4.39 - 4 .30 (m, 1H), 4.25 (d, J = 11.6 Hz, 2H), 3.94 - 3.86 (m, 1H), 3.85 - 3.77 (m, 1H), 3.69-3.57 (m, 2H), 3.31 (s, 3H), 2.43 (m, 2H), 1.18 (d, J=6.4 Hz, 6H).

Preparation of 1-(methylsulfonyl)-1H-pyrrole-3-carboxylic acid (Intermediate K)

1-(Methylsulfonyl)-1H-pyrrole-3-carboxylic acid was synthesized as shown in Scheme 2 below.

Scheme 2

<img file="CO2022010547A2_D0022.tif" />

Step A: Preparation of tert-butyl 1H-pyrrole-3-carboxylate (Intermediate N)

<img file="CO2022010547A2_D0023.tif" />

No.

To a mixture of tert-butyl-prop-2-enoate (78.6 mL, 542 mmol) and 1-(¡ocyanomethylsulfon¡l)-4-methylbenzene (106 g, 542 mmol) in THF (1300 mL) was added NaH 60% in mineral oil (25.97 g, 649 mmol) slowly at 30 °C for 1 hour, then heated to 70 °C. After 2 h, the reaction mixture was poured into saturated aqueous NH4Cl solution and extracted three times with ethyl acetate. The combined organic phase was washed twice with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 20:1 to 3:1) to give Intermediate N (41.5 g, 236 mmol, 43% yield) as a yellow solid.

LCMS (ESI) m/z [M+Na]⁺ = 180,4.

Ή NMR (400 MHz, CDCI3) δ 8.36 (br s, 1H), 7.35 - 7.25 (m, 1H), 6.71 - 6.62 (m, 1H), 6.59 - 6 .49 (m, 1H), 1.48 (s, 9H).

Step B: Preparation of tert-butyl 1-methylsulfonylpyrrole-3-carboxylate (Intermediate O)

<img file="CO2022010547A2_D0024.tif" />

either

To a cooled (0 °C) solution of Intermediate N (40.5 g, 242 mmol) in THF (1500 mL) was added 1 M NaHMDS solution (484 mL, 484 mmol). After stirring at 0 °C for 30 min, methanesulfonyl chloride (28.1 mL, 363 mmol) was added slowly and the mixture was warmed to 30 °C. After 16 h, the reaction mixture was slowly poured into saturated aqueous NH4Cl solution and extracted three times with ethyl acetate. The combined organic layers were washed twice with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography (petroleum ether:ethyl acetate = 10:1) to give a yellow solid. The yellow solid was triturated with methyl tert-butyl ether at room temperature, stirred for 20 minutes, filtered, and dried in vacuo to give Intermediate O (25.7 g, 105 mmol, 43% yield) as a white solid.

¹H NMR (400 MHz, CDCb) δ 7.66-7.64 (m, 1H), 7.10-7.08 (m, 1H), 6.73-6.71 (m, 1H), 3, 21 (s, 3H), 1.56 (s, 9H).

Step C: Preparation of 1-methylsulfonylpyrrole-3-carboxylic acid (Intermediate K)

<img file="CO2022010547A2_D0025.tif" />

To a mixture of Intermediate O (25.7 g, 105 mmol) in 1,4-dioxane (100 mL) was added a 4 M solution of HCl in 1,4-dioxane (400 mL, 1.6 mol) at 15°C After stirring at 15 °C for 14 h, the reaction mixture was concentrated under reduced pressure to give a residue. The residue was triturated with methyl tert-butyl ether at 15^eitherC for 16h. The mixture was filtered and dried in vacuo to provide Intermediate K (18.7 g, 98.8 mmol, 94% yield) as a white solid.

LCMS (ESI) m/z [M+H]+ = 189.8.

¹H NMR (400 MHz, methanol-d4) δ 7.78 - 7.77 (m, 1H), 7.25 - 7.23 (m, 1H), 6.72 - 6.70 (m, 1H), 3.37 (s, 3H).

Step 7: Preparation of N-((S)-1-((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)-3methoxy-1-oxopropan -2-yl)-1 -(methylsulfonyl)-1H-pyrrole-3-carboxamide

<img file="CO2022010547A2_D0026.tif" />

To a solution of 1-methylsulfonylpyrrole-3-carboxylic acid (Intermediate K) (2.43 g, 12.9 mmol), EDCI (2.69 g, 14.0 mmol), HOBt (1.89 g, 14, 0 mmol) and DIPEA (10.2 mL, 58.4 mmol) in dichloromethane (50 mL) was added Intermediate J (5.00 g, 11.7 mmol). After stirring at room temperature for 4 h, the reaction mixture was concentrated under reduced pressure. The residue was diluted with water and extracted three times with ethyl acetate. The combined organic layers were washed three times with saturated aqueous NH4Cl, once with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1 to 1:2). The residue was triturated with methyl tert-butyl ether. After 0.5 h, the suspension was filtered, the filter cake washed with methyl tert-butyl ether and dried in vacuo. The solid was dissolved in dimethyl sulfoxide (12 mL) and added dropwise to water (800 mL). The suspension was filtered to give a moist filter cake. The filter cake was slurried in water and stirred at room temperature. After 1 hour, the solid was collected by filtration, washed three times with water, and dried in vacuo to give N-((S)-1-((4-(6-(cis-2,6-dimethylmorpholino) pyridine -2-yl)thiazol-2yl)amino)-3-methoxy-1-oxopropan-2-yl)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamide (3.9 g, 6.93 mmol, 59.3% yield) as a white solid.

LCMS (ESI) m/z: [M+H]+=563.1.

¹H NMR (400 MHz, DMSO-de) δ 12.49 (br s, 1H), 8.51 (d, J = 7.2 Hz, 1H), 7.98-7.97 (m, 1H), 7.78 (s, 1H), 7.67 - 7.57 (m, 1H), 7.29 - 7.27 (m, 1H), 7.26 (d, J = 7.2 Hz, 1H) , 6.88 - 6.74 (m, 2H), 4.94 - 4.91 (m, 1H), 4.25 (d, J = 11.6 Hz, 2H), 3.77 - 3.67 (m, 2H), 3.63 - 3.62 (m, 2H), 3.57 (s, 3H), 3.31 (s, 3H), 2.44 - 2.38 (m, 2H), 1.18 (d, J=6.0Hz, 6H).

Example 2. Preparation of N-((S)-1-((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)3-(methoxy-d3 )-1-oxopropan-2-yl-3,3-d2)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamide

<img file="CO2022010547A2_D0027.tif" />

N-((S)-1-((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)-3-(methoxy-d3)-1-oxopropan- 2-yl-3,3-d2)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamide was prepared according to the synthetic protocol described in Example 1 with Intermediate H replaced with N-(tert-Butoxycarbonyl) -O-(methyld3)-L-serine-3,3-d2. N-(tert-butoxycarbonyl)-O-(methyl-d3)-L-serine-3,3-d2 was prepared from isotopically enriched material according to the synthetic procedures described in A. Yang et al, Org. Process Res. Dev. 2019, 23, 818-824.

LCMS (ESI) m/z: [M+H]⁺= 568,2.

¹H NMR (400 MHz, DMSO-de) δ 12.45 (s, 1H), 8.47 (d, J = 7.2 Hz, 1H), 7.98 (dd, J = 2.3, 1, 7 Hz, 1H), 7.78 (s, 1H), 7.62 (dd, J = 8.5, 7.4 Hz, 1H), 7.29 (dd, J = 3.2, 2.3 Hz, 1H), 7.26 (d, J = 7.3 Hz, 1H), 6.84 - 6.75 (m, 2H), 4.91 (d, J = 7.2 Hz, 1H), 4.25 (dd, J = 13.1,2.3 Hz, 2H), 3.69 - 3.59 (m, 2H), 3.56 (s, 3H), 2.42 (dd, J = 12.8, 10.5Hz, 2H), 1.18 (d, J = 6.2Hz, 6H).

Example 3. Preparation of N-((R)-1-((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)3-(methoxy)- 1 -oxopropan-2-yl)-1 -(methylsulfonyl)-1H-pyrrolo-3-carboxamide

N-((R)-1-((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)-3-(methoxy)-1-oxopropan- 2yl)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamide was prepared according to the synthetic protocol described in Example 1 with Intermediate H replaced with (2R)-2-(tert-butoxycarbonylamino)-3-methoxypropanoic acid.

LCMS (ESI) m/z: [M+H]⁺= 563,1.

¹H NMR (400 MHz, DMSO-de) δ 12.5 (s, 1H), 8.50 (d, J = 7.2 Hz, 1H), 7.98 (t, J = 1.6 Hz, 1H ), 7.78 (s, 1H), 7.62 (dd, J = 7.2, 8.4 Hz, 1H), 7.29 (dd, J = 2.0, 3.2 Hz, 1H) , 7.26 (d, J = 7.2 Hz, 1H), 6.79 - 6.81 (m, 2H), 4.92 (q, J = 6.4, 12.8 Hz, 1H), 4.25 (d, J = 11.2 Hz, 2H), 3.69 - 3.75 (m, 2H), 3.59 - 3.66 (m, 2H), 3.56 (s, 3H) , 3.31 (s, 3H), 2.41 (dd, J = 10.8, 12.8 Hz, 2H), 1.18 (d, J = 6.0 Hz, 6H).

Example 4. Preparation of N-((R)-1-((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)41

3-(methoxy-d3)-1-oxopropan-2-yl-3,3-d2)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamide

N-((R)-1-((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)-3-(methoxy-d3)-1-oxopropan- 2-yl-3,3-d2)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamide was prepared according to the synthetic protocol described in Example 1 with Intermediate H replaced with N-(tert-Butoxycarbonyl) -O-(methyld3)-D-serine-3,3-d2. N-(tert-butoxycarbonyl)-O-(methyl-d3)-D-serine-3,3-d2 was prepared from isotopically enriched material according to the synthetic procedures described in A. Yang et al, Org. Process Res. Dev. 2019_yes 23, 818-824.

LCMS (ESI) m/z: [M+H]⁺= 568,3.

¹H NMR (400 MHz, DMSO-d6) δ 12.46 (s, 1H), 8.52 - 8.38 (m, 1H), 7.97 (t, J = 1.9 Hz, 1H), 7 .76 (s, 1H), 7.62 (dd, J = 8.5, 7.3 Hz, 1H), 7.29 (dd, J = 3.3, 2.3 Hz, 1H), 7, 26 (d, J = 7.4 Hz, 1H), 6.79 (dt, J = 5.1, 1.8 Hz, 2H), 4.89 (d, J = 5.2 Hz, 1H), 4.31 - 4.20 (m, 2H), 3.63 (ddd, J = 10.5, 6.2, 2.5 Hz, 2H), 3.56 (s, 3H), 2.41 ( dd, J = 12.8, 10.5 Hz, 2H), 1.18 (d, J = 6.2 Hz, 6H).

Example 5. Assay of ATPase catalytic activity of BRM and BRG-1

The ATPase catalytic activity of BRM or BRG-1 was measured by an in vitro biochemical assay using ADP-Glo™ (Promega, V9102). The ADP-Glo™ kinase assay was performed in two steps after the reaction was complete. The first step is to use up any ATP not consumed in the reaction. The second step was to convert the ADP reaction product to ATP, which will be used by luciferase to generate luminescence and will be detected by a luminescence reader, such as Envision.

The assay reaction mix (10 µL) contained 30 nM BRM or BRG-1.20 nM salmon sperm DNA (from Invitrogen, UltraPure™ Salmon Sperm DNA Solution, cat. no. 15632011) and 400 µM ATP in ATPase assay buffer, comprising 20 mM Tris, pH 8, 220 mM MgCl, 50 mM NaCl, 0.1% Tween-20, and 1 mM fresh DTT (Pierce™ DTT (dithiothreitol), catalog #20290). The reaction was started by adding 2.5 μL of ATPase solution to 2.5 μL of ATP/DNA solution in a white low-volume Proxiplate-384 plus plate (PerkinElmer, cat. no. 6008280) and incubated at room temperature for 1 hour. Then, after the addition of 5 μL of the ADP-Glo™ reagent provided in the kit, the reaction was incubated at room temperature for 40 minutes. Next, 10 µL of the kinase detection reagent provided in the kit was added to convert ADP to ATP and the reaction was incubated at room temperature for 60 minutes. Finally, the luminescence measurement is collected with a plate-reading luminometer, such as

Envision.

BRM and BRG-1 were synthesized from five insect cell lines with a purity greater than 90%.

It was found that N-((S)-1 -((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)-3-methoxy5 1-oxopropan- 2-yl)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamide had an IP50 of 3.9 nM against BRM and 5.2 nM against BRG1 in the assay. It was found that N-((R)-1-((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2yl)thiazol-2-yl)amino)-3-(methoxy-d3)-1 -oxopropan-2-yl-3,3-d2)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamide had an IP50 of 443 nM against BRM and 777 nM against BRG1 in the assay. It was found that N-((S)-1((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)-3-(methoxy-d3)- 1-oxopropan-2-yl-3,3-d2)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamide had an IP50 of 4.6 nM against BRM and 7.4 nM against BRG1 in the assay.

Example 6. Synthesis of Compound A

BRG1/BRM inhibitor compound A has the structure:

TO

Compound A was synthesized as shown in Scheme 3 below.

Scheme 3. Synthesis of Compound A

<img file="CO2022010547A2_D0028.tif" />

<img file="CO2022010547A2_D0029.tif" />

<img file="CO2022010547A2_D0030.tif" />

Compound A

The ATPase catalytic activity of BRM or BRG-1 in the presence of Compound A was measured by the in vitro biochemical assay using ADP-Glo™ (Promega, V9102) described above. Compound was found to have an IPso of 10.4 nM against BRM and 19.3 nM against BRG1 in the assay.

Example 7. Effects of BRG1/BRM ATPase inhibition on the growth of uveal melanoma and hematological cancer cell lines

Procedure: Uveal melanoma cell lines (92-1, MP41, MP38, MP46), prostate cancer cell lines (LNCAP), lung cancer cell lines (NCI-H1299), and immortalized embryonic kidney lines (HEK293T) were seeded. ) in 96-well plates with culture medium (see Table 1). The BRG1/BRM ATPase inhibitor, Compound A, was dissolved in DMSO and added to the cells in a concentration gradient from 0 to 10 µM at the time of seeding. The cells were incubated at 37 °C for 3 days. After three days of treatment, the medium was removed from the cells and 30 microliters of TrypLE (Gibco) was added to the cells over 10 minutes. The cells were detached from the plates and resuspended with the addition of 170 microliters of culture medium. Cells from two DMSO-treated control wells were counted and the initial number of cells seeded at the beginning of the experiment were replated onto plates containing fresh compound for an additional four days at 37°C. On day 7, cells were harvested as described above. On day 3 and day 7, relative cell growth was measured by addition of Cell-titer glo (Promega), and luminescence was measured on an Envision plate reader (Perkin Elmer). The concentration of compound at which the growth of each cell line was inhibited by 50% (Gbo), was calculated using Graphpad Prism and is plotted below. For multiple myeloma cell lines (OPM2, MM1S, LP1), ALL cell lines (TALL1, JURKAT, RS411), DLBCL cell lines (SUDHL6, SUDHL4, DB, WSUDLCL2, PFEIFFER), AML cell lines (OCIAML5), cell lines MDS (SKM1), ovarian cancer cell lines (OV7, TYKNU), esophageal cancer cell lines (KYSE150), rhabdoid tumor lines (RD, G402, G401, HS729, A204), liver cancer cell lines ( HLF, HLE, PLCRPF5), and lung cancer cell lines (SW1573, NCIH2444), the above methods were performed with the following modifications: Cells were seeded in 96-well plates, and the following day, the BRG1/BRM ATPase inhibitor, Compound A, was dissolved in DMSO and added to the cells in a concentration gradient from 0 to 10 μΜ. At the time of cell division on days 3 and 7, cells were split into new 96-well plates and fresh compound was added four hours after replating. Table 1 lists the cell lines tested and the growth media used.

Table 1. Cell lines and culture media.

<td>Cellphone line</td><td>Fountain</td><td>culture medium</td>

<td> 92-1</td><td>SIGMA</td><td>RPMI1640+20% FBS</td>

<td>to 204·</td><td>ATCC</td><td>McCoy's 5A +10% FBS</td>

<td>DB</td><td>ATCC</td><td>RPMI164O + 10% FBS</td>

<td>G4O1</td><td>ATCC</td><td>McCoy's SA+10% FBS</td>

<td>GW2</td><td>ATCC</td><td>McCoy's 5A+10% FBS</td>

<td>HEK293T</td><td>ATCC</td><td>DMEM+ 10% FBS</td>

<td>HLE</td><td>JCRB</td><td>MSDF+10%FBS</td>

<td>hlf</td><td>JCRB</td><td>DMEM+1O%FBS</td>

<td>HS729</td><td>ATCC</td><td>□ MEM+ 10% FBS</td>

<td>JURKAT</td><td>ATCC</td><td>RPMI1640+10% FBS</td>

<td>KYSE15O</td><td>dsmz</td><td>RPMU&amp;WHam's F12 +10% FBS</td>

<td>LNCAP</td><td>ATCC</td><td>RPMI1&amp;W+ 10% FBS</td>

<td>LP1</td><td>dsmz</td><td>IMDM + 20% FBS</td>

<td>MM1.S</td><td>ATCC</td><td>RPMI1&amp;W + 10% FBS</td>

<td>MP3 to</td><td>ATCC</td><td>RPMI164O+ 20% FBS</td>

<td>ΜΡΨ1</td><td>ATCC</td><td>RPMI1&amp;W+ 20% FBS</td>

<td>PM+6</td><td>ATCC</td><td>RPMI164O + 20% FBS</td>

<td>NCIH1299</td><td>ATCC</td><td>RPMI1640+10% FBS</td>

<td>NCIH 24-44</td><td>ATCC</td><td>RPMI164O + 20% FBS</td>

<td>□ CIAML5</td><td>dsmz</td><td>alpha-MEM + 20% FBS+lOng/mlGM-CSF</td>

<td>□ PM2</td><td>dsmz</td><td>RPMI1&amp;W+ 10% FBS</td>

<td>OV7</td><td>ecac</td><td>DMEM/Hann's F12 (1:1) + 2mM Glutamine +10% FB5+0.5 ug/τΙ hydrocortisone + 10ug/mlinsulin</td>

<td>pfeiffer</td><td>ATCC</td><td>RPMI164O + 10% FBS</td>

<td>PLCPRF5</td><td>ATCC</td><td>EMEM + 10% FBS</td>

<td>DR</td><td>ATCC</td><td>MSDF+10%FBS</td>

<td>RS411</td><td>ATCC</td><td>RPMI1640+10% FBS</td>

<td>SKM1</td><td>JCRB</td><td>RPMI164O + 10% FBS</td>

<td>SUDHL4</td><td>dsmz</td><td>RPMI1640+10% FBS</td>

<td>YOUR DHLS</td><td>ATCC</td><td>RPMI1&amp;W+20% FBS</td>

<td>SW1573</td><td>ATCC</td><td>DMEM+ 10% FBS</td>

<td>TALL1</td><td>JCRB</td><td>RPMI164O + 10% FBS</td>

<td>TYKNU</td><td>JCRB</td><td>EMEM + 20% FBS</td>

<td>WSUDLCL2</td><td>dsmz</td><td>RPMI164O + 10% FBS</td>

Results: As shown in FIG. 1, uveal melanoma and hematologic cancer cell lines were more sensitive to BRG1/BRM inhibition than the other cell lines tested. Inhibition of uveal melanoma and hematologic cancer cell lines was maintained through day 7.

Example 8. Comparison of BRG1/BRM inhibitors with clinical PKC and MEK inhibitors in uveal melanoma cell lines

Procedure: Uveal melanoma cell lines, 92-1 or MP41, were seeded in 96-well plates in the presence of culture medium (see Table 1). BAF ATPase inhibitors (Compound A), PKC inhibitor (LXS196; MedChemExpress) or MEK inhibitor (Selumetinib; Selleck Chemicals) were dissolved in DMSO and added to cells in a concentration gradient from 0 to 10 pM at the time of sowing. The cells were incubated at 37^eitherC for 3 days. After three days of treatment, cell growth was measured with Cell-titer glow (Promega) and luminescence was read on an Envision plate reader (Perkin Elmer).

Results: As shown in FIG. 2 and FIG. 3, Compound A showed inhibition of uveal melanoma cell growth comparable to that of clinical PKC and MEK inhibitors. Furthermore, Compound A was found to result in a more rapid onset of inhibition than clinical PKC and MEK inhibitors.

Example 9. Synthesis of Compound B

The BRG1/BRM inhibitor compound B has the structure:

<img file="CO2022010547A2_D0031.tif" />

Compound B

Compound B was synthesized as shown in Scheme 4 below.

Scheme 4. Synthesis of Compound B

<img file="CO2022010547A2_D0032.tif" />

HOST.EOCI.DIEA.DMF

<img file="CO2022010547A2_D0033.tif" />

<img file="CO2022010547A2_D0034.tif" />

To a mixture of (2S)-2-amino-4-methylsulfanyl-N-[4-[3-(4-pyridyl)phenyl]thiazol-2-yl]butanamide (2 g, 4.75 mmol, HCI salt) and 1-methylsulfonylpyrrole-3-carboxylic acid (898.81 mg, 4.75 mmol) in DMF (20 mL) was added EDCI (1.37 g, 7.13 mmol), HOBt (962.92 mg, 7, 13 mmol) and DIEA (2.46 g, 19.00 mmol, 3.31 mL) and the mixture was stirred at 25 °C for 3 h. The mixture was poured into H2O (100 mL) and the precipitate was collected by filtration. The solid was triturated in MeOH (20 mL) and the precipitate collected by filtration. The solid was dissolved in DMSO (10 mL) and then the mixture was poured into MeOH (50 mL), and the precipitate formed was collected by filtration and lyophilized to give Compound B (2.05 g, 3.66 mmol, 77 0.01% yield) as a white solid.

LCMS (ESI) m/z [M+H]⁺=555,9.

Ή NMR (400 MHz, DMSO) δ 12.49 (s, 1H), 8.68 - 8.66 (m, 2H), 8.46 (d, J = 7.2 Hz, 1H), 8.31 - 8.30 (m, 1H), 8.02 - 8.00 (m, 1H), 7.94 - 7.96 (m, 1H), 7.83 (s, 1H), 7.73 - 7 .74 (m, 3H), 7.61 - 7.57 (m, 1H), 7.31 - 7.29 (m, 1H), 6.79 - 6.77 (m, 1H), 4.74 - 4.69 (m, 1H), 3.57 (s, 3H), 2.67-2.53 (m, 2H), 2.13-2.01 (m, 5H). ee% = 100%.

Compound B was found to have an IP50 of 3.6 nM against BRM and 5.7 nM against BRG1 in the described ATPase assay.

Example 10. Effects of BRG1/BRM ATPase inhibition on the growth of cell lines of uveal melanoma, hematological cancer, prostate cancer, breast cancer and Ewing's sarcoma

Procedure: All cell lines described above in Example 7 were also tested as described above with Compound B. In addition, the following cell lines were also tested as below. Briefly, for Ewing sarcoma cell lines (CADOES1, RDES, SKES1), retinoblastoma cell lines (WERIRB1), ALL (REH) cell lines, AML cell lines (KASUMI1), prostate cancer cell lines (PC3,

DU145, 22RV1), melanoma cell lines (SH4, SKMEL28, WM115, COLO829, SKMEL3, A375), breast cancer cell lines (MDAMB415, CAMA1, MCF7, BT474, HCC1419, DU4475, BT549), B-ALL cell lines (SUPB15), CML cell lines (K562, MEG01), Burkitt lymphoma cell lines (RAMOS2G64C10, DAUDI), Mantle cell lymphoma cell lines (JEKO1, 5 REC1), Bladder cancer cell lines (HT1197) and lung cancer cell lines (SBC5), The above methods were performed with the following modifications: Cells were seeded in 96-well plates, and the following day, the BRG1/BRM ATPase inhibitor, Compound B, was dissolved in DMSO and added to the cells in a gradient of concentration from 0 to 10 μΜ. At the time of cell division on days 3 and 7, cells were split into new 96-well plates and fresh compound was added four hours after replating. Table 2 lists the cell lines tested and the culture media used.

Table 2. Cell lines and culture media.

<td>Cellphone line</td><td>Fountain</td><td>culture medium</td>

<td>22RV1</td><td>ATCC</td><td>RPMI1640+ 10¾ FBS</td>

<td>at 375</td><td>ATCC</td><td>□ MEM+ 10¾ FBS</td>

<td>BT474</td><td>ATCC</td><td>Hybricare medium + 1.5g/L sodium bicarbonate+10% FEIS</td>

<td>BT549</td><td>ATCC</td><td>RRMI1640 + 0.023 »U/ml insulin* 10® FBS</td>

<td>CADO ESI</td><td>dsmz</td><td>RPMI1640+ 10¾ FBS</td>

<td>BED1</td><td>ATCC</td><td>EMEM + 10% FBS</td>

<td>COLOB29</td><td>ATCC</td><td>RPMI1640+ 10% FBS</td>

<td>□AUDI</td><td>ATCC</td><td>RPMI1640+ 10¾ FBS</td>

<td>DU145</td><td>ATCC</td><td>EMEM + 10¾ FBS</td>

<td>DU4475</td><td>ATCC</td><td>RPMI1640+ 10¾ FBS</td>

<td>HCC1419</td><td>ATCC</td><td>RPMI1640+lü% FBS</td>

<td>HT1197</td><td>ATCC</td><td>EMEM + 10¾ FBS</td>

<td>JEK01</td><td>ATCC</td><td>RPMI1640+ 20¾ FBS</td>

<td>K562</td><td>ATCC</td><td>IMDM + 10¾ FBS</td>

<td>KASUMI1</td><td>ATCC</td><td>RPMI1640+ 10¾ FBS</td>

<td>MCF7</td><td>ATCC</td><td>EMEM +0.01 mg/ml bovine insulin + 10% FBS</td>

<td>MDAMB415</td><td>ATCC</td><td>Leibcwitz's L-15 + 2ιίίΜ L- qlutamine +10 mcg/nnl ¡insulin+ 10 m-cg/m 1 Qlutathion +15¾ FBS</td>

<td>MEGO1</td><td>ATCC</td><td>RPMI1640+ 10% FBS</td>

<td>PC3</td><td>ATCC</td><td>F-12K+ 10% FBS</td>

<td>RAMOS2G64C10</td><td>ATCC</td><td>RPMI1640+10% FBS</td>

<td>ROES</td><td>ATCC</td><td>RPMI1640+ 15% FBS</td>

<td>REC1</td><td>ATCC</td><td>RPMI1640+ 10% FBS</td>

<td>REH</td><td>ATCC</td><td>RPMI1640+ 10% FBS</td>

<td>SBC5</td><td>JCRB</td><td>EMEM + 10¾ FB5</td>

<td>SH4</td><td>ATCC</td><td>MSDF + 10% FBS</td>

<td>5KE51</td><td>ATCC</td><td>McCoy's 5A + 15% FBS</td>

<td>SKMEL28</td><td>ATCC</td><td>EMEM + 10¾ FBS</td>

<td>SKMEL5</td><td>ATCC</td><td>McCoy's5A + 15% FBS</td>

<td>SUPB15</td><td>ATCC</td><td>IMDM +4 mM L- glutamine +1.5 g/sodium LtjcartwnatD + 0.05 mM 2- mercaptpetanol i-20% FBS</td>

<td>WERIRB1</td><td>ATCC</td><td>RPMI1640+ 10% FBS</td>

<td>WM115</td><td>ATCC</td><td>EMEM + 10% FBS</td>

Results: As shown in FIG. 4, uveal melanoma, hematologic cancer, prostate cancer, breast cancer, and Ewing's sarcoma cell lines were more sensitive to BRG1/BRM inhibition than the other cell lines tested. Inhibition of uveal melanoma, hematologic cancer, prostate cancer, breast cancer, and Ewing's sarcoma cell lines was maintained through day 7.

Example 11. Effects of BRG1/BRM ATPase inhibition on the growth of cancer cell lines.

Procedure: A pooled cell viability assay was performed using PRISM (profiling of simultaneous relative inhibition in mixtures) as previously described (Highthroughput identification of genotype-specific cancer vulnerabilities in mixtures of barcoded tumor cell lines, Yu et al, Nature Biotechnology 34 , 419-423, 2016), with the following modifications. Cell lines were obtained from the Cancer Cell Line Encyclopedia (CCLE) collection and adapted to RPMI-1640 medium without phenol red, supplemented with 10% heat-inactivated fetal bovine serum (FBS), to apply a unique protocol of infection and combination for a large compendium of cell lines. A lentiviral spin infection protocol was executed to introduce a 24 nucleotide barcode into each cell line, with an estimated multiplicity of infection (MOI) of 1 for all cell lines, using blasticidin as the selectable marker. More than 750 stable barcoded PRISM cancer cell lines were pooled according to doubling time into sets of 25. For the screening run, instead of plating a set of 25 cell lines in each well as previously described (Yu et al.), all sets of adherent or suspension cell lines were plated together using T25 flasks (100 000 cells/flask) or 6-well plates (50,000 cells/well), respectively. Cells were treated with DMSO or compound in an 8-point triple dose response in triplicate, starting from a maximum concentration of 10 μΜ. As a robustness control of the assay, cells were treated in parallel with two previously validated compounds, the pan-Raf inhibitor AZ-628 and the proteasome inhibitor bortezomib, using a maximum concentration of 2.5 μΜ and 0.039 μΜ, respectively. .

After 3 days of compound treatment, cells were lysed, genomic DNA extracted, barcodes amplified by PCR, and detected with next generation sequencing. Cell viability was determined by comparing the cell line-specific barcode counts in the treated samples with those of the DMSO control and the Day 0 control. Dose-response curves were fitted for each cell line and the corresponding area under the curves (AUC) was calculated and compared to the median AUC of all cell lines (FIG. 5).

Results: Cell lines with AUC below the median were considered the most sensitive.

Example 12. Effects of BRG1/BRM ATPase inhibitors on the growth of uveal melanoma cell lines.

Procedure: Uveal melanoma cell lines (92-1, MP41, MP38, MP46) and non-small cell lung cancer cells (NCIH1299) were seeded in 96-well plates with culture medium (see Table 2). . The BRG1/BRM ATPase inhibitor, Compound B, was dissolved in DMSO and added to the cells in a concentration gradient from 0 to 10 pM at the time of seeding. The cells were incubated at 37 °C for 3 days. After three days of treatment, cell growth was measured with Cell-titer glow (Promega) and luminescence was read on an Envision plate reader (Perkin Elmer).

Results: As shown in FIG. 6, Compound B resulted in potent growth inhibition in cell lines.

Example 13. Comparison of BRG1/BRM inhibitors with clinical PKC and MEK inhibitors in uveal melanoma cell lines

Procedure: Uveal melanoma cell lines, 92-1 or MP41, were seeded in 96-well plates in the presence of culture medium (see Table 2). BAF ATPase inhibitor (Compound B), PKC inhibitor (LXS196; MedChemExpress), and MEK inhibitor (Selumetinib; Selleck Chemicals) were dissolved in DMSO and added to cells in a concentration gradient from 0 to 10 pM at the time of sowing. The cells were incubated at 37 °C for 3 days. After three days of treatment, cell growth was measured with Cell-titer glow (Promega) and luminescence was read on an Envision plate reader (Perkin Elmer).

Results: As shown in FIG. 7 and FIG. 8, Compound B showed more potent effects on growth inhibition of uveal melanoma cells compared to clinical PKC and MEK inhibitors. In addition, Compound B was found to result in a more rapid onset of growth inhibition than clinical PKC and MEK inhibitors.

Example 14. BRG1/BRM ATPase inhibitors are effective in inhibiting the growth of cells resistant to PKC inhibitors.

Procedure: MP41 uveal melanoma cells were rendered resistant to the PKC inhibitor (LXS196; MedChemExpress) by long-term cultivation in culture media (see Table 2) containing increasing concentrations of the compound, up to 1 μΜ. After 3 months, parental MP41 cells and cells resistant to PKC inhibitor (PKCi) were tested for sensitivity to PKC inhibitor (LXS196) or BRG1/BRM ATPase inhibitor (Compound B) in an inhibition assay. of 7 day growth as described above in Example 6.

Results: While PKCi-resistant cells could tolerate growth at higher concentrations of LXS196 than the parent cell line MP41 (FIG. 9), the BRG1/BRM ATPase inhibitor (Compound B) resulted in strong growth inhibition. of both parental and PKCi resistant cell lines (FIG. 10). PKCi resistant cells were more sensitive to Compound B than parental MP41 cells (FIG. 10).

Example 15. Synthesis of Compound C

BRG1/BRM inhibitor compound C has the structure:

<img file="CO2022010547A2_D0035.tif" />

C.

Compound C was synthesized as shown in Scheme 5 below.

Scheme 5. Synthesis of Compound C

<img file="CO2022010547A2_D0036.tif" />

Compound C

Compound C was found to have an IP₅or 5.3 nM against BRM and 1.3 nM against BRG1 in the described ATPase assay.

Example 16. BRG1/BRM ATPase inhibitors cause inhibition of uveal melanoma tumor growth in vivo.

Procedure: Unlabeled mice (Envigo) were grafted subcutaneously in the axillary region 5x10⁶ 92-1 uveal melanoma cells on 50% Matrigel. Tumors grew to a mean of ~200 mm³, at which time the mice were pooled and dosing started. Mice were dosed once daily by oral gavage with vehicle (20% 2-hydroxypropyl-P-cyclodextrin) or increasing doses of Compound C. Tumor volumes and body weights were measured over the course of 3 weeks, and doses were adjusted by body weight to achieve the appropriate dose in terms of mg/kg. At this time, the animals were sacrificed and the tumors were dissected and imaged.

Results: As shown in FIG. 11 and FIG. 12, treatment with Compound C led to inhibition of tumor growth in a dose-dependent manner with tumor regression being observed at the highest dose (50 mg/kg). As shown in FIG. 13, all treatments were well tolerated with no observed loss of body weight (FIG. 13).

Example 17. Effects of BRG1/BRM ATPase inhibition on the growth of uveal melanoma and hematological cancer cell lines.

Procedure: Uveal melanoma cell lines (92-1, MEL202, MP41, MP38, MP46), prostate cancer cells (22RV1), acute leukemia cells (EOL1, THP1) and histocytic lymphoma cells (U937) were seeded in 96-well plates with culture media (see Table 2). BRG1/BRM ATPase Inhibitor 5, N-((S)-1-((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)-3- methoxy1-oxopropan-2-yl)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamide, dissolved in DMSO and added to cells in a concentration gradient from 0 to 2 pM (for uveal melanoma cell lines ), or 0 to 1 pM (for other cell lines), at the time of seeding. The cells were incubated at 37 °C for 3 days. After three days of treatment, cell growth was measured with Cell-titer glow 10 (Promega) and luminescence was read on an Envision plate reader (Perkin Elmer).

Results: As shown in FIG. 14, N-((S)-1-((4-(6-(cis-2,6-dimethylmorpholino)pyridin-2yl)thiazol-2-yl)amino)-3-methoxy-1-oxopropan-2- yl)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamide resulted in potent growth inhibition in all cell lines. As shown in Table 3, the absolute measured IC50 values ​​were below 350 nanomolar for all cell lines tested.

Table 3 lists the cell lines tested, the culture media used and the absolute values ​​of IC50 (nM) after 3 days of treatment with the compound.

Table 3. Cell lines, culture media and absolute IC50 values.

<td>Cellphone line</td><td>Fountain</td><td>culture media</td><td>cancer type</td><td>IC50 absolute (nM)</td>

<td>22RV1</td><td>ATCC</td><td>RPMI1640 + 10% fbs</td><td>Prostate</td><td> 29,7</td>

<td> 92-1</td><td>SIGMA</td><td>RPMI1640 + 10% fbs</td><td>uveal melanoma</td><td> 0,3</td>

<td>EOL1</td><td>dsmz</td><td>RPMI1640 + 10% fbs</td><td>myeloid leukemia acute</td><td> 75,5</td>

<td>MEL202</td><td>SIGMA</td><td>RPMI1640 + 10% fbs</td><td>uveal melanoma</td><td> 62,3</td>

<td>Cellphone line</td><td>Fountain</td><td>culture media</td><td>cancer type</td><td>IC50 absolute (nM)</td>

<td>MP38</td><td>ATCC</td><td>RPMI1640 + 20% fbs</td><td>uveal melanoma</td><td> 31,5</td>

<td>MP41</td><td>ATCC</td><td>RPMI1640 + 20% fbs</td><td>uveal melanoma</td><td> 11,8</td>

<td>MP46</td><td>ATCC</td><td>RPMI1640 + 20% fbs</td><td>uveal melanoma</td><td> 112,6</td>

<td>THP1</td><td>ATCC</td><td>RPMI1640 + 10% fbs</td><td>monocytic leukemia acute</td><td> 344,9</td>

<td>U937</td><td>ATCC</td><td>RPMI1640 + 10% fbs</td><td>histiocytic lymphoma</td><td> 14,8</td>

Example 18. Inhibition of BRG1/BRM ATPase causes inhibition of uveal melanoma tumor growth in vivo.

Procedure: Unlabeled mice (Envigo) were grafted subcutaneously in the axillary region with 5x10 6 92-1 uveal melanoma cells in 50% Matrigel. Tumors grew to a mean of ~200 mm³, at which time the mice were pooled and dosing started. Mice were dosed once daily by oral gavage with vehicle (20% 2-hydroxypropyl-ecyclodextrin) or increasing doses of N-((S)-1-((4-(6-(cis-2, 6-dimethylmorpholino)pyridin-2-yl)thiazol-2yl)amino)-3-methoxy-1-oxopropan-2-yl)-1-(methylsulfonyl)-1H-pyrrole-3-carboxamide. Tumor volumes and body weights were measured over the course of 3 weeks, and doses were adjusted based on body weight to achieve the appropriate dose in mg/kg terms.

Results: As shown in FIG. 15, treatment with N-((S)-1-((4-(6-(cis-2,6dimethylmorpholino)pyridin-2-yl)thiazol-2-yl)amino)-3-methoxy-1-oxopropan -2-yl)-1-(methylsulfonyl)-1H-pyrrolo-3-carboxamide led to inhibition of tumor growth in a dose-dependent manner and tumor regression was observed with the highest dose (1.5 mg/kg). As shown in FIG. 16, all treatments were well tolerated based on the % body weight change observed.

Other modalities

While the invention has been described in connection with specific embodiments thereof, it will be understood that the invention is susceptible to further modification and that this application is intended to cover any variations, uses, or adaptations of the invention by generally following the principles of the invention and including such deviations from the present description that are within the known or usual practice within the art to which the invention pertains and can be applied to the essential characteristics established above, and remains within the scope of The claims.

Other modalities are found in the claims.

The following is claimed: