Trim21-targeted compounds and uses thereof

Abstract

Described herein are compounds, pharmaceutical compositions, and methods for treating TRIM21-associated proliferative disorders, including cancers such as pancreatic cancer and head and neck cancers.

Description

[0001] STDU2-44700.601

[0002] TRIM21 -TARGETED COMPOUNDS AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U. S. Provisional Patent Application No. 63 / 774,409, filed on March 1, 2025, and U. S. Provisional Patent Application No. 63 / 935,565, filed on December 10, 2025, the disclosures of which are incorporated herein by reference in their entireties.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0004] This invention was made with Government support under contract CA230220 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0005] FIELD

[0006] Disclosed herein are compounds, pharmaceutical compositions, and methods for treating TRIM21 -associated proliferative disorders, including cancers such as pancreatic cancer and head and neck cancers.

[0007] BACKGROUND PRLX-93936 is a compound that was evaluated in phase I / II clinical trials for the treatment of multiple myeloma and advanced solid tumors, but development of the drug was discontinued and a mechanism of action for the compound was not conclusively identified.

[0008] SUMMARY

[0009] In one aspect, disclosed herein is a compound of formula (I):

[0010]

[0011] Rb(I)

[0012] or a pharmaceutically acceptable salt thereof, wherein:

[0013] Q is O or NH;STDU2-44700.601

[0014] R1is Ci-C6alkyl, C3-C6 cycloalkyl, or monocyclic heterocyclyl, wherein the alkyl is unsubstituted or substituted with 1 substituent selected from hydroxy, amino, amido, and cyano; or R1is taken together with R2aand the atoms to which they are attached to form a ring;

[0015] X1is CH or N;

[0016] X2is CR2aor N;

[0017] X3is CR2bor N;

[0018] X4is CR2Cor N;

[0019] X5is CR2dor N;

[0020] X6is CR3aor N;

[0021] X7is CR3bor N;

[0022] X8is CR3cor N;

[0023] X9is CR3dor N;

[0024] R2a, R2b, R2C, R2d, R3a, R3b, R3C, and R3dare each independently selected from hydrogen, halo, Ci-Ce alkyl, Ci-Ce haloalkyl, Ci-Ce alkoxy, -NRxRy, -C(O)(Ci-Ce alkyl), cyano, aryl, C3-C6 cycloalkyl, heteroaryl, and heterocyclyl; wherein R2aand R2b, or R2band R2C, or R2Cand R2dare optionally taken together with the carbon atoms to which they are attached to form an optionally substituted ring; and wherein R3aand R3b, or R3band R3c, or R3Cand R3dare optionally taken together with the carbon atoms to which they are attached to form an optionally substituted ring;

[0025] Rxand Ryare each independently selected from hydrogen, Ci-Ce alkyl, and Ci-Ce hydroxyalkyl;

[0026] pl is 1, 2, or 3;

[0027] p2 is 1, 2, or 3;

[0028] n is 0, 1, 2, 3, or 4;

[0029] each R4is independently selected from Ci-Ce alkyl, halo, oxo, Ci-Ce haloalkyl, C3-C6 cycloalkyl, cyano, and -(CH2)q-Y, wherein q is 1, 2, or 3, and Y is selected from hydroxy, amino, C1-C4 alkoxy, C1-C4 alkylsulfonyl, and C1-C4 alkylsulfinyl; or two R4are taken together to form a bond, or two R4are taken together with the atom(s) to which they are attached to form a bond or a 3- to 6-membered ring that is unsubstituted or substituted with one Ci-Ce alkyl group;

[0030] R3is hydrogen or C1-C4 alkyl;

[0031] Z is CR6aR6b, O, or NH; andSTDU2-44700.601

[0032] R6aand R6bare each independently selected from hydrogen and Ci-Ce alkyl, or R6aand R6bare taken together with the carbon atom to which they are attached to form an optionally substituted ring.

[0033] In some embodiments, Q is O.

[0034] In some embodiments, R1is C1-C4 alkyl, C3-C4 cycloalkyl, or a monocyclic 4- or 5-membered heterocyclyl having one heteroatom selected from N and O, wherein the alkyl is unsubstituted or substituted with 1 substituent selected from -OH, -NH2, -NH(CH3), -N(CH3)2, and -C(0)NH2. In some embodiments, R1is unsubstituted C2-C4 alkyl. In some embodiments, R1is ethyl. In some embodiments, R1is C2-C4 alkyl that is substituted with 1 substituent selected from -OH, -NH2, -NH(CH3), and -N(CH3)2. In some embodiments, R1is -CH2CH2NH2.

[0035] In some embodiments, X1is N.

[0036] In some embodiments, X2is CR2a. In some embodiments, R2ais hydrogen.

[0037] In some embodiments, X3is CR2b. In some embodiments, R2bis selected from hydrogen, halo, C1-C3 alkyl, C1-C3 haloalkyl, -C(O)O(Ci-C3 alkyl), and a monocyclic 5- or 6-membered heterocyclyl having 1 or 2 heteroatoms independently selected from O and N. In some embodiments, R2bis halo. In some embodiments, R2bis chloro.

[0038] In some embodiments, X4is CR2c. In some embodiments, R2cis hydrogen, halo, Ci-C3 haloalkyl, C1-C3 alkoxy, -NRxRy, or cyano, wherein Rxis hydrogen and Ryis C1-C3 hydroxyalkyl. In some embodiments, R2cis hydrogen.

[0039] In some embodiments, X3is CR2d. In some embodiments, R2dis hydrogen.

[0040] In some embodiments, X6is CR3a. In some embodiments, R3ais hydrogen.

[0041] In some embodiments, X7is CR3b. In some embodiments, R3bis selected from hydrogen, halo, C1-C3 alkyl, cyano, phenyl, and monocyclic 5- or 6-membered heteroaryl having 1 or 2 heteroatoms independently selected from N, O, and S. In some embodiments, R3bis halo. In some embodiments, R3bis fluoro.

[0042] In some embodiments, X8is CR3c. In some embodiments, R3cis selected from hydrogen and -NRxRy, wherein Rxand Ryare each independently selected from hydrogen and C1-C3 alkyl. In some embodiments, R3cis hydrogen.

[0043] In some embodiments, X9is CR3d. In some embodiments, R3dis hydrogen.

[0044] In some embodiments, each R4is independently selected from Ci-Ce alkyl, halo, and oxo; or two R4are taken together to form a bond, or two R4are taken together with the atom(s) to which they are attached to form a bond or a ring.

[0045] In some embodiments, n is 0.STDU2-44700.601

[0046] In some embodiments, n is 1 or 2, and each R4is independently selected from C1-C3 alkyl, halo, and oxo. In some embodiments, n is 1 and R4is fluoro, methyl, or oxo. In some embodiments, n is 2 and each R4is fluoro, methyl, or oxo.

[0047] In some embodiments, n is 2, and wherein the two R4are taken together to form a bond or are taken together with the atom(s) to which they are attached to form a ring.

[0048] In some embodiments, R5is hydrogen.

[0049] M2

[0050] 7 < R4)n

[0051] In some embodiments, the group

[0052]

[0053] r5has a formula selected from:

[0054]

[0055] In some embodiments, Z is CR6aR6band R6aand R6bare each hydrogen.

[0056] In some embodiments, Z is O.

[0057] In some embodiments, the compound is selected from:

[0058]

[0059] STDU2-44700.601

[0060]

[0061] STDU2-44700.601

[0062]

[0063]

[0064] , and pharmaceutically acceptable salts thereof.

[0065] Also disclosed herein is a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0066] Also disclosed herein is a method of treating a proliferative disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof.

[0067] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0068] In some embodiments, the proliferative disease is associated with tripartite motifcontaining 21 (TRIM21). In some embodiments, the proliferative disease is associated with a mutation in and / or increased activity of TRIM21. In some embodiments, the proliferative disease is cancer. In some embodiments, the cancer is selected from pancreatic cancer, head and neck cancer, breast cancer, colorectal cancer, lung cancer, melanoma, ovarian cancer, kidney cancer, multiple myeloma, esophageal cancer, gastric cancer, liver cancer, bile duct cancer, leukemia, lymphoma, prostate cancer, and sarcoma. In some embodiments, the cancer is pancreatic cancer.

[0069] In some embodiments, the method further comprises treating the subject with one or more additional therapies selected from chemotherapy, surgery, radiation therapy, hormone therapy, immunotherapy, cryotherapy, T cell transfer therapy, and thermotherapy, or any combination thereof.STDU2-44700.601

[0070] In another aspect, disclosed herein is a compound of formula (I), or a pharmaceutically acceptable salt thereof, for use as a medicament.

[0071] In another aspect, disclosed herein is a compound of formula (I), or a pharmaceutically acceptable salt thereof, for use in treating a proliferative disease. In some embodiments, the proliferative disease is associated with tripartite motif-containing 21 (TRIM21). In some embodiments, the proliferative disease is associated with a mutation in and / or increased activity of TRIM21. In some embodiments, the proliferative disease is cancer. In some embodiments, the cancer is selected from pancreatic cancer, head and neck cancer, breast cancer, colorectal cancer, lung cancer, melanoma, ovarian cancer, kidney cancer, multiple myeloma, esophageal cancer, gastric cancer, liver cancer, bile duct cancer, leukemia, lymphoma, prostate cancer, and sarcoma. In some embodiments, the cancer is pancreatic cancer.

[0072] In another aspect, disclosed herein is a kit comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof.

[0073] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the chemical structures of erastin and PRLX-93936, a drug with unknown mechanism of action that was previously tested in early-stage human clinical trials.

[0074] FIG. 2 shows the results from a genome-scale CRISPR / Cas9 drug modifier knockout screen to identify functional mediators required for PRLX-93936 anti-cancer activity. Screen was performed over 21 days using the Brunello sgRNA library. CRISPR guide abundance is summarized to the gene level using MAGeCK. The volcano plot depicts the adjusted q- value on the y-axis and the gene-level log2 fold change on the x-axis. TRIM21 was the top rescue hit.

[0075] FIG. 3 shows the results from a genome-scale CRISPR / Cas9 drug modifier activation screen to identify functional mediators sufficient for PRLX-93936 anti-cancer activity.

[0076] Screen was performed over 13 days using the Calabrcsc-B sgRNA library. CRISPR guide abundance is summarized to the gene level using MAGeCK. The volcano plot depicts the adjusted q-value on the y-axis and the gene- level log2 fold change on the x-axis. TR1M21 was a top sensitization hit.

[0077] FIGS 4A-4B show: (FIG. 4A) the results from a 5-day cancer cell viability assay (PRISM) for 816 diverse cancer cell lines. Area under the dose-response curve (AUC) for each cancer cell line is indicated and grouped by cancer lineage. Boxplots illustrate the median AUC and the 25th / 75th percentiles. Whiskers illustrate the 10th / 90th percentiles;STDU2-44700.601

[0078] (FIG. 4B) a statistical test for anti-cancer activity by lineage (Fisher exact test for more sensitive quartile with Benjamini-Hochberg adjustment).

[0079] FIG. 5 shows baseline cell line gene expression (RNA-seq) versus PRLX-93936 activity across more than 800 cancer cell lines. Linear modeling was employed to estimate effect size. Each point is a gene. TRIM21 expression is highlighted as the top feature predicting sensitivity. Also shown is a scatter plot comparing TRIM21 mRNA expression versus PRLX-93936 viability AUG. Each point is a cancer cell line.

[0080] FIG. 6 shows PRLX-93936 cancer cell viability assays (72H, CellTiterGlo) for two cell lines with high baseline TRIM21 expression and intrinsic sensitivity. Dose response curves are shown for intact TRIM21 (Control sgRNA) versus TRIM21 knockout (TRIM21 sgRNAs).

[0081] FIG. 7 shows PRLX-93936 cancer cell viability assays (72H, CellTiterGlo) for three cell lines with low baseline TRIM21 expression and intrinsic resistance. Dose response curves arc shown for low TRIM21 (Wild type) versus TRIM21 overexpression (TRIM21 OE #1 and TRIM21 OE#2).

[0082] FIG. 8 shows PRLX-93936 cancer cell viability assays (72H, CellTiterGlo) for PANC1 cells with TRIM21 knockout + / - TRIM21 cDNA restoration of expression. A wildtype TRIM21 cDNA is compared with a point mutation expected to reduce TRIM21 activity (R55E).

[0083] FIG. 9 shows induction of luminescent caspase 3 / 7 reporter (CaspaseGlo) following 12 hours of PRLX-93936 treatment of PANCI cells. Cells with intact TRIM21 expression (Control sgRNA) are compared with TRIM21 knockout (KO) cells. Also shown are immunoblots of PANCI cells treated with the indicated concentration of PRLX-93936 for 16 hours. Wild-type (WT) cells are compared with TRIM21 knockout (KO) cells.

[0084] FIG. 10 shows differentially expressed genes in PANCI cells treated with 0.5 uM PRLX-93936 for 2 hours by RNA-seq.

[0085] FIG. 11 shows immunoblots of nuclear and cytoplasmic extracts from PANCI cells treated with 0.5 uM PRLX-93936 for the indicated timepoints.

[0086] DETAILED DESCRIPTION

[0087] As disclosed herein, using CRISPR drug modifier screens and systematic viability profiling, PRLX-93936 was unexpectedly found to act via the Tripartite motif containing-21 (TRIM21) E3 ubiquitin ligase, to simultaneously activate immune signaling and apoptotic pathways. TRIM21 is a RING-family E3 ligase enzyme. E3 ligase enzymes recognizeSTDU2-44700.601

[0088] substrate proteins and facilitate conjugation of ubiquitin and poly-ubiquitin chains (Buetow et al. Nat. Rev. Mol. Cell Biol. 17, 626-642 (2016)). There is strong precedent for stimulating E3 ligase activities via small molecules for therapeutic benefit. For example, thalidomide derivatives act as molecular glues to stimulate CRBN-mediated ubiquitination and degradation of essential substrates such as IKZF1 (Fischer, E. S. et al. Nature 512, 49-53 (2014)) or CSNK1 Al (Petzold et al. Nature 532, 127-130 (2016)). Heterobifunctional ligase recruiting PROTACs are also being developed to expand available substrates (Jevtic et al. Cell Chem Biol 28, 1000-1013 (2021)). TRIM21 has dual actions: it can simultaneously i) bind and target antibody-conjugated proteins for proteasomal degradation via lysine 48 polyubiquitination (Clift, D. et al. Cell 171, 1692-1706.el8 (2017)), and ii) stimulate immune activation via lysine 63 polyubiquitination (Fletcher et al. Proc. Natl. Acad. Sci. U. S. A. 112, 10014-10019 (2015)). By recognizing antibody-bound cargo, TRIM21 is believed to play an important role in pathogen clearance by the immune system (Kiss, L. et al. Nat. Commun. 10, 4502 (2019)).

[0089] Accordingly, disclosed herein are compounds, pharmaceutical compositions, and methods for treating TR1M21 -associated cancers.

[0090] Definitions

[0091] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0092] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

[0093] As used herein, the term “and / or” includes any and all combinations of listed items, including any of the listed items individually. For example, “A, B, and / or C” encompasses A, B, C, AB, AC, BC, and ABC, each of which is to be considered separately described by the statement “A, B, and / or C.”STDU2-44700.601

[0094] For the recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0095] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Sorrell, Organic Chemistry, 2ndedition, University Science Books, Sausalito, 2006; Smith, March’s Advanced Organic Chemistry: Reactions, Mechanism, and Structure, 7thEdition, John Wiley & Sons, Inc., New York, 2013; Larock, Comprehensive Organic Transformations, 3rdEdition, John Wiley & Sons, Inc., New York, 2018; and Carruthers, Some Modem Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

[0096] As used herein, the term “alkyl” refers to a radical of a straight or branched saturated hydrocarbon chain. The alkyl chain can include, e.g., from 1 to 24 carbon atoms (C1-C24 alkyl), 1 to 16 carbon atoms (C1-C16 alkyl), 1 to 14 carbon atoms (C1-C14 alkyl), 1 to 12 carbon atoms (C1-C12 alkyl), 1 to 10 carbon atoms (C1-C10 alkyl), 1 to 8 carbon atoms (Ci-Cs alkyl), 1 to 6 carbon atoms (Ci-Ce alkyl), 1 to 4 carbon atoms (C1-C4 alkyl), 1 to 3 carbon atoms (C1-C3 alkyl), or 1 to 2 carbon atoms (C1-C2 alkyl). Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl

[0097] As used herein, the term “alkoxy” refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, and tert-butoxy.

[0098] As used herein, the term “alkylsulfinyl” refers to an alkyl group, as defined herein, appended to the parent molecular moiety through a sulfinyl group (i.e., -S(O)-).

[0099] As used herein, the term “alkylsulfonyl” refers to an alkyl group, as defined herein, appended to the parent molecular moiety through a sulfonyl group (i.e., -S(O)2-).STDU2-44700.601

[0100] As used herein, the term “amido” refers to a group -C(O)NR2, wherein each R is independently selected from hydrogen and alkyl (e.g., C1-C4 alkyl). A group -C(O)NH(alkyl) may be referred to herein as “alkylamido” and a group - C(O)N(alkyl)2 may be referred to herein as “dialkylamido.”

[0101] As used herein, the term “amino” refers to a group -NR2, wherein each R is independently selected from hydrogen and alkyl (e.g., C1-C4 alkyl). A group -NH(alkyl) may be referred to herein as “alkylamino” and a group -N(alkyl)2 may be referred to herein as “dialkylamino.”

[0102] As used herein, the term “aryl” refers to a radical of a monocyclic, bicyclic, or tricyclic 4n+2 aromatic ring system (e.g., having 6, 10, or 14π electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms (“C6-C14 aryl”). In some embodiments, an aryl group has six ring carbon atoms (“Ce aryl,” i.e., phenyl). In some embodiments, an aryl group has ten ring carbon atoms (“C10 aryl,” e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms (“C14 aryl,” e.g., anthracenyl and phenanthrenyl).

[0103] As used herein, the term “cycloalkyl” refers to a radical of a saturated carbocyclic ring system containing three to ten carbon atoms and zero heteroatoms. The cycloalkyl may be monocyclic, bicyclic, bridged, fused, or spirocyclic. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, adamantyl, bicyclo[2.2.1]heptanyl, bicyclo[3.2.1]octanyl, and bicyclo[5.2.0]nonanyl.

[0104] As used herein, the term “halogen” or “halo” refers to F, Cl, Br, or I.

[0105] As used herein, the term “haloalkyl” refers to an alkyl group, as defined herein, in which at least one hydrogen atom (e.g., one, two, three, four, five, six, seven or eight hydrogen atoms) is replaced with a halogen. In some embodiments, each hydrogen atom of the alkyl group is replaced with a halogen (“perhaloalkyl”). Representative examples of haloalkyl include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, and 3,3,3-trifluoropropyl.

[0106] As used herein, the term “heteroaryl” refers to a radical of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 n electrons shared in a cyclic array) having ring carbon atoms and 1 -4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur (“5-10 membered heteroaryl”). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl bicyclic ring systems can include oneSTDU2-44700.601

[0107] or more heteroatoms in one or both rings. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused (aryl / heteroaryl) ring system. Bicyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, and the like) the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl). Exemplary 5-membered heteroaryl groups containing one heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, without limitation, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, without limitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.

[0108] As used herein, the term “heterocyclyl” refers to a radical of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“3-10 membered heterocyclyl”). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”), and can be saturated or can be partially unsaturated. Heterocyclyl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heterocyclyl” also includes ring systems whereinSTDU2-44700.601

[0109] the heterocyclyl ring, as defined above, is fused with one or more cycloalkyl groups wherein the point of attachment is either on the cycloalkyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system. A heterocyclyl group may be described as, e.g., a 3-7-membered heterocyclyl, wherein the term “membered” refers to the non-hydrogen ring atoms, i.e., carbon, nitrogen, oxygen, sulfur, boron, phosphorus, and silicon, within the moiety.

[0110] Exemplary 3 -membered heterocyclyl groups containing one heteroatom include, without limitation, azirdinyl, oxiranyl, and thiiranyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, without limitation, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, without limitation, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, without limitation, piperidinyl (e.g., 2,2,6,6-tetramethylpiperidinyl), tetrahydropyranyl, dihydropyridinyl, pyridinonyl (e.g., l-methylpyridin-2-onyl), and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, piperazinyl, morpholinyl, pyridazinonyl (2-methylpyridazin-3-onyl), pyrimidinonyl (e.g., 1-methylpyrimidin-2-onyl, 3-methylpyrimidin-4-onyl), dithianyl, dioxanyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, triazinanyl. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary 5 -membered heterocyclyl groups fused to a Ce aryl ring (also referred to herein as a 5,6-bicyclic heterocyclyl ring) include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, and the like. Exemplary 5-membered heterocyclyl groups fused to a heterocyclyl ring (also referred to herein as a 5,5-bicyclic heterocyclyl ring) include, without limitation, octahydropyrrolopyrrolyl (e.g., octahydropyrrolo[3,4-c]pyrrolyl), and the like. Exemplary 6-membered heterocyclyl groups fused to a heterocyclyl ring (also referred to as a 4,6-STDU2-44700.601

[0111] membered heterocyclyl ring) include, without limitation, diazaspirononanyl (e.g., 2,7-diazaspiro[3.5]nonanyl). Exemplary 6-membered heterocyclyl groups fused to an aryl ring (also referred to herein as a 6,6-bicyclic heterocyclyl ring) include, without limitation, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like. Exemplary 6-membered heterocyclyl groups fused to a cycloalkyl ring (also referred to herein as a 6,7-bicyclic heterocyclyl ring) include, without limitation, azabicyclooctanyl (e.g., (l,5)-8-azabicyclo[3.2.1]octanyl). Exemplary 6-membered heterocyclyl groups fused to a cycloalkyl ring (also referred to herein as a 6,8-bicyclic heterocyclyl ring) include, without limitation, azabicyclononanyl (e.g., 9-azabicyclo[3.3.1]nonanyl).

[0112] When a group or moiety can be substituted, the term “substituted" indicates that one or more (e.g., 1, 2, 3, 4, 5, or 6; in some embodiments 1, 2, or 3; and in other embodiments 1 or 2) hydrogens on the group indicated in the expression using “substituted” can be replaced with a selection of recited indicated groups or with a suitable substituent group known to those of skill in the art (e.g., one or more of the groups recited below), provided that the designated atom’s normal valence is not exceeded. Substituent groups include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, acyl, amino, amido, amidino, aryl, azido, carbamoyl, carboxyl, carboxyl ester, cyano, cycloalkyl, cycloalkenyl, guanidino, halo, haloalkyl, haloalkoxy, heteroaryl, heterocyclyl, hydroxy, hydrazino, imino, oxo, nitro, phosphate, phosphonate, sulfonic acid, thiol, thione, or combinations thereof.

[0113] As used herein, in chemical structures the indication:

[0114]

[0115] represents a point of attachment of one moiety to another moiety (e.g., a substituent group to the rest of the compound).

[0116] For compounds described herein, groups and substituents thereof may be selected in accordance with permitted valence of the atoms and the substituents, such that the selections and substitutions result in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

[0117] When substituent groups are specified by their conventional chemical formulae, written from left to right, such indication also encompass substituent groups resulting from writing the structure from right to left. For example, if a bivalent group is shown as -CH2O-, such indication also encompasses -OCH2-; similarly, -OC(O)NH- also encompasses -NHC(O)O-. When linker moieties are shown, the linkers can be attached to other moieties of the compound in either direction.STDU2-44700.601

[0118] The terms “administer,” “administering,” or “administration,” as used herein refer to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a compound or a pharmaceutical composition.

[0119] As used herein, the terms “condition,” “disease,” and “disorder” are used interchangeably.

[0120] An “effective amount” of a compound or composition refers to an amount sufficient to elicit a desired biological response (e.g., treating a condition). As will be appreciated by those skilled in the art, the effective amount of a compound may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health of the subject. An effective amount encompasses therapeutic and prophylactic treatment. For example, in treating cancer, an effective amount of a compound or composition may reduce tumor burden or stop the growth or spread of a tumor.

[0121] A “therapeutically effective amount” of a compound or composition is an amount sufficient to provide a therapeutic benefit in the treatment of a condition, or to delay or minimize one or more symptoms associated with the condition. In some embodiments, a therapeutically effective amount is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to minimize one or more symptoms associated with the condition. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment of the condition. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the condition, or enhances the therapeutic efficacy of another therapeutic agent.

[0122] A “subject” to which administration is contemplated includes, but is not limited to, a human (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) and / or other non-human animals, for example, mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys); commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and / or dogs) and birds (e.g., commercially relevant birds such as chickens, ducks, geese, and / or turkeys).

[0123] As used herein, the terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or condition, or one or more signs or symptoms thereof. In some embodiments, “treatment,” “treat,” and “treating” require that signs or symptoms of the disease disorder or condition have developedSTDU2-44700.601

[0124] or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease or condition. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence.

[0125] Compounds

[0126] Disclosed herein are compounds of formula (I):

[0127] R1

[0128]

[0129] R5(I)

[0130] and pharmaceutically acceptable salts thereof, wherein:

[0131] Q is O or NH;

[0132] R1is Ci-Ce alkyl, C3-C6 cycloalkyl, or monocyclic heterocyclyl, wherein the alkyl is unsubstituted or substituted with 1 substituent selected from hydroxy, amino, amido, and cyano; or R1is taken together with R2aand the atoms to which they are attached to form a ring;

[0133] X1is CH or N;

[0134] X2is CR2aor N;

[0135] X3is CR2bor N;

[0136] X4is CR2Cor N;

[0137] X5is CR2dor N;

[0138] X6is CR3aor N;

[0139] X7is CR3bor N;

[0140] X8is CR3Cor N;

[0141] X9is CR3dor N;

[0142] R2a, R2b, R2C, R2d, R3a, R3b, R3C, and R3dare each independently selected from hydrogen, halo, Ci-Ce alkyl, Ci-Ce haloalkyl, C1-C6alkoxy, -NRxRy, -C(O)(Ci-Ce alkyl),STDU2-44700.601

[0143] cyano, aryl, C3-C6 cycloalkyl, heteroaryl, and heterocyclyl; wherein R2aand R2b, or R2band R2C, or R2Cand R2dare optionally taken together with the carbon atoms to which they are attached to form an optionally substituted ring; and wherein R3aand R3b, or R3band R3c, or R3Cand R3dare optionally taken together with the carbon atoms to which they are attached to form an optionally substituted ring;

[0144] Rxand Ryare each independently selected from hydrogen, Ci-Ce alkyl, and Ci-Ce hydroxyalkyl;

[0145] pl is 1, 2, or 3;

[0146] p2 is 1, 2, or 3;

[0147] n is 0, 1, 2, 3, or 4;

[0148] each R4is independently selected from Ci-Ce alkyl, halo, oxo, Ci-Ce haloalkyl, C3-C6 cycloalkyl, cyano, and -(CH2)q-Y, wherein q is 1, 2, or 3, and Y is selected from hydroxy, amino, C1-C4 alkoxy, C1-C4 alkylsulfonyl, and C1-C4 alkylsulfinyl; or two R4are taken together to form a bond, or two R4arc taken together with the atom(s) to which they are attached to form a bond or a 3- to 6-membered ring that is unsubstituted or substituted with one Ci-Ce alkyl group;

[0149] R3is hydrogen or C1-C4 alkyl;

[0150] Z is CR6aR6b, O, or NH; and

[0151] R6aand R6bare each independently selected from hydrogen and Ci-Ce alkyl, or R6aand R6bare taken together with the carbon atom to which they are attached to form an optionally substituted ring.

[0152] In some embodiments, Q is O. In some embodiments, Q is NH.

[0153] In some embodiments, R1is C1-C4 alkyl, C3-C4 cycloalkyl, or a monocyclic 4- or 5-membered heterocyclyl having one heteroatom selected from N and O, wherein the alkyl is unsubstituted or substituted with 1 substituent selected from -OH, -NH2, -NH(CH3), -N(CH3)2, and -C(O)NH2. In some embodiments, R1is selected from methyl, ethyl, cyclopropyl, azetidinyl, -CH2CH2NH2, -CH2CH2N(CH3)2, -CH2CH2OH, and -CH2C(O)NH2. In some embodiments, R1is C -C4 alkyl. In some embodiments, R1is ethyl. In some embodiments, R1is n-propyl. In some embodiments, R1is isopropyl. In some embodiments, R1is butyl (e.g., n-butyl, sec-butyl, isobutyl, or tert-butyl). In some embodiments, R1is C2-C4 alkyl that is substituted with 1 substituent selected from -OH, -NH2, -NH(CH3), and -N(CIl3)2. In some embodiments, R1is -CH2CH2NH2.STDU2-44700.601

[0154] In particular embodiments, Q is O and R1is C2-C4 alkyl that is substituted with 1 substituent selected from -OH, -NH2, -NH(CH3), and -N(CH3)2. In other particular embodiments, Q is O and R1is -CH2CH2NH2.

[0155] In some embodiments, X1is N. In some embodiments, X1is CH.

[0156] In some embodiments, X2is CR2a. In some embodiments, R2ais hydrogen or halo. In some embodiments, R2ais hydrogen. In some embodiments, R2ais chloro.

[0157] In some embodiments, X2is N.

[0158] In some embodiments, X3is CR2b. In some embodiments, R2bis selected from hydrogen, halo, C1-C3 alkyl, C1-C3 haloalkyl, -C(O)O(Ci-C3 alkyl), and a monocyclic 5- or 6-membered heterocyclyl having 1 or 2 heteroatoms independently selected from O and N. In some embodiments, R2bis hydrogen, chloro, bromo, methyl, trifluoromethyl, -C(O)OCH3, or morpholino. In some embodiments, R2bis hydrogen or halo. In some embodiments, R2bis hydrogen. In some embodiments, R2bis halo. In some embodiments, R2bis chloro.

[0159] In some embodiments, X4is CR2c. In some embodiments, R2cis hydrogen, halo, Ci-C3 haloalkyl, C1-C3 alkoxy, -NRxRy, or cyano, wherein Rxis hydrogen and Ryis C1-C3 hydroxyalkyl. In some embodiments, R2cis hydrogen, fluoro, chloro, bromo, trifluoromethyl, ethoxy, -NHCH2CH2OH, or cyano. In some embodiments, R2cis hydrogen.

[0160] In some embodiments, X4is N.

[0161] In some embodiments, X3is CR2b, X4is CR2c, and R2band R2care taken together with the carbon atoms to which they are attached to form a 5- or 6-membered heterocyclic ring having 1 or 2 heteroatoms independently selected from N and O.

[0162] In some embodiments, X3is CR2d. In some embodiments, R2dis hydrogen or halo. In some embodiments, R2dis hydrogen. In some embodiments, R2dis halo. In some embodiments, R2dis bromo. In some embodiments, X5is N.

[0163] X3

[0164] II

[0165] ., X4

[0166] X

[0167] In some embodiments, the group

[0168]

[0169] 5

[0170] has a structure selected from:STDU2-44700.601

[0171]

[0172] In some embodiments, X6is CR3aand R3ais hydrogen. In some embodiments, X6is N.

[0173] In some embodiments, X7is CR3b. In some embodiments, R3bis selected from hydrogen, halo, C1-C3 alkyl, cyano, phenyl, and monocyclic 5- or 6-membered heteroaryl having 1 or 2 heteroatoms independently selected from N, O, and S. In some embodiments, R3bis selected from hydrogen, fluoro, chloro, iodo, methyl, cyano, phenyl, and pyridyl. In some embodiments, R3bis halo. In some embodiments, R3bis fluoro.

[0174] In some embodiments, X8is CR3c. In some embodiments, R3cis selected from hydrogen, halo, C1-C3 alkoxy, and -NRxRy, wherein Rxand Ryare each independently selected from hydrogen and C1-C3 alkyl. In some embodiments, R3cis selected from hydrogen and -NRxRy, wherein Rxand Ryare each independently selected from hydrogen and C1-C3 alkyl. In some embodiments, R3cis selected from hydrogen, chloro, methyl, -NH2, -NHCH3, and -N(CH3)2. In some embodiments, R3cis selected from hydrogen, -NH2, -NHCH3, and -N(CH3)2. In some embodiments, R3cis hydrogen.

[0175] In some embodiments, X8is N.

[0176] In some embodiments, X9is CR3d. In some embodiments, R3dis selected from hydrogen, halo, C1-C3 alkyl, and -NRxRy, wherein Rxand Ryarc each hydrogen. In some embodiments, R3dis selected from hydrogen, chloro, methyl, and -NH2. In some embodiments, R3dis hydrogen.STDU2-44700.601

[0177] In some embodiments, X9is N.

[0178] In some embodiments, pl is 1. In some embodiments, pl is 2. In some embodiments, pl is 3. In some embodiments, p2 is 1. In some embodiments, p2 is 2. In some embodiments, p2 is 3. In some embodiments, pl is 1 and p2 is 1. In some embodiments, pl is 1 and p2 is 2. In some embodiments, pl is 2 and p2 is 2. In some embodiments, pl is 2 and p2 is 3. In some embodiments, pl is 3 and p2 is 3.

[0179] In some embodiments, n is 0.

[0180] In some embodiments, n is 1 or 2 and each R4is independently selected from Ci-Ce alkyl, halo, oxo, Ci-Ce haloalkyl, C3-C6 cycloalkyl, cyano, and -(CH2)q-Y, wherein q is 1, 2, or 3, and Y is selected from hydroxy, amino, C1-C4 alkoxy, C1-C4 alkylsulfonyl, and C1-C4 alkylsulfinyl. In some embodiments, n is 1 or 2 and each R4is independently selected from methyl, ethyl, isopropyl, tert-butyl, cyclopropyl, trifluoromethyl, cyano, -CH2OH, -CH2OCH3, -CH2NH2, -CH2NHCH3, -CH2N(CH3)2, -CH2S(O)CH3, -CH2S(O)2CH3, and -CH2C(O)CH3. In some embodiments, n is 1 or 2 and each R4is independently selected from C1-C3 alkyl, halo, and oxo. In some embodiments, n is 1 and R4is fluoro, methyl, or oxo. In some embodiments, n is 2 and each R4is halo (e.g„ fluoro), C1-C3 alkyl (e.g„ methyl), or oxo.

[0181] In some embodiments, n is 2, and the two R4are taken together to form a bond or are taken together with the atom(s) to which they are attached to form a ring. In some embodiments, n is 2, and the two R4are taken together to form a bond. In embodiments in which two R4are taken together to form a bond, the bond can result in a fused ring system as shown below:

[0182]

[0183] In some embodiments, n is 2, and the two R4are taken together with the atom(s) to which they are attached to form a ring. In such embodiments, the bond can result in a bicyclic ring system as shown below:

[0184]

[0185] In some embodiments, n is 2, and the two R4, together with the atom(s) to which they are attached, are taken together to form a 3-, 4-, or 5-membered cycloalkyl or a heterocyclylSTDU2-44700.601

[0186] having one heteroatom selected from N and O, wherein the cycloalkyl or heterocyclyl is unsubstituted or substituted with one Ci-Ce alkyl. In some embodiments, the two R4, together with the atom(s) to which they are attached, are taken together to form a cyclopropyl, a cyclobutyl, a cyclopentyl, an oxetanyl, or an azetidinyl ring, wherein the ring is unsubstituted or substituted with one methyl group.

[0187] In some embodiments, n is 3, and each R4is independently selected from Ci-Ce alkyl. In some embodiments, each R4is methyl.

[0188] In some embodiments, R3is hydrogen.

[0189]

[0190] STDU2-44700.601

[0191]

[0192] M2

[0193] XR4)n

[0194] In some embodiments, the group

[0195]

[0196] r5isH.

[0197] In some embodiments, Z is CR6aR6b. In some embodiments, R6aand R6bare each hydrogen. In some embodiments, R6ais hydrogen and R6bis methyl. In some embodiments, R6aand R6bare taken together with the carbon atom to which they are attached to form a C3-C4 cycloalkyl ring. In some embodiments, Z is O. In some embodiments, Z is NH.

[0198] In some embodiments, the compound of formula (I) is a compound of formula (la):STDU2-44700.601

[0199]

[0200] wherein R1, R2b, R3b, R3c, p1, p2, n, R4, and R5have any of the meanings defined or described herein for formula (I).

[0201] In some embodiments, the compound of formula (I) is selected from:

[0202]

[0203] STDU2-44700.601

[0204]

[0205] STDU2-44700.601

[0206] a

[0207]

[0208] ndH, and pharmaceutically acceptable salts thereof.

[0209] Additional compounds of formula (I) include:

[0210]

[0211] STDU2-44700.601

[0212]

[0213] and pharmaceutically acceptable salts thereof.

[0214] Certain compounds described herein may have at least one asymmetric center.

[0215] Additional asymmetric centers may be present depending upon the nature of the various substituents on the molecule. Compounds with asymmetric centers give rise to enantiomers (optical isomers), diastereomers (configurational isomers) or both, and it is intended that all of the possible enantiomers and diastereomers, in mixtures and as pure or partially purified compounds, are included within the scope of this disclosure.

[0216] The independent syntheses of the enantiomerically or diastereomerically enriched compounds, or their chromatographic separations, may be achieved as known in the art by appropriate modification of the methodology disclosed herein. Their absolute stereochemistry may be determined by the x-ray crystallography of crystalline products or crystalline intermediates that are derivatized, if necessary, with a reagent containing an asymmetric center of known absolute configuration.

[0217] If desired, racemic mixtures of the compounds may be separated so that the individual enantiomers are isolated. The separation can be carried out by methods well known in the art, such as the coupling of a racemic mixture of compounds to an enantiomerically pure compound to form a diastereomeric mixture, followed by separation of the individual diastereomers by standard methods, such as fractional crystallization or chromatography. The coupling reaction is often the formation of salts using an enantiomerically pure acid or base. The diastereomeric derivatives may then be converted to the pure enantiomers by cleavage of the added chiral residue. The racemic mixture of the compounds can also be separated directly by chromatographic methods using chiral stationary phases, which methods are well known in the art. Alternatively, any enantiomer of a compound may be obtained by stereoselective synthesis using optically pure starting materials or reagents of known configuration by methods well known in the art.STDU2-44700.601

[0218] Compounds may also possess tautomeric forms, and all tautomers also constitute embodiments of the disclosure.

[0219] Compounds described herein may also exhibit atropisomerism. Atropisomers are conformers that arise from restricted rotation about a single bond, and individual atropisomers can be isolated as separate chemical species. Examples of compounds exhibiting atropisomerism are ortho-substituted biphenyl and 1,1,2,2-re / ra-tert-butylethane. Compounds disclosed herein may exhibit atropisomerism about the single bond marked with an asterisk in the depiction of formula (I) below. When a compound disclosed herein is shown without identifying the specific atropisomer, both atropisomers are included. Certain exemplary compounds described herein may be separated into their individual atropisomers, and in some embodiments, one atropisomer may have increased potency compared to the other. All atropisomers and mixtures thereof are within the scope of this disclosure.

[0220] R1

[0221]

[0222] R5

[0223] The present disclosure also includes an isotopically-labeled compound, which is identical to those recited in formula (I), but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion in the compounds of the invention are hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as, but not limited to2H,3H,13C,14C,15N,18O,170,31P,32P,35S,1SF, and36C1, respectively. Substitution with heavier isotopes such as deuterium (2H) can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. The compound may incorporate positron-emitting isotopes for medical imaging and positron-emitting tomography (PET) studies for determining the distribution of receptors. Suitable positron-emitting isotopes that can be incorporated in compounds of formula (I) arenC,13N,15O, and1SF. Isotopically-labeled compounds of formula (I) canSTDU2-44700.601

[0224] generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples using appropriate isotopically-labeled reagent in place of non-isotopically-labeled reagent.

[0225] Compounds disclosed herein can exist in solvated as well as unsolvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like, and it is intended that the disclosure encompass both solvated and unsolvated forms. In one embodiment, the compound is amorphous. In one embodiment, the compound is a single polymorph. In another embodiment, the compound is a mixture of polymorphs. In another embodiment, the compound is in a crystalline form.

[0226] a. Methods of Synthesis

[0227] Compounds disclosed herein can be prepared by a variety of methods, including those illustrated in the Examples.

[0228] Compounds and intermediates may be isolated and purified by methods well-known to those skilled in the art of organic synthesis. Examples of conventional methods for isolating and purifying compounds can include, but are not limited to, chromatography on solid supports such as silica gel, alumina, or silica derivatized with alkylsilane groups, by recrystallization at high or low temperature with an optional pretreatment with activated carbon, thin-layer chromatography, distillation at various pressures, sublimation under vacuum, and trituration, as described for instance in “Vogel's Textbook of Practical Organic Chemistry,” 5th edition (1989), by Fumiss, Hannaford, Smith, and Tatchell, pub. Longman Scientific & Technical, Essex CM202JE, England.

[0229] Reaction conditions and reaction times for each individual step can vary depending on the particular reactants employed and substituents present in the reactants used. Reactions can be worked up in a conventional manner, e.g., by eliminating the solvent from the residue and further purified according to methodologies generally known in the art such as, but not limited to, crystallization, distillation, extraction, trituration and chromatography. Unless otherwise described, the starting materials and reagents are either commercially available or can be prepared by one skilled in the art from commercially available materials using methods described in the chemical literature.

[0230] Standard experimentation, including appropriate manipulation of the reaction conditions, reagents and sequence of the synthetic route, protection of any chemical functionality that cannot be compatible with the reaction conditions, and deprotection at a suitable point in the reaction sequence of the method are included in the scope of the disclosure. Suitable protecting groups and the methods for protecting and deprotectingSTDU2-44700.601

[0231] different substituents using such suitable protecting groups are well known to those skilled in the art; examples of which can be found in PGM Wuts and TW Greene, in Greene's book titled Protective Groups in Organic Synthesis (4thed.), John Wiley & Sons, NY (2006).

[0232] When an optically active form of a disclosed compound is required, it can be obtained by carrying out one of the procedures described herein using an optically active starting material (prepared, for example, by asymmetric induction of a suitable reaction step), or by resolution of a mixture of the stereoisomers of the compound or intermediates using a standard procedure (such as chromatographic separation, recrystallization, or enzymatic resolution).

[0233] Similarly, when a pure geometric isomer of a compound is required, it can be obtained by carrying out one of the procedures described herein using a pure geometric isomer as a starting material, or by resolution of a mixture of the geometric isomers of the compound or intermediates using a standard procedure such as chromatographic separation.

[0234] The synthetic schemes and specific examples as described arc illustrative and arc not to be read as limiting the scope of the disclosure or the claims. Alternatives, modifications, and equivalents of the synthetic methods and specific examples are contemplated.

[0235] b. Pharmaceutically Acceptable Salts

[0236] The disclosed compounds may exist as pharmaceutically acceptable salts. The term “pharmaceutically acceptable salt” refers to salts or zwitterions of the compounds which are water or oil-soluble or dispersible, suitable for treatment of disorders without undue toxicity, irritation, and allergic response, commensurate with a reasonable benefit / risk ratio and effective for their intended use. The salts may be prepared during the final isolation and purification of the compounds or separately by reacting an amino group of the compound with a suitable acid. For example, a compound may be dissolved in a suitable solvent, such as but not limited to methanol and water and treated with at least one equivalent of an acid, like hydrochloric acid. The resulting salt may precipitate out and be isolated by filtration and dried under reduced pressure. Alternatively, the solvent and excess acid may be removed under reduced pressure to provide a salt. Representative salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, isethionate, fumarate, lactate, maleate, methanesulfonate, naphthylenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, oxalate, maleate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, glutamate, para-toluenesulfonate, undecanoate, hydrochloric, hydrobromic, sulfuric, phosphoric, and theSTDU2-44700.601

[0237] like. Amino groups of the compounds may also be quatemized with alkyl chlorides, bromides and iodides such as methyl, ethyl, propyl, isopropyl, butyl, lauryl, myristyl, stearyl and the like.

[0238] Basic addition salts may be prepared during the final isolation and purification of the disclosed compounds by reaction of a carboxyl group with a suitable base such as the hydroxide, carbonate, or bicarbonate of a metal cation such as lithium, sodium, potassium, calcium, magnesium, or aluminum, or an organic primary, secondary, or tertiary amine. Quaternary amine salts can be prepared, such as those derived from methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N, N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N, N-dibenzylphenethylamine, 1 -ephenamine, N, N’ -dibenzylethylenediamine, ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like.

[0239] Pharmaceutical C

[0240]

[0241] The disclosed compounds may be incorporated into pharmaceutical compositions suitable for administration to a subject (such as a patient, which may be a human or nonhuman). The pharmaceutical compositions may include pharmaceutically acceptable carriers. The term “pharmaceutically acceptable carrier,” as used herein, means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as, but not limited to, lactose, glucose and sucrose; starches such as, but not limited to, corn starch and potato starch; cellulose and its derivatives such as, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as, but not limited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols; such as propylene glycol; esters such as, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents such as, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.STDU2-44700.601

[0242] Thus, the compounds and their pharmaceutically acceptable salts may be formulated for administration by, for example, solid dosing, eye drop, in a topical oil-based formulation, injection, inhalation (either through the mouth or the nose), implants, or oral, buccal, parenteral, or rectal administration. Techniques and formulations may generally be found in “Remington’s Pharmaceutical Sciences,’’ (Meade Publishing Co., Easton, Pa.). Therapeutic compositions must typically be sterile and stable under the conditions of manufacture and storage.

[0243] The route by which the disclosed compounds are administered and the form of the composition will dictate the type of carrier to be used. The composition may be in a variety of forms, suitable, for example, for systemic administration (e.g., oral, rectal, nasal, sublingual, buccal, implants, or parenteral) or topical administration (e.g., dermal, pulmonary, nasal, aural, ocular, liposome delivery systems, or iontophoresis).

[0244] Carriers for systemic administration typically include at least one of diluents, lubricants, binders, disintegrants, colorants, flavors, sweeteners, antioxidants, preservatives, glidants, solvents, suspending agents, wetting agents, surfactants, combinations thereof, and others. All carriers are optional in the compositions.

[0245] Suitable diluents include sugars such as glucose, lactose, dextrose, and sucrose; diols such as propylene glycol; calcium carbonate; sodium carbonate; sugar alcohols, such as glycerin; mannitol; and sorbitol. The amount of diluent(s) in a systemic or topical composition is typically about 50 to about 90% by weight of the composition.

[0246] Suitable lubricants include silica, talc, stearic acid and its magnesium salts and calcium salts, calcium sulfate; and liquid lubricants such as polyethylene glycol and vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, com oil and oil of theobroma. The amount of lubricant(s) in a systemic or topical composition is typically about 5 to about 10% by weight of the composition.

[0247] Suitable binders include polyvinyl pyrrolidone; magnesium aluminum silicate; starches such as corn starch and potato starch; gelatin; tragacanth; and cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, methylcellulose, microcrystalline cellulose, and sodium carboxymethylcellulose. The amount of binder(s) in a systemic composition is typically about 5 to about 50% by weight of the composition.

[0248] Suitable disintegrants include agar, alginic acid and the sodium salt thereof, effervescent mixtures, croscarmellose, crospovidone, sodium carboxymethyl starch, sodium starch glycolate, clays, and ion exchange resins. The amount of disintegrant(s) in a systemic or topical composition is typically about 0.1 to about 10% by weight of the composition.STDU2-44700.601

[0249] Suitable colorants include a colorant such as an FD& C dye. When used, the amount of colorant in a systemic or topical composition is typically about 0.005 to about 0.1% by weight of the composition.

[0250] Suitable flavors include menthol, peppermint, and fruit flavors. The amount of flavor(s), when used, in a systemic or topical composition is typically about 0.1 to about 1.0%.

[0251] Suitable sweeteners include aspartame and saccharin. The amount of sweetener(s), when used, in a systemic or topical composition is typically about 0.001 to about 1 % by weight of the composition.

[0252] Suitable antioxidants include butylated hydroxyanisole (“BHA”), butylated hydroxytoluene (“BHT”), and vitamin E. The amount of antioxidant(s) in a systemic or topical composition is typically about 0.1 to about 5% by weight of the composition.

[0253] Suitable preservatives include benzalkonium chloride, methyl paraben, and sodium benzoate. The amount of preservative(s) in a systemic or topical composition is typically about 0.01 to about 5% by weight of the composition.

[0254] Suitable glidants include silicon dioxide. The amount of glidant(s) in a systemic or topical composition is typically about 1 to about 5% by weight of the composition.

[0255] Suitable solvents include water, isotonic saline, ethyl oleate, glycerin, hydroxylated castor oils, alcohols such as ethanol, and phosphate buffer solutions. The amount of solvent(s) in a systemic or topical composition is typically from about 0 to about 100% by weight of the composition.

[0256] Suitable suspending agents include AVICEL RC-591 (from FMC Corporation of Philadelphia, PA) and sodium alginate. The amount of suspending agent(s) in a systemic or topical composition is typically about 1 to about 8% by weight of the composition.

[0257] Suitable surfactants include lecithin. Polysorbate 80, and sodium lauryl sulfate, and the TWEENS from Atlas Powder Company of Wilmington, Delaware. Suitable surfactants include those disclosed in the C. T. F. A. Cosmetic Ingredient Handbook, 1992, pp.587-592; Remington’s Pharmaceutical Sciences, 15th Ed. 1975, pp. 335-337; and McCutcheon’s Volume 1, Emulsifiers & Detergents, 1994, North American Edition, pp. 236-239. The amount of surfactant(s) in the systemic or topical composition is typically about 0.1% to about 5% by weight of the composition.

[0258] Although the amounts of components in the systemic compositions may vary depending on the type of systemic composition prepared, in general, systemic compositions include 0.01% to 50% by weight of an active compound and 50% to 99.99% by weight ofSTDU2-44700.601

[0259] one or more carriers. Compositions for parenteral administration typically include 0.1% to 10% by weight of actives and 90% to 99.9% by weight of a carrier including a diluent and a solvent.

[0260] Compositions for oral administration can have various dosage forms. For example, solid forms include tablets, capsules, granules, and bulk powders. These oral dosage forms include a safe and effective amount, usually at least about 5% by weight, and more particularly from about 25% to about 50% by weight of actives. The oral dosage compositions include about 50% to about 95% by weight of carriers, and more particularly, from about 50% to about 75% by weight.

[0261] Tablets can be compressed, tablet triturates, enteric-coated, sugar-coated, film-coated, or multiple-compressed. Tablets typically include an active component, and a carrier comprising ingredients selected from diluents, lubricants, binders, disintegrants, colorants, flavors, sweeteners, glidants, and combinations thereof. Specific diluents include calcium carbonate, sodium carbonate, mannitol, lactose and cellulose. Specific binders include starch, gelatin, and sucrose. Specific disintegrants include alginic acid and croscarmellose. Specific lubricants include magnesium stearate, stearic acid, and talc. Specific colorants are the FD& C dyes, which can be added for appearance. Chewable tablets preferably contain sweeteners such as aspartame and saccharin, or flavors such as menthol, peppermint, fruit flavors, or a combination thereof.

[0262] Capsules (including implants, time release and sustained release formulations) typically include an active compound (e.g., a compound of formula (I)), and a carrier including one or more diluents disclosed above in a capsule comprising gelatin. Granules typically comprise a disclosed compound, and preferably glidants such as silicon dioxide to improve flow characteristics. Implants can be of the biodegradable or the non-biodegradable type.

[0263] The selection of ingredients in the carrier for oral compositions depends on secondary considerations like taste, cost, and shelf stability, which are not critical for the purposes of this disclosure.

[0264] Solid compositions may be coated by conventional methods, typically with pH or time-dependent coatings, such that a disclosed compound is released in the gastrointestinal tract in the vicinity of the desired application, or at various points and times to extend the desired action. The coatings typically include one or more components selected from the group consisting of cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropylSTDU2-44700.601

[0265] methyl cellulose phthalate, ethyl cellulose, EUDRAGIT® coatings (available from Evonik Industries of Essen, Germany), waxes and shellac.

[0266] Compositions for oral administration can have liquid forms. For example, suitable liquid forms include aqueous solutions, emulsions, suspensions, solutions reconstituted from non-effervescent granules, suspensions reconstituted from non-effervescent granules, effervescent preparations reconstituted from effervescent granules, elixirs, tinctures, syrups, and the like. Liquid orally administered compositions typically include a disclosed compound and a carrier, namely, a carrier selected from diluents, colorants, flavors, sweeteners, preservatives, solvents, suspending agents, and surfactants. Peroral liquid compositions preferably include one or more ingredients selected from colorants, flavors, and sweeteners.

[0267] Other compositions useful for attaining systemic delivery of the subject compounds include sublingual, buccal and nasal dosage forms. Such compositions typically include one or more of soluble filler substances such as diluents including sucrose, sorbitol and mannitol; and binders such as acacia, microcrystalline cellulose, carboxymethyl cellulose, and hydroxypropyl methylcellulose. Such compositions may further include lubricants, colorants, flavors, sweeteners, antioxidants, and glidants.

[0268] The disclosed compounds can be topically administered. Topical compositions that can be applied locally to the skin may be in any form including solids, solutions, oils, creams, ointments, gels, lotions, shampoos, leave-on and rinse-out hair conditioners, milks, cleansers, moisturizers, sprays, skin patches, and the like. Topical compositions include: a disclosed compound (e.g., a compound of formula (I)), or a pharmaceutically acceptable salt thereof), and a carrier. The carrier of the topical composition preferably aids penetration of the compounds into the skin. The carrier may further include one or more optional components.

[0269] The amount of the carrier employed in conjunction with a disclosed compound is sufficient to provide a practical quantity of composition for administration per unit dose of the compound. Techniques and compositions for making dosage forms useful in the methods of this disclosure are described in the following references: Modem Pharmaceutics, Chapters 9 and 10, Banker & Rhodes, eds. (1979); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms, 2nd Ed., (1976).

[0270] A carrier may include a single ingredient or a combination of two or more ingredients. In the topical compositions, the carrier includes a topical carrier. Suitable topical carriers include one or more ingredients selected from phosphate buffered saline, isotonic water, deionized water, monofunctional alcohols, symmetrical alcohols, aloe vera gel, allantoin, glycerin, vitamin A and E oils, mineral oil, propylene glycol, PPG-2 myristyl propionate,STDU2-44700.601

[0271] dimethyl isosorbide, castor oil, combinations thereof, and the like. More particularly, carriers for skin applications include propylene glycol, dimethyl isosorbide, and water, and even more particularly, phosphate buffered saline, isotonic water, deionized water, monofunctional alcohols, and symmetrical alcohols.

[0272] The carrier of a topical composition may further include one or more ingredients selected from emollients, propellants, solvents, humectants, thickeners, powders, fragrances, pigments, and preservatives, all of which are optional.

[0273] Suitable emollients include stearyl alcohol, glyceryl monoricinoleate, glyceryl monostearate, propane- 1,2-diol, butane- 1,3-diol, mink oil, cetyl alcohol, isopropyl isostearate, stearic acid, isobutyl palmitate, isocetyl stearate, oleyl alcohol, isopropyl laurate, hexyl laurate, decyl oleate, octadecan-2-ol, isocetyl alcohol, cetyl palmitate, di-n-butyl sebacate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, butyl stearate, polyethylene glycol, triethylene glycol, lanolin, sesame oil, coconut oil, arachis oil, castor oil, acetylated lanolin alcohols, petroleum, mineral oil, butyl myristate, isostearic acid, palmitic acid, isopropyl linoleate, lauryl lactate, myristyl lactate, decyl oleate, myristyl myristate, and combinations thereof. Specific emollients for skin include stearyl alcohol and polydimethylsiloxane. The amount of emollient(s) in a skin-based topical composition is typically about 5% to about 95% by weight of the composition.

[0274] Suitable propellants include propane, butane, isobutane, dimethyl ether, carbon dioxide, nitrous oxide, and combinations thereof. The amount of propellant(s) in a topical composition is typically about 0% to about 95% by weight of the composition.

[0275] Suitable solvents include water, ethyl alcohol, methylene chloride, isopropanol, castor oil, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, dimethylsulfoxide, dimethyl formamide, tetrahydrofuran, and combinations thereof. Specific solvents include ethyl alcohol and homotopic alcohols. The amount of solvent(s) in a topical composition is typically about 0% to about 95% by weight of the composition.

[0276] Suitable humectants include glycerin, sorbitol, sodium 2-pyrrolidone-5 -carboxylate, soluble collagen, dibutyl phthalate, gelatin, and combinations thereof. Specific humectants include glycerin. The amount of humect ant(s) in a topical composition is typically 0% to 95% by weight of the composition.

[0277] The amount of thickener(s) in a topical composition is typically about 0% to about 95% by weight of the composition.STDU2-44700.601

[0278] Suitable powders include beta-cyclodextrins, hydroxypropyl cyclodextrins, chalk, talc, fullers earth, kaolin, starch, gums, colloidal silicon dioxide, sodium polyacrylate, tetra alkyl ammonium smectites, trialkyl aryl ammonium smectites, chemically-modified magnesium aluminum silicate, organically-modified montmorillonite clay, hydrated aluminum silicate, fumed silica, carboxy vinyl polymer, sodium carboxymethyl cellulose, ethylene glycol monostearate, and combinations thereof. The amount of powder(s) in a topical composition is typically 0% to 95% by weight of the composition.

[0279] The amount of fragrance in a topical composition is typically about 0% to about 0.5%, particularly, about 0.001% to about 0.1% by weight of the composition.

[0280] Suitable pH adjusting additives include HC1 or NaOH in amounts sufficient to adjust the pH of a topical pharmaceutical composition.

[0281] Methods of Use

[0282] The compounds disclosed herein have TRIM21 -dependent anticancer activity.

[0283] Accordingly, the compounds and pharmaceutical compositions disclosed herein can be used for treatment of proliferative diseases such as cancer, including TRIM21 -dependent cancers.

[0284] Accordingly, in some embodiments, the disclosure provides a method of treating a proliferative disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound described herein (e.g., a compound of formula (I), or a pharmaceutically acceptable salt thereof), or a pharmaceutical composition described herein (e.g., a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof). In some embodiments, the proliferative disease is associated with TRIM21. In some embodiments, the proliferative disease is associated with a mutation in and / or increased activity of TRIM21, e.g., due to increased expression of TRIM21. In some embodiments, the proliferative disease is cancer (e.g., a TRIM21-associated cancer). In some embodiments, the cancer is pancreatic cancer, head and neck cancer, breast cancer, colorectal cancer, lung cancer, melanoma, ovarian cancer, kidney cancer, multiple myeloma, esophageal cancer, gastric cancer, liver cancer, bile duct cancer, leukemia, lymphoma, prostate cancer, and sarcoma. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is head and neck cancer.

[0285] In some embodiments, the disclosed methods comprise detecting and / or quantifying the amount of TRIM21 in a sample obtained from the subject, and administering a therapeutically effective amount of a compound disclosed herein, or a pharmaceuticallySTDU2-44700.601

[0286] acceptable salt thereof, based on the presence or amount of TRIM21 detected and / or quantified in the sample.

[0287] In some embodiments, the methods comprise comparing the amount of TRIM21 to a reference level and identifying the subject as having a TRIM21 -associated cancer when the amount of TRIM21 is greater than or equal to the reference level.

[0288] “Reference level” as used herein refers to a value that is used to assess diagnostic, prognostic, or therapeutic efficacy and that has been linked or is associated herein with various clinical parameters (e.g., TRIM21 -associated cancer). It is well-known that reference levels may vary depending on the nature of the assay used to determine the concentration, level, or amount of the analyte (e.g., antibodies employed, reaction conditions, sample purity, etc.) and that assays can be compared and standardized. It further is well within the ordinary skill of one in the art to adapt the disclosure herein for other assays to obtain assay-specific reference levels for those other assays based on the description provided by this disclosure. Whereas the precise value of the reference level may vary between assays, the findings as described herein should be generally applicable and capable of being extrapolated to other assays. The reference level may be based on positive controls from individuals identified as having a TRIM21 -associated cancer. Alternatively, the reference level may be based on negative controls from a healthy subject (e.g., a subject not diagnosed with TRIM21-associated cancer).

[0289] In some embodiments, the reference level is the reference level of TRIM21 in a sample or compiled samples from a subject(s) known to be afflicted with TRIM21 -associated cancer. For example, a reference value may be based on the lowest value for the level of TRIM21 in known subjects with TRIM21 -associated cancer, such that values for the level of TRIM21 equal to or higher than that value are indicative of TRIM21 -associated cancer. Alternatively, in some embodiments, the reference level is the reference level of TRIM21 in a sample or compiled samples from a subject(s) known to not have TRIM21 -associated cancer. For example, a reference value may be based on the highest value for the level of TRIM21 in known subjects without a TRIM21 -associated cancer (e.g., healthy subjects or subjects with cancer not associated with TRIM21), such that values for the level of TRIM21 higher than that value are indicative of TRIM21 -associated cancer.

[0290] The “sample” broadly refers to any material, biological fluid, tissue, or cell obtained or otherwise derived from a subject. This includes blood and blood products (including whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, and serum), mucosal biopsy tissue and brushed cells, sputum, tears, mucus, nasal washes, nasal aspirate,STDU2-44700.601

[0291] breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, lymph fluid, nipple aspirate, bronchial aspirate (e.g., bronchoalveolar lavage), bronchial brushing, synovial fluid, joint aspirate, organ secretions, cells, a cellular extract, and cerebrospinal fluid. This also includes experimentally separated fractions of all of the foregoing. For example, a blood sample can be fractionated into serum, plasma, or into fractions containing particular types of blood cells, such as red blood cells or white blood cells (leukocytes). In some embodiments, a sample can be a combination of samples from an individual, such as a combination of a tissue and fluid sample. The term “sample” also includes materials containing homogenized solid material, such as from a stool sample, a tissue sample, or a tissue biopsy, for example. In some embodiments, the biological sample may comprise tumor tissue, suspected tumor tissue, or lymph node tissue. Any suitable methods for obtaining a biological sample can be employed; exemplary methods include, e.g., phlebotomy, swab (e.g., buccal swab), and a fine needle aspirate biopsy procedure. Exemplary tissues susceptible to fine needle aspiration include lymph node, lung, lung washes, BAL (bronchoalveolar lavage), thyroid, breast, pancreas, and liver. Samples can also be collected, e.g., by micro dissection (e.g., laser capture micro dissection (LCM) or laser micro dissection (LMD)), bladder wash, smear (e.g., a PAP smear), or ductal lavage.

[0292] In some embodiments, the methods further comprise obtaining the sample(s) from the subject. The sample(s) can be obtained using techniques known to those skilled in the art, and the sample(s) may be used directly as obtained from the source or following a pretreatment to modify the character of the sample. Such pretreatment may include, for example, preparing plasma from blood, diluting viscous fluids, filtration, precipitation, dilution, distillation, mixing, concentration, inactivation of interfering components, the addition of reagents, lysing, and the like. It will be appreciated that obtaining a sample from a subject may comprise extracting the sample directly from the subject or receiving the biological sample from a third party.

[0293] The sample(s) may be obtained in a medical facility, e.g., at an Emergency Room, urgent care clinic, walk-in clinic, a long term care facility, or another appropriate site of medical practice. The sample(s) may be obtained in a home or residential setting (e.g., a senior living or hospice setting) or during transportation to a medical facility (e.g., ambulance).

[0294] The present disclosure is not limited by the type of assay used to detect and / or quantify the level of TRIM21. The nature of methods and the test can be any assay known in the art such as, for example, immunoassays, point-of-care assays, clinical chemistry assay,STDU2-44700.601

[0295] protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis, or protein immunostaining, a protein assay, a competitive binding assay, a lateral flow assay, a functional protein assay, or chromatography or spectrometry methods, such as high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS). Also, the assay can be employed in a clinical chemistry format such as would be known by one of ordinary skill in the art.

[0296] i. Dosages

[0297] It will be appreciated that appropriate dosages of the compounds, and compositions comprising the compounds, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments described herein. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and / or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient. The amount of compound and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects.

[0298] Administration in vivo can be effected in one dose, continuously or intermittently (e.g. in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be earned out with the dose level and pattern being selected by the treating physician.

[0299] ii. Combination Therapies

[0300] A compound or composition described herein may be used in combination with other known therapies. Administered “in combination,” as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject’s affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms ofSTDU2-44700.601

[0301] administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

[0302] A compound or composition described herein and the at least one additional therapeutic agent can be administered simultaneously, in the same or in separate compositions, or sequentially. For sequential administration, the compound described herein can be administered first, and the additional agent can be administered subsequently, or the order of administration can be reversed.

[0303] In some embodiments, a compound or composition described herein is administered in combination with at least one of chemotherapy, surgery, radiation therapy, hormone therapy, immunotherapy, cryotherapy, T cell transfer therapy, and thermotherapy, or any combination thereof. In some embodiments a compound or composition described herein is administered in combination with two or more of chemotherapy, surgery, radiation therapy, hormone therapy, immunotherapy, cryotherapy, T cell transfer therapy, and thermotherapy. For example, in some embodiments, a compound or composition described herein is administered in combination with an immunotherapy, such as an immune checkpoint inhibitor. In some embodiments, a compound or composition described herein is administered in combination with a T cell transfer therapy, such as CAR T-cell therapy.

[0304] In some embodiments, a compound or composition described herein is used in combination with chemotherapy. In some embodiments, a compound or composition described herein is used in combination with a chemotherapeutic agent identified on the “A to Z List of Cancer Drugs” published by the National Cancer Institute.

[0305] KitsSTDU2-44700.601

[0306] For use in the therapeutic applications described herein, kits and articles of manufacture are also provided, which include a compound or pharmaceutical composition described herein (e.g., a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof ). In some embodiments, such kits comprise a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers are formed from a variety of materials such as glass or plastic.

[0307] The articles of manufacture provided herein contain packaging materials. Packaging materials for use in packaging pharmaceutical products include those found in, e.g., U. S. Patent Nos. 5,323,907, 5,052,558 and 5,033,252. Examples of pharmaceutical packaging materials include, but arc not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment. For example, in some embodiments the container(s) includes a compound of formula (I), or a pharmaceutically acceptable salt thereof, optionally in a composition or in combination with another agent as disclosed herein. The container(s) optionally have a sterile access port (for example the container is an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Such kits optionally comprising a compound with an identifying description or label or instructions relating to its use in the methods described herein.

[0308] For example, a kit typically includes one or more additional containers, each with one or more of various materials (such as reagents, optionally in concentrated form, and / or devices) desirable from a commercial and user standpoint for use of a compound described herein. Non-limiting examples of such materials include, but not limited to, buffers, diluents, filters, needles, syringes; carrier, package, container, vial and / or tube labels listing contents and / or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included. A label is optionally on or associated with the container. For example, a label is on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself, a label is associated with a container when it is present within a receptacle or earner that also holds the container, e.g., as a package insert. In addition, a label is used to indicate that the contents are to be used for aSTDU2-44700.601

[0309] specific therapeutic application. In addition, the label indicates directions for use of the contents, such as in the methods described herein. In certain embodiments, the pharmaceutical composition is presented in a pack or dispenser device which contains one or more unit dosage forms containing a compound provided herein. The pack, for example, contains metal or plastic foil, such as a blister pack. Or, the pack or dispenser device is accompanied by instructions for administration. Or, the pack or dispenser is accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, is the labeling approved by the U. S. Food and Drug Administration for prescription drugs, or the approved product insert. In some embodiments, compositions containing a compound provided herein formulated in a compatible pharmaceutical carrier are prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

[0310] Examples

[0311] All solvents used were commercially available and were used without further purification. Reactions were typically run using anhydrous solvents under an inert atmosphere of nitrogen. Proton NMR spectra were recorded using a Bruker plus 400 NMR Spectrometer unless stated otherwise. All deuterated solvents contained typically 0.03% to 0.05% v / v tetramethylsilane, which was used as the reference signal (set at 50.00 for both

[0312]

[0313] and13C). Preparative reverse-phase HPLC was performed using Varian HPLC system. The column used was XB ridge Prep C18 OBD Column, 5 pm, 19 x 150 mm. The instrument using reverse-phase conditions (acetonitrile I water, containing 0.1 % Ammonium hydrogen carbonate or formic acid).

[0314] Example 1

[0315] Compound Syntheses and Characterization

[0316] Synthesis of 2-(l-(tert-butoxycarbonyl)-2,2-dimethylpiperidin-4-yl) acetic acid

[0317]

[0318] Boo Boc Boc Step 1: tert-butyl 4-(2-ethoxy-2-oxoethylidene)-2,2-dimethylpiperidine-l -carboxylate. To a stirred solution of triethyl phosphonoacetate (473 mg, 2.11 mmol, 1.60STDU2-44700.601

[0319] equiv) in tetrahydrofuran (3 mL) was added n-butyllithium (126 mg, 1.98 mmol, 1.50 equiv) drop wise at -78 °C under nitrogen atmosphere. The resulting mixture was stirred at -78 °C for 45 min under nitrogen atmosphere. To the above mixture was added tert-butyl 2,2-dimethyl-4-oxopiperidine-l -carboxylate (300 mg, 1.32 mmol, 1.00 equiv) in tetrahydrofuran (2 mL) dropwise at -78 °C. The resulting mixture was slowly warmed to room temperature and stirred for additional 16 h. The resulting mixture was poured into water and extracted with ethyl acetate. After washed with brine, dried over anhydrous sodium sulfate and filtered, the filtrate was concentrated to get tert-butyl 4-(2-ethoxy-2-oxoethylidene)-2,2-dimethylpiperidine- 1 -carboxylate (400 mg, ciude product) which was used directly into next step. MS (ESIpos): m / z = 298.

[0320] Step 2: tert-butyl 4-(2-ethoxy-2-oxoethyl)-2,2-dimethylpiperidine-l -carboxylate. A mixture of tert-butyl 4-(2-ethoxy-2-oxoethylidene)-2,2-dimethylpiperidine-l -carboxylate (400 mg, 1.34 mmol, 1.00 equiv) and Pd / C (10%, 580 mg, 5.45 mmol, 4.05 equiv) in ethanol (6 mL) was stirred at room temperature for overnight under hydrogen atmosphere. The resulting mixture was filtered, the filter cake was washed with ethanol. The filtrate was concentrated under reduced pressure to get tert-butyl 4-(2-ethoxy-2-oxoethyl)-2,2-dimethylpiperidine-l-carboxylate (460 mg, crude product). MS (ESIpos): m / z = 300.

[0321] Step 3: 2-(l-(tert-butoxycarbonyl)-2,2-dimethylpiperidin-4-yl) acetic acid. A mixture of tert-butyl 4-(2-ethoxy-2-oxoethyl)-2,2-dimethylpiperidine-l -carboxylate (430 mg, 1.44 mmol, 1.00 equiv) and lithium hydroxide (103 mg, 4.31 mmol, 3.00 equiv) in tetrahydrofuran (4 mL) and water (4 mL) was stirred at room temperature for overnight. The mixture was acidified to pH 6 with cone, hydrochloric acid. The resulting mixture was poured into water and extracted with ethyl acetate. After washed with brine, dried over anhydrous sodium sulfate and filtered, the filtrate was concentrated to get 2-[l-(tert-butoxycarbonyl)-2,2-dimethylpiperidin-4-yl] acetic acid (400 mg, crude product). MS (ESIpos): m / z = 272.

[0322] Synthesis of 3-(4-chloro-2-ethoxyphenyI)-6-fluoro-2-(piperidin-4-ylmethyl)quinazolin- 4(3H)-one (Compound 1)STDU2-44700.601

[0323]

[0324] Step 1: tert-butyl 4-((6-fluoro-4-oxo-4H-benzo[d][l,3]oxazin-2-yl)methyl)piperidine-1 -carboxylate. To a solution of 2-amino-5 -fluorobenzoic acid (500 mg, 3.223 mmol, 1 equiv) and [l-(tert-butoxycarbonyl)piperidin-4-yl]acetic acid (941 mg, 3.868 mmol, 1.2 equiv) in pyridine (8 mL) was added diphenyl phosphonate (3020 mg, 12.892 mmol, 4 equiv) and the resulting mixture was stirred for 3 h at 70 °C. The resulting mixture was concentrated under vacuum to get a residue. The residue was dissolved in ethyl acetate and washed with sat. sodium bicarbonate and brine in sequence. After dried over anhydrous sodium sulfate, filtered and concentrated, the crude product was purified via prep-TLC eluting with 30% ethyl acetate in petroleum ether to afford tert-butyl 4-[(6-fluoro-4-oxo-3,l -benzoxazin-2-yl)methyl]piperidine-1-carboxylate (800 mg, 68.49% yield, 75% purity). MS (ESIpos): m / z = 363 (M+H)+.

[0325] Step 2: tert-butyl 4-((3-(4-chloro-2-ethoxyphenyl)-6-fluoro-4-oxo-3,4-dihydroquinazolin-2-yl)methyl)piperidine-l -carboxylate. To a solution of tert-butyl 4-[(6-fluoro-4-oxo-3,l-benzoxazin-2-yl)methyl]piperidine-l -carboxylate (200 mg, 0.552 mmol, 1 equiv) and 4-chloro-2-ethoxyaniline (115 mg, 0.662 mmol, 1.2 equiv) in pyridine (4 mL) was added diphenyl phosphonate (516.98 mg, 2.208 mmol, 4 equiv) and the resulting mixture was stirred for 17 h at room temperature. The reaction mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with sat. sodium bicarbonate, sat. copper(TI) sulfate, dried over anhydrous sodium sulfate, filtered and concentrated to get a residue. The crude product was purified via prep-TLC eluting with 50% ethyl acetate in petroleum ether to get tert-butyl 4- { [3-(4-chloro-2-ethoxyphenyl)-6-fluoro-4-oxoquinazolin-2-yl]methyl}piperidine-l -carboxylate (200 mg, 70.23% yield, 90% purity). MS (ESIpos): m / z = 516 (M+H)+.

[0326] Step 3: 3-(4-chloro-2-ethoxyphenyl)-6-fluoro-2-(piperidin-4-ylmethyl)quinazolin-4-onc. To a solution of tert-butyl 4-{[3-(4-chloro-2-cthoxyphcnyl)-6-fluoro-4-oxoquinazolin-2-yl]methyl}piperidine-l -carboxylate (200 mg, 0.388 mmol, 1 equiv) in dichloromethane (4mL) was added trifluoroacetic acid (1 mL) and the reaction mixture was stirred for 2 h at room temperature. The extra solvent was evaporated and the crude product was purified by prep-STDU2-44700.601

[0327] HPLC with the following conditions: Column: XB ridge Prep OBD Cl 8 Column, 30*150 mm, 5pm; Mobile Phase A: Water(10mmol / L NH4HCO3), Mobile Phase B: ACN; Flow rate: 60 mL / min mL / min; Gradient (B%): 21% B to 51% B in 8min; Wave Length: 254nm / 220nm nm; RTl(min): 6.48 to get 3-(4-chloro-2-ethoxyphenyl)-6-fluoro-2-(piperidin-4- ylmethyl)quinazolin-4-one (123.1 mg, 76.37% yield, 99.9% purity) as a white solid. MS (ESIpos): m / z = 416.05 (M+H)+.'H-NMR (400 MHz, DMSO-rfe): 5 [ppm] = 7.78-7.68 (m, 3H), 7.46 (d, 1H), 7.36 (d, 1H), 7.19 (dd, 1H), 4.09 (q, 2H), 3.84 (br, s, 1H), 2.83-2.67 (m, 2H), 2.52-2.20 (m, 4H), 1.84-1.82 (m, 1H), 1.58-1.44 (m, 2H), 1.14 (t, 3H), 0.98-0.82 (m, 2H).

[0328] Synthesis of 3-(4-chloro-2-(2-(dimethylamino)ethoxy)phenyl)-6-fluoro-2-(piperidin-4- ylmethyl)quinazolin-4(3H)-one (Compound 16)

[0329]

[0330] Step 1: 2-(5-chloro-2-nitrophenoxy)-N, N-dimethylethan-l-amine. A mixture of 4- chloro-2-fluoro- 1 -nitrobenzene (3 g, 17.090 mmol, 1.00 equiv), dimethylaminoethanol (2.29 g, 25.635 mmol, 1.50 equiv) and CS2CO3 (11.14 g, 34.180 mmol, 2.00 equiv) in N, N- dimethylformamide (40 mL) was stirred at 60 °C for 2 h. The mixture was allowed to cool down to room temperature. The resulting mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to get |2-(5-chloro-2-nitrophenoxy) ethyl] dimethylamine (4.8 g, crude Product). LC-MS: MS (ESIpos): m / z = 245 (M)+.

[0331] Step 2: 4-chloro-2-(2-(dimethylamino)ethoxy)aniline. A mixture of [2-(5-chloro-2- nitrophenoxy) ethyl] dimethylamine (4.4 g, 17.983 mmol, 1.00 equiv) and SnCL (17.05 g,STDU2-44700.601

[0332] 89.915 mmol, 5.00 equiv) in ethanol (70 mL) was slirred at 70 °C for 2 h. The mixture was allowed to cool down to room temperature. The resulting mixture was concentrated under reduced pressure. The resulting mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to get 4-chloro-2-[2-(dimethylamino) ethoxy] aniline (2.6 g, crude Product). LC-MS: MS (ESIpos): m / z = 215 (M)+.

[0333] ii. Synthesis of 3-(4-chloro-2-(2-(dimethylamino)ethoxy)phenyl)-6-fluoro-2-(piperidin-4- ylmethyl)quinazolin-4(3H)-one (Compound 16)

[0334]

[0335] Step 1: tert-butyl 4-((6-fluoro-4-oxo-4H-benzo[d][l,3]oxazin-2-yl)methyl)piperidine-1 -carboxylate. To a solution of 2-amino-5-fluorobenzoic acid (3 g, 19.34 mmol, 1 equiv) and [l-(tert-butoxycarbonyl)piperidin-4-yl]acetic acid (5.65 g, 23.20 mmol, 1.2 equiv) in pyridine (40 mL) was added diphenyl phosphonate (18.12 g, 77.36 mmol, 4 equiv) and then the reaction mixture was stirred for 6 hours at 70°C. The resulting mixture was concentrated under vacuum. The residue was diluted with ethyl acetate. The combined organic layers were washed with NaHCCh (aq.), brine and then dried over anhydrous Na2SC>4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with ethyl acetate in petroleum ether from 0 to 30% to get tert-butyl 4- [(6-fluoro-4-oxo-3,l-benzoxazin-2-yl)methyl]piperidine-l -carboxylate (3.5 g, 49.94%yield, 80%purity). MS (ESIpos): m / z = 362.

[0336] Step 2: tert-butyl 4-((3-(4-chloro-2-(2-(dimethylamino)ethoxy)phenyl)-6-fluoro-4-oxo-3, 4-dihydroquinazolin-2-yl)methyl)piperidine-l -carboxylate. To a solution of tert-butyl 4-[(6-fluoro-4-oxo-3,l-benzoxazin-2-yl)methyl]piperidine-l-carboxylate (300 mg, 0.83 mmol, 1 equiv) and 4-chloro-2-[2-(dimethylamino)ethoxy]aniline (213 mg, 0.99 mmol, 1.2 equiv) in pyridine (12 mL) was added diphenyl phosphonate (775 mg, 3.31 mmol, 4 equiv) and then the reaction mixture was stirred for 20 hours at 70 °C. The resulting mixture was concentrated under vacuum. The resulting mixture was added NaHCCh (aq.) and then extracted with ethyl acetate. The combined organic layers were dried over anhydrous Na2SC>4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by prep-TLC (ethyl acetate) to get tert-butyl 4-[(3-{4-chloro-2-[2-(dimethylamino)ethoxy]phenyl}-6-fluoro-4-STDU2-44700.601

[0337] oxoquinazolin-2-yl)methyl]piperidine-l -carboxylate (400 mg, 86.43% yield, 85% purity). MS (ESIpos): m / z = 559.

[0338] Step 3: 3-(4-chloro-2-(2-(dimethylamino)ethoxy)phenyl)-6-fluoro-2-(piperidin-4-ylmethyl)quinazolin-4(3H)-one (Compound 16). To a solution of tert-butyl 4-[(3-{4-chloro-2-[2-(dimethylamino)ethoxy]phenyl}-6-fluoro-4-oxoquinazolin-2-yl)methyl]piperidine-l-carboxylate (250 mg, 0.40 mmol, 1 equiv, 90%) in DCM (4 mL) was added TFA (1 mL) and then the reaction mixture was stirred for 2 hours at room temperature. The resulting mixture was concentrated under vacuum. The residue was purified by prep-HPLC [ Column: Xselect CSH Prep C18 OBD Column, 30*150 mm, 5pm; Mobile Phase A: Water(0.1% FA), Mobile Phase B: ACN; Flow rate: 60 mL / min mL / min; Gradient (B%): 2% B to 21% B in 7 min; Wave Length: 254nm / 220nm nm; RTl(min): 6.3] to get 3-{4-chloro-2-[2- (dimethylamino)ethoxy]phenyl}-6-fluoro-2-(piperidin-4-ylmethyl)quinazolin-4-one (180 mg, 97.45%yield, 99.6%purity) as a white solid. LC-MS: MS (ESIpos): m / z = 459. 'll NMR (400 MHz, DMSO) 58.35 (s, br, 1H), 7.82 - 7.71 (m, 3H), 7.55 (d, J = 8.3 Hz, 1H), 7.48 (d, J = 2.2 Hz. 1H), 7.29 (d, J = 8.4 Hz, 1H), 4.44 (s, 2H), 3.36 (s, 2H), 3.23 (d, J = 12.4 Hz, 2H), 2.86 (s, 2H), 2.68 - 2.53 (m, 6H), 2.36 - 2.22 (m, 2H), 2.12 (s, 1H),1.81 (t, J = 14.7 Hz, 2H), 1.30 -1.10 (m, 2H).

[0339] The compounds in Table 1 were synthesized according to the general methods shown above for Compounds 1 and 16, using appropriate starting materials.

[0340] Table 1. Additional Compounds and Characterization Data

[0341] MS

[0342] Entry (ESIpos)

[0343] StructureJH NMR (400 MHz, DMSO-A)

[0344] m / z

[0345] (M+H)+

[0346] . Cl

[0347] 57.76-7.62 (m, 2H), 7.58 (d, 1H), 7.40 (d, 1H). 7.31 (d, 1H), 7.15 (d, 1H), 5.28-2.25 2 tn 418.15 (m, 1H), 4.15-3.98 (m, 2H), 2.61-2.52 (m,

[0348] 4H), 2.13 (s, 1H), 1.80-1.76 (m, 2H), 1.41- 1.38 (m, 2H), 1.13 (t, 3H).

[0349] 0

[0350]

[0351] HSTDU2-44700.601

[0352] 57.82-7.70 (m, 3H), 7.47 (d, 1H), 7.37 (s, ° °^rci1H), 7.20 (d, 1H), 4.17-4.05 (m, 2H), 3.60- 3.45 (m, 1H), 3.28-3.12 (m, 2H), 2.97-2.60 402.15

[0353] (m, 2H), 2.60-2.40 (m, 2H), 2.30-2.22 (m, 1H), 2.03-1.75 (m, 1H), 1.53-1.15 (m, 1H), X 1.12 (1, 3H).

[0354] NH

[0355] Y "o ■■

[0356] o 68.44 (s, 1H), 7.80-7.70 (m, 3H), 7.47 (d,

[0357] 1H), 7.39 (d, 1H), 7.22 (dd, 1H), 4.20-4.05 388.10 (m, 2H), 3.97-3.85 (m, 2H), 3.62 (t, 1H),

[0358] 3.53 (t, 1H), 3.28-3.12 (m, 1H), 2.80-2.75 X (m, 1H), 2.59-2.56 (m, 1H), 1.14 (t, 3H).

[0359] N

[0360] H

[0361] 57.81 - 7.69 (m, 3H), 7.45 (d, 1H), 7.35 (d,Fwjn 1H), 7.19 (dd, 1H), 4.09 (q, 2H), 3.40-3.35 Xl X 430.10 (m, 2H), 3.26-3.08 (m, 2H), 2.50-2.17 (m,

[0362] 3H), 2.10-1.88 (m, 1H), 1.68-1.56 (d, 3H), 1.50-1.32 (m, 1H), 1.30-1.02 (m, 5H). N— '

[0363] H

[0364] 57.80-7.76 (m, 3H), 7.44 (d, 1H), 7.36 (dd, III), 7.20 (dd, III), 4.10 (q, 211), 3.50-3.46 (m, 1H), 3.10-3.00 (m, 1H), 2.98-2.79 (m, 444.20

[0365] 3H), 2.34-2.11 (m, 3H), 1.68 (s, 2H), 1.61- 1.33 (m, 4H), 1.32-1.22 (m, 2H), 1.12 (t,

[0366] 3H).

[0367] 57.84 - 7.70 (m, 3H), 7.47 (t, 1H), 7.35 (d, w 3 1H), 7.19 (dd, 1H), 4.09 (q, 2H), 3.41 (d, XXI 414.00 1H), 3.23 (d, 1H), 2.79 (d, 1H), 2.58-2.56

[0368] (m, 1H), 2.31-2.21 (m, 2H), 1.38-1.25 (m, 1H), 1.16-1.02 (m, 3H), 1.02-1.00 (m, 1H),

[0369] 0.76-0.70 (m, 1H).

[0370]

[0371] HSTDU2-44700.601

[0372] 57.82-7.70 (m, 3H), 7.46 (dd. 1H), 7.37 (d, 1H), 7.21 (dd, 1H), 4.50 (dd, 1H), 4.16-4.03 (m, 2H), 3.07-2.96 (m, 1H), 2.83 (d, 1H), YjT X 434.20 2.64 (d, 1H), 2.56-2.55 (m, 1H), 2.47-2.39

[0373] (m, 1H), 2.20-2.18 (m, 2H), 2.99 (s, 1H), 1.41-1.39 (m, 1H), 1.34-1.17 (m, 1H), 1.13

[0374] (t, 3H).

[0375] H

[0376] 67.83-7.70 (m, 3H), 7.49 (dd, 1H), 7.38 (t, 3 1H), 7.22 (dt, 1H), 4.18-4.08 (m, 2H), 3.05- Yr X 452.05 2.98 (m, 1H), 2.80-2.76 (m, 2H), 2.76-2.58

[0377] (m, 1H), 2.50-2.30 (m, 2H), 2.25-2.06 (m,?tF1H), 1.93-1.83 (m, 1H), 1.30-1.09 (m, 4H). H

[0378] 5 8.42-8.24 (s, III), 7.87-7.72 (m, 311), 7.41-, J 3 7.35 (m, 2H), 7.24-7.14 (m, 1H), 4.10 (q, Tri 434.05 2H), 3.10-2.99 (m, 2H), 2.90-2.72 (m, 4H),

[0379] 2.16-2.12 (m, 2H), 2.01-1.86 (m, 1H), 1.80- 1.65 (m, 1H), 1.13 (t, 3H).

[0380] H

[0381] 57.79-7.76 (m, 3H), 7.53-7.43 (m, 1H), 7.38 (d, 1H), 7.21 (d, 1H), 4.10 (q, 2H), Ti l 3.05-2.94 (m, 1H), 2.90-2.80 (m, 1H), 2.79- 430.20

[0382] 2.50 (m, 1H), 2.46-2.14 (m, 2H), 2.05-1.90 (m, 1H), 1.70-1.62 (m, 2H), 1.40-1.30 (m, 1H), 1.17-0.94 (m, 7H), 0.79-0.76 (m, 1H). H

[0383] ?o. _CI 57.78-7.75 (m, 3H), 7.51-7.43 (m, 1H),

[0384] 7.37 (d, 1H), 7.19 (dd, 1H), 4.14-4.03 (in, YIX 2H), 3.12 (t, 1H), 2.78-2.76 (m, 1H), 2.54- 444.15 2.51 (m, 1H), 2.26-2.10 (m, 2H), 2.05-1.92

[0385] (m, 1H), 1.53-1.50 (m, 1H), 1.38-1.20 (m, 2H), 1.13 (t, 3H), 1.03 (t, 3H), 0.84 (t, 3H),

[0386] 0.60-0.57 (m, 1H).

[0387]

[0388] HSTDU2-44700.601

[0389] 57.80-7.68 (m, 3H), 7.47 (d, 1H), 7.36 (d, 1H), 7.19 (dd, 1H), 4.14-4.04 (m, 2H), 2.65- 'Ou x 2.51 (m, 2H), 2.30-2.15 (m, 2H), 1.99 (s,

[0390] 444.15

[0391] 1H), 1.60-1.40 (m, 2H), 1.40-1.25 (m, 1H), OO Z= / / 1.18-1.08 (m, 3H), 0.98 - 0.91 (m, 6H), 0.86-0.65 (m, 2H).

[0392] W

[0393] H

[0394] ^0.. oCl

[0395] F57.81-7.68 (m, 3H), 1A1 (d, 1H), 7.37 (s,. O 3 1H), 7.20 (d, 1H), 4.10 (q, 2H), 3.28-3.26 'Ou 1 442.20 (m, 1H), 2.46 (d, 1H), 2.30-2.01 (m, 3H),

[0396] 1.88-1.86 (m, 1H), 1.60-1.56 (m, 2H), 1.46- < H \ 1.30 (m, 3H), 1.17-1.05 (m, 5H).

[0397] \N /

[0398] 5 10.73 (s, 1H), 7.90-7.65 (m, 3H), 7.47 (d, 444.15 1H), 7.37 (s, 1H), 7.24 -7.12 (m, 1H), 4.10

[0399] (q, 2H), 2.75-2.53 (m, 3H). 2.46-2.44 (m, 1H), 2.38-2.23 (m, 3H), 1.13 (t, 3H).

[0400] XN’x^

[0401] 1~ 0., CI

[0402] 57.82 - 7.70 (m, 3H), 7.57 - 7.43 (m, 2H), 7.32 p^JuO 473.20 - 7.23 (m, 1H), 4.45 - 4.27 (m, 2H), 3.25 - 3.06 TjU X (m, 4H), 3.06 - 2.91 (m, 2H), 2.47 (s, 6H), 2.37 ^^'N'^i -2.11 (m, 3H), 1.95 - 1.56 (m,4H), 1.54- 1.37

[0403] (m, 1H), 1.28 - 1.13 (m, 1H).

[0404] NH

[0405] ''N'^0

[0406] 1~ XI

[0407] 58.13 (s, 1H), 7.89 - 7.68 (m, 3H), 7.56 (d, J =F.o,o 8.3 Hz, 1H), 7.48 (d, J = 2.2 Hz, 1H), 7.32 - Xrx 495.05 7.22 (m, 1H), 4.43 - 4.25 (m, 2H), 3.35 - 3.27

[0408] (m, 1H), 3.16 - 2.98 (m, 4H), 2.88 - 2.65 (m, 3H), 2.40 (s, 6H), 2.24 - 2.09 (m, 1H), 2.03 (d, J = 13.7 Hz, 1H), 1.40- 1.18 (m, 1H).

[0409]

[0410] HSTDU2-44700.601

[0411] 1o YCI88.13 (s, 1H), 7.84 - 7.71 (m, 3H), 7.52 (d, J =

[0412] 8.3 Hz, 1H), 7.44 (d, J = 2.3 Hz, 1H), 7.31 - 7.20 (m, 1H), 4.28 - 4.13 (m, 2H), 3.06 (d, J = 495.05

[0413] ■ F 11.0 Hz, 2H), 2.93 - 2.62 (m, 5H), 2.57 (d, J = XA 11.1 Hz, 1H), 2.35 -2.20 (m, 1H), 2.15 (s, 6H),

[0414] 1.94 (d, J = 13.8 Hz, 1H), 1.33 - 1.18 (m, 1H).ki\r

[0415] H

[0416] _CI 37.53 (d, J = 11.2 Hz, 1H), 7.40 (dd, 7 = 8.4,

[0417] 4.9 Hz, 1H), 7.34 (d, J = 2.2 Hz, 1H), 7.17 (dt, J A X = 8.4, 2.1 Hz, 1H), 6.86 (d, J = 8.1 Hz, 1H),

[0418] 6.29 (s, 2H), 4.13-4.04 (m, 2H), 3.02 - 2.91 (m, H2N X'^JXAC A'A 466.14

[0419] 1H), 2.81 (d, J = 13.2 Hz, 1H), 2.78 - 2.62 (m, XVF 2H), 2.47 - 2.36 (m, 2H), 2.19-1.96 (m, 1H),

[0420] 1.86 (d, J = 13.0 Hz, 1H), 1.28 - 1.16 (m, 1H),

[0421] 1.13 (t, 7= 6.9 Hz, 3H).

[0422] H

[0423] 1

[0424] o Oy-yCl 87.81 - 7.69 (m, 3H), 7.48 (d, 7 = 8.3 Hz, 1H),

[0425] 7.36 (d, 7= 2.2 Hz, 1H), 7.20 (dd, 7= 8.3, 2.1 A A JI Hz, 1H), 4.14 - 3.94 (m, 2H), 2.83 (s, 2H), 2.38 XXX 473.20 (s, 2H), 2.29 -2.17 (m, 2H), 2.08 - 1.95 (m,

[0426] 2H), 1.89 (s, 6H), 1.86 - 1.76 (m, 1H), 1.65 - 1.55 (m, 2H), 1.48 (d, 7= 11.6 Hz, 2H), 1.01 - 0.86 (m, 2H).

[0427] X H

[0428] X'X

[0429] 87.80 - 7.67 (m, 3H), 7.47 (d, 7 = 8.4 Hz, 1H), A X 7.40 (d, 7 = 2.2 Hz, 1H), 7.21 (dd, 7 = 8.3, 2.1 XXX Hz, 1H), 4.17 - 4.05 (m, 2H), 2.83 (d, 7 = 12.0

[0430] 459.19

[0431] Hz, 2H), 2.48 - 2.32 (m, 4H), 2.26 (d, 7 = 6.8 Hz, 2H), 1.96 (s, 6H), 1.82 (d, 7 = 17.7 Hz, 1H), 1.50 (t, 7= 12.7 Hz, 2H), 1.01 - 0.85 (m, 2H). J H

[0432] X'MX'X]

[0433] 10 O^YCI87.80 - 7.68 (m, 3H), 7.47 (d, 7 = 8.4 Hz, 1H),

[0434] 7.40 (d, 7 = 2.2 Hz, 1H), 7.21 (dd,7= 8.4, 2.2 Hz, 1H), 4.21 - 4.02 (m, 2H), 2.83 (d, 7 = 12.0 A XA 459.19

[0435] Hz, 2H), 2.47 - 2.32 (m, 4H), 2.26 (d, 7 = 6.8 Hz, 2H), 1.96 (s, 6H), 1.82 (d, 7 = 17.8 Hz, 1H), 1.50 (t, 7= 12.5 Hz, 2H), 1.01 -0.86 (m, 2H). X

[0436]

[0437] HSTDU2-44700.601

[0438] 57.80 - 7.68 (m, 3H), 7.47 (dd, J = 8.4, 3.1 Hz,1~ O^s^CI 1H), 7.39 (dd, J = 9.0, 2.2 Hz, 1H), 7.23 - 7.17 XT (m, 1H), 4.18 - 4.01 (m, 2H), 3.39 (s, 1H), 3.23

[0439] - 3.05 (m, 2H), 2.79 - 2.66 (m, 1H), 2.66 - 2.55 w. 473.20 (m, 1H), 2.44 - 2.36 (m, 2H), 2.33 - 2.24 (m,

[0440] 2H), 2.10 (d, J = 19.0 Hz, 1H), 1.96 (s, 6H), 1.60 (dd, J = 34.0, 15.0 Hz, 3H), 1.40 (s, 1H), NH 1.27 -0.99 (m, 2H).

[0441] 57.80 - 7.68 (m, 3H), 7.47 (d, J = 8.3 Hz, 1H), 7.39 (dd, J = 4.8, 2.2 Hz, 1H), 7.21 (dd, J = 8.4, < 'VX 2.3 Hz, 1H), 4.20-4.00 (m, 2H), 3.30-3.05 (m,

[0442] 473.15

[0443] ^'NA \ 2H), 2.77 - 2.51 (m, 3H), 2.49 - 2.34 (m, 2H),

[0444] 2.29-2.22 (m, 2H), 2.08 (s, 1H), 1.96 (s, 6H), 1.67 - 1.40 (m, 4H), 1.27 - 1.09 (m, 2H). NH

[0445] 1o °^vcl57.80 - 7.68 (m, 3H), 7.47 (dd, J = 8.4, 3.0 Hz,

[0446] 1H), 7.38 (dd, J = 8.9, 2.2 Hz, 1H), 7.23 - 7.17 YGJG (m, 1H), 4.18 -4.01 (m, 2H), 3.36 (d, J = 4.4

[0447] 473.20 Hz, 3H), 3.23 - 3.10 (m, 1H), 2.77 - 2.55 (m,

[0448] 2H), 2.45 - 2.37 (m, 2H), 2.34 - 2.24 (m, 2H), 1.96 (s, 6H), 1.72 - 1.51 (m, 3H), 1.41 (d, J = N 10.4 Hz, 1H), 1.28 - 0.98 (m, 2H).

[0449] H

[0450] 1o °^ vci57.80 - 7.68 (m, 3H), 7.47 (dd, J = 8.4, 1.2 Hz,

[0451] 1H), 7.39 (dd, J = 4.6, 2.2 Hz, 1H), 7.20 (dt, J = YGG 8.4, 2.6 Hz, 1H), 4.20-4.00 (m, 2H), 3.27 - 3.05

[0452] 473.20 (m, 3H), 3.02 - 2.75 (s, 1 H), 2.77 - 2.52 (m,

[0453] 211), 2.49 -2.35 (m, 211), 2.32 -2.18 (m, 211), 1.97 (s, 6H), 1.70-1.50 (m, 3H), 1.50 - 1.30 (m,

[0454] 1H), 1.26- 1.00 (m, 2H).

[0455] H HO"^

[0456] O^^x^CI 57.80 - 7.70 (m, 3H), 7.45 (d, J = 8.3 Hz, 1H),

[0457] 7.41 (d, J = 2.2 Hz, 1H), 7.20 (dd, J = 8.3, 2.2 F^z O Hz, 1H), 4.72 (s, 1H), 4.07 (t, J = 5.0 Hz, 2H), Xrx 432.14 3.52 (dd, J = 5.9, 4.2 Hz, 2H), 2.81 (d, J = 12.0

[0458] Hz, 2H), 2.45 - 2.31 (m, 2H), 2.31 - 2.17 (m, 2H), 1.86 (s, 1H), 1.50 (t, J= 13.4 Hz, 2H), 1.03 6 - 0.80 (m, 2H).

[0459]

[0460] HSTDU2-44700.601

[0461] HO^l

[0462] OVYCI67.81 - 7.70 (m, 3H), 7.49 - 7.39 (m, 2H), 7.23

[0463] -7.15 (m, 1H), 4.72 (s, 1H), 4.14-3.97 (m, 2H), 3.61 - 3.46 (m, 2H), 2.72 - 2.55 (m, 2H), 29 460.17 2.34 - 2.14 (m, 2H), 2.02 (s, 1H), 1.52 (dd, J =

[0464] 322, 12.3 Hz, 1H), 1.37 - 1.22 (m, 1H), 1.04 - 0.89 (m, 6H), 0.85 - 0.63 (m, 2H).

[0465] AA- H

[0466] 87.81 - 7.68 (m, 3H), 7.49 - 7.35 (m, 2H), 7.23 - 7.14 (m, 1H), 4.13 - 4.00 (m, 2H), 3.57 - 3.47 AT (m, 2H), 3.42 - 3.34 (m, 2H), 3.16 (dd, J = 27.4, 30 446.16 13.9 Hz, 2H), 2.68 (d, J = 47.3 Hz, 1H), 2.41 - 2.23 (m, 2H), 2.04 (d, J = 46.5 Hz, 1H), 1.74 - 1.53 (m, 3H), 1.43 (d, J = 13.0 Hz, 1H), 1.29 - 0.95 (m, 2H).

[0467] NH H

[0468] O °Y^YCI 87.80 - 7.70 (m, 3H), 7.47 (d, J = 8.3 Hz, 1H), F.. ^. A A JI 7.37 (d, J = 2.2 Hz, 1H), 7.20 (dd, J = 8.3, 2.2

[0469] Hz, 1H), 4.15 - 3.97 (m, 2H), 2.83 (s, 2H), 2.46 31 459.19

[0470] - 2.35 (m, 2H), 2.34 - 2.17 (m, 4H), 2.05 (s, 3H), 1.85 (s, 1H), 1.70 - 1.57 (m, 2H), 1.50 (t, J = 13.3 Hz, 2H), 1.04 - 0.83 (m, 2H). A

[0471]

[0472] H

[0473] Example 2

[0474] Biological Assays

[0475] Materials and Methods

[0476] Transfection and Lentivirus Production. HEK293T cells were seeded at a density of 35,000 per well in 1 mL of DMEM medium supplemented with 10% FBS and 1% penicillin streptomycin. The following day, a packaging mix was prepared by combining 800 ng psPAX2, 300 ng pMD2. G and 900 ng sgRNA backbone in 125 ul OptiMEM (Invitrogcn). This mixture was then combined with 6 ul TransIT-LTl (Minis) in 125 ul OptiMEM, incubated for 25 min at room temperature and then added to cells. After two days of transfection, the medium containing lentivirus was collected and stored at -80 °C before use. To transduce target cells, the lentivirus was added to the cells in the presence of 1 ug / ml Polybrene.

[0477] CRISPR knockout ofTRIM21. sgRNAs targeting TRIM21 were designed utilizing the CrisPick design tool (https: / / portals.broadinstitute.org / gppx / crispick / public). A total of 5STDU2-44700.601

[0478] pairs of oligonucleotides targeting TRIM21, and a control pair of CHR2-2, were annealed and cloned into the pXPR_023 all-in-one Cas9 vector. In brief, vector was linearized using BsmBI (New England Biolabs). The annealed oligonucleotides were ligated into the linearized and purified vector backbone with T4 DNA ligase (New England Biolabs).

[0479] Constructs were transformed, purified using the Miniprep Kit (Qiagen), and the guide sequence was confirmed by Sanger sequencing. PANCI cells were stably transduced with lentivirus for TRIM21 pXPR_023 or the negative control CHR2-2 pXPR_023 in 12- well plates via centrifugation at 2,000 rpm and 30 °C for two hours. The next day, cells were split into flasks and selected with 2 ug / ml puromycin for 3 days. Gene knockout was confirmed by immunoblot.

[0480] Gene overexpression. The TRIM21 gene cDNA was cloned into the pDONR_221 vector using BP Clonase (Thermo Fisher Scientific), and then the transferred into the pLX_311 mammalian expression vector using LR Clonase (Thermo Fisher Scientific).

[0481] Lentivirus was generated as described above. Cell lines were transduced with the harvested virus, and infected cells were selected with 2 ug / mL blasticidin (Gibco) for 10 days.

[0482] PANCJ CRISPR-Cas9 genome-wide knockout screen. PANCI cells were stably transduced with pLX_311-Cas9. The Brunello genome-scale sgRNA library was obtained from the Broad Institute GPP. Virus was titrated to a goal infection efficiency of 30-50%. PANCI -Cas9 cells were infected with Brunello library virus in 12-well plates via centrifugation at 2,000 rpm and 30 °C for 2 hours. The following day, cells were split into two replicate flasks and selected with 2 ug / ml puromycin for 5 days. After selection, replicates were seeded into 110 nM PRLX-93936 or DMSO control. Cells were maintained at 37 °C and 5% CO2 in CellSTACK 1,272 cm22-STACK flasks (Coming) in RPMI with 10% FBS. Cells were reseeded approximately every 3 days at a minimum of 40 million cells per passage to maintain approximately 500x library representation. Media and drugs were refreshed every 3-4 days for a total of 3 weeks. PRLX-93936 dose was gradually increased to 250 nM over the course of the screen. Genomic DNA was isolated from cell pellets using the NucleoSpin Blood XL columns (MACHEREY-NAGEL). Genomic DNA PCR and nextgeneration sequencing by standard Illumina methods were performed to determine guide abundance.

[0483] PANCI CRISPR-dCas9 genome-wide activation screen. PANCI cells were stably transduced with pXPR_109 to express dCas9-VP64. The Calabrese B genome-scale vims library was obtained from the Broad Institute GPP. PANCI -dCas9-VP64 cells were infected with this library via centrifugation as described earlier. The following day, cells were splitSTDU2-44700.601

[0484] into two replicates and reseeded. One day later, 2 ug / ml of puromycin was added. After selection for 5 days, replicates were split into DMSO or 110 nM PRLX-93936 drug arms in duplicate and cultured for 2 weeks as described followed by genomic DNA isolation and sequencing as described earlier.

[0485] CRISPR screen analysis. Guides targeting multiple genes or with <50 reads in the plasmid DNA pool were filtered. Counts were normalized against total library size. The guide-level log2 fold change was computed using the ratio between treatment versus vehicle control counts. The median log2 fold change from all guides targeting each gene was calculated. Statistical significance for each gene-level result was calculated using the MAGeCK-RRA method (MAGeCK version 0.5.9.5), using 10,000 permutations. Two-sided P values were corrected for multiple hypothesis testing using the Benjamini-Hochberg method.

[0486] RNA-seq. PANCI cells were seeded in 6- well plates. The following day, cells were treated in triplicate with 0.5 uM PRLX-93936 or DMSO vehicle control for 2 hours. RNA was isolated using the RNeasy Mini Kit (QIAGEN) with DNase treatment. RNA QC, library preparation, and Novaseq sequencing were performed by Genewiz. Gene-level expression values were obtained from RNA-seq using the nf-core / maseq pipeline (version 3.12.0). Salmon (version 1.10.1) was used to generate gene-level read counts. Differential gene expression was calculated using the DESeq2 package (version 1.28.0). These tools were run using the Stanford Sherlock computing cluster.

[0487] Multiplexed cell line screening. The established PRISM cell proliferation assay was carried out as previously described. In brief, approximately 800 barcoded cell lines in pools of 20-25 were thawed and plated into 384- well plates (1,250 cells per well for adherent cells, 2,000 cells per well for suspension or mixed suspension-adherent pools). Cells were treated with an eight -point dose curve starting at 10 uM with threefold dilutions in triplicate and incubated for 120 hours. Following lysis, the barcode for each cell was detected by mRNA-based Luminex detection as previously described and input to a standardized R pipeline (https: / / github.com / broadinstitute / prism_data_processing) to generate viability estimates relative to vehicle treatment and fit dose-response curves. The area under the dose-response curve (AUC), which is correlated with drug potency, was used as a metric of dmg potency for each cell line. Reference publicly available gene expression data were retrieved from the Cancer Cell Line Encyclopedia. Predictive biomarker relationships were determined by comparing each cell line baseline molecular feature (e.g., RNA-seq gene expression) versus the observed AUC models by univariate linear modeling.STDU2-44700.601

[0488] Results and Discussion

[0489] To determine functional mediators of PRLX-93936, genome-scale CRISPR / Cas9 drug modifier knockout and activation screens were performed in the PANCI pancreatic cancer cell line. In the knockout screen, conducted using the Brunello CRISPR guide RNA library, TRIM21 knockout was the top hit that rescued from PRLX-93936. Other rescue hits included genes necessary for NFkB (IKK-alpha and IKK-gamma) and interferon signaling (IRF2) (FIG.

[0490] 2). IKK enzymes have been reported to be direct substrates of TRIM21 (Wada et al. J. Biochem.

[0491] 146, 821-832 (2009).

[0492] The results of the CRISPR gene activation screen, conducted with the Calabrese-B CRISPR guide RNA library, were highly complementary (FIG. 3). TRIM21 overexpression sensitized to PRLX-93936 treatment, along with IRF2 and IRF overexpression.

[0493] Next, predictive biomarkers for PRLX-93936 response were sought via large-scale proliferation assays across 816 cancer cell lines (FIG. 4A). Sensitive cell lines were identified across multiple cancer types, including lineage-specific enrichment for activity against pancreatic cancer and head and neck cancer (upper aerodigestive cancers) (FIG. 4B). Next, cell line molecular features were systematically compared with PRLX-93936 drug response by linear modeling. High expression of TRIM21 was the top predictive biomarker of cell line response (FIG. 5). Thus, in addition to functionally mediating the anti-cancer activity of PRLX-93936, TRIM21 expression is a promising predictive biomarker for the drug.

[0494] To validate the functional role of TRIM21 in PRLX-93936 response, multiple isogenic cancer cell lines were generated. First, TRIM21 was knocked out via CRISPR Cas9 in TRIM21 -expressing PANCI and KP3 pancreatic cancer cells. Both cell lines are sensitive to PRLX-93936 at baseline. TRIM21 knockout completely rescues from PRLX-93936 activity in a dose response viability assay (FIG. 6). Next, experiments were conducted to determine whether ectopic expression of TRIM21 in resistant cell lines would alter sensitivity to the drug. Across three resistant cell lines of various lineages (ovarian cancer, lung cancer, and melanoma) all become highly sensitive (>500x sensitization) to PRLX-93936 upon TRIM21 overexpression by lentiviral transduction (FIG. 7).

[0495] To confirm on-target activity of our TRIM21 CRISPR guides, knockout-restoration studies were performed in PANCI cells. Re-expression of wild-type TRIM21 in knockout cells via guide-resistant cDNA restored sensitivity (FIG. 8). RING E3 ligases rely on a critical “linchpin” residue, most commonly arginine, to catalyze transfer of a ubiquitin molecule from an E2 enzyme to a substrate (Lips et al. EMBO J. 39, el04863 (2020)). The TRIM21 R55ESTDU2-44700.601

[0496] linchpin mutation does not fully restore sensitivity to cells. Thus, functional TRIM21 is necessary for PRLX-93936 activity.

[0497] PRLX-93936 was previously reported to induce apoptosis (unlike the parent compound, erastin, which is a prototypic ferroptosis inducer). Here, PRLX-93936 was found to induce caspase 3 / 7 activation in a TRIM21 -dependent manner (FIG. 9). In addition, multiple hallmarks of apoptosis including cleavage of PARP, caspase 3, and caspase 7 are detectable by immunoblot at 16 hours (FIG. 9). TRIM21 knockout prevents PARP and caspase cleavage.

[0498] To identify downstream effectors of PRLX-93936, RNA-Seq was performed using drug-treated cells. Results demonstrated rapid induction of multiple NFkB and interferon gene targets including IRF1, RNFAIP3, NFKBIA, CD83, CXCL3, and other genes (FIG. 10). PRLX-93936-induced increases in protein expression and nuclear localization of NFkB (RelA) and IRF1 were confirmed by immunoblot (FIG. 11)

[0499] Compounds shown in Example 1 were tested against PANCI (intact TRIM21) and PANC1-TRIM21 knockout cells to verify TRIM21 dependency. Cell line IC50 values (CellTiterGlo at 72 hours) for each compound against PANCI cells are shown Table 2.

[0500] Table 2. ICso Values

[0501] PANC1- PANCI ICso

[0502] Compound TRIM21 KO

[0503] (J1M)

[0504] ICso (pM)

[0505] 1 0.01775 >10

[0506] 2 ND ND

[0507] 3 0.560 >10

[0508] 4 >10 >10

[0509] 5 0.011 >10

[0510] 6 0.028 >10

[0511] 7 1.415 >10

[0512] 8 0.084 >10

[0513] 9 0.071 >10

[0514] 10 0.041 >10

[0515] 11 0.023 >10

[0516] 12 0.042 >10

[0517] 13 0.015 >10

[0518] 14 0.637 >10

[0519] 15 >10 >10

[0520] 16 0.0155 > 10.0

[0521]

[0522] 17 0.007 > 10.0STDU2-44700.601

[0523] 0.194 > 30.0

[0524] 0.0101 > 30.0

[0525] 0.0438 > 30.0

[0526] 0.0356 > 30.0

[0527] 0.005 > 30.0

[0528] 1.59 > 30.0

[0529] 0.00427 > 30.0

[0530] 0.00408 > 30.0

[0531] 0.685 > 30.0

[0532] 1.42 > 30.0

[0533] 0.0478 > 30.0

[0534] 0.0421 > 30.0

[0535] 0.0216 > 30.0

[0536]

[0537] 0.312 > 30.0

Claims

STDU2-44700.601CLAIMS1. A compound of formula (I):R1R5(I)or a pharmaceutically acceptable salt thereof, wherein:Q is O or NH;R1is Ci-Ce alkyl, C3-C6 cycloalkyl, or monocyclic heterocyclyl, wherein the alkyl is unsubstituted or substituted with 1 substituent selected from hydroxy, amino, amido, and cyano; or R1is taken together with R2aand the atoms to which they are attached to form a ring;X1is CH or N;X2is CR2aor N;X3is CR2bor N;X4is CR2Cor N;X5is CR2dor N;X6is CR3aor N;X7is CR3bor N;X8is CR3Cor N;X9is CR3dor N;R2S p2b p>2<;|Cd. j^3banj R3JAREincjepencjentiy selected from hydrogen, halo, Ci-Ce alkyl, Ci-Ce haloalkyl, Ci-Ce alkoxy, -NRxRy, -C(O)(Ci-Ce alkyl), cyano, aryl, C3-C6 cycloalkyl, heteroaryl, and heterocyclyl; wherein R2aand R2b, or R2band R2C, or R2Cand R2dare optionally taken together with the carbon atoms to which they are attached to form an optionally substituted ring; and wherein R3aand R3b, or R3band R3c, or R3Cand R3dare optionally taken together with the carbon atoms to which they are attached to form an optionally substituted ring;STDU2-44700.601Rxand Ryare each independently selected from hydrogen, Ci-C6 alkyl, and Ci-Ce hydroxyalkyl;pl is 1, 2, or 3;p2 is 1, 2, or 3;n is 0, 1, 2, 3, or 4;each R4is independently selected from Ci-Ce alkyl, halo, oxo, Ci-Ce haloalkyl, Cj-Cc, cycloalkyl, cyano, and -(CH2)q-Y, wherein q is 1, 2, or 3, and Y is selected from hydroxy, amino, C1-C4 alkoxy, C1-C4 alkylsulfonyl, and C1-C4 alkylsulfinyl; or two R4are taken together to form a bond, or two R4are taken together with the atom(s) to which they are attached to form a bond or a 3- to 6-membered ring that is unsubstituted or substituted with one C1-C6 alkyl group;R5is hydrogen or C1-C4 alkyl;Z is CR6aR6b, O, or NH; andR6aand R6bare each independently selected from hydrogen and Ci-Ce alkyl, or R6aand R6bare taken together with the carbon atom to which they are attached to form an optionally substituted ring.

2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein Q is O.

3. The compound of claim 1 or claim 2, or a pharmaceutically acceptable salt thereof, wherein R1is C1-C4 alkyl, C3-C4 cycloalkyl, or a monocyclic 4- or 5-membered heterocyclyl having one heteroatom selected from N and O, wherein the alkyl is unsubstituted or substituted with 1 substituent selected from -OH, -NH2, -NI I(C'[ h), -N(CH3)2, and -C(O)NH2.

4. The compound of any one of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein R1is unsubstituted C2-C4 alkyl.

5. The compound of claim 4, or a pharmaceutically acceptable salt thereof, wherein R1is ethyl.

6. The compound of any one of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein R1is C2-C4 alkyl that is substituted with 1 substituent selected from -OH, -NH2, -NH(CH3), and -N(CH3)2.STDU2-44700.6017. The compound of claim 6, or a pharmaceutically acceptable salt thereof, wherein R1is -CH2CH2NH2.

8. The compound of any one of claims 1-7, or a pharmaceutically acceptable salt thereof, wherein X1is N.

9. The compound of any one of claims 1 -8, or a pharmaceutically acceptable salt thereof, wherein X2is CR2a.

10. The compound of claim 9, or a pharmaceutically acceptable salt thereof, wherein R2ais hydrogen.

11. The compound of any one of claims 1-10, or a pharmaceutically acceptable salt thereof, wherein X is CR2b.

12. The compound of claim 11, or a pharmaceutically acceptable salt thereof, wherein R2bis selected from hydrogen, halo, C1-C3 alkyl, C1-C3 haloalkyl, -C(O)O(Ci-C3 alkyl), and a monocyclic 5- or 6-membered heterocyclyl having 1 or 2 heteroatoms independently selected from O and N.

13. The compound of claim 12, or a pharmaceutically acceptable salt thereof, wherein R2bis halo.

14. The compound of claim 13, or a pharmaceutically acceptable salt thereof, wherein R2bis chloro.

15. The compound of any one of claims 1-14, or a pharmaceutically acceptable salt thereof, wherein X4is CR2c.

16. The compound of claim 15, or a pharmaceutically acceptable salt thereof, wherein R2cis hydrogen, halo, C1-C3 haloalkyl, C1-C3 alkoxy, -NRxRy, or cyano, wherein Rxis hydrogen and Ryis C1-C3 hydroxyalkyl.STDU2-44700.60117. The compound of claim 16, or a pharmaceutically acceptable salt thereof, wherein R2cis hydrogen.

18. The compound of any one of claims 1-17, or a pharmaceutically acceptable salt thereof, wherein X5is CR2d.

19. The compound of claim 18, or a pharmaceutically acceptable salt thereof, wherein R2dis hydrogen.

20. The compound of any one of claims 1-19, or a pharmaceutically acceptable salt thereof, wherein X6is CR3a.

21. The compound of claim 20, or a pharmaceutically acceptable salt thereof, wherein R3ais hydrogen.

22. The compound of any one of claims 1-21, or a pharmaceutically acceptable salt thereof, wherein X7is CR3b.

23. The compound of claim 22, or a pharmaceutically acceptable salt thereof, wherein R3bis selected from hydrogen, halo, C1-C3 alkyl, cyano, phenyl, and monocyclic 5- or 6-membered heteroaryl having 1 or 2 heteroatoms independently selected from N, O, and S.

24. The compound of claim 23, or a pharmaceutically acceptable salt thereof, wherein R3bis halo.

25. The compound of claim 24, or a pharmaceutically acceptable salt thereof, wherein R3bis fluoro.

26. The compound of any one of claims 1-25, or a pharmaceutically acceptable salt thereof, wherein X8is CR3c.

27. The compound of claim 26, or a pharmaceutically acceptable salt thereof, wherein R3cis selected from hydrogen and -NRxRy, wherein Rxand Ryare each independently selected from hydrogen and C1-C3 alkyl.STDU2-44700.60128. The compound of claim 27, or a pharmaceutically acceptable salt thereof, wherein R3cis hydrogen.

29. The compound of any one of claims 1 -28, or a pharmaceutically acceptable salt thereof, wherein X9is CR3d.

30. The compound of claim 29, or a pharmaceutically acceptable salt thereof, wherein R3dis hydrogen.

31. The compound of any one of claims 1-30, or a pharmaceutically acceptable salt thereof, wherein each R4is independently selected from Ci-Cs alkyl, halo, and oxo; or two R4are taken together to form a bond, or two R4are taken together with the atom(s) to which they are attached to form a bond or a ring.

32. The compound of any one of claims 1-31, or a pharmaceutically acceptable salt thereof, wherein n is 0.

33. The compound of any one of claims 1-31, or a pharmaceutically acceptable salt thereof, wherein n is 1 or 2, and each R4is independently selected from C1-C3 alkyl, halo, and oxo.

34. The compound of claim 33, or a pharmaceutically acceptable salt thereof, wherein n is 1 and R4is fluoro, methyl, or oxo.

35. The compound of claim 33, or a pharmaceutically acceptable salt thereof, wherein n is 2 and each R4is fluoro, methyl, or oxo.

36. The compound of any one of claims 1-31, or a pharmaceutically acceptable salt thereof, wherein n is 2, and wherein the two R4are taken together to form a bond or are taken together with the atom(s) to which they are attached to form a ring.

37. The compound of any one of claims 1-36, or a pharmaceutically acceptable salt thereof, wherein R3is hydrogen.STDU2-44700.60138. The compound of any one of claims 1-37, or a pharmaceutically acceptable saltthereof, wherein the groupR5has a formula selected from:

39. The compound of any one of claims 1-38, or a pharmaceutically acceptable salt thereof, wherein Z is CR6aR6band R6aand R6bare each hydrogen.

40. The compound of any one of claims 1-38, or a pharmaceutically acceptable salt thereof, wherein Z is O.

41. The compound of claim 1, wherein the compound is selected from:STDU2-44700.601STDU2-44700.601and pharmaceutically acceptable salts thereof.

42. A pharmaceutical composition comprising a compound of any one of claims 1-41, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

43. A method of treating a proliferative disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1-41, or a pharmaceutically acceptable salt thereof.

44. The method of claim 43, wherein the subject is a mammal.

45. The method of claim 43, wherein the subject is a human.

46. The method of any one of claims 43-45, wherein the proliferative disease is associated with tripartite motif-containing 21 (TRIM21).

47. The method of claim 46, wherein the proliferative disease is associated with a mutation in and / or increased activity of TRIM21.

48. The method of any one of claims 43-47, wherein the proliferative disease is cancer.STDU2-44700.60149. The method of claim 48, wherein the cancer is selected from pancreatic cancer, head and neck cancer, breast cancer, colorectal cancer, lung cancer, melanoma, ovarian cancer, kidney cancer, multiple myeloma, esophageal cancer, gastric cancer, liver cancer, bile duct cancer, leukemia, lymphoma, prostate cancer, and sarcoma.

50. The method of claim 49, wherein the cancer is pancreatic cancer.

51. The method of any one of claims 43-50, further comprising treating the subject with one or more additional therapies selected from chemotherapy, surgery, radiation therapy, hormone therapy, immunotherapy, cryotherapy, T cell transfer therapy, and thermotherapy, or any combination thereof.

52. A compound of any one of claims 1-41, or a pharmaceutically acceptable salt thereof, for use as a medicament.

53. A compound of any one of claims 1-41, or a pharmaceutically acceptable salt thereof, for use in treating a proliferative disease.

54. The compound for use of claim 53, wherein the proliferative disease is associated with tripartite motif-containing 21 (TRIM21).

55. The compound for use of claim 54, wherein the proliferative disease is associated with a mutation in and / or increased activity of TRIM21.

56. The compound for use of any one of claims 53-55, wherein the proliferative disease is cancer.

57. The compound for use of claim 56, wherein the cancer is selected from pancreatic cancer, head and neck cancer, breast cancer, colorectal cancer, lung cancer, melanoma, ovarian cancer, kidney cancer, multiple myeloma, esophageal cancer, gastric cancer, liver cancer, bile duct cancer, leukemia, lymphoma, prostate cancer, and sarcoma.

58. The compound for use of claim 57, wherein the cancer is pancreatic cancer.STDU2-44700.60159. A kit comprising a compound of any one of claims 1-41, or a pharmaceutically acceptable salt thereof.

Images