PDHK inhibitors and uses thereof

Abstract

Disclosed herein are compounds that inhibit pyruvate dehydrogenase kinases (PDHKs), including compounds that are selective for PDHK1 over other PDHK isoforms. Also disclosed herein are pharmaceutical compositions comprising the compounds and methods of using the compounds in treating disorders, such as cancers.

Description

[0001] UM-43663.601

[0002] PDHK INHIBITORS AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to and benefit of U.S. Provisional Patent Application No.63 / 699,360, filed on September 26, 2024, the disclosure of which is incorporated herein 5 by reference in its entirety.

[0004] FIELD

[0005] Provided herein are compounds that inhibit pyruvate dehydrogenase kinases (PDHKs), including compounds that are selective for PDHK1 over other PDHK isoforms. Also disclosed herein are pharmaceutical compositions comprising the compounds and 10 methods of using the compounds in treating disorders, such as cancers.

[0006] BACKGROUND

[0007] Pyruvate dehydrogenase kinases (PDHKs) regulate the pyruvate dehydrogenase complex and control the flux of acetyl-CoA into the Krebs cycle. Dysregulation of PDHK activity has been implicated in a broad range of metabolic and proliferative diseases. For 15 example, elevated PDHK activity contributes to the glycolytic phenotype known as the Warburg effect, which is a common feature of tumor metabolism. Although PDHK inhibitors have been developed, including dichloroacetic acid, no PDHK inhibitors are currently approved for human therapy and compounds undergoing clinical studies inhibit multiple PDHK isoforms.

[0008] 20 SUMMARY

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

[0010] (I)

[0011] or a pharmaceutically accepta

[0012]

[0013] wherein:

[0014] R1is selected from hydrogen, halo, C1-C6 alkoxy, and C1-C6 thioalkoxy; 25 R2is selected from hydrogen, halo, C1-C6alkoxy, and C3-C6cycloalkyl;

[0015] R3is selected from hydrogen and halo;

[0016] R4is selected from -COOR5a, -CONHR5b, -CN, and C1-C6alkoxy;

[0017] 1 UM-43663.601

[0018] R5aand R5bare each independently selected from hydrogen and C1-C6alkyl; and R6is phenyl or a monocyclic 5- or 6-membered heteroaryl, each of which is independently unsubstituted or substituted with 1, 2, or 3 substituents independently selected from halo, C1-C6alkyl, C1-C6alkoxy, and C3-C6cycloalkyl.

[0019] 5 In some embodiments, R1is selected from hydrogen, halo, and C1-C3 thioalkoxy. In some embodiments, R1is selected from hydrogen, fluoro, chloro, bromo, and methylthio.

[0020] In some embodiments, R2is selected from hydrogen, halo, C1-C4 alkoxy, and C3-C4 cycloalkyl. In some embodiments, R2is selected from hydrogen, chloro, bromo, methoxy, ethoxy, isopropoxy, and cyclopropyl.

[0021] 10 In some embodiments, R3is selected from hydrogen and chloro. In some embodiments, R3is hydrogen.

[0022] In some embodiments, R4is selected from -COOR5a, -CONHR5b, C1-C3 alkoxy, and - CN, wherein R5aand R5bare each independently selected from hydrogen and methyl. In some embodiments, R4is -COOR5aand R5ais hydrogen.

[0023] 15

[0024]

[0025] 2 UM-43663.601

[0026] .

[0027] In some embodiments, R6i with 2 substituents independently

[0028]

[0029] selected from halo and C1-C4 alkyl. In some embodiments, R6is a monocyclic 5-membered heteroaryl having one heteroatom selected from S, O, and N, which is substituted with 1 5 substituent selected from halo and C1-C4 alkyl. In some embodiments, R6has a structure:

[0030] wherein:

[0031]

[0032] R7ais halo;

[0033] R7bis hydrogen or halo;

[0034] 10 R7cis C1-C4 alkyl or hydrogen;

[0035] R7dis halo or C1-C4alkyl; and

[0036] X is S, O, or NH.

[0037] In some embodiments, R6has a structure selected from:

[0038] .

[0039] 15

[0040]

[0041]

[0042] 3 UM-43663.601

[0043] 5

[0044]

[0045] 4 UM-43663.601

[0046] an

[0047]

[0048] 5 In another aspect, disclosed herein is a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0049] In another aspect, disclosed herein is a method of treating a disorder associated with a pyruvate dehydrogenase kinase (PDHK) in a subject in need thereof, comprising

[0050] 10 administering to the subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof.

[0051] In some embodiments, the disorder is associated with overexpression of pyruvate dehydrogenase kinase 1 (PDHK1).

[0052] In some embodiments, the disorder is cancer. In some embodiments, the cancer is 15 selected from acute myeloid leukemia, breast cancer, esophageal cancer, gastric cancer, glioblastoma, head and neck cancer, multiple myeloma, nasopharyngeal cancer, ovarian cancer, retinoblastoma, prostate cancer, bladder cancer, glioma, hepatocellular carcinoma, non-small cell lung cancer, pancreatic cancer, and transitional cell carcinoma.

[0053] 5 UM-43663.601

[0054] In some embodiments, the disorder is selected from Alzheimer's disease, multiple sclerosis, hepatic steatosis, Charcot-Marie-Tooth disease, Barth Syndrome, heart failure, inflammatory bowel disease, metabolic syndrome, metabolic dysfunction-associated steatohepatitis, diabetes, endometriosis, heart fibrosis, lactic acidosis, macular degeneration, 5 myalgic encephalomyelitis, nonalcoholic fatty liver disease, pyruvate dehydrogenase deficiency, and pulmonary arterial hypertension.

[0055] In another aspect, disclosed herein is a compound of formula (I), or a pharmaceutically acceptable salt thereof, for use as a medicament.

[0056] In another aspect, disclosed herein is a compound of formula (I), or a

[0057] 10 pharmaceutically acceptable salt thereof, for use in treating a disorder associated with a pyruvate dehydrogenase kinase (PDHK).

[0058] In some embodiments, the disorder is associated with overexpression of pyruvate dehydrogenase kinase 1 (PDHK1).

[0059] In some embodiments, the disorder is cancer. In some embodiments, the cancer is 15 selected from acute myeloid leukemia, breast cancer, esophageal cancer, gastric cancer, glioblastoma, head and neck cancer, multiple myeloma, nasopharyngeal cancer, ovarian cancer, retinoblastoma, prostate cancer, bladder cancer, glioma, hepatocellular carcinoma, non-small cell lung cancer, pancreatic cancer, and transitional cell carcinoma.

[0060] In some embodiments, the disorder is selected from Alzheimer's disease, multiple 20 sclerosis, hepatic steatosis, Charcot-Marie-Tooth disease, Barth Syndrome, heart failure, inflammatory bowel disease, metabolic syndrome, metabolic dysfunction-associated steatohepatitis, diabetes, endometriosis, heart fibrosis, lactic acidosis, macular degeneration, myalgic encephalomyelitis, nonalcoholic fatty liver disease, pyruvate dehydrogenase deficiency, and pulmonary arterial hypertension.

[0061] 25 Other aspects and embodiment of the disclosure will become apparent in light of the following description.

[0062] BRIEF DESCRIPTION OF THE DRAWINGS FIG.1 shows data for inhibition of PDH phosphorylation in A549 cells measured by Western Blot.

[0063] 30 FIG.2 shows data for PDHK Target engagement in live HEK293 cells as determined by NanoBRET using the lipoamide site probe molecule N-[(p-{[(2R,5S)-2,5-dimethyl-4- (3,3,3-trifluoro-2-hydroxy-2-methylpropionyl)-1-piperazinyl]carbonyl}phenyl)methyl]3-[4,4- difluoro-5-(2-pyrrolyl)-3a,4aλ⁴-diaza-4λ⁴-bora-4H-s-indacen-2-yl]propionamide.

[0064] 6 UM-43663.601

[0065] FIG.3 shows data for a cell viability assay in A549 cells using CellTiter-Glo.

[0066] FIG.4 shows a scatter plot of tabulated results for the selectivity panel showed in Table 2. Proteins with activity reduced by ≥20% are displayed in blue.

[0067] FIG.5 shows NanoBRET target engagement studies of compounds disclosed herein 5 in permeabilized HEK293 cells in comparison to a pan-isoform PDHK inhibitor, VER- 246608. All compounds were assessed at a single concentration of 30 µM except for VER- 246608 (1 µM). Data was collected in technical triplicate and is normalized to a DMSO control.

[0068] FIG.6 shows NanoBRET titration curves of PTM-130 against (top, left to right) 10 PDHK1 and PDHK2 and (bottom, left to right) PDHK3 and PDHK4. Data were collected in triplicate for PDHK1–3 and duplicate for PDHK4.

[0069] FIG.7 shows target modulation by PTM-130 in A549 cells. Levels of PDHK1, total PDH (tPDH), and pPDH at the Ser232 and Ser293 sites were assessed at various time points after treatment with PTM-130. Data are representative of at least two independent biological 15 replicates.

[0070] FIG.8 shows cytotoxicity of PTM-130 in A549 cells as determined by CellTiter-Glo. Cells were exposed to PTM-130 for 72 h at concentrations up to 50 µM. Data are displayed as the average of technical triplicates ± standard deviation.

[0071] DETAILED DESCRIPTION

[0072] 20 Disclosed herein are PDHK inhibitors, including compounds that are selective inhibitors of PDHK1. PDHK1 and its isoforms, PDHK2, 3 and 4 play critical roles in cellular bioenergetics by negatively regulating the pyruvate dehydrogenase complex (PDHc) (see, e.g., Roche & Hiromasa Cell. Mol. Life Sci.2007, 64:830-849). The PDHc catalyzes the irreversible oxidative carboxylation of pyruvate to produce acetyl-CoA, thereby influencing 25 the balance between glycolysis and the tricarboxylic acid cycle, which supports oxidative phosphorylation (see, e.g., Wieland, Rev. Physiol., Biochem. Pharmcol.1983, 96: 123-170). Since the first report of congenital pyruvate dehydrogenase deficiency in 1970 (see, e.g., Blass et al. J. Clin. Invest.1970, 49:423-432), and subsequent findings linking PDHKs with diabetes (see, e.g., Jeoung, Diabetes Metab. J.2015, 39(3):188-97), PDHKs have been 30 extensively studied as potential therapeutic targets for a broad range of different diseases, notably including diabetes and cancer (see, e.g., Wang et al. Biosci. Rep.2021, 41(4):

[0073] BSR20204402). Multiple small molecule PDHK inhibitors have been described in the literature (see e.g., Li et al. Bioorg. Chem.2024, 144: 107160; Zhang et al. Drug Disc. Today

[0074] 7 UM-43663.601

[0075] 2015, 20(9): 1112-1119; Anwar et al. Biochim. Biophys. Acta 2021, 1876(1):188568).

[0076] However, most of these compounds inhibit two (see, e.g., Carbone et al. Int. J. Mol Sci.2023, 24(4): 3679), three (see, e.g., Sakimura et al. Alzheimer’s Res.& Therapy 2024, 16(1): 197), or four (see, e.g., Moore et al. Oncotarget 2014, 5(24): 12862-12876) PDHK isoforms and 5 very few examples have been reported of isoform-selective PDHK inhibitors (see, e.g., Liu et al. J. Med. Chem.2017, 60(6): 2227-2244).

[0077] Compounds that selectively inhibit a single PDHK isoform may offer advantages over unselective PDHK inhibitors. PDHK protein expression is tissue specific (see, e.g., Klyuyeva et al. J. Biol. Chem.2019, 294(3): 828-851) and different isoforms are elevated in different 10 diseases states (see, e.g., Park et al. Diabetes Metab. J.2018, 42(4): 270-281), suggesting that it may only be necessary to target individual isoforms for therapeutic purposes. In support of this notion, single isoform si- and sh- RNA PDHK knockdown experiments have shown anti- tumor effects in mouse models (see, e.g., McFate et al. J. Biol. Chem.2008, 283(33): 22700- 22708). In contrast, knocking out all four PDHK isoforms results in mice not surviving 15 beyond the first few days after birth (see, e.g.,. Heinemann-Yerushalmi, et al. Dev. Cell.

[0078] 2021, 56: 1182-1194). Unfortunately, to date effective tool molecules for interrogating the biology of individual PDHK isoforms have been severely lacking. Thus, the need exists to identify isoform selective PDHK inhibitors both to increase our understanding of PDHK biology and as potential therapeutics.

[0079] 20 Definitions

[0080] 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, 25 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.

[0081] 30 As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise.

[0082] 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,

[0083] 8 UM-43663.601

[0084] B, C, AB, AC, BC, and ABC, each of which is to be considered separately described by the statement “A, B, and / or C.”

[0085] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6- 5 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.

[0086] 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 10 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; 15 Larock, Comprehensive Organic Transformations, 3rdEdition, John Wiley & Sons, Inc., New York, 2018; and Carruthers, Some Modern Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

[0087] As used herein, the term “alkyl” refers to a radical of a straight or branched saturated 20 hydrocarbon chain. The alkyl chain can include, e.g., from 1 to 24 carbon atoms (C1-C24 alkyl), 1 to 16 carbon atoms (C1-C16alkyl), 1 to 14 carbon atoms (C1-C14alkyl), 1 to 12 carbon atoms (C1-C12 alkyl), 1 to 10 carbon atoms (C1-C10 alkyl), 1 to 8 carbon atoms (C1-C8 alkyl), 1 to 6 carbon atoms (C1-C6alkyl), 1 to 4 carbon atoms (C1-C4alkyl), 1 to 3 carbon atoms (C1-C3 alkyl), or 1 to 2 carbon atoms (C1-C2 alkyl). Representative examples of alkyl 25 include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso- butyl, 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.

[0088] 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 30 of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, and tert-butoxy.

[0089] 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 14 π electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms (“C6-C14aryl”). In some

[0090] 9 UM-43663.601

[0091] embodiments, an aryl group has six ring carbon atoms (“C6aryl,” i.e., phenyl). In some embodiments, an aryl group has ten ring carbon atoms (“C10aryl,” e.g., naphthyl such as 1- naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms (“C14aryl,” e.g., anthracenyl and phenanthrenyl).

[0092] 5 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,

[0093] 10 bicyclo[3.2.1]octanyl, and bicyclo[5.2.0]nonanyl.

[0094] As used herein, the term “halogen” or “halo” refers to F, Cl, Br, or I.

[0095] 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 π electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom 15 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 one 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 20 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., 25 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

[0096] 30 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

[0097] 10 UM-43663.601

[0098] 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, 5 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.

[0099] As used herein, the term “thioalkoxy” refers to an alkyl group, as defined herein, 10 appended to the parent molecular moiety through a sulfur atom. Representative examples of alkoxy include, but are not limited to, methylthio, ethylthio, and propoxythio.

[0100] 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 15 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, 20 haloalkyl, haloalkoxy, heteroaryl, heterocyclyl, hydroxy, hydrazino, imino, oxo, nitro, phosphate, phosphonate, sulfonic acid, thiol, thione, or combinations thereof.

[0101] As used herein, in chemical structures the indication:

[0102] represents a point of attachment of one mo

[0103]

[0104] o another moiety (e.g., a substituent group to 25 the rest of the compound).

[0105] 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.

[0106] 30 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-,

[0107] 11 UM-43663.601

[0108] such indication also encompasses -OCH2-; similarly, -OC(O)NH- also encompasses - NHC(O)O-.

[0109] The terms “administer,” “administering,” or “administration,” as used herein refer to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a compound or 5 a pharmaceutical composition.

[0110] As used herein, the terms “condition,” “disease,” and “disorder” are used interchangeably.

[0111] 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 10 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 15 or stop the growth or spread of a tumor.

[0112] 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 20 treatment of a condition or to minimize one or more symptoms associated with the condition.

[0113] 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

[0114] 25 condition, or enhances the therapeutic efficacy of another therapeutic agent.

[0115] 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, 30 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).

[0116] 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

[0117] 12 UM-43663.601

[0118] 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 developed 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

[0119] 5 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. Compounds

[0120] Disclosed herein are compounds of formula (Ia):

[0121] 10 a)

[0122] and pharmaceutically accepta wherein:

[0123]

[0124] R1is selected from hydrogen, halo, C1-C6 alkoxy, C1-C6 thioalkoxy, and C1-C6 alkyl; R2is selected from hydrogen, halo, C1-C6 alkoxy, C3-C6 cycloalkyl, hydroxy, and - NR2aR2b, wherein R2aand R2bare each independently C1-C6 alkyl; or

[0125] 15 R1and R2are taken together with the atoms to which they are attached to form a heteroaryl that is unsubstituted or is substituted with one C1-C6 alkyl group;

[0126] R3is selected from hydrogen, halo, -COOH, and C1-C6alkoxy;

[0127] R4is selected from hydrogen, -COOR5a, -CONR5bR5c, -CN, and C1-C6 alkoxy;

[0128] R5a, R5b, and R5care each independently selected from hydrogen and C1-C6alkyl; and 20 R6is phenyl or a monocyclic 5- or 6-membered heteroaryl, each of which is independently unsubstituted or substituted with 1, 2, or 3 substituents independently selected from halo, C1-C6 alkyl, C1-C6 alkoxy, and C3-C6 cycloalkyl.

[0129] Also disclosed herein are compounds of formula (I):

[0130] a)

[0131] 25 and pharmaceutically accepta

[0132]

[0133] , wherein:

[0134] 13 UM-43663.601

[0135] R1is selected from hydrogen, halo, C1-C6alkoxy, and C1-C6thioalkoxy;

[0136] R2is selected from hydrogen, halo, C1-C6alkoxy, and C3-C6cycloalkyl;

[0137] R3is selected from hydrogen and halo;

[0138] R4is selected from -COOR5a, -CONHR5b, -CN, and C1-C6alkoxy;

[0139] 5 R5aand R5bare each independently selected from hydrogen and C1-C6 alkyl; and R6is phenyl or a monocyclic 5- or 6-membered heteroaryl, each of which is independently unsubstituted or substituted with 1, 2, or 3 substituents independently selected from halo, C1-C6alkyl, C1-C6alkoxy, and C3-C6cycloalkyl.

[0140] In some embodiments, R1is selected from hydrogen, halo, and C1-C6 thioalkoxy. In 10 some embodiments, R1is selected from hydrogen, halo, and C1-C3thioalkoxy. In some embodiments, R1is selected from hydrogen, fluoro, chloro, bromo, and methylthio. In some embodiments, R1is halo. In some embodiments, R1is chloro. In some embodiments, R1is bromo. In some embodiments, R1is C1-C6 alkyl. In some embodiments, R1is selected from methyl and ethyl. In some embodiments, R1is selected from methylthio and ethylthio. In 15 some embodiments, R1is methoxy.

[0141] In some embodiments, R2is selected from hydrogen, halo, C1-C4 alkoxy, and C3-C4 cycloalkyl. In some embodiments, R2is selected from hydrogen, chloro, bromo, methoxy, ethoxy, isopropoxy, and cyclopropyl. In some embodiments, R2is hydrogen. In some embodiments, R2is halo. In some embodiments, R2is C1-C4alkoxy. In some embodiments, 20 R2is C3-C4 cycloalkyl. In some embodiments, R2is hydroxy. In some embodiments, R2is and -NR2aR2b, wherein R2aand R2bare each independently C1-C6alkyl. In some embodiments, R2is -N(CH3)2.

[0142] In some embodiments, R1and R2are taken together with the atoms to which they are attached to form a heteroaryl that is unsubstituted. In some embodiments, R1and R2are taken 25 together with the atoms to which they are attached to form a 5- or 6-membered monocyclic heteroaryl having 1 or 2 nitrogen atoms that is unsubstituted. In some embodiments, R1and R2are taken together with the atoms to which they are attached to form a heteroaryl that is substituted with one methyl group. In some embodiments, R1and R2are taken together with the atoms to which they are attached to form a 5- or 6-membered monocyclic heteroaryl 30 having 1 or 2 nitrogen atoms that is substituted with one methyl group.

[0143] In some embodiments, R3is selected from hydrogen and chloro. In some embodiments, R3is hydrogen. In some embodiments, R3is halo. In some embodiments, R3is chloro. In some embodiments, R3is -COOH. In some embodiments, R3is C1-C6 alkoxy. In some embodiments, R3is methoxy.

[0144] 14 UM-43663.601

[0145] In some embodiments, R4is selected from -COOR5a, -CONHR5b, C1-C3alkoxy, and - CN. In some embodiments, R4is selected from -COOR5a, -CONHR5b, C1-C3alkoxy, and - CN, wherein R5aand R5bare each independently selected from hydrogen and methyl. In some embodiments, R4is selected from -COOR5a, -CONHR5b, methoxy, and -CN, wherein R5ais 5 hydrogen and R5bis selected from hydrogen and methyl. In some embodiments, R4is - COOR5aand R5ais hydrogen. In some embodiments, R4is hydrogen. In some embodiments, R4is -CONR5bR5c, wherein R5band R5care each methyl.

[0146] 10

[0147]

[0148] 15 UM-43663.601

[0149] 5

[0150] ,

[0151]

[0152] yl 10 having 1 or 2 heteroatoms independently selected from S, O, and N, wherein the phenyl or heteroaryl is unsubstituted or substituted with 1, 2, or 3 substituents independently selected

[0153] 16 UM-43663.601

[0154] from halo, C1-C6alkyl, C1-C6alkoxy, and C3-C6cycloalkyl. In some embodiments, R6is phenyl or a monocyclic 5- or 6-membered heteroaryl having 1 or 2 heteroatoms independently selected from S, O, and N, wherein the phenyl or heteroaryl is unsubstituted or substituted with 1, 2, or 3 substituents independently selected from halo, C1-C4alkyl, C1-C45 alkoxy, and C3-C6 cycloalkyl. In some embodiments, R6is phenyl or a monocyclic 5- membered heteroaryl having one heteroatom selected from S, O, and N, wherein the phenyl or heteroaryl is substituted with 1 or 2 substituents independently selected from halo and C1- C4alkyl. In some embodiments, R6is phenyl substituted with 2 substituents independently selected from halo and C1-C4 alkyl. In some embodiments, R6is a monocyclic 5-membered 10 heteroaryl having one heteroatom selected from S, O, and N, which is substituted with 1 substituent selected from halo and C1-C4 alkyl.

[0155] In some embodiments, R6has a structure:

[0156] wherein:

[0157]

[0158] 15 R7ais halo;

[0159] R7bis hydrogen or halo;

[0160] R7cis C1-C4 alkyl or hydrogen;

[0161] R7dis halo or C1-C4alkyl; and

[0162] X is S, O, or NH.

[0163] 20 In some embodiments, R7ais chloro or bromo. In some embodiments, R7ais chloro. In some embodiments, R7ais bromo. In some embodiments, R7bis hydrogen or chloro. In some embodiments, R7bis hydrogen. In some embodiments, R7cis methyl or hydrogen. In some embodiments, R7cis methyl. In some embodiments, R7cis hydrogen. In some embodiments, R7bis chloro. In some embodiments, R7dis halo. In some embodiments, R7dis chloro. In 25 some embodiments, X is S.

[0164] In some embodiments, R6has a structure:

[0165] wherein:

[0166]

[0167] 17 UM-43663.601

[0168] R7ais halo;

[0169] R7bis hydrogen or halo; and

[0170] R7cis C1-C4 alkyl or hydrogen.

[0171] In some embodiments, R7ais chloro or bromo. In some embodiments, R7ais chloro. In 5 some embodiments, R7ais bromo. In some embodiments, R7bis hydrogen or chloro. In some embodiments, R7bis hydrogen. In some embodiments, R7cis methyl or hydrogen. In some embodiments, R7cis methyl. In some embodiments, R7cis hydrogen. In some embodiments, R7bis chloro.

[0172] In some embodiments, R7cis C1-C4 alkoxy. In some embodiments, R7cis methoxy.

[0173] 10 In some embodiments, R6has a structure:

[0174] wherein:

[0175]

[0176] R7dis halo or C1-C4 alkyl; and

[0177] X is S, O, or NH.

[0178] 15 In some embodiments, R7dis halo. In some embodiments, R7dis chloro. In some embodiments, X is S.

[0179] In some embodiments, R6has a structure selected from:

[0180] .

[0181]

[0182] 20 .

[0183] In some emb

[0184]

[0185] ,

[0186] .

[0187]

[0188] n some embodiments, the compound of formula (I) is selected from:

[0189] 18 UM-43663.601

[0190] 5

[0191]

[0192] 19 UM-43663.601

[0193] 5 an

[0194]

[0195] In some embodiments, the compound of formula (I) is selected from:

[0196] ,

[0197]

[0198] 20 UM-43663.601

[0199] ,

[0200]

[0201] 5 lly

[0202]

[0203] Certain compounds described herein may have at least one asymmetric center. 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 10 (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.

[0204] 21 UM-43663.601

[0205] 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 5 intermediates that are derivatized, if necessary, with a reagent containing an asymmetric center of known absolute configuration.

[0206] 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 10 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 15 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.

[0207] Compounds may also possess tautomeric forms, and all tautomers also constitute 20 embodiments of the disclosure.

[0208] 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

[0209] 25 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,17O,31P,32P,35S,18F, and36Cl, 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 30 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) are11C,13N,15O, and18F. Isotopically-labeled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by

[0210] 22 UM-43663.601

[0211] processes analogous to those described in the accompanying Examples using appropriate isotopically-labeled reagent in place of non-isotopically-labeled reagent.

[0212] 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 5 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.

[0213] a. Methods of Synthesis

[0214] 10 Compounds disclosed herein can be prepared by a variety of methods, including those illustrated in the Examples.

[0215] 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 15 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 Furniss, Hannaford, Smith, and Tatchell, pub. Longman 20 Scientific & Technical, Essex CM202JE, England.

[0216] 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 25 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.

[0217] Standard experimentation, including appropriate manipulation of the reaction 30 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 deprotecting different substituents using such suitable protecting groups are well known to those skilled in

[0218] 23 UM-43663.601

[0219] 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).

[0220] 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 5 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).

[0221] Similarly, when a pure geometric isomer of a compound is required, it can be 10 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.

[0222] The synthetic schemes and specific examples as described are illustrative and are not to be read as limiting the scope of the disclosure or the claims. Alternatives, modifications, 15 and equivalents of the synthetic methods and specific examples are contemplated.

[0223] b. Pharmaceutically Acceptable Salts

[0224] 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, 20 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 25 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, 30 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 the like. Amino groups of the compounds may also be quaternized with alkyl chlorides, bromides

[0225] 24 UM-43663.601

[0226] and iodides such as methyl, ethyl, propyl, isopropyl, butyl, lauryl, myristyl, stearyl and the like.

[0227] 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 5 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,

[0228] 10 dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine and N,N’-dibenzylethylenediamine, ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like.

[0229] Pharmaceutical Compositions

[0230] The disclosed compounds may be incorporated into pharmaceutical compositions 15 suitable for administration to a subject (such as a patient, which may be a human or non- human). The pharmaceutical compositions may include a therapeutically effective amount or a prophylactically effective amount of the agent.

[0231] The pharmaceutical compositions may include pharmaceutically acceptable carriers. The term “pharmaceutically acceptable carrier,” as used herein, means a non-toxic, inert 20 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 25 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; 30 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,

[0232] 25 UM-43663.601

[0233] flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

[0234] 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, 5 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.

[0235] 10 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).

[0236] 15 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.

[0237] Suitable diluents include sugars such as glucose, lactose, dextrose, and sucrose; diols 20 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.

[0238] Suitable lubricants include silica, talc, stearic acid and its magnesium salts and calcium salts, calcium sulfate; and liquid lubricants such as polyethylene glycol and

[0239] 25 vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn 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.

[0240] Suitable binders include polyvinyl pyrrolidone; magnesium aluminum silicate; starches such as corn starch and potato starch; gelatin; tragacanth; and cellulose and its 30 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.

[0241] Suitable disintegrants include agar, alginic acid and the sodium salt thereof, effervescent mixtures, croscarmellose, crospovidone, sodium carboxymethyl starch, sodium

[0242] 26 UM-43663.601

[0243] 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.

[0244] 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 5 weight of the composition.

[0245] 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%.

[0246] Suitable sweeteners include aspartame and saccharin. The amount of sweetener(s), 10 when used, in a systemic or topical composition is typically about 0.001 to about 1% by weight of the composition.

[0247] 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.

[0248] 15 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.

[0249] 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.

[0250] 20 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.

[0251] Suitable suspending agents include AVICEL RC-591 (from FMC Corporation of 25 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.

[0252] 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; 30 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.

[0253] 27 UM-43663.601

[0254] 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 of one or more carriers. Compositions for parenteral administration typically include 0.1% to 5 10% by weight of actives and 90% to 99.9% by weight of a carrier including a diluent and a solvent.

[0255] 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

[0256] 10 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.

[0257] Tablets can be compressed, tablet triturates, enteric-coated, sugar-coated, film-coated, or multiple-compressed. Tablets typically include an active component, and a carrier 15 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 20 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.

[0258] Capsules (including implants, time release and sustained release formulations) typically include an active compound (e.g., a compound of formula (I)), and a carrier 25 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.

[0259] The selection of ingredients in the carrier for oral compositions depends on secondary 30 considerations like taste, cost, and shelf stability, which are not critical for the purposes of this disclosure.

[0260] 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

[0261] 28 UM-43663.601

[0262] desired action. The coatings typically include one or more components selected from the group consisting of cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methyl cellulose phthalate, ethyl cellulose, EUDRAGIT® coatings (available from Evonik Industries of Essen, Germany), waxes and shellac.

[0263] 5 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 10 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.

[0264] Other compositions useful for attaining systemic delivery of the subject compounds include sublingual, buccal and nasal dosage forms. Such compositions typically include one 15 or more of soluble filler substances such as diluents including sucrose, sorbitol and mannitol;

[0265] and binders such as acacia, microcrystalline cellulose, carboxymethyl cellulose, and hydroxypropyl methylcellulose. Such compositions may further include lubricants, colorants, flavors, sweeteners, antioxidants, and glidants.

[0266] The disclosed compounds can be topically administered. Topical compositions that 20 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 25 compounds into the skin. The carrier may further include one or more optional components.

[0267] 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: Modern Pharmaceutics, Chapters 30 9 and 10, Banker & Rhodes, eds. (1979); Lieberman et al. Pharmaceutical Dosage Forms:

[0268] Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms, 2nd Ed., (1976).

[0269] 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,

[0270] 29 UM-43663.601

[0271] deionized water, monofunctional alcohols, symmetrical alcohols, aloe vera gel, allantoin, glycerin, vitamin A and E oils, mineral oil, propylene glycol, PPG-2 myristyl propionate, 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 5 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] 10 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,

[0274] 15 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 20 typically about 5% to about 95% by weight of the composition.

[0275] 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.

[0276] Suitable solvents include water, ethyl alcohol, methylene chloride, isopropanol, castor 25 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.

[0277] 30 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 humectant(s) in a topical composition is typically 0% to 95% by weight of the composition.

[0278] 30 UM-43663.601

[0279] The amount of thickener(s) in a topical composition is typically about 0% to about 95% by weight of the composition.

[0280] Suitable powders include beta-cyclodextrins, hydroxypropyl cyclodextrins, chalk, talc, fullers earth, kaolin, starch, gums, colloidal silicon dioxide, sodium polyacrylate, tetra 5 alkyl ammonium smectites, trialkyl aryl ammonium smectites, chemically-modified magnesium aluminum silicate, organically-modified montmorillonite clay, hydrated aluminum silicate, fumed silica, carboxyvinyl 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.

[0281] 10 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.

[0282] Suitable pH adjusting additives include HCl or NaOH in amounts sufficient to adjust the pH of a topical pharmaceutical composition.

[0283] Methods of Use

[0284] 15 Compounds disclosed herein are inhibitors of PDHKs, and in some embodiments, compounds disclosed herein are selective inhibitors of PDHK1. Accordingly, compounds and pharmaceutical compositions disclosed herein may be used in methods for treatment of disorders, including proliferative diseases such as cancers. In some embodiments, the disorder is characterized or mediated by the activity of a PDHK, such as PDHK1. In some 20 embodiments, the disorder is one in which a PDHK, such as PDHK1, is mutated or overexpressed.

[0285] Accordingly, in some embodiments, disclosed herein is a method of treating a disorder associated with a PDHK in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of a compound disclosed 25 herein (e.g., a compound of formula (I) or a pharmaceutically acceptable salt thereof), or a pharmaceutical composition comprising a compound disclosed herein (e.g., a compound of formula (I) or a pharmaceutically acceptable salt thereof).

[0286] In some embodiments, the disorder is a proliferative disease, i.e., a disease that occurs due to abnormal growth or extension by the multiplication of cells. In some embodiments, the 30 proliferative disease is cancer. The term “cancer” refers to a class of diseases characterized by development of abnormal cells that proliferate uncontrollably and have the ability to infiltrate and destroy normal body tissues. See, e.g., Stedman’s Medical Dictionary, 25th ed.; Hensyl ed.; Williams & Wilkins: Philadelphia, 1990. Accordingly, in some embodiments, the

[0287] 31 UM-43663.601

[0288] compounds and pharmaceutical compositions disclosed herein are used for treating cancer in a subject in need thereof. In some embodiments, the cancer is a cancer in which a PDHK, such as PDHK1, is mutated. In some embodiments, the cancer is a cancer in which a PDHK, such as PDHK1, is highly expressed, or is overexpressed relative to non-cancerous cells. 5 In some embodiments, the cancer is a solid tumor, such as a sarcoma or a carcinoma.

[0289] In some embodiments, the cancer is a hematologic malignancy.

[0290] In some embodiments, the cancer is one known to be associated with PDHKs, in particular PDHK1. For example, such cancers include acute myeloid leukemia (see, e.g., Erdem et al. Nat. Commun.2002, 13(1):1105), breast cancer (see, e.g., Peng et al. Oncogene 10 2018, 37(8):1062-1074), esophageal cancer (see, e.g., Dong et al. Oncotarget 2016,

[0291] 7(42):68170-68178), gastric cancer (see, e.g., Hur et al. Int. J. Oncol.2013, 42(1):44-54), glioblastoma (see, e.g., Larrieu et al. Cancers 2022, 14(15): 3769), head and neck cancer (see, e.g., Hsu et al. FASEB J.2017, 31(10):4265-4276), multiple myeloma (see, e.g., Fujiwara et al. Brit. J. Cancer 2013, 108(1):170-178), nasopharyngeal cancer (see, e.g., 15 Xiang et al. Pathol. Res. Pract.2016, 212(12):1102-1107), ovarian cancer (see, e.g., Siu et al. Oncogenesis 2020, 9(2):24), retinoblastoma (see, e.g., Sradhanjali et al. PLoS One 2017, 12(5):e0177744), prostate cancer (see, e.g., Nunes-Xavier et al. Front. Oncol.2022, 12:873516), bladder cancer (see, e.g., Lee et al. Int. J. Mol. Sci.2022, 23(21):13240), glioma (see, e.g., Jha et al. Neurosci. Biobehav. R.2016, 68:1-19), hepatocellular carcinoma (see, 20 e.g., Shen et al. Brit. J. Cancer 2013, 108(1):72-81), non-small cell lung cancer (see, e.g., Guo et al. Bioorg. Med. Chem. Lett.2019, 29(2):291-296), pancreatic cancer (see, e.g., Tataranni et al. Cells 2019, 8(5):478), and transitional cell carcinoma (see, e.g., Harting et al. Int. J. Oncol.2016, 49(6):2341-2350). Accordingly, in some embodiments, the methods of treatment disclosed herein include cancers selected from acute myeloid leukemia, breast 25 cancer, esophageal cancer, gastric cancer, glioblastoma, head and neck cancer, multiple myeloma, nasopharyngeal cancer, ovarian cancer, retinoblastoma, prostate cancer, bladder cancer, glioma, hepatocellular carcinoma, non-small cell lung cancer, pancreatic cancer, and transitional cell carcinoma, in a subject in need thereof.

[0292] In some embodiments, the cancer is a relapsed or refractory cancer, such as a cancer 30 described herein. In some embodiments, the cancer is a metastasized cancer, such as a cancer described herein.

[0293] In addition to proliferative diseases such as cancers, PDHKs including PDHK1 have also been associated with other disorders, including neurodegeneration such as that associated with Alzheimer’s disease (see, e.g., Pal et al. Front. Aging Neurosci.2023 Nov

[0294] 32 UM-43663.601

[0295] 14:15:1282855), multiple sclerosis (see, e.g., Gerriets et al. J. Clin. Invest.2015, 125(1):194- 20 ), hepatic steatosis (see, e.g., Go et al. Diabetes 2016, 65(10):2876-87), Charcot-Marie- Tooth disease (see, e.g., Narayanan et al. Hum. Mol. Genet.2021 Dec 17;31(1):133-145), Barth Syndrome (see, e.g., Liang et al. Sci. Rep.2024;14(1):11497), heart failure (see, e.g., 5 Aizawa et al. Cells 2023 Dec 30;13(1):8), inflammatory bowel diseases including Crohn’s disease (see, e.g., Lee et al. Cell Mol. Gastroenterol. Hepatol.2023, 15(2):439-46), metabolic syndrome (see, e.g., Lee et al. J. Med. Chem.2019, 62(2):575-58), metabolic dysfunction- associated steatohepatitis (see, e.g., Zhang et al. Biochem. Biophys. Res. Commun.2018, 495(1):582-586), diabetes (see, e.g., Jeoung, Diabetes Metab. J.2015, 39(3):188-97), 10 endometriosis (see, e.g., Horne et al. Proc. Natl. Acad. Sci. USA 2019, 116(51):25389- 25391), heart fibrosis (see, e.g., Tian et al. Circ. Res.2020, 126(12):1723-1745), lactic acidosis (see, e.g., Kuroda et al. J. Inerit. Metab. Dis.1986, 9(3):244-52), macular degeneration (see, e.g., Lambert et al. J. Mol. Med.2020, 98(12):1737-1751), myalgic encephalomyelitis / long COVID (see, e.g., Fluge et al. JCI Insight 2016, 1(21):e89376), 15 nonalcoholic fatty liver disease (see, e.g., Saed et al. ACS Pharmacol. Transl. Sci.2021, 4(2):

[0296] 582–588), pyruvate dehydrogenase deficiency (see, e.g., Patel et al. Mol. Genet. Metab.2012, 106(3):385-94.), and pulmonary arterial hypertension (see, e.g., Michelakis et al. Sci. Transl. Med.2017, 9(413):eaao4583). Accordingly, in some embodiments, the methods of treatment disclosed herein include methods of treating a disorder selected from Alzheimer's disease, 20 multiple sclerosis, hepatic steatosis, Charcot-Marie-Tooth disease, Barth Syndrome, heart failure, inflammatory bowel disease, metabolic syndrome, metabolic dysfunction-associated steatohepatitis, diabetes, endometriosis, heart fibrosis, lactic acidosis, macular degeneration, myalgic encephalomyelitis, nonalcoholic fatty liver disease, pyruvate dehydrogenase deficiency, and pulmonary arterial hypertension, in a subject in need thereof.

[0297] 25 In the methods of treatment disclosed herein, a compound or pharmaceutical composition may be administered to the subject by any convenient route of administration, whether systemically / peripherally or at the site of desired action, including but not limited to, oral (e.g., by ingestion); topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., an aerosol, e.g., 30 through mouth or nose); rectal; vaginal; parenteral (e.g., by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal injection); or by implant of a depot, for example, subcutaneously or intramuscularly. In some embodiments, the administration comprises oral

[0298] 33 UM-43663.601

[0299] administration. In some embodiments, the administration comprises parenteral administration. Additional modes of administration may include adding the compound and / or a composition comprising the compound to a food or beverage, including a water supply for an animal, to supply the compound as part of the animal’s diet.

[0300] 5 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 of the present disclosure. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular 10 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

[0301] 15 concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects.

[0302] 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 20 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 carried out with the dose level and pattern being selected by the treating physician. In general, a suitable dose of the compound is in the range of about 100 μg to about 250 mg per kilogram body weight of the subject per day.

[0303] 25 The compound or composition may be administered once, on a continuous basis (e.g.

[0304] by an intravenous drip), or on a periodic / intermittent basis, including about once per hour, about once per two hours, about once per four hours, about once per eight hours, about once per twelve hours, about once per day, about once per two days, about once per three days, about twice per week, about once per week, and about once per month. The composition may 30 be administered until a desired reduction of symptoms is achieved.

[0305] A compound 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

[0306] 34 UM-43663.601

[0307] 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 of administration. This is sometimes referred to herein as “simultaneous” or “concurrent 5 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 10 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 15 effect of the first treatment delivered is still detectable when the second is delivered.

[0308] 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 20 order of administration can be reversed.

[0309] In some embodiments, a compound described herein is administered in combination with other therapeutic treatment modalities, including chemotherapy, surgery, radiation therapy, hormone therapy, immunotherapy, transplantation (e.g., stem cell transplantation, bone marrow transplantation), cryotherapy, T cell transfer therapy, and thermotherapy, or any 25 combination thereof. Such combination therapies may allow for lower dosages of the administered agent and / or other agent (e.g., chemotherapeutic agent), thus avoiding possible toxicities or complications associated with the various therapies.

[0310] In some embodiments, the compound described herein is administered with at least one additional therapeutic agent, such as a chemotherapeutic agent. In certain embodiments, 30 the compound described herein is administered in combination with one or more additional chemotherapeutic agents. The chemotherapeutic agent may be a chemotherapeutic agent identified on the “A to Z List of Cancer Drugs” published by the National Cancer Institute. Kits

[0311] 35 UM-43663.601

[0312] 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 comosition comprising a compound of formula (I), or a 5 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 can be formed from a variety of 10 materials such as glass or plastic.

[0313] 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 are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, 15 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 20 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.

[0314] For example, a kit typically includes one or more additional containers, each with one 25 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 30 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 carrier 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 a

[0315] 36 UM-43663.601

[0316] 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, 5 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 10 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.

[0317] 15 Examples

[0318] Abbreviations used in the Examples include the following: Ac is acetyl; AcCl is acetyl chloride; BOP is benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate; BSA is bovine serum albumin; DCM is dichloromethane; DIAD is diisopropyl azodicarboxylate; DIPEA is N,N-diisopropylethylamine; DMAP is N,N-20 dimethylaminopyridine; DMF is N,N-dimethylformamide; DMSO is dimethyl sulfoxide;

[0319] EGTA is ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; ESI-TOF is electrospray ionization time-of-flight; EtOAc is ethyl acetate; EtOH is ethanol; h is hours; HRMS is high resolution mass spectrometry; iPrOH is isopropanol; MeOH is methanol; min is minutes; OAc is acetate; PBS is phosphate-buffered saline; RIPA is

[0320] 25 radioimmunoprecipitation assay; r.t. is room temperature; TBST is Tris-buffered saline with Tween 20; THF is tetrahydrofuran; and Xantphos is (9,9-Dimethyl-9H-xanthene-4,5- diyl)bis(diphenylphosphane).

[0321] Example 1

[0322] Compound Syntheses

[0323] 30 General Procedure A: Nitroaryl reduction. The appropriate nitroaryl (1 eq), iron powder (4 eq), and ammonium chloride (6 eq) were combined in 2:1 methanol / water (0.2 M) and stirred at 60 ºC for 18–24 h. Once cooled to r.t., the reaction mixture was passed through a plug of Celite. The filtrate was extracted with DCM (3x) and the combined organic portions

[0324] 37 UM-43663.601

[0325] were washed with brine (1x), dried over sodium sulfate, filtered, and concentrated in vacuo to afford the product. The product was purified by column chromatography if required.

[0326] General Procedure B: Sulfonamide Coupling. The appropriate aniline (1 eq), the appropriate sulfonyl chloride (1–1.2 eq), and DMAP (0.05–0.25 eq) were dissolved in 5 anhydrous THF (0.1–0.3 M). Pyridine (3 eq) was added and the reaction mixture was stirred at 60 ºC for 16–26 h. In some cases, acetyl chloride (0.75–2.2 eq) was added to react out remaining aniline starting material and aid in separation during purification. The reaction mixture was diluted with water and extracted with ethyl acetate (3x). Combined organic portions were washed with brine (1x), dried over sodium sulfate, decanted, and concentrated 10 in vacuo. The product was purified by column chromatography in ethyl acetate / hexanes.

[0327] General Procedure C: Ester Hydrolysis. The appropriate methyl ester was dissolved in methanol or ethanol and an equal volume of 1 M aqueous sodium hydroxide to achieve an ester concentration of 0.1 M. The reaction mixture was stirred at 55 ºC for 4–26 h. The solvent was removed in vacuo and the remaining residue was dissolved in water. The 15 resulting aqueous mixture was acidified with hydrochloric acid solution (1 M in water) and the precipitate was filtered, rinsed with water, and dried to afford the product.

[0328] Scheme 1. General synthetic route toward decorated biarylsulfonamide analogs i) Fe, NH4Cl, 2:1 MeOH / H2O, 60 ºC. ii) R3–SO2Cl, DMAP, pyridine, THF, 60 ºC, then, if necessary, AcCl, 20 r.t. iii) 1 M aq. NaOH, MeOH, 55 ºC.

[0329]

[0330] Synthesis of 3-Bromo-5-((5-chloro-2-methylphenyl)sulfonamido)benzoic acid (PTM-159)

[0331]

[0332] 38 UM-43663.601

[0333] Step i: Methyl 3-amino-5-bromobenzoate). Methyl 3-bromo-5-nitrobenzoate (260 mg, 1.0 mmol) was reacted according to General Procedure A. Purification by flash chromatography (0–40% EtOAc / hexanes gradient over 18 min, 25 g column) afforded the title compound as a white solid (150 mg, 65%).1H NMR (400 MHz, CDCl3) δ 7.53 (s, 1H), 7.26 5 (s, 1H), 6.99 (s, 1H), 3.89 (s, 3H), 3.84 (s, br, 2H).13C NMR (101 MHz, CDCl3) δ 166.16, 147.82, 132.74, 123.06, 122.45, 121.80, 114.72, 52.48.

[0334] Step ii: Methyl 3-bromo-5-((5-chloro-2-methylphenyl)sulfonamido)benzoate. Methyl 3-amino-5-bromobenzoate (25 mg, 0.11 mmol) was reacted according to General Procedure B. Purification by flash chromatography (0–40% EtOAc / hexanes gradient over 16 min, 12 g 10 column) afforded the title compound as a white solid (17 mg, 37%).1H NMR (499 MHz, CD3OD) δ 7.94 (d, J = 2.3 Hz, 1H), 7.77 (s, 1H), 7.68 (s, 1H), 7.50 (dd, J = 8.2, 2.3 Hz, 1H), 7.47 (s, 1H), 7.35 (d, J = 8.2 Hz, 1H), 3.89 (s, 3H), 2.60 (s, 3H).13C NMR (126 MHz, CD3OD) δ 166.30, 140.54, 140.26, 137.40, 135.52, 134.26, 134.10, 133.06, 130.63, 128.56, 127.06, 123.73, 119.65, 53.11, 19.65.

[0335] 15 Step iii: 3-Bromo-5-((5-chloro-2-methylphenyl)sulfonamido)benzoic acid (PTM-159).

[0336] Following General Procedure C, methyl 3-bromo-5-((5-chloro-2- methylphenyl)sulfonamido)benzoate (17 mg, 41 µmol) was subjected to ester hydrolysis to yield the title compound as a white solid (12 mg, 73%).1H NMR (499 MHz, CD3OD) δ 7.94 (d, J = 2.3 Hz, 1H), 7.78 (s, 1H), 7.70 (s, 1H), 7.50 (dd, J = 8.2, 2.3 Hz, 1H), 7.46 (s, 1H), 20 7.35 (d, J = 8.2 Hz, 1H), 2.60 (s, 3H).13C NMR (126 MHz, CD3OD) δ 167.35, 140.42,

[0337] 140.26, 137.37, 135.50, 134.92, 134.25, 133.08, 130.62, 128.86, 126.94, 123.60, 120.07, 19.66. HRMS (ESI / TOF) m / z for C14H11BrClNO4S [M-H]-: 401.9208 (calc), 401.9199 (observed), 403.9178 (observed, halogen peak).

[0338] Additional Compounds. The following additional compounds were synthesized in an25 analogous manner to that shown for PTM-159, and in Scheme 1.

[0339] Cpd Structure Mass Spec1H NMR

[0340] , , , , 8

[0341]

[0342] 39 UM-43663.601

[0343] PTM-1241H NMR (400 MHz,

[0344] HRMS (ESI / TOF) CD3OD) δ 8.04 (d, J 21 H 1H 780 , 3 7

[0345] ,

[0346] J 0 = , 2

[0347] ,

[0348] J 7

[0349] ,

[0350] J , 3 2

[0351]

[0352] 40 UM-43663.601

[0353] PTM-1431H NMR (499 MHz,

[0354] CD3OD) δ 8.11 (dd, 77 22 H 1H), 3 , ,

[0355] , , , ,

[0356] , J = d, 2

[0357] , ), ,

[0358] , J 1 0 = ,

[0359]

[0360] 41 UM-43663.601

[0361] PTM-157

[0362] HRMS (ESI / TOF)1H NMR (499 MHz, m / z for CD3OD) δ 8.06 (d J 0

[0363] ,

[0364] 2 , 9 J

[0365] , , 3 = ,

[0366] ,

[0367] J , ), , 7

[0368]

[0369] 42 UM-43663.601

[0370] PTM-1711H NMR (499 MHz,

[0371] CD3OD) δ 8.13 (d, J HRMS ESITOF 21 H 1H 770 d, 0 5

[0372] ,

[0373] , 3

[0374] ,

[0375] ,

[0376] ,

[0377] 2

[0378]

[0379] 43 UM-43663.601

[0380] PTM-3551H NMR (499 MHz,

[0381] HRMS (ESI / TOF) CD3OD) δ 7.94 (d, J 23 H 1H 766

[0382] , J , ,

[0383] , d, 6

[0384] , J 3 = d, 8

[0385] ).

[0386] , , 2

[0387]

[0388] 44 UM-43663.601

[0389] PTM-1281H NMR (499 MHz,

[0390] CD3OD) δ 7.76 (dd, 79 18 H 1H), 7 9

[0391] ,

[0392] , , 9 0 = , )

[0393]

[0394] 45 UM-43663.601

[0395] PTM-1741H NMR (499 MHz, CD3OD) δ 7.84 (s, 1H 768 d 23 s,

[0396] , J 5 1 = ,

[0397]

[0398] Scheme 2. Synthetic route toward thioether analogs. i) appropriate thiol, Pd2(dba)3, Xantphos, DIPEA, 1,4-dioxane, 100 ºC. ii) Fe, NH4Cl, 2:1 MeOH / H2O, 60 ºC. iii) R2–SO2Cl, DMAP, pyridine, THF, 60 ºC iv) 1 M aq. NaOH, MeOH, 55 ºC.

[0399] 5

[0400]

[0401] 46 UM-43663.601

[0402] Synthesis of 3-((5-Chloro-2-methylphenyl)sulfonamido)-4-(methylthio)benzoic acid (PTM-75)

[0403]

[0404] 5 Step i: Methyl 4-(methylthio)-3-nitrobenzoate. Under a nitrogen atmosphere, methyl 4- bromo-3-nitrobenzoate (100 mg, 0.39 mmol), Xantphos (45 mg, 77 µmol), and Pd2(dba)3(35 mg, 39 µmol) were dissolved in degassed 1,4-dioxane (2.20 mL). DIPEA (0.17 mL, 0.96 mmol) was added followed by sodium methanethiolate (0.27 mL, 0.58 mmol, 15% solution in water). The reaction mixture was refluxed and allowed to stir for 22.5 h. The reaction mixture 10 was cooled to r.t., diluted with diethyl ether, and filtered through a short plug of Celite, which was also rinsed with diethyl ether. The solvent was removed in vacuo to afford a brown oil. The oil was dissolved in DCM, dry loaded onto silica gel, and purified via column chromatography (0–30% EtOAc / hexanes gradient over 16 min, 25 g column) to yield the title compound as a yellow solid (44 mg, 50%).1H NMR (499 MHz, CDCl3) δ 8.88 (s, 1H), 8.19 15 (d, J = 8.5 Hz, 1H), 7.43 (d, J = 8.6 Hz, 1H), 3.96 (s, 3H), 2.55 (s, 3H).

[0405] Step ii: Methyl 3-amino-4-(methylthio)benzoate. Methyl 4-(methylthio)-3- nitrobenzoate (44 mg, 0.19 mmol) was reacted according to General Procedure A to afford the title compound as a yellow oil (25 mg, 65%).1H NMR (499 MHz, CDCl3) δ 7.40 (dd, J = 7.8, 1.9 Hz, 1H), 7.36 (d, J = 2.0 Hz, 1H), 7.28 (d, J = 8.1 Hz, 1H), 3.88 (s, 3H), 2.44 (s, 3H). 20 Step iii: Methyl 3-((5-chloro-2-methylphenyl)sulfonamido)-4-(methylthio)benzoate).

[0406] Methyl 3-amino-4-(methylthio)benzoate (25 mg, 0.13 mmol) was reacted according to General Procedure B. Purification by flash chromatography (0–30% EtOAc / hexanes gradient over 16 min, 12 g column) afforded the title compound as a yellow oil (13 mg, 27%).1H NMR (400 MHz, CDCl3) δ 8.02 (d, J = 2.3 Hz, 1H), 7.98 (d, J = 1.8 Hz, 1H), 7.74 (dd, J = 8.2, 1.8 Hz, 25 1H), 7.41 (dd, J = 8.2, 2.3 Hz, 1H), 7.35 (d, J = 8.2 Hz, 1H), 7.22 (d, J = 8.1 Hz, 1H), 3.90 (s, 3H), 2.62 (s, 3H), 2.40 (s, 3H).

[0407] Step iv: 3-((5-Chloro-2-methylphenyl)sulfonamido)-4-(methylthio)benzoic acid (PTM-75). Following General Procedure C, methyl 3-((5-chloro-2-methylphenyl)sulfonamido)-4-

[0408] 47 UM-43663.601

[0409] (methylthio)benzoate (13 mg, 0.034 mmol) was subjected to ester hydrolysis to yield the title compound as a yellow solid (9 mg, 72%).1H NMR (400 MHz, DMSO-d6) δ 12.93 (s, 1H), 10.08 (s, 1H), 7.77 (dd, J = 8.3, 1.9 Hz, 1H), 7.64 – 7.57 (m, 2H), 7.45 (d, J = 7.9 Hz, 1H), 7.40 (d, J = 1.9 Hz, 1H), 7.29 (d, J = 8.4 Hz, 1H), 2.55 (s, 3H), 2.35 (s, 3H).13C NMR (101 MHz, 5 DMSO-d6) δ 166.80, 145.29, 141.12, 136.49, 135.02, 133.02, 132.45, 130.87, 128.97, 128.91, 128.36, 127.33, 125.45, 20.00, 14.58. HRMS (ESI / TOF) m / z for C15H14ClNO4S2[M-H]-: 369.9980 (calc), 369.9972 (observed), 371.9946 (observed, 3:1 Cl peak).

[0410] Additional Compounds. The following additional compounds were synthesized in ananalogous manner to that shown for PTM-75 and in Scheme 2.

[0411] 10

[0412] Cpd Structure Mass Spec1H NMR

[0413] PTM-1471H NMR (499 MHz,

[0414] , 3

[0415] 2

[0416] , , , J d,

[0417] ,

[0418]

[0419] 48 UM-43663.601

[0420] PTM-981H NMR (400 MHz,

[0421] CD3OD) δ 8.00 (d, J = 19 H 1H 784 d J

[0422] 6

[0423] =

[0424] , 2

[0425]

[0426] cee . yec oues o ay ee aaogs. a3, , - –.., e H2SO4, MeOH, 60 ºC. ii) EtOH or iPrOH, DIAD, PPh3, THF, 0 ºC–r.t. iii) Fe, NH4Cl, 2:1 MeOH / H2O, 60 ºC. iv) 5-chloro-2-methylbenzenesulfonyl chloride, DMAP, pyridine, THF, 60 ºC. v) 1 M 5 aq. NaOH, MeOH, 55 ºC.

[0427]

[0428] zoic acid (PTM-211)

[0429] 10

[0430]

[0431] 49 UM-43663.601

[0432] Step i: Methyl 4-chloro-3-hydroxy-5-nitrobenzoate. A solution of boron tribromide (6.5 mL, 6.5 mmol, 1 M in DCM) was added dropwise to a solution of methyl 4-chloro-3-methoxy- 5-nitrobenzoate (400 mg, 1.63 mmol) in DCM (1.0 mL) at 0 ºC under a nitrogen atmosphere. The reaction mixture was allowed to warm to r.t. as it stirred for 18 h. The reaction was 5 quenched slowly with methanol at 0 ºC and the solvent was removed in vacuo. The remaining dark brown oil was dissolved in methanol (4 mL), concentrated sulfuric acid (0.2 mL) was added, and the reaction mixture was stirred in open air at 60 ºC for 24 h before being cooled to r.t. and concentrated in vacuo to afford a brown residue. The residue was dissolved in water and the aqueous layer was extracted with DCM (1x). The aqueous layer was basified with 10 aqueous saturated sodium bicarbonate solution and extracted with DCM (2x). The combined organic portions were washed with additional aqueous saturated sodium bicarbonate solution (1x) and brine (1x), dried over sodium sulfate, decanted, dry loaded onto silica gel, and purified via column chromatography (0–40% EtOAc / hexanes gradient over 18 min, 25 g column) to yield the title compound as a pale yellow solid (132 mg, 35%).1H NMR (400 MHz, CDCl3) δ 15 8.15 (d, J = 1.9 Hz, 1H), 7.92 (d, J = 1.9 Hz, 1H), 6.26 (s, br, 1H), 3.97 (s, 3H). HRMS (ESI / TOF) m / z for C8H6ClNO5 [M-H]-: 229.9862 (calc), 229.9862 (observed), 231.9836 (observed, 3:1 Cl peak).

[0433] Step ii: Methyl 4-chloro-3-isopropoxy-5-nitrobenzoate. Under a nitrogen atmosphere, triphenylphosphine (56 mg, 0.21 mmol) and methyl 4-chloro-3-hydroxy-5-nitrobenzoate (43 20 mg, 0.19 mmol) were dissolved in anhydrous THF (1.2 mL) and isopropanol (16 µL, 0.19 mmol) was added. The mixture was cooled to 0 ºC and DIAD (36 µL, 0.19 mmol) was added dropwise. The reaction mixture was stirred at r.t. for 4 h before being diluted with water and extracted with DCM (3x). The combined organic portions were washed with 1 M aqueous hydrochloric acid solution (3x) and brine (1x), dried over sodium sulfate, decanted, dry loaded 25 onto silica gel, and purified via column chromatography (0–30% EtOAc / hexanes gradient over 12 min, 12 g column) to afford the title compound as a white solid (39 mg, 77%).1H NMR (499 MHz, CDCl3) δ 7.97 (d, J = 1.8 Hz, 1H), 7.74 (d, J = 1.8 Hz, 1H), 4.73 (hept, J = 6.1 Hz, 1H), 3.95 (s, 3H), 1.42 (d, J = 6.0 Hz, 6H).13C NMR (126 MHz, CDCl3) δ 164.68, 155.28, 149.81, 129.73, 121.83, 117.61, 117.19, 73.46, 53.04, 21.90.

[0434] 30 Step iii: Methyl 3-amino-4-chloro-5-isopropoxybenzoate. Methyl 4-chloro-3-isopropoxy-5-nitrobenzoate (39 mg, 0.14 mmol) was reacted according to General ProcedureA. Due to only partial reduction observed by1H NMR after 18 h, the resulting mixture of desired product and partially reduced product was resubjected to General Procedure A for an additional 22 h to afford the title compound as a yellow oil (27 mg, 78%).1H NMR (400 MHz,

[0435] 50 UM-43663.601

[0436] CDCl3) δ 7.08 (d, J = 1.9 Hz, 1H), 7.01 (d, J = 1.8 Hz, 1H), 4.62 (hept, J = 6.1 Hz, 1H), 4.19 (s, br, 2H), 3.88 (s, 3H), 1.38 (d, J = 6.1 Hz, 6H).

[0437] Step iv: Methyl 4-chloro-3-((5-chloro-2-methylphenyl)sulfonamido)-5- isopropoxybenzoate. Methyl 3-amino-4-chloro-5-isopropoxybenzoate (27 mg, 0.11 mmol) was 5 reacted according to General Procedure B. Purification by flash chromatography (0–30% EtOAc / hexanes gradient over 16 min, 12 g column) afforded a mixture of aniline and desired product inseparable by normal-phase chromatography. To remedy this, mixture was dissolved in DCM (0.60 mL), acetyl chloride (17 µL, 0.24 mmol, 2.2 eq) was added to acetylate the unreacted aniline, and the reaction mixture was stirred at r.t. for 3 h. The reaction mixture was 10 diluted with water and extracted with DCM (3x). The combined organic portions were washed with brine (1x), dried over sodium sulfate, decanted, dry loaded onto silica gel, and purified via column chromatography (10–70% EtOAc / hexanes gradient over 12 min, 12 g column), but still afforded a 1.4:1 mixture of desired product and aniline. Thus, the mixture was, again, dissolved in DCM (0.60 mL) and acetyl chloride (20 µL, 0.28 mmol, 2.5 eq) was added. The 15 reaction mixture stirred at r.t. for 18 h before being diluted with water and extracted with DCM (3x). The combined organic portions were washed with brine (1x), dried over sodium sulfate, decanted, dry loaded onto silica gel, and purified via column chromatography (10–60% EtOAc / hexanes gradient over 12 min, 12 g column) to afford the pure title compound as a yellow oil (5 mg, 10%).1H NMR (499 MHz, CDCl3) δ 8.05 (s, 1H), 7.70 (s, 1H), 7.41 (dd, J = 20 8.2, 2.3 Hz, 1H), 7.32 (s, 1H), 7.22 (d, J = 8.2 Hz, 1H), 4.62 (hept, J = 6.0 Hz, 1H), 3.91 (s, 3H), 2.63 (s, 3H), 1.35 (d, J = 6.0 Hz, 6H).13C NMR (126 MHz, CDCl3) δ 165.92, 154.14, 138.28, 135.99, 134.53, 134.22, 133.62, 132.36, 130.45, 129.80, 118.02, 112.39, 110.99, 72.56, 52.77, 22.02, 19.93.

[0438] Step v: 4-Chloro-3-((5-chloro-2-methylphenyl)sulfonamido)-5-isopropoxybenzoic acid25 (PTM-211). Following General Procedure C, methyl 4-chloro-3-((5-chloro-2-methylphenyl)sulfonamido)-5-isopropoxybenzoate (5 mg, 12 µmol) was subjected to ester hydrolysis to yield the title compound as a white solid (4 mg, 83%).1H NMR (499 MHz, CD3OD) δ 7.77 (d, J = 2.3 Hz, 1H), 7.74 (d, J = 1.8 Hz, 1H), 7.49 – 7.45 (m, 2H), 7.34 (d, J = 8.2 Hz, 1H), 4.65 (hept, J = 6.1 Hz, 1H), 2.61 (s, 3H), 1.32 (d, J = 5.9 Hz, 6H).13C NMR (126 30 MHz, CD3OD) δ 168.30, 155.46, 141.01, 137.64, 136.31, 135.40, 133.99, 132.81, 131.16, 130.40, 124.04, 119.73, 113.77, 73.60, 22.10, 20.06. HRMS (ESI / TOF) m / z for C17H17Cl2NO5S [M-H]-: 416.0131 (calc), 416.0127 (observed), 418.0104 (observed, halogen peak).

[0439] 51 UM-43663.601

[0440] Additional Compounds. The following additional compounds were synthesized in ananalogous manner to that shown for PTM-211 and in Scheme 3

[0441] Cpd Structure Mass Spec1H NMR

[0442] PTM-172

[0443] HRMS (ESI / TOF)

[0444] ,

[0445] – s,

[0446] z, J 3 = d, 1 ,

[0447] , J 3 = d, 1

[0448] )

[0449]

[0450] 5 Scheme 4. Synthetic route to cyclopropyl analog. i) cyclopropylboronic acid, Pd(OAc)2, PPh3, Cs2CO3, 10:1 toluene / water, 80 ºC. ii) Fe, NH4Cl, 2:1 MeOH / H2O, 60 ºC. iii) 5-chloro- 2-methylbenzenesulfonyl chloride, DMAP, pyridine, THF, 60 ºC. iv) 1 M aq. NaOH, MeOH, 55 ºC.

[0451] 52 UM-43663.601

[0452]

[0453] Synthesis of 3-((5-Chloro-2-methylphenyl)sulfonamido)-5-cyclopropylbenzoic acid (PTM-201)

[0454] 5 Step i: Methyl 3-cyclopropyl-5-nitrobenzoate. Under a nitrogen atmosphere, methyl 3- bromo-5-nitrobenzoate (130 mg, 0.50 mmol), cyclopropylboronic acid (64 mg, 0.75 mmol), Pd(OAc)2 (13 mg, 0.058 mmol), triphenylphosphine (13 mg, 0.050 mmol), and cesium carbonate (407 mg, 1.25 mmol) were dissolved in a degassed solution of 10:1 toluene / water (2.2 mL) and stirred at 80 ºC for 18 h. The reaction mixture was cooled to r.t. and diluted with 10 ethyl acetate. The organic mixture was washed with water (3x) and brine (1x), dried over sodium sulfate, and concentrated in vacuo to afford a cloudy yellowish brown oil. The oil was dissolved in DCM, dry loaded onto silica gel, and purified via column chromatography (0– 40% EtOAc / hexanes gradient over 16 min, 25 g column) to afford the title compound as a white solid (82 mg, 74%).1H NMR (400 MHz, CDCl3) δ 8.60 (s, 1H), 8.08 (s, 1H), 8.04 (s, 15 1H), 3.98 (s, 3H), 2.19 – 1.91 (m, 1H), 1.24 – 1.07 (m, 2H), 0.95 – 0.75 (m, 2H).13C NMR (101 MHz, CDCl3) δ 165.24, 148.46, 147.24, 132.54, 131.63, 124.48, 121.55, 52.80, 15.47, 10.39.

[0455] Step iii: Methyl 3-amino-5-cyclopropylbenzoate. Methyl 3-cyclopropyl-5- nitrobenzoate (82 mg, 0.37 mmol) was reacted according to General Procedure A. Purification 20 by flash chromatography (10–50% EtOAc / hexanes gradient over 16 min, 12 g column) afforded the title compound as a yellow oil (65 mg, 92%).1H NMR (400 MHz, CDCl3) δ 7.16 – 7.11 (m, 2H), 6.59 (s, 1H), 3.87 (s, 3H), 3.71 (s, br, 2H), 1.90 – 1.79 (m, 1H), 0.99 – 0.89 (m, 2H), 0.73 – 0.66 (m, 2H).13C NMR (101 MHz, CDCl3) δ 167.53, 146.53, 145.78, 131.21, 117.26, 117.08, 113.29, 52.15, 15.43, 9.28.

[0456] 25 Step iv: Methyl 3-((5-chloro-2-methylphenyl)sulfonamido)-5-cyclopropylbenzoate.

[0457] Methyl 3-amino-5-cyclopropylbenzoate (25 mg, 0.13 mmol) was reacted according to General Procedure B. Purification by flash chromatography (0–40% EtOAc / hexanes gradient over 16 min, 12 g column) afforded the title compound as a white solid (29 mg, 58%).1H NMR (499 MHz, CDCl3) δ 8.00 (d, J = 2.3 Hz, 1H), 7.49 (s, 1H), 7.44 – 7.39 (m, 2H), 7.23 (d, J = 8.2 Hz, 30 1H), 7.01 (s, 1H), 6.83 (s, br, 1H), 3.89 (s, 3H), 2.60 (s, 3H), 1.91 – 1.83 (m, 1H), 1.03 – 0.97

[0458] 53 UM-43663.601

[0459] (m, 2H), 0.69 – 0.63 (m, 2H).13C NMR (126 MHz, CDCl3) δ 166.54, 146.66, 138.72, 136.40, 135.69, 134.12, 133.36, 132.40, 131.41, 130.15, 123.91, 122.21, 118.71, 52.54, 20.02, 15.47, 9.82.

[0460] Step vi: 3-((5-Chloro-2-methylphenyl)sulfonamido)-5-cyclopropylbenzoic acid (PTM- 5 201). Following General Procedure C, methyl 3-((5-chloro-2-methylphenyl)sulfonamido)-5- cyclopropylbenzoate (25 mg, 66 µmol) was subjected to ester hydrolysis to yield the title compound as a white solid (21 mg, 87%).1H NMR (499 MHz, CD3OD) δ 7.90 (d, J = 2.3 Hz, 1H), 7.49 (t, J = 1.8 Hz, 1H), 7.46 (dd, J = 8.2, 2.3 Hz, 1H), 7.43 (t, J = 1.6 Hz, 1H), 7.32 (d, J = 8.2 Hz, 1H), 6.97 (t, J = 2.0 Hz, 1H), 2.58 (s, 3H), 1.93 – 1.84 (m, 1H), 1.02 – 0.95 (m, 2H), 10 0.64 – 0.58 (m, 2H).13C NMR (126 MHz, CD3OD) δ 169.12, 147.41, 140.51, 138.87, 137.36, 135.33, 133.94, 132.94, 132.90, 130.74, 124.04, 121.90, 119.16, 19.69, 15.94, 9.98. HRMS (ESI / TOF) m / z for C17H16ClNO4S [M-H]-: 364.0416 (calc), 364.0408 (observed), 366.0385 (observed, 3:1 Cl peak).

[0461] 15 Synthesis of 3-((5-Chloro-2-methylphenyl)sulfonamido)-5-(dimethylamino)benzoic acid (PTM-235)

[0462]

[0463] Step 1: Methyl 3-(dimethylamino)-5-nitrobenzoate. Under a nitrogen atmosphere, 20 methyl 3-bromo-5-nitrobenzoate (150 mg, 0.58 mmol), dimethylamine hydrochloride (61 mg, 0.75 mmol), Pd2(dba)3(26 mg, 0.029 mmol), Xantphos (50 mg, 0.087 mmol), and cesium carbonate (839 mg, 2.6 mmol) were dissolved in a toluene (2.3 mL) and stirred at 90 ºC for 21 h. The reaction mixture was diluted with water and filtered through a plug of Celite. The filtrate was extracted with ethyl acetate (3x) and the combined organic portions were washed with 25 brine (1x), dried over sodium sulfate, and concentrated in vacuo. The crude product was dissolved in DCM, dry loaded onto silica gel, and purified by column chromatography (0–20% EtOAc / hexanes gradient over 16 min, 12 g column) to afford the title compound as a yellow solid (84 mg, 65%).1H NMR (400 MHz, CDCl3) δ 8.13 (s, 1H), 7.63 (s, 2H), 3.95 (s, 3H), 3.09

[0464] 54 UM-43663.601

[0465] (s, 6H).13C NMR (101 MHz, CDCl3) δ 165.91, 150.61, 149.31, 131.85, 117.97, 111.26, 109.47, 52.62, 40.39.

[0466] Step 2: Methyl 3-amino-5-(dimethylamino)benzoate. Methyl 3-(dimethylamino)-5- nitrobenzoate (84 mg, 0.38 mmol) was reacted according to General Procedure A. Purification 5 by flash chromatography (0–50% EtOAc / hexanes gradient over 16 min, 12 g column) afforded the title compound as a yellow oil (74 mg, quant.).1H NMR (400 MHz, CDCl3) δ 6.86 (s, 1H), 6.75 (s, 1H), 6.21 (s, 1H), 3.87 (s, 3H), 3.69 (s, br, 2H), 2.95 (s, 6H).13C NMR (101 MHz, CDCl3) δ 167.89, 151.51, 147.32, 131.56, 104.96, 104.68, 103.18, 51.98, 40.56.

[0467] Step 3: Methyl 3-((5-chloro-2-methylphenyl)sulfonamido)-5-10 (dimethylamino)benzoate. Methyl 3-amino-5-(dimethylamino)benzoate (79 mg, 0.41 mmol) was reacted according to General Procedure B. Purification by flash chromatography (0–40% EtOAc / hexanes gradient over 16 min, 12 g column) afforded the title compound as a yellow solid (93 mg, 60%).1H NMR (400 MHz, CDCl3) δ 8.03 (d, J = 2.3 Hz, 1H), 7.38 (dd, J = 8.1, 2.3 Hz, 1H), 7.20 (d, J = 8.5 Hz, 2H), 7.07 (dd, J = 2.5, 1.3 Hz, 1H), 6.95 (dd, J = 2.0, 1.3 Hz, 15 1H), 6.63 (t, J = 2.3 Hz, 1H), 3.88 (s, 3H), 2.92 (s, 6H), 2.60 (s, 3H).13C NMR (101 MHz, CDCl3) δ 167.14, 151.04, 138.70, 137.17, 135.69, 133.93, 133.04, 132.02, 131.57, 130.19, 109.78, 108.68, 107.31, 52.39, 40.32, 19.81.

[0468] Step 4: 3-((5-Chloro-2-methylphenyl)sulfonamido)-5-(dimethylamino)benzoic acid (PTM-235). Following General Procedure C, methyl 3-((5-chloro-2-20 methylphenyl)sulfonamido)-5-(dimethylamino)benzoate (93 mg, 0.24 mmol) was subjected to ester hydrolysis to yield the title compound as a white solid (73 mg, 81%).1H NMR (400 MHz, CD3OD) δ 7.94 (d, J = 2.3 Hz, 1H), 7.45 (dd, J = 8.1, 2.3 Hz, 1H), 7.31 (d, J = 8.3 Hz, 1H), 7.09 (dd, J = 2.5, 1.3 Hz, 1H), 7.04 (dd, J = 2.0, 1.3 Hz, 1H), 6.66 (t, J = 2.2 Hz, 1H), 2.91 (s, 6H), 2.59 (s, 3H).13C NMR (101 MHz, CD3OD) δ 169.74, 152.25, 140.62, 139.55, 137.42, 25 135.30, 133.87, 133.35, 132.79, 130.91, 110.72, 110.21, 108.57, 40.71, 19.69. HPLC: retention time 9.040 min (Method B); Purity (AUC): >95%. HRMS (ESI / TOF) m / z for C16H17ClN2O4S [M+H]+: 369.0671 (calculated), 369.0737 (observed), 371.0649 (observed, 3:1 Cl peak).

[0469] Scheme 5. Synthetic route to terminal amide analogs. i) appropriate amine, DIPEA, BOP, 30 DMF, r.t.

[0470]

[0471] 55 UM-43663.601

[0472] Synthesis of 4-Bromo-3-((5-chloro-2-methylphenyl)sulfonamido)-N-methylbenzamide (PTM-222)

[0473]

[0474] 5 A solution of methanamine hydrochloride (10 mg, 0.15 mmol), PTM-130 (30 mg, 0.074 mmol), and DIPEA (39 µL, 0.22 mmol) in anhydrous DMF (200 µL) was stirred at r.t. for 5 min before addition of ((1H-benzo[d][1,2,3]triazol-1- yl)oxy)tris(dimethylamino)phosphonium hexafluorophosphate(V) (BOP, 43 mg, 0.096 mmol). The reaction mixture was stirred at r.t. for 4 h and then crushed ice was added to the mixture.

[0475] 10 The resulting white precipitate was collected by filtration and rinsed with cold water to afford a white solid. The solid was dissolved in DCM, dry loaded onto silica gel, and purified via column chromatography (70% EtOAc / hexanes isocratic for 5 min, 12 g column) to afford the title compound as a white solid (20 mg, 65%).1H NMR (499 MHz, CD3OD) δ 7.95 (d, J = 2.2 Hz, 1H), 7.71 (d, J = 2.3 Hz, 1H), 7.61 (d, J

[0476]

[0477] 1H), 7.52 (dd, J = 8.4, 2.1 Hz, 1H), 7.47 15 (dd, J = 8.2, 2.3 Hz, 1H), 7.34 (d, J = 8.2 Hz, 1H), 2.91 (s, 3H), 2.59 (s, 3H).13C NMR (126 MHz, CD3OD) δ 168.84, 141.44, 137.73, 136.58, 135.99, 135.46, 134.44, 133.90, 132.81, 130.14, 128.14, 127.30, 124.13, 26.98, 20.40. HRMS (ESI / TOF) m / z for C15H14BrClN2O3S [M-H]-: 414.9524 (calc), 414.9526 (observed), 416.9517 (observed, halogen peak).

[0478] Additional Compounds. The following additional compounds were synthesized in an20 analogous manner to that shown for PTM-222 and in Scheme 5.

[0479] Cpd Structure Mass Spec1H NMR

[0480] ,

[0481] 4 = d, 8

[0482]

[0483] 56 UM-43663.601

[0484] PTM-2231H NMR (499 MHz, CD3OD) δ 7.71 (d, J 23 H 1H 762

[0485] s,

[0486]

[0487] Scheme 6. Synthesis of chlorothiophene analog. i) AcCl, CHCl3, r.t. ii) NaH, 5- chlorothiophene-2-sulfonyl chloride, THF, 0 ºC–r.t.. iii) 1 M aq. NaOH, MeOH, 55 ºC.

[0488] 5 Synthes

[0489]

[0490] s o -romo-3-((5-coroopene)--suonamdo)benzoc acd (M-158)

[0491] Step i: Methyl 3-acetamido-4-bromobenzoate. Acetyl chloride (0.31 mL, 4.35 mmol) was added to a solution of methyl 3-amino-4-bromobenzoate (400 mg, 1.74 mmol) in chloroform (6.5 mL). The reaction mixture was stirred at r.t. for 16 h before being quenched with water. The organic phase was separated and the aqueous phase was extracted with DCM 10 (3x). The combined organic portions were washed with brine (1x), dried over sodium sulfate, dry loaded onto silica gel, and purified by column chromatography (20–50% EtOAc / hexanes gradient over 8 min, 12 g column) to afford the title compound as a light orange solid (250 mg, 53%).1H NMR (499 MHz, CDCl3) δ 8.94 (s, 1H), 7.66 (d, J = 8.2 Hz, 1H), 7.62 (d, J = 8.3 Hz, 1H), 7.60 (s, br, 1H), 3.91 (s, 3H), 2.26 (s, 3H).13C NMR (126 MHz, CDCl3) δ 168.27, 166.30, 15 135.98, 132.46, 130.70, 126.24, 122.96, 118.46, 52.53, 24.96.

[0492] 57 UM-43663.601

[0493] Step ii: Methyl 4-bromo-3-((5-chlorothiophene)-2-sulfonamido)benzoate. Under anitrogen atmosphere, a solution of methyl 3-acetamido-4-bromobenzoate (20 mg, 74 µmol) inTHF (0.20 mL) was added dropwise to a stirring solution of sodium hydride (10 mg, 0.42 mmol, 60% dispersion in mineral oil) in THF (0.30 mL) at 0 ºC. The reaction mixture stirred 5 at 0 ºC for 30 min before addition of a solution of 5-chlorothiophene-2-sulfonyl chloride (16 mg, 74 µmol) in THF (0.20 mL). The reaction mixture was allowed to warm to r.t. as it stirred for 18 h. The reaction mixture was cooled to 0 ºC and quenched with iced water. The aqueous mixture was extracted with ethyl acetate (3x) and the combined organic portions were washed with water (1x) and brine (1x), dried over sodium sulfate, filtered, and concentrated in vacuo to 10 afford an oil. The crude oil was dissolved in DCM, dry loaded onto silica gel, and purified by column chromatography (0–40% EtOAc / hexanes gradient over 16 min, 12 g column) to afford the deacetylated sulfonamide product as a white solid (6 mg, 20%).1H NMR (499 MHz, CD3OD) δ 8.12 (s, 1H), 7.78 (dd, J = 8.3, 2.1 Hz, 1H), 7.71 (d, J = 8.4 Hz, 1H), 7.28 (d, J = 4.1 Hz, 1H), 7.03 (d, J = 4.1 Hz, 1H), 3.93 (s, 3H).

[0494] 15 Step iii: 4-Bromo-3-((5-chlorothiophene)-2-sulfonamido)benzoic acid (PTM-158).

[0495] Following General Procedure C, methyl 4-bromo-3-((5-chlorothiophene)-2- sulfonamido)benzoate (6 mg, 15 µmol) was subjected to ester hydrolysis to yield the title compound as a white solid (4 mg, 69%).1H NMR (499 MHz, CD3OD) δ 8.13 (d, J = 2.0 Hz, 1H), 7.78 (dd, J = 8.3, 2.0 Hz, 1H), 7.70 (d, J = 8.3 Hz, 1H), 7.29 (d, J = 4.1 Hz, 1H), 7.03 (d, 20 J = 4.1 Hz, 1H).13C NMR (126 MHz, CD3OD) δ 168.14, 140.25, 136.42, 134.61, 133.49, 132.53, 130.17, 130.00, 128.54, 126.12, 125.31. HRMS (ESI / TOF) m / z for C11H7BrClNO4S2[M-H]-: 393.8616 (calc), 393.8610 (observed), 395.8585 (observed, halogen peak).

[0496] Synthesis of 4-((5-Chloro-2-methylphenyl)sulfonamido)-1H-indole-6-carboxylic acid 25 (PTM-205)

[0497]

[0498] PTM-205

[0499] 58 UM-43663.601

[0500] Step 1: 1-(tert-Butyl) 6-methyl 4-bromo-1H-indole-1,6-dicarboxylate. Methyl 4- bromo-1H-indole-6-carboxylate (100 mg, 0.39 mmol), DMAP (5 mg, 0.039 mmol), and Boc2O (112 mg, 0.51 mmol) were dissolved in DCM (4.0 mL). Triethylamine (71 µL, 0.51 mmol) was added and the reaction mixture was stirred at r.t. for 18 h. The reaction mixture was diluted 5 with water and extracted with DCM (3x). The combined organic portions were washed with brine (1x), dried over sodium sulfate, decanted, dry loaded onto silica gel, and purified by column chromatography (30% EtOAc / hexanes isocratic for 8 min, 12 g column) to afford the title compound as a yellow solid (109 mg, 78%).1H NMR (400 MHz, CDCl3) δ 8.77 (s, 1H), 8.05 (d, J = 1.3 Hz, 1H), 7.74 (d, J = 3.7 Hz, 1H), 6.62 (d, J = 3.7 Hz, 1H), 3.93 (s, 3H), 1.68 10 (s, 9H).13C NMR (101 MHz, CDCl3) δ 166.51, 149.05, 134.73, 134.60, 129.31, 127.09, 126.55, 116.30, 114.37, 107.14, 85.04, 52.39, 28.16.

[0501] Steps 2 and 3: Methyl 4-amino-1H-indole-6-carboxylate. Under a nitrogen atmosphere, 1-(tert-Butyl) 6-methyl 4-bromo-1H-indole-1,6-dicarboxylate (109 mg, 0.31 mmol), tert-butyl carbamate (43 mg, 0.37 mmol), Pd2(dba)3 (14 mg, 0.015 mmol), Xantphos (27 mg, 0.046 15 mmol), and cesium carbonate (301 mg, 0.92 mmol) were dissolved in toluene (1.2 mL) and stirred at 90 ºC for 20 h. The reaction mixture was cooled to r.t., filtered through Celite, and concentrated in vacuo to afford an orangish oil. Open to air, the oil was dissolved in DCM (2.4 mL) and TFA (0.60 mL) was added. The reaction mixture was stirred at r.t. for 3 h then washed with saturated aqueous sodium bicarbonate solution (2x) and brine (1x). The organic portion 20 was dried over sodium sulfate, decanted, dry loaded onto silica gel, and purified by column chromatography (20–80% EtOAc / hexanes gradient over 18 min, 12 g column) to afford the globally deprotected title compound as a brown solid (26 mg, 44% over 2 steps).1H NMR (400 MHz, CD3OD) δ 7.58 (s, 1H), 7.28 (d, J = 3.2 Hz, 1H), 7.02 (d, J = 1.3 Hz, 1H), 6.56 (d, J = 3.2 Hz, 1H), 3.86 (s, 3H).13C NMR (101 MHz, CD3OD) δ 170.57, 140.83, 137.67, 127.05, 25 124.93, 123.19, 106.15, 105.01, 99.93, 52.24.

[0502] Step 4: Methyl 4-((5-chloro-2-methylphenyl)sulfonamido)-1H-indole-6-carboxylate. Methyl 4-amino-1H-indole-6-carboxylate (26 mg, 0.14 mmol) was reacted according to General Procedure B. Purification by flash chromatography (10–50% EtOAc / hexanes gradient over 16 min, 12 g column) afforded the title compound as a yellow oil (42 mg, 81%).1H NMR 30 (400 MHz, CD3OD) δ 7.90 (s, 1H), 7.82 (d, J = 2.3 Hz, 1H), 7.56 (d, J = 1.3 Hz, 1H), 7.35 (d, J = 3.2 Hz, 1H), 7.27 (dd, J = 8.2, 2.3 Hz, 1H), 7.11 (d, J = 8.2 Hz, 1H), 6.55 (d, J = 2.1 Hz, 1H), 3.85 (s, 3H), 2.51 (s, 3H).13C NMR (101 MHz, CD3OD) δ 169.29, 141.03, 137.69, 137.32, 135.04, 133.52, 132.64, 130.54, 129.42, 129.31, 127.54, 124.22, 114.42, 112.40, 100.47, 52.50, 19.88.

[0503] 59 UM-43663.601

[0504] Step 5: 4-((5-chloro-2-methylphenyl)sulfonamido)-1H-indole-6-carboxylic acid (PTM-205). Following General Procedure C, methyl 4-((5-chloro-2- methylphenyl)sulfonamido)-1H-indole-6-carboxylate (42 mg, 0.11 mmol) was subjected to ester hydrolysis to yield the title compound as a tan solid (26 mg, 64%).1H NMR (499 MHz, 5 CD3OD) δ 11.06 (s, br, 1H), 7.93 (s, 1H), 7.82 (d, J = 2.3 Hz, 1H), 7.57 (d, J = 1.3 Hz, 1H), 7.38 – 7.33 (m, 2H), 7.21 (d, J = 8.2 Hz, 1H), 6.53 (s, 1H), 2.53 (s, 3H).13C NMR (126 MHz, CD3OD) δ 170.65, 141.17, 137.96, 137.41, 135.07, 133.54, 132.73, 130.56, 129.32, 129.15, 127.84, 124.83, 115.30, 112.81, 100.57, 19.94. HPLC retention time: 8.682 min (Method B); Purity (AUC): 95%. HRMS (ESI / TOF) m / z for C16H13ClN2O4S [M-H]-: 363.0212 (calc), 10 363.0209 (observed), 363.0245 (observed, 3:1 Cl peak).

[0505] Synthesis of 4-((5-Chloro-2-methylphenyl)sulfonamido)-1-methyl-1H-indole-6- carboxylic acid (PTM-202)

[0506] 15

[0507]

[0508] Step 1: Methyl 4-bromo-1-methyl-1H-indole-6-carboxylate. Methyl iodide (80 µL, 1.3 mmol) was added to a solution of methyl 4-bromo-1H-indole-6-carboxylate (250 mg, 0.98 mmol) and potassium carbonate (246 mg, 1.8 mmol) in anhydrous DMF (5.5 mL). The reaction mixture was stirred at r.t. for 24 h before being quenched with water and extracted with ethyl 20 acetate (3x). The combined organic portions were washed with water (3x) and brine (1x), dried over sodium sulfate, filtered, and concentrated in vacuo to afford a brown oil. The oil was dissolved in DCM, dry loaded onto silica gel, and purified by column chromatography (0–40% EtOAc / hexanes gradient over 12 min, 12 g column) to afford the title compound as a yellow solid (200 mg, 76%).1H NMR (400 MHz, CDCl3) δ 7.97 (s, 1H), 7.94 (s, 1H), 7.20 (s, 1H), 25 6.51 (s, 1H), 3.93 (s, 3H), 3.79 (s, 3H).13C NMR (101 MHz, CDCl3) δ 167.05, 136.02, 132.57, 132.45, 124.20, 123.06, 114.27, 111.04, 101.92, 52.23, 33.43.

[0509] 60 UM-43663.601

[0510] Steps 2 and 3: Methyl 4-amino-1-methyl-1H-indole-6-carboxylate (111). Under anitrogen atmosphere, Methyl 4-bromo-1-methyl-1H-indole-6-carboxylate (200 mg, 0.75mmol), tert-butyl carbamate (105 mg, 0.90 mmol), Pd2(dba)3 (34 mg, 0.037 mmol), Xantphos (65 mg, 0.11 mmol), and cesium carbonate (729 mg, 2.2 mmol) were dissolved in toluene (3 5 mL) and stirred at 90 ºC for 18 h. The reaction mixture was filtered through Celite and concentrated in vacuo to afford an orange oil. Open to air, the oil was dissolved in DCM (6 mL) and TFA (1.5 mL) was added. The reaction mixture was stirred at r.t. for 2.5 h then washed with saturated aqueous sodium bicarbonate solution (2x) and brine (1x). The organic portion was dried over sodium sulfate, decanted, dry loaded onto silica gel, and purified by column 10 chromatography (20–60% EtOAc / hexanes gradient over 16 min, 12 g column) to afford the title compound as a brown oil (62 mg, 41% over 2 steps).1H NMR (400 MHz, CDCl3) δ 7.59 (d, J = 1.1 Hz, 1H), 7.13 – 7.06 (m, 2H), 6.42 (d, J = 3.2 Hz, 1H), 4.02 (s, br, 2H), 3.92 (s, 3H), 3.79 (s, 3H).13C NMR (101 MHz, CDCl3) δ 168.49, 139.18, 137.07, 130.10, 124.52, 121.41, 104.26, 103.44, 97.83, 52.00, 33.30.

[0511] 15 Step 4: Methyl 4-((5-chloro-2-methylphenyl)sulfonamido)-1-methyl-1H-indole-6-carboxylate. Methyl 4-amino-1-methyl-1H-indole-6-carboxylate (20 mg, 0.10 mmol) wasreacted according to General Procedure B. Purification by flash chromatography (10–50% EtOAc / hexanes gradient over 16 min, 12 g column) afforded the title compound as a pale yellow solid (16 mg, 42%).1H NMR (499 MHz, CDCl3) δ 8.01 (d, J = 2.2 Hz, 1H), 7.93 (s, 20 1H), 7.55 (s, 1H), 7.38 (dd, J = 8.2, 2.3 Hz, 1H), 7.20 – 7.17 (m, 2H), 6.84 (s, br, 1H), 6.43 (d, J = 3.2 Hz, 1H), 3.93 (s, 3H), 3.84 (s, 3H), 2.62 (s, 3H).13C NMR (126 MHz, CDCl3) δ 167.50, 139.14, 137.06, 135.82, 133.94, 133.12, 132.38, 132.27, 130.24, 127.65, 126.20, 124.12, 113.34, 110.17, 98.03, 52.32, 33.49, 20.11.

[0512] Step 5: 4-((5-Chloro-2-methylphenyl)sulfonamido)-1-methyl-1H-indole-6-carboxylic 25 acid (PTM-202). Following General Procedure C, Methyl 4-((5-chloro-2- methylphenyl)sulfonamido)-1-methyl-1H-indole-6-carboxylate (15 mg, 38 µmol) was subjected to ester hydrolysis to yield the title compound as a pale brown solid (10 mg, 69%).

[0513] 1H NMR (400 MHz, CD3OD) δ 7.91 (t, J = 1.1 Hz, 1H), 7.81 (d, J = 2.3 Hz, 1H), 7.61 (d, J = 1.2 Hz, 1H), 7.37 (dd, J = 8.2, 2.3 Hz, 1H), 7.29 (d, J = 3.2 Hz, 1H), 7.22 (d, J = 8.3 Hz, 1H), 30 6.52 (dd, J = 3.2, 0.9 Hz, 1H), 3.81 (s, 3H), 2.54 (s, 3H).13C NMR (101 MHz, CD3OD) δ 170.47, 141.08, 138.30, 137.41, 135.10, 133.58, 133.47, 132.68, 130.60, 129.54, 128.04, 124.98, 115.19, 110.68, 99.80, 33.21, 19.94. HPLC retention time: 9.402 min (Method B); Purity (AUC): >95%. HRMS (ESI / TOF) m / z for C17H15ClN2O4S [M-H]-: 377.0368 (calc), 377.0358 (observed), 379.0335 (observed, 3:1 Cl peak).

[0514] 61 UM-43663.601

[0515] Synthesis of 4-Bromo-3-((5-chloro-N,2-dimethylphenyl)sulfonamido)benzoic acid (PTM- 195)

[0516] 5

[0517]

[0518] Step 1: Methyl 4-bromo-3-((5-chloro-N,2-dimethylphenyl)sulfonamido)benzoate. Methyl iodide (4 µL, 67 µmol) was added to a solution of Methyl 4-bromo-3-((5-chloro-2-methylphenyl)sulfonamido)benzoate (20 mg, 48 µmol) and potassium carbonate (10 mg, 72µmol) in anhydrous DMF (0.30 mL). The reaction mixture was stirred at r.t. for 3 h before 10 being quenched with water and extracted with ethyl acetate (4x). The combined organic portions were washed with water (2x) and brine (1x), dried over sodium sulfate, decanted, and concentrated in vacuo to afford the desired product as an orange oil (22 mg, quant.).1H NMR (499 MHz, CDCl3) δ 7.99 (d, J = 2.0 Hz, 1H), 7.86 (dd, J = 8.4, 2.1 Hz, 1H), 7.83 (d, J = 2.3 Hz, 1H), 7.68 (d, J = 8.4 Hz, 1H), 7.43 (dd, J = 8.2, 2.3 Hz, 1H), 7.25 (d, J = 8.2 Hz, 1H), 3.92 15 (s, 3H), 3.29 (s, 3H), 2.47 (s, 3H).13C NMR (126 MHz, CDCl3) δ 165.49, 140.04, 138.96, 136.63, 134.33, 134.30, 133.04, 132.75, 132.16, 130.84, 130.80, 130.13, 129.90, 52.67, 38.94, 20.92.

[0519] Step 2: 4-Bromo-3-((5-chloro-N,2-dimethylphenyl)sulfonamido)benzoic acid. Following General Procedure C, methyl 4-bromo-3-((5-chloro-N,2-20 dimethylphenyl)sulfonamido)benzoate (21 mg, 49 µmol) was subjected to ester hydrolysis to yield the title compound as a white solid (17 mg, 84%).1H NMR (499 MHz, DMSO-d6) δ 13.38 (s, br, 1H), 7.87 (d, J = 8.3 Hz, 1H), 7.82 (dd, J = 8.3, 2.0 Hz, 1H), 7.71 – 7.66 (m, 3H), 7.50 (d, J = 8.0 Hz, 1H), 3.22 (s, 3H), 2.39 (s, 3H).13C NMR (126 MHz, DMSO-d6) δ 165.67, 139.26, 138.23, 136.38, 135.04, 134.30, 133.19, 131.50, 131.31, 130.90, 130.77, 129.19, 25 128.89, 38.59, 20.04. HPLC retention time: 11.545 min (Method B); Purity (AUC): >95%.

[0520] HRMS (ESI / TOF) m / z for C15H13BrClNO4S [M-H]-: 415.9364 (calc), 415.9362 (observed), 417.9337 (observed, halogen peak).

[0521] 62 UM-43663.601

[0522] Synthesis of 4-Bromo-3-(N-(5-chloro-2-methylphenyl)sulfamoyl)benzoic acid (PTM-191)

[0523]

[0524] A mixture of 5-chloro-2-methylaniline (17 mg, 0.12 mmol) and 4-bromo-3- 5 (chlorosulfonyl)benzoic acid (30 mg, 0.10 mmol) in anhydrous THF (0.50 mL) was stirred at 60 ºC for 21 h. The reaction mixture was diluted with ethyl acetate and washed with saturated aqueous sodium bicarbonate solution (2x). The combined basic aqueous portions were acidified to pH 1 with 1 M aqueous hydrochloric acid solution. The acidic aqueous mixture was extracted with ethyl acetate (3x) and the combined organic portions were dried over 10 sodium sulfate, filtered, and concentrated in vacuo to afford the title compound as a yellow oil (8 mg, 20%).1H NMR (499 MHz, CD3OD) δ 8.51 (d, J = 2.0 Hz, 1H), 8.07 (dd, J = 8.2, 2.1 Hz, 1H), 7.97 (d, J = 8.2 Hz, 1H), 7.14 (d, J = 8.1 Hz, 1H), 7.11 – 7.06 (m, 2H), 2.20 (s, 3H).

[0525] 13C NMR (126 MHz, CD3OD) δ 167.30, 141.43, 137.30, 136.79, 135.61, 134.49, 133.55, 133.14, 132.53, 131.99, 127.88, 127.28, 126.10, 17.77. HPLC retention time: 9.793 min 15 (Method B); Purity (AUC): >95%. HRMS (ESI / TOF) m / z for C14H11BrClNO4S [M-H]-:

[0526] 401.9208 (calc), 401.9202 (observed), 403.9183 (observed, halogen peak).

[0527] Synthesis of 4-Bromo-3-(5-chloro-2-methylbenzamido)benzoic acid (PTM-197)

[0528] 20

[0529]

[0530] Step 1: Methyl 4-bromo-3-(5-chloro-2-methylbenzamido)benzoate. Methyl 3-amino-4- bromobenzoate (50 mg, 0.22 mmol) and 5-chloro-2-methylbenzoic acid (31 mg, 0.18 mmol) were dissolved in anhydrous acetonitrile (0.50 mL).1-Methyl-1H-imidazole (NMI, 51 µL, 0.63 mmol) was added followed by N- (chloro(dimethylamino)methylene)-N-

[0531] 63 UM-43663.601

[0532] methylmethanaminium hexafluorophosphate(V) (TCFH, 61 mg, 0.22 mmol). The reaction mixture was stirred at r.t. for 22 hours and then diluted with water. The resulting light brown precipitate was filtered, rinsed with 2:1 H2O / acetonitrile, and dried to afford the title compound as a light brown solid (27 mg, 40%).1H NMR (400 MHz, CDCl3) δ 9.08 (s, 1H), 7.95 (s, 1H), 5 7.77 – 7.65 (m, 2H), 7.56 (s, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.24 (s, 1H), 3.95 (s, 3H), 2.53 (s, 3H).13C NMR (126 MHz, CDCl3) δ 166.32, 166.07, 136.92, 135.69, 135.31, 132.93, 132.51, 131.86, 130.78, 130.66, 126.85, 126.64, 122.93, 119.02, 52.47, 19.57.

[0533] Step 2: 4-Bromo-3-(5-chloro-2-methylbenzamido)benzoic acid. Following General Procedure C, methyl 4-bromo-3-(5-chloro-2-methylbenzamido)benzoate (14 mg, 35 µmol) 10 was subjected to ester hydrolysis at r.t. for 2 days to yield the title compound as a white solid (10 mg, 78%).1H NMR (400 MHz, DMSO-d6) δ 13.32 (s, br, 1H), 10.25 (s, 1H), 8.11 (s, 1H), 7.83 (d, J = 8.3 Hz, 1H), 7.73 (dd, J = 8.3, 2.1 Hz, 1H), 7.63 (d, J = 2.3 Hz, 1H), 7.46 (dd, J = 8.2, 2.3 Hz, 1H), 7.34 (d, J = 8.2 Hz, 1H), 2.41 (s, 3H).13C NMR (101 MHz, DMSO-d6) δ 166.61, 166.36, 137.82, 136.34, 134.76, 133.22, 130.89, 130.06, 129.64, 129.25, 128.45, 15 127.15, 127.09, 125.32, 18.90. HPLC retention time: 10.546 min (Method B); Purity (AUC):

[0534] >95%. HRMS (ESI / TOF) m / z for C15H11BrClNO3 [M-H]-: 365.9538 (calc), 365.9534 (observed), 367.9511 (observed, halogen peak).

[0535] Example 2

[0536] Biological Assays

[0537] 20 Assay for inhibition of PDHK

[0538] Compounds were tested using a HotSpot assay (Anastassiadis et al. Nature Biotechnol.2011, 29(11), 1039-45). PDHK protein was purchased from SignalChem.

[0539] Substrate was either maltose binding protein or PDHKtide. The Reaction Buffer was composed of 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.01% Brij35, 0.03 25 mg / mL BSA, 0.1 mM Na3VO4, 2 mM DTT and 1% DMSO. Inhibitors were dissolved in 100% DMSO and added to the enzyme mixture by acoustic technology (Echo550, nanoliterrange). Following incubation for 20 min at room temperature, [ɣ-33P]-ATP was added toinitiate the reaction and the mixture was incubated for a further 2 h at room temperature. Kinase activity was detected using the P81 filter-binding method. All compounds were tested 30 in dose response with 3-fold dilutions. Data are presented in Table 1.

[0540] CellTiter-Glo luminescent cell viability assay

[0541] CellTiter-Glo (Promega, Cat. # G7571) is used to monitor ATP levels and is conducted according to the manufacturer’s recommended protocol. Briefly, cells are plated at 90 µL per

[0542] 64 UM-43663.601

[0543] well in a 96 well plate (Corning, Cat. # 3917). Control wells contain 90 ^L Opti-MEM assay media. Following overnight incubation, 5 ^L tracer dilution buffer is added followed by 5 ^L of Opti-MEM media to each well. The plate is rotated to mix contents and incubated at 37 ^C, 5% CO2for 2 h. Following the incubation, 100 ^L of CellTiter-Glo Reagent is added to each 5 well. The plate is rotated to mix the contents and incubated for 10 min in the dark at r.t.

[0544] Luminescence is measured using a Promega Discover GloMax plate reader.

[0545] Western blot for PDH phosphorylation

[0546] A549 cells are seeded into 60 mm culture dishes at a density of 5x104and treated with the indicated concentrations of test compound. The cells are washed with PBS, collected and 10 pelleted. The cells are chemically lysed using RIPA cell lysis buffer (Thermo Fisher, Cat. # 89900) with 1X protease inhibitor cocktail (Thermo Fisher, Cat. # 87786) and phosphatase inhibitor (Thermo Fisher, Cat. # 78420). The cells are also subjected to sonication 4 x 10 seconds per sample to ensure complete lysis. Following cell lysis, a Bradford assay is conducted to ensure that equal amounts of lysate are loaded onto NuPAGE 4-12% Bis-Tris 15 gels (Invitrogen) followed by transfer to PVDF membrane. The membranes are blocked for 1 h followed by overnight incubation with the relevant primary antibody: PDH phospho(Ser293)E1α (Cell Signaling, Cat. # 31866), 1:1000; PDH (Cell Signaling, Cat. # 2784), 1:1000, GAPDH, 1:1000 (Cell Signaling, Cat. # 2118). The membranes are washed 3 x 10 min with TBST, incubated with anti-rabbit secondary antibodies (Cell Signaling, Cat. # 20 7074) for 1 h, then washed again 3 x 10 min with TBST. The membranes are imaged using an ECL prime (Sigma, Cat. # GERPN2236).

[0547] NanoBRET assay

[0548] For target engagement analysis, to a 96-well plate with 85 μL of transfected HEK cells per well, is added 5 μL of 20X NanoBRET Tracer Reagent and 10 μL of 10X Test Compound 25 in Opti-MEM I reduced medium (Thermo Fisher Scientific, Cat. #11058-021). The cells are incubated for 2 h at 37 ^C, 5% CO2. To measure a BRET signal NanoGlo Substrate and NanoLuc Inhibitor (Promega, Cat. # N2160) are added according to manufacturer’s recommended protocol. BRET is measured by luminescence on a GloMax Discover Plate Reader (Promega, Cat. # GM3000) at 450 nm BP filter (donor) and 600 nm LP filter (acceptor) 30 with 0.5 s integration time. Milli-BRET units (mBU) were background corrected (Equation 1).

[0549] mBu = ((Acceptorsample / Donorsample)-( Acceptorno-tracer control / Donorno-tracer control))*1000 (Equation 1)

[0550] 65 UM-43663.601

[0551] Mitochondrial respirometry assay

[0552] C2C12 cells are plated in 24-well XF cell culture microplates at 50,000 cells per well in Seahorse XF assay medium. The plate is incubated at 37 °C in a CO2 incubator overnight. Stock solutions of oligomycin, FCCP and rotenone / antimycin are diluted in Seahorse XF 5 assay medium to working concentrations of 1 ^M (oligomycin and FCCP) or 0.5 ^M (rotenone). These solutions are loaded into the injection ports of a XF sensor cartridge. The C2C12 cells are gently washed with fresh, pre-warmed Seahorse XF assay medium, test compound is added and the cells are equilibrated in a non-CO2, 37 °C incubator for 1 h. Analysis is performed on a Seahorse XFe24 Analyzer following the manufacturer’s 10 instructions.

[0553] Protein Selectivity Panel

[0554] A custom panel of potential off-targets was specified by us and the in vitro assays were performed at Reaction Biology Corporation (Malvern, PA, USA) using a similar workflow as described above for the HotSpot Kinase Assay for all selected proteins apart 15 from Hsp90a. Compound binding to Hsp90a was determined by a fluorescence polarization assay using a fluorescently labeled ATP-competitive inhibitor. (see Kim, J., et al. SLASDiscov.2004, 9 (5), 375–381; Howes, R. et al., Anal. Biochem.2006, 350 (2), 202–213).Briefly, PTM-130 was delivered in 100% DMSO using Acoustic technology (Echo550; nanoliter range) into a solution containing 30 nM Hsp90a in buffer consisting of 20 mM 20 Hepes (pH 7.5), 10 mM magnesium chloride, 50 mM sodium chloride, 0.02% Brij35, 0.02 mg / mL BSA, 2 mM DTT, and 1.1% DMSO. The mixture was incubated at r.t. for 30 min before fluorescently labeled geldanamycin (FITC-geldanamycin) was added. The mixture was incubated at r.t. for 3 h before probe binding was measured by fluorescent signal.

[0555] 25 Table 1. IC50 Dataa

[0556] Molecule PDHK1: [IC50] PDHK2: [IC50] PDHK3: [IC50] PDHK4: [IC50]>> > >>> > >> >>>>

[0557]

[0558] 66 UM-43663.601

[0559] PTM-155 > 30.0 ND ND NDPTM-156 2.13 > 100 99 14> >>>>>> >> >>>> >> >> >>> >>>>>>>>>>>>>>>> >

[0560]

[0561] bThe PDHK1 IC50 for PTM-366 was also measured at 1 mM ATP to be 0.160 M.cVER-246608 is Vernalis, a potent pan-isoform PDHK inhibitor.

[0562] dDCA is dichloroacetic acid.

[0563] 5

[0564] 67 UM-43663.601

[0565] Selectivity Profile of PTM-130

[0566] To interrogate the broader selectivity of compounds disclosed herein, a panel of 15 potential off-targets was constructed consisting of both canonical and atypical kinases as wellas proteins previously identified (see Cho, H., et al. J. Med. Chem.2019, 62 (18), 8461–8479;5 Moore, J. D., et al. Oncotarget 2014, 5 (24), 12862–12876) as off-targets for other PDHKinhibitors (Table 2, FIG.4). PTM-130 did not inhibit other atypical kinases such as AMPK, mTOR, or TRPM7 / CHAK1 nor does it bind to Hsp90, a non-kinase chaperone protein and fellow member of the GHKL ATPase superfamily with an ATP binding site similar to that of the PDHKs. However, 30 µM PTM-130 did weakly inhibit the activity of the canonical 10 tyrosine kinases BTK (20%) and c-Src (30%), even though it does not contain a conventional hinge-binding motif. These results demonstrate that PTM-130 is a highly selective inhibitor of PDHK1 in biochemical assays.

[0567] Table 2. Selectivity panel for PTM-130 against an assortment of kinases and proteins 15 structurally related to PDHK.

[0568] Protein % enzyme activity

[0569] (relative to DMSO control)a

[0570] aPTM-130 was d yed as the average of duplicate measurem

[0571]

[0572] ents ± standard deviation. % fluorescent probe binding relative to a DMSO control.

[0573] 68 UM-43663.601

[0574] PDHK1 Target Engagement

[0575] Using a NanoBRET assay, PDHK1 target engagement was evaluated in permeabilized HEK293 cells for PTM-130 and other compounds disclosed herein. It was observed that PTM-130 inhibited a NanoBRET probe binding to PDHK1 yet had no effect 5 on the same probe molecule binding to PDHK2, 3, or 4 (FIG 5, single concentration and FIG.

[0576] 6, titration). These data are generally consistent with the results of the biochemical assays and provide further evidence that this series of compounds is selective for PDHK1. The results are especially striking when compared to the effects of a potent pan-isoform inhibitor, VER- 246608, which greatly reduced probe binding to all four isoforms. PTM-130 exhibited no 10 cytotoxicity at concentrations up to 30 µM in HEK293 cells.

[0577] Next, effect of PTM-130 was examined on live A549 lung carcinoma cells, a line thathas been shown to express high levels of PDHK1 (see Chen, Y., et al. Mol. Oncol.2019, 13(7), 1490–1502). In these cells, PTM-130 reduces PDH phosphorylation in a time-dependent manner without affecting levels of PDHK1 protein or total PDH (tPDH) (FIG.7). Notably, 15 PTM-130 primarily modulates phosphorylation of Ser232, a site that is only phosphorylatedby PDHK1 (see Korotchkina, L. G., et al. J. Biol. Chem.2001, 276 (40), 37223–37229;Kolobova, E., et al. Biochem. J.2001, 358 (Pt 1), 69). In comparison, phosphorylation levelsat Ser293 are only weakly affected and remain consistent over the time course, suggesting that PDHK2–4 activity is maintained, and providing further evidence that PTM-130 is highly 20 selective for PDHK1, including in a cellular context. Strikingly, 50 µM PTM-130 displayed no effect on cell viability as determined using CellTiter-Glo (FIG.8).

[0578] 69

Claims

UM-43663.601CLAIMS1. A compound of formula (I):(I)5 or a pharmaceutically accepta wherein:R1is selected from hydrogen, halo, C1-C6 alkoxy, and C1-C6 thioalkoxy;R2is selected from hydrogen, halo, C1-C6alkoxy, and C3-C6cycloalkyl;R3is selected from hydrogen and halo;R4is selected from -COOR5a, -CONHR5b, -CN, and C1-C6alkoxy;10 R5aand R5bare each independently selected from hydrogen and C1-C6alkyl; and R6is phenyl or a monocyclic 5- or 6-membered heteroaryl, each of which is independently unsubstituted or substituted with 1, 2, or 3 substituents independently selected from halo, C1-C6 alkyl, C1-C6 alkoxy, and C3-C6 cycloalkyl.15 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R1is selected from hydrogen, halo, and C1-C3thioalkoxy.

3. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein R1is selected from hydrogen, fluoro, chloro, bromo, and methylthio.

204. The compound of any one of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein R2is selected from hydrogen, halo, C1-C4 alkoxy, and C3-C4 cycloalkyl.

5. The compound of claim 4, or a pharmaceutically acceptable salt thereof, wherein R2is 25 selected from hydrogen, chloro, bromo, methoxy, ethoxy, isopropoxy, and cyclopropyl.

6. The compound of any one of claims 1-5, or a pharmaceutically acceptable salt thereof, wherein R3is selected from hydrogen and chloro.70UM-43663.6017. The compound of claim 6, or a pharmaceutically acceptable salt thereof, wherein R3is hydrogen.

8. The compound of any one of claims 1-7, or a pharmaceutically acceptable salt 5 thereof, wherein R4is selected from -COOR5a, -CONHR5b, C1-C3 alkoxy, and -CN, wherein R5aand R5bare each independently selected from hydrogen and methyl.

9. The compound of claim 8, or a pharmaceutically acceptable salt thereof, wherein R4is -COOR5aand R5ais hydrogen.1010. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein thegroup has a structure selected from:1571UM-43663.60111. The compound of any one of claims 1-10, or a pharmaceutically acceptable salt thereof, wherein R6is phenyl substituted with 2 substituents independently selected from halo and C1-C4alkyl.

512. The compound of any one of claims 1-10, or a pharmaceutically acceptable salt thereof, wherein R6is a monocyclic 5-membered heteroaryl having one heteroatom selected from S, O, and N, which is substituted with 1 substituent selected from halo and C1-C4alkyl.10 13. The compound of any one of claims 1-12, or a pharmaceutically acceptable salt thereof, wherein R6has a structure:wherein:R7ais halo;15 R7bis hydrogen or halo;R7cis C1-C4alkyl or hydrogen;R7dis halo or C1-C4 alkyl; andX is S, O, or NH.20 14. The compound of any one of claims 1-12, or a pharmaceutically acceptable salt thereof, wherein R6has a structure selected from:.

15. The compound of claim 1, wherein the compound is selected from:72UM-43663.601573UM-43663.6015 an16. A pharmaceutical composition comprising a compound of any one of claims 1-15, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.10 17. A method of treating a disorder associated with a pyruvate dehydrogenase kinase (PDHK) in a subject in need thereof, comprising administering to the subject a74UM-43663.601therapeutically effective amount of a compound of any one of claims 1-15, or a pharmaceutically acceptable salt thereof.

18. The method of claim 17, wherein the disorder is associated with overexpression of 5 pyruvate dehydrogenase kinase 1 (PDHK1).

19. The method of claim 17, wherein the disorder is cancer.

20. The method of claim 19, wherein the cancer is selected from acute myeloid leukemia, 10 breast cancer, esophageal cancer, gastric cancer, glioblastoma, head and neck cancer, multiple myeloma, nasopharyngeal cancer, ovarian cancer, retinoblastoma, prostate cancer, bladder cancer, glioma, hepatocellular carcinoma, non-small cell lung cancer, pancreatic cancer, and transitional cell carcinoma.15 21. The method of claim 17, wherein the disorder is selected from Alzheimer's disease, multiple sclerosis, hepatic steatosis, Charcot-Marie-Tooth disease, Barth Syndrome, heart failure, inflammatory bowel disease, metabolic syndrome, metabolic dysfunction-associated steatohepatitis, diabetes, endometriosis, heart fibrosis, lactic acidosis, macular degeneration, myalgic encephalomyelitis, nonalcoholic fatty liver disease, pyruvate dehydrogenase 20 deficiency, and pulmonary arterial hypertension.

22. A compound of any one of claims 1-15, or a pharmaceutically acceptable salt thereof, for use as a medicament.25 23. A compound of any one of claims 1-15, or a pharmaceutically acceptable salt thereof, for use in treating a disorder associated with a pyruvate dehydrogenase kinase (PDHK).

24. The compound for use of claim 23, wherein the disorder is associated with overexpression of pyruvate dehydrogenase kinase 1 (PDHK1).3025. The compound for use of claim 23, wherein the disorder is cancer.

26. The compound for use of claim 25, wherein the cancer is selected from acute myeloid leukemia, breast cancer, esophageal cancer, gastric cancer, glioblastoma, head and neck75UM-43663.601cancer, multiple myeloma, nasopharyngeal cancer, ovarian cancer, retinoblastoma, prostate cancer, bladder cancer, glioma, hepatocellular carcinoma, non-small cell lung cancer, pancreatic cancer, and transitional cell carcinoma.5 27. The compound for use of claim 23, wherein the disorder is selected from Alzheimer's disease, multiple sclerosis, hepatic steatosis, Charcot-Marie-Tooth disease, Barth Syndrome, heart failure, inflammatory bowel disease, metabolic syndrome, metabolic dysfunction- associated steatohepatitis, diabetes, endometriosis, heart fibrosis, lactic acidosis, macular degeneration, myalgic encephalomyelitis, nonalcoholic fatty liver disease, pyruvate10 dehydrogenase deficiency, and pulmonary arterial hypertension.76

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