UPR modulators to treat hearing loss

Abstract

Disclosed herein are compound of structural formula (I'): (I'). Pharmaceutical compositions comprising the same, methods of treating hearing loss using the same, and method of making the compounds of structural formula (I') are also disclosed.

Description

[0001] Docket No. 136840-00220

[0002] UPR MODULATORS TO TREAT HEARING LOSSRELATED APPLICATIONS

[0003] This application claims the benefit of priority of U. S. Application No. 63,773,920, filed on March 18, 2025 and U. S. Application No. 63 / 904,946, filed on October 24, 2025. The entire teachings of each of the aforementioned applications are incorporated herein by reference.

[0004] FIELD OF THE INVENTION

[0005] Provided herein are novel compounds, pharmaceutical compositions thereof, and methods of use for treating hearing loss (HL).

[0006] GOVERNMENT FUNDING

[0007] This invention was made with government support under Grant No. 1R43DC021398-01A1, awarded by the National Institute of Health. The United States government has certain rights to the invention.

[0008] BACKGROUND OF THE INVENTION

[0009] Noise-induced hearing loss (NIHL) is a profound public health problem, affecting over 40 million Americans and causing the loss of 4 million disability-adjusted life years worldwide from occupational exposure annually. HL is also a significant risk factor for dementia, underscoring the expanded morbidity of this disorder. 27% of NIHL is from acute sound exposure, whereas 23% occurs from sub-acute or chronic exposure to sound, suggesting that interventions that target the mechanisms that underlie acute or sub-acute HL, can make an immediate and transformative clinical impact for this large and inadequately treated patient population.

[0010] Hearing loss can also be mediated by drug exposure. This includes the following classes of drags, aminoglycosides (e.g. tobramycin and amikacin), anti-cancer drugs (cisplatin) and loop diuretics (furosemide) and phosphodiesterase 5 inhibitors (tadalafil) (Prescrire Int. 2014, 23, 290-294). Individuals who must take these medications for critical medical needs should not have to suffer such a morbid side-effect from the treatment.

[0011] 1

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[0013] Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) are key drivers of NIHL pathophysiology (Li, et al., J. Clin. Invest. 2018, 128, 5150-5162; Rouse, et al., Sci. Rep. 2020, 10, 18063). In addition to NIHL, other modes of hearing loss, such as side effects from drugs such as cisplatin, can lead to hearing loss (Kros & Steyger, Cold Spring Harb. Perspect. Med. 2019, 9, a033548). Both NIHL and drug toxicity may be mediated through the UPR, which is a component of ER responses. The ER is the site of nearly all synthesis, folding, and maturation of transmembrane and secreted proteins before transit through the Golgi for additional post-translational modification and delivery to different cellular compartments. The UPR is a series of highly conserved intra-ER processes that assess in real time the capacity of the ER for protein folding and processing. These signaling cascades can be activated to increase the capacity of the cell to handle an extra load of protein synthesis, to downregulate the rate of protein synthesis to immediately ameliorate the excess load of unfolded proteins or, if the load is too great, to activate pathways leading to cell apoptosis. Three main parallel signaling pathways constitute the UPR, which are labeled by the initial protein in the pathway that senses change in protein synthesis regulation: PERK (double stranded RNA-activated protein kinase (PKR)-like ER kinase), IRE1 (inositol requiring enzyme 1), and ATF6 (activating transcription factor 6). PERK activation upregulates transcription of CHOP, a transcription factor that controls genes involved in apoptosis in addition to the UPR. IRE1 activation affects ER homeostasis through sXBPl, leading to upregulation of molecular chaperones (that aid in folding proteins) and proteins involved in ERAD (endoplasmic reticulum associated protein degradation). Similarly, activation of ATF6 upregulates target genes that mitigate ER stress, including BiP (binding immunoglobulin protein; GRP78), an ER chaperone that translocates newly synthesized proteins across the ER membrane, enhances protein folding, directs mis-folded proteins to ERAD and helps regulate Ca2+ homeostasis. Thus, the UPR comprises a wide range of interacting pathways that facilitate homeostatic control over many ER processes but can lead to cell death if excessively perturbed.

[0014] Modulation of the UPR has been shown to affect multiple forms of hearing loss, including NIHL (Li, et al., J. Clin. Invest. 2018, 128, 5150-5162; Rouse, etal., Sci. Rep. 2020, 10, 18063). Modulating the protein CHOP (transcription factor C / EBP homologous protein) and proteins in its signaling pathway (including PERK and eiF2B) can participate in the pathway that inhibits multiple forms of hearing loss (Li, et al., J. Clin. Invest. 2018, 128, 5150-5162). The link between hearing loss and the UPR has been demonstrated by genetic

[0015] 2

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[0017] inhibition of the key UPR pathways (Li, et al., J. Clin. Invest. 2018, 128, 5150-5162).

[0018] Additionally, it has been shown that cochlear synaptopathy (diminished wave I amplitude and loss of synapses between the cochlear nerve and inner hair cells) is prevented by ISRIB (integrated stress response inhibitor), a compound that mediates the UPR (Summers, et al., J. Mol. Biol. 1987, 196, 175-198). ISRIB can modulate the UPR in cell lines, cochlear hair cells and can prevent hearing loss in mice from noise exposure (permanent threshold shift; Li, et al., J. Clin. Invest. 2018, 128, 5150-5162) and can prevent cochlear synatopathy after a transient threshold shift (Rouse, et al., Sci. Rep. 2020, 10, 18063).

[0019] SUMMARY OF THE INVENTION

[0020] Disclosed herein are a series of compounds useful for treating one or more symptoms of hearing loss or slowing the onset of hearing loss in a subject in need thereof. The compounds disclosed herein have been shown to significantly reduce CHOP signaling from ER stress and limit apoptosis as a result of ER stress (See Examples 1 and 2).

[0021] One embodiment of the invention is a compound represented by structural formula (I’):

[0022]

[0023] or a pharmaceutically acceptable salt thereof, wherein:

[0024] Xi is CH or N;

[0025] X2is O, S, or NH;

[0026] X3 is selected from the group consisting of O, -OCH2-*, -O(CH2)2-*, S, and NH, wherein * indicates the point of attachment to Ring A;

[0027] X4 is absent or O;

[0028] Y is -(CH2)P-, Z is CH2and is a single bond; or Y is CH, Z is CH and =^= is a double bond;

[0029] Ring A is phenyl or 5- to 9-membered heteroaryl;

[0030] each Ri and R2are each independently selected from the group consisting of hydrogen, halogen, -CN, -OH, -COOH, -NR8R9, -NO2, C₁₋₆alkyl, and Ci-ehaloalkyl;

[0031] 3

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[0033] each R3 is independently selected from hydrogen, halogen, -CN, -OH, -NO2, Ci-6alkyl, and C1-6haloalkyl;

[0034] R4, Rs, Re, and R7 are each independently selected from hydrogen, Ci-ealkyl, and C1-6haloalkyl;

[0035] R8 and R9 are each independently selected from hydrogen and C1-3alkyl;

[0036] m, n, and 0 are each independently 1, or 2; and

[0037] p is 1 or 2.

[0038] Another embodiment of the invention is a compound represented by structural formula (I):

[0039]

[0040] or a pharmaceutically acceptable salt thereof, wherein:

[0041] Xi is CH or N;

[0042] X2 and X3 are each independently O, S, or NH;

[0043] Y is -(CH2)P-, Z is CH2 and === is a single bond; or Y is CH, Z is CH and === is a double bond;

[0044] each R1 and R2 are each independently selected from the group consisting of hydrogen, halogen, -CN, -OH, -NO2, C1-6alkyl, and C1-6haloalkyl;

[0045] each R3 is independently selected from hydrogen, halogen, -CN, -OH, -NO2, Ci-ealkyl, and Ci-ehaloalkyl;

[0046] R4, R5, R6, and R7 are each independently selected from hydrogen, C1-6alkyl, and C1-6haloalkyl;

[0047] m, n, and 0 are each independently 1, or 2; and

[0048] p is 1 or 2.

[0049] Another embodiment of the invention is a pharmaceutical composition comprising a compound disclosed herein or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0050] Another embodiment of the invention is a method of treating hearing loss in a subject in need thereof or slowing the onset of hearing loss in a subject at risk of developing hearing loss. The hearing loss can be, but is not limited to, age-related hearing loss, noise-induced 4

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[0052] hearing loss, genetic or inherited hearing loss, hearing loss due to ototoxic exposure, disease-induced hearing loss, trauma-induced hearing loss, and cochlear synaptopathy. The method comprises administering to the subject an effective amount of a compound disclosed herein or a pharmaceutically acceptable salt thereof.

[0053] DETAILED DESCRIPTION

[0054] / . Compounds of the Inven tion

[0055] Disclosed herein are a series of compounds that demonstrate the ability to reduce CHOP signaling protein in cells and protect cells for endoplasmic reticulum stress-induced cell death. As such, the disclosed compounds can be used to treat hearing loss or to slow the onset of hearing loss in a subject at risk of hearing loss. Compounds of the invention are described herein below.

[0056] A first embodiment of the invention is a compound represented by structural formula (I’) or (I) or a pharmaceutically acceptable salt thereof, wherein the variables are as described above.

[0057] A second embodiment of the invention is a compound represented by structural formula (I’) or (I) or a pharmaceutically acceptable salt thereof, wherein X2 and X3 are O; and the remainder of the variables are as described in the first embodiment.

[0058] A third embodiment of the invention is a compound represented by structural formula (I’) or (I) or a pharmaceutically acceptable salt thereof, wherein each R3is hydrogen; and the remainder of the variables are as described in the first or second embodiment.

[0059] A fourth embodiment of the invention is a compound represented by structural formula (F) or (I) or a pharmaceutically acceptable salt thereof, wherein R 4, R5, Re, and R? are each hydrogen: and the remainder of the variables are as described in the first, second, or third embodiment.

[0060] A fifth embodiment of the invention is a compound represented by structural formula (I)’, wherein the compound is represented by structural formula (Ila’), (lib’), or (lie’):

[0061] H 'Z N /

[0062] N

[0063]

[0064] 5

[0065] ME1\60300477.v1Docket No. 136840-00220

[0066]

[0067] or a pharmaceutically acceptable salt thereof, wherein the remainder of the variables are as described in the first embodiment.

[0068] A sixth embodiment of the invention is a compound represented by structural Formula (F), (I), or (Ila’) wherein the compound represented by structural formula (II):

[0069]

[0070] or a pharmaceutically acceptable salt thereof; wherein the remainder of the variables are as described in the first embodiment.

[0071] A seventh embodiment of the invention is a compound represented by structural formula (I’), (I), (Ila’), (lib’), (lie’) or (II), or a pharmaceutically acceptable salt thereof, wherein Xj is CH; and the remainder of the variables are as described in the first, second, third, or fourth embodiment.

[0072] An eighth embodiment of the invention is a compound represented by structural formula (I’), (I), (Ila’), (lib’), (lie’) or (II), or a pharmaceutically acceptable salt thereof, wherein Xj is N; and the remainder of the variables are as described in the first, second, third, or fourth embodiment.

[0073] A ninth embodiment of the invention is a compound represented by structural formula (F), (I), (Ila’), (lib’), (He’) or (II), or a pharmaceutically acceptable salt thereof, wherein Y and Z are CH; and the remainder of the variables are as described in the first, second third, fourth, seventh or eighth embodiment.

[0074] A tenth embodiment of the invention is a compound represented by structural formula (I’), (1), (Ila’), (lib’), (He’) or (II), or a pharmaceutically acceptable salt thereof, wherein Y

[0075] 6

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[0077] and Z are CH2; and the remainder of the variables are as described in the first, second third, fourth, seventh or eighth embodiment.

[0078] An eleventh embodiment of the invention is a compound represented by structural formula (T), (I), (Ila’), (lib’), (He’) or (II), or a pharmaceutically acceptable salt thereof, wherein Y is (CH2)2 and Z is CH2; and the remainder of the variables are as described in the first, second third, fourth, seventh or eighth embodiment.

[0079] A twelfth embodiment of the invention is a compound represented by structural formula (F), (Ila’), (lib’ ), or (lie’), wherein the compound is represented by one of the following structures:

[0080]

[0081] 7

[0082] ME1\60300477.v1Docket No. 136840-00220

[0083]

[0084] or a pharmaceutically acceptable salt thereof, and the remainder of the variables are as described in the first or second embodiment.

[0085] A thirteenth embodiment of the invention is a compound represented by structural formula (I’), (Ila’), (lib’), (lie’), (Illa’), (Illb’). (IIIc’), (Illd’), (life’), (Illf’), (Illg’), (Illh’ ), or (Illi’), or a pharmaceutically acceptable salt thereof, wherein X3 is selected from O, -OCH2-*, and -O(CH2)2-*, and the remainder of the variables are as described in the first, second, third, fourth, seventh, eighth, ninth, tenth or eleventh embodiment.

[0086] A fourteenth embodiment of the invention is a compound represented by structural formula (I’), (Ila’), (lib’), (He’), (Illa’), (Illb’). (IIIc’), (Hid’), (life’), (Illf’), (Illg’), (Illh’), or (Illi’), or a pharmaceutically acceptable salt thereof, wherein X4 is absent, and the remainder of the variables are as described in the first, second, third, fourth, seventh, eighth, ninth, tenth, eleventh or thirteenth embodiment.

[0087] A fifteenth embodiment of the invention is a compound represented by structural formula (I’), (Ila’), (lib’), (He’), (Illa’), (Illb’), (IIIc’), (Hid’), (life’), (Illf’), (Illg’), (Illh’), or (Illi’), or a pharmaceutically acceptable salt thereof, wherein X4 is O, and the remainder of the variables are as described in the first, second, third, fourth, seventh, eighth, ninth, tenth, eleventh or thirteenth embodiment.

[0088] A sixteenth embodiment of the invention is a compound represented by structural formula (I’), (I), (II), (Ila’), (lib’), (lie’), (Hla’), (Illb’), (IIIc’), (Hid’), (life’), or (Illf’), wherein the compound is represented by one of the following structures:

[0089] 8

[0090] ME1\60300477.v1Docket No. 136840-00220

[0091] , (Hie); and

[0092]

[0093] or a pharmaceutically acceptable salt thereof; and the remainder of the variables are as described in the first embodiment.

[0094] A seventeenth embodiment of the invention is a compound represented by structural formula (I); (I), (II), (Ila’), (lib’), (lie’), (Illa’), (Illb’), (IIIc’), (Hid’), (Hie’), (Ulf ), (Illa), (Illb), (IIIc), (Illd), (Ille), or (lilt), wherein the compound is represented by one of the following structural formula:

[0095]

[0096] 9

[0097] ME1\60300477.v1Docket No. 136840-00220

[0098]

[0099] wherein q is 0 or 1, or a pharmaceutically acceptable salt thereof; and the remainder of the variables are as described in the first embodiment.

[0100] An eighteenth embodiment of the invention is a compound represented by structural formula (IVa), (IVb), (IVc), (IVd), (IVe), or (IVf), or a pharmaceutically acceptable salt thereof, wherein q is 0, and the remainder of the variables are as described in the first embodiment.

[0101] A nineteenth embodiment of the invention is a compound represented by structural formula (IV a), (IVb), (IVc), (IVd), (IVe), or (IVf), or a pharmaceutically acceptable salt thereof, wherein q is 1, and the remainder of the variables are as described in the first embodiment.

[0102] A twentieth embodiment of the invention is a compound represented by structural formula (F), (I), (Ila’), (lib’), (He’), (II), (Illa’), (Illb’), (IIIc’ ), (Hid’), (Hie’), (Ulf’), (Illg’), (Illh’). (Illi’), (Illa), (Illb), (IIIc), (Hid), (Ille), (Ulf), (IVa), (IVb), (IVc), (IVd), (IVe), or (IVf), or a pharmaceutically acceptable salt thereof, wherein each Ri and R2 are

[0103] 10

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[0105] independently selected from hydrogen, halogen, -COOH, -N(CH₃)₂, C₁₋₃alkyl, and C₁₋₃haloalkyl; and the remainder of the variables are as described in the first, second, third, fourth, seventh, eighth, ninth, tenth, eleventh, thirteenth, fourteenth, fifteenth, seventeenth, eighteenth or nineteenth embodiment.

[0106] A twenty-first embodiment of the invention is a compound represented by structural formula (I’), (I). (Ila’), (lib’), (lie’), (II), (Illa’). (Illb’), (IIIc’), (Illd’ ), (Ille’), (Ulf’ ). (Illg’), (Illh’), (Illi’), (Illa), (Illb), (IIIc), (Hid), (life), (Ulf), (IVa), (IVb), (IVc), (IVd), (IVe), or (IVf), or a pharmaceutically acceptable salt thereof, wherein each Ri and R2 are independently selected from halogen, C₁₋₆alkyl, and C₁₋₆haloalkyl; and the remainder of the variables are as described in the first, second, third, fourth, seventh, eighth, ninth, tenth, eleventh, thirteenth, fourteenth, fifteenth, seventeenth, eighteenth or nineteenth embodiment.

[0107] A twenty-second embodiment of the invention is a compound represented by structural formula (I’), (I), (Ila’), (lib’), (He’), (II), (Illa’), (Illb’), (IIIc’), (Illd’), (life’). (IIIF), (Illg’), (Illh’), (ini’), (Illa), (Illb), (IIIc), (Hid), (Hie), (Ulf), (IVa), (IVb), (IVc), (IVd), (IVe), or (IVf), or a pharmaceutically acceptable salt thereof, wherein each Ri and R2 are independently selected from halogen and C₁₋₃haloalkyl; and the remainder of the variables are as described in the first, second, third, fourth, seventh, eighth, ninth, tenth, eleventh, thirteenth, fourteenth, fifteenth, seventeenth, eighteenth or nineteenth embodiment.

[0108] A twenty-third embodiment of the invention is a compound represented by structural formula (I’), (I), (Ila’), (lib’), (He’), (II), (Illa’), (Illb’), (IIIc’), (Hid’), (Ille’), (IIIF). (Illg’), (Illh’), (Illi’), (Illa), (Illb), (IIIc), (Hid), (life), (Ulf), (IVa), (IVb), (IVc), (IVd), (IVe), or (IVf), or a pharmaceutically acceptable salt thereof, wherein each Ri and R2 are independently selected from -F, -Cl, and –CF₃; and the remainder of the variables are as described in the first, second, third, fourth, seventh, eighth, ninth, tenth, eleventh, thirteenth, fourteenth, fifteenth, seventeenth, eighteenth, or ninteenth embodiment.

[0109] A twenty-fourth embodiment of the invention is a compound, or a pharmaceutically acceptable salt thereof, wherein the compound is represented by a compound in Table 1, or a pharmaceutically acceptable salt thereof. The neutral form and pharmaceutically acceptable salts of compounds disclosed herein are included in the invention.

[0110] Table 1. Compounds of the Invention

[0111] No No

[0112] Structure Structure

[0113]

[0114] 11

[0115] ME1\60300477.v1Docket No. 136840-00220

[0116]

[0117] ME1\60300477.v1Docket No. 136840-00220

[0118] £ AA> o

[0119] 17 18 | J H (

[0120] fA- £..... A. A^'A " F &

[0121] 19 0 < 20

[0122] AA / J O J: H

[0123] is i H

[0124] .. Ay-S;* r f A

[0125] . A... 0.,:.-x. A;....9 72 o' ■•■" 21

[0126] cA o

[0127] |i 1 t:

[0128] o AY\ ■■■ ■..,..•■■■•■.,.. X:....,• • 23...<:>......,. O„....,. X... &.(. X,....;X 6 X... X. W 24

[0129] J j £

[0130] :?*3V

[0131] ..••••X.,.f5s ■■......< X....--A:-... Z

[0132] O ■ G -;'-:

[0133] 0 * 0

[0134] 25 26...;... X. X. X. X d

[0135] A _• H f A - h - £

[0136] . A.,,&....■■■?>...x...- C X 0 X' 27........c>.... A*SAA ■.. S k. ■.x. -..... OH 28

[0137] x " -AHo

[0138] £

[0139] i A i N 4:-..&. > U, 4 a A ‘A- Y ° h 29 30 a

[0140] .. O. A J W 0

[0141] >> > M ar " x "

[0142] 31 0 32::.. -%; Y.. O....>..N.. A...,:? A ■ i H o tf: H

[0143]

[0144] 13

[0145] ME1\60300477.v1Docket No. 136840-00220

[0146]

[0147] II. Definitions

[0148] To facilitate understanding of the disclosure set forth herein, a number of terms are defined below.

[0149] Generally, the nomenclature used herein and the laboratory procedures in organic chemistry, medicinal chemistry, and pharmacology described herein are those well-known and commonly employed in the art. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0150] The term “alkyl” refers to a saturated monovalent hydrocarbon radical. The term “alkyl” encompasses both linear and branched alkyl. Unless otherwise specified, alkyl is a linear saturated monovalent hydrocarbon radical that has 1 to 10 (C₁₋₁₀), or 1 to 6 (C₁₋₆) carbon atoms, or a branched saturated monovalent hydrocarbon radical of 3 to 10 (C3-10), or 3 to 6 (C3-6) carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl (including all isomeric forms), n-propyl, isopropyl, butyl (including all isomeric forms), n-butyl, isobutyl, sec-butyl, t-butyl, pentyl (including all isomeric forms), and hexyl (including all isomeric forms). Alternatively, C1-6 alkyl refers to a linear saturated monovalent hydrocarbon radical of 1 to 6 carbon atoms or a branched saturated monovalent hydrocarbon radical of 3 to 6 carbon atoms.

[0151] The terms “halogen”, “halide” or “halo” refers to fluorine, chlorine, bromine, and / or iodine.

[0152] The term “haloalkyl” means alkyl substituted with one or more halogen atoms.

[0153] Examples of haloalkyl, include, but are not limited to, trifluoromethyl, trichloromethyl, pentafluoroethyl and the like. In certain aspects, the haloalkyl is represented by a fluoroalkyl. In other aspects, the haloalkyl is represented by a perfluoroalkyl.

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[0156] The term "heteroaryl" refers to an aromatic 5- to 9-membered monocyclic or fused bicyclic ring system, having 1 to 4 (i.e., 1, 2, 3, or 4) heteroatoms independently selected from O, N and S. Examples of 5- to 9-membered heteroaryls include, but are not limited to, furanyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridyl, pyrimidinyl, pyrrolyl, thiazolyl, thienyl, benzofuranyl, benzimidazolyl, benzoisoxazolyl, benzopyranyl, benzothienyl, benzoxazolyl, indolyl, indazolyl, isoquinolinyl, naphthyridinyl, quinolinyl, quinoxalinyl, and quinazolinyl.

[0157] The term ‘‘subject” refers to an animal in need of treatment for hearing loss or at risk for developing hearing loss, including, but not limited to, a primate (e.g., human), cow, pig, sheep, goat, horse, dog, cat, rabbit, rat, or mouse. The terms “subject” and “patient” are used interchangeably herein in reference, for example, to a mammalian subject. In one embodiment, the subject is a human.

[0158] The term “effective amount” is meant to include the amount of a compound that, when administered, is sufficient to alleviate, at least to some extent, one or more symptoms of hearing loss or slow the onset or development of hearing loss in a subject at risk of developing hearing loss. “An effective amount” of the disclosed compounds or pharmaceutically acceptable salts thereof, is determined by the physician on the basis of the patient-specific parameters, such as age, weight, sex, severity of the disease, etc. The dosage is preferably between 0.0001 mg to 1000 mg / kg body weight.

[0159] Another embodiment of the invention is a compound disclosed herein, wherein one or more hydrogen atoms are optionally replaced with deuterium. The deuterium enrichment at any one of the sites where hydrogen has been replaced by deuterium is at least 10%, 25%, 50%, 75%, 85%, 90%, 95%, 98% or 99%. Deuterium enrichment is a mole percent and is obtained by dividing the number of compounds with deuterium enrichment at the site of enrichment with the number of compounds having hydrogen or deuterium at the site of enrichment. With respect to the compounds disclosed herein, all hydrogen atoms are present in natural abundance unless otherwise specified.

[0160] Compounds of the present disclosure are generally administered as part of a pharmaceutical compositions, which comprises the compound and a pharmaceutically acceptable carrier or excipient. The term “pharmaceutically acceptable carrier,” “pharmaceutically acceptable excipient,” “physiologically acceptable carrier,” or “physiologically acceptable excipient” refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating

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[0163] material. In one embodiment, each component is “pharmaceutically acceptable” in the sense of being compatible with other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio. See, Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 7th Edition, Rowe et al., Eds., The Pharmaceutical Press and the American Pharmaceutical Association: 2012; Handbook of Pharmaceutical Additives, 3rd Edition, Ash and Ash Eds., Gower Publishing Company: 2007; and Pharmaceutical Preformulation and Formulation, 2nd Edition, Gibson Ed., CRC Press LLC: Boca Raton, FL, 2009.

[0164] Pharmaceutical compositions of the present disclosure may be in solid forms (e.g., powders, suspensions, tablets, capsules, wafers, patches, and the like), liquid forms (e.g., solutions, suspensions, elixirs, syrups, sprays, and the like) and semi-solid forms (e.g., lotions, gels, emulsions, and the like). Powders may include amorphous powders, crystalline powders, and mixtures thereof. Tablets may include chewable tablets, non-chewable tablets, ingestible tablets, buccal tablets, troches, lozenges, suppositories, and the like. Capsules may include hard-shell capsules and soft-shell capsules in the form of troches, lozenges, suppositories, and the like. Solutions include aqueous solutions, non-aqueous solutions and mixed (aqueous / non-aqueous) solutions in the form of parenteral solutions, injectable solutions, infusible solutions, and the like.

[0165] The composition can be formulated to contain a daily dose or an appropriate portion of the daily dose in a dosage unit, which can be a single tablet or capsule or a liquid of a suitable volume.

[0166] In one embodiment, the solution is prepared from a water-soluble salt, such as hydrochloride. Generally, all compositions are prepared according to known methods in pharmaceutical chemistry. Capsules can be prepared by mixing the compound with a suitable carrier or diluent and filling an appropriate amount of the mixture into the capsule.

[0167] Commonly used carriers and diluents include, but are not limited to, inert powdered substances, such as a variety of different starches, powdered cellulose, especially crystalline and microcrystalline cellulose, sugars like fructose, mannitol and sucrose, cereal flour, and similar edible powder.

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[0170] Tablets can be prepared by direct compression, wet granulation, or dry granulation. The preparation usually adds diluent, binder, lubricant and disintegrant and the compound. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts (such as sodium chloride) and powdered sugar. Powdered cellulose derivatives are also useful. Typical tablet binders are the following substances, such as starch, gelatin, and sugar (such as lactose, fructose, glucose, etc.). Natural and synthetic gums are also suitable, including gum acacia, alginate, methylcellulose, polyvinylpyrrolidone, etc. Polyethylene glycol, ethylcellulose and wax can also act as binders.

[0171] Lubricants can be selected from such slippery solids such as talc, magnesium stearate and calcium stearate, stearic acid and hydrogenated vegetable oils. A tablet disintegrant swells when wet to break the tablet and release the compound. They include starch, clay, cellulose, algin and gum. More specifically, for example, corn and potato starch, methylcellulose, agar, bentonite, lignocellulose, powdered natural sponge, anion exchange resin, alginic acid, guar gum, citrus pomace, carboxymethylcellulose and sodium lauryl sulfate can be used. Tablets can be coated with sugar as a flavoring and sealing agent or coated with a film-forming protective agent to optimize the dissolution performance of the tablet. The composition can also be formulated into chewable tablets, for example, by adding some substances to the formulation, such as mannitol.

[0172] When a compound provided herein contains an acidic or basic moiety, it may also be provided as a pharmaceutically acceptable salt (See, Berge et al., J. Pharm. Sci. 1977, 66, 1- 19; and “Handbook of Pharmaceutical Salts, Properties, and Use,” Stahl and Wermuth, Ed.; Wiley- VCH and VHCA, Zurich, 2002).

[0173] Suitable acids for use in the preparation of pharmaceutically acceptable salts include, but are not limited to, acetic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, citric acid, ethanesulfonic acid, 2-hydroxy-ethanesulfonic acid, fumaric acid, L-glutamic acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, (+)-L-lactic acid, (+)-DL-lactic acid, maleic acid, (-)-L-malic acid, malonic acid, methanesulfonic acid, nicotinic acid, nitric acid, oxalic acid, perchloric acid, phosphoric acid, saccharic acid, salicylic acid, succinic acid, sulfuric acid, (+)-L-tartaric acid, and p-toluenesulfonic acid.

[0174] Suitable bases for use in the preparation of pharmaceutically acceptable salts, include, but are not limited to, inorganic bases, such as magnesium hydroxide, calcium hydroxide, potassium hydroxide, or sodium hydroxide; and organic bases, such as primary’, secondary,

[0175] 17

[0176] ME1\60300477.v1Docket No. 136840-00220

[0177] tertiary, and quaternary, aliphatic and aromatic amines, including, but not limited to, L-arginine, choline, methylamine, dimethylamine, trimethylamine, diethylamine, triethylamine, isopropylamine, diisopropylamine, ethanolamine, diethanolamine, ethylenediamine, morpholine, piperidine, piperazine, pyrrolidine, pyridine, quinuclidine, and N-methyl-D-glucamine.

[0178] / / / . Methods of Treatment

[0179] The terms “treat,” “treating,” and “treatment” include alleviating or attenuating hearing loss, or one or more symptoms of hearing loss, e.g., restoring hearing at least partially. Subjects which can be therapeutically treated include those with partial hearing loss or those with complete hearing loss. “Slowing the onset of hearing loss” means that the at risk subject develops hearing loss at a later point in time when treated than in the absence of treatment. “Slowing the onset of hearing loss” can also mean that, once hearing loss develops, the subject develops a milder form of hearing loss.

[0180] An “at-risk population” is meant to include subjects that possess characteristics that result in the subjects being likely to develop the disorder, disease, or condition. For example, an at-risk population for hearing loss may include subjects who are elderly (e.g., greater than sixty years of age), exposed to loud environments, exposed to ototoxic substances, and / or exposed to trauma resulting in hearing loss. Non limiting examples of ototoxic substances include aminoglycosides (e.g. tobramycin and amikacin), anti-cancer drugs (cisplatin) and loop diuretics (furosemide) and phosphodiesterase 5 inhibitors (tadalafil). At risk populations include those who have not developed hearing loss as well as those who have developed some degree of hearing loss, i.e., treating subjects with partial hearing loss to reduce the likelihood of developing further hearing loss.

[0181] The term “endoplasmic reticulum stress” or “ER stress” refers to perturbation of endoplasmic reticulum homeostasis, e.g., perturbation of the protein folding functionality of the endoplasmic reticulum.

[0182] The compounds or pharmaceutical compositions provided herein can be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracistemal injection or infusion, subcutaneous injection, or implant), inhalation, nasal, vaginal, rectal, sublingual, or topical (e.g., transdermal or local) routes of administration and can be formulated, alone or together, in suitable dosage unit with pharmaceutically acceptable excipients, carriers, and vehicles appropriate for each route of administration. Also provided

[0183] 18

[0184] ME1\60300477.v1Docket No. 136840-00220

[0185] is administration of the compounds or pharmaceutical compositions provided herein in a depot formulation, in which the active ingredient is released over a predefined time period.

[0186] For oral administration, a disclosed compound may be incorporated with an excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Pharmaceutical compositions provided herein can be formulated to contain from about 1.0 to about 1,000 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. The pharmaceutical compositions can be administered on a regimen of 1 to 4 times per day, including once, twice, three times, and four times per day.

[0187] For parenteral administration, solutions of a compound used in the disclosed methods can generally be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0188] The compound provided herein, e.g., a compound of Formula I,; or a pharmaceutically acceptable salt thereof; can also be combined or used in combination with other agents or therapies useful in the treatment, prevention, or amelioration of hearing loss.

[0189] Suitable other therapeutic agents can also include, but are not limited to, (1) alpha- adrenergic agents; (2) antiarrhythmic agents; (3) anti-atherosclerotic agents, such as ACAT inhibitors; (4) antibiotics, such as an thracyc lines, bleomycins, mitomycin, dactinomycin, and plicamycin; (5) anticancer agents and cytotoxic agents, e.g., alkylating agents, such as nitrogen mustards, alkyl sulfonates, nitrosoureas, ethylenimines, and triazenes; (6) anticoagulants, such as acenocoumarol, argatroban, bivalirudin, lepirudin, fondaparinux, heparin, phenindione, warfarin, and ximelagatran; (7) anti-diabetic agents, such as biguanides (e.g., metformin), glucosidase inhibitors (e.g., acarbose), insulins, meglitinides (e.g., repaglinide), sulfonylureas (e.g., glimepiride, glyburide, and glipizide), thiozolidinediones (e.g., troglitazone, rosiglitazone, and pioglitazone), and PPAR-gamma agonists; (8) antifungal agents, such as amorolfine, amphotericin B, anidulafungin, bifonazole, butenafine, butoconazole, caspofungin, ciclopirox, clotrimazole, econazole, fenticonazole, filipin, fluconazole, isoconazole, itraconazole, ketoconazole, micafungin, miconazole, naftifine, natamycin, nystatin, oxyconazole, ravuconazole, posaconazole, rimocidin, sertaconazole, sulconazole, terbinafine, terconazole, tioconazole, and voriconazole; (9) antiinflammatories.

[0190] 19

[0191] ME1\60300477.v1Docket No. 136840-00220

[0192] e.g., non-steroidal anti-inflammatory agents, such as aceclofenac, acemetacin, amoxiprin, aspirin, azapropazone, benorilate, bromfenac, carprofen, celecoxib, choline magnesium salicylate, diclofenac, diflunisal, etodolac, etoricoxib, faislamine, fenbufen, fenoprofen, flurbiprofen, ibuprofen, indometacin, ketoprofen, ketorolac, lornoxicam, loxoprofen, lumiracoxib, meclofenamic acid, mefenamic acid, meloxicam, metamizole, methyl salicylate, magnesium salicylate, nabumetone, naproxen, nimesulide, oxyphenbutazone, parecoxib, phenylbutazone, piroxicam, salicyl salicylate, sulindac, sulfinpyrazone, suprofen, tenoxicam, tiaprofenic acid, and tolmetin; (10) antimetabolites, such as folate antagonists, purine analogues, and pyrimidine analogues; (11) anti-platelet agents, such as GPIIb / IIIa blockers (e.g., abciximab, eptifibatide, and tirofiban), P2Y(AC) antagonists (e.g., clopidogrel, ticlopidine and CS-747), cilostazol, dipyridamole, and aspirin; (12) antiproliferatives, such as methotrexate, FK506 (tacrolimus), and mycophenolate mofetil; (13) anti-TNF antibodies or soluble TNF receptor, such as etanercept, rapamycin, and leflunimide; (14) aP2 inhibitors; (15) beta-adrenergic agents, such as carvedilol and metoprolol; (16) bile acid sequestrants, such as questran; (17) calcium channel blockers, such as amlodipine besylate; (18) chemotherapeutic agents; (19) cyclooxygenase-2 (COX-2) inhibitors, such as celecoxib and rofecoxib; (20) cyclosporins; (21) cytotoxic drags, such as azathioprine and cyclophosphamide; (22) diuretics, such as chlorothiazide, hydrochlorothiazide, flumethiazide, hydroflumethiazide, bendroflumethiazide, methylchloro thiazide, trichloromethiazide, polythiazide, benzothiazide, ethacrynic acid, ticrynafen, chlorthalidone, furosenide, muzolimine, bumetanide, triamterene, amiloride, and spironolactone; (23) endothelin converting enzyme (ECE) inhibitors, such as phosphoramidon; (24) enzymes, such as L-asparaginase; (25) Factor Vila Inhibitors and Factor Xa Inhibitors; (26) farnesyl-protein transferase inhibitors; (27) fibrates; (28) growth factor inhibitors, such as modulators of PDGF activity; (29) growth hormone secretagogues; (30) HMG Co A reductase inhibitors, such as pravastatin, lovastatin, atorvastatin, simvastatin, NK-104 (a.k.a. itavastatin, nisvastatin, or nisbastatin), and ZD-4522 (also known as rosuvastatin, atavastatin, or visastatin); neutral endopeptidase (NEP) inhibitors; (31) hormonal agents, such as glucocorticoids (e.g., cortisone), estrogens / antiestrogens, androgens / antiandrogens, progestins, and luteinizing hormone-releasing hormone antagonists, and octreotide acetate; (32) immunosuppressants; (33) mineralocorticoid receptor antagonists, such as spironolactone and eplerenone; (34) micro tubule-disruptor agents, such as ecteinascidins; (35) microtubule-stabilizing agents, such as pacitaxel, docetaxel, and epothilones A-F; (36) MTP Inhibitors; (37) niacin; (38) phosphodiesterase inhibitors, such as PDE III inhibitors 20

[0193] ME1\60300477.v1Docket No. 136840-00220

[0194] (e.g., cilostazol) and PDE V inhibitors (e.g., sildenafil, tadalafil, and vardenafil); (39) plant-derived products, such as vinca alkaloids, epipodophyllotoxins, and taxanes; (40) platelet activating factor (PAF) antagonists; (41) platinum coordination complexes, such as cisplatin, satraplatin, and carboplatin; (42) potassium channel openers; (43) prenyl-protein transferase inhibitors; (44) protein tyrosine kinase inhibitors; (45) renin inhibitors; (46) squalene synthetase inhibitors; (47) steroids, such as aldosterone, beclometasone, betamethasone, deoxycorticosterone acetate, fludrocortisone, hydrocortisone (cortisol), prednisolone, prednisone, methylprednisolone, dexamethasone, and triamcinolone; (48) TNF-alpha inhibitors, such as tenidap; (49) thrombin inhibitors, such as hirudin; (50) thrombolytic agents, such as anistreplase, reteplase, tenecteplase, tissue plasminogen activator (tPA), recombinant tPA, streptokinase, urokinase, prourokinase, and anisoylated plasminogen streptokinase activator complex (APSAC); (51) thromboxane receptor antagonists, such as ifetroban; (52) topoisomerase inhibitors; (53) vasopeptidase inhibitors (dual NEP-ACE inhibitors), such as omapatrilat and gemopatrilat; and (54) other miscellaneous agents, such as, hydroxyurea, procarbazine, mitotane, hexamethylmelamine, and gold compounds.

[0195] In certain embodiments, the other therapies that may be used in combination with the compounds provided herein include, but are not limited to, surgery, endocrine therapy, biologic response modifiers (e.g., interferons, interleukins, and tumor necrosis factor (TNF)), hyperthermia and cryotherapy, and agents to attenuate any adverse effects (e.g., antiemetics).

[0196] Such other agents, or drugs, can be administered, by a route and in an amount commonly used therefor, simultaneously or sequentially with the compound provided herein, e.g., a compound of Formula I, or a pharmaceutically acceptable salt thereof. When a compound provided herein is used contemporaneously with one or more other drugs, a pharmaceutical composition containing such other drugs in addition to the compound provided herein can be utilized, but is not required. In another embodiment, pharmaceutical compositions provided herein include one or more other active ingredients or therapeutic agents, in addition to a compound provided herein.

[0197] The compounds provided herein can be prepared, isolated, or obtained by any methods known to one of skill in the art, and the following examples are only representative and do not exclude other related procedures.

[0198] EXEMPLIFICATION

[0199] I. Definitions

[0200] 21

[0201] ME1\60300477.v1Docket No. 136840-00220

[0202] The disclosure will be further understood by the following non-limiting examples. As used herein, the symbols and conventions used in these processes, schemes and examples, regardless of whether a particular abbreviation is specifically defined, are consistent with those used in the contemporary scientific literature, for example, the Journal of the American Chemical Society or the Journal of Biological Chemistry. Specifically, but without limitation, the following abbreviations may be used in the examples and throughout the specification: g (grams); mg (milligrams); mL (milliliters); pL (microliters); M (molar); mM (millimolar); pM (micromolar); mol (moles); mmol (millimoles); hr or hrs (hour or hours); and min (minutes).

[0203] For all of the following examples, standard procedures and methods known to those skilled in the art can be utilized. Unless otherwise indicated, all temperatures are expressed in °C (degrees Centigrade). All procedures are conducted at room temperature unless otherwise noted.

[0204] II. Biological Studies

[0205] Example 1: ER Stress Induction in HEK Cells Treated With Compounds of the Invention HEK 293 cells were cultured in DMEM with 10% FBS, Pen / Strep in 5% CO2, at 37 °C in incubator. Upon reaching about 80% confluency, cells were split and seeded at approximately 2x104 cells in each well of 96-well plate one day before treatment. Cells were grown in good shape and passaged one or two times before testing.

[0206] Cells were treated with 0.5 pM of the ER stress inducer thapsigargin and 1 pM of each drug or vehicle (DMSO) for 2 hours. The cells were then harvested, pelleted and treated to ready for testing mRNA abundance for specific UPR genes from cells in the PCR reaction using the TaqMan™ Gene Expression Cells- to-CT™ Kit (Thermo Fisher). Prescreened primers were ordered from Bio-Rad. The gene tested is CHOP, (the C / EBP Homologous Protein). These markers were compared against GAPDH. The qPCR system we used was CFX 384 Real time (Bio-Rad). Samples were measured in triplicates and relative quantification, by the ΔΔCt-method, using GAPDH as reference.

[0207] The biological results are summarized in Table 2, where A represents a greater than 50% reduction in CHOP signal in compound-treated versus untreated cells, B represents a 20% to 50% reduction in CHOP signal, and C represents a 1% to 20% reduction in CHOP signal.

[0208] Table 2. Results of ER Stress Induction Assay

[0209] 22

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[0211] Compound No. CHOP Reduction Compound No. CHOP Reduction

[0212] 1 A 2 B 3 B 4 C 5 C 6 C 7 C 8 c 9 c 10 A 11 A 12 B 13 B 14 B 15 B 16 B 17 C 18 B

[0213]

[0214] Example 2: Protection of HEK Cells from ER Stress-induced Cell Death

[0215] HEK 293 cells were cultured in DMEM supplemented with 10% FBS and Pen / Strep in a 5% CO2 incubator at 37 °C. Upon reaching 80% confluency, cells were split and seeded at approximately 2×104cells / well in 96- well white- walled plates one day before treatment.

[0216] Cells were then treated for 24 hours with 0.5 pM of ER stress inducer thapsigargin and 1 p M of each JBI-compound or vehicle control (DMSO). Before adding the CellTiter-Glo® reagent, plates were incubated at room temperature for 30 minutes to allow cells to equilibrate. The assay quantifies ATP levels, which correlate to metabolically active and viable cells. 100 pL of CellTiter-Glo® reagent was then added directly to each well (1:1 ratio with the existing medium). The plate was shaken on an orbital shaker for 2 minutes to ensure complete cell lysis, followed by a 10-minute incubation at room temperature to stabilize the luminescent signal. Luminescence was measured using a Varioskan™ plate reader with an integration time of 200 ms. Each plate was read three times, and all readings were used for data analysis. Cell viability (%) was calculated relative to the DMSO control using the formula: % Viability= (Luminescence of treated sample / Luminescence of control) ×100.

[0217] The biological results are summarized in Table 3 where A represents a cell viability greater than 90% and B represents a cell viability of 80% to 90%.

[0218] Table 3. Results of ER Stress Cell Survival Assay Compound No. Cell Viability Compound No. Cell Viability

[0219] 1 B 2 B

[0220]

[0221] 23

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[0223] Compound No. Cell Viability Compound No. Cell Viability

[0224] 5 B 7 A 10 A 11 A 12 A 13 B

[0225]

[0226] Example 3: Reduction of ER Stress-Induced ATF4 Expression

[0227] Activating Transcription Factor 4 (ATF4) is a stress-responsive basic leucine zipper transcription factor that plays a central role in the integrated stress response (ISR). During endoplasmic reticulum (ER) stress, the PERK branch of the unfolded protein response (UPR) phosphorylates eIF2a, which selectively enhances translation of ATF4. Once induced, ATF4 upregulates genes involved in a variety of cellular processes including apoptosis, thereby initiating cell-death if cells encounter prolonged or unresolvable stress such as ototoxic agents or excessive noise. Through this regulated transcriptional program, ATF4 functions as a key mediator linking ER stress signals to adaptive and, when necessary, pro-apoptotic outcomes.

[0228] Method:

[0229] Compounds were tested for reduction of ATF4 expression using the ATF4 Luciferase Reporter HEK 293 Stable Cell Line. Cells were cultured in DMEM with 10% FBS, Pen / Strep in 5% CO2, at 37 °C in incubator. Cells were split and seeded at approximately 2×104cells in each well of 96-well plate before treatment. Cells were treated to provide a final concentration of 0.2 pM thapsigargin and 10 pM of each compound for 4 hours. The cells were then harvested, pelleted and tested with the Bright-GLo luminescence assay kit (Promega). Samples were measured in triplicates and a change in fluorescence intensity when compared to vehicle control correlated to a reduction in ATF4 expression. The biological results are summarized in Table 4 where A represents a greater than 75% reduction in ATF4 signal in compound-treated versus vehicle-treated cells, B represents a 50% to 74% reduction in ATF4 signal, C represents a 25% to 49% reduction in ATF4, and D represents a 24% or less reduction in ATF4 signal.

[0230] Table 4. Results of ER Stress Cell Survival Assay Compound No. Ceil Viability Compound No. Cell Viability

[0231] 19 B 20 B 21 B 22 B

[0232]

[0233] 24

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[0235] Compound No. Cell Viability Compound No. Cell Viability

[0236] 23 B 24 C 25 C 26 D 27 C 28 B 29 D 30 B 31 D 32 D 33 A 34 D 35 D 36 C

[0237]

[0238] III. Synthesis of Compounds

[0239]

[0240] Compound 1: 2-(4-chlorophenoxy)--(1-(2-(4-chlorophenoxy)acetyl)-1-indol-5-yl)acetamide. A solution of lH-indol-5-amine (200 mg, 1.513 mmol, 1 equiv) in DMF (4 mL) was treated with NaH (108.94 mg, 4.539 mmol, 3 equiv) at 0 °C for 40 min under nitrogen atmosphere followed by the addition of 2-(4-chlorophenoxy)acetyl chloride (768.20 mg, 3.782 mmol, 2.5 equiv) at 0 °C. The resulting mixture was stirred at room temperature for an additional 45 min. The reaction was quenched with water at 0 °C. The resulting mixture was extracted with EtOAc (3 x 20mL). The combined organic layers were washed with brine (3x20 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by reversed-phase flash chromatography with the following conditions: column, C18 silica gel; mobile phase, MeCN in Water (0.1% NH4HCO3), 5% to 70% gradient in 30 min: detector, UV 254 nm. The crude product (450mg) was purified by Prep-HPLC with the following conditions (Column: XBridge Prep OBD Cl 8 Column, 30*150 mm, 5 pm; Mobile Phase A: Water(10 mmol / L NH4HCO3), Mobile Phase B: ACN; Flow rate: 60 mL / min; Gradient: 57% B to 67% B in 10 min; Wave Length: 254 nm; RTl(min): 10.14) to afford the title compound (73 mg, 10.28%yield, 98.9%purity) as a white solid. LC-MS: (ES, m / z): 469.05 [M+H]+. 1H NMR (400 MHz, DMSO-6) 8 10.17 (s, 1H), 8.24 (d, J = 9.0 Hz, 1H), 8.06 (d, J = 2.0 Hz, 1H), 7.92 (d,.7= 3.8 Hz, 1H), 7.52 - 7.45 (m, 1H), 7.41 - 7.32 (m, 4H), 7.14 - 7.02 (m, 4H), 6.82

[0241] 25

[0242] ME1\60300477.v1Docket No. 136840-00220

[0243] (d, J= 3.8 Hz, 1H), 5.54 (s, 2H), 4.74 (s, 2H).

[0244]

[0245] Compound 2: 2-(4-chlorophenoxy)--(1-(2-(4-chlorophenoxy)acetyl)-1-pyrrolo[2,3-c]pyridin-5-yl)acetamide. A mixture of lH-pyrrolo[2,3-c]pyridin-5-amine (120 mg, 0.901 mmol, 1 equiv), 2-(4-chlorophenoxy)acetic acid (420.39 mg, 2.252 mmol, 2.5 equiv), TCFH (328.72 mg, 1.171 mmol, 1.3 equiv), and 1 -methyl- IH-imidazole (369.97 mg, 4.505 mmol, 5 equiv) in MeCN (15 mL) was stirred at room temperature for 1 hour. The residue was purified by reversed-phase flash chromatography with the following conditions: column, C18 silica gel; mobile phase, MeCN in Water, 10% to 50% gradient in 15 min; detector, UV 254 nm. This resulted in title compound (51.8 mg, 12.22% yield, 97.7% purity) as a white solid. LC-MS: (ES, m / z): 470.20 [M+H]+. 1H NMR (400 MHz, DMSO-6) δ 10.59 (s, 1H), 9.22 (s, 1H), 8.34 (s, 1H), 8.15 (d,. / = 3.8 Hz, 1H), 7.41 - 7.32 (m, 4H), 7.18 - 7.09 (m, 2H), 7.07 - 6.99 (m, 2H), 6.92 (d, J = 3.7 Hz, 1H), 5.58 (s, 2H), 4.82 (s, 2H).

[0246] Compounds 4, 8, 9 and 14 were prepared following a similar procedure above with the corresponding pyrrolo[2,3-c]pyridin-5-amine and substituted 2-phenoxy acetic acid. Compound

[0247] Structure Characterization Data

[0248] No.

[0249] LC-MS: (ES, m / z): 472.05 [M+H]+Al NMR (400 MHz, DMSO-r / 6) 8 10.57 f* A.0

[0250] (s, 1H), 8.85 (s, 1H), 8.02 (s, 1H), 7.40 - 4 <..a, -' AN JLN ■') 7 \;

[0251] j \. / 7.27 (m, 4H), 7.07 - 6.96 (m. 4H), 4.98 (s, cr ' ■'%;

[0252] 2H), 4.78 (s, 2H), 4.19 (t,. / = 8.5 Hz, 2H),

[0253] 3.27 (t. J = 8.4 Hz, 2H).

[0254]

[0255] 26

[0256] ME1\60300477.v1Docket No. 136840-00220

[0257] LC-MS: (ES, m / z): 538.25 [M+H]+1H NMR (400 MHz, DMSO-rfc) 5 10.70 (s, 1H), 9.22 (s, 1H), 8.34 (s, 1H), 8.17 (d, = 3.8 Hz, 1H), 7.73 - 7.65 (m, 4H), 7.30

[0258] (d, J = 8.6 Hz, 2H), 7.18 (d, J = 8.7 Hz, 2H), 6.93 (d, J = 3.7 Hz, 1H), 5.70 (s, 2H),

[0259] 4.93 (s, 2H).

[0260] LC-MS: (ES, m / z): 539.15 [M+H]+1H NMR (400 MHz, DMSO-6) 5 10.10 (s, 1H), 7.94 (d, 7= 8.6 Hz, 1H), 7.73 -- 9., I f l

[0261] A si y 7.62 (m, 5H), 7.34 (dd, 7= 8.7, 2.2 Hz,

[0262] 1H), 7.18 (dd, 7= 8.6, 5.0 Hz, 4H), 5.06 (s, 2H), 4.80 (s, 2H), 4.17 (t, 7 = 8.4 Hz, y^

[0263] / > V / ■"" 2H), 3.20 (t, 7= 8.4 Hz, 2H).

[0264] LC-MS: (ES, m / z): 506.20 [M+H]+ / \ O / u zx

[0265] !H NMR (400 MHz, DMSO-X) 6 10.63 (s, 1H), 9.22 (s, 1H), 8.33 (s, 1H), 8.13 (d, X A

[0266] 9-? p '-0

[0267] 14 y-,.?7 = 3.8 Hz, 1H), 7.56 - 7.47 (m, 2H), 7.40

[0268] C:' y ■' \s

[0269] F - 7.29 (m, 1H), 7.20 - 7.11 (m, 1H), 7.06

[0270] - 6.97 (m, 1H), 6.95 - 6.87 (m, 2H), 5.61

[0271] (s, 2H). 4.87 (s, 2H).

[0272]

[0273]

[0274] Compound 3: 2-(4-chlorophenoxy)--(1-(2-(4-chlorophenoxy)acetyl)indolin-5-yl)acetamide. A solution of 2,3-dihydro-lH-indol-5-amine (1 g, 7.453 mmol, 1 equiv), 2-(4-chlorophenoxy) acetic acid (3.48 g, 18.633 mmol, 2.5 equiv), DIEA (4.82 g, 37.265 mmol, 5 equiv), and HATU (7.08 g, 18.633 mmol, 2.5 equiv) in DMF (10 mL) was stirred at room temperature overnight. The resulting mixture was extracted with EtOAc (3 x 30mL). The combined organic layers were washed with brine (2x30 mL) and dried over

[0275] anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography with the following conditions:

[0276] 27

[0277] ME1\60300477.v1Docket No. 136840-00220

[0278] column, C18 silica gel; mobile phase, MeCN in water (0.1% FA), 10% to 50% gradient in 10 min; detector, UV 254 nm. This resulted in 741 mg as a yellow oil crude product. The crude product (741mg) was purified by Prep-HPLC with the following conditions (Column:

[0279] XSelect CSH Prep C18 OBD Column, 30*150 mm, 5 μm; Mobile Phase A: Water(10 mmol / L NH4HCO3), Mobile Phase B: ACN; Flow rate: 60 mL / min; Gradient: 45% B to 65% B in 12min; Wave Length: 254 / 220 nm; RTl(min): 10.9) to afford title compound (59.7 mg, 1.70%yield, 99.1%purity) as a white solid. LC-MS: (ES, m / z): 471.05 [M+H]+.1H NMR (400 MHz, DMSO-6) δ 10.03 (s, 1H), 7.94 (d, = 8.7 Hz, 1H), 7.64 (d, J= 2.1 Hz, 1H), 7.41 - 7.29 (m, 5H), 7.08 - 6.96 (m, 4H), 4.94 (s, 2H), 4.69 (s, 2H), 4.15 (t,.7= 8.4 Hz, 2H), 3.19 (t, = 8.4 Hz, 2H)

[0280] Compounds 5, 6, 10-12, and 15-18 were prepared following a similar procedure above with the corresponding diamine and the substituted 2-phenoxy acetic acid.

[0281] Compound

[0282] Structure Characterization Data

[0283] No.

[0284] LCMS (ESI, m / z): 485.15 [M+H]+JH NMR (400 MHz, DMSO-6) δ 10.06 A zC (s. 1H), 7.54 (s. 2H), 7.37 (d, 8.0 Hz, 9 9 " T F"

[0285] % 3H). 7.31 (d, J = 8.0 Hz. 2H), 7.03 (d, J = 5 J I

[0286] 8.0 Hz, 2H), 6.89 (s, 2H), 4.97 (s, 2H), 4.70 (s, 2H), 3.68 (t, J= 7.8 Hz, 2H), 2.68 OS

[0287] (d, 7.8 Hz, 2H), 1.89 (d, J = 8.1 Hz,

[0288] 2H).

[0289] LC-MS: (ES, m / z): 486.05 [M+H]+^-NMR (400 MHz, DMSO-J6) 8 10.51 P rMy0

[0290] ,r...s.,0.vjl„ „A -3 < -o(s, 1H), 8.72 (s, 1H), 7.90 (s, 1H), 7.42 - 6

[0291] 7.29 (m, 4H). 7.06 - 6.87 (m, 4H), 5.07 (s, 'V 2H), 4.79 (s, 2H), 3.73 (t, J= 6.0 Hz, 2H), C:

[0292] 2.77 (t, 2H), 1.98 - 1.83 (m, 2H)

[0293]

[0294] 28

[0295] ME1\60300477.v1Docket No. 136840-00220

[0296] LC-MS: (ES, m / z): 540.10 [M+H]+JH NMR (400 MHz, DMSO-6) δ 10.67 / J / (s, IH), 8.85 (s, IH), 8.02 (s, IH), 7.67 10 A i x (dd, J = 8.8, 6.9 Hz, 4H), 7.17 (dd, J = o ~ tr * t 1

[0297] ' ' X 13.0, 8.6 Hz, 4H), 5.10 (s, 2H), 4.90 (s.

[0298] 2H), 4.21 (t,.7= 8.5 Hz, 2H), 3.29 (t,. / =

[0299] 8.4 Hz, 2H)

[0300] LCMS (ESI, m / z): 553.15 [M+H]+’H NMR (400 MHz, DMSO-6) δ 10.13,, j.y..,z;o

[0301] (s, 1H), 7.69 (d, J= 8.6 Hz, 2H), 7.64 (d, J 11 si J H 8 ------ 8.5 Hz, 2H), 7.55 (s, 2H), 7.38 (s, IH),

[0302] Z. X / '"■

[0303] 7.18 (d, J= 8.5 Hz, 2H), 7.06 (s, 2H), 5.09 (s, 2H), 4.82 (s, 2H), 3.69 (t, J 6.2 Hz, 2H), 2.71 (L. / = 6.8 Hz, 2H), 1.89 (s, 2H) LC-MS: (ES, m / z): 554.20 [M+H]+, L A,ojH NMR (400 MHz, DMSO-6) δ 10.62 X " 'a (s, IH), 8.76 (s, IH), 7.90 (s, IH), 7.66 (t, 12 [;,s H 3

[0304] 9.5 Hz, 4H), 7.21 - 7.04 (m, 4H), 5.19 < X (s, 2H), 4.91 (s, 2H), 3.74 (t,. / = 6.0 Hz,

[0305] 2H), 2.78 (t, 2H), 2.02 - 1.83 (m, 2H) LC-MS: (ES, m / z): 507.05 [M+H]+NMR (400 MHz, DMSO-&) 6 10.04 (s, IH), 7.94 (d, 8.6 Hz, IH), 7.64 (d, J = 2.2 Hz, IH). 7.40-7.60 (m, 2H), 7.34 15 1 I J..

[0306] J 1 '« ( v

[0307] cr X(dd, J= 8.7, 2.2 Hz, IH), 7.25-7.01 (m, T " Ai

[0308] F

[0309] 2H), 7.00-6.80 (m, 2H). 4.99 (s, 2H). 4.74 (s, 2H), 4.14 (t, J = 8.4 Hz, 2H), 3.19 (t, J = 8.4 Hz, 2H)

[0310]

[0311] 29

[0312] ME1\60300477.v1Docket No. 136840-00220

[0313] LC-MS: (ES, m / z): 508.20 [M+H]+JH NMR (300 MHz, DMSO-6) δ 10.57 T~\ / P (s, 1H), 8.85 (s, 1H), 7.99 (d, J = 16.0 Hz, 16 i IYK. 1H), 7.49 (td,.7= 8.9, 5.8 Hz, 2H), 7.15

[0314] " Y "ci

[0315] F (ddd, J= 14.0, 11.4, 2.8 Hz, 2H), 6.96 - 6.84 (m, 2H), 5.03 (s, 2H), 4.83 (s, 2H), 4.19 (t, J = 8.5 Hz, 2H) LCMS (ESI, m / z): 521.15 [M+H]+JH NMR (400 MHz, DMSO-&) 5 10.07 (s, 1H), 7.69 - 7.51 (m, 3H), 7.46 (t, J = 8.9 Hz, 1I-I), 7.37 (d, 7 = 8.5 Hz, 1H), 7.18 17:: j j

[0316] - 7.12 (m, 1H), 7.04 (s, 1H), 6.96 -- 6.86 (m, 1H), 6.78 (s, 1H), 5.03 (s, 2H), 4.75 (s, 6;

[0317] 2H), 3.68 (t,. / = 6.2 Hz, 2H), 2.70 (t,.7 = 6.6 Hz, 2H), 1.91 (t, 7= 6.1 Hz, 2H) LC-MS: (ES, m / z): 552.10 [M+H]+{H NMR (400 MHz, DMSO-6) δ 10.55 o (s, 1H), 8.76 (s, 1H), 7.89 (s, 1H), 7.55 - y "'O

[0318] 18 r JHI 7.43 (m, 2H), 7.12 (dd,.7= 11.4, 2.9 Hz,

[0319] 2H), 6.91 - 6.80 (m, 2H), 5.12 (s, 2H), OS 4.84 (s, 2H), 3.72 (t, 2H), 2.78 (t, 7= 6.6

[0320] Hz, 2H), 2.01 - 1.85 (m, 2H)

[0321]

[0322]

[0323] Compound 7: 2-(4-(trifluoromethyl)phenoxy)--(1-(2-(4-(trifluoromethyl)phenoxy)acetyl)-1-indol-5-yl)acetamide. A solution of 2-(4-(trifluoromethyl)phenoxy)acetic acid (1.30 g, 5.903 mmol, 2.5 equiv) in DCM (5 mL) was treated with DMF (34.51 mg, 0.472 mmol, 0.2 equiv) at 0 °C for 5 min. This was followed by the addition of oxalic dichloride (1.50 g, 11.805 mmol, 5 equiv) in portions at 0 °C. The resulting mixture was stirred at room temperature for an additional hour. The resulting mixture was concentrated under reduced pressure, and the residue was dissolved with DMF

[0324] 30

[0325] ME1\60300477.v1Docket No. 136840-00220

[0326] (solution A) and used directly. A separate solution of lH-indol-5-amine (312 mg, 2.361 mmol, 1.00 equiv) in DMF (4 mL) was treated with NaH (0.28 g, 11.805 mmol, 5 equiv) in portions at 0 °C. The resulting mixture was stirred at room temperature for 1 hour. Then, solution A was added to this mixture at 0 °C. The resulting mixture was stirred at room temperature for an additional hour. The reaction was quenched with water at 0 °C. The resulting mixture was extracted with EtOAc (3 x 25mL), and the combined organic layers were washed with brine (3x20 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reversed -phase flash chromatography with the following conditions: column, C18 silica gel; mobile phase, MeCN in Water (0.1% NH4HCO3), 5% to 70% gradient in 30 min; detector, UV 254 nm. The crude product was further purified by Prep-HPLC with the following conditions (Column: XBridge Prep OBD C18 Column, 30*150 mm, 5 pm; Mobile Phase A: Water(10 mmol / L NH4HCO3), Mobile Phase B: ACN; Flow rate: 60 mL / min; Gradient: 24% B to 34% B in 10 min; Wave Length: 254 nm; RTl(min): 10.23) to afford the title compound (53.3 mg, 97.7% purity) as a white solid. LC-MS: (ES, m / z): 537.15 [M+H]+.

[0327]

[0328] NMR (400 MHz, DMSO-6) 8 10.24 (s, 1H), 8.24 (d, J= 8.9 Hz, 1H), 8.06 (d, J = 2.0 Hz, 1H), 7.93 (d, J = 3.8 Hz, 1H), 7.70 (dd, 8.7, 6.1 Hz, 4H), 7.48 (dd, 8.9, 2.1 Hz, 1H), 7.28 (d, 8.6 Hz, 2H), 7.21 (d, J = 8.6 Hz, 2H), 6.84 (d,.7= 3.8 Hz, 1 H), 5.66 (s, 2H), 4.85 (s, 2H).

[0329]

[0330] Compound 13: 2-(4-chloro-3-fluorophenoxy)-A-(l-(2-(4-chIoro-3-fluorophenoxy)acetyI)-lH-indoI-5-yl)acetamide. A solution of lH-indol-5-amine (300 mg, 2.270 mmol, 1 equiv), TCFH (955.32 mg, 3.405 mmol, 1.5 equiv), 1 -methyl- IH-imid azole (298.19 mg, 3.632 mmol, 1.6 equiv) and 2-(4-chloro-3-fluorophenoxy)acetic acid (557.24 mg, 2.724 mmol, 1.2 equiv) in MeCN (5 mL) was stirred at room temperature for

[0331] 2 hours under air atmosphere. The resulting mixture was concentrated, and the residue was purified by silica gel column chromatography, eluted with PE / EA (3:7) to afford 2-(4-chloro-3-fluorophenoxy)-jV-(17 / -indol-5-yl)acetamide (550 mg, 76.02% yield, 96% purity) as a white solid. A solution of 2-(4-chloro-3-fluorophenoxy)acetic acid (882.56 mg, 4.315 mmol, 2.5 equiv) in DCM (5 mL) was treated with DMF (25.23 mg, 0.345 mmol, 0.2 equiv) at 0 °C for 5 min under air atmosphere followed by the addition of oxalic dichloride

[0332] 31

[0333] ME1\60300477.v1Docket No. 136840-00220

[0334] (1095.06 mg, 8.630 mmol, 5 equiv) in portions at 0 °C. The resulting mixture was stirred at room temperature for an additional hour and then concentrated under reduced pressure. The residue was dissolved in DMF (solution A) and was used directly. A separate solution of 2-(4-chloro-3-fluorophenoxy)-N-(lH-indol-5-yl)acetamide (550 mg, 1.726 mmol, 1 equiv) in DMF (5 mL) was treated with NaH (207.05 mg, 8.630 mmol, 5 equiv) in portions at 0 °C. The resulting mixture was stirred at room temperature for 1 hour. To this mixture was added solution A at 0 °C. The resulting solution was stirred at room temperature for an additional Ihour. The reaction was quenched with water at 0 °C. The resulting mixture was extracted with EtOAc (3 x 25mL), and the combined organic layers were washed

[0335] with brine (3x20 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography with the following conditions: column, C18 silica gel; mobile

[0336] phase, MeCN in Water (0.1% NH4HCO3), 5% to 70% gradient in 30 min; detector, UV 254 nm and further purified by Prep-HPLC with the following conditions (Column: XBridge Prep OBD C18 Column, 30*150 mm, 5 pm; Mobile Phase A: Water(10 mmol / L NH4HCO3), Mobile Phase B: ACN; Flow rate: 60 mL / min; Gradient: 58% B to 68% B in 10 min; Wave Length: 254 / 220 nm; RTl(min): 10.23) to afford the title compound (58.6 mg, 98.5% purity) as a white solid. LC-MS: (ES, m / z): 505.05 [M+H]+.1H NMR (400 MHz, DMSO-6) 8 10.19 (s, 1H), 8.24 (d,

[0337]

[0338] 8.8 Hz, 1H), 8.05 (d, 7= 2.0 Hz, 1H), 7.90 (d. 7= 3.8 Hz, 1H).

[0339] 7.60 - 7.45 (m, 3H), 7.31 (dd,. / = 11.5, 2.9 Hz, 1H), 7.17 (dd,. / = 11.4, 2.9 Hz, 1H), 7.03 - 6.90 (m, 2H), 6.83 (d, 7= 3.8 Hz, 1H), 5.58 (s, 2H), 4.79 (s, 2H).

[0340]

[0341] Compound 19: N-(l-(2-(4-chlorophenoxy)ethyI)indolin-5-yI)-2-(4-(trifluoromethyI) phenoxy)acetamide. To a solution of 2-[4-(trifluoromethyl)phenoxy]acetic acid and 1,1- dimethylethyl 5-amino-2,3-dihydro-lH-indole-l-carboxylate (0.75 mmol) in DMF (5.0 mL) were added l-|3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) (0.75 mmol), 1 -hydroxybenzotriazole hydrate (HOBT) (0.75 mmol), and N, N-disopropylethylamine (DIPEA) (0.75 mmol). The reaction mixture was stirred at room temperature overnight and then partitioned between ethyl acetate and brine. The organic phase was dried (MgSCU), and concentrated. Purification by silica gel column

[0342] 32

[0343] ME1\60300477.v1Docket No. 136840-00220

[0344] chromatography (CH2Cl2:MeOH) gave the Boc intermediate compound. This compound was stirred in HCl / dioxane (25.0 mL) at 25 °C for 16 hrs. The reaction mixture was concentrated under reduced pressure and resuspended in 40 mL of ethanol to which was added l-chloro-4-(2-chloroethoxy)benzene (0.75 mmol). The mixture was sealed in a sealed tube and heated at 100°C for 10 hours and concentrated. The residue was purified by preparative HPLC [water (formic acid) - ACN] to give to give the title compound as an off-white solid. LCMS: 491.1 [M+H]+

[0345] Compound 33 was prepared following a similar procedure above using 2-(4-chloro-3- fluorophen oxy) acetic acid in lieu of 2-[4-(trifluoromethyl)phenoxy]acetic acid.

[0346] Compound No. Structure Characterization Data

[0347] ... CS

[0348] £

[0349] 33 LCMS: [M+HJ+: 474.5

[0350]

[0351] a

[0352]

[0353] Compound 22: N-(l-(2-(4-chlorophenoxy)ethyI)-l,2,3,4-tetrahydroquiiiolm-6-yl)-2-(4-(trifluoromethyl)phenoxy)acetamide. To a solution of 2-[4-(trifluoromethyl)phenoxy]acetic acid and 1,1 -dimethyl ethyl 6-amino-3,4-dihydro-l (2 / / )-quinolinecarboxylate (0.75 mmol) in DMF (5.0 mL) were added l-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) (0.75 mmol), 1 -hydroxybenzotri azole hydrate (HOBT) (0.75 mmol), andN, N-disopropylethylamine (DIPEA) (0.75 mmol). The reaction mixture was stirred at room temperature overnight and then partitioned between ethyl acetate and brine. The organic phase was dried (MgSCh), and concentrated. Purification by silica gel column chromatography (CH2Cl2:MeOH) gave the Boc intermediate compound. This compound was stirred in HCl / dioxane (25.0 mL) at 25 °C for 16 hrs. The reaction mixture was concentrated under reduced pressure and resuspended in 40 mL of ethanol to which was added l-chloro-4-(2-chloroethoxy)benzene (0.75 mmol). The mixture was sealed in a sealed tube and heated at 100°C for 10 hours and concentrated. The residue was purified by preparative HPLC [water (formic acid) - ACN] to give to give the title compound as an off-white solid. LCMS: 504.9 [M+H]+

[0354] Compound 25 was prepared following a similar procedure above using 2-(4-chloro-3- 33

[0355] ME1\60300477.v1Docket No. 136840-00220

[0356] fluorophenoxy) acetic acid in lieu of 2-[4-(trifluoromethyl)phenoxy]acetic acid.

[0357] Compound No. Structure Characterization Data

[0358] 25 LCMS: 488.8 [M+H]+

[0359] •i H

[0360]

[0361] f

[0362]

[0363] Compound 20: N-(l-(2-((6-methylpyridin-2-yl)methoxy)acetyl)indoIin-5-yl)-2-(4-(trifluoromethyl)phenoxy)acetamide. To a solution of 2-[4-(trifluoromethyl)phenoxy]acetic acid and 1,1 -dimethylethyl 5-amino-2,3-dihydro-lH-indole-l-carboxylate (0.75 mmol) in DMF (5.0 mL) were added l-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) (0.75 mmol), 1 -hydroxybenzotriazole hydrate (HOBT) (0.75 mmol), and N, N-disopropyl ethyl amine (DIPEA) (0.75 mmol). The reaction mixture was stirred at room temperature overnight and then partitioned between ethyl acetate and brine. The organic phase was dried (MgSC ), and concentrated. Purification by silica gel column chromatography (CH2Cl2:MeOH) gave the Boc intermediate compound. This compound was stirred in HCl / dioxane (25.0 mL) at 25 °C for 16 hrs. The reaction mixture was concentrated under reduced pressure and resuspended in DCM (15.0 mL) to which was added 2-[(6-methyi-2-pyridinyl)methoxy]acetic acid (1.0 mmol), (COC1)2 (2.83 eq), and DMF (0.10 eq). The mixture reaction was stirred at 25 °C for 2 hrs. The reaction mixture was concentrated under reduced pressure and purified by preparative HPLC [water (formic acid) - ACN] to give to give the title compound as an off-white solid. LCMS: 500.5 [M+H]

[0364] Compound 36 was prepared following a similar procedure above using 2-(4-chloro-3-fluorophenoxy) acetic acid in lieu of 2-[4-(trifluoromethyl)phenoxy]acetic acid.

[0365] Compound No. Structure Characterization Data

[0366] C A V+ + A /

[0367] 36 LCMS: 483.9 [M+H]

[0368] J 8+

[0369] Bi

[0370] £

[0371]

[0372] 34

[0373] ME1\60300477.v1Docket No. 136840-00220

[0374] . M

[0375]

[0376] Compound 24: N-(l-(2-((6-methylpyridin-2-yl)methoxy)acetyl)-l, 2,3,4-tetrahydroquinolin-6-yI)-2-(4-(trifluoromethyI)phenoxy)acetamide. To a solution of 2-[4-(trifluoromethyl)phenoxy]acetic acid or 2-(4-chloro-3-fluorophenoxy)acetic acid (0.5 mmol) and 1,1 -dimethylethyl 6-amino-3,4-dihydro-l(277 -quinolinecarboxylate (0.75 mmol) in DMF (5.0 mL) were added l-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) (0.75 mmol), 1 -hydroxybenzotriazole hydrate (HOBT) (0.75 mmol), and N, N-disopropylethylamine (DIPEA) (0.75 mmol). The reaction mixture was stirred at room temperature overnight and then partitioned between ethyl acetate and brine. The organic phase was dried (MgSCL), and concentrated. Purification by silica gel column chromatography (CH2Cl2:MeOH) gave the Boc intermediate compound. This compound was stirred in HCI / dioxane (25.0 mL) at 25 °C for 16 hrs. The reaction mixture was concentrated under reduced pressure and resuspended in DCM (15.0 mL) to which w'as added 2-[(6-methyl-2-pyridinyl)methoxy]acetic acid (1.0 mmol), (COC1)2 (2.83 eq), and DMF (0.10 eq). The mixture reaction was stirred at 25 °C for 2 hrs. The reaction mixture was concentrated under reduced pressure and purified by preparative HPLC [water (formic acid) - ACN] to give to give the title compound as an off-white solid. LCMS: 514.6 [M+H]’ Compound 28 was prepared following a similar procedure above using 2-(4-chloro-3- fluorophenoxy) acetic acid in lieu of 2-[4-(trifluoromethyl)phenoxy]acetic acid.

[0377] Compound No. Structure Characterization Data

[0378] 28 LCMS: 498.0 [M+H]+

[0379] H

[0380] F

[0381]

[0382] I J It

[0383]

[0384] Compound 21: N-(1-(2-((1H-indazol-6-yl)oxy)acetyl)-1,2,3,4-tetrahydroquinolin-6-yl)-2-(4-(trifluoromethyl)phenoxy)acetamide. To a solution of 2-[4-(trifluoromethyl)phenoxy]acetic acid and 1, 1 -dimethylethyl 6-amino-3,4-dihydro- 1(277)- quinolinecarboxylate (0.75 mmol) in DMF (5.0 mL) were added l-[3- 35

[0385] ME1\60300477.v1Docket No. 136840-00220

[0386] (dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) (0.75 mmol), 1-hydroxybenzotriazole hydrate (HOBT) (0.75 mmol), andN, N-disopropylethylamine (DIPEA) (0.75 mmol). The reaction mixture was stirred at room temperature overnight and then partitioned between ethyl acetate and brine. The organic phase was dried (MgSC ), and concentrated. Purification by silica gel column chromatography (CH2Cl2:MeOH) gave the Boc intermediate compound. This compound was stirred in HCl / di oxane (25.0 mL) at 25 °C for 16 hrs. The reaction mixture was concentrated under reduced pressure and resuspended in DCM (15.0 mL) to which was added 2-(1-indazol-6-yloxy)-acetic acid (1.0 mmol), (COCl)2(2.83 eq), and DMF (0.10 eq). The mixture reaction was stirred at 25 °C for 2 hrs. The reaction mixture was concentrated under reduced pressure and purified by preparative HPLC [water (formic acid) - ACN] to give to give the title compound as an off-white solid. LCMS: 525.3 [M+H]+

[0387] Compound 30 was prepared following a similar procedure above using 2-(4-chloro-3-fluorophenoxy) acetic acid in lieu of 2-[4-(trifluoromethyl)phenoxy]acetic acid.

[0388] Compound No. Structure Characterization Data

[0389] 30 LCMS: 509.3 [M+H]+

[0390] £

[0391]

[0392] *v. V

[0393] &

[0394] 0

[0395]

[0396] Compound 23: 4-((2-oxo-2-(6-(2-(4-(trifluoroiriethyl)phenoxy)acetamido)-3,4-dihydroquino!in-l(2H)-yI)ethoxy)methyl)benzoic acid. To a solution of 2-[4-(trifluoromethyl)phenoxy]acetic acid and 1,1 -dimethylethyl 6-amino-3,4-dihydro- 1(277)-quinolinecarboxylate (0.75 mmol) in DMF (5.0 L) were added l-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) (0.75 mmol), 1-hydroxy benzotriazole hydrate (HOBT) (0.75 mmol), and N, N-disopropylethylamine (DIPEA) (0.75 mmol). The reaction mixture was stirred at room temperature overnight and then partitioned between ethyl acetate and brine. The organic phase was dried (MgSO4), and concentrated. Purification by silica gel column chromatography (CH2Cl2:MeOH) gave the Boc intermediate compound. This compound was stirred in HCl / dioxane (25.0 mL) at 25 °C for 16 hrs. The reaction mixture was concentrated under reduced pressure and resuspended in

[0397] 36

[0398] ME1\60300477.v1Docket No. 136840-00220

[0399] DCM (15.0 mL) to which was added 1 -methyl 4-[(carboxymethoxy)methyl]benzoate (1.0 mmol), (COC1)2 (2.83 eq), and DMF (0.10 eq). The mixture reaction was stirred at 25 °C for 2 hrs. The reaction mixture was concentrated under reduced pressure, resuspended in methanol, and treated with NaOH (1.60 eq). The mixture was stirred at 40 °C for 12 hrs then the reaction mixture was adjusted to pH=5 by IM HC1. The aqueous phase was extracted with ethyl acetate (15 mL *3). The combined organic phase was dried over anhydrous Na2SO4, filtered and concentrated give a residue which was purified by preparative HPLC [water (formic acid) - ACN] to give to give the title compound as an off-white solid. LCMS: 543.3 [M+H]+

[0400] Compound 27 was prepared following a similar procedure above using 2-(4-chloro-3-fluorophenoxy)acetic acid in lieu of 2-[4-(trifluoromethyl)phenoxy]acetic acid.

[0401] Compound No. Structure Characterization Data

[0402] 27 C-..... A X;. A,.. ci.. LCMS: 527.0 [M+H]+

[0403]

[0404] ..., A A d

[0405]

[0406] Compound 32: 4-((2-oxo-2-(5-(2-(4-(trifluoromethyl)phenoxy)acetamido)indoIin-l-yl)ethoxy)methyl)benzoic acid. To a solution of 2-[4-(trifluoromethyl)phenoxy]acetic acid and 1,1 -dimethylethyl 5-amino-2,3-dihydro-lH-indole-l-carboxylate (0.75 mmol) in DMF (5.0 mL) were added l-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) (0.75 mmol), 1 -hydroxybenzotriazole hydrate (HOBT) (0.75 mmol), andN, N-disopropyl ethyl amine (DIPEA) (0.75 mmol). The reaction mixture was stirred at room temperature overnight and then partitioned between ethyl acetate and brine. The organic phase was dried (MgSO4), and concentrated. Purification by silica gel column chromatography (CH2C12: MeOH) gave the Boc intermediate compound. This compound was stirred in HCl / dioxane (25.0 mL) at 25 °C for 16 hrs. The reaction mixture was concentrated under reduced pressure and resuspended in DCM (15.0 mL) to which was added 1 -methyl 4-[(carboxymethoxy)methyl]benzoate (1.0 mmol), (COC1)2 (2.83 eq), and DMF (0.10 eq). The mixture reaction was stirred at 25 °C for 2 hrs. The reaction mixture was concentrated under reduced pressure, resuspended in methanol, and treated with NaOH (1.60 eq). The mixture was stirred at 40 °C for 12 hrs then the reaction mixture was adjusted to pH=5 by IM HC1.

[0407] 37

[0408] ME1\60300477.v1Docket No. 136840-00220

[0409] The aqueous phase was extracted with ethyl acetate (15 mL *3). The combined organic phase was dried over anhydrous Na2SO4, filtered and concentrated give a residue which was purified by preparative HPLC [water (formic acid) - ACN] to give to give the title compound as an off-white solid. LCMS: 529.0 [M+H]’

[0410] Compound 35 was prepared following a similar procedure above using 2-(4-chloro-3-fluorophenoxy)acetic acid in lieu of 2-[4-(tritluoromethyl)phenoxy]acetic acid.

[0411] Compound No. Structure Characterization Data

[0412] 35 LCMS: 512.9 [M+H]+

[0413] a

[0414] A,.

[0415]

[0416] ? - -•V'- Compound 29: N-(l-(2-(4-(dimethylamino)phenethoxy)acetyl)-l, 2,3,4- tetrahydroquinolin-6-yI)-2-(4-(trifluoromethyI)phenoxy)acetamide. To a solution of 2-[4-(trifluoromethyl)phenoxy]acetic acid and 1,1 -dimethylethyl 6-amino-3,4-dihydro-l(2 / / )-quinolinecarboxyiate (0.75 mmol) in DMF (5.0 mL) were added l-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) (0.75 mmol), 1-hydroxybenzotriazole hydrate (HOBT) (0.75 mmol), and N, N-disopropylethylamine (DIPEA) (0.75 mmol). The reaction mixture was stirred at room temperature overnight and then partitioned between ethyl acetate and brine. The organic phase was dried (MgSCE), and concentrated. Purification by silica gel column chromatography (CH2Cl2:MeOH) gave the Boc intermediate compound. This compound was stirred in HCl / dioxane (25.0 mL) at 25 °C for 16 hrs. The reaction mixture was concentrated under reduced pressure and resuspended in DCM (15.0 mL) to which was added 2-[2-[4-(dimethylamino)phenyl]ethoxy]acetic acid (1.0 mmol), (COC1)2 (2.83 eq), and DMF (0.10 eq). The mixture reaction was stirred at 25 °C for 2 hrs. The reaction mixture was concentrated under reduced pressure and purified by preparative HPLC [water (formic acid) - ACN] to give to give the title compound as an off- white solid. LCMS: 556.5 [M+H]+

[0417] Compound 26 was prepared following a similar procedure above using 2-(4-chloro-3-fluorophenoxy) acetic acid in lieu of 2-[4-(trifluoromethyl)phenoxy]acetic acid.

[0418] Compound No. Structure Characterization Data

[0419]

[0420] 38

[0421] ME1\60300477.v1Docket No. 136840-00220

[0422] X A........+

[0423] g £ ’’T ¥ ¥ ■-■■■ -:-'

[0424] 26.....o... A A,. A 6 LCMS: 540.1 [M+H]4A -■ &

[0425] A: ***

[0426]

[0427] :< •■••■:•',o. ■■■. A K A A O

[0428] S J

[0429]

[0430] '": S"

[0431] Compound 31: N-(l-(2-(4-(dimethylamino)phenethoxy)acetyl)indolin-5-yl)-2-(4-(trifluoromethyl)phenoxy)acetamide. To a solution of 2-[4-(trifluoromethyl)phenoxy]acetic acid and 1,1 -dimethylethyl 5-amino-2,3-dihydro-lH-indole-l-carboxylate (0.75 mmol) in DMF (5.0 mL) were added l-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) (0.75 mmol), 1 -hydroxybenzotriazole hydrate (HOBT) (0.75 mmol), and N, N-disopropylethylamine (DIPEA) (0.75 mmol). The reaction mixture was stirred at room temperature overnight and then partitioned between ethyl acetate and brine. The organic phase was dried (MgSO4), and concentrated. Purification by silica gel column chromatography (CH2Cl2:MeOH) gave the Boc intermediate compound. This compound was stirred in HCl / dioxane (25.0 mL) at 25 °C for 16 hrs. The reaction mixture was concentrated under reduced pressure and resuspended in DCM (15.0 mL) to which was added 2-[2-[4-(dimethylamino)phenyl]ethoxy]acetic acid (1.0 mmol), (COC1)2 (2.83 eq), and DMF (0.10 eq). The mixture reaction was stirred at 25 °C for 2 hrs. The reaction mixture was concentrated under reduced pressure and purified by preparative HPLC [water (formic acid) - ACN] to give to give the title compound as an off-white solid. LCMS: 542.4 [M+H]4Compound 34 was prepared following a similar procedure above using 2-(4-chloro-3-fluorophenoxy) acetic acid in lieu of 2-[4-(trifluoromethyl)phenoxy]acetic acid.

[0432] Compound No. Structure Characterization Data

[0433] 34 LCMS: 526.3 [M+H]4

[0434]

[0435] 39

[0436] ME1\60300477.v1

Claims

Docket No. 136840-00220CLAIMS1. A compound represented by structural formula (I’):(R3)O Yor a pharmaceutically acceptable salt thereof, wherein:Xi is CH or N;X2is (). S, or NH;X3 is selected from the group consisting of O. -OCH2-*, -O(CH2)2-*, S, and NH, wherein * indicates the point of attachment to Ring A;X4 is absent or O;Y is -(CH2)P-, Z is CH2and is a single bond; or Y is CH, Z is CH and is a double bond;Ring A is phenyl or 5- to 9-membered heteroaryl;each Ri and R2 are each independently selected from the group consisting of hydrogen, halogen, -CN, -OH, -COOH, -NRsRp, -NO2, Ci-ealkyl, and Ci-ehaloalkyl; each R3 is independently selected from hydrogen, halogen, -CN, -OH, -NO2, C1-6alkyl, and Ci-ehaloalkyl;R4, R5, R6, and R7are each independently selected from hydrogen, Cj -ealkyl, and Ci-ehaloalkyl;R8 and R9 are each independently selected from hydrogen and C1-3alkyl; m, n, and 0 are each independently 1, or 2; andp is 1 or 2.2, A compound represented by structural formula (I):or a pharmaceutically acceptable salt thereof, wherein:40ME1\60300477.v1Docket No. 136840-00220X1 is CH or N;X2 and X3 are each independently O. S, or NH;Y is -(CH2)P-, Z is CH2 and — is a single bond; or Y is CH, Z is CH and ----- is a double bond;each Ri and R2 are each independently selected from the group consisting of hydrogen, halogen, -CN, -OH, -NO2, Ci-ealkyl, and Ci-ehaloalkyl;each R3 is independently selected from hydrogen, halogen, -CN, -OH, -NO2, Ci-ealkyl, and C₁₋₆haloalkyl;R4, Rs, Re, and R? are each independently selected from hydrogen, Ci-ealkyl, and C₁₋₆haloalkyl;m, n, and 0 are each independently 1, or 2; andp is 1 or 2.

3. The compound of claim 1 or 2, wherein X2 and X3 are O.

4. The compound of any one of claims 1-3, wherein each R3is hydrogen.

5. The compound of any one of claims 1-4, wherein R4, Rs, Re, and R7 are each hydrogen.

6. The compound of claim 1, wherein the compound is represented by structural formula (Ila’), (lib’), or (IIc’):41ME1\60300477.v1Docket No. 136840-00220or a pharmaceutically acceptable salt thereof.

7. The compound of any one of claims 1-6, wherein the compound i s represented by structural formula (II):or a pharmaceutically acceptable salt thereof.

8. The compound of any one of claims 1-7, wherein Xj is CH.

9. The compound of any one of claims 1-7, wherein Xi is N.

10. The compound of any one of claims 1-9, wherein Y and Z are CH.

11. The compound of any one of claims 1-9, wherein Y and Z are CHz.

12. The compound of any one of claims 1-9, wherein Y is (CH2)2 and Z is CH2.

13. The compound of any one of claims 1, 4-6, and 8-12, wherein the compound is represented by one of the following structures:ME1\60300477.v1Docket No. 136840-0022014. The compound of any one of claims 1, 4-6, and 8-13, wherein X3 is selected from O, - OCH2-*, and -O(CH2)2-*15. The compound of any one of claims 1, 4-6, and 8-14, wherein X4 is absent.

16. The compound of any one of claims 1, 4-6, and 8-14, wherein X4 is O.

17. The compound of claim 1 or 2, wherein the compound is represented by one of the following structures:44ME1\60300477.v1Docket No. 136840-0022018. The compound of claim 1 or 2, wherein the compound is represented by one of the following structural formula:wherein q is 0 or 1.

19. The compound of claim 18, wherein q is 0.

20. The compound of claim 18, wherein q is 1.45ME1\60300477.v1Docket No. 136840-0022021. The compound of any one of claims 1 and 3-20, wherein each Ri and R2 are independently selected from hydrogen, halogen, -COOH, -N(CH₃)₂, Cj-salkyl, and Cj. shaloalkyl.

22. The compound of any one of claims 1-21, wherein each Ri and R2 are independently selected from halogen, Cnsalkyl, and Ci-ehaloalkyl.

23. The compound of any one of claims 1-22, wherein each Ri and R2 are independently selected from halogen and C₁₋₃haloalkyl.

24. The compound of any one of claims 1, and 3-21, wherein each Ri and R2 are independently selected from H, -F, -Cl, -CH3, -CF3, -COOH, and -N(CF13)2.

25. The compound of any one of claims 1-24, wherein each Ri and R2 are independently selected from -F, -Cl, and -CF3.

26. The compound of any one of claims 1-25, wherein the compound is represented by a compound in Table 1, or a pharmaceutically acceptable salt thereof.

27. A pharmaceutical composition comprising the compound of any one of claims 1 to 26, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

28. A method of treating hearing loss in a subject in need thereof or slowing the onset of hearing loss in a subject at risk of developing hearing loss, comprising administering to the subject an effective amount of the compound of any one of claims 1 to 26 or a pharmaceutically acceptable salt thereof.

29. The method of claim 28 wherein the hearing loss in age-related.

30. The method of claim 28, wherein the hearing loss is noise induced.

31. The method of claim 28, wherein the hearing loss is genetic or inherited.46ME1\60300477.v1Docket No. 136840-0022032. The method of claim 28, wherein the hearing loss is due to ototoxicity.

33. The method of claim 28, wherein the hearing loss is due to disease.

34. The method of claim 28, wherein the hearing loss is due to trauma.

35. The method of claim 28, wherein the hearing loss is due to cochlear synaptopathy.47MEl\603(X)477.vl

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