((pyrrolopyridine-3-carbonyl) imino)-2,3-dihydrothiazol-4-YL) methyl phosphates for activating yap

Abstract

(2-((1H-pyrrolo[2,3-b]pyridine-3-carbonyl)imino)-2,3-dihydrothiazol-4-yl)methyl dihydrogen phosphates and (2-((1H-pyrrolo[2,3-b]pyridine-3-carbonyl)imino)thiazolidin-4-yl)methyl dihydrogen phosphates are disclosed. The compounds activate Yap and inhibit Lats kinases. They are therefore useful for treating hearing loss.

Description

Docket No.2877.041AWO ((PYRROLOPYRIDINE-3-CARBONYL)IMINO)-2,3-DIHYDROTHIAZOL-4- YL)METHYL PHOSPHATES FOR ACTIVATING YAP Cross Reference to Related Application

[0001] This application claims priority of US provisional application 63 / 666,481, filed July 1, 2024, the disclosure of which is hereby incorporated herein by reference in its entirety. Government Rights Statement

[0002] This invention was made with government support under grant number T32GM007739 awarded by National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE INVENTION Technical Field

[0003] The present application relates generally to [((pyrrolopyridine-3- carbonyl)imino)-2,3-dihydrothiazol-4-yl]methyl phosphates that inhibit Lats kinases and thus increase Yap activity. The compounds are useful for inducing the proliferation of supporting cells in the inner ear, and thus potentially for treating hearing loss. Background Information

[0004] Initiated in response to injury, regeneration is a complex process that can restore the structure and function of damaged tissue. Some adult mammalian tissues retain a gradually declining regenerative capability beyond development. Regeneration occurs either by activation and amplification of resident stem cells, as in the epithelia of the skin and intestine, or through cellular dedifferentiation and proliferation, as in the liver. In other instances, such as central nervous and cardiac-muscle tissues, cells exhibit little or no potential for regeneration after injury.{H2424053.1}1 4922-2197-3586, v.1Docket No.2877.041AWO

[0005] In view of its fundamental roles in development, proliferation, stem-cell maintenance, and dedifferentiation, Hippo signaling is an inviting target for driving regeneration. The regenerative potential of the Hippo pathway has become abundantly clear in numerous organs, including the heart, retina, liver, and intestine. Hippo signaling limits the size of the developing murine utricle, a sensory organ in the vestibular portion of the inner ear, and the Yap-Tead complex is active during—and necessary for— proliferative regeneration in the neonatal utricle. These observations suggest that chemical activation of Yap signaling might engender supporting-cell proliferation in adult tissue, a key missing step in the regeneration of the mammalian inner ear.

[0006] Described herein is a small genus of compounds that function as inhibitors of Lats kinases. Based on the results in vitro described below, and by analogy with the experiments described in US published application 2023-0137893, the disclosure of which is incorporated herein by reference in its entirety, the person of skill would expect that the compounds of the invention would cause supporting cells to reenter the cell cycle, resulting in hair-cell regeneration. SUMMARY OF THE INVENTION

[0007] The invention is directed to to [((pyrrolopyridine-3-carbonyl)imino)-2,3- dihydrothiazol-4-yl]methyl phosphates, pharmaceutical compositions and methods for inhibiting Lats or activating Yap, and thereby stimulating regeneration of target cells, particularly hair-cells.

[0008] The present invention relates, in a first aspect, to compounds of formula I:{H2424053.1}2 4922-2197-3586, v.1Docket No.2877.041AWOR1and R2are independently selected from the group consisting of hydrogen, halogen, hydroxy, (C1-C3)alkyl, halo(C1-C3)alkyl, (C1-C3)alkoxy, and halo(C1-C3)alkoxy; R4is chosen from hydrogen, fluoro, chloro and methyl; the dashed line represents an optionally present double bond; and n is zero or one.

[0009] In another aspect, the invention relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a compound as described herein.

[0010] In another aspect, the invention relates to a method of for activating YAP in a cell expressing YAP comprising exposing the cell to a compound as described herein.

[0011] In another aspect, the invention relates to a method for LATS inhibition in a cell population expressing LATS comprising exposing the cell population to a compound as described herein.

[0012] In another aspect, the invention relates to a method for stimulating hair cell regeneration comprising exposing a supporting-cell population to a compound as described herein.

[0013] In another aspect, the invention relates to a method of treating a subject having, or at risk of developing, hearing loss, comprising administering to the subject an effective amount of a compound as described herein.{H2424053.1}3 4922-2197-3586, v.1Docket No.2877.041AWO BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In the accompanying drawings like reference numerals indicate like elements and features in the various figures. For clarity, not every element may be labeled in every figure. In addition, the drawings are not necessarily complete when viewed without reference to the text, emphasis instead being placed upon illustrating the principles of the invention.

[0015] FIG.1 presents a bar graph showing ratio of phosphorylated Yap to total Yap for control and the average of three concentrations (270 mM, 360 mM, and 450 mM) of administration of a compound of the invention. DETAILED DESCRIPTION OF THE INVENTION

[0016] The invention relates to compounds of formula I:R1and R2are independently selected from the group consisting of hydrogen, halogen, hydroxy, (C1-C3)alkyl, halo(C1-C3)alkyl, (C1-C3)alkoxy, and halo(C1-C3)alkoxy; R4is chosen from hydrogen, fluoro, chloro and methyl; a dashed line represents an optionally present double bond; and{H2424053.1}4 4922-2197-3586, v.1Docket No.2877.041AWO n is zero or one.

[0017] In some embodiments, R4is hydrogen. In some embodiments, R1and R2are both hydrogen. In some embodiments, n is one; in others, n is zero.

[0018] In the formula the dashed line represents an optional double bond. In other words, the compounds can be either (dihydrothiazolyl)methyl phosphates or (thiazolidinyl)methyl phosphates:

[0019] It has been found that compounds of formula I are useful for inhibiting Lats or activating Yap and are therefore potential therapeutic agents for stimulating regeneration of target cells, particularly hair-cells. Such compounds would be useful for treating hearing loss.

[0020] It is to be understood that in various embodiments, the pharmaceutical compositions of the present inventions comprise one or more pharmaceutically acceptable excipients, including, but not limited to, one or more binders, bulking agents, buffers, stabilizing agents, surfactants, wetting agents, lubricating agents, diluents, disintegrants, viscosity enhancing or reducing agents, emulsifiers, suspending agents, preservatives, antioxidants, opacifying agents, glidants, processing aids, colorants, sweeteners, taste-masking agents, perfuming agents, flavoring agents, diluents, polishing agents, polymer matrix systems, plasticizers and other known additives to provide an elegant presentation of the drug or aid in the manufacturing of a medicament or pharmaceutical product comprising a composition of the present inventions. Examples of carriers and excipients well known to those skilled in the art and are described in detail {H2424053.1}5 4922-2197-3586, v.1Docket No.2877.041AWO in, e.g., Ansel, Howard C., et al., Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R., et al. Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe, Raymond C. Handbook of Pharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005.

[0021] In various embodiments, non-limiting examples of excipients include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose, (e.g., Nos.2208, 2906, 2910), hydroxypropyl cellulose, titanium dioxide, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, silicic acid, sorbitol, starch, pre-gelatinized starch, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pre-gelatinized starch, other starches, clays, other algins, other celluloses, gums, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, a syloid silica gel (AEROSIL200, manufactured by W.R. Grace Co. of Baltimore, MD), a coagulated aerosol of synthetic silica (marketed by Degussa Co. of Plano, TX), CAB-O- SIL (a pyrogenic silicon dioxide product sold by Cabot Co. of Boston, MA), colorants and mixtures thereof.

[0022] The terms "subject" or "subject in need thereof" are used interchangeably herein. These terms refer to a patient who has been diagnosed with the underlying disorder to be treated. Ordinarily, the patient will be a human. The subject may currently be experiencing symptoms associated with the disorder or may have experienced{H2424053.1}6 4922-2197-3586, v.1Docket No.2877.041AWO symptoms in the past. Additionally, a "subject in need thereof" may be a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological systems of a disease, even though a diagnosis of this disease may not have been made.

[0023] As used herein, the terms “treatment” or “treating" are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including, but not limited to, therapeutic benefit. Therapeutic benefit includes eradication or amelioration of the underlying disorder being treated; it also includes the eradication or amelioration of one or more of the symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder.

[0024] As used herein, and as would be understood by the person of skill in the art, the recitation of “a compound” - unless expressly further limited - is intended to include salts of that compound. In particular, as shown below, the recitation “a compound of formula I” as applied to Example 1, would include both the free base and its salts:Docket No.2877.041AWO in which M+is any counterion. In a particular embodiment, the term “compound of formula I” refers to the compound or a pharmaceutically acceptable salt thereof. The term "pharmaceutically acceptable salt" refers to salts prepared from pharmaceutically acceptable non-toxic bases including inorganic bases and organic bases. Suitable pharmaceutically acceptable base addition salts for the compounds of the present invention include, but are not limited to, metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from lysine, arginine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium cations. Methods of Treatment

[0025] The compositions described herein may be administered to a subject having or at risk of developing hearing loss (e.g., sensorineural hearing loss) and / or vestibular dysfunction by a variety of routes, such as local administration to the middle or inner ear (e.g., administration to or through the oval window, round window, or semicircular canal (e.g., the horizontal canal), or by transtympanic or intratympanic injection), intravenous, parenteral, intradermal, transdermal, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage, and oral administration. The most suitable route for administration in any given case will depend on the particular composition administered, the patient, pharmaceutical formulation methods, administration methods (e.g., administration time and administration route), the patient's age, body weight, sex, severity of the disease being treated, the patient’s diet, and the patient’s excretion rate. Compositions may be administered once, or more than once (e.g., once annually, twice annually, three times annually, bi-monthly, monthly, or bi-weekly).

[0026] Subjects that may be treated as described herein are subjects having or at risk of developing hearing loss and / or vestibular dysfunction (e.g., subjects having or at risk of developing hearing loss, vestibular dysfunction, or both). The compositions and methods{H2424053.1}8 4922-2197-3586, v.1Docket No.2877.041AWO described herein can be used to treat subjects having or at risk of developing damage to cochlear hair cells (e.g., damage related to acoustic trauma, disease or infection, head trauma, ototoxic drugs, or aging), subjects having or at risk of developing damage to vestibular hair cells (e.g., damage related to disease or infection, head trauma, ototoxic drugs, or aging), subjects having or at risk of developing sensorineural hearing loss, deafness, or auditory neuropathy, subjects having or at risk of developing vestibular dysfunction (e.g., dizziness, vertigo, loss of balance, bilateral vestibulopathy, oscillopsia, or a balance disorder), subjects having tinnitus (e.g., tinnitus alone, or tinnitus that is associated with sensorineural hearing loss or vestibular dysfunction), subjects having a genetic mutation associated with hearing loss and / or vestibular dysfunction, or subjects with a family history of hereditary hearing loss, deafness, auditory neuropathy, tinnitus, or vestibular dysfunction. In some embodiments, the subject has or is at risk of developing hearing loss and / or vestibular dysfunction that is associated with or results from loss of hair cells (e.g., cochlear or vestibular hair cells). The methods described herein may include a step of screening a subject for one or more mutations in genes known to be associated with hearing loss and / or vestibular dysfunction prior to treatment with or administration of the compositions described herein. A subject can be screened for a genetic mutation using standard methods known to those of skill in the art (e.g., genetic testing). The methods described herein may also include a step of assessing hearing and / or vestibular function in a subject prior to treatment with or administration of the compositions described herein. Hearing can be assessed using standard tests, such as audiometry, auditory brainstem response (ABR), electrocochleography (ECOG), and otoacoustic emissions. Vestibular function may be assessed using standard tests, such as eye movement testing (e.g., electronystagmogram (ENG) or videonystagmogram (VNG)), tests of the vestibulo-ocular reflex (VOR) (e.g., the head impulse test (Halmagyi–Curthoys test), which can be performed at the bedside or using a video- head impulse test (VHIT), or the caloric reflex test), posturography, rotary-chair testing, ECOG, vestibular evoked myogenic potentials (VEMP), and specialized clinical balance tests, such as those described in Mancini and Horak, Eur J Phys Rehabil Med, 46:239 (2010). These tests can also be used to assess hearing and / or vestibular function in a subject after treatment with or administration of the compositions described herein. The{H2424053.1}9 4922-2197-3586, v.1Docket No.2877.041AWO compositions and methods described herein may also be administered as a preventative treatment to patients at risk of developing hearing loss and / or vestibular dysfunction, e.g., patients who have a family history of hearing loss or vestibular dysfunction (e.g., inherited hearing loss or vestibular dysfunction), patients carrying a genetic mutation associated with hearing loss or vestibular dysfunction who do not yet exhibit hearing impairment or vestibular dysfunction, or patients exposed to risk factors for acquired hearing loss (e.g., acoustic trauma, disease or infection, head trauma, ototoxic drugs, or aging) or vestibular dysfunction (e.g., disease or infection, head trauma, ototoxic drugs, or aging).

[0027] The compositions and methods described herein can be used to induce or increase hair cell regeneration in a subject (e.g., cochlear and / or vestibular hair cell regeneration). Subjects that may benefit from compositions that induce or increase hair cell regeneration include subjects suffering from hearing loss or vestibular dysfunction as a result of loss of hair cells (e.g., loss of hair cells related to trauma (e.g., acoustic trauma or head trauma), disease or infection, ototoxic drugs, or aging), and subjects with abnormal hair cells (e.g., hair cells that do not function properly when compared to normal hair cells), damaged hair cells (e.g., hair cell damage related to trauma (e.g., acoustic trauma or head trauma), disease or infection, ototoxic drugs, or aging), or reduced hair cell numbers due to genetic mutations or congenital abnormalities.

[0028] The compositions and methods described herein can also be used to prevent or reduce hearing loss and / or vestibular dysfunction caused by ototoxic drug-induced hair cell damage or death (e.g., cochlear hair cell and / or vestibular hair cell damage or death) in subjects who have been treated with ototoxic drugs, or who are currently undergoing or soon to begin treatment with ototoxic drugs. Ototoxic drugs are toxic to the cells of the inner ear, and can cause sensorineural hearing loss, vestibular dysfunction (e.g., vertigo, dizziness, imbalance, bilateral vestibulopathy, oscillopsia, or a balance disorder), tinnitus, or a combination of these conditions. Drugs that have been found to be ototoxic include aminoglycoside antibiotics (e.g., gentamycin, neomycin, streptomycin, tobramycin, kanamycin, vancomycin, and amikacin), viomycin, antineoplastic drugs{H2424053.1}10 4922-2197-3586, v.1Docket No.2877.041AWO (e.g., platinum-containing chemotherapeutic agents, such as cisplatin, carboplatin, and oxaliplatin), loop diuretics (e.g., ethacrynic acid and furosemide), salicylates (e.g., aspirin, particularly at high doses), and quinine. In some embodiments, the methods and compositions described herein can be used to treat bilateral vestibulopathy or oscillopsia. Bilateral vestibulopathy and oscillopsia can be induced by aminoglycosides (e.g., the methods and compositions described herein can be used to promote or increase hair cell regeneration in a subject having or at risk of developing aminoglycoside-induced bilateral vestibulopathy or oscillopsia).

[0029] Treatment may include administration of a composition containing a compound described herein in various unit doses. Each unit dose will ordinarily contain a predetermined-quantity of the therapeutic composition. The quantity to be administered, and the particular route of administration and formulation, are within the skill of those in the clinical arts. A unit dose need not be administered as a single injection but may include continuous infusion over a set period of time. Dosing may be performed using a syringe pump to control infusion rate in order to minimize damage to the inner ear (e.g., the cochlea and / or vestibular system).

[0030] The compositions described herein are administered in an amount sufficient to improve hearing, improve vestibular function (e.g., improve balance or reduce dizziness or vertigo), reduce tinnitus, treat bilateral vestibulopathy, treat oscillopsia, treat a balance disorder, increase or induce hair cell regeneration (e.g., cochlear and / or vestibular hair cell regeneration), increase hair cell numbers, activate YAP, and / or inhibit LATS. Hearing may be evaluated using standard hearing tests (e.g., audiometry, ABR, electrocochleography (ECOG), and otoacoustic emissions) and may be improved compared to hearing measurements obtained prior to treatment. Vestibular function may be evaluated using standard tests for balance and vertigo (e.g., eye movement testing (e.g., ENG or VNG), posturography, VOR testing (e.g., head impulse testing (Halmagyi– Curthoys testing, e.g., VHIT), or caloric reflex testing), rotary-chair testing, ECOG, VEMP, and specialized clinical balance tests) and may be improved compared to measurements obtained prior to treatment. In some embodiments, the compositions are{H2424053.1}11 4922-2197-3586, v.1Docket No.2877.041AWO administered in an amount sufficient to improve the subject’s ability to understand speech. The compositions described herein may also be administered in an amount sufficient to slow or prevent the development or progression of sensorineural hearing loss and / or vestibular dysfunction (e.g., in subjects who carry a genetic mutation associated with hearing loss or vestibular dysfunction, who have a family history of hearing loss or vestibular dysfunction (e.g., hereditary hearing loss or vestibular dysfunction), or who have been exposed to risk factors associated with hearing loss or vestibular dysfunction (e.g., ototoxic drugs, head trauma, disease or infection, or acoustic trauma) but do not exhibit hearing impairment or vestibular dysfunction (e.g., vertigo, dizziness, or imbalance), or in subjects exhibiting mild to moderate hearing loss or vestibular dysfunction). These effects may occur, for example, within 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, or more, following administration of the compositions described herein. The patient may be evaluated 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or more following administration of the composition depending on the dose and route of administration used for treatment. Depending on the outcome of the evaluation, the patient may receive additional treatments. Preparation of Compounds

[0031] The following abbreviations are used in the synthetic routes: THF (tetrahydrofuran), MeOH (methanol), DCM (dicholoromethane), DMF (N,N- dimethylformamide), ACN (acetonitrile), EtOH (ethanol), EtOAc (ethyl acetate), IPA (2- propanol), DMSO (dimethyl sulfoxide), MTBE (methyl tert-butyl ether), TEA (triethylamine), DIPEA (N,N-diisopropylethylamine), TMEDA (tetramethylethylenediamine), DMAP (N,N-dimethylpyridin-4-amine), EDCI (N-(3- Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride), HOBt (1- Hydroxybenzotriazole hydrate), HBTU ((2-(1H-benzotriazol-1-yl)-1,1,3,3- tetramethyluronium hexafluorophosphate), T3P (propanephosphonic acid anhydride), TBAI (tetrabutylammonium iodide), LAH (lithium aluminum hydride), TFA (trifluoroacetic acid).{H2424053.1}12 4922-2197-3586, v.1Docket No.2877.041AWO

[0032] Preparative HPLC purification refers to the use of a water / acetonitrile gradient with or without the use of additives such as HCl, formic acid, TFA, or NH4HCO3 using an appropriate hydrophobic stationary phase. Preparation of (Z)-(2-((1H-pyrrolo[2,3-b]pyridine-3-carbonyl)imino)-3-benzyl-2,3- dihydrothiazol-4-yl)methyl dihydrogen phosphate [Example 1]{H2424053.1}13 4922-2197-3586, v.1Docket No.2877.041AWODocket No.2877.041AWO Step 1. Ethyl N-(benzylcarbamothioyl)carbamate

[0033] To a mixture of benzylamine (4.00 g, 37.3 mmol) and O-ethyl carbonisothiocyanatidate (4.90 g, 37.3 mmol) in EtOAc (50 mL) was added TMEDA (0.430 g, 3.73 mmol) at 25 °C. The mixture was stirred at 25 °C for 1 hour and then concentrated under reduced pressure. The residue was triturated with 2% EtOAc in petroleum ether (50 mL) to afford 7.60 g of ethyl N-(benzylcarbamothioyl) carbamate as a solid.1H NMR (400 MHz, CDCl3) δ 9.97 (br. s, 1H), 7.40 - 7.28 (m, 5H), 4.87 (d, J = 5.2 Hz, 2H), 4.24 - 4.19 (m, 2H), 1.31 (t, J = 6.8 Hz, 3H) LCMS (m / z [M+H]+): 239.1 Step 2. (Z)-methyl 3-((N-benzyl-N'-(ethoxycarbonyl)carbamimidoyl)thio)- 2- oxopropanoate

[0034] To a mixture of N-(benzylcarbamothioyl)carbamate (7.60 g, 31.8 mmol) and cesium carbonate (20.7 g, 63.7 mmol) in acetonitrile (100 mL) was added methyl 3- bromo-2-oxo -propanoate (7.50 g, 41.4 mmol) at 25 °C. The reaction mixture was stirred at 25 °C for 2 hours then quenched with water (100 mL) and extracted with EtOAc (4 x 100 mL). The combined organic phases were washed with brine (3 x 50 mL), dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated. The residue was purified by silica gel column chromatography, eluting with 10 to 30% EtOAc in petroleum ether to afford 5.50 g of the title compound as a solid.1H NMR (400 MHz, CDCl3) δ 7.30 - 7.21 (m, 5H), 5.28 (d, J = 15.6 Hz, 1H), 4.73 (br s, 1H), 4.33 (d, J = 15.6 Hz, 1H), 4.29 - 4.20 (m, 2H), 3.58 (d, J = 12.4 Hz, 1H), 3.29 - 3.23 (m, 4H), 1.34 (t, J = 7.2 Hz, 3H) LCMS (m / z [M+H]+): 339.1 Step 3. Methyl (Z)-3-benzyl-2-((ethoxycarbonyl)imino)-2,3-dihydrothiazole-4- carboxylate

[0035] To a solution of (Z)-methyl 3-((N-benzyl-N'- (ethoxycarbonyl)carbamimidoyl)thio)- 2-oxopropanoate (5.50 g, 16.2 mmol) in THF (80 mL) was added DIPEA (4.20 g, 32.5 mmol) and thionyl chloride (2.10 g, 17.8 mmol) at 0{H2424053.1}15 4922-2197-3586, v.1Docket No.2877.041AWO °C. The mixture was stirred at 25 °C for 1 hour then quenched with water (100 mL) and extracted with EtOAc (4 x 100 mL). The combined organic layers were washed with brine (3 x 50 mL), dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated. The residue was purified by silica gel column chromatography, eluting with 10 to 30% EtOAc in petroleum ether to afford 4.00 g of the title compound as an oil.1H NMR (400 MHz, CDCl3) δ 7.55 (s, 1H), 7.33 - 7.16 (m, 5H), 5.86 (s, 2H), 4.30 - 4.25 (m, 2H), 3.81 (s, 3H), 1.36 (t, J = 7.2 Hz, 3H) LCMS (m / z [M+H]+): 321.5 Step 4. Ethyl (Z)-(3-benzyl-4-(hydroxymethyl)thiazol-2(3H)-ylidene)carbamate

[0036] To a solution of methyl (Z)-3-benzyl-2-((ethoxycarbonyl)imino)-2,3- dihydrothiazole -4-carboxylate (3.90 g, 12.2 mmol) in THF (40 mL) was added lithium aluminum hydride (0.560 g, 14.6 mmol) at 0 °C and the reaction mixture was continued to stir at 0 °C for 15 minutes. The reaction was diluted with THF (150 mL), quenched with water (4 mL), and followed by the addition of a 15% solution of NaOH (4 mL) and water (12 mL) at 0 °C. The mixture was filtered, and the filter cake was washed with EtOAc (50 mL). The filtrate was dried with anhydrous Na2SO4, filtered, and concentrated. The residue was triturated with EtOAc (80 mL) to afford 2.50 g of the title compound as a solid.1H NMR (400 MHz, DMSO-d6) δ 7.38 - 7.25 (m, 3H), 7.12 (d, J = 7.2 Hz, 2H), 6.85 (s, 1H), 5.77 (br. s, 1H), 5.40 (s, 2H), 4.30 (s, 2H), 4.07 - 4.02 (m, 2H), 1.18 (t, J = 7.2 Hz, 3H) LCMS (m / z [M+H]+): 292.9 Step 5. (3-Benzyl-2-imino-2,3-dihydrothiazol-4-yl)methanol

[0037] To a solution of (Z)-ethyl (3-benzyl-4-(hydroxymethyl)thiazol-2(3H)- ylidene)carbamate (1.00 g, 3.42 mmol) in EtOH (15 mL) was added NaOH (2.74 g, 68.4 mmol). The mixture was stirred at 30 °C for 2 hours, and then concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography (0.1% NH3.H2O / MeCN condition) to afford 0.35 g of (3-benzyl-2-imino-2,3- dihydrothiazol-4-yl)methanol as a solid.{H2424053.1}16 4922-2197-3586, v.1Docket No.2877.041AWO Step 6. (Z)-N-(3-Benzyl-4-(hydroxymethyl)thiazol-2(3H)-ylidene)-1H-pyrrolo[2,3- b]pyridine-3-carboxamide

[0038] To a mixture of (3-benzyl-2-imino-2,3-dihydrothiazol-4-yl)methanol (0.300 g, 1.36 mmol) and 1H-pyrrolo[2,3-b]pyridine-3-carboxylic acid (0.220 g, 1.36 mmol) in DMF (5 mL) was added DIPEA (0.530 g, 4.09 mmol), HOBt (0.110 g, 0.820 mmol) and EDCI (0.390 g, 2.04 mmol) at 25 °C. The reaction mixture was stirred at 25 °C for 3 hours and purified by reversed-phase flash chromatography (0.1% FA / MeCN condition) to afford 0.12 g of the title compound as a solid.1H NMR (400 MHz, DMSO-d6) δ 12.16 (br. s, 1H), 8.44 (dd, J = 1.6 Hz, 8.0 Hz, 1H), 8.23 (dd, J = 1.6 Hz, 4.4 Hz, 1H), 8.11 (d, J = 2.8 Hz, 1H), 7.40 - 7.23 (m, 5H), 7.12 (dd, J = 4.8 Hz, 8.0 Hz, 1H), 6.86 (s, 1H), 5.66 - 5.60 (m, 3H), 4.42 (d, J = 5.6 Hz, 2H) LCMS (m / z [M+H]+): 365.3 An alternative route to the hydroxymethyl thiazole A proceeds as shown below:{H2424053.1}17 4922-2197-3586, v.1Docket No.2877.041AWO[2,3- b]pyridine-3-carbonyl)imino)-3-benzyl-2,3-dihydrothiazol-4-yl)methyl dihydrogen phosphate [Example 1] by reaction with dibenzylphosphoryl chloride, followed by hydrogenolysis over palladium on carbon: {H2424053.1}18 4922-2197-3586, v.1Docket No.2877.041AWO

[0040] To test the solubility of compound 1, 40 mg samples were placed with rapid agitation into 200 µL volumes of various aqueous solutions. The object was to produce a soluble sample at 200 mg / mL or 450 mM. The pure compound 1 formed a white emulsion with pH < 4 in distilled water. One sample largely cleared—with a few particles—at pH < 6.6 with 1.5 equivalent of NaOH, then cleared competedly at pH ≈ 8 with 1.8 equivalents. Another sample with 1.75 equivalents of NaOH, cleared well and almost completely at pH ≈ 7.

[0041] In a repeat of the two foregoing trials, 100 mg in 500 µL of solution, again amounting to 200 mg / mL—cleared completely at pH ≈ 6.8 after some crushing of particles. Solubilization was accomplished without titration in 400 µL of 1 M NaOH and 100 µL of distilled water, or 1.78 equivalents of NaOH. The solution was slightly straw- colored. After two days at room temperature, samples with less than 1.5 eq precipitated to a compact pellet or became a densely cloudy slurry. Samples above 1.5 eq, however,{H2424053.1}19 4922-2197-3586, v.1Docket No.2877.041AWO remained solvated and clear. All samples precipitated immediately and extensively when an excess of HCl was added, suggesting that the water-insoluble parent compound likely remained intact. In sum, it appears that a stable, approximately neutral solution of the compound of example 1 can be created at 200 mg / mL or 450 mM. The compound congeals if left in acidic solution for more than a few minutes. Even near-neutrality—pH ≈ 7 and pH ≈ 6.8 resulted in coagulation after two days, so it appears that a moderately alkaline solution is necessary to maintain solubility at a desired concentration.

[0042] Further studies suggested that solubilization is precarious with anything less than two equivalents of NaOH and that the maximal solubility is between 200 mg / mL and 300 mg / mL. Reverse-phase liquid chromatography and mass spectrometry (LC MS) indicated that 1 is resistant to basic hydrolysis and is unlikely to suffer any other molecular rearrangement. Even neutralization, and perhaps a mild degree of acidification, does not cause hydrolysis.

[0043] Six Swiss-Webster mice, all six weeks of age, were obtained from the breeding colony of the CBC. Animal 1 (24.2 g) and animal 2 (21.9 g) were used as uninjected controls; the sample masses from the apices of the hearts were both 50 mg. The others were treated identically as pairs: Animals 3 and 4 had a 2.0 hr exposure to Example 1 prepared at 20 mg / mL (45 mM; 10 mg in 450 µL water, titration to pH ≈ 10 with 24 µL of 2 M NaOH, or 2.1 equivalents, and adjustment to 500 µL total). After injection with 150 µL apiece, and based on the animals' actual weights after sacrifice, the doses were 125 mg / kg (animal 3, 24.0 g, sample mass 50 mg) and 119 mg / kg (animal 4, 25.2 g, sample mass 53 mg). Both animals were quite "squinty" and showed moderate inhibition of exploratory movements. Animals 5 and 6 had a 2.5 hr exposure to 1 prepared at 20 mg / mL (10 mg with 50 mg Kolliphor HS 15 in 450 µL PBS). After injection with 200 µL apiece, and based on the animals' actual weights after sacrifice, the doses were 134 mg / kg (animal 5, 29.9 g, sample mass 51 mg) and 159 mg / kg (animal 6, 25.2 g, sample mass 50 mg). Animal 6 was slightly "squinty" but showed little inhibition of exploratory movements; animal 5 was essentially unaffected.{H2424053.1}20 4922-2197-3586, v.1Docket No.2877.041AWO

[0044] About half of each heart—probably somewhat more than 50 mg—was simply dried on a Kimwipe, placed into a 1.5 mL plastic tube, wrapped with aluminum foil, and submerged in liquid nitrogen. The processed samples were maintained on ice throughout the procedures, then shipped with the raw samples on dry ice.

[0045] The initial analysis of the processed samples showed moderate suppression of phosphorylated Yap (pYap) in samples 3 and 4, but above-control levels in samples 5 and 6. Sample 3 had a lower level of total Yap (tYap) as well as pYap, probably an artifact of some loss during preparation. Repetition of the blotting and labeling yielded similar results for samples 3 and 4, but showed a slight decline in pYap in the Kolliphor samples 5 and 6.

[0046] Compound 1 was formulated at 450 mM by placing 480 mg into 1.22 mL water plus 1.08 mL of 2 M NaOH (900 mM or 2.0 eq., based on a final volume of 2.4 mL), yielding 2.30 mL at pH ≈ 7 with extensive remaining particles. Adding 20 µL of 2 M NaOH (17 mM or 0.038 eq.) and crushing the particles led to pH ≈ 8 with no remaining particles. Adding a further 30 µL of NaOH (25 mM or 0.056 eq.) caused complete solubilization at pH ≈ 10 with a total of 1.13 mL of base (942 mM or 2.09 eq.). The volume was made up to 2.4 mL with 50 µL of water.

[0047] Twelve Alzet 2001 pumps, each filled with 200 µL of solution, were primed overnight at 37 ˚C in PBS. The animal numbers, injection concentrations, amount injected, animat weights, and calculated dosages in mg / kg.day for the subsequent experiment were as follows: 1 Operated / implanted control 32 g 2 Operated / implanted control 33 g 3 Operated / implanted control 31 g 4 120 mg / mL 270 mM 38 g 76 mg / kg / day 5 120 mg / mL 270 mM 33 g 87 mg / kg / day 6 120 mg / mL 270 mM 34 g 85 mg / kg / day{H2424053.1}21 4922-2197-3586, v.1Docket No.2877.041AWO 7 160 mg / mL 360 mM 33 g 116 mg / kg / day 8 160 mg / mL 360 mM 34 g 113 mg / kg / day 160 mg / mL 360 mM 32 g 120 mg / kg / day 10 200 mg / mL 450 mM 36 g 133 mg / kg / day 11 200 mg / mL 450 mM 32 g 150 mg / kg / day 12 200 mg / mL 450 mM 33 g 145 mg / kg / day

[0048] The preparative and surgical procedures were as follows: Buprenorphine at 150 µL per animal, or about 0.13 mg / kg was administered preoperatively and subcutaneously at 200 µL per animal, or about 0.17 mg / kg. The wound area was inflitrated before surgery with 200 µL of bupivacaine at 2.5 mg / mL. Animals recovered and were maintained in cages set one-half over a hotpad at a medium setting. Twenty-four hours after implantation, all the animals were alive and without restricted movement or squintiness. The small leftover amounts of the two highest-concentration samples remained clear and liquid at room temperature for at least 17 days.

[0049] The animals were sacrificed after a total of 4.2 days. Upon aspiration with the loading needle, all 12 pumps seemed nearly empty, though the dead volume in the needle and syringe neck likely represented 12 drops or 30- 60 µL. All the observed residue was clear, highly liquid, and free of particles. The treated animals demonstrated a reduced ratio of phosphorylated Yap relative to total Yap, as shown in Fig.1.{H2424053.1}22 4922-2197-3586, v.1

Claims

Docket No.2877.041AWO CLAIMS 1. A compound of formula I:R1and R2are independently selected from the group consisting of hydrogen, halogen, hydroxy, (C1-C3)alkyl, halo(C1-C3)alkyl, (C1-C3)alkoxy, and halo(C1-C3)alkoxy; R4is chosen from hydrogen, fluoro, chloro and methyl; a dashed line represents an optionally present double bond; and n is zero or one.

2. A compound according to claim 1 wherein R4is hydrogen.

3. A compound according to claim 1 wherein R1and R2are both hydrogen.

4. A compound according to claim 1 wherein the dashed line represents a double bond.

5. A compound according to any of claims 1-4 wherein n is one.

6. A compound according to any of claims 1-4 wherein n is zero.

7. The compound of formula{H2424053.1}23 4922-2197-3586, v.1Docket No.2877.041AWO according to claim 1.

8. A pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a compound according to any of claims 1-4 or 7.

9. A method of for activating YAP in a cell expressing YAP comprising exposing the cell to a compound according to any of claims 1-4 or 7.

10. A method for LATS inhibition in a cell population expressing LATS comprising exposing the cell population to a compound according to any of claims 1-4 or 7.

11. A method for stimulating hair cell regeneration comprising exposing a supporting-cell population to a compound according to any of claims 1-4 or 7.

12. A method of treating a subject having, or at risk of developing, hearing loss, comprising administering to the subject an effective amount of a compound according to any of claims 1-4 or 7.{H2424053.1}24 4922-2197-3586, v.1

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