BR112017026612B1 - NOTCH SIGNALING PATHWAY INHIBITORS, THEIR USES, AND PHARMACEUTICAL COMPOSITION - Google Patents

Abstract

NOTCH SIGNALING PATHWAY INHIBITOR COMPOUNDS. The present invention provides the following compounds, or pharmaceutically acceptable salt thereof, and a pharmaceutical composition containing said compounds, useful as an inhibitor of the Notch signaling pathway for the treatment of the aforementioned cancers, of sensorineural hearing loss caused by the loss of auditory hair cells and for the generation of auditory hair cells.

Description

[0001] The loss of sensory hair cells in the inner ear through aging, noise exposure, chemical exposure, medications, disease, and genetic disorders causes hearing impairment in many people each year. Regardless of the etiology, death or dysfunction of mechanosensory hair cells located within the organ of Corti of the cochlea is the leading cause of sensorineural hearing loss (SNHL). Prophylactic measures to inhibit hearing loss are primarily limited to peripheral protection, such as earplugs or noise reduction or cancellation headphones. Current treatments for SNHL rely primarily on electronic technologies such as sound amplification using hearing aids or hair cell bypassing by electrical stimulation of surviving spiral ganglion neurons using cochlear implants. Steroid administration, to allow systemic therapy or intratympanic therapy, has also been suggested for sudden sensorineural hearing loss.

[0002] The cochlear sensory epithelium contains hair cells adapted for the detection of sound vibration, which is transduced by stereocilia on the apical surfaces of the hair cell into electrical impulses and transmitted to the brain via cranial nerve VIII. The auditory hair cells produced during development are postmitotic and are not replaced after loss or as part of normal cell movement in mammals. As a result, SNHL due to loss of auditory hair cells is irreversible. The development of auditory hair cells during the embryonic period involves a differentiation in which the prosensory epithelial cells acquire different fates, the hair cell or the supporting cell, through a process of lateral inhibition that is mediated by Notch signaling. The prosensory epithelial cells are prevented from differentiating into hair cells by active ligand-stimulated Notch signaling in adjacent hair cells. These cells become supporting cells.

[0003] Notch signaling is an evolutionary conserved pathway that plays a fundamental role in development and tissue homeostasis in mammals. Notch receptors and ligands contain single-pass transmembrane domains and are expressed on the cell surface, and for this reason, Notch signaling is particularly important in mediating communication between adjacent cells expressing the receptors and ligands. There are four known Notch receptors found in rodents and humans, designated Notch 1 through Notch 4. Notch receptors are heterodimeric proteins composed of extracellular and intracellular domains that are initially synthesized as a single polypeptide. Receptor-ligand interaction activates a series of proteolytic cleavages of the Notch receptor polypeptide in which Y-secretase activity is involved. Y-secretase activity cleaves the Notch intracellular domain from the inner side of the plasma membrane, which translocates to the nucleus to form a transcription factor complex. The Notch intracellular domain (NICD) is the active form of the protein. Diverse functions of Notch signaling include proliferation, differentiation, apoptosis, angiogenesis, migration, and self-renewal. These diverse roles of Notch signaling during normal tissue development and maintenance can be aberrantly activated in different forms of cancer. Oncogenic functions of Notch signaling include inhibition of apoptosis and promotion of cell proliferation.

[0004] Y-Secretase plays an essential role in the Notch activation cascade. As a result, Y-secretase inhibitors have been actively investigated for their potential to block Notch receptor activation for the treatment of cancer. No Notch inhibitor chemotherapeutic drugs have emerged, although clinical trials are ongoing.

[0005] Y-Secretase, via the Notch signaling pathway, also plays an essential role in the differentiation of prosensory cells. As a result, Y-secretase inhibitors have been actively investigated for their potential to block Notch receptor activation for the treatment of SNHL. Rather than administering a Notch inhibitory agent through various methodologies to enter the systemic circulation to induce atonal homolog 1 (Atoh1 or Math1) expression at abnormally high levels and for extended periods of time, it would be useful and desirable to induce Atoh1 expression, in the cochlear environment, at a more targeted expression level and time frame. The inability to modulate Atoh1 continues to be an obstacle to auditory hair cell research and the development of therapeutics for auditory hair cell regeneration. Although research continues, as of WO 2014/039781, no commercial Notch inhibitor for the treatment of sensorineural hearing loss caused by loss of auditory hair cells has emerged.

[0006] There is a need to find compounds having Notch signaling pathway inhibitory activity. There is a further need to find compounds that inhibit the Notch signaling pathway through Y-secrease inhibitory activity. There is also a need to find compounds that have distinct structural features that may contribute to the Notch signaling pathway and Y-secrease inhibitory activity. Another need is to find compounds that induce Atoh1 expression by inhibiting the Notch signaling pathway. There is also a need to find compounds that demonstrate desirable absorption, distribution, metabolism and excretion properties.

[0007] One aspect of the invention is to provide a compound of the structure: Compound 1 4,4,4-trifluoro-N-((2S)-1-((9-methoxy-3,3-dimethyl-5-oxo-2,3,5,6-tetrahydro-1H-benzo[f]pyrrolo[1,2-a]azepin-6-yl)amino)-1-oxopropan-2-yl)butanamide, or substantially pure diastereomeric isomer thereof, or a pharmaceutically acceptable salt of any of the above.

[0008] Another aspect of the invention is a compound of the structure: Compound 2 N-((2S)-1-((8,8-dimethyl-6-oxo-6,8,9,10-tetrahydro-5H-pyrido[3,2-f]pyrrolo[1,2-a]azepin-5-yl)amino)-1-oxopropan-2-yl)-4,4,4-trifluorobutanamide, or substantially pure diastereomeric isomer thereof, or a pharmaceutically acceptable salt of any of the above.

[0009] Another aspect of the present invention provides a pharmaceutical composition comprising Compound 1, or a substantially pure diastereomer thereof; or Compound 2, or a substantially pure diastereomer thereof; or a pharmaceutically acceptable salt of any of the above, and a pharmaceutically acceptable carrier.

[0010] Another aspect of the present invention provides a method of treating a cancer that is T-cell acute lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, erythroleukemia, triple negative breast cancer, breast cancer, ovarian cancer, melanoma, lung cancer, non-small cell lung cancer, pancreatic cancer, glioblastoma, colorectal cancer, head and neck cancer, cervical cancer, prostate cancer, liver cancer, oral squamous cell carcinoma, skin cancer, medulloblastoma, angiosarcoma, rhabdomyosarcoma, liposarcoma, malignant fibrous histiocytoma, hepatocellular carcinoma, intrahepatic and extrahepatic cholangiocarcinoma, and adenoid cystic carcinoma in a patient comprising administering to a patient in need thereof a therapeutically effective amount of Compound 1, a substantially pure diastereomer thereof or its pharmaceutically acceptable salt or of Compound 2, a substantially pure diastereomer thereof or its pharmaceutically acceptable salt.

[0011] Another aspect of the present invention provides a method of treating lung cancer in a patient comprising administering to a patient in need thereof a therapeutically effective amount of Compound 1, a substantially pure diastereomer thereof or a pharmaceutically acceptable salt thereof, or of Compound 2, a substantially pure diastereomer thereof or a pharmaceutically acceptable salt thereof.

[0012] Another aspect of the present invention provides a method of treating sensorineural hearing loss caused by loss of auditory hair cells in a patient in need thereof, comprising administering a therapeutically effective amount of Compound 1, a substantially pure diastereomer thereof or a pharmaceutically acceptable salt thereof, or Compound 2, a substantially pure diastereomer thereof or a pharmaceutically acceptable salt thereof, to said patient.

[0013] Another aspect of the present invention provides a method for inducing the generation of auditory hair cells in a patient in need thereof, comprising administering a therapeutically effective amount of Compound 1, a substantially pure diastereomer thereof or a pharmaceutically acceptable salt thereof, or Compound 2, a substantially pure diastereomer thereof or a pharmaceutically acceptable salt thereof, to said patient.

[0014] Another aspect of the present invention provides Compound 1, a substantially pure diastereomer thereof, or a pharmaceutically acceptable salt thereof, or Compound 2, a substantially pure diastereomer thereof, or a pharmaceutically acceptable salt thereof, for use in therapy.

[0015] Another aspect of the present invention provides Compound 1, a substantially pure diastereomer thereof, or a pharmaceutically acceptable salt thereof, or Compound 2, a substantially pure diastereomer thereof, or a pharmaceutically acceptable salt thereof, for use in the treatment of T-cell acute lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, erythroleukemia, triple negative breast cancer, breast cancer, ovarian cancer, melanoma, lung cancer, non-small cell lung cancer, pancreatic cancer, glioblastoma, colorectal cancer, head and neck cancer, cervical cancer, prostate cancer, liver cancer, oral squamous cell carcinoma, skin cancer, medulloblastoma, angiosarcoma, rhabdomyosarcoma, liposarcoma, malignant fibrous histiocytoma, hepatocellular carcinoma, intrahepatic or extrahepatic cholangiocarcinoma or adenoid cystic carcinoma.

[0016] A further aspect of the present invention provides Compound 1, a substantially pure diastereomer thereof, or a pharmaceutically acceptable salt thereof, or Compound 2, a substantially pure diastereomer thereof, or a pharmaceutically acceptable salt thereof, for use in the treatment of lung cancer.

[0017] Another aspect of the present invention provides Compound 1, a substantially pure diastereomer thereof, or a pharmaceutically acceptable salt thereof, or Compound 2, a substantially pure diastereomer thereof, or a pharmaceutically acceptable salt thereof for use in the treatment of sensorineural hearing loss caused by loss of auditory hair cells.

[0018] Another aspect of the present invention provides Compound 1, a substantially pure diastereomer thereof, or a pharmaceutically acceptable salt thereof, or Compound 2, a substantially pure diastereomer thereof, or a pharmaceutically acceptable salt thereof, for use in inducing the generation of auditory hair cells.

[0019] Another aspect of the present invention provides the use of Compound 1, a substantially pure diastereomer thereof, or a pharmaceutically acceptable salt thereof, or of Compound 2, a substantially pure diastereomer thereof, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of T-cell acute lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, erythroleukemia, triple negative breast cancer, breast cancer, ovarian cancer, melanoma, lung cancer, non-small cell lung cancer, pancreatic cancer, glioblastoma, colorectal cancer, head and neck cancer, cervical cancer, prostate cancer, liver cancer, oral squamous cell carcinoma, skin cancer, medulloblastoma, angiosarcoma, rhabdomyosarcoma, liposarcoma, malignant fibrous histiocytoma, carcinoma hepatocellular, intrahepatic or extrahepatic cholangiocarcinoma or adenoid cystic carcinoma.

[0020] Another aspect of the present invention provides the use of Compound 1, a substantially pure diastereomer thereof, or a pharmaceutically acceptable salt thereof, or of Compound 2, a substantially pure diastereomer thereof, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of lung cancer.

[0021] Another aspect of the present invention provides the use of Compound 1, a substantially pure diastereomer thereof, or a pharmaceutically acceptable salt thereof, or of Compound 2, a substantially pure diastereomer thereof, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of sensorineural hearing loss caused by the loss of auditory hair cells.

[0022] Another aspect of the present invention provides the use of Compound 1, a substantially pure diastereomer thereof, or a pharmaceutically acceptable salt thereof, or of Compound 2, a substantially pure diastereomer thereof, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for inducing the generation of auditory hair cells.

[0023] Another aspect of the invention provides a method of treating sensorineural hearing loss caused by loss of auditory hair cells in a canine companion animal comprising administering a therapeutically effective amount of Compound 1, a substantially pure diastereomer thereof, or a pharmaceutically acceptable salt thereof, or Compound 2, a substantially pure diastereomer thereof, or a pharmaceutically acceptable salt thereof, to said canine companion animal.

[0024] Another aspect of the invention provides a method of inducing the generation of auditory hair cells in a canine companion animal in need thereof, comprising administering a therapeutically effective amount of Compound 1, a substantially pure diastereomer thereof, or a pharmaceutically acceptable salt thereof, or Compound 2, a substantially pure diastereomer thereof, or a pharmaceutically acceptable salt thereof, to said canine companion animal.

[0025] The phrase "Compound 1, a diastereomer thereof" means 4,4,4-trifluoro-N-((2S)-1-((9-methoxy-3,3-dimethyl-5-oxo-2,3 ,5,6-tetrahydro-1H-benzo[f]pyrrolo[1,2-a]azepin-6-yl)amino)-1-oxopropan-2-yl)butanamide; or a diastereomer 4,4,4-trifluoro-N-((S)-1-(((S)-9-methoxy-3,3-dimethyl-5-oxo-2,3,5,6-tetra- hydro-1H-benzo[f]pyrrolo[1,2- a]azepin-6-yl)amino)-1-oxopropan-2-yl)butanamide or 4,4,4-trifluoro-N- ((S)- 1-(((R)-9-methoxy-3,3-dimethyl-5-oxo-2,3,5,6-tetrahydro-1H-benzo[f]pyrrolo[1,2-a]azepin- 6-yl)amino)-1-oxopropan-2-yl)butanamide. Likewise, the phrase "Compound 2, a diastereomer thereof" means N-((2S)-1-((8,8-dimethyl-6-oxo-6,8,9,10-tetrahydro-5H- pyrido[3,2-f]pyrrolo[1,2-a]azepin-5-yl)amino)-1-oxopropan-2-yl)-4,4,4-trifluorobutanamide; or a diastereomer N-((S)-1-(((S)-8,8-dimethyl-6-oxo-6,8,9,10-tetrahydro-5H-pyrido[3,2-f] pyrrolo[1,2-a]azepin-5-yl)amino)-1-oxopropan-2-yl)-4,4,4-trifluorobutanamide or N-((S)-1-(((R)-8 ,8-dimethyl-6-oxo-6,8,9,10-tetrahydro-5H-pyrido[3,2-f]pyrrolo[1,2-a]azepin-5-yl)amino)-1- oxopropan-2-yl)-4,4,4-trifluorobutanamide.

[0026] The term "patient" means mammal and "mammal" includes, but is not limited to, a human being after the postnatal period. The postnatal period in a human being is the period beginning immediately after parturition and continuing for 30 days.

[0027] "Effective therapeutic amount" or "effective amount" means the dosage of Compound 1, a substantially pure diastereomer thereof that is Isomer 1 or Isomer 2, or a pharmaceutically acceptable salt thereof, or of Compound 2, a substantially pure diastereomer thereof that is Isomer 1 or Isomer 2, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing any of the foregoing, necessary to inhibit Notch signaling in a cancer patient and destroy the target cancer cells or slow or halt the progression of cancer in a patient. Similarly, "therapeutically effective amount" or "effective amount" means the dosage of Compound 1, a substantially pure diastereomer thereof that is Isomer 1 or Isomer 2, or a pharmaceutically acceptable salt thereof, or of Compound 2, a substantially pure diastereomer that is Isomer 1 or Isomer 2, or a pharmaceutically effective salt thereof, or a pharmaceutical composition containing any of the foregoing, necessary to inhibit Notch signaling in a patient with sensorineural hearing loss caused by loss or damage of auditory hair cells or to induce the generation of auditory hair cells.

[0028] A "substantially pure diastereomer of Compound 1 or Compound 2" means Isomer 1 or Isomer 2 substantially free of the other Isomer. Compound 1 or Compound 2 is "substantially diastereomerically pure" when the isomeric purity at the 6-position of Compound 1 or at the 5-position of Compound 2 is greater than 90% enantiomeric excess. In another embodiment, the isomeric purity is greater than 95% enantiomeric excess at the 6-position of Compound 1 or at the 5-position of Compound 2. In yet another embodiment, the isomeric purity is greater than 98% enantiomeric excess at the 6-position of Compound 1 or at the 5-position of Compound 2. In yet another embodiment, the isomeric purity is greater than 99% enantiomeric excess at the 6-position of Compound 1 or at the 5-position of Compound 2. All stereoisomers, including diastereomeric mixtures of Compound 1 or Compound 2, are contemplated within the present invention.

[0029] Predicted dosages of Compound 1, a substantially pure diastereomer thereof, or a pharmaceutically acceptable salt thereof, or Compound 2, a substantially pure diastereomer thereof, or a pharmaceutically acceptable salt thereof for the treatment of cancer are in the range of 0.1 to 100 mg/patient/day. Preferred dosages are anticipated to be in the range of 1.0 to 75 mg/patient/day. Most preferred dosages are anticipated to be in the range of 2.0 to 50 mg/patient/day. Predicted dosages of Compound 1, a diastereomer thereof, or a pharmaceutically acceptable salt thereof, or Compound 2, a diastereomer thereof, or a pharmaceutically acceptable salt thereof for the treatment of sensorineural hearing loss caused by loss or damage of auditory hair cells or for the induction of auditory hair cell generation are in the range of 0.01 to 100 mg/patient/day. Preferred dosages are anticipated to be in the range of 0.1 to 10 mg/patient/day. Most preferred dosages are anticipated to be in the range of 0.2 to 1.0 mg/patient/day.

[0030] For cancer, sensorineural hearing loss, or induction of auditory hair cell generation, the exact dosage required to treat a patient and the duration of treatment time will be determined by a physician in view of the stage and severity of the disease, as well as the specific needs and response of the individual patient. Although expressed as dosing on a daily basis, the administration regimen may be adjusted to provide a more optimal therapeutic benefit for a patient and to control and ameliorate drug-related toxicities. In addition to daily administration, administration every other day (Q2D); every other day for a five-day period followed by two days without dosing (T.I.W.); every third day (Q3D); or once weekly (Q.I.W.) over a 21-day dosing cycle; or other administration regimens may be appropriate.

[0031] The terms "treatment", "treat" and "treating" are meant to include the full spectrum of intervention for cancer, sensorineural hearing loss caused by loss or damage of auditory hair cells, or induction of the generation of auditory hair cells, such as the administration of the active compound to alleviate, delay or reverse one or more of the symptoms and delay the progression of the cancer or loss or damage of auditory hair cells from which the patient is suffering, or to induce the generation of auditory hair cells in the patient.

[0032] "Canine companion animals" means domesticated and domestically bred canines, including, but not limited to, pets, service dogs, rescue dogs, herding dogs, and livestock guardian dogs.

[0033] "Auditory hearing loss", "sensorineural hearing loss" or "hearing loss" in a human means that the hearing threshold (the softest sound heard (intensity) at a specific frequency) in a patient is 21 to 40 dB for mild; 41 to 55 dB for moderate; 56 to 70 dB for moderately severe; 71 to 90 dB for severe; and 91 dB and above for loud. The intensities tested typically range from 0 dB to 120 dB. Frequencies tested are typically 250, 500, 1000, 2000, 4000, and 8000 Hz. Diagnostic hearing evaluations are performed by routine tests known to and used by those of skill in the art including pure tone audiometry including pure tone average, speech audiometry including speech reception threshold, auditory brainstem response (ABR or BAER) assessments, transtympanic electrocochleography (ECOG), and otoacoustic emission (OAE) testing. Pediatric and infant evaluations are also performed by routine tests known to and used by those of skill in the art including visual reinforcement audiometry, activity audiometry, otoacoustic emissions (OAEs), and auditory brainstem response assessments.

[0034] A compound of the present invention is preferably formulated as a pharmaceutical composition with a pharmaceutically acceptable carrier and administered by a variety of routes. Preferably, for the treatment of cancer, such compositions are for oral administration.

[0035] To treat sensorineural hearing loss caused by loss or damage of auditory hair cells or to induce the generation of auditory hair cells, a pharmaceutical composition suitable for oral or parenteral administration to provide local or systemic therapy can be formulated and administered. Oral compositions include tablets, coated tablets, solid or soft gelatin capsules, solutions, emulsions or suspensions. Parenteral compositions can be formulated to allow for administration including subcutaneous, intravenous, intramuscular, intraperitoneal, intrapleural, intrasternal, transtympanic, intralabyrinthine or intracochlear injections, or infusion. Pharmaceutical compositions can be formulated to allow a compound of the invention to be bioavailable following administration of the composition to a patient, or formulated to provide a controlled or sustained release of the active pharmaceutical ingredient. A pharmaceutical composition suitable for transtympanic administration to the middle ear cavity is preferred, and administration to the vestibular window niche of the middle ear is also preferred. Intralabirinthine injection and intracochlear injection are also contemplated. The preferred route of administration for the treatment of hearing loss is local transtympanic injection, however, this does not preclude pharmaceutical compositions suitable for alternative routes of administration to provide systemic delivery and may include compositions and dosage regimens alternative to or in addition to local transtympanic administration. Also preferred is a sustained release local delivery composition where the release interval may range from three (3) days to ninety (90) days depending on the delivery vehicle or delivery vehicle agent mixture in which Compound 1, or a substantially pure diastereomer, or pharmaceutically acceptable salt thereof, is associated.

[0036] Pharmaceutical compositions, processes for their preparation, and delivery systems for targeted drug delivery are well known in the art. See, for example, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (A. Gennaro, et al., eds., 19th ed., Mack Publishing Co., 1995); Salt and Plontke, “Principles of Local Drug Delivery to the Inner Ear,” Audiol Neurotol, 2009, (14); 350-360; Rhee, et al., “Sustained-Release Injectable Drug Delivery,” Pharmaceutical Technology, Special Issue Drug Delivery, 01 Nov. 2010. The pharmaceutical compositions may contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colors, flavors, salts for varying osmotic pressure, buffers, masking agents, or antioxidants.

[0037] A compound of the present invention is capable of reacting with various inorganic and organic acids to form pharmaceutically acceptable acid addition salts. Such pharmaceutically acceptable salts and the common methodology for their preparation are well known in the art. See, for example, P. Stahl, et al., HANDBOOK OF PHARMACEUTICAL SALTS: PROPERTIES, SELECTION AND USE, (VCHA/Wiley-VCH, 2002); S. M. Berge, et al., “Pharmaceutical Salts,” Journal of Pharmaceutical Sciences, Vol. 66, No. 1, January 1977.

[0038] Compound 1, a substantially pure diastereomer thereof, or a pharmaceutically acceptable salt thereof, or Compound 2, a substantially pure diastereomer thereof, or a pharmaceutically acceptable salt thereof, can be prepared by a variety of procedures known in the art, such as those described below. Specific synthetic steps can be combined in different ways to prepare Compound 1, a diastereomer thereof, or a pharmaceutically acceptable salt thereof, or Compound 2, a diastereomer thereof, or a pharmaceutically acceptable salt thereof.

[0039] Compound 1 is designated: 4,4,4-trifluoro-N-((2S)-1-((9-methoxy-3,3-dimethyl-5-oxo-2,3,5,6-tetrahydro-1H-benzo[f]pyrrolo[1,2-a]azepin-6-yl)amino)-1-oxopropan-2-yl)butanamide and may also be designated 4,4,4-trifluoro-N-{(1S)-2-[(9-methoxy-3,3-dimethyl-5-oxo-2,3,5,6-tetrahydro-1H-pyrrolo[1,2-a][1]benzazepin-6-yl)amino]-1-methyl-2-oxoethyl}butanamide; and may also be designated: butanamide, 4,4,4-trifluoro-N-[(1S)-1-methyl-2-oxo-2-[(2,3,5,6-tetrahydro-9-methoxy-3,3-dimethyl-5-oxo-1H-pyrrolo[1,2-a][1]benzazepin-6-yl)amino]ethyl]-; and other names may be used to unambiguously identify Compound 1. The diastereomers are designated 4,4,4-trifluoro-N-((S)-1-(((S)-9-methoxy-3,3-dimethyl-5-oxo-2,3,5,6-tetrahydro-1H-benzo[f]pyrrolo[1,2-a]azepin-6-yl)amino)-1-oxopropan-2-yl)butanamide and 4,4,4-trifluoro-N-((S)-1-(((R)-9-methoxy-3,3-dimethyl-5-oxo-2,3,5,6-tetrahydro-1H-benzo[f]pyrrolo[1,2-a]azepin-6-yl)amino)-1-oxopropan-2-yl)butanamide. Other names may be used to unequivocally identify each of the diastereomers.

[0040] Compound 2 is designated N-((2S)-1-((8,8-dimethyl-6-oxo-6,8,9,10-tetrahydro-5H-pyrido[3,2-f]pyrrolo[1,2-a]azepin-5-yl)amino)-1-oxopropan-2-yl)-4,4,4-trifluorobutanamide and may also be designated N-{(1S)-2-[(8,8-dimethyl-6-oxo-6,8,9,10-tetrahydro-5H-pyrido[3,2-f]pyrrolo[1,2-a]azepin-5-yl)amino]-1-methyl-2-oxoethyl}-4,4,4-trifluorobutanamide; and may also be designated: butanamide, 4,4,4-trifluoro-N-[(1S)-1-methyl-2-oxo-2-[(6,8,9,10-tetrahydro-8,8-dimethyl-6-oxo-5H-pyrido[3,2-f]pyrrolo[1,2-a]azepin-5-yl)amino]ethyl]-; and other names may be used to unambiguously identify Compound 2. The diastereomers are designated N-((S)-1-(((S)-8,8-dimethyl-6-oxo-6,8,9,10-tetrahydro-5H-pyrido[3,2-f]pyrrolo[1,2-a]azepin-5-yl)amino)-1-oxopropan-2-yl)-4,4,4-trifluorobutanamide and N-((S)-1-(((R)-8,8-dimethyl-6-oxo-6,8,9,10-tetrahydro-5H-pyrido[3,2-f]pyrrolo[1,2-a]azepin-5-yl)amino)-1-oxopropan-2-yl)-4,4,4-trifluorobutanamide. Other names may be used to unequivocally identify each of the diastereomers.

[0041] It will be understood that Compound 1 and Compound 2 are depicted with one of the two chiral centers fixed. Herein, the Cahn-Ingold-Prelog designations of (R)- and (S)- are used to refer to the specific isomers. The specific stereoisomers can be prepared through stereospecific synthesis using enantiomerically pure or enriched starting materials. Specific stereoisomers of starting materials, intermediates, or racemic mixtures including Compound 1 or Compound 2 can be separated by techniques well known in the art, such as those found in Stereochemistry of Organic Compounds, E. I. Eliel and S. H. Wilen (Wiley 1994) and Enantiomers, Racemates, and Resolutions, J., Jacques, A. Collet, and S. H. Wilen (Wiley 1991), including chromatography on chiral stationary phases, enzymatic resolutions, or crystallization or fractional chromatography of diastereomers formed for this purpose, such as the diastereomeric salts. Where a chiral compound is isolated or separated into its isomers, but the absolute configurations or auricular rotations are not determined, the isomers are arbitrarily designated as Isomer 1 and Isomer 2 corresponding to the order in which each elutes from chiral chromatography, and if chiral chromatography is initiated at the beginning of the synthesis, the same designation is applied to the intermediates and subsequent examples. Although all mixtures containing the compounds of the present invention are contemplated within the present invention, the preferred embodiment is Compound 1, Isomer 2 or Compound 2, Isomer 2.

[0042] The compounds employed as initial starting materials in the synthesis of the compounds of the present invention are well known and, to the extent that they are not commercially available, are readily synthesized using the specific references provided, by standard procedures commonly employed by those of skill in the art, or are found in general reference texts.

[0043] Examples of known procedures and methods include those described in general reference texts such as Comprehensive Organic Transformations, VCH Publishers Inc, 1989; Compendium of Organic Synthetic Methods, Volumes 1-10, 1974 2002, Wiley Interscience; Advanced Organic Chemistry, Reactions Mechanisms, and Structure, 5th Edition, Michael B. Smith and Jerry March, Wiley Interscience, 2001; Advanced Organic Chemistry, 4th Edition, Part B, Reactions and Synthesis, Francis A. Carey and Richard J. Sundberg, Kluwer Academic / Plenum Publishers, 2000, etc., and references cited.

[0044] As used herein, the following terms have the meanings indicated: "mAtoh1" refers to rat atonal homologous protein 1; "hAtoh 1 refers to human atonal homologous protein 1; "Atoh1" refers to human atonal gene homolog 1; "Basic Medium" refers to 500 mL DMEM/F12 medium + 5 mL N2 100X stock and 10 mL B27 50X stock plus 500 μl Ampicillin (1000X, 50 mg/mL) and 1667μl Fungizone (300X); "BFGF" refers to basic fibroblast growth factor; "DMEM" refers to Dulbecco's Modified Eagle's Medium; "DMSO" refers to dimethyl sulfoxide; "EDTA" refers to ethylenediaminetetraacetic acid; "EGF" refers to epidermal growth factor; "EGTA" refers to ethylene glycol glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid; ES/MS refers to electrospray mass spectroscopy; "FBS" refers to fetal bovine serum; "GFP" refers to green fluorescent protein; "h" refers to hour or hours; "HBSS" refers to Hank's balanced salt solution; "HEK" refers to human embryonic kidney; "IC50" refers to the concentration of an agent that produces 50% of the maximal possible inhibitory response to that agent; "IgG" refers to immunoglobulin G; "Math1" refers to human atonal gene homolog 1; "Medium A" refers to 200 mL of basic medium + EGF bFGF + IGF-1 + heparan sulfate at 20, 10, 50, and 50 ng/mL, respectively; "MEM" refers to minimum essential medium; "Min" refers to minutes; "MS" refers to mass spectroscopy; "N1ICD" refers to Notch1 intracellular domain; "nGFP" refers to nuclear green fluorescent protein; "OC" refers to organ of corti; "PBS" refers to phosphate buffered saline; "PBST" refers to phosphate buffered saline + Tween®; "qPCR" refers to quantitative polymerase chain reaction; "qRT-PCR" refers to quantitative reverse transcription polymerase chain reaction; "rpm" refers to revolutions per minute; "RLT buffer" refers to RNeasyLysis buffer; "RT" refers to room temperature; "Tbp" refers to TATA binding protein. Preparation 1 1-Bromo-3-(2-bromo-4-methoxy-phenyl)propan-2-one

[0045] Add trimethylsilyldiazomethane (118.4 mL, 236.80 mmol, 2M in hexanes) dropwise to a stirred 0 °C solution of 2-(2-bromo-4-methoxy-phenyl)acetyl chloride (52 g, 197.3 mmol) in tetrahydrofuran (197 mL) and acetonitrile (197 mL) under nitrogen. After 15 min allow to warm to RT and stir for 2 h under nitrogen. Concentrate to obtain a thick red oil (61 g). Add hydrogen bromide (36 mL, 197 mmol, 33% in acetic acid) dropwise to a stirred 5 °C solution of 2-(2-bromo-4-methoxy-phenyl)acetyl azide from the previous step in acetic acid (265 mL). After addition, allow to warm to RT under nitrogen. After 45 min, quench with ice/water (500 mL) resulting in a brown precipitate. Filter the solids and wash with water (100 mL) and hexanes (100 mL) and dry under vacuum at 45 °C for 2 h to afford the title compound as an orange oil (63.0 g, 99%). 1H NMR (300 MHz, CDCl 3 ): 7.17−7.12 (m, 2H), 6.85 (dd, J = 2.5, 8.5 Hz, 1H), 4.03 (s, 2H), 3.97 (s, 2H), 3.79 (s, 3H). Preparation 2 (3Z)-1-(2-Bromo-4-methoxy-phenyl)-3-(3,3-dimethylpyrrolidin-2-ylidene)propan-2-one

[0046] Add 3,3-dimethylpyrrolidine-2-thione (26.5 g, 205 mmol) to a suspension of the intermediate provided by Preparation 1,1-bromo-3-(2-bromo-4-methoxy-phenyl)propan-2-one (60.00 g, 186 mmol), and potassium iodide (31 g, 86 mmol) in tetrahydrofuran (600 mL) at RT all at once and stir for 1 h. English:Add methyl tert-butyl ether (200 mL), discard the solids, and rinse the filter cake with methyl tert-butyl ether (100 mL) to afford 1-(2-bromo-4-methoxy-phenyl)-3-[(4,4-dimethyl-2,3-dihydropyrrole-1-ium-5-yl)sulfanyl]propan-2-one iodide as a light yellow solid (100 g). To a suspension of the solid (100 g, 201 mmol) in acetonitrile (1 L), add triethylamine (56 mL, 401 mmol) and triphenylphosphine (58 g, 221 mmol) and stir at 65 °C for 2.5 h. Add methyl tert-butyl ether (200 mL) and discard the solids. Evaporate the filtrate, triturate the residue with methyl tert-butyl ether (20 mL), and discard the solids (twice). Evaporate the combined filtrates to obtain 50 g of crude material. Purify the residue by flash column chromatography on silica eluting with a 20 to 50% ethyl acetate in hexanes gradient to obtain the title compound as an off-white solid (41 g, 70%). MS (m/z): 338.0/340.0 (M+/M+2). Preparation 3 9-Methoxy-3,3-dimethyl-2,3-dihydro-1H-pyrrolo[1,2-a][1]benzazepin-5(6H)-one

[0047] Degas/flush with nitrogen (x3) a mixture of the intermediate provided by Preparation 2, (3Z)-1-(2-bromo-4-methoxyphenyl)-3-(3,3-dimethylpyrrolidin-2-ylidene)propan-2-one (40.0 g, 118 mmol), palladium acetate (2.7 g, 12 mmol), cesium carbonate (77 g, 237 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (13.7 g, 24 mmol), and N-methyl-2-pyrrolidone (1.2 L). Heat the suspension at 150 °C for 4 h. Cool to RT, filter off the solids, wash with ethyl acetate, and discard the solids. Add ethyl acetate (1 L) and water (500 mL) to the filtrate, filter off the solids through diatomaceous earth, and discard. Separate the filtrate layers, extract from the aqueous phase with ethyl acetate (2 × 20 mL) followed by dichloromethane (3 × 100 mL). Wash the combined organic layers with 5% aqueous sodium bicarbonate solution twice, dry over sodium sulfate, filter, and concentrate to obtain 100 mL of dark brown oil. Filter the material over a silica plug eluting with dichloromethane followed by 1% methanol/dichloromethane and concentrate the filtrate. Partition the residue between ethyl acetate and 5% aqueous sodium bicarbonate solution, extract back from the aqueous phase with ethyl acetate, dry over sodium sulfate, filter, and concentrate to obtain the crude product as a dark brown foam. Dissolve the resulting foam in ethyl acetate (250 mL), add SiliaBond Thiol® (80 g), and stir at RT overnight. Discard the solids, wash filter cake with ethyl acetate and dichloromethane, concentrate the filtrate to obtain the title compound as a light brown solid (19.6 g, 65%). MS (m/z): 258.0 (M + H). Preparation 4 6-Hydroxyimino-9-methoxy-3,3-dimethyl-2,3-dihydro-1H-pyrrolo[1,2- a ][1]benzazepin-5(6H)-one

[0048] Add potassium tert-butoxide (11 g, 98 mmol) in several portions to a stirred 0 °C solution of the intermediate provided by Preparation 3, 9-methoxy-3,3-dimethyl-2,3-dihydro-1H-pyrrolo[1,2-a][1]benzazepin-5(6H)-one (16.8 g, 65 mmol), in tetrahydrofuran (336 mL) and stir for 15 min. Add amyl nitrite (12.2 mL, 91 mmol) dropwise and stir the mixture for 30 min at 0 °C. Pour the reaction mixture into ice/water (200 mL) and extract the mixture with ethyl acetate (2 x 200 mL). Discard the crystallized solids, extract the filtrate with dichloromethane (2 x 100 mL). Wash the combined organic components with brine (100 mL), dry over sodium sulfate, filter, and concentrate to afford a light brown solid. Triturate the solid with 1:1 methyl tert-butyl ether/hexane, combine with the previously collected solid to obtain the title compound as a light yellow solid (16.5 g, 88%). MS (m/z): 287.1 (M + H). Preparation 5 6-Amino-9-methoxy-3,3-dimethyl-2,3-dihydro-1H-pyrrolo[1,2- a ][1]benzazepin-5(6H)-one

[0049] Add trifluoroacetic acid (17 mL, 231 mmol) dropwise over 10 min to a stirred suspension at 5°C to 10°C of the intermediate provided by Preparation 4, 6-Hydroxy-imino-9-methoxy-3,3-dimethyl-2,3-dihydro-1H-pyrrolo[1,2-a][1]benzazepin-5(6H)-one (16.5 g, 58 mmol), and zinc dust (11.3 g, 173 mmol) in dichloromethane (248 mL) then allow to warm to RT. Filter the mixture through a plug of diatomaceous earth, pour the filtrate over a mixture of ice and saturated aqueous sodium carbonate (1:1, 500 mL). Filter the resulting suspension through diatomaceous earth and rinse the filter plug with dichloromethane. Extract the aqueous layer with dichloromethane (100 mL), dry the combined organic layers over sodium sulfate, filter, and concentrate to obtain the title compound as a light brown foam (14.7 g, 94%). MS (m/z): 273.1 (M + H). Preparation 6 Benzyl (2S)-2-(4,4,4-trifluorobutanoylamino)propanoate

[0050] Add L-alanine benzyl ester hydrochloride (7 g, 32.5 mmol), diisopropylethylamine (28 mL, 162 mmol), hydroxybenzotriazole hydrate (7.5 g, 49 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (9.3 g, 49 mmol) to a stirred solution of 4,4,4-trifluorobutyric acid (7.1 g, 49 mmol) in dichloromethane (162 mL) and stir at RT under N2 for 20 h. Quench with 20% aqueous citric acid solution (150 mL), stir the mixture for 5 min, separate the layers, and extract the aqueous phase with dichloromethane (100 mL). Wash the combined organic layers with saturated aqueous sodium bicarbonate solution (150 mL), dry over magnesium sulfate, filter, and concentrate to obtain 10.6 g of light yellow solid. Purify the residue by flash column chromatography on silica eluting with a 25 to 50% ethyl acetate in hexanes gradient to obtain the title compound as a white solid (9.2 g, 94%). MS (m/z): 304.2 (M + H). Preparation 7 (2S)-2-(4,4,4-Trifluorobutanoylamino)propanoic acid

[0051] Add palladium (1.8 g, 0.8 mmol, 5% on C) to a stirred solution of the intermediate provided by Preparation 6, (2S)-2-(4,4,4-trifluorobutanoylamino)benzylpropanoate (8.8 g, 29 mmol), in methanol (88 mL) at RT. Degas the mixture and stir under hydrogen (balloon atmosphere) for 5 h. Filter the mixture through diatomaceous earth, wash the plug with methanol, and concentrate the filtrate to obtain a white solid. Triturate the solids with dichloromethane and dry under vacuum overnight at RT to afford the title compound as a white solid (6.11 g, 99% yield). MS (m/z): 214.1 (M + H). Preparation 8 Methyl 3-(3,3-dimethyl-2-thioxopyrrolidin-1-yl)propanoate

[0052] Dissolve 3,3-dimethylpyrrolidine-2-thione (15.7 g, 122 mmol) and methyl acrylate (12 mL, 134 mmol) in tetrahydrofuran (100 mL) and stir at RT under nitrogen. Add sodium hydroxide (0.8 g, 20 mmol) and stir overnight at RT under nitrogen. Dilute with brine, extract with ethyl acetate, separate the layers, wash the organic layer with brine, dry over sodium sulfate, filter, and concentrate to yield 27.5 g of crude solids. Purify the residue by flash column chromatography on silica eluting with a gradient of 20 to 50% ethyl acetate in hexanes to afford the title compound as a white solid (25.9 g, 99%). MS (m/z): 216.2 (M + H). Preparation 9 Methyl 3-[(2Z)-2-(2-ethoxy-2-oxo-ethylidene)-3,3-dimethyl-pyrrolidin-1-yl]propanoate

[0053] Add tetrakis(acetate)dirhodium(II) (3.74 g, 8.5 mmol) to a stirred solution of the intermediate provided by Preparation 8, methyl 3-(3,3-dimethyl-2-thioxopyrrolidin-1-yl)propanoate (41.4 g, 192 mmol), in toluene (156 mL) and heat to 110 °C under nitrogen. Add ethyl diazoacetate (89 mL, 844 mmol) dropwise over approximately 18 h, then heat overnight at 110 °C. Concentrate and purify the residue by flash column chromatography on silica eluting with 30% ethyl acetate in hexanes to give the title compound as a yellow oil (34.5 g, 67%). MS (m/z): 270.2 (M + H). Preparation 10 (2Z)-2-(3,3-dimethylpyrrolidin-2-ylidene)ethyl acetate

[0054] Add potassium hexamethyldisilazide (301 mL, 0.5 M in toluene, 151 mmol) dropwise over 45 min to a stirred solution of the intermediate provided by Preparation 9, methyl 3-[(2Z)-2-(2-ethoxy-2-oxo-ethylidene)-3,3-dimethyl-pyrrolidin-1-yl]propanoate (33.8 g, 126 mmol), in tetrahydrofuran (430 mL) under nitrogen using an ice/water bath to maintain the temperature <30°C. Stir for 40 min after addition is complete. Quench with saturated aqueous sodium bicarbonate solution (250 mL) then concentrate. Partition between diethyl ether and brine solution, extract the aqueous phase with ethyl acetate (x 4), dry over sodium sulfate, filter, and concentrate to obtain 37 g of material. Purify the residue by flash column chromatography on silica eluting with 10% ethyl acetate in hexanes to give the title compound (9.1 g, 40%). MS (m/z): 184.2 (M + H). Preparation 11 (2Z)-2-[3,3-dimethyl-1-(3-methyl-2-pyridyl)pyrrolidin-2-ylidene]ethyl acetate

[0055] Combine 2-Bromo-3-methylpyridine (1.5 g, 8.7 mmol), the intermediate provided by Preparation 10, (2Z)-2-(3,3-dimethylpyrrolidin-2-ylidene)ethyl acetate (1.6 g, 8.7 mmol), and sym-dimethylethylene diamine (0.77 mL, 8.7 mmol) in 1,4-dioxane (15 mL) in a reaction vessel. Bubble nitrogen through the solution with stirring for 10 minutes. Add potassium carbonate (3.6 g, 26 mmol) and copper(I) iodide (0.83 g, 4.4 mmol) all at once, seal, and heat the stirred mixture at 120 °C for 2 days. Dilute with dichloromethane, filter through diatomaceous earth, and concentrate the filtrate. Purify the residue by flash column chromatography on silica eluting with a gradient of 5 to 100% ethyl acetate in hexanes to obtain the title compound as a light yellow oil (1.58 g, 66%). MS (m/z): 275.0 (M + H). Preparation 12 8,8-Dimethyl-9,10-dihydro-5H-pyrido[3,2-f]pyrrolo[1,2-a]azepin-6(8H)-one

[0056] Add sodium bis(trimethylsilyl)amide (22 mL, 1 M in tetrahydrofuran, 22 mmol) via syringe to a stirred solution of the intermediate provided by Preparation 11, (2Z)-2-[3,3-dimethyl-1-(3-methyl-2-pyridyl)pyrrolidin-2-ylidene]ethyl acetate (2.88 g, 10.5 mmol), and tetrahydrofuran (60 mL) in a reaction vessel. Seal the vessel and heat at 70 °C with stirring for 3 days. Cool to RT, quench with brine, extract with ethyl acetate, dry over sodium sulfate, filter, and concentrate to obtain a brown oil. Purify the residue by flash column chromatography on silica eluting with a gradient of 50 to 100% ethyl acetate in hexanes to obtain the title compound as a yellow solid (1.70 g, 71%). MS (m/z): 229.2 (M + H). Preparation 13 6-Azido-10-aza-3,3-dimethyl-2,6-dihydro-1H-pyrrolo[1,2-a][1]benzazepin-5-one

[0057] Add lithium diisopropylamide (7.0 mL, 10.5 mmol, 1.5 M in hexanes) over 10 min to a stirred -78°C solution of the intermediate provided by Preparation 12, 8,8-dimethyl-9,10-dihydro-5H-pyrido[3,2-f]pyrrolo[1,2-a]azepin-6(8H)-one (1.85 g, 8.1 mmol), in tetrahydrofuran (50 mL) under nitrogen. After 30 min add acetic acid (2.3 mL, 41 mmol) via syringe then allow to warm to RT under nitrogen. Quench with saturated aqueous sodium bicarbonate solution. Partition the mixture between brine and ethyl acetate, separate the layers, wash the ethyl acetate layer with brine, dry over sodium sulfate, filter, and concentrate to obtain a brown solid. Purify the residue by flash column chromatography on silica eluting with a gradient of 5 to 60% ethyl acetate in hexanes to give the title compound (1.2 g, 55%). MS (m/z): 270.2 (M + H). Preparation 14 5-Amino-3,3-dimethyl-9,10-dihydro-5H-pyrido[3,2-f]pyrrolo[1,2-a]azepin-6(8H)-one

[0058] Add zinc dust (1.2 g, 18 mmol) followed by ammonium chloride (5 g, 66 mmol) to a stirred solution of the intermediate provided by Preparation 13, 6-azido-10-aza-3,3-dimethyl-2,6-dihydro-1H-pyrrolo[1,2-a][1]benzazepin-5-one (1.2 g, 4.5 mmol), in ethanol (50 mL) and water (15 mL) at RT. After 1 h dilute with ethyl acetate, discard the solids and concentrate the filtrate. Suspend the residue between ethyl acetate and brine, separate the layers, dry over sodium sulfate, filter and concentrate. Purify the residue by flash column chromatography on silica eluting with a gradient of 5 to 40% [10% 2M ammonia in methanol/dichloromethane] in dichloromethane to give the title compound as a yellow solid (0.72 g, 67%). MS (m/z): 244.2 (M + H). Preparation 15 N-[(1S)-2-[(10-aza-3,3-dimethyl-5-oxo-2,6-dihydro-1H-pyrrolo[1,2- a ][1]benzazepin-6-yl)amino]-1-methyl-2-oxo-ethyl] tert-butyl carbamate

[0059] Add (2S)-2-(tert-butoxycarbonylamino)propanoic acid (0.69 g, 3.6 mmol), 1-hydroxybenzotriazole hydrate (0.55 g, 3.6 mmol), and diisopropylethylamine (0.67 mL, 3.9 mmol) to a stirred 0 °C solution of the intermediate provided by Preparation 14, 5-amino-3,3-dimethyl-9,10-dihydro-5H-pyrido[3,2-f]pyrrolo[1,2-a]azepin-6(8H)-one (0.72 g, 3.0 mmol), in tetrahydrofuran (10 mL) under nitrogen. Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.68 g, 3.6 mmol) and allow the mixture to warm to RT under nitrogen. After 2 h, dilute with water and extract with ethyl acetate. Wash the organic layer with brine, dry over sodium sulfate, filter, and concentrate. Purify the residue by flash column chromatography on silica eluting with a gradient of 50 to 100% ethyl acetate in hexanes to give the title compound as a yellow solid (1.04 g, 84%). MS (m/z): 415.0 (M + H). Preparation 16 (2S)-2-Amino-N-(10-aza-3,3-dimethyl-5-oxo-2,6-dihydro- 1H -pyrrolo[1,2-a][1]benzazepin-6-yl)propanamide hydrochloride

[0060] Add hydrogen chloride (12.5 mL, 50 mmol, 4M in dioxane) to a stirred solution of the intermediate provided by Preparation 15 tert-butyl N-[(1S)-2-[(10-aza-3,3-dimethyl-5-oxo-2,6-dihydro-1H-pyrrolo[1,2-a][1]benzazepin-6-yl)amino]-1-methyl-2-oxo-ethyl] carbamate (1.04 g, 2.5 mmol) in 1,4-dioxane (40 mL) and heat to 45 °C with stirring. When LC/MS indicates complete reaction, concentrate to obtain the title compound as a yellow solid (0.93 g, crude). MS (m/z): 315.2 (M+1). Example 1 4,4,4-trifluoro-N-((2S)-1-((9-methoxy-3,3-dimethyl-5-oxo-2,3,5,6-tetrahydro-1H-benzo[f]pyrrolo[1,2-a]azepin-6-yl)amino)-1-oxopropan-2-yl)butanamide Part 1 Diastereomeric 4,4,4-Trifluoro-N-((2S)-1-((9-methoxy-3,3-dimethyl-5-oxo-2,3,5,6-tetrahydro-1H-benzo[f]pyrrolo[1,2-a]azepin-6-yl)amino)-1-oxopropan-2-yl)butanamide

[0061] Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (12.4 g, 65 mmol) to a stirred solution of the intermediate provided by Preparation 5, 6-Amino-9-methoxy-3,3-dimethyl-2,3-dihydro-1H-pyrrolo[1,2-a][1]benzazepin-5(6H)-one, and the intermediate provided by Preparation 7, (2S)-2-(4,4,4-trifluorobutanoylamino)propanoic acid (10.9 g, 51 mmol), in dichloromethane (294 mL) at RT. Cool the mixture to 5 °C, add 1-hydroxybenzotriazole (8.75 g, 65 mmol) and continue stirring at RT for 10 min. Wash the mixture with water (3 × 100 mL), dry over sodium sulfate, filter, and concentrate to afford a dark solid. Triturate with methyl tert-butyl ether to afford the title compound (mixture of diastereomers) as an off-white solid (22.0 g, 87%). MS (m/z): 468.1 (M + H). Part 2 4,4,4-trifluoro-N-((2S)-1-((9-methoxy-3,3-dimethyl-5-oxo-2,3,5,6-tetrahydro-1H-benzo[f]pyrrolo[1,2-a]azepin-6-yl)amino)-1-oxopropan-2-yl)butanamide Isomers 1 and 2

[0062] Separate a mixture of diastereomers from Example 1, Part 1 on a Chiralpak AD column eluting with 10% acetonitrile/ethanol (0.2% dimethylethylamine) to obtain compound Isomer 1 (Rt = 3.20 mins.) as a white solid (9.8 g, 38%) and compound Isomer 2 (RT = 7.37 mins.) as a white solid (9.0 g, 36%). MS (m/z): 468.2 (M + H) for both isomers. Example 2 N-((2S)-1-((8,8-dimethyl-6-oxo-6,8,9,10-tetrahydro-5H-pyrido[3,2-f]pyrrolo[1,2-a]azepin-5-yl)amino)-1-oxopropan-2-yl)-4,4,4-trifluorobutanamide Part 1 Diastereomeric N-{(1S)-2-[(8,8-Dimethyl-6-oxo-6,8,9,10-tetrahydro-5H-pyrido[3,2- f]pyrrolo[1,2-a]azepin-5-yl)amino]-1-methyl-2-oxoethyl}-4,4,4-trifluorobutanamide

[0063] Add 4,4,4-trifluorobutanoic acid (0.45 g, 3.2 mmol), 1-hydroxybenzotriazole hydrate (0.49 g, 3.2 mmol), and diisopropylethylamine (2.3 mL, 13.2 mmol) to a stirred 0°C solution of the intermediate provided by Preparation 16, (2S)-2-amino-N-(10-aza-3,3-dimethyl-5-oxo-2,6-dihydro-1H-pyrrolo[1,2-a][1]benzazepin-6-yl)propanamide hydrochloride (0.93 g, 2.6 mmol), in tetrahydrofuran (50 mL) under nitrogen. Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.61 g, 3.2 mmol) and allow the mixture to warm to RT under nitrogen for 16 h. Dilute with water and extract with ethyl acetate. Wash the organic layer with brine, dry over sodium sulfate, filter, and concentrate. Purify the residue by flash column chromatography on silica eluting with a gradient of 75 to 100% ethyl acetate in hexanes to give the title compound (mixture of diastereomers) as an off-white solid (0.82 g, 71%). MS (m/z): 439.2 (M + 1). Part 2 N-((2S)-1-((8,8-dimethyl-6-oxo-6,8,9,10-tetrahydro-5H-pyrido[3,2- f]pyrrolo[1,2-a]azepin-5-yl)amino)-1-oxopropan-2-yl)-4,4,4-trifluorobutanamide Isomers 1 and 2

[0064] Separate a mixture of diastereomers from Example 2, Part 1 on a Chiralpak AD-H column eluting with 15% MeOH/CO2(gas) to obtain Isomer 1 (Rt = 1.60 mins.) as a white solid (194 mg, 24%, epimerizes to 32% DE (diastereomeric excess)) and Isomer 2 (Rt = 2.31 mins.) as a white solid (469 mg, 57%). MS (m/z): 439.0 (M + H) for both isomers.

[0065] Cancer is increasingly recognized as a heterogeneous collection of diseases whose initiation and progression are induced by aberrant function of one or more genes that regulate DNA repair, genome stability, cell proliferation, cell death, adhesion, angiogenesis, invasion, and metastasis in cellular and tissue microenvironments. Variant or aberrant function of "cancer" genes can result from naturally occurring DNA polymorphism, changes in genome copy number (through amplification, deletion, chromosome loss, or duplication), changes in gene and chromosome structure (through chromosomal translocation, inversion, or other rearrangement leading to dysregulated gene expression), and point mutations. Cancerous neoplasms can be induced by an aberrant gene function and maintained by the same aberrant gene function, or maintenance and progression exacerbated by the functions of additional aberrant genes.

[0066] In addition to the genetic chromosomal aberrations mentioned above, each of the cancers may also include epigenetic modifications of the genome including DNA methylation, genomic imprinting and histone modification through acetylation, methylation or phosphorylation. An epigenetic modification may play a role in the induction and/or maintenance of malignancy.

[0067] Comprehensive catalogs of cytogenetic aberrations in human cancer have been compiled and are maintained and regularly updated online (see The Mitelman Database of Chromosome Aberrations in Cancer at the US National Cancer Institute (NCI) Cancer Genome Anatomy Project (CGAP). The database includes the chromosomal aberrations for at least some of the malignancies of the present invention. The Wellcome Trust Sanger Institute Cancer Genome Project maintains a detailed online "Cancer Gene Census" of all human genes that have been causally linked to tumorigenesis, as does the COSMIC (Catalog of Somatic Mutations in Cancer) database of somatic mutations in human cancer. Another source containing abundant information on cytogenetic changes causally related to various types of cancer is the Atlas of Genetics and Cytogenetics in Oncology and Haematology.

[0068] Diagnosis of cancerous malignancies by biopsy, immunophenotyping, and other tests are known and routinely used. In addition to high-resolution chromosome linkage and advanced chromosome imaging technologies, chromosomal aberrations in suspected cancer cases can be determined by cytogenetic analysis such as fluorescence in situ hybridization (FISH), karyotyping, spectral karyotyping (SKY), multiple FISH (M-FISH), comparative genomic hybridization (CGH), single nucleotide polymorphism arrays (SNP Chips), and other diagnostic and analytical tests known and used by those skilled in the art.

[0069] The oncogenic role of Notch was first reported in human T-cell leukemia involving a translocation of the Notch1 intracellular domain to the T-cell receptor-β promoter region, resulting in overexpression of the Notch1 intracellular domain (Grabher et al. Nature Review Cancer, 2006(6):347-359; Weng et al. Science, 2004(306):269-271). Overexpression of the Notch1 intracellular domain in mouse hematopoietic progenitor cells caused the mice to have T-cell acute lymphoblastic leukemia similar to that in humans. In addition to T-cell acute lymphoblastic leukemia, there is increasing evidence that Notch signals are oncogenic in other cancers through multiple mechanisms, including receptor amplification and overexpression of ligands and/or receptors including acute lymphoblastic leukemia, chronic lymphoblastic leukemia (Rosati et al, Blood, 2009(113): 856-865), acute myeloid leukemia (Sliwa et al. Int J Clin Exp Pathol, 2014(7(3)): 882-889), chronic myeloid leukemia (Nakahara et al. Blood, 2010(115(14)): 2872-2881), and erythroleukemia (Robert-Moreno et al, Leukemia, 2007(21): 1496-1503). Aberrant constitutive Notch signaling due to mutation or during expression of ligands and/or receptors is also implicated in several solid tumor malignancies, including triple-negative breast cancer (Stoeck et al. Cancer Discovery, 2014(4): 1154–1167), breast cancer, ovarian cancer (Park et al. Cancer Research, 2006(66):6312–6318), melanoma (Gast et al. Genes, Chromosomes & Cancer, 2010(49):733–745), lung cancer, non-small cell lung cancer (Westhoff et al. PNAS, 2009(106):22293– 22298), pancreatic cancer, glioblastoma, colorectal cancer, head and neck cancer, cervical cancer, prostate cancer, liver cancer, squamous cell carcinoma (oral), skin cancer, and medulloblastoma (Rangathan et al., Nature Review Cancer, 2011(11):338–351 and supplementary information S1 (table)). Aberrant constitutive Notch signaling due to mutation or during expression of ligands and/or receptors is also involved in angiosarcoma (Ravi et al, J Clin Oncol, 2007, (25(18S, June 20 Supplement)): Abstract 10030), rhabdomyosarcoma (Belyea et al, Clin Cancer Res, 2011(17(23)): 7324-7336; Roma et al, Clin Cancer Res, 2011(17(3)): 505-513), liposarcoma (J Clin Oncol, 2009, (27(15S, Supplement)): Abstract 10526), malignant fibrous histiocytoma (Wang et al, Cancer Res, 2012, (72): 1013-1022), hepatocellular carcinoma (Villanueva et al, Gastroenterology, 2012, (143): 1660-1669), intrahepatic and extrahepatic cholangiocarcinoma (Wu et al, Int J Exp Pathol, 2014, (7(6)): 3272-3279; Sekiya et al, J Clin Invest, 2012, (122(11)): 3914-3918; Yoon et al, World J stroenterol, 2011, (17(35)): 4023-4030), and adenoid cystic carcinoma (Bell et al, Annals of Diagnostic Pathology, 2014, (18): 10-13; Stoeck et al, Cancer Discov, 2014, (4): 1154-1167). Inhibition of Notch signaling presents an attractive target to provide therapeutic benefits to cancer patients whose disease was induced by aberrant activation of the constitutive Notch signaling pathway. Shih et al. Cancer Research, 2007(67)1879-1882.

[0070] One of the definitive genes for inner ear hair cell development is the mammalian homologue of the basic helix-loop-helix transcription factor atonal-1 (Atohl). Expression of Atohl in cochlear cells is required for the genesis of auditory hair cells. Prosensory epithelial cells within the developing organ of Corti that express Atohl will differentiate into auditory hair cells (Helms et al, Development 2000, (127(6)): 1185-1196), and Atohl is one of the early markers of auditory hair cell differentiation. The supporting cells of the organ of Corti retain the potential to develop hair cell characteristics, including cilia formation (Zheng et al, Nature Neuroscience, 2000,(3(6)): 580-586; Kawamoto et al, J Neurosci, 2003, (23(11)): 4395-4400; Izumikawa et al, Nat Med, 2005, (11(3)):271-276; and proper hair cell function (Kawamoto et al, 2003).

[0071] Each hair cell in the cochlea is surrounded by non-sensory supporting cells that provide trophic and structural support for the hair cells and ganglia and are essential in maintaining appropriate ionic concentrations in the organ of Corti through gap junction intercellular communication. There are two types of hair cells: inner and outer hair cells. Cochlear hair cells in mammals, including humans, consist of one row of inner hair cells and three rows of outer hair cells. The inner hair cells are the actual sensory receptors, and 95% of the auditory nerve fibers that project to the brain arise from this subpopulation. The endings on the outer hair cells are almost all from efferent axons arising from cells in the brain, and these cells function as acoustic preamplifiers. Supporting cells play a key role in the generation of auditory hair cells after loss or damage of auditory hair cells. During development, hair and supporting cells develop from a common progenitor and the appearance of hair cells signals surrounding cells to develop into supporting cells through contact inhibition mediated by the Notch signaling pathway (Kelley, Nat Rev Neurosci, 2006, (11): 837-849.

[0072] Based on the ability of supporting cells to transdifferentiate into auditory hair cells, together with their shared developmental pathway, it has been postulated that supporting cells may function as hair cell progenitors (Parker et al, Audiology and Neurootology, 2004, (9(2)): 72-80). Several studies have demonstrated that bypassing cell cycle inhibition in supporting cells can result in the generation of auditory hair cells in mammals (Lowenheim et al, Proc Natl Acad Sci USA, 1999, (96): 4084-4088; Torchinsky et al, J Neurocytol, 1999, (28(10-11)): 913-924; Minoda et al, Hear Res, 2007, (232): 44-51). Therefore, adult mammalian supporting cells retain the ability to transdifferentiate into auditory hair cells once they are free to enter the cell cycle.

[0073] Drug therapy for the restoration of auditory hair cells is a novel approach and intratympanic administration into the middle ear fluid, preferably to the vestibular window niche, without actual injection into the cochlea can effectively induce Atoh1 expression at a therapeutic dose and for an adequate period of time to elicit transdifferentiation of auditory hair cells from supporting cells in the cochlea. Mizutari et al, Neuron, 2013, (77(1)): 58-69 showed that middle ear delivery of a gamma-secretase inhibitor (LY411575) can be used to regenerate auditory hair cells lost to acoustic trauma in mice and that this generation of new hair cells from supporting cells resulted in significant, measurable, auditory recovery.

[0074] This work provided conceptual evidence for a therapeutic effect of Notch signaling pathway inhibition on auditory hair cell generation and hearing restoration in a mouse model and provided a mechanistic explanation (differentiation of supporting cells to auditory hair cells) for the physiological effect. Dose-limiting toxicity was shown during systemic administration of LY411575.

[0075] The following in vitro and in vivo studies demonstrate the Notch signaling pathway inhibitory activity and efficacy of Compounds 1 and 2, or a substantially pure diastereomer thereof, against a specific cancer cell line. These assays are generally recognized by those skilled in the art as indicative of human clinical chemotherapeutic activity. Inhibition of the cleavage of the Notch intracellular domain by Y-secretase is expected to be effective against each of the receptors Notch 1, Notch 2, Notch 3, and Notch 4. Assays demonstrating Notch signaling pathway inhibitory activity and efficacy can be performed substantially as follows or by similar assays providing similar data.

Cellular Imaging Assay of N1ICD Notch1 Nuclear Accumulation

[0076] HEK293ΔE12 cells (HEK293 cells are engineered to stably express mouse Notch1 cDNA encoding amino acid 1703-2183, NP_032740.3, (Full-length mouse Notch 1 protein precursor: SEQ ID NO: 1) with the 23 amino acid signal peptide sequence, MPRLLTPLLCLTLLPALAARGLR (SEQ ID NO: 2), at its N-terminus) are plated at 5000 cells/well in 96-well plates, incubated in Dulbecco's modified Eagle's medium (DMEM)-high glucose with 5% fetal bovine serum (FBS) at 37°C, 5% CO2 for 24 hours. Cells are treated with test compound by dosing 10 point dilutions of 1:3 across the range of 1000 nM to 0.05 nM and with a final concentration of dimethyl sulfoxide (DMSO) at 0.2%. After 24 h of treatment, cell plates are processed through the following steps sequentially: fix cells with 100 μL/well PREFER™ fixative for 30 min at room temperature (RT); permeabilize cells with 100 μL/well 0.1% TRITON® X100 in phosphate-buffered saline (PBS) for 20 min at RT; wash 3 times with 100 μL/well PBS each; add 50 μl/well of rabbit anti-N1ICD antibody (Notch1 Intracellular Domain), at 1:2000 in PBS with 1% bovine serum albumin and incubate for 1.5 hours at 37°C; wash 3 times with 100 μl/well of PBS each; incubate with 50 μl/well of goat anti-rabbit IgG (Immunoglobulin G) Alexa 488 at a dilution of 1:1000 in PBS with 1% bovine serum albumin and incubate for 1 hour at 37°C; wash 3 times with 100 μl/well of PBS each and add 100 μl/well of 15 μM propidium iodide with 50 μg/mL of RNAse for 30 minutes to stain the nuclei. Plates are imaged with the ACUMEN EXPLORER™ laser scanning fluorescence microplate cytometer (TTP LABTECH LTD) to measure total cell nuclear count/well and total nuclear area/well with fluorescence at 655 nm to 705 nm (DNA-bound propidium iodide emission) and antibody binding fluorescence to N1ICD in the nuclear region at 505 nm to 530 nm. The primary assay output is a ratio of total nuclear N1ICD fluorescence to total nuclear area, the normalized nuclear N1ICD signal. A relative cytotoxicity profile was collected as % cell number to 0.2% DMSO control cells. Antibody recognizing Notch1 or cleaved N1ICD is raised to a human peptide corresponding to the amino terminal cleavage site of human Notch1 at Val1744. In untreated control cells, N1ICD generated from Notch1 will translocate and accumulate in the nucleus. When cells are treated with a Notch cleavage inhibitor compound, the nuclear N1ICD signal will decrease. The concentration response and IC50 are determined by curve fitting to a four-parameter logistic for the nuclear N1ICD signal, while the % cell number is plotted on the same graph for cytotoxicity profiling. Performing the assay essentially as described above, the mean IC50 for Compound 1, Isomer 2 is 0.37 nM (+/- 0.16; n = 2) and Compound 2, Isomer 2 is 1.55 nM (+/- 1.12; n = 2). Neither compound affects cell number up to a concentration of 1000 nM. These data demonstrate that Compound 1, Isomer 2 and Compound 2, Isomer 2 each have affinity for Notch 1 and inhibit the intracellular accumulation of the Notch 1 intracellular domain cell signaling peptide.

In vivo target inhibition studies Animal studies

[0077] To evaluate the in vivo effect of Compound 1, Isomer 2, and Compound 2, Isomer 2 on the inhibition of Notch processing (PD) pharmacodynamics, animal studies were conducted in tumor-bearing Balb/C mice (Charles River). A total of 5 mice are used for each group. Mice are fed ad libitum regular chow. Treatment is initiated with oral administration (gavage) of compound or vehicle (1% Na-CMC in 0.25% Tween-80) in 0.2 mL volume. At designated time points (4 or 8 hours post-dose) following treatment, animals are sacrificed by CO2 asphyxiation and cervical dislocation. Tissues (lungs) are removed and used for analysis of PD response as measured by cleaved N1ICD.

N1ICD Analysis

[0078] To assess N1ICD levels in the lung, approximately 75 mg is cut from the frozen tissue and minced prior to homogenization (actual mass recorded). Frozen tumor samples are transferred to Lysing Matrix-D™ tubes and resuspended in ice-cold XY lysis buffer (25 mM Tris pH 7.5, 10 μg/mL trypsin/chymotrypsin inhibitor, 10 μg/mL Aprotinin, 60 mM glycerol beta-phosphate, 1% Triton® X100, 10 mM NaF, 2.5 mM pyrophosphate, 150 mM NaCl, 15 mM ethylene diamine tetraacetic acid (EDTA) pH 8.0, 5 mM ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) pH 8.0, 1 mM Na vanadate, 10 μg/mL Leupeptin, 1 mM dithiothreitol, 1 μM microcystin LR, 10 μg/mL Np-tosyl-L-phenylalanine chloromethyl ketone (TPCK), 2 mM Nα-p-tosyl-L-arginine methyl ester hydrochloride (TAME), 15 mM 4-nitrophenyl di(tris)phosphate salt (PNPP), 0.1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), 5 mM Benzamidine, 1 μM okadaic acid containing 1X Complete tablet (Roche Complete™ No. 11697 498 001) and 1X Protease Inhibitor Cocktail (Sigma-Aldrich P8340) in a mass:volume ratio of 75 mg/mL buffer. Tissues are homogenized in a Fast Prep FP120 homogenizer (Thermo Scientific, Rockford, IL) at a speed of 6.0 for 30 seconds at 4°C, followed by 15 minutes of incubation on ice. This is repeated for a total of 2 to 3 cycles until homogenization is complete. Lysates are centrifuged in an eppendorf centrifuge at 4°C at 30,000 rpm for 15 minutes to remove debris. 400 μL of supernatant is removed and transferred to a new eppendorf tube and subjected to a freeze/thaw cycle. Samples are centrifuged again in an eppendorf centrifuge at 4°C at 30,000 rpm for 30 minutes, and 120 μL of supernatant is collected for analysis. Total protein concentration is determined using Pierce BCA Protein Assay Kit™ (Thermo Scientific, Rockford, IL) using a Thermomax™ plate reader (Molecular Devices, Sunnyvale, CA). N1ICD levels are determined using a custom N1ICD ELISA. The analyte is captured with a custom Notch1-specific rabbit monoclonal antibody (Val1744) cleaved and detected with a C-terminal Notch1 SULFO-TAG™ sheep polyclonal antibody (Meso Scale Discovery, Gaithersburg, Maryland) (R&D Systems, Minneapolis, MN). Lysates are diluted to 2 μg/μL in ice-cold ELISA Tris lysis buffer (R6OTX) (Meso Scale Discovery, Gaithersburg, Maryland) containing 1X Complete tablet (Roche Complete™ mini No. 11 836 153 001) and 1X Protease Inhibitor cocktail (Sigma-Aldrich P8340) and 25 μL is added to the ELISA plate. Incubation of 50 μg of protein lysate is performed at RT for one hour each to capture the analyte and with detection antibody. Plates are read on a Sector Imager 6000™ (Meso Scale Discovery, Gaithersburg, Maryland). Background-subtracted N1ICD is normalized to total protein and presented as % inhibition relative to the vehicle-treated group. N1ICD % inhibition and statistical significance (p-value) as measured by the Dunett method in tumors harvested 4 hours after the last dose for Compound 1, Isomer 2 or Compound 2, Isomer 2 is analyzed essentially as described above and summarized in Table 1. Table 1.

[0079] The data in Table 1 demonstrate inhibition of N1ICD cleavage by Compound 1, Isomer 2 and Compound 2, Isomer 2 in mouse lung tissue. The data in Table 1 further provide an in vivo correlation with the functional activity data described above.

Induction of mAtoh1 Expression in Auricular Spheres

[0080] Generally, organ of Corti cell isolation is performed on day 0, largely by the method of Oshima et al., Methods Mol Biol., 2009; 493: 141-162. Cell culture and propagation are performed for 3 to 4 days. Cell plating and test compound treatment occur on day 3 or 4. Attachment and differentiation can occur over the next 7 days. On day 10 or 11, cell lysis and RNA isolation are performed. TaqMan qRT-PCR is performed on day 12 and results analyzed on days 13 to 15 for Atoh1 and Tbp1 expression.

Cell isolation

[0081] The surrounding tissue of the pars dura of the temporal bone is removed from postpartum mice at 1 to 4 days (transgenic strain: mAtoh1 driven nGFP, Lumpkin EA, et al., Gene Expr Patterns, 2003; (36): 389-95) of both sexes. The cup-shaped cochlea is separated from the vestibular system using forceps. At this stage of development, the bony labyrinth is not completely calcified and is easily dissected using forceps. Carefully open the bony labyrinth of the cochlea and remove the spiral ligament and attached organ of Corti coiled together along the spiral of the aster by apically releasing the aster. Starting at the base, separate the spiral ligament from the organ of Corti using fine forceps.

[0082] Transfer the dissected organ of Corti (OC) into individual 1.5 mL tubes filled with 850 μL of ice-cold HBSS. Transfer up to 12 OC tissues into the same tube. Quickly spin the OC tissues and remove the HBSS. Dissociate the cells by adding 100 μL of pre-warmed TrypLE™ Select (Life Technologies) and incubating at 37°C for 13 min. Quickly spin to disaggregate the OC tissues. Remove the TrypLE™ Select. Add 100 μL of Medium A (DMEM/F12, N2 supplement (LifeTechnologies), B27 supplement (LifeTechnologies), ampicillin (50 μg/mL) and Fungizone (LifeTechnologies), EGF (20 ng/mL), bFGF (10 ng/mL) IGF-1 50 ng/mL) and heparan sulfate (50 ng/mL)). The dissected and digested tissue is then triturated with a P200 pipette tip. Remove all cell aggregates and debris; transfer the cell suspension and wash through a pre-wetted 70 μm cell strainer to pool the cell suspension in a single new culture tube. Rinse the dissociation tubes and cell strainer with sufficient Medium A and pool with the cell suspension. Calculate the cell density with a hemocytometer or Countess® (Life Technologies). Add fresh Medium A to the cell suspension to obtain a final plating density of 1.0E5 cells/mL and plate onto ultra-low attachment culture ware, T-75 (Greiner).

Suspension Culture to Propagate OC Spheres

[0083] Dissociated isolated cells are cultured in an ultra-low attachment culture article (Greiner) in Medium A for 3 to 4 days in a humidified 5% CO2 incubator at 37°C to obtain clonally cultured spheres, called first generation spheres, from floating cells. If cells adhere to the non-adherent plates during the culture period, they can be dislodged by gentle mixing.

Culture and Treatment of Connections

[0084] After culturing the spheres for 3 to 4 days, visually count the spheres under a microscope to standardize the seeding density of ear spheres per mL for this step. Harvest the cells by centrifugation and resuspend with basic medium at a concentration of approximately 2000 spheres per mL. Seed the spheres in a volume of 150 μL per well of a cell culture-treated 96-well plate to obtain approximately 300 spheres per well. Treat the spheres with experimental agents in quadruplicate in a final volume of 200 μL in basic medium (DMEM/F12, N2 supplement (LifeTechnologies), B27 supplement (LifeTechnologies), ampicillin (50 μg/mL), and Fungizone (LifeTechnologies)). Cultivate the treated spheres for 7 days in a temperature and humidity controlled incubator in 5% CO2, 37°C. Remove the medium and proceed with RNA extraction.

RNA extraction

[0085] Add Buffer RLT plus (Qiagen) supplemented with 1% Beta-mercaptoethanol, 175 μl per well. Add 2.5 μl of 4 ng/μl RNA vehicle (RNeasy® Plus Micro Kit, Qiagen) diluted in Buffer RLT Plus to each well prior to RNA extraction. Total RNA is extracted using the RNeasy® Plus Micro Kit (Qiagen) according to the manufacturer's instructions.

cDNA synthesis

[0086] cDNA synthesis is performed using the SuperScript® III First-Line Synthesis System (LifeTechnologies, catalog 18080-051) following the manufacturer's protocol using random hexamers for cDNA synthesis. Dilute the cDNA 50% by adding 21 μL of ultra-pure nuclease-free water. 5 μL of diluted cDNA is used for qPCR.

qPCR

[0087] To quantify the expression of hair cell markers from treated sphere cultures, qPCR with probes to detect a gene of interest mAtoh1 and an endogenous control gene is performed in a single two-color multiplex reaction. TaqMan Gene Expression Master Mix (LifeTechnologies, 4369016) and a probe of interest (Atoh1; Mm00476035_s1 and an endogenous control probe (Tbp1; Mm00446971_m1, LifeTechnologies) are used according to the manufacturer's instructions. Conditions are kept constant for each probe. Relative gene expression between treatment groups is analyzed using the ΔΔCT method and replicate measurements are averaged. Experiments are performed in triplicate, at a minimum, and are reported as a grand mean. Table 2

[0088] The data in Table 2 show relative quantification (RQ) values of Atoh1 expression, a marker of hair cell generation, in ear beads treated with each of Compound 1, Isomer 2, and Compound 2, Isomer 2 at three concentrations (10 nM, 100 nM, and 1 μM). Data (± standard error of the mean, SEM) represent fold induction of gene expression relative to samples treated with negative control (DMSO vehicle). (*, significant difference from negative control, p < 0.05).

Induction of hAtoh1 Expression in Human Tumor Cell Lines

[0089] To evaluate the potency of Compounds 1, Isomer 2 and 2, Isomer 2 in their ability to induce hAtoh1 expression, a human tumor cell line is utilized. The regulation of endogenous Notch signaling for this cell line is similar to that of the inner ear. Generally, this assay is performed following the principles in Kazanjian et al., Gastroenterology, 2010, 139(3): 918-928 and Supplementary Materials and Methods.

[0090] Generally, standard cell culture techniques are used. The low passage human colorectal adenocarcinoma cell line LS174T (ATCC CL-188) maintained in growth medium (MEM 10% FBS) is plated 10,000 cells in 100 μL of assay medium (MEM 1% FBS) per well in 96-well tissue culture treated plates.

[0091] The following day, half-log serial dilutions of 10 mM stock test compounds (diluted in 100% DMSO) were performed to obtain eleven decreasing doses of drug compound. Final concentrations should range from 10 μM to 0.127 nM. A 3.16-fold serial dilution is used, with DMSO concentrations in each well of 100%. A further 1:10 dilution of the initial DMSO dilution is performed in Assay Medium. Another 1:100 dilution again in Assay Medium is performed.

[0092] 100 μL of the test compound dilutions are added to each well containing cells and growth medium and cultured for 72 hours at 37°C in 5% CO2.

[0093] Cells are harvested from plates on day 4, and RNA extracted and purified using RNeasy® Plus 96 RNA (Qiagen) following the manufacturer's protocol.

[0094] cDNA synthesis is performed using the SuperScript® III First-Strand Synthesis System (LifeTechnologies, 18080-051) following the manufacturer's protocol using random hexamers for cDNA synthesis. The cDNA is diluted 50% by adding 21 μl of ultra-pure nuclease-free water. 5 μl of the diluted cDNA is used for qPCR.

qPCR

[0095] To quantify the expression of hair cell markers from the treated sphere cultures, qPCR probes to detect the expression of hAtoh1 or TBP in a single two-color multiplex reaction are performed. To measure relative gene expression for each sample, TaqMan Gene Expression Master Mix (LifeTechnologies, 4369016) and a probe of interest (human Atoh1; LifeTechnologies Assay Id Hs00944192_s1) plus an endogenous control (human TBP, LifeTechnologies Assay Id Hs00427620_m1) are used according to the manufacturer's instructions. Conditions are kept constant for each probe. Relative gene expression between treatment groups is analyzed using the ΔΔCT method and replicate measurements are averaged.

[0096] Four separate assays are performed to obtain IC50 values for each test compound. The IC50 value is determined by fitting concentration-response data to the “log(antagonist) vs. response model (three parameters)” using GraphPad Prism® software for 12 doses over a concentration range of 4.0E-11 to 1.0E-6 M with 108 total points analyzed for each compound. The IC50 value for Compounds 1, Isomer 2 and 2, Isomer 2 is shown in Table 3. Table 3

[0097] The data in Table 3 show the IC50 values of Compound 1, Isomer 2 and Compound 2, Isomer 2 as calculated by the relative expression of hAtoh1, a marker of auditory hair cell generation in this assay.

Response to Compounds in an Organ of Corti Explant Assay

[0098] Generally, organotypic explant culture of organ of Corti is performed on Day 0 according to the method described in Parker et al., Journal of Visualized Experiments, 2010, (36), e1685. Explant culture is performed for 4 to 7 days at which point the culture is prepared with respect to cell lysis for qRT-PCR, or tissue fixation for immunohistochemistry.

Isolation and Culture of Organs of Corti Explants

[0099] The bulla and surrounding tissue of the pars dura of the temporal bone are removed from 1- to 4-day postpartum mice (transgenic strain: nGFP driven by Atoh1, Lumpkin EA, et al., Gene Expr Patterns, 2003, (36): 389-95) of both sexes. The organs of Corti are dissected in Hanks' solution, the spiral ligament is removed, and the explants are placed on cover slips coated with poly-L-ornithine (0.01%, Sigma) and laminin (50 mg/mL, Becton Dickinson) to obtain a preparation with a flat cochlear surface. Each cochlear explant is then placed in a single well of a 24-well plate (Falcon) and cultured in DMEM (Invitrogen) with 5% fetal bovine serum, 5% horse serum, and penicillin-streptomycin (LifeTechnologies). Experimental compounds are added with medium on day 0 and replaced every 2 to 3 days. All cultures are maintained in a humidified 5% CO2 incubator at 37°C.

Gene Expression Analysis by qRT-PCR

[00100] RNA extraction is performed by lysing explant tissue with 350 μl RLT Plus buffer (Qiagen) per well, and total RNA is purified by RNeasy® Plus Mini Kit (Qiagen). cDNA synthesis is performed with the SuperScript® III First-Strand Synthesis System (LifeTechnologies) using random hexamers. To quantify the induction of hair cell marker expression, qPCR is performed with TaqMan Gene Expression Master Mix (LifeTechnologies, 4369016) and probes to detect the genes of interest (e.g., hAtoh1; Assay ID Mm00476035_s1, Pou4f3; Assay ID Mm04213795_s1, and Myo7a; Assay ID Mm01274015_m1, LifeTechnologies) and an endogenous control gene, Tbp (Assay ID Mm00446971_m1, LifeTechnologies). Relative gene expression between treatment groups is analyzed using the ΔΔCT method, and replicate measurements are averaged. Experiments are performed in triplicate, at a minimum, and reported as a grand mean. Table 4

[00101] The data in Table 4 show the relative quantification (RQ) values of expression of hair cell generation markers; Atoh1, Pou4f3, and Myo7a, in organ of Corti explants treated with Compound 1, Isomer 2 and Compound 2, Isomer 2 at 1 μM. Data (± SEM) represent fold induction in gene expression relative to negative control treated samples.

Immunostaining of New Atoh1-Expressing Hair Cells

[00102] After explant culture, tissues are fixed in the 24-well plate with 200 μL of Cytofix/Cytoperm (BD) for 30 min, rinsed with PBS containing TritonX100 (PBST), and stained with 200 μL of primary antibody solution consisting of 4% normal donkey serum in PBST with goat anti-GFP (1:2500; AbCAM, ab5450) and rabbit anti-myosin VIIa (1:1000; Proteus Bioscience #25-6790) for 1 hour. Tissues are then thoroughly washed with PBST and stained with a secondary antibody solution consisting of Alexa Fluor 488 donkey anti-goat IgG (1:1000; Invitrogen, A-11055) and Alexa Fluor 568 donkey anti-rabbit IgG (1:1000 Invitrogen, A10042) for 1 hour. They are then thoroughly washed with PBST and mounted on microscope slides. Individual hair cells are identified by expression of the Atoh1 surrogate marker GFP (green) and myosin VIIa (red), imaged, and then counted within a 100 μM span of the apex to examine nascent hair cell formation in compound-treated experimental samples in contrast to hair cell numbers in untreated or negative control (biologically inactive compound)-treated samples. Blind cell counts are measured in five separate experiments. Table 5

[00103] The data in Table 5 show the numbers of outer hair cells in organ of Corti explants cultured for four days in the presence of Compound 1, Isomer 2 (n = 13) or Compound 2, Isomer 2 (n = 15) at 1 μM and immunoblotted for GFP (a surrogate Atoh1 marker) and myosin VIIa. The percent increase in hair cells is relative to negative control-treated samples (n = 18).

Analysis of Gene Expression after Ototoxic Damage

[00104] A variation of the organ of Corti organotypic explant culture is used to model hair cell regeneration in response to subsequent compound treatment following ototoxic injury. This injury model is performed according to the method described in Korrapati et al., PLOSOne, 2013, 8(8), e73276 where a 95% reduction in inter-apical hair cells was reported. On day 0, to damage and selectively neutralize hair cells within the organ of Corti explant, treatment with aminoglycosides (gentamicin (100 μM) is added for 18 to 24 hours. On day 1, gentamicin is removed and treatment with Compound 1, Isomer 2 and Compound 2, Isomer 2 at 5 μM is initiated. Explant culture is continued for 4 days total at which point the culture is prepared for the qRT-PCR protocol described above. Visible confirmation of gentamicin-induced damage is readily observed among cultures treated with negative control compounds. However, quantitative measurement for the response to Compound 1, Isomer 2 and Compound 2, Isomer 2 treatment following ototoxic damage obtained by qRT-PCR measured increases in gene expression for the prosensory hair cell markers Atoh1 and Pou4f3 and the hair cell marker Myo7A. Table 6

[00105] Data in Table 6 show relative increases in gene expression by qRT-PCR of hair cell generation markers Atoh1, Pou4f3, and Myo7a in organ of Corti explants treated with Compound 1, Isomer 2 and Compound 2, Isomer 2 at 5 μM following gentamicin injury (100 μM). Data represent percent increase in gene expression relative to negative control samples treated with gentamicin-induced hair cell injury. (*, significant difference from negative control, p < 0.05)

Induction of hair cell marker in whole ear capsule culture

[00106] The ear capsule (ear capsule explant), comprised of the cochlear and vestibular bony labyrinth systems, is dissected from 1- to 4-day-old postnatal mice (transgenic strain: nGFP driven by Atoh 1, Lumpkin EA, et al., Gene Expr. Patterns, 2003, 3(4): 389-95) of both sexes generally according to the method described in Parker et al., Journal of Visualized Experiments, 2010, (36), e1685. The entire ear capsule is isolated from the parietal bone and maintained ex vivo in a roller tube culture system for up to 3 days in medium containing DMEM, high glucose, 5% FBS, 5% horse serum, 1 μg/mL ampicillin.

[00107] The ear capsule explant is used to evaluate test compound, in various delivery vehicles, for inner ear penetrance. Tests are performed with compositions of Compound 1, Isomer 2, (7.2 mM) in various delivery vehicles. Small volumes (100 to 200 nL) are directly applied to the vestibular window niche of the cochlea. The cochlea is dissected, treated, incubated for 1 to 2 hours, and then introduced into roll culture. After 48 h, the ear capsule explant is opened to remove the organ of Corti from the bony labyrinth of the cochlea and analyzed by RT-PCR.

[00108] The composition is allowed to remain for 1 to 2 hours, then removed with the culture medium. The ear capsule explant is then cultured for 48 hours in untreated medium. The cochlea is opened and the entire organ of Corti is then dissected and prepared for quantitative RT-PCR to measure expression levels of Atoh1 hair cell markers (as described in the organ of Corti explant assay, above). Table 7 shows the induction of Atoh1 in the specified delivery vehicle. The upregulation of Atoh1 by Compound 1, Isomer 2, in all compositions tested (2- to 3-fold, n from 12 to 17) is measured in comparison to vehicle control treated ear capsule explants after 48 hours in culture. Table 7: Atoh1 Gene Expression (RT-PCR)

Absorption of test compound into perilymph in vivo in the inner ear

[00109] Female albino Hartley guinea pigs are anesthetized with a ketamine/xylazine mixture before 70 μL of Compound 1, Isomer 2, in compositions as described above for the whole ear capsule culture assay is injected transtympanic to completely fill the middle ear. Perilymph (inner ear fluid) samples are extracted at 0.5, 1, 6, 24, and 48 hours post-injection and analyzed by LCMS (n of 5) for test compound concentration. Table 8 shows that the test compound is absorbed into the inner ear following transtympanic administration into the middle ear with these delivery vehicles. Table 8: IN VIVO Perilymph Levels Amino Acid Sequence SEQ ID NO: 1 (Mouse Notch protein 1 precursor; full length) MPRLLTPLLCLTLLPALAARGLRCSQPSGTCLNGGRCEVANGTEACV CSGAFVGQRCQDSNPCLSTPCKNAGTCHVVDHGGTVDYACSCPLG FSGPLCLTPLDNACLANPCRNGGTCDLLTLTEYKCRCPPGWSGKSC QQADPCASNPCANGGQCLPFESSYICRCPPGFHGPTCRQDVNECS QNPGLCRHGGTCHNEIGSYRCACRATHTGPHCELPYVPCSPSPCQ NGGTCRPTGDTTHECACLPGFAGQNCEENVDDCPGNNCKNGGACV DGVNTYNCRCPPEWTGQYCTEDVDECQLMPNACQNGGTCHNTHG GYNCVCVNGWTGEDCSENIDDCASAACFQGATCHDRVASFYCECP HGRTGLLCHLNDACISNPCNEGSNCDTNPVNGKAICTCPSGYTGPA CSQDVDECALGANPCEHAGKCLNTLGSFECQCLQGYTGPRCEIDVN ECISNPCQNDATCLDQIGEFQCICMPGYEGVYCEINTDECASSPCLH NGHCMDKINEFQCQCPKGFNGHLCQYDVDECASTPCKNGAKCLDG PNTYTCVCTEGYTGTHCEVD IDECDPDPCHYGSCKDGVATFTCLCQ PGYTGHHCETNINECHSQPCRHGGTCQDRDNSYLCLCLKGTTGPNC EINLDDCASNPCDSGTCLDKIDGYECACEPGYTGSMCNVNIDECAGS PCHNGGTCEDGIAGFTCRCPEGYHDPTCLSEVNECNSNPCIHGACR DGLNGYKCDCAPGWSGTNCDINNNECESNPCVNGGTCKDMTSGYV CTCREGFSGPNCQTNINECASNPCLNQGTCIDDVAGYKCNCPLPYT GATCEVVLAPCATSPCKNSGVCKESEDYESFSCVCPTGWQGQTCE VDINECVKSPCRHGASCQNTNGSYRCLCQAGYTGRNCESDIDDCRP NPCHNGGSCTDGINTAFCDCLPGFQGAFCEEDINECASNPCQNGAN CTDCVDSYTCTCPVGFNGIHCENNTPDCTESSCFNG GTCVDGINSFT CLCPPGFTGSYCQYDVNECDSRPCLHGGTCQDSYGTYKCTCPQGY TGLNCQNLVRWCDSAPCKNGGRCWQTNTQYHCECRSGWTGVNCD VLSVSCEVAAQKRGIDVTLLCQHGGLCVDEGDKHYCHCQAGYTGSY CEDEVDECSPNPCQNGATCTDYLGGFSCKCVAGYHGSNCSEEINEC LSQPCQNGGTCIDLTNSYKCSCPRGTQGVHCEINVDDCHPPLDPAS RSPKCFNNGTCVDQVGGYTCTCPPGFVGERCEGDVNECLSNPCDP RGTQNCVQRVNDFHCECRAGHTGRRCESVINGCRGKPCKNGGVCA VASNTARGFICRCPAGFEGATCENDARTCGS LRCLNGGTCISGPRSP TCLCLGSFTGPECQFPASSPCVGSNPCYNQGTCEPTSENPFYRCLC PAKFNGLLCHILDYSFTGGAGRDIPPPQIEEACELPECQVDAGNKVC NLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDS QCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSA ECEWDGLDCAEHVPERLAAGTLVLVVLLPPDQLRNNSFHFLRELSHV LHTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKRSTVGWATSSLLPG TSGGRQRRELDPMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAFL GALASLGSLNIPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVG CGVLLSRKRRRQHG QLWFPEGFKVSEASKKKRREPLGEDSVGLKPL KNASDGALMDDNQNEWGDEDLETKKFRFEEPVVLPDLSDQTDHRQ WTQQHLDAADLRMSAMAPTPPQGEVDADCMDVNVRGPDGFTPLMI ASCSGGGLETGNSEEEEDAPAVISDFIYQGASLHNQTDRTGETALHL AARYSRSDAAKRLLEASADANIQDNMGRTPLHAAVSADAQGVFQILL RNRATDLDARMHDGTTPLILAARLAVEGMLEDLINSHADVNAVDDLG ARKPSTKGLAC GSKEAKDLKARRKKSQDGKGCLLDSSSMLSPVDSLESPHGYLSDVA SPPLLPSPFQQSPSMPLSHLPGMPDTHLGISHLNVAAKPEMAALAGG SRLAFEPPPPRLSHLPVASSASTVLSTNGTGAMNFTVGAPASLNGQC EWLPRLQNGMVPSQYNPLRPGVTPGTLSTQAAGLQHSMMGPLHSS LSTNTLSPIIYQGLPNTRLATQPHLVQTQQVQPQNLQLQPQNLQPPS QPHLSVSSAANGHLGRSFLSGEPSQADVQPLGPSSLPVHTILPQESQ ALPTSLPSSMVPPMTTTQFLTPPSQHSYSSSPVDNTPSHQLQVPEHP FLTPSPESPDQWSSSSPHSNISDWSEGISSPPTTMPSQITHIPEAFK SEQ ID NO: 2 (Signal peptide) MPRLLTPLLCLTLLPALAARGLR

Claims (4)

1. Compound, characterized by the fact that it presents the structure: 4,4,4-trifluoro-N-((2S)-1-((9-methoxy-3,3-dimethyl-5-oxo-2,3,5,6-tetrahydro-1H-benzo[f]pyrrolo[1,2-a]azepin-6-yl)amino)-1-oxopropan-2-yl)butanamide or a pharmaceutically acceptable salt of any of the above. 2. Compound, characterized by the fact that it presents the structure: N-((2S)-1-((8,8-dimethyl-6-oxo-6,8,9,10-tetrahydro-5H-pyrido[3,2-f]pyrrolo[1,2-a]azepin-5-yl)amino)-1-oxopropan-2-yl)-4,4,4-trifluorobutanamide or a pharmaceutically acceptable salt of any of the above. 3. Pharmaceutical composition, characterized by the fact that it comprises a compound, as defined in claim 1 or 2, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. 4. Use of a compound as defined in claim 1 or 2, or a pharmaceutically acceptable salt thereof, characterized in that it is for the manufacture of a medicament for treating T-cell acute lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, erythroleukemia, triple negative breast cancer, breast cancer, ovarian cancer, melanoma, lung cancer, non-small cell lung cancer, pancreatic cancer, glioblastoma, colorectal cancer, head and neck cancer, cervical cancer, prostate cancer, liver cancer, oral squamous cell carcinoma, skin cancer, medulloblastoma, angiosarcoma, rhabdomyosarcoma, liposarcoma, malignant fibrous histiocytoma, hepatocellular carcinoma, intrahepatic or extrahepatic cholangiocarcinoma or adenoid cystic carcinoma.