HK1155652B - Compounds that enhance atoh-1 expression - Google Patents

Description

Compounds that increase the expression of Atoh1

Require priority

Priority of U.S. provisional patent application No. 61/027,032, filed 2, 7, 2008, under 35USC § 119(e), the entire content of which is incorporated herein by reference.

Technical Field

The present invention generally provides compounds, pharmaceutical compositions, and methods of their use, including methods that result in increased expression of the Atoh1 gene (e.g., Hath1) in a biological cell. More particularly, the invention relates to the treatment of diseases and/or conditions which benefit from increased expression of Atoh 1.

Background

One of the most common types of hearing loss is sensorineural deafness, which is caused by the loss of hair cells, the sensory cells in the cochlea that are responsible for transducing sound into electrical signals. Human beings are born with inner ear containing only about 15,000 hair cells per cochlea, however these cells are lost due to various genetic or environmental factors and such lost or damaged cells cannot be replaced. However, overexpression of the transcription factor Atoh1 induced differentiation of epidermal cells into hair cells on the Corti organ of the sensory organ of the cochlea (Zheng and Gao, Nat Neurosci 2000; 3: 580-. Expression of Atoh-1 also serves to push other cells, such as intestinal cells, into a differentiated state (Aragaki et al, biochem. Biophys. Res. Comm.2008. 4 months; 368 (4): 923-) -929), and overexpression of Atoh-1 reduces proliferation of colon cancer cells (Leow et al, Ann N Y Acad Sci.2005 11 months; 1059: 174-83).

Summary of The Invention

The invention features compounds described herein, and compositions containing such compounds. For example, the invention features pharmaceutical compositions comprising one or more compounds disclosed herein, or a pharmaceutically acceptable salt thereof, capable of increasing expression of Atoh1 in a cell, and a pharmaceutically acceptable carrier. The composition can be formulated for administration to a patient. Thus, the invention encompasses such pharmaceutical compositions, as well as concentrated stock solutions and compositions suitable for preservation of cells or tissues in tissue culture, and methods of use of the compounds and compositions.

If the composition is pharmaceutically acceptable (i.e., non-toxic), it can include a pharmaceutically acceptable carrier, such as a buffer (e.g., phosphate buffer), amino acids, urea, alcohol, ascorbic acid, phospholipids, polypeptides, EDTA, sodium chloride (e.g., saline), liposomes, mannitol, sorbitol, water, glycerol, or a combination thereof. Preservatives and dyes may also be included. In some embodiments, the composition is sterile.

The compounds described herein can be used to alter the characteristics of cells maintained in cell culture (e.g., in vitro), and the compounds and/or treated cells can be administered to a patient in need of treatment. For example, a method of treating a patient may be performed as a method comprising the steps of: (a) selecting a patient in need of treatment, and (b) administering to the patient a therapeutically effective amount of a compound described herein (e.g., a compound manufactured for administration). The pharmaceutical composition may be administered systemically (e.g., orally or parenterally). In particular, the composition can be administered intravenously, intramuscularly, intraperitoneally, sublingually, rectally, vaginally, transdermally, subcutaneously, or by inhalation. When administered orally, the composition can be formulated as a tablet (e.g., compressed tablet), pill, syrup, suspension, emulsion, or capsule. When administered parenterally, the composition may be formulated as a lozenge, drop (e.g. ear drops), solution, enema, suppository or spray. The composition may also be administered using a catheter or pump.

The composition may also be administered topically (e.g., to the ear or other site where cell differentiation and/or Atoh1 expression is desired). For administration to the ear, the pharmaceutical composition may be administered by injection into the cochlea's cavity (luminae), into the internal ear canal auditory nerve trunk, and/or into the scala tympani. More specifically, the pharmaceutical composition may be administered by intratympanic injection, applied to (i.e., injected into) the outer, middle or inner ear, by round window injection, or by cochlear capsule injection. The pharmaceutical composition may also be administered to the patient using a catheter or pump (e.g., topically to the middle and/or inner ear).

A patient in need of treatment may have or be at risk of developing a hearing disorder or imbalance associated with loss of auditory hair cells. Although the present invention is not limited to compounds that act by any particular mechanism, the compositions can be used where the treatment is effective to increase the expression of the Atoh1 gene in cells in the inner ear (or other target tissue (e.g., tumor)) of the patient, or to increase the number of cells in the inner ear of the patient that are characteristic of auditory hair cells. The auditory hair cells can be external or internal auditory hair cells.

Patients in need of treatment may also have or be at risk of developing cancer. The cancer may be gastrointestinal cancer (e.g., esophageal cancer, gallbladder cancer, liver cancer, pancreatic cancer, gastric cancer, small bowel cancer, large bowel cancer, colon cancer, or rectal cancer).

Patients in need of treatment may also suffer from or be at risk of developing cerebellar granule neuron deficiency, arthropathy and/or osteoarthritis.

In a particular embodiment, the method of treating a patient suffering from a hearing disorder or imbalance can be performed by a method comprising the steps of: (a) optionally selecting a patient in need of treatment, (b) obtaining a population of cells capable of differentiating into auditory hair cells, (c) contacting the population of cells in vitro with an effective amount of one or more compounds described herein for a time sufficient to allow an increase in the number of cells in the population that are characteristic of differentiated auditory hair cells, and (d) administering the population of cells or a subset thereof (e.g., a more highly differentiated subset of cells) to the ear of the patient. Cell populations capable of differentiating into auditory hair cells may include stem cells, induced pluripotent stem cells (IPS), progenitor cells, support cells, Deiters cells, column cells, inner finger cells, cap cells (protective cells), Hensen cells, and germ cells. The stem cells can be adult stem cells (e.g., stem cells derived from the inner ear, bone marrow, stroma, skin, fat, liver, muscle, or blood), embryonic stem cells, or stem cells obtained from the placenta or umbilical cord. Like stem cells, progenitor cells can be from the inner ear, bone marrow, stroma, skin, fat, liver, muscle, or blood. Administration of the cell population may be accomplished by (a) injecting the cells into the cochlear cavity, into the auditory nerve stem in the inner ear, or into the scala tympani, or (b) implanting the cells within a cochlear implant. In any method of treating a patient with cells, they may also be treated with one or more of the present compounds, and vice versa. As previously mentioned, the pharmaceutical composition may be administered systemically or locally.

Other methods of the invention include methods of increasing the number of cells characteristic of auditory hair cells in a population of cells in vitro. These methods may be implemented as follows: obtaining a population of cells capable of differentiating into auditory hair cells, contacting the population of cells in vitro (e.g., in cell culture) with an effective amount of one or more compounds described herein for a time sufficient to increase the number of cells in the population of cells characteristic of auditory hair cells. The population of cells capable of differentiating into hair cells comprises cells selected from the group consisting of: stem cells, iPS cells, inner ear stem cells, adult stem cells, bone marrow-derived stem cells, embryonic stem cells, mesenchymal stem cells, skin stem cells, adipose-derived stem cells, progenitor cells, inner ear progenitor cells, supporting cells, Deiters cells, column cells, inner finger cells, roof cells, Hensen cells, and germ cells.

The invention also includes the use of a compound described herein as a medicament, and in the manufacture of a medicament for the treatment or prevention of a condition described herein. For example, the medicament may be used in a method for treating or preventing hearing loss or imbalance associated with hair cell loss, or a condition associated with undesired cell proliferation. In addition, the invention includes the use of the compounds in the treatment of conditions described herein, such as hearing loss associated with loss of auditory cells, or a condition associated with undesired cellular proliferation. The drug may be in any of the forms described herein and may be administered alone or in combination with additional therapeutic or active agents.

Further, provided herein are kits (e.g., kits comprising the pharmaceutical compositions described above and the informational material, or kits comprising the compounds described herein and the informational material). The cells in the kit may be prepared according to the methods described above, and any of the kits may contain additional materials, such as devices suitable for administration of the pharmaceutical composition or cell population, e.g., a sterile flexible cannula suitable for insertion into the inner ear of a subject.

In addition, the invention includes cells or cell populations prepared by the methods described herein.

The invention also includes the use of one or more of the compounds described herein as a medicament, e.g., a medicament that can be used to treat a hearing disorder or imbalance condition associated with auditory hair cell loss, and/or a condition associated with abnormal cell proliferation.

The invention also includes the use of one or more of the compounds described herein for treating a hearing disorder or imbalance condition associated with auditory hair cell loss and/or a condition associated with abnormal cell proliferation.

Definition of

The term "abnormal proliferation" as used herein is defined as any undesired over-proliferation of any type of cell, wherein said cell is not restricted to the development of a normal cell cycle, and wherein said proliferation may lead to the development of a tumor or any cancer.

The term "treatment" as used herein refers to any method in which one or more symptoms of a disease or disorder are ameliorated or otherwise beneficially altered. As used herein, amelioration of a symptom of a particular disorder refers to any alleviation, whether permanent or temporary, persistent or transient, that may be attributed to, or associated with, treatment by the compositions and methods of the present invention.

The terms "effective amount" and "therapeutically effective" as used herein, refer to an amount or concentration of one or more compounds or pharmaceutical compositions described herein that is effective to produce the desired effect or physiological result in the condition of administration, after a period of action, including acute or chronic administration, and periodic or continuous administration.

Effective amounts of one or more compounds or pharmaceutical compositions for use in the present invention include: (iii) an amount that promotes increased expression of Atoh 1; promoting differentiation of all or a portion of one or more cells to treat a disease that would benefit from increased expression of Atoh1, e.g., preventing or delaying the onset of one or more diseases that would benefit from increased expression of Atoh1, delaying the progression of one or more diseases that benefit from increased expression of Atoh1, ameliorating the effects of one or more diseases that benefit from increased expression of Atoh1, or generally improving the prognosis of a patient diagnosed with one or more diseases that benefit from increased expression of Atoh1, e.g., one or more of the diseases described herein. For example, in the treatment of hearing loss, compounds that increase hearing to any degree or arrest any symptom of hearing loss would be therapeutically effective. For example, in the treatment of abnormal cell proliferation, compounds that reduce cell proliferation, reduce tumor size, reduce metastasis, and reduce vascular proliferation to the tumor are all therapeutically effective. A therapeutically effective amount of a compound is not required to cure a disease but to provide treatment for a disease.

Throughout the specification, the term "patient" is used to describe an animal, human or non-human, which is to be treated according to the methods of the invention. Including veterinary and non-veterinary applications. The term includes, but is not limited to, birds, reptiles, amphibians, and mammals, e.g., humans, other primates, pigs, rodents such as mice and rats, rabbits, guinea pigs, hamsters, cows, horses, cats, dogs, sheep, and goats. Typical patients include humans, livestock and domestic pets such as cats and dogs.

The term "halo" or "halogen" refers to any group of fluorine, chlorine, bromine or iodine.

In general, and unless otherwise specified, the substituent (group) prefix name is derived from the parent hydride by: (i) replacement of the "alkane (ane)" in the parent hydride with the suffix "yl", "diyl", "triyl", "tetrayl", etc.; or (ii) replacing "e" in the parent hydride with the suffix "yl", "diyl", "triyl", "tetrayl", etc. (where an atom having a free valence is designated, the given valence of that atom is as low as any of the defined valences in the parent hydride). Acceptable nomenclature (conjugated names) such as adamantyl, naphthyl, anthracyl, phenanthryl, furyl, pyridyl, isoquinolyl, quinolinyl, and piperidinyl, as well as common names such as vinyl, allyl, phenyl, and thienyl, are also used throughout this document. Numbering and naming of substituents of fused, bicyclic, tricyclic, polycyclic rings is in keeping with the use of conventional numbering/coding systems.

The term "alkyl" refers to a saturated hydrocarbon chain, which may be straight or branched, containing the indicated number of carbon atoms. E.g. C₁-C₆Alkyl means that the group may contain 1 to 6 (inclusive of 1 and 6) carbon atoms. Any atom may be, for example, substituted with one or more substituents (e.g., any R such as described herein^aThose described in the definition of (a) are optionally substituted. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, and tert-butyl.

The term "haloalkyl" refers to an alkyl group wherein at least one hydrogen atom is substituted with halo. In some embodiments, more than one hydrogen atom (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) is substituted with halo. In some embodiments, the hydrogen atoms may each be substituted with the same halogen (e.g., fluorine), or the hydrogen atoms may be substituted with a combination of different halogens (e.g., fluorine and chlorine). "haloalkyl" also includes alkyl moieties in which all hydrogens are substituted with halo (sometimes referred to herein as perhaloalkyl, e.g., perfluoroalkylSuch as trifluoromethyl). Any atom may, for example, be substituted by one or more substituents (e.g., as any R described herein)^bThose described in the definition of (a) are optionally substituted.

The term "aralkyl" refers to an alkyl moiety in which an alkyl hydrogen atom is replaced with an aryl group. One of the alkyl moiety carbons serves as the point of attachment of the aralkyl moiety to the other moiety. Any ring or chain atom may, for example, be substituted by one or more substituents (e.g., as any R described herein)^cThose described in the definition of (a) are optionally substituted. Non-limiting examples of "aralkyl" groups include benzyl, 2-phenylethyl and 3-phenylpropyl.

The term "heteroaralkyl" refers to an alkyl moiety in which an alkyl hydrogen atom is replaced with a heteroaryl. One of the alkyl carbons serves as the point of attachment of the aralkyl group to the other moiety. Heteroaralkyl includes groups in which more than one hydrogen atom on the alkyl moiety is replaced by a heteroaryl group. Any ring or chain atom may, for example, be substituted by one or more substituents (e.g., as any R described herein)^cThose described in the definition of (a) are optionally substituted. Heteroaralkyl groups may include, for example, 2-pyridylethyl.

The terms "alkoxy" and "haloalkoxy" refer to the-O-alkyl and-O-haloalkyl groups, respectively. The term "phenoxy" refers to an-O-phenyl group.

The term "heterocyclyl" refers to a fully saturated monocyclic, bicyclic, tricyclic, or other polycyclic ring system containing one or more (e.g., 1-4) heterocyclic atoms independently selected from O, N or S. A heteroatom or a ring carbon atom is the point of attachment of a heterocyclyl substituent to another moiety. Any atom may, for example, be substituted by one or more substituents (e.g., as any R described herein)^cThose described in the definition of (a) are optionally substituted. Heterocyclic groups may include, for example, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl (piperidino), piperazinyl, morpholinyl (morpholino), pyrrolinyl, and pyrrolidinyl.

The term "cycloalkyl" refers to a fully saturated monocyclic, bicyclic, tricyclic ring or mixtures thereof(iii) a polycyclic hydrocarbon group. Any atom may, for example, be substituted by one or more substituents (e.g., as any R described herein)^cThose described in the definition of (a) are optionally substituted. One ring carbon atom serves as the point of attachment of one cycloalkyl group to another moiety. Cycloalkyl moieties may include, for example, cyclopropyl, butyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, and norbornyl (bicyclo [2.2.1 ] groups]Heptyl) group.

The term "cycloalkenyl" refers to partially unsaturated monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon groups. A ring carbon (e.g., saturated or unsaturated) is the point of attachment for a cycloalkenyl substituent. Any atom may, for example, be substituted by one or more substituents (e.g., as any R described herein)^cThose described in the definition of (a) are optionally substituted. Cycloalkenyl moieties can include, for example, cyclohexenyl, cyclohexadienyl, or norbornenyl.

The term "aryl" refers to an aromatic monocyclic or bicyclic hydrocarbon ring system in which any ring atom may be, for example, substituted by one or more substituents (e.g., as in any R described herein)^dThose described in the definition of (a) are optionally substituted. Aryl moieties may include phenyl and naphthyl.

The term "heteroaryl" refers to compounds containing a residue independently selected from O, N or S (and mono-and dioxides thereof, e.g., N → O)^-、S(O)、SO₂) And (c) aromatic monocyclic or bicyclic hydrocarbon groups of one or more (e.g., 1-6) heteroatom ring atoms. Any atom may, for example, be substituted by one or more substituents (e.g., as any R described herein)^dThose described in the definition of (a) are optionally substituted. Heteroaryl groups may include pyridyl, thienyl, furyl (furyl), imidazolyl, isoquinolyl, quinolyl, and pyrrolyl.

The symbol C (O) denotes a carbon atom which is bonded to an oxygen atom via a double bond. The term "oxo" refers to double bond oxygen, i.e., ═ O.

The term "substituent" refers to a group that is "substituted" on any atom of, for example, an alkyl, haloalkyl, cycloalkyl, aralkyl, heteroaralkyl, heterocyclyl, cycloalkenyl, aryl, or heterocyclyl group. In general, when the definition of a particular variable includes the possibility of hydrogen and non-hydrogen (halo, alkyl, aryl, etc.), the term "non-hydrogen substituent" is intended to collectively refer to the non-hydrogen possibility in that particular variable.

Symbols, e.g. "being from 1 to 5R^dOptionally substituted C₆-C₁₀Aryl "(and the like) is intended to include unsubstituted C₆-C₁₀Aryl and substituted with 1-5R^dSubstituted C₆-C₁₀And (4) an aryl group. The use of a prefix name for a substituent (group) without the modifier "optionally substituted" or "substituted", such as alkyl, is to be understood as meaning that the particular substituent is unsubstituted. However, the use of "haloalkyl" without the modifier "optionally substituted" or "substituted" is still to be understood as an alkyl in which at least one hydrogen atom is halo-substituted.

For convenience of explanation, it is also to be understood that if in this specification (including the claims) a group is defined as "defined anywhere herein" (or the like), that definition of that particular group includes the first-appearing and most general definition in this specification, as well as any sub-general and specific definitions described anywhere.

This application relates to U.S. provisional patent application No. 60/605,746 filed on day 31 of 2004, international application No. PCT/US2005/030714 filed on day 30 of 8/2005, U.S. application No. 10/989,649 filed on day 15 of 11/2004, U.S. application No. 11/953,797 filed on day 12 of 2007, U.S. application No. 12/187,543 filed on day 7 of 2008, U.S. application No. 60/859,041 filed on day 15 of 11/2006, international application No. PCT/US2007/084654 filed on day 14 of 11/2007, U.S. application No. 12/233.017 filed on day 18 of 2008, U.S. provisional application No. 60/859,041 filed on day 24 of 11/2008, the entire contents of which are incorporated herein by reference.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for use in the invention are described herein; in addition, suitable methods and materials well known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, paragraphs, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims.

Drawings

FIGS. 1A-1H are general structures of phenolic compounds or derivatives thereof.

FIGS. 1I-1K are structures of specific phenolic compounds and derivatives thereof.

FIGS. 2A-2F are general structures of benzamide compounds or related compounds.

FIGS. 2G-2I are structures of specific benzamide compounds or related compounds.

FIGS. 3A-3X are general structures of compounds containing one or more heterocyclic rings.

FIGS. 3Y-3ZZ are structures of specific compounds containing one or more heterocyclic rings.

FIGS. 4A-4G are general structures of compounds containing one or more benzene rings.

FIGS. 4H-4I are structures of specific compounds containing one or more benzene rings.

FIGS. 5A-5E are general structures of compounds comprising an amide group attached to a five-membered heterocyclic ring system.

Fig. 5F and 5G are structures of specific compounds comprising an amide group attached to a five-membered heterocyclic ring system.

FIGS. 6A-6O are general structures of compounds comprising a five-membered heterocyclic ring system fused to another ring system.

FIGS. 6P-6V are structures of specific compounds containing a five-membered heterocyclic ring system fused to another ring system.

FIGS. 7A-7C are general structures of pyridine compounds, and FIG. 7D is a general structure of pyrimidine compounds.

FIGS. 7E-7F are structures of specific pyridine or pyrimidine compounds.

Fig. 8A and 8B are general structures of aniline compounds or aniline derivatives.

Fig. 8C is a structure of a specific structure of an aniline compound or an aniline derivative.

FIGS. 9-116 are line graphs showing expression of Math1 (see example 1) in HEK cells with stably expressing luciferase gene controlled by the Math1 enhancer and minimal promoter. The compound numbers shown in the figures correspond to the compound structures identified in figures 1 through 8. Math1 activity was determined using the high throughput screening method described in example 1 and example 2.

Fig. 117A and 117B are graphs showing the results of experiments performed with optimized high throughput screening.

Fig. 118A and 118B are scatter plots showing replicate experiments with optimized high throughput screening.

Figure 119 is a bar graph showing activation of Math1 assessed in cells exposed to the indicated compounds using the Math1 luciferase reporter assay described in example 1 and example 2. The structures of these compounds are provided in figures 1 to 8. The initial Atoh1 activation results for these compounds are shown in figures 9-114.

Figure 120A is a bar graph showing activation of Math1 assessed in cells exposed to the indicated compounds using the Math1 luciferase reporter assay described in example 1 and example 2. The structures of these compounds are provided in figures 1 to 8. The initial Atoh1 activity results for these compounds are shown in figures 9-114.

FIG. 120B shows the structure of the compound shown in FIG. 117A.

FIG. 120C shows Atoh1mRNA expression in cells exposed to the indicated compounds.

FIGS. 121A and 121B are photographs of untreated cells (A) and cells (B) exposed to Compound (Cp) Cp. -0000540. The cell population for the positive staining hair cell specific markers Math1-GFP and myosin 7a is indicated by arrows.

Detailed Description

In addition, the present invention provides compounds and methods relating to compounds and/or pharmaceutical compositions for treating patients for the conditions described herein. While the methods of treatment are not limited to those in which a particular potential cellular event occurs, the present compounds and compositions can increase the expression of the Atoh1 gene in the subject and/or cells, thereby causing the cells to differentiate into, for example, auditory hair cells.

Atoh-1

Atoh1 protein homolog 1(Atoh1 or atonal) is a proto-neural gene encoding a protein comprising a basic helix-loop-helix (bHLH) domain that appears to play an important role in Cell fate in Drosophila (Drosophila) nervous system development (Jarman et al, Cell, 73: 1307. sup. 1321, 1993). Atoh1 is evolutionarily conserved as a homolog identified in tribolium castaneum (red flower beetle), Fugu rubripes (puffer fish), chicken (Cath1), mouse (Math1), and human (Hath1) (Ben-Arie et al, hum. mol. gene., 5: 1207-. These homologues each comprise a bHLH domain of the same length and with a high degree of sequence homology to the Atoh1 bHLH domain. For example, the length of the Hath1 and Math1 genes are nearly the same. These molecules also have highly similar nucleotide sequences (86% homology) and highly similar bHLH amino acid sequences (89%). The bHLH domain of Cath1 was 97% and 95% homologous to the bHLH domains of Hath1 and Math1, respectively. The cathh 1 bHLH is 67% homologous to the Atoh1 bHLH domain. In contrast, the bHLH domains of other encoded proteins of drosophila share only 40% -50% sequence homology.

Each mammalian Atoh1 homologue functions as a transcription factor that activates E box (CANNTG (SEQ ID NO: 1)) dependent transcription (Arie et al, hum. mol. Genet., 9: 1207-Bu 1216, 1996; Akazawa et al, J.biol. chem., 270: 8730-Bu 8738, 1995), and functions as an important positive regulator in neural tissue and Gastrointestinal (GI) tract that determines cell fate.

Zoghbi et al (U.S. publication No. 2004/0237127) describe the use of the above-described nucleic acids encoding the Atoh1 homologs for the treatment of deafness, osteoarthritis and abnormal cell proliferation.

As used herein, "Atoh 1" refers to any and all Atoh1 related nucleic acid or protein sequences and includes any sequence that is orthologous or homologous (homologous) to an Atoh1 nucleic acid or amino acid sequence, respectively, or has significant sequence similarity to an Atoh1 nucleic acid or amino acid sequence, and thus, the term "Atoh 1" includes other mammalian homologs, e.g., human, mouse, rat, and the like. The sequence may be present in any animal, including mammals (e.g., humans). Examples of Atoh1 nucleic acid and amino acid sequences include, but are not limited to, Atoh1 (e.g., NM _001012432.1 and NP _001012434.1) (chimpanzees (pantroglodytes)), Hath1 (e.g., NM _005172.1 and NP _005163.1) (Homo sapiens), Math1 (e.g., NM _007500.4 and NP _031526.1) (Mus musculus), Atoh1(NM _001109238.1 and NP _001102708.1) (brown mice (Rattus norvegicus)), Atoh1(XM _001102247.1 and XP _001102247.1) (macaque (Macaca mulatta)), Atoh1(NM _001098099.1 and NP _001091568.1) (cattle (bostaurus)), Atoh 001091568.1 (XM _001091568.1 and 001091568.1) (Canis familiaris) and camilia (camilia) and other proteins such as proteins, e.g., proteins 001091568.1, and proteins equivalent proteins, such as proteins, proteins 001091568.1, and proteins equivalent proteins, such as proteins. Furthermore, multiple homologous or similar sequences may be present in the animal. See, for example, GeneID: 474 (homo sapiens), GeneID: 11921 (mus musculus), GeneID: 461380 (chimpanzee), GeneID: 500156 (brown mouse), GeneID: 704893 (macaque), GeneID: 539158 (cattle) and GeneID: 487864 (domestic dog).

Any sequence with significant sequence similarity (i.e., greater than 80% sequence similarity across the entire sequence, e.g., at least 85%, 90%, 95%, 99% or more) to the human Atoh1 sequence (see Genbank acc. nos. nm _005172.1 and NP _005163.1) can be used in the present methods. To determine the percent homology of two nucleic acid sequences, an alignment of the sequences is performed for optimal comparison purposes (gaps are introduced in either or both of the first and second amino acid or nucleic acid sequences as needed for optimal alignment purposes, and non-homologous sequences may be discarded for comparison purposes). At least 80% (in some embodiments, about 85%, 90%, 95%, or 100%) of the length of the reference sequence aligned for comparison purposes is aligned. The nucleotides at the corresponding nucleotide positions are then compared. When the nucleotide occupying a position in the first sequence is the same as the nucleotide at the corresponding position in the second sequence, the molecules are considered to be identical at that position. The percent homology of two sequences is a function of the number of identical positions shared by the sequences, and the number of gaps and the length of each gap are also taken into account in order to obtain an optimal alignment of the two sequences.

Sequence comparison of two sequences and determination of percent homology can be accomplished using mathematical algorithms. For example, the percent homology between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J.mol.biol.48: 444-.

Compound (I)

The present invention provides compounds capable of increasing expression of Atoh1 in a cell. In some embodiments, the increase in Atoh1 expression is a significant increase. In some embodiments, the increase in Atoh1 expression can be, for example, between about 1-10%, 11-20%, 21-30%, 31-40%, 41-50%, 51-60%, 61-70%, 71-80%, 81-90%, 91-100%, 101-200%, 201-300%, 301-400%, 401-500%, 501-1000%, 1001-10000%, 10001-100000% above baseline or more. An increase in Atoh1 may also be expressed as a fold increase, for example, where a 100% increase is a one-fold increase, a 1000% increase is a 10-fold increase, and so forth. Alternatively or additionally, an increase in Atoh1 expression is sufficient to promote differentiation of cells, e.g., non-auditory hair cells (i.e., cells that are not auditory hair cells, e.g., progenitor or stem cells) differentiate into or tend towards auditory hair cells.

The compounds useful in any of the methods described herein are phenolic compounds (or their sulfur analogs, e.g., phenyl thiols), or compounds derived from these compounds, such as phenyl ethers (or thioethers), e.g., linear or cyclic phenyl ethers. For example, these compounds can be represented generally by those structures shown in FIGS. 1A-1H, and specifically exemplified by those structures shown in FIGS. 1I-1K. Any phenolic compound (or sulphur analogue) may be in neutral or salt form, for example, a lithium, sodium, potassium or calcium salt thereof.

These phenolic compounds and derivatives (or their sulfur analogs) are represented by the structure in FIG. 1A, wherein R is₂、R₃、R₄、R₅And R₆Each independently is H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally comprising one or more N, O, S or F atoms; x is O (see FIG. 1B) or S (see FIG. 1C); and R₁Is H or a moiety comprising up to 16 carbon atoms and optionally comprising one or more N, O, S or F atoms. For example, a moiety containing up to 16 carbon atoms and optionally containing one or more atoms which is N, O, S or F may be an alkoxy group or a trifluoromethyl group.

For FIG. 1D, in a particular implementationIn the scheme, R₁And R₂Together define one or more ring systems, each ring system comprising up to 16 carbon atoms and optionally comprising one or more N, O, S or F atoms.

For FIG. 1E, in certain embodiments, R₁Is H; i.e. the compound is a phenol.

For fig. 1F-1H, in certain embodiments, the phenolic derivative is a cyclic ether derivative. For example, the cyclic ether portion of the molecule can be made rigid by introducing a carbonyl group (see fig. 1F) and/or a carbon-carbon double bond (see fig. 1G, which contains both). In other embodiments, these cyclic ether derivatives can be made rigid by introducing a second ring system on the cyclic ether system. In certain embodiments, the structure of FIG. 1F represents a cyclic ether derivative, wherein R₈、R₉、R₁₀And R₁₁Each independently is H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally including one or more N, O, S or F atoms. In other particular embodiments, the structure of FIG. 1G represents a cyclic ether derivative, wherein R₁₂And R₁₃Each independently H, F, Cl, Br, I, OH, or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms. In other particular embodiments, the structure of FIG. 1H represents a cyclic ether derivative, wherein R₁₄And R₁₅Together define one or more ring systems, each ring system comprising a moiety of up to 16 carbon atoms and optionally one or more N, O, S or F atoms, R₁₆、R₁₇And R₁₈Each independently H, F, Cl, Br, I, OH, or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms.

The present compounds are amide compounds and/or related compounds thereof. These compounds can be generally represented by those structures shown in FIGS. 2A-2F, and specifically exemplified by those structures shown in FIGS. 2G-2I. Any of the amides or related compounds thereof may be in neutral or salt form.

The benzylThe amide compound and/or derivative thereof may be represented by the structure of FIG. 2A, wherein R₂₀、R₂₁、R₂₂、R₂₃And R₂₄Each independently is H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms; and R is₂₄And R₂₆Each independently H or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms.

In the compound of FIG. 2A, R₂₄And R₂₅One or more ring systems may be collectively defined, each ring system comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms. These compounds can be represented by the structure in FIG. 2B. For example, these compounds may have the structure shown in FIGS. 2C and 2D, wherein R₂₇、R₂₈、R₂₉、R₃₁And R₃₂Each independently is H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms; and R is₂₆Is H or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms.

Other benzamide related compounds and derivatives thereof are described by the structures in FIGS. 2E and 2F, wherein R is₃₃、R₃₄、R₃₅、R₃₆And R₃₇Each independently is H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms; r₃₉、R₄₀、R₄₁And R₄₂Each independently H or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms.

The present compounds are or may include one or more heterocyclic ring systems, such as a3, 4, 5, 6 or 7 membered ring system, which ring system includes more than one heteroatom, such as O, S, or N. For example, one or more ring systems may include 1, 2, 3, 4, or even 5 heteroatoms, such as O, S or N. In many embodiments, the ring system is aromatic. For example, these compounds may be represented generally by the structures shown in FIGS. 3A-3X, and specifically exemplified by those shown in FIGS. 3Y-3 ZZ. Any of the compounds described, which are or comprise one or more ring systems, may be in neutral or salt form.

Compounds which are or include one or more heterocyclic ring systems are represented by the structure of FIG. 3A, wherein R is₄₃And R₄₄Each independently is H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms; and X is O (FIG. 3B) or S (FIG. 3C).

Compounds which are or comprise one or more heterocyclic ring systems are represented by the structure of FIG. 3D, wherein R is₄₅，R₄₆And R₄₈Each independently is H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms; and R₄₇And R₄₉Each independently H, or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms.

Compounds that are or include one or more heterocyclic ring systems are represented by the structure of FIG. 3E, where R is₅₀、R₅₁、R₅₂And R₅₃Each independently is H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms; and X is O (FIG. 3F) or S (FIG. 3G).

Compounds that are or include one or more heterocyclic ring systems are represented by the structure of FIG. 3H, where R₅₅、R₅₆And R₅₇Each independently is H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms; and R₅₄Is H, or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms. For example, in particular embodiments, R₅₄And R₅₅May collectively define one orA plurality of ring systems, each ring system comprising up to 16 carbon atoms and optionally one or more being N, O, S or F atoms, as shown in figure 3I.

Compounds that are or include one or more heterocyclic ring systems are represented by the structure of FIG. 3J, where R is₅₇、R₅₉And R₆₀Each independently is H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms; and R₅₈Is H, or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms. For example, in particular embodiments, R₅₈And R₅₉One or more ring systems may be collectively defined, each ring system comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms, as shown in FIG. 3K. For example, in particular embodiments, R₅₇And R₆₀One or more ring systems may be collectively defined, each ring system comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms, as shown in FIG. 3L. For example, in particular embodiments, R₅₇And R₆₀And R₅₈And R₅₉May collectively each define one or more ring systems, each ring system comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms, as shown in figure 3M.

Compounds that are or include one or more heterocyclic ring systems are represented by the structure of FIG. 3N, where R is₆₁、R₆₂And R₆₄Each independently is H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms; and R₆₃Is H, or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms. For example, in particular embodiments, R₆₁And R₆₂One or more ring systems may be collectively defined, each ring system comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms, as shown in FIG. 3O.

Is or contain one or more heterocyclic ring systemsThe compounds of the system are represented by the structure in FIG. 3P, where R₆₅And R₆₆Each independently is H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms; and X is O (FIG. 3Q) or S (FIG. 3R).

Compounds that are or contain one or more heterocyclic ring systems are represented by the structures in figure 3S. Wherein R is₆₇、R₆₈And R₆₉Each independently is H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms; and X is O (FIG. 3T) or S (FIG. 3U).

Compounds that are or include one or more heterocyclic ring systems are represented by the structure in FIG. 3V, where R₇₀、R₇₁And R₇₂Each independently is H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms; and X is O (FIG. 3W) or S (FIG. 3X).

The present compounds, which may be used in any of the methods described herein, are or comprise one or more benzene rings, such as fused benzene ring systems, e.g., fused benzene ring systems that are part of a flavonoid, coumarin, or other similar system. For example, such compounds may be represented generally by the structures shown in fig. 4A-4G, and specifically exemplified by those shown in fig. 4H and 4I. Any of the compounds described which are or comprise one or more ring systems may be in neutral or salt form.

Compounds that are or contain one or more benzene ring systems are represented by the structure in FIG. 4A, where R is₇₄、R₇₅、R₇₆、R₇₇、R₇₈And R₇₉Each independently is H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms. For example, in particular embodiments, R₇₆And R₇₇May collectively define one or more ring systems, each ring system containing up to 16The carbon atoms and optionally one or more are N, O, S or F atoms, as shown in fig. 4B. For example, the compound in FIG. 4B can be represented by the compounds in FIGS. 4C and 4D, where R is₈₁、R₈₂、R₈₃And R₈₄Each independently is H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms. For example, in other specific embodiments, wherein R₇₆And R₇₇Collectively defining one or more ring systems, which compounds may be represented by those structures in FIGS. 4E-4G, wherein R is₈₅、R₈₆、R₈₇And R₈₈Each independently is H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms; and R is₈₉Is H or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms.

In some embodiments, a compound useful in any of the methods described herein comprises an amide group bonded to a five-membered heterocyclic system, e.g., a heterocyclic system comprising more than one heteroatom (e.g., O, S or N). For example, one or more ring systems may contain 1, 2, 3, 4, or even 5 heteroatoms, such as O, S or N. In many embodiments, the ring system is aromatic. For example, such compounds can be represented generally by those structures shown in fig. 5A-5E, and specifically exemplified by those structures shown in fig. 5F-5G. Any of the compounds described which are or comprise one or more ring systems may be in neutral or salt form.

In certain embodiments, the compound comprising an amide group bonded to a five-membered heterocyclic ring system is represented by the structure in fig. 5A, wherein R is₉₂、R₉₃And R₉₄Each independently is H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms; and R is₉₀And R₉₁Each independently H, or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms; and X is O (FIG. 5B) or S(FIG. 5C). For example, in some embodiments, R₉₀And R₉₁May together define one or more ring systems comprising moieties of up to 16 carbon atoms and optionally one or more N, O, S or F atoms (see FIG. 5D).

In other certain embodiments, the compound comprising an amide group bonded to a five-membered heterocyclic ring system is represented by the structure in figure 5E, wherein R is₉₇And R₉₈Each independently is H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms; and R₉₅、R₉₆And R₉₉Each independently H, or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms.

In some embodiments, compounds useful in any of the methods described herein comprise a five-membered heterocyclic ring system fused to one or more other ring systems, e.g., ring systems defining one or more 4-, 5-, 6-, 7-, or 8-membered ring systems. For example, such compounds may be represented generally by those structures shown in FIGS. 6A-6O, and specifically illustrated in those structures shown in FIGS. 6P-6V. Any of the compounds described, which are or comprise one or more ring systems, may be in neutral or salt form.

In some embodiments, compounds comprising a five-membered heterocyclic ring system fused to one or more other ring systems are represented by figure 6A, wherein R is₁₀₀、R₁₀₁、R₁₀₂、R₁₀₃、R₁₀₄And R₁₀₅Each independently is H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms; and X is S (see FIG. 6B) or O (see FIG. 6C).

In other embodiments, compounds comprising a five-membered heterocyclic ring system fused to one or more other ring systems are represented by figure 6D, wherein R is₁₀₆、R₁₀₇、R₁₀₈、R₁₀₉、R₁₁₀、R₁₁₁、R₁₁₂、R₁₁₃、R₁₁₄And R₁₁₅Each independently is H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms; and X is S (see FIG. 6E) or O (see FIG. 6F).

In certain embodiments, compounds comprising a five-membered heterocyclic ring system fused to one or more other ring systems are represented by figure 6G, wherein R is₁₁₈、R₁₁₉、R₁₂₀、R₁₂₁、R₁₂₃Each independently is H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms; and R is₁₂₃Is H, or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms.

In certain embodiments, compounds comprising a five-membered heterocyclic ring system fused to one or more other ring systems are represented by fig. 6H, wherein R is₁₂₄、R₁₂₅、R₁₂₆And R₁₂₇Each independently is H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms; and R is₁₂₈And R₁₂₉Together define one or more rings, each ring containing up to 16 carbon atoms and optionally one or more N, O, S or F atoms. For example, the compound in FIG. 6H can be represented by FIG. 6I, where R₁₃₀、R₁₃₁、R₁₃₂And R_128’Each independently is H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms; and R is_129’Is H, or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms.

In other certain embodiments, compounds comprising a five-membered heterocyclic ring system fused to one or more other ring systems are represented by fig. 6J, wherein R is₁₃₄、R₁₃₅、R₁₃₆、R₁₃₇、R₁₄₀And R₁₄₁Each is independentThe three sites are H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms; and R is₁₃₉Is H or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms. For example, and with reference to FIG. 6K, in particular embodiments, R₁₄₀And R₁₄₁Together define one or more rings, each ring including up to 16 carbon atoms and optionally one or more N, O, S or F atoms.

In some embodiments, compounds comprising a five-membered heterocyclic ring system fused to one or more other ring systems are represented by fig. 6L, wherein R is₁₄₃、R₁₄₄、R₁₄₅、R₁₄₆And R₁₄₇Each independently is H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms; and X is O (see FIG. 6M) or S (see FIG. 6N).

In still other embodiments, compounds comprising a five-membered heterocyclic ring system fused to one or more other rings are represented by figure 6O, wherein R is₁₄₉、R₁₅₀、R₁₅₁And R₁₅₂Each independently is H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms; and R is₁₅₃Is H, or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms.

In some embodiments, the compound useful in any of the methods described herein is a pyridine or pyrimidine. For example, such compounds may be represented generally by the structures shown in fig. 7A-7D, and specifically exemplified by those structures shown in fig. 7E and 7D. Any of the compounds, which are pyridines or pyrimidines, may be in neutral or salt form, e.g., as the hydrochloride salt thereof.

In some embodiments, fig. 7A depicts a pyridine compound, wherein R is₁₅₅、R₁₅₆、R₁₅₇、R₁₅₉And R₁₆₀Each of which isIndependently H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms. In some particular embodiments, R₁₅₆And R₁₅₇(see FIG. 7B), or R₁₅₆And R₁₅₇And R₁₅₉And R₁₆₀(see FIG. 7C) together define one or more rings, each ring comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms.

In some embodiments, fig. 7D depicts pyrimidine compounds, wherein R is₁₆₁、R₁₆₂、R₁₆₃And R₁₆₄Each independently is H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms.

In some embodiments, the compound useful in any of the methods described herein is aniline or an aniline derivative. For example, such compounds may be represented generally by those structures shown in fig. 8A and 8B, and specifically exemplified by those structures shown in fig. 8C. Any of the compounds, which are pyridines or pyrimidines, may be in neutral or salt form.

In some embodiments, figure 8A depicts an aniline compound, where R is₁₇₀、R₁₇₁、R₁₇₂、R₁₇₃And R₁₇₄Each independently is H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms; and R is₁₇₅And R₁₇₆Each independently H, or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms.

In some embodiments, figure 8B depicts an aniline derivative compound, wherein R is₁₇₈、R₁₇₉、R₁₈₀And R₁₈₁Each independently is H, F, Cl, Br, I, OH, CN, NO₂Or a moiety comprising up to 16 carbon atoms and optionally one or more N, O, S or F atoms; and R is₁₇₇Is H, or is comprised ofA moiety of up to 16 carbon atoms and optionally one or more N, O, S or F atoms.

In some embodiments, these compounds may have the formula depicted in fig. 6G:

in some embodiments:

R₁₁₈、R₁₁₉、R₁₂₀and R₁₂₁Each of which is independently selected from H, halo, OH, CN, NO₂、C₁-C₃Alkyl radical, C₁-C₃Haloalkyl, C₁-C₃Alkoxy and C₁-C₃A haloalkoxy group;

R₁₂₂is hydrogen or-Z-R^a(ii) a Wherein:

z is O or a bond; and is

R^aThe method comprises the following steps:

(i)C₁-C₆alkyl or C₁-C₆Haloalkyl groups each consisting of 1 to 3R^bOptionally substituted; or

(ii)C₃-C₁₀Cycloalkyl radical, C₃-C₁₀Cycloalkenyl radicals each of which is substituted by 1 to 5R^cOptionally substituted; or

(iii)C₇-C₁₁Aralkyl, or heteroaralkyl containing 6 to 11 atoms, each of which consists of 1 to 5R^cOptionally substituted;

(iv)C₆-C₁₀aryl or heteroaryl containing 5 to 10 atoms, each of which consists of 1 to 5R^dOptionally substituted;

R₁₂₃the method comprises the following steps:

(i) hydrogen; or

(ii)C₆-C₁₀Alkyl or C₆-C₁₀Haloalkyl groups each consisting of 1 to 3R^bSubstitution; or

(iii)C₆-C₁₀Aryl or heteroaryl containing 5 to 10 atoms, each of which consists of 1 to 5R^dOptionally substituted; or

(iv)C₇-C₁₁Aralkyl, or heteroaralkyl containing 6 to 11 atoms, each of which consists of 1 to 5R^cOptionally substituted; or

(v)-(C₁-C₆Alkyl) -Z¹-(C₆-C₁₀Aryl) in which Z is¹Is O, S, NH or N (CH)₃) (ii) a The alkyl moiety consisting of 1-3R^bOptionally substituted; and the aryl moiety consists of 1-5R^dOptionally substituted; or

(vi)-(C₁-C₆Alkyl) -Z²- (heteroaryl group containing 5 to 10 atoms), in which Z²Is O, S, NH or N (CH)₃) (ii) a The alkyl moiety consisting of 1-3R^bOptionally substituted; and the heteroaryl moiety consists of 1-5R^dOptionally substituted; or

-(C₁-C₆Alkyl) -Z³-(C₃-C₁₀Cycloalkyl) in which Z is³Is O, S, NH or N (CH)₃) (ii) a The alkyl moiety consisting of 1-3R^bOptionally substituted; and the cycloalkyl moiety consists of 1-5R^cOptionally substituted;

R_bat each occurrence, independently:

(i)NH₂；NH(C₁-C₃alkyl groups); n (C)₁-C₃Alkyl radical)₂(ii) a A hydroxyl group; c₁-C₆Alkoxy, or C₁-C₆A haloalkoxy group; or

(ii)C₃-C₇Cycloalkyl is optionally substituted with 1-3 substituents independentlyIs selected from C₁-C₆Alkyl, NH₂；NH(C₁-C₃Alkyl groups); n (C)₁-C₃Alkyl radical)₂(ii) a A hydroxyl group; c₁-C₆Alkoxy or C₁-C₆A haloalkoxy group;

R^cat each occurrence, independently:

(i) halogenating; NH (NH)₂；NH(C₁-C₃Alkyl groups); n (C)₁-C₃Alkyl radical)₂(ii) a A hydroxyl group; c₁-C₆An alkoxy group; c₁-C₆A haloalkoxy group; or oxo; or

(ii)C₁-C₆Alkyl or C₁-C₆A haloalkyl group; and

R^dat each occurrence, independently:

(i) halogenating; NH (NH)₂；NH(C₁-C₃Alkyl groups); n (C)₁-C₃Alkyl radical)₂(ii) a A hydroxyl group; c₁-C₆Alkoxy or C₁-C₆A haloalkoxy group; a nitro group; -NHC (O) (C)₁-C₃Alkyl groups); or cyano; or

(ii)C₁-C₆Alkyl or C₁-C₆A haloalkyl group.

Implementations may include one or more of the following features.

Variable R ₁₁₈ 、R ₁₁₉ 、R ₁₂₀ And R ₁₂₁

In certain embodiments, R₁₁₈、R₁₁₉、R₁₂₀And R₁₂₁Each being hydrogen. In other embodiments, each R is₁₁₈、R₁₁₉、R₁₂₀And R₁₂₁Independently selected from H, halo and NO₂. In yet other embodiments, R₁₁₈、R₁₁₉、R₁₂₀And R₁₂₁One (e.g., R)₁₂₀) Is halo, OH, CN, NO₂、C₁-C₃Alkyl radical, C₁-C₃Haloalkyl, C₁-C₃Alkoxy or C₁-C₃Haloalkoxy (e.g. halo, such as chloro; or NO)₂) (ii) a And others are hydrogen (e.g. R)₁₁₈、R₁₁₉、R₁₂₀And R₁₂₁One (e.g. R)₁₂₀) Is halo and NO₂And the others are hydrogen).

Variable R ₁₂₂

In certain embodiments, R₁₂₂May be-Z-R^a. Implementations may include one or more of the following features.

Z may be O.

Z may be a bond.

R^aCan be as follows:

(i)C₁-C₆alkyl or C₁-C₆Haloalkyl groups, each of which may consist of 1 to 3R^bOptionally substituted; or

(iii)C₇-C₁₁Aralkyl or heteroaralkyl containing 6 to 11 atoms, each of which consists of 1 to 5R^cOptionally substituted.

For example, R^aCan be as follows:

(i)C₁-C₆alkyl, each of which may be substituted by 1-3R^bOptionally substituted; or

(iii)C₇-C₁₁Aralkyl radicalA group consisting of 1 to 5R^cOptionally substituted.

R^aMay be C₇-C₁₁Aralkyl, or heteroaralkyl containing 6 to 11 atoms, each of which consists of 1 to 5R^cOptionally substituted (e.g., with 1-5R^cOptionally substituted C₇-C₁₁Aralkyl). For example, R^aCan be benzyl or phenethyl, wherein the phenyl moiety is substituted with 1-5 (e.g., 1-4, 1-3, 1-2, or 1 (e.g., 1-2 or 1)) R^c(e.g., halo (e.g., chloro); C₁-C₆Alkoxy (e.g., OCH)₃) (ii) a Or C₁-C₆Alkyl (e.g., CH)₃) Is optionally substituted. In certain embodiments, Z may be O.

R^aMay be C₁-C₆Alkyl or C₁-C₆Haloalkyl groups each consisting of 1 to 3R^bOptionally substituted (e.g., with 1-3R^bOptionally substituted C₁-C₆Alkyl groups). For example, R^aMay be CH₃. In certain embodiments, Z may be a bond.

In certain embodiments, R₁₂₂May be hydrogen.

Variable R ₁₂₃

In some embodiments, R₁₂₃Can be as follows:

(iii)C₆-C₁₀aryl or heteroaryl containing 5 to 10 atoms, each of which consists of 1 to 5R^dOptionally substituted; or

(iv)C₇-C₁₁Aralkyl, or heteroaralkyl containing 6 to 11 atoms, each of which consists of 1 to 5R^cOptionally substituted; or

(v)-(C₁-C₆Alkyl) -Z¹-(C₆-C₁₀Aryl) in which Z is¹Is O, S, NH or N (CH)₃) (ii) a The alkyl moiety consisting of 1-3R^bOptionally substituted; and the aryl moiety consists of 1-5R^dOptionally substituted.

For example, R₁₂₃Can be as follows:

(iii) from 1 to 5R^dOptionally substituted C₆-C₁₀An aryl group; or

(iv) From 1 to 5R^cOptionally substituted C₇-C₁₁Aralkyl group; or

(v)-(C₁-C₆Alkyl) -Z¹-(C₆-C₁₀Aryl) in which Z is¹Is O, S, NH or N (CH)₃) (ii) a The alkyl moiety consisting of 1-3R^aOptionally substituted; and the aryl moiety consists of 1-5R^dOptionally substituted.

In embodiments, R₁₂₃May be C₆-C₁₀Aryl or heteroaryl containing 5 to 10 atoms, each of which consists of 1 to 5R^dOptionally substituted (e.g., with 1-5R^dOptionally substituted C₆-C₁₀Aryl). For example, R₁₂₃Can be a mixture of 1-5 (e.g., 1-4, 1-3, 1-2, or 1) R^d(e.g., C)₁-C₆Alkoxy (e.g., OCH3)) optionally substituted phenyl.

In embodiments, R₁₂₃May be C₇-C₁₁Aralkyl, or heteroaralkyl containing 6 to 11 atoms, each of which consists of 1 to 5R^cOptionally substituted (e.g., with 1-5R^cOptionally substituted C₇-C₁₁Aralkyl). For example, R₁₂₃Can be benzyl or phenethyl, wherein the phenyl moiety consists of 1-5 (e.g., 1-4, 1-3, 1-2 or 1) R^c(e.g. halo (e.g. chloro); C₁-C₆Alkoxy (e.g., OCH)₃)；C₁-C₆Alkyl (e.g., CH)₃)；NH₂(ii) a Or hydroxy) is optionally substituted.

In embodiments, R₁₂₃May be- (C)₁-C₆Alkyl) -Z¹-(C₆-C₁₀Aryl) in which Z is¹Is O, S, NH or N (CH)₃) (ii) a The alkyl moiety consisting of 1-3R^bOptionally substituted; and the aryl moiety consists of 1-5R^dOptionally substituted. For example, R₁₂₃May be- (CH)₂)-Z¹- (phenyl) in which the phenyl moiety consists of 1-5 (e.g. 1-4, 1-3, 1-2 or 1) R^d(e.g. halo (e.g. chloro); C₁-C₆Alkoxy (e.g., OCH)₃)；C₁-C₆Alkyl (e.g., CH)₃)；NH₂Or hydroxy) is optionally substituted.

A subset of the compounds include those wherein:

R₁₂₂is-Z-R^aWherein R is^aMay be C₇-C₁₁Aralkyl, or heteroaralkyl containing 6 to 11 atoms, each of which consists of 1 to 5R^c(e.g., from 1-5R^cOptionally substituted C₇-C₁₁Aralkyl, such as benzyl or phenethyl) optionally substituted; and

R₁₂₃may be C₆-C₁₀Aryl or heteroaryl containing 5 to 10 atoms, each of which consists of 1 to 5R^d(e.g., from 1-5R^dOptionally substituted C₆-C₁₀Aryl, such as phenyl) is optionally substituted.

Embodiments may include one or more of the following features:

R^cand R^dMay be as defined anywhere herein.

Z may be O.

R₁₁₈、R₁₁₉、R₁₂₀And R₁₂₁Each of which is hydrogen. In other embodiments, R₁₁₈、R₁₁₉、R₁₂₀And R₁₂₁Each independently selected from H, halo and NO₂. In still other embodiments, R₁₁₈、R₁₁₉、R₁₂₀And R₁₂₁Of (e.g., R)₁₂₀) Is halo, OH, CN, NO₂、C₁-C₃Alkyl radical, C₁-C₃Haloalkyl, C₁-C₃Alkoxy or C₁-C₃Haloalkoxy (e.g., halo, such as chloro; or NO)₂) (ii) a And others are all hydrogen (e.g., R)₁₁₈、R₁₁₉、R₁₂₀And R₁₂₁(e.g., R)₁₂₀) Is halo or NO₂And others are hydrogen); for example, one of which is halo (e.g. chloro) or nitro, for example, halo (e.g. chloro), and the other is hydrogen.

For example:

R₁₂₂is-Z-R^aWherein Z is O, and R^aIs composed of 1-5R^cOptionally substituted C₇-C₁₁Aralkyl group; and is

R₁₂₃Is composed of 1-5R^dOptionally substituted to give C₆-C₁₀Aryl, and

R₁₁₈、R₁₁₉、R₁₂₀and R₁₂₁Each of (a) may be hydrogen; or R₁₁₈、R₁₁₉、R₁₂₀And R₁₂₁Each of which may be independently halo (e.g., chloro) or nitro, for example, halo (e.g., chloro); or R₁₁₈、R₁₁₉、R₁₂₀And R₁₂₁One of which may be halo (e.g. chloro) or NO₂For example, halo (such as chloro); and others are hydrogen.

As another example, Z is a bond, and the definitions in the above examples apply.

Examples of compounds having the formula shown in figure 6G include: CP-0000489, CP-0000540, CP-0000550, CP-0000553, CP-0000554, CP-0000557, CP-0000571, CP-0047659, CP-0064483, CP-0066829, CP-0069961, CP-0074806, CP-0080773, CP-0091818, CP-0109953, CP-0105772 and CP-0193184.

Other examples of compounds having the formula shown in figure 6G include:

in some embodiments, the compound may have the formula shown in fig. 6L:

in some embodiments:

x is O or S;

R₁₄₃、R₁₄₄、R₁₄₅and R₁₄₆Each of which is independently selected from H, halo, OH, CN, NO₂、C₁-C₃Alkyl radical, C₁-C₃Haloalkyl, C₁-C₃Alkoxy radical, C₁-C₃A haloalkoxy group; and-NHC (O) (C)₁-C₃Alkyl groups);

R₁₄₇is NR^eR^fWherein R is^eAnd R^fIs hydrogen or C₁-C₃An alkyl group; and R is^eAnd R^fThe other of (1) is:

(i)-C(O)R^g(ii) a Wherein R is^gIs C₆-C₁₀Aryl or heteroaryl containing 5 to 10 atoms, each of which consists of 1 to 5R^hOptionally substituted; or

(ii)C₁-C₃An alkyl group;

or

R₁₄₇Is C₆-C₁₀Aryl or heteroaryl containing 5 to 10 atoms, each of which consists of 1 to 5R^hOptionally substituted;

or

R₁₄₇is-SCH₂RⁱWherein R isⁱThe method comprises the following steps:

(i)C₆-C₁₀aryl or heteroaryl containing 5 to 10 atoms, each of which consists of 1 to 5R^hOptionally substituted; or

(ii)-C(O)NR^eR^fWherein R is^eAnd R^fIs hydrogen or C₁-C₃An alkyl group; and R is^eAnd R^fThe other of (A) is-C (O) R^g(ii) a Wherein R is^gIs C₆-C₁₀Aryl or heteroaryl containing 5 to 10 atoms, each of which consists of 1 to 5R^hOptionally substituted; and is

R^hAt each occurrence, independently:

(i) halogenating; NH (NH)₂；NH(C₁-C₃Alkyl groups); n (C)₁-C₃Alkyl radical)₂(ii) a A hydroxyl group; c₁-C₆Alkoxy or C₁-C₆A haloalkoxy group; a nitro group; or cyano; or

(ii)C₁-C₆Alkyl or C₁-C₆A haloalkyl group.

Implementations may include one or more of the following features.

Variable X

X may be S.

X may be O.

Variable R ₁₄₃ 、R ₁₄₄ 、R ₁₄₅ And R ₁₄₆

In certain embodiments, R₁₄₃、R₁₄₄、R₁₄₅And R₁₄₆Are each hydrogen. In other embodiments, R₁₄₃、R₁₄₄、R₁₄₅And R₁₄₆One of them is halogen, OH, CN, NO₂、C₁-C₃Alkyl radical, C₁-C₃Haloalkyl, C₁-C₃Alkoxy radical, C₁-C₃A haloalkoxy group; or-NHC (O) (C)₁-C₃Alkyl groups); and others are all hydrogen.

Variable R ₁₄₇

In certain embodiments, R₁₄₇May be NR^eR^fWherein R is^eAnd R^fIs hydrogen or C₁-C₃Alkyl (e.g., hydrogen); and R is^eAnd R^fThe other of (1) is:

(i)-C(O)R^g(ii) a Wherein R is^gIs C₆-C₁₀Aryl or heteroaryl containing 5 to 10 atoms, each of which consists of 1 to 5R^hOptionally substituted; or

(ii)C₁-C₃An alkyl group.

In embodiments, R₁₄₇May be NR^eR^fWherein R is^eAnd R^fIs hydrogen or C₁-C₃Alkyl (e.g., hydrogen); and R is^eAnd R^fThe other of (A) is-C (O) R^g(ii) a Wherein R is^gIs C₆-C₁₀Aryl or heteroaryl containing 5 to 10 atoms, each of which consists of 1 to 5R^hOptionally substituted.

By way of example, R^gCan be phenyl consisting of 1-5 (e.g., 1-4, 1-3, 1-2 or 1) R^h(e.g., halo (e.g., chloro)；C₁-C₆Alkoxy (e.g., OCH)₃) (ii) a Or C₁-C₆Alkyl (e.g., CH)₃) Is optionally substituted.

By way of example, R^gMay be a heteroaryl group containing 5 to 6 (e.g., 5) atoms consisting of 1 to 2 (e.g., 1) R^h(e.g., C)₁-C₆Alkyl (e.g., CH)₃) Is optionally substituted.

In certain embodiments:

x may be S; and is

R₁₄₇May be NR^eR^fWherein R is^eAnd R^fIs hydrogen or C₁-C₃Alkyl (e.g., hydrogen); and R is^eAnd R^fThe other of (1) is:

(i)-C(O)R^g(ii) a Wherein R is^gIs C₆-C₁₀Aryl or heteroaryl containing 5 to 10 atoms, each of which consists of 1 to 5R^hOptionally substituted; or

(ii)C₁-C₃An alkyl group;

(e.g., R)^eAnd R^fIs hydrogen or C₁-C₃Alkyl (e.g., hydrogen); and R is^eAnd R^fThe other of (A) is-C (O) R^g(ii) a Wherein R is^gIs C₆-C₁₀Aryl or heteroaryl containing 5 to 10 atoms, each of which consists of 1 to 5R^hOptionally substituted).

In certain embodiments, R₁₄₃、R₁₄₄、R₁₄₅And R₁₄₆Each being hydrogen. In other embodiments, R₁₄₃、R₁₄₄、R₁₄₅And R₁₄₆One of them is halogen, OH, CN, NO₂、C₁-C₃Alkyl radical, C₁-C₃Haloalkyl, C₁-C₃Alkoxy radical, C₁-C₃A haloalkoxy group; or-NHC (O) (C)₁-C₃Alkyl groups); and others are all hydrogen.

Examples of compounds having the formula shown in figure 6L include: CP-0064917, CP-0067233, CP-0068578, CP-0103014, CP-0105777, CP-0107060, CP-0029300, CP-0079983, and CP-0103978.

In some embodiments, the compound may have the formula shown in fig. 6A:

in some embodiments:

x is O or S;

R₁₀₀、R₁₀₁、R₁₀₂and R₁₀₃Each of which is independently selected from H, halo, OH, CN, NO₂、C₁-C₃Alkyl radical, C₁-C₃Haloalkyl, C₁-C₃Alkoxy radical, C₁-C₃A haloalkoxy group; or

R₁₀₀、R₁₀₁、R₁₀₂And R₁₀₃Any two adjacent pairs thereof, together with the carbon atoms to which they are attached, form a fused heterocyclic ring containing a total of 5 or 6 ring atoms; wherein the heterocyclic ring is independently selected from C₁-C₃1-3 substituents of alkyl and oxo are optionally substituted.

R₁₀₄is-C (O) NR^jR^kWherein R is^jAnd R^kIs hydrogen or C₁-C₃An alkyl group; and R is^jAnd R^kThe other of (1) is:

(i) c optionally substituted by 5-6 heterocyclyl₁-C₈An alkyl group; or

(ii) Heteroaryl comprising 5 to 6 atoms, optionally substituted with 1 to 5 substituents independently selected from halo、OH、CN、NO₂、C₁-C₃Alkyl radical, C₁-C₃Haloalkyl, C₁-C₃Alkoxy radical, C₁-C₃A haloalkoxy group; -C (O) NH₂；-NHC(O)(C₁-C₃Alkyl groups); and a fused C₅-C₆A cycloalkyl ring;

or

R₁₀₄Is a heteroaryl group containing 5 to 6 atoms optionally substituted with 1 to 5 substituents independently selected from halo, OH, CN, NO₂、C₁-C₃Alkyl radical, C₁-C₃Haloalkyl, C₁-C₃Alkoxy radical, C₁-C₃A haloalkoxy group; -C (O) NH₂；-NHC(O)(C₁-C₃Alkyl groups); and is

R₁₀₅Is halo or C₁-C₃An alkyl group.

Implementations include one or more of the following features.

Variable X

X may be S.

X may be O.

Variable R ₁₀₀ 、R ₁₀₁ 、R ₁₀₂ And R ₁₀₃

In certain embodiments, R₁₀₀、R₁₀₁、R₁₀₂And R₁₀₃Are each hydrogen. In other embodiments, R₁₀₀、R₁₀₁、R₁₀₂And R₁₀₃One of them is halo, OH, CN, NO₂、C₁-C₃Alkyl radical, C₁-C₃Haloalkyl, C₁-C₃Alkoxy radical, C₁-C₃A haloalkoxy group; or-NHC (O) (C)₁-C₃Alkyl groups); and the others are hydrogen.

Variable R ₁₀₄

In certain embodiments, R₁₀₄is-C (O) NR^jR^kWherein R is^jAnd R^kIs hydrogen or C₁-C₃Alkyl (e.g., hydrogen); and R is^jAnd R^kThe other of (1) is:

(i) c optionally substituted by 5-6 heterocyclyl₁-C₈An alkyl group; or

(ii) Heteroaryl comprising 5 to 6 atoms optionally substituted with 1 to 5 substituents independently selected from halo, OH, CN, NO₂、C₁-C₃Alkyl radical, C₁-C₃Haloalkyl, C₁-C₃Alkoxy radical, C₁-C₃A haloalkoxy group; a cyano group; -C (O) NH₂；-NHC(O)(C₁-C₃Alkyl groups); c₁-C₃An alkyl group; c₁-C₃An alkyl group; and a fused C₅-C₆A cycloalkyl ring.

By way of example, R^jAnd R^kIs hydrogen or C₁-C₃Alkyl (e.g., hydrogen); and R is^jAnd R^kIs a heteroaryl group comprising 5 to 6 atoms, optionally substituted with 1 to 5 substituents independently selected from halo, OH, CN, NO₂、C₁-C₃Alkyl radical, C₁-C₃Haloalkyl, C₁-C₃Alkoxy radical, C₁-C₃A haloalkoxy group; -C (O) NH₂；-NHC(O)(C₁-C₃Alkyl groups); and a fused C₅-C₆A cycloalkyl ring.

Variable R ₁₀₅

R₁₀₅Can be chlorine or methyl.

Examples of compounds having the formula shown in figure 6A include: CP-0079175, CP-0087336, CP-0064314, CP-0068577, and CP-0102404.

In some embodiments, the compound may have the formula shown in fig. 3A:

in certain embodiments:

R₄₃is C₆-C₁₀Aryl or heteroaryl containing 5 to 10 atoms, each of which consists of 1 to 5R^mOptionally substituted.

R₄₄The method comprises the following steps:

(i)C₆-C₁₀aryl or heteroaryl containing 5 to 10 atoms, each of which consists of 1 to 5R^mOptionally substituted; or

(ii)-Z⁴-(C₁-C₆Alkyl), wherein:

Z⁴is a bond or NH; and is

C₁-C₆The alkyl group is substituted with one of:

(a) a heterocycle comprising 5-6 atoms optionally substituted with 1-3 substituents independently selected from oxo and C₁-C₆An alkyl group; or

(b) From 1 to 5R^mOptionally substituted phenoxy; and is

R^mAt each occurrence, independently:

(i) halogenating; NH (NH)₂；NH(C₁-C₃Alkyl groups); n (C)₁-C₃Alkyl radical)₂(ii) a A hydroxyl group; c₁-C₆Alkoxy or C₁-C₆A haloalkoxy group; a nitro group; or cyano; or

(ii)C₁-C₆Alkyl or C₁-C₆A haloalkyl group.

Implementations include one or more of the following features.

Variable R ₄₃

In certain embodiments, R₄₃May be C₆-C₁₀Aryl consisting of 1-5R^mOptionally substituted. For example, R₄₃Can be phenyl consisting of 1-5 (e.g., 1-4, 1-3, 1-2, or 1) R^m(e.g., C)₁-C₆Alkyl (e.g. CH)₃) Is optionally substituted.

In certain embodiments, R₄₃May be a heteroaryl group containing 5 to 6 atoms, each of which consists of 1 to 5R^mOptionally substituted.

Variable R ₄₄

In certain embodiments, R₄₄May be C₆-C₁₀Aryl consisting of 1-5R^mOptionally substituted. For example, R₄₄Can be phenyl consisting of 1-5 (e.g., 1-4, 1-3, 1-2, or 1) R^m(e.g. halo (e.g. chloro); C₁-C₆Alkoxy (e.g. OCH)₃) (ii) a Or C₁-C₆Alkyl (e.g. CH)₃) Is optionally substituted.

Compounds having the formula shown in figure 3A include: CP-0067108, CP-0067246, CP-0068395, CP-0068929, CP-0068961, CP-0070164, CP-0070367, CP-0079642, CP-0104904, and CP-0130665.

In certain embodiments, the compound may have the formula shown in fig. 3U:

in some embodiments:

R₆₇and R₆₈Each is independently:

(i) hydrogen; or

(ii)C₆-C₁₀Aryl or heteroaryl containing 5 to 10 atoms, each of which consists of 1 to 5RⁿOptionally substituted; or

(iii)NH₂(ii) a Or

(iv)-C(O)(C₁-C₆Alkyl groups);

R⁶⁹is NR^oR^pWherein R is^oAnd R^pIs hydrogen or C₁-C₃An alkyl group; and R is^oAnd R^pThe other of (1) is:

(i) hydrogen; or

(ii)C₆-C₁₀Aryl or heteroaryl containing 5 to 6 atoms, from 1 to 5RⁿOptionally substituted; or

(iii)-C(O)(C₁-C₆Alkyl) in which C₁-C₆Alkyl is substituted by phenoxy consisting of 1-5RⁿOptionally substituted;

Rⁿat each occurrence, independently:

(i) halogenating; NH (NH)₂；NH(C₁-C₃Alkyl groups); n (C)₁-C₃Alkyl radical)₂(ii) a A hydroxyl group; c₁-C₆Alkoxy or C₁-C₆A haloalkoxy group; a nitro group; or cyano; or

(ii)C₁-C₆Alkyl or C₁-C₆A haloalkyl group; or

(iii) A phenyl group.

Implementations may include one or more of the following features.

Variable R ₆₇ And R ₆₈

In certain embodiments, R₆₇And R₆₈Is one of C₆-C₁₀Aryl or heteroaryl containing 5 to 10 atoms, each of which consists of 1 to 5RⁿOptionally substituted; and the other is hydrogen.

Variable R ₆₉

In certain embodiments, R^oAnd R^pIs hydrogen or C₁-C₃Alkyl (e.g., hydrogen); and R is^oAnd R^pIs another of C₆-C₁₀Aryl or heteroaryl containing 5 to 6 atoms consisting of 1 to 5RⁿOptionally substituted.

Examples of compounds having the formula shown in figure 3U include: CP-0063182, CP-0071862, CP-0072036, CP-0105343, CP-0122949, and CP-0134381.

In certain embodiments, the compound may have the formula shown in fig. 3E:

in some embodiments:

x is O or S;

R₅₀and R₅₃Each is independently:

(i) hydrogen; or

(ii)-C(O)R^q(ii) a Or

(iii)C₆-C₁₀Aryl or heteroaryl containing 5 to 10 atoms, each of which consists of 1 to 5R^rOptionally substituted;

provided that R is₅₀And R₅₃At least one of which is not hydrogen;

R₅₁and R₅₂Each is independently hydrogen or halo;

R^qthe method comprises the following steps:

(i)C₁-C₆an alkyl group; or

(ii)-NR^sR^tWherein:

(a)R^sand R^tOne of which is hydrogen and the other is C₆-C₁₀Aryl or heteroaryl containing 5 to 10 atoms, each of which consists of 1 to 5R^rOptionally substituted; c substituted by phenoxy₁-C₆Alkyl, the phenoxy radical consisting of 1-5R^rOptionally substituted; or-O-N ═ C (NH)₂)(C₆-C₁₀Aryl) wherein the aryl moiety is substituted with 1 to 5R^rOptionally substituted; or

(b)R^sAnd R^tTogether with the nitrogen atom to which each is attached form a heterocyclic ring containing 5 to 6 atoms; or

(iii)-NH-C(O)(C₆-C₁₀Aryl) wherein the aryl moiety is substituted with 1 to 5R^rOptionally substituted; and is

R^rAt each occurrence, independently:

(i) halogenating; NH (NH)₂；NH(C₁-C₃Alkyl groups); n (C)₁-C₃Alkyl radical)₂(ii) a A hydroxyl group; c₁-C₆Alkoxy or C₁-C₆A haloalkoxy group; a nitro group; or cyano; or

(ii)C₁-C₆Alkyl or C₁-C₆A haloalkyl group.

In certain embodiments, R₅₀And R₅₃One of which is-C (O) R^q(ii) a And R₅₀And R₅₃Is hydrogen or C₆-C₁₀Aryl or heteroaryl containing 5 to 10 atoms, each of which consists of 1 to 5R^rOptionally substituted. In some embodiments, R^qMay be-NR^sR^t.

Examples of compounds having the formula shown in figure 3E include: CP-0061777, CP-0066008, CP-0072253, CP-0099289, CP-0008545, CP-0060852, CP-0072156, CP-0072271, CP-0104766, and CP-0110352.

In certain embodiments, the compound has the formula shown in figure 3N:

in some embodiments:

R₆₁、R₆₂and R₆₄Each is independently:

(i) hydrogen; or

(ii)C₆-C₁₀Aryl or heteroaryl containing 5 to 10 atoms, each of which consists of 1 to 5R^uOptionally substituted; or

(iii)-NH-C(O)(C₆-C₁₀Aryl) wherein the aryl moiety is substituted with 1 to 5R^uOptionally, optionallySubstitution; or

(iv)-C(O)NR^vR^wWherein R is^vAnd R^wOne of which is hydrogen; and R is^vAnd R^wIs another of C₆-C₁₀Aryl consisting of 1-5R^uOptionally substituted; or C₇-C₁₁Aralkyl is optionally substituted by oxo; or

(v)NH₂Or a hydroxymethyl group;

R₆₃the method comprises the following steps:

(i) hydrogen; or

(ii)C₆-C₁₀Aryl or heteroaryl containing 5 to 10 atoms, each of which consists of 1 to 5R^uOptionally substituted; or

(iii)C₁-C₆An alkyl group; and

R^uat each occurrence, independently:

(i) halogenating; NH (NH)₂；NH(C₁-C₃Alkyl groups); n (C)₁-C₃Alkyl radical)₂(ii) a A hydroxyl group; c₁-C₆Alkoxy or C₁-C₆A haloalkoxy group; a nitro group; or cyano; or

(ii)C₁-C₆Alkyl or C₁-C₆A haloalkyl group.

In certain embodiments, R₆₁、R₆₂And R₆₄Two of which are not hydrogen.

Examples of compounds having the formula shown in figure 3N include: CP-0000477, CP-0063375, CP-0064231, CP-0065105, CP-0070844, CP-0070886, and CP-0104765.

In certain embodiments, the compound may have the formula shown in figure 3V.

In certain embodiments, R₇₀May be as described anywhere hereinAn amide (i.e., having the formula-C (O) NRR ') or a trans-amide (i.e., having the formula-NR "C (O) R').

In certain embodiments, R₇₁May be hydrogen.

In certain embodiments, R₇₂Can be as follows:

(i)C₁-C₆an alkyl group; or

(ii)C₆-C₁₀Aryl or heteroaryl containing 5 to 10 atoms, each of which is optionally substituted with 1 to 5 substituents independently selected from halo; NH (NH)₂；NH(C₁-C₃Alkyl groups); n (C)₁-C₃Alkyl radical)₂(ii) a A hydroxyl group; c₁-C₆Alkoxy or C₁-C₆A haloalkoxy group; a nitro group; a cyano group; c₁-C₆An alkyl group; and C₁-C₆A haloalkyl group.

Examples of compounds having the formula shown in figure 3V include: CP-0065665, CP-0075627, and CP-0075656.

In certain embodiments, the compound can have the formula shown in figure 7D.

In certain embodiments, the pyrimidine ring may be substituted with 1-2 substituents independently selected from the group consisting of:

(i) heterocyclyl containing 5 to 6 atoms; or

(ii)C₆-C₁₀Aryl or heteroaryl containing 5 to 10 atoms, each of which is optionally substituted with 1 to 5 substituents independently selected from halo; NH (NH)₂；NH(C₁-C₃Alkyl groups); n (C)₁-C₃Alkyl radical)₂(ii) a A hydroxyl group; c₁-C₆Alkoxy or C₁-C₆A haloalkoxy group; a nitro group; a cyano group; c₁-C₆An alkyl group; and C₁-C₆A haloalkyl group.

In other embodiments, the pyrimidine rings may be substituted with fused rings.

Examples of compounds having the formula shown in figure 7D include: CP-0059547, CP-0059563, CP-0059642, CP-0064382, CP-0067053, CP-0072720, and CP-0079810.

In certain embodiments, the compound may have the formula shown in figure 7A.

In certain embodiments, the pyridine ring may be substituted with an amide or a trans-amide as described anywhere herein.

In other embodiments, the pyrimidine rings may be substituted with one or more fused rings.

Examples of compounds having the formula shown in figure 7A include: CP-0060729, CP-0066751, CP-0069934, CP-0076627, CP-0080276, CP-0089966, CP-0029278, and CP-0130586.

Mixtures of any of the compounds described herein may also be used in any of the methods described herein.

Synthesis method

The compounds of the invention are commercially available from suppliers such as Bionet, Maybrid, Chemdiv, ChemBridge, Peakdale, IFLAB/Life Chemicals, amine, Microsource or Timtec. Alternatively or additionally, commercially available starting materials and reagents can be synthesized according to the methods described herein (or variations thereof) and/or conventional methods, or the compounds described herein can be synthesized according to starting materials and reagents prepared by conventional chemical synthesis methods. The compounds described herein can be isolated from the reaction mixture and further purified by, for example, column chromatography, High Performance Liquid Chromatography (HPLC), or recrystallization. Further methods of synthesizing the compounds of the formulae herein will be apparent to those skilled in the art, as will be appreciated by those skilled in the art. In addition, the various synthetic steps may be performed in alternating order or sequence in order to provide the desired compounds. For the synthesis ofMethods of synthetic chemical transformation of compounds and protecting groups (protection and deprotection) are known in the art and include, for example, those described by the following references, Larock,Comprehensive Organic Transformations(Integrated organic transformations), 2 nd edition, Wiley-VCH Publishers (1999); (ii) Wuts and Greene,Protective Groups in Organic Synthesis(protecting groups in organic synthesis), 4 th edition, John Wiley and Sons (2007); fieser and Fieser, Fieser and Fieser's Reagents for Organic Synthesis (Fieser's Reagents for Organic Synthesis), John Wiley and Sons (1994); and Paquette et al, encyclopedia of Reagents for Organic Synthesis (encyclopedia of Organic Synthesis Reagents), John Wiley and sons (1995), and its successors.

Benzimidazole-containing compounds

Compounds having the formula shown in figure 6G can be obtained commercially or synthesized using conventional synthetic methods. For example, compounds CP-0000489, CP-0000540, CP-0000550, CP-0000553, CP-0000554, CP-0000557, CP-0000571, CP-0047659, CP-0064483, CP-0066829, CP-0069961, CP-0074806, CP-0080773, CP-0091818, CP-0105772, and CP-0109953 are commercially available from the suppliers provided in Table 1 (entries 1-17). Other benzimidazoles 1-14 (scheme 1) described in this disclosure are commercially available.

Other compounds having the formula shown in FIG. 6G can be prepared, for example, by using Kokare et al, proteins&Peptide Letters, 14: 259-263, 2007, which describes the synthesis of CP-0000540. Benzimidazole analogs, which introduce variations into specific portions of the molecule, can be prepared according to scheme 2 using well-established chemistry. Where the 1H-benzimidazole intermediate is commercially available, the alkylation reaction may be carried outTo introduce said R₁Substituents (scheme A). Other processes of commercial benzimidazoles can be used to incorporate different substituents at different positions on the molecule. However, for asymmetrically substituted 1H-benzimidazoles, both other routes may be used. At R₃In the case of electron withdrawing groups, nucleophilic aromatic substitution of 2-fluoronitrobenzene can be used to provide the 2-aminonitrobenzene intermediate (scheme B). For other analogues, introduction of R may be carried out₁Alkylation of the group 2-aminonitrobenzene (scheme C) to provide the same intermediate. The reduction of the nitro group to the amino group can be carried out by well-established reduction methods. Oxidative cyclization of such 1, 2-diamines with aldehydes or condensation with carboxylic acids will provide the desired benzimidazole analogs.

Error! The target cannot be generated by editing the domain code.

Scheme 2: synthesis of benzimidazole analogs

Benzothiazole-containing compounds

Compounds having the formula shown in fig. 6L (X ═ S) can be obtained commercially or synthesized using conventional synthetic methods. For example, compounds CP-0064917, CP-0067233, CP-0068578, CP-0103014, CP-0105777, and CP-0107060 are commercially available from the suppliers provided in Table 1 (entries 18-23). For example, compounds having the formula shown in fig. 6L (X ═ S) can be obtained by cyclization of an o-halobenzamide with lawson' S reagent or by oxidation of a thioaniline. For example, with Song et al, eur.j.med.chem.43 (7): 1519 ═ S (X ═ S) can also be obtained by the chemical method described in 1519-1524, 2008.

Benzoxazole-containing compounds

Compounds having the formula shown in fig. 6L (X ═ O) can be obtained commercially or synthesized using conventional synthetic methods. For example, compounds CP-0029300, CP-0079983, and CP-0103978 were obtained commercially from the suppliers provided in Table 1 (entries 24-27). For example, with Boyd, sci. synth.11: 481-492, 2002, can also give other compounds having the formula shown in fig. 6L (X ═ O).

Quinazolinone-containing compounds

The quinazolinone derivatives included in fig. 2G may be obtained commercially or synthesized using conventional synthetic methods. For example, compounds CP-0034360 and CP-0036187 were obtained commercially from the suppliers provided in Table 1 (entries 27-28). For example, with Connolly et al, Tetrahedron61 (43): 10153 other compounds having the formula shown in FIG. 2C (G) can also be obtained by the chemical method described in 10202, 2005.

Compounds comprising benzimidazolopyrimidines

The benzimidazolopyrimidine compounds having the formula shown in fig. 6I can be obtained commercially or synthesized using conventional synthetic methods. For example, compounds CP-0050095 and CP-0131763 were obtained commercially from the suppliers provided in Table 1 (entries 29-30).

Benzofuran-containing compounds

Compounds having the formula shown in fig. 6A (X ═ O) can be obtained commercially or synthesized using conventional synthetic methods. For example, compounds CP-0079175 and CP-0087336 were obtained commercially from the suppliers provided in Table 1 (entries 31-32). For example, with Hou et al, Progress in heterocyclic chemistry 17: 142-171, 2005, other compounds having the formula shown in fig. 6A (X ═ O) can also be obtained.

Benzothiophene-containing compounds

Compounds having the formula shown in fig. 6A (X ═ S) can be obtained commercially or synthesized using conventional synthetic methods. For example, compounds CP-0064314, CP-0068577, and CP-0102404 are commercially available from the suppliers provided in Table 1 (entries 33-35). For example, the compounds are prepared by using Bravo et al, j.heterocyclic chem., 7 (4): 967-8, 1970, or Rayner et al, sci. synth.10: 155 ═ S, another compound having the formula shown in fig. 6A can also be obtained by the chemical method described in fig. 181, 2005.

Indole-containing compounds

Compounds having the formula shown in fig. 6J can be obtained commercially or synthesized using conventional synthetic methods. For example, compounds CP-0010539, CP-0072096, CP-0078448, and CP-0103978 are commercially available from the suppliers provided in Table 1 (entries 36-38). Other compounds having the formula shown in fig. 6J may also be obtained, for example, using hummphrey et al, chem.rev., 106 (7): 2875 2911, 2006.

Quinoline-containing compounds

The quinoline derivatives included in fig. 7E and 7F can be obtained commercially or synthesized using conventional synthetic methods. For example, compounds CP-0072092 and CP-0087799 were obtained commercially from the suppliers provided in Table 1 (entries 39-40). Other quinoline compounds are also available, for example, using Larsen et al, sci. 389 + 549, 2005.

Compounds comprising benzotriazole

The compounds having the formula shown in fig. 6O can be obtained commercially or synthesized using conventional synthetic methods. For example, compounds CP-0009883 and CP-0070871 were obtained commercially from the suppliers provided in Table 1 (entries 41-42). Other compounds having the formula shown in fig. 6O may also be obtained, for example, using Katritzky et al, chem.rev.98 (2): 409, 548, 1998.

The compounds of the present invention comprising coumarin, benzopyran, tetrahydroquinoline, benzopyranone and benzopyrazine may be obtained commercially or synthesized using conventional synthetic methods. For example, the compounds CP-0063508, CP-0000928, CP-0005069, CP-0096433, and CP-0045061 contained in FIG. 1 were obtained commercially from the suppliers provided in Table 1 (entries 43-47). Other compounds including coumarin, benzopyran, tetrahydroquinoline, benzopyranone and benzopyrazine are available, for example, using Borges et al, curr.med.chem.12 (8): 887 916, 2005; schweizer et al, Chemistry of Heterocyclic Compounds 31: 11-139, 1977; katritzky et al, Tetrahedron 52 (48): 15031-15070, 1996; williams et al, sci. synth.14: 347 638, 2003; kress et al, Progress in Heterocyclic Chemistry 4: 186-203, 1992.

Pyridine-containing compound

The compounds having the formula shown in fig. 7A can be obtained commercially or synthesized using conventional synthetic methods. For example, compounds CP-0060729, CP-0066751, CP-0069934, CP-0076627, CP-0080276, CP-0089966, CP-0029278, and CP-0130586 are commercially available from the suppliers provided in Table 1 (entries 48-55). Other compounds having the formula shown in fig. 7A may also be obtained, for example, using Li et al, Bioorg. & med. chem.lett.17 (8): 2347-: 11-255, 2005.

Pyrimidine-containing compounds

The compounds having the formula shown in fig. 7D can be obtained commercially or synthesized using conventional synthetic methods. For example, compounds CP-0059547, CP-0059563, CP-0059642, CP-0064382, CP-0067053, CP-0072720, and CP-0079810 are commercially available from the suppliers provided in Table 1 (entries 56-62). Other compounds having the formula shown in FIG. 7D may also be obtained, for example, using Luo et al Tetrahedron Lett.43(33), 5739-5742, 2002, or von anger et al Sci. Synth.16: 379-572, 2004.

Furan-containing compounds

Compounds having the formula shown in fig. 3E (X ═ O) can be obtained commercially or synthesized using conventional synthetic methods. For example, compounds CP-0061777, CP-0066008, CP-0072253, and CP-0099289 were obtained commercially from the suppliers provided in Table 1 (entries 63-66). Other compounds having the formula shown in fig. 3E (X ═ O) can also be obtained, for example, using Kort et al, j.med.chem.51 (3): 407 + 416, 2008, or Konig et al, sci. synth.9: 183-286, 2001.

Thiophene-containing compounds

Compounds having the formula shown in fig. 3E (X ═ S) can be obtained commercially or synthesized using conventional synthetic methods. For example, the compounds CP-0008545, CP-0060852, CP-0072156, CP-0072271, CP-0104766 and CP-0110352 are commercially available from the suppliers provided in Table 1 (entries 67-72). Other compounds having the formula shown in fig. 3E (X ═ S) can also be obtained, for example, using Kaizerman et al, j.med.chem.46 (18): 3914 3929, 2003 or Schatz et al, sci. synth.10: 287-392, 2001.

Thiazole-containing compounds

Compounds having the formula shown in figure 3U can be obtained commercially or synthesized using conventional synthetic methods. For example, compounds CP-0063182, CP-0071862, CP-0072036, CP-0105343, CP-0122949, and CP-0134381 are commercially available from the suppliers provided in Table 1 (entries 73-78). Other compounds having the formula shown in FIG. 3U may also be obtained, for example, using Narayana et al, phosphorous, Sulfur and Silicon and the Related Elements 181 (6): 1381-1389, 2006, or Kikelj et al, sci. synth.11: 627-806, 2002.

Pyrazole-containing compounds

The compounds having the formula shown in fig. 3N can be obtained commercially or synthesized using conventional synthetic methods. For example, compounds CP-0000477, CP-0063375, CP-0064231, CP-0065105, CP-0070844, CP-0070886, and CP-0104765 are commercially available from the suppliers provided in Table 1 (entries 79-85). Other compounds having the formula shown in fig. 3N may also be obtained, for example, using McKeown et al, Bioorg. & med. chem.lett., 16 (18): 4767-: 15-226, 2003.

Isoxazole-containing compounds

Compounds having the formula shown in figure 3V can be obtained commercially or synthesized using conventional synthetic methods. For example, compounds CP-0065665, CP-0075627, and CP-0075656 were obtained commercially from the suppliers provided in Table 1 (entries 86-88). Other compounds having the formula shown in fig. 3V may also be obtained, for example, using Wakefield, sci. 229-288, 2002.

Compounds comprising oxadiazoles

The compounds having the formula shown in fig. 3A can be obtained commercially or synthesized using conventional synthetic methods. For example, compounds CP-0067108, CP-0067246, CP-0068395, CP-0068929, CP-0068961, CP-0070164, CP-0070367, CP-0079642, CP-0104904, and CP-0130665 are commercially available from the suppliers provided in Table 1 (entries 89-98). Other compounds having the formula shown in fig. 3A may also be obtained, for example, using Grant et al, j.org.chem.73 (18): 7219-: 127, 184, 2004.

Compounds comprising benzamide

The compounds having the formula shown in fig. 2A can be obtained commercially or synthesized using conventional synthetic methods. For example, compounds CP-0005186, CP-0007991, and CP-0061566 were obtained commercially from the suppliers provided in Table 1 (entries 99-101). Other compounds having the formula shown in figure 2A may also be obtained using methods well known to those skilled in the art, for example, by condensation of the corresponding benzoic acid and amine.

Compounds containing 1, 3, 4-oxadiazole, triazoline, pyrazoline, dihydropyridinone, triazole, indoline and imidazotriazine are commercially available or can be synthesized using conventional synthetic methods. For example, the compounds CP-0062030, CP-0007994, CP-0039073, CP-0004116, CP-0061401, CP-0064286, CP-0110644, and CP-0051092 were commercially available from the suppliers provided in Table 1 (entry 102-.

TABLE 1

^AThese system names provided in table 1 were generated using ChemDraw Ultra software 9.0.1 version as follows. The system name was generated by entering the chemical structure shown in table 1 in the ChemDraw drawing window, selecting the compound, and selecting the "convert structure to name" tool in the structure menu.

Method of treatment

The present invention provides methods relating to the treatment of diseases and/or conditions that would benefit from increased expression of Atoh1 using the compounds described herein. In general, the methods of treatment comprise increasing the level of Atoh1 expression using one or more of the compounds described herein, and thereby promoting partial or total differentiation of the target cell. Diseases that may benefit from such treatment are those in which one or more symptoms of the disease are treated by increasing the expression level of Atoh1, for example, those in which fully or partially differentiated cells are caused by increased expression of Atoh1, (1) used to replace lost or damaged cells or tissue, e.g., functional cells such as, e.g., auditory hair cells, and/or (2) to prevent expansion of damaged cell populations, e.g., cancer cells.

In general, the present invention provides a step of administering one or more compounds described herein to a patient. Alternatively or additionally, the invention provides the step of contacting one or more target cells, e.g., stem cells, iPS cells, progenitor cells and/or support cells, with one or more compounds described herein (e.g., in vitro) to promote complete or partial differentiation of these cells into or towards mature cell types (e.g., hair cells); a step of administering to the patient one or more cells, e.g., cells, progenitor cells, and/or supporting cells, that have been contacted (e.g., in vitro) with one or more compounds described herein; and/or administering one or more cells, e.g., cells, progenitor cells, and/or supporting cells, that have been contacted with one or more compounds described herein (e.g., in vitro) to a patient in combination with one or more compounds.

Loss of auditory hair cells

It is widely accepted that although cells capable of producing hair cells are present in the inner ear, natural hair cell regeneration in the inner ear is low (Li et al, Trends mol. Med., 10, 309-. Thus, lost or damaged hair cells may not be sufficiently replaced by natural physiological processes (e.g., cell differentiation) and thus loss of hair cells occurs. In many individuals, such hair cell loss can result in, for example, sensorineural hearing loss, hearing impairment, and imbalance disorders. Therapeutic strategies that increase hair cell numbers in the inner ear would be beneficial to patients with hair cell loss, e.g., patients suffering from one or more of these conditions.

The importance of Atoh1 in hair cell production is well documented. For example, Math1 is required for hair cell development and differentiation of inner ear progenitor cells into inner ear support cells and/or hair cells (Bermingham et al, Science, 284: 1837-. Furthermore, adenovirus-mediated overexpression of Math1 in the endolymph of mature guinea pigs resulted in differentiation of immature hair cells from non-sensory cells in the mature cochlea (Kawamoto et al, J.Neurosci., 23: 4395-Asca 4400, 2003). The implications of these studies are twofold. First, they demonstrate that non-sensory cells of the mature cochlea retain the ability to differentiate into sensory cells, such as hair cells. Second, they indicate that overexpression of Math1 is necessary and sufficient to direct differentiation of hair cells from non-sensory cells. Subsequent studies have prompted these findings by demonstrating that adenovirus-mediated Atoh1 overexpression induces hair cell regeneration and dramatically increases the hearing threshold in experimental deafness animal models. (IZUmikawa et al, nat. Med., 11: 271-276, 2005)

The compounds provided herein are capable of increasing the level of Atoh1 in a subject and/or in a cell or tissue. As described herein, these compounds promote increased expression of Atoh1, thereby promoting differentiation or trending of the target cell or cells into inner ear sensory cells or cells, e.g., hair cells. The use of these compounds to promote differentiation into hair cells from cells residing in the ear or cells competent to differentiate into hair cells is supported by the laboratory data described above by Bermingham et al, supra, Kawamoto et al, supra, and Izumikawa et al, supra. Accordingly, the compounds described herein are useful for treating those diseases and conditions caused by hair cell loss in a patient.

The present invention provides compounds and methods for treating patients suffering from or at risk of developing an auditory disorder caused by hair cell loss. In some embodiments, the method of treatment comprises the steps of: one or more compounds described herein are administered to a patient to, for example, promote auditory hair cell formation in the patient's ear (e.g., inner ear) and/or increase the number of auditory hair cells in the patient's ear (e.g., inner ear) by promoting the complete or partial differentiation of naturally occurring non-hair cell types in the patient's inner ear into auditory hair cells.

In some embodiments, the method of treatment comprises the steps of: one or more compounds described herein are administered to a patient to promote formation of auditory hair cells (e.g., internal and/or external auditory hair cells) in the inner ear of the patient and/or to increase the number of auditory hair cells (e.g., internal and/or external auditory hair cells) in the inner ear of the patient by promoting the complete or partial differentiation of naturally occurring non-hair cell types in the inner ear of the patient into auditory hair cells.

Examples of cells (e.g., inner and/or outer hair cells) that can differentiate into hair cells include, but are not limited to: inner ear stem cells, iPS cells, progenitor cells, and/or support cells (e.g., Deiters cells, pillar cells, inner finger cells, roof cells, and Hensen cells).

The invention also includes the step of contacting one or more cells having the ability to fully or partially differentiate into hair cells with one or more compounds described herein (e.g., in vitro) to promote full or partial differentiation of these cells into or towards mature cell types of the inner ear, e.g., hair cells (e.g., inner and/or outer hair cells). Exemplary cells having differentiation into hair cells include, but are not limited to: stem cells (e.g., inner ear stem cells, adult stem cells, bone marrow-derived stem cells, embryonic stem cells, mesenchymal stem cells, skin stem cells, iPS cells, and adipose-derived stem cells), progenitor cells (e.g., inner ear progenitor cells), supporting cells (e.g., Deiters cells, pillar cells, inner finger cells, roof cells, and Hensen cells), and/or germ cells.

Alternatively or additionally, the method comprises the step of administering to the ear (e.g., inner ear) of the patient (cell therapy) one or more cells (e.g., inner and/or outer hair cells) that have the ability to differentiate into hair cells and have been contacted (e.g., in vitro) with one or more compounds described herein. Finally, the method includes the step of administering to the ear (e.g., inner ear) of the patient (combination therapy) one or more cells (e.g., inner and/or outer hair cells) that have the ability to differentiate into hair cells and have been contacted (e.g., in vitro) with one or more compounds described herein in combination with one or more compounds.

The invention is useful for treating hair cell loss resulting from loss of cells in the ear, as well as any condition, e.g., the presence of hearing impairment, deafness, and vestibular disorders, by, e.g., promoting differentiation (e.g., complete or partial differentiation) of one or more cells into one or more cells having a function, e.g., sensory cells in the ear, such as hair cells.

In some embodiments, the method comprises the step of selecting a patient at risk of and/or having hair cell loss. Alternatively or additionally, the method comprises the step of selecting a patient at risk for and/or suffering from sensorineural hearing loss. For example, any person who is experiencing or at risk of developing hearing loss is a candidate for the treatment methods described herein. A person suffering from or at risk of developing a hearing loss may have less hearing than the average level of normal persons, or than the person before experiencing the hearing loss. For example, hearing may be reduced by at least 5%, 10%, 30%, 50%, or more.

The subject may be hearing loss associated with hair cell loss due to any cause or as a result of any type of event. For example, a person may be deaf due to genetic or congenital defects; for example, a person may be deaf since birth, or may be deaf or have difficulty hearing due to a gradual loss of hearing due to genetic or congenital defects. In another example, the person may be deaf or have difficulty hearing due to a traumatic event, such as physical trauma to an ear structure, or sudden loud noise, or prolonged exposure to loud noise. For example, prolonged exposure to concert venues, airport runways, and building areas can cause inner ear damage and subsequent hearing loss. Humans may experience chemical-induced ototoxicity, among which otoxins include: therapeutic agents including antineoplastic agents, salicylates, quinones, aminoglycoside antibiotics, contaminants in food or pharmaceuticals, and environmental or industrial contaminants. A person may have an aging-induced hearing disorder, or a person may have tinnitus (characterized by a ringing in the ear).

Humans suitable for treatment using the compounds and methods described herein include humans with vestibular dysfunction, including both bilateral and unilateral vestibular dysfunction. Vestibular dysfunction is a disorder of the inner ear whose symptoms are characterized by dizziness, imbalance, vertigo, nausea and blurred vision, and may be accompanied by hearing problems, fatigue and changes in cognitive function. Vestibular dysfunction may be due to genetic or congenital defects; infections, such as viral or bacterial infections; or an injury, such as a traumatic or non-traumatic injury. The most common detection of vestibular dysfunction is by detecting individual symptoms of the disorder (e.g., vertigo, nausea, and blurred vision).

Alternatively or additionally, the compounds and methods described herein may be used prophylactically, such as to prevent, reduce or delay the progression of hearing loss, deafness, or other auditory disorders associated with loss of inner ear function. For example, a composition comprising one or more compounds can be administered with (e.g., before, after, or simultaneously with) a second therapy, such as a therapy that can affect an auditory disorder. These ototoxic drugs include antibiotics such as neomycin, kanamycin, amikacin, erythromycin, gentamicin, tobramycin, erythromycin, vancomycin, and streptomycin; chemotherapeutic agents, such as cisplatin; non-steroidal anti-inflammatory drugs (NSAIDs) such as choline magnesium trisalicylate, diclofenac, diflunisal, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, salsalate, sulindac, and tolmetin; a diuretic; salicylates, such as aspirin; and certain malaria treatments such as quinine and chloroquine. For example, a human receiving chemotherapy may be treated using the compounds and methods described herein. For example, the chemotherapeutic agent cisplatin is known to cause hearing loss. Thus, a composition comprising one or more compounds can be administered with (e.g., before, after, or simultaneously with) cisplatin treatment to prevent or reduce the severity of the cisplatin side effects. The composition may be administered before, after and/or simultaneously with the second therapeutic agent. The two agents may be administered by different routes of administration.

The compounds and methods described herein are useful for treating hearing disorders caused by sensorineural hair cell loss. Patients with sensorineural hair cell loss experience deterioration of cochlear hair cells, which often results in loss of spiral ganglion neurons in the area of hair cell loss. These patients may also experience loss of support cells in the organ of Corti, and degeneration of limbus, spiral ligaments and vascular lines in the temporal bone material. These patients may receive treatment with agents that trigger differentiation of cells into hair cells, or with tissue grafts including hair cells that are transplanted or injected into the inner ear.

Methods of producing inner ear cells are provided below. The ear cells or ear cell progenitors can be produced from stem cells isolated from a mammal, e.g., a mouse or a human, and the cells can be embryonic stem cells or stem cells taken from a mature (e.g., adult) tissue, such as the inner ear, the central nervous system, blood, skin, eye, or bone marrow. Any of the methods described below for culturing stem cells and inducing their differentiation into ear cells (e.g., hair cells) can be used.

In general, the compounds and methods described herein can be used to produce growth of hair cells in the ear and/or increase the number of hair cells in the ear (e.g., in the inner ear, middle ear, and/or outer ear). For example, the number of hair cells in the ear can be increased by 2, 3, 4, 6, 8, 10 fold or more compared to the number of hair cells prior to treatment. The new hair cell growth can be effective to restore hearing in the subject, or establish at least partial improvement in hearing in the subject. For example, administration of the agent may improve hearing loss by about 5%, 10%, 15%, 20%, 40%, 60%, 80%, 100% or more.

Where appropriate, following treatment, an improvement in human hearing or other symptoms associated with an inner ear disorder can be detected. Methods of detecting hearing are well known and include pure tone audiometry, air conduction, and bone conduction tests. These checks measure the limits of loudness (intensity) and pitch (frequency) that a person can hear. Human hearing tests, including behavioral observation hearing tests (for infants up to 7 months), visual enhancement directed hearing tests (for children up to 7 months and 3 years), and game audiometry for children over 3 years. Otoacoustic emission testing can be used to test the function of cochlear hair cells, and electrocochlear electrogram provides information about cochlear function and the first portion of the neural pathway into the brain. In some embodiments, treatment with or without modification may be continued, or treatment may be discontinued.

Abnormal cell proliferation

Cell proliferation is a tightly regulated process that is usually controlled through multiple checkpoints and safeguards. When one or more of these checkpoints or safeguards are bypassed or disrupted, for example, by genetic mutation, abnormal cell proliferation occurs. The result of abnormal cell proliferation is the formation of cancerous growths or tumors. The most aggressive cancerous growths are usually invasion and metastasis. A few aggressive benign growths are not invasive or metastatic, although they tend to retain the possibility of metastasis.

In some embodiments, the present invention relates to methods and compositions for treating abnormal cell proliferation and/or cancer that would benefit from increased expression of Atoh 1. Identification of abnormally proliferating cells or cancer cells that would benefit from increased expression of Atoh1 can be accomplished by, for example, determining intracellular levels of Atoh1 expression using real-time PCR and other techniques readily performed by those skilled in the art. Such an assay may be performed by obtaining a sample of abnormally proliferating cells or cancer cells from a subject; isolating genetic material (e.g., DNA and RNA) from the sample; reverse transcription of mRNA from the sample; and amplified the Atoh1 sequence using an oligonucleotide that had been designed to hybridize to the Atoh1 sequence. Abnormally proliferating cells or cancer cells, which benefit from increased expression of Atoh1, have undetectable expression of Atoh1 in the cell sample. Alternatively or additionally, the above assay will be repeated using a non-cancer cell control. The expression of Atoh1 in the control was then compared to the expression of Atoh1 in the cancerous samples. Abnormally proliferating cells or cancer cells that would benefit from increased expression of Atoh1 have lower Atoh1 expression than non-cancer controls. In general, abnormally proliferating cells or cancer cells that would benefit from increased expression of Atoh1 will have low or undetectable levels of Atoh1 expression.

In some embodiments, the present invention relates to methods and compositions for treating abnormal cell proliferation and/or cancer in the gastrointestinal system. Exemplary cancers include, but are not limited to, esophageal cancer, gallbladder cancer, liver cancer, pancreatic cancer, gastric cancer, small intestine cancer, large intestine cancer (colon cancer), and rectal cancer.

The treatment of abnormal cell proliferation and/or cancer of the gastrointestinal system using the present invention is supported by the following studies: generally, the intestinal epithelium comprises four major cell types, which are derived from pluripotent stem cells during embryogenesis. The first cell type is absorptive intestinal epithelial cells or columnar cells; the second cell type is a mucus secreting goblet cell; the third cell type is an enteroendocrine cell that secretes a regulatory peptide; and the fourth cell type is a Pan cell that secretes antimicrobial peptides. Healthy animals have each of these four cell types. However, Math 1-free transgenic mice had deficient goblet cells, enteroendocrine cells, and panne cells. This observation leads to the following conclusions: math1 is required for cell fate determination (e.g., differentiation) in intestinal development for these three cell types (Yang et al, Science, 294: 2155-. It was also demonstrated that Hath1 expression was absent in five gastric cancer cell lines compared to normal gastric mucosa. These support the following facts: deletion of Hath1 expression may play a role in gastric carcinogenesis (Sekine et al, biochem. Biophys., Res. Comm., 344: 1166-one 1171, 2006). Reduction of Hath1 and Math1 expression in colon Cancer cell lines has also been reported (Leow et al, Cancer Res., 64: 6050-. However, these studies also showed that overexpression of Hath1 in aggressive colon cancer cell lines resulted in a significant inhibition of cell proliferation, and that this decrease in proliferation occurred due to the differentiation of aggressive colon cancer cells into or towards non-cancerous goblet cells. Thus, these data clearly indicate that gastrointestinal cancer would benefit from increased expression of Atoh1, for example, by promoting differentiation of these cells into or towards non-cancerous cells of the intestinal epithelium, thereby reducing the number of proliferating gastric cancer cells. Accordingly, gastrointestinal cancer patients may be treated with one or more compounds described herein.

In general, the present invention provides compounds and methods for treating a patient suffering from or at risk of developing gastrointestinal cancer. The method of identifying such patients is as follows. The method of treatment includes the step of administering one or more compounds described herein to a patient to treat gastrointestinal cancer (direct therapy).

In some embodiments, the methods comprise a method of selecting a patient at risk for and/or having gastrointestinal cancer.

Methods of identifying patients with gastrointestinal cancer are well known in the art. For example, screening may also include the use of endoscopy (e.g., oral and/or rectal). Screening may also include testing to detect various immunohistochemical markers including, but not limited to: for example, CK20, MUC2, MUC5A, MUC6, DAS-1, and CDX 2.

The present invention uses one or more compounds described herein for providing treatment to a patient suffering from or at risk of developing gastrointestinal cancer. The method of treatment includes the step of administering one or more compounds described herein to the patient to promote the complete or partial differentiation of gastric cancer cells.

In some embodiments, the present invention relates to methods and compositions for treating colorectal cancer. Screens for distinguishing individuals with colorectal cancer are well known in the art. For example, screening for colorectal cancer includes: fecal Occult Blood Test (FOBT), which detects blood in the feces; digital Rectal Examination (DRE), which examines tactile abnormalities in the rectum; sigmoidoscopy, which looks for visual abnormalities in the rectum and lower parts of the colon; colonoscopy, which provides visualization of the rectum and the entire colon; and Dual Contrast Barium Enema (DCBE), which provides X-ray examination of the rectum and colon. Typically, a biopsy or polypectomy of abnormal colorectal tissue is examined to confirm that the tissue is cancerous.

Individuals with colorectal cancer can be classified according to the stage of cancer, such as Dukes, Astler-Coller, and AJCC/TNM classifications. The individual's cancer grade indicates the degree of cancer cell dedifferentiation experienced, i.e., the number of tumor cells that still retain the characteristics of colon or rectal cells. The phase groupings indicate the overall disease phase of the individual. In some systems, the stage groupings are represented as roman numerals from 0 (earliest) to IV (most advanced). At stage 0, cancer can only be found in the lining of the colon or rectum. In stage I, the cancer has spread to more of the inner wall of the colon or rectum. In stage II, the cancer has spread to the outside of the colon or rectum to nearby tissues, but has not spread to lymph nodes. In stage III, the cancer has spread to nearby lymph nodes, but not to other parts of the body. In stage IV, the cancer has spread to other parts of the body. Colorectal cancer tends to spread to the liver and/or lungs. (stages 0 and IV, just described, correspond to stages a and D, respectively, in Duke's fractionation). For further information on the screening, diagnosis and staging of colorectal cancer, see Frei et al,Cancer Medicine，BC Decker Inc.，Hamilton，Ontario(2003)。

the present invention uses one or more compounds described herein for providing treatment to a patient suffering from (e.g., stage 0 through stage IV) or at risk of developing colorectal cancer. The method of treatment comprises the step of administering one or more compounds described herein to a patient to promote the complete or partial differentiation of colorectal cancer cells.

In some embodiments, for example, a patient receiving treatment or having completed treatment for colon cancer may be re-evaluated using the methods described above to determine the effect of the treatment. In some embodiments, treatment with or without modification may be continued, or treatment may be discontinued.

Other disorders

Atoh1 expression in the cerebellum and dorsal spinal cord has also been reported, as well as its important role in, for example, development (Bermingham et al, supra and Helms et al, supra). It is clear that Atoh1 plays a role in promoting cell differentiation in nerve cells and tissues, beyond its role in the inner ear. Thus, the compounds and pharmaceutical compositions described herein may also be useful in the treatment of diseases and/or conditions of this tissue that would benefit from increased expression of Atoh 1.

Alternatively or additionally, the invention may be used to treat cerebellar granule neuronal deficiency, arthropathy and osteoarthritis.

Identification of conditions that may benefit from increased expression of Atoh1 may be determined by measuring intracellular levels of Atoh1 expression using, for example, the RT-PCR method described above. In general, disorders that may benefit from increased expression of Atoh1 have low or undetectable levels of Atoh1 expression.

Routes of administration for treating loss of auditory cells

Direct therapy

The route of administration will vary depending on the disease being treated. Hair cell loss and/or sensorineural hearing loss can be treated using direct therapy, using systemic administration and/or topical administration. In some embodiments, the route of administration can be determined by the patient's health care provider or clinician, e.g., upon evaluation of the patient. In some embodiments, treatment of an individual patient can be tailored, for example, one or more compounds, routes of administration, and frequency of administration, all of which can be individualized. Alternatively, treatment may be administered using standard courses of treatment, e.g., using one or more preselected compounds and a preselected route and frequency of administration.

In some embodiments, one or more compounds described herein can be administered to a patient using a systemic route of administration, e.g., identified as a patient in need of hair cell loss therapy. Systemic routes of administration may include, but are not limited to: parenteral routes of administration, e.g., intravenous, intramuscular, and intraperitoneal; enteral routes of administration, for example, oral routes of administration, lozenges, compressed tablets, pills, tablets, capsules, drops (such as ear drops), syrups, suspensions and emulsions; rectal administration, e.g., rectal suppositories or enemas; vaginal suppositories; urethral suppositories; administration by the transdermal route; and inhalation (e.g., nasal spray).

Alternatively or additionally, one or more compounds described herein can be administered to a patient using a topical route of administration, e.g., identified as a patient in need of hair cell loss therapy. Such a topical route of administration includes administering one or more compounds described herein to the ear of the patient and/or the inner ear of the patient, e.g., by injection and/or using a pump.

In some embodiments, the pharmaceutical composition can be injected into the ear (e.g., otic administration), such as into the cochlear cavity (e.g., the cochlear canal, Sc vestibule, and Sc tympanic cavity), for example, using a syringe (e.g., a single dose syringe). For example, administration of one or more compounds described herein can be by intratympanic injection (e.g., into the middle ear), and/or injection into the outer, middle, and/or inner ear. Such methods are commonly used in the art, for example, for administering steroids and antibiotics to the human ear. The injection may, for example, be through the round ear window or through the cochlear capsule. Other methods of inner ear administration are well known in the art (see, e.g., Salt and Plottke, Drug Discovery Today, 10: 1299-.

In another mode of administration, the pharmaceutical composition may be administered in situ by means of a catheter or pump. For example, a catheter or pump into the ear can introduce the composition into the cochlear cavity, or the round window of the ear, and/or the colon cavity. Exemplary drug delivery devices and methods suitable for administering one or more compounds described herein to an ear (e.g., a human ear) are described by McKenna et al (U.S. publication No. 2006/0030837) and Jacobsen et al (U.S. patent No. 7206639). In some embodiments, the catheter or pump can be placed, for example, in the ear of the patient (e.g., the outer ear, middle ear, and/or inner ear) during the surgical procedure. In some embodiments, the catheter or pump can be placed, for example, in the ear of the patient (e.g., the outer ear, middle ear, and/or inner ear) without surgery.

Alternatively or additionally, one or more compounds described herein may be administered in conjunction with a medical device, such as a cochlear implant or a hearing aid, that wears out in the outer ear. An exemplary cochlear implant suitable for use with the present invention is described by Edge et al (U.S. publication No. 2007/0093878).

In some embodiments, the modes of administration described above may be combined in any order, and may be administered simultaneously or interspersed.

Alternatively or additionally, the invention may be administered according to any method approved by the food and drug administration, as described in the CDER data standards manual, version number 004 (available at fda. give/CDER/dsm/DRG 00301. htm).

Cell therapy

In general, the cell therapy methods described herein can be used to promote complete or partial differentiation of cells into mature cell types (e.g., hair cells) or toward the inner ear in vitro. The cells harvested by this method can then be re-transplanted or implanted into a patient in need of such treatment. Cell culture methods are described below that require the practice of these methods, including methods of identifying and selecting appropriate cell types, methods of promoting full or partial differentiation of selected cells, methods of identifying fully or partially differentiated cell types, and methods of implanting fully or partially differentiated cells.

Selection of cells

Cells suitable for use in the present invention include, but are not limited to, cells that are capable of fully or partially differentiating into mature cells of the inner ear (e.g., hair cells, e.g., inner and/or outer hair cells) upon contact with one or more compounds described herein (e.g., in vitro). Exemplary cells capable of differentiating into hair cells include, but are not limited to, stem cells (e.g., inner ear stem cells, adult stem cells, bone marrow-derived stem cells, embryonic stem cells, mesenchymal stem cells, skin stem cells, iPS cells, and adipose stem cells), progenitor cells (e.g., inner ear progenitor cells), supporting cells (e.g., Deiters cells, column cells, inner finger cells, roof cells, and Hensen cells), and/or germ cells. Li et al, (U.S. publication No. 2005/0287127) and Li et al, (U.S. patent No. 11/953,797) describe the use of stem cells for replacing inner ear sensory cells. Edge et al, PCT/US2007/084654, describe the use of bone marrow-derived stem cells for replacing inner ear auditory cells. iPS cells are described, for example, in Takahashi et al, Cell, Vol.131, No. 5, pp.861-872 (2007); takahashi and Yamanaka, Cell 126, 663-76 (2006); okita et al, Nature 448, 260-262 (2007); yu, j. et al, Science 318 (5858): 1917-1920 (2007); nakagawa et al, nat. biotechnol.26: 101-; and Zaehres and，Cell 131(5)：834-835(2007).

such suitable cells can be identified by analyzing (e.g., qualitatively or quantitatively) for the presence of one or more tissue-specific genes. For example, by detecting the protein product of one or more tissue-specific genes, expression of the gene can be detected. Protein detection techniques include staining proteins with antibodies against the appropriate antigen (e.g., using cell extracts or whole cells). In this case, the appropriate antigen is the protein product of tissue-specific gene expression. Although, in theory, the primary antibody (i.e., the antibody that binds the antigen) may be labeled, it is more common (and improves its visibility) to use a secondary antibody (e.g., anti-IgG) directed against the primary antibody. The second antibody binds to a fluorescent dye, or to a suitable enzyme for colorimetric reactions, or to gold beads (of an electron microscope), or to a biotin-avidin system, so that the position of the primary antibody, and hence the antibody, can be identified.

Tissue-specific gene expression can also be detected by detecting RNA transcribed from the gene. RNA detection methods include reverse transcription coupled polymerase chain reaction (RT-PCR), northern blot analysis, and RNase protection assay.

Exemplary tissue-specific genes that can be used to identify stem cells (e.g., undifferentiated cells) include, but are not limited to, for example, nestin, sox1, sox2, or musashi, NeuroD, Atoh1, and neurogenin 1. Alternatively or additionally, stem cells may be selected for one or more of the unique characteristics that occur in vitro with that cell type. For example, in vitro, stem cells often exhibit the unique potential to form spheres through proliferation of individual cells. Thus, the identification and isolation of the spheres can aid in the process of isolating stem cells from the mature tissue used to make the differentiated cells of the inner ear. For example, stem cells can be cultured in serum-free DMEM/high sugar and F12 medium (1: 1 mix) and supplemented with N2 and B27 solutions and growth factors. Growth factors, such as EGF, IGF-1 and bFGF, have been shown to increase sphere formation in culture.

Exemplary tissue-specific genes that can be used to identify progenitor cells and/or inner ear progenitor cells (e.g., less than fully or partially differentiated cells) include, but are not limited to, for example, nestin, sox2, and musashi, as well as certain inner ear-specific marker genes, such as Brn3c, islet1, and Pax 2.

Exemplary tissue-specific genes that can be used to identify fully differentiated cells (e.g., supporting cells) include, but are not limited to, e.g., p27kip, p75, S100A, Jagged-1, and Prox 1.

Exemplary tissue-specific genes that can be used to identify fully differentiated cells (capable of functioning as inner ear sensory cells) include, but are not limited to, e.g., myosin VIIa, Math1, alpha 9 acetylcholine receptor, espin albumin 3, and F-actin (a muscarinic cyclic peptide).

Alternatively or additionally, cells suspected of being fully differentiated (e.g., cells capable of functioning as inner ear sensory cells) may be subjected to physiological tests to determine whether a conduction channel (conductance channel) present in mature hair cells is present or active.

Alternatively or additionally, inner ear hair cells can be distinguished from other fully differentiated cells of the inner ear (e.g., the spiral ganglion) by analyzing the expression of markers specific for the spiral ganglion, including but not limited to ephrinB2, ephrinB3, trkB, trkC, GATA3, and BF 1.

In some embodiments, suitable cells may be derived from a mammal, such as a human, mouse, rat, pig, sheep, goat, or non-human primate. For example, stem cells have been identified and isolated from the oval bursa of mice (Li et al, Nature Medicine)9: 1293-1299, 2003). These cells may also be obtained from the patient who will then be re-administered.

In some embodiments, suitable cells (e.g., stem cells, progenitor cells, and/or support cells) can be isolated from the inner ear of an animal. More specifically, suitable cells may be obtained from the cochlear Corti organ, the modiolus (center) of the cochlea, the spiral ganglion of the cochlea, the vestibular sensory epithelial cells of the saccular plaques, the crest of the ellipsoidal capsule or the semicircular canal. However, stem cells, progenitor cells, and/or supporting cells may also be obtained from other tissues, such as bone marrow, blood, skin, or the eye. The cells used may be obtained from a single source (e.g., the ear, or a structure or tissue within the ear), or a combination of sources (e.g., the ear and one or more surrounding tissues (e.g., bone marrow, blood, skin, or eye)).

Alternatively or additionally, the method comprises obtaining tissue from an inner ear of the animal, wherein the tissue comprises at least a portion of an elliptical sac. The animal can be a mammal, such as a mouse, rat, pig, rabbit, goat, horse, cow, dog, cat, primate, or human. The separated tissue may be suspended in a neutral buffer, such as Phosphate Buffered Saline (PBS), and subsequently exposed to tissue digesting enzymes (e.g., trypsin, leupeptin, chymotrypsin, etc.), or combinations of enzymes, or mechanical (e.g., physical) forces, such as grinding, to break the tissue into smaller pieces. Alternatively or additionally, both tissue destruction mechanisms may be used. For example, the tissue can be incubated in about 0.05% enzyme (e.g., about 0.001%, 0.01%, 0.03%, 0.07%, or 1.0% enzyme) for about 5, 10, 15, 20, or 30 minutes, after which the cells are mechanically disrupted. The disrupted tissue may be subjected to a device, such as a filter or a bore pipette (bore pipette), which separates stem or progenitor cells from differentiated cells or cell debris. The separation of the cells may comprise passing the cells through a series of filters having progressively decreasing pore sizes. For example, the filter pore size range can be about 80 μm or less, about 70 μm or less, about 60 μm or less, about 50 μm or less, about 40 μm or less, about 30 μm or less, about 35 μm or less, or about 20 μm or less.

The cells obtained may constitute an abundant population of stem and/or progenitor cells; isolation from all (or almost all) differentiated cells or other cellular material within a tissue can be achieved, but need not be consistent with the definition of "isolated". Absolute purity is not required. The invention encompasses cells obtained by the isolation process described herein. The cells may be mixed with a cryoprotectant and stored, or packaged into a kit. Once obtained, the stem cells and/or progenitor cells can be expanded in culture.

If a mixed cell population is used, the proportion of stem cells in the test population can vary. For example, the population can comprise a small number of stem cells (e.g., about 1-10%), a moderate proportion of stem cells (e.g., about 10-90% (e.g., about 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 85% of stem cells)), or a large number of stem cells (e.g., at least 90% of the population (e.g., 92%, 94%, 96%, 97%, 98%, or 99%) can be stem cells). These cells have the potential to differentiate into fully or partially differentiated cells in the inner ear (e.g., the cells may be pluripotent stem cells that differentiate into cells expressing one or more auditory proteins). Partially differentiated cells are functional in therapeutic methods (whether therapeutic or prophylactic) as long as they express a sufficient amount and type of hearing specific protein to confer a benefit (e.g., increased hearing) to the patient.

Differentiation method

In general, the promotion of differentiation can be by contacting a suitable target cell and/or population of cells with one or more compounds described herein for a time sufficient to promote the complete or partial differentiation or toward mature sensory cells (e.g., hair cells) in the inner ear.

For example, suitable cells identified according to the methods described above can be cultured in vitro. Generally, the methods described herein use standard culture methods. Suitable media are described in the art, e.g., in Li et al, Nature Medicine9: 1293 and 1299, 2003. The growth medium used for the cultured stem cells may comprise one or more growth factors or any combination of growth factors. For example, the growth medium may contain leukemia growth inhibitory factor (LIF), which will prevent differentiation of stem cells.

The cells may be dispensed into individual wells of a culture dish and cultured. The formation of spheres (clonal floating colonies) from the isolated cells can be monitored and the spheres can be expanded by disrupting to isolate the cells (e.g., by physical means) and the cells can be cultured repeatedly to form additional spheres. These cultured cells can then be contacted with one or more compounds described herein.

Alternatively or additionally, the cell may be contacted with one or more compounds described herein, and an additional induction procedure is incorporated. There are many induction procedures well known in the art for inducing differentiation of stem cells with neurogenesis potential into neural progenitor cells, including growth factor therapy (e.g., with EGF, FGF, and IGF as described herein) and neurotrophic factor therapy (e.g., with NT3 and BDNF as described herein). Other differentiation procedures are well known in the art; see, e.g., corales et al, j.neurobiol.66 (13): 1489-500 (2006); kim et al, Nature 418: 50-6 (2002); lee et al, nat. biotechnol.18: 675-9 (2000); and Li et al, nat. biotechnol.23: 215-21(2005)

As an example of an additional induction procedure, suitable cells are grown in the presence of a supplemental growth factor that induces differentiation into progenitor cells. These supplemental growth factors were added to the medium. The type and concentration of supplemental growth factors are adjusted to modulate the growth characteristics of the cell (e.g., to stimulate or sensitize the cell to differentiation) and allow the survival of differentiated cells, such as a neuron, glial cell, supporting cell, or hair cell.

Exemplary supplemental growth factors include, but are not limited to, basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), and Epidermal Growth Factor (EGF). Alternatively, the supplemental growth factors may include the neurotrophic factors neurotrophin 3(NT3) and brain-derived neurotrophic factor (BDNF). Exemplary concentration ranges for growth factors can be, for example, from about 100ng/mL to about 0.5ng/mL (e.g., from about 80ng/mL to about 3ng/mL, such as about 60ng/mL, about 50ng/mL, about 40ng/mL, about 30ng/mL, about 20ng/mL, about 10ng/mL, or about 5 ng/mL).

Alternatively or additionally, the medium may be replaced with a medium lacking growth factors. For example, the medium may be serum-free DMEM/high sugar and F12 medium (1: 1 mix) supplemented with N2 and B27 solutions. Equivalent alternative media and nutrients may also be used. The culture conditions can be optimized using methods well known in the art.

In some embodiments, stem cells designed to express a reporter gene that facilitates the transformation of test cells into inner ear cells can be used to test compounds for their ability to promote differentiation. These engineered stem cells constitute a reporter cell line. A reporter gene is any gene whose expression can be detected; such genes include, but are not limited to, Green Fluorescent Protein (GFP), α -Glucuronidase (GUS), luciferase, Chloramphenicol Acetyltransferase (CAT), horseradish peroxidase (HRP), alkaline phosphatase, acetylcholinesterase, and β -galactosidase. Other alternative fluorescent reporter genes include, but are not limited to, Red Fluorescent Protein (RFP), blue-green fluorescent protein (CFP), and Blue Fluorescent Protein (BFP), or any paired combination thereof, provided that the paired proteins emit light within a distinguishable wavelength.

The reporter gene may be under the control of a promoter which is active in cells of the inner ear (including progenitor cells and cells differentiated to varying degrees) but inactive in stem cells. Ideally, the promoter is stably upregulated in differentiated cells or progenitor cells in order to assess partially or fully differentiated phenotypes (e.g., expression of the reporter gene and further identification of genes known to be expressed in inner ear cells).

Method for analyzing complete or partial differentiation

Cells that have been contacted with one or more compounds disclosed herein can be analyzed to determine whether intact partial differentiation has occurred. This assay can be performed by analyzing the presence or absence of tissue-specific genes, as described above (see, cell selection). Alternatively or additionally, hair cells can be identified by physiological tests to determine whether the cells produce the conductive channel characteristic of mature hair cells or spiral ganglion cells. Using the markers described above, the cells can be distinguished from spiral ganglion cells.

A second assay can be used to confirm that the cells have differentiated into inner ear cells, or to provide additional evidence therefor. For example, a gene used as a marker for identifying inner ear cells may be expressed only in a particular cell type (e.g., only in hair cells or only in cells of the spiral ganglion), or the cell may be expressed in a few other cell types (preferably no more than one, two, three, four, or five other cell types). For example, ephrinB1 and ephrinB2 are expressed in spiral ganglion cells, and also in retinal cells. Thus, detection of ephrinB1 or ephrinB2 expression does not conclusively demonstrate that the stem cell has differentiated into a spiral ganglion cell. A second assay can be used to confirm that the cells have developed into spiral ganglion cells. The assay involves detecting a plurality of genes known to be expressed in suspect cell types. For example, for cells expressing ephrinB1 and/or ephrinB2, expression of one or more of GATA3, trkB, trkC, BF1, FGF10, FGF3, CSP, GFAP, and Islet1 can be detected. The assay to express these additional genes is a supplemental evidence that the stem cells have differentiated into spiral ganglion cells.

The second assay also includes detecting a loss of gene expression or a loss of a protein that is not normally expressed in hair cells. The negative markers include a whole cell keratin gene that is not expressed in mature hair cells but is expressed in the supporting cells of the inner ear (Li et al, nat. Med.9: 1293-1299, 2003).

Cells that have been shown to have undergone complete or partial differentiation into inner ear sensory cells (e.g., hair cells) can be transplanted or implanted into a patient.

Implantation method

For example, partially and/or fully differentiated cells produced by the above methods may be transplanted or implanted into the ear, such as into the cochlear cavity, e.g., by injection, e.g., in the form of a cell suspension. The injection may, for example, be through the round window of the ear or through the pericochlear bone capsule (bony capsule). These cells may be injected through the round window of the ear into the auditory nerve trunk of the inner ear canal or into the scala tympani.

To increase the engraftment capacity of the transplanted or implanted cells, the cells may be modified prior to differentiation. For example, the design may be fineThe cells overexpress one or more anti-apoptotic genes in the progenitor cells or differentiated cells. FAK tyrosine kinase or Akt genes are candidate anti-apoptotic genes that can be used for this purpose; overexpression of FAK or Akt can prevent cell death in spiral ganglion cells and promote engraftment when transplanted into another tissue, such as an explanted Corti device (see, e.g., Mangi et al, nat. Med. 9: 1195-. Overexpression of alpha_vβ₃Neuronal progenitors of integrins may have an enhanced ability to extend axons into tissue explants, as integrins have been shown to mediate axon extension from spiral ganglion neurons to the laminin matrix (Aletse et al, Audio. neurootol.6: 57-65, 2001). In another example, expression of ephrinB2 and ephrinB3 can be altered, such as by RNAi silencing or by overexpression of exogenously expressed cDNA, thereby improving the EphA4 signaling event. It has been shown that spiral ganglion neurons are guided by signals from EphA4, and that EphA4 is mediated by cell surface expression of ephrin-B2 and-B3 (Brors et al, J.Comp.Neurol.462: 90-100, 2003). Inactivation of this guide signal increases the number of neurons that reach neuronal targets in the inner ear of an adult. Exogenous factors (such as neurotrophic factors BDNF and NT3) and LIF can be added to the tissue graft to enhance axonal extension and growth to target tissues in vivo and ex vivo tissue cultures. Axonal elongation of sensory neurons can be increased by the addition of neurotrophic factors (BDNF, NT3) and LIF (Gillespie et al, NeuroReport 12: 275-279, 2001).

In some embodiments, the cells described herein can be used in cochlear implants, for example, as described by Edge et al (U.S. publication 2007/0093878). A cochlear implant is an electronic device used to improve the hearing of a person suffering from hearing loss, particularly severe to severe hearing loss. These devices typically include "external" and "internal" portions. The outer part comprises a microphone which can be placed behind the ear to detect sounds in the environment. The sound is then digitized and processed by a small computer called a speech processor. The external elements may be referred to as processor elements. In addition to the microphone and speech processor, the external portion of the implant may include a power source, such as a battery, and an external antenna transmitter coil. The inner part is an electronic device placed under the skin in the vicinity of the ear and is commonly referred to as a stimulator/receiving element (see fig. 1). The encoded signal output by the speech processor is transmitted subcutaneously to an implanted stimulator/receiver element located in the temporal bone groove of an implant (inplante). This transcutaneous transmission occurs through the use of inductive coupling (inductive coupling) provided between an external antenna transmitter coil positioned in communication with an implanted antenna receiver coil provided with the stimulator/receiving element. This communication is typically provided by a Radio Frequency (RF) link, but other such links have been proposed and implanted with varying degrees of success.

The implanted stimulator/receiver element typically includes an antenna receiver coil that receives the encoded signal and energy from an external processor element, and a stimulator that processes the encoded signal and outputs a stimulation signal to the electrode assembly, which processor applies electrical stimulation directly to the auditory nerve that produces the sense of hearing, based on the originally detected sound.

Electrodes connected to the electronic device are inserted into the inner ear. The electrode may be a bundle of wires that openly contact and represent different sound frequencies along the length of the cochlea. The number of electrodes may vary from 1 to about 30 electrodes, such as about 5, 10, 15, 18, 20, 22, 24, 26, or 28 electrodes.

Combination therapy

In some embodiments, the invention provides methods of treating a patient with one or more compounds described herein using direct administration and the cell therapies described above.

Routes of administration for treating abnormal cell proliferation

The route of administration will vary depending on the disease being treated. Abnormal cell proliferation and/or cancer may be treatable using direct therapy, for example, systemic administration and/or topical administration using one or more methods approved by the food and drug administration, such as described in the CDER data standards manual, version number 004 (available from fda.

In some embodiments, the route of administration may be determined by the patient's health care provider or clinician, for example, upon evaluation of the patient. In some embodiments, treatment of an individual patient is customizable, e.g., one or more compounds, routes of administration, and frequency of administration can be individualized. Alternatively, treatment may be administered using standard treatment regimens, e.g., using one or more preselected compounds and a preselected route and frequency of administration.

In some embodiments, one or more compounds described herein can be administered to a patient using systemic administration, e.g., identified as a patient in need of treatment for hair cell loss. Systemic routes of administration may include, but are not limited to, parenteral routes of administration such as intravenous, intramuscular, and intraperitoneal injection; enteral routes of administration, such as oral routes of administration, lozenges, compressed tablets, pills, tablets, capsules, drops, syrups, suspensions and emulsions; rectal administration, such as rectal suppositories or enemas; vaginal suppositories; urethral suppositories; the transdermal route of administration; and inhalation (e.g., nasal spray).

Alternatively or additionally, one or more compounds described herein can be administered to a patient using a topical route of administration, e.g., identified as a patient in need of treatment for hair cell loss. For example, one or more compounds can be administered intraoperatively, e.g., to resect a tumor, and can be administered by injection or topically at one or more sites within and around the cancerous site.

Pharmaceutical preparation

Pharmaceutical compositions comprising one or more compounds described herein (i.e., as active ingredients) will be formulated according to the desired method of administration.

One or more of the compounds described herein can be formulated as a pharmaceutical composition for direct administration to a subject. Pharmaceutical compositions comprising one or more compounds described herein may be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. For example, the pharmaceutical composition may be formulated for topical or systemic administration, e.g., by instillation or injection into the ear, insufflation (e.g., into the ear), intravenous, topical, or oral administration.

The nature of the pharmaceutical composition administered is based on the mode of administration and can be readily determined by one skilled in the art. In some embodiments, the pharmaceutical composition is sterile or sterilizable. The pharmaceutical compositions described in the present invention may include carriers or excipients, many of which are well known to those skilled in the art. Excipients that may be used include buffer media (e.g., citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, polypeptides (e.g., serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, water, and glycerol. Nucleic acids, polypeptides, small molecules, and other regulatory compounds described herein can be administered by any standard route of administration. For example, administration can be parenteral, intravenous, subcutaneous, or oral. The modulating compounds may be formulated in a variety of ways depending on the corresponding route of administration. For example, liquid solutions may be formulated for administration to the ear by drops, for injection, or for ingestion; the gel or powder may be formulated for ingestion or topical use. Methods for making such formulations are well known and can be found, for example,remington's Pharmaceutical Sciences (Remington pharmacy)18 th edition, Gennaro, ed., Mack Publishing co., Easton, Pa., 1990.

One or more compounds described herein can be administered directly and/or topically, e.g., to the inner ear and/or colon, e.g., by injection or by surgical placement, as a pharmaceutical composition. The amount of the pharmaceutical composition may be described as an effective amount, or the amount of the cell-based composition may be described as a therapeutically effective amount. The compositions of the present invention may be placed in a sustained release formulation or an implantable device (e.g., a pump) if the action is appropriate or desired for a period of time.

Alternatively or additionally, the pharmaceutical composition may be formulated for systemic parenteral administration by injection, for example by bolus injection or continuous instillation. The formulations may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an additional preservative. The composition may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in a powdered form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

In addition to the formulations previously described, the compositions may also be formulated as depot preparations. Such long acting formulations may be administered by implantation (subcutaneously). Thus, for example, the composition may be formulated with a suitable polymeric or hydrophobic material (e.g., an acceptable emulsion in oil) or ion exchange resin, or as a sparingly soluble derivative, e.g., a sparingly soluble salt.

Pharmaceutical compositions for systemic oral administration may take the form of tablets or capsules prepared in a conventional manner with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or dibasic calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The tablets may be coated by methods well known in the art. Liquid formulations for oral administration may take the form of, for example, solutions, syrups or suspensions, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared in a conventional manner using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifiers (e.g., lecithin or gum arabic); water insoluble carriers (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl paraben, or sorbic acid). If desired, the formulations may also contain buffer salts, flavoring agents, coloring agents, and sweetening agents. Formulations for oral administration may be suitably formulated to provide controlled release of the active compound.

In some embodiments, the pharmaceutical compositions described herein may include one or more compounds formulated according to any of the above methods, as well as one or more cells obtained from the methods described herein.

Effective/therapeutic dose

Toxicity and therapeutic efficacy of the compounds and pharmaceutical compositions described herein can be determined by standard pharmaceutical procedures using cultured cells or experimental animals to determine LD₅₀(lethal dose of 50% of the population) and ED₅₀(therapeutically effective dose in 50% of body). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as LD₅₀/ED₅₀The ratio of (a) to (b). Polypeptides or other compounds that exhibit a greater therapeutic index are preferred.

The data obtained from cell culture assays and further animal studies can be used to formulate a range of dosage for human use. The dose of the compound is preferably within the circulating concentration range, including the ED, which is relatively little or non-toxic₅₀And has relatively few or no side effects on human hearing. The dosage will vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods described herein, a therapeutically effective dose can be initially evaluated from cell culture assays. The doses can be formulated in animal models to achieve and include IC as determined in cell culture₅₀(i.e., the concentration of test compound at which half-maximal inhibition of symptoms is achieved). This information can be used to more accurately determine the dosage to be administered to a human. Exemplary dosages of the differentiating agent are from at least about 0.01mg to 3000mg per kilogram per day, e.g., at least about 0.01mg per kilogram per dayAbout 0.00001mg, 0.0001mg, 0.001mg, 0.01mg, 0.1mg, 1mg, 2mg, 5mg, 10mg, 25mg, 50mg, 100mg, 200mg, 500mg, 1000mg, 2000mg, or 3000mg or more.

The formulation and route of administration may be adapted to the disease or condition being treated, and to the particular human being treated. The subject may receive a dose once or two or more times per day for a week, month, six months, year, or more. Treatment may continue indefinitely, such as for the entire life of the person. The treatment may be administered at regular or irregular intervals (twice every other day or week), and the dosage or time of administration may be adjusted throughout the treatment. The dosage may remain constant throughout the treatment regimen, or may be reduced or increased during the course of treatment.

Generally, the dosage is useful for the intended purpose of prophylaxis and therapy without undesirable side effects such as toxicity, irritation, or allergic response. Although individual requirements may vary, the determination of the optimal range for an effective amount of a formulation is well within the skill of the art. Human doses can be readily extrapolated from animal experiments (Katocs et al Remington’s Pharmaceutical Sciences (Remington pharmaceuticals) Chapter 2718 th edition, Gennaro eds, Mack Publishing co., Easton, Pa., 1990). In general, the required dosage to provide an effective amount of a formulation, which may be adjusted by one of skill in the art, will vary depending on several factors including the age, health, physical condition, body weight, type and extent of the disease or disorder, frequency of treatment, nature of concurrent treatment if necessary, and the nature and scope of the effect desired (Nies et al, Goodman)& Gilman’s The Pharmacological Basis of Therapeutics Chapter 39 th edition, compiled by Hardman et al, McGraw-Hill, New York, N.Y., 1996).

Reagent kit

The compounds and pharmaceutical compositions described herein can be provided in a kit, as can cells that have induced differentiation (e.g., stem cells, progenitor cells, and/or supporting cells that have differentiated into, e.g., hair cells or hair-like cells) and/or cells that have the ability to differentiate into hair cells. The kit may also include a combination of the compounds and pharmaceutical compositions described herein and the cells. The kit may comprise: (a) one or more compounds, such as in a composition comprising the compounds, (b) cells that have induced differentiation (e.g., stem cells, progenitor cells, and/or supporting cells that have differentiated into, e.g., hair cells or hair-like cells) and/or cells that have the ability to differentiate into hair cells, (c) information material, and any combination of (a) - (c). In some embodiments, (a) and/or (b) may be provided in a syringe (e.g., a pre-filled single-dose disposable syringe) adapted for direct administration of (a) and/or (b) to the ear (e.g., middle or inner ear) of a patient. In some embodiments, (a) and/or (b) may be provided in a catheter and pump system suitable for direct administration of (a) and/or (b) to an ear (e.g., middle or inner ear) of a patient as described above. The informational material may be descriptive, marketing, or other material relating to the use of the methods described herein and/or medicaments for use in the methods described herein. For example, the informational material relates to the treatment of a subject with a compound that has or is at risk of developing auditory hair cell loss and/or abnormal cell proliferation. The kit may also include an accessory for administering the differentiating agent to the cells (in culture or in vivo) and/or administering the cells to the patient.

In one embodiment, the informational material may include instructions for administering the pharmaceutical composition and/or cells in a suitable therapeutic human manner, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein). In another embodiment, the informational material can include instructions for administering the pharmaceutical composition to a suitable subject, e.g., a human, such as a human having or at risk of developing auditory hair cell loss and/or abnormal cell proliferation.

The form of the information material in the kit is not limited. In many cases, informational material (e.g., instructions) is provided in printed form, such as printed text, graphics, and/or photographs, such as labels or printed pages. However, the information material may also be provided in other forms, such as braille, computer readable material, video recording, or audio-visual recording. Of course, the informational material may also be provided in any combination of forms.

In addition to the differentiating agent, the composition of the kit may include other ingredients, such as solvents or buffers, stabilizers, preservatives, fragrances or other cosmetic ingredients, and/or a second agent for treating the conditions or disorders described herein. In addition, other ingredients may be included in the kit, but in compositions and containers different from those in which the agent is present. In such embodiments, the kit may include instructions for mixing the agent with the other ingredients, or instructions for using one or more compounds with the other ingredients.

The kit may comprise one or more containers for pharmaceutical compositions. In some embodiments, the kit comprises separate containers, barriers, or compartments for the composition and the informational material. For example, the composition may be contained in a bottle (e.g., a dropper bottle, such as for applying drops to the ear), a vial or a syringe, and the informational material may be contained in a plastic sleeve or bag. In other embodiments, the various elements of the kit are contained in an integral container. For example, the composition is contained in a bottle, vial or syringe, which is labeled with the informational material in the form of a label. In some embodiments, the kit comprises a plurality (e.g., a plurality) of individual containers, each comprising one or more unit dosage forms of a pharmaceutical composition (e.g., dosage forms described herein). For example, the kit may comprise a plurality of syringes, ampoules, foil packs or blister packs, each containing a single unit dose of the pharmaceutical composition. The container of the kit may be airtight and/or waterproof and the container may be labeled for a particular use. For example, the container may be marked for treatment of a hearing disorder.

As described above, the kit optionally includes a device suitable for application of the composition (e.g., a syringe, pipette, forceps, dropper (e.g., ear dropper), swab (e.g., cotton or wooden swab), or any such delivery device).

Examples

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1 high throughput screening optimization

Human Embryonic Kidney (HEK) cell lines stably expressing a luciferase gene regulated by the Math1 enhancer and minimal promoter were used for High Throughput Screening (HTS) of 144,000 small molecules to identify compounds (i.e., transcription and/or translation) that increase expression of Math 1. These compounds can be used to increase the conversion of stem cells, progenitor cells, and supporting cells into or toward hair cells. Screens for identifying these compounds are described in Li et al (U.S. publication No. 2005/0287127) and Li et al (U.S. application No. 11/953,797).

HTS was optimized using 1 μ M retinoic acid as a positive control for the activated Math 1-luciferase reporter construct. Retinoic acid is a well-known general inducer of differentiation into mature cell types.

HEK cells stably expressing the Math 1-luciferase reporter were placed in 384-well plates and cultured overnight in medium containing 10% Fetal Calf Serum (FCS). The activity of the Math1 enhancer was measured by the increase in fluorescence from the plate reader after addition of the luciferase detector. The number of cells, incubation time, medium volume, cell lysis reagent and luciferase reagent of the assay were optimized. The luminescence levels of cells with the Math 1-luciferase construct with and without retinoic acid (1. mu.M) and/or cells with the luciferase construct with and without the Math1 enhancer and minimal promoter region were compared.

The data show that the Math 1-luciferase reporter is sensitive to retinoic acid and that this assay has a low background. Luciferase activity of cells treated with 1 μ M retinoic acid steadily increased 1.8-fold compared to untreated cells. The promoter without enhancer has a low level of luminescence.

To improve the sensitivity of the assay and reduce the Coefficient of Variation (CV), a more sensitive luciferase reagent (Britelite luciferase reporter assay reagent, Perkin Elmer) was used, and Triton-X-100 was added to the lysis reagent to ensure adequate lysis of the cells. After these changes, a CV of 4.2% was recorded. These conditions were used for all high throughput screens. The fluorescence threshold of positive compounds is defined as the increase of more than twice that of controls (e.g., cells exposed to DMSO).

The exposure time and cell density in the compound were optimized as follows. The optimal exposure time was determined to provide the time at which the activity of Math1 was maximal and cell loss was minimal. This was performed using different retinoic acid concentrations.

After 60 hours of incubation in the presence of retinoic acid, maximum luminescence was observed, however, a significant reduction in signal to 50% was observed at the 72 hour time point. At the 48 hour time point, the luminescence was close to the maximum and had the highest signal to background ratio for all detected times. Therefore, 48 hours was selected as the end point of the measurement.

The optimal cell density was determined by a cell titration experiment in which the activity of the Math 1-luciferase reporter was compared in wells seeded with 2000 to 40000 cells per well in 384-well plates. Cells exposed to retinoic acid were then compared to cells not exposed to retinoic acid. Cell survival assays were also used to determine the survival of cells expressing the Math 1-luciferase reporter.

Cell viability assays showed a linear increase in cell number in the range of 2000 to 10000 cells per well. There was no difference in the signals generated in wells from 8000 to 15000 cells per well, indicating a possible reduction in cell survival during experiments with densities exceeding 10000 cells per well. The greatest difference between untreated and retinoic acid treated cells was observed at a density of 8000 cells per well. Based on these results, assay parameters were selected to be 8000 cells per well and incubated for 48 hours in the presence and absence of test compound or known activator.

Example 2: high throughput screening to identify activators of Atoh-1 expression

Cells were seeded in 384-well plates and can be grown in the absence of growth factors at 37 degrees celsius and 5% CO₂Attach overnight at concentration. Activation of Math1 was measured by the increase in luminescence on the plate reader after addition of luciferase detection reagent. Luminescence was evaluated at 24 hours, 48 hours, 72 hours. These conditions were used to screen 144,000 compounds contained in a small molecule library in the neurodevelopmental drug development Laboratory (LDDN) at harvard university.

HTS was performed using HEK-Math1 cells seeded in 384-well plates. Compounds were added to each well using a needle transfer. The final concentration of each compound was 100. mu.M. Cells in the presence of compounds at 5% CO₂And incubation for 48 hours at neutralization 37 ℃. Then, the cell lysate was collected and bioluminescence was measured. Luminescence values were compared to DMSO on a normalized basis.

Approximately 20000 compounds were screened weekly in 50 384-well plates with the assistance of an automated system (Beckman Biomek FX). Compounds were screened at a mean final concentration of 0.7 μ M (in 0.04% DMSO), each plate including 16 wells of 1 μ M retinoic acid as a positive control and 16 wells of 0.04% DMSO as a vehicle control. The percent luminescence activation of the test compound was determined by comparison of the compound-treated cells to the DMSO-treated cells.

Initial screening was performed using duplicate plates to detect libraries of more than 10,000 compounds. As shown in figure 117B, 40 compounds were observed to enhance the activity of the Math1 enhancer at least two-fold compared to the observed expression level of Math1 in DMSO (i.e., 40 compounds were positive). The Z factor that varies between different wells with positive controls is shown in figure 117A. The Z factor is a HTS statistic that reflects the data quality of each assay plate based on the magnitude of the signal window between the positive and negative controls. The variation between control groups was also calculated. (Zhang et al, J.Biomol. Screen.4: 67-73, 1999). Any plate with a Z value less than 0.4 was repeated.

Similar results were observed in duplicate experiments as shown in fig. 118A and 118B. This demonstrates the reliability and repeatability of the HTS process.

Of 144,000 compounds screened, 921 were found to promote an increase of more than 60% in expression of the Math1 luciferase reporter. The hit rate was 0.47%. The highest activation observed was 160% (e.g., compared to DMSO).

Each of these compounds was then retested for dose dependence on response at final assay concentrations of 0.1. mu.M, 1. mu.M and 5. mu.M. Of the 921 positive compounds identified, 789 induced an increased dose response at concentrations of 0.1 μ M, 1 μ M, 10 μ M. This observation supports the specificity of hits. Overall, of the 921 compounds tested, 82% reproducibly activated the Math1 luciferase reporter, and 29% showed partial toxicity.

After these experiments, these compounds were re-evaluated and further investigated for compounds with good physiochemical properties (i.e., low molecular weight, lack of reactive side chain groups or other unwanted molecular motifs) that exhibited potent activation of the Math1 luciferase and were non-toxic.

The total number of compounds selected for further evaluation was 110 compounds. The structures of these compounds are shown in FIGS. 1 to 8. The enhanced enhancement of Math1 expression by these compounds is shown in FIGS. 9-116.

Example 3 evaluation of Positive Compounds by RT-PCR

Further evaluation was performed on 10 compounds randomly selected from the 110 compounds identified in example 2, as shown below. HEK cells were seeded in 96-well plates at a density of 100000 cells per well. One day after plating, 0.1. mu.M, 1. mu.M and 5. mu.M of positive compound in DMEM solution with 10% Fetal Bovine Serum (FBS) was added to each well. Cells were lysed 48 hours after addition of compound and analyzed for Math1 expression using fluorescein reporter and real-time PCR as described above.

As shown in figure 119, 4 of the 10 randomly selected compounds promoted more than a two-fold increase in expression of Math 1.

As shown in figure 120A, all 4 randomly chosen compounds promoted more than a two-fold increase in expression of Math1 as measured using the fluorescein reporter assay, as with HTS.

RNA was also isolated and Math1 transcript was amplified using high throughput RT-PCR. As shown in figure 120C, all 4 randomly chosen compounds promoted at least a two-fold increase in Math1mRNA expression compared to DMSO control. CP. -0193184 and CP. -0000540 promoted the maximal increase in Math1mRNA expression. Fig. 120B shows the structure of 4 randomly chosen samples.

These data confirm the data provided in example 2.

Example 4: evaluation of Positive Compounds by Hair cell differentiation

The inner ear stem cells were exposed to compound cp. -0000540 randomly selected from the group of 110 positive compounds described in example 2.

As shown in figure 121B, cp. -0000540 increased the number of cells co-labeled with hair cell specific marker Math1-GFP and myosin 7a compared to cells not exposed to cp. -0000540 as shown in figure 121A. Differentiation of hair cells increased to 5.1% of total cells compared to 1.6% of control.

Example 5: second stage evaluation of Positive Compounds

All positive compounds were evaluated for their ability to upregulate Math1mRNA to confirm the observations made in example 2.

Math1mRNA expression levels were analyzed using RT-PCR as described in example 3.

Example 6: third stage evaluation of Positive Compounds

The ability of positive compounds to increase stem cell-derived hair cell yield from the inner ear of mice was evaluated. Positive compounds were also evaluated in vivo in the inner ear of a mouse model of sensory neurological disease.

In vitro, isolated cells were exposed to 0.1. mu.M, 1. mu.M and 5. mu.M of each positive compound identified in examples 3, 4, 5.

The compounds were also added to the inner ear of mouse models of inner ear injury, e.g., gentamicin-treated mouse models and/or flox caspase transgenic mouse models. These models are used to test the ability of the compound to regenerate hair cells following loss due to toxic injury as occurs in human deafness.

In vitro results were assessed by detecting one or more of the hair cell specific markers myosin VIIa, Math1, espin, brn3.1, F-actin (phalloidin), alpha-9-acetylcholine receptor and/or p27kip 1. The antibodies were incubated with the cultured cells and detected by binding a second antibody conjugated to FITC and rhodamine. Individual cells were observed for fluorescence using confocal microscopy and the percentage of positive cells was quantified. The effect of three different concentrations of each compound was also determined.

The cells are also subjected to physiological tests to identify the channels that should be present in mature hair cells as (1) present and (2) activated.

In vivo results were evaluated at time points of weeks 4, 8 and 12. Regeneration of hair cells was assessed using immunocytochemistry, as described above. Functional recovery was assessed using methods routinely performed in specialized procedures in small animal physiology.

Example 7: evaluation of inner ear progenitor cell differentiation

The ability of a selected number of positive compounds to promote differentiation of bone marrow-derived inner ear progenitor cells into hair cells was evaluated. The experiment was initially performed using bone marrow derived inner ear progenitor cells (e.g., Mesenchymal Stem Cells (MSCs)) and a luciferase reporter construct in which luciferase expression is driven by the myosin VII1 enhancer region and promoter. This is a strong promoter in hair cells. The myosin VIIa enhancer region and promoter are also operably linked to GFP. Positive results were determined using RT-PCR and immunocytochemistry using the method for modulating myosin VIIa described in examples 3 to 6.

Example 8: pharmacological Properties of the Compounds

Half maximal Inhibitory Concentration (IC) of each of the compounds identified in example 2 was measured using standard laboratory techniques₅₀) And a median lethal dose or 50% lethal dose (LD 50). IC (integrated circuit)₅₀Is a measure of the effect of a compound to inhibit a biological or biochemical function. LD50 is the dose required for a compound to kill half of the members of the test population.

Each compound IC₅₀And LD50 are shown in table 2.

TABLE 2

CP-0000550	0.03	2	＞30
				CP-0000553	0.01	2	＞30
CP-0000554	0.4	2	＞30
				CP-0000557	0.02	2.6	30
CP-0000571	0.3	2	＞30
				CP-0000928	0.3	2	＞10
CP-0005186	1	1.8	＞10
				CP-0007991	0.6	2	＞10
CP-0007994	1	1.9	＞10
				CP-0008545	0.4	2.5	＞10
CP-0009883	2.5	2.3	＞10
				CP-0010539	0.5	2.1	＞10
CP-0029278	0.5	1.9	＞10
				CP-0029300	1.6	2	＞10
CP-0034360	1	2	＞10
				CP-0036187	1	2.3	＞10
CP-0039073	1.5	2.4	＞10
				CP-0045061	3	1.9	＞10
CP-0047659	1.3	2.3	＞10
				CP-0050095	1.5	2.6	＞10
CP-0059547	1	2	＞10
				CP-0059563	＞10	1.9	＞10
CP-0059642	1	2.2	＞10
				CP-0060729	0.2	1.6	＞10
CP-0060852	2.5	1.8	＞10
				CP-0061401	0.6	1.8	＞10
CP-0061566	0.2	2	＞10
				CP-0061777	＞10	2.1	＞10
CP-0062030	2	1.6	＞10
				CP-0063182	0.2	1.7	＞10

CP-0063375	2.2	1.9	＞10
				CP-0063508	＞10	1.7	＞10
CP-0064231	1	1.5	＞10
				CP-0064314	＞10	1.7	＞10
CP-0064917	＞10	2	＞10
				CP-0065665	0.8	1.7	＞10
CP-0066751	1	1.9	＞10
				CP-0066829	1	1.8	＞10
CP-0067108	3	1.8	＞10
				CP-0067233	2	1.8	＞10
CP-0067246	1.8	1.5	＞10
				CP-0068395	3	1.6	＞10
CP-0068577	1	1.6	＞10
				CP-0068929	0.4	2	＞10
CP-0069934	2	1.7	＞10
				CP-0069961	2	1.7	＞10
CP-0070164	1.6	1.7	＞10
				CP-0070367	2	1.7	＞10
CP-0070844	2	1.7	＞10
				CP-0070871	3	2	＞10
CP-0070886	1	2.1	＞10
				CP-0071862	0.7	1.8	＞10
CP-0072036	1.5	1.7	＞10
				CP-0072092	1	1.7	＞10
CP-0072096	6	2.2	＞10
				CP-0072156	2	2	＞10
CP-0072253	1.3	2	＞10
				CP-0072271	1	2.2	＞10
CP-0072720	3	1.8	＞10
				CP-0074806	0.7	1.8	＞10

CP-0075627	8	2	＞10
				CP-0076627	5	2.2	＞10
CP-0078448	＞10	1.9	＞10
				CP-0079810	3	2.3	＞10
CP-0079983	3	1.8	＞10
				CP-0080276	0.3	2	＞10
CP-0080773	＞10	2.2	＞10
				CP-0087336	0.08	2.2	＞10
CP-0087799	1	2.1	＞10
				CP-0089966	＞10	2.2	＞10
CP-0091818	0.9	2.1	＞10
				CP-0096433	3	2	＞10
CP-0099289	＞10	1.6	＞10
				CP-0102404	＞10	1.6	＞10
CP-0103978	1	2	＞10
				CP-0104765	0.4	2	＞10
CP-0104766	3	3	＞10
				CP-0104904	＞10	2.2	＞10
CP-0105343	3	2	＞10
				CP-0105777	0.3	2	＞10
CP-0107060	0.1	2.2	＞10
				CP-0109953	0.1	1.8	＞10
CP-0110352	0.05	1.8	＞10
				CP-0110644	1	1.8	＞10
CP-0130586	0.5	2.2	＞10
				CP-0130665	0.3	2	＞10
CP-0131763	2	2.4	＞10
				CP-0134381	2	2.2	＞10
CP-0193184	＞10	N/A	＞30

Example 9: characterization of compounds using inner ear progenitor cells isolated from mouse cochlea

The ability of compounds identified by the method described in example 2 to promote differentiation of mouse inner ear progenitor cells isolated from mouse cochlea into hair cells was examined.

Cochlear stem cells were isolated from Atoh1-nGFP mice as described previously (Oshima et al, supra). As described above, these animals expressed enhanced nuclear versions of Green Fluorescent Protein (GFP) when the Atoh1 enhancer element was activated (Chen et al and Lumpkin et al, supra). Thus, cells obtained from these animals can be used to follow the differentiation of inner ear progenitor cells into hair cells using fluorescence microscopy.

Briefly, inner ear progenitor cells are obtained from second or third generation animals, and are between 1 and 3 days of age. Then, the cells were seeded at a density of 300 spheres per well (Oshima et al, JAssoc Res Otolarynggol 8: 18-31, 2007, and Martinez-Monedero et al, JNeurobiol 66: 319-331, 2008), and allowed to stick on the surface of 6-well plates and cultured in the presence of growth factors. Cells cultured in DMEM medium containing N2 and B27 but no growth factors were exposed to the compounds and maintained in culture for 3 to 10 days. Cell differentiation was monitored by detecting the expression of green fluorescence by nuclear GFP from Atoh1 reporter and by staining with mature hair cell markers myosin VIIa and espin in compound treated cultures and comparing to controls at 24, 72m, 108 hour time points.

Positive results were confirmed using the methods described in examples 3-6 and 7, using RT-PCR and immunocytochemistry.

Example 10: using miceCharacterization of Corti device explants Compounds

The compounds identified in example 2 were tested for their ability to promote the formation of new hair cells in mouse Corti organ explants.

Briefly, explants were obtained by dissection from Atoh1-GFP mice. The Corti apparatus was cultured on collagen-coated plates and incubated overnight in serum-containing medium. The compounds were added to the culture at the time of implantation, as described previously (Shi et al, and Martinez-Monedero et al, supra). Cultures were maintained in DMEM media containing B27 supplement (Invitrogen) for 3 to 10 days prior to analysis.

Hair cells are formed from epithelial cells outside the row of hair cells in culture, which are monitored using automated systems and quantitative immunohistochemistry to detect the presence of GFP positive cells.

Example 11: optimization of compounds

The compounds are optimized to provide efficacy in the nanomolar range and reduced cytotoxicity.

The compounds are modified using the pharmacochemistry methods described above. Absorption, distribution, metabolism and excretion (ADME) studies were performed to evaluate Log P assays, water solubility assessments, mouse liver microsome stability assays and plasma protein binding assays, as described below.

Subsequent testing was performed using synthetic or purified compounds that were at least 95% pure as determined by 1H NMR. Other analytical techniques (i.e., 13C NMR, IR, melting point, MS, and/or elemental analysis) are also applied to determine structure and purity. Optically pure substances were also evaluated by chiral stationary phase high performance liquid chromatography. The structure of the compounds was evaluated using 2-D NMR and X-ray crystallography.

Log P values of compounds were determined by adding 15mL of a stock of the compound (in 10mM DMSO) to the test tube750 ml of 1-octanol buffer (pH 7.4). 3mL testosterone (in 50mM DMSO) was used as a control. The sample was shaken at room temperature for one hour and then allowed to stand for one hour to allow the different layers to separate. Each layer of 400ml was transferred and placed in a different container. Each sample was then serially diluted with 50% methanol. Standard curves of compound prepared with 50% methanol and testosterone, and subsequently, samples were analyzed using LC/MS monitoring. The calculated concentration ratio of the test compound in each phase was calculated independently using the minimum dilution of the sample in each phase that met each standard curve of two replicates. Log by taking the average of 2 calculated ratios₁₀To calculate Log P.

1mL of 0.07M NaH adjusted to pH 7.4₂PO₄The buffered solution was combined with at least 1mg of each compound tested to determine an estimate of the water solubility of the compound. Then, the sample was shaken for 2 hours and then allowed to stand at room temperature for 12 hours. The sample was then filtered through a nylon syringe filter saturated with sample having a pore size of 0.45 microns. The resulting filtrate was detected by LC/MS using electrospray (N ═ 2).

Chemical metabolic stability of the compounds was determined using mixed mouse liver microparticles. The compounds bound to 1mg/mL of liver microprojectile protein and 1mM NADPH were incubated for 0, 15, 30 and 60 minutes. Testosterone and propranolol were used as positive controls. Compounds without NADPH and microsomes were used as negative controls. The sample was quenched with acetonitrile and centrifuged at 10,000RPM for 10 minutes to precipitate the protein. The sample supernatant was analyzed by LC/MS (N ═ 3). Standard curves were generated at 4 concentrations (100%, 30%, 10% and 3%) and the remaining amount of test compound remaining was determined at 4 time points.

Plasma protein binding studies were performed by preparing solutions containing compound (5 μm, 0.5% final DMSO concentration), buffer, and 10% plasma (volume of buffer). A96-well dialysis plate was assembled, in each of which a semipermeable cellulose membrane was used to divide it into two (molecular weight cut-off value: 10,000). Buffer was added to one side of the membrane and plasma solution was added to the other side. The plates were then sealed and placed on an orbital shaker and incubated at 37 degrees celsius. Standards prepared in plasma and buffer were incubated with the dialysis plates at 37 degrees celsius. The corresponding solution for each compound was detected in a cartridge using tandem mass spectrometry (LC-MS). Each compound was tested in duplicate.

After equilibration, samples were taken from both sides of the membrane. Solutions of each batch of compound were pooled into two groups (plasma-free and plasma-containing) and the cartridges were analyzed by LC-MS using two calibration standards, plasma-free (6 points) and plasma (7 points). The samples were quantified to determine the amount of bound compound using a standard curve prepared in an equivalent matrix.

Example 12: in vivo pharmacokinetics, toxicity and formulation studies

The compound was administered using intraventricular (icv) injection into the brain of mice, and a dose study was conducted to determine the maximum dose of compound that could be administered by this route.

Pharmacokinetic studies were performed using Intraperitoneal (IP) or intracochlear administration of 3mg/kg of compound to determine the dose and concentration of the compound in the cochlea, relevant tissues. Compound levels were measured in plasma and cochlear tissue at nine time points spanning 24 hours.

The compound for oral administration is dissolved in a formulation such as 2% hydroxypropyl-beta-cyclodextrin at a concentration of 3mg/kg body weight. Intracochlearly administered compounds are administered as described previously (Chen et al, J.Neurosci.methods, 150: 67-73, 2006). Briefly, after anesthesia of the mice, a tube is inserted into the cochleostomy to provide access to the scala tympani. The compound solution was then delivered by a syringe pump over 6 hours at a flow rate of 1 μ Ι _ per hour. The surgical site was then sutured and the animals were examined at various time points.

Example 13: in vivo studies using animal deafness models

Mice of 10 weeks mouse age were exposed to octave-band (8-16 kilohertz) noise at-116 dB SPL for 2 hours. In CBA/CaJ mice, this noise dose can damage the outer hair cells in the bottom half of the cochlea, as well as the inner hair cells and supporting cells in a more limited area in the middle of the cochlea. (Wang et al, J.Ass.Res.Otolarynggol., 3: 248-268, 2002). In addition, in areas where inner hair cells are damaged, nerves can degenerate within 7 days. After the recovery phase, these mice were tested for ABR and DPOAE. The cochlea was dissected and immunostained after cutting the frozen portion or embedding the whole specimen. Cell division was assessed in animals by staining frozen sections with BrdU and antibody. Cell death was assessed by TUNEL. The number of hair cells, supporting cells and spiral ganglion neurons were counted.

One week after noise treatment, noise-treated mice were injected with compound (1mg/10g body weight). Samples were analyzed at time points of day 4, 8, 14 and 21, and hair cell counts were performed for the entire length of the cochlear spiral.

Functional assessment was achieved by using measurements of amplitude versus level functions of DPOAE and ABR as described previously (Kujawa and Liberman, J.Neuropysiol, 78: 3095-.

Other embodiments

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

Claims (12)

1. Use of a compound having the formula:

wherein:

R₁₁₈、R₁₁₉and R₁₂₁Each is independently H;

R₁₂₀selected from H, halo, NO₂、C₁-C₃An alkyl group;

R₁₂₂is hydrogen or-Z-R^a(ii) a Wherein:

z is O or a bond; and is

R^aThe method comprises the following steps:

(i)C₁-C₆an alkyl group; or

(ii) Benzyl or phenethyl, wherein the phenyl moiety of each of them is independently selected from 1-2 halo, C₁-C₆Alkyl and C₁-C₆The substituent of alkoxy is optionally substituted;

R₁₂₃the method comprises the following steps:

(i) phenyl optionally substituted with 1-3 independently selected from C₁-C₆Alkyl radical, C₁-C₆Alkoxy, hydroxy and-NHC (O) (C)₁-C₃Alkyl) substituents; or

(ii) Benzyl or phenethyl, in which the phenyl part of each of them is replaced by NH₂Optionally substituted; or

(iii)-(C₁-C₆Alkyl) -Z¹- (phenyl) in which Z¹Is O, S, NH or N (CH)₃) (ii) a The phenyl moiety is selected from 1-5 independently from halo, C₁-C₆Alkyl and C₁-C₆The substituents of the alkoxy group are optionally substituted.

2. The use of claim 1, wherein the medicament is administered topically to the inner ear of a patient in need of treatment and increases the number of cells with auditory hair cell properties in the inner ear of the patient.

3. The use of claim 2, wherein the medicament is administered by injection into one or more of a cochlear cavity, into an auditory nerve trunk of an internal ear canal, or into the scala tympani.

4. The use of claim 2, wherein the auditory hair cells are external auditory hair cells or internal auditory hair cells.

5. The use of claim 1, wherein R₁₂₂is-Z-R^a。

6. The use of claim 5, wherein Z is O.

7. The use of claim 1, wherein R₁₂₃Is phenyl, consisting of 1-3 substituents selected from C₁-C₆Alkyl radical, C₁-C₆Alkoxy, hydroxy and-NHC (O) (C)₁-C₃Alkyl) is optionally substituted.

8. The use of claim 1, wherein R₁₂₀Is halo or NO₂。

9. The use of claim 1, wherein:

R₁₂₂is-Z-R^aWherein R is^aIs benzyl or phenethyl, wherein the phenyl moiety of each of them is independently selected from 1-2 halo, C₁-C₆Alkyl and C₁-C₆The substituent of alkoxy is optionally substituted; and is

R₁₂₃Is phenyl, consisting of 1-3 independently selected from C₁-C₆Alkyl radical, C₁-C₆Alkoxy, hydroxy and-NHC (O) (C)₁-C₃Alkyl) is optionally substituted.

10. The use of claim 1, wherein:

R₁₂₂is-Z-R^aWherein Z is O, and R^aIs benzyl or phenethyl, wherein the phenyl moiety of each of them is independently selected from 1-2 halo, C₁-C₆Alkyl and C₁-C₆The substituent of alkoxy is optionally substituted; and is

R₁₂₃Is phenyl, consisting of 1-3 substituents selected from C₁-C₆Alkyl radical, C₁-C₆Alkoxy, hydroxy and-NHC (O) (C)₁-C₃Alkyl) is optionally substituted; and is

R₁₂₀Selected from H, halo and NO₂。

11. The use of claim 1, wherein the compound is selected from the group consisting of:

6-chloro-1- (2-chlorobenzyloxy) -2-phenyl-1H-benzo [ d ] imidazole;

6-chloro-1- (2-chlorobenzyloxy) -2- (4-methoxyphenyl) -1H-benzo [ d ] imidazole;

6-chloro-2- (4-methoxyphenyl) -1- (4-methylbenzyloxy) -1H-benzo [ d ] imidazole;

6-chloro-1- (3, 5-dimethylbenzyloxy) -2- (4-methoxyphenyl) -1H-benzo [ d ] imidazole;

6-chloro-1- (4-methoxybenzyloxy) -2- (4-methoxyphenyl) -1H-benzo [ d ] imidazole;

1- (4-methylbenzyloxy) -6-nitro-2-phenyl-1H-benzo [ d ] imidazole;

2, 5-dichloro-N- ((1-methyl-1H-benzo [ d ] imidazol-2-yl) methyl) aniline;

4- (2- (1-methyl-1H-benzo [ d ] imidazol-2-yl) ethyl) aniline;

2- ((4-fluorophenoxy) methyl) -1-methyl-1H-benzo [ d ] imidazole;

2- (4-methoxyphenyl) -1-phenethyl-1H-benzo [ d ] imidazole;

or a pharmaceutically acceptable salt thereof.

12. The use of claim 1, wherein the compound is 6-chloro-1- (2-chlorobenzyloxy) -2-phenyl-1H-benzo [ d ] imidazole, or a pharmaceutically acceptable salt thereof.