HEARING LOSS-PROTECTIVE COMPOUNDS AND METHODS THEREOF

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Patent Application No. 62/715,169, filed August 6, 2018, the disclosure of which is hereby incorporated by reference in its entirety.

STATEMENT OF GOVERNMENT LICENSE RIGHTS

This invention was made with government support under DC013688, DC005987, and NS074506, awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

Aminoglycosides are clinically used drugs that cause dose-dependent sensorineural hearing loss (Smith et al ., New Engl J Med, (1977) 296:349-53) and are known to kill hair cells in the mammalian inner ear (Theopold, Acta Otolaryngol (1977) 84:57-64). In the U.S., over 2,000,000 people receive treatment with aminoglycosides per year. The clinical efficacy of these drugs in treating resistant bacterial infections and their low cost globally account for their continued use and need.

Cisplatin, a chemotherapeutic agent is used for its benefit to life despite its toxic effects on the hair cells of the inner ear. High frequency hearing loss (>8 kHz) has been reported to be as high as 90% in children undergoing cisplatin therapy (Allen, et al, Otolaryngol Head Neck Surg (1998) 118:584-588). The incidence of vestibulotoxic effects of such drugs on patient populations has been less well studied. Estimates range between 3% and 6% with continued reports in the literature of patients with aminoglycoside induced vestibulotoxicity (Dhanireddy et al. , Arch Otolarngol Head Neck Surg (2005) 131 :46-48).

Other clinically important and commonly used drugs also have documented ototoxic effects, including loop diuretics (Greenberg, Am J Med Sci, (2000) 319: 10-24), antimalarial sesquiterpene lactone endoperoxides (i.e., artemesinins) (Toovey and Jamieson, Trans R Soc Trop Med Hyg (2004) 98:261-7), antimalarial quinines (Claessen, et al., Trop Med Int Health, (1998) 3:482-9), salicylates (Matz, Ann Otol Rhinol Laryngol Suppl (1990) 148:39-41), and interferon polypeptides (Formann, et al, Am J Gastroenterol (2004) 99:873-77). There is a need for compounds that can protect against the ototoxic effects of drugs. The present disclosure seeks to fulfill these needs and provides further related advantages.

SUMMARY

This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

In one aspect, the present disclosure features a compound of Formula (A)

or a pharmaceutically acceptable salt thereof, wherein:

Y is halo, C _3-6 alkyl, CN, N0 ₂ , C _3-6 haloalkyl, aryl, or heteroaryl,

Z is S or O;

R ₃ is H, Ci_ ₆ alkyl, C _2-6 alkenyl, or C _2.g alkynyl;

R ₂ is C(0)-U-X, wherein U is C _3-3 alkylene, C _2-6 alkenylene, or C _2-6 alkynylene; and X is a leaving group, or

R ₂ is C(0)CH=CR ₆ R ₇ , wherein R ₆ and R ₇ are each independently H, C \ alkyl, C^. ₆ haloalkyl, or aryl, each optionally substituted with 1, 2, or 3 substituents selected from halo, OH, C \ alkyl, C \ haloalkyl, CN, and N0 ₂ , provided that R ₆ and R ₇ are not both H; or

R ₃ , R ₃ ', R ₄ , and R ₄ ' are each H, C _3.6 alkyl, C _3.6 haloalkyl, or aryl,

R ₃ and R ₄ together with the C atoms to which they are attached form a 5 or 6 membered bridged heterocycle;

L is C alkylene or C heteroalkylene, each optionally substituted with 1, 2, 3, 4, 5, or 6 halo;

n is 0 or 1; and

m is 1 or 2;

provided that when n is 1 and m is 1, R ₃ is H and R ₄ is H, and provided that when n is 0 and m is 2, R ₃ and R4 together with the C atoms to which they are attached form a 5- or 6-membered heterocycloalkyl ring, optionally substituted by 1, 2, or 3 substituents independently selected from halo, OH, C ₃ _6 alkyl, C _3-6 haloalkyl, CN, and N0 _2.

In another aspect, the present disclosure features a pharmaceutical composition that includes a compound of the present disclosure, or a pharmaceutically acceptable salt thereof.

In yet another aspect, the present disclosure features a method of treating hearing loss, including administering to a subject in need thereof a therapeutically effective amount of a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of this disclosure will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

FIGEIRE 1 is a graph showing protective properties over time of embodiments of compounds of the present disclosure vs. comparative compounds.

DETAILED DESCRIPTION

Disclosed herein are compounds, and pharmaceutical compositions that include such compounds, for preventing, treating, and/or protecting against sensory hair cell death. Methods of using the compounds, alone or in combination with other therapeutic agents, are also disclosed.

The present disclosure describes compounds that can protect against drug (e.g., aminoglycoside antibiotic, platinum chemotherapeuticj-induced hearing loss. Contrary to therapeutic agents that act by a reversible mechanism of action, where mechanosensory hair cells are no longer protected against the toxic effects of ototoxins when the protective compound is removed from the medium, the compounds of the present disclosure incorporate chemically reactive functionality(ies) (e.g., halo-acetamide) that can react with nucleophilic functional groups near the compound's binding site on a molecular target to provide extended long-term protection. For example, pretreatment of a subject with the irreversible hearing protective compounds of the present disclosure can provide long-term protection. Definitions

At various places in the present specification, substituents of compounds of the disclosure are disclosed in groups or in ranges. It is specifically intended that the disclosure include each and every individual subcombination of the members of such groups and ranges. For example, the term "C^ alkyl" is specifically intended to individually disclose methyl, ethyl, C ₃ alkyl, C ₄ alkyl, C ₅ alkyl, and C ₆ alkyl.

It is further appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment.

Conversely, various features of the disclosure which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination.

As used herein, the term "substituted" or "substitution" refers to the replacing of a hydrogen atom with a substituent other than H. For example, an "N-substituted piperidin-4- yl" refers to replacement of the H atom from the NH of the piperidinyl with a non hydrogen substituent such as, for example, alkyl.

As used herein, the term "alkyl" refers to a saturated hydrocarbon group which is straight-chained (e.g., linear) or branched. Example alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like. An alkyl group can contain from 1 to about 30, from 1 to about 24, from 2 to about 24, from 1 to about 20, from 2 to about 20, from 1 to about 10, from 1 to about 8, from 1 to about 6, from 1 to about 4, or from 1 to about 3 carbon atoms.

As used herein, the term "aryl" refers to monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings) aromatic hydrocarbons such as, for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, and indenyl. In some embodiments, aryl groups have from 6 to about 20 carbon atoms. In some embodiments, aryl groups are monocyclic aromatic hydrocarbons, such as phenyl.

As used herein, "arylene" refers to a linking aryl group.

As used herein, the term "halo" or "halogen" includes fluoro, chloro, bromo, and iodo.

As used herein, the term "alkylene" refers to a linking alkyl group. As used herein, "alkenyl" refers to an alkyl group having one or more double carbon-carbon bonds. The alkenyl group can be linear or branched. Example alkenyl groups include ethenyl, propenyl, and the like. An alkenyl group can contain from 2 to about 30, from 2 to about 24, from 2 to about 20, from 2 to about 10, from 2 to about 8, from 2 to about 6, or from 2 to about 4 carbon atoms.

As used herein, "alkenylene" refers to a linking alkenyl group.

As used herein, "alkynyl" refers to an alkyl group having one or more triple carbon-carbon bonds. The alkynyl group can be linear or branched. Example alkynyl groups include ethynyl, propynyl, and the like. An alkynyl group can contain from 2 to about 30, from 2 to about 24, from 2 to about 20, from 2 to about 10, from 2 to about 8, from 2 to about 6, or from 2 to about 4 carbon atoms.

As used herein, "alkynylene" refers to a linking alkynyl group.

As used herein, "alkoxy" refers to an -O-alkyl group. Example alkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), t-butoxy, and the like.

As used herein, "haloalkyl" refers to an alkyl group having one or more halogen substituents. Example haloalkyl groups include CF ₃ , C ₂ F ₅ , CHF ₂ , CCl ₃ , CHCl ₂ , C ₂ Cl ₅ , and the like.

As used herein, "haloalkenyl" refers to an alkenyl group having one or more halogen substituents.

As used herein, "haloalkynyl" refers to an alkynyl group having one or more halogen substituents.

As used herein, "haloalkoxy" refers to an -O-(haloalkyl) group.

As used herein, "cycloalkyl" refers to non-aromatic carbocycles including cyclized alkyl, alkenyl, and alkynyl groups. Cycloalkyl groups can include mono- or polycyclic (e.g., having 2, 3 or 4 fused rings) ring systems, including spirocycles. In some embodiments, cycloalkyl groups can have from 3 to about 20 carbon atoms, 3 to about 14 carbon atoms, 3 to about 10 carbon atoms, or 3 to 7 carbon atoms. Cycloalkyl groups can further have 0, 1, 2, or 3 double bonds and/or 0, 1, or 2 triple bonds. Also included in the definition of cycloalkyl are moieties that have one or more aromatic rings fused (i.e., having a bond in common with) to the cycloalkyl ring, for example, benzo derivatives of pentane, pentene, hexane, and the like. A cycloalkyl group having one or more fused aromatic rings can be attached though either the aromatic or non-aromatic portion. One or more ring-forming carbon atoms of a cycloalkyl group can be oxidized, for example, having an oxo or sulfido substituent. Example cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbomyl, norpinyl, norcamyl, adamantyl, and the like.

As used herein, "cycloalkylene" refers to a linking cycloalkyl group.

As used herein, "heteroalkyl" refers to an alkyl group having at least one heteroatom such as sulfur, oxygen, or nitrogen.

As used herein, "heteroalkylene" refers to a linking heteroalkyl group.

As used herein, a "heteroaryl" refers to an aromatic heterocycle having at least one heteroatom ring member such as sulfur, oxygen, or nitrogen. Heteroaryl groups include monocyclic and polycyclic (e.g., having 2, 3 or 4 fused rings) systems. Any ring forming N atom in a heteroaryl group can also be oxidized to form an N-oxo moiety. Examples of heteroaryl groups include without limitation, pyridyl, N-oxopyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furyl, quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrryl, oxazolyl, benzofuryl, benzothienyl, benzthiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, l,2,4-thiadiazolyl, isothiazolyl, benzothienyl, purinyl, carbazolyl, benzimidazolyl, indolinyl, and the like. In some embodiments, the heteroaryl group has from 1 to about 20 carbon atoms, and in further embodiments from about 3 to about 20 carbon atoms. In some embodiments, the heteroaryl group contains 3 to about 14, 3 to about 7, or 5 to 6 ring-forming atoms. In some embodiments, the heteroaryl group has 1 to about 4, 1 to about 3, or 1 to 2 heteroatoms.

As used herein, " heteroaryl ene" refers to a linking heteroaryl group.

As used herein, "heterocycloalkyl" refers to non-aromatic heterocycles including cyclized alkyl, alkenyl, and alkynyl groups where one or more of the ring-forming carbon atoms are replaced by a heteroatom such as an O, N, or S atom. Heterocycloalkyl groups can be mono- or polycyclic (e.g., having 2, 3,4 or more fused rings or having a 2-ring, 3- ring, 4-ring spiro system (e.g., having 8 to 20 ring-forming atoms). Heterocycloalkyl groups include monocyclic and polycyclic groups. Example "heterocycloalkyl" groups include morpholino, thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, 2,3-dihydrobenzofuryl, l,3-benzodioxole, benzo-I,4-dioxane, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, and the like. Ring-forming carbon atoms and heteroatoms of a heterocycloalkyl group can be optionally substituted by oxo or sulfido. Also included in the definition of heterocycloalkyl are moieties that have one or more aromatic rings fused (i.e., having a bond in common with) to the nonaromatic heterocyclic ring, for example phthalimidyl, naphthalimidyl, and benzo derivatives of heterocycles such as indolene and isoindolene groups. In some embodiments, the heterocycloalkyl group has from 1 to about 20 carbon atoms, and in further embodiments from about 3 to about 20 carbon atoms. In some embodiments, the heterocycloalkyl group contains 3 to about 14, 3 to about 7, or 5 to 6 ring-forming atoms. In some embodiments, the heterocycloalkyl group has 1 to about 4, 1 to about 3, or 1 to 2 heteroatoms. In some embodiments, the heterocycloalkyl group contains 0 to 3 double bonds. In some embodiments, the heterocycloalkyl group contains 0 to 2 triple bonds.

As used herein, "heterocycloalkylene" refers to a linking heterocycloalkyl group.

As used herein, "amino" refers to NH _2.

As used herein, "alkylamino" refers to an amino group substituted by an alkyl group.

As used herein, "dialkylamino" refers to an amino group substituted by two alkyl groups.

As used herein, the term "leaving group" is a group that can be displaced by nucleophiles. Examples of leaving groups include halogen, mesyloxy, tosyloxy, and anhydride residues of carbonic acids such as t-butoxy-carbonyloxy.

As used herein, the term "individual," "subject," or "patient," used interchangeably, refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans.

As used herein, the phrase "therapeutically effective amount" refers to the amount of a therapeutic agent (i.e., drug, or therapeutic agent composition) that elicits the biological or medicinal response that is being sought in a tissue, system, animal, individual or human by a researcher, veterinarian, medical doctor or other clinician, which includes one or more of the following:

(1) preventing the disease; for example, preventing a disease, condition or disorder in an individual who can be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease; (2) inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder; and

(3) ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology) such as decreasing the severity of disease.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Compounds

The compounds of the present disclosure can form covalent bonds with their biological binding sites and can continue to provide protection even after the compound is removed from the medium.

In one embodiment, the present disclosure features a compound of Formula (A)

or a pharmaceutically acceptable salt thereof, wherein:

Y is halo, C _j.g alkyl, CN, N0 ₂ , C^ _g haloalkyl, aryl, or heteroaryl,

Z is S or O;

R- _! is H, Ci_ ₆ alkyl, C _2-6 alkenyl, or C _2.g alkynyl;

R ₂ is C(0)-U-X, wherein U is C^ alkylene, C _2-6 alkenylene, or C _2-6 alkynylene; and X is a leaving group, or R ₂ is C(0)CH=CRgR7, wherein R ₆ and R ₇ are each independently H, C _| . alkyl, Ci_6 haloalkyl, or aryl, each optionally substituted with 1, 2, or 3 substituents selected from halo, OH, C^g alkyl, C _| . haloalkyl, CN, and NO2, provided that R6 and R7 are not both H; or

R3, R3', R4, and R4' are each H, C _| . alkyl, C _| . haloalkyl, or aryl,

R ₃ and R ₄ together with the C atoms to which they are attached form a 5 or 6 membered bridged heterocycle;

L is C _2- 5 alkylene or C _2-5 heteroalkylene each optionally substituted with 1, 2, 3, 4, 5, or 6 halo;

n is 0 or 1; and

m is 1 or 2;

provided that when n is 1 and m is 1, R3 is H and R4 is H, and

provided that when n is 0 and m is 2, R ₃ and R ₄ together with the C atoms to which they are attached form a bridged 7- or 8-membered heterocycloalkyl ring, optionally substituted by 1, 2, or 3 substituents independently selected from halo, OH, C _j _ 6 alkyl, C^g haloalkyl, CN, and N0 _2.

In some embodiments, R ₃ , R ₃ ', R ₄ , and R ₄ ' are each H.

In some embodiments, when n is 0 and m is 2, R ₃ and R4 together with the C atoms to which they are attached form a 7- or 8-membered bridged heterocycloalkyl ring, having a [3,2,l]heterocyclooctenyl structure In some embodiments, the [3,2,l]heterocyclooctenyl structure is bridged by N-L-N(R _l )(R ₂ ). For example, the bridged heterocycle can have the following structure:

In some embodiments, the compounds of Formula (A) have Formula (A-l)

(A-l)

or pharmaceutically acceptable salt thereof. In some embodiments, the compounds of Formula (A) have Formula (A-2)

or pharmaceutically acceptable salt thereof.

In any of the compounds of Formula (A), (A-l), or (A-2), in some embodiments, Y is halo or C _j.g alkyl. In some embodiments, Y is halo. For example, Y can be Cl, Br, or I. In certain embodiments, Y is Cl. In some embodiments, Y is C^g alkyl. For example, Y can be methyl. As another example, Y is ethyl.

In any of the compounds of Formula (A), (A-l), or (A-2), in some embodiments,

Z is O.

In any of the compounds of Formula (A), (A-l), or (A-2), in some embodiments, R _l is H or Ci_ ₆ alkyl. For example, can be H. As another example, R^ can be methyl. As another example, R _j can be ethyl.

In any of the compounds of Formula (A), (A-l), or (A-2), in some embodiments, R ₂ is C(0)-U-X, wherein U is C^ alkylene, C _2-6 alkenylene, or C _2-6 alkynylene; and X is a leaving group. For example, U can be -CH _2- ; and X can be a leaving group (e.g., halogen, mesyloxy, tosyloxy, or anhydride residues of carbonic acids such as t-butoxy- carbonyloxy). In some embodiments, X is Cl, Br or I.

In any of the compounds of Formula (A), (A-l), or (A-2), in some embodiments, R ₂ is C(0)CH=CRgR ₇ , wherein R ₆ and R ₇ are each independently C _{| .} alkyl or C _{| .} haloalkyl. For example, R ₂ can be C(0)CH=CRgR ₇ , wherein R ₆ and R ₇ are each independently C \ alkyl. In certain embodiments, R ₂ is C(0)CH=CR ₆ R ₇ , wherein R ₆ and R ₇ are each CH _3.

In any of the compounds of Formula (A), (A-l), or (A-2), in some embodiments, L is a linear C _2-5 alkylene or C _2-5 haloalkylene. For example, L can be a linear C ₃ alkylene. In certain embodiments, L is a linear C ₂ alkylene. In certain embodiments, L is a linear C ₄ alkylene. As an example, L can be a linear C ₃ haloalkylene. In certain embodiments, L is a linear C ₂ haloalkylene. In certain embodiments, L is a linear C ₄ haloalkylene. In some embodiments, L is C ₂ alkylene connected to N, S or O. In some embodiments, the compounds of the present disclosure are:

or pharmaceutically acceptable salt thereof, wherein:

X is a leaving group selected from Cl, Br, and I;

and Y is halo, C^g alkyl, CN, N0 ₂ , C _j.g haloalkyl, aryl, or heteroaryl. In some embodiments, and as will be described in greater detail below, the present disclosure features a pharmaceutical composition that includes a compound of the present disclosure. The pharmaceutical composition can be in the form of a liquid, a gel, a suspension, an aerosol, a tablet, a powder, a pill, a capsule, or an implant.

In some embodiments, and as will be described in greater detail below, the present disclosure features a method of treating hearing loss, and/or a method of decreasing the likelihood of hearing loss, including administering to a subject in need thereof a therapeutically effective amount of a compound the present disclosure, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure.

The treatment methods can further include administering a therapeutically effective amount of the compound the present disclosure, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure, to the subject before, concurrently with, and/or after treatment of the subject with an aminoglycoside antibiotic agent, a platinum-based chemotherapeutic agent, or an ototoxic therapeutic agent. For example, administering the therapeutically effective amount of a compound the present disclosure, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure comprises topical administration to an inner ear of ( e.g ., intratympanic delivery to) the subject. In some embodiments, the present disclosure features topical administration of a therapeutically effective amount of a compound the present disclosure, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure, to a hair cell in the inner ear of the subject.

In some embodiments, the methods include oral administration, intravenous administration, intramuscular administration, and/or subcutaneous administration of a therapeutically effective amount of a compound the present disclosure, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure.

Pharmaceutical Compositions and Methods of Administration

Pharmaceutical compositions of the present disclosure can be formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing of the active compounds into preparations which are optionally used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Additional details about suitable excipients for pharmaceutical compositions described herein can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and

Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins, 1999), herein incorporated by reference in its entirety.

A pharmaceutical composition, as used herein, refers to a mixture of a compound of the present disclosure, having Formula (A), (A-l), (A-2) described herein, or

or pharmaceutically acceptable salt thereof, wherein:

X and Y are as defined above,

with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients. The pharmaceutical composition facilitates administration of the compound to an organism. In practicing the methods of treatment or use provided herein, therapeutically effective amounts of compounds described herein are administered in a pharmaceutical composition to a mammal having a disease, disorder, or condition to be treated. In some embodiments, the mammal is a human. A therapeutically effective amount depends on the severity of the disease, the age and relative health of the subject, the potency of the compound used and other factors. The compounds of the present disclosure are optionally used singly or in combination with one or more therapeutic agents as components of mixtures (as in combination therapy).

The pharmaceutical formulations are optionally administered to a subject by multiple administration routes, including but not limited to, oral, parenteral (e.g., intravenous, subcutaneous, intramuscular), intranasal, buccal, topical, rectal, or transdermal administration routes. Moreover, the pharmaceutical compositions, which include a compound of the present disclosure, are optionally formulated into any suitable dosage form, such as aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions, aerosols, controlled release formulations, fast melt formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate release and controlled release formulations.

One can administer the compounds and/or compositions in a local rather than systemic manner, for example, via injection of the compound directly into an organ or tissue, often in a depot preparation or sustained release formulation. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Furthermore, one can administer the drug in a targeted drug delivery system, for example, in a liposome coated with organ-specific antibody. The liposomes will be targeted to and taken up selectively by the organ. In some embodiments, the drug can be provided in the form of a rapid release formulation, in the form of an extended release formulation, or in the form of an intermediate release formulation.

Pharmaceutical compositions including a compound described herein can be manufactured, for example, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes.

The pharmaceutical compositions includes at least one compound of the present disclosure, as an active ingredient in free-acid or free-base form, or in a pharmaceutically acceptable salt form. In addition, the methods and pharmaceutical compositions described herein include the use of crystalline forms (also known as polymorphs), as well as active metabolites of these compounds having the same type of activity. In some situations, compounds can exist as tautomers. All tautomers are included within the scope of the compounds presented herein. Additionally, many of the compounds described herein exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. The solvated forms of the compounds presented herein are also considered to be disclosed herein.

In certain embodiments, the compositions can include one or more preservatives to inhibit microbial activity. Suitable preservatives include quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride.

Pharmaceutical preparations for oral use are optionally obtained by mixing one or more solid excipient with one or more of the compounds of the present disclosure, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets, pills, or capsules. Suitable excipients include, for example, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or others such as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. If desired, disintegrating agents can be added, such as the cross- linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions can be used, which can optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical preparations that are used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules optionally contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers can be added.

In some embodiments, the solid dosage forms disclosed herein can be in the form of a tablet, (including a suspension tablet, a fast-melt tablet, a bite-disintegration tablet, a rapid-disintegration tablet, an effervescent tablet, or a caplet), a pill, a powder (including a sterile packaged powder, a dispensable powder, or an effervescent powder), a capsule (including both soft or hard capsules, e.g., capsules made from animal-derived gelatin or plant-derived HPMC, or "sprinkle capsules"), solid dispersion, solid solution, bioerodible dosage form, controlled release formulations, pulsatile release dosage forms, multiparticulate dosage forms, pellets, granules, or an aerosol. In other embodiments, the pharmaceutical formulation is in the form of a powder. In still other embodiments, the pharmaceutical formulation is in the form of a tablet, including but not limited to, a fast- melt tablet. The pharmaceutical formulations of the compounds described herein can be administered as a single capsule or in multiple capsule dosage form. In some embodiments, the pharmaceutical formulation is administered in two, or three, or four, capsules or tablets.

In some embodiments, solid dosage forms, e.g., tablets, effervescent tablets, and capsules, are prepared by mixing particles of a compound of the present disclosure, with one or more pharmaceutical excipients to form a bulk blend composition. When referring to these bulk blend compositions as homogeneous, it is meant that the particles of the compound of present disclosure, are dispersed evenly throughout the composition so that the composition can be subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. The individual unit dosages can also include film coatings, which disintegrate upon oral ingestion or upon contact with diluent. These formulations are optionally manufactured by conventional pharmacological techniques.

The pharmaceutical solid dosage forms described herein include a compound of the present disclosure, and optionally one or more pharmaceutically acceptable additives such as a compatible carrier, binder, filling agent, suspending agent, flavoring agent, sweetening agent, disintegrating agent, dispersing agent, surfactant, lubricant, colorant, diluent, solubilizer, moistening agent, plasticizer, stabilizer, penetration enhancer, wetting agent, anti-foaming agent, antioxidant, preservative, or one or more combination thereof. In still other aspects, using standard coating procedures, such as those described in Remington's Pharmaceutical Sciences, 20th Edition (2000), a film coating is provided around the formulation of the compound described herein. In one embodiment, some or all of the particles of the compound described herein are coated. In another embodiment, some or all of the particles of the compound described herein are microencapsulated. In still another embodiment, the particles of the compound described herein are not microencapsulated and are uncoated.

Suitable carriers for use in the solid dosage forms described herein include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerin, magnesium silicate, sodium caseinate, soy lecithin, sodium chloride, tricalcium phosphate, dipotassium phosphate, sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, pregelatinized starch, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, sucrose, microcrystalline cellulose, lactose, mannitol and the like.

Suitable filling agents for use in the solid dosage forms described herein include, but are not limited to, lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, hydroxypropylmethycellulose (HPMC), hydroxypropylmethycellulose phthalate, hydroxypropylmethylcellulose acetate stearate (HPMCAS), sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like. In order to release the compounds of the present disclosure from a solid dosage form matrix as efficiently as possible, disintegrants are often used in the formulation, especially when the dosage forms are compressed with binder. Disintegrants help rupturing the dosage form matrix by swelling or capillary action when moisture is absorbed into the dosage form. Suitable disintegrants for use in the solid dosage forms described herein include, but are not limited to, natural starch such as corn starch or potato starch, a pregelatinized starch such as National 1551 or Amijel®, or sodium starch glycolate such as Promogel® or Explotab®, a cellulose such as a wood product, methylcrystalline cellulose, e.g., Avicel®, Avicel® PH101, Avicel® PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Ming Tia®, and Solka-Floc®, methylcellulose, croscarmellose, or a cross-linked cellulose, such as cross-linked sodium carboxymethylcellulose (Ac-Di-Sol®), cross-linked carboxymethylcellulose, or cross- linked croscarmellose, a cross-linked starch such as sodium starch glycolate, a cross- linked polymer such as crospovidone, a cross-linked polyvinylpyrrolidone, alginate such as alginic acid or a salt of alginic acid such as sodium alginate, a clay such as Veegum® HV (magnesium aluminum silicate), a gum such as agar, guar, locust bean, Karaya, pectin, or tragacanth, sodium starch glycolate, bentonite, a natural sponge, a surfactant, a resin such as a cation-exchange resin, citrus pulp, sodium lauryl sulfate, sodium lauryl sulfate in combination starch, and the like.

Binders impart cohesiveness to solid oral dosage form formulations: for powder filled capsule formulation, they aid in plug formation that are optionally filled into soft or hard shell capsules and for tablet formulation, they ensure the tablet remaining intact after compression and help assure blend uniformity prior to a compression or fill step. Materials suitable for use as binders in the solid dosage forms described herein include, but are not limited to, carboxymethylcellulose, methylcellulose (e.g., Methocel®), hydroxypropylmethylcellulose (e.g. Hypromellose ETSP Pharmacoat-603, hydroxypropylmethylcellulose acetate stearate (Aqoate HS-LF and HS), hydroxyethylcellulose, hydroxypropylcellulose (e.g., Klucel®), ethylcellulose (e.g., Ethocel®), and microcrystalline cellulose (e.g., Avicel®), microcrystalline dextrose, amylose, magnesium aluminum silicate, polysaccharide acids, bentonites, gelatin, polyvinylpyrrolidone/vinyl acetate copolymer, crospovidone, povidone, starch, pregelatinized starch, tragacanth, dextrin, a sugar, such as sucrose (e.g., Dipac®), glucose, dextrose, molasses, mannitol, sorbitol, xylitol (e.g., Xylitab®), lactose, a natural or synthetic gum such as acacia, tragacanth, ghatti gum, mucilage of isapol husks, starch, polyvinylpyrrolidone (e.g., Povidone® CL, Kollidon® CL, Polyplasdone® XL- 10, and Povidone® K-12), larch arabogalactan, Veegum®, polyethylene glycol, waxes, sodium alginate, and the like.

In general, binder levels of 20-70% are used in powder-filled gelatin capsule formulations. Binder usage level in tablet formulations varies whether direct compression, wet granulation, roller compaction, or usage of other excipients such as fillers which itself can act as moderate binder. In some embodiments, binder can form up to 70% in tablet formulations.

Suitable lubricants or glidants for use in the solid dosage forms described herein include, but are not limited to, stearic acid, calcium hydroxide, talc, corn starch, sodium stearyl fumerate, alkali-metal and alkaline earth metal salts, such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearates, magnesium stearate, zinc stearate, waxes, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, a polyethylene glycol or a methoxypolyethylene glycol such as Carbowax™, PEG 4000, PEG 5000, PEG 6000, propylene glycol, sodium oleate, glyceryl behenate, glyceryl palmitostearate, glyceryl benzoate, magnesium or sodium lauryl sulfate, and the like.

Suitable diluents for use in the solid dosage forms described herein include, but are not limited to, sugars (including lactose, sucrose, and dextrose), polysaccharides (including dextrates and maltodextrin), polyols (including mannitol, xylitol, and sorbitol), cyclodextrins and the like.

Suitable wetting agents for use in the solid dosage forms described herein include, for example, oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, quaternary ammonium compounds (e.g., Polyquat 10®), sodium oleate, sodium lauryl sulfate, magnesium stearate, sodium docusate, triacetin, vitamin E TPGS and the like.

Suitable surfactants for use in the solid dosage forms described herein include, for example, sodium lauryl sulfate, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g., Pluronic® (BASF), and the like. Suitable suspending agents for use in the solid dosage forms described here include, but are not limited to, polyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, polyethylene glycol, e.g., the polyethylene glycol optionally is selected to have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 5400 to about 7000, vinyl pyrrolidone/vinyl acetate copolymer (S630), sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, polysorbate-80, hydroxy ethylcellulose, sodium alginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum, sugars, cellulosics, such as, e.g., sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone and the like.

Suitable antioxidants for use in the solid dosage forms described herein include, for example, e.g., butylated hydroxytoluene (BHT), sodium ascorbate, and tocopherol.

There is considerable overlap between additives used in the solid dosage forms described herein. Thus, the above-listed additives should be taken as exemplary, and not limiting, of the types of additives that can be included in solid dosage forms of the pharmaceutical compositions described herein.

In other embodiments, one or more layers of the pharmaceutical formulation are plasticized. Illustratively, a plasticizer is generally a high boiling point solid or liquid. Suitable plasticizers can be added from about 0.01% to about 50% by weight (w/w) of the coating composition. Plasticizers include, but are not limited to, diethyl phthalate, citrate esters, polyethylene glycol, glycerol, acetylated glycerides, triacetin, polypropylene glycol, polyethylene glycol, triethyl citrate, dibutyl sebacate, stearic acid, stearol, stearate, and castor oil.

Compressed tablets are solid dosage forms prepared by compacting the bulk blend of the formulations described above. In various embodiments, compressed tablets which are designed to dissolve in the mouth will include one or more flavoring agents. In other embodiments, the compressed tablets will include a film surrounding the final compressed tablet. In some embodiments, the film coating can provide a delayed release of the compounds of the present disclosure from the formulation. In other embodiments, the film coating aids in patient compliance (e.g., Opadry® coatings or sugar coating). Film coatings including Opadry® typically range from about 1% to about 3% of the tablet weight. In other embodiments, the compressed tablets include one or more excipients.

A capsule can be prepared, for example, by placing the bulk blend of the formulation of the compound described above, inside of a capsule. In some embodiments, the formulations (non-aqueous suspensions and solutions) are placed in a soft gelatin capsule. In other embodiments, the formulations are placed in standard gelatin capsules or non-gelatin capsules such as capsules comprising HPMC. In other embodiments, the formulation is placed in a sprinkle capsule, wherein the capsule can be swallowed whole or the capsule can be opened and the contents sprinkled on food prior to eating. In some embodiments, the therapeutic dose is split into multiple (e.g., two, three, or four) capsules. In some embodiments, the entire dose of the formulation is delivered in a capsule form.

In various embodiments, the particles of the compound of the present disclosure described herein and one or more excipients are dry blended and compressed into a mass, such as a tablet, having a hardness sufficient to provide a pharmaceutical composition that substantially disintegrates within less than about 30 minutes, less than about 35 minutes, less than about 40 minutes, less than about 45 minutes, less than about 50 minutes, less than about 55 minutes, or less than about 60 minutes, after oral administration, thereby releasing the formulation into the gastrointestinal fluid.

In another aspect, dosage forms can include microencapsulated formulations. In some embodiments, one or more other compatible materials are present in the microencapsulation material. Exemplary materials include, but are not limited to, pH modifiers, erosion facilitators, anti-foaming agents, antioxidants, flavoring agents, and carrier materials such as binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents.

Microencapsulated compounds described herein can be formulated by methods that include, e.g., spray drying processes, spinning disk-solvent processes, hot melt processes, spray chilling methods, fluidized bed, electrostatic deposition, centrifugal extrusion, rotational suspension separation, polymerization at liquid-gas or solid-gas interface, pressure extrusion, or spraying solvent extraction bath. In addition to these, several chemical techniques, e.g., complex coacervation, solvent evaporation, polymer- polymer incompatibility, interfacial polymerization in liquid media, in situ polymerization, in-liquid drying, and desolvation in liquid media could also be used. Furthermore, other methods such as roller compaction, extrusion/spheronization, coacervation, or nanoparticle coating can also be used.

The pharmaceutical solid oral dosages forms including formulations described herein, which include a compound described herein, can be further formulated to provide a controlled release of the compound of present disclosure. Controlled release refers to the release of the compounds described herein from a dosage form in which it is incorporated according to a desired profile over an extended period of time. Controlled release profiles include, for example, sustained release, prolonged release, pulsatile release, and delayed release profiles. In contrast to immediate release compositions, controlled release compositions allow delivery of an agent to a subject over an extended period of time according to a predetermined profile. Such release rates can provide therapeutically effective levels of agent for an extended period of time and thereby provide a longer period of pharmacologic response while minimizing side effects as compared to conventional rapid release dosage forms. Such longer periods of response provide for many inherent benefits that are not achieved with the corresponding short acting, immediate release preparations.

In other embodiments, the formulations described herein, which include a compound of the present disclosure, are delivered using a pulsatile dosage form. A pulsatile dosage form is capable of providing one or more immediate release pulses at predetermined time points after a controlled lag time or at specific sites. Pulsatile dosage forms can be administered using a variety of pulsatile formulations including, but are not limited to, those described in U.S. Pat. Nos. 5,011,692; 5,017,381; 5,229,135; 5,840,329; 4,871,549; 5,260,068; 5,260,069; 5,508,040; 5,567,441 and 5,837,284.

Many other types of controlled release systems are suitable for use with the formulations described herein. Examples of such delivery systems include, e.g., polymer- based systems, such as polylactic and polyglycolic acid, polyanhydrides and polycaprolactone; porous matrices, nonpolymer-based systems that are lipids, including sterols, such as cholesterol, cholesterol esters and fatty acids, or neutral fats, such as mono-, di- and triglycerides; hydrogel release systems; silastic systems; peptide-based systems; wax coatings, bioerodible dosage forms, compressed tablets using conventional binders and the like. See, e.g., Liberman et al., Pharmaceutical Dosage Forms, 2 Ed., Vol. 1, pp. 209-214 (1990); Singh et al., Encyclopedia of Pharmaceutical Technology, 2nd Ed., pp. 751-753 (2002); U.S. Pat. Nos. 4,327,725; 4,624,848; 4,968,509; 5,461,140; 5,456,923; 5,516,527; 5,622,721; 5,686,105; 5,700,410; 5,977,175; 6,465,014; and

6,932,983.

In some embodiments, pharmaceutical formulations are provided that include particles of the compounds described herein, e.g. compounds of present disclosure, and at least one dispersing agent or suspending agent for oral administration to a subject. The formulations can be a powder and/or granules for suspension, and upon admixture with water, a substantially uniform suspension is obtained.

Liquid formulation dosage forms for oral administration can be aqueous suspensions selected from the group including, but not limited to, pharmaceutically acceptable aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups. See, e.g., Singh et al., Encyclopedia of Pharmaceutical Technology, 2nd Ed., pp. 754-757 (2002).

The aqueous suspensions and dispersions described herein can remain in a homogenous state, as defined in The LTSP Pharmacists' Pharmacopeia (2005 edition, chapter 905), for at least 4 hours. The homogeneity should be determined by a sampling method consistent with regard to determining homogeneity of the entire composition. In one embodiment, an aqueous suspension can be re-suspended into a homogenous suspension by physical agitation lasting less than 1 minute. In another embodiment, an aqueous suspension can be re-suspended into a homogenous suspension by physical agitation lasting less than 45 seconds. In yet another embodiment, an aqueous suspension can be re-suspended into a homogenous suspension by physical agitation lasting less than 30 seconds. In still another embodiment, no agitation is necessary to maintain a homogeneous aqueous dispersion.

The pharmaceutical compositions described herein can include sweetening agents such as, but not limited to, acacia syrup, acesulfame K, alitame, anise, apple, aspartame, banana, Bavarian cream, berry, black currant, butterscotch, calcium citrate, camphor, caramel, cherry, cherry cream, chocolate, cinnamon, bubble gum, citrus, citrus punch, citrus cream, cotton candy, cocoa, cola, cool cherry, cool citrus, cyclamate, cylamate, dextrose, eucalyptus, eugenol, fructose, fruit punch, ginger, glycyrrhetinate, glycyrrhiza (licorice) syrup, grape, grapefruit, honey, isomalt, lemon, lime, lemon cream, monoammonium glyrrhizinate (MagnaSweet®), maltol, mannitol, maple, marshmallow, menthol, mint cream, mixed berry, neohesperidine DC, neotame, orange, pear, peach, peppermint, peppermint cream, Prosweet® Powder, raspberry, root beer, rum, saccharin, safrole, sorbitol, spearmint, spearmint cream, strawberry, strawberry cream, stevia, sucralose, sucrose, sodium saccharin, saccharin, aspartame, acesulfame potassium, mannitol, talin, sucralose, sorbitol, swiss cream, tagatose, tangerine, thaumatin, tutti frutti, vanilla, walnut, watermelon, wild cherry, wintergreen, xylitol, or any combination of these flavoring ingredients, e.g., anise-menthol, cherry-anise, cinnamon-orange, cherry-cinnamon, chocolate-mint, honey-lemon, lemon-lime, lemon-mint, menthol- eucalyptus, orange-cream, vanilla-mint, and mixtures thereof.

For intravenous injections, compounds described herein can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally recognized in the field. For other parenteral injections, appropriate formulations can include aqueous or nonaqueous solutions, preferably with physiologically compatible buffers or excipients. Such excipients are generally recognized in the field.

Parenteral injections can involve bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The pharmaceutical composition described herein can be in a form suitable for parenteral injection as a sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water- soluble form. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension can also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. In certain embodiments, delivery systems for pharmaceutical compounds can be employed, such as, for example, liposomes and emulsions. In certain embodiments, compositions provided herein also include a mucoadhesive polymer, selected from among, for example, carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylic acid/butyl acrylate copolymer, sodium alginate and dextran.

Generally, an agent, such as a compound of the present disclosure, is administered in an amount effective for amelioration of, or prevention of the development of symptoms of, the disease or disorder (i.e., a therapeutically effective amount). Thus, a therapeutically effective amount can be an amount that is capable of at least partially preventing or reversing a disease or disorder. The dose required to obtain an effective amount can vary depending on the agent, formulation, disease or disorder, and individual to whom the agent is administered.

Determination of effective amounts can also involve in vitro assays in which varying doses of agent are administered to cells in culture and the concentration of agent effective for ameliorating some or all symptoms is determined in order to calculate the concentration required in vivo. Effective amounts can also be based in in vivo animal studies.

An agent can be administered prior to, concurrently with and subsequent to the appearance of symptoms of a disease or disorder. In some embodiments, an agent is administered to a subject with a family history of the disease or disorder, or who has a phenotype that can indicate a predisposition to a disease or disorder, or who has a genotype which predisposes the subject to the disease or disorder.

Methods of Dosing and Treatment Regimens

The compositions containing the compound(s) described herein can be administered for prophylactic and/or therapeutic treatments. In therapeutic applications, the compositions are administered to a patient already suffering from a disease or condition, in an amount sufficient to cure or at least partially arrest the symptoms of the disease or condition. Amounts effective for this use will depend on the severity and course of the disease or condition, previous therapy, the patient's health status, weight, and response to the drugs, and the judgment of the treating physician.

In prophylactic applications, compositions containing the compounds described herein are administered to a patient susceptible to or otherwise at risk of a particular disease, disorder or condition. In this use, the precise amounts also depend on the patient's state of health, weight, and the like. When used in a patient, effective amounts for this use will depend on the severity and course of the disease, disorder or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician.

In the case wherein the patient's condition does not improve, upon the doctor's discretion the administration of the compounds can be administered chronically, that is, for an extended period of time, including throughout the duration of the patient's life in order to ameliorate or otherwise control or limit the symptoms of the patient's disease or condition.

Once improvement of the patient's conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, can be reduced, as a function of the symptoms, to a level at which the improved disease, disorder or condition is retained. Patients can, however, require intermittent treatment on a long-term basis upon any recurrence of symptoms.

The amount of a given agent that will correspond to such an amount will vary depending upon factors such as the particular compound, disease or condition and its severity, the identity (e.g., weight) of the subject or host in need of treatment, but can nevertheless be determined in a manner recognized in the field according to the particular circumstances surrounding the case, including, e.g., the specific agent being administered, the route of administration, the condition being treated, and the subject or host being treated. In general, however, doses employed for adult human treatment will typically be in the range of about 0.02-about 5000 mg per day, in some embodiments, about 1 -about 1500 mg per day. The desired dose can conveniently be presented in a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example as two, three, four or more sub-doses per day.

The pharmaceutical composition described herein can be in unit dosage forms suitable for single administration of precise dosages. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of one or more compound. The unit dosage can be in the form of a package containing discrete quantities of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions can be packaged in single-dose non-reclosable containers. Alternatively, multiple-dose reclosable containers can be used and can, for example, include a preservative. By way of example only, formulations for parenteral injection can be presented in unit dosage form, which include, but are not limited to ampoules, or in multi-dose containers, with an added preservative.

The daily dosages appropriate for the compounds described herein are from about 0.01 mg/kg to about 20 mg/kg. In one embodiment, the daily dosages are from about 0.1 mg/kg to about 10 mg/kg. An indicated daily dosage in the larger mammal, including, but not limited to, humans, is in the range from about 0.5 mg to about 1000 mg, conveniently administered in a single dose or in divided doses, including, but not limited to, up to four times a day or in extended release form. Suitable unit dosage forms for oral administration include from about 1 to about 500 mg active ingredient. In one embodiment, the unit dosage is about 1 mg, about 5 mg, about, 10 mg, about 20 mg, about 50 mg, about 100 mg, about 200 mg, about 250 mg, about 400 mg, or about 500 mg. Such dosages can be altered depending on a number of variables, not limited to the activity of the compound used, the disease or condition to be treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

Toxicity and therapeutic efficacy of such therapeutic regimens can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, the determination of the LD ₅₀ (the dose lethal to 50% of the population) and the ED ₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD ₅₀ and ED ₅₀ . Compounds exhibiting high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED ₅₀ with minimal toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.

Combination Treatments

In some embodiments, the compounds of the present disclosure, and compositions thereof, can be used in combination with other therapeutic agents that are selected for their therapeutic value for the condition to be treated. In general, the compositions described herein and the other therapeutic agents do not have to be administered in the same pharmaceutical composition or at the same time, and because of different physical and chemical characteristics, can be administered by different routes at different times. The determination of the mode of administration and the advisability of administration, where possible, in the same pharmaceutical composition, is within the knowledge of the clinician. The initial administration can be made according to established protocols recognized in the field, and then, based upon the observed effects, the dosage, modes of administration and times of administration can be modified by the clinician.

In some embodiments, a compound of the present disclosure is administered in combination with an aminoglycoside antibiotic agent, a platinum-based chemotherapeutic agent, or an ototoxic therapeutic agent. A therapeutically effective amount of the compound the present disclosure, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure, can be administered to the subject before, concurrently with, and/or after treatment of the subject with an aminoglycoside antibiotic agent, a platinum-based chemotherapeutic agent, or an ototoxic therapeutic agent. For example, the compounds of the present disclosure can be topically administered to an inner ear of ( e.g ., intratympanic delivery to) the subject, and/or to a hair cell in the inner ear of the subject. In some embodiments, a compound of the present disclosure is administered before, concurrently with, and/or after treatment with an aminoglycoside antibiotic agent selected from streptomycin, neomycin, framycetin, paromomycin, paromomycin sulfate, ribostamycin, kanamycin, amikacin, arbekacin, bekanamycin, dibekacin, tobramycin, spectinomycin, hygromycin B, gentamicin, netilmicin, sisomicin, isepamicin, verdamicin, and astromicin. In some embodiments, a compound of the present disclosure is administered before, concurrently with, and/or after treatment with streptomycin. In some embodiments, a compound of the present disclosure is administered in combination with amikacin. In some embodiments, a compound of the present disclosure is administered in combination with neomycin. In some embodiments, a compound of the present disclosure is administered before, concurrently with, and/or after treatment with kanamycin. In some embodiments, a compound of the present disclosure is administered before, concurrently with, and/or after treatment with gentamicin. In some embodiments, a compound of the present disclosure is administered before, concurrently with, and/or after treatment with tobramycin.

In some embodiments, a compound of the present disclosure is administered in a first treatment phase for 1-7 days, and then the compound of the present disclosure is administered in combination with an aminoglycoside antibiotic agent, a platinum-based chemotherapeutic agent, or an ototoxic therapeutic agent in a second treatment phase. In some embodiments, a compound of the present disclosure is administered for 7 days, and then the compound of the present disclosure is administered in combination with an aminoglycoside antibiotic agent, a platinum-based chemotherapeutic agent, or an ototoxic therapeutic agent. In some embodiments, a compound of the present disclosure is administered for 6 days, and then the compound of the present disclosure is administered in combination with an aminoglycoside antibiotic agent, a platinum-based chemotherapeutic agent, or an ototoxic therapeutic agent. In some embodiments, a compound of the present disclosure is administered for 5 days, and then the compound of the present disclosure is administered in combination with an aminoglycoside antibiotic agent, a platinum-based chemotherapeutic agent, or an ototoxic therapeutic agent. In some embodiments, a compound of the present disclosure is administered for 4 days, and then the compound of the present disclosure is administered in combination with an aminoglycoside antibiotic agent, a platinum-based chemotherapeutic agent, or an ototoxic therapeutic agent. In some embodiments, a compound of the present disclosure is administered for 3 days, and then the compound of the present disclosure is administered in combination with an aminoglycoside antibiotic agent, a platinum-based chemotherapeutic agent, or an ototoxic therapeutic agent. In some embodiments, a compound of the present disclosure is administered for 2 days, and then the compound of the present disclosure is administered in combination with an aminoglycoside antibiotic agent, a platinum-based chemotherapeutic agent, or an ototoxic therapeutic agent. In some embodiments, a compound of the present disclosure is administered for 1 day, and then the compound of the present disclosure is administered in combination with an aminoglycoside antibiotic agent, a platinum-based chemotherapeutic agent, or an ototoxic therapeutic agent.

In some embodiments, rather than administering a compound of the present disclosure in combination with an aminoglycoside antibiotic agent, a platinum-based chemotherapeutic agent, or an ototoxic therapeutic agent in the second phase of the treatment program as described above, the aminoglycoside antibiotic agent, platinum- based chemotherapeutic agent, or ototoxic therapeutic agent is administered without a compound of the present disclosure. In some embodiments, the compound of the present disclosure is administered an additional 7 days following the administration of the aminoglycoside antibiotic agent, platinum-based chemotherapeutic agent, or ototoxic therapeutic agent. In some embodiments, the compound of the present disclosure is administered an additional 6 days following the administration of the aminoglycoside antibiotic agent, platinum-based chemotherapeutic agent, or ototoxic therapeutic agent. In some embodiments, the compound of the present disclosure is administered an additional 5 days following the administration of the aminoglycoside antibiotic agent, platinum-based chemotherapeutic agent, or ototoxic therapeutic agent. In some embodiments, the compound of the present disclosure is administered an additional 4 days following the administration of the aminoglycoside antibiotic agent, platinum-based chemotherapeutic agent, or ototoxic therapeutic agent. In some embodiments, the compound of the present disclosure is administered an additional 3 days following the administration of the aminoglycoside antibiotic agent, platinum-based chemotherapeutic agent, or ototoxic therapeutic agent. In some embodiments, the compound of the present disclosure administered an additional 2 days following the administration of the aminoglycoside antibiotic agent, platinum-based chemotherapeutic agent, or ototoxic therapeutic agent. In some embodiments, the compound of the present disclosure is administered an additional 1 day following the administration of the aminoglycoside antibiotic agent, platinum-based chemotherapeutic agent, or ototoxic therapeutic agent.

In some embodiments the compound of the present disclosure and the aminoglycoside antibiotic agent, platinum-based chemotherapeutic agent, or ototoxic therapeutic agent are administered in combination in a single dosage form. In some embodiments the compound of the present disclosure and the aminoglycoside antibiotic agent, platinum-based chemotherapeutic agent, or ototoxic therapeutic agent are administered in combination in separate dosage forms.

In some embodiments, a compound of the present disclosure is administered before, concurrently with, and/or after treatment with a chemotherapeutic agent. In some embodiments, a compound of the present disclosure is administered before, concurrently with, and/or after treatment with a chemotherapeutic agent selected from cisplatin and carboplatin. In some embodiments, a compound of the present disclosure is administered before, concurrently with, and/or after treatment with cisplatin. In some embodiments, a compound of the present disclosure is administered before, concurrently with, and/or after treatment with carboplatin.

The particular choice of compounds used will depend upon the diagnosis of the attending physicians and their judgment of the condition of the patient and the appropriate treatment protocol. The compounds can be administered concurrently (e.g., simultaneously, essentially simultaneously or within the same treatment protocol), or sequentially (e.g., before or after), depending upon the nature of the disease, disorder, or condition, the condition of the patient, and the actual choice of compounds used. The determination of the order of administration, and the number of repetitions of administration of each therapeutic agent during a treatment protocol, is within the knowledge of the physician after evaluation of the disease being treated and the condition of the patient.

Therapeutically-effective dosages can vary when the drugs are used in treatment combinations. Methods for experimentally determining therapeutically-effective dosages of drugs and other agents for use in combination treatment regimens are described in the literature. For example, the use of metronomic dosing, i.e., providing more frequent, lower doses in order to minimize toxic side effects, has been described extensively in the literature. Combination treatment further includes periodic treatments that start and stop at various times to assist with the clinical management of the patient.

For combination therapies described herein, dosages of the co-administered compounds will vary depending on the type of co-drug employed, on the specific drug employed, on the disease or condition being treated and so forth. In addition, when co- administered with one or more biologically active agents, the compound provided herein can be administered simultaneously with the biologically active agent(s), or sequentially (e.g., before or after administration with the biologically active agent(s)). If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein in combination with the biologically active agent(s).

In any case, the multiple therapeutic agents (one of which is a compound of the present disclosure) can be administered in any order or even simultaneously. If simultaneously, the multiple therapeutic agents can be provided in a single, unified form, or in multiple forms (by way of example only, either as a single pill or as two separate pills). One of the therapeutic agents can be given in multiple doses, or both can be given as multiple doses. If not simultaneous, the timing between the multiple doses can vary from more than zero weeks to less than four weeks. In addition, the combination methods, compositions and formulations are not to be limited to the use of only two agents; the use of multiple therapeutic combinations is also envisioned.

It is understood that the dosage regimen to treat, prevent, or ameliorate the condition(s) for which relief is sought, can be modified in accordance with a variety of factors. These factors include the disorder or condition from which the subject suffers, as well as the age, weight, sex, diet, and medical condition of the subject. Thus, the dosage regimen actually employed can vary widely and therefore can deviate from the dosage regimens set forth herein.

The pharmaceutical agents which make up the combination therapy disclosed herein can be a combined dosage form or in separate dosage forms intended for substantially simultaneous administration. The pharmaceutical agents that make up the combination therapy can also be administered sequentially, with either therapeutic compound being administered by a regimen calling for two-step administration. The two- step administration regimen can call for sequential administration of the active agents or spaced-apart administration of the separate active agents. The time period between the multiple administration steps can range from, a few minutes to several hours, depending upon the properties of each pharmaceutical agent, such as potency, solubility, bioavailability, plasma half-life and kinetic profile of the pharmaceutical agent. Circadian variation of the target molecule concentration can also determine the optimal dose interval.

In addition, the compounds described herein also can be used in combination with procedures that can provide additional or synergistic benefit to the patient. By way of example only, patients are expected to find therapeutic and/or prophylactic benefit in the methods described herein, wherein pharmaceutical composition of a compound disclosed herein and/or combinations with other therapeutics are combined with genetic testing to determine whether that individual is a carrier of a mutant gene that is known to be correlated with certain diseases or conditions.

The compounds described herein and combination therapies can be administered before, during or after the occurrence of a disease or condition, and the timing of administering the composition containing a compound can vary. Thus, for example, the compounds can be used as a prophylactic and can be administered continuously to subjects with a propensity to develop conditions or diseases in order to prevent the occurrence of the disease or condition. The initial administration can be via any route practical, such as, for example, an intravenous injection, a bolus injection, infusion over about 5 minutes to about 5 hours, a pill, a capsule, transdermal patch, buccal delivery, and the like, or combination thereof. A compound is preferably administered as soon as is practicable before the onset of a disease or condition, or after the onset of a disease or condition is detected or suspected, and for a length of time necessary for the treatment of the disease or condition. The length of treatment can vary for each subject, and the length can be determined using the known criteria.

The following examples are provided to illustrate, not limit, the invention.

Examples 1 and 2 describe the synthesis and characterization of embodiments of compounds of the present disclosure. Example 3 describes the testing of the protective properties of embodiments of compounds of the present disclosure.

EXAMPLES

Example 1. _ Synthesis of 5-r3(2-chloroacetamido)propyl1-2-(r(4- chlorophenyDcarbamoyll amino i-4H.5 H.6H.7H-thi enor2.3 -clpyridine-3 -carboxamide

Step One. fer/-Butyl 2-amino-3-carbamoyl-4.5-dihvdrothieno[2.3-c]pyridine- 6 -carboxylate (B)

A round bottom flask fitted with a stir bar, was charged with sulfur (196 mg, 6.114 mmol) followed by morpholine (704 pL, 8.14 mmol) and the suspension was stirred at 40 °C for 1 h or until sulfur dissolved in morpholine. The solution was diluted with anhydrous ethanol (EtOH), whereupon 4-N-Boc-piperidinone (1.20 g, 6.02 mmol) and cyanamide (506 mg, 6.02 mmol) were added and the reaction mixture stirred at room temperature overnight. Upon completion, the reaction mixture was concentrated in vacuo to afford viscous orange oil. The resultant product was diluted with methanol (MeOH) and then 10% ethyl acetate (EtOAc) in Hexane was added until precipitation of the product was complete. The product was filtered to afford tan solid (1.8 g, quantitative yield). 1H NMR (300 MHz, MeOH-i¾) d 4.35 (bs, 2H), 3.68-3.55 (m, 2H), 2.71-2.68 (m, 2H), 1.49 (s, 9H). LRMS [M+H] 298.

Step Two. _ tert- Butyl 3-carbamoyl-2-[3-(4-chlorophenvOureido1-4.5- dihvdrothieno[2.3-c1pyridine-6 -carboxylate (D)

To a stirred solution of 4-chlorophenyl isocyanate (325 mg, 2.12 mmol) and triethylamine (444 pL, 3.18 mmol) in anhydrous tetrahydrofuran (THF) (10 mL) was added /er/-butyl 2-amino-3-carbamoyl-4,5-dihydrothieno[2,3-c]pyridine-6(7f/)- carboxylate (632 mg, 2.12 mmol) and the reaction mixture was stirred at room temperature (rt) for 8 h. The reaction mixture was concentrated in vacuo to afford oil, which was taken up dichloromethane (DCM) and washed with water (x2). The organics were then dried with anhydrous Na ₂ S0 ₄ and concentrated in vacuo. The product was purified by Isolera system (Si0 ₂ gel as stationary phase, 25 g HP column, dry loading) using DCM-DCM/MeOH (0%- 8% MeOH in DCM) to afford tan solid (633 mg, 57% yield). LRMS [M+H] 451.

Step _ Three. _ 2-G3 -(4-Chl orophenyl )ureido]-4.5.6.7-tetrahvdrothieno[2 - c]pyridine-3-carboxamide trifluoroacetate (E)

A stirred suspension of /tvV-butyl 3-carbamoyl-2-[3-(4-chlorophenyl)ureido]-4,5- dihydrothieno[2,3-6]pyridine-6(7//)-carboxylate (697 mg, 1.55 mmol) in 6 mL of DCM at room temperature (rt) was treated with 3 mL of trifluoroacetic acid (TFA). The resultant solution was stirred at rt for 25 min whereupon it was concentrated in a rotary evaporator. The product was taken up in MeOH and the solution concentrated in vacuo (x2) to afford a tan solid in quantitative yield. LRMS [M+H] 351. Step Four tert-butyl N-r3-(3-carbamoyl-2-(r(4-

4H.5H.6H.7H-thienor2.3-c1pyridin-6-yl ipropyllcarbamate (F)

A solution of 2-[3-(4-Chlorophenyl)ureido]-4,5,6,7-tetrahydrothieno[2,3- c]pyridine-3 -carboxamide trifluoroacetate (0.48 mmol) in anhydrous N,N'dimethylformamide (DMF) was treated with tri ethyl amine (200 pL, 1.43 mmol) and N-Boc-bromopropylamine (114 mg, 0.48 mmol) and the reaction mixture was allowed to stir at room temperature for 14 h. Analytical HPLC showed close to 50% reaction progression. Another 100 pL of triethyl amine and 70 mg of N-boc-bromopropylamine were added and the reaction mixture was stirred at room temperature. Upon completion (48 h), DMF was removed under vacuo and the resultant crude mixture was diluted with DCM and washed with water (x2). The organic layer was dried (anhydrous Na ₂ S0 ₄ ) and concentrated to afford an oil which was purified by flash chromatography Isolera system (Si0 ₂ gel as stationary phase, 40 g HP column, dry loading) using DCM-DCM/MeOH (0%- 15% MeOH in DCM) to afford light brown solid (144 mg, 60% yield). 1H NMR (300 MHz, dmso-i¾) d 10.95 (s, 1H), 10.18 (s, 1H), 7.55-7.43 (m, 2H), 7.37-7.27 (m, 2H), 6.82 (s, 1H), 3.53-3.43 (m, 2H), 3.03-2.90 (m, 2H), 2.85-2.57 (m, 4H), 2.50-2.40 (m, 2H), 1.69-1.53 (m, 2H), l.37(s, 9H). LRMS [M+H] 508.

Step _ Five. _ 5-(3-aminopropyD-2-(r(4-

4H.5 H.6H.7H-thienor2.3 -clpyridine-3 -carboxamide (G)

A stirred suspension of tert-butyl-N-[3-(2-{[(4-benzoylphenyl)carbamoyl]amino}- 3-carbamoyl-4H,5H,6H,7H-thieno[2,3-c]pyridin-6-yl)propyl]car bamate (70 mg, 0.14 mmol) in 4 mL of DCM at rt was treated with 2 mL of TFA. The resultant solution was stirred at rt for 40 min whereupon it was concentrated in a rotary evaporator. The product was taken up in MeOH and the solution concentrated in vacuo (x2) to afford a light tan solid in quantitative yield. LRMS [M+H] 408.

Step _ Six. _ 5-r3(2-chloroacetamido)propyl1-2-(r(4- chlorophenyl icarbamoyllamino i-4H.5 H.6H.7H-thi enor2.3 -clpyridine-3 -carboxamide (H) To a stirred solution of 6-(3-aminopropyl)-2-{[(4- chlorophenyl)carbamoyl]amino}-4H,5H,6H,7H-thieno[2,3-c]pyrid ine-3-carboxamide (0.59 mmol) in anhydrous THF (4.5) at 0 °C were added triethyl amine (254 pL, 1.77 mmol) and chloroacetyl chloride (1.1 equiv. 51 pL). The resultant mixture was stirred for 8 h with gradual warm up to room temperature. Upon completion, the solvent was removed in vacuo and the oil was taken up in DCM and washed with brine (xl), dried over anhydrous Na ₂ S0 ₄ and concentrated in vacuo to afford the desired product (purity >90%) which was used without further purification. LRMS [M+H] 484.1.

Example 2 (TR.8SV1 l-r3-(2-chloroacetamido)propyl1-4-(r(4-chlorophenvncarbamoyl ] 5-thia-l lazatricvclor6.2.L0 ² . ⁶ 1undeca-2 diene-3-carboxamide

Step _ One. _ (Ί R.8S)- 1 1 -tert-butyl _ N-r3-(3-carbamoyl)1-4-(r(4- chlorophenvncarbamoyl1aminol-5-thia-l lazatricvclor6.2.l.0 ² . ⁶ 1undeca-2 (64-3 -diene-3 - carboxamide (T)

To a stirred suspension of I prepared according to published procedure ¹ (70 mg, 0.16 mmol) in anhydrous THF (4 mL) was added tert-butyl 3-oxopropylcarbamate (163 mg, 5 equiv.), acetic acid (5 uL) and anhydrous magnesium sulfate (0.4 g). The resultant mixture was stirred at rt for 10 min upon which sodium triacetoxyborohydride (4.0 equiv., 140 mg) was added and the mixture was stirred at room temperature for 2 h. The reaction was quenched with NaHCO, solution and filtered through a fritted funnel under vacuum. The filtrate was extracted with DCM (x2) and dried over anhydrous Na ₂ S0 ₄ , concentrated in vacuo to afford the target compound as pale-yellow oil (purity >95% as judged by LC-MS). LRMS [M+H] 534.2.

The product was forwarded to the next step without further purification.

Step Two. (1R.8SV1 l-(3-aminopropyD-4-(IY4-

5-thia-l lazatricvclor6.2.l.0 ² . ⁶ 1undeca-2 -diene-3 -carboxamide (K)

A stirred solution of J (0.08 mmol) in 1.5 mL of DCM at rt was treated with 0.6 mL of TFA. The resultant solution was stirred at rt for 20 min whereupon analytical LCMS showed the reaction to be complete. The reaction mixture was concentrated; the product was taken up in MeOH and the solution concentrated in vacuo (x3) to afford an oil with the desired mass which was forwarded to the next step without further purification (purity >95%). LRMS [M+H] 434.1.

-l l-r3-(2-chloroacetamido)propyl1-4-(r(4- ricvclor6.2.l.0 ² . ⁶ 1undeca-2 (64-3 -diene-3 -

carboxamide (L)

To a stirred solution of K (0.04 mml) in anhydrous THF, trimethylamine (5.0 equiv., 0.2 mmol, 28 uL) was added followed by neat chloroacetyl chloride (7.2 uL). The reaction mixture was stirred at room temperature for 6 hours upon which analytical HPLC revealed only 64% conversion. Another 28 uL of triethylamine and 5 uL of chloroacetyl chloride were added and the reaction mixture was stirred at room temperature overnight. The reaction mixture was then concentrated and purified by reversed-phase preparatory HPLC using Deltapack C18 column using water (with 0.1% TFA) and acetonitrile (with 0.1% TFA) isocratic elution at 15% B for 15 minutes followed by gradient elution from 15% B to 90%B in 10 minutes. The eluted product was lyophilized to afford TFA salt of the desired product as white powder (7 mg, 28% yield). HRMS [M+H] 510.11382.

Synthesis of urea-thiophene carboxamides are described, for example, in Chowdhury, S.; Owens, K.N.; Herr, R.J.; Jiang, Q.; Chen, X.; Johnson; G.; Groppi; V.E.; Raible, D.W.; Rubel, E.W.; Simon, J.A. Phenotypic optimization of urea-thiophene carboxamides to yield potent, well tolerated, and orally active protective agents against aminoglycoside-induced hearing loss. J Med. Chem. 2018, 61, 84, herein incorporated by reference in its entirety.

Example 3 Assessment of protective effects.

FIGURE 1 illustrates the chemical structures of compounds PROTO- 1, ORC- 13661, PROTO-183, PROTO-185, and PROTO-200. The bar graph demonstrates that protection persists even 24 hours after PROTO-200 is washed out.

To test the compounds, newly hatched free-swimming AB zebrafish larvae were raised at 28.5°C in petri dishes and transferred to cell culture baskets placed in 6-well culture plates in groups of ten fish per basket. Fish were pre-treated with test compound (control, PROTO- 1 50 mM or PROTO-200 5 mM) for 1 hour followed by treatment with 200 mM neomycin. Following neomycin exposure, fish were rinsed and exposed to 200 mM neomycin with no protective compound (neo) for 2 hours, 200 mM neomycin for 2 hours with or without PROTO 1 (Pl + neo) or PROTO200 (P200 + neo) as well as to 200 mM neomycin for 2 hours ("Pl + 2h + neo" and "P200 + 2h + neo") or 24 hours ("Pl + 24h + neo" and "P200 + 24h + neo"). Following final exposure, fish were rinsed briefly with a staining agent (DASPEI), anesthetized and mounted on the stage of an epifluorescence dissecting microscope. Hair cell staining of ten neuromasts on one side of each animal was evaluated visually each neuromast was scored for presence of a normal compliment of hair cells, with reduced or absent DASPEI staining indicating a reduction in hair cell number. Composite scores were calculated for animals in each treatment group, normalized to the control group and expressed as % hair cell survival.

Procedures for testing compounds are described, for example, in Chowdhury el al, J Med Chem. 2018 Jan 1 l;6l(l):84-97, herein incorporated by reference in its entirety.

While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.