WO2010141680A2 - Hedgehog pathway antagonists and methods of use - Google Patents

Abstract

The present disclosure provides for a compound, pharmaceutical preparations, kits and methods for the inhibition of the Hh pathway and the alleviation of cancer and developmental disorders associated with the Hh pathway.

Description

HEDGEHOG PATHWAY ANTAGONISTS AND METHODS OF USE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application Serial No.: 61/183,587 entitled "HEDGEHOG PATHWAY ANTAGONISTS AND METHODS OF USE" filed on June 3, 2009, the entirety of which is hereby incorporated by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This disclosure was made with government support under NIH Grant Nos.: R01 CA136574, R01 GM61269, and R01 NS045727 awarded by the U.S. National Institutes of Health of the United States government. The government has certain rights in the disclosure.

TECHNICAL FIELD

The present disclosure is generally related to a compound antagonistic to the Hedgehog pathway, and derivatives of said compound. The disclosure further relates to pharmaceutical compositions and methods of using said compositions to modulate cell growth and tissue development.

BACKGROUND

The Hedgehog (Hh) pathway was first discovered in a Drosophila genetic screen because of its role in patterning the body of the animal (Nusslein-Volhard et al., Nature (1980) 287: 795-801). Shortly afterward, mammalian homologues of the Hedgehog gene of chick and mouse were cloned and characterized (Echelard et al., Cell (1993) 75: 1417-1430; Riddle et al., Ce// (1993) 75: 1401-1416; Roelink et al., Ce// (1994) 76: 761-775). There are three Hh vertebrate homologues, named Sonic Hedgehog (Shh), Desert Hedgehog (Dhh) and Indian Hedgehog (Ihh), with Shh being implicated in limb development and neural tube development (Bitgood et al., Curr. Biol. (1996) 6: 298-304; Chiang et al., Nature (1996) 383: 407-413; St. Jacques et al., Genes Dev. (1999) 13: 2072-2086; Zhang et al., Cell (2001) 106: 781-792). Using both the Drosophila and the mouse models to define how the Hh pathway transduced its signal, it was revealed that this is a multifactorial and unconventional pathway (Hooper et al., Nat. Rev. MoI. Cell. Biol. (2005) 6: 306-317; Huangfu et al., Dev. (2006) 133:3-14). In vertebrates, signal transduction in Hh pathway begins by the Shh ligand binding to Patched (Ptchi) a 12 transmembrane receptor. Ptchi is coupled to and represses a signaling polypeptide Smoothened (Smo), a 7 transmembrane, G-protein coupled receptor-like molecule. In the presence of Shh, the Ptchi inhibition on Smo is released, and Smo transduces the Shh signal, activating downstream pathway components. In the absence of Shh, Ptchi represses Smo signaling, and no signal transduction takes place. The final downstream effector of the Hh pathway is the transcription factor GIi. There are 3 GIi proteins in vertebrates, GIM , Gli2 and Gli3, discovered as genes amplified in glioblastoma (Kinzler ef al., Science (1987) 236: 70-73; Bai ef al., Dei/. Cell (2004) 6: 103- 115; Motoyama et al., Dev. Biol. (2003) 259: 150-161). The three GIi proteins have sequence identity in the zinc finger domain, but have limited similarity outside of this region (Matise & Joyner Oncogene (1999) 18: 7852-7859). GIiI is a transcriptional activator, while Gli2 and Gli3 are bifunctional and can function as a transcriptional activator or, when proteolytically processed, a transcriptional repressor (Dai et al., J.Biol. Chem. (1999) 12: 8143-8152). In general, GIiI expression is restricted to proliferating cells adjacent to tissues expressing Shh. GN2 and Gli3 are broadly expressed in proliferating cells exposed to lower concentrations of Shh (Hui et al., Dev. Biol. (1994) 162: 402-413).

In the cytoplasm, GIi is complexed with the protein Suppressor of Fused and may be tethered to the microtubule cytoskeleton (Methot & Basler, Dev. (2000) 127: 4001-4010; Chen et al., MoI. Cell. Biol. (2005) 25: 7042-7053; Preat, Genetics (1992) 132: 725-736). Upon transduction of a Shh signal, GIi is released from the complex and migrates to the nucleus. There GIi binds to specific sites in the genome and induces gene expression. Some of the transcripts produced by the GIi transcription factors are components of the Hh pathway itself, such as GIM , Ptchi and Hedgehog interacting protein (Hip).

Disruption of the Hh pathway causes developmental abnormalities in embryogenesis and cancer in the adult. Shh mutations in a mouse model have a dramatic embryonic phenotype with lack of anterior and posterior limb polarity, lack of lung mesoderm and most ventral CNS motor neurons (Chiang et al., Nature (1996) 383: 407-413). Mouse models of Ptchi mutations can be embryonic lethal when homozygous, and display severe developmental defects similar to that of the Shh mutants when heterozygous (Goodrich ef a/., Science (1997) 277: 1109-1113). For the GIi genes, a mouse that is homozygous for a GIM mutant with deleted zinc-finger domains develops normally (Matise ef al, Development (1998) 125: 2759). In contrast, Gli2 mutants without zinc-finger domains show developmental defects in the ventral CNS, lung, vertebrae and bones (Matise et al, supra; Motoyama ef al., Nat. Genet. (1998) 20: 54-57). Double mutants that are GIM-/- and Gli2 -/+ have a milder phenotype than Gli2-/- and have relatively normal limb development, but die at birth or shortly after. (Park ef al., Development (2000) 127: 1593-1605). Mice having the Gli2 +/-; GN3-/- double mutant genotype have skeletal abnormalities that are more severe than if having the Gli2 +/- mutation alone, or the Gli3-/- mutation alone (Mo et al., Development (1997) 124: 113-123). In Basal Cell Nevus Syndrome or Gorlin's syndrome, loss of function of Ptchi leads to a predisposition to pediatric medulloblastoma and basal cell carcinoma (BCC) of the skin, the most common pediatric brain tumor and the most common type of skin cancer in the Caucasian population (Goodrich & Scott, Neuron (1998) 21 : 1243-1257). GH1 overexpression is frequently found in BCC patients with Gorlin's syndrome and in non Gorlin's patients where the BCC has arisen spontaneously (Fan et al., Nat. Med. (1997) 3: 788-792). Disruption of the Shh-Gli pathway is has also been described in a number of adult cancers which are discussed in more detail below.

The primary cilium is a hair-like appendage extending from the surface of a cell. This specialized structure, with a unique microtubular cytoskeleton (axoneme) and a surrounding membrane, is assembled and maintained by the intraflagellar transport machinery. Recent work has shown that primary cilia concentrate receptors and signal transduction components that have vital roles in development. In particular, evidence in the developing neural tube and limb bud has shown that genes encoding the IFT motors and the IFT particle subunits are required for Shh signaling. It has been found that the Shh signaling components, Patched 1 (Ptchi), Smo, Suppressor of fused (Su(fu)), and GIi transcription factors, concentrate in primary cilia. Compounds which specifically affect the Hh pathway are few. Cyclopamine is a steroidal alkaloid derived from plants. Both it and derivatives of cyclopamine are being investigated as Smo antagonists. Three other compounds have been described to act downstream of Smo in the Hh pathway, but their targets and mechanism of action are currently unclear (Lauth et al., Proc. Natl. Acad. Sci. USA (2007) 104: 8455-8460; Lee et al., Chembiochem (2007) 8: 1916-1919).

Thus the Hh pathway is important for normal development of the embryo and carcinogenesis in the adult, indicating there is a great need for compounds and methods of using such compounds of the present disclosure for the alleviation and study of cancers and developmental disorders of the Hh pathway. SUMMARY

The present disclosure provides for compounds, pharmaceutical preparations, kits and methods for the inhibition of the Hh pathway and the alleviation of cancer and developmental disorders associated with the Hh pathway. Briefly described, embodiments of this disclosure, among others, encompass compounds and pharmaceutical formulations thereof are provided for the inhibition of the Hh pathway.

One aspect of the disclosure, therefore, provides a compound having the formula A:

or a pharmaceutically acceptable salt thereof, wherein: R_{1 1}R₂, and R₃ are each independently H, an acyl, an alkanoyl, an alkyl, an unsaturated alkyl. a heteroalkyl, an amino group, an amine group, an aryl, a substituted aryl group, an alkoxy, an alkylamino, an alkylthio (or thioalkoxy), a heteroalkyl, a heteroalkylene, a cycloalkyl, an aryl group, a heteroaryl group, or an arylalkyl, or a substituted or a unsubstituted form thereof, or a pharmaceutically acceptable salt thereof.

One embodiment of this aspect of the disclosure provides a compound having the formula I:

or a pharmaceutically acceptable salt thereof.

Another aspect of the disclosure provides embodiments of a pharmaceutical composition comprising a compound having the formula A:

or a pharmaceutically acceptable salt thereof, wherein:

R₁ , R₂, and R₃ are each independently H, an acyl, an alkanoyl, an alkyl, an unsaturated alkyl. a heteroalkyl, an amino group, an amine group, an aryl, a substituted aryl group, an alkoxy, an alkylamino, an alkylthio (or thioalkoxy), a heteroalkyl, a heteroalkylene, a cycloalkyl, an aryl group, a heteroaryl group, or an arylalkyl, or a substituted or a unsubstituted form thereof, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.

An embodiment of this aspect of the disclosure is a pharmaceutical composition comprising a compound having the formula I:

I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. Yet another aspect of the disclosure provides for the use of a compound having the formula A:

or a pharmaceutically acceptable salt thereof, in the manufacture of the pharmaceutical composition for the treatment of a cell or tissue of a subject human or animal having a cell proliferative disorder, where the pharmaceutical composition modulates cell proliferation by modulating the Hedgehog pathway signaling, wherein:

R₁, R₂, and R₃ are each independently H, an acyl, an alkanoyl, an alkyl, an unsaturated alkyl. a heteroalkyl, an amino group, an amine group, an aryl, a substituted aryl group, an alkoxy, an alkylamino, an alkylthio (or thioalkoxy), a heteroalkyl, a heteroalkylene, a cycloalkyl, an aryl group, a heteroaryl group, or an arylalkyl, or a substituted or a unsubstituted form thereof, or a pharmaceutically acceptable salt thereof.

In an embodiment of this aspect of the disclosure the compound has the formula I:

I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, or a pharmaceutically acceptable salt thereof.

Yet another aspect of the disclosure provides embodiments of a method of modulating Hedgehog pathway signaling in a cell, the method comprising: (i) providing a target cell or population of target cells; (ii) contacting the cell with the compound having the formula A:

or a pharmaceutically acceptable salt thereof, wherein:

R₁, R₂, and R₃ are each independently H, an acyl, an alkanoyl, an alkyl, an unsaturated alkyl. a heteroalkyl, an amino group, an amine group, an aryl, a substituted aryl group, an alkoxy, an alkylamino, an alkylthio (or thioalkoxy), a heteroalkyl, a heteroalkylene, a cycloalkyl, an aryl group, a heteroaryl group, or an arylalkyl, or a substituted or a unsubstituted form thereof, or a pharmaceutically acceptable salt thereof, in an amount effective to modulate Hedgehog signaling in the cell, thereby modulating Hedgehog pathway signaling in the target cell or population of target cells.

In the embodiment of this aspect of the disclosure, the compound can have the formula I:

I or a pharmaceutically acceptable salt thereof.

In embodiments of this aspect of the disclosure, the Hedgehog pathway signaling can be associated with cell proliferation, and wherein reducing the Hedgehog pathway signaling in the target cell or population of target cells reduces the proliferation of the target cell or population of target cells. In embodiments of this aspect of the disclosure, the target cell or population of target cells can be present in a cultured cell or population of cells or can be present in a subject animal or human.

In embodiments of this aspect of the disclosure, the target cell or population of target cells can be associated with a Hedgehog-related disorder of the subject animal or human. In some embodiments of this aspect of the disclosure, the Hedgehog-related disorder of the subject animal or human is a tumor or a disorder of a tissue development.

In the embodiments of this aspect of the disclosure, the compound can be in a pharmaceutically acceptable composition. In these embodiments, the pharmaceutically acceptable composition may further comprise a pharmaceutically acceptable carrier. In embodiments of the method of this aspect of the disclosure, the method may further comprise administering to the subject animal or human an effective amount of a second therapeutic agent.

Still another aspect of the disclosure provides embodiments of kits comprising a container and a dose or plurality of doses of the compound having the formula A:

or a pharmaceutically acceptable salt thereof, wherein:

R₁ , R₂, and R₃ are each independently H, an acyl, an alkanoyl, an alkyl, an unsaturated alkyl. a heteroalkyl, an amino group, an amine group, an aryl, a substituted aryl group, an alkoxy, an alkylamino, an alkylthio (or thioalkoxy), a heteroalkyl, a heteroalkylene, a cycloalkyl, an aryl group, a heteroaryl group, or an arylalkyl, or a substituted or a unsubstituted form thereof, or a pharmaceutically acceptable salt thereof, , and a package insert describing the use of said compound, or a pharmaceutical composition comprising said compound, in treating a pathological condition of interest in a subject animal or human.

In the embodiment of this aspect of the disclosure, the compound can have the formula I:

I or a pharmaceutically acceptable salt thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present disclosure will be more readily appreciated upon review of the detailed description of its various embodiments, described below, when taken in conjunction with the accompanying figures. The drawings are described in greater detail in the description and examples below.

Fig. 1 schematically illustrates the Hh signaling pathway in its activated state and possible sites of HPI action. Upon Shh-Ptch1 binding, Smo accumulates in the primary cilium and promotes the stabilization and activation of full-length GIi proteins. The HPIs block this process through distinct mechanisms.

Fig. 2 shows structural formulas of compounds 1-18 selected as agonists of the Hedgehog pathway.

Fig. 3 shows Table 1 presenting the assay data for the compounds 1-18, the structures of which are shown in Fig. 2.

Fig. 4 shows the formula for compound G008-3201 (listed as compound 12 in Figs, 2 and 3) (I). Fig. 5 is a scheme illustrating the synthesis of compounds of the disclosure. Fig. 6 is a digital representation of an NMR output for compound G008-3201 (I).

DETAILED DESCRIPTION

Before the present disclosure described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the exemplary methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supercedes any disclosure of an incorporated publication to the extent there is a contradiction.

It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a sample" includes a plurality of such samples and reference to "the molecule" includes reference to one or more molecules and equivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible. Definitions

In describing and claiming the disclosed subject matter, the following terminology will be used in accordance with the definitions set forth below. As used herein, compounds of the disclosure are "Hedgehog Pathway Inhibitors" or

"HPIs." It is understood that there is no limitation placed on the term as to a particular Hh pathway gene or polypeptide either known or yet to be discovered, nor is it limited to a Hedgehog pathway gene or polypeptide in a particular species (e.g., human).

The terms "GIi transcription factors" and "GIi" as used herein are genes and polypeptides which are effectors of the Hh pathway, and includes GIiI , Gli2 and Gli3,and all variants thereof In certain cases, examples of GIi genes include, but are not limited to, at least the GenBank accession numbers AF316573.1 , NM_005270, NM_000168, NM_010296, NM_001081125, X95255.

As used herein, "hedgehog pathway" or "Hh pathway" is not limited to any species, and refers to a signal transduction pathway where a signal may be initiated by Smoothened (Smo), inhibition of Su(fu), and the like and which result in the translocation of the GIi transcription factor to the cell nucleus to initiate gene transcription.

The term "Hedgehog-related disorder(s)" as used herein refers to disorders associated with disruption or aberrance of the Hedgehog pathway, as well as disorders associated with normal but undesired growth states relating to activation of the Hedgehog pathway. "Hedgehog-related disorder(s)" include, but are not limited to, tumor formation, cancer, neoplasia, malignant hyperproliferative disorders, and non-malignant hyperproliferative disorders. "Hedgehog-related disorder(s)" also include benign prostate hyperplasia, psoriasis, wet macular degeneration, osteoporosis, and unwanted hair growth. The term "antagonist" is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of an Hh pathway- associated polypeptide.

As used herein, "methods known to one of ordinary skill in the art" may be identified though various reference books and databases. Suitable reference books and treatise that detail the synthesis of reactants useful in the preparation of compounds of the present disclosure, or provide references to articles that describe the preparation, include for example, "Synthetic Organic Chemistry", John Wiley & Sons, Inc., New York; S. R. Sandler et al., "Organic Functional Group Preparations," 2nd Ed., Academic Press, New York, 1983; H. O. House, "Modern Synthetic Reactions", 2nd Ed., W.A. Benjamin, Inc. Menlo Park, Calif. 1972; T.L. Gilchrist, "Heterocyclic Chemistry", 2nd Ed., John Wiley & Sons, New York, 1992; J. March, "Advanced Organic Chemistry: Reactions, Mechanisms and Structure", 4th Ed., Wiley-lnterscience, New York, 1992. Specific and analogous reactants may also be identified through the indices of known chemicals prepared by the Chemical Abstract Service of the American Chemical Society, which are available in most public and university libraries, as well as through on-line databases. Chemicals that are known but not commercially available in catalogs may be prepared by custom chemical synthesis houses, where many of the standard chemical supply houses (e.g., those listed above) provide custom synthesis services.

The compounds described herein may be prepared as a single isomer (e.g., enantiomer, cis-trans, positional, diastereomer) or as a mixture of isomers. In a preferred embodiment, the compounds are prepared as substantially a single isomer. Methods of preparing substantially isomerically pure compounds are known in the art. For example, enantiomerically enriched mixtures and pure enantiomeric compounds can be prepared by using synthetic intermediates that are enantiomerically pure in combination with reactions that either leave the stereochemistry at a chiral center unchanged or result in its complete inversion. Alternatively, the final product or intermediates along the synthetic route can be resolved into a single stereoisomer. Techniques for inverting or leaving unchanged a particular stereocenter, and those for resolving mixtures of stereoisomers are well known in the art and it is well within the ability of one of skill in the art to choose and appropriate method for a particular situation. See, generally, Furniss et al., (eds.), Vogel's Encyclopedia of Organic Chemistry 5^th ed., Longman Scientific and Technical Ltd., Essex, 1991 , pp. 809- 816; Heller, Ace. Chem. Res. (1990) 23: 128.

Where a disclosed compound includes a conjugated ring system, resonance stabilization may permit a formal electronic charge to be distributed over the entire molecule. While a particular charge may be depicted as localized on a particular ring system, or a particular heteroatom, it is commonly understood that a comparable resonance structure can be drawn in which the charge may be formally localized on an alternative portion of the compound.

Where substituent groups are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical substituents, which would result from writing the structure from right to left, e.g., -CH₂O- is intended to also recite -OCH₂-.

The term "acyl" or "alkanoyl" by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and an acyl radical on at least one terminus of the alkane radical. The "acyl radical" is the group derived from a carboxylic acid by removing the -OH moiety therefrom.

The term "alkyl," by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include divalent ("alkylene") and multivalent radicals, having the number of carbon atoms designated (i.e. C₁-C₁₀ means one to ten carbons). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n- pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1 ,4- pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The term "alkyl," unless otherwise noted, is also meant to include those derivatives of alkyl defined in more detail below, such as "heteroalkyl." Alkyl groups that are limited to hydrocarbon groups are termed "homoalkyl".

Exemplary alkyl groups of use in the present disclosure contain between about one and about twenty five carbon atoms (e.g., methyl, ethyl and the like). Straight, branched or cyclic hydrocarbon chains having eight or fewer carbon atoms will also be referred to herein as "lower alkyl". In addition, the term "alkyl" as used herein further includes one or more substitutions at one or more carbon atoms of the hydrocarbon chain fragment.

The term "amino" or "amine group" refers to the group -NR¹R" (or NRR¹R") where R, R' and R" are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl. A substituted amine being an amine group wherein R' or R" is other than hydrogen. In a primary amino group, both R' and R" are hydrogen, whereas in a secondary amino group, either, but not both, R' or R" is hydrogen. In addition, the terms "amine" and "amino" can include protonated and quaternized versions of nitrogen, comprising the group -NRR¹R" and its biologically compatible anionic counterions.

The term "aryl" as used herein refers to cyclic aromatic carbon chain having twenty or fewer carbon atoms, e.g., phenyl, naphthyl, biphenyl, and anthracenyl. One or more carbon atoms of the aryl group may also be substituted with, e.g., alkyl; aryl; heteroaryl; a halogen; nitro; cyano; hydroxyl, alkoxyl or aryloxyl; thio or mercapto, alkyl-, or arylthio; amino, alkylamino, arylamino, dialkyl-, diaryl-, or arylalkylamino; aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl, or arylalkylaminocarbonyl; carboxyl, or alkyl- or aryloxycarbonyl; aldehyde; aryl- or alkylcarbonyl; iminyl, or aryl- or alkyliminyl; sulfo; alkyl- or alkylcarbonyl; iminyl, or aryl- or alkyliminyl; sulfo; alkyl- or arylsulfonyl; hydroximinyl, or aryl- or alkoximinyl. In addition, two or more alkyl or heteroalkyl substituents of an aryl group may be combined to form fused aryl-alkyl or aryl-heteroalkyl ring systems (e.g., tetrahydronaphthyl). Substituents including heterocyclic groups (e.g., heteroaryloxy, and heteroaralkylthio) are defined by analogy to the above-described terms.

The terms "alkoxy," "alkylamino", and "alkylthio" (or thioalkoxy) are used in their conventional sense, and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively. The term "heteroalkyl," by itself or in combination with another term, means, unless otherwise stated, a straight or branched chain, or cyclic carbon-containing radical, or combinations thereof, consisting of the stated number of carbon atoms and at least one heteroatom selected from the group consisting of O, N, Si, P, S, and Se, and wherein the nitrogen, phosphorous, sulfur, and selenium atoms are optionally oxidized, and the nitrogen heteroatom is optionally be quaternized. The heteroatom(s) O, N, P, S, Si, and Se may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH₂-CH₂-O-CH₃, -CH₂-CH₂-NH-CH₃, -CH₂-CH₂-N(CH₃)-CH₃, -CH₂-S-CH₂-CH₃, -CH₂-CH₂, - S(O)-CH₃, -CH₂-CH₂-S(O)₂-CH₃, -CH=CH-O-CH₃, -Si(CH₃)₃, -CH₂-CH=N-OCH₃, and - CH=CH-N(CH₃)-CH₃. Up to two heteroatoms may be consecutive, such as, for example, - CH₂-NH-OCH₃ and -CH₂-O-Si(CH₃)₃. Similarly, the term "heteroalkylene" by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH₂-CH₂-S-CH₂-CH₂- and -CH₂-S-CH₂-CH₂-NH-CH₂-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)₂R'- represents both -C(O)₂R¹- and -R¹C(O)₂-.

The terms "cycloalkyl" and "heterocycloalkyl", by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of "alkyl" and "heteroalkyl", respectively. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1- (1 ,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3- morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien- 3-yl, 1-piperazinyl, 2-piperazinyl, and the like. The term "aryl" means, unless otherwise stated, a polyunsaturated, aromatic moiety that can be a single ring or multiple rings (preferably from 1 to 3 rings), which are fused together or linked covalently. The term "heteroaryl" refers to aryl groups (or rings) that contain from one to four heteroatoms selected from N, O, S, and Se, wherein the nitrogen, sulfur, and selenium atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4- imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, A- isoxazolyl, 5-isoxazolyi, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2- benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3- quinolyl, tetrazolyl, benzo[b]furanyl, benzo[b]thienyl, 2,3-dihydrobenzo[1 ,4]dioxin-6-yl, benzo[1 ,3]dioxol-5-yl and 6-quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below.

For brevity, the term "aryl" when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the term "arylalkyl" is meant to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl and the like) including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., "alkyl," "heteroalkyl," "aryl" and "heteroaryl") includes both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) are generically referred to as "alkyl group substituents," and they can be one or more of a variety of groups selected from, but not limited to: -OR¹, =O, =NR', =N-OR\ -NR¹R", -SR¹, -halogen, -SiR¹R¹¹R¹", -OC(O)R¹, - C(O)R¹, -CO₂R¹, -CONR¹R", -OC(O)NR¹R", -NR¹¹C(O)R', -NR'-C(O)NR"R"', -NR¹¹C(O) ₂R', - NR-C(NR¹R¹¹R¹¹¹J=NR"¹¹, -NR-C(NR'R")=NR"\ -S(O)R¹, -S(O) ₂R¹, -S(O) ₂NR¹R", -NRSO₂R¹, - CN and -NO₂ in a number ranging from zero to (2m'+1 ), where m¹ is the total number of carbon atoms in such radical. R', R", R'" and R"" each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g., aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound includes more than one R group, for example, each of the R groups is independently selected as are each R', R", R'" and R"" groups when more than one of these groups is present. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7- membered ring. For example, -NR¹R" is meant to include, but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term "alkyl" is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF₃ and -CH₂CF₃) and acyl (e.g., -C(O)CH₃, -C(O)CF₃, -C(O)CH₂OCH₃, and the like). Similar to the substituents described for the alkyl radical, substituents for the aryl and heteroaryl groups are generically referred to as "aryl group substituents." The substituents are selected from, for example: halogen, -OR', =O, =NR', =N-OR', -NR¹R", -SR¹, -halogen, - SiR¹R¹¹R¹", -OC(O)R¹, -C(O)R', -CO2R¹, -CONR¹R", -OC(O)NR¹R", -NR¹¹C(O)R¹, -NR¹- C(O)NR¹¹R'", -NR"C(0)₂R^f, -NR-C(NR¹R¹¹R¹^=NR"", -NR-C(NR'R")=NR"\ -S(O)R', -S(O)₂R¹, - S(O)₂NR¹R", -NRSO₂R¹, -CN and -NO₂, -R¹, -N₃, -CH(Ph)₂, fluoro(C_rC₄)alkoxy, and fluoro(C₁-C₄)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and where R¹, R", R'" and R"" are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl. When a compound includes more than one R group, for example, each of the R groups is independently selected as are each R', R", R¹" and R"" groups when more than one of these groups is present. In the schemes that follow, the symbol X represents "R" as described above.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -T-C(O)-(CRR')q-U-, wherein T and U are independently -NR-, -0-, -CRR'- or a single bond, and q is an integer of from O to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH₂)r-B-, wherein A and B are independently -CRR'-, -0-, -NR-, -S-, -S(O)-, -S(O)₂-, -S(O)₂NR'- or a single bond, and r is an integer of from 1 to 4. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula - (CRR')s-X-(CR"R'")d-, where s and d are independently integers of from O to 3, and X is -0-, -NR¹-, -S-, -S(O)-, -S(O)₂-, or -S(O)₂NR'-. The substituents R, R', R" and R"¹ are preferably independently selected from hydrogen or substituted or unsubstituted (C1-C6)alkyl.

As used herein, the term "heteroatom" includes oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), silicon (Si), and selenium (Se). The term "amino" or "amine group" refers to the group -NR¹R" (or N⁺RR¹R") where R, R¹ and R" are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl. A substituted amine being an amine group wherein R¹ or R" is other than hydrogen. In a primary amino group, both R¹ and R" are hydrogen, whereas in a secondary amino group, either, but not both, R' or R" is hydrogen. In addition, the terms "amine" and "amino" can include protonated and quaternized versions of nitrogen, comprising the group -N⁺RR¹R" and its biologically compatible anionic counterions.

The term "aqueous solution" as used herein refers to a solution that is predominantly water and retains the solution characteristics of water. Where the aqueous solution contains solvents in addition to water, water is typically the predominant solvent.

The term "carboxyalkyl" as used herein refers to a group having the general formula - (CH₂)_nCOOH, where n is 1-18.

The term "pharmaceutically acceptable" as used herein refers to a compound or combination of compounds that while biologically active will not damage the physiology of the recipient human or animal to the extent that the viability of the recipient is comprised. Preferably, the administered compound or combination of compounds will elicit, at most, a temporary detrimental effect on the health of the recipient human or animal is reduced. "Pharmaceutically acceptable salt" refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly useful inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Organic bases include, but are not limited to, such as isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. The term "IC₅₀" as used herein refers to half maximal inhibitory concentration, for example, representing the concentration of an inhibitor that is necessary for 50% inhibition of the target (i.e. the activity of an enzyme, transcription factor, receptor or ligand). The term "purified" as used herein refers to an HPI removed from an environment in which it was produced and is about 60% free, about 75% free, and most usefully about 90% free from other components with which it is naturally associated or with which it was otherwise associated with during production. The phrases "operably associated" and "operably linked" refer to functionally related nucleic acid sequences. By way of example, a regulatory sequence is operably linked or operably associated with a protein encoding nucleic acid sequence if the regulatory sequence can exert an effect on the expression of the encoded protein. In another example, a promoter is operably linked or operably associated with a protein encoding nucleic acid sequence if the promoter controls the transcription of the encoded protein. While operably associated or operably linked nucleic acid sequences can be contiguous with the nucleic acid sequence that they control, the phrases "operably associated" and "operably linked" are not meant to be limited to those situations in which the regulatory sequences are contiguous with the nucleic acid sequences they control. The term "subject animal or human" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc and human. The terms "proliferating" and "proliferation" as used herein refers to cells undergoing mitosis. The term "apoptosis" as used herein refers to a process of self-destruction in certain cells, for example, epithelial cells and erythrocytes, that are genetically programmed to have a limited life span or are damaged. Apoptosis can be induced either by a stimulus, such as irradiation or toxic drugs, by removal of a repressor agent, or by activation of a pro-apoptotic pathway. The cells disintegrate into membrane-bound particles that are then eliminated by phagocytosis. Apoptosis is also known as programmed cell death.

The term "conjugate" as used herein refers to an HPI that is covalently or non- covalently associated with a molecule or moiety that alters the physical properties of the HPI such as increasing stability and/or facilitate cellular uptake or efficacy of the HPI. The conjugated HPI may have a molecule or moiety attached directly or indirectly through a linker.

Conjugates may contain, for example, amino acids, peptides, polypeptides, proteins, antibodies, antigens, toxins, hormones, lipids, nucleotides, nucleosides, sugars, carbohydrates, polymers such as polyethylene glycol and polypropylene glycol, as well as analogs or derivatives of all of these classes of substances. Additional examples of conjugates are steroids, such as cholesterol, phospholipids, di- and tri-acylglycerols, fatty acids, hydrocarbons that may or may not contain unsaturation or substitutions, enzyme substrates, biotin, digoxigenin, and polysaccharides. Still other examples include thioethers such as hexyl-S-tritylthiol, thiocholesterol, acyl chains such as dodecandiol or undecyl groups, phospholipids such as di-hexadecyl-rac-glycerol, triethylammonium 1 ,2-di-O- hexadecyl-rac-glycer- o-3-H-phosphonate, polyamines, polyethylene glycol, adamantane acetic acid, palmityl moieties, octadecylamine moieties, hexylaminocarbonyl-oxyc- holesterol, farnesyl, geranyl and geranylgeranyl moieties.

Conjugates can also comprise a detectable label. The term "label" when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the HPI so as to generate a "labeled" HPI. The label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable. For example, conjugates can be a HPI covalently attached to a fluorophore. Conjugates may include fluorophores such as, but not limited to, TAMRA, BODIPY, Cyanine derivatives such as Cy3 or Cy5, Dabsyl, or any other suitable fluorophore known in the art.

The term "effective amount" as used herein refers to a concentration of HPI which results in achieving a particular stated purpose, for example, to cause a decrease in transcription of a gene of interest in the cell. Of particular interest is an effective concentration that provides a decrease greater than or equal to at least about 45% or further decrease, including about 50% or more, about 60% or more, about 70% or more, about 75% or more, about 80% or more decrease in target activity (i.e., transcription) relative to a basal expression level. Target activity may be measured by any method known in the art. For example, where the target is a GIi transcription factor, the target activity may be measured by level of transcription (i.e. of the Hh pathway genes GIH or Ptchi), level of the protein whose transcription is operably linked or activity of the protein whose transcription is operably linked. Alternatively, activity of a HPI of the disclosure may be measured by detection of a marker gene, for example, lacZ, one of the family of fluorescent polypeptides (e.g., GFP, YFP, BFP, RFP etc), or luciferase which is operably linked to GIi DNA binding sites. Description

Inappropriate activation of the Hedgehog (Hh) signaling pathway (illustrated in Fig. 1 ) has been implicated in a diverse spectrum of cancers, and its pharmacological blockade has emerged as an anti-tumor strategy. While nearly all known Hh pathway antagonists target the transmembrane protein Smoothened (Smo), small molecules that suppress downstream effectors could more comprehensively remediate Hh pathway-dependent tumors.

Certain neoplasms require Hh ligand function, either through autocrine or paracrine signaling mechanisms, including small-cell lung cancers, pancreatic adenocarcinomas, and prostate tumors. In other cases, ligand-independent Hh target gene expression can lead to tumorigenesis, exemplified by Gorlin's syndrome patients who are heterozygous for Ptchi and highly susceptible to basal cell carcinomas, medulloblastomas, and rhabdomyosarcomas. Oncogenic mutations in Smo have also been identified, and tumors can arise from loss of Su(fu).

A link between Hh target gene expression and oncogenesis indicates that pharmacological inhibitors of the Hh pathway may have therapeutic value. For example, the Smo antagonist cyclopamine can block tumor progression in a variety of mouse cancer models. While small molecules may be effective against Hh ligand-dependent tumors and those that involve a loss of Ptchi function, cancers that result from downstream lesions within the Hh pathway are unlikely to be remediated; the oncogenic Smo mutant SmoM2 is resistant to cyclopamine, and medulloblastomas that arise in Su(fu) heterozygous mice are unresponsive to Smo inhibitors.

Screens of 1 ,990 synthetic chemicals and 94 natural products identified a few compounds that could antagonize Hh target gene expression induced by GIiI or Gli2 over- expression, including GANT-58, GANT-61 , zerumbone, arcyhaflavin C, and physalin F. How these compounds antagonize GIi function has not been determined, although GANT-61 appears to attenuate the DNA-binding activity of GIiI in vivo, and it has been suggested that arcyriaflavin C and physalin F indirectly antagonize GIi function through PKC/MAPK pathway blockade. Similarly, the natural product forskolin can inhibit Hh signaling by activating adenylate cyclase and consequently PKA, but its mechanism of action impacts multiple signaling pathways.

To discover Hh pathway inhibitors that do not directly target Smo, a large-scale, high- throughput screen was conducted for compounds that can abrogate Hh target gene expression induced by the Smo agonist SAG, as described in PCT Publication WO 2009/102864A1 incorporated herein by reference in its entirety. The screening assays minimized the inhibitory activities of Smo-targeting compounds, since most known Smo antagonists are functionally and biochemically competitive with SAG. Hh pathway inhibitors (HPIs) that differentially perturb biochemical and cellular processes associated with Hh signaling, including several that involve the primary cilium were identified. The structural formulas of representative HPIs (1-18) are shown in Fig. 2. Their phenotypes revealed multiple pharmacologically targetable events within the Hh pathway (see Table 1 , Fig. 3), and a subset of these compounds can block the SmoM2-dependent proliferation of medulloblastoma progenitors. The present disclosure encompasses one of the identified compounds (having formula I, Fig. 4) of the large-scale screening, and derivatives thereof.

Disruption of the Hh pathway may result in cellular proliferative disease characterized by the undesired propagation of cells, including, but not limited to, neoplastic disease conditions, such as a cancer. Examples of cellular proliferative disease include, but are not limited to, skin cancer, (including Basal Cell carcinoma), brain cancer, (including glioma and medulloblastoma), colon cancer, mesothelioma, lung cancer, renal cell carcinoma, breast cancer, prostate cancer, sarcoma, ovarian cancer, esophageal cancer, stomach cancer, gastric cancer, hepatocellular cancer, rhabdomyosarcoma, nasopharyngeal cancer and pancreatic cancer. Subjects seeking alleviation according to the methods of the disclosure include any individual having any of the above-mentioned disorders.

In certain cases the disclosure provides compounds and methods of antagonizing, or inhibiting, the Hh pathway (i.e., an activated Hh pathway) by administering an HPI to a mammalian cell. Antagonism of the Hh pathway can be useful in the context of a tumor, for example, inhibition of cellular proliferation, inhibition of cellular transformation, and inhibition of cellular migration (as an anti-cancer agent). Another aspect of the disclosure provides compounds and methods of antagonizing the Hh pathway by administering an HPI to a mammalian cell useful in the context of developmental disorders, where in the developing cell or tissue, inhibition of the Hh pathway would be beneficial. The disclosure should not be construed to be limited solely to the treatment of patients having a cellular proliferative disease. Rather, the disclosure should be construed to include the alleviation of conditions or disease associated with increased expression of Hh pathway genes that would benefit from the compounds and methods of the subject disclosure.

The HPI and methods of the instant disclosure can be used for prophylactic or therapeutic purposes. As used herein, the term "alleviate" or "alleviation" is used to refer to both prevention of disease, and reduction of pre-existing conditions. The reduction of ongoing disease (e.g. cancer), in order to stabilize or improve the clinical symptoms of the patient, is of particular interest. Such alleviation is desirably performed prior to loss of function in the affected tissues. Evidence of alleviation may be any diminution in the severity of disease, particularly measuring the severity of such symptoms as found in development and tumorigenic transformation of the skin, brain, lung, breast, prostate, gut, and blood.

Such subjects may be tested in order to assay the activity and efficacy of the subject HPI. A significant improvement in one or more of parameters is indicative of efficacy. It is well within the skill of the ordinary healthcare worker (e.g., clinician) to adjust dosage regimen and dose amounts to provide for optimal benefit to the patient according to a variety of factors (e.g., patient-dependent factors such as the severity of the disease and the like, the compound administered, and the like). Pharmaceutical preparations containing compounds of the disclosure.

The disclosure further encompasses pharmaceutical preparations of the subject HPI. The subject HPI can be incorporated into a variety of formulations for therapeutic administration by a variety of routes. More particularly, the HPI of the present disclosure can be formulated into pharmaceutical compositions by combination with appropriate pharmaceutically acceptable carriers, diluents, excipients and/or adjuvants, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols, in a sterile vial or in a syringe. Where the formulation is for transdermal administration, the compounds are preferably formulated either without detectable DMSO or with a carrier in addition to DMSO. The formulations may be designed for administration to subjects or patients in need thereof via a number of different routes, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, etc. The administration can be systemic or localized delivery of the formulation to a site in need of treatment, e.g., localized delivery to a tumor or application directly to the skin. Pharmaceutically acceptable excipients usable with the disclosure, such as vehicles, adjuvants, carriers or diluents, are readily available. Moreover, pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985; Remington: The Science and Practice of Pharmacy, A.R. Gennaro, (2000) Lippincott, Williams & Wilkins. The HPI or HPI formulation to be administered will, in any event, contain a quantity of the agent adequate to achieve the desired state in the subject being treated.

In pharmaceutical dosage forms, the subject HPI of the disclosure may be administered in the form of their pharmaceutically acceptable salts, or may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are in no way limiting.

Prodrugs of the HPI of the disclosure can be provided by altering functional groups on the HPI in such a manner that the alterations are removed in the body of a mammal. In certain cases, the alterations/modifications are made during or after synthesis of the effective HPI. Actual methods of preparing prodrugs are known, or will be apparent to the skilled artisan. See Bundgaard, in Design of Prodrugs, ed. H. Bundgaard, Elsevier Science Publishers, New York (1985).

The HPI can be administered to a host using any available conventional methods and routes suitable for delivery of conventional drugs, including systemic or localized routes. In general, routes of administration contemplated by the disclosure include, but are not necessarily limited to, enteral, parenteral, or inhalational routes, such as intrapulmonary or intranasal delivery.

Conventional and pharmaceutically acceptable routes of administration include intranasal, intrapulmonary, intramuscular, intratracheal, intratumoral, subcutaneous, intradermal, topical application, intravenous, rectal, nasal, oral and other parenteral routes of administration. Routes of administration may be combined, if desired, or adjusted depending upon the agent and/or the desired effect. The composition can be administered in a single dose or in multiple doses.

For oral preparations, the subject HPI of the disclosure can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

Parenteral routes of administration include, but are not necessarily limited to; inhalation administration, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, intravenous routes, i.e., any route of administration other than through the alimentary canal, and local injection, with intra or peritumoral injection being of interest, especially where a tumor is a solid or semi-solid tumor (e.g., Hodgkins lymphoma, non-Hodgkins lymphoma, and the like). Local injection into a tissue defining a biological compartment ((e.g., prostate, ovary, regions of the heart (e.g., pericardial space defined by the pericardial sac), intrathecal space, synovial space, and the like)) is also of interest. Parenteral administration can be carried to effect systemic or local delivery of the agent. Where systemic delivery is desired, administration typically involves invasive or systemically absorbed topical or mucosal administration of pharmaceutical preparations.

Methods of administration of the HPI through the skin or mucosa include, but are not necessarily limited to, topical application of a suitable pharmaceutical preparation, transdermal transmission, injection and epidermal administration. For transdermal transmission, absorption promoters or iontophoresis are suitable methods, lontophoretic transmission may be accomplished using commercially available "patches" which deliver their product continuously via electric pulses through unbroken skin for periods of several days or more.

The subject HPI of the disclosure can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol, collagen, cholesterol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives. The HPI of the disclosure can also be delivered to the subject by enteral administration. Enteral routes of administration include, but are not necessarily limited to, oral and rectal (e.g., using a suppository) delivery. Furthermore, the subject HPI can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds of the present disclosure can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature. Dosages of the compounds of the disclosure

Depending on the subject and condition being treated and on the administration route, the subject HPI may be administered in dosages of, for example, 0.1 μg to 100 mg/kg body weight per day. In certain embodiments, the therapeutic administration is repeated until a desired effect is achieved. Similarly the mode of administration can have a large effect on dosage. Thus, for example, oral dosages may be about ten times the injection dose. Higher doses may be used for localized routes of delivery.

A typical dosage may be a solution suitable for intravenous administration; a tablet taken from two to six times daily, or one time release capsule or tablet taken once a day and containing a proportionally higher content of active ingredient, etc. The time release effect may be obtained by capsule materials that dissolve at different pH values, by capsules that release slowly by osmotic pressure, or by any other known means of controlled release.

Those of skill in the art will readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects. Dosages for a given HPI are readily determinable by those of skill in the art by a variety of means. Although the dosage used will vary depending on the clinical goals to be achieved, a suitable dosage range is one which provides up to about 1 μg to about 1 ,000 μg or about 10,000 μg of subject HPI to alleviate a symptom in a subject animal.

Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing the HPI of the disclosure. Similarly, unit dosage forms for injection or intravenous administration may comprise the HPI in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier. Combination therapy using the compounds of the disclosure. For use in the subject methods, the subject HPI may be formulated with or otherwise administered in combination with other pharmaceutically active agents, including other agents that activate or suppress a biochemical activity, such as a chemotherapeutic agent. The subject compounds may be used to provide an increase in the effectiveness of another chemical, such as a pharmaceutical, or a decrease in the amount of another chemical, such as a pharmaceutical that is necessary to produce the desired biological effect.

Examples of chemotherapeutic agents for use in combination therapy include, but are not limited to, daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis- chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6- mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4- hydroxyperoxycyclophosphor- amide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). In certain cases, the HPI of the current disclosure may also be used with an anti- angiogenic factor, for example, the anti-VEGF antibody AVASTIN ™.

Furthermore, the HPI of the present disclosure may also be used in combination therapy with other Hh pathway antagonists, including another HPI of the disclosure. Examples of agents for use in combination therapy include, but are not limited to; cyclopamine, SANT 1-4, antibodies to Shh, Ihh, Dhh, Ptchi , Ptc2 or Smo. For example, the HPI of the disclosure may be administered to antagonize a GIi gene or polypeptide, and the second Hh pathway antagonist may be administered to reduce Smo signaling. In an additional example, a HPI of the disclosure may be administered in combination with a second HPI of the disclosure. Other therapeutics for use in combination therapy with the HPI (formula I) described herein may be administered by the same route of administration (e.g. intrapulmonary, oral, enteral, etc.) as that of the HPI of the disclosure is administered. In the alternative, the therapeutics for use in combination therapy with the HPI of the present disclosure may be administered by a different route of administration that the HPI of the disclosure are administered. Kits

Kits with unit doses of the subject compounds, usually in oral or injectable doses, are provided by the present disclosure. In such kits, in addition to the containers containing the unit doses will be an informational package insert describing the use and attendant benefits of the drugs in treating pathological condition of interest. Representative compounds and unit doses are those described herein above. In one embodiment, the kit comprises a HPI formulation in a sterile vial or in a syringe, which formulation can be suitable for injection in a mammal, particularly a human.

One aspect of the disclosure, therefore, provides a compound having the formula A:

or a pharmaceutically acceptable salt thereof, wherein:

R₁ , R₂, and R₃ are each independently H, an acyl, an alkanoyl, an alkyl, an unsaturated alkyl. a heteroalkyl, an amino group, an amine group, an aryl, a substituted aryl group, an alkoxy, an alkylamino, an alkylthio (or thioalkoxy), a heteroalkyl, a heteroalkylene, a cycloalkyl, an aryl group, a heteroaryl group, or an arylalkyl, or a substituted or a unsubstituted form thereof, or a pharmaceutically acceptable salt thereof.

One embodiment of this aspect of the disclosure provides a compound having the formula I:

or a pharmaceutically acceptable salt thereof.

Another aspect of the disclosure provides embodiments of a pharmaceutical composition comprising a compound having the formula A:

or a pharmaceutically acceptable salt thereof, wherein:

R₁ , R₂, and R₃ are each independently H, an acyl, an alkanoyl, an alkyl, an unsaturated alkyl. a heteroalkyl, an amino group, an amine group, an aryl, a substituted aryl group, an alkoxy, an alkylamino, an alkylthio (or thioalkoxy), a heteroalkyl, a heteroalkylene, a cycloalkyl, an aryl group, a heteroaryl group, or an arylalkyl, or a substituted or a unsubstituted form thereof, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier. An embodiment of this aspect of the disclosure is a pharmaceutical composition comprising a compound having the formula I:

I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

Yet another aspect of the disclosure provides for the use of a compound having the formula A:

or a pharmaceutically acceptable salt thereof, in the manufacture of the pharmaceutical composition for the treatment of a cell or tissue of a subject human or animal having a cell proliferative disorder, where the pharmaceutical composition modulates cell proliferation by modulating the Hedgehog pathway signaling, wherein:

R₁, R₂, and R₃ are each independently H, an acyl, an alkanoyl, an alkyl, an unsaturated alkyl. a heteroalkyl, an amino group, an amine group, an aryl, a substituted aryl group, an alkoxy, an alkylamino, an alkylthio (or thioalkoxy), a heteroalkyl, a heteroalkylene, a cycloalkyl, an aryl group, a heteroaryl group, or an arylalkyl, or a substituted or a unsubstituted form thereof, or a pharmaceutically acceptable salt thereof,.

In an embodiment of this aspect of the disclosure the compound has the formula I:

I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, or a pharmaceutically acceptable salt thereof.

Yet another aspect of the disclosure provides embodiments of a method of modulating Hedgehog pathway signaling in a cell, the method comprising: (i) providing a target cell or population of target cells; (ii) contacting the cell with the compound having the formula A:

or a pharmaceutically acceptable salt thereof, wherein:

R₁ , R₂, and R₃ are each independently H, an acyl, an alkanoyl, an alkyl, an unsaturated alkyl. a heteroalkyl, an amino group, an amine group, an aryl, a substituted aryl group, an alkoxy, an alkylamino, an alkylthio (or thioalkoxy), a heteroalkyl, a heteroalkylene, a cycloalkyl, an aryl group, a heteroaryl group, or an arylalkyl, or a substituted or a unsubstituted form thereof, or a pharmaceutically acceptable salt thereof, in an amount effective to modulate Hedgehog signaling in the cell, thereby modulating Hedgehog pathway signaling in the target cell or population of target cells.

In the embodiment of this aspect of the disclosure, the compound can have the formula I:

I or a pharmaceutically acceptable salt thereof.

In embodiments of this aspect of the disclosure, the Hedgehog pathway signaling can be associated with cell proliferation, and wherein reducing the Hedgehog pathway signaling in the target cell or population of target cells reduces the proliferation of the target cell or population of target cells.

In embodiments of this aspect of the disclosure, the target cell or population of target cells can be present in a cultured cell or population of cells.

In embodiments of this aspect of the disclosure, the target cell or population of target cells can be present in a subject animal or human.

In embodiments of this aspect of the disclosure, the target cell or population of target cells can be associated with a Hedgehog-related disorder of the subject animal or human.

In some embodiments of this aspect of the disclosure, the Hedgehog-related disorder of the subject animal or human is a tumor. In other embodiments of this aspect of the disclosure, the Hedgehog-related disorder of the subject animal or human is a disorder of a tissue development.

In the embodiments of this aspect of the disclosure, the compound can be in a pharmaceutically acceptable composition. In these embodiments, the pharmaceutically acceptable composition may further comprise a pharmaceutically acceptable carrier.

In embodiments of the method of this aspect of the disclosure, the method may further comprise administering to the subject animal or human an effective amount of a second therapeutic agent.

Still another aspect of the disclosure provides embodiments of kits comprising a container and a dose or plurality of doses of the compound having the formula A:

or a pharmaceutically acceptable salt thereof, wherein:

R₁ , R₂, and R₃ are each independently H, an acyl, an alkanoyl, an alkyl, an unsaturated alkyl. a heteroalkyl, an amino group, an amine group, an aryl, a substituted aryl group, an alkoxy, an alkylamino, an alkylthio (or thioalkoxy), a heteroalkyl, a heteroalkylene, a cycloalkyl, an aryl group, a heteroaryl group, or an arylalkyl, or a substituted or a unsubstituted form thereof, or a pharmaceutically acceptable salt thereof, , and a package insert describing the use of said compound, or a pharmaceutical composition comprising said compound, in treating a pathological condition of interest in a subject animal or human.

In the embodiment of this aspect of the disclosure, the compound can have the formula I:

I or a pharmaceutically acceptable salt thereof.

The specific examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present disclosure to its fullest extent. All publications recited herein are hereby incorporated by reference in their entirety.

It should be emphasized that the embodiments of the present disclosure, particularly, any "preferred" embodiments, are merely possible examples of the implementations, merely set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described embodiment(s) of the disclosure without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure, and the present disclosure and protected by the following claims. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the compositions and compounds disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, efc), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a concentration range of "about 0.1 % to about 5%" should be interpreted to include not only the explicitly recited concentration of about 0.1 wt% to about 5 wt%, but also include individual concentrations (e.g., 1 %, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5%, 1 .1 %, 2.2%, 3.3%, and 4.4%) within the indicated range. The term "about" can include ±1 %, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, or ±10%, or more of the numerical value(s) being modified.

EXAMPLES Example 1 Identification of Hh pathway inhibitors (HPIs) that do not directly target Smo: To facilitate the identification of antagonists that act downstream of Smo, screening conditions were established that exploited competitive interactions between Smo agonists and antagonists. Shh-LIGHT2 cells, an NIH-3T3-derived cell line stably transfected with Gli-dependent firefly luciferase and constitutive Renilla luciferase reporters were stimulated with 0.5 μM SAG (N- Methyl-Λ/'-(3-Pyridinylbenzyl)-Λ/'-(3-Chlorobenzo[b]thiophene-2-carbonyl)-1 ,4-

Diaminocyclohexane. 122,755 compounds were surveyed for the ability to block the resulting firefly luciferase expression without affecting Renilla luciferase activity. These assay conditions were resistant to inhibition by cyclopamine and similarly acting Smo inhibitors, whereas forskolin was equipotent against Shh and SAG-dependent Hh pathway activation, as described in PCT Publication WO 2009/102864A1 incorporated herein by reference in its entirety Through this screen, Hh pathway inhibitors with median inhibitory concentrations

(IC₅₀S) less than 10 μM, and mechanisms of action that are distinct from cyclopamine, were identified, as shown in Table 1 , Fig. 3, including the compound having the formula I shown in Fig. 4(and as compound 12, in Table 1 , Fig. 3). This inhibitor, like others as listed in Table 1 , did not exhibit differential inhibition of Shh- and SAG-induced firefly luciferase expression in Shh-LIGHT2 cells, as is observed with cyclopamine and other Smo antagonists such as the SANTs. Nor did the compounds, including the compound having the formula I, attenuate the binding of a fluorescent cyclopamine derivative (BODIPY-cyclopamine) to Smo-over- expressing HEK-293T cells.

The compounds HPI-1 through HPI-4 were tested in other cell lines competent for Hh target gene expression. A stable NIH-3T3 cell line was generated with a Gli-dependent enhanced green fluorescent protein reporter to provide a non-luciferase-based assay for compound activity (Shh-EGFP cells).

The ability of these small molecules to block Shh-induced differentiation of C3H10T(1/2) cells into alkaline phosphatase-positive osteoblasts or the constitutive Hh target gene expression in embryonic fibroblasts derived from Ptch1^~'^~ mice were also evaluated as assessed by the β-galactosidase reporter replacing the Ptchi coding region. The compounds exhibited inhibitory activities in all three of these contexts, as shown in Table 1 (Fig. 3), although some variability with respect to specific IC₅₀S was observed. In contrast, none of the compounds were able to block Wnt signaling in L cells stably transfected with a TCF/LEF-dependent firefly luciferase (Wnt-LIGHT cells) and treated with Wnt3a-conditioned medium. Example 2

Cell lines: Shh-EGFP cells were generated by co-transfecting NIH- 3T3 cells with the Gli- dependent EGFP reporter and pVGRXR (Invitrogen), followed by isolation of Shh-responsive cells by fluorescence-activated cell sorting (FACS) and clonal selection in medium containing 400 μg/mL zeocin. Wnt-LIGHT cells were generated by cotransfecting L cells with the TCF/LEF-dependent firefly luciferase reporter and pcDNA3, followed by selection in medium containing 1 mg/mL geneticin, and the isolation of Wnt3a-responsive clones. FLAG-GN1- and FLAG-Gli2-expressing stable lines were generated by infecting Shh-LIGHT2 and Shh-EGFP cells, respectively, with the corresponding retroviral expression vectors, followed by FACS-based isolation of infected cells and immunofluorescence-based selection of individual clones with low levels of FLAG-GIiI or FLAG-GH2 expression. Primary cultures of cerebellar GNPs were obtained by triturating tumors derived from Math 1 -ere :SmoM2 mice and plating the dissociated cells onto poly-Dornithine- coated plates. Example 3

Shh-LIGHT2 assay for Hh pathway activation and library screening conditions: Shh-N- conditioned medium was prepared. Shh-LIGHT2 cells, an NIH-3T3-derived line stably transfected with Gli-dependent firefly luciferase (8XGIiBS-FL) and constitutive Renilla luciferase (pRLTK, Promega) reporters, were cultured in DMEM (Invitrogen) containing 10% calf serum (CS, Hyclone), 400 μg/mL geneticin, 200 μg/mL zeocin, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. For 96-well plate assays, Shh-LIGHT2 cells were seeded into each plate (10,000 cells/well) and cultured to confluency. The Shh-LIGHT2 cells were then grown in DMEM containing 0.5% CS, 100 U/mL penicillin, 0.1 mg/mL streptomycin, either 5% Shh-N-conditioned medium or 500 nM SAG, and various concentrations of the HPIs. After the cells were cultured for another 30 h, the resulting firefly and Renilla luciferase activities were measured using a Dual Luciferase Reporter kit (Promega) and a Veritas microplate luminometer (Turner Biosystems).

To screen 122,755 compounds from the Stanford High-Throughput Bioscience Center collection, the Shh-LIGHT2 assay was modified as follows. Shh-LIGHT2 cells were cultured in ten 10-cm tissue culture dishes using DMEM containing 10% CS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. When the cells reached >90% confluency, they were removed by trypsinization and diluted to a final volume of 50 mL medium. This cell suspension was then plated into clear-bottom, white-walled 384-well plates using a Titertek Multidrop dispenser (50 μL/well) and the cells were cultured until they reached confluency (3-4 days). The culture medium was then manually removed from each plate, and DMEM containing 0.5% CS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin was added using the Multidrop dispenser (40 μL/well). The plates were then placed into an automated Staccato system (Caliper Life Sciences), which includes an automated CO₂ incubator. An automated protocol was then run, in 3 which a Multidrop dispenser added 10 μL of a SAG solution in 0.5% CS medium (final SAG concentration of 500 nM) to columns 1-22 and 100 nL of each compound solution was added to the plates using a V&P Scientific pin tool, resulting in a final compound screening concentration of 10 μM. The SAG-free wells were used as negative controls. After 28-32 h, the assay medium was manually removed and the plates were frozen. To assay the firefly luciferase activities, the plates were thawed and placed into a Twister Il stacker in the Staccato system. Bright-Glo luciferase substrate (Promega) was added by the Multidrop dispenser and the plates were analyzed on an Analyst GT microplate reader (2 min in the dark, 0.2 sec integration/well; Molecular Devices). Hits were defined as compounds that reduce the firefly luciferase signal by at least 50% compared to inhibitor-free controls. Primary hits were then re-tested in duplicate in an eight-point, 1 :1 serially diluted dose response curve with a starting concentration of 20 μM. In these experiments, the median inhibitory concentration (IC₅o) for both firefly and Renilla luciferase were determined using a Dual-Glo kit (Promega). Compounds that exhibited IC50s <10 μM (Table 1) and did not inhibit Renilla activity were selected for further study. Example 4

BODIPY-cyclopamine/Smo binding assay. Smo-binding assays were conducted with BODIPY-cyclopamine and Smo over-expressing and HEK-293T cells as previously described, using a CMV-promoter based SV40 origin-containing expression construct for Smo-Myc3 (murine Smo containing three consecutive Myc epitopes at the C-terminus). HEK-293T cells were seeded into 24-well plates (30,000 cells/well) containing poly-D-lysine- treated 12-mm glass coverslips and cultured in 8 DMEM containing 10% fetal bovine serum (FBS, Invitrogen), 100 U/mL penicillin, and 0.1 mg/mL streptomycin. The cells were cultured until they reached 55 to 65% confluency (14-18 h), after which they were transfected with the Smo-Myc3 expression construct and FuGENE (Roche) according to the manufacturer's protocols. 24 h after transfection, the cells were washed with phosphate-buffered saline (PBS) and cultured in DMEM containing 0.5% FBS, 5 nM BODIPY-cyclopamine, and individual HPIs (20 μM). After 30 min, 10 μM Hoescht 33342 was added to each well, and the HPIs were incubated with the cells for an additional 60 min. The cells were then washed two times with PBS buffer, mounted in Prolong Gold (Invitrogen) and immediately imaged using a DM4500B compound microscope (Leica). Example 5

Shh-EGFP assay for Hh pathway activation: A Gli-dependent enhanced green fluorescent protein reporter (Shh-EGFP) was generated by excising firefly luciferase cDNA from the 8XGIiBs vector using Λ/col/Hpal, and ligating in EGFP cDNA excised from the pEGFP-C1 vector (Clontech) by NcoMAflW digestion. NIH- 3T3 cells were seeded into a 6-well plate (150,000 cells/well), cultured in DMEM containing 10% CS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin for 24 h, and then transfected with 1 μg/well of Shh-EGFP plasmid, 50 ng/well of the zeocin resistance-conveying vector pVGRXR (Invitrogen), and FuGENE according to the manufacturer's protocols. The cells were grown to confluency and treated with DMEM containing 0.5% CS, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 5% Shh- N-conditioned medium for 30 h. Following Shh-N treatment, the adherent cultures were dissociated into single cells with 0.05% Trypsin-EDTA (300 μL/well; Invitrogen) for 5 min. The cells were then suspended in 1 mL of PBS containing 1 % CS and sorted on a BD FACSAria (excitation: 488 nm; emission: 530/30 nm) to enrich for cells expressing EGFP in a Shh-N-responsive manner. Clonal populations were then cultured from single cells in DMEM containing 10% CS, 400 μg/mL zeocin, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. To identify Shh-EGFP clones with maximum Shh-N-responsiveness, individual lines were seeded into 24-well plates (45,000 cells/well) and cultured for 48 h. The cells were then treated with DMEM containing 0.5% CS, 100 U/mL pencillin, 0.1 mg/mL streptomycin or the culture medium plus 5% Shh-N-conditioned medium. After 30 h, the adherent cultures were treated with 0.05% trypsin-EDTA (150 μL/well; Invitrogen) for 5 min to dissociate them into single cells. The cells were then resuspended in 500 μl_ of PBS containing 1 % CS and analyzed on a BD FACSCalibur (excitation: 488 nm; emission: 530/30 nm). 10,000 cells were used to assess EGFP expression levels as a measure of Hh pathway activation, and the data was analyzed using FlowJo software (Tree Star). Clones with low basal EGFP levels and maximum Shh-Ninduced EGFP expression were selected for further use. To assess the inhibitory activities of the HPIs in the Shh-EGFP cells, a clonal line with maximum Shh-responsiveness was cultured in 24-well plates as described above. During treatment with Shh-N-conditioned medium, the cells were incubated either DMSO or individual HPIs (each at a concentration ten-fold greater than its IC50 in the Shh-LIGHT2 assay or 30 μM, whichever was lower: 15 μM HPI-1 , 20 μM HPI-2, 30 μM HPI-3, and 30 μM HPI-4). The resulting EGFP levels were then evaluated by FACS and quantified using the FlowJo software. Example 6

C3H10T(1/2) assay for Hh pathway activation: C3H10T(1/2) cells (ATCC) were plated into 96-well plates using DMEM containing 10% FBS, 100 U/mL penicillin, 0.1 mg/mL streptomycin, 5% Shh-N-conditioned medium, and various concentrations of the Hh pathway inhibitors. After approximately 40 h, the cells were washed with PBS and lysed in 50 μl_ of buffer containing 50 mM Tris-HCI, pH 9.5, 150 mM NaCI, 5 mM MgCI₂, and 1 % Triton X-100. Alkaline phosphatase activities in the cell lysates were quantified by adding 10 μl_ of the lysate to 50 μl_ of CDP-Star chemiluminescence reagent (Perkin Elmer) and measuring the resulting chemiluminescence on a Veritas microplate luminometer. Example 7

PtcM^~'^~ fibroblast assay for Hh pathway activation: Hh pathway activation in PtchT^1' fibroblasts was assayed as previously described, using the knocked-in β-galactosidase gene as a reporter for Hh target gene expression. PtchT'^' fibroblasts were grown to 70-80% confluence in a 15-cm dish, trypsinized, and then resuspended in 40 ml. of DMEM containing 10% FBS, 100 U/mL penicillin, and 0.1 mg/mL DMSO. This cell suspension was aliquoted into 96-well plates (150 μL/well), cultured overnight, and then treated with DMSO or various concentrations of the HPIs for 28 h. Cell viability was measured with CellTiter 96 AQ (Promega) according to the manufacturer's protocols. The cells were then lysed in Tropix lysis solution (30 μL/well; Applied Biosystems) and β-galactosidase levels were quantified by a Tropix Galacto-Star kit (Applied Biosystems) on a Veritas microplate luminometer. Example 8

SmoM2-LIGHT assay for Hh pathway activation. SmoM2-LIGHT cells (previously named SmoA1 -LIGHT cells) were grown to 70- 80% confluence in a 15-cm dish, trypsinized, and then resuspended in 40 mL of DMEM containing 10% FBS, 100 U/mL penicillin, and 0.1 mg/mL DMSO. This cell suspension was aliquoted into 96-well plates (150 μL/well), cultured overnight, and then treated with DMSO or various concentrations of the HPIs for 28 h. Cells were then washed once with PBS then treated with 50 μl_ of passive lysis buffer (Promega). Hh pathway-dependent firefly luciferase activity in the lysates was quantified using Bright- GIo reagent and a Veritas microplate luminometer. Constitutive β-galactosidase activity was measured using the Tropix Galacto-Star kit. Example 9 Wnt-LIGHT assay for Wnt pathway activation. Wnt3a-conditioned medium was prepared by culturing L cells stably expressing Wnt3a (ATCC) in DMEM containing 10% FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. After the cells reached 70% confluency, they were cultured in fresh medium, and the resulting Wnt3a-condition medium was collected 30 h later. To generate a Wnt pathway-reporter cell line (Wnt-LIGHT cells), L cells were seeded into a 6-well plate and cultured in DMEM containing 10% FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin until they reached 50% confluency. The cells were then transfected with 1 μg/well of SuperTopFlash reporter, which contained seven TCF/LEF enhancer sites upstream of a basal promoter and firefly luciferase cDNA, 50 ng/well of constitutive Renilla reporter pRLSV40 (Invitrogen), 50 ng/well of the geneticin resistance-conveying vector pcDNA3 (Invitrogen), and FuGENE 6 according to the manufacturer's protocols. Clonal populations were obtained by culturing the transfected cells in DMEM containing 10% FBS, 1 mg/mL geneticin, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. Individual clones were isolated by ring-cloning. To quantitatively assess Wnt pathway activation in the Wnt-LIGHT cells clones, the cells were then cultured in 48-well plates using DMEM containing 10% FBS, 400 μg/mL geneticin, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. After the cells reached confluency, they were grown for another 24 h with either fresh culture medium or Wnt3a-conditioned medium. Through this process, a clone exhibiting maximum Wnt3a responsiveness and minimum basal pathway activation was identified. To determine whether the Hh pathway inhibitors affect Wnt signaling in the Wnt-LIGHT cells, the cells were cultured to 70-80% confluence in a 15-cm dish, trypsinized, and then resuspended in 40 mL of DMEM containing 10% FBS, 100 U/mL penicillin, and 0.1 mg/mL DMSO. This cell suspension was then aliquoted into 96-well plates (150 μL/well) and cultured overnight. The following day, the growth medium was replaced with Wnt3a-conditioned medium containing either DMSO or various concentrations of the HPIs. The cells were cultured further for 24-28 h, after which their firefly and Renilla luciferase activities were measured using a dual luciferase kit and Veritas microplate luminometer. Example 10 Suffu)^''^' fibroblasts assay for Hh pathway activation: Su(fu)^~'^~ fibroblasts were seeded into 24-well plates (60,000 cells/well) and cultured in DMEM containing 10% FBS, 10 μg/mL gentamicin, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. After 24 h, the cells were transfected with Fugene HD (Roche), 8xGliBS-FL (95 ng/well), phRLSV40 (5 ng/well; Promega), and pEGFP-C1 (300 ng/well as a carrier; Clontech) according to the manufacturer's protocols. The cells were then treated with DMSO, 15 μM HPI-1 , 20 μM HPI- 2, 30 μM HPI-3, or 30 μM HPI-4 for 24 h. The resulting firefly and Renilla luciferase activities were measured using a dual luciferase kit and Veritas microplate luminometer. Example 11 Hh pathway activation mediated by the over-expression of GIi, GH2, GH2αPKA, or GH2αGSK: NIH-3T3 cells were seeded into 24-well plates (35,000 cells/well) and cultured in DMEM containing 10% CS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. The cells were co- transfected the following day with 220 ng/well pcDNA-derived GIiI ₁ Gli2, or Gli2 phosphosite mutant expression vectors and 80 ng/well of a 1 :15 mixture of phRLTK (Promega) and 8XGIiBS-FL. After transfection, cells were grown to confluence (approximately 48 h). The cell were then incubated for an additional 28-32 h in DMEM containing 0.5% CS, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and either DMSO, 50 μM forskolin, 50 μM LY294002, or various concentrations of the HPIs. Firefly and Renilla luciferase activities were measured using a dual luciferase kit and a Veritas microplate luminometer. Example 12 Synthesis of HPI: Synthesis of the HPI having the formula I:

I was as follows (and schematically shown in Fig. 5):

A solution of the 4,4,4-trifluoro-1-(thiophen-2-yl)butane-1 ,3-dione derivative (1) and 1.1 molar equivalents of hydroxylamine hydrochloride in MeOH was refluxed for 2-4 h. The solvent was removed in vacuo, and the residue was dissolved in CHCI₃, washed with water, and dried over Na₂SO₄. Solvent evaporation then yielded the desired isoxazole. The 3-(thiophen-2-yl)-5-(trifluoromethyl)isoxazole derivative (3) was added to a pre- prepared solution of SO₂CI₂ (1.3 molar equivalents) in ice-cooled DMF. The resulting mixture was stirred at 95-98 ⁰C for 1 h, and the reaction was cooled, poured into ice water, and extracted with CHCI₃. The organic layer was subsequently washed with water, 5% sodium bicarbonate, and dried over sodium sulfate. Solvent evaporation and silica gel chromatography then yielded the thiophenesulfonyl chloride (5).

The 5-(5-(trifluoromethyl)isoxazol-3-yl)thiophene-2-sulfonyl chloride derivative (5)was dissolved in THF and treated with the desired amine (6) (1.1 molar equivalents). After the reaction was stirred for 1 h at room temperature, the solvent was removed in vacuo. The residue was dissolved in CHCI₃, washed with 5% citric acid, washed with 5% sodium bicarbonate and dried over sodium sulfate. Solvent evaporation and silica gel chromatography then yielded the sulfonamide (7) having the formula I (Fig. 4) as the final product. Example 13 One embodiment of the disclosure is a compound having the formula I as shown in

Fig. 4, and having the NMR according to Fig. 6. The synthetic pathway for this compound is as shown in Fig. 5.

Claims

CLAIMSWhat is claimed is:

1. A compound having the formula A:

or a pharmaceutically acceptable salt thereof, wherein:

R_{1 1}R₂, and R₃ are each independently H, an acyl, an alkanoyl, an alkyl, an unsaturated alkyl. a heteroalkyl, an amino group, an amine group, an aryl, a substituted aryl group, an alkoxy, an alkylamino, an alkylthio (or thioalkoxy), a heteroalkyl, a heteroalkylene, a cycloalkyl, an aryl group, a heteroaryl group, or an arylalkyl, or a substituted or a unsubstituted form thereof, or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1 , wherein the compound has the formula I:

or a pharmaceutically acceptable salt thereof.

3. A pharmaceutical composition comprising a compound having the formula A:

or a pharmaceutically acceptable salt thereof, wherein: Ri ,R₂, and R₃ are each independently H, an acyl, an alkanoyl, an alkyl, an unsaturated alkyl. a heteroalkyl, an amino group, an amine group, an aryl, a substituted aryl group, an alkoxy, an alkylamino, an alkylthio (or thioalkoxy), a heteroalkyl, a heteroalkylene, a cycloalkyl, an aryl group, a heteroaryl group, or an arylalkyl, or a substituted or a unsubstituted form thereof, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.

4. The pharmaceutical composition of claim 3, wherein the compound has formula I:

I or a pharmaceutically acceptable salt thereof.

5. Use of a compound having the formula A:

or a pharmaceutically acceptable salt thereof, in the manufacture of the pharmaceutical composition for the treatment of a cell or tissue of a subject human or animal having a cell proliferative disorder, where the pharmaceutical composition modulates cell proliferation by modulating the Hedgehog pathway signaling, wherein:

Ri ,R₂, and R₃ are each independently H, an acyl, an alkanoyl, an alkyl, an unsaturated alkyl. a heteroalkyl, an amino group, an amine group, an aryl, a substituted aryl group, an alkoxy, an alkylamino, an alkylthio (or thioalkoxy), a heteroalkyl, a heteroalkylene, a cycloalkyl, an aryl group, a heteroaryl group, or an arylalkyl, or a substituted or a unsubstituted form thereof, or a pharmaceutically acceptable salt thereof.

6. The use of the compound of claim 5, wherein the compound has the formula I:

I or a pharmaceutically acceptable salt thereof.

7. A method of reducing Hedgehog pathway signaling in a cell, the method comprising:

(i) providing a target cell or population of target cells; (ii) contacting the cell with the compound as set forth in claim 1 in an amount effective to reduce Hedgehog signaling in the cell, thereby reducing Hedgehog pathway signaling in the target cell or population of target cells.

8. The method of claim 7, wherein the Hedgehog pathway signaling is associated with cell proliferation, and wherein reducing the Hedgehog pathway signaling in the target cell or population of target cells reduces the proliferation of the target cell or population of target cells.

9. The method of claim 7, wherein the target cell or population of target cells is present in a subject animal or human.

10. The method of claim 7, wherein the target cell or population of target cells are associated with a Hedgehog-related disorder of the subject animal or human.

11. The method of claim 7, wherein the Hedgehog-related disorder of the subject animal or human is a tumor.

12. The method of claim 7, wherein the Hedgehog-related disorder of the subject animal or human is a disorder of a tissue development.

13. The method of claim 7, wherein the inhibition of the hedgehog pathway signaling in the cell induces apoptosis of the cell.

14. The method of claim 7, wherein the target cell or population of target cells is a cultured cell.

15. The method of claim 7, wherein the compound is in a pharmaceutically acceptable composition.

16. The method of Claim 15, wherein the pharmaceutically acceptable composition further comprises a pharmaceutically acceptable carrier.

17. The method of claim 15, further comprising administering to the subject animal or human an effective amount of a second therapeutic agent.

18. A kit comprising a container and a dose or plurality of doses of the compound having the formula A:

or a pharmaceutically acceptable salt thereof, wherein:

R₁₁R₂, and R₃ are each independently H, an acyl, an alkanoyl, an alkyl, an unsaturated alkyl. a heteroalkyl, an amino group, an amine group, an aryl, a substituted aryl group, an alkoxy, an alkylamino, an alkylthio (or thioalkoxy), a heteroalkyl, a heteroalkylene, a cycloalkyl, an aryl group, a heteroaryl group, or an arylalkyl, or a substituted or a unsubstituted form thereof, or a pharmaceutically acceptable salt thereof, , and a package insert describing the use of said compound, or a pharmaceutical composition comprising said compound, in treating a pathological condition of interest in a subject animal or human.

19. The kit of claim 18, wherein the the compound has the formula I

I or a pharmaceutically acceptable salt thereof.