WO2016040951A1

WO2016040951A1 - Compounds and methods for inhibition of hedgehog signaling and phosphodiesterase

Info

Publication number: WO2016040951A1
Application number: PCT/US2015/050024
Authority: WO
Inventors: Charles C. Hong; Charles H. Williams; Jonathan HEMPEL; Tk Feaster; Audrey FRIST; Don H. RUBIN; Gary SULIKOWSKI; Jijun Hao
Original assignee: Vanderbilt University
Current assignee: Vanderbilt University
Priority date: 2014-09-12
Filing date: 2015-09-14
Publication date: 2016-03-17
Anticipated expiration: 2017-03-12

Abstract

Compounds and compositions, and methods of use thereof, are provided and have utility in inhibiting hedgehog signaling and/or phosphodiesterase-4 activity.

Description

COMPOUNDS AND METHODS FOR INHIBITION OF

HEDGEHOG SIGNALING AND PHOSPHODIESTERASE

by

Charles C. Hong, of 1400 Wolf Creek Drive, Nolensville, TN, a U.S. citizen;

Charles H. Williams, of 2116 Farley Place, Nashville, TN, a U.S. citizen;

Jonathan Hempel, of 2310 Elliott Avenue #209, Nashville, TN, a U.S. Citizen TK Feaster, of 2602 Franklin Pike #118, Nashville TN 37204, a U.S. Citizen;

Don H. Rubin, of 112 Harding Hill Lane, Nashville, TN, a U.S. Citizen;

Gary Sulikowski, of 804 Turnbridge Drive, Brentwood, TN, a U.S. Citizen;

Jijun Hao, of 1122 Cleghorn Dr. Unit B, Biamond Bar, CA, a citizen of China; and Audrey Frist, of 105 Copperas Court, Murfreesboro, TN, a U.S. citizen. Assignee: Vanderbilt University

Attorney Docket No.: 11672N/0939WO RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Application Serial No. 62/049,735 filed September 12, 2014, and U.S. Provisional Application Serial No. 62/199,442 filed July 31, 2015, the entire disclosures of which are incorporated herein by this reference. GOVERNMENT INTEREST

[0001] This invention was made with government support under RO1HL104040 awarded by National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD

[0002] The presently-disclosed subject matter relates to compounds, compositions, and methods for inhibiting Hedgehog signaling. The presently-disclosed subject matter further relates to compounds, compositions, and methods for inhibiting

phosphodiesterase 4. INTRODUCTION [0003] Hedgehog (Hh) signaling is one of the key regulators of both invertebrate and vertebrate development. During development, Hh signaling regulates a wide variety of processes, including patterning of body segments, organs, and appendages;

chondrogenesis; myotome induction; and floor plate differentiation. In adult animals, Hh signaling regulates the survival of a variety of differentiated cell types, the proliferation of variety of adult stem cells, and the development of hair follicles.

[0004] In these various developmental processes, members of the Hh family of extracellular signaling molecules activate a membrane receptor complex. Initially, the binding of Hh to the transmembrane receptor Patched (Ptc) releases its inhibition of Smoothened (Smo), a distant cousin of the 7-transmembrane G-couple protein receptor family. The activation of Smo by Hh then initiates an intracellular signaling pathway that ultimately results in activation of Gli zinc-finger transcription factors, which are thought to mediate much of the cellular effects of Hh signaling.

[0005] In most subjects, the Hh signaling pathway is normally tightly regulated, becoming activated only in precise locations and at precise times. However, in other subjects, the aberrant activation of the Hh signaling pathway is associated with numerous types of malignancies, including basal cell carcinomas, medulloblastomas, melanomas, fibrosarcomas, rhabdomyosarcomas, glioblastomas, multiple myelomas and pancreatic cancers. Indeed, Hh signaling has been observed to promote tumorigenesis through both cell-autonomous and paracrine effects, and there is increasing recognition that Hh may play a key role in transforming adult stem cells into tumor stem cells and in maintaining tumor cell compartments. Consequently, in recent years, significant efforts have been spent developing small molecule inhibitors of the Hh pathway that are capable of being used in the treatment of cancer.

[0006] Despite the recent efforts, however, the large majority of Hedgehog signaling inhibitors target Smo and are subject to significant inhibitor-driven resistance

mechanisms. Additionally, a large proportion of driver mutations of tumorigenesis occur at signaling nodes downstream of Smo, for which Smo antagonists are not predicted to show efficacy. Therefore, the development of Hedgehog signaling inhibitors that function downstream of Smo would be of great significance to the clinical areas of Hedgehog-driven malignancies. SUMMARY [0007] The presently-disclosed subject matter meets some or all of the above- identified needs, as will become evident to those of ordinary skill in the art after a study of information provided in this document.

[0008] This Summary describes several embodiments of the presently-disclosed subject matter, and in many cases lists variations and permutations of these

embodiments. This Summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently-disclosed subject matter, whether listed in this Summary or not. To avoid excessive repetition, this Summary does not list or suggest all possible combinations of such features.

[0009] The presently-disclosed subject matter includes a compound. In some embodiments, the compound is of the formula:

or pharmaceutically-acceptable salts thereof, wherein

X is selected from C, N, O, and S; R₁ is selected from CH₂CH_3, (CH₂)₂CH₃,

so long as when R₂ is

R₁ is not

and so long as when R₂ is

, X is C, and R3 is H, R₁ is not ,

so long as when R2 is

X is C and R3 is H, R1 is not

, and

so long as when R2 is

, X is C and R3 is H, R1 is not

[0010] In some embodiments, the compound is according to a formula selected from the group consisting of:

, , , and or pharmaceutically-acceptable salts thereof.

[0011] In some embodiments, the compound is according to a formula selected from the group consisting of:

, ,

or pharmaceutically-acceptable salts thereof.

[0012] In some embodiments, the compound is according to the formula:

or pharmaceutically-acceptable salts thereof.

[0013] In some embodiments, the compound is according to the formula::

or pharmaceutically-acceptable salts thereof.

[0014] In some embodiments, the compound is according to the formula:

or pharmaceutically-acceptable salts thereof.

[0015] In some embodiments, the compound is according to the formula:

or pharmaceutically-acceptable salts thereof. [0016] In some embodiments, the compound is according to the formula:

, or pharmaceutically-acceptable salts thereof.

[0017] In some embodiments, the compound is according to the formula:

or pharmaceutically-acceptable salts thereof.

[0018] In some embodiments, the compound is according to the formula:

, or pharmaceutically-acceptable salts thereof.

[0019] In some embodiments, the compound is according to the formula:

, or pharmaceutically-acceptable salts thereof.

[0020] In some embodiments, the compound is according to the formula: or pharmaceutically-acceptable salts thereof.

[0021] In some embodiments, the compound is according to the formula:

, or pharmaceutically-acceptable salts thereof. [0022] In some embodiments, the compound is of the formula: or pharmaceutically-acceptable salts

thereof, wherein

R₄ is selected from

R₅ is selected from CH₃

, a d ; a d

R₆ is selected from H,

[0023] In some embodiments, the compound is of the formula:

pharmaceutically-acceptable salts thereof, wherein

R₇ is selected from

[0024] In some embodiments, the compound is of the formula:

,

, or a pharmaceutically-acceptable salt thereof. [0025] In some embodiments, the compound is of the

formula:

wherein

R₁ is selected from H, and

and R₂ is selected from

.

[0026] In some embodiments, the compound is of the formula:

R₁ is selected from

[0027] In some embodiments, the compound according to the formula selected from the group consisting of:

,

.

[0028] The presently-disclosed subject matter further includes a pharmaceutical composition. In some embodiments, the pharmaceutical composition includes a pharmaceutically-acceptable carrier; and a compound as disclosed herein.

[0029] In some embodiments, the pharmaceutical composition further includes a second compound or composition having Hh signaling inhibition activity, PDE4 inhibition activity, anti-cancer or anti-tumor activity, anti-angiogenic activity, anti- metastatic activity, anti-heart failure activity, and/or anti-inflammation activity, or wherein the second compound or composition is useful for treating a condition of interest.

[0030] The presently-disclosed subject matter further includes a kit that comprises a compound or a pharmaceutical composition, as described herein, and a device for administration of the compound or composition. The presently-disclosed subject matter further provides a kit that comprises a compound or a pharmaceutical composition, as disclosed herein; and further comprising a second compound or composition having Hh signaling inhibition activity, PDE4 inhibition activity, anti-cancer or anti-tumor activity, anti-angiogenic activity, anti-metastatic activity, anti-heart failure activity, and/or anti- inflammation activity, or wherein the second compound or composition is useful for treating a condition of interest.

[0031] In some embodiments, the kit further comprises a second compound or composition and a device for administration of the compound or composition and/or a device for administration of the second compound or composition. [0032] The presently-disclosed subject matter further includes methods. A method of inhibiting hedgehog signaling in a cell is provided and includes contacting a cell with an effective amount of a compound or pharmaceutical composition, as disclosed herein. In some embodiments, contacting the cell with the compound comprises administering the compound or composition to a subject.

[0033] In some embodiments, the administration is to a subject in need of treatment for a condition of interest. In some embodiments the condition of interest is related to heart failure. In other embodiments, the condition of interest is related to PDE4 activity, cancer, angiogenesis, tumorigenisis or tumor activity, metastasis and/or inflammation.

[0034] A method of inhibiting phosphodiesterase-4 (PDE-4) in a cell is provided and includes contacting a cell with an effective amount of a compound or pharmaceutical composition, as disclosed herein. In some embodiments, contacting the cell with the compound comprises administering the compound or composition to a subject. In some embodiments, administration is to a subject in need of treatment for a condition of interest.

[0035] A method of treating a condition of interest is provided and includes contacting a cell with an effective amount of a compound or pharmaceutical

composition, as disclosed herein. In some embodiments, contacting the cell with the compound comprises administering the compound or composition to a subject. In some embodiments, the administration is to a subject in need of treatment for a condition of interest.

BRIEF DESCRIPTION OF THE DRAWINGS [0036] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are used, and the accompanying drawings of which:

[0037] Figure 1 includes data and results of studies showing that Eggmanone inhibits Hedgehog signaling via inhibition of PDE4. Zebrafish embryos treated with 2 ^M Eggmanone (Egm) starting at 4-hours post fertilization (hpf) exhibited range of phenotypes found in Hh pathway mutants, including ventral tail curvature, loss of pectoral fins (Fig.1a), smaller eyes and (Fig.1d) enlarged somites in place of normal chevron-shaped somites. Egm treatment abolished Hh-responsive ptch1 expression in adaxial cells at 12-hpf Fig.1(b; arrow), in the pectoral fin bud at 48-hpf (Fig.1c;

arrow). Egm inhibited Sonic hedgehog (SHH)-responsive Gli-luciferase reporter activity (Fig.1e) and Purmorphamine (Purm, 3 ^M)-induced reporter activity Fig.1(f) (n=4 for each condition, RLU, relative luciferase units, +/- standard error; P-value <0.0184 starting at 1 ^M for I; P-value <0.0054 starting at 0.5 ^M for J. Cyclopamine (Cyc) 5uM). (Fig.1g) Left, Eggmanone inhibited PDE4 isoforms with the IC₅₀ range of 0.8 to 3.75 ^M, except the super-short PDE4D2. Right, representation of PDE4 isoform structures. (Fig.1h) Overexpression of wild-type PDE4D3 (D3WT) induced Hh reporter activity (*P=0.0026 versus pCS2 control), which was abolished by 5 ^M

Eggmanone (p<0.0001 versus D3WT). Overexpression of dominant-negative PDE4D3 (D3DN) decreased Hh reporter activity (**P=0.0121 versus pCS2).

[0038] Figure 2 includes data and results of studies showing that Eggmanone causes local perturbations in cAMP levels resulting in PKA activation restricted to the basal bodies. (Fig.2a) Rolipram increased total cellular cAMP levels, whereas Egm only caused small increase at concentrations above those required to inhibit Hh signaling. (Fig.2b) Left, still images from high-speed video of zebrafish otic kino-cilia. Middle, kymograph visualization demonstrates that cilia movement is markedly reduced following 2 ^M Egm treatment. Right, schematic of motile kino-cilia (green, line of capture for kymograph). (Fig.2c) Immunostaining for the basal body marker gamma- Tubulin (green) and the autophosphorylated PKA catalytic subunit (Phospho^Y197-PKA- C; red) in NIH3T3 cells stimulated with SAG (top) demonstrates a low baseline PKA activation; co-treatment with 5 ^M Egm (middle) increases local PKA activation at the basal body and in areas immediately surrounding it; co-treatment with 10 ^M Rolipram increases PKA activation more diffusely. (Fig.2d) Intensity plot of immunostaining along a line bisecting the basal body and nucleus. (Fig.2e) Correlation plot of p-PKA and gamma-Tubulin staining intensities.

[0039] Figure 3 includes data and results of studies showing that Eggmanone causes dysregulation of cilia-to-nuclear trafficking of Gli2 and selectively kills Hh-dependent cells. (Fig.3a) Immunostaining for the cilia marker Arl13b (green) and Gli2 (red) of NIH3T3 cells stimulated with SAG (20nM) in the presence of 5 ^M Egm or DMSO control. Egm treatment increased co-localization of Gli2 (yellow) in the primary cilia, arrows. (Fig.3b) Quantitative analysis reveals that Egm significantly increased Gli2 localization in the cilia (n=10 for each condition; p=0.026, versus DMSO). (Fig.3c) Representative western blot for Gli2 in nuclear fractions of NIH3T3 cells. Neg, unstimulated. SAG, stimulated with SAG (20nM) for 60 minutes. SAG+FSK, co-treated with SAG and FSK (30 ^M). SAG+EGM, co-treated with SAG and Egm (10 ^M).

Bottom, corresponding western blot for nuclear Lamin-A/C as loading controls. FL, full-length, active form of Gli2. R, proteolytically processed, repressor form of Gli2. (Fig.3d) Quantitative analysis of the ratio of full length Gli2 to lamin-A in the nucleus reveals that SAG treatment increased abundance of full-length Gli2 in the nucleus, and this increase was abrogated by co-treatment with either FSK or Egm. (Fig.3e) SAG treatment increased the nuclear ratio of full-length Gli2 (FL) to repressor Gli2 (R), which was abrogated by co-treatment with either FSK or Egm (For D and E, n=4 for each condition; p<0.05, versus SAG; ratio for each condition was normalized to the ratio of unstimulated controls). (Fig.3f) Egm treatment (10 ^M) led to rapid (within 24-hrs) decline in viability of SmoM2 cells but not NIH3T3 cells (n=3 for each data point; cell viability relative to DMSO-treated cells; *P<0.0001; **P=0.0021) (Fig.3g) Relative cell viability of Daoy (medulloblastoma), RKO (colon cancer) and PC3 (prostate cancer) cells following 72-hour treatment with increasing concentrations of Egm (n=4, for each data point). Egm (10 ^M) treatment of Daoy cells for 48-hours decreased cell proliferation, based on phospho-histone H3 (PH3) staining (Fig.3h) and increased apoptosis, based on TUNEL staining (Fig.3i).

[0040] Figure 4 includes the structure of Eggmanone identified in zebrafish-based screen for compounds that phenocopy hedgehog pathway mutants. Left, eggmanone, with IC50s for inhibition of Hh reporter activity and for PDE4D3 inhibition.

[0041] Figure 5 includes a mass spectrometry analysis of Eggmanone.

[0042] Figure 6 includes an NMR spectra analysis of eggmanone. ¹H NMR (600 MHz, CDCl₃): ^ 7.94 (dd, J = 3.8, 1.0 Hz, 1H), 7.73 (dd, J = 5.0, 1.0 Hz, 1H), 7.20 (dd, J = 5.0, 3.9 Hz, 1H), 4.92 (s, 1H), 4.70 (s, 2H), 4.64 (s, 1H), 4.57 (s, 2H), 2.96 (t, J = 6.0 Hz, 2H), 2.69 (t, J = 6.0 Hz, 2H), 1.83 (s, 3H), 1.83 (m, 4H); LCMS, single peak, 1.42 min, m/e = 416.8 [M+1].

[0043] Figure 7 includes data and results of studies showing that Eggmanone does not recapitulate all hedgehog signaling defects. Zebrafish embryos treated with 2 ^M Eggmanone (Egm) starting at 4-hours post fertilization (hpf) (Fig.7a) smaller eyes, (Fig. 7b) defects in neurocranium chondrogenesis. Egm treatment abolished Hh-responsive ptch1 expression in somites at 24-hpf (Fig.7c;*). Egm did not abolish ptch1 expression in myotome cells (Fig.7c; arrow) and in ventral neural tube (Fig.7c; arrowhead) nor abolish nkx2.2:eGFP expression (Fig.7d; arrowhead)

[0044] Figure 8 includes data and results of studies showing that Eggmanone affects hedgehog signaling but does not affect BMP signaling. (Fig.8a) Eggmanone

significantly inhibited ptch1 expression in response to purmorphamine in Nih3T3 fibroblasts. (Fig.8b) Eggmanone had no significant effects on BMP4-responsive reporter (BRE-luc) activity in C2C12BRA reporter cells⁷. BRE-luc (BMP responsive element driven luciferase) cells were stimulated with BMP4 ligand. Eggmanone had no agonist or antagonist activity.

[0045] Figure 9 includes results from LASSO algorithm. Top, Molecular surface descriptor of eggmanone, with search algorithm results. Bottom, Results of search.

[0046] Figure 10 includes data and results of studies showing Eggmanone’s ability to inhibit different isoforms of PDE4. (Fig.10a) In vitro PDE activity assays across 11 PDE families reveals that Egm (10 ^M) significantly inhibited only the PDE4 class. (Fig.10b) Dose response curves of in vitro PDE assays.

[0047] Figure 11 includes data and results of studies showing that Eggmanone does not disrupt PDE4D3 localization to the peri-ciliary region at the base of the primary cilium. (Fig.11a) Left, vsv-tagged PDE4D3 (green). Middle, Arl13b immunostaining marks the primary cilium (red). Right, merged images. (Fig.11b) NIH3T3 cells transfected with either VSV-PDE4D3 vector or empty vector control were treated with either DMSO or 5uM eggmanone. Lysates were incubated with anti-AKAP450 antibody and complexes bound to Protein A/G beads. After immunoprecipitation, western blot probed with anti-VSV antibody demonstrated physical interaction between AKAP450 and PDE4D3. There is no difference between control and eggmanone treated cells.

[0048] Figure 12 includes data and results of studies showing that Eggmanone increases activation of cAMP-dependent protein kinase (PKA) at the cilium base, but not globally. (Fig.12a) Immunostaining for the cilia marker Arl13b (green) and the autophosphorylated form of the PKA catalytic subunit (red) shows Egm treatment increases local PKA activation at the base of the primary cilia, corresponding to the basal body (n=16 for each condition, p=0.00014, versus SAG alone). (Fig.12b)

Quantitative analysis of (Fig.12a); Immmunostaining of autophosphorylated (Thr-197) form of the PKA catalytic subunit (red) costained with cilia specific Arl13b (green) show that eggmanone treatment increases levels of phospho-PKA only in the periciliary domain, but not the cilium. (Fig.12c) Immunostaining for the basal body marker gamma-Tubulin (green) and the autophosphorylated form of the PKA catalytic subunit (Phospho^Y197-PKA-C; red) in NIH3T3 cells stimulated with Hh pathway activator SAG demonstrates that co-treatment with Egm (5 ^M) treatment increases local PKA activation in the basal body (yellow, merged). (Fig.12d) Quantitative analysis of autophosphorylated PKA reveals that Egm treatment significantly increased PKA activation in the basal body (n=10 for each condition; p<0.05, versus SAG alone). (Fig. 12e) Correlation coefficients from studies in (Fig.12c). (Fig.12f) Graphic comparison of correlation coefficients found in Fig 6e.

[0049] Figure 13 depicts a model for Eggmanone mechanism of action. Left, in the absence of Hh ligand (SHH), Gli transiently enters and subsequently exits the primary cilia without getting activated. A proportion of Gli is proteolytically cleaved into the repressor form (Gli-R), which translocates to the nucleus to repress Hh target gene transcription. Middle, in the presence of Hh ligand (SHH), Gli becomes activated in the cilium by a still uncharacterized modification, then translocates to the nucleus as the full- length activator (Gli-activ.) to activate Hh target gene transcription. PDE4, which is localized to the basal body along with AKAP and PKA, functions as a“barrier” to isolate the primary cilium from the cAMP fluctuations occurring in the rest of the cell and serves to prevent aberrant PKA activation. Right, eggmanone (Egm) treatment selectively targets PDE4 isoforms localized to the basal body, leading to local elevations in the cAMP levels in the peri-ciliary microdomain and to local PKA activation. This in turn impedes Gli-activ. from translocating to the nucleus, resulting in down regulation of Hh signaling.

[0050] Figure 14 is a graph showing anticancer effect of Eggmanone on various cancer cell lines.

[0051] Figure 15 includes results of a BVDV (Bovine Viral Diarrhea Virus, surrogate for Hepatitis C virus) CPE (cytotoxic effect) Assay with Eggmanone, where the compound was tested in half-log concentrations, and the data for the highest 3 concentrations is normalized to the respective DMSO concentrations.

[0052] Figure 16 includes the results of a plaque assay of respiratory syncytial virus (RSV), where 10 ^M Eggmanone was added to cells 1 hour prior to the assay in serial 10 fold dilutions with each dilution performed in triplicate (shown), where the three columns to the left contained vehicle (DMSO) without drug, the three columns to the left are treated with drug, and dilutions are most concentrated in the uppermost wells and serially decrease through the rows.

[0053] Figure 17 includes data and images showing that Eggmanone specifically inhibits Hedgehog signaling. Zebrafish embryos treated with 2 ^M EGM (Egm) starting at 4-hours post fertilization (hpf) exhibited range of phenotypes found in Hh pathway mutants, including ventral tail curvature, loss of pectoral fins (Fig.17a), smaller eyes and when treated at 10hpf (Fig.17b) enlarged somites in place of normal chevron- shaped somites. Egm treatment abolished Hh-responsive ptch1 expression in adaxial cells at 12-hpf (Fig.17c; arrow), and in the pectoral fin bud at 48-hpf (Fig.17d;

arrow). (Fig.17e) Egm inhibited Sonic hedgehog (SHH)-responsive Gli-luciferase (Gli-Luc) reporter activity in a dose-dependent manner. Cyclopamine (Cyc) 5uM for comparison (n=4 for each condition, results represented as mean RLU, relative luciferase units, +/- standard error; P-value <0.0184, starting at 1 ^M). (Fig.17f) Egm inhibited purmorphamine (Purm, 3 ^M)-induced Gli-Luc reporter activity in a dose-dependent manner. (n=4; P-value <0.0054, starting at 0.5 ^M). (Fig.17g) Egm significantly inhibited ptch1 expression in response to purmorphamine in NIH3T3 fibroblasts (n=3 for each condition, expression normalized to GAPDH, P-value <0.003, starting at 1 ^M) (Fig.17h) Egm had no significant effects on BMP4-responsive reporter (BRE-luc) activity in C2C12BRA reporter cells. BRE-luc (BMP responsive element driven luciferase) cells were stimulated with BMP4 ligand. (Fig.17i) Egm had no significant effect on Gli-luciferase reporter activity under Gli2 overexpression conditions.

[0054] Figure 18 includes data and images showing that Eggmanone is a selective PDE4 inhibitor. (Fig.18a) In vitro PDE activity assays across 11 PDE families reveal that Egm (10 and 50 ^M) significantly inhibited only the PDE4 class (bold faced, highlighted). (Fig.18b) Dose response curve for Egm inhibition of indicated PDE isoforms on in vitro assays. (Fig.18c) Left, EGM inhibited PDE4 isoforms with the IC₅₀ range of 0.8 to 73.46 ^M. Right, representation of PDE4 isoform structures. (Fig.18d) Double reciprocal (Lineweaver-burke) plot indicates a competitive mode of inhibition.

[0055] Figure 19 includes a chart of a PDE 4D3 enzyme linearity study showing that inhibition of PDE4 with Egm occurs in a linear manner.

[0056] Figure 20 includes a Eadie Hofstee plot showing that Egm acts in a competitive manner. [0057] Figure 21 includes a graph showing Km versus Egm concentration, wherein the linear relationship suggests that Egm acts in a competitive manner.

[0058] Figure 22 includes data and showing that Hh inhibition requires PDE4 antagonism. (Fig.22a) Results of Hh signaling reporter assays, and of PDE4D3 activity assay for eggmanone (EGM) and 12 analogs. A compound’s ability to antagonize PDE4 correlates with it’s ability to inhibit Hh signaling. (Fig.22b) Overexpression of wildtype PDE4D3 (D3WT) induced Hh reporter activity (*P=0.0026 versus pCS2 control), which was abolished by 5 uM EGM (p<0.0001 versus D3WT). Overexpression of dominant negative PDE4D3 (D3DN) decreased Hh reporter activity (** P=0.0121 versus pCS2)

[0059] Figure 23 includes graphs showing the effects of known PDE4 inhibitors rolipram and D159153 on Hedgehog signaling. (Fig.23a) The competitive PDE4 inhibitor rolipram (beige bars) inhibited Sonic hedgehog (SHH)-responsive Gli- luciferase (Gli-Luc) reporter activity, but, unlike eggmanone (Egm, blue bars), rolipram did not bring the reporter activity down to the baseline even at very high concentrations. (Fig.23b) The allosteric PDE4 inhibitor D159153 (beige bars) did not inhibit Sonic hedgehog (SHH)-responsive Gli-luciferase (Gli-Luc) reporter activity even at very high concentrations (n=3 for each condition, results represented as mean RLU, relative luciferase units, +/- standard error).

[0060] Figure 24 includes data and graphs showing that Eggmanone causes local perturbations in cAMP levels without affecting global cellular cAMP content. (Fig.24a) Eggmanone (EGM) treatment had no effect on total cellular cAMP content in NIH 3T3 cells, and the competitive PDE4 inhibitor rolipram and the allosteric PDE4 inhibitor D159153 substantially and moderately increased total cellular cAMP levels,

respectively. (Fig.24b) Left, still images from high-speed video of zebrafish otic kino- cilium. Middle, kymograph visualization demonstrates that cilium movement is markedly reduced following 2 ^M EGM treatment. Right, schematic of motile kino- cilium (green, line of capture for kymograph). (Fig.24c) Top, NIH3T3 cells expressing mTurquoiseΔ-Epac(CD, ΔDEP)-cp173 Venus-Venus; Bottom, normalized mean kinetics of FRET change detected in response to 5µM Rolipram or 5µM EGM (n = 3). (Fig. 24d) Top, NIH3T3 cells expressing PKAC-YFP and PKARII-CFP; Bottom, normalized mean kinetics of FRET change detected in response to 5µM EGM (n = 2). FRET values are the mean calculated within an ROI drawn to include the entire cytosolic area or the centrosome.

[0061] Figure 25 includes images and graphs showing that Eggmanone (EGM) treatment results in PKA activation restricted to the basal bodies. (Fig.25a)

Immunostaining for the cilia marker Arl13b (green) and the autophosphorylated form of the PKA catalytic subunit (Phospho^Y197-PKA-C; red) in NIH3T3 cells stimulated with the Smo agonist SAG (left) demonstrates a low baseline PKA activation; co-treatment with 5 ^M EGM (left) increases local PKA activation at the base of the primary cilia (n=16 for each condition, p=0.00014, versus SAG alone). (Fig.25b) Quantitative analysis of (Fig.25a). (Fig.25c) Immunostaining for the basal body marker ^-Tubulin (green) and the autophosphorylated PKA catalytic subunit (Phospho^Y197-PKA-C; red) in NIH3T3 cells stimulated with SAG demonstrates that co-treatment with EGM (5 ^M) dramatically increases PKA activation in the basal body (n=10 for each condition;

p<0.05, versus SAG alone). (Fig.25d) Quantitative analysis of (Fig.25c).

[0062] Figure 26 includes images showing that allosteric PDE4 inhibitor D159153 and cAMP analog dibutyril cAMP (DBA) induce spatially localized PKA activation in the basal body.

[0063] Figure 27 includes images and graphs showing that Eggmanone (EGM) causes selective dysregulation of Gli trafficking. (Fig.25a) Immunostaining for the cilium marker Arl13b (green) and Gli2 (red) of NIH3T3 cells stimulated with SAG (20nM) in the presence of 5 ^M EGM or DMSO control. EGM treatment increased co- localization of Gli2 (yellow) in the primary cilium, arrows. (Fig.25b) Quantitative analysis reveals that EGM significantly increased Gli2 localization in the cilium (n=10 for each condition; p=0.026, versus DMSO). (Fig.25c) Representative western blot for Gli2 in nuclear fractions of NIH3T3 cells. Neg, unstimulated. SAG, stimulated with SAG (20nM) for 60 minutes. SAG+FSK, co-treated with SAG and FSK (30 ^M).

SAG+EGM, co-treated with SAG and EGM (10 ^M). Bottom, corresponding western blot for nuclear Lamin-A/C as loading controls. FL, full-length, active form of Gli2. R, proteolytically processed, repressor form of Gli2. (Fig.25d) Quantitative analysis of the ratio of full length Gli2 to lamin-A in the nucleus reveals that SAG treatment increased abundance of full-length Gli2 in the nucleus, and this increase was abrogated by co- treatment with either FSK or EGM. (Fig.25e) SAG treatment increased the nuclear ratio of full-length Gli2 (FL) to repressor Gli2 (R), which was abrogated by co-treatment with either FSK or EGM (For d and e, n=4 for each condition; p<0.05, versus SAG; ratio for each condition was normalized to the ratio of unstimulated controls). (Fig.25f) Immunostaining for the cilium marker Arl13b (red) and IFT88 (green) of NIH3T3 cells stimulated with SAG (20 nM) in the presence of DMSO control (top), 100 ^M ciliobrevin D (middle), or 5 ^M EGM (bottom). Ciliobrevin D perturbed the localization of IFT88 in the cilium, but EGM did not affect IFT88 localization.

[0064] Figure 28 includes an echocardiogram of a mouse after having been administered 20mg/kg Egm via an intraperitoneal injection.

[0065] Figure 29 includes data of the effects on the heart of a mouse after having been administered lvels of from 5mg/kg to 20mg/kg Egm via an intraperitoneal injection. It includes data that, in both healthy wild type mice and mice with heart failure, EGM increases fractional shortening (FS) and decreases end-diastolic left ventricular internal dimension (LVIDd) without increasing heart rate.

[0066] Figure 30 includes images showing that addition of Egm causes local activation of PKA around PDE4 localization.

[0067] Figure 31 includes a graph showing the concentration of total cAMP levels after administration with DMSO, Rolipram (Rol), and an embodied Egm (HI913).

[0068] Figure 32 includes a graph showing the effects of Egm on the contractibility of isolated mouse cardiomyocytes in comparison to a vehicle control (VEH).

[0069] Figure 33 includes a graph showing calcium handling results from mice that had been administered with VEH, EGM, or ISO.

[0070] Figure 34 includes a graph showing the effects of Egm on the contractibility of human cardiomyocytes derived from induced pluripotent stem cells (iPSCs).

[0071] Figure 35 includes a myograph of a cannulated mouse aorta showing that the addition of Egm results in little to no contraction or dilation of the vessel.

[0072] Figure 36 includes a graph of an ascending aorta myography.

[0073] Figure 37 includes a graph of relative cytotoxic effect in BT cells with Bovine Viral Diarrhea Virus, a surrogate for human hepatitis C virus in the present of H1913.

[0074] Figure 38 includes a schematic of the Hedgehog Signaling Pathway.

[0075] Figure 39 includes data and images from discovery of EGM1 inhibiting Hedgehog signaling from an in vivo zebrafish phenotypic screen. (Fig.39 (a)) includes images of zebrafish embryos treated with EGM1 exhibiting ventral tail curvature and loss of pectoral fins (Fig.39 (b)) Egm treatment abolished Hh-responsive ptc1 expression in adaxial cells, and in the pectoral fin bud (Fig.39 (c); arrow). (Fig.39 (d)) graphs the concentration of EGM1 versus percent Hh activity. (Fig.39 (e)) provides data of the relative percent of mRNA

[0076] Figure 40 Includes Synthesis and Characterization of EGM1 Compounds. (Fig.40 (a)) includes a general reaction scheme for the synthesis and derivitization of EGM1. (Fig.40 (b)) includes the Structure Activity Relationship (SAR) of Outer EGM1 Appendages. (Fig.40 (c)) includes compounds with modifications to the EGM1 Core Scaffold. (Fig.40 (d)) SAR-Informed Analog Evaluations.

[0077] Figure 41 includes results of the mechanism of action validation for several EGM1 compounds. Fig.41(a) charts percent Hh Activity (Pct1) based on administration of EGM1 compounds (4), (22), (23) and (24) as provided in Fig.40. Fig.41(b) includes the percent zebrafish displaying phenotype based on compound concentration. Fig.41 (c) includes images of zebrafish and EC₅₀ based on compounds administered.

[0078] Figure 42 provides a schematic of scaffold hopping via virtual screening. 98,000 compounds were screened against EGM13D hypothesis via the Suflex-Sim algorithm. DESCRIPTION OF EXEMPLARY EMBODIMENTS [0079] The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom. In case of conflict, the specification of this document, including definitions, will control.

[0080] The presently-disclosed subject matter meets some or all of the above- identified needs, as will become evident to those of ordinary skill in the art after a study of information provided in this document. To avoid excessive repetition, this Description does not list or suggest all possible combinations of such features.

[0081] The presently-disclosed subject matter includes compounds, pharmaceutical compositions, kits, and methods for using same. In some embodiments the compounds, pharmaceutical compositions, kits, and methods are useful for inhibiting hedgehog (Hh) signaling and/or inhibiting phosphodiesterase 4.

[0082] Chemical compounds having the structures set forth in Table 1A may be referred to herein with reference to the associated formula numbers, also set forth in Table 1A. Formula (1) is also referred to herein as Eggmanone.

[0083] Compound

[0084] The presently-disclosed subject matter includes a compound having a structure represented by the formula:

, or pharmaceutically-acceptable salts thereof, wherein

R₂ is selected from CH₃,

, ,

, ,

[0085] In some embodiments, the compound has a formula selected from the group set forth in Table 2, or pharmaceutically-acceptable salts thereof.

Formula (16)

[0086] In some embodiments, the compound has a formula selected from the group set forth in Table 1B, or pharmaceutically-acceptable salts thereof.

Formula (55) [0087] In some embodiments, the compound has a formula selected from the group consisting

and

, or pharmaceutically-acceptable salts thereof.

[0088] In some embodiments, the compound has the formula

or pharmaceutically-acceptable salts thereof. In some embodiments, the compound has the formula: or pharmaceutically-acceptable salts thereof. In

some embodiments, the compound has the formula: , or

pharmaceutically-acceptable salts thereof. In some embodiments, the compound has the formula:

or pharmaceutically-acceptable salts thereof. In some embodiments, the compound has the formula: , or pharmaceutically-

acceptable salts thereof. In some embodiments, the compound has the formula:

or pharmaceutically-acceptable salts thereof. In some

embodiments, the compound has the formula:

, or

pharmaceuticall -acce table salts thereof. In some embodiments, the compound has the

formula:

or pharmaceuticall -acce table salts thereof. In some

embodiments, the compound has the formula:

or pharmaceutically- acce table salts thereof. In some embodiments, the compound has the formula:

or pharmaceutically-acceptable salts thereof.

[0089] In yet other embodiments, the compound has a structure of the formula:

or pharmaceutically-acceptable salts thereof, wherein

R₄ is selected from

; ,

[0090] In some embodiments, the compound has a formula selected from the group set forth in Table 1C, or pharmaceutically-acceptable salts thereof.

Formula (70)

[0091] In other embodiments, the compound has a structure of the formula:

pharmaceutically-acceptable salts thereof, wherein

R₇ is selected from

.

[0092] In yet further embodiments, the compound has a formula selected from the group set forth in Table 1D, or pharmaceutically-acceptable salts thereof.

Formula (82) Formula (83)

[0093] In yet further embodiments, the compound has a formula selected from the group set forth in Table 1E, or pharmaceutically-acceptable salts thereof.

Formula (99)

0094 In et further embodiments the com ound has a formula selected from the

thereof.

[0095] In yet further embodiments, the compound has a formula set forth herein, including in the Examples.

[0096] Pharmaceutical Compositions

[0097] The presently-disclosed subject matter further includes pharmaceutical compositions of the compounds as disclosed herein, and further includes a pharmaceutically-acceptable carrier. In this regard, the term “pharmaceutically acceptable carrier” refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose.

[0098] Suitable formulations include aqueous and non-aqueous sterile injection solutions that can contain antioxidants, buffers, bacteriostats, bactericidal antibiotics and solutes that render the formulation isotonic with the bodily fluids of the intended recipient; and aqueous and non-aqueous sterile suspensions, which can include suspending agents and thickening agents.

[0099] The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[00100] The formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a frozen or freeze-dried (lyophilized) condition requiring only the addition of sterile liquid carrier immediately prior to use.

[00101] For oral administration, the compositions can take the form of, for example, tablets or capsules prepared by a conventional technique with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycollate); or wetting agents (e.g., sodium lauryl sulphate). The tablets can be coated by methods known in the art.

[00102] Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional techniques with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g. lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate. Preparations for oral administration can be suitably formulated to give controlled release of the active compound. For buccal administration the compositions can take the form of tablets or lozenges formulated in conventional manner.

[00103] The compounds can also be formulated as a preparation for

implantation or injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).

[00104] The compounds can also be formulated in rectal compositions (e.g., suppositories or retention enemas containing conventional suppository bases such as cocoa butter or other glycerides), creams or lotions, or transdermal patches.

[00105] In some embodiments, the pharmaceutical composition includes a compound as disclosed herein or pharmaceutically-acceptable salts thereof.

[00106] In some embodiments, the pharmaceutical composition includes a the compound of Formula (1), or pharmaceutically-acceptable salts thereof. In some embodiments, the pharmaceutical composition includes a the compound of Formula (3), or pharmaceutically-acceptable salts thereof. In some embodiments, the pharmaceutical composition includes a the compound of Formula (5), or pharmaceutically-acceptable salts thereof. In some embodiments, the pharmaceutical composition includes a the compound of Formula (6), or pharmaceutically-acceptable salts thereof. In some embodiments, the pharmaceutical composition includes a the compound of Formula (7), or pharmaceutically-acceptable salts thereof. In some embodiments, the pharmaceutical composition includes a the compound of Formula (8), or pharmaceutically-acceptable salts thereof. In some embodiments, the pharmaceutical composition includes a the compound of Formula (9), or pharmaceutically-acceptable salts thereof. In some embodiments, the pharmaceutical composition includes a the compound of Formula (11), or pharmaceutically-acceptable salts thereof. In some embodiments, the pharmaceutical composition includes a the compound of Formula (12), or pharmaceutically-acceptable salts thereof. In some embodiments, the pharmaceutical composition includes a the compound of Formula (13), or pharmaceutically-acceptable salts thereof. In some embodiments, the pharmaceutical composition includes a the compound of Formula (15), or pharmaceutically-acceptable salts thereof. In some embodiments, the pharmaceutical composition includes a the compound of Formula (16), or pharmaceutically-acceptable salts thereof. In some embodiments, the pharmaceutical composition includes a the compound of any of Formula (1) to Formula (83). [00107] As disclosed herein, compounds and compositions of the presently- disclosed subject matter are inhibitors of hedgehog signaling and inhibitors of PDE4. Such inhibitors have further utilities as described herein, which include, but are not limited to, anti-cancer or anti-tumor activity, anti-angiogenic activity, anti-metastatic activity, and/or anti-inflammation activity, and utility for treating certain conditions of interest. In this regard, in some embodiments, the pharmaceutical composition can further include a second compound or composition having Hh signaling inhibition activity, PDE4 inhibition activity, anti-cancer or anti-tumor activity, anti-angiogenic activity, anti-metastatic activity, and/or anti-inflammation activity, or wherein the second compound or composition is useful for treating a condition of interest. In some embodiments, the addition of the second compound or composition provides for a synergistic response.

[00108] Kits

[00109] The presently-disclosed subject matter further includes kits, including a compound or pharmaceutical composition. In some embodiments, the kit can include a compound or pharmaceutical composition, as described herein, packaged together with a second compound or composition, a treatment device, and/or an administration device. [00110] In some embodiments, the kit includes a compound, or a

pharmaceutical composition including a compound as disclosed herein.

[00111] In some embodiments, a kit can include a compound or pharmaceutical composition as described herein, packaged together with a device useful for

administration of the compound or composition. As will be recognized by those or ordinary skill in the art, the appropriate administration aiding device will depend on the formulation of the compound or composition that is selected and/or the desired administration site. For example, if the formulation of the compound or composition is appropriate for injection in a subject, the device could be a syringe. For another example, if the desired administration site is cell culture media, the device could be a sterile pipette.

[00112] As disclosed herein, compounds and compositions of the presently- disclosed subject matter are inhibitors of hedgehog signaling and inhibitors of PDE4. Such inhibitors have further utilities as described herein, which include, but are not limited to, anti-cancer or anti-tumor activity, anti-angiogenic activity, anti-metastatic activity, and/or anti-inflammation activity, and utility for treating certain conditions of interest. In this regard, in some embodiments, the kit can further include a second compound or composition having Hh signaling inhibition activity, PDE4 inhibition activity, anti-cancer or anti-tumor activity, anti-angiogenic activity, anti-metastatic activity, and/or anti-inflammation activity, or wherein the second compound or composition is useful for treating a condition of interest. In some embodiments, the addition of the second compound or composition provides for a synergistic response.

[00113] The presently-disclosed subject matter further includes kits comprising a reagent to carry out a method as described hereinbelow.

[00114] Methods

[00115] The presently-disclosed subject matter further includes methods. A method of inhibiting hedgehog signaling is provided. In some embodiments, the method includes contacting a cell with an effective amount of a compound or pharmaceutical composition as disclosed herein. In some embodiments, contacting the cell with the compound or composition comprises administering the compound or composition to a subject. In some embodiments, the administration is to a subject in need of treatment for a condition of interest. Examples of relevant conditions of interest associated with inhibition of hedgehog signaling are set forth hereinbelow. [00116] Also provided is a method of inhibiting phosphodiesterase-4. In some embodiments, the method includes contacting a cell with an effective amount of a compound or pharmaceutical composition as disclosed herein. In some embodiments, contacting the cell with the compound or composition comprises administering the compound or composition to a subject. In some embodiments, the administration is to a subject in need of treatment for a condition of interest. Examples of relevant conditions of interest associated with inhibition of PDE4 activity are set forth hereinbelow.

[00117] Also provided is a method of treating a condition of interest. In some embodiments, the method includes contacting a cell with an effective amount of a compound or pharmaceutical composition as disclosed herein. In some embodiments, contacting the cell with the compound or composition comprises administering the compound or composition to a subject. In some embodiments, the administration is to a subject in need of treatment for a condition of interest. Examples of relevant conditions of interest associated with inhibition of Hh signaling and/or inhibition of PDE4 activity are set forth hereinbelow.

[00118] As will be recognized by one of ordinary skill in the art, the term “inhibiting” or“inhibition” does not refer to the ability to completely inactivate all target biological activity in all cases. Rather, the skilled artisan will understand that the term “inhibiting” refers to decreasing biological activity of a target, such as a decreasing Hh signaling or decreasing PDE4 activity, such as can occur with a ligand binding site of the target, or protein in a biochemical pathway of the target, is blocked, or when a non- native complex with the target, or protein in a biochemical pathway of the target, is formed. Such decrease in biological activity can be determined relative to a control, wherein an inhibitor is not administered and/or placed in contact with the target. For example, in some embodiments, a decrease in activity relative to a control can be about a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% decrease. The term“inhibitor” refers to a compound of composition that inactivates or decreases the biological activity of a target, such as Hh signaling pathway or PDE4 activity.

[00119] The terms“treatment” or“treating” refer to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[00120] The terms“subject” or“subject in need thereof” refer to a target of administration, which optionally displays symptoms related to a particular disease, pathological condition, disorder, or the like. The subject of the herein disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Thus, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. A patient refers to a subject afflicted with a disease or disorder. The term“patient” includes human and veterinary subjects.

[00121] The term“administering” refers to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal

administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.

[00122] The term“effective amount” refers to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition. For example, a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be

administered in one or more dose administrations daily, for one or several days.

Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, a preparation can be administered in a“prophylactically effective amount”; that is, an amount effective for prevention of a disease or condition.

[00123] Uses and Conditions of Interest

[00124] As disclosed herein, compounds and compositions of the presently- disclosed subject matter are inhibitors of hedgehog signaling via inhibition of PDE4, without global peturbations in cAMP levels. Rather, surprisingly and unexpectedly, the compounds and compositions disclosed herein selectively raise cAMP levels in the basal body, such that the compounds and compositions might be considered organelle- targeted. As such, the compounds and compositions of the presently-disclosed subject matter have utilities in connection with inhibition of the hedgehog pathway, and utilities in connection with inhibition of PDE4 activity.

[00125] Inhibiting Hedgehog Signaling

[00126] The presently-disclosed subject matter includes methods of inhibiting hedgehog signaling in a cell, comprising contacting a cell with an effective amount of a Hh signaling inhibitor. In some embodiments, the presently-disclosed subject matter includes methods of inhibiting Hh signaling in a cell, comprising administering an effective amount of a Hh signaling inhibitor to a subject. In some embodiments, the subject is in need of a treatment for a condition of interest. In some embodiments, the Hh signaling inhibitor is a compound or pharmaceutical composition as disclosed hereinabove. In some embodiments, the presently-disclosed subject matter includes methods of treating a condition of interest, including conditions as identified herein.

[00127] With regard to targeting hedgehog signaling, methods of the presently- disclosed invention can be useful in treating conditions involving neoplastic or hyperplastic transformations, conditions related to tissue homeostasis, and anti- angiogenesis treatment to target cancers.

[00128] Treatment of neoplastic or hyperplastic transformations.

[00129] Constitutive Hh signal activation, due to mutations that activate the pathway, is implicated in numerous neoplastic or hyperplastic conditions. For instance, constitutive activation of Hh pathway has been shown to play critical roles in

tumorigenesis in malignant medulloblastoma (the most common brain tumor in children), neuroectodermal tumors, ependymomas, tumors associated with Gorlin syndrome (also known as Basal Cell Nevus Syndrome, a hereditary syndrome conferring high risk of skin and brain cancers, including basal cell carcinoma, medulloblastoma, and meningioma), sporadic basal cell carcinoma (the most common form of skin cancer), rhabdomyosarcoma, glioblastoma, renal carcinoma, thyroid carcinoma, bone cancers, chondrosarcoma, breast cancer, urogenital cancers (including prostate cancer), adrenal cancers, gastrointestinal cancers, pancreatic cancers, and lung cancers (small cell lung cancer, squamous cell cancer, and adenocarcinomas). With regard to medulloblastoma, for example, the compounds and compositions disclosed herein have particular utility because they are hedgehog signaling inhibitors that do not target smoothened. These compounds and compositions can selectively kill cells over-expressing oncogenic, drug- resistant forms of smoothened. In the medulloblastoma field, drug resistance to smoothened antagonists are quickly becoming recognized as an important problem.

[00130] Proliferation of these cancer cells requires Hh signaling, and blocking Hh pathways has been shown to inhibit cancer cell proliferation and to reduce tumor size in Xenograft models. In addition to direct promotion of tumorigenesis, Hh pathway has been shown to be required in tissue mesenchyme surrounding pancreatic cancers to support tumor growth by a paracrine effects. Moreover, in animal models, blocking Hh signaling has been shown to suppress metastasis of pancreatic and prostate cancers. [00131] As such, compounds and composition disclosed herein, which are inhibitors of Hh signaling, can have utility in treating cancers in which underlying the neoplastic transformation is caused, maintained or characterized by persistent Hh activation.

[00132] In some embodiments, methods of the presently-disclosed subject matter make use of compounds and composition disclosed herein for treatment of a cancer, such as a cancer identified above. In some embodiments, the cancer can be basal cell carcinoma, breast, cervical, colon, melanoma, prostate, pancreatic,

medulloblastoma, small cell lung, or squamous lung. The status of Hh activation in particular tumor types can be found in publically-available resource, such as the Broad- Novartis Cancer Cell Line Encyclopedia, which can be accessed online

(http://www.broadinstitute.org/ccle/). In some embodiments, the cancer can be: acute B- cell, acute myeloid leukemia (AML), B-cell acute lymphoblastic (ALL-B cell), bile duct cancer, Burkitt’s lyphoma, chondrosarcoma, chronic myeloid leukemia (CML), colorectal, DLBCL lymphoma, endometrial, esophageal, Ewings sarcoma, glioma, Hodgkin’s lymphoma, leukemia, liver, lung (including small cell (SCLC) and non-small cell type (NSCLC)), medulloblastoma, melanoma, mesothelioma, multiple myeloma, neuroblastoma, osteosarcoma, ovarian, pancreatic, prostate, renal, stomach, thyroid, T- cell acute lymphoblastic leukemia (ALL-T cell), or urinary tract.

[00133] In some embodiment, the cancer can be a cancer in which tumor profiling indicates Hh signal activation. Such cancers can be identified, for example, based on the overexpression of Hh pathway markers such as Gli1, Gli2, Gli3, Ptch1, and Ptch2 genes. The status of Hh activation in tumors of an individual subject can be determined, for example, by molecular profiling and accessed through portals such as My Cancer Genome (http://www.mycancergenome.org/). As such, some embodiments of the presently-disclosed subject matter provide for a personalized approach to determining a pathway signature of an individual subject’s neoplasm. In some embodiments, for example, if sequence and expression profile analysis indicate that Hh signaling is activated in a particular subject’s tumor, Hh inhibitors, including compounds and compositions of the presently-disclosed subject matter, can be a used to treat the cancer.

[00134] Anti-angiogenesis therapy.

[00135] An important hallmark of cancer cells is rapid accumulation of mutations within rapidly dividing cell populations. These mutations allow subpopulation of cancer cells to develop resistance to chemotherapeutic agents and thus escape therapy. In the absence of angiogenesis, the growth of tumors is limited by mismatch between oxygen/nutrient supply and demand such that tumors cannot grow beyond a certain size (typically < 2mm³). Tumor angiogenesis is essential for transition into clinically significant large tumors as well as metastasis. Since blood vessels within tumors are typically comprised of noncancerous endothelial cells, targeting endothelial cells with anti-angiogenic molecules is an attractive method to block tumor growth, metastasis and drug resistance. Because Hh signaling plays a critical paracrine role in promoting angiogenesis, Hh signaling inhibitors, such as the compound and compositions as disclosed herein, can also be used as an anti-angiogenesis therapy for variety of cancers.

[00136] Conditions related to tissue homeostasis.

[00137] The Hh pathway plays a key role in postnatal tissue homeostasis and regeneration. For example, in animal models, Hh pathway has been shown become activated after tissue injury, for instance of retina, bile duct, lung, bone and prostate. Hh pathway plays an important role regulating hair follicle, bone marrow, CNS, and benign prostate hyperplasia. As such, Hh signaling inhibitors, such as the compound and compositions as disclosed herein, can also be used as a part of treatment for

neuroproliferative diseases, benign prostate hyperplasia, bone marrow proliferative disease and leukemia, osteopetrosis and hair overgrowth.

[00138] Furthermore, compounds and compositions as disclosed herein can also be useful in methods of stem cell differentiation.

[00139] Inhibiting PDE4 Activity

[00140] The presently-disclosed subject matter includes methods of inhibiting PDE4 Activity in a cell, comprising contacting a cell with an effective amount of a PDE4 inhibitor. In some embodiments, the presently-disclosed subject matter includes methods of inhibiting PDE4 in a cell, comprising administering an effective amount of a PDE4 inhibitor to a subject. In some embodiments, the subject is in need of a treatment for a condition of interest. In some embodiments, the PDE4 inhibitor is a compound or pharmaceutical composition as disclosed hereinabove. In some embodiments, the presently-disclosed subject matter includes methods of treating a condition of interest, including conditions as identified herein.

[00141] With regard to targeting PDE4 activity, methods of the presently- disclosed invention can be useful in treating conditions involving inflammation, making use of PDE4 inhibitors as an anti-tumor, anti-angiogenic, or anti-metastatic agents, making use of PDE4 inhibitors to target the central nervous system, and making use of PDE4 inhibitors as anti-viral agents.

[00142] Targeting inflammation.

[00143] TNF-a is an important target in numerous diseases including rheumatoid arthritis, Crohn’s disease and psoriasis inhibition of PDE4 in monocytes and T-cells prevents TNF-a production. Furthermore inhibition of PDE4 in neutrophils, which play a pivotal role in chronic obstructive pulmonary disease (COPD) and severe asthma, prevents multiple neutrophil responses, including chemotaxis, adhesion and production of IL-8. Furthermore PDE4 inhibitor CP80,633 suppressed T cell

proliferation and production of IL-2, IL-5 and TNF-a. As such, the compounds and compositions disclosed herein can be used in anti-inflammatory treatment.

[00144] Anti-tumor, anti-angiogenic, anti-metastatic agents.

[00145] As disclosed herein, compounds and compositions of the presently- disclosed subject matter have anti-proliferative effects in various cancer cell lines. It is also documented that PDE4 inhibitors have antiproliferative activity against murine carcinoma cells. In addition to anti proliferative effects inhibition of PDE4 has been linked to inhibition of VEGF (Vascular endothelial growth factor) which is essential for angiogenesis. Furthermore, PDE4 inhibition could have anti-metastatic effects due to its inhibition of Rho-driven migration of fibroblasts. PDE4 inhibition can also find utility in the context of pathological angiogenesis, including macular degeneration and diabetic retinopathy. As such, the compounds and compositions disclosed herein can be used as anti-tumor, anti-angiogenic, anti-metastatic, agents.

[00146] Targeting central nervous system .

[00147] PDE4 is expressed in various neuronal cell types in the CNS. Indeed, Rolipram does show some efficacy in several preclinical models for depression, memory deficit, Alzheimer’s disease, and spinal cord injury. Furthermore PDE4 inhibition has been shown to be beneficial and effective in the MPTP mouse model of Parkinson’s disease via a direct neuroprotective effect. Additionally inhibition of PDE4 improves both the working memory and reference memory caused by NMDA receptor antagonists. As such, the compounds and compositions disclosed herein can be used in the treatment of CNS disorders and neuropsychiatric disorders, such as depression, memory deficits, Alzheimers’ disease, spinal cord injury, and Parkinson’s disease.

[00148] Anti-viral agents. [00149] PDE4 was found to be functionally up-regulated in human T- lymphotropic virus-infected T-cells and may contribute to the virus-induced

proliferation. Furthermore, selective blocking of PDE4 activity inhibited IL-2R expression and thereby led to abolishing HIV-1 DNA nuclear import in memory T cells. Additionally there have been recent implications of PDE4 playing major important roles in the infection process of respiratory syncytial virus (RSV), Dengue, and cowpox. As disclosed herein, compounds and compositions of the presently-disclosed subject matter have antiviral effects on, RSV, Influenza, Dengue, and Bovine Viral Diarrhea Virus (BVDV). As such, the compounds and compositions disclosed herein can be used as anti-viral agents.

[00150] The compounds and compositions disclosed herein can also be used in the treatment of conditions in which side effects of existing competitive PDE4 inhibitors have limited treatment options and have prompted need for development of alternative PDE4 inhibitors.

[00151] Treatments related to heart failure

[00152] Heart failure (HF) is a common condition affecting over 5.8 million Americans, and the prevalence of HF is expected increase dramatically over the next 20 years. Presently, one in 5 Americans has lifetime risk of HF. HF is primary reason for hospitalization in US, and a leading cause of death in US (over 300,000 deaths a year). Despite recent medical advances, the HF prognosis remains poor with over 50% mortality within 5 years of diagnosis. Currently, apart from heart transplantation, treatment options are largely palliative. There are no drugs approved for treatment of systolic heart failure. In critical ill patients with end-stage heart failure, positive inotropes like milrinone and dobutamine, which increase heart contractility, augment function of failing heart in the ICU setting. However, long-term administration of inotropes is curtailed by tachyphylaxis and increased risk of arrhythmias, heart failure progression and death.

[00153] The etiology of systolic heart failure, is multifactorial, involving complex interplay between genetic susceptibility and acquired insults, such as myocardial infarction, long-standing hypertension, cardiotoxins, or myocarditis. Disease progression involves maladaptive phenotypic alterations in myocardial structure and function, resulting from neurohormonal and cytokine activation. Despite the multitude of pathways leading to heart failure, cAMP regulation of PKA is emerging as a major regulator of cardiac contraction. [00154] Calcium cycling, which drives the contractile mechanics of

cardiomyocytes, is modulated by PKA phosphorylation of the ryanodine receptor, CREB, NCX1, KCNQ1, troponin I, and phospholamban (PLB) (an endogenous SERCA inhibitor). While short-term increases in cellular cAMP levels– either via stimulation of beta-adrenergic receptor or inhibition of phosphodiesterases (typically PDE3) - enhance cardiac function initially, chronic cAMP elevation results in tachyphylaxis and heart failure progression via adrenergic receptor desensitization and other maldaptive responses.

[00155] However, the present PDE4 inhibitors (e.g., EGM), can be used for the treatment of subjects with systolic heart failure. As described herein, Eggmanone increases fractional shortening (FS) and ejection fraction (EF) of heart without increasing heart rate. In comparison to the traditional inotropes, which increase total cAMP levels in the cardiomyocyte, the unique advantage of the present invention is that the EGM class of PDE4 inhibitors raise cAMP levels locally to wherever PDE4 is localized within specific subcellular compartments, but not globally. Hence,

maladaptive responses to chronic stimulation, such as tachyphylaxis and heart failure progression, can be reduced or avoided.

[00156] Various treatments related to heart treatment can be implemented with the present compounds. In some embodiments the present compounds will comprise a pharmaceutical composition that can be administered to acutely improve cardiac function. This can be particularly beneficial with critically ill subjects with systolic heart failure (e.g, in ICU or inpatient setting). In other embodiments the present compounds can provide inotropic support following surgery (e.g., myocardial surgery), in critically ill subjects with inadequate cardiac output, regardless of etiology (i.e., cardiogenic shock, septic shock, hemorrhagic shock, etc.), and/or in pediatric subjects. In some embodiments the present compositions can be administered to improve or stabilize (i.e., treat) long-term cardiac function, to promote beneficial cardiac remodeling, to provide symptomatic relief and survival benefits in subjects with advanced systolic heart failure as a chronic therapy, and the like.

[00157] Additional Conditions of Interest

[00158] Additional conditions of interest include, but are not limited to, asthma, COPD, bronchitis and bronchiectasis, allergic rhinitis and sinusitis, rheumatoid arthritis, osteoarthritis, gout, eosinophil-related disorders, including chronic eosinophilic pneumonia, chronic interstitial lung disease, allergic granulomatous angiitis/Churg- Strauss syndrome, polyarteritis nodosa, atopic dermatitis, urticaria, conjunctivitis, uveitis, psoriasis, multiple sclerosis and other inflammatory autoimmune diseases, inflammatory bowel disease, including ulcerative colitis and Crohn's disease, septic shock, renal failure, cachexia and infection, liver injury, pulmonary hypertension, bone loss disease , CNS disorders: cognitive and memory defects in Parkinson's dieseas, Huntington's chorea, Wilson's disease, paralysis agitans and thalamic atrophies, arteriosclerotic dementia, improved learning in general, depression, ischemia-reperfusion injury in stroke, diabetes prevention, chronic lymphocytic leukemia, HIV-1 replication, prostate disease, pemphigus, pemphigoid, antiviral: HIV-1, HIV-2, HIV-3,

cytomegalovirus, CMV, influenza, adenovirus, Herpes virus, yeast and fungal infections.

Conditions of interest include anti-viral applications, including applications related to enveloped RNA viruses, such as respiratory syncytial virus, and bronchiolitis (RSV is a leading cause of bronchiolitis), ebola virus, hepatitis C virus, Bovine Viral Diarrhea Virus, Dengue virus, west nile virus, yellow fever virus, measles virus, mumps virus.

[00159] Conditions of interest include improved learning in neurofibromatosis type 1 (http://www.ncbi.nlm.nih.gov/pubmed/25176649), Behcet’s syndrome

(https://www.rareconnect.org/en/community/behcet-s-syndrome/forum/topic/apremilast- a-novel-pde4-inhibitor), and psoriatic arthritis, psoriasis

(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3680635/,

http://www.ncbi.nlm.nih.gov/pubmed/22257911).

[00160] While the terms used herein are believed to be well understood by one of ordinary skill in the art, definitions are set forth to facilitate explanation of the presently-disclosed subject matter.

[00161] 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 the presently-disclosed subject matter belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently-disclosed subject matter, representative methods, devices, and materials are now described.

[00162] Following long-standing patent law convention, the terms“a”,“an”, and“the” refer to“one or more” when used in this application, including the claims. Thus, for example, reference to“a cell” includes a plurality of such cells, and so forth. [00163] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term“about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.

[00164] As used herein, the term“about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.

[00165] As used herein, ranges can be expressed as from“about” one particular value, and/or to“about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then“about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[00166] The presently-disclosed subject matter is further illustrated by the following specific but non-limiting examples. The following examples may include compilations of data that are representative of data gathered at various times during the course of development and experimentation related to the present invention. EXAMPLES

[00167] Example 1

[00168] Cyclic AMP (cAMP) is a ubiquitous secondary messenger which mediates diverse signals with extraordinary functional precision. Functional specificity is thought to involve compartmentalized signaling centers, or‘cAMP microdomains,’ inside which cAMP levels are tightly controlled. By restricting cAMP changes to specific microdomains, a cell can manage multiple cAMP-dependent signals without undesired signal“leakage” between pathways. These cAMP microdomains arise from dynamic process of localized cAMP synthesis via adenyl cyclase (AC) and degradation via phosphodiesterases (PDEs). Consequently, a global loss of PDE activity results in the loss of signal specificity.

[00169] cAMP plays an important, evolutionarily conserved role in Hh regulation. In Drosophila, Hh activation of the Smoothened (Smo) transmembrane protein results in inhibition of cAMP production via G ^i, whereas the loss of PDE4 activity results in a Hh loss-of-function phenotype. Furthermore, PKA (cAMP-activated protein kinase) has a negative role on Hh activity. In vertebrates, where transient trafficking of the transcription factor Gli through the primary cilia is essential for Hh activation, PKA is localized to the basal body at the base of the cilium, and treatment with forskolin, an AC activator, disrupts the Gli trafficking to the cilia. However, whether the basal body might constitute a cAMP microdomain important for Hh regulation was not directly tested since forskolin causes a global PKA activation as well as non-PKA dependent pleiotropic effects.

[00170] In a phenotypic screen for small molecule modulators of zebrafish pattern formation the present inventors identified a series of structurally related compounds, represented by the prototype named Eggmanone (3-(2-methylallyl)-2-((2- oxo-2-(thiophen-2-yl)ethyl)thio)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine- 4(3H)-one) (Fig.4-6), which caused a number of phenotypes resembling those of Hh- deficient mutant embryos; ventral tail curvature, absent pectoral fins, small eyes, loss of neurocranial chondrogenesis, and enlarged, rounded somites (Fig.1a,d; Fig.7a,b). Eggmanone abrogated the expression of the Hh target gene patched-1 (ptch1) in the bud stage adaxial cells, the pectoral fin fields, and the somites (Fig.1b,c), but it did not eliminate ptch1 expression in the ventral neural tube or myotome cells immediately adjacent to the notochord (Fig.7c). Consistent with the context-dependent inhibition of Hh signals in the embryo, the nkx2.2-expressing neurons in the ventral neural tube were not abolished in Eggmanone-treated embryos (Fig.7d).

[00171] In the mouse Hh reporter cell line Shh-Light2, Eggmanone inhibited Hh-inducible Gli-responsive luciferase (Gli-Luc) activity in a dose dependent manner, confirming that the molecular target is conserved in mammals (Fig.1e). Eggmanone also blocked Gli-Luc reporter and ptch1 induction by purmorphamine, a Smo agonist, indicating that Eggmanone targeted Hh pathway at or downstream of Smo activation (Fig.1f, Fig.8a). By contrast, Eggmanone did not affect BMP-responsive luciferase reporter activity, indicating that its Hh reporter inhibition was not due to nonspecific effects on luciferase activity (Fig.8b).

[00172] To elucidate the mechanism of Hh inhibition by Eggmanone, the present inventors utilized the LASSO (“Ligand Activity by Surface Similarity Order”) algorithm to virtually screen for potential targets. As this algorithm implicated PDE5 (Fig.9), the present inventors assayed Eggmanone for in vitro activity against eleven different PDE families and found that it significantly inhibited only the PDE4 family (Fig.10a-b). Eggmanone significantly inhibited isoforms from each gene within the PDE4 (A-D) family (Fig.1g), with an IC₅₀ (concentration causing 50% of maximal inhibition) range of 0.8-3 ^M. Of the seven isoforms of PDE4s tested, only the super- short isoform PDE4D2 was not inhibited by Eggmanone. The naturally occurring N- terminal truncation found in PDE4D2 allowed us to infer that the first 33 residues of the UCR2 domain were essential for Eggmanone inhibition. Moreover, since the UCR2 domain is unique to the PDE4 family, this result also provided a molecular explanation for Eggmanone’s selectivity toward PDE4 isoforms. Interestingly, even at high

Eggmanone concentrations, the enzymatic activities of the PDE4s did not reach 0% (Fig. 8b). Taken together, these results suggested that Eggmanone is a selective allosteric inhibitor of PDE4 that targets the UCR2 domain.

[00173] To rule out other potential targets, the present inventors tested

Eggmanone against other pharmacologically relevant classes of biomolecules using a comprehensive panel of 442 kinases, 158 GPCRs and 21 phosphatases; remarkably, Eggmanone did not exhibit significant agonist or antagonist activity against any of them (Tables 4-6). To confirm the interaction between PDE4 and the Hh pathway in vertebrates, the long isoform PDE4D3 was transfected into Shh-Light2 reporter cells and was found to increase Hh signaling, which was abrogated in the presence of Eggmanone (Fig.1h). Furthermore, a dominant negative construct consisting of a catalytically inactive PDE4D3 inhibited Hh signaling.

[00174] Surprisingly, Eggmanone did not significantly increase total cAMP levels in cells at the concentrations that abolish Hh signaling (Fig.2a). Together with the fact that Eggmanone did not abolish neural tube patterning, which is relatively refractory to cilia disruption in zebrafish , this led us to consider a selective perturbation of local cAMP levels in a microdomain associated with the primary cilium. While there is no known technique to directly measure local cAMP levels within cilia, the frequency and the amplitude of beating cilia are modulated by cAMP levels. When zebrafish embryos were treated with 2 ^M Eggmanone, the otic kino-cilia became markedly less motile (Fig.2b). Since this concentration does not elicit a global cAMP change, this result suggests that Eggmanone selectively affects the local cAMP levels within a microdomain in or near the cilium.

[00175] A subset of PDE4 isoforms, notably PDE4D3, is localized to the centrosome, which also forms the basal body of the cilium and plays a central role in cilia biogenesis and function. Consistent with prior reports, the present inventors found that in NIH3T3 cells over-expressing a VSV-tagged PDE4D3, PDE4D3 co-localized to the base of the cilium (Fig.11a). Eggmanone treatment did not disrupt PDE4D3 localization or physical association with AKAP450 (Fig.11b), a scaffolding protein which anchors PKA to the cilium base. Interestingly, immunostaining for

autophosphorylated, active form of the PKA catalytic subunit demonstrated that Eggmanone significantly increased the intensity of PKA activation almost exclusively at the basal body (Fig.2c; Fig.12a-f). This differs from a more diffuse increase in cytoplasmic phospho-PKA levels using the competitive PDE4 inhibitor rolipram (Fig. 2c-e) and from earlier findings in cerebellar granule neuron precursors using the cAMP analog dibutyril cAMP, which induced the dispersion of PKA from the centrosome and uniform PKA activation in the cell³. Taken together, the results indicated that

Eggmanone selectively targets PDE4s localized to the basal body, leading to localized increases in cAMP levels and PKA activity. Moreover, because Eggmanone does not target the super-short PDE4D2, the most abundant PDE4 isoform present in the cytoplasm, the cAMP levels are largely unaffected outside the peri-ciliary microdomain.

[00176] Eggmanone represents a unique class of selective small molecules to inhibit Hh signaling and a potentially new way to treat diseases caused by aberrant Hh activation. Eggmanone efficiently and selectively killed SmoM2-Light cells, which stably overexpress the constitutively active, oncogenic Smo mutant, which is resistant to cyclopamine (Fig.3f), but not the parental NIH3T3 cells. Moreover, Eggmanone potently and preferentially reduced the viability of human medulloblastoma Daoy cells (Fig.3g), which are known to be hedgehog and PDE4 dependent, by blocking proliferation and inducing apoptosis (Fig.3h,i).

[00177] In vertebrate cells, forskolin prevents the ciliary localization of Gli and subsequent Gli-mediated transcription, but this may be mediated via a PKA-independent mechanism as Gli2 traffics to the cilia of PKA-null embryonic fibroblasts. Eggmanone did not prevent Gli2 localization to the primary cilium (Fig.3a). Quantification of the intensity of Gli2 staining within the primary cilia revealed that significantly more Gli2 accumulated in Eggmanone-treated cilia than in controls (Fig.3b). Moreover,

Eggmanone blunted the nuclear accumulation of the full-length Gli2 (Gli2FL) induced by SAG, a Smo agonist, indicating that cAMP accumulation at basal body blocked Gli2 trafficking from the primary cilium to the nucleus (Fig.3c-e).

[00178] The precise roles of cAMP and PKA with respect to Hh regulation are not fully understood, but based on the findings and those of others, the present inventors propose the following model (Fig.13): Hh activation requires the transport of Gli in and out of primary cilium, where it becomes activated. Eggmanone specifically targets the PDE4s localized to the basal body, resulting in locally elevated cAMP levels. This in turn prevents trafficking of activated Gli from the cilium to the nucleus via local PKA activation in the basal body. The present inventors postulate that the supramolecular complex consisting of PKA and PDE4 functions as a“cAMP barrier” to functionally isolate the peri-ciliary signal transduction events from cAMP fluctuations in the rest of the cell.

[00179] In summary, Eggmanone is an extraordinarily selective allosteric inhibitor of PDE4 whose effects on cAMP levels are spatially restricted to a cellular microdomain encompassing the basal body. The chemical genetic study underscores the importance of the basal body PDE4 activity and cAMP levels in Hh regulation.

Considering there are over 29 PDE4 isoforms transcribed from 4 genes, it seems unlikely that traditional genetic and pharmacological approaches would have revealed these cell biological insights. The ability to selectively manipulate cAMP levels within a specific subcellular microdomain provides a new paradigm for molecular medicine.

[00180] Materials and Methods

[00181] Chemical screen. [00182] All zebrafish experiments were approved by Vanderbilt University Institutional Animal Care and Use Committee. Wild-type zebrafish of AB strain were maintained using standard protocols. Chemical screen for small molecules was performed as previously described. Briefly, pairs of zebrafish were mated, and fertilized eggs were arrayed in 96-well microtiter plates (5 embryos/well) containing 250μl E3 water. At ~4-hpf, small molecule library from Vanderbilt High Throughput Screening Facility was added to each well to the final concentration of 5 ^M. Embryos were incubated at 28.5 ^C until 24 and 48-hpf, when they were examined for gross

morphologic changes indicative of disruption in embryonic patterning. A total of ~30,000 compounds were screened.

[00183 E manone S nthesis

[00184] Cyclohexanone was reacted with methyl cyanoacetate, S₈ and diethylamine in ethanol as previously reported to provide the 2-aminothiophene in 49% yield. Formation of the dithiocarbamate was effected with C₂S and NaOH in DMSO followed by reaction with dimethylsulfate to give the methyl dithiocarbamate, as previously reported.

[00185] To a solution of 3 (1.00 g, 3.32 mmol, 1.0 eq) in CH₃CN (2.2 mL) under argon atmosphere was added methylallylamine•HCl (446 mg, 4.15 mmol, 1.25 eq) then triethylamine (578 µL, 4.15 mmol, 1.25 eq) and the reaction was heated at 80 ºC for 24 hours. The reaction mixture was diluted with CH₂Cl₂, washed with H₂O (2 x 10 mL), and the combined aqueous layers were extracted with CH₂Cl₂ (2 x 10 mL). The combined organic layers were dried over MgSO₄, filtered and concentrated. The crude solid was recrystallized from CH₃CN to provide 4 (591 mg, 2.02 mmol, 61%).

[00186] To a solution of 4 (50 mg, 0.171 mmol, 1.0 eq) in CH₃CN (2.0 mL) was added 2-(chloroacetyl)thiophene (42 mg, 0.260 mmol, 1.5 eq) and Cs₂CO₃ (139 mg, 0.260 mmol, 1.5 eq) and the reaction was heated via microwave irradiation at 70 ºC for 10 minutes. Addition of water caused precipitation of the desired product (30 mg, 0.0720 mmol, 42%). ¹H NMR (600 MHz, CDCl₃): ^ 7.94 (dd, J = 3.8, 1.0 Hz, 1H), 7.73 (dd, J = 5.0, 1.0 Hz, 1H), 7.20 (dd, J = 5.0, 3.9 Hz, 1H), 4.92 (s, 1H), 4.70 (s, 2H), 4.64 (s, 1H), 4.57 (s, 2H), 2.96 (t, J = 6.0 Hz, 2H), 2.69 (t, J = 6.0 Hz, 2H), 1.83 (s, 3H), 1.83 (m, 4H); LCMS, single peak, 1.42 min, m/e = 416.8 [M+1].

[00187] Wholemount zebrafish in situ hybridization

[00188] In situ hybridization was performed as previously described. Zebrafish ptch1 probes were produced as previously described.

[00189] Whole mount immunofluorescence

[00190] Unless otherwise stated, manipulations were performed at RT.

Embryos were fixed in 4% PFA at 4°C overnight. Embryos were blocked with 1x PBS, 1%BSA, 1%Triton-X100, 0.1% DMSO for 2 hours. Embryos were incubated with primary antibodies diluted in block solution overnight at 4°C. Embryos were washed in 1xPBS with 1%Triton-X100 for 60 min. Embryos were incubated with secondary antibodies diluted in block solution for two hours. Primary antibodies specific against Myh1/2/4/6 (F-59) were obtained from Santa Cruz (1:50 dilution). Fluorescence immunocytochemistry was performed using anti-mouse secondary antibody Alexa 488 (1:500 dilution, Invitrogen).

[00191] Zebrafish lines and Maintenance

[00192] Wild-type zebrafish lines of AB and TL; and transgenic line

Tg(nkx2.2:egfp) were maintained using standard protocols.

[00193] Luciferase reporter assays

[00194] For Hh signaling assays, Shh-Light2 cells stably transfected with Gli- Luciferase reporter construct were used along with Shh-conditioned media, as previously described⁷. Alternatively, 3 ^M purmorphamine or 20 nM Smoothened agonist (SAG) (Santa Cruz Biotechnology, Santa Cruz, CA) was used to induce Hh signaling. Reporter cells were seeded in 96-well plates and incubated overnight with the various

concentrations of eggmanone and Shh-conditioned media. To assess the effects of overexpression of Gli-2, PDE4D3 and DN-PDE4D3 on Hh signaling, mammalian expression vectors containing these constructs were transfected into Shh-Light2 cells in 96-well plates using Fugene6 (Roche), according to manufacturer’s instructions. The transfected or Shh-stimulated cells were incubated overnight with the various concentrations of compound. The cells were then lysed, and cell extracts were subjected to Steady-Glo luciferase assay (Promega) according to manufacturer’s instructions. The results were normalized to cell titer, as determined using Cell Titer-Glo luminescence assay (Promega).

[00195] Immunocytochemistry

[00196] NIH3T3 cells were plated on Poly-D-Lysine-coated glass coverslips and were cultured at 37°C, 5% CO₂ in DMEM medium containing 10% fetal bovine serum until reaching 75% confluency. For one set of experiments, cells were then transfected with VSV-tagged PDE4D3 plasmid (gift from Miles Houslay, University of Glasgow, Scotland, UK) using Fugene6 transfection reagent (Roche, Indianapolis, IN) per manufacturer’s protocol. Afterward, cell medium was replaced with DMEM/ 0.5% FBS containing either 5 ^M eggmanone or DMSO and incubated overnight at 37°C, 5% CO₂. Cells were fixed in 4% PFA at room temperature for 10 minutes prior to

permeabilization, blocking, and staining with primary antibodies against Arl13b (gift of Tamary Caspary, Emory University, Atlanta, GA) and VSV (AbCam, Cambridge, MA). Fluorescent immunocytochemistry was performed using species-specific, secondary antibodies (Jackson Immunoresearch, West Grove, PA). For additional

immunocytochemistry experiment, cells were treated with 20 nM SAG in the presence or absence of 5uM Eggmanone. After overnight incubation, cells were washed with PBS, fixed for 10 minutes in 4% PFA, permeabilized 20 minutes at -20C with cold methanol, blocked with PBS/ 1%BSA, and incubated with primary antibodies to phospho-PKA catalytic domain Thr197 (Cell Signaling, Danvers, MA) and then to Arl13b. An additional overnight blocking step using unconjugated rabbit IgG was required between primary antibody incubations since both antibodies were produced in rabbit. Fluorescent conjugated secondary antibodies were used for visualization. Data analysis was performed in part through the use of the VUMC Cell Imaging Shared Resource.

[00197] Quantitative analysis of Gli and phospho-PKA intensity

[00198] Using ImageJ software (National Institutes of Health, developed by W. Rasband), a region of interest was created using the magic wand tool on Arl13b channel and transposed to the Gli2 channel, and integrated density was measured and reported as arbitrary units (a.u.). For phospho-PKA, using ImageJ, a line selection tool was used to select a line projected through the length of the primary cilia and an equal length beyond. The intensity values were potted and the cumulative florescence (area under the curve) was calculated for three cilia, blindly, for each treatment. These values for cilia and pericilia domain were analyzed by a two tailed students t-test. For correlation analysis, correlation coefficient for intensity of gamma-tubulin and phosphor-PKA were calculated and compared among treatments with students t-test.

[00199] Nuclear fraction western blotting

[00200] Cells were fractionated using NE-PER Nuclear and Cytoplasmic extraction reagents (Thermo Scientific, Rockford, IL) per the manufacturer’s protocol. For western blotting, goat anti-Gli2 (R & D Systems) and rabbit anti-Lamin-A/C (Cell Signaling Technology) antibodies were used as primary antibodies.

[00201] Co- immunoprecipitation

[00202] NIH3T3 cells were transfected with VSV-tagged PDE4D3 plasmid (gift from Miles Houslay, University of Glasgow, Scotland, UK) using Fugene6 transfection reagent (Roche, Indianapolis, IN) per manufacturer’s protocol. Afterward, cell medium was replaced with medium containing either 5 ^M eggmanone or DMSO and incubated overnight. Cells were then lysed in CellLytic M Cell Lysis reagent supplemented with 1x Complete Mini Protease Inhibitor Cocktail (Roche). Cell lysate was incubated with mouse anti-AKAP450 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 4˚C overnight. Antibody-antigen complex was conjugated to Protein A/G agarose beads (Thermo Scientific) for 2 hours rocking at 4˚C, followed by five cold 1x TBS washes. The beads were centrifuged, and bound protein was eluted in 1X LDS buffer (Invitrogen). Eluted protein was resolved in SDS-PAGE and transferred onto nitrocellulose membrane for Western blotting. Western blot analysis was performed using an anti-VSV antibody (AbCam, Cambridge, MA).

[00203] Video-microscopy

[00204] For visualizing ciliary beating, live embryos (20 hpf) were removed from their chorion, mounted in SeaPlaque low-melting agarose (Biowhittaker Molecular Applications) (1.0% in embryo medium) in microwells of glass-bottom culture dishes (MatTek), and covered with embryo medium. Movies were acquired by using

OPENLAB software (Improvision) at 55 frames per second with a 63x DIC objective on a Zeiss Axiovert 200 inverted fluorescence microscope equipped with a Retiga Exi Fast camera (Qimaging). Kymographs were obtained by drawing a line across a ciliary trajectory by using ImageJ software (National Institutes of Health, developed by W. Rasband) and Multiple- Kymograph plugin (developed by J. Rietdorf and A. Seitz).

[00205] RT-PCR

[00206] NIH3T3 cells were stimulated with 3 ^M purmorphamine in the presence of eggmanone or DMSO for 24-hours. Cells were collected and RNA isolated with RNeasy kit (Qiagen, Valencia, CA). After subsequent cDNA amplification using Superscript III (Invitrogen, Carlsbad, CA), samples were quantified by comparing Q- PCR cycle thresholds (Ct) for gene expression normalized to GAPDH. The following TaqMan probe and primer sets (Applied Biosystems) were used: GAPDH

(Mm99999915_g1), and Patch1 (Mm01306905_mi).

[00207] cAMP assay

[00208] Shh-Light2 cells were seeded in a 96-well plate and incubated overnight. Varying concentrations of Rolipram or Eggmanone (0.013uM– 30uM) or DMSO only were added to the cells in the absence of serum and incubated 30 minutes, at which time forskolin was added for a final concentration of 1uM. After an additional 15 minutes, cells were washed with PBS and assayed for cAMP levels using EIA based chemiluminescence kit according to the manufacturer’s protocol (Cell Signaling Technologies, Danvers, MA).

[00209] Target profiling assays for kinases, GPCRs and phosphatases

[00210] Profiling assays were not performed in-house. Compounds were shipped to the following companies for possible target identification: Kinase profiling assays were performed by DiscoverRx (San Diego, CA) using a phage display model; GPCR profiling assays were performed by Millipore (St. Louis, MO) using in cells expressing G_α15, a promiscuous G protein that enhances GPCR coupling to downstream Ca²⁺ signaling pathways; phosphatase profiling assay was performed by Millipore (Dundee, UK).

[00211] PDE assays

[00212] In vitro PDE profiling and dose-response assays were performed by BPS Biosciences (San Diego, CA). Kinetic Mechanism of inhibition studies were conducted by Millipore (St. Louis, MO). In brief, the PDE assay measures fluorescent polarization of FAM-AMP as FAM-cAMP is converted to FAM-AMP by PDE), the binding agent. [00213] A series of dilutions of the test compound were prepared with 10% DMSO in assay buffer and 5µl of the dilution was added to a 50µl reaction so that the final concentration of DMSO is 1% in all of reactions. All of the PDE enzymatic reactions were conducted in duplicate at room temperature for 60 minutes in a 50µl mixture containing PDE assay buffer (10mM Tris-HCl, pH7.4, 10mM MaCl2, 0.05% Tween 20), 100nM FAM-cAMP, a PDE enzyme and a test compound.

[00214] After the enzymatic reaction, 100 µl of a binding solution (1:100 dilution of the binding agent, which contains the nano beads that recognize FAM-AMP, with the binding agent diluent) was added to each reaction and the reaction was performed at room temperature for 60 minutes. Fluorescence intensity was measured at an excitation of 485 nm and an emission of 528 nm using a Tecan Infinite M1000 microplate reader. Assays done by Millipore were conducted similiarly with changes noted below:

[00215] Cell viability assay

[00216] Cancer cell lines were seeded in 96 well tissue culture plate at a low density (10,000 cells per well) and treated with varying concentrations of Eggmanone. After 72hr incubation, CellTiter-Blue Cell Viability Assay (Promega, Madison, WI) was then performed according to manufacturer’s protocol. Absorbance was then measured in a Modulus Microplate reader (Promega, Madison, WI) at 590nm and compared to cells treated with DMSO. [00217] Example 2

[00218] Anti-Cancer Effect

[00219] Hedgehog signaling has been implicated in cancer formation and progression; therefore the present inventors assayed the effect of Eggmanone on various cancer lines. With reference to Fig.14, the present inventors found that the prostate cancer cell line PC3 is affected, and the medulloblastoma cell line DAOY and colon cancer cell lines HCT116 and RKO are significantly inhibited. [00220] It has been shown that Eggmanone has anti-proliferative effects in multiple cancer cell lines. There is growing literature that suggests that PDE4 would make an attractive target in a variety of cancers including brain, lung, and even chemo resistant colon cancers. In addition to anti proliferative effects inhibition of PDE4 has been linked to inhibition of VEGF (Vascular endothelial growth factor) which is essential for angiogenesis. As such Eggmanone could serve as an anti-tumor, anti- angiogenic, anti-metastatic, agent in the treatment of cancer. To this end, the present inventors assayed a series of clinically relevant cancer lines and assayed the anti- proliferative properties of a small cohort of eggmanone analogs. These gave a range of EC50s from 4nM-8.4uM.

[00221] Cancer cell lines were seeded in 96well tissue culture plate at a low density and treated with varying concentrations of compounds identified in Table 7. After 72hr incubation, CellTiter-Blue Cell Viability Assay (Promega, Madison, WI) was then performed according to manufacturer’s protocol. Absorbance was then measured in a Modulus Microplate reader (Promega, Madison, WI) at 590nm and compared to cells treated with DMSO.

Adenocarcinoma MCF7 0.483 uM inhibition 27 uM

[00222] Example 3

[00223] Anti-Viral Effect

[00224] PDE4 was found to be functionally up-regulated in human T- lymphotropic virus infected T-cells and may contribute to the virus-induced

proliferation. Furthermore selective blocking of PDE4 activity inhibited IL-2R expression and thereby led to abolishing HIV-1 DNA nuclear import in memory T cells. Additionally there have been recent implications of PDE4 playing major important roles in the infection process of respiratory syncytial virus (RSV), Dengue, and cowpox. With reference to Figs.15 and 16, the present inventors have experimentally shown that Eggmanone has antiviral effects on, RSV, Influenza, Dengue, and BVDV. [00225] Example 4 [00226] Hh Signaling Inhibition and PDE4 Inhibition of Various Compounds.

[00227] Hedgehog signaling inhibition and PDE4 inhibition of various compounds disclosed herein was assayed as described herein above. The following data, provided in Table 8, were obtained.

Formula (11) Formula (12)

Formula (19) Formula (20) [00228] Example 5

[00229 General S nthesis of Meth lall lamine Com ounds

[00230] Cyclohexanone was reacted with methyl cyanoacetate, S₈ and diethylamine in ethanol as previously reported to provide the 2-aminothiophene in 49% yield.¹ Formation of the dithiocarbamate was effected with C₂S and NaOH in DMSO followed by reaction with dimethylsulfate to give the methyl dithiocarbamate, as previously reported.^2,3 Treatment with methylallylamine•HCl effected cyclization to 4 in 61% yield. S-alkylation was performed with one of two methods, where X = aryl, heteroaryl, dialkylamine.

[00231] Method 1. To a solution of 4 (0.171 mmol, 1.0 eq) in CH₃CN (2.0 mL) was added 2-(chloroacetyl)X (0.260 mmol, 1.5 eq) and Cs₂CO₃ (0.260 mmol, 1.5 eq) and the reaction was heated via microwave irradiation at 70 ºC for 10 minutes. Addition of water caused precipitation of the desired product.

[00232] Method 2. To a solution of chloroacetyl chloride (0.26 mmol, 1.0 eq) in CH₂Cl₂ (1.5 mL) under argon atmosphere was added amine (0.26 mmol, 1.0 eq) and Et₃N (0.31 mmol, 1.2 eq) and the reaction was stirred at RT for 3 hours. Solvent was removed in-vacuo. The crude product (0.260 mmol, 1.5 eq) was added as a solution in CH₃CN (1.0 mL) to a solution of 4 (0.171 mmol, 1.0 eq) in CH₃CN (1.0 mL). To the mixture was added Cs₂CO₃ (0.260 mmol, 1.5 eq) and the reaction was heated via microwave irradiation at 70 ºC for 10 minutes. Addition of water caused precipitation of the desired product, which if necessary, was purified by flash column chromatography.

[00233] Example 6

[00234] General S nthesis of All lamine Com ounds

[00235] Cyclohexanone was reacted with methyl cyanoacetate, S₈ and diethylamine in ethanol as previously reported to provide the 2-aminothiophene in 49% yield.¹ Formation of the dithiocarbamate was effected with C₂S and NaOH in DMSO followed by reaction with dimethylsulfate to give the methyl dithiocarbamate, as previously reported.^2,3 Treatment with allylamine effected cyclization to 4 in 61% yield. S-alkylation was performed with one of two methods, where X = aryl, heteroaryl, dialkylamine.

[00236] Method 1. To a solution of 4 (0.171 mmol, 1.0 eq) in CH₃CN (2.0 mL) was added 2-(chloroacetyl)X (0.260 mmol, 1.5 eq) and Cs₂CO₃ (0.260 mmol, 1.5 eq) and the reaction was heated via microwave irradiation at 70 ºC for 10 minutes. Addition of water caused precipitation of the desired product.

[00237] Method 2. To a solution of chloroacetyl chloride (0.26 mmol, 1.0 eq) in CH₂Cl₂ (1.5 mL) under argon atmosphere was added amine (0.26 mmol, 1.0 eq) and Et₃N (0.31 mmol, 1.2 eq) and the reaction was stirred at RT for 3 hours. Solvent was removed in-vacuo. The crude product (0.260 mmol, 1.5 eq) was added as a solution in CH₃CN (1.0 mL) to a solution of 4 (0.171 mmol, 1.0 eq) in CH₃CN (1.0 mL). To the mixture was added Cs₂CO₃ (0.260 mmol, 1.5 eq) and the reaction was heated via microwave irradiation at 70 ºC for 10 minutes. Addition of water caused precipitation of the desired product, which if necessary, was purified by flash column chromatography. [00238] Example 7

[00239] This Examples describes further procedures conducted to synthesize and characterize Eggmanone. Unless stated otherwise, the methods utilized in this Example are the same as the methods described in Example 1. Thus, to avoid undue repetition, the methods described in Example 1 are not restated in this Example.

[00240] A phenotypic screen for small molecule modulators of zebrafish pattern formation identified a series of structurally related compounds, represented by the prototype named eggmanone (3-(2-methylallyl)-2-((2-oxo-2-(thiophen-2- yl)ethyl)thio)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine-4(3H)-one) (Figs.5 and 6), which caused a number of phenotypes resembling those of Hh-deficient mutant embryos: ventral tail curvature, absent pectoral fins, small eyes, loss of neurocranial chondrogenesis, impaired slow muscle formation, and enlarged, rounded somites (Fig. 17a, b; Fig.7a-c). Eggmanone (EGM) abrogated the expression of the Hh target gene patched-1 (ptch1) in the bud-stage adaxial cells, pectoral fin fields, and the somites (Fig. 17c, d). However, eggmanone did not eliminate ptch1 expression in the ventral neural tube or myotome cells adjacent to the notochord (Fig.7d). Moreover, nkx2.2-expressing neurons in the ventral neural tube were not abolished in eggmanone-treated embryos, indicating that Hh inhibition was context-dependent (Fig.7e). Since the zebrafish ventral neural tube patterning is relatively insensitive to ciliary dysfunction, these selective effects of eggmanone suggest a mechanism of action that is cilia dependent.

[00241] In the mouse Hh reporter cell line Shh-Light2, eggmanone inhibited Hh-inducible Gli-responsive luciferase (Gli-Luc) activity in a dose dependent manner, confirming that the molecular target is conserved in mammals (Fig.17e). Eggmanone also blocked Gli-Luc reporter and ptch1 induction by purmorphamine, a Smo agonist, indicating that eggmanone targeted the Hh pathway at or downstream of Smo activation (Fig.17f,g). By contrast, eggmanone did not affect BMP-responsive luciferase reporter activity, indicating that Hh reporter inhibition was not due to nonspecific effects on luciferase activity (Fig.17h). Additionally, eggmanone did not block Gli-Luc reporter activity in cells transiently overexpressing Gli2 (Fig.17i), thus ruling out indirect, non- Hh related effects downstream of Gli function.

[00242] To identify the molecular target of eggmanone, we utilized the LASSO (“Ligand Activity by Surface Similarity Order”) algorithm to virtually screen for potential targets. This algorithm implicated cGMP-specific PDE5 (Fig.9a), presumably based on the similarity of the eggmanone’s core structure to guanine (Fig.9b, c). We assayed eggmanone for in vitro activity against eleven different PDE family members and found to our surprise that it significantly inhibited only the cAMP specific PDE4 family (Fig.18). Eggmanone significantly inhibited isoforms from each gene within the PDE4 (A-D) family (Fig.18b, c), with an IC₅₀ (concentration causing 50% of maximal inhibition) range of 0.80-3.75 ^M. The enzymatic activities of specific PDE4 isoforms did not reach 0% even at high eggmanone concentrations (Fig.18c). Eggmanone had minimal effect (<10% inhibition at 50 ^M) on PDE1A1, PDE5A1, PDE6C, PDE7A1, PDE8A1, PDE9A1, and PDE10A2, and the IC50s for PDE2A, PDE3A and PDE11A4 were well above 50 ^M (Fig.18a, b), indicating that EGM is highly selective for the PDE4 family. Based on these measurements, eggmanone is at least 60-fold more potent against PDE4D3 than any of the tested PDE not belonging to the PDE4 family.

[00243] The PDE4 gene family consists of 4 genes (PDE4A, B, C, D), each containing upstream conserved regions, UCR1 (55 A.A) and UCR2 (78 A.A) that are unique to the PDE4 family. Of the seven isoforms of PDE4s tested, only the super-short isoform PDE4D2, which contains a truncated UCR2 domain, was not inhibited by eggmanone (Fig.18b). Since the UCR2 domain is unique to all of the PDE4 family, this result provides a molecular explanation for eggmanone’s selectivity toward PDE4 isoforms, and suggested that eggmanone might interact with an allosteric site on the UCR2 domain. To ascertain the mode of inhibition, kinetic studies were undertaken using purified PDE4D3, and the results were plotted in the double reciprocal

Lineweaver-Burk plot (Fig 18d; Figs.19-21). Eggmanone exhibited a competitive mode of inhibition on PDE4D3. As discussed below, the results indicate that eggmanone is a selective PDE4 inhibitor with a unique mechanism of action that interacts with both the catalytic and the UCR2 domains.

[00244] To rule out other potential targets, we tested eggmanone against other pharmacologically relevant classes of biomolecules using a comprehensive panel of 442 kinases, 158 GPCRs and 21 phosphatases; remarkably, eggmanone exhibited no significant inhibition (> 10 μM) against any of these targets (Tables 4-6). Additionally, we conducted a small-scale structure activity relationship (SAR) study of eggmanone analogs. Of 12 analogs tested in both Hh-reporter assay and in vitro PDE4 assay, we found a strong correlation between each analog’s ability to inhibit PDE4 and its ability to block Hh (Fig.22a). Consistent with the idea that PDE4 antagonism was responsible for Hh signal inhibition, we found that Rolipram, a structurally unrelated competitive PDE4 inhibitor, could block Hh signaling as well (Fig. 23). Interestingly, even though Rolipram is a far more potent PDE4 inhibitor than eggmanone in vitro ²⁹, Rolipram’s effect on Hh signaling was incomplete even at high concentrations. Furthermore, to confirm the interaction between PDE4 and the Hh pathway in vertebrates, the long isoform PDE4D3 was transfected into Shh-Light2 reporter cells and was found to increase Hh signaling, which was abrogated by the presence of eggmanone (Fig.22b). Finally, a dominant negative construct consisting of a catalytically inactive PDE4D3 inhibited Hh signaling (Fig.22b). Taken together, these results indicate the

pharmacological inhibition of PDE4 activity is central to Hh inhibition by eggmanone and its analogs.

[00245] Although eggmanone and its analogs block the hydrolytic activity of PDE4 in purified enzyme assays (Fig.18), eggmanone surprisingly did not increase total cAMP levels in cells at the concentrations that abolish Hh signaling (Fig 24a). By contrast, rolipram elicited robust cAMP accumulation and the allosteric PDE4 inhibitor D159153 elicited moderate cAMP accumulation (Fig.24a). These observations, together with the fact that eggmanone did not abolish neural tube patterning, led us to consider whether eggmanone only increases local cAMP levels in or near the cilium. While there is no known technique to directly visualize local cAMP levels within cilium, the frequency and the amplitude of beating cilium are modulated by cAMP levels.

When zebrafish embryos were treated with 2 ^M eggmanone, the otic kinocilium became markedly less motile (Fig.24b). Since this concentration does not elicit a global cAMP change, this result suggests that eggmanone selectively modulates the cAMP levels localized within a microdomain associated with the cilium.

[00246] Without being bound by theory or mechanism, the centrosome, which also forms the basal body of the primary cilium and plays a central role in cilium biogenesis and function, was the cAMP microdomain targeted by eggmanone.

Consistent with prior reports, in NIH3T3 cells over-expressing a VSV-tagged PDE4D3, PDE4D3 co-localized to the base of the cilium in physical association with AKAP450, a scaffolding protein which also anchors PKA to the basal body (Fig.11). Eggmanone treatment did not disrupt PDE4D3 localization or physical association with AKAP450 (Fig.11b). These results support the notion that eggmanone promotes local cAMP accumulation by specifically inhibiting the PDE4s, such as PDE4D3, which are localized to the basal body.

[00247] To visualize changes in cAMP concentrations in individual cells and cellular regions, we utilized two distinct FRET (fluorescence resonance energy transfer)- based cAMP sensors: the Epac-FRET sensor (mTurquoiseΔ-Epac(CD, ΔDEP)-cp173 Venus-Venus)³⁵, which detects cytosolic cAMP concentration and the PKA-based cAMP sensor (PKAC-YFP and PKARII-CFP combination), which has been used to document changes in local cAMP levels in the centrosome and basal body. In accordance with the cell lysate data, we found by using the Epac-FRET sensor that rolipram treatment (2 ^M) significantly increased the FRET signal throughout the cell (Fig.4c). By contrast, eggmanone treatment (2 ^M) had no effect on the cytosolic FRET signal (Fig.24c). Using the PKA-based cAMP sensor we found that eggmanone treatment (2 ^M) increased cAMP levels only at discrete regions, presumably

corresponding to the centrosome/basal body, without affecting cAMP levels elsewhere in the cell (Fig.24d).

[00248] Since PKA is a critical downstream mediator activated by cAMP, we next examined the spatial distribution of PKA activation following eggmanone treatment. Immunostaining for the autophosphorylated active form of the PKA catalytic subunit demonstrated that eggmanone significantly increased the intensity of PKA activation almost exclusively in the basal body, which was marked with the ^-tubulin antibody (Figs.2, 12, and 25-26). This differed dramatically from a more diffuse increase in phospho-PKA staining following treatment with the competitive PDE4 inhibitor rolipram, the allosteric inhibitor D159153³⁰ (Fig.26), and the cAMP analog dibutyril cAMP, which induced the dispersion of PKA from the centrosome and more uniform PKA activation in the cell (Fig.26). Thus, eggmanone is functionally unique in its ability to increase cAMP levels and PKA activation precisely in the basal body.

[00249] In vertebrate cells, forskolin antagonizes Hh signaling by preventing ciliary localization of Gli and subsequent Gli-mediated transcription¹⁵. While this effect was attributed to PKA activation, it may be mediated via a PKA-independent mechanism as forskolin blocked ciliary translocation of Gli2 in PKA-null embryonic fibroblasts. By contrast, eggmanone did not prevent Gli2 localization to the primary cilium (Fig.27a). In fact, quantification of the intensity of Gli2 staining within the primary cilium revealed that more Gli2 accumulated in eggmanone-treated cilium than in controls (Fig.27b). Importantly, eggmanone blunted the nuclear accumulation of the full-length Gli2 (Gli2FL) induced by SAG, a Smo agonist, indicating that cAMP accumulation at the basal body blocked Gli2 trafficking from the primary cilium to the nucleus (Figs.27c-e).

[00250] To investigate whether the disruption of the cilium-to-nucleus trafficking of Gli2 by eggmanone was due to a general defect in the retrograde transport within the primary cilium, we compared the effect of the cytoplasmic dynein motor inhibitor ciliobrevin D with the effect of eggmanone on the intraflagellar transport protein 88 (IFT88) trafficking¹⁹. Unlike ciliobrevin D, which severely disrupted the IFT88 localization in the cilium and is known to disrupt cilium morphology, eggmanone had no effect on IFT88 localization or cilium morphology (Fig.27c). Thus, the effects of eggmanone on Gli2 trafficking is specific, rather than an indirect consequence of a global defect in ciliary transport machinery.

[00251] Eggmanone represents a novel class of selective small molecules that inhibit Hh signaling and is a potentially new way to treat diseases caused by aberrant Hh activation³⁷. Eggmanone efficiently and selectively killed SmoM2-Light cells, which stably overexpress the constitutively active, oncogenic Smo mutant, and are resistant to the Smo antagonist cyclopamine (Fig.3f). Eggmanone had no effect on parental NIH3T3 cells. Moreover, eggmanone potently and preferentially reduced the viability of hedgehog and PDE4 dependent human medulloblastoma Daoy cells (Fig.3g) by blocking proliferation and inducing apoptosis (Fig.3h, i).

[00252] Based on the findings, it is proposed that (Fig.13): Hh activation requires trafficking of Gli through the primary cilium, where Gli becomes activated. Eggmanone targets PDE4s localized to the basal body, preventing the normal clearance of cAMP resulting in elevated cAMP levels at or near the cilium base. This in turn leads to the local activation of PKA in the basal body, where it prevents trafficking of Gli activator from the cilium to the nucleus. We postulate that the basal body, which contains the supramolecular complex comprised of both the mediator PKA and the negative regulator PDE4, functions as a“cAMP barrier” and a“signaling rheostat”: as a barrier, the basal body functionally isolates periciliary signal transduction events from cAMP fluctuations in the rest of the cell ³³, and as a rheostat, the basal body sets the threshold cAMP levels required for transduction or suppression of upstream signals emanating from the primary cilium. Eggmanone, by selectively raising the cAMP levels in the basal body, resets the“rheostat” to turn off Hh signaling.

[00253] PDE4 possesses a flexible structure, in which the UCR2 domain folds across the catalytic pocket, in essence to form a“cap” which modulates access to and binding efficiency in the catalytic pocket ⁴⁸. Interestingly, the UCR2-capped and uncapped states appear to be mediated by the phosphorylation status mediated by PKA, with phosphorylation by PKA favoring the uncapped (fully open) state, promoting cAMP degradation and conferring a negative feedback regulation on the PKA activity. While rolipram’s affinity for the catalytic pocket is independent of the UCR2-uncapped or capped states, eggmanone may exhibit a tighter affinity in the UCR2-capped state, abrogating negative feedback regulation of PKA.

[00254] PDE4 also exists as a multimeric complex with the potential for both intramolecular and intermolecular capping and that association with scaffold proteins promote the monomeric conformation⁴⁹. Since eggmanone causes cAMP accumulation only at the basal body, to which various PDE4 isoforms are found in associations with scaffold proteins, we propose that eggmanone is an unusual conditional PDE4 inhibitor whose in vivo activity is dependent on enzyme confirmations conferred by subcellular localization. [00255] Example 8

[00256] This Example describes procedures conducted to evaluate the effectiveness of the present compounds and composition for treating heart failure and the like.

[00257] As shown below (Figs.28 and 29), ionotropic effects are seen within 30 minutes of eggmanone administration to a mouse (20mg/kg IP injection). The ionotropic effects to Eggmanone treatment were observed in the absence of a

chronotropic response (Fig 28). This compound also does not increase the heart rate in mice, and mice treated with EGM exhibited no significant side effects and returned back to baseline heart function within 24 hrs of treatment (Fig.29). Mechanistically compound EGM targets the hydrolase PDE4. In Human adult myocardium, PDE4 localizes strictly to the z-bands.

[00258] In fibroblasts, PDE4 localized to the subcellular organelle called the centrosome (Fig.30). The addition of EGM to fibroblasts caused a spatially restricted activation of PKA around the centrosome without raising total cellular cAMP content (Fig.31). Likewise, allosteric inhibition of PDE4 in the heart lead to localized activation of PKA around the Z-disc without raising total cellular cAMP content.

[00259] To observe whether the effects of eggmanone administration are cardiomyocyte specific or due to off target effects, the contractility of individual mouse cardiomyocytes and the tone of ascending/descending aorta was observed. In mouse cardiomyocytes, Egm caused a 50% increase in contractility over vehicle control (Fig. 32). The substantial increase in contractility with EGM (10 ^M) was not associated with alterations in calcium handling in isolated mouse cardiomyocytes (Fig.33). EGM also increased contractile function in human induced pluripotent stem cell derived

cardiomyocytes (hiPSC-CMs) indicating that EMG will increase contractility in human myocytes (Fig.34).

[00260] For myography, to test the vascular tone for presense of downstream or off target effects, mouse aorta was mounted and cannulated on a closed system. A physiological buffer (with respect to pH, CO_2, and temperature) was circulated through the vessel. Drugs or compounds known to cause vessel constriction (e.g., KCl) or dilation were added to buffer, and the vessel was observed for change in diameter (Fig. 35). After pre-constriction, Egm administration had no effect on the vessel. However, Rolipram caused the vessel to dilate (Figs.35 and 36). These data illustrate that Egm may be acting directly on cardiomyocytes to cause left ventricular constriction rather than acting upon the vascularature leading to a pre-load effect.

[00261] Thus, allosteric PDE4 inhibitors can be used to cause localized activation of PKA without increasing total cAMP content, and the use of a novel class of PDE4 inhibitors with unique mechanism of action to increase cardiac inotropy without chronotropy. Moreover, as this approach does not involve increase in total cAMP content and global PKA activation, the proposed invention of the use of allosteric PDE4 inhibitors for heart failure will increase cardiac output without tachycardia, and without concern for tachyphylaxis and heart failure progression upon chronic administration. [00262] Example 9

[00263] Compounds in Tables 9A and 9B were generated according to schemes set forth herein, in the specification.

[00264] Hh EC₅₀ Gli-Luc refers to treatment of stably transfected NIH-3T3 cells incorporating a Gli promoter-driven firefly luciferase and constitutively active renilla luciferase with multiple concentrations of inhibitor compound from a 10 mM DMSO stock solution and estimation of half-maximal effective inhibitory concentration.

[00265] ZF refers to wild-type embryonic zebrafish phenotypic assay involving dosing n = ~10 embryos in E3 egg water with compound from either a stock of 1 mM or 10 mM in DMSO at 5 hours post-fertilization and observing at 24, 48, and 72 hours post- fertilization. The 50% maximal effective concentration was determined by the concentration of compound at which embryos exhibited the identical phenotype compared to eggmanone-treated embryos.

[00266] Hh % Inh. refers to assaying C3H10T1/2 cells for reduction in SAG- induced (100 nM) Gli1 expression caused by inhibitors after 24 hours at either 10 µM, 1 µM, or five concentrations to determine EC₅₀. Compounds are dosed from 10 mM DMSO stock solutions, and mRNA is isolated after 24 hours of compound treatment. mRNA is reverse transcribed to produce cDNA which is quantified by quantitative polymerase chain reaction (qPCR) in triplicate and levels are normalized to GAPDH levels. Data is presented as percent inhibition compared to positive control (SAG).

[00267] TM3 Gli Luciferase, C3H10T_1/2 qPCR, Gli1 mRNA; Sufu Null (Ptc), PDE4D3, and PDE4D2 data is included for compounds where analyzed. Methods utilized are according to the methods and procedures discussed herein, in the

specification.

[00268] Example 10

[00269] General synthesis of Thienopyrimidine Compounds

[00270] Thienopyrimidines with general structure 1 were synthesized in approximately 5 steps from commercially available starting materials. R₁ and R₂ most commonly exist as a fused cyclohexyl ring.

Figure 1 describes the general synthetic scheme for 1 where R₁ = R₂ = cyclohexyl, Boc- piperidine, or Y = O, S. Where Y=O,S, the R3-NCS procedure was utilized.

Figure 1. General synthesis of 1.

[00271] In Scheme 1, where Y = NBoc, the Boc group was removed with trifluoroacetic acid in DCM. The secondary amine was functionalized through either reaction with a sulfonyl chloride in the presence of base, through amide formation with the R₆-carboxylic acid, or through reductive amination with the R₈-aldehyde.

[00272] Where R₁ = R₂ = H, scheme 2 was utilized, and Scheme 1 was followed upon formation of the 2-aminothiophene shown in Scheme 2, through the route employing dithiourea synthesis and amine substitution.

Scheme 2. General synthesis of 1 where R₁ = R₂ = H or R₁ = Ar, R₂ = H.

[00273] Where R₁ = Ar, R₂ = H, Scheme 2 was followed, involving mono-Boc protection of the 2-aminothiophene, 2-position bromination and Suzuki cross coupling during which Boc group deprotection also occurred. All examples of R₁ = Ar employed R₃-NCS formation of the R₃-thiourea, and Scheme 1 was followed for the remainder of the synthesis.

[00274] Synthesis of 2 followed the general scheme 3. In each case, reaction with the isothiocyanate directly formed the cyclic thiourea.

Scheme 3. General synthesis of 2 where X = C, N.

[00275] R₃ derived from either the free amine through cyclization with the dithiourea of Scheme 1 or from the isothiocyanate through direct reaction with the 2- aminothiophene.

[00276] R₄ derived from S-alkylation of the cyclic thiourea with primary alkyl halides. Where R₄ derives from a 2-haloacetyl starting material, the starting material was purchased from commercial suppliers. Where R₄ derives from a substituted 2- haloacetamide, the 2-haloacetamide was synthesized from 2-chloroacetyl chloride and either a primary or secondary amine.

[00277] Compound 3-159 was synthesized as shown in Scheme 4 from the 2- aminocyclohexylthiophene by reacting with the cyanoacetate with 4 M HCl in dioxane. No other compounds were synthesized using this method.

Scheme 4. Cyclization of to provide 3-159.

[00278] Benzothiophenes were synthesized following Scheme 5 and upon aromatization and deprotection, were elaborated according to Scheme 1.

Scheme 5.2-Aminobenzothiophene synthesis. [00279] Substitutions for the thiopyrimidinone S-linkage were performed by nucleophilic substitution with the requisite chloropyrimidinone to provide O-linked and N-linked analogs as shown in Scheme 6. Conditions slightly varied depending on the nature of the X group.

Scheme 6. Synthesis of S-linked substitution analogs.

[00280] Chemical characterization and biological data are included for representative compounds in the specification.

[00281] Example 11

[00282] It is anticipated that compounds disclosed herein could serve as an anti-tumor, anti-angiogenic, anti-metastatic, agent in the treatment of cancer. To this end, a series of clinically relevant cancer lines were assayed and the cell-killing EC₅₀s for compounds according to the subject matter disclosed herein are provided in Table 10.

[00283] Example 12

[00284] PDE4 as a target for RSV

[00285] The small molecule PDE4 inhibitors of the presently disclosed subject matter are actively anti-viral in viral CPE (cytopathic effect) assays versus RSV

(respiratory syncytial virus), Dengue (1 experiment), and BVDV (bovine viral diarrhea virus, surrogate for human hepatitis C virus). Of note, PDE4 inhibitors are now approved for COPD, for which RSV may be an exacerbating factor).

[00286] As a treatment for Hepatitis C Virus:

[00287] Provided in Fig.37 are the results from BVDV (Bovine Viral Diarrhea Virus, surrogate for Hepatitis C virus) CPE (cytotoxic effect) testing done. The assay was repeated with H1913 (a PDE4B and PDE4D inhibitor ). Hi913 (our prototypic PDE4 inhibitor) was tested in half-log concentrations ranging from 100 µM to 0.33 µM. As the stock solution of H1913 was 10 mM, this meant that the final DMSO

concentrations for the highest Hi913 concentrations were 1%, 0.33%, and 0.1%. The normal final DMSO concentrations used is 0.1%, so additional DMSO controls of 1% and 0.33% were included. The Hi913 data for the highest 3 concentrations is normalized to the respective DMSO concentrations. Note that at 3.3 to 10 µM, our PDE4 inhibitor blocked cytopathic effects of BVDV by ~60 and ~75%, respectively. The outlier effects at 100 µM are probably due to cytotoxicity at the high drug concentration.

[00288] Anti-RSV Effects of PDE4 inhibitor

[00289] RSV is an enveloped single (-) stranded RNA virus, which is the most common cause of severe respiratory illness in children, responsible for majority (70%) of bronchiolitis. RSV infection is the most common cause of hospitalization in USA of young children up to the first year of life. Globally, there are 33 million new cases of RSV each year, responsible for deaths of 66,000 to 199,000 children each year. In addition, elderly over 65-years old and immunocompromised individuals are at increased risk for severe respiratory disease from RSV. In the elderly, symptomatic respiratory illness due to RSV is associated with high morbidity and mortality (11.9%), responsible for 10,000 deaths each year in US alone. Currently, there is no targeted therapy against RSV and treatment remains supportive.

[00290] In Table 11, the results of CPE assays following infection of human epidermoid cancer cells (Hep-2) with RSV. Even at 10,000 higher viral titers, our compound achieved complete inhibition at 10 µM. At 1 and 3 µM, our compound achieved over 98% reduction. Our compound alone caused no apparent cytotoxicity at these concentrations.

[00291] Example 13

[00292] An unbiased zebrafish in vivo chemical genetic screen for small molecule developmental patterning modulators identified EGM1, which phenocopied the loss of Hh zebrafish mutant. In vitro, EGM1 inhibited Hh target gene transcription downstream of SMo and functioned epistatic to the Gli transcription factor regulator Suppressor of Fused (SuFu), as provided in Fig.39. The SAR and hit to lead efforts, as presented in Figs.40and 41 and target identification campaign, are positioned to identify an improved downstream of Smo probe of Hh signaling. Initial appendage and core scaffold SAR indicated narrow parameters for potency improvement while focusing on optimization of solubility properties and elimination of metabolic liabilities. However, a series of cyclopropanes exhibited up to three-fold EC₅₀ reduction and slight solubility optimization. These compounds can serve as intermediates toward identification of a downstream Smo Hh inhibitor, which will be useful for treatment of non-Gorlin syndrome oncogenic mutations and Smo inhibitor resistance. [00293] Throughout this document, various references are mentioned. All such references are incorporated herein by reference, including the references set forth in the following list: REFERENCES

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Claims

What is claimed is : 1. A com ound of the formula:

or pharmaceutically-acceptable salts thereof, wherein

X is selected from C, N, O, and S;

,

,

; so long as when R₂ is

2. The compound of claim 1, according to a formula selected from the group consisting of:

, or pharmaceutically-acceptable salts thereof.

3. The compound of claim 1, according to a formula selected from the group consisting of:

pharmaceutically-acceptable salts thereof.

4. The compound of claim 1, according to the formula:

or pharmaceutically-acceptable salts thereof.

5. The com ound of claim 1 according to the formula:

or pharmaceutically-acceptable salts thereof.

6. The com ound of claim 1 according to the formula:

or pharmaceutically-acceptable salts thereof.

7. The com ound of claim 1 accordin to the formula:

acceptable salts thereof.

8. The com ound of claim 1 according to the formula:

or pharmaceutically-acceptable salts thereof.

9. The com ound of claim 1 according to the formula:

pharmaceutically-acceptable salts thereof.

10. The compound of claim 1, according to the formula:

, or pharmaceutically-acceptable salts thereof.

11. The com ound of claim 1 according to the formula:

or pharmaceutically-acceptable salts thereof.

12. The compound of claim 1, according to the formula:

or pharmaceutically-acceptable salts thereof.

13. The com ound of claim 1 accordin to the formula:

ceptable salts thereof.

14. A compound of the formula:

15. A compound of the formula:

,

R₇ is selected from

16. A compound according to the formula

pharmaceutically-acceptable salt thereof.

17. A compound of the formula:

or pharmaceutically-acceptable salts thereof, wherein R₁ is se

H, and

and

R₂ is selected from

18. A compound of the formula:

19. The compound of claim 14, according to a formula selected from the group consisting of:

,

.

20. A pharmaceutical composition, comprising a pharmaceutically-acceptable carrier; and a compound of any one of claims 1-19 or pharmaceutically-acceptable salts thereof.

21. The pharmaceutical composition of claim 20, and further comprising a second compound or composition having Hh signaling inhibition activity, PDE4 inhibition activity, anti-cancer or anti- tumor activity, anti-angiogenic activity, anti-metastatic activity, anti-heart failure activity, and/or anti-inflammation activity, or wherein the second compound or composition is useful for treating a condition of interest.

22. A kit, comprising a compound according to any one of claims 1-19; and a device for administration of the compound or composition.

23. A kit, comprising a compound according to any one of claims 1-19; and further comprising a second compound or composition having Hh signaling inhibition activity, PDE4 inhibition activity, anti-cancer or anti-tumor activity, anti-angiogenic activity, anti-metastatic activity, anti-heart failure activity, and/or anti-inflammation activity, or wherein the second compound or composition is useful for treating a condition of interest.

24. The kit of claim 23, and further comprising a device for administration of the

compound or composition and/or a device for administration of the second compound or composition.

25. A method of inhibiting hedgehog signaling in a cell, comprising contacting a cell with an effective amount of a compound of any one of claims 1-19 or a compound of the formula:

, or pharmaceutically-acceptable salts thereof, wherein

R

₂ is group consisting of:

H, R₁ is and when R₂ is

26. The method of claim 25, wherein contacting the cell with the compound comprises administering the compound or composition to a subject.

27. The method of claim 26, wherein the administration is to a subject in need of treatment for a condition of interest.

28. A method of inhibiting phosphodiesterase-4 (PDE-4) in a cell, comprising contacting a cell with an effective amount of a compound of any one of claims 1-19.

29. The method of claim 28, wherein contacting the cell with the compound comprises administering the compound or composition to a subject.

30. The method of claim 29, wherein the administration is to a subject in need of treatment for a condition of interest.

31. A method of treating a condition of interest, comprising contacting a cell with an effective amount of a compound of any one of claims 1-19.

32. The method of claim 31, wherein contacting the cell with the compound comprises administering the compound or composition to a subject.

33. The method of claim 32, wherein the administration is to a subject in need of treatment for a condition of interest.

34. The method of any one of claims 25-33, and further comprising making use of a kit of any one of claims 22-24.