WO2016040951A1 - Composés et procédés d'inhibition de la signalisation hedgehog et de la phosphodiestérase - Google Patents

Composés et procédés d'inhibition de la signalisation hedgehog et de la phosphodiestérase Download PDF

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WO2016040951A1
WO2016040951A1 PCT/US2015/050024 US2015050024W WO2016040951A1 WO 2016040951 A1 WO2016040951 A1 WO 2016040951A1 US 2015050024 W US2015050024 W US 2015050024W WO 2016040951 A1 WO2016040951 A1 WO 2016040951A1
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Prior art keywords
compound
formula
activity
eggmanone
cell
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PCT/US2015/050024
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English (en)
Inventor
Charles C. Hong
Charles H. Williams
Jonathan HEMPEL
Tk Feaster
Audrey FRIST
Don H. RUBIN
Gary SULIKOWSKI
Jijun Hao
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Vanderbilt University
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Priority to US15/452,551 priority Critical patent/US10329304B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4365—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system having sulfur as a ring hetero atom, e.g. ticlopidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim

Definitions

  • 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
  • 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;
  • 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.
  • Hh signaling pathway is normally tightly regulated, becoming activated only in precise locations and at precise times.
  • 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.
  • 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.
  • the presently-disclosed subject matter includes a compound.
  • the compound is of the formula:
  • X is selected from C, N, O, and S;
  • R 1 is selected from CH 2 CH 3, (CH 2 ) 2 CH 3 ,
  • the compound is according to a formula selected from the group consisting of:
  • the compound is according to a formula selected from the group consisting of: , ,
  • the compound is according to the formula:
  • the compound is according to the formula::
  • the compound is according to the formula:
  • the compound is according to the formula:
  • the compound is according to the formula:
  • the compound is according to the formula:
  • the compound is according to the formula:
  • the compound is according to the formula:
  • the compound is according to the formula: or pharmaceutically-acceptable salts thereof.
  • the compound is according to the formula:
  • the compound is of the formula: or pharmaceutically-acceptable salts thereof.
  • R 4 is selected from
  • R 5 is selected from CH 3
  • R 6 is selected from H
  • the compound is of the formula:
  • R 7 is selected from
  • the compound is of the formula:
  • the compound is of the
  • R 1 is selected from H, and and R 2 is selected from
  • the compound is of the formula:
  • R 1 is selected from
  • the presently-disclosed subject matter further includes a pharmaceutical composition.
  • the pharmaceutical composition includes a pharmaceutically-acceptable carrier; and a compound as disclosed herein.
  • 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.
  • 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.
  • 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.
  • the presently-disclosed subject matter further includes methods.
  • a method of inhibiting hedgehog signaling in a cell includes contacting a cell with an effective amount of a compound or pharmaceutical composition, as disclosed herein.
  • contacting the cell with the compound comprises administering the compound or composition to a subject.
  • the administration is to a subject in need of treatment for a condition of interest.
  • the condition of interest is related to heart failure.
  • the condition of interest is related to PDE4 activity, cancer, angiogenesis, tumorigenisis or tumor activity, metastasis and/or inflammation.
  • a method of inhibiting phosphodiesterase-4 (PDE-4) in a cell includes contacting a cell with an effective amount of a compound or pharmaceutical composition, as disclosed herein.
  • contacting the cell with the compound comprises administering the compound or composition to a subject.
  • administration is to a subject in need of treatment for a condition of interest.
  • a method of treating a condition of interest includes contacting a cell with an effective amount of a compound or pharmaceutical
  • composition as disclosed herein.
  • contacting the cell with the compound comprises administering the compound or composition to a subject.
  • the administration is to a subject in need of treatment for a condition of interest.
  • 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;
  • 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.
  • 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.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).
  • 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).
  • PH3 phospho-histone H3
  • 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.
  • Figure 5 includes a mass spectrometry analysis of Eggmanone.
  • Figure 6 includes an NMR spectra analysis of eggmanone.
  • 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)
  • FIG. 8 includes data and results of studies showing that Eggmanone affects hedgehog signaling but does not affect BMP signaling. (Fig.8a) Eggmanone
  • Figure 9 includes results from LASSO algorithm. Top, Molecular surface descriptor of eggmanone, with search algorithm results. Bottom, Results of search.
  • 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.
  • 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.
  • 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.
  • PKA cAMP-dependent protein kinase
  • 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. 12e Correlation coefficients from studies in (Fig.12c).
  • Fig.12f Graphic comparison of correlation coefficients found in Fig 6e.
  • Figure 13 depicts a model for Eggmanone mechanism of action.
  • SHH Hh ligand
  • Gli-R repressor form
  • Gli-activ. full- length activator
  • 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.
  • 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.
  • Figure 14 is a graph showing anticancer effect of Eggmanone on various cancer cell lines.
  • 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.
  • BVDV Bovine Viral Diarrhea Virus, surrogate for Hepatitis C virus
  • CPE cytotoxic effect
  • 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.
  • RSV respiratory syncytial virus
  • 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;
  • 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
  • FIG. 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 IC 50 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.
  • Figure 19 includes a chart of a PDE 4D3 enzyme linearity study showing that inhibition of PDE4 with Egm occurs in a linear manner.
  • Figure 20 includes a Eadie Hofstee plot showing that Egm acts in a competitive manner.
  • Figure 21 includes a graph showing Km versus Egm concentration, wherein the linear relationship suggests that Egm acts in a competitive manner.
  • FIG. 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.
  • 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.
  • SHH Sonic hedgehog
  • Gli-Luc Gli- luciferase reporter activity
  • Figure 24 includes data and graphs showing that Eggmanone causes local perturbations in cAMP levels without affecting global cellular cAMP content.
  • ECM Eggmanone
  • 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,
  • 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).
  • FRET values are the mean calculated within an ROI drawn to include the entire cytosolic area or the centrosome.
  • Figure 25 includes images and graphs showing that Eggmanone (EGM) treatment results in PKA activation restricted to the basal bodies. (Fig.25a)
  • 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.
  • DBA cAMP analog dibutyril cAMP
  • 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.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).
  • 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.
  • Figure 28 includes an echocardiogram of a mouse after having been administered 20mg/kg Egm via an intraperitoneal injection.
  • 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.
  • FS fractional shortening
  • LVIDd end-diastolic left ventricular internal dimension
  • Figure 30 includes images showing that addition of Egm causes local activation of PKA around PDE4 localization.
  • Figure 31 includes a graph showing the concentration of total cAMP levels after administration with DMSO, Rolipram (Rol), and an embodied Egm (HI913).
  • Figure 32 includes a graph showing the effects of Egm on the contractibility of isolated mouse cardiomyocytes in comparison to a vehicle control (VEH).
  • VH vehicle control
  • Figure 33 includes a graph showing calcium handling results from mice that had been administered with VEH, EGM, or ISO.
  • Figure 34 includes a graph showing the effects of Egm on the contractibility of human cardiomyocytes derived from induced pluripotent stem cells (iPSCs).
  • iPSCs induced pluripotent stem cells
  • 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.
  • Figure 36 includes a graph of an ascending aorta myography.
  • 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.
  • Figure 38 includes a schematic of the Hedgehog Signaling Pathway.
  • 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
  • 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.
  • 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 EC 50 based on compounds administered.
  • 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.
  • the presently-disclosed subject matter includes compounds, pharmaceutical compositions, kits, and methods for using same.
  • the compounds, pharmaceutical compositions, kits, and methods are useful for inhibiting hedgehog (Hh) signaling and/or inhibiting phosphodiesterase 4.
  • the presently-disclosed subject matter includes a compound having a structure represented by the formula: , or pharmaceutically-acceptable salts thereof, wherein
  • X is selected from C, N, O, and S;
  • R 1 is selected from CH 2 CH 3, (CH 2 ) 2 CH 3 ,
  • R 2 is selected from CH 3 , , ,
  • the compound has a formula selected from the group set forth in Table 2, or pharmaceutically-acceptable salts thereof.
  • the compound has a formula selected from the group set forth in Table 1B, or pharmaceutically-acceptable salts thereof.
  • the compound has a formula selected from the group consisting of:
  • the compound has the formula
  • the compound has the formula: or pharmaceutically-acceptable salts thereof.
  • the compound has the formula: , or
  • the compound has the formula: or pharmaceutically-acceptable salts thereof. In some embodiments, the compound has the formula: , or pharmaceutically-
  • 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
  • the compound has the formula: , or
  • the compound has the
  • the compound has the formula: or pharmaceutically- acce table salts thereof. In some embodiments, the compound has the formula:
  • the compound has a structure of the formula:
  • R 4 is selected from ; ,
  • the compound has a formula selected from the group set forth in Table 1C, or pharmaceutically-acceptable salts thereof.
  • the compound has a structure of the formula:
  • R 7 is selected from .
  • the compound has a formula selected from the group set forth in Table 1D, or pharmaceutically-acceptable salts thereof.
  • the compound has a formula selected from the group set forth in Table 1E, or pharmaceutically-acceptable salts thereof.
  • the com ound has a formula selected from the
  • the compound has a formula set forth herein, including in the Examples.
  • the presently-disclosed subject matter further includes pharmaceutical compositions of the compounds as disclosed herein, and further includes a pharmaceutically-acceptable carrier.
  • 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.
  • Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides).
  • 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.
  • 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.
  • 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.
  • the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • 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.
  • 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).
  • 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 star
  • 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).
  • 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
  • compositions 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.
  • buccal administration the compositions can take the form of tablets or lozenges formulated in conventional manner.
  • the compounds can also be formulated as a preparation for
  • 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).
  • 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.
  • rectal compositions e.g., suppositories or retention enemas containing conventional suppository bases such as cocoa butter or other glycerides
  • creams or lotions e.g., cocoa butter or other glycerides
  • transdermal patches e.g., transdermal patches.
  • the pharmaceutical composition includes a compound as disclosed herein or pharmaceutically-acceptable salts thereof.
  • the pharmaceutical composition includes a the compound of Formula (1), or pharmaceutically-acceptable salts thereof.
  • the pharmaceutical composition includes a the compound of Formula (3), or pharmaceutically-acceptable salts thereof.
  • the pharmaceutical composition includes a the compound of Formula (5), or pharmaceutically-acceptable salts thereof.
  • the pharmaceutical composition includes a the compound of Formula (6), or pharmaceutically-acceptable salts thereof.
  • the pharmaceutical composition includes a the compound of Formula (7), or pharmaceutically-acceptable salts thereof.
  • the pharmaceutical composition includes a the compound of Formula (8), or pharmaceutically-acceptable salts thereof.
  • 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).
  • 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.
  • 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.
  • the addition of the second compound or composition provides for a synergistic response.
  • kits including a compound or pharmaceutical composition.
  • 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.
  • the kit includes a compound, or a
  • composition including a compound as disclosed herein.
  • kits can include a compound or pharmaceutical composition as described herein, packaged together with a device useful for
  • the appropriate administration aiding device will depend on the formulation of the compound or composition that is selected and/or the desired administration site.
  • the device could be a syringe.
  • the device could be a sterile pipette.
  • kits 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.
  • 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.
  • the addition of the second compound or composition provides for a synergistic response.
  • kits comprising a reagent to carry out a method as described hereinbelow.
  • the presently-disclosed subject matter further includes methods.
  • a method of inhibiting hedgehog signaling is provided.
  • the method includes contacting a cell with an effective amount of a compound or pharmaceutical composition as disclosed herein.
  • contacting the cell with the compound or composition comprises administering the compound or composition to a subject.
  • 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.
  • Also provided is a method of inhibiting phosphodiesterase-4.
  • the method includes contacting a cell with an effective amount of a compound or pharmaceutical composition as disclosed herein.
  • contacting the cell with the compound or composition comprises administering the compound or composition to a subject.
  • 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.
  • the method includes contacting a cell with an effective amount of a compound or pharmaceutical composition as disclosed herein.
  • contacting the cell with the compound or composition comprises administering the compound or composition to a subject.
  • 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.
  • 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.
  • a target such as a decreasing Hh signaling or decreasing PDE4 activity
  • 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
  • treatment refers 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.
  • 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.
  • 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.
  • 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.
  • 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
  • a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition.
  • a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.
  • an “effective amount” refers to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition.
  • 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.
  • 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
  • a preparation can be administered in a“prophylactically effective amount”; that is, an amount effective for prevention of a disease or condition.
  • 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.
  • 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.
  • 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.
  • 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.
  • Hh signaling has been shown to inhibit cancer cell proliferation and to reduce tumor size in Xenograft models.
  • Hh pathway has been shown to be required in tissue mesenchyme surrounding pancreatic cancers to support tumor growth by a paracrine effects.
  • blocking Hh signaling has been shown to suppress metastasis of pancreatic and prostate cancers.
  • 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.
  • 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.
  • 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
  • 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.
  • AML acute myeloid leukemia
  • ALL-B cell B-cell acute lymph
  • 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/).
  • some embodiments of the presently-disclosed subject matter provide for a personalized approach to determining a pathway signature of an individual subject’s neoplasm.
  • Hh inhibitors including compounds and compositions of the presently-disclosed subject matter, can be a used to treat the cancer.
  • 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.
  • Hh pathway plays a key role in postnatal tissue homeostasis and regeneration.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • the compounds and compositions disclosed herein can be used in anti-inflammatory treatment.
  • 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.
  • VEGF Vascular endothelial growth factor
  • 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.
  • PDE4 was found to be functionally up-regulated in human T- lymphotropic virus-infected T-cells and may contribute to the virus-induced
  • 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.
  • Heart failure 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.
  • treatment options are largely palliative.
  • positive inotropes like milrinone and dobutamine, which increase heart contractility, augment function of failing heart in the ICU setting.
  • long-term administration of inotropes is curtailed by tachyphylaxis and increased risk of arrhythmias, heart failure progression and death.
  • 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.
  • cAMP regulation of PKA is emerging as a major regulator of cardiac contraction.
  • 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.
  • PKA phosphorylation of the ryanodine receptor
  • CREB ryanodine receptor
  • NCX1, KCNQ1 phospholamban
  • PLB phospholamban
  • the present PDE4 inhibitors can be used for the treatment of subjects with systolic heart failure.
  • EGM Eggmanone increases fractional shortening (FS) and ejection fraction (EF) of heart without increasing heart rate.
  • FS fractional shortening
  • EF ejection fraction
  • 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.
  • maladaptive responses to chronic stimulation such as tachyphylaxis and heart failure progression, can be reduced or avoided.
  • 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).
  • 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.
  • 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.
  • 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
  • cytomegalovirus CMV
  • influenza adenovirus
  • adenovirus Herpes virus
  • yeast 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.
  • enveloped RNA viruses such as respiratory syncytial virus, and bronchiolitis (RSV is a leading cause of bronchiolitis)
  • ebola virus ebola virus
  • hepatitis C virus hepatitis C virus
  • Bovine Viral Diarrhea Virus Dengue virus
  • west nile virus west nile virus
  • yellow fever virus measles virus
  • measles virus measles virus
  • Conditions of interest include improved learning in neurofibromatosis type 1 (http://www.ncbi.nlm.nih.gov/pubmed/25176649), Behcet’s syndrome
  • 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.
  • 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.
  • Cyclic AMP 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.
  • AC adenyl cyclase
  • PDEs phosphodiesterases
  • cAMP plays an important, evolutionarily conserved role in Hh regulation.
  • 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.
  • PKA cAMP-activated protein kinase
  • Gli transcription factor
  • PKA cAMP-activated protein kinase
  • PDE4D3 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
  • 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.
  • 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.
  • Fig.3f human medulloblastoma Daoy cells
  • Fig.3g human medulloblastoma Daoy cells
  • Fig.3h human medulloblastoma Daoy 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).
  • 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).
  • 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.
  • 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.
  • 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).
  • Tg(nkx2.2:egfp) were maintained using standard protocols.
  • Shh-Light2 cells stably transfected with Gli- Luciferase reporter construct were used along with Shh-conditioned media, as previously described 7 .
  • 3 ⁇ M purmorphamine or 20 nM Smoothened agonist (SAG) was used to induce Hh signaling.
  • Reporter cells were seeded in 96-well plates and incubated overnight with the various SAGs (Santa Cruz Biotechnology, Santa Cruz, CA)
  • 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).
  • NIH3T3 cells were plated on Poly-D-Lysine-coated glass coverslips and were cultured at 37°C, 5% CO 2 in DMEM medium containing 10% fetal bovine serum until reaching 75% confluency.
  • DMEM medium containing 10% fetal bovine serum
  • 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.
  • cell medium was replaced with DMEM/ 0.5% FBS containing either 5 ⁇ M eggmanone or DMSO and incubated overnight at 37°C, 5% CO 2 .
  • Cells were fixed in 4% PFA at room temperature for 10 minutes prior to
  • Cells were fractionated using NE-PER Nuclear and Cytoplasmic extraction reagents (Thermo Scientific, Rockford, IL) per the manufacturer’s protocol.
  • NE-PER Nuclear and Cytoplasmic extraction reagents Thermo Scientific, Rockford, IL
  • goat anti-Gli2 R & D Systems
  • rabbit anti-Lamin-A/C Cell Signaling Technology
  • 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).
  • 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).
  • 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
  • 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).
  • 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 ⁇ 15 , a promiscuous G protein that enhances GPCR coupling to downstream Ca 2+ signaling pathways; phosphatase profiling assay was performed by Millipore (Dundee, UK).
  • PDE assay buffer 10mM Tris-HCl, pH7.4, 10mM MaCl2, 0.05% Tween 20
  • 100nM FAM-cAMP 100nM FAM-cAMP
  • PDE enzyme 100nM PDE enzyme and a test compound.
  • 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
  • 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.
  • VEGF Vascular endothelial growth factor
  • Eggmanone could serve as an anti-tumor, anti- angiogenic, anti-metastatic, agent in the treatment of cancer.
  • 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.
  • 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.
  • PDE4 was found to be functionally up-regulated in human T- lymphotropic virus infected T-cells and may contribute to the virus-induced
  • Method 1 To a solution of 4 (0.171 mmol, 1.0 eq) in CH 3 CN (2.0 mL) was added 2-(chloroacetyl)X (0.260 mmol, 1.5 eq) and Cs 2 CO 3 (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.
  • 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.
  • 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.
  • PDE4A, B, C, D genes that are unique to the PDE4 family.
  • UCR1 55 A.A
  • UCR2 78 A.A
  • Fig.18b the super-short isoform
  • 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.
  • kinetic studies were undertaken using purified PDE4D3, and the results were plotted in the double reciprocal
  • 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.
  • eggmanone is functionally unique in its ability to increase cAMP levels and PKA activation precisely in the basal body.
  • forskolin antagonizes Hh signaling by preventing ciliary localization of Gli and subsequent Gli-mediated transcription 15 . 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).
  • 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 activation 37 .
  • 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.
  • 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).
  • 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.
  • 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.
  • 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 .
  • 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.
  • 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.
  • 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 conformation 49 . 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
  • hiPSC-CMs cardiomyocytes
  • 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 Example 9
  • Hh EC 50 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.
  • the 50% maximal effective concentration was determined by the concentration of compound at which embryos exhibited the identical phenotype compared to eggmanone-treated embryos.
  • 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 ⁇ M, 1 ⁇ M, or five concentrations to determine EC 50 .
  • 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).
  • 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
  • R 1 Ar
  • R 2 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 R 1 Ar employed R 3 -NCS formation of the R 3 -thiourea, and Scheme 1 was followed for the remainder of the synthesis.
  • R 3 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.
  • R 4 derived from S-alkylation of the cyclic thiourea with primary alkyl halides.
  • R 4 derives from a 2-haloacetyl starting material, the starting material was purchased from commercial suppliers.
  • R 4 derives from a substituted 2- haloacetamide, the 2-haloacetamide was synthesized from 2-chloroacetyl chloride and either a primary or secondary amine.
  • the small molecule PDE4 inhibitors of the presently disclosed subject matter are actively anti-viral in viral CPE (cytopathic effect) assays versus RSV
  • Fig.37 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 ⁇ M to 0.33 ⁇ M. As the stock solution of H1913 was 10 mM, this meant that the final DMSO
  • Hi913 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 ⁇ M, our PDE4 inhibitor blocked cytopathic effects of BVDV by ⁇ 60 and ⁇ 75%, respectively. The outlier effects at 100 ⁇ M are probably due to cytotoxicity at the high drug concentration.
  • 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.
  • elderly over 65-years old and immunocompromised individuals are at increased risk for severe respiratory disease from RSV.
  • 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.
  • EGM1 An unbiased zebrafish in vivo chemical genetic screen for small molecule developmental patterning modulators identified EGM1, which phenocopied the loss of Hh zebrafish mutant.
  • 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.
  • endothelial growth factor act upstream of the Notch pathway during arterial endothelial differentiation.
  • Hedgehog-Gli signaling in neural stem cells and brain tumors J Neurobiol, 2005.64(4): p.476-90.
  • Thayer, S.P., et al., Hedgehog is an early and late mediator of pancreatic cancer
  • Phosphodiesterase 4 inhibitors prevent cytokine secretion by T lymphocytes by inhibiting nuclear factor-kappaB and nuclear factor of activated T cells activation. J Pharmacol Exp Ther.2001 Nov;299(2):753-9.
  • Favot L Keravis T
  • Lugnier C Modulation of VEGF-induced endothelial cell cycle protein expression through cyclic AMP hydrolysis by PDE2 and PDE4. Thromb Haemost.2004 Sep;92(3):634-45.
  • mutations are alleles of protein kinase A that modulate hedgehog signaling. Genetics 167, 783–796 (2004).
  • Hedgehog signaling is required for cranial neural crest morphogenesis and chondrogenesis at the midline in the zebrafish skull. Development 132, 3977– 3988 (2005).
  • Fabian MA Biggs WH,maschineer DK, et al. A small molecule-kinase interaction map for clinical kinase inhibitors. Nat Biotech.2005;23(3):329-336.

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Abstract

L'invention concerne des composés et des compositions, et leurs procédés d'utilisation, qui s'avèrent utiles dans l'inhibition de la signalisation Hedgehog et/ou de l'activité de phosphodiestérase-4.
PCT/US2015/050024 2014-09-12 2015-09-14 Composés et procédés d'inhibition de la signalisation hedgehog et de la phosphodiestérase WO2016040951A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10329304B2 (en) 2014-09-12 2019-06-25 Vanderbilt University Compounds and methods for inhibition of hedgehog signaling and phosphodiesterase
WO2019234237A1 (fr) 2018-06-07 2019-12-12 Forschungsverbund Berlin E.V. Composés hétérocycliques en tant qu'inhibiteurs de phosphoinositide 3-kinase de classe ii
CN113135891A (zh) * 2021-04-15 2021-07-20 南方医科大学 N-芳基-n＇-（2-噻吩基）硫脲（脲）衍生物及其制备方法和用途
JP2022511500A (ja) * 2018-12-06 2022-01-31 コリア リサーチ インスティチュート オブ ケミカル テクノロジー Ｐｄｅ９ａ阻害活性を有する化合物及びその医薬用途

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US20130225610A1 (en) * 2010-04-06 2013-08-29 Marc Hansen Antimetastatic compounds

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10329304B2 (en) 2014-09-12 2019-06-25 Vanderbilt University Compounds and methods for inhibition of hedgehog signaling and phosphodiesterase
WO2019234237A1 (fr) 2018-06-07 2019-12-12 Forschungsverbund Berlin E.V. Composés hétérocycliques en tant qu'inhibiteurs de phosphoinositide 3-kinase de classe ii
JP2022511500A (ja) * 2018-12-06 2022-01-31 コリア リサーチ インスティチュート オブ ケミカル テクノロジー Ｐｄｅ９ａ阻害活性を有する化合物及びその医薬用途
JP7224464B2 (ja) 2018-12-06 2023-02-17 コリア リサーチ インスティチュート オブ ケミカル テクノロジー Ｐｄｅ９ａ阻害活性を有する化合物及びその医薬用途
CN113135891A (zh) * 2021-04-15 2021-07-20 南方医科大学 N-芳基-n＇-（2-噻吩基）硫脲（脲）衍生物及其制备方法和用途
CN113135891B (zh) * 2021-04-15 2022-05-10 南方医科大学 N-芳基-n′-(2-噻吩基)硫脲(脲)衍生物及其制备方法和用途

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