Modulators of exchange proteins directly activated by cAMP (EPACS)

Embodiments of the invention are directed to compounds that inhibit an activity of EP AC proteins and methods of using the same. The inventors have developed a sensitive and robust high throughput screening (HTS) assay for the purpose of identifying EPAC specific inhibitors (Tsalkova et al. (2012) PLOS ONE 7(1):e30441).

TECHNICAL FIELD

Embodiments of the invention are directed to pharmacology, medicine, and medicinal chemistry. Certain embodiments are directed to compounds the modulate EPAC1 and/or EPAC2. Additional embodiments are directed to methods or medicaments using such compounds.

BACKGROUND

Identification and development of compounds capable of selectively targeting components of complex cell-signaling networks in a cell is a major effort of modern pharmacology. Cyclic adenosine monophosphate (cAMP), a prototypic second messenger, is an important component of cell-signaling networks that control numerous biological processes. In addition to its regulatory functions under physiological conditions, cAMP has been implicated in playing a major role in multiple human diseases, including cancer, diabetes, heart failure, and neurological disorders, such as Alzheimer's disease (AD). Therefore, it is not surprising that current pharmacological therapeutics target the cAMP signaling pathway more than any other pathway.

The major physiological effects of cAMP in mammalian cells are transduced by two ubiquitously expressed intracellular cAMP receptor families: the classic protein kinase A/cAMP-dependent protein kinases (PKAs/cAPKs) and the more recently discovered exchange proteins directly activated by cAMP/cAMP regulated guanine nucleotide exchange factors (EPACs/cAMP-GEFs). While a number of pharmacological inhibitors of PKA are available, only a few EPAC specific antagonists/inhibitors have been described. Thus, there remains a need for additional compositions and methods for selectively modulating EPAC1 and/or EPAC2.

SUMMARY

The inventors have developed a sensitive and robust high throughput screening (HTS) assay for the purpose of identifying EPAC specific inhibitors (Tsalkova et al. (2012)PLOS ONE7 (1):e30441). Using this EPAC HTS assay, the inventors have successfully identified several isoform-specific EPAC inhibitors that are capable of blocking biochemical and cellular cAMP-induced EPAC activation (Tsalkova et al. (2012)Proc. Acad. Natl. Sci. USA.109:18613-18618). In addition, the inventors have synthesized and characterized a number of chemical analogs of these EPAC specific inhibitors (ESI) (Chen et al. (2012)Bioorganic&Medicinal Chemistry Letters.22:4038-4043; Chen et al. (2013)J. Med. Chem. In press; Chen et al. (2013)Tetrahedron Lett. In press). Some of these chemical analogs displayed more potent EPAC inhibition activity and better pharmacological properties than the parental compound. These EPAC specific inhibitors will not only provide a powerful pharmacological tool for dissecting the physiological functions of EPAC and for further elucidating the molecular mechanism of cAMP signaling, but also have important impacts on designing potential therapeutics targeting EPAC in diseases where cAMP signaling and EPAC proteins have been implicated. Studies using EPAC1 knockout mouse and ESIs suggest that EPAC1 plays important roles in obesity/diabetes (Yan et al. (2013)Molecular Cellular Biology33:918-926), cancer (Almahariq et al. (2013)Molecular Pharmacology.83:122-128), immune response, infection etc.

Certain embodiments are directed to an isolated Exchange Protein Activated by cAMP (EPAC) modulating compound having a general formula of Formula I:

where L′ is —SO2—, —NH—, or —C(O)—C(CN)═N—NH—; and W′ and W″ are independently substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

Further embodiments are directed to an isolated Exchange Protein Activated by cAMP (EPAC) modulating compound having a general formula of Formula II:

where R1, R2, R3, R4, and R5are independently hydrogen, hydroxyl, halogen, C1-C4alkoxy; substituted or unsubstituted C1-C10alkyl, substituted or unsubstituted C1-C10heteroalkyl, substituted or unsubstituted C5-C7cycloakyl, substituted or unsubstituted C5-C7heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or C1-C5alkylamine; L is —SO2— or —NH—; and W′ is as described above for Formula I. In a further aspect, L is —SO2—. In certain aspects W′ is substituted phenyl or N-containing heteroaryl. In yet another aspect, a nitrogen in the N-containing heteroaryl is attached to L.

An isolated Exchange Protein Activated by cAMP (EPAC) modulating compound having a general formula of Formula III:

Still a further embodiment is directed to an isolated Exchange Protein Activated by cAMP (EPAC) modulating compound having a general formula of Formula IV:

In certain aspect the compound of formula IV is (3,5-Dichloro-phenyl)-(2,4,6-trimethyl-phenyl)-amine (HJC-2-83); p-Tolyl-(2,4,6-trimethyl-phenyl)-amine (HJC-2-89); or (2,5-Dichloro-phenyl)-(2,4,6-trimethyl-phenyl)-amine (HJC-3-38).

Certain embodiments are directed to an isolated Exchange Protein Activated by cAMP (EPAC) modulating compound having a general formula of Formula V:

where R1, R2, R3, R4, and R5are as described in Formula III above; and W′ is as described in Formula I above. In certain aspects, R1, R2, R3, R4, and R5are independently hydrogen, halogen, C1-C10alkyl, or C1-C10heteroalkyl. In certain aspects, W′ is substituted or unsubstituted indole, substituted or unsubstituted azaindole, or substituted or unsubstituted pyrrole. In certain aspects, W′ is unsubstituted indole or unsubstituted azaindole. In a further aspect, W′ is pyrrole substituted with one or more C1-C10alkyl groups. In certain aspects, W′ is 1-ethylpyrrole or 2,4-dimethylpyrrole.

Certain embodiments are directed to compounds of Formula V where R1, R3, and R5are C1-C10alkyl; R2and R4are hydrogen; and W′ is substituted or unsubstituted indole, substituted or unsubstituted azaindole, or substituted or unsubstituted pyrrole. In certain aspects, W′ is unsubstituted indole or unsubstituted azaindole. In a further aspect, W′ is pyrrole substituted with one or more C1-C10alkyl groups. In certain aspects, W′ is 1-ethylpyrrole or 2,4-dimethylpyrrole.

Certain embodiments are directed to compounds of Formula V where R1, R3, and R5are methyl; R2and R4are hydrogen; and W′ is substituted or unsubstituted indole, substituted or unsubstituted azaindole, or substituted or unsubstituted pyrrole. In certain aspects, W′ is unsubstituted indole or unsubstituted 4-, 5-, 6-, or 7-azaindole. In a further aspect, W′ is pyrrole substituted with one or more methyl or ethyl. In certain aspects, W′ is 1-ethylpyrrole or 2,4-dimethylpyrrole.

Certain embodiments are directed to compounds of Formula V where R1and R3are C1-C10alkyl; R2, R4, and R5are hydrogen; and W′ is substituted or unsubstituted indole, substituted or unsubstituted azaindole, or substituted or unsubstituted pyrrole. In certain aspects, W′ is unsubstituted indole or unsubstituted azaindole. In a further aspect, W′ is pyrrole substituted with one or more C1-C10alkyl groups. In certain aspects, W′ is 1-ethylpyrrole or 2,4-dimethylpyrrole.

Certain embodiments are directed to compounds of Formula V where R1and R3are methyl; R2, R4, and R5are hydrogen; and W′ is substituted or unsubstituted indole, substituted or unsubstituted azaindole, or substituted or unsubstituted pyrrole. In certain aspects, W′ is unsubstituted indole or unsubstituted 4-, 5-, 6-, or 7-azaindole. In a further aspect, W′ is pyrrole substituted with one or more methyl or ethyl. In certain aspects, W′ is 1-ethylpyrrole or 2,4-dimethylpyrrole.

Certain embodiments are directed to compounds of Formula V where R2and R4are C1-C10alkyl; R1, R3, and R5are hydrogen; and W′ is substituted or unsubstituted indole, substituted or unsubstituted azaindole, or substituted or unsubstituted pyrrole. In certain aspects, W′ is unsubstituted indole or unsubstituted azaindole. In a further aspect, W′ is pyrrole substituted with one or more C1-C4alkyl groups. In certain aspects, W′ is 1-ethylpyrrole or 2,4-dimethylpyrrole.

Certain embodiments are directed to compounds of Formula V where R2and R4are methyl; R1, R3, and R5are hydrogen; and W′ is substituted or unsubstituted indole, substituted or unsubstituted azaindole, or substituted or unsubstituted pyrrole. In certain aspects, W′ is unsubstituted indole or unsubstituted 4-, 5-, 6-, or 7-azaindole. In a further aspect, W′ is pyrrole substituted with one or more methyl or ethyl. In certain aspects, W′ is 1-ethylpyrrole or 2,4-dimethylpyrrole.

Certain embodiments are directed to compounds of Formula V where R3is C1-C10alkyl; R1, R2, R4, and R5are hydrogen; and W′ is substituted or unsubstituted indole, substituted or unsubstituted azaindole, or substituted or unsubstituted pyrrole. In certain aspects, W′ is unsubstituted indole or unsubstituted azaindole. In a further aspect, W′ is pyrrole substituted with one or more C1-C10alkyl groups. In certain aspects, W′ is 1-ethylpyrrole or 2,4-dimethylpyrrole.

Certain embodiments are directed to compounds of Formula V where R3is methyl; R1, R2, R4, and R5are hydrogen; and W′ is substituted or unsubstituted indole, substituted or unsubstituted azaindole, or substituted or unsubstituted pyrrole. In certain aspects, W′ is unsubstituted indole or unsubstituted 4-, 5-, 6-, or 7-azaindole. In a further aspect, W′ is pyrrole substituted with one or more methyl or ethyl. In certain aspects, W′ is 1-ethylpyrrole or 2,4-dimethylpyrrole.

Certain embodiments are directed to compounds of Formula V where R1is C1-C10alkyl; R2, R3, R4, and R5are hydrogen; and W′ is substituted or unsubstituted indole, substituted or unsubstituted azaindole, or substituted or unsubstituted pyrrole. In certain aspects, W′ is unsubstituted indole or unsubstituted azaindole. In a further aspect, W′ is pyrrole substituted with one or more C1-C10alkyl groups. In certain aspects, W′ is 1-ethylpyrrole or 2,4-dimethylpyrrole.

Certain embodiments are directed to compounds of Formula V where R1is methyl; R2, R3, R4, and R5are hydrogen; and W′ is substituted or unsubstituted indole, substituted or unsubstituted azaindole, or substituted or unsubstituted pyrrole. In certain aspects, W′ is unsubstituted indole or unsubstituted 4-, 5-, 6-, or 7-azaindole. In a further aspect, W′ is pyrrole substituted with one or more methyl or ethyl. In certain aspects, W′ is 1-ethylpyrrole or 2,4-dimethylpyrrole.

Certain embodiments are directed to an isolated Exchange Protein Activated by cAMP (EPAC) modulating compound having a formula of:

where R16is substituted or unsubstituted C1-C10alkyl, substituted or unsubstituted C3-C6heteroalkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted C3-C6heterocyclyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R17is hydrogen, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; X is sulfur or nitrogen; and Y is a direct bond, —CH2—, —CH2C(O)O—, or —CH2C(O)N—. Formula VI represents an alternative embodiment of Formula I, where W′ is a substituted pyrimidine, and L is a particular linker designated by —X—Y—.

Certain embodiments are directed to compounds of Formula VI where X is sulfur; Y is —CH2—; R16is as described above for Formula VI; and R17is as described above for Formula VI. In certain aspects R17is as described above for Formula VI; and R16is (a) C3-C6cycloakyl, (b) C6cycloakyl, (c) C5cycloalkyl, (d) C4cycloalkyl, (e) C3cycloalkyl, (f) branched or linear C1-C10alkyl, or (g) branched C3alkyl. In certain aspects, R17is substituted phenyl. In certain aspects, R17is a C1-C10alkyl substituted phenyl. In further aspects, the substituted phenyl has 1, 2, or 3 C1-C10alkyl substituents. In certain aspects the C1-C10alkyl substituents are at positions 1, 3, and 5; 2 and 5; 2 and 4; 1 and 3; or 3 of the phenyl group. In a further aspect, R17is 3,6-dimethylphenyl; 3,5-dimethylphenyl; or 2,4-dimethylphenyl. In yet a further aspect, R17is 2,4,6-trimethylphenyl.

Certain embodiments are directed to compounds of Formula VI where X is sulfur; Y is —CH2C(O)N—; R16is as described above for Formula VI; and R17is as described above for Formula VI. In certain aspects R17is as described above for Formula VI; and R16is (a) C3-C6cycloakyl, (b) C6cycloakyl, (c) C5cycloalkyl, (d) C4cycloalkyl, (e) C3cycloalkyl, (f) branched or linear C1-C10alkyl, or (g) branched C3alkyl. In certain aspects, R17is substituted phenyl. In certain aspects, R17is a C1-C10alkyl substituted phenyl. In further aspects, the substituted phenyl has 1, 2, or 3 C1-C10alkyl substituents. In certain aspects the C1-C10alkyl substituents are at positions 1, 3, and 5; 2 and 5; 2 and 4; 1 and 3; or 3 of the phenyl group. In a further aspect, R17is 3,6-dimethylphenyl; 3,5-dimethylphenyl; or 2,4-dimethylphenyl. In yet a further aspect, R17is 2,4,6-trimethylphenyl.

Certain embodiments are directed to compounds of Formula VI where X is nitrogen; Y is —CH2—; R16is as described above for Formula VI; and R17is as described above for Formula VI. In certain aspects R17is as described above for Formula VI; and R16is (a) C3-C6cycloakyl, (b) C6cycloakyl, (c) C5cycloalkyl, (d) C4cycloalkyl, (e) C3cycloalkyl, (f) branched or linear C1-C10alkyl, or (g) branched C3alkyl. In certain aspects, R17is substituted phenyl. In certain aspects, R17is a C1-C10alkyl substituted phenyl. In further aspects, the substituted phenyl has 1, 2, or 3 C1-C10alkyl substituents. In certain aspects the C1-C10alkyl substituents are at positions 1, 3, and 5; 2 and 5; 2 and 4; 1 and 3; or 3 of the phenyl group. In a further aspect, R17is 3,6-dimethylphenyl; 3,5-dimethylphenyl; or 2,4-dimethylphenyl. In yet a further aspect, R17is 2,4,6-trimethylphenyl.

Certain embodiments are directed to compounds of Formula VI where X is nitrogen; Y is a direct bond; R16is as described above for Formula VI; and R17is as described above for Formula VI. In certain aspects R17is as described above for Formula VI; and R16is (a) C3-C6cycloakyl, (b) C6cycloakyl, (c) C5cycloalkyl, (d) C4cycloalkyl, (e) C3cycloalkyl, (f) branched or linear C1-C10alkyl, or (g) branched C3alkyl. In certain aspects, R17is substituted phenyl. In certain aspects, R17is a C1-C10alkyl substituted phenyl. In further aspects, the substituted phenyl has 1, 2, or 3 C1-C10alkyl substituents. In certain aspects the C1-C10alkyl substituents are at positions 1, 3, and 5; 2 and 5; 2 and 4; 1 and 3; or 3 of the phenyl group. In a further aspect, R17is 3,6-dimethylphenyl; 3,5-dimethylphenyl; or 2,4-dimethylphenyl. In yet a further aspect, R17is 2,4,6-trimethylphenyl.

Certain embodiments are directed to an isolated Exchange Protein Activated by cAMP (EPAC) modulating compound having a formula of:

in certain aspects W′ and W″ are as described for Formula I above.

In certain embodiments W′ is an unsubstituted or substituted isoxazole. In certain aspects the isoxazole is attached via the 3 position. In certain aspects the substituted isoxazole is a 4-substituted isoxazole, a 5-substituted isoxazole, or a 4,5-substituted isoxazole. In a particular aspect the substituted isoxazole is a 5-substituted isoxazole. In certain aspects the substituent is independently a branched or unbranched C1to C10alkyl. In certain aspect the alkyl is a methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, neo-pentyl, n-pentyl, or isopenyl. In certain embodiments the isoxazole is a 5-methyl or 5 tert-butyl isoxazole. In a further aspect W′ can be a substituted to unsubstituted phenyl.

Certain embodiments are directed to using one or more EPAC modulators to treat or enhance a therapy for a disease or condition associated with EPAC activity.

Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. Each embodiment described herein is understood to be embodiments of the invention that are applicable to all aspects of the invention.

As used herein, the term “IC50” refers to an inhibitory dose that results in 50% of the maximum response obtained.

The term half maximal effective concentration (EC50) refers to the concentration of a drug that presents a response halfway between the baseline and maximum after some specified exposure time.

The terms “inhibiting,” “reducing,” or “prevention,” or any variation of these terms, when used in the claims and/or the specification includes any measurable decrease or complete inhibition to achieve a desired result.

As used herein, an “inhibitor” as described herein, for example, can inhibit directly or indirectly the activity of a protein. The term “EPAC inhibitor” refers to a compound that decreases the activity of EPAC in a cell. In certain aspects an EPAC inhibitor decreases cancer cell or carcinoma migration by any measurable amount, as compared to such a cell in the absence of such an inhibitor. EPAC inhibitors include EPAC1 inhibitors and/or EPAC2 inhibitors.

As used herein, an “activator” as described herein, for example, can increase the activity of a protein. The term “EPAC activator” refers to a compound that increases the activity of EPAC in a cell. EPAC activators include EPAC1 activators and/or EPAC2 activators.

An “effective amount” of an agent in reference to treating a disease or condition means an amount capable of decreasing, to some extent, a pathological condition or symptom resulting from a pathological condition. The term includes an amount capable of invoking a growth inhibitory, cytostatic and/or cytotoxic effect and/or apoptosis of the cancer or tumor cells.

The phrases “treating cancer” and “treatment of cancer” mean to decrease, reduce, or inhibit the replication of cancer cells; decrease, reduce or inhibit the spread (formation of metastases) of cancer; decrease tumor size; decrease the number of tumors (i.e. reduce tumor burden); lessen or reduce the number of cancerous cells in the body; prevent recurrence of cancer after surgical removal or other anti-cancer therapies; or ameliorate or alleviate the symptoms of the disease caused by the cancer.

As used herein, the term “patient” or “subject” refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dogs, cat, mouse, rat, guinea pig, or species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human subjects are adults, juveniles, infants and fetuses.

DESCRIPTION

cAMP-mediated signaling regulates a myriad of important biological processes under both physiological and pathological conditions. In multi-cellular eukaryotic organisms, the effects of cAMP are transduced by the protein kinase A/cAMP-dependent protein kinase (PKA/cAPK) and the exchange protein directly activated by cAMP/cAMP-regulated guanine nucleotide exchange factor (EPAC/cAMP-GEF) (de Rooij et al. (1998)Nature396: 474-477; Kawasaki et al. (1998)Science282: 2275-2279). Since both PKA and EPAC are ubiquitously expressed in all tissues, an increase in intracellular cAMP levels will lead to the activation of both PKA and EPAC. Net physiological effects of cAMP entail the integration of EPAC- and PKA-dependent pathways in a spatial and temporal manner. Depending upon their relative abundance, distribution and localization, as well as the precise cellular environment, the two intracellular cAMP receptors may act independently, converge synergistically, or oppose each other in regulating a specific cellular function (Cheng et al. (2008)Acta Biochim Biophys Sin(Shanghai) 40: 651-662). Therefore, careful dissections of the individual role and relative contribution of EPAC and PKA within the overall cAMP signaling in various model systems are critical for further elucidating the mechanism of cAMP signaling, as well as essential for developing novel mechanism-based therapeutic strategies targeting specific cAMP-signaling components.

Cyclic AMP is a second messenger that induces physiological responses ranging from growth and differentiation to hormonal, neuronal, and immunological regulation (Tasken and Aandahl (2004)Physiol Rev84:137-167; Holz (2004)Diabetes53:5-13). In the brain, it is involved in memory (Huang et al. (1995)Cell83:1211-1222) and cognitive functions (Sur and Rubenstein (2005)Science310:805-810). There are two forms of EPAC, EPAC1 and EPAC2, which are encoded by separate genes, EPAC1 and EPAC2, respectively. EPAC1 is expressed ubiquitously with predominant expression in the thyroid, kidney, ovary, skeletal muscle, and specific brain regions. EPAC2 is predominantly expressed in the brain and adrenal gland (de Rooij et al. (1998)Nature396:474-477; Kawasaki et al. (1998)Science282:2275 2279).

Embodiments described herein are directed to compounds that modulate EPAC1 and/or EPAC2. Certain embodiments are directed to compounds that specifically modulate EPAC2 or EPAC1. Further embodiments are directed to methods and medicaments for treating EPAC associated diseases or conditions.

I. High Throughput EPAC Assay

The inventors developed a fluorescence-based high throughput assay for screening EPAC specific antagonists (Tsalkova et al. (2012)PLoS. ONE.7: e30441). The assay is highly reproducible and simple to perform using the “mix and measure” format. A pilot screening led to the identification of small chemical compounds capable of specifically inhibiting cAMP-induced EPAC activation while not affecting PKA activity, i.e., EPAC specific inhibitors (ESI).

Primary Screen Assay—

Fluorescence intensity of 8-NBD-cAMP in complex with EPAC2 is used as the readout in the primary screen assay. Primary screen is performed in black 96-well or 384-well microplates. As an example, 50 nM EPAC2 solution is prepared in 20 mM Tris buffer, pH 7.5, containing 150 mM NaCl, 1 mM EDTA, and 1 mM DDT. 8-NBD-cAMP is added to EPAC2 solution up to 60 nM from a stock solution in water. Sample is dispensed into plate and test compounds added from 96-well mother plates. Samples with cAMP addition and no additions are used as a positive and a negative control. Fluorescence intensity signal from 8-NBD was recorded at room temperature (rt) before and after tested compounds are added using SpectaMaxM2 microplate reader (Molecular Devices, Silicon Valley, Calif., USA) with excitation/emission wavelengths set at 470/540 nm.

Secondary Confirmation Assay—

Measurement of in vitro guanine nucleotide exchange factor (GEF) activity of EPAC was adapted from a well known fluorescence-based assay using a fluorescent guanine nucleotide analog (van den Berghe et al. (1997)Oncogene15: 845-850), and used as a functional confirmation assay for the compounds identified from primary screen. Briefly, 0.2 μM of Rap1B(1-167) loaded with the fluorescent GDP analog (Mant-GDP), was incubated with EPAC in 50 mM Tris buffer pH 7.5, containing 50 mM NaCl, 5 mM MgCl2, 1 mM DTT, and a 100-fold molar excess of unlabeled GDP (20 μM) in the presence of various concentrations of test compound and 25 μM cAMP. Exchange of Mant-GDP by GDP was measured as a decrease in fluorescence intensity over time using a FluoroMax-3 spectrofluorometer with excitation/emission wavelengths set at 366/450 nm. Typically, decay in the fluorescence intensity was recorded over a time course of 6000 s with data points taken every 60 s.

Counter Screening Assay—

Kinase activity of the type I and II PKA holoenzyme are measured spectrophotometrically in a 96-well plate with a coupled enzyme assay as described previously (Cook et al. (1982)Biochemistry21: 5794-5799). In this assay, the formation of ADP is coupled to the oxidation of NADH by the pyruvate kinase/lactate dehydrogenase reactions so the reaction rate can be determined by following the oxidation of NADH, reflected by a decrease in absorbance at 340 nm. The kinase reaction mixture (100 μl) contains 50 mM Mops (pH 7.0), 10 mM MgCl2, 1 mM ATP, 1 mM PEP, 0.1 mM NADH, 8 U of pyruvate kinase, 15 U of lactate dehydrogenase, fixed amount of type I or type II PKA holoenzyme, and 0.1 mM cAMP, with or without 25 μM of test compound. Reactions are pre-equilibrated at room temperature and initiated by adding the Kemptide substrate (final concentration 0.26 mM). PKA activity measured in the presence of 25 μM H89, a selective PKA inhibitor, are used as a positive control of PKA inhibition.

Once a compound is identified as having an EPAC modulating activity, a number of analogs and variations are designed to produce an EPAC inhibitor with appropriate pharmacologic characteristics.

II. Chemical Definitions

Various chemical definitions related to EPAC modulating compounds are provided as follows.

As used herein, “predominantly one enantiomer” means that the compound contains at least 85% of one enantiomer, or more preferably at least 90% of one enantiomer, or even more preferably at least 95% of one enantiomer, or most preferably at least 99% of one enantiomer. Similarly, the phrase “substantially free from other optical isomers” means that the composition contains at most 5% of another enantiomer or diastereomer, more preferably 2% of another enantiomer or diastereomer, and most preferably 1% of another enantiomer or diastereomer. In certain aspects, one, both, or the predominant enantiomer forms or isomers are all covered.

As used herein, the term “nitro” means —NO2; the term “halo” or “halogen” designates —F, —Cl, —Br or —I; the term “mercapto” means —SH; the term “cyano” means —CN; the term “azido” means —N3; the term “silyl” means —SiH3, and the term “hydroxy” means —OH.

The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a linear (i.e. unbranched) or branched carbon chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbons, which may be fully saturated, monounsaturated, or polyunsaturated. An unsaturated alkyl group includes those having one or more carbon-carbon double bonds (alkenyl) and those having one or more carbon-carbon triple bonds (alkynyl). The groups, —CH3(Me, methyl), —CH2CH3(Et, ethyl), —CH2CH2CH3(n-Pr, n-propyl), —CH(CH3)2(iso-Pr, iso-propyl), —CH2CH2CH2CH3(n-Bu, n-butyl), —CH(CH3)CH2CH3(sec-butyl), —CH2CH(CH3)2(iso-butyl), —C(CH3)3(tert-butyl), —CH2C(CH3)3(neo-pentyl), are all non-limiting examples of alkyl groups.

The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a linear or branched chain having at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, S, P, and Si. In certain embodiments, the heteroatoms are selected from the group consisting of O, S, and N. The heteroatom(s) may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Up to two heteroatoms may be consecutive. The following groups are all non-limiting examples of heteroalkyl groups: trifluoromethyl, —CH2F, —CH2Cl, —CH2Br, —CH2OH, —CH2OCH3, —CH2OCH2CF3, —CH2OC(O)CH3, —CH2NH2, —CH2NHCH3, —CH2N(CH3)2, —CH2CH2Cl, —CH2CH2OH, CH2CH2OC(O)CH3, —CH2CH2NHCO2C(CH3)3, and —CH2Si(CH3)3.

The term “alkoxy” means a group having the structure —OR′, where R′ is an optionally substituted alkyl or cycloalkyl group. The term “heteroalkoxy” similarly means a group having the structure —OR, where R is a heteroalkyl or heterocyclyl.

The term “amino” means a group having the structure —NR′R″, where R′ and R″ are independently hydrogen or an optionally substituted alkyl, heteroalkyl, cycloalkyl, or heterocyclyl group. The term “amino” includes primary, secondary, and tertiary amines.

The term “oxo” as used herein means oxygen that is double bonded to a carbon atom.

The term “pharmaceutically acceptable salts,” as used herein, refers to salts of compounds of this invention that are substantially non-toxic to living organisms. Typical pharmaceutically acceptable salts include those salts prepared by reaction of a compound of this invention with an inorganic or organic acid, or an organic base, depending on the substituents present on the compounds of the invention.

Suitable pharmaceutically acceptable salts may also be formed by reacting the agents of the invention with an organic base, such as methylamine, ethylamine, ethanolamine, lysine, ornithine and the like. Pharmaceutically acceptable salts include the salts formed between carboxylate or sulfonate groups found on some of the compounds of this invention and inorganic cations, such as sodium, potassium, ammonium, or calcium, or such organic cations as isopropylammonium, trimethylammonium, tetramethylammonium, and imidazolium.

It should be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable.

Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, Selection and Use (2002), which is incorporated herein by reference.

An “isomer” of a first compound is a separate compound in which each molecule contains the same constituent atoms as the first compound, but where the three dimensional configuration of those atoms differs. Unless otherwise specified, the compounds described herein are meant to encompass their isomers as well. A “stereoisomer” is an isomer in which the same atoms are bonded to the same other atoms, but where the configuration of those atoms in three dimensions differs. “Enantiomers” are stereoisomers that are mirror images of each other, like left and right hands. “Diastereomers” are stereoisomers that are not enantiomers.

It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

III. Methods of Using EPAC Modulators

Cyclic adenosine monophosphate (cAMP) is an important component of cell-signaling networks that control numerous biological processes. More than a decade of extensive studies have now firmly established that many cAMP-related cellular processes, previously thought to be controlled by PKA alone, are also mediated by EPAC (Gloerich and Bos, (2010)Annu Rev Pharmacol Toxicol50:355-375). For example, EPAC proteins have been implicated in regulating exocytosis and secretion (Ozaki et al. (2000)Nat Cell Biol2:805-811; Seino and Shibasaki (2005)Physiol Rev85:1303-1342; Maillet et al. (2003)Nat Cell Biol5:633-639; Li et al. (2007)Mol Endocrinol21:159-171), cell adhesion (Enserink et al. (2004)J Biol Chem279:44889-44896; Rangarajan et al. (2003)J Cell Biol160:487-493), endothelial barrier junctions (Cullere et al. (2005)Blood105:1950-1955; Kooistra et al. (2005)FEBS Lett579:4966-4972), leptin signaling, and cardiac functions (Metrich et al. (2010)Pflugers Arch459:535-546). In addition to its regulatory functions under physiological conditions, cAMP has been implicated in playing a major role in multiple human diseases, including cancer, diabetes, heart failure, and neurological disorders, such as Alzheimer's disease (AD). The EPAC1 and/or EPAC2 modulating compounds described herein can be used to provide treatment for a variety of diseases or conditions associated with EPAC activation or inhibition.

A. Cancer Therapy

Certain aspects are directed to treating cancer or cancer metastasis in a subject by administering an EPAC inhibitor.

Like PKA, EPAC contains an evolutionarily conserved cAMP-binding domain that acts as a molecular switch for sensing intracellular levels of the second messenger cAMP, and activates the down-stream signaling molecules small GTPases Rap1 and Rap2 (de Rooij et al. (1998)Nature396:474-477; Kawasaki et al. (1998)Science282:2275-2279). In addition, EPAC proteins exert their functions through interactions with other cellular partners at specific cellular locations. For example, EPAC1 is known to associated with mitotic spindle, plasma membrane and nuclear membrane by interacting with tubulin (Qiao et al. (2002)J Biol Chem277:26581-26586; Mei and Cheng (2005)J Biol Chem277:11497-11504), ezrin-radixin-moesin (ERM) proteins (Gloerich et al. (2010)Mol Cell Biol30:5421-5431; Ross et al. (2011)J Cell Sci124:1808-1818) and nucleoporin RanBP2 (Liu et al. (2010)Mol Cell Biol30:3956-3969; Gloerich et al. (2011)J Cell Biol193:1009-1020), respectively. On the other hand, EPAC2 can interact with Rim (Rab3 interacting molecule) and Rim2 (Kashima et al. (2001)J Biol Chem276:46046-46053; Ozaki et al. (2000)Nat Cell Biol2:805-811), as well as a structurally related calcium sensor Piccolo (Fujimoto et al. (2002)J Biol Chem277:50497-50502). In pancreatic beta cells, interactions among EPAC2, Rim2 and Piccolo are critical for cAMP-mediated insulin secretion (Ozaki et al. (2000)Nat Cell Biol2:805-811; Kashima et al. (2001)J Biol Chem276:46046-46053; Fujimoto et al. (2002)J Biol Chem277:50497-50502).

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human diseases, largely due to the fact that pancreatic cancer is resistant to treatments that are usually effective for other types of cancer. A better understanding of the molecular mechanism of PDAC development and metastasis and effective therapeutics are desperately needed. Recently, it has been shown that EPAC1 is markedly elevated in human PDAC cells as compared with normal pancreas or surrounding tissue (Lorenz et al. (2008)Pancreas37:102-103). EPAC1 has been implicated in promoting cellular proliferation in prostate cancer (Misra and Pizzo (2009)J Cell Biochem108:998-1011; Misra and Pizzo (2011)J Cell Biochem112(6):1685-95) and migration and metastasis in melanoma (Baljinnyam et al. (2011)Pigment Cell Melanoma Res24:680-687; Baljinnyam et al. (2009)Am J Physiol Cell Physiol297:C802-C813; Baljinnyam et al. (2010)Cancer Res70:5607-5617).

EPAC inhibitor ESI-09 is used to demonstrate a functional role for EPAC1 overexpression in pancreatic cancer cell migration and invasion. These findings are consistent with similar results based on RNAi silencing techniques, suggesting that EPAC 1 is a target for therapeutic strategies in PDAC and other cancers.

In certain embodiments an EPAC inhibitor can be administered for the treatment of cancer. In certain aspects the cancer is pancreatic cancer, prostate cancer, melanoma, bladder cancer, blood cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, liver cancer, lung cancer, nasopharynx cancer, ovarian cancer, stomach cancer, testicular cancer, or uterine cancer. In still a further aspect the cancer is pancreatic cancer, particularly pancreatic ductal adenocarcinoma (PDAC). In certain aspects the EPAC inhibitor is selected from the EPAC inhibitors described herein. In a further aspect the EPAC inhibitor is an EPAC 1 inhibitor.

Certain methods are directed to modulating the innate or adaptive immune system of a subject by administering an EPAC modulator. In a further aspect, methods include enhancing an immune response in a subject by administering an EPAC inhibitor. The immune response can be directed to microbes (fungi, virus, bacteria, and the like); abnormal or aberrantly functioning cells, such as cancer cells or hypersensitive immune effectors; or other pathological conditions that would benefit from an enhanced immune response. Immune modulation is a critical aspect for the treatment of a number of diseases and disorders. T cells in particular play a vital role in fighting infections and have the capability to recognize and destroy cancer cells. Enhancing T cell mediated responses is a key component to enhancing responses to a number of therapeutic agents.

Several findings support this notion. Transgenic mice expressing a constitutively active Rap1 had lower levels of pro-inflammatory cytokines and an increased fraction of the CD4+CD103+ Tregs subset (including CD4+CD103+CD25+), which suppressed CD4+CD25− (Tconv) cells more potently than their WT counterparts (Li et al. (2005)J Immunol175, 3133-9). More recently, it was shown that Tregs suppress effector T-cells by direct transfer of cAMP through gap junctions (Fassbender et al. (2010)Cell Immunol265, 91-6; Vignali et al. (2008)Nat Rev Immunol8, 523-32; Somekawa et al. (2005)Circ Res97, 655-62), whose formation in cardiac cells is enhanced by EPAC1 as it facilitates the accumulation of connexons at the site of gap junction formation (Collison and Vignali (2011)Methods Mol Biol707, 21-37). These findings suggest that EPAC1 might play a direct role in contact dependent Treg suppression. To study the in vivo functions of EPAC1, the inventors generated Epac1 knockout (KO) mice. Epac1−/−mice were orally immunized with ovalbumin (OVA) alone or with cholera toxin (CT). In each case Epac1 KO mice had a significantly higher level of serum OVA-specific IgG1 antibodies than that of wild-type (WT) mice as determined by ELISA (FIG. 15). Furthermore, when WT mice were orally immunized with OVA alone or with an EPAC specific antagonist (ESI-09) the mice receiving ESI-09 (oral gavage 50 mg/kg) had a significantly higher level of serum OVA-specific IgG1 antibodies than that of the control group treated with vehicle (FIG. 16).

Based on the amplified immune response in Epac1 KO mice, both antigen-challenged and naïve, it was suggested that a role for Epac1 in mediating the function of CD4+CD25+ regulatory T-cells (Tregs), which are known suppressors of the adaptive and humoral immune responses. The suppressive potency of WT and Epac1 KO Tregs was examined using an in vitro assay that examines CD4+CD25− (Tconv) proliferation in the presence Tregs. Epac1 KO Tconv and WT Tconv proliferated at the same rate when cultured alone. The addition of WT Tregs suppressed the proliferation of both cell populations to the same extent, while the addition of Epac1 KO Tregs suppressed the proliferation of Epac1 KO Tconv to a much lesser degree than it did WT Tconv's. To confirm the specificity of Epac1's impact on Tregs mediated suppression of Tconv, the suppression assay was repeated in the presence of ESI-09 and the outcome was similar. Taken together, these results suggest that presence of Epac1 in Tregs and Tconv sensitizes the latter to suppression by the former.

These findings show that EPAC1 antagonists are effective adjuvants and can be used in conjunction with vaccines and immune-modulators for immunotherapies. Such immunotherapies include those for cancer or other diseases. EPAC1 is a viable target for immune-modulation. In particular EPAC1 inhibitors, can be used as adjuvants for vaccines and/or modulators of immunotherapies.

Certain aspects are directed to administering to a subject an EPAC1 inhibitor in conjunction with an antigen. In certain aspects the EPAC1 inhibitor is administered before, during, or after administration of an antigen. In one embodiment, the antigen is a viral protein. In another embodiment, the antigen is a bacterial protein or a portion thereof. In yet another embodiment, the antigen is a mammalian protein or a portion thereof, e.g., a cancer antigen. The antigen can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or days before or after EPAC inhibitor administration. The antigen and/or inhibitor can be administered 1, 2, 3, 4, 5, 6, 7, 8 or more times over various time periods. In certain aspects more than one antigen can be administered. In certain aspects the subject is a human subject. In a further embodiment additional immune modulators can be administered.

In certain aspects an EPAC inhibitor is administered in combination with an antibody therapy, which can enhance the efficacy of antibody therapy for treatment of cancer or infectious diseases. The EPAC inhibitor can be administered in combination with antibodies such as rituximab, herceptin or erbitux. In some embodiments, the antibody is an anti-cancer antibody. Monoclonal antibodies, including human and humanized monoclonal antibodies work by targeting tumor specific antigens, thus enhancing the host's immune response to tumor cells. Other antibody therapies include use of polyclonal antibodies and use of antibody fragments or regions. Examples of such therapies are trastuzumab (Herceptin), cetuximab, and rituximab (Rituxan or Mabthera).

In certain aspects EPAC specific inhibitors can be used for attenuating or preventing uptake of a microbe by a vascular endothelial cell. Endothelial and epithelial cell-cell junctions and barriers play a critical role in the dissemination of microbe infection. EPAC and its down-stream effector Rap1 have been shown to play an important role in cellular functions related to endothelial cell junctions and barrier (Kooistra et al. (2005)FEBS Lett579:4966-4972; Baumer et al. (2009)J Cell Physiol.220:716-726; Noda et al. (2010)Mol Biol Cell21:584-596; Rampersad et al.J. Biol. Chem.285:33614-33622; Spindler et al (2011)Am J Pathol178:2424-2436). In addition, EPAC is known to be involved in phagocytosis (Yeager et al (2009)Infect Immun77:2530-2543; Shirshev (2011)Biochemistry(Mosc) 76:981-998).

Cyclic AMP is a universal second messenger that is evolutionally conserved in diverse form of lives, including human and pathogens such as bacterial, fungi and protozoa. It has been well recognized that cAMP play major roles in microbial virulence, ranging from a potent toxin to a master regulator of virulence gene expression. (MaDonough & Rodriguez (2012)Nature Rev Microbiol10:27-38). As a major intracellular cAMP receptor, it is likely that EPAC proteins are important cellular targets for microbe infection.

To determine if EPAC 1 plays a role in rickettsia infection, WT and EPAC 1−/−C57BL/6 mice were challenged with sublethal dose ofR. australia. As shown inFIG. 20. All WT mice became severely ill 5 days post infection and a few WT mice died. On the other hand, none of the EPAC1−/−mice became severely sick. These results suggest that deletion of EPAC1 protects mice fromR. australiainfection.

To test if EPAC inhibitors are capable of protecting mice from lethal-dose infection ofR. australia. WT C57BL/6 mice were treated with vehicle or ESI-09 (10 mg/kg, IP) daily. Five days after the treatment, mice were challenged with lethal dose ofR. australiaand continued ESI-09 daily treatment. Similar to EPAC1 genetic deletion, pharmacological inhibition of EPAC1 also led to a striking protection ofR. Australiainfection (FIG. 21). 100% control group became severely sick while only 10% of the treatment group showed sign of sickness.

To investigate the mechanism of EPAC1 inhibition-mediated protection ofR. australiainfection, HUVEC cells treated with vehicle or ESI-09 were infected withR. australia. As shown inFIG. 22, the number of intracellularR. australiawas dramatically reduced in ESI-09 treated HUVEC cells. These data demonstrate that inhibition of EPAC by ESI-09 treatment suppresses cellular entry ofR. australia.

Certain embodiments are directed to methods of suppressing microbe infection comprising administering an EPAC specific inhibitor to a subject having or under the risk of microbe infection. In certain aspects the microbe is a bacteria, virus, or fungi. In other aspects the EPAC specific inhibitor is selected from the EPAC inhibitors described herein.

In certain aspects, compounds described herein can be used to enhance leptin sensitivity and reduce adiposity in a subject.

The adipocyte hormone leptin plays a central role in energy homeostasis. It was discovered in obese mice missing a serum factor, which when replaced decreased food intake and body weight (Zhang et al. (1994)Nature372:425-32). Because of these initial observations, much of the earlier therapeutic attempt using this hormone has been in the treatment of obesity. Serum leptin concentrations in the majority of humans with obesity are high, and a state of leptin resistance is thought to exist (Mantzoros et al. (2000)J Clin Endocrinol Metab85:4000-4002). Thus far, the effect of recombinant human leptin has been limited in causing weight loss in obese individuals except in the state of congenital leptin deficiency (Heymsfield et al. (1999)Jama282:1568-75; Farooqi et al. (1999)N Engl J Med341:879-84).

Activation of EPAC has been shown to impair leptin signaling. Central infusion of an EPAC activator has been shown to blunt the anorexigenic actions of leptin (Fukuda et al. (2011)Cell Metab13:331-339). The present invention provides for the use of EPAC modulators for the treatment of diseases related to abnormalities in the leptin pathway, such as obesity and lipoatrophy and its associated metabolic abnormalities (e.g., hyperglycemia, dyslipidemia, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, atherosclerosis, vascular restenosis, and insulin resistance).

Global Epac1 knockout mice were generated using the Cre-loxP system. Epac1 null mutant mice in general appear healthy without any obvious physical abnormalities. They have similar body weights compared to wild-type mice at birth and up to three weeks of age, when the mice were weaned and started on the high fat diet or standard rodent chow. The body weight gain of Epac1 KO mice on standard chow diet slowed down and became significantly lower than that of the wild-type mice around week 5, eventually reaching approximately 85% of wild-type mice. Similar observations were made for the HFD-fed mice. The body weight of Epac1 KO mice became significantly lower around week 7, and maintained at about 90% of wild-type mice. The average daily food intake of Epac1 KO mice on chow diet was significantly lower than that of the wild-type. On the other hand, while the average daily food intake of Epac1 KO mice on HFD was lower than that of the wild-type mice on HFD, the difference was not statistically significant. Whole body micro-CT scans were performed on HFD-fed mice and body length was measured from C1 to L6 vertebrae to confirm that body weight gain lag in Epac1 KO mice is not the result of overall growth retardation.

To determine why body weight gain is reduced in Epac1 KO mice, the adiposity of the animals was examined. The epididymal fat pads from Epac1 KO mice were visually smaller and weighted significantly less than those from wild-type. When analyzed by Micro-CT imaging, the total fat mass of Epac1 null mice on HFD was significantly less than wild-type. This difference was significant even after fat mass was normalized to body weight. In fact, the difference in total fat mass was larger than the difference in body weight, suggesting that the reduced body weight of Epac1 KO mice is mainly due to a reduction in fat mass. A decrease in adipose tissue mass can be the result of impaired adipocyte differentiation, a reduction of adipocyte size, or both. Histological analysis of epididymal white fat tissue (EWAT) revealed that adipocytes from Epac1 null mice were much smaller than those from the wild-type counterparts. On the other hand, ex vivo adipocyte differentiation analyses using MEF cells isolated from both wild-type and Epac1 KO mice revealed that the Epac1 KO MEFs were as competent as wild-type MEFs to differentiate into adipocytes, suggesting that Epac1 deficiency did not impede normal adipogenesis.

Leptin is secreted by adipose tissue and the plasma levels of leptin are known to correlate with adipose tissue mass while plasma leptin falls in both humans and mice after weight loss (Considine et al. (1996)N. Engl. J. Med.441 334:292-295; Friedman and Halaas (1998)Nature395:763-770; Maffei et al. (1995)Nat. Med.1:1155-1161). To investigate if a reduced fat mass is correlated with reduced plasma leptin in Epac1 null mice, the plasma leptin levels of Epac1 KO and wild-type mice on standard chow and HFD were compared, respectively. For mice on the standard chow diet, the average plasma leptin level of 16-week-old wild-type mice was around 3.97±0.78 ng/ml which is consistent with previous publications (Bates et al. (2003)Nature421:856-859; Kievit et al. (2006)Cell Metab4:123-132). However, the plasma leptin level of the age and gender matched Epac1 KO mice was significantly lower, at about 1.01±0.26 ng/ml. For the mice on HFD at 28-weeks of age (25 weeks on HFD), the average leptin concentration was about 83.16±5.76 ng/ml, whereas the average leptin level of Epac1 KO mice was about 66.15±3.52 ng/ml. These results corroborate the anatomical and morphological observation that Epac1 deficiency reduces white fat tissue adiposity in the standard chow diet as well as HFD fed mouse.

To determine if the apparent decreases in plasma leptin levels are merely the result of decreased adiposity, the leptin levels of 3-week-old mice were measured before significant body weight and adiposity difference can be observed between the wild-type and Epac1 mull mice. Leptin levels in Epac1 KO mice were already reduced significantly compared to those in age and gender match wild-type mice.

Loss of Epac1 Heightens Leptin Signaling Activity and Sensitivity In Vivo.

In light of a recent finding by Fukuda, et al. that activation of Epac-RAP1 with Epac selective agonist, 8-pCPT-2′-O-Me-cAMP (8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic mono-phosphorothioate) blunts leptin signaling in hypothalamus and causes central leptin resistance (Fukuda et al. (2011)Cell Metab13:331-339), the pSTAT3 Y705 localization and immunoactivity in the arcuate nucleus (AN), with or without Epac1, was compared to determine the consequence of reduced plasma leptin levels associated with the loss of Epac1 on leptin sensitivity in vivo. The proopiomelanocortin neurons are direct targets of leptin in the hypothalamus and the leptin-induced STAT3 Y705 phosphorylation and nuclear translocation in the AN is involved in body weight regulation (Bates et al. (2003)Nature421:856-859; Cheung et al. (1997)Endocrinology138:4489-4492; Hubschle et al. (2001)J. Neurosci.21:2413-2424; Schwartz et al. (1996)J. Clin. Invest98:1101-1106). The Epac1 KO AN tissue displayed a slightly enhanced baseline level (PBS vehicle injection) of pSTAT3 Y705 immunoractivity, and a markedly increased nuclear immunostaining of pSTAT3 Y705 in response to ICV injection of leptin than that of the wild-type. To further compare the total pSTAT3 Y705 in the hypothalamus upon stimulation with leptin, we repeated ICV leptin injections and excised the hypothalami for immunoblotting analysis. Consistent with our immunofluorescence study, the basal and the stimulated levels of pSTAT3 Y705 were both increased in the Epac1 KO hypothalamic tissue, suggesting that loss of Epac1 enhances central leptin signaling and sensitivity while decreases peripheral (plasma) leptin levels in vivo.

To investigate if this increased leptin sensitivity associated with loss of Epac1 translates into decreased food intake and body weight in response to leptin in vivo, leptin was injected intraperitoneally to 20-week-old mice. The mice were individually housed for one week to acclimate them with the environment. Escalation of leptin was utilized to cover a wide range of doses (Heymsfield et al. (1999)JAMA282:1568-1575). Due to the nocturnal activity of mice and the short half-life of leptin (Ahren et al. (2000)Int. J. Obes. Relat Metab Disord.24:1579-1585; Hill et al. (1998)Int. J. Obes. Relat Metab Disord.22:765-770), food intake was measured during the first 4-hours of the dark cycle, food intake during the entire 24-hour period, and body weight at the beginning of each dark cycle. It was found that food intake over the first four hours of the dark cycle decreased in a dose-dependent manner in response to leptin administration. Epac1 KO mice displayed a significant reduction in food intake at the higher doses of leptin versus wild-type mice during the first 4-hour dark cycle. Although the 24-hour food intake also decreased with leptin administration, the magnitude of decrease was not statistically significant. Interestingly, leptin injection induced a transient body weight decrease in the wild-type mice which recovered quickly even with highest dose of leptin. In contrast, leptin induced a persistent and dose-dependent body weight reduction in Epac1 KO mice. These results demonstrate that Epac1 deficiency enhances leptin signaling in hypothalamus and that Epac1 KO mice are more sensitive to leptin treatment in vivo in regard to the reduction of food intake and body weight.

To explore the feasibility of increasing leptin sensitivity by targeting Epac1 using small molecules, organotypic brain slice cultures were prepared from 11-day old wild-type C57BL/6 mice. After 7 days ex vivo culture, treatment of the brain tissue with the Epac specific antagonist ESI-09 led to an enhanced pSTAT3 Y705 level both at the basal state and in response to leptin stimulation. Moreover, Epac specific inhibitors also increased the cellular level of SHP2 as observed in the Epac1 KO mice (FIG. 17A). These pharmacological data are in agreement with results obtained using Epac1 KO mice and further confirm that inhibition of Epac1 enhances leptin signaling in the hypothalamus. To further investigate the therapeutic potential of this small molecule, wild-type mice were with ESI-09 (50 mg/kg) or vehicle (corn oil) by oral gavage for 3 weeks. Plasma leptin was significantly reduced after ESI-09 relative to vehicle treatment (FIG. 17B).

Epac1 KO Mice are Protected Against HFD Induced Glucose Intolerance.

It has been well documented that enhanced leptin sensitivity confers resistance to HFD-induced obesity and improved glucose tolerance (Berglund et al. (2012)J. Clin. Invest122:1000-1009; Howard et al. (2004)Nat. Med.10:734-738; Kievit et al. (2006)Cell Metab4:123-132; Mori (2004)Nat. Med.522 10:739-743). The glucose handling capability of wild-type and Epac1 KO mice were compared using the oral glucose tolerance test (OGTT). While similar OGTT results were obtained for wild-type and Epac1 KO mice on the standard chow diet (FIG. 18A), the Epac1 KO mice displayed a markedly enhanced glucose handling capability after 15 weeks on HFD. Firstly, the fasting glucose levels of HFD Epac1 KO mice were significantly lower than those of wild-type; secondly, Epac1 KO mice cleared glucose from blood significantly faster than wild-type mice at every time point after glucose administration. The blood glucose levels of Epac1 KO mice dropped back to baseline in 2 hours while the wild-type blood glucose levels remained elevated (FIG. 18B). In parallel, insulin levels were monitored after overnight fasting and 15 min after glucose administration. No significant differences were observed between wild-type and Epac1 KO mice on the standard chow diet: both showed similar low fasting insulin levels that increased to a similar extent in response to glucose challenge (FIG. 18C). On the other hand, while HFD Epac KO mice showed a slightly decreased fasting insulin level, both HFD wild-type and Epac1 KO mice maintained the ability to increasing plasma insulin in response to blood glucose concentration elevation (FIG. 18D). These data suggest that Epac1 KO mice are resistant to HFD-induced insulin insensitivity as in the case of the wild-type mice. These studies show that Epac1 KO mutant mice are largely protected from the HFD-induced glucose intolerance and insulin resistance.

In certain aspects, an EPAC inhibitor is administered to a leptin-resistant subject. The administration of an EPAC inhibitor increases sensitivity of the subject to endogenous leptin. In a further aspect, leptin or leptin analog can be administered in combination with an EPAC inhibitor to overcome leptin resistance or deficiency. In another aspect, an overweight subject is administered an EPAC inhibitor reducing body weight of the subject. In yet another aspect, an EPAC inhibitor is administered to increase systemic insulin sensitivity. Other aspects include administering an EPAC activator to a subject having anorexic or cachexic symptoms or syndromes, or a hypersensitivity to leptin.

IV. Pharmaceutical Formulations and Administration

In certain embodiments, the invention also provides compositions comprising one or more EPAC modulator with one or more of the following: a pharmaceutically acceptable diluent; a carrier; a solubilizer; an emulsifier; a preservative; and/or an adjuvant. Such compositions may contain an effective amount of at least one EPAC modulator. Thus, the use of one or more EPAC modulators as provided herein for the preparation of a medicament is also included. Such compositions can be used in the treatment of a variety of EPAC associated diseases or conditions such as cancer or leptin associated disease or conditions.

An EPAC modulator may be formulated into therapeutic compositions in a variety of dosage forms such as, but not limited to, liquid solutions or suspensions, tablets, pills, powders, suppositories, polymeric microcapsules or microvesicles, liposomes, and injectable or infusible solutions. The preferred form depends upon the mode of administration and the particular disease targeted. The compositions also preferably include pharmaceutically acceptable vehicles, carriers, or adjuvants, well known in the art.

Formulation components are present in concentrations that are acceptable to the site of administration. Buffers are advantageously used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 4.0 to about 8.5, or alternatively, between about 5.0 to 8.0. Pharmaceutical compositions can comprise TRIS buffer of about pH 6.5-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute therefor.

The pharmaceutical composition to be used for in vivo administration is typically sterile. Sterilization may be accomplished by filtration through sterile filtration membranes. If the composition is lyophilized, sterilization may be conducted either prior to or following lyophilization and reconstitution. The composition for parenteral administration may be stored in lyophilized form or in a solution. In certain embodiments, parenteral compositions are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle, or a sterile pre-filled syringe ready to use for injection.

The above compositions can be administered using conventional modes of delivery including, but not limited to, intravenous, intraperitoneal, oral, intralymphatic, subcutaneous administration, intraarterial, intramuscular, intrapleural, intrathecal, and by perfusion through a regional catheter. Local administration to an organ or a tumor is also contemplated by the present invention. When administering the compositions by injection, the administration may be by continuous infusion or by single or multiple boluses. For parenteral administration, the EPAC modulating agents may be administered in a pyrogen-free, parenterally acceptable aqueous solution comprising the desired EPAC modulating agents in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which one or more EPAC modulating agents are formulated as a sterile, isotonic solution, properly preserved.

Once the pharmaceutical composition of the invention has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.

If desired, stabilizers that are conventionally employed in pharmaceutical compositions, such as sucrose, trehalose, or glycine, may be used. Typically, such stabilizers will be added in minor amounts ranging from, for example, about 0.1% to about 0.5% (w/v). Surfactant stabilizers, such as TWEEN®-20 or TWEEN®-80 (ICI Americas, Inc., Bridgewater, N.J., USA), may also be added in conventional amounts.

To determine the bioavailability of EPAC inhibitors, an IP injection formulation was developed in which the compounds were dissolved in ethanol and then diluted 1:10 with a 10% Tween 80 in normal saline solution. This formulation was determined suitable by passing the simulated in vivo blood dilution assay. In vivo pharmacokinetic studies were performed in four week old female C57BL6/N mice. As shown inFIG. 19, following one single intraperitoneal (IP) injection of the ESI-09 compound (10 mg/kg) in mice (n=5 for each time point), blood levels of ESI-09 were determined to be rapidly elevated reaching maximal values of 42,520 ng/ml (128 μM) at 0.5 hr with a half-life of 3.5 hrs. These results suggest that ESI-09 has an excellent bioactivity in vivo.

For the compounds of the present invention, alone or as part of a pharmaceutical composition, such doses are between about 0.001 mg/kg and 1 mg/kg body weight, preferably between about 1 and 100 μg/kg body weight, most preferably between 1 and 10 μg/kg body weight.

Therapeutically effective doses will be easily determined by one of skill in the art and will depend on the severity and course of the disease, the patient's health and response to treatment, the patient's age, weight, height, sex, previous medical history and the judgment of the treating physician.

Methods of treating cancer may further include administering to the patient chemotherapy or radiotherapy, which may be administered more than one time. Chemotherapy includes, but is not limited to, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, taxotere, taxol, transplatinum, 5-fluorouracil, vincristin, vinblastin, methotrexate, gemcitabine, oxaliplatin, irinotecan, topotecan, or any analog or derivative variant thereof. Radiation therapy includes, but is not limited to, X-ray irradiation, UV-irradiation, γ-irradiation, electron-beam radiation, or microwaves. Moreover, a cell or a patient may be administered a microtubule stabilizing agent, including, but not limited to, taxane, as part of methods of the invention. It is specifically contemplated that any of the compounds or derivatives or analogs, can be used with these combination therapies.

To a solution of cyclohexanecarbaldehyde (1.12 g, 10.0 mmol), methyl cyanoacetate (0.99 g, 10 mmol), and thiourea (0.76 g, 10 mmol) in absolute ethanol (50 mL) was added piperidine (1.70 g, 20 mmol). The mixture was heated under reflux for 6 h and then cooled to room temperature. The solution was concentrated and then the residue was extracted with ethyl acetate (100 mL) and 2N HCl (aq.) (20 mL). The organic layer was isolated, washed with brine, and dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure, and the product was washed with EtOAc (30 mL) to obtain the pure product as a white solid (1.2 g, 51%).1H NMR (600 MHz, DMSO-d6) δ 13.04 (s, 1H), 12.73 (s, 1H), 2.73-2.71 (m, 1H), 1.86-1.79 (m, 4H), 1.73-1.71 (m, 2H), 1.66-1.63 (m, 1H), 1.29-1.20 (m, 3H).

To a solution of 1-methylpiperidine-4-carbaldehyde (600 mg, 4.72 mmol), methyl cyanoacetate (468 mg, 4.72 mmol), and thiourea (359 mg, 4.72 mmol) in absolute ethanol (25 mL) was added piperidine (803 mg, 9.44 mmol). The mixture was heated under reflux for 6 h and then cooled to room temperature. The precipitate was collected by filtration and washed with DCM (10 mL) and EtOAc (10 mL). The desired product was obtained as a pale yellow solid (820 mg, 69%) and used directly for the next step without further characterization.

To a solution of 2-methyl-propionaldehyde (1.0 g, 13.9 mmol), methyl cyanoacetate (1.37 g, 13.9 mmol), and thiourea (1.06 g, 13.9 mmol) in absolute ethanol (45 mL) was added piperidine (2.37 g, 27.8 mmol). The mixture was heated under reflux for 6 h and then cooled to room temperature. The solution was concentrated and then the residue was extracted with ethyl acetate (100 mL) and 2N HCl (aq., 20 mL). The organic layer was isolated, washed with brine, and dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure, and the product was washed with EtOAc (20 mL) to obtain the pure product as a pale yellow solid (1.5 g, 55%).1H NMR (600 MHz, DMSO-d6) δ 13.07 (s, 1H), 12.78 (s, 1H), 3.05-3.01 (m, 1H), 1.30 (d, 6H, J=7.2 Hz).

To a solution of cyclopentanecarbaldehyde (500 mg, 5.1 mmol), methyl cyanoacetate (504 mg, 5.1 mmol), and thiourea (388 mg, 5.1 mmol) in absolute ethanol (20 mL) was added piperidine (868 mg, 10.2 mmol). The mixture was heated under reflux for 6 h and then cooled to room temperature. The solution was concentrated and then the residue was extracted with ethyl acetate (100 mL) and 2N HCl (aq., 20 mL). The organic layer was isolated, washed with brine, and dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure, and the product was washed with EtOAc (10 mL) to obtain the pure product as a pale yellow solid (700 mg, 62%).1H NMR (600 MHz, DMSO-d6) δ 13.05 (s, 1H), 12.86 (s, 1H), 3.09-3.06 (m, 1H), 1.99-1.96 (m, 2H), 1.89-1.82 (m, 4H), 1.65-1.62 (m, 2H).

To a solution of cyclopropanecarbaldehyde (500 mg, 7.13 mmol), methyl cyanoacetate (706 mg, 7.13 mmol), and thiourea (543 mg, 7.13 mmol) in absolute ethanol (15 mL) was added piperidine (1.21 g, 14.27 mmol). The mixture was heated under reflux for 6 h and then cooled to room temperature. The solution was concentrated and then the residue was extracted with ethyl acetate (100 mL) and 2N HCl (aq., 20 mL). The organic layer was isolated, washed with brine, and dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure, and the product was washed with EtOAc (5 mL) to obtain the pure product as a yellow solid (250 mg, 18%).1H NMR (600 MHz, DMSO-d6) δ 12.84 (s, 1H), 12.50 (bs, 1H), 2.01-1.99 (m, 1H), 1.32-1.30 (m, 2H), 1.17-1.16 (m, 2H).

To a solution of CH3CN (0.41 g, 10.0 mmol) in anhydrous THF (5 mL) was added 1.6 M methyl lithium in diethyl ether (3.1 mL, 5.0 mmol) at −78° C. under nitrogen. The mixture was stirred at −78° C. for 0.5 h, and 5-tert-butylisoxazole-3-carboxylic acid ethyl ester (0.5 g, 2.5 mmol) in THF (5 mL) was then added dropwise. The solution was stirred at −78° C. for 1 h and then quenched with acetic acid (0.3 g, 5.0 mmol). The mixture was warmed to 0° C. and poured onto ice/water (10 mL) and extracted with ethyl acetate (50 mL). The organic lay was dried over Na2SO4, filtered and concentrated under reduced pressure. The crude residue (490 mg) was obtained as a yellow oil and directly used for next step without further purification.

Discovery of EPAC Specific Inhibitors

Biochemical Characterization of EPAC Antagonists—

To determine the relative binding affinity of the EPAC antagonist identified in an initial screen (FIG. 1), dose-dependent titrations were performed to test the ability of these compounds to compete with the binding of 8-NBD-cAMP to EPAC2. When various concentrations of cAMP or EPAC2 antagonists were added to reaction mixture with fixed concentrations of EPAC2 and 8-NBD-cAMP, a dose-dependent decrease in 8-NBD-cAMP fluorescence was observed (FIG. 7A). While cAMP competed with 8-NBD-cAMP binding with an apparent IC50of 39 μM, all selected EPAC2 antagonists showed an increased potency with apparent IC50ranging from 0.48 to 18 μM (Table 1). To determine if this apparent high affinity binding of EPAC2 antagonists can be translated to comparative potencies in suppressing the GEF activity of EPAC2, the inventors also determined the inhibition curves of Rap1-GDP exchange activity for three of these EPAC2 antagonists. As shown inFIG. 7B, compounds ESI-05, ESI-07 and ESI-09 inhibited cAMP-mediated EPAC2 GEF activity with apparent IC50of 1.4, 0.43 or 0.7 μM, respectively (Table 2). Since these antagonists were identified using EPAC2 as a target, the inventors tested if these compounds were also effective in suppressing cAMP-mediated EPAC1 GEF activity. While compound ESI-09 inhibited EPAC1-mediated Rap1-GDP exchange activity in a dose-dependent manner similar to that of EPAC2 with an apparent IC50of 3.2 μM, compounds ESI-05 and ESI-07 were completely ineffective in suppressing EPAC1 GEF activity (FIG. 7B). To test the specificity of ESI-05, ESI-07 and ESI-09, counter-screening assays were performed that measure type I and type II PKA holoenzyme activity, respectively. 25 μM of ESI-05, ESI-07 and ESI-09 did not significantly alter cAMP-induced type I and II PKA holoenzymes activation while H89, a selective PKA inhibitor, blocked the type I or II PKA activities completely (FIG. 8).

Cellular Characterization of EPAC Antagonists—

To test if the newly identified EPAC antagonists were capable of modulating EPAC activation in living cells, the ability of these compounds in suppressing EPAC-mediated Rap1 cellular activation is monitored. As shown inFIG. 8A, when HEK293 cells that ectopically express full-length EPAC2 proteins were treated with a EPAC selective cAMP analog 8-(4-Chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate, acetoxymethyl ester (007-AM), an increase in the fraction of GTP-bound cellular Rap1 was observed. Pre-treatment of HEK293/EPAC2 cells with 10 μM of compounds ESI-05, ESI-07, and ESI-09 led a significant reduction of 007-AM induced Rap1 activation while ESI-08 was much less effective. On the other hand, when HEK293 cells that ectopically express full-length EPAC1 proteins were used, only compound ESI-09 was effective in blocking 007-AM induced Rap1 activation while compound ESI-05 and ESI-07 was ineffective (FIG. 8B). These results are consistent with the biochemical Rap1 exchange data shown inFIG. 7Band further confirm that compounds ESI-05 and ESI-07 are EPAC2-specific antagonists while compounds ESI-09 is a pan-EPAC antagonist.

In addition to mediate cAMP-induced Rap1 activation, EPAC proteins are also known to activate the Akt/PKB signaling pathways while PKA inhibits Akt/PKB activation (Mei et al. (2002) J. Biol. Chem. 277: 11497-11504). To determine if ESI-09 is capable of blocking EPAC1- or EPAC2-mediated Akt activation, the phosphorylation status of T308 and S473 of Akt in HEK293/EPAC1 or HEK293/EPAC2 cells, as well as in vascular smooth muscle cell (VSMC) expressing endogenous levels of EPACs, was followed using anti phospho-Akt antibodies. 007-AM led to an increase in Akt phosphorylation for both T308 and S473 as expected. Pretreatment with 10 μM of ESI-09 for 5 min before the administration of 007-AM completely blocked EPAC1 and EPAC2-mediated Akt phosphorylation. Similar results were obtained using endogenously expressed EPAC1 and EPAC2 in human vascular smooth muscle cells (FIG. 10). These results demonstrate that ESI-09 is capable of suppressing EPAC1 and EPAC2 mediated cellular functions.

The discovery of a novel EPAC specific inhibitor provides a new tool for manipulating cAMP signaling pathways and for studying physiological functions of EPAC proteins. It has been recently reported that EPAC1 is over-expressed in pancreatic adenocarcinoma (Lorenz et al. (2008) Pancreas 37: 102-103). However, the functional role of EPAC1 elevation in this neoplasm is not clear. The inventors sought to employ ESI-09 to determine the role of EPAC1 signaling in pancreatic cancer. Treatment of pancreatic cancer cells with ESI-09 did not significantly affect cell proliferation and viability (FIG. 11). On the other hand, when pretreated with 10.0 μM of ESI-09, a significant decrease in cell migration was observed for three pancreatic cancer cell lines, AsPC-1, BxPC-3, and PANC-1 using both trans-well migration/invasion and wound healing migration assays (FIGS. 12A & 12B). In order to determine if the observed impact on cell migration is EPAC1 specific, the effect of suppressing EPAC1 expression on AsPC-1 and PANC-1 migration using RNAi was examined. As shown inFIGS. 12C & 12D, shEPAC1 clone C28 led to a near complete knockdown of EPAC1 expression and significantly inhibited migration of both cell lines, while slight reduction of EPAC1 expression by shEPAC1 clone C32 had no influence on their migratory capability. These results, combined with the fact that ESI-09 inhibited pancreatic cancer migration, suggest EPAC1 promotes pancreatic cancer cell migration. To further determine how ESI-09 inhibits PDA cell migration and invasion, a cell adhesion assay was performed using a collagen I matrix. As shown inFIG. 13, 007-AM led to an increase in cell adhesion for both AcPC-1 and PANC-1 cells, while pre-treatment with ESI-09 decreased 007-AM induced cell adhesion dose-dependently. To determine the in vivo anti-metastatic effect of ESI-09, MIA PaCa-2 stably expressing luciferase were orthotopically implanted into the pancreas of athymic nude mice. The mice were randomly divided into two groups and treated with vehicle or ESI-09 (50 mg/kg per day, oral gavage), respectively. The growth and metastasis of the tumors were monitored by weekly bioluminescence imaging using the IVIS bioluminescence imaging system. As shown inFIG. 14, ESI-09 treatment reduced PDA metastasis.

B. Experimental Procedures

Cellular activation of Rap1 was determined by pull-down of lysates derived from human vascular smooth muscle cell and HEK293 cells stably expressing EPAC1 or EPAC2 employing Ral-GDS-RBD-GST affinity beads as described earlier (Mei and Cheng (2005) Molecular Biosystems 1: 325-331).

Phosphorylation of Akt—

Cellular proteins from cell lysates treated with various reagents were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane. The levels of Akt activation were probed by immuno-blotting analyses using anti-phosphate T308 PKB antibodies (1:1000) and anti-phosphate S473 PKB antibodies (1:1000). At least three independent experiments were performed for each Western blot.

INS-1 cells were plated into 96-well plates pre-coated with poly-lysine at a density of 1×105cells/well. After overnight incubation, the medium was replaced with Krebs-Ringer buffer (KRB) containing 2.9 mM glucose. After an additional two-hour incubation, the cells were pre-treated with testing compounds or DMSO vehicle as a control in fresh KRB containing 16.7 mM glucose for 10 min, followed by a 30 min stimulation by 10 μM of 007-am. The supernatant was collected and subjected to insulin qualification using an Ultra Sensitive Rat Insulin ELISA kit from Crystal Chem. Inc.

The top chamber of 8 micron inserts (Costar Inc) were coated with BD Matrigel™ Basement Membrane Matrix (50 g/mL). Cells (2×105) pretreated with 10.0 μM of ESI-09 for 24 hours were added to the top chamber of the inserts in serum free RPMI medium containing 0.25% BSA. The bottom chamber was filled with 600.0 μL of RPMI containing 10% FBS and 10.0 μM ESI-09. The cells were then incubated at 37° C. in 5% CO2for 20 hours. Cells were removed from the top chamber and migrated cells were fixed in methanol and stained with crystal violet. The number of migrated cells were counted from four different fields.

Cells were grown to 95-100% confluency before a scratch wound was made. The medium was changed to RPMI 10% FBS containing 10.0 μM ESI-09. The cells were then incubated at 37° C. in 5% CO2. The wound was imaged at 0 hours and 22 hours after changing the medium. Healing rate was determined by calculating the percentage of wound closure normalized to a 1.0 mm wound according to the following equation: % wound closure=(distance between the edges of the wound before treatment with ES-09−distance between the edges of the wound 22 hours post treatment with ES-09)/1.0×100.