EPAC1 inhibitors for the treatment of idiopathic pulmonary fibrosis

Abstract

To provide a method for treating idiopathic pulmonary fibrosis.SOLUTION: To provide EPAC1 inhibitors for use in the treatment, prevention and / or amelioration, preferably treatment, of idiopathic pulmonary fibrosis.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to EPAC1 inhibitors for the treatment of idiopathic pulmonary fibrosis.

[0002] This invention was made with United States government support under HL133554 awarded by the National Institutes of Health. The United States government has certain rights in this invention. [Background technology]

[0003] Idiopathic pulmonary fibrosis (IPF) is a rare disease characterized by chronic and progressive fibrosing interstitial pneumonia of unknown etiology, for which no effective treatment is available. To date, despite extensive research in experimental and clinical studies, IPF remains a cause of increasing morbidity and mortality, with a median survival from diagnosis of less than three years. Unfortunately, there is no cure for IPF.

[0004] Therefore, there is an urgent need to identify new therapeutic targets and strategies for treating patients with IPF.

[0005] The objective of this invention is to provide a new therapeutic target and to provide a safe and effective strategy by using small molecule compounds that can prevent and reverse this progressive, fatal lung disease.

[0006] It is therefore an object of the present invention to provide small molecules for treating IPF.

[0007] Thus, the present invention relates to an EPAC1 inhibitor for use in the treatment, prevention and / or amelioration, preferably the treatment, of idiopathic pulmonary fibrosis.

[0008] The present invention is based on the fact that the expression of cAMP-regulated exchange protein 1 (Epac1) was significantly increased in lung tissue from IPF patients and bleomycin-treated mice compared to controls.

[0009] Pharmacological inhibition of Epac1 significantly reduced proliferation and blocked the expression of several fibrotic markers. Notably, pharmacological inhibition of Epac1 was also found to significantly reduce lung fibrosis in vivo using a bleomycin-induced PF mouse model.

[0010] Preferably, the EPAC1 inhibitor for use in the present invention is selected from EPAC1 selective inhibitors.

[0011] In one embodiment, EPAC1 selective inhibitors are compounds that exhibit inhibitory activity against the EPAC1 isoform. More particularly, they generally exhibit inhibitory activity against EPAC1 and little or no inhibitory activity against the EPAC2 isoform.

[0012] By "selective EPAC1 inhibitor" is meant the ability of an EPAC1 inhibitor to act on a specific EPAC1 isoform in preference to the other isoform, EPAC2. An EPAC1 selective inhibitor may have the ability to distinguish between these two isoforms, and therefore essentially acts on the EPAC1 isoform.

[0013] The term "inhibitor" should be understood as "antagonist".

[0014] According to one embodiment, the EPAC1 inhibitor for use in the present invention has formula (I):

[0015] [ka] [In the formula, R'9 is H or a group of the formula:

[0016] [ka] and is a group represented by the formula:

[0017] [ka] is the bond to the nitrogen atom of the tetrahydroquinoline; R'1, R'2, R'3, R'4, and R'8 are H, (C1-C 10 ) alkyl, (C3-C 10 ) cycloalkyl, (C6-C 10 )aryl, (C1-C6)alkylene-(C6-C 10 ) aryl and (C3-C 10 ) heteroaryl, wherein the aryl and heteroaryl groups are optionally substituted with at least one substituent selected from OH, NH, NO, (C-C) alkyl, and halogen; R'5 is a halogen atom; - R'6 and R'7 are independently selected from the group consisting of H and halogen atoms, or a pharmaceutically acceptable salt, hydrate or hydrated salt thereof, or a polymorphic crystalline structure, racemate, diastereomer or enantiomer thereof.

[0018] In one embodiment, the compound of formula (I) has the formula:

[0019] [ka] This is different from the compound represented by the formula:

[0020] In the context of the present invention, the term "treating" or "treatment" as used herein means to ameliorate, alleviate, or inhibit the progression of the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.

[0021] In the context of the present invention, the term "preventing" or "prevention" as used herein means avoiding the appearance or progression of the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.

[0022] Thus, a first family of EPAC1 inhibitors for use in the present invention consists of tetrahydroquinoline derivatives.

[0023] According to the present invention, "(C1-C 10 The term "alkyl" means a saturated or unsaturated aliphatic hydrocarbon group which may be straight or branched and has 1 to 10 carbon atoms in the chain. Preferred alkyl groups have 1 to 4 carbon atoms in the chain, and preferred alkyl groups are, in particular, methyl or ethyl groups. "Branched" means that one or more lower alkyl groups, such as methyl, ethyl or propyl, are attached to a linear alkyl chain.

[0024] The term "(C1-C6) alkylene-" means a saturated or unsaturated aliphatic hydrocarbon divalent radical, which may be straight or branched, having 1 to 6 carbon atoms in the chain. For example, the preferred (C1-C6) alkylene-(C6-C 10 ) Aryl is a benzyl group.

[0025] "(C3-C 10 The term "cycloalkyl" refers to a cyclic saturated hydrocarbon group having 3 to 10 carbon atoms, in particular a cyclopropyl or cyclohexyl group.

[0026] "(C6-C 10 The term "aryl" refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system in which any ring atom capable of substitution may be substituted with a substituent. Examples of aryl moieties include, but are not limited to, phenyl.

[0027] "(C3-C 10The term "heteroaryl" refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system in which any substitutable ring atom may be substituted, and in which one or more carbon atoms are replaced by one or more heteroatoms, such as nitrogen, oxygen, and sulfur atoms; for example, one or two nitrogen atoms, one or two oxygen atoms, one or two sulfur atoms, or a combination of different heteroatoms, such as one nitrogen atom and one oxygen atom. Preferred heteroaryl groups are pyridyl, pyrimidyl, and oxazyl groups.

[0028] The term "halogen" refers to atoms in Group 17 of the periodic table and particularly includes fluorine, chlorine, bromine, and iodine atoms, more preferably fluorine, chlorine, and bromine atoms.

[0029] By "tetrahydroquinoline" is meant the following group:

[0030] [ka] is understood.

[0031] The compounds described herein may have asymmetric centers. Compounds of the present invention containing asymmetrically substituted atoms can be isolated as optically active or racemic forms. Methods for preparing optically active forms, such as by resolving racemic forms or by synthesis from optically active starting materials, are well known in the art. Unless stereochemistry or isomerism is specifically indicated, all chiral forms, diastereomers, racemates, and all geometric isomers of the compounds are intended.

[0032] In one embodiment, in formula (I) having R'2 to R'9 defined above, * ) may be either (R) or (S):

[0033] [ka]

[0034] In certain embodiments, it is (R). In certain embodiments, the enantiomer (R) of the compound of formula (I) is preferred, and more particularly the following enantiomer:

[0035] [ka]

[0036] In another embodiment, the (R)-enantiomer of the compound of Formula (I) is a more potent cAMP antagonist than the racemate and (S)-enantiomer of the compound of Formula (I). In one embodiment, the (R)-enantiomer of the compound of Formula (I) is a selective inhibitor of EPAC1. The (R)-enantiomer can inhibit the GEF activity of EPAC1 10 times more efficiently than the (S)-enantiomer.

[0037] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness and properties of the compounds of the present invention and is not biologically or otherwise undesirable. Pharmaceutically acceptable acid addition salts can be prepared from inorganic and organic acids, while pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. For a review of pharmaceutically acceptable salts, see Berge et al. ((1977) J. Pharm. Sd, vol. 66, 1). For example, salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitric acid, as well as organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, fumaric acid, methanesulfonic acid, and toluenesulfonic acid.

[0038] In certain embodiments, the compounds of the present invention have the following formula (I-1):

[0039] [ka] which is included in formula (I), and R'9 has the following formula:

[0040] [ka] It is shown as follows.

[0041] In certain embodiments, the EPAC1 inhibitor for use in the present invention has the following formula (I-1):

[0042] [ka] [In the formula, R'1, R'2, R'3, R'4 and R'8 are H, (C1-C 10 ) alkyl, (C3-C 10 ) cycloalkyl, (C6-C 10 )aryl, (C1-C6)alkylene-(C6-C 10 ) aryl and (C3-C 10 ) heteroaryl, wherein the aryl and heteroaryl groups are optionally substituted with at least one substituent selected from OH, NH, NO, (C-C) alkyl, and halogen; R'5 is a halogen atom; - R'6 and R'7 are independently selected from the group consisting of H and halogen atoms] or a pharmaceutically acceptable salt, hydrate or hydrated salt thereof, or a polymorphic crystalline structure, racemate, diastereomer or enantiomer thereof.

[0043] In certain embodiments, R'1 is H in formula (I-1) as defined above. In certain embodiments, in formula (I) or (I-1), R'2 is H or (C1-C 10 ) alkyl. In certain embodiments, in formula (I) or (I-1), R'2 is (C1-C 10 ) alkyl. Preferably, R'2 is (C1-C4) alkyl. More preferably, in formula (I) or (I-1), R'2 is a methyl group. In another embodiment, R'2 is H. Preferably, in formula (I) or (I-1), R'2 is H or a methyl group.

[0044] In certain embodiments, in formula (I) or (I-1), R'3 is H. In another embodiment, in formula (I) or (I-1), R'4 is H. In another embodiment, in formula (I) or (I-1), R'8 is H. In one embodiment, in formula (I) or (I-1), R'3, R'4 and R'8 are H.

[0045] In certain embodiments, in Formula (I) or (I-1), (C3-C 10 ) The heteroaryl group is selected from the group consisting of pyridyl, pyrimidyl and oxazyl groups.

[0046] In another embodiment, in formula (I) or (I-1), (C3-C 10 ) The aryl group is a phenyl group.

[0047] In another embodiment, in Formula (I) or (I-1), (C1-C6) alkylene-(C6-C 10 ) Aryl is a benzyl group.

[0048] In certain embodiments, in formula (I) or (I-1), R'5 is selected from the group consisting of F, Cl, Br, and I. Preferably, R'5 is Br.

[0049] In certain embodiments, in formula (I) or (I-1), R'6 is selected from the group consisting of H, F, Cl, Br, and I. In certain embodiments, in formula (I) or (I-1), R'6 is selected from the group consisting of F, Cl, Br, and I. In another embodiment, in Formula (I) or (I-1), R'6 is F. In another embodiment, in Formula (I) or (I-1), R'6 is H. Preferably, in Formula (I) or (I-1), R'6 is H or F.

[0050] In certain embodiments, in formula (I) or (I-1), R'7 is selected from the group consisting of H, F, Cl, Br, and I. In certain embodiments, in formula (I) or (I-1), R'7 is selected from the group consisting of F, Cl, Br, and I. In another embodiment, R'7 is Br. In another embodiment, R'7 is H. Preferably, R'7 is H or Br.

[0051] In a preferred embodiment, in formula (I) or (I-1), R'1 is H and R'5 is Br. In another preferred embodiment, in formula (I) or (I-1), at least two of R'5, R'6 and R'7 are halogen.

[0052] The above specific embodiments can be combined with each other.

[0053] Some specific EPAC1 inhibitors for use as defined above have the formula:

[0054] [ka] (Herein designated CE3F4)

[0055] [ka] It has.

[0056] More specifically, some specific compounds for use as defined above have the formula:

[0057] [ka] It has.

[0058] In one embodiment, the EPAC1 inhibitor for use in the present invention has the formula: [ka] It is a compound having the formula:

[0059] Other EPAC1 inhibitors include those of the following formula:

[0060] [ka] Examples of such tetrahydroquinoline derivatives include those represented by the following formula:

[0061] Compounds of formula (I) can be synthesized by the previously published method of P. Bouyssou et al., J. Heterocyclic Chem., 29, 895, 1992. Methods for preparing compounds of formula (I) are well known.

[0062] According to one embodiment, the EPAC1 inhibitor for use in the present invention has the formula (II):

[0063] [ka] [In the formula, R1 is - H; - (C2-C 20 ) alkyl; - (C3-C 10 ) cycloalkyl; - 3-10 membered heterocycloalkyl; - (C6-C 10 ) aryl; and - 5-10 membered heteroaryl selected from the group consisting of: wherein said alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl groups are optionally substituted; R2 is - H; - (C1-C 20 ) alkyl; - (C3-C 10 ) cycloalkyl; - 3-10 membered heterocycloalkyl; - (C6-C 10 ) aryl; and - 5-10 membered heteroaryl or R2 and R4 together with the carbon atoms carrying them (C3-C 10 ) forming a cycloalkyl group, wherein said alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl groups are optionally substituted; R3 is - H; - (C3-C 10 ) cycloalkyl; - 3-10 membered heterocycloalkyl; - (C6-C 10 ) aryl; and - 5-10 membered heteroaryl selected from the group consisting of: wherein said cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups are optionally substituted; and R4 is selected from the group consisting of H, -OH, -NRxRy and -C(O)ORz; Rx, Ry and Rz are independently H or (C1-C 10 ) alkyl; or R2 and R4 together with the carbon atoms carrying them (C3-C 10) forming a cycloalkyl group], or a pharmaceutically acceptable salt, hydrate or hydrated salt thereof, or a polymorphic crystal structure, racemate, diastereomer or enantiomer thereof.

[0064] Thus, a second family of EPAC1 inhibitors for use in the present invention consists of thieno[2,3-b]pyridine derivatives.

[0065] In formula (II), "(C1-C 20 ) alkyl" or "(C2-C 20 The term "alkyl" refers to a saturated or unsaturated aliphatic hydrocarbon group which may be straight-chained or branched, having 1 to 20 carbon atoms or 2 to 20 carbon atoms in the chain, respectively. Preferred alkyl groups have 1 to 5 carbon atoms in the chain, and preferred alkyl groups are, in particular, methyl or ethyl groups. "Branched" means that one or more lower alkyl groups, such as methyl, ethyl or propyl, are attached to a linear alkyl chain. The alkyl group may be substituted.

[0066] In formula (II), "(C3-C 10 The term "cycloalkyl" refers to a cyclic saturated hydrocarbon group having 3 to 10 carbon atoms, in which any available carbon atom may be substituted with a substituent. In particular, a cycloalkyl group is a cyclopropyl or cyclohexyl group.

[0067] "3- to 10-membered heterocycloalkyl" refers to a cyclic saturated hydrocarbon group having 3 to 10 carbon atoms, in which one or more carbon atoms are replaced by one or more heteroatoms, such as nitrogen, oxygen, and sulfur atoms; for example, one or two nitrogen atoms, one or two oxygen atoms, one or two sulfur atoms, or a combination of different heteroatoms, such as one nitrogen atom and one oxygen atom. Any ring atom that can be substituted may be substituted by a substituent. Preferred 3- to 10-membered heterocycloalkyls are nitrogen rings such as furan, thiophene, pyrrole, and pyrazole, or fluorophenyl rings.

[0068] In formula (II), "(C6-C 10 The term "aryl" refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system in which any ring atom capable of substitution may be substituted with a substituent. Examples of aryl moieties include, but are not limited to, phenyl.

[0069] The term "5- to 10-membered heteroaryl" refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system in which any substitutable ring atom may be substituted, and in which one or more carbon atoms are replaced by one or more heteroatoms, such as nitrogen, oxygen, and sulfur atoms; for example, one or two nitrogen atoms, one or two oxygen atoms, one or two sulfur atoms, or a combination of different heteroatoms, such as one nitrogen atom and one oxygen atom. Preferred heteroaryl groups are thienyl, pyridyl, pyrimidyl, and oxazyl groups, and more preferably thienyl groups.

[0070] The term "halogen" refers to atoms in Group 17 of the periodic table and particularly includes fluorine, chlorine, bromine, and iodine atoms, more preferably fluorine, chlorine, and bromine atoms, e.g., fluorine.

[0071] "Optionally substituted" means that the alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups of the compounds of the present invention are substituted with any of the following: -OH, halogen atoms, -C(O)OH, -(C 10 ) alkyl, -(C1-C 10 )alkoxy, and —NR7R8 groups, where R7 and R8 are each independently (C1-C 10 ) alkyl or H.

[0072] Preferably, the alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl groups are optionally substituted with one or more halogen atoms, more preferably with fluorine atoms.

[0073] The term "thieno[2,3-b]pyridine derivative" refers to the following chemical structure:

[0074] [ka] It refers to a compound derived from

[0075] The compounds described herein may have asymmetric centers. Compounds of the present invention containing asymmetrically substituted atoms can be isolated as optically active or racemic forms. Methods for preparing optically active forms, such as by resolving racemic forms or by synthesis from optically active starting materials, are well known in the art. Unless stereochemistry or isomerism is specifically indicated, all chiral forms, diastereomers, racemates, and all geometric isomers of the compounds are intended.

[0076] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness and properties of the compounds of the present invention and is not biologically or otherwise undesirable. Pharmaceutically acceptable acid addition salts can be prepared from inorganic and organic acids, while pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. For a review of pharmaceutically acceptable salts, see Berge et al. ((1977) J. Pharm. Sd, vol. 66, 1). For example, salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitric acid, as well as organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, fumaric acid, methanesulfonic acid, and toluenesulfonic acid.

[0077] A preferred family of EPAC1 inhibitors is of formula (II) above, wherein: R1, - (C2-C 20 ) alkyl; - (C3-C 10 ) cycloalkyl; - 3-10 membered heterocycloalkyl; - (C6-C 10 ) aryl; and - 5-10 membered heteroaryl selected from the group consisting of: wherein said alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl groups are optionally substituted; R2, - H; - (C1-C 20 ) alkyl; - (C3-C 10 ) cycloalkyl; - (C6-C 10 ) aryl; and - 5-10 membered heteroaryl or R2 and R4 together with the carbon atoms carrying them (C3-C 10 ) forming a cycloalkyl group, wherein said alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl groups are optionally substituted; R3, - H; - (C3-C 10 ) cycloalkyl; - 3-10 membered heterocycloalkyl; - (C6-C 10 ) aryl; and - 5-10 membered heteroaryl selected from the group consisting of: wherein said cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups are optionally substituted; and R4 is selected from the group consisting of H, -OH, -NRxRy and -C(O)ORz; Rx, Ry and Rz are independently H or (C1-C 10 ) alkyl; or R2 and R4 together with the carbon atoms carrying them (C3-C 10 ) forming a cycloalkyl group, or a pharmaceutically acceptable salt, hydrate or hydrated salt thereof, or a polymorphic crystal structure, racemate, diastereomer or enantiomer thereof.

[0078] A preferred family of EPAC1 inhibitors is of formula (II) above, wherein: R1, - (C2-C 20 ) alkyl; - (C3-C 10 ) cycloalkyl; - 3-10 membered heterocycloalkyl; - (C6-C 10 ) aryl; and - 5-10 membered heteroaryl selected from the group consisting of: wherein said alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl groups are optionally substituted; R2, - H; - (C1-C 20 ) alkyl; - (C3-C 10 ) cycloalkyl; - (C6-C 10 ) aryl; and - 5-10 membered heteroaryl or R2 and R4 together with the carbon atoms carrying them (C3-C 10 ) forming a cycloalkyl group, wherein said alkyl, cycloalkyl, aryl and heteroaryl groups are optionally substituted; R3, - H; - (C3-C 10 ) cycloalkyl; - 3-10 membered heterocycloalkyl; - (C6-C 10 ) aryl; and - 5-10 membered heteroaryl selected from the group consisting of: wherein said cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups are optionally substituted; and R4 is selected from the group consisting of H, -OH, -NRxRy and -C(O)ORz; Rx, Ry and Rz are independently H or (C1-C 10 ) alkyl; or R2 and R4 together with the carbon atoms carrying them (C3-C 10 ) forming a cycloalkyl group, or a pharmaceutically acceptable salt, hydrate or hydrated salt thereof, or a polymorphic crystal structure, racemate, diastereomer or enantiomer thereof.

[0079] In one embodiment, in formula (II), R3 is - (C3-C 10 ) cycloalkyl; - 3-10 membered heterocycloalkyl; - (C6-C 10 ) aryl; and - 5-10 membered heteroaryl selected from the group consisting of: wherein said cycloalkyl, heterocycloalkyl, aryl and heteroaryl groups are optionally substituted.

[0080] In certain embodiments, in Formula (II), R3 is selected from the group consisting of one or more substituents (preferably (C1-C 10 ) alkyl and halogen atoms) (C-C 10 In one embodiment, R3 is H or (C1-C 10) optionally substituted with one or more substituents selected from the group consisting of alkyl and halogen atoms (C-C 10 ) aryl. In certain embodiments, R3 is H or phenyl optionally substituted with one or more halogen atoms. In certain embodiments, R3 is phenyl optionally substituted, preferably with one or more halogen atoms.

[0081] In one embodiment, in formula (II), R1 is - H; - (C6-C 10 ) aryl; and - 5-10 membered heteroaryl selected from the group consisting of: wherein the aryl and heteroaryl groups are —NR7R8, (C1-C 10 ) alkyl and halogen atoms; wherein R7 and R8 are each independently (C1-C 10 ) alkyl or H.

[0082] In one embodiment, in formula (II), R is H or is substituted by one or more substituents (e.g., (C-C 10 ) optionally substituted (C6-C) by a substituent selected from the group consisting of alkyl, halogen atoms and -NR7R8 groups 10 )aryl; wherein R7 and R8 are each independently (C1-C 10 ) alkyl or H. In certain embodiments, R1 is H or phenyl optionally substituted with one or more halogen atoms (e.g., with one fluorine atom, preferably at the para position).

[0083] In another embodiment, R1 is - (C6-C 10 ) aryl; and - 5-10 membered heteroaryl selected from the group consisting of: wherein the aryl and heteroaryl groups are (C1-C 10 ) optionally substituted by one or more substituents selected from the group consisting of alkyl, halogen atoms and -NR7R8 groups; wherein R7 and R8 are each independently (C1-C 10 ) alkyl or H.

[0084] Preferably, in formula (II), R is substituted by one or more substituents (e.g., (C-C 10 ) optionally substituted (C6-C) by a substituent selected from the group consisting of alkyl, halogen atoms and -NR7R8 groups 10 )aryl; wherein R7 and R8 are each independently (C1-C 10 ) alkyl or H. In certain embodiments, R1 is phenyl optionally substituted with one or more halogen atoms (e.g., with one fluorine atom, preferably at the para position).

[0085] In one embodiment, in formula (II), R2 is - H; - (C1-C 20 ) alkyl; - (C6-C 10 ) aryl; and - 5-10 membered heteroaryl or R2 and R4 together with the carbon atoms carrying them (C3-C 10 ) forming a cycloalkyl group, wherein the alkyl, cycloalkyl, aryl and heteroaryl groups are (C1-C 10 Optionally substituted with one or more substituents selected from the group consisting of alkyl and halogen atoms.

[0086] In certain embodiments, in Formula (II), R2 is (C1-C 10or R2 and R4 together with the carbon atoms carrying them form a (C3-C6) cycloalkyl group, wherein the alkyl, cycloalkyl, and heteroaryl groups are preferably selected from the group consisting of (C1-C 10 ) optionally substituted with one or more substituents selected from the group consisting of alkyl and halogen atoms.

[0087] In certain embodiments, in formula (II), R2 is selected from the group consisting of 5-6 membered heteroaryl groups, said heteroaryl groups preferably being (C1-C 10 ) optionally substituted with one or more substituents selected from the group consisting of alkyl and halogen atoms. In certain embodiments, R2 is a thienyl group.

[0088] In certain embodiments, in Formula (II), R2 is (C1-C 10 ) alkyl and thienyl rings, or R2 and R4 together with the carbon atoms carrying them form a (C5-C6) cycloalkyl group, such as a cyclohexyl group.

[0089] In certain embodiments, in formula (II), R3 is preferably (C1-C 10 ) optionally substituted by one or more substituents selected from the group consisting of alkyl and halogen atoms (C-C 10 ) aryl.

[0090] In certain embodiments, in Formula (II), R4 is selected from the group consisting of H, -OH, -NH2, and -C(O)OH, or R2 and R4 together with the carbon atom bearing them are (C3-C 10 ) cycloalkyl group. In one embodiment, in formula (II), R4 is H, or R2 and R4 together with the carbon atoms carrying them form a (C5-C6) cycloalkyl group. Preferably, R4 is H.

[0091] In certain embodiments, in Formula (II), R4 is H, or R2 and R4 together with the carbon atoms carrying them form a (C5-C6)cycloalkyl group.

[0092] In certain embodiments, in Formula (II), R 1 is a phenyl group and / or R 2 is a thienyl group, said phenyl and thienyl groups being optionally substituted.

[0093] In certain embodiments, in Formula (II), at least one of R and R is (C-C 10 ) an aryl group or a 5- to 10-membered heteroaryl group.

[0094] In certain embodiments, the EPAC1 inhibitor for use in the present invention is a compound having formula (II), wherein: R1, - (C2-C 20 ) alkyl; - (C3-C 10 ) cycloalkyl; - 3-10 membered heterocycloalkyl; - (C6-C 10 ) aryl; and - 5-10 membered heteroaryl selected from the group consisting of: wherein said alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups are optionally substituted; and R2, - H; - (C1-C 20 ) alkyl; - (C3-C 10 ) cycloalkyl; - (C6-C 10 ) aryl; and - 5-10 membered heteroaryl or R2 and R4 together with the carbon atoms carrying them (C3-C 10 ) forming a cycloalkyl group, The compounds comprise alkyl, cycloalkyl, aryl, and heteroaryl groups, wherein the alkyl, cycloalkyl, aryl, and heteroaryl groups are optionally substituted.

[0095] A preferred family of EPAC1 inhibitors for use in the present invention is represented by the following formula (III):

[0096] [ka] [In the formula, - Ra, Rb, Rc, Rd, Re, Rx, Ry and Rz are H, -OH, halogen atoms, -C(O)OH, (C1-C 10 ) alkyl, (C1-C 10 ) alkoxy, and -NR5R6; wherein R5 and R6 are each independently (C1-C 10 ) alkyl or H; - R4 is selected from the group consisting of H, -OH, -NH2 and -C(O)OH; and - R3 has the same meaning as in formula (II) above.

[0097] In certain embodiments, in Formula (III), Ra, Rb, Rc, Rd, Re, Rx, Ry, and Rz are H, a halogen atom, or (C1-C 10 ) alkyl. In one embodiment, in formula (III), Rx, Ry and Rz are H, and / or Ra, Rb, Rd and Re are H. Preferably, in formula (III), Rc is H or a halogen atom, for example, a fluorine atom.

[0098] According to one embodiment, the EPAC1 inhibitor for use in the present invention has the following formula:

[0099] [ka] It has one of the following.

[0100] Preferably, the EPAC1 inhibitor for use in the present invention has the formula:

[0101] [ka] and is designated herein as AM-001.

[0102] The present invention also relates to an EPAC1 inhibitor for use as defined above, said EPAC1 inhibitor being used in combination with pirfenidone or nintedanib.

[0103] The present invention also relates to a method for the prevention and / or treatment and / or amelioration of idiopathic pulmonary fibrosis, comprising administering to a patient in need thereof a pharmaceutically acceptable amount of an EPAC1 inhibitor, optionally in combination with pirfenidone or nintedanib. [Brief explanation of the drawings]

[0104] [Figure 1] Epac1 expression is elevated in pulmonary fibrosis. A) Epac1 mRNA expression levels quantified by RTq-PCR (n=8 / group) in human non-IPF and IPF patients. B) and C) Epac1 mRNA and protein expression quantified by qRT-PCR (n=8 / group) and Western blot (n=3 / group) in an experimental model of idiopathic pulmonary fibrosis induced by intratracheal instillation of bleomycin (4 U / kg; 14 days). Data are shown as mean ± SEM, and p values ​​were calculated using a t-test; *=p<0.05, ***=p<0.001. [Figure 2]The potent Epac1 inhibitor CE3F4 significantly reduced fibroblast proliferation and suppressed the expression of fibrotic markers induced by TGF-β1. A) Schematic diagram of the experimental design to study the role of the Epac1 inhibitor CE3F4 in normal human lung fibroblasts (NHLFs). B) NHLF cell proliferation was determined by labeling cells with the BrdU assay. Relative proliferation under specific conditions is shown (n = 4). C) NHLF cells were treated with TGF-β1 (2 ng / mL; 48 h) alone or in combination with the Epac1 inhibitor CE3F4 (20 μM; 48 h). Fibrotic marker mRNA levels of procollagen type I, α1 [COL1], type III collagen [COL3], connective tissue growth factor [CTGF], and transforming growth factor β [TGFβ] were quantified by qPCR (n = 3). Data are presented as mean±SEM and p-values ​​were calculated using one-way ANOVA; ns=not significant, *=p<0.05, **=p<0.01, and ***=p<0.001. [Figure 3] AM-001, a novel Epac1-selective inhibitor, blocked lung fibroblast proliferation and reduced the expression of fibrotic markers in NHLFs in vitro. A) Schematic of the experimental strategy to evaluate the role of AM-001 in normal human lung fibroblasts. B) Proliferation was determined by labeling cells with the BrdU assay. Relative proliferation under specific conditions is shown (n = 4). C) NHLF cells were treated with TGF-β1 alone or in combination with AM-001 (20 μM; 48 h), and mRNA levels of fibrotic markers (procollagen type I, α1 [COL1], type III collagen [COL3], connective tissue growth factor [CTGF], and transforming growth factor β [TGFβ]) were quantified by qPCR (n = 3). Data are presented as mean ± SEM, and p values ​​were calculated using one-way ANOVA; ns = not significant, * = p < 0.05, ** = p < 0.01, and *** = p < 0.001. [Figure 4]A) Schematic of the experimental design to evaluate the therapeutic effect of the Epac1 inhibitor AM-001 in a mouse model of bleomycin-induced pulmonary fibrosis. B) Right ventricular systolic pressure (RVSP) and C) right ventricular hypertrophy were assessed by Fulton's index (RV / (LV + septum)) (n = 3–7 per group). Data are shown as mean ± SEM, and p values ​​were calculated using one-way ANOVA; * = p < 0.05, ** = p < 0.01, and *** = p < 0.001. [Figure 5] The Epac1 inhibitor AM-001 reduces lung fibrosis in mice with bleomycin-induced pulmonary fibrosis. A) Representative Masson's trichrome-stained lung sections from the indicated mice (upper panel). The graph shows quantification of fibrosis. B) Levels of fibrotic markers COL1, COL3, CTGF, and TGFβ were quantified by qPCR in lung tissue (n = 3–7 / group). C) Representative hematoxylin and eosin-stained lung sections (top) and quantification of medial wall thickness (bottom) are shown (n = 3–7 / group). D) RV sections were stained with fluorescently labeled wheat germ agglutinin to examine the cross-sectional area of ​​RV cardiomyocytes (top) and quantification of cardiomyocyte cross-sectional area (bottom). E) fibrotic marker mRNA levels of COL1, COL3, CTGF, and TGFβ were quantified by qPCR in RV tissue (n = 3–7 / group). F) mRNA levels of RV cardiac hypertrophy-related transcripts (atrial natriuretic factor [ANF], brain natriuretic peptide [BNP], β-myosin heavy chain [β-MHC]) assessed by qPCR. [Figure 6] Epac1- / - mice are protected from bleomycin-induced pulmonary fibrosis. A) Representative Masson's trichrome-stained lung sections from the indicated mice (left panel). The graph shows quantification of fibrosis (right panel). B) Levels of fibrotic markers COL1A1, COL3A1, CTGF, and TGFβ were quantified by qPCR in lung tissue (n = 3-7 / group). Data are shown as mean ± SEM, and p values ​​were calculated using t-test or one-way ANOVA; * = p < 0.05, ** = p < 0.01, and *** = p < 0.001. [Figure 7]Epac1 inhibition reduced FGFR1 and TNC expression. A-B) FGFR1 and TNC mRNA expression in RNA isolated from human lung and C-D) BLM Epac1 KO-treated mice (28 days) compared to controls. E-F) FGFR1 and TNC mRNA levels in BLM + vehicle vs. BLM + AM-001-treated mice (n = 3-7 / group). Data are shown as mean ± SEM, and p values ​​were calculated using t-test or one-way ANOVA; * = p < 0.05, ** = p < 0.01, and *** = p < 0.001. [Figure 8] FOXO3 transcription factor as a target of Epac1. A) FOXO3 mRNA expression in fibroblasts after Epac1 silencing or B) Ad.Epac overexpression in NHLF cells (n=3). [Figure 9] Epac1 silencing reduces proliferation, IL-6, and SMA expression, and restores FOXO3 in IPF fibroblasts. A) Epac1 mRNA expression in NHLFs and IPF FBs transfected with lenti.sh.Epac1 for 72 hours. B) Proliferation of NHLFs and IPF FB cells as determined by BrdU assay under the indicated conditions. C-E) IL-6, α-SMA, and FOXO3 mRNA expression (n=2). [Figure 10] AM-001 reduced IPF fibroblast proliferation and decreased IL-6 and α-SMA expression. A) Epac1 mRNA expression in NHLF and IPF FBs transfected with lenti.sh.Epac1 for 72 hours. B) NHLF cell proliferation determined by BrdU assay under the indicated conditions. C-E) IL-6, α-SMA, and FOXO3 mRNA expression levels in NHLF and IPF fibroblasts treated with AM-001 (20 μM for 48 hours, n=2). F) Representative immunoblots under the indicated conditions in NHLF and IPF fibroblasts treated with AM-001. [Example]

[0105] Background: Exchange protein directly activated by cAMP (Epac) is a PKA-independent signaling molecule activated by adrenergic stimulation (de Rooij et al., 1998, Nature, 396, 474-7; Kawasaki et al., 1998, Science, 282, 2275-9). Two isoforms, Epac1 and Epac2, have been identified. Epac1 is ubiquitously expressed. The two EPAC isoforms, EPAC1 and EPAC2, are guanine nucleotide exchange factors for the Ras-like GTPases Rap1 and Rap2, which are activated independently of protein kinase A, the classical effector of cAMP. With the development of pharmacological modulators of EPAC, numerous reports in the literature have demonstrated the important role of EPAC in regulating various cardiovascular and renal diseases (Yang et al., 2013, Am J Physiol Renal Physiol, 304, F831-9; Laudette et al., 2018, J Cardiovasc Dev Dis, 5). Epac1-selective inhibitory compounds have been investigated for their potential properties in cardiovascular diseases (Fazal et al., 2017, Circ Res, 120, 645-657). The AM-001 compound has been identified and characterized as a novel selective pharmacological inhibitor of Epac1. This small molecule, a thieno[2,3-b]pyridine derivative (3-amino-N-(4-fluorophenyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridine-2-carboxamide), selectively inhibits the catalytic activity of Epac1. AM-001 exhibits cardioprotective properties against myocardial ischemia / reperfusion injury and the deleterious effects of chronic β-AR activation (Laudette et al., 2019, Cardiovasc Res, 115, 1766-1777).

[0106] Materials and Methods This study used RNA, protein samples, and formalin-fixed, paraffin-embedded sections from human IPF and healthy control donors. Human lung tissue was obtained from IPF patients (n = 8) undergoing surgery for organ transplant programs, and healthy control lung explant samples were obtained from the organ transplant program of the University General Consortium Hospital of Valencia (n = 8). Samples were anonymous and archival. The protocol was approved by the Regional Research and Independent Ethics Committee of the University General Consortium Hospital of Valencia (CEIC / 2013). Written informed consent was obtained from each participant.

[0107] Animals. Mice were housed in a pathogen-free facility, and all animal experiments were approved by the Animal Care and Use Committees of the University of Toulouse. Epac1-deficient mice (Epac1- / -) were engineered in the laboratory of Dr. Frank Lezoualc'h. Briefly, Epac1 knockout mice were generated by inserting loxP sequences into introns 7 and 15 of the RAPGEF3 gene by Genoway. Desmin-Cre transgenic females (C57BL / 6 background) that sporadically express Cre recombinase in oocytes were crossed with Epac1 floxed / floxed males (C57BL / 6-SV129 background) to generate Desmin-Cre-Epac1- / - and then Epac1- / - mice. Genotypes were confirmed by PCR before use. In this study, 14-week-old mice (Epac1 knockout and control littermates on a C57BL / 6 or C57BL / 6-SV129 background) were used.

[0108] Intratracheal bleomycin animal model. All animal experiments and procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Animals were anesthetized by IP injection of xylazine / ketamine and secured in a supine position on a tray. After intubation, the board was tilted 45 degrees, and an IA-1C Microsprayer tip (PennCentury, Wyndmoor, PA) was inserted into the lumen of the angiocatheter. Animals received a single intratracheal injection of BLM (50 μL; 4 U / kg) for 28 days. AM-001, a pharmacological inhibitor of Epac1, was intraperitoneally injected (10 mg / kg) every other day for 2 weeks.

[0109] Cardiac Hemodynamic Study. Mice were anesthetized with isoflurane (2–4%), intubated via tracheotomy, and mechanically ventilated with 1–2% isoflurane and oxygen (tidal volume, 6 mL / kg; respiratory rate, 100 breaths / min). The thoracic cavity was opened, and organs were accessed via a sternotomy. Once the pericardium was opened and the heart was fully accessible, an ultrasound flowmeter probe (Flowmeter Probe 2.5S176; Transonic Systems Inc., Ithaca, NY) was inserted into the RV to collect right ventricular systolic pressure (RVSP). Hemodynamic data were recorded using a Scisense PV Control Unit (Scisense, Ontario, Canada).

[0110] Right ventricular weight measurement. After collecting hemodynamic data, the mice were harvested and the heart and lungs were collected. The heart was removed from the chest and perfused with PBS to remove blood and clots. Both the atria and connecting vessels were separated. Next, the RV was separated from the heart and weighed. Finally, the remaining left ventricle (LV) and septum were weighed. The Fulton index was calculated by the weight ratio of the RV weight to the LV + septum weight (RV weight / LV + septum weight) to specifically account for RV hypertrophy.

[0111] Hematoxylin & Eosin and Masson's Trichrome Staining. Lung tissue was excised, filled with PBS / OCT (50:50), and fixed in OCT (frozen at -80°C). Sections were cut at 8 μm and attached to color-frosted glass slides (ThermoFisher). Lung tissue sections were stained with hematoxylin and eosin (H&E) and Masson's Trichrome (Sigma-Aldrich) and visualized using a light microscope. Medial wall thickness and collagen deposition were then quantified using ImageJ software.

[0112] Wheat germ agglutinin (WGA) immunostaining RV sections were fixed with 1% paraformaldehyde (PFA), stained with fluorescently labeled wheat germ agglutinin (WGA) (Invitrogen) overnight at 4°C, and imaged under a Zeiss Observer Z.1 microscope (Carl Zeiss) at 160x magnification. Cardiomyocyte outlines were traced, and cardiomyocyte area was calculated using ImageJ software.

[0113] Cell Culture. Normal human lung fibroblasts (NHLF) and lung fibroblasts from IPF patients were purchased from Lonza, Inc. (Allendale, NJ), cultured as recommended in FGM-2 medium supplemented with 5% fetal bovine serum (FBS) at 37°C in 5% CO2, and passaged upon reaching confluence. All cell lines were tested by the manufacturer and were negative for HIV-1, HBV, HCV, mycoplasma, bacteria, yeast, and fungi.

[0114] shRNA and lentivirus production. Epac1 shRNA (TRCN0000047228) cloned into the pLKO.1 lentiviral expression vector was obtained from Dharmacon. For lentivirus production, the construct and the viral packaging plasmids pSPAX2 and pMD2.G were cotransfected into 293T cells using Effectene® (Qiagen) according to the manufacturer's recommendations. Virus was concentrated by incubation with a Lenti-X Concentrator (Clontech) as recommended by the supplier. Concentrated viral particles were used to infect NHLF cells for 72 hours. RNA and protein expression were measured by RT-qPCR and immunoblotting, respectively, to verify Epac1 knockdown.

[0115] Pharmacological treatment. Cells were seeded at 250,000 cells / well in a 6-well plate and maintained in a 37°C incubator with 5% CO for 24 hours before use. Cells were treated with TGF-β1 (2 ng / mL; 48 hours) alone or in combination with the Epac1 inhibitors CE3F4 (20 μM; 48 hours) or AM-001 (20 μM; 48 hours).

[0116] Cell proliferation NHLF proliferation was measured by 5-bromo-2′-deoxyuridine (BrdU) incorporation using the Cell Proliferation ELISA, BrdU (colorimetric) assay (Roche, Indianapolis, IN) according to the manufacturer's instructions.

[0117] Total RNA isolation, cDNA preparation, and quantitative RT-PCR analysis Total RNA was isolated using TRIzol™ (Invitrogen) and purified using RNeasy mini-columns (Qiagen) according to the manufacturer's instructions. cDNA was synthesized using a cDNA synthesis kit (Applied Biosystems, Foster City, CA) as described by the manufacturer. Quantitative RT-PCR was performed using the PerfeCTa SYBR™ Green FastMix kit (Quantabio, Beverly, MA) and specific primers for the indicated genes according to the manufacturer's instructions. Fold changes in gene expression were determined using the relative comparison method, normalized to GAPDH as an internal loading control.

[0118] SDS-PAGE and immunoblot analysis. Protein lysates were prepared using RIPA lysis buffer (Boston BioProducts) containing a protease / phosphatase inhibitor cocktail (Pierce). After centrifugation at 15,000 × g for 20 min, protein concentrations were determined using a bicinchoninic acid (BCA) assay (Sigma-Aldrich). Proteins were then separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk and hybridized overnight at 4°C with the following primary antibodies: Epac1 (Cell Signaling), phospho-SMAD2 / 3 (Cell Signaling), phospho-STAT3 (Cell Signaling), phospho-AKT (Cell Signaling), total AKT (Cell Signaling), and GAPDH (Thermofisher). The membranes were then incubated with the appropriate secondary HRP-conjugated antibodies (Cell Signaling) for 1 h, and the blots were developed using the ECL system (Thermofisher).

[0119] result 1. Increased Epac1 Expression in Human IPF and a Mouse Model of BLM-Induced Pulmonary Fibrosis. To determine the contribution of Epac protein to the pathogenesis of IPF, Epac1 and Epac2 mRNA expression levels were measured in lung biopsies from patients diagnosed with IPF and in normal lungs. We found a significant increase in Epac1 mRNA expression, whereas Epac2 mRNA levels were unchanged in human fibrotic lung tissue compared with healthy lung samples (Figure 1A). These findings support the finding of increased Epac1a mRNA and protein expression in lung samples from an experimental mouse model of PF induced by intratracheal delivery of bleomycin (BLM) (Figure 1B-C).

[0120] 2. Selective pharmacological inhibition of Epac1 reduced proliferation and suppressed the expression of several fibrotic markers. To further investigate the role of Epac1a in pulmonary fibrosis, we examined the effect of Epac1 inhibition on the proliferation of normal human lung fibroblasts (NHLFs) using bromodeoxyuridine (BrdU). NHLF cells were treated with TGFβ alone or in combination with the Epac1 inhibitor CE3F4 for 48 hours using medium containing either 0.1% or 5% FBS (Figure 2A). The profibrotic TGFβ1 is a key mediator of fibrosis and is thought to contribute to the pathogenesis of IPF (Fernandez et al., 2012). TGFβ treatment and serum stimulation increased NHLF proliferation, whereas CE3F4 treatment significantly reduced this response (Figure 2B). We further sought to determine whether Epac1 inhibition modulated the expression of several TGFβ1-regulated genes in vitro. Therefore, NHLF cells were treated with TGFβ1 alone or in combination with the CE3F4 compound for 48 hours. We then analyzed the mRNA expression of several fibrotic markers, including collagen IAI (COLIA1), COLIII, as well as connective tissue growth factor (CTGF) and transforming growth factor beta (TGFβ1). Notably, Epac1 inhibition by the CE3F4 compound impaired the induction of fibrotic markers induced by TGFβ treatment (Figure 2C).

[0121] 3. AM-001, a novel Epac1-selective inhibitor, inhibited lung fibroblast proliferation and reduced the expression of fibrotic markers in NHLFs in vitro. CE3F4 was identified as a selective pharmacological inhibitor of Epac1 in vitro, but its low bioavailability precludes its use in future in vivo applications. Therefore, we investigated the effects of AM-001, a novel Epac1-selective inhibitory compound designated AM-001, on NHLF proliferation and the expression of fibrotic markers in vitro. Indeed, the small molecule AM-001, a thieno[2,3-b]pyridine derivative (3-amino-N-(4-fluorophenyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridine-2-carboxamide), exhibited selective inhibitory activity against Epac1, while no antagonistic activity against Epac2 or protein kinase A (PKA) activity was reported (Laudette et al., Cardiovasc. Res. 2019, 115(12):1766-1777). This newly characterized and identified compound showed promising cardioprotective properties against myocardial ischemia / reperfusion injury and pathological cardiac remodeling during chronic β-AR activation in preclinical studies. Similar to previous experiments performed in the presence of CE3F4, NHLF cells were treated with TGFβ alone or in combination with the novel Epac1 inhibitor AM-001 for 48 hours using medium containing either 0.1% or 5% FBS (Figure 3A). Our results showed that AM-001 inhibited NHLF proliferation induced by high serum or TGFβ stimulation (Figure 3B). Consistent with previous results, AM-001 impaired the upregulation of fibrotic gene markers induced by TGFβ in vitro (Figure 3C).

[0122] 4. Pharmacological inhibition of Epac1 as a novel therapeutic strategy for inhibiting pulmonary fibrosis and pulmonary dysfunction in a bleomycin-induced PF mouse model. To further support the involvement of Epac1 upregulation in the pathogenesis of pulmonary fibrosis, the potential therapeutic effects of Epac1 inhibition were next evaluated in vivo using the Epac1-selective inhibitor AM-001 (Laudette et al., Cardiovasc. Res. 2019, 115(12):1766-1777). The BLM mouse model is frequently used to induce pulmonary fibrosis and remains the most commonly used rodent animal model for studying interstitial lung disease (Liu et al., 2017, Methods Mol Biol, 1627, 27-42; Leach et al., 2013, Am J Respir Cell Mol Biol, 49, 1093-101). BLM aerosol administration induces increased production of reactive oxygen species, which causes cellular damage to endothelial cells and other cell types, leading to the production of cytokines and profibrotic mediators such as TGFβ and IL-6, resulting in lung injury with a subsequent fibroproliferative response in mice (Adamson, 1976, Environ Health Perspect, 16, 119-26; Yamamoto and Nishioka, 2005, Exp Dermatol, 14, 81-95; Leach et al., 2013).

[0123] Using a treatment strategy, mice were randomly assigned to either a sham control group receiving intratracheal administration of saline or a PF group receiving a single intratracheal aerosol dose of BLM (4 U / kg). Two weeks later, the BLM-treated group was randomly assigned to receive either vehicle or the Epac1 inhibitor AM-001 for two weeks (Figure 4A). Vehicle or AM-001 was injected intraperitoneally (10 mg / kg) every other day for two weeks. Interestingly, the AM-001-treated group showed reduced right ventricular systolic pressure (RVSP) compared with the control group (Figure 4B). Furthermore, our results also showed that AM-001 treatment significantly reduced RV hypertrophy in BLM-treated mice, as revealed by a reduction in Fulton's index (Figure 4C).

[0124] 5. AM-001 Effectively Reverses Interstitial Lung Fibrosis and Vascular Remodeling. To further explore the potential therapeutic effects of Epac1 inhibition in PF, we next assessed interstitial fibrosis levels and vascular remodeling by histological analysis after 2 weeks of treatment. The results showed that both interstitial and perivascular fibrosis increased in response to bleomycin injection, resulting in substantial histological tissue damage in the vehicle-treated group compared with the AM-001-treated group (Figure 5A). Interestingly, these results showed that both interstitial and perivascular fibrosis increased in the vehicle control group and significantly decreased in the AM-001-treated BLM group (Figure 5A). We also measured the expression of several markers of fibrosis in lung samples by RT-qPCR. We found that Epac1 inhibition significantly reduced the mRNA levels of COLI, COLIII, CTGF, and TGFβ in lung samples (Figure 5B). Furthermore, morphometric analysis of the distal pulmonary artery demonstrated a significant increase in medial wall thickness in BLM-treated mice (Figure 5C). Similarly, AM-001 significantly reduced pulmonary vascular remodeling compared with vehicle-treated BLM mice (Figure 5C). Histological analysis demonstrated a reduction in RV cardiomyocyte size when treated with the Epac1 inhibitor AM-001 (Figure 5D). Furthermore, the upregulation of fibrosis- and hypertrophy-related gene markers, such as atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and beta-myosin heavy chain (β-MHC), was impaired in AM-001-treated mice (Figure 5E).

[0125] Overall, these preclinical data indicated that pharmacological inhibition of Epac1 significantly inhibited pulmonary fibrosis, vascular remodeling, and RV hypertrophy while improving hemodynamic parameters in a bleomycin-induced IPF model. Collectively, the in vitro and in vivo results further support the pharmacological inhibition of Epac1 as a promising therapeutic strategy for treating patients with pulmonary fibrosis.

[0126] 6. Epac1 deficiency protects mice from BLM-induced fibrosis. We attempted to address the impact of Epac1 deficiency on PF. Epac1 KO juvenile mice (gift from Dr. Lezoualch) and WT mice were randomly assigned to two groups: an uninjured control saline group (n = 10) and a BLM group (n = 10) administered a single intratracheal injection of BLM (4 U / kg) for 28 days. Notably, 28 days after BLM, fibrotic lesions assessed by Masson's trichrome staining were significantly reduced in Epac1 KO mice compared with WT BLM-treated mice (Figure 6A). Consistent with this, we found that mRNA levels of fibrotic markers, such as procollagen type I α1 [COL1A1], type III collagen [COL3A1], connective tissue growth factor [CTGF], and transforming growth factor β [TGFβ], were significantly reduced in BLM Epac KO mice (Figure 6B). These data suggest that Epac1 is required for the development of pulmonary fibrosis and that its deletion is sufficient to confer marked protection from fibrosis.

[0127] 7. RNA-Seq Identifies Epac1 Target Genes Involved in Key Fibrotic Pathways. To comprehensively identify Epac1 targets, we performed RNA-Seq in NHLFs treated with either DMSO or 20 μM AM-001 (an Epac1 inhibitor) for 48 hours. Analysis of our preliminary data sets in control and AM-001-treated NHLF cells identified key IPF gene signatures involved in fibrosis, ECM remodeling, and proliferation during lung injury.

[0128] From this extensive dataset, TGFβ signaling, FGFR1 (fibroblast growth factor receptor 1), and TNC (tenascin-C) were selected as candidate genes. TGF-β is a regulator of the FGF / FGFR signaling cascade in human lung fibroblasts (1, 2), demonstrating complex interactions among many growth factors. TGF-β also upregulated TNC (3-5). Collectively, this evidence suggests a critical role for Epac1 in the disease pathogenesis of pulmonary fibrosis. FGFR1 and TNC are established triggers of pulmonary fibrosis (5, 6), and both are upregulated in our human lung samples (Figure 7A-B). We also validated the RNA-seq results in Epac1 KO mice. The data showed that BLM significantly increased FGFR1 and TNC mRNA levels in treated WT mice compared to Epac1 KO mice (Figure 7C-D). Interestingly, AM-001 treatment reversed the effects of BLM by significantly reducing FGFR1, along with a trend toward a decrease in TNC mRNA in WT mice (Figure 7E-F). We further analyzed the RNA-Seq dataset obtained using AM-001-treated NHLFs and identified the top 50 transcription factors (TFs) using ENCODE and ChEA libraries. We identified Forkhead Box O3 (FOXO3) as a promising candidate. Forkhead Box O (FOXO) is an evolutionarily conserved TF that is a target of the PI3-kinase / protein kinase B-AKT signaling pathway and is involved in a series of fundamental biological processes.

[0129] Studies have shown that loss of FOXO3 leads to a transdifferentiation and hyperproliferative phenotype of lung fibroblasts (7, 8). Furthermore, FOXO3 KO mice exhibited enhanced sensitivity to BLM administration, increased fibrosis, loss of lung function, and increased mortality (7). Notably, shRNA-mediated Epac1 silencing enhanced FOXO3 mRNA expression, whereas Epac1 overexpression reversed these effects in NHLF cells (Figure 8A-B).

[0130] 8. Effect of Epac1 Loss or Gain of Function in Normal and IPF Fibroblasts. Epac1-deficient IPF fibroblasts or those treated with AM-001 were assessed for proliferation under basal conditions using a BrdU incorporation assay (22). Preliminary data indicate that IPF fibroblasts (FB) exhibit higher Epac1 mRNA levels compared with NHLFs under basal conditions, and both Epac1 knockdown (Figure 9A) and AM-001 inhibition reduce IPF fibroblast proliferation (Figures 9B–10B). Epac1 has emerged as an essential regulator of the proinflammatory IL-6 signaling pathway (9, 10). Deletion or inhibition of Epac1 in IPF fibroblasts attenuates IL-6 and α-SMA uptake and restores FOXO3 mRNA expression (Figures 9C–E and 10C–E). Furthermore, the phosphorylation of SMAD2 / 3, STAT3, and AKT was decreased in AM-001-treated IPF fibroblasts (Fig. 10E), suggesting that AM-001 targets TGF-β1 signaling, AKT, and IL-6.

[0131] In summary, these data provide preliminary evidence for the potential of Epac1 as a key player in profibrotic gene expression and fibroblast activation and proliferation in PF. Thus, Epac1 may be a promising therapeutic target for the development of AM-001 as a novel antifibrotic drug for PF. [Prior art documents] [Non-patent literature]

[0132] [Table 1]

Claims

1. An EPAC1 inhibitor for use in the treatment, prevention and / or amelioration, preferably the treatment, of idiopathic pulmonary fibrosis.

2. 2. An EPAC1 inhibitor for use according to claim 1, comprising the formula (I): 【Chemistry 21】 [In the formula, R'9 is H or a group of the formula: 【Chemistry 22】 and is a group represented by the following formula: 【Chemistry 23】 is the bond to the nitrogen atom of the tetrahydroquinoline; R'1, R'2, R'3, R'4, and R'8 are H, (C １ -C １０ ) alkyl, (C ３ -C １０ ) cycloalkyl, (C ６ -C １０ ) aryl, (C １ -C ６ ) alkylene-(C ６ -C １０ ) aryl and (C ３ -C １０ ) heteroaryl, wherein the aryl and heteroaryl groups are independently selected from the group consisting of OH, NH ２ , NO ２ , (C １ -C ６ ) optionally substituted with at least one substituent selected from alkyl, and halogen; R'5 is a halogen atom; - R'6 and R'7 are independently selected from the group consisting of H and a halogen atom], or a pharmaceutically acceptable salt, hydrate or hydrated salt thereof, or a polymorphic crystal structure, racemate, diastereomer or enantiomer thereof, with the proviso that The compound of formula (I) has the following formula: 【Chemistry 24】 is different from the compound represented by the formula:

3. The EPAC1 inhibitor for use according to claim 2, wherein in formula (I), R'9 is H.

4. 3. The EPAC1 inhibitor for use according to claim 1 or 2, which is represented by the following formula (I-1): 【Chemistry 25】 The EPAC1 inhibitor having the formula:

5. EPAC1 inhibitor for use according to claim 1, which is of formula (II): 【Chemistry 26】 [In the formula, R １ teeth, - (C ２ -C ２０ ) alkyl; - (C ３ -C １０ ) cycloalkyl; 3- to 10-membered heterocycloalkyl; - (C ６ -C １０ ) aryl; and 5- to 10-membered heteroaryl selected from the group consisting of: wherein said alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl groups are optionally substituted; R ２ teeth, - H; - (C １ -C ２０ ) alkyl; - (C ３ -C １０ ) cycloalkyl; - (C ６ -C １０ ) aryl; and 5- to 10-membered heteroaryl or R ２ and R ４ together with the carbon atoms that carry them (C ３ -C １０ ) forming a cycloalkyl group; wherein said alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl groups are optionally substituted; R ３ teeth, - H; - (C ３ -C １０ ) cycloalkyl; 3- to 10-membered heterocycloalkyl; - (C ６ -C １０ ) aryl; and 5- to 10-membered heteroaryl selected from the group consisting of: wherein said cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups are optionally substituted; and R ４ is selected from the group consisting of H, —OH, —NRxRy, and —C(O)ORz; Rx, Ry and Rz are each independently H or (C １ -C １０ ) alkyl; or R ２ and R ４ together with the carbon atoms that carry them (C ３ -C １０ ) forming a cycloalkyl group], or a pharmaceutically acceptable salt, hydrate or hydrated salt thereof, or a polymorphic crystal structure, racemate, diastereomer or enantiomer thereof.

6. In formula (II), R ３ but, - (C ３ -C １０ ) cycloalkyl; 3- to 10-membered heterocycloalkyl; - (C ６ -C １０ ) aryl; and 5- to 10-membered heteroaryl selected from the group consisting of:

6. The EPAC1 inhibitor for use according to claim 5, wherein the cycloalkyl, heterocycloalkyl, aryl and heteroaryl groups are optionally substituted.

7. In formula (II), R １ but, - (C ６ -C １０ ) aryl; and 5- to 10-membered heteroaryl selected from the group consisting of: wherein the aryl and heteroaryl groups are —NR ７ R ８ , (C １ -C １０ ) optionally substituted with one or more substituents selected from the group consisting of alkyl and halogen atoms; ７ and R ８ are independently (C １ -C １０ 7. The EPAC1 inhibitor for use according to claim 5 or 6, wherein said EPAC1 inhibitor is selected from alkyl or H.

8. In formula (II), R ２ but, - H; - (C １ -C ２０ ) alkyl; - (C ６ -C １０ ) aryl; and 5- to 10-membered heteroaryl or R ２ and R ４ together with the carbon atoms having these (C ３ -C １０ ) forming a cycloalkyl group; wherein the alkyl, cycloalkyl, aryl and heteroaryl groups are selected from the group consisting of (C １ -C １０ 8. The EPAC1 inhibitor for use according to any one of claims 5 to 7, wherein the EPAC1 inhibitor is optionally substituted with one or more substituents selected from the group consisting of alkyl and halogen atoms.

9. In formula (II), R ３ is one or more substituents (preferably (C １ -C １０ ) alkyl and halogen atoms) (C ６ -C １０ 9. The EPAC1 inhibitor for use according to any one of claims 5 to 8, wherein the EPAC1 inhibitor is aryl.

10. In formula (II), R ４ is H or R ２ and R ４ together with the carbon atoms having these (C ５ -C ６ 10. The EPAC1 inhibitor for use according to any one of claims 5 to 9, wherein the EPAC1 inhibitor forms a cycloalkyl group.

11. In formula (II), R １ is a phenyl group, and / or R ２ The EPAC1 inhibitor for use according to any one of claims 4 to 9, wherein is a thienyl group, and said phenyl and thienyl groups are optionally substituted.

12. The following formula (III): 【Chemistry 27】 [In the formula, Ra, Rb, Rc, Rd, Re, Rx, Ry, and Rz are each independently H, —OH, a halogen atom, —C(O)OH, (C １ -C １０ ) alkyl, (C １ -C １０ ) alkoxy, and —NR ５ R ６ selected from the group consisting of: Here, R ５ and R ６ are independently (C １ -C １０ ) alkyl or H; R ４ is H, -OH, -NH ２ and —C(O)OH; and R ３ 6. An EPAC1 inhibitor for use according to claim 5, characterized in that:

13. The following formula: 【Chemistry 28】 6. The EPAC1 inhibitor for use according to claim 5, wherein the EPAC1 inhibitor has one of the following structure:

14. The following formula: 【Chemistry 29】 6. The EPAC1 inhibitor for use according to claim 5, having the formula:

15. 15. The EPAC1 inhibitor for use according to any one of claims 1 to 14 in combination with pirfenidone or nintedanib.

16. A method for the prevention and / or treatment and / or amelioration of idiopathic pulmonary fibrosis, comprising administering to a patient in need thereof a pharmaceutically acceptable amount of an EPAC1 inhibitor, optionally in combination with pirfenidone or nintedanib.

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