Cell-protective compounds and their uses

Abstract

The present invention relates to cytoprotective compounds and their uses. The present invention relates to cytoprotective, particularly cardioprotective and renoprotective organic compounds, preferably organic compounds that inhibit substrate phosphorylation caused by G protein-coupled receptor kinase 2 (GRK2, ADRBK1). Preferably, the organic compounds inhibit GRK2-mediated phosphorylation of serine / arginine-rich splicing factor 1 (SRSF1, ASF-1, SF2) and / or photosensory factors for the treatment of hypertension, heart disease, cardiac dysfunction or failure, and heart disease-related conditions such as cardiomyocyte necrosis, ischemic heart disease and / or ischemic heart injury or aging. In addition, the present invention relates to methods for identifying inhibitors of (GRK2)-mediated (SRSF1) and / or photosensory factor phosphorylation.

Description

[0001] This application is a divisional application of the patent application for invention with the application date of January 10, 2018, application number 201880017149.7, and invention name "Cell Protective Compounds and Their Uses". Technical Field

[0002] The present invention relates to cell protective, particularly heart and kidney protective organic compounds, preferably to organic compounds that inhibit substrate phosphorylation caused by G protein-coupled receptor kinase 2 (GRK2, ADRBK1). Preferably, the organic compound inhibits the phosphorylation of serine / arginine-rich splicing factor 1 (SRSF1, ASF-1, SF2) and / or photosensory factor mediated by GRK2 for the treatment of heart diseases, hypertension, cardiac dysfunction or failure, and heart disease-related conditions, such as cardiomyocyte necrosis, ischemic heart disease and / or ischemic heart injury and aging. In addition, the present invention relates to a method for identifying inhibitors of phosphorylation of SRSF1 and / or photosensory factor mediated by GRK2. Background Art

[0003] Cardiovascular diseases and heart failure are the most common causes of death, so there is an urgent need for new treatment methods. Established pharmacological therapies for cardiovascular diseases include, for example, β-blockers, inhibitors of the angiotensin II AT1 system, ACE inhibitors, mineralocorticoid receptor blockers, and the combined neprilysin-AT1 receptor blocker sacubitril-valsartan. Despite these available treatment methods, the prognosis of patients with end-stage heart failure is worse than that of most cancer patients.

[0004] In experimental models of heart diseases and heart failure, the inhibition of G protein-coupled receptor kinase 2 (GRK2, ADRBK1, βARK) is associated with cardioprotection (Hullmann et al., Pharmacol. Res. 110, 52 - 64 (2016); Abd Alla et al., J. Biol. Chem. 291, 2583 - 2600 (2016)), and GRK2 is upregulated in patients with cardiovascular diseases and hypertension (Ungerer et al., Circulation 87, 454 - 463 (1993); Gros et al., J. Clin. Invest. 99, 2087 - 2093 (1997)).

[0005] One method uses the βARK-C-terminus, which inhibits GRK2-mediated receptor phosphorylation by scavenging the activated βγ subunits of heterotrimeric G proteins (Koch et al., Science 268, 1350 - 1353 (1995); US 7,060,871).

[0006] However, the βARK-C-terminus did not reach clinical studies because it is a large protein of 194 amino acids, difficult to administer and requires gene therapy approaches to reach its intracellular target GRK2. Additionally, the βATRK-C-terminus also inhibits the activity of the G-βγ subunits, which have multiple other related functions in vivo. Consistent with this statement, the cardioprotective effects of the βARK-C-terminus in vivo also involve GRK2-inhibition independent mechanisms (Volkers M et al., Circ.Res. 108, 27-39 (2011)).

[0007] Compounds identified by computer screening that interact with the Gβ subunit of the heterotrimeric G protein also show increased myocardial contractility similar to the βARK-C-terminus (WO 2004 / 101495 A1). However, this approach is not specific for GRK2 either and is expected to interfere with other Gβ-γ mediated functions similar to the βARK-C-terminus. These compounds have not been further characterized and have also not reached clinical studies.

[0008] Another scientific study involved GRKInh, a peptide inhibitor of GRK2 (18 amino acids), which is derived from the first intracellular loop of the β2-adrenergic receptor (Winstel et al., Biochem.Pharmacol. 70, 1001-1008 (2005)). GRKinh is cardioprotective in vivo (Abd Alla et al., J.Biol.Chem. 291, 2583-2600 (2016); Fu et al., J.Biol.Chem. 288, 7739-7755 (2013)). However, so far, the binding site of GRKInh on GRK2 has not been determined and GRKInh is a peptide that is difficult to administer and requires gene therapy approaches to reach its intracellular target GRK2. Additionally, GRKInh is an immunogenic peptide that can trigger an autoimmune response against the β2-adrenergic receptor.

[0009] Paroxetine is an ATP-site directed kinase inhibitor of GRK2 (Thal et al., ACS Chem.Biol. 7, 1830-1839 (2012); Schumacher et al., Sci.Transl.Med. 7, 277ra31 (2015)). The facts that paroxetine (i) is not specific for GRK2, (ii) has low inhibitory activity against GRK2, (iii) has a different major high-affinity target, the serotonin transporter, and (iv) causes weight gain make paroxetine unsuitable for cardioprotective therapy (Uguz et al., Gen Hosp Psychiatry 46-48, 37 (2015)).

[0010] Other ATP-site directed GRK2 inhibitors, such as the Takeda compound (WO 2007 / 034846 A1) which has high affinity and selectivity for GRK2, have not been tested in vivo and their cardioprotective properties are unknown. In addition, Waldschmidt et al. (J. Med. Chem. 59, 3793-3807 (2016)) reported GRK2 inhibitors. However, all of these high-affinity GRK2-specific ATP-site directed kinase inhibitors block all functions of GRK2 and may have major side effects because GRK2 plays an indispensable role in vivo, which cannot be compensated for, i.e., knockout of GRK2 is lethal (Jaber et al., PNAS USA 93, 12974-12979 (1996)). In addition, the in vivo effects of the Takeda or Waldschmidt compounds are not documented, and the off-target effects and bioavailability of the compounds are unknown.

[0011] In summary, so far, no new cardioprotective treatments have reached clinical studies. The lack of suitable targets, the lack of specificity for a given target, and / or the occurrence of side effects of available inhibitors are the main problems. In addition, the mechanisms underlying many treatments, such as the cardioprotective effect of GRK2 inhibition and its practical applicability to cardioprotective therapy, are still controversially discussed in the prior art.

[0012] The problem underlying the present invention is to provide new and / or improved cytoprotective, preferably cardioprotective and renoprotective compounds, which are preferably used for medical and / or cytoprotective, preferably cardioprotective and / or renoprotective therapy. Summary of the Invention

[0013] In a first aspect, the problem underlying the present invention is solved by a compound of formula (I) or (II) and its pharmaceutically acceptable salts or solvates for medical treatment, preferably cytoprotection, more preferably cardioprotective and / or renoprotective therapy:

[0014]

[0015] wherein:

[0016] The dotted lines between positions (2), (3), (4) and (5) in formula (I) and between positions (1), (2), (3), (4), (5) and (6) in formula (II) represent single or double bonds between the respective positions;

[0017] X is selected from N and C;

[0018] Y is selected from S and C, provided that when Y is C, X is N;

[0019] a is an integer from 0 to 15, preferably from 0 to 10, more preferably from 0 to 5, and most preferably 0 or 1;

[0020] R 1 is selected from

[0021] (i) hydrogen, hydroxyl, F, Cl, Br, and oxo, preferably, if X is not N, or if X is N and a is not 0, then R 1 is selected from hydroxyl, F, Cl, Br, and oxo;

[0022] (ii) linear or branched substituted or unsubstituted (C 1-10 ) alkyl ethers, (C 2-10 ) alkenyl ethers, (C 2-10 ) alkynyl ethers, and (C 4-10 ) carbocyclic ethers;

[0023] (iii) linear or branched substituted or unsubstituted (C 1-10 ) alkyls, preferably (C 1-5 ) alkyls, more preferably methyl, ethyl, and propyl, and most preferably methyl, (C 2-10 ) alkenyls, and (C 2-10 ) alkynyls;

[0024] (iv) substituted or unsubstituted carbocycles selected from (C 3-10 ) carbocycles, preferably (C3) carbocycles and (C 5-6 ) carbocycles, preferably aromatic (C6) carbocycles, more preferably unsubstituted phenyl and phenyl substituted with a substituent at the para position, the substituent being selected from Cl, F, Br, substituted or unsubstituted methyl, preferably -(CF3), ethyl, propyl, and cyclopropyl; and

[0025] (v) substituted or unsubstituted (C 3-6 ) heterocycles and (C7 - C 10 ) carbobicycles or heterobicycles, wherein the heterocycles and heterobicycles have 1 - 3 heteroatoms each independently selected from N, O, and S, preferably substituted or unsubstituted (C7) heterobicycles having 2 heteroatoms selected from N and S, more preferably substituted or unsubstituted indazolyl, benzimidazolyl, and benzodioxolyl, preferably indazolyl, benzimidazolyl, and benzodioxolyl connected through the (5) or (6) position, more preferably indazolyl or benzodioxolyl through the (6) position or benzimidazolyl through the (5) position;

[0026] R 2 is selected from

[0027] (i) hydrogen, hydroxyl, O - R 14 , -O - C(=O) - R 14 , F, Cl, Br, and oxo, wherein R 14Selected from

[0028] (aa) a linear or branched, substituted or unsubstituted (C 1-10 ) alkyl group, preferably a (C 1-5 ) alkyl group, more preferably methyl, ethyl and propyl, most preferably methyl, (C 2-10 ) alkenyl group and (C 2-10 ) alkynyl group;

[0029] (bb) a substituted or unsubstituted aromatic or non-aromatic (C 3-10 ) carbocyclic ring, preferably a (C 3-6 ) cycloalkyl group, more preferably a (C3) carbocyclic ring and a (C6) carbocyclic ring, preferably a (C6) carbocyclic ring, more preferably a phenyl group mono-substituted at the para position by a (C3) carbocyclic ring or -(CF3) or a phenyl group di-substituted at the meta position by a (C3) carbocyclic ring or -(CF3); and

[0030] (cc) a substituted or unsubstituted aromatic or non-aromatic (C 3-6 ) heterocyclic ring, preferably an aromatic (C 3-6 ) heterocyclic ring having 1-3 heteroatoms each independently selected from N, O and S;

[0031] (ii) a linear or branched, substituted or unsubstituted (C 1-10 ) alkyl group, preferably a (C 1-5 ) alkyl group, more preferably methyl, ethyl and propyl, most preferably methyl, (C 2-10 ) alkenyl group, (C 2-10 ) alkynyl group and (C 3-10 ) carbocyclic ring, preferably a (C 3-6 ) cycloalkyl group;

[0032] (iii) a linear or branched, substituted or unsubstituted (C 1-10 ) alkyl ether, (C 2-10 ) alkenyl ether, (C 2-10 ) alkynyl ether and (C 4-10 ) carbocyclic ether; and

[0033] (iv) a substituted or unsubstituted (C 3-6 ) heterocyclic ring and a (C7-C 10 ) carbobicyclic or heterobicyclic ring, wherein the heterocyclic ring and the heterobicyclic ring have 1-3 heteroatoms each independently selected from N, O and S, preferably a substituted or unsubstituted (C7) heterobicyclic ring having 2 heteroatoms selected from N and S, more preferably a substituted or unsubstituted indazolyl group, benzimidazolyl group and benzodioxolyl group, preferably an indazolyl group, benzodioxolyl group or benzimidazolyl group linked through the (5) or (6) position of the indazolyl group, benzimidazolyl group and benzodioxolyl group;

[0034] R 3 and R4 independently selected from

[0035] (i) hydrogen, -O-R 14 , -O-C(=O)-R 14 , F, Cl, Br and oxo, wherein R 14 is selected from

[0036] (aa) linear or branched substituted or unsubstituted (C 1-10 )alkyl, preferably (C 1-5 )alkyl, more preferably methyl, ethyl and propyl, most preferably methyl, (C 2-10 )alkenyl and (C 2-10 )alkynyl;

[0037] (bb) substituted or unsubstituted aromatic or non-aromatic (C 3-10 )carbocycle, preferably (C 3-6 )cycloalkyl, more preferably (C3)carbocycle and (C6)carbocycle, preferably (C6)carbocycle, more preferably phenyl mono-substituted at the para position by (C3)carbocycle or -(CF3) or phenyl di-substituted at the meta position by (C3)carbocycle or -(CF3); and

[0038] (cc) substituted or unsubstituted aromatic or non-aromatic (C 3-6 )heterocycle, preferably aromatic (C 3-6 )heterocycle having 1-3 heteroatoms each independently selected from N, O and S;

[0039] (ii) linear or branched substituted or unsubstituted (C 1-10 )alkyl, preferably (C 1-5 )alkyl, more preferably methyl, ethyl and propyl, most preferably methyl, (C 2-10 )alkenyl, (C 2-10 )alkynyl and (C 3-10 )carbocycle, preferably substituted or unsubstituted (C 3-6 )cycloalkyl and (C 3-6 )heterocycle having 1-3 heteroatoms each independently selected from N, O and S;

[0040] (iii) linear or branched substituted or unsubstituted (C 1-10 )alkyl ether, (C 2-10 )alkenyl ether, (C 2-10 )alkynyl ether and (C 4-10 )carbocycle ether;

[0041] (iv) wherein R 12 is selected from

[0042] (aa) hydrogen, hydroxyl, substituted or unsubstituted N, F, Cl and Br;

[0043] (bb) straight-chain or branched substituted or unsubstituted (C 1-10 ) alkyl, preferably (C 1-5 ) alkyl, more preferably methyl, ethyl and propyl, most preferably methyl, (C 2-10 ) alkenyl and (C 2-10 ) alkynyl;

[0044] (cc) substituted or unsubstituted aromatic or non-aromatic (C 3-10 ) carbocycle, preferably (C 3-6 ) cycloalkyl, more preferably (C3) carbocycle and (C6) carbocycle, preferably (C6) carbocycle, more preferably phenyl mono-substituted at the para position by (C3) carbocycle or -(CF3), or phenyl di-substituted at the meta position by (C3) carbocycle or -(CF3); and

[0045] (dd) substituted or unsubstituted aromatic or non-aromatic (C 3-6 ) heterocycle, preferably aromatic (C 3-6 ) heterocycle having 1-3 heteroatoms each independently selected from N, O and S; and

[0046] (v) wherein X is N or C, a is an integer from 0 to 15, preferably 0 to 10, more preferably 0 to 5, most preferably 0 or 1, and R 13 is selected from

[0047] (aa) hydrogen, hydroxy, F, Cl and Br;

[0048] (bb) straight-chain or branched substituted or unsubstituted (C 1-10 ) alkyl, preferably (C 1-5 ) alkyl, more preferably methyl, ethyl and propyl, most preferably methyl, (C 2-10 ) alkenyl and (C 2-10 ) alkynyl;

[0049] (cc) substituted or unsubstituted (C 3-10 ) carbocycle, preferably (C 3-6 ) cycloalkyl, (C7-C 10 ) carbobicyclic or heterobicyclic and (C 3-6 ) heterocycle, wherein the heterobicyclic and heterocyclic have 1-3 heteroatoms each independently selected from N, O and S,

[0050] For R 3 More preferably, R 13is a (C7) heterobicycle having 2 heteroatoms selected from N and S, most preferably a substituted or unsubstituted indazolyl, benzimidazolyl and benzodioxolyl, preferably an indazolyl, benzimidazolyl and benzodioxolyl linked through the (5) or (6) position, more preferably linked through the (5) position of the indazolyl and benzodioxolyl or the (6) position of the benzimidazolyl, and

[0051] For R 4 More preferably, R 13 is a substituted or unsubstituted aromatic (C6) carbocycle, preferably a (C6) carbocycle mono- or di-substituted at the meta position by a (C3)-carbocycle or -(CF3), or a (C6) carbocycle mono-substituted at the para position by a (C3)-carbocycle or -(CF3); and

[0052] (dd) a straight-chain or branched substituted or unsubstituted (C 1-10 ) alkyl ether, (C 2-10 ) alkenyl ether, (C 2-10 ) alkynyl ether and (C 4-10 ) carbocyclic ether;

[0053] wherein, if the (2), (3) and / or (4) positions of the ring of formula (I) are sp 3 -hybridized, R 2 and R 4 and / or R 3 and R 4 are preferably in a cis or trans configuration with respect to each other, more preferably in a trans configuration, preferably, R 2 is in the (R)- or (S)-configuration, R 3 is in the (R)- or (S)-configuration and / or R 4 is in the (R)- or (S)-configuration, more preferably, R 2 is in the (R)-configuration, R 3 is in the (R)-configuration and / or R 4 is in the (R)-configuration, or R 2 is in the (S)-configuration, R 3 is in the (S)-configuration and / or R 4 is in the (S)-configuration;

[0054] R 5 and R 9 are selected from

[0055] (i) hydrogen, hydroxy, F, Cl, Br and oxo, provided that if X is N and Y is C, then R 9 is not oxo;

[0056] (ii) a straight-chain or branched substituted or unsubstituted (C 1-10 ) alkyl ether, (C 2-10 ) alkenyl ether, (C2-10 ) alkynyl ethers and (C 4-10 ) carbocyclic ethers;

[0057] (iii) straight-chain or branched substituted or unsubstituted (C 1-10 ) alkyl, preferably (C 1-5 ) alkyl, more preferably methyl, ethyl and propyl, most preferably methyl, (C 2-10 ) alkenyl and (C 2-10 ) alkynyl;

[0058] (iv) substituted or unsubstituted (C 3-10 ) carbocycle, preferably substituted or unsubstituted (C3) carbocycle, substituted or unsubstituted aromatic (C 5-6 ) carbocycle, more preferably cyclopenta-2,4-dien-1-yl and aromatic (C6) carbocycle, most preferably phenyl unsubstituted or substituted at the para position by a substituent selected from Cl, F, Br, substituted or unsubstituted methyl, preferably -(CF3), ethyl, propyl and cyclopropyl; and

[0059] (v) (C 3-6 ) heterocycle having 1-3 heteroatoms each independently selected from N, O and S, preferably substituted or unsubstituted imidazolyl and pyrazolyl, more preferably imidazolyl and pyrazolyl linked to the ring of formula (I) through the imidazolyl- / pyrazolyl-position-(1)-nitrogen;

[0060] wherein, if the (5) position of the ring of formula (I) is sp 3 -hybridized, then R 5 is preferably in the (S)- or (R)-configuration, more preferably in the (R)-configuration;

[0061] and wherein, if the (3) position of the ring of formula (II) is sp 3 -hybridized, then R 9 is preferably in the (S)- or (R)-configuration, more preferably in the (S)-configuration;

[0062] R 6 and R 11 are independently selected from

[0063] (i) straight-chain or branched substituted or unsubstituted (C 1-10 ) alkyl ethers, (C 2-10 ) alkenyl ethers, (C 2-10 ) alkynyl ethers and (C 4-10 ) carbocyclic ethers;

[0064] (ii) straight-chain or branched substituted or unsubstituted (C 1-10 ) alkyl, preferably (C 1-5 ) alkyl, more preferably methyl, ethyl and propyl, most preferably methyl, (C 2-10) alkenyl and (C 2-10 ) alkynyl;

[0065] (iii) a substituted or unsubstituted carbocycle selected from (C 3-10 ) carbocycles, preferably (C3) carbocycles and (C 5-6 ) carbocycles, more preferably an aromatic (C6) carbocycle, most preferably an unsubstituted phenyl or phenyl mono- or di-substituted at the meta and para positions by substituents selected from Cl, F, Br, substituted or unsubstituted methyl, ethyl, propyl and cyclopropyl; and

[0066] (iv) a substituted or unsubstituted (C 3-6 ) heterocycle and a (C7-C 10 ) carbobicyclic or heterobicyclic ring, wherein the heterocycle and heterobicyclic ring have 1-3 heteroatoms each independently selected from N, O and S, preferably a substituted or unsubstituted (C7) heterobicyclic ring having 2 heteroatoms selected from N and S, most preferably a substituted or unsubstituted indazolyl, benzimidazolyl and benzodioxolyl group, preferably an indazolyl, benzimidazolyl and benzodioxolyl group linked through the (5) or (6) position, more preferably linked through the (6) position of the indazolyl or benzodioxolyl group or the (5) position of the benzimidazolyl group,

[0067] wherein if Y is S, then R 6 is absent; and / or

[0068] wherein if the ring of formula (II) has a double bond between the (4) and (5) positions or between the (3) and (4) positions of the ring of formula (II), then R 11 is absent,

[0069] and provided that if X is N, Y is C and the ring of formula (II) is aromatic, then R 6 is not 1,2,4-triazolyl;

[0070] R 7 is selected from

[0071] (i) hydrogen, hydroxy, F, Cl, Br and oxo;

[0072] (ii) a straight-chain or branched-chain substituted or unsubstituted (C 1-10 ) alkyl group, preferably a (C 1-5 ) alkyl group, more preferably methyl, ethyl and propyl, most preferably methyl, (C 2-10 ) alkenyl, (C 2-10 ) alkynyl and (C 3-10 ) carbocycle, preferably (C 3-6 ) cycloalkyl and a (C 3-6 ) heterocycle having 1-3 heteroatoms each independently selected from N, O and S;

[0073] (iii) linear or branched substituted or unsubstituted (C 1-10 ) alkyl ethers, (C 2-10 ) alkenyl ethers, (C 2-10 ) alkynyl ethers and (C 4-10 ) carbocyclic ethers; and

[0074] (iv) wherein R 12 is selected from

[0075] (aa) hydrogen, hydroxyl, substituted or unsubstituted N, F, Cl and Br;

[0076] (bb) linear or branched substituted or unsubstituted (C 1-10 ) alkyl groups, preferably (C 1-5 ) alkyl groups, more preferably methyl, ethyl and propyl, most preferably methyl, (C 2-10 ) alkenyl, (C 2-10 ) alkynyl and aromatic or non-aromatic (C 3-10 ) carbocycles, preferably (C 3-6 ) cycloalkyl groups, more preferably (C3) carbocycles, most preferably aromatic (C6) carbocycles mono-substituted at the para position by a (C3) carbocycle or -(CF3) or di-substituted at the meta position by a (C3) carbocycle or -(CF3); and

[0077] (cc) substituted or unsubstituted aromatic or non-aromatic (C 3-6 ) heterocycles, preferably aromatic (C 3-6 ) heterocycles having 1-3 heteroatoms each independently selected from N, O and S;

[0078] R 8 is selected from the group defined above for R 7 which group further includes

[0079] (i) wherein X is N or C, a is an integer from 0-15, preferably 0-10, more preferably 0-5, most preferably 0 or 1, and R 13 is selected from

[0080] (aa) hydrogen, hydroxyl, F, Cl and Br;

[0081] (bb) linear or branched substituted or unsubstituted (C 1-10 ) alkyl groups, preferably (C 1-5 ) alkyl groups, more preferably methyl, ethyl and propyl, most preferably methyl, (C 2-10 ) alkenyl and (C 2-10 ) alkynyl;

[0082] (cc) substituted or unsubstituted (C 3-10 ) carbocycles, preferably (C 3-6)cycloalkyl, (C7-C 10 )carbocyclic ring or heterocyclic ring having two rings and (C 3-6 )heterocyclic ring, wherein said heterocyclic ring having two rings and heterocyclic ring having 1-3 heteroatoms each independently selected from N, O and S,

[0083] More preferably, a substituted or unsubstituted (C7) heterocyclic ring having two heteroatoms selected from N and S, most preferably a substituted or unsubstituted indazolyl, benzimidazolyl and benzodioxolyl, preferably indazolyl and benzodioxolyl linked through the (5) or (6) position, more preferably linked through the (5) position of indazolyl and benzodioxolyl or the (6) position of benzimidazolyl,

[0084] Most preferably, a substituted or unsubstituted aromatic (C6) carbocyclic ring, preferably a (C6) carbocyclic ring mono- or disubstituted by a (C3)-carbocyclic ring or -(CF3) at the meta position or mono-substituted by a (C3)-carbocyclic ring or -(CF3) at the para position; and

[0085] (dd) a straight-chain or branched-chain substituted or unsubstituted (C 1-10 ) alkyl ether, (C 2-10 ) alkenyl ether, (C 2-10 ) alkynyl ether and (C 4-10 ) carbocyclic ether;

[0086] Provided that if X is N, Y is C and the ring of formula (II) is aromatic, then R 8 is not 1,2,4-triazolyl, and wherein if the (5) position of the ring of formula (II) is sp 3 -hybridized, then R 8 is preferably in the (R)- or (S)-configuration, more preferably in the (R)-configuration;

[0087] R 10 is absent or selected from

[0088] (i) hydrogen;

[0089] (ii) methyl; and

[0090] (iii) cyclopropyl or phenyl mono-substituted at the para position by a substituent selected from H, Cl, F, Br, methyl, -(CF3) and cyclopropyl;

[0091] wherein R 2 、R 3 、R 4 、R 5 、R 6 、R 7 、R 8 、R 9 、R 10 and R 11One or more of them are directly connected to the ring of formula (I) or (II), or are connected to the linking group between R 2 、R 3 、R 4 、R 5 、R 6 、R 7 、R 8 、R 9 、R 10 and / or R 11 and the ring of formula (I) or (II), wherein the linking group is selected from linear or branched substituted or unsubstituted (C 1-10 ) alkyl ethers, (C 2-10 ) alkenyl ethers, (C 2-10 ) alkynyl ethers, (C 4-10 ) carbocyclic ethers, linear or branched substituted or unsubstituted (C 1-10 ) alkyls, (C 2-10 ) alkenyls and (C 2-10 ) alkynyls.

[0092] The above compounds target the activation phosphorylation of the non-receptor substrate SRSF1 mediated by GRK2, and SRSF1 is a target that has not been considered before in cell protection, especially in cardiac and / or renal protection and cardiac treatment. SRSF1 is a non-receptor GRK2 substrate that promotes the symptoms of heart failure, and the phosphorylation of SRSF1 mediated by GRK2 leads to irreversible cardiomyocyte necrosis and cardiac dysfunction. Examples 1 to 3 further described below using transgenic animal models and Figures 1A to 3 demonstrate that GRK2-mediated phosphorylation of SRSF1 promotes the symptoms of heart failure and cardiomyocyte necrosis, which is the main cause of irreversible heart injury. The compounds of the present invention interfere with this previously unrecognized main heart failure promoting mechanism triggered by GRK2 (see Examples 9 to 12 below and Figures 9 to 12 ). GRK2 is upregulated in patients with cardiovascular diseases and hypertension (see above). Transgenic mice with overexpression of human GRK2 that mimic the upregulation of GRK2 found in the patient's heart develop enhanced SRSF1-mediated cardiomyocyte necrosis and cardiac dysfunction. For example, this is due to the enhanced splicing of the CAMK2D isoform B / C that promotes cardiomyocyte necrosis and heart failure (see Example 3 below and Figure 3)。It should be noted that irreversible necrotic cardiomyocyte death is a major feature of myocardial infarction and ischemic heart disease, which are caused by major cardiovascular risk factors, such as essential hypertension and other hypertensive disorders, atherosclerosis, chronic heart failure, hyperlipidemia, diabetes, stroke, depression, stress, and aging. For example, inhibition of GRK2-mediated SRSF1 phosphorylation that promotes heart failure by the compounds of the present invention exerts a cardioprotective effect on the symptoms induced by chronic pressure overload of heart failure in vivo (see Examples 11, 12, and Figure 11 and 12 ), for example, by delaying cardiomyocyte necrosis (see Examples 9 to 12 below and Figures 9 to 12 ), heart dysfunction (see Examples 11, 12, and Figure 11 and 12 ), and the development of cardiovascular disease-induced aging (see Example 12 and Figure 12 ) to exert a cardioprotective effect. It should be noted that paroxetine also inhibits GRK2-mediated SRSF1 phosphorylation in vitro and in vivo (see Examples 8 and Figure 8 ), but inhibits in a non-specific manner and has all the above-mentioned disadvantages that make paroxetine unsuitable for cardioprotective therapy in patients (Uguz et al., Gen Hosp Psychiatry 46-48, 37 (2015)). DETAILED DESCRIPTION

[0093] In a first aspect, the present invention relates to the compounds themselves disclosed above, i.e., within the scope of the substance claims. In another aspect, the present invention relates to these compounds for medical treatment, preferably for cytoprotection, particularly for cardioprotective and renoprotective treatment.

[0094] As used herein, the term "cardioprotective therapy" refers to the prevention or treatment of any disease or disorder affecting the heart, i.e., treatment that protects the heart from disease or treats existing heart diseases. Cardioprotective therapy includes, for example, reducing the risk of cardiovascular mortality and / or morbidity, preventing and treating irreversible necrotic cardiomyocyte death caused by diseases such as myocardial infarction, ischemic cardiovascular disease, angina pectoris, atherosclerosis, hypertensive disorders, chronic heart failure, chronic renal failure, cerebrovascular disease, decompensated heart failure, stress, aging, and / or depression. Irreversible necrotic cardiomyocyte death may or usually is the main cause of cardiac remodeling, ultimately leading to heart dysfunction, myocardial hypertrophy with dilation, myocardial thinning, and end-stage heart failure.

[0095] In a preferred embodiment, the compound for use according to the invention is a compound in which, in formula (I), a double bond is present between positions (3) and (4), or between positions (2) and (3) and between positions (4) and (5), or no double bond is present in the ring; and in formula (II), no double bond is present in the ring or the ring is aromatic;

[0096] and / or

[0097] a is 0 or 1; and / or

[0098] R 1 is selected from

[0099] (i) hydrogen;

[0100] (ii) a straight-chain or branched, substituted or unsubstituted (C 1-5 )alkyl group, more preferably methyl, ethyl and propyl, most preferably methyl;

[0101] (iii) a substituted or unsubstituted cyclopropyl and phenyl group, preferably a substituted phenyl group, more preferably a phenyl group mono-substituted at the para position by a substituent selected from H, Cl, F, Br, methyl, -(CF3) and cyclopropyl; and

[0102] (iv) a substituted or unsubstituted, preferably unsubstituted indazolyl, benzimidazolyl and benzodioxolyl group, preferably attached through the (6) position of the indazolyl and benzodioxolyl groups or the (5) position of the benzimidazolyl group.

[0103] In a further preferred embodiment, the compound for use according to the invention is a compound in which

[0104] R 2 is selected from

[0105] (i) hydrogen or oxo;

[0106] (ii) a straight-chain or branched, substituted or unsubstituted (C 1-5 )alkyl group, more preferably methyl, ethyl and propyl, most preferably methyl; and

[0107] (iii) a substituted or unsubstituted indazolyl, benzimidazolyl and benzodioxolyl group, preferably an indazolyl, benzimidazolyl and benzodioxolyl group attached through the (5) or (6) position of the indazolyl, benzodioxolyl and benzimidazolyl groups; and / or

[0108] R 3 is selected from

[0109] (i) hydrogen;

[0110] (ii) straight-chain or branched substituted or unsubstituted (C 1-5 ) alkyl, more preferably methyl, ethyl and propyl, most preferably methyl;

[0111] (iii) wherein R 12 is selected from

[0112] (aa) N; and

[0113] (bb) substituted or unsubstituted cyclopropyl and phenyl, preferably phenyl mono-substituted at the para-position by cyclopropyl or -(CF3) or di-substituted at the meta-positions by cyclopropyl or -(CF3) at each meta-position; and

[0114] (iv) wherein X is N, a is 1 and R 13 is selected from substituted or unsubstituted, preferably unsubstituted indazolyl, benzimidazolyl and benzodioxolyl groups attached through the (6) or (5) position of the indazolyl, benzimidazolyl and benzodioxolyl groups, preferably attached through the (5) position of the indazolyl and benzodioxolyl groups or the (6) position of the benzimidazolyl group;

[0115] and / or

[0116] R 4 is selected from

[0117] (i) hydrogen and hydroxyl;

[0118] (ii) straight-chain or branched substituted or unsubstituted (C 1-5 ) alkyl, more preferably methyl, ethyl and propyl, most preferably methyl;

[0119] (iii) wherein R 12 is selected from

[0120] (aa) N; and

[0121] (bb) substituted or unsubstituted cyclopropyl and phenyl, preferably phenyl mono-substituted at the para-position or meta-position by cyclopropyl or -(CF3) or di-substituted at the meta-positions by cyclopropyl or -(CF3) at each meta-position; and

[0122] (iv) wherein X is N, a is 1 and R 13 is phenyl mono- or di-substituted at each meta-position by cyclopropyl or -(CF3) or mono-substituted at the para-position by cyclopropyl or -(CF3);

[0123] wherein, if the (2), (3) and / or (4) positions of the ring of formula (I) are sp 3 -hybridized, R 2 and R 4 and / or R3 and R 4 are preferably in the cis or trans configuration with respect to each other, more preferably in the trans configuration. Preferably, R 2 is in the (R)- or (S)-configuration, and R 3 is in the (R)- or (S)-configuration and / or R 4 is in the (R)- or (S)-configuration. More preferably, R 2 is in the (R)-configuration, and R 3 is in the (R)-configuration and / or R 4 is in the (R)-configuration, or R 2 is in the (S)-configuration, and R 3 is in the (S)-configuration and / or R 4 is in the (S)-configuration; and / or

[0124] R 5 is selected from

[0125] (i) hydrogen;

[0126] (ii) a linear or branched, substituted or unsubstituted (C 1-5 )alkyl group, more preferably methyl, ethyl and propyl, most preferably methyl;

[0127] (iii) a substituted or unsubstituted cyclopropyl and phenyl group, preferably a substituted phenyl group, more preferably a phenyl group monosubstituted at the para position by a substituent selected from H, Cl, F, Br, methyl, -(CF3) and cyclopropyl;

[0128] (iv) cyclopenta-2,4-dien-1-yl; and

[0129] (v) a substituted or unsubstituted, preferably unsubstituted imidazolyl and pyrazolyl group attached to the ring of formula (I) via the imidazolyl- / pyrazolyl-position-(1)-nitrogen;

[0130] wherein, if the (5) position of the ring of formula (I) is sp 3 -hybridized, R 5 is preferably in the (R)- or (S)-configuration, more preferably in the (R)-configuration; and / or

[0131] R 6 and R 11 are independently selected from substituted or unsubstituted, preferably unsubstituted indazolyl, benzimidazolyl and benzodioxolyl groups attached via the (6) or (5) position, preferably attached via the (6) position of indazolyl and benzodioxolyl or the (5) position of benzimidazolyl;

[0132] wherein if Y is S, then R 6 is absent; and / or

[0133] Wherein, if the ring of formula (II) has a double bond between the (4) and (5) positions or between the (3) and (4) positions of the ring of formula (II), then R 11 is absent; and / or

[0134] R 7 and R 8 are independently selected from the group consisting of

[0135] (i) hydrogen;

[0136] (ii) a straight-chain or branched, substituted or unsubstituted (C 1-5 )alkyl, more preferably methyl, ethyl and propyl, most preferably methyl; and

[0137] (iii) wherein R 12 is selected from

[0138] (aa) N; and

[0139] (bb) a substituted or unsubstituted cyclopropyl and phenyl, preferably phenyl mono-substituted at the para position by cyclopropyl or -(CF3) or phenyl di-substituted at each meta position by cyclopropyl or -(CF3) at the meta positions;

[0140] wherein, for R 8 , the group further includes

[0141] wherein X is N, a is 1 and R 13 is selected from substituted or unsubstituted, preferably unsubstituted indazolyl, benzimidazolyl and benzodioxolyl groups linked through the (6) or (5) position, preferably linked through the (5) position of indazolyl and benzodioxolyl or the (6) position of benzimidazolyl; and

[0142] wherein, if the (5) position of the ring of formula (II) is sp 3 -hybridized, then R 8 is preferably in the (R)- or (S)-configuration, more preferably in the (R)-configuration; and / or

[0143] R 9 is selected from

[0144] (i) hydrogen, methyl and cyclopropyl; and

[0145] (ii) a substituted or unsubstituted phenyl, preferably phenyl substituted at the para position by a substituent selected from H, Cl, F, Br, methyl, -(CF3) and cyclopropyl; and

[0146] wherein, if the (3) position of the ring of formula (II) is sp 3 -hybridized, then R 9Preferably, it is in the (R)- or (S)-configuration, more preferably in the (S)-configuration; and / or

[0147] R 10 is absent or selected from

[0148] (i) hydrogen;

[0149] (ii) methyl; and

[0150] (iii) cyclopropyl and phenyl mono-substituted at the para-position by a substituent selected from H, Cl, F, Br, methyl, -(CF3) and cyclopropyl,

[0151] wherein if X is C and / or if the ring of formula (II) has a double bond between positions (1) and (2) or between positions (2) and (3) of the ring of formula (II), then R 10 is absent.

[0152] In a further preferred embodiment, the compound for use according to the invention is a compound of formula (I) (formula Ia) in which the double bond is between positions (3) and (4)

[0153]

[0154] wherein X is N and a is 0, and wherein

[0155] R 1 is selected from indazolyl, benzimidazolyl and benzodioxolyl groups attached via the (6) or (5) position, preferably attached via the (6) position of indazolyl and benzodioxolyl or the (5) position of benzimidazolyl; and / or

[0156] R 2 is oxo; and / or

[0157] R 3 is selected from

[0158] (i) hydrogen;

[0159] (ii) methyl; and

[0160] (iii) wherein R 12 is selected from

[0161] (aa) N; and

[0162] (bb) cyclopropyl and phenyl mono-substituted at the para-position by cyclopropyl or -(CF3) or di-substituted at each meta-position by cyclopropyl or -(CF3) at the meta-positions; and / or

[0163] R 4 is hydroxy; and / or

[0164] R5 Selected from

[0165] (i) hydrogen;

[0166] (ii) methyl;

[0167] (iii) cyclopropyl and phenyl mono-substituted at the para position by a substituent selected from H, Cl, F, Br, methyl, -(CF3) and cyclopropyl;

[0168] (iv) cyclopenta-2,4-dien-1-yl; and

[0169] (v) imidazolyl and pyrazolyl linked to the ring of formula (I) through imidazolyl- / pyrazolyl-position-(1)-nitrogen;

[0170] wherein R 5 is preferably in the (R)- or (S)-configuration, more preferably in the (R)-configuration.

[0171] In a further preferred embodiment, the compound for use according to the invention is a compound of formula (I) (formula Ib) in which the two double bonds are located between positions (2) and (3) and between positions (4) and (5) respectively

[0172]

[0173] wherein X is N or C, preferably N, a is 0 or 1, preferably, if X is C, then a is 0, and wherein

[0174] R 1 is selected from indazolyl, benzimidazolyl and benzodioxolyl linked through the (5) or (6) position, preferably linked through the (6) position of indazolyl and benzodioxolyl or the (5) position of benzimidazolyl; and / or

[0175] R 2 is selected from

[0176] (i) hydrogen; and

[0177] (ii) methyl; and / or

[0178] R 3 is selected from

[0179] (i) hydrogen;

[0180] (ii) methyl; and

[0181] (iii) wherein R 12 is selected from

[0182] (aa) N; and

[0183] (bb) Cyclopropyl and phenyl which is monosubstituted by cyclopropyl or -(CF3) at the para position or disubstituted by cyclopropyl or -(CF3) at each meta position; and / or

[0184] R 4 is hydrogen; and / or

[0185] R 5 is selected from

[0186] (i) hydrogen;

[0187] (ii) methyl; and

[0188] (iii) cyclopropyl and phenyl monosubstituted at the para position by a substituent selected from H, Cl, F, Br, methyl, -(CF3) and cyclopropyl.

[0189] In a further preferred embodiment, the compound for use according to the invention is a compound of formula (I) in which the bond in the five-membered ring of formula (I) is fully saturated (formula Ic)

[0190]

[0191] wherein X is N, a is 0, and wherein

[0192] R 1 is selected from

[0193] (i) hydrogen;

[0194] (ii) methyl; and

[0195] (iii) cyclopropyl and phenyl monosubstituted at the para position by a substituent selected from H, Cl, F, Br, methyl, -(CF3) and cyclopropyl; and / or

[0196] R 2 is selected from

[0197] (i) hydrogen; and

[0198] (ii) indazolyl, benzimidazolyl and benzodioxolyl, preferably indazolyl, benzimidazolyl and benzodioxolyl linked through the (5) or (6) position, if R 3 is not methyl, more preferably linked through the (5) position of indazolyl and benzodioxolyl or the (6) position of benzimidazolyl, if R 3 is methyl, most preferably linked through the (6) position of indazolyl and benzodioxolyl or the (5) position of benzimidazolyl; and / or

[0199] R 3 is selected from

[0200] (i) hydrogen or methyl; and

[0201] (ii) wherein X is N, a is 1 and R 13 is selected from indazolyl, benzimidazolyl and benzodioxolyl groups linked at the (6) or (5) position of indazolyl, benzimidazolyl and benzodioxolyl groups, preferably linked through the (5) position of indazolyl and benzodioxolyl groups or the (6) position of benzimidazolyl groups; and / or

[0202] R 4 is selected from

[0203] (i) hydrogen;

[0204] (ii) methyl;

[0205] (iii) wherein R 12 is selected from

[0206] (aa) N; and

[0207] (bb) cyclopropyl and phenyl groups monosubstituted by cyclopropyl or -(CF3) at the para position or disubstituted by cyclopropyl or -(CF3) at each meta position; and

[0208] (iv) wherein X is N, a is 1 and R 13 is phenyl monosubstituted by cyclopropyl or -(CF3) at the meta position or disubstituted by cyclopropyl or -(CF3) at each meta position or monosubstituted by cyclopropyl or -(CF3) at the para position;

[0209] wherein, R 2 and R 4 and / or R 3 and R 4 are preferably in a trans configuration with each other, preferably, R 2 is in the (S)-configuration, R 3 is in the (S)-configuration and R 4 is in the (R)-configuration; and / or

[0210] R 5 is hydrogen.

[0211] In a further preferred embodiment, the compound for use according to the invention is a compound of formula (II) wherein the bond within the ring of formula (II) is completely saturated (formula IIa)

[0212]

[0213] wherein X is C, Y is S, and wherein

[0214] R 6 is absent; and / or

[0215] R 7 selected from

[0216] (i) hydrogen;

[0217] (ii) methyl; and

[0218] (iii) wherein R 12 selected from

[0219] (aa) N; and

[0220] (bb) cyclopropyl or phenyl mono - substituted at the para - position by cyclopropyl or -(CF3) or di - substituted at each meta - position by cyclopropyl or -(CF3); and / or

[0221] R 8 selected from

[0222] (i) hydrogen and methyl; and

[0223] (ii) wherein X is N, a is 1 and R 13 selected from indazolyl, benzimidazolyl and benzodioxolyl groups linked through the (6) or (5) position, preferably linked through the (5) position of indazolyl and benzodioxolyl or the (6) position of benzimidazolyl;

[0224] wherein R 8 is preferably in the (R) - or (S) - configuration, more preferably the (R) - configuration; and / or

[0225] R 9 selected from

[0226] (i) hydrogen, methyl and cyclopropyl; and

[0227] (ii) phenyl mono - substituted at the para - position by a substituent selected from H, Cl, F, Br, methyl, -(CF3) and cyclopropyl;

[0228] wherein R 9 is preferably in the (R) - or (S) - configuration, more preferably the (S) - configuration; and / or

[0229] R 10 is hydrogen; and / or

[0230] R 11 is hydrogen or selected from indazolyl, benzimidazolyl and benzodioxolyl groups linked through the (6) or (5) position, preferably linked through the (6) position of indazolyl and benzodioxolyl or the (5) position of benzimidazolyl.

[0231] In a further preferred embodiment, the compound for use according to the invention is a compound of formula (II) in which the ring of formula (II) is aromatic (formula IIb)

[0232]

[0233] wherein X is N, Y is C, and wherein

[0234] R 6 is selected from indazolyl, benzimidazolyl and benzodioxolyl groups attached via the (6) or (5) position, preferably attached via the (6) position of indazolyl and benzodioxolyl or the (5) position of benzimidazolyl; and / or

[0235] R 7 and R 8 are independently selected from

[0236] (i) hydrogen;

[0237] (ii) methyl; and

[0238] (iii) wherein R 12 is selected from

[0239] (aa) N; and

[0240] (bb) cyclopropyl and phenyl mono-substituted with cyclopropyl or -(CF3) in the para position or phenyl di-substituted with cyclopropyl or -(CF3) in each meta position in the meta position;

[0241] and / or

[0242] R 9 is hydrogen; and / or

[0243] R 10 and / or R 11 is absent.

[0244] In a further preferred embodiment, the compound for use according to the invention is a compound of formula (II) in which the two double bonds are located between the (1) and (6) positions and between the (4) and (5) positions, respectively (formula IIc)

[0245]

[0246] wherein X is N, Y is C, and wherein

[0247] R 6 is selected from indazolyl, benzimidazolyl and benzodioxolyl groups attached via the (6) or (5) position, preferably attached via the (6) position of indazolyl and benzodioxolyl or the (5) position of benzimidazolyl; and / or

[0248] R 7 and R 8 are independently selected from hydrogen and methyl;

[0249] and / or

[0250] R 9 is hydrogen; and / or

[0251] R 10 is selected from

[0252] (i) hydrogen;

[0253] (ii) methyl; and

[0254] (iii) cyclopropyl and phenyl mono-substituted at the para position by a substituent selected from H, Cl, F, Br, methyl, -(CF3) and cyclopropyl; and / or

[0255] R 11 is absent.

[0256] In a further preferred embodiment, the compound for use according to the invention is a compound wherein

[0257] R 1 is selected from hydrogen, methyl,

[0258]

[0259] and / or

[0260] R 2 is selected from hydrogen, oxo, methyl,

[0261]

[0262] and / or

[0263] R 3 is selected from hydrogen, methyl,

[0264]

[0265] and / or

[0266] R 4 is selected from hydrogen, hydroxy, methyl,

[0267]

[0268] wherein, R 4 and / or R 3 and R 4 are preferably in a cis or trans configuration with respect to each other, more preferably in a trans configuration, preferably, R 2 is in the (R)- or (S)-configuration, R 3is in the (R)- or (S)-configuration and / or R 4 is in the (R)- or (S)-configuration, more preferably, R 2 is in the (R)-configuration, R 3 is in the (R)-configuration and / or R 4 is in the (R)-configuration, or R 2 is in the (S)-configuration, R 3 is in the (S)-configuration and / or R 4 is in the (S)-configuration, and / or

[0269] R 5 and R 9 is selected from hydrogen, methyl, cyclopenta-2,4-dien-1-yl,

[0270]

[0271] wherein R 9 is preferably not cyclopenta-2,4-dien-1-yl,

[0272] wherein, if the (5) position of the ring of formula (I) is sp 3 -hybridized, then R 5 is preferably in the (R)- or (S)-configuration, more preferably the (R)-configuration, and

[0273] wherein R 9 is preferably in the (R)- or (S)-configuration, more preferably the (R)-configuration, and / or

[0274] if Y is S, then R 6 is absent, and if Y is C, then R 6 is selected from

[0275] and / or

[0276] R 7 and R 8 is selected from hydrogen, methyl,

[0277]

[0278] wherein R 8 is additionally selected from

[0279]

[0280] wherein R 8 is preferably in the (R)- or (S)-configuration, more preferably the (R)-configuration; and / or

[0281] R 10 is absent or is selected from hydrogen, methyl,

[0282]

[0283] wherein if X is C, and / or if the ring of formula (II) has a double bond between positions (1) and (2) or between positions (2) and (3) of the ring of formula (II), then R 10 is absent, and / or

[0284] R 11 is absent or is selected from hydrogen,

[0285]

[0286] wherein if the ring of formula (II) has a double bond between positions (4) and (5) or between positions (3) and (4) of the ring of formula (II), then R 11 is absent.

[0287] In a further preferred embodiment, the compound for use according to the invention is a compound selected from:

[0288] (i) a first residue covalently bound to a second residue, said first residue being selected from

[0289] 1-(1,3-benzodioxol-5-yl)-3-hydroxy-5-oxo-2-methyl-2H-pyrrol-4-yl,

[0290] 1-(1,3-benzodioxol-5-yl)-2-cyclopropyl-3-hydroxy-5-oxo-2H-pyrrol-4-yl,

[0291] 1-(1,3-benzodioxol-5-yl)-3-hydroxy-5-oxo-2H-pyrrol-4-yl,

[0292] 1-(1,3-benzodioxol-5-yl)-2-(cyclopenta-2,4-dien-1-yl)-5-oxo-3-hydroxy-2H-pyrrol-4-yl,

[0293] 1-(1,3-benzodioxol-5-yl)-3-hydroxy-5-oxo-2-(pyrazol-1-yl)-2H-pyrrol-4-yl,

[0294] 1-(1,3-benzodioxol-5-yl)-3-hydroxy-5-oxo-2-(imidazol-1-yl)-2H-pyrrol-4-yl,

[0295] 1-(1,3-benzodioxol-5-yl)-3-hydroxy-5-oxo-2-phenyl-2H-pyrrol-4-yl,

[0296] 1-(1,3-benzodioxol-5-yl)-3-hydroxy-5-oxo-2-(p-tolyl)-2H-pyrrol-4-yl,

[0297] 1-(1,3-Benzodioxol-5-yl)-2-(4-chlorophenyl)-3-hydroxy-5-oxo-2H-pyrrol-4-yl,

[0298] 1-(1,3-Benzodioxol-5-yl)-2-(4-fluorophenyl)-3-hydroxy-5-oxo-2H-pyrrol-4-yl,

[0299] 1-(1,3-Benzodioxol-5-yl)-2-(4-bromophenyl)-3-hydroxy-5-oxo-2H-pyrrol-4-yl,

[0300] 1-(1,3-Benzodioxol-5-yl)-2-(4-cyclopropylphenyl)-3-hydroxy-5-oxo-2H-pyrrol-4-yl,

[0301] 1-(1,3-Benzodioxol-5-yl)-3-hydroxy-5-oxo-2-[4-(trifluoromethyl)phenyl]-2H-pyrrol-4-yl,

[0302] 3-Hydroxy-1-(1H-indazol-6-yl)-5-oxo-2-[4-(trifluoromethyl)phenyl]-2H-pyrrol-4-yl,

[0303] 3-Hydroxy-1-(1H-indazol-6-yl)-5-oxo-2-phenyl-2H-pyrrol-4-yl,

[0304] 3-Hydroxy-1-(1H-indazol-6-yl)-5-oxo-2-(p-tolyl)-2H-pyrrol-4-yl,

[0305] 2-(4-Chlorophenyl)-3-hydroxy-1-(1H-indazol-6-yl)-5-oxo-2H-pyrrol-4-yl,

[0306] 2-(4-Fluorophenyl)-3-hydroxy-1-(1H-indazol-6-yl)-5-oxo-2H-pyrrol-4-yl,

[0307] 2-(4-Bromophenyl)-3-hydroxy-1-(1H-indazol-6-yl)-5-oxo-2H-pyrrol-4-yl,

[0308] 2-(4-Cyclopropylphenyl)-3-hydroxy-1-(1H-indazol-6-yl)-5-oxo-2H-pyrrol-4-yl,

[0309] 2-Cyclopropyl-3-hydroxy-1-(1H-indazol-6-yl)-5-oxo-2H-pyrrol-4-yl,

[0310] 3-Hydroxy-1-(1H-indazol-6-yl)-5-oxo-2-methyl-2H-pyrrol-4-yl,

[0311] 3-Hydroxy-1-(1H-indazol-6-yl)-5-oxo-2H-pyrrol-4-yl,

[0312] 3-Hydroxy-1-(1H-indazol-6-yl)-5-oxo-2-(pyrazol-1-yl)-2H-pyrrol-4-yl,

[0313] 2-(Cyclopenta-2,4-dien-1-yl)-3-hydroxy-1-(1H-indazol-6-yl)-5-oxo-2H-pyrrol-4-yl,

[0314] 3-Hydroxy-2-(imidazol-1-yl)-1-(1H-indazol-6-yl)-5-oxo-2H-pyrrol-4-yl,

[0315] 1-(1H-Benzimidazol-5-yl)-3-hydroxy-5-oxo-2-dimethyl-2H-pyrrol-4-yl,

[0316] 1-(1H-Benzimidazol-5-yl)-2-cyclopropyl-3-hydroxy-5-oxo-2H-pyrrol-4-yl,

[0317] 1-(1H-Benzimidazol-5-yl)-3-hydroxy-5-oxo-2-(pyrazol-1-yl)-2H-pyrrol-4-yl,

[0318] 1-(1H-Benzimidazol-5-yl)-3-hydroxy-5-oxo-2H-pyrrol-4-yl,

[0319] 1-(1H-Benzimidazol-5-yl)-3-hydroxy-5-oxo-2-(imidazol-1-yl)-2H-pyrrol-4-yl,

[0320] 1-(1H-Benzimidazol-5-yl)-2-(cyclopenta-2,4-dien-1-yl)-3-hydroxy-5-oxo-2H-pyrrol-4-yl,

[0321] 1-(1H-Benzimidazol-5-yl)-2-(4-fluorophenyl)-3-hydroxy-5-oxo-2H-pyrrol-4-yl,

[0322] 1-(1H-Benzimidazol-5-yl)-3-hydroxy-5-oxo-2-[4-(trifluoromethyl)phenyl]-2H-pyrrol-4-yl,

[0323] 1-(1H-Benzimidazol-5-yl)-3-hydroxy-5-oxo-2-phenyl-2H-pyrrol-4-yl,

[0324] 1-(1H-benzimidazol-5-yl)-3-hydroxy-5-oxo-2-(p-tolyl)-2H-pyrrol-4-yl,

[0325] 1-(1H-benzimidazol-5-yl)-2-(4-chlorophenyl)-3-hydroxy-5-oxo-2H-pyrrol-4-yl,

[0326] 1-(1H-benzimidazol-5-yl)-2-(4-bromophenyl)-3-hydroxy-5-oxo-2H-pyrrol-4-yl,

[0327] 1-(1H-benzimidazol-5-yl)-2-(4-cyclopropylphenyl)-3-hydroxy-5-oxo-2H-pyrrol-4-yl,

[0328] wherein the 2H-pyrrole ring is numbered as follows:

[0329]

[0330] 1-(1,3-benzodioxol-5-ylmethyl)-2-methyl-5-(p-tolyl)pyrrol-3-yl,

[0331] 1-(1,3-benzodioxol-5-ylmethyl)-5-(4-chlorophenyl)-2-methyl-pyrrol-3-yl,

[0332] 1-(1,3-benzodioxol-5-ylmethyl)-5-(4-fluorophenyl)-2-methyl-pyrrol-3-yl,

[0333] 1-(1,3-benzodioxol-5-ylmethyl)-2-methyl-5-[4-(trifluoromethyl)phenyl]pyrrol-3-yl,

[0334] 1-(1,3-benzodioxol-5-ylmethyl)-5-(4-bromophenyl)-2-methyl-pyrrol-3-yl,

[0335] 1-(1,3-benzodioxol-5-ylmethyl)-5-(4-cyclopropylphenyl)-2-methyl-pyrrol-3-yl,

[0336] 1-(1,3-benzodioxol-5-ylmethyl)-2-methyl-5-phenyl-pyrrol-3-yl,

[0337] 1-(1,3-benzodioxol-5-ylmethyl)-5-phenyl-pyrrol-3-yl,

[0338] 1-(1,3-benzodioxol-5-ylmethyl)-5-(p-tolyl)pyrrol-3-yl,

[0339] 1-(1,3-Benzodioxol-5-ylmethyl)-5-(4-fluorophenyl)pyrrol-3-yl,

[0340] 1-(1,3-Benzodioxol-5-ylmethyl)-5-[4-(trifluoromethyl)phenyl]pyrrol-3-yl,

[0341] 1-(1,3-Benzodioxol-5-ylmethyl)-5-(4-chlorophenyl)pyrrol-3-yl,

[0342] 1-(1,3-Benzodioxol-5-ylmethyl)-5-(4-bromophenyl)pyrrol-3-yl,

[0343] 1-(1,3-Benzodioxol-5-ylmethyl)-5-(4-cyclopropylphenyl)pyrrol-3-yl,

[0344] 1-(1,3-Benzodioxol-5-ylmethyl)-2,5-dimethyl-pyrrol-3-yl,

[0345] 1-(1,3-Benzodioxol-5-ylmethyl)-2-methyl-pyrrol-3-yl,

[0346] 1-(1,3-Benzodioxol-5-ylmethyl)-5-cyclopropyl-2-methyl-pyrrol-3-yl,

[0347] 1-(1,3-Benzodioxol-5-ylmethyl)pyrrol-3-yl,

[0348] 1-(1,3-Benzodioxol-5-ylmethyl)-5-methyl-pyrrol-3-yl,

[0349] 1-(1,3-Benzodioxol-5-ylmethyl)-5-cyclopropyl-pyrrol-3-yl,

[0350] 1-(1H-Indazol-6-ylmethyl)-2-methyl-5-phenyl-pyrrol-3-yl,

[0351] 1-(1H-Indazol-6-ylmethyl)-2-methyl-5-(p-tolyl)pyrrol-3-yl,

[0352] 5-(4-Chlorophenyl)-1-(1H-indazol-6-ylmethyl)-2-methyl-pyrrol-3-yl,

[0353] 5-(4-Fluorophenyl)-1-(1H-indazol-6-ylmethyl)-2-methyl-pyrrol-3-yl,

[0354] 1-(1H-Indazol-6-ylmethyl)-2-methyl-5-[4-(trifluoromethyl)phenyl]pyrrol-3-yl,

[0355] 5-(4-bromophenyl)-1-(1H-indazol-6-ylmethyl)-2-methyl-pyrrol-3-yl,

[0356] 5-(4-cyclopropylphenyl)-1-(1H-indazol-6-ylmethyl)-2-methyl-pyrrol-3-yl,

[0357] 1-(1H-indazol-6-ylmethyl)-5-phenyl-pyrrol-3-yl,

[0358] 1-(1H-indazol-6-ylmethyl)-5-(p-tolyl)pyrrol-3-yl,

[0359] 5-(4-fluorophenyl)-1-(1H-indazol-6-ylmethyl)pyrrol-3-yl,

[0360] 1-(1H-indazol-6-ylmethyl)-5-[4-(trifluoromethyl)phenyl]pyrrol-3-yl,

[0361] 5-(4-chlorophenyl)-1-(1H-indazol-6-ylmethyl)pyrrol-3-yl,

[0362] 5-(4-bromophenyl)-1-(1H-indazol-6-ylmethyl)pyrrol-3-yl,

[0363] 5-(4-cyclopropylphenyl)-1-(1H-indazol-6-ylmethyl)pyrrol-3-yl,

[0364] 5-cyclopropyl-1-(1H-indazol-6-ylmethyl)-2-methyl-pyrrol-3-yl,

[0365] 1-(1H-indazol-6-ylmethyl)-2-methyl-pyrrol-3-yl,

[0366] 1-(1H-indazol-6-ylmethyl)-2,5-dimethyl-pyrrol-3-yl,

[0367] 1-(1H-indazol-6-ylmethyl)pyrrol-3-yl,

[0368] 5-cyclopropyl-1-(1H-indazol-6-ylmethyl)pyrrol-3-yl,

[0369] 1-(1H-indazol-6-ylmethyl)-5-methyl-pyrrol-3-yl,

[0370] 1-(1H-benzimidazol-5-ylmethyl)-2-methyl-5-phenyl-pyrrol-3-yl,

[0371] 1-(1H-benzimidazol-5-ylmethyl)-2-methyl-5-(p-tolyl)pyrrol-3-yl,

[0372] 1-(1H-benzoimidazol-5-ylmethyl)-2-methyl-5-(p-tolyl)pyrrol-3-yl,

[0373] 1-(1H-benzoimidazol-5-ylmethyl)-5-(4-fluorophenyl)-2-methyl-pyrrol-3-yl,

[0374] 1-(1H-benzoimidazol-5-ylmethyl)-5-(4-cyclopropylphenyl)-2-methyl-pyrrol-3-yl,

[0375] 1-(1H-benzoimidazol-5-ylmethyl)-5-(4-bromophenyl)-2-methyl-pyrrol-3-yl,

[0376] 1-(1H-benzoimidazol-5-ylmethyl)-2-methyl-5-[4-(trifluoromethyl)phenyl]pyrrol-3-yl,

[0377] 1-(1H-benzoimidazol-5-ylmethyl)-5-phenyl-pyrrol-3-yl,

[0378] 1-(1H-benzoimidazol-5-ylmethyl)-5-(p-tolyl)pyrrol-3-yl,

[0379] 1-(1H-benzoimidazol-5-ylmethyl)-5-[4-(trifluoromethyl)phenyl]pyrrol-3-yl,

[0380] 1-(1H-benzoimidazol-5-ylmethyl)-5-(4-chlorophenyl)pyrrol-3-yl,

[0381] 1-(1H-benzoimidazol-5-ylmethyl)-5-(4-fluorophenyl)pyrrol-3-yl,

[0382] 1-(1H-benzoimidazol-5-ylmethyl)-5-(4-bromophenyl)pyrrol-3-yl,

[0383] 1-(1H-benzoimidazol-5-ylmethyl)-5-(4-cyclopropylphenyl)pyrrol-3-yl,

[0384] 1-(1H-benzoimidazol-5-ylmethyl)-2,5-dimethyl-pyrrol-3-yl,

[0385] 1-(1H-benzoimidazol-5-ylmethyl)-2-methyl-pyrrol-3-yl,

[0386] 1-(1H-benzoimidazol-5-ylmethyl)-5-cyclopropyl-2-methyl-pyrrol-3-yl,

[0387] 1-(1H-benzoimidazol-5-ylmethyl)-5-methyl-pyrrol-3-yl,

[0388] 1-(1H-benzoimidazol-5-ylmethyl)pyrrol-3-yl,

[0389] 1-(1H-benzoimidazol-5-ylmethyl)-5-cyclopropyl-pyrrol-3-yl,

[0390] 1-(1,3-benzodioxol-5-yl)-5-(p-tolyl)pyrrol-3-yl,

[0391] 1-(1,3-benzodioxol-5-yl)-5-phenyl-pyrrol-3-yl,

[0392] 1-(1,3-benzodioxol-5-yl)-5-(4-fluorophenyl)pyrrol-3-yl,

[0393] 1-(1,3-benzodioxol-5-yl)-5-[4-(trifluoromethyl)phenyl]pyrrol-3-yl,

[0394] 1-(1,3-benzodioxol-5-yl)-5-(4-bromophenyl)pyrrol-3-yl,

[0395] 1-(1,3-benzodioxol-5-yl)-5-(4-chlorophenyl)pyrrol-3-yl,

[0396] 1-(1,3-benzodioxol-5-yl)-5-(4-cyclopropylphenyl)pyrrol-3-yl,

[0397] 1-(1,3-benzodioxol-5-yl)-2-methyl-5-(p-tolyl)pyrrol-3-yl,

[0398] 1-(1,3-benzodioxol-5-yl)-2-methyl-5-phenyl-pyrrol-3-yl,

[0399] 1-(1,3-benzodioxol-5-yl)-5-(4-fluorophenyl)-2-methyl-pyrrol-3-yl,

[0400] 1-(1,3-benzodioxol-5-yl)-2-methyl-5-[4-(trifluoromethyl)phenyl]pyrrol-3-yl,

[0401] 1-(1,3-benzodioxol-5-yl)-5-(4-chlorophenyl)-2-methyl-pyrrol-3-yl,

[0402] 1-(1,3-benzodioxol-5-yl)-5-(4-bromophenyl)-2-methyl-pyrrol-3-yl,

[0403] 1-(1,3-Benzodioxol-5-yl)-5-(4-cyclopropylphenyl)-2-methyl-pyrrol-3-yl,

[0404] 1-(1,3-Benzodioxol-5-yl)-2,5-dimethyl-pyrrol-3-yl,

[0405] 1-(1,3-Benzodioxol-5-yl)-2-methyl-pyrrol-3-yl,

[0406] 1-(1,3-Benzodioxol-5-yl)-5-cyclopropyl-2-methyl-pyrrol-3-yl,

[0407] 1-(1,3-Benzodioxol-5-yl)-5-methyl-pyrrol-3-yl,

[0408] 1-(1,3-Benzodioxol-5-yl)pyrrol-3-yl,

[0409] 1-(1,3-Benzodioxol-5-yl)-5-cyclopropyl-pyrrol-3-yl,

[0410] 1-(1H-Indazol-6-yl)-5-(p-tolyl)pyrrol-3-yl,

[0411] 5-(4-Chlorophenyl)-1-(1H-indazol-6-yl)pyrrol-3-yl,

[0412] 5-(4-Bromophenyl)-1-(1H-indazol-6-yl)pyrrol-3-yl,

[0413] 5-(4-Fluorophenyl)-1-(1H-indazol-6-yl)pyrrol-3-yl,

[0414] 1-(1H-Indazol-6-yl)-5-phenyl-pyrrol-3-yl,

[0415] 5-(4-Cyclopropylphenyl)-1-(1H-indazol-6-yl)pyrrol-3-yl,

[0416] 1-(1H-Indazol-6-yl)-5-[4-(trifluoromethyl)phenyl]pyrrol-3-yl,

[0417] 1-(1H-Indazol-6-yl)-2-methyl-5-(p-tolyl)pyrrol-3-yl,

[0418] 5-(4-Chlorophenyl)-1-(1H-indazol-6-yl)-2-methyl-pyrrol-3-yl,

[0419] 5-(4-Bromophenyl)-1-(1H-indazol-6-yl)-2-methyl-pyrrol-3-yl,

[0420] 5-(4-Fluorophenyl)-1-(1H-indazol-6-yl)-2-methyl-pyrrol-3-yl,

[0421] 1-(1H-indazol-6-yl)-2-methyl-5-phenyl-pyrrol-3-yl,

[0422] 5-(4-Cyclopropylphenyl)-1-(1H-indazol-6-yl)-2-methyl-pyrrol-3-yl,

[0423] 1-(1H-indazol-6-yl)-2-methyl-5-[4-(trifluoromethyl)phenyl]pyrrol-3-yl,

[0424] 1-(1H-indazol-6-yl)-2,5-dimethyl-pyrrol-3-yl,

[0425] 1-(1H-indazol-6-yl)-2-methyl-pyrrol-3-yl,

[0426] 5-Cyclopropyl-1-(1H-indazol-6-yl)-2-methyl-pyrrol-3-yl,

[0427] 1-(1H-indazol-6-yl)-5-methyl-pyrrol-3-yl,

[0428] 1-(1H-indazol-6-yl)pyrrol-3-yl,

[0429] 5-Cyclopropyl-1-(1H-indazol-6-yl)pyrrol-3-yl,

[0430] 1-(1H-benzimidazol-5-yl)-5-(p-tolyl)pyrrol-3-yl,

[0431] 1-(1H-benzimidazol-5-yl)-5-(4-chlorophenyl)pyrrol-3-yl,

[0432] 1-(1H-benzimidazol-5-yl)-5-(4-bromophenyl)pyrrol-3-yl,

[0433] 1-(1H-benzimidazol-5-yl)-5-(4-fluorophenyl)pyrrol-3-yl,

[0434] 1-(1H-benzimidazol-5-yl)-5-phenyl-pyrrol-3-yl,

[0435] 1-(1H-benzimidazol-5-yl)-5-(4-cyclopropylphenyl)pyrrol-3-yl,

[0436] 1-(1H-benzimidazol-5-yl)-5-[4-(trifluoromethyl)phenyl]pyrrol-3-yl,

[0437] 1-(1H-benzimidazol-5-yl)-2-methyl-5-(p-tolyl)pyrrol-3-yl,

[0438] 1-(1H-benzimidazol-5-yl)-5-(4-chlorophenyl)-2-methyl-pyrrol-3-yl,

[0439] 1-(1H-benzimidazol-5-yl)-5-(4-bromophenyl)-2-methyl-pyrrol-3-yl,

[0440] 1-(1H-benzimidazol-5-yl)-5-(4-fluorophenyl)-2-methyl-pyrrol-3-yl,

[0441] 1-(1H-benzimidazol-5-yl)-2-methyl-5-phenyl-pyrrol-3-yl,

[0442] 1-(1H-benzimidazol-5-yl)-5-(4-cyclopropylphenyl)-2-methyl-pyrrol-3-yl,

[0443] 1-(1H-benzimidazol-5-yl)-2-methyl-5-[4-(trifluoromethyl)phenyl]pyrrol-3-yl,

[0444] 1-(1H-benzimidazol-5-yl)-2,5-dimethyl-pyrrol-3-yl,

[0445] 1-(1H-benzimidazol-5-yl)-2-methyl-pyrrol-3-yl,

[0446] 1-(1H-benzimidazol-5-yl)-5-cyclopropyl-2-methyl-pyrrol-3-yl,

[0447] 1-(1H-benzimidazol-5-yl)-5-methyl-pyrrol-3-yl,

[0448] 1-(1H-benzimidazol-5-yl)pyrrol-3-yl,

[0449] 1-(1H-benzimidazol-5-yl)-5-cyclopropyl-pyrrol-3-yl,

[0450] wherein the pyrrole ring is numbered as follows:

[0451]

[0452] (3R)-3-(1,3-benzodioxol-5-ylmethylcarbamoyl)-5-phenyl-thiomorpholin-2-yl,

[0453] (3R)-3-(1,3-benzodioxol-5-ylmethylcarbamoyl)-5-(p-tolyl)thiomorpholin-2-yl,

[0454] (3R)-3-(1,3-Benzodioxol-5-ylmethylcarbamoyl)-5-(4-fluorophenyl)thiomorpholin-2-yl,

[0455] (3R)-3-(1,3-Benzodioxol-5-ylmethylcarbamoyl)-5-(4-bromophenyl)thiomorpholin-2-yl,

[0456] (3R)-3-(1,3-Benzodioxol-5-ylmethylcarbamoyl)-5-(4-chlorophenyl)thiomorpholin-2-yl,

[0457] (3R)-3-(1,3-Benzodioxol-5-ylmethylcarbamoyl)-5-(4-cyclopropylphenyl)thiomorpholin-2-yl,

[0458] (3R)-3-(1,3-Benzodioxol-5-ylmethylcarbamoyl)-5-[4-(trifluoromethyl)phenyl]thiomorpholin-2-yl,

[0459] (3R)-3-(1H-Indazol-5-ylmethylcarbamoyl)-5-phenyl-thiomorpholin-2-yl,

[0460] (3R)-3-(1H-Indazol-5-ylmethylcarbamoyl)-5-(p-tolyl)thiomorpholin-2-yl,

[0461] (3R)-5-(4-Fluorophenyl)-3-(1H-indazol-5-ylmethylcarbamoyl)thiomorpholin-2-yl,

[0462] (3R)-5-(4-Bromophenyl)-3-(1H-indazol-5-ylmethylcarbamoyl)thiomorpholin-2-yl,

[0463] (3R)-5-(4-Chlorophenyl)-3-(1H-indazol-5-ylmethylcarbamoyl)thiomorpholin-2-yl,

[0464] (3R)-5-(4-Cyclopropylphenyl)-3-(1H-indazol-5-ylmethylcarbamoyl)thiomorpholin-2-yl,

[0465] (3R)-3-(1H-Indazol-5-ylmethylcarbamoyl)-5-[4-(trifluoromethyl)phenyl]thiomorpholin-2-yl,

[0466] (3R)-3-(3H-Benzimidazol-5-ylmethylcarbamoyl)-5-phenyl-thiomorpholin-2-yl,

[0467] (3R)-3-(3H-Benzimidazol-5-ylmethylcarbamoyl)-5-(p-tolyl)thiomorpholin-2-yl,

[0468] (3R)-3-(3H-benzimidazol-5-ylmethylcarbamoyl)-5-(4-fluorophenyl)thiomorpholin-2-yl,

[0469] (3R)-3-(3H-benzimidazol-5-ylmethylcarbamoyl)-5-[4-(trifluoromethyl)phenyl]thiomorpholin-2-yl,

[0470] (3R)-3-(3H-benzimidazol-5-ylmethylcarbamoyl)-5-(4-bromophenyl)thiomorpholin-2-yl,

[0471] (3R)-3-(3H-benzimidazol-5-ylmethylcarbamoyl)-5-(4-chlorophenyl)thiomorpholin-2-yl,

[0472] (3R)-3-(3H-benzimidazol-5-ylmethylcarbamoyl)-5-(4-cyclopropylphenyl)thiomorpholin-2-yl,

[0473] 4-(1,3-benzodioxol-5-yl)-5-(4-fluorophenyl)thiomorpholin-2-yl,

[0474] 4-(1,3-benzodioxol-5-yl)-5-(4-bromophenyl)thiomorpholin-2-yl,

[0475] 4-(1,3-benzodioxol-5-yl)-5-(4-chlorophenyl)thiomorpholin-2-yl,

[0476] 4-(1,3-benzodioxol-5-yl)-5-methyl-thiomorpholin-2-yl,

[0477] 4-(1,3-benzodioxol-5-yl)-5-cyclopropyl-thiomorpholin-2-yl,

[0478] 4-(1,3-benzodioxol-5-yl)-5-(4-cyclopropylphenyl)thiomorpholin-2-yl,

[0479] 4-(1,3-benzodioxol-5-yl)-5-(p-tolyl)thiomorpholin-2-yl,

[0480] 4-(1,3-benzodioxol-5-yl)-5-phenyl-thiomorpholin-2-yl,

[0481] 4-(1,3-benzodioxol-5-yl)-5-[4-(trifluoromethyl)phenyl]thiomorpholin-2-yl,

[0482] 4-(1,3-benzodioxol-5-yl)thiomorpholin-2-yl,

[0483] 4-(1,3-Benzodioxol-5-yl)-5-(4-fluorophenyl)-3-methyl-thiomorpholin-2-yl,

[0484] 4-(1,3-Benzodioxol-5-yl)-5-(4-bromophenyl)-3-methyl-thiomorpholin-2-yl,

[0485] 4-(1,3-Benzodioxol-5-yl)-5-(4-chlorophenyl)-3-methyl-thiomorpholin-2-yl,

[0486] 4-(1,3-Benzodioxol-5-yl)-3,5-dimethyl-thiomorpholin-2-yl,

[0487] 4-(1,3-Benzodioxol-5-yl)-5-cyclopropyl-3-methyl-thiomorpholin-2-yl,

[0488] 4-(1,3-Benzodioxol-5-yl)-5-(4-cyclopropylphenyl)-3-methyl-thiomorpholin-2-yl,

[0489] 4-(1,3-Benzodioxol-5-yl)-3-methyl-5-(p-tolyl)thiomorpholin-2-yl,

[0490] 4-(1,3-Benzodioxol-5-yl)-3-methyl-5-phenyl-thiomorpholin-2-yl,

[0491] 4-(1,3-Benzodioxol-5-yl)-3-methyl-5-[4-(trifluoromethyl)phenyl]thiomorpholin-2-yl,

[0492] 4-(1,3-Benzodioxol-5-yl)-3-methyl-thiomorpholin-2-yl,

[0493] 5-(4-Fluorophenyl)-4-(1H-indazol-6-yl)thiomorpholin-2-yl,

[0494] 5-(4-Bromophenyl)-4-(1H-indazol-6-yl)thiomorpholin-2-yl,

[0495] 5-(4-Chlorophenyl)-4-(1H-indazol-6-yl)thiomorpholin-2-yl,

[0496] 4-(1H-Indazol-6-yl)-5-methyl-thiomorpholin-2-yl,

[0497] 5-Cyclopropyl-4-(1H-indazol-6-yl)thiomorpholin-2-yl,

[0498] 5-(4-Cyclopropylphenyl)-4-(1H-indazol-6-yl)thiomorpholin-2-yl,

[0499] 4-(1H-Indazol-6-yl)-5-(p-tolyl)thiomorpholin-2-yl,

[0500] 4-(1H-Indazol-6-yl)-5-phenyl-thiomorpholin-2-yl,

[0501] 4-(1H-Indazol-6-yl)-5-[4-(trifluoromethyl)phenyl]thiomorpholin-2-yl,

[0502] 4-(1H-Indazol-6-yl)thiomorpholin-2-yl,

[0503] 5-(4-Fluorophenyl)-4-(1H-indazol-6-yl)-3-methyl-thiomorpholin-2-yl,

[0504] 5-(4-Bromophenyl)-4-(1H-indazol-6-yl)-3-methyl-thiomorpholin-2-yl,

[0505] 5-(4-Chlorophenyl)-4-(1H-indazol-6-yl)-3-methyl-thiomorpholin-2-yl,

[0506] 4-(1H-Indazol-6-yl)-3,5-dimethyl-thiomorpholin-2-yl,

[0507] 5-(4-Cyclopropylphenyl)-4-(1H-indazol-6-yl)-3-methyl-thiomorpholin-2-yl,

[0508] 4-(1H-Indazol-6-yl)-3-methyl-5-(p-tolyl)thiomorpholin-2-yl,

[0509] 4-(1H-Indazol-6-yl)-3-methyl-5-phenyl-thiomorpholin-2-yl,

[0510] 5-Cyclopropyl-4-(1H-indazol-6-yl)-3-methyl-thiomorpholin-2-yl,

[0511] 4-(1H-Indazol-6-yl)-3-methyl-thiomorpholin-2-yl,

[0512] 4-(1H-Indazol-6-yl)-3-methyl-5-[4-(trifluoromethyl)phenyl]thiomorpholin-2-yl,

[0513] 4-(1H-Benzimidazol-5-yl)-5-(4-fluorophenyl)thiomorpholin-2-yl,

[0514] 4-(1H-Benzimidazol-5-yl)-5-(4-bromophenyl)thiomorpholin-2-yl,

[0515] 4-(1H-benzimidazol-5-yl)-5-(4-chlorophenyl)thiomorpholin-2-yl,

[0516] 4-(1H-benzimidazol-5-yl)-5-methyl-thiomorpholin-2-yl,

[0517] 4-(1H-benzimidazol-5-yl)-5-cyclopropyl-thiomorpholin-2-yl,

[0518] 4-(1H-benzimidazol-5-yl)-5-(4-cyclopropylphenyl)thiomorpholin-2-yl,

[0519] 4-(1H-benzimidazol-5-yl)-5-(p-tolyl)thiomorpholin-2-yl,

[0520] 4-(1H-benzimidazol-5-yl)-5-phenyl-thiomorpholin-2-yl,

[0521] 4-(1H-benzimidazol-5-yl)-5-[4-(trifluoromethyl)phenyl]thiomorpholin-2-yl,

[0522] 4-(1H-benzimidazol-5-yl)thiomorpholin-2-yl,

[0523] 4-(1H-benzimidazol-5-yl)-5-(4-fluorophenyl)-3-methyl-thiomorpholin-2-yl,

[0524] 4-(1H-benzimidazol-5-yl)-5-(4-bromophenyl)-3-methyl-thiomorpholin-2-yl,

[0525] 4-(1H-benzimidazol-5-yl)-5-(4-chlorophenyl)-3-methyl-thiomorpholin-2-yl,

[0526] 4-(1H-benzimidazol-5-yl)-3,5-dimethyl-thiomorpholin-2-yl,

[0527] 4-(1H-benzimidazol-5-yl)-5-cyclopropyl-3-methyl-thiomorpholin-2-yl,

[0528] 4-(1H-benzimidazol-5-yl)-5-(4-cyclopropylphenyl)-3-methyl-thiomorpholin-2-yl,

[0529] 4-(1H-benzimidazol-5-yl)-3-methyl-5-(p-tolyl)thiomorpholin-2-yl,

[0530] 4-(1H-benzimidazol-5-yl)-3-methyl-5-phenyl-thiomorpholin-2-yl,

[0531] 4-(1H-Benzimidazol-5-yl)-3-methyl-5-[4-(trifluoromethyl)phenyl]thiomorpholin-2-yl,

[0532] 4-(1H-Benzimidazol-5-yl)-3-methyl-thiomorpholin-2-yl,

[0533] wherein the thiomorpholine ring is numbered as follows:

[0534]

[0535]

[0536] 6-(1,3-Benzodioxol-5-yl)pyrimidin-4-yl,

[0537]

[0538] 6-(1H-Benzimidazol-5-yl)pyrimidin-4-yl,

[0539]

[0540] 6-(1,3-Benzodioxol-5-yl)-5-methyl-pyrimidin-4-yl,

[0541]

[0542] 4-(1,3-Benzodioxol-5-yl)pyrimidin-5-yl,

[0543]

[0544] 6-(1H-Benzimidazol-5-yl)-5-methyl-pyrimidin-4-yl,

[0545]

[0546] 4-(1H-Benzimidazol-5-yl)pyrimidin-5-yl,

[0547]

[0548] 6-(1H-Indazol-6-yl)pyrimidin-4-yl,

[0549]

[0550] 6-(1H-Indazol-6-yl)-4-methyl-pyrimidin-5-yl, and

[0551]

[0552] 6-(1H-Indazol-6-yl)pyrimidin-5-yl;

[0553] The second residue is selected from hydrogen, methyl,

[0554] and

[0555] (ii) a first residue covalently bound to the second residue, said first residue being selected from

[0556] 6-(1,3-benzodioxol-5-yl)-2H-pyrimidin-1-yl,

[0557] 6-(1,3-benzodioxol-5-yl)-4-methyl-2H-pyrimidin-1-yl,

[0558] 6-(1H-benzimidazol-5-yl)-2H-pyrimidin-1-yl,

[0559] 6-(1H-benzimidazol-5-yl)-4-methyl-2H-pyrimidin-1-yl,

[0560] 6-(1,3-benzodioxol-5-yl)-4,5-dimethyl-2H-pyrimidin-1-yl,

[0561] 6-(1,3-benzodioxol-5-yl)-5-methyl-2H-pyrimidin-1-yl,

[0562] 6-(1H-benzimidazol-5-yl)-4,5-dimethyl-2H-pyrimidin-1-yl,

[0563] 6-(1H-indazol-6-yl)-2H-pyrimidin-1-yl,

[0564] 6-(1H-indazol-6-yl)-4-methyl-2H-pyrimidin-1-yl,

[0565] 6-(1H-benzimidazol-5-yl)-5-methyl-2H-pyrimidin-1-yl,

[0566] 6-(1H-indazol-6-yl)-4,5-dimethyl-2H-pyrimidin-1-yl,

[0567] 6-(1H-indazol-6-yl)-5-methyl-2H-pyrimidin-1-yl,

[0568] wherein the 2H-pyrimidine ring is numbered as follows:

[0569]

[0570] (4R)-3-(1,3-benzodioxol-5-ylmethylcarbamoyl)-4-[[3-(trifluoromethyl)phenyl]methyl-aminocarbonyl]pyrrolidin-1-yl,

[0571] (4R)-3-(1,3-Benzodioxol-5-ylmethylcarbamoyl)-4-[[3,5-bis(trifluoromethyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0572] (4R)-3-(1,3-Benzodioxol-5-ylmethylcarbamoyl)-4-[[4-(trifluoromethyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0573] (4R)-3-(1,3-Benzodioxol-5-ylmethylcarbamoyl)-4-[[3-(cyclopropylphenyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0574] (4R)-3-(1,3-Benzodioxol-5-ylmethylcarbamoyl)-4-[[3,5-bis(cyclopropylphenyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0575] (4R)-3-(1,3-Benzodioxol-5-ylmethylcarbamoyl)-4-[[4-(cyclopropylphenyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0576] (4R)-3-(1H-Indazol-5-ylmethylcarbamoyl)-4-[[3-(trifluoromethyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0577] (4R)-3-(1H-Indazol-5-ylmethylcarbamoyl)-4-[[3,5-bis(trifluoromethyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0578] (4R)-3-(1H-Indazol-5-ylmethylcarbamoyl)-4-[[4-(trifluoromethyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0579] (4R)-3-(1H-Indazol-5-ylmethylcarbamoyl)-4-[[3-(cyclopropyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0580] (4R)-3-(1H-Indazol-5-ylmethylcarbamoyl)-4-[[3,5-bis(cyclopropyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0581] (4R)-3-(1H-Indazol-5-ylmethylcarbamoyl)-4-[[4-(cyclopropyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0582] (4R)-3-(3H-Benzimidazol-5-ylmethylcarbamoyl)-4-[[3-(trifluoromethyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0583] (4R)-3-(3H-Benzimidazol-5-ylmethylcarbamoyl)-4-[[3,5-bis(trifluoromethyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0584] (4R)-3-(3H-Benzimidazol-5-ylmethylcarbamoyl)-4-[[4-(trifluoromethyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0585] (4R)-3-(3H-Benzimidazol-5-ylmethylcarbamoyl)-4-[[3-(cyclopropyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0586] (4R)-3-(3H-Benzimidazol-5-ylmethylcarbamoyl)-4-[[3,5-bis(cyclopropyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0587] (4R)-3-(3H-Benzimidazol-5-ylmethylcarbamoyl)-4-[[4-(cyclopropyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0588] (4R)-2-(1,3-Benzodioxol-5-yl)-4-[[3-(trifluoromethyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0589] (4R)-2-(1,3-Benzodioxol-5-yl)-4-[[3,5-bis(trifluoromethyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0590] (4R)-2-(1,3-Benzodioxol-5-yl)-4-[[4-(trifluoromethyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0591] (4R)-2-(1,3-Benzodioxol-5-yl)-4-methylpyrrolidin-1-yl,

[0592] (4R)-2-(1,3-Benzodioxol-5-yl)-4-[[3-(cyclopropyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0593] (4R)-2-(1,3-Benzodioxol-5-yl)-4-[[3,5-bis(cyclopropyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0594] (4R)-2-(1,3-Benzodioxol-5-yl)-4-[[4-(cyclopropyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0595] (4R)-2-(1H-Indazol-5-yl)-4-[[3-(trifluoromethyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0596] (4R)-2-(1H-Indazol-5-yl)-4-[[3,5-bis(trifluoromethyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0597] (4R)-2-(1H-Indazol-5-yl)-4-[[4-(trifluoromethyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0598] (4R)-2-(1H-Indazol-5-yl)-3-methylpyrrolidin-1-yl,

[0599] (4R)-2-(1H-Indazol-5-yl)-4-[[3-(cyclopropyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0600] (4R)-2-(1H-Indazol-5-yl)-4-[[3,5-bis(cyclopropyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0601] (4R)-2-(1H-Indazol-5-yl)-4-[[4-(cyclopropyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0602] (4R)-2-(3H-Benzimidazol-5-yl)-4-[[3-(trifluoromethyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0603] (4R)-2-(3H-Benzimidazol-5-yl)-4-[[3,5-bis(trifluoromethyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0604] (4R)-2-(3H-Benzimidazol-5-yl)-4-[[4-(trifluoromethyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0605] (4R)-2-(3H-Benzimidazol-5-yl)-3-methylpyrrolidin-1-yl,

[0606] (4R)-2-(3H-Benzimidazol-5-yl)-4-[[3-(cyclopropyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0607] (4R)-2-(3H-benzimidazol-5-yl)-4-[[3,5-bis(cyclopropyl)phenyl]methyl-carbamoyl]pyrrolidin-1-yl,

[0608] (4R)-2-(3H-benzimidazol-5-yl)-4-[[4-(cyclopropyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0609] (4R)-2-(1,3-benzodioxol-5-yl)-3-methyl-4-[[3-(trifluoromethyl)phenyl]methyl-carbamoyl]pyrrolidin-1-yl,

[0610] (4R)-2-(1,3-benzodioxol-5-yl)-3-methyl-4-[[3,5-bis(trifluoromethyl)phenyl]methyl-carbamoyl]pyrrolidin-1-yl,

[0611] (4R)-2-(1,3-benzodioxol-5-yl)-3-methyl-4-[[4-(trifluoromethyl)phenyl]methyl-carbamoyl]pyrrolidin-1-yl],

[0612] (4R)-2-(1,3-benzodioxol-5-yl)-3-methyl-pyrrolidin-1-yl,

[0613] (4R)-2-(1,3-benzodioxol-5-yl)-3-methyl-4-[[3-(cyclopropyl)phenyl]methyl-carbamoyl]pyrrolidin-1-yl,

[0614] (4R)-2-(1,3-benzodioxol-5-yl)-3-methyl-4-[[3,5-bis(cyclopropyl)phenyl]methyl-carbamoyl]pyrrolidin-1-yl,

[0615] (4R)-2-(1,3-benzodioxol-5-yl)-3-methyl-4-[[4-(cyclopropyl)phenyl]methyl-carbamoyl]pyrrolidin-1-yl,

[0616] (4R)-2-(1H-indazol-6-yl)-3-methyl-4-[[3-(trifluoromethyl)phenyl]methyl-carbamoyl]pyrrolidin-1-yl,

[0617] (4R)-2-(1H-indazol-6-yl)-3-methyl-4-[[3,5-bis(trifluoromethyl)phenyl]methyl-carbamoyl]pyrrolidin-1-yl,

[0618] (4R)-2-(1H-indazol-6-yl)-3-methyl-4-[[4-(trifluoromethyl)phenyl]methyl-carbamoyl]pyrrolidin-1-yl,

[0619] (4R)-2-(1H-indazol-6-yl)-3-methyl-pyrrolidin-1-yl

[0620] (4R)-2-(1H-indazol-6-yl)-3-methyl-4-[[3-(cyclopropyl)phenyl]methyl-carbamoyl]pyrrolidin-1-yl,

[0621] (4R)-2-(1H-indazol-6-yl)-3-methyl-4-[[3,5-bis(cyclopropyl)phenyl]methyl-carbamoyl]pyrrolidin-1-yl,

[0622] (4R)-2-(1H-indazol-6-yl)-3-methyl-4-[[4-(cyclopropyl)phenyl]methyl-carbamoyl]pyrrolidin-1-yl,

[0623] (4R)-2-(1H-benzimidazol-5-yl)-4-[[3-(trifluoromethyl)phenyl]methyl-carbamoyl]pyrrolidin-1-yl,

[0624] (4R)-2-(1H-benzimidazol-5-yl)-4-[[3,5-bis(trifluoromethyl)phenyl]methyl-carbamoyl]pyrrolidin-1-yl,

[0625] (4R)-2-(1H-benzimidazol-5-yl)-4-[[4-(trifluoromethyl)phenyl]methyl-carbamoyl]pyrrolidin-1-yl,

[0626] (4R)-2-(1H-benzimidazol-5-yl)-3-methyl-pyrrolidin-1-yl,

[0627] (4R)-2-(1H-benzimidazol-5-yl)-4-[[3-(cyclopropyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0628] (4R)-2-(1H-benzimidazol-5-yl)-4-[[3,5-bis(cyclopropyl)phenyl]methyl-carbamoyl]pyrrolidin-1-yl, and

[0629] (4R)-2-(1H-benzimidazol-5-yl)-4-[[4-(cyclopropyl)phenyl]methylcarbamoyl]pyrrolidin-1-yl,

[0630] wherein the pyrrolidine ring is numbered as follows:

[0631]

[0632] the second residue is selected from hydrogen, methyl,

[0633]

[0634] wherein the first residue is covalently bonded to the second residue at the - position of the first residue;

[0635] preferably a compound selected from the following:

[0636]

[0637] 1-(1,3-Benzodioxol-5-yl)-4-(cyclopropanecarbonyl)-3-hydroxy-2-phenyl-2H-pyrrol-5-one;

[0638]

[0639] 1-(1,3-Benzodioxol-5-ylmethyl)-5-(4-fluorophenyl)-2-methyl-pyrrole-3-carboxamide;

[0640]

[0641] (3R)-N-(1,3-Benzodioxol-5-ylmethyl)-5-(4-fluorophenyl)thiomorpholine-3-carboxamide;

[0642]

[0643] 4-(1,3-Benzodioxol-5-yl)pyrimidine;

[0644]

[0645] (4R)-N3-(1,3-Benzodioxol-5-ylmethyl)-N4-[[3-(trifluoromethyl)phenyl]methyl]pyrrolidine-3,4-dicarboxamide;

[0646]

[0647] 1-(1,3-Benzodioxol-5-ylmethyl)-2-methyl-5-phenyl-pyrrole-3-carboxamide;

[0648]

[0649] 1-(1,3-Benzodioxol-5-ylmethyl)-2-methyl-5-(p-tolyl)pyrrole-3-carboxamide; and

[0650]

[0651] 1-(1,3-Benzodioxol-5-ylmethyl)-5-(4-chlorophenyl)-2-methyl-pyrrole-3-carboxamide.

[0652] In a further aspect, the invention relates to compounds selected from:

[0653]

[0654] wherein R 4 is selected from hydroxy, -O-R 14 and -O-C(=O)-R 14 , wherein R 14 is selected from

[0655] (aa) straight-chain or branched, substituted or unsubstituted (C 1-10 )alkyl, preferably (C 1-5 )alkyl, more preferably methyl, ethyl and propyl, most preferably methyl, (C 2-10 )alkenyl and (C 2-10 )alkynyl;

[0656] (bb) substituted or unsubstituted aromatic or non-aromatic (C 3-10 )carbocyclic, preferably (C 3-6 )cycloalkyl, more preferably (C3)carbocyclic and (C6)carbocyclic, preferably (C6)carbocyclic, more preferably phenyl mono-substituted at the para position by (C3)carbocyclic or -(CF3) or di-substituted at the meta position by (C3)carbocyclic or -(CF3); and

[0657] (cc) substituted or unsubstituted aromatic or non-aromatic (C 3-6 )heterocyclic, preferably aromatic (C 3-6 )heterocyclic having 1-3 heteroatoms each independently selected from N, O and S;

[0658] Preferably compounds selected from:

[0659]

[0660] 1-(1,3-Benzodioxol-5-yl)-4-(cyclopropanecarbonyl)-3-hydroxy-2-phenyl-2H-pyrrol-5-one;

[0661]

[0662] 1-(1,3-Benzodioxol-5-ylmethyl)-5-(4-fluorophenyl)-2-methyl-pyrrole-3-carboxamide;

[0663]

[0664] 4-(1,3-Benzodioxol-5-yl)pyrimidine; and

[0665]

[0666] (4R)-N3-[(1,3-benzodioxol-5-yl)methyl]-N4-[[3-(trifluoromethyl)phenyl]methyl]pyrrolidine-3,4-dicarboxamide.

[0667] The compounds described herein are generally named using the nomenclature calculated based on the structural diagrams by the software ACD / Chemsketch 2015 provided by Advanced Chemistry Development, Inc., Canada and BIOVIA Draw 2016 provided by BIOVIA, USA. For each molecule described herein, this description provides the structural formula, which specifically numbers the residues of the rings of Formulas I and II for the purpose of naming. It is further noted that the structural formula is binding on the chemical name, rather than being estimated from the chemical name; in other words, if the name and the structural formula are in conflict, the structural formula shall prevail.

[0668] For compounds having an asymmetric center, it is understood that unless otherwise specified, all optical isomers and their mixtures are encompassed. If not indicated differently, each stereogenic carbon can be in the (R)- or (S)-configuration or a combination of configurations. Moreover, compounds having two or more asymmetric elements can exist as a mixture of diastereoisomers. In addition, the compounds of the present invention preferably have a diastereoisomer purity of at least 50%, preferably at least 60%, 70%, 80%, 85%, more preferably at least 90%, 95%, 96%, 97%, and most preferably at least 98%, 99% or 100%. In addition, compounds having a carbon-carbon double bond can exist in the Z- and E-forms, and unless otherwise specified, all isomeric forms of the compounds are included in the present invention. When the compounds exist in various tautomeric forms, the listed compounds are not limited to any particular tautomer, but are intended to include all tautomeric forms.

[0669] For example, the compounds described below:

[0670] encompass tautomeric forms:

[0671] The listed compounds are also intended to include compounds in which one or more atoms are replaced by isotopes (i.e., atoms having the same atomic number but different mass numbers). As a general example, but not limited to, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include 11 C, 13 C, and 14 C.

[0672] Compounds of the formula provided herein having one or more stereogenic centers have an enantiomeric excess of at least 50%. For example, such compounds can have an enantiomeric excess of at least 60%, 70%, 80%, 85%, preferably at least 90%, 95% or 98%. Some embodiments of the compounds have an enantiomeric excess of at least 99%. It is evident that a single enantiomer (optically active form) can be obtained by asymmetric synthesis, synthesis from an optically pure precursor, biosynthesis, for example using modified CYP102 (CYP BM-3), or resolution of a racemate, for example enzymatic resolution or resolution by conventional methods such as crystallization in the presence of a resolving agent, or chromatography using, for example, a chiral HPLC column.

[0673] As used herein, "substituent" or "residue" or "R" refers to a molecular moiety that is covalently bonded to an atom within a target molecule. For example, a "substituent", "R" or "residue" can be a moiety such as a halogen, an alkyl, a haloalkyl or any other substituent described herein that is covalently bonded to an atom (preferably a carbon or nitrogen atom) and forms part of the target molecule. The term "substituted" as used herein means that any one or more hydrogens on a specified atom are replaced by a substituent selected from the specified substituents, provided that the normal valence of the specified atom is not exceeded and the substitution results in a stable compound, i.e., a compound that can be isolated and characterized using conventional methods. For example, the substitution can be in the form of an oxygen bonded to any other chemical atom that is not carbon, such as a hydroxyl group, or an oxyanion. When the substituent is oxo, i.e., =O, then 2 hydrogens on the atom are replaced. An oxo group as a substituent on an aromatic carbon atom results in the conversion of -CH- to -C(=O)- and loss of aromaticity. For example, a pyridyl group substituted with oxo is a pyridone.

[0674] The term "heteroatom" as used herein should be understood to mean an atom other than carbon and hydrogen, such as and preferably O, N, S and P.

[0675] If a first compound, substituent or residue ends, for example, with the name "-3-yl", then this ending indicates that the first compound, substituent or residue is covalently bonded to a second compound, substituent or residue at the atom in the 3-position of the first compound. Of course, this definition applies to any given integer before the "-yl" terminus of the compound, substituent or residue name. For example, if 1-(1,3-benzodioxol-5-ylmethyl)pyrrol-3-yl is selected as the first residue covalently bonded to a second residue, then the following compound is formed:

[0676] In the context of the present invention, it should be understood that prior terms such as "linear or branched", "substituted or unsubstituted" mean that each of the subsequent terms will be construed as being modified by the prior term. For example, the scope of the term "linear or branched substituted or unsubstituted alkyl, alkenyl, alkynyl, carbocycle" includes linear or branched substituted or unsubstituted alkyl; linear or branched substituted or unsubstituted alkenyl; linear or branched substituted or unsubstituted alkynyl; linear or branched substituted or unsubstituted alkylene; and linear or branched substituted or unsubstituted carbocycle. For example, the term "(C 2-10 ) alkenyl, alkynyl or alkylene" means a group of a compound having 2 to 10 carbons and an alkenyl, alkynyl or alkylene functional group.

[0677] The expression "alkyl" refers to a saturated linear or branched hydrocarbon group containing the indicated number of carbon atoms, for example, "(C 1-10 ) alkyl" means a hydrocarbon residue containing 1 to 10 carbon atoms, for example, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, 2,2-dimethylbutyl, etc.

[0678] The expression "alkenyl" refers to an at least partially unsaturated, substituted or unsubstituted linear or branched hydrocarbon group containing the indicated number of carbon atoms, for example, "(C 2-10 ) alkenyl" means a hydrocarbon residue containing 2 - 10 carbon atoms, for example, ethenyl (or vinyl), propenyl (allyl), isopropenyl, butenyl, isoprenyl or hex-2-enyl, or for example a hydrocarbon group containing a methylene chain interrupted by one double bond, for example, a hydrocarbon group found in monounsaturated fatty acids, or a hydrocarbon group containing a polyene interrupted by methylene, for example, a hydrocarbon group containing two or more of the following structural units -[CH=CH-CH2]-, for example, a hydrocarbon group found in polyunsaturated fatty acids. The alkenyl has one or more, preferably 1, 2, 3, 4, 5 or 6 double bonds.

[0679] The expression "alkynyl" refers to an at least partially unsaturated, substituted or unsubstituted linear or branched hydrocarbon group containing the indicated number of carbon atoms, for example, "(C 2-10 ) alkynyl" means a hydrocarbon residue containing 2 to 10 carbon atoms, for example, ethinyl, propynyl, butynyl, acetylenyl or propargyl. Preferably, the alkynyl has one or two (particularly preferably one) triple bonds.

[0680] In addition, the terms "alkyl", "alkenyl" and "alkynyl" refer to groups in which one or more hydrogen atoms have been replaced by, for example, a halogen atom, preferably F or Cl, for example, 2,2,2-trichloroethyl or trifluoromethyl.

[0681] The term "carbocyclic ring" should be understood to mean a substituted or unsubstituted aliphatic hydrocarbon ring containing the indicated number of carbon atoms, such as "(C 3-10 ) carbocyclic ring", or 3 to 20, preferably 3 to 12, more preferably 5 or 6 carbon atoms. These carbocyclic rings can be aromatic or non-aromatic systems. The non-aromatic ring systems can be mono-unsaturated or poly-unsaturated.

[0682] The term "carbobicyclic ring" refers to a carbocyclic ring as defined above that contains more than 1 ring, preferably two rings. Preferred carbocyclic and carbobicyclic rings include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, phenyl, indanyl, indenyl, benzocyclobutyl, dihydronaphthyl, tetrahydronaphthyl, naphthyl, decahydronaphthyl, benzocycloheptyl, benzocycloheptenyl, spiro[4,5]decyl, norbornyl, decahydronaphthyl, bicyclo[4.3.0]nonyl, tetrahydronaphthalene or cyclopentylcyclohexyl. The carbocyclic- and / or carbobicyclic ring residue can be attached to the remaining structure of the complete molecule through any atom of the ring, thereby producing a stable structure.

[0683] The term "carbocyclic ring" should also include "cycloalkyl", which should be understood to mean a ring containing an aliphatic hydrocarbon preferably having 3 - 12 carbon atoms. These non-aromatic ring systems can be mono-unsaturated or poly-unsaturated, i.e., the term includes cycloalkenyl and cycloalkynyl.

[0684] The term "heterocyclic ring" refers to a stable substituted or unsubstituted, aromatic or non-aromatic, preferably 3 to 20-membered, more preferably 3 - 12-membered, most preferably 5 or 6-membered, monocyclic, heteroatom-containing ring. Each heterocyclic ring is composed of carbon atoms and one or more, preferably 1 to 4, more preferably 1 to 3 heteroatoms, which are preferably selected from nitrogen, oxygen and sulfur. In addition to the indicated non-carbon atoms, the heterocyclic ring may also contain the number of carbon atoms: "(C 3-6 ) heterocyclic ring" means having 3 to 6 carbon atoms in addition to the given number of heteroatoms.

[0685] The term "heterobicyclic ring" refers to a heterocyclic ring as defined above that contains more than 1 ring, preferably two rings.

[0686] A hetero- and / or heterobicyclic residue may be attached to the remainder of the intact molecule through any atom of the ring, thereby resulting in a stable structure. Exemplary heterocycles and heterobicycles include, but are not limited to, pyrrolidinyl, pyrrolinyl, morpholinyl, thiomorpholinyl, thiomorpholine sulfoxide, thiomorpholine sulfone, dioxolanyl, piperidinyl, piperazinyl, tetrahydrofuranyl, 1-oxo-λ4-thiomorpholinyl, 13-oxa-11-aza-tricyclo[7.3.1.0-2,7]trideca-2,4,6-triene, tetrahydropyranyl, 2-oxo-2H-pyranyl, tetrahydrofuranyl, 1,3-dioxolane-2-one, 1,3-dioxan-2-one, 1,4-dioxanyl, 8-oxa-3-aza-bicyclo[3.2.1]-octyl, 2-oxa-5-aza-bicyclo[2.2.1]heptyl, 2-thia-5-aza-bicyclo[2.2.1]heptyl, piperidone, tetrahydro-pyrimidinone, pentamethylene sulfide, pentamethylene sulfoxide, pentamethylene sulfone, tetramethylene sulfide, tetramethylene sulfoxide and tetramethylene sulfone, indazolyl, benzimidazolyl, benzodioxolyl, imidazolyl, 1,3-benzodioxolyl and pyrazolyl.

[0687] The expression "alkyl / alkenyl / alkynyl ether" refers to a saturated or unsaturated straight-chain or branched hydrocarbon group containing the indicated number of carbon atoms. For example, "(C 1-10 ) alkyl ether" represents a hydrocarbon residue containing from 1 to 10 carbon atoms and any suitable number of oxygen atoms which will result in an ether structure. As used herein, the alkyl / alkenyl / alkynyl ether group is to be understood to mean any straight-chain or branched substituted or unsubstituted alkyl / alkenyl / alkynyl chain containing an oxygen atom as the ether moiety (i.e., an oxygen bonded through two carbons). The ether residue may be attached to the formula provided by the present invention through a carbon atom or through the oxygen atom of the ether residue.

[0688] As used herein, "substituent" or "residue" or "R", preferably R 2 、R 3 、R 4 、R 5 、R 6 、R 7 、R 8 、R 9 、R 10 and / or R 11 may be attached directly or through a linking group to the formula provided by the present invention. The linking group may also be in the form of polyethylene glycol. As used herein, the term polyethylene glycol refers to a chain of substituted or unsubstituted ethylene oxide monomers.

[0689] As used herein, the terms "nitrogen" or "N" and "sulfur" or "S" include any oxidized form of nitrogen and sulfur and any quaternized form of any basic nitrogen, provided that the resulting compound is chemically stable. For example, for -S-C 1-6An alkyl group is to be understood as including -S(O)-C 1-6 alkyl groups and -S(O)2-C 1-6 alkyl groups.

[0690] A residue linked to a second target compound at a given position is to be understood as a residue covalently bound to the second compound at the specified atomic position. For example, an indazolyl group linked through the (5) position of the indazolyl group represents the following residue: In this case, the numbering starts - as is customary in the art - at the 1H-nitrogen. However, it should be noted that some nomenclatures may provide different starting points for the numbering. For example, as will be understood by those skilled in the art, the 1H-benzimidazol-6-yl residue is the same as the 3H-benzimidazol-5-yl residue.

[0691] As used herein, words defining the limits of a length range, for example, "from 1 to 5" or "(C 1-5 )" mean any integer from 1 to 5, i.e., 1, 2, 3, 4, and 5. In other words, any range defined by two explicitly mentioned integers is intended to include and disclose any integer defining the limits and any integer included within the range.

[0692] By way of example, as used herein, the term "mono- or di-substituted in the meta position or mono-substituted in the para position" means that the compound is substituted by at least one given substituent in the para position of the position at which the compound is linked to another compound or residue, or is substituted by at least one substituent in its two meta positions. For example, the term "di-substituted in the meta position by a (C3) carbocycle or -(CF3)" means that the compound is substituted by a (C3) carbocycle or -(CF3) in each meta position, or is substituted by a (C3) carbocycle in one meta position and by -(CF3) in the other meta position. Preferably, the term means that the compound is substituted by a (C3) carbocycle in each meta position or by a -(CF3) in each meta position, i.e., is substituted by the same substituent in the two meta positions. As shown above for the para position, the meta position means the meta position of the position at which the compound is linked to another compound or residue.

[0693] For example, the term "phenyl, preferably mono-substituted in the para or meta position by cyclopropyl or -(CF3), or di-substituted in each meta position by cyclopropyl or -(CF3)" preferably represents the following structures:

[0694]

[0695] The residue R 1 , R 5 and / or R 9 is preferably a phenyl group mono-substituted in the para position by the group consisting of Cl, F, Br, substituted or unsubstituted methyl, preferably -(CF3), ethyl, propyl, and cyclopropyl. The residue R12 and R 13 is preferably a (C6) carbocyclic ring, more preferably a phenyl group mono-substituted at the para-position by a (C3) carbocyclic ring, preferably cyclopropyl or -(CF3), or a phenyl group di-substituted at the meta-position by a (C3) carbocyclic ring, preferably cyclopropyl or (-CF3). Further preferably, R 1 , R 5 , R 9 , R 12 and / or R 13 is a phenyl group mono-, di- or tri-substituted at the ortho-, meta- and / or para-positions by a group consisting of Cl, F, Br, substituted or unsubstituted methyl, preferably -(CF3), ethyl, propyl and cyclopropyl. Representing R 1 , R 5 , R 9 , R 12 and / or R 13 The di-substituted or tri-substituted phenyl group may be a di-substituted or tri-substituted phenyl group which is substituted by the same substituent at the corresponding ortho-, meta- and para-positions or is substituted by different substituents at the corresponding ortho-, meta- and / or para-positions, wherein the substituents are selected from Cl, F, Br, substituted or unsubstituted methyl, preferably -(CF3), ethyl, propyl and cyclopropyl. Each combination and number of substituents selected from Cl, F, Br, substituted or unsubstituted methyl, preferably -(CF3), ethyl, propyl and cyclopropyl, at the ortho-, meta- and / or para-positions of the phenyl group representing R 1 , R 5 , R 9 , R 12 and / or R 13 are expressly disclosed herein.

[0696] The scope of the present invention includes those analogues of the compounds as described above and in the claims, for example, for reasons of metabolic stability, which are characterized by the exchange of one or more carbon-bonded hydrogens, preferably one or more aromatic carbon-bonded hydrogens, with a halogen atom such as F, Cl or Br, preferably F. For example, Compound-1 may be characterized by replacing one or more aromatic carbon-bonded hydrogens in the benzene ring or replacing one or more aromatic or non-aromatic carbon-bonded hydrogens in the 1,3-benzodioxol-5-yl moiety with one or more halogen atoms, preferably F. In addition, for example, Compound-4 may be characterized by replacing one or more aromatic carbon-bonded hydrogens in the pyrimidine ring or replacing one or more aromatic or non-aromatic carbon-bonded hydrogens in the benzodioxole moiety with one or more halogen atoms, preferably F.

[0697] In a preferred embodiment, the present invention relates to a compound for the above use or the above compound itself, wherein the compound inhibits the phosphorylation of serine / arginine-rich splicing factor 1 (SRSF1, ASF-1, SF2) or a photosensory factor, preferably by G protein-coupled receptor kinase 2 (GRK2, ADRBK1).

[0698] On the other hand, the present invention relates to pharmaceutical compositions comprising, as an active substance, a compound for the above-mentioned use or the above-mentioned compound itself or a pharmaceutically acceptable derivative thereof, optionally in combination with excipients and / or carriers.

[0699] The present invention includes pharmaceutically acceptable salts or solvates of the compounds of formula (I) and (II) of the present invention. "Pharmaceutically acceptable salts or solvates" means any pharmaceutically acceptable salt, solvate or ester or any other compound which, when administered to a patient, is capable of (directly or indirectly) providing a compound of the present invention or a pharmacologically active metabolite or pharmacologically active residue thereof. Pharmacologically active metabolites should be understood to mean any compound of the present invention capable of being metabolized enzymatically or chemically. This includes, for example, hydroxylated or oxidized derivative compounds of the present invention.

[0700] Pharmaceutically acceptable salts include salts derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, p-toluenesulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid and benzenesulfonic acid. Other acids, such as oxalic acid, although not pharmaceutically acceptable per se, may be used in the preparation of salts which can be used as intermediates in obtaining the compounds and their pharmaceutically acceptable acid addition salts. Salts derived from suitable bases include alkali metals (e.g. sodium), alkaline earth metals (e.g. magnesium), ammonium and N-(C1-C4 alkyl)4 + salts.

[0701] Furthermore, the scope of the present invention also includes prodrugs of the compounds of the present invention. Prodrugs include those compounds which are modified after a simple chemical transformation to produce a compound of the present invention. Simple chemical transformations include hydrolysis, oxidation and reduction. Specifically, when a prodrug is administered to a patient, the prodrug can be converted into the compounds disclosed above, thereby imparting the desired pharmacological effect.

[0702] In a preferred embodiment, the compounds for use according to the present invention are used for cardioprotective treatment selected from

[0703] (i) reducing the risk of cardiovascular mortality and / or morbidity in the condition of essential hypertension and / or chronic hypertension;

[0704] (ii) reducing the risk of aging caused by cardiovascular diseases;

[0705] (iii) reducing the risk of cardiovascular mortality and / or morbidity in the condition of left ventricular dysfunction and signs of heart failure after recent myocardial infarction

[0706] (iv) reducing the risk of cardiovascular mortality and / or morbidity in the condition of chronic heart failure and left ventricular dysfunction;

[0707] (v) reducing the risk of cardiovascular mortality and / or morbidity in the condition of dilated cardiomyopathy;

[0708] (vi) reducing the risk of cardiovascular mortality and / or morbidity in the condition of left ventricular dysfunction;

[0709] (vii) reducing the risk of cardiovascular mortality and / or morbidity in the condition of cardiomyocyte necrosis;

[0710] (viii) reducing the risk of cardiovascular mortality and / or morbidity in the condition of myocardial fibrosis;

[0711] (ix) preventing, preferably primary prevention, of cardiomyocyte necrosis and / or dilated cardiomyopathy, preferably in the condition of ischemic heart disease and increased risk of ischemic heart injury, said increased risk being preferably due to cardiovascular risk factors selected from hypertension, atherosclerosis, chronic and acute stress, depression, diabetes, chronic heart failure, angina pectoris, atrial fibrillation, chronic renal failure, and aging;

[0712] (x) preventing, preferably secondary prevention, of cardiomyocyte necrosis and / or dilated cardiomyopathy in patients with a previous event selected from acute cardiovascular diseases, myocardial infarction, ischemic heart disease, angina pectoris, atrial fibrillation, decompensated and chronic heart failure, and cerebrovascular stroke; and

[0713] (xi) treating the acute disease state of cardiovascular diseases, said cardiovascular diseases being preferably selected from myocardial infarction, angina pectoris, ischemic heart disease, cerebrovascular diseases, and decompensated heart failure;

[0714] wherein the compound or pharmaceutical composition as described above is used for treating animals or humans, preferably mammals, more preferably humans.

[0715] Furthermore, the compounds of the present invention inhibit cytopathic SRSF1 phosphorylation in human renal cells, resulting in, for example, pro-survival activity towards human renal cells. Thus, in a preferred embodiment, the compounds of the present invention can be used to treat patients suffering from various forms of kidney diseases and nephropathies, such as nephropathies caused by hypertension, renal artery stenosis, heart failure, ischemia, diabetes, and / or toxins.

[0716] For therapeutic use, the compounds of the present invention can be administered in any conventional dosage form in any conventional manner. Routes of administration include oral, intravenous, intramuscular, and subcutaneous injection. Preferred modes of administration are oral, intravenous, or subcutaneous administration.

[0717] The compounds may be administered alone or in combination with adjuvants (including other active ingredients) that enhance compound stability, facilitate administration of pharmaceutical compositions containing them in certain embodiments, provide increased dissolution or dispersion, increase activity, provide adjuvant therapy (e.g., beta-adrenergic receptor antagonists, ACE inhibitors, angiotensin receptor antagonists, mineralocorticoid receptor antagonists, ivabradine, calcium channel antagonists, and / or diuretics), etc. Advantageously, such combination therapies use lower doses of conventional therapeutic agents, thereby avoiding the toxicity and adverse side effects that may occur when those agents are used as monotherapies. The above-described compounds may be physically combined with conventional therapeutic agents or other adjuvants into a single pharmaceutical composition. In this regard, reference may be made to U.S. Patent Application Nos. 09 / 902,822, PCT / US 01 / 21860, and U.S. Provisional Application No. 60 / 313,527 by Cappola et al., each of which is incorporated herein by reference in its entirety. Advantageously, the compounds may then be administered together in a single dosage form. In some embodiments, pharmaceutical compositions containing such combinations of compounds contain at least about 5%, but more preferably at least about 20% of the compounds of the present invention (w / w). The optimal percentage (w / w) of the compounds of the present invention may vary and is within the capabilities of those skilled in the art. Alternatively, the compounds may be administered separately (sequentially or in parallel). Separate administration allows for greater flexibility in the dosing regimen.

[0718] As described above, dosage forms of the compounds described herein include pharmaceutically acceptable carriers and adjuvants known to those of ordinary skill in the art. Methods for preparing such dosage forms are known (see, e.g., H.C. Ansel and N.G. Popovish, Pharmaceutical Dosage Forms and Drug Delivery Systems, 5 th ed., Lea and Febiger (1990)). Dosage levels and requirements are well recognized in the art and can be selected by those of ordinary skill in the art from the available methods and techniques suitable for a particular patient. In some embodiments, for a 70 kg patient, the dosage level ranges from 1 - 300 mg / dose. While one dose per day may be sufficient, up to 5 doses per day may be administered. For oral doses, up to 2000 mg / day may be required. Reference may also be made to U.S. Provisional Application No. 60 / 339,249 in this regard. As those skilled in the art will appreciate, lower or higher doses may be required depending on specific factors. For example, the specific dose and treatment regimen will depend on factors such as the general health of the patient, the severity and course of the patient's condition or its disposition, and the judgment of the treating physician.

[0719] On the other hand, the present invention relates to a method for cytoprotective, preferably cardioprotective or nephroprotective treatment of a patient, preferably a mammalian patient, more preferably a human patient, the method comprising the step of administering to a patient in need of such treatment a therapeutically effective amount of a compound according to the invention or an effective amount of a pharmaceutical composition according to the invention.

[0720] On the other hand, the present invention relates to a method for identifying a G protein-coupled receptor kinase 2 (GRK2) inhibitor, preferably by measuring the phosphorylation of serine / arginine-rich splicing factor 1 (SRSF1) and / or a photosensory factor mediated by GRK2, the method comprising the following steps

[0721] (i) providing and incubating GRK2 and SRSF1 or a photosensory factor under physiological conditions suitable for the phosphorylation of SRSF1 or a photosensory factor in the presence and absence of a test compound;

[0722] (ii) measuring the phosphorylation of SRSF1 or a photosensory factor in the presence and absence of a test compound;

[0723] (iii) identifying the test compound as an inhibitor or non-inhibitor based on the phosphorylation of SRSF1 or a photosensory factor in the presence of the test compound relative to the phosphorylation of SRSF1 or a photosensory factor in the absence of the test compound.

[0724] In a preferred embodiment, the method for identification as described above is a method wherein

[0725] (a) in step (i), the incubation is carried out in the presence of radiolabeled ATP, preferably [γ- 32 P]ATP, preferably at about 25 to 40 °C for about 30 to 90 minutes; and / or

[0726] (b) the incubation of step (i) is stopped by dilution at a temperature below 30 °C, preferably at a temperature of about 0 to 10 °C; and / or

[0727] (c) the phosphorylation of SRSF1 or a photosensory factor in step (ii) is measured by (A) filtering the product of (i) through a filter, preferably a glass fiber filter; (B) washing the filter; and (C) preferably measuring the radioactivity bound to the filter with a β-counter.

[0728] Non-limiting and representative examples of methods for identifying inhibitors of (GRK2)-mediated phosphorylation of (SRSF1) and (light-sensing factor) are provided in Example 13 and Example 5 below. For example, to identify small molecule inhibitors of (GRK2)-mediated phosphorylation of (SRSF1) and (light-sensing factor), phosphorylation assays can be performed in a reaction buffer supplemented with ATP, preferably about 5 μM of [γ-32P]-ATP (e.g., 1x10 6 DPM, specific activity of about 3000 Ci / mmol)) and about 300 - 500 nM of SRSF1 (or light-sensing factor) (e.g., 20 mM Tris, 2 mM EDTA, 5 mM MgCl2, 0.05% BSA, pH 7.5 or 20 mM Hepes, 2 mM MgCl2, 0.025% DDM (= n-dodecyl-β-D-maltoside), pH 7.4). The reaction mixture is added to GRK2 or GRK2-S670A (e.g., about 60 nM, in the reaction buffer with or without increasing concentrations of small molecule compounds) to obtain a final reaction volume of, for example, about 50 μL. After incubation at about 30 °C for, for example, about 30 - 60 minutes, phosphorylation can be terminated by adding ice-cold reaction buffer, preferably about 5 volumes of reaction buffer. The reaction mixture is immediately applied to a filter, preferably a glass fiber filter. After three washing steps using, for example, about 5 ml of reaction buffer, the radioactivity bound to the filter can be measured in a β-counter.

[0729] Moreover, the compounds of the present invention can be used as markers for altered GRK2 activity. Brief Description of the Drawings

[0730] The following drawings and examples are used to illustrate the present invention and are not intended to limit the scope of the present invention described in the appended claims.

[0731] In the drawings and examples, the following names are assigned to the various compounds and control compounds of the present invention:

[0732] Compound-1: 1-(1,3-benzodioxol-5-yl)-4-(cyclopropanecarbonyl)-3-hydroxy-2-phenyl-2H-pyrrol-5-one;

[0733] Compound-2: 1-(1,3-benzodioxol-5-ylmethyl)-5-(4-fluorophenyl)-2-methyl-pyrrole-3-carboxamide;

[0734] Compound-3: (3R)-N-(1,3-benzodioxol-5-ylmethyl)-5-(4-fluorophenyl)thiomorpholine-3-carboxamide;

[0735] Compound-4: 4-(1,3-Benzodioxol-5-yl)pyrimidine;

[0736] Compound-5: (4R)-N3-(1,3-Benzodioxol-5-ylmethyl)-N4-[[3-(trifluoromethyl)phenyl]methyl]pyrrolidine-3,4-dicarboxamide;

[0737] Compound-22: 1-(1,3-Benzodioxol-5-ylmethyl)-2-methyl-5-phenyl-pyrrole-3-carboxamide;

[0738] Compound-23: 1-(1,3-Benzodioxol-5-ylmethyl)-2-methyl-5-(p-tolyl)pyrrole-3-carboxamide;

[0739] Compound-24: 1-(1,3-Benzodioxol-5-ylmethyl)-5-(4-chlorophenyl)-2-methyl-pyrrole-3-carboxamide;

[0740] Compound-6: Ibuprofen ((±)-2-(4-isobutylphenyl)propanoic acid) (reference compound, Sigma-Aldrich, St. Louis, USA);

[0741] Figure 1A -E: Identification of SRSF1 as a novel non-receptor GRK2 substrate

[0742] A, left panel: GRK2 was enriched from heart biopsy samples of heart failure patients by immunoaffinity chromatography, and enriched GRK2 and co-enriched SRSF1 were detected in immunoblotting. Right panel: Nano-LC-ESI-MS / MS analysis identified SRSF1 (serine / arginine-rich splicing factor 1, ASF / SF2, SEQ ID NO: 7) as a previously unidentified GRK2 interacting protein from heart biopsy samples of heart failure patients. B, GRK2 phosphorylated SRSF1 in an in vitro kinase assay. Nano-LC-ESI-MS / MS analysis identified phosphopeptides with serine 199 / 201 as the GRK2 phosphorylation sites (SEQ ID NO: 8 and SEQ ID NO: 9). C, Paroxetine inhibited GRK2-mediated SRSF1 phosphorylation with a half-maximal inhibitory concentration (IC50 value) of 2.38 μM (n = 4). D, Another cardioprotective GRK2 inhibitor, GRKInh, inhibited GRK2-mediated SRSF1 phosphorylation. E, Immunoblot detection of GRK2-activated SRSF1 phosphorylation with the phospho-specific SR antibody 1H4 (p-SRSF1).

[0743] Figure 2: GRK2 induces the activation of Srsf1 phosphorylation in vivo

[0744] A, B, Tg-GRK2 mice with cardiac-specific expression of GRK2 under the control of the α-MHC promoter (A) and immunoblot detection of increased GRK2 protein in heart lysates from Tg-GRK2 mice relative to non-transgenic B6 controls (B; n = 4 hearts / group). C, Tg-GRK2 mice have reduced left ventricular ejection fraction as measured by echocardiography (±s.d., n = 6). D, E, Immunoblot detection of increased p-Srsf1 (right) and Camk2d isoforms B and C content in heart lysates from Tg-GRK2 mice relative to non-transgenic controls (n = 4 / group). F, Myocardial necrosis in Tg-GRK2 hearts was determined by von Kossa staining (n = 6; ±S.d.), bar: 100 μm. G, H, Immunoblot detection of pSrsf1 (G) and Camk2d isoforms B / C (H) in Tg-GRK2 hearts transduced with control lentivirus or lentivirus targeting Srsf1 via miRNA (+miSrsf1). I, Downregulation of Srsf1 by miSrsf1 delays the development of cardiac dysfunction in Tg-GRK2 mice (n = 4, ±s.d.). J, Myocardial necrosis was determined by von Kossa staining (n = 6; ±s.d.).

[0745] Figure 3 : Transgenic overexpression of SRSF1 induces enhanced Camk2d splicing and signs of heart failure

[0746] A, Transgenic mice with cardiac-specific expression of SRSF1 were generated under the control of the cardiac-specific α-MHC promoter. B, Increased SRSF1 / Srsf1 protein in the hearts of Tg-SRSF1 mice was detected by immunoblot (n = 4 hearts / group). C, Enhanced cardiac splicing of Camk2d protein isoforms B and C in Tg-SRSF1 mice (n = 4 hearts / group). D, Myocardial necrosis in Tg-SRSF1 hearts was determined by von Kossa staining (n = 5, ±s.d.). E, Histological evaluation of Tg-SRSF1 hearts showed dilated cardiac hypertrophy relative to non-transgenic B6 controls. Heart sections were stained with hematoxylin-eosin (HE) and represent 4 mice / group. F, Cardiac dysfunction in Tg-SRSF1 mice relative to non-transgenic B6 controls was determined by echocardiography (n = 5; ±s.d.).

[0747] Figure 4 : Inhibition of GRK2 in vivo delays the activation of Srsf1 phosphorylation and signs of heart failure in a chronic pressure overload model of cardiac dysfunction

[0748] A, Generation of transgenic mice with myocardial - specific expression of dominant - negative GRK2 - K220R and immunoblot detection of GRK2 - K220R in Tg - GRK2 - K220R hearts (n = 3 (B6 control) and n = 5 (Tg - GRK2 - K220R hearts)). B, Immunoblot detection of cardiac pSrsf1 and Camk2d isoforms B / C in 4 - month - old B6 control (B6), Tg - GRK2 - K220R, and Tg - GRKInh mouse hearts subjected to chronic pressure overload by abdominal aortic constriction (AAC) for 8 weeks; n = 4 mice / group. C, Determination of myocardial necrosis by von Kossa staining (n = 6 / group; ±s.d.). D, Cardiac dysfunction in B6 mice with delayed GRK2 inhibition by GRK2 - K220R or GRKInh for 8 weeks of AAC (n = 6 / group; ±s.d.).

[0749] Figure 5 : The GRK2 inhibitor RKIP does not inhibit the activation of Srsf1 phosphorylation and induces signs of heart failure in vivo

[0750] A, Human RKIP does not inhibit GRK2 - mediated phosphorylation of SRSF1 in vitro. Instead, human RKIP inhibits the phosphorylation of the light - sensing factor by GRK2 (IC50 = 950 nM, n = 6 - 7). B, C, Immunoblot detection of cardiac RKIP in Tg - RKIP2 and Tg - RKIP3 lines with myocardial - specific expression of human RKIP (B; n = 3 mice / group), and reduced left ventricular ejection fraction in Tg - RKIP2 and Tg - RKIP3 lines (n = 5 / group; ±s.d.). D, Histological evaluation and heart weight / body weight determination of 8 - month - old Tg - RKIP2 hearts relative to B6 and Tg - GRK2 - K220R hearts (left) (n = 6, ±s.d). Tissue sections represent 4 hearts / group. E, F, Determination of cardiac fibrosis by Sirius red staining (E) and necrosis by von Kossa staining (F) in Tg - RKIP2 hearts, B6, and Tg - GRK2 - K220R hearts. G, Immunoblot detection of cardiac Srsf1 (total Srsf1) and activated phosphorylated p - Srsf1 in heart lysates from mice of different groups (n = 3 / group).

[0751] Figure 6 : Signs of heart failure also appear in the Tg - RKIP mouse line in the FVB background

[0752] A, Immunoblot detection of human / mouse RKIP in the Tg-RKIP mouse strain generated in the FVB background (n = 8 / group, ±s.d.). B, Immunohistological detection of RKIP protein in the hearts of Tg-RKIP1 relative to non-transgenic FVB controls. Immunohistology represents 4 hearts / group. C, D, Left ventricular ejection fraction (C; n = 5) and heart weight / body weight ratio (n = 6; ±s.d.) of Tg-RKIP1 and Tg-RKIP2 mice relative to non-transgenic FVB controls. E, Histological analysis of Tg-RKIP hearts relative to non-transgenic FVB controls. Tissue sections represent four mice / group. F, G, Left ventricular ejection fraction of Tg-RKIP1 and TgRKIP2 mice relative to non-transgenic FVB controls, and RNAi-mediated downregulation of human RKIP by lentiviral transduction of miRKIP (F; n = 5; ±s.d.). Downregulation of human RKIP was confirmed by real-time qRT-PCR (G; n = 5; ±s.d.).

[0753] Figure 7 : Transgenic RKIP expression induces a gene expression signature associated with GRK2 inhibition

[0754] A, Immunoblot detection of serine-153 phosphorylation (pS153) of RKIP in Tg-RKIP and B6 control hearts (n = 5 mice / group). B, Depletion of GRK2 protein from Tg-RKIP heart lysates by affinity purification (AP) using immobilized RKIP-specific antibody. Control affinity purification with an irrelevant antibody neither depletes RKIP nor GRK2. The upper panel shows a representative experiment and the lower panel shows the quantification data assessment (±s.d., n = 4; *, p = 0.0286; Mann Whitney test). C, Isoproterenol-stimulated cAMP responses in neonatal cardiomyocytes from Tg-RKIP and Tg-GRK2-K220R mice (n = 6; ±s.d.). D, Expression of the cAMP-inducible gene Ttc14 is significantly increased in Tg-RKIP and Tg-GRK2-K220R hearts relative to B6 controls (n = 3; ±s.d.). E, F, Genome-wide gene expression profiles show concordant regulation of a 45% significantly altered probe set between Tg-RKIP and Tg-GRK2-K220R hearts. Panel F shows the concordantly regulated probe set in Tg-RKIP and Tg-GRK2-K220R hearts (two gene chips / group; **, p < 0.01 and ***, p < 0.001, t-test relative to B6 controls).

[0755] Figure 8 : The cardioprotective GRK2 inhibitor paroxetine delays the heart failure phenotype in Tg-RKIP mice

[0756] A-C, Paroxetine (5 mg / kg body weight, in drinking water, daily) treatment for 8 weeks decreased the cardiac contents of Srsf1 and phosphorylated, activated Srsf1 in the hearts of 5-month-old Tg-RKIP mice (A) and the splicing of Camk2d isoform B / C (B), as determined by immunoblotting (B, n = 5 hearts / group) and real-time qRT-PCR (C, n = 6; ±s.d). D, Myocardial necrosis associated with calcium overload was determined on cardiac sections by von Kossa staining (n = 6 hearts / group; ±s.d.). E, Left ventricular ejection fraction was determined by echocardiography (n = 6 mice / group; ±s.d.). F, Inactivation phosphorylation of the heart failure-promoting transcription factor Pparg at serine 273 (pS273-Pparg) was determined by immunoblotting with a phosphoserine-273-Pparg-specific antibody (IB: pS273-Pparg; left panel). Paroxetine treatment enhanced the inactivation of Pparg in the hearts of Tg-RKIP mice (left panel), whereas paroxetine treatment did not alter the total cardiac Pparg content (right panel) (n = 4 mice / group).

[0757] Figure 9 : Development of small molecule compounds that inhibit GRK2-mediated SRSF1 phosphorylation.

[0758] GRK2-mediated phosphorylation of SRSF1 was determined with purified protein in the presence of increasing concentrations of paroxetine or different small molecule compounds. SRSF1 phosphorylation was expressed as % of control, which was SRSF1 phosphorylation in the absence of inhibitor. The IC50 value (0.45 μM) of 1-(1,3-benzodioxol-5-yl)-4-(cyclopropanecarbonyl)-3-hydroxy-2-phenyl-2H-pyrrol-5-one (“Compound-1”) was less than 1 / 5 of the paroxetine IC50 value (±s.d., n = 4).

[0759] Figure 10 : Small molecule compounds inhibit GRK2-mediated SRSF1 phosphorylation in intact Tg-SRSF1 cardiomyocytes and human HEK cells.

[0760] A, Immunoblotting of GRK2-mediated SRSF1 / Srsf1 phosphorylation in lysates of isolated neonatal cardiomyocytes from Tg-SRSF1 mice after incubation with 10 μM paroxetine, 1-(1,3-benzodioxol-5-yl)-4-(cyclopropanecarbonyl)-3-hydroxy-2-phenyl-2H-pyrrole-5-one (“Compound-1”), or 4-(1,3-benzodioxol-5-yl)pyrimidine (“Compound-4”) for 60 hours, compared to vehicle-treated control (“C”) cardiomyocytes. B, Immunoblot detection of GRK2-mediated SRSF1 phosphorylation (pSRSF1) using lysates from HEK (human embryonic kidney) cells incubated with 10 μM of 4-(1,3-benzodioxol-5-yl)pyrimidine (“Compound-4”), 1-(1,3-benzodioxol-5-ylmethyl)-5-(4-chlorophenyl)-2-methyl-pyrrole-3-carboxamide (“Compound-24”), or (4R)-N3-(1,3-benzodioxol-5-ylmethyl)-N4-[[3-(trifluoromethyl)phenyl]methyl]pyrrolidine-3,4-dicarboxamide (“Compound-5”) for 60 hours, compared to vehicle-treated control (“C”).

[0761] Figure 11 : Cardioprotective effects of small molecule compounds in vivo.

[0762] A, Immunoblot determination of GRK2-mediated Srsf1 phosphorylation in heart lysates prepared from non-transgenic B6 mice hearts after 5 hours of treatment with 10 mg / kg of 1-(1,3-benzodioxol-5-yl)-4-(cyclopropanecarbonyl)-3-hydroxy-2-phenyl-2H-pyrrole-5-one (″Compound-1″), 4-(1,3-benzodioxol-5-yl)pyrimidine (″Compound-4″), 1-(1,3-benzodioxol-5-ylmethyl)-5-(4-fluorophenyl)-2-methyl-pyrrole-3-carboxamide (″Compound-2″) or (4R)-N3-(1,3-benzodioxol-5-ylmethyl)-N4-[[3-(trifluoromethyl)phenyl]methyl]pyrrolidine-3,4-dicarboxamide (″Compound-5″) (i.p.), compared to vehicle-treated B6 controls (″con″). The right panel shows the quantitative immunoblot assessment. B, Left panel: Determination of AAC-induced cardiac p-Srsf1 content in heart lysates of B6 mice treated with 5 mg / kg / d of 1-(1,3-benzodioxol-5-yl)-4-(cyclopropanecarbonyl)-3-hydroxy-2-phenyl-2H-pyrrole-5-one (″Compound-1″) or (4R)-N3-(1,3-benzodioxol-5-ylmethyl)-N4-[[3-(trifluoromethyl)phenyl]methyl]pyrrolidine-3,4-dicarboxamide (″Compound-5″) for 7 days after AAC, compared to vehicle-treated B6 controls undergoing AAC (″AAC-con″). The middle panel shows the quantitative immunoblot assessment. Right and bottom panels: Echocardiographic determination of cardiac function parameters in B6 mice treated with ″Compound-1″ or ″Compound-5″ for 7 days after AAC, compared to vehicle-treated B6 controls undergoing AAC ((±s.d.; n = 5 (A) and n = 3 (B); Dunnett's multiple comparison test versus con (A) or AAC-con (B)).

[0763] Figure 12 Oral treatment with Compound-1 and Compound-4 abrogated heart failure and aging caused by cardiovascular disease.

[0764] A. Left ventricular ejection fraction was measured in 6-month-old male B6 mice that had undergone 3 months of chronic pressure overload by AAC and were orally treated with compound-1 (AAC + compound-1) and compound-4 (AAC + compound-4), compared to untreated AAC controls. Treatment began one month after AAC induction and continued for 2 months. Only mice with heart failure (ejection fraction < 34% one month after AAC) were included in the study (±s.d., n = 5 / group, ***p = 0.0006, **p = 0.0051, compared to untreated AAC, Dunnett's multiple comparison test). B. Histological evaluation of hearts with 3 months of AAC with no (control, upper panel) or 2 months of compound-1 treatment (lower panel) showed that compound-1 delayed cardiac hypertrophy induced by chronic pressure overload (n = 4 hearts / group). C, D. Treatment with compound-1 also delayed aging caused by cardiovascular diseases, such as aging caused by chronic pressure overload, i.e., the appearance of grey hair was significantly delayed after 2 months of treatment with compound-1 (left (D) and right (D) panels, n = 5 different mice).

[0765] Figure 13A -J: Identification of test compound molecules and exemplary chemical synthesis routes according to the present invention

[0766] A-H. Compounds -1 to -5 and compounds -22 to -24 were analyzed by electrospray mass spectrometry and HPLC analysis. I, J. Exemplary chemical synthesis routes for compound-1 and compound-4.

[0767] Examples

[0768] Example 1: Identification of SRSF1 as a new non-receptor GRK2 substrate

[0769] Given the pathophysiological importance of GRK2 and its yet unknown targets, new GRK2 interacting proteins have been sought. A GRK2-specific antibody was covalently coupled to an affinity matrix. Protein lysates from biopsy samples of heart failure patients were applied, the bound proteins were eluted, and enriched GRK2 and co-enriched proteins were separated by SDS-PAGE. The stained protein bands were excised, and Nano-LC-ESI-MS / MS analysis identified SRSF1 (serine / arginine-rich splicing factor 1, ASF / SF2) as a previously unrecognized GRK2 interacting protein ( Figure 1A , right panel). As a control, immunoblot detection confirmed the protein enrichment of GRK2 and the co-enrichment of SRSF1 ( Figure 1A , left panel). SRSF1 was found to be a kinase substrate of GRK2. In an in vitro kinase assay, GRK2 phosphorylated recombinant SRSF1 protein ( Figure 1B)。 Nano-LC-ESI-MS / MS analysis identified phosphopeptides, where Serine 199 / 201 were the phosphorylation sites of GRK2( Figure 1B )。 GRK2 phosphorylation of SRSF1 at Serine 199 / 201 was relevant because these residues and the phosphorylation of these residues were substantially related to the splicing function of SRSF1 (Zuo P and Manley JL, EMBO J 12, 4727-4737 (1993); Xiao SH and Manley JL, Genes Dev 11, 334-344 (1997)). As a control for GRK2 specificity in in vitro phosphorylation assays, the cardioprotective ATP-site directed GRK2 inhibitor paroxetine was applied (Thal DM et al., ACS Chem Biol 7, 1830-1839 (2012); Schumacher SM et al., Sci.Transl.Med. 7, 277ra31 (2015)). Paroxetine inhibited GRK2-mediated SRSF1 phosphorylation with a half-maximal inhibitory concentration (IC50 value) of 2.38 μM (Figure 1C). This value was comparable to the inhibition of other GRK2 substrates mediated by paroxetine (Thal DM et al., ACS Chem Biol 7, 1830-1839 (2012); Schumacher SM et al., Sci.Transl.Med. 7, 277ra31 (2015)). Additionally, another cardioprotective GRK2 inhibitor, GRKInh(6,7), also inhibited GRK2-mediated in vitro phosphorylation of SRSF1 (Figure 1D). The phosphorylation of SRSF1 activated by GRK2 was also confirmed in immunoblots with the phosphorylation-specific SR antibody 1H4( Figure 1E ), which detects phosphorylated RS repeats in the carboxyl-terminal domain of SRSF1 (Neugebauer KM et al., Genes Dev. 11, 1148-1159 (1997)).

[0770] Example 2: GRK2 Induces Activation of Srsf1 Phosphorylation in Vivo

[0771] It was investigated whether GRK2 phosphorylated Srsf1 in vivo and Tg-GRK2 mice were generated with myocardial-specific human GRK2 (ADRBK1) expression under the control of the myocardial-specific α-MHC promoter( Figure 2 in A). The hearts of Tg-GRK2 mice had increased GRK2 protein levels (2-fold that of non-transgenic B6 controls, Figure 2In B). Consistent with the pathophysiological role of increased cardiac GRK2 protein levels (Hullmann J et al., Pharmacol. Res. 110, 52 - 64 (2016)), Tg - GRK2 mice develop cardiac dysfunction with increasing age, as demonstrated by the significant reduction in cardiac ejection fraction in 8 - month - old Tg - GRK2 mice ( Figure 2 In C). The cardiac content of activated phospho - Srsf1 was determined to analyze whether the increased GRK2 protein levels in transgenic Tg - GRK2 mice lead to increased Srsf1 phosphorylation and activation. Immunoblotting assays showed that Tg - GRK2 hearts had increased levels of activated Srsf1 phosphorylation ( Figure 2 In D). The splicing factor activity of activated Srsf1 is required for the splicing of Camk2d (calcium / calmodulin - dependent kinase II isoform δ) isoforms B and C (Xu X et al., Cell 120, 59 - 72 (2005)). Consistent with enhanced Srsf1 activation, Tg - GRK2 hearts had increased cardiac - specific Camk2d isoforms B and C, as detected by immunoblotting ( Figure 2 In E). The increase in Camk2d isoforms B and C is sufficient to promote cardiac dysfunction and heart failure (Zhang T et al., Circ. Res. 92, 912 - 919 (2003); Zhang T et al., J. Biol. Chem. 277, 1261 - 1267 (2002)).

[0772] In addition, Camk2d promotes cardiac necrosis (Zhang T et al., Nat. Med. 22, 175 - 182 (2016)). As determined by von Kossa staining, Tg - GRK2 hearts showed an increase in necrotic areas with calcium overload ( Figure 2 In F). The causal relationship between GRK2 - mediated Srsf1 activation and the cardiac phenotype of Tg - GRK2 hearts was also demonstrated by lentiviral transduction of miRNA targeting Srsf1 to downregulate Srsf1 by RNAi ( Figure 2 In G). Downregulation of Srsf1 delayed the induction of Camk2d isoforms B and C, the development of heart failure with calcium overload, and the signs of cardiac necrosis in Tg - GRK2 hearts ( Figure 2 In H - J).

[0773] Example 3: Transgenic overexpression of SRSF1 induces enhanced Camk2d splicing and signs of heart failure

[0774] Tg - SRSF1 mice with increased cardiac SRSF1 protein due to myocardial - specific SRSF1 expression were generated under the control of the myocardial - specific α - MHC promoter ( Figure 3In A). Tg-SRSF1 mice have increased cardiac SRSF1 protein ( Figure 3 In B) and show enhanced splicing of Camk2d isoforms B and C ( Figure 3 In C). At the same time, Tg-SRSF1 mice develop myocardial necrosis with calcium overload, accompanied by dilated cardiac hypertrophy (indicating cardiomyocyte loss) and cardiac dysfunction ( Figure 3 In D-F).

[0775] Example 4: Inhibition of GRK2 in vivo delays the activation of Srsf1 phosphorylation and signs of heart failure in a chronic pressure overload model of cardiac dysfunction

[0776] Srsf1 and GRK2 are upregulated in experimental models of pressure overload-induced cardiac dysfunction (Hullmann J et al., Pharmacol. Res. 110, 52-64 (2016); Kim T et al., Mol Cells 37, 81-87 (2014)). Transgenic expression of a dominant-negative GRK2-K220R mutant delays the induction of Srsf1 phosphorylation activation triggered by pressure overload through GRK2 inhibition ( Figure 4 In B). Transgenic Tg-GRK2-K220R mice with myocardial-specific expression of GRK2-K220R were generated ( Figure 4 In A). The cardioprotective GRK2 inhibitor GRKInh (Abd Alla, J et al., J. Biol. Chem. 291, 2583-2600 (2016); Fu X et al., J. Biol. Chem. 288, 7738-7755 (2013)) (which inhibits SRSF1 phosphorylation in vitro (see Figure 1)) also delays GRK2 activation of Srsf1 phosphorylation induced by pressure overload. At the same time, two different modes of GRK2 inhibition delay Camk2d isoform B / C splicing, myocardial necrosis with calcium overload, and signs of heart failure ( Figure 4 In C, D). Therefore, cardioprotective GRK2 inhibition attenuates pressure overload-induced Srsf1 activation, cardiomyocyte death, and signs of heart failure.

[0777] Example 5: The GRK2 inhibitor RKIP does not inhibit the activation of Srsf1 phosphorylation and induces signs of heart failure in vivo

[0778] The effects of another GRK2 inhibitor, namely raf kinase inhibitor protein RKIP (Lorenz K et al., Nature 426, 574 - 579 (2003)), which is a dual - specific GRK2 and Raf kinase inhibitor, were analyzed. RKIP was shown to be upregulated in heart biopsy samples from heart failure patients (Schmid E et al., Nat. Med. 21, 1298 - 1306 (2015)). Human RKIP does not inhibit the phosphorylation of SRSF1 by GRK2 in vitro ( Figure 5 in A). As a control, human RKIP is an effective GRK2 inhibitor and inhibits the phosphorylation of a light - sensing factor ( Figure 5 in A), which is another non - receptor substrate of GRK2 (Ruiz - Gomez A et al., J. Biol. Chem. 275, 29724 - 29730 (2000)). The IC50 value for human RKIP - mediated inhibition of GRK2 - induced phosphorylation of the light - sensing factor was 950 nM ( Figure 5 in A), comparable to the reported IC50 value of 460 nM for RKIP - mediated inhibition of GPCR phosphorylation (Lorenz K et al., Nature 426, 574 - 579 (2003)). Tg - RKIP mice with cardiac - specific expression of human RKIP developed signs of heart failure in a dose - dependent manner ( Figure 5 in B, C). Meanwhile, dilated cardiac hypertrophy, cardiac fibrosis, and necrosis were evident ( Figure 5 in D - F). Consistent with in vitro experiments demonstrating that human RKIP cannot inhibit the phosphorylation of SRSF1 by activated GRK2 (see Figure 5 in A), the cardiac content of phosphorylated Srsf1 was higher in the hearts of Tg - RKIP mice than in non - transgenic B6 controls ( Figure 5 in G).

[0779] Example 6: The Tg - RKIP mouse strain in the FVB background also exhibits signs of heart failure

[0780] In a transgenic mouse strain expressing human RKIP generated on an FVB background, a heart failure phenotype induced by transgenic human RKIP expression was similarly detected in a dose - dependent manner ( Figure 6 in A - D). Histological analysis showed that Tg - RKIP mice in the FVB background developed severe dilated cardiac hypertrophy and cardiomyocyte loss ( Figure 6 in E). Lentiviral transduction of miRNA targeting RKIP (PEBP1) partially reversed the cardiac dysfunction of Tg - RKIP mice by downregulating transgenic RKIP (PEBP1) via RNAi ( Figure 6In F, G). Therefore, the heart failure phenotype of Tg-RKIP mice is attributed to transgenic RKIP expression.

[0781] Example 7: Transgenic RKIP interacts with GRK2 and induces a gene expression signature associated with GRK2 inhibition

[0782] The GRK2 inhibitory activity of transgenic RKIP (human RKIP~PEBP1) is controlled in vivo. Phosphorylation of RKIP at serine-153 converts RKIP from Raf1 inhibition to GRK2 inhibition (Lorenz K et al., Nature 426, 574-579 (2003)). Abundant serine-153 phosphorylation of RKIP was demonstrated by immunoblotting in Tg-RKIP hearts ([[]] Figure 7 In A). Serine-153 phosphorylated RKIP is sufficient to bind and neutralize 90.4% (±3.6%, n = 4) of GRK2 protein in Tg-RKIP hearts Figure 7 In B). We determined the resensitization of isoproterenol-stimulated cAMP responses in neonatal cardiomyocytes mediated by GRK2 inhibition as a readout of GRK2 inhibition by RKIP and GRK2-K220R, respectively (Abd Alla, J et al., J. Biol. Chem. 291, 2583-2600 (2016); Kong KC et al., Biochemistry 47, 9279-9288 (2008)). Neonatal cardiomyocytes from Tg-RKIP and Tg-GRK2-K220R mice showed comparable signs of GRK2 inhibition, as demonstrated by significantly enhanced β-adrenergic receptor-mediated cAMP responses ( Figure 7 In C). Resensitized cAMP signaling was confirmed in vivo by a significant increase in the expression of the cAMP-inducible gene Ttc14 in Tg-RKIP and Tg-GRK2-K220R hearts Figure 7 In D). Genome-wide microarray gene expression profiles further revealed a GRK2 inhibition-associated gene expression signature in Tg-RKIP hearts Figure 7 In E, F). Notably, there was a 45% consistent regulation of significantly altered probe sets between Tg-RKIP and Tg-GRK2-K220R hearts Figure 7 In E, F).

[0783] Example 8: The cardioprotective GRK2 inhibitor paroxetine delays the heart failure phenotype in Tg-RKIP mice

[0784] It was analyzed whether the failure of RKIP to inhibit the activation of Srsf1 phosphorylation by GRK2 led to the heart failure phenotype in Tg-RKIP mice. Tg-RKIP mice were treated with the GRK2 inhibitor paroxetine, which was cardioprotective in an experimental model of myocardial infarction (Schumacher SM et al., Sci. Transl. Med. 7, 277ra31 (2015)). Paroxetine also inhibited SRSF1 phosphorylation in vitro (see Figure 1C). Treatment with paroxetine for 8 weeks decreased the cardiac content of activated Srsf1 phosphorylation and the splicing of Camk2d isoforms B / C in the Tg-RKIP heart ( Figure 8 in A-C). At the same time, it delayed the occurrence of myocardial necrosis and signs of heart failure ( Figure 8 in D-F). In summary, data from transgenic mice and different GRK2 inhibitors showed that cardioprotective GRK2 inhibition depends on the inhibition of GRK2 on the activation of Srsf1 phosphorylation.

[0785] Example 9: Development of small molecule compounds that inhibit GRK2-mediated SRSF1 phosphorylation.

[0786] Small molecule compounds that inhibit GRK2-mediated SRSF1 phosphorylation were developed ( Figure 9 ). 1-(1,3-Benzodioxol-5-yl)-4-(cyclopropanecarbonyl)-3-hydroxy-2-phenyl-2H-pyrrole-5-one (“Compound-1”) inhibited GRK2-mediated SRSF1 phosphorylation with an IC50 value of 0.45 μM. The IC50 value of 1-(1,3-Benzodioxol-5-yl)-4-(cyclopropanecarbonyl)-3-hydroxy-2-phenyl-2H-pyrrole-5-one (“Compound-1”) was less than 1 / 5 of the IC50 value of paroxetine ( Figure 9) Paroxetine is the only available small molecule GRK2 inhibitor with proven cardioprotective activity under in vivo experimental conditions (Schumacher SM et al., Sci. Transl. Med. 7, 277ra31 (2015)). Four compounds, 1-(1,3-benzodioxol-5-ylmethyl)-5-(4-fluorophenyl)-2-methyl-pyrrole-3-carboxamide, (3R)-N-(1,3-benzodioxol-5-ylmethyl)-5-(4-fluorophenyl)thiomorpholine-3-carboxamide, 4-(1,3-benzodioxol-5-yl)pyrimidine, and (4R)-N3-(1,3-benzodioxol-5-ylmethyl)-N4-[[3-(trifluoromethyl)phenyl]methyl]pyrrolidine-3,4-dicarboxamide (“Compounds 2 - 5”) inhibit SRSF1 phosphorylation with IC50 values ranging from 1.87 μM (4-(1,3-benzodioxol-5-yl)pyrimidine, “Compound - 4”) to 12.87 μM ((3R)-N-(1,3-benzodioxol-5-ylmethyl)-5-(4-fluorophenyl)thiomorpholine-3-carboxamide, “Compound - 3”) ( Figure 9 ) The untreated control compound ibuprofen ((±)-2-(4-isobutylphenyl)propanoic acid (“Compound - 6”, Sigma - Aldrich, St. Louis, USA) showed no inhibition at up to 1 mM ( Figure 9 ).

[0787] Example 10: Small molecule compounds inhibit GRK2 - mediated SRSF1 / Srsf1 phosphorylation in intact cardiomyocytes and human kidney cells.

[0788] 1-(1,3 - benzodioxol - 5 - yl)-4-(cyclopropanecarbonyl)-3 - hydroxy - 2 - phenyl - 2H - pyrrole - 5 - one (“Compound - 1”) and 4-(1,3 - benzodioxol - 5 - yl)pyrimidine (“Compound - 4”) also reduced SRSF1 / Srsf1 phosphorylation promoting heart failure in isolated Tg - SRSF1 cardiomyocytes ( Figure 10A). GRK2 inhibition promotes the survival of human renal cells (Fu X et al., J. Biol. Chem. 288, 7738 - 7755 (2013)). 4-(1,3-Benzodioxol-5-yl)pyrimidine (“Compound-4”), 1-(1,3-benzodioxol-5-ylmethyl)-5-(4-chlorophenyl)-2-methyl-pyrrole-3-carboxamide (“Compound-24”), and (4R)-N3-(1,3-benzodioxol-5-ylmethyl)-N4-[[3-(trifluoromethyl)phenyl]methyl]pyrrolidine-3,4-dicarboxamide (“Compound-5”) inhibit SRSF1 phosphorylation of damaged cells in human renal cells ( Figure 10 B).

[0789] Example 11: Cardioprotective effects of small molecule compounds in vivo.

[0790] Short-term treatment for 5 hours with small molecule compounds (1-((1,3-benzodioxol-5-yl)-4-(cyclopropanecarbonyl)-3-hydroxy-2-phenyl-2H-pyrrole-5-one (“Compound-1”), 4-(1,3-benzodioxol-5-yl)pyrimidine (“Compound-4”), 1-(1,3-benzodioxol-5-ylmethyl)-5-(4-fluorophenyl)-2-methyl-pyrrole-3-carboxamide (″Compound-2″), and (4R)-N3-(1,3-benzodioxol-5-ylmethyl)-N4-[[3-(trifluoromethyl)phenyl]methyl]pyrrolidine-3,4-dicarboxamide (″Compound-5″)) reduced GRK2-mediated Srsf1 phosphorylation that promotes heart failure under in vivo basal conditions ( Figure 11 A). Treatment with “Compound-1” or “Compound-5” also delayed the cardiac p-Srsf1 content induced by chronic pressure overload and improved the cardiac performance of B6 mice after chronic pressure overload imposed by AAC. Figure 11 B).

[0791] Example 12: Oral treatment with Compound-1 and Compound-4 alleviates heart failure and aging caused by cardiovascular diseases

[0792] We investigated whether the novel small molecule GRK2 inhibitor Compound-1 (1-(1,3-benzodioxol-5-yl)-4-(cyclopropanecarbonyl)-3-hydroxy-2-phenyl-2H-pyrrole-5-one)) and Compound-4 (4-(1,3-benzodioxol-5-yl)pyrimidine) could delay heart failure symptoms in a chronic pressure overload model of heart failure imposed by abdominal aortic constriction (AAC). Compound-1 (3 mg / kg / d) and Compound-4 (5 mg / kg / d) were orally administered in the AAC-induced heart failure model. Oral treatment with Compound-1 and Compound-4 was initiated in B6 mice with heart failure symptoms (left ventricular ejection fraction < 34%) induced by four weeks of AAC. Treatment with Compound-1 and Compound-4 for two months resolved AAC-induced cardiac dysfunction, as demonstrated by the significant improvement in left ventricular ejection fraction to 41.4 ± 4.8% and 38.2 ± 3.7% after treatment with Compound-1 and Compound-4, respectively, compared to an ejection fraction of 28.6% ± 3.5% in untreated AAC controls ( Figure 12 Panel A). In addition to the improved cardiac function, histological analysis showed a reduction in AAC-induced cardiac hypertrophy after oral treatment with Compound-1 ( Figure 12 Panel B). Meanwhile, due to the improvement in cardiac function, the aging caused by cardiovascular disease in mice was significantly delayed, i.e., the appearance of gray hair was significantly delayed after 8 weeks of treatment with Compound-1 compared to untreated B6 control animals ( Figure 12 Panels C, D).

[0793] Example 13: Materials and Methods

[0794] Generation of Transgenic Mice

[0795] The study used the following transgenic mouse lines, which were generated by our team: Tg-RKIP(PEBP1) mice in B6(C57BL / 6J) and FVB backgrounds, Tg-GRK2(ADRBK1) mice in B6 background, Tg-SRSF1 mice in B6 background, Tg-GRK2-K220R and Tg-GRKInh mice in B6 background. All transgenes were expressed under the control of the cardiac-specific α-MHC promoter (the MyHC plasmid was kindly provided by James Gulick, Gulick J et al., J. Biol. Chem. 266, 9180-9185 (1991)). Transgenic mice were generated according to standard procedures. Briefly, the DNA encoding the indicated protein / peptide was inserted into the α-MHC (MyHC) plasmid, the plasmid sequence was removed by Not I digestion, and the purified DNA (2 ng / μL) was injected into the fertilized oocytes of superovulated B6(C57BL / 6J) and FVB(FVB / N) mice. The injected embryos were transferred into pseudopregnant CD-1 mice by oviduct transfer according to standard procedures. Genomic DNA of the F0 generation was isolated from ear punch biopsies collected at 3-4 weeks of age, and the integration of the transgene was analyzed by PCR (Fu X et al., J. Biol. Chem. 288, 7738-7755 (2013)).

[0796] Experimental heart failure model induced by chronic pressure overload and transthoracic echocardiography

[0797] Chronic pressure overload imposed by abdominal aortic constriction (AAC) was used as an experimental model to induce signs of cardiac hypertrophy and heart failure. As described (AbdAlla S et al., Cardiovasc. Hematological Agents Med. Chem. 9, 190-206 (2011)), aortic constriction of the abdominal aorta was performed in 8-12-week-old transgenic mice or non-transgenic B6 controls anesthetized with tribromoethanol. The abdominal aorta was constricted above the suprarenal artery by tying a 7-0 silk suture ligature around a blunt 26-gauge needle. The same surgical procedure was performed on age-matched controls, except for aortic ligation (sham-operated mice).

[0798] Cardiac function parameters were determined by transthoracic echocardiography, which was performed on anesthetized mice using a Vivid 7 echocardiography device (GE Healthcare, Glattbrugg, Switzerland) and a 12 MHz linear array transducer. Left ventricular ejection fraction was calculated in the parasternal long-axis view in M-mode using the Teichholz formula. M-mode imaging was performed according to the recommendations of the American Heart Association applicable to mice (Sahn DJ et al., Circulation 58, 1072-1083 (1978)). Recordings were interpreted offline using EchoPac Pc 3.0 software (GE Healthcare, Glattbrugg, Switzerland).

[0799] Animal experiments were conducted in accordance with NIH guidelines and were reviewed and approved by the local animal care and use committee (University of Zurich).

[0800] Nano-LC-ESI-MS / MS

[0801] To enrich proteins that interact with human cardiac GRK2 protein, protein lysates were prepared from small myocardial biopsy samples of patients with signs of heart failure who underwent mitral valve replacement. Informed consent was obtained from all participants. The study was conducted in accordance with the principles of the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Ain Shams University. Patient characteristics of the study participants have been previously published (AbdAlia S et al., Cardiovasc. Hematological Agents Med. Chem. 9, 190 - 206 (2011)). Enrichment of GRK2 and co - enrichment of GRK2 - interacting proteins were performed similarly as described (Fu X et al., J. Biol. Chem. 288, 7738 - 7755 (2013)). Briefly, myocardial proteins were solubilized at 4 °C for 30 min in solubilization buffer (PBS solution of 1% sodium deoxycholate, 0.05% SDS, 0.05% Tween 20, pH 7.4, supplemented with protease inhibitors), insoluble materials were removed by centrifugation, the supernatant was diluted 1:5 in PBS supplemented with protease inhibitors, and subjected to affinity chromatography with anti - GRK2 antibody (6 mg of affinity - purified IgG was conjugated to 1 ml of Affigel 10, Bio - Rad Gmbh, München, Germany; polyclonal anti - GRK2 antibody was raised in rabbits against full - length recombinant GRK2 protein expressed in Sf9 insect cells). After incubation overnight at 4 °C, unbound proteins were removed by washing with PBS (20 column volumes), and bound proteins were eluted with 0.25 M NH4OH, 10% dioxane, pH 11. The pH of the eluate was immediately adjusted to pH 7.4, the eluted proteins were concentrated by acetone precipitation, dissolved in 8 M urea, and subjected to SDS - PAGE containing 8% urea under reducing conditions. After Coomassie blue staining, the enriched protein bands were excised and subjected to nano - LC - ESI - MS / MS. The SRSF1 protein was identified in gel slices encompassing the 30 - 40 kDa protein range. Protein identification was performed by nano - LC - ES - MS / MS (ProteomeFactory AG, Berlin). The MS system consisted of an Agilent 1100 nano - LC system (Agilent, Boeblingen, Germany), a PicoTip emitter (New Objective, Woburn, MA), and an Esquire 3000plus ion trap MS (Bruker, Bremen, Germany). The excised protein bands were subjected to in - gel digestion with trypsin (Promega, Mannheim, Germany) and applied to non - LC - MS / MS.After capturing and desalting peptides for 5 minutes using 1% acetonitrile and 0.5% formic acid solution on an enrichment column (Zorbax SB C18, 0.3x5mm, Agilent Boeblingen, Germany), the peptides were separated on a Zorbax 300SB C18, 75μm x 150mm column (Agilent Boeblingen, Germany) using an acetonitrile, 0.1% formic acid gradient (from 5% to 40% acetonitrile in 40 minutes). MS spectra were automatically acquired by an Esquire 3000plus according to the instrument settings for nan-LC-MS / MS analysis by the manufacturer. Proteins were identified using MS / MS ion searches of the Mascot search engine (MatrixScience, London, England) and the nr protein database (National Center for Biotechnology Information, Bethesda, MD). According to the common charge state distribution of the instrument and method, the ion charge in the search parameters for ion form ESI-MS / MS data acquisition was set to "1+, 2+ or 3+" (Fu X et al., J. Biol. Chem. 288, 7738-7755 (2013)).

[0802] To identify the residues in SRSF1 phosphorylated by GRK2, nano-LC-ES-MS / MS analysis was performed on purified recombinant SRSF1 after in vitro phosphorylation assays. The procedure was as described above, but the MS system consisted of an Agilent 1100 nanoLC system (Agilent, Waldbronn, Germany), a Nanomate 100 electrospray system (Advion, Ithaca, USA), and a Finnigan LTQ-FT mass spectrometer (ThermoFisher, Bremen, Germany). The settings of the Mascot search engine were adjusted to identify variable modifications, namely deamidation (NQ), oxidation (M), phosphorylation (ST), and phosphorylation (Y).

[0803] Expression and purification of recombinant proteins

[0804] Recombinant human GRK2 protein and GRK2-S670A protein were expressed and purified in Spodoptera frugiperda (Sf9) cells by a baculovirus expression system. The cDNA encoding hexahistidine-tagged human GRK2 (ADRBK1) GRK2-S670A was subcloned into the pFastBac1 expression plasmid (Invitrogen TM, in Thermo Fisher Scientific, Waltham, MA, USA), recombinant baculovirus was generated using the Bac-To-Bac baculovirus expression system (Thermo Fisher Scientific, Waltham, MA, USA). Sf9 cells were infected with the recombinant baculovirus at an MOI of 2 - 3. At 48 hours post-infection, the cells were harvested by centrifugation, lysed with lysis buffer (300 mM NaCl, 50 mM HEPES, pH 7.5, supplemented with 1% NP40, 1 mM PMSF, and protease inhibitor mixture) and applied to Ni-NTA chromatography. After incubation overnight at 4 °C, unbound proteins were removed by washing with lysis buffer (20x column volume), followed by washing with 1x column volume of 30 mM imidazole-containing lysis buffer. The bound GRK2 was eluted with 300 mM imidazole / lysis buffer, desalted by PD10 column chromatography, supplemented with 20% glycerol and stored at -80 °C for further use.

[0805] Under the control of the T7 promoter, the cDNAs encoding human His6-SRSF1 and the carboxyl-terminal domain of the photosensing factor-His6 and GRK2 (SEQ ID NO: 10, His-6 tagged) were subcloned into the PET-3d expression plasmid ( In EMD Millipore, Merck KGaA, Darmstadt, Germany), this allows for protein expression induction by IPTG via T7 RNA polymerase in BL21(DE3)pLysS bacteria. Bacteria expressing the protein are collected by centrifugation, frozen in liquid nitrogen and thawed on ice in lysis buffer (8 M urea, 300 mM NaCl, 50 mM HEPES, 10 mM imidazole, pH 7.5) freshly supplemented with 2-mercaptoethanol (0.7 ml / L) before use (10 ml of lysis buffer is used for the bacterial pellet from 200 ml of culture medium). The bacterial lysate is incubated for 1 hour at room temperature, sonicated and centrifuged at 4000×g for 15 minutes at 4 °C. The supernatant is applied to a Ni-NTA column matrix pre-washed with 20 ml of lysis buffer. After incubation overnight at 4 °C, the flow-through is discarded and the Ni-NTA affinity matrix is subjected to 3 different washing steps with wash buffer-1 (4 M urea, 300 mM NaCl, 50 mM HEPES, 20 mM imidazole, pH 7.5, freshly supplemented with 0.7 ml / l 2-mercaptoethanol), wash buffer-2 (2 M urea, 300 mM NaCl, 50 mM Hepes, 20 mM imidazole, pH 7.5, freshly supplemented with 0.7 ml / L 2-mercaptoethanol) and wash buffer-3 (300 mM NaCl, 50 mM HEPES, 20 mM imidazole, pH 7.5, freshly supplemented with 0.7 ml / L 2-mercaptoethanol) (incubated for 30 minutes at 4 °C, where 20 ml of each wash buffer is used for 1 ml of 5% Ni-NTA matrix). Finally, the protein is eluted with elution buffer (300 mM NaCl, 50 mM HEPES, 500 mM imidazole, pH 7.5, freshly supplemented with 0.7 ml / L 2-mercaptoethanol). The buffer of the eluted protein is changed to 150 mM NaCl, 50 mM HEPES (pH 7.5) by PD10 column chromatography.

[0806] In vitro phosphorylation assay

[0807] In vitro phosphorylation assays of GRK2 and GRK2-S670A were performed using recombinant proteins. GRK2 and GRK2-S670A were expressed and purified in Spodoptera frugiperda (Sf9) cells by the baculovirus expression system. In the presence of the indicated increasing concentrations of GRK2 inhibitors, substrate phosphorylation was carried out in reaction buffers (50 μL of 20 mM Tris, 2 mM EDTA, 5 mM MgCl2 pH 7.5; or 20 mM Hepes, 2 mM MgCl2, 0.025% DDM (n-dodecyl-β-D-maltoside), pH 7.4) supplemented with 50 μM or 5 μM of ATP and [γ-32P]-ATP (1×10 6 DPM, specific activity 3000 Ci / mmol) and 300 - 500 nM substrate (SRSF1, light-sensing factor), respectively. The reaction was initiated by adding GRK2-S670A or GRK2 (50 nM or 130 nM). After incubation at 30 °C for 30 minutes, the reaction was terminated by adding 5× SDS-Laemmli buffer. Proteins were separated by SDS-PAGE and autoradiography was performed. For the analysis of small molecule inhibitors, phosphorylation assays were carried out in reaction buffers (20 mM Tris, 2 mM EDTA, 5 mM MgCl2, 0.05% BSA, pH 7.5; or 20 mM Hepes, 2 mM MgCl2, 0.025% DDM (n-dodecyl-β-D-maltoside), pH 7.4) supplemented with 5 μM of ATP, [γ-32P]-ATP (1×106 DPM, specific activity 3000 Ci / mmol) and 300 - 500 nM substrate (SRSF1, light-sensing factor) (= reaction mixture). The reaction mixture was added to GRK2-S670A or GRK2 (50 nM or 60 nM, in reaction buffers with or without increasing concentrations of small molecule compounds) to give a final reaction volume of 50 μL. After incubation at 30 °C for 30 - 60 minutes, phosphorylation was terminated by adding 5 volumes of ice-cold reaction buffer. The reaction mixture was immediately applied to glass fiber filters (GF / C, Whatman, GE Healthcare LifeSciences, Glattbrugg, Switzerland). After three washing steps with 5 ml of reaction buffer, the radioactivity bound to the filters was measured in a β-counter.

[0808] Immunoblot detection of proteins

[0809] For immunoblot detection of proteins, heart tissues were pulverized in liquid nitrogen and extracted with RIPA buffer supplemented with protease / phosphatase inhibitor mixture as previously described (Fu X et al., J. Biol. Chem. 288, 7738 - 7755 (2013)) with minor modifications. Particulate materials were removed by centrifugation at 20,000 x g for 15 minutes at 4°C. The dissolved protein precipitate was defatted with acetone / methanol (12:2; final concentration 83%) for 90 minutes at 4°C. The precipitate was collected by centrifugation (5000 x g, 10 minutes, 4°C) and then subjected to three washing steps with 0.2 ml cold acetone. The pellet was dissolved in SDS-sample buffer containing 2% SDS, 0.1 M DTT and 6 M urea for 90 minutes at room temperature. After addition of iodoacetamide (10 mM), the samples were stored at -70°C for further use. After separation of proteins by SDS-PAGE (10% gel for proteins < 100 kDa; 7.5% gel for proteins > 100 kDa), proteins were detected with affinity-purified antibodies or F(ab)2 fragments of each antibody, and then electrophoretically transferred to PVDF membranes by semi-dry blotting ( SD Semi-Dry Transfer Cell, Bio-Rad GmbH, München, Germany). For electrophoretic transfer of Fasn protein, a tank transfer cell (Mini cell, Bio-Rad GmbH, München, Germany) was used. Bound antibodies were visualized with F(ab)2 fragments of enzyme-conjugated secondary antibodies (Dianova GmbH, Hamburg, Germany) or, where appropriate, by enzyme-conjugated protein A (Calbiochem, EMD Millipore, Merck KGaA, Darmstadt, Germany), followed by enhanced chemiluminescence detection (ECL Prime, Amersham, GE Healthcare Life Sciences, Glattbrugg, Switzerland).

[0810] Antibody

[0811] The following antibodies were used for immunoblotting of proteins: anti-Gnb antibody was generated in rabbits against purified Gnb (AbdAlla, J et al., J. Biol. Chem. 291, 2583-2600 (2016)); anti-GRK2 antibody was generated in rabbits against recombinant GRK2 expressed in Sf9 cells (Abd Alla, J et al., J. Biol. Chem. 291, 2583-2600(2016)); anti-pRKIP antibody was generated in rabbits against a short amino acid sequence from human RKIP containing phosphorylated serine-153 (sc-32623, Santa Cruz Biotechnology Inc., Dallas, TX, USA); anti-SRSF1 antibody is a mouse monoclonal antibody epitope mapped near the N-terminus of the SF2 / ASF protein (sc-33652, Santa Cruz Biotechnology Inc. USA); SR (1H4) antibody is a mouse monoclonal antibody generated against full-length SR from Xenopus laevis (sc-13509 from Santa Cruz Biotechnology Inc. USA); anti-CAMK2D polyclonal antibody was generated in rabbits against full-length human protein (purified polyclonal antibody, catalog number H00000817-D01P; Abnova, Taipei, Taiwan, China), and a monoclonal anti-CAMK2D antibody generated in mice against a partial recombinant protein of CAMK2D (amino acids 301-410) with a GST tag (WH0000817M2; Sigma-Aldrich, St. Louis, MO, USA) was used to detect CAMK2D / Camk2d isoform B / C.

[0812] Compound synthesis

[0813] The compounds were synthesized by EMC microcollections GmbH, Tübingen, Germany and ChiroBlock GmbH, Wolfen, Germany. The compounds were synthesized on a small scale by solid-phase chemical synthesis methods, which were adapted from established protocols (for "Compound-1": Poncet J et al., J. Chem. Soc. Perkin Trans I., 611 - 616 (1990); for "Compound-2", "Compound-22", "Compound-23" and "Compound-24": Trautwein AW et al., Bioorg. Med. Chem. Lett. 8, 2381 - 2384 (1998); for Compound-3: Sakai K et al., Chem. Pharm. Bull. 29(6) 1554 - 1560 (1981); for "Compound-4": Coombs TC et al., Bioorg. Med. Chem. Lett. 23, 3654 - 3661 (2013); and for "Compound-5": Baber JC et al., Bioorg. Med. Chem. 20, 3565 - 3574 (2012)). Additionally, Compound-1 and Compound-4 were synthesized on a larger scale as described in detail below.

[0814] Synthesis of Compound-1

[0815] The synthesis of Compound-1 (1-(1,3-benzodioxol-5-yl)-4-(cyclopropanecarbonyl)-3-hydroxy-2-phenyl-2H-pyrrol-5-one) was carried out in a 6-step chemical reaction process (ChiroBlock GmbH, Wolfen, Germany). Step-1 involves the synthesis of methyl 2-(1,3-benzodioxol-5-ylamino)-2-phenylacetate. A mixture of methyl 2-oxo-2-phenylacetate (96 g, 584 mmol, 4.0 equiv), 1,3-benzodioxol-5-amine (20 g, 146 mmol, 1.0 equiv) and Na2SO4 / cyclohexane (800 ml) was refluxed under N2 for 21 h. 5% Pd / C (7.8 g) was added and the resulting suspension was hydrogenated at 20 bar and 20 °C for 48 h. The resulting heterogeneous mixture was diluted with EtOAc (ca. 800 ml) and filtered through diatomaceous earth. The filtrate was concentrated in vacuo (40 °C, 100 mbar) to give a brown oil (135 g), which was purified by flash chromatography (silica gel, ethyl acetate - petroleum ether 12∶88 to 30∶70) to give the target 3 as an off-white solid (18.46 g; purity 95%, yield 44%).

[0816] Step - 2 is the synthesis of S-tert-butyl thioacetate. A solution of pyridine (87.0 g, 1.1 mol, 1.1 equiv) in chloroform (800 ml) was cooled in an ice bath and treated with acetyl chloride (86.4 g, 1.1 mol, 1.1 equiv) while maintaining the reaction temperature below 11 °C. 2-Methylpropane-2-thiol (90.2 g, 1.0 mol, 1.0 equiv) was added dropwise to the resulting orange suspension over 40 minutes. The mixture was stirred for 48 hours and then quenched with water (500 ml). The phases were separated and the aqueous phase was extracted with chloroform (400 ml). The combined organic extracts were washed with 400 ml of water, 10% H2SO4, saturated NaHCO3 and water, and then dried over Na2SO4. The resulting chloroform solution was fractionated to give the target S-tert-butyl thioacetate as a clear liquid (55.8 g, 95% purity, 45% yield).

[0817] In Step - 3, the synthesis of S-(2-pyridyl) cyclopropanecarbothioate was carried out. At 20 °C, cyclopropanecarbonyl chloride (23.5 g, 225 mmol, 1.0 equiv) was added dropwise to a solution of pyridine-2-thiol (25.0 g, 225 mmol, 1.0 equiv) in THF (250 ml). The mixture was stirred for 10 minutes, filtered, and the filter cake was washed with 1:4 Et2O / petroleum ether (250 ml). The solid thus obtained was dissolved in water (250 ml) and treated with NaHCO3 (19 g, 225 mmol, 1.0 equiv). The aqueous solution was extracted with 2 × 250 ml of EtOAc. The combined organic fractions were dried over Na2SO4 and concentrated in vacuo to give S-(2-pyridyl) cyclopropanecarbothioate as a yellow oil (37 g, 95% purity; 92% yield).

[0818] Step - 4 is the synthesis of S - tert - butyl 3 - cyclopropyl - 3 - oxo - thiopropionate. Charge HMDS (83.3 g, 516 mmol, 2.5 eq) and freshly distilled THF (800 ml) into a 2 L three - necked round - bottom flask. Cool the resulting mixture in an acetone / dry ice bath and add dropwise 1.6 M nBuLi / hexane (323 ml, 516 mmol, 2.5 eq) while maintaining the temperature below - 50 °C. Then, successively treat the resulting mixture with S - (2 - pyridyl) cyclopropanethioate (37.0 g, 206 mmol, 1.0 eq) and S - tert - butyl thioacetate (23.4 g, 214 mmol, 1.04 eq). Stir the resulting solution at - 30 °C for 1 h and quench the reaction with 1 N H2SO4 (800 ml) (under TLC process control). Extract the resulting suspension with EtOAc (3 * 900 ml), combine the organic portions, wash with brine (2 L), dry over Na2SO4, and concentrate in vacuo. Purify the crude product by flash chromatography (silica gel, ethyl acetate - petroleum ether 25∶75) to obtain the target S - tert - butyl 3 - cyclopropyl - 3 - oxo - thiopropionate as a brown oil (29.5 g, purity 83%, yield: 59%).

[0819] In step - 5, the synthesis of methyl 2 - [1,3 - benzodioxol - 5 - yl - (3 - cyclopropyl - 3 - oxo - propionyl) amino] - 2 - phenyl - acetate is carried out. Charge methyl 2 - (1,3 - benzodioxol - 5 - yl amino) - 2 - phenyl - acetate (18.5 g, 61 mmol, 1.0 eq), S - tert - butyl 3 - cyclopropyl - 3 - oxo - thiopropionate (15.9 g, 66 mmol, 1.073 eq), CF3COOAg (814.6 g, 66 mmol, 1.073 eq) and distilled THF (400 ml) into a 1 L round - bottom flask. Stir the resulting mixture at 20 °C for 36 h (control the process by TLC). Concentrate the dark brown reaction mixture in vacuo and purify by flash chromatography (silica gel, ethyl acetate - petroleum ether 25:75 to 50:50) to obtain the target methyl 2 - [1,3 - benzodioxol - 5 - yl - (3 - cyclopropyl - 3 - oxo - propionyl) amino] - 2 - phenyl - acetate as a brown oil (21.0 g, purity: 90%, yield: 78%).

[0820] The final step - 6 gives the final target 1-(1,3-benzodioxol-5-yl)-4-(cyclopropanecarbonyl)-3-hydroxy-2-phenyl-2H-pyrrol-5-one (Compound - 1). Charge a 500 ml round-bottom flask with methyl 2-[1,3-benzodioxol-5-yl-(3-cyclopropyl-3-oxopropanoyl)amino]-2-phenylacetate (20.0 g; 45.5 mmol, 1.0 equivalent), CsF (6.9 g, 45.5 mmol, 1.0 equivalent) and DMF (140 ml), and stir the resulting mixture at 60 °C for 20 h (monitor the process by TLC). Concentrate the dark brown reaction mixture in vacuo, and treat the residue with 1N H2SO4 (400 ml). Extract the resulting mixture with EtOAc (500 ml), wash the organic phase with brine (2 * 300 ml), dry over Na2SO4, and concentrate in vacuo to obtain crude 1-(1,3-benzodioxol-5-yl)-4-(cyclopropanecarbonyl)-3-hydroxy-2-phenyl-2H-pyrrol-5-one as a brown solid (19 g). Wash the above solid with EtOAc on a filter until it becomes colorless to give the target compound - 1 (1-(1,3-benzodioxol-5-yl)-4-(cyclopropanecarbonyl)-3-hydroxy-2-phenyl-2H-pyrrol-5-one) as an off-white solid (5.0 g, purity: 98%, yield: 30%).

[0821] Synthesis of Compound - 4

[0822] Compound - 4 (4-(1,3-benzodioxol-5-yl)pyrimidine) was synthesized by the following procedure (ChiroBlock GmbH, Wolfen, Germany). Charge a 250 ml round-bottom flask with 1-(1,3-benzodioxol-5-yl)ethanone (10.0 g, 60.9 mmol, 1.0 equivalent), (EtO)3CH (27 g, 183 mmol, 3.0 equivalents), ZnCl2 (0.83 g, 6.1 mmol, 0.1 equivalent), NH4CH3COO (0.4 g, 122 mmol, 2.0 equivalents) and toluene (120 ml), and stir the resulting mixture at reflux for 48 h, then at 20 °C for 48 h (monitor the process by TLC). Quench the reaction mixture with saturated NaHCO3 (400 ml) and extract with chloroform (400 ml). Dry the organic phase over Na2SO4 and concentrate in vacuo, and purify the resulting crude product by flash chromatography (silica gel, MeOH-CHCl3 (0:100 to 5:95)) to give the target compound - 4 (4-(1,3-benzodioxol-5-yl)pyrimidine) as an off-white solid (3.0 g, purity 97%; yield 25%).

[0823] Isolation of neonatal cardiomyocytes and cell experiments

[0824] Neonatal mouse and rat cardiomyocytes were isolated as described (Fu X et al., J. Biol. Chem. 288, 7738 - 7755 (2013); Lorenz K et al., Nature 426, 574 - 579 (2003)). Briefly, hearts were dissected from 2 - 3 - day - old mice, the atria and aorta were removed, and the hearts were transferred to sterile buffer A (137 mM NaCl, 5.36 mM KCl, 0.81 mM MgSO4, 5.55 mM dextrose, 0.44 mM KH2PO4, 0.34 mM Na2HPO4, 20 mM HEPES, 100 U / ml penicillin, 100 μg / ml streptomycin, pH 7.4). The hearts were cut into small pieces and incubated (on a magnetic stirrer) in buffer A supplemented with 150 mg / L trypsin (Becton & Dickinson, Franklin Lakes, NJ, USA) for 15 minutes. The supernatant was discarded and the process was repeated once. Thereafter, the heart tissue was digested by continuous incubation in buffer A supplemented with trypsin for 5 minutes at room temperature until the heart tissue was completely digested. The cells in the supernatant were collected by centrifugation (10 minutes, 700×g, room temperature), the cells were suspended in MEM containing 5% FCS, and filtered through a nylon mesh (40 μm). Fibroblasts were removed by pre - plating for 1 hour at 37°C. Non - adherent cardiomyocytes were collected and cultured in MEM (supplemented with 5% FCS and 25 mg / l BrdU). Similar to that described (Abd Alla, J et al., J. Biol. Chem. 291, 2583 - 2600 (2016)), after stimulation of β - adrenergic receptors with 100 nM isoproterenol, the cellular cAMP levels of the isolated cardiomyocytes were measured using a cAMP enzyme immunoassay kit (CA200, Sigma Aldrich, St. Louis, MO, USA). Human embryonic kidney cells were cultured as described (Fu X et al., J. Biol. Chem. 288, 7738 - 7755 (2013)).

[0825] Histological techniques

[0826] For histological analysis, paraffin sections of mouse heart samples were used. Immunohistological detection of RKIP was performed using affinity-purified polyclonal antibodies raised in rabbits against recombinant RKIP. After antigen retrieval, the sections were incubated with the primary antibody (1:200 dilution) in blocking buffer [PBS, pH 7.4, supplemented with 5% (w / v) bovine serum albumin, 0.05% Tween-20] at 37 °C for 1 h. Unbound antibodies were removed by three washing steps with PBS supplemented with 0.05% Tween-20. After incubation with a peroxidase-conjugated secondary antibody (Dianova, Dianova GmbH, Hamburg, Germany; 1:500 dilution) followed by a washing step, an enzyme substrate reaction (DAB Enhanced Liquid Substrate System, Sigma-Aldrich, St. Louis, MO, USA) was performed. Immunohistological sections were imaged using a Leica DMI6000 microscope equipped with a DFC420 camera. Myocardial necrosis was determined by von Kossa staining (Calcium Staining Kit, Modified Von Kossa No. KT028, Diagnostic Biosystems, Pleasanton, CA, USA).

[0827] In vivo lentivirus-mediated downregulation of Srsf1 by RNAi

[0828] For in vivo downregulation of Srsf1 expression, Tg-GRK2 mice were transduced by i.p. administration of replication-defective lentivirus (in PBS, 1x10 8 copies / mouse) that downregulated Srsf1 by RNAi through polymerase II (Pol II)-dependent expression of a miRNA precursor targeting Srsf1 RNA. By inserting the specified double-stranded oligonucleotides (miSrsf1 upstream strand (top strand) 5'-TGC TGT TTA AGT CCT GCC AGC TTC CAG TTT TGG CCA CTG ACT GACTGG AAG CTC AGG ACT TAA A-3` (SEQ ID NO: 1); and miSrsf1 downstream strand (bottom strand) 5′-CCT GTT TAA GTC CTG AGC TTC CAG TCA GTC AGT GGC CAA AAC TGG AAG CTG GCA GGACTT AAA C-3′ (SEQ ID NO: 2)) into pLenti6 / V5-Dest vector (Invitrogen Invitrogen TM, in Thermo Fisher Scientific, Waltham, MA, USA) to generate a lentiviral expression plasmid, the double-stranded oligonucleotide encoding an engineered miRNA precursor sequence targeting the murine Srsf1 gene by RNAi interference. Pseudotyped lentiviruses were generated by co-transfecting 293FT cells with a mixture of the lentiviral plasmid and the packaging plasmids pLP1, pLP2, and pLP / VSVG (Invitrogen TM , Thermo Fisher Scientific, Waltham, MA, USA). Downregulation of Srsf1 protein expression was confirmed by immunoblotting after transduction of mice with miSrsf1-lentivirus.

[0829] Genome-wide microarray gene expression analysis

[0830] Basically as described (Abd Alla, J et al., J. Biol. Chem. 291, 2583 - 2600 (2016)), whole-genome microarray gene expression analysis of cardiac tissues from Tg-RKIP and Tg-GRK2-K220R mice was performed using Affymetrix GeneChip Mouse Genome MG430 2.0 Arrays. Using GeneSpring GX software (Agilent, Santa Clara, CA, USA), GO analysis of microarray data was performed on data processed with GCOS and / or RMA. Unpaired two-tailed Student's t-test was used to compare data between the two groups. Probe sets with significant upregulation (fold change ≥ 2 relative to their respective control groups, P ≤ 0.01) were used for GO classification. As described (Abd Alla, J et al., J. Biol. Chem. 291, 2583 - 2600 (2016); Fu X et al., J. Biol. Chem. 288, 7738 - 7755 (2013)), real-time qRT-PCR for Camk2d isoform splicing was performed using a LightCycler 480 (Roche Molecular Diagnostics, Pleasanton, CA, USA). The following primers were used: Camk2d-forward 5`-ACG AGA AAT TTT TCA GCA GCC-3` (SEQ ID NO: 3); Camk2d-reverse-A 5`-ACAGT AGT TTG GGG CTC CAG C-3` (SEQ ID NO: 4); Camk2d-reverse-B 5`-T CAT CTG AAC ACT CGA ACT GG-3` (SEQ ID NO: 5); Camk2d-reverse-C 5`-CTC AGT TGA CTC CTT TAC CCC-3` (SEQ ID NO: 6).

[0831] Statistical analysis

[0832] Unless otherwise specified, results are expressed as mean ± s.d. P values were calculated using Student's t-test. Analysis of variance was performed for comparisons between more than two groups, followed by post hoc tests, and statistical significance was set at P value < 0.05 unless otherwise stated.

Claims

1. Use of a compound and its pharmaceutically acceptable salts in the preparation of a medicament for cardioprotective and / or renoprotective treatment, wherein the compound is selected from: 1-(1,3-Benzodioxol-5-yl)-4-(cyclopropanecarbonyl)-3-hydroxy-2-phenyl-2H-pyrrol-5-one; 1-(1,3-Benzodioxol-5-ylmethyl)-5-(4-fluorophenyl)-2-methyl-pyrrole-3-carboxamide; (3R)-N-(1,3-Benzodioxol-5-ylmethyl)-5-(4-fluorophenyl)thiomorpholine-3-carboxamide; 4-(1,3-Benzodioxol-5-yl)pyrimidine; (4R)-N3-(1,3-Benzodioxol-5-ylmethyl)-N4-[[3-(trifluoromethyl)phenyl]methyl]pyrrolidine-3,4-dicarboxamide; 1-(1,3-Benzodioxol-5-ylmethyl)-2-methyl-5-phenyl-pyrrole-3-carboxamide; 1-(1,3-Benzodioxol-5-ylmethyl)-2-methyl-5-(p-tolyl)pyrrole-3-carboxamide; and 1-(1,3-Benzodioxol-5-ylmethyl)-5-(4-chlorophenyl)-2-methyl-pyrrole-3-carboxamide.

2. A compound according to the following formula: 1-(1,3-Benzodioxol-5-yl)-4-(cyclopropanecarbonyl)-3-hydroxy-2-phenyl-2H-pyrrol-5-one.

3. The use according to claim 1, wherein the compound inhibits the phosphorylation of serine / arginine-rich splicing factor 1 and / or photoreceptor factor caused by G protein-coupled receptor kinase 2.

4. Use of a pharmaceutical composition in the preparation of a medicament for cardioprotective and / or renoprotective treatment, the pharmaceutical composition comprising as an active substance a compound as defined in claim 1 or a compound according to claim 2, optionally in combination with excipients and / or carriers.

5. The use according to claim 1 or claim 4, wherein the cardioprotective treatment is selected from (i) reducing the risk of cardiovascular mortality and / or morbidity in the context of essential hypertension and / or chronic hypertension; (ii) reducing the risk of aging caused by cardiovascular diseases; (iii) reducing the risk of cardiovascular mortality and / or morbidity in the context of left ventricular dysfunction and signs of heart failure after recent myocardial infarction; (iv) reducing the risk of cardiovascular mortality and / or morbidity in the context of chronic heart failure and left ventricular dysfunction; (v) reducing the risk of cardiovascular mortality and / or morbidity in the context of dilated cardiomyopathy; (vi) reducing the risk of cardiovascular mortality and / or morbidity in the context of left ventricular dysfunction; (vii) reducing the risk of cardiovascular mortality and / or morbidity in the context of cardiomyocyte necrosis; (viii) reducing the risk of cardiovascular mortality and / or morbidity in the context of myocardial fibrosis; (ix) Preventing, optionally primary prevention, of cardiomyocyte necrosis and / or dilated cardiomyopathy, optionally in the context of ischemic heart disease and conditions with an increased risk of ischemic heart injury, said increased risk optionally due to cardiovascular risk factors selected from hypertension, atherosclerosis, chronic and acute stress, depression, diabetes, chronic heart failure, angina, atrial fibrillation, chronic renal failure, and aging; (x) Preventing, optionally secondary prevention, of cardiomyocyte necrosis and / or dilated cardiomyopathy in patients with a previous event selected from acute cardiovascular disease, myocardial infarction, ischemic heart disease, angina, atrial fibrillation, decompensated and chronic heart failure, and cerebrovascular stroke; and (xi) Treating an acute disease state of a cardiovascular disease, said cardiovascular disease optionally selected from myocardial infarction, angina, ischemic heart disease, cerebrovascular disease, and decompensated heart failure.

6. The use according to claim 1 or claim 4, wherein the renoprotective treatment is the treatment of kidney disease.

7. The use according to claim 6, wherein the renoprotective treatment is the treatment of kidney disease caused by hypertension, renal artery stenosis, ischemia, diabetes, and / or toxins.

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