CANNABIDIOL DERIVATIVES AS INHIBITORS OF THE HIF PROLYL HYDROXYLASES ACTIVITY

Abstract

Cannabidiol quinol derivatives of Formula (I) and compositions comprising the same for use in the treatment of conditions that benefit from the inhibition of the HIF prolyl hydroxylases (PHDs) activity are described. Said cannabidiol quinol derivatives of Formula (I), and compositions comprising the same, show thus capacity to inhibit PHD activities and, as a result, stabilize the HIF-1α and HIF-2α levels, activate the HIF pathway in different cell types, induce angiogenesis in human endothelial vascular cell, regulate HIF-dependent gene expression in different cell types and induce collagen contraction. Said cannabidiol quinol derivatives of Formula (I) are useful in the treatment of conditions that benefit from the inhibition of the HIF prolyl hydroxylases (PHDs) activity such as stroke, traumatic injuries anemia, myocardial ischaemia-reperfusion injury, acute lung injury, infectious diseases, diabetic and chronic wounds, organ transplantation, acute kidney injury or arterial diseases.

Claims

1. A method for treating or preventing a neurological and cognitive disease or disorder, the method comprising administering a composition comprising an inhibitor of ACSS2 to a subject in need thereof.

2. The method of claim 1, wherein neurological and cognitive disease or disorder is selected from the group consisting of post-traumatic stress disorder (PTSD), bipolar disorder, depression, Tourette's Syndrome, schizophrenia, obsessive-compulsive disorder, anxiety disorder, panic disorders, and phobias.

3. The method of claim 1, wherein the neurological and cognitive disease or disorder is PTSD.

4. The method of claim 1, wherein the inhibitor of ACSS2 is at least one of the group consisting of a chemical compound, a protein, a peptide, a peptidomemetic, an antibody, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, an antisense nucleic acid molecule.

5. The method of claim 1, wherein the inhibitor of ACSS2 is a small molecule.

6. The method of claim 5, wherein the small molecule is a compound according to one of Formula (1) to Formula (4): ##STR00032## wherein, X.sub.11 is selected from the group consisting of C(R.sub.14)(R.sub.15), O, S and NR.sub.15; each occurrence of X.sub.12 is selected from the group consisting of C(R.sub.14)(R.sub.15), O, S and NR.sub.15; R.sub.11 is selected from the group consisting of hydrogen, —OR.sub.15, alkyl, cycloalkyl, —C.sub.4-C.sub.6 heterocyclyl, aryl, and —C.sub.4-C.sub.6 heteroaryl, wherein R.sub.11 is optionally substituted; R.sub.12 and R.sub.13 are each independently selected from the group consisting of hydrogen, alkyl, aryl, and —C.sub.4-C.sub.6 heteroaryl, wherein R.sub.12 and R.sub.13 are optionally substituted; each occurrence of R.sub.14 and R.sub.15 are independently selected from the group consisting of hydrogen, halogen, —OH, and C.sub.1-C.sub.6 alkyl; and n is an integer from 0-8; ##STR00033## wherein, R.sub.21 is selected from the group consisting of —C(R.sub.23).sub.m, cycloalkyl, heterocycyl, cycloalkyl-one, and heterocycyl-one; R.sub.22 is selected from the group consisting of alkyl, aryl, heteroaryl, —C.sub.1-C.sub.3 alkyl-(C.sub.3-C.sub.6 aryl), and —C.sub.1-C.sub.3 alkyl-(C.sub.3-C.sub.6 heteroaryl); each occurrence of R.sub.23 is independently selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, heterocycyl, —OH, and —CN; and m is an integer from 1 to 3; ##STR00034## wherein R.sub.31 is selected from the group consisting of —C(R.sub.35).sub.p, cycloalkyl, heterocycyl, cycloalkyl-one, heterocycyl-one; R.sub.32 is selected from the group consisting of alkyl, aryl, heteroaryl, —C.sub.1-C.sub.3 alkyl-(C.sub.3-C.sub.6 aryl), and —C.sub.1-C.sub.3 alkyl-(C.sub.3-C.sub.6 heteroaryl); R.sub.33 and R.sub.34 are each independently selected from the group consisting of hydrogen, halogen, alkyl, aryl, heteroaryl; each occurrence of R.sub.35 is independently selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, heterocycyl, —OH, and —CN; and p is an integer from 1 to 3; ##STR00035## wherein, X.sub.41 is selected from the group consisting of O and S; R.sub.41 is selected from the group consisting of alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and combinations thereof, wherein R.sub.41 may be optionally substituted; and R.sub.42 and R.sub.43 are each independently selected from the group consisting of phenyl, thiophenyl and furanyl.

7. The method of claim 6, wherein the compound of Formula (1) is selected from the group consisting of ##STR00036## ##STR00037## ##STR00038##

8. The method of claim 6, wherein the compound of Formula (2) is selected from the group consisting of ##STR00039##

9. The method of claim 6, wherein the compound of Formula (3) is selected from the group consisting of ##STR00040##

10. The method of claim 6, wherein the compound of Formula (4) is selected from the group consisting of ##STR00041## ##STR00042## ##STR00043##

11. A method for treating or preventing an addiction or addiction-related disease or disorder the method comprising administering a composition comprising an inhibitor of ACSS2 to a subject in need thereof.

12. The method of claim 11, wherein addiction is alcoholism.

13. The method of claim 11, wherein the addiction-related disease or disorder is acute and/or chronic alcohol induced memory deficit.

14. The method of claim 11, wherein the inhibitor of ACSS2 is at least one of the group consisting of a chemical compound, a protein, a peptide, a peptidomemetic, an antibody, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, an antisense nucleic acid molecule.

15. The method of claim 14, wherein the small molecule is a compound according to one of Formula (1) to Formula (4): ##STR00044## wherein, X.sub.11 is selected from the group consisting of C(R.sub.14)(R.sub.15), O, S and NR.sub.15; each occurrence of X.sub.12 is selected from the group consisting of C(R.sub.14)(R.sub.15), O, S and NR.sub.15; R.sub.11 is selected from the group consisting of hydrogen, —OR.sub.15, alkyl, cycloalkyl, —C.sub.4-C.sub.6 heterocyclyl, aryl, and —C.sub.4-C.sub.6 heteroaryl, wherein R.sub.11 is optionally substituted; R.sub.12 and R.sub.13 are each independently selected from the group consisting of hydrogen, alkyl, aryl, and —C.sub.4-C.sub.6 heteroaryl, wherein R.sub.12 and R.sub.13 are optionally substituted; each occurrence of R.sub.14 and R.sub.15 are independently selected from the group consisting of hydrogen, halogen, —OH, and C.sub.1-C.sub.6 alkyl; and n is an integer from 0-8; ##STR00045## wherein, R.sub.21 is selected from the group consisting of —C(R.sub.23).sub.m, cycloalkyl, heterocycyl, cycloalkyl-one, and heterocycyl-one; R.sub.22 is selected from the group consisting of alkyl, aryl, heteroaryl, —C.sub.1-C.sub.3 alkyl-(C.sub.3-C.sub.6 aryl), and —C.sub.1-C.sub.3 alkyl-(C.sub.3-C.sub.6 heteroaryl); each occurrence of R.sub.23 is independently selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, heterocycyl, —OH, and —CN; and m is an integer from 1 to 3; ##STR00046## wherein R.sub.31 is selected from the group consisting of —C(R.sub.35).sub.p, cycloalkyl, heterocycyl, cycloalkyl-one, heterocycyl-one; R.sub.32 is selected from the group consisting of alkyl, aryl, heteroaryl, —C.sub.1-C.sub.3 alkyl-(C.sub.3-C.sub.6 aryl), and —C.sub.1-C.sub.3 alkyl-(C.sub.3-C.sub.6 heteroaryl); R.sub.33 and R.sub.34 are each independently selected from the group consisting of hydrogen, halogen, alkyl, aryl, heteroaryl; each occurrence of R.sub.35 is independently selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, heterocycyl, —OH, and —CN; and p is an integer from 1 to 3; ##STR00047## wherein, X.sub.41 is selected from the group consisting of O and S; R.sub.41 is selected from the group consisting of alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and combinations thereof, wherein R.sub.41 may be optionally substituted; and R.sub.42 and R.sub.43 are each independently selected from the group consisting of phenyl, thiophenyl and furanyl.

16. The method of claim 15, wherein the compound of Formula (1) is selected from the group consisting of ##STR00048## ##STR00049## ##STR00050##

17. The method of claim 15, wherein the compound of Formula (2) is selected from the group consisting of ##STR00051##

18. The method of claim 15, wherein the compound of Formula (3) is selected from the group consisting of ##STR00052##

19. The method of claim 15, wherein the compound of Formula (4) is selected from the group consisting of ##STR00053## ##STR00054## ##STR00055##

20. A compound according to one of Formula (1) to Formula (4): ##STR00056## wherein, X.sub.11 is selected from the group consisting of C(R.sub.14)(R.sub.15), O, S and NR.sub.15; each occurrence of X.sub.12 is selected from the group consisting of C(R.sub.14)(R.sub.15), O, S and NR.sub.15; R.sub.11 is selected from the group consisting of hydrogen, —OR.sub.15, alkyl, cycloalkyl, —C.sub.4-C.sub.6 heterocyclyl, aryl, and —C.sub.4-C.sub.6 heteroaryl, wherein R.sub.11 is optionally substituted; R.sub.12 and R.sub.13 are each independently selected from the group consisting of hydrogen, alkyl, aryl, and —C.sub.4-C.sub.6 heteroaryl, wherein R.sub.12 and R.sub.13 are optionally substituted; each occurrence of R.sub.14 and R.sub.15 are independently selected from the group consisting of hydrogen, halogen, —OH, and C.sub.1-C.sub.6 alkyl; and n is an integer from 0-8; ##STR00057## wherein, R.sub.21 is selected from the group consisting of —C(R.sub.23).sub.m, cycloalkyl, heterocycyl, cycloalkyl-one, and heterocycyl-one; R.sub.22 is selected from the group consisting of alkyl, aryl, heteroaryl, —C.sub.1-C.sub.3 alkyl-(C.sub.3-C.sub.6 aryl), and —C.sub.1-C.sub.3 alkyl-(C.sub.3-C.sub.6 heteroaryl); each occurrence of R.sub.23 is independently selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, heterocycyl, —OH, and —CN; and m is an integer from 1 to 3; ##STR00058## wherein R.sub.31 is selected from the group consisting of —C(R.sub.35).sub.p, cycloalkyl, heterocycyl, cycloalkyl-one, heterocycyl-one; R.sub.32 is selected from the group consisting of alkyl, aryl, heteroaryl, —C.sub.1-C.sub.3 alkyl-(C.sub.3-C.sub.6 aryl), and —C.sub.1-C.sub.3 alkyl-(C.sub.3-C.sub.6 heteroaryl); R.sub.33 and R.sub.34 are each independently selected from the group consisting of hydrogen, halogen, alkyl, aryl, heteroaryl; each occurrence of R.sub.35 is independently selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, heterocycyl, —OH, and —CN; and p is an integer from 1 to 3; ##STR00059## wherein, X.sub.41 is selected from the group consisting of O and S; R.sub.41 is selected from the group consisting of alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and combinations thereof, wherein R.sub.41 may be optionally substituted; and R.sub.42 and R.sub.43 are each independently selected from the group consisting of phenyl, thiophenyl and furanyl.

21. The method of claim 20, wherein the compound of Formula (1) is selected from the group consisting of ##STR00060## ##STR00061## ##STR00062##

22. The method of claim 20, wherein the compound of Formula (2) is selected from the group consisting of ##STR00063##

23. The method of claim 20, wherein the compound of Formula (3) is selected from the group consisting of ##STR00064##

24. The method of claim 20, wherein the compound of Formula (4) is selected from the group consisting of ##STR00065## ##STR00066## ##STR00067##

Description

BRIEF DESCRIPTION OF THE FIGURES

[0106] FIG 1. Cannabidiol derivatives of Formula (I) induce HIF-1α level stabilization in oligodendrocytes. Stimulation of human oligodendrocyte MO13.3 cells for 6 hours with either 150 of μDFX or 1 μM of Cannabidol (CBD), of VCE-004, of compounds II, III, IV or V (FIG. 1A), or of compounds VI, VII, VIII, IX or X (FIG. 1B), to determine the expression of HIF-1α and α-tubulin by Western blots.

[0107] FIG. 2. HIF-1α level stabilization in oligodendrocytes: FIG. 2A shows the stimulation of MO3.13 cells with increasing concentrations of compound VIII or with DFX (150 μM) during 6 h. The steady state levels of the proteins HIF-1α and α-tubulin were determined by Western blots. FIG. 2B shows the time course induction of HIF-1α stabilization in MO3.13 cells stimulated with 2.5 μM compound VIII or 150 μM DFX (1 to 12 hours). The steady state levels of the proteins HIF-1α and α-tubulin were determined by Western blots.

[0108] FIG. 3. HIF-1α and HIF-2α levels stabilization in oligodendrocytes. Stabilization of the levels of HIF-1α and HIF-2α induced by compound VIII without affecting the expression of PHDs. MO3.13 cells were stimulated with increasing concentrations of compound VIII or 150 μM of DFX during six hours. The steady state levels of the proteins HIF-1α, HIF-2α, PHD1, PHD2, PHD3, and actin, were determined by Western blots.

[0109] FIG. 4. Cannabidiol quinol derivatives inhibit PHD activity. Inhibition of the HIF-1α hydroxylation activity of PHDs, and HIF-1α stabilization by compound VIII. MO3.13 cells were stimulated with increasing concentrations of compound VIII or 150 μM of DFX during six hours in the presence of the proteasome inhibitor MG132. The steady state levels of the proteins hydroxylated HIF-1α (OH-HIF-1α), HIF-1α, and actin, were determined by Western blots.

[0110] FIG. 5. HIF-1α and HIF-2α levels stabilization in Human Microvascular Endothelial Cells (HMEC). FIG. 5A: Stimulation of HMECs with increasing concentrations of compound VIII or with DFX (150 μM) during 3 hours. The steady state levels of the proteins HIF-1α and α-tubulin were determined by Western blots. FIG. 5B: Time coarse induction of HIF-1α stabilization in HMEC stimulated with 2.5 μM of compound VIII or 150 μM of DFX. The steady state levels of the proteins HIF-1α and α-tubulin were determined by Western blots. FIG. 5C: The stabilization of the levels of HIF-1α and HIF-2α induced by compound VIII without affecting the expression of PHDs in HMIEC. HMEC were stimulated with increasing concentrations of compound VIII or 150 μM of DFX for six hours. The steady state levels of the proteins HIF-1α, HIF-2α, PHD1, PHD2, PDH3, and actin were determined by Western blots.

[0111] FIG. 6. HIF-1α level stabilization in neuronal cells. Stimulation of SK-N-SH cells with increasing concentrations of compound VIII or with DFX (150 μM) during 6 h. The steady state levels of the proteins HIF-1α and α-tubulin were determined by Western blots.

[0112] FIG. 7. Compound VIII induces angiogenesis. Measurements of endothelial cell tube formation as a model of angiogenesis in green fluorescent Human endothelial vascular cells (HUVEC) co-cultured with primary fibroblasts and stimulated separately with compound VIII (1 μM) or VEGFA (10 ng/ml) for 7 days. Values of network length (in mm/mm.sup.2) represent the mean±SEM (n=3).

[0113] FIG. 8. Compound VIII induces the expression of the HIF-1α-dependent genes VGFA and EPO. MO13.3 cells (FIG. 8A) and HBMEC cells (FIG. 8B) were stimulated with increasing concentrations of compound VIII for 12 h and the expression of VGFA and EPO mRNAs determined by qPCR. Data are expressed as mean±SEM (n=3).

[0114] FIG. 9. Influence of compound VIII on collagen gel contraction. NIH 3T3-EPO-Luc fibroblasts were incorporated into collagen gels with and without indicated concentrations of compound VIII. FIG. 9A: Images of contracted of gel matrices in response to either compound VIII (1, 2.5 and 5 μM) or DMGO for 24 h are shown. FIG. 9B: Gel surface area quantified in terms of total pixel number using ImageJ, where indicated *p<0.025 and **p<0.01.

EXAMPLES

[0115] The examples of the present invention described below aim to illustrate its embodiments without limiting its scope of protection.

Example 1

Activation of the HIF Pathway

[0116] To investigate the biological activities of the different compounds, HIF-1α transactivation assays were performed either in NIH-3T3-EPO-Luc cells (Table 1) or in HaCaT-EPO-luc cells (Table 2). The NIH3T3-EPO-luc and HaCaT-EPO-luc cells have been stably transfected with the plasmid Epo-Luc plasmid. The EPO-Hypoxia Response Element (HRE)-luciferase reporter plasmid contains three copies of the HRE consensus sequence from the promoter of the erythropoietin gene fused to the luciferase gene.

[0117] NIH3T3-EPO-luc cells were maintained at 37° C. in a humidified atmosphere containing 5% CO.sub.2 in DMEM supplemented with 10% fetal calf serum (FBS), and 1% (v/v) penicillin/streptomycin. Deferoxamine (DFX) was purchased from Sigma-Aldrich (USA). Cells (1×10.sup.4/well in 96-well plates) were seeded the day before the assay. The next day, the cells were stimulated with increasing concentrations of either Cannabidiol (CBD), VCE-004 or compounds II to X. After six hours of stimulation the cells were lysed in 25 mM Tris-phosphate pH 7.8, 8 mM MgCl.sub.2, 1 mM DTT, 1% Triton X-100, and 7% glycerol during 15 min at RT in a horizontal shaker. Luciferase activity was measured using a microplate luminometer (Berthold) following the instructions of the luciferase assay kit (Promega, Madison, Wis., USA).

[0118] HaCaT-EPO-Luc cells were maintained at 37° C. in a humidified atmosphere containing 5% CO.sub.2 in DMEM supplemented with 10% fetal calf serum (FBS), and 1% (v/v) penicillin/streptomycin. The cells (1×10.sup.5/well in 24-well plates) were seeded the day before the assay and then stimulated with increasing concentrations of either Cannabidiol (CBD), VCE-004 or compounds II to X. After six hours of stimulation the cells were lysed in 25 mM Tris-phosphate pH 7.8, 8 mM MgCl.sub.2, 1 mM DTT, 1% Triton X-100, and 7% glycerol during 15 min at RT in a horizontal shake. Luciferase activity was measured in the cell lysates as indicated for NIH3T3-EPO-Luc cells. The RLUs are calculated and the EC50 and IRA (Intrinsic relative activity) values in both cell lines were determined relative to 150 μM deteroxamine (DFX) using the following equation: IRA coefficient=(EC.sub.50−DFX×E.sub.max)/(EC.sub.50×E.sub.max−DFX), where EC.sub.50 and E.sub.max denote EC.sub.50 and E.sub.max of the agonist, and EC.sub.50−DFX and E.sub.max−DFX denote EC.sub.50 and E.sub.max values of the standard agonist DFX (Table 1 and 2).

TABLE-US-00001 TABLE 1 HIF-1α transactivation assays in NIH-3T3-EPO Luc fibroblast cells. NIH3T3-EPO-luc cell line stably transfected with the Epo-Luc plasmid, which contains three copies of the Hypoxia Response Element consensus sequence from the promoter of the erythropoietin gene fused to luciferase gene. The efficacy and potency for HIF-1α activation is shown. Efficacy HIF-1α Potency EC.sub.50 Compound (IRA coefficient).sup.a HIF-1α (μM) CBD — — VCE-004 — — II 0.46 4.3 III 0.28 3.6 IV 0.61 3.2 V 0.85 1.8 VI 0.61 2.8 VII 0.68 2.9 VIII 0.63 2.6 IX 0.8 2.5 X 0.29 3.3

TABLE-US-00002 TABLE 2 HIF-1α transactivation assays in HaCaT-EPO Luc fibroblast cells. NIH3T3-EPO-luc cell line stably transfected with the Epo-Luc plasmid, which contains three copies of the Hypoxia Response Element consensus sequence from the promoter of the erythropoietin gene fused to luciferase gene. The efficacy and potency for HIF-1α activation is shown. Efficacy HIF-1α Potency EC.sub.50 Compound (IRA coefficient).sup.a HIF-1α (μM) CBD — — VCE-004 — — II 3.87 3.2 III 5.84 1.7 IV 5.64 1.6 V 5.01 1.9 VI 3.02 4 VII 2.3 5.5 VIII 6.27 1.4 IX 8.64 1.4 X 10.35 1.2

[0119] A significant increase in luciferase activity was seen with all cannabinoid derivatives as compared with untreated cells. Thus, it can be concluded that the chemical modifications in position 3 of VCE-004 are critical to activate the HIF pathway.

Example 2

Cannabinoid Derivatives Stabilize the Levels of HIF-1α and HLF-2α in Different Cell Types and Inhibit PHDs Prolyl Hydrolase Activity

[0120] To gain insight into the regulation of HIF-1α stabilization by the compounds of Formula (I), the effect on HIF-1α expression in different cell types was investigated. Human oligodendrocyte MO13.3 cells were stimulated for 6 h with either 150 μDFX or 1 μM of Cannabidiol (CBD), VCE-004, compounds II to V (FIG. 1A), compounds VI to X (FIG. 1B). After that, the cells were washed with PBS and incubated in 50 μl of NP-40 buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and 1% NP-40) supplemented with 10 mM NaF, 1 mM Na.sub.3VO.sub.4, 10 μg/ml leupeptine, 1 μg/ml pepstatin and aprotinin, and 1 μl/ml PMSF saturated. After centrifugation, the supernatants were mixed with SDS sample buffer and boiled at 95° C. Proteins were eleetrophoresed in 8-10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (20 V and 30 min per membrane). After blocking with non-fat milk or BSA in TBST buffer, primary antibodies were added. The washed membranes were incubated with appropriate secondary antibodies coupled to horseradish peroxidase that were detected by an enhanced chemiluminescence system (USB). The antibody against HIF-1α (610959) was purchased from BD Biosciences and the antibody anti-β-tubulin (clone AA2) was purchased from Sigma-Aldrich (Saint Louis, Mo., USA).

[0121] All the compounds described in the present invention elevated HIF-1α protein level under normoxia conditions (21% O.sub.2). The extent of induction was comparable to that of desferrioxamine (DFX), an iron chelator known to stabilize the levels of HIF-1α (FIG. 1A and FIG. 1B).

[0122] Next, MO13.3 cells (oligodendrocyte cell line) were stimulated with the increasing concentrations of compound VIII or with DFX (150 μM) during 6 h. After that, proteins isolation and western blots were performed as in FIG. 1. The results show that compound VIII induces stabilization of the levels of HIF-1α in a concentration dependent manner (FIG. 2A). FIG. 2B shows the time-course for stabilization of the levels of HIF-1α in MO13.3 treated with compound VIII.

[0123] The explanation to the stabilization of the levels of HIF-1α and HIF-2α proteins may be due to either the reduction of the expression of PHD proteins or the inhibition of its prolyl hydrolase activity. Thus, to identify which of said mechanisms is responsible for said stabilization, the expression of HIF-1α, HIF-2α and PDHs (PDH1, PDH2 and PDH3) proteins were analyzed by western blots. MO13.3 cells were stimulated with the increasing concentrations of compound VIII or with DFX (150 μM) during 6 h. After that, proteins isolation and western blots were performed as in FIG. 1. The antibodies anti-HIF-2α (ab8365), anti-PHD3 (ab30782) anti-PHD1 (ab80361) and anti-PHD2 (ab109088) were purchased from Abcam (Cambrigde, UK).

[0124] The results clearly show that compound VIII stabilized HIF-1α and HIF-2α expression without affecting the expression of PDH1, PDH2 and PDH3 (FIG. 3).

[0125] To study the activity of PDHs on the stabilization of the HIF-1α levels, MO13.3 cells were treated with increasing concentrations of compound VIII and the steady state levels of hydroxylated HIF-1α (OH-HIF-1α) and total HIF-1α proteins were identified by western blot. As depicted in FIG. 4 compound VIII led a decreased in the expression of OH-HIF-1α that paralleled with an increase in the expression of total HIF-1α protein.

[0126] Altogether, results indicate that compound VIII inhibits the PDHs prolyl hydrolase activity and as consequence HIF-1α and HIF-2α protein levels are stabilized.

[0127] The stabilization of HIF-1α and HIF-2α protein levels by the compounds of Formula (I) was also shown in other cell types:

[0128] Human Microvascular Endothelial Cells (HMEC) were treated with increasing concentrations of compound VIII (FIG. 5A), and also treated with compound VIII (2.5 μM) at different times (FIG. 5B). It is shown that compound VIII induces the stabilization of HIF-1α levels in MO13.3 cells in a concentration dependent manner (FIG. 5A). Moreover, compound VIII also induces stabilization of the HIF-2α levels in this type of cells without affecting the expression of PDH1, PHD2 and PHD3 (FIG. 5C). It is also shown that the maximal expression of HIF-1α was achieved after 3 h of treatment with compound VIII (FIG. 5B). Similarly, compound VIII also induces stabilization of the levels of HIF-1α in SK-N-SH, a neuronal cell line (FIG. 6).

Example 3

Angiogenesis Induced by Compounds of Formula (I)

[0129] To test the functional consequences of compound VIII stimulation in a physiological model, endothelial cell tube formation was measured as a model of angiogenesis. CellPlayer™ GFP AngioKit-96 (Essen BioScience Inc., Welwyn Garden City, UK) was supplied as growing co-cultures of human matrix (normal human dermal fibroblast, NHDF) and endothelial cells (HUVEC) at the earliest stages of tubule formation. CellPlayer 96-well kinetic angiogenesis assay was performed according to the manufacturer's protocol. Briefly, lentivirally infected green fluorescent protein (GFP)-HUVECs were cocultured with normal human dermal fibroblasts in a 96-well microplate. The plate was placed in IncuCyte, and images were automatically acquired in both phase and fluorescence every 6 hours for 7 days. At day 1, compound VIII (1 μM) or rhVEGFA (10 ng/ml) were added on the endothelial tube networks and kept throughout the experiment. Tube formation over the 7-day assay was quantified using the Essen BioScience Angiogenesis Analysis Module. This module provides multiple assay metrics, including tube length and branch point formation, which are used to assess angiogenic effects on network formation. Briefly, the fluorescent images were analyzed to generate a segmentation mask closely resembling the in vitro network. The mask was then refined to specifically identify tube-forming events, and the kinetic response was plotted using the IncuCyte and GraphPad Prism software (La Jolla, Calif.).

[0130] In FIG. 7 it is shown that compound VIII 1 μM as well as the positive control VrhEGFA (10 ng/ml) increased significantly the network length in HUVEC cells.

Example 4

Compounds of Formula (I) Modulate the Expression of Genes Modulated by the HIF-1 Transcription Factor

[0131] In order to understand the molecular mechanisms underlying the effects of compound VIII, HMEC cells were treated with compound VIII (5 μM) for 12 hours, and then mRNA was and the expression of 84 genes involved in the hypoxia was analyzed using the Human Hypoxia Signaling Pathway RT.sup.2 Profiler PCR Array following the manufacturer's instructions (Qiagen Iberia, Madrid Spain). This array contains 84 key genes involved in Fibrosis development. Data were analyzed using the 2.sup.−ΔΔCt method and normalized with five housekeeping genes.

[0132] It is shown in Table 3 that compound VIII clearly upregulated the expression of a set of genes including ANGPTL4 and VEGFA that are known to be upregulated in response to hypoxia and in response to HIF PDHs inhibitors:

TABLE-US-00003 TABLE 3 Expression of HIF related genes in Human primary microvascular endothelial cells. Human primary microvascular endothelial cells were stimulated with compound VIII (5 μM) for 12 h and the expression analysis of genes involved in the human hypoxia-signaling pathway determined by PCR array. Gen Symbol, reference sequence number, description of each gene and fold induction or repression of gene expression compared to untreated control cells is shown. Refseq is referring to Human Hypoxia Signaling Pathway RT.sup.2 Profiler PCR Array (Qiagen Iberia, Madrid Spain). Fold Regulation Symbol Refseq Description (Comp. VIII 5 μm) A01 ADM NM_001124 Adrenomedullin 4.27 A08 BLM NM_000057 Bloom syndrome, RecQ helicase-like −4.07 B03 EGLN1 NM_022051 Egl nine homolog 1 (C. elegans) 4.25 B05 EGR1 NM_001964 Early growth response 1 7.09 B09 ERO1A NM_014584 ERO1-like (S. cerevisiae) 5.60 B12 FOS NM_005252 FBJ murine osteosarcoma viral oncogene 8.04 homolog D01 LOX NM_002317 Lysyl oxidase 5.21 D06 MXI1 NM_005962 MAX interactor 1 5.69 E01 PDK1 NM_002610 Pyruvate dehydrogenase kinase, isozyme 1 4.89 E03 PFKFB3 NM_004566 6-phosphofructo-2-kinase/fructose-2,6- 8.56 biphosphatase E09 PLAU NM_002658 Plasminogen activator, urokinase 6.86 E12 SERPINE1 NM_000602 Serpin peptidase inhibitor, clade E member 1 6.00 F01 SLC16A3 NM_004207 Solute carrier family 16, member 3 4.72 (monocarboxylic acid transporter 4) F02 SLC2A1 NM_006516 Solute carrier family 2 (facilitated 19.73 glucose transporter), member 1 F03 TFRC NM_003234 Transferrin receptor (p90, CD71) 3.26 F09 ALDOC NM_005165 Aldolase C, fructose-bisphosphate 7.67 F10 ANGPTL4 NM_001039667 Angiopoietin-like 4 145.63 F11 ANKRD37 NM_181726 Ankyrin repeat domain 37 6.63 F12 BHLHE40 NM_003670 Basic helix-loop-helix family, member e40 8.22 G01 BNIP3 NM_004052 BCL2/adenovirus E1B 19 kDa interacting 6.46 protein 3 G02 BNIP3L NM_004331 BCL2/adenovirus E1B 19 kDa interacting 5.35 protein 3-like G05 HK2 NM_000189 Hexokinase 2 8.43 G07 NDRG1 NM_006096 N-myc downstream regulated 1 25.25 G08 P4HA1 NM_000917 Prolyl 4-hydroxylase, alpha polypeptide I 10.56 G09 PFKFB4 NM_004567 6-phosphofructo-2-kinase/fructose-2,6- 31.26 biphosphatase 4 G11 SLC2A3 NM_006931 Solute carrier family 2 (facilitated glucose 12.19 transporter), member 3 G12 VEGFA NM_003376 Vascular endothelial factor A 18.09

[0133] To further extend the analysis of gene expression regulated by compounds of Formula (I), human brain microvascular cells (HBMEC) and MO13.3 cells were treated with increasing concentrations of compound VIII for 12 and the mRNA isolated. Single-stranded complementary DNA was synthesized from up to 1 μg of total RNA using iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, Calif., USA). The reaction mixture was kept frozen at −20° C. until enzymatic amplification. The iQ™ SYBR Green Supermix (Bio-Rad) was used to quantify mRNA levels for VEGFA and EPO. Real-time PCR was performed using a CFX96 Real-Time PCR Detection System (Bio-Rad). The GAPDH housekeeping gene was used to standardize the mRNA expression levels in every sample. Expression levels were calculated using the 2.sup.−ΔΔCt method. Sequences of oligonucleotide primers are given in Table 4.

[0134] Compound VIII upregulated the expression of EPO and VEGFA in both MO13.3 and HBMEC cells (FIG. 8A and FIG. 8B).

TABLE-US-00004 TABLE 4 List of human primer sequences used in quantitative Polymerase Chain Reaction. Gene Forward Reverse EPO 5′-ctccgaacaatcactgct-3′ 5′-ggtcatctgtcccctgtcct-3′ VEGFA 5′-cgaagtggtgaagttcatggatg-3′ 5′-ttctgtatcagtctttcctggtg-3 GAPDH 5′-tggcaaagtggagattgttgcc- -3′ 5′-aagatggtgatgggcttcccg-3′

Example 5

Compounds of Formula (I) Induce Collagen Contraction

[0135] We explored whether the compounds of Formula (I) influence wound healing and tissue remodeling. For this purpose, a model of wound healing was used to assess the influence of compound VIII on fibroblast collagen gel contraction. NIH3T3-EPO-Luc were lifted from culture plates with trypsin, washed with PBS, and resuspended in complete medium at 500,000 cells/ml. Collagen gels were made as previously described (Phillips and Bonassar. Exp Cell Res. 2005. 310:79-81). All gels contained a final concentration of 150,000 cells/ml and 1.0 mg/ml collagen I with or without indicated concentrations of either compound VIII or 1 mM DMOG (dimethyloxaloylglycine). Gels were digitally imaged after release (t=0) and at various time points thereafter. Gel surface area was quantified in terms of pixel number using ImageJ (http://rsb.info.nih.gov/ij/). Relative changes in surface area are reported as a percent of the original surface area. As shown in FIG. 9 exposure of fibroblast embedded gels to compound VIII enhanced contraction of collagen gels to the same tenet than DMOG, which was used as a positive control. These findings directly implicate compound VIII in tissue remodeling and wound contraction.

[0136] The present results substantiate the therapeutic use of the compounds described in the present inventions, for the clinical management of conditions that benefit from the inhibition of the PHDs activity or the stabilization of the HIF-1α and HIF-1β, such as stroke, traumatic injuries anemia, myocardial ischaemia-reperfusion injury, acute lung injury, infectious diseases, diabetic and chronic wounds, organ transplantation, acute kidney injury and arterial diseases.