Hydroxamate triterpenoid derivatives

Abstract

Triterpenoid derivatives and compositions comprising said triterpenoids derivatives of Formula (I) are described, wherein RC(O)NHOH. Said triterpenoids and compositions show capacity to bind PHD2, stabilize HIF-1 and HIF-2 proteins, activate the HIF pathway in different cell types, induce angiogenesis in human endothelial vascular cell, show neuroprotective activity in vitro and in vivo, antidiabetic activity and reduce the levels of lipids in vivo, and increase the plasma levels of Erythropoietin in vivo. The triterpenoid derivatives described act also in a selective manner and do not induce Nrf2 activation, NF-B inhibition, STAT3 inhibition, and TGR5 activation, which are known activities of the natural triterpenoid precursors. Said triterpenoid derivatives are useful in the treatment of conditions and diseases which are responsive to HIF activation such as stroke, cerebral palsy, traumatic injuries and neurodegenerative diseases; and also IBD, myocardial ischaemia-reperfusion injury, acute lung injury, diabetic and chronic wounds, organ transplantation, acute kidney injury or arterial diseases. ##STR00001##

Claims

1. A triterpene derivative of Formula (Ia) or stereoisomers, pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof, ##STR00044## wherein independently, A-B is a single carbon-carbon bond or a double carbon-carbon bond; B is a methylene (CH.sub.2), an olefin methine (CH), a hydroxymethine [CH(OH)], or a hydroxylated olefin carbon [C(OH)]; B-C is a single carbon-carbon bond or a double carbon-carbon bond; or is part of a heterocyclic ring comprising one or more heteroatoms wherein at least one of said heteroatoms is nitrogen; and wherein said heterocyclic ring is a five-membered ring comprising one nitrogen and one oxygen; C is a hydroxymethine [CH(OH)], an acyloxymethine [CH(OCOR)], an olefin methine (CH), a carbonyl [C(O)], an oxime [C(NOH)] or an hydrazone [C(NNH.sub.2)], wherein R is methyl; D-E is a single or a double carbon-carbon bond; F is F.sub.1a, F.sub.2a or F.sub.3a; ##STR00045## G is a methylene (CH.sub.2) or a carbonyl [C(O)]; and R is a hydroxamate group (CONHOH); and wherein, when C is an acyloxymethine [CH(OCOR)], the triterpene derivative of Formula (Ia) is ##STR00046## when B is a methylene (CH.sub.2), C is a hydroxymethine [CH(OH)], D-E is a double carbon-carbon bond, G is a methylene (CH.sub.2) and R is a hydroxamate (CONHOH), F is F.sub.3a; when B is a methylene (CH.sub.2), C is a hydroxymethine [CH(OH)], D-E is a single carbon-carbon bond, G is a methylene (CH.sub.2) and R is a hydroxamate (CONHOH), F is F.sub.1a or F.sub.2a; when B is a methylene (CH.sub.2), C is an oxime [C(NOH)], D-E is a double carbon-carbon bond, G is a methylene (CH.sub.2) and R is a hydroxamate (CONHOH), F is F.sub.1a or F.sub.3a.

2. The triterpene derivative according to claim 1, wherein said triterpene derivative is selected from the group consisting of: ##STR00047## ##STR00048##

3. A compound selected from the group consisting of XVIII and XIX: ##STR00049##

4. A pharmaceutical composition comprising at least one triterpene derivative of Formula (Ia) of claim 1 as a first active ingredient and at least one excipient or carrier.

5. A method of treating a condition or disease responsive to the activation of a HIF pathway, wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of the triterpene derivative of claim 1.

6. The method of claim 5, wherein the condition or disease responsive to the activation of the HIF pathway is selected from the group consisting of stroke, cerebral palsy, traumatic injuries and neurodegenerative diseases.

7. The method of claim 5, wherein the condition or disease responsive to the activation of the HIF pathway is selected from the group consisting of IBD, myocardial ischaemia-reperfusion injury, acute lung injury, diabetic and chronic wounds, organ transplantation, acute kidney injury and arterial diseases.

8. The method of claim 5, wherein the condition or disease responsive to the activation of the HIF pathway is diabetes, hyperlipidemia or hypertriglyceridemia.

9. The method, according to claim 5, wherein said method produces an increase in the erythropoietin plasma levels.

10. A method of treating a condition or disease wherein the treatment of said condition or disease benefits from HIF-1 or HIF-2 activation, the method comprising administering to a subject in need thereof a triterpene derivative of Formula (I) or, stereoisomers, pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof, ##STR00050## wherein independently, A-B is a single carbon-carbon bond or a double carbon-carbon bond; B is a methylene (CH.sub.2), an olefin methine (CH), a hydroxymethine [CH(OH)], or a hydroxylated olefin carbon [C(OH)]; B-C is a single carbon-carbon bond or a double carbon-carbon bond; or is part of a heterocyclic ring comprising one or more heteroatoms wherein at least one of said heteroatoms is nitrogen; and wherein said heterocyclic ring is a five membered ring comprising one nitrogen and one oxygen; C is a hydroxymethine [CH(OH)], an acyloxymethine [CH(OCOR)], an olefin methine (CH), a carbonyl [C(O)], an oxime [C(NOH)] or an hydrazone [C(NNH.sub.2)], wherein R is methyl; D-E is a single or a double carbon-carbon bond; F is F.sub.1, F.sub.2 or F.sub.3; ##STR00051## G is a methylene (CH.sub.2) or a carbonyl [C(O)]; and R is a hydroxamate group (CONHOH).

11. The method according to claim 10, wherein said triterpene derivative is selected from the group consisting of: ##STR00052## ##STR00053## ##STR00054##

12. The method of claim 10, wherein said condition or disease is selected from the group consisting of stroke, cerebral palsy, traumatic injuries and neurodegenerative diseases.

13. The method of claim 10, wherein said condition or disease is selected from the group consisting of IBD, myocardial ischaemia-reperfusion injury, acute lung injury, diabetic and chronic wounds, organ transplantation, acute kidney injury and arterial diseases.

14. The method of claim 10, wherein the condition or disease is diabetes, hyperlipidemia or hypertriglyceridemia.

15. The method according to claim 10, wherein said method produces an increase in the erythropoietin plasma levels.

16. The method of claim 10, wherein said triterpene derivative of Formula (I) is a triterpene derivative of Formula (Ib): ##STR00055##

17. The method according to claim 15, wherein said triterpene derivative is selected from (II), (III), (IV), (V), (VI), (VII), (VIII), (X), (XI), (XII), (XIII), (XIV) or (XV): ##STR00056## ##STR00057## ##STR00058##

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1. Top scored conformation of betulinic acid (FIG. 1A) and compound VII (FIG. 1B) bound to PHD2 (PDB 4BQW).

(2) FIG. 2. HIF-1 transactivation assays in HaCaT-EPO Luc keratinocyte cells.

(3) Hypoximimetic effects of DFX and compounds II to XV in HaCaT-EPO-Luc cells. The concentration of the tested compound (M) is shown at the x-axis and the percentage of HIF-1 activation is shown at the y-axis. This figure shows the effect of DFX versus compounds (FIG. 2A) II, III, IV, V, VI, VII, XI, (FIG. 2B) VIII, IX, X, XII, XIII, XIV and XV on EPO-luc activity considering the induction mediated by DFX (150 M) as 100% activation over untreated cells.

(4) FIG. 3. HIF-1 stabilization in oligodendrocytes.

(5) Stimulation of human oligodendrocyte MO13.3 cells for 3 hours with either 150 DFX or 10 M of Olenaolic acid (OA), compounds II, III, IV, V, VI (FIG. 3A), betulinic acid (BA), compounds VII, VIII, IX, X, XI (FIG. 3B), ursolic acid (UA), maslinic acid (MA), compounds XII, XIII, XIV and XV (FIG. 3C) to determine the expression of HIF-1 and -actin by Western blots.

(6) FIG. 4. HIF-1 stabilization in 293T cells.

(7) Stimulation of human Embryonic Kidney 293 cells with increasing concentrations of compound VII or with DFX (150 M) during 3 h. The steady state levels of the proteins HIF-1, PHD1, PHD2 and -actin were determined by Western blots.

(8) FIG. 5. HIF-2 stabilization in hIPCs.

(9) Stimulation of human Islet-Derived Precursor Cells (hIPCs) with increasing concentrations of compound VII or with DFX (150 M) during 3 h, and the expression of HIF-2, PHD3 and -actin determined by Western blots.

(10) FIG. 6. Compound VII induces angiogenesis.

(11) 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 VII (1 M), rhFGF (10 ng/ml) and VEGFA (10 ng/ml) for 7 days. Values represent the meanSEM (n=3).

(12) FIG. 7. Influence of compound VII on erythropoietin (EPO) in vivo.

(13) EPO in plasma measured using a mouse EPO ELISA kit in C57BL/6 male mice administered with compound VII (30 mg/kg or 60 mg/kg) or betulinic acid (BA) (60 mg/kg) intraperitoneally. Control group did not receive any treatment. Data are expressed as meanSEM (n=3).

(14) FIG. 8. Compound VII abrogates 3-NP toxicity in Q7 and Q11 striatal cells. Q7 and Q111 Striatal cells were pretreated with increasing concentrations of compound VII for 6 h and then exposed to 3-NP (10 mM) for an additional 24 h, after which YOYO-1 staining was used to investigate cell death. Cell death was determined using the IncuCyte HD software and the treatment with 3-NP alone in Q111 cells was considered as 100% of cell death.

(15) FIG. 9. Behavioral score after 3NP intoxication.

(16) Intoxicated mice with 3-Nitropropionic acid (3-NP) were subjected to behavioral tests for determining their neurological status after the treatment with compound VII (30 mg/kg) and Betulinic acid (BA) (30 mg/kg) versus a control of non-intoxicated mice. Hind limb clasping, Locomotor activity, Hind limb dystonia and Truncal Dystonia were rated from 0 to 2 based on severity: a score of 0 typically indicates normal function and 2 seriously affected. Values are expressed as meansSEM for 6 animals per group.

(17) FIG. 10. Effect of compound VII on neuronal loss.

(18) Intoxicated mice with 3-Nitropropionic acid (3-NP) for determining degeneration in the striatum including Control (non-intoxicated mice), 3NP, 3NP+BA and 3NP+Compound VII). Quantification of Nissl-positive cells in the mouse striatum. Total average number of neurons (100 magnification) is shown. Values are expressed as meansSEM for 6 animals per group. Data were subjected to one-way analysis of variance followed by the Student-Newman-Keuls test. ***P<0.001 when comparing the control group with the 3NP and control group. #P<0.05 when comparing the 3NP group with the 3NP+compound VII group

(19) FIG. 11. Effect of compound VII on microgliosis (Iba1.sup.+) and astrogliosis (GFAP.sup.+) induced by 3NP

(20) Intoxicated mice with 3-Nitropropionic acid (3-NP) for determining microglia activation and astrogliosis including Control (non-intoxicated mice), 3NP, 3NP+BA and 3NP+Compound VII). Iba-1 and glial fibrillary acidic protein (GFAP) expression were determined by immunostaining of brain sections through the different group of mice and quantification of the different markers was performed with Image J software. Total average number of microglia (Iba1.sup.+) and astrocytes (GFAP.sup.+) is shown.

(21) FIG. 12. Compound VII reduces the expression on inflammatory marker mRNAs in the striatum.

(22) Gene expression of inflammatory markers including COX-2 (FIG. 12D), IL-1 (FIG. 12B), IL-6 (FIG. 12A) and iNOS (FIG. 12C), was down regulated in 3NP+compound VII (30 mg/kg) treated mice compared with 3NP+Vehicle mice. Betulinic acid (BA) treatment (30 mg/kg) also inhibited the expression of inflammatory markers. Expression levels were calculated using the 2.sup.Ct method. Values are expressed as meansSEM for 6 animals per group.

(23) FIG. 13. Effect of compound VII on body weight, fat mass and adiposity in mice subjected to high fat diet (HFD animals).

(24) Mice were fed with HFD for 13 weeks and the treated daily with compound VII (30 mg/Kg) for 21 days and the effect on body weight was monitored every 3-4 days (FIG. 13A). % Lean mass (FIG. 13B) is calculated as the proportion of entire weight of the body without the proportion due to fat. % Fat mass (FIG. 13C) is calculated as the proportion of fat to the total body weight. Fat mass and the percentage of adiposity was calculated at the week 15. Body composition was assessed by Magnetic Resonance Imaging (MRI). Values are expressed as meansSEM for 10 animals per group.

(25) FIG. 14. Compound VII improves glucose tolerance (GTT) in HFD animals.

(26) GTT of the HFD-fed mice after the 3-week vehicle (Control, CD) or compound VII administration (30 mg/Kg). The sum of the trapezoidal areas between the 0, 15, 30, 45, 60, 90 and 120 min time points corresponding to each animal were summed to obtain the area under the curve (AUC). The relative area values are expressed as a percentage relative to the average AUC of the vehicle cohort, which is defined as 100%. Values are expressed as meanS.E.M (n=6).

(27) FIG. 15. Compound VII prevents hypertriglyceridemia in HFD animals.

(28) Plasma triglycerides levels in control and HFD-fed mice after the 3-week vehicle (Control, CD) or compound VII administration (30 mg/Kg). Values are expressed as meanS.E.M (n=6).

EXAMPLES

(29) The examples of the present invention described below aim to illustrate its preferred embodiments without limiting its scope of protection.

Example 1. Synthesis of the Triterpenoid Derivatives of Formula (I) and of Comparative Compound XX

(30) General Synthesis of the N-Hydroxy-Triterpen-28-Amide Derivatives of Formula (I) (Hydroxamate Formation):

(31) To an ice-cold solution of a triterpenic acid precursor (1 eq/mol) in dry dichloromethane (DCM), oxalyl chloride (6 eq/mol) was added dropwise, and the mixture was heated at 40 C. for 1.5 hours. The solvent was removed in vacuum, the residue was dissolved in dry pyridine or N,N-diisopropylethylamine (DiPEA), and hydroxylammonium chloride (6 eq/mol) were added. The reaction was heated at 40 C. for 3 hours, quenched with 2 N H.sub.2SO.sub.4 sol. and extracted with EtOAc. The organic phases were washed with brine, dried over Na.sub.2SO.sub.4 and evaporated under vacuum. The crude compound was purified over silica gel.

(32) Known compounds are identified when possible with means of their CAS number.

(3) 3-Hydroxy-N-hydroxy-olean-12-en-28-amide (Compound II)

(33) CAS num.: 1854922-22-7

(34) Off-white solid (60%). .sup.1H NMR (300 MHz, CDCl.sub.3): d: .sup.1H NMR (300 MHz, CDCl.sub.3): d=5.41 (brt, 1H, H-12), 3.19 (dd, J=10.0, 4.89 Hz, 1H), 2.44 (d, J=10.8 Hz, 1H), 2.05-1.86 (m, 3H), 1.16 (s, 3H), 0.98 (s, 3H), 0.92 (s, 3H), 0.90 (s, 3H), 0.88 (s, 3H), 0.81 (s, 3H), 0.78 (s, 3H) (only readily peaks are reported); .sup.13C NMR (75 MHz, CDCl.sub.3): d=176.7, 144.9, 123.9, 78.9, 55.2, 47.6, 46.4, 45.5, 42.0, 40.8, 39.5, 38.8, 38.6, 37.0, 34.0, 32.9, 32.3, 32.0, 30.7, 28.1, 27.3, 27.2, 25.84, 23.7, 23.6, 23.5, 18.3, 16.7, 15.6, 15.4.

3-Hydroxyimino-N-hydroxy-olean-12-en-28-amide (Compound III)

(35) Compound III was obtained according to the general synthetic scheme described above herein (hydroxamate formation), starting from the known precursor CAS num. 17990-42-0:

(36) ##STR00030##

(37) The oxime formation featured in position C of compound III was carried out as well as the hydroxamate formation during the same general synthetic path.

(38) Off-white solid (70%). .sup.1H NMR (300 MHz, CDCl.sub.3): d=5.43 (brt, 1H), 3.07 (bdt, J=14.9, 1H), 2.46 (m, 1H), 1.98 (m, 3H), 1.13 (s, 10H), 1.07 (s, 6H), 1.05 (s, 3H), 0.86 (s, 6H), 0.82 (s, 3H) (only readily peaks are reported); .sup.13C NMR (75 MHz, CDCl.sub.3) d=176.7, 167.5, 144.9, 123.8, 55.7, 47.1, 46.2, 45.5, 42.0, 40.8, 40.3, 39.4, 38.4, 37.0, 33.9, 32.9, 31.9, 30.7, 29.7, 29.2, 27.2, 25.7, 25.5, 23.7, 23.5, 23.4, 19.0, 17.3, 16.7, 14.9.

(3) 3-Acetyloxy-11-oxo-olean-12-en-N-hydroxy-28-amide (Compound V)

(39) Compound V was obtained according to the general synthetic scheme described above herein (hydroxamate formation), from the known precursor CAS num. 14605-17-5:

(40) ##STR00031##

(41) Pale yellow solid (75%). .sup.1H NMR (300 MHz, CDCl.sub.3): d=5.65 (s, 1H), 4.47 (dd, J=4.8 J=11.0 Hz, 1H), 3.31 (m, 1H), 2.76 (t, J=12.5 Hz, 2H), 2.32 (s, 1H), 2.00 (s, 3H), 1.12 (s, 3H), 0.91 (s, 3H), 0.88 (s, 6H), 0.82 (s, 9H) (only readily peaks are reported); .sup.13C NMR (75 MHz, CDCl.sub.3) d=200.5, 174.4, 171.2, 168.4, 127.8, 80.7, 61.9, 55.0, 45.3, 44.8, 43.6, 43.0, 40.7, 38.7, 38.0, 37.1, 33.8, 32.8, 32.6, 32.1, 30.7, 28.1, 27.3, 23.6, 23.5, 23.4, 23.1, 21.3, 19.0, 17.3, 16.7, 16.3.

(3) 3-Hydroxy-11-oxo-olean-12-en-N-hydroxy-28-amide (Compound IV)

(42) To obtain compound IV, the deacetylation of compound V was carried out as follows:

(43) ##STR00032##

(44) To a solution of compound V, (3) 3-Acetyloxy-11-oxo-olean-12-en-N-hydroxy-28-amide (1 eq/mol) in THF/MeOH 1:1 was added NaOH 4N (50 eq/mol). The mixture was heated at 40 C. overnight, quenched with H.sub.2SO.sub.4 sol. 2N and extracted with EtOAc. The organic phases were washed with brine, dried over Na.sub.2SO.sub.4 and evaporated under vacuum. The crude compound was purified over silica gel.

(45) Pale yellow solid (65%). .sup.1H NMR (300 MHz, CDCl.sub.3): d=5.67 (s, 1H), 3.20 (t, J=6.1 Hz, 1H), 2.74 (d, J=12.2 Hz, 2H), 2.32 (s, 1H), 2.10-2.02 (m, 1H), 1.18 (s, 3H), 0.96 (s, 3H), 0.94 (s, 3H), 0.90 (s, 9H), 0.77 (s, 3H) (only readily peaks are reported); .sup.13C NMR (75 MHz, CDCl.sub.3) d=200.2, 175.8, 167.9, 128.0, 78.8, 62.1, 55.0, 45.2, 44.7, 43.6, 40.9, 39.2, 37.3, 33.7, 32.8, 32.7, 32.1, 30.7, 29.7, 28.1, 27.4, 27.3, 23.7, 23.4, 23.3, 19.0, 17.5, 16.2, 15.6, 14.2.

Oleana-2,12-dien-N-hydroxy-28-amide (Compound VI)

(46) Compound VI was obtained following the general synthetic scheme described above herein (hydroxamate formation), starting from the known precursor CAS num. 272108-04-0:

(47) ##STR00033##

(48) Yellow oil (48%). .sup.1H NMR (300 MHz, CDCl.sub.3): d=5.43-5.32 (m, 3H), 2.44 (d, J=11.3 Hz, 1H), 1.14 (s, 3H), 0.97 (s, 6H), 0.87 (s, 12H) (only readily peaks are reported); .sup.13C NMR (75 MHz, CDCl.sub.3) d=176.4, 144.5, 138.0, 124.1, 121.3, 51.9, 46.4, 46.1, 45.5, 42.1, 41.0, 40.7, 39.5, 36.1, 34.5, 34.0, 33.0, 31.9, 31.8, 31.6, 30.7, 27.2, 25.9, 25.7, 23.8, 23.5, 22.9, 19.6, 16.3, 15.6.

(3) 3-Hydroxy-N-hydroxy-lup-20(29)-en-28-amide (Compound VII)

(49) CAS num.: 1822375-07-4

(50) Off-white solid (55%). .sup.1H NMR (300 MHz, CDCl.sub.3): d: 4.73 (s, 1H), 4.60 (s, 1H), 3.20-3.15 (m, 1H), 3.05 (ddd, J=11.4, 6, 4.49 Hz, 1H), 1.67 (s, 3H), 0.95 (s, 6H), 0.92 (s, 3H), 0.80 (s, 3H), 0.74 (s, 3H) (only readily peaks are reported); .sup.13C NMR (75 MHz, CDCl.sub.3): d: 175.2, 150.9, 108.5, 78.4, 55.7, 54.3, 50.8, 50.7, 46.7, 42.3, 40.7, 38.7, 38.2, 37.8, 37.1, 36.9, 34.3, 32.6, 30.7, 29.3, 27.8, 26.9, 25.5, 20.8, 19.2, 18.2, 16.0, 15.9, 15.2, 14.5.

Lup-2-eno[2,3-d]isoxazol-N-hydroxy-28-amide (Compound VIII)

(51) Compound VIII was obtained following the general synthetic scheme described above herein (hydroxamate formation), starting from the known precursor CAS num. 620958-43-2:

(52) ##STR00034##

(53) White solid. .sup.1H NMR (300 MHz, CD.sub.3OD): d=10.36 (s, 1H, NH), 8.32 (s, 1H, OH), 8.26 (s, 1H), 4.67 (s, 1H), 4.55 (s, 1H), 3.00 (t, J=9.3 Hz, 1H), 2.61 (t, J=12.0 Hz, 1H), 1.64 (s, 3H), 1.40 (s, 3H), 1.22 (s, 3H), 1.11 (s, 3H), 0.93 (s, 3H), 0.74 (s, 3H) (only readily peaks are reported); .sup.13C NMR (75 MHz, CDCl.sub.3) d=172.6, 151.3, 151.0, 109.9, 109.4, 54.0, 53.3, 50.6, 49.0, 48.9, 46.7, 42.4, 38.9, 37.3, 35.6, 34.8, 33.4, 32.6, 30.9, 29.0, 25.7, 21.7, 19.5, 18.7, 16.4, 16.2, 14.8.

1H-Lup-20(29)-eno[3,2-c]pyrazol-N-hydroxy-28-amide (Compound IX)

(54) Compound IX was obtained according to the general synthetic scheme described above herein (hydroxamate formation), starting from the known precursor CAS num. 1334386-31-0:

(55) ##STR00035##

(56) Pale yellow solid. .sup.1H NMR (300 MHz, (CD.sub.3).sub.2CO): d=7.17 (s, 1H), 4.72 (s, 1H), 4.58 (s, 1H), 3.20-3.13 (m, 1H), 2.69-2.61 (m, 1H), 1.69 (s, 3H), 1.28 (s, 3H), 1.18 (s, 3H), 1.02 (s, 3H), 0.98 (s, 3H), 0.80 (s, 3H) (only readily peaks are reported); .sup.13C NMR (75 MHz, (CD.sub.3).sub.2CO) d=172.4, 151.0, 149.1, 132.8, 111.9, 109.0, 59.7, 50.0, 53.7, 50.5, 49.2, 46.8, 42.2, 40.7, 38.6, 37.9, 37.7, 36.6, 33.5, 33.4, 32.3, 30.8, 30.6, 25.7, 23.3, 21.4, 19.1, 18.7, 15.6, 14.2, 13.7.

Lupa-2,20(29)-dien-N-hydroxy-28-amide (Compound X)

(57) Compound X was obtained according to the general synthetic scheme described above herein (hydroxamate formation), starting from the known precursor CAS num. 173106-19-9:

(58) ##STR00036##

(59) Pale yellow solid (54%) .sup.1H NMR (300 MHz, CDCl.sub.3): d=5.38-5.28 (m, 2H), 4.69 (s, 1H), 4.56 (s, 1H), 3.01 (t, J=10.7 Hz, 1H), 2.37 (t, J=12.1 Hz, 1H), 1.63 (s, 3H), 1.21 (s, 3H), 0.93 (s, 3H), 0.89 (s, 3H), 0.82 (s, 3H), 0.81 (s, 3H) (only readily peaks are reported); .sup.13C NMR (75 MHz, CDCl.sub.3) d=175.0, 150.5, 137.9, 121.6, 109.65, 54.3, 52.1, 50.4, 49.2, 42.3, 40.8, 38.4, 37.9, 36.4, 34.6, 33.5, 32.8, 31.7, 30.9, 30.8, 29.7, 29.3, 25.6, 22.6, 19.5, 16.4, 15.8, 14.6, 14.5, 14.3.

3-Hydroxyimino-N-hydroxy-lup-20(29)-en-28-amideStep (b)(Compound XI)

(60) Prior to the general synthetic scheme described above herein (hydroxamate formation), the conversion of the hydroxyl group of the C-3 position of betulinic acid into a carbonyl group was carried out as follows:

(61) ##STR00037##

(62) To a solution of betulinic acid (1 gr, 2.19 mmol) in acetone/EtOAc 5:5 (10 mL) was added Jones reagent until the disappearance of the starting material (control by TLC). The reaction was washed with brine and extracted with EtOAc. The organic phases were dried over Na.sub.2SO.sub.4 and evaporated under vacuum and the crude was purified over silica gel (PE/EtOAc 9:1), 3-Oxo-lup-20(29)-en-28-acid (compound XVI; CAS num.: 4481-62-3) (89%) as an off-white solid.

(63) Compound XVI was then used to carry out the hydroxamate formation following the general synthetic scheme described above herein to obtain compound XI.

(64) Off-white solid (65%). .sup.1H NMR (300 MHz, CDCl.sub.3): d=4.73 (s, 1H), 4.61 (t, J=6.1 Hz, 1H), 3.04-3.00 (m, 2H), 2.32 (s, 1H), 1.68 (s, 3H), 1.24 (s, 6H), 1.22 (s, 3H), 1.12 (s, 3H), 0.97 (s, 3H), 0.96 (s, 3H), 0.91 (s, 3H) (only readily peaks are reported); 177.3, 167.3, 150.5, 109.6, 55.6, 55.0, 50.3, 50.2, 46.8, 42.5, 40.8, 40.2, 38.7, 38.2, 37.9, 37.2, 34.0, 33.3, 30.8, 29.4, 27.4, 25.6, 22.9, 21.5, 21.2, 19.4, 19.1, 16.1, 15.8, 14.6.

(3) 3-Hydroxy-N-hydroxy-urs-12-en-28-amide (Compound XII)

(65) CAS num.: 915415-61-1

(66) Off-white solid (55%). .sup.1H NMR (300 MHz, CDCl.sub.3): d=5.34 (brt, 1H, H-12), 3.18 (dd, J=9.5, 3.9 Hz, 1H), 2.09-1.92 (m, 3H), 1.06 (s, 3H), 0.94 (s, 3H), 0.90 (d, J=3.2 Hz, 3H), 0.87 (d, J=7.8 Hz, 3H), 0.80 (s, 3H), 0.78 (s, 3H), 0.75 (s, 3H) (only readily peaks are reported); .sup.13C NMR (75 MHz, CDCl.sub.3): d=176.8, 140.1, 126.6, 126.6, 79.0, 55.1, 52.2, 47.5, 42.4, 39.5, 39.5, 39.0, 38.8, 36.9, 28.1, 25.7, 23.3, 21.2, 17.2, 16.7, 15.7, 15.5.

3 Hydroxyimino-N-hydroxy-urs-12-en-28-amide (Compound XIII)

(67) Compound disclosed in patent CN102180939. Said compound was prepared from ursolic acid carrying out an analogous synthesis to compound XI, wherein in a first step the conversion of the C-3 hydroxyl to carbonyl using Jones reagent is carried out, followed by the hydroxamate formation according to the general synthetic scheme described above herein to obtain compound XIII.

(68) Pale yellow solid (70%). .sup.1H NMR (300 MHz, CDCl.sub.3): d=5.40 (brt, 1H), 3.07 (bdt, J=15.6, 1H), 2.12 (m, 1H), 1.24 (s, 3H), 1.15 (s, 3H), 1.08 (s, 3H), 1.06 (s, 3H), 1.03 (s, 3H), 0.94 (s, 3H), 0.81 (s, 3H) (only readily peaks are reported); .sup.13C NMR (75 MHz, CDCl.sub.3) d=177.3, 167.7, 140.6, 126.5, 55.7, 52.1, 47.0, 42.5, 40.2, 39.6, 39.4, 39.0, 38.5, 37.0, 36.7, 32.2, 30.6, 29.7, 27.7, 27.4, 24.8, 23.5, 23.4, 23.3, 21.1, 19.0, 17.3, 17.2, 16.8, 15.1.

(2,3) 2,3-Dihydroxy-N-hydroxy-olean-12-en-28-amide (Compound XIV)

(69) Compound XIV was obtained according to the general synthetic scheme described above herein (hydroxamate formation), starting from the known precursor CAS num.: 6089-92-5

(70) ##STR00038##

(71) Compound XIX was deacetylated to obtain compound XIV:

(72) ##STR00039##

(73) Off-white solid (45%). .sup.1H NMR (300 MHz, CDCl.sub.3): d=5.44 (brt, 1H), 3.72-3.61 (m, 1H), 2.99 (d, J=9.5 Hz, 1H), 2.45 (d, J=12.2 Hz, 1H), 1.15 (s, 3H), 1.02 (s, 3H), 0.98 (s, 3H), 0.90 (s, 3H), 0.87 (s, 3H), 0.82 (s, 3H), 0.78 (s, 3H) (only readily peaks are reported); .sup.13C NMR (75 MHz, CDCl.sub.3): d=175.9, 144.0, 123.6, 83.7, 68.2, 55.0, 47.3, 46.2, 46.1, 45.6, 41.2, 41.0, 39.0, 38.7, 37.8, 33.6, 32.6, 32.4, 32.3, 31.4, 28.5, 27.4, 27.3, 23.2, 23.1, 22.8, 18.6, 16.3, 16.5, 16.1.

2-Hydroxy-3-oxo-oleana-1,12-dien-N-hydroxy-28-amide (Compound XV)

(74) Prior to the general synthetic scheme described above herein (hydroxamate formation), the acetylation of the hydroxyl group of the C-2 position of a known maslinic acid derivative with CAS num. 1382923-75-2 was carried out as follows:

(75) ##STR00040##

(76) To a solution of the known maslinic acid derivative (1 eq/mol) in dry pyridine (10 mL per gr of acid) were sequentially added acetic anhydride (2 eq/mol) and DMAP (0.1 eq/mol). The reaction is stirred at room temperature until the disappearance of the starting material (control by TLC), quenched with methanol, diluted with H.sub.2SO.sub.4 sol. 2N and extracted with EtOAc. The organic phases were washed with brine, dried over Na.sub.2SO.sub.4 and evaporated under vacuum to give compound XVII without further purification.

(77) Compound XVII was then used to carry out the hydroxamate conversion following the general synthetic scheme described above herein as follows:

(78) ##STR00041##

(79) Compound XVII was then deacetylated to obtain compound XV as follows:

(80) ##STR00042##

(81) To a solution of compound XVII (1 eq/mol) in THF/MeOH 1:1 was added NaOH 4N (50 eq/mol). The mixture was heated at 40 C. overnight, quenched with H.sub.2SO.sub.4 sol. 2N and extracted with EtOAc. The organic phases were washed with brine, dried over Na.sub.2SO.sub.4 and evaporated under vacuum. The crude compound was purified over silica gel.

(82) Pale yellow solid (69%). .sup.1H NMR (300 MHz, CDCl.sub.3): d=6.31 (s, 1H), 5.47 (brt, 1H), 2.46 (d, J=11.3 Hz, 1H), 1.20 (s, 3H), 1.19 (s, 3H), 1.13 (s, 3H), 1.10 (s, 3H), 0.88 (s, 3H), 0.86 (s, 6H), (only readily peaks are reported); .sup.13C NMR (75 MHz, CDCl.sub.3) d=201.0, 176.4, 145.2, 143.8, 127.8, 123.2, 53.7, 46.1, 45.5, 44.0, 43.1, 42.4, 40.9, 40.1, 38.4, 33.9, 32.9, 32.0, 30.7, 27.1, 27.0, 25.8, 23.6, 23.4, 21.9, 20.8, 19.6, 18.7, 17.1, 14.2.

(83) As a comparative compound XX was synthesized from glycyrrhetinic acid (GA) by using a T3P/triethylamine protocol without the need of protecting groups (Ech-Chahad et al., Tetrahedron Letters 2005, 46: 5113-5115).

(84) ##STR00043##

(85) In Silico Assays

Example 2. Calculation of PHD2-Binding Affinity in Silico

(86) Molecular structures were obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/) or designed with MarvinSketch (ChemAxon, Cambridge, Mass.). The receptor model used was the PDB reference 4BQW. Binding properties were calculated by using the AutoDock4 (Morris et al., J. Comp. Chem. 2009 December; 30(16)2785-91) and the Vina software (Trott and Olson, J. Comp. Chem. 2010 Jan. 30; 31(2):455-61) with the virtual screening tool PyRx (Wolf L K. Chem. & Eng. News 2009, 87) The search space for the docking, around the receptor molecule surface, was set according to previous findings about several binding sites for different PHD2 ligands (Rabinowitz M H, J. Med. Chem. 2013 Dec. 12; 56(23):9369-4025).

(87) Once analysis has been performed, AutoDock Vina provides the estimated binding affinity value, which is the sum of the intermolecular energy, due to the interaction between both molecules, and the torsional free-energy penalty, due to the conformation adopted by these molecules to properly fit the interaction surface. A negative value indicates that bond is thermodynamically stable, whereas a positive value means instability.

(88) Table I shows the PHD2-binding energy (Kcal/mol) for the compounds described in the present invention and also for positive (IOX-2) and negative (glycyrrethinic acid) controls. Mean values from AutoDock-Vina using the structure 4BQW (from Protein Data Bank) are shown. Average values from three independent experiments are shown.

(89) TABLE-US-00001 TABLE I Energy binding of triterpenoids and derivatives to PDH2. COMPOUND B.E. AutoDock (Kcal/mol) Oleanolic acid 6.35 Compound II 6.33 Compound III 3.30 Compound IV 6.41 Compound V 3.35 Compound VI 5.27 Betulinic acid 4.76 Compound VII 6.55 Compound VIII 5.77 Compound IX 4.33 Compound X 7.08 Compound XI 6.09 Ursolic acid 5.74 Compound XII 6.01 Compound XIII 5.45 Maslinic acid 3.20 Compound XIV 4.30 Compound XV 3.57 IOX 2 9.05 Glycyrrethinic acid 1.24 PHD2-binding energy (Kcal/mol). Mean values from AutoDock/Vina using the structure 4BQW (from Protein Data Bank) are shown. Average values from three independent experiments are shown.

(90) Search space was restricted to a 300 volume around residues H313, D315, H374, R383, Y303, Y310, Y329, I327, I256 and M299, proposed as main binding sites by building a grid surpassing these space in 10 Amstrong along the three axes by (Rabinowitz M H, J. Med. Chem. 2013 Dec. 12; 56(23):9369-4025) (FIG. 1). The resulting binding energy found for Compound VII was 6.55 Kcal/mol, Ki 15.74 uM (at 298.15 K), intermolecular energy 7.38 Kcal/mol and root mean square deviation from atomic position 0.0 , thus improving the binding features found for its natural precursor betulinic acid, with a binding energy of 4.76 Kcal/mol.

(91) In Vitro and In Vivo Assays

Example 3. Selective Induction of HIF-1 Activity

(92) To investigate the biological activities of the different compounds, HIF-1 transactivation assays were performed either in NIH-3T3-EPO-Luc cells (Table II) or in HaCaT-EPO-luc cells (FIG. 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. 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. DFX was purchased from Sigma-Aldrich (USA). Cells (110.sup.4/well in 96 well plates) were seeded the day before the assay. The next day, the cells were stimulated either with increasing concentrations of either Oleanolic acid (OA), Betulinic acid (BA), Ursolic acid (UA), Maslinic acid (MA) Glycyrrethinic acid (GA), compounds of Formula (I), II to XV, or comparative compounds XX and CDDO-Me. 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 is measured using a microplate luminometer (Berthold) following the instructions of the luciferase assay kit (Promega, Madison, Wis., USA). The RLUs are calculated and the EC50 and IRA (Intrinsic relative activity) values were determined relative to 150 M deferoxamine (DFX) using the following equation: IRA coefficient=(EC.sub.50-DFXE.sub.max)/(EC.sub.50E.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 II).

(93) None of the natural triterpenoids (OA; Olanolic acid, BA; Betulinic acid; UA, Ursolic acid, and MA; Maslimic acid) used as templates for the synthesis of the compounds included in the present invention were able to activate the EPO promoter as a surrogate marker of HIF-1 activation. In contrast all the triterpenoid derivatives of present invention clearly activated the HIF-1 pathway (Table II). Moreover, as shown in Table II below, compounds II to VI which are oleanolic acid derivatives of Formula I provide activation of the HIF pathway, while in contrast, an oleanolic derivative such as CDDO-Me, comprising a cyanide group at position 2 (corresponding to position B of Formula I) does not result in activation of the HIF pathway.

(94) Thus, it can be concluded that the chemical modifications introduced in the compounds described are critical to inhibit the enzymatic activity of PHD2, and as a consequence, to activate the HIF pathway, but not for the binding of the compounds to the protein.

(95) Table II also shows that GA and its hydroxamate derivative compound XX, do not activate the HIF-1 pathway. Thus, particularly, it can be concluded that the chemical modifications introduced at the position 28 of the backbone defined in the present invention is critical to inhibit the enzymatic activity of PHD2, and as a consequence, to activate the HIF pathway, but not for the binding of the compounds to the protein.

(96) TABLE-US-00002 TABLE II HIF-1 transactivation assays in NIH-3T3-EPO Luc fibroblast cells. Efficacy HIF-1 Potency EC.sub.50 Compound (IRA coefficient) HIF-1 (M) OA (>50) II 0.39 16.37 III 0.43 3.83 IV 0.15 11.39 V 0.16 6.71 VI 0.16 5.00 BA (>50) VII 0.36 4.81 VIII 0.55 2.58 IX 0.34 2.41 X 0.48 3.24 XI 0.31 2.58 UA (>50) XII 0.17 7.69 XIII 0.10 8.93 MA (>50) XIV 0.10 7.10 XV 0.06 11.22 GA (>50) XX (>50) CDDOMe (>50) 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-1 activation is shown.

(97) Next, the activity of the compound in another cell type such as the keratinocyte cell line HaCaT-EPO-Luc was studied. The 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 (110.sup.5/well in 24 well plates) were seeded the day before the assay and then stimulated with either DFX (150 M) or with increasing concentrations of compounds II to XV for 6 h. Then, 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. Luciferase activity was measured in the cell lysate using a TriStar LB 941 multimode microplate reader (Berthold) following the instructions of the Luciferase Assay Kit (Promega, Madison, Wis., USA). The above assay is illustrated by FIG. 2, which shows the hypoximimetic effects of DFX and compounds II to XV in HaCaT-EPO-Luc cells. Data are given as percentage of activation considering DFX (150 M) as 100% induction over untreated cells. A significant increase in luciferase activity was seen with all triterpenoid derivatives as compared with untreated cells.

(98) To further study the target selectivity of the compounds described in the present invention the effect of the natural triterpenoids (OA; BA, UA, and MA), compounds II to XV of Formula (I) disclosed in present invention, and of comparative compound CDDO-Me on NF-B inhibition, STAT-3 inhibition, Nrf2 activation and TGR5 activation was analyzed. For this study cell lines NIH-3T3-KBF-Luc, HeLa-STAT3-Luc, HaCaT-ARE-Luc and CHO-TGR5-CRE-luc were used respectively. The NIH3T3-KBF-Luc cell line stably transfected with the plasmid KBF-Luc plasmid, which contains three copies of NF-B binding site (from major histocompatibility complex promoter), fused to a minimal simian virus 40 promoter driving the luciferase gene. Cells (110.sup.4/well) were seeded in 96-well plates, treated with increasing concentrations of the compounds II to XV for 15 min and then stimulated with 30 ng/ml TNF. After 6 h the luciferase activity in the cell lysates was measured as indicated above. The RLU is calculated and the results are expressed as percentage of inhibition of NF-B activity induced by TNF (100% activation) (Table III).

(99) TABLE-US-00003 TABLE III Effects on NF-B, STAT-3, Nrf2 and TGR5 pathways. IC.sub.50 IC.sub.50 EC.sub.50 IC.sub.50 EC.sub.50 NF-B STAT3 NRF2 NRF2 TGR5 Compound (M) (M) (M) (M) (M) OA (>50) (>50) (>50) (>50) 18.90 II (>50) (>50) (>50) (>50) (>50) III (>50) (>50) 23.93 (>50) (>50) IV (>50) (>50) 10.04 (>50) (>50) V (>50) (>50) (>50) (>50) (>50) VI (>50) (>50) (>50) (>50) (>50) BA (>50) (>50) 9.02 (>50) 22.15 VII (>50) (>50) (>50) (>50) (>50) VIII (>50) (>50) (>50) (>50) 9.62 XI (>50) (>50) (>50) (>50) 17.50 X (>50) (>50) (>50) (>50) (>50) XI (>50) (>50) (>50) (>50) 17.50 UA (>50) (>50) 38.36 (>50) 11.46 XII (>50) (>50) (>50) (>50) 5.91 XIII (>50) (>50) (>50) (>50) (>50) MA (>50) (>50) (>50) (>50) 10.19 XIV (>50) (>50) (>50) (>50) (>50) XV (>50) (>50) (>50) (>50) (>50) CDDOMe 1.20 2.38 40.94 0.06 (>50) Effect of compounds II to XV, OA; BA, UA, MA and of comparative compound CDDOMe, on NF-B inhibition (IC50), STAT-3 inhibition (IC50), Nrf2 activation (EC50) and inhibition (IC50) and TGR5 activation (EC50) we used the cell lines NIH-3T3-KBF-Luc, HeLa-STAT3-Luc, HaCaT-ARE-Luc and CHO-TGR5-CRE-luc respectively. The IC50 and EC50 data are shown.

(100) The HeLa-STAT3-luc cells stably transfected with the plasmid 4M67 pTATA TK-Luc. Cells (2010.sup.3 cells/ml) were seeded in 96-well plates, treated with increasing concentrations of the compounds II to XIV, and of comparative compound CDDO-Me, for 15 min and then stimulated with IFN- 25 IU/ml. After 6 h the luciferase activity in the cell lysates was measured as indicated above. The RLU was calculated and the results expressed as percentage of inhibition of STAT3 activity induced by IFN- (100% activation) (Table III). The HaCaT-ARE-Luc cell line contains the Nqo1 Antioxidant Response Element (ARE)-Luc reporter plasmid. ARE is activated by all members of the CNC family of factors (Nrf1, Nrf2, Nrf3 and p45 NF-E2). The cells were cultivated in 96-well plates at the concentration of 2510.sup.3 cells/well in a CO.sub.2 incubator at 37 C. For induction of Nrf2 activation the cells were treated with increasing concentrations of the compounds II to XV, and of comparative compound CDDO-Me, for 6 h. As a positive control the cells were treated with 0.02 mM of the antioxidant Tert-butyl-hydroquinone (TBHQ). Luciferase activity in the cell lysates was measured as indicated above and the EC50 calculated (Table III). The CHO-TGR5-CRE-Luc cells stably transfected with the pTGR5 and CRE-Luc. The CRE-responsive luciferase construct encodes the firefly luciferase reporter gene under the control of a minimal (m)CMV promoter and tandem repeats of the CRE transcriptional response element (TRE) and is useful to monitor cAMP signaling pathways activated by TGR5 agonists. Cells (110.sup.4/well) were seeded in 96-well plates, treated with increasing concentrations of the compounds II to XV and CDDO-Me, for 6 h. As a positive control the cells were treated with 10 M of LCA (lithocholic acid). Luciferase activity in the cell lysates was measured as indicated above and the EC50 calculated (Table III).

(101) None of the compounds inhibited the NF-B and STAT3 pathways induced by TNF and IFN respectively. In addition, none of the compounds activated the Nrf2 pathway and only compounds VIII, XI and XII showed agonistic TGR5 activity (Table III). In contrast, CDDO-Me clearly inhibited NF-B and STAT-3 signaling pathways and activated the Nrf2 pathway. This result further demonstrated that the cyanide group is critical for some biological activities but is not required to activate the HIF-1 pathway (Table III).

Example 4. Triterpenoid Derivatives Stabilize HIF-1 and HIF-2

(102) To gain insight into the regulation of HIF-1 stabilization by the compounds described in the present invention the effect on HIF-1 expression in different cell types was investigated. Human oligodendrocyte MO13.3 cells were stimulated for 3 h with either 150 DFX or 10 M of Olenaolic acid (OA), compounds II, III, IV, V, VI (FIG. 3A), betulinic acid (BA), compounds VII, VIII, IX, X, XI (FIG. 3B), ursolic acid (UA), maslinic acid (MA), compounds XII, XIII, XIV and XV (FIG. 3C). After that, the cells were washed with PBS and incubated in 50 l 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 g/ml leupeptine, 1 g/ml pepstatin and aprotinin, and 1 l/ml PMSF saturated. After centrifugation the supernatants were mixed with SDS sample buffer and boiled at 95 C. Proteins were electrophoresed 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-1 (610959) was from BD Biosciences and the antibody anti--actin (AC-74) was purchased from Sigma-Aldrich (Saint Louis, Mo., USA).

(103) All the compounds described in the present invention elevated HIF-1 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 HIF-1 (FIGS. 3A, 3B and 3C).

(104) Next, Human Embryonic Kidney 293 cells (293T) were stimulated with the increasing concentrations of compound VII or with DFX (150 M) during 3 h. After that, proteins isolation and western blots were performed as in FIG. 3. The antibody against HIF-1 (610959) was from BD Biosciences (USA), the antibodies anti-PHD1 (ab80361) and anti-PHD2 (ab109088) were from Abcam (Cambrigde, UK), and the antibody anti--actin (AC-74) was purchased from Sigma-Aldrich (Saint Louis, Mo., USA).

(105) The results clearly show that compound VII, as a representative of the compounds described in the present invention stabilized HIF-1 expression without affecting the expression of PDH1 and PDH2 (FIG. 4).

(106) Since PDH2 and PDH3 also regulate the expression of HIF-2, the effect of compound VII on HIF-2 stabilization in Human Islet-Derived Precursor Cells (hIPCs) obtained from Innoprot SL (Spain) (reference p10472) was investigated. hIPCs were stimulated with the increasing concentrations of compound VII or with DFX (150 M) during 3 h. After that, proteins isolation and western blots were performed as in FIG. 3. The antibodies against HIF-2 (ab8365) and PHD3 (ab30782) were from Abcam (Cambrigde, UK), and the antibody anti--actin (AC-74) was purchased from Sigma-Aldrich (Saint Louis, Mo., USA). (FIG. 5).

(107) The results clearly show that compound VII, as a representative of the compounds described in the present invention stabilized HIF-2 expression without affecting the expression of PHD3 (FIG. 5).

(108) Altogether, results indicate that compound VII binds PDH2 inhibiting its activity and as consequence HIF-1 and HIF-2 protein levels are stabilized.

Example 5. Compound VII Induces Angiogenesis

(109) To test the functional consequences of compound VII 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 VII (1 and 2.5 M) or VEGF (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.).

(110) In FIG. 6 it is shown that compound VII 1 M as well as the positive controls (rFGF; 10 ng/ml and VEGFA; 10 ng/ml) increased significantly the network length in HUVEC cells.

Example 6. Compound VII Increase the Plasma Levels of Erythropoietin (EPO)

(111) Erythropoietin (EPO) is one of the earliest described and most sensitive HIF target genes; being positively regulated at the transcription level. Here, the ability of compound VII to regulated EPO levels in vivo was examined. Sixteen-week-old C57BL/6 male mice were treated intraperitoneally (i.p.) with Betulinic Acid (60 mg/kg) or compound VII (30 mg/kg or 60 mg/kg). Blood samples were taken under general anaesthesia 4 hours after treatment and the circulating levels of EPO in plasma were quantified using a mouse EPO ELISA kit (R&D Systems) according to manufacturer's instructions. EPO values represent the meanSEM (n=3).

(112) As shown in FIG. 7 in vivo administration of compound VII, as a representative of the compounds described in the present invention, in mice (30 and 60 mg/kg/day) strongly increased circulating EPO plasma levels. In contrast Betulinic acid (BA), the parental of compound VII, did not influence the levels of EPO in plasma.

Example 7. Effect of Compound VII on 3-NP-Induced Cytotoxicity in Striatal Cells, and Prevention of 3-NP Induced Huntington's Disease in Mice

(113) 7.1: Effect of Compound VII on 3-NP-Induced Cytotoxicity in Striatal Cells

(114) To investigate whether HIF activation in striatal Q7 and Q111 cells is sufficient to protect striatal neurons (provide neuroprotection), the effect of compound VII on 3-NP induced death was examined. In particular, the effect of 6 h pre-treatment with compound VII on 24 h of 3-Nitropropionic acid (3-NP) exposure in STHdh.sup.Q7/Q7 (striatal Q7) and STHdh.sup.Q111/Q111 (striatal Q111) cells was studied.

(115) STHdh.sup.Q111/Q111 cells express a mutated form of the huntingtin protein and STHdh.sup.Q7/Q7 cells express the wild type form of this protein. Clonal striatal cell lines established from E14 striatal primordia of Hdh.sup.Q111/Q111 (mutant) and Hdh.sup.Q7/Q7 (wild-type) knock-in mouse littermates were immortalized using a replication defective retrovirus transducing the tsA58/U19 large T-antigen (Trettel F. et al., Hum Mol Genet. 2000; 9:2799-2809). Striatal Q7 and Q111 cells were maintained in Dulbecco's modified Eagle medium (DMEM) containing 25 mM D-glucose, 1 mM L-glutamine, 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, and 400 g/mL Geneticin and were incubated at 33 C. with 5% CO.sub.2.

(116) 3-Nitropropionic acid (3-NP) is a potent irreversible inhibitor of mitochondrial complex II enzyme and leads to mitochondrial dysfunction and oxidative stress.

(117) STHdh.sup.Q7/Q7 and STHdh.sup.Q111/Q111 cells (10.sup.4 cells/well in 96-well plates) were incubated with YOYO-1 (Life Technologies) and then treated with 3-NP (10 mM) and or DFX (50 M) as a positive control for PHDs inhibition. YOYO-1 is diluted in cell culture medium and added to a final concentration of 0.1 M to both experimental and control wells. YOYO-1 is a cell impermeant cyanine dimer nucleic acid stain that can only enter cells with a compromised plasma membrane and fluorescently stain the nuclear DNA. The uptake of YOYO-1 by damaged cells correlates with the increase in YOYO-1 fluorescence. Treated cells are placed in an Incucyte FLR imaging system, and the YOYO-1 fluorescence is measured after 24 h of 3-NP treatment. Following the incubation period object counting analysis was performed using the Incucyte FLR software to calculate the total number of YOYO-1 fluorescence positive cells and total DNA containing objects (end point). The cytotoxicity index is calculated by dividing the number of YOYO-1 fluorescence positive objects by the total number of DNA containing objects for each treatment group and converted to percentage of cell death.

(118) STHdh.sup.Q111/Q111 cells were more sensitive than STHdh.sup.Q7/Q7 cells to the exposure to 3-NP and compound VII clearly provided a significant level of neuroprotection to 3-NP-induced cytotoxicity (FIG. 8). Although to a lesser extent, DFX also protected the cells from the cytotoxic activity of 3-NP.

(119) 7.2: Prevention of 3-NP Induced Huntington's Disease in Mice

(120) The intoxication of mice with 3-Nitropropionic acid (3-NP), results in a myriad of neurological, biochemical and histological effects that are reminiscent of some aspects of Huntington disease (HD) pathology. 3NP-treated mice exhibited high scores in hindlimb clasping, dystonia, kyphosis and in general locomotor activity compared to control animals (non-intoxicated with 3-NP).

(121) Striatal neurodegeneration was induced in 16-week-old C57BL/6 male mice (Harlan Ibrica, Barcelona, Spain) by six intraperitoneal (i.p.) injections of 3-nitropropionic acid (3NP) (30 mg/kg; one injection each every 12 h prepared in PBS). 3NP-treated animals and the non-lesioned control group (injected with PBS) were used for pharmacological studies with Betulinic acid (30 mg/Kg) and compound VII (30 mg/Kg). Treatments consisted of 5 i.p. injections every 24 h with 50 mg/kg the test compounds or vehicle (10% DMSO plus 6.2% Tween 20 in saline buffer), with the first injection 24 h prior the first 3NP injection and the rest of doses 30 min before the injections of 3NP. Mice were subjected to behavioral tests for determining their neurological status. General locomotor activity, the hindlimb clasping and dystonia, and the truncal dystonia were evaluated. All behavioral tests were conducted prior to drug injections to avoid acute effects of the compounds under investigation. All behavioral tests were conducted prior to drug injections to avoid acute effects of the compounds under investigation and all animals were euthanized 12 h after the last injection of 3NP. Once euthanized, animals were dissected and their brains were rapidly removed. The right hemisphere was used to dissect the striatum, which was quickly frozen in RNAlater (Sigma-Aldrich, Germany) to analyzed inflammatory markers were by Real Time PCR. The left hemisphere was fixed in fresh 4% paraformaldehyde (in 0.1M phosphate buffered-saline) for 48 hours at 4 C. and embedded in paraffin wax for histological analysis.

(122) FIG. 9 shows that compound VII clearly alleviated the clinical symptoms induced by 3-NP intoxication. Betulinic acid (BA) also showed some neuroprotective activity although to a lesser extent.

(123) Next, striatal parenchyma of 3NP-lesioned mice was also used for analysis of some histological and molecular markers related to inflammation and neurodegeneration, which are affected in this experimental model. The striatal parenchyma of these 3NP-treated animals showed an important reduction in Nissl-stained cells, which indicates an important degree of neuronal death caused by 3NP, that was clearly prevented by treatment with compound VII but not by Betulinic acid (BA) (FIG. 10).

(124) In addition, compound VII-mediated neuroprotection was associated with reduced 3NP-induced microgliosis and astrogliosis as determined by Iba1 and GFAP immunohistochemistry (FIG. 11). For this test, intoxicated mice with 3-Nitropropionic acid (3-NP) were used for determining microglia activation and astrogliosis including a Control with non-intoxicated mice (3NP, 3NP+BA and 3NP+Compound VII). Iba-1 and glial fibrillary acidic protein (GFAP) expression were determined by immunostaining of brain sections through the different group of mice. Quantification of the different markers was performed with Image J software. Total average number of microglia (Iba1.sup.+) and astrocytes (GFAP.sup.+) is shown in FIG. 11.

(125) Finally, the expression of inflammatory enzymes COX-2 and iNOs was significantly up regulated in 3NP-lesioned mice in parallel to increased expression of proinflammatory cytokines IL-1 and IL-6. The mRNA expression for COX-2 (FIG. 12D), IL-1 (FIG. 12B), IL-6 (FIG. 12A) and iNOS (FIG. 12C), was down regulated in 3NP+compound VII (30 mg/kg) treated mice compared with 3NP+Vehicle mice. Betulinic acid (BA) treatment (30 mg/kg) also inhibited the expression of inflammatory markers (as shown in FIG. 12). Expression levels were calculated using the 2.sup.Ct method. Values are expressed as meansSEM for 6 animals per group

Example 8. Real-Time Quantitative PCR Used in the Invention (Example 7)

(126) Total RNA was isolated from striata (3NP model) using RNeasy Lipid Tissue Mini Kit (Qiagen, GmbH). The total amount of RNA extracted was quantitated by spectrometry at 260 nm and its purity from the ratio between the absorbance values at 260 and 280 nm. Genomic DNA was removed to eliminate DNA contamination. Single-stranded complementary DNA was synthesized from up to 1 g of total RNA (pool from at least 3 animals per group) 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 COX-2, IL-6, IL-1, and iNOS. 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.Ct method. Sequences of oligonucleotide primers are given in Table IV.

(127) TABLE-US-00004 TABLEIV Listofmouseprimersequencesusedin quantitativePolymeraseChainReaction. Gene Forward Reverse IL-6 5-GAACAACGATG 5-TCCAGGTAGCT ATGCACTTGC-3 ATGGTACTCC-3 iNOS 5-AACGGAGAAC 5-CAGCACAAGG GTTGGATTTG-3 GGTTTTCTTC-3 COX-2 5-TGAGCAACTAT 5-GCACGTAGTCTT TCCAAACCAGC-3 CGATCACTATC-3 IL-I 5-CTCCACCTCA 5-GCCGTCTTTC ATGGACAGAA-3 ATTACACAGG-3 GAPDH 5-TGGCAAAGTGG 5-AAGATGGTGAT AGATTGTTGCC-3 GGGCTTCCCG-3

(128) The present results substantiate the therapeutic use of the compounds described in the present inventions, for the clinical management of conditions and diseases the treatment of which is responsive to HIF activation such as stroke, cerebral palsy, traumatic injuries, neurodegenerative diseases such as Multiple Sclerosis, Huntington disease, Alzheimer disease and Parkinson disease, IBD, myocardial ischaemia-reperfusion injury, acute lung injury, organ transplantation, acute kidney injury and arterial diseases.

Example 9. Effect of Compound VII on a High Fat Diet (HFD) Model of Diabetes and Metabolic Pathologies

(129) High-fat diet-fed mouse is a widely used model for impaired glucose tolerance (IGT) and type 2 diabetes. Eight-week-old male C57BL/6 mice (Charles River-France) were used for experiments. Mice were maintained under controlled conditions [12 h light/dark cycle; temperature 20 C. (2 C.) and 40-50% relative humidity] with free access to tap water and standard rodent chow ad libitum. After 1 week of acclimatization, mice were divided into two groups and fed ad libitum with a standard dietCD- (Code U8220G10R, SAFE Diets, Augy, France) or a High-Fat dietHFD- (D12451; Research Diets, New Brunswick, N.J.) in order to induce obesity for 15 weeks (diet-induced obesity, DIO). In order to assess the potential beneficial metabolic effects of compound VII in this model of DIO, pharmacological administration of the compound (30 mg/kg body weight) was performed by intraperitoneal (i.p.) injection every 24 h from 12.sup.th to 15.sup.th week of diet exposure. Control animals received the corresponding vehicle injections (1:1:18 Ethanol:Cremophor:Saline).

(130) To ensure the effectiveness of HFD feeding regimen and assess the efficacy of compounds in terms of amelioration of the metabolic phenotype, body composition analyses of fat and lean masses were performed by QMR (quantitative magnetic resonance) using the EchoMRI 700 analyzer (Houston, Tex., software v. 2.0). In this sense, MRI scans were taken in three time points during the experimental period: before starting diet exposure, after 12 week of feeding regimen (coinciding with start of treatment) and at the end of experimental procedures (on the 15.sup.th week of diet exposure). Additionally, five days before sacrifice, glucose tolerance test (GTT) was performed in overnight food-deprived mice. To this end, mice were injected intraperitoneally with 2 mg of glucose/g body weight and blood glucose was measured before and 30, 60 and 120 min after injection utilizing an Accu-Chek Advantage (Roche) glucometer. At the end of the experiment, mice were sacrificed and blood samples were collected using EDTA-coated tubes and plasma stored at 80 C. Plasma triglycerides were measured using and enzymatic colorimetric kit (QCA, Barcelona, Spain).

(131) We found that compound VII alleviated weight gain (FIG. 13A), increase in Fat Mass (FIG. 13B) and adiposity (FIG. 13C). Moreover, compound VII improved glucose tolerance (FIG. 14) and normalize the plasma levels of triglycerides (FIG. 15) in HFD mice. Our results are in agreement with previous report showing that PDH inhibitors improves glucose and lipid metabolism in murine models of obesity and diabetes induced by HFD (Rahtu-Korpela et al., Diabetes. 2014 October; 63(10):3324-33). Therefore, said results indicate that the compounds of the invention are useful in the treatment of diabetes or other related metabolic pathologies such as hypertriglyceridemia.