Geranyl geranyl acetone analogs and uses thereof

Abstract

The invention relates to novel therapeutic compounds, more in particular to biologically active analogs and uses thereof as medicament, for instance for the treatment of atrial fibrillation. Provided is a compound of the general formula (formula I) wherein R.sub.1 is H or a saturated or unsaturated aliphatic moiety comprising 1 to 8 C-atoms; and X is selected from the group consisting of moieties X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5 and X.sub.6. Exemplary uses include the prevention or therapeutic treatment of a HSF1-mediated disease. ##STR00001##

Claims

1. A pharmaceutical composition comprising a compound of the general formula I ##STR00023## wherein R.sub.1 is H or a saturated or unsaturated aliphatic moiety comprising 1 to 8 C-atoms; and wherein R.sub.10 is a C.sub.1-C.sub.5 alkyl group, optionally substituted with one or more selected from the group consisting of halogen, oxo, sulfo, hydroxyl, CN, alkoxy, amino, amido, aryl, substituted aryl, hetero-aryl, substituted hetero-aryl, cyclo-alkyl, heterocycloalkyl, nitro, carboxylic acid and carboxylic ester; or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier and/or adjuvant.

2. A method of treatment of a disease which is associated with loss of proteostatic control, including the accumulation of misfolded proteins and/or the presence of damaged proteins, comprising administering to a subject in need thereof a pharmaceutical composition according to claim 1.

3. A method of treatment of a disease which is associated with ischemia/reperfusion injury, comprising administering to a subject in need thereof a pharmaceutical composition according to claim 1.

4. A method for the treatment of a disease which is associated with loss of proteostatic control, including the accumulation of misfolded proteins and/or the presence of damaged proteins or a disease which is associated with ischemia/reperfusion injury, comprising administering to a subject in need thereof a pharmaceutical composition according to claim 1.

5. The pharmaceutical composition of claim 1, wherein R.sub.10 is a C.sub.1-C.sub.5 alkyl group, optionally substituted with one, two, or three halogens.

6. The pharmaceutical composition of claim 1, wherein the compound of general formula (I) is compound 51 or compound 52: ##STR00024##

7. The method of claim 4, wherein the disease is supraventricular arrhythmia.

8. The method of claim 4, wherein the pharmaceutical composition comprises compound 51 or compound 52: ##STR00025##

Description

LEGEND TO THE FIGURES

(1) FIG. 1: Exemplary compounds according to Formula I wherein X is X.sub.1.

(2) FIG. 2: Exemplary compounds according to Formula I wherein X is X.sub.2.

(3) FIG. 3: Exemplary compound according to Formula I wherein X is X.sub.3.

(4) FIG. 4: Exemplary compounds according to Formula I wherein X is X.sub.4.

(5) FIG. 5: Exemplary compounds according to Formula I wherein X is X.sub.5.

(6) FIG. 6: Exemplary compounds according to Formula I wherein X is X.sub.6.

(7) FIG. 7: Exemplary compounds according to Formula II.

(8) FIG. 8: Hsp70 induction in renal tubular cells by various GGA-like compounds. GGA was used as positive control. For details see Example 3. Panel A: Exemplary Western blot analysis. Panel B: densitometric analysis of the signal intensity for Hsp70 normalized to actin.

EXAMPLES

Example 1: Synthesis of Exemplary Analogs

(9) Synthetic Route for Compound 76 (Taken as an Example)

(10) ##STR00018##

Synthesis of (E)-1-bromo-3,7-dimethylocta-2,6-diene

(11) Geraniol (10 g, 11.4 mL, 64.8 mmol, 1.0 eq) was dissolved in DCM (50 mL) under a nitrogen atmosphere. The solution was cooled to 20 C. A solution of PBr.sub.3 (3.0 mL, 32.4 mmol, 0.5 eq) in DCM (10 mL) was added drop-wise, keeping the temperature <16 C. The color changed to green/blue during addition. The reaction mixture was stirred for 3 h at 20 C. Water (50 mL) was added carefully at 40 C. The water layer was extracted with Et.sub.2O (350 mL). The combined organic layers were washed with sat. aq. NH.sub.4Cl. sol. (350 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo to provide a brown oil (14.5 g, 66.6 mmol, quant.)

Synthesis of (E)-Ethyl 7,11-dimethyl-3-oxododeca-6,10-dienoate (Compound 21)

(12) NaH (60% in oil, 2.63 g, 65.8 mmol, 1.0 eq) was suspended in THF (20 mL), under a nitrogen atmosphere. The solution was cooled to 0 C. with an ice/water bath. Ethylacetoacetate (8.3 mL, 65.8 mmol, 1.0 eq) was added drop-wise in 1 h. During addition a thick suspension was formed which later became a yellow solution. Gas formation was visible and an exothermic reaction was observed. The temperature was kept <10 C. The reaction mixture was stirred for 10 min at 0 C. n-BuLi (2.5M in hexanes, 26.3 mL, 65.8 mmol, 1.0 eq) was added drop-wise in 10 min. An exothermic reaction was observed. During addition the flask was cooled with an ice/MeOH bath. A bright yellow suspension was formed which changed into a yellow solution. The temperature was kept at 0 C. for 10 min. Geranylbromide (9.1 mL, 46.1 mmol, 0.7 eq) was added drop-wise and a suspension was formed. The reaction mixture was warmed to room temperature and stirred for 1 h. The reaction mixture was poured in sat. aq. NH.sub.4Cl sol. (100 mL) and the water layer was extracted with TBME (3100 mL). The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated in vacuo to provide a yellow oil (17.1 g). The crude product was purified by automated column chromatography (eluents, 0 to 100% DCM in heptanes) affording a colorless oil (6.0 g, 22.4 mmol, 49%).

(13) .sup.1H-NMR (CDCl.sub.3, in ppm)): 1.30 (t, 3H), 1.60 (s, 6H), 1.69 (s, 3H), 2.02 (m, 4H), 2.31 (m, 2H), 2.60 (t, 2H), 3.44 (s, 2H), 4.10 (q, 2H), 5.06 (t, 2H). M+=267.

Synthesis of (E)-ethyl 2,2,7,11-tetramethyl-3-oxododeca-6,10-dienoate (Compound 76)

(14) NaH (70 mg, 1.8 mmol, 2.6 eq) was suspended in TFH (6 mL), under a nitrogen atmosphere, and cooled to 0 C. with an ice/water bath. A solution of compound 21 (180 mg, 0.68 mmol, 1.0 eq) in THF (20 mL) was added and allowed to warm to room temperature and subsequently stirred for 30 min. A solution of MeI (101 L, 1.6 mmol, 2.4 eq) in THF (20 mL) was added and the reaction mixture was heated to reflux temperature and stirred overnight. The solvents were removed in vacuo. Water (30 mL) was added and the water layer was extracted with TBME (330 mL) and EtOAc (130 mL). The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude product was purified by automated column chromatography (eluens 0 to 100% DCM in heptanes) affording a colorless oil (20.1 mg, 0.068 mmol, 10%)

(15) .sup.1H-NMR (CDCl.sub.3, in ppm)): 1.25 (t, 3H), 1.35 (s, 6H), 1.60 (s, 6H), 1.67 (s, 3H), 2.01 (m, 4H), 2.25 (m, 2H), 2.49 (t, 2H), 4.20 (q, 2H), 5.04 (t, 2H). M+=295.

(16) Synthetic Route for Compound 28 (Taken as an Example)

(17) ##STR00019##

Synthesis of (E)-8,12-Dimethyltrideca-7,11-Diene-2,4-Dione (Compound 28)

(18) Sodium hydride (60%, 2 g, ca. 50 mmol) was added under nitrogen atmosphere to THF (125 mL) while stirring. The suspension was cooled to 0 C. for 10 minutes and 2,4-pentanedione (5 g, 50 mmol) was added drop-wise over ca. 3 minutes. A slightly exothermic reaction took place, resulting in some gas evolution and a thick white suspension was obtained. nBuLi (19 mL, 2.5 M in hexanes) was added in ca. 15 seconds with a plastic syringe. A slightly yellow, clear solution was obtained. After 20 minutes geranyl bromide (7.3 g, 33.62 mmol) was added. The resulting suspension was stirred while warming to room temperature for 1 hour. The reaction mixture was quenched with sat. aq. NH.sub.4Cl (70 mL). Extraction with TBME (2150 mL), drying of the organic fractions with Na.sub.2SO.sub.4 and concentration under vacuum gave the crude product (9.66 g, >100%). Purification of a small sample by ISCO chromatography afforded (E)-8,12-dimethyltrideca-7,11-diene-2,4-dione (63 mg) as an oil.

(19) .sup.1H-NMR (CDCl.sub.3, in ppm)): 1.60 (s, 6H), 1.69 (s, 3H), 2.01 (m, 4H), 2.10 (s, 3H), 2.30 (m, 4H), 3.60 (s, 2H), 5.08 (t, 2H). M+=237.

(20) Synthetic Route for Compound 62 (Taken as an Example)

(21) ##STR00020##

Synthesis of (E)-5-(4,8-dimethylnona-3,7-dien-1-yl)-1H-pyrazol-3-ol (Compound 62)

(22) Compound 21 (500 mg, 1.88 mmol, 1.0 eq) was dissolved in EtOH (10 mL), under a nitrogen atmosphere. The solution was cooled to 0 C. with an ice/water bath. Hydrazine (64% water, 0.12 mL, 2.44 mmol, 1.3 eq) was added and the solution was warmed to room temperature overnight. The solvents were removed in vacuo and Et.sub.2O (10 mL) was added to the residue. The solids were isolated by filtration and purified by automated column chromatography (eluens, 50 to 100% EtOAc in heptanes) yielding a white solid (81 mg, 0.35 mmol, 18%).

(23) .sup.1H-NMR (CDCl.sub.3, in ppm)): 1.61 (s, 6H), 1.65 (s, 3H), 2.05 (m, 4H), 2.30-2.65 (m, 4H), 3.60 (s, 2H), 5.06 (m, 2H), 5.48 (s, 1H). M+=235.

(24) Synthetic Route for Compound 51 and 52 (Compound 51 is Taken as an Example).

(25) ##STR00021##

Synthesis of (E)-N-(3,7-Dimethylocta-2,6-dien-1-yl)methanesulfonamide (Compound 51)

(26) A solution of geranylamine (306 mg, 2.0 mmol) and triethylamine (360 mg, 3.6 mmol) in dichloromethane (2 mL) was cooled to 0 C. with an ice bath and methanesulphonyl chloride (229 mg, 2 mmol) was added. After stirring overnight while warming to room temperature water (10 mL) was added. Extraction with dichloromethane (210 mL), drying of the combined organic layers with Na.sub.2SO.sub.4 and concentration under vacuum provided crude compound 51. Purification by ISCO chromatography afforded (E)-N-(3,7-dimethylocta-2,6-dien-1-yl)methanesulfonamide (208 mg, 0.90 mmol, 45%).

(27) .sup.1H-NMR (CDCl.sub.3, in ppm)): 1.60 (s, 3H), 1.66 (s, 6H), 2.08 (m, 4H), 2.97 (s, 3H), 3.79 (t, 2H), 5.04 (t, 1H), 5.21 (t, 1H). M+=232.

(28) Synthetic Route for Compound 61 (Taken as an Example)

(29) ##STR00022##

Synthesis of Ethyl 3-(2-hydroxyphenyl)-3-oxopropanoate (Compound 60)

(30) A solution of LHMDS (1M, 220 mL, 220 mmol, 3.0 eq) was under N.sub.2 atmosphere cooled to 78 C. A solution of 2-hydroxyacetophenone (8.84 mL, 73.5 mmol, 1.0 eq) in THF (300 mL) was added drop-wise in 30 min. The temperature was maintained at 78 C. for 1 h and 2 h at 10 C. The solution was cooled again to 78 C. and subsequently a solution of diethylcarbonate (9.8 mL, 80.8 mmol 1.1 eq) in THF (30 mL) was added. The reaction mixture was allowed to warm to room temperature over the weekend and poured in a mixture of HCl (37%, 50 mL) and ice (1.5 L). The layers were separated and the water layer was extracted with DCM (2500 mL). The combined organic layers were washed with brine (10.5 L), dried over Na.sub.2SO.sub.4 and concentrated in vacuo yielding a yellow oil (16.4 g). The crude product was stirred in DCM and the solids formed were removed by filtration. The filtrate was purified by automated column chromatography (eluens, 0 to 30% EtOAc in heptanes) affording a colorless oil (11.6 g, 55.6 mmol, 76%).

(31) .sup.1H-NMR (CDCl.sub.3, in ppm)): 1.27 (t, 3H), 4.01 (s, 2H), 4.23 (q, 2H), 6.97 (t, 1H), 7.00 (d, 1H), 7.55 (t, 1H), 7.64 (d, 1H). M+=209.

Synthesis of (E)-Ethyl 2-(2-hydroxybenzoyl)-5,9-dimethyldeca-4,8-dienoate (Compound 61)

(32) Diispropylamine (3.6 mL, 41.4 mmol, 4.5 eq) was dissolved in THF (25 mL), under a nitrogen atmosphere. The solution was cooled to 20 C. n-BuLi (2.5M, 14.7 mL, 36.8 mmol, 4.0 eq) was added subsequently. After stirring for 5 min at 20 C. the reaction mixture was cooled to 78 C. A solution of compound 60 (3.8 g, 18.4 mmol, 2.0 eq) in THF (20 mL) was added. The reaction mixture was stirred for 1 h at <70 C. and 2 h at <10 C. and cooled again to 78 C. A solution of geranylbromide in THF (10 mL) was added. The reaction mixture was allowed to warm to room temperature overnight and poured in a mixture of brine (100 mL) and sat. aq. NH.sub.4Cl sol. (100 mL). The water layer was extracted with DCM (1100 mL and 250 mL). The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated in vacuo yielding a brown oil (5.9 g). The crude product was purified by automated column chromatography (eluens 0 to 30% EtOAc in heptanes) furnishing a yellow oil (0.69 g, 2.0 mmol, 22%).

(33) .sup.1H-NMR (CDCl.sub.3, in ppm)): 1.21 (t, 3H), 1.57 (s, 3H), 1.62 (s, 6H), 2.00 (m, 4H), 2.78 (m, 2H), 4.18 (q, 2H), 4.38 (t, 1H), 5.07 (m, 2H), 6.98 (t, 1H), 7.02 (d, 1H), 7.55 (t, 1H), 7.81 (d, 1H). M+=345.

Example 2: Biological Activity of Novel Analogs

(34) This Example demonstrates the biological activity of various representative compounds of the invention. Compounds were screened for their ability to 1) boost HSP protein and mRNA expression in a mammalian cell-line, 2) induce programmed cell death in a human cancer cell-line, 3) protect against loss of contractile function induced by tachypacing.

(35) Materials and Methods

(36) Cell Culture and Heat Shock Conditions:

(37) HL-1 Cardiomyocytes

(38) HL-1 atrial myocytes, a cell line derived from adult mouse atria were obtained from Dr. William Claycomb (Louisiana State University, New Orleans, La., USA). The myocytes were maintained in Complete Claycomb Medium (JRH, UK) supplemented with 100 M norepinephrine stock (consisting of 10 mM norepinephrine (Sigma, The Netherlands) dissolved in 0.3 mM L-ascorbic acid (Sigma)), 4 mM L-glutamine (Gibco, The Netherlands) and 10% FBS (Sigma). The myocytes were cultured in flasks coated with 0.02% gelatin (Sigma), in a 5% CO.sub.2 atmosphere at 37 C.

(39) HL-1 cardiomyocytes were subjected to a mild heat shock of 44 C. for 10 minutes. After a recovery of 10 minutes (37 degrees 5% CO.sub.2), compounds were added in a final concentration of 10 M (dissolved in DMSO, final concentration 0.1%). To control myocytes, 0.1% DMSO was added. After six hours the cells were lysed in RIPA buffer and used for Western blotting as described previously..sup.3, 10

(40) HL-1 Cardiomyocyte Tachypacing

(41) HL-1 cardiomyocytes were cultured on coverslips and showed spontaneous contraction at 1 Hz. The cardiomyocytes were tachypaced with a C-Pace100-culture pacer in C-Dish100-culture dishes (IonOptix Corporation, The Netherlands), at 4 Hz with square-wave 20-msec pulses (40 V). Capture was ascertained by microscopic examination of cell shortening. We required a capture efficiency of >90% of cardiomyocytes throughout stimulation. HL-1 cardiomyocytes were pretreated with test compound (10 M) for 8 hours, followed by tachypacing at 4.5 Hz or normal pacing at 1 Hz. After pacing, HL-1 cardiomyocytes were used to measure the calcium transient (CaT) as a read out for contractile function.

(42) Live Imaging and Measurement of CaT

(43) To measure CaT, 2 M of the Ca.sup.2+-sensitive Fluo-4-AM dye (Invitrogen, The Netherlands) was loaded into HL-1 cardiomyocytes by 45 min incubation, followed by 3 times washing with DMEM solution. Ca.sup.2+ loaded myocytes were excited by 488 nm and light emitted at 500-550 nm and visually recorded with a 40-objective, using a Solamere-Nipkow-Confocal-Live-Cell-Imaging system (based on a Leica DM IRE2 Inverted microscope). The live recording of CaT in HL-1 cardiomyocytes was performed at 1 Hz of stimulation in a temperature (37 C.) controlled system. By use of the software ImageJ (National Institutes of Health, USA), the absolute value of fluorescent signals in live cardiomyocytes were recorded and analyzed. To compare the fluorescent signals between experiments, the following calibration was utilized: F.sub.cal=F/F0, in which (F) is fluorescent dye at any given time and (F0) is fluorescent signal at rest [10]. Mean values from each experimental condition were based on 7 consecutive CaT in at least 50 myocytes.

(44) MCF-7 Cells

(45) MCF-7 breast cancer cells were cultured in DMEM supplemented with 10% FCS. Test compounds were added to the medium in a final concentration of 10 for 16-20 hours. Ratio death and living cells was determined by using trypan blue staining of death cells. The cell pellets were lysed in RIPA buffer and used for Western blot analysis.

(46) Protein-Extraction and Western Blot Analysis

(47) Western-blot analysis was performed as described previously.sup.3, 10. Equal amount of protein in SDS-PAGE sample buffer was sonicated before separation on 10% PAA-SDS gels. After transfer to nitrocellulose membranes (Stratagene, The Netherlands), membranes were incubated with primary antibodies against HSP25 (Stressgen, The Netherlands, SPA-801, 1:1000), HSP27 (Stressgen, SPA-800, 1:1000), HSP70 (Stressgen, SPA-810, 1:1000), GAPDH (Affinity Reagents, The Netherlands) or PARP (Santa Cruz, The Netherlands, sc-7150, 1:500). Horseradish peroxidase-conjugated anti-mouse or anti-rabbit (Santa-Cruz Biotechnology, The Netherlands) was used as secondary antibody. Signals were detected by the ECL-detection method (Amersham, The Netherlands) and quantified by densitometry using GeneTools software from Syngene (The Netherlands).

(48) Statistical Analysis

(49) Results are expressed as meanSEM. All experimental procedures were performed in at least duplicate series. ANOVA was used for multiple-group comparisons. Student t tests were used for comparisons involving only 2 groups, and 1 tests with Bonferroni correction were used to compare individual group differences when multiple-comparison ANOVA was significant. All P values were two-sided. P<0.05 was considered statistically significant. SPSS version 16.0 was used for statistical evaluation.

(50) Results

(51) Screening for Analogs Capable of Boosting HSP70 Levels in HL-1 Cardiomyocytes.

(52) Compounds were also screened for HSP70 boosting effects. Hereto, HL-1 cardiomyocytes were first subjected to a mild heat shock which activates the Heat Shock Factor. After the mild heat shock, compound (10 M) was added to the medium of HL-1 cardiomyocytes for 6 hours, followed by determination of the HSP70 amount. We observed that exemplary analogs 23, 28, 46, 49, 51, 52, 60 61, 62, 76 and 95 are potent inducers of mammalian HSP70 protein expression (Table 1).

(53) TABLE-US-00001 TABLE 1 Biological activity of GGA-analogs. HSP boosting HL-1 cardiomyocytes Contractile mRNA (qPCR) Cell Death function Grp78 MCF-7 cancer cells HL-1 Com- Protein HSP HSP HSP HSP Not Death/living HSP HSP Protection pound HSP70 70 25 90 40 HSF1 100 M 27 70 PARP CaT No drug 0 1 1 1 1 1 4.5 1 1 1 50% GGA ++ + ++ ++ + 0 0 + 0 0 ++++ FA 0 ND GA ++ ND 21 0 22 0 23 ++++ ++ + + + 0 24 0 26 0 0 0 0 + 28 ++++ + + + + 0 0 ++ 0 0 ++++ 32 + 0 + 0 0 33 + 34 0 35 0 36 0 0 0 39 0 0 + 0 0 40 0 41 0 + + 0 + 43 0 44 0 45 0 ++ 0 ++ 46 ++ + ++ ++ + 0 49 +++ ++++ +++ +++ +++ 0 0 0 0 0 51 ++++ + + + + 0 0 ++ + 0 ++++ 52 ++++ + + + + 0 ++++ 53 + 56 0 0 0 0 0 61 ++ + + + + 0 0 0 0 0 ++++ 62 ++ + 0 + + 0 ++++ 64 0 65 0 ++ +++ ++ ++ 66 0 0 + ++ + 67 0 0 0 0 0 68 0 0 +++ ++ + 69 0 70 0 0 +++ ++ 0 76 +++ +++ ++ ++ + 0 ND ++++ 78 0 0 ++ 0 + 79 + ND 81 0 82 0 ND 84 + 85 0 0 + 89 + 0 0 0 0 91 + 95 +++ ++++ + ++ + 0 97 0 98 0 0 ++ 0 0 99 0 0 0 0 100 0 0 + 0 101 0 ++ 0 ++ 103 0 0 0 0 0 104 = 124 + 125 0 127 0 0 0 0 128 0 Induction of MCF-7 Cell Death: 0 = no dead cells; + = P < 0.05; ++ = P < 0.01; +++ = P < 0.001; ND = not determined. HSP boosting effect in HL-1; 0 = no induction HSP levels; + = 2-10-fold induction; ++ = 10-30-fold induction; +++ = > 30-fold induction
Upregulation of Mammalian HSP mRNA Levels

(54) Compounds were also screened for their ability to upregulate HSP gene expression. Hereto, HL-1 cardiomyocytes were first subjected to a mild heat shock which activates the Heat Shock Factor. After the mild heat shock, compound (10 M) was added to the medium of HL-1 cardiomyocytes for 6 hours, followed by determination of HSP70, HSP25, HSP90 and HSP40 which are targets of the heat shock factor 1 (HSF1) transcription factor. In addition, we determined mRNA levels of the gene encoding the glucose-responsive protein 78 (Grp78) which is HSF1-independent. We observed that exemplary GGA analogs 23, 28, 46, 51, 52, 60, 61, 62 and 76 are strong inducers of HSP gene expression, while no upregulation of Grp78 mRNA was observed (Table 1). This indicates that regulation of HSP gene expression by a compound of the invention is mediated via HSF-1.

(55) Screening for Compounds Able to Induce Programmed Cell Death.

(56) Since we observed that some compounds aggravate tachypacing-induced contractile dysfunction, we next investigated if these compounds can induce programmed cell death in a human breast cancer cell-line MCF-7. Hereto, MCF-7 cells were incubated for 16 hours with 10 M of a test compound after which the number of death and living cells was determined. In total 28 compounds were tested for cell death. Four compounds (41, 45, 65 and 101) showed a significant increase in cell death, which correlated with an aggravation of contractile dysfunction. GGA was used as a control as well as non-treated MCF-7 cells. Compounds 26, 41, 45, 65, 66, 68, 78, 85, and 101 induced PARP cleavage, indicative of pro-apoptotic caspase activation. The findings indicate that GGA-like compounds which aggravate heart failure can induce programmed cell death in MCF-7 breast cancer cell-line.

Example 3

Effect of GGA-Like Compounds on Renal Cells

(57) This example illustrates that GGA-like compounds also find their use in the prevention or therapy of renal injury.

(58) It has been shown by Wang et al. (Kidney International (2011) 79, 861-870) that the kidney cortical Hsp70 content inversely correlates with tubular injury, apoptosis, and organ dysfunction after injury. It was also found that increased Hsp70 expression mice reduces both ischemic tubular injury and organ dysfunction. When administered after ischemia, this inducer also decreased tubular injury and organ failure. Thus, increasing Hsp70 either before or after ischemic injury preserves renal function by attenuating acute kidney injury.

(59) The present inventors therefore evaluated the effect of various GGA-like compounds on the induction of Hsp70 expression in murine proximal tubule cells.

(60) Murine proximal tubule (MPT) cells were isolated and cultured as described previously (Borkan et al. Heat stress ameliorates ATP depletion-induced sublethal injury in mouse proximal tubule cells. Am. J. Physiol. 1997; F347-55).

(61) The MPT cells were exposed to 40 M of compound for 5 hours, after which the cells were harvested in RIPA buffer followed by Western blotting.

(62) For Western-blot analysis, equal amount of protein in SDS-PAGE sample buffer was separated on 10% PAA-SDS gels. After transfer to nitrocellulose membranes (Stratagene, USA), membranes were incubated with primary antibodies against HSP70 (Enzo C92F3A/StressGen SP810) or beta-actin (Sigma A541). Horseradish peroxidase-conjugated anti-mouse or anti-sheep (Jackson ImmunoRes) was used as secondary antibody. Signals were detected by the ECL-detection method (Amersham).

(63) It was found that compounds 32, 46, 47, 49, 51, 52, 61, 62, 69, 95 and 106 were strong inducers of HSP70 expression in renal cells, and even outperformed the inducing capacity of the positive control GGA. FIG. 8 shows exemplary Western blot analysis and densitometric quantification of HSp70 induction.

(64) These data indicate that the compounds find their use in the prevention of injury to renal tubular cells, for example attenuating acute kidney injury (AKI), by increasing Hsp70 either before or after ischemic injury.

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