Patent Application
20080058412
a,
1
b,
1
c illustrate the antioxidant characteristics of inventive compound 2 (Cpd 2) tested at a concentration of 10−4M.
a shows the kinetics of conjugated diene formation over time. The lag phase was 120 minutes when LDL were incubated with copper alone as compared with 314 minutes when the medium also contained compound 2.
b illustrates the rate of diene formation, which was 1.8 nmol/min/mg of LDL in the presence of copper alone and only 0.1 nmol/min/mg of LDL when compound 2 was present in the medium.
c represents the maximum amount of conjugated dienes formed over time. Copper alone induced the formation of 372 nmol/mg of conjugated dienes, compared with 35 nmol/mg when the medium also contained compound 2, which corresponds to a 90% decrease in the amount of conjugated dienes formed.
a,
2
b,
2
c illustrate the antioxidant characteristics of inventive compound 4 (Cpd 4), compound 6 (Cpd 6) and compound 8 (Cpd 8) tested at a concentration of 10−4M.
a shows the kinetics of conjugated diene formation.
The lag phase was 132 minutes when LDL were incubated with copper alone as compared with 401, 205 and 169 minutes in the presence of compounds 4, 6 and 8, respectively.
b illustrates the rate of diene formation, which was 2.2 nmol/min/mg of LDL in the presence of copper alone. The presence of compounds 4, 6 and 8 slowed the rate of the diene oxidation reaction to 0.2 nmol/min/mg in the presence of compound 4 and 1.7 nmol/min/mg in the presence of compounds 6 or 8.
The total amount of dienes formed (
a illustrates the antioxidant characteristics of inventive compound 11 (Cpd 11).
The antioxidant character of compound 11 was demonstrated for different concentrations comprised between 10−6 M and 3.5×10−5 M.
In the absence of compound 11, the lag phase was 87.2 minutes. Starting at the 10−6 M concentration, the lag phase increased relative to the control to 101.5 minutes. The lag phase increased in a dose-related manner to reach a maximum of 210 minutes at the concentration of 3.3×10−5M.
a,
4
b,
4
c illustrate the antioxidant characteristics of inventive compound 19 (Cpd 19) and compound 23 (Cpd 23) tested at a concentration of 10−4M.
a shows the kinetics of conjugated diene formation.
The lag phase was 61 minutes in the presence of copper alone as compared with 92.5 and 96.4 minutes in the presence of compounds 19 and 23, respectively.
The antioxidant character of compounds 19 and 23 was also manifested as a decrease in the rate of diene formation and by a decrease in the total amount of dienes formed.
In the absence of the compounds, the diene formation rate was 1.9 nmol/min/mg of LDL (
a,
5
b,
5
c illustrate the antioxidant characteristics of inventive compound 25 (Cpd 25), compound 27 (Cpd 27), compound 29 (Cpd 29) and compound 31 (Cpd 31) tested at a concentration of 10−4M.
a shows the kinetics of LDL oxidation in the presence of the different compounds, which increased in the presence of the different antioxidant compounds. It was 54.9 minutes in the presence of compound 29, increasing to 87.6 minutes with compound 25, 124.5 minutes with compound 31 and reaching 170.8 minutes in the presence of compound 27.
The antioxidant character of said compounds was also illustrated by the LDL oxidation rate (
The LDL oxidation rate was 2 nmol/min/mg of LDL in the absence of the compounds (
The total amount of dienes formed was 386 nmol/mg of LDL in the absence of the compounds (
a,
6
b,
6
c illustrate the antioxidant characteristics of inventive compound 37 (Cpd 37) tested at a concentration of 10−4M.
a shows the kinetics of LDL oxidation. The presence of the compound in the medium induced an increase in the lag phase, reaching 106 minutes in the presence of compound 37 whereas in the absence of said compound it was only 56 minutes.
The decrease in the rate of LDL oxidation and the decrease in the amount of dienes formed also illustrate the antioxidant character of the test compound. In the absence of the compound, the oxidation rate was 2 nmol/min/mg of LDL compared with 1.8 nmol/min/mg of LDL when the compound was present (
a,
7
b,
7
c illustrate the antioxidant characteristics of inventive compound 13 (Cpd 13), compound 33 (Cpd 33), compound 41 (Cpd 41), compound 47 (Cpd 47), tested at a concentration of 10−4M.
a shows the kinetics of LDL oxidation. In the absence of the antioxidant compounds, the lag phase was 67.3 minutes, increasing in the presence of the different compounds to a value of 100 minutes in the presence of compound 41, 126.5 minutes for compound 47, 148 minutes for compound 33 and 219 minutes for compound 13.
The presence of the compounds in the medium also had an effect on the LDL oxidation rate and on the total amount of dienes formed.
Compounds 13 and 33 induced a marked decrease in the diene oxidation rate (
Only compounds 33 and 41 induced a decrease in the total amount of dienes formed (
a,
8
b,
8
c illustrate the antioxidant characteristics of inventive compound 17 (Cpd 17), compound 21 (Cpd 21), compound 39 (Cpd 39) and compound 43 (Cpd 43) tested at a concentration of 10−4M.
The kinetics of LDL oxidation are shown in
In the absence of the compounds, the lag phase was 67.3 minutes, increasing to 97, 148 and 133 minutes for compound 43, 17 and 39, respectively.
b represents the rate of LDL oxidation, which was 2.5 nmol/min/mg in the absence of the compounds as compared with 1.8, 1.2 and 2.2 nmol/min/mg in the presence of compound 17, 39 and 43, respectively.
c shows the total amount of dienes formed during oxidation. Only compound 39 induced a significant decrease in the total amount of dienes formed, which was 432.3 nmol/mg in the absence of the compound and 371.2 nmol/mg in the presence of compound 39.
The longer lag phase of conjugated diene formation, the reduction in the rate of diene formation and the decrease in the total amount of dienes formed are three parameters which confirm the antioxidant characteristics of the inventive compounds.
a and 9b show the evaluation of PPARα and PPARγ agonist properties of the inventive compounds using the PPARα/Gal4 and PPARγ/Gal4 transactivation system in RK13 cells.
RK13 cells were incubated with the compound 2 at concentrations comprised between 0.01 and 10 μM for 24 hours. The results are expressed as the induction factor (ratio of luminescent signal obtained with the compound and that observed without the compound) after the different treatments. The higher the induction factor the more potent the PPARα or PPARγ agonist activity.
a shows the induction factors for compound 2 with the PPARα/Gal4 transactivation system. The values of these induction factors are given in the following table.
The induction factor for compound 2 was maximum at the 10 μM concentration, reaching a value of 18.49.
b shows the induction factors for compound 2 with the PPARγ/Gal4 transactivation system. The values of these induction factors are given in the following table:
In the PPARγ/Gal4 system, the induction factors ranged from 1.31 to 31.00, increasing with the concentration of compound 2 in the medium.
a
10
b,
11
a,
11
b,
11
c,
12
a,
12
b,
13
a,
13
b,
14
a,
14
b,
14
c,
15
a,
15
b,
15
c,
16
a,
17
a,
18
a,
18
b show the evaluation of PPARα, PPARγ and PPARδ agonist properties of the inventive compounds in the PPARα/Gal4, PPARγ/Gal4 and PPARδ/Gal4 transactivation system in COS-7 cells.
COS-7 cells were incubated with different concentrations of the inventive compounds for 24 hours. The results are expressed as the induction factor (ratio of luminescent signal obtained with the compound and that observed without the compound) after the different treatments.
a and 10b show the induction factors for inventive compound 4 (Cpd4), compound 6 (Cpd6) and compound 8 (Cpd8).
a shows the induction factors for compound 4 (Cpd4), compound 6 (Cpd6) and compound 8 (Cpd8) with the PPARα/Gal4 transactivation system. The values of these induction factors are given in the following table:
The maximum induction factor was 9.92 for compound 4 at a concentration of 10 μM, 7.01 for compound 6 (10 μM) and 15.67 for compound 8 (1 μM).
b shows the induction factors for compound 4, compound 6 and compound 8 with the PPARγ/Gal4 transactivation system. The values of these induction factors are given in the following table:
Compound 4 had a maximum induction factor of 5.82 at the 10 μM concentration. The maximum induction factors were 6.83 for compound 6 (10 μM) and 2.74 for compound 8 (10 μM).
a,
11
b and 11c illustrate the induction factors for inventive compound 13 (Cpd13).
a shows the induction factors for compound 13 with the PPARα/Gal4 transactivation system. The values of these induction factors are given in the following table:
The maximum induction factor of 17.05 was observed at a concentration of 1 μM.
b shows the induction factors of compound 13 with the PPARγ/Gal4 transactivation system. The values of these induction factors are given in the following table:
The maximum value of 4.03 was seen at the 1 μM concentration.
The maximum value of 28.75 was seen at the 1 μM concentration.
a and 12b illustrate the induction factors of inventive compound 23 (Cpd23).
a shows the induction factors of compound 23 with the PPARγ/Gal4 transactivation system. The values of these induction factors are given in the following table:
The maximum value of 8.35 was seen at the 1 μM concentration.
b shows the induction factors for compound 23 with the PPARγ/Gal4 transactivation system. The values of these induction factors are given in the following table:
The maximum value of 7.24 was seen at the 1 μM concentration.
a and 13b illustrate the induction factors of inventive compound 29 (Cpd29).
a shows the induction factors of compound 29 with the PPARα/Gal4 transactivation system. The values of these induction factors are given in the following table:
The maximum value of 15.75 was seen at the 3 μM concentration.
b shows the induction factors for compound 29 with the PPARδ/Gal4 transactivation system. The values of these induction factors are given in the following table:
The maximum value of 87.56 was seen at the 10 μM concentration.
a,
14
b and 14c show the induction factors for inventive compound 31 (Cpd31).
a shows the induction factors for compound 31 with the PPARα/Gal4 transactivation system. The values of these induction factors are given in the following table:
The maximum value of 6.03 was seen at the 10 μM concentration.
b shows the induction factors for compound 31 with the PPARγ/Gal4 transactivation system. The values of these induction factors are given in the following table:
The maximum value of 7.79 was seen at the 3 μM concentration.
c shows the induction factors for compound 31 with the PPARδ/Gal4 transactivation system. The values of these induction factors are given in the following table:
The maximum value of 11.70 was seen at the 10 μM concentration.
a,
15
b and 15c illustrate the induction factors for inventive compound 33 (Cpd33).
a shows the induction factors for compound 33 with the PPARα/Gal4 transactivation system. The values of these induction factors are given in the following table:
The maximum value of 20.82 was seen at the 0.3 μM concentration.
b shows the induction factors for compound 33 with the PPARγ/Gal4 transactivation system. The values of these induction factors are given in the following table:
The maximum value of 7.99 was seen at the 3 μM concentration.
c shows the induction factors for compound 33 with the PPARδ/Gal4 transactivation system. The values of these induction factors are given in the following table:
The maximum value of 90.84 was seen at the 3 μM concentration.
a illustrates the induction factors for inventive compound 35 (Cpd35).
a shows the induction factors for compound 35 with the PPARα/Gal4 transactivation system. The values of these induction factors are given in the following table:
The maximum value of 24.33 was seen at the 1 μM concentration.
a shows the induction factors for inventive compound 37 (Cpd37) with the PPARα/Gal4 transactivation system. The values of these induction factors are given in the following table:
The maximum value of 19.77 was seen at the 1 μM concentration.
a and 18b show the induction factors for inventive compound 39 (Cpd39).
a shows the induction factors for compound 39 with the PPARα/Gal4 transactivation system. The values of these induction factors are given in the following table:
The maximum value of 10.42 was seen at the 1 μM concentration.
b shows the induction factors for compound 39 with the PPARγ/Gal4 transactivation system. The values of these induction factors are given in the following table:
The maximum value of 4.86 was seen at the 0.3 μM concentration.
These results shown in the figures demonstrate that the inventive compounds tested exhibit PPARα, PPARγ and/or PPARδ ligand activity and therefore enable the transcriptional activation of these nuclear receptors.
a,
19
b,
19
c,
19
d,
20
a,
20
b,
20
c and 20d illustrate the effects of treatment with compound 2 (Cpd2), compound 13 (Cpd13), compound 33 (Cpd33) and compound 39 (Cpd39) on triglyceride and cholesterol metabolism in Apo E2/E2 transgenic mice treated by gavage with the compound at a dose of 50 mg/kg/day, for seven days.
a and 19b illustrate the decrease in plasma triglycerides and cholesterol induced by compound 2.
a and 20b illustrate the decrease in plasma triglycerides and cholesterol induced by compounds 13, 33 and 39.
c and 19d illustrate the distribution of triglycerides and cholesterol in lipoparticles evaluated by exclusion chromatography, induced by treatment with compound 2.
c and 20d illustrate the distribution of triglycerides and cholesterol in lipoparticles evaluated by exclusion chromatography, induced by treatment with compounds 13, 33 and 39.
A typical distribution of triglycerides and cholesterol primarily in large lipoparticles was observed. A decrease in triglycerides and cholesterol in this lipoparticle class was seen after treatment with the different test compounds.
Compound 39 (10−6 M) was added at D0 of differentiation of monocytes to dendritic cells. After six days of differentiation (in the presence of cytokines GM-CSF and IL-4), the dendritic cells were analyzed by flow cytometry.
(---): Fluorochrome-coupled Ab with control isotype
(in black): FITC (fluorescein isothiocyanate)-coupled anti-CD1a Ab or PE (phycoerythrin)-coupled anti-CD86 Ab.
Dendritic cells were incubated for 4 hours with compounds 31, 13 or 39, then stimulated with LPS for 16 hours. CCR7 and ELC transcripts were analyzed by quantitative RT-PCR and the cytokine TNFalpha was analyzed by ELISA.
Other aspects and advantages of the invention will become apparent in the following examples, which are given for purposes of illustration and not by way of limitation.
The compounds according to the invention were prepared according to the general methods outlined below.
Synthesis of 1,3-diphenylprop-2-en-1-ones in acidic medium:
The ketone (1 eq) and the aldehyde (1 eq) were dissolved in ethanol solution saturated with gaseous hydrochloric acid. The reaction was stirred at room temperature for 6 hours and the solvent was then eliminated by vacuum evaporation. The 1,3-diphenylprop-2-en-1-one was purified by chromatography on silica gel or by recrystallization.
Synthesis of 1,3-diphenylprop-2-en-1-ones in the presence of sodium hydroxide:
The ketone (1 eq) and the aldehyde (1 eq) were dissolved in a hydroalcoholic solution of sodium hydroxide (20 eq). The mixture was stirred at room temperature for 18 hours. The medium was acidified to pH=2 with hydrochloric acid.
The 1,3-diphenylprop-2-en-1-one was obtained by precipitation or solid/liquid extraction after evaporation of the reaction medium. It was purified by silica gel chromatography or by recrystallization.
Synthesis of substituted 1,3-diphenylprop-2-en-1-ones in the presence of sodium ethylate:
Sodium (1 eq) was dissolved in absolute ethanol. The ketone (1 eq) and the aldehyde (1 eq) were added. The reaction mixture was stirred at room temperature for 12 hours and 2 N sodium hydroxide (5 eq) was then added. The mixture was kept at 100° C. for 12 hours. The reaction medium was acidified by adding 6 N aqueous hydrochloric acid solution. The solvent was eliminated by vacuum evaporation. The residue was purified by chromatography on silica gel or by recrystallization.
The phenol (1 eq) or the thiophenol (1 eq) was dissolved in acetonitrile and the halogenated derivative (1 to 10 eq) and potassium carbonate (5 eq) were added. The reaction medium was briskly stirred under reflux for approximately 10 hours. The salts were eliminated by filtration, the solvent and excess reagent were eliminated by vacuum evaporation, and the expected product was purified by silica gel chromatography.
The alcohol (1 eq), the phenol (1 eq) and the triphenylphosphine were dissolved in dichloromethane. Diisopropylazodicarboxylate (1 eq) was added and the mixture was stirred for 12 hours at room temperature.
The reaction medium was washed with water, dried on magnesium sulfate and vacuum evaporated. The evaporation residue was purified by silica gel chromatography.
General Method 6:
The tert-butyl ester (1 eq) was dissolved in dichloromethane, trifluoroacetic acid (10 eq) was added, and the mixture was stirred at room temperature for 12 hours. The resulting product was purified by chromatography on silica gel or by recrystallization.
This compound was synthesized from 4′-hydroxyacetophenone and dibromoethane according to general method 4 described earlier.
It was purified by chromatography on silica gel (elution: cyclohexane/ethyl acetate 9:1).
1H NMR CDCl3 δppm: 2.55 (s, 3H), 3.66 (t, 2H, J=6.50 Hz), 4.35 (t, 2H, J=6.50 Hz), 6.94 (d, 2H, J=7.23 Hz), 7.94 (d, 2H, J=7.23 Hz)
Starting material 1 (1 eq) and penthanethiol (1 eq) were dissolved in methanol in the presence of triethylamine (2 eq). The reaction medium was refluxed for 18 hours and the solvent eliminated by vacuum evaporation. The oil was taken up in ethyl acetate, washed with aqueous 2N hydrochloric acid solution. 4′-(pentylthioethyloxy)acetophenone was obtained after purification on silica gel (elution: cyclohexane/ethyl acetate 9:1).
1H NMR CDCl3 δppm: 0.85 (m, 3H), 1.24-1.39 (m, 4H), 1.52-1.62 (m, 2H), 2.50 (s, 3H), 2.64 (t, 2H, J=7.2 Hz), 2.94 (t, 2H, J=6.8 Hz), 4.14 (t, 2H, J=6.8 Hz), 6.88 (d, 2H, J=8.7 Hz), 7.89 (d, 2H, J=8.7 Hz)
This compound was synthesized from 4-hydroxy-3,5-dimethylbenzaldehyde and tert-butyl bromoisobutyrate according to general method 4.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 8:2).
1H NMR CDCl3 δppm: 1.43 (s, 6H), 1.49 (s, 9H), 2.28 (s, 6H), 7.53 (s, 2H), 9.88 (s, 1H)
2,6-dimethylphenol (1 eq) was dissolved in methylene chloride and the solution was cooled to 0° C. Aluminium chloride (3 eq) and acetyl bromide (2 eq) were then added. The mixture was stirred for 3 hours at room temperature, then poured on ice. The aqueous phase was extracted with dichloromethane, the organic phase was washed with water until neutrality, dried on magnesium sulfate and the solvent was eliminated by vacuum evaporation. The intermediate ester obtained was purified by silica gel chromatography (elution: cyclohexane/ethyl acetate 9:1) then taken up in aqueous 2N sodium hydroxide (2.5 eq). The mixture was stirred for 48 hours at room temperature then acidified with dilute hydrochloric acid. The precipitate was washed with water until the wash water reached a neutral pH.
1H NMR CDCl3 δppm: 2.30 (s, 6H), 2.54 (s, 3H), 7.65 (s, 2H)
This compound was synthesized from 4′-hydroxyacetophenone and (R,S)-5-[1,2]dithiolan-3-ylpentanol according to general method 5 described earlier. Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 95:5).
1H NMR CDCl3 δppm: 1.42-1.62 (m, 4H), 1.62-1.75 (m, 2H), 1.75-1.89 (m, 2H), 1.89-1.98 (m, 1H), 2.42-2.51 (m, 1H), 2.56 (s, 3H), 3.08-3.21 (m, 2H), 3.55-3.61 (m, 1H), 4.06 (t, 2H, J=6.2 Hz), 6.92 (d, 2H, J=8.7 Hz), 7.93 (d, 2H, J=8.7 Hz)
This compound was synthesized from 4-hydroxy-3,5-dimethylbenzaldehyde and 2-hydroxy-2-phenyl ethyl acetate according to general method 5 described earlier.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 9:1).
1H NMR CDCl3 δppm: 1.22 (t, 3H, J=7.35 Hz), 2.20 (s, 6H), 4.16-4.28 (m, 2H), 5.3 (s, 1H), 7.38-7.51 (m, 7H), 9.87 (s, 1H)
This compound was synthesized from 4′-hydroxyacetophenone and 2-cyclohexylethanol according to general method 5 described earlier.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 9:1).
1H NMR CDCl3 δppm: 0.90-1.80 (m, 13H), 2.56 (s, 3H), 4.07 (t, 2H, J=6.45 Hz), 6.92 (d, 2H, J=8.80 Hz), 7.93 (d, 2H, J=8.80 Hz)
This compound was synthesized from 2,6-dimethylthiophenol and methyl iodide according to general method 4 described earlier.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 9:1).
1H NMR CDCl3 δppm: 2.28 (s, 3H), 2.62 (s, 6H), 7.16 (m, 3H)
3′,5′-Dimethyl-4′-methylthioacetophenone
Starting material 8 (1 eq) was dissolved in methylene chloride, the solution was cooled to 0° C. and aluminium chloride (2.5 eq) and acetyl bromide (2 eq) were then added. The mixture was stirred for 72 hours at room temperature, then poured on ice. The aqueous phase was extracted with dichloromethane, the organic phase was washed with water until neutrality, dried on magnesium sulfate and the solvent was eliminated by vacuum evaporation.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 9:1).
1H NMR CDCl3 δppm: 2.23 (s, 3H), 2.54 (s, 3H), 2.56 (s, 6H), 7.63 (s, 2H)
This compound was synthesized from starting material 4 and methyl iodide according to general method 4 described earlier.
The crude product obtained after elimination of the potassium carbonate by filtration and elimination of the solvents by vacuum evaporation was used for the synthesis of the corresponding intermediate compound.
1H NMR CDCl3 δppm: 2.31 (s, 6H), 2.54 (s, 3H), 3.74 (s; 3H), 7.63 (s, 2H)
This compound was synthesized from starting material 4 and 2-cyclohexylethanol according to general method 5 described earlier.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 85:15).
1H NMR CDCl3 δppm: 0.92-1.80 (m, 13H), 2.31 (s, 6H), 2.55 (s, 3H), 3.86 (t, 2H, J=7.05 Hz), 7.63 (s, 2H)
This compound was synthesized from starting material 4 and dibromoethane according to general method 4 as described above.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 85:15).
1H NMR CDCl3 δppm: 2.36 (s, 6H), 2.56 (s, 3H), 3.68 (t, 2H, J=6.21 Hz), 4.14 (t, 2H, J=6.21 Hz), 7.65 (s, 2H)
This compound was synthesized from starting material 1 and cyclohexane thiol according to general method 4 as described above.
1H NMR CDCl3 δppm: 1.08 (m, 5H), 1.40 (m, 1H), 1.56 (m, 2H), 1.80 (m, 2H), 2.30 (s, 3H), 2.53 (m, 1H), 2.69 (t, 2H, J=6.96 Hz), 3.95 (t, 2H, J=6.96 Hz), 6.68 (d, 2H, J=8.88 Hz), 7.69 (d, 2H, J=8.88 Hz)
This compound was synthesized from starting material 12 and cyclohexane thiol according to general method 4 as described above.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 9:1).
1H NMR CDCl3 δppm: 1.26-1.42 (m, 5H), 1.59-1.65 (m, 1H), 1.80 (m, 2H), 2.00 (m, 2H), 2.35 (s, 6H), 2.56 (s, 3H), 2.75 (m, 1H), 2.95 (t, 2H, J=6.81 Hz), 3.96 (t, 2H, J=6.81 Hz), 7.64 (s, 2H)
This compound was synthesized from 4′-hydroxy-3-methylacetophenone and methyl iodide according to general method 4 as described above.
The crude product obtained after elimination of the potassium carbonate by filtration and elimination of the solvents by vacuum evaporation was used for the synthesis of the corresponding intermediate compound.
1H NMR CDCl3 δppm: 2.53 (s, 3H), 2.56 (s, 3H), 3.90 (s, 3H), 6.85 (d, 1H, J=8.46 Hz), 7.78 (s, 1H), 7.82 (d, 1H, J=8.46 Hz)
This compound was synthesized from 2,6-dimethylthiophenol and hexyl bromide according to general method 4 as described above.
Purification was by chromatography on silica gel (elution: cyclohexane).
1H NMR CDCl3 δppm: 0.90 (t, 3H, J=6.57 Hz), 1.27-1.58 (m, 8H), 2.57 (s, 6H), 2.66 (t, 2H, J=7.11 Hz), 7.12 (m, 3H)
Starting material 16 (1 eq) was dissolved in methylene chloride, the solution was cooled to 0° C. and aluminium chloride (1 eq) and acetyl bromide (1 eq) were then added. The mixture was stirred for 2 hours at room temperature, then poured on ice. The aqueous phase was extracted with dichloromethane, the organic phase was washed with water until neutrality, dried on magnesium solvent and the solvent was eliminated by vacuum evaporation.
Purification was by chromatography on silica gel (elution: cyclohexane).
1H NMR CDCl3 δppm: 0.87 (t, 3H, J=6.72 Hz), 1.22-1.53 (m, 8H), 2.58 (s, 3H), 2.59 (s, 6H), 2.68 (t, 2H, J=7.23 Hz), 7.66 (s, 2H)
Starting material 12 (1 eq) and morpholine (0.7 eq) were dissolved in acetone and potassium carbonate (1 eq) was added. The mixture was refluxed for 72 hours. Potassium carbonate was eliminated by filtration, the solvent was eliminated by vacuum evaporation. The residual oil was taken up in aqueous 1N hydrochloric acid solution and washed with ethyl acetate. The aqueous phase was basified (pH 9) by addition of potassium carbonate, then extracted with ethyl acetate. The organic phase was dried on magnesium sulfate and the solvent was eliminated by vacuum evaporation.
1H NMR CDCl3 δppm: 2.33 (s, 6H), 2.54 (s, 3H), 2.60 (t, 4H, J=4.70 Hz), 2.81 (t, 2H, J=5.76 Hz), 3.76 (t, 4H, J=4.70 Hz) 3.93 (t, 2H, J=5.76 Hz), 7.62 (s, 2H)
2,6-difluorophenol (1 eq) and hexamethylenetetramine (2 eq) were dissolved in a water/acetic acid mixture (10:90). The reaction mixture was refluxed for 18 hours then cooled to room temperature.
The reaction mixture was extracted with dichloromethane, the organic phases were pooled, dried on magnesium sulfate, and the solvent was eliminated by vacuum evaporation.
1H NMR CDCl3 δppm: 7.35 (dd, 2H, J=6.57 Hz, J=2.82 Hz), 9.67 (s, 1H)
4-methoxy-3-trifluoromethylbenzonitrile (1 eq) was dissolved in anhydrous THF. Magnesium methyl chloride in solution in THF (1 eq) was added and the reaction mixture was stirred for 16 hours at room temperature then one hour under reflux after adding more magnesium methyl chloride (1 eq).
The reaction mixture was poured on an aqueous 1N hydrochloric acid solution and extracted with dichloromethane. The organic phase was neutralized with aqueous potassium bicarbonate solution then washed with water and dried on magnesium sulfate. The solvent was eliminated by vacuum evaporation.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 9:1).
1H NMR CDCl3 δppm: 2.60 (s, 3H), 3.99 (s, 3H), 7.07 (d, 1H, J=8.79 Hz), 8.14 (d, 1H, J=8.79 Hz, J=1.77 Hz), 8.19 (s, 1H)
This compound was synthesized from starting material 2 and 4-hydroxy-3,5-dimethylbenzaldehyde according to general method 1 as described above.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 85:15).
1H NMR CDCl3 δppm: 0.91 (m, 3H), 1.33-1.42 (m, 4H), 1.59-1.67 (m, 2H), 2.29 (s, 6H), 2.64 (t, 2H, J=7.60 Hz), 2.96 (t, 2H, J=6.80 Hz), 4.24 (t, 2H, J=6.80 Hz), 6.97 (d, 2H, J=8.70 Hz), 7.31 (s, 2H), 7.37 (d, 1H, J=15.54 Hz), 7.72 (d, 1H, J=15.54 Hz), 8.03 (d, 2H, J=8.70 Hz)
This compound was synthesized from starting material 5 and 4-hydroxy-3,5-dimethylbenzaldehyde according to general method 1 as described above.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 8:2).
1H NMR CDCl3 δppm: 1.45-1.65 (m, 4H), 1.65-1.77 (m, 2H), 1.77-1.87 (m, 2H), 1.87-2.0 (m, 1H), 2.30 (s, 6H), 2.43-2.51 (m, 1H), 3.09-3.22 (m, 2H), 3.56-3.62 (m, 1H), 4.04 (t, 2H, J=6.40 Hz), 6.96 (d, 2H, J=8.50 Hz), 7.31 (s, 2H), 7.41 (d, 1H, J=15.40 Hz), 7.73 (d, 1H, J=15.40 Hz), 8.04 (d, 2H, J=8.50 Hz)
This compound was synthesized from 4′-methylthioacetophenone and 3,5-dibromo-4-hydroxybenzaldehyde according to general method 1 as described above.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 8:2).
1H NMR CDCl3 δppm: 2.55 (s, 3H), 6.19 (s, 1H), 7.32 (d, 2H, J=8.70 Hz), 7.41 (1H, J=15.40 Hz), 7.63 (d, 1H, J=15.40 Hz), 7.75 (s, 2H), 7.96 (d, 2H, J=8.70 Hz)
This compound was synthesized from starting material 7 and 3,5-dimethyl-4-hydroxybenzaldehyde according to general method 1 as described above.
The product crystallized in the reaction medium and was drained, washed with ethanol previously cooled to 0° C. and vacuum dried.
1H NMR CDCl3 δppm: 0.90-1.80 (m, 13H), 2.30 (s, 6H), 4.08 (t, 2H, J=6.54 Hz), 6.97 (d, 2H, J=9.00 Hz), 7.30 (s, 2H), 7.42 (d, 1H, J=15.50 Hz), 7.73 (d, 1H, J=15.50 Hz), 8.03 (d, 2H, J=9.00 Hz)
This compound was synthesized from starting material 9 and 3,5-dimethyl-4-hydroxybenzaldehyde according to general method 1 as described above.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 8:2).
1H NMR CDCl3 δppm: 2.28 (s, 3H), 2.30 (s, 6H), 2.64 (s, 6H), 7.32 (s, 2H), 7.36 (d, 1H, J=15.76 Hz), 7.72 (s, 2H), 7.73 (d, 1H, J=15.76 Hz)
This compound was synthesized from starting material 10 and 3,5-dimethyl-4-hydroxybenzaldehyde according to general method 1 as described above.
The product crystallized in the reaction medium and was drained, washed with ethanol previously cooled to 0° C. and vacuum dried.
1H NMR CDCl3 δppm: 2.28 (s, 6H), 2.35 (s, 6H), 3.77 (s, 3H), 7.30 (s, 2H), 7.35 d, 1H, J=15.63 Hz), 7.70 (d, 1H, J=15.63 Hz), 7.72 (s, 2H)
This compound was synthesized from starting material 11 and 3,5-dimethyl-4-hydroxybenzaldehyde according to general method 1 as described above.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 8:2).
1H NMR CDCl3 δppm: 0.94-1.05 (m, 2H), 1.16-1.31 (m, 4H), 1.57-1.82 (m, 7H), 2.30 (s, 6H), 2.35 (s, 6H), 3.86 (t, 2H, J=7.08 Hz), 7.32 (s, 2H), 7.38 (d, 1H, J=15.81 Hz), 7.71 (s, 2H), 7.72 (d, 1H, J=15.81 Hz)
This compound was synthesized from starting material 13 and 3,5-dimethyl-4-hydroxybenzaldehyde according to general method 1 as described above.
The product crystallized in the reaction medium and was drained and washed with ethanol previously cooled to 0° C.
1H NMR CDCl3 δppm: 1.23-1.42 (m, 5H), 1.63-1.65 (m, 1H), 1.79-1.81 (m, 2H), 2.01-2.08 (m, 2H), 2.29 (s, 6H), 2.73-2.81 (m, 1H), 2.96 (t, 2H, J=7.08 Hz), 4.20 (t, 2H, J=7.08 Hz), 6.97 (d, 2H, J=8.73 Hz), 7.30 (s, 2H), 7.41 (d, 1H, J=15.53 Hz), 7.73 (d, 1H, J=15.53 Hz), 8.04 (d, 2H, J=8.73 Hz)
This compound was synthesized from 2′,4′,5′-trimethylacetophenone and 3,5-dimethyl-4-hydroxybenzaldehyde according to general method 1 described earlier.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 7:3).
1H NMR CDCl3 δppm: 2.27 (s, 9H), 2.29 (s, 3H), 2.38 (s, 3H), 7.00 (d, 1H, J=15.90 Hz), 7.04 (s, 1H), 7.23 (s, 2H), 7.27 (s, 1H), 7.39 (d, 1H, J=15.90 Hz)
This compound was synthesized from starting material 14 and 3,5-dimethyl-4-hydroxybenzaldehyde according to general method 1 described earlier.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 7:3).
1H NMR CDCl3 δppm: 1.32 (m, 5H), 1.63 (m, 1H), 1.79 (m, 2H), 2.03 (m, 2H), 2.29 (s, 6H), 2.37 (s, 6H), 2.75 (m, 1H), 2.97 (t, 2H, J=7.05 Hz), 3.97 (t, 2H, J=7.05 Hz), 7.30 (s, 2H) 7.37 (d, 1H, J=15.70 Hz), 7.70 (d, 1H, J=15.70 Hz), 7.71 (s, 2H)
This compound was synthesized from 4′-methylthioacetophenone and 3-fluoro-4-hydroxybenzaldehyde according to general method 1 described earlier.
The product crystallized in the reaction medium and was drained and vacuum dried.
1H NMR CDCl3 δppm: 2.55 (s, 3H), 7.04 (t, 1H, J=8.37 Hz), 7.30-7.42 (m, 5H), 7.73 (d, 1H, J=15.54 Hz), 7.95 (d, 2H, J=8.40 Hz)
This compound was synthesized from pentamethylacetophenone and 3,5-dimethyl-4-hydroxybenzaldehyde according to general method 1 described earlier.
The product crystallized in the reaction medium and was drained and purified by recrystallization in ethanol.
1H NMR CDCl3 δppm: 2.09 (s, 6H), 2.20 (s, 6H), 2.22 (s, 6H), 2.26 (s, 3H), 6.83 (d, 1H, J=16.11 Hz), 7.05 (d, 1H, J=16.11 Hz), 7.16 (s, 2H)
This compound was synthesized from 4′-phenoxyacetophenone and 3,5-dimethyl-4-hydroxybenzaldehyde according to general method 1 described earlier.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 7:3).
1H NMR CDCl3 δppm: 2.30 (s, 6H), 7.05 (d, 2H, J=8.67 Hz), 7.1 (d, 2H, J=8.47 Hz), 7.21 (t, 1H, J=7.30 Hz), 7.31 (s, 2H), 7.43-7.38 (m, 3H), 7.75 (d, 1H, J=15.36 Hz), 8.05 (d, 2H, J=8.47 Hz)
This compound was synthesized from 4′-methoxy-3′-fluoroacetophenone and 3,5-dimethyl-4-hydroxybenzaldehyde according to general method 1 described earlier.
The product crystallized in the reaction medium and was drained, then washed with heptane.
1H NMR CDCl3 δppm: 2.30 (s, 6H), 3.98 (s, 3H), 7.04 (t, 1H, J=8.30 Hz), 7.31 (s, 2H), 7.35 (d, 1H, J=15.69 Hz), 7.74 (d, 1H, J=15.69 Hz), 7.79-7.87 (m, 2H)
This compound was synthesized from starting material 15 and 3,5-dimethyl-4-hydroxybenzaldehyde according to general method 1 described earlier.
The product crystallized in the reaction medium and was drained, then washed with heptane.
1H NMR CDCl3 δppm: 2.30 (s, 9H), 3.92 (s, 3H), 6.90 (d, 1H, J=8.45 Hz), 7.31 (s, 2H), 7.43 (d, 1H, J=15.52 Hz), 7.73 (d, 1H, J=15.52 Hz), 7.88 (s, 1H), 7.93 (d, 1H, J=8.45 Hz)
This compound was synthesized from starting material 17 and 3,5-dimethyl-4-hydroxybenzaldehyde according to general method 1 described earlier.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 8:2).
1H NMR CDCl3 δppm: 0.88 (t, 3H, J=6.90 Hz), 1.20-1.50 (m, 8H), 2.30 (s, 6H), 2.63 (s, 6H), 2.70 (t, 2H, J=6.9 Hz), 7.32 (s, 2H), 7.36 (d, 1H, J=15.51 Hz), 7.72 (s, 2H), 7.73 (d, 1H, J=15.51 Hz)
This compound was synthesized from 2′,5′-dimethoxyacetophenone and 3,5-dimethyl-4-hydroxybenzaldehyde according to general method 1 described earlier.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 7:3).
1H NMR CDCl3 δppm: 2.27 (s, 6H), 3.74 (s, 3H), 3.82 (s, 3H), 6.93 (d, 1H, J=8.73 Hz), 7.02 (dd, 1H, J=8.73 Hz, J=3.27 Hz), 7.14 (d, 1H, J=3.27 Hz), 7.22 (d, 1H, J=15.81 Hz), 7.25 (s, 2H), 7.53 (d, 1H, J=15.81 Hz)
This compound was synthesized from 4′-bromoacetophenone and starting material 19 according to general method 1 described earlier.
The ethanol was eliminated by vacuum evaporation and the solid was washed with absolute ethanol.
1H NMR CDCl3 δppm: 5.97 (s, 1H), 7.18 (d, 2H, J=8.30 Hz), 7.35 (d, 1H, J=15.36 Hz), 7.65 (m, 3H), 7.89 (d, 2H, J=8.30 Hz)
This compound was synthesized from starting material 20 and 3,5-dimethyl-4-hydroxybenzaldehyde according to general method 1 described earlier.
The ethanol was eliminated by vacuum evaporation and the solid was washed with absolute ethanol.
1H NMR DMSOd6 δppm: 2.22 (s, 6H), 4.01 (s, 3H), 7.41 (d, 1H, J=9.00 Hz), 7.52 (s, 2H), 7.64 (d, 1H, J=15.40 Hz), 8.96 (s, 1H), 7.76 (d, 1H, J=15.40 Hz), 8.29 (d, 1H, J=1.60 Hz), 8.49 (dd, 1H, J=9.00 Hz, J=1.60 Hz)
This compound was synthesized from Intermediate compound 1 and tert-butyl bromoisobutyrate according to general method 4 described earlier.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 9:1).
1H NMR CDCl3 δppm: 0.91 (t, 3H, J=7.10 Hz), 1.37-1.69 (m, 21H) 2.27 (s, 6H), 2.63 (t, 2H, J=7.10 Hz), 2.93 (t, 2H, J=7.10 Hz), 4.21 (t, 2H, J=7.10 Hz), 6.97 (d, 2H, J=8.70 Hz), 7.28 (s, 2H), 7.44 (d, 1H, J=15.81 Hz), 7.70 (d, 1H, J=15.81 Hz), 8.03 (d, 2H, J=8.70 Hz)
This compound was synthesized from compound 1 according to general method 6 described earlier.
Purification was by chromatography on silica gel (elution: dichloromethane-methanol: 98:2).
1H NMR CDCl3 δppm: 0.84-0.89 (m, 3H), 1.39-1.24 (m, 4H), 1.39 (s, 6H), 1.50-1.57 (m, 2H), 2.22 (s, 6H), 2.61 (t, 2H, J=7.40 Hz), 2.90 (t, 2H, J=6.20 Hz), 4.26 (t, 2H, J=6.20 Hz), 7.09 (d, 2H, J=8.50 Hz), 7.57 (s, 2H), 7.59 (d, 1H, J=15.40 Hz), 7.83 (d, 1H, J=15.40 Hz), 8.15 (d, 2H, J=8.50 Hz), 12.90 (s, 1H)
MS (ES-MS): 483.2 (m−1)
MP° C.=85.2-89.8
This compound was synthesized from starting material 3 and starting material 4 according to general method 1 described earlier.
Purification was by chromatography on silica gel (elution: dichloromethan/methanol: 95:5).
1H NMR CDCl3 ppm: 1.46 (s, 6H), 1.53 (s, 9H), 2.27 (s, 6H), 2.33 (s, 6H), 7.28 (s, 2H), 7.43 (d, 1H, J=15.81 Hz), 7.69 (d, 1H, J=15.81 Hz), 7.74 (s, 2H)
This compound was synthesized from compound 3 according to general method 6 described earlier.
Purification was by chromatography on silica gel (elution: dichloromethane/methanol: 98:2).
1H NMR CDCl3 δppm: 1.39 (s, 6H), 2.22 (s, 6H), 2.25 (s, 6H), 7.33 (s, 2H), 7.45 (d, 1H, J=15.5 Hz), 7.69 (d, 1H, J=15.5 Hz), 7.75 (s, 2H)
MS (ES-MS): 381.3 (m−1)
MP° C.=199.3-199.8
This compound was synthesized from intermediate compound 2 and tert-butyl bromoisobutyrate according to general method 4 described earlier.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 85:15).
1H NMR CDCl3 δppm: 1.43 (s, 6H), 1.53 (m, 13H), 1.65-1.75 (m, 2H), 1.75-1.85 (m, 2H), 1.85-1.97 (m, 1H), 2.28 (s, 6H), 1.46-1.52 (m, 1H), 3.12-3.21 (m, 2H), 3.58-3.63 (m, 1H), 4.05 (t, 2H, J=6.21 Hz), 6.97 (d, 2H, J=8.30 Hz), 7.29 (s, 2H), 7.45 (d, 1H, J=15.50 Hz), 7.70 (d, 1H, J=15.50 Hz), 8.03 (d, 2H, J=8.30 Hz)
This compound was synthesized from compound 5 according to general method 6 described earlier.
Purification was by chromatography on silica gel (elution: dichloromethane/methanol: 98:2).
1H NMR CDCl3 δppm: 1.56 (m, 10H), 1.67-1.77 (m, 2H), 1.77-1.90 (m, 2H), 1.90-1.97 (m, 1H), 2.30 (s, 6H), 2.43-2.52 (m, 1H), 3.11-3.22 (m, 2H), 3.58-3.63 (m, 1H), 4.05 (t, 2H, J=6.20 Hz), 6.98 (d, 2H, J=8.80 Hz), 7.31 (s,2H), 7.46 (d, 1H, J=15.80 Hz), 7.71 (d, 1H, J=15.80 Hz), 8.03 (d, 2H, J=8.80 Hz)
MS (ES-MS): 529.1 (M+1)
MP° C.: 182.7-186.6° C.
This compound was synthesized from intermediate compound 3 and tert-butyl bromoisobutyrate according to general method 4 described earlier.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 9:1).
1H NMR CDCl3 δppm: 1.54 (s, 9H), 1.63 (s, 6H), 2.56 (s, 3H), 7.33 (d, 2H, J=8.50 Hz), 7.44 (d, 1H, J=15.70 Hz), 7.62 (d, 1H, J=15.70 Hz), 7.78 (s, 2H), 7.96 (d, 2H, J=8.50 Hz)
This compound was synthesized from compound 7 according to general method 6 described earlier.
Purification was by chromatography on silica gel (elution: dichloromethane/methanol: 98:2).
1H NMR CDCl3 δppm: 1.54 (s, 6H), 2.51 (s, 3H), 7.41 (d, 2H, J=8.5 Hz), 7.64 (d, 1H, J=15.4 Hz), 8.04 (d, 1H, J=15.4 Hz), 8.15 (d, 2H, J=8.5 Hz), 8.29 (s, 2H), 12.93 (s, 1H)
MS (ES-MS): 513.2 (m−1)
This compound was synthesized from intermediate compound 4 and tert-butyl bromoisobutyrate according to general method 4 described earlier.
Purification was by chromatography on silica gel (elution: dichloromethane/cyclohexane: 7:3).
1H NMR CDCl3 δppm: 0.90-1.30 (m, 5H), 1.50 (m, 16H), 1.73 (m, 7H), 2.28 (s, 6H), 4.08 (t, 2H, J=6.54 Hz), 6.97 (d, 2H, J=8.70 Hz), 7.29 (s, 2H), 7.45 (d, 1H, J=15.75 Hz), 7.70 (d, 1H, J=15.75 Hz), 8.03 (d, 2H, J=8.70 Hz)
This compound was synthesized from compound 10 according to general method 6 described earlier.
Purification was by precipitation in a mixture of dichloromethane/heptane.
1H NMR CDCl3 δppm: 0.90-1.30 (m, 5H), 1.56 (m, 7H), 1.70 (m, 7H), 2.30 (s, 6H), 4.09 (t, 2H, J=6.57 Hz), 6.98 (d, 2H, J=9.09 Hz), 7.32 (s, 2H), 7.4 (d, 1H, J=15.60 Hz), 7.71 (d, 1H, J=15.60 Hz), 8.04 (d, 2H, J=9.09 Hz)
MS (ES-MS): 465.3 (m+1)
MP° C.: 134.8-135.3
This compound was synthesized from intermediate compound 5 and tert-butyl bromoisobutyrate according to general method 4 described earlier.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 8:2).
1H NMR CDCl3 δppm: 1.50 (s, 6H), 1.51 (s, 3H), 1.53 (s, 9H), 2.28 (s, 6H), 2.63 (s, 6H), 7.30 (s, 2H), 7.39 (d, 1H, J=15.69 Hz), 7.69 (d, 1H, J=15.69 Hz), 7.72 (s, 2H)
This compound was synthesized from compound 12 according to general method 6 described earlier.
Purification was by chromatography on silica gel (elution: dichloromethane/methanol 98:2).
1H NMR DMSOd6 δppm: 1.39 (s, 6H), 2.22 (s, 6H), 2.28 (s, 3H), 2.59 (s, 6H), 7.56 (s, 2H), 7.62 (d, 1H, J=15.37 Hz), 7.79 (d, 1H, J=15.37 Hz), 7.89 (s, 2H), 12.95 (s,1H)
MS (ES-MS): 412.9 (m+1)
MP° C.: 177.0-179.0
This compound was synthesized from compound 3 and propyl bromide according to general method 4 described earlier. The crude product obtained after elimination of the potassium carbonate and elimination of the solvents by vacuum evaporation was used for the synthesis of compound 15.
1H NMR CDCl3 δppm: 1.09 (t, 3H, J=7.41 Hz), 1.46 (s, 6H), 1.58 (s, 9H), 1.83 (m, 2H), 2.27 (s, 6H), 2.35 (s, 6H), 3.78 (t, 2H, J=6.09 Hz), 7.29 (s, 2H), 7.41 (d, 1H, J=15.32 Hz), 7.68 (d, 1H, J=15.32 Hz), 7.70 (s, 2H)
This compound was synthesized from compound 14 according to general method 6 described earlier.
Purification was by chromatography on silica gel (elution: dichloromethane/methanol 95:5).
1H NMR CDCl3 δppm: 1.05 (t, 3H, J=7.29 Hz), 1.39 (s, 6H), 1.78 (m, 2H), 2.23 (s, 6H), 2.32 (s, 6H), 3.78 (m, 2H), 7.56 (s, 2H), 7.58 (d, 1H, J=16.26 Hz), 7.80 (d, 1H, J=16.26 Hz), 7.86 (s, 2H)
MS (ES-MS): 424.9 (m+1)
MP° C.: 188.5-189.7
This compound was synthesized from intermediate compound 6 and tert-butyl bromoisobutyrate according to general method 4 described earlier.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 95:5).
1H NMR CDCl3 δppm: 1.47 (s, 9H), 1.53 (s, 6H), 2.29 (s, 6H), 2.31 (s, 6H), 3.79 (s, 3H), 7.30 (s, 2H), 7.40 (d, 1H, J=15.50 Hz), 7.70 (d, 1H, J=15.50 Hz), 7.71 (s, 2H)
This compound was synthesized from compound 16 according to general method 6 described earlier.
Purification was by chromatography on silica gel (elution: dichloromethane/methanol 98:2).
1H NMR CDCl3 δppm: 1.57 (s, 6H), 2.31 (s, 6H), 2.38 (s, 6H), 3.79 (s, 3H), 7.33 (s, 2H), 7.43 (d, 1H, J=15.81 Hz), 7.71 (d, 1H, J=15.81 Hz), 7.72 (s, 2H)
MS (ES-MS): 396.9 (m+1)
MP° C.: 166.6-168.8
This compound was synthesized from compound 3 and hexyl bromide according to general method 4 described earlier. The crude product obtained after elimination of the potassium carbonate and elimination of the solvents by vacuum evaporation was used for the synthesis of compound 19.
1H NMR CDCl3 δppm: 0.93 (t, 3H, J=8.58 Hz), 1.37 (m, 4H), 1.47 (s, 6H), 1.53 (m, 1H), 1.83 (m, 2H), 2.28 (s, 6H), 2.36 (s, 6H), 3.82 (t, 2H, J=6.54 Hz), 7.29 (s, 2H), 7.40 (d, 1H, J=15.57 Hz), 7.70 (d, 1H, J=15.57 Hz), 7.71 (s, 2H)
This compound was synthesized from compound 18 according to general method 6 described earlier.
Purification was by chromatography on silica gel (elution: dichloromethane/methanol 95:5).
1H NMR CDCl3 δppm: 0.93 (t, 3H, J=7.02 Hz), 1.37 (m, 4H), 1.50 (m, 2H), 1.56 (s, 6H), 1.83 (m, 2H), 2.30 (s, 6H), 2.34 (s, 6H), 3.82 (t, 2H, J=6.57 Hz), 7.32 (s, 2H), 7.42 (d, 1H, J=15.48 Hz), 7.69 (d,1H, J=15.48 Hz), 7.71 (s, 2H)
MS (ES-MS): 466.9 (m+1)
MP° C.: 171.0-172.0
This compound was synthesized from intermediate compound 7 and tert-butyl bromoisobutyrate according to general method 4 described earlier.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 85:15).
1H NMR CDCl3 δppm: 0.94-1.53 (m, 28H), 2.28 (s, 6H), 2.35 (s, 6H), 3.86 (t, 2H, J=6.75 Hz), 7.29 (s, 2H), 7.41 (d, 1H, J=15.76 Hz), 7.70 (d, 1H, J=15.76 Hz), 7.71 (s, 2H)
This compound was synthesized from compound 20 according to general method 6 described earlier.
Purification was by chromatography on silica gel (elution: dichloromethane/methanol 98:2).
1H NMR CDCl3 δppm: 0.97-1.04 (m, 2H), 1.16-1.34 (m, 4H), 1.56 (s, 6H), 1.63-1.82 (m, 7H), 2.30 (s, 6H), 2.35 (s, 6H), 3.86 (t, 2H, J=6.60 Hz), 7.32 (s, 2H), 7.43 (d, 1H, J=15.81 Hz), 7.70 (d, 1H, J=15.81 Hz), 7.71 (s, 2H)
MS (ES-MS): 492.9 (m+1)
MP° C.: 166.4-167.7
This compound was synthesized from intermediate compound 8 and tert-butyl bromoisobutyrate according to general method 4 described earlier.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 7:3).
1H NMR CDCl3 δppm: 1.29 (m, 5H), 1.46 (s, 6H), 1.53 (s, 9H), 1.62 (m, 1H), 1.80 (m, 2H), 2.03 (m, 2H), 2.27 (s, 6H), 2.75 (m, 1H), 2.95 (t, 2H, J=6.81 Hz), 4.20 (t, 2H, J=6.81 Hz), 6.97 (d, 2H, J=9.24 Hz), 7.28 (s, 2H), 7.43 (d, 1H, J=15.78 Hz), 7.70 (d, 1H, J=15.78 Hz), 8.03 (d, 2H, J=9.24 Hz)
This compound was synthesized from compound 22 according to general method 6 described earlier.
Purification was by chromatography on silica gel (elution: dichloromethane/methanol 98:2).
1H NMR CDCl3 δppm: 1.27-1.38 (m, 4H), 1.56 (s, 6H), 1.63-1.66 (m, 2H), 1.79-1.81 (m, 2H), 2.01-2.04 (m, 2H), 2.30 (s, 6H), 2.76-2.77 (m, 1H), 2.96 (t, 2H, J=7.08 Hz), 4.21 (t, 2H, J=7.08 Hz), 6.97 (d, 2H, J=8.61 Hz), 7.31 (s, 2H), 7.41 (d, 1H, J=15.60 Hz), 7.73 (d, 1H, J=15.60 Hz), 8.04 (d, 2H, J=8.61 Hz)
MS (Maldi-Tof): 496.67 (m+1)
MP° C.: 112.3-114
This compound was synthesized from intermediate compound 9 and tert-butyl bromoisobutyrate according to general method 4 described earlier.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 8:2).
1H NMR CDCl3 δppm: 1.40-1.65 (m, 15H), 2.22 (s, 6H), 2.25 (s, 3H), 2.28 (s, 3H), 2.35 (s, 3H), 7.00 (s, 1H), 7.01 (d, 1H, J=15.70 Hz), 7.18 (s, 2H), 7.24 (s, 1H), 7.35 (d, 1H, 15.70 Hz)
This compound was synthesized from compound 24 according to general method 6 described earlier.
Purification was by chromatography on silica gel (elution: dichloromethane/methanol 98:2).
1H NMR CDCl3 δppm: 1.55 (s, 6H), 2.27 (s, 6H), 2.27-2.30 (m, 6H), 2.39 (s, 3H), 7.05 (s, 1H), 7.07 (d, 1H, J=15.24 Hz), 7.24 (s, 2H), 7.28 (s, 1H), 7.4 (d, 1H, J=15.78 Hz)
MS (ES-MS): 381.2 (m+1)
MP° C.: 168.7-173.3
This compound was synthesized from intermediate compound 10 and tert-butyl bromoisobutyrate according to general method 4 described earlier.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 95:5).
1H NMR CDCl3 δppm: 1.27-2.04 (m, 10H), 1.47 (s, 6H), 1.53 (s, 9H), 2.29 (s, 6H), 2.38 (s, 6H), 2.75 (m, 1H), 2.98 (t, 2H, J=6.84 Hz), 3.98 (t, 2H, J=6.84 Hz), 7.29 (s, 2H), 7.40 (d, 1H, J=15.63 Hz), 7.70 (d, 1H, J=15.63 Hz), 7.71 (s, 2H)
This compound was synthesized from compound 26 according to general method 6 described earlier.
Purification was by chromatography on silica gel (elution: dichloromethane/methanol 98:2).
1H NMR CDCl3 δppm: 1.26-1.42 (m, 5H), 1.56 (s, 6H), 1.62-1.64 (m, 1H), 1.79-1.81 (m, 2H), 2.03-2.00 (m, 2H), 2.3 (s, 6H), 2.38 (s, 6H), 2.71-2.78 (m, 1H), 2.97 (t, 2H, J=7.00 Hz), 3.98 (t, 2H, J=7.00 Hz), 7.32 (s, 2H), 7.43 (d, 1H, J=15.78 Hz), 7.7 (d, 1H, J=15.24 Hz), 7.71 (s, 2H)
MS (MALDI-TOF): 524.78 (m+1)
MP° C.: 156.0-158.0
This compound was synthesized from intermediate compound 11 and tert-butyl bromoisobutyrate according to general method 4 described earlier.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 8:2).
1H NMR CDCl3 δppm: 1.43 (s, 9H), 1.62 (s, 6H), 2.53 (s, 3H), 6.95 (t, 1H, J=8.07 Hz), 7.32 (d, 2H, J=8.64 Hz), 7.39 (m, 3H), 7.72 (d, 1H, J=15.50 Hz), 7.95 (d, 2H, J=8.64 Hz)
This compound was synthesized from compound 28 according to general method 6 described earlier.
It was purified by precipitation in a 70:30 mixture of dichloromethane/heptane.
1H NMR CDCl3 δppm: 1.67 (s, 6H), 2.56 (s, 3H), 7.09 (t, 1H, J=8.19 Hz), 7.32 (m, 3H), 7.43 (m, 2H), 7.73 (d, 1H, J=15.24 Hz), 8.73 (d, 2H, J=8.73 Hz)
MS (ES-MS): 375.1 (m+1)
MP° C.: 142.2-144.6
This compound was synthesized from intermediate compound 12 and tert-butyl bromoisobutyrate according to general method 4 described earlier.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 95:5).
1H NMR CDCl3 δppm: 1.44 (s, 6H), 1.53 (s, 9H), 2.11 (s, 6H), 2.22 (s, 6H), 2.23 (s, 6H), 2.28 (s, 3H), 6.84 (d, 1H, J=16.26 Hz), 7.06 (d, 1H, J=16.26 Hz), 7.16 (s, 2H)
This compound was synthesized from compound 30 according to general method 6 described earlier.
Purification was by chromatography on silica gel (elution: dichloromethane/methanol 98:2).
1H NMR CDCl3 ppm: 1.53 (s, 6H), 2.11 (s, 6H), 2.22 (s, 6H), 2.24 (s, 6H), 2.28 (s, 3H), 6.87 (d, 1H, J=16.20 Hz), 7.08 (d, 1H, J=16.20 Hz), 7.19 (s, 2H)
MS (ES-MS): 409.1 (m+1)
MP° C.: 192.8-194.2
This compound was synthesized from intermediate compound 13 and tert-butyl bromoisobutyrate according to general method 4 described earlier.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 7:3).
1H NMR CDCl3 δppm: 1.47 (s, 6H), 1.53 (s, 9H), 2.28 (s, 6H), 7.02 (d, 2H, J=8.70 Hz), 7.1 (d, 2H, J=7.92 Hz), 7.21 (t, 1H, J=7.35 Hz), 7.29 (s, 2H), 7.39-7.46 (m, 3H), 7.73 (d, 1H, J=16.20 Hz), 8.04 (d, 2H, J=8.70 Hz)
This compound was synthesized from compound 32 according to general method 6 described earlier.
Purification was by chromatography on silica gel (elution: dichloromethane/methanol 98:2).
1H NMR DMSOd6 δppm: 1.39 (s, 6H), 2.22 (s, 6H), 7.08 (d, 2H, J=8.55 Hz), 7.15 (d, 2H, J=8.01 Hz), 7.25 (t, 1H, J=7.41 Hz), 7.47 (t, 2H, J=7.44 Hz), 7.55 (s, 2H), 7.62 (d, 1H, J=15.70 Hz), 7.82 (d, 1H, J=15.70 Hz), 8.19 (d, 2H, J=8.55 Hz)
MS (ES-MS): 430.9 (m+1)
MP° C.: 154.0-156.0
This compound was synthesized from intermediate compound 14 and tert-butyl bromoisobutyrate according to general method 4 described earlier.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 8:2).
1H NMR CDCl3 δppm: 1.50 (s, 6H), 1.53 (s, 9H), 2.28 (s, 6H), 3.98 (s, 3H), 7.04 (t, 1H, J=8.07 Hz), 7.29 (s, 2H), 7.39 (d, 1H, J=15.70 Hz), 7.73 (d, 1H, J=15.70 Hz), 7.78-7.86 (m, 2H)
This compound was synthesized from compound 34 according to general method 6 described earlier.
Purification was by chromatography on silica gel (elution: dichloromethane/methanol 98:2).
1H NMR DMSOd6 δppm: 1.39 (s, 6H), 2.22 (s, 6H), 3.95 (s, 3H), 7.31 (t, 1H, J=7.35 Hz), 7.57 (s, 2H), 7.60 (d, 1H, J=15.78 Hz), 7.83 (d, 1H, J=15.78 Hz), 7.99-8.06 (m, 2H)
MS (ES-MS): 387.1 (m+1)
MP° C.: 167.0-169.0
This compound was synthesized from intermediate compound 15 and tert-butyl bromoisobutyrate according to general method 4 described earlier.
1H NMR CDCl3 δppm: 1.46 (s, 6H), 1.52 (s, 9H), 2.27 (s, 9H), 3.90 (s, 3H), 6.88 (d, 1H, J=8.73 Hz), 7.28 (s, 2H), 7.45 (d, 1H, J=16.11 Hz), 7.70 (d, 1H, J=16.11 Hz), 7.87 (s, 1H), 7.92 (dd, 1H, J=8.73 Hz, J=1.65 Hz)
This compound was synthesized from compound 36 according to general method 6 described earlier.
Purification was by chromatography on silica gel (elution: dichloromethane/methanol 98:2) followed by recrystallization in acetonitrile.
1H NMR CDCl3 δppm: 1.39 (s, 6H), 2.22 (s, 6H), 2.24 (s, 3H), 3.90 (s, 3H), 7.08 (d, 1H, J=8.55 Hz), 7.56 (s, 2H), 7.58 (d, 1H, J=16.71 Hz), 7.82 (d, 1H, J=15.51 Hz), 7.99 (s, 1H), 8.06 (d, 1H, 8.55), 12.95 (s, 1H)
MS (ES-MS): 383.2 (m+1)
MP° C.: 157.0-159.0
This compound was synthesized from intermediate compound 16 and tert-butyl bromoisobutyrate according to general method 4 described earlier.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 9:1).
1H NMR CDCl3 δppm: 0.88 (t, 3H, J=6.84 Hz), 1.25-1.62 (m, 8H), 1.47 (s, 6H), 1.53 (s, 9H), 2.29 (s, 6H), 2.62 (s, 6H), 2.70 (t, 2H, J=6.96 Hz), 7.30 (s, 2H), 7.39 (d, 1H, J=15.90 Hz), 7.70 (d, 1H, J=15.51 Hz), 7.71 (s, 2H)
This compound was synthesized from compound 38 according to general method 6 described earlier.
Purification was by chromatography on silica gel (elution: dichloromethane/methanol 98:2).
1H NMR DMSOd6 δppm: 0.84 (m, 3H), 1.22-1.40 (m, 8H), 2.08 (s, 6H), 2.22 (s, 6H), 2.58 (s, 6H), 2.73 (t, 2H, J=6.90 Hz), 7.57 (s, 2H), 7.63 (d, 1H, J=15.35 Hz), 7.8 (d, 1H, J=15.35 Hz), 7.89 (s, 2H)
MS (ES-MS): 483.2 (m+1)
MP° C.: 130.0-132.0
This compound was synthesized from intermediate compound 17 and tert-butyl bromoisobutyrate according to general method 4 described earlier.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 7:3).
1H NMR CDCl3 ppm: 1.45 (s, 6H), 1.52 (s, 9H), 2.25 (s, 6H), 3.81 (s, 3H), 3.86 (s, 3H), 6.93 (d, 1H, J=9.24 Hz), 7.01 (dd, 1H, J=8.82 Hz, J=2.7 Hz), 7.14 (d, 1H, J=2.8 Hz), 7.22 (s, 2H), 7.26 (d, 1H, J=15.60 Hz), 7.52 (d, 1H, J=15.60 Hz)
This compound was synthesized from compound 40 according to general method 6 described earlier.
Purification was by chromatography on silica gel (elution: dichloromethane/methanol 98:2).
1H NMR DMSOd6 δppm: 1.38 (s, 6H), 2.19 (s, 6H), 3.75 (s, 3H), 3.8 (s, 3H), 7.00 (d, 1H, J=2.16 Hz), 7.12 (m, 2H), 7.26 (d, 1H, J=16.2 Hz), 7.37 (d, 1H, J=13.5 Hz), 7.4 (s, 2H)
MS (ES-MS): 398.3 (m−1)
MP° C.: oily product
This compound was synthesized from starting materials 18 and 3 according to general method 1 described earlier. After elimination of the ethanol by vacuum evaporation compound 42 was obtained after trituration of the residual oil in diethyl ether.
1H NMR CDCl3 δppm: 1.36 (t, 3H, J=6.84 Hz), 1.49 (s, 6H), 2.24 (s, 6H), 2.38 (s, 6H), 3.20 (s, 2H), 3.50 (s, 2H), 3.73 (d, 2H, J=11.04 Hz), 4.03 (d, 2H, J=11.04 Hz), 4.30-4.45 (m, 6H), 7.36 (d, 1H, J=15.75 Hz), 7.28 (s, 2H), 7.66 (m, 3H), 13.39 (s, 1H, N.HCl, exchange/D2O)
Compound 42 was dissolved in ethanol and 2M sodium hydroxide was added. The mixture was stirred for 18 hours at room temperature, then poured in water. The aqueous phase was washed with ethyl acetate, neutralized by addition of acetic acid, then extracted with diethyl ether. The precipitate which formed in the ether phase was drained and recrystallized in absolute ethanol.
1H NMR CDCl3 δppm: 1.50 (s, 6H), 2.28 (s, 6H), 2.36 (s, 6H), 2.89 (m, 4H), 3.06 (t, 2H, J=5.46 Hz), 3.87 (m, 4H), 4.06 (t, 2H, J=5.46 Hz), 6.50 (s, 1H), 7.40 (d, 1H, J=15.78 Hz), 7.27 (s, 2H), 7.68 (m, 3H).
MS (MALDI-TOF): 496 (m+1)
MP° C.: 167-169
This compound was synthesized from intermediate compound 18 and tert-butyl bromoisobutyrate according to general method 4 described earlier.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 95:5).
1H NMR CDCl3 δppm: 1.50 (s, 9H), 1.57 (s, 6H), 7.38 (d, 2H, J=8.50 Hz), 7.65 (d, 1H, J=15.78 Hz), 7.66 (m, 3H), 7.89 (d, 2H, J=8.50 Hz)
This compound was synthesized from compound 44 according to general method 6 described earlier.
Purification was by recrystallization in diisopropyl ether.
1H NMR CDCl3 δppm: 1.65 (s, 6H), 7.24 (d, 2H, J=8.50 Hz), 7.41 (d, 1H, J=15.84 Hz), 7.66 (m, 3H), 7.89 (d, 2H, J=8.50 Hz)
MS (ES-MS): 425 (m+1)
MP° C.: 142
This compound was synthesized from intermediate compound 19 and tert-butyl bromoisobutyrate according to general method 4 described earlier.
Purification was by chromatography on silica gel (elution: cyclohexane/ethyl acetate 9:1).
1H NMR CDCl3 δppm: 1.47 (s, 6H), 1.53 (s, 9H), 2.29 (s, 6H), 4.00 (s, 3H), 7.10 (d, 1H, J=8.65 Hz), 7.30 (s, 2H), 7.40 (d, 1H, J=15.27 Hz), 7.75 (d, 1H), 8.25 (d, 1H, J=8.65 Hz), 8.28 (s, 1H)
This compound was synthesized from compound 46 according to general method 6 described earlier. Compound 47 in pure state was obtained after eliminating the solvents by vacuum evaporation.
1H NMR DMSOd6 δppm: 1.40 (s, 6H), 2.23 (s, 6H), 4.03 (s, 3H), 7.43 (d, 1H, J=8.7 Hz), 7.60 (s, 2H), 7.65 (d, 1H, J=15.40 Hz), 7.88 (d, 1H, J=15.40 Hz), 8.31 (s, 1H), 8.51 (d, 1H, J=8.70 Hz), 12.80 (s, 1H).
MS (ES-MS): 437.3 (m+1)
MP° C.: 182.5
The inventive compounds which were tested are the compounds whose preparation is described in the above examples.
LDL oxidation is an important alteration and plays a predominant role in the establishment and development of atherosclerosis (Jurgens, Hoff et al. 1987) The following protocol allows to demonstrate the antioxidant properties of compounds. Unless otherwise indicated, the reagents were from Sigma (St Quentin, France). LDL were prepared according to the method described by Lebeau et al. (Lebeau, Furman et al. 2000).
The solutions of test compounds were prepared at 10−2 M concentration in bicarbonate buffer (pH 9) and diluted in PBS to obtain final concentrations ranging from 0.1 to 100 μM
Prior to oxidation, EDTA was removed from the LDL preparation by dialysis. Oxidation then took place at 30° C. by adding 100 μl of 16.6 μM CuSO4 solution to 160 μL of LDL (125 μg protein/ml) and 20 μl of a test compound solution. The formation of dienes, the species under observation, was followed by measuring optical density at 232 nm in the samples treated with the compounds in the presence or absence of copper. Optical density at 232 nm was measured every 10 minutes for 8 hours in a thermostated spectrophotometer (Tecan Ultra 380). The analyses were performed in triplicate. The compounds were considered to have antioxidant activity when they induced a longer lag phase and reduced the rate of oxidation and the amount of dienes formed in comparison with the control sample. The inventors demonstrate that the inventive compounds have at least one of the aforementioned antioxidant properties indicating that the inventive compounds have intrinsic antioxidant activity.
Typical results are given in
Neuronal, neuroblastoma (human) and PC12 cells (rat) were the cell lines used for this type of study. PC12 cells were prepared from a rat pheochromocytoma and have been characterized by Greene and Tischler (Greene and Tischler, 1976). These cells are commonly used in studies of neuron differentiation, signal transduction and neuronal death. PC12 cells were grown as previously described (Farinelli, Park et al. 1996), in complete RPMI medium (Invitrogen) supplemented with 10% horse serum and 5% fetal calf serum.
(Primary) cultures of endothelial and smooth muscle cells were also used. Cells were obtained from Promocell (Promocell GmBH, Heidelberg) and cultured according to the supplier's instructions.
The cells were treated with different doses of the compounds ranging from 5 to 300 μM for 24 hours. The cells were then recovered and the increase in expression of the target genes was evaluated by quantitative PCR.
mRNA Measurement:
mRNA was extracted from the cultured cells treated or not with the inventive compounds. Extraction was carried out with the reagents of the Absolutely RNA RT-PCR miniprep kit (Stratagene, France) as directed by the supplier. mRNA was then assayed by spectrometry and quantified by quantitative RT-PCR with a Light Cycler Fast Start DNA Master Sybr Green I kit (Roche) on a Light Cycler System (Roche, France). Primer pairs specific for the genes encoding the antioxidant enzymes superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) were used as probes. Primer pairs specific for the Mactin and cyclophilin genes were used as control probes.
An increase in mRNA expression of the antioxidant enzyme genes, measured by quantitative RT-PCR, was demonstrated in the different cell types used, when the cells were treated with the inventive compounds.
The antioxidant properties of the compounds were also evaluated by means of a fluorescent tag the oxidation of which is followed by appearance of a fluorescence signal. The reduction in the intensity of the emitted fluorescence signal was determined in cells treated with the compounds in the following manner : PC12 cells cultured as described earlier (black 96-well plates, transparent bottom, Falcon) were incubated with increasing doses of H2O2 (0.25 mM-1 mM) in serum-free medium for 2 and 24 hours. After incubation, the medium was removed and the cells were incubated with 10 μM dichlorodihydrofluorescein diacetate solution (DCFDA, Molecular Probes, Eugene, USA) in PBS for 30 min at 37° C. in a 5% CO2 atmosphere. The cells were then rinsed with PBS. The fluorescence emitted by the oxidation tag was measured on a fluorimeter (Tecan Ultra 384) at an excitation wavelength of 495 nm and an emission wavelength of 535 nm. The results are expressed as the percentage of protection relative to the oxidized control.
Fluorescence intensity was lower in the cells incubated with the inventive compounds than in untreated cells. These findings indicate that the inventive compounds promote inhibition of the production of oxidative species in cells subjected to oxidative stress. The previously described antioxidant properties are also effective at inducing antiradical protection in cultured cells.
The protective effect of the compounds on lipid peroxidation in cultured cells (cell models noted hereinabove) was determined as follows: the different cell lines and the primary cell cultures were treated as described earlier, the cell supernatant was recovered after treatment and the cells were lysed and recovered for determination of protein concentration. Lipid peroxidation was detected as follows:
Lipid peroxidation was measured by using thiobarbituric acid (TBA) which reacts with lipid peroxidation of aldehydes such as malondialdehyde (MDA). After treatment, the cell supernatant was collected (900 μl) and 90 μl of butylated hydroxytoluene were added (Morliere, Moysan et al. 1991). One milliliter of 0.375% TBA solution in 0.25 M HCl containing 15% trichloroacetic acid was also added to the reaction medium. The mixture was heated at 80° C. for 15 min, cooled on ice and the organic phase was extracted with butanol. The organic phase was analysed by spectrofluorimetry (λexc=515 nm and λem=550 nm) on a Shimazu 1501 spectrofluorimeter (Shimadzu Corporation, Kyoto, Japan). TBARS are expressed as MDA equivalents using tetra-ethoxypropane as standard. The results were normalized for protein concentration.
The decrease in lipid peroxidation observed in the cells treated with the inventive compounds confirms the previous results.
The inventive compounds advantageously exhibit intrinsic antioxidant properties allowing to slow and/or inhibit the effects of an oxidative stress. The inventors also show that the inventive compounds are capable of inducing the expression of genes encoding antioxidant enzymes. These particular features of the inventive compounds allow cells to more effectively fight against oxidative stress and therefore be protected against free radical-induced damage.
The inventive compounds which were tested are compounds having a carboxylic acid function, whose preparation is described in the above examples.
Nuclear receptors of the PPAR subfamily which are activated by two major pharmaceutical classes—fibrates and glitazones, widely used in the clinic for the treatment of dyslipidemias and diabetes—play an important role in lipid and glucose homeostasis. The following experimental data show that the inventive compounds activate PPARα, PPARγ et PPARδ in vitro.
PPAR activation was tested in vitro in RK13 epitheloid or COS-7 cell lines by measuring the transcriptional activity of chimeras composed of the DNA binding domain of the yeast gal4 transcription factor and the ligand binding domain of the different PPARs. These latter results were then confirmed in cell lines according to the following protocols:
The example is given for RK13 cells and for COS-7 cells.
RK13 cells were from ECACC (Porton Down, UK), COS-7 cells were from the ATCC (American Type Culture Collection) and were grown in DMEM medium supplemented with 10% (VN) fetal calf serum, 100 U/ml penicillin (Gibco, Paisley, UK) and 2 mM L-glutamine (Gibco, Paisley, UK). The culture medium was changed every two days. Cells were kept at 37° C. in a humidified 95% air/5% CO2 atmosphere.
The plasmids pG5TkpGL3, pRL-CMV, pGal4-hPPARα, pGal4-hPPAR7, pGal4-hPPARδ and pGal4-φ have been described by Raspe, Madsen et al. (1999). The pGal4-mPPARα, pGal4-hPPARγ and pGal4-hPPARδ constructs were obtained by cloning into the pGal4-φ vector of PCR-amplified DNA fragments corresponding to the DEF domains of the human PPARα, PPARγ and PPARδ nuclear receptors.
RK13 cells were seeded in 24-well culture dishes at 5×104 cells/well, COS-7 cells in 96-well culture dishes at 5×104 cells/well and transfected for 2 hours with the reporter plasmid pG5TkpGL3 (50 ng/well), the expression vectors pGal4-φ, pGal4-mPPARα, pGal4-hPPARα, pGal4-hPPARγ, pGal4-hPPARδ (100 ng/well) and the transfection efficiency control vector pRL-CMV (1 ng/well) according to the previously described protocol (Raspe, Madsen et al. 1999), then incubated for 36 hours with the test compounds. At the end of the experiment, the cells were lysed (Gibco, Paisley, UK) and luciferase activity was determined with a Dual-Luciferase™ Reporter Assay System kit (Promega, Madison, Wis., USA) for RK13 cells and Steady Glow Luciferase (Promega) for COS-7 cells according to the supplier's instructions as previously described. The protein content of the cell extracts was then measured with the Bio-Rad Protein Assay (Bio-Rad, Munich, Germany) as directed by the supplier.
The inventors demonstrate an increase in luciferase activity in cells treated with the inventive compounds and transfected with the pGal4-hPPARα plasmid. Said induction of luciferase activity indicates that the inventive compounds are activators of PPARα. The results are given in
The inventors demonstrate an increase in luciferase activity in cells treated with the inventive compounds and transfected with the pGal4-hPPARγ plasmid. Said induction of luciferase activity indicates that the inventive compounds are activators of PPARγ. The results are given in
The inventors demonstrate an increase in luciferase activity in cells treated with the inventive compounds and transfected with the pGal4-hPPARδ plasmid. Said induction of luciferase activity indicates that the inventive compounds are activators of PPARδ.
The results are given in
An inflammatory response is observed in many neurological disorders, including multiple sclerosis, Alzheimer's disease and Parkinson's disease, cerebral ischemia and head trauma, and inflammation is also an important factor in neurodegeneration. In stroke, one of the first reactions of glial cells is to release cytokines and free radicals. This release of cytokines and free radicals results in an inflammatory response in the brain which can lead to neuron death (Rothwell, 1997).
Cell lines and primary cells were cultured as described hereinabove.
LPS (lipopolysaccharide) bacterial endotoxin (Escherichia coli 0111: B4) (Sigma, France) was reconstituted in distilled water and stored at 4° C. Cells were treated with LPS 1 μg/ml for 24 hours. To avoid interference from other factors the culture medium was completely changed.
TNF-α is an important factor in the inflammatory response to stress (oxidative stress for example). To evaluate TNF-α secretion in response to stimulation by increasing doses of LPS, the culture medium of stimulated cells was removed and TNF-α was assayed with an ELISA-TNF-α kit (Immunotech, France). Samples were diluted 50-fold so as to be in the range of the standard curve (Chang, Hudson et al. 2000).
The anti-inflammatory property of the compounds was characterized as follows: the cell culture medium was completely changed and the cells were incubated with the test compounds for 2 hours, after which LPS was added to the culture medium at 1 μg/ml final concentration. After a 24-hour incubation, the cell supernatant was recovered and stored at 80° C. when not treated directly. Cells were lysed and protein was quantified with the Bio-Rad Protein Assay kit (Bio-Rad, Munich, Germany) according to the supplier's instructions.
The measurement of the decrease in TNF-α secretion induced by treatment with the test compounds is expressed as pg/ml/μg protein and as the percentage relative to the control. These results show that the inventive compounds have anti-inflammatory properties.
The inventive compounds which were tested are the compounds whose preparation is described in the above examples.
Fibrates, widely used in human medicine for the treatment of dyslipidemiae involved the development of atherosclerosis, one of the leading causes of morbidity and mortality in industrialized countries, are potent activators of the PPARα nuclear receptor. The latter regulates the expression of genes involved in the transport (apolipoproteins such as Apo AI, ApoAII and ApoC-III, membrane transporters such as FAT) or catabolism of lipids (ACO, CPT-I or CPT-II). In rodents and humans, treatment with PPARα activators therefore leads to a decrease in plasma cholesterol and triglyceride levels.
The following protocols were designed to demonstrate a decrease in circulating triglyceride and cholesterol levels, and also highlight the interest of the inventive compounds for preventing and/or treating cardiovascular diseases.
Apo E2/E2 transgenic mice were housed in a 12-hour light/dark cycle at a constant temperature of 20±3° C. After a 1-week acclimatization period, the mice were weighed and divided into groups of 6 animals selected such that the distribution of body weight was uniform. The test compounds were suspended in carboxymethylcellulose and administered by gastric lavage at the indicated doses, once a day for 7 days. Animals had access to food and water ad libitum. At the end of the experiments, animals were weighed and sacrificed under anesthesia. Blood was collected on EDTA. Plasma was isolated by centrifugation at 3000 rpm for 20 minutes. Liver samples were removed and stored frozen in liquid nitrogen for later analysis.
Lipid concentrations in plasma (total cholesterol and free cholesterol, triglycerides and phospholipids) were determined by a colorimetric assay (Boehringer, Mannheim, Germany) according to the supplier's instructions. Plasma concentrations of apolipoproteins AI, AII and CIII were determined as previously described (Raspe et al. 1999, Asset G et al., Lipids, 1999).
a,
19
b,
19
c and 19d give an example of the results where the activity of compound 2 on triglyceride and cholesterol metabolism is illustrated.
a,
20
b,
20
c and 20d illustrate the activity of compounds 13, 33 and 39 on triglyceride and cholesterol metabolism.
Total RNA was isolated from the liver fragments by extraction with a mixture of guanidine thiocyanate/phenol acid/chloroform as previously described (Raspe et al. 1999). Messenger RNA was quantified by semi-quantitative or quantitative RT-PCR with the Light Cycler Fast Start DNA Master Sybr Green I kit (Hoffman-La Roche, Basel, Switzerland) on a Light Cycler System (Hoffman-La Roche, Basel, Switzerland). Primer pairs specific for the ACO, Apo CIII and Apo II genes were used as probes. Primer pairs specific for the 36B4, β-actin and cyclophilin genes were used as control probes. Alternatively, total RNA was analyzed by Northern Blot or Dot Blot according to the previously described protocol (Raspe et al., 1999).
C57 black/6 mice (wild-type) were used for this experiment.
Animals were maintained on a 12 hour light-dark cycle at a temperature of 20° C.±3° C. Water and food were available ad libitum. Food intake and weight gain were recorded.
The inventive compounds or the vehicle (0.5% carboxycellulose) were administered to the animals by gavage, for 14 days before ischemia induction in the middle cerebral artery.
Animals were anesthetized by intraperitoneal injection of 300 mg/kg chloral hydrate. A rectal probe was inserted and body temperature was maintained at 37° C.±0.5° C. Blood pressure was monitored throughout the experiment.
Under a surgical microscope, the right carotid artery was exposed by a median incision in the neck. The pterygopalatine artery was ligated at its origin and an arteriotomy was fashioned in the external carotid artery so as to insert a nylon monofilament, which was gently advanced to the common carotid artery and then into the internal carotid artery so as to occlude the origin of the middle cerebral artery. The filament was withdrawn one hour later to allow reperfusion.
Twenty-four hours after reperfusion, animals previously treated or not with the compounds were euthanized by pentobarbital overdose.
Brains were rapidly frozen and sliced. Sections were stained with cresyl violet. Unstained zones of the brain sections were considered to be damaged by the infarct. Areas were measured and the volume of the infarct and the two hemispheres was calculated by the following formula: (corrected infarct volume=infarct volume−(volume of right hemisphere−volume of left hemisphere)) to compensate for cerebral oedema.
Analysis of the brain sections from treated animals revealed a marked decrease in infarct volume as compared with untreated animals. When the inventive compounds were administered to the animals before the ischemia (prophylactic effect), they were capable of inducing neuroprotection.
3/ Measurement of Antioxidant Activity:
The mouse brains were frozen, crushed and reduced to powder, then resuspended in saline solution. The different enzyme activities were then measured as described by the following authors: superoxide dismutase (Flohe and Otting 1984); glutathione peroxidase (Paglia and Valentine 1967); glutathione reductase (Spooner, Delides et al. 1981); glutathione-S-transferase (Habig and Jakoby 1981); catalase (Aebi 1984).
Said different enzyme activities were increased in brain preparations from animals treated with the inventive compounds.
1/ Ischemia Induction/Reperfusion by Intraluminal Occlusion of the Middle Cerebral Artery.
Animals such as those described previously were used for this experiment. Animals were anesthetized by intraperitoneal injection of 300 mg/kg chloral hydrate. A rectal probe was inserted and body temperature was maintained at 37° C.±0.5° C. Blood pressure was monitored throughout the experiment. Under a surgical microscope, the right carotid artery was exposed by a median incision in the neck. The pterygopalatine artery was ligated at its origin and an arteriotomy was fashioned in the external carotid artery so as to insert a nylon monofilament, which was gently advanced to the common carotid artery and then into the internal carotid artery so as to occlude the origin of the middle cerebral artery. The filament was withdrawn one hour later to allow reperfusion.
Animals first subjected to ischemia-reperfusion were treated with the inventive compounds by the oral route (gavage) for 24 or 72 hours, twice a day.
24 or 72 hours after reperfusion, animals previously treated or not with the compounds were euthanized by pentobarbital overdose.
Brains were rapidly frozen and sliced. Sections were stained with cresyl violet. Unstained zones of the brain sections were considered to be damaged by the infarct. Areas were measured and the volume of the infarct and the two hemispheres was calculated by the following formula: (corrected infarct volume=infarct volume−(volume of right hemisphere−volume of left hemisphere)) to compensate for cerebral oedema.
In the case of curative treatment (treatment of the acute phase), animals treated with the inventive compounds had fewer brain lesions than untreated animals. In fact, the infarct volume was smaller when the inventive compounds were administered one or more times after ischemia-reperfusion.
By virtue of their PPAR activator and antioxidant properties, the inventive compounds have a beneficial effect on the progression of atheromatous plaque.
Female Apo E2/E2 transgenic mice aged approximately 2 months were maintained on a 12 hour light-dark cycle at a constant temperature of 20° C.±3° C. throughout the acclimatization period and throughout the experiment.
After a 1-week acclimatization period, the mice were weighed and divided into groups of 8 animals selected such that the distribution of body weight was uniform. Animals had access to food and water ad libitum. They were fed a western-style diet containing 21% fat and 0.15% cholesterol for 2 weeks prior to treatment.
After this period, the test compounds were added to the feed at the indicated doses. The duration of treatment was 6 weeks.
The animals were weighed and sacrificed under anesthesia by cervical dislocation.
Krebs Ringer solution was added for 10 minutes. The tissues were fixed overnight with 4% PAF in 10 mM PBS at −4° C. The samples were then washed with 100 mM PBS. The hearts were placed in 30% sucrose-Tris for one day then immersed in OCT (Tissue Teck) under vacuum for 30 minutes, then in a mould containing OCT, immersed in isopentane and cooled in liquid nitrogen. The samples were stored at −80° C.
10 μm-thick cryosections were cut from the aortic arch until disappearance of the valves and collected on gelatin-coated slides.
The slides were stained with red oil and hematoxylin so as to differentiate the media from the intima. The different morphogenic parameters were determined with the help of an Olympus microscope and a color camera hooked up to an image analysis system. Damaged areas were quantified manually with a graphic panel hooked up to the same computer system.
The overall area of the atheromatous lesions was expressed in μM2, and compared with the controls. The inventive compounds which were tested induced a significant decrease in lesion area, reflecting a reduction in lesion progression.
Compound 39 was tested for its effects on the differentiation of monocyte-derived dendritic cells (by monitoring the acquisition of the dendritic cell phenotype).
For these experiments, blood samples from volunteer donors (Etablissement Francais du Sang) and monocytes were isolated by a standard protocol using anti-CD14-conjugated magnetic beads (Miltenyi Biotec). Monocytes isolated in this manner were then induced to differentiate by incubation for 6 days in culture medium containing a mixture of cytokines GM-CSF and IL-4 (20 ng/ml for each cytokine).
Compound 39 was added at t=0 and acquisition of the dendritic cell phenotype was followed by expression of the cell surface marker CD1a. The inventors thereby show that compound 39 markedly interfered with differentiation to dendritic cells by almost totally inhibiting the expression of CD1a at the cell surface (
The effects of said compounds were then studied on dendritic cell maturation induced by LPS (lipopolysaccharide). For these experiments, monocyte-derived dendritic cells obtained at D6 of differentiation were pretreated with the inventive compounds for 4 hours, then stimulated with LPS for 16 hours. In this manner it was shown that the compounds significantly interfered with LPS-induced transcription of the CCR7 receptor and the ELC ligand thereof (
The decrease in the expression of ELC and CCR7—key genes in dendritic cell motility—suggests that the inventive compounds inhibit the migration of dendritic cells to secondary lymphoid organs and thereby interfere with the initiation of the immune response triggered by said cells.
The inventors thus demonstrate that monocyte-derived dendritic cells treated with compound 31 had a lower capacity to induce the proliferation of naive CD4+ T cells, by a Mixed Lymphocyte Reaction (MLR) (
The effects of the inventive compounds were then analyzed in vivo in a mouse model of ovalbumin (OVA)-induced allergic asthma.
For these experiments, the mice were sensitized by intraperitoneal injections of ovalbumin in the presence of aluminium hydroxides, at D0 and D10 of the experiment. From D18 to D22, the mice received the inventive compounds (50 mg/kg to 200 mg/kg) daily by gavage. Three consecutive administrations of ovalbumin in aerosol form were given on D20, D21 and D22. The compound was administered approximately 1 hour before each administration. The mice were sacrificed on D24 and the bronchoalveolar lavage fluid (BAL) was collected to determine cellularity (macrophages, eosinophils, lymphocytes, neutrophils) and to measure cytokines IL-5, IL-13, IL-4.
The results show that the inventive compounds interfered with the differentiation and maturation of dendritic cells and inhibited the migration of said cells to secondary lymphoid organs. Moreover, the inventive compounds had a lower capacity to induce the proliferation of naive CD4+ T cells. The inventive compounds therefore interfere with the initiation of the immune response and hence represent an advantageous therapeutic tool for the treatment of asthma.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0400123 | Jan 2004 | FR | national |
| 0409257 | Sep 2004 | FR | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/FR05/00040 | 1/7/2005 | WO | 00 | 7/6/2006 |
