Chromene derivatives substituted by alkoxide as inhibitors of the TCR-Nck interaction

Abstract

The present invention relates to a group of compounds of formula (I) containing a chromene nucleus: ##STR00001##
and that present the capacity to inhibit the proliferation of lymphocytes mediated by the Nck interaction with TCR, so that the present invention also relates to the use of these compounds for the treatment of diseases or conditions where said interaction triggers a complication such as transplant rejection reactions, immune or autoimmune diseases, inflammatory diseases or proliferative diseases.

Claims

1. A compound having the structure of formula (I) ##STR00010## or a pharmaceutically acceptable salt thereof, wherein: R.sub.1 is C.sub.1-C.sub.4 alkyl substituted by C.sub.3-C.sub.6 cycloalkyl; R.sub.2 and R.sub.3 are each independently selected from hydrogen, and substituted or not substituted C.sub.1-C.sub.6 alkyl; or R.sub.2 and R.sub.3 form, together with the nitrogen atom they are bound to, substituted or not substituted saturated 5- or 6-membered heterocycle; R.sub.4 is halogen.

2. The compound according to claim 1 wherein R.sub.1 is methyl substituted by C.sub.3-C.sub.6 cycloalkyl.

3. The compound according to claim 1 wherein R.sub.1 is C.sub.1-C.sub.4 alkyl substituted by cyclopropyl.

4. The compound according to claim 1 wherein R.sub.1 is CH.sub.2-cyclopropyl.

5. The compound according to claim 1 wherein R.sub.2 and R.sub.3 form, together with the nitrogen atom they are bound to, a not substituted saturated 5-membered heterocycle.

6. The compound according to claim 1 wherein R.sub.2 and R.sub.3 form, together with the nitrogen atom they are bound to, a not substituted saturated 6-membered heterocycle.

7. The compound according to claim 6 wherein the not substituted saturated 6-membered heterocycle contains an additional N or O atom.

8. The compound according to claim 1 wherein R.sub.2 is H.

9. The compound according to claim 8 wherein R.sub.3 is substituted or not substituted C.sub.1-C.sub.4 alkyl.

10. The compound according to claim 9 wherein R.sub.3 is CH.sub.2CH.sub.3.

11. The compound according to claim 9 wherein R.sub.3 is a C.sub.1-C.sub.4 alkyl group substituted by an NRR group, wherein R and R are independently H or C.sub.1-C.sub.4 alkyl.

12. The compound according to claim 11 wherein R.sub.3 is CH.sub.2CH.sub.2N(CH.sub.3).sub.2.

13. The compound according to claim 5 wherein the not substituted saturated 5-membered heterocycle is pyrrolidinyl.

14. The compound according to claim 1 wherein R.sub.2 and R.sub.3 form, together with the nitrogen atom they are bound to, substituted saturated 6-membered heterocycle.

15. The compound according to claim 14 wherein the saturated 6-membered heterocycle is substituted by C.sub.1-C.sub.4 alkyl in at least one of its positions.

16. The compound according to claim 14 wherein the 6-membered saturated heterocycle contains an additional N or O atom.

17. The compound according to claim 16 wherein the N atom is substituted by C.sub.1-C.sub.4 alkyl.

18. The compound according to claim 16 wherein the 6-membered saturated heterocycle is ##STR00011##

19. The compound according to claim 1 wherein R.sub.4 is fluorine.

20. The compound of formula (I) according to claim 1 which is selected from the group consisting of: 1-((6(cyclopropylmethoxy)4-(4-fluorophenyl)-2H-chromen-3-yl)methyl)pyrrolidine, 1-(6-(cyclopropylmethoxy)-4-(4-fluorophenyl)-2H-chromen-3-yl)methyl)-4-methylpiperazine, N.sup.1-((6-(cyclopropylmethoxy)-4-(4-fluorophenyl)-2H-chromen-3-yl)methyl)-N.sup.2,N.sup.2-dimethylethane-1,2-diamine, 4-((6-(cyclopropylmethoxy)-4-(4-fluorophenyl)-2H-chromen-3-yl)methyl)morpholine, and N-((6-(cyclopropylmethoxy)-4-(4-fluorophenyl)-2H-chromen-3-yl)methyl)ethanamine.

21. A pharmaceutical composition comprising a compound of formula (I) according to claim 1 and one or more pharmaceutically acceptable excipients.

22. The compound according to claim 7 wherein the not substituted saturated 6-membered heterocycle is ##STR00012##

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1. Represents the ability of inhibiting the proliferation of T lymphocytes for each of the tested compounds of the invention.

(2) FIG. 2. Represents an estimation of the level of neuronal damage during the study among the subjects receiving placebo and those receiving the compound AX-104.

(3) FIG. 3. Represents weight progression during the study among the subjects receiving placebo and those receiving the compound AX-104.

EXAMPLES

(4) The invention will be illustrated by tests performed by the inventors, which shows the effectiveness of the compounds of the invention.

Example 1: Synthesis of the Compounds of the Invention

(5) Synthesis Scheme of AX-101

(6) ##STR00004##

(7) Synthesis of AX-101 (free base): To a solution of AX-24.HCl (1 g, 1 eq.) in dry DCM (10 ml), BBr.sub.3 (0.79 g, 1.2 eq.) was added drop by drop at ?78? C. and stirred at RT overnight. The reaction was monitored by TLC. After completion, the reaction mixture was poured into a cold solution of saturated sodium bicarbonate and extracted with DCM (2?20 ml). The combined organic layer was washed with water (20 ml), saline solution (10 ml) and dried over Na.sub.2SO.sub.4. Purification of the crude product by column chromatography (100% ethyl acetate) yielded 0.5 g of the desired product (light brown solid) with a purity of 97.8% by HPLC. .sup.1NMR (400 MHz, DMSO-d.sup.6): ? 8.80 (s, 1H), 7.31-727 (m, 2H), 7.20-7.17 (m, 2H), 6.68-6.66 (d, 2H), 6.51-6.28, (dd, 1H), 5.93 (s, 1H), 473 (s, 1H), 2.96 (s, 2H), 2.29 (b, 4H), 1.61 (b, 4H). Theoretic MS for C.sub.20H.sub.20FNO.sub.2: 325.4; M.sup.++1 found: 326.1.

(8) Synthesis Scheme of AX-104

(9) ##STR00005##

(10) Synthesis of AX-104: To the mixture of AX-101 (0.7 g, 1 eq.), K.sub.2CO.sub.3 (1 g, 3 eq.), cyclopropyl methyl bromide (1.16 g, 4 eq.) in DMF (10 ml) and acetone (20 ml), and tetramethylammonium bromide (0.1 g, catalytic) were added. The resulting reaction mixture was heated at 80? C. overnight. The reaction was controlled by TLC. After completion, the reaction mixture was poured into cold water (40 ml) and extracted with ethyl acetate (3?20 ml). The combined organic layer was washed with water (2?15 ml), saline solution (10 ml) and dried over anhydrous sodium sulfate. Purification of the crude product by column chromatography (20% ethyl acetate in hexane) yielded 0.1 g of the desired product with a purity of 96% by HPLC, .sup.1H NMR (400 MHz, DMSO-d.sup.6): ? 7.31-7.27 (m, 2H), 7.21-7.19 (m, 2H), 6.78-6.76 (d, 2H), 6.70-6.67 (dd, 1H), 5.97-5.96 (d, 1H), 3.58-3.56 (d, 2H), 2.97 (s, 2H), 2.3 (b, 4H), 1.61 (b, 4H), 1.8-12 (m, 4H), 0.49-0.44 (m, 2H), 0.21-0.18 (m, 2H), Theoretic MS for C.sub.24H.sub.26FNO.sub.2: 379.5, M.sup.++ found 1, 380.1.

(11) Synthesis Scheme for AX-133 to AX-136

(12) ##STR00006##

(13) Synthesis of Compound B: To a solution of compound A (10 g, 1 eq,) in dry DCM (150 ml), BBr.sub.3 (11.4 g. 1.3 eq.) was added drop by drop at 0? C. and stirred at RT overnight. The reaction was controlled by TLC. After completion, the reaction mixture was poured into cold water and extracted with DCM (2?150 ml). The combined organic layer was washed with water (2?70 ml), saline solution (20 ml) and dried over Na.sub.2SO.sub.4. Purification of the crude product by column chromatography (40% ethyl acetate in hexane) yielded 4.7 g of the desired product. Theoretic MS for C.sub.16H.sub.11FO.sub.3: 324.12, M.sup.++1 found, 325.2.

(14) Synthesis of Compound C: To the mixture of Compound B (5.5 g, 1 eq), K.sub.2CO.sub.3 (11.2 g 4 eq.), cyclopropylmethyl bromide (5.5 g, 2 eq.) in DMF (50 ml), and tetramethylammonium bromide (0.1 g, catalytic) was added. The resulting reaction mixture was heated at 75? C. overnight. The reaction was controlled by TLC. After completion the mixture was poured into cold water (100 ml) and extracted with ethyl acetate (3?70 ml). The combined organic layer was washed with water (2?40 ml), saline solution (20 ml) and dried over anhydrous sodium sulfate. Purification of the crude product by column chromatography (10% ethyl acetate in hexane) yielded 1.7 g of the desired product. Theoretic MS for C.sub.20H.sub.17FO.sub.3: 324.12, M.sup.++1 found, 325.2.

(15) Synthesis of Compound D: To the mixture of Compound C (3 g, 1 eq), sodium borohydride (0.17 g, 0.5 eq.) was added dropwise in toluene (20 ml), methanol (2 ml) to RT and stirred at this temperature for 2 h. The reaction was monitored by TLC. After completion, the mixture was poured into cold water (100 ml) and extracted with ethyl acetate (2?30 ml). The combined organic layer was washed with water (20 ml), saline solution (10 ml) and dried over anhydrous sodium sulfate. Evaporation of the organic layer under reduced pressure yielded 2.7 g of the desired product. This material was taken directly to the next step without purification and analysis.

(16) Synthesis of Compound E: To a mixture of Compound D (3.5 g, 1 eq), sodium borohydride was added dropwise in toluene (30 ml), thionyl chloride (1.78 g, 1.4 eq) at 0? C. and stirred at RT for 2 h. The reaction was monitored by TLC. After completion, the mixture was poured into iced water (25 ml) and extracted with ethyl acetate (2?25 ml). The combined organic layer was washed with water (30 ml), saline solution (10 ml) and dried over anhydrous sodium sulfate. Evaporation of the organic layer under reduced pressure (vacuum evaporation) yielded 3.4 g of raw product. This material was taken directly to the next step without purification and analysis.

(17) Synthesis of AX-133: Mixture of Compound E (0.3 g, 1 eq), K.sub.2CO.sub.3 (0.36 g of 3 eq), N-methyl piperazine (0.13 g, 1.5 eq) in diisopropyl ether (15 ml) was was stirred at RT overnight. The reaction was monitored by TLC. After completion, the mixture was poured into cold water (20 ml) and extracted with ethyl acetate (2?30 ml). The combined organic layer was washed with water (20 ml), saline solution (10 ml) and dried over anhydrous sodium sulfate. Purification of the crude product by column chromatography (10% methanol in DCM) yielded 40 mg of the desired product with a purity of 92.9% by HPLC. .sup.1H NMR (400 MHz, CDCl.sub.3): ? 7.11-7.2 (m, 4H), 6.78-6.75 (d, 1H), 6.65-6.63 (dd, 1H), 6.15-6.14 (d, 1H), 4.78 (s, 2H), 3.57-3.55 (d, 2H), 2.97 (s, 2H), 2.52 (b, 8H), 1.23 (s, 3H), 1.14-1.8 (m, 1H), 0.56-0.52 (m, 2H), 0.24-0.020 (m, 2H). Theoretic MS for C.sub.25H.sub.29FN.sub.2O.sub.2: 408.51, M.sup.++1 found 409.2.

(18) Synthesis of AX-134: Mixture of Compound E (0.3 g, 1 eq.), K.sub.2CO.sub.3 (0.55 g 5 eq.), N,N-dimethylethylenediamine (0.28 g, 4 eq.) in diisopropyl ether (15 mL) was refluxed for 2 h. The reaction was monitored by TLC. After completion, the mixture was poured into cold water (20 ml) and extracted with ethyl acetate (2?30 ml). The combined organic layer was washed with water (20 ml), saline solution (10 ml) and dried over anhydrous sodium sulfate. Purification of the crude product by column chromatography (12% methanol in DCM) yielded 80 mg of the desired product with a purity of 93.2% by HPLC. .sup.1H NMR (CDCl.sub.3): ? 7.16-7.8 (m, 4H), 6.80-677 (d, 1H), 6.68-6.63 (dd, 1H), 6.18-6.17 (d, 1H), 4.85 (s, 2H), 3.60-3.58 (d, 2H), 3.19 (s, 2H), 2.55-2.52 (t, 2H), 2.34-231 (t, 2H), 2.18 (b, 5H), 1.14-1.12 (m, 1H), 0.57-0.55 (m, 2H), 0.25-0.24 (m, 2H). Theoretic MS for C.sub.24H.sub.29FN.sub.2).sub.2: 3965, M.sup.++1 found, 397.2.

(19) Synthesis of AX-135: Mixture of Compound E (0.2 g, 1 eq), K.sub.2CO.sub.3 (0.24 g of 3 eq), morpholine (0.1 g, 2 eq) in diisopropyl ether (15 ml) was refluxed for 2 h. The reaction was monitored by TLC. After completion, the mixture was poured into cold water (20 ml) and extracted with ethyl acetate (2?20 ml). The combined organic layer was washed with water (20 ml), saline solution (10 ml) and dried over anhydrous sodium sulfate. Purification of the crude product by column chromatography (30% ethyl acetate in hexane) yielded 45 mg of the desired product with a purity of 95.8% by HPLC. .sup.1H NMR (DMSO-d.sup.6): ? 7.37-7.26 (t, 2H), 7.21-7.16 (m, 2H), 6.78-6.76 (d, 1H), 671-6.68 (dd, 1H), 5.97-5.96 (d, 1H), 4.75 (s, 2H), 3.58-3.56 (d, 2H), 3.52-3.50 (m, 2H), 2.87 (s, 2H), 2.21 (b, 4H), 1.8-1.3 (m, 1H), from 0.49 to 0.44 (m, 2H), 0.21 to 0.17 (m, 2H). Theoretic MS for C.sub.24H.sub.26FNO.sub.3: 395.47, M.sup.++1 found, 396.1.

(20) Synthesis of AX-136; Mixture of Compound E (0.3 g, 1 eq), K.sub.2CO.sub.3 (0.36 g of 3 eq), ethylamine hydrochloride (0.22 g, 3 eq) in DMF (10 ml) was stirred at RT overnight. The reaction was monitored by TLC. After completion, the mixture was poured into cold water (20 ml) and extracted with ethyl acetate (3?20 ml). The combined organic layer was washed with water (2?15 ml), saline solution (10 ml) and dried over anhydrous sodium sulfate. Purification of the crude product by column chromatography (5% methanol in DCM) yielded 50 mg of the desired product with a purity of 92.2% by HPLC. .sup.1H NMR (CDCl.sub.3): ? 7.16-7.9 (m, 4H), 6.80-6.78 (d, 1H), 6.68-6.63 (dd, 1H), 6.19-6.17 (s, 1H), 4.83 (s, 2H), 3.60-3.58 (d, 2H), 3.2 (s, 2H), 2.54-2.49 (q, 2H), 1.16-1.12 (m, 1H), 1.02-0.98 (t, 3H), 0.59 to 0.54 (m, 2H), 0.27-0.23 (m, 2H). Theoretic MS for C.sub.24H.sub.26FNO.sub.3; 353.43; M-44.3 found, 309.1 (Ethyl amine group cleaves mass).

(21) Synthesis Scheme for AX-102, AX-117 and AX-118

(22) ##STR00007##

(23) Synthesis of Compound B: A mixture of Compound A (3 g, 1 eq), K.sub.2CO.sub.3 (3.06 g, 2 eq), ethyl chlorodifluoroacetate (2.2 g, 1.3 eq) in DMF (20 ml), was heated at 75? C. overnight. The reaction was monitored by TLC. After completion, the mixture was poured into cold water (50 ml) and extracted with ethyl acetate (3?50 ml). The combined organic layer was washed with water (2?30 ml), saline solution (20 ml) and dried over anhydrous sodium sulfate. Purification of the crude product by column chromatography (10% ethyl acetate in hexane) yielded 1 g of the desired product, .sup.1H NMR (DMSO-d.sup.6) Theoretical MS for C.sub.17H.sub.11F.sub.3NO.sub.3: 320.26; M.sup.++1 found, 321.1.

(24) Synthesis of Compound C: To the mixture of Compound B (1 g, 1 eq), sodium borohydride (0.05 g, 0.5 eq) in toluene (10 ml) and methanol (2 ml) was added dropwise at RT and stirred at this temperature for 2 h. The reaction was monitored by TLC. After completion, the mixture was poured into cold water (30 ml) and extracted with ethyl acetate (2?30 ml). The combined organic layer was washed with water (15 ml), saline solution (10 ml) and dried over anhydrous sodium sulfate. Evaporation of the organic layer under reduced pressure (vacuum evaporation) yielded 0.9 g of the desired product. This material was taken directly to the next step without purification and analysis.

(25) Synthesis of Compound D: To a mixture of Compound C (0.9 g, 1 eq) in toluene (20 ml), thionyl chloride (0.43 g, 1.3 eq) was added drop by drop at 0? C. and stirred at RT for 2 h. The reaction was monitored by TLC. After completion, the mixture was poured into cold water (20 ml) and extracted with ethyl acetate (2?30 ml). The combined organic layer was washed with water (20 ml), saline solution (10 ml) and dried over anhydrous sodium sulfate. Evaporation of the organic layer under reduced pressure (vacuum evaporation) yielded 0.9 g of raw product. This material was taken directly to the next step without purification and analysis.

(26) Synthesis of AX-102: Mixture of Compound D (0.25 g, 1 eq), K.sub.2CO.sub.3 (0.5 g, 5 eq), and pyrrolidine (0.15 g, 3 eq) in diisopropyl ether (15 ml) was heated at 60? C. for 2 h. The reaction was monitored by TLC. After completion the mixture was poured into cold water (20 ml) and extracted with ethyl acetate (2?25 ml). The combined organic layer was washed with water (20 ml), saline solution (10 ml) and dried over anhydrous sodium sulfate. Purification of the crude product by column chromatography (40% ethyl acetate in hexane) gave 50 mg of the desired product with 94.4% HPLC purity 30. .sup.1H NMR (DMSO-d.sup.6).sup.1H NMR (CDCl.sub.3) ? 7.12-7.11 (d, 4H), 6.88-6.81 (m, 2H), 6.47-6.10 (q, 1H), 4.9 (s, 2H), 3.0 (s, 2H), 2.38 (b, 2H), 1.70 (b, 4H). Theoretic MS for C.sub.21H.sub.20F.sub.3NO.sub.2: 375.4; M.sup.++1 found, 376.1.

(27) Synthesis of AX-117: Mixture of Compound D (0.25 g, 1 eq), K.sub.2CO.sub.3 (0.3 g, 3 eq), and N-methyl piperazine (0.11 g, 1.5 eq) in diisopropyl ether (15 ml) was heated at 60? C. for 2 h. The reaction was monitored by TLC. After completion, the mixture was poured into cold water (20 ml) and extracted with ethyl acetate (2?30 ml). The combined organic layer was washed with water (20 ml), saline solution (10 ml) and dried over anhydrous sodium sulfate. Purification of the crude product by column chromatography (8% methanol in DCM) yielded 40 mg of the desired product with a purity of 96.3% by HPLC. .sup.1H NMR (DMSO-d.sup.6) ? 7.32-7.30 (b, 2H), 7.22 (b, 2H), 7.13-6.76 (1H), 6.98-9.89 (2H), 6.2 (s, 1H), 4.88 (s, 2H), 2.91 (s, 3H), 2.67) b, 4H), 2.41 (b, 8H). Theoretic MS for C.sub.22H.sub.23F.sub.3N.sub.2O.sub.2: 404.43; M.sup.++1 found, 405.1.

(28) Synthesis of AX-118.HCl: Mixture of Compound D (0.2 g, 1 eq), K.sub.2CO.sub.3 (0.32 g, 4 eq), and N,N-dimethylethylenediamine (0.15 g, 3 eq) in diisopropyl ether (10 mL) was heated at 60? C. for 2 h. The reaction was monitored by TLC. After completion, the mixture was poured into cold water (20 ml) and extracted with ethyl acetate (2?25 ml). The combined organic layer was washed with water (20 ml), saline solution (10 ml) and dried over anhydrous sodium sulfate. Purification of the crude product by column chromatography (7% methanol in DCM) yielded 50 mg of the desired product with a purity of 92% by HPLC. The previous product was dissolved in dioxane (4 ml) and 0.5 ml of 4M HCl was added and stirred at RT. After 2 h, the filtration of the resulting solid, washed with 10 ml of n-pentane and vacuum dried, yielded 40 mg of pure product with a purity of 93% by HPLC. .sup.1H NMR (DMSO-d.sup.6) ? 10.7 (b, 1H), 9.76 (b, 2H), 7.42-7.34 (m, 4H), 7.17-6.98 (m, 3H), 6.26 (s, 1H), 5.04 (s, 2H), 3.6 (b, 2H), 3.3 (b, 2H), 2.79 (b, 6H). Theoretic MS for C.sub.21H.sub.24ClF.sub.3N.sub.2O.sub.2: 428.88; M.sup.++1 found, 393.1 (HCl).

(29) Synthesis Scheme for AX-109 to AX-112

(30) ##STR00008##

(31) Synthesis of Compound B: A mixture of Compound A (3 g, 1 eq) sodium borohydride (0.17 g, 0.5 eq) in toluene (20 ml), methanol (2 ml) was added dropwise at RT and stirred at this temperature for 2 h. The reaction was monitored by TLC. After completion, the mixture was poured into cold water (100 ml) and extracted with ethyl acetate (2?30 ml). The combined organic layer was washed with water (20 ml), saline solution (10 ml) and dried over anhydrous sodium sulfate. Evaporation of the organic layer under reduced pressure yielded 2.7 g of the desired product. This material was taken directly to the next step without purification and analysis.

(32) Synthesis of Compound C: To the mixture of Compound B (3.5 g, 1 eq), in toluene (30 ml), thionyl chloride (1.78 g, 1.4 eq) was added drop by drop at 0? C. and stirred for 2 h at RT 15, The reaction was monitored by TLC. After completion, the mixture is poured into iced water (25 ml) and extracted with ethyl acetate (2?25 ml). The combined organic layer was washed with water (30 ml), saline solution (10 ml) and dried over anhydrous sodium sulfate. Evaporation of the organic layer under reduced pressure (vacuum evaporation) yielded 34 g of crude product. This material was taken directly to the next step without purification and analysis.

(33) Synthesis of AX-109: Mixture of Compound C (0.4 g, 1 eq), K.sub.2CO.sub.3 (0.5 g, 3 eq), and N-methyl piperazine (0.15 g, 1.2 eq) in ether diisopropyl (4 ml) was stirred at RT for 15 h. The reaction was monitored by TLC. Then poured into cold water (20 ml) and extracted with ethyl acetate (2?30 ml). The combined organic layer was washed with water (20 ml), saline solution (10 ml) and dried over anhydrous sodium sulfate. Purification of the crude product by column chromatography (5% methanol in DCM) yielded 170 mg of the desired product with a purity of 94.5% by HPLC. .sup.1H NMR (CDCl.sub.3) ? 7.10-7.09 (d, 4H), 6.82-6.79 (d, 1H), 6.68-6.65 (dd, 1H), 6.13 (d, 1H), 4.79 (s, 2H), 3.61 (s, 3H), 2.95 (s, 2H), 2.51-2.44 (b, 8H), 2.08 (s, 2H). Theoretic MS for C.sub.22H.sub.25FN.sub.2O.sub.2: 368.4; M.sup.++1 found, 369.2.

(34) Synthesis of AX-110: Mixture of Compound C (0.4 g, 1 eq), K.sub.2CO.sub.3 (0.5 g 3 eq), and 10 N,N-dimethylethylenediamine (0.13 g, 1.2 eq.) in diisopropyl ether (4 ml) was stirred at RT for 15 h. The reaction was monitored by TLC. After completion the mixture was poured into cold water (20 ml) and extracted with ethyl acetate (2?30 ml). The combined organic layer was washed with water (20 ml), saline solution (10 ml) and dried over anhydrous sodium sulfate. Purification of the crude product made by the formation of HCl salt followed by neutralization using saturated sodium bicarbonate yielded 130 mg of the desired product with a purity of 94% by HPLC. NMR (CDCl.sub.3) ? 7.17-7.08 (m, 4H), 6.82-6.80 (d, 1H), 6.67-6.65 (dd, 1H), 6.15-6.14 (d, 1H), 4.82 (s, 2H), 3.62 (s, 3H), 3.21 (s, 2H), 2.55-2.53 (t, 2H), 2.35-2.32 (t, 2H), 2.19 (s, 6H). Theoretic MS for C.sub.21H.sub.25FN.sub.2O.sub.2: 356.4; M.sup.++1 found, 357.1.

(35) Synthesis of AX 111: Mixture of Compound C (0.4 g, 1 eq), K.sub.2CO.sub.3 (0.5 g, 3 eq) and morpholine (0.13 g, 1.2 eq) in diisopropyl ether (4 ml) was stirred at RT for 15 h. The reaction was monitored by TLC. After completion, the mixture was poured into cold water (20 ml) and extracted with ethyl acetate (2?30 ml). The combined organic layer was washed with water (20 ml), saline solution (10 ml) and dried over anhydrous sodium sulfate, Purification of the crude product made by crystallization using diethyl ether yielded 160 mg of the desired product with a purity of 96.5% purity by HPLC. .sup.1H NMR (CDCl.sub.3) ? 7.12-7.10 (d, 4H), 6.82-6.80 (d, 11-1), 6.68-6.65 (dd, 1H), 6.13 (d, 1H), 4.82 (s, 2H), 3.65-3.63 (m, 4H), 3.62 (s, 3H), 2.91 (s, 2H), 2.31 (b, 4H). Theoretic MS for C.sub.21H.sub.22FNO.sub.3: 355.4; M.sup.++1 found, 356.1.

(36) Synthesis of AX-112: Mixture of Compound C (0.4 g, 1 eq), K.sub.2CO.sub.3 (0.5 g, 3 eq), and ethylamine. HCl (0.12 g, 1.2 eq) in diisopropyl ether (4 ml) was stirred at RT for 15 h. The reaction was monitored by TLC. After completion the mixture was poured into cold water (20 ml) and extracted with ethyl acetate (2?30 ml). The combined organic layer was washed with water (20 ml), saline solution (10 ml) and dried over anhydrous sodium sulfate. Purification of the crude product by column chromatography (6% methanol in DCM) yielded 120 mg of the desired product with a purity of 95.0% by HPLC. NMR (CDCl.sub.3) ? 7.26-7.09 (m, 4H), 6.82-6.80 (d, 1H), 6.68-6.65 (dd, 1H), 6.14 (d, 1H), 4.86 (s, 2H), 3.62 (s, 3H), 3.25 (s, 2H), 2.58-2.53 (m, 2H), 1.05-1.01 (t, 3H). Theoretic MS for C.sub.19H.sub.20FNO.sub.2: 313.4; M.sup.++1 found, 269.2 (NHEt).

(37) Synthesis Scheme for AX-103 and AX-126 to AX-128

(38) ##STR00009##

(39) Synthesis of Compound B: To mixture of Compound A (3.5 g, 1 eq) in toluene (30 ml), thionyl chloride (1.78 g, 1.4 eq) chloride was added drop by drop at 0? C. and stirred at RT for 2 h. The reaction was monitored by TLC. After completion, the mixture was poured into iced water (25 ml) and extracted with ethyl acetate (2?25 ml). The combined organic layer was washed with water (30 ml), saline solution (10 ml) and dried over anhydrous sodium sulfate. Evaporation of the organic layer under reduced pressure yielded 2.4 g of crude product. This material was taken directly to the next step without purification and analysis.

(40) Synthesis of AX-103.HCl; The mixture of Compound B (0.2 g, 1 eq), K.sub.2CO.sub.3 (0.2 g 3 eq) and pyrrolidine (0.05 g, 1.2 eq) in diisopropyl ether (4 ml) was stirred at RT for 15 h. The reaction was monitored by TLC. After completion the mixture was poured into cold water (20 ml) and extracted with ethyl acetate (2?30 ml). The combined organic layer was washed with water (20 ml), saline solution (10 ml) and dried over anhydrous sodium sulfate. The crude product was converted into its HCl salt at a scale of 45 mg with a purity of 98.2% by HPLC. .sup.1H NMR (CDCl.sub.3) ? 7.26-7.20 (m, 2H), 7.17-7.14 (m, 2H), 7.14-7.06 (d, 1H), 6.95-6.93 (d, 1H), 6.41-6.40 (d, 1H), 5.29 (s, 2H), 3.72-3.66 (m, 4H). Theoretic MS for C.sub.21H.sub.23ClF.sub.4N.sub.2O.sub.2: 429.84; M.sup.++1 found, 394.1 (HCl).

(41) Synthesis of AX-126.HCl: The mixture of Compound B (0.4 g, 1 eq). K.sub.2CO.sub.3 (0.5 g, 3 eq) and N,N-dimethylethylenediamine (0.13 g, 1.2 eq.) in diisopropyl ether (4 ml) was stirred at RT for 15 h. The reaction was monitored by TLC. After completion the mixture was poured into cold water (20 ml) and extracted with ethyl acetate (2?30 ml). The combined organic layer was washed with water (20 ml), saline solution (10 ml) and dried over anhydrous sodium sulfate. Purification of the crude product made by formation of the HCl salt yielded 60 mg of the desired product with a purity of 93.7% purity by HPLC. .sup.1H NMR (DMSO-d.sup.6) ? 10.8 (b, 1H), 9.70 (b, 1H), 7.93-7.92 (b, 2H), 7.30-7.24 (m, 2H), 7.19-7.11 (m, 2H), 7.01-6.96 (m, 2H), 6.87-6.85 (d, 1H), 6.3 (s, 1H), 5.0 (s, 2H), 3.55 (s, 2H), 3.40 (b, 2H), 2.78 (b, 6H) Theoretic MS for C.sub.21H.sub.23ClF.sub.4N.sub.2O.sub.2: 446.87; M.sup.++1 found, 411.1 (HCl).

(42) Synthesis of AX-127; The mixture of Compound B (0.4 g, 1 eq), K.sub.2CO.sub.3 (0.5 g, 3 eq) and morpholine (0.13 g, 1.2 eq) in diisopropyl ether (4 ml) was stirred at RT for 15 h. The reaction was monitored by TLC. After completion the mixture was poured into cold water (20 ml) and extracted with ethyl acetate (2?30 ml). The combined organic layer was washed with water (20 ml), saline solution (10 ml) and dried over anhydrous sodium sulfate. It was evaporated under reduced pressure (vacuum evaporation) to yield 110 mg of the desired product with a purity of 96% by HPLC. .sup.1H NMR (CDCl.sub.3) ? 7.16-7.08 (m, 4H), 6.97-6.95 (d, 1H), 6.85-6.83 (d, 1H), 6.40-6.39 (b, 1H), 4.90 (s, 2H), 3.66-3.64 (t, 4H), 2.92 (s, 2H), 2.31 (b, 4H). Theoretic MS for C.sub.21H.sub.19F.sub.4NO.sub.3: 409.3; M.sup.++1 found, 410.1.

(43) Synthesis of AX-128,HCl: The mixture of Compound B (0.4 g, 1 eq,), K.sub.2CO.sub.3 (0.5 g, 3 eq) and ethylamine. HCl (0.12 g, 1.2 eq.) in diisopropyl ether (4 ml) was stirred at RT for 1 h. The reaction was monitored by TLC. After completion, the mixture was poured into cold water (20 ml) and extracted with ethyl acetate (2?30 ml). The combined organic layer was washed with water (20 ml), saline solution (10 ml) and dried over anhydrous sodium sulfate. Purification of the crude product made by formation of HCl salt yielded 65 mg of the desired product with a purity of 98.2% by HPLC. .sup.1H NMR (CDCl.sub.3) ? 7.27-7.24 (m, 2H), 7.21-7.16 (m, 2H), 7.04-7.01 (m, 1H), 6.86-6.84 (d, 1H), 6.44-6.43 (b, 1H), 5.13 (s, 2H), 3.58-3.55 (t, 2H), 2.91-2.87 (m, 2H). Theoretic MS for C.sub.19H.sub.18ClF.sub.4NO.sub.2: 405.8; M.sup.++1 found, 368.1 (HCl).

Example 2: Inhibition of T Cell Proliferation Induced by TCR Stimulation

(44) The effect of compounds AX-101, AX-103HCl; AX-104, AX-109, AX-110, AX-111, AX-127, AX-133, AX-135 and AX-136 on the capacity of the TCR to induce T lymphocyte proliferation was assessed in primary T lymphocytes obtained from blood of healthy human donors (PBMC, peripheral blood mononuclear cells). PBMC of the volunteers were isolated from venous blood by centrifugation in Ficoll-Paque Plus density gradients. Purified cells (NWT; Nylon Wood T cells) were cultured in triplicate in 96-well plates (0.5?10.sup.5/well) in 200 ul of complete medium and stimulated with OKT3 (10 ug/m|) or with OKT3 (10 ug/ml) plus CD28 in the presence or absence of different concentrations of compounds 1 and 10 uM. The cultures were incubated for three days and analyzed after addition of 0.5 uCi [3H]TdR/well for the last 12 h of the culture. The radioactivity incorporated into DNA was determined by liquid scintillation counting. As cells divide, radioactivity is incorporated into daughter cells which it gives an idea of the degree of cell proliferation. The inhibition capacity of the tested compounds is shown in FIG. 1.

Example 3: Improving the Bioavailability of the Compound AX-104 after Oral Administration in Rodents

(45) The pharmacokinetic properties of the compound AX-104 were analyzed in relation to those observed for compound ECRA-24 (described in WO2012/042078). For this, intravenous administration of the compound (bolus) and oral gavage of a solution of the compounds was performed separately in rats (mean?SD; n=3). As seen in the data shown in Tables 1 and 2, the total bioavailability of the compound AX-104 after oral administration was 24% compared to that of 2% of ECRA-24.

(46) TABLE-US-00001 TABLE 1 Pharmacokinetic parameters means of ECRA-24 in Swiss albino mice. Dose T.sub.max C.sub.max AUC.sub.last AUC.sub.int CL V.sub.ss T.sub.1/2 Route (mg/kg) (h) (ng/mL) (ng .Math. h/mL) (ng .Math. h/mL) (mL/min/kg) (L/kg) (h) F .sup.a IV 5 NA 1168.89 838.43 840.27 99.17 11.37 3.36 PO 50 0.25 171.56 297.03 299.69 NA NA 4.93 4

(47) TABLE-US-00002 TABLE 2 Pharmacokinetic parameters means of AX-104 in Swiss albino mice. Route T.sub.max C.sub.max AUC.sub.last AUC.sub.inf CL V.sub.ss T.sub.1/2 (Dosis) Analite (h) (ng/mL) (ng .Math. h/mL) (ng .Math. h/mL) (mL/min/kg) (L/kg) (h) F .sup.b IV AX-04 NA 1281.10 .sup.c 723.92 749.72 111.15 20.64 8.45 NA (5 mg/kg) PO AX-104 0.25 930.20.sup. 1759.86 1793.43 NA NA NA 24 (50 mg/kg)

Example 4: In Vivo Test on Model of EAE (Experimental Autoimmune Encephalomyelitis)

(48) The model of chronic EAE was induced in 10 C57BL/6 female mice per treatment group (6-8 weeks old, 20 g body weight) by subcutaneous injection of a total of 150 ?g of peptide MOG (Myelin Oligodendrocyte Glycoprotein; MOG35-55, Espikem, Germany) emulsified in Freund's Complete Adjuvant (CFA, Sigma-Aldrich) and supplemented with 5 mg/ml of Mycobacterium tuberculosis (H37Ra strain of Difco) in both femoral regions. The mice were immediately injected intraperitoneally with 150 ng of pertussis toxin (Sigma-Aldrich) and once more, 48 h after immunization. The compound indicated (AX-104) was prepared in a saline buffer and administered orally during the first 10 days starting on the same day of immunization. Subjects on the placebo group received an equivalent oral dose containing simply saline buffer. Animals were weighed and clinical signs of the disease were analyzed by an external observer out of the process by means of visual analysis of the symptoms based on the following scale; 0=normal; 1=weak limp tail or slight limp of hind limbs; 2=moderate hind limb weakness or mild ataxia; 3=moderately severe weakness in hind limbs: 4=severe hindlimb weakness or mild forelimb weakness or moderate ataxia; 5=paraplegia with nothing more than moderate forelimb weakness; and 6=paraplegia with severe forelimb weakness or severe ataxia, moribund condition or death. FIG. 2 shows how the animals receiving compound AX-104 present a reduced score when compared to the placebo group.

(49) In parallel, the weight of the animals was also monitored as a measurement indicative of the general welfare of the animals and evolution of the disease. FIG. 3 shows that treated animals present a weight loss significantly reduced when compare to animals on the placebo group.

(50) At the end of the study, the animals were anesthetized and an intra-cardiac perfusion was performed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.6). The brain and spinal cords of mice was dissected and fixed.

(51) Results overall show that compound AX-104 is capable of significantly reducing the impact of the symptoms associated to the model.

Example 5: In Vitro Test on Blood Cells

(52) Human peripheral blood mononuclear cells (PBMC) from healthy adult volunteer donors were isolated from venous blood by centrifugation in Ficoll-Paque Plus density gradients. Purified T cells (NWT-0.5?10.sup.5/well) are cultured in triplicate in 96-well plates in 200 ul of complete medium and stimulated with OKT3 (10 ug/ml) or with OKT3 (10 ug/ml) and CD28 in presence or absence of test compounds at the desired concentrations. The cultures were grown for three days and a pulse of 0.5 uCi of [.sup.3H] TdR/well is administered during the last 12 h of culture. The radioactivity incorporated in the DNA was assessed by liquid scintillation counting.