Phenyl-guanidine derivatives

Abstract

Provided herein are phenyl-guanidine derivatives for the inhibition of Rac1 which blocks its interaction with guanosine exchange factors (GEFs) belonging to the DBL family as agents for the treatment of aggressive and/or resistant tumors, as well as pharmaceutical compositions comprising them, their use in therapy and processes for their preparation.

Claims

1. A method for treating a cancer mediated by Rho-GTPase cell proteins in a human which comprises administering to said human an effective amount of a compound of formula (I): ##STR00015## or a salt thereof, or any of its stereoisomeric forms or a mixture thereof wherein: R.sub.1 is CF.sub.3 and R.sub.1′ is H; wherein A is selected from: A is a radical selected from linear or branched (C1-C6)alkyl or one of the known carbocyclic or heterocyclic ring systems with 1-2 rings, wherein each of the rings forming the ring system: has 5-7 members, each member independently selected from C, N, O, S, CH, CH.sub.2, NH; and is saturated, partially unsaturated or aromatic; wherein A is substituted by one or more radicals selected from the group consisting of H, halogen, nitro, cyano, linear or branched (C.sub.1-C.sub.6)alkyl, halo-(C.sub.1-C.sub.6)alkyl, linear or branched (C.sub.2-C.sub.6)alkenyl, —OR.sub.2, —COR.sub.2, —COOR.sub.2, —OC(O)R.sub.2, —C(O)NR.sub.3R.sub.4, —NR.sub.3R.sub.4, —R.sub.5NHR.sub.6, —SR.sub.2, —SO—R.sub.2, —SO.sub.2—R.sub.2, and —SO.sub.2NR.sub.3R.sub.4; wherein each R.sub.2 independently represents H or linear or branched (C.sub.1-C.sub.4)alkyl, each R.sub.3 independently represents H or linear or branched (C.sub.1-C.sub.4)alkyl, each R.sub.4 independently represents H, linear or branched (C.sub.1-C.sub.6)alkyl, phenyl, pyridine or quinoline; wherein the phenyl, pyridine and quinoline ring system is substituted by one or more radical selected from H, linear or branched (C.sub.1-C.sub.4)alkyl, and NH.sub.2; R.sub.5 and R.sub.6 are independently selected from H, linear or branched (C.sub.1-C.sub.4)alkyl, together with pharmaceutical excipients or carriers.

2. The method according to claim 1, wherein A is a ring system selected from ##STR00016## wherein the wavy line indicates the point of attachment of the ring to the adjacent nitrogen, and wherein A is substituted as defined in claim 1.

3. The method according to claim 2, wherein A is a ring system selected from ##STR00017## wherein the wavy line indicates the point of attachment of the ring to the adjacent nitrogen; and wherein A is substituted by one or more radicals as defined in claim 1.

4. The method according to claim 3, wherein the ring system of A is substituted by one or more radicals selected from the group consisting of H, F, Cl, Br, I, nitro, cyano, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, —CF.sub.3, —CH.sub.2CF.sub.3, linear or branched (C.sub.2-C.sub.6)alkenyl, —OH, —CH.sub.3, —OCH.sub.2CH.sub.3, —COH, —COCH.sub.3, —COOH, —COOCH.sub.3, —COOCH.sub.2CH.sub.3, —OC(O)H, —OC(O)CH.sub.3, —C(O)NH.sub.2, —NH.sub.2, —NHCH.sub.3, —N(CH.sub.3).sub.2, —CH.sub.2NH.sub.2, —CH.sub.2NHCH.sub.3, —SH.sub.2, —SO—CH.sub.3, —SO.sub.2—CH.sub.3, —SO.sub.2NH.sub.2, —SO.sub.2NHCH.sub.3, —SO.sub.2NHCH.sub.2CH.sub.3, —SO.sub.2N(CH.sub.3).sub.2, —SO.sub.2N(CH.sub.2CH.sub.3).sub.2, —SO.sub.2N(CH.sub.2CH.sub.2CH.sub.3).sub.2, —SO.sub.2N(CH.sub.2CH.sub.2CH.sub.2CH.sub.3).sub.2 and —SO.sub.2N(CH.sub.2CH.sub.2(CH.sub.3).sub.2).sub.2.

5. The method according to claim 1, wherein the compound is of formula Ia: ##STR00018## or a salt thereof, or any of its stereoisomeric forms or a mixture thereof; wherein R.sub.1 is CF.sub.3 and R.sub.1′ is H.

6. The method according to claim 5, wherein A is a radical of one of the known heterocyclic ring systems with 1-2 rings, wherein each of the rings forming the ring system has 5-7 members, each member independently selected from C, N, O, S, CH, CH.sub.2, NH; is saturated, partially unsaturated or aromatic; wherein A is substituted by one or more radical selected from the group consisting of H, F, Br, I, nitro, cyano, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, —CF.sub.3, —CH.sub.2CF.sub.3, linear or branched (C.sub.2-C.sub.6)alkenyl, —OH, —OCH.sub.3, —OCH.sub.2CH.sub.3, —COH, —COCH.sub.3, —COOH, —COOCH.sub.3, —COOCH.sub.2CH.sub.3, —OC(O)H, —OC(O)CH.sub.3, —C(O)NH.sub.2, —NH.sub.2, —NHCH.sub.3, —N(CH.sub.3).sub.2, —CH.sub.2NH.sub.2, —CH.sub.2NHCH.sub.3, —SH.sub.2, —SO—CH.sub.3, —SO.sub.2—CH.sub.3, —SO.sub.2NH.sub.2, —SO.sub.2NHCH.sub.3, —SO.sub.2NHCH.sub.2CH.sub.3, —SO.sub.2N(CH.sub.3).sub.2, —SO.sub.2N(CH.sub.2CH.sub.3).sub.2, —SO.sub.2N(CH.sub.2CH.sub.2CH.sub.3).sub.2, —SO.sub.2N(CH.sub.2CH.sub.2CH.sub.2CH.sub.3).sub.2 and —SO.sub.2N(CH.sub.2CH.sub.2(CH.sub.3).sub.2).sub.2.

7. The method according to claim 5, wherein A is a phenyl radical substituted by one or more radicals selected from the group consisting of H, F, Cl, Br, I, nitro, cyano, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, —CF.sub.3, —CH.sub.2CF.sub.3, linear or branched (C.sub.2-C.sub.6)alkenyl, —OH, —OCH.sub.3, —OCH.sub.2CH.sub.3, —COH, —OCH.sub.3, —COOH, —COOCH.sub.3, —COOCH.sub.2CH.sub.3, —OC(O)H, —OC(O)CH.sub.3, —(O)NH.sub.2, —NH.sub.2, —NHCH.sub.3, —N(CH.sub.3).sub.2, —CH.sub.2NH.sub.2, —CH.sub.2NHCH.sub.3, —SH.sub.2, —SO—CH.sub.3, —SO.sub.2—CH.sub.3, —SO.sub.2NH.sub.2, —SO.sub.2NHCH.sub.3, —SO.sub.2NHCH.sub.2CH.sub.3, —SO.sub.2N(CH.sub.3).sub.2, —SO.sub.2N(CH.sub.2CH.sub.3).sub.2, —SO.sub.2N(CH.sub.2CH.sub.2CH.sub.3).sub.2, —SO.sub.2N(CH.sub.2CH.sub.2CH.sub.2CH.sub.3).sub.2 and —SO.sub.2N(CH.sub.2CH.sub.2(CH.sub.3).sub.2).sub.2.

8. The method according to claim 1, wherein the compound is selected from: N-pyrimidin-2-yl-N′-[2-(trifluoromethyl)phenyl]guanidine (1); N-(4-ethyl-6-methylpyrimidin-2-yl)-N′-[2-(trifluoromethyl)phenyl]guanidine (2); N-(4-methyl-6-propylpyrimidin-2-yl)-N′-[2-(trifluoromethyl)phenyl]guanidine (3); N-(4-isopropyl-6-methylpyrimidin-2-yl)-N′-[2-(trifluoromethyl)phenyl]guanidine (4); N-(4-butyl-6-methylpyrimidin-2-yl)-N′-[2-(trifluoromethyl)phenyl]guanidine (5); N-(4-tert-butyl-6-methylpyrimidin-2-yl)-N′-[2-(trifluoromethyl)phenyl]guanidine (6); N-(4,6-diaminopyrimidin-2-yl)-N′-[2-(trifluoromethyl)phenyl]guanidine (7); N-(4,6-dichloropyrimidin-2-yl)-N′-[2-(trifluoromethyl)phenyl]guanidine (8); N-(4,6-difluoropyrimidin-2-yl)-N′-[2-(trifluoromethyl)phenyl]guanidine (9); N-[4-methyl-6-(trifluoromethyl)pyrimidin-2-yl]-N′-[2-(trifluoromethyl)phenyl]guanidine (10); N-(4-cyano-6-methylpyrimidin-2-yl)-N′-[2-(trifluoromethyl)phenyl]guanidine (11); N-(5-methylpyrimidin-2-yl)-N′-[2-(trifluoromethyl)phenyl]guanidine (12); N-(4-chloro-6-methylpyrimidin-2-yl)-N′-[2-(trifluoromethyl)phenyl]guanidine (13); N-(4-fluoro-6-methylpyrimidin-2-yl)-N′-[2-(trifluoromethyl)phenyl]guanidine (14); N-(4-fluoropyrimidin-2-yl)-N′-[2-(trifluoromethyl)phenyl]guanidine (15); N-(5-fluoropyrimidin-2-yl)-N′-[2-(trifluoromethyl)phenyl]guanidine (16); N-[4,6-bis(trifluoromethyl)pyrimidin-2-yl]-N′-[2-(trifluoromethyl)phenyl]guanidine (17); N-(4,6-dicyanopyrimidin-2-yl)-N′-[2-(trifluoromethyl)phenyl]guanidine (18); N-pyridin-2-yl-N′-[2-(trifluoromethyl)phenyl]guanidine (19); N-pyridin-3-yl-N′-[2-(trifluoromethyl)phenyl]guanidine (20); N-pyridin-4-yl-N′-[2-(trifluoromethyl)phenyl]guanidine (21); N-pyrimidin-4-yl-N′-[2-(trifluoromethyl)phenyl]guanidine (22); N-pyrimidin-5-yl-N-[2-(trifluoromethyl)phenyl]guanidine (23); N-(4,6-dimethylpyridin-2-yl)-N′-[2-(trifluoromethyl)phenyl]guanidine (24); N-(3,5-dimethylphenyl)-N-[2-(trifluoromethyl)phenyl]guanidine (25); N-(2,6-dimethylpyridin-4-yl)-N′-[2-(trifluoromethyl)phenyl]guanidine (26); N-phenyl-N′-[2-(trifluoromethyl)phenyl]guanidine (27); 2-[(imino{[2-(trifluoromethyl)phenyl]amino}methyl)amino]-N,N-dimethylbenzenesulfonamide (28); 2-[(imino{[2-(trifluoromethyl)phenyl]amino}methyl)amino]-N,N-diethylbenzenesulfonamide (29); 2-[(imino{[2-(trifluoromethyl)phenyl]amino}methyl)amino]-N,N-dipropylbenzenesulfonamide (30); 2-[(imino{[2-(trifluoromethyl)phenyl]amino}methyl)amino]-N,N-dibutylbenzenesulfonamide (31); 3-[(imino{[2-(trifluoromethyl)phenyl]amino}methyl)amino]-N,N-dimethylbenzenesulfonamide (32); 3-[(imino{[2-(trifluoromethyl)phenyl]amino}methyl)amino]-N,N-diethylbenzenesulfonamide (33); 3-[(imino{[2-(trifluoromethyl)phenyl]amino}methyl)amino]-N,N-dipropylbenzenesulfonamide (34); 3-[(imino{[2-(trifluoromethyl)phenyl]amino}methyl)amino]-N,N-dibutylbenzenesulfonamide (35); 4-[(imino{[2-(trifluoromethyl)phenyl]amino}methyl)amino]-N,N-dimethylbenzenesulfonamide (36); 4-[(imino{[2-(trifluoromethyl)phenyl]amino}methyl)amino]-N,N-diethylbenzenesulfonamide (37); 4-[(imino{[2-(trifluoromethyl)phenyl]amino}methyl)amino]-N,N-dipropylbenzenesulfonamide (38); 4-[(imino{[2-(trifluoromethyl)phenyl]amino}methyl)amino]-N,N-dibuthylbenzenesulfonamide (39); N-(2-nitrophenyl)-N′-[2-(trifluoromethyl)phenyl]guanidine (40); N-(3-nitrophenyl)-N′-[2-(trifluoromethyl)phenyl]guanidine (41); N-(4-nitrophenyl)-N′-[2-(trifluoromethyl)phenyl]guanidine (42); N-2-thienyl-N′-[2-(trifluoromethyl)phenyl]guanidine (43); N-3-thienyl-N′-[2-(trifluoromethyl)phenyl]guanidine (44); N-1H-pyrrol-2-yl-N′-[2-(trifluoromethyl)phenyl]guanidine (45); N-1H-pyrrol-3-yl-N′-[2-(trifluoromethyl)phenyl]guanidine (46); N-2-furyl-N′-[2-(trifluoromethyl)phenyl]guanidine (47); N-3-furyl-N′-[2-(trifluoromethyl)phenyl]guanidine (48); N-1,3-oxazol-2-yl-N′-[2-(trifluoromethyl)phenyl]guanidine (49); N-1,3-thiazol-2-yl-N′-[2-(trifluoromethyl)phenyl]guanidine (50); N-1H-imidazol-2-yl-N′-[2-(trifluoromethyl)phenyl]guanidine (51); N-isoxazol-5-yl-N′-[2-(trifluoromethyl)phenyl]guanidine (52); N-1H-benzimidazol-2-yl-N′-[2-(trifluoromethyl)phenyl]guanidine (53); N-(3,4-dimethylisoxazol-5-yl)-N′-[2-(trifluoromethyl)phenyl]guanidine (54); 1-(4-(4-amino-2-methylquinolin-7-ylamino)pyrimidin-2-yl)-3-(2-(trifluoromethyl)phenyl)guanidine (57); N-(4-amino-2-methylquinolin-7-yl)-N′-[2-(trifluoromethyl)phenyl]guanidine (58); N-quinolin-7-yl-N′-[2-(trifluoromethyl)phenyl]guanidine (59); N-methyl-N′-[2-(trifluoromethyl)phenyl]guanidine (60); N-ethyl-N′-[2-(trifluoromethyl)phenyl]guanidine (61); N-propyl-N′-[2-(trifluoromethyl)phenyl]guanidine (62); N-buthyl-N′-[2-(trifluoromethyl)phenyl]guanidine (63); N-(2-methylphenyl)-N′-[2-(trifluoromethyl)phenyl]guanidine (64); and N-[4,6-bis(methyl)pyrimidin-2-yl]-N′-[2-(trifluoromethyl)phenyl]guanidine (65).

9. The method according to claim 1, wherein the compound is selected from: N-[4,6-bis(methyl)pyrimidin-2-yl]-N′-[2-(trifluoromethyl)phenyl]guanidine (65), N-(3,5-dimethylphenyl)-N′-[2-(trifluoromethyl)phenyl]guanidine (25), N-phenyl-N′-[2-(trifluoromethyl)phenyl]guanidine (27), N-(3-nitrophenyl)-N′-[2-(trifluoromethyl)phenyl]guanidine (41), N-[4-methyl-6-(trifluoromethyl)pyrimidin-2-yl]-N′-[2-(trifluoromethyl)phenyl]guanidine (10), and N-(2-methylphenyl)-N′-[2-(trifluoromethyl)phenyl]guanidine (64).

10. The method according to claim 1, wherein the cancer mediated by Rho-GTPase cell proteins is a proliferative disorder selected from the group consisting of precancerosis; dysplasia; metaplasia; carcinomas of the gastrointestinal or colorectal tract, liver, pancreas, kidney, bladder, prostate, endometrium, ovary, testes, melanoma, dysplastic oral mucosa, invasive oral cancers, small cell and non-small cell lung carcinomas, hormone-dependent breast cancers, hormone-independent breast cancers, transitional and squamous cell cancers, neurological malignancies including neuroblastoma, gliomas, glioblastoma, astrocytomas, osteosarcomas, soft tissue sarcomas, hemangioamas, endocrinological tumors, hematologic neoplasias including leukemias, lymphomas, and other myeloproliferative and lymphoproliferative diseases, carcinomas in situ, hyperplastic lesions, adenomas, fibromas, histiocytosis, chronic inflammatory proliferative diseases, vascular proliferative diseases, virus-induced proliferative diseases, and skin diseases characterized by hyperproliferation of keratinocytes and/or T cells.

11. The method according to any claim 1, wherein the method comprises administering to a subject simultaneously, sequentially or separately a compound of formula I and i) one or more anticancer agents; ii) radiotherapy; iii) conventional surgery; iv) or mixtures thereof.

12. A compound of formula (II) ##STR00019## or a salt thereof, or any of its stereoisomeric forms or a mixture thereof wherein: A is a radical of one of the known carbocyclic or heterocyclic ring systems with 1-2 rings, wherein each of the rings forming the ring system has 5-7 members, each member independently selected from C, N, O, S, CH, CH.sub.2, NH; is saturated, partially unsaturated or aromatic; wherein A is substituted by one or more radicals selected from the group consisting of H, F, Cl, Br, I, nitro, cyano, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, linear or branched (C.sub.2-C.sub.6)alkenyl, —OH, —OCH.sub.3, —OCH.sub.2CH.sub.3, —COH, —COCH.sub.3, —COOH, —COOCH.sub.3, —COOCH.sub.2CH.sub.3, —OC(O)H, —OC(O)CH.sub.3, —C(O)NH.sub.2, —NH.sub.2, —NHCH.sub.3, —N(CH.sub.3).sub.2, —CH.sub.2NH.sub.2, —CH.sub.2NHCH.sub.3, —SH.sub.2, —SO—CH.sub.3, —SO.sub.2—CH.sub.3, —SO.sub.2NH.sub.2, —SO.sub.2NHCH.sub.3, —SO.sub.2NHCH.sub.2CH.sub.3, —SO.sub.2N(CH.sub.3).sub.2, —SO.sub.2N(CH.sub.2CH.sub.3).sub.2, —SO.sub.2N(CH.sub.2CH.sub.2CH.sub.3).sub.2, —SO.sub.2N(CH.sub.2CH.sub.2CH.sub.2CH.sub.3).sub.2 and —SO.sub.2N(CH.sub.2CH.sub.2(CH.sub.3).sub.2).sub.2; with the proviso that the compound is other than N-(4-methyl-6-hydroxy-pyrimidin-2-yl)-N′-(2-trifluoromethylphenyl)guanidine, N-[4,6-bis(methyl)pyrimidin-2-yl]-N′-[2-(trifluoromethyl)phenyl]guanidine or N-[(4-methyl)pyrimidin-2-yl]-N′-[2-(trifluoromethyl)phenyl]guanidine.

13. The compound according to claim 12, wherein A is a phenyl radical substituted by one or more radicals selected from the group consisting of H, F, Cl, Br, I, nitro, cyano, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, linear or branched (C.sub.2-C.sub.6)alkenyl, —OH, —OCH.sub.3, —OCH.sub.2CH.sub.3, —COH, —COCH.sub.3, —COOH, —COOCH.sub.3, —COOCH.sub.2CH.sub.3, —OC(O)H, —OC(O)CH.sub.3, —C(O)NH.sub.2, —NH.sub.2, —NHCH.sub.3, —N(CH.sub.3).sub.2, —CH.sub.2NH.sub.2, —CH.sub.2NHCH.sub.3, —SH.sub.2, —SO—CH.sub.3, —SO.sub.2—CH.sub.3, —SO.sub.2NH.sub.2, —SO.sub.2NHCH.sub.3, —SO.sub.2NHCH.sub.2CH.sub.3, —SO.sub.2N(CH.sub.3).sub.2, —SO.sub.2N(CH.sub.2CH.sub.3).sub.2, —SO.sub.2N(CH.sub.2CH.sub.2CH.sub.3).sub.2, —SO.sub.2N(CH.sub.2CH.sub.2CH.sub.2CH.sub.3).sub.2 and —SO.sub.2N(CH.sub.2CH.sub.2(CH.sub.3).sub.2).sub.2.

14. The compound according to claim 12, wherein A is a heterocyclic ring systems with 1-2 rings, wherein each of the rings forming the ring system has 5-7 members, each member independently selected from C, N, O, S, CH, CH.sub.2, NH; is saturated, partially unsaturated or aromatic; wherein A is substituted by one or more radicals selected from the group consisting of H, F, Cl, Br, I, nitro, cyano, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, linear or branched (C.sub.2-C.sub.6)alkenyl, —OH, —OCH.sub.3, —OCH.sub.2CH.sub.3, —COH, —COCH.sub.3, —COOH, —COOCH.sub.3, —COOCH.sub.2CH.sub.3, —OC(O)H, —OC(O)CH.sub.3, —C(O)NH.sub.2, —NH.sub.2, —NHCH.sub.3, —N(CH.sub.3).sub.2, —CH.sub.2NH.sub.2, —CH.sub.2NHCH.sub.3, —SH.sub.2, —SO—CH.sub.3, —SO.sub.2—CH.sub.3, —SO.sub.2NH.sub.2, —SO.sub.2NHCH.sub.3, —SO.sub.2NHCH.sub.2CH.sub.3, —SO.sub.2N(CH.sub.3).sub.2, —SO.sub.2N(CH.sub.2CH.sub.3).sub.2, —SO.sub.2N(CH.sub.2CH.sub.2CH.sub.3).sub.2, —SO.sub.2N(CH.sub.2CH.sub.2CH.sub.2CH.sub.3).sub.2 and —SO.sub.2N(CH.sub.2CH.sub.2(CH.sub.3).sub.2).sub.2; with the proviso that the compound is other than N-(4-methyl-6-hydroxy-pyrimidin-2-yl)-N′-(2-trifluoromethylphenyl)guanidine, N-[4,6-bis(methyl)pyrimidin-2-yl]-N′-[2-(trifluoromethyl)phenyl]guanidine or N-[(4-methyl)pyrimidin-2-yl]-N′-[2-(trifluoromethyl)phenyl]guanidine.

15. A pharmaceutical composition comprising at least one compound of claim 12 and one or more pharmaceutically acceptable excipients or carriers.

16. The pharmaceutical composition according to claim 15, which further comprises another therapeutically active substance.

17. A process for preparing the compound of claim 1, which comprises reacting a compound of formula (III) with a compound of formula (IV): ##STR00020##

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Antiproliferative effect of compounds (65) and (25) over LN229 (A), MDA-MB-231 (B) and F3II (C).

(2) FIG. 2. Effect of compound (65) over Rac-Tiam complex.

(3) FIG. 3: Pull down assay of compounds (65) and (25) with LN229 human glioblastoma cells. Western Blot shows levels of intracellular Rac activation at 10 μM.

(4) FIG. 4: Phosphorilation leves of PAK after treatment with compound (65) at different doses.

(5) FIG. 5: Actin cytoskeleton reorganization over LN229 human glioblastoma cells.

(6) FIG. 6: Trans-well migration assay with LN229 human glioblastoma cells.

(7) FIG. 7: The effect of compound (65) on the cell cycle of LN229 human glioblastoma cells. **p<0.001 ANOVA cont. Dunnett's Multiple Comparison Test

(8) FIG. 8: Antiproliferative effect of compounds (65), (25), (27), (41) and (64) over LN229 human glioblastoma cells.

(9) FIG. 9: Antimetastatic effect of compound (25) on F3II cells. Results from three independent experiments are presented together. *P<0.05 Mann-Whitney test.

EXAMPLES

(10) All reagents were commercially available. Melting points were measured with an Electrothermal IA9000 Series (with a temperature gradient of 1° C./minute) and were uncorrected. Chromatograpy purifications were performed in a flash column chromatograpy apparatus Teledyne Isco CombiFlash Companion equipment with Redisep detachable columns, using mixtures of solvents with ascending polarity as mobile phase. .sup.1H and .sup.13C NMR spectra were recorded on a Bruker ADVANCE DPX-400. Microanalysis was carried out by UNYMFOR (CONICET-FCEyN). Low resolution mass spectra were recorded on a Shimadzu QP2010 apparatus. IR spectra were recorded on a Nicolet Impact 400 apparatus.

Example 1

Preparation of N-(3,5-dimethylphenyl)-N′-[2-(trifluoromethyl)phenyl]guanidine (25)

(11) The title compound was obtained according to the following method:

(12) ##STR00009##

(13) An equimolar amount of compounds chlorohydrate of 3,5-dimethylaniline solution (126 mg, 0.80 mmol) and N-(2-(trifluoromethyl)phenyl)cianamide (149 mg, 0.80 mmol) in absolute ethanol (2.5 mL) was heated to reflux with stirred for 16 h. An aqueous solution of NaOH (0.5 M, 2.2 mL) was added until pH 9. The mixture was extracted with dichloromethane (3×3 mL). Organic phases were dried with Na.sub.2SO.sub.4 and filtered. The solvent was evaporated to obtain 225 mg (92% yield) of the title compound. Crude product was purified by column chromatography with a hexane:ethyl acetate gradient (1:4 to 0:10) in presence of 0.01% of triethylamine to obtaining 138 mg (56%) of pure compound (25) as a white solid, m.p. 127° C.

(14) .sup.1H RMN (400 MHz, CDCl.sub.3) δ 7.63 (d, J=6.5 Hz, 1H), 7.44 (m, 1H), 7.08 (m, 2H), 6.84 (s, 2H), 6.77 (s, 1H), 4.27 (sa, 2H), 2.28 (s, 6H). .sup.13C RMN (100 MHz, CDCl.sub.3) δ 149.43, 147.59, 139.12, 132.79, 126.90 (q, J=5 Hz), 125.97, 125.31, 124.39 (q, J=272 Hz), 123.79 (q, J=29 Hz), 122.12, 120.36, 21.28. IR (cm.sup.−1), 3476, 3372, 1648, 1560, 1316. MS (m/z, relative intensity) 307 (M.sup.+, 23), 238 (M.sup.+-CF.sub.3, 6), 121 (100). Anal. Calcd. for C.sub.16H.sub.16F.sub.3N.sub.3: % C, 62.35; % H, 5.07; % N, 13.24. Found: % C, 62.53; % H, 5.25; % N, 13.67.

(15) Compounds (27), (41) and (64) were prepared in a similar way reacting the corresponding aniline with the corresponding cianamide.

N-phenyl-N′-[2-(trifluoromethyl)phenyl]guanidine (27)

(16) ##STR00010##

(17) Yield: 66%. White solid, m.p.=108-109° C. .sup.1H RMN (400 MHz, CDCl.sub.3) δ 7.62 (d, J=6.5 Hz, 1H), 7.44 (m, 1H), 7.30-7.21 (m, 4H), 7.08 (m, 2H), 4.92 (sa, 2H). .sup.13C RMN (100 MHz, CDCl.sub.3) δ 149.12, 147.04, 139.40, 132.86, 129.42, 126.94 (q, J=5 Hz), 125.18, 124.34 (q, J=272 Hz), 124.23, 123.79 (q, J=29 Hz), 122.55, 122.37. IR (cm.sup.−1), 3379, 1647, 1549, 1318. MS (m/z, relative intensity) 279 (M.sup.+, 26), 210 (M.sup.+-CF.sub.3, 7), 93 (100). Anal. Calcd. for C.sub.14H.sub.12F.sub.3N.sub.3.0.25H.sub.2O: % C, 59.26; % H, 4.44; % N, 14.81. Found: % C, 59.68; % H, 4.14; % N, 14.42.

N-(3-nitrophenyl)-N′-[2-(trifluoromethyl)phenyl]guanidine (41)

(18) ##STR00011##

(19) Yield: 60%. Yellow solid. .sup.1H RMN (400 MHz, CDCl.sub.3) δ 8.16 (s, 1H), 7.87 (d, J=7.9 Hz, 1H), 7.70 (m, 1H), 7.66 (d, J=7.6 Hz, 1H), 7.50 (t, J=7.7 Hz, 1H), 7.45 (t, J=7.9 Hz, 1H), 7.21-7.14 (m, 2H), 4.18 (sa, 2H). .sup.13C RMN (100 MHz, CD.sub.3OD CDCl.sub.3) δ 148.48, 148.29, 146.28, 142.09, 132.35, 128.96, 126.28 (q, J=5 Hz), 125.25, 124.45, 123.98 (q, J=272 Hz), 123.15 (q, J=29 Hz), 122.02, 115.98, 113.96. IR (cm.sup.−1) 3437, 3336, 1656, 1520, 1318, 1105. MS (m/z, relative intensity) 324 (M.sup.+, 75), 255 (M.sup.+-CF.sub.3, 48), 138 (100)

N-(2-methylphenyl)-N′-[2-(trifluoromethyl)phenyl]guanidine (64)

(20) ##STR00012##

(21) Yield: 87%. White solid, m.p.=129° C. .sup.1H RMN (400 MHz, CDCl.sub.3) δ 7.84 (d, J=7.9 Hz, 0.5H), 7.62 (d, J=7.8 Hz, 1H), 7.58 (d, J=7.9 Hz, 0.5H), 7.48 (m, 1.5H), 7.33 (d, J=7.6 Hz, 1H), 7.23-7.10 (m, 3.5H), 4.34 (sa, 2H), 2.29 (s, 3H). .sup.13C RMN (100 MHz, CD.sub.3OD-CDCl.sub.3) δ 158.08, 151.61, 146.84, 137.71, 136.55, 134.10, 133.18, 132.83, 131.26, 127.12, 127.06 (q, J=5 Hz), 126.44, 126.32, 124.61 (q, J=29 Hz), 124.60 (q, J=272 Hz), 124.31, 123.06, 17.52. IR (cm.sup.−1), 3438, 3409, 1645, 1592, 1316. MS (m/z, relative intensity) 293 (M.sup.+, 35), 224 (M.sup.+-CF.sub.3, 6), 107 (100).

(22) *Signals of two conformers are listed.

(23) Preparation of N-[4,6-bis(methyl)pyrimidin-2-yl]-N′-[2-(trifluoromethyl)phenyl]guanidine (65) was performed following the method described in Chin. J. Chem. 2008, 1481.sup.1.

(24) ##STR00013##

(25) Cyanoguanidine (10 g, 0.12 mol), acetylacetone (17.2 mL, 0.17 mol) were treated with 2M NaOH (5.7 mL) in water (74 mL). The reaction mixture was stirred at 100° C. for 18 hs. The mixture was cooled down to room temperature and then to 0° C. under stirring for 2 hs. The crystallized solid was collected and dried at 50° C. to give 14.2 g of a pink solid. The crude solid was recrystallized from ethanol to give 10.2 g (58% yield) of N-(4.6-dimethylpyrimidin-2-yl)cyanamide as a white solid: mp 230-231° C., (lit.sup.1. 230-231° C.), .sup.1H RMN (400 MHz, CDCl.sub.3) δ 6.43 (s, 1H), 4.36 (bs, 1H), 3.26 (s, 6H), This intermediate was reacted with an equimolar amount of 2-(trifluoromethyl)aniline hydrochloride (13.6 g, 69.12 mmol) by refluxing the mixture in ethanol (150 mL) for 20 h. The mixture was allowed to reach room temperature and the pH was adjusted to 12-13 by addition of 0.5M NaOH aqueous solution (45 mL). The obtained solid was collected and recrystallized from ethanol:water (1:1) to yield 5.4 g of pure 65 as a white crystalline solid: m.p.=161-162° C. (lit.sup.1. 149-151° C.) .sup.1H RMN (400 MHz, CDCl.sub.3) δ 7.63 (d, J=7.6 Hz, 1H), 7.45 (t, J=7.6 Hz, 1H), 7.08 (m, 2H), 6.57 (s, 1H), 2.35 (s, 6H). .sup.13C RMN (100 MHz, CDCl.sub.3) δ 149.43, 147.59, 139.12, 132.79, 126.90 (q, J=5 Hz), 125.97, 125.31, 124.39 (q, J=272 Hz), 123.79 (q, J=29 Hz), 122.12, 120.36, 21.28. IR (cm.sup.−1): 3443, 3213, 1662. HRMS (ESI) calcd for C.sub.14H.sub.15F.sub.3N.sub.5 (MH.sup.+): 310.1274. found: 310.1265. Anal. Calcd. for C.sub.14H.sub.14F.sub.3N.sub.5.0.2H.sub.2O: % C, 53.74; % H, 4.64; % N, 22.38. Found: % C, 54.03; % H, 4.57; % N, 21.98.

1-(4-methyl-6-(trifluoromethyl)pyrimidin-2-yl)-3-(2-trifluoromethyl)phenyl)guanidine (10)

(26) ##STR00014##

(27) This compound was prepared following the procedure described for compound (65) employing 1,1,1-trifluoro-2,4-pentanedione instead of 2,4-pentanedione (Chin. J. Chem. 2008, 1481). .sup.1H RMN (400 MHz, CDCl.sub.3) δ 7.67 (d, J=6.5 Hz, 1H), 7.51 (t, J=7.7 Hz, 1H), 7.19 (m, 2H), 6.96 (s, 1H), 2.43 (s, 3H). .sup.13C RMN (100 MHz, CDCl.sub.3) δ 171.23, 60.05, 155.21, 150.63, 146.03, 132.79, 126.97 (q, J=5 Hz), 125.52, 123.96 (q, J=272 Hz), 123.21 (m), 120.30 (q, J=275 Hz), 108.48, 24.30. IR (cm.sup.−1) 3492, 3311, 1668, 1557, 1316. MS (m/z, relative intensity) 363 (M.sup.+, 91), 362 (M.sup.+-H, 100), 294 (M.sup.+-CF.sub.3, 92). Elemental Anal. Calcd. for C.sub.14H.sub.11F.sub.6N.sub.5: % C, 46.29, % H, 3.05, % N, 19.27. Found: % C, 46.32, % H, 3.04, % N, 18.98.

Example 2

Proliferation Assay

(28) Cells were maintained in monolayer culture with the corresponding media, supplemented with 5% fetal bovine serum (FBS) previously inactivated with heat, 2 mM glutamine and 80 mg/ml gentamicine.

(29) The cells were treated for 72 hours in the presence of FBS 10% with different doses of the compounds in 96 wells at a 2500 cell/well density. The cell growth was estimated by MTT test, for which to the cell monolayers was added MTT, incubated and resuspended in DMSO. Finally, the number of cells was estimated by measuring the absorbance values at 570 nm.

(30) Compound (65) as a representative example of Rac1 inhibitor compounds according to the present invention was tested in cell proliferation assays performed with following cancer cell lines F3II (murine mammary carcinoma), 3LL (murine lung carcinoma), LN229 (human glioblastoma), MCF7 (human mammary carcinoma), H125 (human lung carcinoma), PC3 (human prostate adenocarcinoma) and MDA-MB-231 (human mammary carcinoma).

(31) The level of proliferation was measured at 72 hours (h) after in vitro induction of compound (65) at different concentrations (200 μM, 100 μM, 50 μM, 25 μM, and 1 μM in presence of 10% fetal bovine serum (FBS) with the aim of determining the inhibitory concentration 50% (IC.sub.50). The concentration producing 50% inhibition (IC50) was determined by non-linear regression function of GraphPad Prism5®. Results shown correspond to the average of three separate experiments. The IC.sub.50 values are depicted in Table 1.

(32) TABLE-US-00001 TABLE 1 IC.sub.50 values (μM) of compound (65) tested in F3II, MCF7, 3LL, H125, LN229, PC3 and MDA-MB-231 cells F3II MCF7 3LL H125 LN229 PC3 MDA-MB-231 (65) 61 44 68 127 73 138 48

(33) Other compounds according to formula I were also tested in LN229 cells. IC.sub.50 values (μM) obtained are shown in Table 2.

(34) TABLE-US-00002 TABLE 2 IC.sub.50 values (μM) of compounds (25), (27), (41) and (64) tested in LN229 cells Cpd. (25) Cpd (27) Cpd (41) Cpd (64) IC50 (μM) 73 211 88 175

(35) Compound (25) was also tested in F3II and MDA-MB-231 cells. IC.sub.50 values (μM) obtained are shown in Table 3.

(36) TABLE-US-00003 TABLE 3 IC.sub.50 values (μM) of compound (25) tested in F3II and MDA-MB-231 cells. F3II MDA-MB-231 (25) 36 40

(37) FIG. 1 show the doses-response curves of compounds (65) and (25) on the proliferative capacity of three cancer cell lines: (A) F3II (murine mammary carcinoma), (B) LN229 (human glioblastoma), (C) MDA-MB-231 (human mammary carcinoma).

Example 3

Inhibition of the Rac1 Activation Levels (Pull-Down)

(38) This assay consists in the determination of inhibition power of the compounds of the invention over the intracellular active Rac1 levels (Rac-GTP). For determining the levels of Rac-GTP, the “Pull-Down” assay was used, which is based in the conformation bond of Rac-GTP to the p21 domain of PAK1 protein, which is the direct effector of Rac-GTP (Wanf H. et al.; J. Biol. Che. 2002, 277: 4541-4550).

(39) Compounds (65) and (25) were tested in order to determine its inhibition power over the interaction Rac-Tiam. Thus, the precipitation affinity of Tiam with the recombinant protein GST-Rac was evaluated in presence of compound (65). FIG. 2 shows the effect of compound (65) over Rac-Tiam complex. It is shown the decrease of Rac interaction with the activator in presence of compound (65) in a Western Blot anti Tiam1 vs. control.

(40) The inhibitory effect of compounds (25) and (65) over the intracellular active Rac levels were evaluated (Rac-GTP) by the “Pull-Down” assay.

(41) LN229 cells (human glioblastoma) were seeded in 6-well cell culture plaques and were kept in absence of FBS for 48 hours (starvation). Afterwards, they were treated with compounds (25) and (65) and they were stimulated for 15 minutes with EGF (100 ng/ml), washed with phosphate buffered saline (PBS) at low temperature and lysated in a buffer containing 8 μg of the fusion protein GST-PBD, 20 nM Tris-HCl, pH 7.5, 1 nM DTT, 5 mM MgCl.sub.2, 150 mM NaCl, 0.5% NP-40, 5 mM β-glicerophosphate and protease inhibitors (5 μg/ml 4-(2-aminoethyl)bencenesulfonyl fluoride, 5 μg (ml leupeptin, 5 μg/ml aprotinin and 1 μg/ml pepstatin A). The lysates were centrifuged at 14,000×g (4° C., 10 min) and then incubated with Glutation-Agarose Beads (GAB, Amersham Pharmacia) previously fitted with GST-Pak at 4° C. for 1 hour. After washing, the GABs were boiled for 5 minutes in loading buffer. The samples were separated in a 12% SDS-polyacrilamide gel and electro-trasferred to a PVDF membrane for its ulterior “Western Blot” analysis using an anti-Rac1 antibody (Sigma). The total Rac levels were analyzed in a similar way from aliquots taken from the cell lysate.

(42) FIG. 3 shows the inhibitory effect of compounds (25) and (65) over the intracellular active Rac levels at 10 μM.

(43) Following, the effect of compound (65) over the PAK1 activation was evaluated. Starvated cells for 48 hours were treated with compound (65) for 1 hour and they were actived with EGF 100 ng/ml. FIG. 4 shows the decrease of PAK phosphorilation with the increase of compound (65) concentration.

(44) The effect of compound (65) over the polymerization of actin filaments was evaluated by immunofluorescence with conjugated faloidine with the fluorochrome AlexaFluor (Invitrogen). LN229 human glioblastoma cells were starvated for 24 hours, treated with compound (65) for 1 hour, and stimulated with EGF. As expected, EGF induced the polymerization of actin filaments in no-treated cells, whereas cells treated with compound (65) shown a decreasing of intracellular fibrilar actin levels and a diffuse sign in the cellular citoplasm (c.f. FIG. 5). An accurate sign of cellular boundaries is observed in all cells due to the cortical actin marking.

Example 4

Antimigration Effect

(45) Cell motility is a key process in the invasion and tumor metastasis processes and it is closely regulated by the Rho-GTPases family, particularly by Rac. This assay consists in the determination of the antimigration effect of compound (65) on LN229 human glioblastoma cells.

(46) The cell migration in vitro was measured by the trans-well migration assay, wherein LN229 cells previously treated with different concentrations (10 μM, 50 μM and 100 μM) of compound (65) and serum deprived were placed on the upper layer of a cell permeable membrane of a trans-well plaque. Following an incubation perior of 24 hours, the cells migrated through the 8 μm pores of the membrane to the lower layer.

(47) FIG. 6 shows that all the treatments significantly decreased the cell migration in a dose dependent manner

Example 5

Cell Cycle Effect

(48) The effect of compound (65) on the cell cycle was studied by flow citometry. It is known that Rac1 GTPase is related with transcription of molecules such as cyclin D1, which are required to progress from G1 phase to S phase. Inhibition of Rac1 induces cell arrest in G0-G1 phase.

(49) LN229 cells synchronized in G0-G1 phase and stimulated for 24 hours with FBS were treated with compound (65). FIG. 7 shows that in presence of 50 μM of compound (65), 60% of cell population is in G0-G1 phase, whereas controls shows only 25% of cell population in this phase. Therefore, it is shown that compound (65) arrests tumoral cells in G0-G1 phase in a dose dependent manner.

Example 6

Antiproliferative Assay

(50) The antiproliferative effect of compounds (65), (25), (27), (41) and (64) was tested over LN229 human glioblastoma cells (c.f FIG. 8).

(51) Cells were maintained in monolayer culture with the corresponding media, supplemented with 5% fetal bovine serum (FBS) previously inactivated with heat, 2 mM glutamine and 80 mg/ml gentamicine.

(52) The cells were trated for 72 hours in the presence of FBS 10% with different doses of the compounds in 96 wells at a 2500 cell/well density. The cell growth was estimated by MTT test, for which to the cell monolayers was added MTT, incubated and resuspended in DMSO. Finally, the number of cells was estimated by measuring the absorbance values at 570 nm.

Example 7

Antimetastatic Effect of (25) on F3II Mammary Carcinoma Cells

(53) Specific pathogen-free female BALB/c inbred mice from UNLP (Buenos Aires, Argentina), with an age of 8-10 weeks and a average weight of 20 g, were used. They were housed in plastic cages under standard conditions and had access to rodent chow and water ad libitum. On the designated day 0 of the experiment, F3II mammary carcinoma cells were injected into the lateral tail vein of unanesthetized mice at a concentration of 2×10.sup.5 viable cells/0.3 ml DMEM/mice. At day 21, animals were sacrificed by cervical dislocation and necropsied. To investigate the presence of lung metastasis, lungs were removed, fixed in Bouin's solution and the number of surface nodules was determined under a dissecting microscope.

(54) To study the effect of compound (25) on metastatic lung colonization, mice were injected i.p at daily doses of 25 mg/kg body weight from days 0 to 21. The results are presented in FIG. 9. Daily treatment of mice with compound (25) at (25 mg/kg/day) significantly reduced by about 35% the formation of metastatic lung colonies.

(55) As expected, compound (25) was well tolerated in adult female BALB/c mice. In all cases, treatment caused no significant changes in animal weight when compared to the control group.