Dual inhibitor compounds for use in the treatment of neurodegenerative disorders and alzheimer's disease

Abstract

The present invention relates to Compounds of Formula (I) and pharmaceutical compositions containing the same. It further relates to their use in the prevention or treatment of central nervous system diseases or disorders, in particular, cognitive, neurodegenerative or neuronal diseases or disorders. ##STR00001##

Claims

1. A compound of Formula (I) ##STR00012## wherein: R.sub.1 is hydrogen or linear C.sub.1-6alkyl, unsubstituted or substituted with one or more R.sub.6 substituents; R.sub.2 is selected from the group consisting of linear or branched C.sub.1-6alkyl, unsubstituted or substituted with one or more R.sub.6 substituents; C.sub.3-6cycloalkyl, unsubstituted or substituted with one or more R.sub.6 substituents; C.sub.3-6cycloalkylC.sub.1-6alkyl, unsubstituted or substituted with one or more R.sub.6 substituents; aryl, unsubstituted or substituted with one or more R.sub.6 substituents; heteroaryl, unsubstituted or substituted with one or more R.sub.6 substituents; arylC.sub.1-6alkyl, unsubstituted or substituted with one or more R.sub.6 substituents; aryloxyC.sub.1-6alkyl, unsubstituted or substituted with one or more R.sub.6 substituents; heteroarylC.sub.1-6alkyl, unsubstituted or substituted with one or more R.sub.6 substituents; heteroaryloxyC.sub.1-6alkyl, unsubstituted or substituted with one or more R.sub.6 substituents; heterocycloalkylC.sub.1-6alkyl, unsubstituted or substituted with one or more R.sub.6 substituents; or COR.sub.5; R.sub.5 is selected from the group consisting of linear or branched C.sub.1-9alkyl, unsubstituted or substituted with one or more R.sub.6 substituents; C.sub.3-6cycloalkyl, unsubstituted or substituted with one or more R.sub.6 substituents; C.sub.3-6cycloalkylC.sub.1-6alkyl, unsubstituted or substituted with one or more R.sub.6 substituents; aryl, unsubstituted or substituted with one or more R.sub.6 substituents; heteroaryl, unsubstituted or substituted with one or more R.sub.6 substituents; arylC.sub.1-6alkyl, unsubstituted or substituted with one or more R.sub.6 substituents; aryloxyC.sub.1-6alkyl, unsubstituted or substituted with one or more R.sub.6 substituents; heteroarylC.sub.1-6alkyl, unsubstituted or substituted with one or more R.sub.6 substituents; heteroaryloxyC.sub.1-6alkyl, unsubstituted or substituted with one or more R.sub.6 substituents; heterocycloalkylC.sub.1-6alkyl, unsubstituted or substituted with one or more R.sub.6 substituents; R.sub.6 is selected from the group consisting of halogen and dialkylamino; or, R.sub.1 and R.sub.2 together with the nitrogen atom to which they are attached may form a 4- to 7-membered azacyclic ring containing up to two nitrogen atoms; Y.sub.1 and Y.sub.2 are independently selected from C or N; R.sub.3 and R.sub.4 are independently selected from the group consisting of hydrogen, halogen, linear or branched unsubstituted or substituted C.sub.1-6alkyl, unsubstituted or substituted C.sub.1-6alkoxy, hydroxy, trifluoromethyl, amino, monoalkylamino, dialkylamino, or a pharmaceutically acceptable salt or solvate thereof.

2. The compound according to claim 1, wherein: R.sub.1 is hydrogen or linear C.sub.1-4alkyl, unsubstituted or substituted with one or more R.sub.6 substituents; R.sub.2 is selected from the group consisting of linear or branched C.sub.1-4alkyl, unsubstituted or substituted with one or more R.sub.6 substituents; C.sub.3-6cycloalkyl, unsubstituted or substituted with one or more R.sub.6 substituents; C.sub.3-6cycloalkylC.sub.1-4alkyl, unsubstituted or substituted with one or more R.sub.6; aryl, unsubstituted or substituted with one or more R.sub.6 substituents; heteroaryl, unsubstituted or substituted with one or more R.sub.6 substituents; arylC.sub.1-4alkyl, unsubstituted or substituted with one or more R.sub.6 substituents; heteroarylC.sub.1-4alkyl, unsubstituted or substituted with one or more R.sub.6 substituents; heterocycloalkylC.sub.1-4alkyl, unsubstituted or substituted with one or more R.sub.6 substituents; and COR.sub.5; R.sub.5 is selected from the group consisting of linear or branched C.sub.1-7alkyl, unsubstituted or substituted with one or more R.sub.6 substituents; arylC.sub.1-4alkyl, unsubstituted or substituted with one or more R.sub.6 substituents; aryloxyC.sub.1-4alkyl, unsubstituted or substituted with one or more R.sub.6 substituents; heteroarylC.sub.1-4alkyl, unsubstituted or substituted with one or more R.sub.6 substituents; heteroaryloxyC.sub.1-4alkyl, unsubstituted or substituted with one or more R.sub.6 substituents; heterocyclylC.sub.1-4alkyl, unsubstituted or substituted with one or more R.sub.6 substituents; R.sub.6 is selected from the group consisting of halogen and dialkylamino; or, R.sub.1 and R.sub.2 together with the nitrogen atom to which they are attached may form a 4- to 6-membered azacyclic ring containing one nitrogen atom; Y.sub.1 and Y.sub.2 are independently selected from C or N; R.sub.3 and R.sub.4 are independently selected from the group consisting of hydrogen, halogen, linear or branched unsubstituted or substituted C.sub.1-3alkyl, unsubstituted or substituted C.sub.1-3alkoxy, hydroxy, trifluoromethyl.

3. The compound according to claim 1 wherein: R.sub.1 is hydrogen, methyl, ethyl, and n-propyl unsubstituted or substituted with one R.sub.6 substituent; R.sub.2 is selected from the group consisting of methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, cyclopropyl, cyclopropylmethyl, phenyl, benzyl, 4-pyridylmethyl, piperidin-1-ylpropyl, morpholin-4-yl-propyl, 4-methylpiperazin-1-yl-propyl, COR.sub.5; R.sub.5 is selected from the group consisting of methyl, ethyl, n-propyl, i-propyl, i-butyl, 1-propylbutyl, phenoxymethyl unsubstituted or substituted with one R.sub.6 substituent, heteroaryloxyethyl unsubstituted or substituted with one R.sub.6 substituent, heterocycloalkylbutyl unsubstituted or substituted with one R.sub.6 substituent; R.sub.6 is selected from the group consisting of fluorine and dialkylamino group; or, R.sub.1 and R.sub.2 together with the nitrogen atom to which they are attached may form an azacyclic ring selected from an azetidine, a pyrrolidine or a piperidine ring system; Y.sub.1 and Y.sub.2 are independently selected from C or N; R.sub.3 and R.sub.4 are independently selected from the group consisting of hydrogen, fluorine, methyl.

4. A compound selected from the group consisting of: 4-(4-fluorophenyl)-6-(methylamino)-3,4-dihydro-1,3,5-triazin-2(1H)-one; 6-(ethylamino)-4-(o-tolyl)-3,4-dihydro-1,3,5-triazin-2(1H)-one; 6-(ethylamino)-4-(4-fluorophenyl)-3,4-dihydro-1,3,5-triazin-2(1H)-one; 6-(ethylamino)-4-(4-fluorophenyl)-3,4-dihydro-1,3,5-triazin-2(1H)-one; 6-(ethylamino)-4-(4-fluorophenyl)-3,4-dihydro-1,3,5-triazin-2(1H)-one; 4-(4-fluorophenyl)-6-(propylamino)-3,4-dihydro-1,3,5-triazin-2(1H)-one; 4-(4-fluorophenyl)-6-(isopropylamino)-3,4-dihydro-1,3,5-triazin-2(1H)-one; 4-(4-fluorophenyl)-6-(isobutylamino)-3,4-dihydro-1,3,5-triazin-2(1H)-one; 4-(4-fluorophenyl)-6-(phenylamino)-3,4-dihydro-1,3,5-triazin-2(1H)-one; 6-(dimethylamino)-4-(4-fluorophenyl)-3,4-dihydro-1,3,5-triazin-2(1H)-one; 6-(diethylamino)-4-(4-fluorophenyl)-3,4-dihydro-1,3,5-triazin-2(1H)-one; 4-(4-fluorophenyl)-6-(piperidin-1-yl)-3,4-dihydro-1,3,5-triazin-2(1H)-one; 6-(butylamino)-4-(4-fluorophenyl)-3,4-dihydro-1,3,5-triazin-2(1H)-one; 6-(ethylamino)-4-(pyridin-3-yl)-3,4-dihydro-1,3,5-triazin-2(1H)-one; 4-(4-fluorophenyl)-6-(pyrrolidin-1-yl)-3,4-dihydro-1,3,5-triazin-2(1H)-one; N-(4-(4-fluorophenyl)-6-oxo-1,4,5,6-tetrahydro-1,3,5-triazin-2-yl)acetamide; N-(4-(4-fluorophenyl)-6-oxo-1,4,5,6-tetrahydro-1,3,5-triazin-2-yl)-2-propylpentanamide; 5-(1,2-dithiolan-3-yl)-N-(4-(4-fluorophenyl)-6-oxo-1,4,5,6-tetrahydro-1,3,5-triazin-2-yl)pentanamide; 3-((1H-benzo[d][1,2,3]triazol-1-yl)oxy)-N-(4-(4-fluorophenyl)-6-oxo-1,4,5,6-tetrahydro-1,3,5-triazin-2-yl)propanamide; N-(4-(4-fluorophenyl)-6-oxo-1,4,5,6-tetrahydro-1,3,5-triazin-2-yl)-2-phenoxyacetamide; 2-(4-fluorophenoxy)-N-(4-(4-fluorophenyl)-6-oxo-1,4,5,6-tetrahydro-1,3,5-triazin-2-yl)acetamide; 6-((3-(dimethylamino)propyl)amino)-4-(4-fluorophenyl)-3,4-dihydro-1,3,5-triazin-2(1H)-one; 4-(4-fluorophenyl)-6-((3-(piperidin-1-yl)propyl)amino)-3,4-dihydro-1,3,5-triazin-2(1H)-one; 6-(benzylamino)-4-(4-fluorophenyl)-3,4-dihydro-1,3,5-triazin-2(1H)-one; 4-(4-fluorophenyl)-6-((pyridin-4-ylmethyl)amino)-3,4-dihydro-1,3,5-triazin-2(1H)-one; 6-(dipropylamino)-4-(4-fluorophenyl)-3,4-dihydro-1,3,5-triazin-2(1H)-one, 6-(cyclopropylamino)-4-(4-fluorophenyl)-3,4-dihydro-1,3,5-triazin-2(1H)-one; 6-((cyclopropylmethyl)amino)-4-(4-fluorophenyl)-3,4-dihydro-1,3,5-triazin-2(1H)-one; 6-((3-(diethylamino)propyl)amino)-4-(4-fluorophenyl)-3,4-dihydro-1,3,5-triazin-2(1H)-one; 4-(4-fluorophenyl)-6-((3-morpholinopropyl)amino)-3,4-dihydro-1,3,5-triazin-2(1H)-one; 4-(4-fluorophenyl)-6-((3-(4-methylpiperazin-1-yl)propyl)amino)-3,4-dihydro-1,3,5-triazin-2(1H)-one; 6-(azetidin-1-yl)-4-(4-fluorophenyl)-3,4-dihydro-1,3,5-triazin-2(1H)-one; N-(4-(4-fluorophenyl)-6-oxo-1,4,5,6-tetrahydro-1,3,5-triazin-2-yl)propionamide; N-(4-(4-fluorophenyl)-6-oxo-1,4,5,6-tetrahydro-1,3,5-triazin-2-yl)butyramide; N-(4-(4-fluorophenyl)-6-oxo-1,4,5,6-tetrahydro-1,3,5-triazin-2-yl)isobutyramide; N-(4-(4-fluorophenyl)-6-oxo-1,4,5,6-tetrahydro-1,3,5-triazin-2-yl)-3-methylbutanamide.

5. Pharmaceutical composition comprising a compound of Formula (I) according to claim 1 and a pharmaceutically acceptable carrier, stabilizer, diluent, or excipient thereof.

6. The pharmaceutical composition according to claim 5 further comprising a second therapeutic agent.

7. The pharmaceutical composition according to claim 5 wherein the second therapeutic agent is selected from the group consisting of a neuroprotectant and an agent for Alzheimer's disease treatment.

8. A method of treating a central nervous system disease or disorder by dual inhibition of BACE-1 and GSK-3 comprising administrating a therapeutically effective amount of a compound of Formula (I) to an individual in need thereof, ##STR00013## wherein: R.sub.1 is hydrogen, linear or branched, unsubstituted or substituted, C.sub.1-6 alkyl; R.sub.2 is selected from the group consisting of linear or branched, unsubstituted or substituted, C.sub.1-6alkyl, C.sub.2-6 alkenyl, C.sub.2-6alkynyl, unsubstituted or substituted C.sub.3-6 cycloalkyl, unsubstituted or substituted C.sub.3-6cycloalkylC.sub.1-6 alkyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted aryl C.sub.1-6alkyl, unsubstituted or substituted aryloxyC.sub.1-6alkyl, unsubstituted or substituted heteroarylC.sub.1-6alkyl, unsubstituted or substituted heteroaryloxyC.sub.1-6alkyl, unsubstituted or substituted heterocycloalkylC.sub.1-6alkyl, COR.sub.5; R.sub.5 is selected from the group consisting of linear or branched unsubstituted or substituted C.sub.1-9alkyl, unsubstituted or substituted C.sub.3-6cycloalkyl, unsubstituted or substituted C.sub.3-6cycloalkylC.sub.1-6alkyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted arylC.sub.1-6alkyl, unsubstituted or substituted aryloxyC.sub.1-6alkyl, unsubstituted or substituted heteroarylC.sub.1-6alkyl, unsubstituted or substituted heteroaryloxyC.sub.1-6alkyl, unsubstituted or substituted heterocycloalkylC.sub.1-6alkyl; or R.sub.1 and R.sub.2 together with the nitrogen atom to which they are attached may form a 4- to 7-membered azacyclic ring containing up to three heteroatoms selected from nitrogen and oxygen; Y.sub.1 and Y.sub.2 are independently selected from C or N; R.sub.3 and R.sub.4 are independently selected from the group consisting of hydrogen, halogen, linear or branched unsubstituted or substituted C.sub.1-6alkyl, unsubstituted or substituted C.sub.1-6alkoxy, unsubstituted or substituted hydroxyC.sub.1-6alkyl, hydroxy, cyano, nitro, unsubstituted or substituted fluoroC.sub.1-6alkyl, unsubstituted or substituted fluoroC.sub.1-6alkoxy, amino, monoalkylamino, dialkylamino; or pharmaceutically acceptable salt or solvate thereof; wherein the disease or condition is selected from the group consisting of Alzheimer's diseases, AIDS-related dementia, cancer-related dementia, frontotemporal dementia, inflammation and chronic inflammatory diseases, and tauopathies.

9. The pharmaceutical composition according to claim 7 wherein the second therapeutic agent is selected from the group consisting of galantamine, rivastigmine, donepezil, and memantine.

Description

PREPARATIONS AND EXAMPLES

Preparation I (Examples 1-15, 22-26)

(1) General synthesis of compounds of Formula (I) wherein R.sub.1 is hydrogen, linear or branched unsubstituted or substituted C.sub.1-6alkyl, R.sub.2 is linear or branched unsubstituted or substituted C.sub.1-6alkyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted arylC.sub.1-6alkyl, unsubstituted or substituted heteroarylC.sub.1-6alkyl, unsubstituted or substituted heterocyclylC.sub.1-6alkyl, or R.sub.1 and R.sub.2 together with the nitrogen atom to which they are attached may form a 4 to 7 membered azacyclic ring containing up to three heteroatoms selected from nitrogen and oxygen, and R.sub.3, R.sub.4, Y.sub.1 and Y.sub.2 are as defined in Formula (I).

General Procedure (A): Synthesis of Intermediates of Formula (IV)Step 1, Scheme 1

(2) ##STR00005##

(3) To a suspension of sodium dicyanamide (V) (1.2 eq.) in 1-butanol (1.2 M), was added the proper amine hydrochloride (VI) (1.0 eq.). The resulting mixture was heated at reflux for 6-8 hours, affording a white precipitate, which was filtered off. The filtrate was concentrated under vacuum to yield crude cyanoguinidine (IV). Further purification by flash chromatography was performed when required.

(IVa) N-cyano-N-methylguanidine

(4) The title compound was obtained according to general procedure A using methylamine hydrochloride (0.63 g, 9.30 mmol). The crude material was triturated with Et.sub.2O, affording IVa as a yellow oil, which was used in the next step without further purification: 0.72 g (77%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 2.74 (s, 3H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 28.4, 120.2, 163.4.

(IVb) N-cyano-N-ethylguanidine

(5) The title compound was obtained according to general procedure A using ethylamine hydrochloride (2.29 g, 27.07 mmol). Elution with DCM/MeOH/33% aqueous NH.sub.3 solution (9:1:0.1) afforded IVb as a yellow oil: 2.5 g (73%). .sup.1H-NMR (D.sub.2O, 400 MHz) 1.19 (t, J=6.8, 3H), 3.23 (q, J=6.8, 2H). .sup.13C-NMR (D.sub.2O, 100 MHz) 13.3, 36.5, 120.5, 161.1.

(IVc) N-cyano-N-propylguanidine

(6) The title compound was obtained according to general procedure A using propylamine hydrochloride (2.30 g, 24.53 mmol). Elution with DCM/MeOH/33% aqueous NH.sub.3 solution (9:1:0.1) afforded IVc as a waxy solid: 1.9 g (63%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 0.90 (t, J=7.2, 3H), 1.49-1.54 (m, 2H), 3.09 (t, J=6.8, 2H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 10.1, 22.2, 42.8, 118.8, 161.7.

(IVd) N-cyano-N-isopropylguanidine

(7) The title compound was obtained according to general procedure A using isopropylamine hydrochloride (0.89 g, 9.30 mmol). The crude material was triturated with Et.sub.2O and DCM, affording IVd as a white solid, which was used in the next step without further purification: 0.80 g (69%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) (4.5:1 mixture of rotamers) 1.12 (d, J=6.0, 1.1H, 2CH.sub.3 minor), 1.29 (d, J=6.0, 4.9H, 2CH.sub.3 major), 3.37-3.43 (m, 0.8H, CH major), 3.52-3.58 (m, 0.2, CH minor). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 19.6 (2CH.sub.3 major), 21.6 (2CH.sub.3 minor), 43.3 (CH minor), 43.9 (CH major), 119.1, 165.3.

(IVe) N-cyano-N-isobutylguanidine

(8) The title compound was obtained according to general procedure A using isobutylamine hydrochloride (1.02 g, 9.30 mmol). The crude material was triturated with Et.sub.2O and DCM, affording IVe as a woxy solid, which was used in the next step without further purification: 1.23 g (95%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 0.88 (d, J=6.8, 6H), 1.75-1.78 (m, 1H), 2.95 (d, J=7.2, 2H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 19.0, 28.2, 48.4, 119.1, 161.7.

(IVf) N-cyano-N-phenylguanidine

(9) The title compound was obtained according to general procedure A using aniline hydrochloride (0.65 g, 5.05 mmol). The crude material was triturated with H.sub.2O, affording IVf as a white solid, which was used in the next step without further purification: 0.80 g (quantitative yield). .sup.1H-NMR (DMSO-d.sub.6, 400 MHz) 6.98 (br s, exch, 2H), 7.05-7.09 (m, 1H), 7.28-7.35 (m, 2H), 9.03 (br s, exch, 1H). .sup.13C-NMR (DMSO-d.sub.6, 100 MHz) 117.6, 121.7, 124.2, 129.2, 138.4, 159.9.

(IVg) N-cyano-N,N-dimethylguanidine

(10) The title compound was obtained according to general procedure A using dimethylamine hydrochloride (1.52 g, 18.72 mmol). The crude material was triturated with DCM, affording IVg as a white solid, which was used in the next step without further purification: 0.40 g (18%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 2.77 (s, 6H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 36.5, 119.0, 161.1.

(IVh) N-cyano-N,N-diethylguanidine

(11) The title compound was obtained according to general procedure A using diethylamine hydrochloride (1.02 g, 9.30 mmol). The crude material was triturated with Et.sub.2O, affording IVh as a yellow solid, which was used in the next step without further purification: 0.95 g (73%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) (1.5:1 mixture of rotamers) 1.13 (t, J=6.8, 3.6H, 2CH.sub.3 major), 1.30 (t, J=6.8, 2.4H, 2CH.sub.3 minor), 3.04 (q, J=6.8, 1.8H, 2CH.sub.2 minor), 3.5 (q, J=6.8, 2.2H, 2CH.sub.2 major). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 10.3 (2CH.sub.3 minor), 12.0 (2CH.sub.3 major), 42.2 (2CH.sub.2 minor), 42.5 (2CH.sub.2 major), 119.3, 159.8.

(IVi) N-cyano-1-piperidinecarboximidamide

(12) The title compound was obtained according to general procedure A using piperidine hydrochloride (1.13 g, 9.30 mmol). The crude material was triturated with DCM, affording IVi as a white solid, which was used in the next step without further purification: 0.46 g (33%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 1.52-1.57 (m, 4H), 1.62-1.67 (m, 2H), 3.44-3.46 (m, 4H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 23.7, 25.2, 45.7, 119.2, 159.9.

(IVj) N-cyano-N-butylguanidine

(13) The title compound was obtained according to general procedure A using butylamine hydrochloride (1.02 g, 9.30 mmol). The crude material was triturated with DCM, affording IVj as a gummy solid, which was used in the next step without further purification: 1.12 g (86%). .sup.1H-NMR (DMSO-d.sub.6, 400 MHz) 0.83-0.88 (m, 3H), 1.22-1.27 (m, 2H), 1.29-1.39 (m, 2H), 3.00-3.02 (m, 2H). .sup.13C-NMR (DMSO-d.sub.6, 100 MHz) 14.0, 19.8, 31.5, 40.11, 118.8, 161.6.

(IVk) N-cyano-1-pyrrolidinecarboximidamide

(14) The title compound was obtained according to general procedure A using pyrrolidine hydrochloride (1.00 g, 9.30 mmol). The crude material was triturated with Et.sub.2O, affording IVk as a light yellow solid, which was used in the next step without further purification: 0.97 g (76%). .sup.1H-NMR (DMSO-d.sub.6, 400 MHz) 1.76-1.78 (m, 4H), 3.20-3.23 (m, 4H). .sup.13C-NMR (DMSO-d.sub.6, 100 MHz) 25.3, 47.2, 118.8, 159.0.

(IVl) N-cyano-N-(dimethylpropan-3-amino)guanidine

(15) The title compound was obtained according to general procedure A using N.sup.1,N.sup.1-dimethylpropane-1,3-diamine dihydrochloride (0.51 g, 2.90 mmol). The crude material was taken up with 40 mL of MeOH/2N aqueous HCl solution (1:0.5), split in two parts, loaded onto two 2 g ISOLUTE SCX-2 columns, and eluted with MeOH/33% aqueous NH.sub.3 solution (1:0.1) (215 mL). The organic phase was concentrated under vacuum, affording Ivl as a gummy solid, which was used in the next step without further purification: 0.10 g (21%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 1.68-1.76 (m, 2H), 2.24 (s, 6H), 2.33-2.39 (m, 2H), 3.16-3.22 (m, 2H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 26.8, 38.4, 44.0, 56.1, 118.7, 160.1.

(IVm) N-cyano-N-(3-(piperidin-1-yl)propan)guanidine

(16) The title compound was obtained according to general procedure A using 3-(piperidin-1-yl)propan-1-amine dihydrochloride (0.51 g, 2.36 mmol). The crude material was taken up with 20 mL of saturated aqueous Na.sub.2CO.sub.3 solution and extracted with EtOAc (320 mL). The organic layers were collected, dried over anhydrous Na.sub.2SO.sub.4, filtered and evaporated under vacuum. The residue was purified with flash chromatography, eluting with DCM/MeOH/33% aqueous NH.sub.3 solution (8:2:0.2). IVm was obtained as a gummy solid: 0.34 g (45%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 1.44-1.48 (m, 2H), 1.59-1.65 (m, 4H), 1.70-1.77 (m, 2H), 2.44-2.56 (m, 6H), 3.17 (t, J=6.4, 2H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 23.4, 24.7, 25.5, 39.2, 53.8, 55.5, 118.7, 161.8.

(IVn) N-cyano-N-benzylguanidine

(17) The title compound was obtained according to general procedure A using benzylamine hydrochloride (1.34 g, 9.36 mmol). Elution with DCM/MeOH/33% aqueous NH.sub.3 solution (9:1:0.1) afforded IVn as a gummy solid: 0.81 g (50%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 4.30 (s, 2H), 7.18-7.29 (m, 5H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 44.7, 119.0, 127.0, 127.2, 128.4, 138.1, 161.7.

(IVo) N-cyano-N-pyridin-4-ylmethylguanidine

(18) The title compound was obtained according to general procedure A using pyridin-4-ylmethanamine dihydrochloride (1.69 g, 9.36 mmol). Elution with DCM/MeOH/33% aqueous NH.sub.3 solution (8:2:0.2) afforded IVo as a gummy solid: 0.93 g (57%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 4.40 (s, 2H), 7.25 (d, J=5.6, 2H), 8.39 (d, J=5.6, 2H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 43.4, 118.7, 122.2, 147.6, 148.8, 161.9.

(IVp) N-cyano-N,N-dipropylguanidine

(19) The title compound was obtained according to general procedure A using dipropylamine hydrochloride (0.94 g, 9.30 mmol). The crude material was taken up with EtOAc and washed with water. The organic layer was concentrated under vacuum, affording IVp as a colourless oil, which was used in the next step without further purification: 0.76 g (50%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 0.88 (t, J=7.2, 6 H), 1.55-1.60 (m, 4H), 3.24 (t, J=7.6, 4H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 10.2, 20.6, 49.8, 119.4, 160.3.

General Procedure (B): Synthesis of Intermediates of Formula (III)Step 2, Scheme 1

(20) ##STR00006##

(21) To a solution of cyanoguanidine (IV) (1 eq.) in water (1.2 M) was added 70% aqueous sulfuric acid (2 eq.). The resulting mixture was stirred at rt for 15 minutes and heated at reflux for 1 hours. The reaction mixture was then cooled to rt and basified with Na.sub.2CO.sub.3. The aqueous phase was evaporated in vacuo, and the crude material was taken up with MeOH. The residue was filtered off and the organic solvent was concentrated under vacuum, to afford compound III, which was used in the next step without further purification.

(IIIa) (N-methylcarbamimidoyl)urea

(22) The title compound was obtained according to general procedure B using IVa (0.77 g, 7.12 mmol). IIIa was obtained as a yellow oil: 0.66 g (83%). .sup.1H-NMR (CD.sub.3OD, 200 MHz) 2.80 (s, 3H). .sup.13C-NMR (CD.sub.3OD, 50 MHz) 26.1, 161.2, 166.9.

(IIIb) (N-ethylcarbamimidoyl)urea

(23) The title compound was obtained according to general procedure B using IVb (2.50 g, 20.46 mmol). IIIb was obtained as a gummy solid: 3.90 g (quantitative yield). .sup.1H-NMR (D.sub.2O, 400 MHz) 0.48 (t, J=6.8, 3H), 2.58 (q, J=6.8, 2H). .sup.13C-NMR (D.sub.2O, 100 MHz) 12.1, 35.8, 152.8, 155.2.

(IIIc) (N-propylcarbamimidoyl)urea

(24) The title compound was obtained according to general procedure B using IVc (1.80 g, 14.26 mmol). IIIc was obtained as a gummy solid: 2.40 g (quantitative yield). .sup.1H-NMR (D.sub.2O, 400 MHz) 0.49-0.54 (m, 3H), 1.14-1.25 (m, 2H), 2.71 (t, J=6.8, 1H), 2.83 (t, J=6.8, 1H). .sup.13C-NMR (D.sub.2O, 100 MHz) 10.1, 21.1, 42.6, 155.7, 156.3.

(IIId) (N-isopropylcarbamimidoyl)urea

(25) The title compound was obtained according to general procedure B using IVd (0.80 g, 6.39 mmol). IIId was obtained as a gummy solid: 0.50 g (54%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) (1.1:1 mixture of rotamers) 1.25 (d, J=6.8, 3.1H, 2CH.sub.3 major), 1.29 (d, J=6.8, 2.9H, 2CH.sub.3 minor), 3.39-3.46 (m, 0.48H, CH minor), 3.81-3.88 (m, 0.52H, CH major). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 19.6 (2CH.sub.3 major), 21.0 (2CH.sub.3 minor), 43.9 (CH major), 44.0 (CH minor), 153.3, 155.6.

(IIIe) (N-isobutylcarbamimidoyl)urea

(26) The title compound was obtained according to general procedure B using IVe (1.23 g, 8.77 mmol). IIIe was obtained as a gummy solid: 1.39 g (quantitative yield). .sup.1H-NMR (CD.sub.3OD, 400 MHz) (2.3:1 mixture of rotamers) 0.23 (d, J=5.2, 4.2H, 2CH.sub.3 major), 0.42 (d, J=5.2, 1.8H, 2CH.sub.3 minor), 0.51-0.53 (m, 0.3H, CH minor), 1.16-1.18 (m, 0.7H, CH major), 2.39 (d, J=6.8, 0.6H, CH.sub.2 minor) 2.87 (d, J=6.8, 1.4H, CH.sub.2 major). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 16.2 (2CH.sub.3 minor), 18.0 (2CH.sub.3 major), 26.7 (CH major+minor), 47.4 (CH.sub.2 minor), 47.5 (CH.sub.2 major) 153.6, 154.6.

(IIIf) (N-phenylcarbamimidoyl)urea

(27) The title compound was obtained according to general procedure B using IVf (0.80 g, 4.90 mmol). IIIf was obtained as a white solid: 0.72 g (82%). .sup.1H-NMR (CDCl.sub.3, 400 MHz) 7.01-7.06 (m, 3H), 7.21-7.25 (m, 2H). .sup.13C-NMR (CDCl.sub.3, 100 MHz) 124.0, 125.0, 129.5, 140.0, 155.6, 163.6.

(IIIg) (N,N-dimethylcarbamimidoyl)urea

(28) The title compound was obtained according to general procedure B using IVg (0.72 g, 5.89 mmol). IIIg was obtained as a white solid: 0.46 g (60%). .sup.1H-NMR (DMSO, 400 MHz) 2.86 (s, 6H). .sup.13C-NMR (DMSO, 100 MHz) 36.5, 160.8, 167.0.

(IIIh) (N,N-dimethylcarbamimidoyl)urea

(29) The title compound was obtained according to general procedure B using IVh (0.95 g, 6.76 mmol). IIIh was obtained as a yellow oil: 0.73 g (68%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) (4.2:1 mixture of rotamers) 1.11 (t, J=6.8, 4.8H, 2CH.sub.3 major), 1.18 (t, J=6.8, 1.2H, 2CH.sub.3 minor), 3.36 (q, J=6.8, 3.2H, 2CH.sub.2 major), 3.46 (q, J=6.8, 0.8H, 2CH.sub.2 major). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 12.0 (2CH.sub.3 minor), 12.6 (2CH.sub.3 major), 41.4 (2CH.sub.2 major), 43.1 (2CH.sub.2 minor), 158.9, 167.1.

(IIIi) (piperidine-1-carboximidoyl)urea

(30) The title compound was obtained according to general procedure B using IVi (0.46 g, 3.02 mmol). IIIi was obtained as a gummy solid: 0.51 g (quantitative yield). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 1.65-1.73 (m, 6H), 3.50-3.59 (m, 4H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 23.1, 24.9, 46.9, 153.2, 155.3.

(IIIj) (N-butylcarbamimidoyl)urea

(31) The title compound was obtained according to general procedure B using IVj (1.07 g, 7.64 mmol). IIIj was obtained as a gummy solid: 1.15 g (95%). .sup.1H-NMR (DMSO-d.sub.6, 400 MHz) 0.85-0.89 (m, 3H), 1.27-1.33 (m, 2H), 1.36-1.43 (m, 2H), 3.04-3.08 (m, 2H). .sup.13C-NMR (DMSO-d.sub.6, 100 MHz) 13.7, 19.5, 31.3, 39.6, 160.3, 166.8.

(IIIk) (pyrrolidine-1-carboximidoyl)urea

(32) The title compound was obtained according to general procedure B using IVk (0.97 g, 7.04 mmol). IIIk was obtained as a light yellow solid: 0.80 g (73%). .sup.1H-NMR (DMSO-d.sub.6, 400 MHz) 1.76-1.79 (m, 4H), 3.25-3.28 (m, 4H). .sup.13C-NMR (DMSO-d.sub.6, 100 MHz) 25.1, 46.2, 155.9, 158.1.

(IIIl) (N-(dimethylpropan-3-amino)carbamimidoyl)urea

(33) The title compound was obtained according to general procedure B using IVl (0.10 g, 0.61 mmol). IIIl was obtained as a yellow gummy solid: 0.11 g (95%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 1.71-1.76 (m, 2H), 2.25 (s, 6H), 2.37 (t, J=7.6, 2H), 3.20 (t, J=7.6, 2H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 26.7, 38.3, 43.9, 56.3, 155.3, 156.4.

(IIIm) (N-(3-(piperidin-1-yl)propan))carbamimidoyl) urea

(34) The title compound was obtained according to general procedure B using IVm (0.34 g, 1.62 mmol). IIIm was obtained as a yellow gummy solid: 0.23 g (93%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 1.42-1.48 (m, 2H), 1.59-1.63 (m, 4H), 1.76-1.83 (m, 2H), 2.48-2.52 (m, 6H), 3.24-3.31 (m, 2H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 23.6, 24.9, 25.3, 38.7, 53.7, 54.8, 157.4, 159.5.

(IIIn) (N-benzylcarbamimidoyl)urea

(35) The title compound was obtained according to general procedure B using IVn (0.80 g, 4.60 mmol). IIIn was obtained as a white solid: 0.82 g (93%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 4.35 (s, 2H), 7.21-7.30 (m, 5H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 44.1, 126.9, 127.0, 128.1, 128.3, 160.9, 167.5.

(IIIo) (N-pyridin-4-ylmethylcarbamimidoyl)urea

(36) The title compound was obtained according to general procedure B using IVo (0.93 g, 5.31 mmol). IIIo was obtained as a gummy solid: 0.86 g (85%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 4.49 (s, 2H), 7.34 (d, J=5.6, 2H), 8.44 (d, J=5.6, 2H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 42.7, 122.2, 147.6, 148.7, 159.8, 165.6.

(IIIp) (N,N-dipropylcarbamimidoyl)urea

(37) The title compound was obtained according to general procedure B using IVp (0.77 g, 4.60 mmol). IIIp was obtained as a white solid: 0.68 g (79%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 0.79 (t, J=7.2, 6H), 1.42-1.48 (m, 4H), 3.16 (t, J=7.6, 4H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 11.5, 21.2, 48.2, 159.7, 167.2.

General Procedure (C): Synthesis of Compounds of Formula (I)Step 3, Scheme 1

(38) ##STR00007##

(39) To a solution of guanylurea (III) (1 eq) in concentrated H.sub.2SO.sub.4 (5.5 M) was added the proper benzaldehyde (II) (1.2 eq). After stirring at rt for 72 hours, the reaction mixture was diluted with a small amount of cold H.sub.2O. The solution was then made basic with Na.sub.2CO.sub.3, affording a precipitate which was collected by filtration. Whereas no precipitation was observed, the aqueous phase was concentrated in vacuo. The crude triazinone (I) was either purified by trituration with organic solvents or by flash chromatography.

Example 1

4-(4-fluorophenyl)-6-(methylamino)-3,4-dihydro-1,3,5-triazin-2(1H)-one

(40) The title compound was obtained according to general procedure C using 4-fluoro-benzaldheyde (IIa) (0.73 mL, 6.84 mmol) and IIIa (0.66 g, 5.7 mmol). Elution with DCM/MeOH/33% aqueous NH.sub.3 solution (8.5:1.5:0.1) afforded the compound of example 1 as a white solid: 0.33 g (26%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 2.76 (s, 3H), 5.68 (s, 1H), 7.07-7.12 (m, 2H), 7.42-7.45 (m, 2H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 26.5, 66.0, 115.0 (d, J=16.0), 128.1 (d, J=8.3), 138.1, 150.7, 154.2, 162.9 (d, J=244). MS (ESI) m/z: 223 [M+H].sup.+; MS (ESI) m/z: 221 [MH].sup..

Example 2

6-(ethylamino)-4-(o-tolyl)-3,4-dihydro-1,3,5-triazin-2(1H)-one

(41) The title compound was obtained according to general procedure C using o-tolyl-benzaldheyde-benzaldheyde (IIb) (0.36 mL, 3.19 mmol) and IIIb (0.34 g, 2.65 mmol). Elution with DCM/MeOH/33% aqueous NH.sub.3 solution (9:1:0.1) afforded the compound of example 2 as a white solid: 0.26 g (43%). .sup.1H-NMR (DMSO-d.sub.6, 400 MHz) 1.02 (t, J=8.0, 3H), 2.37 (s, 3H), 3.05 (q, J=8.0, 2H), 5.32 (br s, exch, 1H), 5.77 (s, 1H), 7.14-7.16 (m, 3H), 7.25-7.27 (m, 1H), 7.35 (br s, exch, 1H), 8.48 (br s, exch, 1H). .sup.13C-NMR (DMSO-d.sub.6, 100 MHz) 15.4, 19.1, 35.3, 67.1, 126.4, 126.6, 130.7, 131.3, 135.7, 141.9, 148.3, 153.5. MS (ESI) m/z: 233 [M+H].sup.+.

Example 3

6-(ethylamino)-4-(4-fluorophenyl)-3,4-dihydro-1,3,5-triazin-2(1H)-one

(42) The title compound was obtained according to general procedure C using 4-fluoro-benzaldheyde (IIa) (0.50 mL, 4.60 mmol) and IIIb (0.50 g, 3.84 mmol). Trituration with a mixture of organic solvents (DCM/MeOH) afforded the compound of example 3 as a white solid: 0.46 g (50%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 1.11 (t, J=8.0, 3H), 3.20 (q, J=8.0, 2H), 5.65 (s, 1H), 7.06-7.10 (m, 2H), 7.40-7.43 (m, 2H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 14.9, 36.7, 67.5, 116.3 (d, J=22.0), 129.5 (d, J=8.0), 139.6, 156.6, 158.5, 164.2 (d, J=244.0). MS (ESI) m/z: 237 [M+H].sup.+; MS (ESI) m/z: 235 [MH].sup..

Example 4

(+)6-(ethylamino)-4-(4-fluorophenyl)-3,4-dihydro-1,3,5-triazin-2(1H)-one

(43) The title compound was obtained, as a white solid, by chiral HPLC enantiomeric separation of the racemic compound of Example 3: 0.046 g (61%). ee>99.5% (detector UV 240 nm). Retention time on analytical chiral HPLC: 8.475 min. .sup.1H-NMR (CD.sub.3OD, 400 MHz) 1.11 (t, J=8.0, 3H), 3.20 (q, J=8.0, 2H), 5.65 (s, 1H), 7.06-7.10 (m, 2H), 7.40-7.43 (m, 2H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 14.9, 36.7, 67.5, 116.3 (d, J=22.0), 129.5 (d, J=8.0), 139.6, 156.6, 158.5, 164.2 (d, J=244.0). MS (ESI) m/z: 237 [M+H].sup.+; MS (ESI) m/z: 235 [MH].sup.. [].sup.20.sub.D=+2.1 (c 0.13, MeOH) [calculated for the corresponding trifluoroacetate salt].

Example 5

()6-(ethylamino)-4-(4-fluorophenyl)-3,4-dihydro-1,3,5-triazin-2(1H)-one

(44) The title compound was obtained, as a white solid, by chiral HPLC enantiomeric separation of the racemic compound of Example 3: 0.043 g (57%). ee>99.4% (detector UV 240 nm). Retention time on analytical chiral HPLC: 16.479 min. .sup.1H-NMR (CD.sub.3OD, 400 MHz) 1.11 (t, J=8.0, 3H), 3.20 (q, J=8.0, 2H), 5.65 (s, 1H), 7.06-7.10 (m, 2H), 7.40-7.43 (m, 2H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 14.9, 36.7, 67.5, 116.3 (d, J=22.0), 129.5 (d, J=8.0), 139.6, 156.6, 158.5, 164.2 (d, J=244.0). MS (ESI) m/z: 237 [M+H].sup.+; MS (ESI) m/z: 235 [MH].sup.. [].sup.20.sub.D=0.9 (c 0.13, MeOH) [calculated for the corresponding trifluoroacetate salt].

Example 6

4-(4-fluorophenyl)-6-(propylamino)-3,4-dihydro-1,3,5-triazin-2(1H)-one

(45) The title compound was obtained according to general procedure C using 4-fluoro-benzaldheyde (IIa) (0.36 mL, 3.32 mmol) and IIIc (0.40 g, 2.77 mmol). Elution with DCM/MeOH/33% aqueous NH.sub.3 solution (9:1:0.1) afforded the compound of example 6 as a white solid: 0.11 g (16%). .sup.1H-NMR (DMSO-d.sub.6, 400 MHz) 0.84 (t, J=7.2, 3H), 1.40-1.49 (m, 2H), 3.03 (t, J=6.0, 2H), 5.59 (s, 1H), 5.75 (br s, exch, 1H), 7.14-7.18 (m, 2H), 7.34-7.38 (m, 2H), 7.55 (br s, exch, 1H), 8.55 (br s, exch, 1H). .sup.13C-NMR (DMSO-d.sub.6, 100 MHz) 11.8, 22.7, 42.3, 68.1, 115.4 (d, J=22.0), 128.6 (d, J=8.0), 141.2, 149.0, 153.8, 161.6 (d, J=244.0). MS (ESI) m/z: 251 [M+H].sup.+.

Example 7

4-(4-fluorophenyl)-6-(isopropylamino)-3,4-dihydro-1,3,5-triazin-2(1H)-one

(46) The title compound was obtained according to general procedure C using 4-fluoro-benzaldheyde (IIa) (0.45 mL, 4.15 mmol) and IIId (0.50 g, 3.46 mmol). Trituration with a mixture of organic solvents (DCM/Et.sub.2O) afforded the compound of example 7 as a white solid: 0.19 g (22%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 1.11 (m, 6H), 3.81-3.92 (m, 1H), 5.65 (s, 1H), 7.08-7.12 (m, 2H), 7.41-7.45 (m, 2H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 21.6, 21.7, 42.1, 65.5, 114.9 (d, J=20.5), 128.1 (d, J=8.3), 138.6, 150.5, 154.6, 164.2 (d, J=242.0). MS (ESI) m/z: 251 [M+H].sup.+; MS (ESI) m/z: 249 [MH].sup..

Example 8

4-(4-fluorophenyl)-6-(isobutylamino)-3,4-dihydro-1,3,5-triazin-2(1H)-one

(47) The title compound was obtained according to general procedure C using 4-fluoro-benzaldheyde (IIa) (1.13 mL, 10.52 mmol) and IIIe (1.34 g, 8.77 mmol). Elution with DCM/MeOH/33% aqueous NH.sub.3 solution (9:1:0.05) afforded the compound of example 8 as a white solid: 0.33 g (14%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 0.91 (d, J=6.8, 6H), 1.79-1.82 (m, 1H), 3.00-3.05 (m, 2H), 5.69 (s, 1H), 7.08-7.12 (m, 2H), 7.43-7.46 (m, 2H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 19.1, 28.1, 66.0, 115.0 (d, J=21.2), 128.1 (d, J=8.3), 138.2, 149.2, 153.8, 162.8 (d, J=243.8). MS (ESI) m/z: 265 [M+H].sup.+; MS (ESI) m/z: 263 [MH].sup..

Example 9

4-(4-fluorophenyl)-6-(phenylamino)-3,4-dihydro-1,3,5-triazin-2(1H)-one

(48) The title compound was obtained according to general procedure C using 4-fluoro-benzaldheyde (IIa) (0.30 mL, 2.70 mmol) and IIIf (0.40 g, 2.24 mmol). Elution with DCM/MeOH (9:1) afforded the compound of example 9 as a white solid: 0.06 g (10%). .sup.1H-NMR (DMSO-d.sub.6, 400 MHz) 5.78 (s, 1H), 6.89-6.92 (m, 1H), 7.17-7.24 (m, 4H), 7.41-7.45 (m, 2H), 7.52-7.54 (m, 2H), 7.82 (br s, exch, 1H), 8.02 (br s, exch, 1H), 8.52 (br s, exch, 1H). .sup.13C-NMR (DMSO-d.sub.6, 100 MHz) 68.3, 114.6 (d, J=21.0), 118.1, 121.3, 128.2 (d, J=9.0), 128.6, 140.0, 145.11, 145.6, 152.1, 162.0 (d, J=240.2). MS (ESI) m/z: 285 [M+H].sup.+; MS (ESI) m/z: 283 [MH].sup..

Example 10

6-(dimethylamino)-4-(4-fluorophenyl)-3,4-dihydro-1,3,5-triazin-2(1H)-one

(49) The title compound was obtained according to general procedure C using 4-fluoro-benzaldheyde (IIa) (0.45 mL, 4.25 mmol) and IIIg (0.46 g, 3.54 mmol). Elution with DCM/MeOH/33% aqueous NH.sub.3 solution (8.5:1.5:0.1) afforded the compound of example 10 as a white solid: 0.11 g (13%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 3.02 (s, 6H), 5.67 (s, 1H), 7.11-7.15 (m, 2H), 7.45-7.49 (m, 2H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 36.0, 66.2, 115.0 (d, J=21.2), 128.1 (d, J=9.1), 139.2, 153.6, 155.6, 162.9 (d, J=244.0). MS (ESI) m/z: 237 [M+H].sup.+; MS (ESI) m/z: 235 [MH].sup..

Example 11

6-(diethylamino)-4-(4-fluorophenyl)-3,4-dihydro-1,3,5-triazin-2(1H)-one

(50) The title compound was obtained according to general procedure C using 4-fluoro-benzaldheyde (IIa) (0.59 mL, 5.52 mmol) and IIIh (0.73 g, 4.60 mmol). Trituration with a mixture of organic solvents (DCM/Et.sub.2O) afforded the compound of example 11 as a white solid: 0.25 g (21%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 1.12 (t, J=7.0, 6H), 3.42 (q, J=7.0, 4H), 5.66 (s, 1H), 7.07-7.12 (m, 2H), 7.43-7.46 (m, 2H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 12.3, 41.5, 64.7, 115.0 (d, J=21.2), 128.0 (d, J=8.4), 137.9, 150.8, 153.7, 162.9 (d, J=243.3). MS (ESI) m/z: 265 [M+H].sup.+; MS (ESI) m/z: 263 [MH].sup..

Example 12

4-(4-fluorophenyl)-6-(piperidin-1-yl)-3,4-dihydro-1,3,5-triazin-2(1H)-one

(51) The title compound was obtained according to general procedure C using 4-fluoro-benzaldheyde (IIa) (0.40 mL, 3.62 mmol) and IIIi (0.51 g, 3.02 mmol). Elution with DCM/MeOH/33% aqueous NH.sub.3 solution (9:1:0.05) afforded the compound of example 12 as a white solid: 0.23 g (28%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 1.55-1.58 (m, 4H), 1.64-1.68 (m, 2H), 3.49-3.52 (m, 4H), 5.67 (s, 1H), 7.10-7.14 (m, 2H), 7.44-7.48 (m, 2H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 23.9, 25.4, 45.6, 64.5, 115.0 (d, J=22.0), 128.1 (d, J=8.3), 137.5 (d, J=3.9), 151.8, 156.0, 164.3 (d, J=243.8). MS (ESI) m/z: 277 [M+H].sup.+; MS (ESI) m/z: 275 [MH].sup..

Example 13

6-(butylamino)-4-(4-fluorophenyl)-3,4-dihydro-1,3,5-triazin-2(1H)-one

(52) The title compound was obtained according to general procedure C using 4-fluoro-benzaldheyde (IIa) (0.94 mL, 8.76 mmol) and IIIj (1.15 g, 7.30 mmol). Elution with DCM/MeOH/33% aqueous NH.sub.3 solution (9:1:0.1) afforded the compound of example 13 as a white solid: 0.71 g (39%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 0.88-0.92 (m, 3H), 1.32-1.36 (m, 2H), 1.48-1.54 (m, 2H), 3.17-3-18 (m, 2H), 5.70 (s, 1H), 7.06-7.11 (m, 2H), 7.43-7.46 (m, 2H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 14.2, 21.0, 32.4, 41.6, 67.1, 116.3 (d, J=21.9), 129.4 (d, J=8.4), 139.3, 151.3, 155.2, 163.7 (d, J=243.8). MS (ESI) m/z: 265 [M+H].sup.+; MS (ESI) m/z: 263 [MH].sup..

Example 14

6-(ethylamino)-4-(pyridin-3-yl)-3,4-dihydro-1,3,5-triazin-2(1H)-one

(53) The title compound was obtained according to general procedure C using 3-pyridinecarboxaldehyde (IIc) (0.43 mL, 4.61 mmol) and IIIb (0.50 g, 3.84 mmol). Elution with DCM/MeOH/33% aqueous NH.sub.3 solution (8:2:0.2) afforded the compound of example 14 as a white solid: 0.08 g (10%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 1.40 (t, J=6.8, 3H), 3.19 (q, J=6.8, 2H), 5.79 (s, 1H), 7.44-7.47 (m, 1H), 7.89 (d, J=8.5, 1H), 8.50 (d, J=5.0, 1H), 8.58 (s, 1H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 13.4, 35.3, 65.0, 124.0, 134.9, 138.5, 147.2, 148.8, 151.3, 155.2. MS (ESI) m/z: 220 [M+H].sup.+; MS (ESI) m/z: 218 [MH].sup..

Example 15

4-(4-fluorophenyl)-6-(pyrrolidin-1-yl)-3,4-dihydro-1,3,5-triazin-2(1H)-one

(54) The title compound was obtained according to general procedure C using 4-fluoro-benzaldheyde (IIa) (0.65 mL, 6.10 mmol) and IIIk (0.80 g, 5.08 mmol). Elution with DCM/MeOH/33% aqueous NH.sub.3 solution (9:1:0.05) afforded the compound of example 15 as a white solid: 0.29 g (22%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 1.91-1.93 (m, 4H), 3.40-3.43 (m, 4H), 5.69 (s, 1H), 7.08-7.13 (m, 2H), 7.46-7.49 (m, 2H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 24.7, 46.3, 64.8, 115.0 (d, J=21.2), 128.3 (d, J=8.3), 137.7, 155.3, 160.4, 163.5 (d, J=244.4). MS (ESI) m/z: 263 [M+H].sup.+.

Preparation II (Examples 16-21)

(55) General synthesis of compounds of Formula (I) wherein R.sub.1 is hydrogen, linear or branched unsubstituted or substituted C.sub.1-6alkyl, R.sub.2 is COR.sub.5, and R.sub.3, R.sub.4, R.sub.5, Y.sub.1 and Y.sub.2 are as defined in Formula (I).

General Procedure (D): Synthesis of Intermediates of Formula (VIII)Steps 1-3, Scheme 1

(56) Intermediates of Formula (VIII) were obtained according to the procedures reported in preparation I, following Steps 1-3 in Scheme 1.

(VIIIa) 6-amino-4-(4-fluorophenyl)-3,4-dihydro-1,3,5-triazin-2(1H)-one

(57) The title compound was obtained according to general procedure D using 4-fluoro-benzaldheyde (IIa) (2.5 mL, 23.80 mmol) and the commercially available guanylurea sulphate (3.0 g, 19.80 mmol). Trituration with a mixture of organic solvents (DCM/MeOH) afforded VIIIa as a white solid: 2.90 g (88%). .sup.1H-NMR (DMSO-d.sub.6, 400 MHz) 5.56 (s, 1H), 5.75 (br s, exch, 1H) 7.15-7.19 (m, 2H), 7.35-7.38 (m, 2H), 7.49 (br s, exch, 1H), 8.51 (br s, exch, 1H). .sup.13C-NMR (DMSO-d.sub.6, 100 MHz) 67.5, 115.4 (d, J=22.0), 128.6 (d, J=8.0), 140.2, 150.8, 153.5, 161.2 (d, J=244.0). MS (ESI) m/z: 209 [M+H].sup.+.

General Procedure (E): Synthesis of Compounds of Formula (I)Scheme 2

(58) ##STR00008##

(59) Method (a): To an ice-cold solution/suspension of VIII (1 eq.) in a mixture of organic solvent such as pyridine/DCM, DMF/DCM, and 2,6-lutidine/DMF (0.4 M) was added dropwise the proper acyl chloride (VII) (1 eq.). After stirring at 0 C. for 3-4 hours, the reaction mixture was diluted with DCM or EtOAc and washed with 2N HCl. The organic phase was dried over anhydrous Na.sub.2SO.sub.4 and evaporated under reduced pressure. The crude triazinone (I) was purified by flash chromatography.

(60) Method (b): A solution of the proper carboxylic acid (VII) (1 eq.) and EDCI.HCl (1.3), in DCM or DMF (0.15 M), was treated with HOBt (1.3 eq.). The mixture was stirred for 1 h at rt, and then added dropwise to an ice-cold suspension of VIII (1.1 eq.) in DCM or DMF (0.12 M). After adding Et.sub.3N or DIPEA (1.1 eq.), the resulting mixture was allowed to stir overnight at rt.

(61) The reaction mixture was then either diluted with DCM and washed with H.sub.2O, or diluted with EtOAc and washed with 5% aqueous LiCl solution, in case the reaction solvent was DCM or DMF respectively. The organic phase was dried over anhydrous Na.sub.2SO.sub.4 and evaporated under reduced pressure. The crude triazinone (I) was purified by flash chromatography.

(62) Method (c): A solution of the proper carboxylic acid (VII) (1.2 eq.) and EDCI.HCl (1.56), in DMF (0.4 M), was stirred for 1 hour at rt, and then added dropwise to an ice-cold suspension of VIII (1 eq.) in DMF (0.12 M). After stirring overnight at rt, the reaction mixture was diluted with EtOAc and washed with 5% aqueous LiCl solution. The organic phase was dried over anhydrous Na.sub.2SO.sub.4 and evaporated under reduced pressure. The residue was then triturated with organic solvents, affording the compound of interest without further purification.

Example 16

N-(4-(4-fluorophenyl)-6-oxo-1,4,5,6-tetrahydro-1,3,5-triazin-2-yl)acetamide

(63) The title compound was obtained according to general procedure E, method (a), from Villa (0.08 g, 0.38 mmol) and AcCl (27 L, 0.38 mmol) in 2,6-lutidine/DMF (3:1). Elution with DCM/MeOH (9.5:0.5) afforded the compound of example 15 as a white solid: 24 mg (25%). .sup.1H-NMR (DMSO-d.sub.6, 400 MHz) 1.98 (s, 3H), 5.74 (s, 1H), 7.13-7.17 (m, 2H), 7.33-7.36 (m, 2H), 7.96 (br s, exch, 1H). .sup.13C-NMR (DMSO-d.sub.6, 100 MHz) 23.8, 67.2, 114.8 (d, J=22.0), 127.3 (d, J=8.0), 139.6, 146.9, 152.3, 161.2 (d, J=244.0), 174.8. MS (ESI) m/z: 251 [M+H].sup.+; MS (ESI) m/z: 249 [MH].sup..

Example 17

N-(4-(4-fluorophenyl)-6-oxo-1,4,5,6-tetrahydro-1,3,5-triazin-2-yl)-2-propylpentanamide

(64) The title compound was obtained according to general procedure E, method (b), from VIIIa (0.15 g, 0.72 mmol) and valproic acid (104 L, 0.65 mmol) in DMF. Elution with DCM/MeOH (9.5:0.5) afforded the compound of example 16 as a white solid: 80 mg (37%). .sup.1H-NMR (DMSO-d.sub.6, 400 MHz) 0.81-0.85 (m, 6H), 1.19-1.25 (m, 4H) 1.26-1.35 (m, 2H), 1.45-1.54 (m, 2H), 2.35-2.37 (m, 1H), 5.78 (s, 1H), 7.18-7.20 (m, 2H), 7.35-7-37 (m, 2H), 7.95 (br s, exch, 1H), 9.84 (br s, exch, 1H), 10.76 (br s, exch, 1H). .sup.13C-NMR (DMSO-d.sub.6, 100 MHz) 14.0, 20.5, 34.7, 47.3, 68.2, 115.5 (d, J=21.0), 127.5 (d, J=8.3), 139.3, 145.6, 152.3, 162.5 (d, J=247.3), 180.0. MS (ESI) m/z: 335 [M+H].sup.+; MS (ESI) m/z: 333 [MH].sup..

Example 18

5-(1,2-dithiolan-3-yl)-N-(4-(4-fluorophenyl)-6-oxo-1,4,5,6-tetrahydro-1,3,5-triazin-2-yl)pentanamide

(65) The title compound was obtained according to general procedure E, method (b), from VIIIa (0.15 g, 0.72 mmol) and (+/) lipoic acid (0.13 g, 0.65 mmol) in DCM. Elution with DCM/MeOH (9.5:0.5) afforded the compound of example 17 as a white solid: 77 mg (31%). .sup.1H-NMR (DMSO-d.sub.6, 400 MHz) 1.33-1.39 (m, 2H), 1.49-1.59 (m, 3H), 1.62-1.70 (m, 1H), 1.82-1.90 (m, 1H), 2.31 (t, J=4.0, 2H), 2.36-2.44 (m, 1H), 3.08-3.14 (m, 1H), 3.15-3.21 (m, 1H), 3.58-3.66 (m, 1H) 5.80 (s, 1H), 7.18-7.23 (m, 2H), 7.39-7.42 (m, 2H) 8.00 (br s, exch, 1H), 9.88 (br s, exch, 1H), 10.65 (br s, exch, 1H). .sup.13C-NMR (DMSO-d.sub.6, 100 MHz) 24.2, 27.9, 33.9, 36.2, 38.0, 39.7, 55.9, 67.5, 115.1 (d, J=21.0), 128.0 (d, J=8.3), 139.2, 145.6, 151.2, 162.1 (d, J=243.0), 176.8. MS (ESI) m/z: 397 [M+H].sup.+; MS (ESI) m/z: 395 [MH].sup.

Example 19

3-((1H-benzo[d][1,2,3]triazol-1-yl)oxy)-N-(4-(4-fluorophenyl)-6-oxo-1,4,5,6-tetrahydro-1,3,5-triazin-2-yl)propanamide

(66) The title compound was obtained according to general procedure E, method (b), from VIIIa (0.10 g, 0.48 mmol) and acrylic acid (33 L, 0.48 mmol) in DMF. After stirring at rt overnight, precipitation of a white solid was observed. The precipitate was collected by filtration and triturate with a mixture of DCM/Et.sub.2O, affording the compound of interest as a white solid: 45 mg (25%). .sup.1H-NMR (DMSO-d.sub.6, 400 MHz) 2.98-3.01 (m, 2H), 4.69-4.71 (m, 2H), 5.79 (s, 1H), 7.19-7.24 (m, 2H), 7.35-7.38 (m, 2H), 7.41-7.45 (m, 1H), 7.66-7.70 (m, 1H), 7.86 (d, J=8.8, 1H), 7.93 (d, J=8.4, 1H), 8.15 (br, exch, 1H), 9.74 (br, exch, 1H), 10.62 (br, exch, 1H). .sup.13C-NMR (DMSO-d.sub.6, 100 MHz) 30.5, 43.6, 67.5, 112.5, 114.7, 115.3 (d, J=21.0), 124.4, 128.1 (d, J=9.0), 130.3, 133.9, 134.0, 137.8, 147.8, 150.5, 161.8 (d, J=246), 175.3. MS (ESI) m/z: 398 [M+H].sup.+; MS (ESI) m/z: 396 [MH].sup..

Example 20

N-(4-(4-fluorophenyl)-6-oxo-1,4,5,6-tetrahydro-1,3,5-triazin-2-yl)-2-phenoxyacetamide

(67) The title compound was obtained according to general procedure E, method (c), from VIIIa (0.20 g, 0.96 mmol) and phenoxy acetic acid (0.18 g, 1.15 mmol). Trituration with a mixture of DCM/Et.sub.2O afforded the compound of example 19 as a white solid: 11 mg (3%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 4.64 (s, 2H), 5.90 (s, 1H), 6.94-6.96 (m, 3H), 7.13-7.17 (m, 2H), 7.25-7.28 (m, 2H), 7.45-7.48 (m, 2H). MS (ESI) m/z: 343 [M+H].sup.+; MS (ESI) m/z: 341 [MH].sup..

Example 21

2-(4-fluorophenoxy)-N-(4-(4-fluorophenyl)-6-oxo-1,4,5,6-tetrahydro-1,3,5-triazin-2-yl)acetamide

(68) The title compound was obtained according to general procedure E, method (c), from VIIIa (0.16 g, 0.78 mmol) and 4-fluorophenoxy acetic acid (0.16 g, 0.94 mmol). Trituration with a mixture of DCM/Et.sub.2O afforded the compound of example 20 as a white solid: 34 mg (12%). .sup.1H-NMR (DMSO-d.sub.6, 400 MHz) 4.57 (s, 2H), 5.78 (s, 1H), 6.84-6.87 (m, 2H), 7.04-7.08 (m, 2H), 7.20-7.24 (m, 2H), 7.37-7.40 (m, 2H), 8.42 (br s, exch, 1H), 10.38 (br s, exch, 1H). .sup.13C-NMR (DMSO-d.sub.6, 100 MHz) 64.4, 69.0, 115.3, 115.6, 115.7, 127.8, 137.1, 147.6, 151.0, 154.6, 155.8 (d, J=260), 162.0 (d, J=244), 172.7. MS (ESI) m/z: 361 [M+H].sup.+; MS (ESI) m/z: 359 [MH].sup..

Example 22

6-((3-(dimethylamino)propyl)amino)-4-(4-fluorophenyl)-3,4-dihydro-1,3,5-triazin-2(1H)-one

(69) The title compound was obtained according to general procedure C using 4-F-benzaldehyde (IIa) (78 L, 0.72 mmol) and IIIl (0.10 g, 0.60 mmol). Elution with DCM/MeOH/33% aqueous NH.sub.3 solution (7.5:2.5:0.2.5) afforded the compound of example 22 as a white solid: 30 mg (17%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 1.69-1.73 (m, 2H), 2.22 (s, 6H), 2.38 (t, J=7.2, 2H), 3.21-3.28 (m, 2H), 5.67 (s, 1H), 7.08-7.13 (m, 2H), 7.42-7.46 (m, 2H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 25.8, 38.6, 46.2, 55.8, 66.0, 115.3 (d, J=21.6) 128.5 (d, J=8.7), 139.5, 150.6, 152.7, 162.3 (d, J=242.8). MS (ESI) m/z 294 [M+H].sup.+; MS (ESI) m/z 292 [MH].sup..

Example 23

4-(4-fluorophenyl)-6-((3-(piperidin-1-yl)propyl)amino)-3,4-dihydro-1,3,5-triazin-2(1H)-one

(70) The title compound was obtained according to general procedure C using 4-F-benzaldehyde (IIa) (0.20 mL, 1.80 mmol) and IIIm (0.34 g, 1.50 mmol). Elution with DCM/MeOH/33% aqueous NH.sub.3 solution (7.5:2.5:0.2.5) afforded the compound of example 23 as a white solid: 0.12 g (24%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 1.44-1.46 (m, 2H), 1.56-1.60 (m, 4H), 1.70-1.74 (m, 2H), 2.37-2.44 (m, 6H), 3.20-3.23 (m, 2H), 5.65 (s, 1H), 7.07-7.11 (m, 2H), 7.40-7.44 (m, 2H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 23.6, 24.9, 25.8, 38.6, 53.9, 55.8, 66.0, 115.0 (d, J=21.2), 128.1 (d, J=8.4), 138.2, 150.3, 153.2, 162.7 (d, J=243.8). MS (ESI) m/z 334 [M+H].sup.+; MS (ESI) m/z 332 [MH].sup..

Example 24

6-(benzylamino)-4-(4-fluorophenyl)-3,4-dihydro-1,3,5-triazin-2(1H)-one

(71) The title compound was obtained according to general procedure C using 4-F-benzaldehyde (IIa) (0.56 mL, 5.20 mmol) and IIIn (0.82 g, 4.30 mmol). Elution with DCM/MeOH/33% aqueous NH.sub.3 solution (9:1:0.1) afforded the compound of example 24 as a white solid: 0.12 g (36%). .sup.1H-NMR (DMSO-d.sub.6, 400 MHz) 4.40-4.44 (m, 2H), 5.71 (s, 1H), 7.06-7.11 (m, 2H), 7.26-7.31 (m, 7H). .sup.13C-NMR (DMSO-d.sub.6, 100 MHz) 44.5, 67.3, 114.9 (d, J=21.2), 126.7, 127.0 (d, J=8.3), 128.06, 128.1, 138.3, 142.7, 150.6, 152.8, 162.5 (d, J=242.6). MS (ESI) m/z 299 [M+H].sup.+; MS (ESI) m/z 297 [MH].sup..

Example 25

4-(4-fluorophenyl)-6-((pyridin-4-ylmethyl)amino)-3,4-dihydro-1,3,5-triazin-2(1H)-one

(72) The title compound was obtained according to general procedure C using 4-F-benzaldehyde (IIa) (0.58 mL, 5350 mmol) and IIIo (0.86 g, 4.46 mmol). Elution with DCM/MeOH/33% aqueous NH.sub.3 solution (8:2:0.2) afforded the compound of example 25 as a white solid: 0.18 g (14%). .sup.1H-NMR (DMSO-d.sub.6, 400 MHz) 4.35 (s, 2H), 5.59 (s, 1H), 7.08-7.13 (m, 2H), 7.24-7.26 (m, 4H), 8.47 (d, J=4.8, 2H). .sup.13C-NMR (DMSO-d.sub.6, 100 MHz) 42.4, 67.1, 114.5 (d, J=21.3), 121.8, 127.9 (d, J=8.2), 139.4, 148.6, 149.1, 152.5, 153.2, 161.3 (d, J=250.0). MS (ESI) m/z 300 [M+H].sup.+; MS (ESI) m/z 298 [MH].sup..

Example 26

6-(dipropylamino)-4-(4-fluorophenyl)-3,4-dihydro-1,3,5-triazin-2(1H)-one

(73) The title compound was obtained according to general procedure C using 4-fluoro-benzaldheyde (IIa) (0.47 mL, 4.38 mmol) and IIIp (0.68 g, 3.64 mmol). Trituration with a mixture of organic solvents (DCM/Et.sub.2O) afforded the compound of example 26 as a white solid: 0.23 g (22%). .sup.1H-NMR (CD.sub.3OD, 400 MHz) 0.88 (t, J=7.2, 6H), 1.55-1.61 (m, 4H), 3.27-3.30 (m, 2H), 3.36-3.41 (m, 2H), 5.65 (s, 1H), 7.10-7.14 (m, 2H), 7.43-7.47 (m, 2H). .sup.13C-NMR (CD.sub.3OD, 100 MHz) 9.8, 20.8, 48.8, 64.6, 115.0 (d, J=22.0), 128.0 (d, J=9.0), 137.9, 156.7, 161.2, 162.9 (d, J=243). MS (ESI) m/z 293 [M+H].sup.+; MS (ESI) m/z 291 [MH].sup..

(74) Methods to Assess the Biochemical Activity of the Compounds of the Invention

(75) Inhibition of BACE-1

(76) -secretase (BACE-1, Sigma-Aldrich) inhibition studies were performed by employing a peptide mimicking APP sequence as substrate (Methoxycoumarin-Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys-dinitrophenyl, M-2420, Bachem, Germany). The following procedure was employed: 5 L of test compounds (or DMSO, if preparing a control well) were pre-incubated with 175 L of enzyme (in 20 mM sodium acetate containing 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) 0.1% w/v) for 1 hour at rt. The substrate (3 M, final concentration) was then added and left to react for 15 minutes at 37 C. The fluorescence signal was read at em=405 nm (exc=320 nm) using a Fluoroskan Ascent. The DMSO concentration in the final mixture maintained below 5% (v/v) guaranteed no significant loss of enzyme activity. The fluorescence intensities with and without inhibitor were compared and the percent inhibition due to the presence of test compounds was calculated. The background signal was measured in control wells containing all the reagents, except BACE-1 and subtracted. The % inhibition due to the presence of increasing test compound concentration was calculated by the following expression: 100(IF.sub.i/IF.sub.o100) where IF.sub.i and IF.sub.o are the fluorescence intensities obtained for BACE-1 in the presence and in the absence of inhibitor, respectively. Inhibition curves were obtained by plotting the % inhibition versus the logarithm of inhibitor concentration in the assay sample, when possible. The linear regression parameters were determined and the IC.sub.50 extrapolated (GraphPad Prism 4.0, GraphPad Software Inc.). To demonstrate inhibition of BACE-1 activity a peptido-mimetic inhibitor (-secretase inhibitor IV, Calbiochem, IC.sub.50=20 nM) was serially diluted into the reactions' wells. The results are illustrated in Table 1.

(77) Inhibition of GSK-3

(78) Human recombinant GSK-3 was purchased from Millipore (Millipore Iberica S.A.U.) The prephosphorylated polypeptide substrate was purchased from Millipore (Millipore Iberica SAU). Kinase-Glo Luminescent Kinase Assay was obtained from Promega (Promega Biotech Iberica, SL). ATP and all other reagents were from Sigma-Aldrich. Assay buffer contained 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.5), 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethylene glycol tetraacetic acid (EGTA), and 15 mM magnesium acetate. The method developed by Baki was followed [Baki et al. Assay and Drug Development Technologies 2007, 5(1),75-83] to analyze the inhibition of GSK-3. Kinase-Glo assays were performed in assay buffer using white 96-well plates. In a typical assay, 10 L (10 M) of test compound (dissolved in DMSO at 1 mM concentration and diluted in advance in assay buffer to the desired concentration) and 10 L (20 ng) of enzyme were added to each well followed by 20 L of assay buffer containing 25 M substrate and 1 M adenosine triphosphate (ATP). The final DMSO concentration in the reaction mixture did not exceed 1%. After a 30 minutes incubation at 30 C., the enzymatic reaction was stopped with 40 L of Kinase-Glo reagent. Glow-type luminescence was recorded after 10 minutes using a Fluoroskan Ascent multimode reader.

(79) The activity is proportional to the difference of the total and consumed ATP. The inhibitory activities were calculated on the basis of maximal kinase (AVERAGE positive) and luciferase activities (AVERAGE negative) measured in the absence of inhibitor and in the presence of reference compound inhibitor (SB415826 [Coghlan et al. Chem Biol 2000, 7, 793-803], Merck Millipore, IC.sub.50=54 nM) at total inhibition concentration (5 M), respectively.

(80) The linear regression parameters were determined and the IC.sub.50 extrapolated (GraphPad Prism 4.0, GraphPad Software Inc.). The results are illustrated in Table 1.

(81) TABLE-US-00001 TABLE 1 Example BACE-1 IC.sub.50 (M) GSK-3 IC.sub.50 (M) 2 50.31 6.61 40.71 2.70 3 16.05 0.64 7.11 0.37 4 8.13 12.57 5 12.55 15.67 6 36.83 9.85 4.34 0.63 9 n.a. 6.93 0.14 n.a.: not available

(82) Methods to Assess the Neuroprotective Activity of the Compounds of the Invention

(83) Primary Cell Cultures

(84) Astrocytes were prepared from neonatal (P2) rat cerebral cortex, as previously described by Luna-Medina et al. [Luna-Medina, R.; Cortes-Canteli, M.; Alonso, M.; Santos, A.; Martinez, A.; Perez-Castillo, A. Regulation of inflammatory response in neural cells in vitro by thiadiazolidinones derivatives through peroxisome proliferator-activated receptor gamma activation. J. Biol. Chem. 2005, 280, 21453-21462].

(85) Briefly, after removal of the meninges, the cerebral cortex was dissected, dissociated, and incubated with 0.25% trypsin/EDTA at 37 C. for 1 hour. After centrifugation, the pellet was washed 3 times with Hank's balanced salt solution (HBSS) (Gibco) and the cells were placed on noncoated flasks and maintained in HAMS/Dulbecco's modified eagle's medium (DMEM) (1:1) medium containing 10% of fetal bovine serum (FBS). After 15 days, the flasks were agitated on an orbital shaker for 4 hours at 240 rpm at 37 C., the supernatant was collected, centrifuged, and the cellular pellet containing the microglial cells resuspended in complete medium (HAMS/DMEM (1:1) containing 10% FBS) and seeded on uncoated 96-well plates. Cells were allowed to adhere for 2 hours, and the medium was removed to eliminate nonadherent oligodendrocytes. New fresh medium containing 10 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF) was added. The remaining astroglial cells adhered on the flasks were then trypsinized, collected, centrifugated, and plated onto 96-well plates with complete medium. The purity of cultures obtained by this procedure was >98% as determined by immunofluorescence with the OX42 (microglial marker) and the glial fibrillary acidic protein (GFAP, astroglial marker) antibodies.

(86) Nitrites Measurement

(87) An overexpression and/or over activity of GSK-3 results in increased level of microglia activation in different neurodegenerative diseases. Furthermore, GSK-3 is proved to regulate inflammatory tolerance in astrocytes [Beurel, E.; Jope, R. S. Glycogen synthase kinase-3 regulates inflammatory tolerance in astrocytes. Neuroscience 2011, 169, 1063-1070]. Accordingly, we explored whether these new BACE-1/GSK-3 dual inhibitors, might exert anti-inflammatory effect in cell-based assays.

(88) The potential anti-inflammatory activity of the selected compounds was tested by evaluating the production of nitrites from primary cultured glial cells. Primary cultures of astrocytes and microglia were incubated with the selected compounds (10 M) for 1 hour, and then cells were cultured for another 24 hours with a potent inflammatory agent, such as lipopolysaccharide (LPS) (10 g/mL). LPS is able to induce nitrite production and accumulation in the culture medium, which was assayed by the standard Griess reaction. Supernatants were collected from the media and mixed with an equal volume of Griess reagent (Sigma). Samples were then incubated at rt for 15 minutes and absorbance read using a plate reader at 492/540 nm.

(89) When primary astrocytes and microglial cells were stimulated with LPS, we observed a significant induction of nitrite production, which was generally reduced by triazinones treatment (FIGS. 2A, 2B). Particularly, the 6-(alkylamino) and 6-(phenylamino) compounds of examples 3, 6 and 9 normally showed a higher potency than the 6-amino triazinones, used as reference compounds (compounds A-C in Table 2 below). More interestingly, compounds of example 3 and 6, showing a balanced GSK-3/BACE-1 inhibitory profile, resulted the most potent among the tested derivatives, and they were able to restore nitrite production to basal level in astrocyte cells (FIG. 2A).

(90) Methods to Assess the Neurogenic Activity of the Compounds of the Invention

(91) Neurosphere Cultures

(92) Neurosphere (NS) cultures were derived from the hippocampus of adult rats and induced to proliferate using established passaging methods to achieve optimal cellular expansion according to published protocols [Ferron, S. R.; Andreu-Agullo, C.; Mira, H.; Sanchez, P.; Marques-Torrejon, M. A.; Farinas, I. A combined ex/in vivo assay to detect effects of exogenously added factors in neural stem cells. Nature Protoc. 2007, 2, 849-859]. Rats were decapitated, brains removed, and the hippocampus dissected as described [Morales-Garcia, J. A.; Luna-Medina, R.; Alfaro-Cervello, C.; Cortes-Canteli, M.; Santos, A.; Garcia-Verdugo, J. M.; Perez-Castillo, A. Peroxisome proliferator-activated receptor gamma ligands regulate neural stem cell proliferation and differentiation in vitro and in vivo. Glia 2011, 59, 293-307]. Briefly, cells were seeded into 12-well dishes and cultured in DMEM/F12 (1:1, Invitrogen) containing 10 ng/mL epidermal growth factor (EGF, Peprotech, London, UK), 10 ng/mL fibroblast growth factor (FGF, Peprotech), and B27 medium (Gibco). After 3 days in culture, NS were cultivated in the presence or absence of the indicated compounds (10 M) during a week. After that, NS from 10-day old cultures were plated for 72 hours onto 100 g/mL poly-L-lysine-coated coverslips in the absence of exogenous growth factors.

(93) Immunocytochemistry

(94) Neurogenesis is a crucial property for new AD-modifying drugs, since it confers the potential to increase endogenous regeneration as a repair mechanism in the damaged brain, and reduce neuronal loss and degeneration. Considering that GSK-3 inhibition is reported to regulate and increase neurogenesis [Lange, C.; Mix, E.; Frahm, J.; Glass, A.; Muller, J.; Schmitt, O.; Schmole, A. C.; Klemm, K.; Ortinau, S.; Hubner, R.; Frech, M. J.; Wree, A.; Rolfs, A. Small molecule GSK-3 inhibitors increase neurogenesis of human neural progenitor cells. Neurosci. Lett. 2011, 488, 36-40; Morales-Garcia, J. A.; Luna-Medina, R.; Alonso-Gil, S.; Sanz-SanCristobal, M.; Palomo, V.; Gil, C.; Santos, A.; Martinez, A.; Perez-Castillo, A. Glycogen Synthase Kinase 3 Inhibition Promotes Adult Hippocampal Neurogenesis in Vitro and in Vivo. ACS Chem. Neurosci. 2012, 3, 963-971], it was on interest to verify whether addition of compounds of example 3 and 6 to NS cultures of primary rat neural stem cells could regulate cell differentiation toward a neuronal phenotype. Thus, cells were processed for immunocytochemistry to detect neural markers, such as -tubulin and the microtule-associated protein (MAP2), which demonstrate the potential neurogenic effect of the compounds of the invention.

(95) Cells were processed for immunocytochemistry as previously described [Luna-Medina, R.; Cortes-Canteli, M.; Alonso, M.; Santos, A.; Martinez, A.; Perez-Castillo, A. Regulation of inflammatory response in neural cells in vitro by thiadiazolidinones derivatives through peroxisome proliferator-activated receptor gamma activation. J. Biol. Chem. 2005, 280, 21453-21462]. Briefly, at the end of the treatment period, NS cultures were grown on glass coverslips in 24-well cell culture plates. Cultures were then washed with phosphate-buffered saline (PBS) and fixed for 30 minutes with 4% paraformaldehyde at 25 C. and permeabilized with 0.1% Triton X-100 for 30 minutes at 37 C. After 1 hour incubation with the selected primary antibodies (polyclonal anti--tubulin (clone Tuj1; Abcam) and mouse monoclonal anti-MAP2 (Sigma)) cells were washed with phosphate-buffered saline and incubated with the corresponding Alexa-labeled secondary antibody (Alexa-488 and Alex-647 to reveal -tubulin and MAP2 respectively; Molecular Probes; Leiden, The Netherlands) for 45 minutes at 37 C. Later on, images were obtained using a TCS SP5 laser scanning spectral confocal microscope (Leica Microsystems). Confocal microscope settings were adjusted to produce the optimum signal-to-noise ratio. Dapi staining was used as a nuclear marker.

(96) As shown in FIG. 3, some neurogenic effects were observed when neurospheres were treated with triazinones. Particularly, when compared to control, the number of -tubulin positive cells (immature neuronal marker) was significantly increased in cultures treated with compound of example 3. Notably, compound of example 6 significantly amplified the number of MAP-2 positive cells (mature neuronal marker), whereas only a marginal increase was appreciable in cells treated with the reference compound A.

(97) Methods to Assess the Neurotoxic Effects and the Glial Modulatory Activity of the Compounds of the Invention

(98) Primary Cell Cultures

(99) Mixed glial cell cultures were prepared from cerebral cortex of newborn Wistar rats (Rattus norvegicus), as previously described [Monti, B.; D'Alessandro, C.; Farini, V.; Bolognesi, A.; Polazzi, E.; Contestabile, A.; Stirpe, F.; Battelli, M. G. In vitro and in vivo toxicity of type 2 ribosome-inactivating proteins lanceolin and stenodactylin on glial and neuronal cells. Neurotoxicology 2007, 28, 637-644]. Briefly, brain tissue was cleaned from meninges, trypsinized for 15 minutes at 37 C. and, after mechanical dissociation, the cell suspension was washed and plated on poly-L-lysine (Sigma-Aldrich, St. Louis, Mo., USA, 10 g/mL) coated flasks (75 cm.sup.2). Mixed glial cells were cultured for 10-13 days in Basal Medium Eagle (BME, Life technologies Ltd, Paisley, UK) supplemented with 100 mL/L heat-inactivated FBS (Life technologies), 2 mmol/L glutamine (Sigma-Aldrich) and 100 mol/L gentamicin sulfate (Sigma-Aldrich).

(100) Microglial cells were harvested from mixed glial cell cultures by mechanical shaking, resuspended in fresh medium without serum and plated on uncoated 35 mm dishes at a density of 1.510.sup.6 cells/1.5 mL medium/well for western blot analysis or on 96 wells at 110.sup.5 cells/0.2 mL medium/well for MTT assay. Cells were allowed to adhere for 30 minutes and then washed to remove non-adhering cells. These primary cultures are pure microglial cells, being more than 99% of adherent cells positive for isolectin B4 and negative for astrocyte and oligodendrocyte markers.

(101) For the preparation of purified astrocyte cultures, 10-day-old primary mixed glial cultures were vigorously shaken to detach microglia and oligodendrocytes growing on top of the astrocytic layer. The remaining adherent cells were detached with trypsin (0.25%)/EDTA (Life technologies), and the resulting cell suspension was left at rt in uncoated flasks to allow adherence of microglia to the plastic surface. After 20-30 minutes, non-adherent or loosely adherent cells were collected after mild shaking of the flasks, and the adhesion step was performed once more. Supernatants containing non-adherent cells were collected and centrifuged; cells were resuspended in fresh BME medium without serum (Life technologies) and reseeded on poly-L-lysine-coated (Sigma-Aldrich) 35 mm dishes at a density of 1.510.sup.6 cells/1.5 mL medium/well for western blot analysis or on 96 wells at 1105 cells/0.2 mL medium/well for MTT assay. Afterwards, western blot analysis was performed.

(102) Primary cultures of cerebellar granular neurons (CGNs) were prepared from 7 day-old rats of Wistar strain, as previously described [Monti, B.; D'Alessandro, C.; Farini, V.; Bolognesi, A.; Polazzi, E.; Contestabile, A.; Stirpe, F.; Battelli, M. G. In vitro and in vivo toxicity of type 2 ribosome-inactivating proteins lanceolin and stenodactylin on glial and neuronal cells. Neurotoxicology 2007, 28, 637-644]. Briefly, cells were dissociated from cerebella and plated on 35 mm dishes or in 24 well plates, previously coated with 10 g/mL poly-L-lysine, at a density of 2105 cells/cm.sup.2 in BME supplemented with 100 mL/L heat-inactivated FBS (Life technologies), 2 mmol/L glutamine, 100 mol/L gentamicin sulphate and 25 mmol/L KCl (all from Sigma-Aldrich). 16 hours later, 10 M cytosine arabino-furanoside (Sigma-Aldrich) was added to avoid glial proliferation. After 7 days in vitro (7 DIV), differentiated neurons were shifted to serum free BME medium containing 25 mmol/L KCl and used for MTT assay.

(103) MTT Assay

(104) The viability of the different brain cells in primary cultures exposed to increasing concentrations of compounds of example 3 and 6 (0, 10, 20 and 50 M) for 24 hours was evaluated by MTT assay [Monti, B.; D'Alessandro, C.; Farini, V.; Bolognesi, A.; Polazzi, E.; Contestabile, A.; Stirpe, F.; Battelli, M. G. In vitro and in vivo toxicity of type 2 ribosome-inactivating proteins lanceolin and stenodactylin on glial and neuronal cells. Neurotoxicology 2007, 28, 637-44]. Briefly, thiazolyl blue was added to culture medium at a final concentration of 0.1 mg/mL. Following a 20 minutes incubation for CGNs and 2 hours for glial cells at 37 C. in the dark, the MTT precipitate was dissolved in 0.1 M Tris-HCl buffer containing 5% Triton X-100 (all from Sigma-Aldrich) and absorbance was read at 570 nm in a multiplate spectophotometric reader (Bio-Rad).

(105) Importantly, compounds of example 3 and 6 did not display any toxicity in glial and neuronal cells up to 50 M (FIGS. 4a, 4b and 4c).

(106) Western Blot Analysis.

(107) The transformation from the neuroprotective M2 to the cytotoxic M1 glial cells is considered a crucial step in the progression of AD [Boche, D.; Perry, V. H.; Nicoll, J. A. Review: activation patterns of microglia and their identification in the human brain. Neuropathol. Appl. Neurobiol. 2013, 39, 3-18]. This M1/M2 phenotypic classification is based on a specific pattern of pro- or anti-inflammatory cytokines and receptors, whose release and expression is regulated, among others, by GSK-3 activity [Goldmann, T.; Prinz, M. Role of microglia in CNS autoimmunity. Clin. Dev. Immunol. 2013, 2013, 208093]. In this respect, GSK-3 activation has been reported to foster and maintain the pro-inflammatory state. On this basis, we evaluated the ability of compounds of example 3 and 6 to modulate the expression level of the inducible nitric oxide synthase (iNOS) as M1 marker, and the triggering receptor expressed on myeloid cells 2 (TREM2) as M2 marker on glial cells.

(108) Microglial and astrocyte cells exposed to LPS (100 ng/mL) in presence or absence of different concentrations (0, 5, 10 and 20 M) of compounds of example 3 and 6 for 24 hours were directly in ice-cold lysis buffer (Tris 50 mM, SDS 1%, protease inhibitor cocktail 0.05%) and protein content was determined by using the Lowry method [Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951, 193, 265-275]. 20 g of protein extracts were resuspended in 20 L of loading buffer (0.05M Tris-HCl pH 6.8; 40 g/L sodium dodecyl sulfate; 20 mL/L glycerol; 2 g/L bromophenol blue and 0.02 M dithiothreitol; all chemicals were from Sigma-Aldrich) and loaded onto 10% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE; Bio-Rad Laboratories SrL, Segrate, MI, IT). After electrophoresis and transfer onto nitrocellulose membranes (GE Healthcare Europe GmbH, Milano, MI, IT), membranes were blocked for 1 hour in 5% non-fat milk (Bio-Rad)/0.1% Tween-20 in phosphate buffered saline (PBS, Sigma-Aldrich), pH 7.4, and incubated overnight) at 4 C. with primary antibodies (rabbit polyclonal anti-iNOS or anti-TREM2 1:1000, both from Santa Cruz Biotechnology Inc., Santa Cruz, Calif., USA, or mouse monoclonal anti-actin, 1:2000, Sigma-Aldrich) in 0.1% Tween-20/PBS. Membranes were then incubated with an anti-rabbit or anti-mouse secondary antibody conjugated to horseradish peroxidase (1:2000; Santa Cruz), for 90 minutes at rt in 0.1% Tween-20/PBS. Labelled proteins were detected by using the enhanced chemiluminescence method (ECL; Bio-RAD). Densitometric analysis was performed by using Scion Image software from NIH.

(109) As shown in FIG. 5, when cultures of primary rat glial cells were stimulated with LPS, we observed the expected iNOS induction, which was reduced by a 24 hours co-treatment with compounds of example 3 and 6 in a dose-dependent manner (FIGS. 5a and b). Furthermore, along the same line, in microglia cells treated with LPS we observed a reduction of TREM2 expression, which was restored by 24 hours co-treatment with compounds of example 3 and 6 (FIG. 5c).

(110) TABLE-US-00002 TABLE 2 Entry Structure Compound A Compound B 0 Compound C