2,3-dihydroquinazolin-4(1H)-one derivatives for use in the treatment of viral infections

Abstract

Use of 2,3-dihydroquinazolin-4(1H)-one cyclic derivatives of formula (I) for the treatment of infection with viruses entering cells by endocytosis, especially filovirus such as Ebolavirus. ##STR00001##

Claims

1. A method of treating a viral infection in a subject where the virus is a filovirus or an adenovirus, the method comprising administering to the subject a therapeutically effective amount of a compound of formula (I): ##STR00079## or a pharmaceutically acceptable salt thereof, wherein: R.sup.1 is a hydrogen atom, a halogen atom or a C.sub.1-C.sub.3 alkoxy group; R.sup.2 is: a phenyl radical, a phenyl radical substituted by: a phenyl radical; a group CF.sub.3; a halogen atom; a group SO.sub.2-phenyl; a group SX or OX, X being a C.sub.1-C.sub.4 alkyl; a C.sub.1-C.sub.4 alkoxy group; a PEG substituent of general formula (CH.sub.2CH.sub.2O).sub.nR, in which n=1 to 10 and R is a hydrogen atom or a methyl group; or a group COOY or COY, Y being an hydrogen atom, a C.sub.1-C.sub.4 alkyl group, a phenyl radical or an allyl group, single or fused aromatic heterocyclic groups having 5 to 10 atoms and including one or two nitrogen atom(s), an adamantyl group, or an adamantyl group substituted by OH; R.sup.3 is: a thiophene group substituted by: a phenyl group; a phenyl group substituted by one or more of: a C.sub.1-C.sub.3 alkoxy group, and a group CN, NO.sub.2, COX and COOX, X being a C.sub.1-C.sub.4 alkyl radical; a group SY, Y being a C.sub.1-C.sub.4 alkyl group or a phenyl group; an aromatic heterocyclic group selected from the group consisting of furan, thiophene, pyrrole, pyrroline, pyrrolidine, dioxolane, oxazole, thiazole, imidazole, imidazoline, imidazolidine, pyrazole, isoxazole, isothiazole, pyran, pyridine, piperidine, dioxane, morpholine, pyridazine, pyrimidine and pyrazine, each of which is optionally substituted by at least one C.sub.1-C.sub.3 alkyl radical and/or a group CH.sub.2N.sub.3 thereof; and R.sup.4 is: a hydrogen atom or a methyl group.

2. The method according to claim 1, wherein the compound of formula (I) is selected from the group consisting of: 3-phenyl-2-(5-phenylthiophen-2-yl)-2,3-dihydroquinazolin-4(1H)-one, 2-(5-(methylthio)thiophen-2-yl)-3-phenyl-2,3-dihydroquinazolin-4(1H)-one, 2-([2,2-bithiophen]-5-yl)-3-phenyl-2,3-dihydroquinazolin-4(1H)-one, 3-phenyl-2-(5-(pyridin-2-yl)thiophen-2-yl)-2,3-dihydroquinazolin-4(1H)-one, 2-(5-(furan-2-yl)thiophen-2-yl)-3-phenyl-2,3-dihydroquinazolin-4(1H)-one, 2-(5-(2-methylthiazol-4-yl)thiophen-2-yl)-3-phenyl-2,3-dihydroquinazolin-4(1H)-one, 6-fluoro-2-(5-(2-methylthiazol-4-yl)thiophen-2-yl)-3-phenyl-2,3-dihydroquinazolin-4(1H)-one, 1-methyl-3-phenyl-2-(5-phenylthiophen-2-yl)-2,3-dihydroquinazolin-4(1H)-one, 1-methyl-3-phenyl-2-(5-(pyridin-2-yl)thiophen-2-yl)-2,3-dihydroquinazolin-4(1H)-one, 1-methyl-2-(5-(methylthio)thiophen-2-yl)-3-phenyl-2,3-dihydroquinazolin-4(1H)-one, 2-([2,2-bithiophen]-5-yl)-1-methyl-3-phenyl-2,3-dihydroquinazolin-4(1H)-one, 2-(5-(furan-2-yl)thiophen-2-yl)-1-methyl-3-phenyl-2,3-dihydroquinazolin-4(1H)-one, 1-methyl-2-(5-(2-methylthiazol-4-yl)thiophen-2-yl)-3-phenyl-2,3-dihydroquinazolin-4(1H)-one, 1-methyl-3-phenyl-2-(5-(phenylthio)thiophen-2-yl)-2,3-dihydroquinazolin-4(1H)-one, 2-(5-(3,4-dimethoxyphenyl)thiophen-2-yl)-1-methyl-3-phenyl-2,3-dihydroquinazolin-4(1H)-one, 1-methyl-2-(5-(3-nitrophenyl)thiophen-2-yl)-3-phenyl-2,3-dihydroquinazolin-4(1H)-one, 2-(5-(furan-3-yl)thiophen-2-yl)-1-methyl-3-phenyl-2,3-dihydroquinazolin-4(1H)-one, methyl 4-(5-(1-methyl-4-oxo-3-phenyl-1,2,3,4-tetrahydroquinazolin-2-yl)thiophen-2-yl)benzoate, 2-(5-(4-acetylphenyl)thiophen-2-yl)-1-methyl-3-phenyl-2,3-dihydroquinazolin-4(1H)-one, 1-methyl-3-phenyl-2-(5-(3,4,5-trimethoxyphenyl)thiophen-2-yl)-2,3-dihydroquinazolin-4(1H)-one, 1-methyl-2-(5-(4-methyl-3-nitrophenyl)thiophen-2-yl)-3-phenyl-2,3-dihydroquinazolin-4(1H)-one, 4-(5-(1-methyl-4-oxo-3-phenyl-1,2,3,4-tetra hydroquinazolin-2-yl)thiophen-2-yl)benzonitrile, 6-fluoro-1-methyl-2-(5-(2-methylthiazol-4-yl)thiophen-2-yl)-3-phenyl-2,3-dihydroquinazolin-4(1H)-one, 3-(2-benzoylphenyl)-1-methyl-2-(5-(2-methylthiazol-4-yl)thiophen-2-yl)-2,3-dihydroquinazolin-4(1H)-one, 3-(2-benzoylphenyl)-6-fluoro-1-methyl-2-(5-(2-methylthiazol-4-yl)thiophen-2-yl)-2,3-dihydroquinazolin-4(1H)-one, and a pharmaceutically acceptable salt thereof.

3. The method according to claim 1 wherein: R.sup.1 is a hydrogen atom, a halogen atom or a C.sub.1-C.sub.3 alkoxy group; R.sup.2 is a phenyl radical, a phenyl radical substituted by: a phenyl radical; a group CF.sub.3; a halogen atom, a group SO.sub.2-phenyl; a group SX or OX, X being a C.sub.1-C.sub.4 alkyl; a C.sub.1-C.sub.4 alkoxy group; a PEG substituent of general formula (CH.sub.2CH.sub.2O).sub.nR, in which n=1 to 10, and R is a hydrogen atom or a methyl group; or a group COOY or COY, Y being an hydrogen atom, a C.sub.1-C.sub.4 alkyl group, a phenyl radical or an allyl group; R.sup.3 is a thiophene substituted on the C5 carbon of the thiophene, the quinazolinone being linked to the C2 carbon of the thiophene by: a phenyl group; a phenyl group substituted by one or more of: a C.sub.1-C.sub.3 alkoxy group, a group CN, NO.sub.2, COX or COOX, X being a C.sub.1-C.sub.4 alkyl radical; a group SY, Y being a C.sub.1-C.sub.4 alkyl group or a phenyl group; or an aromatic heterocyclic group selected from the group consisting of furan, thiophene, pyrrole, pyrroline, pyrrolidine, dioxolane, oxazole, thiazole, imidazole, imidazoline, imidazolidine, pyrazole, isoxazole, isothiazole, pyran, pyridine, piperidine, dioxan, morpholine, pyridazine, pyrimidine, and pyrazine; and R.sup.4 is a hydrogen atom or a methyl group.

4. The method according to claim 1 wherein: R.sup.1 is a hydrogen or a fluorine atom; R.sup.2 is a phenyl group or a phenyl group substituted by a chlorine, iodine or fluorine atom, a OCH.sub.3, SCH.sub.3, SO.sub.2-phenyl or phenyl group; R.sup.3 is a thiophene group substituted by a phenyl, SCH.sub.3, 2-thiophene, 2-furan, 3-furan, 2-pyridine, 4-(2-Me thiazole) or a S-phenyl group, and R.sup.4 is a hydrogen atom or a methyl group.

5. The method according to claim 1, wherein R.sup.2 is a phenyl radical substituted by the group SX or OX, X being a methyl.

6. The method according to claim 1, wherein R.sup.2 is the phenyl radical substituted by a PEG substituent of general formula (CH.sub.2CH.sub.2O).sub.nR, in which n=1 and R is a hydrogen atom or a methyl group.

7. The method according to claim 1, wherein R.sup.2 is a phenyl radical substituted by a PEG substituent of general formula (CH.sub.2CH.sub.2O).sub.nR, in which n=1 to 10 and R is a methyl group.

8. The method according to claim 1, wherein R.sup.2 is pyridine.

9. The method according to claim 1, wherein R.sup.3 is substituted by a phenyl group substituted by a group CN, NO.sub.2, COX or COOX, X being a methyl.

10. The method according to claim 1, wherein the filovirus is Ebolavirus.

11. A method of treating a viral infection in a subject, comprising administering to the subject a therapeutically effective amount of A pharmaceutical composition comprising a compound of formula (I): ##STR00080## or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable vehicle, wherein: R.sup.1 is a hydrogen atom, a halogen atom or a C.sub.1-C.sub.3 alkoxy group; R.sup.2 is: a phenyl radical, a phenyl radical substituted by: a phenyl radical; a group CF.sub.3; a halogen atom; a group SO.sub.2-phenYl; a group SX or OX, X being a C.sub.1-C.sub.4 alkyl; a C.sub.1-C.sub.4 alkoxy group; a PEG substituent of general formula (CH.sub.2CH.sub.2O).sub.nR, in which n=1 to 10 and R is a hydrogen atom or a methyl group; or a group COOY or COY, Y being an hydrogen atom, a C.sub.1-C.sub.4 alkyl group, a phenyl radical or an allyl group, single or fused aromatic heterocyclic groups having 5 to 10 atoms and including one or two nitrogen atom(s), an adamantyl group, or an adamantyl group substituted by OH; R.sup.3 is: a thiophene group substituted by: a phenyl group: a phenyl group substituted by one or more of: a C.sub.3-C.sub.3 alkoxy group, and a group CN, NO.sub.2, COX and COOX, X being a C.sub.1-C.sub.4 alkyl radical; a group SY, Y being a C.sub.1-C.sub.4 alkyl group or a phenyl group; an aromatic heterocyclic group selected from the group consisting of furan, thiophene, pyrrole, pyrroline, pyrrolidine, dioxolane, oxazole, thiazole, imidazole, imidazoline, imidazolidine, pyrazole, isoxazole, isothiazole, pyran, pyridine, piperidine, dioxane, morpholine, pyridazine, pyrimidine and pyrazine, each of which is optionally substituted by at least one C.sub.1-C.sub.3 alkyl radical and/or a group CH.sub.2N.sub.3 thereof; and R.sup.4 is: a hydrogen atom or a methyl group, wherein the virus is selected from the group consisting of a filovirus and an adenovirus.

12. The method according to claim 11, wherein the filovirus is Ebolavirus.

13. The method according to claim 11, wherein the pharmaceutical composition is administered nasally, through the lung, orally or parenterally.

Description

EXAMPLES

(1) I. Synthesis of Compounds According to the Invention

(2) General Procedure for the Synthesis of Compounds with R.sup.4H

(3) To a solution of 2-aminobenzamides (4.71 mmol) in tetrahydrofuran (23 mL, 0.2M) was added the aldehyde (4.71 mmol) and p-toluene sulfonic acid (10 mol %, 0.5 mmol) in a sealed tube. The solution was warmed to 75 C. and stirred until complete consumption of starting materials (monitored by thin layer chromatography, usually overnight). After cooling to room temperature, silica was added and THF was evaporated. Purification by silica gel chromatography of the crude mixture yielded the expected compounds as solids.

(4) ##STR00077##
General Procedure for the Synthesis of Compounds with R.sup.4=Me

(5) To a mixture of NaH (1.5 equiv.) in anhydrous THF (0.3M) was added dropwise a solution of NH tetrahydroquinazolin-4-one derivatives (1 equiv.) in anhydrous THF (0.5M) at 0 C. After 30 min, iodomethane (1.1 equiv.) was added dropwise. After 10 min, the solution was warmed to room temperature and stirred for 3 h. The reaction was then quenched with a saturated solution of NaHCO.sub.3. The aqueous layer was then repeatedly extracted with CH.sub.2Cl.sub.2. The combined organic layers were then washed with a 1M solution of HCl (210 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuum. The purification by silica gel chromatography of the crude mixture yielded the expected compounds as solids.

(6) ##STR00078##

(7) The different cyclic molecules, yields and purities obtained are listed in the Table I below:

(8) TABLE-US-00002 TABLE I 2,3-dihydroquinazolin-4(1H)-one cyclic compounds (R.sup.4 = H) code compound Name yield LC/MS .sup.1H .sup.13C RN-1- 1 2-(3-Methoxyphenyl)-3-phenyl- 88% x x x 013 2,3-dihydroquinazolin-4(1H)-one RN-1- 2 2-(4-(Dimethylamino)phenyl)-3- 77% x x x 019 phenyl-2,3-dihydroquinazolin- 4(1H)-one RN-1- 3 2,3-Diphenyl-2,3- 84% x x x 021 dihydroquinazolin-4(1H)-one RN-1- 4 2-(4-Methoxyphenyl)-3-phenyl- 78% x x x 027 2,3-dihydroquinazolin-4(1H)-one RN-1- 5 2-(3-Methylthiophen-2-yl)-3- 85% x x x 066 phenyl-2,3-dihydroquinazolin- 4(1H)-one RN-1- 6 2-(4-Methylthiophen-2-yl)-3-phenyl- 65% x x x 067 2,3-dihydroquinazolin-4(1H)-one RN-1- 7 2-(5-Ethylthiophen-2-yl)-3-phenyl- 77% x x x 068 2,3-dihydroquinazolin-4(1H)-one RN-1- 8 3-((5R,7S)-3- 81% x x x 086 Hydroxyadamantan-1-yl)-2-(5- methylthiophen-2-yl)-2,3- dihydroquinazolin-4(1H)-one RN-1- 9 2-(5-Bromothiophen-2-yl)-3- 91% x x x 100 phenyl-2,3-dihydroquinazolin- 4(1H)-one RN-1- 10 2-(5-Methylthiophen-2-yl)-3- 34% x x x 118 (pyridin-3-yl)-2,3- dihydroquinazolin-4(1H)-one RN-1- 11 2-(5-Methylthiophen-2-yl)-3- 64% x x x 120 (pyridin-4-yl)-2,3- dihydroquinazolin-4(1H)-one RN-1- 12 2-(5-Methylthiophen-2-yl)-3- 53% x x x 132 (pyrimidin-4-yl)-2,3- dihydroquinazolin-4(1H)-one RN-1- 13 3-Phenyl-2-(5-phenylthiophen-2- 86% x x x 148 yl)-2,3-dihydroquinazolin- 4(1H)-one RN-1- 14 3-([1,1-Biphenyl]-4-yl)-2-(5- 73% x x x 149 methylthiophen-2-yl)-2,3- dihydroquinazolin-4(1H)-one RN-1- 15 2-(5-Methylthiophen-2-yl)-3-(3- 68% x x x 151 (trifluoromethyl)phenyl)-2,3- dihydroquinazolin-4(1H)-one RN-1- 16 8-Methyl-2-(5-methylthiophen- 99% x x x 162 2-yl)-3-phenyl-2,3- dihydroquinazolin-4(1H)-one RN-1- 17 2-(5-(Methylthio)thiophen-2-yl)- 82% x x 173 3-phenyl-2,3-dihydroquinazolin- 4(1H)-one RN-1- 18 2-([2,2-Bithiophen]-5-yl)-3- 86% x x x 174 phenyl-2,3-dihydroquinazolin- 4(1H)-one RN-1- 19 3-Phenyl-2-(5-(pyridin-2- 72% x x x 175 yl)thiophen-2-yl)-2,3- dihydroquinazolin-4(1H)-one RN-1- 20 2-(5-(Furan-2-yl)thiophen-2-yl)- 64% x x x 176 3-phenyl-2,3-dihydroquinazolin- 4(1H)-one RN-1- 21 2-(5-(2-Methylthiazol-4- 68% x x x 177 yl)thiophen-2-yl)-3-phenyl-2,3- dihydroquinazolin-4(1H)-one RN-2- 22 3-(4-Fluorophenyl)-2-(5- 80% x x x 120 methylthiophen-2-yl)-2,3- dihydroquinazolin-4(1H)-one RN-2- 23 3-(2-Fluorophenyl)-2-(5- 93% x x x 137 methylthiophen-2-yl)-2,3- dihydroquinazolin-4(1H)-one RN-2- 24 6-Methoxy-2-(5-methylthiophen- 89% x x x 161 2-yl)-3-phenyl-2,3- dihydroquinazolin-4(1H)-one RN-2- 25 6-Fluoro-2-(5-methylthiophen-2- 70% x x x 162 yl)-3-phenyl-2,3- dihydroquinazolin-4(1H)-one RN-2- 26 7-Fluoro-2-(5-methylthiophen-2- 80% x x x 163 yl)-3-phenyl-2,3- dihydroquinazolin-4(1H)-one RN-2- 27 3-(4-Bromophenyl)-2-(5- 80% x x x 177 methylthiophen-2-yl)-2,3- dihydroquinazolin-4(1H)-one RN-2- 28 3-(3-Bromophenyl)-2-(5- 91% x x x 178 methylthiophen-2-yl)-2,3- dihydroquinazolin-4(1H)-one RN-2- 29 3-(4-Chlorophenyl)-2-(5- 89% x x x 180 methylthiophen-2-yl)-2,3- dihydroquinazolin-4(1H)-one RN-2- 30 3-(3-Chlorophenyl)-2-(5- 91% x x x 181 methylthiophen-2-yl)-2,3- dihydroquinazolin-4(1H)-one RN-2- 31 3-(2-(Methylthio)phenyl)-2-(5- 79% x x x 183 methylthiophen-2-yl)-2,3- dihydroquinazolin-4(1H)-one RN-2- 32 3-(2-Chlorophenyl)-2-(5- 92% x x x 182 methylthiophen-2-yl)-2,3- dihydroquinazolin-4(1H)-one RN-2- 33 6-Iodo-2-(5-methylthiophen-2- 92% x 199 yl)-3-phenyl-2,3- dihydroquinazolin-4(1H)-one RN-3- 34 6-fluoro-2-(5-(2-methylthiazol- 36% x x x 121 4-yl)thiophen-2-yl)-3-phenyl- 2,3-dihydroquinazolin-4(1H)- one

(9) TABLE-US-00003 TABLE II 2,3-dihydroquinazolin-4(1H)-one compounds (R.sup.4 = methyl). code compound name yield LC/MS .sup.1H .sup.13C RN-1- 35 1-Methyl-2-(5-methylthiophen-2- 85% partir de x x x 181 yl)-3-phenyl-2,3- RN-1-001 dihydroquinazolin-4(1H)-one RN-1- 36 1-Methyl-3-phenyl-2-(5- 69% partir de x x x 186 phenylthiophen-2-yl)-2,3- RN-1-069 dihydroquinazolin-4(1H)-one RN-1- 37 1-Methyl-3-phenyl-2-(5-(pyridin- 97% partir de x x x 192 2-yl)thiophen-2-yl)-2,3- RN-1-101 dihydroquinazolin-4(1H)-one RN-1- 38 1-Methyl-2-(5- 91% partir de x x x 196 (methylthio)thiophen-2-yl)-3- RN-1-077 phenyl-2,3-dihydroquinazolin- 4(1H)-one RN-1- 39 2-([2,2-Bithiophen]-5-yl)-1- 95% partir de x x x 197 methyl-3-phenyl-2,3- RN-1-080 dihydroquinazolin-4(1H)-one RN-2- 40 2-(5-(Furan-2-yl)thiophen-2-yl)- 97% partir de x x x 001 1-methyl-3-phenyl-2,3- RN-1-104 dihydroquinazolin-4(1H)-one RN-2- 41 2-(5-Ethylthiophen-2-yl)-1- 84% partir de x x x 005 methyl-3-phenyl-2,3- RN-1-068 dihydroquinazolin-4(1H)-one RN-2- 42 1-Methyl-2-(5-(2-methylthiazol- 83% partir de x x x 015 4-yl)thiophen-2-yl)-3-phenyl-2,3- RN-1-105 dihydroquinazolin-4(1H)-one RN-2- 43 2-(5-Bromothiophen-2-yl)-1- 85% partir de x x x 016 methyl-3-phenyl-2,3- RN-1-100 dihydroquinazolin-4(1H)-one RN-2- 44 1-Methyl-3-phenyl-2-(5- 91% partir de x x x 019 (phenylthio)thiophen-2-yl)-2,3- RN-2-016 dihydroquinazolin-4(1H)-one RN-2- 45 2-(5-(3,4- 88% partir de x x x 029 Dimethoxyphenyl)thiophen-2-yl)- RN-2-016 1-methyl-3-phenyl-2,3- dihydroquinazolin-4(1H)-one RN-2- 46 1-Methyl-2-(5-(3- 90% partir de x x x 032 nitrophenyl)thiophen-2-yl)-3- RN-2-016 phenyl-2,3-dihydroquinazolin- 4(1H)-one RN-2- 47 2-(5-(Furan-3-yl)thiophen-2-yl)- 80% partir de x x x 034 1-methyl-3-phenyl-2,3- RN-2-016 dihydroquinazolin-4(1H)-one RN-2- 48 Methyl 4-(5-(1-methyl-4-oxo-3- 80% partir de x x x 049 phenyl-1,2,3,4- RN-2-016 tetrahydroquinazolin-2- yl)thiophen-2-yl)benzoate RN-2- 49 2-(5-(4-Acetylphenyl)thiophen-2- 89% partir de x x x 050 yl)-1-methyl-3-phenyl-2,3- RN-2-016 dihydroquinazolin-4(1H)-one RN-2- 50 1-Methyl-3-phenyl-2-(5-(3,4,5- 73% partir de x x x 053 trimethoxyphenyl)thiophen-2-yl)- RN-2-016 2,3-dihydroquinazolin-4(1H)-one RN-2- 51 1-methyl-2-(5-(4-methyl-3- 76% partir de x x x 057 nitrophenyl)thiophen-2-yl)-3- RN-2-016 phenyl-2,3-dihydroquinazolin- 4(1H)-one RN-2- 52 4-(5-(1-Methyl-4-oxo-3-phenyl- 78% partir de x x x 059 1,2,3,4-tetrahydroquinazolin-2- RN-2-016 yl)thiophen-2-yl)benzonitrile RN-3- 53 5-(1-Methyl-4-oxo-3-phenyl- 68% partir de x 012 1,2,3,4-tetrahydroquinazolin-2- RN-2-016 yl)thiophene-2-carbonitrile RN-3- 54 3-(2-chlorophenyl)-1-methyl-2- 67% x x x 066 (5-methylthiophen-2-yl)-2,3- dihydroquinazolin-4(1H)-one RN-3- 55 3-(2-iodophenyl)-1-methyl-2-(5- 46% x x x 067 methylthiophen-2-yl)-2,3- dihydroquinazolin-4(1H)-one RN-3- 56 3-(2-methoxyphenyl)-1-methyl-2- 28% x x x 068 (5-methylthiophen-2-yl)-2,3- dihydroquinazolin-4(1H)-one RN-3- 57 1-methyl-3-(2- 73% x x x 069 (methylthio)phenyl)-2-(5- methylthiophen-2-yl)-2,3- dihydroquinazolin-4(1H)-one RN-3- 58 3-(2-fluorophenyl)-1-methyl-2-(5- 51% x x x 070 methylthiophen-2-yl)-2,3- dihydroquinazolin-4(1H)-one RN-3- 59 3-([1,1-biphenyl]-2-yl)-1-methyl- 74% x x x 089 2-(5-methylthiophen-2-yl)-2,3- dihydroquinazolin-4(1H)-one RN-3- 60 1-methyl-2-(5-methylthiophen-2- 65% x x x 098 yl)-3-(2-(phenylsulfonyl)phenyl)- 2,3-dihydroquinazolin-4(1H)-one RN-3- 61 6-fluoro-1-methyl-2-(5-(2- 71% x x x 122 methylthiazol-4-yl)thiophen-2- yl)-3-phenyl-2,3- dihydroquinazolin-4(1H)-one
II. Measurement of the Protective Activity of Compounds Against Ebola

(10) Experimental Protocol

(11) Hela cells were seeded at a density of 50% into 96-well plates in MEM medium supplemented with 10% fetal bovine serum and incubated at 37 C., 5% CO.sub.2. The cells were allowed to recover overnight. Drugs were dissolved in DMSO to 20 mM each. The following day each drug was diluted 100-fold into MEM with serum, serially diluted and then added in equal volume to the cell medium in each well. The final concentration of each drug is indicated in the figures. After 1 hour preincubation virus was added to a multiplicity of infection of 0.1. The virus used was a recombinant Ebolavirus that encodes green fluorescent protein (Towner et al. 2005. Virology 332:20-7). This virus is replication competent and shows normal pathogenesis in animals but expresses GFP 24 hours after infection has taken place. For this reason, the experiment was halted at 24 hours after virus challenge which was sufficient time for a single round of virus infection and expression of the GFP infection marker. The cells were then fixed in 10% Protocol Formalin for 24 hours. Cell nuclei were stained with DAPI following standard methods. The infection was then analyzed by photographing each well using an epifluorescent microscope. Total and infected cells were counted by counting DAPI stained cells and green fluorescent cells respectively using Cell Profiler software. The fraction of infected cells was calculated by dividing the number of green fluorescent cells by the total number of cells.

(12) Results

(13) IC50 is defined as the concentration of each drug that results in 50% inhibition of infection. Inhibition of infection was calculated as shown below:

(14) $\mathrm{fraction}\mathrm{infection}=\left(\frac{\mathrm{number}\mathrm{of}\mathrm{green}\mathrm{fluorescent}\mathrm{cells}}{\mathrm{number}\mathrm{of}\mathrm{DAPI}\mathrm{stained}\mathrm{cell}\mathrm{nuclei}}\right)$$\mathrm{inhibition}\left(\%\right)=100\left(1-\frac{\mathrm{fraction}\mathrm{infection}\mathrm{after}\mathrm{drug}\mathrm{treatment}}{\mathrm{fraction}\mathrm{infection}\mathrm{without}\mathrm{treatment}}\right)$

(15) Each drug was tested at least four times and combined data used in calculations. Three parameter dose response curves were fitted to data by non-linear regression and IC50 values determined using Graphpad Prism 5 software (Table 3). The equation used is below:
Y=lower plateau+(upperlower plateau)(1+10.sup.(Xlog IC50))

(16) Table III presents the results (compound IC50 in micromolar concentrations): the most potent compounds present the lower IC.sub.50.

(17) TABLE-US-00004 TABLE III Evaluation of biological activities of compounds according to the present invention. molecule compound IC.sub.50 (M) control Retro-2.sup.cycl 42.5 RN-1-068 7 11.0 RN-1-148 13 4.2 RN-1-173 17 7.4 RN-1-174 18 4.7 RN-1-175 19 5.6 RN-1-176 20 5.4 RN-1-177 21 2.3

(18) The Figure represents inhibition of infection curves for quinazolinones represented in Table III. Hela cells were treated for 1 hour with the indicated concentrations of each compound. Each compound was initially dissolved in DMSO to 20 mM and then diluted to a starting concentration of 100 M and then by serial 2-fold dilution. This gave the highest concentration of DMSO as 0.5% (v/v) and was included as control and data shows the highest final concentration present with each compound at right (0.5% v/v) with serial 2-fold dilutions to the left. Three parameter dose response curves were fitted to data by non-linear regression using Graphpad Prism software (La Jolla, Calif., USA). The x-axis shows the concentrations of each compound as a Log.sub.2 scale. Two graphs are shown to simplify data presentation. The same DMSO data is shown for each. A single representative experiment is shown.

(19) All tested compounds of general formula (I) show a better activity compared to Retro-2.sup.cycl.