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COMPOUNDS FOR TREATING CYSTIC FIBROSIS

20170362239 · 2017-12-21

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention relates to compounds of Formula (I) or pharmaceutically acceptable enantiomers, salts, solvates or prodrugs thereof. The invention further relates to the use of the compounds of Formula (I) for the treatment of cystic fibrosis. The invention also relates to a process for manufacturing compounds of Formula (I).

    ##STR00001##

    Claims

    1-14. (canceled)

    15. A compound of general Formula Ib-1: ##STR00204## or a pharmaceutically acceptable enantiomer, salt, solvate or prodrug thereof, wherein: X represent CR.sup.9 or N; wherein R.sup.9 represents H; OH; halo; C1-C4-alkyl; SO.sub.mR.sup.10 wherein m is 0, 1, 2 or 3 and wherein R.sup.10 represents H, NH.sub.2 or C1-C4-alkyl; or NR.sup.11R.sup.12 wherein R.sup.11 and R.sup.12 represent each independently H or C1-C4-alkyl; R.sup.4 represents H; halo; NH.sub.2; OH; alkyloxy; aryloxy; heteroaryloxy; —O—CO—NH—CHPh.sub.2; NR.sup.13R.sup.14 wherein R.sup.13 and R.sup.14 represent each independently an alkyl group; —NH-Ph-CONH.sub.2; —NH—CH.sub.2—R.sup.15 wherein R.sup.15 represents COOH, —COOalkyl, alkyloxyaryl, arylalkyl or heteroarylalkyl; —NH—CHR.sup.16R.sup.17 wherein R.sup.16 represents hydroxymethyl or methyl and R.sup.17 represents hydroxymethyl, COOH or COOalkyl; or —NHCO—R.sup.18 wherein R.sup.18 represents H, alkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl or alkyloxyaryl; R.sup.5 represents H; halo; alkyl; OH; alkyloxy; aryloxy; arylalkyloxy; heteroarylalkyloxy; SO.sub.pR.sup.19 wherein p is 0, 1, 2 or 3 and wherein R.sup.19 represents H, NH.sub.2, alkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl or alkyloxyaryl; or NR.sup.20R.sup.21 wherein R.sup.20 and R.sup.21 represents each independently a group selected from H, alkyl, arylalkyl, heteroarylalkyl, aryl, heteroaryl, formyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, arylalkylcarbonyl, heteroarylalkylcarbonyl and alkyloxyarylcarbonyl; R.sup.6 represents H; arylalkyl wherein the aryl group is optionally substituted by one or more group selected from halo and carboxyl; heteroarylalkyl; heteroarylaminoalkyl wherein the heteroaryl group is optionally substituted by one or more group selected from halo and NH.sub.2; aminoalkyl; aryl substituted by one or more group selected from halo, alkyloxy, hydroxymethyl, CONH.sub.2, COOH, COOalkyl, SO.sub.3H and SO.sub.2NH.sub.2; provided that: R.sup.4 and R.sup.5 are not both H; R.sup.5 and R.sup.6 are not both H; R.sup.5 and R.sup.4 are not both Cl; and provided that compound of Formula I″ is not 4-(2-amino-6-hydroxy-9H-purin-9-yl)benzamide; N-2-phenethyl-9H-purine-2,6-diamine; 9-benzyl-9H-purin-6-amine; 9-(3-phenylpropyl)-9H-purin-6-amine; 4-(2-amino-6-hydroxy-9H-purin-9-yl)benzoic acid; 9-benzyl-2-chloro-N-(4-methoxybenzyl)-9H-purin-6-amine; 2-chloro-9H-purin-6-amine; 2-amino-9H-purin-6-ol; 3-(2-amino-6-hydroxy-9H-purin-9-yl)benzoic acid.

    16. The compound according to claim 15 of Formula Ib-1a ##STR00205## or a pharmaceutically acceptable enantiomer, salt, solvate or prodrug thereof, wherein R.sup.4, R.sup.5, R.sup.6 and R.sup.9 are as defined in claim 15.

    17. The compound according to claim 15, of Formula Ib-1a1 ##STR00206## or a pharmaceutically acceptable enantiomer, salt, solvate or prodrug thereof, wherein R.sup.4, R.sup.1 and R.sup.6 are as defined in claim 15.

    18. The compound according to claim 15, of Formula Ib-1b ##STR00207## or a pharmaceutically acceptable enantiomer, salt, solvate or prodrug thereof, wherein R.sup.4, R.sup.5 and R.sup.6 are as defined in claim 15.

    19. The compound according to claim 15, selected from the group consisting of 9-benzyl-2-chloro-9H-purin-6-amine; 9-benzyl-N.sup.2-(3-phenylpropyl)-9H-purine-2,6-diamine; 9-benzyl-N.sup.6-(4-methoxybenzyl)-N.sup.2-(3-phenylpropyl)-9H-purine-2,6-diamine; (S)-2-((9-benzyl-2-((3-phenylpropyl)amino)-9H-purin-6-yl)amino)propanoic acid; N.sup.2-(3-phenylpropyl)-9H-purine-2,6-diamine; 2-chloro-N-(3-phenylpropyl)-9H-purin-6-amine; 2-chloro-N-(4-methoxybenzyl)-9H-purin-6-amine; N.sup.6-(4-methoxybenzyl)-N.sup.2-(3-phenylpropyl)-9H-purine-2,6-diamine; N-(4-methoxybenzyl)-2-(3-phenylpropoxy)-9H-purin-6-amine; N-(4-methoxybenzyl)-2-phenethoxy-9H-purin-6-amine; (S)-tert-butyl 2-((2-chloro-9H-purin-6-yl)amino)propanoate; (S)-2-((2-chloro-9-(4-(hydroxymethyl)phenyl)-9H-purin-6-yl)amino)propanoic acid; (S)-tert-butyl 2-((2-chloro-9-(4-(hydroxymethyl)phenyl)-9H-purin-6-yl)amino)propanoate; 4-(6chloro-9H-purin-9-yl)benzamide; 4-(2-chloro-6-(diethylamino)-9H-purin-9-yl)benzamide; 4-(6-amino-2-chloro-9H-purin-9-yl)benzamide; 4-(6-amino-9H-purin-9-yl)benzamide; 4-(2,6-diamino-9H-purin-9-yl)benzamide; 4-(2-chloro-6-((1,3-dihydroxypropan-2-yl)amino)-9H-purin-9-yl)benzamide; (S)-2-((9-(4-carbamoylphenyl)-2chloro-9H-purin-6-yl)amino)-3-hydroxypropanoic acid; 2-((9-(4-carbamoylphenyl)-2-chloro-9H-purin-9-yl)amino)acetic acid; tert-butyl 2-((9-(4-carbamoylphenyl)-2-chloro-9H-purin-6-yl)amino)acetate; 4-(2-chloro-6-((4-methoxybenzyl)amino)-9H-purin-9-yl)benzamide; (S)-2-((9-(4-carbamoylphenyl)-2chloro-9H-purin-6-yl)amino)propanoic acid; (S)-tert-butyl 2-((9-(4-carbamoylphenyl)-2-chloro-9H-purin-6-yl)amino)propanoate; 4-(2-amino-6-((4-carbamoylphenyl)amino)-9H-purin-9-yl)benzamide; methyl 4-(2-acetamido-6-((benzhydrylcarbaramoyl)oxy)-9H-purin-9-yl)benzoate, methyl 4-(2-amino-6-hydroxy-9H-purin-9-yl)benzoate; methyl 4-(2-acetamido-6-hydroxy-9H-purin-9-yl)benzoate; 4-(7chloro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)benzamide; tert-butyl 2-((5-amino-3-(4 carbamoylphenyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-yl)amino)acetate; 2-(3-phenylpropoxy)-9H-purin-6-amine; (4-(6-amino-2-chloro-9H-purin-9-yl)phenyl)methanol; (S)-2-((9-(4-(hydroxymethyl)phenyl)-2-((3-phenylpropyl)amino)-9H-purin-6-yl)amino)propanoic acid; N6-(4-methoxybenzyl)-N2-(4-phenylbutyl)-9H-purine-2,6-diamine; 4-(6-((4-methoxybenzyl)amino)-2-((3-phenylpropyl)amino)-9H-purin-9-yl)benzamide; (4-(2,6-diamino-9H-purin-9-yl)phenyl)methanol; 4-(2-amino-6-methoxy-9H-purin-9-yl)benzamide; 4-(2-amino-9H-purin-9-yl)benzamide; 4-(7-chloro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)benzenesulfonamide; N-(6-chloro-9H-purin-2-yl)-3-phenylpropanamide; N2-(4-phenylbutyl)-9H-purine-2,6-diamine; 2-chloro-9H-purin-6-amine; 4-(6-chloro-9H-purin-9-yl)benzenesulfonamide; and 4-(6-chloro-8-methyl-9H-purin-9-yl)benzamide.

    20. A pharmaceutical composition comprising a compound of general Formula Ib-1 ##STR00208## or a pharmaceutically acceptable enantiomer, salt, solvate or prodrug thereof, wherein: X represent CR.sup.9 or N, wherein R.sup.9 represents H; OH; halo; C1-C4-alkyl; SO.sub.mR.sup.10 wherein m is 0, 1, 2 or 3 and wherein R.sup.10 represents H, NH.sub.2 or C1-C4-alkyl; or NR.sup.11R.sup.12 wherein R.sup.11 and R.sup.12 represent each independently H or C1-C4-alkyl; R.sup.4 represents H; halo; NH.sub.2; OH; alkyloxy; aryloxy; heteroaryloxy; —O—CO—NH—CHPh.sub.2; NR.sup.13R.sup.14 wherein R.sup.13 and R.sup.14 represent each independently an alkyl group; —NH-Ph-CONH.sub.2; —NH—CH.sub.2—R.sup.15 wherein R.sup.15 represents COOH, —COOalkyl, alkyloxyaryl, arylalkyl or heteroarylalkyl; —NH—CHR.sup.16R.sup.17 wherein R.sup.16 represents hydroxymethyl or methyl and R.sup.17 represents hydroxymethyl, COOH or COOalkyl; or —NHCO—R.sup.18 wherein R.sup.18 represents H, alkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl or alkyloxyaryl; R.sup.5 represents H; halo; alkyl; OH; alkyloxy; aryloxy; arylalkyloxy; heteroarylalkyloxy; SO.sub.pR.sup.19 wherein p is 0, 1, 2 or 3 and wherein R.sup.19 represents H, NH.sub.2, alkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl or alkyloxyaryl; or NR.sup.20R.sup.21 wherein R.sup.20 and R.sup.21 represents each independently a group selected from H, alkyl, arylalkyl, heteroarylalkyl, aryl, heteroaryl, formyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, arylalkylcarbonyl, heteroarylalkylcarbonyl and alkyloxyarylcarbonyl; R.sup.6 represents H; arylalkyl wherein the aryl group is optionally substituted by one or more group selected from halo and carboxyl; heteroarylalkyl; heteroarylaminoalkyl wherein the heteroaryl group is optionally substituted by one or more group selected from halo and NH.sub.2; aminoalkyl; aryl substituted by one or more group selected from halo, alkyloxy, hydroxymethyl, CONH.sub.2, COOH, COOalkyl, SO.sub.3H and SO.sub.2NH.sub.2; provided that: R.sup.4 and R.sup.5 are not both H; R.sup.5 and R.sup.6 are not both H; R.sup.5 and R.sup.4 are not both Cl; and provided that compound of Formula Ib-1 is not 4-(2-amino-6-hydroxy-9H-purin-9-yl)benzamide; N.sup.2-2-phenethyl-9H-purine-2,6-diamine; 9-benzyl-2-chloro-N-(4-methoxybenzyl)-9H-purin-6-amine; and at least one pharmaceutically acceptable carrier.

    21. A medicament comprising a compound of general Formula Ib-1 ##STR00209## or a pharmaceutically acceptable enantiomer, salt, solvate or prodrug thereof, wherein: X represent CR.sup.9 or N, wherein R.sup.9 represents H; OH; halo; C1-C4-alkyl; SO.sub.mR.sup.10 wherein m is 0, 1, 2 or 3 and wherein R.sup.10 represents H, NH.sub.2 or C1-C4-alkyl; or N.sup.11R.sup.12 wherein R.sup.11 and R.sup.12 represent each independently H or C1-C4-alkyl; R.sup.4 represents H; halo; NH.sub.2; OH; alkyloxy; aryloxy; heteroaryloxy; —O—CO—NH—CHPh.sub.2; NR.sup.13R.sup.14 wherein R.sup.13 and R.sup.14 represent each independently an alkyl group; —NH-Ph-CONH.sub.2; —NH—CH.sub.2—R.sup.15 wherein R.sup.15 represents COOH, —COOalkyl, alkyloxyaryl, arylalkyl or heteroarylalkyl; —NH—CHR.sup.16R.sup.17 wherein R.sup.16 represents hydroxymethyl or methyl and R.sup.17 represents hydroxymethyl, COOH or COOalkyl; or —NHCO—R.sup.18 wherein R.sup.18 represents H, alkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl or alkyloxyaryl; R.sup.5 represents H; halo; alkyl; OH; alkyloxy; aryloxy; arylalkyloxy; heteroarylalkyloxy; SO.sub.pR.sup.19 wherein p is 0, 1, 2 or 3 and wherein R.sup.19 represents H, NH.sub.2, alkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl or alkyloxyaryl; or NR.sup.20R.sup.21 wherein R.sup.20 and R.sup.21 represents each independently a group selected from H, alkyl, arylalkyl, heteroarylalkyl, aryl, heteroaryl, formyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, arylalkylcarbonyl, heteroarylalkylcarbonyl and alkyloxyarylcarbonyl; R.sup.6 represents H; arylalkyl wherein the aryl group is optionally substituted by one or more group selected from halo and carboxyl; heteroarylalkyl; heteroarylaminoalkyl wherein the heteroaryl group is optionally substituted by one or more group selected from halo and NH.sub.2; aminoalkyl; aryl substituted by one or more group selected from halo, alkyloxy, hydroxymethyl, CONH.sub.2, COOH, COOalkyl, SO.sub.3H and SO.sub.2NH.sub.2; provided that: R.sup.4 and R.sup.5 are not both H; R.sup.5 and R.sup.6 are not both H; R.sup.5 and R.sup.4 are not both Cl; and provided that compound of Formula I′ is not 4-(2-amino-6-hydroxy-9H-purin-9-yl)benzamide; N.sup.2-2-phenethyl-9H-purine-2,6-diamine; 9-benzyl-2-chloro-N-(4-methoxybenzyl)-9H-purin-6-amine.

    22. A method of treatment of a disease or disorder associated with chloride channels comprising the administration to a patient in need thereof of a compound of general Formula Ib-1: ##STR00210## or a pharmaceutically acceptable enantiomer, salt, solvate or prodrug thereof, wherein: X represent CR.sup.9 or N, wherein R.sup.9 represents H; OH; halo; C1-C4-alkyl; SO.sub.mR.sup.10 wherein m is 0, 1, 2 or 3 and wherein R.sup.10 represents H, NH.sub.2 or C1-C4-alkyl; or NR.sup.11R.sup.12 wherein R.sup.11 and R.sup.12 represent each independently H or C1-C4-alkyl; R.sup.4 represents H; halo; NH.sub.2; OH; alkyloxy; aryloxy; heteroaryloxy; —O—CO—NH—CHPh.sub.2; NR.sup.13R.sup.14 wherein R.sup.13 and R.sup.14 represent each independently an alkyl group; —NH-Ph-CONH.sub.2; —NH—CH.sub.2—R.sup.15 wherein R.sup.15 represents COOH, —COOalkyl, alkyloxyaryl, arylalkyl or heteroarylalkyl; —NH—CHR.sup.16R.sup.17 wherein R.sup.16 represents hydroxymethyl or methyl and R.sup.17 represents hydroxymethyl, COOH or COOalkyl; or —NHCO—R.sup.18 wherein R.sup.18 represents H, alkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl or alkyloxyaryl; R.sup.5 represents H; halo; alkyl; OH; alkyloxy; aryloxy; arylalkyloxy; heteroarylalkyloxy; SO.sub.pR.sup.19 wherein p is 0, 1, 2 or 3 and wherein R.sup.19 represents H, NH.sub.2, alkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl or alkyloxyaryl; or NR.sup.20R.sup.21 wherein R.sup.20 and R.sup.21 represents each independently a group selected from H, alkyl, arylalkyl, heteroarylalkyl, aryl, heteroaryl, alkyloxyaryl, formyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, arylalkylcarbonyl, heteroarylalkylcarbonyl and alkyloxyarylcarbonyl; R.sup.6 represents H; arylalkyl wherein the aryl group is optionally substituted by one or more group selected from halo and carboxyl; heteroarylalkyl; heteroarylaminoalkyl wherein the heteroaryl group is optionally substituted by one or more group selected from halo and NH.sub.2; aminoalkyl; aryl substituted by one or more group selected from halo, alkyloxy, hydroxymethyl, CONH.sub.2, COOH, COOalkyl, SO.sub.3H and SO.sub.2NH.sub.2; provided that: R.sup.4 and R.sup.5 are not both H; R.sup.5 and R.sup.6 are not both H; R.sup.5 and R.sup.4 are not both Cl.

    23. The method according to claim 22, wherein the disease is associated with the CFTR protein, preferably the disease is cystic fibrosis.

    24. The method according to claim 23, wherein cystic fibrosis is due to a mutation of the gene encoding the CFTR protein, preferably cystic fibrosis is due to the deletion of the phenylalanine residue at the position 508.

    Description

    BRIEF DESCRIPTION OF THE DRAWINGS

    [0400] FIG. 1 is a photograph of an immunoblot of F508del-CFTR in presence of compounds of the invention. B represents the B-band and C represents the C-band.

    [0401] FIG. 2 is a histogram showing the iodide efflux of HeLa cells expressing F508del-CFTR incubated with the compound 49 of the invention, Isolab, VX-809, or combinations thereof.

    [0402] FIG. 3 is a histogram showing the iodide efflux of HeLa cells expressing F508del-CFTR incubated with the compound 49 during 4, 8, 12, 24 or 48 hours.

    [0403] FIG. 4A is a histogram showing the activation of F508del-CFTR of cells incubated with the compound 49 of the invention during the time necessary for an optimal correction and then rinsed. The iodide efflux was measured after the time indicated on the x-axis.

    [0404] FIG. 4B is a histogram showing the activation of F508del-CFTR of cells incubated with the compound VX-508 during the time necessary for an optimal correction and then rinsed. The iodide efflux was measured after the time indicated on the x-axis.

    [0405] In all figures, the term “Cpd” means compound.

    EXAMPLES

    [0406] The present invention will be better understood with reference to the following examples. These examples are intended to representative of specific embodiments of the invention, and are not intended as limiting the scope of the invention.

    I. Chemistry

    1.1. Material

    [0407] All starting materials were commercially available research grade chemicals and used without further purification. They were purchased from Sigma-Aldrich, Fisher, Tokyo Chemical Industry, or Alfa Aesar. Reactions were monitored by analytical TLC on silica gel (Alugram Sil G/UV254) from Macherey-Nagel with fluorescent indicator UV254. LRMS were achieved with a NERMAG spectrometer for the FAB, DCI and EI techniques and with a ZQ Waters for the ESI. HRMS were obtained from the Mass Spectrometry Service of ICOA, at the University of Orleans, France. .sup.1H NMR spectra were recorded on a Bruker Avance 400 at 400 MHz using the residual solvent signal as internal standard. Chemical shifts are reported in ppm (parts per million) relative to the residual signal of the solvent, and the signals are described as singlet (s), broad singlet (bs), doublet (d), triplet (t), doublet of doublet (dd), quartet (q), sextuplet (sext), septuplet (sept), multiplet (m); coupling constants are reported in Hertz (Hz). Columns chromatography were performed on silica gel (MN Kieselgel 60, 0.063e0.2 mm/70e230 mesh, Machereye-Nagel) or on C18 reversed phase (Macherey-Nagel Polygoprep 60e50 C18). Flash chromatography were performed on Grace Reveleris apparatus using Grace Flash Cartridges.

    1.2. General Methods of Synthesis

    General Synthesis Protocol I.

    [0408] To a solution of copper(II) acetate (1 to 2 equivalents) in DMF (5 to 10 mL/mmol) were successively added the purine derivative (1 equivalent), boronic acid derivative (1 to 2 equivalents), triethylamine (2 equivalents) and eventually molecular sieves 3 Å. The solution was stirred for 24 to 96 h, then concentrated under reduced pressure.

    [0409] A saturated aqueous solution of ethylenediaminetetraacetic acid (EDTA) was added and the resulting mixture was extracted with dichloromethane or ethyl acetate. The organic layer was washed with water and brine, then dried over magnesium sulfate. After filtration and concentration under reduced pressure the crude product was purified by chromatography on silica gel to afford the pure compound.

    General Synthesis Protocol II.

    [0410] To a solution of the purine derivative (1 equivalent) dissolved in the amine derivative (20 equivalents) was added p-toluenesulfonic acid (0.1 equivalent). The solution was stirred for 5 min under microwave irradiation at 180° C. The crude product in solution in the amine derivative was purified by chromatography on silica gel.

    General Synthesis Protocol III.

    [0411] A solution of the purine derivative in TFA (10 mL/mmol) was refluxed for 24 h and then concentrated under reduced pressure. The crude product was purified by precipitation or by chromatography on silica gel.

    General Synthesis Protocol IV.

    [0412] To a suspension of purine derivative in THF (2 mL/mmol) were added TBAF (1 M in THF, 2 equivalents) and the aryl bromide derivative (2 equivalents). The resulting solution was stirred for 10 to 60 min, and then concentrated under reduced pressure. The crude product was purified by precipitation or by chromatography on silica gel.

    General Synthesis Protocol V.

    [0413] To a solution of the purine derivative in DMF (2 mL/mmol) were added aryl bromide derivative (1.1 equivalent) and K.sub.2CO.sub.3 (1.1 equivalent). The resulting suspension was stirred for 16 h, then the solid was filtrated and washed with methanol. The filtrate was concentrated under reduced pressure. The crude product was purified by chromatography on silica gel.

    General Synthesis Protocol VI.

    [0414] To a solution of the purine derivative in dichloromethane (40 mL/mmol) was added TFA (10 mL/mmol). The solution was stirred for 16 h, and then concentrated under reduced pressure. The crude product was purified by precipitation and by washing with water or by chromatography on silica gel.

    General Synthesis Protocol VII.

    [0415] To a solution of the purine derivative (1 equivalent) in DMF (20 mL/mmol) were added amino acid ester (1.5 equivalent) and triethylamine (3 equivalents). The solution was stirred for 8 h at 80° C., and then was concentrated under reduced pressure. The crude residue was dissolved in dichloromethane and purified by extraction with water, by precipitation or by chromatography on silica gel to afford pure compound.

    General Synthesis Protocol VIII.

    [0416] To a solution of purine derivative (1 equivalent) in water (10 mL/mmol) were added the amino acid (6 equivalents) and K.sub.2CO.sub.3 (6 equivalents). The solution was stirred for 8 h at 70° C. After cooling to room temperature, the solution was neutralized by addition of aqueous hydrochloric acid (5%). The resulting solution was purified by extraction with dichloromethane and then the aqueous layer was concentrated under reduced pressure. The crude residue was purified by reverse phase chromatography eluting with water/methanol.

    General Synthesis Protocol IX.

    [0417] To a solution of the purine derivative in acetonitrile (0.3 mL/mmol) or in a mixture methanol/dichloromethane (1/1 v/v, 0.3 mL/mmol) were added NaBH.sub.3CN (6 equivalents) and the corresponding aldehyde (8 equivalents). The solution was stirred for 3 days at room temperature and then concentrated under reduced pressure. The crude product was purified by chromatography on silica gel.

    General Synthesis Protocol X.

    [0418] To a solution of the chloropyrimidine derivative in a mixture water/dioxane (1/1 v/v, 4 mL/mmol) were added the amine (1 equivalent) and Na.sub.2CO.sub.3 (2 equivalents). The solution was refluxed overnight. After concentration under reduced pressure, the crude product was purified by precipitation.

    General Synthesis Protocol XI.

    [0419] To a suspension of the pyrimidine derivative in a mixture of dichloromethane/aqueous acetic acid (37%) (1/1 v/v, 10 mL/mmol) was added NaNO.sub.2 (1.1 equivalent). The suspension was stirred for 30 min at room temperature and then methanol (10 mL/mmol) was added. The suspension was stirred for 24 h at room temperature and the resulting precipitate was filtrated and washed with methanol to afford the pure product.

    General Synthesis Protocol XII.

    [0420] To a suspension of the pyrimidine derivative in trimethyl orthoformate (5 mL/mmol) was added ethanesulfonic acid (25 μL/mmol). The suspension was stirred for 1 h under microwave irradiation at 120° C. The resulting precipitate was filtrated and washed with methanol to afford the pure product.

    General Synthesis Protocol XIII.

    [0421] To a suspension of the purine derivative in the alcohol derivative (2.5 mL/mmol) was added NaOH (250 mg/mmol). The suspension was stirred for 3 h at 85° C. The crude product in solution in the alcohol derivative was purified by chromatography on silica gel to afford the pure product.

    General Synthesis Protocol XIV.

    [0422] To a suspension of the purine derivative in absolute ethanol (30 mL/mmol) was added aqueous hydrochloric acid (10%, v/v, 3 mL/mmol). The suspension was stirred for 24 h at room temperature and then the solvents were removed under reduced pressure to afford the pure product.

    General Synthesis Protocol XV.

    [0423] To a solution of the chloropyrimidine derivative in a mixture water/dioxane (1/1 v/v, 4 mL/mmol) was added the amine (3 equivalent). The solution was refluxed overnight. After cooling at room temperature, the crude product was purified by precipitation.

    General Synthesis Protocol XVI.

    [0424] To a solution of the nitrochloropyrimidine derivative in tetrahydrofuran (4 mL/mmol) were added the amine (1 equivalent) and NaHCO.sub.3 (1.1 equivalents). The solution was stirred at room temperature overnight. After concentration under reduced pressure, the crude product was purified by chromatography on silica gel to afford the pure product.

    General Synthesis Protocol XVII.

    [0425] To a solution of the chloropyrimidine derivative in a mixture water/dioxane (1/1 v/v, 2 mL/mmol) was added the amine (1 or 2 equivalents) and paratoluenesulfonic acid (0.5). The solution was refluxed overnight. After cooling at room temperature, the crude product was purified by precipitation.

    1.3. Synthesis of Intermediates

    Intermediate Int-1.

    [0426] This compound was synthesized through general synthesis protocol I from compound 9 and phenylboronic acid, and was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound Int-1 (42%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.99 (s, 1H, CH.sub.Ar), 7.68-7.62 (m, 2H, 2 CH.sub.Ar), 7.60-7.54 (m, 2H, 2 CH.sub.Ar), 7.49-7.43 (m, 1H, CH.sub.Ar), 7.38-7.32 (m, 2H, 2 CH.sub.Ar), 6.93-6.88 (m, 2H, 2 CH.sub.Ar), 6.35 (bs, 1H, NH), 4.79 (bs, 2H, CH.sub.2), 3.82 (s, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.19H.sub.17ClN.sub.5O: [M+H].sup.+ 366.11194, found 366.1116.

    Intermediate Int-2.

    [0427] This compound was synthesized through general synthesis protocol I from 2,6-dichloropurine and 4-(hydroxymethyl)phenylboronic acid, and was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound Int-2 (7%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.37 (s, 1H, CH.sub.Ar), 7.74-7.64 (m, 2H, 2 CH.sub.Ar), 7.64-7.58 (m, 2H, 2 CH.sub.Ar), 4.83 (s, 2H, CH.sub.2), 1.85 (bs, 1H, OH); HRMS (ESI) calc. for C.sub.12H.sub.9Cl.sub.2N.sub.4O: [M+H].sup.+ 295.01479, found 295.0150.

    Intermediate Int-3.

    [0428] This compound was synthesized through general synthesis protocol V from 2,6-dichloropurine and benzyle bromide, to afford pure compound Int-3 (19%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.04 (s, 1H, CH.sub.Ar), 7.45-7.36 (m, 3H, 3 CH.sub.Ar), 7.34-7.29 (m, 2H, 2 CH.sub.Ar), 5.41 (s, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.12H.sub.9Cl.sub.2N.sub.4: [M+H].sup.+, 279.01988, found 279.0201.

    Intermediate Int-4.

    [0429] This compound was synthesized through general synthesis protocol VII from compound Int-3 and L-alanine tert-butyl ester, was purified by extraction dichloromethane/water and then by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound Int-4 (47%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.65 (s, 1H, CH.sub.Ar), 7.42-7.32 (m, 3H, 3 CH.sub.Ar), 7.29-7.24 (m, 2H, 2 CH.sub.Ar), 6.41 (bs, 1H, NH), 5.31 (s, 2H, CH.sub.2), 4.81 (bs, 1H, CH), 1.53 (d, J=7.1 Hz, 3H, CH.sub.3), 1.48 (s, 9H, CH.sub.3); HRMS (ESI) calc. for C.sub.19H.sub.23ClN.sub.5O.sub.2: [M+H].sup.+ 388.15348, found 388.1538.

    Intermediate Int-5.

    [0430] This compound was synthesized through general synthesis protocol II from compound Int-4 and 3-phenylpropyl-1-amine, and was purified by chromatography on silica gel (eluent dichloromethane/ethyl acetate) to afford pure compound Int-5 (75%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.46 (s, 1H, CH.sub.Ar), 7.40-7.30 (m, 8H, 8 CH.sub.Ar), 7.27-7.21 (m, 2H, 2 CH.sub.Ar), 6.05 (bs, 1H, NH), 5.23 (s, 2H, CH.sub.2), 4.98-4.90 (m, 1H), 4.81 (bs, 1H, CH), 3.63-3.43 (m, 2H, CH.sub.2), 2.80-2.74 (m, 2H, CH.sub.2), 2.06-1.95 (m, 2H, CH.sub.2), 1.55 (d, J=7.1 Hz, 3H, CH.sub.3), 1.52 (s, 9H, 3 CH.sub.3); HRMS (ESI) calc. for C.sub.28H.sub.35N.sub.6O.sub.4: [M+H].sup.+ 487.28160, found 487.2811.

    Intermediate Int-6.

    [0431] This compound was synthesized through general synthesis protocol I from 6-diBoc-adenine and 4-carbamoylphenylboronic acid, and was purified by chromatography on silica gel (elution with dichloromethane/methanol) to afford pure compound Int-6 (39%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.95 (s, 1H, CH.sub.Ar), 8.42 (s, 1H, CH.sub.Ar), 8.06 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.92 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 6.12 (bs, 1H, NH), 5.76 (bs, 1H, NH), 1.50 (s, 18H, 6 CH.sub.3); HRMS (ESI) calc. for C.sub.22H.sub.27N.sub.6O.sub.5: [M+H].sup.+ 455.20374, found 455.2033.

    Intermediate Int-7.

    [0432] To a solution of 2,6-dichloropurine in ethyl acetate (3 mL/mmol) was added para-toluenesulfonic acid (2 mg/mmol). The solution was stirred at 50° C., and a solution of 3,4-dihydropyrane (1.3 equivalent) in ethyl acetate (0.5 mL/mmol) was added for 30 min. The solution was stirred at 50° C. for 15 min, cooled to room temperature, washed with water and brine. The organic layer was evaporated under reduced pressure to afford pure compound Int-7 (99%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.95 (s, 1H, CH.sub.Ar), 5.74 (dd, J=10.8 Hz, J=2.2 Hz, 1H, CH), 4.02 (m, 1H, CH), 3.74 (m, 1H, CH), 2.26 (m, 1H, CH), 1.98 (m, 2H, CH.sub.2), 1.43-1.75 (m, 3H, CH+CH.sub.2); HRMS (ESI) calc. for C.sub.10H.sub.11Cl.sub.2N.sub.4O: [M+H].sup.+ 273.03044, found 273.0303.

    Intermediate Int-8.

    [0433] This compound was synthesized through general synthesis protocol II from compound 15 and 3-phenylpropyl-1-amine, and was purified by chromatography on silica gel (eluent dichloromethane/ethyl acetate) to afford pure compound Int-8 (87%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.68 (s, 1H, CH.sub.Ar), 7.65 (d, J=8.4 Hz, 2H, 2 CH.sub.Ar), 7.48 (d, J=8.4 Hz, 2H, 2 CH.sub.Ar), 7.36-7.27 (m, 2H, 2 CH.sub.Ar), 7.25-7.20 (m, 3H, 3 CH.sub.Ar), 6.27 (bs, 1H, NH), 5.12-5.00 (m, 1H, CH), 4.75 (s, 2H, CH.sub.2), 3.59-3.38 (m, 2H, CH.sub.2), 2.78-2.71 (m, 2H, CH.sub.2), 2.02-1.92 (m, 2H, CH.sub.2), 1.57 (d, J=7.1 Hz, 3H, CH.sub.3), 1.53 (s, 9H, 3 CH.sub.3); HRMS (ESI) calc. for C.sub.28H.sub.35N.sub.6O.sub.3: [M+H].sup.+ 503.27652, found 503.2767.

    Intermediate Int-9.

    [0434] This compound was synthesized through general synthesis protocol X from 4-aminobenzamide and 2,5-diamino-4,6-dichloropyrimidine to afford pure compound Int-9 (97%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 9.28 (bs, 1H, NH), 7.89 (d, J=8.9 Hz, 2H, 2 CH.sub.Ar), 7.84 (d, J=8.9 Hz, 2H, 2 CH.sub.Ar), 7.24 (bs, 1H, NH.sub.2), 4.81 (bs, 5H, NH.sub.2); HRMS (ESI) calc. for C.sub.11H.sub.12ClN.sub.6O: [M+H].sup.+ 279.07556, found 279.0755.

    Intermediate Int-10.

    [0435] This compound was synthesized through general synthesis protocol XII from compound Int-9 (11%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.62 (s, 1H, CH.sub.Ar), 8.09 (bs, 1H, NH.sub.2), 8.06 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.98 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.50 (bs, 1H, NH.sub.2), 7.10 (s, 2H, NH.sub.2); HRMS (ESI) calc. for C.sub.12H.sub.10ClN.sub.6O: [M+H].sup.+289.05991, found 289.0599.

    Intermediate Int-11.

    [0436] Compound 106 (1 equivalent) was suspended in NH.sub.4OH 30% (20 mL/mmol), heated to 40° C. for 40 min, concentrated and the resulting solid was suspended in boiling methanol (50 mL) then filtrated. This operation was done twice to afford pure compound Int-11 as an orange solid (59%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 10.89 (bs, 1H, NH), 8.69 (bs, 2H, NH.sub.2), 8.08 (s, 1H, CH.sub.Ar), 7.94 (bs, 1H, NH.sub.2), 7.88 (d, J=8.1 Hz, 2H, 2 CH.sub.Ar), 7.74 (d, J=8.1 Hz, 2H, 2 CH.sub.Ar), 7.33 (bs, 1H, NH.sub.2); HRMS (ESI) calc. for C.sub.11H.sub.11N.sub.6O.sub.3: [M+H].sup.+ 275.08871, found 275.0889.

    1.4. Synthesis of Compounds

    [0437] Compounds 8, 29, 31, 32, II-1 and II-2 were synthesized through specific protocol described hereafter.

    [0438] Compound 1. This compound was synthesized through general synthesis protocol V from adenine and 1-bromo-3-phenylpropane, and was purified by chromatography on silica gel (elution with dichloromethane/methanol) to afford pure compound 1 (57%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.39 (bs, 1H, CH.sub.Ar), 7.76 (bs, 1H, CH.sub.Ar), 7.36-7.14 (m, 5H, 5 CH.sub.Ar), 5.63 (bs, 2H, NH.sub.2), 4.23 (t, J=7.2 Hz, 2H, CH.sub.2), 2.69 (t, J=7.3 Hz, 2H, CH.sub.2), 2.28 (m, 2H, CH.sub.2); MS (ESI [+]) m/z 254 [M+H].sup.+.

    [0439] Compound 2. This compound was synthesized through general synthesis protocol III from compound 4, and was purified by chromatography on silica gel (elution with dichloromethane/methanol) to afford pure compound 2 (50%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.25 (s, 1H, CH.sub.Ar), 7.78 (bs, 2H, NH.sub.2), 7.26-7.38 (m, 5H, 5 CH.sub.Ar), 5.33 (s, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.12H.sub.11ClN.sub.5: [M+H].sup.+ 260.06975, found 260.0703.

    [0440] Compound 3. This compound was synthesized through general synthesis protocol III from compound 5, and was purified by chromatography on silica gel (elution with dichloromethane/methanol) to afford pure compound 3 (98%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.04 (s, 1H, CH.sub.Ar), 7.78 (bs, 1H, NH), 7.35-7.15 (m, 10H, CH.sub.Ar), 7.06 (bs, 2H, NH.sub.2), 5.20 (bs, 2H, CH.sub.2), 2.67-2.59 (m, 2H, CH.sub.2), 1.90-1.80 (m, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.21H.sub.23N.sub.6: [M+H].sup.+ 359.19787, found 359.1979.

    [0441] Compound 4. This compound was synthesized through general synthesis protocol IV from compound 9 benzyle bromide, and was purified by precipitation in methanol to afford pure compound 4 (78%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.59 (s, 1H, CH.sub.Ar), 7.28-7.37 (m, 7H, 7 CH.sub.Ar), 6.88 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 6.22 (bs, 1H, NH), 5.32 (s, 2H, CH.sub.2), 4.75 (bs, 2H, CH.sub.2), 3.81 (s, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.20H.sub.19ClN.sub.5O: [M+H].sup.+ 380.12726, found 380.1281.

    [0442] Compound 5. This compound was synthesized through general synthesis protocol II from compound 4 and 3-phenylpropyl-1-amine, and was purified by precipitation in methanol to afford pure compound 5 (67%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.38 (s, 1H, CH.sub.Ar), 7.15-7.35 (m, 12H, 12 CH.sub.Ar), 6.86 (d, J=8.6 Hz, 2H, 2 CH.sub.Ar), 5.74 (bs, 1H, NH), 5.20 (s, 2H, CH.sub.2), 4.87 (bs, 1H, NH), 4.71 (bs, 2H, CH.sub.2), 3.80 (s, 3H, CH.sub.3), 3.50 (q, J=7.0 Hz, 2H, CH.sub.2), 2.73 (t, J=7.0 Hz, 2H, CH.sub.2), 1.96 (quint., J=7.0 Hz, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.29H.sub.31N.sub.6O: [M+H].sup.+ 479.25539, found 479.2553.

    [0443] Compound 6. This compound was synthesized through general synthesis protocol VI from compound Int-5 to afford pure compound 6 (99%). .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.72 (bs, 1H, CH.sub.Ar), 7.32-7.10 (m, 10H, 10 CH.sub.Ar), 5.22 (s, 2H, CH.sub.2), 4.71 (bs, 1H, CH), 3.39 (t, J=7.0 Hz, 2H, CH.sub.2), 2.70-2.62 (m, 2H, CH.sub.2), 1.96-1.84 (m, 2H, CH.sub.2), 1.55 (d, J=7.1 Hz, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.24H.sub.27N.sub.6O.sub.2: [M+H].sup.+ 431.21900, found 431.2195.

    [0444] Compound 7. This compound was synthesized through general synthesis protocol III from compound 10, and was purified by chromatography on silica gel (elution with dichloromethane/methanol) to afford pure compound 7 (79%). .sup.1H NMR (400 MHz, d.sub.6-DMSO, 60° C.) δ 8.05 (s, 1H, CH.sub.Ar), 7.94 (bs, 2H, NH.sub.2), 7.16-7.30 (m, 5H, 5 CH.sub.Ar), 3.36 (t, J=7.0 Hz, 2H, CH.sub.2), 2.66 (t, J=7.0 Hz, 2H, CH.sub.2), 1.89 (quint., J=7.0 Hz, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.14H.sub.17N.sub.6: [M+H].sup.+ 269.15092, found 269.1507.

    [0445] Compound 8. To a solution of 2,6-dichloropurine (1 equivalent) in DMF (4 mL/mmol) were added 3-phenylpropylamine (1.2 equivalent) and diisopropylethylamine (2 equivalents). The solution was stirred for 6 h at 80° C., and then evaporated under reduced pressure. The crude product was dissolved in dichloromethane and purified by extraction with water to afford pure compound 8 (37%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 12.95 (s, 1H, NH), 8.16 (bs, 1H, NH), 8.11 (s, 1H, CH.sub.Ar), 7.33-7.12 (m, 5H, 5 CH.sub.Ar), 3.44 (s, 2H, CH.sub.2), 2.69-2.61 (m, 2H, CH.sub.2), 1.97-1.83 (m, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.14H.sub.15ClN.sub.5: [M+H].sup.+ 288.10105, found 288.1011.

    [0446] Compound 9. To a solution of 2,6-dichloropurine in DMF (1.5 mL/mmol) were added 4-methoxybenzylamine (1.1 equivalent) and triethylamine (2 equivalents). The solution was stirred overnight at 80° C. After concentration under reduced pressure, the crude product was purified by precipitation in dichloromethane to afford pure compound 9 (95%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 13.06 (bs, 1H, NH), 8.62 (bs, 1H, NH), 8.12 (s, 1H, CH.sub.Ar), 7.28 (d, J=8.3 Hz, 2H, 2 CH.sub.Ar), 6.88 (d, J=8.3 Hz, 2H, 2 CH.sub.Ar), 5.08 (bs, 0.4H, CH.sub.2); 4.56 (s, 1.6H, CH.sub.2), 3.71 (s, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.13H.sub.13ClN.sub.5O: [M+H].sup.+ 290.08077, found 290.0803.

    [0447] Compound 10. This compound was synthesized through general synthesis protocol II from compound 9 and 3-phenylpropyl-1-amine, and was purified by chromatography on silica gel (elution with dichloromethane/methanol) to afford pure compound 10 (6%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 12.12 (bs, 1H, NH), 7.62 (s, 1H, CH.sub.Ar), 7.59 (bs, 1H, NH), 7.14-7.28 (m, 7H, 7 CH.sub.Ar), 6.82 (d, J=8.5 Hz, 2H, 2 CH.sub.Ar), 6.25 (bs, 1H, NH), 4.54 (bs, 2H, CH.sub.2), 3.69 (s, 3H, OCH.sub.3), 3.23 (q, J=7.0 Hz, 2H, CH.sub.2), 2.60 (t, J=7.0 Hz, 2H, CH.sub.2), 1.80 (quint., J=7.0 Hz, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.22H.sub.25N.sub.6O: [M+H].sup.+ 389.20844, found 389.2092.

    [0448] Compound 11. This compound was synthesized through general synthesis protocol XIV from compound 99 (35%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 9.15 (bs, 1H, NH), 8.46 (s, 1H, CH.sub.Ar), 7.16-7.31 (m, 7H, 7 CH.sub.Ar), 6.88 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 4.63 (s, 2H, CH.sub.2), 4.35 (s, 2H, CH.sub.2), 3.72 (s, 3H, CH.sub.3), 2.71 (m, 2H, CH.sub.2), 2.03 (m, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.22H.sub.24N.sub.5O.sub.2: [M+H].sup.+ 390.19245, found 390.1922.

    [0449] Compound 12. This compound was synthesized through general synthesis protocol XIV from compound 98 (100%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 9.36 (bs, 1H, NH), 8.56 (s, 1H, CH.sub.Ar), 7.21-7.33 (m, 7H, 7 CH.sub.Ar), 6.89 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 4.67 (bs, 2H, CH.sub.2), 4.57 (t, J=6.9 Hz, 2H, CH.sub.2), 3.72 (s, 3H, CH.sub.3), 3.04 (t, J=6.9 Hz, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.21H.sub.22N.sub.5O.sub.2: [M+H].sup.+ 376.17680, found 376.1769.

    [0450] Compound 14. This compound was obtained in two steps through general synthesis protocol VII from compound Int-2 and 1-alanine tert-butyl ester (purification by extraction dichloromethane/water) then through general synthesis protocol VI to afford pure compound 14 (64%). .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.39 (s, 1H, CH.sub.Ar), 7.74 (d, J=8.4 Hz, 2H, 2 CH.sub.Ar), 7.60 (d, J=8.6 Hz, 2H, 2 CH.sub.Ar), 4.72 (s, 2H, CH.sub.2), 1.61 (t, J=7.0 Hz, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.15H.sub.15ClN.sub.5O.sub.3: [M+H].sup.+ 348.08579, found 348.0856.

    [0451] Compound 15. This compound was synthesized through general synthesis protocol VII from compound Int-2 and 1-alanine tert-butyl ester, and was purified by extraction to afford pure compound 15 (97%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.02 (s, 1H, CH.sub.Ar), 7.64 (d, J=8.5 Hz, 2H, 2 CH.sub.Ar), 7.55 (d, J=8.5 Hz, 2H, 2 CH.sub.Ar), 6.48 (bs, 1H, NH), 4.83 (bs, 1H, CH), 4.78 (s, 2H, CH.sub.2), 1.57 (d, J=7.0 Hz, 3H, CH.sub.3), 1.50 (s, 9H, 3 CH.sub.3); HRMS (ESI) calc. for C.sub.19H.sub.23ClN.sub.5O.sub.3: [M+H].sup.+ 404.14839, found 404.1487.

    [0452] Compound 16. This compound was synthesized through general synthesis protocol XII from compound 49 (56%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 9.20 (s, 1H, CH.sub.Ar), 8.89 (s, 1H, CH.sub.Ar), 8.13 (bs, 1H, NH), 8.12 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 8.05 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.54 (bs, 1H, NH); HRMS (ESI) calc. for C.sub.12H.sub.9ClN.sub.5O: [M+H].sup.+ 274.04901, found 274.0487.

    [0453] Compound 17. This compound was obtained by addition of triethylamine on compound 79 during reaction through general synthesis protocol I. It was purified by extraction dichloromethane/water and then by chromatography on silica gel (elution with dichloromethane/methanol) to afford pure compound 17 (1%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.63 (s, 1H, CH.sub.Ar), 8.13-8.05 (m, 3H, 2 CH.sub.Ar+NH), 7.90 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.51 (s, 1H, NH), 4.21 (bs, 2H, CH.sub.2), 3.69 (bs, 2H, CH.sub.2), 1.24 (bs, 6H, 2 CH.sub.3); HRMS (ESI) calc. for C.sub.16H.sub.18ClN.sub.6O: [M+H].sup.+ 345.12251, found 345.1223.

    [0454] Compound 18. This compound was synthesized through general synthesis protocol III from compound 25, and was purified by precipitation in a mixture dichloromethane and methanol to afford pure compound 18 (87%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.65 (s, 1H, CH.sub.Ar), 8.10 (bs, 1H, NH), 8.07 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.94 (bs, 2H, NH.sub.2), 7.93 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.50 (bs, 1H, NH); HRMS (ESI) calc. for C.sub.12H.sub.10ClN.sub.6O: [M+H].sup.+ 289.05991, found 289.0602.

    [0455] Compound 19. This compound was synthesized through general synthesis protocol VI from compound Int-6 to afford pure compound 19 (99%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.83 (s, 1H, CH.sub.Ar), 8.41 (bs, 3H, 2 CH.sub.Ar+NH.sub.2), 8.19-8.06 (m, 3H, CH.sub.Ar+NH), 8.00 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.53 (bs, 1H, NH); HRMS (ESI) calc. for C.sub.12H.sub.11N.sub.6O: [M+H].sup.+ 255.09888, found 255.0985.

    [0456] Compound 20. This compound was obtained in two steps through general synthesis protocol I and VI from 2,6-bis(diBoc)-diaminopurine, and purified by precipitation in dichloromethane to afford pure compound 20 (4%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.58 (bs, 2H, NH.sub.2), 8.47 (s, 1H, CH.sub.Ar), 8.10 (bs, 1H, NH), 8.06 (d, J=8.6 Hz, 2H, 2 CH.sub.Ar), 7.91 (d, J=8.6 Hz, 2H, 2 CH.sub.Ar), 7.52 (bs, 1H, NH), 7.24 (bs, 2H, NH.sub.2); HRMS (ESI) calc. for C.sub.12H.sub.12N.sub.7O: [M+H].sup.+ 270.10978, found 270.1096.

    [0457] Compound 21. This compound was synthesized through general synthesis protocol VII from compound 79 and 2-aminopropane-1,3-diol to afford pure compound 21 (87%). .sup.1H NMR (400 MHz, d.sub.6-DMSO, 60° C.) δ 8.60 (s, 1H, CH.sub.Ar), 8.12-8.05 (m, 2H, 2 CH.sub.Ar), 7.99-7.69 (m, 3H, 2 CH.sub.Ar+NH), 7.51 (d, J=8.6 Hz, 1H, NH), 7.31 (bs, 1H, NH), 4.59 (t, J=5.3 Hz, 2H, 2 OH), 4.31 (bs, 1H, CH), 3.64 (t, J=5.7 Hz, 4H, CH.sub.2); HRMS (ESI) calc. for C.sub.15H.sub.16ClN.sub.6O.sub.3: [M+H].sup.+ 363.09669, found 363.0967.

    [0458] Compound 22. This compound was synthesized through general synthesis protocol VIII from compound 79 and 1-serine to afford pure compound 22 (82%). .sup.1H NMR (400 MHz, D.sub.2O) δ 7.86 (s, 1H, CH.sub.Ar), 7.34 (d, J=8.1 Hz, 2H, 2 CH.sub.Ar), 7.08 (d, J=8.1 Hz, 2H, 2 CH.sub.Ar), 4.28 (bs, 1H, CH), 3.90 (bs, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.15H.sub.14ClN.sub.6O.sub.4: [M+H].sup.+ 377.07608, found 377.0759.

    [0459] Compound 23. This compound was synthesized in two steps through general synthesis protocol VII from compound 79 and glycine tert-butyl ester (purification by extraction dichloromethane/water) and through general synthesis protocol VI to afford pure compound 23 (68%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 12.71 (bs, 1H, COOH), 8.70 (s, 1H, CH.sub.Ar), 8.64 (bs, 0.8H, NH), 8.46 (bs, 0.2H, NH), 8.14-8.04 (m, 3H, 2 CH.sub.Ar+NH), 7.93 (d, J=8.6 Hz, 2H, 2 CH.sub.Ar), 7.51 (s, 1H, NH), 4.56 (d, J=6.5 Hz, 0.4H, CH.sub.2), 4.11 (d, J=6.1 Hz, 1.6H, CH.sub.2); HRMS (ESI) calc. for C.sub.14H.sub.12ClN.sub.6O.sub.3: [M+H].sup.+ 347.06539, found 347.0651.

    [0460] Compound 24. This compound was synthesized through general synthesis protocol VII from compound 79 and glycine tert-butyl ester, and was purified by chromatography on silica gel (elution with dichloromethane/methanol) to afford pure compound 24 (69%). .sup.1H NMR (400 MHz, d.sub.6-DMSO, 60° C.) δ 8.63 (s, 1H, CH.sub.Ar), 8.45 (bs, 1H, NH), 8.10-8.06 (m, 2H, 2 CH.sub.Ar), 7.93 (d, J=8.6 Hz, 3H, 2 CH.sub.Ar+NH), 7.32 (bs, 1H, NH), 4.09 (bs, 2H, CH.sub.2), 1.44 (s, 9H, 3 CH.sub.3); HRMS (ESI) calc. for C.sub.18H.sub.20ClN.sub.6O.sub.3: [M+H].sup.+ 403.12799, found 403.1279.

    [0461] Compound 25. This compound was synthesized through general synthesis protocol I from compound 9 and 4-carbamoylphenylboronic acid, and was purified by chromatography on silica gel (elution with dichloromethane/methanol) to afford pure compound 25 (7%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.96 (t, J=6.0 Hz, 1H, NH), 8.66 (s, 1H, CH.sub.Ar), 8.09 (s, 1H, NH), 8.08 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.92 (d, J=8.5 Hz, 2H, 2 CH.sub.Ar), 7.50 (s, 1H, NH), 7.30 (d, J=8.5 Hz, 2H, 2 CH.sub.Ar), 6.89 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 4.60 (d, J=6.0 Hz, 2H, CH.sub.2), 3.72 (s, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.20H.sub.18ClN.sub.6O.sub.2: [M+H].sup.+ 409.11743, found 409.1179.

    [0462] Compound 26. This compound was synthesized through general synthesis protocol VI from compound 27 to afford pure compound 26 (99%). .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.51 (bs, 1H, CH.sub.Ar), 8.17-8.07 (m, 2H, 2 CH.sub.Ar), 8.01-7.91 (m, 2H, 2 CH.sub.Ar), 1.63 (d, J=7.3 Hz, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.15H.sub.14ClN.sub.6O.sub.3: [M+H].sup.+ 361.08104, found 361.0809.

    [0463] Compound 27. This compound was synthesized through general synthesis protocol VII from compound 79 and 1-alanine tert-butyl ester, and was purified by extraction to afford pure compound 27 (61%). .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.51 (bs, 1H, CH.sub.Ar), 8.51 (d, J=8.6 Hz, 2H, 2 CH.sub.Ar), 7.97 (d, J=8.6 Hz, 2H, 2 CH.sub.Ar), 4.73-4.55 (m, 1H, CH), 1.59 (d, J=7.3 Hz, 3H, CH.sub.3), 1.52 (s, 9H, 3 CH.sub.3); HRMS (ESI) calc. for C.sub.19H.sub.22ClN.sub.6O.sub.3: [M+H].sup.+ 417.14364, found 417.1438.

    [0464] Compound 28. This compound was synthesized through general synthesis protocol XV from 4-aminobenzamide and N-(2-amino-4,6-dichloropyrimidin-5-yl)formamide to afford pure compound 28 (79%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 10.29 (bs, 1H, NH), 8.60 (s, 1H, CH.sub.Ar), 8.21-7.96 (m, 7H, 6 CH.sub.Ar+NH), 7.95-7.78 (m, 3H, 2 CH.sub.Ar+NH), 7.51 (bs, 1H, NH), 7.25 (bs, 1H, NH), 4.08 (bs, 2H, NH.sub.2); HRMS (ESI) calc. for C.sub.19H.sub.17N.sub.8O.sub.2: [M+H].sup.+ 389.14690, found 389.1469.

    [0465] Compound 29. The compound 30 was added to a mixture of LiOH (1M) and THF (1/1, v/v, 30 mL/mmol). The resulting solution was stirred for 4 h at 60° C. After evaporation under reduced pressure, the crude residue was dissolved in water and washed by ethyl acetate. Aqueous hydrochloric acid (1M) was added to the aqueous layer until pH 2-3. The resulting precipitate was filtrated and washed with water to afford pure compound 29 (89%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 13.13 (bs, 1H, COOH), 10.82 (bs, 1H, NH), 8.17 (s, 1H, CH.sub.Ar), 8.07 (d, J=8.2 Hz, 2H, CH.sub.Ar), 7.94 (d, J=8.2 Hz, 2H, CH.sub.Ar), 6.63 (bs, 2H, NH.sub.2); HRMS (ESI) calc. for C.sub.12H.sub.10N.sub.5O.sub.3: [M+H].sup.+ 272.07782, found 272.0778.

    [0466] Compound 30. This compound was synthesized through general synthesis protocol I from 2-N-acetyl-6-O-diphenylcarbamoylguanine and 4-(methoxycarbonyl)-phenylboronic acid, and was purified by chromatography on silica gel (elution with dichloromethane/methanol) to afford pure compound 30 (13%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.30-8.22 (m, 3H, 3 CH.sub.Ar), 8.04 (bs, 1H, NH), 7.83 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.51-7.27 (m, 11H, 10 CH.sub.Ar+NH), 3.97 (s, 3H, CH.sub.3), 2.53 (s, 3H, CH.sub.3).

    [0467] Compound 31. The compound 30 was added to a solution of NH.sub.3 (2M) in methanol. The resulting solution was stirred for 4 h at 70° C. After evaporation under reduced pressure, the crude product was washed with dichloromethane to afford pure compound 31 (89%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 10.60 (bs, 1H, OH), 8.20 (s, 1H, CH.sub.Ar), 8.12 (d, J=8.6 Hz, 2H, 2 CH.sub.Ar), 8.00 (d, J=8.6 Hz, 2H, 2 CH.sub.Ar), 6.61 (bs, 2H, NH.sub.2), 3.91 (s, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.13H.sub.12N.sub.5O.sub.3: [M+H].sup.+ 286.09347, found 286.0934.

    [0468] Compound 32. To a solution of compound 30 in dichloromethane (20 mL/mmol) was added triethylsilane (5 equivalents). The solution was stirred at 0° C. and TFa (5 mL/mmol) was added during 15 min. The solution was then stirred at room temperature for 4 h. After evaporation under reduced pressure, the crude product was suspended in dichloromethane, filtrated and washed with dichloromethane to afford pure compound 32 (80%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 12.16 (bs, 1H, OH or NH), 11.69 (bs, 1H, OH or NH), 8.45 (s, 1H, CH.sub.Ar), 8.14 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.96 (d, J=8.7 Hz, 2H, CH.sub.Ar), 3.90 (s, 3H, CH.sub.3), 2.18 (s, 3H, CH.sub.3).

    [0469] Compound 33. This compound was synthesized through general synthesis protocol XI from compound 49 (65%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 9.22 (s, 1H, CH.sub.Ar), 8.28 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 8.20 (bs, 1H, NH), 8.19 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.60 (bs, 1H, NH); HRMS (ESI) calc. for C.sub.11H.sub.8ClN.sub.6O: [M+H].sup.+ 275.04426, found 275.0440.

    [0470] Compound 34. This compound was synthesized through general synthesis protocol VII from compound 33 and glycine tert-butyl ester to afford pure compound 34 (64%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.68 (t, J=6.0 Hz, 1H, NH), 8.28 (d, J=8.6 Hz, 2H, 2 CH.sub.Ar), 8.12-8.05 (m, 3H, 2 CH.sub.Ar+NH), 7.48 (bs, 1H, NH), 6.80 (bs, 2H, NH.sub.2), 4.09 (d, J=6.0 Hz, 2H, CH.sub.2), 1.43 (s, 9H, 3 CH.sub.3); HRMS (ESI) calc. for C.sub.17H.sub.21N.sub.8O.sub.3: [M+H].sup.+ 385.17311, found 385.1730.

    [0471] Compound 35. This compound was synthesized through general synthesis protocol X from 4-phenylbutylamine and 5-amino-4,6-dichloropyrimidine and was purified by chromatography on silica gel (elution with dichloromethane/methanol) to afford pure compound 35 (78%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 7.72 (s, 1H, CH.sub.Ar), 7.31-7.23 (m, 2H, 2 CH.sub.Ar), 7.22-7.13 (m, 3H, 3 CH.sub.Ar), 6.77 (t, J=5.2 Hz, 1H, NH), 4.99 (bs, 2H, NH.sub.2), 3.43-3.37 (m, 2H, CH.sub.2), 2.61 (t, J=7.3 Hz, 2H, CH.sub.2), 1.66-1.52 (m, 4H, 2 CH.sub.2); HRMS (ESI) calc. for C.sub.14H.sub.18ClN.sub.4: [M+H].sup.+ 277.12145, found 277.1414.

    [0472] Compound 36. This compound was synthesized through general synthesis protocol X from 1,3-diaminopropane and 5-amino-4,6-dichloropyrimidine and was purified by chromatography on silica gel (elution with dichloromethane/methanol) to afford pure compound 36 (81%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 7.72 (s, 1H, CH.sub.Ar), 7.03 (bs, 1H, NH), 5.08 (bs, 2H, NH.sub.2), 3.70 (bs, 3H, NH.sub.3), 3.47-3.37 (m, 2H, CH.sub.2), 2.72 (t, J=7.1 Hz, 2H, CH.sub.2), 1.81-1.67 (m, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.7H.sub.13ClN.sub.5: [M+H].sup.+ 202.08540, found 202.0855.

    [0473] Compound 37. This compound was synthesized through general synthesis protocol X from 3-phenylpropylamine and 5-amino-4,6-dichloropyrimidine to afford pure compound 37 (64%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 7.72 (s, 1H, CH.sub.Ar), 7.32-7.25 (m, 2H, 2 CH.sub.Ar), 7.25-7.14 (m, 3H, 3 CH.sub.Ar), 6.80 (t, J=5.0 Hz, 1H, NH), 5.02 (bs, 2H, NH.sub.2), 3.39 (dd, J=12.4, 7.0 Hz, 2H, CH.sub.2), 2.69-2.63 (m, 2H, CH.sub.2), 1.92-1.83 (m, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.13H.sub.16ClN.sub.4: [M+H].sup.+ 263.10580, found 263.1058.

    [0474] Compound 38. This compound was synthesized through general synthesis protocol X from 1,3-diaminopropane and 5-amino-4,6-dichloropyrimidine and was purified by chromatography on silica gel (elution with dichloromethane/methanol) to afford pure compound 38 (11%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 7.73 (s, 2H, 2 CH.sub.Ar), 6.91-6.69 (m, 2H, 2 NH), 5.01 (bs, 4H, 2 NH.sub.2), 3.46 (dd, J=12.3, 7.0 Hz, 4H, 2 CH.sub.2), 1.88 (p, J=7.0 Hz, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.11H.sub.15Cl.sub.2N.sub.8: [M+H].sup.+ 329.07912, found 329.0790.

    [0475] Compound 39. This compound was synthesized through general synthesis protocol X from 2-phenylethylamine and 5-amino-4,6-dichloropyrimidine to afford pure compound 39 (76%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 7.75 (s, 1H, CH.sub.Ar), 7.34-7.27 (m, 2H, CH.sub.Ar), 7.27-7.16 (m, 3H, CH.sub.Ar), 6.92 (t, J=5.2 Hz, 1H, NH), 5.00 (bs, 2H, NH.sub.2), 3.66-3.55 (m, 2H, CH.sub.2), 2.87 (t, J=7.4 Hz, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.12H.sub.14ClN.sub.4: [M+H].sup.+ 249.09015, found 249.0901.

    [0476] Compound 40. This compound was synthesized through general synthesis protocol X from 2-(4-fluorophenyl)-ethylamine and 5-amino-4,6-dichloropyrimidine to afford pure compound 40 (89%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 7.75 (s, 1H, CH.sub.Ar), 7.34-7.23 (m, 2H, 2 CH.sub.Ar), 7.17-7.05 (m, 2H, CH.sub.Ar), 6.89 (t, J=5.0 Hz, 1H, NH), 4.99 (s, 2H, NH.sub.2), 3.65-3.54 (m, 2H, CH.sub.2), 2.86 (t, J=7.2 Hz, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.12H.sub.13ClFN.sub.4: [M+H].sup.+ 267.08073, found 267.0807.

    [0477] Compound 41. This compound was synthesized through general synthesis protocol X from 4-(aminoethyl)-pyridine and 5-amino-4,6-dichloropyrimidine to afford pure compound 41 (67%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.50-8.41 (m, 2H, 2 CH.sub.Ar), 7.75 (s, 1H, CH.sub.Ar), 7.32-7.21 (m, 2H, 2 CH.sub.Ar), 6.91 (t, J=5.1 Hz, 1H, NH), 4.99 (bs, 2H, NH.sub.2), 3.70-3.62 (m, 2H, CH.sub.2), 2.90 (t, J=7.1 Hz, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.11H.sub.13ClN.sub.5: [M+H].sup.+ 250.08540, found 250.0855.

    [0478] Compound 42. This compound was synthesized through general synthesis protocol X from 1,2-diaminoethane (1.5 equivalents) and 5-amino-4,6-dichloropyrimidine to afford pure compound 42 (32%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 7.75 (s, 2H, 2 CH.sub.Ar), 7.05 (bs, 2H, 2 NH), 5.00 (bs, 4H, 2 NH.sub.2), 3.67-3.50 (m, 4H, 2 CH.sub.2).

    [0479] Compound 43. This compound was synthesized through general synthesis protocol X from benzylamine and 5-amino-4,6-dichloropyrimidine and recrystallised in a mixture water/methanol (13/3, v/v) to afford pure compound 43 (81%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 7.72 (s, 1H, CH.sub.Ar), 7.37-7.29 (m, 5H, CH.sub.Ar), 7.28-7.21 (m, 1H, NH), 5.08 (bs, 2H, NH.sub.2), 4.63 (d, J=5.7 Hz, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.11H.sub.12ClN.sub.4: [M+H].sup.+ 235.07450, found 235.0746.

    [0480] Compound 44. This compound was synthesized through general synthesis protocol X from 4-(aminomethyl)-benzoic acid and 5-amino-4,6-dichloropyrimidine to afford pure compound 44 (89%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 7.82 (d, J=8.2 Hz, 2H, 2 CH.sub.Ar), 7.76 (t, J=5.6 Hz, 1H, NH), 7.70 (s, 1H, CH.sub.Ar), 7.22 (d, J=8.2 Hz, 2H, 2 CH.sub.Ar), 5.25 (bs, 2H, NH.sub.2), 4.61 (d, J=5.6 Hz, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.12H.sub.12ClN.sub.4O.sub.2: [M+H].sup.+279.06433, found 279.0644.

    [0481] Compound 45. This compound was synthesized through general synthesis protocol X from 3-(aminomethyl)-pyridine and 5-amino-4,6-dichloropyrimidine to afford pure compound 45 (68%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.56 (d, J=1.7 Hz, 1H, CH.sub.Ar), 8.46 (dd, J=4.8, 1.7 Hz, 1H, CH.sub.Ar), 7.74 (s, 1H, CH.sub.Ar), 7.71 (dt, J=7.8, 2.0 Hz, 1H, CH.sub.Ar), 7.41-7.33 (m, 2H, CH.sub.Ar+NH), 5.08 (bs, 2H, NH.sub.2), 4.64 (d, J=5.6 Hz, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.10H.sub.11ClN.sub.5: [M+H].sup.+ 236.06975, found 236.0697.

    [0482] Compound 46. This compound was synthesized through general synthesis protocol X from 4-(aminomethyl)-pyridine and 5-amino-4,6-dichloropyrimidine to afford pure compound 46 (76%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.48 (d, J=3.6 Hz, 2H, 2 CH.sub.Ar), 7.70 (s, 1H, CH.sub.Ar), 7.48 (bs, 1H, NH), 7.28 (d, J=3.6 Hz, 2H, 2 CH.sub.Ar), 5.12 (s, 2H, NH.sub.2), 4.65 (d, J=4.8 Hz, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.10H.sub.11ClN.sub.5: [M+H].sup.+ 236.06975, found 236.0697.

    [0483] Compound 47 was purchased from Tokyo Chemical Industry Co., Ltd.

    [0484] Compound 48 was purchased from Tokyo Chemical Industry Co., Ltd.

    [0485] Compound 49. This compound was synthesized through general synthesis protocol X from 4-aminobenzamide and 5-amino-4,6-dichloropyrimidine to afford pure compound 49 (27%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.79 (s, 1H, NH), 7.93 (s, 1H, CH.sub.Ar), 7.85 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.84 (bs, 1H, NH), 7.78 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.21 (bs, 1H, NH), 5.52 (bs, 2H, NH.sub.2); HRMS (ESI) calc. for C.sub.11H.sub.11ClN.sub.5O: [M+H].sup.+ 264.06466, found 264.0644.

    [0486] Compound 50. This compound was synthesized through general synthesis protocol XV from 4-aminobenzamide and 5-amino-4,6-dichloro-2-methylpyrimidine to afford pure compound 50 (71%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 9.44 (bs, 2H, 2 NH), 7.92-7.83 (m, 8H, 8 CH.sub.Ar), 7.59 (bs, 4H, 4 NH), 7.24 (bs, 2H, 2 NH), 2.43 (s, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.19H.sub.20N.sub.7O.sub.2: [M+H].sup.+ 378.16730, found 378.1672.

    [0487] Compound 51. This compound was synthesized through general synthesis protocol X from 3-aminobenzamide and 5-amino-4,6-dichloropyrimidine, was purified by chromatography on silica gel (elution with dichloromethane/methanol) and then recrystallised from isopropanol to afford pure compound 51 (51%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.76 (bs, 1H, NH), 8.10 (s, 1H, CH.sub.Ar), 8.03-7.84 (m, 3H, 2 CH.sub.Ar+NH), 7.53 (d, J=7.7 Hz, 1H, CH.sub.Ar), 7.45-7.27 (m, 2H, CH.sub.Ar+NH), 5.46 (bs, 2H, NH.sub.2); HRMS (ESI) calc. for C.sub.11H.sub.11ClN.sub.5O: [M+H].sup.+ 264.06466, found 264.0648.

    [0488] Compound 52. This compound was synthesized through general synthesis protocol X from 4-aminosulfanilic acid and 5-amino-4,6-dichloropyrimidine to afford pure compound 52 (45%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.77 (bs, 1H, NH), 7.91 (s, 1H, CH.sub.Ar), 7.67-7.63 (m, 2H, CH.sub.Ar), 7.60-7.53 (m, 2H, CH.sub.Ar), 5.10 (bs, 2H, NH.sub.2); HRMS (ESI) calc. for C.sub.10H.sub.8ClN.sub.4O.sub.3S: [M−H].sup.− 299.00111, found 299.0011.

    [0489] Compound 53. This compound was synthesized through general synthesis protocol III from 99, and was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 53 (94%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.16 (s, 1H, CH.sub.Ar), 7.74 (bs, 2H, NH.sub.2), 7.33-7.11 (m, 5H, 5 CH.sub.Ar), 4.25 (t, J=6.5 Hz, 2H, CH.sub.2), 2.78-2.69 (m, 2H, CH.sub.2), 2.07-1.94 (m, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.14H.sub.16N.sub.5O: [M+H].sup.+ 270.13494, found 270.1349.

    [0490] Compound 54. Compound Int-2 was dissolved in a saturated solution of NH.sub.3 in ethanol. The resulting solution was stirred in a sealed tube at 60° C. for 24 h. After evaporation under reduced pressure, the crude residue was washed several times with water to afford pure compound 54 (70%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.54 (s, 1H, CH.sub.Ar), 7.90 (bs, 2H, NH.sub.2), 7.73 (d, J=8.4 Hz, 2H, 2 CH.sub.Ar), 7.52 (d, J=8.4 Hz, 2H, 2 CH.sub.Ar), 5.33 (bs, 1H, OH), 4.58 (bs, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.12H.sub.11ClN.sub.5O: [M+H].sup.+ 276.06466, found 276.0645.

    [0491] Compound 55. To a solution of 5-amino-4,6-dichloropyrimidine (1 equivalent) in DMF (3 mL/mmol), 4-hydroxybenzamide (1.2 equivalents) and K.sub.2CO.sub.3 (3 equivalents) were added. The resulting solution was stirred overnight under argon at 60° C. After evaporation under reduced pressure, the crude residue was purified by extraction with ethyl acetate/water and then with ethyl acetate/brine to afford pure compound 55 (59%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 7.99 (bs, 1H, NH.sub.2), 7.97-7.90 (m, 2H, 2 CH.sub.Ar), 7.82 (s, 1H, CH.sub.Ar), 7.37 (bs, 1H, NH.sub.2), 7.34-7.27 (m, 2H, 2 CH.sub.Ar), 5.83 (bs, 2H, NH.sub.2); HRMS (ESI) calc. for C.sub.11H.sub.10ClN.sub.4O.sub.2: [M+H].sup.+ 265.04868, found 265.0485.

    [0492] Compound 56. This compound was synthesized through general synthesis protocol XVII from 5-amino-4,6-dichloropyrimidine and 4-aminobenzenesulfonamide to afford pure compound 56 (99%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.90 (bs, 1H, NH), 7.94 (s, 1H, CH.sub.Ar), 7.92-7.85 (m, 2H, 2 CH.sub.Ar), 7.80-7.74 (m, 2H, 2 CH.sub.Ar), 7.23 (bs, 2H, NH.sub.2), 5.55 (bs, 2H, NH.sub.2); HRMS (ESI) calc. for C.sub.10H.sub.11ClN.sub.5O.sub.2S: [M+H].sup.+ 300.03165, found 300.0315.

    [0493] Compound 57. This compound was synthesized through general synthesis protocol VI from compound Int-8 and was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 57 (45%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.00 (s, 1H, CH.sub.Ar), 7.73 (d, J=8.2 Hz, 2H, 2 CH.sub.Ar), 7.52 (d, J=8.2 Hz, 2H, 2 CH.sub.Ar), 7.27-7.10 (m, 5H, 5 CH.sub.Ar), 4.71-4.59 (m, 3H, CH.sub.2+CH), 3.38 (dd, J=14.6, 7.4 Hz, 2H, CH.sub.2), 2.68 (t, J=7.6 Hz, 2H, CH.sub.2), 1.95-1.86 (m, 2H, CH.sub.2), 1.53 (d, J=7.0 Hz, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.24H.sub.27N.sub.6O.sub.3: [M+H].sup.+ 447.21392, found 447.2136.

    [0494] Compound 58. This compound was synthesized through general synthesis protocol II from compound 9 and 4-phenylbutylamine (10 equivalents), and was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 58 (61%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 12.11 (bs, 1H, NH), 7.68-7.45 (m, 2H, NH+CH.sub.Ar), 7.35-7.21 (m, 4H, 4 CH.sub.Ar), 7.21-7.07 (m, 3H, 3 CH.sub.Ar), 6.83 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 6.19 (bs, 1H, NH), 4.55 (bs, 2H, CH.sub.2), 3.69 (s, 3H, CH.sub.3), 3.23 (dd, J=12.7, 6.5 Hz, 2H, CH.sub.2), 2.56 (t, J=7.3 Hz, 2H, CH.sub.2), 1.65-1.45 (m, 4H, 2 CH.sub.2); HRMS (ESI) calc. for C.sub.23H.sub.27N.sub.6O: [M+H].sup.+ 403.22409, found 403.2242.

    [0495] Compound 59. To a suspension of Int-11 (1 equivalent) in a mixture of water/THF (2/3 v/v, 7 mL/mmol) were added palladium acetate (0.1 equivalent) and potassium fluoride (40% on alumina, 2 equivalents). The reaction mixture was kept at room temperature for 40 min, concentrated and purified by reverse phase chromatography (eluent water/methanol) to afford pure compound 59 (76%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.10 (s, 1H, NH), 7.79-7.69 (m, 4H, 3 CH.sub.Ar+NH.sub.2), 7.64 (d, J=8.8 Hz, 2H, 2 CH.sub.Ar), 7.08 (bs, 1H, NH.sub.2), 6.01 (bs, 2H, NH.sub.2), 4.29 (bs, 2H, NH.sub.2); HRMS (ESI) calc. for C.sub.11H.sub.12N.sub.5O: [M+H].sup.+ 230.10363, found 230.1034.

    [0496] Compound 60. To a solution of compound 10 (1 equivalent) in DMF (1 mL/mmol), CuI (1.1 equivalents), trans-i 1,2-diaminocyclohexane (2.2 equivalents), K.sub.3PO.sub.4 (4 equivalents) and 4-iodobenzamide (1.3 equivalents) were added. The solution was stirred overnight at 90° C. under argon. After concentration under reduced pressure, the reaction mixture was diluted with dichloromethane and extracted with a saturated aqueous solution of ethylenediaminetetraacetic acid (EDTA). The organic layer was washed with water and brine, then dried over magnesium sulfate. After filtration and concentration under reduced pressure the crude product was purified by chromatography on silica gel (eluent dichloromethane/methanol) then by crystallization from ethanol to afford pure compound 60 (11%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 10.50 (bs, 1H, NH), 8.33 (bs, 1H, NH), 8.21-8.08 (m, 3H, 3 CH.sub.Ar), 7.94-7.83 (m, 3H, NH.sub.2+2 CH.sub.Ar), 7.35-7.12 (m, 7H, 7 CH.sub.Ar), 6.84 (d, J=8.4 Hz, 2H, 2 CH.sub.Ar), 6.66 (bs, 1H, NH.sub.2), 4.58 (bs, 2H, CH.sub.2), 3.70 (s, 3H, CH.sub.3), 3.30-3.26 (m, 2H, CH.sub.2), 2.66-2.59 (m, 2H, CH.sub.2), 1.92-1.78 (m, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.29H.sub.30N.sub.7O.sub.2: [M+H].sup.+ 508.2456, found 508.2486.

    [0497] Compound 61. To a solution of 2,6-diaminopurine (1 equivalent) in DMF (1 mL/mmol), CuI (1.1 equivalents), N,N′-dimethylethylenediamine (2.2 equivalents), K.sub.3PO.sub.4 (4 equivalents) and 4-iodobenzylalcool (1.3 equivalents) were added. The solution was stirred overnight at 90° C. under argon. After concentration under reduced pressure, the reactionnal mixture was diluted with dichloromethane and extracted with a saturated aqueous solution of ethylenediaminetetraacetic acid (EDTA). The organic layer was washed with water and brine, then dried over magnesium sulfate. After filtration and concentration under reduced pressure the crude product was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 61 (5%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.11 (s, 1H, CH.sub.Ar), 7.78 (d, J=8.5 Hz, 2H, 2 CH.sub.Ar), 7.45 (d, J=8.5 Hz, 2H, 2 CH.sub.Ar), 6.78 (bs, 2H, NH.sub.2), 5.87 (bs, 2H, NH.sub.2), 5.26 (t, J=5.7 Hz, 1H, OH), 4.55 (d, J=5.7 Hz, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.12H.sub.13Cl.sub.zN.sub.6O: [M+H].sup.+ 257.11454, found 257.1144.

    [0498] Compound 62. To a suspension of Int-10 (1 equivalent) in methanol (3 mL/mmol), was added sodium methoxide (10 equivalents). The suspension was stirred under reflux for 10 h, under argon. After concentration under reduced pressure, the crude product was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 62 (59%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.39 (s, 1H, CH.sub.Ar), 8.08 (bs, 1H, NH.sub.2), 8.07-7.96 (m, 4H, 4 CH.sub.Ar), 7.48 (bs, 1H, NH.sub.2), 6.63 (bs, 2H, NH.sub.2), 4.00 (s, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.13H.sub.13N.sub.6O: [M+H].sup.+ 285.10945, found 285.1099.

    [0499] Compound 63. To a suspension of Int-10 (1 equivalent) in methanol (4 ml/mmol) were added Pd/C 10% (0.2 equivalent) and ammonium formate (11 equivalents). The reaction mixture was bubbled with argon and then refluxed for 17 h. After concentration under reduced pressure, the crude product was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 63 (77%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.71 (s, 1H, CH.sub.Ar), 8.59 (s, 1H, CH.sub.Ar), 8.09 (bs, 1H, NH.sub.2), 8.07-7.96 (m, 4H, 4 CH.sub.Ar), 7.49 (bs, 1H, NH.sub.2), 6.72 (bs, 2H, NH.sub.2); HRMS (ESI) calc. for C.sub.12H.sub.11N.sub.6O: [M+H].sup.+ 255.09889, found 255.0989.

    [0500] Compound 64. This compound was synthesized through general synthesis protocol XI from compound 56. After precipitation, it was further purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 64 (58%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 9.24 (s, 1H, CH.sub.Ar), 8.41 (d, J=8.8 Hz, 2H, 2 CH.sub.Ar), 8.16 (d, J=8.8 Hz, 2H, 2 CH.sub.Ar), 7.60 (bs, 2H, NH.sub.2); HRMS (ESI) calc. for C.sub.10H.sub.8ClN.sub.6O.sub.2S: [M+H].sup.+ 311.01125, found 311.0112.

    [0501] Compound 65. To a solution of 5-amino-4,6-dichloro-2-methylpyrimidine (1 equivalent) in a mixture water/dioxane (1/1 v/v, 4 mL/mmol) was added 4-aminobenzamide (1 equivalent). The solution was refluxed overnight. After cooling down to room temperature, the suspension was filtered and the precipitate was washed with 1,4-dioxane. The crude product was then triturated in an aqueous solution of NaHCO.sub.3 5% and filtered to afford pure compound 65 (44%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.72 (bs, 1H, NH), 7.90-7.76 (m, 5H, 4 CH.sub.Ar+NH.sub.2), 7.21 (bs, 1H, NH.sub.2), 5.27 (bs, 2H, NH.sub.2), 2.34 (s, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.12H.sub.13ClN.sub.5O: [M+H].sup.+ 278.08031, found 278.0808.

    [0502] Compound 66. To a solution of 3-phenylpropanol (1 equivalent) in THF (5 mL/mmol), NaH (1.5 equivalent) was added. The resulting solution was stirred under argon for 40 min at room temperature. After cooling to 0° C., 5-amino-4,6-dichloropyrimidine was added and the solution was stirred under argon for 1 h 30 at 0° C. and then at room temperature overnight. After evaporation under reduced pressure, the crude residue was purified by extraction dichloromethane/water and then by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 66 (95%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 7.87 (s, 1H, CH.sub.Ar), 7.32-7.21 (m, 4H, 4 CH.sub.Ar), 7.18 (ddd, J=6.2, 3.4, 1.6 Hz, 1H, CH.sub.Ar), 5.38 (bs, 2H, NH.sub.2), 4.33 (t, J=6.4 Hz, 2H, CH.sub.2), 2.82-2.71 (m, 2H, CH.sub.2), 2.05 (dq, J=9.1, 6.4 Hz, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.13H.sub.15ClN.sub.3O: [M+H].sup.+ 264.08982, found 264.0901.

    [0503] Compound 67 was purchased from Tokyo Chemical Industry Co., Ltd.

    [0504] Compound 68 was purchased from Sigma-Aldrich Co., Ltd.

    [0505] Compound 69. 2,5-diamino-4,6-dichloropyrimidine (1 equivalent) was suspended in NH.sub.4OH (18 mL/mmol), heated in a sealed tube to 135° C. for 24 h, concentrated in vacuo then purified by chromatography on silica gel (eluent dichloromethane/methanol/NH.sub.4OH) to afford pure compound 69 (93%). .sup.13C NMR (101 MHz, d.sub.6-DMSO) δ 156.0, 155.5, 113.3, 39.5; HRMS (ESI) calc. for C.sub.4H.sub.7ClN.sub.5: [M+H].sup.+ 160.03845, found 160.0385.

    [0506] Compound 70. To a solution of freshly prepared 3-phenylpropanic anhydride (1 equivalent) in pyridine (4.5 mL/mmol) was added 2-amino-6-chloropurine (1 equivalent). The resulting solution was stirred at room temperature for 5 h, concentrated in vacuo then purified by chromatography on silica gel (eluent dichloromethane/methanol). Fractions containing the final product were collected and concentrated in vacuo. The residual solid was dissolved in dichloromethane (1 ml) and precipitated with cyclohexane (6 mL). The precipitate was filtrated, washed with cyclohexane and dried in vacuo to afford pure compound 70 (13%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.57 (s, 1H, NH), 7.34-7.15 (m, 7H, 6 CH.sub.Ar+NH), 3.63 (t, J=7.6 Hz, 2H, CH.sub.2), 3.02 (t, J=7.6 Hz, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.14H.sub.13ClN.sub.5O: [M+H].sup.+ 302.08031, found 302.0804.

    [0507] Compound 71. To a suspension of 157 (1 equivalent) in methanol (10 ml/mmol) was added Pd/C 10% (0.1 equivalent). The reaction mixture was bubbled with argon and then with dihydrogen. The suspension was kept overnight under dihydrogen (1 atm), and was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 71 (45%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 12.12 (bs, 1H, OH), 8.07 (bs, 1H, NH), 7.84-7.67 (m, 3H, 2 CH.sub.Ar+NH.sub.2), 7.61 (s, 1H, CH.sub.Ar), 7.42 (d, J=8.8 Hz, 2H, 2 CH.sub.Ar), 7.09 (bs, 1H, NH.sub.2), 4.43 (bs, 2H, NH.sub.2). HRMS (ESI) calc. for C.sub.11H.sub.12N.sub.5O.sub.2: [M+H].sup.+ 246.09855, found 246.0985.

    [0508] Compound 72. This compound was synthesized through general synthesis protocol III from compound 58 and was purified by washing with water to afford pure compound 72 (97%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 12.11 (bs, 1H, NH), 7.63 (bs, 1H, CH.sub.Ar), 7.26 (t, J=7.3 Hz, 2H, 2 CH.sub.Ar), 7.23-7.10 (m, 3H, 3 CH.sub.Ar), 6.51 (bs, 2H, NH.sub.2), 6.14-6.05 (m, 1H, NH), 3.23 (dd, J=12.8, 6.5 Hz, 2H, CH.sub.2), 2.59 (t, J=7.4 Hz, 2H, CH.sub.2), 1.64-1.47 (m, 4H, 2 CH.sub.2); HRMS (ESI) calc. for C.sub.15H.sub.19N.sub.6: [M+H].sup.+ 283.16657, found 283.1666.

    [0509] Compound 74 was purchased from Tokyo Chemical Industry Co., Ltd.

    [0510] Compound 75. To a suspension of 155 (1 equivalent) in methanol (8 ml/mmol) was added Pd/C 10% (0.04 equivalent). The reaction mixture was bubbled with argon and then with dihydrogen. The suspension was kept overnight under dihydrogen (1 atm), and concentrated under reduced pressure. The crude product was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 75 (74%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.38 (bs, 1H, NH), 7.91 (s, 1H, CH.sub.Ar), 7.83-7.74 (m, 3H, 2 CH.sub.Ar+NH.sub.2), 7.71 (d, J=8.8 Hz, 2H, CH.sub.Ar), 7.13 (bs, 1H, NH.sub.2), 4.71 (bs, 2H, NH.sub.2), 3.92 (s, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.12H.sub.14N.sub.5O.sub.2: [M+H].sup.+ 260.11420, found 260.1140.

    [0511] Compound 76. To a suspension of 56 in trimethyl orthoformate (3 mL/mmol) was added ethanesulfonic acid (25 μL/mmol). The suspension was heated under microwave irradiation at 120° C. for 1 h. After cooling down to room temperature, it was diluted with methanol and filtered. The precipitate was then triturated in water (6 mL/mmol) for 24 h and filtered to afford pure compound 76 (48%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 9.21 (s, 1H, CH.sub.Ar), 8.90 (s, 1H, CH.sub.Ar), 8.16 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 8.07 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.55 (bs, 2H, NH.sub.2); HRMS (ESI) calc. for C.sub.11H.sub.9ClN.sub.5O.sub.2S: [M+H].sup.+ 310.01600, found 310.0161.

    [0512] Compound 77. To a suspension of 49 in triethyl orthoacetate (3 mL/mmol) was added ethanesulfonic acid (25 μL/mmol). The suspension was heated at 130° C. for 48 h. After cooling down to room temperature, the suspension was filtered. The precipitate was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 77 (36%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.68 (s, 1H, CH.sub.Ar), 8.17 (bs, 1H, NH.sub.2), 8.10 (d, J=8.6 Hz, 2H, 2 CH.sub.Ar), 7.71 (d, J=8.6 Hz, 2H, 2 CH.sub.Ar), 7.59 (bs, 1H, NH.sub.2), 2.55 (s, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.13H.sub.11ClN.sub.5O: [M+H].sup.+ 288.06466, found 288.0650.

    [0513] Compound 78. To a solution of 158 (1 equivalent) in methanol (7 ml/mmol) was added Pd/C 10% (0.04 equivalent). The reaction mixture was bubbled with argon and then with dihydrogen. The suspension was kept overnight under dihydrogen (1 atm), and concentrated under reduced pressure. The crude product was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 78 (61%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 9.32 (bs, 1H, NH), 8.03 (s, 1H, CH.sub.Ar), 7.52 (s, 1H, CH.sub.Ar), 7.42 (bs, 1H, OH), 7.14 (d, J=8.5 Hz, 2H, 2 CH.sub.Ar), 6.71 (d, J=8.5 Hz, 2H, 2 CH.sub.Ar), 5.13 (bs, 2H, NH.sub.2), 4.52 (d, J=5.5 Hz, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.11H.sub.13N.sub.4O: [M+H].sup.+217.10839, found 217.1085.

    [0514] Compound 79. This compound was synthesized through general synthesis protocol I from 2,6-dichloropurine and 4-carbamoylphenylboronic acid, and was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 79 (16%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 9.19 (s, 1H, CH.sub.Ar), 8.18-8.09 (m, 3H, 2 CH.sub.Ar+NH), 7.97-7.93 (m, 2H, 2 CH.sub.Ar), 7.55 (bs, 1H, NH); HRMS (ESI) calc. for C.sub.12H.sub.8Cl.sub.2N.sub.5O: [M+H].sup.+ 308.01001, found 308.0100.

    [0515] Compound 80. This compound was synthesized through general synthesis protocol I from 2,6-dichloropurine and phenylboronic acid, and was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 80 (14%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.38 (s, 1H, CH.sub.Ar), 7.70-7.65 (m, 2H, 2 CH.sub.Ar), 7.65-7.59 (m, 2H, 2 CH.sub.Ar), 7.56-7.51 (m, 1H, CH.sub.Ar); HRMS (ESI) calc. for C.sub.11H.sub.7Cl.sub.2N.sub.4: [M+H].sup.+ 265.00454, found 265.0042.

    [0516] Compound 81. This compound was synthesized through general synthesis protocol VII from compound 80 and 1-alanine tert-butyl ester, and was precipitated in diethyl ether, filtrated and washed to afford pure compound 81 (97%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.00 (s, 1H, CH.sub.Ar), 7.67-7.63 (m, 2H, 2 CH.sub.Ar), 7.57-7.52 (m, 2H, 2 CH.sub.Ar), 7.47-7.40 (m, 1H, CH.sub.Ar), 6.45 (bs, 1H, NH), 4.84 (bs, 1H, CH), 1.57 (d, J=7.1 Hz, 3H, CH.sub.3), 1.50 (s, 9H, 3 CH.sub.3); HRMS (ESI) calc. for C.sub.18H.sub.21N.sub.5O.sub.2: [M+H].sup.+ 374.138090, found 374.1378.

    [0517] Compound 82. This compound was synthesized through general synthesis protocol II from compound 81 and 3-phenylpropyl-1-amine, and was purified by chromatography on silica gel (eluent dichloromethane/ethyl acetate) to afford pure compound 82 (22%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.81 (s, 1H, CH.sub.Ar), 7.79-7.71 (m, 2H, 2 CH.sub.Ar), 7.55 (t, J=7.9 Hz, 2H, 2 CH.sub.Ar), 7.42 (t, J=7.4 Hz, 1H, CH.sub.Ar), 7.38-7.19 (m, 5H, 5 CH.sub.Ar), 6.10 (bs, 1H, NH), 4.95 (bs, 1H, CH), 4.82 (bs, 1H, NH), 3.65-3.40 (m, 2H, CH.sub.2), 2.87-2.65 (m, 2H, CH.sub.2), 2.04-1.89 (m, 2H, CH.sub.2), 1.58 (d, J=7.1 Hz, 3H, CH.sub.3), 1.53 (s, 9H, 3 CH.sub.3); HRMS (ESI) calc. for C.sub.27H.sub.33N.sub.6O.sub.2: [M+H].sup.+ 473.26597, found 473.2660.

    [0518] Compound 83. This compound was synthesized through general synthesis protocol VI from compound 92, was purified by extraction dichloromethane/water and then was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 83 (93%). .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.00 (s, 1H, CH.sub.Ar), 7.79-7.71 (m, 2H, 2 CH.sub.Ar), 7.57-7.50 (m, 2H, 2 CH.sub.Ar), 7.47-7.39 (m, 1H, CH.sub.Ar), 7.27-7.11 (m, 5H, 5 CH.sub.Ar), 4.72 (bs, 1H, CH), 3.45-3.35 (m, 2H, CH.sub.2), 2.74-2.62 (m, 2H, CH.sub.2), 1.96-1.84 (m, 2H, CH.sub.2), 1.58 (d, J=7.2 Hz, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.23H.sub.25N.sub.6O.sub.2: [M+H].sup.+ 417.20347, found 417.2034.

    [0519] Compound 85. This compound was synthesized through general synthesis protocol II from compound Int-1 and 2-(4-methoxyphenyl)ethyl-1-amine, and was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 85 (31%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.75 (m, 3H, 3 CH.sub.Ar), 7.53 (m, 2H, 2 CH.sub.Ar), 7.41 (tt, J=7.4 Hz, J=1.1 Hz, 1H, CH.sub.Ar), 7.33 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.15 (d, J=8.6 Hz, 2H, 2 CH.sub.Ar), 6.88 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 6.85 (d, J=8.6 Hz, 2H, 2 CH.sub.Ar), 5.86 (bs, 1H, NH), 4.97 (bs, 1H, NH), 4.77 (bs, 2H, CH.sub.2), 3.81 (s, 3H, CH.sub.3), 3.80 (s, 3H, CH.sub.3), 3.64 (q, J=7.0 Hz, 2H, CH.sub.2), 2.87 (t, J=7.0 Hz, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.28H.sub.29N.sub.6O.sub.2: [M+H].sup.+ 481.23465, found 481.2348.

    [0520] Compound 86. This compound was synthesized through general synthesis protocol I from 2,6-dichloropurine and 4-acetylphenylboronic acid, and was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 86 (16%). .sup.1H NMR (400 MHz, CD.sub.3OD) δ 9.00 (s, 1H, CH.sub.Ar), 8.32-8.25 (m, 2H, 2 CH.sub.Ar), 8.09-8.04 (m, 2H, 2 CH.sub.Ar), 2.70 (s, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.13H.sub.9Cl.sub.2N.sub.4O: [M+H].sup.+ 307.01479, found 307.0149.

    [0521] Compound 88. This compound was synthesized through general synthesis protocol II from 2,6-dichloropurine and 4-methoxybenzylamine, and was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 88 (66%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 12.99 (bs, 1H, NH), 8.11 (bs, 1H, CH.sub.Ar), 7.92 (bs, 2H, 2 NH); 7.23 (d, J=8.5 Hz, 4H, 4 CH.sub.Ar), 6.85 (d, J=8.5 Hz, 2H, 2 CH.sub.Ar), 6.84 (d, J=8.5 Hz, 2H, 2 CH.sub.Ar), 4.64 (bs, 2H, CH.sub.2), 4.50 (d, J=5.5 Hz, 2H, CH.sub.2), 3.72 (s, 3H, OCH.sub.3), 3.71 (s, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.21H.sub.23N.sub.6O.sub.2: [M+H].sup.+ 391.18770, found 391.1877.

    [0522] Compound 89. This compound was synthesized through general synthesis protocol V from compound 9 and 4-(bromomethyl)benzamide, and was purified by precipitation in methanol to afford pure compound 89 (50%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.81 (t, J=5.4 Hz, 1H, NH), 8.29 (s, 1H, CH.sub.Ar), 7.93 (bs, 1H, NH.sub.2), 7.83 (d, J=8.1 Hz, 2H, 2 CH.sub.Ar), 7.35 (bs, 1H, NH.sub.2), 7.32 (d, J=8.1 Hz, 2H, 2 CH.sub.Ar), 7.28 (d, J=8.6 Hz, 2H, 2 CH.sub.Ar), 6.87 (d, J=8.6 Hz, 2H, 2 CH.sub.Ar), 5.40 (s, 2H, CH.sub.2), 4.56 (d, J=5.4 Hz, 2H, CH.sub.2), 3.71 (s, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.21H.sub.20ClN.sub.6O.sub.2: [M+H].sup.+ 423.13308, found 423.1334.

    [0523] Compound 90. This compound was synthesized through general synthesis protocol I from 2,6-dichloropurine and 4-(methoxycarbonyl)phenylboronic acid, and was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 90 (7%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.44 (s, 1H, CH.sub.Ar), 8.29 (d, J=8.9 Hz, 2H, 2 CH.sub.Ar), 7.84 (d, J=8.9 Hz, 2H, 2 CH.sub.Ar), 3.99 (s, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.13H.sub.9Cl.sub.2N.sub.4O.sub.2: [M+H].sup.+ 323.00971, found 323.0998.

    [0524] Compound 91. This compound was obtained in two steps through general synthesis protocol I from 2-N-acetyl-6-O-diphenylcarbamoylguanine and 3,4-dimethoxyphenylboronic acid and was purified by chromatography on silica gel (eluent dichloromethane/methanol). The intermediate compound was added to a solution of NH.sub.3 (2M) in methanol. The resulting solution was stirred for 4 h at 70° C. After evaporation under reduced pressure, the crude product was washed with dichloromethane to afford pure compound 91 (72%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 10.69 (bs, 1H, OH), 8.00 (s, 1H, CH.sub.Ar), 7.38 (s, 1H, CH.sub.Ar), 7.23 (m, 1H, CH.sub.Ar), 7.08 (m, 1H, CH.sub.Ar), 6.51 (bs, 2H, NH.sub.2), 3.83 (s, 6H, 2 CH.sub.3).

    [0525] Compound 92. This compound was obtained in two steps through general synthesis protocol I from 2-N-acetyl-6-O-diphenylcarbamoylguanine and phenylboronic acid and was purified by chromatography on silica gel (eluent dichloromethane/methanol). The intermediate compound was added to a solution of NH.sub.3 (2M) in methanol. The resulting solution was stirred for 4 h at 70° C. After evaporation under reduced pressure, the crude product was washed with dichloromethane to afford pure compound 92 (86%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 10.56 (bs, 1H, OH), 8.08 (s, 1H, CH.sub.Ar), 7.77 (m, 2H, 2 CH.sub.Ar), 7.58 (m, 2H, 2 CH.sub.Ar), 7.46 (m, 1H, CH.sub.Ar), 6.53 (bs, 2H, NH.sub.2).

    [0526] Compound 93. This compound was synthesized through general synthesis protocol V from 2,6-dichloropurine and 4-(bromomethyl)benzamide, and was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 93 (64%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.85 (s, 1H, CH.sub.Ar), 7.95 (bs, 1H, NH.sub.2), 7.84 (d, J=8.4 Hz, 2H, 2 CH.sub.Ar), 7.39 (d, J=8.4 Hz, 2H, 2 CH.sub.Ar), 7.38 (bs, 1H, NH.sub.2), 5.55 (s, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.13H.sub.10Cl.sub.2N.sub.5O: [M+H].sup.+ 322.02569, found 322.0259.

    [0527] Compound 94. This compound was synthesized through general synthesis protocol I from 2,6-dichloropurine and (pyridin-3-yl)boronic acid, and was purified by chromatography on silica gel (eluent dichloromethane/ethyl acetate) to afford pure compound 94 (2%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.95 (bs, 1H, CH.sub.Ar), 8.76 (bs, 1H, CH.sub.Ar), 8.43 (s, 1H, CH.sub.Ar), 8.15-8.08 (m, 1H, CH.sub.Ar), 7.58 (dd, J=8.2, 4.8 Hz, 1H, CH.sub.Ar); HRMS (ESI) calc. for C.sub.10H.sub.6Cl.sub.2N.sub.5: [M+H].sup.+ 265.99948, found 265.9994.

    [0528] Compound 95. This compound was synthesized through general synthesis protocol I from 2,6-dichloropurine and 3-carbamoylphenylboronic acid, and was purified by chromatography on silica gel (eluent dichloromethane/methanol/ethyl acetate) to afford pure compound 95 (3%). .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.94 (s, 1H, CH.sub.Ar), 8.31 (t, J=1.9 Hz, 1H, CH.sub.Ar), 8.10-8.03 (m, 2H, 2 CH.sub.Ar), 7.77 (t, J=7.9 Hz, 1H, CH.sub.Ar); HRMS (ESI) calc. for C.sub.12H.sub.8Cl.sub.2N.sub.5O: [M+H].sup.+ 308.01004, found 308.0099.

    [0529] Compound 96. This compound was synthesized through general synthesis protocol XV from 4-aminobenzamide and 6-chlorouracil to afford pure compound 96 (64%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 10.58 (s, 1H, NH), 10.30 (s, 1H, NH), 8.58 (s, 1H, CH.sub.Ar), 7.92 (bs, 1H, NH.sub.2), 7.88-7.84 (m, 2H, 2 CH.sub.Ar), 7.30 (bs, 1H, NH.sub.2), 7.25-7.19 (m, 2H, 2 CH.sub.Ar), 4.88 (s, 1H, NH); HRMS (ESI) calc. for C.sub.11H.sub.11N.sub.4O.sub.3: [M+H].sup.+ 247.08257, found 247.0822.

    [0530] Compound 97. To a suspension of compound Int-7 in isopropanol (5 mL/mmol) were added 4-methoxybenzylamine (1.1 equivalent) and DIPEA (2.4 equivalents). The suspension was stirred at 60° C. for 30 min. After cooling to room temperature, the resulting precipitate was filtrated and washed with isopropanol to afford pure compound 97 (75%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.90 (s, 1H, CH.sub.Ar), 7.30 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 6.88 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 6.17 (bs, 1H, NH), 5.70 (dd, J=10.7 Hz, J=2.2 Hz, 1H, CH), 4.74 (bs, 2H, CH.sub.2), 4.15 (m, 1H, CH), 3.78 (m, 1H, CH), 1.63-2.12 (m, 6H, 2 CH+2 CH.sub.2); HRMS (ESI) calc. for C.sub.18H.sub.21ClN.sub.5O.sub.2: [M+H].sup.+ 374.13783, found 374.1378.

    [0531] Compound 98. This compound was synthesized through general synthesis protocol XIII from compound 97 and 2-phenylethanol, and was purified by chromatography on silica gel (elution with dichloromethane/ethyl acetate) to afford pure compound 98 (99%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.80 (s, 1H, CH.sub.Ar), 7.24-7.32 (m, 7H, 7 CH.sub.Ar), 6.86 (d, J=8.6 Hz, 2H, 2 CH.sub.Ar), 6.01 (bs, 1H, NH), 5.64 (dd, J=10.5 Hz, J=2.1 Hz, 1H, CH), 4.75 (bs, 2H, CH.sub.2), 4.56 (t, J=7.8 Hz, 2H, CH.sub.2), 4.14 (m, 1H, CH), 3.80 (s, 3H, CH.sub.3), 3.75 (m, 1H, CH), 3.16 (t, J=7.8 Hz, 2H, CH.sub.2), 1.62-2.09 (m, 6H, 2 CH+2 CH.sub.2); HRMS (ESI) calc. for C.sub.26H.sub.30N.sub.5O.sub.3: [M+H].sup.+ 460.23432, found 460.2342.

    [0532] Compound 99. This compound was synthesized through general synthesis protocol XIII from compound 97 and 3-phenylpropanol, and was purified by chromatography on silica gel (elution with dichloromethane/methanol) to afford pure compound 99 (20%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.70 (s, 1H, CH.sub.Ar), 7.16-7.29 (m, 7H, 7 CH.sub.Ar), 6.83 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 6.45 (bs, 1H, NH), 5.63 (dd, J=10.5 Hz, J=2.3 Hz, 1H, CH), 4.71 (bs, 2H, CH.sub.2), 4.39 (t, J=6.5 Hz, 2H, CH.sub.2), 4.12 (m, 1H, CH), 3.78 (s, 3H, CH.sub.3), 3.73 (m, 1H, CH), 2.81 (t, J=6.5 Hz, 2H, CH.sub.2), 2.13 (m, 2H, CH.sub.2), 1.59-2.09 (m, 6H, 2 CH+2 CH.sub.2).

    [0533] Compound 100. This compound was synthesized through general synthesis protocol X from 4-aminobenzamide and 2-amino-4,6-dichloropyrimidine and was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 100 (17%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 9.55 (bs, 1H, NH), 7.87-7.74 (m, 5H, 4 CH.sub.Ar+NH.sub.2), 7.18 (bs, 1H, NH.sub.2), 6.82 (bs, 2H, NH.sub.2), 6.06 (s, 1H, CH.sub.Ar); HRMS (ESI) calc. for C.sub.11H.sub.11ClN.sub.5O: [M+H].sup.+ 264.06466, found 264.0647.

    [0534] Compound 101 was purchased from Tokyo Chemical Industry Co., Ltd.

    [0535] Compound 102 was purchased from Sigma-Aldrich Co., Ltd.

    [0536] Compound 103 was purchased from ABCR GmbH & Co. KG.

    [0537] Compound 104. This compound was synthesized through general synthesis protocol III from 131, and was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 104 (99%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 12.94 (bs, 1H, NH), 8.37 (bs, 2H, NH.sub.2), 8.15 (s, 1H, CH.sub.Ar), 7.61 (bs, 1H, NH), 7.40-7.23 (m, 2H, 2 CH.sub.Ar), 7.12 (t, J=8.9 Hz, 2H, 2 CH.sub.Ar), 3.57-3.51 (m, 2H, CH.sub.2), 2.86 (t, J=7.2 Hz, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.13H.sub.14FN.sub.6: [M+H].sup.+ 273.12585, found 273.1257.

    [0538] Compound 105. To a solution of 4-aminobenzamide (1 equivalent) in DMF (1 ml/mmol) was added 4-chloro-3-nitropyridine (1 equivalent). The solution was stirred at 50° C. under argon for 2 h. After cooling, water was added and the resulting precipitate was filtrated, washed with water, toluene and dried in vacuo to afford pure compound 105 (86%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 9.88 (bs, 1H, NH), 9.10 (s, 1H, CH.sub.Ar), 8.26 (d, J=6.1 Hz, 1H, CH.sub.Ar), 7.99 (bs, 1H, NH.sub.2), 7.96 (d, J=8.4 Hz, 2H, 2 CH.sub.Ar), 7.41 (d, J=8.4 Hz, 2H, 2 CH.sub.Ar), 7.37 (bs, 1H, NH.sub.2), 7.02 (d, J=6.1 Hz, 1H, CH.sub.Ar); HRMS (ESI) calc. for C.sub.12H.sub.11N.sub.4O.sub.3: [M+H].sup.+ 259.08256, found 259.0824.

    [0539] Compound 106. This compound was synthesized through general synthesis protocol XVI from 4,6-dichloro-5-nitropyrimidine and 4-aminobenzamide to afford pure compound 106 as yellow solid (89%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 10.21 (bs, 1H, NH), 8.58 (s, 1H, CH.sub.Ar), 7.93 (bs, 1H, NH.sub.2), 7.88 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.62 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.33 (bs, 1H, NH.sub.2); HRMS (ESI) calc. for C.sub.11H.sub.9ClN.sub.5O.sub.3: [M+H].sup.+294.03884, found 294.0387.

    [0540] Compound 107. To a suspension of 49 (1 equivalent) in methanol (5 ml/mmol) and diethyl ether (5 ml/mmol) was added Pd/C (0.05 equivalent). The reaction mixture was bubbled with argon and then with dihydrogen. The suspension was kept overnight under dihydrogen (1 atm), concentrated in vacuo and then purified by chromatography on silica gel (eluent dichloromethane/methanol/NH.sub.4OH) to afford pure compound 107 (78%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.72 (bs, 1H, NH), 8.09 (s, 1H, CH.sub.Ar), 7.90-7.77 (m, 6H, 5 CH.sub.Ar+NH.sub.2), 7.16 (bs, 1H, NH.sub.2), 5.37 (bs, 2H, NH.sub.2); HRMS (ESI) calc. for C.sub.11H.sub.12N.sub.5O: [M+H].sup.+ 230.10363, found 230.1036.

    [0541] Compound 108. To a solution of 4-mercaptobenzamide (1 equivalent) in ethanol (2 mL/mmol) were added 5-amino-4,6-dichloropyrimidine (1 equivalent) and potassium carbonate (1.2 equivalents). The suspension was refluxed for 4 h, concentrated and purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 108 (6%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.06 (bs, 1H, NH.sub.2), 8.01 (s, 1H, CH.sub.Ar), 7.90 (d, J=8.4 Hz, 2H, CH.sub.Ar), 7.58 (d, J=8.4 Hz, 2H, 2 CH.sub.Ar), 7.46 (bs, 1H, NH.sub.2), 5.84 (bs, 2H, NH.sub.2); HRMS (ESI) calc. for C11H.sub.10ClN.sub.4OS: [M+H].sup.+ 281.02584, found 281.0257.

    [0542] Compound 109. This compound was synthesized through general synthesis protocol XVII from 5-amino-4,6-dichloropyrimidine and 4-methylaniline (1 equivalent) and was purified by precipitation in water to afford pure compound 109 (86%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.72 (bs, 1H, NH), 7.84 (s, 1H, CH.sub.Ar), 7.58 (d, J=8.3 Hz, 2H, 2 CH.sub.Ar), 7.14 (d, J=8.3 Hz, 2H, 2 CH.sub.Ar), 5.86 (bs, 2H, NH.sub.2), 2.27 (s, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.11H.sub.12ClN.sub.4: [M+H].sup.+ 235.07450, found 235.0745.

    [0543] Compound 110. This compound was synthesized through general synthesis protocol XVII from 5-amino-4,6-dichloropyrimidine and 4-fluoroaniline (1 equivalent) and was purified by precipitation to afford pure compound 110 (74%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.71 (bs, 1H, NH), 7.85 (s, 1H, CH.sub.Ar), 7.73-7.66 (m, 2H, 2 CH.sub.Ar), 7.22-7.12 (m, 2H, 2 CH.sub.Ar), 4.17 (bs, 2H, NH.sub.2); HRMS (ESI) calc. for C.sub.10H.sub.9ClFN.sub.4: [M+H].sup.+ 239.04943, found 239.0493.

    [0544] Compound 111 was purchased from Sigma-Aldrich Co., Ltd.

    [0545] Compound 112. Argon was bubbled through a suspension of 6-chloro-7-deazapurine (1 equivalent), potassium carbonate (1.5 equivalents), trans-1,2-diaminocyclohexane (1.1 equivalents) and 4-iodobenzamide (1 equivalent) in 1,4-dioxane (6.5 mL/mmol). After 10 min, copper iodide (1 equivalent) was added and the mixture was flushed again with argon. The grey suspension was refluxed for 14 h under argon. After cooling, the suspension was diluted with ethyl acetate and washed with a saturated aqueous solution of EDTA. The aqueous layer was extracted with ethyl acetate (four times). The combined organic layer was washed with brine, dried over MgSO.sub.4, filtrated and concentrated. The residue was purified by chromatography on silica gel (eluent cyclohexane/ethyl acetate) to afford pure compound 112 (22%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.75 (s, 1H, CH.sub.Ar), 8.25 (d, J=3.8 Hz, 1H, CH.sub.Ar), 8.13-8.05 (m, 3H, 2 CH.sub.Ar, NH.sub.2), 8.00 (d, J=8.8 Hz, 2H, 2 CH.sub.Ar), 7.49 (bs, 1H, NH.sub.2), 6.95 (d, J=3.8 Hz, 1H, CH.sub.Ar); HRMS (ESI) calc. for C.sub.13H.sub.10ClN.sub.4O: [M+H].sup.+ 273.05376, found 273.0537.

    [0546] Compound 113. To a solution of 2-amino-6-chloropurine (0.8 equivalent) in a mixture of acetonitrile and water (1/3 v/v, 4 mL/mmol), palladium(II) diacetate (0.04 equivalent), triphenylphosphine-3,3′,3″-trisulfonic acid trisodium salt (0.2 equivalent), Cs.sub.2CO.sub.3 (2.4 equivalents) and 4-carbamoylphenylboronic acid (1 equivalent) were added. The solution was stirred for 5 min under microwave irradiation at 150° C. After cooling to 4° C., the reaction mixture was filtrated and the crude solid was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 113 (23%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 12.70 (bs, 1H, NH), 8.79 (d, J=8.5 Hz, 2H, 2 CH.sub.Ar), 8.14 (s, 1H, CH.sub.Ar), 8.07 (bs, 1H, NH.sub.2), 8.02 (d, J=8.5 Hz, 2H, 2 CH.sub.Ar), 7.45 (bs, 1H, NH.sub.2), 6.43 (bs, 2H, NH.sub.2); HRMS (ESI) calc. for C.sub.12H.sub.11N.sub.6O: [M+H].sup.+ 255.09889, found 255.0988.

    [0547] Compound 114. To a suspension of 3-fluoro-4-nitropyridine-N-oxide (1 equivalent) in absolute ethanol (1 mL/mmol) was added 4-aminobenzamide (3 equivalents). The suspension was refluxed for 8 h under argon. After cooling, methanol was added (15 mL/mmol) and the suspension was stirred for 1 h then filtrated. The precipitate was suspended in methanol (15 mL/mmol). The suspension was stirred for 1 h, filtrated, and the precipitate was washed with methanol to afford pure compound 114 (96%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 9.59 (bs, 1H, NH), 8.12 (d, J=7.3 Hz, 1H, CH.sub.Ar), 7.97 (bs, 1H, NH.sub.2), 7.95-7.89 (m, 3H, 3 CH.sub.Ar), 7.71 (dd, J=7.3, 1.9 Hz, 1H, CH.sub.Ar), 7.45 (d, J=8.6 Hz, 2H, CH.sub.Ar), 7.36 (bs, 1H, NH.sub.2); HRMS (ESI) calc. for C.sub.12H.sub.11N.sub.4O.sub.4: [M+H].sup.+ 275.07803, found 275.0775.

    [0548] Compound 115. To a suspension of 148 (1 equivalent) in methanol (10 ml/mmol) was added Pd/C 10% (0.05 equivalent). The reaction mixture was bubbled with argon and then with dihydrogen. The suspension was kept overnight under dihydrogen (1 atm) and was purified by chromatography on silica gel (eluent dichloromethane/methanol). The pure fractions were then evaporated and the residual oil was precipitated with HCl in diethyl ether 1 M which after filtration afforded pure compound 115 (62%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 10.14 (bs, 1H, NH), 7.98 (bs, 1H, NH.sub.2), 7.94 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.46-7.38 (m, 3H, 3 CH.sub.Ar), 7.38-7.29 (m, 2H, 1 CH.sub.Ar+NH.sub.2), 7.02 (dd, J=7.9, 5.9 Hz, 1H, CH.sub.Ar); HRMS (ESI) calc. for C.sub.12H.sub.13N.sub.4O: [M+H].sup.+ 229.10838, found 229.1083.

    [0549] Compound 116. To a suspension of 16 (1 equivalent) in methanol (10 mL/mmol) was added Pd/C 10% (0.05 equivalent). The reaction mixture was bubbled with argon and then with dihydrogen. The suspension was kept under dihydrogen (1 atm) at 50° C. for four days. It was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 116 (6 mg, 6%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 9.31 (s, 1H, CH.sub.Ar), 9.14 (s, 1H, CH.sub.Ar), 9.05 (s, 1H, CH.sub.Ar), 8.16-8.06 (m, 5H, 4 CH.sub.Ar+NH.sub.2), 7.53 (s, 1H, NH.sub.2); HRMS (ESI) calc. for C.sub.12H.sub.10N.sub.5O: [M+H].sup.+ 240.08853, found 240.0884.

    [0550] Compound 117. To a solution of 2-6-diaminopurine (1 equivalent) in DMF (1 mL/mmol), CuI (1.1 equivalents), N,N′-dimethylethylenediamine (2.2 equivalents), K.sub.3PO.sub.4 (4 equivalents) and 4-iodobenzyl alcohol (1.3 equivalents) were added. The solution was stirred overnight at 90° C. under argon. After concentration under reduced pressure, the reaction mixture was diluted with dichloromethane and extracted with a saturated aqueous solution of ethylenediaminetetraacetic acid (EDTA). The organic layer was washed with water and brine, then dried over magnesium sulfate. After filtration and concentration under reduced pressure the crude product was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 117 (7%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 9.40 (bs, 1H, NH), 8.25 (s, 1H, CH.sub.Ar), 7.98-7.94 (m, 2H, 2 CH.sub.Ar), 7.84-7.78 (m, 2H, 2 CH.sub.Ar), 7.48 (d, J=8.6 Hz, 2H, 2 CH.sub.Ar), 7.23 (d, J=8.6 Hz, 2H, 2 CH.sub.Ar), 6.22 (bs, 2H, NH.sub.2), 5.29 (t, J=5.8 Hz, 1H, OH), 5.05 (t, J=5.7 Hz, 1H, OH), 4.57 (d, J=5.8 Hz, 2H, CH.sub.2), 4.45 (d, J=5.7 Hz, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.19H.sub.19N.sub.6O.sub.2: [M+H].sup.+ 363.15640, found 363.1564.

    [0551] Compound 118. This compound was synthesized through general synthesis protocol VI from compound 81 and was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 118 (47%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.58 (bs, 1H, CH.sub.Ar), 7.91 (bs, 1H, NH), 7.79 (d, J=7.8 Hz, 2H, 2 CH.sub.Ar), 7.65-7.57 (m, 2H, 2 CH.sub.Ar), 7.48 (t, J=7.4 Hz, 1H, CH.sub.Ar), 4.26 (bs, 1H, CH), 1.42 (d, J=7.0 Hz, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.14H.sub.13ClN.sub.5O.sub.2: [M+H].sup.+ 318.07523, found 318.0753.

    [0552] Compound 119. This compound was synthesized through general synthesis protocol VIII from 93 and 1-serine to afford pure compound 119 (91%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.27 (s, 1H, CH.sub.Ar), 7.94 (bs, 1H, NH.sub.2), 7.82 (d, J=8.4 Hz, 2H, 2 CH.sub.Ar), 7.40 (d, J=5.1 Hz, 1H, NH), 7.34 (bs, 1H, NH.sub.2), 7.29 (d, J=8.3 Hz, 2H, 2 CH.sub.Ar), 5.85 (bs, 1H, OH), 5.40 (s, 2H, CH.sub.2), 3.93-3.79 (m, 2H, CH.sub.2), 3.44-3.37 (m, 1H, CH); HRMS (ESI) calc. for C.sub.16H.sub.16ClN.sub.6O.sub.4: [M+H].sup.+ 391.09161, found 391.0922.

    [0553] Compound 120. This compound was synthesized through general synthesis protocol XVI from 4,6-dichloro-5-nitropyrimidine (1 equivalent) and 4-aminoacetanilide (1 equivalent). It was stirred at room temperature under argon for 7 h. The suspension was then filtered and the precipitate was washed with ethyl acetate. The filtrate was concentrated until precipitation. The precipitate was filtrated to afford pure compound 120 (25%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 10.02 (bs, 1H, NH), 9.99 (bs, 1H, NH), 8.47 (s, 1H, CH.sub.Ar), 7.57 (d, J=8.9 Hz, 2H, 2 CH.sub.Ar), 7.39 (d, J=8.9 Hz, 2H, 2 CH.sub.Ar), 2.04 (s, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.12H.sub.11ClN.sub.5O.sub.3: [M+H].sup.+ 308.05449, found 308.0543.

    [0554] Compound 121. This compound was synthesized through general synthesis protocol VII from compound 93 and 1-alanine tert-butyl ester. After concentration, the crude product was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 121 (79%). .sup.1H NMR (400 MHz, d.sub.6-DMSO, 60° C.) δ 8.25 (s, 1H, NH), 8.20 (bs, 1H, CH.sub.Ar), 7.83 (d, J=8.3 Hz, 2H, 2 CH.sub.Ar), 7.33 (d, J=8.3 Hz, 2H, 2 CH.sub.Ar), 5.41 (s, 2H CH.sub.2), 4.53 (bs, 1H, CH), 1.46 (d, J=7.3 Hz, 3H, CH.sub.3), 1.43-1.35 (m, 9H, 3 CH.sub.3); HRMS (ESI) calc. for C.sub.20H.sub.24ClN.sub.6O.sub.3: [M+H].sup.+ 431.15929, found 431.1600.

    [0555] Compound 122. A mixture of 49 (1 equivalent) and urea (43 equivalents) was heated to 160° C. Once the temperature reached the melting point of urea, ethanesulfonic acid (25 μL/mmol) was added. The resulting solution was stirred at 160° C. for 15 h. After cooling down to room temperature, it was diluted with water (13 mL/mmol), filtered, washed with water and dried under reduced pressure. The crude product was purified by chromatography on silica gel (eluent dichloromethane/methanol). The purest fractions were combined and purified by reverse phase chromatography (eluent water/methanol) to afford pure compound 122 (13%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 12.40 (bs, 1H, NH), 8.46 (s, 1H, CH.sub.Ar), 8.08 (bs, 1H, NH.sub.2), 8.03 (d, J=8.6 Hz, 2H, 2 CH.sub.Ar), 7.74 (d, J=8.6 Hz, 2H, 2 CH.sub.Ar), 7.49 (bs, 1H, NH.sub.2); HRMS (ESI) calc. for C.sub.12H.sub.9ClN.sub.5O.sub.2: [M+H].sup.+ 290.04393, found 290.0441.

    [0556] Compound 123. This compound was synthesized through general synthesis protocol XVI from 4,6-dichloro-5-nitropyrimidine (1 equivalent) and 4-methylaminobenzoic acid (1.05 equivalent) to afford pure compound 123 (68%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 13.16 (bs, 1H, COOH), 8.74 (s, 1H, CH.sub.Ar), 7.91 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.48 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 3.57 (s, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.12H.sub.10ClN.sub.4O.sub.4: [M+H].sup.+ 309.03851, found 309.0385.

    [0557] Compound 124. To a suspension of 106 (1 equivalent) in ethanol (5 ml/mmol) was added sodium thiomethoxide (2 equivalents). The suspension was stirred for 1 h at room temperature and then diethyl ether (45 mL/mmol) was added. After filtration the solid was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 124 (78%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 10.61 (bs, 1H, NH), 8.56 (s, 1H, CH.sub.Ar), 7.97 (bs, 1H, NH.sub.2), 7.90 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.69 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.36 (bs, 1H, NH.sub.2), 2.51 (s, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.12H.sub.12N.sub.5O.sub.3S: [M+H].sup.+ 306.06554, found 306.0654.

    [0558] Compound 125. To a solution of 123 (1 equivalent) in anhydrous dichloromethane 5 (8 mL/mmol) was added thionyl chloride (8 equivalents). The solution was stirred for 2 h at room temperature and then evaporated. The residual solid was suspended in anhydrous dichloromethane (5 mL/mmol) and NH.sub.3 in 1,4-dioxane (0.5 mol/L, 5 equivalents) was added. After 2 h, water was added and the reactionnal mixture separated. The aqueous layer was extracted with dichloromethane. The organic layer was washed with brine and was dried over MgSO.sub.4. After evaporation under reduced pressure the crude product was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 125 (83%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.72 (s, 1H, CH.sub.Ar), 8.05 (bs, 1H, NH.sub.2), 7.87 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.47 (bs, 1H, NH.sub.2), 7.44 (d, J=8.6 Hz, 2H, 2 CH.sub.Ar), 3.55 (s, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.12H.sub.11ClN.sub.5O.sub.3: [M+H].sup.+ 308.05449, found 308.0545.

    [0559] Compound 126. To a suspension of 125 (1 equivalent) in a mixture of ethyl acetate (4 mL/mmol) and ethanol (4 mL/mmol) was added tin chloride dihydrate (4 equivalents).

    [0560] After 7 h under reflux, the suspension was concentrated to dryness and the crude product was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 126 (54%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.14 (s, 1H, CH.sub.Ar), 7.83 (bs, 1H, NH.sub.2), 7.80 (d, J=8.9 Hz, 2H, 2 CH.sub.Ar), 7.18 (bs, 1H, NH.sub.2), 6.86 (d, J=8.8 Hz, 2H, 2 CH.sub.Ar), 5.05 (bs, 2H, NH.sub.2), 3.34 (s, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.12H.sub.13ClN.sub.5O: [M+H].sup.+ 278.08031, found 278.0802.

    [0561] Compound 127. This compound was synthesized through general synthesis protocol II from 9 and [3-(3-fluorophenyl)propyl]amine (10 equivalents) to afford pure compound 127 (41%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 12.13 (bs, 1H, NH), 7.62 (bs, 2H, CH.sub.Ar+NH), 7.34-7.23 (m, 3H, 3 CH.sub.Ar), 7.08-7.01 (m, 2H, 2 CH.sub.Ar), 7.01-6.93 (m, 1H, CH.sub.Ar), 6.82 (d, J=8.6 Hz, 2H, CH.sub.Ar), 6.28 (bs, 1H, NH), 4.54 (bs, 2H, CH.sub.2), 3.69 (s, 3H, CH.sub.3), 3.27-3.17 (m, 2H, CH.sub.2), 2.62 (t, J=6.5 Hz, 2H, CH.sub.2), 1.84-1.75 (m, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.22H.sub.24FN.sub.6O: [M+H].sup.+ 407.19901, found 407.1995.

    [0562] Compound 128. This compound was synthesized through general synthesis protocol III from compound 127, and was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 128 (17%). .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.76 (s, 1H, CH.sub.Ar), 7.26 (td, J=8.2, 6.1 Hz, 1H, CH.sub.Ar), 7.03 (d, J=7.8 Hz, 1H, CH.sub.Ar), 6.98-6.92 (m, 1H, CH.sub.Ar), 6.88 (td, J=8.2, 1.9 Hz, 1H, CH.sub.Ar), 3.37 (t, J=7.0 Hz, 2H, CH.sub.2), 2.75-2.65 (m, 2H, CH.sub.2), 1.96-1.84 (m, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.14H.sub.16FN.sub.6: [M+H].sup.+ 287.14150, found 287.1416.

    [0563] Compound 129. To a suspension of 124 (1 equivalent) in methanol (10 ml/mmol) was added Pd/C 10% (0.1 equivalent). The reaction mixture was bubbled with argon and then with dihydrogen. The suspension was kept overnight under dihydrogen (1 atm), and was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 129 (16%). .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.11 (s, 1H, CH.sub.Ar), 7.85 (d, J=8.9 Hz, 2H, 2 CH.sub.Ar), 7.75 (d, J=8.9 Hz, 2H, 2 CH.sub.Ar), 2.59 (s, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.12H.sub.14N.sub.5OS: [M+H].sup.+ 276.09135, found 276.0913.

    [0564] Compound 130. To a solution of 6-amino-2-chloropurine (0.8 equivalent) in a mixture of acetonitrile and water (1/3 v/v, 4 mL/mmol), palladium(II) diacetate (0.04 equivalent), triphenylphosphine-3,3′,3″-trisulfonic acid trisodium salt (0.2 equivalent), Cs.sub.2CO.sub.3 (2.4 equivalents) and 4-carbamoylphenylboronic acid (1 equivalent) were added. The solution was stirred for 10 min under microwave irradiation at 150° C. After cooling to 4° C., the reaction mixture was filtrated and the crude solid was purified by chromatography on silica gel (eluent dichloromethane/methanol) and crystallized from ethanol to afford compound 130 (3%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 12.93 (s, 1H, NH), 8.38 (d, J=8.5 Hz, 2H, 2 CH.sub.Ar), 8.12 (bs, 1H, NH.sub.2), 8.02 (bs, 1H, NH.sub.2), 7.95 (d, J=8.6 Hz, 2H, 2 CH.sub.Ar), 7.23 (bs, 2H, NH.sub.2); HRMS (ESI) calc. for C.sub.12H.sub.11N.sub.6O: [M+H].sup.+ 255.09889, found 255.0988.

    [0565] Compound 131. This compound was synthesized through general synthesis protocol II from compound 9 and 4-fluorophenylethylamine (10 equivalents) to afford pure compound 131 (74%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 12.13 (bs, 1H, NH), 7.64 (bs, 2H, NH+CH.sub.Ar), 7.27 (d, J=8.6 Hz, 2H, 2 CH.sub.Ar), 7.20 (bs, 2H, 2 CH.sub.Ar), 7.08 (t, J=8.9 Hz, 2H, 2 CH.sub.Ar), 6.83 (d, J=8.6 Hz, 2H, 2 CH.sub.Ar), 6.26 (bs, 1H, NH), 4.56 (bs, 2H, CH.sub.2), 3.50-3.38 (m, 2H, CH.sub.2), 2.79 (t, J=7.3 Hz, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.12H.sub.22FN.sub.6O: [M+H].sup.+ 393.18336, found 393.1831.

    [0566] Compound 132 was purchased from Fisher Scientific SAS.

    [0567] Compound 133 was purchased from Tokyo Chemical Industry Co., Ltd.

    [0568] Compound 134 was purchased from Sigma-Aldrich Co., Ltd.

    [0569] Compound 135 was purchased from Sigma-Aldrich Co., Ltd.

    [0570] Compound 136 was purchased from Fluorochem, Ltd.

    [0571] Compound 137 was purchased from Sigma-Aldrich Co., Ltd.

    [0572] Compound 138 was purchased from Tokyo Chemical Industry Co., Ltd.

    [0573] Compound 139 was purchased from Tokyo Chemical Industry Co., Ltd.

    [0574] Compound 140. This compound was synthesized through general synthesis protocol XV from 4-amino-2,6-dichloropyrimidine and 4-aminobenzamide (1.1 equivalents) to afford pure compound 140 (39%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 10.56 (s, 1H, NH), 10.21 (s, 1H, NH), 8.13-7.73 (m, 8H, 2 NH.sub.2+4 CH.sub.Ar), 7.66 (d, J=8.5 Hz, 2H, 2 CH.sub.Ar), 7.55 (d, J=8.2 Hz, 2H, 2 CH.sub.Ar), 7.30 (m, 2H, 2 NH.sub.2), 5.64 (s, 1H, CH.sub.Ar); HRMS (ESI) calc. for C.sub.18H.sub.18N.sub.7O.sub.2: [M+H].sup.+ 364.15195, found 364.1518.

    [0575] Compound 141. To a suspension of 105 (1 equivalent) in a mixture of methanol (4 ml/mmol) and diethyl ether (4 ml/mmol) was added Pd/C 10% (0.04 equivalent). The reaction mixture was bubbled with argon and then with dihydrogen. The suspension was kept overnight under dihydrogen (1 atm), and was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 141 (62%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 7.97 (s, 1H, CH.sub.Ar), 7.88 (bs, 1H, NH), 7.81 (d, J=8.6 Hz, 2H, 2 CH.sub.Ar), 7.77 (bs, 1H, NH.sub.2), 7.71 (d, J=5.3 Hz, 1H, CH.sub.Ar), 7.12 (bs, 1H, NH.sub.2), 7.06 (d, J=8.8 Hz, 2H, 2 CH.sub.Ar), 7.04 (d, J=5.6 Hz, 1H, CH.sub.Ar), 4.99 (bs, 2H, NH.sub.2). HRMS (ESI) calc. for C.sub.12H.sub.13N.sub.4O: [M+H].sup.+ 229.10838, found 229.1084.

    [0576] Compound 142. This compound was synthesized through general synthesis protocol XV from 5-amino-4,6-dichloropyrimidine and 4-(aminomethyl)benzamide to afford pure compound 142 (51%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 7.91 (bs, 1H, NH.sub.2), 7.82 (d, J=8.3 Hz, 2H, 2 CH.sub.Ar), 7.72 (s, 1H, CH.sub.Ar), 7.48-7.40 (m, 1H, NH), 7.37 (d, J=8.3 Hz, 2H, 2 CH.sub.Ar), 7.30 (bs, 1H, NH.sub.2), 5.11 (bs, 2H, NH.sub.2), 4.67 (d, J=5.7 Hz, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.12H.sub.13ClN.sub.5O: [M+H].sup.+ 278.08031, found 278.0802.

    [0577] Compound 143. To a solution of 4-aminobenzamide (1 equivalent) in anhydrous DMF (1 ml/mmol) were successively added 2,4-dichloro-3-nitropyridine (1 equivalent) and potassium carbonate (1.2 equivalents). The resulting suspension was stirred for 8 h at 100° C. under argon, concentrated in vacuo and then purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 143 (42%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 9.43 (s, 1H, NH), 8.12 (d, J=6.0 Hz, 1H, CH.sub.Ar), 7.97 (bs, 1H, NH.sub.2), 7.93 (d, J=8.6 Hz, 2H, 2 CH.sub.Ar), 7.47-7.27 (m, 3H, NH.sub.2+2 CH.sub.Ar), 7.16 (d, J=6.0 Hz, 1H, CH.sub.A). HRMS (ESI) calc. for C.sub.12H.sub.10ClN.sub.4O.sub.3: [M+H].sup.+ 293.04359, found 293.0433.

    [0578] Compound 144. This compound was synthesized through general synthesis protocol XVII from 5-amino-4,6-dichloropyrimidine and 4-chlorobenzamide (1 equivalent) and was purified by crystallization from methanol to afford pure compound 144 (73%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.92 (bs, 1H, NH), 7.88 (s, 1H, CH.sub.Ar), 7.82-7.75 (m, 2H, 2 CH.sub.Ar), 7.41-7.33 (m, 2H, 2 CH.sub.Ar), 6.67 (bs, 2H, NH.sub.2); HRMS (ESI) calc. for C.sub.10H.sub.9Cl.sub.2N.sub.4: [M+H].sup.+ 255.01988, found 255.0199.

    [0579] Compound 145. To a solution of compound 106 (1 equivalent) in methanol (10 mL/mmol) were added glycine (1 equivalent) and diisopropylethylamine (2 equivalents). The solution was refluxed for 1 h, concentrated under reduced pressure then suspended in water. The suspension was filtrated and the solid was washed several times with water to afford pure compound 145 (99%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 12.85 (bs, 1H, COOH), 10.96 (bs, 1H, NH), 9.58 (t, J=5.6 Hz, 1H, NH), 8.18 (s, 1H, CH.sub.Ar), 7.94 (bs, 1H, NH.sub.2), 7.88 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.72 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.33 (bs, 1H, NH.sub.2), 4.23 (d, J=5.6 Hz, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.13H.sub.13N.sub.6O.sub.5: [M+H].sup.+ 333.09416, found 333.0941.

    [0580] Compound 146. This compound was synthesized through general synthesis protocol III from compound Int-1 and was purified by precipitation in water to afford pure compound 146 (63%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.56 (s, 1H, CH.sub.Ar), 7.91 (bs, 2H, NH.sub.2), 7.85-7.77 (m, 2H, 2 CH.sub.Ar), 7.65-7.57 (m, 2H, 2 CH.sub.Ar), 7.52-7.45 (m, 1H, CH.sub.Ar); HRMS (ESI) calc. for C.sub.11H.sub.9ClN.sub.5: [M+H].sup.+ 246.05410, found 246.0540.

    [0581] Compound 147. To a refluxed suspension of 143 (1 equivalent) in ethyl acetate (5 ml/mmol) was added portionwise tin chloride dihydrate (5.7 equivalents) over 2 h 30.

    [0582] After cooling, the reaction was quenched by the addition of a saturated aqueous solution of NaHCO.sub.3. The aqueous layer was extracted four times with ethyl acetate. The combined organic layers were washed with brine, dried over MgSO.sub.4, filtrated, concentrated in vacuo, and purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 147 (51%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.05 (bs, 1H, NH), 7.82 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.79 (s, 1H, NH.sub.2), 7.52 (d, J=5.2 Hz, 1H, CH.sub.Ar), 7.15 (bs, 1H, NH.sub.2), 7.10 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.06 (d, J=5.3 Hz, 1H, CH.sub.Ar), 5.14 (bs, 2H, NH.sub.2); HRMS (ESI) calc. for C.sub.12H.sub.12ClN.sub.4O: [M+H].sup.+ 263.06942, found 263.0692.

    [0583] Compound 148. To a solution of 4-aminobenzamide (1 equivalent) in absolute ethanol (0.6 ml/mmol) were added 2-chloro-3-nitropyridine (1 equivalent) and potassium carbonate (1.2 equivalents). The suspension was heated under microwave irradiation at 170° C. for 1 h 30, then purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 148 (19%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 10.06 (s, 1H, NH), 8.59-8.53 (m, 2H, 2 CH.sub.Ar), 7.89 (bs, 1H, NH.sub.2), 7.88 (d, J=8.8 Hz, 2H, CH.sub.Ar), 7.78 (d, J=8.8 Hz, 2H, 2 CH.sub.Ar), 7.27 (m, 1H, NH.sub.2), 7.09-7.03 (m, 1H, CH.sub.Ar); HRMS (ESI) calc. for C.sub.12H.sub.11N.sub.4O.sub.3: [M+H].sup.+ 259.08257, found 259.0823.

    [0584] Compound 149. To a solution of 4-aminobenzamide (1 equivalent) in ethanol (2 mmol/mL) was added 4,6-dichloropyrimidine (1 equivalent) and the solution was refluxed for 7 h. After cooling, the precipitate was filtrated and recrystallized from methanol (60 mL) to afford pure compound 149 (37%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 10.09 (bs, 1H, NH), 8.55 (s, 1H, CH.sub.Ar), 7.91-7.81 (m, 3H, 2 CH.sub.Ar+NH.sub.2), 7.72 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.24 (bs, 1H, NH.sub.2), 6.88 (s, 1H, CH.sub.Ar); HRMS (ESI) calc. for C.sub.11H.sub.10ClN.sub.4O: [M+H].sup.+ 249.05376, found 249.0536.

    [0585] Compound 150. This compound was synthesized through general synthesis protocol XVII from 5-amino-4,6-dichloropyrimidine and aniline (1 equivalent), and was filtrated from the reaction mixture to afford pure compound 150 (50%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.58 (bs, 1H, NH), 7.86 (s, 1H, CH.sub.Ar), 7.73-7.65 (m, 2H, 2 CH.sub.Ar), 7.37-7.29 (m, 2H, 2 CH.sub.Ar), 7.08-7.01 (m, 1H, CH.sub.Ar), 5.44 (bs, 2H, NH.sub.2); HRMS (ESI) calc. for C.sub.10H.sub.10ClN.sub.4: [M+H].sup.+ 221.05885, found 221.0589.

    [0586] Compound 151. To a solution of 4,6-dichloro-5-nitropyrimidine (1 equivalent) in THF (2.5 mL/mmol), NaHCO.sub.3 (1.5 equivalents) and 4-aminoacetanilide (1 equivalent) were added and stirred at room temperature under argon for 7 h. The suspension was filtered and the precipitate washed with ethyl acetate. The precipitate was then triturated in warm methanol and filtered to afford pure compound 151 (18%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 10.87 (bs, 2H, NH), 9.99 (bs, 2H, NH), 8.10 (s, 1H, CH.sub.Ar), 7.59 (d, J=8.8 Hz, 4H, 2 CH.sub.Ar), 7.51 (d, J=8.8 Hz, 4H, 2 CH.sub.Ar), 2.05 (s, 6H, 2 CH.sub.3); HRMS (ESI) calc. for C.sub.20H.sub.20N.sub.7O.sub.4: [M+H].sup.+ 422.15713, found 422.1570.

    [0587] Compound 152. To a suspension of 147 in trimethyl orthoformate (5 mL/mmol) was added ethanesulfonic acid (25 μL/mmol). The suspension was stirred for 24 h under reflux. The crude mixture was concentrated under reduced pressure, to afford pure compound 152 (82%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.90 (s, 1H, CH.sub.Ar), 8.26 (d, J=5.6 Hz, 1H, CH.sub.Ar), 8.17 (bs, 1H, NH.sub.2), 8.14 (d, J=8.6 Hz, 2H, 2 CH.sub.Ar), 7.83 (d, J=8.6 Hz, 2H, 2 CH.sub.Ar), 7.76 (d, J=5.6 Hz, 1H, CH.sub.Ar), 7.56 (bs, 1H, NH.sub.2); HRMS (ESI) calc. for C.sub.13H.sub.10ClN.sub.4O: [M+H].sup.+ 273.05377, found 273.0535.

    [0588] Compound 153. This compound was synthesized through general synthesis protocol VI from compound 121, was purified by extraction dichloromethane/water to afford pure compound 153 (95%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 12.63 (bs, 1H, COOH), 8.51 (d, J=7.3 Hz, 1H, NH), 8.33 (s, 1H, CH.sub.Ar), 7.93 (bs, 1H, NH.sub.2), 7.83 (d, J=8.3 Hz, 2H, 2 CH.sub.Ar), 7.36 (bs, 1H, NH.sub.2), 7.31 (d, J=8.3 Hz, 2H, 2 CH.sub.Ar), 5.42 (s, 2H, CH.sub.2), 4.68-4.52 (m, 1H, CH), 1.46 (d, J=7.3 Hz, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.16H.sub.16ClN.sub.6O.sub.3: [M+H].sup.+ 375.09669, found 375.0965.

    [0589] Compound 154. A suspension of 112 in NH.sub.4OH 30% (22 mL/mmol), was heated to 100° C. for 1 h 30 under microwave irradiation. After concentration under reduced pressure, the reaction mixture was purified by chromatography on silica gel (eluent dichloromethane/methanol), to afford pure compound 154 (67%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.16 (s, 1H, CH.sub.Ar), 8.04 (bs, 1H, NH.sub.2), 8.03-7.97 (m, 4H, 4 CH.sub.Ar), 7.67 (d, J=3.7 Hz, 1H, CH.sub.Ar), 7.41 (bs, 1H, NH.sub.2), 7.20 (bs, 2H, NH.sub.2), 6.81 (d, J=3.7 Hz, 1H, CH.sub.Ar); HRMS (ESI) calc. for C.sub.13H.sub.12N.sub.5O: [M+H].sup.+ 254.10364, found 254.1033.

    [0590] Compound 155. To a suspension of 106 (1 equivalent) in methanol (8 mL/mmol), sodium methoxide (4 equivalents) was added. The suspension was stirred at 50° C. for 40 min under argon. After concentration under reduced pressure, the crude product was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 155 (99%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 9.96 (bs, 1H, NH), 8.45 (s, 1H, CH.sub.Ar), 7.92 (bs, 1H, NH.sub.2), 7.86 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.63 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.31 (bs, 1H, NH.sub.2), 4.02 (s, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.12H.sub.12N.sub.5O.sub.4: [M+H].sup.+ 290.08838, found 290.0886.

    [0591] Compound 156. To a suspension of 120 (1 equivalent) in a mixture of ethyl acetate (2 mL/mmol) and ethanol (2 mL/mmol) was added tin chloride dihydrate (5 equivalents). After 24 h under reflux, the suspension was concentrated to dryness and the crude product was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 156 (29%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 9.87 (bs, 1H, NH), 8.53 (bs, 1H, NH), 7.82 (s, 1H, CH.sub.Ar), 7.58 (d, J=9.1 Hz, 2H, 2 CH.sub.Ar), 7.52 (d, J=9.1 Hz, 2H, 2 CH.sub.Ar), 5.38 (bs, 2H, NH.sub.2), 2.02 (s, 3H, CH.sub.3); HRMS (ESI) calc. for C.sub.12H.sub.13ClN.sub.5O: [M+H].sup.+ 278.08031, found 278.0808.

    [0592] Compound 157. To a suspension of sodium hydride (3 equivalents) in benzyl alcohol (5.5 mL/mmol) was added 106 (1 equivalent). The suspension was stirred for 2 h at room temperature and then diethyl ether (27 mL/mmol) was added. The suspension was filtered and the solid was purified by chromatography on silica gel (eluent dichloromethane/methanol) to afford pure compound 157 (81%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 9.98 (bs, 1H, NH), 8.46 (s, 1H, CH.sub.Ar), 7.92 (bs, 1H, NH.sub.2), 7.86 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.63 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 7.49-7.33 (m, 5H, 5 CH.sub.Ar), 7.32 (bs, 1H, NH.sub.2), 5.55 (s, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.18H.sub.16N.sub.5O.sub.4: [M+H].sup.+ 366.11968, found 366.1197.

    [0593] Compound 158. To a solution of 5-amino-4,6-dichloropyrimidine (1 equivalent) in a mixture of 1,4-dioxane (3.3 mL/mmol) and water (0.33 mL/mmol) were added 4-benzyloxybenzylamine (1.3 equivalent) and NaHCO.sub.3 (2 equivalents). The solution was refluxed for 24 h. After concentration under reduced pressure, the crude product was triturated in water and filtered. The resulting precipitate was then triturated in methanol and filtered to afford pure compound 158 (88%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 7.74 (s, 1H, CH.sub.Ar), 7.47-7.36 (m, 4H, 4 CH.sub.Ar), 7.36-7.30 (m, 1H, CH.sub.Ar), 7.29-7.21 (m, 3H, 2 CH.sub.Ar+NH), 6.98 (d, J=8.7 Hz, 2H, 2 CH.sub.Ar), 5.10 (s, 2H, CH.sub.2), 5.08 (bs, 2H, NH.sub.2), 4.55 (d, J=5.5 Hz, 2H, CH.sub.2); HRMS (ESI) calc. for C.sub.18H.sub.18ClN.sub.4O: [M+H].sup.+ 341.11637, found 341.1167.

    [0594] Compound II-1. To a solution of 4-chloroquinoline (1 equivalent) in DMF (7 mL/mmol), palladium(0) tetrakis(triphenylphosphine) (0.1 equivalent) was added. To a solution of potassium carbonate (2.1 equivalents) in DMF (2 mL/mmol), 4-carbamoylphenylboronic acid (1.05 equivalent) was added. After 10 min of stirring the two solutions were combined and refluxed overnight. After filtration on celite, and evaporation under reduced pressure, the crude product was purified by chromatography on silica gel (elution with dichloromethane/methanol) to afford pure compound II-1 (52%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.98 (d, J=4.4 Hz, 1H, CH.sub.Ar), 8.25-8.18 (m, 1H, CH.sub.Ar), 8.02-7.96 (m, 2H, 2 CH.sub.Ar), 7.88-7.82 (m, 1H, CH.sub.Ar), 7.76 (ddd, J=8.3, 6.9, 1.3 Hz, 1H, CH.sub.Ar), 7.64-7.59 (m, 2H, 2 CH.sub.Ar), 7.53 (ddd, J=8.3 Hz, J=6.9 Hz, J=1.3 Hz, 1H, CH.sub.Ar), 7.35 (d, J=4.4 Hz, 1H, CH.sub.Ar), 6.16 (bs, 1H, NH), 5.71 (bs, 1H, NH); HRMS (ESI) calc. for C.sub.16H.sub.13N.sub.2O: [M+H].sup.+ 249.10224, found 249.1022.

    [0595] Compound II-2. To a solution of 5-chloroquinoline (1 equivalent) in DMF (7 mL/mmol), palladium(0) tetrakis(triphenylphosphine) (0.1 equivalent) was added. To a solution of potassium carbonate (2.1 equivalents) in DMF (2 mL/mmol), 4-carbamoyl-phenylboronic acid (1.05 equivalent) was added. After 10 min of stirring the two solutions were combined and refluxed overnight. After filtration on celite, and evaporation under reduced pressure, the crude product was purified by chromatography on silica gel (elution with dichloromethane/methanol) to afford pure compound II-2 (28%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.95 (dd, J=4.1, 1.6 Hz, 1H, CH.sub.Ar), 8.22-8.16 (m, 1H, CH.sub.Ar), 8.14-8.07 (m, 2H, 2 CH.sub.Ar), 8.07-8.02 (m, 2H, 2 CH.sub.Ar), 7.85 (dd, J=8.5, 7.1 Hz, 1H, CH.sub.Ar), 7.63-7.56 (m, 3H, 2 CH.sub.Ar+NH), 7.54 (dd, J=8.6, 4.1 Hz, 1H, CH.sub.Ar), 7.46 (bs, 1H, NH).

    II. Biology

    Materials and Methods

    Material

    Pharmacology, Chemicals and Reagents.

    [0596] The CFTR activator forskolin and potentiator genistein (Illek et al., 1995) were purchased from LC Laboratories (PKC Pharmaceuticals, Woburn, Mass.). We purchased DIDS (5-Isothiocyanato-2-[2-(4-isothiocyanato-2-sulfophenyl)ethenyl]benzene-1-sulfonic acid) and VX809 (3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid) from Selleckchem (Houston, USA). Stock solutions of F (10 mM), G (30 mM), CFTRinh-172 (10 mM), VX809 (10 mM), were prepared in DMSO. We prepared stock solution of iminosugars, miglustat and IsoLAB ([1,4-dideoxy-2-hydroxymethyl-1,4-imino-L-threitol]) dissolved in water (100 mM) before further dilution.

    Methods

    Cell Culture.

    [0597] HeLa cells expressing wild-type CFTR (spTCF-wt) or F508del-CFTR (spTCF-ΔF) were cultured in Dulbecco's modified Eagle's medium+GlutaMAX™-I (Invitrogen) supplemented with 8% (v/v) fetal bovine serum (FBS), 1% (v/v) penicillin/streptomycin and were selected using Zeocin (50 μg/mL). Both cell lines were grown in standard culture conditions (37° C., 5% C02). Cells were plated in 35 mm plastic dishes for whole-cell patch-clamp recordings and western blot analysis and in 96-well plates for cytotoxicity assay. For all cell culture, culture media were renewed every 2 days.

    Western Blot Analysis.

    [0598] Cell lysates (10 mM Tris, 1% Nonidet P-40, 0.5% sodium deoxycholate, pH 7.5) were separated 72 h after seeding for HeLa cells by 5% SDS-PAGE (50 μg of protein/well). After saturation, nitrocellulose membrane was incubated overnight at 4° C. in phosphate-buffered saline, 0.1% Tween 20 with 1 μg/ml mouse anti-CFTR monoclonal antibody (clone MAB3480; Chemicon International, Millipore Bioscience Research Reagents, Temecula, Calif.). After washing, goat peroxidase-conjugated anti-mouse IgG (1:10 000; Sigma-Aldrich) was used as secondary antibody. CFTR was visualized by chemiluminescence with ECL Western blotting detection reagent (GE Healthcare, Buckinghamshire, UK).

    Iodide Efflux.

    [0599] CFTR chloride channel activity was assayed by measuring the rate of iodide (.sup.125I) efflux from living cells. All experiments were performed with a MultiPROBE®IIex robotic liquid handling system (Perkin Elmer Life Sciences, Courtaboeuf, France). At the beginning of each experiment, cells were washed twice with efflux buffer containing (in mM) 136.9 NaCl, 5.4 KCl, 0.3 KH.sub.2PO.sub.4, 0.3 NaH.sub.2PO.sub.4, 1.3 CaCl.sub.2, 0.5 MgCl.sub.2, 0.4 MgSO.sub.4, 5.6 glucose and 10 HEPES, pH 7.4. Cells were incubated in efflux buffer containing Na.sup.125I (1 μCi Na.sup.125I/ml, NEN, Boston, Mass.) during 1 h at 37° C., then washed with efflux medium to remove extracellular .sup.125I. The loss of intracellular .sup.125I was determined by removing the medium with efflux buffer every 1 min for up to 10 min. The first three aliquots were used to establish a stable baseline in efflux buffer alone. A medium containing the activators of CFTR (Fsk 10 μM+Gst 30 μM) and the appropriate drug was used for the remaining aliquots. Residual radioactivity was extracted with 0.1 N NaOH/0.1% SDS, and determined using a Packard Cobra™II gamma counter (Perkin Elmer life Sciences, Courtaboeuf, France). The fraction of initial intracellular .sup.125I lost during each time point was collected and time-dependent rates of .sup.125I efflux calculated from: ln (.sup.125It.sub.1/.sup.125It.sub.2)/(t.sub.1−t.sub.2) where .sup.125It is the intracellular .sup.125I at time t, and t.sub.1 and t.sub.2 successive time points. Curves were constructed by plotting rate of .sub.125I versus time. All comparisons were based on maximal values for the time-dependent rates (k=peak rates, min−1) excluding the points used to establish the baseline (k.sub.peak−k.sub.basal, min.sup.−1), and histograms were presented as percentage of activation.

    Time Dependence.

    [0600] Time dependence of the compounds was assayed by measuring the time necessary for an optimal correction of the protein F508del-CFTR. HeLa cells expressing F508del-CFTR were incubated with the compound in acute treatment or during 4, 8, 12, 24 or 48 hours and the iodide efflux was measured as described previously. In some experiments, the incubation with the compound and the measure of iodide efflux are separated by a rinse and a waiting period of 4, 6, 8, 12 or 24 hours before activation with the medium containing the activators of CFTR (Fsk 10 μM+Gst 30 μM).

    Statistical Analysis.

    [0601] Results are expressed as means±SE of n observations. Statistical analysis was carried out using GraphPad (Prism, La Jolla, Calif.) version 5.0 for Windows (GraphPad Software). To compare sets of data, we used one-way Anova followed by Dunnett multiple-comparison test or Student's t test. Differences were considered statistically significant when P<0.05.

    Results

    Maturation Profile

    [0602] In order to evaluate the effect of the compounds on the correction of the mutant protein, we assessed the maturation F508del-CFTR maturation profile. WT core-glycosylated immature CFTR (referred here as B-band CTFR) reaches the plasma membrane after a modification process that allows the protein to become mature and fully glycosylated (referred here as C-band CFTR). On the contrary, F508del-CFTR remains trapped in the endoplasmic reticulum (ER) as B-band CFTR.

    [0603] We thus performed a series of western blot experiments mimicking all the experimental conditions to monitor the C-band appearance.

    [0604] Immunoblots are presented in FIG. 1. As shown in FIG. 1, we observed mature, fully glycosylated C-band F508del-CFTR in the presence of the compounds 1, 7, 10, 14, 20 and 49, compared with untreated cells. Therefore, the compounds of the invention correct the trafficking of F508del-CFTR to the plasma membrane, where the protein can play its role of chloride channel.

    Iodide Transport

    [0605] Next we investigated the effect of the compounds on the activity of F508del-CFTR by performing iodide efflux experiments. Iodide efflux of cells treated with the compounds was measured and compared with iodide efflux obtained with incubation with VX809, a known corrector of CFTR, normalized at 100%. Results were classified in 3 groups: +++≧60%; 35%≦++<60% and 10%≦+<35%.

    [0606] The results are shown in Table 3. Iodide efflux of untreated cells expressing F508del-CFTR is below 10% (3.6%).

    TABLE-US-00003 TABLE 3 Iodide efflux of cells expressing F508del and incubated with different compounds of the invention Compound Iodide number efflux II-1 +++ 1 ++ 2 + 4 +++ 5 ++ 6 + 7 +++ 9 + 10 ++ 11 +++ 12 + 14 +++ 15 + 16 + 17 + 18 ++ 20 ++ 21 +++ 22 ++ 23 +++ 25 + 26 + 27 +++ 28 ++ 29 +++ 30 ++ 31 + 33 +++ 47 +++ 48 +++ 49 +++ 53 + 54 +++ 55 +++ 56 +++ 57 +++ 58 ++ 59 ++ 60 ++ 61 ++ 62 + 63 + 64 + 65 ++ 79 + 80 + 81 + 82 + 83 + 85 + 86 + 88 + 89 + 90 + 91 + 92 + 93 + 94 + 95 + 96 + 97 + 98 + 101 ++ 102 +++ 103 + 104 ++ 105 +++ 106 + 107 +++ 108 + 109 ++ 110 + 111 ++ 112 + 113 +++ 114 +++ 115 +++ 116 ++ 117 ++ 118 +++ 119 ++ 120 ++ 121 + 122 + 123 + 124 + 125 +++ 126 +++ 127 ++ 128 ++ 129 +++ 130 +++

    [0607] The results show that the tested compounds restore the activation of a chloride conductance dependent of AMPc on cells expressing F508del-CFTR, compared with untreated cells. Thus these compounds have a corrector activity on F508del-CFTR.

    [0608] Taken together, these results demonstrate that the compounds of the invention are good candidates for the treatment of cystic fibrosis.

    Combination of Compounds

    [0609] Then we tested combinations of compounds of the invention with other correctors of F508del-CFTR, especially with Isolab and VX-809, by performing the same iodide efflux experiments. The results, presented in FIG. 2, show that the combination of the tested compounds enhances the conductance of F508del-CFTR more than the compounds alone.

    [0610] These results demonstrate the interest to use a combination of compounds to treat cystic fibrosis.

    Time Dependence

    [0611] To evaluate the relevancy of the correction of F508del-CFTR by the compound of the invention, we then tested its “time dependence” by studying the period of incubation necessary for an optimal correction. The results presented in FIG. 3 show that the optimal correction is reached after a treatment of 12 hours and is conserved after a treatment of 24 or 48 hours.

    [0612] We then wondered if the compound could modify the stability of the protein. After the incubation with the compound during the necessary time for an optimal correction, the cells were rinsed. After 4, 6, 8, 12 or 24 hours the activation of CFTR was realized and the activity of F508del-CFTR measured. As shown in FIG. 4A, the corrector activity of the compound 49 remains after 8 hours and reaches 50% of its maximum after 12 hours. Besides, the same experiments performed with the corrector VX-809 presented in FIG. 4B show less than 50% of activity after 8 hours.