KINASE INHIBITORS
20170209445 ยท 2017-07-27
Inventors
Cpc classification
C07D409/12
CHEMISTRY; METALLURGY
A61K31/517
HUMAN NECESSITIES
A61P29/00
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
C07F9/65128
CHEMISTRY; METALLURGY
A61K31/496
HUMAN NECESSITIES
C07D403/12
CHEMISTRY; METALLURGY
C07F9/65583
CHEMISTRY; METALLURGY
A61K31/5355
HUMAN NECESSITIES
A61K31/541
HUMAN NECESSITIES
International classification
A61K31/517
HUMAN NECESSITIES
A61K31/541
HUMAN NECESSITIES
A61K31/496
HUMAN NECESSITIES
Abstract
Compounds of formula I,
##STR00001##
have anti-inflammatory activity. Exemplary mechanisms of action include inhibition of one or more of members of: the family of p38 mitogen-activated protein kinase enzymes; Syk kinase; and members of the Src family of tyrosine kinases. The compounds have use in therapy, including in pharmaceutical combinations, especially in the treatment of inflammatory diseases, including inflammatory diseases of the lung, eye and intestines.
Claims
1. A compound of formula I, ##STR00084## wherein R.sup.1 represents -L.sup.1-C(O)NR.sup.XR.sup.Y, -L.sup.2-S(O).sub.2R.sup.Y1, -L.sup.3-P(O)R.sup.Y1R.sup.Y2, -L.sup.4-S(O).sub.2NR.sup.XR.sup.Y, -L.sup.5-C(O)R.sup.Y, -L.sup.6-S(O).sub.0-1R.sup.Y1, -L.sup.7-C(O)OR.sup.Y, NS(O)(CH.sub.3).sub.2 or S(O)(NR.sup.X)CH.sub.3 NR.sup.XR.sup.X1, Het.sup.1 optionally substituted with one or more substituents selected from the group consisting of halo, hydroxy, C.sub.1-4 alkyl and C.sub.1-4 alkoxy, [C.sub.1-4 alkylene].sub.0-1-Het.sup.2, which Het.sup.2 group is optionally substituted with one or more substituents selected from the group consisting of halo, hydroxy, oxo, C.sub.1-4 alkyl and C.sub.1-4 alkoxy, [C.sub.1-4 alkylene].sub.0-1CN, C.sub.1-4 alkylene-OH C.sub.1-4 alkylene-OP(O)(OH).sub.2 or halo or, when either one or none of R.sup.4A to R.sup.4C represents H, then R.sup.1 may alternatively represent H; L.sup.1, L.sup.2, L.sup.3, L.sup.4, L.sup.5, L.sup.6 and L.sup.7 independently represent a bond, [C(R.sup.a)(R.sup.b)].sub.1-2, C(R.sup.a)(R.sup.b)N(R.sup.X2), where the C-atom is connected to the phenyl ring, or L.sup.1, L.sup.2, L.sup.3, L.sup.4, L.sup.5 or L.sup.7 represents N(R.sup.X2), or L.sup.1, L.sup.2 or L.sup.4 represents O, or L.sup.3 or L.sup.7 represents OC(R.sup.a)(R.sup.b), wherein the O-atom of the latter substituent is attached to the phenyl ring; R.sup.X and R.sup.X1 independently represent H or C.sub.1-6 alkyl optionally substituted by hydroxy or OP(O)(OH).sub.2, or R.sup.X1 represents Het.sup.1 optionally substituted with one or more substituents selected from the group consisting of halo, hydroxy, C.sub.1-4 alkyl and C.sub.1-4 alkoxy; R.sup.Y, R.sup.Y1 and R.sup.Y2 independently represent C.sub.1-6 alkyl, C.sub.3-7 cycloalkyl, phenyl, benzyl, Het.sup.1 or Het.sup.2, which latter six groups are optionally substituted by one or more substituents selected from the group consisting of halo, hydroxy, OP(O)(OH).sub.2, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, C(O)OH, N(R.sup.c)(R.sup.d) and Het.sup.3, which latter group is optionally substituted by C.sub.1-4 alkyl, or R.sup.Y represents H, each R.sup.X2 independently represents H or C.sub.1-4 alkyl optionally substituted by hydroxy or OP(O)(OH).sub.2; R.sup.c and R.sup.d independently represent H, methyl or C(R.sup.e)(R.sup.f)C.sub.1-3 alkyl, the C.sub.1-3 alkyl portion of which latter group is optionally substituted by one or more hydroxy substituents; R.sup.a, R.sup.b, R.sup.e and R.sup.f represent, independently at each occurrence, H or methyl; R.sup.1A represents C.sub.1-6 alkoxy, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, which latter four groups are optionally substituted by one or more substituents selected from the group consisting of C.sub.1-2 alkyl, halo, hydroxy, OP(O)(OH).sub.2, C.sub.1-2 alkoxy and N(R.sup.c)(R.sup.d) or Het.sup.4, H, halo, cyano, phenyl or Het.sup.1, which latter two groups are optionally substituted with one or more substituents selected from the group consisting of C.sub.1-4 alkyl and C.sub.1-4 alkoxy; R.sup.1C and R.sup.1E independently represent H, halo, cyano or methyl; R.sup.1D represents C.sub.2-7 alkyl, C.sub.2-7 alkenyl, C.sub.2-7 alkynyl, C.sub.3-7 cycloalkyl, phenyl, Het.sup.1 or Het.sup.2, which latter seven groups are optionally substituted by one or more substituents selected from the group consisting of C.sub.1-4 alkyl, halo, cyano, hydroxy, OP(O)(OH).sub.2 and C.sub.1-4 alkoxy, or R.sup.1D represents trimethylsilyl or trifluoromethyl; R.sup.2 represents H, C.sub.1-4 alkyl, C.sub.3-4 cycloalkyl or halo; R.sup.3 represents C.sub.1-2 alkyl optionally substituted by one or more halo atoms, or R.sup.3 represents H or halo; L represents a direct bond or C.sub.1-2 alkylene; X represents CH or N; one of R.sup.4A, R.sup.4B and R.sup.4C represents R.sup.5a, and each of the other two of R.sup.4A, R.sup.4B and R.sup.4C independently represents R.sup.5b; R.sup.5a represents -Q.sup.1-C(R.sup.6c)(R.sup.6d)[C.sub.1-5 alkylene]-R.sup.6a, which C.sub.1-5 alkylene group is optionally substituted by oxo and/or by one or more substituents selected from the group consisting of halo and hydroxy, -Q.sup.2-[C(R.sup.6c)(R.sup.6d)(CH.sub.2).sub.0-1CH.sub.2O].sub.1-12CH.sub.2(CH.sub.2).sub.0-1CH.sub.2R.sup.6a, -Q.sup.3-J-Het.sup.x, which Het.sup.x group is optionally substituted by one or more substituents selected from the group consisting of halo, hydroxyl, oxo, CO.sub.2H, C.sub.1-3 alkyl, C.sub.1-3 alkoxy and C.sub.1-3 hydroxyalkyl, -J-S(O).sub.nR.sup.6b, -J-P(O)R.sup.6eR.sup.6f, -J-CO.sub.2H, OS(O).sub.2R.sup.6e, OS(O).sub.2NH.sub.2, NS(O)R.sup.6eR.sup.6f, -J-COR.sup.6b, S(O)(NR.sup.6c)CH.sub.3, OCONH.sub.2, CH.sub.2OH or CH.sub.2OP(O)(OH).sub.2; R.sup.5b represents C.sub.1-3 alkoxy or C.sub.1-3 alkyl, which latter two groups are optionally substituted by hydroxy, OP(O)(OH).sub.2 or one or more halo atoms, or R.sup.5b represents S(O).sub.1-2R.sup.6e, OS(O).sub.2R.sup.6e, N(R.sup.9)(R.sup.10), C.sub.2-3 alkynyl, H, cyano, C(O)N(R.sup.9)(R.sup.10), hydroxy or halo; R.sup.6a represents OR.sup.7a, S(O).sub.0-2R.sup.7aa, N(R.sup.7b)(R.sup.7c) or CO.sub.2H; R.sup.6b represents C.sub.1-8 alkyl, C.sub.3-8 cycloalkyl, phenyl, Het.sup.1 or Het.sup.2, which latter five groups are optionally substituted by one or more substituents selected from the group consisting of halo, hydroxyl, OP(O)(OH).sub.2, C.sub.1-4 alkyl and C.sub.1-4 alkoxy and which Het.sup.2 group is optionally substituted or further substituted by one or more oxo groups, or, when n is 2 or R.sup.6b is attached to -J-CO, in which J is C.sub.1-4 alkylene, R.sup.6b may alternatively represent N(R.sup.7b)(R.sup.7c); R.sup.7a to R.sup.7c independently represent H, C(R.sup.7d)(R.sup.7e)C.sub.1-3 alkylene-OH, C(R.sup.7d)(R.sup.7e)C.sub.1-3 alkylene-OP(O)(OH).sub.2 or C.sub.1-4 alkyl optionally substituted by one or more halo atoms, or R.sup.7b and R.sup.7c, together with the N-atom to which they are attached, form a 4- to 7-membered heterocyclic group that is fully saturated, partially unsaturated or fully aromatic and which heterocyclic group contains one N atom (the atom to which R.sup.7b and R.sup.7c are attached) and, optionally, one or more further heteroatoms selected from the group consisting of O, S and N, and which heterocyclic group is optionally substituted by one or more substituents selected from halo, hydroxy, oxo, C.sub.1-4 alkyl, C.sub.1-4 alkoxy and C.sub.1-4 hydroxyalkyl, or R.sup.7a represents P(O)(OH).sub.2; R.sup.7aa represents C(R.sup.7d)(R.sup.7e)C.sub.1-3 alkylene-OH or C.sub.1-4 alkyl optionally substituted by one or more halo atoms; R.sup.6c, R.sup.6d, R.sup.7d and R.sup.7e independently represent H or methyl; R.sup.6e and R.sup.6f represent, independently at each occurrence, C.sub.1-4 alkyl optionally substituted with one or more halo atoms, or, when R.sup.5a represents -J-P(O)R.sup.6eR.sup.6f, R.sup.6e and R.sup.6f may additionally represent hydroxy or C.sub.1-4 alkoxy; Q.sup.1, Q.sup.2, and Q.sup.3 independently represent C(O)N(R.sup.8), O, S(O).sub.2N(R.sup.8) or S(O).sub.p; J represents a direct bond or C.sub.1-4 alkylene; n and p independently represent 0, 1 or 2; R.sup.8, R.sup.9 and R.sup.10 independently represent H or methyl; Het.sup.x represents Het.sup.1 or Het.sup.3; Het.sup.1 represents, independently upon each occurrence, a 5- or 6-membered heterocyclic group that is fully aromatic, which group contains one or more heteroatoms selected from the group consisting of N, O and S; and Het.sup.2 to Het.sup.4 represent, independently upon each occurrence, a 4- to 7-membered heterocyclic group that is fully saturated or partially unsaturated, which group contains one or more heteroatoms selected from the group consisting of N, O and S; or a pharmaceutically acceptable salt, solvate or isotopic derivative thereof.
2. A compound according to claim 1 that is a compound of formula Ia, Ib, Ic, Id or Ie, ##STR00085## wherein R.sup.5b1 and R.sup.5b2 independently represent R.sup.5b and R.sup.1, R.sup.1A, R.sup.1c, R.sup.3, R.sup.5a and R.sup.5b are as defined in claim 1.
3. A compound according to claim 1, wherein R.sup.1 represents C(O)N(H)R.sup.Y, NHS(O).sub.2CH.sub.3 or P(O)(CH.sub.3).sub.2.
4. A compound according to claim 1, wherein R.sup.Y represents H.
5. A compound according to claim 1, wherein R.sup.1A represents methoxy or ethoxy.
6. A compound according to claim 1, wherein R.sup.1D represents tert-butyl.
7. A compound according to claim 1, wherein R.sup.3 represents methyl.
8. A compound according to claim 1, wherein R.sup.5a represents C(O)NHC(H)(R.sup.6c)[C.sub.1-4 alkylene]-R.sup.6a, which C.sub.1-3 alkylene group is optionally substituted by oxo or by one or two hydroxy, -Q.sup.2-[C(H)(R.sup.6c)CH.sub.2O].sub.1-6CH.sub.2CH.sub.2R.sup.6a, -Q.sup.3-J-Het.sup.x, -J-P(O)(C.sub.1-2 alkyl).sub.2, CO.sub.2H, OS(O).sub.2CH.sub.3, NS(O)(CH.sub.3).sub.2, CO-Het.sup.2, which Het.sup.2 group is optionally substituted by one or two oxo groups, or S(O)(NH)CH.sub.3.
9. A compound according to claim 1, wherein R.sup.5b represents halo, cyano, CCH, OS(O).sub.2CH.sub.3, methyl or methoxy, which latter two groups are optionally substituted by one or more fluoro atoms.
10. A compound according to claim 1, wherein: Q.sup.2 represents O or C(O)NH; Q.sup.3 represents O, C(O)N(H), C(O)N(CH.sub.3) or S(O).sub.2N(CH.sub.3); and/or J represents a direct bond or C.sub.1-3 alkylene.
11. A compound according to claim 1, wherein R.sup.6c represents H or methyl.
12. A compound according to claim 1, wherein R.sup.6a represents OH, OCH.sub.3S(O).sub.2CH.sub.3, CO.sub.2H or N(R.sup.7b)(R.sup.7c).
13. A compound according to claim 1, wherein: R.sup.7b and R.sup.7c both represent methyl, or R.sup.7b and R.sup.7c, together with the N-atom to which they are attached, form a morpholinyl, thiomorpholinyl, homomorpholinyl or pyrrolidinyl group, a piperazinyl group optionally substituted by one or more substituents selected from the group consisting of methyl and 2-hydroxyethyl, a piperidinyl group optionally substituted by hydroxy or a thiomorpholinyl group optionally substituted by one or more oxo groups; and/or Het.sup.2 represents a 5- or 6-membered heterocyclic group that is fully saturated or partially unsaturated, which group contains one or two heteroatoms selected from the group consisting of N, O and S.
14. A compound as claimed in claim 1, which is a compound selected from the group consisting of: 3-(2-((3-methoxy-5-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)phenyl)amino)quinazolin-6-yl)-4-methyl-N-(3-(trifluoromethyl)phenyl)benzamide; N-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-methoxy-5-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide; N-(5-(tert-butyl)-3-carbamoyl-2-methoxyphenyl)-3-(2-((3-methoxy-5-((2-morpholinoethyl)carbamoyl)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide; N-(5-(tert-butyl)-3-(dimethylphosphoryl)-2-methoxyphenyl)-3-(2-((3-methoxy-5-((2-morpholinoethyl)carbamoyl)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide; N-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-methoxy-5-((2-morpholinoethyl)carbamoyl)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide; N-(5-tert-butyl-2-methoxy-3-methylsulfonyl-phenyl)-3-[2-[3-methoxy-5-(2-morpholinoethyl-carbamoyl)anilino]quinazolin-6-yl]-4-methyl-benzamide; N-(5-tert-butyl-2-methoxy-3-methylsulfinyl-phenyl)-3-[2-[3-methoxy-5-(2-morpholinoethyl-carbamoyl)anilino]quinazolin-6-yl]-4-methyl-benzamide; N-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((4-((dimethyl(oxo)-I6-sulfanylidene)amino)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide; 4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxybenzoic acid; 4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxy-N-methyl-N-(2-morpholinoethyl)benzamide; 3-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)-quinazolin-2-yl)amino)-5-((2-morpholinoethyl)carbamoyl)phenyl methanesulfonate; 4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)-quinazolin-2-yl)amino)-2-methoxy-N-(3-(1-oxidothiomorpholino)propyl)benzamide; N-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((4-(1,1-dioxidothiomorpholine-4-carbonyl)-3-methoxyphenyl)amino)quinazolin-6-yl)-4-methylbenzamide; 4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)-quinazolin-2-yl)amino)-N-(2-(4-(2-hydroxyethyl)piperazin-1-yl)ethyl)-2-methoxybenzamide; 3-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)phenyl methanesulfonate; N-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-cyano-5-(2-morpholinoethoxy)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide; N-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-methoxy-5-(N-methyl-N-(2-morpholinoethyl)sulfamoyl)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide; N-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-4-methyl-3-(2-((3-((2-morpholinoethyl)carbamoyl)phenyl)amino)quinazolin-6-yl)benzamide; N-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-methoxy-5-(N-methyl-N-(3-morpholinopropyl)sulfamoyl)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide; N-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-cyano-5-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide; N-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-ethynyl-5-((2-(2-(2-methoxyethoxy)ethoxy)ethyl)carbamoyl)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide; N-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-(dimethylphosphoryl)phenyl)amino)quinazolin-6-yl)-4-methyl benzamide; N-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-((dimethylphosphoryl)methyl)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide; 4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)phenyl methanesulfonate; N-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-ethynyl-5-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide; N-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-fluoro-5-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide; N-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-ethynyl-5-((2-(2-methoxyethoxy)ethyl)carbamoyl)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide; N-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-ethynyl-5-((2-methoxyethyl)carbamoyl)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide; 3-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-5-methoxybenzoic acid; 4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)-quinazolin-2-yl)amino)-2-methoxy-N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)benzamide; N-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-((dimethyl(oxo)-I6-sulfanylidene)amino)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide; N-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-(2-(2-(2-methoxyethoxy)-ethoxy)ethoxy)-5-(trifluoromethoxy)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide; N-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-5-(trifluoromethyl)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide; N-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-4-methyl-3-(2-((4-(S-methylsulfonimidoyl)phenyl)amino)quinazolin-6-yl)benzamide; N-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-4-methyl-3-(2-((3-((2-morpholinoethyl)carbamoyl)-5-(trifluoromethyl)phenyl)amino)quinazolin-6-yl)benzamide; 4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-N-(2-(2-(2-hydroxyethoxy)ethoxy)ethyl)-2-methoxybenzamide; 4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxy-N-(2-(2-methoxyethoxy)ethyl)benzamide; 4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)-quinazolin-2-yl)amino)-N-(3-hydroxy-2,2-bis(hydroxymethyl) propyl)-2-methoxybenzamide; N-(2-(1,4-oxazepan-4-yl)ethyl)-4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)-phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxybenzamide; 4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methyl-phenyl)quinazolin-2-yl)amino)-2-methoxy-N-(2-(4-methylpiperazin-1-yl)ethyl)benzamide; 4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methyl-phenyl)quinazolin-2-yl)amino)-2-methoxy-N-(2-(1-methylpiperidin-4-yl)ethyl)benzamide; N-(3-(1,4-oxazepan-4-yl)propyl)-4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)-phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxybenzamide; 4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxy-N-(1-methylpiperidin-4-yl)benzamide; 4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxy-N-(2-(methylsulfonyl)ethyl)benzamide; 4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methyl-phenyl)quinazolin-2-yl)amino)-2-methoxy-N-(2-(2-methyl-1H-imidazol-1-yl)ethyl)benzamide; N-(2-(1H-imidazol-4-yl)ethyl)-4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)-phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxybenzamide; 4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxy-N-(2-(pyridin-3-yl)ethyl)benzamide; 4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)-quinazolin-2-yl)amino)-N-(2-(4-hydroxypiperidin-1-yl)ethyl)-2-methoxybenzamide; 4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)-quinazolin-2-yl)amino)-2-methoxy-N-((1-methylpiperidin-4-yl)methyl)benzamide; 4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)-quinazolin-2-yl)amino)-N-(2-(4-hydroxy-1-methylpiperidin-4-yl)ethyl)-2-methoxybenzamide; 4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxy-N-(2-(pyrrolidin-1-yl)ethyl)benzamide; 4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)-quinazolin-2-yl)amino)-2-methoxy-N-(2-(3,3,4-trimethylpiperazin-1-yl)ethyl)benzamide; 4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxy-N-(2-morpholinoethyl)benzamide; 4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-N-(1,1-dioxidotetrahydro-2H-thiopyran-4-yl)-2-methoxybenzamide; [4-[[6-[5-[[5-tert-butyl-3-(methanesulfonamido)-2-methoxy-phenyl]carbamoyl]-2-methyl-phenyl]quinazolin-2-yl]amino]-2-methoxy-phenyl]-methyl-phosphinic acid; 4-[[6-[5-[[5-tert-butyl-3-(methanesulfonamido)-2-methoxy-phenyl]carbamoyl]-2-methyl-phenyl]quinazolin-2-yl]amino]-2,6-dimethoxy-N-[2-(4-methylpiperazin-1-yl)ethyl]benzamide; 4-[[6-[5-[[5-tert-butyl-3-(methanesulfonamido)-2-methoxy-phenyl]carbamoyl]-2-methyl-phenyl]quinazolin-2-yl]amino]-N-(2-dimethylaminoethyl)-2,6-dimethoxy-benzamide; 2-[5-tert-butyl-2-methoxy-3-[[3-[2-[3-methoxy-4-[2-(1-oxo-1,4-thiazinan-4-yl)ethylcarbamoyl]-anilino]quinazolin-6-yl]-4-methyl-benzoyl]amino]-N-methylsulfonyl-anilino]ethyl dihydrogen phosphate; 2-[[4-[[6-[5-[[5-tert-butyl-3-(methanesulfonamido)-2-methoxy-phenyl]carbamoyl]-2-methyl-phenyl]quinazolin-2-yl]amino]-2,6-dimethoxy-benzoyl]amino]ethyl dihydrogen phosphate; 2-[2-[2-[[4-[[6-[5-[[5-tert-butyl-3-(methanesulfonamido)-2-methoxy-phenyl]carbamoyl]-2-methyl-phenyl]quinazolin-2-yl]amino]-2,6-dimethoxy-benzoyl]amino]ethoxy]ethoxy]ethyl dihydrogen phosphate; and [2-[5-tert-butyl-2-methoxy-3-[[3-[2-[3-methoxy-4-[2-(1-oxo-1,4-thiazinan-4-yl)ethylcarbamoyl]anilino]quinazolin-6-yl]-4-methyl-benzoyl]amino]anilino]-2-oxo-ethyl]dihydrogen phosphate, or a pharmaceutically acceptable salt, solvate or isotopic derivative thereof.
15. A pharmaceutical formulation comprising a compound as defined in claim 1, or pharmaceutically acceptable salt, solvate or isotopic derivative thereof, in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.
16. A combination product comprising: (A) a compound as defined in claim 1, or pharmaceutically acceptable salt, solvate or isotopic derivative thereof, and (B) another therapeutic agent, wherein each of components (A) and (B) is formulated in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier.
17. (canceled)
18. (canceled)
19. (canceled)
20. A method of treating or preventing an inflammatory disease, said method comprising administering to a subject an effective amount of: a compound as defined in claim 1, or pharmaceutically acceptable salt, solvate or isotopic derivative thereof, a pharmaceutical formulation comprising a compound as defined in claim 1, or pharmaceutically acceptable salt, solvate or isotopic derivative thereof, in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, or a combination product comprising: (A) a compound as defined in claim 1, or pharmaceutically acceptable salt, solvate or isotopic derivative thereof, and (B) another therapeutic agent, wherein each of components (A) and (B) is formulated in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier.
21. The method according to claim 20, wherein the inflammatory disease is selected from the group consisting of ulcerative colitis, Crohn's disease, gluten sensitive enteropathy (coeliac disease), eosinophilic eosophagitis, intestinal graft versus host disease, conjunctivitis, allergic conjunctivitis, keratoconjunctivitis sicca (dry eye), glaucoma, diabetic retinopathy, macular oedema, diabetic macular oedema, central retinal vein occlusion (CRVO), dry and/or wet age related macular degeneration (AMD), post-operative cataract inflammation, uveitis, posterior uveitis, anterior uveitis pan uveitis, corneal graft and limbal cell transplant rejection, cystic fibrosis, pulmonary hypertension, lung sarcoidosis, idiopathic pulmonary fibrosis, COPD, chronic bronchitis emphysema, asthma, paediatric asthma, atopic dermatitis, allergic dermatitis, contact dermatitis, psoriasis, allergic rhinitis, rhinitis and sinusitis.
22. The method according to claim 10, wherein the inflammatory disease is ulcerative colitis, Crohn's disease, keratoconjunctivitis sicca (dry eye) or uveitis.
23. The method according to claim 20, wherein the inflammatory disease is asthma or COPD.
24. A process for the preparation of a compound of formula I which process comprises: (a) reaction of a compound of formula II, ##STR00086## wherein LG.sup.1 represents a leaving group and R.sup.1, R.sup.1A, R.sup.1C, R.sup.1D, R.sup.1E, R.sup.2 and R.sup.3 are as defined in claim 1 with a compound of formula III, ##STR00087## wherein L, X, R.sup.4A, R.sup.4B and R.sup.4C are as defined in claim 1; (b) reaction of a compound of formula IV, ##STR00088## wherein LG.sup.2 represents a leaving group and R.sup.2, R.sup.3, L, X, R.sup.4A, R.sup.4B and R.sup.4C are as defined in claim 1, with a compound of formula V, ##STR00089## wherein R.sup.1, R.sup.1A, R.sup.1C, R.sup.1D and R.sup.1E are as defined in claim 1; (c) for compounds of formula I in which R.sup.1 represents -L.sup.1-C(O)NR.sup.XR.sup.Y, -L.sup.2-S(O).sub.2R.sup.Y1, -L.sup.3-P(O)R.sup.Y1R.sup.Y2, -L.sup.4-S(O).sub.2NR.sup.XR.sup.Y, -L.sup.5-C(O)R.sup.Y or -L.sup.7-C(O)OR.sup.Y, in which L.sup.1, L.sup.2, L.sup.3, L.sup.4, L.sup.5 and L.sup.7 represent N(R.sup.X2) or C(R.sup.a)(R.sup.b)N(R.sup.X2), reaction of a compound of formula VI, ##STR00090## wherein L.sup.1 represents a direct bond or C(R.sup.a)(R.sup.b) and R.sup.X2, R.sup.1A, R.sup.1C, R.sup.1D, R.sup.1E, R.sup.2, R.sup.3, L, X, R.sup.4A, R.sup.4B and R.sup.4C are as defined in claim 1, with a compound of formula VIIa, VIIb, VIIc, VIId, VIIe or VIIf,
LG.sup.3-C(O)NR.sup.XR.sup.YVIIa
LG.sup.3-S(O).sub.2R.sup.Y1VIIb
LG.sup.3-P(O)R.sup.Y1R.sup.Y2VIIc
LG.sup.3-S(O).sub.2NR.sup.XR.sup.YVIId
LG.sup.3-C(O)R.sup.YVIIe
LG.sup.3-C(O)OR.sup.YVIIf wherein LG.sup.3 represents a leaving group and R.sup.X, R.sup.Y, R.sup.Y1 and R.sup.Y2 are as defined in claim 1; (d) for compounds of formula I in which R.sup.1 represents -L.sup.1-C(O)NR.sup.XR.sup.Y in which L.sup.1 represents a bond or [C(R.sup.a)(R.sup.b)].sub.1-2, reaction of a compound of formula VIII, ##STR00091## wherein LG.sup.2 represents a leaving group, L.sup.a represents a bond or [C(R.sup.a)(R.sup.b)].sub.1-2- and R.sup.1A, R.sup.1C, R.sup.1D, R.sup.1E, R.sup.2, R.sup.3, L, X, R.sup.4A, R.sup.4B and R.sup.4C are as defined in claim 1, with a compound of formula IX,
HNR.sup.XR.sup.YIX wherein R.sup.X and R.sup.Y are as defined in claim; (e) for compounds of formula I where Q.sup.1, Q.sup.2 or Q.sup.3 represents C(O)N(R.sup.8) or S(O).sub.2N(R.sup.8) or R.sup.5a represents C.sub.1-4-alkylene-CON(R.sup.7b)R.sup.7c or S(O).sub.2N(R.sup.7b)R.sup.7c, condensation of a compound of formula VIIIa ##STR00092## wherein one of R.sup.4AA, R.sup.4BB and R.sup.4CC represents [C.sub.1-4-alkylene].sub.0-1C(O)LG.sup.2 or S(O).sub.2LG.sup.2 and each of the other two of R.sup.4AA, R.sup.4BB and R.sup.4CC independently represents R.sup.5b and R.sup.1, R.sup.1A, R.sup.1C, R.sup.1D, R.sup.1E, R.sup.2, R.sup.3, R.sup.5b, L and X are as defined in claim 1 and LG.sup.2 is as defined above, with an amine of formula IXa, IXb, IXc or IXd,
HNR.sup.8C(R.sup.6c)(R.sup.6d)[C.sub.1-5 alkylene]-R.sup.6a1IXa
HNR.sup.8[C(R.sup.6c)(R.sup.6d)(CH.sub.2).sub.0-1CH.sub.2O].sub.1-12CH.sub.2(CH.sub.2).sub.0-1CH.sub.2R.sup.6a1IXb
HNR.sup.8-J-Het.sup.XIXc
HN(R.sup.7b)R.sup.7cIXd which C.sub.1-5 alkylene and Het.sup.X groups are optionally substituted as described in claim 1, wherein R.sup.6c, R.sup.6d, R.sup.8, J and Het.sup.X are as defined in claim 1, and R.sup.6a1 takes the same definition as R.sup.6a in claim 1, except that CO.sub.2H is only present in protected form; (f) for compounds of formula I in which R.sup.1 represents C.sub.1-4 alkylene-OP(O)(OH).sub.2, R.sup.X and/or R.sup.X1 represents C.sub.1-6 alkyl substituted by OP(O)(OH).sub.2, R.sup.Y, R.sup.Y1 and/or R.sup.Y2 represents C.sub.1-6 alkyl, C.sub.3-7 cycloalkyl, phenyl, benzyl, Het.sup.1 or Het.sup.2, which latter six groups are substituted by OP(O)(OH).sub.2 and are optionally further substituted by one or more substituents selected from the group consisting of halo, hydroxy, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, C(O)OH, N(R.sup.c)(R.sup.d) and Het.sup.3, which latter group is optionally substituted by C.sub.1-4 alkyl, R.sup.X2 represents C.sub.1-4 alkyl substituted by OP(O)(OH).sub.2, R.sup.1A represents C.sub.1-6 alkoxy, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, which latter four groups are substituted by OP(O)(OH).sub.2 and are optionally further substituted by one or more substituents selected from the group consisting of C.sub.1-2 alkyl, halo, hydroxy, C.sub.1-2 alkoxy and N(R.sup.c)(R.sup.d) or Het.sup.4, R.sup.1D represents C.sub.2-7 alkyl, C.sub.2-7 alkenyl, C.sub.2-7 alkynyl, C.sub.3-7 cycloalkyl, phenyl, Het.sup.1 or Het.sup.2, which latter seven groups are substituted by OP(O)(OH).sub.2 and are optionally further substituted by one or more substituents selected from the group consisting of C.sub.1-4 alkyl, halo, cyano, hydroxy and C.sub.1-4 alkoxy, or R.sup.1D represents trimethylsilyl or trifluoromethyl, R.sup.5a represents CH.sub.2OP(O)(OH).sub.2, R.sup.5b represents C.sub.1-3 alkoxy or C.sub.1-3 alkyl, which latter two groups are substituted by OP(O)(OH).sub.2, R.sup.6b represents C.sub.1-8 alkyl, C.sub.3-8 cycloalkyl, phenyl, Het.sup.1 or Het.sup.2, which latter five groups are substituted by OP(O)(OH).sub.2 and are optionally further substituted by one or more substituents selected from the group consisting of halo, hydroxyl, C.sub.1-4 alkyl and C.sub.1-4 alkoxy and which Het.sup.2 group is optionally substituted or further substituted by one or more oxo groups, R.sup.7a, R.sup.7b and/or R.sup.7c represents C(R.sup.7d)(R.sup.7e)C.sub.1-3 alkylene-OP(O)(OH).sub.2 or R.sup.7a represents P(O)(OH).sub.2, reaction of a hydroxy group on a corresponding compound of formula I in which, respectively R.sup.1 represents C.sub.1-4 alkylene-OH, R.sup.X and/or R.sup.X1 represents C.sub.1-6 alkyl substituted by hydroxy, R.sup.Y, R.sup.Y1 and/or R.sup.Y2 represents C.sub.1-6 alkyl, C.sub.3-7 cycloalkyl, phenyl, benzyl, Het.sup.1 or Het.sup.2, which latter six groups are substituted by hydroxy and are optionally further substituted by one or more substituents selected from the group consisting of halo, hydroxy, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, C(O)OH, N(R.sup.c)(R.sup.d) and Het.sup.3, which latter group is optionally substituted by C.sub.1-4 alkyl, R.sup.X2 represents C.sub.1-4 alkyl substituted by hydroxy, R.sup.1A represents C.sub.1-6 alkoxy, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, which latter four groups are substituted by hydroxy and are optionally further substituted by one or more substituents selected from the group consisting of C.sub.1-2 alkyl, halo, hydroxy, C.sub.1-2 alkoxy and N(R.sup.c)(R.sup.d) or Het.sup.4, R.sup.1D represents C.sub.2-7 alkyl, C.sub.2-7 alkenyl, C.sub.2-7 alkynyl, C.sub.3-7 cycloalkyl, phenyl, Het.sup.1 or Het.sup.2, which latter seven groups are substituted by hydroxy and are optionally further substituted by one or more substituents selected from the group consisting of C.sub.1-4 alkyl, halo, cyano, hydroxy and C.sub.1-4 alkoxy, or R.sup.1D represents trimethylsilyl or trifluoromethyl, R.sup.5a represents CH.sub.2OH, R.sup.5b represents C.sub.1-3 alkoxy or C.sub.1-3 alkyl, which latter two groups are substituted by hydroxy, R.sup.6b represents C.sub.1-8 alkyl, C.sub.3-8 cycloalkyl, phenyl, Het.sup.1 or Het.sup.2, which latter five groups are substituted by hydroxy and are optionally further substituted by one or more substituents selected from the group consisting of halo, hydroxyl, C.sub.1-4 alkyl and C.sub.1-4 alkoxy and which Het.sup.2 group is optionally substituted or further substituted by one or more oxo groups, R.sup.7a, R.sup.7b and/or R.sup.7c represents C(R.sup.7d)(R.sup.7e)C.sub.1-3 alkylene-OH or R.sup.7a represents H, with di-tert-butyl diethylphosphoramidite or dibenzyl-N,N-diisopropylphosphoramidite, followed by reaction with an oxidant and then removal of the respective tert-butyl or benzyl protecting groups by hydrolysis using an acid or hydrogenolysis with a palladium catalyst; (g) deprotection of a protected derivative of a compound of formula I.
Description
PREPARATION OF COMPOUNDS OF THE INVENTION
Example 1
3-(2-((3-Methoxy-5-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)phenyl)amino)quinazolin-6-yl)-4-methyl-N-(3-(trifluoromethyl)phenyl)benzamide
[0578] ##STR00023##
(i) Methyl 3-(2-chloroquinazolin-6-yl)-4-methylbenzoate
[0579] A mixture of 6-bromo-2-chloroquinazoline (780 mg, 3.20 mmol) and methyl 4-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (1000 mg, 3.62 mmol) in 1,2-dimethoxyethane (3 mL) and 1 M NaHCO.sub.3 solution (10 mL, 10.00 mmol) was purged with nitrogen for 5 minutes. The mixture was heated to 90 C. before adding Pd(Ph.sub.3P).sub.4 (200 mg, 0.173 mmol) and stirring for 1 h. The mixture was diluted with water (150 mL) and extracted with diethyl ether (3150 mL). The combined organic phases were washed with water (150 mL), 20% brine (150 mL) and saturated brine (150 mL). The organic phase was dried (MgSO.sub.4) and concentrated to give a yellow oil which was purified by chromatography on the Companion (80 g column, 0-30% EtOAc/isohexane) to afford the sub-title compound (510 mg) as a cream solid.
[0580] LCMS m/z 313 (M+H).sup.+ (ES.sup.+)
(ii) 3-Methoxy-5-nitrophenol
[0581] A mixture of KOH (29.0 g, 517 mmol) and 1-bromo-3-methoxy-5-nitrobenzene (30 g, 129 mmol) in water (70 mL) and dioxane (70 mL) was degassed for 5 minutes prior to the addition of di-tert-butyl(2,4,6-triisopropyl-[1,1-biphenyl]-2-yl)phosphine (1.263 g, 2.97 mmol) and Pd.sub.2(dba).sub.3 (1.184 g, 1.293 mmol). The resulting mixture was degassed for a further 2 minutes then heated under a nitrogen atmosphere at 100 C. for 2 h. The mixture was cooled, then acidified with 5M HCl to pH 1 and extracted with EtOAc (2500 mL). The organic layer was washed with saturated brine (200 mL), dried (MgSO.sub.4), filtered and concentrated under reduced pressure. The crude product was purified through a pad of silica eluting with 30% EtOAc/isohexane to afford the sub-title compound (20.76 g) as a yellow solid.
[0582] .sup.1H NMR (400 MHz; DMSO-d.sub.6) 10.46 (s, 1H), 7.20 (s, 1H), 7.19 (s, 1H), 6.76 (s, 1H), 3.82 (s, 3H).
[0583] LCMS m/z 168 (MH).sup. (ES.sup.)
(iii) 1-Methoxy-3-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-5-nitrobenzene
[0584] To a stirred suspension of the product from step (ii) above (8.14 g, 45.7 mmol) and K.sub.2CO.sub.3 (12.64 g, 91 mmol) in acetone (150 mL) was added 1-bromo-2-(2-(2-methoxy-ethoxy)ethoxy)ethane (8.85 mL, 48.0 mmol). The resulting mixture was refluxed overnight, cooled and filtered. The filtrate was evaporated under reduced pressure and the residue purified by chromatography on silica gel (220 g column, 0-60% EtOAc/isohexane) to afford the sub-title compound (13.41 g) as a yellow oil.
[0585] .sup.1H NMR (400 MHz, DMSO-d.sub.6) : 7.34-7.32 (m, 2H), 6.98 (t, 1H), 4.22-4.20 (m, 2H), 3.85 (s, 3H), 3.77-3.74 (m, 2H), 3.60-3.57 (m, 2H), 3.54-3.50 (m, 4H), 3.44-3.40 (m, 2H), 3.23 (s, 3H).
[0586] LCMS m/z 316 (M+H).sup.+ (ES.sup.+)
(iv) 3-Methoxy-5-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)aniline
[0587] The product from step (iii) above (13.4 g, 42.5 mmol) was dissolved in ethanol (150 mL) and Fe powder (13 g, 233 mmol) was added followed by a solution of NH.sub.4Cl (2.3 g, 43.0 mmol) in water (150 mL). The resulting suspension was heated at 80 C. for 3 h. The reaction was cooled to rt and filtered through Celite. The filtrate was concentrated in vacuo then partitioned between water (250 mL) and EtOAc (400 mL). The organic layer was separated, dried (MgSO.sub.4), filtered and concentrated under reduced pressure. The crude product was purified by chromatography on silica gel (120 g column, 0-4% MeOH/DCM) to afford the sub-title compound (10.95 g) as an oil.
[0588] .sup.1H NMR (400 MHz, DMSO-d.sub.6) 5.76-5.73 (m, 2H), 5.68 (t, 1H), 5.07 (s, 2H), 3.98-3.89 (m, 2H), 3.72-3.65 (m, 2H), 3.63 (s, 3H), 3.60-3.48 (m, 6H), 3.47-3.40 (m, 2H), 3.24 (s, 3H)
[0589] LCMS m/z 286 (M+H).sup.+ (ES.sup.+)
(v) Methyl 3-(2-((3-methoxy-5-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)phenyl)amino)-quinazolin-6-yl)-4-methylbenzoate
[0590] The product from step (i) above (375 mg, 1.199 mmol), the product from step (iv) above (411 mg, 1.440 mmol) and Cs.sub.2CO.sub.3 (600 mg, 1.842 mmol) were stirred in 1,4-dioxane (9 mL) whilst degassing with nitrogen. A solution of Pd.sub.2(dba).sub.3 (60 mg, 0.066 mmol) and BINAP (80 mg, 0.128 mmol) in degassed 1,4-dioxane (1 mL) was added and the mixture was heated to 80 C. for 20 h. The mixture was diluted with water (100 mL) and extracted with ethyl acetate (350 mL). The combined organic phases were washed with water (50 mL), saturated brine (50 mL), dried (MgSO.sub.4) and concentrated under reduced pressure to yield an orange oil. The crude product was purified by chromatography on the Companion (12 g column, 25-100% EtOAc/isohexane) to afford the sub-title compound (525 mg) as a orange oil.
[0591] LCMS m/z 562 (M+H).sup.+ (ES.sup.+)
(vi) 3-(2-((3-Methoxy-5-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)phenyl)amino)quinazolin-6-yl)-4-methylbenzoic acid
[0592] A solution of the product from step (v) above (560 mg, 0.997 mmol) in THF (6 mL) and methanol (2 mL) was stirred with 2 M NaOH soln (1.6 mL, 3.20 mmol) and water (10 mL) at rt over a weekend. The mixture was concentrated under reduced pressure to remove the organic solvents and then diluted with water (50 mL). The aqueous solution was washed with diethyl ether (350 mL) then acidified with 1 M HCl (3.2 mL) and extracted with ethyl acetate (350 mL). The combined ethyl acetate solutions were washed with saturated brine (50 mL), dried (MgSO.sub.4) and concentrated under reduced pressure to afford the sub-title compound (430 mg) as an orange waxy solid.
[0593] LCMS m/z 548 (M+H).sup.+ (ES.sup.+)
(vii) 3-(2-((3-Methoxy-5-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)phenyl)amino)quinazolin-6-yl)-4-methyl-N-(3-(trifluoromethyl)phenyl)benzamide
[0594] Et.sub.3N (26.7 L, 0.192 mmol) was added to a stirred solution of 3-(trifluoromethyl)aniline (22 mg, 0.137 mmol), the product from step (vi) above (70 mg, 0.128 mmol) and HATU (58.3 mg, 0.153 mmol) in N,N-dimethylformamide (1 mL) and the mixture was stirred at rt for 30 minutes. Water (10 mL) was added and the mixture was extracted with ethyl acetate (310 mL). The combined organic phases were washed with 20% brine (210 mL), saturated brine (10 mL), dried (MgSO.sub.4) and concentrated under reduced pressure. The crude product was purified by chromatography on the Companion (12 g column, EtOAc) to afford the title compound (54 mg) as a yellow glass.
[0595] .sup.1H NMR (DMSO-d6) 400 MHz, : 10.53 (s, 1H), 9.91 (s, 1H), 9.38 (s, 1H), 8.27-8.22 (m, 1H), 8.12-8.06 (m, 1H), 8.03-7.99 (m, 2H), 7.96 (dd, 1H), 7.92 (dd, 1H), 7.78 (d, 1H), 7.64-7.57 (m, 1H), 7.55 (d, 1H), 7.49-7.43 (m, 1H), 7.37 (dd, 1H), 7.32 (dd, 1H), 6.20 (dd, 1H), 4.14-4.06 (m, 2H), 3.82-3.73 (m, 2H), 3.78 (s, 3H), 3.65-3.59 (m, 2H), 3.59-3.51 (m, 4H), 3.47-3.42 (m, 2H), 3.24 (s, 3H), 2.39 (s, 3H).
[0596] LCMS m/z 691 (M+H).sup.+ (ES.sup.+); 689 (MH).sup. (ES.sup.)
Example 2
N-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-methoxy-5-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide
[0597] ##STR00024##
[0598] Et.sub.3N (26.7 L, 0.192 mmol) was added to a stirred solution of N-(3-amino-5-(tert-butyl)-2-methoxyphenyl)methanesulfonamide (see, for example, Cirillo, P. F. et al., WO 2002/083628, 24 Oct. 2002; 35 mg, 0.129 mmol), 3-(2-((3-methoxy-5-(2-(2-(2-methoxyethoxy)ethoxy)-ethoxy)phenyl)amino)quinazolin-6-yl)-4-methylbenzoic acid (see Example 1(vi) above; 70 mg, 0.128 mmol) and HATU (58.3 mg, 0.153 mmol) in N,N-dimethylformamide (1 mL) and the mixture was stirred at rt for 3 h. Slow conversion was observed so the mixture was heated to 45 C. and stirred over 18 h. Water (10 mL) was added and the mixture was extracted with ethyl acetate (310 mL). The combined organic phases were washed with 20% brine (210 mL), saturated brine (10 mL), dried (MgSO.sub.4) and concentrated under reduced pressure. The crude product was purified by chromatography on the Companion (12 g column, EtOAc) to afford the title compound (56 mg) as a yellow solid.
[0599] .sup.1H NMR (DMSO-d6) 400 MHz, : 9.90 (s, 1H), 9.89 (s, 1H), 9.38 (s, 1H), 9.14 (s, 1H), 8.02-7.97 (m, 2H), 7.97-7.90 (m, 2H), 7.78 (d, 1H), 7.52 (d, 1H), 7.46 (d, 1H), 7.37 (dd, 1H), 7.32 (dd, 1H), 7.25 (d, 1H), 6.20 (dd, 1H), 4.14-4.07 (m, 2H), 3.81-3.75 (m, 2H), 3.78 (s, 3H), 3.71 (s, 3H), 3.65-3.59 (m, 2H), 3.59-3.51 (m, 4H), 3.47-3.42 (m, 2H), 3.24 (s, 3H), 3.05 (s, 3H), 2.39 (s, 3H), 1.28 (s, 9H).
[0600] LCMS m/z 802 (M+H).sup.+ (ES.sup.+); 800 (MH).sup. (ES.sup.)
Example 3
N-(5-(tert-Butyl)-3-carbamoyl-2-methoxyphenyl)-3-(2-((3-methoxy-5-((2-morpholinoethyl)-carbamoyl)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide
[0601] ##STR00025##
(i) 3-Amino-5-methoxy-N-(2-morpholinoethyl)benzamide
[0602] To a stirred mixture of 3-amino-5-methoxybenzoic acid (5.20 g, 31.1 mmol), Et.sub.3N (4.50 mL, 32.3 mmol) and 2-morpholinoethanamine (4.23 mL, 32.3 mmol) in THF (150 mL) and DMF (4 mL) was added HATU (14.72 g, 38.7 mmol) and the reaction stirred at ambient temperature overnight. After this time the mixture was taken up in ethyl acetate (300 mL) and washed with sat NaHCO.sub.3 (aq) (2100 mL). The aqueous was back extracted with further ethyl acetate (450 mL) and organics combined, dried over MgSO.sub.4, filtered and concentrated under reduced pressure. Trituration with isohexanes (100 mL) afforded a pale orange gum (15 g). The crude product was purified by chromatography on the Companion (220 g column, 0-60% IPA in DCM). Fractions were combined as two separate batches to afford the sub-title compound as two separate batches (2.48 g and 2.87 g) as orange solids.
[0603] .sup.1H NMR (400 MHz; CDCl.sub.3) : 6.69-6.64 (m, 3H), 6.35 (t, 1H), 3.81 (br.s, 2H), 3.81 (s, 3H), 3.73 (m, 4H), 3.53 (dd, 2H), 2.62-2.57 (m, 2H), 2.53-2.49 (m, 4H).
[0604] LCMS m/z 280 (M+H).sup.+ (ES.sup.+)
[0605] The first batch (2.0 g) was recrystallised in acetonitrile (18 mL) to yield the sub-title compound (1.70 g) as a white solid which was used in the next step.
(ii) Methyl 3-(2-((3-methoxy-5-((2-morpholinoethyl)carbamoyl)phenyl)amino)quinazolin-6-yl)-4-methylbenzoate
[0606] A solution of methyl 3-(2-chloroquinazolin-6-yl)-4-methylbenzoate (see Example 1(i) above; 1.45 g, 4.64 mmol), the product from step (i) above (1.360 g, 4.87 mmol) and p-TSA monohydrate (1.323 g, 6.95 mmol) in dry DMF (50 mL) was stirred at 85 C. for 18 h. The reaction mixture was cooled to rt and partitioned between EtOAc (200 mL) and sat. NaHCO.sub.3 solution (300 mL). The organics were washed with brine (5200 mL), dried (MgSO.sub.4) and evaporated to dryness. The crude product was purified by chromatography on silica gel (120 g column, 0-10% MeOH/DCM) to afford the sub-title compound (2.0 g) as a yellow solid.
[0607] .sup.1H NMR (400 MHz, DMSO-d6) 10.11 (s, 1H), 9.40 (d, 1H), 8.34 (t, 1H), 8.02 (d, 1H), 7.99 (d, 1H), 7.95-7.85 (m, 4H), 7.76 (d, 1H), 7.54 (d, 1H), 7.02 (dd, 1H), 3.87 (s, 3H), 3.86 (s, 3H), 3.59 (t, 4H), 3.40 (q, 2H), 2.44 (m, 6H), 2.38 (s, 3H).
[0608] LCMS m/z 556 (M+H).sup.+ (ES.sup.+); 554 (MH).sup. (ES.sup.)
(iii) 3-(2-((3-Methoxy-5-((2-morpholinoethyl)carbamoyl)phenyl)amino)quinazolin-6-yl)-4-methylbenzoic acid
[0609] A suspension of the product from step (ii) above (2.0 g, 3.60 mmol) in THF (50 mL) and MeOH (25 mL) was treated with LiOH (0.431 g, 18.00 mmol) followed by water (10 mL) and stirred at rt for 18 h. The reaction mixture was acidified with 1 M HCl (to pH 3) and extracted with EtOAc (350 mL). LCMS showed desired product in aqueous phase with small amount in organics. Both phases were passed through SAX resin (loading MeOH) and then eluted with 5% AcOH/MeOH. The washings were evaporated to afford a yellow solid that was treated with MeOH (100 mL) and the white solid removed by filtration. The filtrate was evaporated and the residue adsorbed on to silica and purified by chromatography on silica gel (80 g column, 0-20% MeOH/DCM with 1% NH.sub.4OH). The product was dried overnight under vacuum at 40 C. to afford the sub-title compound (918 mg) as a yellow solid.
[0610] .sup.1H NMR (400 MHz, DMSO-d6) 10.09 (s, 1H), 9.39 (d, 1H), 8.34 (t, 1H), 8.03 (t, 1H), 7.96 (d, 1H), 7.92 (t, 1H), 7.89-7.78 (m, 3H), 7.75 (d, 1H), 7.40 (d, 1H), 7.01 (dd, 1H), 3.86 (s, 3H), 3.59 (t, 4H), 3.45-3.36 (m, 2H), 2.45 (d, 6H), 2.34 (s, 3H).
[0611] LCMS m/z 542 (M+H).sup.+ (ES.sup.+); 540 (MH).sup. (ES.sup.)
(iv) N-(5-(tert-Butyl)-3-carbamoyl-2-methoxyphenyl)-3-(2-((3-methoxy-5-((2-morpholinoethyl)carbamoyl)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide
[0612] A suspension of the product from step (iii) above (200 mg, 0.369 mmol) and 3-amino-5-(tert-butyl)-2-methoxybenzamide (90 mg, 0.406 mmol) in dry DMF (3 mL) was treated with DIPEA (193 L, 1.108 mmol) then HATU (154 mg, 0.406 mmol) and the reaction mixture stirred at rt for 72 h. HATU (154 mg, 0.406 mmol) was added and the reaction mixture stirred for 1 h. Reaction mixture did not change so the solution was loaded directly on to SCX resin (load MeOH; eluted 1% NH.sub.3/MeOH) and the eluted product evaporated. The crude product was purified by chromatography on silica gel (12 g column, 0-10% MeOH/DCM) to afford the title compound (30 mg) as a yellow solid.
[0613] .sup.1H NMR (400 MHz, DMSO-d6) 10.10 (s, 1H), 9.89 (s, 1H), 9.41 (d, 1H), 8.33 (t, 1H), 8.05-7.91 (m, 5H), 7.80-7.75 (m, 2H), 7.72 (s, 1H), 7.57 (s, 1H), 7.53 (d, 1H), 7.50 (d, 1H), 7.02 (dd, 1H), 3.86 (s, 3H), 3.74 (s, 3H), 3.59 (t, 4H), 3.40 (q, 2H), 2.46 (d, 6H), 2.40 (s, 3H), 1.30 (s, 9H).
[0614] LCMS m/z 746 (M+H).sup.+ (ES.sup.+); 744 (MH).sup. (ES.sup.)
Example 4
N-(5-(tert-Butyl)-3-(dimethylphosphoryl)-2-methoxyphenyl)-3-(2-((3-methoxy-5-((2-morpholinoethyl)carbamoyl)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide
[0615] ##STR00026##
(i) 5-(tert-Butyl)-1-iodo-2-methoxy-3-nitrobenzene
[0616] To a stirred solution of 5-(tert-butyl)-2-methoxy-3-nitroaniline (3.7 g, 16.50 mmol) and iodine (2.5 g, 9.85 mmol) in toluene (50 mL) at 0 C. was added tert-butyl nitrite (2.5 mL, 18.92 mmol) and the reaction warmed to rt and stirred overnight. The reaction was diluted with EtOAc (100 ml) and washed with brine (60 mL) then sat. aq. sodium thiosulfate solution (60 mL). The organic phase was dried (MgSO.sub.4), filtered and concentrated in vacuo affording a red oil. The crude product was purified by chromatography on the Companion (120 g column, 0-5% EtOAc in hexane) to afford the sub-title compound (4 g) as a yellow oil.
[0617] .sup.1H NMR (400 MHz, DMSO-d6) 8.11 (d, 1H), 7.89 (d, 1H), 3.85 (s, 3H), 1.29 (s, 9H).
(ii) 5-(tert-Butyl)-3-iodo-2-methoxyaniline
[0618] Fe powder (7.00 g, 125 mmol) was added to a suspension of the product from step (i) above (4.2 g, 12.53 mmol) in ethanol (30 mL) and NH.sub.4Cl (1 g, 18.69 mmol) in water (15 mL). The solution was stirred vigorously (overhead stirrer) at 95 C. (block temperature, internal temperature 75 C.) for 1 h. The reaction mixture was filtered through Celite and the filtrate diluted with water (100 mL). The product was extracted with EtOAc (3100 mL). The organics were bulked, dried (MgSO.sub.4), filtered and evaporated to a dark brown oil. The crude product was purified by chromatography on silica gel (80 g column, 10% EtOAc:isohexane to 20%) to afford the sub-title compound (3.5 g) as cream crystalline solid.
[0619] .sup.1H NMR (400 MHz, DMSO-d6) 6.88 (d, 1H), 6.73 (d, 1H), 5.07 (s, 2H), 3.60 (s, 3H), 1.19 (s, 9H).
[0620] LCMS m/z 306 (M+H).sup.+ (ES.sup.+)
(iii) (3-Amino-5-(tert-butyl)-2-methoxyphenyl)dimethylphosphine oxide
[0621] Dimethylphosphine oxide (1 mL, 15.89 mmol) was added to a degassed suspension of the product from step (ii) above (3.5 g, 11.47 mmol), Pd(OAc).sub.2 (250 mg, 1.114 mmol), xantphos (1.3 g, 2.247 mmol) and finely powdered potassium phosphate tribasic (6 g, 28.3 mmol) in DMF (50 mL) and stirred vigorously under nitrogen at 150 C. block temperature for 1 h. The mixture was diluted with DCM (100 mL) and filtered through Celite. The filtrate was evaporated to a dark gum. The crude product was purified by chromatography on silica gel (80 g column, 2% MeOH:EtOAc to 10%) to afford the sub-title compound (2 g) as a yellow solid.
[0622] .sup.1H NMR (400 MHz, DMSO-d6) 6.97 (d, 1H), 6.89 (dd, 1H), 5.00 (s, 2H), 3.72 (s, 3H), 1.64 (d, 6H), 1.24 (s, 9H).
[0623] LCMS m/z 256 (M+H).sup.+ (ES.sup.+)
(iv) N-(5-(tert-Butyl)-3-(dimethylphosphoryl)-2-methoxyphenyl)-3-(2-((3-methoxy-5-((2-morpholinoethyl)carbamoyl)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide
[0624] A suspension of 3-(2-((3-methoxy-5-((2-morpholinoethyl)carbamoyl)phenyl)amino)quinazolin-6-yl)-4-methylbenzoic acid (see Example 3(iii) above; 150 mg, 0.277 mmol) and the product from step (iii) above (78 mg, 0.305 mmol) in dry DMF (2 mL) was treated with DIPEA (145 L, 0.831 mmol) then HATU (116 mg, 0.305 mmol) and the reaction mixture stirred at rt for 72 h. Reaction mixture was partitioned between EtOAc (20 mL) and sat. NaHCO.sub.3 solution (20 mL). The organics were washed with brine (520 mL) and dried (MgSO.sub.4). The crude product was purified by chromatography on silica gel (12 g column, 0-10% MeOH/DCM with 1% NH.sub.3) followed by preparative HPLC (Gilson, Basic (0.1% Ammonium Bicarbonate), Basic, Waters X-Bridge Prep-C18, 5 m, 1950 mm column, 5-95% MeCN in Water) then dried at 40 C. under vacuum for 24 h to afford the title compound (34 mg) as a yellow solid.
[0625] .sup.1H NMR (400 MHz, DMSO-d6) 10.14 (s, 1H), 10.08 (s, 1H), 9.41 (s, 1H), 8.31 (d, 1H), 8.08-7.90 (m, 6H), 7.78 (d, 1H), 7.68 (d, 1H), 7.64 (d, 1H), 7.53 (d, 1H), 7.02 (s, 1H), 3.86 (s, 3H), 3.78 (s, 3H), 3.59 (t, 4H), 3.37 (m, 2H), 2.48-2.41 (m, 6H), 2.40 (s, 3H), 1.68 (d, 6H), 1.31 (s, 9H).
[0626] LCMS m/z 779 (M+H).sup.+ (ES.sup.+); 777 (MH).sup. (ES.sup.)
Example 5
N-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-methoxy-5-((2-morpholinoethyl)carbamoyl)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide
[0627] ##STR00027##
[0628] A solution of 3-(2-((3-methoxy-5-((2-morpholinoethyl)carbamoyl)phenyl)amino)quinazolin-6-yl)-4-methylbenzoic acid (see Example 3(iii) above; 200 mg, 0.369 mmol), N-(3-amino-5-(tert-butyl)-2-methoxyphenyl)methanesulfonamide (see, for example, Cirillo, P. F. et al., WO 2002/083628, 24 Oct. 2002; 101 mg, 0.369 mmol) and DIPEA (193 L, 1.108 mmol) in dry DMF (5 mL) was treated with HATU (154 mg, 0.406 mmol) and the reaction mixture stirred at 60 C. for 18 h and then cooled to rt and stirred for 48 h. Reaction mixture was partitioned between EtOAc (40 mL) and sat. NaHCO.sub.3 soln (40 mL). The organics were washed with brine (540 mL) and dried (MgSO.sub.4). The crude product was purified by chromatography on silica gel (12 g column, 0-10% MeOH/DCM with 1% NH.sub.3) followed by preparative HPLC (Gilson, Basic (0.1% Ammonium Bicarbonate), Basic, Waters X-Bridge Prep-C18, 5 m, 1950 mm column, 5-95% MeCN in water) then dried at 40 C. under vacuum for 24 h to afford the title compound (60 mg) as a yellow solid.
[0629] .sup.1H NMR (400 MHz, DMSO-d6) 10.08 (s, 1H), 9.89 (s, 1H), 9.44-9.39 (m, 1H), 9.14 (s, 1H), 8.33-8.27 (m, 1H), 8.06-7.88 (m, 6H), 7.80-7.75 (m, 1H), 7.56-7.50 (m, 1H), 7.49-7.44 (m, 1H), 7.27-7.22 (m, 1H), 7.06-6.98 (m, 1H), 3.86 (s, 3H), 3.71 (s, 3H), 3.63-3.54 (m, 4H), 3.45-3.36 (m, 2H), 3.05 (s, 3H), 2.49-2.42 (m, 6H), 2.40 (s, 3H), 1.28 (s, 9H).
[0630] LCMS m/z 796 (M+H).sup.+ (ES.sup.+); 794 (MH).sup. (ES.sup.)
Example 6
[0631] The following compounds are prepared by methods analogous to those described above.
(a) N-(5-tert-Butyl-2-methoxy-3-methylsulfonyl-phenyl)-3-[2-[3-methoxy-5-(2-morpholinoethyl-carbamoyl)anilino]quinazolin-6-yl]-4-methyl-benzamide
[0632] ##STR00028##
(b) N-(5-tert-Butyl-2-methoxy-3-methylsulfinyl-phenyl)-3-[2-[3-methoxy-5-(2-morpholinoethyl-carbamoyl)anilino]quinazolin-6-yl]-4-methyl-benzamide
[0633] ##STR00029##
Example 7
N-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((4-((dimethyl(oxo)-I6-sulfanylidene)amino)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide
[0634] ##STR00030##
(i) 6-Bromo-2-(methylthio)quinazoline
[0635] A suspension of 6-bromo-2-chloroquinazoline (22.52 g, 92 mmol) in dry DMF (200 mL) was cooled to 0 C. and treated dropwise with a solution/slurry of sodium thiomethoxide (6.45 g, 92 mmol) in dry DMF (100 mL). The reaction mixture was slowly warmed to rt and stirred for 2 h. The reaction mixture was diluted with EtOAc (1 L) and partitioned with water (1 L). The organics were washed with brine (4500 mL), dried (MgSO.sub.4), and evaporated. The yellow solid was triturated from 50% diethyl ether/iso-hexanes, washing with fresh diethyl ether and drying under suction to afford the sub-title compound (16.59 g) as a yellow solid.
[0636] .sup.1H NMR (400 MHz, Chloroform-d) 9.10 (d, 1H), 8.01 (dd, 1H), 7.92 (dd, 1H), 7.77 (dt, 1H), 2.70 (s, 3H).
(ii) Methyl 4-methyl-3-(2-(methylthio)quinazolin-6-yl)benzoate
[0637] A suspension of the product from step (i) above (12.93 g, 50.7 mmol), methyl 4-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (14.0 g, 50.7 mmol) and cesium carbonate (49.75 g, 153 mmol) in THF/dioxane/water (200 mL; 2:2:1) was degassed with N.sub.2 (under sonication) and then treated with PdCl.sub.2(PPh.sub.3).sub.2 (3.56 g, 5.07 mmol). The suspension was further degassed and then stirred at 70 C. for 24 h. The reaction mixture was then held at rt for 48 h. The reaction mixture was partitioned between EtOAc (1 L) and 20% brine (1 L) and the organics washed with brine (21 L), dried (MgSO.sub.4) and evaporated to afford a brown oil. The crude product was purified by chromatography on silica gel (330 g column, 0-20% EtOAc/iso-hexanes). The resulting yellow solid was triturated from 30% diethyl ether/iso-hexanes to afford the sub-title compound (9.04 g) as an off-white solid. The liquours were evaporated to afford a second crop of the sub-title compound (3.02 g) as a yellow solid.
[0638] .sup.1H NMR (400 MHz, DMSO-d6) 9.46 (d, 1H), 8.12 (dd, 1H), 8.01 (dd, 1H), 7.97-7.86 (m, 3H), 7.58-7.50 (m, 1H), 3.87 (s, 3H), 2.66 (s, 3H), 2.36 (s, 3H).
(iii) 4-Methyl-3-(2-(methylthio)quinazolin-6-yl)benzoic acid
[0639] A solution of the product from step (ii) above (9.04 g, 27.9 mmol) in THF (150 mL) and MeOH (60 mL) was treated with aq. sodium hydroxide (16.72 ml, 33.4 mmol) and the reaction mixture stirred at rt for 18 h. Additional aq. sodium hydroxide (2.79 ml, 5.57 mmol) was added and the reaction mixture stirred at rt for 72 h. The reaction mixture was acidified with 1M HCl (to pH 1) and the white solid filtered, washed with water, diethyl ether, dried under suction and then dried under vacuum at 40 C. for 24 h to afford the sub-title compound (8.66 g) as a white solid.
[0640] .sup.1H NMR (400 MHz, DMSO-d6) 12.98 (s, 1H), 9.46 (d, 1H), 8.12 (dd, 1H), 8.01 (dd, 1H), 7.97-7.84 (m, 3H), 7.51 (d, 1H), 2.66 (s, 3H), 2.36 (s, 3H).
(iv) N-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-4-methyl-3-(2-(methylthio)quinazolin-6-yl)benzamide
[0641] A suspension of the product from step (iii) above (7.63 g, 24.58 mmol) and N-(3-amino-5-(tert-butyl)-2-methoxyphenyl)methanesulfonamide (8.01 g, 29.4 mmol) in dry DMF (130 mL) was cooled to 0 C. and treated slowly with DIPEA (12.88 ml, 73.8 mmol). The reaction was stirred for 10 mins until a solution formed. HATU (11.22 g, 29.5 mmol) was added portionwise and the reaction mixture allowed to reach rt and stirred for 18 h. The reaction mixture was treated with water (500 mL) and the slurry stirred for 30 mins. The resulting suspension was filtered and dried under suction. The solid was dissolved in DCM (80 mL) and partitioned with water (80 mL). The organics were separated through a hydrophobic frit, and evaporated and the crude product was purified by chromatography on silica gel (330 g column, 0-30% then to 45% EtOAc/iso-hexanes) to afford the sub-title compound (8.73 g) as a pale yellow solid.
[0642] .sup.1H NMR (400 MHz, DMSO-d6) 9.90 (s, 1H), 9.47 (d, 1H), 9.15 (s, 1H), 8.16 (d, 1H), 8.07 (dd, 1H), 8.01-7.92 (m, 3H), 7.54 (d, 1H), 7.45 (d, 1H), 7.25 (d, 1H), 3.70 (s, 3H), 3.04 (s, 3H), 2.66 (s, 3H), 2.38 (s, 3H), 1.28 (s, 9H).
[0643] LCMS m/z 565 (M+H).sup.+ (ES.sup.+); 563 (MH).sup. (ES.sup.)
(v) N-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-4-methyl-3-(2-(methylsulfinyl)quinazolin-6-yl)benzamide
[0644] A solution of the product from step (iv) above (11.0 g, 19.48 mmol) in dry DCM (375 mL) was cooled to 0 C. and treated portionwise with mCPBA (3.70 g, 21.43 mmol). The reaction mixture was slowly warmed to rt and stirred for 18 h. The reaction mixture was cooled to 0 C. and mCPBA (1.008 g, 5.84 mmol) was added and the reaction mixture warmed to rt and stirred for 2 h. The reaction mixture was partitioned with water (500 mL) and the organics washed with brine (2500 mL), dried (MgSO.sub.4) and evaporated. The residue was adsorbed onto silica and the crude product was purified by chromatography on silica gel (330 g column, 0-100% iso-hexanes/EtOAc then 0-8% MeOH/DCM) to afford the sub-title compound (8.89 g) as a pale yellow solid.
[0645] .sup.1H NMR (400 MHz, DMSO-d6) 9.92 (s, 1H), 9.83 (d, 1H), 9.15 (s, 1H), 8.38 (dd, 1H), 8.31-8.23 (m, 2H), 8.03 (s, 2H), 7.57 (d, 1H), 7.47 (d, 1H), 7.25 (d, 1H), 3.71 (s, 3H), 3.05 (s, 3H), 2.99 (s, 3H), 2.40 (s, 3H), 1.28 (s, 9H).
(vi) N-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((4-((dimethyl(oxo)-I6-sulfanylidene)amino)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide
[0646] A solution of the product from step (v) above (90 mg, 0.155 mmol) in dry DMF (1 mL) was treated with tosic acid (8.0 mg, 0.047 mmol. solution in 0.5 mL dry DMF) followed by ((4-aminophenyl)imino)dimethyl-I6-sulfanone (57 mg, 0.310 mmol) and the reaction mixture warmed to 70 C. and stirred for 18 h. The crude product was purified by preparative HPLC (Waters, Acidic (0.1% Formic acid), Acidic, Waters X-Select Prep-C18, 5 m, 1950 mm column, 35-65% MeCN in Water) to afford the title compound (18 mg) as a yellow solid.
[0647] .sup.1H NMR (400 MHz, DMSO-d6) 9.89 (s, 1H), 9.73 (s, 1H), 9.31 (d, 1H), 9.14 (s, 1H), 8.02-7.91 (m, 3H), 7.88 (dd, 1H), 7.81 (d, 2H), 7.70 (d, 1H), 7.51 (d, 1H), 7.46 (d, 1H), 7.25 (d, 1H), 6.97-6.90 (m, 2H), 3.71 (s, 3H), 3.20 (s, 6H), 3.05 (s, 3H), 2.40 (s, 3H), 1.28 (s, 9H).
[0648] LCMS m/z 701 (M+H).sup.+ (ES.sup.+)
Example 8
4-((6-(5-((5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxybenzoic acid
[0649] ##STR00031##
(i) Methyl 4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxybenzoate
[0650] A solution of N-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-4-methyl-3-(2-(methylsulfinyl)quinazolin-6-yl)benzamide (see Example 7(v) above; 2.02 g, 3.48 mmol), methyl 4-amino-2-methoxybenzoate (1.29 g, 7.12 mmol) and tosic acid (0.198 g, 1.044 mmol) in dry DMF (30 mL) was warmed to 70 C. and stirred for 18 h. The reaction mixture was partitioned between EtOAc (50 mL) and water (50 mL). The organics were washed with brine (550 mL), dried (MgSO.sub.4) and evaporated. The crude product was purified by chromatography on silica gel (80 g column, 0-100% EtOAc/iso-hexanes. Product eluted at 70%) to afford the sub-title compound (1.45 g) as a pale yellow solid.
[0651] .sup.1H NMR (400 MHz, DMSO-d6) 10.35 (s, 1H), 9.88 (s, 1H), 9.46 (d, 1H), 9.14 (s, 1H), 8.22 (d, 1H), 8.05 (d, 1H), 7.98 (dd, 3H), 7.86 (d, 1H), 7.74 (d, 1H), 7.56-7.50 (m, 2H), 7.47 (d, 1H), 7.25 (d, 1H), 3.91 (s, 3H), 3.77 (s, 3H), 3.71 (s, 3H), 3.05 (s, 3H), 2.39 (s, 3H), 1.28 (s, 9H).
[0652] LCMS m/z 698 (M+H).sup.+ (ES.sup.+); 696 (MH).sup. (ES.sup.)
(ii) 4-((6-(5-((5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxybenzoic acid
[0653] A suspension of the product from step (i) above (1.45 g, 2.078 mmol) in THF/MeOH (1:1; 12 mL) was treated with aq. sodium hydroxide (1.351 mL, 2.70 mmol) and the resultant solution stirred at rt for 18 h. Additional aq. sodium hydroxide (2.60 ml, 5.19 mmol) was added and the reaction mixture stirred at rt for 72 h. The reaction mixture was cooled to 0 C. and acidified to pH1 with 1M HCl. The resulting yellow solid was filtered, washed with water, diethyl ether and dried under suction. The solid was further dried for 18 h under vacuum at 40 C. to afford the title compound (1.24 g) as an orange solid.
[0654] .sup.1H NMR (400 MHz, DMSO-d6) 10.31 (s, 1H), 9.89 (s, 1H), 9.45 (d, 1H), 9.14 (s, 1H), 8.19 (d, 1H), 8.05 (d, 1H), 8.02-7.93 (m, 3H), 7.85 (d, 1H), 7.75 (d, 1H), 7.55-7.49 (m, 2H), 7.47 (d, 1H), 7.25 (d, 1H), 3.92 (s, 3H), 3.71 (s, 3H), 3.05 (s, 3H), 2.39 (s, 3H), 1.28 (s, 9H).
[0655] LCMS m/z 684 (M+H).sup.+ (ES.sup.+); 682 (MH).sup. (ES.sup.)
Example 9
4-((6-(5-((5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxy-N-methyl-N-(2-morpholinoethyl)benzamide
[0656] ##STR00032##
[0657] HATU (49 mg, 0.129 mmol) was added to a stirred solution of 4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxybenzoic acid (see Example 8 above; 80 mg, 0.117 mmol), N-methyl-2-morpholinoethanamine (20 mg, 0.139 mmol) and DIPEA (62 L, 0.355 mmol) in DMF (3 mL) at rt. The mixture was stirred for 3 h. The mixture was poured into water (10 mL). The organic layer was extracted with DCM (10 mL) and dried via a hydrophobic phase separator. The crude product was purified by preparative HPLC (Varian, Basic (0.1% Ammonium Bicarbonate), Basic, Waters X-Bridge Prep-C18, 5 m, 1950 mm column, 20-50% MeCN in Water) to afford the title compound (21 mg) as a yellow solid.
[0658] .sup.1H NMR (400 MHz, DMSO-d6) 10.10 (s, 1H), 9.87 (s, 1H), 9.40 (s, 1H), 9.14 (s, 1H), 8.07-7.92 (m, 5H), 7.80 (d, 1H), 7.54-7.50 (m, 2H), 7.45 (d, 1H), 7.24 (d, 1H), 7.11 (dd, 1H), 3.86 (s, 3H), 3.70 (s, 3H), 3.59 (t, 2H), 3.55 (m, 1H), 3.47 (t, 2H), 3.25 (bs, 1H), 3.04 (s, 3H), 2.97 (s, 1.5H), 2.82 (s, 1.5H), 2.45 (m, 3H), 2.39 (s, 3H), 2.19 (m, 2H), 1.27 (s, 9H). (1 proton under DMSO peak)
[0659] LCMS m/z 810 (M+H).sup.+ (ES.sup.+)
Example 10
3-((6-(5-((5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)-quinazolin-2-yl)amino)-5-((2-morpholinoethyl)carbamoyl)phenyl methanesulfonate
[0660] ##STR00033##
(i) Methyl 3-amino-5-((methylsulfonyl)oxy)benzoate
[0661] Methyl 3-amino-5-hydroxybenzoate (239 mg, 1.430 mmol) and triethylamine (239 L, 1.716 mmol) were stirred in dichloromethane (15 mL). Methanesulfonyl chloride (122 L, 1.573 mmol) was added and the mixture was stirred at room temperature for 18 h. The mixture was diluted with water (20 mL), and extracted with dichloromethane (340 mL). The combined organic phases were washed with saturated brine (20 mL), dried (MgSO.sub.4) and concentrated to yield a colourless oil. The crude product was purified by chromatography on the Companion (12 g column, 0-50% EtOAc:iso-hexanes) to afford the sub-title compound (342 mg) as a colourless oil.
[0662] .sup.1H NMR (DMSO-d6) 400 MHz, : 7.18 (t, 1H), 6.95 (t, 1H), 6.72 (t, 1H), 5.81 (s, 2H), 3.83 (s, 3H), 3.37 (s, 3H).
[0663] LCMS m/z 246 (M+H).sup.+ (ES.sup.+)
(ii) 3-Amino-5-((methylsulfonyl)oxy)benzoic acid
[0664] 1M Sodium hydroxide solution (1.464 mL, 1.464 mmol) was added to a solution of the product from step (i) above (0.342 g, 1.394 mmol) in THF (1 mL) at rt then stirred for 4 h. The mixture was acidified with 1 M HCl (1.5 mL) and extracted with EtOAc (310 mL). The combined organic phases were washed with saturated brine (10 mL), dried (MgSO.sub.4) and concentrated to yield the sub-title compound (287 mg).
[0665] .sup.1H NMR (DMSO-d6) 400 MHz, :7.06 (t, 1H), 6.94 (t, 1H), 6.70 (t, 1H), 5.75 (brs, 2H), 3.36 (s, 3H).
[0666] LCMS m/z 232 (M+H).sup.+ (ES.sup.+); 230 (MH).sup. (ES.sup.)
(iii) 3-Amino-5-((2-morpholinoethyl)carbamoyl)phenyl methanesulfonate
[0667] 2-Morpholinoethanamine (0.261 mL, 1.986 mmol) was added to an ice cold suspension of T3P (50% in EtOAc, 0.739 mL, 1.241 mmol), the product from step (ii) above (0.287 g, 0.993 mmol) and TEA (0.415 mL, 2.98 mmol) in EtOAc (5 mL). The mixture was allowed to warm to room temperature and stir overnight. Sat. NaHCO.sub.3 solution (20 mL) was added and the mixture was extracted with EtOAc (310 mL). The combined organic phases were washed with saturated brine (20 mL), dried (MgSO.sub.4) and concentrated under reduced pressure to yield a sticky solid. The solid was triturated in diethyl ether to yield the sub-title compound (268 mg) as an off-white solid.
[0668] .sup.1H NMR (400 MHz, DMSO) : 8.28 (t, 1H), 6.99 (t, 1H), 6.83 (t, 1H), 6.62 (t, 1H), 5.66 (br s, 2H), 3.61-3.53 (m, 4H), 3.37-3.30 (m, 2H), 3.36 (s, 3H), 2.49-2.38 (m, 6H).
[0669] LCMS m/z 344 (M+H).sup.+ (ES.sup.+)
(iv) 3-((6-(5-((5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-5-((2-morpholinoethyl)carbamoyl)phenyl methanesulfonate
[0670] A solution of N-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-4-methyl-3-(2-(methylsulfinyl)quinazolin-6-yl)benzamide (see Example 7(v) above; 80 mg, 0.138 mmol) and the product from step (iii) above (95 mg, 0.276 mmol) in dry DMF (1 mL) and treated with tosic acid (60 mg, 0.315 mmol). Reaction mixture was warmed to 70 C. and stirred for 24 h and then cooled to rt and stirred for 48 h. The reaction mixture was basified with aq. ammonia and purified by preparative HPLC (Waters, Basic (0.1% Ammonium Bicarbonate), Basic, Waters X-Bridge Prep-C18, 5 m, 1950 mm column, 35-65% MeCN in Water) and dried for 24 h under vacuum at 45 C. to afford the title compound (34 mg) as an off-white solid.
[0671] .sup.1H NMR (400 MHz, DMSO-d6) 10.39 (s, 1H), 9.91 (s, 1H), 9.45 (s, 1H), 9.16 (s, 1H), 8.50 (s, 1H), 8.46 (d, 1H), 8.28 (t, 1H), 8.05 (d, 1H), 8.04-7.93 (m, 3H), 7.80 (d, 1H), 7.53 (d, 1H), 7.46 (d, 1H), 7.40-7.36 (m, 1H), 7.25 (d, 1H), 3.71 (s, 3H), 3.59 (t, 4H), 3.51 (s, 3H), 3.42 (d, 2H), 3.05 (s, 3H), 2.44 (s, 4H), 2.39 (s, 3H), 1.28 (s, 9H). 2 aliphatic signals obscured by DMSO peak.
[0672] LCMS m/z 860 (M+H).sup.+ (ES.sup.+)
Example 11
4-((6-(5-((5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)-quinazolin-2-yl)amino)-2-methoxy-N-(3-(1-oxidothiomorpholino)propyl)benzamide
[0673] ##STR00034##
[0674] A solution of 4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxybenzoic acid (see Example 8 above; 78 mg, 0.114 mmol) in dry DMF (0.5 mL) was treated with 4-(3-aminopropyl)thiomorpholine 1-oxide (22 mg, 0.125 mmol) and DIPEA (40 L, 0.228 mmol). A solution of HATU (48 mg, 0.125 mmol) in dry DMF (0.5 mL) was added and the reaction mixture shaken at rt for 18 h. The crude product was purified by preparative HPLC (Waters, Basic (0.1% Ammonium Bicarbonate), Basic, Waters X-Bridge Prep-C18, 5 m, 1950 mm column, 35-65% MeCN in Water) to afford the title compound (12 mg) as a yellow solid.
[0675] .sup.1H NMR (400 MHz, DMSO-d6) 10.26 (s, 1H), 9.90 (s, 1H), 9.44 (s, 1H), 9.15 (s, 1H), 8.23 (d, 1H), 8.09 (t, 1H), 8.04 (d, 1H), 8.03-7.92 (m, 3H), 7.84 (dd, 2H), 7.59-7.50 (m, 2H), 7.47 (d, 1H), 7.25 (d, 1H), 4.01 (s, 3H), 3.71 (s, 3H), 3.32 (t, 2H), 3.05 (s, 3H), 2.93-2.82 (m, 4H), 2.78-2.61 (m, 4H), 2.44 (t, 2H), 2.39 (s, 3H), 1.71 (t, 2H), 1.28 (s, 9H).
[0676] LCMS m/z 842 (M+H).sup.+ (ES.sup.+)
Example 12
N-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((4-(1,1-dioxidothiomorpholine-4-carbonyl)-3-methoxyphenyl)amino)quinazolin-6-yl)-4-methylbenzamide
[0677] ##STR00035##
[0678] A solution of 4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxybenzoic acid (see Example 8 above; 78 mg, 0.114 mmol) in dry DMF (0.5 mL) was treated with thiomorpholine 1,1-dioxide (15 mg, 0.114 mmol) and DIPEA (40 L, 0.228 mmol). A solution of HATU (48 mg, 0.125 mmol) in dry DMF (0.5 mL) was added and the reaction mixture shaken at rt for 18 h. The crude product was purified by preparative HPLC (Waters, Basic (0.1% Ammonium Bicarbonate), Basic, Waters X-Bridge Prep-C18, 5 m, 1950 mm column, 35-65% MeCN in Water) to afford the title compound (60 mg) as a yellow solid.
[0679] .sup.1H NMR (400 MHz, DMSO-d6) 10.18 (s, 1H), 9.89 (s, 1H), 9.42 (s, 1H), 9.15 (s, 1H), 8.13 (d, 1H), 8.06-7.90 (m, 4H), 7.83 (d, 1H), 7.59-7.50 (m, 2H), 7.47 (d, 1H), 7.29 (d, 1H), 7.26 (d, 1H), 3.90 (s, 5H), 3.72 (s, 3H), 3.64 (s, 2H), 3.05 (s, 7H), 2.40 (s, 3H), 1.28 (s, 9H).
[0680] LCMS m/z 801 (M+H).sup.+ (ES.sup.+)
Example 13
4-((6-(5-((5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)-quinazolin-2-ylamino)-N-(2-(4-(2-hydroxyethyl)piperazin-1-yl)ethyl)-2-methoxybenzamide
[0681] ##STR00036##
[0682] A solution of 4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxybenzoic acid (see Example 8 above; 47 mg, 0.069 mmol), 2-(4-(2-aminoethyl)piperazin-1-yl)ethanol (13 mg, 0.076 mmol) and DIPEA (24 L, 0.137 mmol) in dry DMF (0.3 mL) was treated with a solution of HATU (29 mg, 0.076 mmol) in dry DMF (0.3 mL) and the reaction mixture shaken at rt for 18 h. The crude product was purified by preparative HPLC (Waters, Basic (0.1% Ammonium Bicarbonate), Basic, Waters X-Bridge Prep-C18, 5 m, 1950 mm column, 35-65% MeCN in Water) to afford the title compound (39 mg) as a yellow solid.
[0683] .sup.1H NMR (400 MHz, DMSO-d6) 10.28 (s, 1H), 9.88 (s, 1H), 9.44 (d, 1H), 9.15 (s, 1H), 8.32 (s, 1H), 8.26 (d, 1H), 8.08-7.81 (m, 6H), 7.59-7.50 (m, 2H), 7.47 (d, 1H), 7.25 (d, 1H), 4.38 (t, 1H), 4.05 (s, 3H), 3.71 (s, 3H), 3.51 (q, 2H), 3.41 (d, 2H), 3.05 (s, 3H), 2.48-2.37 (m, 15H), 1.28 (s, 9H). Aliphatics obscured under DMSO signal.
[0684] LCMS m/z 839 (M+H).sup.+ (ES.sup.+); 837 (MH).sup. (ES.sup.)
Example 14
[0685] The following compounds were prepared by methods analogous to those described above. Where chemical shifts from 1H NMR spectra are reported, these were obtained at 400 MHz and ambient temperature, unless otherwise specified.
(a) 3-((6-(5-((5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)phenyl methanesulfonate
[0686] ##STR00037##
[0687] .sup.1H NMR (400 MHz, DMSO-d6) 10.25 (s, 1H), 9.88 (s, 1H), 9.43 (d, 1H), 9.14 (s, 1H), 8.28 (t, 1H), 8.03 (d, 1H), 7.97 (dd, 3H), 7.88 (d, 1H), 7.80 (d, 1H), 7.53 (d, 1H), 7.51-7.42 (m, 2H), 7.25 (d, 1H), 6.98 (ddd, 1H), 3.71 (s, 3H), 3.45 (s, 3H), 3.04 (s, 3H), 2.40 (s, 3H), 1.28 (s, 9H).
[0688] LCMS m/z 704 (M+H).sup.+ (ES.sup.+); 702 (MH).sup. (ES.sup.)
(b) N-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-cyano-5-(2-morpholinoethoxy)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide
[0689] ##STR00038##
[0690] .sup.1H NMR (400 MHz, DMSO-d6) 10.30 (s, 1H), 9.89 (s, 1H), 9.45 (d, 1H), 9.14 (s, 1H), 8.08-8.03 (m, 2H), 8.03-7.92 (m, 4H), 7.84 (d, 1H), 7.53 (d, 1H), 7.47 (d, 1H), 7.25 (d, 1H), 7.09 (dd, 1H), 4.20 (t, 2H), 3.71 (s, 3H), 3.62-3.58 (m, 4H), 3.05 (s, 3H), 2.75 (t, 2H), 2.39 (s, 3H), 1.28 (s, 9H). 4 protons under DMSO signal.
[0691] LCMS m/z 764 (M+H).sup.+ (ES.sup.+)
(c) N-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-methoxy-5-(N-methyl-N-(2-morpholinoethyl)sulfamoyl)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide
[0692] ##STR00039##
[0693] .sup.1H NMR (400 MHz, DMSO-d6) 10.30 (s, 1H), 9.89 (s, 1H), 9.44 (d, 1H), 9.14 (s, 1H), 8.18 (t, 1H), 8.05 (d, 1H), 8.04-7.91 (m, 4H), 7.76 (d, 1H), 7.53 (d, 1H), 7.47 (d, 1H), 7.25 (d, 1H), 6.89 (dd, 1H), 3.88 (s, 3H), 3.71 (s, 3H), 3.56-3.51 (m, 4H), 3.19 (t, 2H), 3.05 (s, 3H), 2.83 (s, 3H), 2.39 (s, 7H), 1.28 (s, 9H). 2 protons under DMSO signal.
[0694] LCMS m/z 846 (M+H).sup.+ (ES.sup.+)
(d) N-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-4-methyl-3-(2-((3-((2-morpholinoethyl)carbamoyl)phenyl)amino)quinazolin-6-yl)benzamide
[0695] ##STR00040##
[0696] .sup.1H NMR (400 MHz, DMSO-d6) 10.08 (s, 1H), 9.89 (s, 1H), 9.40 (d, 1H), 9.14 (s, 1H), 8.42 (s, 1H), 8.32 (d, 1H), 8.21-8.13 (m, 1H), 8.04-7.97 (m, 2H), 7.98-7.90 (m, 2H), 7.77 (d, 1H), 7.52 (d, 1H), 7.48-7.42 (m, 3H), 7.25 (d, 1H), 3.71 (s, 3H), 3.59 (t, 4H), 3.41 (m, 2H), 3.05 (s, 3H), 2.46 (d, 6H), 2.40 (s, 3H), 1.28 (s, 9H).
[0697] LCMS m/z 766 (M+H).sup.+ (ES.sup.+)
(e) N-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-methoxy-5-(N-methyl-N-(3-morpholinopropyl)sulfamoyl)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide
[0698] ##STR00041##
[0699] .sup.1H NMR (400 MHz, DMSO-d6) 10.31 (s, 1H), 9.89 (s, 1H), 9.44 (d, 1H), 9.14 (s, 1H), 8.18 (t, 1H), 8.05 (d, 1H), 8.02-7.92 (m, 4H), 7.76 (d, 1H), 7.53 (d, 1H), 7.47 (d, 1H), 7.25 (d, 1H), 6.87 (dd, 1H), 3.88 (s, 3H), 3.71 (s, 3H), 3.52 (m, 4H), 3.08 (t, 2H), 3.05 (s, 3H), 2.78 (s, 3H), 2.39 (s, 3H), 2.29 (t, 6H), 1.71-1.61 (m, 2H), 1.28 (s, 9H).
[0700] LCMS m/z 860 (M+H).sup.+ (ES.sup.+)
(f) N-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-cyano-5-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide
[0701] ##STR00042##
[0702] .sup.1H NMR (400 MHz, DMSO-d6) 10.31 (s, 1H), 9.88 (s, 1H), 9.46 (d, 1H), 9.14 (s, 1H), 8.07-8.02 (m, 3H), 7.97 (ddd, 3H), 7.87-7.83 (m, 1H), 7.53 (d, 1H), 7.46 (d, 1H), 7.25 (d, 1H), 7.08 (dd, 1H), 4.25-4.17 (m, 2H), 3.83-3.77 (m, 2H), 3.71 (s, 3H), 3.65-3.59 (m, 2H), 3.59-3.50 (m, 4H), 3.47-3.40 (m, 2H), 3.23 (s, 3H), 3.04 (s, 3H), 2.39 (s, 3H), 1.28 (s, 9H).
[0703] LCMS m/z 797 (M+H).sup.+ (ES.sup.+)
(g) N-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-ethynyl-5-((2-(2-(2-methoxyethoxy)ethoxy)ethyl)carbamoyl)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide
[0704] ##STR00043##
[0705] .sup.1H NMR (400 MHz, DMSO-d6) 10.22 (s, 1H), 9.89 (s, 1H), 9.43 (d, 1H), 9.14 (s, 1H), 8.56 (s, 1H), 8.51 (t, 1H), 8.30 (dd, 1H), 8.04 (d, 1H), 8.02-7.91 (m, 3H), 7.78 (dt, 1H), 7.58 (t, 1H), 7.53 (d, 1H), 7.46 (d, 1H), 7.25 (d, 1H), 4.27 (s, 1H), 3.71 (s, 3H), 3.61-3.49 (m, 8H), 3.48-3.38 (m, 4H), 3.22 (s, 3H), 3.05 (s, 3H), 2.40 (s, 3H), 1.28 (s, 9H).
[0706] LCMS m/z 823 (M+H).sup.+ (ES.sup.+)
(h) N-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-(dimethylphosphoryl)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide
[0707] ##STR00044##
[0708] .sup.1H NMR (400 MHz, DMSO-d6) 10.13 (s, 1H), 9.89 (s, 1H), 9.41 (d, 1H), 9.15 (s, 1H), 8.46 (dt, 1H), 8.21-8.13 (m, 1H), 8.04-7.90 (m, 4H), 7.76 (d, 1H), 7.56-7.44 (m, 3H), 7.38 (ddt, 1H), 7.25 (d, 1H), 3.71 (s, 3H), 3.05 (s, 3H), 2.40 (s, 3H), 1.70 (d, 6H), 1.28 (s, 9H).
[0709] LCMS m/z 686 (M+H).sup.+ (ES.sup.+)
(i) N-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-((dimethylphosphoryl)methyl)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide
[0710] ##STR00045##
[0711] .sup.1H NMR (400 MHz, DMSO-d6) 9.93 (s, 1H), 9.89 (s, 1H), 9.37 (d, 1H), 9.14 (s, 1H), 8.03-7.87 (m, 6H), 7.77 (d, 1H), 7.52 (d, 1H), 7.46 (d, 1H), 7.28-7.17 (m, 3H), 3.71 (s, 3H), 3.11 (d, 2H), 3.05 (s, 3H), 2.40 (s, 3H), 1.35 (d, 6H), 1.28 (s, 9H).
[0712] LCMS m/z 700 (M+H).sup.+ (ES.sup.+)
(j) 4-((6-(5-((5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)phenyl methanesulfonate
[0713] ##STR00046##
[0714] .sup.1H NMR (400 MHz, DMSO-d6) 10.16 (s, 1H), 9.88 (s, 1H), 9.41 (d, 1H), 9.15 (s, 1H), 8.15-8.08 (m, 2H), 8.05-7.91 (m, 4H), 7.84-7.74 (m, 1H), 7.52 (d, 1H), 7.46 (d, 1H), 7.37-7.32 (m, 2H), 7.25 (d, 1H), 3.71 (s, 3H), 3.37 (s, 3H), 3.05 (s, 3H), 2.40 (s, 3H), 1.28 (s, 9H).
[0715] LCMS m/z 704 (M+H).sup.+ (ES.sup.+)
(k) N-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-ethynyl-5-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide
[0716] ##STR00047##
[0717] .sup.1H NMR (400 MHz, DMSO-d6) 10.07 (s, 1H), 9.89 (s, 1H), 9.41 (d, 1H), 9.14 (s, 1H), 8.05-7.98 (m, 2H), 7.95 (ddd, 2H), 7.88 (t, 1H), 7.79 (d, 1H), 7.67 (s, 1H), 7.52 (d, 1H), 7.46 (d, 1H), 7.25 (d, 1H), 6.69 (dd, 1H), 4.16 (m, 3H), 3.82-3.76 (m, 2H), 3.71 (s, 3H), 3.65-3.60 (m, 2H), 3.59-3.51 (m, 4H), 3.48-3.40 (m, 2H), 3.23 (s, 3H), 3.05 (s, 3H), 2.39 (s, 3H), 1.28 (s, 9H).
[0718] LCMS m/z 796 (M+H).sup.+ (ES.sup.+)
(l) N-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-fluoro-5-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide
[0719] ##STR00048##
[0720] .sup.1H NMR (400 MHz, DMSO-d6) 10.15 (s, 1H), 9.88 (s, 1H), 9.42 (d, 1H), 9.14 (s, 1H), 8.04-7.98 (m, 2H), 7.95 (dd, 2H), 7.82 (d, 1H), 7.61 (d, 1H), 7.55-7.49 (m, 1H), 7.46 (d, 2H), 7.25 (d, 1H), 6.48 (d, 1H), 4.17-4.10 (m, 2H), 3.78 (t, 2H), 3.71 (s, 3H), 3.62 (dd, 2H), 3.60-3.51 (m, 4H), 3.47-3.40 (m, 2H), 3.24 (s, 3H), 3.04 (s, 3H), 2.39 (s, 3H), 1.28 (s, 9H).
[0721] LCMS m/z 790 (M+H).sup.+ (ES.sup.+)
(m) N-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-ethynyl-5-((2-(2-methoxyethoxy)ethyl)carbamoyl)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide
[0722] ##STR00049##
[0723] .sup.1H NMR (400 MHz, DMSO-d6) 10.22 (s, 1H), 9.88 (s, 1H), 9.43 (d, 1H), 9.15 (s, 1H), 8.56 (t, 1H), 8.50 (t, 1H), 8.32-8.28 (m, 1H), 8.04 (d, 1H), 8.02-7.90 (m, 3H), 7.78 (d, 1H), 7.58 (t, 1H), 7.53 (d, 1H), 7.46 (d, 1H), 7.25 (d, 1H), 4.27 (s, 1H), 3.71 (s, 3H), 3.60-3.53 (m, 4H), 3.50-3.39 (m, 4H), 3.25 (s, 3H), 3.04 (s, 3H), 2.40 (s, 3H), 1.28 (s, 9H).
[0724] LCMS m/z 779 (M+H).sup.+ (ES.sup.+)
(n) N-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-ethynyl-5-((2-methoxyethyl)carbamoyl)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide
[0725] ##STR00050##
[0726] .sup.1H NMR (400 MHz, DMSO-d6) 10.22 (s, 1H), 9.89 (s, 1H), 9.43 (d, 1H), 9.14 (s, 1H), 8.57 (t, 1H), 8.51 (t, 1H), 8.31 (s, 1H), 8.04 (d, 1H), 8.02-7.92 (m, 3H), 7.78 (d, 1H), 7.58 (d, 1H), 7.53 (d, 1H), 7.46 (d, 1H), 7.25 (d, 1H), 4.27 (s, 1H), 3.71 (s, 3H), 3.52-3.42 (m, 4H), 3.30 (s, 3H), 3.04 (s, 3H), 2.40 (s, 3H), 1.28 (s, 9H).
[0727] LCMS m/z 735 (M+H).sup.+ (ES.sup.+)
(o) 3-((6-(5-((5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-5-methoxybenzoic acid
[0728] ##STR00051##
[0729] .sup.1H NMR (400 MHz, DMSO-d6) 12.92 (s, 1H), 10.16 (s, 1H), 9.94 (s, 1H), 9.42 (d, 1H), 9.17 (s, 1H), 8.17-8.09 (m, 2H), 8.05-7.91 (m, 4H), 7.80-7.74 (m, 1H), 7.52 (d, 1H), 7.46 (d, 1H), 7.25 (d, 1H), 7.11 (dd, 1H), 3.85 (s, 3H), 3.71 (s, 3H), 3.05 (s, 3H), 2.39 (s, 3H), 1.27 (s, 9H).
[0730] LCMS m/z 684 (M+H).sup.+ (ES.sup.+); 682 (MH).sup. (ES.sup.)
(p) 4-((6-(5-((5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxy-N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)benzamide
[0731] ##STR00052##
[0732] .sup.1H NMR (400 MHz, DMSO-d6) 10.28 (s, 1H), 9.86 (s, 1H), 9.43 (s, 1H), 9.13 (s, 1H), 8.24 (d, 1H), 8.19 (dd, 1H), 8.03 (d, 1H), 7.98-7.94 (m, 3H), 7.88 (d, 1H), 7.84 (d, 1H), 7.55-7.51 (m, 2H), 7.45 (s, 1H), 7.24 (s, 1H), 4.01 (s, 3H), 3.70 (s, 3H), 3.58-3.42 (m, 12H), 3.23 (s, 3H), 3.02 (bs, 3H), 2.39 (s, 3H), 1.27 (s, 9H).
[0733] LCMS m/z 829 (M+H).sup.+ (ES.sup.+)
(q) N-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-((dimethyl(oxo)-I6-sulfanylidene)amino)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide
[0734] ##STR00053##
[0735] .sup.1H NMR (400 MHz, DMSO-d6) 9.92 (s, 1H), 9.85 (s, 1H), 9.36 (s, 1H), 7.99 (s, 1H), 7.99-7.89 (m, 3H), 7.83 (s, 1H), 7.71 (d, 1H), 7.52 (d, 1H), 7.44 (d, 2H), 7.25 (d, 1H), 7.13 (t, 1H), 6.58 (d, 1H), 3.70 (s, 3H), 3.29 (s, 6H), 3.04 (s, 3H), 2.39 (s, 3H), 1.27 (s, 9H).
[0736] LCMS m/z 701 (M+H).sup.+ (ES.sup.+)
(r) N-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-(2-(2-(2-methoxy-ethoxy)ethoxy)ethoxy)-5-(trifluoromethoxy)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide
[0737] ##STR00054##
[0738] .sup.1H NMR (400 MHz, DMSO-d6) 10.24 (s, 1H), 9.90 (s, 1H), 9.44 (d, 1H), 9.14 (s, 1H), 8.04 (d, 1H), 8.02-7.92 (m, 3H), 7.86-7.76 (m, 2H), 7.70 (t, 1H), 7.53 (d, 1H), 7.47 (d, 1H), 7.25 (d, 1H), 6.58 (s, 1H), 4.17 (t, 2H), 3.80 (dd, 2H), 3.71 (s, 3H), 3.65-3.60 (m, 2H), 3.59-3.50 (m, 4H), 3.47-3.41 (m, 2H), 3.23 (s, 3H), 3.05 (s, 3H), 2.39 (s, 3H), 1.28 (s, 9H).
[0739] LCMS m/z 856 (M+H).sup.+ (ES.sup.+)
(s) N-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-3-(2-((3-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-5-(trifluoromethyl)phenyl)amino)quinazolin-6-yl)-4-methylbenzamide
[0740] ##STR00055##
[0741] .sup.1H NMR (400 MHz, DMSO-d6) 10.30 (s, 1H), 9.90 (s, 1H), 9.45 (d, 1H), 9.14 (s, 1H), 8.05 (d, 2H), 8.03-7.91 (m, 4H), 7.80 (d, 1H), 7.53 (d, 1H), 7.47 (d, 1H), 7.25 (d, 1H), 6.89 (s, 1H), 4.23 (t, 2H), 3.84-3.78 (m, 2H), 3.71 (s, 3H), 3.66-3.59 (m, 2H), 3.59-3.49 (m, 4H), 3.45-3.40 (m, 2H), 3.23 (s, 3H), 3.05 (s, 3H), 2.39 (s, 3H), 1.28 (s, 9H).
[0742] LCMS m/z 840 (M+H).sup.+ (ES.sup.+)
(t) N-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-4-methyl-3-(2-((4-(S-methylsulfonimidoyl)phenyl)amino)quinazolin-6-yl)benzamide
[0743] ##STR00056##
[0744] .sup.1H NMR (400 MHz, DMSO-d6) 9.90 (s, 1H), 9.30 (d, 1H), 8.00-7.90 (m, 3H), 7.90-7.83 (m, 1H), 7.65 (dd, 3H), 7.50 (d, 1H), 7.44 (d, 1H), 7.24 (d, 1H), 6.69-6.62 (m, 2H), 6.11 (s, 2H), 3.69 (s, 3H), 3.50 (s, 3H), 3.04 (s, 3H), 2.36 (s, 3H), 1.27 (s, 9H).
[0745] LCMS m/z 687 (M+H)+ (ES+)
(u) N-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-4-methyl-3-(2-((3-((2-morpholinoethyl)carbamoyl)-5-(trifluoromethyl)phenyl)amino)quinazolin-6-yl)benzamide
[0746] ##STR00057##
[0747] .sup.1H NMR (400 MHz, DMSO-d6) 10.49 (s, 1H), 9.91 (s, 1H), 9.47 (d, 1H), 9.16 (s, 1H), 8.71 (s, 1H), 8.71-8.60 (m, 2H), 8.07 (d, 1H), 8.04-7.92 (m, 3H), 7.79 (d, 2H), 7.53 (d, 1H), 7.46 (d, 1H), 7.25 (d, 1H), 3.71 (s, 3H), 3.59 (t, 4H), 3.44 (d, 2H), 3.05 (s, 3H), 2.44 (d, 4H), 2.40 (s, 3H), 1.28 (s, 9H). 2 aliphatic signals obscured by DMSO signal.
[0748] LCMS m/z 834 (M+H).sup.+ (ES.sup.+)
(v) 4-((6-(5-((5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-N-(2-(2-(2-hydroxyethoxy)ethoxy)ethyl)-2-methoxybenzamide
[0749] ##STR00058##
[0750] .sup.1H NMR (400 MHz, DMSO-d6) 10.29 (s, 1H), 9.89 (s, 1H), 9.44 (s, 1H), 9.15 (s, 1H), 8.26 (d, 1H), 8.21 (t, 1H), 8.04 (d, 1H), 8.03-7.92 (m, 3H), 7.88 (dd, 2H), 7.57-7.50 (m, 2H), 7.47 (d, 1H), 7.25 (d, 1H), 4.60 (t, 1H), 4.02 (s, 3H), 3.72 (s, 3H), 3.63-3.43 (m, 12H), 3.05 (s, 3H), 2.40 (s, 3H), 1.28 (s, 9H).
[0751] LCMS m/z 815 (M+H).sup.+ (ES.sup.+)
(w) 4-((6-(5-((5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxy-N-(2-(2-methoxyethoxy)ethyl)benzamide
[0752] ##STR00059##
[0753] .sup.1H NMR (400 MHz, DMSO-d6) 10.29 (s, 1H), 9.89 (s, 1H), 9.44 (s, 1H), 9.15 (s, 1H), 8.26 (d, 1H), 8.21 (t, 1H), 8.04 (d, 1H), 8.03-7.93 (m, 3H), 7.88 (dd, 2H), 7.57-7.50 (m, 2H), 7.47 (d, 1H), 7.25 (d, 1H), 4.02 (s, 3H), 3.72 (s, 3H), 3.63-3.52 (m, 4H), 3.52-3.43 (m, 4H), 3.28 (s, 3H), 3.05 (s, 3H), 2.39 (s, 3H), 1.28 (s, 9H).
[0754] LCMS m/z 785 (M+H).sup.+ (ES.sup.+)
(x) 4-((6-(5-((5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-N-(3-hydroxy-2,2-bis(hydroxymethyl)propyl)-2-methoxybenzamide
[0755] ##STR00060##
[0756] .sup.1H NMR (400 MHz, DMSO-d6) 10.29 (s, 1H), 9.88 (s, 1H), 9.44 (s, 1H), 9.15 (s, 1H), 8.60 (t, 1H), 8.26 (d, 1H), 8.04 (d, 1H), 8.02-7.93 (m, 3H), 7.88 (dd, 2H), 7.57-7.50 (m, 2H), 7.46 (d, 1H), 7.25 (d, 1H), 4.54 (t, 3H), 4.00 (s, 3H), 3.72 (s, 3H), 3.36 (d, 6H), 3.34 (d, 2H), 3.04 (s, 3H), 2.39 (s, 3H), 1.28 (s, 9H).
[0757] LCMS m/z 801 (M+H).sup.+ (ES.sup.+)
(y) N-(2-(1,4-Oxazepan-4-yl)ethyl)-4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)-phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxybenzamide
[0758] ##STR00061##
[0759] .sup.1H NMR (400 MHz, DMSO-d6) 10.28 (s, 1H), 9.90 (s, 1H), 9.44 (s, 1H), 9.15 (s, 1H), 8.31 (t, 1H), 8.25 (d, 1H), 8.04 (d, 1H), 8.02-7.94 (m, 3H), 7.91 (d, 1H), 7.86 (d, 1H), 7.59-7.51 (m, 2H), 7.48 (d, 1H), 7.26 (d, 1H), 4.04 (s, 3H), 3.71 (m, 5H), 3.69-3.64 (m, 2H), 3.40 (q, 2H), 3.06 (s, 3H), 2.71 (dt, 4H), 2.66 (t, 2H), 2.40 (s, 3H), 1.84 (p, 2H), 1.28 (s, 9H).
[0760] LCMS m/z 810 (M+H).sup.+ (ES.sup.+)
(z) 4-((6-(5-((5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methyl-phenyl)quinazolin-2-yl)amino)-2-methoxy-N-(2-(4-methylpiperazin-1-yl)ethyl)benzamide
[0761] ##STR00062##
[0762] .sup.1H NMR (400 MHz, DMSO-d6) 10.29 (s, 1H), 9.89 (s, 1H), 9.44 (s, 1H), 9.16 (s, 1H), 8.31 (t, 1H), 8.26 (d, 1H), 8.06-7.84 (m, 6H), 7.58-7.50 (m, 2H), 7.48 (d, 1H), 7.26 (d, 1H), 4.05 (s, 3H), 3.72 (s, 3H), 3.41 (q, 2H), 3.06 (s, 3H), 2.44 (m, 13H), 2.19 (s, 3H), 1.28 (s, 9H).
[0763] LCMS m/z 809 (M+H).sup.+ (ES.sup.+)
(aa) 4-((6-(5-((5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methyl-phenyl)quinazolin-2-yl)amino)-2-methoxy-N-(2-(1-methylpiperidin-4-yl)ethyl)benzamide
[0764] ##STR00063##
[0765] .sup.1H NMR (400 MHz, DMSO-d6) 10.26 (s, 1H), 9.89 (s, 1H), 9.44 (d, 1H), 9.19 (s, 1H), 8.23 (d, 1H), 8.08-7.91 (m, 5H), 7.84 (t, 2H), 7.58-7.49 (m, 2H), 7.47 (d, 1H), 7.26 (d, 1H), 4.00 (s, 3H), 3.72 (s, 3H), 3.33 (dt, 2H), 3.05 (s, 3H), 2.74 (dt, 2H), 2.39 (s, 3H), 2.13 (s, 3H), 1.82 (td, 2H), 1.66 (dd, 2H), 1.47 (q, 2H), 1.28 (s, 12H).
[0766] LCMS m/z 808 (M+H).sup.+ (ES.sup.+)
(ab) N-(3-(1,4-Oxazepan-4-yl)propyl)-4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methyl-sulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxybenzamide
[0767] ##STR00064##
[0768] .sup.1H NMR (400 MHz, DMSO-d6) 10.26 (s, 1H), 9.89 (s, 1H), 9.46-9.42 (m, 1H), 9.15 (s, 1H), 8.23 (d, 1H), 8.13-7.91 (m, 5H), 7.84 (dd, 2H), 7.58-7.49 (m, 2H), 7.48 (d, 1H), 7.26 (d, 1H), 4.01 (s, 3H), 3.72 (s, 3H), 3.67 (t, 2H), 3.64-3.58 (m, 2H), 3.34 (q, 2H), 3.06 (s, 3H), 2.66-2.60 (m, 3H), 2.54-2.48 (m, 3H), 2.39 (s, 3H), 1.80 (p, 2H), 1.68 (p, 2H), 1.28 (s, 9H).
[0769] LCMS m/z 824 (M+H).sup.+ (ES.sup.+)
(ac) 4-((6-(5-((5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxy-N-(1-methylpiperidin-4-yl)benzamide
[0770] ##STR00065##
[0771] .sup.1H NMR (400 MHz, DMSO-d6) 10.27 (s, 1H), 9.89 (s, 1H), 9.44 (s, 1H), 9.16 (s, 1H), 8.25 (d, 1H), 8.06-7.93 (m, 4H), 7.90 (d, 1H), 7.84 (dd, 2H), 7.58-7.50 (m, 2H), 7.48 (d, 1H), 7.26 (d, 1H), 4.02 (s, 3H), 3.87-3.75 (m, 1H), 3.72 (s, 3H), 3.05 (s, 3H), 2.66 (d, 2H), 2.39 (s, 3H), 2.18 (s, 3H), 2.14-2.00 (m, 2H), 1.89-1.79 (m, 2H), 1.64-1.50 (m, 2H), 1.28 (s, 9H).
[0772] LCMS m/z 780 (M+H).sup.+ (ES.sup.+)
(ad) 4-((6-(5-((5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxy-N-(2-(methylsulfonyl)ethyl)benzamide
[0773] ##STR00066##
[0774] .sup.1H NMR (400 MHz, DMSO-d6) 10.31 (s, 1H), 9.89 (s, 1H), 9.44 (d, 1H), 9.15 (s, 1H), 8.53 (d, 1H), 8.26 (d, 1H), 8.06-7.84 (m, 6H), 7.59-7.50 (m, 2H), 7.47 (d, 1H), 7.25 (d, 1H), 4.01 (s, 3H), 3.77 (t, 2H), 3.42 (t, J=6.6 Hz, 2H), 3.34 (s, 3H), 3.08 (s, 3H), 3.05 (s, 3H), 2.39 (s, 3H), 1.28 (s, 9H).
[0775] LCMS m/z 789 (M+H).sup.+ (ES.sup.+)
(ae) 4-((6-(5-((5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methyl-phenyl)quinazolin-2-yl)amino)-2-methoxy-N-(2-(2-methyl-1H-imidazol-1-yl)ethyl)benzamide
[0776] ##STR00067##
[0777] .sup.1H NMR (400 MHz, DMSO-d6) 10.29 (s, 1H), 9.90 (s, 1H), 9.44 (s, 1H), 9.16 (s, 1H), 8.23 (d, 1H), 8.14 (t, 1H), 8.07-7.90 (m, 4H), 7.85 (dd, 2H), 7.59-7.50 (m, 2H), 7.47 (d, 1H), 7.25 (d, 1H), 7.06 (d, 1H), 6.76 (d, 1H), 4.08 (t, 2H), 3.96 (s, 3H), 3.72 (s, 3H), 3.60 (q, 2H), 3.06 (s, 3H), 2.39 (s, 3H), 2.30 (s, 3H), 1.28 (s, 9H).
[0778] LCMS m/z 791 (M+H).sup.+ (ES.sup.+)
(af) N-(2-(1H-Imidazol-4-yl)ethyl)-4-((6-(5-((5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)-phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxybenzamide
[0779] ##STR00068##
[0780] .sup.1H NMR (400 MHz, DMSO-d6) 11.87 (s, 1H), 10.27 (s, 1H), 9.90 (s, 1H), 9.44 (s, 1H), 9.20 (s, 1H), 8.35 (s, 1H), 8.22 (d, 1H), 8.07-7.92 (m, 4H), 7.87 (dd, 2H), 7.67-7.58 (m, 1H), 7.59-7.48 (m, 2H), 7.48 (d, 1H), 7.26 (d, 1H), 6.90 (s, 1H), 3.97 (s, 3H), 3.72 (s, 3H), 3.56 (q, 2H), 3.06 (s, 3H), 2.77 (t, 2H), 2.39 (s, 3H), 1.28 (s, 9H).
[0781] LCMS m/z 777 (M+H).sup.+ (ES.sup.+)
(ag) 4-((6-(5-((5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxy-N-(2-(pyridin-3-yl)ethyl)benzamide
[0782] ##STR00069##
[0783] .sup.1H NMR (400 MHz, DMSO-d6) 10.27 (s, 1H), 9.90 (s, 1H), 9.44 (s, 1H), 9.16 (s, 1H), 8.54-8.48 (m, 1H), 8.46 (dd, 1H), 8.21 (d, 1H), 8.10 (t, 1H), 8.07-7.92 (m, 4H), 7.84 (dd, 2H), 7.71 (dt, 1H), 7.58-7.50 (m, 2H), 7.47 (d, 1H), 7.36 (ddd, 1H), 7.25 (d, J=2.4 Hz, 1H), 3.94 (s, 3H), 3.72 (s, 3H), 3.58 (q, 2H), 3.06 (s, 3H), 2.89 (t, 2H), 2.39 (s, 3H), 1.28 (s, 9H).
[0784] LCMS m/z 788 (M+H).sup.+ (ES.sup.+)
(ah) 4-((6-(5-((5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-N-(2-(4-hydroxypiperidin-1-yl)ethyl)-2-methoxybenzamide
[0785] ##STR00070##
[0786] .sup.1H NMR (400 MHz, DMSO-d6) 10.28 (s, 1H), 9.89 (s, 1H), 9.44 (d, 1H), 9.14 (s, 1H), 8.32 (t, 1H), 8.25 (d, 1H), 8.04 (d, 1H), 8.01-7.82 (m, 5H), 7.58-7.49 (m, 2H), 7.47 (d, 1H), 7.25 (d, 1H), 4.59 (d, 1H), 4.04 (s, 3H), 3.71 (s, 3H), 3.49 (d, 1H), 3.40 (q, 2H), 3.05 (s, 3H), 2.77 (d, 2H), 2.47 (t, 2H), 2.40 (s, 3H), 2.09 (t, 2H), 1.77 (d, 2H), 1.44 (d, 2H), 1.28 (s, 9H).
[0787] LCMS m/z 810 (M+H).sup.+ (ES.sup.+); 808 (MH).sup. (ES.sup.)
(ai) 4-((6-(5-((5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxy-N-((1-methylpiperidin-4-yl)methyl)benzamide
[0788] ##STR00071##
[0789] .sup.1H NMR (400 MHz, DMSO-d6) 10.26 (s, 1H), 9.89 (s, 1H), 9.44 (s, 1H), 9.14 (s, 1H), 8.23 (d, 1H), 8.09-7.91 (m, 5H), 7.88-7.79 (m, 2H), 7.53 (d, 2H), 7.47 (d, 1H), 7.25 (d, 1H), 4.00 (s, 3H), 3.71 (s, 3H), 3.21 (t, 2H), 3.05 (s, 3H), 2.77 (s, 2H), 2.39 (s, 3H), 2.16 (s, 3H), 1.84 (s, 2H), 1.65 (d, 2H), 1.51 (s, 1H), 1.28 (s, 11H).
[0790] LCMS m/z 794 (M+H).sup.+ (ES.sup.+); 792 (MH).sup. (ES.sup.)
(aj) 4-((6-(5-((5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-N-(2-(4-hydroxy-1-methylpiperidin-4-yl)ethyl)-2-methoxybenzamide
[0791] ##STR00072##
[0792] .sup.1H NMR (400 MHz, DMSO-d6) 10.26 (s, 1H), 9.88 (s, 1H), 9.44 (d, 1H), 9.15 (s, 1H), 8.32 (t, 1H), 8.22 (d, 1H), 8.04 (d, 1H), 8.03-7.93 (m, 3H), 7.86 (dd, 2H), 7.56-7.50 (m, 2H), 7.46 (d, 1H), 7.25 (d, 1H), 4.26 (s, 1H), 3.99 (s, 3H), 3.71 (s, 3H), 3.42 (q, 2H), 3.04 (s, 3H), 2.43-2.25 (m, 7H), 2.16 (s, 3H), 1.65 (t, 2H), 1.52 (d, 4H), 1.28 (s, 9H).
[0793] LCMS m/z 824 (M+H).sup.+ (ES.sup.+)
(ak) 4-((6-(5-((5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxy-N-(2-(pyrrolidin-1-yl)ethyl)benzamide
[0794] ##STR00073##
[0795] .sup.1H NMR (400 MHz, DMSO-d6) 10.28 (s, 1H), 9.88 (s, 1H), 9.44 (s, 1H), 9.15 (s, 1H), 8.31 (t, 1H), 8.23 (d, 1H), 8.04 (d, 1H), 8.02-7.94 (m, 3H), 7.87 (dd, 2H), 7.57-7.50 (m, 2H), 7.46 (d, 1H), 7.25 (d, 1H), 4.00 (s, 3H), 3.71 (s, 3H), 3.41 (q, 2H), 3.05 (s, 3H), 2.61 (t, 2H), 2.52 (s, 4H), 2.39 (s, 3H), 1.74 (p, 4H), 1.28 (s, 9H). 4 aliphatic signals obscured under DMSO signal.
[0796] LCMS m/z 780 (M+H).sup.+ (ES.sup.+); 778 (MH).sup. (ES.sup.)
(al) 4-((6-(5-((5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methyl-phenyl)quinazolin-2-yl)amino)-2-methoxy-N-(2-(3,3,4-trimethylpiperazin-1-yl)ethyl)benzamide
[0797] ##STR00074##
[0798] .sup.1H NMR (400 MHz, DMSO-d6) 10.28 (s, 1H), 9.88 (s, 1H), 9.44 (d, 1H), 9.15 (s, 1H), 8.26 (d, 1H), 8.19 (t, 1H), 8.04 (d, 1H), 8.01-7.84 (m, 5H), 7.57-7.51 (m, 2H), 7.46 (d, 1H), 7.25 (d, 1H), 4.02 (s, 3H), 3.71 (s, 3H), 3.41 (q, 2H), 3.30 (s, 2H), 3.04 (s, 3H), 2.49-2.36 (m, 9H), 2.12 (s, 3H), 1.28 (s, 9H), 1.00 (s, 6H).
[0799] LCMS m/z 837 (M+H).sup.+ (ES.sup.+); 835 (MH).sup. (ES.sup.)
(am) 4-((6-(5-((5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-2-methoxy-N-(2-morpholinoethyl)benzamide
[0800] ##STR00075##
[0801] .sup.1H NMR (400 MHz, DMSO-d6) 10.28 (s, 1H), 9.88 (s, 1H), 9.44 (d, 1H), 9.15 (s, 1H), 8.31 (s, 1H), 8.24 (d, 1H), 8.04 (d, 1H), 8.03-7.93 (m, 3H), 7.88 (dd, 2H), 7.59-7.50 (m, 2H), 7.46 (d, 1H), 7.25 (d, 1H), 4.04 (s, 3H), 3.71 (s, 3H), 3.64 (t, 4H), 3.43 (d, 2H), 3.05 (s, 3H), 2.51-2.42 (m, 6H), 2.40 (s, 3H), 1.28 (s, 9H).
[0802] LCMS m/z 796 (M+H).sup.+ (ES.sup.+); 794 (MH).sup. (ES.sup.)
(an) 4-((6-(5-((5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamoyl)-2-methylphenyl)quinazolin-2-yl)amino)-N-(1,1-dioxidotetrahydro-2H-thiopyran-4-yl)-2-methoxybenzamide
[0803] ##STR00076##
[0804] .sup.1H NMR (400 MHz, DMSO-d6) 10.27 (d, 1H), 9.89 (s, 1H), 9.44 (s, 1H), 9.14 (s, 1H), 8.22 (d, 1H), 8.05 (d, 2H), 8.02-7.92 (m, 3H), 7.84 (d, 1H), 7.75 (d, 1H), 7.53 (d, 2H), 7.47 (d, 1H), 7.25 (d, 1H), 4.19 (d, 1H), 4.00 (s, 3H), 3.71 (s, 3H), 3.11 (d, 4H), 3.05 (s, 3H), 2.39 (s, 3H), 2.22-2.06 (m, 4H), 1.28 (s, 9H).
[0805] LCMS m/z 815 (M+H).sup.+ (ES.sup.+)
Example 15
[0806] The following compounds are prepared by methods analogous to those described above.
(a) [4-[[6-[5-[[5-tert-Butyl-3-(methanesulfonamido)-2-methoxy-phenyl]carbamoyl]-2-methyl-phenyl]quinazolin-2-yl]amino]-2-methoxy-phenyl]-methyl-phosphinic acid
[0807] ##STR00077##
(b) 4-[[6-[5-[[5-tert-Butyl-3-(methanesulfonamido)-2-methoxy-phenyl]carbamoyl]-2-methyl-phenyl]quinazolin-2-yl]amino]-2,6-dimethoxy-N-[2-(4-methylpiperazin-1-yl)ethyl]benzamide
[0808] ##STR00078##
(c) 4-[[6-[5-[[5-tert-Butyl-3-(methanesulfonamido)-2-methoxy-phenyl]carbamoyl]-2-methyl-phenyl]quinazolin-2-yl]amino]-N-(2-dimethylaminoethyl)-2,6-dimethoxy-benzamide
[0809] ##STR00079##
(d) 2-[5-tert-Butyl-2-methoxy-3-[[3-[2-[3-methoxy-4-[2-(1-oxo-1,4-thiazinan-4-yl)ethylcarbamoyl]anilino]quinazolin-6-yl]-4-methyl-benzoyl]amino]-N-methylsulfonyl-anilino]ethyl dihydrogen phosphate
[0810] ##STR00080##
(e) 2-[[4-[[6-[5-[[5-tert-Butyl-3-(methanesulfonamido)-2-methoxy-phenyl]carbamoyl]-2-methyl-phenyl]quinazolin-2-yl]amino]-2,6-dimethoxy-benzoyl]amino]ethyl dihydrogen phosphate
[0811] ##STR00081##
(f) 2-[2-[2-[[4-[[6-[5-[[5-tert-Butyl-3-(methanesulfonamido)-2-methoxy-phenyl]carbamoyl]-2-methyl-phenyl]quinazolin-2-yl]amino]-2,6-dimethoxy-benzoyl]amino]ethoxy]ethoxy]ethyl dihydrogen phosphate
[0812] ##STR00082##
(g) [2-[5-tert-Butyl-2-methoxy-3-[[3-[2-[3-methoxy-4-[2-(1-oxo-1,4-thiazinan-4-yl)ethylcarbamoyl]anilino]quinazolin-6-yl]-4-methyl-benzoyl]amino]anilino]-2-oxo-ethyl]dihydrogen phosphate
[0813] ##STR00083##
Biological Testing: Experimental Methods
Enzyme Binding Assays (Kinomescan)
[0814] Kinase enzyme binding activities of compounds disclosed herein may be determined using a proprietary assay which measures active site-directed competition binding to an immobilized ligand (Fabian, M. A. et al., Nature Biotechnol., 2005, 23:329-336). These assays may be conducted by DiscoverX (formerly Ambit; San Diego, Calif.). The percentage inhibition produced by incubation with a test compound may be calculated relative to the non-inhibited control.
Enzyme Inhibition Assays
[0815] The enzyme inhibitory activities of compounds disclosed herein are determined by FRET using synthetic peptides labelled with both donor and acceptor fluorophores (Z-LYTE, Invitrogen Ltd., Paisley, UK).
p38 MAPK Enzyme Inhibition
[0816] The following two assay variants can be used for determination of p38 MAPK inhibition.
Method 1
[0817] The inhibitory activities of test compounds against the p38 MAPK isoform (MAPK14: Invitrogen), are evaluated indirectly by determining the level of activation/phosphorylation of the down-stream molecule, MAPKAP-K2. The p38 MAPK protein (80 ng/mL, 2.5 L) is mixed with the test compound (2.5 L of either 4 g/mL, 0.4 g/mL, 0.04 g/mL or 0.004 g/mL) for 2 hr at RT. The mix solution (2.5 L) of the p38 inactive target MAPKAP-K2 (Invitrogen, 600 ng/mL) and FRET peptide (8 M; a phosphorylation target for MAPKAP-K2) is then added and the kinase reaction is initiated by adding ATP (40 M, 2.5 L). The mixture is incubated for 1 hr at RT. Development reagent (protease, 5 L) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan Flash, ThermoFisher Scientific).
Method 2
[0818] This method follows the same steps as Method 1 above, but utilises a higher concentration of the p38 MAPK protein (2.5 L of 200 ng/mL protein instead of 2.5 L of 80 ng/mL protein) for mixing with the test compound.
p38 MAPK Enzyme Inhibition
[0819] The inhibitory activities of compounds of the invention against p38MAPK (MAPK12: Invitrogen), are evaluated in a similar fashion to that described hereinabove. The enzyme (800 ng/mL, 2.5 L) is incubated with the test compound (2.5 L at either 4 g/mL, 0.4 g/mL, 0.04 g/mL, or 0.004 g/mL) for 2 hr at RT. The FRET peptides (8 M, 2.5 L), and appropriate ATP solution (2.5 L, 400 M) is then added to the enzymes/compound mixtures and incubated for 1 hr. Development reagent (protease, 5 L) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan Flash, Thermo Scientific).
c-Src and Syk Enzyme Inhibition
[0820] The inhibitory activities of compounds of the invention against c-Src and Syk enzymes (Invitrogen), are evaluated in a similar fashion to that described hereinabove. The relevant enzyme (3000 ng/mL or 2000 ng/mL respectively, 2.5 L) is incubated with the test compound (either 4 g/mL, 0.4 g/mL, 0.04 g/mL, or 0.004 g/mL, 2.5 L each) for 2 hr at RT. The FRET peptides (8 M, 2.5 L), and appropriate ATP solutions (2.5 L, 800 M for c-Src, and 60 M ATP for Syk) are then added to the enzymes/compound mixtures and incubated for 1 hr. Development reagent (protease, 5 L) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan Flash, ThermoFisher Scientific).
GSK 3 Enzyme Inhibition
[0821] The following two assay variants can be used for determination of GSK 3 inhibition.
Method 1
[0822] The inhibitory activities of compounds of the invention against the GSK 3 enzyme isoform (Invitrogen), are evaluated by determining the level of activation/phosphorylation of the target peptide. The GSK3- protein (500 ng/mL, 2.5 L) is mixed with the test compound (2.5 L at either 4 g/mL, 0.4 g/mL, 0.04 g/mL, or 0.004 g/mL) for 2 hr at RT. The FRET peptide (8 M, 2.5 L), which is a phosphorylation target for GSK3, and ATP (40 M, 2.5 L) are then added to the enzyme/compound mixture and the resulting mixture incubated for 1 hr. Development reagent (protease, 5 L) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan Flash, ThermoFisher Scientific).
[0823] In all cases, the site-specific protease cleaves non-phosphorylated peptide only and eliminates the FRET signal. Phosphorylation levels of each reaction are calculated using the ratio of coumarin emission (donor) over fluorescein emission (acceptor), for which high ratios indicate high phosphorylation and low ratios indicate low phosphorylation levels. The percentage inhibition of each reaction is calculated relative to non-inhibited control and the 50% inhibitory concentration (IC.sub.50 value) is then calculated from the concentration-response curve.
Method 2
[0824] This method follows the same steps as Method 1 above, but utilises a shorter period of mixing of the test compound (105 minutes instead of 2 hours) with the GSK3- protein.
Cellular Assays
[0825] The compounds of the invention were studied using one or more of the following assays.
(a) LPS-Induced TNF/IL-8 Release in d-U937 Cells
[0826] U937 cells, a human monocytic cell line, are differentiated to macrophage-type cells by incubation with phorbol myristate acetate (PMA; 100 ng/mL) for 48 to 72 hr. Cells are pre-incubated with final concentrations of test compound for 2 hr and are then stimulated with 0.1 g/mL of LPS (from E. Coli: O111:B4, Sigma) for 4 hr. The supernatant is collected for determination of TNF and IL-8 concentrations by sandwich ELISA (Duo-set, R&D systems). The inhibition of TNF production is calculated as a percentage of that achieved by 10 g/mL of BIRB796 at each concentration of test compound by comparison against vehicle control. The relative 50% effective concentration (REC.sub.50) is determined from the resultant concentration-response curve. The inhibition of IL-8 production is calculated at each concentration of test compound by comparison with vehicle control. The 50% inhibitory concentration (IC.sub.50) is determined from the resultant concentration-response curve.
(b) LPS-Induced TNF/IL-8 Release in PBMC Cells
[0827] Peripheral blood mononuclear cells (PBMCs) from healthy subjects are separated from whole blood using a density gradient (Lymphoprep, Axis-Shield Healthcare). The PBMCs are seeded in 96 well plates and treated with compounds at the desired concentration for 2 hours before addition of 1 ng/mL LPS (Escherichia Coli 0111:B4 from Sigma Aldrich) for 24 hours under normal tissue culture conditions (37 C., 5% CO.sub.2). The supernatant is harvested for determination of IL-8 and TNF concentrations by sandwich ELISA (Duo-set, R&D systems) and read on the fluorescence microplate reader (Varioskan Flash, ThermoFisher Scientific). The concentration at 50% inhibition (IC.sub.50) of IL-8 and TNF production is calculated from the dose response curve.
(c) IL-2 and IFN Gamma Release in CD3/CD28 Stimulated PBMC Cells
[0828] PBMCs from healthy subjects are separated from whole blood using a density gradient (Lymphoprep, Axis-Shield Healthcare). Cells are added to a 96 well plate pre-coated with a mixture of CD3/CD28 monoclonal antibodies (0.3 g/mL eBioscience and 3 g/mL BD Pharmingen respectively). Compound at the desired concentration is then added to the wells and the plate left for 3 days under normal tissue culture conditions. Supernatants are harvested and IL-2 and IFN gamma release determined by Sandwich ELISA (Duo-set, R&D System). The IC.sub.50 is determined from the dose response curve.
(d) IL-1-Induced IL-8 Release in HT29 Cells
[0829] HT29 cells, a human colon adenocarcinoma cell line, are plated in a 96 well plate (24 hrs) and pre-treated with compounds at the desired concentration for 2 hours before addition of 5 ng/mL of IL-1 (Abcam) for 24 hours. Supernatants are harvested for IL-8 quantification by Sandwich ELISA (Duo-set, R&D System). The IC.sub.50 is determined from the dose response curve.
(e) LPS-Induced IL-8 and TNF Release in Primary Macrophages
[0830] PBMCs from healthy subjects are separated from whole blood using a density gradient (Lymphoprep, Axis-Shield Healthcare). Cells are incubated for 2 hrs and non-adherent cells removed by washing. To differentiate the cells to macrophages the cells are incubated with 5 ng/mL of GM-CSF (Peprotech) for 7 days under normal tissue culture conditions. Compounds are then added to the cells at the desired concentration for a 2 hour pre-treatment before stimulation with 10 ng/mL LPS for 24 hours. Supernatants are harvested and IL-8 and TNF release determined by Sandwich ELISA (Duo-set, R&D System). The IC.sub.50 is determined from the dose response curve.
(f) Poly I:C-Induced ICAM-1 Expression in BEAS2B Cells
[0831] Poly I:C is used in these studies as a simple, RNA virus mimic. Poly I:C-Oligofectamine mixture (1 g/mL Poly I:C, 2% Oligofectamine, 25 L; Invivogen Ltd., San Diego, Calif., and Invitrogen, Carlsbad, Calif., respectively) is transfected into BEAS2B cells (human bronchial epithelial cells, ATCC). Cells are pre-incubated with final concentrations of test compounds for 2 hr and the level of ICAM1 expression on the cell surface is determined by cell-based ELISA. At a time point 18 hr after poly I:C transfection, cells are fixed with 4% formaldehyde in PBS and then endogenous peroxidase is quenched by the addition of washing buffer (100 L, 0.05% Tween in PBS: PBS-Tween) containing 0.1% sodium azide and 1% hydrogen peroxide. Cells are washed with wash-buffer (3200 L) and after blocking the wells with 5% milk in PBS-Tween (100 L) for 1 hr, the cells are incubated with anti-human ICAM-1 antibody (50 L; Cell Signalling Technology, Danvers, Mass.) in 1% BSA PBS overnight at 4 C.
[0832] The cells are washed with PBS-Tween (3200 L) and incubated with the secondary antibody (100 L; HRP-conjugated anti-rabbit IgG, Dako Ltd., Glostrup, Denmark). The cells are then incubated with of substrate (50 L) for 2-20 min, followed by the addition of stop solution (50 L, 1N H.sub.2SO.sub.4). The ICAM-1 signal is detected by reading the absorbance at 450 nm against a reference wavelength of 655 nm using a spectrophotometer. The cells are then washed with PBS-Tween (3200 L) and total cell numbers in each well are determined by reading absorbance at 595 nm after Crystal Violet staining (50 L of a 2% solution in PBS) and elution by 1% SDS solution (100 L) in distilled water. The measured OD 450-655 readings are corrected for cell number by dividing with the OD595 reading in each well. The inhibition of ICAM-1 expression is calculated at each concentration of test compound by comparison with vehicle control. The 50% inhibitory concentration (IC.sub.50) is determined from the resultant concentration-response curve.
(g) Cell Mitosis Assay
[0833] Peripheral blood mononucleocytes (PBMCs) from healthy subjects are separated from whole blood (Quintiles, London, UK) using a density gradient (Histopaque-1077, Sigma-Aldrich, Poole, UK). The PBMCs (3 million cells per sample) are subsequently treated with 2% PHA (phytohaemagglutinin, Sigma-Aldrich, Poole, UK) for 48 hr, followed by a 20 hr exposure to varying concentrations of test compounds. At 2 hr before collection, PBMCs are treated with demecolcine (0.1 g/mL; Invitrogen, Paisley, UK) to arrest cells in metaphase. To observe mitotic cells, PBMCs are permeabilised and fixed by adding Intraprep (50 L; Beckman Coulter, France), and stained with anti-phospho-histone 3 (0.26 ng/L; #9701; Cell Signalling, Danvers, Mass.) and propidium iodide (1 mg/mL; Sigma-Aldrich, Poole, UK) as previously described (Muehlbauer P. A. and Schuler M. J., Mutation Research, 2003, 537:117-130). Fluorescence is observed using an ATTUNE flow cytometer (Invitrogen, Paisley, UK), gating for lymphocytes. The percentage inhibition of mitosis is calculated for each treatment relative to vehicle (0.5% DMSO) treatment.
(h) Rhinovirus-Induced IL-8 Release and ICAM-1 Expression
[0834] Human rhinovirus RV16 is obtained from the American Type Culture Collection (Manassas, Va.). Viral stocks are generated by infecting Hela cells with HRV until 80% of the cells are cytopathic.
[0835] BEAS2B cells are infected with HRV at an MOI of 5 and incubated for 2 hr at 33 C. with gentle shaking for to promote absorption. The cells are then washed with PBS, fresh media added and the cells are incubated for a further 72 hr. The supernatant is collected for assay of IL-8 concentrations using a Duoset ELISA development kit (R&D systems, Minneapolis, Minn.).
[0836] The level of ICAM1 expressing cell surface is determined by cell-based ELISA. At 72 hr after infection, cells are fixed with 4% formaldehyde in PBS. After quenching endogenous peroxidase by adding 0.1% sodium azide and 1% hydrogen peroxide, wells are washed with wash-buffer (0.05% Tween in PBS: PBS-Tween). After blocking well with 5% milk in PBS-Tween for 1 hr, the cells are incubated with anti-human ICAM-1 antibody in 5% BSA PBS-Tween (1:500) overnight. Wells are washed with PBS-Tween and incubated with the secondary antibody (HRP-conjugated anti-rabbit IgG, Dako Ltd.). The ICAM-1 signal is detected by adding substrate and reading at 450 nm with a reference wavelength of 655 nm using a spectrophotometer. The wells are then washed with PBS-Tween and total cell numbers in each well are determined by reading absorbance at 595 nm after Crystal Violet staining and elution by 1% SDS solution. The measured OD.sub.450-655 readings are corrected for cell number by dividing with the OD.sub.595 reading in each well. Compounds are added 2 hr before HRV infection and 2 hr after infection when non-infected HRV is washed out.
(i) Assessment of HRV16 Induced CPE in MRC5
[0837] MRC-5 cells are infected with HRV16 at an MOI of 1 in DMEM containing 5% FCS and 1.5 mM MgCl.sub.2, followed by incubation for 1 hr at 33 C. to promote adsorption. The supernatants are aspirated, and then fresh media added followed by incubation for 4 days. Where appropriate, cells are pre-incubated with compound or DMSO for 2 hr, and the compounds and DMSO added again after washout of the virus.
[0838] Supernatants are aspirated and incubated with methylene blue solution (100 L, 2% formaldehyde, 10% methanol and 0.175% Methylene Blue) for 2 hr at RT. After washing, 1% SDS in distilled water (100 L) is added to each well, and the plates are shaken lightly for 1-2 hr prior to reading the absorbance at 660 nm. The percentage inhibition for each well is calculated. The IC.sub.50 value is calculated from the concentration-response curve generated by the serial dilutions of the test compounds.
(j) In Vitro RSV Virus Load in Primary Bronchial Epithelial Cells
[0839] Normal human bronchial epithelial cells (NHBEC) grown in 96 well plates are infected with RSV A2 (Strain A2, HPA, Salisbury, UK) at an MOI of 0.001 in the LHC8 Media:RPMI-1640 (50:50) containing 15 mM magnesium chloride and incubated for 1 hr at 37 C. for adsorption. The cells are then washed with PBS (3200 L), fresh media (200 L) is added and incubation continued for 4 days. Where appropriate, cells are pre-incubated with the compound or DMSO for 2 hr, and then added again after washout of the virus.
[0840] The cells are fixed with 4% formaldehyde in PBS solution (50 L) for 20 min, washed with WB (3200 L), (washing buffer, PBS including 0.5% BSA and 0.05% Tween-20) and incubated with blocking solution (5% condensed milk in PBS) for 1 hr. Cells are then washed with WB (3200 L) and incubated for 1 hr at RT with anti-RSV (2F7) F-fusion protein antibody (40 L; mouse monoclonal, lot 798760, Cat. No. ab43812, Abcam) in 5% BSA in PBS-tween. After washing, cells are incubated with an HRP-conjugated secondary antibody solution (50 L) in 5% BSA in PBS-Tween (lot 00053170, Cat. No. P0447, Dako) and then TMB substrate added (50 L; substrate reagent pack, lot 269472, Cat. No. DY999, R&D Systems, Inc.). This reaction is stopped by the addition of 2N H.sub.2SO.sub.4 (50 L) and the resultant signal is determined colourimetrically (OD: 450 nm with a reference wavelength of 655 nm) in a microplate reader (Varioskan Flash, ThermoFisher Scientific).
[0841] Cells are then washed and a 2.5% crystal violet solution (50 L; lot 8656, Cat. No. PL7000, Pro-Lab Diagnostics) is applied for 30 min. After washing with WB, 1% SDS in distilled water (100 L) is added to each well, and plates are shaken lightly on the shaker for 1 hr prior to reading the absorbance at 595 nm. The measured OD.sub.450-655 readings are corrected to the cell number by dividing the OD.sub.450-655 by the OD.sub.595 readings. The percentage inhibition for each well is calculated and the IC.sub.50 value is calculated from the concentration-response curve generated from the serial dilutions of compound.
(k) Cell Viability Assay: MTT Assay
[0842] Differentiated U937 cells are pre-incubated with each test compound (final concentration 1 g/mL or 10 g/mL in 200 L media indicated below) under two protocols: the first for 4 hr in 5% FCS RPMI1640 media and the second in 10% FCS RPMI1640 media for 24 h. The supernatant is replaced with new media (200 L) and MTT stock solution (10 L, 5 mg/mL) is added to each well. After incubation for 1 hr the media are removed, DMSO (200 L) is added to each well and the plates are shaken lightly for 1 hr prior to reading the absorbance at 550 nm. The percentage loss of cell viability is calculated for each well relative to vehicle (0.5% DMSO) treatment. Consequently an apparent increase in cell viability for drug treatment relative to vehicle is tabulated as a negative percentage.
(l) Human Biopsy Assay
[0843] Intestinal mucosa biopsies are obtained from the inflamed regions of the colon of IBD patients. The biopsy material is cut into small pieces (2-3 mm) and placed on steel grids in an organ culture chamber at 37 C. in a 5% CO.sub.2/95% O.sub.2 atmosphere in serum-free media. DMSO control or test compounds at the desired concentration are added to the tissue and incubated for 24 hr in the organ culture chamber. The supernatant is harvested for determination of IL-6, IL-8, IL-1 and TNF levels by R&D ELISA. Percentage inhibition of cytokine release by the test compounds is calculated relative to the cytokine release determined for the DMSO control (100%).
(m) Accumulation of Catenin in d-U937 Cells
[0844] U937 cells, a human monocytic cell line, are differentiated into macrophage-type cells by incubation with PMA; (100 ng/mL) for between 48 to 72 hr. The cells are then incubated with either final concentrations of test compound or vehicle for 18 hr. The induction of -catenin by the test compounds is stopped by replacing the media with 4% formaldehyde solution. Endogenous peroxide activity is neutralised by incubating with quenching buffer (100 L, 0.1% sodium azide, 1% H.sub.2O.sub.2 in PBS with 0.05% Tween-20) for 20 min. The cells are washed with washing buffer (200 L; PBS containing 0.05% Tween-20) and incubated with blocking solution (200 L; 5% milk in PBS) for 1 hr, re-washed with washing buffer (200 L) and then incubated overnight with anti--catenin antibody solution (50 L) in 1% BSA/PBS (BD, Oxford, UK).
[0845] After washing with washing buffer (3200 L; PBS containing 0.05% Tween-20), cells are incubated with an HRP-conjugated secondary antibody solution (100 L) in 1% BSA/PBS (Dako, Cambridge, UK) and the resultant signal is determined colourimetrically (OD: 450 nm with a reference wavelength of 655 nm) using TMB substrate (50 L; R&D Systems, Abingdon, UK). This reaction is stopped by addition of 1N H.sub.2SO.sub.4 solution (50 L). Cells are then washed with washing buffer and 2% crystal violet solution (50 L) is applied for 30 min. After washing with washing buffer (3200 L), 1% SDS (100 L) is added to each well and the plates are shaken lightly for 1 hr prior to measuring the absorbance at 595 nm (Varioskan Flash, Thermo-Fisher Scientific).
[0846] The measured OD.sub.450-655 readings are corrected for cell number by dividing the OD.sub.450-655 by the OD.sub.595 readings. The percentage induction for each well is calculated relative to vehicle, and the ratio of induction normalised in comparison with the induction produced by a standard control comprising of Reference Compound A (N-(4-(4-(3-(3-tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)ureido)naphthalen-1-yloxy)pyridin-2-yl)-2-methoxyacetamide) (1 g/mL) which is defined as 100%.
(n) T Cell Proliferation
[0847] PBMCs from healthy subjects are separated from whole blood using a density gradient (Lymphoprep, Axis-Shield Healthcare). The lymphocyte fraction is first enriched for CD4+ T cells by negative magnetic cell sorting as per the manufacturer's instructions (Miltenyi Biotec 130-091-155). Nave CD4+ T cells are then separated using positive magnetic selection of CD45RA+ cells using microbeads as per the manufacturer's instructions (130-045-901). Cells are plated at 210.sup.5 cells per well in 100 L RPMI/10% FBS on 96 well flat bottomed plate (Corning Costar). 25 L of test compound are diluted to the appropriate concentration (8 final conc.) in normal medium and added to duplicate wells on the plate to achieve a dose response range of 0.03 ng/mL-250 ng/mL. DMSO is added as a negative control. Plates are allowed to pre-incubate for 2 hours before stimulation with 1 g/mL anti-CD3 (OKT3; eBioscience). After 72 h, the medium in each well is replaced with 150 L of fresh medium containing 10 M BrdU (Roche). After 16 h, the supernatant is removed, the plate is dried and the cells fixed by adding 100 L of fix/denature solution to each well for 20 min as per the manufacturer's instructions (Roche). Plates are washed once with PBS before addition of the anti-BrdU detection antibody and incubated for 90 mins at room temperature. Plates are then washed gently 3 with the wash buffer supplied and developed by addition of 100 L of substrate solution. The reaction is stopped by addition of 50 L of 1 M H.sub.2SO.sub.4, and read for absorbance at 450 nm on a plate reader (Varioskan Flash, ThermoFisher Scientific). The IC.sub.50 is determined from the dose response curve.
(o) IL-2 and IFN Release in CD3/CD28 Stimulated LPMC Cells from IBD Patients
[0848] Lamina propria mononuclear cells (LPMCs) are isolated and purified from inflamed IBD mucosa of surgical specimens or from normal mucosa of surgical specimens as follows:
[0849] The mucosa is removed from the deeper layers of the surgical specimens with a scalpel and cut in fragments 3-4 mm size. The epithelium is removed by washing the tissue fragments three times with 1 mM EDTA (Sigma-Aldrich, Poole, UK) in HBSS (Sigma-Aldrich) with agitation using a magnetic stirrer, discarding the supernatant after each wash. The sample is subsequently treated with type 1A collagenase (1 mg/mL; Sigma-Aldrich) for 1 h with stirring at 37 C. The resulting cell suspension is then filtered using a 100 m cell strainer, washed twice, resuspended in RPMI-1640 medium (Sigma-Aldrich) containing 10% fetal calf serum, 100 U/mL penicillin and 100 g/mL streptomycin, and used for cell culture.
[0850] Freshly isolated LPMCs (210.sup.5 cells/well) are stimulated with 1 g/mL -CD3/-CD28 for 48 h in the presence of either DMSO control or appropriate concentrations of compound. After 48 h, the supernatant is removed and assayed for the presence of TNF and IFN by R&D ELISA. Percentage inhibition of cytokine release by the test compounds is calculated relative to the cytokine release determined for the DMSO control (100%).
(p) Inhibition of Cytokine Release from Myofibroblasts Isolated from IBD Patients
[0851] Myofibroblasts from inflamed IBD mucosa are isolated as follows:
[0852] The mucosa is dissected and discarded and 1 mm-sized mucosal samples are cultured at 37 C. in a humidified CO.sub.2 incubator in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich) supplemented with 20% FBS, 1% non-essential amino acids (Invitrogen, Paisley, UK), 100 U/mL penicillin, 100 g/mL streptomycin, 50 g/mL gentamycin, and 1 g/mL amphotericin (Sigma-Aldrich). Established colonies of myofibroblasts are seeded into 25-cm.sup.2 culture flasks and cultured in DMEM supplemented with 20% FBS and antibiotics to at least passage 4 to provide a sufficient quantity for use in stimulation experiments.
[0853] Subconfluent monolayers of myofibroblasts are then seeded in 12-well plates at 310.sup.5 cells per well are starved in serum-free medium for 24 h at 37 C., 5% CO.sub.2 before being cultured for 24 h in the presence of either DMSO control or appropriate concentrations of compound. After 24 h the supernatant is removed and assayed for the presence of IL-8 and IL-6 by R&D ELISA. Percentage inhibition of cytokine release by the test compounds is calculated relative to the cytokine release determined for the DMSO control (100%).
(q) Human Neutrophil Degranulation
[0854] Neutrophils are isolated from human peripheral blood as follows:
[0855] Blood is collected by venepuncture and anti-coagulated by addition of 1:1 EDTA:sterile phosphate buffered saline (PBS, no Ca+/Mg+). Dextran (3% w/v) is added (1 part dextran solution to 4 parts blood) and the blood allowed to stand for approximately 20 minutes at rt. The supernatant is carefully layered on a density gradient (Lymphoprep, Axis-Shield Healthcare) and centrifuged (15 mins, 2000 rpm, no brake). The supernatant is aspirated off and the cell pellet is re-suspended in sterile saline (0.2%) for no longer than 60 seconds (to lyse contaminating red blood cells). 10 times volume of PBS is then added and the cells centrifuged (5 mins, 1200 rpm). Cells are re-suspended in HBSS+ (Hanks buffered salt solution (without phenol red) containing cytochalasin B (5 g/mL) and 1 mM CaCl.sub.2) to achieve 510.sup.6 cells/mL.
[0856] 510.sup.4 cells are added to each well of a V-bottom 96 well plate and incubated (30 mins, 37 C.) with the appropriate concentration of test compound (0.3-1000 ng/mL) or vehicle (DMSO, 0.5% final conc). Degranulation is stimulated by addition of fMLP (final conc 1 M) which after a further incubation (30 mins, 37 C.) the cells are removed by centrifugation (5 mins, 1500 rpm) and the supernatants transferred to a flat bottom 96 well plate. An equal volume of tetramethylbenzidine (TMB) is added and after 10 mins the reaction terminated by addition of an equal volume of sulphuric acid (0.5 M) and absorbance read at 450 nm (background at 655 nm subtracted). The 50% inhibitory concentration (IC.sub.50) is determined from the resultant concentration-response curve.
(r) Cell Cytotoxicity Assay
[0857] 110.sup.5 Jurkat cells (immortalised human T lymphocytes) are added to the appropriate number of wells of a 96 well plate in 100 L of media (RPMI supplemented with 10% foetal bovine serum). 1 L of DMSO control (final concentration 1.0% v/v) or test compound (final concentration 20, 5 or 1 g/mL) is added to the wells and incubated at 37 C., 5% CO.sub.2. After 24 hours, the plate is centrifuged at 1200 rpm for 3 minutes and the supernatant discarded. Cells are then resuspended in 150 L (final concentration 7.5 g/mL) of propidium iodide (PI) in PBS and incubated at 37 C., 5% CO.sub.2 for 15 minutes. After 15 minutes, cells are analysed by flow cytometry (BD accuri) using the FL3 window. The % viability is calculated as the % of cells that are PI negative in the test wells normalised to the DMSO control.
In Vivo Screening: Pharmacodynamics and Anti-Inflammatory Activity
(i) LPS-Induced Neutrophil Accumulation in Mice
[0858] Non-fasted Balb/c mice are dosed by the intra tracheal route with either vehicle, or the test substance at the indicated times (within the range 2-8 hr) before stimulation of the inflammatory response by application of an LPS challenge. At T=0, mice are placed into an exposure chamber and exposed to LPS (7.0 mL, 0.5 mg/mL solution in PBS) for 30 min. After a further 8 hr the animals are anesthetized, their tracheas cannulated and BALF extracted by infusing and then withdrawing from their lungs 1.0 mL of PBS via the tracheal catheter. Total and differential white cell counts in the BALF samples are measured using a Neubauer haemocytometer. Cytospin smears of the BALF samples are prepared by centrifugation at 200 rpm for 5 min at RT and stained using a DiffQuik stain system (Dade Behring). Cells are counted using oil immersion microscopy. Data for neutrophil numbers in BAL are shown as meanS.E.M. (standard error of the mean). The percentage inhibition of neutrophil accumulation is calculated for each treatment relative to vehicle treatment.
(ii) Cigarette Smoke Model
[0859] A/J mice (males, 5 weeks old) are exposed to cigarette smoke (4% cigarette smoke, diluted with air) for 30 min/day for 11 days using a Tobacco Smoke Inhalation Experiment System for small animals (Model SIS-CS; Sibata Scientific Technology, Tokyo, Japan). Test substances are administered intra-nasally (35 L of solution in 50% DMSO/PBS) once daily for 3 days after the final cigarette smoke exposure. At 12 hr after the last dosing, each of the animals is anesthetized, the trachea cannulated and bronchioalveolar lavage fluid (BALF) is collected. The numbers of alveolar macrophages and neutrophils are determined by FACS analysis (EPICS ALTRA II, Beckman Coulter, Inc., Fullerton, Calif., USA) using anti-mouse MOMA2 antibody (macrophage) or anti-mouse 7/4 antibody (neutrophil).
(iii) DSS-Induced Colitis in Mice
[0860] Non-fasted, 10-12 week old, male BDF1 mice are dosed by oral gavage twice daily with either vehicle, reference item (5-ASA) or test compound one day before (Day 1) stimulation of the inflammatory response by treatment with dextran sodium sulphate (DSS). On Day 0 of the study DSS (5% w/v) is administered in the drinking water followed by BID dosing of the vehicle (5 mL/kg), reference (100 mg/kg) or test compound (5 mg/kg) for 7 days. The drinking water with DSS is replenished every 3 days. During the study animals are weighed every day and stool observations are made and recorded as a score, based on stool consistency. At the time of sacrifice on Day +6 the large intestine is removed and the length and weight are recorded. Sections of the colon are taken for either MPO analysis to determine neutrophil infiltration or for histopathology scoring to determine disease severity.
(iv) TNBS-Induced Colitis in Mice
[0861] Non-fasted, 10-12 week old, male BDF1 mice are dosed by oral gavage twice daily with either vehicle (5 mL/kg), reference item (Budesonide 2.5 mg/kg) or test compound (1 or 5 mg/kg) one day before (Day 1) stimulation of the inflammatory response by treatment with 2,4,6-trinitrobenzenesulphonic acid (TNBS) (15 mg/mL in 50% ethanol/50% saline). On Day 0 of the study TNBS (200 L) is administered intra-colonically via a plastic catheter with BID dosing of the vehicle, reference or test compound continuing for 2 or 4 days. During the study animals are weighed every day and stool observations are made and recorded as a score, based on stool consistency. At the time of sacrifice on Day 2 (or Day 4) the large intestine is removed and the length and weight recorded. Sections of the colon are taken for histopathology scoring to determine disease severity.
(v) Adoptive Transfer in Mice
[0862] On Study day 0, female Balb/C mice are terminated and spleens obtained for CD45RB.sup.high cell isolation (Using SCID IBD cell Separation protocol). Approximately 410.sup.5 cells/mL CD45RB.sup.high cells are then injected IP (100 L/mouse) into female SCID animals. On study day 14, mice are weighed and randomized into treatment groups based on body weight. On Day 14, compounds are administered BID, via oral gavage, in a dose volume of 5 mL/kg. Treatment continues until study day 42, at which point the animals are necropsied 4 hours after am administration. The colon length and weight is recorded and used as a secondary endpoint in the study as a measurement of colon oedema. The colon is then divided into six cross-sections, four of which are used for histopathology scoring (primary endpoint) and two are homogenised for cytokine analysis. Data shown is the % inhibition of the induction window between nave animals and vehicle animals, where higher inhibition implies closer to the non-diseased, nave, phenotype.
(vi) Endotoxin-Induced Uveitis in Rats
[0863] Male, Lewis rats (6-8 weeks old, Charles River UK Limited) are housed in cages of 3 at 19-21 C. with a 12 h light/dark cycle (07:00/19:00) and fed a standard diet of rodent chow and water ad libitum. Non-fasted rats are weighed, individually identified on the tail with a permanent marker and receive a single intravitreal administration into the right vitreous humor (5 L dose volume) of 100 ng/animal, i.v.t. of LPS (Escherichia coli 0111:B4 prepared in PBS, Sigma Aldrich, UK) using a 32-gauge needle. Untreated rats are injected with PBS. Test compound, dexamethasone (Dex) or vehicle (20% hydroxypropyl--cyclodextrin, 0.1% HPMC, 0.01% Benzalconium chloride, 0.05% EDTA, 0.7% NaCl in deionised water) are administered by the topical route onto the right eye (10 L) of animals 30 minutes prior to LPS, at the time of LPS administration, and 1, 2 and 4 hours post LPS administration. Before administration, the solution or suspension to be administered is agitated for 5 minutes to ensure a uniform suspension. 6 hours after LPS dosing, animals are euthanized by overdose with pentobarbitone (i.v.). Following euthanasia, the right eye of each animal is enucleated and dissected into front (anterior) and back (posterior) sections around the lens. Each section is weighed and homogenised in 500 L of sterile phosphate buffered saline followed by 20 minutes centrifugation at 12000 rpm at 4 C. The resulting supernatant is divided into 3 aliquots and stored at 80 C. until subsequent cytokine analysis by R&D DuoSet ELISA.
Summary of In Vitro and In Vivo Screening Results
[0864]
TABLE-US-00001 TABLE 1a Dissociation constants for selected kinases determined by LeadHunter Discover Services (DiscoveRx Corporation, Fremont, CA), using the KINOMEscan technology. Test Compound Dissociation Constant (nM) Example No. Lck p38 MAPK Syk 2 13 15 68 3 6.7 3.9 50
TABLE-US-00002 TABLE 1b Results from in vitro p38 MAPK (Method 2), c-Src, Syk and GSK3 (Method 2) inhibition assays Test Compound IC50 Values for Enzyme Inhibition (nM) Example No. p38 MAPK c-Src Syk GSK3 1 776 14 >1448 >14478 2 32 27 >1247 >12470 3 12 18 781 >13066 4 >12839 5 >12564 10 24 20 >1163 12 8 4 190
TABLE-US-00003 TABLE 2 Results from cellular assays in PBMCs and HT29 cells (the protocols for which are described by assays (b) to (d) above). IC.sub.50 Values for Inhibition of Cytokine Release (nM) Test Compound PBMCs HT29 cells Example No. IL-8 IL-2 IFN IL-8 1 133.7 2 26.0 125.9 53.3 78.7 3 8.0 16.5 18.4 4 14.8 5 10.3 38.7 30.4 7 3.8 8 4.0 9 3.1 10 3.5 13.1 11 7.6 12 2.3 12.6 13 6.7 14(a) 31.1 14(b) 15.2 14(c) 18.6 14(d) 8.3 14(e) 20.6 14(f) 10.4 14(g) 17.3 14(h) 9.4 14(i) 6.9 14(j) 9.1 14(k) 16.7 14(l) 16.6 14(m) 10.4 14(n) 11.0 14(o) 28.4 14(p) 7.5 14(q) 6.1 14(r) 49.1 14(s) 35.6 14(t) 67.6 14(u) 20.2 14(v) 4.4 14(w) 8.4 14(x) 7.7 14(y) 4.6 14(z) 6.0 14(aa) 9.5 14(ab) 5.4 14(ac) 5.1 14(ad) 7.0 14(ae) 6.5 14(af) 13.2 14(ag) 11.6 14(ah) 8.0 14(ai) 7.9 14(aj) 9.3 14(ak) 14.8 14(al) 8.3 14(am) 19.5 14(an) 12.3
[0865] As illustrated in Table 3 below, the compound of Example 3 of the present invention is markedly less active than the Reference Compound A (N-(4-(4-(3-(3-tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)ureido)naphthalen-1-yloxy)pyridin-2-yl)-2-methoxyacetamide; WO 2010/112936) in assay (g) above, which measures impact on cell division (mitosis) in PBMCs. Furthermore, compounds of the examples of the present invention are substantially less cytotoxic than Reference Compound A, displaying enhanced viabilities in cell cytotoxicity assay (r) above (Table 3).
TABLE-US-00004 TABLE 3 Effect of compounds of the examples on cell division in PBMCs and on Jurkat cell viability (NT = not tested). % Inhibition Test compound of mitosis % Viability % Viability % Viability Example No. at 5 g/mL at 1 g/mL at 5 g/mL at 20 g/mL Reference 87.8.sup.a 23.5 18.9 17.3 Compound A 3 66.3 100.4 82.9 77.5 5 NT 116.5 48.8 44.6 7 NT 79.0 54.7 49.7 8 NT 91.1 65.8 30.6 9 NT 63.0 38.4 28.7 10 NT 91.5 82.5 77.8 12 NT 86.4 79.3 81.0 14(g) NT 110.1 75.5 65.4 14(i) NT 69.8 8.4 2.1 .sup.aSee, for example, the value reported in WO 2013/050757.
Summary of Additional Studies
Hydrolytic Stability Study
[0866] Chemical stability of compounds of the invention was assessed in a mixture of DMSO and water (3:1) at a test compound concentration of 1 mg/mL
[0867] General HPLC Procedure [0868] Agilent, Waters X-Select C18, 2.5 m, 4.630 mm column, 4 min method, 5-95% [0869] MeCN/water (0.1% formic acid). [0870] Flow rate 2.5 ml/min. [0871] Column Oven Temperature 40 C. [0872] Detection 254 nm.
[0873] Sample Preparation [0874] A 1.0 mg sample of test compound was dissolved in 1 mL of DMSO/water (3:1) and the resulting mixture was stirred at rt overnight.
[0875] Recording Stability [0876] A 50 L aliquot of the test solution was removed and analysed in duplicate by 5 L HPLC injections. The peak area for the test compound (taken to be 100% product, 0% degradation) was recorded following manual integration of the corresponding UV trace. The area retention time was also recorded. [0877] The test solution was heated to 60 C. internal temperature (65 C. block temperature), with stirring, and timing commenced once the target temperature had been reached. After 5 and 24 h, samples were removed and analysed by HPLC (5 L injections). In all cases, 5 L injections were used and the samples analysed in duplicate. [0878] The peak areas for the test compounds were recorded at both subsequent timepoints and the % decomposition calculated from the % change in peak area over time. The remaining product was calculated as a percentage of the original value. [0879] Reference Compound B (3-ethynyl-5-((4-((4-(3-(3-isopropyl-1-(p-tolyl)-1H-pyrazol-5-yl)ureido)naphthalen-1-yl)oxy)pyrimidin-2-yl)amino)-N-(2-morpholinoethyl)benzamide; Cariou, C. A. M., et al, WO 2014/027209) was included in each stability study as a control to validate the study.
[0880] The results of the study are reported in Table 4 below. In contrast to the compound of Example 2, the Reference Compound underwent substantial decomposition under the conditions of the experiment.
TABLE-US-00005 TABLE 4 Results from hydrolytic stability study. Test Compound Time (min) % Parent Remaining Reference Compound B 0 100.sup.a (100.sup.b) 300 93.3.sup.a (71.0.sup.b) 1440 52.3.sup.a (26.0.sup.b) Example 2 0 100 300 104.2 1440 102.9 Key .sup.aResults for Reference Compound B from the experiment in which the compound of Example 2 was tested. .sup.bCumulative average of results for Reference Compound B from multiple experiments.
Abbreviations
[0881] AcOH glacial acetic acid [0882] aq aqueous [0883] 5-ASA 5-aminosalicylic acid [0884] ATP adenosine-5-triphosphate [0885] BALF bronchioalveolar lavage fluid [0886] BID bis in die (twice-daily) [0887] BINAP 2,2-bis(diphenylphosphino)-1,1-binaphthyl [0888] BOP (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate [0889] br broad [0890] BrdU 5-bromo-2-deoxyuridine [0891] BSA bovine serum albumin [0892] CatCart catalytic cartridge [0893] CDI 1,1-carbonyl-diimidazole [0894] COPD chronic obstructive pulmonary disease [0895] d doublet [0896] dba dibenzylideneacetone [0897] DBU 1,8-diazabicyclo[5.4.0]undec-7-ene [0898] DCC dicyclohexylcarbodiimide [0899] DCM dichloromethane [0900] DIAD diisopropyl azodicarboxylate [0901] DIPEA diisopropylethylamine [0902] DMAP 4-dimethylaminopyridine [0903] DMEM Dulbecco's modified eagle medium [0904] DMF N,N-dimethylformamide [0905] DMSO dimethyl sulfoxide [0906] DPPA diphenylphosphoryl azide [0907] d-U937 cells PMA differentiated U-937 cells [0908] EDTA ethylenediaminetetraacetic acid [0909] ELISA enzyme-linked immunosorbent assay [0910] (ES.sup.) electrospray ionization, negative mode [0911] (ES.sup.+) electrospray ionization, positive mode [0912] Et ethyl [0913] Et.sub.3N triethylamine [0914] EtOAc ethyl acetate [0915] EtOH ethanol [0916] FACS fluorescence-activated cell sorting [0917] FBS foetal bovine serum [0918] FCS foetal calf serum [0919] fMLP formyl-methionyl-leucyl-phenylalanine [0920] FRET fluorescence resonance energy transfer [0921] GSK3 glycogen synthase kinase 3 [0922] HBEC primary human bronchial epithelial cells [0923] HBSS Hank's balanced salt solution [0924] HPLC high performance liquid chromatography [0925] HPMC hydroxypropylmethylcellulose [0926] h or hr hour(s) [0927] HATU 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate [0928] HOAt 1-hydroxy-7-azabenzotriazole [0929] HOBt hydroxybenzotriazole [0930] HRP horseradish peroxidase [0931] HRV human rhinovirus [0932] ICAM-1 inter-cellular adhesion molecule 1 [0933] IFN interferon- [0934] IL interleukin [0935] iPrOAc isopropyl acetate [0936] JNK c-Jun N-terminal kinase [0937] LC liquid chromatography [0938] Lck lymphocyte-specific protein tyrosine kinase [0939] LPS lipopolysaccharide [0940] m multiplet [0941] (M+H).sup.+ protonated molecular ion [0942] MAPK mitogen-activated protein kinase [0943] MAPKAP-K2 mitogen-activated protein kinase-activated protein kinase-2 [0944] mCPBA meta-chloroperbenzoic acid [0945] Me methyl [0946] MeCN acetonitrile [0947] MeOH methanol [0948] MHz megahertz [0949] min or mins minute(s) [0950] MMAD mass median aerodynamic diameter [0951] MOI multiplicity of infection [0952] MPO myeloperoxidase [0953] MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide [0954] MS mass spectrometry [0955] m/z mass-to-charge ratio [0956] NMP N-methyl pyrrolidinone [0957] NMR nuclear magnetic resonance (spectroscopy) [0958] OD optical density [0959] PBMC peripheral blood mononuclear cell [0960] PBS phosphate buffered saline [0961] Ph phenyl [0962] PHA phytohaemagglutinin [0963] PMA phorbol myristate acetate [0964] pTSA 4-methylbenzenesulfonic acid (para-toluenesulfonic acid) [0965] PyBOP (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate [0966] q quartet [0967] rt or RT room temperature [0968] RP HPLC reverse phase high performance liquid chromatography [0969] rpm revolutions per minute [0970] RPMI Roswell Park Memorial Institute [0971] RSV respiratory syncytial virus [0972] s singlet [0973] sat or satd saturated [0974] SCID severe combined immunodeficiency [0975] SCX solid supported cation exchange (resin) [0976] SDS sodium dodecyl sulfate [0977] S.sub.NAr nucleophilic aromatic substitution [0978] Syk Spleen tyrosine kinase [0979] t triplet [0980] T3P 1-propanephosphonic acid cyclic anhydride [0981] TBAF tetrabutylammonium fluoride [0982] TBDMS tert-butyldimethylsilyl [0983] TCID.sub.50 50% tissue culture infectious dose [0984] TEA triethylamine [0985] THF tetrahydrofuran [0986] TFA trifluoroacetic acid [0987] TGF transforming growth factor beta [0988] TIPS triisopropylsilyl [0989] TMB 3,3,5,5-tetramethylbenzidine [0990] TMS-Cl trimethylsilyl chloride [0991] TNF tumor necrosis factor alpha
[0992] Prefixes n-, s-, i-, t- and tert- have their usual meanings: normal, secondary, iso, and tertiary.