KINASE INHIBITORS
20170291917 · 2017-10-12
Inventors
Cpc classification
C07D409/12
CHEMISTRY; METALLURGY
A61P1/04
HUMAN NECESSITIES
A61P29/00
HUMAN NECESSITIES
A61K31/4412
HUMAN NECESSITIES
A61K31/443
HUMAN NECESSITIES
A61K31/4439
HUMAN NECESSITIES
A61K31/4436
HUMAN NECESSITIES
A61K31/505
HUMAN NECESSITIES
A61K31/4412
HUMAN NECESSITIES
A61K31/4436
HUMAN NECESSITIES
A61K31/4439
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K31/505
HUMAN NECESSITIES
C07D405/12
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
A61K31/675
HUMAN NECESSITIES
A61K31/44
HUMAN NECESSITIES
A61K31/501
HUMAN NECESSITIES
C07D239/47
CHEMISTRY; METALLURGY
C07D403/12
CHEMISTRY; METALLURGY
C07D213/74
CHEMISTRY; METALLURGY
C07D401/12
CHEMISTRY; METALLURGY
A61K31/443
HUMAN NECESSITIES
A61K31/501
HUMAN NECESSITIES
A61K31/506
HUMAN NECESSITIES
International classification
A61K31/44
HUMAN NECESSITIES
C07D239/47
CHEMISTRY; METALLURGY
A61K31/675
HUMAN NECESSITIES
A61K31/443
HUMAN NECESSITIES
C07D409/12
CHEMISTRY; METALLURGY
A61K31/4439
HUMAN NECESSITIES
A61K31/505
HUMAN NECESSITIES
A61K31/4436
HUMAN NECESSITIES
C07D401/12
CHEMISTRY; METALLURGY
C07D405/12
CHEMISTRY; METALLURGY
C07D413/12
CHEMISTRY; METALLURGY
Abstract
There are provided compounds of formula I,
##STR00001##
wherein T, A, Q, Z, G, R.sup.4, R.sup.5a, R.sup.5b and n have meanings given in the description, which compounds have antiinflammatory activity (e.g. through 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) and 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, ##STR00100## wherein: T represents ##STR00101## W represents O, S or NCH.sub.3; V represents N or CR.sup.1; R.sup.1 represents C.sub.1-3 alkoxy, C.sub.1-3 alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl, which latter four groups are optionally substituted by one or more substituents selected from the group consisting of halo, hydroxy and C.sub.1-2 alkoxy, or R.sup.1 represents H; R.sup.2 represents —NR.sup.A1S(O).sub.2R.sup.B1, —S(O).sub.1-2R.sup.B2, —P(O)R.sup.B3R.sup.B4, —C(O)NR.sup.A2R.sup.A3 or —CH.sub.2NR.sup.A4C(O)R.sup.A5; R.sup.A1 to R.sup.A5 independently represent H or C.sub.1-3 alkyl optionally substituted by one or more substituents selected from the group consisting of halo, hydroxy, NR.sup.CR.sup.D and C.sub.1-2 alkoxy, or R.sup.A2 and R.sup.A3 together represent C.sub.3-6 n-alkylene or C.sub.4-5 n-alkylene interrupted between C2 and C3 by —O—, —S(O).sub.q— or —N(R.sup.E)—; R.sup.B1 to R.sup.B4 independently represent C.sub.1-3 alkyl or C.sub.3-6 cycloalkyl, which latter two groups are optionally substituted by one or more halo substituents; R.sup.C and R.sup.D independently represent H or C.sub.1-3 alkyl, which latter substituent is optionally substituted by hydroxyl or C.sub.1-2 alkoxy, or R.sup.C and R.sup.D together combine to form C.sub.4-6 alkylene optionally interrupted between C2 and C3 by —O—, —S(O).sub.q— or —N(R.sup.E); R.sup.E represents H or methyl; q represents 0, 1 or 2; R.sup.3 represents C.sub.2-7 alkyl, C.sub.2-7 alkenyl, C.sub.2-7 alkynyl or C.sub.3-7 cycloalkyl, which latter four groups are optionally substituted by hydroxyl, C.sub.1-2 alkoxy or halo, or R.sup.3 represents morpholinyl or trimethylsilyl; A represents CH or N; R.sup.4 represents C.sub.1-3 alkoxy, C.sub.3-5 cycloalkoxy, or C.sub.1-3 alkyl, which latter three groups are optionally substituted by one or more halo substituents, or R.sup.4 represents ethynyl, cyano, S(O).sub.2CH.sub.3, halo or H; Q represents O, S(O).sub.p, SO.sub.2N(R.sup.6) or C(O)N(R.sup.6); n represents 1, 2 or 3; p represents 0, 1 or 2; R.sup.5a and R.sup.5b independently represent H, methyl or halo, or R.sup.5a and R.sup.5b together represent C.sub.2-6 n-alkylene; when n represents 1, Z represents O, S or NR.sup.7 or, when n represents 2 or 3, Z represents either an O-atom on each occurrence, or either an S-atom or NR.sup.7 on one occurrence and an O-atom on each other occurrence; R.sup.6 and R.sup.7 independently represent H or methyl; G represents —[(CH.sub.2).sub.r-Het.sup.1].sub.0-1-C(O).sub.2H or a carboxylic acid isostere; r represents 0 or, when Het.sup.1 is attached to (CH.sub.2).sub.r via a ring heteroatom, r may alternatively represent 1; and Het.sup.1 represents 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 or a 4- to 7-membered heterocyclic group that is fully saturated or partially unsaturated, and is monocyclic or is fused or bridged bicyclic, which group contains one or more heteroatoms selected from the group consisting of N, O and S, wherein Het.sup.1 is optionally substituted by one or more substituents selected from the group consisting of C.sub.1-3 alkyl, C.sub.1-3 alkoxy, halo, hydroxyl and oxo, or a pharmaceutically acceptable salt thereof.
2. A compound of formula Iy, ##STR00102## or a pharmaceutically acceptable salt thereof.
3. A compound according to claim 2 that is 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid.
4. A compound as claimed in claim 1 that is a compound of formula Ix or Ia, ##STR00103## wherein R to R.sup.4, R.sup.5a, R.sup.5b, A, Q and n are defined in claim 1, or a pharmaceutically acceptable salt thereof.
5. A compound as claimed in claim 1 that is a compound of formula Ib, ##STR00104## wherein R.sup.2, A, Q and n are defined in claim 1, or a pharmaceutically acceptable salt thereof.
6. A compound as claimed in claim 1, wherein: R.sup.1 represents methoxy or deuterated methoxy; R.sup.3 represents trimethylsilyl or —C(CH.sub.3).sub.2—R, wherein R represents ethynyl or methyl; and/or R.sup.4 represents cyclopropoxy or methoxy, which latter group is optionally substituted by one or more halo substituents.
7. A compound as claimed in claim 1, wherein R.sup.2 represents —C(O)NH.sub.2, —C(O)NHCH.sub.3, —S(O).sub.1-2CH.sub.3, —S(O).sub.1-2CH.sub.2CH.sub.3, —P(O)(CH.sub.3).sub.2, —N(CH.sub.3)S(O).sub.2CH.sub.3, —NHS(O).sub.2CH.sub.2CH.sub.3 or —NHS(O).sub.2CH.sub.3.
8. A compound as claimed in claim 1, wherein A represents CH.
9. A compound as claimed in claim 1, wherein Q represents C(O)NH, S(O), S(O).sub.2 or O.
10. A compound as claimed in claim 1, wherein: n represents 2; R.sup.5a and R.sup.5b independently represent H or methyl or R.sup.5a and R.sup.5b together represent —(CH.sub.2).sub.2—; and/or G represents —CO.sub.2H or -Het.sup.1-CO.sub.2H, wherein the -Het.sup.1-CO.sub.2H moiety is a structural fragment selected from the group consisting of: ##STR00105## or G represents a carboxylic acid isostere selected from the group consisting of tetrazolyl, —C(O)N(H)—S(O).sub.2CH.sub.3, —C(O)N(H)—S(O).sub.2N(CH.sub.3).sub.2, ##STR00106## or a tautomer of any of the latter three groups.
11. A compound as claimed in claim 1, wherein: R.sup.2 represents —NHS(O).sub.2CH.sub.3; Q represents C(O)NH or O; and n represents 2.
12. A compound as claimed in claim 1 which is a compound selected from the group consisting of: 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxybenzamido)ethoxy)ethoxy)acetic acid; 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyrimidin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid; 2-(2-(2-((3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenyl) sulfonyl)ethoxy)ethoxy)acetic acid; 2-(2-(2-((3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenyl) sulfinyl)ethoxy)ethoxy)acetic acid; 2-(2-(2-((3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonyl)phenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenyl) sulfonyl)ethoxy)ethoxy)acetic acid; 2-(2-(2-((3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfinyl)phenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenyl) sulfonyl)ethoxy)ethoxy)acetic acid; 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-(trifluoromethyl)phenoxy)ethoxy)ethoxy)acetic acid; 6-((2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)methyl)pyridazine-3-carboxylic acid; 5-((2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)methyl)-1,2,4-oxadiazole-3-carboxylic acid; 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-cyclopropoxyphenoxy)ethoxy)ethoxy)acetic acid; 1-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)cyclopropane-1-carboxylic acid; 4-((2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)methyl)thiophene-2-carboxylic acid; 1-((2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)methyl)-3-methyl-H-pyrazole-4-carboxylic acid; 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-ethylphenoxy)ethoxy)ethoxy)acetic acid; 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-(methoxy-d3)-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid; 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)-N-(methylsulfonyl)acetamide; 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylcarbamoyl)phenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid; 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfinyl)phenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid; 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonyl)phenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid; 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-3-(dimethylphosphoryl)-2-methoxyphenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid; 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(N-methylmethylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid; 5-((2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)methyl)furan-3-carboxylic acid; 5-((2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)methyl)tetrahydrofuran-3-carboxylic acid; 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)propanoic acid; 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-3-(ethylsulfonyl)-2-methoxyphenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid; 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-3-(ethylsulfonamido)-2-methoxyphenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid; 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)isoxazol-3-yl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid; N-(5-(tert-butyl)-2-methoxy-3-(3-(4-((2-((3-methoxy-5-(2-(2-((5-oxo-2,5-dihydroisoxazol-3-yl)methoxy)ethoxy)ethoxy)phenyl)amino)pyridin-4-yl)oxy)naphthalen-1-yl)ureido)phenyl)-methanesulfonamide; 2-((2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethyl)thio)acetic acid; 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)propanoic acid, (R)-isomer; 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)propanoic acid, (S)-isomer; 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)-2-methylpropanoic acid; 1-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethyl)-1H-pyrazole-4-carboxylic acid; N-(3-(3-(4-((2-((3-(2-(2-((1H-tetrazol-5-yl)methoxy)ethoxy)ethoxy)-5-methoxyphenyl)amino)-pyridin-4-yl)oxy)naphthalen-1-yl)ureido)-5-(tert-butyl)-2-methoxyphenyl)methanesulfonamide; 2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)acetic acid; 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)-N—(N,N-dimethylsulfamoyl)acetamide; 5-((2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)methyl)thiophene-2-carboxylic acid; 5-((2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)methyl)thiophene-3-carboxylic acid; 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-(difluoromethoxy)phenoxy)ethoxy)ethoxy)acetic acid; 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-ethynylphenoxy)ethoxy)ethoxy)acetic acid; N-(5-(tert-butyl)-2-methoxy-3-(3-(4-((2-((3-methoxy-5-(2-(2-((5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)methoxy)ethoxy)ethoxy)phenyl)amino)pyridin-4-yl)oxy)naphthalen-1-yl)ureido)phenyl)methanesulfonamide; 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-(trifluoromethoxy)phenoxy)ethoxy)ethoxy)acetic acid; N-(5-(tert-butyl)-2-methoxy-3-(3-(4-((2-((3-methoxy-5-(2-(2-((3-oxo-2,3-dihydroisoxazol-5-yl)methoxy)ethoxy)ethoxy)phenyl)amino)pyridin-4-yl)oxy)naphthalen-1-yl)ureido)phenyl)-methanesulfonamide; and 5-((2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)methyl)-1-methyl-1H-pyrrole-2-carboxylic acid, and a pharmaceutically acceptable salt thereof.
13. A pharmaceutical formulation comprising a compound as defined in claim 1, or a pharmaceutically acceptable salt thereof, in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.
14. A pharmaceutical formulation comprising a compound as defined in claim 2, or a pharmaceutically acceptable salt thereof, in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.
15. A combination product comprising (A) a compound as defined in claim 1, or a pharmaceutically acceptable salt 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.
16. A combination product comprising (A) a compound as defined in claim 2, or a pharmaceutically acceptable salt 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. 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 thereof, or a pharmaceutical formulation comprising a compound as defined in claim 1, or a pharmaceutically acceptable salt 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 a pharmaceutically acceptable salt 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.
18. 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 2, or pharmaceutically acceptable salt thereof, or a pharmaceutical formulation comprising a compound as defined in claim 2, or a pharmaceutically acceptable salt thereof, in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, or a combination product comprising: (A) a compound as defined in claim 2, or a pharmaceutically acceptable salt 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.
19. A method according to claim 17, wherein the inflammatory disease is selected from the group consisting of cystic fibrosis, pulmonary hypertension, lung sarcoidosis, idiopathic pulmonary fibrosis, COPD, chronic bronchitis, emphysema, asthma, paediatric asthma, atopic dermatitis, allergic dermatitis, contact dermatitis or psoriasis, allergic rhinitis, rhinitis, sinusitis, conjunctivitis, allergic conjunctivitis, keratoconjunctivitis sicca (dry eye or xerophthalmia), 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, gluten sensitive enteropathy (coeliac disease), eosinophilic esophagitis, intestinal graft versus host disease, Crohn's disease and ulcerative colitis.
20. A method according to claim 18, wherein the inflammatory disease is selected from the group consisting of cystic fibrosis, pulmonary hypertension, lung sarcoidosis, idiopathic pulmonary fibrosis, COPD, chronic bronchitis, emphysema, asthma, paediatric asthma, atopic dermatitis, allergic dermatitis, contact dermatitis or psoriasis, allergic rhinitis, rhinitis, sinusitis, conjunctivitis, allergic conjunctivitis, keratoconjunctivitis sicca (dry eye or xerophthalmia), 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, gluten sensitive enteropathy (coeliac disease), eosinophilic esophagitis, intestinal graft versus host disease, Crohn's disease and ulcerative colitis.
21. A method according to claim 19, wherein the inflammatory disease is uveitis, keratoconjunctivitis sicca (dry eye or xerophthalmia), Crohn's disease or ulcerative colitis.
22. A method according to claim 20, wherein the inflammatory disease is uveitis, keratoconjunctivitis sicca (dry eye or xerophthalmia), Crohn's disease or ulcerative colitis.
23. A process for the preparation of a compound of formula I, as defined in claim 1, which process comprises: (a) for compounds of formula I in which G represents —[(CH.sub.2).sub.r-Het.sup.1].sub.0-1-C(O).sub.2H, hydrolysis or hydrogenolysis of an ester of formula I(P), ##STR00107## wherein R.sup.x represents C.sub.1-6 alkyl or benzyl, respectively, and T, R.sup.4, R.sup.5a, R.sup.5b, A, Q, Z, n, r and Het.sup.1 are as defined in claim 1; (b) reaction of a compound of formula II, ##STR00108## with a compound of formula III, ##STR00109## wherein one of Z.sup.1 and Z.sup.2 is a structural fragment of formula IVa or IVb, ##STR00110## and the other of Z.sup.1 and Z.sup.2 is a structural fragment of formula V, ##STR00111## wherein W, V, R.sup.1 to R.sup.4, R.sup.5a, R.sup.5b, A, Q, Z, G and n are as defined in claim 1; (c) reaction of a compound of formula IIa, ##STR00112## wherein Z.sup.1 is as defined above, with a suitable azide-forming agent, which reaction is followed, without isolation, by thermal rearrangement of the intermediate acyl azide (of formula Z.sup.1—C(O)—N.sub.3) to provide, in situ, a compound of formula II, which compound is then reacted with a compound of formula III as defined above; (d) reaction of a compound of formula IIb, ##STR00113## wherein LG.sup.1 represents a leaving group and Z.sup.1 is as defined above, with a compound of formula III, as defined above; (e) reaction of a compound of formula VI, ##STR00114## wherein LG.sup.2 represents a leaving group and R.sup.1 to R.sup.3 and A are as defined in claim 1, with a compound of formula VII, ##STR00115## wherein R.sup.4, R.sup.5a, R.sup.5b, Q, Z, G and n are as defined in claim 1; (f) for compounds of formula I in which Q represents S(O).sub.1-2, oxidation of a corresponding compound of formula I in which Q represents S; (g) for compounds of formula I in which Q represents C(O)NH, reaction of a compound of formula VIII, ##STR00116## wherein LG.sup.3 represents OH, OR.sup.x or a leaving group, R.sup.x is as defined above and R.sup.1 to R.sup.3, and A are as defined in claim 1, with a compound of formula IX, ##STR00117## wherein R.sup.5a, R.sup.5b, Z, G and n are as defined in claim 1; (h) for compounds of formula I in which G represents —[(CH.sub.2).sub.r-Het.sup.1].sub.0-1-C(O).sub.2H, oxidation of an alcohol of formula Xa, ##STR00118## wherein T, R.sup.4, R.sup.5a, R.sup.5b, A, Q, Z, n, r and Het.sup.1 are as defined in claim 1; or (i) for compounds of formula I in which G represents —C(O)N(H)OH, —C(O)N(H)OCH.sub.3, —C(O)N(H)—S(O).sub.2CH.sub.3 or —C(O)N(H)—S(O).sub.2N(CH.sub.3).sub.2, coupling of a corresponding compound of formula I in which G represents —CO.sub.2H with hydroxylamine, methoxyamine, methanesulfonamide or dimethylsulfamide, respectively; (k) for compounds of formula I in which G represents a hydroxy-substituted isoxazole having the structure: ##STR00119## reaction of a compound of formula Xb, ##STR00120## wherein T, A, R.sup.4, R.sup.5a, R.sup.5b, Q, Z and n are as defined in claim 1 and R.sup.x is as defined above, with hydroxylamine; (l) for compounds of formula I in which G represents tetrazol-5-yl, reaction of a compound of formula Xc, ##STR00121## wherein T, A, R.sup.4, R.sup.5a, R.sup.5b, Q, Z, and n are as defined in claim 1, with a source of azide; (m) for compounds of formula I in which G represents 5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl, reaction of a compound of formula Xc, as defined above, with hydroxylamine, followed by reaction of the resulting N-hydroxyamidine (amidoxime) compound with a —C(O)— source; (n) deprotection of a protected derivative of a compound of formula I, wherein the protected derivative bears a protecting group on an O- or N-atom of the compound of formula I.
24. A compound of formula I(P) as defined in claim 23, or a salt thereof.
25. A compound of formula I(P) as claimed in claim 24, wherein: R.sup.1 represents methoxy; R.sup.2 represents —NHS(O).sub.2CH.sub.3; R.sup.3 represents tert-butyl; A represents CH; R.sup.4 represents methoxy; R.sup.5a and R.sup.5b both represent H; Q represents O; Z represents O; n represents 2; and/or R.sup.x represents ethyl.
26. A compound as claimed in claim 24 which compound is: ##STR00122## (ethyl 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetate), or a salt thereof.
27. A compound: (a) of formula II or IIb, as defined in claim 23, or a salt or protected derivative thereof, wherein Z.sup.1 is a structural fragment of formula V, as defined in claim 23 wherein the protected derivative of the compound of formula II or IIb is a compound in which, when G represents —[(CH.sub.2).sub.r-Het.sup.1].sub.0-1-C(O).sub.2H, the C(O).sub.2H moiety is protected as an ester of formula C(O)OR.sup.x, wherein R.sup.x is as defined in claim 23; or (b) of formula III, as defined in claim 23, or a salt or protected derivative thereof, wherein Z.sup.2 is a structural fragment of formula V, as defined in claim 23, wherein the protected derivative of the compound of formula III is a compound (i) in which the NH.sub.2 group of formula III is replaced by nitro, or in which a H-atom of the NH.sub.2 group of formula III is replaced by R′—C(O)—, wherein R′ is C.sub.1-8 alkyl optionally substituted by one or more fluoro groups or R′ is H, phenyl or benzyl, which latter two groups are optionally substituted by one or more groups selected from the group consisting of halo, hydroxy, methyl and methoxy; or R″—O—C(O)—, wherein R″ is tert-butyl, phenyl, benzyl or fluorenyl, which latter three groups are optionally substituted by one or more groups selected from the group consisting of halo, hydroxy, methyl and methoxy; and/or (ii) in which, when G represents —[(CH.sub.2).sub.r-Het.sup.1].sub.0-1-C(O).sub.2H, the C(O).sub.2H moiety of formula III is protected as an ester of formula C(O)OR.sup.x, wherein R.sup.x is as defined in claim 23.
28. A compound of formula II, IIb or III as claimed in claim 27, or a salt or protected derivative thereof, wherein: A represents CH; R.sup.4 represents methoxy; R.sup.5a and R.sup.5b both represent H; Q represents O; Z represents O; and/or n represents 2.
29. A compound of formula III, or a salt thereof, as claimed in claim 27, which compound is ##STR00123## (ethyl 2-(2-(2-(3-((4-((4-((tert-butoxycarbonyl)amino)naphthalen-1-yl)oxy)pyridin-2-yl)-amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetate) or ##STR00124## (ethyl 2-(2-(2-(3-((4-((4-aminonaphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)-ethoxy)ethoxy)acetate).
30. A compound of formula IIb, or a salt or protected derivative thereof, as claimed in claim 27, wherein LG.sup.1 represents imidazolyl, chloro, or aryloxy.
31. A compound of formula IIb, or a salt or protected derivative thereof, as claimed in claim 27, wherein: LG.sup.1 represents phenoxy; and G represents C(O).sub.2H, wherein the protected derivative of the compound of formula IIb is a compound in which the C(O).sub.2H moiety is protected as an ester of formula C(O)OR.sup.x, wherein R.sup.x is as defined in claim 23.
32. A compound of formula IIb, or a salt or protected derivative thereof, as claimed in claim 31, wherein A represents CH; R.sup.4 represents methoxy; R.sup.5a and R.sup.5b both represent H; Q represents O; Z represents O; and/or n represents 2.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0442]
TABLE-US-00002 TABLE A peak listing for XRPD profile of product of Example 31, Method 1 Pos. Height FWHM d-spacing Rel. Int. [°2Th.] [cts] [°2Th.] [Å] [%] 3.9317 997.91 0.1023 22.47367 68.42 4.4389 376.68 0.1279 19.90685 25.83 5.9292 224.18 0.1279 14.90639 15.37 8.9406 240.27 0.1535 9.89115 16.47 9.4882 279.74 0.1023 9.32147 19.18 11.2243 804.76 0.1279 7.88327 55.17 14.6967 1458.55 0.2303 6.02757 100.00 15.8103 156.22 0.1535 5.60546 10.71 18.0348 732.39 0.1791 4.91876 50.21 18.4303 539.12 0.1023 4.81408 36.96 18.9396 749.55 0.3582 4.68577 51.39 21.0357 301.56 0.1535 4.22334 20.68 22.6909 254.38 0.1535 3.91888 17.44 26.2933 130.65 0.1279 3.38956 8.96 27.5888 51.44 0.4093 3.23328 3.53 27.9150 40.67 0.1279 3.19623 2.79 28.9822 37.73 0.3070 3.08092 2.59 33.3548 18.18 0.1535 2.68635 1.25
TABLE-US-00003 TABLE B peak listing for XRPD profile of product of Example 31, Method 2 Pos. Height FWHM d-spacing Rel. Int. [°2Th.] [cts] [°2Th.] [Å] [%] 3.9435 937.49 0.1023 22.40637 92.78 4.4734 508.47 0.2047 19.75372 50.32 5.8834 347.72 0.2558 15.02234 34.41 9.0053 212.93 0.1791 9.82024 21.07 9.5071 332.04 0.1535 9.30301 32.86 11.3151 633.79 0.2047 7.82025 62.72 14.7734 1010.46 0.2558 5.99645 100.00 15.6873 215.33 0.5117 5.64914 21.31 18.0549 555.83 0.2558 4.91331 55.01 19.0099 725.20 0.3582 4.66858 71.77 21.1976 433.24 0.8187 4.19144 42.88 22.7798 354.68 0.4093 3.90378 35.10 26.3822 80.59 0.3070 3.37834 7.98 27.6970 45.38 0.6140 3.22089 4.49 31.6934 43.32 0.8187 2.82328 4.29
[0443]
EXPERIMENTAL METHODS
General Procedures
[0444] All starting materials and solvents were obtained either from commercial sources or prepared according to the literature citation. Unless otherwise stated all reactions were stirred. Organic solutions were routinely dried over anhydrous magnesium sulfate. Hydrogenations were performed on a Thales H-cube flow reactor under the conditions stated or under a balloon of hydrogen. Microwave reactions were performed in a CEM Discover and Smithcreator microwave reactor, heating to a constant temperature using variable power microwave irradiation.
[0445] Normal phase column chromatography was routinely carried out on an automated flash chromatography system such as CombiFlash Companion or CombiFlash RF system using pre-packed silica (230-400 mesh, 40-63 μm) cartridges. SCX was purchased from Supelco and treated with 1M hydrochloric acid prior to use. Unless stated otherwise the reaction mixture to be purified was first diluted with MeOH and made acidic with a few drops of AcOH. This solution was loaded directly onto the SCX and washed with MeOH. The desired material was then eluted by washing with 1% NH.sub.3 in MeOH.
Analytical Methods
[0446] Analytical HPLC was carried out using a Waters Xselect CSH C18, 2.5 μm, 4.6×30 mm column eluting with a gradient of 0.1% Formic Acid in MeCN in 0.1% aqueous Formic Acid or a Waters Xbridge BEH C18, 2.5 μm, 4.6×30 mm column eluting with a gradient of MeCN in aqueous 10 mM Ammonium Bicarbonate. UV spectra of the eluted peaks were measured using either a diode array or variable wavelength detector on an Agilent 1100 system.
[0447] Analytical LCMS was carried out using a Waters Xselect CSH 018, 2.5 μm, 4.6×30 mm column eluting with a gradient of 0.1% Formic Acid in MeCN in 0.1% aqueous Formic Acid or a Waters Xbridge BEH C18, 2.5 μm, 4.6×30 mm column eluting with a gradient of MeCN in aqueous 10 mM Ammonium Bicarbonate. UV and mass spectra of the eluted peaks were measured using a variable wavelength detector on either an Agilent 1200 or an Agilent Infinity 1260 LCMS with 6120 single quadrupole mass spectrometer with positive and negative ion electrospray.
[0448] Preparative HPLC was carried out using a Waters Xselect CSH C18, 5 μm, 19×50 mm column using either a gradient of either 0.1% Formic Acid in MeCN in 0.1% aqueous Formic Acid or a gradient of MeCN in aqueous 10 mM Ammonium Bicarbonate or employing a Waters Xbridge BEH C18, 5 μm, 19×50 mm column using a gradient of MeCN in aqueous 10 mM Ammonium Bicarbonate. Fractions were collected following detection by UV at a single wavelength measured by a variable wavelength detector on a Gilson 215 preparative HPLC or Varian PrepStar preparative HPLC or by mass and UV at a single wavelength measured by a ZQ single quadrupole mass spectrometer, with positive and negative ion electrospray, and a dual wavelength detector on a Waters FractionLynx LCMS.
[0449] .sup.1H NMR Spectroscopy.
[0450] .sup.1H NMR spectra were acquired on a Bruker Avance III spectrometer at 400 MHz. Either the central peaks of chloroform-d, dirmethylsulfoxide-d.sub.6 or an internal standard of tetramethylsilane were used as references.
Preparation of Compounds of the Invention
Example 1
2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxybenzamido)ethoxy)ethoxy)acetic acid, hydrochloride salt
[0451] ##STR00055##
(i) Methyl 3-amino-5-methoxybenzoate
[0452] To a stirred suspension of 3-amino-5-methoxybenzoic acid (47 g, 281 mmol) in MeOH (1 L) at 0° C. was added thionyl chloride (123 mL, 1687 mmol) dropwise. The reaction was warmed to rt and stirred for 72 h. The resulting solid was isolated by filtration, washing with diisopropyl ether, affording the product as the HCl salt. The filtrate was evaporated, the residue triturated with MeOH/ether, filtered and washed with ether. The combined solid was suspended in DCM (500 mL) and basified with sat. aq. NaHCO.sub.3 solution (300 mL) with vigorous stirring. The organic phase was separated, washed with brine (200 mL), dried (MgSO.sub.4), filtered and evaporated under reduced pressure to give a solid. The solid was triturated with ether/isohexane to afford the sub-title compound (46 g) as a solid.
[0453] .sup.1H NMR (400 MHz, DMSO-d6) δ: 6.83 (dd, 1H), 6.63 (dd, 1H), 6.37 (t, 1H), 5.42 (s, 2H), 3.79 (s, 3H), 3.70 (s, 3H).
[0454] LCMS m/z 182 (M+H).sup.+ (ES.sup.+)
(ii) Methyl 3-((4-((4-((tert-butoxycarbonyl)amino) naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxybenzoate
[0455] A mixture of the product from step (i) above (10.8 g, 59.6 mmol), tert-butyl (4-((2-chloropyridin-4-yl)oxy)naphthalen-1-yl)carbamate (see, for example, WO 2014/162126; 20 g, 53.9 mmol), finely ground potassium carbonate (15.2 g, 110 mmol) and BrettPhos G3 precatalyst (800 mg, 0.883 mmol) in tBuOH (400 mL) was extensively degassed with N.sub.2. The reaction was heated under nitrogen at 90° C. (block temperature) for 2 h. The reaction mixture was diluted with DCM (500 mL), filtered through Celite and concentrated in vacuo to afford a brown foam. The foam was triturated with Et.sub.2O (500 mL). The resultant solid was filtered, washing with further Et.sub.2O (100 mL), and dried in vacuo to affording the sub-title compound (25.6 g) as an off-white/pale-grey solid.
[0456] .sup.1H NMR (400 MHz, DMSO-d6) δ: 9.37 (s, 1H), 9.19 (s, 1H), 8.13-8.14 (m, 2H), 7.84 (d, 1H), 7.77 (bs, 1H), 7.69 (t, 1H), 7.55-7.65 (m, 3H), 7.36 (d, 1H), 6.96 (bs, 1H), 6.62 (dd, 1H), 6.09 (d, 1H), 3.82 (s, 3H), 3.75 (s, 3H), 1.53 (s, 9H).
[0457] LCMS m/z 516 (M+H).sup.+ (ES.sup.+)
(iii) Methyl 3-((4-((4-aminonaphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxybenzoate
[0458] HCl (5 N in iPrOH) (79 mL, 395 mmol) was added to a solution of the product from step (ii) above (20 g, 38.8 mmol) in DCM (250 mL) and the reaction left stirring overnight. Et.sub.2O (300 mL) was added and the resulting precipitate was isolated by filtration, washing with further Et.sub.2O. The solid was partitioned between DCM (400 mL) and sat. aq. NaHCO.sub.3 solution (600 mL). The organic layer was dried via hydrophobic frit and concentrated in vacuo to afford the sub-title compound (15.5 g) as a beige foam.
[0459] .sup.1H NMR (400 MHz, DMSO-d6) δ: 9.09 (s, 1H), 8.15-8.18 (m, 1H), 8.08 (d, 1H), 7.75 (t, 1H), 7.69 (t, 1H), 7.63-7.65 (m, 1H), 7.43-7.47 (m, 2H), 7.11 (d, 1H), 6.95 (t, 1H), 6.72 (d, 1H), 6.56 (dd, 1H), 6.04 (d, 1H), 5.83 (bs, 2H), 3.82 (s, 3H), 3.75 (s, 3H).
[0460] LCMS m/z 416 (M+H).sup.+ (ES.sup.+)
(iv) Methyl 3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxybenzoate
[0461] Triethylamine (28 μL, 0.201 mmol) was added to a solution of phenyl (5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamate (see, for example, WO 2014/162126; 480 mg, 1.223 mmol) and the product from step (iii) above (412 mg, 0.992 mmol) in iPrOAc (15 mL) at 60° C. (block temperature) and the mixture stirred for 24 h. The solution was cooled to rt and concentrated in vacuo affording a red oil. The crude product was purified by chromatography on the Companion (40 g column, 1-5% MeOH in DCM) to afford the sub-title compound (580 mg) as a pale pink foam.
[0462] LCMS m/z 714 (M+H).sup.+ (ES.sup.+)
(v) 3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamidophenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxybenzoic acid, hydrochloride salt
[0463] To a stirred solution of the product from step (iv) above (580 mg, 0.813 mmol) in THF (20 mL) was added NaOH (6M aq.) (2 mL, 12.00 mmol). MeOH (5 mL) was added and the reaction stirred overnight. The reaction was concentrated in vacuo affording a yellow solid. The solid was suspended in 1 M HCl (20 mL) and the resulting gel-like solid filtered, washing with water. The resulting solid was dried for 1 h on the frit then further dried at 40° C. under vacuum affording the sub-title compound (526 mg) as an off-white solid.
[0464] LCMS m/z 699.77 (M+H).sup.+ (ES.sup.+)
(vi) Methyl 2-(2-(2-aminoethoxy)ethoxy)acetate, trifluoroacetic acid salt
[0465] To a stirred solution of methyl 2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-oate (see, for example, WO 2011/037610; 195 mg, 0.703 mmol) in DCM (2 mL) was added TFA (500 μL, 6.49 mmol) and the mixture stirred at rt for 1 h. The reaction was concentrated in vacuo then re-concentrated from toluene affording the sub-title compound (230 mg) as a colourless oil.
[0466] LCMS m/z 178 (M+H).sup.+ (ES.sup.+)
(vii) Methyl 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-ureido)naphthalen-1-yl)oxy)pyridin-2-amino)-5-methoxybenzamido)ethoxy)ethoxy)acetate
[0467] HATU (120 mg, 0.316 mmol) was added to a stirred solution of the product from step (v) above (150 mg, 0.204 mmol), the product from step (vi) above (100 mg, 0.343 mmol) and Hünig's Base (250 μL, 1.431 mmol) in NMP (2 mL) at rt. The mixture was stirred for 2 h. The reaction was partitioned between water (15 mL) and EtOAc (10 mL). The aqueous phase was extracted with EtOAc (5 mL) and the combined organics washed with water and brine, then dried via hydrophobic frit and concentrated in vacuo. The crude product was purified by chromatography on the Companion (12 g column, 1-5% MeOH in DCM) to afford the sub-title compound (173 mg) as a colourless gum.
[0468] LCMS m/z 859 (M+H).sup.+ (ES.sup.+)
(viii) 2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxybenzamido)ethoxy)ethoxy)acetic acid, hydrochloride salt
[0469] To a stirred solution of the product from step (vii) above (173 mg, 0.201 mmol) in THF (3 mL) was added NaOH (6 M aq.) (0.30 mL, 1.800 mmol). MeOH (1 mL) was added and the resulting solution stirred at rt for 2 h. The reaction was concentrated in vacuo affording a yellow solid. The solid was suspended in 1M HCl (10 mL) and the mixture sonicated. The resulting suspension was filtered and the recovered solid washed with water then dried in vacuo at 40° C. overnight affording the title compound (140 mg) as a colourless solid.
[0470] .sup.1H NMR (400 MHz, DMSO-d6) δ: 9.61 (bs, 1H), 9.53 (s, 1H), 9.15 (s, 1H), 8.99 (s, 1H), 8.46 (t, 1H), 8.35 (d, 1H), 8.18 (d, 1H), 8.15 (d, 1H), 8.07 (d, 1H), 7.87 (d, 1H), 7.71-7.74 (m, 1H), 7.62-7.66 (m, 1H), 7.43-7.45 (m, 2H), 7.35 (s, 1H), 7.08 (s, 1H), 7.03 (d, 1H), 6.73 (bs, 1H), 6.22 (s, 1H), 4.01 (s, 2H), 3.81 (s, 3H), 3.77 (s, 3H), 3.51-3.60 (m, 6H), 3.40 (q, 2H), 3.10 (s, 3H), 1.27 (s, 9H).
[0471] LCMS m/z 845 (M+H).sup.+ (ES.sup.+)
Example 2
2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyrimidin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid
[0472] ##STR00056##
(i) Ethyl 2-(2-(2-hydroxyethoxy)ethoxy)acetate
[0473] A solution of ethyl diazoacetate (1.9 mL, 18.32 mmol) in DCM (20 mL) was added dropwise to a solution of diethylene glycol (5.0 mL, 52.7 mmol) and rhodium(II) acetate dimer (160 mg, 0.362 mmol) in DCM (300 mL) over 1 h. The reaction was stirred at rt overnight. The mixture was evaporated under reduced pressure and the residue purified by chromatography on silica gel (220 g column, 0-100% EtOAc/isohexane) to afford the sub-title compound (2.38 g) as a dark blue oil.
[0474] .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 4.23 (q, 2H), 4.19 (s, 2H), 3.60-4.05 (m, 8H), 1.28 (t, 3H).
(ii) Ethyl 2-(2-(2-((methylsulfonyl)oxy)ethoxy)ethoxy)acetate
[0475] Methanesulfonyl chloride (0.6 mL, 7.70 mmol) was added dropwise to a solution of the product from step (i) above (1.20 g, 6.24 mmol) and Et.sub.3N (1.7 mL, 12.20 mmol) in DCM (20 mL) at 0-5° C., warmed to rt and stirred for 2 h. The mixture was partitioned between DCM (50 mL) and water (30 mL), the organic layer washed with sat. aq. NaHCO.sub.3 (30 mL), dried (MgSO.sub.4), filtered and evaporated under reduced pressure. The crude product was purified by chromatography on silica gel (40 g column, 0-70% EtOAc/isohexane) to afford the sub-title compound (1.494 g) as an oil.
[0476] .sup.1H NMR (400 MHz; CDCl.sub.3) δ 4.42-4.39 (m, 2H), 4.24 (q, 2H), 4.15 (s, 2H), 3.82-3.79 (m, 2H), 3.77-3.72 (m, 4H), 3.10 (s, 3H), 1.31 (t, 3H).
(iii) Ethyl 2-(2-(2-(3-methoxy-5-nitrophenoxy)ethoxy)ethoxy)acetate
[0477] A mixture of 3-methoxy-5-nitrophenol (1.973 g, 11.67 mmol), the product from step (ii) above (2.92 g, 10.80 mmol) and K.sub.2CO.sub.3 (4.48 g, 32.4 mmol) in DMF (60 mL) was heated at 60° C. for 20 h. The reaction was cooled to rt then partitioned between ether (200 mL) and water (200 mL). The organic layer was washed with sat. aq. NaHCO.sub.3 solution (100 mL) and brine (100 mL) then dried (MgSO.sub.4), filtered and evaporated under reduced pressure. The crude product was purified by chromatography on silica gel (80 g column, 0-50% EtOAc/isohexane) to afford the sub-title compound (3.35 g) as a yellow oil.
[0478] LCMS m/z 344 (M+H).sup.+ (ES.sup.+)
(iv) Ethyl 2-(2-(2-(3-amino-5-methoxyphenoxy)ethoxy)ethoxy)acetate
[0479] To a solution of the product from step (iii) above (3.35 g, 9.76 mmol) in EtOH (20 mL) and EtOAc (5 mL) was added Pd/C (5 wt %) (0.5 g, 0.235 mmol). The resulting suspension was stirred under a 5 bar (0.5 MPa) atmosphere of H.sub.2 for 8 h. The reaction was purged with N.sub.2 then filtered through Celite. The filtrate was concentrated in vacuo affording the sub-title compound (3.0 g) as a pale yellow oil.
[0480] .sup.1H NMR (400 MHz, DMSO-d6) δ: 5.75 (d, 2H), 5.68 (t, 1H), 5.06 (s, 2H), 4.13 (s, 2H), 4.12 (q, 2H), 3.93-3.96 (m, 2H), 3.68-3.70 (m, 2H), 3.58-3.64 (m, 4H), 3.62 (s, 3H), 1.20 (s, 3H).
[0481] LCMS m/z 314 (M+H).sup.+ (ES.sup.+)
(v) Ethyl 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-oxy)pyrimidin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetate
[0482] A mixture of N-(5-(tert-butyl)-3-(3-(4-((2-chloropyrimidin-4-yl)oxy)naphthalen-1-yl)ureido)-2-methoxyphenyl)methanesulfonamide (see, for example, WO 2014/162126; 300 mg, 0.526 mmol), the product from step (iv) above (247 mg, 0.789 mmol) and pTSA hydrate (30 mg, 0.158 mmol) in THF (5 mL) was heated at 65° C. for 40 h. The mixture was cooled, partitioned between EtOAc (100 mL) and sat. aq. NaHCO.sub.3 (50 mL), the organic layer washed with 1 M HCl (50 mL), water (50 mL), dried (MgSO.sub.4), filtered and evaporated under reduced pressure. The crude product was purified by chromatography on silica gel (40 g column, 0-5% MeOH/DCM) to afford the sub-title compound (378 mg) as a foam.
[0483] LCMS m/z 847 (M+H).sup.+ (ES.sup.+)
(vi) 2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-methylsulfonamido phenyl)ureido)-naphthalen-1-oxy)pyrimidin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid
[0484] A mixture of the product from step (v) above (376 mg, 0.444 mmol) and 2 M aq. NaOH (700 μL, 1.400 mmol) in THF (5 mL) and MeOH (2 mL) was stirred at rt for 20 h. The solvent was removed in vacuo, the residue dissolved in water (5 mL) and acidified with AcOH. The mixture was evaporated and the residue purified by chromatography on the Companion (RP Flash C18) (40 g column, 15-75% MeCN/10 mM Ammonium Bicarbonate) to afford the title compound (72 mg) as a white solid.
[0485] .sup.1H NMR (400 MHz; DMSO-d6) δ 12.57 (s, 1H), 9.43 (s, 1H), 9.37 (s, 1H), 9.15 (s, 1H), 8.94 (s, 1H), 8.42 (d, 1H), 8.28 (d, 1H), 8.19 (d, 1H), 8.11 (d, 1H), 7.85 (d, 1H), 7.70-7.65 (m, 1H), 7.61-7.56 (m, 1H), 7.42 (d, 1H), 7.02 (d, 1H), 6.80 (brs, 2H), 6.54 (d, 1H), 6.04 (s, 1H), 4.00 (s, 2H), 3.89-3.83 (m, 2H), 3.81 (s, 3H), 3.69-3.64 (m, 2H), 3.60-3.54 (m, 4H), 3.51 (s, 3H), 3.10 (s, 3H), 1.27 (s, 9H).
[0486] LCMS m/z 819 (M+H).sup.+ (ES.sup.+)
Example 3
2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy)-3-(methylsulfonamido)phenyl)ureido-naphthalen-1-yl)oxy)pyridin-2-yl)amino-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid
[0487] ##STR00057##
Method 1
(i) Ethyl 2-(2-(2-(3-((4-((4-((tert-butoxycarbonyl)amino)naphthalen-1-yl)oxy)pyridin-2-yl)-amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetate
[0488] A mixture of tert-butyl (4-((2-chloropyridin-4-yl)oxy)naphthalen-1-yl)carbamate (see, for example, WO 2014/162126; 497 mg, 1.340 mmol), ethyl 2-(2-(2-(3-amino-5-methoxyphenoxy)ethoxy)ethoxy)acetate (see Example 2(iv) above; 420 mg, 1.340 mmol) and K.sub.2CO.sub.3 (556 mg, 4.02 mmol) in DMF (6 mL) was degassed under vacuum, back-filling with N.sub.2 three times. BrettPhos G3 precatalyst (37 mg, 0.041 mmol) was added and the mixture heated to 80° C. for 1 h. The mixture was cooled to rt and partitioned between EtOAc (70 mL) and water (50 mL). The organic layer washed with water (50 mL) and brine (30 mL) then dried (MgSO.sub.4), filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel (40 g column, 0-100% EtOAc/isohexane) to afford the sub-title compound (870 mg) as a colourless foam.
[0489] LCMS m/z 648 (M+H).sup.+ (ES.sup.+)
(ii) Ethyl 2-(2-(2-(3-((4-((4-aminonaphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxy-phenoxy)ethoxy) ethoxy)acetate
[0490] A mixture of the product from step (i) above (690 mg, 1.065 mmol) and TFA (1 mL, 12.98 mmol) in DCM (5 mL) was stirred at rt for 20 h then evaporated. The residue was partitioned between EtOAc (60 mL) and sat. aq. NaHCO.sub.3 solution (40 mL), the organic layer was separated, washed with water (40 mL), dried (MgSO.sub.4), filtered and evaporated under reduced pressure to afford the sub-title compound (572 mg) as a brown gum.
[0491] LCMS m/z 548 (M+H).sup.+ (ES.sup.+)
(iii) Ethyl 2-(2-(2-(3-((4-((4-(3-(tert-butyl-2-methoxy-3-(methylsulfonamido)phenyl) ureido)-naphthalen-1-oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy acetate
[0492] A mixture of phenyl (5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamate (see, for example, WO 2014/162126; 490 mg, 1.249 mmol), the product from step (ii) above (570 mg, 1.041 mmol) and Et.sub.3N (50 μL, 0.359 mmol) in THF (10 mL) was heated under reflux for 24 h. The solvent was removed and the residue purified by chromatography on silica gel (40 g column, 0-5% MeOH/DCM) to afford the sub-title compound (736 mg) as a foam.
[0493] LCMS m/z 844 (M+H).sup.+ (ES.sup.+)
(iv) 2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid
[0494] A mixture of the product from step (iii) above (803 mg, 0.892 mmol) and aq. 2 M NaOH (1.3 mL, 2.60 mmol) in THF (8 mL) and MeOH (3 mL) was stirred at rt for 20 h. The reaction was acidified with AcOH (3 mL) then concentrated in vacuo affording a pale solid. The residue was purified by chromatography on the Companion (RP Flash C18) (40 g column, 15-75% MeCN/10 mM Ammonium Bicarbonate) to afford the title compound (653 mg) as an off-white solid.
[0495] .sup.1H NMR (400 MHz, DMSO-d6) δ: 9.55 (5, 1H), 9.02 (s, 1H), 8.90 (s, 1H), 8.32 (d, 1H), 8.19 (d, 1H), 8.12 (s, 1H), 8.10 (s, 1H), 7.86 (d, 1H), 7.67-7.71 (m, 1H), 7.58-7.62 (m, 1H), 7.38 (d, 1H), 7.03 (d, 1H), 6.86 (s, 1H), 6.78 (s, 1H), 6.59 (dd, 1H), 6.09 (d, 1H), 6.03 (t, 1H), 3.93-3.96 (m, 2H), 3.93 (s, 2H), 3.81 (s, 3H), 3.69-3.71 (m, 2H), 3.65 (s, 3H), 3.55-3.61 (m, 4H), 3.10 (s, 3H), 1.27 (s, 9H).
[0496] LCMS m/z 818 (M+H).sup.+ (ES.sup.+)
Method 2
(I) Ethyl 2-[2-(2-benzyloxyethoxy)ethoxy]acetate
[0497] To a 5 L flask under nitrogen was added 60% NaH (73.5 g, 1.8375 mol) and THF (2.3 L). The resulting slurry was cooled to 0-5° C. 2-(2-Benzyloxyethoxy)ethanol (300 g, 1.5287 mol) dissolved in THF (700 mL) was then added dropwise over 1 h. Exotherm and gas evolution was observed throughout addition and as the reaction proceeded. The reaction was stirred for 40 mins. Ethyl bromoacetate (207 mL, 1.8375 mol) was then added, dropwise over 1 h, maintaining the temperature<5° C. As the reaction proceeded the mixture turned yellow in colour. The reaction was stirred for 2 h and allowed to warm to rt. LC showed 68% product and 1.6% starting material. To the reaction was added TBME (1 L) and water (1 L). The organics were separated and the aqueous phase re-extracted with TBME (2 L and 1 L). The combined organics were dried, filtered and concentrated in vacuo. The residue (462 g) was purified on silica (3 kg) eluting with 10% EtOAc:heptane (20 L), 20% EtOAc:heptane (10 L), 25% EtOAc:heptane (20 L) and 30% EtOAc:heptane (10 L). The product-containing fractions were concentrated in vacuo to give 322.6 g (74% yield) of the sub-title compound, for which .sup.1H NMR analysis indicated a purity of >95%.
[0498] .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 7.25-7.37 (m, 5H), 4.58 (s, 2H), 4.22 (q, 2H), 4.16 (s, 2H), 3.63-3.76 (m, 8H), 1.28 (t, 3H).
(II) Ethyl 2-[2-(2-hydroxyethoxy)ethoxy]acetate
[0499] To a 5 L flask under nitrogen was charged 10% Pd/C (32 g), this was followed by the addition of the product of step (I) above (320 g, 1.133 mol) dissolved in EtOH (3.2 L). The reaction was purged with hydrogen for 4 h and then stirred under a hydrogen atmosphere overnight. .sup.1H NMR indicated 2.5% starting material remaining, and so the reaction was purged with hydrogen for 2 h. .sup.1H NMR then indicated complete reaction. The reaction mixture was filtered through Celite and washed with ethanol (1 L). The filtrate was concentrated in vacuo to give the sub-title compound. The residue was then concentrated in vacuo from toluene (300 mL) and DCM (2×300 mL) to remove any traces of ethanol which may react in the next stage. A total of 217.8 g of the sub-title compound (100% yield) was obtained, accounting for solvent.
[0500] Alternatively, the sub-title compound was prepared by the following method:
[0501] To a 20 L vessel under nitrogen was charged 10% Pd/C (100 g), this was followed by the addition of the product of step (I) above (1003 g), dissolved in DCM (10.3 L). The reaction was stirred under a hydrogen atmosphere overnight, after which NMR analysis indicated complete reaction. The mixture was filtered through celite and washed with DCM (3×1 L). This product thereby obtained was used directly in the next step (mesylation reaction) without further purification (to give an 86% yield over steps (II) and (III)).
[0502] .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 4.23 (q, 2H), 4.13 (s, 2H), 3.68-3.77 (m, 6H), 3.59-3.63 (m, 2H), 2.51-2.57 (br m, 1H), 1.28 (t, 3H).
(II) Ethyl 2-[2-(2-methylsulfonyloxyethoxy)ethoxy]acetate
[0503] To a 10 L flask under nitrogen was added the product of step (II) above (219 g, 1.139 mol), DCM (4.4 L) and triethylamine (326 mL, 2.349 mol). The solution turned yellow on addition of the triethylamine. The solution was cooled to 0-5° C. and methanesulfonyl chloride (108.5 mL, 1.4 mol) was added dropwise. The reaction was allowed to warm to 8° C., at which point TLC of the reaction mixture indicated complete reaction. The reaction was concentrated in vacuo. The residue was partitioned between ethyl acetate (4.4 L) and water (2 L). The organics were separated and washed with sat. aqueous NaHCO.sub.3 (2 L) and brine (2 L). The aqueous phase was back extracted with ethyl acetate (1 L). The combined organics were dried, filtered and concentrated in vacuo to give the sub-title compound as a red oil (297 g, 97%).
(IV) Ethyl 2-(2-(2-(3-methoxy-5-nitrophenoxy)ethoxy)ethoxy)acetate
[0504] To a 5 L flange flask under nitrogen was added 3-methoxy-5-nitrophenol (126.8 g, 0.749 mol), potassium carbonate (288 g, 2.086 mol), DMF (2536 mL) and the product of step (III) above (198.4 g active, 0.7348 mol). The reaction was heated to 60° C. overnight. LC indicated 95.8% product and 1.45% starting material (3-methoxy-5-nitrophenol). The reaction was cooled to rt and the reaction mixture transferred to a 20 L flask. To the mixture was added TBME (10 L) and water (6 L). The organics were separated and washed with sat. aqueous NaHCO.sub.3 (6 L) and sat. brine (6 L) before drying, filtering and concentrating in vacuo to yield a total of 243 g of the sub-title compound, accounting for solvent (95% yield). LC indicated a purity of 97.7% (254 nm).
(V) Ethyl-2-(2-(2-(3-amino-5-methoxyphenoxy)ethoxy)ethoxy)acetate
[0505] To a 5 L hydrogenation vessel was charged 5% Pd/C (48.6 g), the product of step (IV) above (243 g, 0.708 mol) and ethanol (2.5 L). The vessel was purged with nitrogen three times and then stirred under a hydrogen atmosphere at 5 bar (0.5 MPa) (purged three times with hydrogen) for 6 h. LC indicated complete reaction. The mixture was filtered and washed with ethanol (1200 mL). The organics were then concentrated in vacuo. The residue was then concentrated from heptane (2×500 mL). This gave 212 g (96% yield) of the sub-title compound, for which LC indicated a purity of 98.1% (254 nm). .sup.1H NMR indicated a purity of >95%.
(VI) Ethyl 2-(2-(2-(3-((4-((4-((tert-butoxycarbonyl)amino)naphthalen-1-yl)oxy)pyridin-2-yl)-amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetate
[0506] To a 5 L flange flask under nitrogen was added the product of step (V) above (190 g, 0.6065 mol), tert-butyl (4-((2-chloropyridin-4-yl)oxy)naphthalen-1-yl)carbamate (see, for example, WO 2014/162126; 246.9 g, 0.6658 mol), DMF (3.4 L) and potassium carbonate (272 g, 1.97 mol). The reaction was vacuum degassed three times and released to nitrogen each time. The resulting slurry was heated to 40° C. and then Brettphos G3 Pd (13.66 g, 0.015 mol) was added. The mixture was then heated to 70° C. and stirred for 30 mins. LC indicated <1% of the product of step (V) above remaining. This was confirmed by NMR. The reaction was cooled to rt and filtered, then the solid was washed with DMF (700 mL). The filtrate was then concentrated in vacuo. The residue was dissolved in ethyl acetate (6 L) and washed with sat. brine (2×4 L). The organics were then dried, filtered and concentrated in vacuo. This gave 393 g (100% yield, accounting for solvent) of the sub-title compound, for which NMR indicated a purity of ˜95% and LC indicated a purity of 94.5% (254 nm).
[0507] Alternatively, the sub-title compound was prepared by the following method:
[0508] To a 50 L vessel under nitrogen was charged the product of step (V) above (967 g) and THF (17425 mL). This was followed by tert-butyl (4-((2-chloropyridin-4-yl)oxy)naphthalen-1-yl)carbamate (see, for example, WO 2014/162126; 1254 g) and potassium carbonate (1387 g). The vessel was vacuum degassed (×3) and released to nitrogen (×3). The reaction was then charged with Pd-173 (39.2 g). The reaction was heated to reflux overnight, after which LC analysis indicated trace starting material. The reaction was cooled to 60° C. and further Pd-173 (6.13 g) was added. The reaction was heated to reflux for 1 h, after which LC analysis indicated complete reaction. The reaction mixture was filtered and the residue washed with THF (9.2 L). The filtrate was concentrated in vacuo and the residue concentrated from ethyl acetate and heptane to remove the residual THF. The material was then purified via chromatography (12 kg silica), eluting with 50% ethyl acetate:heptane (60 L), 70% ethyl acetate:heptane (60 L) and 85% ethyl acetate:heptane (20 L). This gave 1896 g (95% yield, accounting for solvent (EtOAc)) of the sub-title compound, for which NMR indicated a purity of >95%, excluding solvent, and LC indicated a purity of 97.4%.
[0509] .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 8.06 (d, 1H), 7.96 (d, 2H), 7.80-7.88 (br d, 1H), 7.48-7.60 (m, 2H), 7.20 (d, 1H), 6.98 (br s, 1H), 6.61 (s, 1H), 6.38-6.41 (m, 4H), 6.13 (m, 1H), 4.10-4.24 (m, 4H), 3.90-3.94 (m, 2H), 3.77-3.83 (m, 6H), 3.68 (s, 3H), 1.57 (s, 9H), 1.24-1.30 (m, 3H).
(VII) Ethyl 2-(2-(2-(3-((4-((4-aminonaphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxy)-phenoxy)ethoxy) ethoxy)acetate
[0510] To a 5 L flange flask under nitrogen was added the product of step (VI) above (393 g, 0.6067 mol), DCM (1.742 L) and TFA (350 mL, 4.57 mol). The reaction was heated to 30° C. overnight, after which LC analysis indicated 18% starting material remaining. TFA (100 mL) was added to the reaction, which was then heated to reflux for 2 h. LC indicated 1.2% starting material remaining. The reaction was cooled to rt and concentrated in vacuo. The residue was azeotroped with toluene (1 L). The residue was diluted with ethyl acetate (3 L) and treated carefully with sat aqueous NaHCO.sub.3 (4 L), at which point off-gassing was observed. The organics were separated and washed with more sat. aqueous NaHCO.sub.3 (2 L). The combined aqueous phase was basic as required. The aqueous phase was extracted with ethyl acetate (1 L). The combined organics were dried, filtered and concentrated in vacuo. The residue was subjected to chromatography (4.5 kg silica), eluting with DCM to 5% MeOH:DCM. The product-containing fractions were combined and concentrated in vacuo. A total of 292 g of the sub-title compound (accounting for DCM) was obtained (80% yield), for which .sup.1H NMR analysis indicated >95% purity and LC indicated a purity of 98.5% (254 nm).
(VIII) Ethyl 2-(2-(2-(3-methoxy-5-((4-((4-(phenoxycarbonyl)amino)naphthalen-1-yl)oxy)pyridin-2-yl)amino)phenoxy)ethoxy)ethoxy)acetate
[0511] To a 50 L vessel was charged the product of step (VII) above (1478 g of that product contained ˜10% THF) and THF (20.825 L). This was followed by the addition of NaHCO.sub.3 (339.7 g). The mixture was cooled to −10° C. and charged with phenyl chloroformate (338.5 mL). The reaction was stirred for 1 h, after which LC indicated 97.5% product and 0.9% starting material. The reaction was then warmed to rt, at which point LC indicated 98% product and 0.32% starting material. The reaction mixture was filtered and washed with THF (2.5 L). The residue was concentrated to 2.6 kg before being dissolved in ethyl acetate:THF (2.9 L:262 mL) and then added by vacuum transfer to a 50 L vessel containing heptane (26.715 L). This led to precipitation of the product. The mixture was stirred for 2 h and filtered. The solids were then dried under vacuum at 40° C. overnight. A total of 1843 g (102% yield, accounting for solvent) of the sub-title compound was obtained, for which NMR indicated a purity of >95%, excluding solvents, and LC indicated a purity of 95.9%.
[0512] .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO) δ: 10.00-10.40 (br, 2H), 8.28 (d, 1H), 8.01 (d, 1H), 7.83 (d, 1H), 7.77 (d, 1H), 7.60-7.71 (m, 2H), 7.39-7.50 (m, 3H), 7.22-7.28 (m, 3H), 6.78 (dd, 1H), 6.56 (s, 1H), 6.49 (s, 1H), 6.24-6.29 (m, 2H), 4.02-4.09 (4H), 3.96-3.99 (m, 2H), 3.62-3.69 (m, 5H), 3.50-3.58 (m, 4H), 1.14 (t, 3H).
(IX) Ethyl 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamidophenyl)-ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetate
[0513] To a 10 L flask under nitrogen was added the product of step (VII) above (262.8 g, 0.4799 mol), dissolved in iPrOAc (3.15 L). The mixture was heated to 40° C. and phenyl N-(5-(tert-butyl)-2-methoxy-3-((methylsulfonyl)amino)phenyl)carbamate (see, for example, WO 2014/162126; 197.1 g, 0.5022 mol) was added. The mixture was further heated to 50° C. to give a solution which was subsequently treated with triethylamine (13.14 mL, 0.094 mol) and the reaction heated to 68° C. overnight. The reaction was cooled to rt and concentrated in vacuo. A portion of the residue (38 g) was purified via chromatography eluting with 25% EtOAc:DCM to EtOAc. The product containing fractions were concentrated in vacuo, and LC analysis (at 254 nm) indicated a purity of 98.6% for the remainder. This material was combined with the bulk and purified on silica (10 kg) eluting with 25% EtOAc:DCM (80 L), DCM (20 L), 1% MeOH:DCM (20 L), 3% MeOH:DCM (20 L) and 5% MeOH:DCM (50 L). The product containing fractions were combined and concentrated in vacuo. This gave 375 g of material with a LC purity of 95.4% and NMR purity of 90%. This material further purified via chromatography (10 kg silica). The column was eluted with 1% MeOH:DCM (60 L), 1.5% MeOH:DCM (20 L), 2% MeOH:DCM (20 L), 2.5% MeOH:DCM (20 L), 3% MeOH:DCM (40 L) then 5% MeOH:DCM (40 L). The purest fractions were combined and concentrated in vacuo to give 324 g of the sub-title compound, for which LC analysis (at 254 nm) indicated a purity of 98.9% and NMR indicated a purity of ˜95%.
[0514] Alternatively, the sub-title compound was prepared by the following method:
[0515] The product of step (VIII) above (1902 g) and THF (19.02 L) were charged to a reaction vessel. The reaction was then charged with N-(3-amino-5-(tert-butyl)-2-methoxyphenyl)methanesulfonamide (see, for example, Cirillo, P. F. et al., WO 2002/083628, 24 Oct. 2002; 815 g) and triethylamine (380.4 mL). The reaction was heated to reflux overnight, after which LC analysis indicated complete reaction (86% product and 0.4% starting material). The reaction was cooled to rt and filtered to remove triethylamine hydrochloride. The solids were washed with THF (3.8 L). The filtrate was split into 3 equal portions and concentrated. The portions were then concentrated from 40% ethyl acetate:heptane (3 L) to remove the majority of THF, which would affect column chromatography. Each of the three portions was purified via chromatography (10 kg silica per portion, with the crude material loaded on to the column with 2 L of DCM), eluting with 75% ethyl acetate:heptane (20 L), 80% ethyl acetate:heptane (120 L) and then 85% ethyl acetate:heptane (40 L). This gave material with a 98.0% purity by LC and a purity by NMR analysis of >95%, excluding solvents. The sub-title compound was isolated in 83% yield (687 g).
[0516] .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO) δ: 9.38 (s, 1H), 9.15 (s, 1H), 8.92 (s, 1H), 8.88 (s, 1H), 8.29 (d, 1H), 8.19 (d, 1H), 8.09-8.13 (m, 2H), 7.86 (d, 1H), 7.70 (t, 1H), 7.60 (t, 1H), 7.38 (d, 1H), 7.02 (d, 1H), 6.90 (s, 1H), 6.79 (s, 1H), 6.57 (dd, 1H), 6.02-6.08 (m, 2H), 4.09-4.13 (m, 4H), 3.96-3.99 (m, 2H), 3.80 (s, 3H), 3.69-3.72 (m, 2H), 3.58-3.65 (m, 7H), 3.10 (s, 3H), 1.27 (s, 9H), 1.18 (t, 3H).
(X) 2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid
[0517] To a 10 L flask under nitrogen was added the product of step (IX) above (317 g, 0.374 mol), THF (2.54 L) and methanol (950 mL). This was followed by the addition of 2 M NaOH (633 mL, 1.266 mol), at which point a small exotherm was noted. The reaction was stirred for 1 h. LC analysis indicated complete reaction. To the reaction was added acetic acid (633 mL), which again caused a small exotherm to be noted. The reaction mixture was then concentrated in vacuo to give a viscous oil. Water (3.2 L) was added and the mixture stirred for 20 mins. Initially an oily solid stuck to side of flask, this was scraped from the side of the vessel with a spatula, the solid became a mobile, flocculent solid. The solid was filtered and washed with water (500 mL) and heptane (1.5 L). The solid was then dried overnight under vacuum at 50° C., before being dissolved in 10% methanol:DCM and subjected to chromatography (6 kg silica) eluting with 10% methanol:DCM (60 L), 20% methanol:DCM (60 L) then methanol. The cleanest fractions were combined and concentrated in vacuo to give a viscous oil. The residue was concentrated from THF (2×2 L) to give a foamy solid. The solid (297 g) contained 8.55% THF and 2.29% AcOH. The material was slurried in water (900 mL) overnight twice and filtered to give 268 g of the title compound (262 g, accounting for solvent, 85% yield) with a purity of 98.2% by LC analysis and a purity of >95% by NMR. The material contained 2.11% THF and 0.26% AcOH.
Example 4
[0518] The following compounds are prepared by methods analogous to those described above.
(a) 2-(2-(2-((3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenyl)sulfonyl)ethoxy)ethoxy)acetic acid
[0519] ##STR00058##
(b) 2-(2-(2-((3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyPhenyi)sulfinyl)ethoxy)ethoxy)acetic acid
[0520] ##STR00059##
(c) 2-(2-(2-((3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonyl)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenyl)sulfonyl)ethoxy)ethoxy)acetic acid
[0521] ##STR00060##
(d) 2-(2-(2-((3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfinyl)phenyl) ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenyl)sulfonyl)ethoxy)ethoxy)acetic acid
[0522] ##STR00061##
(e) 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-(trifluoromethyl)phenoxy)ethoxy)ethoxy)acetic acid
[0523] ##STR00062##
(f) 6-((2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamide)phenyl) ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)methyl)pyridazine-3-carboxylic acid
[0524] ##STR00063##
(g) 5-((2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl) ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-(methoxyphenoxy)ethoxy)methyl)-1,2,4-oxadiazole-3-carboxylic acid
[0525] ##STR00064##
(h) 2-(2-(2-(3-((4-((4-(3-(5=(tert-Butyl)-2-(methoxy-3-(methylsulfonamido)phenyl) ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid
[0526] ##STR00065##
(i) 1-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)cyclopropane-1-carboxylic acid
[0527] ##STR00066##
(j) 4-((2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl) ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)methyl)thiophene-2-carboxylic acid
[0528] ##STR00067##
(k) 1-((2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)methyl)-3-methyl-1H-pyrazole-4-carboxylic acid
[0529] ##STR00068##
(l) 2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl) ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-ethylphenoxy)ethoxy)ethoxy)acetic acid
[0530] ##STR00069##
Example 5
2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-2-(methoxy-d3)-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid
[0531] ##STR00070##
(i) 5-(tert-butyl)-2-(methoxy-d3)-1,3-dinitrobenzene
[0532] A mixture of 4-(tert-butyl)-2,6-dinitrophenol (5 g, 20.81 mmol), caesium carbonate (13.56 g, 41.6 mmol) and iodomethane-d3 (1.6 mL, 25.7 mmol) in DMF (50 mL) was stirred at rt for 4 days then partitioned between ether (300 mL) and water (300 mL). The organic layer was separated, washed with water (200 mL), dried (MgSO.sub.4), filtered and evaporated under reduced pressure to afford the sub-title compound (4.3 g) as a yellow solid.
[0533] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.04 (s, 2H), 1.40 (s, 9H).
(ii) 5-(tert-Butyl)-2-(methoxy-d3)-3-nitroaniline
[0534] 10% Pd/C (500 mg, Type 39, 50% w/w paste with water) was added to a solution of the product from step (i) above (4.25 g, 16.52 mmol) and cyclohexene (2.5 mL, 24.68 mmol) in EtOH (70 mL). The reaction mixture was heated at 70° C. for 1 h then a further portion of cyclohexene (5 mL) was added. After heating for 1 h, a third portion of cyclohexene (5 mL) was added, heated for 2 h then the reaction mixture cooled and filtered through celite. The filtrate was evaporated under reduced pressure and the residue dissolved in EtOAc/ether (300 mL, 1/1), washed with 0.2M aq HCl (2×150 mL), brine (200 mL), dried (MgSO.sub.4), filtered and evaporated under reduced pressure to afford the sub-title compound (3.43 g) as a yellow oil.
[0535] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.27 (d, 1H), 7.05 (d, 1H), 1.31 (s, 9H).
[0536] m/z 228 (M+H).sup.+ (ES.sup.+)
(iii) N-(5-(tert-Butyl)-2-(methoxy-d3)-3-nitrophenyl)methanesulfonamide
[0537] To a stirred solution of the product from step (ii) above (3.42 g, 15.05 mmol) in DCM (25 mL) at 0-5° C., was added pyridine (7 mL, 87 mmol) then MsCl (1.9 mL, 24.38 mmol). The mixture was warmed to rt and stirred for 3 days. The mixture was poured into 1 M HCl (200 mL) and extracted with DCM (200 mL). The organic phase was washed with 1 M HCl (100 mL) and brine (100 mL), then dried (MgSO.sub.4), filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel (120 g column, 0-40% EtOAc/isohexane) to afford the sub-title compound (3.9 g) as a solid.
[0538] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.88 (s, 1H), 7.66 (s, 1H), 7.06 (s, 1H), 3.09 (s, 3H), 1.36 (s, 9H).
(iv) N-(3-Amino-5-(tert-butyl)-2-(methoxy-d3)phenyl)methanesulfonamide
[0539] A mixture of the product from step (iii) above (3.85 g, 12.61 mmol) and 10% Pd—C (500 mg) in EtOH (40 mL) was hydrogenated at 5 bar for 4 h. The mixture was filtered through celite, washing with EtOAc. The filtrate was evaporated under reduced pressure to give a solid that was triturated with ether/isohexane. The solid was filtered and dried to afford the sub-title compound (2.92 g) as a white solid.
[0540] .sup.1H NMR (400 MHz, DMSO-d6) δ 8.70 (s, 1H), 6.58 (s, 2H), 4.91 (s, 2H), 3.00 (s, 3H), 1.20 (s, 9H).
[0541] m/z 276 (M+H).sup.+ (ES.sup.+)
(v) Phenyl (5-(tert-butyl)-2-(methoxy)-d3)-3-(methylsulfonamido)phenylcarbamate
[0542] Phenyl chloroformate (470 μL, 3.75 mmol) was added to a mixture of the product from step (iv) above (1 g, 3.63 mmol) and NaHCO.sub.3 (0.610 g, 7.26 mmol) in DCM (20 mL) and THF (10 mL). The mixture was stirred for 20 h then THF (10 mL) was added followed by phenyl chloroformate (150 μL). The mixture was stirred for 5 h then partitioned between DCM (100 mL) and water (50 mL). The organic layer was separated, dried (MgSO.sub.4), filtered and evaporated under reduced pressure. The residue was triturated with ether/isohexane, filtered and dried to afford the sub-title compound (1.415 g, 3.54 mmol, 98% yield) as a white solid.
[0543] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.01 (bs, 1H), 7.46-7.42 (m, 2H), 7.35 (bs, 1H), 7.32-7.27 (m, 2H), 7.25-7.21 (m, 2H), 6.78 (s, 1H), 3.11 (s, 3H), 1.32 (s, 9H).
[0544] m/z 396 (M+H).sup.+ (ES.sup.+)
(vi) Ethyl 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-(methoxy-d3)-3-(methylsulfonamido)phenyl)-ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxy) phenoxy)ethoxy)ethoxy)acetate
[0545] Ethyl 2-(2-(2-(3-((4-((4-aminonaphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)-ethoxy)ethoxy)acetate (see Example 3(ii) above; 200 mg, 0.365 mmol) and the product from step (v) above (152 mg, 0.383 mmol) were dissolved in iPrOAc (3 mL, 25.6 mmol) and NEt.sub.3 (10.1 μL, 0.073 mmol) added. The mixture was stirred at 75° C. for 16 h and concentrated in vacuo. Crude LCMS showed the sub-title compound to be the major component.
[0546] m/z 849.3 (M+H).sup.+ (ES.sup.+)
[0547] The crude product was purified by chromatography on silica gel (12 g column, 0-5% MeOH/DCM) to afford a white solid (213 mg) that was used directly in step (ii) below.
(vii) 2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-2-(methoxy-d3)-3-(methylsulfonamidophenyl) ureido)-naphthalen-1-yloxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid
[0548] A stirred solution of the product from step (vi) above (213 mg, 0.251 mmol) in THF (10 mL) and EtOH (4 mL) was treated with NaOH (2M aq.) solution (0.452 mL, 0.903 mmol) and stirred at rt overnight. the mixture was treated with AcOH (0.5 mL, 8.73 mmol) and concentrated in vacuo. The residue was triturated with water (10 mL) and filtered. The filtrate was treated with formic acid (0.2 mL) and left to precipitate for 48 h then filtered. The combined solids were taken on to purification.
[0549] A total of 208 mg crude product was purified by chromatography (RP Flash C18, 26 g column, 15-50% MeCN/10 mM Ammonium Bicarbonate) to afford the title compound (128 mg) as a light pink solid.
[0550] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.39 (s, 1H), 9.14 (s, 1H), 8.92 (s, 1H), 8.88 (s, 1H) 8.30 (d, 1H), 8.19 (d, 1H), 8.11 (dd, 2H), 7.87 (dd, 1H), 7.70 (ddd, 1H), 7.61 (ddd, 1H), 7.39 (d, 1H), 7.03 (d, 1H), 6.91 (t, 1H), 6.79 (t, 1H), 6.58 (dd, 1H), 6.08 (d, 1H), 6.04 (t, 1H), 4.05-3.95 (m, 4H), 3.71 (dd, 2H), 3.66 (s, 3H), 3.60 (s, 4H), 3.10 (s, 3H), 1.27 (s, 9H).
[0551] m/z 821.3 (M+H).sup.+ (ES.sup.+)
Example 6
2-(2-(2-(3-((4-((4-(3-(5-tert-Butyl-2-methoxy-3-(methylsulfonamidophenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)-N-(methylsulfonyl)acetamide
[0552] ##STR00071##
[0553] DIPEA (71.1 μL, 0.407 mmol) was added to a solution of 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid (see Example 3 above; 111 mg, 0.136 mmol) and methanesulfonamide (19.36 mg, 0.204 mmol) in dry DMF (2 mL) at rt, followed by HATU (77 mg, 0.204 mmol). The resulting yellow coloured solution was stirred at rt for 3 h. Further portions of methanesulfonamide (19.36 mg, 0.204 mmol), DIPEA (71.1 μL, 0.407 mmol) and HATU (77 mg, 0.204 mmol) were added to the reaction and the resulting solution stirred at rt for 1.5 h. The reaction was then partioned between EtOAc (10 mL) and water (10 mL). The aqueous layer was extracted with EtOAc (2×10 mL). The organic layers were combined, dried (MgSO.sub.4) and concentrated in vacuo. The crude product was purified by chromatography (RP Flash C18, 12 g column, 15-50% MeCN/10 mM Ammonium Bicarbonate). The product-rich fractions were combined, the pH adjusted to 4 with formic acid and the solvent removed in vacuo The resulting solid was dried at 40° C. under vacuum overnight to afford the title compound (11 mg) as a white solid.
[0554] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.40 (s, 1H), 8.92 (s, 1H), 8.87 (s, 1H), 8.30 (d, 1H), 8.19 (d, 2H), 8.12 (d, 1H), 8.10 (d, 1H), 7.87 (d, 1H), 7.70 (ddd, 1H), 7.61 (dd, 1H), 7.38 (d, 1H), 7.02 (d, 1H), 6.90-6.75 (m, 2H), 6.57 (dd, 1H), 6.09 (d, 1H), 6.05 (t, 1H), 3.96 (dd, 2H), 3.81 (s, 3H), 3.71-3.66 (m, 7H), 3.56 (s, 3H), 3.10 (s, 3H), 2.74 (s, 3H), 1.27 (s, 9H).
[0555] m/z 895.5 (M+H).sup.+ (ES.sup.+)
Example 7
2-(2-(2-(3-(4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylcarbamoyl)phenyl)ureido) naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid
[0556] ##STR00072##
(i) Ethyl 2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl-2-methoxy)-3-(methylcarbamoyl)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetate
[0557] Ethyl 2-(2-(2-(3-((4-((4-aminonaphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)-ethoxy)ethoxy)acetate (see Example 3(ii) above; 125 mg, 0.228 mmol) was dissolved in iPrOAc (3 mL) at 50° C., and phenyl (5-(tert-butyl)-2-methoxy-3-(methylcarbamoyl)phenyl)carbamate (see WO 2014/162126; 85 mg, 0.240 mmol) added to the solution. The resulting mixture was stirred at 50° C. until the mixture became a solution (ca. 5 min) then NEt.sub.3 (6.36 μL, 0.046 mmol) added. The resulting solution was heated to 75° C. (block temperature) and left to stir for 16 h. The reaction was cooled to rt and the solvent removed in vacuo. The crude product was purified by chromatography on silica gel (12 g column, 0-5% MeOH/DCM to afford the sub-title compound (161 mg) as a colourless glass.
[0558] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.47 (s, 1H), 8.88 (d. 2H), 8.44 (d, 1H). 8.29 (d, 1H), 8.17 (q, 1H), 8.14-7.99 (m, 2H), 7.87 (d, 1H), 7.71 (ddd, 1H), 7.61 (ddd, 1H), 7.39 (d, 1H), 7.11 (d, 1H), 6.91 (t, 1H), 6.79 (t, 1H), 6.57 (dd, 1H), 6.09 (d, 1H), 6.04 (t, 1H), 4.16-4.04 (m, 4H), 4.03-3.94 (m, 2H), 3.80 (s, 3H), 3.76-3.68 (m, 2H), 3.65 (s, 3H), 3.64-3.47 (m, 4H), 2.82 (d, 3H), 1.28 (s, 9H), 1.18 (t, 3H).
[0559] m/z 810.6 (M+H).sup.+ (ES.sup.+)
(ii) 2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl-2-methoxy-3-(methylcarbamoyl)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid
[0560] NaOH (2M aq.) (350 μL, 0.700 mmol) was added to a solution of the compound from step (i) (161 mg, 0.199 mmol) in THF (1.6 mL) and MeOH (0.6 mL) and the resulting yellow solution stirred at rt for 3 h. The reaction was acidified with AcOH (82 μL, 1.427 mmol) and concentrated in vacuo. The crude product was purified by chromatography (RP Flash C18 24 g column, 15-75% MeCN/10 mM Ammonium Bicarbonate). The product-rich fractions were combined and the pH adjusted to pH 6 with formic acid. The volatile solvent was removed in vacuo. A precipitate formed and was collected by filtration to afford the title compound (70 mg) as a white solid.
[0561] .sup.1H NMR (400 MHz, DMSO-d6) δ 12.57 (s, 1H), 9.46 (s, 1H), 8.88 (d, 2H), 8.44 (d, 1H), 8.29 (d, 1H), 8.17 (q, 1H), 8.11 (d, 1H), 8.08 (d, 1H), 7.87 (dd, 1H), 7.71 (ddd, 1H), 7.61 (ddd, 1H), 7.39 (d, 1H), 7.11 (d, 1H), 6.90 (t, 1H), 6.79 (t, 1H), 6.57 (dd, 1H), 6.09 (d, 1H), 6.04 (t, 1H), 4.03 (s, 2H), 3.98 (dd, 2H), 3.80 (s, 3H), 3.75-3.68 (m, 2H), 3.65 (s, 3H), 3.63-3.52 (m, 4H), 2.82 (d, 3H), 1.28 (s, 9H).
[0562] m/z 782.0 (M+H).sup.+ (ES.sup.+)
Example 8
2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy)-3-(methylsulfinyl)phenyl ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid
[0563] ##STR00073##
(i) 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy)-3-(methylsulfinyl)phenyl ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid
[0564] Ethyl 2-(2-(2-(3-((4-((4-aminonaphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)-ethoxy)ethoxy)acetate (see Example 3(ii) above; 125 mg, 0.228 mmol) was dissolved in iPrOAc (2 mL) at 50 C, and phenyl (5-(tert-butyl)-2-methoxy-3-(methylsulfinyl)phenyl)carbamate (see WO 2015/092423; 87 mg, 0.240 mmol) added to the solution. The resulting mixture was stirred at 50° C. until the mixture became a solution (ca. 2 min) then NEt.sub.3 (6.36 μL, 0.046 mmol) added. The resulting solution was heated to 75° C. (block temperature) and left to stir for 16 h. The reaction was cooled to rt and the solvent removed in vacuo. The crude product was purified by chromatography on silica gel (12 g column, 0-5% MeOH/DCM) to afford the sub-title compound (172 mg) as a colourless glass.
[0565] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.41 (s, 1H), 8.96 (s, 1H), 8.87 (s, 1H), 8.50 (d, 1H), 8.28 (d, 1H), 8.18-8.05 (m, 2H), 7.89-7.83 (m, 1H), 7.71 (ddd, 1H), 7.61 (ddd, 1H), 7.46-7.31 (m, 2H), 6.91 (t, 1H), 6.79 (t, 1H), 6.58 (dd, 1H), 6.09 (d, 1H), 6.04 (t, 1H), 4.18-4.06 (m, 4H), 4.03-3.93 (m, 2H), 3.87 (s, 3H), 3.76-3.67 (m, 2H), 3.66 (s, 3H), 3.63-3.55 (m, 4H), 2.79 (s, 3H), 1.32 (s, 9H), 1.18 (t, 3H).
[0566] m/z 815.5 (M+H).sup.+ (ES.sup.+)
(ii) 2-(2-(2-(3-((4-((4-(3-(5-(tert-Buty)-2-methoxy-3-(methylsulfinyl)phenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid
[0567] NaOH (2M aq.) (350 μL, 0.700 mmol) was added to a solution of the compound from step (i) above (172 mg, 0.211 mmol) in THF (1.6 mL) and MeOH (0.6 mL) and the resulting yellow solution stirred at rt for 3 h. The reaction was acidified with AcOH (82 μL, 1.427 mmol) and the solvent removed in vacuo. The crude product was purified by chromatography (RP Flash C18 12 g column, 15-50% MeCN/10 mM Ammonium Bicarbonate). The product-rich fractions were combined and the pH adjusted to pH 5 with formic acid. The solvent was removed to afford the title compound (130 mg) as a white solid.
[0568] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.51 (s, 1H), 9.03 (s, 1H), 8.88 (s, 1H), 8.50 (d, 1H), 8.30 (d, 1H), 8.14-8.03 (m, 2H), 7.87 (dd, 1H), 7.70 (ddd, 1H), 7.65-7.54 (m, 1H), 7.39 (d, 1H), 7.36 (d, 1H), 6.84 (s, 1H), 6.77 (t, 1H), 6.58 (dd, 1H), 6.10 (d, 1H), 6.03 (t, 1H), 3.99 (s, 2H), 3.94 (t, 2H), 3.86 (s, 3H), 3.70 (dd, 2H), 3.65 (s, 3H), 3.62-3.55 (m, 4H), 2.79 (s, 3H), 1.32 (s, 9H).
[0569] m/z 787.0 (M+H).sup.+ (ES.sup.+)
Example 9
2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonyl)phenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxphenoxy)ethoxy)ethoxy)acetic acid
[0570] ##STR00074##
(i) Ethyl 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonyl)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetate
[0571] Ethyl 2-(2-(2-(3-((4-((4-aminonaphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)-ethoxy)ethoxy)acetate (see Example 3(ii) above; 125 mg, 0.228 mmol) was dissolved in iPrOAc (2 mL) at 50° C., and phenyl (5-(tert-butyl)-2-methoxy-3-(methylsulfonyl)phenyl)-carbamate (see WO 2015/092423; 90 mg, 0.240 mmol) added to the solution. The resulting mixture was stirred at 50° C. for ca 5 min, then THF (1 mL) was added. The reactants went into solution then NEt.sub.3 (6.36 μL, 0.046 mmol) added. The resulting solution was heated to 75° C. (block temperature) and left to stir for 16 h. The reaction was cooled to rt and the solvent removed in vacuo. The crude product was purified by chromatography on silica gel (12 g column, 0-5% MeOH/DCM) to afford the sub-title compound (151 mg) as a colourless glass.
[0572] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.46 (s, 1H), 9.08 (s, 1H), 8.87 (s, 1H), 8.68 (d, 1H), 8.29 (d, 1H), 8.12 (s, 1H), 8.10 (d, 1H), 7.88 (dt, 1H), 7.72 (ddd, 1H), 7.62 (ddd, 1H), 7.45 (d, 1H), 7.40 (d, 1H), 6.91 (t, 1H), 6.79 (t, 1H), 6.58 (dd, 1H), 6.09 (d, 1H), 6.04 (t, 1H), 4.19-4.06 (m, 4H), 4.04-3.97 (m, 2H), 3.95 (s, 3H), 3.78-3.69 (m, 2H), 3.66 (s, 3H), 3.64-3.55 (m, 4H), 3.35 (s, 3H), 1.32 (s, 9H), 1.18 (t, 3H).
[0573] LCMS m/z 831.5 (M+H)+ (ES+)
(ii) 2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonyl)phenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid
[0574] NaOH (2M aq.) (350 μL, 0.700 mmol) was added to a solution of the compound from step (i) (151 mg, 0.182 mmol) in THF (1.6 mL) and MeOH (0.6 mL) and the resulting yellow solution stirred at rt for 3 h. The reaction was acidified with AcOH (82 μL, 1.427 mmol) and the solvent removed in vacuo. The crude product was purified by chromatography (RP Flash C18 12 g column, 15-50% MeCN/10 mM Ammonium Bicarbonate). The product-rich fractions were combined and adjusted to pH 5 with formic acid. The solvent was removed to afford the title compound (114 mg) as a white solid.
[0575] .sup.1H NMR (400 MHz, DMSO-d6) δ 12.62 (s, 1H), 9.49 (s, 1H), 9.10 (s, 1H), 8.87 (s, 1H), 8.68 (d, 1H), 8.29 (d, 1H), 8.14-8.05 (m, 2H), 7.95-7.80 (m, 1H), 7.72 (ddd, 1H), 7.62 (ddd, J=8.1, 1H), 7.45 (d, 1H), 7.40 (d, 1H), 6.89 (t, 1H), 6.78 (t, 1H), 6.58 (dd, 1H), 6.10 (d, 1H), 6.04 (t, 1H), 4.02 (s, 2H), 4.00-3.92 (m, 5H), 3.76-3.68 (m, 2H), 3.65 (s, 3H), 3.63-3.56 (m, 4H), 3.34 (s, 3H), 1.32 (s, 9H).
[0576] m/z 803.0 (M+H).sup.+ (ES.sup.+)
Example 10
2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-3-(dimethylphosphoryl)-2-methoxyphenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid
[0577] ##STR00075##
(i) Ethyl 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-3-(dimethylphosphoryl)-2-methoxyphenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetate
[0578] Ethyl 2-(2-(2-(3-((4-((4-aminonaphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)-ethoxy)ethoxy)acetate (see Example 3(ii) above; 125 mg, 0.228 mmol) was dissolved in iPrOAc (2 mL) at 50° C., and phenyl (5-(tert-butyl)-3-(dimethylphosphoryl)-2-methoxyphenyl)-carbamate (see WO 2015/092423; 90 mg, 0.240 mmol) added to the solution. The resulting mixture was stirred at 50° C. for ca. 5 min and THF (1 mL) added. NEt.sub.3 (6.36 μL, 0.046 mmol) was added and the resulting mixture was heated to 75° C. (block temperature) and the resulting solution left to stir for 16 h at 75° C. The reaction was cooled to rt and the solvent removed in vacuo. The crude product was purified by chromatography on silica gel (12 g column, 0-10% MeOH/DCM) to afford the sub-title compound (169 mg) as a pale pink glass.
[0579] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.22 (s, 1H), 8.87 (s, 1H), 8.45 (s, 1H), 8.28 (d, 1H), 8.11 (d. 1H), 8.08-8.03 (m, 2H), 7.87 (d, 1H), 7.71 (t, 1H), 7.65-7.57 (m, 1H), 7.43-7.34 (m, 2H), 6.90 (t, 1H), 6.79 (t, 1H), 6.57 (dd, 1H), 6.09 (d, 1H), 6.04 (t, 1H), 4.19-4.05 (m, 4H), 4.03-3.94 (m, 2H), 3.71 (s, 2H), 3.65 (s, 3H), 3.64-3.53 (m, 4H), 2.58 (s, 3H), 1.78 (s, 3H), 1.75 (s, 3H), 1.30 (s, 9H), 1.18 (t, 3H).
[0580] m/z 829.5 (M+H).sup.+ (ES+)
(ii) 2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl-3-(dimethylphosphoryl)-2-methoxyphenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid
[0581] NaOH (2M aq.) (350 μL, 0.700 mmol) was added to a solution of the compound from step (i) above (169 mg, 0.204 mmol) in THF (1.6 mL) and MeOH (0.6 mL) and the resulting solution stirred at rt for 3 h. The reaction was acidified with AcOH (82 μL, 1.427 mmol) and the solvent removed in vacuo. The crude product was purified by chromatography (RP Flash C18 12 g column, 15-50% MeCN/10 mM Ammonium Bicarbonate). The product-rich fractions were combined and the pH adjusted to pH 5 with formic acid. The solvent was removed to afford the title compound (131 mg) as a white solid.
[0582] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.47 (s, 1H), 9.00 (s, 1H), 8.87 (s, 1H), 8.42 (d, 1H), 8.31 (d, 1H), 8.16-8.06 (m, 2H), 7.87 (dd, 1H), 7.70 (ddd, 1H), 7.61 (ddd, 1H), 7.41-7.31 (m, 2H), 6.85 (t, 1H), 6.77 (t, 1H), 6.58 (dd, 1H), 6.09 (d, 1H), 6.03 (t, 1H), 3.99-3.92 (m, 4H), 3.90 (s, 3H), 3.74-3.67 (m, 2H), 3.65 (s, 3H), 3.62-3.53 (m, 4H), 1.76 (s, 3H), 1.73 (s, 3H), 1.31 (s, 9H).
[0583] m/z 801.0 (M+H).sup.+ (ES.sup.+)
Example 11
2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(N-methylmethylsulfonamido)phenyl) ureido) naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid
[0584] ##STR00076##
(i) Ethyl 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(N-methylmethylsulfonamido)phenyl)-ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetate
[0585] Ethyl 2-(2-(2-(3-((4-((4-aminonaphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)-ethoxy)ethoxy)acetate (see Example 3(ii) above; 125 mg, 0.228 mmol) was dissolved in iPrOAc (2 mL) at 50° C., and phenyl (5-(tert-butyl)-2-methoxy-3-(N-methylmethylsulfonamido)-phenyl)carbamate (see WO 2016/051187; 97 mg, 0.240 mmol) added to the solution. The resulting mixture was stirred at 50° C. for ca 5 min, upon which the reactants dissolved. NEt.sub.3 (6.36 μL, 0.046 mmol) added and the resulting solution was heated to 75° C. (block temperature) and the solution was left to stir for 4 h at 75° C. The solvent was removed and the crude product purified by chromatography on silica gel (12 g column, 0-5% MeOH/DCM) to afford the sub-title compound (83 mg) as a pale pink solid.
[0586] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.39 (s, 1H), 8.93 (s, 1H), 8.87 (s, 1H), 8.33 (d, 1H), 8.29 (d, 1H), 8.11 (d, 1H), 8.10 (d, 1H), 7.87 (dd, 1H), 7.70 (ddd, 1H), 7.61 (ddd, 1H), 7.39 (d, 1H), 7.03 (d, 1H), 6.91 (t, 1H), 6.79 (t, 1H), 6.58 (dd, 1H), 6.08 (d, 1H), 6.04 (t, 1H), 4.19-4.03 (m, 4H), 3.98 (dd, 2H), 3.89 (s, 3H), 3.71 (dd, 2H), 3.65 (s, 3H), 3.64-3.56 (m, 4H), 3.25 (s, 3H), 3.15 (s, 3H), 1.29 (s, 9H), 1.18 (t, 3H).
[0587] m/z 860.5 (M+H).sup.+ (ES+)
(ii) 2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(N-methylmethylsulfonamido)phenyl-ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid
[0588] NaOH (2M aq.) (175 μL, 0.350 mmol) was added to a solution of the compound from step (i) above (83 mg, 0,097 mmol) in THF (1.6 mL) and MeOH (0.6 mL) and the resulting yellow solution stirred at rt for 3 h. The reaction was acidified with AcOH (82 μL, 1.427 mmol) and the solvent removed in vacuo. The crude product was purified by chromatography (RP Flash C18 12 g column, 15-50% MeCN/10 mM Ammonium Bicarbonate). The product-rich fractions were combined and the pH adjusted to pH 5 with formic acid. The solvent was removed to yield the title compound (57 mg) as a pale pink solid.
[0589] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.73 (s, 1H), 9.13 (s, 1H), 8.90 (s, 1H), 8.36 (d, 1H), 8.31 (d, 1H), 8.15-8.04 (m, 2H), 7.90-7.82 (m, 1H), 7.67 (ddd, 1H), 7.59 (ddd, 1H), 7.37 (d, 1H), 7.02 (d, 1H), 6.74 (d, 2H), 6.60 (dd, 1H), 6.12 (d, 1H), 6.02 (t, 1H), 3.93-3.82 (m, 5H), 3.78 (s, 2H), 3.72-3.66 (m, 2H), 3.65 (s, 3H), 3.59-3.51 (m, 4H), 3.25 (s, 3H), 3.14 (s, 3H), 1.29 (s, 9H).
[0590] m/z 832.0 (M+H).sup.+ (ES.sup.+)
Example 12
5-((2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl) ureido)naphthalen-1 yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)methyl)furan-3-carboxylic acid
[0591] ##STR00077##
(i) Methyl 5-(2-hydroxyethoxy)methyl)furan-3-carboxylate
[0592] To a stirred solution of dry ethane-1,2-diol (0.259 mL, 4.58 mmol) in DMSO (3 mL) at 0° C. was added tBuOK (141 mg, 1.260 mmol) slowly, portion-wise over 10 min. The resulting solution was further stirred for 30 min at same temperature before adding TBAl (42.3 mg, 0.115 mmol). A homogeneous solution of methyl 5-(chloromethyl)furan-3-carboxylate (200 mg, 1.146 mmol) in DMSO (1 mL) was added dropwise to the above reaction mixture and stirred at rt overnight. 3 mL MeOH was added and the reaction stirred once again overnight. Water (15 mL) was added, the aqueous layer extracted with ethyl acetate (2×15 mL) and dried over anhydrous sodium sulfate. Ethyl acetate was evaporated under reduced pressure. The crude product was purified by chromatography on silica gel (12 g column, 0-5% MeOH/DCM) to afford the sub-title compound (110 mg) as a colourless oil.
[0593] .sup.1H NMR (400 MHz, DMSO-d6) δ 8.37 (d, 1H), 6.75 (d, 1H), 4.63 (t, 1H), 4.45 (s, 2H), 3.77 (s, 3H), 3.54-3.46 (m, 2H), 3.46-3.40 (m, 2H).
[0594] m/z 218.0 (M+NH.sub.4).sup.+ (ES.sup.+)
(ii) Methyl 5-((2-(3-methoxysulfonyl)oxy)ethoxy)methyl)furan-2-carboxylate
[0595] The product from step (i) above (105 mg, 0.525 mmol) was dissolved in 1 mL DCM and NEt.sub.3 (88 μL, 0.629 mmol) and MsCl (45.0 μL, 0.577 mmol) were added. The reaction was stirred at rt for 2 h after which time LCMS indicated the reaction had gone to completion. The reaction was diluted with DCM (10 mL), washed with water (10 mL), passed through a phase separator and concentrated in vacuo to yield the sub-title compound (130 mg) as a yellow oil that gradually hardened to a yellow solid.
[0596] .sup.1H NMR (400 MHz, DMSO-d6) δ 8.39 (d, 1H), 6.79 (d, 1H), 4.56-4.46 (m, 2H), 4.35-4.28 (m, 2H), 3.77 (s, 3H), 3.71-3.63 (m, 2H), 3.17 (s, 3H).
(iii) Methyl 5-((2-(3-methoxy-5-nitrophenoxy)ethoxy)methyl)furan-3-carboxylate
[0597] 3-Methoxy-5-nitrophenol (75 mg, 0.445 mmol), the product from step (ii) (130 mg, 0.467 mmol) and potassium carbonate (184 mg, 1.335 mmol) were suspended in DMF (0.5 mL) and heated to 85° C. (block temperature) overnight. The reaction was cooled, diluted with water (15 mL) and extracted with TBME (3×15 mL). The combined organic layers were washed with water (20 mL), brine (20 mL), dried (MgSO4), filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel (24 g column, 0-50% EtOAc/isohexane) to afford the sub-title compound (124 mg) as a yellow oil that gradually became a pale yellow waxy solid.
[0598] .sup.1H NMR (400 MHz, DMSO-d6) δ 8.38 (d, 1H), 7.34 (d, 2H), 6.98 (t, 1H), 6.78 (d, 1H), 4.53 (d, 2H), 4.28-4.20 (m, 2H), 3.86 (s, 3H), 3.82-3.71 (m, 5H).
[0599] m/z 369.0 (M+NH.sub.4).sup.+ (ES.sup.+)
(iv) Methyl 5-((2-(3-amino-5-methoxyphenoxy)ethoxy)methyl)furan-3-carboxylate
[0600] A solution of the product from step (iii) above (120 mg, 0.342 mmol) in EtOH (20 mL) was hydrogenated in the H-Cube (10% Pd/C, 30×4 mm, Full hydrogen, rt, 1 mL/min). A blockage resulted in the solution being exposed to overpressure for ca. 45 mins. The reaction mixture was concentrated in vacuo to yield an oil that was used directly in step (v). LCMS revealed a 2:3 mixture of the sub-title compound (m/z 322.0 (M+H).sup.+ (ES.sup.+)) and methyl 5-((2-(3-amino-5-methoxyphenoxy)ethoxyethoxy)methyl)tetrahydrofuran-3-carboxylate (m/z 326.0 (M+H).sup.+ (ES.sup.+) (107 mg).
(v) Methyl 5-((2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)methyl)tetrahydrofuran-3-carboxylate (Product A) and
Methyl 5-((2-(3-((4-((4-(3-(5-(tert-butyl-2-methoxy-3-(methylsulfonamido)phenyl) ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino-5-methoxyphenoxy)ethoxy)methylfuran-3-carboxylate (Product B)
[0601] A suspension of the product mixture from step (iv) above (95 mg, 0.296 mmol), N-(5-(tert-butyl)-3-(3-(4-((2-chloropyridin-4-yl)oxy)naphthalen-1-yl)ureido)-2-methoxyphenyl)-methanesulfonamide (see WO 2014/162126; 168 mg, 0.296 mmol), freshly ground potassium carbonate (123 mg, 0.887 mmol), and BrettPhosG3 precatalyst (13.40 mg, 0.015 mmol) in DMF (3 mL) was evacuated and backfilled with nitrogen three times. The reaction was then heated under nitrogen at 85° C. (block temperature) for 16 h. The mixture was cooled, diluted with EtOAc (15 mL), washed with brine and concentrated onto silica gel. Attempted chromatography on silica gel (12 g column, 0-5% (0.7 M Ammonia/MeOH)/DCM) afforded little separation and an impure mixture of products were obtained after trituration with water (3 mL). The product was further purified by chromatography on RP Flash C18 (27 g column, 15-75% MeCN/10 mM Ammonium Bicarbonate) to yield Product A (30 mg) as an off-white solid that was used in Example 13 without further purification.
[0602] m/z 858.1 (approximately 75% purity at 254 nm)
[0603] Further elution of the RP column yielded Product B (20 mg) as an off-white solid that was taken to the next step without further purification.
[0604] m/z 854.1 (approximately 55% purity at 254 nm)
(vi) 5-((2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-ylamino)-5-methoxyphenoxy)ethoxy)methyl)furan-3-carboxylic acid
[0605] Product B of step (v) above (20 mg, 0.023 mmol) was dissolved in THF (2 mL) and MeOH (0.5 mL). NaOH (2M aq.) (129 μL, 0.258 mmol) was added and the mixture stirred at rt for 1.5 h. The mixture was acidified with AcOH (0.25 mL) and concentrated in vacuo. The crude product was purified by preparative HPLC (Waters, Acidic (0.1% Formic acid), Acidic, Waters X-Select Prep-C18, 5 μm, 19×50 mm column, 35-65% MeCN in Water) to afford the title compound (2.4 mg) as an off-white solid.
[0606] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.49 (s, 1H), 8.99 (s, 1H), 8.89 (s, 1H), 8.30 (d, 1H), 8.17 (d, 1H), 8.13-8.05 (m, 3H), 7.85 (dd, 1H), 7.69 (ddd, 1H), 7.62-7.56 (m, 1H), 7.38 (d, 1H), 7.01 (d, 1H), 6.86 (t, 1H), 6.78 (t, 1H), 6.63 (s, 1H), 6.58 (dd, 1H), 6.05 (d, 1H), 6.01 (t, 1H), 4.46 (s, 2H), 4.01-391 (m, 2H), 3.79 (s, 3H), 3.72-3.66 (m, 2H), 3.64 (s, 3H), 3.09 (s, 3H), 1.26 (s, 9H).
[0607] m/z 840.2 (M+H).sup.+ (ES.sup.+)
Example 13
5-((2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-methylsulfonamido)phenylureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)methyl)tetrahydrofuran-3-carboxylic acid
[0608] ##STR00078##
[0609] Product A of Example 12(v) above (30 mg, 0.035 mmol) was dissolved in 0.4 mL THF and 0.1 mL MeOH. NaOH (2M aq.) (192 μL, 0.385 mmol) was added and the mixture stirred at rt for 1.5 h. The reaction was acidified with AcOH (0.25 mL) and concentrated in vacuo. The crude product was purified by preparative HPLC (Waters, Basic (0.1% Ammonium Bicarbonate), Basic, Waters X-Bridge Prep-C18, 5 μm, 19×50 mm column, 35-65% MeCN in Water) to afford the title compound (7 mg) as an off-white solid.
[0610] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.55 (d, 1H), 8.99 (d, 1H), 8.84 (d, 1H), 8.37 (s, 1H), 8.25 (dd, 1H), 8.09 (t, 1H), 8.06-8.00 (m, 2H), 7.78 (dd, 1H), 7.64-7.58 (m, 1H), 7.53 (ddd, 1H), 7.31 (dd, 1H), 6.95 (d, 1H), 6.78 (dt, 1H), 6.72 (q, 1H), 6.51 (ddd, 1H), 5.97 (dt, 2H), 3.90-3.83 (m, 3H), 3.77-3.69 (m, 4H), 3.66-3.60 (m, 3H), 3.57 (s, 3H), 3.40-3.34 (m, 2H), 3.01 (s, 3H), 2.90-2.82 (m, 1H), 2.07-1.99 (m, 1H), 1.66 (ddd, 1H), 1.19 (s, 9H).
[0611] m/z 844.1 (M+H).sup.+
Example 14
2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl-2-methoxy-3-(methylsulfonamido)phenyl) ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)propanoic acid
[0612] ##STR00079##
(i) Ethyl 2-(2-(2-(benzvloxy)ethoxy)ethoxy) propanoate
[0613] 2-(2-(Benzyloxy)ethoxy)ethanol (5 g, 25.5 mmol) was dissolved in DCM (50 mL), passed through a phase separator and concentrated in vacuo. It was then redissolved in dry THF (50 mL, 25.5 mmol) under nitrogen and cooled in an ice bath. NaH (60% in mineral oil, 1.070 g, 26.8 mmol) was added portionwise over 10 min and the resulting suspension stirred for 30 min. Ethyl 2-bromopropanoate (3.73 mL, 28.0 mmol) was added dropwise over 15 min. The reaction was stirred overnight, quenched with sat. NHa.sub.4C (5 mL) and the resulting mixture concentrated directly onto silica gel. The crude product was purified by chromatography on silica gel (80 g column, 0-50% EtOAc/isohexane) to afford the sub-title compound (1.51 g) as a colourless oil.
[0614] .sup.1H NMR (400 MHz, DMSO-d6) δ 7.51-7.09 (m, 5H), 4.50 (s, 2H), 4.11 (qd, 2H), 4.05 (q, 1H), 3.70-3.41 (m, 8H), 1.27 (d, 3H), 1.20 (t, 3H).
(ii) Ethyl 2-(2-(2-hydroxyethoxy)ethoxy)propanoate
[0615] The product from step (i) above (1.5 g, 5.06 mmol) was dissolved in EtOH (60 mL, 5.06 mmol) and hydrogenated over Pd—C (0.539 g, 0.506 mmol) at 1 bar H.sub.2 for 16 h at rt. The reaction was filtered through celite, the solids washed with EtOH (20 mL) and the mixture concentrated in vacuo to yield the sub-title compound (931 mg) as a colourless oil.
[0616] .sup.1H NMR (400 MHz, DMSO-d6) δ 4.56 (t, 1H), 4.12 (qd, 2H), 4.04 (q, 1H), 3.67-3.56 (m, 1H), 3.56-3.45 (m, 5H), 3.42 (dd, 2H), 1.27 (d, 3H), 1.21 (t, 3H).
(iii) Ethyl 2-(2-(2-(3-methoxy-5-nitrophenoxy)ethoxy)ethoxy)propanoate
[0617] The product from step (ii) above (932 mg, 4.52 mmol) was dissolved in DCM (5 mL) and cooled in an ice bath. NEt.sub.3 (756 μL, 5.42 mmol) and MsCl (387 μL, 4.97 mmol) were added sequentially dropwise and the reaction stirred for 1 h in the ice bath. DCM (50 mL) was added and the organic layer washed with brine (2×50 mL), passed through a phase separator and concentrated in vacuo to yield 1.13 g of a yellow oil. 282 mg of this yellow oil, 3-methoxy-5-nitrophenol (160 mg, 0.946 mmol), and potassium carbonate (392 mg, 2.84 mmol) were suspended in DMF (3 mL) and heated to 80° C. overnight. The reaction was cooled and partitioned between TBME (20 mL) and brine (20 mL). The aqueous layer was extracted with TBME (20 mL) and the combined organic layers washed with brine (40 mL), dried (MgSO.sub.4), filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel (12 g column, 0-50% EtOAc/isohexane) to afford the sub-title compound (200 mg) as a yellow oil.
[0618] .sup.1H NMR (400 MHz, DMSO-d6) δ 7.34 (dt, 2H), 7.00 (t, 1H), 4.29-4.18 (m, 2H), 4.12 (ddq, 2H), 4.05 (q, 1H), 3.86 (s, 3H), 3.81-3.74 (m, 2H), 3.66-3.57 (m, 3H), 3.56-3.46 (m, 1H), 1.26 (d, 3H), 1.19 (t, 3H).
(iv) Ethyl 2-(2-(2-(3-amino-5-methoxyphenoxy)ethoxy)ethoxy)propanoate
[0619] To a solution of the product from step (iii) above (200 mg, 0.560 mmol) in EtOH (30 mL) and EtOAc (7 mL) was added Pd/C (5 wt %, type 87 L) (61.3 mg, 0.029 mmol). The resulting suspension was stirred under 3 bar H.sub.2 for 1 h. The reaction mixture was filtered through celite and concentrated in vacuo to yield the sub-title compound (130 mg) as a red oil.
[0620] .sup.1H NMR (400 MHz, DMSO-d6) δ 5.75 (d, 2H), 5.69 (t, 1H), 5.05 (s, 2H), 4.12 (qd, 2H), 4.06 (q, 1H), 4.00-3.91 (m, 2H), 3.73-3.66 (m, 2H), 3.66-3.55 (m, 6H), 3.55-3.49 (m, 1H), 1.28 (d, 3H), 1.20 (t, 3H).
(v) Ethyl 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxphenoxy)ethoxy)ethoxy)propanoate
[0621] A solution of the product from step (iv) above (130 mg, 0.397 mmol), N-(5-(tert-butyl)-3-(3-(4-((2-chloropyridin-4-yl)oxy)naphthalen-1-yl)ureido)-2-methoxyphenyl)methanesulfonamide (see WO 2014/162126; 226 mg, 0.397 mmol), potassium carbonate (165 mg, 1.191 mmol), and BrettPhosG3 precatalyst (18.00 mg, 0.020 mmol) in DMF (3 mL) was degassed with nitrogen for 10 mins. The reaction was heated under nitrogen at 85° C. (block temperature) for 2 h. The reaction was cooled and partitioned between EtOAc (10 mL) and water (10 mL). The aqueous phase was extracted with EtOAc (5 mL). The combined organic phases were washed with brine (5 mL) dried (MgSO.sub.4), filtered and concentrated in vacuo affording a dark brown solid. The crude product was purified by chromatography on silica gel (12 g column, 1-6% MeOH in DCM) to afford the sub-title compound (125 mg) as a pale beige foam.
[0622] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.38 (s, 1H), 9.13 (s, 1H), 8.91 (s, 1H), 8.87 (s, 1H), 8.29 (d, 1H), 8.19 (d, 1H), 8.15-8.07 (m, 2H), 7.87 (dt, 1H), 7.70 (ddd, 1H), 7.61 (ddd, 1H), 7.38 (d, 1H), 7.03 (d, 1H), 6.91 (t, 1H), 6.78 (t, 1H), 6.58 (dd, 1H), 6.08 (d, 1H), 6.04 (t, 1H), 4.11 (qd, 2H), 4.07-4.02 (m, 1H), 3.98 (dd, 2H), 3.81 (s, 3H), 3.75-3.69 (m, 2H), 3.65 (s, 3H), 3.62-3.55 (m, 3H), 3.55-3.47 (m, 1H), 3.10 (s, 3H), 1.31-1.23 (m, 12H), 1.19 (t, 3H).
[0623] m/z 860.1 (M+H).sup.+ (ES.sup.+)
(vi) 2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamidophenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)propanoic acid
[0624] The product from step (vi) above (120 mg, 0.140 mmol) was dissolved in THF (2 mL) and MeOH (0.5 mL). NaOH (2M aq.) (767 μL, 1.535 mmol) was added and the mixture stirred at rt for 16 h. The reaction was acidified with AcOH (0.25 mL) and concentrated in vacuo. The crude product was purified by chromatography on RP Flash C18 (24 g column, 15-75% MeCN/10 mM Ammonium Bicarbonate). The product containing fractions were combined, acidified with formic acid to ca. pH 4, concentrated in vacuo and the resulting precipitate filtered off washing with water (5 mL) to afford the title compound (64 mg) as an off-white solid.
[0625] .sup.1H NMR (400 MHz, DMSO-d6) δ 12.48 (s, 1H), 9.36-9.30 (m, 1H), 9.04 (s, 1H), 8.85 (s, 1H), 8.80 (s, 1H), 8.22 (d, 1H), 8.11 (d, 1H), 8.04 (s, 1H), 8.02 (d, 1H), 7.79 (dd, 1H), 7.63 (ddd, 1H), 7.53 (ddd, 1H), 7.31 (d, 1H), 6.95 (d, 1H), 6.82 (t, 1H), 6.71 (t, 1H), 6.50 (dd, 1H), 6.01 (d, 1H), 5.96 (t, 1H), 3.93-3.82 (m, 3H), 3.73 (s, 3H), 3.67-3.61 (m, 2H), 3.59-3.53 (m, 4H), 3.50 (dd, 2H), 3.44-3.37 (m, 1H), 3.02 (s, 3H), 1.19 (s, 9H), 1.18 (d, 3H).
[0626] m/z 832.1 (M+H).sup.+ (ES.sup.+)
Example 15
2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl-3-(ethylsulfonyl)-2-methoxyphenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenyl)ethoxy)ethoxy)acetic acid
[0627] ##STR00080##
(i) (5-(tert-Butyl)-2-methoxy-3-nitrophenyl)(ethyl)sulfane
[0628] Tert-butyl nitrite (3.18 mL, 26.8 mmol) was added dropwise at rt to a stirred, dark brown solution of 1,2-diethyldisulfane (3.29 mL, 26.8 mmol) and 5-(tert-butyl)-2-methoxy-3-nitroaniline (2 g, 8.92 mmol) in MeCN (50 mL). The reaction was then heated to reflux (block temp 80° C.) and the dark brown solution was stirred at reflux for 2 h. The reaction was then cooled to rt and the solvent evaporated. The dark red residue was azeotroped with toluene (3×25 mL). The crude product was purified by chromatography on silica gel (80 g column, 0-20% MTBE:isohexane) to afford the sub-title compound (1.007 g) as an orange oil. .sup.1H NMR (400 MHz, DMSO-d6) δ 7.64 (d, 1H), 7.53 (d, 1H), 3.84 (s, 3H), 3.09 (q, 2H), 1.31 (s, 9H), 1.28 (t, 3H).
(ii) 5-(tert-Butyl)-1-(ethylsulfonyl)-2-methoxy-3-nitrobenzene
[0629] m-CPBA (1.747 g, 7.80 mmol) was added to a solution of the compound from step (i) above (1 g, 3.71 mmol) in DCM (40 mL) under nitrogen at 0° C. and the resulting orange slurry stirred at 0° C. for 30 min then warmed to rt and stirred at rt for 2.5 h. The reaction was quenched with a solution of sodium thiosulfate (2.348 g, 14.85 mmol) dissolved in water (10 mL) and stirred at rt for 30 min. The layers were diluted with DCM (50 mL) and separated. The organic layer was washed with sat. aq. NaHCO.sub.3 (3×20 mL) and dried (MgSO.sub.4). The solvent was removed to afford a dark red oil. The crude product was purified by chromatography on silica gel (40 g column, 20-100% DCM:heptane) to afford the sub-title compound (935 mg) as a thick orange oil.
[0630] .sup.1H NMR (400 MHz, DMSO-d6) δ 8.32 (d, 1H), 8.06 (d, 1H), 3.94 (s, 3H), 3.49 (q, 2H), 1.34 (s, 9H), 1.15 (t, 3H).
[0631] m/z 302.2 (M+H).sup.+ (ES.sup.+)
(iii) 5-(tert-butyl)-3-(ethylsulfonyl)-2-methoxyaniline
[0632] NH.sub.4Cl (66.4 mg, 1.241 mmol) was added to a slurry of the compound from step (ii) above (935 mg, 3.10 mmol) and iron (1733 mg, 31.0 mmol) in EtOH (20 mL), water (5 mL) and THF (2 mL). The resulting black slurry was heated to reflux for 1 h. The solution was cooled to rt and filtered through celite, washing with EtOAc (2×20 mL). The solvent was removed in vacuo. The crude product was purified by chromatography on silica gel (40 g column, 0-40% EtOAc:isohexane) to afford the sub-title compound (820 mg) as a thick yellow oil.
[0633] m/z 272.3 (M+H)+ (ES+)
(iv) Phenyl (5-(tert-butyl)-3-(ethylsulfonyl)-2-methoxyphenyl) carbamate
[0634] Phenyl chloroformate (417 μL, 3.32 mmol) was added to a slurry of the compound from step (iii) (820 mg, 3.02 mmol) and NaHCO.sub.3 (762 mg, 9.06 mmol) in DCM (8 mL) and THF (2 mL). The resulting slurry was stirred at rt for 18 h. The reaction was diluted with DCM (10 mL) and washed with water (10 mL) and brine (10 mL). The organic layer was concentrated in vacuo to afford a light orange solid. This was triturated with cyclohexane (10 mL) and the resulting solid collected by filtration, washing with cyclohexane (2×2 mL) to afford the sub-title compound (940 mg) as a beige solid.
[0635] m/z 392.3 (M+H)+ (ES+)
(v) Ethyl 2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-3-(ethylsulfonyl)-2-methoxyphenyl) ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetate
[0636] Ethyl 2-(2-(2-(3-((4-((4-aminonaphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)-ethoxy)ethoxy)acetate (see Example 3(ii) above; 100 mg, 0.183 mmol) was dissolved in iPrOAc (2 mL) at 50° C. and the compound from step (iv) above (75 mg, 0.192 mmol) added to the solution. The resulting mixture was stirred at 50° C. for until the carbamate dissolved (ca 5 min) then NEt.sub.3 (5.09 μL, 0.037 mmol) was added and the resulting solution stirred at 75° C. for 4 h. The solvent was removed. The crude product was purified by chromatography on silica gel (12 g column, 0-5% MeOH/DCM). The product was repurified by chromatography (RP Flash C18 12 g column, 15-75% MeCN/10 mM Ammonium Bicarbonate) to afford the sub-title compound (86 mg) as a light pink solid.
[0637] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.49 (s, 1H), 9.07 (s, 1H), 8.88 (s, 1H), 8.70 (d, 1H), 8.29 (d, 1H), 8.14-8.04 (m, 2H), 7.88 (d, 1H), 7.71 (t, 1H), 7.62 (t, 1H), 7.42 (d, 1H), 7.40 (d, 1H), 6.90 (t, 1H), 6.78 (t, 1H), 6.58 (dd, 1H), 6.09 (d, 1H), 6.04 (t, 1H), 4.19-4.06 (m, 4H), 3.98 (t, 2H), 3.94 (s, 3H), 3.75-3.68 (m, 2H), 3.65 (s, 3H), 3.64-3.55 (m, 4H), 3.44 (q, 2H), 1.31 (s, 9H), 1.22-1.12 (m, 6H).
[0638] m/z 845.5 (M+H)+ (ES+)
(vi) 2-(2-(2-(3-((4-(4-(3-(5-(tert-Butyl)-3-(ethylsulfonyl)-2-methoxyphenoxy)ureido naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid
[0639] NaOH (2M aq.) (175 μL, 0.350 mmol) was added to a solution of the compound from step (v) above (86 mg, 0.102 mmol) in THF (1.6 mL) and MeOH (0.6 mL) and the resulting solution stirred at rt overnight. The reaction was quenched with AcOH (24.14 μL, 0.422 mmol) and the solvent removed in vacuo. The crude product was purified by chromatography (RP Flash C18 12 g column, 15-75% MeCN/10 mM Ammonium Bicarbonate). The product-rich fractions were neutralised with formic acid and concentrated to afford the title compound (67 mg) as a light beige solid.
[0640] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.68 (s, 1H), 9.21 (s, 1H), 8.88 (s, 1H), 8.69 (d. 1H), 8.32 (d, 1H), 8.14-8.07 (m, 2H), 7.87 (d, 1H), 7.76-7.66 (m, 1H), 7.66-7.56 (m, 1H), 7.42 (d, 1H), 7.39 (d, 1H), 6.79 (s, 1H), 6.75 (t, 1H), 6.59 (dd 1H), 6.11 (d, 1H), 6.03 (t, 1H), 3.99-3.86 (m, 7H), 3.69 (dd, 2H), 3.65 (s, 3H), 3.58 (q, 4H), 3.44 (q, 2H), 1.31 (s, 9H), 1.15 (q, 3H).
[0641] m/z 817.5 (M+H).sup.+ (ES.sup.+)
Example 16
2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-3-(ethylsulfonamido-2-methoxyphenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid
[0642] ##STR00081##
(i) N-(5-(tert-butyl)-2-methoxy-3-nitrophenvl)ethanesulfonamide
[0643] Ethanesulfonyl chloride (475 μL, 5.02 mmol) was added at 0° C. to a solution of 5-(tert-butyl)-2-methoxy-3-nitroaniline (750 mg, 3.34 mmol) and pyridine (1082 μL, 13.38 mmol) in DCM (10 mL). The resulting solution was stirred at 000° C. for 5 min, then at rt for 16 h. The reaction was washed with 2 M HCl (10 mL) and brine (10 mL) and concentrated in vacuo. The crude product was purified by chromatography on silica gel (12 g column, 0-20% EtOAc/isohexane) to afford the sub-title compound, 915 mg of a yellow oil that solidified on standing. The solid was triturated with ether:isohexane (1:1 ratio, 10 mL) to afford the sub-title compound (686 mg) as a pale yellow solid.
[0644] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.57 (s, 1H), 7.68 (s, 2H), 3.82 (s, 3H), 3.22 (q, 2H), 1.40-1.18 (m, 12H).
[0645] m/z 315 (M−H)−(ES−)
(ii) N-(3-amino-5-(tert-butyl-2-methoxyphenyl) ethanesulfonamide
[0646] 10% Pd/C, 50% Paste in water, Type 39 (46.2 mg, 0.434 mmol) was added to a solution of the compound from step (i) above (686 mg, 2.168 mmol) in EtOH (5 mL) and EtOAc (2 mL) and the resulting slurry stirred under H.sub.2 at 5 bar pressure overnight. The reaction was filtered through celite, washing with EtOAc (50 mL). The solvent was removed in vacuo to afford the sub-title compound (600 mg) as a light pink solid.
[0647] .sup.1H NMR (400 MHz, DMSO-d6) δ 8.67 (s, 1H), 6.61-6.52 (m, 2H), 4.88 (s, 2H), 3.63 (s, 3H), 3.09 (q, 2H), 1.25 (t, 3H), 1.20 (s, 9H).
[0648] m/z 287.3 (M+H)+ (ES+)
(iii) Phenyl (5-(tert-butyl)-3-(ethylsulfonamido)-2-methoxyphenyl)carbamate
[0649] Phenyl chloroformate (289 μL, 2.305 mmol) was added to a slurry of the compound from step (ii) above (600 mg, 2.095 mmol) and NaHCO.sub.3 (528 mg, 6.29 mmol) in DCM (8 mL) and THF (2 mL). The resulting slurry was stirred at rt for 2 h. The reaction was diluted with DCM (10 mL) and washed with water (10 mL) and brine (10 mL). The organic layer was concentrated in vacuo to afford an orange oil that solidified upon standing. This was triturated with cyclohexane (10 mL) and the resulting solid collected by filtration, washing with cyclohexane (2×2 mL) to afford the sub-title compound (788 mg, 1.842 mmol, 88% yield) as a light beige solid.
[0650] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.49 (s, 1H), 9.12 (s, 1H), 7.54 (s, 1H), 7.47-7.36 (m, 2H), 7.30-7.19 (m, 3H), 7.17 (d, 1H), 3.77 (s, 3H), 3.15 (q, 2H), 1.28 (t, 3H), 1.24 (s, 9H).
[0651] m/z 429.4 (M+Na)+(ES+)
(iv) Ethyl 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-3-(ethylsufonamido)-2-methoxyphenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetate
[0652] Ethyl 2-(2-(2-(3-((4-((4-aminonaphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)-ethoxy)ethoxy)acetate (see Example 3(ii) above; 100 mg, 0.183 mmol) was dissolved in iPrOAc (2 mL) at 50° C. and the compound from step (iii) (97 mg, 0.240 mmol) added to the solution. The resulting mixture was stirred at 50° C. until the carbamate dissolved (ca 5 min). NEt.sub.3 (5.09 μL, 0.037 mmol) was added and the resulting solution stirred at 75° C. for 4 h. The solvent was removed in vacuo. Chromatography on silica gel (12 g column, 0-5% MeOH/DCM) did not afford sufficient purity. The crude product was repurified by chromatography (RP Flash C18 12 g column, 15-75% MeCN/10 mM Ammonium Bicarbonate) to afford the sub-title compound (70 mg) as a light pink solid.
[0653] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.37 (s, 1H), 9.12 (s, 1H), 8.90 (s, 1H), 8.87 (s, 1H), 8.29 (d, 1H), 8.16 (d, 1H), 8.12 (d, 1H), 8.10 (s, 1H), 7.91-7.83 (m, 1H), 7.74-7.65 (m, 1H), 7.61 (ddd, 1H), 7.38 (d, 1H), 7.02 (d, 1H), 6.90 (t, 1H), 6.78 (t, 1H), 6.58 (dd, 1H), 6.08 (d, 1H), 6.04 (t, 1H), 4.14-4.06 (m, 4H), 4.02-3.94 (m, 2H), 3.81 (s, 3H), 3.71 (dd, 2H), 3.65 (s, 3H), 3.64-3.56 (m, 4H), 3.21-3.12 (m, 2H), 1.31 (t, 3H), 1.26 (s, 9H), 1.18 (t, 3H).
[0654] m/z 860.5 (M+H)+ (ES+).
(v) 2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-3-(ethylsulfonamido)-2-methoxyphenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid
[0655] NaOH (2M aq.) (40.7 μL, 0.081 mmol) was added to a solution of the compound from step (iv) above (70 mg, 0.081 mmol) in THF (1.6 mL) and MeOH (0.6 mL) and the resulting solution stirred at rt overnight. The reaction was quenched with AcOH (24.1 μL, 0.422 mmol) and the solvent removed in vacuo. The crude product was purified by chromatography (RP Flash C18 12 g column, 15-75% MeCN/10 mM Ammonium Bicarbonate). The product-rich fractions were neutralised with formic acid and concentrated to afford the title compound (68 mg) as a light beige solid.
[0656] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.47 (s, 1H), 8.96 (s, 1H), 8.88 (s, 1H), 8.31 (d, 1H), 8.17 (d, 1H), 8.12 (s, 1H), 8.10 (d, 1H), 7.86 (dd, 1H), 7.69 (ddd, 1H), 7.60 (ddd, 1H), 7.38 (d, 1H), 7.02 (d, 1H), 6.86 (t, 1H), 6.78 (t, 1H), 6.58 (dd, 1H), 6.09 (d, 1H), 6.03 (t, 1H), 4.01-3.90 (m, 4H), 3.81 (s, 3H), 3.73-3.68 (m, 2H), 3.65 (s, 3H), 3.63-3.55 (m, 4H), 3.20-3.13 (m, 2H), 1.31 (t, 3H), 1.26 (s, 9H).
[0657] m/z 832.5 (M+H).sup.+ (ES.sup.+)
Example 17
2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl) isoxazol-3-yl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid
[0658] ##STR00082##
(i) Phenyl (5-(tert-butyl)isoxazol-3-yl)carbamate
[0659] A slurry of phenyl chloroformate (0.492 mL, 3.92 mmol, 5-(tert-butyl)isoxazol-3-amine (0.5 g, 3.57 mmol) and NaHCO.sub.3 (0.899 g, 10.70 mmol) in DCM (8 mL) and THF (2 mL) was stirred at rt for 4 h. The reaction was diluted with DCM (10 mL), washed with water (10 mL), brine (10 mL) and the solvent evaporated to give a colourless oil that was stirred in cyclohexane (10 mL) for 10 min. A white solid formed that was collected by filtration to afford the sub-title compound (674 mg) as a white solid.
[0660] .sup.1H NMR (400 MHz, DMSO-d6) δ 11.16 (s, 1H), 7.47-7.41 (m, 2H), 7.28 (ddt, 1H), 7.25-7.19 (m, 2H), 6.44 (s, 1H), 1.30 (s, 9H).
[0661] m/z 261.3 (M+H).sup.+ (ES.sup.+)
(ii) Ethyl 2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl)isoxazol-3-yl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetate
[0662] Ethyl 2-(2-(2-(3-((4-((4-aminonaphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)-ethoxy)ethoxy)acetate (see Example 3(ii) above; 100 mg, 0.183 mmol) was dissolved in iPrOAc (2 mL) at 50° C. and the product from (i) (62.4 mg, 0.240 mmol) added to the solution and stirred at 50° C. until the carbamate dissolved (ca 5 min). NEt.sub.3 (5.1 μL, 0.037 mmol) was added and the resulting solution stirred at 75° C. for 4 h. The solvent was removed in vacuo. Chromatography on silica gel (12 g column, 0-5% MeOH/DCM) did not afford sufficient purity and the crude product was further purified by chromatography (RP Flash C18 12 g column, 15-75% MeCN/10 mM Ammonium Bicarbonate) to afford the sub-title compound (86 mg) as a white solid.
[0663] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.93 (s, 1H), 9.12 (s, 1H), 8.87 (s, 1H), 8.18 (d, 1H), 8.11 (d, 1H), 8.06 (d, 1H), 7.87 (d, 1H), 7.71 (ddd, 1H), 7.62 (ddd, 1H), 7.39 (d, 1H), 6.90 (t, 1H), 6.78 (t, 1H), 6.57 (dd, 1H), 6.52 (s, 1H), 6.08 (d, 1H), 6.04 (t, 1H), 4.16-4.05 (m, 4H), 4.02-3.95 (m, 2H), 3.71 (dd, 2H), 3.65 (s, 3H), 3.64-3.57 (m, 4H), 1.32 (s, 9H), 1.19 (t, 3H).
[0664] m/z 714.2 (M+H)+ (ES+)
(iii) 2-(2-(2-(3-((4-((4-(3-(5-(tert-Butl)isoxazol-3-yl) ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid
[0665] NaOH (2M aq.) (175 μL, 0.350 mmol) was added to a solution of the compound from step (i) above (86 mg, 0.120 mmol) in THF (1.6 mL) and MeOH (0.6 mL) and the resulting solution stirred at rt overnight. The reaction was quenched with AcOH (24.1 μL, 0.422 mmol) and the solvent removed in vacuo. The crude product was purified by chromatography (RP Flash C18 12 g column, 15-75% MeCN/10 mM Ammonium Bicarbonate). The product-rich fractions were neutralised with formic acid and concentrated to afford the title compound (59 mg) as a white solid.
[0666] .sup.1H NMR (400 MHz, DMSO-d6) δ 10.23 (s, 1H), 9.45 (s, 1H), 8.87 (s, 1H), 8.21 (t, 1H), 8.11 (d, 1H), 8.03 (d. 1H), 7.87 (dd, 1H), 7.69 (ddd, 1H), 7.65-7.55 (m, 1H), 7.37 (d. 1H), 6.77 (s, 1H), 6.73 (t, 1H), 6.60 (dd, 1H), 6.52 (s, 1H), 6.09 (d, 1H), 6.03 (t, 1H), 3.96 (s, 2H), 3.89 (d, 2H), 3.69 (dd, 2H), 3.65 (s, 3H), 3.62-3.54 (m, 4H), 1.32 (s, 9H) m/z 686.5 (M+H).sup.+ (ES.sup.+).
Example 18
N-(5-(tert-Butl-2-methoxy-3-(3-(4-((2-((3-methoxy-5-(2-(2-((5-oxo-2,5-dihydroisoxazol-3-yl)methoxy)ethoxy)ethoxy)phenyl)amino)pyridin-4-yl)oxy)naphthalen-1-yl)ureido)phenyl)-methanesulfonamide
[0667] ##STR00083##
(i) 1-[5-tert-butyl-3-(methanesulfonamido)-2-methoxy-phenyl]-3-[4-[[2-[3-[2-[2-[2-(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-yl)-2-oxo-ethoxy]ethoxy]ethoxy]-5-methoxy-anilino]-4-pyridyl]oxy]-1-naphthyl]urea
[0668] To a solution of 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid (see Example 3 above; 500 mg, 0.611 mmol) in DCM (3 mL) and THF (3 mL) was added DMAP (74.7 mg, 0.611 mmol). The resulting suspension was cooled to 0° C. and DCC (139 mg, 0.672 mmol) added. The resulting suspension was stirred at 0° C. for 10 min, then 2,2-dimethyl-1,3-dioxane-4,6-dione (26.4 mg, 0.183 mmol) added. The resulting suspension was stirred at 0° C. for 10 min then at rt overnight. Further portions of DCC (37.8 mg, 0.183 mmol) and 2,2-dimethyl-1,3-dioxane-4,6-dione (26.4 mg, 0.183 mmol) were added to the yellow suspension and stirring continued for 4 h. The solvent was removed to afford an orange solid that was suspended in cold DCM (3 mL) and filtered (sinter funnel, Grade 1 Whatman paper), washing with cold DCM (3×2 mL). The filtrate chilled and refiltered through a plug of cotton wool and solvent was removed affording a light yellow solid. The crude product was purified by chromatography on silica gel (12 g column, 0-100% EtOAc/isohexane, then 4% MeOH:DCM) to afford the sub-title compound (771 mg) as a white solid.
[0669] m/z 944.6 (M+H).sup.+ (ES.sup.+)
(ii) Ethyl 4-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamidophenyl)ureido-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)-3-oxobutanoate
[0670] The product from step (i) above (403 mg, 0.324 mmol) was slurried in EtOH (50 mL) and heated to reflux for 24 h. The solvent was removed in vacuo to afford a gum that was product was purified by chromatography on silica gel (40 g column, 0-10% MeOH/DCM,) to afford the sub-title compound (200 mg, 0.205 mmol, 63.2% yield) as a colourless gum.
[0671] m/z 886.5 (M+H).sup.+ (ES.sup.+)
(iii) N-(5-(tert-Butyl)-2-methoxy-3-(3-(4-((2-((3-methoxy-5-(2-(2-((5-oxo-2,5-dihydroisoxazol-3-yl)methoxy)ethoxy)ethoxy)phenyl)amino)pyridin-4-yl)oxy)naphthalen-1-yl)ureido)phenyl)-methanesulfonamide
[0672] A suspension of the product from (ii) (100 mg, 0.113 mmol), hydroxylamine hydrochloride (31.3 mg, 0.450 mmol) and sat. aq. sodium bicarbonate (512 μL, 0.563 mmol) in EtOH (2.5 mL) was heated to reflux for 1 h, after which time a homogeneous solution was obtained. The reaction was cooled to rt and concentrated in vacuo. The crude product was purified by chromatography (RP Flash C18 12 g column, 15-75% MeCN/10 mM Ammonium Bicarbonate). The product-rich fractions were combined and the pH adjusted to 7 with formic acid, the organic solvent was evaporated under a flow of N.sub.2, protecting the sample from light. The aqueous solvent was then removed on a rotary evaporator, at 30° C., to afford the title compound (20 mg) as a light pink solid.
[0673] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.58 (s, 1H), 9.02 (s, 1H), 8.94 (s, 1H), 8.32 (d, 1H), 8.18 (d, 1H), 8.12 (s, 1H), 8.10 (d, 1H), 7.86 (dd, 1H), 7.69 (ddd, 1H), 7.60 (ddd, 1H), 7.38 (d, 1H), 7.02 (d, 1H), 6.88 (t, 1H), 6.78 (t, 1H), 6.58 (dd, 1H), 6.07 (d, 1H), 6.03 (t, 1H), 4.06 (s, 2H), 3.95 (dd, 2H), 3.91 (s, 1H), 3.80 (s, 3H), 3.68 (dd, 2H), 3.65 (s, 3H), 3.59-3.52 (m, 2H), 3.52-3.44 (m, 2H), 3.09 (s, 3H), 1.27 (s, 9H).
[0674] m/z 857.5 (M+H).sup.+ (ES.sup.+)
Example 19
2-((2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl) ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethyl)thio)acetic acid
[0675] ##STR00084##
(i) Methyl 2,2,3,3-tetramethyl-4,7-dioxa-10-thia-3-siladodecan-12-oate
[0676] 2,2′-Oxydiethanol (7.05 mL, 80.5 mmol), NEt.sub.3 (8.36 mL, 60 mmol) and DMAP (0.020 g, 0.164 mmol) were dissolved in DCM (100 mL) and cooled in an ice bath. A solution of TBSCl (7.28 g, 48.3 mmol) in DCM (20 mL) was added dropwise over 15 mins and the mixture allowed to warm to room temperature overnight. The organic layer was washed with saturated NaHCO.sub.3 (100 mL), saturated NH.sub.4Cl (100 mL), and then saturated NaCl (100 mL), passed through a phase separator and concentrated in vacuo to yield a colourless oil (8.95 g). To a solution of this oil (2.0 g) and NEt.sub.3 (1.82 mL, 13.07 mmol) in DCM (20 mL) at 0-5° C. was added MsCl (0.611 mL, 7.84 mmol) dropwise. The resulting mixture warmed to rt and stirred for 2 h, diluted with DCM (30 mL) and the organic layer washed with water (50 mL), brine (50 mL), passed through a phase separator and concentrated in vacuo to yield 2.45 g of a light yellow oil. NaH (60% in mineral oil, 0.731 g, 18.27 mmol) was suspended in DMF (10 mL, 129 mmol), cooled in an ice bath and methyl 2-mercaptoacetate (1.515 mL, 16.60 mmol) added dropwise under nitrogen. After 30 min at rt the mixture was cooled in an ice bath and the light yellow oil from above (2.36 g) was added as a solution in DMF (5 mL) and stirred for 2 h. Sat. NH.sub.4Cl(aq) (50 mL) was added and the aqueous layer extracted with EtOAc (2×50 mL). The combined organic layers were washed with brine, dried (Na2SO4), filtered and concentrated in vacuo onto silica gel. The crude product was purified by chromatography on silica gel (80 g column, 0-20% EtOAc/isohexane) to afford the sub-title compound (1.22 g) as a colourless oil.
[0677] .sup.1H NMR (400 MHz, DMSO-d6) δ 3.71-3.65 (m, 2H), 3.63 (s, 3H), 3.59 (t, 2H), 3.47-3.42 (m, 2H), 3.42-3.37 (m, 2H), 2.74 (t, 2H), 0.86 (s, 9H), 0.04 (s, 6H)
(ii) Methyl 2-((2-(2-hydroxyethoxy)ethyl)thio)acetate
[0678] The product from step (i) above (546 mg, 1.770 mmol) was dissolved in AcOH:water (3 mL; 2:1 ratio) and stirred at rt for 1 h. The solvent was then removed in vacuo to yield the sub-title compound (347 mg) as a colourless oil.
[0679] .sup.1H NMR (400 MHz, DMSO-d6) δ 4.57 (t, 1H), 3.60 (s, 3H), 3.53 (t, 2H), 3.48-3.40 (m, 2H), 3.40-3.33 (m, 4H), 2.70 (t, 2H).
(iii) Methyl 2-((2-(2-((methylsulfonyl)oxy)ethoxy)ethyl)thio)acetate
[0680] The product from step (ii) above (344 mg, 1.771 mmol) was dissolved in DCM (5 mL) and cooled in an ice bath. NEt.sub.3 (370 μL, 2.66 mmol) followed by MsCl (166 μL, 2.125 mmol) were added dropwise and the mixture left to warm to rt overnight. The mixture was diluted with DCM (10 mL) and the organic layer washed with 0.1 M HCl (10 mL). The mixture was passed through a phase separator and concentrated in vacuo to yield the sub-title compound (396 mg) as light yellow oil.
[0681] .sup.1H NMR (400 MHz, DMSO-d6) δ 4.32-4.20 (m, 2H), 3.64-3.61 (m, 2H), 3.60 (s, 3H), 3.58 (t, 2H), 3.37 (s, 2H), 3.15 (s, 3H), 2.73 (t, 2H).
(iv) Methyl 2-((2-(2-(3-methoxy-5-nitrophenoxy)ethoxy)ethyl)thio)acetate
[0682] 3-Methoxy-5-nitrophenol (118 mg, 0.699 mmol), the product from step (iii) above (200 mg, 0.734 mmol) and potassium carbonate (290 mg, 2.098 mmol) were suspended/dissolved in DMF (3 mL) and heated to 80° C. overnight. The reaction was cooled and partitioned between TBME (20 mL) and brine (20 mL). The aqueous layer was extracted with TBME (20 mL) and the combined organic layers washed with brine (40 mL), dried (MgSO.sub.4), filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel (12 g column, 0-50% EtOAc/isohexane) to afford the sub-title compound (170 mg) as a yellow oil.
[0683] .sup.1H NMR (400 MHz, DMSO-d6) δ 7.35 (dt, 2H), 7.00 (t, 1H), 4.29-4.15 (m, 2H), 3.86 (s, 3H), 3.79-3.73 (m, 2H), 3.66 (t, 2H), 3.63 (s, 3H), 3.42 (s, 2H), 2.77 (t, 2H).
[0684] m/z 346.0 (M+H).sup.+ (ES.sup.+)
(v) Methyl 2-((2-(2-(3-amino-5-methoxyphenoxy)ethoxy)ethyl)thio)acetate
[0685] The product from step (iv) above (170 mg, 0.492 mmol) was dissolved in EtOH (4 mL, 68.5 mmol) and H.sub.2O (0.5 mL). Iron (165 mg, 2.95 mmol) and NH.sub.4Cl (211 mg, 3.94 mmol) were added and the flask evacuated and backfilled with nitrogen three times. The reaction mixture was heated to 80° C. with vigorous stirring for 2 h. LCMS revealed complete conversion to the sub-title compound. The mixture was cooled, filtered through celite and the solids washed with EtOH (10 mL). The solution was concentrated in vacuo to yield the sub-title as a light yellow oil (120 mg).
[0686] m/z 316.0 (M+H).sup.+ (ES.sup.+)
(vi) Methyl 2-((2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-ureido)naphthalen-1-yl)oxy)pyridin-2-)amino)-5-methoxyphenoxy)ethoxy)ethyl)thio)acetate
[0687] A suspension of the product from step (v) above (120 mg, 0.380 mmol), N-(5-(tert-butyl)-3-(3-(4-((2-chloropyridin-4-yl)oxy)naphthalen-1-yl)ureido)-2-methoxyphenyl)methanesulfonamide (see WO 2014/162126; 217 mg, 0.380 mmol), Pd-175 (7.43 mg, 9.51 μmol) and freshly ground potassium carbonate (158 mg, 1.141 mmol) in DMF (3 mL) was degassed by 3 cycles of evacuation and backfilling with nitrogen. The reaction was heated to 70° C. (block temperature) for 2 h then concentrated in vacuo. The crude product was purified by chromatography on RP Flash C18 (26 g column, 25-100% MeCN/10 mM Ammonium Bicarbonate) to afford the sub-title compound (152 mg) as a colourless, glassy solid.
[0688] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.40 (s, 1H), 9.16 (s, 1H), 8.93 (s, 1H), 8.89 (s, 1H), 8.34-8.26 (m, 1H), 8.19 (d, 1H), 8.12 (d, 1H), 8.10 (d, 1H), 7.86 (dd, 1H), 7.71 (ddd, 1H), 7.61 (ddd, 1H), 7.39 (d, 1H), 7.02 (d, 1H), 6.90 (t, 1H). 6.79 (t, 1H), 6.58 (dd, 1H), 6.07 (d, 1H), 6.03 (t. 1H), 4.02-3.94 (m, 2H), 3.81 (s, 3H), 3.74-3.68 (m, 2H), 3.65 (s, 3H), 3.62 (s, 3H), 3.41 (s, 2H), 3.10 (s, 3H), 2.77 (t, 2H), 1.27 (s, 9H).
[0689] m/z 848.0 (M+H).sup.+ (ES.sup.+)
(vii) 2-((2-(2-(3-((4-((4-(3-(5-(tert-Butyl-2-methoxy-3-methylsulfonamido phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethyl)thio) acetic acid
[0690] The product from step (vi) (150 mg, 0.177 mmol) was dissolved in THF (4 mL). NaOH (2M aq.) (973 μL, 1.946 mmol) was added and the mixture stirred at rt overnight. The reaction was acidified with AcOH (0.25 mL) and concentrated in vacuo. The crude product was purified by chromatography on RP Flash C18 (24 g column, 15-75% MeCN/10 mM Ammonium Bicarbonate). The product containing fractions were combined, acidified with formic acid to ca. pH 4, concentrated in vacuo and the resulting precipitate filtered off, washed with water (5 mL) and dried in vacuo at 50° C. for 24 h to afford the title compound (78 mg) as a white solid.
[0691] .sup.1H NMR (400 MHz, DMSO-d6) δ 12.54 (s, 1H), 9.40 (s, 1H), 9.17 (s, 1H), 8.93 (s, 1H), 8.89 (s, 1H), 8.29 (d, 1H), 8.19 (d, 1H), 8.12 (d, 1H), 8.10 (d, 1H), 7.86 (dd, 1H), 7.71 (ddd, 1H), 7.61 (ddd, 1H), 7.39 (d, 1H), 7.02 (d, 1H), 6.90 (t, 1H), 6.79 (t, 1H), 6.58 (dd, 1H), 6.07 (d, 1H), 6.03 (t, 1H), 4.03-3.93 (m, 2H), 3.80 (s, 3H), 3.74-3.67 (m, 2H), 3.67-3.58 (m, 5H), 3.29 (s, 2H), 3.10 (s, 3H), 2.76 (t, 2H), 1.27 (s, 9H).
[0692] m/z 833.9 (M+H).sup.+ (ES.sup.+)
Example 20
2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl) ureido)-naphthalen-1-oxy)pyridin-2-yl)amino-5-methoxyphenoxy)ethoxy)ethoxy)propanoic acid (Enantiomers 1 and 2)
[0693] ##STR00085##
[0694] The title compound of Example 14 (40 mg, 0.048 mmol) was submitted to preparative chiral HPLC purification (Gilson, Daicel Chirapak IC column, 30% EtOH in 4:1 hexane:DCM (0.2% diethyl amine) to obtain two enantiontiomers: Enantiomer 1 (9.2 mg) and Enantiomer 2 (5.2 mg). The absolute stereochemistry of the two enantiomers is not known.
(a) Enantiomer 1
[0695] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.59 (s, 1H), 9.04 (s, 1H), 8.89 (s, 1H), 8.33 (d, 1H), 8.15 (d, 1H), 8.13-8.07 (m, 2H), 7.86 (dd, 1H), 7.68 (ddd, 1H), 7.59 (ddd, 1H), 7.36 (d, 1H), 7.02 (d, 1H), 6.80-6.71 (m, 2H), 6.58 (dd, 1H), 6.10 (d, 1H), 6.02 (t, 1H), 3.93-3.85 (m, 2H), 3.80 (s, 4H), 3.68 (dd, 2H), 3.64 (s, 3H), 3.55 (t, 2H), 3.46-3.42 (m, 1H), 3.08 (s, 3H), 1.26 (s, 9H), 1.21 (d, 3H).
[0696] m/z 832.1 (M+H).sup.+
[0697] Chiral HPLC (Daicel Chiralpak IC, 5 um, 4.6×250 mm, 45 min method, 1.0 mL/imin, 30% EtOH in DCM/Hexane (1:4) (0.2% DEA) RT=8.9 min, 84% ee @ 254 nm.
(b) Enantiomer 2
[0698] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.55 (s, 1H), 9.02 (s, 1H), 8.89 (s, 1H), 8.33 (d, 1H), 8.16 (d, 1H), 8.12 (d, 1H), 8.10 (d, 1H), 7.88-7.84 (m, 1H), 7.69 (ddd, 1H), 7.60 (ddd, 1H), 7.37 (d, 1H), 7.02 (d, 1H), 6.81 (t, 1H), 6.76 (t, 1H), 6.59 (dd, 1H), 6.10 (d, 1H), 6.03 (t, 1H), 3.92 (t, 2H), 3.84 (d, 4H), 3.74-3.67 (m, 2H), 3.65 (s, 3H), 3.56 (t, 2H), 3.45-3.42 (m, 1H), 3.09 (s, 3H), 1.27 (s, 9H), 1.23 (d, 3H).
[0699] m/z 832.1 (M+H).sup.+ (ES.sup.+)
[0700] Chiral HPLC (Daicel Chiralpak IC, 5 urn, 4.6×250 mm, 45 min method, 1.0 mL/min, 30% EtOH in DCM/Hexane (1:4) (0.2% DEA) RT=11.2 min, 98% ee @ 254 nm.
Example 21
2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl) ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxyethoxy)-2-methylpropanoic acid
[0701] ##STR00086##
(i) Ethyl 2-(2-(2-(benzyloxy)ethoxy)ethoxy)-2-methylpropanoate
[0702] Ethyl 2-(2-(2-(benzyloxy)ethoxy)ethoxy)propanoate (1.1 g, 3.71 mmol) was dissolved in THF (35 mL) and cooled to −78° C. LiHMDS (1 M in THF, 4.08 mL, 4.08 mmol) was added and the reaction stirred for 1 h. MeI (2M in TBME) (2.04 mL, 4.08 mmol) was added and the mixture warmed to rt and stirred overnight. The reaction was quenched with NH.sub.4Cl (2 mL) and concentrated directly onto silica. The crude product was purified by chromatography on silica gel (80 g column, 0-40% EtOAc/isohexane) to afford the sub-title compound (328 mg) as a colourless oil.
[0703] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.31-7.17 (m, 5H), 4.50 (s, 2H), 4.11 (q, 2H), 3.65-3.53 (m, 6H), 3.53-3.48 (m, 2H), 1.36 (s, 6H), 1.21 (t, 3H).
(ii) Ethyl 2-methyl-2-(2-(2-((methylsulfonyl)oxy)ethoxy)ethoxy) propanoate
[0704] The product from step (i) above (390 mg, 1.25 mmol) was dissolved in EtOH (30 mL, 1.257 mmol) and 5 wt % Pd—C (type 87 L, 134 mg, 0.063 mmol) added. The mixture was stirred at rt under 4 bar of H.sub.2 for 16 h. HPLC confirmed consumption of the starting material. The reaction mixture was filtered through celite, washing the solids with EtOH (50 mL) and concentrated in vacuo to yield a colourless oil. The oil was dissolved in dry DCM (10 mL) and cooled in a water ice bath. NEt.sub.3 (192 μL, 1.378 mmol) and MsCl (98 μL, 1.263 mmol) were added and the mixture allowed to warm to rt with stirring overnight. The reaction was diluted with DCM (30 mL), washed with 0.1 M HCl (20 mL) and the aqueous layer further extracted with DCM (10 mL). The combined organic layers were passed through a phase separator and concentrated in vacuo to afford the sub-title compound (313 mg) as a colourless oil.
[0705] .sup.1H NMR (400 MHz, DMSO-d6) δ 4.34-4.27 (m, 2H), 4.10 (q, 2H), 3.71-3.63 (m, 2H), 3.59-3.50 (m, 2H), 3.48-3.40 (m, 2H), 3.18 (s, 3H), 1.32 (s, 6H), 1.19 (t, 3H).
(iii) Ethyl 2-(2-(2-(3-methoxy-5-nitrophenoxy)ethoxy)ethoxy)-2-methylpropanoate
[0706] 3-Methoxy-5-nitrophenol (188 mg, 1.112 mmol), the product from step (ii) above (316 mg, 1.059 mmol) and freshly-ground potassium carbonate (439 mg, 3.18 mmol) were suspended in DMF (3 mL) and heated to 80° C. overnight. The reaction was cooled and partitioned between TBME (20 mL) and brine (20 mL). The aqueous layer was extracted with TBME (20 mL) and the combined organic layers washed with brine (40 mL), dried (MgSO4), filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel (80 g column, 0-40% EtOAc/isohexane) to afford the sub-title compound (346 mg) as a yellow oil. .sup.1H NMR (400 MHz, DMSO-d6) δ 7.35 (dt, 2H), 7.00 (t, 1H), 4.26-4.19 (m, 2H), 4.11 (q, 2H), 3.86 (s, 3H), 3.80-3.75 (m, 2H), 3.58 (dd, 2H), 3.47 (dd, 2H), 1.33 (s, 6H), 1.19 (t, 3H).
(iv) Ethyl 2-(2-(2-(3-amino-5-methoxyphenoxy)ethoxy)ethoxy)-2-methylpropanoate
[0707] The product from step (iii) above (335 mg, 0.902 mmol) was dissolved in EtOH (5 mL) and Pd/C (5 wt % type 87 L, 28.8 mg, 0.014 mmol) added. The reaction was stirred under 1 bar H.sub.2 for 2 h. The reaction was filtered through celite, washing with EtOH (50 mL) and concentrated in vacuo. The crude product was purified by chromatography on silica gel (12 g column, 0-5% (0.7 M Ammonia/MeOH)/DCM) to afford the sub-title compound (226 mg) as a red oil.
[0708] .sup.1H NMR (400 MHz, DMSO-d6) δ 5.78-5.72 (m, 2H), 5.68 (t, 1H), 5.07 (s, 2H), 4.11 (q, 2H), 3.99-3.87 (m, 2H), 3.75-3.65 (m, 2H), 3.62 (s, 3H), 3.58-3.53 (m, 2H), 3.48-3.43 (m, 2H), 1.33 (s, 6H), 1.19 (d, 3H).
[0709] m/z 342.1 (M+H).sup.+ (ES.sup.+)
(v) Ethyl 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-)oxy)pyridin-2-yl)amino-5-methoxyphenoxy)ethoxyethoxy)-2-methylpropanoate
[0710] A suspension of the product from step (iv) above (210 mg, 0.615 mmol), N-(5-(tert-butyl)-3-(3-(4-((2-chloropyridin-4-yl)oxy)naphthalen-1-yl)ureido)-2-methoxyphenyl)methanesulfonamide (see WO 2014/162126; 350 mg, 0.615 mmol) and freshly ground potassium carbonate (255 mg, 1.845 mmol) in DMF (3 mL) was degassed by 3 cycles of evacuation and backfilling with nitrogen. The mixture was heated to 40° C. for 5 min and BrettPhosG3 precatalyst (13.94 mg, 0.015 mmol) was added as a solution in DMF (1 mL). The flask was evacuated and backfilled with nitrogen and then heated to 75° C. (block temperature) for 4 h. The reaction was cooled to rt and a further portion of freshly ground potassium carbonate (255 mg, 1.845 mmol) and Pd-173 (14 mg) was added. The reaction was degassed by evacuation and backfilling with nitrogen 3 times and heated to 75° C. (block temperature) for 12 h, diluted with DCM (50 mL), washed with brine (50 mL), passed through a phase separator and concentrated in vacuo. The crude product was purified by chromatography on RP Flash C18 (40 g column, 25-100% MeCN/10 mM Ammonium Bicarbonate) to afford the sub-title compound (77 mg) as a brown solid.
[0711] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.39 (s, 1H), 8.90 (d, 2H), 8.31-8.28 (m, 1H), 8.19 (d, 1H), 8.14-8.07 (m, 2H), 7.87 (dd, 1H), 7.71 (ddd, 1H), 7.61 (ddd, 1H), 7.39 (d, 1H), 7.02 (d, 1H), 6.91 (t, 1H), 6.78 (t, 1H), 6.58 (dd, 1H), 6.07 (d, 1H), 6.03 (t, 1H), 4.10 (q, 2H), 4.01-3.95 (m, 2H), 3.81 (s, 3H), 3.74-3.69 (m, 2H), 3.65 (s, 3H), 3.55 (dd, 2H), 3.46 (dd, 2H), 3.10 (s, 3H), 1.32 (s, 6H), 1.27 (s, 9H), 1.19 (t, 3H).
[0712] m/z 848.0 (M+H).sup.+ (ES.sup.+)
(vi) 2-(2-(2-(3-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxyethoxy)-2-methylpropanoic acid
[0713] The product from step (v) (77 mg, 0.088 mmol) was dissolved in THF (2 mL) and MeOH (0.5 mL). NaOH (2M aq.) (485 μL, 0.969 mmol) was added and the mixture stirred at rt overnight. The reaction was acidified with AcOH (0.25 mL) and concentrated in vacuo. The crude product was purified by chromatography on RP Flash C18 (24 g column, 15-75% MeCN/10 mM Ammonium Bicarbonate). The product containing fractions were combined, acidified with formic acid to ca. pH 4 and concentrated in vacuo. The solid was then redissolved in the minimum amount of EtOH (ca. 1 mL) and water (0.5 mL) added dropwise to crash out the white solid. The vial was then centrifuged at 2000 rpm for 2 min and the supernatant decanted to yield the title compound (40 mg) as a white solid.
[0714] .sup.1H NMR (400 MHz, DMSO-d6) δ 12.50 (s, 1H), 9.38 (s, 1H), 9.11 (s, 1H), 8.91 (s, 1H), 8.87 (s, 1H), 8.29 (d, 1H), 8.18 (d, 1H), 8.14-8.07 (m, 2H), 7.87 (dd, 1H), 7.70 (ddd, 1H), 7.61 (ddd, 1H), 7.38 (d, 1H), 7.02 (d, 1H), 6.90 (t, 1H), 6.78 (t, 1H), 6.57 (dd, 1H), 6.08 (d, 1H), 6.03 (t, 1H), 3.97 (dd, 2H), 3.81 (s, 3H), 3.71 (dd, 2H), 3.65 (s, 3H), 3.56 (dd, 2H), 3.48 (dd, 2H), 3.10 (s, 3H), 1.31 (s, 6H), 1.27 (s, 9H).
[0715] m/z 846.1 (M+H).sup.+ (ES.sup.+)
Example 22
1-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethyl-1H-pyrazole-4-carboxylic acid
[0716] ##STR00087##
(i) Methyl 1-(2-(2-(benzyloxy)ethoxy)ethyl)-1H-pyrazole-4-carboxylate
[0717] Potassium carbonate (822 mg, 5.95 mmol) was added to a solution of 2-(2-(benzyloxy)ethoxy)ethyl methanesulfonate (598 mg, 2.181 mmol) and methyl 1H-pyrazole-4-carboxylate (250 mg, 1.982 mmol) in DMF (15 mL) and heated to 60° C. for 2 days. The reaction was cooled to rt, diluted with EtOAc (50 mL) and washed sequentially with water (30 mL), sat. aq. NaHCO.sub.3 (30 mL) and 20% v/v brine (30 mL). The organic layer was dried (MgSO.sub.4) and concentrated in vacuo. The crude product was purified by chromatography on silica gel (12 g column, 0-100% EtOAc/isohexane) to afford the sub-title compound (472 mg) as a colourless oil.
[0718] .sup.1H NMR (400 MHz, DMSO-d6) δ 8.33 (d, 1H), 7.87 (d, 1H), 7.38-7.30 (m, 2H), 7.30-7.24 (m, 3H), 4.44 (s, 2H), 4.32 (t, 2H), 3.80 (t, 2H), 3.72 (s, 3H), 3.58-3.53 (m, 2H), 3.53-3.48 (m, 2H).
[0719] m/z 305.1 (M+H).sup.+ (ES.sup.+)
(ii) Methyl 1-(2-(2-(hydroxyethoxy)ethyl)-1H-pyrazole-4-carboxylate
[0720] Pd/C 10% in 50% paste in water (Type 39) (33.0 mg, 0.310 mmol) was added to a solution of the product from step (i) above (472 mg, 1.551 mmol) in EtOH (4 mL) and the resulting slurry stirred under H.sub.2 at 1 bar pressure overnight. The reaction was filtered through celite, washing with EtOAc (2×20 mL) and the solvent removed to afford the sub-title compound (326 mg) as a colourless oil.
[0721] .sup.1H NMR (400 MHz, DMSO-d6) δ 8.33 (d, 1H), 7.87 (d, 1H), 4.59 (t, 1H), 4.31 (t, 2H), 3.78 (t, 2H), 3.74 (s, 3H), 3.48-3.42 (m, 2H), 3.42-3.37 (m, 2H).
(iii) Methyl 1-(2-(2-(methylsulfonyl)oxy)ethoxy)ethyl)-1H-pyrazole-4-carboxylate
[0722] MsCl (142 μL, 1.826 mmol) was added to a solution of the product from step (ii) above (326 mg, 1.522 mmol) in DCM (10 mL) and TEA (424 μL, 3.04 mmol) at 0° C., and the resulting solution stirred at rt overnight. A second aliquot of NEt.sub.3 (424 μL, 3.04 mmol) and MsCl (142 μL, 1.826 mmol) was added at rt, and the reaction stirred at rt for further 3 h. The reaction was diluted with DCM (50 mL) and washed with 20% v/v brine (50 mL). The solvent was removed to afford an orange oil. The crude product was purified by chromatography on silica gel (12 g column, 0-10% MeOH/DCM) to afford the sub-title compound (431 mg) as an orange oil.
[0723] .sup.1H NMR (400 MHz, DMSO-d6) δ 8.33 (d, 1H), 7.88 (d, 1H), 4.33 (t, 2H), 4.27 (m, 2H), 3.83 (t, 2H), 3.74 (s, 3H), 3.65 (dt, 2H), 3.12 (s, 3H).
[0724] m/z 293.3 (M+H).sup.+ (ES.sup.+)
(iv) Methyl 1-(2-(2-(3-methoxy-5-nitrophenoxy)ethoxy)ethyl)-1H-pyrazole-4-carboxylate
[0725] Potassium carbonate (611 mg, 4.42 mmol) was added to a solution of 3-methoxy-5-nitrophenol (274 mg, 1.622 mmol) and the product from step (iii) above (431 mg, 1.474 mmol) in DMF (15 mL) and heated to 60° C. for 2 days. The reaction was cooled to rt, diluted with EtOAc (50 mL) and washed sequentially with water (30 mL), sat. aq. NaHCO.sub.3 (30 mL) and 20% v/v brine (30 mL). The organic layer was dried (MgSO.sub.4) and concentrated in vacuo. The crude product was purified by chromatography on silica gel (12 g column, 0-100% EtOAc/isohexane) to afford the sub-title compound (387 mg) as a colourless oil.
[0726] .sup.1H NMR (400 MHz, DMSO-d6) δ 8.30 (d, 1H), 7.83 (d, 1H), 7.31 (dt, 2H), 6.94 (t, 1H), 4.33 (t, 2H), 4.22-4.11 (m, 2H), 3.90-3.82 (m, 5H), 3.78-3.72 (m, 2H), 3.71 (s, 3H).
[0727] m/z 366.4 (M+H).sup.+ (ES.sup.+)
(v) Methyl 1-(2-(2-(3-amino-5-methoxyphenoxy)ethoxy)ethyl)-H-pyrazole-4-carboxylate
[0728] A slurry of the product from step (iv) above (378 mg, 1.035 mmol), NH.sub.4Cl (22.14 mg, 0.414 mmol) and iron (578 mg, 10.35 mmol) in EtOH (20 mL), water (2 mL) and THF (3 mL) was heated to reflux for 1 h. The reaction was cooled to rt and filtered through celite, washing with EtOAc (2×20 mL). The solvent was removed in vacuo. The crude product was purified by chromatography on silica gel (12 g column, 0-10% MeOH/DCM) to afford the sub-title compound (300 mg) as a yellow oil.
[0729] .sup.1H NMR (400 MHz, DMSO-d6) δ 8.34 (d, 1H), 7.88 (d, 1H), 5.75 (t, 1H), 5.73 (t, 1H), 5.66 (t, 1H), 5.06 (s, 2H), 4.33 (t, 2H), 3.94-3.87 (m, 2H), 3.84 (t, 2H), 3.73 (s, 3H), 3.70-3.64 (m, 2H), 3.62 (s, 3H).
[0730] m/z 336.3 (M+H).sup.+ (ES.sup.+)
(vi) Methyl 1-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino-5-methoxyphenoxy)ethoxy)ethyl-1H-pyrazole-4-carboxylate
[0731] A suspension of N-(5-(tert-butyl)-3-(3-(4-((2-chloropyridin-4-yl)oxy)naphthalen-1-yl)ureido)-2-methoxyphenyl)methanesulfonamide (see WO 2014/162126; 239 mg, 0.420 mmol), the product from step (v) above (141 mg, 0.420 mmol), freshly ground potassium carbonate (174 mg, 1.261 mmol) in DMF (2 mL) in a vial was evacuated and back-filled with nitrogen 3 times. The mixture was heated to 40° C. and Pd 175 (9.52 mg, 10.51 μmol) added. The reaction mixture was heated at 75° C. for 2 h. The reaction was then cooled and filtered. The filtrate was partitioned between EtOAc (50 mL) and 20% v/v brine (50 mL). The organic layer was dried (MgSO.sub.4), filtered and concentrated. The crude product was purified by chromatography on silica gel (12 g column, 0-10% MeOH/DCM) to afford the sub-title compound (240 mg) as a light brown solid.
[0732] m/z 868.1 (M+H).sup.+ (ES.sup.+)
(vii) Methyl 1-(2-(2-(3-((4-((4-(3-(5-(tert-butyl-2-methoxy-3-(methylsulfonamido)phenyl)-ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethyl)-1H-pyrazole-4-carboxylate
[0733] NaOH (2M aq.) (415 μL, 0.830 mmol) was added to a solution of the product from step (vi) above (240 mg, 0.277 mmol) in THF (1.6 mL) and MeOH (0.6 mL) and the resulting solution stirred at rt overnight. Further NaOH (2M aq.) (415 μL, 0.830 mmol) was added and the reaction stirred at rt for 2 h. The reaction was quenched with AcOH (24.14 μL, 0.422 mmol) and the solvent removed in vacuo. The crude product was purified by chromatography (RP Flash C18 12 g column, 15-75% MeCN/10 mM Ammonium Bicarbonate) to afford the title compound (130 mg, 0.149 mmol, 54.0% yield) as a white solid.
[0734] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.40 (s, 1H), 8.92 (s, 1H), 8.86 (s, 1H), 8.29 (d, 1H), 8.23 (s, 1H), 8.18 (d, 1H), 8.11 (d, 1H), 8.09 (d, 1H), 7.87 (dd, 1H), 7.79 (d, 1H), 7.70 (ddd, 1H), 7.61 (ddd, 1H), 7.38 (d, 1H), 7.02 (d, 1H), 6.87 (t, 1H), 6.80 (t, 1H), 6.57 (dd, 1H), 6.08 (d, 1H), 6.02 (t, 1H), 4.32 (t, 2H), 4.00-3.89 (m, 2H), 3.84 (t, 2H), 3.81 (s, 3H), 3.73-3.67 (m, 2H), 3.65 (s, 3H), 3.10 (s, 3H), 1.27 (s, 9H).
[0735] m/z 854.5 (M+H).sup.+ (ES.sup.+)
Example 23
N-(3-(3-(4-((2-((3-(2-(2-(1H-Tetrazol-5-yl)methoxyethoxy)ethoxy)-5-methoxyphenyl)amino)-pyridin-4-yl)oxy)naphthalen-1-yl)ureido)-5-(tert-butyl)-2-methoxyphenyl)methanesulfonamide
[0736] ##STR00088##
(i) 2,2,3,3-Tetramethyl-4,7,10-trioxa-3-siladodecane-12-nitrile
[0737] A solution of 2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)ethanol (2 g, 6.35 mmol) in THF (10 mL) was added dropwise to a slurry of NaH (60% in oil, 0.356 g, 8.89 mmol) in dry THF (40 mL) at 0° C. under nitrogen. The resulting slurry was stirred at 0° C. for 20 min, and a solution of bromoacetonitrile (0.44 mL, 6.35 mmol) in dry THF (10 mL) added to the reaction mixture. The resulting dark coloured solution was allowed to warm to rt and stirred at rt overnight. The reaction was quenched with MeOH (0.5 mL) and diluted with 20% v/v brine (20 mL) and EtOAc (50 mL). The layers were separated and the aqueous layer extracted with EtOAc (3×20 mL). The combined organic extractions were dried (MgSO.sub.4) and concentrated in vacuo. The crude product was purified by chromatography on silica gel (24 g column, 0-100% EtOAc/isohexane) to afford the sub-title compound (840 mg) as a thick brown oil.
[0738] .sup.1H NMR (400 MHz, DMSO-d6) δ 4.44 (s, 2H), 3.65 (dd, 2H), 3.62-3.56 (m, 2H), 3.56-3.49 (m, 2H), 3.41 (dd, 2H), 0.82 (s, 9H), 0.00 (s, 6H).
[0739] m/z 282 (M+Na).sup.+ (ES.sup.+)
(ii) 2-(2-(2-(Hydroxyethoxy)ethoxy)acetonitrile
[0740] The compound from step (i) above (728 mg, 2.81 mmol) was stirred in AcOH (5 mL) and water (2.5 mL) for 1 h. The solvent was removed and the residue azeotroped with toluene (3×5 mL) to afford the sub-title compound (409 mg) as a thick colourless oil.
[0741] .sup.1H NMR (400 MHz, DMSO-d6) δ 4.53 (bs, 1H), 4.44 (s, 2H), 3.65-3.57 (m, 2H), 3.57-3.49 (m, 2H), 3.49-3.41 (m, 2H), 3.41-3.35 (m, 2H).
(iii) 2-(2-Cyanomethoxy)ethoxy)ethyl methanesulfonate
[0742] MsCl (322 μL, 4.14 mmol) was added to a solution of the compound from step (ii) above (429 mg, 2.96 mmol) and NEt.sub.3 (824 μL, 5.91 mmol) in DCM (15 mL) at 0° C., then the resulting solution stirred at rt overnight. The reaction was diluted with DCM (50 mL) and washed with 20% v/v brine (100 mL). The organic layer was passed through a hydrophobic frit and concentrated. The crude product was purified by chromatography on silica gel (12 g column, 0-100% EtOAc/isohexane) to afford the sub-title compound (640 mg) as a colourless oil.
[0743] .sup.1H NMR (400 MHz, DMSO-d6) 4.50 (s, 2H), 4.39-4.25 (m, 2H), 3.72-3.64 (m, 4H), 3.64-3.59 (m, 2H), 3.19 (s, 3H).
(iv) 2-(2-(2-(3-Methoxy-5-nitrophenoxy)ethoxy)ethoxy)acetonitrile
[0744] Freshly ground potassium carbonate (1189 mg, 8.60 mmol) was added to a solution of 3-methoxy-5-nitrophenol (533 mg, 3.15 mmol) and the compound from step (iii) above (640 mg, 2.87 mmol) in DMF (15 mL) and heated to 60° C. overnight. The reaction was cooled to rt, diluted with EtOAc (100 mL) and washed with 20% v/v brine (100 mL). The organic layer was dried (MgSO.sub.4) and concentrated in vacuo. The crude product was purified by chromatography on silica gel (40 g column, 0-100% EtOAc/isohexane) to afford the sub-title compound (743 mg) as a yellow oil.
[0745] .sup.1H NMR (400 MHz, DMSO-d6) δ 7.35 (t, 1H), 7.33 (t, 1H), 7.00 (t, 1H), 4.49 (s, 2H), 4.26-4.18 (m, 2H), 3.86 (s, 3H), 3.81-3.72 (m, 2H), 3.71-3.59 (m, 4H).
[0746] m/z 319.2 (M+Na).sup.+ (ES.sup.+)
(v) 2-(2-(2-(3-Amino-5-methoxyphenoxy)ethoxy)ethoxy)acetonitrile
[0747] Iron (754 mg, 13.5 mmol) followed by ammonium chloride (28.9 mg, 0.54 mmol) was added to a solution of the compound from step (iv) above (400 mg, 1.35 mmol) in EtOH (13 mL), THF (5 mL) and water (2 mL) and the resulting slurry heated to reflux for 2 h. The reaction was cooled and filtered through celite, washing with EtOAc (2×20 mL). The solvent was removed in vacuo. The crude product was purified by chromatography on silica gel (24 g column, 0-100% EtOAc/isohexane) to afford the sub-title compound (263 mg, 0.938 mmol, 69.5% yield) as a thick light yellow oil.
[0748] .sup.1H NMR (400 MHz, DMSO-d6) δ 5.75 (d, 2H), 5.69 (t, 1H), 5.05 (s, 2H), 4.49 (s, 2H), 4.00-3.91 (m, 2H), 3.71-3.63 (m, 6H), 3.63 (s, 3H).
[0749] m/z 267.3 (M+H).sup.+ (ES.sup.+)
(vi) N-(5-(tert-Butyl)-3-(3-(4-((2-((3-(2-(2-(cyanomethoxy)ethoxy)ethoxy)-5-methoxyphenyl)-amino)pyridin-4-yl)oxy)naphthalen-1-yl)ureido)-2-methoxyphenyl)methanesulfonamide
[0750] A suspension of N-(5-(tert-butyl)-3-(3-(4-((2-chloropyridin-4-yl)oxy)naphthalen-1-yl)ureido)-2-methoxyphenyl)methanesulfonamide (see WO 2014/162126; 310 mg, 0.54 mmol), the compound from step (v) above (145 mg, 0.54 mmol) and freshly ground potassium carbonate (226 mg, 1.63 mmol) in DMF (3 mL) was evacuated, back filling with nitrogen 3 times. The mixture was heated under nitrogen to 40° C. and Pd-175 (10.6 mg, 0.014 mmol) added. The reaction mixture was heated at 75° C. for 2 h, cooled and filtered. The filtrate was partitioned between EtOAc (50 mL) and 20% v/v brine (50 mL). The organic layer was dried (MgSO.sub.4), filtered and concentrated. The crude product was purified by chromatography on silica gel (12 g column, 0-10% MeOH/DCM) to afford a thick brown oil. The material was dissolved in DCM (5 mL) and washed with 20% v/v brine (10 mL). The solvent was removed to afford the sub-title compound (362 mg) as a beige solid.
[0751] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.38 (s, 1H), 9.13 (s, 1H), 8.91 (s, 1H), 8.87 (s, 1H), 8.29 (d, 1H), 8.18 (d, 1H), 8.12 (d, 1H), 8.10 (d, 1H), 7.87 (dd, 1H), 7.70 (ddd, 1H), 7.61 (ddd, 1H), 7.38 (d, 1H), 7.02 (d, 1H), 6.91 (t, 1H), 6.79 (t, 1H), 6.58 (dd. 1H), 6.08 (d, 1H), 6.04 (t, 1H), 4.48 (s, 2H), 4.04-3.92 (m, 2H), 3.81 (s, 3H), 3.72 (dt, 2H), 3.69-3.58 (m, 7H), 3.10 (s, 3H), 1.27 (s, 9H).
[0752] m/z 799.4 (M+H).sup.+ (ES.sup.+)
(vii) N-(3-(3-(4-((2-((3-(2-(2-((1H-Tetrazol-5-yl)methoxy)ethoxy)ethoxy)-5-methoxyphenyl)-amino)pyridin-4-yl)oxy)naphthalen-1-yl)ureido)-5-(tert-butyl)-2-methoxyphenyl)-methanesulfonamide
[0753] TMSN.sub.3 (49.8 μL, 0.37 mmol) was added to a slurry of the compound from step (vi) above (100 mg, 0.12 mmol) and dibutyltin oxide (31 mg, 0.12 mmol) in toluene (2 mL) and the resulting slurry heated to 100° C. for 1 h. The reaction was cooled to rt and quenched with MeOH (2 mL). The solvent was removed and the crude product purified by chromatography (RP Flash C18, 12 g column, 15-75% MeCN/10 mM Ammonium Bicarbonate). The product-rich fractions were combined and the pH adjusted to 7 with formic acid. The solvent was removed to afford an off-white solid. This was dissolved in EtOH (1 mL) and precipitated with water (4 mL). The resulting precipitate was collected by filtration to afford the title compound (31 mg) as an off-white solid.
[0754] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.38 (s, 1H), 9.13 (s, 1H), 8.91 (s, 1H), 8.87 (s, 1H), 8.29 (d, 1H), 8.19 (d, 1H), 8.11 (d, 1H), 8.10 (d, 1H), 7.87 (dd, 1H), 7.70 (ddd, 1H), 7.61 (ddd, 1H), 7.38 (d, 1H), 7.02 (d, 1H), 6.90 (t, 1H), 6.78 (t, 1H), 6.57 (dd. 1H), 6.08 (d, 1H), 6.03 (t, 1H), 4.84 (s, 2H), 4.03-3.93 (m, 2H), 3.81 (s, 3H), 3.74-3.68 (m, 2H), 3.68-3.58 (m, 7H), 3.10 (s, 3H), 1.27 (s, 9H).
[0755] m/z 842.1 (M+H).sup.+ (ES.sup.+)
Example 24
2-(2-(3-((4-((4-(3-(5-(tert-Buty-2-methoxy-3-(methylsulfonamido)phenyl) ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)acetic acid
[0756] ##STR00089##
(i) Ethyl 2-(2-(3-methoxy-5-nitrophenoxy)ethoxy)acetate
[0757] Potassium carbonate (1.226 g, 8.87 mmol) was added to a slurry of 3-methoxy-5-nitrophenol (0.5 g, 2.96 mmol), ethyl 2-(2-chloroethoxy)acetate (0.440 mL, 2.96 mmol) and sodium iodide (0.222 g, 1.478 mmol) in DMF (20 mL) and stirred at 70° C. for 2 h. The heating was increased to 90° C. and the reaction left to stir for 24 h. The reaction was cooled to rt and partitioned between EtOAc (100 mL) and 20% v/v brine (100 mL), the organic layer washed with 20% v/v brine (50 mL), dried (MgSO.sub.4) and concentrated in vacuo. The crude product was purified by chromatography on silica gel (24 g column, 0-50% EtOAc/isohexane). The material obtained was dissolved in EtOAc (50 mL) and washed with NaOH (2 M aq, 2×50 mL). The organic layer was dried (MgSO.sub.4) and concentrated in vacuo to afford the sub-title compound (300 mg) as a light yellow oil.
[0758] .sup.1H NMR (400 MHz, DMSO-d6) δ 7.40-7.30 (m, 2H), 7.00 (t, 1H), 4.30-4.21 (m, 2H), 4.20 (s, 2H), 4.12 (q, 2H), 3.90-3.79 (m, 5H), 1.20 (t, 3H).
[0759] m/z 322.2 (M+Na).sup.+ (ES.sup.+)
(ii) Ethyl 2-(2-(3-amino-5-methoxyphenoxy)ethoxy)acetate
[0760] Iron (560 mg, 10.0 mmol) followed by ammonium chloride (21.4 mg, 0.40 mmol) was added to a solution of the compound from step (i) above (300 mg, 1.00 mmol) in EtOH (13 mL), THF (5 mL) and water (2 mL) and the resulting slurry heated to reflux for 1 h and stirred at rt overnight. The reaction was filtered through celite, washing with EtOAc (2×10 mL) and the filtrate concentrated in vacuo. The crude product was purified by chromatography on silica gel (24 g column, 0-100% EtOAc/isohexane) to afford the sub-title compound (233 mg) as a thick brown oil.
[0761] .sup.1H NMR (400 MHz, DMSO-d6) δ 5.75 (p, 2H), 5.68 (t, 1H), 5.05 (br s, 2H), 4.17 (s, 2H), 4.12 (q, 2H), 4.00-3.94 (m, 2H), 3.83-3.71 (m, 2H), 3.63 (s, 3H), 1.21 (t, 3H).
[0762] m/z 270.3 (M+H).sup.+ (ES.sup.+)
Ethyl 2-(2-(3-((4-((4-(3-(5-tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxyacetate
[0763] A suspension of N-(5-(tert-butyl)-3-(3-(4-((2-chloropyridin-4-yl)oxy)naphthalen-1-yl)ureido)-2-methoxyphenyl)methanesulfonamide (see WO 2014/162126; 211 mg, 0.37 mmol), the compound from step (ii) above (100 mg, 0.37 mmol) and freshly ground potassium carbonate (154 mg, 1.11 mmol) in DMF (3 mL) was evacuated back filling with nitrogen 3 times. The mixture was heated under nitrogen to 40° C. and Pd-175 (7.2 mg, 9.28 μmol) added. The reaction mixture was heated at 75° C. for 2 h. The reaction was then cooled and filtered. The filtrate was partitioned between EtOAc (50 mL) and 20% v/v brine (50 mL). The organic layer was dried (MgSO.sub.4), filtered and concentrated. The crude product was purified by chromatography on silica gel (12 g column, 0-10% MeOH/DCM) to afford the coupling product as a thick brown oil. The material was dissolved in DCM (5 mL) and washed with 20% v/v brine (10 mL). The solvent was removed to afford the sub-title compound (233 mg) as a beige solid.
[0764] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.38 (s, 1H), 9.13 (s, 1H), 8.91 (s, 1H), 8.87 (s, 1H), 8.29 (d, 1H), 8.19 (d, 1H), 8.11 (dd, 2H), 7.87 (dt, 1H), 7.70 (ddd, 1H), 7.61 (ddd, 1H), 7.38 (d, 1H), 7.03 (d, 1H), 6.91 (t, 1H), 6.79 (t, 1H), 6.58 (dd, 1H), 6.08 (d, 1H), 6.03 (t, 1H), 4.18 (s, 2H), 4.11 (q, 2H), 4.06-3.96 (m, 2H), 3.85-3.74 (m, 5H), 3.66 (s, 3H), 3.10 (s, 3H), 1.27 (s, 9H), 1.19 (t, 3H).
[0765] m/z 802.1 (M+H).sup.+ (ES.sup.+)
(iv) 2-(2-(3-((4-((4-(3-(5-tert-Butyl-2-methoxy-3-(methylsulfonamidophenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)acetic acid
[0766] NaOH (2M aq, 462 μL, 0.92 mmol) was added to a solution of the compound from step (iv) above (247 mg) in THF (1.6 mL) and MeOH (0.6 mL) and the resulting solution stirred at rt overnight. The reaction was quenched with AcOH (106 μL, 1.848 mmol) and the solvent removed in vacuo. The crude product was purified by chromatography (RP Flash 018, 12 g column, 15-75% MeCN/10 mM Ammonium Bicarbonate) and product rich fractions combined and the pH adjusted to ca. 7 with formic acid. The solvent was then removed to afford a white solid. This was slurried in hot EtOH (2 mL), then triturated with water (2 mL). The resulting solid was collected by filtration, washing with water (2×1 mL) to afford the title compound (145 mg) as a white solid.
[0767] .sup.1H NMR (400 MHz, DMSO-d6) δ 12.63 (s, 1H), 9.42 (s, 1H), 9.13 (s, 1H), 8.92 (s, 1H), 8.88 (s, 1H), 8.30 (d, 1H), 8.19 (d, 1H), 8.12 (d, 1H), 8.10 (d, 1H), 7.87 (dd, 1H), 7.70 (ddd, 1H), 7.61 (ddd, 1H), 7.38 (d, 1H), 7.02 (d, 1H), 6.89 (s, 1H), 6.78 (t, 1H), 6.58 (dd, 1H), 6.08 (d, 1H), 6.04 (t, 1H), 4.07 (s, 2H), 4.03-3.91 (m, 2H), 3.81 (s, 3H), 3.80-3.72 (m, 2H), 3.66 (s, 3H), 3.10 (s, 3H), 1.27 (s, 9H).
[0768] m/z 774.4 (M+H).sup.+ (ES.sup.+)
Example 25
2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl-2-methoxy-3-(methylsulfonamido)phenyl) ureido)-naphthalen-1-yl)oxy)pyridin-2 amino)-5-methoxyphenoxy)ethoxy) ethoxy)-N—(N,N-dimethylsulfamoyl)acetamide
[0769] ##STR00090##
[0770] CDI (26.2 mg, 0.16 mmol) was added to a solution of 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid (see Example 3 above; 120 mg, 0.14 mmol) in dry DMF (2 mL) at rt and the resulting solution stirred at 50° C. for 1 h. Dimethylsulfamide (36.4 mg, 0.293 mmol) and DBU (44.2 μL, 0.293 mmol) were added to the solution and the reaction stirred at rt overnight. A further portion of dimethylsulfamide (36.4 mg, 0.293 mmol) and DBU (44.2 μL, 0.293 mmol) was added and the reaction stirred at rt for a further 2 h. The reaction was quenched with water (0.1 mL) and the crude reaction solution purified by chromatography (RP Flash C18, 12 g column, 15-50% MeCN/10 mM Ammonium Bicarbonate). The product rich fractions were combined and the volatile solvent removed in vacuo. The pH was then adjusted to 7 with formic acid and the resulting solid collected by filtration, washing with water (2×1 mL), to afford the title compound (48 mg) as a white solid.
[0771] .sup.1H NMR (400 MHz, DMSO-d6) δ 11.26 (s, 1H), 9.38 (s, 1H), 9.13 (s, 1H), 8.91 (s, 1H), 8.87 (s, 1H), 8.29 (d, 1H), 8.19 (d, 1H), 8.12 (d, 1H), 8.10 (s, 1H), 7.87 (dd, 1H), 7.70 (ddd, 1H), 7.61 (ddd, 1H), 7.38 (d, 1H), 7.03 (d, 1H), 6.91 (t, 1H), 6.79 (t, 1H), 6.58 (dd, 1H), 6.08 (d, 1H), 6.04 (t, 1H), 4.06 (s, 2H), 4.02-3.94 (m, 2H), 3.81 (s, 3H), 3.75-3.69 (m, 2H), 3.66 (s, 3H), 3.61 (s, 4H), 3.10 (s, 3H), 2.80 (s, 6H), 1.27 (s, 9H).
[0772] m/z 924.5 (M+H).sup.+ (ES.sup.+)
Example 26
5-((2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamidophenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxphenoxy)ethoxy)methyl)thiophene-2-carboxylic acid
[0773] ##STR00091##
(i) Methyl 5-(hydroxymethyl)thiophene-2-carboxylate
[0774] Methyl 5-formylthiophene-2-carboxylate (311 mg, 1.82 mmol) was dissolved in MeOH (4 mL), cooled in an ice bath and NaBH.sub.4 (68 mg, 1.82 mmol) added portion wise over ten minutes. The reaction was stirred for 2 h after which time sat. aq. ammonium chloride (10 mL) was added. The aqueous phase was extracted with DCM (2×15 mL), passed through a phase separator and concentrated in vacuo to yield the sub-title compound (303 mg) as a colourless oil.
[0775] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.61 (d. 1H), 6.92 (dt, 1H), 4.86-4.69 (m, 2H), 3.81 (s, 3H).
(ii) Methyl 5-(chloromethyl)thiophene-2-carboxylate
[0776] The product from step (i) (287 mg, 1.66 mmol) was dissolved in dry CHCl.sub.3 (3 mL) and cooled to 0° C. DMF (0.05 mL, 3.60 mmol) and thionyl chloride (3 eq, 0.36 mL) were added and the mixture stirred for 2 h. The reaction was quenched at 0° C. with MeOH (0.5 mL), diluted with DCM (15 mL), washed with brine (15 mL), passed through a phase separator and concentrated in vacuo. The sub-title compound (303 mg) was isolated as colourless oil.
[0777] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.58 (d, 1H), 7.00 (dt, 1H), 4.69 (d, 2H), 3.82 (s, 3H).
(iii) Methyl 5-((2-hydroxyethoxy)methyl)thiophene-2-carboxylate
[0778] To a stirred solution of dry ethane-1,2-diol (0.35 mL, 6.29 mmol) in DMSO (1.5 mL) at 0° C. was added potassium tert-butoxide (194 mg, 1.73 mmol) portion wise over 10 min. The resulting solution was stirred for 30 min at same temperature before adding TBAl (58.1 mg, 0.15 mmol). A homogeneous solution of the product from step (ii) above (300 mg, 1.57 mmol) in DMSO (0.5 mL) was added dropwise to the above reaction mixture and stirred at rt overnight. MeOH (3 mL) was added and the reaction stirred overnight. Cold water (25 mL) was added and the aqueous layer extracted with ethyl acetate (2×25 mL) and the combined organic layers concentrated in vacuo. The crude product was purified by chromatography on silica gel (12 g column, 0-5% MeOH/DCM) to afford the sub-title compound (115 mg) as a yellow oil.
[0779] .sup.1H NMR (400 MHz, DMSO-d6) δ 7.70 (d, 1H), 7.14 (dt, 1H), 4.78-4.63 (m, 3H), 3.81 (s, 3H), 3.58-3.44 (m, 4H).
(iv) Methyl 5-((2-((methylsulfonyl)oxy)ethoxy)methyl)thiophene-2-carboxylate
[0780] The product from step (iii) above (115 mg, 0.53 mmol) was dissolved in DCM (5 mL) and cooled in an ice bath. NEt.sub.3 (111 μL, 0.79 mmol) followed by MsCl (49.7 μL, 0.63 mmol) were added dropwise and the mixture left to warm to rt overnight. The mixture was diluted with DCM (10 mL) and the organic layer washed with 0.1 M HCl (10 mL). The mixture was passed through a phase separator and concentrated in vacuo to yield the sub-title compound (125 mg) as light yellow oil.
[0781] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.71 (d, 1H), 7.19-7.14 (m, 1H), 4.77 (d, 2H), 4.41-4.30 (m, 2H), 3.82 (s, 3H), 3.77-3.69 (m, 2H), 3.19 (s, 3H).
(v) Methyl 5-((2-(3-Methoxy-5-nitrophenoxy)ethoxy)methyl)thiophene-2-carboxylate
[0782] 3-Methoxy-5-nitrophenol (65.0 mg, 0.384 mmol), the product from step (iv) above (125 mg, 0.40 mmol) and potassium carbonate (159 mg, 1.15 mmol) were suspended/dissolved in DMF (3 mL) and heated to 80° C. overnight. The reaction was cooled and partitioned between TBME (20 mL) and brine (20 mL). The aqueous layer was extracted with TBME (20 mL) and the combined organic layers washed with brine (40 mL), dried (MgSO.sub.4), filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel (12 g column, 0-50% EtOAc/isohexane) to afford the sub-title compound (105 mg) as a yellow solid.
[0783] .sup.1H NMR (400 MHz, DMSO-d6) δ 7.70 (d, 1H), 7.35 (dt, 2H), 7.15 (dt, 1H), 6.99 (t, 1H), 4.79 (d, 2H), 4.36-4.19 (m, 2H), 3.89-3.78 (m, 9H).
(vi) Methyl 5-((2-(3-amino-5-methoxyphenoxy)ethoxy)methyl)thiophene-2-carboxylate
[0784] The product from step (v) above (105 mg, 0.286 mmol) was dissolved in EtOH (4 mL, 68.5 mmol). Pd—C (type 87 L) (30.4 mg, 0.014 mmol) was added and the reaction stirred under an atmosphere of hydrogen (1 bar) for 1 h. The mixture was filtered through celite and the solids washed with ethanol (10 mL). The solution was concentrated directly onto silica. The crude product was purified by chromatography on silica gel (12 g column, 0-5% (0.7 M Ammonia/MeOH)/DCM) but did not yield a product of sufficient purity. The product was repurified by chromatography on silica gel (12 g column, 0-5% (0.7 M Ammonia/MeOH)/DCM) to afford the sub-title compound (57 mg) as a dark red oil.
[0785] m/z 338.1 (M+H).sup.+ (ES.sup.+)
(vii) Methyl 5-((2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamidophenyl) ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxphenoxy)ethoxy)methyl)thiophene-2-carboxylate
[0786] N-(5-(tert-butyl)-3-(3-(4-((2-chloropyridin-4-yl)oxy)naphthalen-1-yl)ureido)-2-methoxyphenyl)-methanesulfonamide (see WO 2014/162126; 96 mg, 0.169 mmol), the product from step (vi) above (57 mg, 0.169 mmol), Pd-175 (6.60 mg, 8.45 μmol) and freshly ground potassium carbonate (70.0 mg, 0.507 mmol) in DMF (2 mL) were degassed by evacuation and backfilling with nitrogen three times. The resulting mixture was heated to 70° C. for 2 h after which time a further portion of Pd-175 (13.2 mg, 8.45 μmol) was added dropwise as a solution in DMF (2 mL) over 2 h. The reaction was cooled and concentrated in vacuo. The crude product was purified by chromatography (RP Flash C18) (12 g column, 25-100% MeCN/10 mM Ammonium Bicarbonate) to afford the sub-title compound (23 mg) as a dark red solid.
[0787] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.38 (s, 1H), 9.13 (s, 1H), 8.90 (s, 1H), 8.87 (s, 1H), 8.32-8.27 (m, 1H), 8.20-8.14 (m, 1H), 8.13-8.06 (m, 2H), 7.90-7.84 (m, 1H), 7.74-7.66 (m, 2H), 7.61 (ddd, 1H), 7.38 (d, 1H), 7.17-7.11 (m, 1H), 7.03 (d, 1H), 6.90 (t, 1H), 6.80 (t, 1H), 6.58 (dd, 1H), 6.08 (d, 1H), 6.04 (t, 1H), 4.77 (d, 2H), 4.03 (dd, 2H), 3.84-3.74 (m, 7H), 3.65 (s, 3H), 3.08 (s, 3H), 1.27 (s, 9H).
(viii) 5-((2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamidophenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)methylthiophene-2-carboxylic acid
[0788] The product from step (vii) above (22 mg, 0.025 mmol) was dissolved in THF (0.75 mL) and MeOH (0.25 mL). NaOH (2M aq.) (139 μL, 0.27 mmol) was added and the mixture stirred at rt overnight. The reaction was acidified with AcOH (0.25 mL) and concentrated in vacuo. The crude product was purified by chromatography on RP Flash C18 (12 g column, 15-75% MeCN/10 mM Ammonium Bicarbonate). The product-containing fractions were combined, acidified with formic acid to ca. pH 4 and concentrated in vacuo to yield the title compound (7.8 mg) as a white solid.
[0789] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.44 (s, 1H), 8.94 (s, 1H), 8.88 (s, 1H), 8.30 (d, 1H), 8.18 (d, 1H), 8.14-8.07 (m, 2H), 7.89-7.84 (m, 1H), 7.69 (ddd, 1H), 7.60 (ddd, 1H), 7.46 (d, 1H), 7.38 (d, 1H), 7.04 (d, 1H), 7.03 (d, 1H), 6.89 (t, 1H), 6.81 (t, 1H), 6.57 (dd, 1H), 6.08 (d, 1H), 6.04 (t, 1H), 4.71 (s, 2H), 4.02 (dd, 2H), 3.81 (s, 3H), 3.79-3.73 (m, 2H), 3.65 (s, 3H), 3.10 (s, 3H), 1.27 (s, 9H).
[0790] m/z 856.2 (M+H).sup.+ (ES.sup.+)
Example 27
5-((2-(3-((4-((4-(3-(5-tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)methyl)thiophene-3-carboxylic acid
[0791] ##STR00092##
(i) Methyl 5-(hydroxymethyl)thiophene-3-carboxylate
[0792] Methyl 5-formylthiophene-2-carboxylate (311 mg, 1.82 mmol) was dissolved in MeOH (4 mL), cooled to 0° C. and NaBH.sub.4 (114 mg, 2.11 mmol) was added portionwise over ten minutes. The reaction was stirred for 2 h and sat. aq. ammonium chloride (10 mL) added. The aqueous phase was extracted with DCM (2×15 mL), passed through a phase separator and concentrated in vacuo to yield the sub-title compound (209 mg) as a colourless oil.
[0793] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.04 (d, 1H), 7.40 (dt, 1H), 4.82 (dd, 2H), 3.85 (s, 3H), 1.97 (t, 1H).
(ii) Methyl 5-(chloromethyl)thiophene-3-carboxylate
[0794] The product from step (i) above (209 mg, 1.21 mmol) was dissolved in dry CHCl.sub.3 (3 mL) and cooled to 0° C. DMF (0.05 mL, 3.60 mmol) and thionyl chloride (0.26 mL, 3.6 mmol) were added and the mixture stirred for 2 h. The reaction was quenched at 0° C. with MeOH (0.5 mL). The reaction was diluted with DCM (15 mL), washed with brine (15 mL), passed through a phase separator and concentrated in vacuo to yield the sub-title compound (190 mg) as a colourless oil.
[0795] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.07 (d, J=1.4 Hz, 1H), 7.48 (dt, J=1.4, 0.8 Hz, 1H), 4.76 (d, J=0.7 Hz, 2H), 3.86 (s, 3H).
(iii) Methyl 5-((2-hydroxyethoxy)methyl)thiophene-3-carboxylate
[0796] To a stirred solution of dry ethane-1,2-diol (0.22 mL, 3.99 mmol) in DMSO (1.5 mL) at 0° C. was added potassium tert-butoxide (123 mg, 1.09 mmol) portion wise over 10 min. The resulting solution was further stirred for 30 min at the same temperature before adding TBAl (36.8 mg, 0.10 mmol). A homogeneous solution of the product from step (ii) above (190 mg, 0.99 mmol) in DMSO (0.5 mL) was added dropwise and stirred at rt overnight. MeOH (3 mL) was added and the reaction stirred overnight. Cold water (15 mL) was added, the aqueous layer extracted with ethyl acetate (2×25 mL) and the combined organic layers concentrated in vacuo. The crude product was purified by chromatography on silica gel (12 g column, 0-5% MeOH/DCM) to afford the sub-title compound (106 mg) as a yellow oil.
[0797] .sup.1H NMR (400 MHz, DMSO-d6) δ 8.31 (d, 1H), 7.40 (dt, 1H), 4.72-4.62 (m, 3H), 3.79 (s, 3H), 3.55-3.49 (m, 2H), 3.49-3.42 (m, 2H).
(iv) Methyl 5-((2-((methylsulfonyl)oxy)ethoxy)methyl)thiophene-3-carboxylate
[0798] The product from step (iii) above (105 mg, 0.486 mmol) was dissolved in DCM (5 mL) and cooled to 00° C. NEt.sub.3 (102 μL, 0.728 mmol) followed by MsCl (45.4 μL, 0.583 mmol) were added dropwise and the mixture left to warm to rt overnight. The mixture was diluted with DCM (10 mL) and the organic layer washed with 0.1 M HCl (10 mL). The aqueous layer was further extracted with DCM (5 mL), the combined organic layers passed through a phase separator and concentrated in vacuo to yield the sub-title compound (115 mg) as a light yellow oil.
[0799] .sup.1H NMR (400 MHz, DMSO-d6) δ 8.33 (d, 1H), 7.45-7.41 (m, 1H), 4.72 (d, 2H), 4.47-4.26 (m, 2H), 3.79 (s, 3H), 3.75-3.61 (m, 2H), 3.22-3.09 (m, 3H).
(v) Methyl 5-((2-(3-methoxy-5-nitrophenoxy)ethoxy))methyl)thiophene-3-carboxylate
[0800] 3-Methoxy-5-nitrophenol (59.0 mg, 0.349 mmol), the product from step (iv) above (110 mg, 0.366 mmol) and potassium carbonate (145 mg, 1.046 mmol) were suspended/dissolved in DMF (3 mL) and heated to 80° C. overnight. The reaction was cooled and partitioned between TBME (20 mL) and brine (20 mL). The aqueous layer was extracted with TBME (20 mL) and the combined organic layers washed with brine (40 mL), dried (MgSO.sub.4), filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel (12 g column, 0-50% EtOAc/isohexane) to afford the sub-title compound (92 mg) as a yellow solid.
[0801] .sup.1H NMR (400 MHz, DMSO-d) δ 8.30 (d, 1H), 7.45-7.40 (m, 1H), 7.34 (dt, 2H), 6.99 (t, 1H), 4.75 (d, 2H), 4.31-4.20 (m, 2H), 3.86 (s, 3H), 3.83-3.80 (m, 2H), 3.79 (s, 3H).
(vi) Methyl 5-((2-(3-amino-5-methoxyphenoxy)ethoxy)methyl)thiophene-3-carboxylate
[0802] The product from step (v) above (92 mg, 0.25 mmol) was dissolved in EtOH (4 mL, 68.5 mmol) with water (0.5 mL). Iron (84 mg, 1.50 mmol) and ammonium chloride (107 mg, 2.00 mmol) were added and the reaction mixture heated to 70° C. with vigorous stirring for 2 h. The mixture was cooled, filtered through celite and the solids washed with ethanol (10 mL). The solvent was removed in vacuo. The crude product was purified by chromatography on silica gel (12 g column, 0-5% (0.7 M Ammonia/MeOH)/DCM) to afford the sub-title compound (42 mg) as an orange oil.
[0803] .sup.1H NMR (400 MHz, DMSO-d6) δ 8.31 (d, 1H), 7.47-7.40 (m, 1H), 5.79-5.73 (m, 2H), 5.69 (t, 1H), 5.05 (s, 2H), 4.73 (d, 2H), 4.06-3.90 (m, 2H), 3.79 (s, 3H), 3.76-3.71 (m, 2H), 3.62 (s, 3H).
(vii) Methyl 5-((2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl) ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)methyl)thiophene-3-carboxylate
[0804] A suspension of the product from step (vi) above (37 mg, 0.11 mmol), N-(5-(tert-butyl)-3-(3-(4-((2-chloropyridin-4-yl)oxy)naphthalen-1-yl)ureido)-2-methoxyphenyl) methanesulfonamide (see WO 2014/162126; 62.4 mg, 0.110 mmol), Pd-175 (4.28 mg, 5.48 μmol) and freshly ground potassium carbonate (45.5 mg, 0.32 mmol) in DMF (3 mL) was degassed by 3 cycles of evacuation and backfilling with nitrogen. The reaction was heated to 70° C. for 2 h. A further portion of Pd-175 (8.48 mg, 10.9 μmol) was added dropwise in DMF (2 mL) over 2 h. The reaction was concentrated in vacuo. The crude product was purified by chromatography on RP Flash C18 (12 g column, 25-100% MeCN/10 mM Ammonium Bicarbonate) to afford the sub-title compound (36 mg) as a beige solid.
[0805] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.38 (s, 1H), 9.13 (s, 1H), 8.90 (s, 1H), 8.87 (s, 1H), 8.36-8.25 (m, 2H), 8.18 (d, 1H), 8.11 (d, 1H), 8.10 (s, 1H), 7.89-7.84 (m, 1H), 7.70 (ddd, 1H), 7.61 (ddd, 1H), 7.44-7.40 (m, 1H), 7.38 (d, 1H), 7.03 (d, 1H), 6.90 (t, 1H), 6.79 (t, 1H), 6.57 (dd, 1H), 6.08 (d, 1H), 6.04 (t, 1H), 4.73 (d, 2H), 4.06-3.99 (m, 2H), 3.81 (s, 3H), 3.78 (s, 3H), 3.77-3.72 (m, 2H), 3.65 (s, 3H), 3.10 (s, 3H), 1.27 (s, 9H).
(viii) 5-((2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido) naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)methylthiophene-3-carboxylic acid
[0806] The product from step (vii) above (33 mg, 0.038 mmol) was dissolved in THF (2 mL) and MeOH (0.5 mL). NaOH (2M aq., 209 μL, 0.417 mmol) was added and the mixture stirred at rt overnight. The reaction was acidified with AcOH (0.25 mL) and concentrated in vacuo. The crude product was purified by chromatography on RP Flash C18 (24 g column, 15-75% MeCN/10 mM Ammonium Bicarbonate). The product-containing fractions were combined, acidified with formic acid to ca. pH 4, concentrated in vacuo. The resulting solid was redissolved in the minimum amount of ethanol (ca. 0.3 mL) and water (0.2 mL) added dropwise to crash out the white solid. The vial was centrifuged at 2000 rpm for 5 minutes and the supernatant decanted. The resulting solid was dried in vacuo at 55° C. for 48 h to yield the title compound as a white solid.
[0807] .sup.1H NMR (400 MHz, DMSO-d6) δ 12.68 (bs, 1H), 9.38 (s, 1H), 9.12 (bs, 1H), 8.91 (s, 1H), 8.87 (s, 1H), 8.29 (d, 1H), 8.20 (d, 1H), 8.19 (d, 1H), 8.14-8.07 (m, 2H), 7.87 (dd, 1H), 7.70 (ddd, 1H), 7.61 (ddd, 1H), 7.42-7.33 (m, 2H), 7.03 (d, 1H), 6.90 (t, 1H), 6.80 (t, 1H), 6.57 (dd, 1H), 6.08 (d, 1H), 6.04 (t, 1H), 4.72 (d, 2H), 4.06-3.98 (m, 2H), 3.81 (s, 3H), 3.78-3.71 (m, 2H), 3.65 (s, 3H), 3.10 (s, 3H), 1.27 (s, 9H).
[0808] m/z 856.2 (M+H).sup.+ (ES.sup.+)
Example 28
2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl-oxy)pyridin-2-yl)amino)-5-(difluoromethoxy)phenoxy)ethoxy)ethoxy)acetic acid
[0809] ##STR00093##
(i) 1-Bromo-3-(difluoromethoxy)-5-nitrobenzene
[0810] A mixture of 3-bromo-5-nitrophenol (460 mg, 2.11 mmol), sodium 2-chloro-2,2-difluoroacetate (804 mg, 5.28 mmol) and Cs.sub.2CO.sub.3 (1375 mg, 4.22 mmol) in DMF (8 mL) was heated at 100° C. for 1 h. The mixture was partitioned between TBME (50 mL) and water (50 mL), the aqueous layer extracted with TBME (30 mL) and the combined organic layers washed with brine (50 mL). The organic layer was concentrated in vacuo to yield the sub-title compound (400 mg) as a colourless oil.
[0811] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.25 (t, 1H), 7.96 (t, 1H), 7.69-7.62 (m, 1H), 6.60 (t, 1H).
(ii) 3-(Difluoromethoxy)-5-nitrophenol
[0812] A mixture of KOH (197 mg, 2.98 mmol) and the product from step (i) above (200 mg, 0.746 mmol) in water (1.5 mL) and dioxane (1.5 mL) was degassed for 5 minutes prior to the addition of di-tert-butyl(2′,4′,6′-triisopropyl-[1,1′-biphenyl]-2-yl)phosphine (17.4 mg, 0.041 mmol) and Pd.sub.2(dba).sub.3 (17.0 mg, 0.019 mmol). The resulting mixture was degassed for a further 2 minutes and then heated under a nitrogen atmosphere at 100° C. for 3 h. The reaction was cooled and partitioned between 1 M HCl (20 mL) and EtOAc (20 mL). The organic layer was washed with water (20 mL), brine (20 mL), dried (MgSO.sub.4) and concentrated in vacuo to yield the sub-title compound (176 mg) as a dark brown oil.
[0813] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.56 (d, 2H), 6.96 (t, 1H), 6.57 (t, 1H).
(iii) Ethyl 2-(2-(2-(3-(difluoromethoxy)-5-nitrophenoxy)ethoxy)ethoxy)acetate
[0814] The product from step (ii) above (174 mg, 0.71 mmol), ethyl 2-(2-(2-((methylsulfonyl)oxy)-ethoxy)ethoxy)acetate (see Example 2(ii) above; 202 mg, 0.74 mmol) and potassium carbonate (295 mg, 2.13 mmol) were suspended/dissolved in DMF (4 mL) and heated to 60° C. for 16 h. The reaction was cooled and partitioned between TBME (20 mL) and brine (20 mL). The aqueous layer was extracted with TBME (20 mL) and the combined organic layers washed with brine (40 mL) and concentrated onto silica gel. The crude product was purified by chromatography on silica gel (12 g column, 0-50% EtOAc/isohexane) to afford the sub-title compound (147 mg) as a yellow oil.
[0815] .sup.1H NMR (400 MHz, DMSO-d6) δ 7.68-7.61 (m, 1H), 7.61 (t, 1H), 7.45 (t, 1H), 7.29 (t, 1H), 4.33-4.24 (m, 2H), 4.17-4.06 (m, 4H), 3.83-3.73 (m, 2H), 3.62 (s, 4H), 1.19 (t, 3H).
[0816] m/z 397.1 (M+NH.sub.4).sup.+ (ES.sup.+)
(iv) Ethyl 2-(2-(2-(3-amino-5-(difluoromethoxy)phenoxy)ethoxy)ethoxy)acetate
[0817] A solution of the product from step (iii) above (147 mg, 0.36 mmol) and Pd/C (Type 87 L, 5 wt %) (39.2 mg, 0.02 mmol) in EtOH (20 mL) were stirred under 2 bar H.sub.2 for 2 h. The reaction was filtered through celite, washing with EtOH (10 mL) and the mixture concentrated in vacuo. The crude product was purified by chromatography on silica gel (12 g column, 0-5% (0.7 M Ammonia/MeOH)/DCM) to afford the sub-title compound (89 mg) as a yellow oil.
[0818] .sup.1H NMR (400 MHz, DMSO-d6) δ 7.09 (t, 1H), 5.99 (t, 1H), 5.94 (t, 1H), 5.88 (t, 1H), 5.37 (s, 2H), 4.16-4.08 (m, 4H), 4.00-3.95 (m, 2H), 3.74-3.67 (m, 2H), 3.66-3.57 (m, 4H), 1.20 (t, 3H).
[0819] m/z 350.1 (M+H).sup.+ (ES.sup.+)
(v) Ethyl 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy) 3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-(difluoromethoxy)phenoxy)ethoxy)ethoxy)acetate
[0820] N-(5-(tert-Butyl)-3-(3-(4-((2-chloropyridin-4-yl)oxy)naphthalen-1-yl) ureido)-2-methoxyphenyl)-methanesulfonamide (see WO 2014/162126; 128 mg, 0.22 mmol), the product from step (iv) above, Pd-175 (8.7 mg, 0.01 mmol) and freshly ground potassium carbonate (93 mg, 0.67 mmol) were dissolved in DMF (1 mL) and degassed by evacuation and backfilling with nitrogen three times. The resulting mixture was heated to 70° C. for 2 h. The reaction mixture was cooled and loaded directly onto a reverse phase column. The crude product was purified by chromatography (RP Flash C18, 12 g column, 25-100% MeCN/10 mM Ammonium Bicarbonate) to afford the sub-title compound (116 mg) as a yellow solid.
[0821] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.39 (s, 1H), 9.13 (s, 1H), 9.08 (s, 1H), 8.90 (s, 1H), 8.30 (d, 1H), 8.17 (d, 1H), 8.16-8.09 (m, 2H), 7.90-7.84 (m, 1H), 7.71 (ddd, 1H), 7.61 (ddd, 1H), 7.39 (d, 1H), 7.17 (t, 1H), 7.14 (t, 1H), 7.08 (t, 1H), 7.03 (d, 1H), 6.63 (dd, 1H), 6.26 (t, 1H), 6.08 (d, 1H), 4.14-4.06 (m, 4H), 4.06-3.99 (m, 2H), 3.81 (s, 3H), 3.76-3.70 (m, 2H), 3.64-3.57 (m, 4H), 3.09 (s, 3H), 1.27 (s, 9H), 1.18 (t, 3H).
[0822] m/z 882.3 (M+H).sup.+ (ES.sup.+)
(vi) 2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-(difluoromethoxy)phenoxy)ethoxy)ethoxy)acetic acid
[0823] The product from step (v) above (116 mg, 0.13 mmol) was dissolved in THF (3 mL) and MeOH (1 mL). NaOH (2M aq., 723 μL, 1.447 mmol) was added and the mixture stirred at rt overnight. The reaction was acidified with AcOH (0.25 mL) and concentrated in vacuo. The crude product was purified by chromatography on RP Flash C18 (24 g column, 15-75% MeCN/10 mM Ammonium Bicarbonate). The product containing fractions were combined, acidified with formic acid to ca. pH 4, concentrated in vacuo and the resulting precipitate filtered off washing with water (5 mL). The solid was then redissolved in the minimum amount of ethanol (ca. 1 mL) and water (0.5 mL) added dropwise to crash out a white solid. The vial was then centrifuged at 2000 rpm for 5 minutes and the supernatant decanted. The resulting solid was dried in vacuo at 55° C. for 48 h to yield the title compound (58 mg) as a white solid.
[0824] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.42 (s, 1H), 9.09 (s, 1H), 8.93 (s, 1H), 8.30 (d, 1H), 8.18 (d, 1H), 8.14 (d, 1H), 8.11 (d, 1H), 7.86 (dd, 1H), 7.70 (ddd, 1H), 7.61 (ddd, 1H), 7.39 (d, 1H), 7.15 (t, 1H), 7.14 (t, 1H), 7.08 (t, 1H), 7.03 (d, 1H), 6.63 (dd, 1H), 6.27 (t, 1H), 6.09 (d, 1H), 4.08-3.94 (m, 4H), 3.81 (s, 3H), 3.76-3.69 (m, 2H), 3.60 (s, 4H), 3.10 (s, 3H), 1.27 (s, 9H).
[0825] m/z 854.2 (M+H).sup.+ (ES.sup.+)
Example 29
2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl-2-methoxy-3-(methylsulfonamidophenyl) ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-ethynylphenoxy) ethoxy)ethoxy)acetic acid
[0826] ##STR00094##
(i) tert-Butyl 2-(2-(2-benzyloxy)ethoxyethoxy)acetate
[0827] Sodium hydride (4.08 g, 102 mmol) was added portionwise to an ice bath-cooled solution of 2-(2-(benzyloxy)ethoxy)ethanol (9.14 mL, 51.0 mmol) in THF (200 mL) over 15 minutes. The reaction was stirred for 1 h after which time tert-butyl 2-bromoacetate (8.97 mL, 61.1 mmol) in THF (50 mL) was added dropwise over 1 h. The reaction was stirred at ice bath temperature for 3 h and quenched with ammonium chloride (50 mL). TBME (250 mL) was added and the organic layer washed with brine (2×200 mL). The organic layer was concentrated onto silica and the crude product was purified by chromatography on silica gel (220 g column, 0-50% EtOAc/isohexane) to afford the sub-title compound (1.95 g) as a colourless oil.
[0828] .sup.1H NMR (400 MHz, DMSO-d6) δ 7.43-7.20 (m, 5H), 4.50 (s, 2H), 3.99 (s, 2H), 3.66-3.47 (m, 8H), 1.42 (s, 9H).
(ii) tert-Butyl 2-(2-(2-hydroxyethoxy)ethoxy)acetate
[0829] The product from step (i) above (1.83 g, 5.90 mmol) was dissolved in methanol (30 mL) and Pd—C (type 87 L, 5 wt. %, 0.627 g, 0.29 mmol) added. The mixture was stirred at room temperature under 4 bar of hydrogen for 16 h. The reaction mixture was filtered through celite, washing the solids with EtOH (50 mL) and concentrated in vacuo to yield the sub-title compound (1.23 g) as a colourless oil.
[0830] .sup.1H NMR (400 MHz, DMSO-d6) δ 4.57 (t, 1H), 3.99 (s, 2H), 3.60-3.51 (m, 4H), 3.51-3.40 (m, 4H), 1.43 (s, 9H).
(iii) tert-Butyl 2-(2-(2-((methylsulfonyl)oxy)ethoxy)ethoxy)acetate
[0831] The product from step (ii) above (1.23 g, 5.58 mmol) was dissolved in DCM (30 mL) and cooled in an ice bath. EtN.sub.3 (1.16 mL, 8.38 mmol) followed by MsCl (0.52 mL, 6.70 mmol) were added dropwise and the mixture left to warm to room temperature overnight. The mixture was diluted with DCM (10 mL) and the organic layer washed with 0.1 M HCl (10 mL). The mixture was passed through a phase separator and concentrated in vacuo to yield the sub-title compound (1.83 g) as light yellow oil.
[0832] .sup.1H NMR (400 MHz, DMSO-d6) δ 4.35-4.28 (m, 2H), 4.00 (s, 2H), 3.73-3.65 (m, 2H), 3.64-3.54 (m, 4H), 3.18 (s, 3H), 1.43 (s, 9H).
(iv) tert-Butyl 2-(2-(2-(3-bromo-5-nitrophenoxy)ethoxy)ethoxy)acetate
[0833] 3-bromo-5-nitrophenol (0.626 g, 2.87 mmol), the product from step (iii) above (1 g, 3.02 mmol) and potassium carbonate (1.191 g, 8.62 mmol) were suspended/dissolved in 3 mL DMF and heated to 80° C. overnight. The reaction was cooled and partitioned between TBME (20 mL) and brine (20 mL). The aqueous layer was extracted with TBME (20 mL) and the combined organic layers washed with brine (40 mL), dried (MgSO.sub.4), filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel (40 g column, 0-50% EtOAc/isohexane) to afford the sub-title compound (965 mg) as a yellow oil.
[0834] .sup.1H NMR (400 MHz, DMSO-d6) δ 8.04-7.89 (m, 1H), 7.79-7.72 (m, 1H), 7.70 (dd, 1H), 4.32-4.22 (m, 2H), 3.99 (s, 2H), 3.82-3.72 (m, 2H), 3.66-3.54 (m, 4H), 1.42 (s, 9H).
[0835] m/z 439.1 (M+NH.sub.4).sup.+ (ES.sup.+)
(v) tert-Butyl 2-(2-(2-(3-nitro-5-((triisopropylsilyl)ethynyl) phenoxy)ethoxy)ethoxy)acetate
[0836] Pd(PPh).sub.4 (86 mg, 0.075 mmol) was added to a degassed suspension of the product from step (iv) above (314 mg, 0.747 mmol), CuI (7.11 mg, 0.037 mmol), and ethynyltriisopropylsilane (0.268 mL, 1.195 mmol) in triethylamine (1 mL) and DMF (3 mL). The mixture was heated at 85° C. (block temp.) for 1 h then cooled and concentrated directly onto silica gel. The crude product was purified by chromatography on silica gel (24 g column, 0-50% EtOAc/isohexane) to afford the sub-title compound (255 mg) as a light yellow oil.
[0837] .sup.1H NMR (400 MHz, DMSO-d6) δ 7.82-7.71 (m, 2H), 7.47-7.45 (m, 1H), 4.34-4.23 (m, 2H), 3.98 (s, 2H), 3.81-3.73 (m, 2H), 3.66-3.54 (m, 4H), 1.41 (s, 9H), 9H), 1.19-1.03 (m, 21H).
(vi) tert-Butyl 2-(2-(2-(3-amino-5-((triisopropylsilyl)ethynyl)phenoxy)ethoxy)ethoxy)acetate
[0838] The product from step (v) above (255 mg, 0.489 mmol) was dissolved in EtOH (6 mL) and H.sub.2O (0.75 mL). Iron (164 mg, 2.93 mmol) and ammonium chloride (209 mg, 3.91 mmol) were added and the flask evacuated and backfilled with nitrogen three times. The reaction mixture was heated to 80° C. with vigorous stirring for 2 h. The mixture was cooled, filtered through celite and the solids washed with ethanol (10 mL). The resulting crude product was dissolved in DCM (20 mL), washed with water (20 mL), passed through a phase separator and concentrated in vacuo to yield the sub-title compound (203 mg) as an orange oil.
[0839] .sup.1H NMR (400 MHz, DMSO-d6) δ 6.30 (dd, 1H), 6.17 (t, 1H), 6.14 (dd, 1H), 5.24 (s, 2H), 4.03-3.96 (nm, 4H), 3.72-3.67 (m, 2H), 3.60 (s, 4H), 1.42 (s, 9H), 1.09 (s, 21H).
[0840] m/z 492.3 (M+H).sup.+ (ES.sup.+)
(vii) tert-Butyl 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-Methoxy-3-(methylsulfonamido)phenyl)-ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-((triisopropylsilyl)ethynyl)phenoxy)ethoxy)-ethoxy)acetate
[0841] N-(5-(tert-butyl)-3-(3-(4-((2-chloropyridin-4-yl)oxy)naphthalen-1-yl)ureido)-2-methoxyphenyl)-methanesulfonamide (see WO 2014/162126; 231 mg, 0.407 mmol), the product from step (vi) above (200 mg, 0.407 mmol), Pd-175 (15.8 mg, 0.02 mmol) and freshly ground potassium carbonate (169 mg, 1.220 mmol) were dissolved/suspended in DMF (3 mL) and degassed by evacuation and backfilling with nitrogen three times. The resulting mixture was heated to 70° C. for 2 h. The reaction mixture was cooled and partitioned between TBME (30 mL) and water (30 mL). The organic layer was washed with water (20 mL) and concentrated in vacuo to yield the sub-title compound (152 mg) as a brown solid.
[0842] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.38 (s, 1H), 9.13 (s, 1H), 9.00 (5, 1H), 8.91 (s, 1H), 8.30 (d, 1H), 8.18 (d, 1H), 8.15-8.07 (m, 2H), 7.89-7.83 (m, 1H), 7.73-7.68 (m, 1H), 7.63-7.58 (m, 1H), 7.56 (t, 1H), 7.39 (d, 1H), 7.17 (t, 1H), 7.03 (d, 1H), 6.61 (dd, 1H), 6.48 (dd, 1H), 6.07 (d, 1H), 4.10-4.01 (m, 2H), 3.99 (s, 2H), 3.81 (s, 3H), 3.76-3.68 (m, 2H), 3.60 (s, 4H), 3.09 (s, 3H), 1.40 (s, 9H), 1.27 (s, 9H), 1.16-1.02 (m, 21H).
[0843] m/z 1024.3 (M+H).sup.+ (ES.sup.+)
(viii) 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-Methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-ethynylphenoxy)ethoxy)ethoxy)acetic acid
[0844] The product from step (vii) above (152 mg, 0.14 mmol) was dissolved in THF (4 mL) and TBAF (1M in THF) (156 μL, 0.15 mmol) added. The reaction was stirred for 60 h at room temperature then partitioned between DCM (40 mL) and water (40 mL). The organic layer was washed with brine (50 mL), passed through a phase separator and concentrated in vacuo. The crude material (100 mg) was dissolved in DCM (1 mL) and TFA (178 μL, 2.30 mmol) added. The reaction was stirred for 16 h and a further portion of TFA (178 μL, 2.30 mmol) added. The volatiles were removed in vacuo and the crude product purified by chromatography (RP Flash C18, 12 g column, 15-75% MeCN/10 mM Ammonium Bicarbonate). The product was further purified by preparative HPLC (Basic, Waters X-Bridge Prep-C18, 5 μm, 19×50 mm column, 35-65% MeCN in Water) to yield the title compound (6 mg) as a light yellow solid.
[0845] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.61 (s, 1H), 9.08 (s), 9.08 (s, 1H), 9.04 (s, 1H), 8.33 (d, 1H), 8.17 (d, J=2.3 Hz, 1H), 8.15 (d, 1H), 8.10 (d, 1H), 7.86 (dd, 1H), 7.69 (ddd, 1H), 7.60 (ddd, 1H), 7.38 (d, 1H), 7.32 (t, 1H), 7.26 (s, 1H), 7.02 (d, 1H), 6.63 (dd, 1H), 6.51 (dd, 1H), 6.11 (d, 1H), 4.06 (s, 1H), 3.96 (d, 2H), 3.84-3.78 (m, 4H), 3.70 (dd, 2H), 3.57 (s, 4H), 3.09 (s, 3H), 1.27 (s, 9H).
[0846] m/z 812.2 (M+H).sup.+ (ES.sup.+)
Example 30
N-(5-(tert-Butyl-2-methoxy-3-(3-(4-((2-((3-methoxy-5-(2-(2-((5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)methoxy)ethoxy)ethoxy)phenyl)amino)pyridin-4-yl)oxy)naphthalen-1-yl)ureido)phenyl)methanesulfonamide
[0847] ##STR00095##
[0848] A suspension of N-(5-(tert-butyl)-3-(3-(4-((2-((3-(2-(2-(cyanomethoxy)ethoxy)ethoxy)-5-methoxyphenyl)amino)pyridin-4-yl)oxy)naphthalen-1-yl)ureido)-2-methoxyphenyl)methane-sulfonamide (see Example 23(vi) above; 79 mg, 0.01 mmol) in EtOH (2 mL) and hydroxylamine (50% in water) (6.0 μL, 0.198 mmol) was heated to 75° C. and left to stir overnight. The solvent was removed and the residue azeotroped with toluene (2×1 mL) to afford a clear oil; m/z 832.1 (M+H).sup.+ (ES.sup.+). The crude product was dissolved in DMF (2.5 mL) and cooled to 0° C. Pyridine (8.8 μl, 0.109 mmol) was added, followed by isobutyl chloroformate (0.013 mL, 0.099 mmol) and the resulting solution stirred at 0° C. for 30 min, then at rt for 20 min. The reaction was quenched with water (10 mL) and extracted with EtOAc (3×5 mL). The combined organic extractions were washed with brine (5 mL), passed through a hydrophobic frit and concentrated in vacuo to give a brown oil; m/z 932.5 (M+H).sup.+ (ES.sup.+). The crude material was dissolved in a mixture of EtOH (2.5 mL) and sat. aq. NaHCO.sub.3 (0.5 mL) and stirred at 65° C. overnight. The reaction was filtered and diluted with DMF (1 mL). The EtOH was removed under a flow of air, then the crude reaction mixture was purified by chromatography on the Companion (RP Flash C18) (12 g column, 15-75% MeCN/10 mM Ammonium Bicarbonate). The product-rich fractions were combined and the pH adjusted to ca. 7 with formic acid. The solvent was removed in vacuo to afford a dark brown gum. This was repurified by preparative HPLC (Waters, Basic (0.1% Ammonium Bicarbonate), Basic, Waters X-Bridge Prep-C18, 5 μm, 19×50 mm column, 30-60% MeCN in Water) and the product-rich fractions freeze-dried to afford the title compound as a colourless gum (5 mg).
[0849] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.39 (s, 1H), 9.13 (s, 1H), 8.91 (s, 1H), 8.88 (s, 1H), 8.29 (d, 1H), 8.19 (d, 1H), 8.11 (d, 1H), 8.10 (d, 1H), 7.87 (dd, 1H), 7.70 (ddd, 1H), 7.61 (ddd, 1H), 7.38 (d, 1H), 7.03 (d, 1H), 6.89 (t, 1H), 6.80 (t, 1H), 6.58 (d, 1H), 6.53 (s, 1H), 6.08 (d, 1H), 6.04 (t, 1H), 4.34 (s, 2H), 4.03-3.91 (m, 2H), 3.81 (s, 3H), 3.74-3.68 (m, 2H), 3.65 (s, 3H), 3.63-3.54 (m, 4H), 3.10 (s, 3H), 1.27 (s, 9H).
[0850] m/z 857.7 (M+H).sup.+ (ES.sup.+)
Example 31
2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl) ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid, sodium salt
[0851] ##STR00096##
Method 1
[0852] To a 5 L flask under nitrogen was added IPA/water (90:10; 2.56 L, 12 volumes), the solvent was heated to 55° C. at which temperature 2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid (see Example 3 above: 213.9 g, 0.261 mol) was charged gradually over 15 minutes. A formal solution was obtained after an additional 7 minutes agitation. To the solution (pink) was added sodium hydrogen carbonate (1.05 equiv.; 0.274 mol, 274 mL), maintaining the temperature at 53° C. The solution was cooled to 50° C. over 20 minutes and seeded with the sodium salt of 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)acetic acid (1.0 g); the seed maintained and cooling was continued at 10° C./hour to 25° C. after which point 8 volumes of IPA (1.71 L) was charged over 2 hours. The batch was agitated for 18 hours at this temperature and then cooled to 0° C. and aged for 1.5 hours ahead of isolation via filtration. The cake and vessel rinse were performed using the batch liquors, the cake pulled dry and the solids dried in vacuo at 50° C. for 18 hours. A yield of 81% was obtained (177.9 g) for the title compound as a faint red solid; purity by HPLC was reported at 99.24 area % and proton NMR indicated a batch that conformed to structure with 0.58% wt IPA and 0.55% wt water (solvents determined by HRGC and KF respectively). The batch was dried at 50° C. under vacuum to take the IPA level down to 2,238 ppm (0.22%). The sodium content was 2.5% by ion chromatography.
[0853] .sup.1H NMR (400 MHz, DMSO-d6) δ: 9.95 (s, 1H), 9.24 (s, 1H), 8.93 (s, 1H), 8.38 (d, 1H), 8.08-8.12 (m, 3H), 7.83 (d, 1H), 7.55-7.67 (m, 2H), 7.35 (d, 1H), 7.01 (d, 1H), 6.65-6.72 (m, 2H), 6.60 (dd, 1H), 6.11 (d, 1H), 6.00 (t, 1H), 3.77-3.84 (m, 5H), 3.63-3.68 (m, 7H), 3.53 (s, 4H), 3.05 (s, 3H), 1.25 (s, 9H).
Method 2
[0854] Ethyl 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl) ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxyphenoxy)ethoxy)ethoxy)acetate (see Example 3(iii) above; 413 g gross (containing 3.14% ethyl acetate, equivalent to 400 g active, 472.8 mmol, 1 eq.) was mixed with acetone/water (800 mL and 800 mL, 2 vol and 2 vol) and NaOH (37.8 g, 945.7 mmol, 2 eq.) and stirred overnight at 22° C. The reaction was acidified to pH 7.07 (pH range of 6.9 to 7.3 is acceptable) using AcOH (26.6 mL, 465 mmol, 0.9836 eq.), after which IPA (4000 mL, 10 vol) was added (though 12 volumes of IPA have been shown to give similar yields and purity). The resulting mixture was cooled to 10° C. over 1 h and stirred for 1 h (for larger scale preparations, the mixture can instead be stirred overnight at 7° C.) before being filtered to provide a crude product (329 g) 83% yield (for which: purity as determined by LC was 98.9%, with <0.1% starting material; XRD and DSC analysis indicated the form as produced by Method 1 above; and NMR analysis indicated 1.3% NaOAc and 0.4% IPA). The crude product (329 g, 392 mmol, 1 eq.) was slurried in 8 vol of 15% water:IPA (395 mL water:2237 mL IPA) at 22° C. for 2 h. The resulting mixture was then heated to 45° C. for 2 h, before being cooled to 30° C. and then filtered. This gave the title compound (315 g) at 94% recovery from the crude product, for which the NaOAc content was 0.39% and IPA was 0.36%. The overall yield for the reaction was 79%.
Example 32
2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl) ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-(trifluoromethoxy)phenoxy)ethoxy)ethoxy)acetic acid
[0855] ##STR00097##
(i) 3-Hydroxy-5-(trifluoromethoxy)benzoic acid
[0856] A solution of 3-bromo-5-(trifluoromethoxy)benzoic acid (3200 mg, 11.23 mmol) and NaOH (2520 mg, 44.9 mmol) in water (30 mL) and dioxane (30 mL) was degassed for 5 minutes prior to the addition of Pd.sub.2(dba).sub.3 (206 mg, 0.225 mmol) and di-tert-butyl(2′,4′,6′-triisopropyl-[1,1′-biphenyl]-2-yl)phosphine (215 mg, 0.505 mmol). The resulting mixture was degassed for a further 2 minutes and then heated under a nitrogen atmosphere at 100° C. for 2.5 h. The mixture was diluted with water (150 mL) and washed with diethyl ether (3×75 mL). The aqueous layer was then acidified with HCl (1 M, 33 mL) to pH 3 and extracted with ethyl acetate (3×75 mL). The combined organic layers were washed with saturated brine (50 mL), dried over MgSO.sub.4, filtered, and concentrated under reduced pressure to afford a yellow oil. The oil was redissolved in diethyl ether (10 mL) and diluted with isohexane (30 mL). The resulting precipitate was collected by filtration and washed with isohexane (10 mL) to yield the sub-title compound (1.71 g) as a tan solid.
[0857] .sup.1H NMR (400 MHz, DMSO-d6) δ 13.34 (bs, 1H), 10.57 (bs, 1H), 7.36 (dd, 1H), 7.28-7.20 (m, 1H), 6.99-6.90 (m, 1H).
(ii) Benzyl 3-(2-(2-(2-(tert-butoxy)-2-oxoethoxy)ethoxy)ethoxy)-5-(trifluoromethoxy)benzoate
[0858] The product from step (i) above (210 mg, 0.945 mmol) and potassium carbonate (392 mg, 2.84 mmol) was dissolved/suspended in DMF (1.25 mL) and benzyl bromide (0.112 mL, 0.945 mmol) added. The mixture was stirred at room temperature for 4 h. Potassium carbonate (392 mg, 2.84 mmol), DMF (4 mL) and tert-butyl 2-(2-(2-((methylsulfonyl)oxy)ethoxy)ethoxy)acetate (see Example 29(iii) above; 310 mg, 1.040 mmol) were added and the reaction heated to 70° C. for 16 h. The reaction was cooled and partitioned between TBME (50 mL) and water (50 mL). The aqueous layer was extracted with DCM (50 mL), the combined organic layers washed with brine (50 mL) and concentrated in vacuo. The crude product was purified by chromatography on silica gel (12 g column, 0-50% EtOAc/iso-hexane) to afford the sub-title compound (278 mg) as a colourless oil.
[0859] .sup.1H NMR (400 MHz, DMSO-d6) δ 7.52 (dd, 1H), 7.50-7.45 (m, 2H), 7.45-7.34 (m, 4H), 7.34-7.29 (m, 1H), 5.38 (s, 2H), 4.28-4.17 (m, 2H), 3.98 (s, 2H), 3.82-3.72 (m, 2H), 3.65-3.55 (m, 4H), 1.40 (s, 9H).
[0860] m/z 532.2 (M+NH.sub.4)+(ES.sup.+)
(iii) 3-(2-(2-(2-(tert-Butoxy)-2-oxoethoxy)ethoxy)ethoxy)-5-(trifluoromethoxy)benzoic acid
[0861] The product from step (ii) above (278 mg, 0.540 mmol) and Pd—C (57.5 mg, 0.027 mmol) were dissolved/suspended in EtOH (10 mL) and stirred under an atmosphere of H.sub.2 (2 bar) for 16 h. The reaction was filtered and concentrated in vacuo to yield the sub-title compound (214 mg) as a glassy solid.
[0862] .sup.1H NMR (400 MHz, DMSO-d6) δ 13.51 (s, 1H), 7.48 (dd, 1H), 7.42-7.34 (m, 1H), 7.26-7.20 (m, 1H), 4.26-4.18 (m, 2H), 3.99 (s, 2H), 3.82-3.71 (m, 2H), 3.66-3.54 (m, 4H), 1.41 (s, 9H).
(iv) tert-Butyl 2-(2-(2-(3-(((benzyloxy)carbonyl)amino)-5-(trifluoromethoxy)phenoxy)ethoxy)-ethoxy)acetate
[0863] NEt.sub.3 (0.071 ml, 0.513 mmol) was added to a stirred solution of benzyl alcohol (0.355 mL, 3.42 mmol), the product from step (iii) above (145 mg, 0.342 mmol) and diphenyl phosphorylazide (0.081 mL, 0.376 mmol) in toluene (2 mL, 0.342 mmol). The reaction was heated to 80° C. for 2 h and concentrated in vacuo. The crude product was purified by chromatography on silica gel (4 g column, 0-50% EtOAc/isohexane) to afford the sub-title compound (53 mg) as a colourless oil.
[0864] m/z 547.2 (M+NH.sub.4).sup.+ (ES.sup.+)
(v) tert-Butyl 2-(2-(2-(3-amino-5-(trifluoromethoxy)phenoxy)ethoxy)ethoxy)acetate
[0865] The product from step (iv) above (50 mg, 0.094 mmol) and Pd—C (20.1 mg, 9.44 μmol) was dissolved/suspended in EtOH (10 mL) and stirred under an atmosphere of H.sub.2 (2 bar) for 16 h. The reaction was cooled, filtered through celite, washing with EtOH (10 mL) and concentrated in vacuo to yield the sub-title compound (38 mg) as a red oil.
[0866] m/z 396.1 (M+H).sup.+ (ES.sup.+)
(vi) tert-Butyl 2-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-(trifluoromethoxy)phenoxy)ethoxy)ethoxy)-acetate
[0867] N-(5-(tert-butyl)-3-(3-(4-((2-chloropyridin-4-yl)oxy)naphthalen-1-yl)ureido)-2-methoxyphenyl)-methanesulfonamide (see WO 2014/162126; 54.7 mg, 0.096 mmol), the product from step (v) above (38 mg, 0.096 mmol), Pd-175 (7.51 mg, 9.61 μmol) and freshly ground potassium carbonate (39.8 mg, 0.288 mmol) were degassed by evacuation and backfilling with nitrogen three times. The resulting mixture was heated to 70° C. for 2 h. The reaction mixture was cooled and injected directly onto a RP column. The crude product was purified by chromatography (RP Flash C18, 12 g column, 25-100% MeCN/10 mM Ammonium Bicarbonate) to afford the sub-title compound (52 mg) as a light yellow solid.
[0868] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.39 (s, 1H), 9.19 (s, 1H), 9.14 (s, 1H), 8.91 (s, 1H), 8.30 (d, 1H), 8.18 (d, 1H), 8.16 (d, 1H), 8.12 (d, 1H), 7.86 (dd, 1H), 7.71 (ddd, 1H), 7.61 (ddd, 1H), 7.40 (d, 1H), 7.31 (s, 1H), 7.28 (t, 1H), 7.03 (d, 1H), 6.66 (dd, 1H), 6.40 (t, 1H), 6.07 (d, 1H), 4.07-4.02 (m, 2H), 3.98 (s, 2H), 3.81 (s, 3H), 3.76-3.71 (m, 2H), 3.59 (s, 4H), 3.10 (s, 3H), 1.40 (s, 9H), 1.27 (s, 9H).
(vii) 2-(2-(2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl) ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-(trifluoromethoxy)phenoxy)ethoxy)ethoxy)acetic acid
[0869] The product from step (vi) above (50 mg, 0.054 mmol) was dissolved in THF (1 mL) and NaOH (2 M aq, 269 μL, 0.539 mmol) added. The mixture was stirred overnight and then acidified to pH 4 with AcOH (0.25 mL) and concentrated in vacuo. The crude product was purified by chromatography (RP Flash C18, 4 g column, 35-65% MeCN/10 mM Ammonium Bicarbonate), the product containing fractions were acidified to pH 4 with formic acid (0.6 mL) and concentrated in vacuo to afford the title compound (20 mg) as a light beige solid.
[0870] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.59 (s, 1H), 9.23 (s, 1H), 9.02 (s, 1H), 8.33 (d, 1H), 8.17 (d, 1H), 816 (d, 1H), 8.11 (d, 1H), 7.86 (dd, 1H), 7.69 (ddd, J=8.4, 1H), 7.60 (ddd, 1H), 7.39 (d, 1H), 7.33 (s, 1H), 7.24 (t, 1H), 7.03 (d, 1H), 6.65 (dd, 1H), 6.42-6.39 (m, 1H), 6.10 (d, 1H), 4.03 (dd, 2H), 3.84 (s, 2H), 3.81 (s, 3H), 3.75-3.68 (m, 2H), 3.58 (s, 4H), 3.09 (s, 3H), 1.27 (s, 9H).
[0871] m/z 872.2 (M+H).sup.+ (ES.sup.+)
Example 33
N-(5-(tert-Butyl)-2-methoxy-3-(3-(4-((2-((3-methoxy-5-(2-(2-((3-oxo-2,3-dihydroisoxazol-5-yl)methoxy)ethoxy)ethoxy)phenyl)amino)pyridin-4-yl)oxy)naphthalen-1-yl)ureido)phenyl)methanesulfonamide
[0872] ##STR00098##
[0873] A slurry of ethyl 4-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)-ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)-3-oxobutanoate (see Example 18(ii) above; 359 mg, 0.404 mmol) in MeOH (3.5 mL) and water (0.5 mL) was cooled to 0° C. and NaOH (2 M aq) (404 μL, 0.809 mmol) was added. The resulting solution was stirred at 0° C. for 10 min. A solution of hydroxylamine hydrochloride (84 mg, 1.213 mmol) in MeOH (200 μL) was prepared and cooled to 0° C. NaOH (2 M aq) (606 μL, 1.213 mmol) was added to the hydroxylamine solution, and stirred at 0° C. for 10 min. The hydroxylamine solution was then added to the solution of enolate and stirred at 0° C. for 2 h. The solution was then added dropwise to conc HCl (100 μL, 3.29 mmol) at 75° C., and stirred at 75° C. for 1 h. The heating was removed and the pH was adjusted to ca 7 with NaOH (2 M aq.) and diluted with water (20 mL). The aqueous layer was extracted with EtOAc (3×10 mL) and the combined organic extracts washed with 20% v/v brine (10 mL). The organic layer was passed through a hydrophobic frit and concentrated in vacuo. The crude product was purified by preparative HPLC (Waters, Basic (0.1% Ammonium Bicarbonate), Basic, Waters X-Bridge Prep-C18, 5 μm, 19×50 mm column, 35-65% MeCN in Water) and the product rich fractions freeze dried to afford the title compound (38 mg) as a light yellow solid.
[0874] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.47 (s, 1H), 8.94 (s, 1H), 8.70 (s, 1H), 8.34 (d, 1H), 8.13-8.05 (m, 2H), 7.84 (d, 1H), 7.67 (ddd, 1H), 7.63-7.54 (m, 2H), 7.36 (d, 1H), 7.03 (d, 1H), 6.91-6.84 (m, 2H), 6.56 (dd, 1H), 6.09 (d, 1H), 6.04 (t, 1H), 4.05 (s, 1H), 4.00-3.94 (m, 2H), 3.84 (s, 1H), 3.78 (s, 3H), 3.73-3.67 (m, 2H), 3.66 (s, 3H), 3.59-3.54 (m, 2H), 3.52-3.47 (m, 2H), 2.61 (s, 3H), 1.22 (s, 9H).
[0875] m/z 857.2 (M+H).sup.+ (ES.sup.+)
Example 34
5-((2-(3-((4-((4-(3-(5-(tert-Butyl)-2-meth oxy-3-methylsulfonamido)phenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)methyl-1-methyl-1H-pyrrole-2-carboxylic acid
[0876] ##STR00099##
(i) Methyl 5-(hydroxymethyl)-1-methyl-1H-pyrrole-2-carboxylate
[0877] Sodium borohydride (0.249 g, 6.58 mmol) was added to a solution of methyl 5-formyl-1-methyl-1H-pyrrole-2-carboxylate (1.1 g, 6.58 mmol) in a mixture of MeOH (50 mL) and THF (15 mL) at 0° C. and the resulting solution stirred at 0° C. for 2 h. The reaction was slowly quenched with sat. aq. ammonium chloride (50 mL) and a white solid crashed out. The solution was filtered, and the filtrate extracted with DCM (2×50 mL). The solvent was removed in vacuo. The crude material was dissolved in DCM (2 mL) and passed through a plug of silica, washing with 5% MeOH in DCM (200 mL). The solvent was removed in vacuo to afford the sub-title compound as a brown oil (1.12 g).
[0878] m/z 170.6 (M+H).sup.+ (ES.sup.+)
(ii) Methyl 5-((2-(benzyl)oxy)ethoxy)methyl-1-methyl-1H-pyrrole-2-carboxylate
[0879] NaH (60% in oil, 213 mg, 5.32 mmol) was added in two portions over 5 min to a solution of the compound from step (i) above (600 mg, 3.55 mmol), ((2-bromoethoxy)methyl)benzene (0.6 mL, 3.79 mmol) and sodium iodide (532 mg, 3.55 mmol) in dry DMF (50 mL) under nitrogen at 0° C., and the resulting solution stirred at rt overnight. The reaction was quenched with MeOH (3 mL) then diluted with 20% v/v brine (100 mL). The aqueous layer was extracted with EtOAc (3×100 mL), and the combined organic extractions washed with 20% v/v brine (50 mL). The organic layer was dried (MgSO.sub.4) and concentrated in vacuo. The crude product was purified by chromatography on silica gel (40 g column, 0-100% EtOAc/isohexane) to afford the sub-title compound (213 mg) as a thin colourless oil.
[0880] .sup.1H NMR (400 MHz, DMSO-d6) δ 7.39-7.24 (m, 5H), 6.80 (d, 1H), 6.15 (d, 1H), 4.51 (s, 2H), 4.48 (s, 2H), 3.83 (s, 3H), 3.74 (s, 3H), 3.65-3.49 (m, 4H).
[0881] m/z 304.1 (M+H).sup.+ (ES.sup.+)
(iii) Methyl 5-((2-hydroxyethoxy)methyl)-1-methyl-1H-pyrrole-2-carboxylate
[0882] Pd/C 10% in 50% paste in water (Type 39) (14.94 mg, 0.140 mmol) was added to a solution of the compound from step (ii) above (213 mg, 0.702 mmol) in EtOH (4 mL) and the resulting slurry stirred under hydrogen at 1 bar pressure for 4 h. The reaction was filtered through celite, washing with EtOAc (20 mL) and the solvent removed to afford the sub-title compound (137 mg) as a thin colourless oil.
[0883] .sup.1H NMR (400 MHz, DMSO-d6) δ 6.80 (d, 1H), 6.16 (d, 1H), 4.63 (t, 1H), 4.49 (s, 2H), 3.84 (s, 3H), 3.74 (s, 3H), 3.54-3.47 (m, 2H), 3.47-3.41 (m, 2H).
[0884] m/z 236.1 (M+Na).sup.+ (ES.sup.+)
(iv) Methyl 5-((2-(3-methoxy-5-nitrophenoxy)ethoxy)methyl)-1-methyl-1H-pyrrole-2-carboxylate
[0885] DIAD (339 μL, 1.745 mmol) was added to a solution of the compound from step (iii) above (310 mg, 1.454 mmol), 3-methoxy-5-nitrophenol (295 mg, 1.745 mmol) and triphenylphosphine (458 mg, 1.745 mmol) in dry THF (15 mL) at 0° C., and the resulting red solution stirred at rt overnight. The yellow solution was diluted with EtOAc (30 mL) and washed with water (20 mL). The organic layer was dried (MgSO.sub.4) and concentrated in vacuo. The crude product was purified by chromatography on the Companion (40 g column, 0-100% EtOAc/isohexane) to afford the desired Mitsunobu product as a yellow solid. The product was dissolved in EtOAc (50 mL) and washed sequentially with NaOH (2 M aq., 2×50 mL), water (50 mL) and 20% v/v brine (50 mL). The organic layer was dried (MgSO.sub.4) and concentrated in vacuo to afford the sub-title compound (638 mg) as a light yellow solid.
[0886] .sup.1H NMR (400 MHz, DMSO-d6) δ 7.33 (m, 2H), 6.96 (t, 1H), 6.79 (d, 1H), 6.18 (d, 1H), 4.57 (s, 2H), 4.30-4.17 (m, 2H), 3.85 (s, 3H), 3.83 (s, 3H), 3.79-3.74 (m, 2H), 3.73 (s, 3H).
[0887] m/z 387.1 (M+Na).sup.+ (ES.sup.+)
(v) Methyl 5-((2-(3-amino-5-methoxyphenoxy)ethoxy)methyl)-1-methyl-1H-pyrrole-2-carboxylate
[0888] Iron powder (978 mg, 17.51 mmol) followed by ammonium chloride (37.5 mg, 0.700 mmol) was added to a solution of the compound from step (iv) above (638 mg, 0.841 mmol) in EtOH (13 mL), THF (5 mL) and water (2 mL) and the resulting slurry heated to reflux for 2 h. The reaction was cooled and filtered through celite, washing with EtOAc (2×20 mL). The solvent was removed in vacuo. The crude product was purified by chromatography on silica gel (24 g column, 0-100% EtOAc/isohexane) to afford the sub-title compound (238 mg) as a thick colourless oil.
[0889] .sup.1H NMR (400 MHz, DMSO-d6) δ 6.80 (d, 1H), 6.18 (d, 1H), 5.75 (m, 2H), 5.67 (t, 1H), 5.05 (s, 2H), 4.55 (s, 2H), 3.99-3.93 (m, 2H), 3.85 (s, 3H), 3.74 (s, 3H), 3.71-3.65 (m, 2H), 3.62 (s, 3H).
[0890] m/z 335.0 (M+H).sup.+ (ES.sup.+)
(vi) Methyl 5-((2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-methylsulfonamido)phenyl)ureido)-naphthalen-1-)oxy)pyridin-2-yl)amino)-5-methoxyphenoxy)ethoxy)methyl-1-methyl-1H-pyrrole-2-carboxylate
[0891] A suspension of N-(5-(tert-butyl)-3-(3-(4-((2-chloropyridin-4-yl)oxy)naphthalen-1-yl)ureido)-2-methoxyphenyl)methanesulfonamide (see WO 2014/162126; 405 mg, 0.712 mmol), the compound from step (v) above (238 mg, 0.712 mmol), freshly ground potassium carbonate (295 mg, 2.135 mmol) in DMF (3 mL) was evacuated back filling with nitrogen 3 times. The mixture was heated under nitrogen to 40° C. and Pd-175 (13.90 mg, 0.018 mmol) added. The reaction mixture was heated at 75° C. for 2 h. The reaction was then cooled and filtered. The filtrate was partitioned between EtOAc (50 mL) and 20% v/v brine (50 mL). The organic layer was passed through a hydrophobic frit then concentrated. The crude product was purified by chromatography (RP Flash C18) (24 g column, 15-80% MeCN/10 mM Ammonium Bicarbonate) to afford the sub-title compound (350 mg) as a white solid.
[0892] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.38 (s, 1H), 9.13 (s, 1H), 8.91 (s, 1H), 8.87 (s, 1H), 8.29 (d, 1H), 8.18 (d, 1H), 8.11 (d, 1H), 8.10 (d, 1H), 7.87 (dt, 1H), 7.70 (ddd, 1H), 7.61 (ddd, 1H), 7.38 (d, 1H), 7.03 (d, 1H), 6.89 (t, 1H), 6.86-6.77 (m, 2H), 6.58 (dd, 1H), 6.17 (d, 1H), 6.08 (d, 1H), 6.02 (t, 1H), 4.55 (s, 2H), 4.07-3.95 (m, 2H), 3.84 (s, 3H), 3.81 (s, 3H), 3.73 (s, 3H), 3.72-3.69 (m, 2H), 3.65 (s, 3H), 3.10 (s, 3H), 1.27 (s, 9H).
[0893] m/z 867.3 (M+H).sup.+ (ES.sup.+)
(vii) 5-((2-(3-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-5-ethoxyphenoxy)ethoxy)methyl)-1-methyl-1H-pyrrole-2-carboxylic acid
[0894] NaOH (2M aq) (606 μl, 1.211 mmol) was added to a solution of the compound from step (vi) above (350 mg, 0.404 mmol) in THF (3 mL) and MeOH (1.2 mL) and the resulting solution stirred at rt for 8 h. A further portion of NaOH (2M aq) (606 μL, 1.211 mmol) was added and the resulting solution stirred at rt overnight. The solution was quenched with AcOH (139 μL, 2.422 mmol) and the solvent removed in vacuo. The crude product was purified by chromatography (RP Flash 018) (12 g column, 15-75% MeCN/10 mM Ammonium Bicarbonate). The product rich fractions were combined and the pH adjusted to 7 with formic acid. The volatile solvent was removed in vacuo during which a solid crashed out. This was collected by filtration, washing with water (2×2 mL) and the solid dried in vacuo at 40° C. overnight to afford the title compound (165 mg) as a white solid.
[0895] .sup.1H NMR (400 MHz, DMSO-d6) δ 12.19 (s, 1H), 9.38 (s, 1H), 9.13 (s, 1H), 8.91 (s, 1H), 8.87 (s, 1H), 8.29 (d, 1H), 8.19 (d, 1H), 8.12 (d, 1H), 8.10 (d, 1H), 7.87 (dd, 1H), 7.70 (ddd, 1H), 7.61 (ddd, 1H), 7.38 (d, 1H), 7.03 (d, 1H), 6.90 (t, 1H), 6.79 (t, 1H), 6.74 (d, 1H), 6.57 (dd, 1H), 6.13 (d, 1H), 6.08 (d, 1H), 6.03 (t, 1H), 4.53 (s, 2H), 4.06-3.93 (m, 2H), 3.84 (s, 3H), 3.81 (s, 3H), 3.75-3.68 (m, 2H), 3.65 (s, 3H), 3.10 (s, 3H), 1.27 (s, 9H).
[0896] m/z 853.0 (M+H).sup.+ (ES.sup.+)
Biological Testing: Experimental Methods
Enzyme Binding Assays (Kinomescan)
[0897] 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
[0898] 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 MAPKα Enzyme Inhibition
[0899] The following two assay variants can be used for determination of p38 MAPKα inhibition.
Method 1
[0900] The inhibitory activities of test compounds against the p38 MAPKα isoform (MAPK14: Invitrogen) are evaluated indirectly by determining the level of activation/phosphorylation of the down-stream molecule, MAPKAP-K2. The p38 MAPKα protein (80 ng/mL, 2.5 μL) is mixed with the test compound (2.5 μL of either 4 μg/mL, 0.4 μg/mL, 0.04 μg/mL or 0.004 μg/mL) for 2 hr at RT. The mix solution (2.5 μL) of the p38α inactive target MAPKAP-K2 (Invitrogen, 600 ng/mL) and FRET peptide (8 μM; a phosphorylation target for MAPKAP-K2) is then added, then the kinase reaction is initiated by adding ATP (40 μM, 2.5 μL). The mixture is incubated for 1 hr at RT. Development reagent (protease, 5 μL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan® Flash, ThermoFisher Scientific).
Method 2
[0901] This method follows the same steps as Method 1 above, but utilises a higher concentration of the p38 MAPKα protein (2.5 μL of 200 ng/mL protein instead of 2.5 μL of 80 ng/mL protein) for mixing with the test compound (tested at either 1 μg/mL, 0.1 μg/mL, 0.01 μg/mL or 0.001 μg/mL).
p38 MAPKγ Enzyme Inhibition
[0902] The inhibitory activities of compounds of the invention against p38MAPKγ (MAPK12: Invitrogen), are evaluated in a similar fashion to that described hereinabove. The enzyme (800 ng/mL, 2.5 μL) is incubated with the test compound (2.5 μL of either 4 μg/mL, 0.4 μg/mL, 0.04 μg/mL, or 0.004 μg/mL) for 2 hr at RT. The FRET peptides (8 μM, 2.5 μL), and appropriate ATP solution (2.5 μL, 400 μM) are then added to the enzymes/compound mixtures and the whole is incubated for 1 hr. Development reagent (protease, 5 μL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan® Flash, Thermo Scientific).
c-Src and Syk Enzyme Inhibition
[0903] 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 μL) is incubated with the test compound (either 1 μg/mL, 0.1 μg/mL, 0.01 μg/mL, or 0.001 μg/mL, 2.5 μL each) for 2 hr at RT. The FRET peptides (8 μM. 2.5 μL), and appropriate ATP solutions (2.5 μL, 800 μM for c-Src, and 60 μM ATP for Syk) are then added to the enzymes/compound mixtures and the mixture incubated for 1 hr. Development reagent (protease, 5 μL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan® Flash, ThermoFisher Scientific).
GSK 3α Enzyme Inhibition
[0904] The following two assay variants can be used for determination of GSK 3α inhibition.
Method 1
[0905] The inhibitory activities of compounds of the invention against the GSK 3α enzyme isoform (Invitrogen), are evaluated by determining the level of activation/phosphorylation of the target peptide. The GSK3-α protein (500 ng/mL, 2.5 μL) is mixed with the test compound (2.5 μL at either 4 μg/mL, 0.4 μg/mL, 0.04 μg/mL, or 0.004 μg/mL) for 2 hr at RT. The FRET peptide (8 μM, 2.5 μL), which is a phosphorylation target for GSK3α, and ATP (40 μM, 2.5 μL) are then added to the enzyme/compound mixture and the resulting mixture incubated for 1 hr. Development reagent (protease, 5 μL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan® Flash, ThermoFisher Scientific).
[0906] 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
[0907] 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-α protein. In addition, the concentrations of test compound employed are either 10 μg/mL, 1 μg/mL, 0.1 μg/mL, or 0.01 μg/mL
Cellular Assays
[0908] The compounds of the invention were studied using one or more of the following assays.
(a) Inhibition of p38 MAPKα and Lck in Jurkat Cells
[0909] Jurkat T cells were cultured in starve medium (RPMI 1640+5% FBS) for 24 h prior to the experiment. Cells were harvested and resuspended at 10×10.sup.6 cells/mL in starve medium and then plated into round-bottomed 96 well plates at 1×10.sup.6 cells/well. Serial dilutions of test compound were added (1% final DMSO concentration) for 2 h prior to stimulation. Following pre-incubation with compound, cells were stimulated with H.sub.2O.sub.2 (0.05% final) for 5 min. The reaction was stopped by centrifugation at 2000 rpm (3 min, 4° C.), then the supernatant was removed and 100 μL of cold fix/perm solution (BD Fix/Perm kit #554714) added. Plates were incubated for 20 min at 4° C. before centrifugation and washing with supplied 1× wash medium (BD Fix/Perm kit #554714). Cells were stained for either phospho-p38α (T180/182), supplied by Cell Signalling Technology (9211s), or phospho-Lck (Y394), supplied by R&D (MAB7500). Antibodies were diluted to 5 μg/mL (R&D) or 1:200 (Cell Signalling Technology) in wash medium, before being incubated 1 h at 4° C. in the dark. Following 3 repeat washes with ice cold wash buffer, secondary antibody (anti-rabbit-FITC #F1362 or anti-mouse-FITC #F2883, both from Sigma) was added at a dilution of 1:1000 and incubated for 1 h at 4° C. in the dark. Cells were washed 3× times in cold wash buffer then, following a final wash in cold PBS, were resuspended in 150 μL cold PBS. Cells were analysed by flow cytometry using BD Accuri C6.
(Aa) LPS-Induced TNFα/IL-8 Release in d-U937 Cells
[0910] 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 μg/mL of LPS (from E. Coli: 0111:B4, Sigma) for 4 hr. The supernatant is collected for determination of TNFα and IL-8 concentrations by sandwich ELISA (Duo-set, R&D systems). The inhibition of TNFα production is calculated as a percentage of that achieved by 10 μg/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 TNFα/IL-83 Release in PBMC Cells
[0911] 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 TNFα 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 TNFα production is calculated from the dose response curve.
(c) IL-2 and IFN Gamma Release in CD3/CD28 Stimulated PBMC Cells
[0912] 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 μg/mL eBioscience and 3 μg/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-1β-Induced IL-8 Release in HT29 Cells
[0913] HT29 cells, a human colon adenocarcinoma cell line, are plated in a 96 well plate (24 hr) and pre-treated with compounds at the desired concentration for 2 hours before addition of 5 ng/mL of IL-1β (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 TNFα Release in Primary Macrophages
[0914] 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, they 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 TNFα 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
[0915] Poly I:C is used in these studies as a simple, RNA virus mimic. Poly I:C-Oligofectamine mixture (1 μg/mL Poly I:C, ±2% Oligofectamine, 25 μL; 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 μL, 0.05% Tween in PBS: PBS-Tween) containing 0.1% sodium azide and 1% hydrogen peroxide. Cells are washed with wash-buffer (3×200 μL) and after blocking the wells with 5% milk in PBS-Tween (100 μL) for 1 hr, the cells are incubated with anti-human ICAM-1 antibody (50 μL; Cell Signalling Technology, Danvers, Mass.) in 1% BSA PBS overnight at 4° C.
[0916] The cells are washed with PBS-Tween (3×200 μL) and incubated with the secondary antibody (100 μL; HRP-conjugated anti-rabbit IgG, Dako Ltd., Glostrup, Denmark). The cells are then incubated with substrate (50 μL) for 2-20 min, followed by the addition of stop solution (50 μL, 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 (3×200 μL) and total cell numbers in each well are determined by reading absorbance at 595 nm after Crystal Violet staining (50 μL of a 2% solution in PBS) and elution by 1% SDS solution (100 μL) 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
[0917] 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 20% 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 μg/mL; Invitrogen, Paisley, UK) to arrest cells in metaphase. To observe mitotic cells, PBMCs are permeabilised and fixed by adding Intraprep (50 μL; 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
[0918] 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.
[0919] BEAS2B cells are infected with HRV at an MOI of 5 and incubated for 2 hr at 33° C. with gentle shaking 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.).
[0920] The level of ICAM-1 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 with 1% SDS solution. The measured OD.sub.450-855 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 Cytopathic Effect (CPE) in MRC5 Cells
[0921] MRC5 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.
[0922] Supernatants are aspirated and incubated with methylene blue solution (100 μL, 2% formaldehyde, 10% methanol and 0.175% Methylene Blue) for 2 hr at RT. After washing, 1% SDS in distilled water (100 μL) 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
[0923] Normal human bronchial epithelial cells (NHBEC) grown in 96 well plates are infected with RSV A2 (Strain A2, HPA, Salisbury, UK) at a 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 washed with PBS (3×200 μL), then fresh media (200 μL) 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.
[0924] The cells are fixed with 4% formaldehyde in PBS solution (50 μL) for 20 min, washed with WB (3×200 μL) (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 (3×200 μL) and incubated for 1 hr at RT with anti-RSV (2F7) F-fusion protein antibody (40 μL; 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 μL) in 5% BSA in PBS-Tween (lot 00053170, Cat. No. P0447, Dako) and then TMB substrate added (50 μL; 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 μL) 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).
[0925] Cells are then washed and a 2.5% crystal violet solution (50 μL; lot 8656, Cat. No. PL7000, Pro-Lab Diagnostics) is applied for 30 min. After washing with WB, 1% SDS in distilled water (100 μL) 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.450-855 readings are corrected to the cell number by dividing the OD.sub.450-855 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
[0926] Differentiated U937 cells are pre-incubated with each test compound (final concentration 1 μg/mL or 10 μg/mL in 200 μL 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 μL) and MTT stock solution (10 μL, 5 mg/mL) is added to each well. After incubation for 1 hr the media are removed, DMSO (200 μL) 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
[0927] Intestinal mucosa biopsies are obtained from the inflamed regions of the colons 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-1β and TNFα 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 β Catenin in d-U937 Cells
[0928] 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 β-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 μL, 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 μL; PBS containing 0.05% Tween-20) and incubated with blocking solution (200 μL; 5% milk in PBS) for 1 hr, re-washed with washing buffer (200 μL) and then incubated overnight with anti-β-catenin antibody solution (50 μL) in 1% BSA/PBS (BD, Oxford, UK).
[0929] After washing with washing buffer (3×200 μL; PBS containing 0.05% Tween-20), cells are incubated with a HRP-conjugated secondary antibody solution (100 μL) 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 μL; R&D Systems, Abingdon, UK). This reaction is stopped by addition of 1N H.sub.2SO.sub.4 solution (50 μL). Cells are then washed with washing buffer and 2% crystal violet solution (50 μL) is applied for 30 min. After washing with washing buffer (3×200 μL), 1% SDS (100 μL) 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).
[0930] The measured OD.sub.450-855 readings are corrected for cell number by dividing the OD.sub.450-855 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 the Reference compound N-(4-(4-(3-(3-tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)ureido)naphthalen-1-yloxy)pyridin-2-yl)-2-methoxyacetamide (1 μg/mL), which is defined as unity.
(n) T Cell Proliferation
[0931] 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). Naïve 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 2×10.sup.5 cells per well in 100 μL RPMI/10% FBS on 96 well flat bottomed plate (Corning Costar). 25 μL of test compound are diluted to the appropriate concentration (8× final concentration) 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 μg/mL anti-CD3 (OKT3; eBioscience). After 72 h, the medium in each well is replaced with 150 μL of fresh medium containing 10 μM BrdU (Roche). After 16 h, the supernatant is removed, the plate is dried and the cells fixed by adding 100 μL 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 μL of substrate solution. The reaction is stopped by addition of 50 μL 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 IFNγ Release in CD3/CD28 Stimulated LPMC Cells from IBD Patients
[0932] 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: The mucosa is removed from the deeper layers of the surgical specimens with a scalpel and cut in fragments of size 3-4 mm. 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 μm cell strainer, washed twice, resuspended in RPMI-1640 medium (Sigma-Aldrich) containing 10% fetal calf serum, 100 U/mL penicillin and 100 μg/mL streptomycin, and used for cell culture.
[0933] Freshly isolated LPMCs (2×10.sup.5 cells/well) are stimulated with 1 μg/mL α-CD3/α-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 TNFα and IFNγ 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
[0934] Myofibroblasts from inflamed IBD mucosa are isolated as follows:
[0935] 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 μg/mL streptomycin, 50 μg/mL gentamycin, and 1 μg/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.
[0936] Subconfluent monolayers of myofibroblasts, seeded in 12-well plates at 3×10.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
[0937] Neutrophils are isolated from human peripheral blood as follows:
[0938] 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 μg/mL) and 1 mM CaCl.sub.2) to achieve 5×10.sup.6 cells/mL.
[0939] 5×10.sup.4 cells are added to each well of a V-bottom 96 well plate and are 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 concentration 1 μM). 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
[0940] 1×10.sup.5 Jurkat cells (immortalised human T lymphocytes) are added to the appropriate number of wells of a 96 well plate in 100 μL of media (RPMI supplemented with 10% foetal bovine serum). 1 μL of DMSO control (final concentration 1.0% v/v) or test compound (final concentration 20, 5 or 1 μg/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 μL (final concentration 7.5 μg/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 P 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
[0941] 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 represented as mean±S.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
[0942] 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 μL 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 bronchoalveolar 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
[0943] 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
[0944] 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 μL) 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
[0945] 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 4×10.sup.5 cells/mL CD45RB.sup.high cells are then injected intraperitoneally (100 μL/mouse) into female SCID animals. On study day 14, mice are weighed and random/zed into treatment groups based on body weight. On Day 14, compounds are administered BID, via oral gavage, in 5% polyoxyethylene 40 stearate in 20 mM pH 7.8 aqueous phosphate buffer in a dose volume of 5 mL/kg. Treatment continues until study day 49, at which point the animals are necropsied 4 hours after the morning administration. The colon length and weight are 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 naïve animals and vehicle animals, where higher inhibition implies closer to the non-diseased, naïve, phenotype.
(vi) Endotoxin-Induced Uveitis in Rats
[0946] 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 μL dose volume) of 100 ng/animal 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 or vehicle (4% polyoxyl 40 stearate, 4% mannitol in PBS (pH 7.4)) are administered by the topical route onto the right eye (10 μL) of animals 1 hour prior to LPS, at the time of LPS administration, and 1, 2 and 4 hours post LPS administration. Before administration, the solution to be administered is sonicated to ensure a clear solution. 6 hours after LPS dosing, animals are euthanized by overdose with pentobarbitone (via cardiac puncture). Immediately after euthanasia, 10 μL of aqueous humor is collected from the right eye of the rats by puncture of the anterior chamber using a 32 gauge needle under a surgical microscope. The aqueous humor is diluted in 20 μL of PBS and total cell counts are measured immediately using a Countess automated cell counter (Invitrogen). Following collection of the aqueous humour, 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 μL 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
[0947]
TABLE-US-00004 TABLE 1 Results from in vitro p38 MAPKα (Method 2), c-Src and Syk inhibition assays Test Compound IC50 Values for Enzyme Inhibition (nM) Example No. p38 MAPKα c-Src Syk 1 — — — 2 23 14 8 3 23 18 12
TABLE-US-00005 TABLE 2 Inhibition of cytokine release in stimulated cells (assays (b) and (c) above). Test Compound IC50 Values in PBMCs (nM) Example No. IL-8 IFNγ TNFα 1 115.1 — — 2 24.6 39.4 — 3 17.3 31.8 10.8 5 19.5 — — 6 18.1 — — 7 20.8 — — 8 8.0 — — 9 11.7 — — 10 26.5 — — 11 11.7 — — 12 5.6 — — 13 2.8 — — 14 7.3 — — 15 16.4 — — 16 16.3 — — 17 17.2 — — 18 29.3 — — 19 2.9 — — .sup. 20(a) 10.4 — — .sup. 20(b) 19.1 — — 21 14.4 — — 22 18.1 — — 23 29.2 — — 24 61.9 — — 25 26.3 — — 26 15.3 — — 27 7.3 — — 28 20.6 — —
[0948] As illustrated in Table 3 below, compounds of the examples of the present invention are substantially less cytotoxic than the Reference Compound (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), displaying enhanced viabilities in cell cytotoxicity assay (r) above (Table 3). In addition, the compounds of the examples of the present invention are substantially less cytotoxic at 20 μg/mL than the Reference Compound A (3-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)naphthalen-1-yl)oxy)-pyrimidin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)propanoic acid; WO 2014162126).
TABLE-US-00006 TABLE 3 Effect of compounds of the examples on Jurkat cell viability (assay (r) above; NT = not tested). Test % Viability % Viability % Viability compound at 1 μg/mL at 5 μg/mL at 20 μg/mL Reference 29 26 24 compound Reference 96 93 48 compound A 1 NT NT NT 2 99 99 97 3 99 99 96
[0949] As illustrated in Table 4 below, the compound of Example 3 was also screened in the in vivo (adoptive transfer) assay (v) above alongside Reference Compound A, (3-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)naphthalen-1-yl)oxy)pyrimidin-2-yl)amino)-5-methoxyphenoxy)ethoxy)ethoxy)propanoic acid, and Reference Compound B, 3-((4-((4-(3-(3-(tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)ureido)naphthalen-1-yl)oxy)pyrimidin-2-yl)amino)-5-ethynyl-N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)benzamide {Fyfe, M. C. T., WO 2014/140582}. Analysis of the relative ratios of colon weight to length in naïve, control and treated animals at the end of the study revealed that the compound of Example 3 provided superior activity compared to the two Reference Compounds in this T cell driven in vivo model of colonic inflammation using a simple aqueous-based vehicle.
TABLE-US-00007 TABLE 4 Summary of results from adoptive transfer mouse model. Treatment group Dose Colon weight:length % Inhibition Naïve N/A 0.021 ± 0.001 100 Vehicle control N/A 0.043 ± 0.007 0 Reference Compound A 3 mg/kg.sup.[†] 0.047 ± 0.007 −14 Reference Compound B 3 mg/kg 0.041 ± 0.005 11 Example 3 3 mg/kg 0.032 ± 0.004 49 .sup.[†]Dose was lowered to 0.6 mg/kg on day 27 because of poor tolerability (body weight loss).
Summary of Additional Studies
Determination of Solubilities in Fasted-State Simulated Colonic Fluid (FaSSCoF)
[0950] The solubilities of compounds of the invention in FaSSCoF at pH 6.5 are determined using a modification of a previously-reported procedure (Vertzoni, M., et al. Pharm. Res. 2010, 27, 2187-2196). In place of the bile salt extract employed in the original procedure (which extract is no longer available), the modified procedure uses a mixture of sodium taurocholate (0.15 g), glycocholic acid (0.15 g), ursodeoxycholic acid (0.05 g), cholic acid (0.05 g), and glycodeoxycholic acid (0.05 g). These five bile acids are ground together with a mortar and pestle to produce a fine white powder that is incorporated into the FaSSCoF, as outlined below.
[0951] FaSSCoF Medium:
[0952] Tris(hydroxymethyl)aminomethane (Tris; 0.275 g) and maleic acid (0.44 g) are dissolved in water (35 mL) to give a solution whose pH is adjusted to 6.5 by treatment with 0.5M NaOH (ca. 12 mL). The solution is then made up to 50 mL with water. A portion of this Tris/maleate buffer solution (ca. 25 mL) is added to a 0.5 L round-bottomed flask, before being treated with 0.00565 g of the bile acid mixture described above. Solutions of phosphatidylcholine (0.0111 g) in DCM (0.15 mL) and palmitic acid (0.0013 g) in DCM (0.15 mL) are added, then the organic solvent is evaporated off under reduced pressure at 40° C. until a clear solution, with no perceptible DCM odour, is achieved. The volume of the evaporated solution is adjusted to 50 mL by addition of the remainder of Tris/maleate buffer, then BSA (0.115 g) is added, before being dissolved by gentle agitation.
[0953] Solubility Determination:
[0954] Test compounds are suspended in the pH 6.5 FaSSCoF medium to give a maximum final concentration of 2-10 mg/mL. The suspensions are equilibrated at 25° C. for 24 h, before being filtered through a glass fibre C filter. The filtrates are then diluted as appropriate for injection and quantification by HPLC with reference to a standard. Different volumes of the standard, diluted and undiluted sample solutions are injected and the solubilities are calculated using the peak areas determined by integration of the peak found at the same retention time as the principal peak in the standard injection.
[0955] FaSSCoF solubilities are shown in Table 5 below, which reveals that compounds of the Examples (or salts thereof) exhibited solubilities in the FaSSCoF medium at pH 6.5 in excess of 0.03 mg/mL, while some displayed solubilities greater than 1 mg/mL. The pH 6.5 FaSSCoF solubilities measured for compounds of the Examples were superior to those of both Reference Compound A, (3-(2-(2-(3-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methyl-sulfonamido)phenyl)ureido)naphthalen-1-yl)oxy)pyrimidin-2-yl)amino)-5-methoxyphenoxy)-ethoxy)ethoxy)propanoic acid, and Reference Compound B, 3-((4-((4-(3-(3-(tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)ureido)naphthalen-1-yl)oxy)pyrimidin-2-yl)amino)-5-ethynyl-N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)benzamide {Fyfe, M. C. T., WO 2014/140582}.
TABLE-US-00008 TABLE 5 Solubilities measured for certain compounds of the Examples of the present invention, or salts thereof, in FaSSCoF at pH 6.5. Test Compound pH 6.5 FaSSCoF Solubility (mg/mL) Example No. Run 1 Run 2 Run 3 Run 4 Reference Compound A 0.007 0.007 — — Reference Compound A 0.18 0.12 0.03 0.03 (sodium salt) Reference Compound B <0.001 <0.001 — — 2 0.09 0.06 — — 2 (sodium salt) 0.58 — — — 3 0.28 0.20 — — 31 1.90 2.10 — — 8 2.8 3.2 — — 9 0.46 0.54 — — 14 1.2 1.1 — — 17 1.7 1.5 — — 19 0.03 0.03 — — .sup. 20(a) 0.62 0.64 — —
[0956] Further studies also revealed that, in phosphate-buffered saline at either pH 6.5 with 0.5% by weight of simulated intestinal fluid or pH 7.2, the compound of Example 31 (i.e. the sodium of the compound of Example 3) was both more soluble than, and had a faster dissolution rate than, both of Reference Compounds A (hydrochloride salt) and B.
Microcentrifuge Dissolution Tests
[0957] In vitro non-sink dissolution performance of the Compound of Example 3 was evaluated alongside Reference Compounds A and B employing microcentrifuge dissolution tests in which samples were either 1) transferred from intestinal buffer (IB) to colonic buffer (CB) or 2) dosed directly into intestinal media containing simulated bile-salt micelles (IB-SIF). Dissolution performance of the compounds at various timepoints was determined by centrifugation and analysis of the supernatant concentrations by off-line reverse-phase HPLC analysis.
1) Transfer from Intestinal Buffer (IB) to Colonic Buffer (CB) Experiment
[0958] Test compounds (0.45 mg±0.05 mg) were weighed into a microcentrifuge tube, then 0.900 mL of IB receptor solution—phosphate buffered saline (PBS) warmed to 37° C. at pH 6.5—was added. A timer was started and the sample tubes were vortexed at the maximum setting for 1 minute. When the timer read 3, 13 and 23 min—corresponding to the 25, 15 and 5 min timepoints, respectively—the sample tubes were centrifuged for 1 min at 15,800 Relative Centrifugal Force (RCF). At each timepoint, a portion of the supernatant (50 μL) was added to diluent {250 μL of 75/25 THF/Water (v/v)} and the compound concentrations were measured by off-line HPLC analysis (Table 6a). After a further 5 min, 0.900 mL of CB receptor solution—pH 10.7 PBS solution—was added, such that the pH was adjusted to 7.2, and the timer was reset to 0 min. When the timer read 2, 8, 18, 38, 88 and 1,198 min—corresponding to the 4, 10, 20, 40, 90 and 1,200 min timepoints, respectively—the sample tubes were centrifuged for 1 min at 15,800 RCF. At each timepoint, a portion of the supernatant (50 μL) was added to diluent {250 μL of 75/25 THF/Water (v/v)} and the compound concentrations were measured by off-line HPLC analysis (Table 6a). The HPLC samples for the 90 and 1,200 min timepoints were additionally centrifuged for 8 min at 80,000 rpm at 37° C. in an ultracentrifuge (UCF), then the supernatant (50 μL) was added to diluent {250 μL of 75/25 THF/Water (v/v)} and the compound concentrations were measured by off-line HPLC analysis (Table 6b) to determine the concentration of free drug+drug in micelles.
TABLE-US-00009 TABLE 6a Concentrations (μg/mL) measured during dissolution test 1). Test Cpd IB Timepoint (min) CB Timepoint (min) Example No. −30 −25 −15 −5 4 10 20 40 90 1,200 Reference 0.0 2.9 4.6 5.3 11.4 11.5 10.7 10.7 8.4 7.9 Compound A Reference 0.0 1.9 1.0 2.2 5.0 1.9 0.9 0.6 0.0 4.9 Compound B 3 0.0 5.3 7.5 8.5 9.3 9.1 14.3 48.3 60.7 85.4
TABLE-US-00010 TABLE 6b Additional concentrations (μg/mL) measured during dissolution test 1). UCF Timepoint (min) Test Compound Example No. 90 1,200 Reference Compound A 5.1 3.9 Reference Compound B 0.0 1.6 3 34.1 74.9
2) Experiment with Direct Dosing into Intestinal Media Containing Simulated Bile-Salt Micelles (IB-SIF)
[0959] Test compounds (0.45 mg±0.05 mg) were weighed into a microcentrifuge tube then 1.800 mL of IB-SIF receptor solution—prepared previously by dissolution of 0.250 g SIF powder (Biorelevant.com) in 50 mL of pH 6.5 PBS warmed to 37° C.—was added. A timer was started and the sample tubes were vortexed at the maximum setting for 1 minute. When the timer read 2, 8, 18, 38, 88 and 1,198 min—corresponding to the 4, 10, 20, 40, 90 and 1,200 min timepoints, respectively—the sample tubes were centrifuged for 1 min at 15,800 RCF. Then, at each timepoint, the supernatant (50 μL) was added to diluent {250 μL of 75/25 THF/Water (v/v)} and the compound concentrations were measured by off-line HPLC analysis (Table 7a). To determine the concentration of free drug+drug in micelles, the HPLC samples for the 90 and 1,200 min timepoints were additionally centrifuged for 8 min at 80,000 rpm at 37° C. in an ultracentrifuge (UCF), then the supernatant (50 μL) was added to diluent {250 PL of 75/25 THF/Water (v/v)} and the compound concentrations were measured by off-line HPLC analysis (Table 7b).
TABLE-US-00011 TABLE 7a Concentrations (μg/mL) measured during dissolution test 2). Test Compound IB Timepoint (min) Example No. 0 4 10 20 40 90 1,200 Reference 0.0 39.8 63.4 83.5 96.5 99.9 101.0 Compound A Reference 0.0 1.1 0.9 1.3 1.0 0.7 3.0 Compound B 3 0.0 255.7 255.9 251.7 249.2 253.5 239.0
TABLE-US-00012 TABLE 7b Additional concentrations (μg/mL) measured during dissolution test 2). UCF Timepoint (min) Test Compound Example No. 90 1,200 Reference Compound A 87.0 88.8 Reference Compound B 0.7 2.9 3 229.4 169.9
Abbreviations
[0960] AcOH glacial acetic acid [0961] aq aqueous [0962] 5-ASA 5-aminosalicylic acid [0963] ATP adenosine-5′-triphosphate [0964] BALF bronchoalveolar lavage fluid [0965] BID bis in die (twice-daily) [0966] BINAP 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl [0967] BOP (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate [0968] br broad [0969] BrdU 5-bromo-2′-deoxyuridine [0970] BSA bovine serum albumin [0971] CatCart® catalytic cartridge [0972] CDI 1,1-carbonyl-diimidazole [0973] COPD chronic obstructive pulmonary disease [0974] d doublet [0975] dba dibenzylideneacetone [0976] DBU 1,8-diazabicyclo[5.4.0]undec-7-ene [0977] DCC dicyclohexylcarbodiimide [0978] DCM dichloromethane [0979] DIAD diisopropyl azodicarboxylate [0980] DIPEA diisopropylethylamine [0981] DMAP 4-dimethylaminopyridine [0982] DMEM Dulbecco's modified eagle medium [0983] DMF N,N-dimethylformamide [0984] DMSO dimethyl sulfoxide [0985] DPPA diphenylphosphoryl azide [0986] d-U937 cells PMA differentiated U-937 cells [0987] EDTA ethylenediaminetetraacetic acid [0988] ELISA enzyme-linked immunosorbent assay [0989] (ES.sup.−) electrospray ionization, negative mode [0990] (ES.sup.+) electrospray ionization, positive mode [0991] Et ethyl [0992] Et.sub.3N triethylamine [0993] EtOAc ethyl acetate [0994] EtOH ethanol [0995] FACS fluorescence-activated cell sorting [0996] FBS foetal bovine serum [0997] FCS foetal calf serum [0998] fMLP formyl-methionyl-leucyl-phenylalanine [0999] FRET fluorescence resonance energy transfer [1000] GSK3α glycogen synthase kinase 3a [1001] HBEC primary human bronchial epithelial cells [1002] HBSS Hank's balanced salt solution [1003] HPLC high performance liquid chromatography [1004] HPMC hydroxypropylmethylcellulose [1005] h or hr hour(s) [1006] HATU 2-(1H-7-azabenzotriazol-1-yl)-1, 1,3,3-tetramethyl uronium [1007] hexafluorophosphate [1008] HOAt 1-hydroxy-7-azabenzotriazole [1009] HOBt hydroxybenzotriazole [1010] HRP horseradish peroxidise [1011] HRV human rhinovirus [1012] ICAM-1 inter-cellular adhesion molecule 1 [1013] IFNγ interferon-γ [1014] IL interleukin [1015] iPrOAc isopropyl acetate [1016] JNK c-Jun N-terminal kinase [1017] LC liquid chromatography [1018] Lck lymphocyte-specific protein tyrosine kinase [1019] LiHMDS lithium bis(trimethylsilyl)amide [1020] LPS lipopolysaccharide [1021] m multiplet [1022] (M+H).sup.+ protonated molecular ion [1023] MAPK mitogen-activated protein kinase [1024] MAPKAP-K2 mitogen-activated protein kinase-activated protein kinase-2 [1025] mCPBA meta-chloroperbenzoic acid [1026] Me methyl [1027] MeCN acetonitrile [1028] MeOH methanol [1029] MHz megahertz [1030] min or mins minute(s) [1031] MMAD mass median aerodynamic diameter [1032] MOI multiplicity of infection [1033] MPO myeloperoxidase [1034] MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide [1035] MS mass spectrometry [1036] m/z mass-to-charge ratio [1037] NMP N-methyl pyrrolidinone [1038] NMR nuclear magnetic resonance (spectroscopy) [1039] OD optical density [1040] PBMC peripheral blood mononuclear cell [1041] PBS phosphate buffered saline [1042] Ph phenyl [1043] PHA phytohaemagglutinin [1044] PMA phorbol myristate acetate [1045] pTSA 4-methylbenzenesulfonic acid (para-toluenesulfonic acid) [1046] PyBOP (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate [1047] q quartet [1048] rt or RT room temperature [1049] RP HPLC reverse phase high performance liquid chromatography [1050] rpm revolutions per minute [1051] RPMI Roswell Park Memorial Institute [1052] RSV respiratory syncytial virus [1053] s singlet [1054] sat or satd saturated [1055] SCID severe combined immunodeficiency [1056] SCX solid supported cation exchange (resin) [1057] SDS sodium dodecyl sulfate [1058] S.sub.NAr nucleophilic aromatic substitution [1059] Syk Spleen tyrosine kinase [1060] t triplet [1061] T3P 1-propanephosphonic acid cyclic anhydride [1062] TBAl tetrabutylammonium iodide [1063] TBAF tetrabutylammonium fluoride [1064] TBDMS tert-butyldimethylsilyl [1065] TBME tert-butyl methyl ether [1066] TBSCl tert-butyldimethylsilyl chloride [1067] tBuXPhos 2-di-tert-butylphosphino-2′,4′,6′-triisopropylbiphenyl [1068] TCID.sub.50 50% tissue culture infectious dose [1069] TEA triethylamine [1070] THF tetrahydrofuran [1071] TFA trifluoroacetic acid [1072] TGFβ transforming growth factor beta [1073] TIPS triisopropylsilyl [1074] TMB 3,3′,5,5′-tetramethylbenzidine [1075] TMS-Cl trimethylsilyl chloride [1076] TNFα tumor necrosis factor alpha
[1077] Prefixes n-, s-, i-, t- and tert- have their usual meanings: normal, secondary, iso, and tertiary.