USE OF EZH2 INHIBITOR COMBINED WITH BTK INHIBITOR IN PREPARING DRUG FOR TREATING TUMOR
20210030736 ยท 2021-02-04
Inventors
- Ke MA (Lianyungang, Jiangsu, CN)
- Guoqing CAO (Lianyungang, Jiangsu, CN)
- Changyong Yang (Lianyungang, Jiangsu, CN)
- Lianshan ZHANG (Lianyungang, Jiangsu, CN)
Cpc classification
A61K31/4545
HUMAN NECESSITIES
A61K31/5025
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K31/4545
HUMAN NECESSITIES
A61K31/5025
HUMAN NECESSITIES
C07D237/00
CHEMISTRY; METALLURGY
International classification
A61K31/4545
HUMAN NECESSITIES
A61K31/5025
HUMAN NECESSITIES
Abstract
This application describes a use of an EZH2 inhibitor combined with a BTK inhibitor in preparing a drug for treating a tumor is described.
Claims
1.-15. (canceled)
16. A method for treating a tumor in a subject in need thereof, the method comprising administering to the subject a combination of an EZH2 inhibitor and a BTK inhibitor, wherein the EZH2 inhibitor is a compound of formula (I): ##STR00021## or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, wherein, ring A is selected from the group consisting of heterocyclyl and cycloalkyl; each R.sup.1 is identical or different and each is independently selected from the group consisting of hydrogen, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, amino, nitro, hydroxy, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, OR.sup.6, C(O)R.sup.6, C(O)OR.sup.6, S(O).sub.mR.sup.6, S(O).sub.mNR.sup.7R.sup.8 and (CH.sub.2).sub.xR.sup.a, wherein the alkyl, haloalkyl, heterocyclyl, aryl and heteroaryl are each independently and optionally substituted by one or more substituents selected from the group consisting of alkyl, haloalkyl, halogen, amino, nitro, cyano, hydroxy, alkoxy, haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.a is selected from the group consisting of halogen, cycloalkyl, heterocyclyl and NR.sup.7R.sup.8, wherein the cycloalkyl and heterocyclyl are each independently and optionally substituted by one or more substituents selected from the group consisting of alkyl, haloalkyl, halogen, amino, nitro, cyano, hydroxy, alkoxy, haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.2 is hydrogen or alkyl, wherein the alkyl is optionally substituted by one or more substituents selected from the group consisting of halogen, hydroxy, cyano, cycloalkyl and heterocyclyl; R.sup.3 is selected from the group consisting of hydrogen, alkyl, halogen, cyano, alkoxy and haloalkyl; each R.sup.4 is identical or different and each is independently selected from the group consisting of hydrogen, alkyl, haloalkyl, hydroxy, amino, alkoxy, haloalkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, OR.sup.6, C(O)R.sup.6, C(O)OR.sup.6, S(O).sub.mR.sup.6, S(O).sub.mNR.sup.7R.sup.8 and NR.sup.7R.sup.8; each R.sup.5 is identical or different and each is independently selected from the group consisting of hydrogen, alkyl, oxo, halogen, haloalkyl, hydroxy, amino, alkoxy, haloalkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, OR.sup.6, C(O)R.sup.6, C(O)OR.sup.6, S(O).sub.mR.sup.6, S(O).sub.mNR.sup.7R.sup.8 and NR.sup.7R.sup.8; R.sup.6 is selected from the group consisting of hydrogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, amino, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.7 and R.sup.8 are identical or different and each is independently selected from the group consisting of hydrogen, alkyl, alkoxy, hydroxyalkyl, hydroxy, amino, alkoxycarbonyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein the alkyl, amino, cycloalkyl, heterocyclyl, aryl and heteroaryl are each independently and optionally substituted by one or more substituents selected from the group consisting of alkyl, halogen, hydroxy, amino, alkoxycarbonyl, nitro, cyano, alkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; m is 0, 1 or 2; n is 0, 1, 2 or 3; p is 0, 1, 2, 3, 4 or 5; q is 0, 1 or 2; and x is 0, 1, 2 or 3.
17. The method according to claim 16, wherein the EZH2 inhibitor is a compound of formula (IA): ##STR00022## or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, wherein, G is selected from the group consisting of CR.sup.bR.sup.c, CO, NR.sup.d, S(O).sub.m and oxygen; R.sup.b and R.sup.c are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, halogen, amino, nitro, hydroxy, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, OR.sup.6, C(O)R.sup.6, C(O)OR.sup.6, S(O).sub.mR.sup.6 and NR.sup.7R.sup.8; R.sup.d is selected from the group consisting of hydrogen, alkyl, cycloalkyl, haloalkyl, hydroxyalkyl, heterocyclyl, aryl, heteroaryl, C(O)R.sup.6, C(O)OR.sup.6 and S(O).sub.mR.sup.6; and R.sup.1 to R.sup.4, R.sup.6 to R.sup.8, n, m and q are as defined in claim 16.
18. The method according to claim 16, wherein the EZH2 inhibitor is a compound of formula (IB): ##STR00023## or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, wherein, E is CH or nitrogen; F is selected from the group consisting of CR.sup.bR.sup.c, CO, NR.sup.d and oxygen; R.sup.b and R.sup.c are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, halogen, amino, nitro, hydroxy, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, OR.sup.6, C(O)R.sup.6, C(O)OR.sup.6, S(O).sub.mR.sup.6 and NR.sup.7R.sup.8; R.sup.d is selected from the group consisting of hydrogen, alkyl, cycloalkyl, haloalkyl, hydroxyalkyl, heterocyclyl, aryl, heteroaryl, C(O)R.sup.6, C(O)OR.sup.6 and S(O).sub.mR.sup.6; each R.sup.e is identical or different and each is independently selected from the group consisting of hydrogen, alkyl, haloalkyl, halogen, amino, nitro, cyano, hydroxy, alkoxy, haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; t is 0, 1, 2, 3, 4 or 5; x is 0, 1, 2 or 3; y is 0, 1, 2 or 3; and R.sup.2 to R.sup.4, R.sup.6 to R.sup.8, m and n are as defined in claim 16.
19. The method according to claim 16, wherein the EZH2 inhibitor is a compound of formula (IC): ##STR00024## or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, wherein, each R.sup.e is identical or different and is independently selected from the group consisting of hydrogen, alkyl and halogen; t is 0, 1, 2, 3, 4 or 5; and R.sup.2 to R.sup.4 and n are as defined in claim 16.
20. The method according to claim 16, wherein the EZH2 inhibitor is a compound of formula (ID): ##STR00025## or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, wherein, R.sup.e is selected from the group consisting of hydrogen, alkyl, haloalkyl, halogen, amino, nitro, cyano, hydroxy, alkoxy, haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; and R.sup.2 to R.sup.4 and n are as defined in claim 16.
21. The method according to claim 16, wherein the BTK inhibitor is a compound of formula (II): ##STR00026## or a pharmaceutically acceptable salt or a stereoisomer thereof, wherein, A is selected from the group consisting of CR.sup.1 and N; R.sup.1 is selected from the group consisting of hydrogen, halogen and optionally substituted alkyl, wherein the substituent is selected from the group consisting of halogen, hydroxy, cyano, nitro, carboxy, amino, alkyl, alkoxy and haloalkyl; R.sup.a, R.sup.b, R.sup.c and R.sup.d are each independently selected from the group consisting of hydrogen, halogen, hydroxy, cyano, nitro, optionally substituted alkyl and optionally substituted alkoxy, wherein the substituent is selected from the group consisting of halogen, hydroxy, cyano, nitro, carboxy, amino, alkyl, alkoxy and haloalkyl; B is selected from the group consisting of hydrogen, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl and optionally substituted heteroaryl, wherein the substituent is selected from the group consisting of halogen, hydroxy, cyano, nitro, carboxy, amino, alkyl, alkoxy and haloalkyl; L is selected from the group consisting of a bond and optionally substituted alkyl; and Y is selected from the group consisting of optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl and optionally substituted heteroaryl, wherein the substituent is selected from the group consisting of halogen, hydroxy, cyano, nitro, carboxy, amino, alkyl, alkylcarbonyl, alkynylcarbonyl and haloalkyl.
22. A method for treating a tumor in a subject in need thereof, the method comprising administering to the subject a combination of an EZH2 inhibitor and a BTK inhibitor, wherein the EZH2 inhibitor is a compound of formula (IE): ##STR00027## or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, and the BTK inhibitor is a compound of formula (IIA): ##STR00028## or a pharmaceutically acceptable salt thereof or a stereoisomer thereof.
23. The method according to claim 20, wherein the pharmaceutically acceptable salt is selected from the group consisting of phosphate, hydrochloride, methanesulfonate, maleate, malate, p-toluenesulfonate and besylate.
24. The method according to claim 16, wherein the BTK inhibitor is selected from the group consisting of Ibrutinib, Acalabrutinib, MSC-2364447, Spebrutinib, HM-71224, Plevitrexed, GS-4059, GDC-0853, SNS-062, CGP-53716, Idoxifene, BTG-511, Banoxantrone, Glucarpidase, Anti-digoxin polyclonal antibody, Crotalidae polyvalent immune Fab (ovine, BTG) and Otelixizumab.
25. The method according to claim 16, wherein the combination optionally comprises a third component selected from the group consisting of an HDAC inhibitor, CDK4/6 inhibitor, ALK inhibitor, JAK2 inhibitor, Bcl-2 inhibitor, Hsp90 inhibitor, glucocorticoid, vinca alkaloid, antimetabolite, DNA damaging agent, Lenalidomide, Rituximab, PKC perturbagen, Lyn/Fyn inhibitor, Syk inhibitor, PI3K inhibitor, PKC inhibitor, IKK inhibitor, 20 s proteasome, IRF-4, IRAK4 antibody, CXCR4 antibody, CXCR5 antibody, GLS antibody, PLK antibody, CD20 antibody, Topo II inhibitor, DNA methyltransferase inhibitor, Ras/MAPK inhibitor and FGFR1 inhibitor.
26. The method according to claim 25, wherein the HDAC inhibitor is selected from the group consisting of Panobinostat Lactate, Belinostat, Chidamide, Romidepsin, Vorinostat, Bexanostat and Entinostat; the CDK4/6 inhibitor is selected from the group consisting of Palbociclib, Blinatumomab, Tiagabine Hydrochloride and Itolizumab; the Bcl-2 inhibitor is selected from the group consisting of Venetoclax, Oblimersen Sodium, ABT-737 and HA14-1; the Hsp90 inhibitor is selected from the group consisting of Sebelipase alfa and Retaspimycin Hydrochloride; the JAK2 inhibitor is selected from the group consisting of Tofacitinib citrate, Ruxolitinib Phosphate, Lestaurtinib, Momelotinib Dihydrochloride, Peficitinib and Filgotinib; the PKC perturbagen is selected from the group consisting of Teprenone, Truheal, HO/03/03, Sotrastaurin, Enzastaurin and GF109203X; the ALK inhibitor is selected from the group consisting of Alectinib hydrochloride, Ceritinib, Crizotinib, Bendamustine, Carmustine, Lumostine, chlormethine hydrochloride and NVP-TAE684; the PI3K inhibitor is selected from the group consisting of GS-1101, IPI-145, BKM120, BEZ235, GDC-0941, AMG319, CAL-101 and A66; and the IKK inhibitor is selected from the group consisting of Auranofin, BAY 86-9766 and RDEA-119.
27. The method according to claim 25, wherein the tumor is lymphoma.
28. The method according to claim 27, wherein the lymphoma is non-Hodgkin lymphoma.
29. The method according to claim 28, wherein the non-Hodgkin lymphoma is B cell proliferative disease.
30. The method according to claim 29, wherein the B cell proliferative disease is selected from the group consisting of diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high-risk CLL or non-CLL/SLL lymphoma, follicular lymphoma (FL), mantle cell lymphoma (MCL), precursor B cell tumor, precursor B lymphoblastic leukemia (or lymphoma), mature (peripheral) B cell tumor, lymphoplasmacytic lymphoma (or immunoblastoma), extranodal mucosa-associated lymphoma, hairy cell leukemia, plasmacytoma (or plasma cell myeloma), Waldenstrom's macroglobulinemia, multiple myeloma, marginal zone lymphoma, Burkitt's lymphoma (BL), non-Burkitt high grade B cell lymphoma or extranodal marginal zone B cell lymphoma, acute or chronic myelogenous (or myeloid) leukemia, myelodysplastic syndrome and acute lymphoblastic leukemia.
31. The method according to claim 27, wherein a ratio of the EZH2 inhibitor to the BTK inhibitor is 0.001-1000.
32. The method according to claim 31, wherein the ratio of the EZH2 inhibitor to the BTK inhibitor is 0.1-10.
33. The method according to claim 32, wherein an administration dose of the EZH2 inhibitor is 1-2000 mg, and an administration dose of the BTK inhibitor is 1-1000 mg.
34. A pharmaceutical composition comprising an EZH2 inhibitor, a BTK inhibitor, and one or more pharmaceutically acceptable excipients, diluents or carriers, wherein the EZH2 inhibitor is a compound of formula (I): ##STR00029## or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, wherein, ring A is selected from the group consisting of heterocyclyl and cycloalkyl; each R.sup.1 is identical or different and each is independently selected from the group consisting of hydrogen, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, amino, nitro, hydroxy, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, OR.sup.6, C(O)R.sup.6, C(O)OR.sup.6, S(O).sub.mR.sup.6, S(O).sub.mNR.sup.7R.sup.8 and (CH.sub.2).sub.xR.sup.a, wherein the alkyl, haloalkyl, heterocyclyl, aryl and heteroaryl are each independently and optionally substituted by one or more substituents selected from the group consisting of alkyl, haloalkyl, halogen, amino, nitro, cyano, hydroxy, alkoxy, haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.a is selected from the group consisting of halogen, cycloalkyl, heterocyclyl and NR.sup.7R.sup.8, wherein the cycloalkyl and heterocyclyl are each independently and optionally substituted by one or more substituents selected from the group consisting of alkyl, haloalkyl, halogen, amino, nitro, cyano, hydroxy, alkoxy, haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.2 is hydrogen or alkyl, wherein the alkyl is optionally substituted by one or more substituents selected from the group consisting of halogen, hydroxy, cyano, cycloalkyl and heterocyclyl; R.sup.3 is selected from the group consisting of hydrogen, alkyl, halogen, cyano, alkoxy and haloalkyl; each R.sup.4 is identical or different and each is independently selected from the group consisting of hydrogen, alkyl, haloalkyl, hydroxy, amino, alkoxy, haloalkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, OR.sup.6, C(O)R.sup.6, C(O)OR.sup.6, S(O).sub.mR.sup.6, S(O).sub.mNR.sup.7R.sup.8 and NR.sup.7R.sup.8; each R.sup.5 is identical or different and each is independently selected from the group consisting of hydrogen, alkyl, oxo, halogen, haloalkyl, hydroxy, amino, alkoxy, haloalkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, OR.sup.6, C(O)R.sup.6, C(O)OR.sup.6, S(O).sub.mR.sup.6, S(O).sub.mNR.sup.7R.sup.8 and NR.sup.7R.sup.8; R.sup.6 is selected from the group consisting of hydrogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, amino, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.7 and R.sup.8 are identical or different and each is independently selected from the group consisting of hydrogen, alkyl, alkoxy, hydroxyalkyl, hydroxy, amino, alkoxycarbonyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein the alkyl, amino, cycloalkyl, heterocyclyl, aryl and heteroaryl are each independently and optionally substituted by one or more substituents selected from the group consisting of alkyl, halogen, hydroxy, amino, alkoxycarbonyl, nitro, cyano, alkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; m is 0, 1 or 2; n is 0, 1, 2 or 3; p is 0, 1, 2, 3, 4 or 5; q is 0, 1 or 2; and x is 0, 1, 2 or 3.
35. The pharmaceutical composition according to claim 34, wherein the BTK inhibitor is a compound of formula (II): ##STR00030## or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, wherein, A is selected from the group consisting of CR.sup.1 and N; R.sup.1 is selected from the group consisting of hydrogen, halogen and optionally substituted alkyl, wherein the substituent is selected from the group consisting of halogen, hydroxy, cyano, nitro, carboxy, amino, alkyl, alkoxy and haloalkyl; R.sup.a, R.sup.b, R.sup.c and R.sup.d are each independently selected from the group consisting of hydrogen, halogen, hydroxy, cyano, nitro, optionally substituted alkyl and optionally substituted alkoxy, wherein the substituent is selected from the group consisting of halogen, hydroxy, cyano, nitro, carboxy, amino, alkyl, alkoxy and haloalkyl; B is selected from the group consisting of hydrogen, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl and optionally substituted heteroaryl, wherein the substituent is selected from the group consisting of halogen, hydroxy, cyano, nitro, carboxy, amino, alkyl, alkoxy and haloalkyl; L is selected from the group consisting of a bond and optionally substituted alkyl; and Y is selected from the group consisting of optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl and optionally substituted heteroaryl, wherein the substituent is selected from the group consisting of halogen, hydroxy, cyano, nitro, carboxy, amino, alkyl, alkylcarbonyl, alkynylcarbonyl and haloalkyl.
Description
DESCRIPTION OF THE DRAWINGS
[0111]
[0112]
[0113]
[0114]
[0115]
DETAILED DESCRIPTION OF THE INVENTION
[0116] The exemplary experimental solutions for the medicinal use of the composition of the present invention in treating diabetes are provided below in order to demonstrate the favorable activity and beneficial technical effects of the composition of the present invention. However, it should be understood that the following experimental solutions are merely examples of the present invention and are not intended to limit the scope of the present invention. A person skilled in the art, based on the teachings of the specification, can make suitable modifications or alterations to the technical solutions of the present invention without departing from the spirit and scope of the present invention.
Comparative Example 1. Preparation of N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-(piperidin-1-ylmethyl)benzofuran-4-carboxamide represented by formula (IE) (compound B)
[0117] ##STR00018## ##STR00019## ##STR00020##
Step 1
3-Bromo-2-ethyl-5-nitrobenzoic acid
[0118] 2-Ethylbenzoic acid 1a (20.0 g, 133 mmol, prepared according to the method disclosed in Journal of the American chemical Society 1991, 113(13), 4931-6) was added to 150 mL of sulfuric acid, and then sodium nitrate (11.3 g, 133 mmol) was added in batches in an ice bath. The reaction solution was stirred for 3 hours, and then N-bromosuccinimide (2.6 g, 14.5 mmol) was added in batches. The reaction mixture was stirred for 1 hour at 60 C. After the reaction was completed, the reaction solution was poured to ice water, stirred well and filtered. The filtrate was washed with water, and concentrated under reduced pressure to obtain the crude title product 3-bromo-2-ethyl-5-nitrobenzoic acid 1b (35 g) as a white solid, which was directly used in the next step without purification.
Step 2
Methyl 3-bromo-2-ethyl-5-nitrobenzoate
[0119] The crude 3-bromo-2-ethyl-5-nitrobenzoic acid 1b (35 g, 128 mmol) was dissolved in 200 mL of N,N-dimethylformamide, and then iodomethane (21.8 g, 153 mmol) and potassium carbonate (35.3 g, 255 mmol) were added. The reaction mixture was stirred for 2 hours at room temperature. After the reaction was completed, the reaction solution was concentrated under reduced pressure. The reaction solution was added with excess water, and extracted with ethyl acetate. The organic phases were combined, washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain the crude title product methyl 3-bromo-2-ethyl-5-nitrobenzoate 1c (36 g) as a yellow oil, which was directly used in the next step without purification.
Step 3
Methyl 5-amino-3-bromo-2-ethylbenzoate
[0120] The crude methyl 3-bromo-2-ethyl-5-nitrobenzoate 1c (35.0 g, 121 mmol) was added to 250 mL of ethanol and 150 mL of water. The reaction solution was heated to 70 C., added with ammonium chloride (52.8 g, 969 mmol), and then added with iron powder (34 g, 606 mmol) in batches. The reaction mixture was stirred for 2 hours at 70 C. After the reaction was completed, the reaction solution was filtered through celite while hot. The filter cake was washed with hot ethanol, and then the filtrate was combined and concentrated under reduced pressure. Ethyl acetate and saturated sodium bicarbonate solution were added. Two phases were separated, and the aqueous phase was extracted with ethyl acetate. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with n-hexane and ethyl acetate as the eluent to obtain the title product methyl 5-amino-3-bromo-2-ethylbenzoate 1d (22.0 g, yield 70%) as a yellow solid.
Step 4
Methyl 3-bromo-2-ethyl-5-hydroxybenzoate
[0121] Methyl 5-amino-3-bromo-2-ethylbenzoate 1d (15.0 g, 58 mmol) was dissolved in 10 mL of acetonitrile, and then 200 mL of 10% sulfuric acid was added. The reaction solution was stirred well and cooled to 3 C. in an ice-salt bath, and then added dropwise with 10 mL of a pre-prepared solution of sodium nitrite (4.4 g, 64 mmol). The reaction solution was stirred for 4 hours at the above temperature, added dropwise with 200 mL of 50% sulfuric acid, and then stirred for 1 hour at 90 C. After the reaction was completed, the reaction solution was extracted three times with ethyl acetate. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with n-hexane and ethyl acetate as the eluent to obtain the title product methyl 3-bromo-2-ethyl-5-hydroxybenzoate 1e (5.5 g, yield 37%) as a brown solid.
Step 5
Methyl 3-bromo-5-(2,2-diethoxyethoxy)-2-ethylbenzoate
[0122] Methyl 3-bromo-2-ethyl-5-hydroxybenzoate 1e (35 g, 135 mmol) was dissolved in 200 mL of N,N-dimethylformamide, and then 2-bromo-1,1-diethoxyethane (40 g, 202 mmol) and potassium carbonate (37 g, 269 mmol) were added. The reaction mixture was stirred at 120 C. for 12 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove N,N-dimethylformamide. The reaction solution was added with water, and extracted three times with ethyl acetate. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with n-hexane and ethyl acetate as the eluent to obtain the title product methyl 3-bromo-5-(2,2-diethoxyethoxy)-2-ethylbenzoate 1f (40 g, yield 80%) as a light yellow oil.
Step 6
Methyl 6-bromo-5-ethylbenzofuran-4-carboxylate
[0123] Polyphosphoric acid (30 g) was added to 400 mL of toluene. The reaction solution was heated to 100 C., and added with 50 mL of a pre-prepared solution of methyl 3-bromo-5-(2,2-diethoxyethoxy)-2-ethylbenzoate 1f (40 g, 107 mmol) in toluene under stirring. The reaction solution was stirred for 16 hours at 100 C. After the reaction was completed, the supernatant was decanted. The residue was added with water and ethyl acetate. Two phases were separated, and the aqueous phase was extracted with ethyl acetate. The organic phases were combined, washed with saturated sodium carbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with n-hexane and ethyl acetate as the eluent to obtain the title product methyl 6-bromo-5-ethylbenzofuran-4-carboxylate 1g (11.8 g, yield 39%) as a yellow solid.
Step 7
Methyl 5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)benzofuran-4-carboxylate
[0124] Methyl 6-bromo-5-ethylbenzofuran-4-carboxylate 1g (11.0 g, 39 mmol), tetrahydro-2H-pyran-4-amine (5.89 g, 58 mmol), tris(dibenzylideneacetone)dipalladium (3.6 g, 3.9 mmol), (0.9 mmol) bis(diphenylphosphino)-1,1-binaphthalene (4.86 g, 7.8 mmol) and cesium carbonate (38 g, 117 mmol) were dissolved in 100 mL of toluene. The reaction solution was stirred for 12 hours at 100 C. After the reaction was completed, the reaction solution was filtered through celite, and the filter cake was washed with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with n-hexane and ethyl acetate as the eluent to obtain the title product methyl 5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)benzofuran-4-carboxylate 1h (10.0 g, yield 85%) as a yellow solid.
Step 8
Methyl 5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)benzofuran-4-carboxylate
[0125] Methyl 5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)benzofuran-4-carboxylate 1h (10.0 g, 0.033 mmol) was dissolved in 150 mL of 1,2-dichloroethane, and then acetaldehyde (7.2 g, 0.165 mmol) and acetic acid (9.9 g, 0.165 mmol) were added. The reaction solution was stirred for 1 hour, and added with sodium triacetoxyborohydride (20.8 g, 0.1 mmol). The reaction solution was stirred for 12 hours at room temperature. After the reaction was completed, the reaction solution was concentrated under reduced pressure, neutralized with saturated sodium bicarbonate solution, and extracted with ethyl acetate. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with n-hexane and ethyl acetate as the eluent to obtain the title product methyl 5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)benzofuran-4-carboxylate 1i (7.8 g, yield 71%) as a white solid.
[0126] MS m/z (LC-MS): 332.4 [M+1]
Step 9
Methyl 5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-formylbenzofuran-4-carboxylate
[0127] Methyl 5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)benzofuran-4-carboxylate 1i (1.6 g, 4.8 mmol) was dissolved in 25 mL of tetrahydrofuran. The reaction solution was cooled to 70 C., and added dropwise with 2.0 M diisopropylamide (3.6 mL, 7.3 mmol) under an argon atmosphere. The reaction solution was stirred for 90 minutes, and added with N,N-dimethylformamide (536 mg, 7.3 mmol). The reaction solution was stirred for 2 hours, and then slowly warmed up to room temperature. The reaction solution was added with excess ammonium chloride, stirred well and extracted three times with ethyl acetate. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with n-hexane and ethyl acetate as the eluent to obtain the title product methyl 5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-formylbenzofuran-4-carboxylate 1j (1.3 g, yield 75%) as a yellow oil.
[0128] MS m/z (ESI):360.2 [M+1]
Step 10
Methyl 5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-(hydroxymethyl)benzofuran-4-carboxylate
[0129] Methyl 5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-formylbenzofuran-4-carboxylate 1j (1.4 g, 3.9 mmol) was dissolved in 5 mL of tetrahydrofuran and 10 mL 4-carboxylate 1j (1.4 g, 3.9 mmol) was dissolved in 5 mL of tetrahydrofuran and 10 mL of methanol, and then sodium borohydride (222 mg, 5.8 mmol) was added. The reaction solution was stirred for 30 minutes at room temperature. After the reaction was completed, the reaction solution was concentrated under reduced pressure, added with water and saturated sodium bicarbonate solution, and extracted three times with ethyl acetate. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with n-hexane and ethyl acetate as the eluent to obtain the title product methyl 5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-(hydroxymethyl)benzofuran-4-carboxylate 1k (1.4 g, yield 99%) as a yellow oil.
Step 11
Methyl 2-(bromomethyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)benzofuran-4-carboxylate
[0130] Methyl 5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-(hydroxymethyl)benzofuran-4-carboxylate 1k (1.0 g, 2.8 mmol) was dissolved in 30 mL of tetrahydrofuran, and then phosphorus tribromide (1.12 g, 4.2 mmol) was added dropwise. The reaction solution was stirred for 12 hours at room temperature. After the reaction was completed, the reaction solution was neutralized with saturated sodium bicarbonate solution, and extracted with ethyl acetate. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain the crude title product methyl 2-(bromomethyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)benzofuran-4-carboxylate 1l (1.15 g) as a yellow oil, which was directly used in the next step without purification.
Step 12
Methyl 5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-(piperidin-1-ylmethyl)benzofuran-4-carboxylate
[0131] The crude methyl 2-(bromomethyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)benzofuran-4-carboxylate 1l (1.15 g, 2.7 mmol) was dissolved in 15 mL of acetonitrile, and then 10 mL of a pre-prepared solution of piperidine (362 mg, 4.3 mmol) in acetonitrile were added dropwise. The reaction solution was stirred for 30 minutes at room temperature. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and added with ethyl acetate and saturated sodium bicarbonate solution. Two phases were separated, and the aqueous phase was extracted with ethyl acetate. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with dichloromethane and methanol as the eluent to obtain the title product methyl 5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-(piperidin-1-ylmethyl)benzofuran-4-carboxylate 1m (1.2 g, yield 99%) as a yellow oil.
[0132] MS m/z (LC-MS): 429.2[M+1]
Step 13
5-Ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-(piperidin-1-ylmethyl)benzofuran-4-carboxylic acid
[0133] Methyl 5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-(piperidin-1-ylmethyl)benzofuran-4-carboxylate 1m (1.2 g, 2.7 mmol) was dissolved in 5 mL of tetrahydrofuran and 20 mL of methanol, and then 5 mL of 4 M sodium hydroxide solution were added. The reaction solution was stirred for 12 hours at 60 C. After the reaction was completed, concentrated hydrochloric acid was added to adjust the pH of the reaction solution to 4. The mixture was concentrated under reduced pressure, and the residue was dissolved in a mixed solvent of dichloromethane and methanol (V:V=5:1) and filtered. The filter cake was washed with a mixed solvent of dichloromethane and methanol (V:V=5:1). The filtrates were combined and concentrated under reduced pressure to obtain the crude title product 5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-(piperidin-1-ylmethyl)benzofuran-4-carboxylic acid 1n (1.1 g) as a yellow solid, which was directly used in the next step without purification.
[0134] MS m/z (LC-MS): 415.2[M+1]
Step 14
Preparation of the Compound of Formula (IE) (Defined as Compound B)
[0135] 5-Ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-(piperidin-1-ylmethyl)benzofuran-4-carboxylic acid 1n (1.0 g, 2.4 mmol) was dissolved in 30 mL of N,N-dimethylformamide, and then 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (696 mg, 3.6 mmol), 1-hydroxybenzotriazole (490 mg, 3.6 mmol) and N,N-diisopropylethylamine (1.56 g, 12.1 mmol) were added. The reaction solution was stirred for 1 hour, and then added with 3-(aminomethyl)-4,6-dimethylpyridin-2(1H)-one hydrochloride 2a (593 mg, 3.0 mmol, prepared according to the method disclosed in the patent application WO2014097041). The reaction solution was stirred for 12 hours at room temperature. After the reaction was completed, the reaction solution was added with excess water, and extracted with a mixed solvent of dichloromethane and methanol (V:V=8:1). The organic phases were combined, washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with dichloromethane and methanol as the eluent to obtain the title product N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-(piperidin-1-ylmethyl)benzofuran-4-carboxamide IE (750 mg, yield 57%) as a white solid.
[0136] MS m/z (ESI): 549.7 [M+1]
[0137] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): 11.48 (s, 1H), 8.15 (t, 1H), 7.39 (s, 1H), 6.46 (s, 1H), 5.86 (s, 1H), 4.32 (d, 2H), 3.83 (d, 2H), 3.54 (s, 2H), 3.21 (t, 2H), 3.01-3.07 (m, 2H), 2.92-2.97 (m, 1H), 2.77-2.82 (m, 2H), 2.39 (brs, 4H), 2.23 (s, 3H), 2.11 (s, 3H), 1.64-1.67 (brd, 2H), 1.47-1.55 (m, 6H), 1.36-1.37 (brd, 2H), 1.02 (t, 3H), 0.82 (t, 3H).
Example 1. Effect of the Composition of the Present Invention on the Proliferation of DOHH-2 Cells In Vitro
[0138] Test compounds: the compound of formula (IE) (defined as compound B, prepared according to the method disclosed in WO2017084494 (patent application PCT/CN2016/104318), see comparative example 1), and the compound of formula (IIA) (defined as compound A, prepared according to the method disclosed in the patent application WO2016007185A1).
[0139] Cell line: human B cell lymphoma DOHH-2 cells (purchased from DSMZ), cultured in vitro in RPMI 1640 medium containing 10% fetal bovine serum (FBS).
[0140] Formulation of Test Compound Solution:
[0141] Each of the test compounds was all formulated with DMSO into a 10 mM stock solution, which was formulated into the desired concentration with serum-free medium when used.
Experimental Method
[0142] A certain number of cells in logarithmic growth phase was inoculated in a 96-well culture plate. After 24 hours, the cells were added with the test compounds in different concentrations (1-100000 nM), and incubated for 72 hours. Each well was added with the MTT working solution. After 4 hours, the cells were lysed with the triple solution, and the OD value was measured at a wavelength of 570 nm by a microplate reader.
[0143] Data Analysis:
[0144] The cell growth inhibition rate was calculated by the following formula:
Inhibition rate=(OD value of control wellOD value of drugadministered well)/OD value of control well100%;
[0145] The half effective concentration IC.sub.50 was calculated using the non-linear regression method according to the inhibition, rate of each concentration.
[0146] In the combined administration, the concentration ratio of compound A to compound B was 1:10. The combination index (CI) was calculated with Calcu-Syn program using the median effect method to evaluate the relationship between the two compounds during the combined administration (CI<1 refers to synergistic effect, CI=1 refers to additive effect, CI>1 refers to antagonistic effect).
Experimental Results
[0147]
TABLE-US-00001 TABLE 1 Effect of single compound on the proliferation of DOHH-2 cells Compound IC.sub.50 (nM) Compound A 324.6 Compound B 5919.0
TABLE-US-00002 TABLE 2 Inhibition effect of the combined administration on the proliferation of DOHH-2 cells CI Compound ED.sub.50 ED.sub.75 ED.sub.90 Compound A + Compound B 0.09 0.09 0.09
Experimental Conclusion
[0148] It can be seen from the above table data that the combined administration of compound A and compound B has a synergistic inhibition effect on the proliferation of DOHH-2 cells in vitro.
Example 2. Effect of the Composition of the Present Invention on the Proliferation of SU-DHL-4 and SU-DHL-6 Cells In Vitro
[0149] Test compounds: the compound of formula (IE) (defined as compound B, prepared according to the method disclosed in WO2017084494 (patent application PCT/CN2016/104318), see comparative example 1), and the compound of formula (IIA) (defined as compound A, prepared according to the method disclosed in the patent application WO2016007185A1).
[0150] Cell line: human B cell lymphoma SU-DHL-4 and SU-DHL-6 cells (purchased from ATCC), cultured in vitro in RPMI 1640 medium containing 10% fetal bovine serum (FBS).
[0151] Formulation of Test Compound Solution:
[0152] Each of the test compounds was all formulated with DMSO into a 10 mM stock solution, which was formulated into the desired concentration with serum-free medium when used.
Experimental Method
[0153] A certain number of cells in logarithmic growth phase was inoculated in a 96-well culture plate. After 24 hours, the cells were added with the test compounds in different concentrations (1-40000 nM), and incubated for 72 hours. Each well was added with the MTT working solution. After 4 hours, the cells were lysed with the triple solution (10% SDS, 5% isobutanol, 0.012 mol/L HCl) at 37 C. overnight, and the OD value was measured at a wavelength of 570 nm by a microplate reader.
[0154] Data Analysis:
[0155] The cell growth inhibition rate was calculated by the following formula:
Inhibition rate=(OD value of control wellOD value of drug-administered well)/OD value of control well100%
[0156] The half effective concentration IC.sub.50 was calculated using the non-linear regression method according to the inhibition rate of each concentration.
[0157] In the combined administration, the concentration ratio of compound A to compound B was 1:2 (for SU-DHL-4) and 1:4 (for SU-DHL-6). The combination index (CI) was calculated with Calcu-Syn program using the median effect method to evaluate the relationship between the two compounds during the combined administration (CI<1 refers to synergistic effect, CI=1 refers to additive effect, CI>1 refers to antagonistic effect).
Experimental Results
[0158]
TABLE-US-00003 TABLE 3 Effect of single compound on the proliferation of SU-DHL-4 and SU-DHL-6 cells IC.sub.50(nM) Cell line Compound A Compound B SU-DHL-4 1059 2169 SU-DHL-6 711 4660
TABLE-US-00004 TABLE 4 Inhibition effect of the combined administration on the proliferation of SU-DHL-4 and SU-DHL-6 cells CI Compound A + Compound B ED50 ED75 ED90 SU-DHL-4 0.77 0.60 0.47 SU-DHL-6 0.57 0.36 0.23
Experimental Conclusion
[0159] It can be seen from the above table date that the combined administration of compound A and compound B has a synergistic inhibition effect on the proliferation of SU-DHL-4 and SU-DHL-6 cells in vitro.
Example 3: Efficacy of the Composition of the Present Invention on the Subcutaneous Transplantation Tumor in Nude Mice Inoculated with the Human Follicular Lymphoma DOHH-2 Cells
[0160] Test compounds: the compound of formula (IE) (defined as compound B, prepared according to the method disclosed in WO2017084494 (patent application PCT/CN2016/104318), see comparative example 1), and the compound of formula (IIA) (defined as compound A, prepared according to the method disclosed in the patent application WO2016007185A1).
[0161] Test animals: BALB/cA-nude nude mice, 5-6 weeks old, female, purchased from Shanghai Lingchang Biotechnology Co., Ltd., with laboratory animals use license No.: SCXK (Shanghai) 2013-0018 and animal certificate No.: 2013001818958, feeding condition: SPF grade.
[0162] Formulation of the Solution of the Test Compound:
[0163] The test compounds were all formulated with 0.2% Tween 80+0.5% CMC solutions, and diluted to the corresponding concentration.
Experimental Method
[0164] (1) The nude mice were subcutaneously inoculated with lymphoma DOHH-2 cells. When the tumors grown to 100-200 mm.sup.3, the animals were grouped randomly (D0). The dose and regimen of the administration are shown in Table 5.
[0165] (2) Observation and recording: the tumor volume was measured 2 to 3 times per week, the mice were weighed, and the data were recorded.
[0166] (3) Tumor measurement and endpoint
[0167] The endpoint is mainly dependent on whether the tumor growth is delayed or whether the mouse is cured. The tumor volume (in mm.sup.3) was measured twice a week with caliper in two dimensions.
[0168] The tumor volume (V) is calculated as:
V=0.5ab.sup.2,
wherein a and b represent length and width, respectively;
T/C (%)=(TT.sub.0)/(CC.sub.0)100,
wherein T and C represent the tumor volume at the end of the experiment; T.sub.0 and C.sub.0 represent the tumor volume at the beginning of the experiment. The T/C value (percentage) is indicative of anti-tumor efficacy.
[0169] (4) Data analysis: the statistics were summarized, including mean and standard error of mean (SEM), statistical analysis of differences in tumor volume between groups, and analysis of data obtained by drug interaction that was carried out at the optimal treatment time point after the last administration (Day 21 after grouping). One-way variance analysis was performed to compare tumor volume and tumor weight between groups. When a non-significant F-statistic was obtained (p<0.001, treatment variance vs. error variance), the comparison between groups was performed using Games-Howell test. All data were analyzed using SPSS17.0, and P<0.05 was considered as statistically significant.
Experimental Results
[0170]
TABLE-US-00005 TABLE 5 Effect of the combined administration on the proliferation of DOHH-2 cells Mean Mean Number of tumor tumor % tumor animals in volume volume inhibition each group at (mm.sup.3) (mm.sup.3) % T/C rate P value the end of the Groups Administration Route D 0 SEM D 21 SEM D 21 D 21 D 21 experiment Solvent D 0-20 PO, BID 106.4 1.6 1347.2 178.2 10 Compound A D 0-20 PO, BID 111.1 2.3 742.0 108.7 51 49 * 0.027 6 50 mg/kg Compound B D 0-20 PO, BID 107.7 4.4 914.1 153.2 65 35 * 0.117 6 50 mg/kg Compound A D 0-20 PO, BID 105.8 3.4 414.2 86.4 25 75 0.002 6 50 mg/kg + compound B 50 mg/kg D 0: the time of the first administration; PO: oral administration; BID: twice a day; P value refers to comparison with solvent; * P < 0.05, comparison with Compound A 50 mg/kg + compound B 50 mg/kg, with Student's t test; thee number of mice at the beginning of the test, solvent group n = 10, treatment group n = 6.
Experimental Conclusion
[0171] It can be seen from the data in Table 5 that compound A (50 mg/kg, PO, QD21) inhibited the growth of the subcutaneous transplantation tumor in nude mice inoculated with DOHH-2 cells, and the tumor growth inhibition rate was 49% (P<0.05, compared with the solvent). Compound B (50 mg/kg, PO, QD21) had a certain inhibition effect on DOHH-2 cells, and the tumor growth inhibition rate was 35% (P>0.05, compared with solvent). When the two compounds were administered in combination, the tumor growth inhibition rate was increased to 75% and the efficacy was significantly stronger than that of compound A or compound B alone (P<0.05, compared with single compound, see
[0172] In summary, the combined effect of the BTK inhibitor compound A and the EZH2 inhibitor compound B of the present invention is better than the effect of single compound, and such a combination has a synergistic effect.
Example 4: Efficacy of the Composition of the Present Invention on the Subcutaneous Transplantation Tumor in Mice Inoculated with the Human B Cell Lymphoma SU-DHL-4 Cells
[0173] Test compounds: the compound of formula (IE) (defined as compound B, prepared according to the method disclosed in WO2017084494 (patent application PCT/CN2016/104318), see comparative example 1), and the compound of formula (IIA) (defined as compound A, prepared according to the method disclosed in the patent application WO2016007185A1).
[0174] Test animals: SCID.BG mice, 5-6 weeks old, female, purchased from Shanghai Lingchang Biotechnology Co., Ltd., with laboratory animals use license No.: SCXK (Shanghai) 2013-0018 and animal certificate No.: 2013001820833, feeding condition: SPF grade.
[0175] Formulation of the Solution of the Test Compound:
[0176] The test compounds were all formulated with 0.2% Tween 80+0.5% CMC solution, and diluted to the corresponding concentration.
Experimental Method
[0177] (1) The mice were subcutaneously inoculated with SU-DHL-4 cells (B cell lymphoma SU-DHL-4 cells were purchased from ATCC). When the tumors grown to 100-150 mm.sup.3, the animals were grouped according to the tumor volume (D0). The dose and regimen of the administration are shown in Table 6.
[0178] (2) Observation and recording: the tumor volume was measured 2 to 3 times per week, the mice were weighed, and the data were recorded.
[0179] (3) Tumor measurement and endpoint
[0180] The endpoint is mainly dependent on whether the tumor growth is delayed or whether the mouse is cured. The tumor volume (in mm.sup.3) was measured twice a week with caliper in two dimensions.
[0181] The tumor volume (V) is calculated as:
V=0.5ab.sup.2,
wherein a and b represent length and width, respectively;
T/C (%)=(TT.sub.0)/(CC.sub.0)100,
wherein T and C represent the tumor volume at the end of the experiment; T.sub.0 and C.sub.0 represent the tumor volume at the beginning of the experiment. The T/C value (percentage) is indicative of anti-tumor efficacy.
Tumor growth inhibition rate (TGI) (%)=100T/C (%);
[0182] When the tumor regressed, the tumor growth inhibition rate
(TGI) (%)=100(TT.sub.0)/T.sub.0100
[0183] If the tumor volume is smaller than the initial volume, i.e. T<T.sub.0 or C<C.sub.0, it is defined as partial regression (PR); if the tumor completely disappears, it is defined as complete regression (CR).
[0184] (4) Data analysis: the statistics were summarized, including mean and standard error of mean (SEM), statistical analysis of differences in tumor volume between groups, and analysis of data obtained by drug interaction that was carried out at the optimal treatment time point after the last administration (Day 14 after grouping). One-way variance analysis was performed to compare tumor volume and tumor weight between groups. When a non-significant F-statistic was obtained (p<0.001, treatment variance vs. error variance), single-tailed Mann-Whitney statistical analysis was performed to compare tumor volumes of the two groups, and P<0.05 was considered as statistically significant.
Experimental Results
[0185]
TABLE-US-00006 TABLE 6 Effect of the combined administration on the subcutaneous transplantation tumor in mice inoculated with the human B cell lymphoma SU-DHL-4 cells Mean Mean Number of tumor tumor % tumor animals in volume volume inhibition each group at (mm.sup.3) (mm.sup.3) % T/C rate Partial Complete the end of the Groups Administration Route D 0 SEM D 14 SEM D 14 D 14 regression regression experiment Solvent D 0-13 PO, BID 113.2 4.8 751.8 146.7 0 0 8 Compound A D 0-13 PO, BID 122.4 4.8 276.9 73.5 24 76 * 1 1 8 50 mg/kg Compound B D 0-13 PO. BID 126.6 5.3 384.7 145.0 40 60 * 2 1 8 50 mg/kg Compound A D 0-13 PO, BID 129.7 3.8 186.2 66.0 9 91 ** 2 1 8 50 mg/kg + compound B 50 mg/kg D 0: the time of the first administration; PO: oral administration; BID: twice a day; * P < 0.05, ** P < 0.01, comparison with the solvent.
Experimental Conclusion
[0186] It can be seen from the data in Table 6 that compound A (50 mg/kg, PO, QD14) inhibited the growth of the subcutaneous transplantation tumor in mice inoculated with SU-DHL-4 cells, and the tumor growth inhibition rate was 76%; the tumor partially regressed in 1/8 of mice, and the tumor completely regressed in 1/8 of mice. Compound B (50 mg/kg, PO, QD14) had a tumor growth inhibition rate of 60% against SU-DHL-4 cells; the tumor partially regressed in 2/8 of mice, and the tumor completely regressed in 1/8 of mice. When compound A and compound B were administered in combination, the tumor growth inhibition rate was increased to 91%; the tumor partially regressed in 2/8 of mice, and the tumor completely regressed in 118 of mice; and the efficacy was significantly stronger than that of compound A or compound B alone (see
[0187] In summary, the combined effect of the BTK inhibitor compound A and the EZH2 inhibitor compound B of the present invention is better than the effect of single compound, and such a combination has a synergistic effect.