COMPOUNDS USEFUL AS KINASE INHIBITORS
20230145305 · 2023-05-11
Inventors
- Chengxi He (Chongqing, CN)
- Xianlong WANG (Chongqing, CN)
- Yue RONG (Chongqing, CN)
- Rui Tan (Chongqing, CN)
- Huajie Zhang (Chongqing, CN)
- Haohan Tan (Chongqing, CN)
- Yanxin Liu (Chongqing, CN)
- Shu Lin (San Leandro, CA, US)
- Tongshuang Li (San Leandro, CA, US)
- Xingdong Zhao (Chongqing, CN)
- Weibo Wang (Moraga, CA)
Cpc classification
A61K45/06
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
Provided are certain BTK inhibitors, pharmaceutical compositions thereof, and methods of use thereof.
Claims
1. A compound of formula (I): ##STR00218## or a pharmaceutically acceptable salt thereof, wherein: is a single bond or a double bond to maintain ring A and ring B being aromatic; A.sup.1 is selected from C and N; A.sup.2 is selected from CR.sup.X, C(O) and N; A.sup.3 is selected from CR.sup.6 and NR.sup.6; A.sup.4 selected from CR.sup.7, C(O), N and NR.sup.XR.sup.5; A.sup.5 is selected from CR.sup.X, NR.sup.X or absent; A.sup.6 is selected from N and O; B.sup.1 is selected from C and N; B.sup.2 is selected from C and N; when A.sup.4 and A.sup.6 are N, B.sup.2 is C, then A.sup.2 is not N; when A.sup.5 is absent, R.sup.5 and R.sup.6 together with the atoms to which they are attached are not form a ring, then A.sup.2 and A.sup.3 are not N at the same time; X is selected from CR.sup.X and N; Y is selected from CR.sup.X and N; Z is selected from aryl, heteroaryl and heterocyclyl, wherein aryl, heteroaryl and heterocyclyl are each unsubstituted or substituted with at least one substituent, independently selected from Rx; R.sup.1 is selected from hydrogen, C.sub.1-10 alkyl and C.sub.3-10 cycloalkyl, wherein alkyl and cycloalkyl are each unsubstituted or substituted with at least one substituent, independently selected from R.sup.X1; R.sup.2 is selected from hydrogen, C.sub.1-10 alkyl and C.sub.3-10 cycloalkyl, wherein alkyl and cycloalkyl are each unsubstituted or substituted with at least one substituent, independently selected from R.sup.X2; or R.sup.1 and R.sup.2 together with the atom(s) to which they are attached form a C.sub.3-10 cycloalkyl or heterocyclic ring of 4 to 12 members containing 1, 2 or 3 heteroatoms independently selected from oxygen, sulfur, nitrogen and phosphorus, and optionally substituted with 1, 2 or 3 R.sup.X1 groups; each R.sup.3 is independently selected from halogen, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.3-10 cycloalkyl, C.sub.3-10 cycloalkyl-C.sub.1-4 alkyl, heterocyclyl, heterocyclyl-C.sub.1-4 alkyl, aryl, aryl-C.sub.1-4 alkyl, heteroaryl, heteroaryl-C.sub.1-4 alkyl, CN, NO.sub.2, —NR.sup.A3R.sup.B3, —OR.sup.A3, —C(O)R.sup.A3, —C(═NR.sup.E3)R.sup.A3, —C(═N—OR.sup.B3)R.sup.A3, —C(O)OR.sup.A3, —OC(O)R.sup.A3, —C(O)NR.sup.A3R.sup.B3, —NR.sup.A3C(O)R.sup.B3, —C(═NR.sup.E3)NR.sup.A3R.sup.B3, —NR.sup.A3C(═NR.sup.E3)R.sup.B3, —OC(O)NR.sup.A3R.sup.B3, —NR.sup.A3C(O)OR.sup.B3, —NR.sup.A3C(O)NR.sup.A3R.sup.B3, —NR.sup.A3C(S)NR.sup.A3R.sup.B3, —NR.sup.A3C(═NR.sup.E3)NR.sup.A3R.sup.B3, —S(O).sub.rR.sup.A3, —S(O)(═NR.sup.E3)R.sup.B3, —N═S(O)R.sup.A3R.sup.B3, —S(O).sub.2OR.sup.A3, —OS(O).sub.2R.sup.A3, —NR.sup.A3S(O).sub.rR.sup.B3, —NR.sup.A3S(O)(═NR.sup.E3)R.sup.B3, —S(O).sub.rNR.sup.A3R.sup.B3, —S(O)(═NR.sup.E3)NR.sup.A3R.sup.B3, —NR.sup.A3S(O).sub.2NR.sup.A3R.sup.B3, —NR.sup.A3S(O)(═NR.sup.E3)NR.sup.A3R.sup.B3, —P(O)R.sup.A3R.sup.B3 and —P(O)(OR.sup.A3)(OR.sup.B3), wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each unsubstituted or substituted with at least one substituent, independently selected from R.sup.X3; or any two of R.sup.3 together with the atoms to which they are attached form a C.sub.3-10 cycloalkyl or heterocyclic ring of 4 to 12 members containing 1, 2 or 3 heteroatoms independently selected from oxygen, sulfur, nitrogen and phosphorus, and optionally substituted with 1, 2 or 3 R.sup.X3 groups; R.sup.5 is selected from hydrogen, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.3-10 cycloalkyl, C.sub.3-10 cycloalkyl-C.sub.1-4 alkyl, heterocyclyl, heterocyclyl-C.sub.1-4 alkyl, aryl, aryl-C.sub.1-4 alkyl, heteroaryl, heteroaryl-C.sub.1-4 alkyl and —C(O)R.sup.A5, wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each unsubstituted or substituted with at least one substituent, independently selected from R.sup.X5; R.sup.6 is selected from hydrogen, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.3-10 cycloalkyl, C.sub.3-10 cycloalkyl-C.sub.1-4 alkyl, heterocyclyl, heterocyclyl-C.sub.1-4 alkyl, aryl, aryl-C.sub.1-4 alkyl, heteroaryl, heteroaryl-C.sub.1-4 alkyl and —C(O)R.sup.A6, wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each unsubstituted or substituted with at least one substituent, independently selected from R.sup.X6; or R.sup.5 and R.sup.6 together with the atoms to which they are attached form a C.sub.3-10 cycloalkyl or heterocyclic ring of 4 to 12 members containing 1, 2 or 3 heteroatoms independently selected from oxygen, sulfur, nitrogen and phosphorus, and optionally substituted with 1, 2, 3 or 4 R.sup.4 groups; each R.sup.4 is independently selected from hydrogen, halogen, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.3-10 cycloalkyl, C.sub.3-10 cycloalkyl-C.sub.1-4 alkyl, heterocyclyl, heterocyclyl-C.sub.1-4 alkyl, aryl, aryl-C.sub.1-4 alkyl, heteroaryl, heteroaryl-C.sub.1-4 alkyl, CN, —NR.sup.A4R.sup.B4, —OR.sup.A4, —C(O)R.sup.A4, —C(O)OR.sup.A4, —OC(O)R.sup.A4, —C(O)NR.sup.A4R.sup.B4, —NR.sup.A4C(O)R.sup.B4, and —S(O).sub.rR.sup.A4, wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each unsubstituted or substituted with at least one substituent, independently selected from R.sup.X4; or any two of R.sup.4 together with the atom(s) to which they are attached form a C.sub.3-10 cycloalkyl or heterocyclic ring of 4 to 12 members containing 1, 2 or 3 heteroatoms independently selected from oxygen, sulfur, nitrogen and phosphorus, and optionally substituted with 1, 2 or 3 R.sup.X4 groups; R.sup.7 is selected from hydrogen, halogen, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.3-10 cycloalkyl, C.sub.3-10 cycloalkyl-C.sub.1-4 alkyl, heterocyclyl, heterocyclyl-C.sub.1-4 alkyl, aryl, aryl-C.sub.1-4 alkyl, heteroaryl, heteroaryl-C.sub.1-4 alkyl, CN, NO.sub.2, —NR.sup.A7R.sup.B7, —OR.sup.A7, —C(O)R.sup.A1, —C(═NR.sup.E7)R.sup.A7, —C(═N—OR.sup.B7)R.sup.A7, —C(O)OR.sup.A7, —OC(O)R.sup.A7, —C(O)NR.sup.A7R.sup.B7, —NR.sup.A7C(O)R.sup.B7, —C(═NR.sup.E7)NR.sup.A7R.sup.B7, —NR.sup.A7C(═NR.sup.E7)R.sup.B7, —OC(O)NR.sup.A7R.sup.B7, —NR.sup.A7C(O)OR.sup.B7, —NR.sup.A7C(O)NR.sup.A7R.sup.B7, —NR.sup.A7C(S)NR.sup.A7R.sup.B7, —NR.sup.A7C(═NR.sup.E7)NR.sup.A7R.sup.B7, —S(O).sub.rR.sup.A7, —S(O)(═NR.sup.E7)R.sup.B7, —N═S(O)R.sup.A7R.sup.B7, —S(O).sub.2OR.sup.A7, —OS(O).sub.2R.sup.A7, —NR.sup.A7S(O).sub.rR.sup.B7, —NR.sup.A7S(O)(═NR.sup.E7)R.sup.B7, —S(O).sub.rNR.sup.A7R.sup.B7, —S(O)(═NR.sup.E7)NR.sup.A7R.sup.B7, —NR.sup.A7S(O).sub.2NR.sup.A7R.sup.B7, —NR.sup.A7S(O)(═NR.sup.E7)NR.sup.A7R.sup.B7, —P(O)R.sup.A7R.sup.B7 and —P(O)(OR.sup.A7)(OR.sup.B7), wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each unsubstituted or substituted with at least one substituent, independently selected from R.sup.X7; or R.sup.6 and R.sup.7 together with the atoms to which they are attached form a C.sub.3-10 cycloalkyl or heterocyclic ring of 4 to 12 members containing 1, 2 or 3 heteroatoms independently selected from oxygen, sulfur, nitrogen and phosphorus, and optionally substituted with 1, 2 or 3 R.sup.X6 groups; each R.sup.A3 and R.sup.B3 are independently selected from hydrogen, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.3-10 cycloalkyl, C.sub.3-10 cycloalkyl-C.sub.1-4 alkyl, heterocyclyl, heterocyclyl-C.sub.1-4 alkyl, aryl, aryl-C.sub.1-4 alkyl, heteroaryl and heteroaryl-C.sub.1-4 alkyl, wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each unsubstituted or substituted with at least one substituent, independently selected from R.sup.X3; or “R.sup.A3 and R.sup.B3”, together with the atom(s) to which they are attached form a heterocyclic ring of 4 to 12 members containing 0, 1 or 2 additional heteroatoms independently selected from oxygen, sulfur, nitrogen and phosphorus and optionally substituted with 1, 2 or 3 R.sup.X3 groups; each R.sup.A4 and R.sup.B4 are independently selected from hydrogen, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.3-10 cycloalkyl, C.sub.3-10 cycloalkyl-C.sub.1-4 alkyl, heterocyclyl, heterocyclyl-C.sub.1-4 alkyl, aryl, aryl-C.sub.1-4 alkyl, heteroaryl, and heteroaryl-C.sub.1-4 alkyl, wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each unsubstituted or substituted with at least one substituent, independently selected from R.sup.X4; or each “R.sup.A4 and R.sup.B4”, together with the atom(s) to which they are attached form a heterocyclic ring of 4 to 12 members containing 0, 1 or 2 additional heteroatoms independently selected from oxygen, sulfur, nitrogen and phosphorus, and optionally substituted with 1, 2 or 3 R.sup.X4 groups; R.sup.A is selected from hydrogen, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.3-10 cycloalkyl, C.sub.3-10 cycloalkyl-C.sub.1-4 alkyl, heterocyclyl, heterocyclyl-C.sub.1-4 alkyl, aryl, aryl-C.sub.1-4 alkyl, heteroaryl, and heteroaryl-C.sub.1-4 alkyl, wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each unsubstituted or substituted with at least one substituent, independently selected from R.sup.X5; R.sup.A6 is selected from hydrogen, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.3-10 cycloalkyl, C.sub.3-10 cycloalkyl-C.sub.1-4 alkyl, heterocyclyl, heterocyclyl-C.sub.1-4 alkyl, aryl, aryl-C.sub.1-4 alkyl, heteroaryl, and heteroaryl-C.sub.1-4 alkyl, wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each unsubstituted or substituted with at least one substituent, independently selected from R.sup.X6; each R.sup.A7 and R.sup.B7 are independently selected from hydrogen, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.3-10 cycloalkyl, C.sub.3-10 cycloalkyl-C.sub.1-4 alkyl, heterocyclyl, heterocyclyl-C.sub.1-4 alkyl, aryl, aryl-C.sub.1-4 alkyl, heteroaryl, and heteroaryl-C.sub.1-4 alkyl, wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each unsubstituted or substituted with at least one substituent, independently selected from R.sup.X7; or each “R.sup.A7 and R.sup.B7” together with the atom(s) to which they are attached form a heterocyclic ring of 4 to 12 members containing 0, 1 or 2 additional heteroatoms independently selected from oxygen, sulfur, nitrogen and phosphorus, and optionally substituted with 1, 2 or 3 R.sup.X7 groups; each R.sup.E3 and R.sup.E7 are independently selected from hydrogen, C.sub.1-10 alkyl, CN, NO.sub.2, —OR.sup.a1, —SR.sup.a1, —S(O).sub.rR.sup.a1, —C(O)R.sup.a1, —C(O)OR.sup.a1, —C(O)NR.sup.a1R.sup.b1 and —S(O).sub.rNR.sup.a1R.sup.b1; each R.sup.X, R.sup.X1, R.sup.X2, R.sup.X3, R.sup.X4, R.sup.X5, R.sup.X6 and R.sup.X7 are independently selected from hydrogen, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.3-10 cycloalkyl, C.sub.3-10 cycloalkyl-C.sub.1-4 alkyl, heterocyclyl, heterocyclyl-C.sub.1-4 alkyl, aryl, aryl-C.sub.1-4 alkyl, heteroaryl, heteroaryl-C.sub.1-4 alkyl, halogen, CN, NO.sub.2, —(CR.sup.c1R.sup.d1).sub.tNR.sup.a1R.sup.b1, —(CR.sup.c1R.sup.d1).sub.tOR.sup.b1, —(CR.sup.c1R.sup.d1).sub.tC(O)R.sup.a1, —(CR.sup.c1R.sup.d1).sub.tC(═NR.sup.e1)R.sup.a1, —(CR.sup.c1R.sup.d1).sub.tC(═N—OR.sup.b1)R.sup.a1, —(CR.sup.c1R.sup.d1).sub.tC(O)OR.sup.b1, —(CR.sup.c1R.sup.d1).sub.tOC(O)R.sup.b1, —(CR.sup.c1R.sup.d1).sub.tC(O)NR.sup.a1R.sup.b1, —(CR.sup.c1R.sup.d1)NR.sup.a1C(O)R.sup.b1, —(CR.sup.c1R.sup.d1).sub.tC(═NR.sup.e1)NR.sup.a1R.sup.b1, —(CR.sup.c1R.sup.d1).sub.tR.sup.a1C(═NR.sup.e1)R.sup.b1, —(CR.sup.c1R.sup.d1).sub.tOC(O)NR.sup.a1R.sup.b1, —(CR.sup.c1R.sup.d1).sub.tR.sup.a1C(O)OR.sup.b1, —(CR.sup.c1R.sup.d1).sub.tR.sup.a1C(O)NR.sup.a1R.sup.b1, —(CR.sup.c1R.sup.d1)NR.sup.a1C(S)NR.sup.a1R.sup.b1, —(CR.sup.c1R.sup.d1)NR.sup.a1C(═NR.sup.e1)NR.sup.a1R.sup.b1, —(CR.sup.c1R.sup.d1).sub.tS(O).sub.rR.sup.b1, —(CR.sup.c1R.sup.d1).sub.tS(O)(═NR.sup.e1)R.sup.b1, —(CR.sup.c1R.sup.d1).sub.tN═S(O)R.sup.a1R.sup.b1, —(CR.sup.c1R.sup.d1).sub.tS(O).sub.2OR.sup.b1, —(CR.sup.c1R.sup.d1).sub.tOS(O).sub.2R.sup.b1, —(CR.sup.c1R.sup.d1).sub.tNR.sup.a1S(O).sub.rR.sup.b1, —(CR.sup.c1R.sup.d1)NR.sup.a1S(O)(═NR.sup.e1)R.sup.b1, —(CR.sup.c1R.sup.d1).sub.tS(O).sub.rNR.sup.a1R.sup.b1, —(CR.sup.c1R.sup.d1).sub.tS(O)(═NR.sup.e1)NR.sup.a1R.sup.b1, —(CR.sup.c1R.sup.d1).sub.tNR.sup.a1S(O).sub.2NR.sup.a1R.sup.b1, —(CR.sup.c1R.sup.d1).sub.tNR.sup.a1S(O)(═NR.sup.e1)NR.sup.a1R.sup.b1, —(CR.sup.c1R.sup.a1).sub.tP(O)R.sup.a1R.sup.b1 and —(CR.sup.c1R.sup.d1).sub.tP(O)(OR.sup.a1)(OR.sup.b1), wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each unsubstituted or substituted with at least one substituent, independently selected from R.sup.Y; each R.sup.a1 and each R.sup.b1 are independently selected from hydrogen, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.3-10 cycloalkyl, C.sub.3-10 cycloalkyl-C.sub.1-4 alkyl, heterocyclyl, heterocyclyl-C.sub.1-4 alkyl, aryl, aryl-C.sub.1-4 alkyl, heteroaryl and heteroaryl-C.sub.1-4 alkyl, wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each unsubstituted or substituted with at least one substituent, independently selected from R.sup.Y; or R.sup.a1 and R.sup.b1 together with the atom(s) to which they are attached form a heterocyclic ring of 4 to 12 members containing 0, 1 or 2 additional heteroatoms independently selected from oxygen, sulfur, nitrogen and phosphorus, and optionally substituted with 1, 2 or 3 R.sup.Y groups; each R.sup.c1 and each R.sup.d1 are independently selected from hydrogen, halogen, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.3-10 cycloalkyl, C.sub.3-10 cycloalkyl-C.sub.1-4 alkyl, heterocyclyl, heterocyclyl-C.sub.1-4 alkyl, aryl, aryl-C.sub.1-4 alkyl, heteroaryl and heteroaryl-C.sub.1-4 alkyl, wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each unsubstituted or substituted with at least one substituent, independently selected from R.sup.Y; or R.sup.c1 and R.sup.d1 together with the carbon atom(s) to which they are attached form a ring of 3 to 12 members containing 0, 1 or 2 heteroatoms independently selected from oxygen, sulfur and nitrogen, and optionally substituted with 1, 2 or 3 R.sup.Y groups; each R.sup.e1 is independently selected from hydrogen, C.sub.1-10 alkyl, C.sub.3-10 cycloalkyl, C.sub.3-10 cycloalkyl-C.sub.1-4 alkyl, CN, NO.sub.2, —OR.sup.a2, —SR.sup.a2, —S(O).sub.rR.sup.a2, —C(O)R.sup.a2, —C(O)OR.sup.a2, —S(O).sub.rNR.sup.a2R.sup.b2 and —C(O)NR.sup.a2R.sup.b2; each R.sup.Y is independently selected from C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.3-10 cycloalkyl, C.sub.3-10 cycloalkyl-C.sub.1-4 alkyl, heterocyclyl, heterocyclyl-C.sub.1-4 alkyl, aryl, aryl-C.sub.1-4 alkyl, heteroaryl, heteroaryl-C.sub.1-4 alkyl, halogen, CN, NO.sub.2, —(CR.sup.c2R.sup.d2).sub.tNR.sup.a2R.sup.b2, —(CR.sup.c2R.sup.d2).sub.tOR.sup.b2, —(CR.sup.c2R.sup.d2).sub.tC(O)R.sup.a2, —(CR.sup.c2R.sup.d2).sub.tC(═NR.sup.e2)R.sup.a2, —(CR.sup.c2R.sup.d2).sub.tC(═N—OR.sup.b2)R.sup.a2, —(CR.sup.c2R.sup.d2).sub.tC(O)OR.sup.b2, —(CR.sup.c2R.sup.d2).sub.tOC(O)R.sup.b2, —(CR.sup.c2R.sup.d2).sub.tC(O)NR.sup.a2R.sup.b2, —(CR.sup.c2R.sup.d2)NR.sup.a2C(O)R.sup.b2, —(CR.sup.c2R.sup.d2).sub.tC(═NR.sup.e2)NR.sup.a2R.sup.b2, —(CR.sup.c2R.sup.d2).sub.tNR.sup.a2C(═NR.sup.e2)R.sup.b2, —(CR.sup.c2R.sup.d2).sub.tOC(O)NR.sup.a2R.sup.b2, —(CR.sup.c2R.sup.d2).sub.tNR.sup.a2C(O)OR.sup.b2, —(CR.sup.c2R.sup.d2).sub.tNR.sup.a2C(O)NR.sup.a2R.sup.b2, —(CR.sup.c2R.sup.d2).sub.tR.sup.a2C(S)NR.sup.a2R.sup.b2, —(CR.sup.c2R.sup.d2)NR.sup.a2C(═NR.sup.e2)NR.sup.a2R.sup.b2, —(CR.sup.c2R.sup.d2).sub.tS(O).sub.rR.sup.b2, —(CR.sup.c2R.sup.d2).sub.tS(O)(═NR.sup.e2)R.sup.b2, —(CR.sup.c2R.sup.d2).sub.tN═S(O)R.sup.a2R.sup.b2, —(CR.sup.c2R.sup.d2).sub.tS(O).sub.2OR.sup.b2, —(CR.sup.c2R.sup.d2).sub.tOS(O).sub.2R.sup.b2, —(CR.sup.c2R.sup.d2).sub.tR.sup.a2S(O).sub.rR.sup.b2, —(CR.sup.c2R.sup.d2).sub.tR.sup.a2S(O)(═NR.sup.e2)R.sup.b2, —(CR.sup.c2R.sup.d2).sub.tS(O).sub.rNR.sup.a2R.sup.b2, —(CR.sup.c2R.sup.d2).sub.tS(O)(═NR.sup.e2)NR.sup.a2R.sup.b2, —(CR.sup.c2R.sup.d2).sub.tNR.sup.a2S(O).sub.2NR.sup.a2R.sup.b2, —(CR.sup.c2R.sup.d2).sub.tNR.sup.a2S(O)(═NR.sup.e2)NR.sup.a2R.sup.b2, —(CR.sup.c2R.sup.d2).sub.tP(O)R.sup.a2R.sup.b2 and —(CR.sup.c2R.sup.d2).sub.tP(O)(OR.sup.a2)(OR.sup.b2), wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each unsubstituted or substituted with at least one substituent, independently selected from OH, CN, amino, halogen, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.3-10 cycloalkyl, C.sub.1-10 alkoxy, C.sub.3-10 cycloalkoxy, C.sub.1-10 alkylthio, C.sub.3-10 cycloalkylthio, C.sub.1-10 alkylamino, C.sub.3-10 cycloalkylamino and di(C.sub.1-10 alkyl)amino; each R.sup.a2 and each R.sup.b2 are independently selected from hydrogen, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.3-10 cycloalkyl, C.sub.3-10 cycloalkyl-C.sub.1-4 alkyl, C.sub.1-10 alkoxy, C.sub.3-10 cycloalkoxy, C.sub.1-10 alkylthio, C.sub.3-10 cycloalkylthio, C.sub.1-10 alkylamino, C.sub.3-10 cycloalkylamino, di(C.sub.1-10 alkyl)amino, heterocyclyl, heterocyclyl-C.sub.1-4 alkyl, aryl, aryl-C.sub.1-4 alkyl, heteroaryl and heteroaryl-C.sub.1-4 alkyl, wherein alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, cycloalkoxy, alkylthio, cycloalkylthio, alkylamino, cycloalkylamino, heterocyclyl, aryl and heteroaryl are each unsubstituted or substituted with at least one substituent, independently selected from halogen, CN, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.3-10 cycloalkyl, OH, C.sub.1-10 alkoxy, C.sub.3-10 cycloalkoxy, C.sub.1-10 alkylthio, C.sub.3-10 cycloalkylthio, amino, C.sub.1-10 alkylamino, C.sub.3-10 cycloalkylamino and di(C.sub.1-10 alkyl)amino; or R.sup.a2 and R.sup.b2 together with the atom(s) to which they are attached form a heterocyclic ring of 4 to 12 members containing 0, 1 or 2 additional heteroatoms independently selected from oxygen, sulfur, nitrogen and phosphorus, and optionally substituted with 1 or 2 substituents, independently selected from halogen, CN, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.3-10 cycloalkyl, OH, C.sub.1-10 alkoxy, C.sub.3-10 cycloalkoxy, C.sub.1-10 alkylthio, C.sub.3-10 cycloalkylthio, amino, C.sub.1-10 alkylamino, C.sub.3-10 cycloalkylamino and di(C.sub.1-10 alkyl)amino; each R.sup.c2 and each R.sup.d2 are independently selected from hydrogen, halogen, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.3-10 cycloalkyl, C.sub.3-10 cycloalkyl-C.sub.1-4 alkyl, C.sub.1-10 alkoxy, C.sub.3-10 cycloalkoxy, C.sub.1-10 alkylthio, C.sub.3-10 cycloalkylthio, C.sub.1-10 alkylamino, C.sub.3-10 cycloalkylamino, di(C.sub.1-10 alkyl)amino, heterocyclyl, heterocyclyl-C.sub.1-4 alkyl, aryl, aryl-C.sub.1-4 alkyl, heteroaryl and heteroaryl-C.sub.1-4 alkyl, wherein alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, cycloalkoxy, alkylthio, cycloalkylthio, alkylamino, cycloalkylamino, heterocyclyl, aryl and heteroaryl are each unsubstituted or substituted with at least one substituent, independently selected from halogen, CN, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.3-10 cycloalkyl, OH, C.sub.1-10 alkoxy, C.sub.3-10 cycloalkoxy, C.sub.1-10 alkylthio, C.sub.3-10 cycloalkylthio, amino, C.sub.1-10 alkylamino, C.sub.3-10 cycloalkylamino and di(C.sub.1-10 alkyl)amino; or R.sup.c2 and R.sup.d2 together with the carbon atom(s) to which they are attached form a ring of 3 to 12 members containing 0, 1 or 2 heteroatoms independently selected from oxygen, sulfur and nitrogen, and optionally substituted with 1 or 2 substituents, independently selected from halogen, CN, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.3-10 cycloalkyl, OH, C.sub.1-10 alkoxy, C.sub.3-10 cycloalkoxy, C.sub.1-10 alkylthio, C.sub.3-10 cycloalkylthio, amino, C.sub.1-10 alkylamino, C.sub.3-10 cycloalkylamino and di(C.sub.1-10 alkyl)amino; each R.sup.e2 is independently selected from hydrogen, CN, NO.sub.2, C.sub.1-10 alkyl, C.sub.3-10 cycloalkyl, C.sub.3-10 cycloalkyl-C.sub.1-4 alkyl, C.sub.1-10 alkoxy, C.sub.3-10 cycloalkoxy, —C(O)C.sub.1-4 alkyl, —C(O)C.sub.3-10 cycloalkyl, —C(O)OC.sub.1-4 alkyl, —C(O)OC.sub.3-10 cycloalkyl, —C(O)N(C.sub.1-4 alkyl).sub.2, —C(O)N(C.sub.3-10 cycloalkyl).sub.2, —S(O).sub.2C.sub.1-4 alkyl, —S(O).sub.2C.sub.3-10 cycloalkyl, —S(O).sub.2N(C.sub.1-4 alkyl).sub.2 and —S(O).sub.2N(C.sub.3-10 cycloalkyl).sub.2; m is selected from 0, 1, 2, 3 and 4; each r is independently selected from 0, 1 and 2; each t is independently selected from 0, 1, 2, 3 and 4.
2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the moiety ##STR00219## in Formula (I) is selected from ##STR00220## wherein each R.sup.4, R.sup.5, R.sup.6 and R.sup.7 are as defined in Formula (I), n is selected from 0, 1, 2, 3 and 4; p is selected from 0, 1, 2, 3 and 4; wherein the symbol indicates the point of attachment to the rest of the molecule.
3.-15. (canceled)
16. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R.sup.6 is selected from C.sub.1-8 alkyl, C.sub.2-10 alkenyl, C.sub.3-10 cycloalkyl, heterocyclyl, aryl, heteroaryl and —C(O)R.sup.A6, wherein alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each unsubstituted or substituted with at least one substituent, independently selected from R.sup.X6.
17. The compound of claim 16 or a pharmaceutically acceptable salt thereof, wherein R.sup.6 is selected from methyl, ethyl, ##STR00221## ##STR00222##
18.-22. (canceled)
23. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein X is CH or N.
24. (canceled)
25. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein Y is CH or N.
26.-30. (canceled)
31. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein m is selected from 1 and 2, each R.sup.3 is F.
32. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the moiety ##STR00223## in Formula (I) is selected from ##STR00224##
33.-34. (canceled)
35. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein Z is selected from 5- to 12-membered aryl and heteroaryl, wherein aryl and heteroaryl are each unsubstituted or substituted with at least one substituent, independently selected from R.sup.X.
36. The compound of claim 35 or a pharmaceutically acceptable salt thereof, wherein Z is selected from phenyl and pyridinyl, which is unsubstituted or substituted with at least one substituent, independently selected from R.sup.X.
37. (canceled)
38. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R.sup.X is selected from C.sub.1-10 alkyl, C.sub.3-10 cycloalkyl, halogen, CN, NO.sub.2, NH.sub.2, OH, methoxy and ethoxy, wherein methoxy, ethoxy alkyl and cycloalkyl are each unsubstituted or substituted with at least one substituent, independently selected from R.sup.Y.
39. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein Z is selected from ##STR00225##
40.-41. (canceled)
42. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R.sup.1 and R.sup.2 are independently selected from H and methyl.
43. (canceled)
44. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R.sup.1 and R.sup.2 together with the atom(s) to which they are attached form a cyclopropyl.
45. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from ##STR00226## ##STR00227## ##STR00228## ##STR00229## ##STR00230## ##STR00231## ##STR00232## ##STR00233## ##STR00234## ##STR00235## ##STR00236## ##STR00237## ##STR00238## ##STR00239## ##STR00240## ##STR00241## ##STR00242## ##STR00243## ##STR00244## ##STR00245## ##STR00246## ##STR00247## ##STR00248## ##STR00249## ##STR00250## and pharmaceutically acceptable salts thereof.
46. A pharmaceutical composition, comprising a compound of claim 1 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier.
47. A method of treating, ameliorating or preventing a condition, which responds to inhibition of BTK, comprising administering to a subject in need of such treatment an effective amount of a compound of claim 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, and optionally in combination with a second therapeutic agent.
48. A method of treating a cell-proliferative disorder, comprising administering to a subject in need of such treatment an effective amount of a compound of claim 1 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof, and optionally in combination with a second therapeutic agent.
49. The method claim 48, wherein the cell-proliferative disorder is B-cell proliferative disorder.
50. The method of claim 49, wherein the B-cell proliferative disorder is selected from B-cell malignancies, B-cell chronic lymphocytic lymphoma, chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, multiple sclerosis, small lymphocytic lymphoma, mantle cell lymphoma, B-cell non-Hodgkin's lymphoma, activated B-cell like diffuse large B-cell lymphoma, multiple myeloma, diffuse large B-cell lymphoma, follicular lymphoma, primary effusion lymphoma, burkitt lymphoma/leukemia, lymphomatoid granulomatosis, and plasmacytoma.
Description
EXAMPLES
[0445] Various methods may be developed for synthesizing a compound of formula (I) or a pharmaceutically acceptable salt thereof. Representative methods for synthesizing a compound of formula (I) or a pharmaceutically acceptable salt thereof are provided in the Examples. It is noted, however, that a compound of formula (I) or a pharmaceutically acceptable salt thereof may also be synthesized by other synthetic routes that others may devise.
[0446] It will be readily recognized that certain compounds of formula (I) have atoms with linkages to other atoms that confer a particular stereochemistry to the compound (e.g., chiral centers). It is recognized that synthesis of a compound of formula (I) or a pharmaceutically acceptable salt thereof may result in the creation of mixtures of different stereoisomers (enantiomers, diastereomers). Unless a particular stereochemistry is specified, recitation of a compound is intended to encompass all of the different possible stereoisomers.
[0447] A compound of formula (I) can also be prepared as a pharmaceutically acceptable acid addition salt by, for example, reacting the free base form of the at least one compound with a pharmaceutically acceptable inorganic or organic acid. Alternatively, a pharmaceutically acceptable base addition salt of the at least one compound of formula (I) can be prepared by, for example, reacting the free acid form of the at least one compound with a pharmaceutically acceptable inorganic or organic base. Inorganic and organic acids and bases suitable for the preparation of the pharmaceutically acceptable salts of compounds of formula (I) are set forth in the definitions section of this Application. Alternatively, the salt forms of the compounds of formula (I) can be prepared using salts of the starting materials or intermediates.
[0448] The free acid or free base forms of the compounds of formula (I) can be prepared from the corresponding base addition salt or acid addition salt form. For example, a compound of formula (I) in an acid addition salt form can be converted to the corresponding free base thereof by treating with a suitable base (e.g., ammonium hydroxide solution, sodium hydroxide, and the like). A compound of formula (I) in a base addition salt form can be converted to the corresponding free acid thereof by, for example, treating with a suitable acid (e.g., hydrochloric acid, etc).
[0449] The N-oxides of a compound of formula (I) or a pharmaceutically acceptable salt thereof can be prepared by methods known to those of ordinary skill in the art. For example, N-oxides can be prepared by treating an unoxidized form of the compound of formula (I) with an oxidizing agent (e.g., trifluoroperacetic acid, permaleic acid, perbenzoic acid, peracetic acid, meta-chloroperoxybenzoic acid, or the like) in a suitable inert organic solvent (e.g., a halogenated hydrocarbon such as dichloromethane) at approximately 0 to 80° C. Alternatively, the N-oxides of the compounds of formula (I) can be prepared from the N-oxide of an appropriate starting material.
[0450] Compounds of formula (I) in an unoxidized form can be prepared from N-oxides of compounds of formula (I) by, for example, treating with a reducing agent (e.g., sulfur, sulfur dioxide, triphenyl phosphine, lithium borohydride, sodium borohydride, phosphorus trichloride, tribromide, and the like) in an suitable inert organic solvent (e.g., acetonitrile, ethanol, aqueous dioxane, and the like) at 0 to 80° C.
[0451] Protected derivatives of the compounds of formula (I) can be made by methods known to those of ordinary skill in the art. A detailed description of the techniques applicable to the creation of protecting groups and their removal can be found in T. W. Greene, Protecting Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, Inc. 1999.
[0452] As used herein the symbols and conventions used in these processes, schemes and examples are consistent with those used in the contemporary scientific literature, for example, the Journal of the American Chemical Society or the Journal of Biological Chemistry. Standard single-letter or three-letter abbreviations are generally used to designate amino acid residues, which are assumed to be in the L-configuration unless otherwise noted. Unless otherwise noted, all starting materials were obtained from commercial suppliers and used without further purification. For example, the following abbreviations may be used in the examples and throughout the specification: g (grams); mg (milligrams); L (liters); mL (milliliters); μL (microliters); psi (pounds per square inch); M (molar); mM (millimolar); i.v. (intravenous); Hz (Hertz); MHz (megahertz); mol (moles); mmol (millimoles); RT (room temperature); min (minutes); h (hours); mp (melting point); TLC (thin layer chromatography); Rt (retention time); RP (reverse phase); MeOH (methanol); i-PrOH (isopropanol); TEA (triethylamine); TFA (trifluoroacetic acid); TFAA (trifluoroacetic anhydride); THF (tetrahydrofuran); DMSO (dimethyl sulfoxide); EtOAc (ethyl acetate); DME (1,2-dimethoxyethane); DCM (dichloromethane); DCE (dichloroethane); DMF (N,N-dimethylformamide); DMPU (N,N′-dimethylpropyleneurea); CDI (1,1-carbonyldiimidazole); IBCF (isobutyl chloroformate); HOAc (acetic acid); HOSu (N-hydroxysuccinimide); HOBT (1-hydroxybenzotriazole); Et.sub.2O (diethyl ether); EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride); BOC (tert-butyloxycarbonyl); FMOC (9-fluorenylmethoxycarbonyl); DCC (dicyclohexylcarbodiimide); CBZ (benzyloxycarbonyl); Ac (acetyl); atm (atmosphere); TMSE (2-(trimethylsilyl)ethyl); TMS (trimethylsilyl); TIPS (triisopropylsilyl); TBS (t-butyldimethylsilyl); DMAP (4-dimethylaminopyridine); Me (methyl); OMe (methoxy); Et (ethyl); tBu (tert-butyl); HPLC (high pressure liquid chromatography); BOP (bis(2-oxo-3-oxazolidinyl)phosphinic chloride); TBAF (tetra-n-butylammonium fluoride); m-CPBA (meta-chloroperbenzoic acid). For example, the following abbreviations in table 1 may be used in the examples and throughout the specification.
[0453] References to ether or Et.sub.2O are to diethyl ether; brine refers to a saturated aqueous solution of NaCl. Unless otherwise indicated, all temperatures are expressed in ° C. (degrees Centigrade). All reactions were conducted under an inert atmosphere at RT unless otherwise noted.
[0454] .sup.1H NMR spectra were recorded on a Varian Mercury Plus 400. Chemical shifts are expressed in parts per million (ppm). Coupling constants are in units of hertz (Hz). Splitting patterns describe apparent multiplicities and are designated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and br (broad).
[0455] Low-resolution mass spectra (MS) and compound purity data were acquired on a Shimadzu LC/MS single quadrupole system equipped with electrospray ionization (ESI) source, UV detector (220 and 254 nm), and evaporative light scattering detector (ELSD). Thin-layer chromatography was performed on 0.25 mm Superchemgroup silica gel plates (60F-254), visualized with UV light, 5% ethanolic phosphomolybdic acid, ninhydrin, or p-anisaldehyde solution. Flash column chromatography was performed on silica gel (200-300 mesh, Branch of Qingdao Haiyang Chemical Co., Ltd).
Synthetic Schemes
[0456] A compound of formula I or pharmaceutically acceptable salt thereof may be synthesized according to a variety of reaction schemes. Some illustrative schemes are provided below and in the examples. Other reaction schemes could be readily devised by those skilled in the art in view of the present disclosure.
[0457] In the reactions described hereinafter it may be necessary to protect reactive functional groups, for example hydroxy, amino, imino, thio or carboxy groups, where these are desired in the final product, to avoid their unwanted participation in the reactions. Conventional protecting groups may be used in accordance with standard practice, for examples see T. W. Greene and P. G. M. Wuts in “Protective Groups in Organic Chemistry” John Wiley and Sons, 1991.
[0458] Synthetic methods for preparing the compounds of the present disclosure are illustrated in the following Schemes and Examples. Starting materials are commercially available or may be made according to procedures known in the art or as illustrated herein.
[0459] The intermediates shown in the following schemes are either known in the literature or may be prepared by a variety of methods familiar to those skilled in the art.
[0460] As shown in the Scheme 1, the compounds of formula I can be synthesized from bromide II and potassium trifluoroborate salt III, which are either known in the literature or may be prepared by a variety of methods familiar to those skilled in the art. Coupling of brominated II with potassium trifluoroborate salt III via a suzuki reaction leads to compounds of formula I.
##STR00089##
[0461] As an illustration of the preparation of intermediates of formula II, a preparation of compound IIa is illustrated in Scheme 2. Starting from aldehyde IIa-A and substituted aromatic amines IIa-B, which are either commercially available or known in the literature, the intermediate IIa-C was prepared by treating of IIa-A with IIa-B in the presence of p-toluenesulfonic acid. IIa-C was treated with t-BuONa in a solvent such as THF to give intramolecular cyclization product IIa-D. The amino group of IIa-D can be converted into an iodine group via a sandmeyer reaction with reagents such as CH.sub.2I.sub.2 and tert-butyl nitrite to give IIa-E. The iodinate IIa-E can be transformed into ester IIa-F by carbonylation with CO(gas). Heating of IIa-F with N.sub.2H.sub.4.H.sub.2O leads to intermediate IIa.
##STR00090##
[0462] As a further illustration of the preparation of intermediates of formula II, a preparation of compound IIb is illustrated in Scheme 3. The reaction of the diazonium salt derived from aromatic amines IIb-B with aldehyde IIa-A in the presence of a base such as NaOAc in a solvent such as EtOH provides phenylhydrazone IIb-C. The 4-aminopyrazole IIb-D can be readily prepared from phenylhydrazone IIb-C through a sequence of alkylation of phenylhydrazone with bromoaectonitrile and intramolecular cyclization effected by a base such as t-BuONa. The compound IIb can be prepared from amine IIb-D in the same way as IIa from amine IIa-D shown in the scheme 2.
##STR00091##
[0463] A preparation of compound IIc is illustrated in Scheme 4 as a further illustration of the preparation of intermediates of formula II. Starting from the commercially available aliphatic amine IIc-A, Boc-hydrazine IIc-C can be prepared by treatment with IIc-B in the presence of inorganic bases such as NaHCO.sub.3 (sat.). Deprotection of the Boc group in IIc-C with TFA/DCM gives IIc-D which is reacted with IIc-E in the presence of organic bases such as TEA to provide amino-pyrazole IIc-F. IIc-F can be converted into IIc through the sequence shown in Scheme 2.
##STR00092##
[0464] In some cases the order of carrying out the foregoing reaction schemes may be varied to facilitate the reaction or to avoid unwanted reaction products. The following examples are provided so that the invention might be more fully understood. These examples are illustrative only and should not be construed as limiting the invention in any way.
Example 1
N-(4-(7-amino-3-cyclopentyl-4-oxo-4,5-dihydro-1H-pyrrolo[2,3-d]pyridazin-1-yl)benzyl)-2-methoxybenzamide (1)
[0465] ##STR00093##
2-cyclopentyl-3-oxopropanenitrile (1a)
[0466] The title compound 2-cyclopentyl-3-oxopropanenitrile (1a) was prepared according to the method described in WO2015/74135.
2-((4-bromophenyl)amino)acetonitrile (1b)
[0467] The title compound 2-((4-bromophenyl)amino)acetonitrile (1b) was prepared according to the method described in WO2005/40110.
3-((4-bromophenyl)(cyanomethyl)amino)-2-cyclopentylacrylonitrile (1c)
[0468] To a solution of 2-cyclopentyl-3-oxopropanenitrile (1a) (140 mg, 1.0 mmol) and 2-((4-bromophenyl)amino)acetonitrile (1b) (210 mg, 1.0 mmol) in toluene (10 ml) was added p-Toluenesulfonic acid (17.2 mg, 0.1 mmol), the mixture was stirred at 110° C. for 12 h. The reaction mixture was filtered and concentrated. The residue was purified by column chromatography on silica gel eluting with PE/EtOAc (10:1˜5:1) to give 3-((4-bromophenyl)(cyanomethyl)amino)-2-cyclopentylacrylonitrile (1c). MS-ESI (m/z): 330 332[M+1].sup.+.
3-amino-1-(4-bromophenyl)-4-cyclopentyl-1H-pyrrole-2-carbonitrile (1d)
[0469] To the suspension of t-BuONa (53 mg, 0.55 mmol) in THF (5 mL) was added a solution of 3-((4-bromophenyl)(cyanomethyl)amino)-2-cyclopentylacrylonitrile (1c) (120 mg, 0.36 mmol) in THF (5 mL) at 0° C. The mixture was heated to 25° C. and stirred for 2 h. The reaction was quenched with water and extracted with EtOAc (2×50 mL). The extracts were washed with water and brine, dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue was purified by column chromatography on silica gel eluting with PE/EtOAc (10:1) to give 3-amino-1-(4-bromophenyl)-4-cyclopentyl-1H-pyrrole-2-carbonitrile (1d). MS-ESI (m/z): 330, 332 (1:1) [M+1].sup.+.
1-(4-bromophenyl)-4-cyclopentyl-3-iodo-1H-pyrrole-2-carbonitrile (1e)
[0470] To a solution of 3-amino-1-(4-bromophenyl)-4-cyclopentyl-1H-pyrrole-2-carbonitrile (1d) (200 mg, 0.6 mmol) and CH.sub.2I.sub.2 (586 mg, 2.18 mmol) in CH.sub.3CN (5 mL) was added tert-butyl nitrite (156 mg, 1.5 mmol) dropwise at 35° C. The mixture was heated to 65° C. and stirred for 0.5 h. The reaction was concentrated and purified by column chromatography on silica gel eluting with PE/EtOAc (15:1) to give 1-(4-bromophenyl)-4-cyclopentyl-3-iodo-1H-pyrrole-2-carbonitrile (1e). MS-ESI (m/z): 441, 443 (1:1) [M+1].sup.+.
Methyl-1-(4-bromophenyl)-2-cyano-4-cyclopentyl-1H-pyrrole-3-carboxylate (1f)
[0471] To a solution of 1-(4-bromophenyl)-4-cyclopentyl-3-iodo-1H-pyrrole-2-carbonitrile (1e) (200 mg, 0.23 mmol) in MeOH was added Pd(dppf)Cl.sub.2 (100 mg, 0.11 mmol) and TEA (70 mg, 0.69 mmol) at RT. The mixture was stirred at 50° C. under CO (15 psi) for 1 h. The mixture was filtered and concentrated. The residue was purified by column chromatography on silica gel eluting with PE/EtOAc (10:1) to give methyl 1-(4-bromophenyl)-2-cyano-4-cyclopentyl-1H-pyrrole-3-carboxylate (if). MS-ESI (m/z): 373, 375 (1:1) [M+1].sup.+.
7-amino-1-(4-bromophenyl)-3-cyclopentyl-1,5-dihydro-4H-pyrrolo[2,3-d]pyridazin-4-one (12)
[0472] To a solution of methyl 1-(4-bromophenyl)-2-cyano-4-cyclopentyl-1H-pyrrole-3-carboxylate (if) (50 mg, 0.13 mmol) in EtOH (2 mL) was added NH.sub.2NH.sub.2.H.sub.2O (1 ml). The mixture was stirred at 90° C. for 12 h. The reaction mixture was concentrated and purified by column chromatography on silica gel eluting with DCM/MeOH (50:1) to give 7-amino-1-(4-bromophenyl)-3-cyclopentyl-1,5-dihydro-4H-pyrrolo[2,3-d]pyridazin-4-one (1g). MS-ESI (m/z): 373, 375 (1:1) [M+1].sup.+.
Potassium trifluoro [(2-methoxybenzamido)methyl] borate (1h)
[0473] The title compound potassium trifluoro [(2-methoxybenzamido)methyl]borate (1h) was prepared according to the method described in WO2017/103611.
N-(4-(7-amino-3-cyclopentyl-4-oxo-4,5-dihydro-1H-pyrrolo[2,3-d]pyridazin-1-yl)benzyl)-2-methoxybenzamide (1)
[0474] To a solution of 7-amino-1-(4-bromophenyl)-3-cyclopentyl-1,5-dihydro-4H-pyrrolo[2,3-d]pyridazin-4-one (1g) (15 mg, 0.040 mmol), potassium trifluoro [(2-methoxybenzamido)methyl] borate (1h) (11 mg, 0.040 mmol) and Cs.sub.2CO.sub.3 (40 mg, 0.52 mol) in THF (2 mL) and H.sub.2O (0.2 mL) was added Pd(OAc).sub.2 (2 mg, 0.008 mmol) and Xantphos (9 mg, 0.016 mmol). The mixture was stirred at 80° C. for 1 h. The mixture was diluted with water and extracted with DCM/MeOH (10:1, 2×20 mL). The extracts were washed with water and brine, dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (15:1) to give N-(4-(7-amino-3-cyclopentyl-4-oxo-4,5-dihydro-1H-pyrrolo[2,3-d]pyridazin-1-yl)benzyl)-2-methoxybenzamide (1). MS-ESI (m/z): 458 [M+1].sup.+.
Example 2
N-(4-(7-amino-3-cyclopentyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyridazin-1-yl)benzyl)-2-methoxybenzamide (2)
[0475] ##STR00094##
(E)-N-(4-bromophenyl)cyclopentanecarbohydrazonoyl cyanide (2a)
[0476] To a solution of 4-bromoaniline (1.72 g, 10.0 mmol) in HCl (6 N, 15 ml) was added NaNO.sub.2 (2.00 g, 30.0 mmol) protions at −10° C., the mixture was stirred at RT for 1 h. Then the mixture was added a solution of 2-cyclopentyl-3-oxopropanenitrile (1a) (2.00 g, 14.6 mmol) and NaOAc (16.0 g, 195 mmol) in EtOH (160 ml). The mixture was stirred at −10° C. for 1 h. The reaction was quenched with H.sub.2O, extracted with EtOAc (2×50 mL). The extracts were washed with water and brine, dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue was purified by column chromatography on silica gel eluting with PE/EtOAc (20:1˜10:1) to give (E)-N-(4-bromophenyl)cyclopentanecarbohydrazonoyl cyanide (2a). MS-ESI (m/z): 292, 294 (1:1) [M+1].sup.+.
4-amino-1-(4-bromophenyl)-3-cyclopentyl-1H-pyrazole-5-carbonitrile (2b)
[0477] To a solution of (E)-N-(4-bromophenyl)cyclopentanecarbohydrazonoyl cyanide (2a) (1.2 g, 4.1 mmol) and 2-bromoacetonitrile (4.9 g, 41 mmol) in t-BuOH (80 mL) was added t-BuONa (3.9 g, 41 mmol) at 0° C. The mixture was warmed to 25° C. and stirred for 2 h. The reaction was quenched with water, extracted with EtOAc (2×50 mL). The extracts were washed with water and brine, dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue was purified by column chromatography on silica gel eluting with PE/EtOAc (10:1) to give 4-amino-1-(4-bromophenyl)-3-cyclopentyl-1H-pyrazole-5-carbonitrile (2b). MS-ESI (m/z): 331, 333 (1:1) [M+1].sup.+.
1-(4-bromophenyl)-3-cyclopentyl-4-iodo-1H-pyrazole-5-carbonitrile (2c)
[0478] To a solution of 4-amino-1-(4-bromophenyl)-3-cyclopentyl-1H-pyrazole-5-carbonitrile (2b) (200 mg, 0.600 mmol) and CH.sub.2I.sub.2 (586 mg, 2.18 mmol) in CH.sub.3CN (5 mL) was added tert-butyl nitrite (156 mg, 1.50 mmol) dropwise at 35° C. The mixture was heated to 65° C. and stirred for 0.5 h. The reaction was concentrated and purified by column chromatography on silica gel eluting with PE/EtOAc (10:1) to give 1-(4-bromophenyl)-3-cyclopentyl-4-iodo-1H-pyrazole-5-carbonitrile (2c). MS-ESI (m/z): 442, 444 (1:1) [M+1].sup.+.
Butyl-1-(4-bromophenyl)-5-cyano-3-cyclopentyl-1H-pyrazole-4-carboxylate (2d)
[0479] To a solution of 1-(4-bromophenyl)-3-cyclopentyl-4-iodo-1H-pyrazole-5-carbonitrile (2c) (80 mg, 0.18 mmol) in BuOH was added Pd(dppf)Cl.sub.2 (80 mg, 0.11 mmol) and TEA (58 mg, 0.57 mmol) at RT. The mixture was stirred at 100° C. under CO (15 psi) for 2 h. The mixture was filtered and concentrated. The residue was purified by column chromatography on silica gel eluting with PE/EtOAc (20:1) to give butyl-1-(4-bromophenyl)-5-cyano-3-cyclopentyl-1H-pyrazole-4-carboxylate (2d)._MS-ESI (m/z): 416, 418 (1:1) [M+1].sup.+.
7-amino-1-(4-bromophenyl)-3-cyclopentyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyridazin-4-one (2e)
[0480] Butyl-1-(4-bromophenyl)-5-cyano-3-cyclopentyl-1H-pyrazole-4-carboxylate (2d) (40 mg, 0.1 mmol) in EtOH (2 mL) was added NH.sub.2NH.sub.2.H.sub.2O (1 ml). The mixture was stirred at 90° C. for 12 h. The reaction mixture was concentrated and purified by column chromatography on silica gel eluting with DCM/MeOH (20:1) to give 7-amino-1-(4-bromophenyl)-3-cyclopentyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyridazin-4-one (2e). MS-ESI (m/z): 374, 376 (1:1) [M+1].sup.+.
N-(4-(7-amino-3-cyclopentyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyridazin-1-yl)benzyl)-2-methoxybenzamide (2)
[0481] The title compound N-(4-(7-amino-3-cyclopentyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyridazin-1-yl)benzyl)-2-methoxybenzamide (2) was prepared according to the synthetic method of 1 by replacing of 7-amino-1-(4-bromophenyl)-3-cyclopentyl-1,5-dihydro-4H-pyrrolo[2,3-d]pyridazin-4-one (1g) with 7-amino-1-(4-bromophenyl)-3-cyclopentyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyridazin-4-one (2e). MS-ESI (m/z): 459 [M+1].sup.+.
Example 3
N-(4-(7-amino-3-cyclopentyl-1H-pyrazolo[3,4-c]pyridin-1-yl)benzyl)-2-methoxybenzamide (3)
[0482] ##STR00095##
cyclopentyl(3-fluoro-2-methoxypyridin-4-yl)methanol (3a)
[0483] To a solution of 3-fluoro-2-methoxypyridine (3.00 g, 23.6 mmol) in THF (60 ml) at −70° C. was added LDA (2.0 M in THF, 18.0 ml, 36.0 mmol) dropwise. The resulting mixture was stirred at −70° C. for 1 h. And then a solution of cyclopentanecarbaldehyde (3.00 ml, 28.3 mmol) in THF (5 ml) was added to the mixture dropwise. The mixture was warmed to RT and stirred at RT for overnight. The mixture was diluted with water and extracted with EA (2×100 mL). The extracts were washed with water and brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel eluting with PE/EA (10:1) to give cyclopentyl(3-fluoro-2-methoxypyridin-4-yl)methanol (3a). MS-ESI (m/z): 226 [M+1].sup.+.
cyclopentyl(3-fluoro-2-methoxypyridin-4-yl)methanone (3b)
[0484] To a solution of cyclopentyl(3-fluoro-2-methoxypyridin-4-yl)methanol (3a) (5.60 g, 24.7 mmol) in MeCN (60 ml) was added DMP (15.8 g, 37.2 mmol). The resulting mixture was stirred at RT for 1 h. The mixture was diluted with water and extracted with EA (2×100 mL). The extracts were washed with water and brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel eluting with PE/EA (30:1) to give cyclopentyl(3-fluoro-2-methoxypyridin-4-yl)methanone (3b). MS-ESI (m/z): 224 [M+1].sup.+.
3-cyclopentyl-7-methoxy-1H-pyrazolo[3,4-c]pyridine (3c)
[0485] A mixture of cyclopentyl(3-fluoro-2-methoxypyridin-4-yl)methanone (3b) (4.70 g, 21.0 mmol) and hydrazine hydrate (15 ml) in EtOH (10 mL) and dioxane (50 ml) was stirred at 95° C. for 1 h. After being cooled to RT, the mixture was concentrated. The residue was purified by column chromatography on silica gel eluting with PE/EA (5:1) to give 3-cyclopentyl-7-methoxy-1H-pyrazolo[3,4-c]pyridine (3c). MS-ESI (m/z): 218 [M+1].sup.+.
1-(4-bromophenyl)-3-cyclopentyl-7-methoxy-1H-pyrazolo[3,4-c]pyridine (3d)
[0486] A mixture of 3-cyclopentyl-7-methoxy-1H-pyrazolo[3,4-c]pyridine (3c) (640 mg, 2.94 mmol), (4-bromophenyl)boronic acid (1.18 g, 5.87 mmol), Cu(OAc).sub.2 (801 mg, 4.40 mmol) and pyridine (464 mg, 5.87 mmol) in DMF (30 ml) was stirred at 40° C. for overnight. The mixture was diluted with water and extracted with EA (2×100 mL). The extracts were washed with water and brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel eluting with PE/EA (20:1) to give 1-(4-bromophenyl)-3-cyclopentyl-7-methoxy-1H-pyrazolo[3,4-c]pyridine (3d). MS-ESI (m/z): 372, 374 (1:1) [M+1].sup.+.
1-(4-bromophenyl)-3-cyclopentyl-1,6-dihydro-7H-pyrazolo[3,4-c]pyridin-7-one (3e)
[0487] A mixture of 1-(4-bromophenyl)-3-cyclopentyl-7-methoxy-1H-pyrazolo[3,4-c]pyridine (3d) (610 mg, 1.64 mmol), NaI (492 mg, 3.28 mmol) and chlorotrimethylsilane (1.10 ml, 8.20 mmol) in MeCN (40 ml) was stirred at 60° C. 1 h. The mixture was diluted with water and extracted with EA (3×100 mL). The extracts were washed with water and brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel eluting with PE/EA (2:1) to give 1-(4-bromophenyl)-3-cyclopentyl-1,6-dihydro-7H-pyrazolo[3,4-c]pyridin-7-one (3e). MS-ESI (m/z): 358, 360 (1:1) [M+1].sup.+.
1-(4-bromophenyl)-7-chloro-3-cyclopentyl-1H-pyrazolo[3,4-c]pyridine (3f)
[0488] A mixture of 1-(4-bromophenyl)-3-cyclopentyl-1,6-dihydro-7H-pyrazolo[3,4-c]pyridin-7-one (3e) (530 mg, 1.48 mmol) and N,N-dimethylaniline (2 drops) in POCl.sub.3 (10 ml) was stirred at 100° C. for 1 h. The mixture was concentrated. The residue was diluted with EA and washed with water, sat. NaHCO.sub.3 and brine, dried over Na.sub.2SO.sub.4 and concentrated to give 1-(4-bromophenyl)-7-chloro-3-cyclopentyl-1H-pyrazolo[3,4-c]pyridine (3f). MS-ESI (m/z): 376, 378 (1:1) [M+1].sup.+.
N-(4-(7-chloro-3-cyclopentyl-1H-pyrazolo[3,4-c]pyridin-1-yl)benzyl)-2-methoxybenzamide (32)
[0489] The title compound N-(4-(7-chloro-3-cyclopentyl-1H-pyrazolo[3,4-c]pyridin-1-yl)benzyl)-2-methoxybenzamide (3g) was prepared according to the synthetic method of 1 by replacing of 7-amino-1-(4-bromophenyl)-3-cyclopentyl-1,5-dihydro-4H-pyrrolo[2,3-d]pyridazin-4-one (1g) with 1-(4-bromophenyl)-7-chloro-3-cyclopentyl-1H-pyrazolo[3,4-c]pyridine (3f). MS-ESI (m/z): 461, 463 (3:1) [M+1].sup.+.
N-(4-(3-cyclopentyl-7-((diphenylmethylene)amino)-1H-pyrazolo[3,4-c]pyridin-1-yl)benzyl)-2-methoxybenzamide (3h)
[0490] A mixture of N-(4-(7-chloro-3-cyclopentyl-1H-pyrazolo[3,4-c]pyridin-1-yl)benzyl)-2-methoxybenzamide (3g) (30 mg, 0.065 mmol), diphenylmethanimine (24 mg, 0.13 mmol), Pd.sub.2(dba).sub.3 (12 mg, 0.013 mmol), Xantphos (15 mg, 0.026 mmol) and Cs.sub.2CO.sub.3 (63.0 mg, 0.195 mmol) in dioxane (2 ml) was stirred at 90° C. under N.sub.2 for overnight. After being cooled to RT, the mixture was diluted with water and extracted with EA (3×100 mL). The extracts were washed with water and brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel eluting with PE/EA (3:2) to give N-(4-(3-cyclopentyl-7-((diphenylmethylene)amino)-1H-pyrazolo[3,4-c]pyridin-1-yl)benzyl)-2-methoxybenzamide (3h). MS-ESI (m/z): 606 [M+1].sup.+.
N-(4-(7-amino-3-cyclopentyl-1H-pyrazolo[3,4-c]pyridin-1-yl)benzyl)-2-meth oxybenzamide (3)
[0491] A mixture of N-(4-(3-cyclopentyl-7-((diphenylmethylene)amino)-1H-pyrazolo[3,4-c]pyridin-1-yl)benzyl)-2-methoxybenzamide (3h) (10.0 mg, 0.0165 mmol and 2 N HCl (0.5 ml) in THF (1 ml) was stirred at RT under N.sub.2 for overnight. The mixture was adjusted to PH=9 with K.sub.2CO.sub.3 (aq) and extracted with EA (3×10 mL). The extracts were washed with water and brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (15:1) to give N-(4-(7-amino-3-cyclopentyl-1H-pyrazolo[3,4-c]pyridin-1-yl)benzyl)-2-methoxybenzamide (3). MS-ESI (m/z): 442 [M+1].sup.+.
Example 4
N-(4-(4-amino-1-cyclopentyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-d]pyridazin-3-yl)benzyl)-2-methoxybenzamide (4)
[0492] ##STR00096##
ethyl 3-cyano-1-cyclopentyl-1H-pyrrole-2-carboxylate (4a)
[0493] The title compound ethyl 3-cyano-1-cyclopentyl-1H-pyrrole-2-carboxylate (4a) was prepared according to the method described in U.S. Pat. No. 7,071,199.
4-amino-1-cyclopentyl-1,6-dihydro-7H-pyrrolo[2,3-d]pyridazin-7-one (4b)
[0494] A mixture of ethyl 3-cyano-1-cyclopentyl-1H-pyrrole-2-carboxylate (4a) (480 mg, 2.07 mmol) and hydrazine hydrate (2 ml) in EtOH (10 mL) was stirred at 80° C. for overnight. After being cooled to RT, The mixture was diluted with water and extracted with EA (5×20 mL). The extracts were washed with water and brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (30:1) to give 4-amino-1-cyclopentyl-1,6-dihydro-7H-pyrrolo[2,3-d]pyridazin-7-one (4b). MS-ESI (m/z): 219 [M+1].sup.+.
4-amino-1-cyclopentyl-3-iodo-1,6-dihydro-7H-pyrrolo[2,3-d]pyridazin-7-one (4c)
[0495] A mixture of 4-amino-1-cyclopentyl-1,6-dihydro-7H-pyrrolo[2,3-d]pyridazin-7-one (4b) (300 mg, 1.37 mmol) and NIS (339 mg, 1.51 mmol) in AcOH (5 mL) was stirred at RT for overnight. The mixture was diluted with water. The solid was collected and purified by column chromatography on silica gel eluting with DCM/MeOH (40:1) to give 4-amino-1-cyclopentyl-3-iodo-1,6-dihydro-7H-pyrrolo[2,3-d]pyridazin-7-one (4c). MS-ESI (m/z): 345 [M+1].sup.+.
4-amino-3-(4-bromophenyl)-1-cyclopentyl-1,6-dihydro-7H-pyrrolo[2,3-d]pyridazin-7-one (4d)
[0496] A mixture of 4-amino-1-cyclopentyl-3-iodo-1,6-dihydro-7H-pyrrolo[2,3-d]pyridazin-7-one (4c) (150 mg, 0.435 mmol), (4-bromophenyl)boronic acid (78.0 mg, 0.392 mmol), Pd(dppf)Cl.sub.2 (32.0 mg, 0.0435 mmol) and K.sub.2CO.sub.3 (120.0 mg, 0.870 mmol) in dioxane (4 mL) and H.sub.2O (1 ml) was stirred at 70° C. under N.sub.2 for 2 h. After being cooled to RT, the mixture was diluted with water and extracted with DCM (3×20 mL). The extracts were washed with water and brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (50:1) to give 4-amino-3-(4-bromophenyl)-1-cyclopentyl-1,6-dihydro-7H-pyrrolo[2,3-d]pyridazin-7-one (4d). MS-ESI (m/z): 373, 375 (1:1) [M+1].sup.+.
N-(4-(4-amino-1-cyclopentyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-d]pyridazin-3-yl)benzyl)-2-methoxybenzamide (4)
[0497] The title compound N-(4-(4-amino-1-cyclopentyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-d]pyridazin-3-yl)benzyl)-2-methoxybenzamide (7) was prepared according to the synthetic method of 1 by replacing of 7-amino-1-(4-bromophenyl)-3-cyclopentyl-1,5-dihydro-4H-pyrrolo[2,3-d]pyridazin-4-one (1g) with 4-amino-3-(4-bromophenyl)-1-cyclopentyl-1,6-dihydro-7H-pyrrolo[2,3-d]pyridazin-7-one (4d). MS-ESI (m/z): 458 [M+1].sup.+.
Example 5
N-(4-(4-amino-1-cyclopentyl-7-oxo-6,7-dihydro-1H-pyrazolo[3,4-d]pyridazin-3-yl)benzyl)-2-methoxybenzamide (5)
[0498] ##STR00097##
ethyl 3-(4-bromophenyl)-1H-pyrazole-5-carboxylate (5a)
[0499] The title compound ethyl 3-(4-bromophenyl)-1H-pyrazole-5-carboxylate (5a) was prepared according to the method described in Chinese Journal of Chemistry, 2011, 2039-2048.
ethyl 3-(4-bromophenyl)-4-iodo-1H-pyrazole-5-carboxylate (5b)
[0500] To a solution of ethyl 3-(4-bromophenyl)-1H-pyrazole-5-carboxylate (5a) (290 mg, 1.00 mmol) and NIS (270 mg, 1.20 mol) in MeCN (10 mL) was added CAN (55 mg, 0.10 mmol) at RT. The mixture was stirred at 90° C. for 4 h. The mixture was diluted with water and extracted with EtOAc (3×20 mL). The extracts were washed with water and brine, dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue was dispersed in DCM and stirred for 1 h. The solid was collected and dried to give ethyl 3-(4-bromophenyl)-4-iodo-1H-pyrazole-5-carboxylate (5b). MS-ESI (m/z): 421, 423 (1:1) [M+1].sup.+.
Ethyl-3-(4-bromophenyl)-1-cyclopentyl-4-iodo-1H-pyrazole-5-carboxylate (5c)
[0501] To a solution of ethyl 3-(4-bromophenyl)-4-iodo-1H-pyrazole-5-carboxylate (5b) (421 mg, 1.00 mmol), cyclopentanol (86 mg, 1.0 mmol) and PPh.sub.3 (314 mg, 1.20 mmol) in THF (5 mL) was added DIAD (244 mg, 1.20 mmol) at 0° C. dropwise. The mixture was stirred at 25° C. under N.sub.2 for 5 h. The mixture was diluted with water and extracted with EtOAc (2×20 mL). The extracts were washed with water and brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel eluting with PE/EA (10:1) to give ethyl 3-(4-bromophenyl)-1-cyclopentyl-4-iodo-1H-pyrazole-5-carboxylate (5c). MS-ESI (m/z): 489, 491 (1:1) [M+1].sup.+.
Ethyl-3-(4-bromophenyl)-4-cyano-1-cyclopentyl-1H-pyrazole-5-carboxylate (5d)
[0502] A mixture of ethyl 3-(4-bromophenyl)-1-cyclopentyl-4-iodo-1H-pyrazole-5-carboxylate (5c) (160 mg, 0.327 mmol), CuCN (44.0 mg, 0.491 mmol) Pd(dppf)Cl.sub.2 (24.0 mg, 0.0327 mmol) and Pd.sub.2(dba).sub.3 (30.0 mg, 0.0327 mmol) in DMF (2 mL) was stirred at 100° C. under N.sub.2 for 7 h. After being cooled to RT, the mixture was diluted with water and extracted with EA (3×20 mL). The extracts were washed with water and brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel eluting with PE/EA (20:1) to give ethyl 3-(4-bromophenyl)-4-cyano-1-cyclopentyl-1H-pyrazole-5-carboxylate (5d). MS-ESI (m/z): 388, 390 (1:1) [M+1].sup.+.
4-amino-3-(4-bromophenyl)-1-cyclopentyl-1,6-dihydro-7H-pyrazolo[3,4-d]pyridazin-7-one (5e)
[0503] A solution of ethyl 3-(4-bromophenyl)-4-cyano-1-cyclopentyl-1H-pyrazole-5-carboxylate (5d) (67.0 mg, 0.173 mmol) and hydrazine hydrate (1 ml) in EtOH (3 mL) was stirred at 80° C. for 9 h. After being cooled to RT, the mixture was diluted with water and extracted with EA (3×20 mL). The extracts were washed with water and brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (50:1) to give 4-amino-3-(4-bromophenyl)-1-cyclopentyl-1,6-dihydro-7H-pyrazolo[3,4-d]pyridazin-7-one (5e). MS-ESI (m/z): 374, 376 (1:1) [M+1].sup.+.
N-(4-(4-amino-1-cyclopentyl-7-oxo-6,7-dihydro-1H-pyrazolo[3,4-d]pyridazin-3-yl)benzyl)-2-methoxybenzamide (5)
[0504] The title compound N-(4-(4-amino-1-cyclopentyl-7-oxo-6,7-dihydro-1H-pyrazolo[3,4-d]pyridazin-3-yl)benzyl)-2-methoxybenzamide (5) was prepared according to the synthetic method of 1 by replacing of 7-amino-1-(4-bromophenyl)-3-cyclopentyl-1,5-dihydro-4H-pyrrolo[2,3-d]pyridazin-4-one (1g) with 4-amino-3-(4-bromophenyl)-1-cyclopentyl-1,6-dihydro-7H-pyrazolo [3,4-d]pyridazin-7-one (5e). MS-ESI (m/z): 459 [M+1].sup.+.
Example 6
N-(4-(4-amino-7-cyclopentylimidazo[5,1-f][1,2,4]triazin-5-yl)benzyl)-2-methoxybenzamide (6)
[0505] ##STR00098##
3-amino-6-(aminomethyl)-1,2,4-triazin-5(4H)-one hydrochloride (6a)
[0506] The title compound 3-amino-6-(aminomethyl)-1,2,4-triazin-5(4H)-one hydrochloride (6a) was prepared according to the method described in WO2016/6975.
2,5-dioxopyrrolidin-1-yl cyclopentanecarboxylate (6b)
[0507] The title compound 2,5-dioxopyrrolidin-1-yl cyclopentanecarboxylate (6b) was prepared according to the method described in J. Med. Chem. 1993, 115, 925-938.
N-((3-amino-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl)methyl)cyclopentanecarboxamide (6c)
[0508] To a solution of 3-amino-6-(aminomethyl)-1,2,4-triazin-5(4H)-one hydrochloride (6a) (450 mg, 2.12 mmol) and 2,5-dioxopyrrolidin-1-yl cyclopentanecarboxylate (6b) (447 mg, 2.12 mmol) in CH.sub.3CN (20 ml) and THF (10 ml) was added NaHCO.sub.3 (356 mg, 4.24 mmol), the mixture was stirred at RT for 12 h. The reaction was diluted with water, extracted with EtOAc (2×50 mL). The extracts were washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue was purified by column chromatography on silica gel eluting with PE/EtOAc (4:1˜2:1) to give N-((3-amino-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl)methyl) cyclopentanecarboxamide (6c). MS-ESI (m/z): 238 [M+1].sup.+.
2-amino-7-cyclopentylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (6d)
[0509] To the solution of N-((3-amino-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl)methyl) cyclopentanecarboxamide (6c) (120 mg, 0.510 mmol) in DCE (10 mL) was added POCl.sub.3 (770 mg, 5.10 mmol) at RT. The mixture was warmed up to 70° C. and stirred for 2 h. The reaction was quenched with H.sub.2O and adjust pH >8 with sat. Na.sub.2CO.sub.3, extracted with DCM: i-PrOH (3:1, 2×30 mL). The extracts were dried over Na.sub.2SO.sub.4 and concentrated to give 2-amino-7-cyclopentylimidazo [5,1-f][1,2,4]triazin-4(3H)-one (6d). MS-ESI (m/z): 220 [M+1].sup.+.
2-amino-7-cyclopentyl-5-iodoimidazo[5,1-f][1,2,4]triazin-4(3H)-one (6e)
[0510] To a solution of 2-amino-7-cyclopentylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (6d) (150 mg, 0.680 mmol) in CH.sub.3CN (5 mL) was added NIS (170 mg, 0.750 mmol) at 40° C. The mixture was stirred for 2 h. The reaction was diluted with water, extracted with EtOAc (2×50 mL). The extracts were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (40:1) to give 2-amino-7-cyclopentyl-5-iodoimidazo[5,1-f][1,2,4]triazin-4(3H)-one (6e). MS-ESI (m/z): 346 [M+1].sup.+.
7-cyclopentyl-5-iodoimidazo[5,1-f][1,2,4]triazin-4(3H)-one (6f)
[0511] To a solution of 2-amino-7-cyclopentyl-5-iodoimidazo[5,1-f][1,2,4]triazin-4(3H)-one (6e) (160 mg, 0.440 mmol) in THF (4 ml) was added tert-butyl nitrite (90 mg, 0.88 mmol) dropwise at RT. The mixture was stirred for 3 h. The reaction was quenched with water, extracted with EtOAc (2×30 mL). The extracts were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (10:1) to give 7-cyclopentyl-5-iodoimidazo[5,1-f][1,2,4]triazin-4(3H)-one (6f). MS-ESI (m/z): 331 [M+1].sup.+.
7-cyclopentyl-5-iodoimidazo[5,1-f][1,2,4]triazin-4-amine (62)
[0512] To the suspension of 1,2,4-triazole (166 mg, 0.240 mmol) and pyridine (0.8 ml) was added POCl.sub.3 (123 mg, 0.8 mmol) dropwise at 0° C. and stirred for 0.5 h. The solution of 7-cyclopentyl-5-iodoimidazo[5,1-f][1,2,4]triazin-4(3H)-one (6f) (130 mg, 0.400 mmol) in pyridine (0.8 ml) was added to the mixture dropwise. Then the mixture was stirred at RT for 2 h. The mixture was cooled to −10° C. and 7 N NH.sub.3./MeOH (0.8 ml) was added, and then the mixture was warmed up to RT and stirred for 0.5 h. The reaction was quenched with HCl (0.1 N), extracted with EtOAc (2×30 mL). The extracts were washed with HCl (0.1 N), sat. NaHCO.sub.3, dried over Na.sub.2SO.sub.4 and concentrated to give 7-cyclopentyl-5-iodoimidazo[5,1-f][1,2,4]triazin-4-amine (6g). MS-ESI (m/z): 330 [M+1].sup.+.
4-(4-amino-7-cyclopentylimidazo[5,1-f][1,2,4]triazin-5-yl)phenol (6h)
[0513] A mixture of 7-cyclopentyl-5-iodoimidazo[5,1-f][1,2,4]triazin-4-amine (6g) (70 mg, 0.21 mmol), (4-hydroxyphenyl)boronic acid (44 mg, 0.32 mmol), Pd(dppf)Cl.sub.2 (16 mg, 0.021 mmol) and K.sub.2CO.sub.3 (60 mg, 0.42 mmol) in dioxane (4 mL) and H.sub.2O (1 ml) was stirred at 90° C. under N.sub.2 for 2 h. After being cooled to RT, the mixture was diluted with water and extracted with EtOAc (3×20 mL). The extracts were washed with water and brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (50:1) to give 4-(4-amino-7-cyclopentylimidazo[5,1-f][1,2,4]triazin-5-yl)phenol (6h). MS-ESI (m/z): 296 [M+1].sup.+.
4-(4-amino-7-cyclopentylimidazo[5,1-f][1,2,4]triazin-5-yl)phenyl trifluoromethanesulfonate (6i)
[0514] To a mixture of 4-(4-amino-7-cyclopentylimidazo[5,1-f][1,2,4]triazin-5-yl)phenol (6h) (90 mg, 0.21 mmol) and TEA (86 mg, 0.84 mmol) in DCM (4 mL) was added trifluoromethanesulfonic anhydride (90 mg, 0.32 mmol) at 0° C. dropwise. The mixture was stirred at 25° C. for overnight. The mixture was diluted with water and extracted with EtOAc (3×20 mL). The extracts were washed with water and brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (100:1) to give 4-(4-amino-7-cyclopentylimidazo[5,1-f][1,2,4]triazin-5-yl)phenyl trifluoromethanesulfonate (6i). MS-ESI (m/z): 428 [M+1].sup.+.
N-(4-(4-amino-7-cyclopentylimidazo[5,1-f][1,2,4]triazin-5-yl)benzyl)-2-meth oxybenzamide (6)
[0515] The title compound N-(4-(4-amino-7-cyclopentylimidazo[5,1-f][1,2,4]triazin-5-yl)benzyl)-2-methoxybenzamide (6) was prepared according to the synthetic method of 1 by replacing of 7-amino-1-(4-bromophenyl)-3-cyclopentyl-1,5-dihydro-4H-pyrrolo[2,3-d]pyridazin-4-one (1g) with 4-(4-amino-7-cyclopentylimidazo[5,1-f][1,2,4]triazin-5-yl)phenyl trifluoromethanesulfonate (6i). MS-ESI (m/z): 443 [M+1].sup.+.
Example 7
N-(4-(8-amino-3-cyclopentylimidazo[1,5-a]pyrazin-1-yl)benzyl)-2-methoxybenzamide (7)
[0516] ##STR00099##
4-(8-amino-3-cyclopentylimidazo[1,5-a]pyrazin-1-yl)phenol (7a)
[0517] The title compound 4-(8-amino-3-cyclopentylimidazo[1,5-a]pyrazin-1-yl)phenol (7a) was prepared according to the method described in WO2015/74138.
4-(8-amino-3-cyclopentylimidazo[1,5-a]pyrazin-1-yl)phenyl trifluoromethanesulfonate (7b)
[0518] To a mixture of 4-(8-amino-3-cyclopentylimidazo[1,5-a]pyrazin-1-yl)phenol (7a) (120 mg, 0.250 mmol) and pyridine (40 mg, 0.50 mmol) in DCM (10 mL) was added trifluoromethanesulfonic anhydride (71 mg, 0.25 mmol) at 0° C. dropwise. The mixture was stirred at 25° C. for overnight. The mixture was diluted with water and extracted with EA (3×20 mL). The extracts were washed with water and brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel eluting with PE/EA (100:1) to give 4-(8-amino-3-cyclopentylimidazo[1,5-a]pyrazin-1-yl)phenyl trifluoromethanesulfonate (7b). MS-ESI (m/z): 427 [M+1].sup.+.
N-(4-(8-amino-3-cyclopentylimidazo[1,5-a]pyrazin-1-yl)benzyl)-2-methoxybenzamide (7)
[0519] The title compound N-(4-(8-amino-3-cyclopentylimidazo[1,5-a]pyrazin-1-yl)benzyl)-2-methoxybenzamide (7) was prepared according to the synthetic method of 1 by replacing of 7-amino-1-(4-bromophenyl)-3-cyclopentyl-1,5-dihydro-4H-pyrrolo[2,3-d]pyridazin-4-one (1g) with 4-(8-amino-3-cyclopentylimidazo[1,5-a]pyrazin-1-yl)phenyl trifluoromethanesulfonate (7b). MS-ESI (m/z): 442 [M+1].sup.+.
Example 8
N-(4-(7-amino-3-cyclopentyl-4-oxo-4,5-dihydro-1H-pyrrolo[2,3-d]pyridazin-1-yl)benzyl)-5-fluoro-2-methoxybenzamide (8)
[0520] ##STR00100##
potassium trifluoro((5-fluoro-2-methoxybenzamido)methyl)borate (8a)
[0521] The title compound potassium trifluoro((5-fluoro-2-methoxybenzamido)-methyl)borate (8a) was prepared according to the method described in WO2017/103611.
N-(4-(7-amino-3-cyclopentyl-4-oxo-4,5-dihydro-1H-pyrrolo[2,3-d]pyridazin-1-yl)benzyl)-5-fluoro-2-methoxybenzamide (8)
[0522] The title compound N-(4-(7-amino-3-cyclopentyl-4-oxo-4,5-dihydro-1H-pyrrolo[2,3-d]pyridazin-1-yl)benzyl)-5-fluoro-2-methoxybenzamide (8) was prepared according to the synthetic method of 1 by replacing of potassium trifluoro [(2-methoxybenzamido)methyl] borate (1h) with potassium trifluoro((5-fluoro-2-methoxybenzamido)-methyl)borate (8a). MS-ESI (m/z): 476 [M+1].sup.+.
Example 9
N-(4-(6-amino-9-cyclopentyl-8-oxo-8,9-dihydro-7H-purin-7-yl)benzyl)-2-methoxybenzamide (9)
[0523] ##STR00101##
N,N-dibenzyl-6-chloro-5-nitropyrimidin-4-amine (9a)
[0524] To a solution of 4,6-dichloro-5-nitropyrimidine (3.00 g, 15.5 mmol) in DCM (30 mL) was added Et.sub.3N (4.3 mL) and dibenzylamine (3.2 g, 16 mmol) at 0° C. dropwise. The mixture was stirred at 0° C. for 1.5 h. The mixture was quenched with water and extracted with DCM. The extracts were washed with water and brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel eluting with PE/EA (10:1˜8:1) to give N,N-dibenzyl-6-chloro-5-nitropyrimidin-4-amine (9a). MS-ESI (m/z): 355 [M+1].sup.+.
N.SUP.4.,N.SUP.4.-dibenzyl-N.SUP.6.-cyclopentyl-5-nitropyrimidine-4,6-diamine (9b)
[0525] The mixture of N,N-dibenzyl-6-chloro-5-nitropyrimidin-4-amine (9a) (934 mg, 2.64 mmol) and cyclopentanamine (0.52 mL, 5.82 mmol) in dioxane was stirred at 50° C. for 1.5 h. The mixture was cooled to RT and concentrated. The residue was extracted with EtOAc. The extracts were washed with water and brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue purified by column chromatography on silica gel eluting with PE/EtOAc (10:1) to give N.sup.4,N.sup.4-dibenzyl-N.sup.6-cyclopentyl-5-nitropyrimidine-4,6-diamine (9b). MS-ESI (m/z): 404 [M+1].sup.+.
N.SUP.4.,N.SUP.4.-dibenzyl-N.SUP.6.-cyclopentylpyrimidine-4,5,6-triamine (9c)
[0526] The mixture of N.sup.4,N.sup.4-dibenzyl-N.sup.6-cyclopentyl-5-nitropyrimidine-4,6-diamine (9b) (1.57 g, 3.90 mmol), Fe (2.2 g, 39 mmol) and NH.sub.4Cl (4.2 g, 78 mmol) in MeOH/H.sub.2O (80/16 mL) was stirred at 50° C. for overnight. The mixture was filtered and concentrated. The residue was extracted with EtOAc. The extracts were washed with water and brine, dried over Na.sub.2SO.sub.4 and concentrated to give N.sup.4,N.sup.4-dibenzyl-N.sup.6-cyclopentylpyrimidine-4,5,6-triamine (9c). MS-ESI (m/z): 374 [M+1].sup.+.
9-cyclopentyl-6-(dibenzylamino)-7,9-dihydro-8H-purin-8-one (9d)
[0527] The mixture of N.sup.4,N.sup.4-dibenzyl-N.sup.6-cyclopentylpyrimidine-4,5,6-triamine (9c) (1.00 g, 2.68 mmol) and CDI (1.73 g, 10.7 mmol) in THF (40 mL) was stirred at 75° C. for overnight. The mixture was cooled to RT and concentrated. The residue was extracted with EtOAc. The extracts were washed with water and brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue purified by column chromatography on silica gel eluting with PE/EtOAc (5:1˜3:1) to give 9-cyclopentyl-6-(dibenzylamino)-7,9-dihydro-8H-purin-8-one (9d). MS-ESI (m/z): 400 [M+1].sup.+.
6-amino-9-cyclopentyl-7,9-dihydro-8H-purin-8-one (9e)
[0528] The mixture of 9-cyclopentyl-6-(dibenzylamino)-7,9-dihydro-8H-purin-8-one (9d) (400 mg, 1.00 mmol), Pd(OH).sub.2/C (20%, 400 mg) and HOAc (0.2 mL) in EtOH (40 mL) was stirred at 60° C. under H.sub.2 (1 atm) for overnight. The mixture was cooled to RT and filtered. The filtrate was extracted with EtOAc. The extracts were washed with water and brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue purified by column chromatography on silica gel eluting with DCM/MeOH (100:1˜15:1) to give 6-amino-9-cyclopentyl-7,9-dihydro-8H-purin-8-one (9e). MS-ESI (m/z): 220 [M+1].sup.+.
(4-((2-methoxybenzamido)methyl)phenyl)boronic acid (9f)
[0529] The title compound (4-((2-methoxybenzamido)methyl)phenyl)boronic acid (9f) was prepared according to the method described in WO2017/046604.
N-(4-(6-amino-9-cyclopentyl-8-oxo-8,9-dihydro-7H-purin-7-yl)benzyl)-2-methoxybenzamide (9)
[0530] A mixture of 6-amino-9-cyclopentyl-7,9-dihydro-8H-purin-8-one (9e) (40 mg, 0.18 mmol), (4-((2-methoxybenzamido)methyl)phenyl)boronic acid (9f) (205 mg, 0.72 mmol), Cu(OAc).sub.2 (67 mg, 0.36 mmol) and pyridine (57 mg, 0.36 mmol) in DMF (3 ml) was stirred at RT for overnight. The mixture was diluted with water and extracted with EA. The extracts were washed with water and brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (40:1) to give N-(4-(6-amino-9-cyclopentyl-8-oxo-8,9-dihydro-7H-purin-7-yl)benzyl)-2-methoxybenzamide (9). MS-ESI (m/z): 459 [M+1].sup.+
Example 10
4-amino-3-cyclopentyl-1-(4-((5-fluoro-2-methoxybenzamido)methyl)phenyl)-1H-pyrazole-5-carboxamide (10)
[0531] ##STR00102##
N-(4-(4-amino-5-cyano-3-cyclopentyl-1H-pyrazol-1-yl)benzyl)-5-fluoro-2-methoxybenzamide (10a)
[0532] The title compound N-(4-(4-amino-5-cyano-3-cyclopentyl-1H-pyrazol-1-yl)benzyl)-5-fluoro-2-methoxybenzamide (10a) was prepared according to the synthetic method of 1 by replacing of 7-amino-1-(4-bromophenyl)-3-cyclopentyl-1,5-dihydro-4H-pyrrolo[2,3-d]pyridazin-4-one (1g) and potassium trifluoro [(2-methoxybenzamido)methyl] borate (1h) with 4-amino-1-(4-bromophenyl)-3-cyclopentyl-1H-pyrazole-5-carbonitrile (2b) and potassium trifluoro((5-fluoro-2-methoxybenzamido)-methyl)borate (8a). MS-ESI (m/z): 434 [M+1].sup.+.
4-amino-3-cyclopentyl-1-(4-((5-fluoro-2-methoxybenzamido)methyl)phenyl)-1H-pyrazole-5-carboxamide (10)
[0533] The mixture of N-(4-(4-amino-5-cyano-3-cyclopentyl-1H-pyrazol-1-yl)benzyl)-5-fluoro-2-methoxybenzamide (10a) (8.00 mg, 0.0185 mmol) and H.sub.2SO.sub.4 (47 mg, 0.185 mmol) in TFA (84 mg, 0.74 mmol) was stirred at 50° C. for 4.5 h. The mixture was diluted with ice-water, adjusted to PH >8 with Na.sub.2CO.sub.3 and extracted with EtOAc. The extracts were washed with water and brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue purified by PTLC eluting with DCM/MeOH (20:1) to give 4-amino-3-cyclopentyl-1-(4-((5-fluoro-2-methoxybenzamido)methyl)phenyl)-1H-pyrazole-5-carboxamide (10). MS-ESI (m/z): 452 [M+1].sup.+.
[0534] Following essentially the same procedures described for Examples 1-10, Examples 11-120 listed in Table 1 were prepared from the appropriate starting materials which are commercially available or known in the literature. The structures and names of Examples 11-120 are given in Table 1.
TABLE-US-00001 TABLE 1 EXAMPLE Structure Name DATA 11
Kinase Assay
[0535] The kinase activity of BTK (C481S) was assayed at Reaction Biology Corporation. The substrate in the BTK (C481S) reaction, pEY (poly[Glu:Tyr] (4:1)) (Sigma, Cat. #P7244-250MG), was prepared in fresh reaction buffer (20 mM Hepes (pH 7.5), 10 mM MgCl.sub.2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mi BSA, 0.1 mM Na.sub.3VO.sub.4, 2 mM DTT, 1% DMSO). BTK(C481S) (SignalChem, Cat. #B10-12CH) was delivered into the substrate solution and mixed gently. The final concentrations of BTK (C481S) and the substrate in the reaction mixture were 6 nM and 0.2 mg/ml, respectively. Compounds were tested in 10-point concentration/response mode with 3-fold serial dilution steps starting at 1 μM.
[0536] Compounds in 100% DMSO were delivered into the kinase reaction mixture by acoustic liquid delivery technology (Echo550; nanoliter range) and incubated for 20 min at room temperature. 10 μM [.sup.33P]-ATP (ATP: Sigma, Cat #: A7699; [.sup.33P]-ATP: Hartmann Analytic, Cat #: SCF-301-12) was delivered into the reaction mixture to initiate the reaction. The mixture was incubated for 120 min at room temperature. Radioactivity was detected utilizing a proprietary filter-binding method. Kinase activity data were expressed as the percent remaining kinase activity in test samples compared to vehicle (DMSO) reactions. IC.sub.50 values were obtained using GraphPad Prism software.
[0537] Select compounds prepared as described above were assayed according to the biological procedures described herein. The results are given in the table 2.
TABLE-US-00002 TABLE 2 BTK(C481S) BTK (C481S) Example IC50 (nM) Example IC50 (nM) 1 5.0 7 3.0 2 3.3 8 0.45 4 6.5 11 0.5 5 8.0 17 0.5 6 7.8 81 0.91
Cell Proliferation Assays
[0538] MTS testing kit was purchased from Promega (Madison, Wis., USA). The RPMI-1640, Fetal bovine serum and Penicillin-Streptomycin were purchased from BI (Biological Industries, Beit Haemek, Israel). Dimethyl sulfoxide (DMSO) was purchased from Sigma (St. Louis., Mo., USA). OCI-LY10 cells were cultured in RPMI1640 supplemented with Penicillin-Streptomycin and 10% FBS.
[0539] To investigate whether a compound is able to inhibit the activity of BTK in cells, a mechanism-based assay using OCI-LY10 cells was developed. In this assay, inhibition of BTK was detected by the inhibition of OCI-LY10 cells proliferation. Cells were plated into 96-well plates at a density of 10000 cells/well. Plates were incubated at 37° C., with 5% CO.sub.2 for 4 h. Compounds were serially diluted and added to the plates with the final compound concentrations as 10000, 3333.3, 1111.1, 270.4, 123.5, 41.2, 13.7, 4.6 and 1.5 nM. Plates were incubated at 37° C., with 5% CO.sub.2 for 72 h. 20 μl MTS was added into each well and the plates were incubated at 37° C., with 5% CO.sub.2 for exactly 2 h. The absorbance was measured by a microplate reader at 490 nm. IC.sub.50 was calculated using GraphPad Prism 5.0 software.
[0540] Select compounds prepared as described above were assayed according to the biological procedures described herein. The results are given in the table 3.
TABLE-US-00003 TABLE 3 OCI-LY10 OCI-LY10 Example IC50 (nM) Example IC50 (nM) 1 28 67 1 2 34 68 2 4 69 69 5 5 68 70 1 8 1 71 23 11 1 72 1 12 9 73 1 13 2 74 1 14 85 75 12 16 1 76 1 17 1 77a 1 18 1 77b 1 22 44 78 26 27 1 79 50 30 1 80 1 31 1 81 1 34 4 82 3 35 1 83 46 36 1 84 4 37 9 85 18 38 35 86 90 40a 7 87 4 40b 83 88 1 41 18 89 1 45 32 90 15 48 48 92a 10 49 9 92b 22 50 19 93 24 53 3 94 1 54 1 96 1 55 28 97 23 56 3 98 92 57 24 99 65 59 62 102 76 60 30 105 4 61 15 106 11 62 1 107 11 63 1 109 1 64 1 110 23 65 1 111 14 66 1 112 2