PYRAZOLE-CONTAINING POLYCYCLIC DERIVATIVE INHIBITOR, PREPARATION METHOD THEREFOR AND APPLICATION THEREOF

Abstract

Provided are a pyrazole-containing polycyclic derivative inhibitor, a preparation method therefor and an application thereof. In particular, provided are a compound as represented by formula (I), a preparation method therefor, a pharmaceutical composition containing the compound, and an application thereof as a P2X3 inhibitor in treatment of P2X3 receptor function disorders, particularly in treatment of neurogenic diseases, wherein the substituents in the formula (I) are the same as those in the description in definition.

##STR00001##

Claims

1. A compound of formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof: ##STR00180## wherein: L.sub.1 is selected from the group consisting of a bond, —(CH.sub.2).sub.n1—, —(CH.sub.2).sub.n1C(O)(CR.sub.aaR.sub.bb).sub.n2—, —(CH.sub.2).sub.n1C(O)NR.sub.aa(CH.sub.2).sub.n2—, —(CH.sub.2).sub.n1(CR.sub.aaR.sub.bb).sub.n2—, —(CR.sub.aaR.sub.bb).sub.n1O(CH.sub.2).sub.n2—, —(CH.sub.2).sub.n1O(CR.sub.aaR.sub.bb).sub.n2—, —(CR.sub.aaR.sub.bb).sub.n1S(CH.sub.2).sub.n2—, —(CH.sub.2).sub.n1S(CR.sub.aaR.sub.bb).sub.n2—, —(CR.sub.aaR.sub.bb).sub.n1(CH.sub.2).sub.n2NR.sub.cc—, —(CH.sub.2).sub.n1NR.sub.aa(CR.sub.bbR.sub.cc).sub.n2—, —(CH.sub.2).sub.n1NR.sub.aaC(O)—, —(CH.sub.2).sub.n1P(O)R.sub.aa—, —(CH.sub.2).sub.n1S(O).sub.n2—, —(CH.sub.2).sub.n1S(O).sub.n2NR.sub.aa— and —(CH.sub.2).sub.n1NR.sub.aaS(O).sub.n2—; R.sub.aa to R.sub.cc are each independently selected from the group consisting of hydrogen, deuterium, halogen, amino, nitro, hydroxy, cyano, alkyl, deuterated alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, heterocyclylalkyl, cycloalkyl, heterocyclyl, aryl, aryloxy, heteroaryl and heteroaryloxy, wherein the amino, alkyl, deuterated alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, heterocyclylalkyl, cycloalkyl, heterocyclyl, aryl, aryloxy, heteroaryl and heteroaryloxy can be each optionally further substituted; or, any two of R.sub.aa to R.sub.cc are bonded to form a cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein the cycloalkyl, heterocyclyl, aryl or heteroaryl can be optionally further substituted; L.sub.2 is selected from the group consisting of a bond, —(CH.sub.2).sub.n3—, —(CH.sub.2).sub.n3C(O)(CR.sub.ddR.sub.ee).sub.n4—, —(CH.sub.2).sub.n3C(O)NR.sub.dd(CH.sub.2).sub.n4—, —(CH.sub.2).sub.n3(CR.sub.ddR.sub.ee).sub.n4—, —(CR.sub.ddR.sub.ee).sub.n3O(CH.sub.2).sub.n4—, —(CH.sub.2).sub.n3O(CR.sub.ddR.sub.ee).sub.n4—, —(CR.sub.ddR.sub.ee).sub.n3S(CH.sub.2).sub.n4—, —(CH.sub.2).sub.n3S(CR.sub.ddR.sub.ee).sub.n4—, —(CR.sub.ddR.sub.ee).sub.n3(CH.sub.2).sub.n4NR.sub.ff—, —(CH.sub.2).sub.n3NR.sub.dd(CR.sub.eeR.sub.ff).sub.n4—, —(CH.sub.2).sub.n3NR.sub.ddC(O)—, —(CH.sub.2).sub.n3P(O)R.sub.dd—, —(CH.sub.2).sub.n3S(O).sub.n4—, —(CH.sub.2).sub.n3S(O).sub.n4NR.sub.dd— and —(CH.sub.2).sub.n3NR.sub.ddS(O).sub.n4—; R.sub.dd to R.sub.ff are each independently selected from the group consisting of hydrogen, deuterium, halogen, amino, nitro, hydroxy, cyano, alkyl, deuterated alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, heterocyclylalkyl, cycloalkyl, heterocyclyl, aryl, aryloxy, heteroaryl and heteroaryloxy, wherein the amino, alkyl, deuterated alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, heterocyclylalkyl, cycloalkyl, heterocyclyl, aryl, aryloxy, heteroaryl and heteroaryloxy can be each optionally further substituted; or, any two of R.sub.dd to R.sub.ff are bonded to form a cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein the cycloalkyl, heterocyclyl, aryl or heteroaryl can be optionally further substituted; ring A is selected from the group consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sub.1 is selected from the group consisting of hydrogen, deuterium, halogen, amino, nitro, hydroxy, cyano, alkyl, deuterated alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, heterocyclylalkyl, cycloalkyl, heterocyclyl, aryl, aryloxy, heteroaryl and heteroaryloxy, wherein the amino, alkyl, deuterated alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, heterocyclylalkyl, cycloalkyl, heterocyclyl, aryl, aryloxy, heteroaryl and heteroaryloxy can be each optionally further substituted; ring B is selected from the group consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sub.2 is selected from the group consisting of hydrogen, deuterium, halogen, amino, nitro, hydroxy, cyano, alkyl, deuterated alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, heterocyclylalkyl, cycloalkyl, heterocyclyl, aryl, aryloxy, heteroaryl and heteroaryloxy, wherein the amino, alkyl, deuterated alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, heterocyclylalkyl, cycloalkyl, heterocyclyl, aryl, aryloxy, heteroaryl and heteroaryloxy can be each optionally further substituted; R.sub.3 is selected from the group consisting of hydrogen, deuterium, halogen, amino, nitro, hydroxy, cyano, alkyl, deuterated alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, heterocyclylalkyl, cycloalkyl, heterocyclyl, aryl, aryloxy, heteroaryl and heteroaryloxy, wherein the amino, alkyl, deuterated alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, heterocyclylalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl can be each optionally further substituted; R.sup.a is selected from the group consisting of hydrogen, deuterium, halogen, amino, nitro, hydroxy, cyano, oxo, thioxo, alkyl, deuterated alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, heterocyclylalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, —(CH.sub.2).sub.n5R.sub.gg, —(CH.sub.2).sub.n5OR.sub.gg, —(CH.sub.2).sub.n5C(O)OR.sub.gg, —(CH.sub.2).sub.n5SR.sub.gg, —(CH.sub.2).sub.n5NR.sub.ggC(O)(CH.sub.2).sub.n6R.sub.hh, —(CH.sub.2).sub.n5NR.sub.ggC(O)OR.sub.hh, —(CH.sub.2).sub.n5NR.sub.ggC(O)NR.sub.hhR.sub.ii, —(CH.sub.2).sub.n5NR.sub.ggR.sub.hh, —NR.sub.gg(CH.sub.2).sub.n5R.sub.hh, —(CH.sub.2).sub.n5C(O)NR.sub.gg(CH.sub.2).sub.n6R.sub.hh, —(CH.sub.2).sub.n5C(O)R.sub.gg, —OC(R.sub.ggR.sub.hh).sub.n5(CH.sub.2).sub.n6R.sub.ii, —(CH.sub.2).sub.n5S(O).sub.n6R.sub.gg, —(CH.sub.2).sub.n5NR.sub.ggS(O).sub.n6R.sub.hh, —CH═CH(CH.sub.2).sub.n5R.sub.gg, —CH═CH(CH.sub.2).sub.n5NR.sub.ggR.sub.hh, —CH═CH(CH.sub.2).sub.n5NR.sub.ggC(O)R.sub.hh and —CH═CH(CH.sub.2).sub.n5NR.sub.ggC(O)NR.sub.hhR.sub.ii, wherein the amino, alkyl, deuterated alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, heterocyclylalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl can be each optionally further substituted; R.sub.gg to R.sub.ii are each independently selected from the group consisting of hydrogen, deuterium, halogen, amino, nitro, hydroxy, cyano, alkyl, deuterated alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, heterocyclylalkyl, cycloalkyl, heterocyclyl, aryl, aryloxy, heteroaryl and heteroaryloxy, wherein the amino, alkyl, deuterated alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, heterocyclylalkyl, cycloalkyl, heterocyclyl, aryl, aryloxy, heteroaryl and heteroaryloxy can be each optionally further substituted; or, any two of R.sub.gg to R.sub.ii are bonded to form a cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein the cycloalkyl, heterocyclyl, aryl or heteroaryl can be optionally further substituted; x is an integer from 0 to 6; e is an integer from 0 to 6; n1, n3, and n5 are each independently an integer from 0 to 3; and n2, n4, and n6 are each independently an integer from 0 to 2.

2. The compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof according to claim 1, wherein L.sub.1 is selected from the group consisting of a bond, —(CH.sub.2).sub.n1—, —(CH.sub.2).sub.n1C(O)(CR.sub.aaR.sub.bb).sub.n2—, —(CH.sub.2).sub.n1C(O)NR.sub.aa(CH.sub.2).sub.n2—, —(CH.sub.2).sub.n1(CR.sub.aaR.sub.bb).sub.n2—, —(CR.sub.aaR.sub.bb).sub.n1O(CH.sub.2).sub.n2—, —(CH.sub.2).sub.n1O(CR.sub.aaR.sub.bb).sub.n2—, —(CR.sub.aaR.sub.bb).sub.n1S(CH.sub.2).sub.n2—, —(CH.sub.2).sub.n1S(CR.sub.aaR.sub.bb).sub.n2—, —(CR.sub.aaR.sub.bb).sub.n1(CH.sub.2).sub.n2NR.sub.cc—, —(CH.sub.2).sub.n1NR.sub.aa(CR.sub.bbR.sub.cc).sub.n2—, —(CH.sub.2).sub.n1C(O)(CR.sub.aaR.sub.bb).sub.n2—, —(CH.sub.2).sub.n1NR.sub.aaC(O)—, —(CH.sub.2).sub.n1P(O)R.sub.aa—, —(CH.sub.2).sub.n1S(O).sub.n2—, —(CH.sub.2).sub.n1S(O).sub.n2NR.sub.aa— and —(CH.sub.2).sub.n1NR.sub.aaS(O).sub.n2—; R.sub.aa to R.sub.cc are each independently selected from the group consisting of hydrogen, deuterium, halogen, amino, hydroxy, cyano, nitro, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, cyano-substituted C.sub.1-6 alkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl, wherein the amino, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, cyano-substituted C.sub.1-6 alkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl are each optionally substituted by one or more substituents selected from the group consisting of deuterium, halogen, amino, hydroxy, cyano, nitro, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, cyano-substituted C.sub.1-6 alkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl; or, any two of R.sub.aa to R.sub.cc are bonded to form a C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl or 5 to 14 membered heteroaryl, wherein the C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl or 5 to 14 membered heteroaryl is optionally substituted by one or more substituents selected from the group consisting of deuterium, halogen, amino, hydroxy, cyano, nitro, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, cyano-substituted C.sub.1-6 alkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl; n1 is an integer from 0 to 3; and n2 is an integer from 0 to 2.

3. The compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof according to claim 1, wherein L.sub.2 is selected from the group consisting of a bond, —(CH.sub.2).sub.n3—, —(CH.sub.2).sub.n3C(O)(CR.sub.ddR.sub.ee).sub.n4—, —(CH.sub.2).sub.n3C(O)NR.sub.dd(CH.sub.2).sub.n4—, —(CH.sub.2).sub.n3(CR.sub.ddR.sub.ee).sub.n4—, —(CR.sub.ddR.sub.ee).sub.n3O(CH.sub.2).sub.n4—, —(CH.sub.2).sub.n3O(CR.sub.ddR.sub.ee).sub.n4—, —(CR.sub.ddR.sub.ee).sub.n3S(CH.sub.2).sub.n4—, —(CH.sub.2).sub.n3S(CR.sub.ddR.sub.ee).sub.n4—, —(CR.sub.ddR.sub.ee).sub.n3(CH.sub.2).sub.n4NR.sub.ff—, —(CH.sub.2).sub.n3NR.sub.dd(CR.sub.eeR.sub.ff).sub.n4—, —(CH.sub.2).sub.n3NR.sub.ddC(O)—, —(CH.sub.2).sub.n3P(O)R.sub.dd—, —(CH.sub.2).sub.n3S(O).sub.n4—, —(CH.sub.2).sub.n3S(O).sub.n4NR.sub.dd— and —(CH.sub.2).sub.n3NR.sub.ddS(O).sub.n4—; R.sub.dd to R.sub.ff are each independently selected from the group consisting of hydrogen, deuterium, halogen, amino, hydroxy, cyano, nitro, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, cyano-substituted C.sub.1-6 alkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl, wherein the amino, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, cyano-substituted C.sub.1-6 alkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl are each optionally substituted by one or more substituents selected from the group consisting of deuterium, halogen, amino, hydroxy, cyano, nitro, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, cyano-substituted C.sub.1-6 alkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl; or, any two of R.sub.dd to R.sub.ff are bonded to form a C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl or 5 to 14 membered heteroaryl, wherein the C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl or 5 to 14 membered heteroaryl is optionally substituted by one or more substituents selected from the group consisting of deuterium, halogen, amino, hydroxy, cyano, nitro, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, cyano-substituted C.sub.1-6 alkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl; n3 is an integer from 0 to 3; and n4 is an integer from 0 to 2.

4. The compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof according to claim 1, wherein ring A is selected from the group consisting of C.sub.3-8 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl.

5. The compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof according to claim 1, wherein R.sub.1 is selected from the group consisting of hydrogen, deuterium, halogen, amino, hydroxy, cyano, nitro, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl, C.sub.6-14 aryloxy, 5 to 14 membered heteroaryl and 5 to 14 membered heteroaryloxy, wherein the amino, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl, C.sub.6-14 aryloxy, 5 to 14 membered heteroaryl and 5 to 14 membered heteroaryloxy are each optionally substituted by one or more substituents selected from the group consisting of deuterium, halogen, amino, hydroxy, cyano, nitro, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, cyano-substituted C.sub.1-6 alkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl, C.sub.6-14 aryloxy, 5 to 14 membered heteroaryl, 5 to 14 membered heteroaryloxy, —(CH.sub.2).sub.m1OR.sub.a, —(CH.sub.2).sub.m1SR.sub.a, —(CH.sub.2).sub.m1C(O)R.sub.a, —(CH.sub.2).sub.m1NR.sub.aR.sub.b, —(CH.sub.2).sub.m1C(O)NR.sub.aR.sub.b, —(CH.sub.2).sub.m1NR.sub.aC(O)R.sub.b and —(CH.sub.2).sub.m1S(O).sub.m2R.sub.a; R.sub.a and R.sub.b are each independently selected from the group consisting of hydrogen, deuterium, halogen, amino, hydroxy, cyano, nitro, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, cyano-substituted C.sub.1-6 alkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl, wherein the amino, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, cyano-substituted C.sub.1-6 alkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl are each optionally substituted by one or more substituents selected from the group consisting of deuterium, halogen, amino, hydroxy, cyano, nitro, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, cyano-substituted C.sub.1-6 alkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl; or, R.sub.a and R.sub.b are bonded to form a C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl or 5 to 14 membered heteroaryl, wherein the C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl or 5 to 14 membered heteroaryl is optionally substituted by one or more substituents selected from the group consisting of deuterium, halogen, amino, hydroxy, cyano, nitro, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, cyano-substituted C.sub.1-6 alkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl; m1 is an integer from 0 to 3; and m2 is an integer from 0 to 2.

6. The compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof according to claim 1, wherein R.sub.2 is selected from the group consisting of hydrogen, deuterium, halogen, amino, hydroxy, cyano, oxo, thioxo, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 hydroxyalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.3-8 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl.

7. The compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof according to claim 1, wherein, R.sub.3 is selected from the group consisting of hydrogen, deuterium, halogen, amino, hydroxy, cyano, oxo, thioxo, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 hydroxyalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.3-8 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl.

8. The compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof according to claim 1, wherein, R.sup.a is selected from the group consisting of hydrogen, deuterium, halogen, amino, hydroxy, cyano, oxo, thioxo, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 hydroxyalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.3-8 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl, 5 to 14 membered heteroaryl, —(CH.sub.2).sub.n5R.sub.gg, —(CH.sub.2).sub.n5OR.sub.gg, —(CH.sub.2).sub.n5C(O)OR.sub.gg, —(CH.sub.2).sub.n5SR.sub.gg, —(CH.sub.2).sub.n5NR.sub.ggC(O)(CH.sub.2).sub.n6R.sub.hh, —(CH.sub.2).sub.n5NR.sub.ggC(O)OR.sub.hh, —(CH.sub.2).sub.n5NR.sub.ggC(O)NR.sub.hhR.sub.ii, —(CH.sub.2).sub.n5NR.sub.ggR.sub.hh, —NR.sub.gg(CH.sub.2).sub.n5R.sub.hh, —(CH.sub.2).sub.n5C(O)NR.sub.gg(CH.sub.2).sub.n6R.sub.hh, —(CH.sub.2).sub.n5C(O)R.sub.gg and —OC(R.sub.ggR.sub.hh).sub.n5(CH.sub.2).sub.n6R.sub.ii; preferably selected from the group consisting of hydrogen, deuterium, R.sub.gg to R.sub.ii are each independently selected from the group consisting of hydrogen, deuterium, halogen, amino, hydroxy, cyano, nitro, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, cyano-substituted C.sub.1-6 alkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl, wherein the amino, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, cyano-substituted C.sub.1-6 alkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl are each optionally substituted by one or more substituents selected from the group consisting of deuterium, halogen, amino, hydroxy, cyano, nitro, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, cyano-substituted C.sub.1-6 alkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl; or, any two of R.sub.gg to R.sub.ii are bonded to form a C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl or 5 to 14 membered heteroaryl, wherein the C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl or 5 to 14 membered heteroaryl is optionally substituted by one or more substituents selected from the group consisting of deuterium, halogen, amino, hydroxy, cyano, nitro, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, cyano-substituted C.sub.1-6 alkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl; n5 is an integer from 0 to 3; and n6 is an integer from 0 to 2.

9. The compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof according to claim 1, wherein ring B is shown as following: ##STR00181## wherein: M.sub.1, M.sub.2, M.sub.3 and M.sub.4 are each independently selected from the group consisting of —CR.sub.A1—, —C(O)—, —N—, —CR.sub.A1R.sub.A2— and —NR.sub.A3—; R.sub.A1 to R.sub.A3 are each independently selected from the group consisting of hydrogen, deuterium, halogen, amino, nitro, hydroxy, cyano, alkyl, deuterated alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, heterocyclylalkyl, cycloalkyl, heterocyclyl, aryl, aryloxy, heteroaryl and heteroaryloxy, wherein the amino, alkyl, deuterated alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, heterocyclylalkyl, cycloalkyl, heterocyclyl, aryl, aryloxy, heteroaryl and heteroaryloxy can be each optionally further substituted.

10. The compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof according to claim 9, wherein, M.sub.1, M.sub.2, M.sub.3 and M.sub.4 are each independently CR.sub.A1; R.sub.A1 is selected from the group consisting of hydrogen, deuterium, halogen, amino, hydroxy, cyano, oxo, thioxo, C.sub.1-3 alkyl, C.sub.2-5 alkenyl, C.sub.2-5 alkynyl, C.sub.1-3 deuterated alkyl, C.sub.1-3 haloalkyl, C.sub.1-3 hydroxyalkyl, C.sub.1-3 alkoxy, C.sub.1-3 haloalkoxy, C.sub.3-6 cycloalkyl, 3 to 8 membered heterocyclyl containing 1 to 3 atoms selected from the group consisting of N, O and S, C.sub.6-10 aryl and 5 to 10 membered heteroaryl containing atoms selected from the group consisting of 1 to 3 N, O and S.

11. The compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof according to claim 9, wherein, at least one of M.sub.1, M.sub.2, M.sub.3 and M.sub.4 is N; or M.sub.1 is N, and M.sub.2, M.sub.3 and M.sub.4 are each independently CR.sub.A1; R.sub.A1 is selected from the group consisting of hydrogen, deuterium, halogen, amino, hydroxy, cyano, oxo, thioxo, C.sub.1-3 alkyl, C.sub.2-5 alkenyl, C.sub.2-5 alkynyl, C.sub.1-3 deuterated alkyl, C.sub.1-3 haloalkyl, C.sub.1-3 hydroxyalkyl, C.sub.1-3 alkoxy, C.sub.1-3 haloalkoxy, C.sub.3-6 cycloalkyl, 3 to 8 membered heterocyclyl containing 1 to 3 atoms selected from the group consisting of N, O and S, C.sub.6-10 aryl and 5 to 10 membered heteroaryl containing 1 to 3 atoms selected from the group consisting of N, O and S.

12. The compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof according to claim 1, wherein ring B is shown as following: ##STR00182## wherein: M.sub.6, M.sub.7 and M.sub.8 are each independently selected from the group consisting of —CR.sub.A4—, —C(O)—, —N—, —O—, —S—, —CR.sub.A4R.sub.A5— and —NR.sub.A6—; R.sub.A4 to R.sub.A6 are each independently selected from the group consisting of hydrogen, deuterium, halogen, amino, nitro, hydroxy, cyano, alkyl, deuterated alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, heterocyclylalkyl, cycloalkyl, heterocyclyl, aryl, aryloxy, heteroaryl and heteroaryloxy, wherein the amino, alkyl, deuterated alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, heterocyclylalkyl, cycloalkyl, heterocyclyl, aryl, aryloxy, heteroaryl and heteroaryloxy can be each optionally further substituted.

13. The compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof according to claim 1, wherein, ring A is selected from ##STR00183## M.sub.5 is selected from the group consisting of —N— and —CR.sub.4—; R.sub.4 is selected from the group consisting of hydrogen, deuterium, halogen, amino, hydroxy, cyano, nitro, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl, wherein the amino, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl are each optionally substituted by one or more substituents selected from the group consisting of deuterium, halogen, amino, hydroxy, cyano, nitro, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, cyano-substituted C.sub.1-6 alkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-12 aryl and 5 to 12 membered heteroaryl.

14. The compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is further shown as formula (II): ##STR00184## wherein e is an integer from 0 to 3.

15. The compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof according to claim 14, wherein the compound is further shown as formula (III): ##STR00185## wherein: R.sub.5 is selected from the group consisting of hydrogen, deuterium, halogen, amino, hydroxy, cyano, oxo, thioxo, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 hydroxyalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.3-8 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl; R.sup.b is selected from the group consisting of hydrogen, deuterium, halogen, amino, hydroxy, cyano, oxo, thioxo, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 hydroxyalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.3-8 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl; y is an integer from 0 to 3.

16. The compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof according to claim 15, wherein the compound is further shown as formula (IV): ##STR00186## wherein: ring C is selected from the group consisting of C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl, or ring C is absent; R.sup.c is selected from the group consisting of hydrogen, deuterium, halogen, amino, hydroxy, cyano, nitro, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl, C.sub.6-14 aryloxy, 5 to 14 membered heteroaryl, 5 to 14 membered heteroaryloxy, —(CH.sub.2).sub.m3OR.sub.c, —(CH.sub.2).sub.m3SR.sub.c, —(CH.sub.2).sub.m3C(O)R.sub.c, —(CH.sub.2).sub.m3NR.sub.cR.sub.d, —(CH.sub.2).sub.m3C(O)NR.sub.cR.sub.d, —(CH.sub.2).sub.m3NR.sub.cC(O)R.sub.d and —(CH.sub.2).sub.m3S(O).sub.m4R.sub.c, wherein the amino, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl, C.sub.6-14 aryloxy, 5 to 14 membered heteroaryl and 5 to 14 membered heteroaryloxy are each optionally substituted by one or more substituents selected from the group consisting of deuterium, halogen, amino, hydroxy, cyano, nitro, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, cyano-substituted C.sub.1-6 alkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl, C.sub.6-14 aryloxy, 5 to 14 membered heteroaryl and 5 to 14 membered heteroaryloxy; R.sub.c and R.sub.d are each independently selected from the group consisting of hydrogen, deuterium, halogen, amino, hydroxy, cyano, nitro, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, cyano-substituted C.sub.1-6 alkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl, wherein the amino, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, cyano-substituted C.sub.1-6 alkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl are each optionally substituted by one or more substituents selected from the group consisting of deuterium, halogen, amino, hydroxy, cyano, nitro, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, cyano-substituted C.sub.1-6 alkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl; or, R.sub.c and R.sub.d are bonded to form a C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl or 5 to 14 membered heteroaryl, wherein the C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl or 5 to 14 membered heteroaryl is optionally substituted by one or more substituents selected from the group consisting of deuterium, halogen, amino, hydroxy, cyano, nitro, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, cyano-substituted C.sub.1-6 alkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl; m3 is an integer from 0 to 3; m4 is an integer from 0 to 2; and z is an integer from 0 to 6.

17. The compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof according to claim 14, wherein the compound is further shown as formula (V): ##STR00187## wherein: R.sub.1 is selected from the group consisting of hydrogen, deuterium, halogen, amino, hydroxy, cyano, nitro, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl, wherein the amino, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl are each optionally substituted by one or more substituents selected from the group consisting of deuterium, halogen, amino, hydroxy, cyano, nitro, oxo, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, cyano-substituted C.sub.1-6 alkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-12 aryl and 5 to 12 membered heteroaryl; R.sub.2 is selected from the group consisting of hydrogen, deuterium, halogen, amino, hydroxy, cyano, nitro, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl, wherein the amino, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl are each optionally substituted by one or more substituents selected from the group consisting of deuterium, halogen, amino, hydroxy, cyano, nitro, oxo, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, cyano-substituted C.sub.1-6 alkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-12 aryl and 5 to 12 membered heteroaryl; R.sub.3 is selected from the group consisting of hydrogen, deuterium, halogen, amino, hydroxy, cyano, oxo, thioxo, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 hydroxyalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.3-8 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl; and e is an integer from 0 to 3.

18. The compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof according to claim 17, wherein: R.sub.1 is selected from the group consisting of hydrogen, deuterium, halogen, amino, hydroxy, cyano, nitro, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, C.sub.3-12 cycloalkyl, 3 to 12 membered heterocyclyl, C.sub.6-14 aryl and 5 to 14 membered heteroaryl, wherein the amino, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 deuterated alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 hydroxyalkyl, C.sub.3-8 cycloalkyl, 3 to 8 membered heterocyclyl, C.sub.6-10 aryl and 5 to 10 membered heteroaryl are each optionally substituted by one or more substituents selected from the group consisting of deuterium, halogen, amino, hydroxy, cyano, nitro, oxo, C.sub.1-3 alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl, C.sub.1-3 deuterated alkyl, C.sub.1-3 haloalkyl, C.sub.1-3 alkoxy, C.sub.1-3 haloalkoxy, C.sub.1-3 hydroxyalkyl, cyano-substituted C.sub.1-3 alkyl, C.sub.3-8 cycloalkyl, 3 to 8 membered heterocyclyl, C.sub.6-10 aryl and 5 to 10 membered heteroaryl; R.sub.2 is selected from the group consisting of hydrogen, deuterium, halogen, amino, hydroxy, cyano, nitro, C.sub.1-3 alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl, C.sub.1-3 deuterated alkyl, C.sub.1-3 haloalkyl, C.sub.1-3 alkoxy, C.sub.1-3 haloalkoxy, C.sub.1-3 hydroxyalkyl, C.sub.3-8 cycloalkyl, 3 to 8 membered heterocyclyl, C.sub.6-10 aryl and 5 to 10 membered heteroaryl; R.sub.3 is selected from the group consisting of hydrogen, deuterium, halogen, amino, hydroxy, cyano, oxo, thioxo, C.sub.1-3 alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl, C.sub.1-3 deuterated alkyl, C.sub.1-3 haloalkyl, C.sub.1-3 hydroxyalkyl, C.sub.1-3 alkoxy, C.sub.1-3 haloalkoxy, C.sub.3-8 cycloalkyl, 3 to 8 membered heterocyclyl, C.sub.6-10 aryl and 5 to 10 membered heteroaryl; and e is an integer from 0 to 3.

19. The compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof according to claim 1, wherein the specific structure of the compound is as follows: ##STR00188## ##STR00189## ##STR00190## ##STR00191## ##STR00192## ##STR00193## ##STR00194## ##STR00195## ##STR00196## ##STR00197## ##STR00198## ##STR00199## ##STR00200## ##STR00201## ##STR00202## ##STR00203## ##STR00204## ##STR00205## ##STR00206## ##STR00207## ##STR00208## ##STR00209## ##STR00210## ##STR00211## ##STR00212## ##STR00213## ##STR00214## ##STR00215##

20. A method for preparing the compound of formula (III), a stereoisomer thereof or a pharmaceutically acceptable salt thereof according to claim 15, comprising the following step of: ##STR00216## reacting a compound of formula (III-2) with a compound of formula (III-3) to obtain the target compound of formula (III); wherein: X.sub.2 is halogen.

21. A method for preparing the compound of formula (V), a stereoisomer thereof or a pharmaceutically acceptable salt thereof according to claim 17, comprising the following step of: ##STR00217## reacting a compound of formula (V-2) with a compound of formula (V-3) to obtain the target compound of formula (V); wherein: X.sub.5 is halogen.

22. A pharmaceutical composition comprising a therapeutically effective dose of the compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof according to claim 1, and one or more pharmaceutically acceptable carriers or excipients.

23. (canceled)

24. A method for preventing and/or treating a neurogenic disease in a patient in need thereof, the method comprising administering to the patient a therapeutically effective dose of the compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof according to claim 1, wherein the neurogenic disease is selected from the group consisting of gynecological diseases, urinary tract disease states, respiratory disorders, and pain related diseases or conditions.

25. The method according to claim 24, wherein the pain related disease or condition is neuropathic pain or pain and discomfort related to uterine fibroid.

26. The compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof according to claim 15, wherein: R.sup.b is selected from the group consisting of hydrogen, deuterium, halogen, amino, hydroxy, cyano, oxo, thioxo, C.sub.1-3 alkyl, C.sub.2-5 alkenyl, C.sub.2-5 alkynyl, C.sub.1-3 deuterated alkyl, C.sub.1-3 haloalkyl, C.sub.1-3 hydroxyalkyl, C.sub.1-3 alkoxy, C.sub.1-3 haloalkoxy, C.sub.3-6 cycloalkyl, 3 to 10 membered heterocyclyl, C.sub.6-12 aryl and 5 to 12 membered heteroaryl.

27. The compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof according to claim 15, wherein: R.sup.b is selected from the group consisting of hydrogen, deuterium, fluorine, chlorine, bromine, amino, hydroxy, cyano, oxo, thioxo, methyl, ethyl, propyl, vinyl, propenyl, allyl, ethynyl, propynyl, propargyl, deuterated methyl, deuterated ethyl, deuterated propyl, fluoromethyl, fluoroethyl, fluoropropyl, chloromethyl, chloroethyl, chloropropyl, bromomethyl, bromoethyl, bromopropyl, hydroxymethyl, hydroxyethyl, hydroxypropyl, methoxy, ethoxy, propoxy, fluoromethoxy, fluoroethoxy, fluoropropoxy, chloromethoxy, chloroethoxy, chloropropoxy, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, epoxypropyl, epoxybutyl, epoxypentyl, epoxyhexyl, epoxyheptyl, aziridinyl, azetidinyl, azacyclopentyl, azacyclohexyl, azacycloheptyl, thienyl, pyrrolyl, pyridyl, pyranyl, piperazinyl, phenyl and naphthyl.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0236] The present invention will be further described with reference to the following examples, but the examples should not be considered as limiting the scope of the present invention.

EXAMPLES

[0237] The structures of the compounds of the present invention were identified by nuclear magnetic resonance (NMR) and/or liquid chromatography-mass spectrometry (LC-MS). NMR shifts (δ) are given in parts per million (ppm). NMR is determined by a Bruker AVANCE-400 instrument. The solvents for determination are deuterated-dimethyl sulfoxide (DMSO-d.sub.6), deuterated-methanol (CD.sub.3OD) and deuterated-chloroform (CDCl.sub.3), and the internal standard is tetramethylsilane (TMS).

[0238] Liquid chromatography-mass spectrometry (LC-MS) is determined on an Agilent 1200 Infinity Series mass spectrometer. High performance liquid chromatography (HPLC) is determined on an Agilent 1200DAD high pressure liquid chromatograph (Sunfire C18 150×4.6 mm column), and a Waters 2695-2996 high pressure liquid chromatograph (Gimini C.sub.18 150×4.6 mm column).

[0239] Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate is used as the thin-layer silica gel chromatography (TLC) plate. The dimension of the silica gel plate used in TLC is 0.15 mm to 0.2 mm, and the dimension of the silica gel plate used in product purification is 0.4 mm to 0.5 mm. Yantai Huanghai 200 to 300 mesh silica gel is generally used as a carrier for column chromatography.

[0240] The raw materials used in the examples of the present invention are known and commercially available, or can be synthesized by or according to known methods in the art.

[0241] Unless otherwise stated, all reactions of the present invention are carried out under continuous magnetic stirring under a dry nitrogen or argon atmosphere. The solvent is dry, and the reaction temperature is in degrees celsius.

Example 1

2-(2-(Tert-butyl)-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0242] ##STR00058##

Step 1: Preparation of N-(3-(tert-butyl)-1H-pyrazol-5-yl)-2-chloronicotinamide

[0243] ##STR00059##

[0244] 3-(Tert-butyl)-1H-pyrazol-5-amine (2.77 g, 19.93 mmol), DIPEA (6.2 g, 49.8 mmol) and HATU (5.4 g, 0.144 mmol) were added successively to a solution of 2-chloronicotinic acid (1.57 g, 9.96 mmol) in DMF (30 mL) under an ice bath condition. The ice bath was removed, and the reaction solution was stirred for 1 h. The mixture was treated to obtain Example 1-1 (2.5 g, 90%).

[0245] MS m/z (ESI): 279.7 [M+H].sup.+.

Step 2: Preparation of 2-(tert-butyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-5(4H)-one

[0246] ##STR00060##

[0247] Potassium carbonate (1.61 g, 11.66 mmol) and 1,4-diazabicyclo[2.2.2]octane (DABCO) (150.9 mg, 1.35 mmol) were added to a solution of Example 1-1 (2.5 g, 8.97 mmol) in DMF (50 mL). The reaction solution was stirred at room temperature for 16 hours. The mixture was treated to obtain Example 1-2 (2.1 g, 97%).

[0248] MS m/z (ESI): 279.7 [M+H].sup.+.

Step 3: Preparation of 2-(2-(tert-butyl)-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0249] ##STR00061##

[0250] Potassium carbonate (4.28 g, 30.96 mmol) and Example 1-3 (4.33 g, 18.57 mmol) were added to a solution of Example 1-2 (1.5 g, 6.19 mmol) in DMF (30 mL) at room temperature. The mixture was heated to 80° C. and stirred for 2 h. The reaction solution was cooled followed by addition of water. The precipitate was filtered, washed with ethyl acetate, and purified to obtain Example 1 (656 mg, yield: 27%).

[0251] MS m/z (ESI): 395.4 [M+H].sup.+.

[0252] .sup.1H NMR (400 MHz, DMSO-d6) δ 11.01 (s, 1H), 8.80-8.78 (m, 1H), 8.47 (d, J=7.6 Hz, 1H), 8.30 (d, J=2.8 Hz, 1H), 8.01-7.94 (m, 1H), 7.73-7.66 (m, 1H), 7.49 (dd, J=8.0, 4.8 Hz, 1H), 6.34 (s, 1H), 4.87 (s, 2H), 1.26 (s, 9H).

Example 2

2-(2-Bromo-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0253] ##STR00062##

[0254] Example 2 was synthesized according to the method of Example 1. The target compound (500 mg, yield: 68%) was obtained by replacing 3-(tert-butyl)-1H-pyrazol-5-amine with 3-bromo-1H-pyrazol-5-amine.

[0255] MS m/z (ESI): 418.2 [M+H].sup.+.

[0256] .sup.1H NMR (400 MHz, DMSO-d6) δ 11.32 (s, 1H), 9.85 (d, J=7.6 Hz, 1H), 8.74 (d, J=6.4 Hz, 1H), 8.40 (d, J=2.8 Hz, 1H), 8.05-8.00 (m, 1H), 7.78-7.73 (m, 1H), 7.23-7.17 (m, 1H), 6.31 (s, 1H), 5.52 (s, 2H).

Example 3

N-(5-Fluoropyridin-2-yl)-2-(2-methyl-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)acetamide

[0257] ##STR00063##

[0258] Example 3 was synthesized according to the method of Example 1. The target compound (20 mg, yield: 26%) was obtained by replacing 3-(tert-butyl)-1H-pyrazol-5-amine with 3-methyl-1H-pyrazol-5-amine.

[0259] MS m/z (ESI): 353.3 [M+H].sup.+.

Example 4

N-(5-Fluoropyridin-2-yl)-2-(2-ethyl-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl) acetamide

[0260] ##STR00064##

[0261] Example 4 was synthesized according to the method of Example 1. The target compound (15 mg, yield: 36%) was obtained by replacing 3-(tert-butyl)-1H-pyrazol-5-amine with 3-ethyl-1H-pyrazol-5-amine.

[0262] MS m/z (ESI): 367.4 [M+H].sup.+.

Example 5

N-(5-Fluoropyridin-2-yl)-2-(2-isopropyl-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)acetamide

[0263] ##STR00065##

[0264] Example 5 was synthesized according to the method of Example 1. The target compound (15 mg, yield: 36%) was obtained by replacing 3-(tert-butyl)-1H-pyrazol-5-amine with 3-isopropyl-1H-pyrazol-5-amine.

[0265] MS m/z (ESI): 381.4 [M+H].sup.+.

Example 6

N-(5-Fluoropyridin-2-yl)-2-(2-isopropenyl-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)acetamide

[0266] ##STR00066##

[0267] Example 2 (100 mg, 0.24 mmol), isopropenylboronic acid (41.2 mg, 0.48 mmol), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (19.2 mg, 0.024 mmol) and cesium carbonate (232.8 mg, 0.72 mmol) were stirred in dioxane (4 mL) and water (1 mL) at 100° C. under microwave for 1 h. The reaction solution was concentrated to dryness by rotary evaporation, and purified by preparative high performance liquid chromatography to obtain Example 6 (54 mg, yield: 60%).

[0268] MS m/z (ESI): 379.4 [M+H].sup.+.

Example 7

N-(5-Fluoropyridin-2-yl)-2-(5-oxo-2-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)acetamide

[0269] ##STR00067##

[0270] Example 7 was synthesized according to the method of Example 1. The target compound (15 mg, yield: 36%) was obtained by replacing 3-(tert-butyl)-1H-pyrazol-5-amine with 3-trifluoromethyl-1H-pyrazol-5-amine.

[0271] MS m/z (ESI): 407.3 [M+H].sup.+.

[0272] .sup.1H NMR (400 MHz, DMSO-d6) δ 11.08 (s, 1H), 8.96 (dd, J=8.0, 1.6 Hz, 1H), 8.65 (dd, J=8.0, 1.6 Hz, 1H), 8.37 (d, J=3.2 Hz, 1H), 8.07-8.02 (m, 1H), 7.78-7.73 (m, 2H), 7.05 (s, 1H), 5.02 (s, 2H).

Example 8

2-(2-Amino-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0273] ##STR00068##

Step 1: Preparation of methyl 5-oxo-4,5-dihydropyrazolo[1,5-a]pyrido[3,2-e]pyrimidine-2-carboxylate

[0274] ##STR00069##

[0275] The synthetic method of Example 8-1 was according to the synthetic method of Example 1-2. Example 8-1 (500 mg, 73%) was obtained by replacing 3-(tert-butyl)-1H-pyrazol-5-amine with methyl 5-amino-1H-pyrazole-3-carboxylate.

[0276] MS: m/z (ESI): 245.2 [M+H].sup.+.

Step 2: Preparation of methyl 4-(2-((5-fluoropyridin-2-yl)amino)-2-oxoethyl)-5-oxo-4,5-dihydropyrazolo[1,5-a]pyrido[3,2-e]pyrimidine-2-carboxylate

[0277] ##STR00070##

[0278] The synthetic method of Example 8-2 was according to the synthetic method of Example 1. The title compound Example 8-2 (500 mg, 51%) was obtained by using Example 8-1 as the starting material.

[0279] MS m/z (ESI): 397.3 [M+H].sup.+.

Step 3: Preparation of 4-(2-((5-fluoropyridin-2-yl)amino)-2-oxoethyl)-5-oxo-4,5-dihydropyrazolo[1,5-a]pyrido[3,2-e]pyrimidine-2-carboxylic acid

[0280] ##STR00071##

[0281] A solution of LiOH (519 mg, 12.36 mmol) in water (2 mL) was added to a solution of Example 8-2 (490 mg, 1.24 mmol) in tetrahydrofuran (10 mL) at room temperature. The mixture was stirred at room temperature for 3 h, then the pH was adjusted to about 3 with 1M HCl. The solution was concentrated to dryness to obtain Example 8-3 (470 mg, 99%).

[0282] MS m/z (ESI): 383.3 [M+H].sup.+.

Step 4: Preparation of 2-(2-amino-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0283] ##STR00072##

[0284] Ammonia was added to a solution of Example 8-3 (450 mg, 1.2 mmol) in 1,4-dioxane (10 mL), Et.sub.3N (33 μL, 0.24 mmol) and BOP reagent (598 mg, 1.35 mmol), and stirred at room temperature for 20 min. Sodium azide (160 mg, 2.46 mmol) and tetrabutylammonium bromide (786 mg, 2.46 mmol) were added, and the reaction solution was stirred for 1 hour. The reaction solution was diluted with 1,4-dioxane (12 mL), followed by addition of 2 M aqueous H.sub.2SO.sub.4 solution (4 mL) and heating at 100° C. for 2 h. The solvent was evaporated, and the residues were diluted with water and extracted with ethyl acetate. The organic layer was washed with brine and dried over anhydrous sodium sulfate, and the solvent was evaporated. The resulting residues were purified by column chromatography to obtain Example 8 (360 mg, 86%).

[0285] MS m/z (ESI): 354.3 [M+H].sup.+.

Example 9

2-(2-Cyclopropyl-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0286] ##STR00073##

[0287] The synthetic method of Example 9 was according to the synthetic method of Example 6. The title compound Example 9 (8 mg, 51%) was obtained by replacing isopropenylboronic acid with cyclopropylboronic acid.

[0288] MS m/z (ESI): 378.4 [M+H].sup.+.

Example 10

2-(2-Cyclopentyl-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0289] ##STR00074##

[0290] The synthetic method of Example 10 was according to the synthetic method of Example 1. The title compound Example 10 (9 mg, 28%) was obtained by replacing 3-bromo-1H-pyrazol-5-amine with 3-cyclopentyl-1H-pyrazol-5-amine.

[0291] MS m/z (ESI): 407.4 [M+H].sup.+.

Example 11

2-(2-Cyclopentenyl-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0292] ##STR00075##

[0293] The synthetic method of Example 11 was according to the synthetic method of Example 6. The title compound Example 11 (15 mg, 81%) was obtained by replacing isopropenylboronic acid with cyclopentenylboronic acid.

[0294] MS m/z (ESI): 405.4 [M+H].sup.+.

Example 12

N-(5-Fluoropyridin-2-yl)-2-(5-oxo-2-(tetrahydro-2H-thiopyran-4-yl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)acetamide

[0295] ##STR00076##

Step 1: Preparation of 2-(2-(3,6-dihydro-2H-thiopyran-4-yl)-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0296] ##STR00077##

[0297] The synthetic method of Example 12-1 was according to the synthetic method of Example 6. The title compound Example 12-1 (20 mg, 81%) was obtained by replacing isopropenylboronic acid with (3,6-dihydro-2H-thiopyran-4-yl)boronic acid.

[0298] MS m/z (ESI): 437.5 [M+H].sup.+.

Step 2: Preparation of N-(5-fluoropyridin-2-yl)-2-(5-oxo-2-(tetrahydro-2H-thiopyran-4-yl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)acetamide

[0299] ##STR00078##

[0300] Example 12-1 (20 mg, 0.045 mmol) was dissolved in methanol (1 mL). 10% wet palladium on carbon (2 mg) was added, and the reaction solution was heated to reflux under a hydrogen atmosphere. After completion of the reaction, the reaction solution was filtered through celite, and purified to obtain Example 12 (13 mg, 65%).

[0301] MS m/z (ESI): 439.5 [M+H].sup.+.

Example 13

2-(2-(2,2-Difluoroacetyl)piperidin-4-yl)-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0302] ##STR00079##

[0303] The synthetic method of Example 13 was according to the synthetic method of Example 6. The title compound Example 13 (6 mg, 11%) was obtained.

[0304] MS m/z (ESI): 500.4 [M+H].sup.+.

Example 14

N-(5-Fluoropyridin-2-yl)-2-(2-(oxetan-3-ylamino)-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)acetamide

[0305] ##STR00080##

[0306] Example 9 (35.3 mg, 0.1 mmol) and oxetanone (7.1 mg, 0.1 mmol) were dissolved in methanol (1 mL). Sodium borohydride (3.8 mg, 0.1 mmol) and p-toluenesulfonic acid monohydrate (0.1 mmol) were added to the resulting mixture, and the reaction solution was heated to reflux for 3 hours. The reaction mixture was quenched with saturated aqueous NaHCO.sub.3 solution (10 mL), and extracted with dichloromethane (3*10 mL). The combined extracts were dried over anhydrous sodium sulfate, filtered and concentrated. The resulting crude product was purified to obtain Example 14 (20 mg, 50%).

[0307] MS m/z (ESI): 410.4 [M+H].sup.+.

Example 15

2-(2-(Cyclopentylamino)-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0308] ##STR00081##

[0309] The synthetic method of Example 15 was according to the synthetic method of Example 14. The title compound Example 15 (7 mg, 13%) was obtained.

[0310] MS m/z (ESI): 422.4 [M+H].sup.+.

Example 16

N-(Cyclopropylmethyl)-4-(2-((5-fluoropyridin-2-yl)amino)-2-oxoethyl)-N-methyl-5-oxo-4,5-dihydropyrazolo[1,5-a]pyrido[3,2-e]pyrimidine-2-carboxamide

[0311] ##STR00082##

[0312] Example 8-3 (36.7 mg, 0.096 mmol) was dissolved in DMF (1 mL) under an ice bath condition, followed by successively adding 1-cyclopropyl-N-methylformamide (16.4 mg, 0.192 mmol), DIPEA (62 mg, 0.48 mmol) and HATU (54 mg, 0.144 mmol). The ice bath was removed, and the reaction solution was stirred for 1 h. The mixture was treated to obtain Example 16 (22 mg, 50%).

[0313] MS m/z (ESI): 450.5 [M+H].sup.+.

Example 17

N-(Cyclopropyl)-4-(2-((5-fluoropyridin-2-yl)amino)-2-oxoethyl)-N-methyl-5-oxo-4,5-dihydropyrazolo[1,5-a]pyrido[3,2-e]pyrimidine-2-carboxamide

[0314] ##STR00083##

[0315] The synthetic method of Example 17 was according to the synthetic method of Example 16. The title compound Example 17 (20 mg, 50%) was obtained.

[0316] MS m/z (ESI): 436.4 [M+H].sup.+.

Example 18

N-(5-Fluoropyridin-2-yl)-2-(5-oxo-2-phenylpyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)acetamide

[0317] ##STR00084##

[0318] The synthetic method of Example 18 was according to the synthetic method of Example 6. The title compound Example 18 (6 mg, 54%) was obtained.

[0319] MS m/z (ESI): 415.4 [M+H].sup.+.

Example 19

N-(5-Fluoropyridin-2-yl)-2-(2-(6-methylpyridin-3-yl)-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)acetamide

[0320] ##STR00085##

[0321] The synthetic method of Example 19 was according to the synthetic method of Example 6. The title compound Example 19 (9 mg, 50%) was obtained.

[0322] MS m/z (ESI): 430.4 [M+H].sup.+.

Example 20

N-(5-Fluoropyridin-2-yl)-2-(2-(2-methylpyridin-4-yl)-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)acetamide

[0323] ##STR00086##

[0324] The synthetic method of Example 20 was according to the synthetic method of Example 6. The title compound Example 20 (13 mg, 50%) was obtained.

[0325] MS m/z (ESI): 430.4 [M+H].sup.+.

Example 21

2-(2,5-Dimethylpyridin-4-yl)-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0326] ##STR00087##

[0327] The synthetic method of Example 21 was according to the synthetic method of Example 6. The title compound Example 21 (18 mg, 56%) was obtained.

[0328] MS m/z (ESI): 444.4 [M+H].sup.+.

[0329] .sup.1H NMR (400 MHz, DMSO-d6) δ 11.21 (s, 1H), 8.90-8.86 (m, 1H), 8.84-8.81 (m, 1H), 8.76-8.74 (m, 1H), 8.38 (s, 1H), 8.30 (s, 1H), 8.06-7.98 (m, 1H), 7.78-7.70 (m, 1H), 7.53-7.47 (m, 1H), 7.35 (s, 1H), 5.44 (s, 2H), 2.78 (s, 3H), 2.74 (s, 3H).

Example 22

2-(2-(Tert-butyl)-8-chloro-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0330] ##STR00088##

[0331] The synthetic method of Example 22 was according to the synthetic method of Example 1. The title compound Example 22 (4 mg, 19%) was obtained.

[0332] MS m/z (ESI): 429.8 [M+H].sup.+.

[0333] .sup.1H NMR (400 MHz, DMSO-d6) δ 11.08 (s, 1H), 8.52 (d, J=8.4 Hz, 1H), 8.38 (d, J=2.8 Hz, 1H), 8.08-8.03 (m, 1H), 7.79-7.74 (m, 1H), 7.62 (d, J=8.4 Hz, 1H), 6.47 (s, 1H), 4.93 (s, 2H), 1.33 (s, 9H).

Example 23

2-(2-(Tert-butyl)-8-methyl-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0334] ##STR00089##

[0335] The synthetic method of Example 23 was according to the synthetic method of Example 1. The title compound Example 23 (8 mg, 19%) was obtained.

[0336] MS m/z (ESI): 409.4 [M+H].sup.+.

[0337] .sup.1H NMR (400 MHz, DMSO-d6) δ 11.07 (s, 1H), 8.40 (d, J=8.0 Hz, 1H), 8.37 (d, J=3.2 Hz, 1H), 8.06-8.02 (m, 1H), 7.75 (td, J=8.4, 2.8 Hz, 1H), 7.42 (d, J=8.0 Hz, 1H), 6.38 (s, 1H), 4.93 (s, 2H), 2.68 (s, 3H), 1.33 (s, 9H).

Example 24

2-(2-(Tert-butyl)-7-methyl-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0338] ##STR00090##

[0339] The synthetic method of Example 24 was according to the synthetic method of Example 1. The title compound Example 24 (7 mg, 16%) was obtained.

[0340] MS m/z (ESI): 409.4 [M+H].sup.+.

[0341] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.09 (s, 1H), 8.70 (d, J=2.3 Hz, 1H), 8.36 (dd, J=8.7, 2.7 Hz, 2H), 8.04 (s, 1H), 7.76 (dt, J=8.9, 4.5 Hz, 1H), 6.38 (s, 1H), 4.93 (s, 2H), 2.45 (s, 3H), 1.31 (s, 9H).

Example 25

2-(2-(Tert-butyl)-6-methyl-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0342] ##STR00091##

[0343] The synthetic method of Example 25 was according to the synthetic method of Example 1. The title compound Example 25 (5 mg, 16%) was obtained.

[0344] MS m/z (ESI): 409.4 [M+H].sup.+.

Example 26

2-(2-(Tert-butyl)-5-oxopyrazolo[1,5-a]quinazolin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0345] ##STR00092##

[0346] The synthetic method of Example 26 was according to the synthetic method of Example 1. The title compound Example 26 (6 mg, 16%) was obtained.

[0347] MS m/z (ESI): 394.4 [M+H].sup.+.

Example 27

2-(2-(Tert-butyl)-5-oxopyrazolo[1,5-a]pyrido[2,3-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0348] ##STR00093##

[0349] The synthetic method of Example 27 was according to the synthetic method of Example 1. The title compound Example 27 (6 mg, 16%) was obtained.

[0350] MS m/z (ESI): 395.4 [M+H].sup.+.

Example 28

2-(2-(Tert-butyl)-7-methyl-5-oxopyrazolo[1,5-a]pyrido[2,3-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0351] ##STR00094##

[0352] The synthetic method of Example 28 was according to the synthetic method of Example 1. The title compound Example 28 (9 mg, 21%) was obtained.

[0353] MS m/z (ESI): 409.4 [M+H].sup.+.

Example 29

2-(2-(Tert-butyl)-7-chloro-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0354] ##STR00095##

[0355] The synthetic method of Example 29 was according to the synthetic method of Example 1. The title compound Example 29 (15 mg, 32%) was obtained.

[0356] MS m/z (ESI): 429.1 [M+H].sup.+.

[0357] .sup.1H NMR (400 MHz, DMSO-d6) δ 11.09 (s, 1H), 8.91 (d, J=2.4, 1H), 8.54 (d, J=2.4 Hz, 1H), 8.38 (d, J=2.8 Hz, 1H), 8.08-8.02 (m, 1H), 7.79-7.74 (m, 1H), 6.47 (s, 1H), 4.94 (s, 2H), 1.32 (s, 9H).

Example 30

2-(2-(Tert-butyl)-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0358] ##STR00096##

[0359] The synthetic method of Example 30 was according to the synthetic method of Example 1. The title compound Example 30 (25 mg, 46%) was obtained by using 2-chloro-6-trifluoromethylnicotinic acid as the starting material.

[0360] MS m/z (ESI): 463.1 [M+H].sup.+.

[0361] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.09 (s, 1H), 7.78 (d, J=8.0 Hz, 1H), 8.38 (d, J=2.8 Hz, 1H), 8.09-8.04 (m, 1H), 8.00 (d, J=8.0 Hz, 1H), 7.79-7.74 (m, 1H), 6.52 (s, 1H), 4.95 (s, 2H), 1.34 (s, 9H).

Example 31

2-(Tert-butyl)-4-(4-chlorobenzyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-5(4H)-one

[0362] ##STR00097##

[0363] The synthetic method of Example 31 was according to the synthetic method of Example 1. The title compound Example 31 (12 mg, 24%) was obtained.

[0364] MS m/z (ESI): 367.1 [M+H].sup.+.

[0365] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.83 (dd, J=4.8, 1.6 Hz, 1H), 8.56 (dd, J=8.0, 1.6 Hz, 1H), 7.55 (dd, J=8.0, 4.8 Hz, 1H), 7.45 (d, J=8.4 Hz, 1H), 7.39 (d, J=8.4 Hz, 1H), 6.31 (s, 1H), 5.25 (s, 2H), 1.30 (s, 9H).

Example 32

2-(2-(Tert-butyl)-7-isopropyl-5,8-dioxo-5,6,7,8-tetrahydro-4H-pyrazolo[1,5-a]pyrrolo[3,4-e]pyrimidin-4-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0366] ##STR00098##

Step 1: Preparation of 4-hydroxy-1-isopropyl-5-oxo-2,5-dihydro-1H-pyrrole-3-carboxylic acid

[0367] ##STR00099##

[0368] LiOH (0.23 g, 9.4 mmol) was added to a solution of Example 32-1 (2.0 g, 9.4 mmol) in CH.sub.3OH (30 mL) under an ice bath condition. The ice bath was removed, and the reaction solution was stirred for 1 h. The reaction solution was adjusted to pH 5 to 6 with 1 mol/L aqueous hydrochloric acid solution, and extracted with ethyl acetate (10 mL*3). The organic phase was dried and concentrated to obtain Example 32-2 (1.5 g, 73%).

[0369] MS m/z (ESI): 184.7 [M−H].sup.+.

Step 2: Preparation of N-(3-(tert-butyl)-1H-pyrazol-5-yl)-4-hydroxy-1-isopropyl-5-oxo-2,5-dihydro-1H-pyrrole-3-carboxamide

[0370] ##STR00100##

[0371] The synthetic method of Example 32-3 was according to the synthetic method of Example 1-1. The title compound Example 32-3 (0.26 g, 44%) was obtained by using Example 32-2 as the starting material.

[0372] MS m/z (ESI): 307.2 [M+H].sup.+.

Step 3: Preparation of 2-(tert-butyl)-7-isopropyl-6,7-dihydro-4H-pyrazolo[1,5-a]pyrrolo[3,4-e]pyrimidine-5,8-dione

[0373] ##STR00101##

[0374] The synthetic method of Example 32-4 was according to the synthetic method of Example 1-2. The title compound Example 32-4 (0.18 g, 78%) was obtained by using Example 32-3 as the starting material.

[0375] MS m/z (ESI): 289.2 [M+H].sup.+.

Step 4: Preparation of 2-(2-(tert-butyl)-7-isopropyl-5,8-dioxo-5,6,7,8-tetrahydro-4H-pyrazolo[1,5-a]pyrrolo[3,4-e]pyrimidin-4-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0376] ##STR00102##

[0377] The synthetic method of Example 32-4 was according to the synthetic method of Example 1. The title compound Example 32-5 (0.12 g, 65%) was obtained by using Example 32-4 as the starting material.

[0378] MS m/z (ESI): 441.2 [M+H].sup.+.

Example 33

N-(5-Fluoropyridin-2-yl)-2-(2-methyl-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)acetamide

[0379] ##STR00103##

[0380] The synthetic method of Example 33 was according to the synthetic method of Example 2. The title compound Example 33 (18 mg, 30%) was obtained.

[0381] MS m/z (ESI): 421.1 [M+H].sup.+.

[0382] .sup.1H NMR (400 MHz, DMSO-d6) δ 11.06 (s, 1H), 8.80 (d, J=8.0 Hz, 1H), 8.37 (s, 1H), 8.07-8.03 (m, 1H), 8.00 (d, J=8.0 Hz, 1H), 7.79-7.73 (m, 1H), 6.28 (s, 1H), 4.95 (s, 2H), 2.33 (s, 3H).

Example 34

2-(2-Ethyl-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0383] ##STR00104##

Step 1: Preparation of tert-butyl 5-amino-3-ethyl-1H-pyrazole-1-carboxylate

[0384] ##STR00105##

[0385] 3-Ethyl-1H-pyrazol-5-amine (2.0 g, 18.0 mmol) was dissolved in anhydrous dichloromethane (50 mL), followed by addition of triethylamine (2.2 g, 21.6 mmol) and di-tert-butyl dicarbonate (4.7 g, 21.6 mmol). The reaction solution was reacted at room temperature for 16 hours. The reaction solution was washed successively with water (50 mL*2) and saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting crude product was purified by column chromatography (ethyl acetate/dichloromethane=0 to 20%) to obtain the title product Example 34-1 (3.4 g), yield: 89.5%.

[0386] MS: m/z (ESI): 212.1 [M+H].sup.+.

Step 2: Preparation of tert-butyl 5-amino-3-ethyl-1H-pyrazole-1-carboxylate

[0387] ##STR00106##

[0388] Example 34-1 (3.4 g, 16.1 mmol) was dissolved in anhydrous dichloromethane (60 mL), followed by addition of triethylamine (5.4 g, 53.1 mmol). A solution (50 mL) of freshly prepared 2-chloro-6-(trifluoromethyl)nicotinoyl chloride (4.3 g, 17.7 mmol) in dichloromethane was added dropwise under a nitrogen atmosphere at 0° C. After completion of the addition, the reaction solution was reacted at room temperature for 30 minutes. The reaction solution was washed successively with water (200 mL*2) and saturated sodium chloride solution (200 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0 to 20%) to obtain Example 34-2 (2.6 g), yield: 38.2%.

[0389] MS: m/z (ESI): 319.1 [M-Boc+H].sup.+.

Step 3: Preparation of N-(3-ethyl-1H-pyrazol-5-yl)-2-chloro-6-(trifluoromethyl)nicotinamide

[0390] ##STR00107##

[0391] Example 34-2 (2.6 g, 6.2 mmol) was dissolved in anhydrous dichloromethane (10 mL), followed by addition of a solution (4 M, 20 mL) of hydrochloric acid in dioxane. The reaction solution was reacted at room temperature for 4 hours. The reaction solution was directly concentrated to dryness by rotary evaporation to obtain Example 34-3 (1.9 g), yield: 96.0%.

[0392] MS: m/z (ESI): 319.0 [M+H].sup.+.

Step 4: 2-Ethyl-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-5(4H)-one

[0393] ##STR00108##

[0394] Example 34-3 (1.9 g, 6.0 mmol) was dissolved in N,N-dimethylformamide (20 mL), followed by addition of potassium carbonate (2.5 g, 18.0 mmol). The reaction solution was heated to 120° C. and reacted for 2 hours. The reaction solution was cooled to room temperature and used directly in the next step.

[0395] MS: m/z (ESI): 283.1[M+H].sup.+.

Step 5: 2-(2-Ethyl-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0396] ##STR00109##

[0397] Potassium carbonate (1.5 g, 10.6 mmol) and 2-bromo-N-(5-fluoropyridin-2-yl)acetamide (0.99 g, 4.2 mmol) were added to the reaction solution of Example 34-4 (1.0 g, 3.5 mmol) in N,N-dimethylformamide (20 mL), and reacted at 40° C. for 2 hours. The reaction solution was cooled to room temperature, poured into 300 mL of water, and extracted with ethyl acetate (200 mL*3). The organic phases were combined, washed successively with water (200 mL*2) and saturated sodium chloride solution (200 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting crude product was recrystallized from ethyl acetate to obtain Example 34.

[0398] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.06 (s, 1H), 8.79 (d, J=7.6 Hz, 1H), 8.37 (s, 1H), 8.07-8.03 (m, 1H), 8.00 (d, J=8.0 Hz, 1H), 7.79-7.72 (m, 1H), 6.36 (s, 1H), 4.96 (s, 2H), 2.70 (q, J=7.6 Hz, 2H), 1.25 (t, J=7.6 Hz, 3H).

[0399] MS m/z (ESI): 435.1 [M+H].sup.+.

Example 35

2-(2-Cyclopropyl-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0400] ##STR00110##

[0401] The synthetic method of Example 35 was according to the synthetic method of Example 1. The title compound Example 35 (17 mg, 28%) was obtained.

[0402] .sup.1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 8.78 (d, J=8.0 Hz, 1H), 8.37 (s, 1H), 8.08-8.02 (m, 1H), 7.98 (d, J=8.4 Hz, 1H), 7.79-7.73 (m, 1H), 6.23 (s, 1H), 4.91 (s, 2H), 2.11-2.04 (m, 1H), 1.04-0.98 (m, 2H), 0.82-0.78 (m, 2H).

[0403] MS m/z (ESI): 447.1 [M+H].sup.+.

Example 36

N-(5-Fluoropyridin-2-yl)-2-(2-isopropyl-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)acetamide

[0404] ##STR00111##

[0405] The synthetic method of Example 36 was according to the synthetic method of Example 4. The title compound Example 36 (10 mg, 22%) was obtained.

[0406] MS m/z (ESI): 449.1 [M+H].sup.+.

[0407] .sup.1H NMR (400 MHz, DMSO-d6) δ 11.06 (s, 1H), 8.79 (d, J=8.0 Hz, 1H), 8.37 (s, 1H), 8.09-8.03 (m, 1H), 8.00 (d, J=8.0 Hz, 1H), 7.79-7.74 (m, 1H), 6.42 (s, 1H), 4.96 (s, 2H), 3.08-3.01 (m, 1H), 1.29 (s, 3H), 1.27 (s, 3H).

Example 37

2-(2-Cyclopentyl-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0408] ##STR00112##

[0409] The synthetic method of Example 37 was according to the synthetic method of Example 1. The title compound Example 37 (18 mg, 30%) was obtained.

[0410] MS m/z (ESI): 475.1 [M+H].sup.+.

[0411] .sup.1H NMR (400 MHz, DMSO) δ 11.07 (s, 1H), 8.79 (d, J=8.0 Hz, 1H), 8.38 (s, 1H), 8.14-7.89 (m, 2H), 7.77 (s, 1H), 6.42 (s, 1H), 4.96 (s, 2H), 3.17 (s, 1H), 2.14-1.93 (m, 3H), 1.67 (m, 5H).

Example 38

2-(2-(4,4-Difluorocyclohexyl)-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0412] ##STR00113##

[0413] The synthetic method of Example 38 was according to the synthetic method of Example 1. The title compound Example 38 (8 mg, 20%) was obtained.

[0414] MS m/z (ESI): 525.1 [M+H].sup.+.

[0415] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.05 (s, 1H), 8.79 (d, J=8.2 Hz, 1H), 8.37 (d, J=3.0 Hz, 1H), 8.01 (d, J=8.0 Hz, 2H), 7.76 (t, J=9.0 Hz, 1H), 6.49 (s, 1H), 4.95 (s, 2H), 2.95 (s, 1H), 2.05 (q, J=19.1, 17.8 Hz, 6H), 1.74 (d, J=13.1 Hz, 2H).

Example 39

N-(5-Fluoropyridin-2-yl)-2-(2-(6-methylpyridin-3-yl)-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)acetamide

[0416] ##STR00114##

[0417] The synthetic method of Example 39 was according to the synthetic method of Example 1. The title compound Example 39 (15 mg, 28%) was obtained.

[0418] MS m/z (ESI): 498.1 [M+H].sup.+.

[0419] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.11 (s, 1H), 9.01 (s, 1H), 8.85 (d, J=8.0 Hz, 1H), 8.37 (d, J=2.8 Hz, 1H), 8.25 (dd, J=8.0, 2.4 Hz, 1H), 8.13-8.03 (m, 2H), 7.79-7.74 (m, 1H), 7.41 (d, J=8.0 Hz, 1H), 7.15 (s, 1H), 5.02 (s, 2H), 2.54 (s, 3H).

Example 40

2-(2,5-Dimethylpyridin-4-yl)-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0420] ##STR00115##

[0421] The synthetic method of Example 40 was according to the synthetic method of Example 1. The title compound Example 40 (22 mg, 45%) was obtained.

[0422] MS m/z (ESI): 512.1 [M+H].sup.+.

[0423] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.09 (s, 1H), 8.86 (d, J=8.4 Hz, 1H), 8.46-8.43 (m, 2H), 8.39-8.36 (m, 1H), 8.12-8.03 (m, 2H), 7.79-7.74 (m, 1H), 7.57 (s, 1H), 7.02 (s, 1H), 5.05 (s, 2H), 2.53 (s, 3H), 2.51 (s, 3H).

Example 41

2-(2-Amino-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0424] ##STR00116##

[0425] The synthetic method of Example 41 was according to the synthetic method of Example 1. The title compound Example 41 (12 mg, 26%) was obtained.

[0426] MS m/z (ESI): 422.1 [M+H].sup.+.

Example 42

2-(2-(Cyclopentylamino)-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0427] ##STR00117##

[0428] The synthetic method of Example 42 was according to the synthetic method of Example 8. The title compound Example 42 (9 mg, 19%) was obtained.

[0429] MS m/z (ESI): 489.2 [M+H].sup.+.

Example 43

N-(5-Fluoropyridin-2-yl)-2-(2-(oxetan-3-ylamino)-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a ]pyrido[3,2-e]pyrimidin-4(5H)-yl)acetamide

[0430] ##STR00118##

[0431] The synthetic method of Example 43 was according to the synthetic method of Example 14. The title compound Example 43 (15 mg, 25%) was obtained.

[0432] MS m/z (ESI): 478.1 [M+H].sup.+.

Example 44

2-(8-Amino-2-(tert-butyl)-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0433] ##STR00119##

Step 1: Preparation of 2-(8-amino-2-(tert-butyl)-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0434] ##STR00120##

[0435] Example 22 (100 mg, 0.234 mmol) and aqueous ammonia (5 mL) were added to a round-bottomed flask at room temperature, and the mixture was stirred at 80° C. for 5 h. After completion of the reaction, the reaction solution was purified by HPLC to obtain Example 44 (52 mg, 54%).

[0436] MS m/z (ESI): 410.2 [M+H].sup.+.

[0437] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.96 (s, 1H), 8.41 (s, 1H), 8.02-7.95 (m, 1H), 7.93 (d, J=8.8 Hz, 1H), 7.68 (s, 1H), 7.45 (s, 2H), 6.43 (d, J=8.7 Hz, 1H), 6.15 (s, 1H), 4.79 (s, 2H), 1.23 (s, 9H).

Example 45

2-(2-(Tert-butyl)-8-cyano-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0438] ##STR00121##

Step 1: Preparation of 2-(2-(tert-butyl)-8-cyano-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0439] ##STR00122##

[0440] Example 22 (80 mg, 0.187 mmol), CuCN (45 mg, 0.5 mmol) and DMF (2 mL) were added to a round-bottomed flask at room temperature, and the mixture was stirred at 150° C. for 5 h under a nitrogen atmosphere. After completion of the reaction, the reaction solution was purified by HPLC to obtain Example 45 (26 mg, 33%).

[0441] MS m/z (ESI): 420.1 [M+H].sup.+.

[0442] .sup.1H NMR (400 MHz, DMSO) δ 11.06 (s, 1H), 8.72 (d, J=8.0 Hz, 1H), 8.37 (s, 1H), 8.17-7.98 (m, 2H), 7.77 (d, J=8.3 Hz, 1H), 6.51 (s, 1H), 4.94 (s, 2H), 1.28 (s, 9H).

Example 46

2-(2-(Tert-butyl)-8-methoxy-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0443] ##STR00123##

Step 1: Preparation of 2-(2-(tert-butyl)-8-methoxy-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0444] ##STR00124##

[0445] Example 22 (80 mg, 0.187 mmol), MeONa (43 mg, 0.8 mmol) and DMF (2 mL) were added to a round-bottomed flask at room temperature, and the mixture was stirred at 80° C. for 3 h under a nitrogen atmosphere. After completion of the reaction, the reaction solution was purified by HPLC to obtain Example 46 (35 mg, 45%).

[0446] MS m/z (ESI): 425.0 [M+H].sup.+.

[0447] .sup.1H NMR (400 MHz, DMSO-d6) δ 11.20 (s, 1H), 8.45 (d, J=8.8 Hz, 1H), 8.37 (d, J=3.2 Hz, 1H), 8.06-8.00 (m, 1H), 7.76-7.71 (m, 1H), 6.75 (d, J=8.8 Hz, 1H), 6.55 (s, 1H), 5.24 (s, 2H), 3.88 (s, 3H), 1.33 (s, 9H).

Example 47

2-(2-(Tert-butyl)-5-oxo-7-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0448] ##STR00125##

[0449] Example 47 was synthesized according to the method of Example 1. Example 47 (36 mg, 52%) was obtained by replacing 2-chloronicotinic acid with 2-chloro-5-(trifluoromethyl)nicotinic acid.

[0450] MS m/z (ESI): 463.1 [M+H].sup.+.

[0451] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.05 (s, 1H), 9.24 (s, 1H), 8.73 (s, 1H), 8.36 (d, J=3.1 Hz, 1H), 8.04 (s, 1H), 7.75 (td, J=8.8, 3.2 Hz, 1H), 6.51 (d, J=2.7 Hz, 1H), 4.96 (s, 2H), 1.33 (s, 9H).

Example 48

2-(2-(Tert-butyl)-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0452] ##STR00126##

[0453] Example 48 was synthesized according to the method of Example 1. Example 48 (52 mg, 46%) was obtained by replacing 2-chloronicotinic acid with 2,4-dichloronicotinic acid.

[0454] MS m/z (ESI): 429.2 [M+H].sup.+.

Example 49

2-(2-(Tert-butyl)-6-isopropyl-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0455] ##STR00127##

Step 1: Preparation of 2-(2-(tert-butyl)-6-isopropyl-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0456] ##STR00128##

[0457] Isopropylmagnesium bromide (1 M, 1 mL) was added dropwise to a solution of Example 48 (100 mg, 0.233 mmol) in THE (5 mL) at −70° C. under a nitrogen atmosphere, and the mixture was stirred at room temperature for 3 h. After completion of the reaction, the reaction solution was purified by HPLC to obtain Example 49 (62 mg, 60%).

[0458] MS m/z (ESI): 437.0 [M+H].sup.+.

Example 50

2-(2-(Tert-butyl)-6-cyclopropyl-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0459] ##STR00129##

[0460] Example 50 was synthesized according to the method of Example 49. Example 50 (36 mg, 58%) was obtained by replacing isopropylmagnesium bromide with cyclopropylmagnesium bromide.

[0461] MS m/z (ESI): 435.2 [M+H].sup.+.

Example 51

2-(2-(Tert-butyl)-5-oxo-6-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0462] ##STR00130##

[0463] Example 51 was synthesized according to the method of Example 1. Example 51 (36 mg, 52%) was obtained by replacing 2-chloronicotinic acid with 2-chloro-4-(trifluoromethylnicotinic acid.

[0464] MS m/z (ESI): 463.1 [M+H].sup.+.

Example 52

2-(6-Amino-2-(tert-butyl)-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0465] ##STR00131##

[0466] Example 52 was synthesized according to the method of Example 44. Example 52 (36 mg, 52%) was obtained by replacing Example 22 with Example 48.

[0467] MS m/z (ESI): 410.2 [M+H].sup.+.

Example 53

2-(7-(Tert-butyl)-4-oxopyrazolo[1,5-a]thiazolo[5,4-e]pyrimidin-5(4H)-yl)-N-(5-fluoropyrid in-2-yl)acetamide

[0468] ##STR00132##

[0469] The synthetic method of Example 53 was according to the synthetic method of Example 1. The title compound (19 mg, 21%) was obtained.

[0470] MS m/z (ESI): 401.4 [M+H].sup.+.

Example 54

2-(7-(Tert-butyl)-3-isopropyl-4-oxo-3,4-dihydro-5H-pyrazolo[5,1-b]purin-5-yl)-N-(5-fluoro pyridin-2-yl)acetamide

[0471] ##STR00133##

[0472] The synthetic method of Example 54 was according to the synthetic method of Example 1. The title compound (11 mg, 28%) was obtained.

[0473] MS m/z (ESI): 426.5 [M+H].sup.+.

Example 55

2-(2-(Tert-butyl)-6-ethyl-5-oxo-5,6-dihydro-4H-dipyrazolo[1,5-a:3′,4′-e]pyrimidin-4-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0474] ##STR00134##

[0475] The synthetic method of Example 55 was according to the synthetic method of Example 1. The title compound (26 mg, 28%) was obtained.

[0476] MS m/z (ESI): 412.4 [M+H].sup.+.

Example 56

2-(7-(Tert-butyl)-3-methyl-4-oxoisoxazolo[4,3-e]pyrazolo[1,5-a]pyrimidin-5(4H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0477] ##STR00135##

[0478] The synthetic method of Example 56 was according to the synthetic method of Example 1. The title compound (23 mg, 25%) was obtained.

[0479] MS m/z (ESI): 399.4 [M+H].sup.+.

Example 57

2-(7-(Tert-butyl)-3-methyl-4-oxoisothiazolo[4,3-e]pyrazolo[1,5-a]pyrimidin-5(4H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0480] ##STR00136##

[0481] The synthetic method of Example 57 was according to the synthetic method of Example 1. The title compound (19 mg, 29%) was obtained.

[0482] MS m/z (ESI): 415.5[M+H].sup.+.

Example 58

2-(2-(Tert-butyl)-5-thioxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0483] ##STR00137##

Step 1: Preparation of 2-(2-(tert-butyl)-5-thioxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0484] ##STR00138##

[0485] Lawson's reagent (158 mg, 0.39 mmol) was added to a solution of Example 1 (50 mg, 0.13 mmol) in toluene (2 mL) at room temperature, and the reaction solution was heated by microwave at 115 degrees for 1 hour. LCMS indicated the completion of the reaction, and the reaction solution was purified by p-HPLC(HCOOH) to obtain Example 58 (5 mg, 10%).

[0486] MS m/z (ESI): 411.13 [M+H].sup.+.

Example 59

N-(5-Fluoropyridin-2-yl)-2-(2-(1-methylcyclopropyl)-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)acetamide

[0487] ##STR00139##

[0488] The synthetic method of Example 59 was according to the synthetic method of Example 1. The title compound Example 59 (21 mg, 40%) was obtained.

[0489] MS m/z (ESI): 461.4 [M+H].sup.+.

[0490] .sup.1H NMR (400 MHz, DMSO) δ 11.04 (s, 1H), 8.78 (d, J=7.6 Hz, 1H), 8.37 (s, 1H), 8.17-7.90 (m, 2H), 7.76 (t, J=8.1 Hz, 1H), 6.35 (s, 1H), 4.93 (s, 2H), 1.47 (s, 3H), 1.03 (s, 2H), 0.85 (s, 2H).

Example 60

N-(5-Fluoropyridin-2-yl)-2-(5-oxo-8-(trifluoromethyl)-2-(1-(trifluoromethyl)cyclopropyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)acetamide

[0491] ##STR00140##

[0492] The synthetic method of Example 60 was according to the synthetic method of Example 1. The title compound Example 60 (15 mg, 31%) was obtained.

[0493] MS m/z (ESI): 515.4 [M+H].sup.+.

Example 61

2-(2-(2,2-Difluoroethyl)azetidin-3-yl)-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0494] ##STR00141##

Step 1: Preparation of tert-butyl 5-amino-3-bromo-1H-pyrazole-1-carboxylate

[0495] ##STR00142##

[0496] 3-Bromo-1H-pyrazol-5-amine (10.0 g, 61.7 mmol) was dissolved in anhydrous dichloromethane (100 mL), followed by addition of triethylamine (7.48 g, 74.1 mmol) and di-tert-butyl dicarbonate (16.0 g, 74.1 mmol). The reaction solution was reacted at room temperature for 16 hours. The reaction solution was washed successively with water (50 mL*2) and saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting crude product was purified by column chromatography (ethyl acetate/dichloromethane=0 to 20%) to obtain the title product tert-butyl 5-amino-3-bromo-1H-pyrazole-1-carboxylate Example 61-1 (14.5 g), yield: 89.5%.

[0497] MS: m/z (ESI): 262.0 [M+H].sup.+

[0498] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 6.62 (s, 2H), 5.41 (s, 1H), 1.56 (s, 9H).

Step 2: Preparation of tert-butyl 5-amino-3-bromo-1H-pyrazole-1-carboxylate

[0499] ##STR00143##

[0500] Tert-butyl 5-amino-3-bromo-1H-pyrazole-1-carboxylate Example 61-1 (14.5 g, 55.3 mmol) was dissolved in anhydrous dichloromethane (200 mL), followed by addition of triethylamine (18.5 g, 183 mmol). A solution (50 mL) of freshly prepared 2-chloro-6-(trifluoromethyl)nicotinoyl chloride (13.0 g, 61.0 mmol) in dichloromethane was added dropwise under a nitrogen atmosphere at 0° C. After completion of the addition, the reaction solution was reacted at room temperature for 30 minutes. The reaction solution was washed successively with water (200 mL*2) and saturated sodium chloride solution (200 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0 to 20%) to obtain tert-butyl 5-amino-3-bromo-1H-pyrazole-1-carboxylate Example 61-2 (9.5 g), yield: 38.2%.

[0501] MS: m/z (ESI): 371.0 [M-Boc+H].sup.+

[0502] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.08 (s, 1H), 8.40 (d, J=7.6 Hz, 1H), 8.14 (d, J=7.6 Hz, 1H), 6.96 (s, 1H), 1.58 (s, 9H).

Step 3: Preparation of N-(3-bromo-1H-pyrazol-5-yl)-2-chloro-6-(trifluoromethyl)nicotinamide

[0503] ##STR00144##

[0504] Tert-butyl 5-amino-3-bromo-1H-pyrazole-1-carboxylate Example 61-2 (8.0 g, 17.1 mmol) was dissolved in anhydrous dichloromethane (20 mL), followed by addition of a solution (4 M, 40 mL) of hydrochloric acid in dioxane. The reaction solution was reacted at room temperature for 4 hours. The reaction solution was directly concentrated to dryness by rotary evaporation to obtain N-(3-bromo-1H-pyrazol-5-yl)-2-chloro-6-(trifluoromethyl)nicotinamide Example 61-3 (6.2 g), yield: 98.4%.

[0505] MS: m/z (ESI): 368.9 [M+H].sup.+

[0506] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.50 (s, 1H), 8.39 (d, J=7.6 Hz, 1H), 8.10 (d, J=7.6 Hz, 1H), 6.53 (s, 1H).

Step 4: Preparation of 2-bromo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-5(4H)-one

[0507] ##STR00145##

[0508] N-(3-Bromo-1H-pyrazol-5-yl)-2-chloro-6-(trifluoromethyl)nicotinamide Example 61-3 (6.2 g, 16.8 mmol) was dissolved in N,N-dimethylformamide (80 mL), followed by addition of potassium carbonate (6.96 g, 50.4 mmol). The reaction solution was heated to 120° C. and reacted for 2 hours. The reaction solution was cooled to room temperature and used directly in the next step.

[0509] MS: m/z (ESI): 333.0 [M+H].sup.+

Step 5: Preparation of 2-(2-bromo-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0510] ##STR00146##

[0511] Potassium carbonate (6.96 g, 50.4 mmol) and 2-bromo-N-(5-fluoropyridin-2-yl)acetamide (4.7 g, 20.2 mmol) were added to the reaction solution of 2-bromo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-5(4H)-one Example 61-4 (NA, 16.8 mmol) in N,N-dimethylformamide (80 mL), and reacted at 40° C. for 2 hours. The reaction solution was cooled to room temperature, poured into 300 mL of water, and extracted with ethyl acetate (200 mL*3). The organic phases were combined, washed successively with water (200 mL*2) and saturated sodium chloride solution (200 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting crude product was recrystallized from ethyl acetate to obtain the title product 2-(2-bromo-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide Example 61-A.

[0512] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.05 (s, 1H), 8.84 (d, J=8.0 Hz, 1H), 8.37 (s, 1H), 8.09 (d, J=8.0 Hz, 1H), 8.07-8.02 (m, 1H), 7.80-7.73 (m, 1H), 6.78 (s, 1H), 4.96 (s, 2H).

[0513] MS m/z (ESI): 486.2 [M+H].sup.+.

Step 6: Preparation of tert-butyl 3-(4-(2-(((5-fluoropyridin-2-yl)amino)-2-oxoethyl)-5-oxo-8-(trifluoromethyl)-4,5-dihydropyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-2-yl)azetidine-1-carboxylate

[0514] ##STR00147##

[0515] Zinc dust (<10 μM, 20.3 g) was stirred with 1 M HCl (100 mL). After 2 hours, the suspension was filtered, and the resulting solid was washed with water (×2), then ethanol (×2) and finally ether (×2). The solid was dried under vacuum and stored under a nitrogen atmosphere. Zinc dust (washed, 0.60 g, 9.16 mmol) was vigorously stirred in dimethylacetamide (4 mL) under a nitrogen atmosphere, and the resulting suspension was heated to 65° C. Trimethylchlorosilane (0.12 g, 0.14 mL, 1.14 mmol) and 1,2-dibromoethane (0.098 mL, 1.14 mmol) were added, and the reaction solution was stirred for 40 minutes. A solution of tert-butyl 3-iodoazetidine-1-carboxylate (2.0 g, 7.06 mmol) in dimethylacetamide (4 mL) was added dropwise to the reaction mixture within 0.5 hour. The resulting suspension was stirred at 65° C. for 0.5 h, and then cooled to room temperature. The reaction mixture was used in the next step without treatment. A solution of Example 2 (200 mg, 0.41 mmol) and Pd(dppf)Cl.sub.2 (33 mg, 0.04 mmol) in DMA (3 mL) were added to the above solution, heated to 85° C. and reacted for 16 h. The mixture was treated to obtain Example 61-B (100 mg, 43%).

[0516] MS m/z (ESI): 562.17 [M+H].sup.+.

Step 7: Preparation of 2-(2-(azetidin-3-yl)-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0517] ##STR00148##

[0518] 4 M/L HCl/methanol (6 mL) was added to a solution of Example 61-B (100 mg, 0.18 mmol) in DCM (2 mL). The reaction solution was stirred at room temperature for 2 hours. The reaction solution was directly concentrated to dryness by rotary evaporation to obtain Example 61-C (80 mg, 97%).

[0519] MS m/z (ESI): 462.17 [M+H].sup.+.

Step 8: Preparation of 2-(2-(1-(2,2-difluoroethyl)azetidin-3-yl)-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0520] ##STR00149##

[0521] Potassium carbonate (46 mg, 0.33 mmol) and difluoroiodoethane (42 mg, 0.22 mmol) were added to a solution of Example 61-C (50 mg, 0.11 mmol) in DMF (5 mL) at room temperature. The mixture was heated to 40° C. and stirred for 2 h. The reaction solution was cooled followed by addition of water. The precipitate was filtered, washed with ethyl acetate, and purified to obtain Example 61 (26 mg, yield: 46%).

[0522] MS m/z (ESI): 526.4 [M+H].sup.+.

Example 62

2-(2-Cyclohexyl-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0523] ##STR00150##

[0524] The synthetic method of Example 62 was according to the synthetic method of Example 4. The title compound Example 62 (15 mg, 31%) was obtained.

[0525] MS m/z (ESI): 489.5 [M+H].sup.+.

[0526] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.01 (s, 1H), 8.74 (d, J=8.0 Hz, 1H), 8.33 (s, 1H), 8.04-7.93 (m, 2H), 7.72 (t, J=9.1 Hz, 1H), 6.36 (s, 1H), 4.91 (s, 2H), 2.69 (s, 1H), 1.90 (d, J=12.5 Hz, 3H), 1.70 (dd, J=34.4, 12.4 Hz, 3H), 1.40 (td, J=24.5, 12.0 Hz, 4H).

Example 63

N-(5-Fluoropyridin-2-yl)-2-(2-(3-methylpyridin-4-yl)-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)acetamide

[0527] ##STR00151##

[0528] The synthetic method of Example 63 was according to the synthetic method of Example 1. The title compound Example 63 (15 mg, 30%) was obtained.

[0529] MS m/z (ESI): 498.4 [M+H].sup.+.

[0530] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.12 (s, 1H), 8.95-8.74 (m, 3H), 8.38 (s, 1H), 8.27-8.00 (m, 3H), 7.76 (t, J=9.0 Hz, 1H), 7.25 (s, 1H), 5.09 (s, 2H), 2.72 (s, 3H).

Example 64

N-(5-Fluoropyridin-2-yl)-2-(2-(2-methylpyridin-3-yl)-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)acetamide

[0531] ##STR00152##

[0532] The synthetic method of Example 64 was according to the synthetic method of Example 1. The title compound Example 64 (15 mg, 30%) was obtained.

[0533] MS m/z (ESI): 498.4 [M+H].sup.+.

[0534] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.13 (s, 1H), 8.90-8.62 (m, 3H), 8.36 (s, 1H), 8.15-7.99 (m, 2H), 7.93-7.68 (m, 2H), 7.11 (s, 1H), 5.08 (s, 2H), 2.96 (s, 3H).

Example 65

2-(2,4-Dimethylpyridin-3-yl)-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0535] ##STR00153##

[0536] The synthetic method of Example 65 was according to the synthetic method of Example 1. The title compound Example 65 (10 mg, 33%) was obtained.

[0537] MS m/z (ESI): 512.4 [M+H].sup.+.

Example 66

N-(5-Fluoropyridin-2-yl)-2-(5-oxo-2,8-bis(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)acetamide

[0538] ##STR00154##

[0539] The synthetic method of Example 66 was according to the synthetic method of Example 1. The title compound Example 66 (10 mg, 33%) was obtained.

[0540] MS m/z (ESI): 475.3 [M+H].sup.+.

[0541] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.07 (s, 1H), 8.91 (d, J=8.0 Hz, 1H), 8.37 (d, J=3.1 Hz, 1H), 8.19 (d, J=8.1 Hz, 1H), 8.14-7.98 (m, 1H), 7.76 (t, J=8.8 Hz, 1H), 7.13 (s, 1H), 5.04 (s, 2H).

Example 68

2-(2-Cyano-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0542] ##STR00155##

Step 1: Preparation of 2-(2-cyano-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0543] ##STR00156##

[0544] Example 61-A (300 mg, 0.619 mmol), Zn(CN).sub.2 (300 mg, 2.56 mmol), Pd.sub.2(dba).sub.3 (20 mg, 0.022 mmol), Pd(dppf)Cl.sub.2 (30 mg, 0.036 mmol) and Zn powder (10 mg, 0.154 mmol) were dissolved in DMA (10 mL) at room temperature, followed by purging nitrogen for 2 minutes. The reaction solution was heated by microwave to 140 degrees and reacted for 8 hours. The reaction solution was cooled to room temperature and extracted with ethyl acetate (50 mL). The organic phase was washed twice with saturated brine. The organic phase was dried (Na.sub.2SO.sub.4), concentrated under reduced pressure, and purified by p-HPLC (FA) to obtain 100 mg of the title compound (yield: 38%).

[0545] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.07 (s, 1H), 8.92 (d, J=8.2 Hz, 1H), 8.37 (d, J=3.1 Hz, 1H), 8.22 (d, J=7.9 Hz, 1H), 8.05 (s, 1H), 7.77 (t, J=8.6 Hz, 1H), 7.24 (s, 1H), 5.01 (s, 2H).

[0546] MS m/z (ESI): 432.3 [M+H].sup.+.

Example 69

N-(5-Fluoropyridin-2-yl)-2-(2-(2-hydroxypropan-2-yl)-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)acetamide

[0547] ##STR00157##

Step 1: Preparation of N-(5-fluoropyridin-2-yl)-2-(2-(2-hydroxypropan-2-yl)-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)acetamide

[0548] ##STR00158##

[0549] Example 69-1 (100 mg, 0.22 mmol) (Example 69-1 was synthesized according to the operation of Example 6) was dissolved in dimethoxyethane (2 ml)/MeOH (2 ml) at 25° C., followed by successively adding cobalt(II) isotetraphenylporphyrin (1.3 mg, 0.002 mmol) and tetraethylammonium borohydride (80.2 mg, 0.55 mmol). The reaction mixture was stirred for 1.25 hours. The reaction was stopped and quenched by saturated aqueous ammonium chloride solution (50 mL). The mixture was extracted with ethyl acetate (3×40 mL). The combined organic phases were washed with saturated aqueous solution of sodium chloride (1×80 mL), dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure to remove the solvent. The crude product was purified to obtain the title compound (42 mg, yield: 42%).

[0550] MS m/z (ESI): 465.1 [M+H].sup.+.

[0551] .sup.1H NMR (400 MHz, DMSO) δ 11.07 (s, 1H), 8.81 (d, J=7.9 Hz, 1H), 8.37 (s, 1H), 8.12-7.92 (m, 2H), 7.76 (s, 1H), 6.44 (s, 1H), 4.99 (s, 2H), 1.51 (s, 6H).

Example 70

N-(5-Fluoropyridin-2-yl)-2-(2-isobutyl-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)acetamide

[0552] ##STR00159##

[0553] Example 70 was synthesized according to the method of Example 1. The target compound (26 mg, yield: 26%) was obtained by replacing 3-(tert-butyl)-1H-pyrazol-5-amine with 3-(isobutyl)-1H-pyrazol-5-amine.

[0554] MS m/z (ESI): 463.4 [M+H].sup.+.

[0555] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.05 (s, 1H), 8.79 (d, J=8.0 Hz, 1H), 8.37 (s, 1H), 8.07-8.03 (m, 1H), 8.00 (d, J=8.0 Hz, 1H), 7.78-7.73 (m, 1H), 6.33 (s, 1H), 4.96 (s, 2H), 2.55 (d, J=8.2 Hz, 2H), 2.04-1.93 (m, 1H), 0.95 (d, J=6.4 Hz, 6H).

Example 71

N-(5-Fluoropyridin-2-yl)-2-(2-morpholino-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)acetamide

[0556] ##STR00160##

[0557] Example 71 was synthesized according to the method of Example 1. The target compound (14 mg, yield: 35%) was obtained by replacing 3-(tert-butyl)-1H-pyrazol-5-amine with 3-(morpholinyl)-1H-pyrazol-5-amine.

[0558] MS m/z (ESI): 492.4 [M+H].sup.+.

Example 74

2-(2-(Azetidine-1-carbonyl)-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0559] ##STR00161##

Step 1: Preparation of 2-(2-(azetidine-1-carbonyl)-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0560] ##STR00162##

[0561] DIPEA (0.1 mL, 0.6 mmol) was added to a solution of Example 74-0 mg, 0.22 mmol) (Example 74-1 was synthesized according to Example 8-3) and HATU (83.4 mg, 0.22 mmol) in DMF (2 mL). The mixture was stirred at room temperature for 30 minutes, followed by addition of azetidine (12.5 mg, 0.22 mmol). The reaction solution was stirred at room temperature for 18 hours. Water (40 mL) was added to the reaction solution. The mixture was extracted with ethyl acetate (2×20 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, concentrated and purified to obtain Example 74 (56 mg, yield: 52%).

[0562] MS m/z (ESI): 490.1 [M+H].sup.+.

[0563] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.04 (s, 1H), 8.86 (d, J=8.1 Hz, 1H), 8.37 (s, 1H), 8.15-8.08 (m, 1H), 8.04 (s, 1H), 7.75 (t, J=9.4 Hz, 1H), 6.81 (s, 1H), 5.04 (s, 2H), 4.68-4.52 (m, 2H), 4.08 (t, J=7.5 Hz, 2H), 2.34 (d, J=9.1 Hz, 2H).

Example 78

N-(5-Fluoropyridin-2-yl)-2-(5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)acetamide

[0564] ##STR00163##

Step 1: Preparation of N-(1H-pyrazol-5-yl)-2-chloro-nicotinamide

[0565] ##STR00164##

[0566] 1H-Pyrazol-5-amine (1.66 g, 19.93 mmol), DIPEA (6.2 g, 49.8 mmol) and HATU (5.4 g, 0.144 mmol) were added successively to a solution of 2-chloronicotinic acid (1.57 g, 9.96 mmol) in DMF (30 mL) under an ice bath condition. The ice bath was removed, and the reaction solution was stirred for 1 h. The mixture was treated to obtain Example 78-1 (2.0 g, 90%).

[0567] MS m/z (ESI): 291.0 [M+H].sup.+.

Step 2: Preparation of pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-5(4H)-one

[0568] ##STR00165##

[0569] Potassium carbonate (1.61 g, 11.66 mmol) and 1,4-diazabicyclo[2.2.2]octane (DABCO) (150.9 mg, 1.35 mmol) were added to a solution of Example 78-1 (2.0 g, 8.97 mmol) in DMF (50 mL). The reaction solution was stirred at room temperature for 16 hours.

[0570] The mixture was treated to obtain Example 78-2 (1.6 g, 97%).

[0571] MS m/z (ESI): 255.0[M+H].sup.+.

Step 3: Preparation of 2-(2-(tert-butyl)-5-oxopyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0572] ##STR00166##

[0573] Potassium carbonate (2.23 g, 16.11 mmol) and Example 1-3 (2.25 g, 9.67 mmol) were added to a solution of Example 78-2 (1.5 g, 8.06 mmol) in DMF (30 mL) at room temperature. The mixture was heated to 80° C. and stirred for 2 h. The reaction solution was cooled followed by addition of water. The precipitate was filtered, washed with ethyl acetate, and purified to obtain Example 78 (2.1 g, yield: 78%).

[0574] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.06 (s, 1H), 8.83 (d, J=8.0 Hz, 1H), 8.37 (d, J=3.2 Hz, 1H), 8.06 (d, J=8.0 Hz, 1H), 8.05-8.02 (m, 1H), 7.98 (d, J=2.0 Hz, 1H), 7.78-7.73 (m, 1H), 6.46 (s, 1H), 5.00 (s, 2H).

[0575] MS m/z (ESI): 407.3 [M+H].sup.+.

Example 79

2-(2-Chloro-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0576] ##STR00167##

[0577] Example 79 was synthesized according to the method of Example 1. The target compound (31 mg, yield: 26%) was obtained by replacing 3-(tert-butyl)-1H-pyrazol-5-amine with 3-chloro-1H-pyrazol-5-amine.

[0578] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.05 (s, 1H), 8.84 (d, J=8.1 Hz, 1H), 8.36 (s, 1H), 8.15-7.99 (m, 2H), 7.76 (t, J=9.0 Hz, 1H), 6.73 (s, 1H), 4.96 (s, 2H).

[0579] MS m/z (ESI): 441.7 [M+H].sup.+.

Example 80

2-(3-Cyano-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0580] ##STR00168##

[0581] Example 80 was synthesized according to the method of Example 1. The target compound was obtained by replacing 3-(tert-butyl)-1H-pyrazol-5-amine with 4-cyano-1H-pyrazol-5-amine.

Step 1: Preparation of tert-butyl 5-amino-4-cyano-1H-pyrazole-1-carboxylate

[0582] ##STR00169##

[0583] 5-Amino-1H-pyrazole-4-carbonitrile (2.0 g, 18.5 mmol) was dissolved in anhydrous dichloromethane (40 mL), followed by addition of triethylamine (3.74 g, 37.0 mmol) and di-tert-butyl dicarbonate (4.44 g, 20.4 mmol). The reaction solution was reacted at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure, and made slurry in petroleum ether (50 mL) to obtain the title product tert-butyl 3-amino-4-cyano-1H-pyrazole-1-carboxylate Example 80-1 (3.5 g), yield: 90.9%.

[0584] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.77 (s, 1H), 7.63 (s, 2H), 1.56 (s, 9H).

Step 2: Preparation of tert-butyl-(2-chloro-6-(trifluoromethyl)nicotinamido)-4-cyano-1H-pyrazole-1-carboxylate

[0585] ##STR00170##

[0586] Tert-butyl 3-amino-4-cyano-1H-pyrazole-1-carboxylate Example 80-1 (3.5 g, 16.8 mmol) was dissolved in anhydrous dichloromethane (50 mL), followed by addition of triethylamine (5.35 g, 7.37 mmol). A solution (50 mL) of freshly prepared 2-chloro-6-(trifluoromethyl)nicotinoyl chloride (4.3 g, 17.6 mmol) in dichloromethane was added dropwise under a nitrogen atmosphere at 0° C. After completion of the addition, the reaction solution was reacted at room temperature for 1 hour. The reaction solution was washed successively with water (50 mL*2) and saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0 to 40%) to obtain tert-butyl 3-(2-chloro-6-(trifluoromethyl)nicotinamido)-4-cyano-1H-pyrazole-1-carboxylate Example 80-2 (2.8 g), yield: 38.2%.

[0587] MS: m/z (ESI): 432.8 [M+NH.sub.4]

[0588] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.87 (s, 1H), 9.23 (s, 1H), 8.43 (d, J=7.6 Hz, 1H), 8.13 (d, J=7.6 Hz, 1H), 1.59 (s, 9H).

Step 3: Preparation of 2-chloro-N-4-cyano-1H-pyrazol-5-yl)-6-(trifluoromethyl)nicotinamide

[0589] ##STR00171##

[0590] Tert-butyl 3-(2-chloro-6-(trifluoromethyl)nicotinamido)-4-cyano-1H-pyrazole-1-carboxylate Example 80-2 (2.8 g, 6.73 mmol) was dissolved in anhydrous dichloromethane (10 mL), followed by addition of a solution (4 M, 30 mL) of hydrochloric acid in dioxane. The reaction solution was reacted at room temperature for 5 hours. The reaction solution was directly concentrated to dryness by rotary evaporation to obtain 2-chloro-N-(4-cyano-1H-pyrazol-5-yl)-6-(trifluoromethyl)nicotinamide Example 80-3 (2.1 g), yield: 98.8%.

[0591] MS: m/z (ESI): 315.8 [M+H].sup.+

Step 4: Preparation of 5-oxo-8-(trifluoromethyl)-4,5-dihydropyrazolo[1,5-a]pyrido[3,2-e]pyrimidine-3-carbonitrile

[0592] ##STR00172##

[0593] 2-Chloro-N-(4-cyano-1H-pyrazol-5-yl)-6-(trifluoromethyl)nicotinamide Example 80-3 (2.1 g, 6.65 mmol) was dissolved in N,N-dimethylformamide (40 mL), followed by addition of potassium carbonate (1.84 g, 13.3 mmol). The reaction solution was heated to 120° C. and reacted for 2 hours. The reaction solution was cooled to room temperature, adjusted to pH 5 to 6 with 1M dilute hydrochloric acid, and extracted with ethyl acetate (100 mL*2). The organic phases were combined, washed successively with water (100 mL*2) and saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and made slurry in ethyl acetate (15 mL) to obtain 5-oxo-8-(trifluoromethyl)-4,5-dihydropyrazolo[1,5-a]pyrido[3,2-e]pyrimidine-3-carbonitrile Example 80-4 (1.3 g), yield: 69.9%.

[0594] MS: m/z (ESI): 279.8 [M+H].sup.+

Step 4: Preparation of 2-(3-cyano-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide

[0595] ##STR00173##

[0596] 5-Oxo-8-(trifluoromethyl)-4,5-dihydropyrazolo[1,5-a]pyrido[3,2-e]pyrimidine-3-carbo nitrile Example 80-4 (500 mg, 1.79 mmol) was dissolved in N,N-dimethylformamide (20 mL), followed by addition of potassium carbonate (371 mg, 2.69 mmol) and 2-bromo-N-(5-fluoropyridin-2-yl)acetamide (501 mg, 2.15 mmol). The reaction solution was reacted at 40° C. and for 2 hours. The reaction solution was cooled to room temperature, poured into 100 mL of water, and extracted with ethyl acetate (50 mL*2). The organic phases were combined, washed successively with water (50 mL*2) and saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting crude product was made slurry in ethyl acetate. The resulting mother liquor was concentrated under reduced pressure, and purified by reverse HPLC to obtain the title product 2-(3-cyano-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)-N-(5-fluoropyridin-2-yl)acetamide Example 80.

[0597] MS m/z (ESI): 432.3[M+H].sup.+.

[0598] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.2 (s, 1H), 8.93 (d, J=8.0 Hz, 1H), 8.58 (s, 1H), 8.38 (d, J=3.2 Hz, 1H), 8.20 (d, J=8.0 Hz, 1H), 8.07-8.04 (m, 1H), 7.81-7.75 (m, 1H), 5.19 (s, 2H).

Example 81

4-(2-((5-Fluoropyridin-2-yl)amino)-2-oxoethyl)-N-methyl-5-oxo-8-(trifluoromethyl)-4,5-dihydropyrazolo[1,5-a]pyrido[3,2-e]pyrimidine-2-carboxamide

[0599] ##STR00174##

[0600] Example 81 was synthesized according to the method of Example 74. The target compound (48 mg, yield: 61%) was obtained by replacing azacyclobutylamine with methylamine.

[0601] MS m/z (ESI): 464.1[M+H].sup.+.

[0602] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.04 (s, 1H), 8.88 (d, J=8.0 Hz, 1H), 8.53 (d, J=5.4 Hz, 1H), 8.37 (s, 1H), 8.13 (d, J=8.1 Hz, 1H), 8.04 (s, 1H), 7.76 (t, J=8.9 Hz, 1H), 6.84 (s, 1H), 5.05 (s, 2H), 2.80 (d, J=4.6 Hz, 3H).

Example 82

N-(5-Fluoropyridin-2-yl)-2-(2-(hydroxymethyl)-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)acetamide

[0603] ##STR00175##

Step 1: Preparation of N-(5-fluoropyridin-2-yl)-2-(2-(hydroxymethyl)-5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)acetamide

[0604] ##STR00176##

[0605] Diisobutylaluminum hydride (1M in toluene, 0.66 mL, 0.66 mmol) was added to a solution of Example 82-1 (100 mg, 0.22 mmol) (Example 82-1 was synthesized according to Example 8-2) in THE (2 mL) at 0° C. The mixture was stirred at room temperature overnight. Rochelle's salt solution (1.0 M, 5 ml) was added, followed by addition of ethyl acetate (5 mL). The resulting suspension was stirred at room temperature until clear phase separation was achieved. The organic phase was separated, and the aqueous phase was extracted with EtOAc (3×40 ml). The combined organic layers were washed with saturated aqueous solution of sodium bicarbonate (50 mL) and saturated brine (50 mL), dried over anhydrous sodium sulfate, concentrated and purified to obtain the target compound (32 mg, yield: 34%).

[0606] MS m/z (ESI): 437.1[M+H].sup.+.

[0607] .sup.1H NMR (400 MHz, DMSO) δ 11.06 (s, 1H), 8.82 (d, J=7.9 Hz, 1H), 8.37 (s, 1H), 8.02 (m, 2H), 7.76 (s, 1H), 6.40 (s, 1H), 5.44 (s, 1H), 5.00 (s, 2H), 4.56 (s, 2H).

Example 83

N-(5-Chloropyridin-2-yl)-2-(5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)acetamide

[0608] ##STR00177##

[0609] Example 83 was synthesized according to the method of Example 78. The target compound (23 mg, yield: 54%) was obtained by replacing 5-fluoropyridin-2-amine with 5-chloropyridin-2-amine.

[0610] MS m/z (ESI): 423.1[M+H].sup.+.

[0611] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.14 (s, 1H), 8.84 (d, J=8.0 Hz, 1H), 8.42 (d, J=2.6 Hz, 1H), 8.05 (t, J=9.3 Hz, 2H), 7.99-7.89 (m, 2H), 6.47 (d, J=2.0 Hz, 1H), 5.02 (s, 2H).

Example 84

N-(5-Chloropyrimidin-2-yl)-2-(5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)acetamide

[0612] ##STR00178##

[0613] Example 84 was synthesized according to the method of Example 78. The target compound (21 mg, yield: 53%) was obtained by replacing 5-fluoropyridin-2-amine with 5-chloropyrimidine-2-amine.

[0614] MS m/z (ESI): 424.1[M+H].sup.+.

[0615] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.29 (s, 1H), 8.95-8.72 (m, 3H), 8.06 (d, J=8.0 Hz, 1H), 7.98 (d, J=2.0 Hz, 1H), 6.46 (d, J=2.0 Hz, 1H), 5.16 (s, 2H).

Example 85

N-(3,5-Difluoropyridin-2-yl)-2-(5-oxo-8-(trifluoromethyl)pyrazolo[1,5-a]pyrido[3,2-e]pyrimidin-4(5H)-yl)acetamide

[0616] ##STR00179##

[0617] Example 85 was synthesized according to the method of Example 78. The target compound (25 mg, yield: 46%) was obtained by replacing 5-fluoropyridin-2-amine with 3,5-difluoropyridine.

[0618] MS m/z (ESI): 425.1[M+H].sup.+.

[0619] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.32 (s, 1H), 8.82 (d, J=8.0 Hz, 1H), 8.56 (dd, J=10.2, 2.2 Hz, 1H), 8.12-7.91 (m, 3H), 6.45 (d, J=2.0 Hz, 1H), 5.01 (s, 2H).

Biological Assay and Evaluation

[0620] The present invention is further illustrated below in combination with the following test examples, which are not intended to limit the scope of the present invention.

Test Example 1. Determination of the Effect of the Compounds of the Present Invention on Calcium Ion Mobility in Cells Stably Expressing 1321N1-hP2X3 Receptors

[0621] Experimental Objective:

[0622] To determine the inhibitory activity of the compounds on 1321N1-hP2X3 receptor.

[0623] Experimental instruments:

[0624] 384-well cell plate (Corning; 3712);

[0625] 384-well compound plate (Corning; 3657);

[0626] 384-well assay plate (LABCYTE; P-05525);

[0627] FLIPR (Molecular Devices).

[0628] Experimental Reagents:

[0629] DMEM (Gibco; 11965);

[0630] FBS (Gibco; 10099-141);

[0631] Hygromycin B (Invitrogen, 10687010);

[0632] Matrix (Thermo; 5416);

[0633] DMSO (Sigma; D2650);

[0634] HBSS (Invitrogen; 14025);

[0635] HEPES (Invitrogen; 15630080);

[0636] Probenecid (Sigma; P8761);

[0637] Versene (Gibco; 15040066);

[0638] G418 (Sigma; G5013);

[0639] FLIPR® Calcium 4 Assay Kit (Molecular Devices; R8141);

[0640] α,β-meATP (Sigma; M6517);

[0641] ATP hydrolytic enzyme (Sigma; A7646);

[0642] Stably transfected cell line: 1321N1-hP2X3 (supplied by Shanghai ChemPartner Chemical Research Co., Ltd.).

[0643] Experimental Method:

[0644] 1. Formulation of the Reagents:

[0645] Assay buffer: 1*HBSS+20 mM HEPES;

[0646] Cell culture medium: DMEM+10% FBS+75 g/mL Hygromycin B+300 g/mL G418;

[0647] Plating medium: DMEM+10% DPBS;

[0648] 0.5*Dye: 10*Dye stock+1.25 Probenecid+1*assay buffer+0.5 U/mL ATP hydrolytic enzyme;

[0649] 2. The cells were cultured to 70%-90% confluency in the cell culture medium at 37° C., 5% CO.sub.2. The medium was discarded, and the cells were added with 2 mL of Versene, and the cells were placed in an incubator at 37° C. for 2 to 5 min. The cells were collected by addition of 10 mL of plating medium and counted. The cells were seeded to the 384-well assay plate by addition of 50 μL solution (a density of 1×10.sup.4 cells/well) to each well, and incubated for 16 to 24 hours (at least overnight).

[0650] 3. The medium was discarded, and 30 μL of 1× dye was added. The cells were incubated at 37° C. in the dark for 60 min.

[0651] 4. The compound powder was dissolved in DMSO to obtain a 20 mM stock solution. 180× compound with required concentration was formulated, and diluted in gradient for concentration points.

[0652] 5. Preparation of compound plate: 500 nL of 180× compound was transferred to the compound plate (source plate for FLIPR) using ECHO. 30 μL of assay buffer was added to each well, and the plate was shaken gently for 20 to 40 minutes.

[0653] 6. Determination: 15 μL of 3× compound was taken from each well and added to the cell plate. The samples were added by FLIPR instrument, and the calcium signals were detected. After 15 minutes, 22.5 μL of 3× agonist (EC.sub.80 concentration) was added to each well and the calcium signals were detected.

[0654] Processing Method of the Experimental Data:

[0655] The calcium signal values were determined by FLIPR. The ratio of the 340/510 nm wavelength signals to 380/510 nm wavelength signals was used as the calculated results for each sampling time point in the experiment. The calculation of maximum minus minimum was derived from the ratio signal curve.

[0656] The percent inhibition rate and ten-point concentration data were fitted to the parametric nonlinear logistic equation by using GraphPad prism to calculate the IC.sub.50 values of the compounds.

[0657] Experimental Results:

[0658] The results of the compounds of the Examples of the present invention in the 1321N1-hP2X3 receptor cell function calcium ion mobility assay are shown in Table 1:

TABLE-US-00001 TABLE 1 Example 1321N1-hP2X3 No. IC.sub.50 (nM)  1 16.19 21 304.8 30 88.20 34 63.38 35 77.69 36 185.0 37 336.7 40 292.5 59 259.2 61-A 76.40 62 384.7 63 238.5 64 149.5 66 130.4 68 32.45 74 203.1 78 49.00 79 34.70 80 64.35 83 136.7

[0659] Experimental Conclusion: The above data indicate that the compounds of the present invention show good inhibitory effect in the 1321N1-hP2X3 receptor cell function calcium ion mobility assay.

Test Example 2. Determination of the Effect of the Compounds of the Present Invention on Calcium Ion Mobility in Cells Stably Expressing 1321N1-hP2X2/3 Receptors

[0660] Experimental Objective:

[0661] To determine the inhibitory activity of the compounds on 1321N1-hP2X2/3 receptor.

[0662] Experimental Instruments:

[0663] 384-well cell plate (Corning; 3712);

[0664] 384-well compound plate (Corning; 3657);

[0665] 384-well assay plate (LABCYTE; P-05525);

[0666] FLIPR (Molecular Devices).

[0667] Experimental Reagents:

[0668] DMEM (Gibco; 11965);

[0669] FBS (Gibco; 10099-141);

[0670] Hygromycin B (Invitrogen, 10687010);

[0671] Matrix (Thermo; 5416);

[0672] DMSO (Sigma; D2650);

[0673] HBSS (Invitrogen; 14025);

[0674] HEPES (Invitrogen; 15630080);

[0675] Probenecid (Sigma; P8761);

[0676] Versene (Gibco; 15040066);

[0677] G418 (Sigma; G5013);

[0678] FLIPR® Calcium 4 Assay Kit (Molecular Devices; R8141);

[0679] α,β-meATP (Sigma; M6517);

[0680] ATP hydrolytic enzyme (Sigma; A7646);

[0681] Stably transfected cell line: 1321N1-hP2X2/3 (supplied by Shanghai ChemPartner Chemical Research Co., Ltd.).

[0682] Experimental Method:

[0683] 1. Formulation of the Reagents:

[0684] Assay buffer: 1*HBSS+20 mM HEPES;

[0685] Cell culture medium: DMEM+10% FBS+75 μg/mL Hygromycin B+150 μg/mL G418;

[0686] Plating medium: DMEM+10% DPBS;

[0687] 0.5*Dye: 10*Dye stock+1.25 Probenecid+1*assay buffer+0.5 U/mL ATP hydrolytic enzyme;

[0688] 2. The cells were cultured to 70%-90% confluency in the cell culture medium at 37° C., 5% CO.sub.2. The medium was discarded, and the cells were added with 2 mL of Versene, and the cells were placed in an incubator at 37° C. for 2 to 5 min. The cells were collected by addition of 10 mL of plating medium and counted. The cells were seeded to the 384-well assay plate by addition of 50 μL solution (a density of 1×10.sup.4 cells/well) to each well, and incubated for 16 to 24 hours (at least overnight).

[0689] 3. The medium was discarded, and 30 μL of 1× dye was added. The cells were incubated at 37° C. in the dark for 60 min.

[0690] 4. The compound powder was dissolved in DMSO to obtain a 20 mM stock solution. 180× compound with required concentration was formulated, and diluted in gradient for concentration points.

[0691] 5. Preparation of compound plate: 500 nL of 180× compound was transferred to the compound plate (source plate for FLIPR) using ECHO. 30 μL of assay buffer was added to each well, and the plate was shaken gently for 20 to 40 minutes.

[0692] 6. Determination: 15 μL of 3× compound was taken from each well and added to the cell plate. The samples were added by FLIPR instrument, and the calcium signals were detected. After 15 minutes, 22.5 μL of 3× agonist (EC.sub.80 concentration) was added to each well and the calcium signals were detected.

[0693] Processing Method of the Experimental Data:

[0694] The calcium signal values were determined by FLIPR. The ratio of the 340/510 nm wavelength signals to 380/510 nm wavelength signals was used as the calculated results for each sampling time point in the experiment. The calculation of maximum minus minimum was derived from the ratio signal curve.

[0695] The percent inhibition rate and ten-point concentration data were fitted to the parametric nonlinear logistic equation by using GraphPad prism to calculate the IC.sub.50 values of the compounds.

[0696] Experimental Results:

[0697] The results of the compounds of the Examples of the present invention in the 1321N1-hP2X2/3 receptor cell function calcium ion mobility assay are shown in Table 2:

TABLE-US-00002 TABLE 2 Example 1321N1-hP2X2/3 No. IC.sub.50 (nM)  1 64390 30 14540 34 25240 35 >30000 40 16660 59 32170 61-A 6363 68 5629 78 4523 80 3037 83 >30000

[0698] Experimental Conclusion:

[0699] The above data indicate that the compounds of the present invention show weak inhibitory effect in the 1321N1-h2X2/3 receptor cell function calcium ion mobility assay.

Test Example 3. Pharmacokinetic Assay in Balb/C Mice

[0700] 1. Study Objective:

[0701] Balb/C mice were used as test animals. The pharmacokinetic behavior of the compounds of Examples was studied in mouse body (plasma) by orally administration at a dose of 5 mg/kg.

[0702] 2. Experimental Protocol

[0703] 2.1 Test Compounds:

[0704] Compounds of the Examples of the present invention, prepared by the applicant.

[0705] 2.2 Test Animals:

[0706] Male Balb/C mice (6 mice per group), purchased from Shanghai Jiesijie Laboratory Animal Co., LTD, with Certificate No.: SCXK (Shanghai) 2013-0006 N0.311620400001794.

[0707] 2.3 Formulation of the Compound:

[0708] 5 g of hydroxyethyl cellulose (HEC, CMC-Na, viscosity: 800-1200 Cps) was weighed and dissolved in 1000 mL of purified water, followed by addition of 10 g of Tween80.

[0709] The mixture was mixed well to obtain a clear solution.

[0710] 2.4 Administration:

[0711] After an overnight fast, male Balb/C mice were administered p.o. with the test compound at a dose of 5 mg/kg and a volume of 10 mL/kg.

[0712] 2.5 Sample Collection:

[0713] 0.04 mL of blood was taken from the orbit of the mouse before administration and at 0, 0.5, 1, 2, 4, 6, 8 and 24 hours after administration. The samples were stored in EDTA-K2 tubes, and centrifuged for 6 minutes at 4° C., 6000 rpm to separate the plasma. The plasma samples were stored at −80° C.

[0714] 2.6 Sample process:

[0715] 1) 160 μL of acetonitrile was added to 20 μL of the plasma sample for precipitation, and then the mixture was centrifuged at 3500×g for 5 to 20 minutes. 2) After the above process, 100 μL of the supernatant was taken to analyze the concentration of the test compound by LC/MS/MS.

[0716] 2.7 Liquid Chromatography Analysis [0717] Liquid chromatography condition: Shimadzu LC-20AD pump [0718] Mass spectrometry condition: AB Sciex API 4000 mass spectrometer [0719] Chromatographic column: phenomenex Gemiu 5 um C18 50×4.6 mm [0720] Mobile phase: Eluent A was 0.1% formic acid in water, and Eluent B was acetonitrile [0721] Flow rate: 0.8 mL/min [0722] Elution time: 0-4.0 minutes the eluent is as follows:

TABLE-US-00003 Time/minute Eluent A Eluent B 0.01 90% 10% 0.5 90% 10% 0.8  5% 95% 2.4  5% 95% 2.5 90% 10% 4.0 Stop

[0723] 3. Experimental Results and Analysis

[0724] The main parameters of pharmacokinetics were calculated by WinNonlin 8.2. The results of pharmacokinetic test in mice are shown in the following Table 3:

TABLE-US-00004 TABLE 3 Results of pharmacokinetic test in mice Pharmacokinetic test (5 mg/kg) Area under Average Peak curve Plasma Half residence Example time AUC.sub.0-t concentration life time No. t.sub.max(h) (ng/mL*h) C.sub.max(ng/mL) t.sub.1/2(h) MRT.sub.0-∞(h) 30 0.50 10943.63 3546.70 1.36 2.16 34 1.00 7584.0 2360.0 1.4 2.3 61-A 2.00 30539.48 3433.3 4.60 5.60 68 2.00 10160.1 1826.7 1.8 3.8 78 1.00 5043.0 1293.3 1.6 2.5 Note: 0.5% CMC-Na (1% Tween 80)

[0725] Experimental Conclusion:

[0726] It can be seen from the results of pharmacokinetic test in mice in the table that the compounds of the Examples of the present invention show good pharmacokinetic properties, and both the exposure AUC and maximum plasma concentration Cmax are good.

Test Example 4. Pharmacokinetic Assay in Rats

[0727] 1. Study Objective:

[0728] SD rats were used as test animals. The pharmacokinetic behavior of the compounds of Examples was studied in rat body (plasma) by orally administration at a dose of 5 mg/kg.

[0729] 2. Experimental Protocol

[0730] 2.1 Test Compounds: Compounds of the Examples of the present invention, prepared by the applicant.

[0731] 2.2 Test animals:

[0732] Male SD rats (3 rats per group), purchased from Shanghai Jiesijie Laboratory Animal Co., LTD, with Certificate No.: SCXK (Shanghai) 2013-0006 N0.311620400001794.

[0733] 2.3 Formulation of the Compound:

[0734] 5 g of hydroxyethyl cellulose (HEC, CMC-Na, viscosity: 800-1200 Cps) was weighed and dissolved in 1000 mL of purified water, followed by addition of 10 g of Tween80.

[0735] The mixture was mixed well to obtain a clear solution.

[0736] 2.4 Administration:

[0737] After an overnight fast, male SD rats (3 rats per group) were administered p.o. with the test compound at a dose of 5 mg/kg and a volume of 10 mL/kg.

[0738] 2.5 Sample Collection:

[0739] 0.2 mL of blood was taken from the jugular vein of the rat before administration and at 0, 0.5, 1, 2, 4, 6, 8 and 24 hours after administration. The samples were stored in EDTA-K2 tubes, and centrifuged for 6 minutes at 4° C., 6000 rpm to separate the plasma. The plasma samples were stored at −80° C.

[0740] 2.6 Sample Process:

[0741] 1) 160 μL of acetonitrile was added to 40 μL of the plasma sample for precipitation, and then the mixture was centrifuged at 3500×g for 5 to 20 minutes.

[0742] 2) After the above process, 100 μL of the supernatant was taken to analyze the concentration of the test compound by LC/MS/MS.

[0743] 2.7 Liquid Chromatography Analysis [0744] Liquid chromatography condition: Shimadzu LC-20AD pump [0745] Mass spectrometry condition: AB Sciex API 4000 mass spectrometer [0746] Chromatographic column: phenomenex Gemiu 5 um C18 50×4.6 mm [0747] Mobile phase: Eluent A was 0.1% formic acid in water, and Eluent B was acetonitrile [0748] Flow rate: 0.8 mL/min [0749] Elution time: 0-4.0 minutes, the eluent is as follows:

TABLE-US-00005 Time/minute Eluent A Eluent B 0.01 90% 10% 0.5 90% 10% 0.8  5% 95% 2.4  5% 95% 2.5 90% 10% 4.0 Stop

[0750] 3. Experimental Results and Analysis

[0751] The main parameters of pharmacokinetics were calculated by WinNonlin 8.2. The results of pharmacokinetic test in rats are shown in the following Table 4:

TABLE-US-00006 TABLE 4 Results of pharmacokinetic test in rats Pharmacokinetic test (5 mg/kg) Area under Average Peak curve Plasma Half residence Example time AUC.sub.0-t concentration life time No. t.sub.max(h) (ng/mL*h) C.sub.max(ng/mL) t.sub.1/2(h) MRT.sub.0-∞(h) 34 4.00 5783 904 3.5 6.2 61-A 2.00 11977 1547 8.0 5.8 68 4.00 9852 1877 1.8 3.5 78 2.00 6811 1217 1.7 3.7 80 4.00 21252 2193 11.3 13.6 Note: 0.5% CMC-Na (1% Tween 80)

[0752] 4. Experimental Conclusion:

[0753] It can be seen from the results of pharmacokinetic test in rats in the table that the compounds of the Examples of the present invention show good pharmacokinetic properties at the dose of 5 mg/kg, and both the exposure AUC and maximum plasma concentration C.sub.max are good.

Test Example 5. Assay of Metabolic Stability in Liver Microsome

[0754] 1. Experimental Objective:

[0755] The objective of the experiment is to determine the stability of the compounds of the Examples in liver microsome of mouse, rat, dog and human.

[0756] 2. Experimental Procedure:

[0757] 2.1 Formulation of the Working Solution of the Compound

[0758] Formulation of the working solution of the compound: The stock solution of the compound was added to phosphate buffer, and the final concentration was 20 μM.

[0759] 2.2 Formulation of the Working Solution of Liver Microsome

[0760] Liver microsome was diluted with 100 mM phosphate buffer to obtain a final concentration of 0.625 mg/mL.

[0761] 2.3 Formulation of NADPH and UDPGA

[0762] NADPH (reduced nicotinamide adenine dinucleotide phosphate) and UDPGA (uridine diphosphate glucuronic acid) were weighed respectively, followed by addition of 100 mM phosphate buffer. The final concentrations were 20 mM.

[0763] 2.4 Formulation of the Channel-Forming Reagent

[0764] 1 mg of Alamethicin was weighed, to which 200 μL of DMSO was added to obtain a 5 mg/mL solution. The solution was diluted with phosphate buffer to obtain a final concentration of 50 μg/mL.

[0765] 2.5 Formulation of the reaction stop solution Stop solution: Cold acetonitrile containing 100 ng/mL labetalol hydrochloride and 400 ng/mL tolbutamide as internal standards.

[0766] 2.6 Incubation Procedure

[0767] 400 μL of the prepared liver microsome, 25 μL of the working solution of the compound and 25 μL of Alamethicin were added to a 96-well plate successively, which was then pre-incubated at 37° C. for 10 min. 50 μL of the prepared NADPH/UDPGA was added to initiate the reaction, and the plate was incubated at 37° C. The total volume of the reaction system was 500 μL. The final contents of the components were as follows:

TABLE-US-00007 Components Content Liver microsome 0.5 mg/mL Compound 1 μM NADPH 2 mM UDPGA 2 mM Alamethicin 2.5 μg/mL

[0768] 2.7 Sample Analysis

[0769] 2.7.1 Chromatographic Conditions:

[0770] Instrument: Shimadzu LC-30 AD;

[0771] Chromatographic column: XBridge® C18 (50*4.6 mm, particle size: 5 μm);

[0772] Mobile phase: A: 0.1% formic acid solution, B: methanol

[0773] Eluent gradient: 0.2-1.6 min 5% A to 95% A, 3.0-3.1 min 95% A to 5% A Running time: 4.0 min.

[0774] 2.7.2 Mass Spectrometry Conditions:

[0775] Instrument: API5500 liquid chromatography-mass spectrometer, AB Sciex;

[0776] Ion source: Electrospray ionization source (ESI);

[0777] Drying gas: N2, temperature: 500° C.;

[0778] Electrospray voltage: 5000V;

[0779] Detection method: Positive ion detection;

[0780] Scanning mode: Mode of reaction monitoring (MRM).

[0781] 3. Experimental Results:

TABLE-US-00008 TABLE 5 Results of the metabolic stability assay of the compounds of the Examples in liver microsome Mouse Rat Dog Human Re- Re- Re- Re- main- main- main- main- Ex- ing ing ing ing am- (%, (%, (%, (%, ple t.sub.1/2 60 t.sub.1/2 60 t.sub.1/2 60 t.sub.1/2 60 No. (min) min) (min) min) (min) min) (min) min) 34   186.6 81.3 1735.0 104.4 / / ∞ 113.9 61-A   409.1 92.5  941.8 102.3  937.5 96.0 ∞ 100.4 68   165.0 91.6 ∞ 106.4 1352.9 99.0 1405.4 101.2 78 34099.6 99.3 1964.7  98.9  671.0 93.4  884.7  97.5

[0782] 4. Experimental Conclusion:

[0783] The above data show that the compounds of the Examples of the present invention have good metabolic stability in liver microsome of mouse, rat, dog and human.

Test Example 6. Assay of Plasma Protein Binding Rate

[0784] 1. Experimental Objective:

[0785] The objective of the experiment is to determine the plasma protein binding of the compounds of the Examples in plasma.

[0786] 2. Experimental Instruments and Materials:

[0787] Liquid chromatography-mass spectrometer, centrifuge, vortex mixer, pipette, continuous pipette, 96-well plate, tissue homogenizer (used for tissue sample analysis), 50% aqueous solution of methanol, acetonitrile solution containing internal standard, blank medium (plasma, urine or tissue homogenate, etc.)

[0788] 3. Experimental Procedure:

[0789] 3.1 Formulation of the Stock Solution a of the Test Compound

[0790] The compound of the Example was formulated into 1 mM solution A with DMSO;

[0791] 3.2 Formulation of the Plasma Solution B

[0792] The solution A was added to the plasma solution to obtain 5 μM solution B;

[0793] 3.3 Operation Procedure

[0794] 1) 200 μL of solution B was added to the inside of the membrane;

[0795] 2) 350 L of PBS was added to the outside of the membrane;

[0796] 3) Incubation in a water bath at 37° C. for 6 h;

[0797] 4) The sample was diluted and analyzed by mass spectrometry.

[0798] 4. Chromatographic conditions:

[0799] Instrument: Shimadzu LC-20 AD;

[0800] Chromatographic column: Phenomenex Gemiu® C18 (50*4.6 mm, particle size: 5 μm);

[0801] Mobile phase: A: acetonitrile, B: 0.1% formic acid solution; 0˜0.5 min: 5% A.fwdarw.90% A, 2.0˜2.1 min: 90% A.fwdarw.5% A; flow rate: 0.8 mL/min; running time: 5.0 min; injection volume: 5 μL.

[0802] 5. Mass Spectrometry Conditions:

[0803] Instrument: API4000 liquid chromatography-mass spectrometer, AB Co., USA;

[0804] The ion source was electrospray ionization source (ESI);

[0805] The temperature of the drying gas (N2) was 500° C.;

[0806] The electrospray voltage was 5500V;

[0807] The detection method was positive ion detection;

[0808] The scanning mode was mode of reaction monitoring (MRM); the scan time was 0.1 is.

[0809] 6. Experimental Results:

TABLE-US-00009 TABLE 6 Results of the plasma protein binding rate assay of the compounds of the Examples Example Mouse Rat Dog Human No. % Unbound % Unbound % Unbound % Unbound 34 4.3 4.4 3.2 1.5 61-A 4.4 2.7 3.7 1.6 68 16.2 11.0 8.4 6.6 78 8.4 6.1 14.7 10.3 80 25.6 23.4 13.5 13.5

[0810] 7. Experimental Conclusion:

[0811] The above data show that the compounds of the Examples of the present invention have high plasma protein binding rate with little species variation.

Test Example 7. CYP Enzyme Single-Point Inhibition Assay

[0812] 1. Experimental Objective

[0813] The inhibition of the compounds on CYP450 enzyme isoformwas rapidly predicted by single-point method using human liver microsome incubation system.

[0814] 2. Experimental Procedure

[0815] 2.1 Solution Formulation

[0816] 2.5 mM NADPH: 4.165 mg of NADPH (reduced nicotinamide adenine dinucleotide phosphate) was weighed, followed by addition of 100 mM phosphate buffer to 2 mL. 0.25 mg/mL microsome solution: 4 mL of 100 mM phosphate buffer was added to 50 μL of 20 mg/mL microsome solution and mixed well.

[0817] Formulation of the reaction solution of the test compound:

[0818] The test compound of the Example was weighed, diluted to 10 mM with DMSO and then to 100 μM with 100 mM phosphate buffer.

[0819] 2.2 Experimental Procedure:

[0820] 1. 40 μL of liver microsome, 10 μL of substrate and 10 μL of the test compound were added to a 96-well plate and pre-incubated for 3 min.

[0821] 2. 40 μL of NADPH was added.

[0822] 3. 300 μL of acetonitrile stop solution containing internal standard was added at 20 min.

[0823] 4. The sample was centrifuged and injected.

[0824] 3. Experimental Results:

TABLE-US-00010 TABLE 7 Results of the CYP enzyme single-point inhibition assay of the compounds of the Examples IC.sub.50 (μM) Compound 1A2 2C9 2C19 2D6 3A4-M 3A4-T 34    24.9 >100 >100 >100 >100 >100 61-A    72.1 >100 >100 >100 >100 >100 68 >100   >100 >100 >100 >100 >100 78 >100   >100 >100 >100 >100 >100 Note: Strong inhibition: IC.sub.50 < 1 μM; moderate inhibition: 1 μM < IC.sub.50 < 10 μM; weak inhibition: IC.sub.50 > 10 μM

[0825] 4. Experimental Conclusion:

[0826] The above data show that the compounds of the Examples of the present invention have no strong inhibition on CYP enzyme isoforms, and the risk of DDI is low.

Test Example 8. hERG Potassium Channel Inhibition Activity Assay

[0827] 1. Cell Preparation

[0828] 7.1.1 CHO-hERG cells were cultured in a 175 cm.sup.2 culture flask. After the cell density reached 60-80%, the culture solution was removed. The cells were washed with 7 mL of PBS once, and dissociated with 3 mL of Detachin.

[0829] 7.1.2 After completion of dissociation, the cells were neutralized with 7 mL of culture solution. The solution was centrifuged, and the supernate was removed. The cells were resuspended in 5 mL of culture solution. The cell indensity is ensured as 2˜5×10.sup.6/mL.

[0830] 2. Solution Formulation

TABLE-US-00011 TABLES 8 Components of intracellular and extracellular fluids Reagents Extracellular fluid (mM) Intracellular fluid (mM) CaCl.sub.2 2 5.374 MgCl.sub.2 1 1.75 KCl 4 120 NaCl 145 — Glucose 10 — HEPES 10 10 EGTA — 5 Na-ATP — 4 pH 7.40 (adjusted with NaOH), 7.25 (adjusted with KOH), Osmolarity~305 mOsm Osmolarity~290 mOsm

[0831] 3. Electrophysiological Recording Process

[0832] Single cell sealing impedance and formation of whole-cell mode were automatically performed by Qpatch instrument. After obtaining the whole-cell recording mode, the cell was clamped at −80 mV. The cell first underwent pre-voltage of −50 mV for 50 msec, then underwent depolarization stimulation at +40 mV for 5 sec, and then underwent repolarization at −50 mV for 5 sec, and then the voltage returned to −80 mV The cell underwent the stimulation at the voltage every 15 sec. The data were recorded for 2 min, then extracellular fluid was administrated, and then the data were recorded for 5 min. Then, the administration process begun. The concentration of the test compound started from the lowest concentration, and each test concentration was administrated for 2.5 min. At least three cells (n≥3) were tested for each concentration.

[0833] 4. Compound Formulation

[0834] 4.1 20 mM mother liquor of the compound was diluted with extracellular fluid. 2495 L of extracellular fluid was added to 5 μL of 20 mM mother liquor of the compound to obtain a concentration of 40 μM (500-fold dilution). The solution was subjected to a 3-fold serial dilution with extracellular fluid containing 0.2% DMSO to obtain a required final concentration.

[0835] 4.2 The highest test concentration was 40 μM. The 6 concentrations were 40, 13.33, 4.44, 1.48, 0.49 and 0.16 μM.

[0836] 4.3 The DMSO content in the final test concentration did not exceed 0.2%. This concentration of DMSO had no effect on hERG potassium channel.

[0837] 5. Data Analysis

[0838] The experimental data was analyzed by XLFit software.

[0839] 6. Quality Control

[0840] Environment: humidity 20˜50%, temperature 22-25° C.

[0841] Reagents: the reagents used were purchased from Sigma, with a purity of >98%

[0842] The experimental data in the report must meet the following criteria:

[0843] Whole cell sealing impedance >100 MΩ

[0844] Tail current amplitude >400 pA

[0845] Pharmacological parameters:

[0846] The inhibition effect of Cisapride at multiple concentrations on hERG channel was used as the positive control.

[0847] 7. Experimental Results:

TABLE-US-00012 TABLE 9 Results of inhibition effect of the compounds of the Examples at multiple concentrations on hERG current Example No. hERG IC.sub.50 (uM) 61-A >10 68 18.38 78 >10 80 >20

[0848] 8. Experimental Conclusion:

[0849] Inhibition of cardiac hERG potassium channel by drug is the main cause of drug-induced QT prolongation syndrome. It can be seen from the experimental results that the compounds of the Examples of the present invention have no obvious inhibition effect on cardiac hERG potassium channel. Cardiotoxic effects at high doses can thus be avoided.

Test Example 9. Taste Sensitivity Assay in BALB/c Mice

[0850] 1. Experimental Objective:

[0851] In this assay, compounds with less toxic and side effects on animal taste were screened by quinine bitter solution experiment.

[0852] 2. Main experimental instruments and materials

[0853] 2.1 Instruments:

[0854] 1. Ultra-clean workbench (CJ-2F, Suzhou Fengshi Laboratory Animal Equipment Co., Ltd);

[0855] 2. Electronic balance (CPA2202D, Sartorius);

[0856] 3. Electronic balance (BSA2202S-CW, Sartorius);

[0857] 4. Pure water maker (Pacific TII, Thermo).

[0858] 2.2 Reagents:

[0859] Quinine monohydrochloride dihydrate (6119-47-7, Adamas).

[0860] 2.3 Animals:

[0861] BALB/c mice, 6 to 8 weeks old, ♂, purchased from Shanghai SIPPR-BK Laboratory Animal Co., Ltd.

[0862] 3. Experimental Procedure:

[0863] 3.1 Animal Screening

[0864] One day before the experiment, all BALB/c mice were weighed, and animals with too high or too low body weight were excluded.

[0865] 3.2 Grouping and Water Deprivation

[0866] BALB/c mice were randomly grouped according to body weight, and were deprived of water 12 to 16 hours before administration with no fasting.

[0867] 3.3 Formulation of Aqueous Solution of Quinine

[0868] An appropriate amount of quinine monohydrochloride dihydrate was weighed and formulated into an aqueous solution quinine hydrochloride (concentration: 3 mmol/L) with ultrapure water for later use.

[0869] 3.4 Formulation of Test Compound

[0870] An appropriate amount of the test compound was weighed and formulated into the target concentration with the corresponding solvent according to the experimental design for later use.

[0871] 3.5 Administration and Quinine Solution Intake Assay in Animal

[0872] Administration and fasting: On the day of the experiment, the animals were weighed and fasted, bedding was changed, and the compounds were administered according to the experimental design.

[0873] Quinine Solution Intake Assay:

[0874] 1. The corresponding clean mouse drinking bottle was rinsed 2 to 3 times with ultrapure water and the formulated 3 mmol/L aqueous solution of quinine hydrochloriderespectively. The bottle was filled and weighed, and the weight was recorded as Wi.sub.0.

[0875] 2. According to the experimental design, a certain period after administration, the filled bottle was gently placed in the corresponding mouse cage, and the timing was started. After 30 min, the bottle was gently taken out and weighed, and the weight was recorded as Wi.sub.30.

[0876] 3. Calculation of solution consumption of animals in each group: ΔWW(g)=Wi.sub.30−Wi.sub.0; calculation of solution consumption of single mouse: ΔpWW(g)=ΔWW/N, N is the number of animals in each group.

[0877] 4. Dysgeusia rate=(ΔpWW of the group in which the drinking water was the aqueous solution of quinine hydrochlorideand the test compound was administered at the same time −ΔpWW of the group in which the drinking water was aqueous solution of quinine hydrochlorideand the solvent control was administered at the same time)/(ΔpWW of the group in which the drinking water was ultrapure water and the solvent control was administered at the same time −ΔpWW of the group in which the drinking water was aqueous solution of quinine hydrochloride and the solvent control was administered at the same time)×100%. Data processing was performed with software such as Excel.

[0878] 5. The animals were euthanized after completion of the experiment.

[0879] 4. Experimental Results:

TABLE-US-00013 TABLE 10 Results of the taste sensitivity assay of the compounds of the Examples Solution consumption of single mouse (g) Ultrapure Quinine Dysgeusia Compound water solution rate Solvent group 0.598 / / (20% HP-B-CD) Solvent group / 0.068 / (20% HP-B-CD) 61-A @30 mpk / 0.048 −3.77% 68 @30 mpk / 0.056 −2.26% 78 @30 mpk / 0.016 −9.81%

[0880] 5. Experimental Conclusion:

[0881] It can be seen from the above results that the compounds of the present application have low toxic and side effects on the taste of mice.

Test Example 10. Pharmacodynamic Study on Citric Acid-Induced Acute Cough of Guinea Pigs

[0882] 1. Experimental Objective

[0883] The objective of this experiment is to evaluate the efficacy of the compounds o a citric acid-induced acute cough model of guinea pigs.

[0884] 2. Experimental Instruments and Reagents

[0885] 2.1 Key Instruments

TABLE-US-00014 Instrument name Manufacturer Model/Specification Device number WBP DSI Whole Body 100301, 100249 Plethysmography Electronic balance Changzhou Tianzhiping EL-2KL 6072710 Instrument Equipment Co., Ltd. Ultrasonic cell Ningbo Scientz SCIENTZ-IID 10192149 poulverizer Biotechnology Co., Ltd. Electronic balance Mettler Toledo MS205DL B844687071 Pipette Eppendorf 5 mL I19578I Pipette Eppendorf 1000 μL Q12774H Pipette Eppendorf 200 μL L33188I Pipette Eppendorf 100 μL R12555H

[0886] 2.2 Key Reagents

TABLE-US-00015 Reagent name Manufacturer Article number Sodium carboxymethyl Sigma C5678 cellulose Tween 80 Sigma P4780 ATP Sigma A2383 Citric acid Sigma C2404

[0887] 3. Experimental Operation and Data Processing:

[0888] 3.1 Animals

[0889] Hartley Guinea Pigs, male, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0890] 3.2 Experimental Procedure

[0891] The animals were adaptively feed. After their body weight reached the standard (300 to 400 g), the animals were serially numbered and randomly grouped according to their body weight.

[0892] Cough induction method: The guinea pig was put into the whole body plethysmography box to adapt for 3-5 minutes. ATP atomization was performed for 2 minutes. After an interval of 3 minutes, citric acid atomization was performed for 5 minutes. From the beginning of citric acid atomization, the number of coughs and the cough latency of the animals were recorded within 10 min.

[0893] 3.3 Administration Regimen and Monitoring of Cough Indicators

[0894] The test compound was administered to the guinea pig by a single gavage 2 hours before citric acid atomization. The guinea pig was put into the respiratory plethysmography chamber of the DSI Buxco whole body plethysmography (WBP) at the predetermined time, and subjected to cough induction by citric acid atomization. From the beginning of citric acid atomization, the total number of coughs (CCnt) and cough latency (CIP) in the guinea pig within 10 minutes were recorded by the WBP system.

[0895] 3.4 Data Processing

[0896] All data were entered into Excel files and expressed as mean±standard error. The data of each group were analyzed and compared by one-way ANOVA. If the statistical analysis results showed p<0.05, then there was a significant difference. Pairwise comparisons were carried out by t-test method to compare the differences.

[0897] The results show that the compounds of the Examples of the present invention can effectively improve cough symptoms in the citric acid-induced acute cough model of guinea pigs, and the reduction rate of the total number of coughs is over 59%.