Substituted morpholines as ATR kinase inhibitors

Abstract

Disclosed is a pyrazolo-heteroaryl derivative, a preparation method therefor, and medical use thereof. In particular, the present invention relates to a pyrazolo-heteroaryl derivative as shown in the general formula (I), a preparation method therefor, a pharmaceutical composition containing the derivative, and a use thereof as a therapeutic agent, particularly as ATR kinase inhibitor and in the preparation of drugs for the treatment and/or prevention of hyperproliferative diseases. The definition of each group in the general formula (I) is identical as in the specification. ##STR00001##

Claims

1. A compound of formula (I): ##STR00099## or a pharmaceutically acceptable salt or tautomer thereof, wherein: ##STR00100## R.sup.1 is alkyl or cyclopropyl, wherein the alkyl or cyclopropyl is substituted with one or more CN substituents; R.sup.2 is H or alkyl, wherein the alkyl is optionally substituted with one or more substituents independently selected from the group consisting of halogen, CN, NO.sub.2, NH.sub.2, OH, O(alkyl), cycloalkyl, heterocyclyl, aryl, and heteroaryl; ring A is pyrazolyl; each R.sup.3 is independently H, halogen, CN, alkyl, alkenyl, NH.sub.2, OH, O(alkyl), O(haloalkyl), cycloalkyl, heterocyclyl, aryl, or heteroaryl; wherein each alkyl and O(alkyl) is optionally and independently substituted with one or more substituents independently selected from the group consisting of halogen, CN, NO.sub.2, NH.sub.2, OH, O(alkyl), cycloalkyl, heterocyclyl, aryl, and heteroaryl; and wherein each cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally and independently substituted with one or more substituents independently selected from the group consisting of halogen, CN, NO.sub.2, alkyl, haloalkyl, hydroxyalkyl, NH.sub.2, OH, O(alkyl), cycloalkyl, heterocyclyl, aryl, and heteroaryl; and n is 0, 1, 2, or 3.

2. The compound according to claim 1, or a pharmaceutically acceptable salt or tautomer thereof, wherein R.sup.1 is C(CH.sub.3).sub.2CN or 1-cyanocyclopropyl.

3. The compound according to claim 1, or a pharmaceutically acceptable salt or tautomer thereof, wherein R.sup.2 is H, CH.sub.3, or CH.sub.2CH.sub.3.

4. The compound according to claim 1, wherein the compound is of formula (II): ##STR00101## or a pharmaceutically acceptable salt or tautomer thereof.

5. The compound according to claim 1, or a pharmaceutically acceptable salt or tautomer thereof, wherein each R.sup.3 is independently H.

6. The compound according to claim 1, wherein the compound is selected from the group consisting of: ##STR00102## ##STR00103## or a pharmaceutically acceptable salt or tautomer thereof.

7. The compound according to claim 6, wherein the compound is: ##STR00104## or a pharmaceutically acceptable salt or tautomer thereof.

8. The compound according to claim 6, wherein the compound is: ##STR00105## or a pharmaceutically acceptable salt or tautomer thereof.

9. A pharmaceutical composition comprising at least one pharmaceutically acceptable carrier, diluent, or excipient and the compound according to claim 1, or a pharmaceutically acceptable salt or tautomer thereof.

10. A method for inhibiting ataxia-telangiectasia and rad3-related (ATR) kinase activity in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of the pharmaceutical composition according to claim 9.

11. The method according to claim 10, wherein the subject has a cancer.

12. The method according to claim 11, wherein the cancer is selected from the group consisting of B-cell lymphoma, bladder cancer, bone cancer, a brain tumor, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, gallbladder cancer, gastric cancer, a head and neck tumor, kidney cancer, leukemia, liver cancer, lung cancer, a melanoma, multiple myeloma, neuroblastoma, neuroglioma, ovarian cancer, pancreatic cancer, prostate cancer, a sarcoma, skin cancer, and a thyroid tumor.

13. The method according to claim 10, wherein the subject has a hyperproliferative disease.

14. A process for preparing a compound of formula (I) according to claim 1: ##STR00106## wherein: ##STR00107## R.sup.1 is alkyl or cyclopropyl, wherein the alkyl or cyclopropyl is substituted with one or more CN substituents; R.sup.2 is H or alkyl, wherein the alkyl is optionally substituted with one or more substituents independently selected from the group consisting of halogen, CN, NO.sub.2, NH.sub.2, OH, O(alkyl), cycloalkyl, heterocyclyl, aryl, and heteroaryl; ring A is pyrazolyl; each R.sup.3 is independently H, halogen, CN, alkyl, alkenyl, NH.sub.2, OH, O(alkyl), O(haloalkyl), cycloalkyl, heterocyclyl, aryl, or heteroaryl; wherein each alkyl and O(alkyl) is optionally and independently substituted with one or more substituents independently selected from the group consisting of halogen, CN, NO.sub.2, NH.sub.2, OH, O(alkyl), cycloalkyl, heterocyclyl, aryl, and heteroaryl; and wherein each cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally and independently substituted with one or more substituents independently selected from the group consisting of halogen, CN, NO.sub.2, alkyl, haloalkyl, hydroxyalkyl, NH.sub.2, OH, O(alkyl), cycloalkyl, heterocyclyl, aryl, and heteroaryl; and n is 0, 1, 2, or 3; wherein the process comprises the following step: reacting a compound of formula (IA): ##STR00108## wherein: ##STR00109## R.sup.1 is alkyl or cyclopropyl, wherein the alkyl or cyclopropyl is substituted with one or more CN substituents; R.sup.2 is H or alkyl, wherein the alkyl is optionally substituted with one or more substituents independently selected from the group consisting of halogen, CN, NO.sub.2, NH.sub.2, OH, O(alkyl), cycloalkyl, heterocyclyl, aryl, and heteroaryl; and X is halogen; with a compound of formula (IB): ##STR00110## wherein: ring A is pyrazolyl; ##STR00111## each R.sup.3 is independently H, halogen, CN, alkyl, alkenyl, NH.sub.2, OH, O(alkyl), O(haloalkyl), cycloalkyl, heterocyclyl, aryl, or heteroaryl; wherein each alkyl and O(alkyl) is optionally and independently substituted with one or more substituents independently selected from the group consisting of halogen, CN, NO.sub.2, NH.sub.2, OH, O(alkyl), cycloalkyl, heterocyclyl, aryl, and heteroaryl; and wherein each cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally and independently substituted with one or more substituents independently selected from the group consisting of halogen, CN, NO.sub.2, alkyl, haloalkyl, hydroxyalkyl, NH.sub.2, OH, O(alkyl), cycloalkyl, heterocyclyl, aryl, and heteroaryl; and n is 0, 1, 2, or 3; to obtain the compound of formula (I) above.

15. A process for preparing a compound of formula (II) according to claim 4: ##STR00112## wherein: ##STR00113## R.sup.1 is alkyl or cyclopropyl, wherein the alkyl or cyclopropyl is substituted with one or more CN substituents; R.sup.2 is H or alkyl, wherein the alkyl is optionally substituted with one or more substituents independently selected from the group consisting of halogen, CN, NO.sub.2, NH.sub.2, OH, O(alkyl), cycloalkyl, heterocyclyl, aryl, and heteroaryl; each R.sup.3 is independently H, halogen, CN, alkyl, alkenyl, NH.sub.2, OH, O(alkyl), O(haloalkyl), cycloalkyl, heterocyclyl, aryl, or heteroaryl; wherein each alkyl and O(alkyl) is optionally and independently substituted with one or more substituents independently selected from the group consisting of halogen, CN, NO.sub.2, NH.sub.2, OH, O(alkyl), cycloalkyl, heterocyclyl, aryl, and heteroaryl; and wherein each cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally and independently substituted with one or more substituents independently selected from the group consisting of halogen, CN, NO.sub.2, alkyl, haloalkyl, hydroxyalkyl, NH.sub.2, OH, O(alkyl), cycloalkyl, heterocyclyl, aryl, and heteroaryl; and n is 0, 1, 2, or 3; wherein the process comprises the following step: deprotecting a compound of formula (IIA) or formula (IIGA): ##STR00114## or a pharmaceutically acceptable salt or tautomer thereof, wherein: ##STR00115## R.sup.1 is alkyl or cyclopropyl, wherein the alkyl or cyclopropyl is substituted with one or more CN substituents; R.sup.2 is H or alkyl, wherein the alkyl is optionally substituted with one or more substituents independently selected from the group consisting of halogen, CN, NO.sub.2, NH.sub.2, OH, O(alkyl), cycloalkyl, heterocyclyl, aryl, and heteroaryl; R.sup.a is CH.sub.2CHCH.sub.2, CH.sub.2-phenyl, CH.sub.2-(p-methoxyphenyl), C(O)CH.sub.3, C(O)OC(CH.sub.3).sub.3, or tetrahydropyranyl (THP); each R.sup.3 is independently H, halogen, CN, alkyl, alkenyl, NH.sub.2, OH, O(alkyl), O(haloalkyl), cycloalkyl, heterocyclyl, aryl, or heteroaryl; wherein each alkyl and O(alkyl) is optionally and independently substituted with one or more substituents independently selected from the group consisting of halogen, CN, NO.sub.2, NH.sub.2, OH, O(alkyl), cycloalkyl, heterocyclyl, aryl, and heteroaryl; and wherein each cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally and independently substituted with one or more substituents independently selected from the group consisting of halogen, CN, NO.sub.2, alkyl, haloalkyl, hydroxyalkyl, NH.sub.2, OH, O(alkyl), cycloalkyl, heterocyclyl, aryl, and heteroaryl; and n is 0, 1, or 2; with an acid selected from the group consisting of acetic acid, formic acid, hydrochloric acid, methanesulfonic acid, nitric acid, phosphoric acid, sulfuric acid, p-toluenesulfonic acid, trifluoroacetic acid, (CH.sub.3).sub.3SiCl, and (CH.sub.3).sub.3SiOTf, to obtain the compound of formula (II) above.

16. The process according to claim 15, wherein R.sup.a is tetrahydropyranyl (THP).

17. A compound of formula (IIA) or formula (IIGA): ##STR00116## or a pharmaceutically acceptable salt or tautomer thereof, wherein: ##STR00117## R.sup.1 is alkyl or cyclopropyl, wherein the alkyl or cyclopropyl is substituted with one or more CN substituents; R.sup.2 is H or alkyl, wherein the alkyl is optionally substituted with one or more substituents independently selected from the group consisting of halogen, CN, NO.sub.2, NH.sub.2, OH, O(alkyl), cycloalkyl, heterocyclyl, aryl, and heteroaryl; R.sup.a is CH.sub.2CHCH.sub.2, CH.sub.2-phenyl, CH.sub.2-(p-methoxyphenyl), C(O)CH.sub.3, C(O)OC(CH.sub.3).sub.3, or tetrahydropyranyl (THP); each R.sup.3 is independently H, halogen, CN, alkyl, alkenyl, NH.sub.2, OH, O(alkyl), O(haloalkyl), cycloalkyl, heterocyclyl, aryl, or heteroaryl; wherein each alkyl and O(alkyl) is optionally and independently substituted with one or more substituents independently selected from the group consisting of halogen, CN, NO.sub.2, NH.sub.2, OH, O(alkyl), cycloalkyl, heterocyclyl, aryl, and heteroaryl; and wherein each cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally and independently substituted with one or more substituents independently selected from the group consisting of halogen, CN, NO.sub.2, alkyl, haloalkyl, hydroxyalkyl, NH.sub.2, OH, O(alkyl), cycloalkyl, heterocyclyl, aryl, and heteroaryl; and n is 0, 1, or 2.

18. The compound according to claim 17, wherein the compound is selected from the group consisting of: ##STR00118## or a pharmaceutically acceptable salt or tautomer thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) Unless otherwise stated, the terms used in the specification and claims have the following meanings.

(2) The term alkyl refers to a saturated aliphatic hydrocarbon group which is a linear or branched group containing 1 to 20 carbon atoms, preferably an alkyl containing 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) carbon atoms, and more preferably an alkyl containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various side-chain isomers thereof, etc. More preferred is a lower alkyl having 1 to 6 carbon atoms, and non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl and the like. The alkyl may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available connection site with one or more substituents preferably independently optionally selected from the group consisting of H, D, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl.

(3) The term alkylene refers to a saturated linear or branched aliphatic hydrocarbon group having 2 residues derived from the parent alkane by removal of two hydrogen atoms from the same carbon atom or two different carbon atoms, which is a linear or branched group containing 1 to 20 carbon atoms, preferably an alkylene group containing 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) carbon atoms, and more preferably an alkylene group containing 1 to 6 carbon atoms. Non-limiting examples of alkylene include, but are not limited to, methylene (CH.sub.2), 1,1-ethylene (CH(CH.sub.3)), 1,2-ethylene (CH.sub.2CH.sub.2), 1,1-propylene (CH(CH.sub.2CH.sub.3)), 1,2-propylene (CH.sub.2CH(CH.sub.3)), 1,3-propylene (CH.sub.2CH.sub.2CH.sub.2), 1,4-butylene (CH.sub.2CH.sub.2CH.sub.2CH.sub.2) and the like. The alkylene may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available connection site with one or more substituents preferably independently optionally selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo.

(4) The term alkenyl refers to an alkyl compound containing at least one carbon-carbon double bond in the molecule, wherein the alkyl is as defined above. The alkenyl may be substituted or unsubstituted. When substituted, the alkenyl may be substituted with one or more substituents preferably independently selected from the group consisting of hydrogen, alkyl, alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl.

(5) The term alkynyl refers to an alkyl compound containing at least one carbon-carbon triple bond in the molecule, wherein the alkyl is as defined above. The alkynyl may be substituted or unsubstituted. When substituted, the alkynyl may be substituted with one or more substituents preferably independently selected from the group consisting of hydrogen, alkyl, alkoxy, halogen, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl.

(6) The term cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent. The cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 8 (e.g., 3, 4, 5, 6, 7 or 8) carbon atoms, and most preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like. Polycyclic cycloalkyl includes spiro cycloalkyl, fused cycloalkyl, and bridged cycloalkyl.

(7) The term spiro cycloalkyl refers to a 5- to 20-membered polycyclic group in which monocyclic rings share one carbon atom (referred to as the spiro atom), wherein the spiro cycloalkyl may contain one or more double bonds. Preferably, the spiro cycloalkyl is 6- to 14-membered, and more preferably 7- to 10-membered (e.g., 7, 8, 9 or 10-membered). According to the number of the spiro atoms shared among the rings, the spiro cycloalkyl may be monospiro cycloalkyl, bispiro cycloalkyl or polyspiro cycloalkyl, preferably monospiro cycloalkyl and bispiro cycloalkyl, more preferably 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered monospiro cycloalkyl. Non-limiting examples of spiro cycloalkyl include:

(8) ##STR00053##

(9) The term fused cycloalkyl refers to a 5- to 20-membered carbon polycyclic group in which each ring shares a pair of adjacent carbon atoms with the other rings in the system, wherein one or more of the rings may contain one or more double bonds. Preferably, the fused cycloalkyl is 6- to 14-membered, and more preferably 7- to 10-membered (e.g., 7, 8, 9 or 10-membered). According to the number of the formed rings, the fused cycloalkyl may be bicyclic, tricyclic, tetracyclic or polycyclic cycloalkyl, preferably bicyclic or tricyclic cycloalkyl, and more preferably 3-membered/4-membered, 3-membered/5-membered, 3-membered/6-membered, 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/4-membered, 5-membered/5-membered, 5-membered/6-membered, 6-membered/3-membered, 6-membered/4-membered, 6-membered/5-membered and 6-membered/6-membered bicyclic cycloalkyl. Non-limiting examples of fused cycloalkyl include:

(10) ##STR00054##

(11) The term bridged cycloalkyl refers to a 5- to 20-membered carbon polycyclic group in which any two rings share two carbon atoms that are not directly connected to each other, wherein the bridged cycloalkyl may contain one or more double bonds. Preferably, the bridged cycloalkyl is 6- to 14-membered, and more preferably 7- to 10-membered (e.g., 7, 8, 9 or 10-membered). According to the number of the formed rings, the bridged cycloalkyl may be bicyclic, tricyclic, tetracyclic or polycyclic, preferably bicyclic, tricyclic or tetracyclic, and more preferably bicyclic or tricyclic. Non-limiting examples of bridged cycloalkyl include:

(12) ##STR00055##

(13) The cycloalkyl ring includes those in which the cycloalkyl described above (including monocyclic, spiro, fused and bridged rings) is fused to an aryl, heteroaryl or heterocycloalkyl ring, wherein the ring connected to the parent structure is cycloalkyl. Non-limiting examples include indanyl, tetrahydronaphthyl, benzocyclopentyl, benzocycloheptanyl, and the like, preferably benzocyclopentyl and tetrahydronaphthyl.

(14) The cycloalkyl may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available connection site with one or more substituents preferably independently optionally selected from the group consisting of hydrogen, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl.

(15) The term alkoxy refers to O-(alkyl) and O-(unsubstituted cycloalkyl), wherein the alkyl is as defined above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy. The alkoxy may be substituted or unsubstituted. When substituted, the alkoxy may be substituted with one or more substituents preferably independently selected from the group consisting of H, D, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl. The term heterocyclyl refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, wherein one or more of the ring atoms are heteroatoms selected from the group consisting of nitrogen, oxygen, S, S(O) and S(O).sub.2, excluding a cyclic portion of OO, OS or SS, and the remaining ring atoms are carbon. The heterocyclyl preferably contains 3 to 12 ring atoms, wherein 1 to 4 (e.g., 1, 2, 3 and 4) are heteroatoms; more preferably, contains 3 to 8 (e.g., 3, 4, 5, 6, 7 or 8) ring atoms, wherein 1 to 3 (e.g., 1, 2 or 3) are heteroatoms; even more preferably, contains 3 to 6 ring atoms, wherein 1 to 3 are heteroatoms; and most preferably, contains 5 or 6 ring atoms, wherein 1 to 3 are heteroatoms. Non-limiting examples of monocyclic heterocyclyl include pyrrolidinyl, tetrahydropyranyl, 1,2,3,6-tetrahydropyridinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Polycyclic heterocyclyl includes spiro heterocyclyl, fused heterocyclyl, and bridged heterocyclyl.

(16) The term spiro heterocyclyl refers to a 5- to 20-membered polycyclic heterocyclyl group in which monocyclic rings share one atom (referred to as the spiro atom), wherein one or more ring atoms are heteroatoms selected from the group consisting of nitrogen, oxygen, S, S(O) and S(O).sub.2, and the remaining ring atoms are carbon atoms. The spiro heterocyclyl may contain one or more double bonds. Preferably, the spiro heterocyclyl is 6- to 14-membered, and more preferably 7- to 10-membered. According to the number of spiro atoms shared among the rings, the spiro heterocyclyl may be monospiro heterocyclyl, bispiro heterocyclyl or polyspiro heterocyclyl, preferably monospiro heterocyclyl and bispiro heterocyclyl, and more preferably 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered monospiro heterocyclyl. Non-limiting examples of spiro heterocyclyl include:

(17) ##STR00056##

(18) The term fused heterocyclyl refers to a 5- to 20-membered polycyclic heterocyclyl group in which each ring shares a pair of adjacent atoms with the other rings in the system, wherein one or more of the rings may contain one or more double bonds. In the fused heterocyclyl, one or more of the ring atoms are heteroatoms selected from the group consisting of nitrogen, oxygen, S, S(O) and S(O).sub.2, and the remaining ring atoms are carbon atoms. Preferably, the fused heterocyclyl is 6- to 14-membered, and more preferably 7- to 10-membered (e.g., 7, 8, 9 or 10-membered). According to the number of the formed rings, the fused heterocyclyl may be bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclyl, preferably bicyclic or tricyclic fused heterocyclyl, and more preferably 3-membered/4-membered, 3-membered/5-membered, 3-membered/6-membered, 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered, 5-membered/6-membered, 6-membered/3-membered, 6-membered/4-membered, 6-membered/5-membered and 5-membered/4-membered, 6-membered/6-membered bicyclic fused heterocyclyl. Non-limiting examples of fused heterocyclyl include:

(19) ##STR00057##

(20) The term bridged heterocyclyl refers to a 5- to 14-membered polycyclic heterocyclyl group in which any two rings share two carbon atoms that are not directly connected to each other, wherein the bridged heterocyclyl may contain one or more double bonds. In the bridged heterocyclyl, one or more of the ring atoms are heteroatoms selected from the group consisting of nitrogen, oxygen, S, S(O) and S(O).sub.2, and the remaining ring atoms are carbon atoms. Preferably, the bridged heterocyclyl is 6- to 14-membered, and more preferably 7- to 10-membered (e.g., 7, 8, 9 or 10-membered). According to the number of the formed rings, the bridged heterocyclyl may be bicyclic, tricyclic, tetracyclic or polycyclic, preferably bicyclic, tricyclic or tetracyclic, and more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclyl include:

(21) ##STR00058##

(22) The heterocyclyl ring includes those in which the heterocyclyl described above (including monocyclic, spiro heterocyclic, fused heterocyclic and bridged heterocyclic rings) is fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring connected to the parent structure is heterocyclyl. Non-limiting examples include:

(23) ##STR00059##

(24) The heterocyclyl may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available connection site with one or more substituents preferably independently optionally selected from the group consisting of hydrogen, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl.

(25) The term aryl refers to a 6- to 14-membered, preferably 6- to 10-membered carbon monocyclic or fused polycyclic (fused polycyclic rings are those sharing a pair of adjacent carbon atoms) group having a conjugated -electron system such as phenyl and naphthyl. The aryl ring includes those in which the aryl ring described above is fused to a heteroaryl, heterocyclyl or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring. Non-limiting examples include:

(26) ##STR00060##

(27) The aryl may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available connection site with one or more substituents preferably independently optionally selected from the group consisting of hydrogen, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl.

(28) The term heteroaryl refers to a heteroaromatic system containing 1 to 4 (e.g., 1, 2, 3 and 4) heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from the group consisting of oxygen, sulfur and nitrogen. The heteroaryl is preferably 5- to 10-membered (e.g., 5, 6, 7, 8, 9 and 10) and more preferably 5- or 6-membered, e.g., furanyl, thienyl, pyridinyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, pyrazolyl, triazolyl, and tetrazolyl. The heteroaryl ring includes those in which the heteroaryl ring described above is fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring. Non-limiting examples include:

(29) ##STR00061##

(30) The heteroaryl may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available connection site with one or more substituents preferably independently optionally selected from the group consisting of hydrogen, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl.

(31) The cycloalkyl, heterocyclyl, aryl and heteroaryl described above have 1 residue derived from the parent ring by removal of one hydrogen atom from a ring atom, or 2 residues derived from the parent ring by removal of two hydrogen atoms from the same ring atom or two different ring atoms, i.e., divalent cycloalkyl, divalent heterocyclyl, arylene, or heteroarylene.

(32) The term amino protecting group refers to a group that can be easily removed and is intended to protect an amino group from being changed when a reaction is conducted elsewhere in the molecule. Non-limiting examples include tetrahydropyranyl, tert-butoxycarbonyl, acetyl, benzyl, allyl, p-methoxybenzyl, and the like. These groups may be optionally substituted with 1 to 3 substituents selected from the group consisting of halogen, alkoxy and nitro. The amino protecting group is preferably tetrahydropyranyl.

(33) The term cycloalkyloxy refers to cycloalkyl-O, wherein the cycloalkyl is as defined above.

(34) The term haloalkyl refers to an alkyl group substituted with one or more halogens, wherein the alkyl group is as defined above.

(35) The term deuterated alkyl refers to an alkyl group substituted with one or more deuterium atoms, wherein the alkyl group is as defined above.

(36) The term hydroxy refers to OH group.

(37) The term hydroxyalkyl refers to an alkyl group substituted with hydroxy, wherein the alkyl group is as defined above.

(38) The term halogen refers to fluorine, chlorine, bromine or iodine.

(39) The term amino refers to NH.sub.2.

(40) The term cyano refers to CN.

(41) The term nitro refers to NO.sub.2.

(42) The term carbonyl refers to CO.

(43) The term carboxy refers to C(O)OH.

(44) The term carboxylate refers to C(O)O(alkyl) or C(O)O(cycloalkyl), wherein the alkyl and cycloalkyl are as defined above.

(45) THP refers to tetrahydropyranyl.

(46) The compounds disclosed herein may be present as tautomers. For the purposes of the present disclosure, reference to a compound of formula (I) refers to the compound itself, or any one tautomer thereof itself, or a mixture of two or more tautomers. For example, reference to pyrazolyl is understood to include any one of the following two structures or a mixture of the two tautomers,

(47) ##STR00062##

(48) The compounds disclosed herein include isotopic derivatives thereof. The term isotopic derivative refers to compounds that differ in structure only by having one or more enriched isotopic atoms. For example, compounds having the structure disclosed herein having deuterium or tritium in place of hydrogen, or .sup.18F-fluorine labeling (.sup.18F isotope) in place of fluorine, or .sup.11C-, .sup.13C- or .sup.14C-enriched carbon (.sup.11C-, .sup.13C- or .sup.14C-carbon labeling; .sup.11C-, .sup.13C- or .sup.14C-isotope) in place of a carbon atom are within the scope of the present disclosure. Such a compound can be used as an analytical tool or a probe in, for example, a biological assay, or may be used as a tracer for in vivo diagnostic imaging of disease, or as a tracer in a pharmacodynamic, pharmacokinetic or receptor study. The present disclosure encompasses various deuterated forms of the compounds of formula (I). Each available hydrogen atom connected to a carbon atom may be independently replaced with a deuterium atom. Those skilled in the art are able to synthesize the compounds of formula (I) in deuterated form with reference to the relevant literature. Commercially available deuterated starting materials can be used in preparing the deuterated forms of the compounds of formula (I), or they can be synthesized using conventional techniques with deuterated reagents including, but not limited to, deuterated borane, tri-deuterated borane in tetrahydrofuran, deuterated lithium aluminum hydride, deuterated iodoethane, deuterated iodomethane, and the like.

(49) The term optional or optionally means that the event or circumstance subsequently described may, but not necessarily, occur, and that the description includes instances where the event or circumstance occurs or does not occur. For example, a heterocyclyl group optionally substituted with alkyl means that alkyl may be, but not necessarily, present, and that the description includes instances where the heterocyclyl group is or is not substituted with alkyl.

(50) The term substituted means that one or more, preferably up to 5, more preferably 1 to 3 hydrogen atoms in the group are independently substituted with a corresponding number of substituents. It goes without saying that a substituent is only in its possible chemical position, and those skilled in the art will be able to determine (experimentally or theoretically) possible or impossible substitution without undue efforts. For example, it may be unstable when an amino or hydroxy group having a free hydrogen is bound to a carbon atom having an unsaturated (e.g., olefinic) bond.

(51) The term pharmaceutical composition refers to a mixture containing one or more of the compounds described herein or a physiologically/pharmaceutically acceptable salt or pro-drug thereof, and other chemical components, for example physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to an organism, which facilitates the absorption of the active ingredient, thereby exerting biological activities.

(52) The term pharmaceutically acceptable salt refers to salts of the disclosed compounds which are safe and effective for use in the body of a mammal and possess the requisite biological activities.

(53) For drugs and pharmacological active agents, the term therapeutically effective amount refers to an amount of a medicament or an agent that is sufficient to provide the desired effect but is non-toxic. The determination of the effective amount varies from person to person. It depends on the age and general condition of a subject, as well as the particular active substance used. The appropriate effective amount in a case may be determined by those skilled in the art in the light of routine tests.

(54) Synthesis of Compounds Disclosed Herein

(55) In order to achieve the purpose of the present disclosure, the following technical schemes are adopted in the present disclosure:

(56) ##STR00063##

(57) A method for preparing the compound of formula (I) or the salt thereof disclosed herein comprises the following steps: subjecting a compound of formula (LA) and a compound of formula (IB) to a coupling reaction in an alkaline condition in the presence of a catalyst to obtain the compound of formula (I), wherein: X is halogen, preferably Br; R.sup.b is

(58) ##STR00064## and ring A, G.sup.1, G.sup.2, R.sup.1, R.sup.2, R.sup.3 and n are as defined in formula (I).

(59) ##STR00065##

(60) A method for preparing the compound of formula (II) or the salt thereof disclosed herein comprises the following steps: subjecting a compound of formula (IA) and a compound of formula (IID) to a coupling reaction in an alkaline condition in the presence of a catalyst to obtain the compound of formula (II), wherein: X is halogen, preferably Br; R.sup.b is

(61) ##STR00066## and G.sup.1, G.sup.2, R.sup.1, R.sup.2, R.sup.3 and n are as defined in formula (II).

(62) ##STR00067##

(63) A method for preparing the compound of formula (II) or the salt thereof disclosed herein comprises the following steps: subjecting a compound of formula (IA) and a compound of formula (IIB) to a coupling reaction in an alkaline condition in the presence of a catalyst to obtain a compound of formula (IIA); and removing an amino protecting group from the compound of formula (IIA) in an acidic condition to obtain the compound of formula (II), wherein: X is halogen, preferably Br; R.sup.b is

(64) ##STR00068## and G.sup.1, G.sup.2, R.sup.1, R.sup.2, R.sup.3 and n are as defined in formula (II).

(65) ##STR00069##

(66) A method for preparing the compound of formula (II) or the salt thereof disclosed herein comprises the following steps: subjecting a compound of formula (IA) and a compound of formula (IIGB) to a coupling reaction in an alkaline condition in the presence of a catalyst to obtain a compound of formula (IIGA); and removing an amino protecting group from the compound of formula (IIGA) in an acidic condition to obtain the compound of formula (II), wherein: X is halogen, preferably Br; R.sup.b is

(67) ##STR00070## and G.sup.1, G.sup.2, R.sup.1, R.sup.2, R.sup.3 and n are as defined in formula (II).

(68) ##STR00071##

(69) A method for preparing the compound of formula (III) or the salt thereof disclosed herein comprises the following steps: subjecting a compound of formula (IIIC) and a compound of formula (IID) to a coupling reaction in an alkaline condition in the presence of a catalyst to obtain the compound of formula (III), wherein: X is halogen, preferably Br; R.sup.b is

(70) ##STR00072## and R.sup.1, R.sup.2, R.sup.3 and n are as defined in formula (III).

(71) ##STR00073##

(72) A method for preparing the compound of formula (III) or the salt thereof disclosed herein comprises the following steps: subjecting a compound of formula (IIIC) and a compound of formula (IIB) to a coupling reaction in an alkaline condition in the presence of a catalyst to obtain a compound of formula (IIIA); and removing an amino protecting group from the compound of formula (IIIA) in an acidic condition to obtain the compound of formula (III), wherein: X is halogen, preferably Br; R.sup.a is the amino protecting group; R.sup.b is

(73) ##STR00074## and R.sup.1, R.sup.2, R.sup.3 and n are as defined in formula (III).

(74) ##STR00075##

(75) A method for preparing the compound of formula (III) or the salt thereof disclosed herein comprises the following steps: subjecting a compound of formula (IIIC) and a compound of formula (IIGB) to a coupling reaction in an alkaline condition in the presence of a catalyst to obtain a compound of formula (IIIGA); and removing an amino protecting group from the compound of formula (IIIGA) in an acidic condition to obtain the compound of formula (III), wherein: X is halogen, preferably Br; R.sup.a is the amino protecting group; R.sup.b is

(76) ##STR00076## and R.sup.1, R.sup.2, R.sup.3 and n are as defined in formula (III).

(77) ##STR00077##

(78) A method for preparing the compound of formula (IV) or the salt thereof disclosed herein comprises the following steps: subjecting a compound of formula (IVC) and a compound of formula (IID) to a coupling reaction in an alkaline condition in the presence of a catalyst to obtain the compound of formula (IV), wherein: X is halogen, preferably Br; R.sup.b is

(79) ##STR00078## and R.sup.1, R.sup.2, R.sup.3 and n are as defined in formula (IV).

(80) ##STR00079##

(81) A method for preparing the compound of formula (IV) or the salt thereof disclosed herein comprises the following steps: subjecting a compound of formula (IVC) and a compound of formula (IIB) to a coupling reaction in an alkaline condition in the presence of a catalyst to obtain a compound of formula (IVA); and removing an amino protecting group from the compound of formula (IVA) in an acidic condition to obtain the compound of formula (IV), wherein: X is halogen, preferably Br; R.sup.a is the amino protecting group; R.sup.b is

(82) ##STR00080## and R.sup.1, R.sup.2, R.sup.3 and n are as defined in formula (IV).

(83) ##STR00081##

(84) A method for preparing the compound of formula (IV) or the salt thereof disclosed herein comprises the following steps: subjecting a compound of formula (IVC) and a compound of formula (IIGB) to a coupling reaction in an alkaline condition in the presence of a catalyst to obtain a compound of formula (IVGA); and removing an amino protecting group from the compound of formula (IVGA) in an acidic condition to obtain the compound of formula (IV), wherein: X is halogen, preferably Br; R.sup.a is the amino protecting group; R.sup.b is

(85) ##STR00082## and R.sup.1, R.sup.2, R.sup.3 and n are as defined in formula (IV).

(86) The reagents that provide alkaline conditions in the above synthesis schemes include organic bases including, but not limited to, triethylamine, N,N-diisopropylethylamine, n-butyllithium, lithium diisopropylamide, lithium bis(trimethylsilyl)amide, potassium acetate, sodium tert-butoxide, potassium tert-butoxide and sodium n-butoxide, and inorganic bases including, but not limited to, sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide and lithium hydroxide.

(87) The catalysts described in the above synthesis schemes include, but are not limited to, palladium on carbon, tetrakis(triphenylphosphine)palladium(0), palladium dichloride, palladium acetate, bis(triphenylphosphine)palladium(II) dichloride, bis(dibenzylideneacetone)palladium, chlorine (2-dicyclohexylphosphino-2,4,6-triisopropyl-1,1-biphenyl) [2-(2-amino-1,1-biphenyl)]palladium, [1,1-bis(diphenylphosphino)ferrocene]palladium(II) dichloride, [1,1-bis(dibenzylphosphino)ferrocene]palladium dichloride or tris(dibenzylideneacetone)dipalladium(0), preferably tetrakis(triphenylphosphine)palladium(0)) or bis(triphenylphosphine)palladium(II) dichloride.

(88) The reagents that provide acidic conditions in the above synthesis schemes include, but are not limited to, hydrogen chloride, hydrogen chloride in 1,4-dioxane, trifluoroacetic acid, formic acid, acetic acid, hydrochloric acid, sulfuric acid, methanesulfonic acid, nitric acid, phosphoric acid, p-toluenesulfonic acid, Me.sub.3SiCl, TMSOTf, preferably trifluoroacetic acid.

(89) The amino protecting groups in the above synthetic schemes include, but are not limited to, tetrahydropyranyl (THP), tert-butyloxycarbonyl, acetyl, benzyl, allyl and p-methoxybenzyl. These groups may be optionally substituted with 1-3 substituents selected from the group consisting of halogen, alkoxy and nitro. Tetrahydropyranyl is preferred.

(90) The above reactions are preferably conducted in a solvent including, but not limited to: ethylene glycol dimethyl ether, acetic acid, methanol, ethanol, n-butanol, toluene, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, n-hexane, dimethyl sulfoxide, 1,4-dioxane, water or N,N-dimethylformamide.

DETAILED DESCRIPTION

(91) The following examples further illustrate the present disclosure, but the present disclosure is not limited thereto.

EXAMPLES

(92) The structure of the compound was determined by nuclear magnetic resonance (NMR) spectroscopy and/or mass spectrometry (MS). NMR shift (8) is given in a unit of 10.sup.6 (ppm). NMR spectra were measured using a Bruker AVANCE-400 nuclear magnetic resonance instrument, with deuterated dimethyl sulfoxide (DMSO-d.sub.6), deuterated chloroform (CDCl.sub.3) and deuterated methanol (CD.sub.3OD) as determination solvents, with tetramethylsilane (TMS) as internal standard.

(93) Mass spectra were measured using Agilent 1200/1290 DAD-6110/6120 Quadrupole MS liquid chromatography-mass spectrometry system (manufacturer: Agilent; MS model: 6110/6120 Quadrupole MS), Waters ACQuity UPLC-QD/SQD (manufacturer: Waters, MS model: Waters ACQuity Qda Detector/waters SQ Detector) and THERMO Ultimate 3000-Q Exactive (manufacturer: THERMO, MS model: THERMO Q Exactive).

(94) High performance liquid chromatography (HPLC) was performed using Agilent HPLC 1200DAD, Agilent HPLC 1200VWD and Waters HPLC e2695-2489 high pressure liquid chromatography.

(95) Chiral HPLC was performed on Agilent 1260 DAD HPLC.

(96) HPLC preparation was performed using Waters 2545-2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP and Gilson GX-281 preparative chromatographs.

(97) Chiral preparation was performed on a Shimadzu LC-20AP preparative chromatograph.

(98) A CombiFlash Rf200 (TELEDYNE ISCO) system was used for rapid preparation.

(99) Huanghai HSGF254 or Qingdao GF254 silica gel plates of specifications 0.15 mm to 0.2 mm were adopted for thin layer chromatography (TLC) analysis and 0.4 mm to 0.5 mm for TLC separation and purification.

(100) The silica gel column chromatography generally used 200 to 300-mesh silica gel (Huanghai, Yantai) as the carrier.

(101) The mean inhibition of kinase and the IC.sub.50 value were measured using a NovoStar microplate reader (BMG, Germany).

(102) Known starting materials described herein may be synthesized using or according to methods known in the art, or may be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc., Chembee Chemicals, and other companies.

(103) In the examples, the reactions were performed in an argon atmosphere or a nitrogen atmosphere unless otherwise specified.

(104) An argon atmosphere or a nitrogen atmosphere means that the reaction flask is connected to a balloon containing about 1 L of argon or nitrogen.

(105) A hydrogen atmosphere means that the reaction flask is connected to a balloon containing about 1 L of hydrogen.

(106) Parr 3916EKX hydrogenator, Qinglan QL-500 hydrogenator or HC2-SS hydrogenator was used for pressurized hydrogenation reactions.

(107) The hydrogenation reaction usually involved 3 cycles of vacuumization and hydrogen purge.

(108) A CEM Discover-S 908860 microwave reactor was used for the microwave reaction.

(109) In the examples, a solution refers to an aqueous solution unless otherwise specified.

(110) In the examples, the reaction temperature was room temperature, i.e., 20 C. to 30 C., unless otherwise specified.

(111) The monitoring of the reaction progress in the examples was conducted by thin layer chromatography (TLC). The developing solvent for reactions, the eluent system for column chromatography purification and the developing solvent system for thin layer chromatography included: A: dichloromethane/methanol system, B: n-hexane/ethyl acetate system, and C: petroleum ether/ethyl acetate system. The volume ratio of the solvents was adjusted according to the polarity of the compound, or by adding a small amount of basic or acidic reagents such as triethylamine, and acetic acid.

(112) THP refers to tetrahydropyranyl.

Example 1

(R)-2-methyl-2-(1-methyl-5-(3-methylmorpholinyl)-3-(1H-pyrazol-3-yl)-1H-pyrazolo[4,3-b]pyridin-7-yl)propanenitrile 1

(113) ##STR00083## ##STR00084## ##STR00085##

Step 1

Methyl(R,E)-1-methyl-4-((1-(3-methylmorpholino)ethylidene)amino)-1H-pyrazole-5-carboxylate 1c

(114) Compound (R)-1-(3-methylmorpholinyl) ethan-1-one 1b (2.5 g, 17.7 mmol, prepared by the method disclosed on page 86 for intermediate-1 in the Example of the Patent Publication No. WO2016020320A1) was dissolved in 1,2-dichloroethane, and cooled in an ice/water bath in an argon atmosphere. Phosphorus oxychloride (7.4 g, 48.3 mmol) was slowly and dropwise added before the mixture was stirred at room temperature for 30 min. Compound methyl 4-amino-1-methyl-1H-pyrazole-5-carboxylate 1a (2.5 g, 16.1 mmol, Jiangsu Aikon) was added. The reaction system was heated to 80 C. and stirred for 2 h. The mixture was cooled to room temperature and concentrated at reduced pressure. The residue was diluted with dichloromethane (200 mL), and cooled in an ice/water bath. Saturated sodium bicarbonate solution was added dropwise to neutralize the dilution to pH 8 to 9. The organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was purified by silica gel column chromatography with eluent system C to obtain the target compound 1c (4.8 g, 94% yield).

(115) MS m/z (ESI): 281.2 [M+1].

Step 2

(R)-1-methyl-5-(3-methylmorpholinyl)-1H-pyrazolo[4,3-b]pyridin-7-ol 1d

(116) Compound 1c (2.6 g, 9.3 mmol) was dissolved in tetrahydrofuran (20 mL) and cooled in an ice/water bath. Lithium bis(trimethylsilyl)amide (27.8 mL, a 1 M solution in tetrahydrofuran, 27.8 mmol) was added slowly and the system was reacted at 0 C. for 1 h. The reaction was quenched by adding methanol (10 mL). The mixture was purified by silica gel column chromatography with eluent system A to obtain the target compound 1d (400 mg, 55.8% yield).

(117) MS m/z (ESI): 249.0 [M+1].

Step 3

(R)-4-(7-chloro-1-methyl-1H-pyrazolo[4,3-b]pyridin-5-yl)-3-methylmorpholine 1e

(118) Compound 1d (400 mg, 1.6 mmol) was dissolved in 3.0 mL of phosphorus oxychloride. The system was heated to 90 C. and stirred for 2.0 h. The reaction mixture was cooled to room temperature and concentrated at reduced pressure. The residue was diluted with dichloromethane (50 mL), and cooled in an ice/water bath. Saturated sodium bicarbonate solution was added to neutralize the dilution to pH 8 to 9. The system was stirred for 0.5 h for reaction and let stand for separation. The organic phase was collected, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was purified by silica gel column chromatography with eluent system C to obtain the target compound 1e (240 mg, 56% yield).

(119) MS m/z (ESI): 267.0 [M+1].

Step 4

(R)-2-methyl-2-(1-methyl-5-(3-methylmorpholinyl)-1H-pyrazolo[4,3-b]pyridin-7-yl)propane nitrile 1g

(120) Compound 1e (240 mg, 0.91 mmol) and compound isobutyronitrile 1f (620 mg, 8.9 mmol, Shanghai Bide) were dissolved in 30 mL of tetrahydrofuran in a nitrogen atmosphere and cooled in a dry ice/acetone bath. Lithium bis(trimethylsilyl)amide (8.9 mL, a 1 M solution in tetrahydrofuran, 8.9 mmol) was added dropwise. The system was stirred at a low temperature for 0.5 h, naturally warmed to room temperature and stirred for 1 h. The reaction was quenched with water. The organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system C to obtain the target compound 1g (200 mg, 74% yield).

(121) MS m/7. (ESI): 300.1 [M+1].

Step 5

(R)-2-(3-bromo-1-methyl-5-(3-methylmorpholinyl)-1H-pyrazolo[4,3-b]pyridin-7-yl)-2-methylpropanenitrile 1h

(122) Compound 1g (200 mg, 0.67 mmol) was dissolved in 5 mL of 1,4-dioxane, and a solution of sodium hydroxide (0.66 mL, 2 M, 1.32 mmol) was added. The mixture was cooled in an ice/water bath before bromine (427 mg, 2.67 mmol) was added. The reaction system was stirred at a low temperature for 10 min, naturally warmed to room temperature and stirred for 1 h for reaction. Ethyl acetate was added for dilution. The organic phase was washed with saturated sodium thiosulfate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system C to obtain the target compound 1h (140 mg, 55% yield).

(123) MS m/z (ESI): 377.9 [M+1].

Step 6

2-methyl-2-(1-methyl-5-((R)-3-methylmorpholinyl)-3-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)-1H-pyrazolo[4,3-b]pyridin-7-yl)propanenitrile 1i

(124) Compound 1h (20 mg, 0.05 mmol), tetrakis(triphenylphosphine)palladium(0) (18 mg, 0.015 mmol), sodium carbonate (11 mg, 0.10 mmol) and 1-(tetrahydro-2H-pyran-2-yl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (29 mg, 0.10 mmol, Shanghai Bide) were dissolved in 4 mL of ethylene glycol dimethyl ether. 1 mL of water was added. In an argon atmosphere, the reaction system was heated by microwave to 120 C. and reacted for 1 h. The reaction mixture was cooled to room temperature before 20 ml of water was added. Ethyl acetate (20 mL3) was added for extraction. The organic phases were combined, concentrated at reduced pressure, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system C to obtain the target compound 1i (20 mg, 84% yield).

(125) MS m/z. (ESI): 450.1 [M+1].

Step 7

(R)-2-methyl-2-(1-methyl-5-(3-methylmorpholinyl)-3-(1H-pyrazol-3-yl)-1H-pyrazolo[4,3-b]pyridin-7-yl)propanenitrile 1

(126) Compound 1i (20 mg, 0.04 mmol) was dissolved in 5 mL of dichloromethane. 5 mL of trifluoroacetic acid was added dropwise before the reaction system was stirred for 4 h for reaction. The reaction mixture was concentrated at reduced pressure and adjusted to pH 8 to 9 by dropwise adding a 7 M solution of ammonia in methanol. The resulting mixture was concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system A to obtain the target compound 1 (7.0 mg, 43% yield).

(127) MS m/z (ESI): 366.0 [M+1].

(128) .sup.1H NMR (400 MHz, CD.sub.3OD): 7.58 (s, 1H), 7.03 (s, 1H), 6.86 (s, 1H), 4.39 (s, 4H), 4.04-3.82 (m, 2H), 3.74 (s, 2H), 3.58 (td, 1H), 3.26 (dd, 1H), 1.88 (d, 6H), 1.19 (d, 3H).

Example 2

(R)-1-(1-methyl-5-(3-methylmorpholinyl)-3-(1H-pyrazol-3-yl)-1H-pyrazolo[4,3-b]pyridin-7-yl)cyclopropanenitrile 2

(129) ##STR00086## ##STR00087##

Step 1

(R)-1-(1-methyl-5-(3-methylmorpholinyl)-1H-pyrazolo[4,3-b]pyridin-7-yl) cyclopropanenitrile 2a

(130) Compound 1e (86 mg, 0.32 mmol), cyclopropanenitrile (65 mg, 0.97 mmol), tris(dibenzylideneacetone)dipalladium(0) (30 mg, 0.03 mmol) and 1,1-binaphthyl-2,2-bis-diphenylphosphine (40 mg, 0.06 mmol) were dissolved in 2 mL of tetrahydrofuran. In an argon atmosphere, lithium bis(trimethylsilyl)amide (1.0 mL, a 1 M solution in tetrahydrofuran, 1.0 mmol) was added. The reaction mixture was sealed, heated to 80 C., stirred for 1 h and cooled to room temperature. 20 mL of water was added. Ethyl acetate (20 mL3) was added for extraction. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system C to obtain the target compound 2a (80 mg, 84% yield).

(131) MS m/z (ESI): 298.3 [M+1].

Step 2

(R)-1-(3-bromo-1-methyl-5-(3-methylmorpholinyl)-1H-pyrazolo[4,3-b]pyridin-7-yl) cyclopropanenitrile 2b

(132) Compound 2a (30 mg, 0.1 mmol) was dissolved in 5 mL of 1,4-dioxane, and a solution of sodium hydroxide (0.1 mL, 2 M, 0.2 mmol) was added. The mixture was cooled in an ice/water bath before bromine (64 mg, 0.4 mmol) was added. The reaction system was stirred at a low temperature for 10 min, naturally warmed to room temperature and stirred for 1 h for reaction. 20 mL of ethyl acetate was added for dilution. The organic phase was washed with saturated sodium thiosulfate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system C to obtain the target compound 2b (30 mg, 80% yield).

(133) MS m/z (ESI): 376.4 [M+1].

Step 3

1-(1-methyl-5-((R)-3-methylmorpholinyl)-3-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)-1H-pyrazolo[4,3-b]pyridin-7-yl) cyclopropanenitrile 2c

(134) Compound 2b (30 mg, 0.08 mmol), tetrakis(triphenylphosphine)palladium(0) (10 mg, 0.08 mmol), sodium carbonate (17 mg, 0.16 mmol) and 1-(tetrahydro-2H-pyran-2-yl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (44 mg, 0.16 mmol) were dissolved in 4.0 mL of ethylene glycol dimethyl ether. 1.0 mL of water was added. In an argon atmosphere, the reaction system was heated by microwave to 120 C., stirred for 1 h for reaction and cooled to room temperature. 20 mL of water was added. Ethyl acetate (20 mL3) was added for extraction. The organic phases were combined, concentrated at reduced pressure, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system C to obtain the target compound 2c (30 mg, 84% yield).

(135) MS m/z (ESI): 448.3 [M+1].

Step 4

(R)-1-(1-methyl-5-(3-methylmorpholinyl)-3-(1H-pyrazol-3-yl)-1H-pyrazolo[4,3-b]pyridin-7-yl) cyclopropanenitrile 2

(136) Compound 2c (30 mg, 0.07 mmol) was dissolved in 5 mL of dichloromethane. 1 mL of trifluoroacetic acid was added dropwise before the reaction system was stirred for 4 h for reaction. The reaction mixture was concentrated at reduced pressure and adjusted to pH 8 to 9 by dropwise adding a 7 M solution of ammonia in methanol. The resulting mixture was concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system A to obtain the target compound 2 (13.5 mg, 55% yield).

(137) MS m/z (ESI): 364.3 [M+1].

(138) .sup.1H NMR (400 MHz, CD.sub.3OD): 7.58 (d, 1H), 7.01 (d, 1H), 6.93 (s, 1H), 4.40 (d, 1H), 4.38 (s, 3H), 3.95 (dd, 2H), 3.79-3.68 (m, 2H), 3.57 (td, 1H), 3.30-3.25 (m, 1H), 1.92-1.80 (m, 2H), 1.72-1.58 (m, 2H), 1.18 (d, 3H).

Example 3

(R)-3-methyl-4-(1-methyl-7-(1-methyl-1H-pyrazol-5-yl)-3-(1H-pyrazol-3-yl)-1H-pyrazolo[4,3-b]pyridin-5-yl) morpholine 3

(139) ##STR00088## ##STR00089##

Step 1

(R)-3-methyl-4-(1-methyl-7-(1-methyl-1H-pyrazol-5-yl)-1H-pyrazolo[4,3-b]pyridin-5-yl) morpholine 3a

(140) Compound 1e (250 mg, 0.94 mmol), bis(triphenylphosphine)palladium(II) dichloride (66 mg, 0.09 mmol), potassium carbonate (260 mg, 1.8 mmol) and 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (390 mg, 1.87 mmol) were dissolved in 8.0 mL of ethylene glycol dimethyl ether. 2.0 mL of water was added. In an argon atmosphere, the reaction system was heated by microwave to 120 C., reacted for 2 h and cooled to room temperature. 20 mL of water was added for dilution. Ethyl acetate (20 mL3) was added for extraction. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system C to obtain the target compound 3a (290 mg, 99% yield).

(141) MS m/z (ESI): 313.2 [M+1].

Step 2

(R)-4-(3-bromo-1-methyl-7-(1-methyl-1H-pyrazol-5-yl)-1H-pyrazolo[4,3-b]pyridin-5-yl)-3-methylmorpholine 3b

(142) Compound 3a (300 mg, 0.97 mmol) was dissolved in 5 mL of N,N-dimethylformamide and cooled in an ice/water bath. N-bromosuccinimide (205 mg, 1.2 mmol) was added. The mixture was stirred at a low temperature for 10 min, naturally warmed to room temperature and stirred for 1 h. 20 mL of ethyl acetate was added for dilution. The organic phase was washed with saturated sodium thiosulfate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system C to obtain the target compound 3b (115 mg, 30% yield).

(143) MS m/z (ESI): 391.1 [M+1].

Step 3

(3R)-3-methyl-4-(1-methyl-7-(1-methyl-1H-pyrazol-5-yl)-3-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)-1H-pyrazolo4,3-bpyridin-5-yl) morpholine 3c

(144) Compound 3b (35 mg, 0.09 mmol), tetrakis(triphenylphosphine)palladium(0) (10 mg, 0.009 mmol), sodium carbonate (19 mg, 0.18 mmol) and 1-(tetrahydro-2H-pyran-2-yl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (50 mg, 0.18 mmol) were dissolved in 5.0 mL of ethylene glycol dimethyl ether. 1 mL of water was added. In an argon atmosphere, the reaction system was heated by microwave to 120 C., reacted for 1 h and cooled. 20 mL of water was added. Ethyl acetate (20 mL3) was added for extraction. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system C to obtain the target compound 3c (20 mg, 48% yield).

(145) MS m/z (ESI): 463.4 [M+1].

Step 4

(R)-3-methyl-4-(1-methyl-7-(1-methyl-1H-pyrazol-5-yl)-3-(1H-pyrazol-3-yl)-1H-pyrazolo[4,3-b]pyridin-5-yl) morpholine 3

(146) Compound 3c (60 mg, 0.13 mmol) was dissolved in 5 mL of dichloromethane. 1 mL of trifluoroacetic acid was added dropwise before the reaction system was stirred for 4 h for reaction. The reaction mixture was concentrated at reduced pressure and adjusted to pH 8 to 9 by dropwise adding a 7 M solution of ammonia in methanol. The resulting mixture was concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system A to obtain the target compound 3 (20 mg, 40.7% yield).

(147) MS m/z (ESI): 379.2 [M+1].

(148) .sup.1H NMR (400 MHz, CD.sub.3OD): 7.58 (d, 1H), 7.57 (d, 1H), 7.03 (d, 1H), 6.90 (s, 1H), 6.48 (d, 1H), 4.38 (d, 1H), 4.02-3.88 (m, 2H), 3.72 (s, 2H), 3.65 (s, 3H), 3.57 (td, 1H), 3.50 (s, 3H), 3.27-3.22 (m, 1H), 1.19 (d, 3H).

Example 4

(R)-2-(1-ethyl-5-(3-methylmorpholinyl)-3-(1H-pyrazol-3-yl)-1H-pyrazolo[4,3-b]pyridin-7-yl)-2-methylpropanenitrile 4

(149) ##STR00090## ##STR00091##

Step 1

Methyl 1-ethyl-4-nitro-1H-pyrazole-5-carboxylate 4b

(150) Compound methyl 1-ethyl-4-nitro-1H-pyrazole-5-carboxylate 4a (5 g, 25.1 mmol, Shanghai Bide) was dissolved in 100 mL of methanol. 10% palladium on carbon (1 g) was added. The system was purged with hydrogen three times and stirred for 14 h. The mixture was filtered and the filtrate was concentrated at reduced pressure to obtain a crude product of the target compound 4b (4.2 g), which was used directly in the next reaction without purification.

(151) MS m/z (ESI): 170.1 [M+1].

Step 2

Methyl (R,E)-1-ethyl-4-((1-(3-methylmorpholino)ethylidene)amino)-1H-pyrazole-5-carboxylate 4c

(152) Compound 1b (3.3 g, 23.0 mmol) was dissolved in 1,2-dichloroethane and cooled in an ice/water bath in an argon atmosphere. Phosphorus oxychloride (5.4 g, 35.2 mmol) was dropwise and slowly added. The mixture was stirred at room temperature for 30 min before compound 4b (2.5 g, 16.1 mmol) was added. The mixture was heated to 80 C. and stirred for reaction for 2 h. The mixture was cooled to room temperature and concentrated at reduced pressure. The residue was diluted with dichloromethane (200 mL), and cooled in an ice/water bath. Saturated sodium bicarbonate solution was added dropwise to neutralize the dilution to pH 8 to 9. The organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was purified by silica gel column chromatography with eluent system C to give the target compound 4c (2.3 g, 66.1% yield).

(153) MS m/z (ESI): 295.2 [M+1].

Step 3

(R)-1-ethyl-5-(3-methylmorpholinyl)-1H-pyrazolo[4,3-b]pyridin-7-ol 4d

(154) Compound 4c (1 g, 3.39 mmol) was dissolved in tetrahydrofuran (20 mL) and cooled in an ice/water bath. Lithium bis(trimethylsilyl)amide (10 mL, a 1 M solution in tetrahydrofuran, 10 mmol) was added slowly and the system was reacted at 0 C. for 1 h. The reaction was quenched by adding methanol (10 mL). The mixture was purified by silica gel column chromatography with eluent system A to give the target compound 4d (250 mg, 28.1% yield).

(155) MS m/z (ESI): 263.1 [M+1].

Step 4

(R)-4-(7-chloro-1-ethyl-1H-pyrazolo[4,3-b]pyridin-5-yl)-3-methylmorpholine 4e

(156) Compound 4d (250 mg, 0.95 mmol) was dissolved in 2.0 mL of phosphorus oxychloride. The system was heated to 90 C. and stirred for 2 h. The reaction mixture was cooled to room temperature and concentrated at reduced pressure. The residue was diluted with dichloromethane (50 mL), and cooled in ice water. Saturated sodium bicarbonate solution was added to neutralize the dilution to pH 8 to 9. The system was stirred for 0.5 h for reaction and let stand for separation. The organic phase was collected and washed with saturated brine (50 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was purified by silica gel column chromatography with eluent system C to give the target compound 4e (120 mg, 42.5% yield).

(157) MS m/z (ESI): 281.3 [M+1].

Step 5

(R)-2-(1-ethyl-5-(3-methylmorpholinyl)-1H-pyrazolo[4,3-b]pyridin-7-yl)-2-methylpropanenitrile 4g

(158) Compound 4e (120 mg, 0.43 mmol) and compound 1f (295 mg, 4.3 mmol, Shanghai Bide) were dissolved in 30 mL of tetrahydrofuran and cooled in a dry ice/acetone bath in an argon atmosphere. Lithium bis(trimethylsilyl)amide (1.7 mL, a 1 M solution in tetrahydrofuran, 1.7 mmol) was added dropwise. The system was stirred at a low temperature for 0.5 h, naturally warmed to room temperature and stirred for 1 h. The reaction was quenched with water. The organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system C to obtain the target compound 4g (95 mg, 74% yield).

(159) MS m/z (ESI): 314.1 [M+1].

Step 6

(R)-2-(3-bromo-1-ethyl-5-(3-methylmorpholinyl)-1H-pyrazolo[4,3-b]pyridin-7-yl)-2-methylpropanenitrile 4h

(160) Compound 4g (95 mg, 0.67 mmol) was dissolved in 5 mL of 1,4-dioxane, and a solution of sodium hydroxide (0.3 mL, 2 M, 0.6 mmol) was added. The mixture was cooled in an ice/water bath before bromine (194 mg, 1.2 mmol) was added. The reaction system was stirred at a low temperature for 10 min, naturally warmed to room temperature and stirred for 1 h for reaction. Ethyl acetate was added for dilution. The organic phase was washed with saturated sodium thiosulfate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system C to obtain the target compound 4h (41 mg, 34% yield).

(161) MS m/z. (ESI): 392.1 [M+1].

Step 7

2-(1-ethyl-5-((R)-3-methylmorpholinyl)-3-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)-1H-pyrazolo[4,3-b]pyridin-7-yl)-2-methylpropanenitrile 4i

(162) Compound 4h (40 mg, 0.1 mmol), tetrakis(triphenylphosphine)palladium(0) (12 mg, 0.01 mmol), sodium carbonate (32 mg, 0.3 mmol) and 1-(tetrahydro-2H-pyran-2-yl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (32 mg, 0.20 mmol, Shanghai Bide) were dissolved in 4 mL of dioxane. 1 mL of water was added. In an argon atmosphere, the reaction system was heated by microwave to 120 C. and reacted for 1 h. The reaction mixture was cooled to room temperature before 20 ml of water was added. Ethyl acetate (20 mL3) was added for extraction. The organic phases were combined, concentrated at reduced pressure, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system C to obtain the target compound 4i (40 mg, 85% yield).

(163) MS m/z. (ESI): 464.1 [M+1].

Step 8

(R)-2-(1-ethyl-5-(3-methylmorpholinyl)-3-(1H-pyrazol-3-yl)-1H-pyrazolo[4,3-b]pyridin-7-yl)-2-methylpropanenitrile 4

(164) Compound 4i (20 mg, 0.04 mmol) was dissolved in 5 ml of dichloromethane. 5 mL of trifluoroacetic acid was added dropwise before the reaction system was stirred for 4 h for reaction. The reaction mixture was concentrated at reduced pressure and adjusted to pH 8 to 9 by dropwise adding a 7 M solution of ammonia in methanol. The resulting mixture was concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system A to obtain the target compound 4 (15 mg, 45% yield).

(165) MS m/z (ESI): 380.2 [M+1].

(166) .sup.1H NMR (400 MHz, CD.sub.3OD): 7.57 (s, 1H), 7.04 (s, 1H), 6.85 (s, 1H), 4.66-4.64 (m, 2H), 4.39-4.38 (m, 1H), 3.97-3.91 (m, 2H), 3.74 (s, 2H), 3.58-3.57 (m, 1H), 3.28-3.27 (m, 1H), 1.86 (d, 6H), 1.46 (t, 3H), 1.18 (d, 3H).

Example 5

(R)-1-(1-ethyl-5-(3-methylmorpholinyl)-3-(1H-pyrazol-3-yl)-1H-pyrazolo[4,3-b]pyridin-7-yl) cyclopropanenitrile 5

(167) ##STR00092##

Step 1

(R)-1-(1-ethyl-5-(3-methylmorpholinyl)-1H-pyrazolo[4,3-b]pyridin-7-yl) cyclopropanenitrile 5a

(168) Compound 4e (500 mg, 1.78 mmol), cyclopropanenitrile (239 mg, 3.56 tris(dibenzylideneacetone)dipalladium(0) (162 mg, 0.18 mmol) and mmol), 1,1-binaphthyl-2,2-bis-diphenylphosphine (222 mg, 0.36 mmol) were dissolved in 2 mL of tetrahydrofuran. In an argon atmosphere, lithium bis(trimethylsilyl)amide (5.3 mL, a 1 M solution in tetrahydrofuran, 5.3 mmol) was added. The reaction mixture was sealed, heated to 80 C., stirred for 1 h and cooled to room temperature. 20 mL of water was added. Ethyl acetate (20 mL3) was added for extraction. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system C to obtain the target compound 5a (360 mg, 64.9% yield).

(169) MS m/z (ESI): 312.2 [M+1].

Step 2

(R)-1-(3-bromo-1-ethyl-5-(3-methylmorpholinyl)-1H-pyrazolo[4,3-b]pyridin-7-yl) cyclopropanenitrile 5b

(170) Compound 5a (377 mg, 1.2 mmol) was dissolved in 5 mL of tetrahydrofuran and cooled in an ice/water bath. N-bromosuccinimide (215 mg, 1.2 mmol) was added. The mixture was stirred at a low temperature for 10 min, naturally warmed to room temperature and stirred for 2 h. 20 mL of ethyl acetate was added for dilution. The organic phase was washed with saturated sodium thiosulfate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system C to obtain the target compound 5b (100 mg, 21% yield).

(171) MS m/z (ESI): 390.3 [M+1].

Step 3

(R)-1-(1-ethyl-5-(3-methylmorpholinyl)-3-(1H-pyrazol-3-yl)-1H-pyrazolo[4,3-b]pyridin-7-yl) cyclopropanenitrile 5

(172) Compound 5b (100 mg, 0.25 mmol), [1,1-bis(diphenylphosphino)ferrocene]palladium(II) dichloride dichloromethane complex (43 mg, 0.05 mmol), sodium carbonate (81 mg, 0.78 mmol) and (1H-pyrazol-3-yl) boronic acid (43 mg, 0.38 mmol, Shanghai Bide) were dissolved in 4.0 mL of dioxane. 1.0 mL of water was added. In an argon atmosphere, the reaction system was stirred at 100 C. for 2 h and cooled to room temperature. 20 mL of water was added. Ethyl acetate (20 mL3) was added for extraction. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system A to obtain the target compound 5 (10 mg, 10% yield).

(173) .sup.1H NMR (400 MHz, CD.sub.3OD): 7.82 (s, 1H), 7.04-7.12 (m, 2H), 4.84-4.82 (m, 2H), 4.51-4.50 (m, 1H), 4.09-4.05 (m, 2H), 3.86-3.85 (m, 2H), 3.69-3.66 (m, 1H), 3.43-3.42 (m, 1H), 1.97 (d, 2H), 1.80-1.78 (m, 2H), 1.67 (t, 3H), 1.32 (d, 3H).

Example 6

(R)-2-methyl-2-(5-(3-methylmorpholinyl)-3-(1H-pyrazol-3-yl)-1H-pyrazolo[4,3-b]pyridin-7-yl)propanenitrile 6

(174) ##STR00093## ##STR00094## ##STR00095##

Step 1

Methyl 1-benzyl-4-nitro-1H-pyrazole-5-carboxylate 6b

(175) Methyl 4-nitro-1H-pyrazole-3-carboxylate 6a (2 g, 11.69 mmol, Meryer) was dissolved in 30 mL of N,N-dimethylformamide. Potassium carbonate (1.75 g, 12.66 mmol) and benzyl bromide (2.04 g, 11.93 mmol) were added. The mixture was stirred at room temperature for 17 h. 80 mL of ethyl acetate was added to the reaction mixture, which was then washed with water (30 mL3) and saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system C to obtain the target compound 6b (710 mg, 23.3% yield).

(176) MS m/z (ESI): 262.0 [M+1].

Step 2

Methyl 4-amino-1-benzyl-1H-pyrazole-5-carboxylate 6c

(177) Compound 6b (710 mg, 2.72 mmol) was dissolved in 20 mL of absolute ethanol, and iron powder (1.52 g, 27.22 mmol) and ammonium chloride (1.46 g, 27.29 mmol) were added. The reaction system was heated at reflux and stirred for 17 h. The reaction mixture was cooled to room temperature, and filtered through a Buchner funnel pre-layered with celite. The solid was washed with ethyl acetate, and the combined filtrates were concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system C to obtain the target compound 6c (650 mg, containing ethanol transesterification products, 100% yield).

(178) MS m/z (ESI): 232.2 [M+1], 246.2 [M+1].

Step 3

(R)-1-(4-amino-1-benzyl-1H-pyrazol-5-yl)-3-(3-methylmorpholinyl)propane-1,3-dione 6d

(179) Lithium bis(trimethylsilyl)amide (11.29 mL, a 1 M solution in tetrahydrofuran, 11.29 mmol) was transferred to a three-necked flask. In a nitrogen atmosphere, the mixture was cooled to an internal temperature of 10 C. to 0 C. and a solution of 1b in 2-methyltetrahydrofuran (0.65 g, 4.54 mmol, 1.6 mL) was added dropwise. The system was incubated for reaction for 40 min. A solution of 6c in 2-methyltetrahydrofuran (0.65 g, 2.81 mmol, 2.6 mL) was then added dropwise. The reaction system was incubated for reaction for 1 h. 10 mL of water was added to the reaction mixture. Ethyl acetate (10 mL3) was added for extraction. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system C to obtain the target compound 6d (440 mg, 45.7% yield).

(180) MS m/7. (ESI): 343.2 [M+1].

Step 4

(R)-1-benzyl-7-chloro-5-(3-methylmorpholinyl)-1H-pyrazolo[4,3-b]pyridine 6e

(181) Compound 6d (0.44 g, 1.29 mmol) was dissolved in 5 mL of acetonitrile. N,N-diisopropylethylamine (0.5 g, 3.87 mmol) was added. The mixture was cooled to an inner temperature of 5 C. to 0 C. Phosphorus oxychloride (0.79 g, 5.15 mmol) was added dropwise. The system was incubated for reaction for 1.5 h, and reacted at 65 C. for 4 h. The reaction solution was cooled to room temperature and poured into 20 mL of saturated sodium carbonate solution. Ethyl acetate (20 mL3) was added for extraction. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system C to obtain the target compound 6e (180 mg, 40.8% yield).

(182) MS m/z (ESI): 343.1 [M+1].

Step 5

(R)-2-methyl-2-(5-(3-methylmorpholinyl)-1H-pyrazolo[4,3-b]pyridin-7-yl)propanenitrile 6g

(183) Compound 6e (180 mg, 0.53 mmol) and isobutyronitrile 6f (218 mg, 3.15 mmol) were dissolved in 5 mL of tetrahydrofuran. In a nitrogen atmosphere, the mixture was cooled to an internal temperature of less than 70 C. Lithium bis(trimethylsilyl)amide (3.15 mL, a 1 M solution in tetrahydrofuran, 3.15 mmol) was added dropwise. The system was incubated for 30 min for reaction, and further reacted at room temperature for 1 h. 10 mL of water was added. Ethyl acetate (10 mL3) was added for extraction. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system C to obtain the target compound 6g (80 mg, 53.3% yield).

(184) MS m/7. (ESI): 286.2 [M+1].

Step 6

(R)-2-(3-bromo-5-(3-methylmorpholinyl)-1H-pyrazolo[4,3-b]pyridin-7-yl)-2-methylpropanenitrile 6h

(185) Compound 6g (80 mg, 0.28 mmol) was dissolved in 2 mL of tetrahydrofuran. N-bromosuccinimide (50 mg, 0.28 mmol) was added. The mixture was stirred at room temperature for 30 min. 10 mL of saturated sodium thiosulfate solution was added. Ethyl acetate (10 mL3) was added for extraction. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system C to obtain the target compound 6h (40 mg, 39.2% yield).

(186) MS m/z (ESI): 364.1 [M+1].

Step 7

2-methyl-2-(5-((R)-3-methylmorpholinyl)-3-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)-1H-pyrazolo[4,3-b]pyridin-7-yl)propanenitrile 6j

(187) Compound 6h (40 mg, 0.11 mmol), 1-(tetrahydro-2H-pyran-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole 6i (61 mg, 0.22 mmol, Shanghai Bide) and sodium carbonate (24 mg, 0.22 mmol) were added to a three-necked flask. 2 mL of 1,4-dioxane and 0.5 mL of water were added. After 3 nitrogen purges, bis(triphenylphosphine)palladium(II) dichloride (16 mg, 22.8 mol) was added, followed by another 3 nitrogen purges. In a nitrogen atmosphere, the external temperature was raised to 85 C., and the system was stirred for 1 h. The reaction solution was cooled to room temperature before 10 mL of water was added to the reaction mixture. Ethyl acetate (10 mL3) was added for extraction. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system C to obtain the target compound 6j (25 mg, 52.3% yield).

(188) MS m/z (ESI): 436.3 [M+1].

Step 8

2-Methyl-2-(5-((R)-3-methylmorpholinyl)-3-(1H-pyrazol-5-yl)-1H-pyrazolo[4,3-b]pyridin-7-yl)propanenitrile 6

(189) Compound 6j (22 mg, 50.51 umol) was dissolved in 3 mL of isopropanol. Trifluoroacetic acid (404 mg, 3.54 mmol) was added. The system was stirred at room temperature for 30 min. The reaction solution was poured into 10 mL of saturated sodium bicarbonate solution. The mixture was concentrated at reduced pressure. Ethyl acetate (10 mL3) was added for extraction. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated at reduced pressure, and the residue was purified by thin layer chromatography with developing solvent system C to obtain the target compound 6 (5 mg, 28.2% yield).

(190) MS m/z (ESI): 352.1 [M+1].

(191) .sup.1H NMR (500 MHz, CDCl.sub.3): 7.77 (s, 1H), 7.18 (s, 1H), 6.93 (s, 1H), 4.42-4.43 (d, 1H), 4.14-4.11 (m, 1H), 4.01-3.98 (m, 1H), 3.88-3.89 (m, 2H), 3.74-3.67 (m, 1H), 3.44-3.42 (m, 1H), 2.00 (s, 6H), 1.36-1.35 (d, 3H).

Comparative Example 1 (Example 7)

(R)-2-(1-methyl-5-(3-methylmorpholinyl)-3-(1H-pyrazol-3-yl)-1H-pyrazolo[4,3-b]pyridin-7-yl)propan-2-ol 7

(192) ##STR00096## ##STR00097## ##STR00098##

Step 1

(R)-1-methyl-5-(3-methylmorpholinyl)-1H-pyrazolo[4,3-b]pyridin-7-yl trifluoromethanesulfonate 7a

(193) Compound 1d (500 mg, 2.0 mmol) was dissolved in 5.0 mL of dichloromethane. N,N-diisopropylethylamine (520 mg, 4.0 mmol) and N-phenyl-bis(trifluoromethanesulfonimide) (790 mg, 2.2 mmol) were added. The system was stirred for 2.0 h. The reaction mixture was concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system C to obtain the target compound 7a (600 mg, 78.3% yield).

(194) MS m/z (ESI): 381.3 [M+1].

Step 2

Methyl (R)-1-methyl-5-(3-methylmorpholinyl)-1H-pyrazolo[4,3-b]pyridine-7-carboxylate 7b

(195) Compound 7a (400 mg, 1.05 mmol), [1,1-bis(diphenylphosphino)ferrocene]palladium(II) dichloride dichloromethane complex (178 mg, 0.21 mmol, Shanghai Bide) and triethylamine (210 mg, 2.09 mmol) were dissolved in 8 mL of methanol. In a carbon monoxide atmosphere, the system was stirred at 65 C. for 15 h. The resulting mixture was cooled to room temperature and filtered. The filtrate was concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system C to obtain the target compound 7b (90 mg, 29.5% yield).

(196) MS m/z (ESI): 291.1 [M+1].

Step 3

Methyl (R)-3-bromo-1-methyl-5-(3-methylmorpholine)-1H-pyrazolo[4,3-b]pyridine-7-carboxylate 7c

(197) Compound 7b (90 mg, 0.31 mmol) was dissolved in 5 mL of tetrahydrofuran. N-bromosuccinimide (110 mg, 0.62 mmol) was added. The mixture was stirred at room temperature for 30 min. 10 mL of saturated sodium thiosulfate solution was added. Ethyl acetate (10 mL3) was added for extraction. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated at reduced pressure to obtain a crude product of the target compound 7c (180 mg), which was directly used in the next step without purification.

(198) MS m/z (ESI): 369.1 [M+1].

Step 4

Methyl 1-methyl-5-((R)-3-methylmorpholinyl)-3-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)-1H-pyrazolo[4,3-b]pyridine-7-carboxylate 7d

(199) Compound 7c (180 mg, 0.316 mmol), [1,1-bis(diphenylphosphino)ferrocene]palladium(II) dichloride dichloromethane complex (30 mg, 0.032 mmol), sodium carbonate (83 mg, 0.783 mmol) and 1-(tetrahydro-2H-pyran-2-yl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (176 mg, 0.632 mmol, Shanghai Bide) were dissolved in 4 mL of dioxane. 1 mL of water was added. In an argon atmosphere, the reaction system was heated by microwave to 120 C. and reacted for 1 h. The reaction mixture was cooled to room temperature before 20 ml of water was added. Ethyl acetate (20 mL3) was added for extraction. The organic phases were combined, concentrated at reduced pressure, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system C to obtain the target compound 7d (45 mg, 32.3% yield).

(200) MS m/z (ESI): 441.2 [M+1].

Step 5

2-(1-methyl-5-((R)-3-methylmorpholinyl)-3-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)-1H-pyrazolo[4,3-b]pyridin-7-yl)propan-2-ol 7e

(201) Compound 7d (45 mg, 0.102 mmol) was dissolved in 5 mL of tetrahydrofuran. In an ice bath, a 1 M solution of methylmagnesium bromide in tetrahydrofuran (36 mg, 0.301 mmol, Shanghai Bide) was added dropwise before the reaction system was stirred for 2 h for reaction. 10 mL of saturated ammonium chloride solution was added to the reaction mixture. Ethyl acetate (10 mL3) was added for extraction. The organic phase was concentrated at reduced pressure, and the residue was purified by silica gel column chromatography with eluent system A to obtain the target compound 7e (40 mg, 88.9% yield).

(202) MS m/z (ESI): 441.6 [M+1].

Step 6

(R)-2-(1-methyl-5-(3-methylmorpholinyl)-3-(1H-pyrazol-3-yl)-1H-pyrazolo[4,3-b]pyridin-7-yl)propan-2-ol 7

(203) Compound 7e (40 mg, 90 umol) was dissolved in 3 mL of methanol. A solution of hydrochloric acid in dioxane (1 mL, 4 N) was added. The system was stirred at room temperature for 30 min. The reaction solution was concentrated at reduced pressure, and the residue was purified by thin layer chromatography with developing solvent system C to obtain the target compound 7 (10 mg, 30.9% yield).

(204) MS m/7. (ESI): 357.6 [M+1].

(205) .sup.1H NMR (400 MHz, CD.sub.3OD): 7.68 (s, 1H), 7.43 (s, 1H), 7.11 (s, 1H), 4.50-4.42 (m, 2H), 4.21 (s, 3H), 4.10-3.99 (m, 4H), 3.84 (d, 2H), 3.68 (td, 2H), 1.28 (d, 6H).

Test Examples

Biological Evaluation

Test Example 1. Inhibitory Effect of Compounds Disclosed Herein on ATR Enzyme

(206) The following method was used to determine the inhibitory effect of the compounds disclosed herein on ATR enzyme. The experimental methodology is briefly described as follows:

I. Materials and Instruments

(207) 1. ATR enzyme (Eurofins Pharma Discovery Services, 14-953-M) 2. GST-tag P53 protein (Eurofins Pharma Discovery Services, 14-952-M) 3. 384-well plate (Thermo Scientific, 267462) 4. U-shaped bottom 96-well plate (Corning, 3795) 5. MAb Anti-phospho p53-Eu cryptate (Cisbio, 61P08KAE) 6. MAb Anti GST-d2 (Cisbio, 61GSTDLF) 7. ATP solution (Promega, V916B) 8. EDTA (Thermo Scientific, AM9260G) 9. HEPES (Gibco, 15630-080) 10. Microplate reader (BMG, PHERAsta)

II. Procedures

(208) 1 nM ATR enzyme, 50 nM P53 protein, 7.435 M ATP and small molecule compounds of different concentrations (serially 3-fold diluted from 1 M to the 11th concentration) were mixed and incubated at room temperature for 2 h. A terminating buffer (12.5 mM HEPES, 250 mM EDTA) was added. The mixture was well mixed before 0.42 ng/well of mAb anti-phospho p53-Eu cryptate and 25 ng/well of mAb anti GST-d2 were added. The mixture was incubated overnight at room temperature, and the fluorescence signals at 620 nm and 665 nm were detected using a PHERAstar system. Data were processed using GraphPad software.

III. Experimental Data

(209) The inhibitory activity of the compounds disclosed herein against ATR enzyme can be determined by the above assay, and the IC.sub.50 values obtained are shown in Table 1.

(210) TABLE-US-00003 TABLE 1 IC.sub.50 for ATR enzyme inhibition by compounds disclosed herein Maximum i. Example ii. IC.sub.50/nM iii. inhibition (%) iv. 1 v. 3 vi. 100 vii. 2 viii. 9 ix. 100 x. 3 xi. 15 xii. 100 xiii. 4 xiv. 6 xv. 100 xvi. 5 xvii. 8 xviii. 100 xix. 6 xx. 3 xxi. 100 xxii. Comparative xxiii. 73 xxiv. 97 Example 1

(211) Conclusions: The compounds disclosed herein had superior inhibitory activities against ATR enzyme than that of Comparative Example 1.

Test Example 2. Cell Proliferation Assay

(212) The following method evaluates the inhibitory effect of the compounds disclosed herein on the proliferation of LoVo cells via IC.sub.50 by measuring the intracellular ATP content. The experimental methodology is briefly described as follows:

I. Materials and Instruments

(213) 1. LoVo, human colon cancer cells (Cobioer, Nanjing, CBP60032) 2. Fetal bovine serum (FBS) (Gibco, 10091-148) 3. F-12K Medium (Gibco, 21127030) 4. CellTite-Glo reagent (Promega, G7573) 5. 96-well cell culture plate (Corning, 3903) 6. Pancreatin (Invitrogen, 25200-072) 7. Microplate reader (BMG, PHERAstar) 8. Cell counter (Countstar, Shanghai, IC1000)

II. Procedures

(214) LoVo cells were cultured in an F-12K culture medium containing 10% of FBS, and passaged twice or thrice a week in a passage ratio of 1:3 or 1:5. During passage, cells were digested by pancreatin, transferred to a centrifuge tube, and centrifuged for 3 min at 1200 rpm. The supernatant was discarded, and fresh culture medium was added to resuspend the cells. To a 96-well cell culture plate, 90 L of the cell suspension was added at a density of 3.8810.sup.4 cells/mL. To peripheral wells of the 96-well plate, only 100 L of complete medium was added. The plate was incubated in an incubator for 24 h (37 C., 5% CO.sub.2).

(215) The test samples were diluted to 2 mM in DMSO and serially 3-fold diluted to the 10.sup.th concentration. Blank and control wells were set. 5 L of the serially diluted test compound solutions was added to 95 L of fresh medium. 10 L of the medium containing the compound above was added to the plate. The plate was incubated in an incubator for 3 days (37 C., 5% CO.sub.2). 50 L of CellTiter-Glo reagent was added into each well of the 96-well cell culture plate. The plate was let stand for 5-10 min in the dark at room temperature. The chemiluminescence signals were read by a PHERAstar system, and the data were processed by GraphPad software.

III. Experimental Data

(216) The inhibitory activity of the compounds disclosed herein against LoVo cell proliferation can be determined by the above assay, and the IC.sub.50 values obtained are shown in Table 2.

(217) TABLE-US-00004 TABLE 2 IC.sub.50 for LoVo cell proliferation inhibition by compounds disclosed herein Example IC.sub.50/nM Maximum inhibition (%) 1 43 93 2 75 90 3 124 87 4 64 89 5 100 93 6 55 95 Comparative Example 1 316 92

(218) Conclusions: The compounds disclosed herein had superior inhibitory activities against LoVo cell proliferation than that of Comparative Example 1.

(219) Pharmacokinetic Evaluation

Test Example 3. Pharmacokinetic Study of Compounds Disclosed Herein

1. Introduction

(220) The plasma concentration of the compounds of Examples 1, 2 and 3 in rats after intragastric administration were measured by LC/MS/MS. The pharmacokinetic performance in rats of the compounds disclosed herein was studied and their pharmacokinetic profile was evaluated.

2. Methodology

(221) 2.1. Test Compounds

(222) The compounds of Examples 1, 2 and 3.

(223) 2.2. Test Animals

(224) 12 healthy adult SD rats (half male and half female; purchased from Vital River) were evenly divided into 3 groups of 4.

(225) 2.3. Pharmaceutical Formulation

(226) A certain amount of the compound was added to a mixed solvent containing 5% of DMSO, 5% of Tween 80 and 90% of normal saline to obtain a colorless and clear solution.

(227) 2.4. Administration

(228) SD rats were intragastrically administered with the compounds after fasting overnight, at a dose of 2 mg/kg and a volume of 10.0 mL/kg.

3. Procedures

(229) Rats were intragastrically administered with the compounds of Examples 1, 2 and 3. 0.2 mL of blood was collected from the orbit pre-dose and at 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 11.0 and 24.0 h post-does. The blood samples were transferred to EDTA-K2kk anticoagulation vacutainers, centrifuged at 4 C. and 11000 rpm for 5 min to separate plasma. The plasma samples were stored at 20 C. The mice were fed 2 h after administration.

(230) The plasma concentration of the compounds in rats after intragastric administration was determined: 25 L of rat plasma at each time point post-dose was mixed with 50 L of internal standard and 175 L of acetonitrile; the mixture was vortexed 5 min, and centrifuged for 10 min at 4000 rpm. 1 L of supernatant was taken for LC/MS/MS analysis.

4. Pharmacokinetics

(231) TABLE-US-00005 TABLE 3 Pharmacokinetics of compounds disclosed herein Pharmaceutical study (2 mg/kg) Plasma Area under Apparent concentration curve Clearance volume of C.sub.max AUC Half life Retention time CL/F distribution No. (ng/mL) (ng/mL*h) T.sub.1/2 (h) MRT (h) (mL/min/kg) Vz/F (mL/kg) 1 1112 394 3203 2747 2.62 2.48 3.08 2.31 17.7 12.4 2058 1222 2 309 219 1592 1392 2.57 1.39 3.99 2.26 51.7 49.5 7804 6135 3 673.57 86.67 2706 807 2.23 0.53 3.26 0.67 13.14 3.7 2437 448

(232) Conclusions: The compounds disclosed herein demonstrated good absorption profile and significant pharmacokinetic superiority.