A cyclic compound with anti-tumor activity and its use

Abstract

Provided are a compound with a new structure as a PRMT5 inhibitor, a method for preparing the compound, and its use in treating diseases mediated by PRMT5 inhibitors. Experiments have confirmed that these compounds have a strong inhibitory effect on the PRMT5 enzyme. Also provided is a method for synthesizing the compound, which has a simple process, convenient operation, and is conducive to large-scale industrial production and application.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a compound used as a PRMT5 inhibitor and a preparation method and use of the compound. Background Art

[0002] PRMT5 is a type of protein arginine methyltransferase, which is the abbreviation of its English name Protein arginine N-methyltransferase 5. It is a new anti-tumor target related to epigenetic modification. It has several aliases, namely Hsl7, Jbp1, Skb1, Capsuleen, or Dart5. PRMT5 is the main enzyme for arginine monomethylation and symmetrical dimethylation. More and more literature has proved that protein arginine methyltransferases (PRMTs) play a key role in different biological processes, such as cell growth and proliferation, apoptosis, metastasis, etc.

[0003] The function of protein arginine methyltransferases (PRMTs) is to transfer a methyl group from S-adenosylmethionine (AdoMet or SAM) to arginine residues on histones or other proteins to form methylarginine and S-adenosylhomocysteine ​​(SAH). Currently, nine members of this family (PRMT1-9) have been identified. According to the different ways in which they catalyze arginine methylation, PRMTs can be divided into three types: Type I PRMTs include PRMT1, PRMI2, PRMT3, PRMT4, PRMT6 and PRM8, which catalyze monomethylarginine (MMA) and asymmetric dimethylarginine (aDMA); Type II PRMTs include PRMT5 and PRMT9, which catalyze MMA and symmetric dimethylarginine (sDMA); Type III PRMTs are PRMT7, which can only methylate monomethylation. As an epigenetic enzyme, PRMT5 can symmetrically methylate arginine residues of histone or non-histone substrates, affecting multiple target genes and multiple signaling pathways. It plays an important role in protein methylation, such as participating in variable splicing, post-transcriptional regulation, RNA processing, cell proliferation, cell differentiation, apoptosis and tumor formation. Selective inhibition of PRMT5 can be used as a potential powerful new anticancer drug. Research on the development of new drugs targeting PRMT5 has a positive role in filling the gap in solving unmet clinical needs.

[0004] In the past few years, there have been many reports on PRMT5 inhibitors, such as WO2014100719A, WO2019102494A, WO2015200677A, WO2015200680A, WO2014100764A, WO2014100730A, WO2014100716A, WO2014100695A, WO2019173804A, CN108570059 A, WO2018167269A, etc. And two compounds, JNJ-64619178 and GSK-3326595, have entered clinical treatment of solid tumors and mantle cell lymphoma.

[0005]

[0006] JNJ-64619178 is a selective PRMT5 inhibitor developed by Johnson & Johnson, which has an inhibitory effect on the growth of various tumor cells in vitro. Johnson & Johnson has selected a large number of xenograft animal models to demonstrate its effective anti-tumor effect, for example: small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), acute myeloid leukemia (AML) and non-Hodgkin lymphoma Xenograft models were selected for anti-tumor experiments. Up to 99% significant tumor growth inhibition was observed in the model, and tumor growth continued to be inhibited within weeks after drug withdrawal. JNJ-64619178 can inhibit the Sym-Arg dimethylation of SMD1 / 3 proteins, the core components of the tumor spliceosome, and inhibit the Sym-Arg dimethylation of serum proteins. These can be used as pharmacodynamic markers for PRMT5 to inhibit tumor growth in the Xenograft model. In the SCLC model, effective and long-term inhibition of SMD1 / 3 dimethylation by PRMT5 was observed during and after administration. Based on these high selectivity and high efficacy, good pharmacokinetics and safety, and significant preclinical efficacy and efficacy results, JNJ-64619178 began Phase I clinical trials in 2018.

[0007] GSK-3326595 is optimized from EPZ015666 (structure as follows). It is a highly selective, orally available small molecule and a first-generation PRMT5 inhibitor. EPZ015666 has shown significant in vitro and in vivo activity in mantle cell lymphoma. After two years of optimization and preclinical studies, GlaxoSmithKline (GSK) announced in September 2016 that GSK-3326595 had entered the clinic for the first time. At the 2019 ESMO conference, GlaxoSmithKline announced the Phase I clinical data of GSK-3326595. The Phase I clinical trial of GSK-3326595 selected adult patients with solid tumors. The main purpose was to conduct safety, tolerability and PK / PD tests, and collect efficacy data (ORR and DCR). The data showed that GSK3326595 PK was dose-dependent in plasma.

[0008]

[0009] Although some PRMT5 inhibitor small molecules have been disclosed, no PRMT5 inhibitor has been developed and marketed yet, so it is still urgent to develop new compounds with potential for marketing and better efficacy and pharmacokinetic results. The present invention designs a series of compounds with new structures shown in the general formula, and finds that compounds with such structures exhibit excellent effects and functions, which has positive significance for the development of PRMT5 inhibitors. Summary of the invention

[0010] The object of the present invention is to provide a compound with a novel structure as a PRMT5 inhibitor, a method for preparing the compound, and use thereof in treating diseases mediated by PRMT5 inhibitors.

[0011] In a first aspect, the present invention provides a compound as shown in the following formula (I), and its stereoisomers, geometric isomers, tautomers, pharmaceutically acceptable salts, prodrugs, hydrates, solvates or isotope-labeled analogs.

[0012]

[0013] in, Indicates whether the bond between two atoms is a double bond or a single bond, making the whole ring structure aromatic or non-aromatic;

[0014] Z may be independently selected from C or N;

[0015] q can be selected from 0 or 1, q=0 represents the ring is a five-membered ring, q=1 represents the ring is a six-membered ring;

[0016] Z1 can be independently selected from CH, N, C(R 2 ), but when q=1, the Z1 of the three positions are not N at the same time; R2 Each occurrence is independently selected from hydrogen, halogen, hydroxy, thiol, amino, cyano, optionally substituted -R 2a , optionally substituted -OR 2a , optionally substituted -NH-R 2a , optionally substituted -N(R 2a )R 2a , where R 2a Each occurrence is independently selected from C 1-6 Alkyl, C2-6 alkenyl, C2-6 alkynyl, 3-6 Cycloalkyl, 4-6 membered heterocyclyl, phenyl, 5-6 membered heteroaryl; the optional substitution means that the H on the substituted group is not substituted or one or more substitutable sites of the substituted group are independently selected from halogen, hydroxyl, thiol, amino, cyano, C 1-6 Alkyl, C2-6 alkenyl, C2-6 alkynyl, 3-6 substituted by cycloalkyl, 4-6 membered heterocyclyl, phenyl, or 5-6 membered heteroaryl;

[0017] Z2 can be independently selected from CH, C(R 2 )、N、O、S、N(R 1 ), where R 1 Can be selected from hydrogen, C 1-6 Alkyl or C 3-6 Cycloalkyl; wherein each C 1-6 Alkyl and C 3-6 The cycloalkyl group may be optionally substituted with halogen, cyano, optionally substituted R 1a , optionally substituted -OR 1a , optionally substituted -SR 1a , optionally substituted -N(R 1a )R 1a , optionally substituted -NHR 1a , optionally substituted 5-6 membered heteroaryl or optionally substituted phenyl; wherein R 1a Each occurrence is independently selected from hydrogen, C 1-6 Alkyl, C 3-6 The optional substitution means that the H on the substituted group is not substituted or one or more substitutable sites of the substituted group are independently selected from halogen, hydroxyl, thiol, amino, cyano, C 1-6 Alkyl, C2-6 alkenyl, C2-6 alkynyl, 3-6 substituted by cycloalkyl, 4-6 membered heterocyclyl, phenyl, or 5-6 membered heteroaryl;

[0018] p can be selected from 0 or 1;

[0019] When p=0, it represents that the ring is a five-membered ring; Z3 and Z5 can be independently selected from N, S, O, CH(R 2 )、C(R 2 )、N(R 1 );

[0020] When p=1, it represents that the ring is a six-membered ring; Z3, Z4, and Z5 can be independently selected from N, S, O, N(R 1 )、CH(R 2 )、C(R 2 );

[0021] X is selected from -O-, -S-, -CO-, -N(R 5 )-、-S(O)N(R 5 )-、-S(O)2N(R 5 )-、-N(R 5 )SO-、-N(R 5 )S(O)2-、-C(O)N(R 5 )-、-N(R 5 )C(O)-、-N(R 5 )C(O)N(R 5 )-、-CH(R 5 )-one of them; where R 5 Each occurrence is independently selected from hydrogen, optionally substituted C 1-6 Alkyl, optionally substituted C 3-6 cycloalkyl, an optionally substituted 4-6 membered heterocyclic group; the optional substitution means that the H on the substituted group is not substituted or one or more substitutable sites of the substituted group are independently selected from halogen, hydroxyl, thiol, amino, cyano, C 1-6 Alkyl, C2-6 alkenyl, C2-6 alkynyl, 3-6 substituted by cycloalkyl, 4-6 membered heterocyclyl, phenyl, or 5-6 membered heteroaryl;

[0022] Y is selected from a bond, -H, -OH, -NH2, halogen, -O-, -S-, -CO-, -C(R 6 )F-, -CF2-, -SO-, -SO2-, -N(R 6 )-、-S(O)N(R 6 )-、-S(O)2N(R 6 )-、-N(R 6 )SO-、-N(R 6 )S(O)2-、-C(O)N(R 6 )-、-N(R 6 )C(O)-、-CH(R 6 )-one of them; where R6 may be selected from hydrogen, optionally substituted C 1-6 Alkyl, optionally substituted C 3-6 cycloalkyl, an optionally substituted 4-6 membered heterocyclic group; the optional substitution means that the H on the substituted group is not substituted or one or more substitutable sites of the substituted group are independently selected from halogen, hydroxyl, thiol, amino, cyano, C 1-6 Alkyl, C2-6 alkenyl, C2-6 alkynyl, 3-6 substituted by cycloalkyl, 4-6 membered heterocyclic group, phenyl, 5-6 membered heteroaryl; when Y is selected from -H, -OH, -NH2, halogen, G 4 does not exist;

[0023] o=0, 1, 2, 3, 4, 5;

[0024] G 1 and G 3 can be selected from hydrogen, halogen, hydroxyl, thiol, amino, cyano, optionally substituted C 1-6 Alkyl, optionally substituted C 3-6 Cycloalkyl, optionally substituted 4-6 membered heterocyclic group; or G 1 and G 3 are linked to form an optionally substituted C 3-8 The optional substitution means that the H on the substituted group is not substituted or one or more substitutable sites of the substituted group are independently selected from halogen, hydroxyl, thiol, amino, cyano, C 1-6 Alkyl, C2-6 alkenyl, C2-6 alkynyl, 3-6 substituted by cycloalkyl, 4-6 membered heterocyclyl, phenyl, or 5-6 membered heteroaryl;

[0025] G 2 R is selected from hydrogen, halogen, hydroxy, mercapto, amino, cyano, optionally substituted 7 , optionally substituted O(R 7 )、S(R 7 ), optionally substituted NH(R 7 ), optionally substituted N(R 7 )(R 7 ) one of which; wherein R 7 Selected from hydrogen, C 1-6 Alkyl, C 3-6 The optional substitution means that the H on the substituted group is not substituted or one or more substitutable sites of the substituted group are independently selected from halogen, hydroxyl, thiol, amino, cyano, C 1-6 Alkyl, C2-6 alkenyl, C2-6 alkynyl, 3-6substituted by cycloalkyl, 4-6 membered heterocyclyl, phenyl, or 5-6 membered heteroaryl;

[0026] G 4 Selected from the following groups: optionally substituted C 1-12 Alkyl, optionally substituted C 2-12 Alkenyl, optionally substituted C 2-12 Alkynyl, optionally substituted C 3-6 Cycloalkyl, optionally substituted 4-6 membered heterocyclic group, optionally substituted C 6-10 aryl, an optionally substituted 5-10 membered heteroaryl; the optional substitution means that the H on the substituted group is not substituted or one or more substitutable sites of the substituted group are independently selected from hydrogen, halogen, hydroxyl, thiol, amino, cyano, -R 3 、-OR 3 、-NH-R 3 、-N(R 3 )R 3 , replaced by, where R 3 Each occurrence is independently selected from hydrogen, halogen, hydroxyl, mercapto, amino, cyano, C 1-6 Alkyl, C 3-6 Cycloalkyl, 4-6 membered heterocycloalkyl, phenyl, 5-6 membered heteroaryl, or one or more substituted C 1-6 Alkyl, C2-6 alkenyl, C2-6 alkynyl, 3-6 Cycloalkyl, 4-6 membered heterocyclyl, phenyl, 5-6 membered heteroaryl; D is selected from one of a bond, NH, N(CH3), O, S, and f = 0, 1, 2, 3, 4, 5, 6, 7, 8;

[0027] A can be selected from -N(R 9 )-、-CH(R 9 )- or -CH(NHR 9 )-, m = 0 or 1; wherein R 9 Can be selected from hydrogen, hydroxyl, halogen, C 1-6 Alkyl, C 3-6 One of cycloalkyl and 4-6 membered heterocyclic group;

[0028] B can be selected from -N- or -CH-;

[0029] C can be selected from -NR 10 -or-CHR 10 -, n = 0 or 1 (or n = 0, 1 or 2); R 10 Selected from hydrogen, hydroxyl, halogen, C 1-6 Alkyl, C 3-6 One of cycloalkyl and 4-6 membered heterocyclic group;

[0030] A, B, C have at least one substituted N atom that meets the respective definitions;

[0031] H ring is selected from C 6-10 An aryl ring, a 5-10 membered heteroaryl ring; the aryl ring or the heteroaryl ring may be independently substituted by one or more of the following substituents: hydrogen, halogen, hydroxyl, thiol, amino, cyano, optionally substituted -R 4 , optionally substituted -OR 4 , optionally substituted -NH-R 4 , optionally substituted -N(R 4 )R 4 ; Among them, R 4 Each occurrence is independently selected from C 1-6 Alkyl, C2-6 alkenyl, C2-6 alkynyl, 3-6 Cycloalkyl, 4-6 membered heterocyclyl, phenyl, 5-6 membered heteroaryl; the optional substitution means that the hydrogen on the substituted group is not replaced or one or more substitutable sites of the substituted group are independently selected from halogen, hydroxyl, thiol, amino, cyano, C 1-6 Alkyl, C2-6 alkenyl, C2-6 alkynyl, 3-6 The cycloalkyl group may be substituted by a 4-6 membered heterocyclic group, a phenyl group or a 5-6 membered heteroaryl group.

[0032] In some embodiments of the present invention, It represents that the connecting bond between two atoms is a double bond or a single bond, making the whole ring structure aromatic.

[0033] In some embodiments of the present invention, Indicates that the bond between the two atoms is a double bond or a single bond, making Z in the ring structure 2 The ring where Z is located is an aromatic ring. 5 The ring where it is located is a non-aromatic ring.

[0034] In some embodiments of the present invention, Indicates that the connecting bond between the two atoms is a double bond or a single bond, making Z in the ring structure 2 The ring where Z is located is a non-aromatic ring. 5 The ring where it is located is an aromatic ring.

[0035] In some embodiments of the present invention, Indicates that the connecting bond between the two atoms is a double bond or a single bond, making Z in the ring structure 2 The ring and Z 5 The rings are all non-aromatic rings.

[0036] In some embodiments of the present invention, X is selected from one of -O-, -S-, -CO-, -NH-, -SONH-, -S(O)2NH-, -NHSO-, -NHS(O)2-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, and -CH2-; for example, it is one of -O-, -S-, -CO-, -NH-, -SONH-, -S(O)2NH-, -NHSO-, -NHS(O)2-, -C(O)NH-, -NHC(O)-, and -NHC(O)NH-; for example, it is one of -O-, -S-, -CO-, -NH-, -S(O)2NH-, -NHS(O)2-, -C(O)NH-, -NHC(O)-, and -NHC(O)NH-.

[0037] In some embodiments of the present invention, Y is selected from a bond, -H, -OH, -NH2, halogen, -O-, -S-, -CO-, -CHF-, -CF2-, -SO-, -SO2-, -NH-, -S(O)NH-, -S(O)2NH-, -NHS(O)-, -NHS(O)2-, -C(O)NH-, -NHC(O)-, -CH2-; for example, a bond, H, OH, NH2, halogen, -O-, -S-, -CO-, -SO2-, -NH-, -S(O)2NH-, -NHS(O)2-, -C(O)NH-, -NHC(O)-, -CH2-.

[0038] In some embodiments of the present invention, o=0,1,2.

[0039] In some embodiments of the present invention, G 1 and G 3 are independently selected from hydrogen or methyl; or G 1 and G 3 are linked to form an optionally substituted C 5-6 carbon ring structure.

[0040] In a further embodiment, G 1 and G 3 are linked to form an optionally substituted C 5-6 Cycloalkyl structure.

[0041] In some embodiments of the present invention, G 2 One selected from hydrogen, fluorine, hydroxyl, amino, methyl, cyclopropyl, methoxy, methylthio, methylamino; for example, one selected from fluorine, hydroxyl, amino.

[0042] In some embodiments of the present invention, R 2Each occurrence is independently selected from hydrogen, halogen, hydroxy, mercapto, amino, cyano, methyl, ethyl, trifluoromethyl, difluoromethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl, phenyl, pyridinyl, pyrimidinyl.

[0043] In some embodiments of the present invention, R 3 independently selected from hydrogen, halogen, hydroxyl, thiol, amino, cyano, C 1-6 Alkyl, C 3-6 methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl, phenyl, pyridyl, pyrimidyl, or one or more substituted cycloalkyl, 4-6 membered heterocycloalkyl, phenyl, or 5-6 membered heteroaryl.

[0044] In some embodiments of the present invention, D is selected from one of a bond, -NH-, and f is selected from 0, 1, 2, and 3.

[0045] In some embodiments of the present invention, G 4 Selected from the following groups: optionally substituted C 1-12 Alkyl, optionally substituted C 2-12 Alkenyl, optionally substituted C 2-12 Alkynyl, optionally substituted C 3-6 Cycloalkyl, optionally substituted 4-6 membered heterocyclic group, optionally substituted C 6-10 The invention relates to one of aryl and optionally substituted 5-10 membered heteroaryl; the optional substitution means that the hydrogen on the substituted group is not substituted or one or more substitutable sites of the substituted group are independently substituted by a substituent selected from halogen, hydroxyl, thiol, amino, cyano, trifluoromethyl, difluoromethyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, sec-butoxy, methylamino, ethylamino, n-propylamino, isopropylamino, n-butylamino, isobutylamino, tert-butylamino and sec-butylamino.

[0046] In some embodiments of the present invention, G 4 Selected from:

[0047]

[0048] Among them, R 11 and R 12 independently selected from hydrogen, halogen, hydroxyl, thiol, amino, cyano, -R 3 、-OR 3 、-NH-R 3 、-N(R 3 )R 3 , Among them, R 3 Each occurrence is independently selected from hydrogen, halogen, hydroxyl, mercapto, amino, cyano, C 1-6 Alkyl, C 3-6 Cycloalkyl, 4-6 membered heterocycloalkyl, phenyl, 5-6 membered heteroaryl, or one or more substituted C 1-6 Alkyl, C2-6 alkenyl, C2-6 alkynyl, 3-6 Cycloalkyl, 4-6 membered heterocyclyl, phenyl, 5-6 membered heteroaryl; D is selected from one of a bond, NH, N(CH3), O, S, and f=0, 1, 2, 3, 4, 5, 6, 7, 8.

[0049] In the above embodiments, R 3 independently selected from hydrogen, halogen, hydroxyl, thiol, amino, cyano, C 1-6 Alkyl, C 3-6 methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl, phenyl, pyridyl, pyrimidyl, or one or more substituted cycloalkyl, 4-6 membered heterocycloalkyl, phenyl, or 5-6 membered heteroaryl.

[0050] In the above embodiments, D is selected from one of a bond and -NH-, and f is selected from 0, 1, 2, and 3.

[0051] In some embodiments of the present invention, D is selected from a bond, -NH-, f is selected from 0, 1, 2, 3; R 3 Selected from hydrogen, halogen, hydroxyl, thiol, amino, cyano, C 1-6 Alkyl, C 3-6 Cycloalkyl, 4-6 membered heterocycloalkyl, phenyl, 5-6 membered heteroaryl, one or more arbitrarily substituted hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, pyridyl.

[0052] In the above embodiment, D is selected from a bond, -NH-, f is selected from 0, 1, 2, 3; R 3 Selected from hydrogen, halogen, hydroxyl, thiol, amino, cyano, C 1-6 Alkyl, C 3-6 One or more arbitrarily substituted hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, pyridyl in the cycloalkyl group.

[0053] In the above embodiment, D is selected from a bond, -NH-, f is selected from 0, 1, 2, 3; R 3It is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl and pyridyl, which may be arbitrarily substituted by one or more of hydrogen, halogen and hydroxyl.

[0054] In some embodiments of the present invention, D is selected from a bond, -NH-, f is selected from 0, 1, 2, 3; R 3 independently selected from hydrogen, halogen, hydroxyl, thiol, amino, cyano, C 1-6 Alkyl, C 3-6 Cycloalkyl, 4-6 membered heterocycloalkyl, phenyl, 5-6 membered heteroaryl, one or more arbitrarily substituted furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl; for example, R 3 independently selected from hydrogen, halogen, hydroxyl, thiol, amino, cyano, C 1-3 One or more arbitrarily substituted furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl in the alkyl group; for example, R 3 Independently selected from pyrazolyl which may be arbitrarily substituted by one or more of hydrogen, halogen and methyl.

[0055] In some embodiments of the present invention, A may be independently selected from -NH-, -CH2- or -CH(NH2)-, m=0 or 1; B may be independently selected from -N- or -CH-; C may be independently selected from -NH- or -CH2-, n=0 or 1; and at least one of A, B, and C is a substituted N atom that meets their respective definitions.

[0056] In some embodiments of the present invention, You can choose from:

[0057]

[0058] In some embodiments of the present invention, You can choose from:

[0059]

[0060] In some embodiments of the invention, the H ring is selected from:

[0061]

[0062] Among them, R 13 is selected from hydrogen, halogen, hydroxy, mercapto, amino, cyano, optionally substituted -R 4 , optionally substituted -OR 4 , optionally substituted -NH-R 4 , optionally substituted -N(R 4 )R 4; Among them, R 4 Each occurrence is independently selected from C 1-6 Alkyl, C2-6 alkenyl, C2-6 alkynyl, 3-6 Cycloalkyl, 4-6 membered heterocyclyl, phenyl, 5-6 membered heteroaryl; the optional substitution means that the H on the substituted group is not substituted or one or more substitutable sites of the substituted group are independently selected from halogen, hydroxyl, thiol, amino, cyano, C 1-6 Alkyl, C2-6 alkenyl, C2-6 alkynyl, 3-6 The cycloalkyl group may be substituted by a 4-6 membered heterocyclic group, a phenyl group or a 5-6 membered heteroaryl group.

[0063] In the above embodiments, R 13 is selected from hydrogen, halogen, hydroxy, mercapto, amino, cyano, optionally substituted -R 4 , optionally substituted -OR 4 , optionally substituted -NH-R 4 , optionally substituted -N(R 4 )R 4 ; Among them, R 4 Each occurrence is independently selected from C 1-6 alkyl, 4-6 membered heterocyclic group, phenyl, 5-6 membered heteroaryl; the optional substitution means that the H on the substituted group is not substituted or one or more substitutable sites of the substituted group are independently selected from halogen, hydroxyl, thiol, amino, cyano, C 1-6 Alkyl, C 3-6 Substituted by cycloalkyl or phenyl.

[0064] In some embodiments of the present invention, Can be selected from formula (II) A , (II) B , (II) C :

[0065]

[0066] In a further embodiment, formula (II) A You can choose from:

[0067]

[0068] In a further embodiment, formula (II) B You can choose from:

[0069]

[0070] In a further embodiment, formula (II) C You can choose from:

[0071]

[0072] In some embodiments of the present invention, formula (II) A Selected from:

[0073] wherein each Rx is independently selected from hydrogen, C optionally substituted by halogen 1-6 Alkyl (such as CH3, -CF3), halogen (such as fluorine), cyano or 6-10 membered aryl (such as phenyl);

[0074] Formula (II) B Selected from

[0075] Formula (II) C Selected from

[0076] Selected from: The H ring is selected from wherein each R13 when present is independently selected from hydrogen or halogen (such as fluorine);

[0077] O = 1 or 2;

[0078] X is selected from -O-, -S-, -CH2-, -N(R 5 )-,R 5 are independently selected from hydrogen or optionally substituted C 1-6 Alkyl (such as methyl);

[0079] G1 is selected from hydrogen,

[0080] G2 is selected from hydroxyl or amino;

[0081] G3 is selected from hydrogen;

[0082] Y is selected from a bond, -NH2, -OH, -NH-; and

[0083] G4 is selected from an optionally substituted 4-6 membered heterocyclic group, an optionally substituted 5-10 membered heteroaryl group (such as ); wherein R11 is selected from hydrogen or C 1-6 Alkyl (such as methyl); R12 is selected from wherein D is selected from a bond; f = 0; each R3 is independently selected from C 1-6 Alkyl, optionally C 1-6 5-6 membered heteroaryl (eg 5 membered nitrogen-containing heteroaryl) substituted by alkyl (eg methyl).

[0084] In some embodiments of the present invention, formula (II) A Selected from wherein each Rx is independently selected from hydrogen, C optionally substituted by halogen 1-6 Alkyl or 6-10 membered aryl;

[0085] Formula (II) B Selected from

[0086] Formula (II) C Selected from

[0087] Selected from: The H ring is selected from m=0;

[0088] O = 1;

[0089] X is selected from S or NH;

[0090] G1 is selected from hydrogen;

[0091] G2 is selected from hydroxyl group;

[0092] G3 is selected from hydrogen;

[0093] Y is selected from -NH-; and

[0094] G4 is selected from Wherein, R11 is selected from hydrogen; R12 is selected from wherein D is selected from a bond; f = 0; R3 is selected from C 1-6 Alkyl, optionally C 1-6 5-6 membered heteroaryl (eg 5 membered nitrogen-containing heteroaryl) substituted by alkyl (eg methyl); R13 is selected from hydrogen or halogen (eg fluorine).

[0095] In some embodiments of the present invention, the compound represented by formula (I), and its stereoisomers, geometric isomers, tautomers, pharmaceutically acceptable salts, prodrugs, hydrates, solvates or isotope-labeled analogs are selected from the following compounds:

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103] The present invention also aims to provide a method for preparing the compound represented by general formula (I), and its stereoisomers, geometric isomers, tautomers, pharmaceutically acceptable salts, prodrugs, hydrates, solvates or isotope-labeled analogs.

[0104] The method can be prepared by the method shown in Scheme 1, for example, by firstly reacting the LG a (leaving group a) and G 4 -YH connection, and then the LG of the ring structure b (Leaving group b) is connected to the side chain of XH to synthesize the target compound. Scheme 1 is as follows:

[0105]

[0106] The method can be prepared by the method shown in Scheme 2, for example, by firstly reacting the LG b (Leaving group b) is connected to the side chain of XH, and then the LG of the ring structure is a (leaving group a) and G 4 -YH connection, the target compound was synthesized. Scheme 2 is as follows:

[0107]

[0108] Wherein, in the above preparation method, the definitions of the substituents in the compounds shown are as described above.

[0109] The present invention also provides a pharmaceutical composition, which comprises the compound (I) shown in the present invention, or its stereoisomer, geometric isomer, tautomer, pharmaceutical salt, prodrug, hydrate, solvate or isotope-labeled analog, and pharmaceutically acceptable excipients. The pharmaceutical composition includes but is not limited to oral dosage forms, parenteral dosage forms, external dosage forms and rectal dosage forms. In some embodiments, the amount of the compound applied is, for example, in the range of about 0.001mg / kg body weight / day to about 10g / kg body weight / day. In other embodiments, a dosage level lower than the lower limit of the above range may be sufficient. In some embodiments, a dosage level higher than the upper limit of the above range may be required. In some embodiments, the individual to whom the pharmaceutical composition is applied is a mammal, such as a human.

[0110] The purpose of the present invention also includes providing the use of the compound (I) shown in the present invention, or its stereoisomers, geometric isomers, tautomers, pharmaceutically acceptable salts, prodrugs, hydrates, solvates or isotope-labeled analogs in the preparation of drugs for treating diseases mediated by PRMT5 inhibitors.

[0111] The purpose of the present invention also includes providing the compound (I) shown in the present invention, or its stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, prodrug, hydrate, solvate or isotope-labeled analog, which is used to treat diseases mediated by PRMT5 inhibitors.

[0112] The purpose of the present invention also includes providing a method for preventing and / or treating diseases mediated by PRMT5 inhibitors, which comprises administering to a patient a therapeutically effective dose of a compound represented by general formula (I), or its stereoisomers, geometric isomers, tautomers, pharmaceutically acceptable salts, prodrugs, hydrates, solvates, isotope-labeled analogs or the pharmaceutical composition of the present invention.

[0113] In some embodiments, the disease mediated by the PRMT5 inhibitors described herein is cancer.

[0114] definition

[0115] Unless otherwise specified, the term "alkyl" refers to a monovalent saturated aliphatic hydrocarbon group, a straight or branched chain group containing 1 to 20 carbon atoms, preferably containing 1 to 10 carbon atoms (i.e., C 1-10 Alkyl), further preferably containing 1 to 8 carbon atoms (C 1-8 Alkyl), more preferably containing 1 to 6 carbon atoms (i.e. C 1-6 Alkyl), such as "C 1-6"Alkyl" means that the group is an alkyl group and the number of carbon atoms in the carbon chain is between 1 and 6 (specifically 1, 2, 3, 4, 5 or 6). Examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, neopentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, n-heptyl, n-octyl, etc.

[0116] Unless otherwise specified, the term "cycloalkyl" refers to a monocyclic saturated aliphatic hydrocarbon group having a specified number of carbon atoms, preferably containing 3 to 12 carbon atoms (i.e., C 3-12 cycloalkyl), more preferably containing 3 to 10 carbon atoms (C 3-10 Cycloalkyl), preferably 3 to 6 carbon atoms (C 3-6 Cycloalkyl), 4-6 carbon atoms (C 4-6 Cycloalkyl), 5-6 carbon atoms (C 5-6 Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methylcyclopropyl, 2-ethyl-cyclopentyl, dimethylcyclobutyl, and the like.

[0117] Unless otherwise specified, the term "alkoxy" refers to -O-alkyl, wherein the alkyl is as defined above, i.e., contains 1-20 carbon atoms, preferably, contains 1-10 carbon atoms, more preferably 1-8 carbon atoms, more preferably 1-6 carbon atoms (specifically 1, 2, 3, 4, 5 or 6). Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, 1-methylpropoxy, 2-methylpropoxy, tert-butoxy, pentyloxy, 1-methylbutoxy, 2-methylbutoxy, 3-methylbutoxy, 1,1-dimethylpropoxy, 1,2-dimethylpropoxy, 2,2-dimethylpropoxy, 1-ethylpropoxy, and the like.

[0118] Unless otherwise specified, the term "alkenyl" refers to a straight or branched unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms and having at least one double bond. The alkenyl group may contain 2 to 20 carbon atoms, preferably 2 to 10 carbon atoms (i.e., C 2-10 Alkenyl), further preferably containing 2 to 8 carbon atoms (C 2-8 Alkenyl), more preferably containing 2 to 6 carbon atoms (i.e., C 2-6 alkenyl), 2-5 carbon atoms (i.e. C 2-5 alkenyl), 2-4 carbon atoms (i.e. C 2-4 alkenyl), 2-3 carbon atoms (i.e. C 2-3 alkenyl), 2 carbon atoms (i.e., C2 alkenyl), for example, "C 2-6"Alkenyl" means that the group is an alkenyl group, and the number of carbon atoms in the carbon chain is between 2 and 6 (specifically 2, 3, 4, 5 or 6). Non-limiting examples of alkenyl include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 1-butenyl, isobutenyl and 1,3-butadienyl, etc.

[0119] Unless otherwise specified, the term "alkynyl" refers to a straight or branched unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms and having at least one triple bond. The alkynyl group may contain 2 to 20 carbon atoms, preferably 2 to 10 carbon atoms (i.e., C 2-10 Alkynyl), further preferably containing 2 to 8 carbon atoms (C 2-8 Alkynyl), more preferably containing 2 to 6 carbon atoms (i.e. C 2-6 Alkynyl), 2-5 carbon atoms (i.e. C 2-5 Alkynyl), 2-4 carbon atoms (i.e. C 2-4 Alkynyl), 2-3 carbon atoms (i.e. C 2-3 alkynyl), 2 carbon atoms (i.e., C2 alkynyl), for example, "C 2-6 "Alkynyl" means that the group is an alkynyl group, and the number of carbon atoms in the carbon chain is between 2 and 6 (specifically 2, 3, 4, 5 or 6). Non-limiting examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl and 1-butynyl, etc.

[0120] Unless otherwise specified, the term "halogen" or "halo" refers to F, Cl, Br, I. The term "haloalkyl" refers to an alkyl group as defined above in which one, two or more hydrogen atoms or all hydrogen atoms are replaced by halogen. Representative examples of haloalkyl include CCl3, CF3, CHCl2, CH2Cl, CH2Br, CH2I, CH2CF3, CF2CF3, etc.

[0121] Unless otherwise specified, the term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic, bicyclic or polycyclic hydrocarbon substituent, which is a non-aromatic structure, containing 3-20 ring atoms, wherein 1, 2, 3 or more ring atoms are selected from N, O or S, and the remaining ring atoms are C. Preferably, it contains 3-12 ring atoms, and further preferably contains 3-10 ring atoms, or 3-8 ring atoms, or 3-6 ring atoms, or 4-6 ring atoms, or 5-6 ring atoms. The heteroatoms are preferably 1-4, more preferably 1-3 (i.e., 1, 2 or 3). Examples of monocyclic heterocyclic groups include pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, dihydropyrrolyl, piperidinyl, piperazinyl, pyranyl, etc. Polycyclic heterocyclic groups include spirocyclic, fused ring and bridged heterocyclic groups. Heterocyclic groups also include aromatic groups (such as phenyl) fused saturated heterocyclic groups. Unless otherwise specified, the term "heterocycloalkyl" refers to a saturated "heterocyclyl" as defined above.

[0122] Unless otherwise specified, the term "carbocyclyl" or "carbocycle" refers to a non-aromatic cyclic hydrocarbon radical ("C 3-14 In some embodiments, the carbocyclyl group has 3-12 ring carbon atoms ("C 3-12 carbocyclyl”), or 4-12 ring carbon atoms (“C 4-12 carbocyclyl"), or 3 to 10 ring carbon atoms ("C 3-10 In some embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms (“C 3-8 In some embodiments, a carbocyclyl group has 3 to 7 ring carbon atoms (“C 3-7 In some embodiments, a carbocyclyl group has 4 to 6 ring carbon atoms (“C 4-6 In some embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms (“C 5-10 carbocyclyl"), or 5 to 7 ring carbon atoms ("C 5-7 "Carbocyclyl"). Exemplary C 3-6 Carbocyclyl groups include, but are not limited to, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. Exemplary C 3-8 Carbocyclyl groups include, but are not limited to, the aforementioned C 3-6 Carbocyclyl groups and cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptanyl (C7), bicyclo[2.2.2]octanyl (C8), etc. Exemplary C 3-10 Carbocyclyl groups include, but are not limited to, the aforementioned C 3-8 Carbocyclic groups and cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthyl (C 10 ), spiro[4.5]decyl (C 10) and the like. As illustrated in the above examples, in certain embodiments, the carbocyclyl group is a monocyclic ("monocyclic carbocyclyl") or a fused (fused cyclyl), bridged (bridged cyclyl) or spiro-fused (spirocyclyl) ring system, such as a bicyclic system ("bicyclic carbocyclyl") and can be saturated or can be partially unsaturated. "Carbocyclyl" also includes a ring system in which the carbocyclyl ring as defined above is fused by one or more aryl or heteroaryl groups, wherein the attachment point is on the carbocyclyl ring, and in such cases, the number of carbons continues to indicate the number of carbons in the carbocyclyl system. In certain embodiments, each example of a carbocyclyl group is independently optionally substituted, for example, unsubstituted (a "unsubstituted carbocyclyl") or substituted by one or more substituents (a "substituted carbocyclyl"). In certain embodiments, the carbocyclyl group is unsubstituted C 3-10 In certain embodiments, the carbocyclyl group is a substituted C 3-10 Carbocyclic group.

[0123] Unless otherwise specified, the term "aryl" refers to a monocyclic, bicyclic and tricyclic aromatic carbocyclic ring system containing 6-16 carbon atoms, or 6-14 carbon atoms, or 6-12 carbon atoms, or 6-10 carbon atoms, preferably 6-10 carbon atoms, and the term "aryl" can be used interchangeably with the term "aromatic ring". Examples of aryl groups may include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthrenyl or pyrenyl, etc.

[0124] Unless otherwise specified, the term "heteroaryl" means an aromatic monocyclic or polycyclic ring system containing 5-12 members, or preferably 5-10 members, 5-8 members, and more preferably 5-6 members, wherein 1, 2, 3 or more ring atoms are heteroatoms and the remaining atoms are carbon, the heteroatoms being independently selected from O, N or S, and the number of heteroatoms being preferably 1, 2 or 3. Examples of heteroaryl include, but are not limited to, furanyl, thienyl, oxazolyl, thiazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, thiodiazolyl, triazinyl, phthalazinyl, quinolyl, isoquinolyl, pteridinyl, purinyl, indolyl, isoindolyl, indazolyl, benzofuranyl, benzothienyl, benzopyridinyl, benzopyrimidinyl, benzo pyrazinyl, benzimidazolyl, benzophthalazinyl, pyrrolo[2,3-b]pyridinyl, imidazo[1,2-a]pyridinyl, pyrazolo[1,5-a]pyridinyl, pyrazolo[1,5-a]pyrimidinyl, imidazo[1,2-b]pyridazinyl, [1,2,4]triazolo[4,3-b]pyridazinyl, [1,2,4]triazolo[1,5-a]pyrimidinyl, [1,2,4]triazolo[1,5-a]pyridinyl, and the like.

[0125] Unless otherwise specified, the term "pharmaceutically acceptable salt", "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to salts that are suitable for contact with mammalian tissues, especially human tissues, within the scope of reasonable medical judgment without excessive toxicity, irritation, allergic response, etc. and commensurate with a reasonable benefit / risk ratio. For example, medically acceptable salts of amines, carboxylic acids and other types of compounds are well known in the art.

[0126] Unless otherwise specified, the term "salt" includes salts derived from inorganic acids as well as salts derived from organic acids. If the compound of the present invention is acidic, pharmaceutically acceptable non-toxic bases include salts prepared from inorganic bases and organic bases.

[0127] Unless otherwise specified, the term "isotopically labeled analogs" means that one or more protons in the compounds of Formula I to Formula II may be replaced by deuterium atoms, thereby providing deuterated analogs that may have improved pharmacological activity.

[0128] Unless otherwise specified, the term "prodrug" refers to a drug that is converted into the parent drug in vivo. Prodrugs are often useful because, in some cases, they may be easier to administer than the parent drug. For example, they may be bioavailable by oral administration, whereas the parent drug is not. Prodrugs also have improved solubility in pharmaceutical compositions compared to the parent drug. An example of a prodrug, but not limited to, would be any compound of Formula I administered as an ester ("prodrug") to facilitate transport across cell membranes, where water solubility is detrimental to mobility, but once inside the cell water solubility is beneficial, which is then metabolically hydrolyzed to a carboxylic acid, the active entity. Another example of a prodrug would be a short peptide (polyamino acid) bound to an acid group, where the peptide is metabolized to reveal the active portion.

[0129] Unless otherwise specified, the terms "solvate" and "solvate" mean a physical association of a compound of the invention with one or more solvent molecules, whether organic or inorganic. The physical association includes hydrogen bonding. In certain cases, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvate will be able to be separated. The solvent molecules in the solvate may exist in a regular arrangement and / or a disordered arrangement. The solvate may contain stoichiometric or non-stoichiometric amounts of solvent molecules. "Solvate" encompasses solution phases and separable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Solvation methods are well known in the art.

[0130] Unless otherwise specified, the term "stereoisomer" refers to compounds with the same chemical constitution but different arrangements of atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformers (rotamers), geometric isomers (cis / trans) isomers, atropisomers, etc. Any resulting mixture of stereoisomers can be separated into pure or substantially pure geometric isomers, enantiomers, diastereomers, for example, by chromatography and / or fractional crystallization based on the differences in the physical and chemical properties of the components.

[0131] Unless otherwise specified, the term "tautomer" refers to structural isomers with different energies that can be interconverted through a low energy barrier. If tautomerism is possible (such as in solution), a chemical equilibrium of tautomers can be achieved. For example, proton tautomers (also known as prototropic tautomers) include interconversions by proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions by reorganization of some of the bonding electrons.

[0132] Unless otherwise indicated, the structural formulas described in the present invention include all isomeric forms (such as enantiomers, diastereomers, and geometric isomers (or conformational isomers)): for example, R, S configurations containing asymmetric centers, (Z), (E) isomers of double bonds, and (Z), (E) conformational isomers. Therefore, single stereochemical isomers of the compounds of the present invention or mixtures of their enantiomers, diastereomers, or geometric isomers (or conformational isomers) are all within the scope of the present invention.

[0133] Unless otherwise specified, the term "optionally substituted" means that the hydrogen at the substitutable position of the group is not replaced, or is replaced by one or more substituents, the substituents being preferably selected from the following group: halogen, hydroxyl, thiol, cyano, nitro, amino, azido, oxo, carboxyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-10 Cycloalkyl, C 3-10 Cycloalkylsulfonyl, 3-10 membered heterocycloalkyl, C 6-14 aryl or 5-10 membered heteroaromatic ring group, wherein the C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-10 Cycloalkyl, C 3-10 Cycloalkylsulfonyl, 3-10 membered heterocycloalkyl, C 6-14 The aryl or 5-10 membered heteroaromatic ring group may be optionally selected from halogen, hydroxy, amino, cyano, C1-6 Alkyl or C 1-6 The alkoxy group is substituted by one or more of the alkoxy groups, and the oxo group means that two H at the same substitution position are replaced by the same O to form a double bond.

[0134] The beneficial effects of the present invention are at least one of the following:

[0135] The present invention designs a class of novel compounds, which provides a new direction for the development of PRMT5 inhibitor drugs. In vitro enzyme inhibition activity studies show that these compounds have a strong inhibitory effect on PRMT5 enzymes and can be used as promising compounds for treating diseases mediated by PRMT5 inhibitors. In addition, the present invention studies a specific synthesis method, which has a simple process, convenient operation, and is conducive to large-scale industrial production and application. DETAILED DESCRIPTION

[0136] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually performed under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise defined, all professional and scientific terms used in the text have the same meanings as those familiar to professionals in the field. In addition, any method and material similar or equivalent to the described content can be applied to the method of the present invention. The preferred implementation methods and materials shown in the text are for demonstration purposes only.

[0137] The structure of the compound of the present invention is determined by nuclear magnetic resonance (NMR) or / and liquid chromatography-mass spectrometry (LC-MS) or / and liquid chromatography (HPLC). The instrument used for NMR determination is Bruker AVANCE NEO 400MHz, the instrument used for LC-MS is LCMS WATERS ACQUITY UPLC H-Class PLUS or / and SQD2; the instrument used for HPLC is WATERS ACQUITY UPLC or / and Agilent 1260.

[0138] The starting materials in the examples of the present invention are known and can be purchased on the market, or can be synthesized by or according to methods known in the art.

[0139] Example 1

[0140] Preparation of 1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((4-(3-oxetanylamino)pyridin[3,4-d]pyrimidin-8-yl)amino)propan-2-ol

[0141]

[0142] Step 1: Preparation of 2-(2-methyl-1-oxirane)-1,2,3,4-tetrahydroisoquinoline (Intermediate 1-a)

[0143]

[0144] The compound tetrahydroisoquinoline (4g, 30.04mmol, 1.0eq) was dissolved in tetrahydrofuran (150ml), cooled to 0°C, and KF (3.48g, 60.08mmol, 2.0eq) was added under nitrogen protection. After 1h at 0°C, the compound glycidyl m-nitrobenzenesulfonate (8.8g, 33.95mmol, 1.13eq) was added, and the reaction solution was warmed to room temperature (20-25°C) and reacted for 18 hours. After the reaction was completed, the mother liquor was filtered and concentrated to obtain 6.02g of crude 2-(oxirane-2-methyl)-1,2,3,4-tetrahydroisoquinoline, with a yield of 100%. 1 H NMR (400MHz, CDCl3) δ: 7.16-7.07 (m, 3H), 7.07-7.00 (m, 1H), 3.85-3.66 (m, 2H), 3.21 (tt, J=6.8, 2.3Hz , 1H), 2.99-2.88 (m, 4H), 2.87-2.77 (m, 2H), 2.56 (dd, J=5.0, 2.7Hz, 1H), 2.46 (dd, J=13.3, 6.7Hz, 1H).

[0145] Step 2: Preparation of 1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)-propan-2-ol (Intermediate 1-b)

[0146]

[0147] 2-(2-Methyl-1-oxirane)-1,2,3,4-tetrahydroisoquinoline (5.6 g, 29.62 mmol, 1.0 eq) was dissolved in methanol (150 ml), and 25% (mass fraction) ammonia water (45.6 ml, 296.2 mmol, 10.0 eq) was added. The reaction solution was heated to 45°C and stirred overnight. After the reaction was completed, dichloromethane was added for extraction three times, 40 ml each time, dried over anhydrous sodium sulfate for 10 minutes, filtered, concentrated, and the crude product was separated by column chromatography to obtain 3 g of the intermediate 1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)-propan-2-ol, with a yield of 49%. LCMS: [M+1] + =207.11.

[0148] Step 3: Preparation of 3-amino-2-chloroisonicotinamide (Intermediate 1-c)

[0149]

[0150] The raw material 3-amino-2-chloroisonicotinic acid methyl ester (3g, 16.1mmol) was dissolved in 7 molar ammonia methanol solution (120mL) and stirred at room temperature (20-25°C) for 2 days. After TLC detection, the crude product was directly concentrated, 15 ml of ethyl acetate was added, and the mixture was beaten at 15°C for 10 minutes, and filtered to obtain 2.1g of white solid, i.e. 3-amino-2-chloroisonicotinamide, with a yield of 76.14%. LCMS: [M+H] + =171.95.

[0151] Step 4: Preparation of 8-chloro-3H-pyrido[3,4-d]pyrimidin-4-one (Intermediate 1-d)

[0152]

[0153] The raw material 3-amino-2-chloroisonicotinamide (2.1 g, 12.3 mmol) was dissolved in trimethyl orthoformate (60 mL), heated under reflux for 16 hours, and after the reaction was completed, cooled to room temperature, filtered to obtain a crude solid, added with 5 ml of ethyl acetate to slurry at room temperature, filtered, and dried to obtain 8-chloro-3H-pyrido[3,4-d]pyrimidin-4-one, 1.4 g of white solid, yield 62.9%. LCMS: [M+H] + =182.06; 1 H NMR (400MHz, DMSO) δ: 12.85 (s, 1H), 8.44 (d, J=5.1Hz, 1H), 8.32 (s, 1H), 7.97 (d, J=5.1Hz, 1H).

[0154] Step 5: Preparation of 8-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)pyridin[3,4-d]pyrimidin-4-ol (Intermediate 1-e)

[0155]

[0156] After dissolving 8-chloro-3H-pyrido[3,4-d]pyrimidin-4-one (200 mg, 1.1 mmol) and 1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)-propan-2-ol (340 mg, 1.65 mmol) in NMP (N-methylpyrrolidone) (6 mL), triethylamine (0.55 g, 5.5 mmol) was added and the reaction solution was heated to 130°C for overnight reaction (18 hours). After the reaction was completed, the reaction solution was poured into 50 ml of water, extracted with ethyl acetate three times, 15 ml each time, the ethyl acetate phases were combined, washed once with 20 ml of water, washed once with 20 ml of saturated brine, dried over anhydrous sodium sulfate for 10 minutes, filtered, concentrated, and separated by column chromatography (MeOH: DCM = 1: 15) to obtain 8-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)pyridin[3,4-d]pyrimidin-4-ol 330 mg of white solid, yield 85.7%. LCMS: [M+H] + =352.14.

[0157] Step 6: Preparation of 1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((4-(3-oxetanylamino)pyridin[3,4-d]pyrimidin-8-yl)amino)propan-2-ol (Compound of Example 1)

[0158]

[0159] Compound 8-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)pyridin[3,4-d]pyrimidin-4-ol (90 mg, 0.255 mmol), oxetane-3-amine (28 mg, 0.385 mmol) and PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate) (172.5 mg, 0.33 mmol) were dissolved in DMF (N,N-dimethylformamide) (1 .5mL), DBU (1,8-diazabicycloundec-7-ene) (58mg, 0.385mmol) was added, stirred at room temperature (10-15°C) for 2 hours, the reaction solution was poured into 10 ml of water, extracted with ethyl acetate three times, 6 ml each time, the ethyl acetate phase was combined, washed once with 10 ml of water, washed once with 10 ml of saturated brine, dried over anhydrous sodium sulfate for 5 minutes, filtered, concentrated, and purified by preparative HPLC to obtain 62 mg of the target compound, with a yield of 59%. LCMS: [M+H]+=407.44; 1H NMR (400MHz, DMSO-d6) δ: 8.63 (d, J=5.3Hz, 1H), 8.38 (s, 1H), 7.88 (s, 1H), 7.43 (t, J=5.5Hz, 1H), 7 .18(d, J=6.0Hz, 1H), 7.12-7.06(m, 3H), 7.04-6.97(m, 1H), 5.22-5.09(m, 1H), 5.09(s, 1H), 4.86(t , J=6.9Hz, 2H), 4.65 (t, J=6.4Hz, 2H), 4.09-3.89 (m, 1H), 3.73-3.55 (m, 3H), 3.49-3.38 (m, 1H), 2. 84 (t, J=5.4Hz, 2H), 2.76 (dt, J=11.2, 5.5Hz, 1H), 2.68 (dd, J=11.3, 6.1Hz, 1H), 2.58-2.52 (m, 2H).

[0160] Example 2

[0161] Preparation of (S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((4-(3-oxetanylamino)pyridin[3,4-d]pyrimidin-8-yl)amino)propan-2-ol

[0162]

[0163] Step 1: Preparation of (R)-2-(2-methyl-1-oxirane)-1,2,3,4-tetrahydroisoquinoline (Intermediate 2-a)

[0164]

[0165] The compound tetrahydroisoquinoline (1 g, 7.51 mmol, 1.0 eq) was dissolved in tetrahydrofuran (38 mL), cooled to 0°C, and KF (870 mg, 15.02 mmol, 2.0 eq) was added under nitrogen protection. After reacting at 0°C for 1 h, the compound (S)-(+)-glycidyl m-nitrobenzenesulfonate (2.2 g, 8.49 mmol, 1.13 eq) was added, and the reaction solution was warmed to room temperature and reacted overnight. After the reaction was completed, the mother liquor was filtered and concentrated to obtain 1.5 g of crude (R)-2-(oxirane-2-methyl)-1,2,3,4-tetrahydroisoquinoline, with a yield of 100%. 1H NMR (400MHz, CDCl3) δ: 7.16-7.07 (m, 3H), 7.07-7.00 (m, 1H), 3.85-3.66 (m, 2H), 3.21 (tt, J=6.8, 2.3Hz , 1H), 2.99-2.88 (m, 4H), 2.87-2.77 (m, 2H), 2.56 (dd, J=5.0, 2.7Hz, 1H), 2.46 (dd, J=13.3, 6.7Hz, 1H).

[0166] Step 2: Preparation of (S)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)-propan-2-ol (Intermediate 2-b)

[0167]

[0168] (R)-2-(2-methyl-2-oxirane)-1,2,3,4-tetrahydroisoquinoline (280 mg, 1.481 mmol, 1.0 eq) was dissolved in methanol (7.4 ml), and 25% (mass fraction) ammonia water (2.3 ml, 14.81 mmol, 10.0 eq) was added. The reaction solution was heated to 45°C and stirred overnight. After the reaction was completed, dichloromethane was added for extraction three times, 10 ml each time, and dried over anhydrous sodium sulfate for 10 minutes, filtered, concentrated, and the crude product was separated by column chromatography to obtain 147 mg of the intermediate (S)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)-propan-2-ol, with a yield of 48.2%. LCMS: [M+1] + =207.11.

[0169] Step 3: Preparation of (S)-8-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)pyridin[3,4-d]pyrimidin-4-ol (Intermediate 2-c)

[0170]

[0171] 8-Chloro-3H-pyrido[3,4-d]pyrimidin-4-one (400 mg, 2.2 mmol) (i.e., intermediate 1-d, see Example 1 for preparation method) and (S)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)-propan-2-ol (680 mg, 3.3 mmol) were dissolved in NMP (N-methylpyrrolidone) (11 mL), and triethylamine (1.1 g, 11 mmol) was added. The reaction solution was heated to 130° C. and reacted overnight. After the reaction was completed, the reaction solution was poured into 50 ml of water, extracted with ethyl acetate three times, each time with 15 ml, the ethyl acetate phases were combined, washed once with 20 ml of water, washed once with 20 ml of saturated brine, dried over anhydrous sodium sulfate for 10 minutes, filtered, concentrated, and separated by column chromatography (MeOH: DCM = 1: 15) to obtain (S)-8-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)pyridin[3,4-d]pyrimidin-4-ol 660 mg of white solid, yield 85.7%. LCMS: [M+H] + =352.14.

[0172] Step 4: Preparation of (S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((4-(3-oxetanylamino)pyridin[3,4-d]pyrimidin-8-yl)amino)propan-2-ol (Compound of Example 2)

[0173]

[0174] Compound (S)-8-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)pyridin[3,4-d]pyrimidin-4-ol (180 mg, 0.51 mmol), oxetane-3-amine (56 mg, 0.77 mmol) and PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate) (345 mg, 0.66 mmol) were dissolved in DMF (N,N-dimethylformamide) (2. 5mL), DBU (1,8-diazabicycloundec-7-ene) (116mg, 0.77mmol) was added, and stirred at room temperature (10-15°C) for 2 hours. The reaction solution was poured into 10 ml of water, extracted with ethyl acetate three times, each time with 6 ml, and the ethyl acetate phases were combined, washed once with 10 ml of water and once with 10 ml of saturated brine, dried over anhydrous sodium sulfate for 5 minutes, filtered, concentrated, and purified by preparative HPLC to obtain 122 mg of the target compound with a yield of 58.6%. LCMS: [M+H] + =407.44; 1H NMR (400MHz, DMSO-d6) δ: 8.63 (d, J=5.3Hz, 1H), 8.38 (s, 1H), 7.88 (s, 1H), 7.43 (t, J=5.5Hz, 1H), 7 .18(d, J=6.0Hz, 1H), 7.12-7.06(m, 3H), 7.04-6.97(m, 1H), 5.22-5.09(m, 1H), 5.09(s, 1H), 4.86(t , J=6.9Hz, 2H), 4.65 (t, J=6.4Hz, 2H), 4.09-3.89 (m, 1H), 3.73-3.55 (m, 3H), 3.49-3.38 (m, 1H), 2. 84 (t, J=5.4Hz, 2H), 2.76 (dt, J=11.2, 5.5Hz, 1H), 2.68 (dd, J=11.3, 6.1Hz, 1H), 2.58-2.52 (m, 2H).

[0175] Example 3

[0176] Preparation of (R)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((4-(3-oxetanylamino)pyridin[3,4-d]pyrimidin-8-yl)amino)propan-2-ol

[0177]

[0178] Step 1: Preparation of (S)-2-(2-(ethylene oxide)-1,2,3,4-tetrahydroisoquinoline (Intermediate 3-a)

[0179]

[0180] The compound tetrahydroisoquinoline (1.8 g, 13.518 mmol, 1.0 eq) was dissolved in tetrahydrofuran (75 mL), cooled to 0°C, and KF (1.566 g, 27.03 mmol, 2.0 eq) was added under nitrogen protection. After reacting at 0°C for 1 hour, the compound (R)-oxyethylene-2-ylmethyl 3-nitrobenzenesulfonate (3.96 g, 15.273 mmol, 1.13 eq) was added, and the reaction solution was warmed to room temperature and reacted overnight. After the reaction was completed, the reaction was filtered and the mother liquor was concentrated to obtain 2.7 g of crude (R)-2-(oxirane-2-methyl)-1,2,3,4-tetrahydroisoquinoline, with a yield of 100%. 1H NMR (400MHz, CDCl3) δ: 7.16-7.07 (m, 3H), 7.07-7.00 (m, 1H), 3.85-3.66 (m, 2H), 3.21 (tt, J=6.8, 2.3Hz , 1H), 2.99-2.88 (m, 4H), 2.87-2.77 (m, 2H), 2.56 (dd, J=5.0, 2.7Hz, 1H), 2.46 (dd, J=13.3, 6.7Hz, 1H).

[0181] Step 2: Preparation of (R)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)-propan-2-ol (Intermediate 3-b)

[0182]

[0183] (S)-2-(2-methyl-1-oxirane)-1,2,3,4-tetrahydroisoquinoline (1.4 g, 7.405 mmol, 1.0 eq) was dissolved in methanol (74 mL), and 25% (mass fraction) ammonia water (11.3 ml, 74.05 mmol, 10.0 eq) was added. The reaction solution was heated to 45°C and stirred overnight. After the reaction was completed, dichloromethane was added for extraction three times, 30 ml each time, dried over anhydrous sodium sulfate for 10 minutes, filtered, concentrated, and the crude product was separated by column chromatography to obtain 735 mg of the intermediate (R)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)-propan-2-ol, with a yield of 48.2%. LCMS: [M+1] + =207.11.

[0184] Step 3: Preparation of (R)-8-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)pyridin[3,4-d]pyrimidin-4-ol (Intermediate 3-c)

[0185]

[0186] 8-Chloro-3H-pyrido[3,4-d]pyrimidin-4-one (400 mg, 2.2 mmol) (i.e., intermediate 1-d, see Example 1 for preparation method) and (R)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)-propan-2-ol (680 mg, 3.3 mmol) were dissolved in NMP (N-methylpyrrolidone) (11 mL), and triethylamine (1.1 g, 11 mmol) was added. The reaction solution was heated to 130° C. and reacted overnight. After the reaction was completed, the reaction solution was poured into 50 ml of water, extracted with ethyl acetate three times, each time with 15 ml, the ethyl acetate phases were combined, washed once with 20 ml of water, washed once with 20 ml of saturated brine, dried over anhydrous sodium sulfate for 10 minutes, filtered, concentrated, and separated by column chromatography (MeOH: DCM = 1: 15) to obtain 660 mg of (R)-8-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)pyridin[3,4-d]pyrimidin-4-ol as a white solid, with a yield of 85.7%. LCMS: [M+H] + =352.14.

[0187] Step 4: Preparation of (R)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((4-(3-oxetanylamino)pyridin[3,4-d]pyrimidin-8-yl)amino)propan-2-ol (Compound of Example 3)

[0188]

[0189] Compound (R)-8-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)pyridin[3,4-d]pyrimidin-4-ol (180 mg, 0.51 mmol), oxetane-3-amine (56 mg, 0.77 mmol) and PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate) (345 mg, 0.66 mmol) were dissolved in DMF (N,N-dimethylformamide) (2. 5mL), DBU (1,8-diazabicycloundec-7-ene) (116mg, 0.77mmol) was added, and stirred at room temperature (10-15°C) for 2 hours. The reaction solution was poured into 10 ml of water, extracted with ethyl acetate three times, each time with 6 ml, and the ethyl acetate phases were combined, washed once with 10 ml of water and once with 10 ml of saturated brine, dried over anhydrous sodium sulfate for 5 minutes, filtered, concentrated, and purified by preparative HPLC to obtain 122 mg of the target compound with a yield of 58.6%. LCMS: [M+H] + =407.44; 1H NMR (400MHz, DMSO-d6) δ: 8.63 (d, J=5.3Hz, 1H), 8.38 (s, 1H), 7.88 (s, 1H), 7.43 (t, J=5.5Hz, 1H), 7 .18(d, J=6.0Hz, 1H), 7.12-7.06(m, 3H), 7.04-6.97(m, 1H), 5.22-5.09(m, 1H), 5.09(s, 1H), 4.86(t , J=6.9Hz, 2H), 4.65 (t, J=6.4Hz, 2H), 4.09-3.89 (m, 1H), 3.73-3.55 (m, 3H), 3.49-3.38 (m, 1H), 2. 84 (t, J=5.4Hz, 2H), 2.76 (dt, J=11.2, 5.5Hz, 1H), 2.68 (dd, J=11.3, 6.1Hz, 1H), 2.58-2.52 (m, 2H).

[0190] Example 4

[0191] Preparation of 1-(4-((8-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)pyridin[3,4-d]pyrimidin-4-yl)amino)piperidin-1-yl)ethan-1-one

[0192]

[0193] Compound 8-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)pyridin[3,4-d]pyrimidin-4-ol (180 mg, 0.51 mmol) (i.e., intermediate 1-e, preparation method see Example 1), 1-(4-aminopiperidin-1-yl)ethan-1-one (56 mg, 0.77 mmol) and PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate) (345 mg, 0.66 mmol) were dissolved in DMSO. After adding F (N, N-dimethylformamide) (2.5 mL), DBU (1,8-diazabicycloundec-7-ene) (116 mg, 0.77 mmol) was added, and stirred at 10-15°C for 2 hours. The reaction solution was poured into 10 ml of water and extracted with ethyl acetate three times, each time with 6 ml. The ethyl acetate phase was combined, washed once with 10 ml of water and once with 10 ml of saturated brine, dried over anhydrous sodium sulfate for 10 minutes, filtered, concentrated, and prepared by HPLC to obtain 24 mg of the target compound with a yield of 9.8%. LCMS: [M+H] + =476.37; 1HNMR (400MHz, DMSO-d6) δ: 8.57 (s, 1H), 8.11 (d, J=6.5Hz, 1H), 7.83 (m, 1H), 7.44-7.08 (m, 6H), 4.58-4.26 (m, 5 H), 3.89 (m, 1H), 3.70-3.49 (m, 4H), 3.42-2.97 (m, 6H), 2.67 (t, J=11.7Hz, 1H), 2.15-1.83 (m, 5H), 1.50 (m, 2H).

[0194] Example 5

[0195] Preparation of (S)-1-(4-((8-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)pyridin[3,4-d]pyrimidin-4-yl)amino)piperidin-1-yl)ethan-1-one

[0196]

[0197]

[0198] Compound (S)-8-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)pyridin[3,4-d]pyrimidin-4-ol (90 mg, 0.255 mmol) (i.e., intermediate 2-c, preparation method see Example 2), 1-(4-aminopiperidin-1-yl)ethan-1-one (28 mg, 0.385 mmol) and PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate) (172 mg, 0. 33mmol) was dissolved in DMF (N,N-dimethylformamide) (2.5mL), and DBU (58mg, 0.385mmol) was added, and the mixture was stirred at 10-15°C for 2 hours. The reaction solution was poured into 10ml water and extracted with ethyl acetate three times, each time with 6ml. The ethyl acetate phases were combined, washed once with 10ml water and once with 10ml saturated brine, dried over anhydrous sodium sulfate for 10 minutes, filtered, concentrated, and prepared by HPLC to obtain 13mg of the target compound in a yield of 10%.

[0199] LCMS: [M+H] + =476.37; 1H NMR (400MHz, DMSO-d6) δ: 8.57 (s, 1H), 8.11 (d, J=6.5Hz, 1H), 7.83 (m, 1H), 7.44-7.08 (m, 6H), 4.58-4.26 (m, 5H) ), 3.89 (m, 1H), 3.70-3.49 (m, 4H), 3.42-2.97 (m, 6H), 2.67 (t, J = 11.7Hz, 1H), 2.15-1.83 (m, 5H), 1.50 (m, 2H).

[0200] Example 6

[0201] Preparation of (R)-1-(4-((8-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)pyridin[3,4-d]pyrimidin-4-yl)amino)piperidin-1-yl)ethan-1-one

[0202]

[0203] Compound (R)-8-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)pyridin[3,4-d]pyrimidin-4-ol (180 mg, 0.51 mmol) (i.e., intermediate 3-c, preparation method see Example 3), 1-(4-aminopiperidin-1-yl)ethan-1-one (56 mg, 0.77 mmol) and PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate) (345 mg, 0.6 6mmol) was dissolved in DMF (N,N-dimethylformamide) (2.5mL), and DBU (116mg, 0.77mmol) was added, and the mixture was stirred at 10-15°C for 2 hours. The reaction solution was poured into 10ml water and extracted with ethyl acetate three times, each time with 6ml. The ethyl acetate phases were combined, washed once with 10ml water and once with 10ml saturated brine, dried over anhydrous sodium sulfate for 10 minutes, filtered, concentrated, and prepared by HPLC to obtain 26mg of the target compound in a yield of 10.5%.

[0204] LCMS: [M+H] + =476.37; 1 H NMR (400MHz, DMSO-d6) δ: 8.57 (s, 1H), 8.11 (d, J=6.5Hz, 1H), 7.83 (m, 1H), 7.44-7.08 (m, 6H), 4.58-4.26 (m, 5H) ), 3.89 (m, 1H), 3.70-3.49 (m, 4H), 3.42-2.97 (m, 6H), 2.67 (t, J = 11.7Hz, 1H), 2.15-1.83 (m, 5H), 1.50 (m, 2H).

[0205] Example 7

[0206] Preparation of 1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((8-(3-oxetanylamino)pyridin[3,4-d]pyrimidin-4-yl)amino)propan-2-ol

[0207]

[0208] Step 1: Preparation of 1-((8-chloropyridin-4-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (Intermediate 7-a)

[0209]

[0210] Compound 8-chloro-3H-pyrido[3,4-d]pyrimidin-4-one (300 mg, 1.65 mmol) (i.e., intermediate 1-d, preparation method see Example 1), 1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)-propan-2-ol (512 mg, 2.49 mmol) (i.e., intermediate 1-b, preparation method see Example 1) and PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate) (1.12 g, 2.15mmol) was dissolved in DMF (N,N-dimethylformamide) (8mL), and DBU (377mg, 2.49mmol) was added. The mixture was stirred at room temperature for 2 hours. The reaction solution was poured into 10ml water and extracted with ethyl acetate three times with 6ml each time. The ethyl acetate phases were combined, washed once with 10ml water and once with 10ml saturated brine, dried over anhydrous sodium sulfate for 10 minutes, filtered and concentrated to obtain a crude product. The crude product was separated by column chromatography (MeOH: DCM = 1: 15) to obtain 550mg of 1-((8-chloropyridin[3,4-d]pyrimidin-4-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol, with a yield of 90%. LCMS: [M+H] + =370.29.

[0211] Step 2: Preparation of 1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((8-(3-oxetanylamino)pyridin[3,4-d]pyrimidin-4-yl)amino)propan-2-ol (Compound of Example 7)

[0212]

[0213] Compounds 1-((8-chloropyridin-4-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (200 mg, 0.54 mmol), oxetane-3-amine (395 mg, 5.42 mmol), Brettphos (dicyclohexyl[3,6-dimethoxy-2',4',6'-triisopropyl[1,1'-biphenyl]-2-yl]phosphine) (30 mg, 0.05 mmol), Brettphos-Pd-G3 (methanesulfonic acid (2-dicyclohexylphosphino-3,6-dimethoxy-2',4',6'-triisopropyl[1,1'-biphenyl]-2-yl]phosphine) After dissolving triisopropyl-1,1'-biphenyl)(2-amino-1,1'-biphenyl-2-yl)palladium(II))(49mg, 0.05mmol) in THF(tetrahydrofuran)(3mL), t-BuONa(sodium tert-butoxide)(78mg, 0.81mmol) was added in sequence, and the temperature was raised to 60°C for two hours. After the reaction, the reaction solution was poured into 10 ml of water, extracted with ethyl acetate three times, 5 ml each time, and the ethyl acetate phases were combined, washed once with 10 ml of saturated brine, dried over anhydrous sodium sulfate for 10 minutes, filtered and concentrated to obtain a crude product, and the target molecule 40 mg was obtained by preparative-HPLC with a yield of 18.2%. LCMS: [M+H] + =407.27; 1 H NMR (400MHz, DMSO-d6) δ: 8.49 (s, 1H), 8.32 (t, J=5.3Hz, 1H), 7.84 (d, J=5.8Hz, 1H), 7.78 (d, J=5. 8Hz, 1H), 7.15-7.08 (m, 3H), 7.04 (t, J=6.3Hz, 2H), 5.06 (dq, J=13.4, 6.7Hz, 1H), 4.98 (d, J=4.7H z, 1H), 4.79 (t, J=6.7Hz, 2H), 4.64 (t, J=6.0Hz, 2H), 4.09 (dd, J=11.2, 5.2Hz, 1H), 3.74-3.66 (m, 2H), 3.61 (m, 1H), 3.50-3.45 (m, 1H), 2.82 (d, J=5.6Hz, 2H), 2.79-2.70 (m, 2H), 2.64-2.53 (m, 2H).

[0214] Example 8

[0215] Preparation of (S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((8-(3-oxetanylamino)pyridin[3,4-d]pyrimidin-4-yl)amino)propan-2-ol

[0216]

[0217] Step 1: Preparation of (S)-1-((8-chloropyridin-4-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (Intermediate 8-a)

[0218]

[0219] Compound 8-chloro-3H-pyrido[3,4-d]pyrimidin-4-one (300 mg, 1.65 mmol) (i.e., intermediate 1-d, preparation method see Example 1), (S)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)-propan-2-ol (512 mg, 2.49 mmol) (i.e., intermediate 2-b, preparation method see Example 2) and PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate) (1.12 g, 2.15 mmol) were dissolved in DMF (N,N-dimethylformamide) (8 mL) ), DBU (377 mg, 2.49 mmol) was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was poured into 10 ml of water, and extracted with ethyl acetate three times, each time with 6 ml. The ethyl acetate phases were combined, washed once with 10 ml of water and once with 10 ml of saturated brine, dried over anhydrous sodium sulfate for 10 minutes, filtered, and concentrated to obtain a crude product. Column chromatography (MeOH: DCM = 1: 15) was used to obtain 550 mg of (S)-1-((8-chloropyridin[3,4-d]pyrimidin-4-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol, with a yield of 90%. LCMS: [M+H] + =370.29.

[0220] Step 2: Preparation of (S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((8-(3-oxetanylamino)pyridin[3,4-d]pyrimidin-4-yl)amino)propan-2-ol (Compound of Example 8)

[0221]

[0222] Compound (S)-1-((8-chloropyridin-4-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (200 mg, 0.54 mmol), oxetane-3-amine (395 mg, 5.42 mmol), Brettphos (dicyclohexyl[3,6-dimethoxy-2',4',6'-triisopropyl[1,1'-biphenyl]-2-yl]phosphine) (30 mg, 0.05 mmol), Brettphos-Pd-G3 (methanesulfonic acid (2-dicyclohexylphosphino-3,6-dimethoxy-2',4',6' -triisopropyl-1,1'-biphenyl)(2-amino-1,1'-biphenyl-2-yl)palladium(II))(49mg, 0.05mmol) was dissolved in THF (tetrahydrofuran) (3mL), and t-BuONa (sodium tert-butoxide) (78mg, 0.81mmol) was added in sequence, and the temperature was raised to 60°C for two hours. After the reaction, the reaction solution was poured into 10 ml of water, extracted with ethyl acetate three times, each time with 5 ml, and the ethyl acetate phases were combined, washed once with 10 ml of saturated brine, dried over anhydrous sodium sulfate for 10 minutes, filtered and concentrated to obtain a crude product, and the target molecule 40 mg was obtained by preparative-HPLC with a yield of 18.2%. LCMS: [M+H] + =407.27; 1 H NMR (400MHz, DMSO-d6) δ: 8.49 (s, 1H), 8.32 (t, J=5.3Hz, 1H), 7.84 (d, J=5.8Hz, 1H), 7.78 (d, J=5. 8Hz, 1H), 7.15-7.08 (m, 3H), 7.04 (t, J=6.3Hz, 2H), 5.06 (dq, J=13.4, 6.7Hz, 1H), 4.98 (d, J=4.7H z, 1H), 4.79 (t, J=6.7Hz, 2H), 4.64 (t, J=6.0Hz, 2H), 4.09 (dd, J=11.2, 5.2Hz, 1H), 3.74-3.66 (m, 2H), 3.61 (m, 1H), 3.50-3.45 (m, 1H), 2.82 (d, J=5.6Hz, 2H), 2.79-2.70 (m, 2H), 2.64-2.53 (m, 2H).

[0223] Example 9

[0224] Preparation of (R)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((8-(3-oxetanylamino)pyridin[3,4-d]pyrimidin-4-yl)amino)propan-2-ol

[0225]

[0226] Step 1: Preparation of (R)-1-((8-chloropyridin-4-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (Intermediate 9-a)

[0227]

[0228] Compound 8-chloro-3H-pyrido[3,4-d]pyrimidin-4-one (300 mg, 1.65 mmol) (i.e., intermediate 1-d, preparation method see Example 1), (R)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)-propan-2-ol (512 mg, 2.49 mmol) (i.e., intermediate 3-b, preparation method see Example 3) and PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate) (1.12 g, 2.15 mmol) were dissolved in DMF (N,N-dimethylformamide) (8 mL) ), DBU (377 mg, 2.49 mmol) was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was poured into 10 ml of water, and extracted with ethyl acetate three times, each time with 6 ml. The ethyl acetate phases were combined, washed once with 10 ml of water and once with 10 ml of saturated brine, dried over anhydrous sodium sulfate for 10 minutes, filtered, and concentrated to obtain a crude product. Column chromatography (MeOH: DCM = 1: 15) was used to obtain 550 mg of (S)-1-((8-chloropyridin[3,4-d]pyrimidin-4-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol, with a yield of 90%. LCMS: [M+H] + =370.29.

[0229] Step 2: Preparation of (R)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((8-(3-oxetanylamino)pyridin[3,4-d]pyrimidin-4-yl)amino)propan-2-ol (Compound of Example 9)

[0230]

[0231] Compound (R)-1-((8-chloropyridin-4-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (200 mg, 0.54 mmol), oxetane-3-amine (395 mg, 5.42 mmol), Brettphos (dicyclohexyl[3,6-dimethoxy-2',4',6'-triisopropyl[1,1'-biphenyl]-2-yl]phosphine) (30 mg, 0.05 mmol), Brettphos-Pd-G3 (methanesulfonic acid (2-dicyclohexylphosphino-3,6-dimethoxy-2',4',6'-triisopropyl[1,1'-biphenyl]-2-yl]phosphine) '-Triisopropyl-1,1'-biphenyl)(2-amino-1,1'-biphenyl-2-yl)palladium(II))(49mg, 0.05mmol) was dissolved in THF (tetrahydrofuran) (3mL), and t-BuONa (sodium tert-butoxide) (78mg, 0.81mmol) was added in sequence, and the temperature was raised to 60°C for two hours. After the reaction, the reaction solution was poured into 10 ml of water, extracted with ethyl acetate three times, each time with 5 ml, and the ethyl acetate phases were combined, washed once with 10 ml of saturated brine, dried over anhydrous sodium sulfate for 10 minutes, filtered and concentrated to obtain a crude product, and prepared by HPLC to obtain 40 mg of the target molecule with a yield of 18.2%. LCMS: [M+H] + =407.27; 1 H NMR (400MHz, DMSO-d6) δ: 8.49 (s, 1H), 8.32 (t, J=5.3Hz, 1H), 7.84 (d, J=5.8Hz, 1H), 7.78 (d, J=5. 8Hz, 1H), 7.15-7.08 (m, 3H), 7.04 (t, J=6.3Hz, 2H), 5.06 (dq, J=13.4, 6.7Hz, 1H), 4.98 (d, J=4.7H z, 1H), 4.79 (t, J=6.7Hz, 2H), 4.64 (t, J=6.0Hz, 2H), 4.09 (dd, J=11.2, 5.2Hz, 1H), 3.74-3.66 (m, 2H), 3.61 (m, 1H), 3.50-3.45 (m, 1H), 2.82 (d, J=5.6Hz, 2H), 2.79-2.70 (m, 2H), 2.64-2.53 (m, 2H).

[0232] Example 10

[0233] Preparation of 1-(4-((4-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)pyridin[3,4-d]pyrimidin-8-yl)amino)piperidin-1-yl)ethan-1-one

[0234]

[0235] According to the synthesis method of the second step of Example 7, 60 mg of the target molecule 1-(4-((4-((3-(3,4-dihydroisoquinolin-2(1H)-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (400 mg, 1.08 mmol) (i.e., intermediate 7-a, see Example 7 for preparation method) and 1-(4-aminopiperidin-1-yl)ethan-1-one (990 mg, 5.42 mmol) were synthesized to obtain 11.6% of the target molecule 1-(4-((4-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)pyridin[3,4-d]pyrimidin-8-yl)amino)piperidin-1-yl)ethan-1-one. The yield was 11.6%. LCMS: [M+H] + =476.34; 1 H NMR (400MHz, DMSO-d6) δ: 8.44 (s, 1H), 8.28 (t, J = 5.3Hz, 1H), 7.81 (d, J = 5.8Hz, 1H), 7.14 -7.06 (m, 3H), 7.05-6.96 (m, 3H), 4.97 (d, J = 4.1Hz, 1H), 4.34 (d, J = 13.6Hz, 1H), 4.25-4. 13 (m, 1H), 4.08 (d, J = 5.4Hz, 1H), 3.83 (d, J = 13.6Hz, 1H), 3.65 (m, 3H), 3.54-3.41 (m, 1H) , 3.16 (t, J=11.7Hz, 1H), 2.75 (m, 5H), 2.57 (m, 2H), 2.05-1.84 (m, 5H), 1.63-1.34 (m, 2H).

[0236] Embodiment 11

[0237] Preparation of (S)-1-(4-((4-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)pyridin[3,4-d]pyrimidin-8-yl)amino)piperidin-1-yl)ethan-1-one

[0238]

[0239] According to the method of Example 8, compound (S)-1-((8-chloropyridin-4-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (200 mg, 0.54 mmol) (i.e., intermediate 8-a, preparation method see Example 8) and 1-(4-aminopiperidin-1-yl)ethan-1-one (485 mg, 2.71 mmol) were used to obtain 22 mg of the target molecule (S)-1-(4-((4-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)pyridin-3,4-d]pyrimidin-8-yl)amino)piperidin-1-yl)ethan-1-one, yield 8.5%. LCMS: [M+H] + =476.34; 1 H NMR (400MHz, DMSO-d6) δ: 8.44 (s, 1H), 8.28 (t, J = 5.3Hz, 1H), 7.81 (d, J = 5.8Hz, 1H), 7.14 -7.06 (m, 3H), 7.05-6.96 (m, 3H), 4.97 (d, J = 4.1Hz, 1H), 4.34 (d, J = 13.6Hz, 1H), 4.25-4. 13 (m, 1H), 4.08 (d, J = 5.4Hz, 1H), 3.83 (d, J = 13.6Hz, 1H), 3.65 (m, 3H), 3.54-3.41 (m, 1H) , 3.16 (t, J=11.7Hz, 1H), 2.75 (m, 5H), 2.57 (m, 2H), 2.05-1.84 (m, 5H), 1.63-1.34 (m, 2H).

[0240] Example 12

[0241] Preparation of (R)-1-(4-((4-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)pyridin[3,4-d]pyrimidin-8-yl)amino)piperidin-1-yl)ethan-1-one

[0242]

[0243]

[0244] According to the method of Example 9, compound (R)-1-((8-chloropyridin-4-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (200 mg, 0.54 mmol) (i.e., intermediate 9-a, preparation method see Example 9) and 1-(4-aminopiperidin-1-yl)ethan-1-one (485 mg, 2.71 mmol) were used to obtain 33 mg of the target molecule (R)-1-(4-((4-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)pyridin-3,4-d]pyrimidin-8-yl)amino)piperidin-1-yl)ethan-1-one, yield 12.8%. LCMS: [M+H] + =476.34; 1 H NMR (400MHz, DMSO-d6) δ: 8.44 (s, 1H), 8.28 (t, J = 5.3Hz, 1H), 7.81 (d, J = 5.8Hz, 1H), 7.14 -7.06 (m, 3H), 7.05-6.96 (m, 3H), 4.97 (d, J = 4.1Hz, 1H), 4.34 (d, J = 13.6Hz, 1H), 4.25-4. 13 (m, 1H), 4.08 (d, J = 5.4Hz, 1H), 3.83 (d, J = 13.6Hz, 1H), 3.65 (m, 3H), 3.54-3.41 (m, 1H) , 3.16 (t, J=11.7Hz, 1H), 2.75 (m, 5H), 2.57 (m, 2H), 2.05-1.84 (m, 5H), 1.63-1.34 (m, 2H).

[0245] Embodiment 13

[0246] Preparation of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethanone

[0247]

[0248] Step 1: Synthesis of 3-iodo-1H-pyrazolo[4,3-d]pyrimidin-7-ol (Intermediate 13-a)

[0249]

[0250] The compound 7-hydroxypyrazolo[4,3-d]pyrimidine (2g, 14.694mmol, 1.0eq) was dissolved in DMF (N,N-dimethylformamide) (40mL), and NIS (N-iodosuccinimide) (4.96g, 22.041mmol, 1.5eq) was added, and the reaction was reacted at 60°C for 5 hours under N2 protection. After the reaction was completed by TLC detection, the reaction solvent was dried, and the mixture was dissolved in methanol and filtered. The filter cake was rinsed with methanol and dried to obtain 3.46g of 3-iodo-1H-pyrazolo[4,3-d]pyrimidine-7-ol, with a yield of 89.9%. LCMS: [M+1] + =262.87; 1 H NMR (400MHz, DMSO-d6) δ: 14.55 (s, 1H), 12.41 (s, 1H), 7.92 (d, J = 3.3Hz, 1H).

[0251] Step 2: Synthesis of 3-iodo-1-(((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol (Intermediate 13-b)

[0252]

[0253] Compound 3-iodo-1H-pyrazolo[4,3-d]pyrimidin-7-ol (1.50 g, 5.725 mmol, 1.0 eq) was dissolved in DMF (N,N-dimethylformamide) (28 mL), cesium carbonate (2.24 g, 6.870 mmol, 1.2 eq) was added, and SEMCl ( 2-(Trimethylsilyl)ethoxymethyl chloride ) (1.0 mL, 5.725 mmol, 1.0 eq), the reaction was allowed to react at 20 ° C overnight for 16 hours. After the reaction was completed by TLC, the reaction solution was extracted with dichloromethane and dried over anhydrous sodium sulfate. The organic phase was concentrated and purified by column chromatography to obtain 1.21 g of 3-iodo-1-(((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol, with a yield of 53.8%. LCMS: [M+1] + =393.12.

[0254] Step 3: Synthesis of 1-(4-((3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethanone (Intermediate 13-c)

[0255]

[0256] The compound 3-iodo-1-(((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol (0.60 g, 1.530 mmol, 1.0 eq) was dissolved in DMF (N,N-dimethylformamide) (8 mL), and the compounds 1-acetylpiperidin-4-amine hydrochloride (0.41 g, 2.294 mmol, 1.5 eq), PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate) (1.03 g, 1.988 mmol, 1.3 eq) were added. and DBU (1,8-diazabicycloundec-7-ene) (0.69 mL, 4.589 mmol, 3.0 eq), reacted at 20 ° C for 1 hour. After the reaction was completed by TLC, the reaction solution was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The organic phase was concentrated and purified by column chromatography to obtain 1-(4-((3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethanone 598 mg, yield 75.7%. LCMS: [M+1] + =517.21.

[0257] Step 4: Synthesis of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethanone (Intermediate 13-d)

[0258]

[0259] Compound 1-(4-((3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethanone (200 mg, 0.387 mmol, 1.0 eq) was dissolved in DMSO (dimethyl sulfoxide) (4 mL), and compound (S)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)-propan-2-ol (160 mg, 0.387 mmol, 1.0 eq) was added. 1.5-(4-(4-(4-nitropropene)-2-yl)-1-nitropropene ... The organic phase was concentrated and purified by column chromatography to obtain 32 mg of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethanone, with a yield of 13.9%. LCMS: [M+1] + =595.39.

[0260] Step 5: Synthesis of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethanone (Compound of Example 13)

[0261]

[0262] The compound obtained in the fourth step (26 mg, 0.044 mmol, 1.0 eq) was dissolved in a 1,4-dioxane solution of hydrochloric acid (4 M, 1.2 mL) and reacted at 20°C for 1.5 hours. After the reaction was completed by TLC, the reaction solution was quenched with a saturated aqueous sodium bicarbonate solution, extracted with dichloromethane, and dried over anhydrous sodium sulfate. The organic phase was concentrated and purified by preparative HPLC to obtain 18 mg of the target compound (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethanone, with a yield of 90.0%. LCMS: [M+1] + =465.20; 1H NMR (400MHz, CDCl3) δ: 11.13 (s, 1H), 8.27 (s, 1H), 7.13 (ddt, J=12.9, 6.7, 3.1Hz, 3H), 7.02-6.97 (m, 1H), 6.83 (s, 1H), 4.63 (d, J=13. 6Hz, 1H), 4.48 (s, 1H), 4.19 (dd, J=9.2, 5.4Hz, 1H), 3.88 (t, J=12.4Hz, 2H), 3.73 (d, J=15.0Hz, 1H), 3.66 (dd, J=13.4, 3.5Hz, 1H), 3.45 (dd, J=13.6, 5.8Hz, 1H), 3.32 (t, J=12.9Hz, 1H), 3.03-2.96 (m, 1H), 2.93 (dd, J=7.3, 4.7Hz, 2H), 2.90-2.82 (m, 2H), 2.79 (dd, J=12.5, 9.6Hz, 1H), 2.69 (dd, J=12.5, 3.9Hz, 1H), 2.35 (d, J=13.1Hz, 2H), 2.14 (s, 3H), 2.08 (d, J=12.5Hz, 2H), 1.38 (dd, J=23.1, 12.0Hz, 2H).

[0263] Embodiment 14

[0264] Preparation of (R)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethanone

[0265]

[0266] According to the method of Example 13, 1-(4-((3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethanone (400 mg, 0.774 mmol, 1.0 eq) (i.e., intermediate 13-c, preparation method see Example 13) and raw material (R)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)-propan-2-ol (320 mg, 1.55 mmol, 2.0 eq) (i.e., intermediate 3-b, preparation method see Example 3) were subjected to coupling reaction to obtain intermediate (R)-1-( 4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethanone (97 mg, yield 21%) (Intermediate 14-a), remove the SEM protecting group to obtain 64 mg of the target molecule (R)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethanone, yield 85%. LCMS: [M+1] + =465.20; 1 H NMR (400MHz, CDCl3) δ: 11.13 (s, 1H), 8.27 (s, 1H), 7.13 (ddt, J=12.9, 6.7, 3.1Hz, 3H), 7.02-6.97 (m, 1H), 6.83 (s, 1H), 4.63 (d, J=13. 6Hz, 1H), 4.48 (s, 1H), 4.19 (dd, J=9.2, 5.4Hz, 1H), 3.88 (t, J=12.4Hz, 2H), 3.73 (d, J=15.0Hz, 1H), 3.66 (dd, J=13.4, 3.5Hz, 1H), 3.45 (dd, J=13.6, 5.8Hz, 1H), 3.32 (t, J=12.9Hz, 1H), 3.032.96 (m, 1H), 2.93 (dd, J=7.3, 4.7Hz, 2H), 2.90-2.82 (m, 2H), 2.79 (dd, J=12.5, 9.6Hz, 1H), 2.69 (dd, J=12.5, 3.9Hz, 1H), 2.35 (d, J=13.1Hz, 2H), 2.14 (s, 3H), 2.08 (d, J=12.5Hz, 2H), 1.38 (dd, J=23.1, 12.0Hz, 2H).

[0267] Embodiment 15

[0268] Preparation of 1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethanone

[0269]

[0270] According to the method of Example 13, 1-(4-((3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethanone (600 mg, 1.161 mmol, 1.0 eq) (i.e., intermediate 13-c, preparation method see Example 13) and 1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)-propan-2-ol (480 mg, 2.322 mmol, 2.0 eq) (i.e., intermediate 1-b, preparation method see Example 1) were coupled to obtain racemic intermediate 1-(4-( (3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethanone (172.7 mg, yield 25%) (Intermediate 15-a), remove the SEM protecting group to obtain 115 mg of the target molecule 1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethanone, yield 85%. LCMS: [M+1] + =465.20; 1H NMR (400MHz, CDCl3) δ: 11.13 (s, 1H), 8.27 (s, 1H), 7.13 (ddt, J=12.9, 6.7, 3.1Hz, 3H), 7.02-6.97 (m, 1H), 6.83 (s, 1H), 4.63 (d, J=13. 6Hz, 1H), 4.48 (s, 1H), 4.19 (dd, J=9.2, 5.4Hz, 1H), 3.88 (t, J=12.4Hz, 2H), 3.73 (d, J=15.0Hz, 1H), 3.66 (dd, J=13.4, 3.5Hz, 1H), 3.45 (dd, J=13.6, 5.8Hz, 1H), 3.32 (t, J=12.9Hz, 1H), 3.03-2.96 (m, 1H), 2.93 (dd, J=7.3, 4.7Hz, 2H), 2.90-2.82 (m, 2H), 2.79 (dd, J=12.5, 9.6Hz, 1H), 2.69 (dd, J=12.5, 3.9Hz, 1H), 2.35 (d, J=13.1Hz, 2H), 2.14 (s, 3H), 2.08 (d, J=12.5Hz, 2H), 1.38 (dd, J=23.1, 12.0Hz, 2H).

[0271] Example 16

[0272] Preparation of (S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-(oxabutan-3-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol

[0273]

[0274] Step 1: Synthesis of 3-iodo-N-(oxabutane-3-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine (Intermediate 16-a)

[0275]

[0276] The compound 3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidine-7-ol (590 mg, 1.504 mmol, 1.0 eq) (i.e., intermediate 13-b, preparation method see Example 13) was dissolved in DMF (N,N-dimethylformamide) (8 mL), and the compound oxadiazine-3-amine (165 mg, 2.256 mmol, 1.5 eq), PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate) (1.02 g, 1.955 mmol, 1.3 eq) and DBU (1,8-diazabicycloundec-7-ene) (0.34 mL, 2.256 mmol, 1.5 eq) were added, and the reaction was carried out at 20° C. for 1 hour. After the reaction was completed by TLC, the reaction solution was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The organic phase was concentrated and purified by column chromatography to obtain 402 mg of 3-iodo-N-(oxetan-3-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine (Intermediate 16-a) in a yield of 59.7%. LCMS: [M+1] + =448.03.

[0277] Step 2: Synthesis of (S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-(oxadiazine-3-ylamino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol (Intermediate 16-b)

[0278]

[0279] The compound 3-iodo-N-(oxetan-3-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine (190 mg, 0.425 mmol, 1.0 eq) was dissolved in DMSO (dimethyl sulfoxide) (4 mL), and the compound (S)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)-propan-2-ol (175 mg, 0.849 mmol, 2.0 eq) (i.e., intermediate 2-b, preparation method see Example 2), CuI (copper iodide) (16 mg, 0.085 mmol, 0.2 cq), potassium carbonate (293 mg, 2.124 mmol, 5.0 cq) and L-Prolinol (17 mg, 0.170 mmol, 0.4 eq) were added, and the reaction was carried out at 80° C. for 18 hours under N2 protection. After the reaction was completed by TLC, the reaction solution was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The organic phase was concentrated and purified by column chromatography to obtain 36 mg of compound (S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-(oxadiazine-3-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol, with a yield of 16.1%. LCMS: [M+1] + =526.32.

[0280] Step 3: Synthesis of (S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-(oxabutan-3-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol (Compound of Example 16)

[0281]

[0282] Compound (S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-(oxadiazine-3-ylamino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol (36 mg, 0.068 mmol, 1.0 eq) was dissolved in a solution of hydrochloric acid in 1,4-dioxane (molar concentration: 4 mol / L) (2 mL) and reacted at 20°C for 1 hour. After the reaction was completed by TLC, the reaction solution was quenched with a saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, extracted with chloroform / isopropanol (7:3), and dried over anhydrous sodium sulfate. The organic phase was concentrated and purified by preparative HPLC to obtain 6 mg of the target compound with a yield of 22.2%. LCMS: [M+1] + =396.18; 1H NMR (400MHz, DMSO-d6) δ: 12.39 (s, 1H), 7.69 (s, 1H), 7.15-7.06 (m, 3H), 7.05-6.9 9 (m, 1H), 5.20 (s, 1H), 4.88-4.67 (m, 2H), 4.25 (dtd, J=10.6, 6.7, 4.0Hz, 1H), 4.1 3-4.04(m, 1H), 3.99-3.89(m, 2H), 3.64-3.54(m, 2H), 3.44-3.35(m, 3H), 3.19-3. 11 (m, 1H), 2.81 (t, J=5.8Hz, 2H), 2.70 (dq, J=17.2, 5.6Hz, 2H), 2.60-2.43 (m, 2H).

[0283] Embodiment 17

[0284] Preparation of (R)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-(oxabutan-3-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol

[0285]

[0286] According to the method of Example 16, the raw material compound 3-iodo-N-(oxabutane-3-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine (380 mg, 0.85 mmol, 1.0 eq) (Intermediate 16-a, preparation method see Example 16) and the raw material (R)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)-propan-2-ol (350 mg, 1.7 mmol, 2.0 eq) (Intermediate 3-b, preparation method see Example 3) were subjected to coupling reaction to obtain the intermediate (R)-1- (3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-(oxadiazol-3-ylamino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol (42.4 mg, yield 19%) (Intermediate 17-a), remove the SEM protecting group to obtain 4.8 mg of the target molecule (R)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-(oxadiazol-3-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol, yield 15%. LCMS: [M+1] + =396.18; 1H NMR (400MHz, DMSO-d6) δ12.39 (s, 1H), 7.69 (s, 1H), 7.15-7.06 (m, 3H), 7.05-6.99 (m, 1H), 5.20 (s, 1H), 4.88-4.67 (m, 2H), 4.25 (dtd, J=10.6, 6.7, 4.0Hz, 1H), 4.13 -4.04(m, 1H), 3.99-3.89(m, 2H), 3.64-3.54(m, 2H), 3.44-3.35(m, 3H), 3.19-3.1 1 (m, 1H), 2.81 (t, J=5.8Hz, 2H), 2.70 (dq, J=17.2, 5.6Hz, 2H), 2.60-2.43 (m, 2H).

[0287] Embodiment 18

[0288] Preparation of 1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-(oxabutan-3-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol

[0289]

[0290] According to the method of Example 16, the raw material compound 3-iodo-N-(oxetan-3-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine (760 mg, 1.7 mmol, 1.0 eq) (i.e., intermediate 16-a, preparation method see Example 16) and the raw material 1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)-propan-2-ol (700 mg, 3.4 mmol, 2.0 eq) (i.e., intermediate 1-b, preparation method see Example 1) were subjected to coupling reaction to obtain racemic intermediate 1 -(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-(oxadiazol-3-ylamino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol (134 mg, yield 15%) (Intermediate 18-a), remove the SEM protecting group to obtain 20 mg of the target molecule 1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-(oxadiazol-3-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol, yield 20%. LCMS: [M+1] + =396.18; 1H NMR (400MHz, DMSO-d6) δ: 12.39 (s, 1H), 7.69 (s, 1H), 7.15-7.06 (m, 3H), 7.05-6.9 9 (m, 1H), 5.20 (s, 1H), 4.88-4.67 (m, 2H), 4.25 (dtd, J=10.6, 6.7, 4.0Hz, 1H), 4.1 3-4.04(m, 1H), 3.99-3.89(m, 2H), 3.64-3.54(m, 2H), 3.44-3.35(m, 3H), 3.19-3. 11 (m, 1H), 2.81 (t, J=5.8Hz, 2H), 2.70 (dq, J=17.2, 5.6Hz, 2H), 2.60-2.43 (m, 2H).

[0291] Embodiment 19

[0292] Preparation of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one

[0293]

[0294] Step 1: Preparation of 3-iodo-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7-ol (Intermediate 19-a) and 3-iodo-2-methyl-2H-pyrazolo[4,3-d]pyrimidine-7-ol (Intermediate 19-b)

[0295]

[0296] Sodium hydride (mass fraction 60%) (230 mg, 5.73 mmol, 1.5 eq) was added to a 100 mL three-necked flask, and 20 mL of anhydrous DMF (N, N-dimethylformamide) was added at 0°C under nitrogen protection. After stirring for 15 min, compound 3-iodo-1-hydrogen-pyrazolo[3,4-d]pyrimidin-4(5H)-one (1 g, 3.82 mmol, 1.0 eq) (i.e., intermediate 13-a, preparation method see Example 13) was slowly added dropwise. After stirring for 30 min under this condition, iodomethane (596 mg, 4.2 mmol, 1.1 eq) was added. After the addition was complete, the mixture was heated to room temperature and stirred for two hours. 5 mL of saturated ammonium chloride solution and 50 mL of water were added to the reaction system. The mixture was extracted with ethyl acetate three times (3×20 mL), the organic phases were combined, washed once with 20 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, the solvent was concentrated, and column chromatography (MeOH: DCM=1:20) was used to obtain two intermediates 3-iodo-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7-ol (440 mg light yellow solid, yield 27.8%, LCMS: [M+H] + =277.09. ) and 3-iodo-2-methyl-2H-pyrazolo[4,3-d]pyrimidin-7-ol (150 mg pale yellow solid, yield 9.5%, LCMS: [M+H] + =277.09).

[0297] Step 2: Preparation of 1-(4-((3-iodo-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one (Intermediate 19-c)

[0298]

[0299] Compound 3-iodo-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7-ol (100 mg, 0.364 mmol, 1.0 eq), 1-(4-aminopiperidin-1-yl)ethan-1-one (97.5 mg, 545 mmol, 1.5 eq) and benzotriazol-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate (246 mg, 0.473 mmol, 1.3 eq) were dissolved in 1.8 mL DMF (N,N-dimethylformamide), and nitrogen was replaced three times. DBU (1,8-diazabicycloundec-7-ene) (166 mg, 1.092 mmol, 3 eq) was then added, and the mixture was stirred at room temperature (20-25° C.) for 1 h. The reaction of the raw materials was complete when detected by TLC. 20 mL of water was added to the reaction system, and the mixture was extracted three times with ethyl acetate (3×20 mL). The organic phases were combined, washed once with 20 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated. The mixture was purified by preparative HPLC to obtain 60 mg of (4-((3-iodo-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one as a white solid in a yield of 41.3%. LCMS: [M+H] + =401.15; 1 H NMR (400MHz, DMSO-d6) δ: 8.31-8.20 (m, 1H), 6.91 (d, J=7.8Hz, 1H), 4.45 (ddd, J=10.3, 8.8, 5.2Hz, 1H), 4.39 (d, J=13.3Hz, 1H), 4.26 (s, 3 H), 3.87 (d, J=14.0Hz, 1H), 3.22-3.12 (m, 1H), 2.69 (td, J=12.8, 2.6Hz, 1H), 2.02 (d, J=10.9Hz, 3H), 2.00-1.88 (m, 2H), 1.7-1.48 (m, 2H).

[0300] Step 3: Preparation of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one (Compound of Example 19)

[0301]

[0302] Compound (4-((3-iodo-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one (60 mg, 0.149 mmol, 1.0 eq) was dissolved in DMSO (dimethyl sulfoxide) (0.7 mL), and compound (S)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (46.2 mg, 0.224 mmol, 1.5 eq) (i.e., intermediate 2-b, preparation method see Example 2), CuI (cuprous iodide) (5.6 mg, 0.0298 mmol, 0.2 eq), potassium carbonate (102.8 mg, 0.745 mmol, 5.0 eq) and L-prolinol (6 mg, 0.0596 mmol, 0.4 eq) were added, and the reaction was carried out at 80° C. for 20 hours under N2 protection. After the reaction was completed by TLC detection, 15 mL of water was added to the reaction system, and the mixture was extracted three times with ethyl acetate (3×20 mL). The organic phases were combined, washed once with 20 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated. The mixture was purified by preparative-HPLC to obtain 12 mg of the target molecule (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethane-1-one, with a yield of 16.8%. LCMS: [M+1] + =479.31; 1 H NMR (400MHz, CDCl3) δ: 8.29 (s, 1H), 7.24-7.10 (m, 3H), 7.02 (dd, J=9.2, 6.9Hz, 1H), 4 .86 (d, J=7.4Hz, 2H), 4.64 (d, J=13.7Hz, 1H), 4.52-4.40 (m, 1H), 4.24 (s, 1H), 4.06-3. 82 (m, 5H), 3.65 (d, J = 13.1Hz, 1H), 3.46 (s, 1H), 3.35-3.24 (m, 1H), 2.88 (dd, J = 39.2, 26.6Hz, 6H), 2.31 (d, J=12.7Hz, 1H), 2.23-2.10 (m, 4H), 1.48 (tt, J=16.6, 8.2Hz, 2H).

[0303] Embodiment 20

[0304] Preparation of (R)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one

[0305]

[0306] According to the method of Example 19, the compound (4-((3-iodo-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one (180 mg, 0.45 mmol, 1.0 eq) (i.e., intermediate 19-c, preparation method see Example 19) and the raw material compound (R)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (1 41mg, 0.672mmol, 1.5eq) (i.e., intermediate 3-b, preparation method see Example 3) was subjected to coupling reaction to obtain 36mg of the target molecule (R)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethane-1-one, yield 17%. LCMS: [M+1] + =479.31; 1 H NMR (400MHz, CDCl3) δ: 8.29 (s, 1H), 7.24-7.10 (m, 3H), 7.02 (dd, J=9.2, 6.9Hz, 1H), 4 .86 (d, J=7.4Hz, 2H), 4.64 (d, J=13.7Hz, 1H), 4.52-4.40 (m, 1H), 4.24 (s, 1H), 4.06-3. 82 (m, 5H), 3.65 (d, J = 13.1Hz, 1H), 3.46 (s, 1H), 3.35-3.24 (m, 1H), 2.88 (dd, J = 39.2, 26.6Hz, 6H), 2.31 (d, J=12.7Hz, 1H), 2.23-2.10 (m, 4H), 1.48 (tt, J=16.6, 8.2Hz, 2H).

[0307] Embodiment 21

[0308] Preparation of (R)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one

[0309]

[0310] According to the method of Example 19, the compound (4-((3-iodo-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one (180 mg, 0.45 mmol, 1.0 eq) (i.e., intermediate 19-c, preparation method see Example 19) and the raw material compound (R)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (1 41mg, 0.672mmol, 1.5eq) (i.e., intermediate 1-b, preparation method see Example 1) was subjected to coupling reaction to obtain 36mg of the target molecule (R)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethane-1-one, yield 17%. LCMS: [M+1] + =479.31; 1 H NMR (400MHz, CDCl3) δ: 8.29 (s, 1H), 7.24-7.10 (m, 3H), 7.02 (dd, J=9.2, 6.9Hz, 1H), 4 .86 (d, J=7.4Hz, 2H), 4.64 (d, J=13.7Hz, 1H), 4.52-4.40 (m, 1H), 4.24 (s, 1H), 4.06-3. 82 (m, 5H), 3.65 (d, J = 13.1Hz, 1H), 3.46 (s, 1H), 3.35-3.24 (m, 1H), 2.88 (dd, J = 39.2, 26.6Hz, 6H), 2.31 (d, J=12.7Hz, 1H), 2.23-2.10 (m, 4H), 1.48 (tt, J=16.6, 8.2Hz, 2H).

[0311] Embodiment 22

[0312] Preparation of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-2-methyl-2H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one

[0313]

[0314] According to the method of Example 19, 3-iodo-2-methyl-2H-pyrazolo[4,3-d]pyrimidine-7-ol (i.e., intermediate 19-b, see Example 19 for the preparation method) was used as the starting material to synthesize the target molecule (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-2-methyl-2H-pyrazolo[4,3-d]pyrimidine-7-yl)amino)piperidin-1-yl)ethan-1-one. LCMS: [M+1] + =479.31.

[0315] Embodiment 23

[0316] Preparation of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-2-methyl-2H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one

[0317]

[0318] According to the method of Example 19, 3-iodo-2-methyl-2H-pyrazolo[4,3-d]pyrimidine-7-ol (i.e., intermediate 19-b, see Example 19 for the preparation method) was used as the starting material to synthesize the target molecule (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-2-methyl-2H-pyrazolo[4,3-d]pyrimidine-7-yl)amino)piperidin-1-yl)ethan-1-one. LCMS: [M+1] + =479.31.

[0319] Embodiment 24

[0320] Preparation of 1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-2-methyl-2H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one

[0321]

[0322] According to the method of Example 19, 3-iodo-2-methyl-2H-pyrazolo[4,3-d]pyrimidine-7-ol (i.e., intermediate 19-b, see Example 19 for preparation method) was used as the starting material to synthesize the target molecule 1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-2-methyl-2H-pyrazolo[4,3-d]pyrimidine-7-yl)amino)piperidin-1-yl)ethan-1-one. LCMS: [M+1] +=479.31.

[0323] Embodiment 25

[0324] Preparation of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[3,4-c]pyridin-7-yl)amino)piperidin-1-yl)ethan-1-one

[0325]

[0326] Step 1: Preparation of 7-chloro-3-iodo-1H-pyrazolo[3,4-c]pyridine (Intermediate 25-a)

[0327]

[0328] The compound 7-chloro-1H-pyrazolo[3,4-c]pyridine (2g, 13mmol, 1.0eq) was dissolved in DMF (N,N-dimethylformamide) (40mL), and NIS (N-iodosuccinimide) (4.4g, 19.5mmol, 1.5eq) was added, and the reaction was reacted at 60°C for 4 hours under N2 protection. After the reaction was completed by TLC detection, the reaction solvent was dried, and the mixture was dissolved in ethanol, stirred at 15-20 degrees for 30 minutes, and filtered. The filter cake was rinsed with ethanol and dried to obtain 7-chloro-3-iodo-1H-pyrazolo[3,4-c]pyridine, a yellow solid of 3g, with a yield of 82.4%. LCMS: [M+H] + =279.84; 1 H NMR (400MHz, DMSO) δ: 14.64 (s, 1H), 8.10 (d, J=5.6Hz, 1H), 7.52 (d, J=5.6Hz, 1H).

[0329] Step 2: Preparation of 7-chloro-3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-c]pyridine (Intermediate 25-b)

[0330]

[0331] The compound 7-chloro-3-iodo-1H-pyrazolo[3,4-c]pyridine (1 g, 3.58 mmol, 1.0 eq) was dissolved in DMF (N, N-dimethylformamide) (4 mL), and added dropwise to a suspension of sodium hydride (215 mg, 5.37 mmol, 1.5 eq) in DMF (N, N-dimethylformamide) (4 mL) at 0°C, and stirred for 30 minutes. SEMCl (2-(trimethylsilyl)ethoxymethyl chloride) (776 mg, 4.65 mmol, 1.3 eq) was added dropwise at 0°C, and stirred at room temperature for 1 hour. After the reaction was completed by TLC detection, the reaction solution was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The organic phase was concentrated and purified by column chromatography to obtain 7-chloro-3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-c]pyridine. c ] Pyridine, transparent crystalline solid 920 mg, yield 62.7%. LCMS: [M+H] + =409.95: 1 H NMR (400MHz, DMSO-d6) δ: 8.38-8.24 (m, 1H), 7.87-7.48 (m, 1H), 6.12 (s, 2H), 3.70 (t, J=7.8Hz, 2H), 0.91 (t, J=7.8Hz, 2H), 0.04--0.06 (m, 9H).

[0332] Step 3: Preparation of (S)-1-((7-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-c]pyridin-3-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (Intermediate 25-c)

[0333]

[0334] Compound 7-chloro-3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-c]pyridine (500 mg, 1.22 mmol, 1.0 eq), compound (S)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (302 mg, 1.46 mmol, 1.2 eq), cuprous iodide (93 mg, 0.49 mmol, 0.4 eq), potassium carbonate (840 mg, 6.1 mmol, 5.0 eq) and L-proline (56 mg, 0.49 mmol, 0.4 eq) were dissolved in DMSO (6 mL) and reacted at 80° C. for 18 hours under N2 protection. After the reaction was completed by TLC, the reaction solution was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The organic phase was concentrated and purified by column chromatography to obtain (S)-1-((7-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-c]pyridin-3-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol as a yellow solid (360 mg, yield 60%). LCMS: [M+H] + =488.24; 1 H NMR (400MHz, DMSO-d6) δ: 8.10 (d, J=5.3Hz, 1H), 8.00 (d, J=5.3Hz, 1H), 7.24 (dd, J= 5.5, 3.6Hz, 3H), 7.17 (d, J=4.5Hz, 1H), 6.80 (s, 1H), 5.91-5.85 (m, 2H), 4.94 (d, J=4 .0Hz, 1H), 4.21 (s, 1H), 3.87-3.72 (m, 2H), 3.68 (dd, J=14.2, 6.3Hz, 3H), 3.38-3.27 (m, 1H), 3.00-2.82 (m, 5H), 2.73 (s, 1H), 0.90 (t, J=7.8Hz, 2H), 0.06--0.15 (m, 9H).

[0335] Step 4: Preparation of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-c]pyridin-7-yl)amino)piperidin-1-yl)ethan-1-one (Intermediate 25-d)

[0336]

[0337] Compound (S)-1-((7-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-c]pyridin-3-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (300 mg, 0.616 mmol, 1.0 eq), compound 1-(4-aminopiperidin-1-yl)ethane-1-one hydrochloric acid (132 mg, 0.74 mmol, 1.2 eq), Ru-phos (29 mg, 0.06 mmol, 0.1 eq), Ru-phos-G3 (52 mg, 0.06 mmol, 0.1 eq) and sodium tert-butoxide (178 mg, 1.85 mmol, 3.0 eq) were dissolved in tetrahydrofuran (3 mL) and reacted at 60° C. for 1 hour under N2 protection. After the reaction was completed by TLC detection, the reaction solution was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The organic phase was concentrated and purified by column chromatography to obtain (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-c]pyridin-7-yl)amino)piperidin-1-yl)ethane-1-one as a yellow solid (170 mg) with a yield of 46.6%. LCMS: [M+1] + =594.37.

[0338] Step 5: Preparation of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[3,4-c]pyridin-7-yl)amino)piperidin-1-yl)ethan-1-one (Compound of Example 25)

[0339]

[0340] Compound (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-c]pyridin-7-yl)amino)piperidin-1-yl)ethane-1-one (170 mg, 0.286 mmol, 1.0 eq) was dissolved in a solution of hydrochloric acid in 1,4-dioxane (4 M, 3 mL) and reacted at room temperature for 2 hours. After the reaction was completed by TLC, the reaction solution was quenched with a saturated aqueous sodium bicarbonate solution, extracted with dichloromethane, and dried over anhydrous sodium sulfate. The organic phase was concentrated and purified by preparative HPLC to obtain 40 mg of the target compound with a yield of 30%. LCMS: [M+H] + =464.31; 1H NMR (600MHz, DMSO-d6) δ: 11.20 (s, 1H), 7.42 (d, J = 5.8Hz, 1H), 7.13-7.07 (m, 3H), 7.02 (d, J = 6.7Hz, 1H), 6. 82 (d, J=5.8Hz, 1H), 6.25 (d, J=7.1Hz, 1H), 5.82 (t, J=5.9Hz, 1H), 4.77 (d, J=4.5Hz, 1H), 4.28-4.12 (m, 2H) , 4.01 (d, J = 5.6Hz, 1H), 3.80 (d, J = 13.1Hz, 1H), 3.63 (m, 2H), 3.46-3.40 (m, 1H), 3.19 (m, 3H), 2.90-2.79 (m , 3H), 2.73 (m, 2H), 2.59 (dd, J=12.5, 5.7Hz, 1H), 2.09-1.95 (m, 5H), 1.48-1.36 (m, 1H), 1.33-1.24 (m, 1H).

[0341] Embodiment 26

[0342] Preparation of 1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[3,4-c]pyridin-7-yl)amino)piperidin-1-yl)ethan-1-one

[0343]

[0344] According to the method of Example 25, the raw material compound 1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (i.e., intermediate 1-b, the preparation method is shown in Example 1) is used as the starting material, and is synthesized with 7-chloro-3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-c]pyridine (i.e., intermediate 25-b, the preparation method is shown in Example 25) for coupling reaction, and then connected with the compound 1-(4-aminopiperidin-1-yl)ethane-1-one hydrochloric acid, and the target molecule 1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[3,4-c]pyridin-7-yl)amino)piperidin-1-yl)ethane-1-one is obtained by removing SEM. LCMS: [M+H] + =464.31; 1H NMR (600MHz, DMSO-d6) δ: 11.20 (s, 1H), 7.42 (d, J = 5.8Hz, 1H), 7.13-7.07 (m, 3H), 7.02 (d, J = 6.7Hz, 1H), 6. 82 (d, J=5.8Hz, 1H), 6.25 (d, J=7.1Hz, 1H), 5.82 (t, J=5.9Hz, 1H), 4.77 (d, J=4.5Hz, 1H), 4.28-4.12 (m, 2H) , 4.01 (d, J = 5.6Hz, 1H), 3.80 (d, J = 13.1Hz, 1H), 3.63 (m, 2H), 3.46-3.40 (m, 1H), 3.19 (m, 3H), 2.90-2.79 (m , 3H), 2.73 (m, 2H), 2.59 (dd, J=12.5, 5.7Hz, 1H), 2.09-1.95 (m, 5H), 1.48-1.36 (m, 1H), 1.33-1.24 (m, 1H).

[0345] Embodiment 27

[0346] Preparation of (R)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[3,4-c]pyridin-7-yl)amino)piperidin-1-yl)ethan-1-one

[0347]

[0348] According to the method of Example 25, the raw material compound (R)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (i.e., intermediate 3-b, preparation method see Example 3) is used as the starting material, and it is synthesized with 7-chloro-3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-c]pyridine (i.e., intermediate 25-b, preparation method see Example 25) for coupling reaction, and then connected with the compound 1-(4-aminopiperidin-1-yl)ethane-1-one hydrochloric acid, and SEM is removed to obtain the target molecule (R)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[3,4-c]pyridin-7-yl)amino)piperidin-1-yl)ethane-1-one. LCMS: [M+H] + =464.31; 1H NMR (600MHz, DMSO-d6) δ: 11.20 (s, 1H), 7.42 (d, J = 5.8Hz, 1H), 7.13-7.07 (m, 3H), 7.02 (d, J = 6.7Hz, 1H), 6. 82 (d, J=5.8Hz, 1H), 6.25 (d, J=7.1Hz, 1H), 5.82 (t, J=5.9Hz, 1H), 4.77 (d, J=4.5Hz, 1H), 4.28-4.12 (m, 2H) , 4.01 (d, J = 5.6Hz, 1H), 3.80 (d, J = 13.1Hz, 1H), 3.63 (m, 2H), 3.46-3.40 (m, 1H), 3.19 (m, 3H), 2.90-2.79 (m , 3H), 2.73 (m, 2H), 2.59 (dd, J=12.5, 5.7Hz, 1H), 2.09-1.95 (m, 5H), 1.48-1.36 (m, 1H), 1.33-1.24 (m, 1H).

[0349] Embodiment 28

[0350] Preparation of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-5-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one

[0351]

[0352] Step 1: Preparation of 5-methyl-1H-pyrazolo[4,3-d]pyrimidine-7-ol (Intermediate 28-a)

[0353]

[0354] Compound 4-amino-1H-pyrazole-5-carboxylic acid ethyl ester (500mg, 3.23mmol, 1.0eq) and compound benzylthioacetimidate hydrochloride (648mg, 3.23mmol, 1.0eq) were added to a 100mL round-bottom flask, 16mL pyridine was added, nitrogen was replaced three times, heated to 115°C, and stirred for 12 hours. Cooled to room temperature (15-20 degrees), pyridine was removed by vacuum distillation, 9mL ethanol and 3mL water were added, and pH was adjusted to neutral with 1M (molar concentration) NaOH solution. Filter to obtain a filter cake, and dry at 78°C to obtain 310mg 5-methyl-1H-pyrazolo[4,3-d]pyrimidine-7-phenol, a white solid, with a yield of 64.04%. 1 H NMR (400MHz, DMSO-d6) δ: 13.97 (s, 1H), 12.10 (s, 1H), 7.94 (s, 1H), 2.31 (s, 3H).

[0355] Step 2: Preparation of 3-iodo-5-methyl-1H-pyrazolo[4,3-d]pyrimidine-7-ol (Intermediate 28-b)

[0356]

[0357] Compound 5-methyl-1H-pyrazolo[4,3-d]pyrimidine-7-ol (200 mg, 1.33 mmol, 1.0 eq) and compound N-iodosuccinimide (447 mg, 1.99 mmol, 1.5 eq) were added to a microwave tube, 6 mL DMF (N, N-dimethylformamide) was added, nitrogen was replaced three times, and the mixture was heated to 60 ° C. The reaction was completed in four hours. The mixture was cooled to room temperature (15-20 degrees), 100 mL of water and 30 mL of ethyl acetate were added, and the mixture was separated after stirring for 2 minutes. The organic phase was washed once with water and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was spin-dried. The mixture was pulped with 10 ml of ethanol to obtain 392 mg of a white solid, 3-iodo-5-methyl-1H-pyrazolo[4,3-d]pyrimidine-7-ol (crude product). 1 H NMR (400MHz, DMSO-d6) δ: 14.43 (s, 1H), 12.32 (s, 1H), 2.35 (s, 3H).

[0358] Step 3: Preparation of 3-iodo-5-methyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidine-7-ol (Intermediate 28-c)

[0359]

[0360] Compound 3-iodo-5-methyl-1H-pyrazolo[4,3-d]pyrimidine-7-ol (100 mg, 0.362 mmol, 1.0 eq) and cesium carbonate (141 mg, 0.434 mmol, 1.2 eq) were added to anhydrous 1.8 mL DMF (N,N-dimethylformamide), and nitrogen was replaced three times. Under ice bath conditions, SEMCl (2-(trimethylsilyl)ethoxymethyl chloride) (60 mg, 0.362 mmol, 1.0 eq) was added dropwise, and the mixture was stirred for ten minutes and then returned to room temperature (15-20 degrees), and stirred for one hour. TLC was used to detect that the raw material reaction was complete. 20 mL of water and 15 mL of ethyl acetate were added. The organic phase was washed once with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was dried by spin drying. The product was purified by preparative HPLC to obtain 52 mg of a white solid, 3-iodo-5-methyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidine-7-ol, with a yield of 35.6%. LCMS: [M+H] + =407.14;1 HNMR (400MHz, DMSO-d6) δ: 5.83 (s, 2H), 3.69-3.63 (m, 2H), 2.44 (s, 3H), 0.92-0.86 (m, 2H), 0.02-0.05 (m, 9H).

[0361] Step 4: Preparation of 1-(4-((3-iodo-5-methyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one (Intermediate 28-d)

[0362]

[0363] Compound 3-iodo-5-methyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidine-7-ol (310 mg, 0.763 mmol, 1.0 eq), 1-(4-aminopiperidin-1-yl)ethane-1-one (204 mg, 1.14 mmol, 1.5 eq) and benzotriazol-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate (516 mg, 0.992 mmol, 1.3 eq) were dissolved in 3.8 mL DMF (N,N-dimethylformamide), and nitrogen was replaced three times. 1,8-diazabicycloundec-7-ene (173 mg, 1.14 mmol, 1.5 eq) was then added, and the mixture was stirred at room temperature for 0.5 h. TLC detected that the reaction of the raw materials was complete. 40 mL of water was added to the reaction system, and the mixture was extracted three times with ethyl acetate (3×20 mL). The organic phases were combined, washed once with 20 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated. Column chromatography was used to purify the mixture to obtain 180 mg of 1-(4-((3-iodo-5-methyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethane-1-one as a white solid in a yield of 44.6%. LCMS: [M+H] + =531.24.

[0364] Step 5: Preparation of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-5-methyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one (Intermediate 28-e)

[0365]

[0366] Compound 1-(4-((3-iodo-5-methyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one (170 mg, 0.32 mmol, 1.0 eq) was dissolved in DMSO (dimethyl sulfoxide) (1.6 mL), and compound (S)-1-amino-3-(3,4-dihydroisoquinoline-2(1H)- 1-[4-(4-yl)propan-2-ol (98.8 mg, 0.48 mmol, 1.5 eq) (i.e., intermediate 2-b, preparation method see Example 2), cuprous iodide (12.2 mg, 0.064 mmol, 0.2 eq), potassium carbonate (221.1 mg, 1.6 mmol, 5.0 eq) and L-proline (14.7 mg, 0.128 mmol, 0.4 eq), the reaction was carried out at 80 ° C for 16 hours under N2 protection. After the reaction was completed by TLC detection, 15 mL of water was added to the reaction system, and the mixture was extracted three times with ethyl acetate (3×20 mL). The organic phases were combined, washed once with 20 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated. The mixture was purified by preparative HPLC to obtain 104 mg of a white solid, (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-5-methyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethane-1-one, with a yield of 53.6%. LCMS: [M+1] + =609.49.

[0367] Step 6: Preparation of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-5-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one (Compound of Example 28)

[0368]

[0369] To compound (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-5-methyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethane-1-one (81 mg) was added 4M (molar concentration) hydrochloric acid solution in dioxane (0.9 mL), and the reaction was carried out at room temperature (15-20 degrees) under N2 protection for 1 hour. After the reaction was completed by TLC, a saturated sodium bicarbonate solution was added to the reaction system until pH = 7-8, and the mixture was extracted with 20 mL of ethyl acetate. The organic phase was washed once with 20 mL of a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The solvent was concentrated and purified by preparative HPLC to obtain 30 mg of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-5-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethane-1-one, with a yield of 47.2%. LCMS: [M+1] + =479.31; 1 H NMR (400MHz, CDCl3) δ: 7.20-7.04 (m, 3H), 7.04-6.95 (m, 1H), 6.71 (s, 1H), 4.61 (d, J=13.5Hz, 1H), 4.51 (s, 1H), 4.15 (d, J=3.8Hz, 1H), 3.84 (d, J=15.0Hz, 2H), 3.68 (d, J=15.0Hz, 1H), 3.61 (dd, J=13.5, 3.3Hz, 1H), 3.42 (dd, J=13.2, 5.7Hz, 1H ), 3.31 (t, J=11.7Hz, 1H), 2.98-2.84 (m, 4H), 2.79 (dd, J=13.4, 7.6Hz, 1H), 2.75-2.69 (m, 1H), 2.64 (dd, J=12.5, 4.0Hz, 1H), 2.55 (d, J=14.0Hz, 3H), 2.33 (d, J=11.9Hz, 1H), 2.13 (s, 3H), 2.06 (d, J=10.1Hz, 1H), 1.35 (dt, J=24.5, 12.2Hz, 2H).

[0370] Embodiment 29

[0371] Preparation of 1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-5-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one

[0372]

[0373] According to the method of Example 28, the raw material compound (1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (i.e., intermediate 1-b, preparation method see Example 1) was used as the starting material, and 1-(4-((3-iodo-5-methyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl )ethane-1-one (i.e., intermediate 28d, preparation method see Example 28) is subjected to coupling reaction, and then SEM is removed to obtain the target molecule 1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-5-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethane-1-one. LCMS: [M+1] + 479.31; 1 H NMR (400MHz, CDCl3) δ: 7.20-7.04 (m, 3H), 7.04-6.95 (m, 1H), 6.71 (s, 1H), 4.61 (d, J=13.5Hz, 1H), 4.51 (s, 1H), 4.15 (d, J=3.8Hz, 1H), 3.84 (d, J=15.0Hz, 2H), 3.68 (d, J=15.0Hz, 1H), 3.61 (dd, J=13.5, 3.3Hz, 1H), 3.42 (dd, J=13.2, 5.7Hz, 1H ), 3.31 (t, J=11.7Hz, 1H), 2.98-2.84 (m, 4H), 2.79 (dd, J=13.4, 7.6Hz, 1H), 2.75-2.69 (m, 1H), 2.64 (dd, J=12.5, 4.0Hz, 1H), 2.55 (d, J=14.0Hz, 3H), 2.33 (d, J=11.9Hz, 1H), 2.13 (s, 3H), 2.06 (d, J=10.1Hz, 1H), 1.35 (dt, J=24.5, 12.2Hz, 2H).

[0374] Embodiment 30

[0375] Preparation of (R)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-5-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one

[0376]

[0377] According to the method of Example 28, the raw material compound (R)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (i.e., intermediate 3-b, preparation method see Example 3) is used as the starting material, and a coupling reaction is carried out with 1-(4-((3-iodo-5-methyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethane-1-one (i.e., intermediate 28d, preparation method see Example 28), and then SEM is removed to obtain the target molecule (R)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-5-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethane-1-one. LCMS: [M+1] + =479.31; 1 H NMR (400MHz, CDCl3) δ7.20-7.04 (m, 3H), 7.04-6.95 (m, 1H), 6.71 (s, 1H), 4.61 (d, J=13.5Hz, 1H), 4.51 (s, 1H), 4.15 (d, J =3.8Hz, 1H), 3.84 (d, J = 15.0Hz, 2H), 3.68 (d, J = 15.0Hz, 1H), 3.61 (dd, J = 13.5, 3.3Hz, 1H), 3.42 (dd, J = 13.2, 5.7Hz, 1H) , 3.31 (t, J=11.7Hz, 1H), 2.98-2.84 (m, 4H), 2.79 (dd, J=13.4, 7.6Hz, 1H), 2.75-2.69 (m, 1H), 2.64 (dd, J=12.5, 4.0Hz, 1 H), 2.55 (d, J = 14.0Hz, 3H), 2.33 (d, J = 11.9Hz, 1H), 2.13 (s, 3H), 2.06 (d, J = 10.1Hz, 1H), 1.35 (dt, J = 24.5, 12.2Hz, 2H).

[0378] Embodiment 31

[0379] Preparation of (S)-1-((1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol

[0380]

[0381] Step 1: Preparation of 1H-pyrazolo[4,3-d]pyrimidine (Intermediate 31-a)

[0382]

[0383] The compound 4-methyl-5-aminopyrimidine (3.17 g, 29.0 mmol, 1.0 eq) was dissolved in toluene (40 mL), and Ac2O (acetic anhydride) (6.23 g, 61.0 mmol, 2.1 eq), HOAc (glacial acetic acid) (6.03 g, 101.5 mmol, 3.5 eq), AcOK (potassium acetate) (5.68 g, 58.0 mmol, 2.0 eq), Isoamyl nitrite (isoamyl nitrite) (4.24 g, 36.0 mmol, 1.25 eq), react at 110 ° C for 3 h, LCMS detection reaction is complete, cooled to room temperature 18 ° C, added with water (300 mL), then added with Na2CO3 (sodium carbonate) solid particles, adjusted pH = 9-10, added with ethyl acetate (60 mL × 3) and extracted three times, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate for 10 minutes, filtered, and the crude product was separated and purified by flash chromatography (Silica gel, ethyl acetate: petroleum ether = 1 / 1) to obtain the title compound 1H-pyrazolo [4, 3-d] pyrimidine (400 mg, 11.5%), as a yellow solid. LCMS (ESI) [M + H] + =120.90; 1 H NMR (400MHz, DMSO-d6) δ: 13.92 (s, 1H), 9.36 (s, 1H), 9.04 (s, 1H), 8.47 (s, 1H).

[0384] Step 2: Preparation of 3-iodo-1H-pyrazolo[4,3-d]pyrimidine (Intermediate 31-b)

[0385]

[0386] The raw material 1H-pyrazolo[4,3-d]pyrimidine (400 mg, 3.33 mmol, 1.0 eq) was dissolved in DMF (N,N-dimethylformamide) (5 mL) at room temperature 20°C, and NIS (N-iodosuccinimide) (970 mg, 4.33 mmol, 1.3 eq) was added, and the reaction was heated to 60°C and stirred for 15 hours. After the reaction was completed by LCMS, it was cooled to room temperature 18°C, water (20 mL) was added, and then dichloromethane: methanol (V / V = 10 / 1) (10 mL × 3) was added to extract three times, washed with saturated brine (5 mL), dried over anhydrous sodium sulfate for 10 minutes, filtered, and the crude product was separated and purified by flash chromatography (Silica gel, ethyl acetate: petroleum ether (V / V = 1 / 1)) to obtain the title compound 3-iodo-1H-pyrazolo[4,3-d]pyrimidine (600 mg, 73.2%) as a yellow solid. LCMS(ESI)[M+H] + =246.9. 1HNMR (400MHz, DMSO-d6) δ: 14.35 (s, 1H), 9.34 (s, 1H), 9.08 (s, 1H).

[0387] Step 3: Preparation of 3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidine (Intermediate 31-c)

[0388]

[0389] The raw material 3-iodo-1H-pyrazolo[4,3-d]pyrimidine (300 mg, 1.22 mmol, 1.0 eq) was dissolved in anhydrous DMF (N, N-dimethylformamide) (5 mL), cooled in an ice-water bath, and NaH (sodium hydride) (mass fraction 60%) (98 mg, 2.44 mmol, 2.0 eq) was added, stirred for 0.5 h, and then the compound SEMCl (2-(trimethylsilyl)ethoxymethyl chloride) (243 mg, 1.46 mmol, 1.2 eq) dissolved in anhydrous DMF (N, N-dimethylformamide) (1 mL) was slowly added dropwise, and the temperature was slowly raised from 0°C to room temperature 20°C and stirred for 4 h. After the reaction was completed by LCMS detection, it was added to water (25 mL), extracted three times with ethyl acetate (10 mL×3), washed with saturated brine (10 mL), dried over anhydrous sodium sulfate for 10 minutes, filtered, and the crude product was separated and purified by flash chromatography (Silica gel, ethyl acetate: petroleum ether (V / V = 1 / 5)) to obtain the title compound 3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidine (400 mg, 87.2%) as a yellow solid. LCMS (ESI) [M+H] + =377.0.

[0390] Step 4: Preparation of (S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol (Intermediate 31-d)

[0391]

[0392] The raw material 3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidine (170 mg, 0.45 mmol, 1.0 eq) was added to DMSO (dimethyl sulfoxide) (4 mL), and then (S)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (112 mg, 0.54 mmol, 1.2 eq) (i.e., intermediate 2-b, preparation method see Example 2), K2CO3 (potassium carbonate) (312 mg, 2.26 mmol, 5.0 eq), L-proline (21 mg, 0.18 mmol, 0.4 eq), CuI (cuprous iodide) (17 mg, 0.09 mmol, 0.2 eq) were added under nitrogen protection, and the mixture was stirred at 80°C for 4 h. After the reaction was completed, the mixture was cooled to room temperature (20°C), added to water (25 mL), extracted three times with ethyl acetate (10 mL×3), washed with saturated brine (5 mL), dried over anhydrous sodium sulfate for 10 minutes, filtered, and the crude product was separated and purified by flash chromatography (Silicagel, ethyl acetate: petroleum ether (V / V=1 / 4)) to obtain the title compound (S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol (160 mg, 78.0%) as a yellow solid. LCMS (ESI) [M+H] + =455.3.

[0393] Step 5: Preparation of (S)-1-((1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (Compound of Example 31)

[0394]

[0395] At room temperature 20°C, the raw material (S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol (150 mg, 0.34 mmol) was dissolved in DCM (dichloromethane) (4 mL), 4 M (molar concentration) HCL-dioxane (2 mL) was added, and the mixture was stirred at room temperature 20°C for 5 h. The reaction solution was adjusted to pH 7-8 with 10% NaHCO3 aqueous solution, and DCM (dichloromethane) (10 mL×3) was added for extraction three times, washed with saturated brine (5 mL), and concentrated. The crude product was separated and purified by Prep-HPLC (C18, 10 mmol / l NH4HCO3 in water, MeCN) to obtain the target compound (S)-1-((1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (9 mg, 8.1%). LCMS (ESI) [M+H] + =325.30; 1 H NMR (400MHz, CD3OD) δ: 8.99 (s, 1H), 8.83 (s, 1H), 7.11-7.03 (m, 4H), 4.27-4.24 (m, 1H), 3 .76(s, 2H), 3.68-3.64(m, 2H), 3.50-3.45(m, 2H), 2.94-2.87(m, 4H), 2.76-2.72(m, 2H).

[0396] Embodiment 32

[0397] Preparation of (R)-1-((1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol

[0398]

[0399] According to the method of Example 31, the raw material compound (R)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (i.e., intermediate 3-b, see Example 3 for preparation method) was used as the starting material, and a coupling reaction was carried out with 3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidine (i.e., intermediate 31-c, see Example 31 for preparation method), and then SEM was removed to prepare the target compound (R)-1-((1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol. LCMS (ESI) [M+H] + =325.30;1 H NMR (400MHz, CD3OD) δ: 8.99 (s, 1H), 8.83 (s, 1H), 7.11-7.03 (m, 4H), 4.27-4.24 (m, 1H), 3 .76(s, 2H), 3.68-3.64(m, 2H), 3.50-3.45(m, 2H), 2.94-2.87(m, 4H), 2.76-2.72(m, 2H).

[0400] Embodiment 33

[0401] Preparation of 1-((1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol

[0402]

[0403] According to the method of Example 31, the raw material compound (1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (i.e., intermediate 1-b, preparation method see Example 1) was used as the starting material, and a coupling reaction was carried out with 3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidine (i.e., intermediate 31-c, preparation method see Example 31), and then SEM was removed to prepare the target compound 1-((1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol. LCMS (ESI) [M+H] + =325.30; 1 H NMR (400MHz, CD3OD) δ: 8.99 (s, 1H), 8.83 (s, 1H), 7.11-7.03 (m, 4H), 4.27-4.24 (m, 1H), 3 .76(s, 2H), 3.68-3.64(m, 2H), 3.50-3.45(m, 2H), 2.94-2.87(m, 4H), 2.76-2.72(m, 2H).

[0404] Embodiment 34

[0405] Preparation of (S)-1-((7-amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol

[0406]

[0407] Step 1: Preparation of 3-iodo-1H-pyrazolo[4,3-d]pyrimidin-7-ol (Intermediate 34-a)

[0408]

[0409] 7-Hydroxypyrazolo[4,3-d]pyrimidine (2.00 g, 14.69 mmol, 1.0 eq) was dissolved in DMF (N,N-dimethylformamide) (40 mL), and NIS (N-iodosuccinimide) (4.96 g, 22.04 mmol, 1.5 eq) was added. The reaction was carried out at 60°C for 5 hours under nitrogen protection. After the reaction was completed by TLC detection, the reaction solvent was dried, and the mixture was dissolved in methanol and filtered. The filter cake was rinsed with methanol and dried to obtain the target compound (3.46 g, 89.9%) as a purple-gray solid. LCMS (ESI) [M+H] + =262.87; 1 H NMR (400MHz, DMSO-d6) δ: 14.55 (s, 1H), 12.41 (s, 1H), 7.92 (d, J=3.3Hz, 1H).

[0410] Step 2: Preparation of 3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol (Intermediate 34-b)

[0411]

[0412] 3-iodo-1H-pyrazolo[4,3-d]pyrimidin-7-ol (1.50 g, 5.72 mmol, 1.0 eq) was dissolved in DMF (N,N-dimethylformamide) (28 mL), Cs2CO3 (cesium carbonate) (2.24 g, 6.87 mmol, 1.2 eq) was added, SEMCl (2-(trimethylsilyl)ethoxymethyl chloride) (0.95 g, 5.72 mmol, 1.0 eq) was added dropwise at 0°C, and the reaction was allowed to react overnight at 20°C for 16 hours. After the reaction was completed by TLC detection, the reaction solution was extracted with dichloromethane and dried over anhydrous sodium sulfate. The organic phase was concentrated and the crude product was separated and purified by flash chromatography (Silica gel, DCM: MeOH = 15: 1) to obtain the target intermediate 3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol (1.21 g, 53.8%) as a yellow solid. LCMS (ESI) [M+H] + =393.12.

[0413] Step 3: Preparation of 7-((1H-benzo[d][1,2,3]triazol-1-yl)oxy)-3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidine (Intermediate 34-c)

[0414]

[0415] 3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol (2.00 g, 5.10 mmol, 1.0 eq) was dissolved in DMF (N,N-dimethylformamide) (26 mL), and PyBOP (1H-benzotriazol-1-yloxytripyrrolidino hexafluorophosphate) (3.45 g, 6.63 mmol, 1.3 eq) and DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) (1.16 g, 7.65 mmol, 1.5 eq) were added, and the reaction was carried out at 20° C. for 0.5 hours. After the reaction was completed by TLC, the reaction solution was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The organic phase was concentrated and the crude product was separated and purified by flash chromatography (Silica gel, PE:EA=5:1) to obtain the target compound (1.25 g, 48.1%) as a white solid. LCMS (ESI) [M+H] + =510.16.

[0416] Step 4: Preparation of 3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine (Intermediate 34-d)

[0417]

[0418] 7-((1H-benzo[d][1,2,3]triazol-1-yl)oxy)-3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidine (300 mg, 0.59 mmol, 1.0 eq) was dissolved in a 0.4 M (molar concentration) ammonia solution in 1,4-dioxane (12 mL) and reacted at 100° C. for 2 hours. After the reaction was completed by TLC, the reaction solution was concentrated to obtain the crude target compound (230 mg, 100.0%) as a yellow solid. LCMS (ESI) [M+H] + =392.09.

[0419] Step 5: Preparation of (S)-1-((7-amino-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (Intermediate 34-e)

[0420]

[0421] 3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine (230 mg, 0.59 mmol, 1.0 eq) was dissolved in DMSO (dimethyl sulfoxide) (6 mL), and compound (S)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (182 mg, 0.88 mmol, 1.5 eq) (i.e., intermediate 2-b, preparation method see Example 2), CuI (cuprous iodide) (22 mg, 0.12 mmol, 0.2 eq), K2CO3 (potassium carbonate) (406 mg, 2.94 mmol, 5.0 eq) and L-Proline (27 mg, 0.24 mmol, 0.4 eq) were added, and the reaction was carried out at 80° C. for 18 hours under nitrogen protection. After the reaction was completed by TLC, the reaction solution was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The organic phase was concentrated and the crude product was separated and purified by flash chromatography (Silica gel, DCM: MeOH = 15: 1) to obtain the target compound (140 mg, 50.5%) as a yellow solid. LCMS (ESI) [M+H] + =470.26.

[0422] Step 6: Preparation of (S)-1-((7-amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (Compound of Example 34)

[0423]

[0424] (S)-1-((7-amino-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (120 mg, 0.26 mmol, 1.0 eq) was dissolved in a 4M (molar concentration) hydrochloric acid solution in 1,4-dioxane (5 mL) and reacted at 20° C. for 1 hour. After the reaction was completed by TLC, the reaction solution was quenched with a saturated sodium bicarbonate aqueous solution, extracted with chloroform: isopropanol (7:3), and dried over anhydrous sodium sulfate. The organic phase was concentrated, and the crude product was separated and purified by preparative HPLC (C18, 10 mmol / L NH4HCO3 in water, MeCN) to obtain the target compound (S)-1-((7-amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (31.6 mg, 36.3%). LCMS (ESI) [M+H] + =340.3; 1H NMR (400MHz, DMSO-d6) δ: 11.19 (s, 1H), 8.04 (s, 1H), 7.13-6.98 (m, 6H), 5.45 (t, J=5. 9Hz, 1H), 4.87 (d, J=4.6Hz, 1H), 4.00 (q, J=5.5Hz, 1H), 3.68-3.56 (m, 2H), 3.48 (ddd, J=12.2, 7.1, 4.7Hz, 1H), 3.21 (ddd, J=12.5, 7.0, 5.0Hz, 1H), 2.81 (t, J=5.9Hz, 2H), 2 .71 (dtd, J=17.5, 11.4, 5.6Hz, 2H), 2.58 (dd, J=12.5, 5.8Hz, 1H), 2.54-2.45 (m, 1H).

[0425] Embodiment 35

[0426] Preparation of (R)-1-((7-amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol

[0427]

[0428] According to the method of Example 34, the raw material compound (R)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (i.e., intermediate 3-b, preparation method see Example 3) was used as the starting material, and a coupling reaction was carried out with 3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine (i.e., intermediate 34-d, preparation method see Example 34), and then SEM was removed, and the mixture was separated and purified by preparative HPLC (C18, 10mmol / L NH4HCO3in water, MeCN) to obtain the target compound (R)-1-((7-amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol. LCMS (ESI) [M+H] + =340.3; 1H NMR (400MHz, DMSO-d6) δ: 11.19 (s, 1H), 8.04 (s, 1H), 7.13-6.98 (m, 6H), 5.45 (t, J=5. 9Hz, 1H), 4.87 (d, J=4.6Hz, 1H), 4.00 (q, J=5.5Hz, 1H), 3.68-3.56 (m, 2H), 3.48 (ddd, J=12.2, 7.1, 4.7Hz, 1H), 3.21 (ddd, J=12.5, 7.0, 5.0Hz, 1H), 2.81 (t, J=5.9Hz, 2H), 2 .71 (dtd, J=17.5, 11.4, 5.6Hz, 2H), 2.58 (dd, J=12.5, 5.8Hz, 1H), 2.54-2.45 (m, 1H).

[0429] Embodiment 36

[0430] Preparation of 1-((7-amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol

[0431]

[0432] According to the method of Example 34, the raw material compound 1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (i.e., intermediate 1-b, the preparation method is shown in Example 1) is used as the starting material, and a coupling reaction is carried out with 3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine (i.e., intermediate 34-d, the preparation method is shown in Example 34), and then SEM is removed, and the mixture is separated and purified by preparative HPLC (C18, 10 mmol / L NH4HCO3in water, MeCN) to obtain the target compound 1-((7-amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol. LCMS (ESI) [M+H] + =340.3; 1H NMR (400MHz, DMSO-d6) δ: 11.19 (s, 1H), 8.04 (s, 1H), 7.13-6.98 (m, 6H), 5.45 (t, J=5. 9Hz, 1H), 4.87 (d, J=4.6Hz, 1H), 4.00 (q, J=5.5Hz, 1H), 3.68-3.56 (m, 2H), 3.48 (ddd, J=12.2, 7.1, 4.7Hz, 1H), 3.21 (ddd, J=12.5, 7.0, 5.0Hz, 1H), 2.81 (t, J=5.9Hz, 2H), 2 .71 (dtd, J=17.5, 11.4, 5.6Hz, 2H), 2.58 (dd, J=12.5, 5.8Hz, 1H), 2.54-2.45 (m, 1H).

[0433] Embodiment 37

[0434] Preparation of (S)-3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7-ol

[0435]

[0436] Step 1: Preparation of 3-iodo-1H-pyrazolo[4,3-d]pyrimidin-7-ol (Intermediate 37-a)

[0437]

[0438] The compound 7-hydroxy-pyrazolo[4,3-d]pyrimidine (2g, 14.694mmol, 1.0eq) was dissolved in N,N-dimethylformamide (40mL), and N-iodosuccinimide (4.96g, 22.041mmol, 1.5eq) was added, and the reaction was reacted at 60°C for 5 hours under nitrogen protection. After the reaction was completed by TLC detection, the reaction solvent was dried, and the mixture was dissolved in methanol and filtered. The filter cake was rinsed with methanol and dried to obtain 3.46g of 3-iodo-1H-pyrazolo[4,3-d]pyrimidine-7-ol, with a yield of 89.9%. LCMS: [M+1] + =262.87; 1 H NMR (400MHz, DMSO-d6) δ 14.55 (s, 1H), 12.41 (s, 1H), 7.92 (d, J = 3.3Hz, 1H).

[0439] Step 2: Preparation of 3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol (Intermediate 37-b)

[0440]

[0441] 3-iodo-1H-pyrazolo[4,3-d]pyrimidin-7-ol (1.50 g, 5.725 mmol, 1.0 eq) was dissolved in N,N-dimethylformamide (28 mL), cesium carbonate (2.24 g, 6.870 mmol, 1.2 eq) was added, 2-(trimethylsilyl)ethoxymethyl chloride (1.0 mL, 5.725 mmol, 1.0 eq) was added dropwise at 0°C, and the reaction was allowed to react at 20°C overnight for 16 hours. After the reaction was completed by TLC detection, the reaction solution was extracted with dichloromethane and dried over anhydrous sodium sulfate. The organic phase was concentrated and purified by column chromatography to obtain 1.21 g of 3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol, with a yield of 53.8%. LCMS: [M+1] + =393.12.

[0442] Step 3: Preparation of (S)-3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol (Intermediate 37-c)

[0443]

[0444] Compound 3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol (600 mg, 1.530 mmol, 1.0 eq) was dissolved in DMSO (dimethyl sulfoxide) (10 mL), and compound (S)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (473 mg, 2.294 mmol, 1.5 eq) (i.e., intermediate 2-b, preparation method see Example 2), cuprous iodide (58 mg, 0.306 mmol, 0.2 eq), potassium carbonate (1.06 g, 7.648 mmol, 5.0 eq) and L-proline (70 mg, 0.612 mmol, 0.4 eq) were added, and the reaction was carried out at 80° C. for 18 hours under nitrogen protection. After the reaction was completed by TLC detection, the reaction solution was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The organic phase was concentrated and purified by column chromatography to give 230 mg of (S)-3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol, yield 31.9%. LCMS: [M+1] + =471.37.

[0445] Step 4: Preparation of (S)-3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7-ol (Compound of Example 37)

[0446]

[0447] Compound (S)-3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol (30 mg, 0.064 mmol, 1.0 eq) was dissolved in a 4M (molar concentration) hydrochloric acid solution in 1,4-dioxane (1 mL) and reacted at 20° C. for 1 hour. After the reaction was completed by TLC, the reaction solution was quenched with a saturated aqueous sodium bicarbonate solution, extracted with dichloromethane, and dried over anhydrous sodium sulfate. The organic phase was concentrated and purified by preparative HPLC to obtain 1.8 mg of the target compound (S)-3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7-ol in a yield of 8.3%. LCMS: [M+1] + =341.2; 1 H NMR (400MHz, DMSO-d6) δ12.65 (s, 1H), 12.07 (s, 1H), 7.70 (s, 1H), 7.15-7.06 (m, 3H), 7.03 (d, J=6.6 Hz, 1H), 5.41 (s, 1H), 4.84 (s, 1H), 4.00 (s, 1H), 3.54 (s, 2H), 3.26-3.13 (m, 2H), 2.89-2.65 (m, 6H).

[0448] Embodiment 38

[0449] Preparation of (R)-3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7-ol

[0450]

[0451] According to the method of Example 37, the raw material compound (R)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (i.e., intermediate 3-b, see Example 3 for preparation method) was used as the starting material, and a coupling reaction was carried out with 3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol (i.e., intermediate 37-b, see Example 37 for preparation method), and then the target molecule (R)-3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7-ol was obtained by removing SEM. LCMS: [M+1] + =341.2; 1 H NMR (400MHz, DMSO-d6) δ12.65 (s, 1H), 12.07 (s, 1H), 7.70 (s, 1H), 7.15-7.06 (m, 3H), 7.03 (d, J=6.6 Hz, 1H), 5.41 (s, 1H), 4.84 (s, 1H), 4.00 (s, 1H), 3.54 (s, 2H), 3.26-3.13 (m, 2H), 2.89-2.65 (m, 6H).

[0452] Embodiment 39

[0453] Preparation of 3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7-ol

[0454]

[0455] According to the method of Example 37, the raw material compound (1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (i.e., intermediate 1-b, preparation method see Example 1) was used as the starting material, and a coupling reaction was carried out with 3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol (i.e., intermediate 37-b, preparation method see Example 37), and then the SEM was removed to obtain the target molecule 3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7-ol. LCMS: [M+1] + =341.2; 1H NMR (400MHz, DMSO-d6) δ: 12.65 (s, 1H), 12.07 (s, 1H), 7.70 (s, 1H), 7.15-7.06 (m, 3H), 7.03 (d, J=6. 6Hz, 1H), 5.41 (s, 1H), 4.84 (s, 1H), 4.00 (s, 1H), 3.54 (s, 2H), 3.26-3.13 (m, 2H), 2.89-2.65 (m, 6H).

[0456] Embodiment 40

[0457] Preparation of (S)-1-(4-(3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)piperazin-1-yl)ethan-1-one

[0458]

[0459] Step 1: Preparation of 3-iodo-1H-pyrazolo[4,3-d]pyrimidin-7-ol (Intermediate 40-a)

[0460]

[0461] 7-Hydroxypyrazolo[4,3-d]pyrimidine (2.00 g, 14.69 mmol, 1.0 eq) was dissolved in DMF (N,N-dimethylformamide) (40 mL), and NIS (N-iodosuccinimide) (4.96 g, 22.04 mmol, 1.5 eq) was added. The reaction was carried out at 60°C for 5 hours under nitrogen protection. After the reaction was completed by TLC detection, the reaction solvent was dried, and the mixture was dissolved in methanol and filtered. The filter cake was rinsed with methanol and dried to obtain the target compound (3.46 g, 89.9%) as a purple-gray solid. LCMS (ESI) [M+H] + =262.87; 1 H NMR (400MHz, DMSO-d6) δ: 14.55 (s, 1H), 12.41 (s, 1H), 7.92 (d, J = 3.3Hz, 1H).

[0462] Step 2: Preparation of 3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol (Intermediate 40-b)

[0463]

[0464] 3-iodo-1H-pyrazolo[4,3-d]pyrimidin-7-ol (1.50 g, 5.72 mmol, 1.0 eq) was dissolved in DMF (N,N-dimethylformamide) (28 mL), Cs2CO3 (cesium carbonate) (2.24 g, 6.87 mmol, 1.2 eq) was added, and SEMC] (2-(trimethylsilyl)ethoxymethyl chloride) (0.95 g, 5.72 mmol, 1.0 eq) was added dropwise at 0°C, and the reaction was allowed to react overnight at 20°C for 16 hours. After the reaction was completed by TLC detection, the reaction solution was extracted with dichloromethane and dried over anhydrous sodium sulfate. The organic phase was concentrated and the crude product was separated and purified by flash chromatography (Silica gel, DCM: MeOH = 15: 1) to obtain the target intermediate 3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol (1.21 g, 53.8%) as a yellow solid. LCMS (ESI) [M+H] + =393.12.

[0465] Step 3: Preparation of 1-(4-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)piperazin-1-yl)ethan-1-one (Intermediate 40-c)

[0466]

[0467] The compound 3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidine-7-ol (100 mg, 0.255 mmol, 1.0 eq) was dissolved in DMF (N,N-dimethylformamide) (1.3 mL), and the compound 1-(piperazine-1-yl)ethane-1-one (49 mg, 0.382 mmol, 1.5 eq), PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate) (173 mg, 0.331 mmol, 1.3 eq) and DBU (1,8-diazabicycloundec-7-ene) (116.2 mg, 0.765 mmol, 3.0 eq) were added, and the reaction was carried out at 20° C. for 0.5 hours. After the reaction was completed by TLC, the reaction solution was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The organic phase was concentrated and purified by column chromatography to obtain 115 mg of 1-(4-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)piperazin-1-yl)ethan-1-one, with a yield of 89.8%. LCMS: [M+1] + =503.23.

[0468] Step 4: Preparation of (S)-1-(4-(3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)piperazin-1-yl)ethan-1-one (Intermediate 40-d)

[0469]

[0470] The compound 1-(4-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)piperazin-1-yl)ethan-1-one (115 mg, 0.229 mmol, 1.0 eq) was dissolved in DMSO (2 mL), and the compound (S)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (71 mg, 0.344 mmol, 1.5 eq), cuprous iodide (17 mg, 0.092 mmol, 0.4 eq), potassium carbonate (158.3 mg, 1.145 mmol, 5.0 eq) and L-proline (10.5 mg, 0.092 mmol, 0.4 eq) were added, and the reaction was carried out at 80° C. for 10 hours under N2 protection. After the reaction was completed by TLC detection, the reaction solution was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The organic phase was concentrated and purified by column chromatography to obtain 81 mg of (S)-1-(4-(3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)piperazin-1-yl)ethan-1-one, with a yield of 61.0%. LCMS: [M+1] + =581.48.

[0471] Step 5: Preparation of (S)-1-(4-(3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)piperazin-1-yl)ethan-1-one (Compound of Example 40)

[0472]

[0473] Compound (S)-1-(4-(3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)piperazin-1-yl)ethane-1-one (80 mg, 0.138 mmol, 1.0 eq) was dissolved in a hydrochloric acid solution of 1,4-dioxane (4 M, 0.7 mL) and reacted at 20° C. for 1.5 hours. After the reaction was completed by TLC, the reaction solution was quenched with a saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, and dried over anhydrous sodium sulfate. The organic phase was concentrated and purified by preparative HPLC to obtain 18.9 mg of the target compound (S)-1-(4-(3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)piperazin-1-yl)ethan-1-one in a yield of 30.4%. LCMS: [M+1] + =451.26;1H NMR (400MHz, CDCl3) δ: 8.23 ​​(s, 1H), 7.20-7.09 (m, 3H), 7.03-6.97 (m, 1H), 5.27 (brs, 1H), 4.15 (s, 5H), 3.87 (d, J=14.9Hz, 1H), 3.78 (t, J=5. 3Hz, 2H), 3.72-3.58 (m, 4H), 3.39 (dd, J=15.3, 4.5Hz, 1H), 3.04-2.92 (m, 3H), 2.85-2.72 (m, 2H), 2.63 (dd, J=12.4, 4.2Hz, 1H), 2.16 (s, 3H).

[0474] Embodiment 41

[0475] Preparation of 1-(4-(3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)piperazin-1-yl)ethan-1-one

[0476]

[0477] According to the method of Example 40, the raw material compound 1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (i.e., intermediate 1-b, the preparation method is shown in Example 1) is used as the starting material, and a coupling reaction is carried out with 1-(4-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)piperazin-1-yl)ethane-1-one (i.e., intermediate 40-c, the preparation method is shown in Example 40), and then the SEM is removed to obtain the target molecule 1-(4-(3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)piperazin-1-yl)ethane-1-one. LCMS: [M+1] + =451.26; 1 H NMR (400MHz, CDCl3) 68.23 (s, 1H), 7.20-7.09 (m, 3H), 7.03-6.97 (m, 1H), 5.27 (brs, 1H), 4.15 (s, 5H), 3.87 (d, J=14.9Hz, 1H), 3.78 (t, J=5. 3Hz, 2H), 3.72-3.58 (m, 4H), 3.39 (dd, J=15.3, 4.5Hz, 1H), 3.04-2.92 (m, 3H), 2.85-2.72 (m, 2H), 2.63 (dd, J=12.4, 4.2Hz, 1H), 2.16 (s, 3H).

[0478] Embodiment 42

[0479] Preparation of (R)-1-(4-(3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)piperazin-1-yl)ethan-1-one

[0480]

[0481] According to the method of Example 40, the raw material compound (R)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (i.e., intermediate 3-b, see Example 3 for preparation method) is used as the starting material, and a coupling reaction is carried out with 1-(4-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)piperazin-1-yl)ethane-1-one (i.e., intermediate 40-c, see Example 40 for preparation method), and then the SEM is removed to obtain the target molecule (R)-1-(4-(3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)piperazin-1-yl)ethane-1-one. LCMS: [M+1] + =451.26; 1 H NMR (400MHz, CDCl3) δ: 8.23 ​​(s, 1H), 7.20-7.09 (m, 3H), 7.03-6.97 (m, 1H), 5.27 (brs, 1H), 4.15 (s, 5H), 3.87 (d, J=14.9Hz, 1H), 3.78 (t, J=5. 3Hz, 2H), 3.72-3.58 (m, 4H), 3.39 (dd, J=15.3, 4.5Hz, 1H), 3.04-2.92 (m, 3H), 2.85-2.72 (m, 2H), 2.63 (dd, J=12.4, 4.2Hz, 1H), 2.16 (s, 3H).

[0482] Embodiment 43

[0483] Preparation of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[3,4-c]pyridin-7-yl)amino)piperidin-1-yl)ethan-1-one

[0484]

[0485] Step 1: Synthesis of 5-chloro-3-iodo-1H-pyrazolo[4,3-d]pyrimidine (Intermediate 43-a)

[0486]

[0487] The compound 5-chloro-1H-pyrazolo[4,3-d]pyrimidine (600 mg, 3.88 mmol, 1.0 eq) was dissolved in DMF (N,N-dimethylformamide) (40 mL), and NIS (N-iodosuccinimide) (1.1 g, 4.66 mmol, 1.2 eq) was added, and the reaction was reacted at 60° C. for 2 hours under nitrogen protection. After the reaction was completed by TLC detection, the reaction solvent was dried, the mixture was dissolved in water and stirred for 30 minutes, and filtered. The filter cake was rinsed with water and dried to obtain 5-chloro-3-iodo-1H-pyrazolo[4,3-d]pyrimidine as a yellow solid 950 mg with a yield of 87.4%. LCMS: [M+H] + =280.90; 1 H NMR (400MHz, DMSO-d6) δ: 14.59 (s, 1H), 9.31 (s, 1H).

[0488] Step 2: Preparation of 5-chloro-3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidine (Intermediate 43-b)

[0489]

[0490] The compound 5-chloro-3-iodo-1H-pyrazolo[4,3-d]pyrimidine (500 mg, 1.78 mmol, 1.0 eq) was dissolved in DMF (N,N-dimethylformamide) (3 mL), and added dropwise to a suspension of NaH (sodium hydride) (110 mg, 2.67 mmol, 1.5 eq) in DMF (N,N-dimethylformamide) (3 mL) at 0°C, and stirred for 30 minutes. SEMCl (2-(trimethylsilyl)ethoxymethyl chloride) (387 mg, 2.32 mmol, 1.3 eq) was added dropwise at 0°C, and stirred at room temperature (15-20 degrees) for 1 hour. After the reaction was completed by TLC detection, the reaction solution was extracted with ethyl acetate three times, 10 ml each time, and the organic phases were combined and dried over anhydrous sodium sulfate. The organic phase was concentrated and purified by column chromatography to obtain 5-chloro-3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidine as a colorless crystalline solid (580 mg) with a yield of 79.2%. LCMS: [M+H] + =410.92; 1 H NMR (400MHz, DMSO-d6) δ9.66 (s, 1H), 5.99 (s, 2H), 3.77-3.61 (m, 2H), 1.02-0.85 (m, 2H), 0.01 (s, 9H).

[0491] Step 3: Preparation of 1-(4-((3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)amino)piperidin-1-yl)ethan-1-one (Intermediate 43-c)

[0492]

[0493] The compound 5-chloro-3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidine (500 mg, 1.22 mmol, 1.0 eq), the compound 1-(4-aminopiperidin-1-yl)ethane-1-one hydrochloric acid (261 mg, 1.46 mmol, 1.2 eq), DIEA (N,N-diisopropylethylamine) (550 mg, 4.27 mmol, 3.5 eq) were dissolved in NMP (N-methylpyrrolidone) (6 mL), and reacted at 180° C. for 1 hour under nitrogen protection. After the reaction was completed by TLC detection, the reaction solution was extracted with ethyl acetate, 10 ml each time, and the organic phases were combined and dried over anhydrous sodium sulfate. The organic phase was concentrated and purified by column chromatography to give 1-(4-((3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)amino)piperidin-1-yl)ethan-1-one as a yellow oil (220 mg, yield 35%). LCMS: [M+H] + =517.14.

[0494] Step 4: Preparation of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)amino)piperidin-1-yl)ethan-1-one (Intermediate 43-d)

[0495]

[0496] Compound 1-(4-((3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)amino)piperidin-1-yl)ethan-1-one (220 ng, 0.42 mmol, 1.0 eq), compound (S)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (105 mg, 0.51 mmol, 1.2 eq), CuI (cuprous iodide) (32 mg, 0.168 mmol, 0.4 eq), potassium carbonate (290 mg, 2.1 mmol, 5.0 eq) and L-proline (20 mg, 0.168 mmol, 0.4 eq) were dissolved in DMSO (dimethyl sulfoxide) (2 mL) and the reaction was carried out at 80° C. for 18 hours under nitrogen protection. After the reaction was completed by TLC detection, the reaction solution was extracted with ethyl acetate, 10 ml each time, and the organic phases were combined and dried over anhydrous sodium sulfate. The organic phase was concentrated and purified by column chromatography to obtain (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)amino)piperidin-1-yl)ethan-1-one as a yellow solid, 90 mg, with a yield of 35.6%. LCMS: [M+1] + =595.43.

[0497] Step 5: Preparation of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[3,4-c]pyridin-7-yl)amino)piperidin-1-yl)ethan-1-one (Compound of Example 43)

[0498]

[0499] Compound (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-c]pyridin-7-yl)amino)piperidin-1-yl)ethane-1-one (80 mg, 0.13 mmol, 1.0 eq) was dissolved in a 4M (molar concentration) hydrochloric acid solution in 1,4-dioxane (2 mL) and reacted at room temperature (15-20 degrees) for 4 hours. After the reaction was completed by TLC, the reaction solution was quenched with a saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, 10 ml each time, and the organic phases were combined and dried over anhydrous sodium sulfate. The organic phase was concentrated and purified by preparative HPLC to obtain the target compound, 19.6 mg, with a yield of 32%. LCMS: [M+H] + =465.41; 1H NMR (600MHz, CDCl3) δ: 8.55 (s, 1H), 7.20-7.12 (m, 3H), 7.03 (d, J=7.5Hz, 1H), 4.92 ( m, 2H), 4.50 (d, J=13.1Hz, 1H), 4.32 (s, 1H), 4.14-3.98 (m, 2H), 3.90-3.76 (m, 2H), 3 .67 (d, J=13.0Hz, 1H), 3.47 (dd, J=12.6, 6.1Hz, 1H), 3.26 (t, J=11.1Hz, 1H), 3.13 (s , 1H), 3.01(s, 3H), 2.88(m, 3H), 2.17(s, 1H), 2.12(s, 3H), 2.09(s, 1H), 1.42(s, 2H).

[0500] Embodiment 44

[0501] Preparation of 1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[3,4-c]pyridin-7-yl)amino)piperidin-1-yl)ethan-1-one

[0502]

[0503] According to the method of Example 43, the raw material compound (1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (i.e., intermediate 1-b, see Example 1 for the preparation method) was used as the starting material, and a coupling reaction was carried out with 1-(4-((3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)amino)piperidin-1-yl)ethane-1-one (i.e., intermediate 43-c, see Example 43 for the synthesis method), and then the SEM was removed to obtain the target molecule 1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[3,4-c]pyridin-7-yl)amino)piperidin-1-yl)ethane-1-one. LCMS: [M+H] + =465.41; 1H NMR (600MHz, CDCl3) δ: 8.55 (s, 1H), 7.20-7.12 (m, 3H), 7.03 (d, J=7.5Hz, 1H), 4.92 ( m, 2H), 4.50 (d, J=13.1Hz, 1H), 4.32 (s, 1H), 4.14-3.98 (m, 2H), 3.90-3.76 (m, 2H), 3 .67 (d, J=13.0Hz, 1H), 3.47 (dd, J=12.6, 6.1Hz, 1H), 3.26 (t, J=11.1Hz, 1H), 3.13 (s , 1H), 3.01(s, 3H), 2.88(m, 3H), 2.17(s, 1H), 2.12(s, 3H), 2.09(s, 1H), 1.42(s, 2H).

[0504] Embodiment 45

[0505] Preparation of (R)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[3,4-c]pyridin-7-yl)amino)piperidin-1-yl)ethan-1-one

[0506]

[0507] According to the method of Example 43, the raw material compound (R)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (i.e., intermediate 3-b, see Example 3 for the preparation method) is used as the starting material, and a coupling reaction is carried out with 1-(4-((3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)amino)piperidin-1-yl)ethane-1-one (i.e., intermediate 43-c, see Example 43 for the synthesis method), and then SEM is removed to obtain the target molecule (R)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[3,4-c]pyridin-7-yl)amino)piperidin-1-yl)ethane-1-one. LCMS: [M+H] + =465.41; 1H NMR (600MHz, CDCl3) δ8.55 (s, 1H), 7.20-7.12 (m, 3H), 7.03 (d, J=7.5Hz, 1H), 4.92 (m , 2H), 4.50 (d, J=13.1Hz, 1H), 4.32 (s, 1H), 4.14-3.98 (m, 2H), 3.90-3.76 (m, 2H), 3. 67 (d, J=13.0Hz, 1H), 3.47 (dd, J=12.6, 6.1Hz, 1H), 3.26 (t, J=11.1Hz, 1H), 3.13 (s, 1H), 3.01(s, 3H), 2.88(m, 3H), 2.17(s, 1H), 2.12(s, 3H), 2.09(s, 1H), 1.42(s, 2H).

[0508] Embodiment 46

[0509] Preparation of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)piperidin-1-yl)ethan-1-one

[0510]

[0511] Step 1: Preparation of 6-chloro-3-iodo-1H-pyrazolo[3,4-d]pyrimidine (Intermediate 46-a)

[0512]

[0513] The compound 6-chloro-1H-pyrazolo[3,4-d]pyrimidine (0.85 g, 5.52 mmol, 1.0 eq) was dissolved in DMF (N,N-dimethylformamide) (10 mL), and NIS (N-iodosuccinimide) (1.48 g, 6.62 mmol, 1.2 eq) was added. The reaction temperature was raised to 120°C and stirred for 1 hour under microwave conditions. After the reaction was completed by LCMS, it was cooled to room temperature, water (30 mL) was added, and then dichloromethane: methanol (V / V = 10 / 1) (20 mL × 3) was added to extract three times, washed with saturated brine (25 mL), dried over anhydrous sodium sulfate for 10 minutes, filtered, concentrated, and the crude product was separated and purified by flash chromatography (Silica gel, DCM: MeOH = 50: 1) to obtain the title compound 6-chloro-3-iodo-1H-pyrazolo[3,4-d]pyrimidine (1.10 g, 71.42%) as a yellow solid. LCMS(ESI)[M+H] + =280.9; 1H NMR (400MHz, DMSO-d6) δ14.64 (s, 1H), 9.02 (s, 1H).

[0514] Step 2: Preparation of 6-chloro-3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidine (Intermediate 46-b)

[0515]

[0516] The raw material 6-chloro-3-iodo-1H-pyrazolo[3,4-d]pyrimidine (1.0 g, 3.58 mmol, 1.0 eq) was dissolved in anhydrous THF (tetrahydrofuran) (15 mL), cooled in an ice-water bath, sodium hydride (mass fraction 60%) (286 mg, 27.16 mmol, 2.0 eq) was added, stirred for 0.5 h, and then the compound SEMCl (2-(trimethylsilyl)ethoxymethyl chloride) (654 mg, 3.94 mmol, 1.1 eq) dissolved in anhydrous THF (tetrahydrofuran) (2 mL) was slowly added dropwise, stirred for 10 h from 0°C to room temperature 20°C, after LCMS detection, the mixture was added to water (25 mL), extracted three times with ethyl acetate (20 mL×3), washed with saturated brine (15 mL), dried over anhydrous sodium sulfate for 10 minutes, filtered, concentrated, and the crude product was separated and purified by flash chromatography (Silica gel, EA:Pe = 1:5) to give the title compound 6-chloro-3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidine (670 mg, yield 45.64%) as a yellow solid. LCMS (ESI) [M+H] + =411.04; 1 H NMR (400MHz, DMSO-d6) δ: 9.08 (s, 1H), 5.71 (s, 2H), 3.63-3.59 (t, J=8.0Hz, 2H), 3.63-3.59 (t, J=8.0Hz, 2H), 0.83-0.87 (t, J=8.4Hz, 2H), -0.07 (s, 9H).

[0517] Step 3: Preparation of 1-(4-((3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)piperidin-1-yl)ethan-1-one (Intermediate 46-c)

[0518]

[0519] The intermediate 6-chloro-3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidine (400 mg, 0.97 mmol, 1.0 eq) was added to ACN (acetonitrile) (10 mL), and then 1-acetylpiperidin-4-amine hydrochloride (208 mg, 1.17 mmol, 1.2 eq) and DIEA (N,N-diisopropylethylamine) (275 mg, 2.13 mmol, 2.2 eq) were added, and the mixture was stirred at 90°C for 5 h. After the reaction was completed by LCMS, the mixture was concentrated, and the residue was added to water (15 mL), extracted three times with ethyl acetate (20 mL×3), and the organic phases were combined, dried over anhydrous sodium sulfate for 10 minutes, filtered, and concentrated. The crude product was separated and purified by flash chromatography (Silica gel, ethyl acetate: petroleum ether = 1:5) to give the title compound 1-(4-((3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)piperidin-1-yl)ethan-1-one (400 mg, 79.9%) as a yellow solid. LCMS (ESI) [M+H] + =517.2.

[0520] Step 4: Preparation of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)piperidin-1-yl)ethan-1-one (Intermediate 46-d)

[0521]

[0522] 1-(4-((3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)piperidin-1-yl)ethan-1-one (52 mg, 0.1 mmol, 1.0 eq) was added to DMSO (dimethyl sulfoxide) (2 mL), and then (S)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (25 mg, 0.12 mmol, 1.2 eq), K2CO3 (potassium carbonate) (55 mg, 0.4 mmol, 4.0 eq), L-Proline (9 mg, 0.08 mmol, 0.8 eq), CuI (cuprous iodide) (8 mg, 0.04 mmol, 0.04 eq) were added under nitrogen protection, and the mixture was stirred at 80°C for 12 h. After the reaction was completed, the mixture was cooled to room temperature (20°C), added to water (10 mL), extracted three times with ethyl acetate (10 mL×3), washed with saturated brine (5 mL), dried over anhydrous sodium sulfate for 10 minutes, filtered, and concentrated. The crude product was separated and purified by flash chromatography (Silica gel, dichloromethane: methanol = 10: 1) to obtain the title compound (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)piperidin-1-yl)ethan-1-one (30 mg, 77.96%) as a yellow solid. LCMS (ESI) [M+H] + =455.3.

[0523] Step 5: Preparation of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)piperidin-1-yl)ethan-1-one (Compound of Example 46)

[0524]

[0525] At room temperature of 20°C, the raw material (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)piperidin-1-yl)ethan-1-one (150 mg, 0.34 mmol) was dissolved in Dioxane (1,4-dioxane) (4 mL), and 4 M (molar concentration) HCl-Dioxane (HCl-1,4-dioxane solution) (2 mL) was added, and the mixture was stirred at room temperature for 5 h. The reaction solution was adjusted to pH 7-8 with 10% NaHCO3 aqueous solution, extracted three times with dichloromethane (10 mL×3), washed with saturated brine (5 mL), concentrated, and the residue was separated and purified by preparative HPLC (C18, 10 mmol / L NH4HCO3 aqueous solution, MeCN) (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)piperidin-1-yl)ethane-1-one (55 mg, 34.7%). LCMS (ESI) [M+H] + =465.4;1H NMR (400MHz, CD3OD) δ: 11.52 (s, 1H), 8.65 (s, 1H), 7.12-7.03 (m, 5H), 6.30-6.27 (t, J=2.6Hz, 1H), 4.80 (s, 1H), 4.30-4.26 (d, J=12.4Hz, 1H), 4.00-3.97 (m, 2H), 3.83-3.79 (d, J=13.6Hz, 1H ), 3.68-3.59(q, J=14.8Hz, J=6.0Hz, 1H), 3.42-3.60(m, 1H), 3.16-3.09(m, 2H), 2.83-2.80(m, 2H), 2.77-2.68(m, 3H), 2.59-2.54(m, 2H), 2.01(s, 3H), 1.93-1.84(m, 2H), 1.43-1.28(m, 2H).

[0526] Embodiment 47

[0527] Preparation of (R)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)piperidin-1-yl)ethan-1-one

[0528]

[0529] According to the method of Example 46, the raw material compound (R)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (i.e., intermediate 3-b, preparation method see Example 3) is used as the starting material, and a coupling reaction is carried out with 1-(4-((3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)piperidin-1-yl)ethane-1-one (i.e., intermediate 46-c, preparation method see Example 46), and then SEM is removed to obtain the target molecule (R)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)piperidin-1-yl)ethane-1-one. LCMS(ESI)[M+H] + =465.4; 1 H NMR (400MHz, CD3OD) δ: 11.52 (s, 1H), 8.65 (s, 1H), 7.12-7.03 (m, 5H), 6.30-6.27 (t, J=2.6Hz, 1H), 4.80 (s, 1H), 4.30-4.26 (d, J=12.4Hz, 1H), 4.00-3.97 (m, 2H), 3.83-3.79 (d, J=13.6Hz, 1H ), 3.68-3.59(q, J=14.8Hz, J=6.0Hz, 1H), 3.42-3.60(m, 1H), 3.16-3.09(m, 2H), 2.83-2.80(m, 2H), 2.77-2.68(m, 3H), 2.59-2.54(m, 2H), 2.01(s, 3H), 1.93-1.84(m, 2H), 1.43-1.28(m, 2H).

[0530] Embodiment 48

[0531] Preparation of 1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)piperidin-1-yl)ethan-1-one

[0532]

[0533] According to the method of Example 46, the raw material compound (1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (i.e., intermediate 1-b, preparation method see Example 1) was used as the starting material, and a coupling reaction was carried out with 1-(4-((3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)piperidin-1-yl)ethane-1-one (i.e., intermediate 46-c, preparation method see Example 46), and then the SEM was removed to obtain the target molecule 1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)piperidin-1-yl)ethane-1-one. LCMS (ESI) [M+H] + =465.4;1H NMR (400MHz, CD3OD) δ: 11.52 (s, 1H), 8.65 (s, 1H), 7.12-7.03 (m, 5H), 6.30-6.27 (t, J=2.6Hz, 1H), 4.80 (s, 1H), 4.30-4.26 (d, J=12.4Hz, 1H), 4.00-3.97 (m, 2H), 3.83-3.79 (d, J=13.6Hz, 1H ), 3.68-3.59(q, J=14.8Hz, J=6.0Hz, 1H), 3.42-3.60(m, 1H), 3.16-3.09(m, 2H), 2.83-2.80(m, 2H), 2.77-2.68(m, 3H), 2.59-2.54(m, 2H), 2.01(s, 3H), 1.93-1.84(m, 2H), 1.43-1.28(m, 2H).

[0534] Embodiment 49

[0535] Preparation of (S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-(pyridazin-4-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol

[0536]

[0537] Step 1: Preparation of 3-iodo-1H-pyrazolo[4,3-d]pyrimidin-7-ol (Intermediate 49-a)

[0538]

[0539] 7-Hydroxypyrazolo[4,3-d]pyrimidine (2.00 g, 14.69 mmol, 1.0 eq) was dissolved in DMF (N,N-dimethylformamide) (40 mL), and NIS (N-iodosuccinimide) (4.96 g, 22.04 mmol, 1.5 eq) was added. The reaction was carried out at 60°C for 5 hours under nitrogen protection. After the reaction was completed by TLC detection, the reaction solvent was dried, and the mixture was dissolved in methanol and filtered. The filter cake was rinsed with methanol and dried to obtain the target compound (3.46 g, 89.9%) as a purple-gray solid. LCMS (ESI) [M+H] + =262.87; 1 H NMR (400MHz, DMSO-d6) δ 14.55 (s, 1H), 12.41 (s, 1H), 7.92 (d, J = 3.3Hz, 1H).

[0540] Step 2: Preparation of 3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol (Intermediate 49-b)

[0541]

[0542] 3-iodo-1H-pyrazolo[4,3-d]pyrimidin-7-ol (1.50 g, 5.72 mmol, 1.0 eq) was dissolved in DMF (N,N-dimethylformamide) (28 mL), Cs2CO3 (cesium carbonate) (2.24 g, 6.87 mmol, 1.2 eq) was added, SEMCl (2-(trimethylsilyl)ethoxymethyl chloride) (0.95 g, 5.72 mmol, 1.0 eq) was added dropwise at 0°C, and the reaction was allowed to react at 20°C overnight for 16 hours. After the reaction was completed by TLC, the reaction solution was extracted with dichloromethane and dried over anhydrous sodium sulfate. The organic phase was concentrated, and the crude product was separated and purified by flash chromatography (Silica gel, DCM: MeOH = 15: 1) to obtain the target compound (1.21 g, 53.8%) as a yellow solid. LCMS (ESI) [M+H] + =393.12.

[0543] Step 3: Preparation of 7-((1H-benzo[d][1,2,3]triazol-1-yl)oxy)-3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidine (Intermediate 49-c)

[0544]

[0545] 3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol (2.00 g, 5.10 mmol, 1.0 eq) was dissolved in DMF (N,N-dimethylformamide) (26 mL), and PyBOP (1H-benzotriazol-1-yloxytripyrrolidino hexafluorophosphate) (3.45 g, 6.63 mmol, 1.3 eq) and DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) (1.16 g, 7.65 mmol, 1.5 eq) were added, and the reaction was carried out at 20° C. for 0.5 hours. After the reaction was completed by TLC, the reaction solution was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The organic phase was concentrated and the crude product was separated and purified by flash chromatography (Silica gel, PE:EA=6:1) to obtain the target compound (1.25 g, 48.1%) as a white solid. LCMS (ESI) [M+H] + =510.16.

[0546] Step 4: Preparation of 3-iodo-N-(pyridazin-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine (Intermediate 49-d)

[0547]

[0548] To a solution of 4-aminopyridazine (140 mg, 1.47 mmol, 1.5 eq) in DMF (N, N-dimethylformamide) (3 mL) was added NaH (sodium hydride) (118 mg, 2.94 mmol, 3.0 eq) at 0°C, and stirred for 10 minutes under nitrogen protection. Then a solution of 7-((1H-benzo[d][1,2,3]triazol-1-yl)oxy)-3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidine (500 mg, 0.98 mmol, 1.0 eq) in DMF (N, N-dimethylformamide) (4 mL) was added, and the mixture was reacted at 20°C for 0.5 hours. After the reaction was completed by TLC, the reaction solution was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The organic phase was concentrated and the crude product was separated and purified by flash chromatography (Silica gel, EA) to obtain the title compound (430 mg, 93.3%) as a yellow solid. LCMS (ESI) [M+H] + =470.21.

[0549] Step 5: Preparation of (S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-(pyridazin-4-ylamino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol (Intermediate 49-e)

[0550]

[0551] 3-iodo-N-(pyridazin-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine (320 mg, 0.68 mmol, 1.0 eq) was dissolved in DMSO (dimethyl sulfoxide) (7 mL), and (S)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (169 mg, 0.82 mmol, 1.2 eq), CuI (copper iodide) (26 mg, 0.14 mmol, 0.2 eq), K2CO3 (potassium carbonate) (471 mg, 3.41 mmol, 5.0 eq) and L-Proline (31 mg, 0.27 mmol, 0.4 eq) were added, and the reaction was carried out at 80° C. for 16 hours under nitrogen protection. After the reaction was completed by TLC, the reaction solution was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The organic phase was concentrated and the crude product was separated and purified by flash chromatography (Silica gel, DCM: MeOH = 30: 1) to obtain the target compound (82 mg, 22.0%) as a yellow solid. LCMS (ESI) [M+H] + =548.34.

[0552] Step 6: Preparation of (S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-(pyridazin-4-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol (Compound of Example 49)

[0553]

[0554] (S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-(pyridazin-4-ylamino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol (62 mg, 0.11 mmol, 1.0 eq) was dissolved in MeOH (methanol) (3 mL), and a 1,4-dioxane solution of hydrochloric acid (3 mL, 4 M) was added, and the reaction was reacted at 40° C. for 3 hours. After the reaction was completed by TLC, the reaction solution was quenched with a saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, and dried over anhydrous sodium sulfate. The organic phase was concentrated and the crude product was separated and purified by preparative HPLC (C18, 10 mmol / L NH4HCO3 aqueous solution, MeCN) to give (S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-(pyridazin-4-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol (14 mg, 29.8%). LCMS (ESI) [M+H] + =418.3; 1 H NMR (400MHz, DMSO-d6) δ: 11.48 (s, 1H), 10.19 (s, 1H), 9.51 (s, 1H), 9.04 (d, J=6.0Hz, 1H), 8. 39 (d, J=48.8Hz, 2H), 7.08 (dd, J=5.1, 3.3Hz, 3H), 7.04-6.97 (m, 1H), 5.80 (s, 1H), 4.89 (s, 1 H), 4.19-3.96 (m, 1H), 3.70-3.57 (m, 2H), 3.56-3.48 (m, 1H), 3.31-3.23 (m, 1H), 2.81 (d, J=5 .8Hz, 2H), 2.72 (td, J=11.3, 5.5Hz, 2H), 2.61 (dd, J=12.6, 5.7Hz, 1H), 2.54 (d, J=6.5Hz, 1H).

[0555] Embodiment 50

[0556] Preparation of (R)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-(pyridazin-4-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol

[0557]

[0558] According to the method of Example 49, the raw material compound (R)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (i.e., intermediate 3-b, see Example 3 for preparation method) was used as the starting material, and a coupling reaction was carried out with 3-iodo-N-(pyridazine-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine (i.e., intermediate 49-d, see Example 49 for preparation method), and then the target molecule (R)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-(pyridazine-4-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol was obtained by removing SEM. LCMS (ESI) [M+H] + =418.3; 1 H NMR (400MHz, DMSO-d6) δ: 11.48 (s, 1H), 10.19 (s, 1H), 9.51 (s, 1H), 9.04 (d, J=6.0Hz, 1H), 8. 39 (d, J=48.8Hz, 2H), 7.08 (dd, J=5.1, 3.3Hz, 3H), 7.04-6.97 (m, 1H), 5.80 (s, 1H), 4.89 (s, 1 H), 4.19-3.96 (m, 1H), 3.70-3.57 (m, 2H), 3.56-3.48 (m, 1H), 3.31-3.23 (m, 1H), 2.81 (d, J=5 .8Hz, 2H), 2.72 (td, J=11.3, 5.5Hz, 2H), 2.61 (dd, J=12.6, 5.7Hz, 1H), 2.54 (d, J=6.5Hz, 1H).

[0559] Embodiment 51

[0560] Preparation of 1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-(pyridazin-4-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol

[0561]

[0562] According to the method of Example 49, the raw material compound (1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (i.e., intermediate 1-b, preparation method see Example 1) was used as the starting material, and a coupling reaction was carried out with 3-iodo-N-(pyridazin-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine (i.e., intermediate 49-d, preparation method see Example 49), and then the SEM was removed to obtain the target molecule 1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-(pyridazin-4-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol. LCMS (ESI) [M+H] + =418.3; 1 H NMR (400MHz, DMSO-d6) δ: 11.48 (s, 1H), 10.19 (s, 1H), 9.51 (s, 1H), 9.04 (d, J=6.0Hz, 1H), 8. 39 (d, J=48.8Hz, 2H), 7.08 (dd, J=5.1, 3.3Hz, 3H), 7.04-6.97 (m, 1H), 5.80 (s, 1H), 4.89 (s, 1 H), 4.19-3.96 (m, 1H), 3.70-3.57 (m, 2H), 3.56-3.48 (m, 1H), 3.31-3.23 (m, 1H), 2.81 (d, J=5 .8Hz, 2H), 2.72 (td, J=11.3, 5.5Hz, 2H), 2.61 (dd, J=12.6, 5.7Hz, 1H), 2.54 (d, J=6.5Hz, 1H).

[0563] Embodiment 52

[0564] Preparation of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one

[0565]

[0566] Step 1: Preparation of benzyl 2,2,2-trifluoroacetimidate (Intermediate 52-a)

[0567]

[0568] Compound 2,2,2-trifluoroacetamide (5.0 g, 44.2 mmol, 1.06 eq) was dissolved in dichloromethane (220 mL), dimethyl sulfoxide (10.4 g, 133.4 mmol, 3.2 eq) was added, and the mixture was cooled to -75°C under nitrogen protection. At this temperature, oxalyl chloride (5.25 g, 41.69 mmol, 1.0 eq) and triethylamine (10.6 g, 105.5 mmol, 2.5 eq) were added dropwise, the internal temperature was controlled at about -75°C, and stirring was continued for 0.5 h. DBU (1,8-diazabicycloundec-7-ene) (4.25 g, 27.93 mmol, 0.67 eq) and benzyl alcohol (4.5 g, 41.7 mmol, 1.0 eq) were added, and the mixture was stirred at -75°C for 15 min, then heated to 15°C and stirred for 16 h. Water (100 mL) was added to the reaction system, and the aqueous phase was extracted three times with ethyl acetate (3×50 mL). The organic phase was washed once with water and a saturated sodium chloride solution, respectively, and dried over anhydrous sodium sulfate. The solvent was concentrated and separated by column chromatography (Silica gel, PE: EA = 100: 0-92: 8) to obtain 1.5 g of benzyl 2,2,2-trifluoroacetimidate as a colorless transparent oil with a yield of 16.7%. 1 H NMR (400MHz, DMSO-d6) δ: 10.13 (s, 1H), 7.45-7.39 (m, 5H), 5.29 (s, 2H).

[0569] Step 2: Preparation of 5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidine-7-ol (Intermediate 52-b)

[0570]

[0571] The compound benzyl 2,2,2-trifluoroacetimidate (65.0 mg, 0.322 mmol, 1.0 eq) and ethyl 4-amino-1H-pyrazole-5-carboxylate (50.0 mg, 0.322 mmol, 1.0 eq) were dissolved in pyridine (1.6 mL), heated to 115°C and stirred for 16 h. After cooling to room temperature, pyridine was removed by distillation under reduced pressure, and 31 mg of 5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol was obtained by slurrying with dichloromethane as a white solid with a yield of 46.9%. LCMS (ESI) [M+H] + =205.05; 1 H NMR (400MHz, DMSO-d6) δ: 13.13 (s, 1H), 7.77 (s, 1H).

[0572] Step 3: Preparation of 3-iodo-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidine-7-ol (Intermediate 52-c)

[0573]

[0574] Compound 5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidine-7-ol (2.0 g, 9.8 mmol, 1.0 eq) and compound N-iodosuccinimide (3.3 g, 14.7 mmol, 1.5 eq) were added to N, N-dimethylformamide (48 mL), nitrogen was replaced three times, and the reaction was heated to 60°C for 16 hours. The reaction was completed. The mixture was cooled to room temperature. The solvent was removed by distillation under reduced pressure, and the crude product was slurried with dichloromethane to obtain 1.9 g of a white solid, i.e., 3-iodo-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidine-7-ol, with a yield of 58.6%. LCMS (ESI) [M+H] + =331.02; 1 H NMR (400MHz, DMSO-d6) δ: 14.92 (s, 1H), 13.73 (s, 1H).

[0575] Step 4: Preparation of 3-iodo-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidine-7-ol (Intermediate 52-d)

[0576]

[0577] Compound 3-iodo-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidine-7-ol (50 mg, 0.151 mmol, 1.0 eq) and p-toluenesulfonic acid (3.9 mg, 0.023 mmol, 0.15 eq) were added to dichloromethane (0.8 mL), and DHP (3,4-dihydro-2H-pyran) (19 mg, 0.226 mmol, 1.5 eq) was added. The mixture was stirred at room temperature for 20 min. The reaction was complete after TLC detection. The filter cake was filtered to obtain 3-iodo-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidine-7-ol, 33 mg of white solid. LCMS (ESI) [M+H] + =415.21; 1 H NMR (400MHz, DMSO-d6) δ: 13.97 (s, 1H), 6.15 (dd, J=10.1, 2.3Hz, 1H), 3.96 (d, J=11.1Hz, 1H), 3.70-3.5 8 (m, 1H), 2.30 (ddd, J=23.0, 12.6, 4.0Hz, 1H), 2.06-1.82 (m, 2H), 1.78-1.63 (m, 1H), 1.61-1.44 (m, 2H).

[0578] Step 5: Preparation of 3-(((S)-3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidine-7-ol (Intermediate 52-e)

[0579]

[0580] The compound 3-iodo-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidine-7-ol (30 mg, 0.073 mmol, 1.0 eq) was dissolved in dimethyl sulfoxide (0.36 mL), and the compound (S)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (15.0 mg, 0.073 mmol, 1.0 eq), cuprous iodide (2.7 mg, 0.0145 mmol, 0.2 eq), potassium carbonate (280 mg, 2.03 mmol, 3.0 eq) and L-proline (31.0 mg, 0.27 mmol, 0.4 eq) were added, and the reaction was carried out at 80° C. for 16 hours under N2 protection. After the reaction was completed by TLC, water (15 mL) was added to the reaction system, and the mixture was extracted three times with ethyl acetate (3×20 mL). The organic phases were combined, washed once with a saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated. The mixture was purified by HPLC (DCM: MeOH=20:1) to obtain 66.0 mg of 3-(((S)-3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol as a light yellow solid with a yield of 53.6%. LCMS (ESI) [M+H] + =493.41.

[0581] Step 6: Preparation of (S)-1-(4-((3-(((S)-3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one (Intermediate 52-f)

[0582]

[0583] Compound 3-(((S)-3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidine-7-ol (60.0 mg, 0.122 mmol, 1.0 eq), 1-(4-aminopiperidin-1-yl)ethan-1-one hydrochloride (32.6 mg, 0.183 mmol, 1.5 eq) and PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate) (82.5 mg, 0.158 mmol, 1.3 eq) were dissolved in N,N-dimethylformamide (0.6 mL) and replaced with nitrogen three times. DBU (1,8-diazabicycloundec-7-ene) (27.8 mg, 0.183 mmol, 1.5 eq) was then added, and the mixture was stirred at room temperature for 0.5 h. TLC analysis showed that the reaction of the raw material was complete. Water (10 mL) was added to the reaction system, and the mixture was extracted three times with ethyl acetate (3×20 mL). The organic phases were combined, washed once with a saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated. Purification by HPLC (DCM: MeOH=20:1) gave 40.0 mg of the target intermediate 1-(4-((3-(((S)-3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethane-1-one as a light yellow oil in a yield of 53.3%. LCMS (ESI) [M+H] + =617.43.

[0584] Step 7: Preparation of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one (Compound of Example 52)

[0585]

[0586] To the compound 1-(4-((3-(((S)-3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one (40 mg, 0.06 mmol, 1.0 eq) was added 4 M (molar concentration) hydrochloric acid solution in dioxane (0.2 mL), and the reaction was reacted at room temperature under N2 protection for 1 hour. After the reaction was completed by TLC, a saturated sodium bicarbonate solution was added to the reaction system until pH = 7-8, and the mixture was extracted with ethyl acetate (20 mL). The organic phase was washed once with a saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, and filtered. The solvent was concentrated and separated and purified by preparative HPLC (C18, 0.08% NH4HCO3 aqueous solution / acetonitrile) to obtain 6 mg of the target product (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethane-1-one, with a yield of 17.8%. LCMS (ESI) [M+1] + =533.25; 1 H NMR (400MHz, DMSO-d6) δ: 11.62 (s, 1H), 7.82 (s, 1H), 7.07 (dd, J=28.1, 5.3Hz, 4H), 5.80 (s, 1H), 4.89 (s, 1H), 4.25 (s, 2H), 4.11-3.97 (m, 1H), 3 .82 (d, J=14.0Hz, 1H), 3.76-3.39 (m, 3H), 3.26 (s, 2H), 3.01-2.53 (m, 7H), 2.06 (d, J=10.1Hz, 1H), 2.03 (s, 3H), 1.44 (dd, J=47.5, 10.2Hz, 2H).

[0587] Embodiment 53

[0588] Preparation of (R)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one

[0589]

[0590] According to the method of Example 52, the raw material compound (R)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (i.e., intermediate 3-b, the preparation method of which is shown in Example 3) was used as the starting material, and 3-iodo-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidine-7-ol (i.e., intermediate 52-d, the preparation method of which is shown in Example 5 2) coupling reaction, followed by condensation reaction with 1-(4-aminopiperidin-1-yl)ethane-1-one hydrochloride, and then removal of THP to obtain the target product (R)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethane-1-one. LCMS (ESI) [M+1] + =533.25; 1 H NMR (400MHz, DMSO-d6) δ: 11.62 (s, 1H), 7.82 (s, 1H), 7.07 (dd, J=28.1, 5.3Hz, 4H), 5.80 (s, 1H), 4.89 (s, 1H), 4.25 (s, 2H), 4.11-3.97 (m, 1H), 3 .82 (d, J=14.0Hz, 1H), 3.76-3.39 (m, 3H), 3.26 (s, 2H), 3.01-2.53 (m, 7H), 2.06 (d, J=10.1Hz, 1H), 2.03 (s, 3H), 1.44 (dd, J=47.5, 10.2Hz, 2H).

[0591] Embodiment 54

[0592] Preparation of 1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one

[0593]

[0594] According to the method of Example 52, the raw material compound (1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (i.e., intermediate 1-b, the preparation method is shown in Example 1) is used as the starting material, and 3-iodo-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidine-7-ol (i.e., intermediate 52-d, the preparation method is shown in Example 52) is coupled to react. The target molecule 1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethane-1-one was obtained by condensation reaction with 1-(4-aminopiperidin-1-yl)ethane-1-one hydrochloride and then removal of THP. LCMS(ESI)[M+1] + =533.25; 1 H NMR (400MHz, DMSO-d6) δ: 11.62 (s, 1H), 7.82 (s, 1H), 7.07 (dd, J=28.1, 5.3Hz, 4H), 5.80 (s, 1H), 4.89 (s, 1H), 4.25 (s, 2H), 4.11-3.97 (m, 1H), 3 .82 (d, J=14.0Hz, 1H), 3.76-3.39 (m, 3H), 3.26 (s, 2H), 3.01-2.53 (m, 7H), 2.06 (d, J=10.1Hz, 1H), 2.03 (s, 3H), 1.44 (dd, J=47.5, 10.2Hz, 2H).

[0595] Embodiment 55

[0596] Preparation of (S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-(pyrazin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol:

[0597]

[0598] Step 1: Synthesis of 3-iodo-1H-pyrazolo[4,3-d]pyrimidin-7-ol:

[0599]

[0600] 7-Hydroxypyrazolo[4,3-d]pyrimidine (2.00 g, 14.69 mmol, 1.0 eq) was dissolved in DMF (N,N-dimethylformamide) (40 mL), and NIS (N-iodosuccinimide, CAS: 516-12-1) (4.96 g, 22.04 mmol, 1.5 eq) was added, and the reaction was reacted at 60°C for 5 hours under nitrogen protection. After the reaction was completed by TLC detection, the reaction solvent was dried, and the mixture was dissolved in methanol and filtered. The filter cake was rinsed with methanol and dried to obtain the target compound (3.46 g, yield 89.9%). LCMS (ESI) [M+H] + =262.87; 1 H NMR (400MHz, DMSO-d6) δ14.55 (s, 1H), 12.41 (s, 1H), 7.92 (d, J=3.3Hz, 1H).

[0601] Step 2: Synthesis of 3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol:

[0602]

[0603] 3-iodo-1H-pyrazolo[4,3-d]pyrimidin-7-ol (1.50 g, 5.72 mmol, 1.0 eq) was dissolved in DMF (N,N-dimethylformamide) (28 mL), Cs2CO3 (2.24 g, 6.87 mmol, 1.2 eq) was added, SEMCl (2-(trimethylsilyl)ethoxymethyl chloride) (0.95 g, 5.72 mmol, 1.0 eq) was added dropwise at 0°C, and the reaction was allowed to react at 20°C overnight for 16 hours. After the reaction was completed by TLC, the reaction solution was extracted with dichloromethane and dried over anhydrous sodium sulfate. The organic phase was concentrated, and the crude product was separated and purified by flash chromatography (Silica gel, DCM: MeOH = 15: 1) to obtain the target compound (1.21 g, yield 53.8%). LCMS (ESI) [M+H] + =393.12.

[0604] Step 3: Synthesis of 7-((1H-benzo[d][1,2,3]triazol-1-yl)oxy)-3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidine:

[0605]

[0606] 3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol (2.00 g, 5.10 mmol, 1.0 eq) was dissolved in DMF (N,N-dimethylformamide) (26 mL), and PyBOP (1H-benzotriazol-1-yloxytripyrrolidino hexafluorophosphate) (3.45 g, 6.63 mmol, 1.3 eq) and DBU (1.16 g, 7.65 mmol, 1.5 eq) were added, and the reaction was carried out at 20°C for 0.5 hours. After the reaction was completed by TLC, the reaction solution was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The organic phase was concentrated, and the crude product was separated and purified by flash chromatography (Silicagel, PE:EA=6:1) to obtain the target compound (1.25 g, yield 48.1%). LCMS (ESI) [M+H] + =510.16.

[0607] Step 4: Synthesis of 3-iodo-N-(pyrazine-2-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine:

[0608]

[0609] To a solution of aminopyrazine (142 mg, 1.50 mmol, 1.5 eq) in DMF (N, N-dimethylformamide) (3 mL) was added NaH (120 mg, 2.99 mmol, 3.0 eq) at 0°C, and the mixture was stirred for 10 minutes under nitrogen protection. A solution of 7-((1H-benzo[d][1,2,3]triazol-1-yl)oxy)-3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidine (508 mg, 1.00 mmol, 1.0 eq) in DMF (N, N-dimethylformamide) (4 mL) was then added, and the mixture was reacted at 20°C for 0.5 hours. After the reaction was completed by TLC, the reaction solution was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The organic phase was concentrated, and the crude product was separated and purified by flash chromatography (Silica gel, PE:EA=2:1) ​​to obtain the target compound (450 mg, yield 96.2%). LCMS (ESI) [M+H] + =470.17.

[0610] Step 5: Synthesis of (S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-(pyrazin-2-ylamino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol:

[0611]

[0612] 3-iodo-N-(pyrazin-2-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine (70 mg, 0.15 mmol, 1.0 eq) was dissolved in DMSO (dimethyl sulfoxide) (2 mL), and (S)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (46 mg, 0.22 mmol, 1.5 eq), CuI (57 mg, 0.30 mmol, 2.0 eq), K2CO3 (103 mg, 0.75 mmol, 5.0 eq) and L-Proline (34 mg, 0.30 mmol, 2.0 eq) were added, and the reaction was carried out at 80° C. for 16 hours under nitrogen protection. After the reaction was completed by TLC, the reaction solution was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The organic phase was concentrated, and the crude product was separated and purified by flash chromatography (Silica gel, DCM: MeOH = 15: 1) to obtain the target compound (18 mg, yield 22.0%). LCMS (ESI) [M+H] + =548.43.

[0613] Step 6: Synthesis of (S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-(pyrazin-2-ylamino)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol:

[0614]

[0615] (S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-(pyrazin-2-ylamino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol (29 mg, 0.05 mmol, 1.0 eq) was dissolved in MeOH (methanol) (1 mL), and a solution of hydrochloric acid in 1,4-dioxane (1 mL, 4 M) was added, and the reaction was reacted at 40°C for 4 hours. After the reaction was completed by TLC, the reaction solution was quenched with a saturated aqueous sodium bicarbonate solution, extracted with dichloromethane, and dried over anhydrous sodium sulfate. The organic phase was concentrated, and the crude product was separated and purified by Prep-HPLC (C18, 10 mmol / L NH4HCO3 in water, MeCN) to obtain the target compound (7 mg, yield 31.8%). LCMS (ESI) [M+H] + =418.3; 1H NMR (400MHz, Chloroform-d) δ11.33 (s, 1H), δ8.90 (s, 1H), 8.44 (s, 1H), 8.23 ​​(d, J=16.9Hz, 2H), 7.17-7.05 (m, 3H), 7.02-6.95 (m, 1H), 5.14 (s, 1H), 4 .25(s, 1H), 3.85(d, J=14.8Hz, 1H), 3.68(dd, J=26.2, 14.2Hz, 2H), 3.51(s , 1H), 3.03-2.87 (m, 3H), 2.82-2.71 (m, 2H), 2.65 (dd, J=12.3, 3.7Hz, 1H).

[0616] Embodiment 56

[0617] Preparation of (R)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)thio)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethanone:

[0618]

[0619] Step 1: Synthesis of (R)-S-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)thioacetate:

[0620]

[0621] Dissolve (R)-2-(Oxiran-2-ylmethyl)-1,2,3,4-tetrahydroisoquinoline (50 mg, 0.26 mmol, 1.0 eq) in DCM (dichloromethane) (1.5 mL), add thioacetic acid (thioacetic acid) (30 mg, 0.40 mmol, 1.5 eq) and potassium thioacetate (potassium thioacetate) (45 mg, 0.40 mmol, 1.5 eq), and react at 20°C for 20 hours. After the reaction is completed by TLC detection, the reaction solution is filtered and the filtrate is concentrated to obtain the crude target compound (70 mg, yield 100%). LCMS (ESI) [M+H] + =266.2.

[0622] Step 2: Synthesis of (R)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)thio)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethanone:

[0623]

[0624] 1-(4-((3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethanone (50 mg, 0.10 mmol, 1.0 eq) was dissolved in i-PrOH(isopropanol) (1 mL), and (R)-S-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)thioacetate (31 mg, 0.12 mmol, 1.2 eq), CuI (18 mg, 0.10 mmol, 1.0 eq), K2CO3 (67 mg, 0.48 mmol, 5.0 eq) and ethylene glycol (12 mg, 0.19 mmol, 2.0 eq) were added. The reaction was carried out at 80°C for 17 hours under nitrogen protection and then at 100°C for 23 hours. After the reaction was completed by TLC, the reaction solution was filtered, the filtrate was concentrated, and the crude product was purified by PLC (Silica gel, DCM: MeOH = 10: 1) to obtain the target compound (11 mg, yield 18.5%). LCMS (ESI) [M+H] + =612.5.

[0625] Step 3: Synthesis of (R)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)thio)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethanone:

[0626]

[0627] (R)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)thio)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethanone (11 mg, 0.02 mmol, 1.0 eq) was dissolved in a solution of hydrochloric acid in 1,4-dioxane (0.5 mL, 4 M) and reacted at 20° C. for 2 hours. After the reaction was completed by TLC, the reaction solution was quenched with a saturated aqueous sodium bicarbonate solution, extracted with dichloromethane, and dried over anhydrous sodium sulfate. The organic phase was concentrated, and the crude product was separated and purified by Prep-HPLC (C18, 10 mmol / L NH4HCO3 in water, MeCN) to obtain the target compound (2.5 mg, yield 28.9%). LCMS (ESI) [M+H] + =482.4; 1H NMR (400MHz, Chloroform-d) δ8.39 (s, 1H), 7.18-7.06 (m, 3H), 6.99 (d, J=6.8Hz, 1H), 6.82 (s, 1H) , 4.65 (d, J = 13.5Hz, 1H), 4.51 (s, 1H), 4.14 (s, 1H), 3.89 (d, J = 13.9Hz, 1H), 3.81 (d, J = 15.0Hz, 1H) , 3.71 (d, J=14.9Hz, 1H), 3.39-3.23 (m, 2H), 3.13 (dd, J=14.4, 6.6Hz, 1H), 2.96-2.80 (m, 5H), 2.73 (d, J=6.7Hz, 2H), 2.37 (d, J=12.9Hz, 1H), 2.17 (s, 3H), 2.11 (d, J=13.4Hz, 1H), 1.51-1.33 (m, 2H).

[0628] Embodiment 57

[0629] Preparation of (S)-1-(4-((3-((3-(6-fluoro-3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one:

[0630] Step 1: Preparation of (R)-6-fluoro-2-(oxapropane-2-ylmethyl)-1,2,3,4-tetrahydroisoquinoline:

[0631]

[0632] 6-Fluoro-1,2,3,4-tetrahydroisoquinoline hydrochloride (400 mg, 2.6 mmol, 1.0 eq) and (R)-2-(chloromethyl)oxadiazine (294 mg, 3.2 mmol, 1.2 eq) were dissolved in isopropanol (10 mL), and potassium carbonate (517 mg, 13.2 mmol, 5 eq) was added. The mixture was stirred at 50°C for 16 hours under nitrogen. Water (100 mL) was added to the reaction system, and the aqueous phase was extracted three times with ethyl acetate (3×50 mL). The organic phase was washed once with water and saturated sodium chloride solution, respectively, and dried over anhydrous sodium sulfate. The solvent was concentrated and separated by column chromatography (Silica gel, 0-4% MeOH in DCM) to obtain (R)-6-fluoro-2-(oxadiazine-2-ylmethyl)-1,2,3,4-tetrahydroisoquinoline (350 mg, yield 63.8%). LCMS (ESI) [M+H] + =208.09.

[0633] Step 2 Preparation of (S)-1-amino-3-(6-fluoro-3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol:

[0634]

[0635] (R)-6-Fluoro-2-(oxapropan-2-ylmethyl)-1,2,3,4-tetrahydroisoquinoline (350 mg, 1.69 mmol, 1.0 eq) was dissolved in NH3·H2O (30 mL), stirred at 70°C for 2 hours, and extracted with (CHCl3:i-PrOH=4:1) (3×20 mL). The organic phase was washed once with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was concentrated to give a crude product (S)-1-amino-3-(6-fluoro-3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (400 mg). LCMS (ESI) [M+H]+=225.13.

[0636] Step 3 Preparation of 3-(((S)-3-(6-fluoro-3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidine-7-ol:

[0637]

[0638] The compound 3-iodo-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol (200 mg, 0.48 mmol, 1.0 equiv.) was dissolved in dimethyl sulfoxide (3 mL), and the compound (S)-1-amino-3-(6-fluoro-3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (130 mg, 0.58 mmol, 1.2 equiv.), cuprous iodide (37 mg, 0.19 mol, 0.4 equiv.), L-proline (22 mg, 0.19 mmol, 0.4 equiv.) and potassium carbonate (334 mg, 2.41 mol, 5.0 equiv.) were added, and the reaction was carried out at 80° C. for 16 hours under N2 protection. After the reaction was completed by TLC detection, water (15 mL) was added to the reaction system, and the mixture was extracted three times with ethyl acetate (3×20 mL). The organic phases were combined, washed once with a saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated. The mixture was separated by column chromatography (Silica gel, 0-4% MeOH in DCM) to obtain 3-(((S)-3-(6-fluoro-3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidine-7-ol (200 mg, yield 81.1%). LCMS (ESI) [M+H] + =511.41.

[0639] Step 4 Preparation of 1-(4-((3-(((S)-3-(6-fluoro-3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one:

[0640]

[0641] Compound 3-(((S)-3-(6-fluoro-3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol (150 mg, 0.29 mmol, 1.0 equiv.), 1-(4-aminopiperidin-1-yl)ethan-1-one hydrochloride (63 mg, 0.44 mmol, 1.5 equiv.) and benzotriazol-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate (199 mg, 0.38 mmol, 1.3 eq) were dissolved in N,N-dimethylformamide (1.5 mL), and nitrogen was replaced three times. 1,8-diazabicycloundec-7-ene (134 mg, 0.88 mmol, 3.0 equiv.) was added, and the mixture was stirred at room temperature for 10 minutes. TLC showed that the reaction of the raw materials was complete. Water (10 mL) was added to the reaction system, and the mixture was extracted with ethyl acetate three times (3×20 mL). The organic phases were combined, washed once with a saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, and filtered. The solvent was concentrated and purified by PLC (DCM: MeOH = 15: 1) to obtain 1-(4-((3-(((S)-3-(6-fluoro-3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one (62 mg, yield 33.2%). LCMS(ESI)[M+H] + =635.60.

[0642] Step 5 Preparation of (S)-1-(4-((3-((3-(6-fluoro-3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one:

[0643]

[0644] To 1-(4-((3-(((S)-3-(6-fluoro-3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one was added 2 mL of hydrochloric acid (4 M dioxane solution), and the reaction was reacted at room temperature under N2 protection for 1 hour. After the reaction was completed by TLC, a saturated sodium bicarbonate solution was added to the reaction system until the pH value was 7-8, and the mixture was extracted with ethyl acetate (20 mL). The organic phase was washed once with a saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, and filtered. The solvent was concentrated, and the crude product was separated and purified by Prep-HPLC (C18, 10 mmol / L NH4HCO3 in water, MeCN) to obtain (S)-1-(4-((3-((3-(6-fluoro-3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethane-1-one (8.9 mg, yield 17.1%). LCMS (ESI) [M+H] + =551.44; 1 H NMR (400MHz, CDCl3) δ6.95 (dd, J=8.3, 5.7Hz, 1H), 6.86-6.73 (m, 2H), 4.67 (d, J=12.7Hz, 1H), 4.53 (d, J=6.7Hz, 1H), 4.17 (td, J=9.6, 3.8Hz, 1H), 3.75-3.94 (m, 2H), 3.69-3.60 (m, 2H), 3.5 0-3.40 (m, 1H), 3.36 (t, J=12.2Hz, 1H), 2.98-2.82 (m, 4H), 2.83-2.71 (m, 2H), 2.65 (dd, J=12. 5, 3.9Hz, 1H), 2.44 (d, J = 12.9Hz, 1H), 2.17 (s, 3H), 2.10 (d, J = 10.3Hz, 1H), 1.51-1.22 (m, 2H).

[0645] Embodiment 58

[0646] Preparation of (S)-1-(4-((3-((3-(7-fluoro-3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one:

[0647] Step 1: Preparation of (R)-7-fluoro-2-(oxapropane-2-ylmethyl)-1,2,3,4-tetrahydroisoquinoline:

[0648]

[0649] 7-Fluoro-1,2,3,4-tetrahydroisoquinoline hydrochloride (500 mg, 2.66 mmol, 1.0 eq) and (R)-2-(chloromethyl)oxadiazine (296 mg, 3.20 mmol, 1.2 eq) were dissolved in isopropanol (10 mL), and potassium carbonate (1.84 g, 13.32 mmol, 5.0 eq) was added. The mixture was stirred at 50 ° C for 16 hours under nitrogen protection. The reaction solution was concentrated and separated by column chromatography (Silica gel, 1% MeOH in DCM) to obtain (R)-7-fluoro-2-(oxadiazine-2-ylmethyl)-1,2,3,4-tetrahydroisoquinoline (360 mg, yield 65.2%). LCMS (ESI) [M+H] + =208.09.

[0650] Step 2: Preparation of (S)-1-amino-3-(7-fluoro-3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol:

[0651]

[0652] (R)-7-Fluoro-2-(oxapropan-2-ylmethyl)-1,2,3,4-tetrahydroisoquinoline (360 mg, 1.74 mmol, 1.0 eq) was dissolved in NH4OH (ammonia water) (30 mL) and stirred at 70°C for 2 hours. The mixture was extracted with (chloroform: isopropanol = 4:1) (3×20 mL). The organic phase was washed once with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product (S)-1-amino-3-(7-fluoro-3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (422 mg). LCMS (ESI) [M+H] + =225.14.

[0653] Step 3: Preparation of 3-(((S)-3-(7-fluoro-3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidine-7-ol:

[0654]

[0655] Compound 3-iodo-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol (300 mg, 0.72 mmol, 1.0 equiv) was dissolved in dimethyl sulfoxide (7 mL), and compound (S)-1-amino-3-(7-fluoro-3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (211 mg, 0.94 mmol, 1.3 0.2 equiv.), cuprous iodide (28 mg, 0.14 mol, 0.2 equiv.), L-proline (33 mg, 0.29 mmol, 0.4 equiv.) and potassium carbonate (501 mg, 3.62 mol, 5.0 equiv.) were added, and the reaction was carried out at 80° C. for 15 hours under N2 protection. After the reaction was completed by TLC detection, water (100 mL) was added to the reaction system, and the mixture was extracted three times with ethyl acetate (3×20 mL). The organic phases were combined, washed once with a saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated. The mixture was separated by column chromatography (Silica gel, 3% MeOH in DCM) to obtain 3-(((S)-3-(7-fluoro-3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidine-7-ol (336 mg, yield 90.9%). LCMS (ESI): [M+H] + =511.2.

[0656] Step 4: Preparation of 1-(4-((3-(((S)-3-(7-fluoro-3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one:

[0657]

[0658] Compound 3-(((S)-3-(7-fluoro-3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidine-7-ol (295 mg, 0.58 mmol, 1.0 eq), 1-(4-aminopiperidin-1-yl)ethane-1-one hydrochloride (155 mg, 0.87 mmol, 1.5 eq), benzotriazol-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate (391 mg, 0.75 mmol, 1.3 eq) and 1,8-diazabicycloundec-7-ene (264 mg, 1.73 mmol, 3.0 eq) were dissolved in N,N-dimethylformamide (4.5 mL) and stirred at 20°C for 10 minutes. The reaction of the raw materials was completed when detected by TLC. Water (60 mL) was added to the reaction system, and the mixture was extracted three times with ethyl acetate (3×20 mL). The organic phases were combined, washed once with a saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated. The mixture was purified by PLC (DCM: MeOH=15:1) to obtain 1-(4-((3-(((S)-3-(7-fluoro-3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethane-1-one (100 mg, yield 27.3%). LCMS (ESI) [M+H] + =635.56.

[0659] Step 5: Preparation of (S)-1-(4-((3-((3-(7-fluoro-3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one:

[0660]

[0661] To 1-(4-((3-(((S)-3-(7-fluoro-3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one (95 mg, 0.15 mmol, 1.0 eq) was added 5 mL of a 1,4-dioxane solution (4 M) of hydrochloric acid and the mixture was reacted at 20° C. for 20 minutes. After the reaction was completed by TLC, the reaction solution was quenched with saturated sodium bicarbonate aqueous solution, extracted with dichloromethane (3×20 mL), the organic phase was concentrated, and the crude product was separated and purified by Prep-HPLC (C18, 10 mmol / L NH4HCO3 in water, MeCN) to obtain (S)-1-(4-((3-((3-(7-fluoro-3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethane-1-one (13.7 mg, yield 16.6%). LCMS (ESI) [M+H] + =551.6; 1 H NMR(400MHz, CDCl3)δ7.12(br s, 1H), 7.04 (dd, J=8.5, 5.6Hz, 1H), 6.85 (td, J=8.5, 2.7Hz, 1H), 6.70 (dd, J=9.3, 2.7Hz, 1H), 4.59 (d, J=13.6Hz, 1H), 4.49 (br s, 1H), 4.21 (br s, 1H), 3.93-3.83 (m, 2H), 3.74 (d, J=15.3Hz, 1H), 3.63 (dd, J=13.7, 3.5Hz, 1H), 3.44 (dd, J=13.7, 6.0Hz, 1H), 3.34 (t, J=12.8 Hz, 1H), 3.05-2.85 (m, 5H), 2.83-2.69 (m, 2H), 2.36 (d, J=12.9Hz, 1H), 2.15 (s, 3H), 2.08 (d, J=11.4Hz, 1H), 1.53-1.33 (m, 2H).

[0662] Embodiment 59

[0663] Preparation of (S)-1-(4-((3-((2-hydroxy-3-(1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)propyl)amino)-5-(trifluoromethyl)-1H-pyrido[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one:

[0664] Step 1: Preparation of (R)-2-(oxapropane-2-methylene)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole:

[0665]

[0666] 2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (1 g, 5.81 mmol, 1.0 eq) and (R)-2-(chloromethyl)oxadiazole (617 mg, 6.68 mmol, 1.15 eq) were dissolved in isopropanol (24 mL), and potassium carbonate (4.01 g, 29.03 mmol, 5.0 eq) was added. The mixture was stirred at 50 ° C for 5 hours under nitrogen protection. The reaction solution was concentrated and extracted with DCM (50 mL x 3). The dichloromethane phase was dried over anhydrous sodium sulfate, filtered and concentrated. Column chromatography (Silica gel, 1% MeOH in DCM) was separated to obtain (R)-2-(oxadiazole-2-methylene)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (200 mg, yield 15%). 1 H NMR (400MHz, Chloroform-d) δ7.78 (s, 1H), 7.47 (dq, J=7.5, 0.8Hz, 1H), 7.30 (dt, J=8.2 , 1.0Hz, 1H), 7.19-7.03 (m, 2H), 3.92 (dt, J=14.7, 1.6Hz, 1H), 3.75 (dt, J=14.8, 1.7Hz, 1H), 3.24 (ddt, J=7.0, 4.1, 2.8Hz, 1H), 3.08 (dd, J=13.3, 2.9Hz, 1H), 2.99 (dd, J=6.5, 5 .7Hz, 2H), 2.90-2.78 (m, 3H), 2.55 (dd, J=5.0, 2.7Hz, 1H), 2.51 (dd, J=13.3, 7.1Hz, 1H).

[0667] Step 2: Preparation of (S)-1-amino-3-(1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)propan-2-ol:

[0668]

[0669] (R)-2-(oxapropane-2-methylene)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (150 mg, 0.657 mmol, 1.0 eq) was dissolved in NH3 / MeOH (7M) (2 mL) and stirred at 50°C for 4 hours. The crude product (S)-1-amino-3-(1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)propan-2-ol (160 mg) was obtained by direct spin drying. LCMS (ESI) [M+H] + =246.2

[0670] Step 3: Preparation of 3-(((S)-2-hydroxy-3-(1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)propyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidine-7-ol:

[0671]

[0672] Compound 3-iodo-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol (200 mg, 0.48 mmol, 1.0 equiv) was dissolved in dimethyl sulfoxide (2 mL), and crude compound (S)-1-amino-3-(1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)propan-2-ol (0.2 g, 0.72 mmol, 1.5 equiv.), cuprous iodide (18 mg, 0.096 mol, 0.2 equiv.), L-proline (22 mg, 0.19 mmol, 0.4 equiv.) and potassium carbonate (333 mg, 2.41 mol, 5.0 equiv.) were added, and the reaction was carried out at 80° C. for 15 hours under N2 protection. After the reaction was completed by TLC detection, water (100 mL) was added to the reaction system, and the mixture was extracted three times with ethyl acetate (3×20 mL). The organic phases were combined, washed once with a saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated. The mixture was separated by column chromatography (Silica gel, 3% MeOH in DCM) to obtain 3-(((S)-2-hydroxy-3-(1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)propyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidine-7-ol (160 mg). LCMS (ESI) [M+H] + =532.7.

[0673] Step 4: Preparation of 1-(4-((3-(((S)-2-hydroxy-3-(1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)propyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethane-1-one:

[0674]

[0675] Compound 3-(((S)-2-hydroxy-3-(1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)propyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidine-7-ol (145 mg, 0.272 mmol, 1.0 eq), 1-(4-aminopiperidin-1-yl)ethan-1-one hydrochloride (CAS: 214 147-48-5) (73 mg, 0.409 mmol, 1.5 eq) and benzotriazole-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate (183 mg, 0.353 mmol, 1.3 eq) and 1,8-diazabicycloundec-7-ene (124 mg, 0.818 mmol, 3.0 eq) were dissolved in N,N-dimethylformamide (1.5 mL) and stirred at 20 ° C for 6 minutes. The reaction of the raw materials was completed by TLC. Water (60 mL) was added to the reaction system, and the mixture was extracted three times with ethyl acetate (3×20 mL). The organic phases were combined, washed once with a saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated. PLC (DCM: MeOH = 10: 1) was used for purification to obtain 1-(4-((3-(((S)-2-hydroxy-3-(1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)propyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethane-1-one (100 mg, yield 67%). LCMS (ESI) [M+H] + =656.7.

[0676] Step 5: Preparation of (S)-1-(4-((3-((2-hydroxy-3-(1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)propyl)amino)-5-(trifluoromethyl)-1H-pyrido[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethane-1-one:

[0677]

[0678] To 1-(4-((3-(((S)-2-hydroxy-3-(1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)propyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethan-1-one (90 mg, 0.137 mmol, 1.0 eq) was added 2 mL of a solution of hydrochloric acid in 1,4-dioxane (4 M) and the mixture was reacted at 20°C for 60 minutes. After the reaction was completed by TLC, the reaction solution was quenched with saturated sodium bicarbonate aqueous solution, extracted with dichloromethane (3×20 mL), the organic phase was concentrated, and the crude product was separated and purified by Prep-HPLC (C18, 10 mmol / L NH4HCO3 in water, MeCN) to obtain (S)-1-(4-((3-((2-hydroxy-3-(1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)propyl)amino)-5-(trifluoromethyl)-1H-pyrido[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethane-1-one (13.4 mg, yield 17.0%). LCMS (ESI) [M+H] + =572.6. 1 H NMR (400MHz, DMSO-d6) δ11.53 (br s, 1H), 10.68 (s, 1H), 7.74 (br s, 1H), 7.34 (d, J=7.7Hz, 1H), 7.26 (d, J=7.9Hz, 1H), 7.00 (ddd, J=8.1, 6.4, 1.3Hz, 1H), 6.93 (td, J= 7.5, 1.1Hz, 1H), 5.77 (s, 1H), 4.86 (s, 1H), 4.35-4.14 (m, 2H), 4.10-3.95 (m, 1H), 3.82 (d, J=13.8Hz , 1H), 3.77-3.61 (m, 2H), 3.48 (dt, J=12.1, 5.9Hz, 1H), 3.26-3.23 (m, 2H), 2.91-2.69 (m, 3H), 2.69- 2.50 (m, 3H), 2.60 (dd, J=12.7, 6.4Hz, 1H), 2.16-1.93 (m, 5H), 1.58-1.44 (m, 1H), 1.44-1.31 (m, 1H).

[0679] Embodiment 60

[0680] Preparation of (S)-1-(4-((3-((3-(4,5-dihydro-1H-benzo[d]azepin-3(2H)-yl)-2-hydroxypropyl)amino)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethanone:

[0681] Step 1: Preparation of (R)-3-(2-oxirane-1-ylmethyl)-2,3,4,5-tetrahydro-1H-benzo[d]azepine:

[0682]

[0683] 2,3,4,5-tetrahydro-1H-benzo[d]azepine (500 mg, 3.40 mmol, 1 equiv.) was dissolved in i-PrOH (isopropanol, 17 ml), and K2CO3 (potassium carbonate) (2.35 g, 17.00 mmol, 5.01 equiv.) and (R)-2-(chloromethyl)oxirane (380 mg, 4.11 mmol, 1.21 equiv.) were added. The reaction was allowed to proceed at 50°C overnight (16 h). The reaction was monitored by TLC and LCMS to completion. The reaction solution was cooled to room temperature and filtered. The filtrate was dried by rotary evaporation, and the crude product was separated and purified by flash chromatography (Silica gel, 100% EA) to give the product (R)-3-(2-oxirane-1H-ylmethyl)-2,3,4,5-tetrahydro-1H-benzo[d]azepine (206 mg, yield 29.8%). LCMS(ESI)[M+1] + =204.06; 1 H NMR (400MHz, Chloroform-d) δ7.15-7.07(m, 4H), 3.17-3.13(m, 1H), 3.01-2.90(m, 4H), 2.89-2.77(m, 4H), 2.76-2.68(m, 2H), 2.52-2.43(m, 2H).

[0684] Step 2: Preparation of (S)-1-amino-3-(4,5-dihydro-1H-benzo[d]azepine-3(2H)-yl)propan-2-ol:

[0685]

[0686] Concentrated aqueous ammonia (2.5 ml) was added to a microwave tube containing (R)-3-(oxiran-2-ylmethyl)-2,3,4,5-tetrahydro-1H-benzo[d]azepine (125 mg, 0.615 mmol, 1 equiv.), and the tube was sealed and reacted at 70°C for 5 hours. The reaction was completed by LCMS monitoring. The reaction solution was cooled to room temperature and extracted three times with 30% isopropanol in chloroform. The organic phases were combined, dried, and spin-dried to give crude (S)-1-amino-3-(4,5-dihydro-1H-benzo[d]azepine-3(2H)-yl)propan-2-ol (134 mg, yield 98.9%), which was used directly in the next step. LCMS (ESI) [M+H] + =221.3.

[0687] Step 3: Preparation of 3-(((S)-3-(4,5-dihydro-1H-benzo[d]azepin-3(2H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol:

[0688]

[0689] (S)-1-Amino-3-(4,5-dihydro-1H-benzo[d]azepin-3(2H)-yl)propan-2-ol (105 mg, 0.477 mmol, 1 equiv.), 3-iodo-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol (198 mg, 0.478 mmol, 1 equiv.), CuI (copper iodide) (36 mg, 0.189 mmol, 0.4 equiv.), L-Proline (22 mg, 0.191 mmol, 0.4 equiv.) and K2CO3 (potassium carbonate) (330 mg, 2.388 mmol, 4.99 equiv.) were weighed and added into a dried microwave tube, which was sealed and replaced with nitrogen conditions, and then anhydrous dimethyl sulfoxide (2.4 ml) was added. The reaction was stirred at 80°C overnight (20 h). The reaction solution was filtered and then purified by reverse column (C18, 0.1% NH4OH inwater, MeCN 0-50%) to obtain the product 3-(((S)-3-(4,5-dihydro-1H-benzo[d]azepin-3(2H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol (87 mg, yield 34.1%). LCMS (ESI) [M+1] + =507.5.

[0690] Step 4: Preparation of 1-(4-((3-(((S)-3-(4,5-dihydro-1H-benzo[d]azepin-3(2H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethanone:

[0691]

[0692] The starting materials 3-(((S)-3-(4,5-dihydro-1H-benzo[d]oxazin-3(2H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol (77 mg, 0.152 mmol, 1 equiv.) and PyBOP (1H-benzotriazol-1-yloxytripyrrolidino hexafluorophosphate) were added. (103 mg, 0.198 mmol, 1.3 equiv.) was dissolved in N,N-dimethylformamide (1.5 ml), DBU (1,8-diazabicycloundec-7-ene) (70 mg, 0.460 mmol, 3.0 equiv.) was added, and 1-acetylpiperidin-4-amine hydrochloride (33 mg, 0.185 mmol, 1.2 equiv.) was added after stirring at room temperature for 5 minutes. The reaction was allowed to react at room temperature for 2 hours, and the reaction was completed after monitoring by LCMS. The reaction solution was dried by rotary evaporation, and the crude product was separated and purified by flash chromatography (Silica gel, MeOH: DCM = 0-5%) to obtain the product 1-(4-((3-(((S)-3-(4,5-dihydro-1H-benzo[d]azepin-3(2H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethanone (102 mg, yield 89.4%). LCMS (ESI) [M+1] + =631.7.

[0693] Step 5: Preparation of (S)-1-(4-((3-((3-(4,5-dihydro-1H-benzo[d]azepin-3(2H)-yl)-2-hydroxypropyl)amino)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethanone:

[0694]

[0695] The raw material 1-(4-((3-(((S)-3-(4,5-dihydro-1H-benzo[d]azepin-3(2H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethanone (102 mg, 0.162 mmol, 1 equiv.) was dissolved in dioxane (1 ml), and dioxane hydrochloride (4 M, 0.5 ml, 2 mmol) was added and reacted at room temperature for 5 minutes. The reaction was monitored by TLC to complete. Saturated sodium bicarbonate aqueous solution was added to adjust the pH to 7-8. The solution was dried by vortexing, and the crude product was dissolved in methanol, filtered, and purified by reverse column (C18, 0.1% NH4OH in water, MeCN) and then by prep-HPLC reverse column (C18, 10mmol / L NH4HCO3 in water, MeCN) to obtain the product (S)-1-(4-((3-((3-(4,5-dihydro-1H-benzo[d]azepin-3(2H)-yl)-2-hydroxypropyl)amino)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)piperidin-1-yl)ethanone (13 mg, yield 14.3%). LCMS (ESI) [M+1] + =547.5; 1 H NMR (400MHz, Chloroform-d) δ11.26 (brs, 1H), 7.18-7.06 (m, 4H), 6.98 (brs, 1H), 5.00 (brs, 1H), 4.67 (d, J = 13.6Hz, 1H), 4.60-4.46 (m, 1H), 4.16-4.05 (m, 1H), 3.89 (d, J = 13 .9Hz, 1H), 3.64(d, J=13.5Hz, 1H), 3.48-3.31(m, 2H), 3.05-2.82(m, 7H), 2.79-2.57(m , 4H), 2.43 (d, J=13.0Hz, 1H), 2.17 (s, 3H), 2.11 (d, J=13.0Hz, 1H), 1.49-1.24 (m, 2H).

[0696] Embodiment 61

[0697] Preparation of (S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-((1-(methylsulfonyl)piperidin-4-yl)amino)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol:

[0698] Step 1: Preparation of 3-(((S)-3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidine-7-ol:

[0699]

[0700] Compound 3-iodo-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol (300 mg, 0.725 mmol, 1.0 equiv.) was dissolved in dimethyl sulfoxide (3.6 mL), and compound (S)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (150 mg, 0.725 mmol, 1.0 equiv.), cuprous iodide (27 mg, 0.145 mmol, 0.2 equiv.), potassium carbonate (300 mg, 2.18 mmol, 3.0 equiv.) and L-proline (34 mg, 0.29 mmol, 0.4 equiv.) were added, and the reaction was carried out at 80° C. for 16 hours under N2 protection. After the reaction was completed by TLC detection, water (36 mL) was added to the reaction system, and the mixture was extracted three times with ethyl acetate (3×30 mL). The organic phases were combined, washed once with a saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated. PLC (DCM: MeOH = 20: 1) was used for purification to obtain 3-(((S)-3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidine-7-ol (180 mg, yield 50.4%). LCMS (ESI) [M+1] + =493.46

[0701] Step 2: Preparation of (2S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-((1-(methylsulfonyl)piperidin-4-yl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol:

[0702]

[0703] Compound 3-(((S)-3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidine-7-ol (100 mg, 0.2 mmol, 1.0 equiv.), 1-(methylsulfonyl)piperidin-4-amine (53 mg, 0.3 mmol, 1.5 equiv.) and benzotriazol-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate (155 mg, 0.3 mmol, 1.5 equiv.) were dissolved in N,N-dimethylformamide (1.0 mL), and nitrogen was replaced three times. 1,8-diazabicycloundec-7-ene (91 mg, 0.6 mmol, 3 equiv.) was then added, and the mixture was stirred at room temperature for 0.2 h. TLC detected that the reaction of the raw materials was complete. Water (10 mL) was added to the reaction system, and the mixture was extracted three times with ethyl acetate (3×20 mL). The organic phases were combined, washed once with a saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated. PLC (DCM: MeOH = 20: 1) was used for purification to obtain (2S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-((1-(methylsulfonyl)piperidin-4-yl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol (103 mg, yield 83.7%). LCMS (ESI) [M+1] + =653.53.

[0704] Step 3: Preparation of (S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-((1-(methylsulfonyl)piperidin-4-yl)amino)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol:

[0705]

[0706] To the compound (2S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-((1-(methylsulfonyl)piperidin-4-yl)amino)-1-(tetrahydro-2H-pyran-2-yl)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol (100 mg, 0.153 mmol) was added hydrochloric acid (4M in 1,4-dioxane, 1.0 mL) and the reaction was carried out at room temperature under N2 protection for 0.5 hour. After the reaction was completed by TLC detection, saturated sodium bicarbonate solution was added to the reaction system until pH = 7-8, and extracted with ethyl acetate (20 mL). The organic phase was washed once with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated. The product was separated and purified by Prep-HPLC (C18, 10 mmol / L NH4HCO3 in water, MeCN) to obtain (S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-((1-(methylsulfonyl)piperidin-4-yl)amino)-5-(trifluoromethyl)-1H-pyrazolo[4,3-d]pyrimidin-3-yl)amino)propan-2-ol (22.4 mg, yield 26.1%). LCMS (ESI) [M+H] + =569.4; 1 H NMR (400MHz, DMSO) δ11.55 (s, 1H), 7.80 (s, 1H), 7.09 (m, 3H), 7.03 (m, 1H), 5.79 (brs, 1H), 4.85 (brs, 1H), 4.09 (d, J=49.8Hz, 2H), 3.58 (m, J=12 .1Hz, 4H), 3.47-3.42 (m, 1H), 3.30 (m, 2H), 2.99 (t, J=10.9Hz, 2H), 2.91 (s, 3H), 2.83 (m, 2H), 2.69-2.63 (m, 3H), 2.13 (m, 2H), 1.65-1.59 (m, 2H)

[0707] Embodiment 62

[0708] Preparation of 1-(3,4-dihydroisoquinolin-2(1H)-yl)-4-(4-(3,4-dihydroisoquinolin-2(1H)-yl)-1H-pyrazolo[3,4-d]pyrimidin-1yl)butan-2-ol:

[0709] Step 1: Preparation of 4-chloro-1-(2-(oxiran-2-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidine:

[0710]

[0711] 4-Chloro-1H-pyrazolo[3,4-d]pyrimidine (100 mg, 0.647 mmol, 1.0 equiv.) was dissolved in N,N-dimethylformamide (1 mL), and cesium carbonate (421.6 mg, 1.29 mmol, 2.0 equiv.) was added. The mixture was stirred at 25°C for 4 hours. The reaction was completed by TLC, the solvent was dried by spin drying, and the title compound was separated and purified by Prep-TLC (PE: EA = 8%) to obtain the title compound (15 mg, yield 10.3%). LCMS (ESI) [M+H] + =225.1

[0712] Step 2: Preparation of 1-(3,4-dihydroisoquinolin-2(1H)-yl)-4-(4-(3,4-dihydroisoquinolin-2(1H)-yl)-1H-pyrazolo[3,4-d]pyrimidin-1yl)butan-2-ol:

[0713]

[0714] 4-Chloro-1-(2-(oxiran-2-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidine (10 mg, 0.045 mmol, 1.0 equiv.) was dissolved in N,N-dimethylformamide (0.5 mL), 1,2,3,4-tetrahydroisoquinoline, N-ethyl-N-propan-2-ylpropan-2-amine (8.7 mg, 0.0675 mmol, 1.5 equiv.) were added and stirred at 100°C for 4 hours. After the reaction was completed by LCMS, it was concentrated to obtain a crude product, which was then prepared to obtain the target compound (1.2 mg, yield 7.5%). LCMS (ESI) [M+H] + =456.

[0715] Embodiment 63

[0716] Preparation of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-indazol-7-yl)amino)piperidin-1-yl)ethan-1-one formate:

[0717] The first step: the preparation of (2S)-1-((7-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol:

[0718]

[0719] 7-Bromo-3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (2.0 g, 4.91 mmol, 1 equiv.) was dissolved in dimethyl sulfoxide (25 mL), and (S)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (1.21 g, 5.89 mmol, 1.2 equiv.), cuprous iodide (1.87 g, 0.982 mmol, 0.2 equiv.), potassium carbonate (3.4 g, 24.55 mmol, 5 equiv.) and L-proline (226 mg, 1.96 mmol, 0.4 equiv.) were added, and the reaction was carried out at 80° C. for 16 hours under N2 protection. After the reaction was completed by TLC detection, water (300 mL) was added to the reaction system, extracted three times with ethyl acetate (3×60 mL), the organic phases were combined, washed once with saturated sodium chloride solution (60 mL), dried over anhydrous sodium sulfate, filtered, the solvent was concentrated, and purified by column chromatography (DCM: MeOH = 98: 2) to obtain the title compound (0.54 g, yield 22.6%). LCMS (ESI) [M+H] + =485.1.

[0720] Step 2: Preparation of 1-(4-((3-(((S)-3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-7-yl)amino)piperidin-1-yl)ethan-1-one:

[0721]

[0722] (2S)-1-((7-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (500 mg, 1.03 mmol, 1.0 equiv.) and 1-(4-aminopiperidin-1-yl)ethan-1-one (219 mg, 1.55 mmol, 1.5 equiv.), Pd2(dba)3 (94 mg, 0.103 mmol, 0.1 equiv.), BINAP (64 mg, 0.103 mmol, 0.1 equiv.) and Cs2CO3 (1.68 g, 5.15 mmol, 5 equiv.) were placed in a microwave tube, and toluene (5 mL) was added after nitrogen replacement. The reaction was heated at 110°C for 16 h. The reaction was completed by LCMS monitoring. Cool to room temperature, add water (30 mL) to the reaction system, extract three times with ethyl acetate (3×30 mL), combine the organic phases, wash once with saturated sodium chloride solution (300 mL), dry over anhydrous sodium sulfate, filter, concentrate the solvent, and purify by column chromatography (DCM: MeOH = 98: 2) to obtain the title compound (0.14 g, yield 25.2%). LCMS (ESI) [M+H] + =547.3.

[0723] Step 3: Preparation of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-indazol-7-yl)amino)piperidin-1-yl)ethane-1-one formate:

[0724]

[0725] 1-(4-((3-(((S)-3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-7-yl)amino)piperidin-1-yl)ethane-1-one (120 mg, 0.22 mmol, 1 equiv.) was added to 1,4-dioxane (1 mL), and then 0.25 mL of 4M hydrochloric acid in 1,4-dioxane was added, and the mixture was stirred at 16°C for 1 h. TLC showed that the reaction of the raw material was complete. The solvent was dried by spin drying, and the phase product was separated and purified by Prep-HPLC (C18, 0.1% (V / V) FA in water, MeCN) to obtain the target product (4.54 mg, yield 4.5%). LCMS (ESI) [M+H] + =463.3; 1HNMR (400MHz, DMSO) δ10.93 (s, 1H), 8.16 (s, 1H), 7.12-7.01 (m, 4H), 6.88 (d, J=7.9Hz, 1H), 6.71 (t, J=7.7Hz, 1H), 6.39 (d, J=7.4Hz, 1H), 5.71 (s, 1H), 5.12 (s, 1H), 4.27-4.19 (m, 1H), 4.07-4.00 (m, 1H), 3.83-3.77 (m, 1H), 3.69-3.61 (m, 3H), 3.44-3.40 (m, 1H), 3.24-3.17 (m, 2H), 2.91-2.71 ( m, 6H), 2.68-2.54 (m, 2H), 2.10-2.03 (m, 1H), 2.02 (s, 3H), 2.00-1.90 (m, 1H), 1.39-1.21 (m, 2H).

[0726] Embodiment 64

[0727] Preparation of (S)-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-indazol-7-yl)amino)piperidin-1-yl)(1-methyl-1H-pyrazol-4-yl)methanone formate:

[0728] Step 1: Preparation of tert-butyl 4-((3-(((S)-3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-7-yl)amino)piperidine-1-carboxylate:

[0729]

[0730] (2S)-1-((7-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (2.25 g, 4.64 mmol, 1 equiv.) and tert-butyl-4-aminopiperidine-1-carboxylate (1.39 g, 6.96 mmol, 1.5 equiv.), Pd2(dba)3 (425 mg, 0.46 mmol, 0.1 equiv.), BINAP (289 mg, 0.46 mmol, 0.1 equiv.) and Cs2CO3 (7.56 g, 23.2 mmol, 5 equiv.) were placed in a microwave tube, and toluene (22 mL) was added after nitrogen replacement. The reaction was heated at 110°C for 6 h. The reaction was completed by LCMS monitoring. Cool to room temperature, add water (30 mL) to the reaction system, extract three times with ethyl acetate (3×30 mL), combine the organic phases, wash once with saturated sodium chloride solution (300 mL), dry over anhydrous sodium sulfate, filter, concentrate the solvent, and purify by column chromatography (DCM: MeOH = 98: 2) to obtain the title compound (1.5 g, yield 53.5%). LCMS (ESI) [M+H] + =605.5.

[0731] Step 2 Preparation of (S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-(piperidin-4-ylamino)-1H-indazol-3-yl)amino)propan-2-ol:

[0732]

[0733] Tert-butyl-4-((3-(((S)-3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-7-yl)amino)piperidine-1-carboxylate (0.75 g, 1.24 mmol, 1 equiv.) was added to dichloromethane (16 mL), and trifluoroacetic acid (2 mL) was added, and stirred at 16°C for 1 h. TLC detected that the raw material reaction was complete. The solvent was spin-dried, and the crude product was separated and purified by Prep-HPLC (C18, 0.1% trifluoroacetic acid aqueous solution, acetonitrile) to obtain the target product (0.33 g, yield 63.3%). LCMS (ESI) [M+H] + =421.3.

[0734] Step 3 Preparation of (S)-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-1H-indazol-7-yl)amino)piperidin-1-yl)(1-methyl-1H-pyrazol-4-yl)methanone formate:

[0735]

[0736] (S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-(piperidin-4-ylamino)-1H-indazol-3-yl)amino)propan-2-ol (0.3 g, 0.71 mmol, 1 equiv.), 1-methyl-1H-pyrazole-4-carboxylic acid (108 mg, 0.852 mmol, 1.2 equiv.), HATU (405 mg, 1.06 mmol, 1.5 equiv.) were dissolved in DMF (4 mL), and DIEA (275 mg, 2.13 mmol, 3 equiv.) was added. The mixture was reacted at 16° C. for 1 hour. After the reaction was completed by LCMS, water and ethyl acetate were added, and the crude product was separated and purified by Prep-HPLC (C18, 0.1% formic acid aqueous solution, acetonitrile) to obtain the title compound (46 mg, yield 12.3%). LCMS (ESI) [M+H] + =529.6, 1 H NMR (400MHz, (CD3)2SO) δ10.93 (s, 1H), 8.15 (s, 1H), 8.05 (s, 1H), 7.65 (s, 1H), 7.12-7.01 (m , 4H), 6.88 (d, J=8.0Hz, 1H), 6.71 (t, J=7.7Hz, 1H), 6.40 (d, J=7.4Hz, 1H), 5.70 (s, 1H), 5.17- 5.11(m, 1H), 4.87(s, 1H), 4.19(s, 2H), 4.06-3.99(m, 1H), 3.85(s, 3H), 3.72-3.58(m, 3H), 3. 46-3.38(m, 2H), 3.21-3.12(m, 2H), 2.84-2.60(m, 6H), 2.14-2.00(m, 2H), 1.44-1.29(m, 2H).

[0737] Embodiment 65

[0738] Preparation of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-5-phenyl-1H-indazol-7-yl)amino)piperidin-1-yl)ethan-1-one:

[0739] Step 1: Preparation of 3-iodo-7-nitro-5-phenyl-1H-indazole:

[0740]

[0741] 7-Nitro-5-phenyl-1H-indazole (900 mg, 3.76 mmol, 1.0 equiv.) and potassium hydroxide (633 mg, 11.3 mmol, 3.0 equiv.) were dissolved in DMF (15 mL), and elemental iodine (1.43 g, 5.64 mmol, 1.5 equiv.) was added to the reaction solution and stirred at 25°C for 2 hours. Water (40 mL) was added to quench the reaction, and ethyl acetate (3*15 mL) was used to extract. The combined organic phases were washed with 10% sodium sulfite solution (3*20 mL) and saturated brine (3*20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was separated and purified by flash chromatography (100-200 mesh silica gel, petroleum ether: ethyl acetate = 0-10%) to obtain the title compound (280 mg, yield 20%). LCMS (ESI) [M+H] + =365.75; 1 H NMR (400MHz, CDCl3): δ8.67 (d, J=1.4Hz, 1H), 8.06 (d, J=1.4Hz, 1H), 7.67 (d, J=7.5Hz, 2H), 7.53 (t, J=7.5Hz, 2H), 7.45 (t, J=7.3Hz, 1H).

[0742] Step 2: Preparation of 3-iodo-7-nitro-5-phenyl-1-(((2-(trimethylsilyl)ethoxy)methyl)-1H-indazole:

[0743]

[0744] 3-iodo-7-nitro-5-phenyl-1H-indazole (230 mg, 63 umol, 1.0 equiv.) was dissolved in DMF (5 mL). NaH (38 mg, 94 umol, 1.5 equiv.) was added to the reaction solution at 0°C, and then the temperature was restored to 25°C and stirred for 0.5 hours. SEMCl (157 mg, 94 umol, 1.5 equiv.) was slowly added dropwise to the reaction solution and stirred at 25°C for 1 hour. Water (20 mL) was added to quench the reaction, and ethyl acetate (3*10 mL) was used to extract the mixture. The organic phases were combined and washed with saturated brine (3*15 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was separated and purified by flash chromatography (100-200 mesh silica gel, petroleum ether: ethyl acetate = 0-5%) to obtain the title compound (220 mg, yield 70%). LCMS (ESI) [M+H] + =496.20; 1H NMR (400MHz, CDCl3): δ8.43 (d, J=1.6Hz, 1H), 7.97 (d, J=1.6Hz, 1H), 7.66 (d, J=7.0Hz, 2H), 7.51 (t, J =7.5Hz, 2H), 7.44 (d, J=7.3Hz, 1H), 5.96 (s, 2H), 3.36-3.24 (m, 2H), 0.81-0.71 (m, 2H), -0.12 (s, 9H).

[0745] Step 3: Preparation of 3-iodo-5-phenyl-1-(((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-7-amine:

[0746]

[0747] 3-iodo-7-nitro-5-phenyl-1-(((2-(trimethylsilyl)ethoxy)methyl)-1H-indazole (1.16 g, 2.34 mmol, 1.0 equiv.), reduced iron powder (655 mg, 11.7 mmol, 5.0 equiv.) and ammonium chloride (1.25 g, 23.4 mmol, 10.0 equiv.) were dissolved in ethanol (15 mL) and water (3 mL) and stirred at 80° C. for 2 hours. The reaction solution was filtered through diatomaceous earth, extracted with ethyl acetate (3*15 mL), and the combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was separated and purified by flash chromatography (100-200 mesh silica gel, petroleum ether: ethyl acetate = 0-2%) to obtain the target compound (370 mg, yield 34%). LCMS (ESI) [M+H] + =466.10; 1 H NMR (400MHz, CDCl3): δ7.64 (d, J=7.6Hz, 1H), 7.52 (s, 1H), 7.47 (d, J=7.0Hz, 4H), 7.36 (d, J=6.4Hz, 3H), 6.01 (s, 2H), 5. 83 (s, 1H), 3.72 (d, J=8.7Hz, 1H), 3.63-3.56 (m, 2H), 1.06-1.01 (m, 1H), 0.95-0.89 (m, 2H), -0.01 (s, 4H), -0.05 (s, 6H).

[0748] Step 4: Preparation of 1-(4-((3-iodo-5-phenyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-7-yl)amino)piperidin-1-yl)ethan-1-one:

[0749]

[0750] The raw materials 3-iodo-5-phenyl-1-(((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-7-amine (155 mg, 0.33 mmol, 1.0 equiv.), N-acetyl-4-piperidone (71 mg, 0.5 mmol, 1.5 equiv.) and sodium triacetylborohydride (211 mg, 1.0 mmol, 3.0 equiv.) were dissolved in acetic acid (10 mL), and then titanium tetrachloride solution (0.67 mL, 0.67 mmol, 2.0 equiv.) was added. v, 1M / DCM) was added dropwise to the reaction solution and stirred at 25°C for 16 hours. Water (20 mL) was added to quench the reaction, and ethyl acetate (3*10 mL) was extracted. The combined organic phases were washed with saturated sodium bicarbonate solution (3*20 mL) and saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was separated and purified by flash chromatography (100-200 mesh silica gel, petroleum ether: ethyl acetate = 0-60%) to obtain the title compound (115 mg, yield 59%). LCMS (ESI) [M+1] + =591.20; 1 H NMR (400MHz, CDCl3): δ7.61 (d, J=7.2Hz, 2H), 7.46 (t, J=7.7Hz, 2H), 7.37 (d, J=7.2Hz, 1H), 7.02 (s, 1H), 6.84 (s, 1H), 5.82 (s, 2H ), 3.76 (s, 2H), 3.72-3.67 (m, 2H), 3.30 (s, 2H), 2.13 (s, 4H), 1.76-1.68 (m, 2H), 1.61 (s, 2H), 0.97-0.91 (m, 2H), -0.01 (s, 10H).

[0751] Step 5: Preparation of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-5-phenyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-7-yl)amino)piperidin-1-yl)ethan-1-one:

[0752]

[0753] The reactants 1-(4-((3-iodo-5-phenyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-7-yl)amino)piperidin-1-yl)ethan-1-one (370 mg, 626 umol, 1.0 equiv.), (S)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (258 mg, 1.25 mmol, 2.0 equiv.), cuprous iodide (48 mg, 250 umol, 0.4 equiv.), L-proline (57 mg, 500 umol, 0.8 equiv.) and anhydrous potassium phosphate (530 mg, 2.5 mmol, 4.0 equiv.) were dissolved in DMSO (5 mL). The reaction solution was stirred at 60° C. for 16 hours under nitrogen protection. Add water (20 mL) to quench the reaction, extract with ethyl acetate (3*10 mL), combine the organic phases, wash with saturated brine (3*15 mL), dry over anhydrous sodium sulfate, filter, and concentrate to obtain a crude product. The crude product was separated and purified by flash chromatography (100-200 mesh silica gel, dichloromethane: methanol = 0-5%) to obtain the title compound (42 mg, yield 10%). LCMS (ESI) [M+H] + =669.25.

[0754] Step 6: Preparation of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-5-phenyl-1H-indazol-7-yl)amino)piperidin-1-yl)ethan-1-one:

[0755]

[0756] The reactant (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)-5-phenyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-7-yl)amino)piperidin-1-yl)ethan-1-one (45 mg, 67 umol, 1.0 equiv.) was dissolved in dichloromethane (5 mL). Trifluoroacetic acid (0.2 mL) was added to the reaction solution and stirred at 25°C for 2 hours. The reaction solution was neutralized with aqueous ammonia, then concentrated and purified on a preparative plate (dichloromethane: methanol = 15 / 1) to obtain the target compound (12 mg, yield 3%). LCMS (ESI) [M+H] + =539.20; 1H NMR (400MHz, CD3OD): δ7.55 (d, J=7.8Hz, 2H), 7.37 (t, J=7.7Hz, 2H), 7.26 (d, J=7.4Hz, 1H) , 7.14 (d, J = 4.7Hz, 4H), 7.07 (s, 1H), 6.76 (s, 1H), 4.39 (d, J = 15.2Hz, 1H), 4.28 (s, 1H), 4. 04(s, 1H), 3.91(s, 1H), 3.82(s, 1H), 3.46(t, J=8.3Hz, 2H), 3.34(d, J=10.9Hz, 1H), 3.14( d, J=26.4Hz, 2H), 3.02 (s, 5H), 2.22 (s, 1H), 2.11 (s, 3H), 2.02 (s, 1H), 1.61-1.33 (m, 4H).

[0757] Embodiment 66

[0758] Preparation of (S)-1-(4-(3-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)benzo[d]isoxazol-7-ylamino)piperidin-1-yl)ethanone:

[0759] The first step: Preparation of 3-tert-butoxycarbonylamino-2-fluorobenzonitrile:

[0760]

[0761] 3-Bromo-2-fluorobenzonitrile (25.00 g, 125.00 mmol, 1.0 equiv.), tert-butyl carbamate (17.60 g, 150.00 mmol, 1.2 equiv.), tris(dibenzylideneacetone)dipalladium (2.30 g, 2.50 mmol, 0.02 equiv.), 4,5-bis(diphenylphosphino-9,9-dimethylxanthene) (3.70 g, 7.50 mmol, 0.06 equiv.) and cesium carbonate (49.00 g, 150.00 mmol, 1.2 equiv.) were dissolved in toluene (400 mL) and stirred at 90° C. for 16 hours. LCMS failed to detect molecular weight, and the reaction was complete after spot plate monitoring. The reaction solution was directly filtered, the filter cake was washed with ethyl acetate (100 mL), the filtrate was extracted with water (500 mL), the aqueous phase was washed with ethyl acetate (3*100 mL), the combined organic phases were washed with saturated brine (3*200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was separated and purified by flash chromatography (100-200 mesh silica gel, petroleum ether: ethyl acetate = 0-20%) to obtain the title compound (6.60 g, yield 22.4%). 1H NMR (400MHz, dmso-d6) δ9.38 (s, 1H), 7.94 (s, 1H), 7.59-7.54 (m, 1H), 7.30 (t, J = 8.0Hz, 1H), 1.43 (s, 9H).

[0762] Step 2: Preparation of tert-butyl (3-aminobenzo [d] isoxazol-7-yl) carbamate:

[0763]

[0764] 3-tert-Butyloxycarbonylamino-2-fluorobenzonitrile (6.60 g, 28.00 mmol, 1.0 equiv.) and acetohydroxamic acid (6.30 g, 84.00 mmol, 3.0 equiv.) were dissolved in tert-butanol (100 mL), stirred at 120°C for 2 hours, then returned to 25°C, potassium tert-butoxide (9.40 g, 84.00 mmol, 3.0 equiv.) was slowly added to the reaction solution, and stirred at 25°C for 16 hours. LCMS monitored the formation of the product, water (200 mL) was added to quench the reaction, and ethyl acetate was extracted (3*100 mL), the organic phases were combined, washed with saturated brine (3*150 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was separated and purified by flash chromatography (100-200 mesh silica gel, petroleum ether: ethyl acetate = 0-20%) to obtain the title compound (1.20 g, yield 17.2%). LCMS(ESI)[M+H] + =250.10.

[0765] Step 3: Preparation of (S)-2-(oxirane-2-ylmethyl)-1,2,3,4-tetrahydroisoquinoline:

[0766]

[0767] Tetrahydroisoquinoline (5.00 g, 37.54 mmol, 1.0 equiv.), (R)-(+)-glycidyl m-nitrobenzenesulfonate (11.70 g, 45.05 mmol, 1.2 equiv.) and potassium fluoride (6.50 g, 112.62 mmol, 3.0 equiv.) were dissolved in tetrahydrofuran (100 mL) and stirred at 25°C for 16 hours. The product showed no molecular weight under LCMS monitoring, new spots were generated under spot plate monitoring, the ultraviolet fluorescence was not strong, and the iodine was obvious. The reaction solution was quenched with water (300 mL), extracted with ethyl acetate (3*150 mL), the organic phases were combined, washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was separated and purified by flash chromatography (100-200 mesh silica gel, petroleum ether: ethyl acetate = 0-20%) to obtain the target compound (2.80 g, yield 39.5%). LCMS(ESI)[M+H] + =190.00.

[0768] Step 4: Preparation of (S)-tert-butyl (3-(3,4-dihydroisoquinolin-2(1h)-yl)-2-hydroxypropyl)amino)benzo[d]isoxazol-7-yl)carbamate:

[0769]

[0770] The raw material tert-butyl (3-aminobenzo [d] isoxazol-7-yl) carbamate (900 mg, 3.60 mmol, 1.0 equiv.), (S)-2-(oxiran-2-ylmethyl)-1,2,3,4-tetrahydroisoquinoline (888 mg, 4.70 mmol, 1.3 equiv.) and potassium tert-butoxide (1212 mg, 10.80 mmol, 3.0 equiv.) were dissolved in N,N-dimethylformamide (10 mL) and stirred at 25°C for 4 hours. The product was generated according to LCMS monitoring, and the raw material was not completely reacted. Water (50 mL) was added to quench the reaction, and ethyl acetate was extracted (3*50 mL). The organic phases were combined and washed with saturated sodium bicarbonate solution (3*50 mL) and saturated brine (50 mL), respectively, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was separated and purified by flash chromatography (100-200 mesh silica gel, dichloromethane: methanol = 0-5%) to obtain the title compound (200 mg, yield 12.7%). LCMS (ESI) [M+1] + =439.20.

[0771] Step 5: Preparation of (S)-1-(7-aminobenzo[d]isoxazol-3-yl)amino)-3-(3,4-dihydroisoquinolin-2(1h)-yl)propan-2-ol:

[0772]

[0773] The reactant (s)-tert-butyl (3-(3,4-dihydroisoquinolin-2(1h)-yl)-2-hydroxypropyl)amino)benzo[d]isoxazol-7-yl)carbamate (200 mg, 0.45 mmol, 1.0 equiv.) was dissolved in ethyl acetate hydrochloric acid gas (4M) (5 mL). The reaction solution was stirred at 25°C for 2 hours under nitrogen protection. LCMS monitored the reaction to be complete, and the reaction solution was directly spin-dried to obtain a crude product. The crude product was separated and purified on a preparative plate (dichloromethane: methanol = 10 / 1) to obtain the title compound (100 mg, yield 65.7%). LCMS (ESI) [M+H] + =339.10.

[0774] Step 6: Preparation of (S)-1-(4-(3-(3-(3,4-dihydroisoquinolin-2(1h)-yl)-2-hydroxypropyl)amino)benzo[d]isoxazol-7-ylamino)piperidin-1-yl)ethanone:

[0775]

[0776] The reactant (S)-1-(7-aminobenzo[d]isoxazol-3-yl)amino)-3-(3,4-dihydroisoquinolin-2(1h)-yl)propan-2-ol (100 mg, 0.29 mmol, 1.0 equiv.), N-acetyl-4-piperidone (63 mg, 0.44 mmol, 1.5 equiv.) and sodium triacetoxyborohydride (189 mg, 0.89 mmol, 3.0 equiv.) were dissolved in acetic acid (5 mL), and titanium tetrachloride solution (0.59 mL, 0.59 mmol, 2.0 equiv, 1 M / DCM) was added under ice-cooling. The mixture was stirred at 25° C. for 16 hours. The reaction solution was added with saturated sodium bicarbonate solution (200 mL) to adjust the pH to alkaline, then extracted with ethyl acetate (3*50 mL), the combined organic phases were washed with saturated brine (50 mL), then concentrated, and purified on a preparative plate (dichloromethane: methanol = 10 / 1) to obtain the target compound (49 mg, yield 36.5%). LCMS (ESI) [M+H] + =464.25; 1H NMR (400MHz, cd3od) δ7.08 (s, 3H), 6.99 (s, 2H), 6.87 (d, J = 7.8Hz, 1H), 6.75 (d, J = 7.6Hz, 1H), 4.57 ( s, 1H), 4.43 (d, J=13.3Hz, 1H), 4.21 (dt, J=11.9, 5.9Hz, 1H), 3.91 (d, J=14.0Hz, 1H), 3.76 (t, J=7.3H z, 1H), 3.73-3.71 (m, 2H), 3.44 (dd, J=13.4, 5.3Hz, 1H), 3.35 (dd, J=13.4, 6.7Hz, 1H), 3.27-3.19 (m, 1H), 2.91-2.88(m, 2H), 2.87-2.80(m, 3H), 2.73-2.63(m, 2H), 2.11-2.00(m, 5H), 1.53-1.34(m, 2H).

[0777] Embodiment 67

[0778] Preparation of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)benzo[d]isothiazol-7-yl)amino)piperidin-1-yl)ethanone

[0779] Step 1: Preparation of 2-chloro-3-nitrobenzoyl chloride:

[0780]

[0781] 2-Chloro-3-nitrobenzoic acid (12.00 g, 59.53 mmol, 1.0 equiv.) was dissolved in dichloromethane (200 mL) and N,N-dimethylformamide (1.0 mL) was added. Oxalyl chloride (15.11 g, 119.07 mmol, 2.0 equiv.) was slowly added at 0°C. The reaction was stirred at room temperature for 2 h. The solution became clear. The solvent was spin-dried to obtain the crude target compound (13.00 g, the yield was not counted).

[0782] Step 2: Preparation of 2-chloro-N-(4-methoxybenzyl)-3-nitrobenzamide:

[0783]

[0784] Dissolve 2-chloro-3-nitrobenzoyl chloride (12.00 g, 54.54 mmol, 1.0 equiv.) in dichloromethane (150 mL) and add p-methoxybenzylamine (11.22 g, 81.81 mmol, 1.5 equiv.) at 0°C. Stir at room temperature for 1 h. LCMS detected that the reaction was complete. Add 100 mL of water and extract with dichloromethane three times, 100 mL each time. Combine the organic phases and concentrate to obtain a crude product, which is purified by column chromatography to obtain the title compound (10.60 g, yield 60.6%). LCMS (ESI) [M+1] + =321.0.

[0785] Step 3: Preparation of 2-(4-methoxybenzyl)-7-nitrobenzo[d]isothiazol-3(2H)-one:

[0786]

[0787] 2-Chloro-N-(4-methoxybenzyl)-3-nitrobenzamide (5.00 g, 15.59 mmol, 1.0 equiv.), sulfur powder (600 mg, 18.71 mmol, 1.2 equiv.), cuprous iodide (742 mg, 3.90 mmol, 0.25 equiv.), 1,10-phenanthroline (702 mg, 3.90 mmol, 0.25 equiv.) and potassium carbonate (2.80 g, 20.27 mmol, 1.3 equiv.) were dissolved in N,N-dimethylformamide (60 mL). The reaction was stirred at 110 ° C under nitrogen protection for 3 h. The reaction was monitored by spotting the plate. Concentrate to remove N,N-dimethylformamide. Add 50 mL of water and extract with dichloromethane three times, 100 mL each time. The organic phases were combined and concentrated to obtain a crude product, which was purified by column chromatography to obtain a pure product (4.50 g, yield 91.2%). 1 H NMR (400MHz, CDCl3) δ 8.52-8.50 (m, 1H), 8.42-8.40 (m, 1H), 7.64-7.60 (t, 1H), 7.35-7.33 (m, 2H), 6.91-6.89 (m, 2H), 5.02 (s, 2H), 3.80 (s, 3H).

[0788] Step 4: Preparation of 7-nitrobenzo[d]isothiazol-3-ol:

[0789]

[0790] 2-(4-methoxybenzyl)-7-nitrobenz[d]isothiazol-3(2H)-one (3.50 g, 11.06 mmol, 1.0 equiv.), cerium nitrate (24.26 g, 44.26 mmol, 4.0 equiv.) were dissolved in acetonitrile (80 mL) and water (20 mL). The reaction was stirred at 25 °C for 5 h. TLC showed that the reaction was complete. The solvent was removed by concentration. 100 mL of water was added and extracted with dichloromethane three times, 100 mL each time. The organic phases were combined and concentrated to obtain a crude product, which was slurried with ethyl acetate to obtain a pure product (800 mg, yield 36.9%). 1 H NMR (400MHz, CDCl3) δ11.93 (s, 1H), 8.66-8.63 (m, 1H), 8.36-8.34 (t, 1H), 8.14-8.10 (m, 1H).

[0791] Step 5: (S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-nitrobenzo[d]isothiazol-3-yl)amino)propanol:

[0792]

[0793] 7-Nitrobenzo[d]isothiazol-3-ol (309 mg, 1.58 mmol, 1.0 equiv.) was dissolved in N,N-dimethylformamide (10 mL). 1,8-diazabicycloundec-7-ene (361 mg, 2.37 mmol, 1.5 equiv.) and benzotriazole-1-oxytris(dimethylamino)phosphine hexafluorophosphate (839 mg, 1.90 mmol, 1.2 equiv.) were added, and the nitrogen atmosphere was replaced and the reaction was stirred at 25°C for half an hour. Then (S)-1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (391 mg, 1.90 mmol, 1.2 equiv.) was added to replace the nitrogen atmosphere, and the mixture was stirred at 25°C for 16 hours, and then at 50°C for 2 hours. The product was detected by LCMS. 100 mL of water was added and extracted with ethyl acetate three times, 50 mL each time. The organic phases were combined and concentrated to give a crude product, which was purified using a preparative plate (dichloromethane: methanol = 20 / 1) to give the title compound (170 mg, yield 27.9%). LCMS (ESI) [M+1] + =385.05.

[0794] Step 6: Preparation of (S)-1-((7-aminobenzo[d]isothiazol-3-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propanol:

[0795]

[0796] (S)-1-(3,4-dihydroisoquinolin-2(1H)-yl)-3-((7-nitrobenzo[d]isothiazol-3-yl)amino)propanol (170 mg, 0.44 mmol, 1.0 equiv.), iron powder (124 mg, 2.21 mmol, 5.0 equiv.) and ammonium chloride (237 mg, 4.43 mmol, 10.0 equiv.) were dissolved in ethanol (8 mL) and water (2 mL). The atmosphere was replaced with nitrogen and the reaction was stirred at 80°C for 4 hours. LCMS detected that the reaction was complete. The reaction solution was filtered, 100 mL of water was added and extracted with ethyl acetate three times, 50 mL each time. The organic phases were combined and concentrated to obtain a crude product, which was purified on a preparative plate (dichloromethane: methanol = 10 / 1) to obtain the title compound (70 mg, yield 44.5%). LCMS (ESI) [M+1] + =355.30.

[0797] Step 7: Preparation of (S)-1-(4-((3-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amino)benzo[d]isothiazol-7-yl)amino)piperidin-1-yl)ethanone:

[0798]

[0799] The reactant (S)-1-((7-aminobenzo[d]isothiazol-3-yl)amino)-3-(3,4-dihydroisoquinolin-2(1H)-yl)propanol (70 mg, 0.19 mmol, 1.0 equiv.), N-acetyl-4-piperidone (42 mg, 0.29 mmol, 1.5 equiv.) and sodium triacetoxyborohydride (126 mg, 0.59 mmol, 3.0 equiv.) were dissolved in acetic acid (4 mL), and titanium tetrachloride solution (0.40 mL, 0.40 mmol, 2.0 equiv, 1 M / DCM) was added under ice-cooling. The mixture was stirred at 25° C. for 16 hours. The reaction solution was added with saturated sodium bicarbonate solution (200 mL) to adjust the pH to alkaline, then extracted with ethyl acetate (3*50 mL), the combined organic phases were washed with saturated brine (50 mL), then concentrated, and purified on a preparative plate (dichloromethane: methanol = 10 / 1) to obtain the target compound (20 mg, yield 21.9%). LCMS (ESI) [M+H] + =480.35; 1H NMR (400MHz, cd3od) δ7.14 (d, J=3.7Hz, 2H), 7.08 (d, J=6.3Hz, 3H), 7.01 (s, 1H), 6.75 (s, 1H), 4.61 (s, 1H), 4.46 (d, J=13.9Hz, 1H), 4.26-4.18 (m, 1H), 3.93 (d, J=12.3Hz, 1H), 3.76 -3.66 (m, 3H), 3.57 (qd, J=13.4, 5.8Hz, 2H), 3.24 (d, J=11.4Hz, 1H), 2.94-2.81 (m, 5H), 2 .70 (qd, J=12.9, 6.0Hz, 2H), 2.08 (t, J=15.3Hz, 5H), 1.46 (ddd, J=30.3, 17.9, 6.2Hz, 2H).

[0800] Biological test evaluation

[0801] The present invention is further described and explained below in conjunction with test examples, but these embodiments are not intended to limit the scope of the present invention.

[0802] Test Example 1 Determination of PRMT5 enzyme inhibitory activity of the compounds of the present invention by radioisotope method

[0803] Experimental principle: Under the catalysis of histone methyltransferase PRMT5, the [3H]-CH3 group on the isotope-labeled [3H]-SAM is transferred to the biotinylated histone polypeptide substrate, which can then bind to the FlashPlate. In this way, the isotope is close enough to the FlashPlate to convert the radiation energy of the radioactive isotope [3H] into photons and be detected.

[0804] Experimental method: Prepare 1x enzyme reaction buffer (10mM Tris 8.0 (Sigma, Cat.No.T2694-1L), 0.01% Tween-20 (Sigma, Cat.No.P2287-100ML), 1mM DTT (Sigma, Cat.No.D0632-10G)). Add PRMT5 (Active Motif, Cat.No.31921) to 1x enzyme reaction buffer to prepare 25 / 15x PRMT5 enzyme solution (PRMT5 final concentration: 5nM), transfer 15μL PRMT5 enzyme solution to 384-well microplates (containing different concentrations of compounds (DMSO final concentration 1%)) -well Polypropylene Storage Microplates, Cat. No. 3657) and incubated at room temperature for 15 minutes. The peptide substrate GL-27 (Ac-SGRGKGGKGLGKGGAKRHRKVGG-K (Biotin) (GL Biochem, Cat. No. 342095) and [3H]-SAM (PerkinElmer, Cat. No. NET155V001MC) were added to 1x enzyme reaction buffer to prepare a 25 / 10x substrate solution, and then 10 μL of the peptide substrate solution (final concentration of the peptide substrate was 100 nM, and the final concentration of [3H]-SAM was 0.25 μM) was added. After reacting at room temperature for 120 minutes, 5 μL of 6x ice-cold SAM (Sigma, Cat. No. A7007-100MG) solution was added to terminate the reaction (final concentration of SAM: 0.125 mM). 25 μL of the reaction system was transferred to FlashPlate (Streptavidin FlashPlate HTS PLUS, High Capacity, 384-well, PerkinElmer, Cat.No.SMP410A001PK), incubated at room temperature for 1 hour, washed three times with distilled water containing 0.1% Tween-20, and then read CPM data (Counts Per Minute) on the MicroBeta instrument. After obtaining the CPM raw data of different concentrations of the compound, the data were standardized according to the formula Inh%=(Max-Sample) / (Max-Min)*100% to obtain the enzyme activity inhibition rate Inh% at each concentration point (where Max is the CPM value of the enzyme-positive well, Min is the CPM value of the enzyme-free negative well, and Sample is the CPM value of the compound-treated sample well). Then, the inhibition rate Inh% (Y) corresponding to each concentration (X) was entered in EXCEL, and the XLfit plug-in was used according to the built-in four-parameter fitting formula Y=Bottom+(Top-Bottom) / (1+(IC 50 / X)*HillSlope) to calculate the half inhibitory concentration IC of each compound 50 value.

[0805] Experimental conclusion: Through the above scheme, it is concluded that some of the compounds of the present invention show biological activities as shown in Table 1 in the PRMT5 enzyme activity inhibition test.

[0806] Table 1: IC values ​​of compounds for inhibition of PRMT5 enzyme activity 50 value

[0807]

[0808]

[0809] Note: A stands for IC 50 <100nM.

[0810] The results shown in the above table indicate that this series of compounds has strong PRMT5 enzyme inhibitory activity.

Claims

1. A compound represented by formula (I), its stereoisomers, geometric isomers, tautomers or pharmaceutically acceptable salts: in, Selected from formula (II) A or (II) C : Formula (II) A Selected from: wherein each Rx is independently selected from hydrogen, C optionally substituted by halogen 1-6 Alkyl, halogen or cyano; Formula (II) c Selected from Selected from The H ring is selected from C 6-10 Aryl; X is selected from -N(R 5 )-,R 5 are independently selected from hydrogen or optionally substituted C 1-6 alkyl; G1 is selected from hydrogen, G2 is selected from hydroxyl or amino; G3 is selected from hydrogen; Y is selected from -NH-; and G4 is selected from an optionally substituted 4-6 membered heterocyclic group or an optionally substituted 5-10 membered heteroaryl group, wherein the substituents are selected from wherein D is selected from a bond; f = 0; R3 is selected from C 1-6 Alkyl; and o＝1。 2. Any of the following compounds, their stereoisomers, geometric isomers, tautomers or pharmaceutically acceptable salts:

3. A pharmaceutical composition, characterized in that The composition comprises the compound shown in any one of claims 1 to 2, its stereoisomer, geometric isomer, tautomer or pharmaceutically acceptable salt, and a pharmaceutically acceptable excipient.

4. Use of the compound according to any one of claims 1 to 2, its stereoisomers, geometric isomers, tautomers or pharmaceutically acceptable salts in the preparation of a medicament for treating a disease mediated by PRMT5.

5. The use according to claim 4, wherein the PRMT5-mediated disease is cancer.

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