Arylamine derivatives, their preparation methods and pharmaceutical uses

Abstract

The present disclosure relates to arylamine derivatives, their preparation methods and pharmaceutical uses. Specifically, it relates to compounds represented by general formula (I), their preparation methods, pharmaceutical compositions containing them, and their use as Toll-like receptor (TLR) agonists for the treatment of diseases related to TLR8 activity. The definitions of the various groups in general formula (I) are the same as those defined in the specification.

Description

Technical Field

[0001] The present invention relates to arylamine derivatives, their preparation methods and pharmaceutical uses. Specifically, the present invention relates to compounds represented by general formula (I), their preparation methods, pharmaceutical compositions containing them, and their use as Toll-like receptor (TLR) agonists for the treatment of diseases related to TLR8 activity. Background Art

[0002] Toll-like receptors (TLRs) are a class of pattern recognition receptors that can recognize microorganisms and respond to them. Members of the TLR family play important roles in the immune system. They are not only important components involved in innate immunity but also the bridge connecting innate immunity and specific immunity. This receptor can specifically recognize microorganisms and initiate an immune response.

[0003] TLRs are all type I transmembrane glycoproteins, composed of an extracellular domain rich in 16 - 28 leucine-rich repeats (LRRs), a transmembrane domain, and a cytoplasmic Toll / IL-1 receptor (TIR) domain. X-ray crystallographic analysis determined that the TLR LRR domains are all horseshoe-shaped structures. So far, 11 members have been discovered in humans, among which TLR1, 2, 4, 5, 6, 10, and 11 are located on the cell surface, and TLR3, 7, 8, 9 are located on the endosomal membrane. TLR8 is phylogenetically close to TLR7 and has a high sequence homology, and they are located on adjacent X chromosomes (Xp22). When the LRR of TLR binds to a ligand, the conformation of the TIR domain changes, and then the activation of the TLR signaling pathway is triggered. The TIR domain of TLR can recruit a variety of signaling molecules, including tumor necrosis factor receptor-associated factor 6 and myeloid differentiation factor 88 (MyD88), etc. Among them, TLR8 depends on the MyD88 signaling pathway to induce the activation of protease-1 (AP-1) and nuclear factor κB (NF-κB) to translocate into the nucleus, induce the expression of related genes in the nucleus, secrete chemokines and inflammatory factors, etc., and play a role in transcriptional regulation. In addition, TLR8 can also activate the mitogen-activated protein kinase (MAPK) signaling pathway, including p38, ERK, and JNK, etc., which are mainly involved in the regulation of cell proliferation, cell differentiation, cell transformation, and cell apoptosis, etc., and are closely related to various diseases such as inflammation and tumors (Journal of Immunology, 2017, 33, 813).

[0004] Hepatitis B virus (HBV) is a particulate double-stranded DNA virus. Activation of TLR8 can effectively inhibit HBV replication in vivo and in vitro, thus becoming a target for the development of treatments for chronic hepatitis B virus. Research has found that the TLR8 agonist ssRNA40 can selectively activate innate immune cells around the liver to produce a large amount of IFN-γ, thereby inhibiting HBV replication, and thus has the potential to be used in the treatment of hepatitis virus infections. Stimulating PBMCs with TLR8 agonists has been found to induce the production of high levels of IFN-γ and TNF-α, thereby inhibiting HBV replication (Current Opinion in Virology, 2018, 30, 9).

[0005] TLRs are not only expressed on immune cells but also in various tumor cells, participating in tumor immune surveillance and playing different roles in tumor growth. Among them, after TLR8 is activated, it enhances the activity of natural killer cells (NK cells), increases antibody-dependent cell-mediated cytotoxicity (ADCC), and induces Th1 polarization. TLR8 agonists are potential adjuvants in cancer treatment, aiming to induce specific immune responses against tumor cells and improve the clinical efficacy of approved monoclonal antibody therapies, especially in individuals with reduced ADCC.

[0006] Given the important potential of TLR-8 agonists in treating various diseases, there is an urgent clinical need for novel TLR-8 agonists with strong activity and high selectivity. SUMMARY OF THE INVENTION

[0007] After painstaking research, the inventors designed and synthesized a series of aromatic amine compounds, which showed excellent TLR8 agonist activity and can be developed into drugs for treating TLR8-related diseases.

[0008] Therefore, the object of the present invention is to provide a compound represented by the general formula (I) or its stereoisomers, tautomers, meso forms, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof,

[0009]

[0010] wherein,

[0011] X1 is CR 1 or N;

[0012] X2 is CR 2 or N;

[0013] X3 is CR 3 or N;

[0014] X4 is CR 4 or N;

[0015] L is selected from a bond, -(CH2) v -, -C(O)(CH2) t -, or -(CH2) t C(O)-;

[0016] R 1 is selected from hydrogen, halogen, amino, nitro, cyano, hydroxy, mercapto, carboxy, alkyl, alkoxy, haloalkyl, haloalkoxy;

[0017] R 2 is selected from hydrogen, halogen, cyano, oxo, alkyl, alkenyl, alkynyl, -OR a , -SR a , -NR a R b , cycloalkyl, heterocycloalkyl, aryl and heteroaryl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl are optionally further substituted by one or more Q1 groups;

[0018] R 3 is selected from hydrogen, halogen, cyano, oxo, alkyl, alkenyl, alkynyl, -OR a , -SR a , -NR a R b , cycloalkyl, heterocycloalkyl, aryl and heteroaryl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl are optionally further substituted by one or more Q2 groups;

[0019] R 4 is selected from hydrogen, halogen, amino, nitro, cyano, hydroxy, mercapto, carboxy, alkyl, alkoxy, haloalkyl, haloalkoxy;

[0020] R 5 and R 6 each independently is selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl; wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl are optionally further substituted by a group selected from deuterium, halogen, nitro, cyano, oxo, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -OR a , -SR a , -NR a R b , -C(O)R a , -O(O)CR a , -C(O)OR a , -C(O)NR a R b , -NR a C(O)Rb 、-S(O) n R a 、-S(O) n NR a R b and-NR a S(O) n R b One or more groups are substituted;

[0021] Q1 and Q2 are each independently selected from halogen, nitro, cyano, oxo, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -OR a 、-SR a 、-(CH2) v -NR a R b 、-NR a R b 、-C(O)R a 、-O(O)CR a 、-C(O)OR a 、-C(O)NR a R b 、-NR a C(O)R b 、-S(O) n R a 、-S(O) n NR a R b and-NR a S(O) n R b wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl groups are optionally further selected from halogen, amino, nitro, cyano, carboxyl, ester, oxo, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -OR c 、-SR c 、-(CH2) v -OR c 、-(CH2) v -NR c R d 、-NR c R d 、-C(O)R c 、-O(O)CR c 、-C(O)OR c 、-C(O)NR c R d 、-NR c C(O)R d 、-S(O) n R a, -S(O) n NR c R d and -NR c S(O) n R d is substituted by one or more groups of;

[0022] R a and R b are each independently selected from hydrogen, halogen, hydroxy, nitro, cyano, oxo, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl are optionally further substituted by one or more groups selected from halogen, amino, nitro, cyano, carboxyl, ester, oxo, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -OR c , -SR c , -(CH2) v -OR c , -(CH2) v , -NR c R d , -NR c R d , -C(O)R c , -O(O)CR c , -C(O)OR c , -C(O)NR c R d , -NR c C(O)R d , -S(O) n R a , -S(O) n , -NR c R d and -NR c S(O) n R d by one or more groups of;

[0023] or R a and R b together with the nitrogen atom to which they are attached form a nitrogen - containing heterocycloalkyl, the nitrogen - containing heterocycloalkyl optionally further contains one or more heteroatoms selected from N, O, S in addition to N, and the nitrogen - containing heterocycloalkyl is optionally further substituted by one or more groups selected from halogen, nitro, cyano, oxo, carboxyl, ester, alkyl, haloalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -OR c , -SR c , -(CH2) v , -OR c , -(CH2) v , -NR c R d , -NRc R d 、 -C(O)R c 、 -O(O)CR c 、 -C(O)OR c 、 -C(O)NR c R d 、 -NR c C(O)R d 、 -S(O) n R a 、 -S(O) n NR c R d and -NR c S(O) n R d is substituted with one or more groups of;

[0024] R c and R d are each independently selected from hydrogen, halogen, hydroxy, nitro, cyano, oxo, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic group, aryl and heteroaryl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic group, aryl, heteroaryl are optionally further substituted with one or more groups selected from halogen, amino, nitro, cyano, hydroxy, mercapto, carboxyl, ester group, oxo group, alkyl, alkoxy, cycloalkyl, heterocyclic group, aryl and heteroaryl;

[0025] Or R c and R d together with the nitrogen atom to which they are attached form a nitrogen - containing heterocyclic group, the nitrogen - containing heterocyclic group, in addition to N, optionally further contains one or more heteroatoms selected from N, O, S, and the nitrogen - containing heterocyclic group is optionally further substituted with groups selected from halogen, nitro, cyano, oxo group, hydroxy, mercapto, carboxyl, ester group, alkyl, alkoxy, cycloalkyl, heterocyclic group, aryl and heteroaryl;

[0026] n is 1 or 2;

[0027] v is an integer from 1 to 6;

[0028] t is from 0 to 6.

[0029] In a specific embodiment, the compound of formula (I) according to the present invention or its stereoisomers, tautomers, mesomers, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, is the compound of formula (II) or its stereoisomers, tautomers, mesomers, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof,

[0030]

[0031] wherein, X1, X2, X3, L, R 4 , R 5 , R 6 are as defined in general formula (I).

[0032] In another specific embodiment, the compound represented by general formula (I) according to the present invention, or its stereoisomers, tautomers, mesomers, racemates, enantiomers, diastereoisomers, or a mixture thereof, or a pharmaceutically acceptable salt thereof, is a compound represented by general formula (III), or its stereoisomers, tautomers, mesomers, racemates, enantiomers, diastereoisomers, or a mixture thereof, or a pharmaceutically acceptable salt thereof,

[0033]

[0034] wherein, L, R 1 , R 3 , R 4 , R 5 , R 6 are as defined in general formula (I).

[0035] In another specific embodiment, the compound represented by general formula (I) according to the present invention, or its stereoisomers, tautomers, mesomers, racemates, enantiomers, diastereoisomers, or a mixture thereof, or a pharmaceutically acceptable salt thereof, is a compound represented by general formula (IV), or its stereoisomers, tautomers, mesomers, racemates, enantiomers, diastereoisomers, or a mixture thereof, or a pharmaceutically acceptable salt thereof,

[0036]

[0037] wherein, L, R 1 , R 2 , R 4 , R 5 , R 6 are as defined in general formula (I).

[0038] In another specific embodiment, the compound represented by general formula (I) according to the present invention, or its stereoisomers, tautomers, mesomers, racemates, enantiomers, diastereoisomers, or a mixture thereof, or a pharmaceutically acceptable salt thereof, is a compound represented by general formula (V), or its stereoisomers, tautomers, mesomers, racemates, enantiomers, diastereoisomers, or a mixture thereof, or a pharmaceutically acceptable salt thereof,

[0039]

[0040] wherein, L, R 2 , R4 、R 5 、R 6 as defined in general formula (I).

[0041] In another specific embodiment, a compound represented by general formula (I)-(V) according to the present invention, or a stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereoisomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, wherein:

[0042] L is selected from a bond or -C(O)-; preferably a bond.

[0043] In another specific embodiment, a compound represented by general formula (I)-(V) according to the present invention, or a stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereoisomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, wherein:

[0044] L is selected from -(CH2) v -; v is 1 or 2, preferably 1.

[0045] In another specific embodiment, a compound represented by general formula (I)-(V) according to the present invention, or a stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereoisomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, which is a compound represented by general formula (I-1), (II-1), (III-1), (IV-1), (V-1), or a stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereoisomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof,

[0046]

[0047] wherein, X1, X2, X3, X4, R 1 、R 2 、R 3 、R 4 、R 5 、R 6 as defined in general formula (I).

[0048] In another specific embodiment, a compound represented by general formula (I)-(V) or general formula (I-1), (II-1), (III-1), (IV-1), (V-1) according to the present invention, or a stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereoisomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, wherein,

[0049] R 3 is selected from hydrogen, halogen, C1-C6 alkyl, C6-C10 Aryl and 5- to 10-membered heteroaryl, wherein the C1-C6 alkyl, C6-C 10 The aryl and 5- to 10-membered heteroaryl are optionally further substituted by one or more Q2 groups;

[0050] Q2 is selected from halogen, C1-C6 alkyl, 4- to 6-membered heterocyclic group, C6-C 10 Aryl, 5- to 10-membered heteroaryl, -NR a R b wherein the C6-C 10 The aryl and 5- to 10-membered heteroaryl are optionally further substituted by one or more groups selected from halogen, C1-C6 alkyl, -(CH2) v -NR c R d ;

[0051] R a and R b are each independently selected from hydrogen, C1-C6 alkyl;

[0052] Or R a and R b together with the nitrogen atom to which they are attached form a 4- to 6-membered nitrogen-containing heterocyclic group, which, in addition to N, optionally further contains one or more heteroatoms selected from N, O, S, and the 4- to 6-membered nitrogen-containing heterocyclic group is optionally further substituted by one or more groups selected from oxo group, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, -OR c -SR c -(CH2) v -OR c -NR c R d ;

[0053] R c and R d are each independently selected from hydrogen, C1-C6 alkyl;

[0054] Or R c and R d together with the nitrogen atom to which they are attached form a 4- to 6-membered nitrogen-containing heterocyclic group, which, in addition to N, optionally further contains one or more heteroatoms selected from N, O, S, and the 4- to 6-membered nitrogen-containing heterocyclic group is optionally further substituted by one or more groups selected from halogen, C1-C6 alkyl;

[0055] v is an integer from 1 to 6.

[0056] In another specific embodiment, a compound represented by general formula (I)-(V) or general formula (I-1), (II-1), (III-1), (IV-1), (V-1) according to the present invention, or a stereoisomer, tautomer, meso form, racemate, enantiomer, diastereoisomer thereof, or a mixture form thereof, or a pharmaceutically acceptable salt thereof, wherein, R 3 is selected from hydrogen, halogen, C1-C6 alkyl, preferably hydrogen.

[0057] In another specific embodiment, a compound represented by general formula (I)-(V) or general formula (I-1), (II-1), (III-1), (IV-1), (V-1) according to the present invention, or a stereoisomer, tautomer, meso form, racemate, enantiomer, diastereoisomer thereof, or a mixture form thereof, or a pharmaceutically acceptable salt thereof, wherein,

[0058] R 3 is selected from C6-C 10 aryl and 5- to 10-membered heteroaryl, preferably phenyl or 5-6-membered heteroaryl; wherein the C6-C 10 aryl and 5- to 10-membered heteroaryl are optionally further substituted by one or more Q2 groups;

[0059] Q2 is selected from halogen, C1-C6 alkyl, 4-6-membered heterocyclic group, C6-C 10 aryl, 5- to 10-membered heteroaryl, -NR a R b wherein the C6-C 10 aryl and 5- to 10-membered heteroaryl are optionally further substituted by one or more groups selected from halogen, C1-C6 alkyl;

[0060] R a and R b are each independently selected from hydrogen, C1-C6 alkyl;

[0061] Or R a and R b together with the nitrogen atom to which they are attached form a 4-6-membered nitrogen-containing heterocyclic group, which, in addition to N, optionally further contains one or more heteroatoms selected from N, O, S, and the 4-6-membered nitrogen-containing heterocyclic group is optionally further substituted by one or more groups selected from oxo group, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, -OR c 、-SR c 、-(CH2) v -OR c 、-NR c R d ;

[0062] Rc and R d are each independently selected from hydrogen, C1-C6 alkyl;

[0063] or R c and R d together with the nitrogen atom to which they are attached form a 4-6 membered nitrogen-containing heterocyclic group, which, in addition to N, optionally further contains one or more heteroatoms selected from N, O, S, and the 4-6 membered nitrogen-containing heterocyclic group is optionally further substituted by one or more groups selected from halogen, C1-C6 alkyl. v is an integer from 1 to 6, preferably 1 or 2.

[0064] In another specific embodiment, a compound represented by the general formula (I)-(V) or general formula (I-1), (II-1), (III-1), (IV-1), (V-1) according to the present invention, or a stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture form thereof, or a pharmaceutically acceptable salt thereof, wherein,

[0065] R 3 is selected from 5- to 10-membered heteroaryl, preferably 5- or 6-membered heteroaryl, more preferably pyridyl, pyrimidinyl, imidazolyl, pyrazolyl, pyrrolyl, pyridinylimidazolyl, pyridinylpyrrolyl, pyridinylpyrazolyl, benzimidazolyl, benzopyrazolyl, benzopyrrolyl; the heteroaryl is optionally further substituted by one or more Q2 groups;

[0066] Q2 is selected from halogen, C1-C6 alkyl, C6-C 10 aryl is preferably phenyl, 5- to 10-membered heteroaryl is preferably 5- or 6-membered heteroaryl, wherein the aryl and heteroaryl are optionally further substituted by one or more groups selected from halogen, C1-C6 alkyl.

[0067] In another specific embodiment, a compound represented by the general formula (I)-(V) or general formula (I-1), (II-1), (III-1), (IV-1), (V-1) according to the present invention, or a stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture form thereof, or a pharmaceutically acceptable salt thereof, wherein,

[0068] R 3 is selected from 5- to 10-membered heteroaryl, preferably 5- or 6-membered heteroaryl, more preferably pyridyl, pyrimidinyl, imidazolyl, pyrazolyl, pyrrolyl, pyridinylimidazolyl, pyridinylpyrrolyl, pyridinylpyrazolyl, benzimidazolyl, benzopyrazolyl, benzopyrrolyl; the heteroaryl is optionally further substituted by one or more Q2 groups;

[0069] Q2 is selected from halogen, C1-C6 alkyl, 4-6 membered heterocyclic group or -NRa R b , preferably -NR a R b ;

[0070] R a and R b are each independently selected from hydrogen, C1-C6 alkyl;

[0071] Or R a and R b together with the nitrogen atom to which they are attached form a 4-6 membered nitrogen-containing heterocyclic group, which 4-6 membered nitrogen-containing heterocyclic group optionally further contains one or more heteroatoms selected from N, O, S in addition to N, and the 4-6 membered nitrogen-containing heterocyclic group is optionally further substituted by one or more groups selected from oxo group, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, -OR c , -SR c , -(CH2) v -OR c , -NR c R d ;

[0072] R c and R d are each independently selected from hydrogen, C1-C6 alkyl;

[0073] Or R c and R d together with the nitrogen atom to which they are attached form a 4-6 membered nitrogen-containing heterocyclic group, which 4-6 membered nitrogen-containing heterocyclic group optionally further contains one or more heteroatoms selected from N, O, S in addition to N, and the 4-6 membered nitrogen-containing heterocyclic group is optionally further substituted by one or more groups selected from halogen, C1-C6 alkyl. v is an integer from 1 to 6, preferably 1 or 2.

[0074] In another specific embodiment, a compound represented by the general formula (I)-(V) or general formula (I-1), (II-1), (III-1), (IV-1), (V-1) according to the present invention, or a stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or a mixture form thereof, or a pharmaceutically acceptable salt thereof, wherein,

[0075] R 3 is selected from C6-C 10 aryl, preferably phenyl; the aryl is optionally further substituted by the Q2 group;

[0076] Q2 is selected from 5- or 6-membered heteroaryl, wherein the 5- or 6-membered heteroaryl is optionally further substituted by one or more groups selected from halogen, C1-C6 alkyl.

[0077] In another specific embodiment, a compound represented by the general formula (I)-(V) or general formula (I-1), (II-1), (III-1), (IV-1), (V-1) according to the present invention, or a stereoisomer, tautomer, meso form, racemate, enantiomer, diastereomer thereof, or a mixture form thereof, or a pharmaceutically acceptable salt thereof, wherein,

[0078] R 3 is selected from C1-C6 alkyl, preferably methyl; the C1-C6 alkyl is optionally further substituted by a Q2 group;

[0079] Q2 is selected from C6-C 10 aryl, preferably phenyl, wherein the C6-C 10 aryl is optionally further substituted by -(CH2) v -NR c R d substituted;

[0080] R c and R d together with the nitrogen atom to which they are attached form a 4-6 membered nitrogen-containing heterocyclic group, which, in addition to N, optionally further contains one or more heteroatoms selected from N, O, S, and the 4-6 membered nitrogen-containing heterocyclic group is optionally further substituted by one or more groups selected from halogen, C1-C6 alkyl;

[0081] v is an integer from 1 to 6, preferably 1 or 2.

[0082] In another specific embodiment, a compound represented by the general formula (I)-(V) or general formula (I-1), (II-1), (III-1), (IV-1), (V-1) according to the present invention, or a stereoisomer, tautomer, meso form, racemate, enantiomer, diastereomer thereof, or a mixture form thereof, or a pharmaceutically acceptable salt thereof, wherein,

[0083] R 2 is selected from hydrogen, halogen, C1-C6 alkyl, -NR a R b , the C1-C6 alkyl is optionally further substituted by Q1;

[0084] Q1 is selected from C6-C 10 aryl, 5-10 membered heteroaryl, wherein the C6-C 10 aryl and 5-10 membered heteroaryl are optionally further substituted by one or more groups selected from halogen, C1-C6 alkyl, -(CH2) v -NR c R d ;

[0085] Ra , R b are each independently selected from hydrogen, C1-C6 alkyl;

[0086] R c and R d are each independently selected from hydrogen, C1-C6 alkyl;

[0087] Or R c and R d together with the nitrogen atom to which they are attached form a 4-6 membered nitrogen-containing heterocyclic group, which, in addition to N, optionally further contains one or more heteroatoms selected from N, O, S, and the 4-6 membered nitrogen-containing heterocyclic group is optionally further substituted by one or more groups selected from halogen, C1-C6 alkyl;

[0088] v is an integer from 1 to 6, preferably 1 or 2.

[0089] In another specific embodiment, the compounds represented by the general formula (I)-(V) or general formula (I-1), (II-1), (III-1), (IV-1), (V-1) according to the present invention, or their stereoisomers, tautomers, meso forms, racemates, enantiomers, diastereomers, or their mixture forms, or their pharmaceutically acceptable salts, wherein R 2 is selected from hydrogen, halogen, C1-C6 alkyl, -NR a R b ;

[0090] R a , R b are each independently selected from hydrogen, C1-C6 alkyl.

[0091] In another specific embodiment, the compounds represented by the general formula (I)-(V) or general formula (I-1), (II-1), (III-1), (IV-1), (V-1) according to the present invention, or their stereoisomers, tautomers, meso forms, racemates, enantiomers, diastereomers, or their mixture forms, or their pharmaceutically acceptable salts, wherein,

[0092] R 2 is selected from C1-C6 alkyl, preferably methyl; the C1-C6 alkyl is optionally further substituted by the Q2 group;

[0093] Q2 is selected from C6-C 10 aryl, preferably phenyl, wherein the C6-C 10 aryl is optionally further substituted by -(CH2) v -NR c R d ;

[0094] Rc and R d Together with the nitrogen atom to which they are attached, form a 4-6 membered nitrogen-containing heterocyclic group, which, in addition to N, optionally further contains one or more heteroatoms selected from N, O, S, and the 4-6 membered nitrogen-containing heterocyclic group is optionally further substituted with one or more groups selected from halogen, C1-C6 alkyl;

[0095] v is an integer from 1 to 6, preferably 1 or 2.

[0096] In another specific embodiment, a compound represented by the general formula (I) to (V) or general formula (I-1), (II-1), (III-1), (IV-1), (V-1) according to the present invention, or a stereoisomer, tautomer, meso form, racemate, enantiomer, diastereomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, wherein, R 1 is selected from hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy; preferably, R 1 is hydrogen or halogen.

[0097] In another specific embodiment, a compound represented by the general formula (I) to (V) or general formula (I-1), (II-1), (III-1), (IV-1), (V-1) according to the present invention, or a stereoisomer, tautomer, meso form, racemate, enantiomer, diastereomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, wherein, R 4 is selected from hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy; preferably, R 4 is hydrogen or halogen.

[0098] In another specific embodiment, a compound represented by the general formula (I) to (V) or general formula (I-1), (II-1), (III-1), (IV-1), (V-1) according to the present invention, or a stereoisomer, tautomer, meso form, racemate, enantiomer, diastereomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, wherein,

[0099] R 5 and R 6 are each independently selected from hydrogen and C1-C 12 alkyl, and the C1-C 12 alkyl is optionally further substituted with one or more groups selected from deuterium, -OR a , -SR a , -NR a R b ;

[0100] R a selected from hydrogen, C1-C6 alkyl;

[0101] R b selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl and 5- to 7-membered heterocyclic group;

[0102] Preferably, R 5 is hydrogen, R 6 is C1-C 12 alkyl, and the C1-C 12 alkyl is optionally further substituted with one or more groups selected from deuteration, -OH.

[0103] Typical compounds of the present invention include, but are not limited to:

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117] or in the form of its stereoisomers, tautomers, mesomers, racemates, enantiomers, diastereomers, or a mixture thereof, or a pharmaceutically acceptable salt thereof.

[0118] The present invention further provides a method for preparing a compound represented by the general formula (III-1) according to the present invention or its stereoisomers, tautomers, mesomers, racemates, enantiomers, diastereomers, or a mixture thereof, or a pharmaceutically acceptable salt thereof, which comprises the following steps:

[0119]

[0120] The compound of formula A3 and a boric acid or boronic acid pinacol ester compound are subjected to a metal-catalyzed cross-coupling reaction (such as Suzuki coupling) to obtain a compound of formula A4; then, the protecting group is removed with a suitable acid (such as trifluoroacetic acid) to obtain a compound represented by the general formula (III-1); catalysts such as Pd(PPh3)4, K2CO3, Cs2CO3;

[0121] Or,

[0122]

[0123] The compound of formula A7 and a boric acid or boronic acid pinacol ester compound are subjected to a metal-catalyzed cross-coupling reaction (such as Suzuki coupling) to obtain a compound represented by the general formula (III-1); catalysts such as Pd(PPh3)4, K2CO3, Cs2CO3;

[0124] Wherein: R 1 、R 3 、R 4 、R 5 、R 6 Are as defined in the general formula (III-1).

[0125] The present invention further provides a method for preparing a compound represented by the general formula (IV-1) according to the present invention or its stereoisomers, tautomers, mesomers, racemates, enantiomers, diastereoisomers, or a mixture thereof, or a pharmaceutically acceptable salt thereof, which comprises the following steps:

[0126]

[0127] The compound of formula B3 and a boric acid or boronic acid pinacol ester compound are subjected to a metal-catalyzed cross-coupling reaction (such as Suzuki coupling) to obtain a compound of formula B4; then, the protecting group is removed with a suitable acid (such as trifluoroacetic acid) to obtain a compound represented by the general formula (IV-1); catalysts such as Pd(PPh3)4, K2CO3, Cs2CO3;

[0128] Or,

[0129]

[0130] The compound of formula B7 and a boric acid or boronic acid pinacol ester compound are subjected to a metal-catalyzed cross-coupling reaction (such as Suzuki coupling) to obtain a compound represented by the general formula (IV-1); catalysts such as Pd(PPh3)4, K2CO3, Cs2CO3;

[0131] Wherein: R1 , R 2 , R 4 , R 5 , R 6 As defined by general formula (IV-1).

[0132] The present invention further provides a method for preparing a compound represented by general formula (V-1) according to the present invention, or its stereoisomers, tautomers, mesomers, racemates, enantiomers, diastereoisomers, or a mixture thereof, or a pharmaceutically acceptable salt thereof, which comprises the following steps:

[0133]

[0134] The compound of formula C3 reacts with a boric acid or boronic acid pinacol ester compound through a metal-catalyzed cross-coupling reaction (such as Suzuki coupling) to obtain a compound of formula C4; then, the protecting group is removed with a suitable acid (such as trifluoroacetic acid) to obtain the compound represented by general formula (V-1); catalysts such as Pd(PPh3)4, K2CO3, Cs2CO3;

[0135] Alternatively, the compound represented by general formula (V-1) can be prepared by the following Scheme 6:

[0136]

[0137] The compound of formula C7 reacts with a boric acid or boronic acid pinacol ester compound or R 2 Y compound through a metal-catalyzed cross-coupling reaction (such as Suzuki coupling) to obtain the compound represented by general formula (V-1); catalysts such as Pd(PPh3)4, K2CO3, Cs2CO3;

[0138] Wherein: R 2 , R 4 , R 5 , R 6 As defined by general formula (V-1).

[0139] The present invention further provides a pharmaceutical composition which comprises a general formula compound according to the present invention, or its stereoisomers, tautomers, mesomers, racemates, enantiomers, diastereoisomers, or a mixture thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0140] The present invention further relates to the use of a general formula compound according to the present invention, or its stereoisomers, tautomers, mesomers, racemates, enantiomers, diastereoisomers, or a mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same in the preparation of a TLR8 agonist.

[0141] The present invention further relates to the use of a general formula compound according to the present invention or its stereoisomers, tautomers, mesomers, racemates, enantiomers, diastereoisomers, or a mixture thereof, or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the same in the preparation of a medicament for preventing or treating TLR8-related diseases, which diseases may be viral infectious diseases or malignancies, such as viral infectious diseases including hepatitis B virus infection, HIV virus infection, and malignancies including breast cancer, cervical cancer, colon cancer, lung cancer, gastric cancer, rectal cancer, pancreatic cancer, brain cancer, skin cancer, oral cancer, prostate cancer, bone cancer, kidney cancer, ovarian cancer, bladder cancer, liver cancer, fallopian tube tumor, ovarian tumor, peritoneal tumor, melanoma, solid tumor, glioma, glioblastoma multiforme, hepatocellular carcinoma, papillary renal tumor, head and neck tumor, leukemia, lymphoma, myeloma, and non-small cell lung cancer.

[0142] The present invention further relates to a general formula compound according to the present invention or its stereoisomers, tautomers, mesomers, racemates, enantiomers, diastereoisomers, or a mixture thereof, or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the same, which is used as a TLR8 agonist.

[0143] The present invention further relates to a general formula compound according to the present invention or its stereoisomers, tautomers, mesomers, racemates, enantiomers, diastereoisomers, or a mixture thereof, or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the same, which is used for preventing or treating TLR8-related diseases, which diseases may be viral infectious diseases or malignancies, such as viral infectious diseases including hepatitis B virus infection, HIV virus infection, and malignancies including breast cancer, cervical cancer, colon cancer, lung cancer, gastric cancer, rectal cancer, pancreatic cancer, brain cancer, skin cancer, oral cancer, prostate cancer, bone cancer, kidney cancer, ovarian cancer, bladder cancer, liver cancer, fallopian tube tumor, ovarian tumor, peritoneal tumor, melanoma, solid tumor, glioma, glioblastoma multiforme, hepatocellular carcinoma, papillary renal tumor, head and neck tumor, leukemia, lymphoma, myeloma, and non-small cell lung cancer.

[0144] The present invention further relates to a method for activating TLR8, which comprises administering an effective amount of a general formula compound according to the present invention or its stereoisomers, tautomers, mesomers, racemates, enantiomers, diastereoisomers, or a mixture thereof, or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the same to a patient in need.

[0145] The present invention further relates to a method for preventing or treating TLR8-related diseases, which comprises administering to a patient in need an effective amount of a compound of the general formula according to the present invention or its stereoisomers, tautomers, mesomers, racemates, enantiomers, diastereoisomers, or a mixture thereof, or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same; the diseases may be viral infectious diseases or malignancies, such as viral hepatitis B, HIV virus infection, and malignancies such as breast cancer, cervical cancer, colon cancer, lung cancer, gastric cancer, rectal cancer, pancreatic cancer, brain cancer, skin cancer, oral cancer, prostate cancer, bone cancer, kidney cancer, ovarian cancer, bladder cancer, liver cancer, fallopian tube tumor, ovarian tumor, peritoneal tumor, melanoma, solid tumor, glioma, glioblastoma multiforme, hepatocellular carcinoma, papillary renal tumor, head and neck tumor, leukemia, lymphoma, myeloma, and non-small cell lung cancer.

[0146] According to the conventional methods in the field to which the present invention pertains, the compounds of the present invention can form pharmaceutically acceptable basic addition salts or acid addition salts with bases or acids. The bases include inorganic bases and organic bases, and acceptable organic bases include diethanolamine, ethanolamine, N-methylglucosamine, triethanolamine, tromethamine, etc., and acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, and sodium hydroxide, etc. The acids include inorganic acids and organic acids, acceptable inorganic acids include hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, etc. Acceptable organic acids include acetic acid, trifluoroacetic acid, formic acid, ascorbic acid, etc.

[0147] The pharmaceutical composition containing the active ingredient may be in a form suitable for oral administration, such as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft gelatin capsules, or syrups or elixirs. Oral compositions can be prepared according to any known method for preparing pharmaceutical compositions in the art, and such compositions may contain one or more of the following ingredients: sweetening agents, flavoring agents, coloring agents, and preservatives to provide an attractive and palatable pharmaceutical preparation. Tablets contain the active ingredient and non-toxic pharmaceutically acceptable excipients suitable for mixing to prepare tablets. These excipients may be inert excipients, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating agents and disintegrating agents, such as microcrystalline cellulose, croscarmellose sodium, corn starch, or alginic acid; binders, such as starch, gelatin, polyvinylpyrrolidone, or gum arabic; and lubricants, such as magnesium stearate, stearic acid, or talc. These tablets may be uncoated or may be coated by known techniques to mask the taste of the drug or to delay disintegration and absorption in the gastrointestinal tract, thus providing a sustained release effect over a longer period. For example, water-soluble taste masking substances, such as hydroxypropylmethylcellulose or hydroxypropylcellulose, or extended release substances such as ethylcellulose, cellulose acetate butyrate, may be used.

[0148] Oral formulations can also be provided as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with a water-soluble carrier such as polyethylene glycol or an oil-soluble vehicle such as peanut oil, liquid paraffin or olive oil.

[0149] Aqueous suspensions contain the active substance and excipients suitable for the preparation of an aqueous suspension for mixing. Such excipients are suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone and gum arabic; dispersing or wetting agents which may be naturally occurring phospholipids such as lecithin, or condensation products of ethylene oxide with fatty acids such as polyoxyethylene stearate, or condensation products of ethylene oxide with long chain fatty alcohols such as heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol such as polyoxyethylene sorbitan monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides such as polyoxyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives such as ethylparaben or propylparaben, one or more coloring agents, one or more flavoring agents and one or more sweetening agents such as sucrose, saccharin or aspartame.

[0150] Oil suspensions may be formulated by suspending the active ingredient in a vegetable oil such as peanut oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oil suspensions may contain thickening agents such as beeswax, hard paraffin or cetyl alcohol. Sweetening and flavoring agents as described above may be added to provide a palatable preparation. These compositions may be preserved by the addition of an antioxidant such as butylated hydroxyanisole or α-tocopherol.

[0151] Dispersible powders and granules suitable for the preparation of an aqueous suspension can provide the active ingredient and a dispersing or wetting agent, a suspending agent or one or more preservatives for mixing by the addition of water. Suitable dispersing or wetting agents and suspending agents are as described above. Other excipients such as sweetening agents, flavoring agents and coloring agents may also be added. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

[0152] The pharmaceutical composition of the present invention may also be in the form of an oil-in-water emulsion. The oil phase may be a vegetable oil such as olive oil or peanut oil, or a mineral oil such as liquid paraffin or a mixture thereof. Suitable emulsifiers may be naturally occurring phospholipids such as soy lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of said partial esters and ethylene oxide such as polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening agents, flavoring agents, preservatives and antioxidants. Syrups and elixirs may be formulated with sweetening agents such as glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain demulcents, preservatives, coloring agents and antioxidants.

[0153] The pharmaceutical composition of the present invention may be in the form of a sterile injectable aqueous solution. Acceptable solvents and vehicles that may be used are water, Ringer's solution, and isotonic sodium chloride solution. The sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in the oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. Then the oil solution is added to a mixture of water and glycerol and treated to form a microemulsion. The injection solution or microemulsion may be injected into the bloodstream of a patient by topical bolus injection. Alternatively, the solution and microemulsion are preferably administered in such a way as to maintain a constant circulating concentration of the compound of the present invention. To maintain such a constant concentration, a continuous intravenous delivery device may be used.

[0154] The pharmaceutical composition of the present invention may be in the form of a sterile injectable aqueous or oily suspension for intramuscular and subcutaneous administration. The suspension may be formulated according to known techniques with those suitable dispersing or wetting agents and suspending agents mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension prepared in a non-toxic parenterally acceptable diluent or solvent, such as a solution prepared in 1,3-butanediol. In addition, a sterile fixed oil may conveniently be used as a solvent or suspending medium. For this purpose, any compatible fixed oil including synthetic mono- or di-glycerides may be used. In addition, fatty acids such as oleic acid may also be used to prepare injectables.

[0155] The compounds of the present invention may be administered in the form of suppositories for rectal administration. These pharmaceutical compositions may be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperature but liquid in the rectum and will thus melt in the rectum to release the drug. Such substances include cocoa butter, glycerol gelatin, hydrogenated vegetable oils, polyethylene glycols of various molecular weights and mixtures of fatty acid esters of polyethylene glycol.

[0156] It is well known to those skilled in the art that the dosage of a drug depends on a variety of factors, including but not limited to the following: the activity of the specific compound used, the age of the patient, the weight of the patient, the health status of the patient, the behavior of the patient, the diet of the patient, the time of administration, the mode of administration, the rate of excretion, the combination of drugs, etc. In addition, the optimal treatment method, such as the mode of treatment, the daily dosage of the general formula compound or the type of pharmaceutically acceptable salt, can be verified according to traditional treatment protocols.

[0157] The present invention may contain a compound of the general formula, and its pharmaceutically acceptable salts, hydrates or solvates as active ingredients, and is mixed with a pharmaceutically acceptable carrier or excipient to prepare a composition, and is prepared into a clinically acceptable dosage form. The derivatives of the present invention can be used in combination with other active ingredients as long as they do not produce other adverse effects, such as allergic reactions, etc. The compounds of the present invention can be used as the sole active ingredient or in combination with other drugs for treating diseases related to tyrosine kinase activity. The combination therapy is achieved by administering the respective therapeutic components simultaneously, separately or successively.

[0158] Term Explanation

[0159] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0160] The term "alkyl" refers to a saturated aliphatic hydrocarbon group which is a straight-chain or branched-chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and their various branched isomers, etc. More preferably, it is a lower alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. The alkyl group can be substituted or unsubstituted. When it is substituted, the substituent can be substituted at any available attachment point. The substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl or carboxylate group.

[0161] The term "alkenyl" refers to an alkyl group as defined above consisting of at least two carbon atoms and at least one carbon-carbon double bond, such as vinyl, 1-propenyl, 2-propenyl, 1-, 2- or 3-butenyl, etc. The alkenyl group can be substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

[0162] The term "alkynyl" refers to an alkyl group as defined above consisting of at least two carbon atoms and at least one carbon-carbon triple bond, such as ethynyl, propynyl, butynyl, etc. The alkynyl group can be substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

[0163] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent. The cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, further preferably 3 to 8 carbon atoms, and most preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, etc.; polycyclic cycloalkyl groups include spiro, fused and bridged cycloalkyl groups.

[0164] The term "spiroalkyl" refers to a polycyclic group in which a 5- to 20-membered monocyclic ring shares a carbon atom (called a spiro atom). It can contain one or more double bonds, but none of the rings has a completely conjugated π-electron system. It is preferably 6 to 14 members, more preferably 7 to 10 members. Spiroalkyl groups are classified into monospiroalkyl, bisspiroalkyl or polyspiroalkyl groups according to the number of spiro atoms shared between the rings, preferably monospiroalkyl and bisspiroalkyl groups. More preferably, they are 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 5-membered or 5-membered / 6-membered monospiroalkyl groups. Non-limiting examples of spiroalkyl groups include:

[0165]

[0166] The term "fused cycloalkyl" refers to a fully carbon polycyclic group having 5 to 20 ring atoms, wherein each ring in the system shares an adjacent pair of carbon atoms with other rings in the system, and one or more of the rings may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. Preferably it has 6 to 14 ring atoms, more preferably 7 to 10 ring atoms. Depending on the number of constituent rings, it can be classified into bicyclic, tricyclic, tetracyclic or polycyclic fused cycloalkyl, preferably bicyclic or tricyclic, more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic alkyl. Non-limiting examples of fused cycloalkyl include:

[0167]

[0168] The term "bridged cycloalkyl" refers to a fully carbon polycyclic group having 5 to 20 ring atoms, wherein any two rings share two non-directly connected carbon atoms, and it may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. Preferably it has 6 to 14 ring atoms, more preferably 7 to 10 ring atoms. Depending on the number of constituent rings, it can be classified into bicyclic, tricyclic, tetracyclic or polycyclic bridged cycloalkyl, preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged cycloalkyl include:

[0169]

[0170] The cycloalkyl ring may be fused to an aryl, heteroaryl or heterocycloalkyl ring, wherein the ring connected to the parent structure is cycloalkyl. Non-limiting examples include indanyl, tetrahydronaphthyl, benzocycloheptyl, etc. The cycloalkyl may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl or carboxylate group.

[0171] The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing 3 to 20 ring atoms, wherein one or more of the ring atoms are selected from nitrogen, oxygen or S(O) ma heteroatom (where m is an integer from 0 to 2), but excluding ring moieties of -O-O-, -O-S- or -S-S-, and the remaining ring atoms are carbon. Preferably, it contains 3 to 12 ring atoms, among which 1 to 4 are heteroatoms; most preferably, it contains 3 to 8 ring atoms, among which 1 to 3 are heteroatoms; most preferably, it contains 5 to 7 ring atoms, among which 1 to 2 or 1 to 3 are heteroatoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothienyl, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, pyranyl, etc., preferably 1,2,5-oxadiazolyl, pyranyl or morpholinyl. Polycyclic heterocyclic groups include spiro, fused and bridged heterocyclic groups.

[0172] The term "spiroheterocyclic group" refers to a polycyclic heterocyclic group with 5 to 20 members, where a single atom (called the spiro atom) is shared between monocyclic rings, and one or more of the ring atoms are selected from nitrogen, oxygen or S(O) m a heteroatom (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. It may contain one or more double bonds, but no ring has a completely conjugated π-electron system. Preferably, it has 6 to 14 members, more preferably 7 to 10 members. Spiroheterocyclic groups are classified into monospiroheterocyclic groups, bisspiroheterocyclic groups or multispiroheterocyclic groups according to the number of spiro atoms shared between rings, preferably monospiroheterocyclic groups and bisspiroheterocyclic groups. More preferably, they are 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 5-membered or 5-membered / 6-membered monospiroheterocyclic groups. Non-limiting examples of spiroheterocyclic groups include:

[0173]

[0174] The term "fused heterocyclic group" refers to a polycyclic heterocyclic group with 5 to 20 members, where each ring in the system shares an adjacent pair of atoms with other rings in the system, and one or more of the rings may contain one or more double bonds, but no ring has a completely conjugated π-electron system, and one or more of the ring atoms are selected from nitrogen, oxygen or S(O) m a heteroatom (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. Preferably, it has 6 to 14 members, more preferably 7 to 10 members. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic groups, preferably bicyclic or tricyclic, more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclic groups. Non-limiting examples of fused heterocyclic groups include:

[0175]

[0176] The term "bridged heterocyclic group" refers to a polycyclic heterocyclic group with 5 to 14 members, where any two rings share two non-directly connected atoms, and it may contain one or more double bonds, but no ring has a completely conjugated π-electron system, and one or more of the ring atoms are selected from nitrogen, oxygen or S(O)m (where m is an integer from 0 to 2) heteroatoms, and the remaining ring atoms are carbon. It is preferably 6 to 14 membered, more preferably 7 to 10 membered. According to the number of rings forming the ring, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclic groups, preferably bicyclic, tricyclic or tetracyclic, and more preferably bicyclic or tricyclic. Non-limiting examples of the bridged heterocyclic group include:

[0177]

[0178] The heterocyclic group ring can be fused to an aryl, heteroaryl or cycloalkyl ring, where the ring connected to the parent structure is the heterocyclic group, and non-limiting examples thereof include:

[0179] etc.

[0180] The heterocyclic group can be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl or carboxylate group.

[0181] The term "aryl" refers to a 6 to 14 membered all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent carbon atom pairs) group having a conjugated π electron system, preferably 6 to 10 membered, such as phenyl and naphthyl. More preferably phenyl. The aryl ring can be fused to a heteroaryl, heterocyclic or cycloalkyl ring, where the ring connected to the parent structure is the aryl ring, and non-limiting examples thereof include:

[0182]

[0183] The aryl can be substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate group.

[0184] The term "heteroaryl" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, where the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl is preferably 5 to 10 membered, containing 1 to 3 heteroatoms; more preferably 5 or 6 membered, containing 1 to 2 heteroatoms; preferably, for example, imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, pyrazinyl, etc., preferably imidazolyl, thiazolyl, pyrazolyl or pyrimidinyl, thiazolyl; more preferably pyrazolyl or thiazolyl. The heteroaryl ring can be fused to an aryl, heterocyclic or cycloalkyl ring, where the ring connected to the parent structure is the heteroaryl ring, and non-limiting examples thereof include:

[0185]

[0186] The heteroaryl can be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate group.

[0187] The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), where the alkyl is defined as above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy. The alkoxy can be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate group.

[0188] The term "haloalkyl" refers to an alkyl substituted by one or more halogens, where the alkyl is defined as above.

[0189] The term "haloalkoxy" refers to an alkoxy substituted by one or more halogens, where the alkoxy is defined as above.

[0190] The term "hydroxyalkyl" refers to an alkyl substituted by one or more hydroxyl groups, where the alkyl is defined as above.

[0191] The term "hydroxy" refers to the -OH group.

[0192] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0193] The term "amino" refers to -NH2.

[0194] The term "cyano" refers to -CN.

[0195] The term "nitro" refers to -NO2.

[0196] The term "oxo group" refers to =O.

[0197] The term "carboxyl group" refers to -C(O)OH.

[0198] The term "mercapto group" refers to -SH.

[0199] The term "ester group" refers to -C(O)O(alkyl) or -C(O)O(cycloalkyl), where alkyl and cycloalkyl are as defined above.

[0200] The term "acyl group" refers to a compound containing a -C(O)R group, where R is alkyl, cycloalkyl, heterocyclic group, aryl, or heteroaryl.

[0201] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and this description includes the instances where the event or circumstance occurs or does not occur. For example, "a heterocyclic group optionally substituted by alkyl" means that alkyl may but need not be present, and this description includes the case where the heterocyclic group is substituted by alkyl and the case where the heterocyclic group is not substituted by alkyl.

[0202] "Substituted" means that one or more hydrogen atoms in a group, preferably up to 5, more preferably 1 - 3 hydrogen atoms, are independently replaced by the corresponding number of substituents. It goes without saying that the substituents are only at their possible chemical positions, and those skilled in the art can determine (by experiment or theory) what substitutions are possible or impossible without undue effort. For example, an amino or hydroxyl group with a free hydrogen may be unstable when bonded to a carbon atom with an unsaturated (such as olefinic) bond.

[0203] "Pharmaceutical composition" means a mixture containing one or more compounds described herein or their physiologically / pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient, and thereby exert biological activity.

[0204] "Pharmaceutically acceptable salt" refers to a salt of the compound of the present invention, which has safety and effectiveness when used in mammals and has the appropriate biological activity.

[0205] Synthesis Method of the Compounds of the Present Invention

[0206] To achieve the object of the present invention, the general formula (I) compound of the present invention is prepared by the following synthetic scheme.

[0207] When the compound represented by the general formula (I) is the compound represented by the general formula (III-1), the compound represented by the general formula (III-1) is prepared by the following Scheme 1:

[0208]

[0209] Scheme 1

[0210] At room temperature, in the presence of a suitable base (such as DIEA), the compound of formula A1 reacts with a nucleophilic amine to obtain the compound of formula A2; then, under heating conditions, the compound of formula A2 reacts with 2,4-dimethoxybenzylamine to obtain the compound of formula A3; the compound of formula A3 can react with a boric acid or boronic acid pinacol ester compound through a metal-catalyzed (such as Pd(PPh3)4, K2CO3, Cs2CO3) cross-coupling reaction (such as Suzuki coupling) to obtain the compound of formula A4; the protecting group is removed with a suitable acid (such as trifluoroacetic acid) to obtain the compound represented by the general formula (III-1);

[0211] Wherein: R 1 、R 3 、R 4 、R 5 、R 6 Are as defined in the general formula (III-1).

[0212] Alternatively, the compound represented by the general formula (III-1) can be prepared by the following Scheme 2:

[0213]

[0214] Scheme 2

[0215] The compound of formula A6 reacts with a nucleophilic amine in the presence of a condensing agent (such as BOP) to obtain the coupled product, the compound of formula A7; the compound of formula A7 can react with a boric acid or boronic acid pinacol ester compound through a metal-catalyzed (such as Pd(PPh3)4, K2CO3, Cs2CO3) cross-coupling reaction (such as Suzuki coupling) to obtain the compound represented by the general formula (III-1);

[0216] Wherein: R 1 、R 3 、R 4 、R 5 、R 6 Are as defined in the general formula (III-1).

[0217] When the compound represented by the general formula (I) is the compound represented by the general formula (IV-1), the compound represented by the general formula (IV-1) is prepared by the following Scheme 3:

[0218]

[0219] Scheme 3

[0220] In the presence of a suitable base (e.g., DIEA) at room temperature, the compound of formula B1 reacts with a nucleophilic amine to obtain a compound of formula B2; then, under heating conditions, the compound of formula B2 reacts with 2,4-dimethoxybenzylamine to obtain a compound of formula B3; the compound of formula B3 can react with a boric acid or boronic acid pinacol ester compound through a metal-catalyzed (e.g., Pd(PPh3)4, K2CO3, Cs2CO3) cross-coupling reaction (e.g., Suzuki coupling) to obtain a compound of formula B4; the protecting group is removed with a suitable acid (e.g., trifluoroacetic acid) to obtain the compound represented by the general formula (IV-1);

[0221] wherein: R 1 、R 2 、R 4 、R 5 、R 6 are as defined in the general formula (IV-1).

[0222] Alternatively, the compound represented by the general formula (IV-1) can be prepared by the following Scheme 4:

[0223]

[0224] Scheme 4

[0225] The compound of formula B6 reacts with a nucleophilic amine in the presence of a condensing agent (e.g., BOP) to obtain a coupled product, a compound of formula B7; the compound of formula B7 can react with a boric acid or boronic acid pinacol ester compound through a metal-catalyzed (e.g., Pd(PPh3)4, K2CO3, Cs2CO3) cross-coupling reaction (e.g., Suzuki coupling) to obtain the compound represented by the general formula (IV-1);

[0226] wherein: R 1 、R 2 、R 4 、R 5 、R 6 are as defined in the general formula (IV-1).

[0227] When the compound represented by the general formula (I) is the compound represented by the general formula (V-1), the compound represented by the general formula (V-1) is prepared by the following Scheme 5:

[0228]

[0229] Scheme 5

[0230] In the presence of a suitable base (e.g., DIEA) at room temperature, the compound of formula C1 reacts with a nucleophilic amine to give a compound of formula C2; then, under heating conditions, the compound of formula C2 reacts with 2,4-dimethoxybenzylamine to give a compound of formula C3; the compound of formula C3 can react with a boric acid or boronic acid pinacol ester compound through a metal-catalyzed (e.g., Pd(PPh3)4, K2CO3, Cs2CO3) cross-coupling reaction (e.g., Suzuki coupling) to give a compound of formula C4; deprotection with a suitable acid (e.g., trifluoroacetic acid) gives a compound represented by the general formula (V-1);

[0231] Wherein: R 2 、R 4 、R 5 、R 6 are as defined in the general formula (V-1).

[0232] Alternatively, the compound represented by the general formula (V-1) can be prepared by the following Scheme 6:

[0233]

[0234] Scheme 6

[0235] The compound of formula C6 reacts with a nucleophilic amine in the presence of a condensing agent (e.g., BOP) to give a coupled product, a compound of formula C7; the compound of formula C7 can react with a boric acid or boronic acid pinacol ester compound through a metal-catalyzed (e.g., Pd(PPh3)4, K2CO3, Cs2CO3) cross-coupling reaction (e.g., Suzuki coupling) to give a compound represented by the general formula (V-1);

[0236] Wherein: R 2 、R 4 、R 5 、R 6 are as defined in the general formula (V-1). BRIEF DESCRIPTION OF THE DRAWINGS

[0237] Figure 1 Are the parameters of the compound of Example 52 in cynomolgus monkey serum. DETAILED DESCRIPTION

[0238] The compounds of the present invention and their preparation are further understood through examples, which illustrate some methods of preparing or using the compounds. However, it is to be understood that these examples do not limit the present invention. Variations of the present invention now known or further developed are considered to fall within the scope of the present invention described and claimed herein.

[0239] The compounds of the present invention are prepared using convenient starting materials and general preparation procedures. The present invention provides typical or preferred reaction conditions, such as reaction temperature, time, solvent, pressure, and molar ratio of reactants. However, unless otherwise specified, other reaction conditions can also be adopted. The optimized conditions may vary depending on the specific reactants or solvents used, but generally, the reaction optimization procedures and conditions can be determined.

[0240] In addition, some protecting groups may be used in the present invention to protect certain functional groups from unnecessary reactions. Protecting groups suitable for various functional groups and their protection or deprotection conditions are well-known to those skilled in the art. For example, "Protecting Groups in Organic Synthesis" (3rd Edition, T.W. Greene and G.M. Wuts, Wiley, New York, 1999 and the cited references therein) describes in detail the protection or deprotection of a large number of protecting groups.

[0241] The separation and purification of compounds and intermediates are carried out by appropriate methods and procedures according to specific requirements, such as filtration, extraction, distillation, crystallization, column chromatography, preparative thin-layer chromatography, preparative high-performance liquid chromatography, or a combination of the above methods. The specific usage methods can be referred to the examples described in the present invention. Of course, other similar separation and purification means can also be adopted. They can be characterized using conventional methods (including physical constants and spectroscopic data).

[0242] The structure of the compound is determined by nuclear magnetic resonance (NMR) or / and mass spectrometry (MS). The NMR chemical shift is given in units of 10 -6 (ppm). The NMR measurement is performed using a Bruker dps 300 nuclear magnetic resonance spectrometer, and the solvents for measurement are deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), deuterated methanol (CD3OD), and the internal standard is tetramethylsilane (TMS).

[0243] The MS measurement is performed using an LC (Waters 2695) / MS (Quattro Premier xE) mass spectrometer (manufacturer: Waters) (Photodiode Array Detector).

[0244] Preparative liquid chromatography is performed using an lc6000 high-performance liquid chromatography instrument (manufacturer: Innovent). The chromatographic column is Daisogel C18 10μm 100A (30mm×250mm), and the mobile phase is acetonitrile / water.

[0245] Thin layer chromatography (TLC) used Qingdao Ocean Chemical GF254 silica gel plate. The silica gel plate used in reaction monitoring thin layer chromatography used a specification of 0.20 mm to 0.25 mm, and the silica gel plate used in preparative thin layer chromatography used a specification of 0.5 mm.

[0246] Silica gel column chromatography uses Qingdao marine silica gel 100-200 mesh, 200-300 mesh and 300-400 mesh silica gel as the carrier.

[0247] The known starting materials of the present invention can be synthesized by methods known in the art, or can be purchased from online shopping malls, Beijing Coupling, Sigma, Bailingwei, Yishiming, Shanghai Shuya, Shanghai Inokai, Anaiji Chemical, Shanghai Bid, etc.

[0248] Unless otherwise specified in the examples, all reactions were carried out under a nitrogen atmosphere.

[0249] Argon atmosphere or nitrogen atmosphere means that the reaction bottle is connected to an argon or nitrogen balloon with a capacity of about 1L.

[0250] Reaction solvent, organic solvent or inert solvent are each expressed as the solvent used that does not participate in the reaction under the described reaction conditions, including, for example, benzene, toluene, acetonitrile, tetrahydrofuran (THF), dimethylformamide (DMF), chloroform, dichloromethane, ether, methanol, nitrogen-methylpyrrolidone (NMP), pyridine, etc. Unless otherwise specified in the examples, the solution refers to an aqueous solution.

[0251] The chemical reactions described in the present invention are generally carried out under normal pressure. The reaction temperature is between -78°C and 200°C. The reaction time and conditions are, for example, between -78°C and 200°C at one atmosphere, and are completed within about 1 to 24 hours. If the reaction is left overnight, the reaction time is generally 16 hours. In the embodiments, unless otherwise specified, the reaction temperature is room temperature, which is 20°C to 30°C.

[0252] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equivalent to the described content can be applied to the method of the present invention.

[0253] Example 1: Preparation of (R)-2-((2-aminopyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (1)

[0254]

[0255] Step 1: Preparation of 1-((tert-butyldimethylsilyl)oxy)propan-2-one (1b)

[0256] At room temperature, hydroxyacetone 1a (100 g, 1.35 mol) was dissolved in dichloromethane (DCM) (1 L). The solution was cooled to 0 °C, and imidazole (175 g, 2.57 mol) and tert-butyldimethylchlorosilane (TBDMSCl) (245 g, 1.63 mol) were added successively. The reaction mixture was stirred at 0 °C for 1 h, then slowly warmed to room temperature and stirred for an additional 12 h. After completion of the reaction, the reaction mixture was washed with water (3 × 1 L), and the organic phase was concentrated under reduced pressure to give a pale yellow liquid compound 1b (200 g, 78.7%).

[0257] 1 1H-NMR (CDCl3) δ: 4.15 (s, 2H), 2.17 (s, 3H), 0.93 (s, 9H), 0.09 (s, 6H).

[0258] Step 2: Preparation of (S,E)-N-(1-((tert-butyldimethylsilyl)oxy)prop-2-ylidene)-2-methylpropane-2-sulfonamide (1c)

[0259] At room temperature, compound 1b (200 g, 1.06 mol) and S-tert-butylsulfinamide (129 g, 1.06 mol) were dissolved in tetrahydrofuran (3.6 L). The reaction mixture was added dropwise to titanium(IV) isopropoxide (800 mL, 2.66 mol). The reaction mixture was stirred at 70 °C for 12 h. After completion of the reaction, it was cooled to room temperature. The reaction mixture was concentrated under reduced pressure, and the resulting brown liquid was added to ice water (1 L). The precipitated solid was removed by filtration, and the mixture was extracted with ethyl acetate (3 × 500 mL). The combined organic phases were washed with saturated brine (500 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting brown oily crude product was separated and purified by silica gel column chromatography (mobile phase: ethyl acetate / petroleum ether = 3% - 20%) to give a pale yellow liquid compound 1c (50 g, 16.1%).

[0260] 1 1H NMR (300 MHz, chloroform-d) δ 4.23 (s, 2H), 2.32 (s, 3H), 1.23 (s, 9H), 0.90 (s, 9H), 0.07 (s, 6H).

[0261] LC-MS: m / z 292.2 [M+H] + 。

[0262] Step 3: Preparation of (S)-N-((R)-1-((tert-butyldimethylsilyl)oxy)-2-methylhexan-2-yl)-2-methylpropane-2-sulfonamide (1d)

[0263] At room temperature, under a nitrogen atmosphere, compound 1c (50 g, 0.171 mol) was dissolved in toluene (500 mL). The reaction solution was added dropwise with a heptane solution of trimethylaluminum (207 mL, 1 mol / L, 0.21 mol) at -78 °C. After the addition was complete, stirring was continued for 0.5 h. Then, a hexane solution of n-butylaluminum (102 mL, 2.5 mol / L, 0.26 mol) was added dropwise at -78 °C. After the addition was complete, stirring was carried out at -78 °C for 4 h. After the reaction was completed, the reaction was quenched with water (500 mL), filtered, extracted with ethyl acetate (3 × 200 mL), the combined organic phases were washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained yellow crude product was separated and purified by silica gel column chromatography (mobile phase: ethyl acetate / petroleum ether = 7% - 10%) to obtain yellow liquid compound 1d (26 g, 43.4%).

[0264] 1 H NMR (400 MHz, chloroform-d) δ 3.66 (s, 1H), 3.51 (d, J = 9.4 Hz, 1H), 3.32 (d, J = 9.4 Hz, 1H), 1.72–1.62 (m, 2H), 1.35–1.24 (m, 4H), 1.18 (s, 9H), 1.14 (s, 3H), 0.92–0.87 (m, 12H), 0.05 (s, 3H), 0.05 (s, 3H).

[0265] LC-MS: m / z 350.3 [M + H] + 。

[0266] Step 4: Preparation of (R)-1-((tert-butyldimethylsilyl)oxy)-2-methylhexan-2-amine (1e)

[0267] At room temperature, compound 1d (26 g, 0.074 mol) was dissolved in tetrahydrofuran (250 mL) and water (50 mL). Iodine (3.78 g, 0.015 mol) was added to the reaction solution, and the mixture was stirred overnight at 50 °C. After the reaction was completed, the reaction was diluted with water (200 mL), the reaction solution was concentrated under reduced pressure to remove tetrahydrofuran, extracted with ethyl acetate (4 × 200 mL), the combined organic phases were washed successively with a sodium thiosulfate solution (400 mL) and saturated brine (400 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain brown oily liquid compound 1e (15 g, 82.1%), which was used directly in the next step without purification.

[0268] 11H NMR (400 MHz, chloroform-d) δ 3.30 (q, J = 9.4 Hz, 2H), 1.54 (s, 2H), 1.40–1.20 (m, 6H), 0.98 (s, 3H), 0.93–0.87 (m, 12H), 0.04 (s, 6H).

[0269] LC-MS: m / z 246.2 [M+H] + 。

[0270] Step 5: Preparation of 2,4-dichloropyrido[4,3-d]pyrimidine (1g)

[0271] At room temperature, pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione 1f (50 mg, 0.31 mmol) was dissolved in phosphorus oxychloride (1.5 ml), and then N,N-diisopropylethylamine (DIEA) (356 mg, 2.75 mmol) was slowly added. The reaction mixture was heated to 100 °C and stirred for 4 h. After completion of the reaction, the reaction mixture was cooled to room temperature, diluted with ice water (30 mL), and rapidly extracted with ethyl acetate (3 × 8 mL). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and the ethyl acetate solution of the obtained compound 1g was directly used for the next reaction.

[0272] LC-MS: m / z 200.0 [M+H] + 。

[0273] Step 6: Preparation of (R)-N-((1-((tert-butyldimethylsilyl)oxy)-2-methylhexan-2-yl)-2-chloropyrido[4,3-d]pyrimidin-4-amine (1h)

[0274] At room temperature, (R)-1-((tert-butyldimethylsilyl)oxy)-2-methylhexan-2-amine 1e (90.4 mg, 0.37 mmol) and N,N-diisopropylethylamine (198 mg, 1.53 mmol) were added to the ethyl acetate solution of 1g above. The reaction mixture was stirred at room temperature for 4 h. After completion of the reaction, the reaction mixture was diluted with water (30 mL), and then extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1). The obtained product was concentrated under reduced pressure to give a yellow oily compound 1h (16 mg, 12.7%).

[0275] LC-MS: m / z 409.2 [M+H] + 。

[0276] Step 7: (R)-N 4-(1-((tert-Butyldimethylsilyl)oxy)-2-methylhexan-2-yl)-N 2 Preparation of (2,4-dimethoxybenzyl)pyrido[4,3-d]pyrimidine-2,4-diamine (1i)

[0277] At room temperature, compound 1h (16 mg, 0.039 mmol) and 2,4-dimethoxybenzylamine (DMB-NH2) (45.7 mg, 0.274 mmol) were dissolved in dioxane (1 mL), and then N,N-diisopropylethylamine (15 mg, 0.117 mmol) was added to the system. The reaction mixture was stirred at 100 °C for 4 hours. After the reaction was completed, the reaction was cooled to room temperature, diluted with water (30 mL), and extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1). The obtained product was concentrated under reduced pressure to give compound 1i as a yellow oil (14 mg, 66.7%).

[0278] LC-MS: m / z 540.3 [M+H] + 。

[0279] Step 8: Preparation of (R)-2-((2-aminopyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (1)

[0280] At room temperature, compound 1i (14 mg, 0.027 mmol) was dissolved in trifluoroacetic acid (TFA) (1 mL) and stirred at 40 °C overnight. After the reaction was complete, the reaction mixture was concentrated under reduced pressure, diluted with saturated sodium bicarbonate solution (20 mL), and extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was separated and purified by a preparative chromatography column (column type: XBridge Shield RP18 OBD column, 5um, 19*150mm; mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 18%-35% acetonitrile in 8 minutes; detection wavelength: 220 nm) to give compound 1 as a white solid (3.4 mg, 44%).

[0281] 11H NMR (400 MHz, methanol-d4) δ 9.04 (s, 1H), 8.24 (d, J = 6.0 Hz, 1H), 7.03 (d, J = 5.9 Hz, 1H), 3.97 (d, J = 11.2 Hz, 1H), 3.62 (d, J = 11.2 Hz, 1H), 2.24–2.07 (m, 1H), 1.74–1.58 (m, 1H), 1.37 (s, 3H), 1.32–1.16 (m, 4H), 0.80 (t, J = 7.0 Hz, 3H).

[0282] LC-MS: m / z 276.2 [M+H] + 。

[0283] Example 2: Preparation of (R)-2-((2-amino-7-(2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (2)

[0284]

[0285] Step 1: Preparation of 4-amino-6-chloronicotinamide (2b)

[0286] At room temperature, 4-amino-6-chloronicotinic acid 2a (1.00 g, 5.795 mmol) was dissolved in 1,4-dioxane (15 mL), and thionyl chloride (8 mL) was slowly added dropwise. The reaction solution was stirred at 90 °C for 2 hours. The reaction solution was concentrated under reduced pressure, and the residue was diluted with tetrahydrofuran (THF) (5 mL). At room temperature, ammonia water (10 mL) was slowly added dropwise to the above reaction solution, and stirring was continued for 1 hour. The resulting mixture was concentrated under reduced pressure to obtain a yellow solid compound 2b (961 mg, 96.65%).

[0287] LC-MS: m / z 172.0 [M+H] + 。

[0288] Step 2: Preparation of 7-chloropyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (2c)

[0289] At room temperature, compound 2b (961 mg, 5.60 mmol) was dissolved in N,N-dimethylformamide (DMF) (10 mL). N,N-Carbonyldiimidazole (CDI) (3.63 g, 22.39 mmol) and 1,8-diazabicycloundec-7-ene (DBU) (2.13 g, 13.99 mmol) were added successively. The reaction mixture was stirred at 80 °C for 1 hour. After completion of the reaction, the reaction was quenched with water (30 mL), and the pH of the reaction mixture was adjusted to 4 with dilute hydrochloric acid. The mixture was extracted with ethyl acetate (3 × 40 mL). The combined organic phases were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain dark yellow solid compound 2c (910 mg, 82.23%).

[0290] LC-MS: m / z 198.0 [M+H] + 。

[0291] Step 3: Preparation of 2,4,7-trichloropyrido[4,3-d]pyrimidine (2d)

[0292] At 0 °C, compound 2c (457 mg, 2.31 mmol) was mixed with phosphorus oxychloride (5 mL), and N,N-diisopropylethylamine (1.49 g, 11.53 mmol) was slowly added dropwise. The reaction mixture was stirred at 100 °C for 2 hours. After completion of the reaction, the reaction mixture was cooled to room temperature and concentrated under reduced pressure to obtain crude compound 2d as a black oil (540 mg, crude), which was used directly in the next step without purification.

[0293] LC-MS: m / z 233.9 [M+H] + 。

[0294] Step 4: Preparation of (R)-N-((1-((tert-butyldimethylsilyl)oxy)-2-methylhexan-2-yl)-2,7-dichloropyrido[4,3-d]pyrimidin-4-amine (2e)

[0295] At room temperature, compound 2d (540 mg, 2.30 mmol) was dissolved in 1,4-dioxane (5 mL). (R)-1-((tert-butyldimethylsilyl)oxy)-2-methylhexan-2-amine 1e (1.13 g, 4.61 mmol) and N,N-diisopropylethylamine (2.98 g, 23.03 mmol) were added successively. The reaction was carried out at room temperature for 1.5 hours. After completion of the reaction, the reaction mixture was diluted with water (30 mL) and extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was separated and purified by silica gel column chromatography (mobile phase: ethyl acetate / petroleum ether = 0 - 5%) to obtain compound 2e as a yellow oil (367 mg, 35.93%).

[0296] LC-MS: m / z 443.2 [M+H] + 。

[0297] Step 5: Preparation of (R)-N 4 -(1-((tert-Butyldimethylsilyl)oxy)-2-methylhexan-2-yl)-7-chloro-N 2 -(2,4-dimethoxybenzyl)pyrido[4,3-d]pyrimidine-2,4-diamine (2f)

[0298] At room temperature, dissolve compound 2e (367 mg, 0.83 mmol) in 1,4-dioxane (5 mL), and successively add 2,4-dimethoxybenzylamine (969 mg, 5.80 mmol) and N,N-diisopropylethylamine (321 mg, 2.48 mmol). Stir the reaction mixture at 100 °C overnight. After the reaction is complete, cool it to room temperature. Dilute the reaction mixture with water (20 mL), and extract it with dichloromethane (3 × 20 mL). Combine the organic phases, wash them with saturated ammonium chloride solution (2 × 30 mL), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify the obtained crude product by silica gel column chromatography (mobile phase: ethyl acetate / petroleum ether = 0 - 8%), to obtain the light yellow solid compound 2f (374 mg, 78.70%).

[0299] LC-MS: m / z 574.3 [M+H] + 。

[0300] Step 6: Preparation of (R)-N 4 -(1-((tert-Butyldimethylsilyl)oxy)-2-methylhexan-2-yl)-N 2 -(2,4-dimethoxybenzyl)-7-(2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)pyrido[4,3-d]pyrimidine-2,4-diamine (2g)

[0301] At room temperature, dissolve compound 2f (204 mg, 0.36 mmol) in 1,4-dioxane (2 mL) and water (0.4 mL). Sequentially add 2-(4-methylpiperazin-1-yl)pyrimidine-5-boronic acid pinacol ester (CAS: 942922-07-8) (2h) (264 mg, 1.05 mmol), potassium carbonate (148 mg, 1.07 mmol), and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (Pd(dppf)Cl2) (26 mg, 0.036 mmol). Stir the reaction mixture at 95 °C under a nitrogen atmosphere overnight. After the reaction is complete, cool to room temperature, dilute with water (20 mL), extract with ethyl acetate (3 × 20 mL), wash the combined organic phases with saturated brine (40 mL), dry over anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify the resulting crude product by preparative thin-layer chromatography (mobile phase: ethyl acetate / petroleum ether = 1:2) to obtain compound 2g as a yellow oil (33 mg, 12.97%).

[0302] LC-MS: m / z 716.4 [M+H] + 。

[0303] Step 7: Preparation of (R)-2-((2-amino-7-(2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (2)

[0304] At room temperature, dissolve compound 2g (33 mg, 0.046 mmol) in trifluoroacetic acid (1 mL). Stir the reaction mixture at 40 °C overnight. After the reaction is complete, concentrate the reaction mixture under reduced pressure, and purify the resulting crude product by preparative thin-layer chromatography (mobile phase: dichloromethane / methanol / triethylamine = 100:20:1) to obtain compound 2 as an off-white solid (6.4 mg, 28.66%).

[0305] 1 1H NMR (300 MHz, methanol-d4) δ 9.19 (d, J = 0.7 Hz, 1H), 8.96 (s, 2H), 7.40 (d, J = 0.7 Hz, 1H), 4.09 (d, J = 11.2 Hz, 1H), 3.96 (t, J = 5.2 Hz, 4H), 3.75 (d, J = 11.3 Hz, 1H), 2.55 (t, J = 5.2 Hz, 4H), 2.37 (s, 3H), 2.31–2.20 (m, 1H), 1.86–1.72 (m, 1H), 1.50 (s, 3H), 1.42–1.32 (m, 4H), 0.96–0.89 (m, 3H).

[0306] LC-MS: m / z 452.2 [M+H] + 。

[0307] Example 3: Preparation of (R)-2-((2-amino-7-(6-(4-methylpiperazin-1-yl)pyridin-3-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (3)

[0308]

[0309]

[0310] The preparation method was the same as that of Example 2, except that 2-(4-methylpiperazin-1-yl)pyrimidine-5-boronic acid pinacol ester (2h) was replaced with 2-(4-methylpiperazin-1-yl)pyridine-5-boronic acid pinacol ester (CAS: 918524-63-7) (3b). The obtained crude product was separated and purified by a preparative chromatographic column (column type: SunFire Prep C18 OBD column, 5um, 19*150mm; mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 6%-17% acetonitrile in 8 minutes; detection wavelength: 220 nm), and the formate of compound 3 was obtained.

[0311] 1 H NMR (300 MHz, methanol-d4) δ 9.41 (s, 1H), 8.95 (d, J = 2.0 Hz, 1H), 8.35 (d, J = 8.9 Hz, 1H), 7.68 (s, 1H), 7.07 (d, J = 8.8 Hz, 1H), 4.21 (d, J = 11.4 Hz, 1H), 4.13–3.88 (m, 4H), 3.74 (d, J = 11.3 Hz, 1H), 3.45–3.35 (m, 4H), 2.96 (s, 3H), 2.38–2.18 (m, 1H), 1.87–1.72 (m, 1H), 1.56 (s, 3H), 1.49–1.29 (m, 4H), 1.02–0.87 (m, 3H).

[0312] LC-MS: m / z 451.3 [M+H] + .

[0313] Example 4: Preparation of (R)-2-((2-amino-7-(5-methyl-6-(4-methylpiperazin-1-yl)pyridin-3-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (4)

[0314]

[0315]

[0316] Step 1: Preparation of (5-Methyl-6-(4-methylpiperazin-1-yl)pyridin-3-yl)boronic acid (4b)

[0317] At room temperature, dissolve compound 1-(5-bromo-3-methylpyridin-2-yl)-4-methylpiperazine 4a (500 mg, 1.85 mmol) in 1,4-dioxane (10.0 mL). Sequentially add bis(pinacolato)diboron (B2(Pin)2) (705 mg, 2.78 mmol), potassium acetate (545 mg, 5.55 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride dichloromethane complex (151 mg, 0.19 mmol). Stir the reaction mixture at 80 °C for 3 hours under a nitrogen atmosphere. After the reaction is complete, dilute the reaction mixture with water (20 mL), and extract with ethyl acetate (2 × 20 mL). Concentrate the aqueous phase obtained under reduced pressure. Purify the crude product by reversed-phase column chromatography (column type: Agela C18 column; mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 5% - 20% acetonitrile within 10 minutes; detection wavelength: 220 / 254 nm) to obtain a grayish-white solid compound 4b (370 mg, 85.04%).

[0318] LC-MS: m / z 236.1 [M+H] + 。

[0319] The remaining steps are the same as the preparation method of Example 2, except that compound 2-(4-methylpiperazin-1-yl)pyrimidine-5-boronic acid pinacol ester (2h) is replaced by (5-methyl-6-(4-methylpiperazin-1-yl)pyridin-3-yl)boronic acid (4b). Purify the obtained crude product by preparative chromatography column (column type: XBridge Prep C18 OBD column, 5um, 19*150mm; mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 22% - 46% acetonitrile within 8 minutes; detection wavelength: 254 / 220 nm) to obtain compound 4.

[0320] 11H NMR (400 MHz, methanol-d4) δ 9.19 (d, J = 0.7 Hz, 1H), 8.69 (d, J = 2.4 Hz, 1H), 8.13–8.08 (m, 1H), 7.44 (d, J = 0.7 Hz, 1H), 4.07 (d, J = 11.2 Hz, 1H), 3.74 (d, J = 11.2 Hz, 1H), 3.34–3.23 (m, 4H), 2.75–2.61 (m, 4H), 2.39 (s, 3H), 2.39 (s, 3H), 2.30–2.18 (m, 1H), 1.83–1.72 (m, 1H), 1.48 (s, 3H), 1.42–1.30 (m, 4H), 0.94–0.86 (m, 3H).

[0321] LC-MS: m / z 465.2 [M+H] + 。

[0322] Example 5: Preparation of (R)-2-((2-amino-7-(6-(pyrrolidin-1-yl)pyridin-3-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (5)

[0323]

[0324] The preparation method was the same as that of Example 2, except that 2-(4-methylpiperazin-1-yl)pyrimidine-5-boronic acid pinacol ester (2h) was replaced with 6-(pyrrolidin-1-yl)pyridine-3-boronic acid (CAS: 1150114-75-2) (5b). The obtained crude product was separated and purified by a preparative chromatographic column (column type: SunFire Prep C18 OBD column, 5um, 19*150mm; mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 7%-21% acetonitrile in 8 minutes; detection wavelength: 220 nm), and the formate of compound 5 was prepared.

[0325] 1 1H NMR (300 MHz, methanol-d4) δ 9.36 (s, 1H), 8.80 (d, J = 1.9 Hz, 1H), 8.33–8.15 (m, 1H), 7.54 (s, 1H), 6.67 (d, J = 8.9 Hz, 1H), 4.21 (d, J = 11.3 Hz, 1H), 3.75 (d, J = 11.4 Hz, 1H), 3.64–3.48 (m, 4H), 2.38–2.20 (m, 1H), 2.18–2.01 (m, 4H), 1.88–1.70 (m, 1H), 1.56 (s, 3H), 1.50–1.25 (m, 4H), 1.03–0.88 (m, 3H).

[0326] LC-MS: m / z 422.2 [M+H] + 。

[0327] Example 6: Preparation of (R)-2-((2-amino-7-(6-(piperidin-1-yl)pyridin-3-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (6)

[0328]

[0329]

[0330] The preparation method was the same as that of Example 2, except that 2-(4-methylpiperazin-1-yl)pyrimidine-5-boronic acid pinacol ester (2h) was replaced with 2-(piperidin-1-yl)pyridine-5-boronic acid pinacol ester (CAS: 852228-08-1) (6b). The obtained crude product was separated and purified by a preparative chromatographic column (column type: SunFire Prep C18 OBD column, 5um, 19*150mm; mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 13%-29% acetonitrile in 8 minutes; detection wavelength: 220 nm) to obtain the formate of compound 6.

[0331] 1 1H NMR (400 MHz, methanol-d4) δ 9.35 (s, 1H), 8.82 (d, J = 2.3 Hz, 1H), 8.20 (dd, J = 9.1, 2.5 Hz, 1H), 7.54 (s, 1H), 6.90 (d, J = 9.1 Hz, 1H), 4.18 (d, J = 11.3 Hz, 1H), 3.72 (d, J = 11.3 Hz, 1H), 3.70–3.64 (m, 4H), 2.34–2.19 (m, 1H), 1.84–1.70 (m, 3H), 1.70–1.61 (m, 4H), 1.53 (s, 3H), 1.45–1.29 (m, 4H), 0.98–0.87 (m, 3H).

[0332] LC-MS: m / z 436.4.1 [M+H] + 。

[0333] Example 7: Preparation of (R)-2-((2-amino-7-(6-(4-hydroxypiperidin-1-yl)pyridin-3-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol formate (7)

[0334]

[0335] The preparation method was the same as that of Example 2, except that 2-(4-methylpiperazin-1-yl)pyrimidine-5-boronic acid pinacol ester (2h) was replaced with 6-(4-hydroxypiperidin-1-yl)pyridine-3-boronic acid pinacol ester (CAS: 1251948-86-3) (7b). The obtained crude product was separated and purified by a preparative chromatographic column (column type: XBridge Shield RP18 OBD column, 5um, 19*150mm; mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 10%-32% acetonitrile within 8 minutes; detection wavelength: 254 nm), and the formate of Compound 7 was prepared.

[0336] 1 H NMR (300 MHz, methanol-d4) δ 9.35 (d, J = 1.6 Hz, 1H), 8.85 (d, J = 2.3 Hz, 1H), 8.22 (d, J = 8.9 Hz, 1H), 7.56 (s, 1H), 6.95 (d, J = 9.2 Hz, 1H), 4.30–4.12 (m, 3H), 4.00–3.84 (m, 1H), 3.74 (d, J = 11.3 Hz, 1H), 3.32–3.23 (m, 2H), 2.37–2.20 (m, 1H), 2.04–1.91 (m, 2H), 1.88–1.71 (m, 1H), 1.65–1.48 (m, 5H), 1.46–1.23 (m, 4H), 1.02–0.88 (m, 3H).

[0337] LC-MS: m / z 452.2 [M+H] + 。

[0338] Example 8: Preparation of (R)-2-((2-amino-7-(imidazo[1,2-a]pyridin-6-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (8)

[0339]

[0340] The preparation method was the same as that of Example 2, except that 2-(4-methylpiperazin-1-yl)pyrimidine-5-boronic acid pinacol ester (2h) was replaced with imidazo[1,2-a]pyridine-6-boronic acid pinacol ester (CAS: 1204742-76-6) (8b). The obtained crude product was separated and purified by a preparative chromatographic column (column type: SunFire Prep C18 OBD column, 5um, 19*150mm; mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 7%-15% acetonitrile in 8 minutes; detection wavelength: 254 nm), and the formate of Compound 8 was prepared.

[0341] 1 H NMR (300 MHz, methanol-d4) δ 9.46 (s, 1H), 9.41 (s, 1H), 8.20–8.12 (m, 1H), 8.07 (s, 1H), 7.87–7.68 (m, 3H), 4.23 (d, J = 11.4 Hz, 1H), 3.76 (d, J = 11.4 Hz, 1H), 2.38–2.21 (m, 1H), 1.90–1.74 (m, 1H), 1.58 (s, 3H), 1.50–1.27 (m, 4H), 1.03–0.88 (m, 3H).

[0342] LC-MS: m / z 392.3 [M+H] + 。

[0343] Example 9: Preparation of (R)-2-((2-amino-7-(6-(4-cyclopropylpiperazin-1-yl)pyridin-3-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (9)

[0344]

[0345] Step 1: Preparation of 2-amino-7-chloropyrido[4,3-d]pyrimidin-4(3H)-one (9b)

[0346] At room temperature, 4,6-dichloronicotinic acid 9a (5.0 g, 26.04 mmol) and guanidine hydrochloride (2.74 g, 28.646 mmol) were dissolved in N,N-dimethylformamide (100 mL). Cesium carbonate (16.97 g, 52.084 mmol) and copper(I) iodide (0.99 g, 5.208 mmol) were successively added to the reaction solution. The reaction solution was stirred at 110 °C overnight under a nitrogen atmosphere. After the reaction was completed, the solid in the reaction solution was filtered off, and the filtrate was concentrated under reduced pressure. The resulting residue was added to saturated sodium carbonate solution, and extracted with n-butanol (5 × 50 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a pale white to pale green solid compound 9b (4.2 g, 82.04%).

[0347] LC-MS: m / z 197.0 [M+H] + 。

[0348] Step 2: Preparation of (R)-2-((2-amino-7-chloropyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (9c)

[0349] At room temperature, compound 9b (500.00 mg, 2.543 mmol), (R)-2-amino-2-methylhexan-1-ol (500.61 mg, 3.815 mmol), benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) (1462.34 mg, 3.306 mmol), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (1161.58 mg, 7.629 mmol) were dissolved in N,N-dimethylformamide (8 mL). The reaction solution was stirred at room temperature overnight under a nitrogen atmosphere. After the reaction was completed, the reaction was quenched by adding water (50 mL), and the system was extracted with ethyl acetate (3 × 40 mL). The combined organic phases were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting crude product was separated and purified by silica gel column chromatography (mobile phase: ethyl acetate / petroleum ether = 1:1) to obtain a yellow semi-solid compound 9c (200 mg, 22.84%).

[0350] LC-MS: m / z 310.1 [M+H] + 。

[0351] Step 3: Preparation of 2-(4-cyclopropylpiperazin-1-yl)pyridine-5-boronic acid pinacol ester (9d)

[0352] At room temperature, 6-chloropyridine-3-boronic acid pinacol ester (50 mg, 0.209 mmol) was dissolved in dimethyl sulfoxide (1 mL). 1-Cyclopropylpiperazine (52.69 mg, 0.418 mmol) was added to the reaction solution. Under a nitrogen atmosphere, the mixture was stirred at 150 °C for 3 hours. After the reaction was completed, the reaction was quenched by adding water (20 mL). The system was extracted with ethyl acetate (3 × 20 mL). The combined organic phases were washed with saturated brine (40 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a brown solid compound 9d (72 mg, crude product), which was used directly in the next step without purification.

[0353] LC-MS: m / z 330.2 [M+H] + 。

[0354] Step 4: Preparation of (R)-2-((2-amino-7-(6-(4-cyclopropylpiperazin-1-yl)pyridin-3-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (9)

[0355] At room temperature, compound 9c (32.00 mg, 0.103 mmol) and compound 9d (68.02 mg, 0.206 mmol) were dissolved in 1,4-dioxane (1 mL) and water (0.1 mL). Potassium carbonate (42.83 mg, 0.309 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (16.83 mg, 0.021 mmol) were added successively. Under a nitrogen atmosphere, the reaction solution was stirred at 95 °C overnight. After the reaction was complete, it was cooled to room temperature, diluted with water (10 mL), and extracted with ethyl acetate (3 × 20 mL). The combined organic phases were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was separated and purified by preparative thin-layer chromatography (mobile phase: ethyl acetate / triethylamine = 20:1) to obtain a crude yellow oil. The product was further separated and purified by a preparative chromatography column (column type: XSelect CSH Prep C18 OBD column, 5um, 19*150mm; mobile phase A: water (0.05% ammonia water), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 32%-52% acetonitrile in 8 minutes; detection wavelength: 254 / 220 nm) to obtain a white solid compound 9 (17.5 mg, 35.55%).

[0356] 11H NMR (300 MHz, methanol-d4) δ 9.18 (d, J = 0.7 Hz, 1H), 8.77 (d, J = 2.4 Hz, 1H), 8.17 (dd, J = 9.0, 2.5 Hz, 1H), 7.41 (d, J = 0.8 Hz, 1H), 6.95 (d, J = 9.1 Hz, 1H), 4.08 (d, J = 11.3 Hz, 1H), 3.75 (d, J = 11.3 Hz, 1H), 3.70–3.60 (m, 4H), 2.84–2.75 (m, 4H), 2.36–2.19 (m, 1H), 1.85–1.69 (m, 2H), 1.50 (s, 3H), 1.43–1.32 (m, 4H), 0.98–0.88 (m, 3H), 0.60–0.47 (m, 4H).

[0357] LC-MS: m / z 477.4 [M+H] + 。

[0358] Example 10: Preparation of (R)-4-(5-(2-amino-4-((1-hydroxy-2-methylhexan-2-yl)amino)pyrido[4,3-d]pyrimidin-7-yl)pyridin-2-yl)-1-methylpiperazin-2-one (10)

[0359]

[0360] The preparation method was the same as that of Example 9, except that 1-cyclopropylpiperazine was replaced by 1-methylpiperazin-2-one to obtain Compound 10.

[0361] 1 1H NMR (300 MHz, methanol-d4) δ 9.20 (s, 1H), 8.82 (d, J = 2.4 Hz, 1H), 8.22 (dd, J = 8.9, 2.5 Hz, 1H), 7.43 (s, 1H), 6.95 (d, J = 9.0 Hz, 1H), 4.26 (s, 2H), 4.09 (d, J = 11.3 Hz, 1H), 4.02–3.94 (m, 2H), 3.75 (d, J = 11.3 Hz, 1H), 3.61–3.53 (m, 2H), 3.06 (s, 3H), 2.34–2.19 (m, 1H), 1.87–1.71 (m, 1H), 1.50 (s, 3H), 1.45–1.31 (m, 4H), 0.99–0.87 (m, 3H).

[0362] LC-MS: m / z 465.1 [M+H] + 。

[0363] Preparation of Example 11: (R)-2-((2-Amino-7-(6-(4-ethylpiperazin-1-yl)pyridin-3-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (11)

[0364]

[0365]

[0366] The preparation method was the same as that of Example 9, except that compound 1-cyclopropylpiperazine was replaced with 1-ethylpiperazine to obtain compound 11.

[0367] 1 H NMR (400 MHz, methanol-d4) δ 9.16 (s, 1H), 8.76 (d, J = 2.4 Hz, 1H), 8.15 (dd, J = 9.0, 2.5 Hz, 1H), 7.39 (s, 1H), 6.94 (d, J = 9.0 Hz, 1H), 4.06 (d, J = 11.3 Hz, 1H), 3.73 (d, J = 11.2 Hz, 1H), 3.71–3.64 (m, 4H), 2.63 (t, J = 5.0 Hz, 4H), 2.53 (q, J = 7.2 Hz, 2H), 2.32–2.18 (m, 1H), 1.84–1.71 (m, 1H), 1.48 (s, 3H), 1.44–1.30 (m, 4H), 1.17 (t, J = 7.3 Hz, 3H), 0.91 (t, J = 6.9 Hz, 3H).

[0368] LC-MS: m / z 465.2 [M+H] + .

[0369] Preparation of Example 12: (R)-2-((2-Amino-7-(6-(4-isopropylpiperazin-1-yl)pyridin-3-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (12)

[0370]

[0371] The preparation method was the same as that of Example 9, except that compound 1-cyclopropylpiperazine was replaced with 1-isopropylpiperazine to obtain compound 12.

[0372] 11H NMR (300 MHz, methanol-d4) δ 9.18 (d, J = 0.7 Hz, 1H), 8.78 (d, J = 2.4 Hz, 1H), 8.17 (dd, J = 9.0, 2.5 Hz, 1H), 7.41 (d, J = 0.7 Hz, 1H), 6.96 (d, J = 9.1 Hz, 1H), 4.08 (d, J = 11.3 Hz, 1H), 3.75 (d, J = 11.3 Hz, 1H), 3.72–3.65 (m, 4H), 2.83–2.77 (m, 1H), 2.74 (t, J = 5.2 Hz, 4H), 2.33–2.19 (m, 1H), 1.84–1.70 (m, 1H), 1.50 (s, 3H), 1.45–1.31 (m, 4H), 1.16 (d, J = 6.5 Hz, 6H), 0.99–0.89 (m, 3H).

[0373] LC-MS: m / z 479.2 [M+H] + 。

[0374] Example 13: Preparation of (R)-2-((2-Amino-7-(6-(4-(2-methoxyethyl)piperazin-1-yl)pyridin-3-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (13)

[0375]

[0376] Compound 13 was prepared in the same manner as in Example 9, except that 1-cyclopropylpiperazine was replaced with 1-(2-methoxyethyl)piperazine.

[0377] 1 1H NMR (400 MHz, methanol-d4) δ 9.16 (s, 1H), 8.75 (d, J = 2.5 Hz, 1H), 8.15 (dd, J = 9.0, 2.5 Hz, 1H), 7.39 (s, 1H), 6.93 (d, J = 9.0 Hz, 1H), 4.07 (d, J = 11.3 Hz, 1H), 3.73 (d, J = 11.3 Hz, 1H), 3.67 (t, J = 5.2 Hz, 4H), 3.60 (t, J = 5.5 Hz, 2H), 3.37 (s, 3H), 2.71–2.62 (m, 6H), 2.32–2.17 (m, 1H), 1.83–1.71 (m, 1H), 1.48 (s, 3H), 1.42–1.30 (m, 4H), 0.91 (t, J = 6.9 Hz, 3H).

[0378] LC-MS: m / z 495.3 [M+H] + 。

[0379] Example 14: Preparation of (2R)-2-((2-amino-7-(6-(3,4-dimethylpiperazin-1-yl)pyridin-3-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (14)

[0380]

[0381] The preparation method was the same as that of Example 9, except that 1-cyclopropylpiperazine was replaced with 1,2-dimethylpiperazine to obtain Compound 14.

[0382] 1 H NMR (400 MHz, methanol-d4) δ 9.19–9.13 (m, 1H), 8.75 (d, J = 2.5 Hz, 1H), 8.19–8.10 (m, 1H), 7.42–7.34 (m, 1H), 6.94 (d, J = 9.1 Hz, 1H), 4.31–4.17 (m, 2H), 4.06 (d, J = 11.3 Hz, 1H), 3.73 (d, J = 11.3 Hz, 1H), 3.18–3.05 (m, 1H), 3.01–2.89 (m, 1H), 2.80–2.66 (m, 1H), 2.44–2.32 (m, 4H), 2.32–2.18 (m, 2H), 1.83–1.70 (m, 1H), 1.48 (s, 3H), 1.43–1.26 (m, 4H), 1.19 (d, J = 6.3 Hz, 3H), 0.91 (d, J = 6.9 Hz, 3H).

[0383] LC-MS: m / z 465.5 [M+H] + .

[0384] Example 15: Preparation of (2R)-2-((2-amino-7-(4-methyl-6-(4-methylpiperazin-1-yl)pyridin-3-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (15)

[0385]

[0386]

[0387] The preparation method was the same as that of Example 9, except that 6-chloropyridine-3-boronic acid pinacol ester was replaced with 4-methyl-6-chloropyridine-3-boronic acid pinacol ester, and 1-cyclopropylpiperazine was replaced with 1-methylpiperazine to obtain Compound 15.

[0388] 11H NMR (400 MHz, methanol-d4) δ 9.18 (s, 1H), 8.12 (s, 1H), 7.13 (s, 1H), 6.78 (s, 1H), 4.08 (d, J = 11.3 Hz, 1H), 3.73 (d, J = 11.3 Hz, 1H), 3.65–3.58 (m, 4H), 2.59 (t, J = 5.1 Hz, 4H), 2.37 (s, 3H), 2.34 (s, 3H), 2.30–2.20 (m, 1H), 1.82–1.70 (m, 1H), 1.48 (s, 3H), 1.40–1.30 (m, 4H), 0.97–0.87 (m, 3H).

[0389] LC-MS: m / z 465.4 [M+H] + 。

[0390] Example 16: Preparation of (R)-2-((2-Amino-7-(2-methyl-6-(4-methylpiperazin-1-yl)pyridin-3-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (16)

[0391]

[0392] The preparation method was the same as that of Example 9, except that 6-chloropyridine-3-boronic acid pinacol ester was replaced by 2-methyl-6-chloropyridine-3-boronic acid pinacol ester, and 1-cyclopropylpiperazine was replaced by 1-methylpiperazine to obtain Compound 16.

[0393] 1 1H NMR (400 MHz, methanol-d4) δ 9.16 (s, 1H), 7.60 (d, J = 8.7 Hz, 1H), 7.12 (s, 1H), 6.72 (d, J = 8.7 Hz, 1H), 4.08 (d, J = 11.2 Hz, 1H), 3.73 (d, J = 11.2 Hz, 1H), 3.68–3.57 (m, 4H), 2.57 (t, J = 5.1 Hz, 4H), 2.43 (s, 3H), 2.35 (s, 3H), 2.31–2.20 (m, 1H), 1.82–1.70 (m, 1H), 1.48 (s, 3H), 1.41–1.27 (m, 4H), 0.91 (t, J = 6.9 Hz, 3H).

[0394] LC-MS: m / z 465.4 [M+H] + 。

[0395] Example 17: Preparation of (R)-2-((2-amino-7-(4-fluoro-6-(4-methylpiperazin-1-yl)pyridin-3-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (17)

[0396]

[0397] Step 1: Preparation of 1-(4-fluoropyridin-2-yl)-4-methylpiperazine (17a)

[0398] At room temperature, 2-bromo-4-fluoropyridine (400 mg, 2.273 mmol) and N-methylpiperazine were dissolved in toluene (10.0 mL), and then Rac-BINAP-Pd-G3 (225.56 mg, 0.227 mmol) and cesium carbonate (2962.20 mg, 9.092 mmol) were added successively. The reaction mixture was stirred overnight at 95 °C under a nitrogen atmosphere. After the reaction was complete, the reaction mixture was diluted with water (30 mL), extracted with ethyl acetate (3 × 20 mL), the organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was separated and purified by column chromatography (mobile phase: petroleum ether / ethyl acetate = 2:1) to obtain a dark yellow oily compound 17a (119 mg, 26.82%).

[0399] LC-MS: m / z 196.1 [M+H] + .

[0400] Step 2: Preparation of 1-(5-bromo-4-fluoropyridin-2-yl)-4-methylpiperazine (17b)

[0401] At room temperature, compound 1-(4-fluoropyridin-2-yl)-4-methylpiperazine 17a (119 mg, 0.610 mmol) was dissolved in acetonitrile (ACN) (3 mL), N-bromosuccinimide (NBS) (130.18 mg, 0.732 mmol) was added, and the reaction was carried out overnight at room temperature in the dark under a nitrogen atmosphere. After the reaction was complete, it was concentrated under reduced pressure. Water (10 mL) was added to the residue for dilution, and then it was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was separated and purified by column chromatography (mobile phase: petroleum ether / ethyl acetate = 2:1) to obtain an orange-yellow semi-solid compound 17b (166 mg, 99.35%).

[0402] LC-MS: m / z 274.0 [M+H] + .

[0403] Step 3: Preparation of 2-(4-methylpiperazin-1-yl)-4-fluoropyridine-5-boronic acid (17c)

[0404] At room temperature, dissolve compound 1-(5-bromo-4-fluoropyridin-2-yl)-4-methylpiperazine 17b (166 mg, 0.606 mmol) in 1,4-dioxane (3.0 mL), then successively add bis(pinacolato)diboron (230.65 mg, 0.909 mmol), potassium acetate (178.29 mg, 1.818 mmol) and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (98.66 mg, 0.121 mmol). The reaction mixture is stirred at 80 °C overnight under a nitrogen atmosphere. After the reaction is complete, the reaction mixture is diluted with water (10 mL) and extracted with ethyl acetate (2 × 10 mL). The aqueous phase obtained is concentrated under reduced pressure to obtain a brown solid compound 17c (150 mg, crude product), which is used directly in the next step without purification.

[0405] LC-MS: m / z 240.1 [M+H] + 。

[0406] The remaining steps are the same as in Example 9, except that 2-(4-cyclopropylpiperazin-1-yl)pyridine-5-boronic acid pinacol ester (9d) is replaced by 2-(4-methylpiperazin-1-yl)-4-fluoropyridine-5-boronic acid (17c) to obtain compound 17.

[0407] 1 1H NMR (400 MHz, methanol-d4) δ 9.08 (s, 1H), 8.58 (d, J = 11.4 Hz, 1H), 7.30 (d, J = 1.0 Hz, 1H), 6.57 (d, J = 14.8 Hz, 1H), 3.97 (d, J = 11.3 Hz, 1H), 3.63 (d, J = 11.3 Hz, 1H), 3.57 (t, J = 5.2 Hz, 4H), 2.46 (t, J = 5.1 Hz, 4H), 2.26 (s, 3H), 2.21–2.08 (m, 1H), 1.72–1.60 (m, 1H), 1.38 (s, 3H), 1.32–1.18 (m, 4H), 0.81 (t, J = 6.9 Hz, 3H).

[0408] LC-MS: m / z 469.3 [M+H] + 。

[0409] Example 18: Preparation of (R)-2-((2-amino-7-(6-(4-(dimethylamino)piperidin-1-yl)pyridin-3-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (18)

[0410]

[0411] The preparation method was the same as that of Example 9, except that 1-cyclopropylpiperazine was replaced with 4-dimethylaminopiperidine to obtain Compound 18.

[0412] 1 H NMR (400 MHz, methanol-d4) δ 9.15 (s, 1H), 8.73 (s, 1H), 8.12 (dd, J = 8.9, 2.2 Hz, 1H), 7.37 (s, 1H), 6.94 (d, J = 9.1 Hz, 1H), 4.50 (d, J = 13.5 Hz, 2H), 4.06 (d, J = 11.3 Hz, 1H), 3.73 (d, J = 11.3 Hz, 1H), 2.93 (t, J = 12.9 Hz, 2H), 2.53 (s, 1H), 2.41–2.30 (m, 6H), 2.30–2.18 (m, 1H), 2.06–1.94 (m, 2H), 1.83–1.71 (m, 1H), 1.61–1.44 (m, 5H), 1.42–1.26 (m, 4H), 0.91 (t, J = 6.9 Hz, 3H).

[0413] LC-MS: m / z 479.3 [M+H] + 。

[0414] Example 19: Preparation of (R)-2-((2-amino-7-(6-(4-(2-hydroxyethyl)piperazin-1-yl)pyridin-3-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (19)

[0415]

[0416] The preparation method was the same as that of Example 9, except that 1-cyclopropylpiperazine was replaced with N-hydroxyethylpiperazine to obtain Compound 19.

[0417] 11H NMR (400 MHz, methanol-d4) δ 9.31 (s, 1H), 8.86 (s, 1H), 8.25 (d, J = 8.9 Hz, 1H), 7.57–7.50 (m, 1H), 6.99 (d, J = 9.0 Hz, 1H), 4.14 (d, J = 11.3 Hz, 1H), 3.91–3.76 (m, 6H), 3.72 (d, J = 11.3 Hz, 1H), 3.08–2.97 (m, 4H), 2.97–2.87 (m, 2H), 2.32–2.20 (m, 1H), 1.82–1.72 (m, 1H), 1.52 (s, 3H), 1.43–1.28 (m, 4H), 0.92 (t, J = 6.9 Hz, 3H).

[0418] LC-MS: m / z 481.4 [M+H] + 。

[0419] Example 20: Preparation of (R)-2-((2-Amino-7-(6-(4-propylpiperazin-1-yl)pyridin-3-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (20)

[0420]

[0421] Step 1: Preparation of 2-(4-Propylpiperazin-1-yl)pyridine-5-boronic acid pinacol ester (20a)

[0422] At room temperature, 1-propylpiperazine dibromide (484.39 mg, 1.670 mmol), potassium carbonate (464.99 mg, 3.340 mmol) were dissolved in dimethyl sulfoxide (5 mL), 2-chloropyridine-5-boronic acid pinacol ester (200 mg, 0.835 mmol) was added, and the mixture was stirred at 150 °C for 2 h under a nitrogen atmosphere. After completion of the reaction, the reaction was quenched with water (30 mL), the system was extracted with ethyl acetate (3 × 15 mL), the combined organic phases were washed with saturated brine (15 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a pale yellow liquid compound 20a (549 mg, crude product), which was used directly in the next step without purification.

[0423] LC-MS: m / z 332.2 [M+H] + 。

[0424] The remaining steps were the same as in Example 9, except that 2-(4-cyclopropylpiperazin-1-yl)pyridine-5-boronic acid pinacol ester (9d) was replaced by 2-(4-propylpiperazin-1-yl)pyridine-5-boronic acid pinacol ester (20a) to obtain compound 20.

[0425] 1 1H NMR (400 MHz, methanol-d4) δ 9.16 (s, 1H), 8.75 (d, J = 2.4 Hz, 1H), 8.15 (dd, J = 9.0, 2.5 Hz, 1H), 7.39 (s, 1H), 6.93 (d, J = 9.0 Hz, 1H), 4.06 (d, J = 11.3 Hz, 1H), 3.73 (d, J = 11.3 Hz, 1H), 3.67 (t, J = 5.1 Hz, 4H), 2.63 (t, J = 5.1 Hz, 4H), 2.48–2.36 (m, 2H), 2.31–2.18 (m, 1H), 1.83–1.70 (m, 1H), 1.69–1.53 (m, 2H), 1.48 (s, 3H), 1.42–1.28 (m, 4H), 0.96 (t, J = 7.4 Hz, 3H), 0.91 (t, J = 6.9 Hz, 3H).

[0426] LC-MS: m / z 479.3 [M+H] + .

[0427] Example 21: Preparation of (R)-2-((2-Amino-7-(6-(4-(2,2-difluoroethyl)piperazin-1-yl)pyridin-3-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (21)

[0428]

[0429]

[0430] Compound 21 was prepared in the same manner as in Example 20, except that 1-propylpiperazine dibromide was replaced with 1-(2,2-difluoroethyl)piperazine hydrochloride.

[0431] 1 1H NMR (300 MHz, methanol-d4) δ 9.28 (s, 1H), 8.82 (s, 1H), 8.21 (d, J = 8.8 Hz, 1H), 7.50 (s, 1H), 6.94 (d, J = 8.9 Hz, 1H), 6.05 (tt, J = 55.9, 4.3 Hz, 1H), 4.15 (d, J = 11.3 Hz, 1H), 3.75 (d, J = 11.3 Hz, 1H), 3.70 (t, J = 5.1 Hz, 4H), 2.84 (td, J = 15.2, 4.3 Hz, 2H), 2.75 (t, J = 5.0 Hz, 4H), 2.36–2.17 (m, 1H), 1.88–1.70 (m, 1H), 1.53 (s, 3H), 1.47–1.26 (m, 4H), 1.00–0.87 (m, 3H).

[0432] LC-MS: m / z 501.4 [M+H] + 。

[0433] Example 22: Preparation of (R)-2-((2-amino-7-(6-(4-(2-fluoroethyl)piperazin-1-yl)pyridin-3-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (22)

[0434]

[0435] Step 1: Preparation of 2-(4-(2-fluoroethyl)piperazin-1-yl)pyridine-5-boronic acid pinacol ester (22a)

[0436] At room temperature, 2-chloropyridine-5-boronic acid pinacol ester (150 mg, 0.626 mmol) and 1-(2-fluoroethyl)piperazine dihydrochloride (256.90 mg, 1.252 mmol) were dissolved in dimethyl sulfoxide (2 mL), N,N-diisopropylethylamine (323.77 mg, 2.504 mmol) was added, and the mixture was stirred at 150 °C for 2 hours under a nitrogen atmosphere. After completion of the reaction, the reaction was quenched with water (20 mL), the system was extracted with ethyl acetate (3 × 20 mL), the combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a brown semi-solid compound 22a (198 mg, crude product), which was used directly in the next step without purification.

[0437] LC-MS: m / z 336.2 [M+H] + 。

[0438] The remaining steps were the same as in Example 9, except that 2-(4-cyclopropylpiperazin-1-yl)pyridine-5-boronic acid pinacol ester (9d) was replaced by 2-(4-(2-fluoroethyl)piperazin-1-yl)pyridine-5-boronic acid pinacol ester (22a) to obtain compound 22.

[0439] 11H NMR (400 MHz, methanol-d4) δ 9.19 (s, 1H), 8.72 (d, J = 2.4 Hz, 1H), 8.11 (dd, J = 9.0, 2.5 Hz, 1H), 7.41 (s, 1H), 6.83 (d, J = 9.1 Hz, 1H), 4.66–4.56 (m, 1H), 4.54–4.44 (m, 1H), 4.05 (d, J = 11.4 Hz, 1H), 3.68–3.56 (m, 5H), 2.79–2.72 (m, 1H), 2.71–2.66 (m, 1H), 2.62 (t, J = 5.1 Hz, 4H), 2.25–2.09 (m, 1H), 1.75–1.61 (m, 1H), 1.42 (s, 3H), 1.35–1.16 (m, 4H), 0.82 (t, J = 6.9 Hz, 3H).

[0440] LC-MS: m / z 483.4 [M+H] + 。

[0441] Example 23: Preparation of (R)-2-((2-Amino-7-(6-(4-(2,2,2-trifluoroethyl)piperazin-1-yl)pyridin-3-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (23)

[0442]

[0443] The preparation method was the same as that of Example 22, except that 1-(2-fluoroethyl)piperazine dihydrochloride was replaced with 1-(2,2,2-trifluoroethyl)piperazine dihydrochloride to obtain Compound 23.

[0444] 1 1H NMR (400 MHz, methanol-d4) δ 9.30 (s, 1H), 8.82 (d, J = 2.4 Hz, 1H), 8.25–8.16 (m, 1H), 7.52 (s, 1H), 6.93 (d, J = 9.0 Hz, 1H), 4.15 (d, J = 11.3 Hz, 1H), 3.76–3.65 (m, 5H), 3.14 (q, J = 9.8 Hz, 2H), 2.80 (t, J = 5.1 Hz, 4H), 2.33–2.19 (m, 1H), 1.83–1.71 (m, 1H), 1.52 (s, 3H), 1.41–1.32 (m, 4H), 0.92 (t, J = 6.9 Hz, 3H).

[0445] LC-MS: m / z 519.5 [M+H] + 。

[0446] Preparation of Example 24: (R)-2-((2-Amino-7-(6-(4-propylpiperidin-1-yl)pyridin-3-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (24)

[0447]

[0448] Compound 24 was prepared in the same manner as in Example 22, except that 1-(2-fluoroethyl)piperazine dihydrochloride was replaced with 4-propylpiperidine hydrochloride.

[0449] 1 H NMR (300 MHz, methanol-d4) δ 9.16 (s, 1H), 8.73 (d, J = 2.4 Hz, 1H), 8.12 (dd, J = 9.0, 2.6 Hz, 1H), 7.39 (s, 1H), 6.92 (d, J = 9.2 Hz, 1H), 4.47–4.35 (m, 2H), 4.08 (d, J = 11.2 Hz, 1H), 3.75 (d, J = 11.3 Hz, 1H), 3.00–2.87 (m, 2H), 2.32–2.20 (m, 1H), 1.89–1.76 (m, 3H), 1.67–1.54 (m, 1H), 1.50 (s, 3H), 1.48–1.19 (m, 10H), 1.00–0.89 (m, 6H).

[0450] LC-MS: m / z 478.5 [M+H] + 。

[0451] Preparation of Example 25: (R)-2-((2-Amino-7-(6-(3-ethoxyazetidin-1-yl)pyridin-3-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (25)

[0452]

[0453]

[0454] Compound 25 was prepared in the same manner as in Example 22, except that 1-(2-fluoroethyl)piperazine dihydrochloride was replaced with 3-ethoxyazetidine hydrochloride.

[0455] 11H NMR (400 MHz, methanol-d4) δ 9.15 (s, 1H), 8.68 (d, J = 2.4 Hz, 1H), 8.14 (dd, J = 8.8, 2.4 Hz, 1H), 7.36 (s, 1H), 6.53 (d, J = 8.8 Hz, 1H), 4.53–4.45 (m, 1H), 4.35–4.27 (m, 2H), 4.05 (d, J = 11.2 Hz, 1H), 3.96–3.88 (m, 2H), 3.73 (d, J = 11.3 Hz, 1H), 3.54 (q, J = 7.0 Hz, 2H), 2.30–2.18 (m, 1H), 1.83–1.70 (m, 1H), 1.47 (s, 3H), 1.42–1.30 (m, 4H), 1.23 (t, J = 7.0 Hz, 3H), 0.91 (t, J = 6.9 Hz, 3H).

[0456] LC-MS: m / z 452.5 [M+H] + 。

[0457] Example 26: Preparation of (R)-2-((2-Amino-7-(6-(4-ethoxypiperidin-1-yl)pyridin-3-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (26)

[0458]

[0459] Compound 26 was prepared in the same manner as in Example 22, except that 1-(2-fluoroethyl)piperazine dihydrochloride was replaced with 4-ethoxypiperidine.

[0460] 1 1H NMR (300 MHz, methanol-d4) δ 9.17 (s, 1H), 8.75 (d, J = 2.4 Hz, 1H), 8.14 (dd, J = 9.0, 2.5 Hz, 1H), 7.39 (s, 1H), 6.95 (d, J = 9.2 Hz, 1H), 4.63–4.59 (m, 1H), 4.19–4.11 (m, 1H), 4.07 (d, J = 11.5 Hz, 1H), 3.75 (d, J = 11.2 Hz, 1H), 3.68–3.54 (m, 3H), 2.32–2.14 (m, 1H), 2.09–1.94 (m, 2H), 1.86–1.74 (m, 1H), 1.68–1.54 (m, 2H), 1.49 (s, 3H), 1.44–1.27 (m, 6H), 1.22 (t, J = 7.0 Hz, 3H), 0.97–0.88 (m, 3H).

[0461] LC-MS: m / z 480.1 [M+H]+ 。

[0462] Example 27: Preparation of (R)-2-((2-amino-7-(6-(3-(dipropylamino)azetidin-1-yl)pyridin-3-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (27)

[0463]

[0464] The preparation method was the same as that of Example 22, except that 1-(2-fluoroethyl)piperazine dihydrochloride was replaced with N,N-dipropylazetidin-3-amine to obtain Compound 27.

[0465] 1 H NMR (300 MHz, methanol-d4) δ 9.18 (s, 1H), 8.74–8.68 (m, 1H), 8.17 (dd, J = 8.8, 2.4 Hz, 1H), 7.39 (s, 1H), 6.57 (d, J = 8.8 Hz, 1H), 4.21 (t, J = 7.8 Hz, 2H), 4.08 (d, J = 11.3 Hz, 1H), 4.01–3.91 (m, 2H), 3.87–3.78 (m, 1H), 3.75 (d, J = 11.3 Hz, 1H), 2.60–2.46 (m, 4H), 2.34–2.16 (m, 1H), 1.87–1.71 (m, 1H), 1.62–1.51 (m, 4H), 1.50 (s, 3H), 1.44–1.32 (m, 4H), 1.01–0.87 (m, 9H).

[0466] LC-MS: m / z 507.4 [M+H] + 。

[0467] Example 28: Preparation of (R)-2-((2-amino-7-(6-(4-(diethylamino)piperidin-1-yl)pyridin-3-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (28)

[0468]

[0469] The preparation method was the same as that of Example 9, except that 1-cyclopropylpiperazine was replaced with 4-(diethylamino)piperidine to obtain Compound 28.

[0470] 11H NMR (300 MHz, methanol-d4) δ 9.37 (s, 1H), 8.87 (d, J = 2.4 Hz, 1H), 8.26 (dd, J = 9.1, 2.5 Hz, 1H), 7.62 (s, 1H), 7.03 (d, J = 9.1 Hz, 1H), 4.74–4.54 (m, overlapped with solvent, 2H), 4.17 (d, J = 11.4 Hz, 1H), 3.78–3.65 (m, 2H), 3.31–3.24 (m, overlapped with solvent, 4H), 3.07 (t, J = 12.7 Hz, 2H), 2.31–2.15 (m, 3H), 1.91–1.71 (m, 3H), 1.55 (s, 3H), 1.49–1.27 (m, 10H), 1.02–0.86 (m, 3H).

[0471] LC-MS: m / z 507.5 [M+H] + 。

[0472] Example 29: Preparation of (R)-2-((2-Amino-7-(6-(3-(diethylamino)azetidin-1-yl)pyridin-3-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (29)

[0473]

[0474]

[0475] Compound 29 was prepared in the same manner as in Example 22, except that 1-(2-fluoroethyl)piperazine dihydrochloride was replaced with 3-(diethylamino)azetidine dihydrochloride.

[0476] 11H NMR (300 MHz, methanol-d4) δ 9.17 (d, J = 0.7 Hz, 1H), 8.71 (d, J = 2.4 Hz, 1H), 8.17 (dd, J = 8.8, 2.4 Hz, 1H), 7.39 (d, J = 0.7 Hz, 1H), 6.58 (d, J = 8.8 Hz, 1H), 4.23 (t, J = 7.8 Hz, 2H), 4.08 (d, J = 11.3 Hz, 1H), 4.03–3.92 (m, 2H), 3.87–3.78 (m, 1H), 3.75 (d, J = 11.2 Hz, 1H), 2.67 (q, J = 7.2 Hz, 4H), 2.35–2.18 (m, 1H), 1.88–1.71 (m, 1H), 1.50 (s, 3H), 1.43–1.28 (m, 4H), 1.10 (t, J = 7.2 Hz, 6H), 0.93 (t, J = 6.9 Hz, 3H).

[0477] LC-MS: m / z 479.4 [M+H] + 。

[0478] Example 30: Preparation of (R)-2-((2-Amino-7-(2-(4-(2-fluoroethyl)piperazin-1-yl)pyrimidin-5-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (30)

[0479]

[0480] Step 1: Preparation of 2-(4-(2-fluoroethyl)piperazin-1-yl)pyrimidine-5-boronic acid (30a)

[0481] At room temperature, 2-chloropyrimidine-5-boronic acid (148 mg, 0.935 mmol) and 1-(2-fluoroethyl)piperazine dihydrochloride (209.92 mg, 1.029 mmol) were dissolved in ethanol (2 mL). Triethylamine (331.02 mg, 3.271 mmol) was added to the reaction solution, and the mixture was stirred at 75 °C for 1 hour under a nitrogen atmosphere. After completion of the reaction, the reaction solution was cooled to room temperature and concentrated under reduced pressure to obtain a yellow solid compound 30a (260 mg, crude product), which was used directly in the next step without purification.

[0482] LC-MS: m / z 255.1 [M+H] + 。

[0483] The remaining steps were the same as those in Example 9, except that the compound 2-(4-cyclopropylpiperazin-1-yl)pyridine-5-boronic acid pinacol ester (9d) was replaced with (30a) to obtain compound 30.

[0484] 11H NMR (300 MHz, methanol-d4) δ 9.22 (s, 1H), 8.98 (s, 2H), 7.44 (s, 1H), 4.76–4.71 (m, 1H), 4.59–4.55 (m, 1H), 4.11 (d, J = 11.2 Hz, 1H), 3.97 (t, J = 5.1 Hz, 4H), 3.75 (d, J = 11.3 Hz, 1H), 2.87–2.82 (m, 1H), 2.78–2.73 (m, 1H), 2.72–2.64 (m, 4H), 2.34–2.19 (m, 1H), 1.86–1.71 (m, 1H), 1.51 (s, 3H), 1.46–1.27 (m, 4H), 0.93 (t, J = 6.9 Hz, 3H).

[0485] LC-MS: m / z 484.3 [M+H] + 。

[0486] Example 31: Preparation of (R)-2-((2-amino-7-(2-(4-propylpiperazin-1-yl)pyrimidin-5-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (31)

[0487]

[0488] Compound 31 was prepared in the same manner as in Example 30, except that 1-(2-fluoroethyl)piperazine dihydrochloride was replaced with 1-n-propylpiperazine dibromide.

[0489] 1 1H NMR (400 MHz, methanol-d4) δ 9.18 (d, J = 0.7 Hz, 1H), 8.95 (s, 2H), 7.39 (d, J = 0.8 Hz, 1H), 4.07 (d, J = 11.3 Hz, 1H), 3.99–3.89 (m, 4H), 3.73 (d, J = 11.3 Hz, 1H), 2.60 (t, J = 5.0 Hz, 4H), 2.46–2.38 (m, 2H), 2.30–2.19 (m, 1H), 1.83–1.71 (m, 1H), 1.66–1.54 (m, 2H), 1.48 (s, 3H), 1.41–1.26 (m, 4H), 0.96 (t, J = 7.4 Hz, 3H), 0.91 (t, J = 7.0 Hz, 3H).

[0490] LC-MS: m / z 480.4 [M+H] + 。

[0491] Preparation of Example 32: (R)-2-((2-Amino-7-(2-(4-Isopropylpiperazin-1-yl)pyrimidin-5-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (32)

[0492]

[0493] The preparation method was the same as that of Example 30, except that 1-(2-Fluoroethyl)piperazine dihydrochloride was replaced with 1-Isopropylpiperazine to obtain Compound 32.

[0494] 1 H NMR (300 MHz, methanol-d4) δ 9.19 (s, 1H), 8.96 (s, 2H), 7.41 (s, 1H), 4.09 (d, J = 11.3 Hz, 1H), 3.99–3.92 (m, 4H), 3.75 (d, J = 11.3 Hz, 1H), 2.83–2.74 (m, 1H), 2.68 (t, J = 5.2 Hz, 4H), 2.32–2.20 (m, 1H), 1.85–1.71 (m, 1H), 1.50 (s, 3H), 1.44–1.26 (m, 4H), 1.16 (s, 3H), 1.14 (s, 3H), 0.93 (t, J = 6.9 Hz, 3H).

[0495] LC-MS: m / z 480.3 [M+H] + .

[0496] Preparation of Example 33: (R)-2-((2-Amino-7-(2-(4-(Dimethylamino)piperidin-1-yl)pyrimidin-5-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (33)

[0497]

[0498]

[0499] The preparation method was the same as that of Example 30, except that 1-(2-Fluoroethyl)piperazine dihydrochloride was replaced with 4-Dimethylaminopiperidine to obtain Compound 33.

[0500] 11H NMR (400 MHz, methanol-d4) δ 9.17 (s, 1H), 8.92 (s, 2H), 7.38 (s, 1H), 4.67–4.54 (m, 2H), 4.07 (d, J = 11.3 Hz, 1H), 3.73 (d, J = 11.3 Hz, 1H), 2.96 (t, J = 12.9 Hz, 2H), 2.71–2.55 (m, 1H), 2.37 (s, 6H), 2.30–2.18 (m, 1H), 2.07–1.96 (m, 2H), 1.82–1.70 (m, 1H), 1.47 (s, 3H), 1.44–1.24 (m, 6H), 0.91 (t, J = 6.7 Hz, 3H).

[0501] LC-MS: m / z 480.4 [M+H] + 。

[0502] Example 34: Preparation of (R)-2-((2-amino-7-(4-(4-methylpiperazin-1-yl)phenyl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (34)

[0503]

[0504] The preparation method was the same as that of Example 9, except that 2-(4-cyclopropylpiperazin-1-yl)pyridine-5-boronic acid pinacol ester (9d) was replaced with 4-(4-methylpiperazin-1-yl)phenylboronic acid pinacol ester (CAS: 747413-21-4) (34a) to obtain Compound 34.

[0505] 1 1H NMR (300 MHz, methanol-d4) δ 9.15 (s, 1H), 7.95–7.84 (m, 2H), 7.42 (s, 1H), 7.14–7.03 (m, 2H), 4.07 (d, J = 11.3 Hz, 1H), 3.75 (d, J = 11.3 Hz, 1H), 3.36–3.33 (m, 4H), 2.65 (t, J = 5.1 Hz, 4H), 2.38 (s, 3H), 2.33–2.19 (m, 1H), 1.87–1.71 (m, 1H), 1.49 (s, 3H), 1.45–1.28 (m, 4H), 0.93 (t, J = 6.8 Hz, 3H).

[0506] LC-MS: m / z 450.2 [M+H] + 。

[0507] Preparation of Example 35: (R)-2-((2-Amino-7-(1-(1-methylpiperidin-4-yl)-1H-pyrazol-4-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (35)

[0508]

[0509] The preparation method was the same as that of Example 9, except that 2-(4-cyclopropylpiperazin-1-yl)pyridine-5-boronic acid pinacol ester (9d) was replaced with (1-methylpiperidin-4-yl)-1H-pyrazole-4-boronic acid pinacol ester (CAS: 1323919-64-7) (35a) to obtain Compound 35.

[0510] 1 H NMR (300 MHz, methanol-d4) δ 9.11 (s, 1H), 8.31 (s, 1H), 8.08 (s, 1H), 7.31 (s, 1H), 4.36–4.18 (m, 1H), 4.08 (d, J = 11.3 Hz, 1H), 3.74 (d, J = 11.3 Hz, 1H), 3.13–2.97 (m, 2H), 2.37 (s, 3H), 2.34–2.06 (m, 7H), 1.86–1.69 (m, 1H), 1.49 (s, 3H), 1.45–1.27 (m, 4H), 0.93 (t, J = 6.9 Hz, 3H).

[0511] LC-MS: m / z 439.2 [M+H] + 。

[0512] Preparation of Example 36: (R)-2-((2-Amino-7-(1-(tetrahydropyran-4-yl)-1H-pyrazol-4-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (36)

[0513]

[0514] The preparation method was the same as that of Example 9, except that 2-(4-cyclopropylpiperazin-1-yl)pyridine-5-boronic acid pinacol ester (9d) was replaced with 1-(tetrahydropyran-4-yl)-1H-pyrazole-4-boronic acid pinacol ester (CAS: 1040377-03-4) (36a) to obtain Compound 36.

[0515] 11H NMR (400 MHz, methanol-d4) δ 9.14 (s, 1H), 8.32 (s, 1H), 8.08 (s, 1H), 7.32 (s, 1H), 4.54–4.42 (m, 1H), 4.14–4.03 (m, 3H), 3.72 (d, J = 11.3 Hz, 1H), 3.65–3.54 (m, 2H), 2.32–2.19 (m, 1H), 2.17–2.06 (m, 4H), 1.83–1.69 (m, 1H), 1.48 (s, 3H), 1.44–1.25 (m, 4H), 0.91 (t, J = 7.0 Hz, 3H).

[0516] LC-MS: m / z 426.2 [M+H] + 。

[0517] Example 37: Preparation of (R)-2-((2-amino-7-(4-(pyridin-3-yl)phenyl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (37)

[0518]

[0519] The preparation method was the same as that of Example 9, except that 2-(4-cyclopropylpiperazin-1-yl)pyridine-5-boronic acid pinacol ester (9d) was replaced with 4-(pyridin-3-yl)phenylboronic acid pinacol ester (CAS: 929203-04-3) (37a) to obtain Compound 37.

[0520] 1 1H NMR (400 MHz, methanol-d4) δ 9.14 (s, 1H), 8.80 (d, J = 2.4 Hz, 1H), 8.45 (dd, J = 4.9, 1.6 Hz, 1H), 8.13–8.06 (m, 1H), 8.06–7.99 (m, 2H), 7.79–7.69 (m, 2H), 7.52–7.42 (m, 2H), 3.99 (d, J = 11.3 Hz, 1H), 3.65 (d, J = 11.3 Hz, 1H), 2.22–2.09 (m, 1H), 1.74–1.62 (m, 1H), 1.40 (s, 3H), 1.34–1.21 (m, 4H), 0.82 (t, J = 6.9 Hz, 3H).

[0521] LC-MS: m / z 429.3 [M+H] + 。

[0522] Example 38: Preparation of (R)-2-((2-amino-7-(6-(piperazin-1-yl)pyridin-3-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (38)

[0523]

[0524]

[0525] Step 1: Preparation of tert-butyl (R)-4-(5-(2-amino-4-((1-hydroxy-2-methylhexan-2-yl)amino)pyrido[4,3-d]pyrimidin-7-yl)pyridin-2-yl)piperazine-1-carboxylate (38b)

[0526] The same as step 4 of Example 9, except that 2-(4-cyclopropylpiperazin-1-yl)pyridine-5-boronic acid pinacol ester (9d) was replaced with 4-[4-(N-Boc)piperazin-1-yl]phenylboronic acid pinacol ester (CAS: 496786-98-2) (38a) to obtain compound 38b.

[0527] Step 2: Preparation of (R)-2-((2-amino-7-(6-(piperazin-1-yl)pyridin-3-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (38)

[0528] At room temperature, compound 38b (50 mg, 0.093 mmol) was dissolved in dichloromethane (1 mL), and trifluoroacetic acid (1 mL) was added to the reaction solution. The mixture was stirred at room temperature for 2 hours under a nitrogen atmosphere. After completion of the reaction, the reaction solution was concentrated under reduced pressure and then separated and purified by preparative thin layer chromatography (mobile phase: dichloromethane / methanol = 10:1) to obtain a crude product. The crude product was further separated and purified by a preparative chromatographic column (column type: Gemini-NX C18 AXAI Packed column, 5um, 21.2*150mm; mobile phase A: water (0.05% ammonia), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 23%-41% acetonitrile in 8 minutes; detection wavelength: 254 / 220 nm) to obtain a white solid compound 38 (2.1 mg, 4.76%).

[0529] 11H NMR (400 MHz, methanol-d4) δ 9.15 (d, J = 0.7 Hz, 1H), 8.75 (dd, J = 2.6, 0.7 Hz, 1H), 8.14 (dd, J = 9.0, 2.5 Hz, 1H), 7.38 (d, J = 0.7 Hz, 1H), 6.92 (d, J = 9.0 Hz, 1H), 4.06 (d, J = 11.3 Hz, 1H), 3.73 (d, J = 11.3 Hz, 1H), 3.64–3.58 (m, 4H), 2.98–2.90 (m, 4H), 2.30–2.22 (m, 1H), 1.83–1.71 (m, 1H), 1.48 (s, 3H), 1.40–1.32 (m, 4H), 0.94–0.90 (m, 3H).

[0530] LC-MS: m / z 437.3 [M+H] + 。

[0531] Example 39: Preparation of (R)-2-((7-([2,3'-bipyridin]-5-yl)-2-aminopyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (39)

[0532]

[0533] Step 1: Preparation of 5-bromo-2,3'-bipyridine (39a)

[0534] At room temperature, 3-pyridineboronic acid (500.09 mg, 4.069 mmol) and 5-bromo-2-iodopyridine (1050 mg, 3.699 mmol) were dissolved in 1,4-dioxane (10.0 mL). Potassium carbonate (1533.49 mg, 11.097 mmol) and tetrakis(triphenylphosphine)palladium (427.39 mg, 0.37 mmol) were added successively. The reaction mixture was stirred overnight at 80 °C under a nitrogen atmosphere. After the reaction was complete, the reaction mixture was diluted with water (30 mL), extracted with ethyl acetate (3 × 30 mL), the organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was separated and purified by column chromatography (mobile phase: petroleum ether / ethyl acetate = 4:1) to obtain the yellow solid compound 39a (148 mg, 17.02%).

[0535] LC-MS: m / z 235.0 [M+H] + 。

[0536] Step 2: Preparation of 2,3'-bipyridine-5-boronic acid (39b)

[0537] At room temperature, compound 39a (148 mg, 0.63 mmol) was dissolved in 1,4-dioxane (3.0 mL). To the reaction solution were successively added bis(pinacolato)diboron (239.81 mg, 0.944 mmol), potassium acetate (185.36 mg, 1.889 mmol), and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane complex (51.29 mg, 0.063 mmol). The reaction solution was stirred at 80 °C for 3 hours under a nitrogen atmosphere. After the reaction was complete, the reaction solution was diluted with water (10 mL) and then extracted with ethyl acetate (3 × 10 mL). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a brown solid compound 39b (314 mg, crude product), which was used directly in the next step without purification.

[0538] LC-MS: m / z 201.1 [M+H] + 。

[0539] The remaining steps were the same as in Example 9, except that compound 2-(4-cyclopropylpiperazin-1-yl)pyridine-5-boronic acid pinacol ester (9d) was replaced with 2,3'-bipyridine-5-boronic acid (39b) to obtain compound 39.

[0540] 1 1H NMR (400 MHz, methanol-d4) δ 9.33–9.30 (m, 1H), 9.29–9.24 (m, 2H), 8.62 (dd, J = 4.9, 1.6 Hz, 1H), 8.56–8.48 (m, 2H), 8.10 (d, J = 8.4 Hz, 1H), 7.63–7.57 (m, 2H), 4.09 (d, J = 11.3 Hz, 1H), 3.74 (d, J = 11.3 Hz, 1H), 2.31–2.22 (m, 1H), 1.83–1.73 (m, 1H), 1.50 (s, 3H), 1.43–1.28 (m, 4H), 0.92 (t, J = 7.0 Hz, 3H).

[0541] LC-MS: m / z 430.3 [M+H] + 。

[0542] Example 40: Preparation of (R)-2-((7-([2,4'-bipyridin]-5-yl)-2-aminopyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (40)

[0543]

[0544] The preparation method was the same as that of Example 39, except that 3-pyridineboronic acid was replaced by 4-pyridineboronic acid to obtain Compound 40.

[0545] 1 H NMR (400 MHz, methanol-d4) δ 9.35 (d, J = 2.2 Hz, 1H), 9.29 (s, 1H), 8.72–8.66 (m, 2H), 8.55 (dd, J = 8.3, 2.3 Hz, 1H), 8.19 (d, J = 8.2 Hz, 1H), 8.17–8.13 (m, 2H), 7.63 (s, 1H), 4.10 (d, J = 11.4 Hz, 1H), 3.74 (d, J = 11.3 Hz, 1H), 2.33–2.21 (m, 1H), 1.83–1.71 (m, 1H), 1.50 (s, 3H), 1.43–1.29 (m, 4H), 0.92 (t, J = 6.9 Hz, 3H).

[0546] LC-MS: m / z 430.4 [M+H] + 。

[0547] Example 41: Preparation of (R)-2-((2-amino-7-(6'-methyl-[2,3'-bipyridin]-5-yl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (41)

[0548]

[0549]

[0550] The preparation method was the same as that of Example 39, except that compound 3-pyridineboronic acid was replaced by 6-methylpyridine-3-boronic acid to obtain Compound 41.

[0551] 1 H NMR (400 MHz, methanol-d4) δ 9.38 (s, 1H), 9.25 (s, 1H), 9.02 (s, 1H), 8.46 (d, J = 7.9 Hz, 1H), 8.34 (d, J = 8.0 Hz, 1H), 7.96 (d, J = 7.9 Hz, 1H), 7.66 (s, 1H), 7.40 (d, J = 8.0 Hz, 1H), 4.18 (d, J = 11.4 Hz, 1H), 3.76 (d, J = 11.4 Hz, 1H), 2.59 (s, 3H), 2.33–2.19 (m, 1H), 1.85–1.71 (m, 1H), 1.54 (s, 3H), 1.47–1.24 (m, 4H), 0.93 (t, J = 6.9 Hz, 3H).

[0552] LC-MS: m / z 444.3 [M+H] + 。

[0553] Example 42: Preparation of (R)-2-((7-([2,2'-bipyridin]-5-yl)-2-aminopyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (42)

[0554]

[0555] The preparation method was the same as that of Example 39, except that compound 5-bromo-2,3'-bipyridine (39a) was replaced with 5-bromo-2,2'-bipyridine (42a) to obtain compound 42.

[0556] 1 1H NMR (300 MHz, Methanol-d4) δ 9.35–9.25 (m, 2H), 8.75–8.65 (m, 1H), 8.60–8.51 (m, 1H), 8.51–8.39 (m, 2H), 8.00 (t, J = 7.5 Hz, 1H), 7.65 (s, 1H), 7.56–7.43 (m, 1H), 4.12 (d, J = 11.3 Hz, 1H), 3.77 (d, J = 11.3 Hz, 1H), 2.37–2.17 (m, 1H), 1.88–1.73 (m, 1H), 1.52 (s, 3H), 1.47–1.30 (m, 4H), 0.94 (t, J = 6.8 Hz, 3H).

[0557] LC-MS: m / z 430.1 [M+H] + 。

[0558] Example 43: Preparation of (R)-2-((2-amino-7-(4-(pyrrolidin-1-ylmethyl)benzyl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (43)

[0559]

[0560] Step 1: (R)-N 4 -(1-((tert-butyldimethylsilyl)oxy)-2-methylhexan-2-yl)-7-(4-chlorobenzyl)-N 2 -(2,4-dimethoxybenzyl)pyrido[4,3-d]pyrimidine-2,4-diamine (43a) Preparation

[0561] At room temperature, dissolve compound 2f (150 mg, 0.26 mmol) in 1,4-dioxane (2 mL) and water (0.5 mL). Sequentially add 4-chlorobenzylboronic acid pinacol ester (CAS: 475250-49-8) (43b) (264 mg, 1.05 mmol), potassium carbonate (255 mg, 1.83 mmol), and tetrakis(triphenylphosphine)palladium (60 mg, 0.052 mmol). Stir the reaction mixture at 95 °C under a nitrogen atmosphere for 3 hours. After completion of the reaction, dilute the reaction mixture with water (15 mL), extract with ethyl acetate (3 × 10 mL), combine the organic phases, wash with saturated brine (30 mL), dry over anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify the obtained crude product by preparative thin-layer chromatography (mobile phase: ethyl acetate / petroleum ether = 1:2) to obtain compound 43a as an orange oil (92 mg, 53.01%).

[0562] LC-MS: m / z 664.3 [M+H] + 。

[0563] Step 2: Preparation of (R)-N 4 -(1-((tert-Butyldimethylsilyl)oxy)-2-methylhexan-2-yl)-N 2 -(2,4-dimethoxybenzyl)-7-(4-(pyrrolidin-1-ylmethyl)benzyl)pyrido[4,3-d]pyrimidine-2,4-diamine (43c)

[0564] At room temperature, dissolve compound 43a (92 mg, 0.14 mmol) in 1,4-dioxane (2 mL) and water (0.5 mL). Sequentially add potassium (1-pyrrolidinylmethyl)trifluoroborate (37 mg, 0.19 mmol), potassium carbonate (57.8 mg, 0.41 mmol), and XPhos Pd G3 (11.7 mg, 0.014 mmol). Stir the reaction mixture at 100 °C under a nitrogen atmosphere overnight. After completion of the reaction, dilute the reaction mixture with water (15 mL), extract with ethyl acetate (3 × 10 mL), combine the organic phases, wash with saturated brine (30 mL), dry over anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify the obtained crude product by preparative thin-layer chromatography (mobile phase: ethyl acetate / petroleum ether = 1:2) to obtain compound 43b as an orange oil (33 mg, 33.4%).

[0565] LC-MS: m / z 713.4 [M+H] + 。

[0566] Step 3: Preparation of (R)-2-((2-amino-7-(4-(pyrrolidin-1-ylmethyl)benzyl)pyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (43)

[0567] At room temperature, compound 43b (33 mg, 0.046 mmol) was dissolved in dichloromethane (1 mL), and trifluoroacetic acid (1 mL) was slowly added dropwise. The reaction mixture was stirred at 40 °C overnight. After completion of the reaction, the reaction mixture was concentrated under reduced pressure, and the obtained crude product was separated and purified by preparative chromatography column (column type: XBridge Shield RP18 OBD column, 5 μm, 19 * 150 mm; mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 11% - 47% acetonitrile in 8 minutes; detection wavelength: 254 / 220 nm), to obtain white solid compound 43 (1.6 mg, 7.41%).

[0568] 1 H NMR (400 MHz, methanol-d4) δ 8.97 (s, 1H), 7.26–7.16 (m, 4H), 6.79 (s, 1H), 4.50 (s, 2H), 4.02 (s, 2H), 3.95 (d, J = 11.2 Hz, 1H), 3.60 (d, J = 11.3 Hz, 1H), 3.42–3.34 (m, 2H), 3.08–2.99 (m, 2H), 2.19–2.08 (m, 1H), 1.82–1.72 (m, 4H), 1.68–1.59 (m, 1H), 1.35 (s, 3H), 1.31–1.19 (m, 4H), 0.80 (t, J = 6.9 Hz, 3H).

[0569] LC-MS: m / z 449.3 [M + H] + 。

[0570] Example 44: N 4 -propylpyrido[3,4-d]pyrimidine-2,4-diamine (44) Preparation

[0571]

[0572] Step 1: Preparation of 2-aminopyrido[3,4-d]pyrimidin-4(3H)-one (44b)

[0573] At room temperature, 3-bromoisonicotinic acid 44a (5.0 g, 24.75 mmol) was dissolved in N,N-dimethylformamide (120 mL). Cesium carbonate (16.1 g, 49.50 mmol), copper(I) iodide (0.94 g, 4.95 mmol), and guanidine hydrochloride (2.60 g, 27.23 mmol) were added successively. The reaction mixture was stirred at 110 °C overnight under a nitrogen atmosphere. After completion of the reaction, the solids in the system were filtered, and the filter cake was washed with methanol (3 × 100 mL). The collected filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (mobile phase: methanol / dichloromethane = 20%-30%) to obtain a pale yellow solid compound 44b (3.2 g, 79.4%).

[0574] LC-MS: m / z 163.1 [M+H] + 。

[0575] Step 2: Preparation of N 4 -propylpyrido[3,4-d]pyrimidine-2,4-diamine (44)

[0576] At room temperature, compound 44b (40 mg, 0.247 mmol) was dissolved in N,N-dimethylformamide (3 mL). Benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) (218 mg, 0.494 mmol), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (75.1 mg, 0.494 mmol), and n-propylamine (29.2 mg, 0.494 mmol) were added successively. The reaction mixture was reacted at room temperature for 4 hours under a nitrogen atmosphere. After completion of the reaction, water (10 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (3 × 20 mL). The combined organic phases were washed with saturated brine (2 × 20 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product. The crude product was purified by preparative reversed-phase chromatography column (column type: XBridge Shield RP18 OBD column, 5 μm, 19*150 mm; mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 47%-53% acetonitrile in 8 minutes; detection wavelength: 254 / 220 nm) to obtain a white solid compound 44 (10.2 mg, 20.8%).

[0577] 1 1H NMR (400 MHz, methanol-d4) δ 8.50 (s, 1H), 8.06 (d, J = 5.6 Hz, 1H), 7.69 (d, J = 5.7 Hz, 1H), 3.49–3.41 (m, 2H), 1.70–1.57 (m, 2H), 0.90 (t, J = 7.4 Hz, 3H).

[0578] LC-MS: m / z 204.2 [M+H]+ .

[0579] Example 45: Preparation of N-Butylpyrido[3,4-d]pyrimidine-2,4-diamine (45) 4

[0580]

[0581] Compound 45 was prepared in the same manner as in Example 44, except that propylamine was replaced with butylamine.

[0582] 1 H NMR (400 MHz, methanol-d4) δ 8.50 (d, J = 0.9 Hz, 1H), 8.06 (d, J = 5.6 Hz, 1H), 7.69 (dd, J = 5.6, 0.9 Hz, 1H), 3.49 (t, J = 7.3 Hz, 2H), 1.67–1.55 (m, 2H), 1.41–1.29 (m, 2H), 0.89 (t, J = 7.4 Hz, 3H).

[0583] LC-MS: m / z 218.2 [M+H] + .

[0584] Example 46: Preparation of N-Pentylpyrido[3,4-d]pyrimidine-2,4-diamine (46) 4

[0585]

[0586] Compound 46 was prepared in the same manner as in Example 44, except that propylamine was replaced with pentylamine.

[0587] 1 H NMR (400 MHz, methanol-d4) δ 8.50 (d, J = 0.9 Hz, 1H), 8.06 (dd, J = 5.6, 0.8 Hz, 1H), 7.69 (dd, J = 5.6, 0.9 Hz, 1H), 3.51–3.44 (m, 2H), 1.69–1.56 (m, 2H), 1.37–1.25 (m, 4H), 0.91–0.78 (m, 3H).

[0588] LC-MS: m / z 232.2 [M+H] + .

[0589] Example 47: Preparation of 2-((2-Aminopyrido[3,4-d]pyrimidin-4-yl)amino)-1-pentanol (47)

[0590]

[0591] ​​The preparation method was the same as that of Example 44, except that n-propylamine was replaced by DL-2-amino-1-pentanol to obtain Compound 47.

[0592] 1 H NMR (400 MHz, methanol-d4) δ 8.50 (s, 1H), 8.07 (d, J = 5.7 Hz, 1H), 7.80 (dd, J = 5.6, 0.9 Hz, 1H), 4.47–4.39 (m, 1H), 3.58 (dd, J = 5.4, 1.4 Hz, 2H), 1.68–1.51 (m, 2H), 1.43–1.28 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H).

[0593] LC-MS: m / z 248.2 [M+H] + 。

[0594] Example 48: Preparation of 2-((2-aminopyrido[3,4-d]pyrimidin-4-yl)amino)-1-hexanol (48)

[0595]

[0596] The preparation method was the same as that of Example 44, except that n-propylamine was replaced by DL-2-amino-1-hexanol to obtain Compound 48.

[0597] 1 H NMR (400 MHz, methanol-d4) δ 8.51 (d, J = 0.9 Hz, 1H), 8.07 (d, J = 5.6 Hz, 1H), 7.80 (dd, J = 5.6, 0.9 Hz, 1H), 4.45–4.37 (m, 1H), 3.58 (dd, J = 5.4, 1.1 Hz, 2H), 1.73–1.62 (m, 1H), 1.62–1.51 (m, 1H), 1.51–1.40 (m, 1H), 1.36–1.22 (m, 3H), 0.85–0.78 (m, 3H).

[0598] LC-MS: m / z 262.2 [M+H] + 。

[0599] Example 49: Preparation of (R)-2-((2-aminopyrido[3,4-d]pyrimidin-4-yl)amino)-2-methylbutan-1-ol (49)

[0600]

[0601] The preparation method was the same as that of Example 44, except that n-propylamine was replaced by (R)-2-amino-2-methylbutan-1-ol (49a) to obtain Compound 49.

[0602] 1 1H NMR (400 MHz, methanol-d4) δ 8.79 (s, 1H), 8.53 (d, J = 5.6 Hz, 1H), 8.19 (d, J = 5.6 Hz, 1H), 4.16 (d, J = 11.4 Hz, 1H), 3.73 (d, J = 11.4 Hz, 1H), 2.37–2.22 (m, 1H), 1.89–1.73 (m, 1H), 1.52 (s, 3H), 0.93 (t, J = 7.5 Hz, 3H).

[0603] LC-MS: m / z 248.3 [M+H] + 。

[0604] Example 50: Preparation of (R)-2-((2-aminopyrido[3,4-d]pyrimidin-4-yl)amino)-2-methylpentan-1-ol (50)

[0605]

[0606] Compound 50 was prepared in the same manner as in Example 44, except that n-propylamine was replaced with (R)-2-amino-2-methylpentan-1-ol (50a).

[0607] 1 1H NMR (400 MHz, methanol-d4) δ 8.59 (d, J = 0.9 Hz, 1H), 8.16 (d, J = 5.6 Hz, 1H), 7.90 (dd, J = 5.6, 0.9 Hz, 1H), 4.05 (d, J = 11.4 Hz, 1H), 3.73 (d, J = 11.3 Hz, 1H), 2.23–2.13 (m, 1H), 1.81–1.69 (m, 1H), 1.47 (s, 3H), 1.44–1.28 (m, 2H), 0.94 (t, J = 7.3 Hz, 3H).

[0608] LC-MS: m / z 262.2 [M+H] + 。

[0609] Example 51: Preparation of (R)-2-((2-aminopyrido[3,4-d]pyrimidin-4-yl)amino)-2-methylheptan-1-ol (51)

[0610]

[0611] Compound 51 was prepared in the same manner as in Example 44, except that n-propylamine was replaced with (R)-2-amino-2-methylheptan-1-ol (51a).

[0612] 11H NMR (400 MHz, methanol-d4) δ 8.59 (s, 1H), 8.16 (d, J = 5.6 Hz, 1H), 7.90 (d, J = 5.3 Hz, 1H), 4.06 (d, J = 11.3 Hz, 1H), 3.72 (d, J = 11.3 Hz, 1H), 2.28–2.19 (m, 1H), 1.80–1.72 (m, 1H), 1.47 (s, 3H), 1.34–1.25 (m, 6H), 0.90–0.83 (m, 3H).

[0613] LC-MS: m / z 290.2 [M+H] + 。

[0614] Example 52 and Example 53: Preparation of (R)-2-((2-aminopyrido[3,4-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (52) and (S)-2-((2-aminopyrido[3,4-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (53)

[0615]

[0616] Step 1: Preparation of (R)-N-((1-((tert-butyldimethylsilyl)oxy)-2-methylhexan-2-yl)-2-chloropyrido[3,4-d]pyrimidin-4-amine (52b)

[0617] At room temperature, 2,4-dichloropyrido[3,4-d]pyrimidine 52a (100 mg, 0.50 mmol) was dissolved in 1,4-dioxane (2.0 mL). To the reaction solution were successively added (R)-1-((tert-butyldimethylsilyl)oxy)-2-methylhexan-2-amine (1e) (123 mg, 0.50 mmol) and N,N-diisopropylethylamine (0.26 μL, 1.49 mmol), and the mixture was stirred at room temperature for 2 hours. After the reaction was complete, it was cooled to room temperature. The reaction solution was diluted with water (20 mL), and then extracted with ethyl acetate (3 × 20 mL). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was separated and purified by silica gel column chromatography (mobile phase: petroleum ether / ethyl acetate = 4 / 1) to obtain a yellow oily compound 52b (110 mg, 53.79%).

[0618] LC-MS: m / z 409.1 [M+H] + 。

[0619] Step 2: (R)-N 4 -(1-((tert-butyldimethylsilyl)oxy)-2-methylhexan-2-yl)-N 2Preparation of -(2,4-dimethoxybenzyl)pyrido[3,4-d]pyrimidine-2,4-diamine (52c)

[0620] At room temperature, compound 52b (110 mg, 0.27 mmol) was dissolved in 2,4-dimethoxybenzylamine (1.0 mL). N,N-Diisopropylethylamine (140 μl, 0.80 mmol) was added to the reaction solution, and the mixture was stirred at 80 °C for 2 hours. After the reaction was complete, it was cooled to room temperature. The reaction solution was diluted with water (20 mL), extracted with ethyl acetate (3 × 20 mL), the combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was separated and purified by silica gel column chromatography (mobile phase: petroleum ether / ethyl acetate = 3:2) to obtain the yellow oily compound 52c (114 mg, 78.53%).

[0621] LC-MS: m / z 508.3 [M+H] + 。

[0622] Step 3: Preparation of (R)-2-((2-aminopyrido[3,4-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (52d)

[0623] At room temperature, compound 52c (114 mg, 0.21 mmol) was dissolved in dichloromethane (0.5 mL). Trifluoroacetic acid (0.5 mL) was added to the reaction solution, and the mixture was stirred at room temperature for 12 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure, and the obtained crude product was separated and purified by a preparative chromatographic column (column type: XBridge Shield RP18 OBD column, 5um, 19*150mm; mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 20%-38% acetonitrile in 8 minutes; detection wavelength: 220 nm) to obtain the white solid compound 52d (12 mg, 20.18%).

[0624] 1 1H NMR (300 MHz, methanol-d4) δ 8.61 (d, J = 0.9 Hz, 1H), 8.18 (d, J = 5.6 Hz, 1H), 7.92 (dd, J = 5.6, 0.9 Hz, 1H), 4.09 (d, J = 11.3 Hz, 1H), 3.74 (d, J = 11.2 Hz, 1H), 2.32–2.18 (m, 1H), 1.86–1.71 (m, 1H), 1.49 (s, 3H), 1.41–1.28 (m, 4H), 0.92 (t, J = 6.9 Hz, 3H).

[0625] LC-MS: m / z 275.9 [M+H]+ .

[0626] Step 4: Preparation of (R)-2-((2-aminopyrido[3,4-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (52) and (S)-2-((2-aminopyrido[3,4-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (53)

[0627] Compound 52d was subjected to chiral separation (column type: OptiChiral A6-5 column, 3 * 25 cm, 5 μm; mobile phase A: carbon dioxide, mobile phase B: methanol (2M ammonia methanol solution with 0.1%); flow rate: 50 mL / min; gradient: 50% B isocratic; detection wavelength: 220 nm), to obtain the white solid product compound 52, t R = 2.052 min, ee value (enantiomeric excess): 100%; and the white solid product compound 53, t R = 2.383 min, ee value (enantiomeric excess): 92.8%.

[0628] Compound 52:

[0629] 1 H NMR (300 MHz, methanol-d4) δ 8.61 (s, 1H), 8.17 (d, J = 5.6 Hz, 1H), 7.91 (d, J = 5.6 Hz, 1H), 4.08 (d, J = 11.3 Hz, 1H), 3.74 (d, J = 11.3 Hz, 1H), 2.33–2.15 (m, 1H), 1.89–1.71 (m, 1H), 1.49 (s, 3H), 1.44–1.21 (m, 4H), 0.98–0.82 (m, 3H).

[0630] LC-MS: m / z 276.3 [M+H] + .

[0631] Compound 53:

[0632] 1 H NMR (400 MHz, methanol-d4) δ 8.64 (s, 1H), 8.26 (d, J = 5.6 Hz, 1H), 7.97 (d, J = 5.6 Hz, 1H), 4.12–4.07 (m, 1H), 3.72 (d, J = 11.2 Hz, 1H), 2.29–2.17 (m, 1H), 1.82–1.71 (m, 1H), 1.49 (s, 3H), 1.38–1.29 (m, 4H), 0.89 (t, J = 6.9 Hz, 3H).

[0633] LC-MS: m / z 276.2 [M+H]+ 。

[0634] Example 54: Preparation of 2-((2-aminopyrido[3,4-d]pyrimidin-4-yl)amino)-1-heptanol (54)

[0635]

[0636] The same procedures as Steps 1 to 3 of Example 52 were carried out, except that (R)-1-((tert-butyldimethylsilyl)oxy)-2-methylhexan-2-amine (1e) was replaced by 2-amino-1-heptanol to obtain compound 54.

[0637] 1 H NMR (300 MHz, methanol-d4) δ 8.62 (d, J = 0.9 Hz, 1H), 8.18 (d, J = 5.6 Hz, 1H), 7.91 (dd, J = 5.6, 0.9 Hz, 1H), 4.59–4.46 (m, 1H), 3.74–3.68 (m, 2H), 1.87–1.59 (m, 2H), 1.50–1.27 (m, 6H), 0.97–0.84 (m, 3H).

[0638] LC-MS: m / z 275.9 [M+H] + 。

[0639] Example 55: Preparation of (R)-2-((2-amino-8-fluoropyrido[3,4-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (55)

[0640]

[0641] Step 1: Preparation of 2-amino-8-fluoropyrido[3,4-d]pyrimidin-4(3H)-one (55b)

[0642] At room temperature, 3-chloro-2-fluoroisonicotinic acid (55a) (1.0 g, 5.70 mmol) was dissolved in N,N-dimethylformamide (15 mL). Cesium carbonate (5.57 g, 17.1 mmol), guanidine carbonate (1.03 g, 5.72 mmol), and copper(I) iodide (217 mg, 1.14 mmol) were successively added, and the reaction mixture was stirred at 110 °C for 4 hours under a nitrogen atmosphere. After completion of the reaction, the reaction was quenched by adding water (100 mL). The system was extracted with chloroform (3 × 50 mL), and the combined organic phases were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was separated and purified by silica gel column chromatography (mobile phase: methanol / dichloromethane = 0-10%), and a pale yellow solid 55b (83.9 mg, 8.2%) was obtained.

[0643] LC-MS: m / z 181.0 [M+H] + .

[0644] Step 2: (R)-N 4 Preparation of -(1-((tert-butyldimethylsilyl)oxy)-2-methylhexan-2-yl)-8-fluoropyrido[3,4-d]pyrimidine-2,4-diamine (55c)

[0645] Compound 55b (30 mg, 0.167 mmol) was dissolved in N,N-dimethylformamide (5 mL) at room temperature, and (R)-1-((tert-butyldimethylsilyl)oxy)-2-methylhexan-2-amine (1e) (122 mg, 0.50 mmol), 1,8-diazabicycloundec-7-ene (76.1 mg, 0.50 mmol), and Carter condensation agent (110 mg, 0.25 mmol) were added in sequence. The reaction solution was stirred at room temperature overnight under nitrogen atmosphere. After the reaction was completed, water (50 mL) was added to quench the reaction, and ethyl acetate (3×20 mL) was added to the system for extraction. The combined organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was separated and purified by silica gel column chromatography (mobile phase: ethyl acetate / petroleum ether = 15-30%) to obtain white solid compound 55c (10 mg, 14.7%).

[0646] LC-MS: m / z 408.3 [M+H] + .

[0647] Step 3: Preparation of (R)-2-((2-amino-8-fluoropyrido[3,4-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (55)

[0648] Compound 55c (10 mg, 0.025 mmol) was dissolved in a mixture of trifluoroacetic acid (2 mL) and dichloromethane (2 mL) at room temperature. The reaction solution was stirred at 45°C for 2 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure, the crude product was diluted with water (30 mL), and dichloromethane (3×15 mL) was added to the system for extraction. The combined organic phase was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by preparative reverse phase chromatography (column type: XBridge ShieldRP18 OBD column, 5um, 19*150mm; mobile phase A: water (10mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 25mL / min; gradient: 25%-42% acetonitrile in 8 minutes; detection wavelength: 254 / 220nm) to obtain a white solid product 55 (1.4 mg, 19.4%).

[0649] 11H NMR (400 MHz, methanol-d4) δ 7.69 (d, J = 5.7 Hz, 1H), 7.59 (dd, J = 5.7, 1.3 Hz, 1H), 3.98 (d, J = 11.2 Hz, 1H), 3.61 (d, J = 11.2 Hz, 1H), 2.20–2.06 (m, 1H), 1.72–1.60 (m, 1H), 1.37 (s, 3H), 1.32–1.14 (m, 4H), 0.80 (t, J = 7.0 Hz, 3H).

[0650] LC-MS: m / z 293.9 [M+H] + 。

[0651] Example 56: Preparation of (R)-2-((2-amino-5-fluoropyrido[3,4-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (56)

[0652]

[0653] Step 1: Preparation of 3-amino-5-fluoropyridine-4-carboxamide (56b)

[0654] At room temperature, ammonia water (30 mL) was added to methyl 3-amino-5-fluoropyridine-4-carboxylate (56a) (900 mg, 5.29 mmol). The reaction was stirred at 60 °C for 3 hours. After the reaction was completed, the reaction solution was cooled to room temperature, diluted with water, and ethyl acetate (3 × 50 mL) was added to the system for extraction. The combined organic phases were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was separated and purified by silica gel column chromatography (mobile phase: ethyl acetate / petroleum ether = 1:1) to obtain the yellow solid compound 56b (700 mg, 84.7%).

[0655] LC-MS: m / z 156.0 [M+H] + 。

[0656] Step 2: Preparation of 5-fluoropyrido[3,4-d]pyrimidine-2,4(1H,3H)-dione (56c)

[0657] At room temperature, compound 56b (700 mg, 4.49 mmol) was dissolved in 1,4-dioxane (15 mL), and phosgene (3.09 g, 15.7 mmol) was added. The reaction mixture was stirred at 120 °C for 16 h. After completion of the reaction, the reaction mixture was cooled to room temperature, quenched with water, and then extracted with ethyl acetate (3 × 40 mL). The combined organic phases were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product obtained was separated and purified by silica gel column chromatography (mobile phase: ethyl acetate / petroleum ether = 1:1) to give yellow solid compound 56c (350 mg, 43.1%).

[0658] LC-MS: m / z 182.0 [M+H] + 。

[0659] Step 3: Preparation of 2,4-dichloro-5-fluoropyrido[3,4-d]pyrimidine (56d)

[0660] At 0 °C, compound 56c (350 mg, 1.93 mmol) was mixed with phosphorus oxychloride (5.0 mL), and N,N-diisopropylethylamine (1.24 g, 9.65 mmol) was added. The reaction mixture was stirred at 100 °C for 0.5 h. After completion of the reaction, the reaction mixture was cooled to room temperature and concentrated under reduced pressure to give brown crude compound 56d (70.0 mg, 16.7%).

[0661] LC-MS: m / z 218.0 [M+H] + 。

[0662] Step 4: Preparation of (R)-N-((1-((tert-butyldimethylsilyl)oxy)-2-methylhexan-2-yl)-2-chloro-5-fluoropyrido[3,4-d]pyrimidin-4-amine (56e)

[0663] At room temperature, compound 56d (70.0 mg, 0.32 mmol) was dissolved in 1,4-dioxane (5.0 mL). (R)-1-((tert-Butyldimethylsilyl)oxy)-2-methylhexan-2-amine (1e) (94.4 mg, 0.39 mmol) and N,N-diisopropylethylamine (144 mg, 1.12 mmol) were successively added to the reaction mixture, and the mixture was reacted at room temperature for 12 h. After completion of the reaction, the reaction mixture was diluted with water (15 mL), and then extracted with ethyl acetate (3 × 10 mL). The combined organic phases were washed with saturated brine (15 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product obtained was separated and purified by preparative thin-layer chromatography (mobile phase: ethyl acetate / petroleum ether = 1:8) to give brown oily compound 56e (55.0 mg, 40.2%).

[0664] LC-MS: m / z 427.2 [M+H] + 。

[0665] Step 5: Preparation of (R)-N 4 -(1-((tert-Butyldimethylsilyl)oxy)-2-methylhexan-2-yl)-N 2 -(2,4-Dimethoxybenzyl)-5-fluoropyrido[3,4-d]pyrimidine-2,4-diamine (56f)

[0666] At room temperature, dissolve compound 56e (55 mg, 0.13 mmol) in 1,4-dioxane (5 mL). Add 2,4-dimethoxybenzylamine (21 mg, 0.15 mmol) and N,N-diisopropylethylamine (58 mg, 0.45 mmol) to the reaction solution in sequence, and stir at 100 °C for 16 hours. After the reaction is complete, dilute the reaction solution with water (15 mL), extract with ethyl acetate (3×10 mL), combine the organic phases, wash with saturated brine (15 mL), dry over anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify the obtained crude product by preparative thin-layer chromatography (mobile phase: ethyl acetate / petroleum ether = 1:10) to obtain compound 56f as a brown oil (30.0 mg, 44.3%).

[0667] LC-MS: m / z 558.3 [M+H] + 。

[0668] Step 6: Preparation of (R)-2-((2-Amino-5-fluoropyrido[3,4-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (56)

[0669] At room temperature, dissolve compound 56f (30 mg, 0.057 mmol) in dichloromethane (3 mL). Add trifluoroacetic acid (1 mL) to the reaction solution and stir at room temperature for 3 hours. After the reaction is complete, concentrate the reaction solution under reduced pressure, dilute with saturated sodium bicarbonate solution (10 mL), extract with ethyl acetate (3×10 mL), combine the organic phases, wash with saturated brine (20 mL), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify the obtained crude product by preparative chromatography column (column type: XBridge ShieldRP18 OBD column, 5um, 19*150mm; mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 25%-45% acetonitrile in 8 minutes; detection wavelength: 254 / 220 nm) to obtain compound 56 as a white solid (7.30 mg, 43.7%).

[0670] 11H NMR (400 MHz, methanol-d4) δ 8.33 (d, J = 1.2 Hz, 1H), 7.92 (d, J = 2.9 Hz, 1H), 3.81 (d, J = 11.1 Hz, 1H), 3.60 (d, J = 11.0 Hz, 1H), 2.02–1.88 (m, 1H), 1.87–1.73 (m, 1H), 1.38 (s, 3H), 1.31–1.19 (m, 4H), 0.87–0.77 (m, 3H).

[0671] LC-MS: m / z 293.9 [M+H] + 。

[0672] Example 57: Preparation of (R)-2-((2-amino-6-fluoropyrido[3,4-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (57)

[0673]

[0674] Step 1: Preparation of 5-amino-2-fluoropyridine-4-carboxamide (57b)

[0675] At room temperature, 5-amino-2-fluoroisonicotinic acid 57a (1.50 g, 9.62 mmol) was dissolved in thionyl chloride (20 mL). The reaction was refluxed at 80 °C for 1 h. After completion of the reaction, it was directly concentrated under reduced pressure. The obtained brown oily liquid compound was dissolved in tetrahydrofuran (5 mL), and slowly added dropwise to ammonia water (15 mL) at 0 °C. The reaction solution was stirred at 0 °C for 1 h. After completion of the reaction, water was added to dilute the reaction, and ethyl acetate (3 × 50 mL) was added to the system for extraction. The combined organic phases were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was separated and purified by silica gel column chromatography (mobile phase: ethyl acetate / petroleum ether = 1:1) to obtain the crude yellow solid compound 57b (960 mg, 64.4%).

[0676] LC-MS: m / z 156.0 [M+H] + 。

[0677] The remaining steps were the same as in Example 56, except that 3-amino-5-fluoropyridine-4-carboxamide (56b) was replaced by 5-amino-2-fluoropyridine-4-carboxamide (57b) to obtain compound 57.

[0678] 11H NMR (300 MHz, methanol-d4) δ 8.29 (s, 1H), 7.74 (s, 1H), 4.11 (d, J = 11.3 Hz, 1H), 3.73 (d, J = 11.3 Hz, 1H), 2.12–2.01 (m, 1H), 1.68–1.56 (m, 1H), 1.40 (s, 3H), 1.36–1.31 (m, 4H), 0.96–0.86 (m, 3H).

[0679] LC-MS: m / z 293.9 [M+H] + .

[0680] Example 58: Preparation of (R)-2-((2-amino-7-fluoropyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (58)

[0681]

[0682] Step 1: Preparation of 2-fluoro-5-iodo-4-aminopyridine (58b) and 2-fluoro-3-iodo-4-aminopyridine (58c)

[0683] At room temperature, dissolve compound 2-fluoro-4-aminopyridine 58a (3.0 g, 26.8 mmol) in ethanol (100 mL), and successively add iodine (6.79 g, 26.8 mmol) and silver sulfate (8.34 g, 26.8 mmol), and stir at room temperature for 16 hours. After the reaction is completed, filter, and directly concentrate the filtrate under reduced pressure to obtain the crude product. The crude product is separated and purified by silica gel column chromatography (mobile phase: ethyl acetate / petroleum ether = 20%-40%) to obtain compound 58b (0.96 g, 15.0%) and compound 58c (3.10 g, 48.7%).

[0684] LC-MS: m / z 58b 238.9 [M+H] + ; LC-MS: m / z 58c 238.9 [M+H] + .

[0685] Step 2: Preparation of methyl 4-amino-6-fluoronicotinate (58d)

[0686] At room temperature, compound 58b (2.33 g, 9.79 mmol) was dissolved in methanol (40 mL). Triethylamine (0.99 g, 9.79 mmol) and [1,1'-bis(diphenylphosphino)ferrocene] dichloropalladium (0.72 g, 0.98 mmol) were added successively, and the reaction was carried out at 20 atmospheres of carbon monoxide and 100 °C for 6 hours. After the reaction was completed, filtration was carried out, and the filtrate was directly concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (mobile phase: ethyl acetate / petroleum ether = 20%-40%) to obtain light yellow solid compound 58d (543 mg, 32.6%).

[0687] LC-MS: m / z 171.0 [M+H] + 。

[0688] The remaining steps were the same as in Example 56, except that 3-amino-5-fluoroisonicotinamide (56b) was replaced with methyl 4-amino-6-fluoronicotinate (58d) to obtain compound 58.

[0689] LC-MS: m / z 294.2 [M+H] + 。

[0690] 1 1H NMR (300 MHz, methanol-d4) δ 7.95 (d, J = 5.7 Hz, 1H), 7.01 (d, J = 6.0 Hz, 1H), 3.93 (d, J = 11.2 Hz, 1H), 3.71 (d, J = 11.0 Hz, 1H), 2.15–1.83 (m, 2H), 1.50 (s, 3H), 1.44–1.31 (m, 4H), 0.94 (t, J = 6.5 Hz, 3H).

[0691] Example 59: Preparation of (R)-2-((2-amino-5-fluoropyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (59)

[0692]

[0693]

[0694] Step 1: Preparation of methyl 4-amino-6-fluoronicotinate (59a)

[0695] At room temperature, 2-fluoro-3-iodo-4-aminopyridine 58c (3.10 g, 13.0 mmol) was dissolved in methanol (60 mL). Triethylamine (1.32 g, 13.0 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (1.06 g, 1.30 mmol) were added successively. The reaction was carried out at 20 atmospheres of carbon monoxide and 100 °C for 6 hours. After the reaction was completed, filtration was carried out, and the filtrate was directly concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (mobile phase: ethyl acetate / petroleum ether = 20%-40%) to obtain a light yellow solid compound 59a (1.6 g, 72.2%).

[0696] LC-MS: m / z 171.0 [M+H] +

[0697] The remaining steps were the same as in Example 56, except that 3-amino-5-fluoroisonicotinamide (56b) was replaced with methyl 4-amino-6-fluoronicotinate (59a) to obtain Compound 59.

[0698] LC-MS: m / z 294.2 [M+H] + 。

[0699] 1 1H NMR (300 MHz, methanol-d4) δ 7.94 (dd, J = 6.0, 0.8 Hz, 1H), 7.01 (dd, J = 6.0, 1.9 Hz, 1H), 3.93 (d, J = 11.1 Hz, 1H), 3.71 (d, J = 11.0 Hz, 1H), 2.14–1.99 (m, 1H), 1.98–1.83 (m, 1H), 1.50 (s, 3H), 1.45–1.25 (m, 4H), 1.02–0.87 (m, 3H).

[0700] Example 60: Preparation of 2-((2-amino-6-(4-(pyrrolidin-1-ylmethyl)benzyl)pyrido[3,4-d]pyrimidin-4-yl)amino)hexan-1-ol (60)

[0701]

[0702]

[0703] Step 1: Preparation of methyl 5-amino-2-chloroisonicotinate (60b)

[0704] At 0 °C, 5-amino-2-chloropyridine-4-carboxylic acid 60a (25 g, 145 mmol) was dissolved in methanol (300 mL), and then thionyl chloride (100 mL) was added dropwise to the above solution (maintaining the internal temperature of the reaction system at 20 - 25 °C). After the addition was completed, the temperature was raised to 70 °C and stirred overnight. The reaction system was cooled to room temperature. The mixture was concentrated under reduced pressure, and the resulting residue was slurried twice with an aqueous sodium bicarbonate solution. After filtration, the filter cake was dried to obtain a yellow solid compound 60b (16 g, 59.1%).

[0705] LC-MS: m / z 187.0 [M+H] + 。

[0706] Step 2: Preparation of 2-amino-6-chloropyrido[3,4-d]pyrimidin-4(3H)-one (60c)

[0707] At room temperature, in a 100 mL glass bottle, compound 60b (3.0 g, 16.1 mmol), chloramidine hydrochloride (3.7 g, 32.2 mmol), and dimethyl sulfone (10 g) were added in sequence. The reaction solution was heated to 150 °C and stirred for 4 hours. After the reaction was completed, the reaction solution was diluted with water (100 mL), filtered, and the obtained filter cake was washed twice with water. The obtained filter cake was dried to obtain a yellow solid compound 60c (3.7 g, crude product).

[0708] LC-MS: m / z 197.0 [M+H] + 。

[0709] Step 3: Preparation of 2-((2-amino-6-chloropyrido[3,4-d]pyrimidin-4-yl)amino)hexan-1-ol (60d)

[0710] At room temperature, compound 60c (130 mg, 0.66 mmol) and DL-2-amino-1-hexanol (155 mg, 1.32 mmol) were dissolved in N,N-dimethylformamide (2 mL). Then, Carter's condensing agent (351 mg, 0.79 mmol) and 1,8-diazabicycloundec-7-ene (302 mg, 1.98 mmol) were added to the system in sequence. The reaction solution was stirred at room temperature for 4 hours. After the reaction was completed, the reaction solution was diluted with water (30 mL), and then extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (mobile phase: petroleum ether:ethyl acetate = 1:1) to obtain a yellow oily compound 60d (120 mg, 61%).

[0711] LC-MS: m / z 296.1 [M+H] + 。

[0712] Step 4: Preparation of 2-((2-amino-6-(4-chlorobenzyl)pyrido[3,4-d]pyrimidin-4-yl)amino)hexan-1-ol (60e)

[0713] At room temperature, compound 60d (120 mg, 0.41 mmol) and 4-chlorobenzylboronic acid pinacol ester (43b) (512 mg, 2.02 mmol) were dissolved in dioxane (2 mL) and water (0.4 mL), and then potassium carbonate (168 mg, 1.21 mmol) and tetrakis(triphenylphosphine)palladium (93 mg, 0.081 mmol) were successively added to the system. The reaction mixture was stirred at 95 °C under a nitrogen atmosphere for 4 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, diluted with water (30 mL), and then extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (mobile phase: petroleum ether: ethyl acetate = 1:1) to give compound 60e as a yellow oil (60 mg, 38.3%).

[0714] LC-MS: m / z 386.2 [M+H] + 。

[0715] Step 5: Preparation of 2-((2-amino-6-(4-(pyrrolidin-1-ylmethyl)benzyl)pyrido[3,4-d]pyrimidin-4-yl)amino)hexan-1-ol (60)

[0716] At room temperature, compound 60e (60 mg, 0.155 mmol) and potassium (1-pyrrolidinylmethyl)trifluoroborate (35 mg, 0.18 mmol) were dissolved in dioxane (1 mL) and water (0.2 mL), and then Xphos Pd G3 (9.9 mg, 0.012 mmol) and potassium carbonate (64 mg, 0.46 mmol) were successively added to the system. The reaction mixture was stirred at 100 °C under a nitrogen atmosphere for 4 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, diluted with water (30 mL), and then extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was separated and purified by preparative chromatography column (column type: XBridge Shield RP18 OBD column, 5um, 19*150mm; mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 6%-18% acetonitrile in 8 minutes; detection wavelength: 220 nm) to obtain compound 60 as an off-white solid (19.5 mg, 26.4%).

[0717] 11H NMR (400 MHz, methanol-d4) δ 8.73 (s, 1H), 8.14 (s, 1H), 7.50–7.36 (m, 4H), 4.68–4.55 (m, 1H), 4.32 (s, 2H), 4.26 (s, 2H), 3.79–3.65 (m, 2H), 3.30–3.27 (m, 2H), 2.15–1.99 (m, 4H), 1.83–1.62 (m, 2H), 1.46–1.32 (m, 4H), 0.96–0.88 (m, 3H).

[0718] LC-MS: m / z 435.2 [M+H] + 。

[0719] Example 61: Preparation of (R)-2-((2-Amino-6-(4-(pyrrolidin-1-ylmethyl)benzyl)pyrido[3,4-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (61)

[0720]

[0721]

[0722] Step 1: Preparation of (R)-N 4 -(1-((tert-Butyldimethylsilyl)oxy)-2-methylhexan-2-yl)-6-chloropyrido[3,4-d]pyrimidine-2,4-diamine (61a)

[0723] At room temperature, compound 60c (300 mg, 1.52 mmol) and (R)-1-((tert-butyldimethylsilyl)oxy)-2-methylhexan-2-amine (1e) (749 mg, 3.05 mmol) were dissolved in N,N-dimethylformamide (2 mL), and then Carter's condensing agent (810 mg, 1.83 mmol) and 1,8-diazabicycloundec-7-ene (697 mg, 4.57 mmol) were added to the system in sequence. The reaction mixture was stirred at room temperature for 4 hours. After completion of the reaction, the reaction mixture was diluted with water (30 mL), and then extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (mobile phase: petroleum ether:ethyl acetate = 1:1) to give the yellow oily compound 61a (102 mg, 76%).

[0724] LC-MS: m / z 424.2 [M+H] + 。

[0725] Step 2: (R)-N 4Preparation of -(1-((tert-Butyldimethylsilyl)oxy)-2-methylhexan-2-yl)-6-(4-chlorobenzyl)pyrido[3,4-d]pyrimidine-2,4-diamine (61b)

[0726] At room temperature, compound 61a (100 mg, 0.24 mmol) and 4-chlorobenzylboronic acid pinacol ester (43b) (297 mg, 1.17 mmol) were dissolved in dioxane (2 mL) and water (0.4 mL), and then tetrakis(triphenylphosphine)palladium (54 mg, 0.04 mmol) and potassium carbonate (97 mg, 0.7 mmol) were successively added to the system. The reaction mixture was stirred at 95 °C under a nitrogen atmosphere for 4 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, diluted with water (30 mL), and then extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (mobile phase: petroleum ether: ethyl acetate = 1:1) to give the yellow oily compound 61b (62 mg, 51%).

[0727] LC-MS: m / z 514.3 [M+H] + 。

[0728] Step 3: Preparation of (R)-N 4 -(1-((tert-Butyldimethylsilyl)oxy)-2-methylhexan-2-yl)-6-(4-(pyrrolidin-1-ylmethyl)benzyl)pyrido[3,4-d]pyrimidine-2,4-diamine (61c)

[0729] At room temperature, compound 61b (60 mg, 0.12 mmol) and potassium (1-pyrrolidinylmethyl)trifluoroborate (26 mg, 0.14 mmol) were dissolved in dioxane (1 mL) and water (0.2 mL), and then potassium carbonate (48 mg, 0.35 mmol) and Xphos PdG3 (7.4 mg, 0.009 mmol) were successively added to the system. The reaction mixture was stirred at 100 °C under a nitrogen atmosphere for 4 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, diluted with water (30 mL), and then extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (mobile phase: petroleum ether: ethyl acetate = 2:1) to give the yellow oily compound 61c (8 mg, 12%).

[0730] LC-MS: m / z 563.4 [M+H] + 。

[0731] Step 4: Preparation of (R)-2-((2-amino-6-(4-(pyrrolidin-1-ylmethyl)benzyl)pyrido[3,4-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (61)

[0732] At room temperature, dissolve compound 61c (8 mg, 0.014 mmol) in dichloromethane (1.0 mL). Add trifluoroacetic acid (0.5 mL) to the reaction solution and stir at room temperature for 1 hour. After the reaction is complete, concentrate the reaction solution under reduced pressure, dilute it with saturated sodium bicarbonate solution (10 mL), extract it with ethyl acetate (3×10 mL), combine the organic phases, wash them with saturated brine (20 mL), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. The obtained crude product is separated and purified by a preparative chromatographic column (column type: XBridgeShield RP18 OBD column, 5 μm, 19*150 mm; mobile phase A: water (0.05% ammonia water), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 30% - 65% acetonitrile in 8 minutes; detection wavelength: 220 nm) to obtain white solid compound 61 (1.2 mg, 8.3%).

[0733] 1 H NMR (400 MHz, methanol-d4) δ 8.73 (s, 1H), 8.24 (s, 1H), 7.42 (q, J = 8.1 Hz, 4H), 4.32 (s, 2H), 4.26 (s, 2H), 4.17 (d, J = 11.4 Hz, 1H), 3.70 (d, J = 11.4 Hz, 1H), 3.30–3.27 (m, 4H), 2.29–2.19 (m, 1H), 2.13–2.01 (m, 4H), 1.83–1.72 (m, 1H), 1.52 (s, 3H), 1.40–1.29 (m, 4H), 0.90 (t, J = 6.9 Hz, 3H).

[0734] LC-MS: m / z 449.2 [M+H] + 。

[0735] Example 62: Preparation of (R)-2-((2-amino-6-(4-((dimethylamino)methyl)benzyl)pyrido[3,4-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (62)

[0736]

[0737] Step 1: Preparation of (R)-2-((2-amino-6-chloropyrido[3,4-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (62a)

[0738] At room temperature, compound 60c (300 mg, 1.56 mmol), (R)-2-amino-2-methylhexan-1-ol (300 mg, 2.27 mmol), Carter's condensing agent (810 mg, 1.83 mmol), 1,8-diazabicycloundec-7-ene (697 mg, 4.59 mmol) were dissolved in N,N-dimethylformamide (5 mL). The reaction mixture was stirred at room temperature for 12 hours. After the reaction was completed, water (20 mL) was added to dilute the reaction, and the system was extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained yellow crude product was separated and purified by silica gel column chromatography (mobile phase: methanol / dichloromethane / triethylamine = 1 / 12 / 0.1) to give yellow solid compound 62a (300 mg, 62.03%).

[0739] LC-MS: m / z 310.1 [M+H] + 。

[0740] Step 2: Preparation of (R)-2-((2-amino-6-(4-chlorobenzyl)pyrido[3,4-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (62b)

[0741] At room temperature, compound 62a (150 mg, 0.48 mmol), 4-chlorobenzylboronic acid pinacol ester (43b) (245 mg, 0.97 mmol), potassium carbonate (201 mg, 1.45 mmol), tetrakis(triphenylphosphine)palladium were dissolved in 1,4-dioxane (2 mL) and water (0.5 mL). The reaction mixture was stirred at 95 °C under a nitrogen atmosphere for 12 hours. After the reaction was completed, water (20 mL) was added to dilute the reaction, and the system was extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained yellow crude product was separated and purified by silica gel column chromatography (mobile phase: methanol / dichloromethane / triethylamine = 1 / 13 / 0.1) to give yellow solid compound 62b (30 mg, 15.53%).

[0742] LC-MS: m / z 400.2 [M+H] + 。

[0743] Step 3: Preparation of (R)-2-((2-amino-6-(4-((dimethylamino)methyl)benzyl)pyrido[3,4-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (62)

[0744] At room temperature, compound 62b (20 mg, 0.05 mmol), potassium (dimethylaminomethyl)trifluoroborate (165 mg, 0.08 mmol), cesium carbonate (49 mg, 0.15 mmol), 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl (5 mg, 0.01 mmol), and palladium(II) acetate (1 mg, 0.005 mmol) were dissolved in 1,4-dioxane (1 mL) and water (0.2 mL). The reaction mixture was purged with nitrogen three times and stirred at 100 °C for 12 h. After completion of the reaction, the reaction was diluted with water (20 mL), and the system was extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting crude product was separated and purified by preparative chromatography (column type: XBridge Shield RP18 OBD column, 10 μm, 19*250 mm; mobile phase A: water (0.05% ammonia), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 30%-55% acetonitrile in 8 min; detection wavelength: 254 / 220 nm). The product was collected and lyophilized to give compound 62 as a white oil (1.1 mg, 5.21%).

[0745] 1 H NMR (400 MHz, methanol-d4) δ 8.44 (d, J = 0.8 Hz, 1H), 7.80 (d, J = 0.8 Hz, 1H), 7.15 (s, 4H), 4.07 (s, 2H), 3.95 (d, J = 11.3 Hz, 1H), 3.61 (d, J = 11.2 Hz, 1H), 3.56–3.53 (m, 1H), 3.40 (s, 2H), 2.16 (s, 6H), 2.14–2.07 (m, 1H), 1.72–1.59 (m, 1H), 1.36 (s, 3H), 1.28–1.15 (m, 4H), 0.79 (t, J = 7.0 Hz, 3H).

[0746] LC-MS: m / z 423.3 [M+H] + 。

[0747] Example 63: Preparation of (R)-2-((2-Amino-6-methylpyrido[3,4-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (63)

[0748]

[0749] At room temperature, compound 62a (50.0 mg, 0.161 mmol), trimethylcyclotriboroxane (50 wt% in THF, 60.78 mg, 0.242 mmol), cesium carbonate (157.76 mg, 0.484 mmol), and tetrakis(triphenylphosphine)palladium (37.30 mg, 0.032 mmol) were dissolved in 1,4-dioxane (1.8 mL) and water (0.2 mL). The reaction mixture was stirred at 100 °C under a nitrogen atmosphere for 12 hours. After completion of the reaction, the reaction mixture was concentrated under reduced pressure, and the residue was separated and purified by preparative thin-layer chromatography (mobile phase: ethyl acetate / methanol = 30:1). The obtained crude product was further separated and purified by a preparative chromatographic column (column type: XSelect CSH Prep C18 OBD column, 5 μm, 19*150 mm; mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 10% - 22% acetonitrile in 8 minutes; detection wavelength: 254 / 220 nm). The product was collected and freeze-dried to obtain the formate of compound 63 as a yellow-green solid (13.8 mg, 25.49%).

[0750] 1 H NMR (400 MHz, methanol-d4) δ 8.88 (br s, 1H), 8.23 (s, 1H), 4.17 (d, J = 11.3 Hz, 1H), 3.70 (d, J = 11.3 Hz, 1H), 2.64 (s, 3H), 2.32–2.18 (m, 1H), 1.83–1.71 (m, 1H), 1.53 (s, 3H), 1.42–1.28 (m, 4H), 0.91 (t, J = 6.9 Hz, 3H).

[0751] LC-MS: m / z 290.3 [M+H] + 。

[0752] Example 64: Preparation of (R)-2-((2-Amino-6-ethylpyrido[3,4-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (64)

[0753]

[0754]

[0755] Compound 64 was prepared in the same manner as in Example 63, except that trimethylcyclotriboroxane was replaced with ethylboronic acid.

[0756] 11H NMR (300 MHz, methanol-d4) δ 8.65 (s, 1H), 8.03 (s, 1H), 4.17 (d, J = 11.3 Hz, 1H), 3.73 (d, J = 11.3 Hz, 1H), 2.92 (q, J = 7.6 Hz, 2H), 2.36–2.19 (m, 1H), 1.86–1.71 (m, 1H), 1.53 (s, 3H), 1.41–1.35 (m, 4H), 0.93 (t, J = 7.1 Hz, 3H).

[0757] LC-MS: m / z 303.9 [M+H] + 。

[0758] Example 65: Preparation of (R)-2-((2-amino-6-(dimethylamino)pyrido[3,4-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (65)

[0759]

[0760] At room temperature, compound 62a (40 mg, 0.129 mmol), dimethylamine hydrochloride (15.79 mg, 0.194 mmol), cesium carbonate (168.27 mg, 0.516 mmol), Pd-PEPPSI-IPent Cl -o-methylpyridine (CAS 1612891-29-8, 10.85 mg, 0.013 mmol) were dissolved in 1,4-dioxane (1.5 mL). The reaction mixture was stirred at 90 °C for 12 h under a nitrogen atmosphere. After completion of the reaction, the reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (3 × 20 mL). The combined organic phases were washed with saturated brine (40 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was separated and purified by a preparative chromatographic column (column type: XBridge Prep C18 OBD column, 5 μm, 19*150 mm; mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 25%-40% acetonitrile in 8 minutes; detection wavelength: 254 / 220 nm). The product was collected and lyophilized to give the yellow-green solid compound 65 (2.3 mg, 5.43%).

[0761] 11H NMR (400 MHz, methanol-d4) δ 8.34 (d, J = 0.9 Hz, 1H), 6.98 (d, J = 0.9 Hz, 1H), 4.04 (d, J = 11.3 Hz, 1H), 3.72 (d, J = 11.3 Hz, 1H), 3.10 (s, 6H), 2.31–2.18 (m, 1H), 1.83–1.72 (m, 1H), 1.47 (s, 3H), 1.43–1.26 (m, 4H), 0.90 (t, J = 7.1 Hz, 3H).

[0762] LC-MS: m / z 319.2 [M+H] + 。

[0763] Examples 66 and 67: Preparation of (R)-2-((2-aminopyrimido[5,4-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (66) and (R)-2-((6-aminopyrimido[5,4-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (67)

[0764]

[0765] Step 1: Preparation of (R)-N-((1-((tert-butyldimethylsilyl)oxy)-2-methylhexan-2-yl)-2,6,8-trichloropyrimido[5,4-d]pyrimidin-4-amine (66b)

[0766] At room temperature, 2,4,6,8-tetrachloropyrimido[5,4-d]pyrimidine 66a (700 mg, 2.59 mmol) was dissolved in anhydrous tetrahydrofuran (5 mL). After cooling to -78 °C, N,N-diisopropylethylamine (402 mg, 3.11 mmol) and (R)-1-((tert-butyldimethylsilyl)oxy)-2-methylhexan-2-amine (1e) (637 mg, 2.59 mmol) were added successively. The reaction mixture was stirred at -78 °C for 1 h under a nitrogen atmosphere. After completion of the reaction, the reaction was quenched with water (20 mL). The system was extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product 66b as a pale yellow solid (510 mg, 41.1%).

[0767] LC-MS: m / z 478.1 [M+H] + 。

[0768] Step 2: Preparation of (R)-N-((1-((tert-butyldimethylsilyl)oxy)-2-methylhexan-2-yl)-2,6-dichloropyrimido[5,4-d]pyrimidin-4-amine (66c)

[0769] At room temperature, compound 66b (480 mg, 1.00 mmol) was dissolved in methanol (5 mL), and 10% palladium on carbon (48 mg, water content 10%) was added successively. The reaction system was stirred at room temperature under a hydrogen atmosphere for 1 hour. After the reaction was completed, the reaction solution was filtered and concentrated to obtain a white solid compound 66c (300 mg, 67.3%).

[0770] LC-MS: m / z 444.2 [M+H] + 。

[0771] Step 3: Preparation of (R)-N 4 -(1-((tert-Butyldimethylsilyl)oxy)-2-methylhexan-2-yl)-6-chloro-N 2 -(2,4-dimethoxybenzyl)pyrimido[5,4-d]pyrimidine-2,4-diamine (66d) and (R)-N 8 -(1-((tert-Butyldimethylsilyl)oxy)-2-methylhexan-2-yl)-6-chloro-N 2 -(2,4-dimethoxybenzyl)pyrimido[5,4-d]pyrimidine-2,8-diamine (67a)

[0772] At room temperature, compound 66c (300 mg, 0.675 mmol) was dissolved in 1,4-dioxane (3 mL), and N,N-diisopropylethylamine (174 mg, 1.35 mmol) and 2,4-dimethoxybenzylamine (113 mg, 0.675 mmol) were added successively. The reaction system was stirred at 100 °C for 3 hours. After the reaction was completed, the reaction was quenched with water (20 mL), and the system was extracted with ethyl acetate (3 × 20 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (mobile phase: ethyl acetate / petroleum ether = 20%-40%) to obtain a mixture of 66d and 67a as a yellow oily liquid (80 mg, 21.8%).

[0773] LC-MS: m / z 575.3 [M+H] + 。

[0774] Step 4: Preparation of (R)-N 4 -(1-((tert-Butyldimethylsilyl)oxy)-2-methylhexan-2-yl)-N 2 -(2,4-dimethoxybenzyl)pyrimido[5,4-d]pyrimidine-2,4-diamine (66e) and (R)-N 8 -(1-((tert-Butyldimethylsilyl)oxy)-2-methylhexan-2-yl)-N 2 -(2,4-dimethoxybenzyl)pyrimido[5,4-d]pyrimidine-2,8-diamine (67b)

[0775] At room temperature, a mixture of compound 66d and 67a (80.0 mg, 0.148 mmol) was dissolved in methanol (3 mL). 10% palladium on carbon (8 mg, 10% water content) and ammonium formate (28.0 mg, 0.444 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour. After completion of the reaction, the reaction was quenched with water (10 mL), and the system was extracted with ethyl acetate (3 × 10 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude mixture of 66e and 67b as a yellow semi-solid (40.0 mg, 53.3%).

[0776] LC-MS: m / z 541.3 [M+H] + 。

[0777] Step 5: Preparation of (R)-2-((2-aminopyrimido[5,4-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (66) and (R)-2-((6-aminopyrimido[5,4-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (67)

[0778] At room temperature, a mixture of compound 66e and 67b (40 mg, 0.0787 mmol) was dissolved in trifluoroacetic acid (1 mL). The reaction mixture was stirred at 40 °C for 4 hours. After completion of the reaction, the mixture was concentrated under reduced pressure to obtain a crude product, which was purified by preparative chromatography column (column type: XSelect CSH Prep C18 OBD column, 5 μm, 19*150 mm; mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 10%-30% acetonitrile in 8 minutes; detection wavelength: 254 / 220 nm) to obtain white solid 66 (1.0 mg, 4.9%) and white solid 67 (5.6 mg, 27.4%).

[0779] Compound 66:

[0780] 1 H NMR (400 MHz, methanol-d4) δ 9.06 (s, 1H), 8.93 (s, 1H), 3.87 (d, J = 11.2 Hz 1H), 3.62 (d, J = 11.2 Hz 1H), 2.05 - 1.97 (m, 1H), 1.87 - 1.81 (m, 1H), 1.45 (s, 3H), 1.29 - 1.18 (m, 4H), 0.85 (t, J = 6.8 Hz, 3H).

[0781] LC-MS: m / z 277.2 [M+H] + 。

[0782] Compound 67:

[0783] 1 1H NMR (400 MHz, methanol-d4) δ 8.74 (s, 1H), 8.13 (s, 1H), 3.83 (d, J = 11.2 Hz, 1H), 3.61 (d, J = 11.2 Hz, 1H), 2.02 - 1.94 (m, 1H), 1.80 - 1.71 (m, 1H), 1.39 (s, 3H), 1.25 - 1.15 (m, 4H), 0.82 (t, J = 6.8 Hz, 3H).

[0784] LC-MS: m / z 277.2 [M+H] + 。

[0785] Example 68: Preparation of (R)-2-((2-aminopyrido[3,4-d]pyrimidin-4-yl)amino)-2-methylhexane-1,1-dideuterio-1-ol (68)

[0786]

[0787] Step 1: Preparation of (R)-3-methyl-5-phenyl-5,6-dihydro-2H-1,4-oxazine-2-one (68a)

[0788] At room temperature, (R)-2-amino-2-phenylethanol (5 g, 36.44 mmol) and ethyl pyruvate (4.23 g, 36.44 mmol) were dissolved in trifluoroethanol (110 mL), and the temperature was raised to 75 °C and reacted for 16 hours. After the reaction was completed, it was cooled to room temperature, filtered, and the filter cake was washed with ethyl acetate (3 × 30 mL). The filtrate was concentrated under reduced pressure, and the obtained yellow crude product was separated and purified by silica gel column chromatography (mobile phase: ethyl acetate / petroleum ether = 0 - 10%), and white solid 68a (2.4 g, 34.8%) was obtained.

[0789] LC-MS: m / z 190.1 [M+H] + 。

[0790] Step 2: Preparation of (3R,5R)-3-butyl-3-methyl-5-phenylmorpholin-2-one (68b)

[0791] At room temperature, compound 68a (2.4 g, 12.68 mmol) was dissolved in dry tetrahydrofuran (80 mL). The reaction solution was cooled to -78 °C, and boron trifluoride diethyl ether solution (3.3 mL) was slowly added dropwise under a nitrogen atmosphere. The reaction was maintained at -78 °C for 1.5 hours. Then, a tetrahydrofuran solution of n-butylmagnesium chloride (13.5 mL, 2 mol / L) was slowly added dropwise, and the reaction was maintained at -78 °C for 2 hours. After the reaction was completed, the temperature was slowly raised to room temperature, and the reaction was quenched with an aqueous ammonium chloride solution. Ethyl acetate (3 × 50 mL) was added to the system for extraction. The combined organic phases were washed with saturated brine (120 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained brown oily crude product was separated and purified by silica gel column chromatography (mobile phase: ethyl acetate / petroleum ether = 0 - 5%), to give white solid 68b (1.35 g, 43.0%).

[0792] LC-MS: m / z 248.2 [M+H] + 。

[0793] Step 3: Preparation of (R)-2-(((R)-2-hydroxy-1-phenylethyl)amino)-2-methylhexane-1,1-dideuterio-1-ol (68c)

[0794] At room temperature, compound 68b (500 mg, 2.02 mmol) was dissolved in dry tetrahydrofuran (5 mL). The reaction solution was placed at 0 °C, and lithium aluminum deuteride (169.7 mg, 4.04 mmol) was added. The reaction solution was stirred at room temperature for 2 hours. After the reaction was completed, the reaction was quenched with water under an ice bath. Ethyl acetate (3 × 6 mL) was added to the system for extraction. The combined organic phases were washed with saturated brine (15 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give pale yellow solid 68c (360 mg, 70.3%).

[0795] LC-MS: m / z 254.3 [M+H] + 。

[0796] Step 4: Preparation of (R)-2-amino-2-methylhexane-1,1-dideuterio-1-ol hydrochloride (68d)

[0797] At room temperature, compound 68c (360 mg, 1.42 mmol) was dissolved in anhydrous ethanol (5 mL), and palladium hydroxide on carbon (144 mg, 0.42 mmol) and a dioxane solution of hydrogen chloride (0.5 mL, 4 mol / L) were added. The reaction mixture was reacted under a hydrogen atmosphere (5 atm) at 70 °C for 16 hours. After the reaction was completed, it was cooled to room temperature, filtered, and the filter cake was washed with ethanol (3 × 3 mL). The filtrate was concentrated under reduced pressure to obtain a yellow crude product. Water (10 mL) was added, and ethyl acetate (3 × 8 mL) was added to the system for extraction. The aqueous phase was concentrated under reduced pressure to obtain a yellow oily product 68d (185 mg, 76.7%)

[0798] LC-MS: m / z 134.0 [M+H] + 。

[0799] Step 5: Preparation of (R)-2-((2-aminopyrido[3,4-d]pyrimidin-4-yl)amino)-2-methylhexane-1,1-dideuterio-1-ol (68)

[0800] At room temperature, compound 68d (90 mg, 0.67 mmol) and 2-aminopyrido[3,4-d]pyrimidin-4(3H)-one (44b) (100 mg, 0.62 mmol) were dissolved in N,N-dimethylformamide (2 mL), and Carter's condensing agent (327 mg, 0.74 mmol) and 1,8-diazabicycloundec-7-ene (282 mg, 1.85 mmol) were added. The reaction was carried out at room temperature for 16 hours. After the reaction was complete, the reaction mixture was diluted with water (10 mL) and then extracted with ethyl acetate (3 × 8 mL). The combined organic phases were washed with saturated brine (3 × 15 mL) and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure, and the resulting crude product was separated and purified by preparative chromatography column (column type: XBridgeShield RP18 OBD Column, 5um, 19*150mm; mobile phase A: water (0.05% ammonia water), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 25%-40% acetonitrile in 8 minutes; detection wavelength: 254 / 220 nm), to obtain a white solid product 68 (13.6 mg, 7.8%).

[0801] 1 1H NMR (300 MHz, methanol-d4) δ 8.61 (d, J = 0.8 Hz, 1H), 8.18 (d, J = 5.6 Hz, 1H), 7.92 (dd, J = 5.7, 0.9 Hz, 1H), 2.32–2.16 (m, 1H), 1.85–1.70 (m, 1H), 1.48 (s, 3H), 1.43–1.29 (m, 4H), 0.97–0.86 (m, 3H).

[0802] LC-MS: m / z 278.4 [M+H] + 。

[0803] Example 69: Preparation of (R)-2-((2-aminopyrido[3,4-d]pyrimidin-4-yl)amino)-2-ethylhexan-1-ol (69)

[0804]

[0805]

[0806] Step 1: Preparation of (R)-3-ethyl-5-phenyl-5,6-dihydro-2H-1,4-oxazine-2-one (69a)

[0807] At room temperature, dissolve compound (R)-2-amino-2-phenylethanol (4.11 g, 29.96 mmol) and methyl 2-oxobutanoate (3.48 g, 29.96 mmol) in trifluoroethanol (90 mL), and heat to 75 °C for reaction for 16 hours. After the reaction is completed, cool to room temperature, filter, and wash the filter cake with ethyl acetate (3 × 30 mL). Concentrate the filtrate under reduced pressure, and purify the obtained yellow crude product by silica gel column chromatography (mobile phase: ethyl acetate / petroleum ether = 0 - 10%), to obtain white solid 69a (2.3 g, 37.8%).

[0808] LC-MS: m / z 204.2 [M+H] + 。

[0809] Step 2: Preparation of (3R,5R)-3-butyl-3-ethyl-5-phenylmorpholin-2-one (69b)

[0810] At room temperature, dissolve compound 69a (2.3 g, 11.32 mmol) in dry tetrahydrofuran (75 mL), cool the reaction solution to -78 °C, and slowly add boron trifluoride diethyl ether solution (3.0 mL) dropwise under a nitrogen atmosphere, and keep the reaction at -78 °C for 1.5 hours. Then slowly add a tetrahydrofuran solution of n-butylmagnesium chloride (11.9 mL, 2 mol / L) dropwise, and keep the reaction at -78 °C for 2 hours. After the reaction is completed, slowly warm to room temperature, quench the reaction with aqueous ammonium chloride solution, add ethyl acetate (3 × 50 mL) to the system for extraction, wash the combined organic phases with saturated brine (120 mL), dry over anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify the obtained brown oily crude product by silica gel column chromatography (mobile phase: ethyl acetate / petroleum ether = 0 - 5%), to obtain white solid 69b (1.23 g, 41.6%).

[0811] LC-MS: m / z 262.3 [M+H] + 。

[0812] Step 3: Preparation of (R)-2-ethyl-2-(((R)-2-hydroxy-1-phenylethyl)amino)hexan-1-ol (69c)

[0813] At room temperature, compound 69b (1.23 g, 4.71 mmol) was dissolved in dry tetrahydrofuran (10 mL). The reaction solution was placed at 0 °C, and a tetrahydrofuran solution of lithium borohydride (4.7 mL, 2 mol / L) was added. The reaction solution was reacted at room temperature for 2 hours. After the reaction was completed, the reaction was quenched by adding water under an ice bath. Ethyl acetate (3 × 12 mL) was added to the system for extraction. The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a pale yellow solid 69c (1.1 g, 88.1%).

[0814] LC-MS: m / z 266.2 [M+H] + 。

[0815] Step 4: Preparation of (R)-2-amino-2-ethylhexan-1-ol hydrochloride (69d)

[0816] At room temperature, compound 69c (1.1 g, 4.15 mmol) was dissolved in anhydrous ethanol (20 mL). Palladium on carbon hydroxide (421 mg, 1.23 mmol) and a dioxane solution of hydrogen chloride (1.5 mL, 4 mol / L) were added. The reaction solution was reacted under a hydrogen atmosphere (5 atm) at 70 °C for 16 hours. After the reaction was completed, it was cooled to room temperature and filtered. The filter cake was washed with ethanol (3 × 8 mL). The filtrate was concentrated under reduced pressure to obtain a yellow crude product. Water (30 mL) was added, and ethyl acetate (3 × 25 mL) was added to the system for extraction. The aqueous phase was concentrated under reduced pressure to obtain a yellow oily product 69d (750 mg, 99.6%)

[0817] LC-MS: m / z 146.0 [M+H] + 。

[0818] Step 5: Preparation of (R)-2-((2-aminopyrido[3,4-d]pyrimidin-4-yl)amino)-2-ethylhexan-1-ol (69)

[0819] At room temperature, compound 69d (202 mg, 1.39 mmol) and 2-aminopyrido[3,4-d]pyrimidin-4(3H)-one (44b) (150 mg, 0.93 mmol) were dissolved in N,N-dimethylformamide (3 mL). Then, HATU (491 mg, 1.11 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (422 mg, 2.78 mmol) were added, and the reaction was carried out at room temperature for 16 hours. After the reaction was complete, the reaction mixture was diluted with water (10 mL) and extracted with ethyl acetate (3×8 mL). The combined organic phases were washed with saturated brine (3×15 mL) and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure, and the resulting crude product was separated and purified by preparative chromatography (column type: XBridgeShield RP18 OBD Column, 5 μm, 19*150 mm; mobile phase A: water (0.05% ammonia water), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 25%-50% acetonitrile in 8 minutes; detection wavelength: 254 / 220 nm), to obtain the white solid product 69 (16.3 mg, 6.0%).

[0820] 1 H NMR (300 MHz, methanol-d4) δ 8.61 (d, J = 0.8 Hz, 1H), 8.18 (d, J = 5.6 Hz, 1H), 7.92 (dd, J = 5.6, 0.9 Hz, 1H), 3.94 (d, J = 1.4 Hz, 2H), 2.15–1.83 (m, 4H), 1.46–1.22 (m, 4H), 0.99–0.85 (m, 6H).

[0821] LC-MS: m / z 290.2 [M+H] + 。

[0822] Biological Test

[0823] Test Example 1: Agonist Activity of the Compounds of the Present Invention against hTLR8 and hTLR7

[0824] In vitro analysis of the receptor binding activity of the compounds of the present invention against hTLR8 and hTLR7 was performed using HEK-Blue TM hTLR8 cells and HEK-Blue TMhTLR7 cells. These cells are co-transfected with the hTLR8 or hTLR7 gene and a reporter gene for secreted alkaline phosphatase (SEAP) in HEK293 cells. The gene of SEAP is placed downstream of the IFN-β minimal promoter, which consists of 5 NF-κB and AP-1 binding sites. Stimulants of hTLR8 or hTLR7 will activate the NF-κB and AP-1 promoters to produce SEAP, and the effect of the compound is evaluated by detecting the SEAP level.

[0825] Test reagents:

[0826] HEK-Blue hTLR8 cells and HEK-Blue hTLR7 cells (from Invivogen)

[0827] HEK-BlueTM detection reagent (from Invivogen)

[0828] DMEM medium (from Gibco)

[0829] Fetal bovine serum (from Gibco)

[0830] Normocin TM 、Zeocin and Blasticidine (from Invivogen)

[0831] Test procedure:

[0832] 1. Collect the cells in the cell culture flask, adjust the cell density to 2.2×10 5 / mL, resuspend the cells with HEK-BlueTM detection reagent, and inoculate 45 μL of the cell suspension into a 384-well plate, with 10,000 cells per well.

[0833] 2. Preparation of the compound plate: The test compound is diluted 3-fold with DMSO from 2 mM for 10 gradients. Take 2 μL of the diluted compound and add it to 38 μL of HEK-BlueTM detection reagent for 20-fold intermediate dilution. The well with cells plus 0.5% DMSO serves as the negative control well with a low reading. The well with cells plus 1 μM GS-9688 (synthesized according to the synthetic route of WO2016141092A1) serves as the positive control well with a high reading.

[0834] 3. Take 5 μL of the intermediate-diluted compound and add it to the 384-well plate already inoculated with 45 μL of cells for 10-fold dilution of the drug, and the final concentration of DMSO is 0.5%.

[0835] 4. Place the 384-well plate containing cells and compounds in an incubator at 37°C and 5% CO2 for 16 hours.

[0836] 5. After 16 hours, take out the culture plate and use the instrument VICTOR Nivo to detect the light absorption of SEAP at 620 nm.

[0837] 6. Analyze the data using GraphPad Prism 8 software to obtain the EC of each compound 50 .

[0838] Calculate the average value of the data for each concentration and the positive and negative controls. Calculate the percentage of activity by the following formula:

[0839] % Activity = (Compound reading - Negative well reading) / (Positive well reading - Negative well reading) × 100.

[0840] Calculate the IC of each compound by fitting the data to a non - linear regression equation 50 :

[0841] Y = Minimum value + (Maximum value - Minimum value) / (1 + 10^((LogEC 50 - X) × Hill slope));

[0842] Where X is the logarithm of the compound concentration and Y is the percentage of activity.

[0843] The agonist activity of the compounds of the present invention against TLR8 / TLR7 is shown in Table 1 below.

[0844] Table 1 Agonist EC of the compounds of the present invention against TLR8 / TLR7 50 Value

[0845]

[0846]

[0847]

[0848] Conclusion: The compounds of the present invention can selectively activate TLR8.

[0849] Test Example 2: Study on the absorption mechanism of the compound of Example 52

[0850] In this experiment, by detecting the permeability coefficient of the test substance in the Caco - 2 cell model, its possible permeation and absorption were investigated. The concentration of the compound in the test sample was detected using LC / MS / MS, and the apparent permeability coefficient (Papp) of the test compound across the Caco - 2 cell membrane was calculated. Caco - 2 cells were purchased from the American Type Culture Collection, Numbered HTB-37, after about 14 days of culture, it was completely confluent and differentiated. The test was a two-way drug administration, that is, the transport rate of the compound from the apical to the basolateral side was detected simultaneously, and 2) the transport rate of the compound from the basolateral to the apical side was measured.

[0851] The information of some main reagents involved in the experiment is shown in the following table:

[0852]

[0853] 1. Preparation of Caco-2 cells

[0854] Dilute Caco-2 cells (American Type Culture Collection, HTB-37) with the medium to 6.86×10 5 cells / mL, and add 50 μL of the cell suspension to the filter wells of a 96-well Transwell plate (Cat.No.3391). Incubate the cell culture plate in a cell culture incubator at 37 °C, 5% CO2, and 95% relative humidity for 14 - 18 days. Replace the cell medium every other day.

[0855] 2. Evaluation of cell monolayer stability

[0856] Remove the old medium and replace it with pre-warmed fresh medium before evaluation. Use a Millicell Epithelial Volt-Ohm measurement system (Millipore, USA) to measure the transepithelial electrical resistance (TEER) across the monolayer. After measurement, return the plate to the incubator.

[0857] The TEER value is calculated according to the following formula:

[0858] Measured TEER value (ohms) × membrane area (cm 2 ) = TEER value (ohm·cm 2 )

[0859] where the TEER value is greater than 230 ohm·cm 2 , indicating that the Caco-2 monolayer membrane is qualified and can be used for subsequent tests.

[0860] 3. Preparation of compound solution

[0861] Accurately weigh the compound of Example 52, dissolve it with DMSO to prepare a stock solution with a concentration of 2 mM, and dilute it with HBSS (Gibico, 10 mM HEPES, pH 7.4) to obtain a 10 μM working solution. Metoprolol and digoxin are used as control compounds.

[0862] 4. Conduct drug transport experiments

[0863] Before the experiment, the monolayer was washed twice with pre-warmed HBSS (10 mM HEPES, pH 7.4). Then the culture plates were incubated at 37 °C for 30 minutes.

[0864] ① Determine the transport rate of the drug from the apical to the basolateral direction: 125 μL of the working solution was added to the Transwell (apical compartment), and immediately 50 μL of the sample was transferred from the apical compartment to a new 96-well plate containing 200 μL of IS (100 nM alprazolam, 200 nM caffeine, and 100 nM tolbutamide) as the initial donor sample (AB). Vortex at 1000 rpm for 10 minutes. The wells in the receiver plate (basolateral compartment) were filled with 235 μL of buffer.

[0865] ② Determine the transport rate of the drug from the basolateral to the apical direction: 285 μL of the working solution was added to the wells of the receiver plate (basolateral compartment), and 50 μL of the sample was transferred.

[0866] The culture plates were incubated at 37 °C for 2 hours. At the end of the incubation, 50 μL of the samples from the donor side (the apical compartment for Ap→Bl, the basolateral compartment for Bl→Ap) and the receiver side (the basolateral compartment for Ap→Bl and the apical compartment for Bl→Ap) were transferred to the wells of a new 96-well plate, and then 200 μL of acetonitrile containing IS (100 nM alprazolam, 200 nM caffeine, and 100 nM tolbutamide) was added. The samples were vortexed for 10 minutes, 50 μL of the sample was transferred to the wells of a new 96-well plate, and then 50 μL of HEPES and 200 μL of IS were added. All the samples were vortexed and mixed for 10 minutes, and then centrifuged at 3,220 g for 40 minutes. Before analysis, 150 μL of the supernatant was mixed with an appropriate amount of ultrapure water and then tested and analyzed using LC-MS / MS.

[0867] The apparent permeability (P app ) was determined using the following equation:

[0868]

[0869] where P app is the apparent permeability (cm / s × 10 -6 ), dQ / dt is the rate of drug transport (pmol / second), A is the surface area of the membrane (cm 2 ), and D0 is the initial donor concentration (nM; pmol / cm 3 ).

[0870] The efflux ratio was determined using the following equation:

[0871]

[0872] where Papp(B-A) Represents the apparent permeability coefficient from the basolateral side to the apical direction, P app(A-B) Represents the apparent permeability coefficient from the apical side to the basolateral direction.

[0873] The test results are shown in Table 2.

[0874] Table 2 Caco-2 test parameters of the compound of Example 52 and the compound of Example 4 of WO2018045144A1

[0875] Parameter Example 52 WO2018045144A1 (Example 4) <![CDATA[P app(A-B) > 19.2 2.72 <![CDATA[P app(B-A) > 31.1 30.0 Effluent Ratio 1.62 11.0 <![CDATA[Recovery rate (%) AP-BL > 107 97.2 <![CDATA[Recovery rate (%) BL-AP > 93.5 102

[0876] Conclusion: The compound of Example 52 has a good permeability coefficient and a low risk of being an efflux transporter substrate. The compound of Example 4 of WO2018045144A1 has low permeability and a high efflux ratio, and is very likely to be an efflux transporter substrate. In comparison, the in vitro Caco-2 test results of the compound of Example 52 of the present invention show better permeability than the compound of Example 4 of WO2018045144A1.

[0877] Test Example 3: Pharmacokinetic study of the compound of Example 52 in rats

[0878] This experiment was designed to evaluate the pharmacokinetic behavior of the compound of Example 52 in rats after intravenous infusion or oral gavage. For intravenous infusion: The test compound was formulated into a clear solution of 0.5 mg / mL, and the solvent was 2% ethanol / 40% polyethylene glycol 300 / 58% 0.01 M hydrochloric acid; for oral gavage: The test compound was formulated into a clear solution of 0.5 mg / mL, and the solvent was 2% ethanol / 40% polyethylene glycol 300 / 58% 0.01 M hydrochloric acid.

[0879] The concentration of the test compound in plasma was determined by high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS). The non-compartmental model of WinNonlin TM Version 8.3 (Pharsight, Mountain View, CA) pharmacokinetic software was used to process plasma and tissue concentrations, and the linear logarithmic trapezoidal method was used to calculate pharmacokinetic parameters.

[0880] The relevant pharmacokinetic parameters of the compound of Example 52 in rats are shown in Table 3 below.

[0881] Table 3 Relevant pharmacokinetic parameters of intravenous infusion and oral gavage of the compound of Example 52 in rats

[0882]

[0883]

[0884] The relevant parameters of the tissue distribution study of the compound in Example 52 in rats at a gavage dose of 5 mg / kg are shown in Table 4 below.

[0885] Table 4 Relevant parameters of the tissue distribution study of the compound in Example 52 in rats by gavage

[0886]

[0887] The relevant parameters of the intestinal bioavailability and hepatic first-pass studies of the compound in Example 52 in rats at a gavage dose of 5 mg / kg are shown in Table 5 below.

[0888] Table 5 Relevant parameters of the intestinal bioavailability and hepatic first-pass studies of the compound in Example 52 in rats by gavage

[0889]

[0890] The relevant parameters of the excretion study of the compound in Example 52 in rats at an intravenous drip dose of 1 mg / kg for 0.5 hours are shown in Table 6 below.

[0891] Table 6 Relevant parameters of the excretion study of the compound in Example 52 in rats by intravenous drip

[0892]

[0893]

[0894] Conclusion: The compound in Example 52 has a low systemic oral bioavailability, but has a high intestinal bioavailability, can specifically enrich in liver tissue, and is mainly excreted through the intestine.

[0895] Test Example 4: Pharmacodynamic study of the compound in Example 52 in cynomolgus monkeys

[0896] This test aims to evaluate the pharmacodynamics of the compound in Example 52 after oral administration in male cynomolgus monkeys.

[0897] Oral administration: The test compound was formulated as 10 mg / mL, and the solvent was 10% ethanol, 40% polyethylene glycol 300, and 50% deionized water. An appropriate amount of the test compound was weighed in an appropriate amount of ethanol, and the mixture was completely dissolved under stirring and / or ultrasonic action. Then, an appropriate volume of polyethylene glycol 300 was added under stirring. Finally, an appropriate volume of deionized water was added under stirring to obtain the formulation of the final concentration. The formulation was stirred at room temperature for at least 10 minutes before and during dosing.

[0898] One cynomolgus monkey of each sex in each group (purchased from Beijing Xie'erxin Biological Resources) was observed twice a day beside the cage. Before drug administration and at 30 min, 1 hr, 2 hr, 4 hr, 8 hr, and 24 hr after drug administration, 0.3 mL of serum was collected from each animal for each sample. The concentrations of biochemical indexes IL12p40, IFN-α, and TNF-α in the serum were measured by a hypersensitive multi-factor electrochemiluminescence analyzer.

[0899] The reagents and instruments involved in the experiment are shown in the following table:

[0900]

[0901] The full name of the supplier MSD: Meso Scale Discovery.

[0902] The test process for serum cytokine concentration is as follows:

[0903] 1) Pre-incubate the U-PLEX 96-well microplate with the coating solution and incubate at room temperature for 1 hour.

[0904] 2) Wash the plate 3 times with 300 μL of 1x wash buffer.

[0905] 3) Add 25 μL of diluent (Diluent43) to each well to be tested, and gently tap the side of the plate to evenly distribute Diluent43 at the bottom of the MSD microplate.

[0906] 4) Add 25 μL of TNF-α, IL-12 or INF-α standard or 25 μL of serum sample to each well, seal the plate with a sealing film and incubate with shaking at room temperature for 1 hour.

[0907] 5) Wash the plate 3 times with 300 ul of 1x wash buffer (PBS-0.05% Tween-20).

[0908] 6) Add 50 μL of detection antibody solution to each well, seal the plate with a sealing film and incubate with shaking at room temperature for 1 hour.

[0909] 7) Wash the plate 3 times with 300 μL of 1x wash buffer.

[0910] 8) Add 150 μL of MSD GOLD Read Buffer B to each well, and read the U-PLEX 96-well microplate with an MSD reader (model: MESO SECTORS600).

[0911] 9) Analyze the data using GraphPad Prism 8 software to obtain the area under the curve (AUC) of the 'cytokine concentration-time' curve.

[0912] Figure 1 Parameters of the compound in Example 52 in cynomolgus monkey serum.

[0913] It can be seen from Figure 1 that after the compound of Example 52 was orally administered at 10 mg / kg, the compound had a significant activating effect on IL-12p40 in the serum of cynomolgus monkeys, but had little effect on TNF-α and INF-α.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

2. A pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt thereof according to claim 1, and a pharmaceutically acceptable carrier or excipient.

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