RU2831403C1 - Compound having kinase inhibitory activity - Google Patents

Abstract

FIELD: organic chemistry.

SUBSTANCE: group of inventions includes compounds of formulas II, IV, as well as individual compounds specified in the claim, a pharmaceutical composition based on them and use thereof. In formula II, X and Y are N and Z is CR; R is H; Ra is selected from the group consisting of H, substituted or unsubstituted C₁-C₆ alkyl group; U is a chemical bond or -NH-; and when U is a chemical bond, then Rc is pyrrolidine; Rc is selected from a group consisting of a substituted or unsubstituted C₁-C₆ alkyl group, a substituted or unsubstituted C₃-C₈ carbocyclic ring, substituted or unsubstituted 3–8-membered heterocyclic ring containing 1–2 heteroatoms selected from oxygen, sulfur and nitrogen atoms; Re is selected from a group consisting of -NHR; wherein R is selected from a group consisting of H; W is substituted or unsubstituted phenyl, and a substituted or unsubstituted 5–6 membered heteroaromatic ring containing 1–2 heteroatoms selected from the group consisting of sulfur and nitrogen atoms; in addition to the common substituent, the W group is further substituted with at least one group with the -MA structure, where M is selected from the group consisting of a chemical bond or -CHR-; other substitutes are as specified in the patent claim. In formula IV, substitutes are as specified in the claim.

EFFECT: pyridino[3,4-b]pyrazine derivatives used to treat diseases associated with abnormal gene levels or abnormal expression of kinases selected from a group consisting of FLT3, ALK and AXL.

16 cl, 2 tbl, 360 ex

Description

AREA OF TECHNOLOGY

The present invention relates to the field of pharmaceutical chemistry and medicine and, in particular, to FLT3 inhibitor compounds, methods for their preparation and their use for the treatment of diseases associated with abnormal activity or expression level of FLT3, for example, for the treatment of acute myeloid leukemia.

PRIOR ART

The occurrence and development of malignant tumors is a complex process. The drugs commonly used for their treatment include alkylating agents, antimetabolites, natural products and antibiotics, but all of these drugs have low efficacy and serious side effects. Small molecule kinase inhibitors are currently actively used in the development of drugs for the treatment of oncological diseases. Protein kinases are catalysts that play a key role in almost all aspects of cell biology and biochemistry. These enzymes generate signaling molecules that regulate cell cycle processes, proliferation, programmed cell death (apoptosis), cytoskeletal function, motility, differentiation, development, transcription and translation. Protein kinases play an important role in various processes, and their precise regulation is critically important, since disruptions in their functions can lead to cancer, cardiovascular diseases, inflammatory processes and diseases of the nervous system. Dysregulation, overexpression, and mutation of protein kinases are responsible for the pathogenesis of human diseases, making these enzymes promising targets for drug therapy. Growth factor receptors with protein tyrosine kinase (PTK) activity are called receptor tyrosine kinases. Receptor protein tyrosine kinases are a class of tightly regulated enzymes, and abnormal activation of various members of this family is one of the markers of cancer. FLT3, as well as KIT, FMS, platelet-derived growth factor receptor (PDGFR), belong to the receptor tyrosine kinase family and play an important role in the regulation of hematopoiesis.

FLT3 (FMS-like tyrosine kinase-3) is a cytokine receptor expressed in hematopoietic stem cells that regulates the survival and proliferation of hematopoietic stem and progenitor cells, dendritic cell maturation, and the maintenance of T-cell homeostasis. FLT3 gene mutations are present in approximately 30% of patients with acute myeloid leukemia (AML), and they include FLT3 internal tandem duplication (FLT3-ITD) and FLT3 tyrosine kinase domain point mutation (FLT3-TKD). FLT3-ITD mutations result in constitutive, ligand-independent tyrosine kinase activation and are known to have a poor prognosis for patient survival, and they are present in approximately 25% of patients with AML, whereas FLT3-TKD occurs in approximately 5-10% of patients. FLT3 can dimerize upon ligand binding and undergo autophosphorylation, thereby initiating diverse intracellular signaling programs. FLT3 mutations can promote cell proliferation and inhibit apoptosis via MAPK, PI3K/AKT/Mtor, and STAT5 signaling pathways in vitro. The high frequency and poor prognosis of FLT3 mutations suggest that it may be an important target for AML therapy.

Thus, there is an urgent need in the art to develop new FLT3 kinase inhibitors.

ESSENCE OF THE INVENTION

The aim of the present invention is to provide a novel FLT3 kinase inhibitor.

In a first aspect the present invention provides a compound of formula I, or pharmaceutically acceptable salts or deuterated products thereof:

Where is the ring? is an aza-(5-6-membered heteroaromatic ring) and a ring is a 5-6 membered aromatic ring or heteroaromatic ring, and the ring together with the ring form a 9-10-membered heteroaromatic ring;

Ra is selected from the group consisting of H, halogen, a substituted or unsubstituted C ₁ -C ₆ alkyl group, a substituted or unsubstituted C ₂ -C ₆ alkenyl group, a substituted or unsubstituted C ₂ -C ₆ alkynyl group, a substituted or unsubstituted C ₃ -C ₈ carbon ring (including saturated and partially unsaturated ring), a substituted or unsubstituted 5- to 9-membered heteroaromatic ring (including monocyclic or condensed ring) containing 1-3 heteroatoms selected from oxygen, sulfur and nitrogen atoms, a substituted or unsubstituted 3- to 8-membered heterocyclic ring containing 1-3 heteroatoms selected from oxygen, sulfur and nitrogen atoms (which is a saturated or partially unsaturated heterocyclic ring, preferably a substituted or unsubstituted C ₃ -C ₆ cycloalkyl group, a substituted or an unsubstituted 3-6 membered heterocyclic ring containing 1-3 heteroatoms selected from oxygen, sulfur and nitrogen atoms), or a substituted and unsubstituted -[L] _m -H group; wherein each L is independently selected from the group consisting of -CH ₂ -, -O-, -NH- or -S-;

m is selected from the group consisting of 1, 2, 3, 4, 5 and 6;

U is selected from the group consisting of a chemical bond or -O-, -CHR-, a carbonyl group, S, -NH-, -NHC(O)-, -NHS(O) _2- , -NHC(O)NH-, -NHC(S)NH-, -COO- and -OS(O) _2- ;

Rc is selected from the group consisting of H, a substituted or unsubstituted _C1 - _C6 alkyl group, a substituted or unsubstituted _C3 - _C8 carbocyclic ring (including saturated and partially unsaturated ring), a substituted or unsubstituted 3-8-membered heterocyclic ring containing 1-3 heteroatoms selected from oxygen, sulfur and nitrogen atoms (saturated and partially unsaturated, including monocyclic, fused, bridged or spirocyclic ring), a substituted or unsubstituted _C6 - _C10 aryl group, a substituted or unsubstituted 5-12-membered heteroaromatic ring containing 1-3 heteroatoms selected from oxygen, sulfur and nitrogen atoms, or a substituted and unsubstituted -[L] _m -H group; wherein L is independently selected from the group consisting of _-CH2- , -O-, -NH-, or -S-;

Re is selected from the group consisting of halogen, -NHR, -OR, a substituted or unsubstituted _C1 - _C6 alkyl group, a substituted or unsubstituted _C1 - _C6 alkyl-NH- group, a substituted or unsubstituted _C1 - _C6 alkoxy group, a substituted and unsubstituted _C3 - _C8 carbocyclic ring (including cases of saturated and partially unsaturated ring) or a substituted and unsubstituted -[L] _m -H- group wherein L is independently selected from the group consisting of _-CH2- , -O-, -NH- or -S-;

W is selected from the group consisting of H, -NHR, -OR, halogen, substituted or unsubstituted _C1 - _C6 alkyl group, substituted or unsubstituted _C1 - _C6 alkoxy group, substituted or unsubstituted _C2 - _C6 alkenyl group, substituted or unsubstituted _C2 - _C6 alkynyl group, substituted or unsubstituted _C3 - _C8 carbocyclic ring (including cases of saturated and partially unsaturated ring), substituted or unsubstituted _C6 - _C10 aryl group, substituted or unsubstituted 4-15-membered heterocyclic ring containing 1-3 heteroatoms selected from oxygen, sulfur and nitrogen atoms (saturated or partially unsaturated ring, including monocyclic, fused, bridged or spirocyclic ring), substituted or unsubstituted 5-12 membered heteroaromatic ring containing 1-3 heteroatoms selected from the group consisting of oxygen, sulfur and nitrogen atoms (including monocyclic and condensed ring), substituted or unsubstituted _-C1 - _C6 alkylphenyl group, substituted or unsubstituted _C3 - _C12 cycloalkyl group (including monocyclic, condensed, bridged or spirocyclic ring), substituted or unsubstituted _C2 - _C10 acyl group, substituted or unsubstituted _C2 - _C10 ester group, substituted or unsubstituted _C6 - _C10 aryloxy group and substituted or unsubstituted _C1 - _C6 amide group;

The W group is substituted by at least one group with the structure -M-A, wherein M is selected from the group consisting of a chemical bond or -CHR-, a carbonyl group, S, O, -NH-, -NHC(O)-, -NHS(O) _2- , -NHC(O)NH-, -NHC(S)NH-, -COO- and -OS(O) _2- ;

A is selected from the group consisting of H, halogen, cyano, amino, nitro, hydroxy, sulfhydryl, aldehyde, carboxyl, sulfonyl, substituted or unsubstituted _C1 - _C6 alkyl, substituted or unsubstituted _C1 - _C6 alkoxy, substituted and unsubstituted _C6 - _C10 aryl, substituted or unsubstituted 4- to 12-membered heterocyclic ring containing 1-3 heteroatoms selected from oxygen, sulfur, and nitrogen atoms (including a monocyclic, fused, bridged, or spirocyclic ring), substituted or unsubstituted 5- to 12-membered heteroaromatic ring containing 1-3 heteroatoms selected from oxygen, sulfur, and nitrogen atoms (including a monocyclic or fused ring), substituted or unsubstituted -C ₁ -C ₆ alkylphenyl group, substituted or unsubstituted C ₃ -C ₁₂ cycloalkyl group, substituted or unsubstituted C ₂ -C ₁₀ acyl group, substituted or unsubstituted C ₂ -C ₁₀ ester group, substituted or unsubstituted C ₆ -C ₁₀ aryloxy group, substituted or unsubstituted C ₁ -C ₆ amide group, substituted or unsubstituted C ₁ -C ₄ alkyl-S(O) ₂ -group, substituted or unsubstituted C ₁ -C ₄ alkyl-SO- group;

In group A, said substitution means substitution with one or more groups selected from group B, wherein group B consists of H, halogen, =O, cyano, amino, nitro, hydroxy, sulfhydryl, aldehyde, carboxyl, sulfonyl, substituted or unsubstituted _C1 - _C6 alkyl, substituted or unsubstituted _C1 - _C6 alkoxy, substituted and unsubstituted _C6 - _C10 aryl, substituted or unsubstituted 3-12-membered (preferably 5-7-membered) heterocyclic ring containing 1-3 heteroatoms selected from oxygen, sulfur and nitrogen atoms, substituted or unsubstituted 5-12-membered heteroaromatic ring containing 1-3 heteroatoms selected from oxygen, sulfur and nitrogen atoms nitrogen, a substituted or unsubstituted -C ₁ -C ₆ alkylphenyl group, a substituted or unsubstituted C ₃ -C ₁₂ cycloalkyl group, a substituted or unsubstituted C ₂ -C ₁₀ acyl group, a substituted or unsubstituted C ₂ -C ₁₀ ester group, a substituted or unsubstituted C ₆ -C ₁₀ aryloxy group, a substituted or unsubstituted C ₁ -C ₆ amide group, a substituted or unsubstituted C ₁ -C ₄ alkyl-S(O) ₂ -group, a substituted or unsubstituted C ₁ -C ₄ alkyl-SO- group; wherein in group B, substitution means substitution with one or more R groups;

R is selected from the group consisting of H, halogen, cyano, amino, nitro, hydroxy, sulfhydryl, aldehyde, carboxyl, sulfonyl, substituted or unsubstituted _C1 - _C6 alkyl, substituted or unsubstituted _C1 - _C6 alkoxy, substituted or unsubstituted _C6 - _C10 aryl, substituted or unsubstituted 5-7-membered heterocyclic ring containing 1-3 heteroatoms selected from oxygen, sulfur and nitrogen atoms, substituted or unsubstituted _-C1 - _C6 alkylphenyl, substituted or unsubstituted _C3 - _C12 cycloalkyl, substituted or unsubstituted _C2 - _C10 acyl, substituted or unsubstituted _C2 - _C10 an ester group, a substituted or unsubstituted C ₆ -C ₁₀ aryloxy group, a substituted or unsubstituted C ₁ -C ₆ amide group, a substituted or unsubstituted C ₁ -C ₄ alkyl-S(O) ₂ group, a substituted or unsubstituted C ₁ -C ₄ alkyl-SO group;

Unless otherwise indicated, in the above formulae, substitution means that the hydrogen atom in the corresponding group is substituted by one or more substituents selected from the group consisting of deuterium, tritium, halogen, hydroxyl group, carboxyl group, sulfhydryl group, benzyl group, C ₁ -C ₁₂ alkoxycarbonyl group, C ₁ -C ₆ aldehyde group, amino group, C ₁ -C ₆ amide group, nitro group, cyano group, unsubstituted or halogenated C ₁ -C ₆ alkyl group, unsubstituted or halogenated C ₃ -C ₈ cyanoalkyl group, C ₂ -C ₁₀ alkenyl group, C ₁ -C ₆ alkoxy group, C ₁ -C ₆ alkylamino group, C ₆ -C ₁₀ aryl group, five- or six-membered a heteroaryl group, a five- or six-membered non-aromatic heterocyclyl group, an -O-(C ₆ -C ₁₀ aryl) group, an -O-(five- and six-membered heteroaryl) group, a C ₁ -C ₁₂ alkylaminocarbonyl group, an unsubstituted or halogenated C ₂ -C ₁₀ acyl group, a sulfonyl (-SO ₂ -OH) group, a phosphoryl (-PO ₃ -OH) group, an unsubstituted or halogenated C ₁ -C ₄ alkyl-S(O) ₂ group, and an unsubstituted or halogenated C ₁ -C ₄ alkyl-SO group.

In another preferred embodiment of the invention, the compound of formula I has a structure represented by the following formula II:

where each group X, Y and Z is independently selected from N or CR.

In another preferred embodiment of the invention, the compound of formula I has a structure represented by the following formula IIa:

Wherein ring W is selected from the group consisting of a substituted and unsubstituted _C6 - _C10 aryl group, a substituted or unsubstituted 4-12-membered heterocyclic ring containing 1-3 heteroatoms selected from oxygen, sulfur and nitrogen atoms, a substituted or unsubstituted 5-12-membered heteroaromatic ring containing 1-3 heteroatoms selected from oxygen, sulfur and nitrogen atoms, a substituted or unsubstituted _-C1 - _C6 alkylphenyl group, a substituted or unsubstituted _C3 - _C12 alkylalkyl group; wherein in group W, said substitution means substitution with one or more groups selected from group A.

In another preferred embodiment of the invention, ring W is selected from the group consisting of a substituted or unsubstituted 4-7-membered heterocyclic ring, a substituted or unsubstituted 5-6-membered heteroaromatic ring, a substituted or unsubstituted 9-10-membered heteroaromatic ring with a bicyclic fused structure, a substituted and unsubstituted phenyl group, and a substituted and unsubstituted _C3 - _C6 cycloalkyl group.

In another preferred embodiment of the invention, ring W is a substituted or unsubstituted ring structure selected from the group consisting of a phenyl group, a cyclopentyl group, a cyclohexyl group,

In another preferred embodiment of the invention, the compound of formula I has a structure represented by the following formula III:

Wherein ring A is selected from the group consisting of a substituted or unsubstituted 4-12-membered heterocyclic ring containing 1-3 heteroatoms selected from oxygen, sulfur and nitrogen atoms, a substituted or unsubstituted 5-12-membered heteroaromatic ring containing 1-3 heteroatoms selected from oxygen, sulfur and nitrogen atoms, a substituted and unsubstituted _C6 - _C10 aryl group, a substituted or unsubstituted _-C1 - _C6 alkylphenyl group, a substituted or unsubstituted _C3 - _C12 cycloalkyl group; M is selected from the group consisting of a chemical bond or -O-, -CHR-, a carbonyl group, S, -NH-, -NHC(O)-, -NHS(O) _2- , -NHC(O)NH-, -NHC(S)NH-, -COO-, -OS(O) _2- .

In another preferred embodiment of the invention, the compound of formula I has a structure represented by the following formula:

Where,

Rf is selected from the group consisting of H, halogen, cyano group, amino group, nitro group, hydroxyl group, sulfhydryl group, aldehyde group, carboxyl group, sulfonyl group, _C1 - _C4 alkyl-S(O) ₂ group, substituted or unsubstituted _C1 - _C6 alkyl group, substituted or unsubstituted _C1 - _C6 alkoxy group;

t is 0, 1, 2, 3, or 4;

ring A is selected from the group consisting of a substituted or unsubstituted 5-12-membered saturated ring (including a spirocyclic and bridged ring), a substituted or unsubstituted 5-12-membered heteroaromatic ring containing 1-3 heteroatoms selected from oxygen, sulfur and nitrogen atoms, a substituted and unsubstituted _C6 - _C10 aryl group containing at least one heteroatom selected from N or O in the ring.

In another preferred embodiment of the invention, at least one Rf is in the meta position of the junction of the benzene ring and the parent core.

In another preferred embodiment of the invention, the compound of formula I has a structure represented by the following formula:

Where,

Rf is selected from the group consisting of H, halogen, cyano group, amino group, nitro group, hydroxyl group, sulfhydryl group, aldehyde group, carboxyl group, sulfonyl group, _C1 - _C4 alkyl-S(O) ₂ group, substituted or unsubstituted _C1 - _C6 alkyl group, substituted or unsubstituted _C1 - _C6 alkoxy group;

t is 0, 1, 2, 3, or 4;

L represents N or CH;

ring A is selected from the group consisting of a substituted or unsubstituted 4-12-membered heterocyclic ring containing 1-3 heteroatoms selected from oxygen, sulfur and nitrogen atoms, a substituted or unsubstituted _C3 - _C12 cycloalkyl group, a substituted or unsubstituted 5-12-membered heteroaromatic ring containing 1-3 heteroatoms selected from oxygen, sulfur and nitrogen atoms, a substituted and unsubstituted _C6 - _C10 aryl group.

Where, when is the nitrogen atom in is a site of the compound, the NH indicated is N (i.e., the hydrogen atom in NH is missing to form the site of the compound).

In another preferred embodiment of the invention, the compound of formula I has a structure represented by the following formula IV:

wherein ring A is selected from the group consisting of a substituted or unsubstituted 4-7-membered heterocyclic ring containing 1-3 heteroatoms selected from oxygen, sulfur and nitrogen atoms, a substituted or unsubstituted 5-12-membered heteroaromatic ring containing 1-3 heteroatoms selected from oxygen, sulfur and nitrogen atoms, a substituted or unsubstituted _C6 - _C10 aryl group, a substituted or unsubstituted _-C1 - _C6 alkylphenyl group, a substituted or unsubstituted _C3 - _C12 cycloalkyl group;

M is selected from the group consisting of a chemical bond or -O-, -CHR-, carbonyl group, S, -NH-, -NHC(O)-, -NHS(O) ₂ -, -NHC(O)NH-, -NHC(S)NH-, -COO-, -OS(O) ₂ -;

ring B is selected from the group consisting of a substituted or unsubstituted 4-12-membered heterocyclic ring containing 1-3 heteroatoms selected from oxygen, sulfur and nitrogen atoms (including a monocyclic, fused, bridged or spirocyclic ring), a substituted or unsubstituted 5-12-membered heteroaromatic ring containing 13 heteroatoms selected from oxygen, sulfur and nitrogen atoms, a substituted and unsubstituted _C6 - _C10 aryl group, a substituted or unsubstituted _C3 - _C12 carbocyclic ring;

V is selected from the group consisting of a chemical bond or -O-, -CHR-, carbonyl group, S, -NH-, -NHC(O)-, -NHS(O) ₂ -, -NHC(O)NH-, -NHC(S)NH-, -СОО-, -OS(O) ₂ -.

In another preferred embodiment of the invention, the compound of formula I has a structure represented by the following formula:

where Rg is selected from the group consisting of halogen, cyano group, amino group, nitro group, hydroxyl group, sulfhydryl group, aldehyde group, carboxyl group, sulfonyl group, substituted or unsubstituted _C1 - _C6 alkyl group, substituted or unsubstituted _C1 - _C6 alkoxy group;

Ring B is selected from the group consisting of a substituted or unsubstituted 4-12-membered heterocyclic ring containing 1-3 heteroatoms selected from oxygen, sulfur and nitrogen atoms (including a monocyclic, fused, bridged or spirocyclic ring), a substituted or unsubstituted 5-12-membered heteroaromatic ring containing 1-3 heteroatoms selected from oxygen, sulfur and nitrogen atoms, a substituted and unsubstituted _C6 - _C10 aryl group, a substituted or unsubstituted _C3 - _C12 carbocyclic ring;

u is 0, 1, 2, 3, or 4.

In another preferred embodiment of the invention, the compound of formula I has a structure represented by the following formula:

where Rg is selected from the group consisting of halogen, cyano group, amino group, nitro group, hydroxyl group, sulfhydryl group, aldehyde group, carboxyl group, sulfonyl group, substituted or unsubstituted _C1 - _C6 alkyl group, substituted or unsubstituted _C1 - _C6 alkoxy group;

u is 0, 1, 2, 3, or 4.

In another preferred embodiment of the invention, ring A contains at least one substituent G, wherein the group G is selected from the group consisting of an amino group, =O, a substituted or unsubstituted 4-7-membered heterocyclic ring containing 1-3 heteroatoms selected from oxygen and nitrogen atoms, a substituted or unsubstituted _C2 - _C10 acyl group, a substituted and unsubstituted _C2 - _C10 ester group, and a substituted and unsubstituted _C1 - _C6 amide group.

Where, when is the nitrogen atom in is a site of the compound, the NH indicated is N (i.e. the hydrogen atom in NH is lost to form the site of the compound).

In another preferred embodiment of the invention, the compound of formula I has a structure selected from the group consisting of:

In another preferred embodiment of the invention, W is selected from the group consisting of:

In another preferred embodiment of the invention, W is selected from the group consisting of:

In another preferred embodiment of the invention, Rc is selected from the group consisting of:

In another preferred embodiment of the invention, URc is selected from the group consisting of:

In another preferred embodiment of the invention, Re is selected from the group consisting of an amino group, a substituted or unsubstituted C ₁ -C ₆ alkyl-NH group.

In a second aspect of the present invention there is provided a pharmaceutical composition comprising a therapeutically effective amount of one or more compounds of formula I, their pharmaceutically acceptable salts, racemates, R-isomers and S-isomers, stereoisomers or tautomers, as described in the first aspect of the invention, as well as one or more pharmaceutically acceptable carriers, excipients, additives, fillers and diluents.

In a third aspect of the present invention there is provided the use of compounds of formula I, their racemates, R-isomers, S-isomers or pharmaceutically acceptable salts, as described in the first aspect of the present invention, in the manufacture of a medicament for the treatment or prevention of diseases associated with abnormal gene levels or abnormal expression of kinases (e.g. due to mutation, deletion or transposition of the corresponding nucleotide sequences, or fusion or overexpression of said kinase), selected from the group consisting of FLT3, ALK, RET, ROS, AXL, EGFR.

In another preferred embodiment of the invention, the disease is selected from the group consisting of acute myeloid leukemia, neurofibroma type I, multiple myeloma, glioblastoma, non-small cell lung cancer, liver cancer, hepatocellular carcinoma, cervical cancer, lymphoma, bone metastases, hormone-refractory prostate cancer, hormone-dependent prostate cancer, thyroid adenoma, medullary thyroid cancer, mesothelioma, glioblastoma, sphincter metastases, Merkel cell carcinoma, urogenital tract tumor, bladder cancer, papillary thyroid cancer, breast cancer, soft tissue sarcoma, glioma, neuroendocrine tumor, renal cell carcinoma, advanced solid tumor, undifferentiated astrocytoma, gastrointestinal stromal tumor tract, von Hippel-Lindau syndrome, small cell lung cancer, pancreatic cancer, endocrine tumor of the pancreas, tumor of the central nervous system, metastatic renal cancer, endometrioid carcinoma, endometrioid adenocarcinoma, lung cancer, colorectal cancer, ovarian cancer, rhabdomyosarcoma, melanoma, retinoblastoma, tumor of the central and peripheral nervous system, acute leukemia, chronic leukemia, cholangiocarcinoma, bronchial carcinoma, esophageal cancer, testicular cancer, skin cancer, oral cancer, neuroblastoma, anaplastic large cell lymphoma.

In another aspect of the present invention, there is provided a conjugate formed between a compound of the present invention and a small biomolecule or a monoclonal antibody by chemical bonding.

It should be understood that, within the scope of the present invention, all technical features of the present invention, indicated above and given below (as examples), can be combined with each other to form new or preferred technical solutions that are not listed here due to limitations of scope.

DETAILED DESCRIPTION OF THE INVENTION

In the course of extensive and in-depth research, the inventors unexpectedly discovered for the first time a class of compounds with inhibitory activity against kinases (such as FLT3, ALK, AXL, EGFR). The present invention was made on this basis.

Terms

In the present invention, halogen is F, Cl, Br or I.

In the present invention, unless otherwise indicated, the terms used herein have the generally accepted meanings known to those skilled in the art. In the present invention, unless otherwise indicated, all chemical formulas are intended to cover all possible optical or geometric isomers (R-, S-isomers or racemates, or cis- and trans-isomers of olefins, etc.)

In the present invention, the term "C ₁ -C ₆ alkyl group" means a straight or branched chain alkyl group containing 16 carbon atoms, including, but not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl groups, etc.; while ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and tert-butyl groups are preferred.

In the present invention, the term "C1-C6 alkoxy group" means a straight or branched chain alkoxy group containing 16 carbon atoms, including, but not limited to, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, and other groups.

In the present invention, the term “C2-C6 alkenyl group” means a straight or branched chain alkenyl group containing a double bond and 2-6 carbon atoms, including, but not limited to, vinyl, propenyl, butenyl, isobutenyl, pentenyl, hexenyl and other groups.

In the present invention, the term "C2-C6 alkynyl group" means a straight or branched chain alkynyl group containing a triple bond and 2-6 carbon atoms, including, but not limited to, ethynyl, propynyl, butynyl, isobutynyl, pentynyl, hexynyl and other groups.

In the present invention, the term "C3-C10 cycloalkyl group" means a cycloalkyl group with a straight or branched chain containing 3-10 carbon atoms in the ring, including, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopeptyl, cyclooctyl, cyclodecyl and other groups. The terms "C3-C8 cycloalkyl group", "C3-C7 cycloalkyl group" and "C3-C6 cycloalkyl group" have similar meanings.

In the present invention, the term "C3-C10 cycloalkenyl group" means a cyclic alkenyl group with 3-10 carbon atoms in the ring, including, but not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, cyclodecylene groups. The term "C3-C7 cycloalkenyl group" has a similar meaning.

In the present invention, the term “C1-C12 alkoxycarbonyl group” means an alkoxycarbonyl group with 1-12 carbon atoms in the alkyl chain, including, but not limited to, methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl and other groups.

In the present invention, the term “C1-C12 alkylaminocarbonyl group” means an alkylaminocarbonyl group with 1-12 carbon atoms in the alkyl chain, including, but not limited to, methylaminocarbonyl, ethylaminocarbonyl, propylaminocarbonyl, isopropylaminocarbonyl, tert-butylaminocarbonyl, benzylaminocarbonyl, dimethylaminocarbonyl and other groups.

In the present invention, the term "C5-C9 furanosyl group" means a furanosyl group with 5-9 carbon atoms, where the 1-position of the glycosyl group is bonded to the main chain, including, but not limited to, ribofuranosyl, deoxyribofuranosyl, galactofuranosyl and other groups.

In the present invention, the term "C5-C9 pyranosyl group" means a pyranosyl group with 5-9 carbon atoms, wherein the 1-position of the glycosyl group is bonded to the main chain, including, but not limited to, glucopyranosyl, glucopyranosyl, rhamnopyranosyl, galactopyranosyl, mannopyranosyl and xylopyranosyl groups.

In the present invention, the terms "aromatic ring" or "aryl group" have a similar meaning, wherein "aryl group" preferably means "C6-C12 aryl group" or "C6-C10 aryl group". The term "C6-C12 aryl group" means an aromatic cyclic group with 6-12 carbon atoms without heteroatoms in the ring, for example, phenyl, naphthyl and other groups. The term "C6-C10 aryl group" has a similar meaning.

In the present invention, the terms "aromatic heterocyclic ring" or "heteroaryl group" have a similar meaning and mean a heteroaromatic group containing one or more heteroatoms. The heteroatoms mentioned herein include oxygen, sulfur and nitrogen. These groups include furyl, thienyl, pyridyl, pyrazolyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl and other groups. The heteroaryl ring can be fused with an aryl ring, a heterocyclyl ring or a cycloalkyl ring, where the ring bound to the parent structure is a heteroaryl ring. The heteroaryl group can be further substituted or unsubstituted.

In the present invention, the term "3- to 12-membered heterocyclyl group" means a saturated or unsaturated 3- to 12-membered cyclic group containing 1-3 heteroatoms selected from oxygen, sulfur and nitrogen atoms in the ring, for example, dioxolanyl and other groups. The term "3- to 7-membered heterocyclyl group" has a similar meaning.

In the present invention, the term "substituted" means that one or more hydrogen atoms in a specified group are replaced by specified substituents. Specified substituents are those described above or exemplified. Unless otherwise indicated, a substituted group may have a substituent selected from the specified group at any substitutable position of the group, and the substituent at each position may be the same or different. A cyclic substituent, such as a heterocyclyl group, may be attached to another ring, such as a cycloalkyl group, to form a spirobicyclic system, such as when two rings share one carbon atom. Those skilled in the art will appreciate that the combinations of substituents contemplated in the present invention are stable combinations or those combinations obtainable by chemical synthesis. Substituents may be (but are not limited to): C1-8 alkyl group, C2-8 alkenyl group, C2-8 alkynyl group, C3-8 cycloalkyl group, 3-12-membered heterocyclyl group, aryl group, heteroaryl group, halogen, hydroxyl group, carboxyl (-COOH) group, C1-8 aldehyde group, C2-10 acyl group, C2-10 ester group, C1-C12 alkoxycarbonyl group, amino group, alkoxy group, C1-10 sulfonyl group and other groups.

FLT3 inhibitor compound

The present invention provides a compound with FLT3 inhibitory activity:

Where each group has the definition given above. Preferred compounds in the present application are compounds selected from the following table 1:

Where compounds 162 and 163 are respectively, but the absolute configuration is unknown. Where compounds 164 and 165 are respectively, but the absolute configuration is unknown. Where compounds 284 and 285 are accordingly, but the absolute configuration is unknown.

Pharmaceutical compositions and routes of administration

Since the compounds of the present invention have high inhibitory activity against kinases, the compounds of the present invention and their various crystalline forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, as well as a pharmaceutical composition containing the compound of the present invention as the main active ingredient, can be used for the treatment, prevention and relief of diseases caused by abnormal activity or abnormal expression of kinases (e.g., FLT3).

The pharmaceutical composition of the present invention comprises a safe and effective amount of the compound of the present invention or a pharmacologically acceptable salt thereof, as well as a pharmacologically acceptable excipient or carrier. The term "safe and effective amount" means that the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dosage unit, more preferably 5-200 mg of the compound of the present invention per dosage unit. The "dosage unit" is preferably a capsule or a tablet.

The term "pharmaceutically acceptable carrier" refers to one or more compatible solid, liquid or gel excipients suitable for use in medicine, and they must be sufficiently pure and of low toxicity. "Compatibility" means that each component of the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the effectiveness of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (e.g. sodium carboxymethylcellulose sodium ethylcellulose cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g. stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g. soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g. propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (e.g. Tween®), wetting agents (e.g. sodium dodecyl sulfate), colorants, flavors, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

The route of administration of the compound or pharmaceutical composition of the present invention is not particularly limited, and common routes of administration include oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous) and local administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active substance is mixed with at least one conventional inert excipient (or carrier), for example, sodium citrate or calcium hydrogen phosphate, or mixed with any of the following: (a) fillers or compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicon dioxide; (b) binders, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) wetting agents, for example, glycerol; (d) with disintegrating agents, for example agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain mixed silicates and sodium carbonate; (e) with release retardants, for example paraffin, (f) with absorption accelerators, for example a quaternary ammonium compound; (g) with wetting agents, for example cetyl alcohol and glycerol monostearate; (h) with adsorbents, for example kaolin; and (i) with lubricants, for example talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or a mixture thereof. Capsule, tablet and pill dosage forms may also contain buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills and granules may be coated, such as enteric coated, and may also contain other materials used in this field. They may contain opacifiers, whereby the release of the active substance or compound in such a composition may occur with a delay in a certain part of the digestive tract. Examples of inclusion components that may be used are polymeric materials and wax materials. If necessary, the active substance may also be in microencapsulated form with one or more of the above-mentioned auxiliary substances.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active substance, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, or mixtures of these substances.

In addition to these inert diluents, the compositions may also contain auxiliary substances, such as wetting, emulsifying and suspending agents, sweeteners, flavoring agents and taste additives.

In addition to the active substance, the suspension may also contain a suspending agent, such as ethoxylated isooctadecanol, polyoxyethylene sorbitol and dehydrated sorbitan ester, microcrystalline cellulose, aluminum methoxide and agar, or a mixture thereof, etc.

Compositions for parenteral administration may be physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions and sterile powders that can be reconstituted into sterile injection solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients are water, ethanol, polyols and any suitable mixtures thereof.

Dosage forms of the compounds of the invention for topical application are ointments, powders, patches, sprays and inhalation dosage forms. The active substance is mixed under sterile conditions with a physiologically acceptable excipient and any preservatives, buffer solutions or nebulizers that may be required if necessary.

The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds. In certain preferred embodiments, the compounds of the present invention may be administered with other small molecule compounds to form PROTACs, or with other large molecule compounds, such as monoclonal antibodies, to form ADCs.

When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is administered to a mammal (e.g., a human) in need of treatment, and the dosage when administered is a pharmaceutically effective dose. For humans weighing 60 kg, the daily dose is usually 1-2000 mg, preferably 50-1000 mg. Of course, for specific doses, factors such as the route of administration, the health condition of the patient, etc., which are within the competence of a qualified physician, should also be taken into account.

The present invention will be further illustrated below with reference to specific examples. It should be understood that these examples are used only to illustrate the invention and are not intended to limit the scope of the invention. In the experimental methods in the following examples, in which specific conditions are not indicated, these conditions generally correspond to those generally accepted or they correspond to those specified in the manufacturer's instructions. Unless otherwise indicated, percentages and values are calculated by weight.

Abbreviations are defined as follows:

The starting materials may be purchased or obtained by methods known or disclosed in the art.

Purification of intermediates and compounds is carried out by conventional methods used in chemical laboratories, such as normal or reversed-phase chromatography, or by recrystallization. Normal-phase chromatography is carried out on a chromatographic column pre-packed with silica gel or by preparative thin-layer chromatography. Silica gel chromatographic columns are mainly glass columns or use a high-performance preparative chromatography system. The mobile phase for normal-phase chromatography is selected from petroleum ether/ethyl acetate, dichloromethane/methanol or other suitable solvents, and elution is carried out at a given ratio. Reversed-phase preparative liquid chromatography is carried out on a C18 column using a preparative liquid chromatograph or a high-performance preparative chromatography system with detection at 214 nm and 254 nm, or using a combined preparative liquid chromatography and mass spectrometry system. For gradient elution, 0.1% hydrochloric acid in water/acetonitrile, water/acetonitrile, 0.1% ammonium bicarbonate in water/acetonitrile, 0.1% formic acid in water/acetonitrile, 0.1% ammonia in water/acetonitrile, 0.1% trifluoroacetic acid in water/acetonitrile or other suitable solvent systems are used as the mobile phase.

The structures of intermediates and compounds are established by nuclear magnetic resonance (NMR) and mass spectrometry (LC-MS). For NMR, a Bruker Ascend 400 or Varian 400 or ZKNJ BIXI-1 300 MHz or Bruker Avance III 400 MHz or Bruker AVANCE Neo 400 MHz NMR spectrometer is used. The solvents used are deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol or other deuterated solvents. Spectral data are presented as follows: chemical shift δ (number of split peaks, spin-spin interaction constant J (Hz), number of hydrogen atoms). Tetramethylsilane is used as an internal standard for chemical shift, and its chemical shift is taken to be zero (δ, 0 ppm). Meanings of several abbreviations: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), brs (broad singlet).

The following representative liquid chromatography/mass spectrometry (LC-MS) methods were used to establish the structural characteristics of the intermediates and compounds:

Method I: Analysis is performed using an Agilent LC1260 system coupled to a 6120 single quadrupole mass spectrometer.

Column: Waters CORTECS C-18, 2.7 μm, 4.6×30 mm. Solvent A: 0.05% aqueous formic acid, solvent B: 0.05% formic acid in acetonitrile, linear increase in acetonitrile concentration from 5% to 95% over one minute, hold for one minute, total 2.5 min; flow rate: 1.8 mL/min; column temperature: 40°C.

Column: XSelect CSH C18, 3.5 μm, 4.6×50 mm. Solvent A: 0.05% aqueous ammonia, solvent B: 0.05% ammonia in acetonitrile, increasing the concentration of acetonitrile from 5% to 95% in one minute, holding for one minute, total 2.5 min; flow rate: 1.8 ml/min; column temperature: 40°C.

Method II: The analysis was performed using an Agilent LC/MSD 1200 system coupled to a quadrupole mass spectrometer. Column: ODS 2000 (50×4.6 mm, 5 μm) (ESI (+) or (-) ionization mode), column temperature: 30°C; flow rate 1.5 ml/min.

General methods: synthesis of compound 8

Example 1: 3-ethyl-8-(3-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)-N2-(tetrahydropyran-4-yl)pyrido[3,4-b]pyrazine-2,5-diaminetrihydrochloride (compound 8)

Step 1: 1-Benzyl-4-piperidone (5.00 g, 26.5 mmol) and 1-tert-butoxycarbonylpiperazine (5.41 g, 29.1 mmol) were dissolved in dichloromethane (100 mL). Acetic acid (2.38 g, 39.7 mmol) was added and the mixture was stirred for 5 h at room temperature. Sodium triacetoxyborohydride (22.4 g, 106 mmol) was then added portionwise and the solution was stirred overnight at room temperature. The solution was concentrated and water (100 mL) was added to the residue. The pH of the solution was adjusted to 10 with 5% sodium hydroxide solution and extracted three times with ethyl acetate (50 mL × 3). The organic phases were combined and washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was purified on a C18 reverse-phase column (increasing the acetonitrile content in water from 50% to 90%) to give 4-(1-benzylpiperidin-4-yl)piperazine-1-carboxylic acid tert-butyl ester as a yellow solid (5.80 g, 61% yield). MS result: 360.4 [M+H] ⁺ .

Step 2: tert-Butyl 4-(1-benzylpiperidin-4-yl)piperazine-1-carboxylate (5.80 g, 16.1 mmol) was dissolved in methanol (100 mL) and 10% palladium on carbon (1.5 g) was added. The mixture was stirred at 50 °C overnight under hydrogen (50 psi). At the end of the reaction, the solution was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure to give tert-butyl 4-(piperidin-4-yl)piperazine-1-formate as a white solid (4.15 g, 96% yield). ¹ H-NMR (300 MHz, CDCl ₃ ): δ 3.44-3.41 (m, 4H), 3.17-3.13 (m, 2H), 2.63-2.58 (m, 2H), 2.55-2.49 (m, 4H), 2.41-2.31 (m, 1H), 1.82-1.78 (m, 2H), 1.46 (s, 9H), 1.43-4.33 (m, 2H).

Step 3: 1-Fluoro-2-methoxy-4-nitrobenzene (2.00 g, 11.7 mmol) and tert-butyl 4-(piperidin-4-yl)piperazine-1-carboxylate (3.46 g, 12.9 mmol) were dissolved in N,N-dimethylformamide (30 mL). Then potassium carbonate (3.23 g, 23.4 mmol) was added. The mixture was stirred at 100 °C for 2 h. At the end of the reaction, the solution was cooled to room temperature, then water (100 mL) was added. The solution was extracted twice with ethyl acetate (30 mL × 2). The organic phases were combined and washed twice with brine (10 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give tert-butyl 4-(1-(2-(2-methoxy-4-nitrophenyl)piperidin-4-yl)piperazine-1-carboxylate as a yellow solid (4.90 g, 99% yield). 421.0 [M+1] ⁺ .

Step 4: tert-Butyl 4-(1-(2-(2-methoxy-4-nitrophenyl)piperidin-4-yl)piperazine-1-carboxylate (4.90 g, 11.7 mmol) was dissolved in methanol (50 mL) and 10% palladium on carbon (0.5 g) was added. The mixture was stirred at 50 °C overnight under hydrogen atmosphere (from a balloon). At the end of the reaction, the mixture was cooled to room temperature, filtered and the filtrate was concentrated under reduced pressure to give tert-butyl 4-(1-(4-amino-2-methoxyphenyl)piperidin-4-yl)piperazine-1-carboxylate as a violet solid (4.50 g, 99% yield). ¹ H NMR (400 MHz, CDCl ₃ ): δ 6.77 (d, J=8.4 Hz, 1H), 6.26-6.23 (multiplet, 2H), 3.81 (singlet, 3H), 3.50-3.38 (multiplet, 8H), 2.55-2.54 (multiplet, 2H), 2.50-2.47 (multiplet, 2H), 2.45-2.37 (multiplet, 1H), 1.83-1.78 (multiplet, 4H), 1.46 (singlet, 9H).

Step 5: tert-Butyl 4-(1-(4-amino-2-methoxyphenyl)piperidin-4-yl)piperazine-1-carboxylate (3.40 g, 8.72 mmol) and diiodomethane (7.00 g, 26.1 mmol) were dissolved in acetonitrile (100 mL). Isoamyl nitrite (1.53 g, 13.1 mmol) was added. The mixture was stirred at 80 °C for 4 h under nitrogen blanket. At the end of the reaction, the mixture was cooled to room temperature and concentrated under reduced pressure. To the resulting residue were added dichloromethane (30 mL), water (20 mL), and saturated sodium carbonate solution (10 mL). The mixture was extracted three times with dichloromethane (20 mL × 3). The organic phases were combined, washed with saturated brine (10 ml), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (petroleum ether/ethyl acetate = 2:1 - ethyl acetate) to give tert-butyl 4-(4-(4-iodo-2-methoxyphenyl)cyclohexyl)piperazine-1-carboxylate as a brown solid (1.85 g, yield 42%). ¹ H-NMR (300 MHz, CDCl ₃ ) δ 7.21 (doublet of doublets, J = 8.1, 1.8 Hz, 1H), 7.09 (doublet, J = 1.5 Hz, 1H), 6.65 (doublet, J = 8.4 Hz, 1H), 3.84 (s, 3H), 3.52-3.48 (m, 2H), 3.46-3.42 (m, 4H), 2.56-2.47 (m, 6H), 2.42-2.38 (m, 1H), 1.87-1.74 (m, 4H), 1.46 (s, 9H). MS Result: 502.5 [M+H] ⁺ .

Step 6: tert-Butyl 4-(4-(4-iodo-2-methoxyphenyl)cyclohexyl)piperazine-1-carboxylate (1.85 g, 3.69 mmol) was dissolved in N,N-dimethylformamide (100 mL). Pinacol diborate (1.13 g, 4.43 mmol), potassium acetate (1.09 g, 11.1 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (135 mg, 0.17 mmol) were added. The mixture was stirred at 100 °C for 5 h under nitrogen blanket. At the end of the reaction, the solution was cooled to room temperature and poured into water (100 mL). Saturated sodium carbonate solution (10 mL) was added. The mixture was extracted three times with ethyl acetate (30 mL × 3). The organic phases were combined, washed with saturated brine (10 ml), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (petroleum ether/ethyl acetate = 1:1 - ethyl acetate) to give 4-(1-(2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)piperidin-4-yl)-1-tert-butoxycarbonylpiperazine as a yellow solid (intermediate A, 1.30 g, yield 70%). ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.39 (d, J=7.8 Hz, 1H), 7.25 (s, 1H), 6.93 (d, J=7.8 Hz, 1H), 3.91 (s, 3H), 3.64-3.60 (m, 2H), 3.46-3.43 (m, 4H), 2.62-2.55 (m, 6H), 2.50-2.41 (m, 1H), 1.89-1.76 (m, 4H), 1.47 (s, 9H), 1.33 (s, 12H). MS Result: 502.6 [M+H] ⁺ .

Step 7: 5-bromo-2-chloropyridine-3,4-diamine (5.00 g, 22.5 mmol) and ethyl 2-oxobutyrate (3.51 g, 27.0 mmol) were dissolved in ethanol (100 mL) and acetic acid (0.2 mL). The mixture was stirred at 90 °C for 36 h under nitrogen blanket. At the end of the reaction, the temperature was lowered to 50 °C. The solution was filtered and the filter cake was washed with ethanol (10 mL×3). The filtrate was concentrated under reduced pressure to give a residue, which was then dissolved in ethanol (20 mL). The solution was refluxed for 10 min, then cooled to 50 °C, filtered, and the filter cake was washed with ethanol (10 mL × 2), dried to give 8-bromo-5-chloro-3-ethylpyrido[3,4-b]pyrazin-2(1H)-one as a yellow solid (4.1 g, 63% yield). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.32 (br s, 1H), 8.50 (s, 1H), 2.86 (quartet, J=7.6 Hz, 2H), 1.25 (t, J=7.6 Hz, 3H).

Step 8: 8-bromo-5-chloro-3-ethylpyrido[3,4-b]pyrazin-2(1H)-one (3.10 g, 10.7 mmol) was suspended in dichloromethane (50 mL) and 5 drops of N,N-dimethylformamide and oxalyl chloride (5.46 g, 43.0 mmol) were added. The mixture was stirred at 40 °C overnight under nitrogen blanket. At the end of the reaction, the mixture was cooled to room temperature and concentrated under reduced pressure. To the resulting residue were added dichloromethane (50 mL), water (30 mL), and saturated sodium carbonate solution (10 mL). The mixture was extracted five times with dichloromethane (30 mL×5). The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 8-bromo-2,5-dichloro-3-ethylpyrido[3,4-b]pyrazine as a yellow solid (2.0 g, 61% yield). ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 8.91 (s, 1H), 3.20 (quartet, J=7.2 Hz, 2H), 1.39 (t, J=7.2 Hz, 3H).

Step 9: Tetrahydropyran-4-amine (1.35 g, 9.77 mmol) was suspended in ethanol (20 mL) and N,N-diisopropylethylamine (2.52 g, 19.5 mmol) was added. The solution was stirred at room temperature for half an hour. 8-Bromo-2,5-dichloro-3-ethylpyrido[3,4-b]pyrazine (2.00 g, 6.51 mmol) was suspended in ethanol (30 mL) and N,N-diisopropylethylamine (1.68 g, 13.0 mmol) was added. The solution was stirred at room temperature for half an hour. The two solutions were combined and stirred at 70 °C overnight. At the end of the reaction, the mixture was cooled to room temperature and concentrated under reduced pressure. Water (30 mL) was added to the residue and the resulting solution was filtered. The filter cake was washed with dichloromethane/methanol = 10/1 (5 ml×3) and dried to give 8-bromo-5-chloro-3-ethyl-N-(tetrahydropyran-4-yl)pyrido[3,4-b]pyrazin-2-amine as a yellow solid (1.2 g, yield 50%). ¹ H-NMR (300 MHz, DMSO-d ₆ ) δ 8.50 (s, 1H), 7.81 (d, J=7.2 Hz, 1H), 4.42-4.31 (m, 1H), 3.98-3.94 (m, 2H), 3.43 (t, J=7.5 Hz, 2H), 2.92 (quartet, J=7.2 Hz, 2H), 1.98-1.93 (m, 2H), 1.80-1.66 (m, 2H), 1.31 (t, J=7.2 Hz, 3H).

Step 10: To a mixture of 8-bromo-5-chloro-3-ethyl-N-(tetrahydropyran-4-yl)pyrido[3,4-b]pyrazin-2-amine (0.960 g, 2.59 mmol) and 4-(1-(2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)phenyl)piperidin-4-yl)-1-tert-butoxycarbonylpiperazine (Intermediate A, 1.30 g, 2.59 mmol) in N,N-dimethylacetamide (50 mL) was added an aqueous solution (5 mL) of sodium carbonate (824 mg, 7.78 mmol) and [11,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (190 mg, 0.26 mmol). The mixture was stirred at 100 °C for 3 h under nitrogen blanket. At the end of the reaction, the mixture was cooled to room temperature and poured into water (150 mL). The mixture was extracted three times with ethyl acetate (30 mL × 3). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (ethyl acetate: dichloromethane/methanol/25% aqueous ammonia = 30:1:0.1) to give the crude product. The crude product was purified by reverse phase C18 column chromatography (increasing the acetonitrile content in water from 50% to 80%) to give tert-butyl 4-(1-(4-(5-chloro-3-ethyl)-2-((tetrahydro-2H-pyran-4-yl)amino)pyridyl[3,4-b]pyrazin-8-yl)-2-methoxyphenyl)piperidin-4-ylpiperazine-1-carboxylate as a yellow solid (0.60 g, 35% yield). ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 8.32 (s, 1H), 7.56 (d, J=7.2 Hz, 1H), 7.30-7.28 (m, 2H), 6.97-6.95 (m, 1H), 4.19-4.11 (m, 1H), 3.93-3.89 (m, 2H), 3.83 (s, 3H), 3.52-3.49 (m, 2H), 3.31-3.30 (m, 4H), 3.25-3.22 (m, 2H), 2.90 (quartet, J=7.2 Hz, 2H), 2.61-2.57 (multiplet, 2H), 2.50-2.41 (multiplet, 4H), 2.38-2.32 (multiplet, 1H), 1.88-1.81 (multiplet, 4H), 1.71-1.55 (multiplet, 4H), 1.40 (singlet, 9H), 1.32 (triplet, J=7.2 Hz, 3H). The result obtained by the MS method: 666.8 [M+H] ⁺ .

Step 11: To a mixture of tert-butyl 4-(1-(4-(5-chloro-3-ethyl-2-((tetrahydro-2H-pyran-4-yl)amino)pyridinyl[3,4-b]pyrazin-8-yl)-2-methoxyphenyl)piperidin-4-ylpiperazine-1-carboxylate (0.30 g, 0.45 mmol), (2,4-dimethoxyphenyl)methylamine (113 mg, 0.680 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (26 mg, 0.045 mmol), and sodium tert-butoxide (130 mg, 1.35 mmol) in toluene (10 mL) was added tris(dibenzylideneacetone)dipalladium (21 mg, 0.024 mmol). The mixture stirred at 110°C for 3 h under nitrogen blanket. At the end of the reaction, the mixture was cooled to room temperature and concentrated under reduced pressure. Ethyl acetate (20 mL) and water (30 mL) were added to the residue. The mixture was extracted three times with ethyl acetate (10 mL×3). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (dichloromethane/methanol = 30:1) to give the crude product. The crude product was purified by reverse phase C18 column chromatography (increasing the acetonitrile content in water from 40% to 90%) to give tert-butyl 4-(1-(4-(5-((2,4)-dimethoxybenzyl)amino)-3-ethyl-2-((tetrahydro-2H-pyran-4-yl)amino)pyridinyl[3,4-b]pyrazin-8-yl)-2-methoxyphenyl)piperidin-4-yl)piperazine-1-carboxylic acid as a yellow solid (0.13 g, 36% yield). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.16 (s, 1H), 7.35-7.33 (m, 1H), 7.30-7.27 (m, 1H), 7.16 (s, 1H), 6.96-6.94 (m, 2H), 6.50-6.49 (m, 1H), 6.46-6.43 (m, 1H), 4.80-4.78 (m, 2H), 4.76-4.74 (m, 1H), 4.25-4.16 (m, 1H), 4.01-3.98 (m, 2H), 3.89-3.88 (m, 6H), 3.80 (singlet, 3H), 3.62-3.59 (multiplet, 2H), 3.50-3.45 (multiplet, 6H), 2.70 (quartet, J=7.2 Hz, 2H), 2.64-2.59 (multiplet, 6H), 2.53-2.45 (multiplet, 1H), 2.08-2.05 (multiplet, 2H), 1.89-1.82 (multiplet, 4H), 1.56-1.52 (multiplet, 2H), 1.47 (singlet, 9H), 1.41 (triplet, J=7.2 Hz, 3H). MS result: 797.5 [M+H] ⁺ .

Step 12: To a mixture of tert-butyl 4-(1-(4-(5-((2,4-dimethoxybenzyl)amino)amino)-3-ethyl-2-((tetrahydro-2H-pyran-4-yl)amino)pyridinyl[3,4-b]pyrazin-8-yl)-2-methoxyphenyl)piperidin-4-yl)piperazine-1-carboxylic acid (0.13 g, 0.16 mmol) in dichloromethane (2 mL) was added trifluoroacetic acid (1 mL). The reaction mixture was stirred at 30 °C for 1 h and concentrated. To the resulting residue was added water (2 mL) and the pH was adjusted to 10 with 5% sodium hydroxide solution. The solution was extracted six times (10 mL×6) with dichloromethane/methanol = 10/1. The organic phases were combined, washed with brine (5 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 3-ethyl-8-(3-methoxy-4-(4-(piperazin-1-yl)piperidin-1-yl)phenyl)-N2-(tetrahydro-2H-pyran-4-yl)pyridine[3,4-b]pyrazine-2,5-diamine as a yellow solid (90 mg, 100% yield). MS result: 547.6 [M+H] ⁺ .

Step 13: To a mixture of 3-ethyl-8-(3-methoxy-4-(4-(piperazin-1-yl)piperidin-1-yl)phenyl)-N2-(tetrahydro-2H-pyran-4-yl)pyridine[3,4-b]pyrazine-2,5-diamine (90 mg, 0.16 mmol) in dichloromethane (2 mL) and methanol (1 mL) were added 37% aqueous formaldehyde (15 mg, 0.18 mmol) and 1 drop acetic acid. After stirring at room temperature for 20 min, sodium cyanoborohydride (31 mg, 0.49 mmol) was added. After stirring at room temperature for 30 min, water (2 mL) was added and the mixture was basified to pH 10 with 5% sodium hydroxide solution. The mixture was extracted six times (10 ml×6) with dichloromethane/methanol = 10/1. The organic phases were combined and concentrated under reduced pressure to give the crude product. The crude product was purified by reversed-phase C18 column chromatography (increasing the acetonitrile content in water from 10% to 50%, the aqueous phase containing 0.1% hydrochloric acid) to give 3-ethyl-8-(3-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)-N2-(tetrahydro-2H-pyran-4-yl)pyridine[3,4-b]pyrazine-2,5-diamine trihydrochloride as a yellow solid (65 g, yield 61%). ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 13.77 (s, 1H), 12.48-12.17 (m, 2H), 8.54 (s, 2H), 7.96-7.94 (m, 2H), 7.55 (broad s, 1H), 7.43-7.32 (m, 2H), 4.14-4.05 (m, 1H). 3.95 (singlet, 3H), 3.92-3.89 (multiplet, 2H), 3.79-3.60 (multiplet, 12H), 3.35-3.27 (multiplet, 1H), 3.25-3.22 (multiplet, 2H), 2.92-2.86 (multiplet, 5H), 2.35-2.25 (multiplet, 4H), 1.83-1.78 (multiplet, 2H), 1.74-1.64 (multiplet, 2H), 1.33 (triplet, J=7.2 Hz, 3H). MS Result: 561.4 [M+H] ⁺ .

The compounds listed in the tables were prepared using the same method and the appropriate starting materials.

The compounds in the following table were prepared by the synthesis of compound 8, with the tetrahydropyran-4-amine in step 9 being replaced by the corresponding starting materials listed in the table:

The compounds in the following table were prepared by the synthesis of compound 8, wherein the tetrahydropyran-4-amine in step 9 was replaced by the corresponding starting materials indicated in the table, and the corresponding product in step 11 was hydrolyzed with alkali to remove the acetyl protecting group:

The compounds in the following table were prepared in a manner similar to the synthesis of compound 8, replacing 1-fluoro-2-methoxy-4-nitrobenzene in step 3 with the corresponding starting materials listed in the table:

The compounds in the following table were prepared by the synthesis of compound 8, replacing intermediate A with the corresponding starting materials listed in the table below for reaction with 8-bromo-5-chloro-3-ethyl-N-(tetrahydropyran-4-yl)pyrido[3,4-b]pyrazin-2-amine:

The product in step 10 of the method for synthesizing compound 8 is compound 12, shown in the following table:

Compound 11 in the following table was prepared by deprotection in step 12 of the synthesis method of compound 8, using compound 12 as the starting material:

The compounds in the following table were prepared by the synthetic route described in Example 11, using the appropriate starting materials listed in the table:

The compound in the following table was prepared by the synthesis of compound 8, replacing (2,4-dimethoxyphenyl)methylamine with trimethylboroxane and using a Suzuki reaction in step 11:

The compounds in the following table were prepared by the method of synthesis of compound 8, with ethyl 2-oxobutyrate being replaced by the corresponding starting materials listed in the table:

The compounds in the following table were prepared by the method of synthesis of compound 8, replacing tert-butyl 4-(piperidin-4-yl)piperazine-1-carboxylate with the corresponding starting materials listed in the table:

Example 2: The intermediate product tert-butyl 4-(1-(4-(5-((2,4-dimethoxybenzyl)amino)-3-ethyl-2-((tetrahydro-2H-pyran-4-yl)amino)pyridino[3,4-b]pyrazin-8-yl)-2-methoxyphenyl)piperidin-4-yl)piperazine-1-carboxylic acid obtained in Step 11 of Example 1 can also be synthesized by the following method, after which compound 8 can be obtained in Steps 12 and 13 in Example 1:

Step 1: A mixture of 8-bromo-5-chloro-3-ethyl-N-(tetrahydropyran-4-yl)pyridino[3,4-b]pyrazin-2-amine (6.00 g, 16.1 mmol) and 2,4-dimethoxybenzamine (15 g, 90 mmol) was stirred at 145 °C overnight. At the end of the reaction, the mixture was cooled to room temperature and purified by silica gel column chromatography (petroleum ether/ethyl acetate = 5:1 - 3:1) to give 8-bromo-N5-(2,4-dimethoxybenzyl)-3-ethyl-N2-(tetrahydro-2H-pyran-4-yl)pyridino[3,4-b]pyrazine-2,5-diamine as a yellow solid (5.5 g, yield 68%). The result obtained by the MS method: 502.5 [M+H] ⁺ .

Step 2: To a mixture of 8-bromo-N5-(2,4-dimethoxybenzyl)-3-ethyl-N2-(tetrahydro-2H-pyran-4-yl)pyridino[3,4-b]pyrazine-2,5-diamine (4.00 g, 7.97 mmol) and intermediate A (4.0 g, 7.97 mmol) in 1,4-dioxane (100 mL) were added cesium carbonate (5.20 g, 15.9 mmol) in water (15 mL) and Pd(dppf) _Cl2 (583 mg, 0.80 mmol). The mixture was stirred and reacted at 110 °C for 3 h under nitrogen blanket. At the end of the reaction, the mixture was cooled to room temperature and concentrated under reduced pressure, and ethyl acetate (50 mL) and water (50 mL) were added. The solution was extracted three times with ethyl acetate (50 ml×3). The combined organic phases were washed with saturated brine (10 ml), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (petroleum ether/ethyl acetate = 1:1-0:1 - ethyl acetate/methanol = 20:1) to give intermediate B as a yellow solid (3.1 g, yield 49%).

The compounds in the following table were prepared in Step 9 of Example 1, whereby tetrahydropyran-4-amine was replaced by the corresponding starting materials listed in the table and reacted with intermediate M according to the method for synthesizing compound 8 in Example 2:

The compounds in the following table were prepared by the method described for synthesizing compound 8 in Example 1, with the tetrahydropyran-4-amine in step 9 being replaced by the corresponding starting materials shown in the table, and then prepared according to the method described in Example 2:

Compound 10 in the following table was prepared by the synthetic route described in Example 2, with 2,4-dimethoxybenzamine replaced by methylamine.

Compound 23 in the following table was prepared by the synthesis method of intermediate A in Example 1 and the synthesis method in Example 2, wherein 1-fluoro-2-methoxy-4-nitrobenzene was replaced by the starting material indicated in the table:

Compound 31 in the following table was prepared by the synthesis method of intermediate A in Example 1 and the synthesis method in Example 2, wherein 1-fluoro-2-methoxy-4-nitrobenzene and tert-butyl 4-(piperidin-4-yl)piperazine-1-carboxylate were replaced by 1-fluoro-2-methyl-4-nitrobenzene and tert-butyl 3,9-diazaspiro[5,5]undecane-3-carboxylate, respectively, shown in the table.

The compounds in the following table were prepared by the synthesis method of intermediate A in Example 1 and the synthesis method in Example 2, wherein 2,4-(piperidin-4-yl)piperazine-1-carboxylic acid tert-butyl ester was replaced by the corresponding starting materials indicated in the table.

The compounds in the following table were prepared according to the synthesis method of intermediate A in Example 1 and the synthesis method in Example 2, with 1-benzyl-4-piperidone being replaced by the starting material indicated in the table.

The compounds in the following table were prepared according to the method described in Example 1, replacing ethyl 2-oxobutyrate ester in step 7 with the starting material shown in the table, followed by the method described in Example 2.

The compounds in the following table were prepared according to the synthesis method of intermediate A in Example 1 and the synthesis method in Example 2, with 4-(4-(4-iodo-2-methoxyphenyl)cyclohexyl)piperazine-1-tert-butyl formate being replaced by the starting material indicated in the table.

Example 3: 3-ethyl-8-(6-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)pyridin-3-yl)-N2-tetrahydro-2H-pyran-4-yl)pyridino [3,4-b]pyrazine-2,5-diamine (compound 21)

Step 1: 5-bromo-2-fluoropyridine (500 mg, 2.84 mmol) and 1-methyl-4-(4-piperidinyl)piperazine (520 mg, 2.84 mmol) were dissolved in N,N-dimethylformamide (10 mL) and potassium carbonate (784 mg, 5.68 mmol) was added. The mixture was stirred and reacted at 110 °C for 6 h. At the end of the reaction, the mixture was cooled to room temperature and water (50 mL) was added. The solution was extracted with ethyl acetate (20 mL×3). 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 resulting residue was purified by silica gel chromatography (dichloromethane:methanol = 20:1 - 10:1) to give a white solid (800 mg, yield 83%). ¹ H-NMR (400 MHz, CDCl ₃ ): δ 8.16 (d, J = 2.0 Hz, 1H), 7.49 (doublet of doublets, J = 8.8, 2.0 Hz, 1H), 6.56 (d, J = 8.8 Hz, 1H), 4.29-4.26 (m, 2H), 2.86-2.79 (m, 2H), 2.71-2.55 (m, 4H), 2.49-2.42 (m, 5H), 2.28 (s, 3H), 1.93-1.85 (m, 2H), 1.57-1.47 (m, 2H).

Step 2: To a mixture of 8-bromo-N5-(2,4-dimethoxybenzyl)-3-ethyl-N2-(tetrahydro-2H-pyran-4-yl)pyridino[3,4-b]pyrazine-2,5-diamine (800 mg, 1.59 mmol) and divaleryl diboron (810 mg, 3.19 mmol) in N,N-dimethylformamide (5 mL) were added potassium acetate (364 mg, 3.19 mmol) and Pd(dppf) _Cl2 (117 mg, 0.16 mmol). The mixture was stirred and reacted at 110 °C for 5 h under nitrogen blanket. At the end of the reaction, the mixture was cooled to room temperature and water (30 mL) was added. The solution was extracted three times with ethyl acetate (20 mL×3). The organic phases were combined, washed with brine (5 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (petroleum ether/ethyl acetate = 1:1-0:1 - ethyl acetate/methanol = 10:1) to afford intermediate C as a brown solid (230 mg, 31% yield). MS result: 468.6 [M+H] ⁺ .

Step 3: Using the synthetic route described in Step 2 of Example 2, 1-(1-(5-bromopyridin-2-yl)piperidin-4-yl)-4-methylpiperazine (87 mg, 0.26 mmol) was reacted with intermediate C (100 mg, 0.21 mmol) to give N5-(2,4-dimethoxybenzyl)-3-ethyl-8-(6-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)pyridin-3-yl)-N2-(tetrahydro-2H)pyran-4-yl)pyridino[3,4-b]pyrazine-2,5-diamine as a yellow solid (70 mg, 49% yield). MS result: 682.6 [M+H] ⁺ .

Step 4: Using the method described for Step 12 in Example 1, N5-(2,4-dimethoxybenzyl)-3-ethyl-8-(6-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)pyridin-3-yl)-N2-(tetrahydro-2H)pyran-4-yl)pyridino[3,4-b]pyrazine-2,5-diamine (70 mg, 0.1 mmol) was reacted to give compound 21 as a yellow solid (70 mg, 0.1 mmol). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 8.38 (d, J=2.0 Hz, 1H), 7.92-7.88 (m, 2H), 7.05 (d, J=6.8 Hz, 1H), 6.85 (d, J=8.8 Hz, 1H), 6.45 (s, 2H), 4.35-4.31 (m, 2H), 4.11-4.03 (m, 1H), 3.93-3.91 (m, 2H), 3.40-3.28 (m, 4H), 2.83-2.78 (m, 4H), 2.42-2.30 (m, 7H), 2.14 (singlet, 3H), 1.89-1.80 (multiplet, 4H), 1.66-1.57 (multiplet, 2H), 1.44-1.34 (multiplet, 2H), 1.31 (triplet, J=7.2 Hz, 3H). The result obtained by the MS method: 532.3[M+H] ⁺ .

Example 4: 3-ethyl-8-(4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)-3-(methylsulfyl)phenyl)-N2-(tetrahydro-2H-pyran-4-yl)pyridino[3,4-b]pyrazine-2,5-diamine (compound 18)

Step 1: Commercially available 1-chloro-2-(methylsulfone)-4-nitrobenzene (1.20 g, 5.08 mmol) and 1-methyl-4-(4-piperidinyl)piperazine (931 mg, 5.08 mmol) were dissolved in dimethyl sulfoxide (20 mL) and potassium carbonate (1.40 g, 10.2 mmol) was added. The mixture was stirred and reacted at 100 °C for 2 h. At the end of the reaction, the mixture was cooled to room temperature and added to water (100 mL). The solution was extracted with ethyl acetate (20 mL×5). The organic phases were combined, washed with saturated brine (10 mL×2), and concentrated under reduced pressure. Dichloromethane (20 ml) was added and the solution was poured into an aqueous solution (30 ml) of concentrated hydrochloric acid (3 ml) and washed with dichloromethane (20 ml). The aqueous phase was basified with 5% sodium hydroxide solution and extracted with dichloromethane (20 ml x 4). The organic phases were combined, washed with saturated brine (5 ml), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give a brown solid (1.70 g, yield 87%). [M+H] ⁺ . ¹ H-NMR (400 MHz, CDCl ₃ ) δ 8.95 (d, J = 2.8 Hz, 1H), 8.42 (d of doublets, J = 8.8, 2.8 Hz, 1H), 7.44 (d, J = 9.2 Hz, 1H), 3.55-3.52 (m, 2H), 3.32 (s, 3H), 2.87-2.81 (m, 2H), 2.73-2.65 (m, 4H), 2.58-2.50 (m, 4H), 2.43-2.34 (m, 1H), 2.30 (s, 1H), 2.08-2.05 (m, 2H), 1.82-1.72 (multiplet, 2H). The result obtained by the MS method: 383.2.

Step 2: Using the method described in Step 4 of Example 1, 1-methyl-4-(1-(2-(methylsulfonyl)-4-nitrophenyl)piperidin-4-yl)piperazine was used as the starting material to give 4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)-3-(methylsulfonyl)aniline as a white solid (1.50 g, 96% yield). The MS result was 353.5 [M+H] ⁺ .

Step 3: 4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)-3-(methylsulfonyl)aniline (600 mg, 1.70 mmol) was dissolved in an aqueous solution (5 mL) of concentrated sulfuric acid (834 mg, 8.51 mmol). The temperature of the solution was brought to 0 °C, and an aqueous solution (0.5 mL) of sodium nitrite (117 mg, 8.51 mmol) was added dropwise to the solution. The solution was stirred at 0-5 °C for 10 min, then an aqueous solution (2 mL) of potassium iodide (565 mg, 3.40 mmol) was added dropwise to the solution. The solution was stirred at 5 °C for 30 min, then poured into water (5 mL). The pH of the solution was adjusted to 10 with 5% sodium hydroxide solution and extracted with dichloromethane (20 mL×3). The organic phases were combined, washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (dichloromethane/methanol = 10:1) on a C18 column (40-80% aqueous acetonitrile solution) to give a yellow solid (250 mg, yield 32%). The MS result was 464.1 [M+H] ⁺ .

Step 4 and 5: The method described for Step 3 and Step 4 of Example 3 was used. 1-(1-(4-iodo-2-(methylsulfone)phenyl)piperidin-4-yl)-4-methylpiperazine (87 mg, 0.26 mmol) was reacted with intermediate C (200 mg, 0.43 mmol) to give compound 18 as a yellow solid (60 mg, 46% yield over two steps). ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 8.46 (d, J=1.2 Hz, 1H), 7.98 (s, 1H), 7.91 (d, J=8.4 Hz, 1H), 7.55 (d, J=8.4 Hz, 1H), 7.06 (d, J=7.6 Hz, 1H), 6.62 (s, 2H), 4.28-4.18 (m, 1H), 3.84-3.82 (m, 2H), 3.40 (s, 3H), 3.38-3.32 (m, 4H), 3.26-3.23 (m, 2H), 2.84-2.77 (m, 5H), 2.73-2.59 (multiplet, 6H), 2.37 (singlet, 3H), 1.93-1.91 (multiplet, 2H), 1.82-1.79 (multiplet, 2H), 1.64-1.57 (multiplet, 4H), 1.31 (triplet, J=7.2 Hz, 3H). The result obtained by the MS method: 609.4 [M+H] ⁺ .

Example 5: 2-(5-(5-amino-3-ethyl-2-((tetrahydro-2H-pyran-4-yl)amino)pyridino[3,4-b]pyrazin-8-yl)-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)propan-2-ol (compound 19)

Step 1: Using the method described for Step 1 of Example 4, 1-(5-bromo-2-fluorophenyl)ethanone (1.20 g, 5.53 mmol) was reacted with 1-methyl-4-(4-piperidinyl)piperazine (1.01 g, 5.53 mmol) to give a yellow solid (1.40 g, 67% yield). The MS result was 380.1 [M+H] ⁺ .

Step 2: To a solution of 1-(5-bromo-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)ethyl-1-one (400 mg, 1.05 mmol) in tetrahydrofuran (10 mL) was added dropwise at 0 °C under a protective nitrogen atmosphere. The reaction mixture was stirred at 0-5 °C for 10 min, then poured into water (10 mL) and extracted with dichloromethane/methanol (10/1, 10 mL×3). The organic phases were combined and concentrated under reduced pressure. The resulting residue was purified on a C18 column (10-60% acetonitrile aqueous solution) to give a white solid (170 mg, 41% yield).

Steps 3 and 4: Using the method described for Step 3 and Step 4 of Example 3, 2-(5-bromo-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)propan-2-ol (102 mg, 0.26 mmol) was reacted with intermediate C (120 mg, 0.26 mmol) to give compound 19 as a white solid (60 mg, 30% yield over two steps). ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ 7.92 (s, 1H), 7.64-7.61 (m, 2H), 7.54 (b s, 1H), 7.39 (d, J=8.0 Hz, 1H), 7.01 (d, J=7.2 Hz, 1H), 6.52 (s, 2H), 4.20-4.12 (m, 1H), 3.90-3.88 (m, 2H), 3.30-3.27 (m, 4H), 2.97-2.88 (m, 4H), 2.81 (quartet, J=7.2 Hz, 2H), 2.74-2.59 (multiplet, 7H), 2.37 (singlet, 3H), 1.95-1.92 (multiplet, 2H), 1.84-1.80 (multiplet, 2H), 1.67-1.58 (multiplet, 2H), 1.57-1.48 (multiplet, 8H), 1.31 (triplet, J=7.2 Hz, 3H). The result obtained by the MS method: 589.4 [M+H] ⁺ .

Example 6: 3-ethyl-8-(3-((4-(4-methylpiperazin-1-yl)piperidin-1-yl)methyl)phenyl)-N2-(tetrahydro-2H-pyran-4-yl)pyrido[3,4-b]pyrazine-2,5-diamine (compound 43)

Step 1: Using the method described for Step 2 in Example 2, 8-bromo-N5-(2,4-dimethoxybenzyl)-3-ethyl-N2-(tetrahydro-2H-pyran-4-yl)pyridino[3,4-b]pyrazine-2,5-diamine (150 mg, 0.3 mmol) was reacted with 3-formylphenylboronic acid (54 mg, 0.36 mmol) to give intermediate B as a yellow solid (110 mg, 69% yield).

Step 2: To a mixture of 3-(5-((2,4-dimethoxybenzyl)amino)-3-ethyl-2-((tetrahydro-2H-pyran-4-yl)amino)pyridino[3,4-b]pyrazin-8-yl)benzaldehyde (110 mg, 0.21 mmol) and 1-methyl-4-(piperidin-4-yl)piperazine (57 mg, 0.31 mmol) in dichloromethane (5 mL) was added 1 drop of acetic acid. The reaction mixture was stirred at room temperature for 5 h and sodium triacetoxyborohydride (133 mg, 0.63 mmol) was added. After stirring at room temperature overnight, water (5 mL) was added. The mixture was extracted with dichloromethane (15 mL×4), the organic phases were combined, washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (dichloromethane/methanol = 30:1-15:1) to give a yellow solid (100 mg, yield 69%). The MS result was 695.7 [M+H] ⁺ .

Step 3: Using the deprotection method described for Step 12 in the method for synthesizing compound 8, N5-(2,4-dimethoxybenzyl)-3-ethyl-8-(3-((4-(4-methylpiperazin-1-yl)piperidin-1-yl)methyl)phenyl)-N2-(tetrahydro-2H-pyran-4-yl)pyridino[3,4-b]pyrazine-2,5-diamine (80 mg, 0.12 mmol) was used as the starting material to give compound 43 as a white solid (40 mg, 61% yield). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.01 (s, 1H), 7.76-7.71 (m, 2H), 7.42-7.38 (m, 2H), 7.16 (d, J=7.2 Hz, 2H), 6.75 (s, 2H), 4.13-4.04 (m, 1H), 3.93-3.90 (m, 3H), 3.32-3.26 (m, 5H), 3.15-3.06 (m, 3H), 2.93-2.72 (m, 10H), 2.54 (s, 3H), 1.86-1.84 (m, 4H), 1.70-1.61 (multiplet, 4H), 1.31 (triplet, J=7.2 Hz, 3H). The result obtained by the MS method: 545.3 [M+H] ⁺ .

Example 7: 3-ethyl-8-(3-methoxy-4-(4-((4-methylpiperazin-1-yl)methyl)piperidin-1-yl)phenyl)-1H-(tetrahydro-2H-pyran-4-yl)pyridino[3,4-b]pyrazine-2,5-diamine (compound 45)

Step 1: To a mixture of 1-methyl-4-(piperidin-4-ylmethyl)piperazine (1.0 g, 5.1 mmol) and 1-bromo-4-chloro-2-methoxybenzene (1.1 g, 5.1 mmol) in 1,4-dioxane (15 mL) were added cesium acetate (5.0 g, 15 mmol), Pd2(dba) ₃ (0.50 g, 0.50 mmol), BINAP (0.60 g, 1.0 mmol). The mixture was stirred and reacted at 110 °C for 3 h under nitrogen blanket. At the end of the reaction, the mixture was cooled to room temperature and water (30 mL) was added. The mixture was extracted three times (20 mL×3) with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (dichloromethane/methanol = 4:1) to give a yellow oily substance (620 mg, yield 35%). (ESI) m/z 338.1. [M+H] ⁺ .

Step 2: To a mixture of 1-((1-(4-chloro-2-methoxyphenyl)piperidin-4-yl)methyl)-4-methylpiperazine (400 mg, 1.2 mmol) and divaleryldiboron (601 mg, 2.4 mmol) in 1,4-dioxane (10 mL) were added potassium acetate (291 mg, 3.6 mmol), pd(dba) ₂ (14 mg, 0.020 mmol) and X-Phos (11 mg, 0.020 mmol). The resulting mixture was stirred at 110 °C for 16 h under nitrogen blanket. At the end of the reaction, the mixture was cooled to room temperature, filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (dichloromethane/methanol = 4:1) to give a yellow oily substance (400 mg, yield 75%). (ESI) m/z 430.2. [M+H] ⁺ .

Step 3: Using the method described in Step 2 of Example 2, 1-((1-(2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxoran-2-yl)phenyl)piperidin-4-yl)methyl)-4-methylpiperazine (137 mg, 0.31 mmol) was reacted with 8-bromo-N5-(2,4-dimethoxybenzyl)-3-ethyl-N2-(tetrahydro-2H-pyran-4-yl)pyridino[3,4-d]pyrazine-2,5-diamine (100 mg, 0.21 mmol) to give a yellow oil (112 mg, 79% yield). (ESI) m/z 725.3. [M+H] ⁺ .

Step 4: N5-(2,4-dimethoxybenzyl)-3-ethyl-8-(3-methoxy-4-(4-((4-methylpiperazin-1-yl)methyl)piperidin-1-yl)phenyl)-N2-(tetrahydro-2H-pyran-4-yl)pyridino[3,4-b]pyrazine-2,5-diamine (112 mg, 0.16 mmol) was dissolved in methanolic solution (4 M, 5 mL) of hydrochloric acid. The solution was stirred at 25 °C for 16 h. At the end of the reaction, the solution was cooled to room temperature and the reaction was quenched with saturated sodium bicarbonate solution (10 mL). The mixture was extracted three times (10 mL×3) with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by preparative liquid chromatography to afford the formate of compound 45 as a yellow solid (19 mg, yield 20%). ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 8.25 (singlet 1H), 7.92 (singlet, 1H), 7.24-7.17 (multiplet, 2H), 7.00 (doublet, J=7.2 Hz, 1H), 6.89 (doublet, J=8.0 Hz, 1H), 6.44 (singlet, 2H), 4.14-4.10 (multiplet, 1H), 3.90 (doublet, J=8.4 Hz, 2H), 3.81 (singlet, 3H), 3.24 (singlet, 6H), 2.83-2.78 (multiplet, 2H), 2.54 (doublet, J=12.8 Hz, 3H), 2.43-2.22 (multiplet, 5H), 2.17 (doublet, J=7.2 Hz, 2H), 2.15 (singlet, 3H), 1.86 (doublet, J=12.4 Hz, 2H), 1.77 (doublet, J=12.0 Hz, 2H), 1.68-1.56 (multiplet, 3H), 1.33-1.22 (multiplet, 5H). (PER) m/z 575.3. [M+H] ⁺ .

Using the method described for Step 1 in Example 7, 1-bromo-4-chloro-2-methoxybenzene was replaced with the starting material shown in the following table to give the corresponding intermediate, then, using the method described in Example 7, 1-methyl-4-(piperidin-4-ylmethyl)piperazine was replaced with 1-methyl-4-(4-piperidinyl) to give the compound shown in the following table.

Using the method described for Step 9 in Example 1 and the synthetic route described for Step 1 in Example 2, the tetrahydropyran-4-amine was substituted with the starting materials from the following table to give the corresponding intermediates, then, using the method described in Example 7, 1-methyl-4-(piperidin-4-ylmethyl)piperazine was substituted with 1-methyl-4-(4-piperidinyl) to give the compounds shown in the following table:

Using the method described in Example 7, 1-methyl-4-(piperidin-4-ylmethyl)piperazine was substituted for the starting materials shown in the following table to give the following compounds:

Using the method described in Example 7 and the catalytic hydrogenation method described for Step 4 of Example 1, 1-((1-(2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxyboran-2-yl)phenyl)piperidin-4-yl)methyl)-4-methylpiperazine was substituted for the starting material shown in the table below to give the compound shown in the table:

Using the method described for Step 2 of Example 2 and the deprotection method described for Step 12 of Example 1, 1-((1-(2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxyboran-2-yl)phenyl)piperidin-4-yl)methyl)-4-methylpiperazine was substituted for the starting material shown in the table below to give the compound shown in the table:

Using the method described in Example 7, 1-((1-(2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxoran-2-yl)phenyl)piperidin-4-yl)methyl)-4-methylpiperazine was substituted for the starting materials shown in the following table to give the following compounds:

Using the method described for step 9 in Example 1, the tetrahydropyran-4-amine was substituted with the following starting material 2 to give the corresponding intermediate, then, using the method described in Example 7 from step 3 to the end, 1-((1-(2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxoran-2-yl)phenyl)piperidin-4-yl)methyl)-4-methylpiperazine was substituted with the following starting material 1 to give the compound shown in the following table:

Example 8: 3-ethyl-8-(4-methylpynradin-1-yl)-N2-(tetrahydro-2H-pyran-4-yl)pyridino[3,4-b]pyrazin-2,5-diamine (compound 53 )

Step 1: To a mixture of 8-bromo-N5-(2,4-dimethoxybenzyl)-3-ethyl-N2-(tetrahydro-2H-pyran-4-yl)pyridino[3,4-b]pyrazine-2,5-diamine (75 mg, 0.15 mmol) and 1-methylpiperazine (30 mg, 0.30 mmol) in 1,4-dioxane (5 mL) were added potassium tert-butoxide (43 mg, 0.45 mmol), tBuXPhos Pd G3 (27 mg, 0.030 mol) and tBuXPhos (28 mg, 0.060 mmol). The mixture was stirred at 110 °C for 14 h under nitrogen blanket. At the end of the reaction, the mixture was cooled to room temperature and water (10 mL) was added. The mixture was extracted three times (10 ml×3) with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (dichloromethane/methanol = 10:1) to give a brown oily substance (50 mg, yield 64%). (ESI) m/z 522.2. [M+1] ⁺ .

Step 2: Using the method described for Step 4 of Example 7, N5-(2,4-dimethoxybenzyl)-3-ethyl-8-(4-methylpiperazin-1-yl)-N2-(tetrahydro-2H-pyran-4-yl)pyridino[3,4-b]pyrazine-2,5-diamine (50 mg, 0.096 mmol) was reacted to give compound 53 as a yellow solid (15 mg, 42 mmol). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.18 (br s, 2H), 7.37 (s, 1H), 7.05 (d, J=7.2 Hz, 1H), 6.16 (br s, 2H), 4.25-4.19 (m, 1H), 3.98-3.94 (m, 2H), 3.41-3.35 (m, 2H), 3.22-3.06 (m, 2H), 2.79 (d, J=7.2 Hz, 2H), 2.59-2.52 (m, 4H), 2.28 (s, 3H), 1.93-1.87 (m, 2H), 1.72-1.62 (multiplet, 2H), 1.28 (triplet, J=7.2 Hz, 4H). (IER) m/z 373.1. [M+1] ⁺ .

Example 9: 3-((5-amino-3-ethyl-8-(1-(1,-methyl-[1,4,-bipiperidin]-4-yl)-1H-pyrazol-4-yl)pyridino[3,4-b]pyrazin-2-yl)amino)cyclopentan-1-ol (compound 134)

Using the method described for Step 2 in Example 6, the starting material from the following table was reacted with compound 206 to give the compound shown in the table:

Example 10: Using the method described for Step 2 in Example 6, the following starting materials were reacted with compound 58 to give the compounds shown in the table:

Using the method described for Step 2 in Example 6, the following starting materials were reacted with compound 58, followed by the deprotection method described for Step 4 in Example 7, to give the compounds shown in the following table:

Using the method described for Step 2 in Example 6, the following starting materials were reacted with compound 58, followed by the deprotection method described for Step 12 in Example 1, then the method described for Step 2 for the preparation of compound 6 was used to react with formaldehyde to give the compound shown in the following table:

Using the method described for Step 2 in Example 6, paraformaldehyde was reacted with compound 157 to give the following compound:

Example 11: 3-ethyl-8-(1-(1-(pent-3-yl)piperidin-4-yl)-1H-pyrazol-4-yl)-N2-(tetrahydro-2H-pyran-4-yl) )pyridino[3,4-b]pyrazine-2,5-diamine (compound 232)

To a solution of compound 58 (100 mg, 0.20 mmol) in acetonitrile (3 mL) were added 3-bromopentane (360 mg, 2.4 mmol) and diisopropylethylamine (90 mg, 0.70 mmol). The mixture was stirred and reacted at 105 °C for 16 h in a Schlenk flask. At the end of the reaction, the mixture was cooled to room temperature and water (10 mL) was added. The mixture was extracted three times (10 mL × 3) with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by preparative liquid chromatography to give the formate of compound 232 as a yellow solid (30 mg, yield 23%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.34 (s, 1H), 8.17 (br s, 4H), 8.01 (s, 1H), 7.05 (d, J=8.0 Hz, 1H), 6.44 (br s, 2H), 4.35-4.26 (m, 1H), 4.17-4.09 (m, 1H), 4.01-3.94 (m, 2H), 3.49-3.43 (m, 2H), 2.83-2.82 (m, 4H), 2.46 (d, J=12.0 Hz, 2H), 2.30-2.23 (m, 1H), 2.06 (doublet, J=8.0 Hz, 2H), 1.99-1.88 (multiplet, 4H), 1.74-1.70 (multiplet 2H), 1.52-1.45 (multiplet, 2H), 1.37-1.24 (multiplet, 5H), 0.91-0.87 (multiplet, 6H). (IER) m/z. 493.2 [M+1] ⁺ .

Example 12: 3-ethyl-8-(5-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)pyridin-2-yl)-N2-(tetrahydro-2H-pyran-4-yl) pyridino[3,4-b]pyrazine-2,5-diamine (compound 20)

Step 1: To a mixture of 3-iodopyridine (1.00 g, 4.88 mmol), 1-methyl-4-(4-piperidinyl)piperazine (893 mg, 4.88 mmol), Xantphos (282 mg, 0.49 mmol), and potassium tert-butoxide (1.09 g, 9.76 mmol) in toluene (20 mL) was added tris(dibenzylideneacetone)dipalladium (447 mg, 0.49 mmol). The mixture was stirred for reaction at 108 °C for 4 h under nitrogen blanket. At the end of the reaction, the mixture was cooled to room temperature and methylene chloride (20 mL) and water (20 mL) were added. The mixture was extracted three times (20 mL x 3) with dichloromethane. The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (dichloromethane/methanol = 10:1 - dichloromethane/methanol/25% aqueous ammonia = 10:1:0.1) to give a yellow solid (750 mg, yield 59%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.30 (d, J=2.8 Hz, 1H), 8.07-8.05 (m, 1H), 7.20-7.17 (m, 1H), 7.15-7.12 (m, 1H), 3.77-3.74 (m, 2H), 2.81-2.74 (m, 2H), 2.65-2.60 (m, 4H), 2.51-2.44 (m, 2H), 2.41-2.38 (m, 1H), 2.31 (s, 3H), 2.19-2.06 (m, 2H), 1.98-1.95 (multiplet, 2H), 1.72-1.62 (multiplet, 2H).

Step 2: To a mixture of 1-methyl-4-(1-(pyridin-3-yl)piperidin-4-yl)piperazine (750 mg, 2.88 mmol) in dichloromethane (30 mL) was added NBS (770 mg, 4.33 mmol) portionwise in an ice bath. The reaction mixture was stirred for 2 h at 5-20 °C and then poured into water (20 mL). The solution was extracted with dichloromethane (20 mL x 4). The organic phases were combined, washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (dichloromethane/methanol = 20:1-10:1) to give a yellow solid (500 mg, yield 51%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.00 (d, J=3.2 Hz, 1H), 7.28-7.16 (m, 1H), 7.07 (doublet of doublets, J=8.8, 3.2 Hz, 1H), 3.71-3.68 (m, 2H), 2.80-2.74 (m, 2H), 2.63-2.36 (m, 9H), 2.30 (s, 3H), 1.97-1.94 (m, 2H), 1.69-1.59 (m, 2H). MS result: 339.1 [M+H] ⁺ .

Steps 3 and 4: Using the method described for Step 3 and Step 4 of Example 3, 1-(1-(6-bromopyridin-3-yl)piperidin-4-yl)-4-methylpiperazine (145 mg, 0.43 mmol) was reacted with intermediate C (200 mg, 0.43 mmol) to give compound 20 as a yellow solid (60 mg, 26% yield over two steps). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.46 (s, 1H), 8.35-8.31 (m, 2H), 7.39-7.36 (m, 1H), 7.24-7.22 (m, 1H), 6.82 (s, 2H), 4.22-4.14 (m, 1H), 3.96-3.94 (m, 2H), 3.88-3.85 (m, 2H), 3.46-3.40 (m, 3H), 3.07-2.73 (m, 12H), 2.58 (s, 3H), 1.94-1.91 (m, 4H), 1.72-1.58 (multiplet, 4H), 1.32 (triplet, J=7.2 Hz, 3H). The result obtained by the MS method: 532.2 [M+H] ⁺ .

Example 13: (1S,3R)-3-((5-amino-8-bromo-3-ethylpyridino[3,4-b]pyrazin-2-yl)amino)cyclopentan-1-ol (compound 151)

Steps 1 and 2, Synthesis of Intermediate D: Using the methods described for Step 9 in Example 1 and for Step 1 in Example 2, (1S,3R)-3-aminocyclopentanol hydrochloride was used to obtain Intermediate D as a yellow solid (yield over two steps 40%). ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 8.01 (singlet, 1H), 7.31-7.17 (multiplet, 2H), 7.07 (doublet, J=8.0 Hz, 1H), 6.56 (singlet, 1H), 6.44-6.38 (multiplet, 1H), 5.76 (singlet, 1H), 4.74 (doublet, J=3.6 Hz, 1H), 4.56 (doublet, J=6.0 Hz, 2H), 4.52-4.41 (multiplet, 1H), 3.83 (singlet, 3H), 3.72 (singlet, 3H), 2.83-2.77 (multiplet, 2H), 2.13-1.99 (multiplet, 2H), 1.83-1.72 (multiplet, 2H), 1.68-1.56 (multiplet, 2H), 1.30 (triplet, J=7.2 Hz, 3H). (IER) m/z 502.0 [M+H] ⁺ .

Step 3: Using the method described for Step 12 in Example 1, intermediate D (100 mg, 0.20 mmol) was used to give compound 151 as a yellow solid (56 mg, 79% yield). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.14 (b s, 1H), 8.00 (s, 1H), 7.43 (d, J=6.8 Hz, 1H), 6.98 (b s, 2H), 4.76 (s, 1H), 4.55-4.45 (m, 1H), 4.22-4.13 (m, 1H), 2.81 (quartet, J=7.2 Hz, 2H), 2.36-2.27 (m, 1H), 2.12-2.01 (m, 1H), 1.87-1.73 (m, 2H), 1.70-1.58 (m, 2H), 1.30 (triplet, J=7.2 Hz, 3H). (IER) m/z 352.1. [M+N] ⁺ .

Example 14: Using the method described for Step 2 in Example 2, the starting materials listed in the following table were reacted with compound 151 to give the corresponding compounds:

Example 15: (1S,3R)-3-((5-amino-3-ethyl-8-(1-(piperidin-4-yl)-1H-pyrrol-3-yl)pyrido[3,4-b]pyrazin-2-yl)amino)cyclopentan-1-ol (compound 152) and (1S,3R)-3-((5-amino-3-ethyl-8-(1-(1-methylpiperidin-4-yl)-1H-pyrrol-3-yl)pyrido[3,4-b]pyrazin-2-yl)amino)cyclopentan-1-ol (compound 153)

Using the method described in Example 3, intermediate D was prepared to intermediate E and then reacted with 4-(3-bromo-1H-pyrrol-1-yl)piperidine-1-carboxylate tert-butyl ester. The resulting product was deprotected using the method described for step 12 in Example 1 to give compound 152 as a yellow solid. Compound 152 was reacted with formaldehyde using the method described for step 2 in Example 6 to give compound 153 as a yellow solid.

Compound 152: ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 7.68 (singlet, 1H), 6.96 (doublet, J=7.2 Hz, 1H), 6.88 (singlet, 1H) , 6.49 (singlet, 2H), 6.07 (triplet, J=4.0 Hz, 1H), 5.87 (singlet, 1H), 4.77 (doublet, J=3.6 Hz, 1H), 4.31 (doublet, J=6.4 Hz, 1H), 4.09 (singlet, 1H), 3.80 (singlet, 1H), 2.92 (doublet, J=12.0 Hz, 2H), 2.77-2.72 (multiplet, 2H), 2.24 (singlet, 1H), 2.00-1.90 (multiplet, 1H), 1.81-1.47 (multiplet, 11H), 1.29 (triplet, J=7.2 Hz, 3H). (IER) m/z. 422.2 [M+1] ⁺ .

Compound 153: ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 7.69 (singlet, 1H), 7.00 (doublet, J=7.2 Hz, 1H), 6.90 (singlet, 1H) , 6.55 (singlet, 2H), 6.10 (triplet, J=4.0 Hz, 1H), 5.90-5.89 (multiplet, 1H), 4.79 (doublet, J=3.6 Hz, 1H), 4.37-4.24 (multiplet, 1H), 4.09 (doublet, J= 4.0 Hz, 1H), 3.80 (singlet, 1H), 2.95 (singlet, 2H), 2.77-2.72 (multiplet, 2H), 2.30 (singlet, 3H), 2.02-1.57 (multiplet, 11H), 1.55-1.49 (multiplet, 1H), 1.30 (triplet, J=7.2 Hz, 3H) . (IER) m/z. 436.2 [M+1] ⁺ .

Example 16: Using the method described for Step 2 in Example 6, the starting material listed in the following table was reacted with compound 154 to give the corresponding compounds:

Example 17: (1S,3R)-3-((5-amino-3-ethyl-8-(1-(1,2,2,6,6-pentamethylpiperidin-4-yl)-1H-pyrazol-4-yl)pyridino[3,4-b]pyrazin-2-yl)amino)cyclopentan-1-ol (compound 169)

Step 1: To a dichloromethane solution (30 mL) containing 4-hydroxy-1,2,2,6,6-pentamethylpiperidine (3 g, 18 mmol) at 0 °C were added triethylamine (8.9 g, 88 mmol) and methanesulfonyl chloride (2.4 g, 21 mmol). The reaction mixture was stirred at 25 °C for 6 h and upon completion, the reaction was quenched with saturated sodium bicarbonate solution. The solution was extracted twice with dichloromethane (30 mL×2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a yellow oily substance (74.2 g, yield 96%), which was used directly in the next reaction. (ESI) m/z 250.2. [M+H] ⁺ .

Step 2: To a solution of N,N-dimethylformamide (20 mL) containing 4-pyrazole borate (2.3 g, 12 mmol) was added sodium hydride (0.48 g, 12 mmol). After stirring at 25 °C for 1 h, the product obtained in step 1, (1,2,2,6,6-pentamethylpiperidin-4-yl)mesylate (2.0 g, 8.0 mol) was added. The solution was stirred at 110 °C for 12 h. The solution was cooled to room temperature and extracted with ethyl acetate (30 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative liquid chromatography to give a yellow oily substance (23 mg, 1.3%). (IER) m/z. 348.2 [M+H] ⁺ .

Steps 3 and 4: Using the method described for Step 2 in Example 2, followed by the deprotection method described for Step 12 in Example 1, Intermediate D (15 mg, 0.03 mmol) was reacted with the product from Step 2 (26 mg, 0.075 mmol) to give compound 169 as a yellow solid (4.5 mg, 28% yield over two steps). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.44 (s, 1H), 8.20 (d, J=8.0 Hz, 4H), 8.02 (s, 1H), 7.10 (d, J=8.0 Hz, 1H), 6.38 (s, 2H), 4.54-4.49 (m, 2H), 4.25-4.20 (m, 1H), 2.81-2.78 (m, 2H), 2.33-2.29 (m, 1H), 2.25 (s, 3H), 2.09-2.05 (m, 1H), 2.00-1.97 (m, 2H), 1.89-1.80 (multiplet, 5H), 1.71-1.61 (multiplet, 2H), 1.32-1.29 (multiplet, 3H), 1.16 (singlet, 6H), 1.11 (singlet, 6H). (ESI) m/z. 493.3 [M+H] ⁺ .

Using the method described in Example 17, starting from step 3, the starting material shown in the following table was reacted with intermediate product D, followed by reaction with methyl iodide using the method described in Example 17 to give the compound shown in the table:

Using the method described in Example 17 from step 3 to the end, the starting materials listed in the following table were used to prepare the corresponding compounds:

Using the method described in Example 17 from step 2 to the end, the starting materials listed in the following table were used to prepare the corresponding compounds: _

Using the method described in Example 17, 4-hydroxy-1,2,2,6,6-pentamethylpiperidine was substituted for the starting materials shown in the following table to give the corresponding compounds:

Example 18:

Step 1: Using the method described for step 2 in Example 2, intermediate D (0.30 g, 0.60 mmol) was used as a starting material to react with tert-butyl 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (0.45 g, 1.2 mmol) to give a yellow solid (180 mg, 45% yield). (ESI) m/z 673.3. [M+H] ⁺ .

Step 2: Using the method described for Step 12 in Example 1, the product from Step 1 (0.18 g, 0.27 mmol) was used as starting material to obtain Intermediate F as a yellow solid (80 mg, 71% yield). (ESI) m/z 423.3. [M+H] ⁺ .

Using the method described for Step 2 in Example 6, the starting materials shown in the following table were reacted with intermediate F, then deprotected using the method described for Step 12 in Example 1, followed by reaction with paraformaldehyde using the method described for Step 2 in Example 6 to give the compound shown in the table:

The following starting materials were reacted with intermediate F using the method described for step 2 in Example 6 to give the compounds shown in the following table:

The following starting materials were reacted with intermediate F using the method described in Example 11 to give the compounds shown in the table:

The following starting material was reacted with intermediate F using the method described for step 3 in Example 35 to give the compound shown in the following table:

Example 19: (1S,3R)-3-((5-amino-3-ethyl-8-(1-(2-hydroxy-2-methylpropyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyridino[3,4-b]pyrazin-2-yl)amino)cyclopentan-1-ol (compound 181)

A solution of intermediate F (90 mg, 0.21 mmol) and 2,2-dimethylethylene oxide (46 mg, 0.64 mmol) in ethanol (10 mL) was stirred at 80 °C for 12 h. At the end of the reaction, the mixture was cooled to room temperature and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (dichloromethane/methanol = 70%:30%) to give compound 181 as a yellow solid (65 mg, yield 59%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.44 (s, 1H), 8.20 (s, 1H), 8.02 (s, 1H), 7.11 (d, J=7.2 Hz, 1H), 6.37 (broad s, 2H), 4.79 (d, J=3.6 Hz, 1H), 4.54-4.44 (m, 1H), 4.28-4.20 (m, 1H), 4.17-4.05 (m, 2H), 3.11-2.99 (m, 2H), 2.80 (quartet, J=7.2 Hz, 2H), 2.38-2.21 (m, 5H), 2.12-1.77 (multiplet, 7H), 1.75-1.60 (multiplet, 2H), 1.31 (triplet, J=7.2 Hz, 3H), 1.11 (singlet, 6H). (IER) m/z. 495.2 [M+1] ⁺ .

The following starting materials were used to prepare the following compound shown in the table using the method described in Example 19:

Example 20: 4-(4-(5-amino-3-ethyl-2-(((1R,3S)-3-hydroxycyclopentyl)amino)pyridino[3,4-b]pyrazin-8-yl)-1H-pyrazol-1-yl)-N-isopropylpiperazine-1-formamide (compound 189)

To a solution of intermediate F (80 mg, 0.19 mmol) and triethylamine (57 mg, 0.57 mmol) in tetrahydrofuran (10 mL) was added isopropyl isocyanate (19 mg, 0.23 mmol). The mixture was stirred at 25 °C for 2 h and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (dichloromethane/methanol = 70%:30%) to give compound 189 as a yellow solid (38 mg, yield 37%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.40 (s, 1H), 8.17 (s, 1H), 8.05 (s, 1H), 7.13 (d, J=6.9 Hz, 1H), 6.47 (broad s, 2H), 6.24 (d, J=7.6 Hz, 1H), 4.79 (d, J=3.6 Hz, 1H), 4.51-4.42 (m, 1H), 4.38-4.28 (m, 1H), 4.25-4.18 (m, 1H), 4.14-41.06 (m, 2H), 3.77 (doublet of doublets, J=13.4, 6.8 Hz, 1H), 2.89-2.73 (multiplet, 4H), 2.27 (doublet of doublets, J=13.4, 6.8 Hz, 1H), 2.10-1.98 (multiplet, 3H), 1.89-1.61 (multiplet, 7H), 1.30 (triplet, J=7.2 Hz, 3H), 1.07 (doublet, J=6.8 Hz, 6H). (IER) m/z 508.3 [M+1] ⁺ .

Example 21: 1-(4-(4-(5-amino-3-ethyl-2-(((1R,3S)-3-hydroxycyclopentyl)amino)pyridino[3,4-b]pyrazin-8-yl)-1H-pyrazol-1-yl)piperadin-1-yl)-2-methylpropyl-1-one (compound 185)

A solution of isobutyric acid (25 mg, 0.28 mmol), EDCI (109 mg, 0.57 mmol), HOBT (77 mg, 0.57 mmol) and diisopropylethylamine (147 mg, 1.1 mmol) in N,N-dimethylformamide (5 mL) was stirred at 0 °C for half an hour, after which intermediate F (80 mg, 0.19 mmol) was added. The mixture was stirred at 25 °C for 2 h and after the reaction, water (20 mL) was added. The solution was extracted three times with ethyl acetate (20 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (dichloromethane/methanol = 95%:5%) to give the compound as a yellow solid (50 mg, yield 51%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.40 (s, 1H), 8.19 (s, 1H), 8.06 (s, 1H), 7.08 (d, J=7.2 Hz, 1H), 6.37 (broad s, 2H), 4.79 (d, J=3.6 Hz, 1H), 4.56-4.40 (m, 3H), 4.25-4.17 (m, 1H), 4.12-4.02 (m, 1H), 3.27-3.18 (m, 1H), 2.96-2.89 (m, 1H), 2.80 (quartet, J=7.2 Hz, 2H), 2.53-2.52 (multiplet, 1H), 2.31-2.22 (multiplet, 1H), 2.17-2.00 (multiplet, 3H), 1.88-1.63 (multiplet, 6H), 1.30 (triplet, J=7.2 Hz, 3H), 1.06-0.99 (multiplet, 6H). (IER) m/z 493.3. [M+1] ⁺ .

Example 22: (1S,3R)-3-((5-amino-3-ethyl-8-(1-(6-methylpyridin-3-yl)-1H-pyrazol-4-yl)pyridino[3,4-b]pyrazin-2-yl)amino)cyclopentan-1-ol (compound 177)

Step 1: To a solution of 2-methyl-5-bromopyridine (2.3 g, 14 mmol) in N,N-dimethylformamide (20 mL) were added 4-bromopyrazole (1.0 g, 6.8 mmol), cesium carbonate (6.6 g, 21 mmol), copper iodide (0.13 g, 0.60 mmol) and trans-N,N-dimethylcyclohexane-1,2-diamine (0.19 g, 1.3 mmol). The mixture was stirred at 110 °C for 12 h under nitrogen blanket. At the end of the reaction, the mixture was cooled to room temperature and water (50 mL) was added. The solution was extracted three times with ethyl acetate (50 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 70%:30%) to give a yellow oily substance (900 mg, yield 29%). (ESI) m/z. 238.1 [M+H] ⁺ .

Step 2 and 3: The method described for Step 6 in Example 1 and the method described for Step 2 in Example 2 were used, respectively. The borate intermediate was prepared using 5-(4-bromo-1H-pyrazol-1-yl)-2-methylpyridine as a starting material, followed by reaction with compound 151 to give compound 177 as a yellow solid (yield over two steps 11%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 9.07 (s, 1H), 8.94 (d, J=2.8 Hz, 1H), 8.46 (s, 1H), 8.30 (s, 1H), 8.12-8.09 (m, 1H), 7.42 (d, J=8.4 Hz, 1H), 7.23 (d, J=6.8 Hz, 1H), 6.66 (s, 2H), 4.82 (d, J=3.6 Hz, 1H), 4.59-4.51 (m, 1H), 4.24-4.18 (m, 1H), 2.85-2.79 (m, 2H), 2.52 (singlet, 3H), 2.32-2.27 (multiplet, 1H), 2.11-2.03 (multiplet, 1H), 1.91-1.76 (multiplet, 2H), 1.71-1.64 (multiplet, 2H), 1.32 (triplet, J=7.2 Hz, 3H). LC-MS: Rt=1.051 min, (ESI) m/z. 431.2 [M+1] ⁺ .

The synthetic method described in Example 22 was used. The borate intermediate was prepared from the starting materials shown in the following table using the methods described for Steps 1 and 2, and then reacted with compound 2 to give the corresponding compounds shown in the table according to the method described for Step 3:

The borate intermediate was prepared according to the methods described for steps 1 and 2 in Example 22, and the corresponding compounds shown in the following table were prepared according to the methods described for steps 3 and 4 in Example 7:

The borate intermediate was prepared according to the methods described for steps 1 and 2 in Example 22, and the corresponding compounds listed in the following table were prepared according to the methods described for steps 3 and 4 in Example 17.

Compound 278 was reacted with the starting material shown in the following table to give the corresponding compound according to the method described for Step 7 in Example 33:

2-Methyl-5-bromopyridine was substituted for the starting materials shown in the following table to give the corresponding compounds according to the method described in Example 22:

The starting materials listed in the following table were used to prepare the corresponding compounds according to the method described in Example 22, starting from step 2.

Using the synthetic route described for compound 151 in Step 1, (1S,3R)-3-aminocyclopentanol hydrochloride was substituted for (1R,3R)-3-aminocyclopentanol to provide the following intermediate 356-A. Then, using the route described in Example 22, 2-methyl-5-bromopyridine was substituted for the starting material shown in the following table, and compound 151 was substituted for intermediate 356-A in Step 3 to provide the corresponding compound:

Example 23: (1S,3R)-3-((5-amino-3-ethyl-8-(1-(2-methylpyrimidin-4-yl)-1H-pyrazol-4-yl)pyridino[3,4-b]pyrazin-2-yl)amino)cyclopentan-1-ol (compound 180)

Step 1. Sodium hydride (112 mg, 4.67 mmol) was added to a solution of 4-pyrazole borate (498 mg, 2.6 mmol) in tetrahydrofuran (20 mL), then 4-chloro-2-methylpyrimidine (300 mg, 2.3 mmol) was added and the solution was stirred at 25 °C for 3 h. After the reaction, water (30 mL) was added and the solution was extracted with ethyl acetate (30 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (dichloromethane/methanol = 9:1) to give a white solid (200 mg, yield 28%). (ESI) m/z. 287.2. [M+H] ⁺ .

Step 2: The product from Step 1 (81 mg, 0.11 mmol) was reacted with compound 151 (50 mg, 0.14 mmol) according to the method described for Step 2 in Example 2 to give compound 180 as a yellow solid (7 mg, 12% yield). ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 9.55 (s 1H), 8.74 (d, J = 5.6 Hz, 1H), 8.58 (s, 1H), 8.43 (s, 1H), 8.17 (br s, 1H), 7.75 (d, J = 5.6 Hz, 1H), 7.15 (d, J = 7.2 Hz, 1H), 6.58 (br s, 2H), 4.62-4.57 (m, 1H), 4.31-4.27 (m, 1H), 2.84-2.80 (m, 2H), 2.64 (s, 3H), 2.40-2.35 (multiplet, 1H), 2.29-2.22 (multiplet, 1H), 1.99-1.81 (multiplet, 2H), 1.79-1.66 (multiplet, 2H), 1.33 (triplet, J=7.2 Hz, 3H). (IER) m/z. 432.2 [M+1] ⁺ .

4-Chloro-2-methylpyrimidine was substituted for the starting materials shown in the following table to give the corresponding compounds according to the method described in Example 23:

Example 24: (1S,3R)-3-((5-amino-3-ethyl-8-(1-(1-(isopropylsulfonyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyridino[3,4b]pyrazin-2-yl)amino)cyclopentan-1-ol (compound 187)

Step 1: tert-Butyl 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxoboran-2-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (1.0 g, 2.65 mmol) was dissolved in 1,4-dioxane (5 mL), followed by the addition of hydrochloric acid in 1,4-dioxane (4 M, 10 mL). The solution was stirred at 25 °C for 1 h. After reaction, the solution was concentrated under reduced pressure to give the compound as a white solid (650 mg, 88%). (ESI) m/z 278.2. [M+H] ⁺ .

Step 2: To a solution of 4-(4,4,5,5-tetramethyl-1,3,2-dioxoboran-2-yl)-1H-pyrazol-1-yl)piperidine (800 mg, 2.9 mmol) and triethylamine (876 mg, 8.7 mmol) in dichloromethane (10 mL) was added 2-propanesulfonyl chloride (494 mg, 3.5 mmol). The mixture was stirred and reacted at 25 °C for 16 h. After the reaction, water (30 mL) was added and the solution was extracted three times with dichloromethane (20 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a yellow oily substance (400 mg, yield 95%). (ESI) m/z 384.3. [M+N] ⁺ .

Steps 3 and 4: The product obtained in step 2 (73 mg, 0.19 mmol) was used as starting material for reaction with intermediate D (80 mg, 0.16 mmol) according to the method described in Example 17 starting from step 3 to give the formate of compound 187 as a yellow solid (30 mg, 43% yield over two steps). ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 8.43 (singlet, 1H), 8.19 (singlet, 1H), 8.14 (singlet, 1H), 8.07 (singlet, 1H), 7.12 (doublet, J=7.2 Hz, 1H), 6.47 (singlet, 2H), 4.79 (singlet, 1H), 4.52-4.46 (multiplet, 1H), 4.44-4.34 (multiplet, 1H), 4.28-4.16 (multiplet, 1H), 3.76 (doublet, J=12.0 Hz, 2H), 3.40 (doublet, J=6.4 Hz, 1H), 3.13 (triplet, J=12.0 Hz, 2H), 2.83-2.77 (multiplet, 2H), 2.32-2.25 (multiplet, 1H), 2.15 (doublet, J=12.0 Hz, 2H), 2.09-1.99 (multiplet, 1H), 1.93-1.76 (multiplet, 4H), 1.73-1.61 (multiplet, 2H), 1.31 (triplet, J=7.2 Hz, 3H), 1.25 (doublet, J=6.4 Hz, 6H). (IER) m/z. 529.3 [M+1] ⁺ .

The starting material shown in the following table was used to prepare the corresponding borate intermediate, and then the borate intermediate was reacted with compound 151 to give the compound shown in the following table according to the method described in Example 24:

2-Propanesulfonyl chloride was substituted for the starting material shown in the following table to give the corresponding compounds according to the method described in Example 24:

Example 25: (S)-3-ethyl-8-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)-N2-(tetrahydrofuran-3-yl)pyridino[3,4-b] pyrazine-2,5-diamine (compound 259)

Steps 1 and 2: Intermediate G was prepared as a yellow solid according to the synthesis method described for intermediate D using (S)-tetrahydrofuran-3-amine (yield over two steps 28%). (ESI) m/z 488.0 [M+H] ⁺ .

Step 3: Intermediate G (200 mg, 0.41 mmol) was used as a starting material for reaction with tert-butyl 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxoboracyclopentan-2)-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (186 mg, 0.49 mmol) according to the method described for Step 2 of Example 2 to give Intermediate H as a yellow oil (280 mg, 94% yield). (ESI) m/z 659.3. [M+H] ⁺ .

Step 4: Intermediate H (280 mg, 0.42 mmol) was used as starting material to afford compound 259 as a yellow solid (88 mg, 48% yield) according to the method described for Step 12 in Example 1. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.34 (s, 1H), 8.18 (s, 1H), 8.03 (s, 1H), 7.25 (d, J=5.6 Hz, 1H), 6.39 (s, 1H), 4.71-4.67 (m, 1H), 4.21-4.01 (m, 1H), 4.04-4.00 (m, 1H), 3.94-3.90 (multiplet, 1H), 3.82-3.76 (multiplet, 1H), 3.74-3.71 (multiplet, 1H), 3.06 (doublet, J=12.0 Hz, 2H), 2.87-2.82 (multiplet, 2H), 2.68-2.59 (multiplet, 2H), 2.54 (singlet, 1H), 2.33-2.25 (multiplet, 1H), 2.14-2.06 (multiplet, 1H), 2.02 (doublet, J=12.0 Hz, 2H), 1.84-1.75 (multiplet, 2H), 1.31 (triplet, J=7.2 Hz, 3H). (ESI) m/z. 409.2 [M+1] ⁺ .

(S)-Tetrahydrofuran-3-amine was substituted for starting material 1 in the following table to obtain the corresponding intermediate according to the method described in Example 25, and then the intermediate was reacted with starting material 2 to obtain the corresponding compound:

(S)-Tetrahydrofuran-3-amine was substituted for the starting material shown in the following table to obtain intermediate 261-A according to the method described in Example 25. Intermediate 261-A was then reacted with methyl iodide to obtain the compound shown in the following table 261 according to the method described in Example 11.

(S)-Tetrahydrofuran-3-amine was substituted for the starting material shown in the following table to give the compound shown in the table according to the method described in Example 25.

The starting material shown in the following table was reacted with compound 258 to give the compound shown in the following table according to the method described for Step 2 in Example b.

The starting material shown in the following table was used to prepare the compound shown in the table according to the method for synthesizing compound 250.

The starting materials in the following table were reacted with compound 259 to give the corresponding compounds shown in the table according to the method described for Step 2 in Example 6.

(S)-Tetrahydrofuran-3-amine was replaced by the starting material shown in the following table to give the compound shown in the table according to the method for the synthesis of compound 225.

Example 26: (1S,3R)-3-((5-amino-3-ethyl-8-(1-(1-(2-hydroxyethyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyridino[3,4-b]pyrazin-2-yl)amino)cyclopentan-1-ol (compound 207)

Step 1: To a solution of intermediate F (50 mg, 0.10 mmol) in N,N-dimethylformamide (5 mL) were added tert-butyl (2-iodoethoxy) dimethylsilane (71 mg, 0.25 mmol) and cesium carbonate (161 mg, 0.50 mmol) and stirred at 45 °C for 12 h. At the end of the reaction, the mixture was cooled to room temperature and water (1 mL) was added. The mixture was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (dichloromethane : methanol = 3:1) to give the compound as a yellow solid (40 mg, yield 41%). (ESI) m/z 581.3. [M+H] ⁺ .

Step 2: The product from Step 1 (40 mg, 0.070 mmol) was used as starting material for deprotection according to the method described for Step 4 in Example 7 to give the formate salt of compound 207 as a yellow solid (5 mg, 31% yield). ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 8.42 (s, 1H), 8.18 (s, 1H), 8.15 (br s, 2H), 8.05 (s, 1H), 7.12 (d, J=7.2 Hz, 1H), 6.42 (br s, 2H), 4.47-4.45 (m, 1H), 4.19-4.15 (m, 2H), 3.55-3.52 (m, 2H), 3.06-3.03 (m, 2H), 2.80-2.75 (m, 2H), 2.55-2.50 (m, 4H), 2.40-2.26 (multiplet, 2H), 2.14-1.92 (multiplet, 6H), 1.83-1.81 (multiplet, 2H), 1.72-1.59 (multiplet, 2H), 1.30 (triplet, J=7.2 Hz, 3H). (IER) m/z 467.2 [M+1] ⁺ .

Intermediate F was replaced by the starting materials shown in the following table to obtain the corresponding compounds according to the method described in Example 26.

Example 27: (S)-3-ethyl-8-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)-1H-(tetrahydrofuran-3-yl)pyridino[3,4-b] pyrazine-2,5-diamine (compound 230)

Intermediate I as a yellow solid (350 mg, 86% yield) was prepared from intermediate G (588 mg, 1.2 mmol) using the method described for step 12 in Example 1. (ESI) m/z 338.1. [M+H] ⁺ .

The starting materials shown in the following table were reacted with intermediate I according to the method described for step 2 in Example 2 to give the corresponding compounds shown in the table.

The starting material shown in the following table was reacted with intermediate G according to the method described in Example 17 from step 3 to the end to obtain the corresponding compound shown in the table.

Example 28: 3-((5-amino-3-ethyl-8-(1-(1,-ethyl-[1,4,-bipiperidin]-4-yl)-1H-pyrazol-4-yl)pyridino[3,4-b]pyrazin-2-yl)amino)cyclohexan-1-ol (compound 246)

(S)-Tetrahydrofuran-3-amine was replaced by 3-aminocyclohexanol to give intermediate J according to the method described in Example 25, followed by use of intermediate J according to the method described for step 2 in Example 6 to give the formate of compound 246. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.47 (s, 1H), 8.22-8.17 (m, 4H), 8.02 (s, 1H), 6.87 (d, J=7.6 Hz, 1H), 6.36 (br s, 2H), 4.63-4.56 (m, 1H), 4.16-4.13 (m, 1H), 4.08 (s, 1H), 3.12-2.93 (multiplet, 6H), 2.80 (quartet, J=7.2 Hz, 2H), 2.35-2.27 (multiplet, 4H), 2.11-1.41 (multiplet, 18H), 1.29 (triplet, J=7.2 Hz, 3H), 1.08-1.01 (multiplet, 3H). (IER) m/z 548.3. [M+1] ⁺ .

Example 29: 3-ethyl-8-(4-(4-(4-ethylpiperazin-1-yl)piperidin-1-yl)-3-methoxyphenyl)-N2-(tetrahydro-2H-pyran-4-yl)pyridino [3,4-b]pyrazine-2,5-diamine (compound 315)

Step 1: To a mixture of 1-ethyl-4-(piperidin-4-ylmethyl)piperazine (8.0 g, 40 mmol) and 1-bromo-4-chloro-2-methoxybenzene (9.6 g, 43 mmol) in 1,4-dioxane (200 mL) were added cesium acetate (28 g, 87 mmol), Pd ₂ (dba) ₃ (4.0 g, 4.3 mmol), BINAP (5.4 g, 8.7 mmol). The mixture was stirred and reacted at 100 °C for 12 h under nitrogen blanket. At the end of the reaction, the mixture was cooled to room temperature and water (100 mL) was added. The solution was extracted three times (200 mL × 3) with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (dichloromethane/methanol = 4:1) to give a yellow oily substance (7.0 g, yield 52%). (ESI) m/z 338.2 [M+H] ⁺ .

Step 2: To a mixture of 1-(1-(4-chloro-2-methoxyphenyl)piperidin-4-yl)-4-ethylpiperazine (7.0 g, 21 mmol) and divaleryldiboron (7.9 g, 31 mmol) in 1,4-dioxane (100 mL) were added potassium acetate (5.1 g, 62 mmol), Pd ₂ (dba) ₃ (1.2 g, 2.1 mmol) and X-Phos (2.0 g, 4.1 mmol). The mixture was stirred for reaction at 100 °C for 12 h under nitrogen blanket. At the end of the reaction, the mixture was cooled to room temperature and water (100 mL) was added. The mixture was extracted three times (300 mL × 3) with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (dichloromethane/methanol = 4:1) to afford intermediate product K as a yellow oily substance (7.0 g, yield 79%). (ESI) m/z 430.3. [M+H] ⁺ .

Step 3: To a 1,4-dioxane/water mixture (8 mL, 4/1) containing 8-bromo-N5-(2,4-dimethoxybenzyl)-3-ethyl-N2-(tetrahydro-2H-pyran-4-yl)pyridino[3,4-b]pyrazine-2,5-diamine (1.4 g, 3.0 mmol) and intermediate K (4.0 g, 7.97 mmol) were added cesium carbonate (2.7 g, 8.0 mmol) and Pd(dppf) _Cl2 (0.20 g, 0.20 mmol). This mixture was reacted in a microwave oven at 100 °C for 1 h under nitrogen blanket. At the end of the reaction, the mixture was cooled to room temperature and water (50 mL) was added. The mixture was extracted three times (30 ml x 3) with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (dichloromethane/methanol = 10:1) to give a yellow solid (900 mg, yield 42%). (ESI) m/z 725.4. [M+H] ⁺ .

Step 4: N5-(2,4-dimethoxybenzyl)-3-ethyl-8-(4-(4-(4-ethylpiperazin-1-yl)piperidin-1-yl)-3-methoxyphenyl)-1H-(tetrahydro-2H-pyran-4-yl)pyridine[3,4-b]pyrazine-2,5-diamine (900 mg, 1.2 mmol) in trifluoroacetic acid/dichloromethane (5 mL, 3/1) solution was stirred at 25 °C for 3 h. At the end of the reaction, diisopropylethylamine (10 mL) was added to stop the reaction, and the mixture was concentrated under reduced pressure. The residue was purified by preparative liquid chromatography to give the formate salt of compound 315 as a yellow solid (255 mg, 35% yield). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.14 (broad s, 1H), 7.91 (s, 1H), 7.25-7.17 (m, 2H), 7.09 (d, J=8.0 Hz, 1H), 6.91 (d, J=8.0 Hz, 1H), 6.62 (broad s, 2H), 4.14-4.10 (m, 1H), 3.91 (d, J=8.0 Hz, 2H), 3.82 (s, 3H), 3.48 (d, J=12.0 Hz, 3H), 3.26-3.24 (m, 2H), 2.82-2.78 (m, 2H), 2.61-2.51 (multiplet, 6H), 2.49-2.44 (multiplet, 6H), 1.88-1.84 (multiplet, 4H), 1.64-1.60 (multiplet, 4H), 1.31 (triplet, J=7.2 Hz, 3H), 1.14 (triplet, J=7.2 Hz, 3H). (IER) m/z. 575.3 [M+1] ⁺ .

1-Ethyl-4-(piperidin-4-ylmethyl)piperazine was substituted for the starting material shown in the following table to give the corresponding intermediate shown in the table, according to the method described in Example 29. The intermediate was then reacted with compound 2 according to the method described for step 3 in Example 29 to give the corresponding compound:

1-Ethyl-4-(piperidin-4-ylmethyl)piperazine was replaced with the starting material 1 shown in the following table according to the method described in Example 29, and 1-bromo-4-chloro-2-methoxybenzene was replaced with the starting material 2 shown in the table to react to give the corresponding compound.

1-Ethyl-4-(piperidin-4-ylmethyl)piperazine was substituted for the starting materials in the following table according to the method described in Example 29 for steps 1 and 2 to give the corresponding intermediates, and then the intermediate was reacted with intermediate D according to the method described in Example 29 for steps 3 and 4 to give the corresponding compound.

1-Ethyl-4-(piperidin-4-ylmethyl)piperazine was substituted with the starting material shown in the following table according to the method described in Example 29 for Step 1 and 2 to give the corresponding intermediate shown in the table, and then the intermediate was reacted with compound 151 according to the method described in Example 29 for Step 3 to give the corresponding compound.

The starting material shown in the following table was used to prepare the corresponding compound shown in the table according to the method described in Example 29 for steps 3 and 4.

The starting materials shown in the following table were used for the reaction with intermediate product K to obtain the corresponding compounds shown in the table according to the method described in Example 29.

Example 30: (1S,3R)-3-((5-amino-3-ethylpyridino[3,4-b]pyrazin-2-yl)amino)cyclopentan-1-ol (compound 263)

Compound 151 (100 mg, 0.28 mmol) was used as a starting material to afford compound 263 as a white solid (20 mg, 25%) according to the method described for step 4 of Example 1. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.14 (br s, 1H), 7.75 (d, J=6.0 Hz, 1H), 7.01 (d, J=7.2 Hz, 1H), 6.59 (d, J=6.0 Hz, 1H), 6.51 (br s, 2H), 4.81 (s, 1H), 4.48-4.44 (m, 1H), 4.19-4.17 (m, 1H). 2.78-2.73 (multiplet, 2H), 2.18-2.14 (multiplet, 1H), 2.01-1.96 (multiplet, 1H), 1.80-1.74 (multiplet, 2H), 1.66-1.59 (multiplet, 2H), 1.28 (triplet, J=7.2 Hz, 3H). (IER) m/z. 274.1 [M+1] ⁺ .

Example 31: (1S,3R)-3-((5-amino-3-ethyl-8-(1-(5-methylpyrimidin-4-yl)-1H-pyrazol-4-yl)pyridino[3,4-b]pyrazin-2-yl)amino)cyclopentan-1-ol (compound 269)

Step 1: To a solution of 4-chloro-5-methylpyrimidine (300 mg, 2.3 mmol) in N,N-dimethylformamide (15 mL) were added 4-(4,4,5,5-tetramethyl-1,3,2-dioxoboran-2-yl)pyrazole (906 mg, 4.6 mmol) and cesium carbonate (2.3 g, 7.0 mmol). The mixture was stirred at 80 °C for 12 h under nitrogen. At the end of the reaction, the mixture was cooled to room temperature and water (50 mL) was added. The solution was extracted three times with ethyl acetate (50 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the product (200 mg, 30% yield). The product was used directly in the next step. (ESI) m/z. 287.2 [M+N] ⁺ .

Steps 2 and 3: The product obtained in step 1 was reacted with intermediate D according to the method described in Example 29 for steps 3 and 4 to give the formate of compound 269 as a yellow solid (yield after two steps 46%). ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 9.61 (singlet, 1H), 8.90 (singlet, 1H), 8.77 (singlet, 1H), 8.56 (singlet, 1H), 8.38 (singlet, 1H), 8.14 (broad singlet, 1H), 7.37 (doublet, J=7.2 Hz, 1H), 6.91 (singlet, 2H), 4.80 (singlet, 1H), 4.57-4.53 (multiplet, 1H), 4.30-4.25 (multiplet, 1H), 2.86-2.81 (multiplet, 2H), 2.66 (singlet, 3H), 2.46-2.40 (multiplet, 2H), 2.23-2.13 (multiplet, 1H), 1.93-1.83 (multiplet, 2H), 1.74-1.64 (multiplet, 2H), 1.32 (triplet, J=7.2 Hz, 3H). (IER) m/z. 432.2 [M+1] ⁺ .

4-Chloro-5-methylpyrimidine was replaced by the starting material shown in the following table according to the method described in Example 31, and the two isomers of the product obtained in Step 1 were separated to give the corresponding compounds shown in the table.

The starting materials shown in the following table were used to prepare the corresponding compound shown in the table according to the method described in Example 6 for Step 2.

4-Chloro-5-methylpyrimidine was substituted for the starting materials shown in the following table to give the corresponding compounds shown in the table according to the method described in Example 31.

4-Chloro-5-methylpyrimidine was substituted for the starting materials in the following table to give the borate intermediate according to the method described for step 1 in Example 31. The intermediate was then reacted with compound 151 according to the method described for step 2 in Example 2 to give the corresponding compounds shown in the following table.

2-Methyl-5-bromopyridine was substituted for the starting materials shown in the following table to give the borate intermediates according to the method described in Example 2. The borate intermediates were then reacted with intermediate D according to the method described in Example 31 for steps 2 and 3 to give the corresponding compounds shown in the following table.

4-Chloro-2-methylpyrimidine was substituted for the starting material shown in the following table to obtain intermediate 335-A according to the method described for step 1 in Example 23. Intermediate 335-A was then reacted with intermediate D according to the method described in Example 31 for steps 2 and 3 to obtain the corresponding compound shown in the following table.

4-Chloro-2-methylpyrimidine was substituted for the starting material shown in the following table to obtain intermediate 346-A according to the method described in Example 23 for Step 1. Intermediate 346-A was then used to prepare the corresponding compound shown in the following table according to the method described in Example 7 for Steps 3 and 4.

Example 32: 8-bromo-3-ethyl-N2-(tetrahydro-2H-pyran-4-yl)pyridino[3,4-b]pyrazine-2,5-diamine (compound 2)

8-Bromo-N5-(2,4-dimethoxybenzyl)-3-ethyl-N2-(tetrahydro-2H-pyran-4-yl)pyridino[3,4-b]pyrazine-2,5-diamine (1.0 g, 2.0 mmol) was used as starting material to obtain compound 2 as a yellow solid (650 mg, 93% yield) according to the method described in Example 1 for Step 12. (ESI) m/z 352.0. [M+H] ⁺ .

The starting materials shown in the following table were reacted with compound 2 according to the method described in Example 23 to give the corresponding compounds shown in the following table.

Example 33: 3-ethyl-8-(3-methoxy-4-(4-(8-methyl-3,8-diazabicyclo[3.2.1]octan-3-yl)piperidin-1-yl)phenyl)-N2 -(tetrahydro-2H-pyran-4-yl)pyridino[3,4-b]pyrazine-2,5-diamine (compound 293) and 8-(4-(4-(3,8-diazabicyclo[3.2.1]octan-3-yl)piperidin-1-yl)-3-methoxyphenyl)-3-ethyl-1H-(tetrahydro-2H-pyran- pyridin-4-yl)pyridino[3,4-b]pyrazin-2,5-diamine (compound 319)

Steps 1 and 2: tert-Butyl 3,8-diazabicyclo[3,2,1]octane-8-carboxylate (3.5 g, 0.016 mol) and benzyl 4-oxopiperidine-1-carboxylate (5.7 g, 0.025 mol) were used as starting materials to give a yellow oily substance (3 g, 61% yield over two steps) according to the methods described in Example 1 for Step 1 and Step 2. (ESI) m/z 296.3. [M+H] ⁺ .

Steps 3 and 4: tert-Butyl 3-(piperidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate was used as starting material to prepare intermediate 293-A as a brown oil (yield over two steps 66%) according to the methods described in Example 7 for Step 1 and Step 2. (ESI) m/z 528.3. [M+H] ⁺ .

Steps 5 and 6: Intermediate 293-A was reacted with 8-bromo-N5-(2,4-dimethoxybenzyl)-3-ethyl-N2-(tetrahydro-2H-pyran-4-yl)pyridino[3,4-b]pyrazine-2,5-diamine according to the method for the synthesis of compound 8 proposed in Example 2 to give compound 319 as a yellow solid (yield after two steps 35%). ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 8.31 (singlet, 2H), 7.92 (singlet, 1H), 7.22-7.18 (multiplet, 2H), 7.01 (doublet, J=7.2 Hz, 1H), 6.89 (doublet, J=8.4 Hz, 1H), 6.45 (singlet, 2H), 4.17-4.07 (multiplet, 1H), 3.92-3.88 (multiplet, 2H), 3.81 (singlet, 3H), 3.77 (singlet, 2H), 3.41 (doublet, J=11.6 Hz, 3H), 3.27 (triplet, J=11.2 Hz, 3H), 2.83-2.74 (multiplet, 4H), 2.60-2.54 (multiplet, 2H), 2.32-2.29 (multiplet, 1H), 1.88-1.75 (multiplet, 8H), 1.66-1.54 (multiplet, 4H), 1.31 (triplet, J=7.2 Hz, 3H). (IER) m/z. 573.3 [M+1] ⁺ .

Step 7: A solution of compound 319 (100 mg, 0.17 mmol) and formaldehyde (8.0 mg, 0.27 mmol) in methanol (10 mL) was stirred at 25 °C for 10 min, followed by the addition of sodium borohydride (20 mg, 0.52 mmol). The mixture was stirred at 25 °C for 30 min and concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (dichloromethane/methanol = 1:1) to give compound 293 as a yellow solid (40 mg, yield 39%). ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 7.92 (s, 1H), 7.27-7.12 (m, 2H), 7.00 (d, J=7.2 Hz, 1H), 6.88 (d, J=8.0 Hz, 1H), 6.44 (broad s, 2H), 4.19-4.01 (m, 1H), 3.92-3.88 (m, 2H), 3.88 (s, 3H), 3.39 (d, J=12.0 Hz, 2H), 3.30-3.22 (m, 2H), 3.10 (s, 2H), 2.83-2.78 (m, 2H), 2.66-2.54 (multiplet, 4H), 2.38 (doublet, J=8.0 Hz, 2H), 2.22 (doublet, J=12.0 Hz, 4H), 1.87-1.77 (multiplet, 6H), 1.70-1.50 (multiplet, 6H), 1.31 (triplet, J=7.2 Hz, 3H). (IER) m/z. 587.3 [M+1] ⁺ .

Example 34: (1S,3R)-3-((5-amino-3-ethyl-8-(3-methoxy-4-(1-(1-methylpiperidin-4-yl)-1H-pyrazol-4-yl)phenyl)pyridino[3,4-b]pyrazin-2-yl)amino)cyclopentan-1-ol (compound 312)

Step 1: To a tetrahydrofuran/water mixture (50 mL) containing 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxoboran-2-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylic acid tert-butyl ester (2.0 g, 5.0 mmol) and 1-bromo-4-chloro-2-methoxybenzene (1.8 g, 7.0 mmol) were added cesium carbonate (5.18 g, 15.0 mmol) and Pd(dppf)Cl ₂ (0.39 g, 0.50 mmol). The resulting mixture was stirred at 70 °C for 1 h under nitrogen blanket. At the end of the reaction, the mixture was cooled to room temperature and water (20 mL) was added. The solution was extracted three times (10 mL × 3) with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (petroleum ether/ethyl acetate = 70%/30%) to give a yellow oily substance (1.8 g, yield 83%). (ESI) m/z 392.1 [M+H] ⁺ .

Steps 2-5: The product obtained in Step 1 was reacted according to the method described in Example 33 for Steps 4-7 to give the formate of compound 312 as a yellow solid (yield after four steps 1.3%). ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 8.18 (doublet, J=4.0 Hz, 3H), 8.00 (singlet, 1H), 7.94 (singlet, 1H), 7.60 (doublet, J=8.0 Hz, 1H), 7.43 (doublet, J=1.2 Hz, 1H), 7.31-7.29 (multiplet, 1H), 7.05 (doublet, J=7.2 Hz, 1H), 6.53 (singlet, 2H), 4.44-4.39 (multiplet, 1H), 4.20-4.09 (multiplet, 2H), 3.91 (singlet, 3H), 2.89 (singlet, 2H), 2.79 (multiplet, 2H), 2.24 (singlet, 3H), 2.19-1.98 (multiplet, 7H), 1.96-1.91 (multiplet, 1H), 1.84-1.54 (multiplet, 5H), 1.31 (triplet, J=7.2 Hz, 3H). (ESI) m/z. 543.3. [M+H] ⁺ .

Example 35: 3-ethyl-8-(3-methoxy-4-(3-methyl-4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)-N2-(tetrahydro-2H-pyran-4-yl)pyridino[3,4-b]pyrazine-2,5-diamine (compound 287)

Step 1: 1-Methylpiperazine (1.5 g, 15 mmol) and N-tert-butoxycarbonyl-3-methyl-4-piperidone (3.2 g, 15 mmol) were used as starting materials to afford intermediate 287-A as a white solid (2.5 g, 53% yield) according to the method described in Example 6 for Step 2. (ESI) m/z 298.3 [M+1] ⁺ .

Step 2: Intermediate 287-A (2.5 g, 8.0 mmol) was used as starting material to prepare intermediate 287-B as a white solid (1.7 g, 87% yield) according to the method described for Step 4 in Example 7. (ESI) m/z 198.3 [M+1] ⁺ .

Step 3: To a mixture of intermediate 287-B (300 mg, 1.5 mmol) and 1-bromo-4-chloro-2-methoxybenzene (336 mg, 1.5 mmol) in 1,4-dioxane solution (10 mL) were added potassium tert-butanol (438 mg, 4.6 mmol), tBuXPhos Pd G3 (120 mg, 0.15 mmol) and tBuXPhos (129 mg, 0.30 mmol). The resulting mixture was stirred at 100 °C for 1 h under nitrogen atmosphere under microwave irradiation. At the end of the reaction, the mixture was cooled to room temperature and water (30 mL) was added. The solution was extracted three times (30 mL × 3) with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (dichloromethane/methanol = 70%:30%) to afford intermediate 287-C as a yellow oily substance (280 mg, yield 54%). (ESI) m/z 338.2. [M+1] ⁺ .

Steps 4 and 5: Intermediate 287-C was reacted according to the method described in Example 7 for Step 2 to give intermediate 287-D, then intermediate 287-D was reacted with compound 2 to give the formate of compound 287 as a yellow solid according to the method described for Step 2 in Example 2 (yield after two steps 37%). ¹ H NR (400 MHz, DMSO-d ₆ ) δ 8.14 (broad s, 1H), 7.91 (s, 1H), 7.22-7.20 (m, 2H), 7.05 (do, J=7.2 Hz, 1H), 6.88-6.85 (m, 1H), 6.55 (s, 2H), 4.14-4.10 (m, 1H), 3.92-3.89 (m, 2H), 3.82 (s, 3H), 3.41-3.37 (m, 5H), 3.30-3.24 (m, 5H), 2.84-2.78 (m, 3H), 2.54-2.52 (m, 2H), 2.51 (s, 3H), 2.17 (s, 3H), 1.88-1.84 (m, 3H), 1.63-1.57 (m, 3H), 1.31 (t, J=7.2 Hz, 3H), 1.09 (d, J=6.8 Hz, 3H). (IER) m/z. 575.3 [M+1] ⁺ .

Example 36: (1S,3R)-3-((5-amino-3-ethyl-8-(3-methoxy-4-(8-methyl-3,8-diazabicyclo[3.2.1]octyl-3-yl)phenyl)pyridino[3,4-b]pyrazin-2-yl)amino)cyclopentan-1-ol (compound 286)

Step 1: tert-Butyl 3,8-diazabicyclo[3,2,1]octane-8-formate (1.1 g, 5.2 mmol) was used to prepare intermediate 286-A as a yellow oil (0.97 g, 50% yield) according to the method described in Example 7 for Step 1. (ESI) m/z 353.3 [M+1] ⁺ .

Step 2: Intermediate 286-A (970 mg, 2.7 mmol) was used as starting material to prepare intermediate 287-B as a white solid (450 mg, 65% yield) according to the method described in Example 7 for Step 4. (ESI) m/z 253.1 [M+1] ⁺ .

Step 3: Intermediate 286-B (450 mg, 1.8 mmol) was used as starting material to give intermediate 286-C as a white solid (370 mg, 74% yield) according to the method described in Example 33 for Step 7. (ESI) m/z 267.1. [M+H] ⁺ .

Steps 4 and 5: Intermediate 286-C was used to prepare intermediate 286-D according to the method described for Step 2 in Example 7, after which intermediate 286-D was reacted with compound 151 according to the method described in Example 2 for Step 2 to give compound 286 as a yellow solid (yield over two steps 2.8%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.90 (s, 1H), 7.32 (d, J=1.6 Hz, 1H), 7.19-7.16 (m, 1H), 7.00 (d, J=7.2 Hz, 1H), 6.81 (d, J=8.4 Hz, 1H), 6.41 (s, 2H), 4.75 (d, J=3.6 Hz, 1H), 4.42-4.37 (m, 1H), 4.13 (d, J=4.4 Hz, 1H), 3.81 (s, 3H), 3.21-3.06 (m, 4H), 2.85-2.72 (multiplet, 4H), 2.21 (singlet, 3H), 2.16-2.11 (multiplet, 1H), 1.96-1.81 (multiplet, 5H), 1.77-1.54 (multiplet, 4H), 1.30 (triplet, J=7.2 Hz, 3H). (IER) m/z. 504.3. [M+N] ⁺ .

Example 37: 3-ethyl-8-(3-methoxy-4-(4-(3-methyl-3,8-diazabicyclo[3.2.1]octyl-8-yl)piperidin-1-yl)phenyl)-N2-(tetrahydro-2H-pyran-4-yl)pyridino[3,4-b]pyrazine-2,5-diamine (compound 327)

Steps 1-3: 1,4-dioxa-8-azanolo[4,5]decane was used to prepare intermediate 327-A as a yellow solid (yield over three steps 20%) according to the method described in Example 7 for Steps 1-3. (ESI) m/z 671.3 [M+1] ⁺ .

Step 4: A solution of intermediate 327-A (1.0 g, 1.5 mmol) in trifluoroacetic acid/dichloromethane (1 mL/10 mL) was stirred at 25 °C for 1.5 h. The resulting residue was purified by silica gel column chromatography (1% ammonia in methanol/dichloromethane = 30%/70%) to afford intermediate 327-B as a yellow solid (750 mg, 92% yield). (ESI) m/z [M+H] ⁺ 521.4.

Step 5: To a solution of intermediate 327-B (600 mg, 1.2 mmol) in 6 N hydrochloric acid (6 mL) was added acetic acid (3 mL), and the solution was stirred at 70 °C for 3 h. After the reaction, the solution was concentrated under reduced pressure and a solution of ammonia in methanol (20 mL) was added to make it basic, and the solution was concentrated again under reduced pressure. The resulting residue was purified by silica gel column chromatography (1% ammonia in methanol/dichloromethane = 30%/70%) to give intermediate L as a yellow solid (350 mg, yield 32%). (ESI) m/z [M+H] ⁺ 477.2.

Step 6: To a mixture of intermediate L (150 mg, 0.30 mmol) and 3-methyl-3,8-diazabicyclo[3.2.1]octane (79 mg, 0.60 mmol) in 1,2-dichloroethane (20 mL) was added sodium triacetoxyborohydride (200 mg, 0.90 mmol). The solution was stirred at 80 °C for 16 h. At the end of the reaction, the mixture was cooled to room temperature and then concentrated under reduced pressure. The resulting residue was purified by preparative liquid chromatography to give the formate salt of compound 327 as a yellow solid (17 mg, 9% yield). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.20 (broad s, 1.8H), 7.92 (s, 1H), 7.26-7.17 (m, 2H), 7.01 (d, J=7.2 Hz, 1H), 6.89 (d, J=8.0 Hz, 1H), 6.45 (broad s, 2H), 4.14-4.10 (m, 1H), 3.91 (d, J=8.4 Hz, 2H), 3.82 (s, 3H), 3.48 (s, 4H), 3.25-3.20 (m, 2H), 2.80 (t, J=7.2 Hz, 2H), 2.64-2.52 (multiplet, 4H), 2.48-2.41 (multiplet, 4H), 2.22 (doublet, J=12.0 Hz, 2H), 1.96 (doublet, J=12.0 Hz, 2H), 1.86 (doublet, J=12.4 Hz, 2H), 1.75-1.57 (multiplet, 6H), 1.50-1.39 (multiplet, 2H), 1.31 (triplet, J=7.2 Hz, 3H). (IER) m/z. 587.4 [M+H] ⁺ .

The starting materials shown in the following table were reacted with intermediate L according to the reductive amination method described in Example 37 for Step 6 to give the corresponding compounds shown in the table.

Example 38: 3-ethyl-8-(3-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)-N2-(tetrahydro-2H-1 shran-4-yl )-1,6-naphthyridine-2,5-diamine (compound 113)

Synthesis of Intermediate M: 1-methyl-4-(4-piperidinyl)piperazine (5.0 g, 30 mmol) was used to obtain intermediate M as a yellow solid according to the method described in Example 7 for steps 1 and 2 (yield over two steps 24%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.39 (d, J=8.0 Hz, 1H), 7.25 (s, 1H), 6.92 (d, J=8.0 Hz, 1H), 3.90 (s, 3H), 2.51-2.34 (m, 13H), 2.34 (s, 3H), 1.89-1.76 (m, 4H), 1.33 (s, 12H). (ESI) m/z. [M+1] ⁺ 416.2.

Step 1: To a solution of 4-amino-2-chloropyridine-3-formaldehyde (200 mg, 1.3 mmol) in N,N-dimethylformamide (5 mL) was added NBS (260 mg, 1.5 mmol). The reaction mixture was stirred at 25 °C for 5 h and then poured into water (10 mL). The solid precipitated, filtered and dried to give intermediate 113-A as a white solid (215 mg, 72% yield). ¹ H NMR (400 MHz, CDCl ₃ ) δ 10.39 (br s, 1H), 9.24 (s, 1H), 8.24 (s, 1H), 5.73 (s, 1H). (IER) m/z 234.9 [M+H] ⁺

Step 2: To a solution of ethyl butyrate (440 mg, 3.8 mmol) in tetrahydrofuran (10 mL) at -78 °C was added FDA solution (1.9 mL, 3.8 mmol, 2 mol/L). The reaction mixture was stirred at -78 °C for 1 h, after which intermediate 113-A (0.21 g, 0.90 mmol) was added. The reaction mixture was stirred at 25 °C for 14 h and added to a saturated solution of ammonium chloride (10 mL). The solution was extracted three times (10 mL × 3) with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 10:1) to afford intermediate 113-B as a white solid (88 mg, 40% yield). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.51 (br s, 1H), 8.50 (s, 1H), 7.85 (s, 1H), 2.59 (doublet of doublets, J = 14.8, 7.2 Hz, 2H), 1.19 (triplet, J = 7.2 Hz, 3H). (ESI) m/z 286.9 [M+H] ⁺

Step 3: Intermediate 113-B (88 mg, 0.31 mmol) was used as starting material to prepare intermediate 113-C as a light yellow solid according to the method described for Step 1 in Example 2 (80 mg, 62% yield). (ESI) m/z 418.0 [M+H] ⁺ .

Step 4: Intermediate 113-C (80 mg, 0.19 mmol) was used as starting material to give intermediate 113-D as a brown solid according to the method described for Step 8 in Example 1 (90 mg, crude product was used directly for the next reaction).

Step 5: A mixture of intermediate 113-D (90 mg) and tetrahydropyran-4-amine (412 mg, 4.1 mmol) was stirred at 145 °C for 1 h. At the end of the reaction, the mixture was cooled to room temperature and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 1:1) to afford intermediate 113-E as a brown solid (60 mg, yield 89%). (ESI) m/z 353.0 [M+H] ⁺ .

Step 6: Intermediate 113-E (60 mg, 0.17 mmol) and intermediate M (142 mg, 0.34 mmol) were used as starting materials to obtain the formate of compound 113 as a yellow solid according to the method described for step 2 in Example 2 (23 mg, 23% yield). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.20 (br s, 3H), 8.00 (s, 1H), 7.81 (s, 1H), 7.23-7.13 (m, 2H), 6.88 (d, J=8.0 Hz, 1H), 6.75 (s, 2H), 6.45 (d, J=7.2 Hz, 1H), 4.19-4.11 (m, 1H), 3.92-3.84 (m, 2H), 3.84-3.77 (m, 3H), 3.45 (d, J=11.2 Hz, 2H), 3.23 (t, J=11.2 Hz, 2H), 2.62-2.54 (multiplet, 6H), 2.53-2.51 (multiplet, 1H), 2.48-2.20 (multiplet, 2H), 2.22 (singlet, 3H), 1.90-1.76 (multiplet, 4H), 1.65-1.48 (multiplet, 4H), 1.25 (doublet of doublets, J=12.0, 4.8 Hz, 3H). (ESI) m/z. 560.3 [M+1] ⁺ .

The starting material shown in the following table was reacted with intermediate M to give the corresponding compound shown in the table according to the synthetic route described in Example 38 for Step 6.

Example 39: 3-ethyl-8-(3-methoxyphenyl)-N2-(tetrahydro-2H-pyran-4-yl)pyrazino[2,3-d]pyridazine-2,5-diamine (compound 116)

Step 1: To a solution of ethanol (40 mL) and saturated ammonium chloride (40 mL) containing 3,6-dichloro-5-nitropyridin-4-amine (4.4 g, 21 mmol) was added iron powder (12 g, 0.21 mol). The reaction mixture was stirred at 25 °C for 18 h, filtered, and the filter cake was washed with ethanol (40 mL). The filtrate was evaporated to remove ethanol, and the residue was extracted three times with ethyl acetate (100 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (dichloromethane:methanol = 20:1) to afford intermediate 116-A as a yellow solid (1.8 g, yield 43%). (ESI) m/z 178.9 [M+H] ⁺

Steps 2-4: According to the method described in Example 1 for Steps 7-9, using intermediate 116-A, intermediate 116-D was obtained as a white solid (yield after three steps 2.2%). (ESI) m/z 312.1 [M+H] ⁺

Step 5: According to the method described in Example 8 for Step 1, using intermediate 116-D (66 mg, 0.21 mmol), intermediate 116-E was obtained as a yellow solid (60 mg, 61% yield). (ESI) m/z 441.1 [M+H] ⁺

Step 6: According to the method described in Example 17 for Step 1, using intermediate 116-E (50 mg, 0.12 mmol) to react with trifluoromethanesulfonic anhydride (40 mg, 0.14 mmol) to give intermediate 116-E as a yellow solid (50 mg, 65% yield). (ESI) m/z 573.0 [M+H] ⁺

Steps 7 and 8: Intermediate 116-E (70 mg, 0.12 mmol) was reacted with (3-methoxyphenyl)boric acid (37 mg, 0.24 mmol) according to the method described in Example 17 for Step 1 to give the formate of compound 116 as a white solid (5 mg, 11% yield over two steps). ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 8.23 (s, 1H), 7.73-7.71 (m, 2H), 7.41 (d, J=7.2 Hz, 1H), 7.38-7.34 (m, 1H), 7.00-6.97 (m, 1H), 6.70 (s, 2H), 4.16-4.12 (m, 1H), 3.96-3.92 (m, 2H), 3.82 (s, 3H), 3.42 (s, 2H), 2.90-2.84 (m, 2H), 1.89 (d, J=12.0 Hz, 2H), 1.67-1.61 (multiplet, 2H), 1.35-1.31 (multiplet, 3H). (IER) m/z. 381.1 [M+1] ⁺ .

Methoxyphenylboronic acid was replaced by intermediate M obtained in Step 7 to give the compound shown in the following table according to the synthetic method described in Example 39.

Example 40: (1S,3R)-3-((5-amino-3-ethyl-8-(3-methoxy-4-(2-methylpyrimidin-4-yl)phenyl)pyrido[3,4-b]pyrazin-2-yl)amino)cyclopentan-1-ol (compound 288)

Step 1: 4-Chloro-2-methylpyrimidine (1.0 g, 7.8 mmol) was reacted with (4-chloro-2-methoxyphenyl)boric acid (1.7 g, 9.3 mmol) according to the method described for Step 2 in Example 2 to give intermediate 288-A as a brown solid (1.6 g, 87% yield).

Steps 2 and 3: Intermediate 288-A was used to prepare intermediate 151 according to the method described for step 2 in Example 7, after which the intermediate was reacted with compound 151 according to the method described for step 2 in Example 2 to give the formate of compound 288 as a yellow solid (yield after two steps was about 59%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.67 (d, J=5.6 Hz, 1H), 8.14 (broad s, 1H), 8.07 (s, 1H), 8.01 (d, J=8.4 Hz, 1H), 7.87 (d, J=5.6 Hz, 1H), 7.58-7.54 (m, 1H), 7.51-7.47 (m, 1H), 7.16 (d, J=7.2 Hz, 1H), 6.81 (broad s, 2H), 4.87-4.65 (m, 1H), 4.46-4.38 (m, 1H), 4.17-4.09 (multiplet, 1H), 3.94 (singlet, 3H), 2.81 (quartet, J=7.2 Hz, 2H), 2.67 (singlet, 3H), 2.20-2.11 (multiplet, 1H), 1.97-1.88 (multiplet, 1H), 1.82-1.58 (multiplet, 4H), 1.32 (triplet, J=7.2 Hz, 3H). (ESI) m/z 472.2. [M+1] ⁺ .

The starting material shown in the following table was reacted according to the method described in Example 40 for steps 1 and 2 to obtain the intermediate product in the form of borate ester, after which the intermediate product in the form of borate ester was reacted with intermediate product D according to the method for the synthesis of compound 8 from step 2 to the end to obtain the corresponding compound shown in the table.

Example 41: cis- and trans-4-(4-(5-amino-3-ethyl-2-(((1R,3S)-3-hydroxycyclopentyl)amino)pyridino[3,4-b]pyrazin-8-yl)-1H-pyrazol-1-yl)-1-methylcyanohexyl-1-ol (compounds 284 and 285)

According to the following method, cis and trans isomers of the starting materials were reacted to give compound 284 and compound 285, respectively.

Step 1: To a solution of cis- or trans-4-(4-iodo-1H-pyrazol-1-yl)-1-methylcyclohexan-1-ol in tetrahydrofuran was added isopropyl magnesium chloride (about 5 equivalents) at 0°C. This mixture was stirred at 0°C under nitrogen atmosphere for 1 h, after which 2-methoxy-4,4,5,5-tetramethyl-1,3,2-dioxapentaborane (about 5.5 equivalents) was added. The mixture was stirred at 0°C for 1 h under nitrogen atmosphere, then heated to 25°C and stirred for 3 h. After the reaction, the mixture was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 3:7 - ethyl acetate) to give intermediate product N as a colorless oily substance (yield about 72%). (ESI) m/z. 307.2. [M+H] ⁺ .

Steps 2 and 3: Intermediate N was reacted with intermediate D according to the method described in Example 31 for Steps 2 and 3 to give compound 284 (yield after two steps 30%) and compound 285 (yield after two steps 6.5%), both as yellow solids. Compound 284: ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 8.45 (s, 1H), 8.17-8.13 (m, 2H), 8.01 (s, 1H), 7.28 (d, 3=6.8 Hz, 1H), 6.82 (s, 2H), 4.92-4.65 (m, 1H), 4.54-4.46 (m, 1H), 4.26-4.07 (m, 3H), 2.82 (quartet, 3=7.2 Hz, 2H), 2.34-2.27 (m, 1H), 2.12-2.03 (m, 3H), 1.88-1.80 (multiplet, 4H), 1.71-1.63 (multiplet, 4H), 1.53-1.46 (multiplet, 2H), 1.31 (triplet, 3=7.2 Hz, 3H), 1.16 (singlet, 3H). (ESI) m/z 452.3. [M+1] ⁺ . Compound 285: ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 8.43 (s, 1H), 8.19 (s, 2H), 8.02 (s, 1H), 7.10 (do, J=7.2 Hz, 1H), 6.38 (broad s, 2H), 4.50-4.47 (m, 1H), 4.28-4.11 (m, 2H), 2.80 (quartet, J=7.2 Hz, 2H), 2.35-2.25 (m, 1H), 2.05-2.00 (m, 3H), 2.00-1.52 (m, 11H), 1.30 (triplet, J=7.2 Hz, 3H), 1.19 (singlet, 3H). (IER) m/z. 452.3 [M+1] ⁺ .

Example 42: (1S,3R)-3-((5-amino-3-ethyl-8-(1-(4-((1-methylpiperidin-4-yl)oxy)phenyl)-1H-pyrazol-4-yl)pyridino[3,4-b]pyrazin-2-yl)amino)cyclopentan-1-ol (compound 292)

Step 1: 4-(4-iodophenoxy)-1-methylpiperidine (800 mg, 2.5 mmol) was reacted with 4-bromo-1H-pyrazole (389 mg, 2.6 mmol) according to the method described in Example 22 for Step 1 to give intermediate 292-A as a yellow solid (410 mg, 48%). (ESI) m/z 336.1 [M+H] ⁺ .

Step 2: To a solution of intermediate 292-A (350 mg, 1.04 mmol) in tetrahydrofuran (10 mL) was added n-butyl solution (0.5 mL, 2.4 N) dropwise at -78 °C under nitrogen atmosphere. The solution was stirred for 20 min, after which 2-methoxy-4,4,5,5-tetramethyl-1,3,2-dioxpentaborane (328 mg, 2.08 mmol) in tetrahydrofuran (5 mL) was added. The mixture was stirred at -78 °C for 1 h under nitrogen atmosphere, then the temperature was brought to 0 °C and stirred for 1 h. At the end of the reaction, methanol (2 mL) was added to stop the reaction, and the solution was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (dichloromethane:methanol = 9:1) to give intermediate product 292-B as a yellow oily substance (150 mg, yield 86%). (ESI) m/z. 302.2. [M+H] ⁺ .

Steps 3 and 4: Intermediate 292-B (114 mg, 0.398 mmol) was reacted with intermediate D (100 mg, 0.199 mmol) according to the method described in Example 31 for Steps 2 and 3 to give the formate of compound 292 as a yellow solid (38 mg, 3.9% yield over two steps). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.77-7.70 (m, 2H), 7.22-7.15 (m, 4H), 6.91 (d, J=8.8 Hz, 2H), 6.52 (d, J=1.6 Hz, 1H), 4.72-4.68 (m, 1H), 4.60-4.54 (m, 1H), 4.09 (s, 1H), 4.00-3.96 (m, 1H), 3.34-3.20 (m, 4H), 2.79 (s, 3H), 2.73-2.66 (m, 2H), 2.14-1.85 (multiplet, 5H), 1.76-1.53 (multiplet, 5H), 1.48-1.38 (multiplet, 1H), 1.25 (triplet, J=7.2 Hz, 3H). (ESI) m/z. 529.3. [M+H] ⁺ .

Example 43: (S,E)-N-(1-((2-((4-(5-amino-3-ethyl-2-((tetrahydro-2H-pyran-4-yl)amino)pyridino[3,4b] pyrazin-8-yl)-2-methoxyphenyl)amino)ethyl)amino)-1-oxopropan-2-yl)-4-(dimethylamino)-N-methylbutyl-2-enamide (connection 358)

Step 1: 4-bromo-2-methoxyaniline (8.0 g, 39 mmol) was reacted with tert-butyl (2-hydroxyethyl)carbamate (6.6 g, 41 mmol) according to the method described for Step 2 of Example 6 to give intermediate 358-A as a yellow solid (4.5 g, 30%). (ESI) m/z 346.1 [M+H] ⁺ .

Steps were used to react intermediate 358-A according to the method described in Example 7 for Steps 2-4 to give intermediate 358-B as a yellow solid (yield after three steps 23%). (ESI) m/z 438.2 [M+H] ⁺ .

Step 5: To a solution of (E)-4-(dimethylamino)butyl-2-enoic acid (500 mg, 3.9 mmol) and (2S)-2-(methylamino)propionate (678 mg, 4.3 mmol) in dichloromethane (10 mL) were added HATU (1.9 g, 5.0 mmol) and triethylamine (0.77 g, 7.7 mmol). The mixture was stirred at 25 °C for 4 h, then concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (methanol (containing 1% ammonia)/dichloromethane = 0% to 100% over 20 min) to give intermediate 358-C as a yellow solid (980 mg, 84% yield). (ESI) m/z. 271.1. [M+N] ⁺ .

Step 6: Intermediate 358-C (500 mg, 1.9 mmol) was reacted according to the method described for Step 1 of Example 24 to give intermediate 358-D as a yellow solid (350 mg, 88% yield). (ESI) m/z 215.1 [M+H] ⁺ .

Step 7: Intermediate 358-D (62 mg, 0.29 mmol) was reacted with intermediate 358-B (120 mg, 0.27 mmol) according to the method described for Step 1 in this Example to give compound 358 as a yellow solid (18.5 mg, 11% yield). ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 8.17 (singlet, 2H), 7.98 (singlet, 1H), 7.88 (singlet, 1H), 7.15 (doublet, J=6.0 Hz, 2H), 6.98 (doublet, J=7.2 Hz, 1H), 6.67-6.59 (multiplet, 2H), 6.52 (singlet, 1H), 6.42 (singlet, 2H), 5.04-5.00 (multiplet, 2H), 4.22-4.11 (multiplet, 1H), 3.96-3.87 (multiplet, 2H), 3.81 (singlet, 3H), 3.31-3.28 (multiplet, 3H), 3.16-3.12 (multiplet, 2H), 3.10-3.00 (multiplet, 2H), 2.90 (singlet, 2H), 2.80-2.75 (multiplet, 3H), 2.16-2.10 (multiplet, 6H), 1.87-1.84 (multiplet, 2H), 1.63-1.60 (multiplet, 2H), 1.31 (triplet, J=7.2 Hz, 1H), 1.23 (doublet, J=7.2 Hz, 1H). (IER) m/z. 634.3. [M+N] ⁺ .

Example of bioassay 1: Experiment to evaluate FLT3 kinase inhibitory activity in vitro:

Reagents and consumables

Equipment

Centrifuge (manufacturer: Eppendorf, model: 5430), Microplate reader (manufacturer: Perkin Elmer, model: Caliper EZ Reader II), Echo 550 (manufacturer: Labcyte, model: Echo 550)

Experimental methodology

1. The process of kinase reaction

(1) 1x kinase buffer was prepared.

(2) Preparation of serial dilutions of compound: Serial 3-fold dilutions of the stock solution (the concentration of the test compound was 1 μM) to 10 concentrations were performed and assayed in separate wells. Compounds were serially diluted to 10 different concentrations (the final concentration was 100-fold dilution) in 384-well plates. 250 nL of the solution were transferred to each well of the 384-well plate using Echo550. 100% DMSO (250 nL) was added to the negative control wells and positive control wells, respectively.

(3) Kinase solution (final concentration was 2.5-fold dilution) was prepared with 1x kinase buffer.

(4) 10 μl of kinase solution (the final concentration was 2.5-fold dilution) was added to the compound wells and positive control wells, respectively; 1x kinase buffer (10 μl) was added to the negative control wells.

(5) The solution was centrifuged at 1000 rpm for 30 s, shaken, mixed and incubated at room temperature for 10 min.

(6) A mixed solution of ATP and kinase substrate 2 (at a final concentration ratio of 25/15) with 1x kinase buffer was prepared.

(7) 15 μl of a mixed solution of ATP and kinase substrate 2 (at a final concentration ratio of 25/15) was added to initiate the reaction.

(8) The 384-well plate was centrifuged at 1000 rpm for 30 s, shaken and mixed, and then incubated at room temperature for 30 min.

(9) To stop the kinase reaction, a stopping solution (30 μl) was added, the solution was centrifuged at 1000 rpm for 30 s, shaken and mixed.

(10) The conversion index was read using Caliper EZ Reader II.

2. Data analysis

Formula for calculation

Where: Conversion(%)_test - conversion rate of the test sample; Conversion(%)_min: negative control well.

Example of bioassay 2: In vitro MV4-11 cell line proliferation inhibition assay:

Experimental materials:

MV4-11 cell lines were purchased from the Cell Bank of Chinese Academy of Sciences, IMDM medium (Gibco, Cat. No. 12440), fetal bovine serum (Gibco, Cat. No. 10099141C), penicillin-streptomycin antibiotics (Gibco, Cat. No. 15140122), DMSO (Sigma, Cat. No. D2650), Celltiter-Glo (CTG) Kit (Promega, Cat. No. G7573), 384-well clear flat-bottom cell culture plate (Corning, Cat. No. 3764), D300e Digital Dispenser (Tecan, Cat. No. D300e), SpectraMax i3x Microplate Reader (MOLECULAR DEVICES, catalog no. i3x)

Experimental methodology:

Cell culture:

MV4-11 cells were cultured in IMDM supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin antibiotics, and the cell density was maintained at less than 1.0 x 10 ⁶ cells/mL to ensure that they were always in the logarithmic growth phase. Cell viability was greater than 95%.

Preparation of test solutions of compounds:

Test compounds were diluted 3-fold from 10 μM in DMSO to 9 concentrations. 60 nL of the diluted compound was added to a cell culture plate and three replicates were set up.

Cells treated with compound:

The prepared MV4-11 cell suspension was introduced into a 384-well plate by adding 20 μl of the cell suspension to each well, i.e., each well contained 2000 MV4-11 cells, with the final DMSO concentration being 0.3%. The cell culture plate was placed in an incubator at 37°C with 5% carbon dioxide and incubated for 72 h.

20 µl of Promega CellTiter-Glo reagent were added to each well and the plate was incubated at room temperature for 10 min to stabilize the luminescent signals. The signals were read using a SpectraMax i3x microplate reader.

Data Analysis

The raw data were converted into an inhibition index, and the _IC50 value (half-maximal inhibitory concentration) of the compound was determined by fitting a nonlinear four-parameter curve.

The following table shows the results obtained in Biological Assay Example 1 and Biological Assay Example 2. The results showed that the compound sample of the present invention has a strong inhibitory effect on FLT3 kinase and has a high inhibitory activity against MV4-11 cell proliferation.

A: _IC50 ≤10 nM;

B: 10 nM<IC ₅₀ ≤100 nM;

C: 100nM<IC ₅₀ ≤500 nM;

D: >500 nM

The results of the activity analysis of some compounds are presented in the following table.

All literature references mentioned in this application are incorporated herein by reference as if each portion of the literature was incorporated by reference. It should also be understood that many changes and modifications may be made by those skilled in the art upon examination of the above description, and such equivalents are also within the scope of the claims appended to this application.

Claims (67)

1. A compound represented by the following formula II, or pharmaceutically acceptable salts thereof: II where X and Y represent N and Z represents CR; wherein R is selected from the group consisting of H; Ra is selected from the group consisting of H, a substituted or unsubstituted C ₁ -C ₆ alkyl group; U represents a chemical bond or -NH-; and when U represents a chemical bond, then Rc represents pyrrolidine; Rc is selected from the group consisting of a substituted or unsubstituted C ₁ -C ₆ alkyl group, a substituted or unsubstituted C ₃ -C ₈ carbocyclic ring, a substituted or unsubstituted 3-8-membered heterocyclic ring containing 1-2 heteroatoms selected from oxygen, sulfur and nitrogen atoms; Re is selected from the group consisting of -NHR; wherein R is selected from the group consisting of H; W is substituted or unsubstituted phenyl, and a substituted or unsubstituted 5-6-membered heteroaromatic ring containing 1-2 heteroatoms selected from the group consisting of sulfur and nitrogen atoms; in addition to the general substituent, the W group is further substituted with at least one group with the structure -M-A, where M is selected from the group consisting of a chemical bond or -CHR-; A is selected from the group consisting of a substituted or unsubstituted _C1 - _C6 alkyl group, a substituted or unsubstituted _C1 - _C6 alkoxy group, a substituted or unsubstituted _C6 aryl group, a substituted or unsubstituted 4-12-membered heterocyclic ring containing 1-2 heteroatoms selected from oxygen and nitrogen atoms, a substituted or unsubstituted 5-12-membered heteroaromatic ring containing 1-2 heteroatoms selected from nitrogen atoms, a substituted or unsubstituted _C3 - _C12 cycloalkyl group; in group A, substitution means substitution with one or more groups selected from group B, where group B consists of H, halogen, =O, cyano, hydroxy, aldehyde, carboxyl, substituted or unsubstituted _C1 - _C6 alkyl, substituted or unsubstituted _C1 - _C6 alkoxy, substituted or unsubstituted 3- to 12-membered heterocyclic ring containing 1 to 2 heteroatoms selected from oxygen and nitrogen atoms, substituted or unsubstituted 5- to 12-membered heteroaromatic ring containing 1 to 2 heteroatoms selected from nitrogen and oxygen atoms, substituted or unsubstituted _C3 - _C12 cycloalkyl, substituted or unsubstituted _C2 - _C10 ester, substituted or unsubstituted _C1 - _C6 amide, substituted or unsubstituted _C1 - _C4 alkyl-S(O) ₂ group, wherein in group B the substitution means substitution with one or more R groups; wherein R is selected from the group consisting of H, halogen, cyano group, hydroxyl group, substituted or unsubstituted _C1 - _C6 alkyl group, substituted or unsubstituted _C1 - _C6 alkoxy group, substituted or unsubstituted _C3 cycloalkyl group, substituted or unsubstituted _C2 - _C10 ester group; unless otherwise indicated, in the above formulae, substitution means that the hydrogen atom in the corresponding group is substituted by one or more substituents selected from the group consisting of deuterium, halogen, hydroxyl group, amino group, _C1 - _C6 amide group, cyano group, unsubstituted or halogenated _C1 - _C6 alkyl group, _C1 - _C6 alkoxy group, _C1 - _C6 alkylamino group, _C1 - _C12 alkylaminocarbonyl group, unsubstituted or halogenated _C2 - _C10 acyl group, unsubstituted or halogenated _C1 - _C4 alkyl-S(O) ₂ group. 2. The compound according to claim 1, or its pharmaceutically acceptable salts, wherein the compound has a structure presented in formula IIa: IIa wherein the ring W is selected from the group consisting of a substituted or unsubstituted phenyl group, a substituted or unsubstituted pyridine, and a substituted or unsubstituted pyrazolyl; wherein in the group W, the said substitution means substitution with one or more groups selected from the group A. 3. The compound according to claim 1, or its pharmaceutically acceptable salts, wherein W is substituted or unsubstituted phenyl. 4. The compound of claim 1, or pharmaceutically acceptable salts thereof, wherein W is a substituted or unsubstituted ring structure selected from the group consisting of 5. The compound according to claim 1, or its pharmaceutically acceptable salts, wherein the compound has a structure presented in formula III: III wherein ring A is selected from the group consisting of a substituted or unsubstituted 4-12-membered heterocyclic ring containing 1-2 heteroatoms selected from oxygen and nitrogen atoms, a substituted or unsubstituted 5-12-membered heteroaromatic ring containing 1-2 heteroatoms selected from nitrogen atoms, a substituted or unsubstituted _C6 aryl group, and a substituted or unsubstituted _C3 - _C12 cycloalkyl group; M is selected from the group consisting of a chemical bond or -CHR-. 6. The compound of claim 5, or its pharmaceutically acceptable salts, wherein the compound has a structure represented in the following formula: Where Rf is selected from the group consisting of H, halogen, cyano group, amino group, nitro group, hydroxyl group, _C1 - _C4 alkyl-S(O) ₂ group, substituted or unsubstituted _C1 - _C6 alkyl group and substituted or unsubstituted _C1 - _C6 alkoxy group; t is 0, 1, 2, 3, or 4; ring A is selected from the group consisting of a substituted or unsubstituted 5-12 membered saturated ring; wherein ring A contains at least one heteroatom selected from N or O. 7. The compound of claim 5, or its pharmaceutically acceptable salts, wherein the compound has a structure represented in the following formula: Where Rf is selected from the group consisting of H; t is 0, 1, 2, 3, or 4; L represents N; ring A is selected from the group consisting of a substituted or unsubstituted 4-12-membered heterocyclic ring containing 1-2 heteroatoms selected from oxygen and nitrogen atoms, a substituted or unsubstituted _C3 - _C12 cycloalkyl group, a substituted or unsubstituted 5-12-membered heteroaromatic ring containing 1-2 heteroatoms selected from nitrogen atoms and a substituted or unsubstituted _C6 aryl group. 8. The compound or pharmaceutically acceptable salts thereof according to claim 5, wherein the compound has a structure represented in the following formula IV: IV wherein ring A is selected from the group consisting of a substituted or unsubstituted 4-12 membered heterocyclic ring containing 1-2 heteroatoms selected from oxygen and nitrogen atoms, a substituted or unsubstituted C ₆ aryl group; M is selected from the group consisting of a chemical bond or -CHR-; ring B is selected from the group consisting of a substituted or unsubstituted 4-12 membered heterocyclic ring containing 1-2 heteroatoms selected from oxygen and nitrogen atoms; V is a chemical bond. 9. The compound of claim 5, or pharmaceutically acceptable salts thereof, wherein ring A contains at least one substituent G, wherein the group G is selected from the group consisting of =O, a substituted or unsubstituted 4-7-membered heterocyclic ring containing 1-2 heteroatoms selected from oxygen and nitrogen atoms, a substituted or unsubstituted _C2 - _C10 ester group, and a substituted or unsubstituted _C1 - _C6 amide group. 10. The compound according to claim 1, or its pharmaceutically acceptable salts, wherein the compound is selected from the following compounds: No structure No structure 3 4 5 6 7 8 14 13 16 15 18 17 20 19 22 21 24 23 26 25 28 27 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 47 46 49 57 63 58 65 64 67 66 69 68 71 70 76 72 78 77 80 79 82 81 84 83 88 85 90 87 92 89 135 91 137 93 139 121 154 134 156 136 160 138 162 155 164 159 166 163 168 165 170 167 172 169 174 171 176 173 178 175 180 177 182 179 184 181 186 183 188 185 190 187 192 189 194 191 196 193 198 195 200 197 202 199 204 201 206 203 208 205 210 207 212 209 214 211 216 213 218 215 220 217 222 219 224 221 226 223 228 225 230 227 232 229 234 231 236 233 238 235 240 237 242 239 244 241 246 243 248 245 250 247 252 249 255 251 257 253 259 256 261 258 266 260 268 265 270 267 272 269 274 271 276 273 278 275 280 277 282 279 284 281 286 285 288 287 290 289 294 291 296 293 298 295 300 297 302 299 304 301 306 303 308 305 310 307 312 309 322 311 324 313 326 323 333 325 335 332 337 334 339 336 341 338 343 340 345 342 347 344 349 346 351 348 353 350 355 352 356 354 327 314 328 315 329 316 330 317 331 318 320 319 321 56 11. A compound of formula IV, or its pharmaceutically acceptable salts, IV where X and Y represent N and Z represents CR; wherein R is selected from the group consisting of H; Ra is selected from the group consisting of H, a substituted or unsubstituted C ₁ -C ₆ alkyl group; U represents -NH-; Rc is selected from the group consisting of a substituted or unsubstituted C ₁ -C ₆ alkyl group, a substituted or unsubstituted C ₃ -C ₈ carbocyclic ring, an unsubstituted 3-8-membered heterocyclic ring containing 1-2 heteroatoms selected from oxygen and nitrogen atoms; Re is selected from the group consisting of -NHR; wherein R is selected from the group consisting of H; W is substituted phenyl, and a substituted or unsubstituted 5-6-membered heteroaromatic ring containing 1-2 heteroatoms selected from the group consisting of sulfur and nitrogen atoms; wherein ring A is selected from the group consisting of a substituted or unsubstituted 4-12 membered heterocyclic ring containing 1-2 heteroatoms selected from oxygen, sulfur and nitrogen atoms, a substituted or unsubstituted C ₆ aryl group; M is selected from the group consisting of a chemical bond or -CHR-; ring B is selected from the group consisting of a substituted or unsubstituted 4-12 membered heterocyclic ring containing 1-2 heteroatoms selected from oxygen, sulfur and nitrogen atoms; V is selected from the group consisting of -CH ₂ -; unless otherwise indicated, in the above formulae, substitution means that the hydrogen atom in the corresponding group is substituted by one or more substituents selected from the group consisting of deuterium, halogen, hydroxyl group, amino group, _C1 - _C6 amide group, cyano group, unsubstituted or halogenated _C1 - _C6 alkyl group, _C1 - _C6 alkoxy group, _C1 - _C6 alkylamino group, _C1 - _C12 alkylaminocarbonyl group, halogenated _C2 - _C10 acyl group, unsubstituted _C1 - _C4 alkyl-S(O) ₂ group. 12. A compound selected from the group consisting of: 45 262 48 264 59 283 61 357 157 358 158 13. A pharmaceutical composition for treating diseases associated with abnormal gene levels or abnormal expression of kinases selected from the group consisting of FLT3, ALK and AXL, comprising a therapeutically effective amount of one or more compounds according to any one of claims 1-12, or their pharmaceutically acceptable salts, as well as one or more pharmaceutically acceptable carriers, excipients, additives, fillers and diluents. 14. Use of a compound according to any one of claims 1 to 12, or pharmaceutically acceptable salts, in the manufacture of medicaments for the treatment of diseases associated with abnormal gene levels or abnormal expression of kinases selected from the group consisting of FLT3, ALK and AXL. 15. Use according to item 14, wherein the disease is selected from the group consisting of lung cancer, acute leukemia, chronic leukemia. 16. The use according to claim 14, wherein the disease is selected from the group consisting of acute myelogenous leukemia, non-small cell lung cancer, and small cell lung cancer.