CN117903171A

CN117903171A - Integrin inhibitors of a class of bicyclic derivatives

Info

Publication number: CN117903171A
Application number: CN202311336242.3A
Authority: CN
Inventors: 郝宇; 黄逸安; 裴成奎
Original assignee: Shanghai Ruling Biomedical Co ltd
Current assignee: Shanghai Ruling Biomedical Co ltd
Priority date: 2022-10-17
Filing date: 2023-10-16
Publication date: 2024-04-19
Also published as: WO2024083086A1

Abstract

A bicyclic αvβ1, αvβ6 and αvβ8 integrin inhibitor compound of formula (I) and racemates, stereoisomers, tautomers, isotopic labels, nitrogen oxides, solvates, polymorphs, metabolites, esters, prodrugs or pharmaceutically acceptable salts thereof, and pharmaceutical compositions containing the same, processes for preparing the same, and pharmaceutical uses thereof. The structure of the formula (I) is as follows.

Description

Integrin inhibitors of a class of bicyclic derivatives

Technical Field

The present invention relates to the field of pharmaceutical chemistry, in particular to a class of bicyclic derivatives as αvβ1, αvβ6 and αvβ8 integrin inhibitors, pharmaceutical compositions comprising such compounds and their use in the treatment and/or prevention of conditions requiring αvβ1, αvβ6 and αvβ8 integrin inhibitors.

Background

Integrins, also known as integrins, zygotes, are a large family of cell surface receptors, heterodimeric transmembrane glycoproteins formed by the binding of an alpha subunit and a beta subunit. In mammals, 18 α and 8 β subunits have been found that non-covalently bind to form at least 24 different integrins, which are differentially expressed by various types of cells and recognize a variety of ligands, mediating information transfer between the intracellular and extracellular matrices as well as between neighboring cells. Integrins are a class of cell adhesion molecules consisting of an extracellular region, a transmembrane region and an intracellular region, wherein the extracellular N-terminal domain is combined with a specific ligand, and the intracellular region is connected with cytoskeletal protein through alpha-actin, ankyrin and focal adhesion proteins to form a ligand-integrins-cytoskeletal transmembrane system. Integrins as cell surface receptors can achieve bi-directional signaling through the cell membrane. One is outside-in signal transfer. Integrin binds to ligands in the ECM, resulting in conformational changes in the integrin and aggregates on the cell membrane, forming focal adhesion, activating multiple intracellular signaling pathways that transduce signals from outside into the cell, thereby regulating cell adhesion, diffusion, migration, proliferation, differentiation, and remodeling. The other is the inside-out signal transmission. The intracellular signal induces the change of the conformation of the talin, which leads to the binding of the talin to the intracellular domain of the β subunit of the integrin, which untwists the links between the tail of the α subunit and the tail of the β subunit in the cytoplasm, and the conformation of the head of the integrin changes, which increases the affinity of the integrin for binding to the extracellular ligand and enhances the adhesion capacity of the cell. Integrins can be classified into 3 kinds according to the specificity of the integrin ligand, namely, binding to laminin-type integrins, binding to collagen-type integrins, and binding to arginine-glycine-aspartic acid sequences (Arg-Gly-Asp, RGD) -type integrins.

RGD-type integrins of different subtypes are involved in the progression of fibrosis at different sites in the body, and among many αv family proteins, integrin αvβ1 is expressed on activated fibroblasts and mesangial cells; integrin αvβ6 is capable of binding to the RGD sequence of TGF- β1, effecting cell-cell contact to activate TGF- β1; αvβ8 binds to the RGD sequence in TGF- β3LAP, presenting LAP complexes to the cell surface matrix metalloproteinases, thereby activating TGF- β1. Avβ6 and avβ1 play a major role in kidney and lung tissue fibrosis, while avβ1 plays a dominant role in liver fibrosis. Integrin avβ1 is a low affinity fibronectin receptor, highly expressed in basal epithelial cells, and has the effect of promoting keratinocyte migration on the underlying fibronectin EDA. Blocking the interaction of integrin avβ1 with TGF- β1 helps to inhibit TGF- β1 activity, blocking fibrosis progression. Integrin αvβ6 expression is reported to be low in normal lung tissue, but avβ6 is rapidly highly expressed when lung injury is inflammatory and fibrotic (HATLEY ET AL, ANGEWANDTE CHEMIE International Edition,2018,57 (13): 3298.). In patients suffering from Primary Biliary Cirrhosis (PBC), alcoholic fatty liver disease, hepatitis b, hepatitis c, and the like, the mRNA expression level of integrin avβ6 is increased. The expression of integrin avβ6 is significantly increased in chronic inflammatory and fibrotic diseases associated with kidney disease compared to normal kidney tissue. In addition, integrin avβ6 is significantly highly expressed in biopsy samples from patients with diabetes, lung hemorrhagic nephritis syndrome, alport syndrome, lupus nephritis, etc. (Koivisto et al, the international journal of biochemistry & cell biology,2018, 99:186). Integrin αvβ8 is extremely important for the regulation of tgfβ, the only binding ligand is L-tgfβ, which is highly expressed in various cancer cells, especially in tumor cells with high Treg, and L-tgfβ is mainly expressed in immune cells such as T cells. Researchers found that αvβ8 promoted differentiation of tumor microenvironment tregs, which in turn promoted tumor growth. The action mechanism is that after the T cell GARP/L-TGFbeta is combined with the alpha v beta 8, conformational change occurs, the activated TGF beta is released, and the activated TGF beta is combined with the TGF beta R on the T cell, so that the differentiation of the T cell into the Treg is promoted. In addition, conditional loss of lung fibroblast αvβ8 can inhibit mouse airway fibrosis in an IL-1β and albumin-induced mouse airway fibrosis model.

Tissue fibrosis can occur in a variety of organs, and is a relatively common fibrotic disease including Idiopathic Pulmonary Fibrosis (IPF), nonalcoholic fatty liver (NASH), cirrhosis, renal fibrosis, scleroderma, myocardial fibrosis, and the like. Tissue damage and inflammation are important contributors to fibrosis. As the inflammation causes necrosis of the parenchymal cells of the organ, local immune cells are activated and various blood cells enter the damaged site. Activated immune cells produce a large number of highly bioactive cytokines and chemokines, resulting in localized activation of mesenchymal cells that produce extracellular matrix (ECM), disrupt the extracellular microenvironment, and further increase production of pro-inflammatory cytokines, chemokines and angiogenic factors. Eventually, the extracellular Matrix abnormally increases and over-deposits to cause lesions to lead to tissue fibrosis (Ricard-Blumet al, matrix Biology,2018, 68:122.). The main feature of fibrosis is the formation and deposition of excess fibrous connective tissue. Chronic concomitant fibrosis damage can cause destruction of tissue structures and organ dysfunction ultimately leading to organ failure.

The integrin family is paid attention as key regulatory factors of chronic inflammation, fibrosis, tumor immunity and the like, and development of a safe and effective integrin inhibitor with novel structure is needed.

Disclosure of Invention

The invention aims to provide an integrin inhibitor of a novel-structure bicyclo derivative, wherein the structure of the compound contains a bicyclo structure, has good inhibition activity and certain drug selectivity on integrin alpha v beta 1, alpha v beta 6 and alpha v beta 8, has excellent oral drug substitution property, and can effectively inhibit the expression quantity of fibronectin and collagen in an in-vitro and in-vivo drug effect model of fibrotic diseases.

In a first aspect of the present invention there is provided a class of bicyclic derivatives of formula I, as well as racemates, stereoisomers, tautomers, isotopic labels, nitroxides, solvates, polymorphs, metabolites, esters, prodrugs or pharmaceutically acceptable salts thereof:

wherein,

Y ₁ is C1-C6 alkylene, -O-, -C1-C6 alkylene) -O-, -NH-, - (C1-C6 alkylene) -NH-;

Y ₂ is C1-C6 alkylene, -O-, -C1-C6 alkylene) -O-, -C (O) - (C1-C6 alkylene) -NH-, -NH-, -NH- (C1-C6 alkylene) -, - (C1-C6 alkylene) -NH-;

R ₁ is a substituted or unsubstituted 6-10 membered aromatic ring, a substituted or unsubstituted 5-8 membered heteroaromatic ring, a substituted or unsubstituted Wherein the C ring, D ring are each independently a substituted or unsubstituted C6-C10 aromatic ring, a substituted or unsubstituted 5-8 membered heteroaromatic ring, a substituted or unsubstituted 5-8 membered cycloalkane ring, or a substituted or unsubstituted 5-8 membered heteroalkane ring;

R ₂ is a hydrogen atom, a substituted or unsubstituted C6-C10 aromatic ring, a substituted or unsubstituted 5-8 membered heteroaromatic ring, a substituted or unsubstituted C8-C16 fused ring, or-L1-L2;

Wherein, -L1-is selected from the group consisting of none, - (substituted or unsubstituted C1-C6 alkylene) -, - (substituted or unsubstituted C1-C6 alkyleneoxy) -, - (substituted or unsubstituted C1-C6 alkylenethio) -, - (substituted or unsubstituted C3-C8 cycloalkyl) -, - (substituted or unsubstituted C3-C8 heterocycloalkyl) -, - (substituted or unsubstituted C6-C10 aryl) -, - (substituted or unsubstituted C5-C8 heteroaryl) -,

L2 is selected from the group consisting of an unsubstituted, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkylthio, substituted or unsubstituted C3-C8 cycloalkyl, - (C1-C3 alkyl) -substituted or unsubstituted C3-C8 cycloalkyl, - (C1-C3 alkoxy) -substituted or unsubstituted C3-C8 cycloalkyl, -O-substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C3-C8 heteroalkyl, - (C1-C3 alkyl) -substituted or unsubstituted C3-C8 heteroalkyl, - (C1-C3 alkoxy) -substituted or unsubstituted C3-C8 heteroalkyl, -O-substituted or unsubstituted C3-C8 heteroalkyl, and substituted or unsubstituted 5-8 membered heteroaryl;

x is an oxygen atom or a nitrogen atom;

Wherein when X is an oxygen atom, R _3a is a hydrogen atom, a C1-C6 alkyl group, a substituted or unsubstituted C6-C10 aromatic ring, R _3b is absent; when X is nitrogen atom, R _3a is hydrogen atom, hydroxyl, C1-C6 alkyl, substituted or unsubstituted C6-C10 aromatic ring, R _3b is hydrogen atom;

Is a substituted or unsubstituted spiro ring or a substituted or unsubstituted parallel ring;

wherein when Is a substituted or unsubstituted spirocyclic ring, as shown in formula Ia:

a is a ring selected from the group consisting of: a substituted or unsubstituted quaternary cycloalkane ring, a substituted or unsubstituted five-membered cycloalkane ring, a substituted or unsubstituted six-membered cycloalkane ring, a substituted or unsubstituted seven-membered cycloalkane ring, a substituted or unsubstituted eight-membered cycloalkane ring, a substituted or unsubstituted quaternary heteroalkyl ring, a substituted or unsubstituted five-membered heteroalkyl ring, a substituted or unsubstituted six-membered heteroalkyl ring, a substituted or unsubstituted seven-membered heteroalkyl ring, a substituted or unsubstituted eight-membered heteroalkyl ring, a substituted or unsubstituted five-membered heteroalkyl ring and a C6-C10 aromatic ring, a substituted or unsubstituted six-membered heteroalkyl ring and a C6-C10 aromatic ring;

B is a ring selected from the group consisting of: a substituted or unsubstituted quaternary cycloalkane ring, a substituted or unsubstituted five-membered cycloalkane ring, a substituted or unsubstituted six-membered cycloalkane ring, a substituted or unsubstituted seven-membered cycloalkane ring, a substituted or unsubstituted eight-membered cycloalkane ring, a substituted or unsubstituted quaternary heteroalkyl ring, a substituted or unsubstituted five-membered heteroalkyl ring, a substituted or unsubstituted six-membered heteroalkyl ring, a substituted or unsubstituted seven-membered heteroalkyl ring, a substituted or unsubstituted eight-membered heteroalkyl ring, a substituted or unsubstituted five-membered heteroalkyl ring and a C6-C10 aromatic ring, a substituted or unsubstituted six-membered heteroalkyl ring and a C6-C10 aromatic ring;

When (when) Is a substituted or unsubstituted fused ring, as shown in formula Ib:

Is a single bond or a double bond;

B is a ring selected from the group consisting of: a substituted or unsubstituted quaternary cycloalkane ring, a substituted or unsubstituted five-membered cycloalkane ring, a substituted or unsubstituted six-membered cycloalkane ring, a substituted or unsubstituted seven-membered cycloalkane ring, a substituted or unsubstituted eight-membered cycloalkane ring, a substituted or unsubstituted C6-C10 aromatic ring, a substituted or unsubstituted quaternary heteroalkyl ring, a substituted or unsubstituted five-membered heteroalkyl ring, a substituted or unsubstituted six-membered heteroalkyl ring, a substituted or unsubstituted seven-membered heteroalkyl ring, a substituted or unsubstituted eight-membered heteroalkyl ring, a substituted or unsubstituted five-membered heteroalkyl ring and a C6-C10 aromatic ring, a substituted or unsubstituted six-membered heteroalkyl ring and a C6-C10 aromatic ring;

n is 0,1, 2 or 3;

Wherein the "substitution" means that 1 to 4 (preferably 1,2,3 or 4) hydrogen atoms on the group are each independently substituted with a substituent selected from the group consisting of: C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, C1-C3 haloalkyl, halogen, nitro, cyano, amino, hydroxy, thiol, = O, C1-C4 carboxyl, C2-C4 ester, C2-C4 amide, C1-C6 alkoxy, carboxylic acid, C1-C4 alcohol, C1-C4 alkylamino, -O- (CH ₂)_m -C3-C8 cycloalkyl, -O- (CH ₂)_m -C3-C8 heteroalkyl), -NH- (CH ₂)_m -C3-C8 cycloalkyl, -NH- (CH ₂)_m -C3-C8 heteroalkyl), wherein each m is independently an integer from 0 to 3;

Wherein the heteroaryl, heteroalkyl, or heteroaryl groups each independently have 1-3 (preferably 1, 2, or 3) heteroatoms selected from N, O and S.

In another preferred embodiment, Y ₁ is absent.

In another preferred embodiment, Y ₁ is C1-C3 alkylene, - (C1-C3 alkylene) -NH-.

In another preferred embodiment, Y ₁ is C1-C3 alkylene.

In another preferred embodiment, Y ₁ is methylene.

In another preferred embodiment, Y ₂ is C1-C3 alkylene, -O-, -C1-C3 alkylene) -O-, -C (O) - (C1-C3 alkylene) -NH-, -NH-, -NH- (C1-C3 alkylene) -, - (C1-C3 alkylene) -NH-. In another preferred embodiment, Y ₂ is C1-C3 alkylene, -C (O) - (C1-C3 alkylene) -NH-, - (C1-C3 alkylene) -NH-.

In another preferred embodiment, Y ₂ is-O-, -C1-C3 alkylene) -O-.

In another preferred embodiment, Y ₂ is C1-C3 alkylene.

In another preferred embodiment, Y ₂ is methylene, ethylene or-O-.

In another preferred embodiment, R ₁ is substituted or unsubstitutedWherein the C ring is a C6-C10 aromatic ring or a 5-8 membered heteroaromatic ring, and the D ring is a substituted or unsubstituted 5-8 membered heteroaromatic ring or a substituted or unsubstituted 5-8 membered heteroalkane ring.

In another preferred embodiment, R ₁ is substituted or unsubstitutedWherein the C ring is a C6 aromatic ring or a 5-6 membered nitrogen containing heteroaromatic ring, and the D ring is a substituted or unsubstituted 5-6 membered nitrogen containing heteroaromatic ring or a substituted or unsubstituted 5-6 membered nitrogen containing heteroalkyl ring.

In another preferred embodiment, the nitrogen-containing heteroaromatic or nitrogen-containing heteroalkyl ring of the C-ring or D-ring optionally further comprises 1 or 2O atoms.

In another preferred embodiment, R ₁ is substituted or unsubstitutedWherein the D ring is a substituted or unsubstituted 5-6 membered nitrogen containing heteroaryl ring or a substituted or unsubstituted 5-6 membered nitrogen containing heteroalkyl ring.

In another preferred embodiment, R ₁ is a substituted or unsubstituted 5-8 membered nitrogen containing heteroaryl ring.

In another preferred embodiment, R ₁ is a substituted or unsubstituted group selected from the group consisting of:

In another preferred embodiment, R ₁ is a substituted or unsubstituted pyridine ring, a substituted or unsubstituted 1,2,3, 4-tetrahydronaphthyridine ring, a substituted or unsubstituted quinoline ring, or a substituted or unsubstituted azaindole ring.

In another preferred embodiment, R ₁ is

In another preferred embodiment, R ₂ is a C6-C10 aromatic ring (preferably a C6 aromatic ring) substituted with R _c, wherein R _c is hydrogen, halogen, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 alkoxy.

In another preferred embodiment, R ₂, the C8-C16 condensed ring means a ring having 8 to 16 ring carbon atoms in which two or three rings are condensed, each ring in the condensed rings being independently a 6-membered aromatic ring, a 5-6-membered heteroaromatic ring, a 5-6-membered cycloalkane ring, or a 5-6-membered heteroalkane ring.

In another preferred embodiment, R ₂ is a C9-C10 fused ring.

In another preferred embodiment, R ₂, the C9-C10 fused ring is preferably a 6 membered aromatic ring fused to a 5-6 membered heteroaromatic ring, more preferably a 6 membered aromatic ring fused to a 6 membered heteroaromatic ring.

In another preferred embodiment, R ₂ is a hydrogen atom or-L1-L2; wherein, -L1-is- (substituted or unsubstituted C6-C10 aryl) -, and L2 is substituted or unsubstituted 5-8 membered heteroaryl.

In another preferred embodiment, R ₂ is-L1-L2.

In another preferred embodiment, -L1-is selected from the group consisting of- (substituted or unsubstituted C3-C8 cycloalkyl) -, - (substituted or unsubstituted C3-C8 heterocycloalkyl) -, - (substituted or unsubstituted C6-C10 aryl) -, - (substituted or unsubstituted C5-C8 heteroaryl) -.

In another preferred embodiment, -L1-is selected from the group consisting of- (substituted or unsubstituted C6-C10 aryl) -, - (substituted or unsubstituted 5-8 membered heteroaryl) -.

In another preferred embodiment, -L1-is selected from the group consisting of- (substituted or unsubstituted C6 aryl) -, - (substituted or unsubstituted 6 membered heteroaryl) -.

In another preferred embodiment, L2 is an unsubstituted, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkylthio, substituted or unsubstituted C3-C8 cycloalkyl, - (C1-C3 alkylene) -substituted or unsubstituted C3-C8 cycloalkyl, - (C1-C3 alkyleneoxy) -substituted or unsubstituted C3-C8 cycloalkyl, -O-substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 3-8 membered heteroalkyl, - (C1-C3 alkylene) -substituted or unsubstituted 3-8 membered heteroalkyl, - (C1-C3 alkyleneoxy) -substituted or unsubstituted 3-8 membered heteroalkyl, -O-substituted or unsubstituted 3-8 membered heteroalkyl, or substituted or unsubstituted 5-8 membered heteroaryl.

In another preferred embodiment, L2 is selected from the group consisting of substituted or unsubstituted 3-8 membered cycloalkyl, - (C1-C3 alkylene) -substituted or unsubstituted C3-C8 cycloalkyl, - (C1-C3 alkyleneoxy) -substituted or unsubstituted 3-8 membered cycloalkyl, -O-substituted or unsubstituted 3-8 membered cycloalkyl, substituted or unsubstituted 3-8 membered heteroalicyclic, - (C1-C3 alkylene) -substituted or unsubstituted 3-8 membered heteroalicyclic, - (C1-C3 alkyleneoxy) -substituted or unsubstituted 3-8 membered heteroalicyclic, -O-substituted or unsubstituted 3-8 membered heteroalicyclic, and substituted or unsubstituted 5-8 membered heteroaryl.

In another preferred embodiment, L2 is selected from the group consisting of a substituted or unsubstituted 3-8 membered heteroalkyl ring, a substituted or unsubstituted 5-8 membered heteroaryl.

In another preferred embodiment, -L1-is- (substituted or unsubstituted C6-C10 aryl) -, L2 is substituted or unsubstituted 5-8 membered heteroaryl.

In another preferred embodiment, -L1-is- (substituted or unsubstituted phenyl) -, L2 is a substituted or unsubstituted 5-6 membered nitrogen containing heteroaryl (preferably pyrazolyl).

In another preferred embodiment, L1 is optionally substituted with one or more R _a, wherein R _a is halogen, C1-C6 alkyl, C3-C6 cycloalkyl.

In another preferred embodiment, L1 is optionally substituted with one or more R _a, wherein R _a is halogen, C1-C6 alkyl.

In another preferred embodiment, L2 is optionally substituted with one or more R _b, wherein R _b is C1-C3 alkyl.

In another preferred embodiment, R ₂ is a structure of formula a:

wherein each Z ₁、Z₂、Z₃、Z₄ is independently CR _c or N;

L ₂ is an unsubstituted, C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkylthio, substituted or unsubstituted 3-8 membered cycloalkyl, substituted or unsubstituted 3-8 membered heteroalicyclic, substituted or unsubstituted 5-8 membered heteroaryl;

Each R _c is independently hydrogen, halogen, C1-C6 alkyl, 3-8 membered cycloalkyl, C1-C6 alkoxy.

In another preferred embodiment, R ₂ is a structure of formula b:

Wherein each Z ₁、Z₂、Z₃、Z₄、Z₅、Z₆、Z₇、Z₈、Z₉ is independently CR _c or N;

Each R _c is independently hydrogen, halogen, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 alkoxy.

In another preferred embodiment, each Z ₁、Z₂、Z₄ is independently CR _c,Z₃ is CR _c or N.

In another preferred embodiment, each Z ₁、Z₂、Z₄ is independently CH and Z ₃ is CR _c or N.

In another preferred embodiment, each Z ₅、Z₆、Z₇ is independently CR _c or N, and at least one of Z ₅、Z₆、Z₇ is N. In another preferred embodiment, R ₂ is of the structure:

Wherein L ₂ is C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkylthio;

In another preferred embodiment, each R _c is independently fluorine, bromine, chlorine, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methoxy, ethoxy.

In another preferred embodiment, R ₂ is substituted or unsubstituted

In another preferred embodiment, R ₂ is

In another preferred embodiment, when X is an oxygen atom, R _3a is a hydrogen atom or a C1-C3 alkyl group and R _3b is absent.

In another preferred embodiment, when X is an oxygen atom, R _3a is a hydrogen atom and R _3b is absent.

In another preferred embodiment, when X is a nitrogen atom, R _3a is a hydrogen atom, a hydroxyl group or a C1-C3 alkyl group, and R _3b is a hydrogen atom.

In another preferred embodiment, when X is a nitrogen atom, R _3a is a hydroxyl group and R _3b is a hydrogen atom.

In a further preferred embodiment of the present invention,Is a substituted or unsubstituted spirocyclic ring, as shown in formula Ia:

In another preferred embodiment, a is a ring selected from the group consisting of: a substituted or unsubstituted quaternary heteroalkyl ring, a substituted or unsubstituted five-membered heteroalkyl ring, a substituted or unsubstituted six-membered heteroalkyl ring, a substituted or unsubstituted seven-membered heteroalkyl ring, a substituted or unsubstituted eight-membered heteroalkyl ring;

b is a ring selected from the group consisting of: a substituted or unsubstituted quaternary heteroalkyl ring, a substituted or unsubstituted five-membered heteroalkyl ring, a substituted or unsubstituted six-membered heteroalkyl ring, a substituted or unsubstituted seven-membered heteroalkyl ring, and a substituted or unsubstituted eight-membered heteroalkyl ring.

In another preferred embodiment A, B are each independently a ring selected from the group consisting of: a quaternary heteroalkyl ring, a substituted or unsubstituted five membered heteroalkyl ring, a substituted or unsubstituted six membered heteroalkyl ring.

In another preferred embodiment, the substitution in the a and B rings means that one or more (e.g. 1,2, 3) hydrogen atoms are substituted with halogen or = O.

In another preferred embodiment, the halogen is F, cl, br, preferably F.

In another preferred embodiment, the heteroalkanes in the a and B rings each independently contain 1-3 (preferably 1, 2, 3) nitrogen atoms.

In another preferred embodiment, the a and B rings are each independently a ring selected from the group consisting of: azetidine, substituted or unsubstituted pyrrolidine, substituted or unsubstituted piperidine ring, substituted or unsubstituted morpholine ring.

In another preferred embodiment, the substitution in the A and B rings is preferably a substitution in the 3-position.

In another preferred embodiment, in the A and B rings, the substitution is preferably at the ortho position of the spiro atom.

In another preferred embodiment, the a and B rings are each independently of the other the following structure:

wherein "×" indicates that the carbon atom is a spiro atom.

In another preferred embodiment, Y ₁ is attached to the A ring through a nitrogen atom in the heteroalkyl ring.

In another preferred embodiment, the B ring is attached to Y ₂ through a nitrogen atom in the heteroalkyl ring.

In another preferred embodiment, the compound is of formula II:

in another preferred embodiment, the number of ring atoms of the ring a is equal to or different from the number of ring atoms of the ring B.

In another preferred embodiment, the number of ring atoms of the a ring is less than, equal to, or greater than the number of ring atoms of the B ring.

In another preferred embodiment, when A is a heteroalkyl ring, Y1 is attached to the A ring through a nitrogen atom in the heteroalkyl ring.

In another preferred embodiment, when A is a cycloalkane ring, Y1 is-O-, - (C1-C6 alkylene) -O-, -NH-or- (C1-C6 alkylene) -NH-.

In another preferred embodiment, when B is a heteroalkyl ring, Y2 is attached to the B ring through a nitrogen atom in the heteroalkyl ring.

In another preferred embodiment, when B is an alkane ring, Y2 is-O-, - (C1-C6 alkylene) -O-, -NH-or- (C1-C6 alkylene) -NH-.

In another preferred embodiment, when A is a 4, 5,6,7,8 membered ring, B is a 4, 5,6,7,8 membered ring.

In another preferred embodiment, the compound is of the formula III-1:

in another preferred embodiment, the compound is of the formula III-2:

in another preferred embodiment, the compound is of the formula III-3:

in another preferred embodiment, the compound is of the formula III-4:

In another preferred embodiment, the compound is of the formula III-5:

in another preferred embodiment, the compound is of the formula III-6:

in another preferred embodiment, in the above formulas III-1 to III-6, R ₁、R₂, A ring, B ring are as described above, and Y ₁、Y₂ is C1-C3 alkylene.

In another preferred embodiment, Y ₁、Y₂、R₁、R₂, X, A, B are each independently of the other the corresponding groups in the compounds 1 to 122 prepared in the examples.

In another preferred embodiment, whenIs a substituted or unsubstituted spirocyclic ring, as shown in formula Ia:

A is independently selected from the following group of rings: a substituted or unsubstituted quaternary heteroalkyl ring, a substituted or unsubstituted five-membered heteroalkyl ring, a substituted or unsubstituted six-membered heteroalkyl ring, a substituted or unsubstituted seven-membered heteroalkyl ring, a substituted or unsubstituted five-membered heteroalkyl ring and a 6-10 membered aromatic ring, a substituted or unsubstituted six-membered heteroalkyl ring and a 6-10 membered aromatic ring;

b is independently selected from the following group of rings: a substituted or unsubstituted quaternary heteroalkyl ring, a substituted or unsubstituted penta-membered heteroalkyl ring, a substituted or unsubstituted hexa-membered heteroalkyl ring, a substituted or unsubstituted hepta-membered heteroalkyl ring, a substituted or unsubstituted penta-membered heteroalkyl ring and a 6-10 membered aromatic ring, and a substituted or unsubstituted hexa-membered heteroalkyl ring and a 6-10 membered aromatic ring.

In another preferred embodiment, whenIs a substituted or unsubstituted fused ring, as shown in formula Ib:

Is a single bond or a double bond;

A is independently selected from the following group of rings: a substituted or unsubstituted quaternary heteroalkyl ring, a substituted or unsubstituted five-membered heteroalkyl ring, a substituted or unsubstituted six-membered heteroalkyl ring, a substituted or unsubstituted seven-membered heteroalkyl ring;

b is independently selected from the following group of rings: a substituted or unsubstituted C6-C10 aromatic ring, a substituted or unsubstituted quaternary heteroalkyl ring, a substituted or unsubstituted five-membered heteroalkyl ring, a substituted or unsubstituted six-membered heteroalkyl ring.

In another preferred embodiment, the "substitution" means that 1 to 4 (preferably 1, 2, 3 or 4) hydrogen atoms on the group are each independently substituted with a substituent selected from the group consisting of: halogen, -OH, -CN, -NO ₂、-NH₂、-NCO、-OCN、-SCN、-NCS、-N₃, - (C1-C3 alkylene) OH, -O (C1-C3 alkyl), -S (C1-C3 alkyl), -C (O) X' R ₄ or -X'C(O)R₅、-SO₃R₄、-OSO₂OR₄、-NR₆SO₂OR₅、-NR₆R₇、-SO₂NR₆R₇、-NH-SO₂-R₆、-N⁺R₆R₇R₈、-C(O)N(R₉)₂、-SO₂R₁₀、-NR₁₁C(O)N(R₁₁)₂、-B(OH)₂、-B(O(C1-C6 alkyl or alkylene)) ₂、-P(O)(OH)₃, -OP (O) (OC 1-C3 alkyl) ₂, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, oxo (=O), 3-12 membered alkyl, 3-12 membered heteroalkyl, 5-10 membered aryl, 5-10 membered aromatic heterocyclic, 3-12 membered heterocyclic, -O- (CH ₂)_p -3-8 membered cycloalkano- (CH ₂)_p -3-8 membered heteroalkyl, -NH- (CH ₂)_p -3-8 membered cycloalkane, -NH- (CH ₂)_p -3-8 membered heteroalkyl);

x' is a bond or is oxygen or sulfur, R ₄ is independently selected from hydrogen, C1-C6 alkyl, 3-12 membered alkyl cyclic group, 5-10 membered aryl, 3-12 membered heterocyclic group, R ₅ is independently selected from hydrogen, C1-C6 alkyl, 3-12 membered alkyl cyclic group, 5-10 membered aryl, 3-12 membered heterocyclic group;

R ₆、R₇ and R ₈ are each independently selected from hydrogen, C1-C6 alkyl, -C (O) X 'R ₄ or-X' C (O) R ₅, or R ₆ and R ₇ together with the N atom to which they are attached form a heterocyclic ring having 4-8 members in the ring structure; wherein R ₆ or R ₇ are not all-C (O) X 'R ₄ or-X' C (O) R ₅;

R ₉ is independently selected from hydrogen or C1-C6 alkyl, or two R ₉ together with the N atom to which they are attached form a 4-8 membered heterocyclic ring;

R ₁₀ is independently selected from hydrogen, C1-C6 alkyl, 3-12 membered alkyl cyclic, 3-12 membered heterocyclic, 5-10 membered aryl;

R ₁₁ is independently selected from hydrogen, C1-C6 alkyl, or two R ₁₁ together with the N atom to which they are attached form a heterocyclic ring having 4-8 members;

p is each independently an integer of 0 to 3.

In another preferred embodiment, the bicyclic derivative is selected from the group consisting of:

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in a second aspect of the present invention there is provided a process for the preparation of a bicyclic derivative as described in the first aspect of the invention, and racemates, stereoisomers, tautomers, isotopic labels, nitroxides, solvates, polymorphs, metabolites, esters, prodrugs or pharmaceutically acceptable salts thereof,

(I) When (when)When substituted or unsubstituted, the method comprises one of the following methods (1) to (5):

(1) Taking a compound i as a starting material, carrying out nucleophilic substitution reaction on the compound i and a reagent 4-bromobut-2-enoate to obtain an intermediate ii, carrying out deprotection on the intermediate ii to obtain an intermediate iii, carrying out reaction on the intermediate iii and a halide or aldehyde derivative to obtain an intermediate iv, carrying out conjugate addition reaction on the intermediate iv under the catalysis of a rhodium catalyst and a chiral ligand to obtain an intermediate v, and finally carrying out hydrolysis reaction on the intermediate v by alkali to obtain a target product, wherein the method is as shown in a scheme I:

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(2) The intermediate ii is subjected to conjugate addition reaction under the catalysis of rhodium catalyst and chiral ligand to obtain an intermediate vi, the intermediate vii is generated after deprotection, the intermediate vii is reacted with halogenide or aldehyde derivative to obtain an intermediate v, and finally, the target product is obtained through hydrolysis reaction of alkali, as shown in a scheme II:

(3) Taking a compound i as a starting material, firstly reacting with a halide or an aldehyde derivative to obtain an intermediate viii, removing a protecting group from the intermediate viii to obtain an intermediate ix, carrying out nucleophilic substitution reaction on the intermediate ix and a reagent 4-bromobut-2-enoate to obtain an intermediate iv, carrying out conjugate addition under the catalysis of a rhodium catalyst and a chiral ligand to obtain an intermediate v, and finally carrying out hydrolysis reaction of alkali to obtain a target product, wherein the method comprises the following steps:

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(4) Taking a compound vii as a starting material, firstly obtaining an intermediate x through nucleophilic substitution reaction, and finally obtaining a target product through hydrolysis reaction of alkali, wherein the method comprises the following steps:

(5) Reacting the compound v with hydroxylamine to obtain a target product, as in scheme four:

n1, n2, n3, n4=0 to 3; ra, rb=x, c=o; rc=x; w=1-2; r' may be methyl or tert-butyl; r ₁ can be R ₂ can be/>

(Ii) When (when)In the case of a substituted or unsubstituted fused ring, the method comprises the steps of:

The method comprises the steps of (1) carrying out nucleophilic substitution reaction on a compound x and 4-bromobut-2-enoate to obtain an intermediate xi, removing a protecting group to obtain the intermediate xii, introducing R ₁CH₂ -into NH of the intermediate xii to obtain the compound xiii, carrying out conjugate addition under the catalysis of a rhodium catalyst and a chiral ligand to obtain the compound xiv, and finally carrying out alkaline condition hydrolysis to obtain a target product, wherein the following route is as follows:

Z is C or N; m is H, a protecting group, an aldehyde group and halogen; n1, n2=0 to 3; ra, rb=x, c=o; w=1-2; r' may be methyl or tert-butyl; r ₁ can be R ₂ can be/>

In a third aspect of the present invention, there is provided a pharmaceutical composition comprising:

(a) A therapeutically effective amount of a bicyclic derivative according to the first aspect of the invention, as well as racemates, stereoisomers, tautomers, isotopic labels, nitroxides, solvates, polymorphs, metabolites, esters, prodrugs or pharmaceutically acceptable salts thereof; and

(B) A pharmaceutically acceptable carrier.

In a fourth aspect of the present invention there is provided a bicyclic derivative of formula (I) as described in the first aspect of the present invention, and racemates, stereoisomers, tautomers, isotopic labels, nitrogen oxides, solvates, polymorphs, metabolites, esters, prodrugs or pharmaceutically acceptable salts thereof, for use in the manufacture of a pharmaceutical composition for the treatment or prophylaxis of a disease, disorder or condition associated with αvβ1, αvβ6 and αvβ8 integrin activity or expression.

In another preferred embodiment, the disease, disorder or condition associated with αvβ1, αvβ6 and αvβ8 integrin activity or expression is selected from the group consisting of: autoimmune diseases, fibrotic diseases, inflammatory Bowel Disease (IBD), relapsing Multiple Sclerosis (RMS), progressive Multifocal Leukoencephalopathy (PML), ulcerative Colitis (UC), crohn's Disease (CD), chronic viral hepatitis b and c, non-alcoholic fatty liver (NAFLD), age-related macular degeneration, diabetic retinopathy, retinal vascular disease, osteoporosis and cell proliferative diseases.

In another preferred embodiment, the fibrotic disease is selected from the group consisting of: pulmonary fibrosis, idiopathic pulmonary fibrosis, nonspecific interstitial pneumonia (NSIP), conventional interstitial lung disease (UIP), radiation-induced pulmonary fibrosis, familial pulmonary fibrosis, airway pulmonary fibrosis, chronic group-progressive pulmonary disease (COPD), interstitial lung disease, liver fibrosis, chronic kidney disease, kidney fibrosis, skin fibrosis, systemic sclerosis, or a combination thereof.

In another preferred example, the fibrotic disease is pulmonary fibrosis (e.g., IPF), liver fibrosis, skin fibrosis, scleroderma, heart fibrosis, kidney fibrosis, intestinal fibrosis, primary Sclerosing Cholangitis (PSC), or biliary fibrosis (e.g., PBC).

In another preferred embodiment, the fibrotic disease Is Pulmonary Fibrosis (IPF).

In another preferred embodiment, the fibrotic disease is primary sclerosing cholangitis or biliary fibrosis (e.g. PBC).

In another preferred embodiment, the fibrotic disease is scleroderma.

In another preferred embodiment, the fibrotic disease is psoriasis.

In another preferred embodiment, the cell proliferative disorder is cancer selected from the group consisting of: breast cancer, cervical cancer, endometrial cancer, ovarian cancer, colon cancer, rectal cancer, pancreatic cancer, liver cancer, lung cancer, non-small cell lung cancer, brain metastases of lung cancer, oral squamous cell carcinoma, head cancer, neck cancer, head and neck squamous cell carcinoma, oral or nasal mucosa cancer, laryngeal cancer, renal cell carcinoma, ovarian cancer, spleen cancer, small intestine cancer, large intestine cancer, stomach cancer, esophageal cancer, lung squamous cell carcinoma, bile duct cancer, gall bladder cancer, endometrial cancer, melanoma, urothelial cancer, genitourinary tract cancer, genital cancer, prostate cancer, testicular cancer, bladder cancer, blood cancer, skin cancer, bone marrow cancer, brain cancer, central nervous system cancer, muscle tissue cancer, thyroid cancer, or a combination thereof.

In a fifth aspect of the present invention, there is provided a method of treating a disease, disorder or condition associated with avβ1, αvβ6 and αvβ8 integrin activity or expression level, the method comprising the steps of: an effective amount of a bicyclic derivative of formula (I), as set forth in the first aspect of the invention, as well as racemates, stereoisomers, tautomers, isotopic labels, nitroxides, solvates, polymorphs, metabolites, esters, prodrugs or pharmaceutically acceptable salts thereof, is administered to a patient in need thereof.

In a sixth aspect of the present invention there is provided a bicyclic derivative, and racemates, stereoisomers, tautomers, isotopic labels, nitroxides, solvates, polymorphs, metabolites, esters, prodrugs or pharmaceutically acceptable salts thereof, having the structure shown in formulas II-a, II-b, II-c:

wherein,

Each Y ₁ is independently C1-C6 alkylene, -O-, - (C1-C6 alkylene) -O-, -NH-, - (C1-C6 alkylene) -NH-;

Each Y ₂ is independently C1-C6 alkylene, -O-, - (C1-C6 alkylene) -NH-, - (C1-C6 alkylene) -NH-;

Each R ₁ is independently a substituted or unsubstituted 6-10 membered aromatic ring, a substituted or unsubstituted 5-8 membered heteroaromatic ring, a substituted or unsubstituted Wherein the C ring, D ring are each independently a substituted or unsubstituted C6-C10 aromatic ring, a substituted or unsubstituted 5-8 membered heteroaromatic ring, a substituted or unsubstituted 5-8 membered cycloalkane ring, or a substituted or unsubstituted 5-8 membered heteroalkane ring;

Each R ₂ is independently a hydrogen atom, a substituted or unsubstituted C6-C10 aromatic ring, a substituted or unsubstituted 5-8 membered heteroaromatic ring, a substituted or unsubstituted C8-C16 fused ring, or-L1-L2;

L2 is selected from the group consisting of unsubstituted, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkylthio, substituted or unsubstituted C3-C8 cycloalkyl, - (C1-C3 alkyl) -substituted or unsubstituted C3-C8 cycloalkyl, - (C1-C3 alkoxy) -substituted or unsubstituted C3-C8 cycloalkyl, -O-substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C3-C8 heteroalkyl, - (C1-C3 alkyl) -substituted or unsubstituted C3-C8 heteroalkyl, - (C1-C3 alkoxy) -substituted or unsubstituted C3-C8 heteroalkyl, -O-substituted or unsubstituted C3-C8 heteroalkyl, and substituted or unsubstituted 5-8 membered heteroaryl;

Each R _3a' is independently C1-C6 alkyl;

Each of which is Each independently is a substituted or unsubstituted spiro ring or a substituted or unsubstituted fused ring;

When (when) Is a substituted or unsubstituted fused ring, as shown in formula Ib: /(I)

Is a single bond or a double bond;

each n is independently 0,1, 2 or 3;

wherein the "substitution" means that 1 to 4 (preferably 1,2,3 or 4) hydrogen atoms on the group are each independently substituted with a substituent selected from the group consisting of: C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, C1-C3 haloalkyl, halogen, nitro, cyano, amino, hydroxy, thiol, = O, C-C4 carboxyl, C2-C4 ester, C2-C4 amide, C1-C6 alkoxy, carboxylic acid, C1-C4 alcohol, C1-C4 alkylamino, -O- (CH ₂)_m -C3-C8 cycloalkyl, -O- (CH ₂)_m -C3-C8 heteroalkyl, -NH- (CH ₂)_m -C3-C8 cycloalkyl, -NH- (CH ₂)_m -C3-C8 heteroalkyl) or-N=Ph ₂, wherein each m is independently an integer from 0 to 3;

Wherein each of said heteroaryl, heteroalkyl, or heteroaryl groups independently has 1-3 (preferably 1, 2, or 3) heteroatoms selected from N, O and S;

wherein, the structures of the formula II-a, the formula II-b and the formula II-c are all chemically stable structures.

In another preferred embodiment, Y ₁、Y₂、R₁、R₂, A, B, n are each independently as described in the first aspect of the invention.

In another preferred embodiment, the "substitution" means that 1 to 4 (preferably 1,2, 3 or 4) hydrogen atoms on the group are each independently substituted with a Boc substituent.

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It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows the results of Western Blotting (WB) experiments on compounds, illustrating the degradation of fibrin α -SMA, fibronectin at different concentrations in examples 1,2, 7, 15, 25, respectively. Pirfenidone (Pirfenidone) and GSK-3008348 were selected as control compounds for this experiment.

FIG. 2 shows rat body weight curves (mean.+ -. SEM) for sham surgery, model, 100mg/kg of Nidamib, 50mg/kg of example 71 low dose and 250mg/kg of example 71 high dose.

FIG. 3 shows the rat weight gain rate curves (mean.+ -. SEM) for sham, model, 100mg/kg of Nidamib, 50mg/kg of example 71 low dose and 250mg/kg of example 71 high dose.

FIG. 4 shows histological changes of bronchioles and pulmonary arterioles in the lesions of left pulmonary fibrosis of the sham-operated group (A), model group (B), 100mg/kg of Nidamib group (C), 50mg/kg of example 71 low dose group (D), 250mg/kg of example 71 high dose group (E) (. Times.200, H & E staining); wherein figure a shows normal pulmonary bronchioles and concomitant pulmonary arterioles; panel B shows bronchiole and pulmonary arteriole lesions; figures C-E show a reduction in the extent of both bronchioles and pulmonary arterioles lesions. In the figure, a is pulmonary arterioles; b is bronchioles. Arrows in panels B-E indicate inflammatory cell infiltration.

Figure 5 shows the lesion and inflammation scores for terminal bronchioles and accompanying small pulmonary arteries in the left pulmonary fibrosis focus in the sham surgery group, model group, 100mg/kg nilamide cloth group, 50mg/kg example 71 low dose group, 250mg/kg example 71 high dose group. In the figure, p <0.001 (compared to model group).

FIG. 6 shows histological changes of the left pulmonary fibrosis focus edge bronchioles and pulmonary arterioles (. Times.200, H & E staining) in sham surgery group (A), model group (B), 100mg/kg Nidamib group (C), 50mg/kg example 71 low dose group (D), 250mg/kg example 71 high dose group (E). Wherein figure a shows normal pulmonary bronchioles and concomitant pulmonary arterioles; panel B shows bronchiole and pulmonary arteriole lesions; figures C-E show a reduction in the extent of both bronchioles and pulmonary arterioles lesions. In the figure, a is pulmonary arterioles; b is bronchioles. Arrows in panels B-E indicate inflammatory cell infiltration.

FIG. 7 shows lesion and inflammation scores for left pulmonary fibrosis focus edge terminal bronchioles and accompanying small pulmonary arteries in sham surgery, model, 100mg/kg Nidamib, 50mg/kg example 71 low dose, 250mg/kg example 71 high dose. In the figure, p <0.001 and p <0.01 (compared to model group).

FIG. 8 shows the histological changes of alveolar tissue (x 200, H & E staining) in the lesions of left lung pulmonary fibrosis in sham surgery (A), model (B), 100mg/kg Nidamib (C), 50mg/kg example 71 low dose (D), 250mg/kg example 71 high dose (E). Wherein figure a shows normal alveolar walls; panel B shows that the sheet alveolar structure within the fibrotic lesion is damaged, disappeared, filled by the massive exuded inflammatory cells and the proliferated connective tissue. Graph C-E shows that the alveolar structure is abnormal, but is significantly better than that of the model group. Group E, i.e., example 71 high dose group, part of the alveoli were close to normal alveolar wall structure. Arrows in panels B-E indicate alveolar structural damage within the fibrotic foci, residual alveolar wall thickening.

FIG. 9 shows intra-lesion alveolar fibrosis in left lung pulmonary fibrosis lesions (×200, masson staining) in sham surgery group (A), model group (B), 100mg/kg Nidamib group (C), 50mg/kg example 71 low dose group (D), 250mg/kg example 71 high dose group (E). Panel A shows normal physiological fibrous tissue without fibrous tissue deposition; panel B shows that alveolar tissue presents with severe fibrotic lesions and significant fibrous tissue deposition. Panels C-E also had fibrotic lesions, but were significantly better than the model group. Arrows in figures B-E indicate fibrous tissue deposition.

FIG. 10 shows left lung fibrosis scores for sham surgery, model, 100mg/kg Nidamib, 50mg/kg example 71 low dose, 250mg/kg example 71 high dose. In the figure, p <0.001 and p <0.05 (compared to model group).

FIG. 11 shows the duty cycle of different pathology scores for left lung fibrosis in sham, model, 100mg/kg Nidamib, 50mg/kg example 71 low dose, 250mg/kg example 71 high dose. In the figure, p <0.001 (compared to model group).

FIG. 12 shows the degree of collagen deposition in the left lung fibrosis foci (. Times.200, masson staining) in sham-operated group (A), model group (B), 100mg/kg Nidamib group (C), 50mg/kg example 71 low dose group (D), 250mg/kg example 71 high dose group (E). Wherein panel a has normal physiological fibrous tissue with collagen area <5%; panel B shows extremely severe collagen deposition. Figures C-E also show increased collagen deposition, but to a lesser extent than the model set. Group E, i.e. example 71 high dose group, had the least amount of collagen deposition, significantly lower than the model group and likewise lower than the nilamide group.

FIG. 13 shows the percentage of collagen deposition area in the sham-operated group, model group, 100mg/kg of Nidamib group, 50mg/kg of example 71 low dose group, 250mg/kg of example 71 high dose group. In the figure, p <0.001 and p <0.05 (compared to model group).

Detailed Description

Through long-term and intensive research, the inventor screens and obtains an integrin inhibitor of a double-ring derivative with a novel structure. The bicyclic derivatives have excellent activity and selectivity to integrins αvβ1, αvβ6 and αvβ8, and are significantly superior to the oral drug generation property of positive control PLN-74809, and show activity effects superior to those of positive reference pirfenidone and nilamide in vitro and in vivo drug effect models of fibrotic diseases, so that the bicyclic derivatives can be used for preparing and preventing pharmaceutical compositions of fibrotic diseases. Based on the above findings, the inventors have completed the present invention.

Terminology

As used herein, the terms "comprising," "including," and "containing" are used interchangeably, and include not only closed-form definitions, but also semi-closed-form and open-form definitions. In other words, the term includes "consisting of … …", "consisting essentially of … …".

As used herein, the term "alkyl" refers to a fully saturated cyclic or acyclic, branched or unbranched carbon chain moiety having the indicated number of carbon atoms, or up to 30 carbon atoms if not indicated, for example having 1 to 3, 1 to 6 carbon atoms. For example, an alkyl group having 1 to 8 carbon atoms refers to moieties such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl, as well as those moieties that are positional isomers of these moieties. Alkyl groups of 10 to 30 carbon atoms include decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, docosyl, triacontyl and tetracosyl, the alkyl groups being optionally substituted.

As used herein, the term "C1-C6 alkyl" or "C1-C3 alkyl" refers to a straight or branched chain alkyl group having 1-6 or 1-3 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or the like.

As used herein, the term "alkylene" refers to an alkyl group having the indicated number of carbon atoms, e.g., 1 to 12 carbon atoms, which contains two points of attachment to the remainder of the compound on its longest carbon chain. Non-limiting examples of alkylene groups include C1-C6 alkylene, C1-C3 alkylene, including in particular methylene- (CH ₂) -, ethylene- (CH ₂CH₂) -, n-propylene- (CH ₂CH₂CH₂) -, isopropylene- (CH ₂CH(CH₃)) -, and the like. The alkylene group may be a cyclic or acyclic, branched or unbranched carbon chain moiety, and may be optionally substituted with one or more substituents.

As used herein, the term "C1-C6 alkoxy" refers to a straight or branched chain alkoxy group having 1 to 6 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, hexyloxy, or the like.

As used herein, the term "C2-C4 ester group" refers to a group having a C1-C3 alkyl-OC (O) -structure or a group having a-OC (O) -C1-C3 alkyl structure, wherein the alkyl group may be linear or branched, such as CH₃COO-、C₂H₅COO-、C₃H₈COO-、(CH₃)₂CHCOO-、-COOCH₃、-COOC₂H₅、-COOC₃H₈, or the like.

As used herein, the term "C2-C4 amide" refers to a group having a C1-C3 alkyl-CO-NH-structure or a group having a-NH-CO-C1-C3 alkyl structure, wherein the alkyl group may be linear or branched, such as CH₃-CO-NH-、C₂H₅-CO-NH-、C₃H₈-CO-NH-、-COOCH₃、-CO-NH-C₂H₅、-CO-NH-C₃H₈, or the like.

As used herein, the term "C2-C4 acyl" refers to a group having a C1-C3 alkyl-CO-structure, wherein the alkyl group may be linear or branched, such as CH ₃-CO-、C₂H₅-CO-、C₃H₈ -CO-, or the like.

As used herein, the term "C1-C3 haloalkyl" refers to a straight or branched alkyl group having 1 to 3 carbon atoms wherein one or more hydrogen atoms are replaced with a halogen group, such as a chloromethyl, dichloroethyl, trichloropropyl, or the like.

As used herein, the term "C1-C4 carboxyl" refers to a group of the C1-C3 alkyl-COOH structure, wherein the alkyl group may be linear or branched, such as CH ₃COOH、C₂H₅COOH、C₃H₈COOH、(CH₃)₂ CHCOOH, or the like.

As used herein, the terms "C6-C10 aromatic ring", "C6-C10 aryl" refer to a monocyclic or bicyclic aromatic hydrocarbon group having 6 to 10 carbon atoms in the ring component, such as a benzene ring, naphthalene ring, or the like.

The term "aryl" includes 3-to 12-membered substituted or unsubstituted monocyclic aromatic groups in which each atom of the ring is carbon (i.e., carbocyclic aryl) or in which one or more atoms are heteroatoms (i.e., heteroaryl). Preferably, the aryl group comprises a 5-to 12-membered ring, more preferably a 6-to 10-membered ring. The term "aryl" also includes polycyclic ring systems having two or more rings in which two or more carbons are common to two adjacent rings, wherein at least one ring is aromatic, e.g., the other rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and/or heterocyclyl. Carbocyclic aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

As used herein, the term "heteroaryl" refers to an optionally substituted aryl group, for example, which is a 5 to 7 membered monocyclic ring system having a ring containing at least one heteroatom and at least one carbon atom, such as pyrrolyl, thienyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, furan, imidazole, thiazole, oxazole, triazole, or the like.

The term "cycloalkyl" refers to a non-aromatic group (including saturated, partially saturated or unsaturated groups) having 3 to 12 carbon atoms (e.g., having 3 to 8, 4,5, or 6 carbon atoms) with single or fused rings (including bridged and spiro ring systems). Thus, the alkyl ring or alkyl ring group may be a saturated alkyl ring, or an unsaturated alkyl ring. In a fused ring system of saturated alkane rings, one or more of the rings are all saturated alkane rings. In a fused ring system of unsaturated alkane rings, one or more of the rings may be saturated or unsaturated.

As used herein, the term "5-8 membered heteroaromatic ring" refers to an aromatic heterocyclic ring system having one to more (preferably 1, 2 or 3) heteroatoms selected from N, O and S, and having 5-8 ring atoms. It will be appreciated that when multiple heteroatoms are present, the heteroatoms may be the same, may be partially the same, or may be entirely different. In the present application, the heteroaromatic ring is preferably a heteroaromatic ring containing 1 to 2 nitrogen atoms. Examples of, for example, 5-membered heteroaryl rings include (but are not limited to): examples of pyrrole rings, furan rings, thiophene rings, imidazole rings, pyrazole rings, oxazole rings, thiazole rings, 6-membered heteroaryl rings include, but are not limited to, pyridine rings, pyrazine rings, pyridazine rings, pyrimidine rings, or the like.

As used herein, the term "5-8 membered heteroaryl" refers to an aromatic group having one to more (preferably 1,2 or 3) heteroatoms selected from N, O and S, and having 5 or 8 ring atoms. It will be appreciated that when multiple heteroatoms are present, the heteroatoms may be the same, may be partially the same, or may be entirely different. Examples of, for example, 5-membered heteroaryl groups include (but are not limited to): pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, thiazolyl, or the like.

The terms "heteroaryl", "heteroaromatic ring", "aromatic heterocyclic" refer to 3-12 membered aromatic groups containing one or more heteroatoms selected from nitrogen, oxygen and sulfur, including monocyclic or multicyclic ring systems which may be fused ring, bridged ring systems and spiro ring systems. The term "fused heteroaromatic ring" refers to a fused ring formed by the fusion of two or more aromatic groups, comprising one or more heteroatoms selected from nitrogen, oxygen and sulfur. Wherein one or more of the fused heteroaryl rings has a heteroatom in the ring. Heteroaryl groups include substituted or unsubstituted aromatic 3-12 membered ring structures, more preferably 5-12 membered rings, more preferably 5-10 membered rings, the ring structures of which include 1-4 heteroatoms. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like. Aryl and heteroaryl groups may be monocyclic, bicyclic or polycyclic. As used herein, C5-C8 heteroaryl means a ring number of 5 to 8.

The term "heteroalkyl" refers to a 3-12 membered non-aromatic group (including saturated, partially saturated or unsaturated groups) containing one or more heteroatoms selected from nitrogen, oxygen and sulfur, including mono-or polycyclic ring systems which may be fused, bridged and spiro ring systems. Thus, the heteroalkyl ring or heteroalkyl ring group may be a saturated heteroalkyl ring, or an unsaturated heteroalkyl ring. In a fused ring system, one or more of the rings may be an alkyl, aryl or heteroaryl group. The number of ring atoms in the heteroalkyl ring may be 3 to 8, 4, 5, or 6. In one embodiment, the nitrogen and/or sulfur atoms of the heterocyclic group are optionally oxidized to provide N-oxide, sulfinyl and sulfonyl moieties. In one embodiment, the carbon atoms of the heterocyclic group are optionally oxidized to form a (c=o) moiety. Herein, C3-C8 means that the number of ring atoms is 3 to 8.

As used herein, the term "5-8 membered heteroalkane ring" is a non-aromatic heterocyclic ring system containing one or more (preferably 1, 2 or 3) heteroatoms selected from N, O and S on any stable ring, and having 5 to 7 ring atoms. The heterocyclic ring may be a saturated, partially unsaturated, unsaturated ring, but cannot be an aromatic ring. In the present application, the heteroalkyl ring is preferably a heteroalkyl ring containing 1 to 2 nitrogen atoms, which optionally may further include 1 or 2 oxygen atoms. It will be appreciated that when multiple heteroatoms are present, the heteroatoms may be the same, may be partially the same, or may be entirely different. In the present application, examples of the 5-membered heteroalkyl ring include, but are not limited to, a pyrrolidine ring, a pyrroline ring, a pyrazolidine ring, a pyrazoline ring, a1, 3-oxapenta ring, examples of the 6-membered heteroalkyl ring include, but are not limited to, a piperidine ring, a morpholine ring, a piperazine ring, a1, 4-dioxane ring, or the like.

As used herein, the term "tetrahydric heteroalkyl" is any stable non-aromatic heterocyclic ring system containing one or more (preferably 1,2 or 3) ring heteroatoms selected from N, O and S, and having 4 ring atoms. In the present application, the tetrahydride ring is preferably a tetrahydride ring having 1 to 2 nitrogen atoms. It will be appreciated that when multiple heteroatoms are present, the heteroatoms may be the same, may be partially the same, or may be entirely different. Examples of quaternary heteroalkane rings include, but are not limited to, oxetane, azetidine, or the like.

The term "heterocyclyl" or "heterocyclic group" refers to a3 to 12 membered ring structure, more preferably a 5 to 12 membered ring, more preferably a 5 to 10 membered ring, the ring structure of which comprises 1 to 4 heteroatoms. The heterocycle may be monocyclic, bicyclic, spiro, or polycyclic. Heterocyclic groups include, for example, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinoline, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenothiazine, furan, phenoxazine, pyrrolidine, oxapenne, thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactams such as azetidinone and pyrrolidone, sudan, sultone, and the like. The heterocyclic ring may be substituted at one or more positions with substituents as described above, such as halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, amino, nitro, mercapto, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, sulfamoyl, sulfinyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocyclic, aromatic or heteroaromatic moiety, -CF ₃, -CN, and the like, where "heterocyclic" includes heteroalkyl and heteroaromatic rings.

As used herein, the term "halogen" is meant to include, for example and without limitation, fluorine, chlorine, bromine, iodine, and the like in both radioactive and non-radioactive forms. In a preferred embodiment, the halogen is selected from fluorine, chlorine and bromine.

As used herein, the term "halo" refers to halogen substituted.

As used herein, the term "autoimmune disease" refers to myasthenia gravis, polymyositis, autoimmune myocarditis, polymyositis rheumatica, psoriatic arthritis, rheumatoid arthritis, sjogren's syndrome, ankylosing spondylitis, recurrent polymyositis, inflammatory bowel disease (e.g., crohn's disease, ulcerative colitis), celiac disease, autoimmune hepatitis, primary Biliary Cirrhosis (PBC), primary Sclerosing Cholangitis (PSC), immune-mediated kidney disease, interstitial cystitis, addison's disease, autoimmune thyroid disease (e.g., hashimoto's thyroiditis, graves ' disease), diabetes, dermatomyositis, psoriasis alopecia areata, autoimmune or immune mediated skin disease, bullous pemphigoid, erythema nodosum, dermatitis herpetiformis, hidradenitis suppurativa, autoimmune urticaria, multiple sclerosis, chronic Inflammatory Demyelinating Polyneuropathy (CIDP), central or peripheral nervous system demyelinating diseases, idiopathic demyelinating polyneuropathy, guillain-barre syndrome, peripheral neuropathy, systemic lupus erythematosus, systemic vasculitis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, allergic diseases (such as allergic purpura), sarcoidosis, fibroalveolar inflammatory diseases.

Pharmaceutical compositions and methods of administration

Because the compounds of the present invention have inhibitory activity and selectivity against integrins αvβ1, αvβ6 and αvβ8, the compounds of the present invention and various crystalline forms thereof, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates thereof, and pharmaceutical compositions containing the compounds of the present invention as a main active ingredient are useful for treating, preventing and alleviating pulmonary fibrosis diseases.

The pharmaceutical compositions of the present invention comprise a safe and effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical compositions contain 0.1 to 1000mg of the compound of the invention per dose, more preferably 0.5 to 500mg of the compound of the invention per dose. Preferably, the "one dose" is a capsule or tablet.

"Pharmaceutically acceptable carrier" means: one or more compatible solid or liquid filler or gel materials which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. "compatible" as used herein means that the components of the composition are capable of blending with and between the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties are 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.), emulsifying agents (e.g., tween), wetting agents (e.g., sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), inhalational and topical. A particularly preferred mode of administration is oral.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is admixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) Fillers or compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) Binders, for example, hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, e.g., glycerin; (d) Disintegrants, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) a slow solvent, such as paraffin; (f) an absorption accelerator, e.g., a quaternary amine compound; (g) Wetting agents, such as cetyl alcohol and glycerol monostearate; (h) an adsorbent, for example, kaolin; and (i) a lubricant, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be released in a delayed manner in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. The active compound may also be in the form of microcapsules with one or more of the above excipients, if desired.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of these substances and the like.

In addition to these inert diluents, the compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar-agar or mixtures of these substances, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.

The compounds of the invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

When a pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is applied to a mammal (e.g., a human) in need of treatment, wherein the dose at the time of administration is a pharmaceutically effective dose, and the daily dose is usually 0.2 to 1000mg, preferably 0.5 to 500mg, for a human having a body weight of 60 kg. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Intermediate a: preparation of tert-butyl 7- (bromomethyl) -3, 4-dihydro-1, 8-naphthyridine-1 (2H) -carboxylate

Step 1: 2-Aminonicotinaldehyde (1 g,8.2 mmol), 1-dimethoxypropan-2-one (1.3 g,10.6 mmol) was dissolved in a 10/1 ethanol/water (22 ml) solution, and sodium hydroxide solution (3M, 3.6 ml) was slowly added dropwise to the solution under nitrogen atmosphere at 0deg.C, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated, the residue was dissolved in ethyl acetate (50 ml) and the organic phase was washed with saturated brine (25 ml x 2), dried, filtered and concentrated to give 2- (dimethoxymethyl) -1, 8-naphthyridine as a yellow solid (A-1)2g.LC-MS:ESI m/z:205.2[M+H]⁺;¹H NMR(400MHz,CDCl3)δ＝9.13(dd,J＝2.0,8.0Hz,1H),8.28(d,J＝13.2Hz,1H)8.23(dd,J＝8.0Hz,10.0Hz,1H),7.79(d,J＝8.0Hz,1H),7.51(dd,J＝4.4,8.0Hz,1H),5.48(s,1H),3.52(s,6H).

Step 2: a-1 (1.6 g,7.8 mmol) was dissolved in ethanol (20 ml), and platinum dioxide (50 mg,0.2 mmol) was slowly added to the solution under nitrogen atmosphere, and the mixture was stirred at room temperature under hydrogen protection (15 psi) for 16 hours. The reaction mixture is filtered, and the filtrate is concentrated to obtain light yellow solid 7- (dimethoxy methyl) -1,2,3, 4-tetrahydro-1, 8-naphthyridine (A-2)1g.LC-MS:ESI m/z:209.2[M+H]⁺;¹H NMR(400MHz,CDCl₃)δ＝7.09(d,J＝7.6Hz,1H),6.63(d,J＝7.6Hz,1H),5.07(s,1H),4.85(br s,1H),3.41-3.24(m,2H),3.33(s,6H)2.65(t,J＝6.0Hz,2H),1.84(quin,J＝6.0Hz,2H).

Step 3: a-2 (1.2 g,5.8 mmol) was dissolved in water (10 ml), 12mmol/ml concentrated hydrochloric acid (1.1 ml,13 mmol) was added to the solution at-5℃and the mixture was stirred at 85℃for 2 hours. The mixture was cooled to room temperature, ph=11 was adjusted by 3M sodium hydroxide solution, the aqueous phase (30 ml x 2) was extracted with ethyl acetate, the organic phase was dried over Na ₂SO₄, filtered and concentrated in vacuo to give the crude product. Purification of the crude product by normal phase silica gel column chromatography (DCM/meoh=20/1) afforded 5,6,7, 8-tetrahydro-1, 8-naphthyridine-2-carbaldehyde as a yellow oily product (A-3)850mg.¹H NMR(400MHz,CDCl₃)δ＝9.82(s,1H),7.30(d,J＝7.2Hz,1H),7.16(d,J＝7.2Hz,1H),5.23(brs,1H),3.47(t,J＝5.6Hz,2H),2.81(t,J＝6.4Hz,2H),2.03-1.87(m,2H).

Step 4: a-3 (3.1 g,19.1 mmol) was dissolved in THF (60 ml), and (Boc) ₂ O (8.3 g,38.2 mmol) and N, N-dimethylaminopyridine (4.7 g,38.2 mmol) were added sequentially to the solution at room temperature, and the mixture was stirred under nitrogen at 80℃for 16 hours. The mixture was washed with saturated aqueous ammonium chloride (20 ml), extracted with ethyl acetate (20 ml x 3), the organic phase dried over Na ₂SO₄, filtered and concentrated in vacuo to give the crude product. Purification of the crude product by normal phase silica gel column chromatography (PE/ea=3/1) gives tert-butyl 7-formyl-3, 4-dihydro-1, 8-naphthyridine-1 (2H) -carboxylate as a pale yellow oil (A-4)1g.LC-MS:ESI m/z:263.3[M+H]⁺.¹H NMR(400MHz,CDCl₃)δ9.97(s,1H)7.65-7.60(m,1H)7.57-7.52(m,1H)3.85-3.81(m,2H)2.85(t,J＝6.50Hz,2H)2.02 -1.96(m,2H)1.55(s,9H).

Step 5: a-4 (1 g,3.8 mmol) was dissolved in THF (20 ml), sodium borohydride (173.1 mg,4.6 mmol) was added at 0deg.C, and the mixture was stirred at 0deg.C under the protection of mixed liquid nitrogen for 1 hour. To the mixture was added water (5 ml) and quenched, extracted with ethyl acetate (10 ml x 3), the organic phase was washed with saturated brine, dried over Na ₂SO₄, filtered and concentrated in vacuo to give the crude product. Purification of the crude product by normal phase silica gel column chromatography (PE/ea=1/0 to 1/1) gives tert-butyl 7- (hydroxymethyl) -3, 4-dihydro-1, 8-naphthyridine-1 (2H) -carboxylate as a pale yellow oil (A-5)750mg.¹H NMR(500MHz,CDCl3)δ7.38(d,J＝8.0Hz,1H)6.83(d,J＝8.0Hz,1H)4.66(s,2H)4.10(brs,1H)3.81-3.77(m,2H)2.78-2.75(m,2H)1.98-1.93(m,2H)1.53(s,9H).

Step 6: a-5 (600 mg,2.3 mmol) was dissolved in DCM (10 ml), triphenylphosphine (893.1 mg,3.4 mmol) and carbon tetrabromide (978.6 mg,3 mmol) were added to the solution at room temperature, and the mixture was stirred at room temperature under nitrogen for 3 hours. Concentrating the reaction solution, purifying the crude product by normal phase silica gel column chromatography (PE/EA=1/1) to obtain light yellow oily product tert-butyl 7- (bromomethyl) -3, 4-dihydro-1, 8-naphthyridine-1 (2H) -carboxylic acid ester (A)450mg.¹H NMR(400MHz,CDCl3)δ7.40(d,J＝7.6Hz,1H)7.12(d,J＝7.6Hz,1H)4.51(s,2H)3.82-3.74(m,2H)2.78(t,J＝6.65Hz,2H)1.95(m,2H)1.56(s,9H).

Intermediate B: preparation of N- (7- (bromomethyl) quinolin-2-yl) -1, 1-diphenylazomethine

Step 1: 3-Phenylacrylic acid (10 g,67.5 mmol) was dissolved in thionyl chloride (50 ml,689.3 mmol), the mixture was stirred at 90℃for 2 hours, concentrated in vacuo to give a residue, the residue was dissolved in DCM (200 ml), sodium bicarbonate (10 g,119. Mmol) was added to the solution at room temperature to neutralize excess thionyl chloride, and 4-dimethylaminopyridine (1 g,8.2 mmol) and m-toluidine (7.2 g,67.5 mmol) were then added to the reaction in sequence and stirred for 14 hours. The reaction solution was washed once with hydrochloric acid solution (5%, 150 ml) and aqueous sodium hydrogencarbonate solution (150 ml), and the organic phase was dried over Na2SO4, filtered and concentrated to give a crude product. Purification of the crude product by normal phase silica gel column chromatography (PE/ea=4/1) gives the 3-phenyl-N- (m-benzyl) acryloylamide as a white solid product (B-1)16g.LC-MS:ESI m/z:238.3[M+H]⁺.¹H NMR(400MHz,CDCl3)δ＝7.75(d,J＝15.2Hz,1H),7.54-7.46(m,3H),7.45-7.38(m,1H),7.38-7.32(m,3H),7.20-7.22(m,1H),6.99-6.90(m,1H),6.59(d,J＝15.2Hz,1H),2.33(s,3H).

Step 2: b-1 (15.7 g,66 mmol) was dissolved in chlorobenzene (200 ml), and aluminum trichloride (44 g,330 mmol) was slowly added under nitrogen atmosphere, and the reaction solution was stirred at 90℃for 2 hours under nitrogen protection. The reaction was cooled, poured into ice water (300 ml), extracted with ethyl acetate (200 ml x 2), the organic phase washed with saturated brine (100 ml), dried over Na ₂SO₄, filtered and concentrated in vacuo to give the crude product. Purification of the crude product by normal phase silica gel column chromatography (PE/ea=1/1) gives 7-methylquinolin-2 (1H) -one as a brown solid product (B-2)10g.1H NMR(400MHz,CDCl3)δ＝12.51(s,1H),8.05(d,J＝9.8Hz,1H),7.79(d,J＝9.3Hz,1H),7.45(d,J＝8.0Hz,1H),7.43-7.37(m,1H),7.34-7.30(m,1H),7.26(s,1H),7.05(d,J＝8.3Hz,2H),6.76(d,J＝9.8Hz,1H),6.68(d,J＝9.5Hz,1H),2.57(s,2H),2.46(s,3H).

Step 3: b-2 (9.8 g,61.4 mmol) was dissolved in phosphorus oxychloride (130 ml,1.4 mol) and the mixture was stirred at 100℃for 2 hours. The mixture was concentrated, slowly poured into ice water (300 ml) and kept in an ice bath, and ph=7 was adjusted by adding sodium hydroxide solid. The mixture was extracted with ethyl acetate (200 ml) and the organic phase was washed with saturated brine, dried over Na ₂SO₄ and concentrated by vacuum filtration to give the crude product. Purification of the crude product by normal phase silica gel column chromatography (PE/ea=10/1) gives 2-chloro-7-methylquinoline as a white solid (B-3)3g.¹H NMR(400MHz,CDCl3)δ＝8.05(d,J＝8.5Hz,1H),7.80(s,1H),7.70(d,J＝8.2Hz,1H),7.39(d,J＝8.1Hz,1H),7.31(d,J＝8.4Hz,1H),2.56(s,3H).

Step 4: b-3 (300 mg,1.7 mmol), diphenylazomethine (459.1 mg,2.5 mmol), palladium catalyst (77.3 mg,0.1 mmol), ligand (105.2 mg,0.2 mmol) and cesium carbonate (1.1 g,3.4 mmol) were dissolved in1, 4-dioxane (5 ml) in this order and stirred for 16 hours at 90℃under the protection of mixed liquid nitrogen. Concentrating the mixture, purifying the crude product by normal phase silica gel column chromatography (PE/EA=19/1) to obtain a pale yellow oily product N- (7-methylquinolin-2-yl) -1, 1-diphenyl azomethine (B-4)570mg.LC-MS:ESI m/z:323.4[M+H]⁺.¹H NMR(400MHz,DMSO-d₆)δ＝8.04(d,J＝8.4Hz,1H),7.75-7.69(m,3H),7.64-7.52(m,5H),7.25-7.20(m,5H),6.83(d,J＝8.4Hz,1H),2.45(s,3H).

Step 5: b-4 (570 mg,1.3 mmol) was dissolved in carbon tetrachloride (28 ml), N-bromosuccinimide (258.3 mg,1.5 mmol) and dibenzoyl peroxide (63.9 mg,0.3 mmol) were added sequentially to the solution at room temperature, and the mixture was stirred at 80℃for 12 hours. After the reaction was diluted with DCM (100 ml), washed once with sodium hydroxide (0.04 mol/ml) and saturated brine (40 ml), the organic phase was dried over Na ₂SO₄, filtered and concentrated in vacuo to give the crude product which was purified by normal phase silica gel column chromatography (PE/ea=10/1) to give 300mg of N- (7- (bromomethyl) quinolin-2-yl) -1, 1-diphenylazone (B) as a yellow oil. LCMS ESI-MS m/z=401.4 [ m+1].

Intermediate C: preparation of (6- ((tert-butoxycarbonyl) amino) pyridin-2-yl) methyl 4-methylbenzenesulfonate

Step 1: referring to the preparation of intermediate A-5, filtration and concentration in vacuo gave tert-butyl (6- (hydroxymethyl) pyridin-2-yl) carbamate (C-1) as a white solid, 100mg.

Step 2: c-1 (100 mg, 445.9. Mu. Mol) and diisopropylethylamine (172.9 mg,1.3 mmol) were dissolved in acetonitrile (4 ml), and p-toluenesulfonyl chloride (47.2 mg, 668.9. Mu. Mol) was added at 0℃and the mixture was stirred at room temperature under nitrogen for 3 hours. The reaction was quenched with saturated aqueous sodium bicarbonate (5 ml), extracted with ethyl acetate (10 ml x 3), the organic phase was dried, filtered and concentrated in vacuo to give the crude product, which was purified by normal phase silica gel column chromatography (PE/ea=1/1) to give the methyl 4-methylbenzenesulfonate as a pale yellow oily product (6- ((tert-butoxycarbonyl) amino) pyridin-2-yl) (C)120mg.¹H NMR(400MHz,CDCl₃)δ7.89-7.76(m,3H)7.62(t,J＝8.0Hz,1H)7.33(d,J＝8.0Hz,2H)7.07(s,1H)7.00(d,J＝8.0Hz,1H)5.00(s,2H)2.45(s,3H)1.51(s,9H).

Intermediate D: preparation of tert-butyl 6- (bromomethyl) -2, 3-dihydro-4H-pyrido [3,2-b ] [1,4] oxazine-4-carboxylic acid ester

Step 1: 6-bromo-2-nitropyridin-3-ol (9 g,41.1 mmol), ethyl bromoacetate (8.2 g,49.3 mmol) and potassium carbonate (11.4 g,82.2 mmol) were dissolved in acetone (90 ml) and stirred at 60℃for 2 hours under nitrogen. The reaction solution was washed with water (200 ml), extracted with ethyl acetate (200 ml), the organic phase was washed with saturated brine, dried over Na ₂SO₄, filtered and concentrated in vacuo to give ethyl 2- ((6-bromo-2-nitropyridin-3-yl) oxo) acetate (D-1) as a brown oily crude product 12g.

Step 2: d-1 (12 g,39.3 mmol), iron powder (11 g,196.7 mmol) and ammonium chloride (10.5 g,196.7 mmol) were successively dissolved in 10/1 methanol/water (110 ml) and stirred at 60℃for 16 hours under nitrogen. The reaction solution was concentrated by filtration, diluted with water (300 ml), extracted with ethyl acetate (300 ml), the organic phase was washed with saturated brine, dried over Na ₂SO₄, filtered and concentrated in vacuo to give a residue which was dissolved in acetic acid (100 ml), stirred at 90 ℃ for 3 hours, then concentrated and filtered to give 7g of the brown solid product 6-bromo-2H-pyrido [3,2-b ] [1,4] oxazin-3 (4H) -one (D-2). LC-MS: ESI m/z 229.1[ M+H ] ⁺.

Step 3: d-2 (1 g,4.4 mmol) was dissolved in THF (10 ml), borane dimethyl sulfide (10M, 1.1 ml) was added at room temperature, stirred at 90℃for 1.5 hours, methanol (1 ml) was added, and stirred at 90℃for 0.5 hours. The reaction was concentrated, washed with saturated sodium bicarbonate (50 ml), extracted with ethyl acetate (50 ml), the organic phase dried over Na ₂SO₄, filtered and concentrated in vacuo to give 757mg of 6-bromo-3, 4-dihydro-2H-pyrido [3,2-b ] [1,4] oxazine (D-3) as a white solid. LC-MS: ESI m/z 214.7[ M+H ] ⁺.

Step 4: d-3 (700 mg,3.3 mmol), potassium vinyltrifluoroborate (264 mg,4.9 mmol), palladium catalyst (119 mg,0.2 mmol) and cesium carbonate (2.7 g,8.1 mmol) were dissolved in 1, 4-dioxane (10 ml) in this order, and stirred at 100℃for 2 hours under nitrogen. The reaction solution was directly concentrated and purified by normal phase silica gel column chromatography (PE/ea=3/1) to give 400mg of 6-vinyl-3, 4-dihydro-2H-pyrido [3,2-b ] [1,4] oxazine (D-4) as a colorless oily product. LC-MS: ESI m/z 163.3[ M+H ] ⁺.

Step 5: referring to the preparation method of intermediate a-4, purifying the crude product by normal phase silica gel column chromatography (PE/ea=5/1) to obtain colorless oily product tert-butyl 6-vinyl-2, 3-dihydro-4H-pyrido [3,2-b ] [1,4] oxazine-4-carboxylate (D-5)500mg.¹H NMR(400MHz,CDCl₃)δ＝7.11(d,J＝8.3Hz,1H),6.99(d,J＝8.2Hz,1H),6.70(dd,J＝10.8,17.2Hz,1H),6.10(d,J＝17.2Hz,1H),5.32(d,J＝10.8Hz,1H),4.25(d,J＝3.3Hz,2H),3.92(br s,2H),1.56(s,9H).

Step 6: d-5 (500 mg,1.9 mmol) was dissolved in 4/1 THF/H ₂ O (12.5 ml), sodium periodate (1.1 g,5.3 mmol) and potassium osmium dihydrate (4.2 mg,0.01 mmol) were added sequentially at room temperature, and stirred at room temperature for 1 hour. The reaction solution was filtered, washed with water (30 ml), extracted with ethyl acetate (30 ml), concentrated, and the crude product was purified by normal phase silica gel column chromatography (PE/ea=3/1) to give 428mg of tert-butyl 6-formyl-2, 3-dihydro-4H-pyrido [3,2-b ] [1,4] oxazine-4-carboxylate (D-6) as a pale yellow solid product. LC-MS: ESI m/z 209.2[ M+H ] ⁺.

Step 7: referring to the preparation of intermediate a-5, the crude product was purified by normal phase silica gel column chromatography (DCM/meoh=20/1) to give tert-butyl 6- (hydroxymethyl) -2, 3-dihydro-4H-pyrido [3,2-b ] [1,4] oxazine-4-carboxylate (D-7) as a colourless oil 400mg. LC-MS: ESI m/z 289.2[ M+23] ⁺.

Step 8: d-7 (50 mg,0.2 mmol) was dissolved in DCM (3 ml), carbon tetrabromide (93.4 mg,0.3 mmol) and triphenylphosphine (74 mg,0.3 mmol) were added sequentially at room temperature and stirred at room temperature for 2 hours. The reaction was quenched with water (20 ml), the organic phase was dried, filtered and concentrated, and the crude product was purified on a silica gel plate (DCM/meoh=20/1) to give the product tert-butyl 6- (bromomethyl) -2, 3-dihydro-4H-pyrido [3,2-b ] [1,4] oxazine-4-carboxylate (D) as a colourless oil (30 mg). LC-MS: ESI m/z 273.2[ M-55] ⁺.

Intermediate E: preparation of 6- (bromomethyl) -2H-pyrido [3,2-b ] [1,4] oxazin-3 (4H) -one

Step 1: referring to the preparation method of intermediate D-4, the crude product was purified by normal phase silica gel column chromatography (PE/ea=3/1) to give 2g of the white solid product 6-vinyl-2H-pyrido [3,2-b ] [1,4] oxazin-3 (4H) -one (E-1). LC-MS: ESI m/z 177.2[ M+H ] ⁺.

Step 2: see methods for preparation of intermediate D-6 to give crude 3-carbonyl-3, 4-dihydro-2H-pyrido [3,2-b ] [1,4] oxazine-6-carbaldehyde (E-2) as a brown solid 900mg. LC-MS: ESI m/z 179.3[ M+H ] ⁺.

Step 3: referring to the preparation of intermediate a-5, the crude product was purified by normal phase silica gel column chromatography (DCM/meoh=10/1) to give 640mg of the yellow solid product 6- (hydroxymethyl) -2H-pyrido [3,2-b ] [1,4] oxazin-3 (4H) -one (E-3). LC-MS: ESI m/z 181.3[ M+H ] +.

Step 4: see procedure for preparation of intermediate D, step 8, crude product was purified on silica gel plate (DCM/meoh=20/1) to give product 6- (bromomethyl) -2H-pyrido [3,2-b ] [1,4] oxazin-3 (4H) -one (E) 34mg as a white solid. LC-MS: ESI m/z 243.2[ M+H ] ⁺.

Intermediate F: preparation of tert-butyl 5-formyl-3H-imidazo [4,5-b ] pyridine-3-carboxylic acid ester

Step 1: referring to the preparation method of intermediate a-4, the crude concentrated reaction solution was purified by normal phase silica gel column chromatography (PE/ea=4/1) to give 550mg of tert-butyl 5-bromo-3H-imidazo [4,5-b ] pyridine-3-carboxylate (F-1) as a pale yellow solid product. ¹ H NMR (400 mhz, cdcl 3) delta 8.63 (s, 1H) 8.15 (d, j=8.44 hz, 1H) 7.52 (d, j=8.44 hz, 1H) 1.71 (s, 9H).

Step 2: referring to the preparation method of the intermediate D-4, the crude product is purified by normal phase silica gel column chromatography (EA) to obtain a white solid product, namely 5-vinyl-3H-imidazo [4,5-b ] pyridine (F-2)160mg.¹H NMR(400MHz,DMSO-d₆)δ12.57-13.20(m,1H)8.41(br s,1H)7.88-8.10(m,1H)7.40(d,J＝8.19Hz,1H)6.89(m,1H)6.20(d,J＝17.36Hz,1H)5.42(br d,J＝10.39Hz,1H).

Step 3: referring to the preparation method of intermediate a-4, the crude product is purified by normal phase silica gel column chromatography (PE/ea=4/1) to obtain a pale yellow solid product tert-butyl 5-vinyl-3H-imidazo [4,5-b ] pyridine-3-carboxylate (F-3)120mg.LC-MS:ESI m/z＝246.4[M+H]⁺.¹H NMR(400MHz,CDCl3)δ8.65(s,1H)8.22(d,J＝8.31Hz,1H)7.42(d,J＝8.44Hz,1H)6.87-7.01(m,1H)6.36(d,J＝17.48Hz,1H)5.53(d,J＝10.88Hz,1H)1.71(s,9H).

Step 4: see preparation of intermediate D-6 to give tert-butyl 5-formyl-3H-imidazo [4,5-b ] pyridine-3-carboxylate as a pale yellow solid (F)120mg.LCMS:ESI-MS m/z＝246.4[M-t-Bu⁺];¹H NMR(400MHz,CDCl3)δ10.12(s,1H)8.73(s,1H)8.37(d,J＝8.31Hz,1H)8.03(d,J＝8.31Hz,1H)1.66(s,9H).

Intermediate G: preparation of (3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) boronic acid

Step 1: (3-bromophenyl) hydrazine hydrochloride (4.4 g,19.7 mmol), potassium acetate (2.5 g,25.6 mmol) and acetylacetone (2.1 g,20.7 mmol) were dissolved in an ethanol solution (20 ml), and stirred for 3 hours at 85℃under nitrogen. Quenching with water, extraction with ethyl acetate, washing the organic phase with saturated brine, drying over Na ₂SO₄, filtering and concentrating under vacuum to give the product 1- (3-bromophenyl) -3, 5-dimethyl-1H-pyrazole as a yellow oil (G-1)5g.LC-MS:ESI m/z:251.1[M+H]⁺.1H NMR(400MHz,CDCl3)δ＝7.65(s,1H),7.48(d,J＝7.9Hz,1H),7.41-7.36(m,1H),7.35-7.28(m,1H),6.01(s,1H),2.31(d,J＝12.6Hz,6H).

Step 2: g-1 (4G, 15.9 mmol) and triisopropyl borate (1.5 eq.) were dissolved in anhydrous THF (40 ml) and n-butyllithium (2.5M, 16 ml) was slowly added dropwise at-65℃under nitrogen, and the mixture was stirred for 2 hours at-65℃to-60 ℃. To the solution was added dropwise dilute hydrochloric acid (3M, 11 ml) at-65 ℃ to quench the reaction, ph=7 was adjusted with sodium hydroxide solution (1M), ea was extracted, the organic phase was washed with saturated brine, dried over Na ₂SO₄, filtered and concentrated in vacuo, and the crude normal phase silica gel column chromatography (DCM/meoh=20/1) was purified to give (3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) boronic acid (G) 3G as a pale yellow solid. LC-MS: ESI m/z 217.3[ M+H ] ⁺.

Intermediate H: preparation of (3-bromo-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) boronic acid

Step 1: 3, 5-dibromoaniline (5 g,19.9 mmol) was dissolved in 40ml of concentrated hydrochloric acid, sodium nitrite (2.1 g,29.9 mmol) was added to the solution at 0℃under a nitrogen atmosphere, stirred at 0℃for 1 hour, then a solution of stannous chloride (7.6 g,40 mmol) in concentrated hydrochloric acid (80 ml) was added dropwise thereto, and stirred at room temperature under a nitrogen atmosphere for 16 hours. After cooling the reaction solution, filtration and concentration in vacuo gave 6g of 3-bromo-5-hydrazinoaniline hydrochloride (H-1) as a yellow solid. LC-MS: ESI m/z 264.9[ M+H ] ⁺.

Step 2: referring to the preparation of intermediate G-1, purification of the crude product by column chromatography (PE/ea=95/5) gives 3G of the yellow solid product 1- (3, 5-dibromophenyl) -3, 5-dimethyl-1H-pyrazole (H-2). LC-MS: ESI m/z 329.3[ M+H ] ⁺.

Step 3: h-2 (500 mg,1.5 mmol), duplex pinacol borate (460 mg,1.8 mmol), potassium acetate (4476 mg,4.6 mmol) and 1, 1-bis (diphenylphosphorus) ferrocene palladium chloride (111 mg, 152. Mu. Mol) were dissolved in 5ml anhydrous Diox and stirred under nitrogen for 2 hours at 90 ℃. The reaction was concentrated, the residue was diluted with 2M dilute hydrochloric acid, extracted with EA, the organic phase was washed with saturated brine, dried over Na ₂SO₄, filtered and concentrated in vacuo to give 400mg of the dark brown oily product 1- (3-bromo-5- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) -3, 5-dimethyl-1H-pyrazole (H-3). LC-MS: ESI m/z 376.8[ M+H ] ⁺.

Step 4: h-3 (400 mg,1.1 mmol) was dissolved in 4/1 THF/H ₂ O (5 ml), and sodium periodate (681 mg,3.2 mmol) and 1M diluted hydrochloric acid (955 μl) were added to the reaction solution and stirred at room temperature for 12 hours. The reaction solution was concentrated, and the crude product was separated by high performance liquid chromatography to give 70mg of (3-bromo-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) boronic acid (H) as a white solid. LC-MS: ESI m/z 295.0[ M+H ] ⁺.

Intermediate I: preparation of (3- (tert-butyl) -5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) boronic acid

Step 1: referring to the preparation of intermediate B-4, the crude product was purified by column chromatography (PE/ea=98/2) to give 5g of the product N- (3-bromo-5- (tert-butyl) phenyl) -1, 1-diphenylazone (I-1) as a yellow oil. LC-MS: ESI m/z 392.1[ M+H ] ⁺.

Step 2: i-1 (4 g,10.2 mmol) was dissolved in 4/1 THF/concentrated HCl (37.5 ml) and stirred at room temperature under nitrogen for 2 hours. The reaction mixture was concentrated, the residue was diluted with 30ml of water, the aqueous phase was adjusted to ph=7-8 with 2M aqueous sodium hydroxide solution, then extracted with EA, the organic phase was washed with saturated brine, dried over Na ₂SO₄, filtered and concentrated in vacuo, and the crude product was purified by column chromatography (PE/ea=95/5) to give 2g of 3-bromo-5- (tert-butyl) aniline (I-2) as a yellow liquid product. LC-MS: ESI m/z 227.7[ M+H ] ⁺.

Step 3: referring to the preparation method of the intermediate H-1, the reaction solution is filtered and concentrated in vacuo to obtain 1g of (3-bromo-5- (tert-butyl) phenyl) hydrazine hydrochloride (I-3) as a yellow solid product. LC-MS: ESI m/z 243.1[ M+H ] ⁺.

Step 4: referring to the preparation of intermediate G-1, the crude product was purified by column chromatography (PE/ea=95/5) to give 600mg of 1- (3-bromo-5- (tert-butyl) phenyl) -3, 5-dimethyl-1H-pyrazole (I-4) as a yellow oil. LC-MS: ESI m/z 306.9[ M+H ] ⁺.

Step 5: see preparation of intermediate H-3 to afford the product 1- (3- (tert-butyl) -5- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) -3, 5-dimethyl-1H-pyrazole (I-5) as a dark brown oil in an amount of 500mg. LC-MS: ESI m/z 355.4[ M+H ] ⁺.

Step 6: referring to the preparation method of the intermediate H, the crude product is separated by high performance liquid chromatography to obtain 55mg of (3- (tert-butyl) -5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) boric acid as a white solid. LC-MS: ESI m/z 273.2[ M+H ] ⁺.

Intermediate J: preparation of tert-butyl 7- (2- (tosyloxy) ethyl) -3, 4-dihydro-1, 8-naphthyridine-1 (2H) -carboxylate

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Step 1: 2-Aminonicotinaldehyde (10 g,81.9 mmol) was dissolved in ethanol (100 ml), acetone (14.3 g,245.7 mmol) and L-proline (10.4 g,90.1 mmol) were added, and stirred at 80℃for 16 hours under nitrogen. Concentrating the reaction solution, purifying the crude product by normal phase silica gel column chromatography (PE/EA=0/1) to obtain white solid product 2-methyl-1, 8-naphthyridine (J-1)11g.¹H NMR(400MHz,CDCl3)δ9.08(d,J＝2.57Hz,1H)8.15(d,J＝8.07Hz,1H)8.08(d,J＝8.19Hz,1H)7.44(dd,J＝8.07,4.28Hz,1H)7.39(d,J＝8.31Hz,1H)2.82(s,3H)

Step 2: j-1 (5 g,34.7 mmol) was dissolved in ethanol (50 ml) and 10% palladium on carbon (1.5 g) was added thereto, and the mixture was stirred at room temperature under a hydrogen atmosphere for 16 hours. Filtering and vacuum concentrating the reaction liquid to obtain white solid 7-methyl-1, 2,3, 4-tetrahydro-1, 8-naphthyridine (J-2)5g.¹H NMR(400MHz,CDCl3)δ7.04(d,J＝7.21Hz,1H)6.35(d,J＝7.34Hz,1H)4.78(m,1H)3.36-3.42(m,2H)2.68(m,2H)2.31(s,3H)1.90(m,2H)

Step 3: referring to the preparation of intermediate a-4, the crude product was purified by normal phase silica gel column chromatography (PE/ea=5/1) to give 4g of the white solid product tert-butyl 7-methyl-3, 4-dihydro-1, 8-naphthyridine-1 (2H) -carboxylate (J-3). LC-MS ESI m/z=249.4 [ m+h ] ⁺.

Step 4: j-3 (3.5 g,14.1 mmol) and dimethyl carbonate (4.4 g,49.3 mmol) were dissolved in THF (30 ml), and a 2M lithium diisopropylamine solution (10.7 ml) was added dropwise at-78℃under nitrogen, and stirred for 1 hour. The reaction mixture was quenched with saturated aqueous ammonium chloride (50 ml), extracted with EA (50 ml x 3), the organic phase was dried, filtered and concentrated, and the crude product was purified by normal phase silica gel column chromatography (PE/ea=4/1) to give tert-butyl 7- (2-methoxy-2-carbonylethyl) -3, 4-dihydro-1, 8-naphthyridine-1 (2H) -carboxylate as a pale yellow solid (J-4)2.9g.¹H NMR(400MHz,CDCl3)δ7.29(d,J＝7.70Hz,1H)6.89(d,J＝7.58Hz,1H)3.71(s,2H)3.66-3.70(m,2H)3.63(s,3H)2.67(t,J＝6.60Hz,2H)1.85(m,2H)1.44(s,9H)

Step 5: referring to the preparation method of intermediate a-4, the crude product is purified by normal phase silica gel column chromatography (PE/ea=4/1) to obtain a pale yellow solid product tert-butyl 7- (2-hydroxyethyl) -3, 4-dihydro-1, 8-naphthyridine-1 (2H) -carboxylic acid ester (J-5)800mg.¹H NMR(400MHz,CDCl3)δ7.31(d,J＝7.58Hz,1H)6.77(d,J＝7.70Hz,1H)5.58(br s,1H)3.98(br s,2H)3.76-3.82(m,2H)2.92(t,J＝5.14Hz,2H)2.72(t,J＝6.48Hz,2H)1.92(m,2H)1.54(s,9H).

Step 6: referring to the preparation method of intermediate C, the crude product was purified by normal phase silica gel column chromatography (PE/ea=4/1) to give a white solid product 260mg.¹H NMR(400MHz,CDCl3)δ7.74(d,J＝8.19Hz,2H)7.27-7.33(m,3H)6.80(d,J＝7.58Hz,1H)4.42(t,J＝7.03Hz,2H)3.70-3.76(m,2H)3.05(t,J＝6.97Hz,2H)2.72(t,J＝6.60Hz,2H)2.44(s,3H)1.91(m,2H)1.45(s,9H).

Intermediate K: preparation of (3- (1- (tert-butoxycarbonyl) -1H-pyrrol-2-yl) phenyl) boronic acid

Step 1: 1, 3-Dibromobenzene (3 g,12.7 mmol), boric acid ester (4.1 g,14 mmol), palladium catalyst (4.7 g) and potassium carbonate (5.3 g,38.2 mmol) were dissolved in Diox/H ₂ O (3/1, 40 ml) and stirred overnight at 85℃under nitrogen. Concentrating the reaction solution, purifying the crude product by normal phase silica gel column chromatography (PE/EA=20/1) to obtain a colorless oily product tert-butyl 2- (3-bromophenyl) -1H-pyrrole-1-carboxylate (K-1)4g.¹H NMR(400MHz,CDCl3)δ＝7.49(s,1H),7.43(br d,J＝7.9Hz,1H),7.39-7.34(m,1H),7.28(br d,J＝7.5Hz,1H),7.25-7.18(m,1H),6.27-6.15(m,2H),1.37(s,9H).

Step 2: see preparation of intermediate G o, purification of the crude product by normal phase silica gel column chromatography (DCM/meoh=20/1) afforded 1.8G of a violet solid product. LC-MS ESI m/z=188.0 [ m-boc+1] ⁺.

Intermediate L: preparation of (3- (5-methyl-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-imidazol-2-yl) phenyl) boronic acid

Step 1: 3-bromobenzene (methylaldehyde) (1 g,5.4 mmol), 2-carbonyl-propanal (14.3 g,79.3 mmol) and aqueous ammonia (5.5 g,43.6 mmol) were dissolved in methanol (15 ml) and stirred at 75℃for 3 hours. The reaction was concentrated and the crude product purified by normal phase silica gel column chromatography (DCM/meoh=10/1) to give 1.5g of 2- (3-bromophenyl) -5-methyl-1H-imidazole (L-1) as a yellow solid. LC-MS ESI m/z=239.1 [ m+h ] ⁺.

Step 2: l-1 (1.5 g,6.3 mmol) and sodium hydride (506.1 mg,12.7 mmol) were dissolved in THF (20 ml), stirred at 25℃for 0.5 h, 2- (trisilyl) ethoxymethyl chloride (1.6 g,9.5 mmol) was slowly added dropwise to the solution at 0℃and stirring was continued for 2.5 h at 0 ℃. The reaction was quenched by addition of 15ml of saturated NH ₄ Cl solution at 0 ℃ and diluted with 20ml of water, extracted with ethyl acetate, the organic phase washed with saturated brine, dried over Na ₂SO₄, filtered and concentrated in vacuo, the crude product purified by normal phase silica gel column chromatography (PE/ea=1/1) to give 1.3g of 2- (3-bromophenyl) -5-methyl-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-imidazole (L-2) as a yellow oil. LC-MS ESI m/z=368.3 [ m+h ] ⁺.

Step 3: l-2 (1.2 g,3.3 mmol), tetrahydroxydiborane (585.7 mg,6.5 mmol), potassium acetate (961.8 mg,9.8 mmol), 2-dicyclohexylphosphine-2 '4' 6-triisopropylbiphenyl (311.5 mg,0.7 mmol) and methanesulfonic acid (2-dicyclohexylphosphine-2 '4' 6-triisopropyl-1, 1' -biphenyl) (2 ' -amino-1, 1' -biphenyl-2-yl) palladium (II) (276.5 mg,0.3 mmol) were dissolved in methanol (20 ml) and stirred under nitrogen for 3 hours at 80 ℃. The reaction solution was concentrated, and the crude product was purified by high performance liquid chromatography to give 670mg of a yellow solid product. LC-MS ESI m/z=334.6 [ m+h ] ⁺.

Intermediate M: preparation of (3- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-imidazol-2-yl) phenyl) boronic acid

Referring to the preparation method of intermediate L, the crude product was purified by high performance liquid chromatography to give 1.5g of a white solid product (M). LC-MS ESI m/z=321.0 [ m+h ] ⁺.

Intermediate N: preparation of (5- (tert-butyl) -2-fluorophenyl) boronic acid

Step 1: referring to the preparation method of the intermediate G, the crude product is purified by normal phase silica gel column chromatography (PE/EA=100/0-3/1) to obtain a pale yellow solid product 760mg.¹H NMR(400MHz,DMSO-d₆)δ＝8.17(s,2H),7.57(dd,J＝2.5,5.7Hz,1H),7.43(ddd,J＝2.7,5.4,8.4Hz,1H),7.00(t,J＝8.8Hz,1H),1.29(s,9H).

General preparation of intermediate P1 (3-cyclopropyl-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) boronic acid, P2 (3-cyclobutyl-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) boronic acid, P3 (3-cyclopentyl-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) boronic acid, P4 (3-cyclohexyl-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) boronic acid:

Step 1: h-2 (1.2 g,3.5 mmol), various cycloalkylboronic acids (1 eq), pd (dppf) Cl ₂ (0.1 eq), potassium carbonate (2 eq), dioxane/water solution (10:1, 7 ml), nitrogen blanket, heating at 93℃for 3H; after the LCMS detection reaction is finished, adding water, quenching, adding ethyl acetate, removing yellow solid by suction filtration, extracting by ethyl acetate, combining organic phases, drying by Na2SO4, filtering, distilling at 50 ℃ under reduced pressure to obtain yellow oily matter, and purifying the crude product by column chromatography (PE/EA (approximately 20/1)) to obtain a corresponding yellow oily product.

Step 2: referring to the preparation of intermediate G, purification of the crude product by reverse phase column chromatography (water/acetonitrile=7/3) gives the corresponding white solid product.

Intermediate Q: preparation of (5- (tert-butyl) - [1,1' -biphenyl ] -3-yl) boronic acid

Referring to the preparation method of intermediate P1 and the like, the crude product was purified by silica gel column chromatography (PE/ea=1/1) to give 450mg of a yellow solid product. LC-MS: ESI m/z 255.1[ M+H ] ⁺.

Intermediate R: preparation of (3, 5-di-tert-butylphenyl) boronic acid

See intermediate P1, step 2, preparation method.

Intermediate 1-INTB: preparation of methyl (S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (2- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-6-yl) butanoate

Step 1: tert-butyl 2, 6-diazaspiro [3.4] octane-2-carboxylate (0.5 g,2.4 mmol) was dissolved in N, N-dimethylformamide (5 ml), diisopropylethylamine (1.ml, 5.9 mmol) and methyl (E) -4-bromobut-2-enoate (506 mg,2.8 mmol) were added at room temperature and stirred for 2 hours. The reaction solution was diluted with water, extracted with ethyl acetate (30 ml x 2), the organic phase was washed with saturated brine, dried over Na ₂SO₄, filtered and concentrated in vacuo, and the crude product was purified by normal phase silica gel column chromatography (DCM/meoh=10/1) to give the product tert-butyl (E) -6- (4-methoxy-4-carbonylbut-2-en-1-yl) -2, 6-diazaspiro [3.4] octane-2-carboxylate as a white solid 580mg.LC-MS:ESI m/z:311.3[M+H]⁺;¹H NMR(400MHz,CDCl3)δ＝6.96(td,J＝6.0,15.6Hz,1H),5.99(d,J＝15.6Hz,1H),3.93-3.79(m,4H),3.75(s,3H),3.23(dd,J＝1.6,6.0Hz,2H),2.73(s,2H),2.61(t,J＝7.0Hz,2H),2.05(t,J＝7.0Hz,2H),1.49(s,9H).

Step 2: tert-butyl (E) -6- (4-methoxy-4-carbonylbut-2-en-1-yl) -2, 6-diazaspiro [3.4] octane-2-carboxylate (0.6 g,1.8 mmol) was dissolved in DCM (5 ml), trifluoroacetic acid (2 ml) was added and stirred at room temperature for 2 hours. The reaction mixture was directly spin-dried to give 550mg of methyl (E) -4- (2, 6-diazaspiro [3.4] octane-6-yl) but-2-enoate as a yellow oily product. LC-MS: ESI m/z 211.3[ M+H ] ⁺.

Step 3: methyl (E) -4- (2, 6-diazaspiro [3.4] oct-6-yl) but-2-enoic acid ester (0.6 g,1.7 mmol), intermediate A-3 (330 mg,2 mmol) and diisopropylethylamine (657 mg,5.1 mmol) were dissolved in 1, 2-dichloroethane (5 ml) in this order and stirred at room temperature for 30 minutes. Sodium cyanoborohydride (160 mg,2.5 mmol) was then added and the reaction continued for 1.5 hours. The reaction was washed with water (30 ml) and extracted with ethyl acetate (30 ml x 2). The organic phase was washed with saturated brine, dried over Na2SO4, filtered and concentrated in vacuo, and the crude product was purified by normal phase silica gel column chromatography (DCM/meoh=10/1) to give the yellow oily product methyl (E) -4- (2- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] oct-6-yl) but-2-enoate (1-INTA) 300mg. LC-MS: ESI m/z 357.4[ M+H ] ⁺.

Step 4: methyl (E) -4- (2- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] oct-6-yl) but-2-enoic acid ester (90 mg,0.3 mmol), intermediate G (191 mg,0.9 mmol), (1, 5-cyclooctadiene) chlororhodium (I) dimer ([ Rh (COD) Cl ] ₂, 6.2mg,0.01 mmol), (R) - (+) -2,2 '-bis (diphenylphosphine) -1,1' -binaphthyl ((R) - (+) -BINAP,15.7mg,0.03 mmol) and cesium carbonate (247 mg,0.8 mmol) were dissolved in dioxane (2 ml) and water (0.2 ml) in this order, heated to 95℃under nitrogen protection and stirred for 2 hours. The reaction solution is directly spin-dried, and the brown oily product is obtained by high performance liquid chromatography separation 50mg.LC-MS:ESI m/z:529.4[M+H]⁺;¹H NMR(400MHz,METHANOL-d₄)δ＝7.78-7.20(m,5H),6.77(br s,1H),6.09(s,1H),4.57-4.06(m,6H),3.90-3.33(m,12H),2.93-2.64(m,4H),2.48-2.43(m,2H),2.27(s,3H)2.24(s,3H),1.95-1.90(m,2H).

Intermediate 2-INTB: preparation of tert-butyl (S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-2-yl) butanoate

Step 1: tert-butyl 2, 6-diazaspiro [3.4] octane-2-carboxylate (500 mg,2.36mmol,1.0 eq) and TEA (595.7 mg,5.9mmol,2.5 eq) were dissolved in DCM (5 mL) at 0deg.C and CbzCl (803.5 mg,4.7mmol,2.0 eq) was added dropwise to react for 16h. Concentrating the reaction solution, and passing the crude product through a column. 302mg of the transparent oily product 6-benzyl 2- (tert-butyl) 2, 6-diazaspiro [3.4] octane-2, 6-dicarboxylic acid ester was obtained. LC-MS: ESI m/z 369.15[ M+Na ] ⁺.

Step 2: 6-benzyl 2- (tert-butyl) 2, 6-diazaspiro [3.4] octane-2, 6-dicarboxylate (295 mg,0.9mmol,1.0 eq) was dissolved in DCM (6 mL) at 0deg.C under nitrogen, TFA (3 mL) was added dropwise, and the reaction was continued for 2h. The reaction solution was neutralized with aqueous ammonia, and the reaction solution was concentrated to obtain 209mg of crude benzyl 2, 6-diazaspiro [3.4] octane-6-carboxylate as a yellow oil. LC-MS: ESI m/z 247.1[ M+H ] ⁺.

Step 3: benzyl 2, 6-diazaspiro [3.4] octane-6-carboxylate (209.7 mg,0.85mmol,1.0 eq) and potassium carbonate (352.8 mg,2.6mmol,3.0 eq) were dissolved in MeCN/DMF (2 mL/2 mL) at room temperature and tert-butyl (E) -4-bromobut-2-enoate (178.7 mg,0.8mmol,1 eq) was added dropwise at 65℃for 2h. The reaction was concentrated, the crude was dissolved in DCM, filtered and the filtrate concentrated. Prep-TLC (DCM/meoh=15/1) gave benzyl (E) -2- (4- (tert-butoxy) -4-carbonylbut-2-en-1-yl) -2, 6-diazaspiro [3.4] octane-6-carboxylate as a pale yellow oil 95mg. LC-MS: ESI m/z 387.52[ M+H ] ⁺.

Wherein tert-butyl (E) -4-bromobut-2-enoate is prepared by dissolving tert-butyl (E) -but-2-enoate (500 mg,3.5mmol,1.0 eq) in CCl4 (6 mL) under nitrogen, adding NBS (501 mg,2.8mmol,0.8 eq) and BPO (26 mg,0.11mmol,0.03 eq) and reacting at 85℃for 16h. TLC showed the reaction was complete, filtered and the filtrate was concentrated. Prep-TLC (PE/ea=10/1) gives the product as a colourless oil 210mg.¹H NMR(400MHz,CDCl₃)δ6.946.86(m,1H),5.95(d,J＝15.6Hz,1H),3.99(dd,J＝7.6,1.2Hz,2H),1.48(s,9H).

Step 4: an aqueous solution of benzyl (E) -2- (4- (tert-butoxy) -4-carbonylbut-2-en-1-yl) -2, 6-diazaspiro [3.4] octane-6-carboxylate (45 mg,0.1mmol,1.0 eq) intermediate G (75.4 mg,0.3mmol,3.0 eq), (1, 5-cyclooctadiene) chlororhodium (I) dimer ([ Rh (COD) Cl ] ₂.7 mg,0.01mmol,0.1 eq), (R) - (+) -2,2 '-bis (diphenylphosphine) -1,1' -binaphthyl ((R) - (+) -BINAP,14.4mg,0.023mmol,0.2 eq) and potassium hydroxide (19.6 mg,0.3mmol,3.0 eq) was dissolved in dioxane (2 mL/2 mL) under nitrogen at room temperature for 16h. Concentrated in vacuo by filtration and crude Prep-TLC (DCM/meoh=17/1) gave benzyl (S) -2- (4- (tert-butoxy) -2- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4-carbonylbutyl) -2, 6-diazaspiro [3.4] octane-6-carboxylate as a colourless oil 25mg. LC-MS: ESI m/z 559.3[ M+H ] ⁺.

Step 5: benzyl (S) -2- (4- (tert-butoxy) -2- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4-carbonylbutyl) -2, 6-diazaspiro [3.4] octane-6-carboxylate (150 mg,0.27mmol,1.0 eq) was dissolved in MeOH (2 mL) under nitrogen at room temperature, pd/C (5 mg) and 1 drop of acetic acid were added, and reacted at room temperature for 16H. The reaction mixture was concentrated by filtration through celite and the crude product was purified by Prep-TLC (DCM/meoh=10/1) to give tert-butyl (S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (2, 6-diazaspiro [3.4] octane-2-yl) butyrate (2-INTA) as a pale yellow oil 90mg. LC-MS: ESI m/z 425.3[ M+H ] ⁺.

Step 6: tert-butyl (S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (2, 6-diazaspiro [3.4] octane-2-yl) butyrate (60 mg,0.1mmol,1.0 eq) and potassium carbonate (58.4 mg,0.4mmol,3.0 eq) were dissolved in DMF (6 mL) at room temperature, and tert-butyl 7- (bromomethyl) -3, 4-dihydro-1, 8-naphthyridine-1 (2H) -carboxylate (A) (30.5 mg,0.13mmol,0.95 eq) was added dropwise to a DMF solution at 65℃for 2H. The reaction was concentrated, the crude was dissolved in DCM, filtered and the filtrate concentrated. Prep-TLC (DCM/meoh=9/1) gave 21mg of product as a colourless oil. LC-MS: ESI m/z 571.4[ M+H ] ⁺.

Intermediate 3-INTB: preparation of methyl (S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (5-carbonyl-6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-2-yl) butanoate

Step 1: tert-butyl 5-carbonyl-2, 6-diazaspiro [3.4] octane-2-carboxylate (220 mg, 972.3. Mu. Mol) was dissolved in THF (10 ml), sodium hydride (110.mg, 2.8 mmol) and intermediate A (450 mg,1.4 mmol) were added at 0deg.C and stirred under nitrogen at 60deg.C for 3 hours. The reaction was added saturated aqueous ammonium chloride (5 ml) and extracted with ethyl acetate (15 ml x 3). The organic phase was washed with saturated brine, dried over Na ₂SO₄, filtered and concentrated in vacuo to give the crude product. Purification of the crude product by normal phase silica gel column chromatography (PE/ea=1/0 to 0/1) afforded the product tert-butyl 7- ((2- (tert-butoxycarbonyl) -5-carbonyl-2, 6-diazaspiro [3.4] octane-6-yl) methyl) -3, 4-dihydro-1, 8-naphthyridine-1 (2H) -carboxylate as a pale yellow oil, 220mg. LC-MS: ESI m/z 373.5[ M-100] ⁺.

Step 2: tert-butyl 7- ((2- (tert-butoxycarbonyl) -5-carbonyl-2, 6-diazaspiro [3.4] octane-6-yl) methyl) -3, 4-dihydro-1, 8-naphthyridine-1 (2H) -carboxylate (220 mg, 465.5. Mu. Mol) was dissolved in methanol (2 ml), 4M dioxane hydrochloride (5 ml) was added at room temperature, and then stirred at room temperature under nitrogen for 1 hour. LCMS detected complete reaction of starting material. The reaction solution is concentrated to obtain a light yellow solid product 6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-5-ketone 200mg.¹H NMR(400MHz,DMSO-d₆)δ9.32(brs,1H)8.90(brs,1H)7.61(d,J＝7.2Hz,1H)6.63(d,J＝7.2Hz,1H)4.48(s,2H)4.15-4.05(m,2H)3.92-3.82(m,2H)3.50(s,2H)3.34-3.28(m,2H)2.80-2.75(m,2H)2.45(t,J＝6.8Hz,2H)1.79-1.85(m,2H).

Step 3: 6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-5-one (150 mg, 485.7. Mu. Mol) was dissolved in acetonitrile (5 ml), methyl (E) -4-bromobut-2-enoate (34.78 mg, 194.3. Mu. Mol) and diisopropylethylamine (188.3 mg,1.5 mmol) were added sequentially at room temperature, and stirred at 80℃for 16 hours under nitrogen. The reaction solution is concentrated to obtain a crude product. Purification by high performance liquid chromatography gave 50mg of the pale yellow oily product methyl (E) -4- (5-carbonyl-6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] oct-2-yl) but-2-enoate (3-INTA). LC-MS: ESI m/z 371.2[ M+H ] ⁺.

Step 4: see intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (PE/ea=1/0 to 0/1, then DCM/meoh=1/0 to 70/30) to give 45mg of the product as a pale yellow oil. LC-MS: ESI m/z 543.7[ M+H ] ⁺.

Intermediate 4-INTB: preparation of methyl (S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) butanoate

Step 1: see intermediate 1-INTB step 3. Purification of the crude product by normal phase silica gel column chromatography (DCM/meoh=10/1) gives tert-butyl 7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonane-2-carboxylate as a yellow oil 2g.LC-MS:ESI m/z:373.2[M+H]⁺;¹H NMR(400MHz,CDCl3)δppm 7.15(d,J＝7.2Hz,1H)6.51(d,J＝7.2Hz,1H)3.75-3.57(m,6H)3.47-3.34(m,4H)2.72(br t,J＝6.2Hz,2H)2.41(brs,2H)1.93-1.86(m,2H)1.81(t,J＝5.6Hz,4H)1.44(s,9H).

Step 2: see intermediate 3-INTB, procedure 2. The reaction mixture was concentrated to give 7- ((2, 7-diazaspiro [3.5] nonan-7-yl) methyl) -1,2,3, 4-tetrahydro-1, 8-naphthyridine as a yellow solid product 1g. LC-MS: ESI m/z 273.2[ M+H ] ⁺.

Step 3: see intermediate 1-INT, procedure 1. Purification of the crude product by normal phase silica gel column chromatography (DCM/meoh=10/1) afforded the product methyl (E) -4- (7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) but-2-enoate (4-INTA) as a red brown oil (600 mg). LC-MS: ESI m/z 371.1[ M+H ] ⁺.

Step 4: see intermediate 1-INTB step 4. Concentrating the reaction solution, purifying by high performance liquid chromatography to obtain reddish brown oily product 45mg.LC-MS:ESI m/z:543.5[M+H]⁺;¹H NMR(400MHz,METHANOL-d4)δppm 7.57-7.41(m,2H)7.39-7.26(m,3H)6.72(d,J＝7.2Hz,1H)6.04(s,1H)4.11(s,2H)3.98-3.78(m,2H)3.65-3.58(m,2H)3.55-3.50(m,3H)3.48-3.40(m,3H)3.35-3.28(m,3H)3.15-3.08(m,3H)2.83-2.73(m,3H)2.72-2.64(m,1H)2.22(s,3H)2.20(s,3H)2.12-1.95(m,4H)1.92-1.87(m,2H).

Intermediate 5-INTB: preparation of methyl (S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (2- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonan-7-yl) butanoate

Step 1: see intermediate 1-INT, procedure 1. Purification of the crude product by normal phase silica gel column chromatography (PE/ea=1/0 to 0/1) gives tert-butyl (E) -7- (4-methoxy-4-carbonylbut-2-en-1-yl) -2, 7-diazaspiro [3.5] nonane-2-carboxylate as a pale yellow oil 1g.¹H NMR(400MHz,CDCl₃)δ6.96(dt,J＝6.0,15.2Hz,1H)5.99(d,J＝15.2Hz,1H)3.72-3.80(s,3H)3.62(s,4H)3.11(dd,J＝1.6,6.0Hz,2H)2.37(s,4H)1.78(t,J＝5.2Hz,4H)1.46(s,9H).

Step 2: see preparation of intermediate 3-INTB, step 2. To obtain the light yellow solid product methyl (E) -4- (2, 7-diazaspiro [3.5] nonan-7-yl) but-2-enoate 810mg.¹H NMR(400MHz,D₂O)δ6.81(dt,J＝7.2,15.2Hz,1H)6.25(d,J＝15.2Hz,1H)4.05-3.95(m,4H)3.87(d,J＝7.2Hz,2H)3.72(s,3H)3.55-3.45(m,2H)3.02-2.92(m,2H)2.44-2.32(m,2H)2.05-1.95(m,2H).

Step 3: intermediate A-3 (604.6 mg,3.0 mmol) and TEA (628.7 mg,6.2 mmol) were dissolved in DCM (20 ml), sodium borohydride acetate (987.5 mg,4.7 mmol) and acetic acid (18.7 mg,0.3 mmol) were added under nitrogen and stirred overnight at room temperature. Adding 20ml of water into the reaction solution, quenching, directly spin-drying to obtain a crude product, purifying by high performance liquid chromatography to obtain a brown product methyl (E) -4- (2- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonan-7-yl) but-2-enoate (5-INTA)600mg.LC-MS:ESI m/z:371.5[M+H]⁺.¹H NMR(400MHz,DMSO-d6)δ7.15(d,J＝7.2Hz,1H)6.81(dt,J＝6.0,15.6Hz,1H)6.51-6.41(m,2H)6.00(d,J＝15.6Hz,1H)4.02(s,2H)3.66(s,6H)3.30-3.25(m,2H)3.12-3.08(m,2H)2.64(t,J＝6.0Hz,2H)2.42-2.20(m,4H)1.85-1.67(m,6H).

Step 4: see intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (PE/ea=1/0 to 0/1, then DCM/meoh=1/0 to 70/30) to give 100mg of product as a pale yellow solid. LC-MS: ESI m/z 543.7[ M+H ] ⁺.

Intermediate 6-INTB: preparation of methyl (S) -4- (7- ((1H-pyrrolo [2,3-b ] pyridin-6-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) butanoate

Step 1: tert-butyl 2, 7-diazaspiro [3.5] nonane-7-carboxylate (500 mg,2.2 mmol) and methyl (E) -4-bromobut-2-enoate (395.5 mg,2.2 mmol) were dissolved in 10ml acetonitrile followed by potassium carbonate (610.7 mg,4.4 mmol). The reaction solution was stirred at 80℃for 16 hours. The reaction solution is filtered and dried to obtain crude product, and the crude product is purified by normal phase silica gel column chromatography (PE/EA from 100 percent to 40 percent) to obtain yellow oily product tert-butyl (E) -2- (4-methoxy-4-carbonyl-but-2-alkene-1-yl) -2, 7-diazaspiro [3.5] nonane-7-carboxylic acid ester 610mg.¹H NMR(400MHz,CDCl₃)δ6.82-6.88(dt,J＝5.6,16Hz,1H),5.95-5.99(d,J＝16Hz,1H),3.73(s,3H),3.28-3.33(m,6H),3.12(s,4H),1.69-1.72(m,4H),1.44(s,9H).

Step 2: see intermediate 1-INTB step 4. Purification of the crude product by normal phase silica gel column chromatography (PE/EA from 100% to 20%) afforded the product tert-butyl (S) -2- (2- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4-methoxy-4-carbonylbutyl) -2, 7-diazaspiro [3.5] nonane-7-carboxylate as a yellow oil 940mg. LC-MS: ESI m/z 497.7[ M+H ] ⁺.

Step 3: see intermediate 3-INTB, procedure 2. The reaction mixture was concentrated and dried to give 1g of crude methyl (S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (2, 7-diazaspiro [3.5] nonan-2-yl) butanoate (6-INTA). LC-MS: ESI m/z 397.5[ M+H ] ⁺.

Step 3: see intermediate 5-INTB, procedure 3. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH from 100% to 90%) to give 30mg of the product as a yellow oil. LC-MS: ESI m/z 527.7[ M+H ] ⁺.

Intermediate 7-INTB: preparation of methyl (3S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [4.4] nonan-2-yl) butanoate

Step 1: see intermediate 3-INTB, procedure 3. Purification of the crude product by normal phase silica gel column chromatography (PE/EA from 100% to 0%) afforded tert-butyl (E) -7- (4-methoxy-4-carbonylbut-2-en-1-yl) -2, 7-diazaspiro [4.4] nonane-2-carboxylate as a yellow oil 1g. LC-MS: ESI m/z 325.5[ M+H ] ⁺.

Step 2: see intermediate 3-INTB, procedure 2. The reaction mixture was directly concentrated to give methyl (E) -4- (2, 7-diazaspiro [4.4] nonan-2-yl) but-2-enoate as a white solid 1g.

Step 3: see intermediate 5-INTB, procedure 3. The crude product was separated by high performance liquid chromatography to give 740mg of methyl (E) -4- (7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [4.4] nonan-2-yl) but-2-enoate (7-INTA) as a yellow oily product. LC-MS: ESI m/z 371.5[ M+H ] ⁺.

Step 4: see intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH from 100% to 70%) to give 77mg of the product as a yellow oil. LC-MS: ESI m/z 542.73[ M+H ] ⁺.

Intermediate 8-INTB: preparation of methyl (S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (9- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -3, 9-diazaspiro [5.5] undec-3-yl) butanoate

Step 1: see intermediate 1-INTB, procedure 1. Purification of the crude product by normal phase silica gel column chromatography (PE/EA from 100% to 30%) afforded tert-butyl (E) -9- (4-methoxy-4-carbonylbut-2-en-1-yl) -3, 9-diazaspiro [5.5] undecane-3-carboxylate as a yellow oil 1g. LC-MS: ESI m/z 352.9[ M+H ] ⁺.

Step 2: see intermediate 3-INTB, procedure 2. To give methyl (E) -4- (3, 9-diazaspiro [5.5] undec-3-yl) but-2-enoic acid ester as a white solid 500mg.

Step 3: see intermediate 5-INTB, procedure 3. The crude product was separated by high performance liquid chromatography (formic acid) to give 60mg of methyl (E) -4- (9- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -3, 9-diazaspiro [5.5] undec-3-yl) but-2-enoate (8-INTA) as a yellow oily product. LC-MS: ESI m/z 399.6[ M+H ] ⁺.

Step 4: see intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH from 100% to 70%) to give 50mg of the product as a yellow oil. LC-MS: ESI m/z 571.7[ M+H ] ⁺.

Intermediate 9-INTB: preparation of methyl-1H-pyrazol-1-yl) phenyl) -4- (8- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 8-diazaspiro [4.5] decan-2-yl) butanoate

Step 1: see intermediate 3-INTB, procedure 3. Purification of the crude product by normal phase silica gel column chromatography (PE/ea=1/0 to 0/1) gives tert-butyl (E) -2- (4-methoxy-4-carbonylbut-2-en-1-yl) -2, 8-diazaspiro [4.5] decane-8-carboxylate as a pale yellow oil 120mg.¹H NMR(400MHz,CDCl₃)δ6.93-6.90(m,1H)5.92(d,J＝17.2Hz,1H)3.68(s,3H)3.29-3.38(m,2H)3.20-3.28(m,2H)3.09-3.18(m,2H)2.56(s,2H)2.36(s,2H)1.57-1.60(m,2H)1.43-1.48(m,4H)1.38(s,9H).

Step 2: see intermediate 3-INTB, procedure 2. To obtain the light yellow solid product methyl (E) -4- (2, 8-diazaspiro [4.5] decan-2-yl) but-2-enoate 100mg.¹H NMR(400MHz,DMSO-d₆)δ8.82(s,2H)6.86-7.02(m,1H)6.29(d,J＝15.6Hz,1H)3.88-4.07(m,2H)3.71(s,3H)3.41-3.62(m,3H)3.06-3.21(m,4H)2.91(m,1H)1.70-2.05(m,6H).

Step 3: see intermediate 5-INTB, procedure 3. Purifying the crude product by high performance liquid chromatography to obtain brown product methyl (E) -4- (8- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 8-diazaspiro [4.5] dec-2-yl) but-2-enoate (9-INTA)70mg.¹H NMR(400MHz,CDCl₃)δ9.57(s,1H)7.27-7.29(m,1H)6.87-7.02(m,1H)6.59(d,J＝7.34Hz,1H)6.01(d,J＝15.2Hz,1H)3.75(s,3H)3.62(s,2H)3.46(s,2H)3.33(d,J＝5.8Hz,2H)2.52-2.67(m,8H)1.91(s,2H)1.64-1.78(m,6H)1.26-1.23(m,2H).

Step 4: see intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (PE/ea=1/0 to 0/1, then DCM/meoh=1/0 to 70/30) to give 40mg of product as a pale yellow oil. LC-MS: ESI m/z 557.7[ M+H ] ⁺.

Intermediate 10-INTB: preparation of methyl (S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (2- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 8-diazaspiro [4.5] decan-8-yl) butanoate

Step 1: see intermediate 3-INTB, procedure 3. Purification of the crude product by normal phase silica gel column chromatography (PE/ea=1/1) gives tert-butyl (E) -8- (4-methoxy-4-carbonylbut-2-en-1-yl) -2, 8-diazaspiro [4.5] decane-2-carboxylate as a yellow oil 78mg.¹H NMR(400MHz,CDCl3)δ＝7.01-6.91(m,1H),5.98(d,J＝15.6Hz,1H),3.74(s,3H),3.43-3.30(m,2H),3.19-3.08(m,4H),2.56-2.24(m,4H),1.72-1.65(m,2H),1.45(s,9H),1.34-1.23(m,3H),0.91-0.78(m,1H).

Step 2 is referred to the preparation method of intermediate 3-INTB step 2. To obtain the white solid product methyl (E) -4- (2, 8-diazaspiro [4.5] decan-8-yl) but-2-enoate 120mg.¹H NMR(400MHz,DMSO-d₆)δ＝9.53-9.27(m,2H),7.02-6.91(m,1H),6.26(d,J＝15.8Hz,1H),3.92(s,2H),3.71(s,3H),3.28-3.20(m,2H),3.13(s,1H),3.06-2.89(m,3H),1.90(s,4H),1.83-1.75(m,2H),1.24-1.20(m,1H),0.99-0.66(m,1H).

Step 3: see intermediate 5-INTB, procedure 3. The crude product was separated by high performance liquid chromatography to give the yellow oily product methyl (E) -4- (2- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 8-diazaspiro [4.5] dec-8-yl) but-2-enoate (10-INTA) 70mg.

Step 4: see intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (DCM/meoh=4/1) to give 10mg of the product as a yellow oil. LC-MS: ESI m/z 557.7[ M+H ] ⁺.

Intermediate 11-INTB: preparation of methyl (S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (1- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) spiro [ indoline-3, 4 '-piperidin ] -1' -yl) butanoate

Step 1: see intermediate 5-INTB, procedure 3. Purification of the crude product by normal phase silica gel column chromatography (PE/ea=5/1) afforded the product tert-butyl 1- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) spiro [ indoline-3, 4 '-piperidine ] -1' -carboxylate as a white solid, 140mg. LC-MS: ESI m/z 435.0[ M+H ] ⁺.

Step 2: tert-butyl 1- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) spiro [ indoline-3, 4 '-piperidine ] -1' -carboxylate (140 mg,0.32 mmol) was dissolved in DCM (2 ml), hydrochloric acid/methanol (2 ml, 4M) was added to the reaction system and stirred at room temperature for 1 hour. The reaction solution was directly concentrated to give 80mg of 1- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) spiro [ indoline-3, 4' -piperidine ] as a yellow solid product. LC-MS: ESI m/z 335.2[ M+H ] ⁺.

Step 3: 1- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) spiro [ indoline-3, 4' -piperidine ] (40 mg,0.15 mmol), methyl (E) -4-bromobut-2-enoate (18.73 mg,0.10 mmol) and potassium carbonate (41 mg,0.30 mmol) were added to DMF and stirred at 85℃for 4 hours. The reaction mixture was added with water (10 ml), followed by extraction with ethyl acetate (10 ml). The organic phase was washed three times with saturated brine (10 ml x 3), dried, concentrated, and purified by normal phase silica gel column chromatography (DCM/meoh=10/1) to give the yellow oily product methyl (E) -4- (1- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) spiro [ indolin-3, 4 '-piperidin ] -1' -yl) but-2-enoate (11-INTA) 45mg. LC-MS: ESI m/z 433.3[ M+H ] ⁺.

Step 4: see intermediate 1-INTB step 4. Purification of the crude product over a silica gel plate (DCM/meoh=10/1) afforded the product as a yellow solid 30mg. LC-MS: ESI m/z 605.2[ M+H ] ⁺.

Intermediate 12-INTB: preparation of methyl (S) -4- (7- ((2-aminoquinolin-7-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) butanoate

Step 1: intermediate B (300 mg,0.5 mmol) was dissolved in acetonitrile (5 ml) and diisopropylethylamine (0.3 ml,1.4 mmol) and intermediate 6-INTA (373.5 mg,0.9 mmol) were added sequentially at room temperature, and the reaction was heated to 80℃and stirred for 6 hours. The reaction solution was directly dried by spin, and the crude product was purified by normal phase silica gel column chromatography (DCM/meoh=10/1) to give the pale yellow oily product methyl (3S) -3- (3, 5-dimethyl-2H-pyrrol-2-yl) phenyl) -4- (7- ((2- ((benzhydryl) amino) quinolin-7-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) butanoate (12-INTA) 220mg. LC-MS: ESI m/z 717.8[ M+H ] ⁺.

Step 2: see intermediate 3-INTB, procedure 2. 230mg of a yellow solid product was obtained. LC-MS: ESI m/z 553.4[ M+H ] ⁺.

Intermediate 13-INTB: preparation of methyl (S) -4- (7- ((6-aminopyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) butanoate

Step 1: see intermediate 12-INTB, procedure 1. The crude product was purified by normal phase silica gel column chromatography (PE/ea=1/0 to 0/1) to give the product methyl (S) -4- (7- ((6- ((tert-butoxycarbonyl) amino) pyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) butanoate as a pale yellow oil 100mg. LC-MS: ESI m/z 603.7[ M+H ] ⁺.

Step 2: see intermediate 3-INTB, procedure 2. 100mg of the product was obtained as a pale yellow oil. LC-MS: ESI m/z 503.6[ M+H ] ⁺.

Intermediate 14-INTB: preparation of methyl (S) -4- (7- ((3, 4-dihydro-2H-pyrido [3,2-b ] [1,4] oxazin-6-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) butanoate

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Step 1: intermediate D (30 mg,0.01 mmol), intermediate 6-INTA (36.1 mg,0.1 mmol) and N, N-diisopropylethylamine (35.3 mg,0.3 mmol) were dissolved in DCM (5 ml) and stirred at room temperature for 2 h. The reaction was quenched with water (20 ml) and extracted with DCM (20 ml). The organic phase was dried by spin and silica gel plate purified (DCM/meoh=20/1) to give tert-butyl (S) -6- ((2- (2- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4-methoxy-4-carbonylbutyl) -2, 7-diazaspiro [3.5] nonan-7-yl) methyl) -2, 3-dihydro-4H-pyrido [3,2-b ] [1,4] oxazine-4-carboxylate as a colourless oil 30mg. LC-MS: ESI m/z 645.8[ M+H ] ⁺.

Step 2: tert-butyl (S) -6- ((2- (2- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4-methoxy-4-carbonylbutyl) -2, 7-diazaspiro [3.5] nonan-7-yl) methyl) -2, 3-dihydro-4H-pyrido [3,2-b ] [1,4] oxazine-4-carboxylate (30 mg,0.05 mmol) was dissolved in DCM (5 ml), methanol hydrochloride (4 m,5 ml) was added at room temperature, and stirred for 1 hour. The reaction solution was directly spin-dried to give 25mg of a yellow solid product. LC-MS: ESI m/z 545.7[ M+H ] ⁺.

Intermediate 19-INTB: preparation of methyl (S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.3] heptan-2-yl) butanoate

Step 1: see intermediate 1-INTB, procedure 1. Purification of the crude product by normal phase silica gel column chromatography (PE/ea=1/0 to 3/7) gives tert-butyl (E) -6- (4-methoxy-4-carbonylbut-2-en-1-yl) -2, 6-diazaspiro [3.3] heptane-2-carboxylate as a pale yellow oil 650mg.¹H NMR(400MHz,CDCl₃-d)δ6.82(m,1H)5.93(m,1H)3.99(s,4H)3.74(s,3H)3.33(s,4H)3.17(m,2H)1.43(s,9H).

Step 2: see intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (PE/ea=1/0 to 0/1) to give the product tert-butyl (S) -6- (2- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4-methoxy-4-carbonylbutyl) -2, 6-diazaspiro [3.3] heptane-2-carboxylate as a yellow oil 160mg. LC-MS ESI m/z=469.4 [ m+h ] ⁺.

Step 3: see intermediate 14-INTB, procedure 2. The product methyl (S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (2, 6-diazaspiro [3.3] heptane-2-yl) butanoate (19-INTA) was obtained as a pale yellow oil in 250mg. LC-MS ESI m/z=369.6 [ m+h ] ⁺.

Step 4: see intermediate 5-INTB, procedure 3. The crude product was purified by normal phase silica gel column chromatography (DCM/meoh=1/0 to 8/2) to give 80mg of the product as a yellow oil. LC-MS ESI m/z=515.7 [ m+h ] ⁺.

Preparation of intermediate 20-INTB methyl (S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (6- (2- (5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) ethyl) -2, 6-diazaspiro [3.3] heptan-2-yl) butanoate

Step 1: intermediate J (45 mg, 104. Mu. Mol) and 19-INTA (50.5 mg, 114.4. Mu. Mol) were dissolved in acetonitrile (5 ml), followed by diisopropylethylamine (40.3 mg, 312.1. Mu. Mol) and sodium iodide (31.2 mg, 208.1. Mu. Mol). The reaction solution was stirred at 60℃for 16 hours. The reaction solution was filtered and dried, and the crude product was purified by normal phase silica gel column chromatography (100% dcm-methanol=5/1) to give tert-butyl (S) -7- (2- (6- (2- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4-methoxy-4-carbonylbutyl) -2, 6-diazaspiro [3.3] heptan-2-yl) ethyl) -3, 4-dihydro-1, 8-naphthyridine-1 (2H) -carboxylate as a pale yellow oil 150mg. LC-MS ESI m/z=629.7 [ m+h ] ⁺.

Step 2: see intermediate 3-INTB, procedure 2. 500mg of the product was obtained as a yellow oil. LC-MS ESI m/z=529.7 [ m+h ] ⁺.

Preparation of methyl 4- (5-bromo-1 '- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) spiro [ indoline-3, 4' -piperidin ] -1-yl) butyrate as intermediate 21-INTB

Step 1: tert-butyl 4-formylpiperidine-1-carboxylate (5 g,23.4 mmol) and 4-bromophenylhydrazine hydrochloride (5.2 g,23.4 mmol) were dissolved in chloroform (50 ml) and ethanol (0.1 ml). Trifluoroacetic acid (8 g,70.3 mmol) was slowly added dropwise under nitrogen protection, the temperature was raised to 50℃and stirring was carried out for 16 hours. The reaction was quenched with water (30 ml) and aqueous hydroxylamine (10 ml) and extracted with DCM (30 ml). The organic phase was spin-dried and purified by normal phase silica gel column chromatography (PE/ea=10/1 to 3/1) to give 2.4g of t-butyl 5-bromospiro [ indole-3, 4 '-piperidine ] -1' -carboxylate as a brown oily product. LC-MS ESI m/z=365.4 [ m+h ] ⁺.

Step 2: tert-butyl 5-bromospiro [ indole-3, 4 '-piperidine ] -1' -carboxylic acid ester (2.4 g,6.6 mmol) was dissolved in ethanol (30 ml) and sodium borohydride (1.2 g,30.2 mmol) was added slowly at room temperature. Stirring was carried out at room temperature for 16 hours. The reaction was dried by spinning, washed with water (50 ml), and extracted with DCM (50 ml). The organic phase was spin-dried and purified by normal phase silica gel column chromatography (PE/ea=10/1 to 3/1) to give 900mg of the brown solid product tert-butyl 5-bromospiro [ indoline-3, 4 '-piperidine ] -1' -carboxylate. LC-MS ESI m/z=367.1 [ m+h ] ⁺.

Step 3: tert-butyl 5-bromospiro [ indoline-3, 4 '-piperidine ] -1' -carboxylate (750 mg,2 mmol) and aldehyde were dissolved in 1, 2-dichloroethane (10 ml), 2 drops of acetic acid were added dropwise at room temperature and stirred for 30 minutes. Sodium borohydride acetate (1.3 g,6.1 mmol) was added and the reaction was allowed to proceed for 16 hours. The reaction solution was directly spin-dried and purified by normal phase silica gel column chromatography (PE/ea=10/1 to 3/1) to give 350mg of t-butyl 5-bromo-1- (4-methoxy-4-carbonylbutyl) spiro [ indoline-3, 4 '-piperidine ] -1' -carboxylate as a brown oily product. LC-MS ESI m/z=467.5 [ m+h ] ⁺.

Step 4: see intermediate 14-INTB, procedure 2. 40mg of methyl 4- (5-bromospiro [ indoline-3, 4' -piperidin ] -1-yl) butyrate was obtained as a brown solid product. LC-MS ESI m/z=367.4 [ m+h ] ⁺.

Step 5: see intermediate 5-INTB, procedure 3. Purification of the crude product over a silica gel plate (DCM/meoh=20/1, rf=0.4) gives the product as a pale yellow oil 25mg.LC-MS:ESI m/z＝513.5[M+H]⁺.¹H NMR(400MHz,CDCl3)δ＝7.21-7.02(m,3H),6.58(d,J＝7.2Hz,1H),6.29(d,J＝8.3Hz,1H),5.03(br s,1H),3.74-3.61(m,3H),3.50-3.34(m,4H),3.24(s,2H),3.09(t,J＝7.0Hz,2H),2.94-2.83(m,2H),2.71(t,J＝6.2Hz,2H),2.49-2.26(m,4H),2.20-2.08(m,2H),1.94-1.89(m,4H),1.65(br d,J＝13.0Hz,2H).

Intermediate 23-INTB: preparation of methyl (S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (7- ((3-carbonyl-3, 4-dihydro-2H-pyrido [3,2-b ] [1,4] oxazin-6-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) butanoate

Step 1: see intermediate 14-INTB, procedure 1. Purification of the crude product over a silica gel plate (DCM/meoh=10/1) afforded the product as a pale yellow solid 40mg. LC-MS: ESI m/z 559.3.3[ M+H ] ⁺.

Intermediate 24-INTB: preparation of methyl (S) -3- (3-bromo-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (7- ((5, 6,7, 8-tetrahydro-1), 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] non-2-yl) butanoate

Step 1: see intermediate 1-INTB step 4. Purification of the crude product via a silica gel plate (DCM/meoh=20/1) afforded the product tert-butyl (S) -2- (2- (3-bromo-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4-methoxy-4-carbonylbutyl) -2, 7-diazaspiro [3.5] nonane-7-carboxylate as a yellow oil 45mg. LC-MS: ESI m/z 575.6[ M+H ] ⁺.

Step 2: see intermediate 14-INTB, procedure 2. To give 40mg of methyl (S) -3- (3-bromo-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (2, 7-diazaspiro [3.5] nonanoic acid-2-yl) butanoate (24-INTA) as a yellow oil. LC-MS: ESI m/z 475.5[ M+H ] ⁺.

Step 3: see intermediate 14-INTB, procedure 1. The crude product was purified by column chromatography (developer (0.2% TEA): 100% DCM-DCM/MeOH=95/5) to give 40mg of the product as a yellow oil. LC-MS: ESI m/z 621.6[ M+H ] ⁺.

Intermediate 25-INTB: preparation of methyl (S) -3- (3- (tert-butyl) -5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (2, 7-diazaspiro [3.5] nonan-2-yl) butanoate

Step 1: see intermediate 1-INTB step 4. Purification of the crude product via a silica gel plate (DCM: methanol=20:1) afforded the product tert-butyl (S) -2- (2- (3- (tert-butyl) -5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4-methoxy-4-oxobutyl) -2, 7-diazaspiro [3.5] nonane-7-carboxylate as a yellow oil 30mg. LC-MS: ESI m/z 553.8[ M+H ] ⁺.

Step 2: see intermediate 14-INTB, procedure 2. The product methyl (S) -3- (3- (tert-butyl) -5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (2, 7-diazaspiro [3.5] non-2-yl) butanoate (25-INTA) was obtained as a yellow oil in 30mg. LC-MS: ESI m/z 453.7[ M+H ] ⁺.

Step 3: see intermediate 14-INTB, procedure 1. Purification of the crude product via a silica gel plate (DCM (0.1% diea): methanol=10:1) afforded the product as a yellow oil, 20mg. LC-MS: ESI m/z 599.8[ M+H ] ⁺.

Intermediate 29-INTB: preparation of methyl (3S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) octahydro-2, 7-naphthyridin-2 (1H) -yl) butanoate

Step 1: 3-bromo-4-methylpyridine (20 g,116.3 mmol), palladium catalyst (5.3 g,5.8 mmol), zinc cyanide (8.2 g,69.8 mmol), zinc powder (0.8 g,11.6 mmol) and DPPF (6.5 g,11.6 mmol) were added to DMF (200 ml), nitrogen was replaced three times, and stirring was performed at 100℃for 16 hours under nitrogen atmosphere. The reaction solution was filtered, and the filtrate was concentrated and purified by normal phase silica gel column chromatography (PE/ea=10/1) to give 7.4g of 4-methylnicotinonitrile as a yellow solid product. LC-MS ESI m/z=119.0 [ m+h ] ⁺.

Step 2: 4-MethylNicotinitrile (7.4 g,62.6 mmol), diethyl carbonate (74 g,626.4 mmol) was dissolved in THF (200 ml), replaced with nitrogen three times, naH (12.5 g,313.2 mmol) was added to the reaction at 0deg.C and stirred at 60deg.C for 16 hours. The reaction mixture was quenched with saturated ammonium chloride (100 ml), and extracted with ethyl acetate (100 ml). The organic phase was washed three times with saturated brine (50 ml x 3), dried, concentrated and purified by normal phase silica gel column chromatography (PE/ea=20/1) to give 11g of ethyl 2- (3-cyanopyridin-4-yl) acetate as a yellow solid. LC-MS ESI m/z=191.1 [ m+h ] ⁺.

Step 3: ethyl 2- (3-cyanopyridin-4-yl) acetate (5 g,26.3 mmol) and Raney-Ni (5 g,26.3 mmol) were added to ethanol (100 ml) and water (100 ml), replaced three times with hydrogen, and stirred under a hydrogen atmosphere at a temperature of 50℃for 16 hours. The reaction solution was filtered. The filtrate was concentrated to give 3.8g of 1, 4-dihydro-2, 7-naphthyridin-3 (2H) -one as a yellow oil. LC-MS ESI m/z=149.1 [ m+h ] ⁺.

Step 4: 1, 4-dihydro-2, 7-naphthyridin-3 (2H) -one (3.8 g,25.7 mmol) was dissolved in THF (300 ml) and DCM (100 ml), borane dimethyl sulfide (10M, 25.7 ml) was added to the reaction system at 0deg.C and the reaction warmed to 80deg.C and stirred for 16 hours. The reaction mixture was quenched by addition of methanol at-78℃and stirred at room temperature for half an hour. Methanol hydrochloride (4M, 200 ml) was added to the reaction solution, and stirred at room temperature for 16 hours. The reaction mixture was concentrated to give 3.4g of 1,2,3, 4-tetrahydro-2, 7-naphthyridine as a yellow oil. LC-MS ESI m/z=135.1 [ m+h ] ⁺.

Step 5: 1,2,3, 4-tetrahydro-2, 7-naphthyridine (3.4 g,25.3 mmol), (Boc) ₂ O (8.3 g,38 mmol), TEA (7.7 g,76 mmol) and DMAP (309.6 mg,2.5 mmol) were dissolved in DCM (50 ml) and stirred at room temperature for 16 hours. The reaction mixture was quenched with water (20 ml) and extracted with DCM (20 ml). The organic phase was washed three times with saturated brine (10 ml x 3), dried, concentrated and purified by normal phase silica gel column chromatography (DCM/meoh=20/1) to give tert-butyl 3, 4-dihydro-2, 7-naphthyridine-2 (1H) -carboxylate as a yellow oil, 1.4g. LC-MS: ES m/z=235.3 [ m+h ] ⁺.

Step 6: tert-butyl 3, 4-dihydro-2, 7-naphthyridine-2 (1H) -carboxylate (1.4 g,6.0 mmol) and palladium hydroxide (557.6 mg,0.8 mmol) were dissolved in acetic acid (100 ml), replaced with hydrogen three times, and stirred under a hydrogen atmosphere at 80℃for 12 hours. The reaction solution was filtered, and after concentrating the filtrate, water (20 ml) was added, the pH was adjusted to 9 with ammonia, and extracted with DCM (20 ml). The organic phase was washed three times with saturated brine (10 ml x 3), dried over organic phase and concentrated to give tert-butyloctahydro-2, 7-naphthyridine-2 (1H) -carboxylate as a pale yellow oil, 800mg. LC-MS ESI m/z=241.2 [ m+h ] ⁺.

Step 7: tert-butyloctahydro-2, 7-naphthyridine-2 (1H) -carboxylate (800 mg,3.3 mmol), potassium carbonate (1.4 g,4.4 mmol) and methyl 4-bromocrotonate (595 mg,3.3 mmol) were dissolved in acetonitrile (15 ml) and stirred at room temperature for 8 hours. The reaction solution was concentrated and purified by normal phase silica gel column chromatography (DCM/meoh=10/1) to give tert-butyl (E) -7- (4-methoxy-4-carbonylbut-2-en-1-yl) octahydro-2, 7-naphthyridine-2 (1H) -carboxylate as a yellow oil 1g.LC-MS:ESI m/z＝339.5[M+H]⁺;¹HNMR(400MHz,CDCl3)δppm 6.84-7.03(m,1H)5.97(br d,J＝15.53Hz,1H)3.46-3.80(m,5H)2.90-3.36(m,4H)2.37(br s,2H)1.84(br s,2H)1.49-1.78(m,6H)1.45(br s,9H).

Step 8: see intermediate 1-INTB step 4. Purification of the crude product by normal phase silica gel column chromatography (DCM/meoh=10/1) afforded the yellow solid product tert-butyl 7- ((S) -2- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4-methoxy-4-carbonylbutyl) octahydro-2, 7-naphthyridine-2 (1H) -carboxylate 115mg. LC-MS ESI m/z=511.7 [ m+h ] ⁺.

Step 9: see intermediate 14-INTB, procedure 2. 80mg of the yellow solid product methyl (3S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (octahydro-2, 7-naphthyridin-2 (1H) -yl) butanoate (29-INTA) was obtained.

Step 10: see intermediate 5-INTB, procedure 3. The crude product was purified by normal phase silica gel column chromatography (DCM/meoh=10/1) to give 40mg of the product as a yellow solid. LC-MS ESI m/z=557.5 [ m+h ] ⁺.

Intermediate 30-INTB: preparation of methyl (S) -4- (2- ((3, 4-dihydro-2H-pyrido [3,2-b ] [1,4] oxazin-6-yl) methyl) -2, 6-diazaspiro [3.4] oct-6-yl) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) butanoate

Step 1: see intermediate 3-INTB, procedure 3. The crude product was purified by silica gel column chromatography (DCM/meoh=15/1) to give tert-butyl (E) -6- (4-methoxy-4-carbonylbut-2-en-1-yl) -2, 6-diazaspiro [3.4] octane-2-carboxylate as a pale yellow oil 170mg. LC-MS: ESI m/z 311.2[ M+H ] ⁺.

Step 2: see intermediate 1-INTB step 4. Purification by normal phase silica gel column chromatography (DCM/meoh=10/1) afforded the yellow solid product tert-butyl (S) -6- (2- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4-methoxy-4-carbonylbutyl) -2, 6-diazaspiro [3.4] octane-2-carboxylate 185mg. LC-MS: ESI m/z 483.3[ M+H ] ⁺.

Step 3: see intermediate 14-INTB, procedure 2. The reaction mixture was concentrated directly to give 150mg of methyl (S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (2, 6-diazaspiro [3.4] octane-6-yl) butanoate (30-INTA) as a yellow oily product.

Step 4: see intermediate 5-INTB, procedure 3. Purification of the crude product by normal phase silica gel column chromatography (DCM/meoh=10/1) afforded tert-butyl (S) -6- ((6- (2- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4-methoxy-4-carbonylbutyl) -2, 6-diazaspiro [3.4] oct-2-yl) methyl) -2, 3-dihydro-4H-pyrido [3,2-b ] [1,4] oxazine-4-carboxylate as a yellow oil 154mg. LC-MS ESI m/z=631.7 [ m+h ] ⁺.

Step 5: see intermediate 14-INTB, procedure 2. 120mg of the product was obtained as a yellow oil. LC-MS ESI m/z=531.6 [ m+h ] ⁺.

Intermediate 31-INTB: preparation of methyl (S) -4- (6- ((3, 4-dihydro-2H-pyrido [3,2-b ] [1,4] oxazin-6-yl) methyl) -2, 6-diazaspiro [3.4] oct-2-yl) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) butanoate

Step 1: see intermediate 12-INTB step 1, wherein the preparation of 31-INTA is seen in the preparation of 30-INTA. The crude product was purified by normal phase silica gel column chromatography (100% dcm-methanol=4/1) to give tert-butyl (S) -6- ((2- (2- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4-methoxy-4-carbonylbutyl) -2, 6-diazaspiro [3.4] oct-6-yl) methyl) -2, 3-dihydro-4H-pyrido [3,2-b ] [1,4] oxazine-4-carboxylate as a yellow oil 160mg. LC-MS ESI m/z=631.7 [ m+h ] ⁺.

Step 2: see intermediate 14-INTB, procedure 2. 200mg of the product was obtained as a yellow oil. LC-MS ESI m/z=531.6 [ m+h ] ⁺.

Intermediate 32-INTB: preparation of methyl (3S) -4- (7- ((3, 4-dihydro-2H-pyrido [3,2-b ] [1,4] oxazin-6-yl) methyl) -2, 7-diazaspiro [4.4] nonan-2-yl) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) butanoate

Step 1: see intermediate 12-INTB step 1, wherein the preparation of 32-INTA is seen in the preparation of 30-INTA. Purification of the crude product by normal phase silica gel column chromatography (DCM/methanol=4/1) afforded tert-butyl 6- ((7- ((S) -2- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4-methoxy-4-carbonylbutyl) -2, 7-diazaspiro [4.4] nonan-2-yl) methyl) -2, 3-dihydro-4H-pyrido [3,2-b ] [1,4] oxazine-4-carboxylate as a yellow oil 150mg. LC-MS ESI m/z=645.7 [ m+h ] ⁺.

Step 2: see intermediate 14-INTB, procedure 2. 150mg of a yellow oily product was obtained. LC-MS ESI m/z=545.7 [ m+h ] ⁺.

Intermediate 34-INTB: preparation of methyl (S) -4- (2- ((1H-pyrrolo [2,3-b ] pyridin-6-yl) methyl) -2, 6-diazaspiro [3.4] oct-6-yl) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) butanoate

Step 1: see intermediate 5-INTB, procedure 3. The crude product was purified by normal phase silica gel column chromatography (DCM/meoh=10/1) to give 85mg of the product as a yellow solid. LC-MS ESI m/z=513.1 [ m+h ] ⁺.

Intermediate 35-INTB: preparation of methyl (S) -4- (6- ((1H-pyrrolo [2,3-b ] pyridin-6-yl) methyl) -2, 6-diazaspiro [3.4] oct-2-yl) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) butanoate

Step 1: see intermediate 5-INTB, procedure 3. The crude product was purified by normal phase silica gel column chromatography (DCM/meoh=1/0 to 8/2) to give 90mg of the product as a yellow oil. LC-MS ESI m/z=513.6 [ m+h ] ⁺.

Intermediate 36-INTB: preparation of methyl (3S) -4- (7- ((1H-pyrrolo [2,3-b ] pyridin-6-yl) methyl) -2, 7-diazaspiro [4.4] nonan-2-yl) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) butanoate

Step 1: see intermediate 5-INTB, procedure 3. The crude product was purified by normal phase silica gel column chromatography (DCM/meoh=1/0 to 8/2) to give 110mg of the product as a yellow oil. LC-MS ESI m/z=527.7 [ m+h ] ⁺.

Intermediate 37-INTB: preparation of methyl (S) -4- (2- ((1H-pyrrolo [2,3-b ] pyridin-6-yl) methyl) -2, 7-diazaspiro [3.5] non-7-yl) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) butanoate

Step 1: see intermediate 1-INTB, procedure 1. Column chromatography (DCM/meoh=95/5) of the crude product gave the yellow liquid product (E) -7- (4-methoxy-4-oxo-but-2-en-1-yl) -2, 7-diazaspiro [3.5] nonane-2-carboxylic acid tert-butyl ester 400mg. LC-MS: ESI m/z 324.7[ M+H ] ⁺.

Step 2: see intermediate 1-INTB step 4. The crude product was chromatographed (DCM/meoh=95/5) to give tert-butyl (S) -7- (2- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4-methoxy-4-oxybutyl) -2, 7-diazaspiro [3.5] nonane-2-carboxylate as a yellow oil, 50mg. LC-MS: ESI m/z 497.7[ M+H ] ⁺.

Step 3: see intermediate 14-INTB, procedure 2. The reaction mixture was dried by spin to give 40mg of methyl (S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (2, 7-diazaspiro [3.5] nonanoic acid-7-yl) butyrate (37-INTA) as a yellow oily product. LC-MS: ESI m/z 397.6[ M+H ] ⁺.

Step 4: see intermediate 5-INTB, procedure 3. The crude product was chromatographed (DCM/meoh=95/5) to give 50mg of the product as a yellow oil. LC-MS: ESI m/z 527.7[ M+H ] ⁺.

Intermediate 38-INTB: preparation of methyl (S) -3- (3-bromo-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.3] heptan-2-yl) butanoate

Step 1: see intermediate 5-INTB step 3, wherein the preparation of 38-INTA is seen in the preparation of 19-INTA. The crude product was purified by normal phase silica gel column chromatography (DCM/meoh=8/2) to give 80mg of the product as a yellow oil. LC-MS ESI m/z=593.6 [ m+h ] ⁺.

Intermediate 39-INTB: preparation of methyl (S) -3- (3- (tert-butyl) -5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.3] heptan-2-yl) butanoate

Step 1: see intermediate 5-INTB step 3, wherein 39-INTA is prepared see 19-INTA. The crude product was purified by normal phase silica gel column chromatography (DCM/meoh=1/0 to 8/2) to give 50mg of the product as a yellow oil. LC-MS ESI m/z=570.8 [ m+h ] ⁺.

Intermediate 40-INTB: preparation of methyl (S) -3- (3- (tert-butyl) -5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (6- ((3, 4-dihydro-2H-pyrido [3,2-b ] [1,4] oxazin-6-yl) methyl) -2, 6-diazaspiro [3.3] heptan-2-yl) butanoate

Step 1: see intermediate 5-INTB, procedure 3. The crude product was purified by normal phase silica gel column chromatography (DCM/meoh=1/0 to 8/2) to give tert-butyl (S) -6- ((6- (2- (3- (tert-butyl) -5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4-methoxy-4-carbonylbutyl) -2, 6-diazaspiro [3.3] heptan-2-yl) methyl) -2, 3-dihydro-4H-pyrido [3,2-b ] [1,4] oxazine-4-carboxylate as a yellow oil 80mg. LC-MS ESI m/z=672.9 [ m+h ] ⁺.

Step 2: see intermediate 14-INTB, procedure 2. 250mg of the product was obtained as a pale yellow oil. LC-MS ESI m/z=572.8 [ m+h ] ⁺.

Intermediate 41-INTB: preparation of methyl (S) -4- (6- ((1H-pyrrolo [2,3-b ] pyridin-6-yl) methyl) -2, 6-diazaspiro [3.3] heptan-2-yl) -3- (3- (tert-butyl) -5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) butanoate

Step 1: see 5-INTB step 3 for preparation. The crude product was chromatographed (DCM/meoh=95/5) to give 50mg of the product as a yellow oil. LC-MS: ESI m/z 554.7[ M+H ] ⁺.

Intermediate 42-INTB: preparation of methyl (S) -3- (3-bromo-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-2-yl) butanoate

Step 1: see intermediate 1-INTB step 4. The reaction was concentrated and purified by normal phase silica gel column chromatography (DCM/meoh=10/1) to give tert-butyl (S) -2- (2- (3-bromo-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4-methoxy-4-carbonylbutyl) -2, 6-diazaspiro [3.4] octane-6-carboxylate as a yellow oil 60mg. LC-MS ESI m/z=563.5 [ m+2h ] ⁺.

Step 2: see intermediate 14-INTB, procedure 2. The product methyl (S) -3- (3-bromo-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (2, 6-diazaspiro [3.4] octane-2-yl) butanoate (42-INTA) was obtained as a yellow oil in 30mg. LC-MS ESI m/z=461.3 [ m+h ] ⁺.

Step 3: see intermediate 5-INTB, procedure 3. The crude product was purified by high performance liquid chromatography to give 15mg of the product as a colourless oil. LC-MS ESI m/z=607.5 [ m+h ] ⁺.

Intermediate 43-INTB: preparation of methyl (S) -3- (3- (tert-butyl) -5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-2-yl) butanoate

Step 1: see intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (100% DCM to DCM/meoh=10/1) to give benzyl (S) -2- (2- (3- (tert-butyl) -5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4-methoxy-4-carbonylbutyl) -2, 6-diazaspiro [3.4] octane-6-carboxylate as a yellow oil 750mg. LC-MS ESI m/z=575.2 [ m+h ] ⁺.

Step 2: see intermediate 2-INTB step 5. The reaction mixture was concentrated by filtration to give 520mg of methyl (S) -3- (3- (tert-butyl) -5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (2, 6-diazaspiro [3.4] octane-2-yl) butanoate (43-INTA), a crude product. LC-MS ESI m/z=439.6 [ m+h ] ⁺.

Step 3: see intermediate 5-INTB, procedure 3. The crude product was purified by high performance liquid chromatography to give 210mg of the product as a yellow oil. LC-MS ESI m/z=585.5 [ m+h ] ⁺.

Intermediate 44-INTB: preparation of methyl (S) -3- (5- (3, 5-dimethyl-1H-pyrazol-1-yl) pyridin-3-yl) -4- (7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) butanoate

Step 1: see intermediate 1-INTB step 4. Purification of the crude product by normal phase silica gel column chromatography (DCM/methanol=4/1) afforded tert-butyl (S) -2- (2- (5-bromopyridine-3-yl) -4-methoxy-4-carbonylbutyl) -2, 7-diazaspiro [3.5] nonane-7-carboxylate as a pale yellow oil 150mg. LC-MS ESI m/z=484.4 [ m+h ] ⁺.

Step 2: tert-butyl (S) -2- (2- (5-bromopyridin-3-yl) -4-methoxy-4-carbonylbutyl) -2, 7-diazaspiro [3.5] nonane-7-carboxylate (65 mg, 134.7. Mu. Mol), 3, 5-dimethyl-1H-pyrazole (19.4 mg, 202.1. Mu. Mol), methanesulfonic acid-2- (di-tert-butylphosphino) -3, 6-dimethoxy-2, 4, 6-triisopropyl-1, 1-biphenyl (2-amino-1, 1-biphenyl-2-yl) palladium (II) (11.5 mg) and cesium carbonate (109.8 mg, 336.9. Mu. Mol) were dissolved in 1, 4-dioxane (4 ml) in this order, heated to 110 ℃ under nitrogen protection and stirred for 16 hours. The reaction solution was directly filtered, spin-dried, and the crude product was separated by high performance liquid chromatography to give 20mg of tert-butyl (S) -2- (2- (5- (3, 5-dimethyl-1H-pyrazol-1-yl) pyridin-3-yl) -4-methoxy-4-carbonylbutyl) -2, 7-diazaspiro [3.5] nonane-7-carboxylate as a pale yellow solid product. LC-MS ESI m/z=498.5 [ m+h ] ⁺.

Step 3: see intermediate 14-INTB, procedure 2. The product methyl (S) -3- (5- (3, 5-dimethyl-1H-pyrazol-1-yl) pyridin-3-yl) -4- (2, 7-diazaspiro [3.5] nonan-2-yl) butanoate (44-INTA) was obtained as a pale yellow oil, 20mg. LC-MS ESI m/z=398.5 [ m+h ] ⁺.

Step 4: see intermediate 5-INTB, procedure 3. The crude product was purified by normal phase silica gel column chromatography (100% dcm-methanol=7/3) to give 20mg of the product as a pale yellow oil. LC-MS ESI m/z=544.3 [ m+h ] ⁺.

Intermediate 45-INTB: preparation of methyl (S) -4- (7- ((1H-pyrrolo [2,3-b ] pyridin-6-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) -3- (3-bromo-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) butanoate

Step 1: see intermediate 5-INTB step 3, wherein 24-INTA is prepared as 6-INTA. The crude product was chromatographed (DCM/meoh=95/5) to give 50mg of the product as a yellow oil. LC-MS: ESI m/z 605.6[ M+H ] ⁺.

Intermediate 46-INTB: preparation of methyl (S) -4- (7- ((1H-pyrrolo [2,3-b ] pyridin-6-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) -3- (3- (tert-butyl) -5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) butanoate

Step 1: see intermediate 5-INTB step 3, wherein 25-INTA is prepared as 6-INTA. The crude product was chromatographed (DCM/meoh=95/5) to give 50mg of the product as a yellow oil. LC-MS: ESI m/z 582.8[ M+H ] ⁺.

Intermediate 47-INTB: preparation of methyl (S) -3- (3-bromo-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (7- ((3, 4-dihydro-2H-pyrido [3,2-b ] [1,4] oxazin-6-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) butanoate

Step 1: see intermediate 5-INTB, procedure 3. The crude product was purified by normal phase silica gel column chromatography (DCM/meoh=1/0 to 8/2) to give tert-butyl (S) -6- ((2- (2- (3-bromo-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4-methoxy-4-carbonylbutyl) -2, 7-diazaspiro [3.5] nonan-7-yl) methyl) -2, 3-dihydro-4H-pyrido [3,2-b ] [1,4] oxazine-4-carboxylate as a yellow oil 80mg. LC-MS ESI m/z=723.3 [ m+h ] ⁺.

Step 2: see intermediate 3-INTB, procedure 2. 50mg of the product was obtained as a pale yellow oil. LC-MS ESI m/z=623.2 [ m+h ] ⁺.

Intermediate 48-INTB: preparation of methyl (S) -3- (3- (tert-butyl) -5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (7- ((3, 4-dihydro-2H-pyrido [3,2-b ] [1,4] oxazin-6-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) butanoate

Step 1: step 1: see intermediate 5-INTB, procedure 3. Purification of the crude product by normal phase silica gel column chromatography (DCM/meoh=1/0 to 8/2) afforded the product tert-butyl (S) -6- ((2- (2- (3- (tert-butyl) -5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4-methoxy-4-carbonylbutyl) -2, 7-diazaspiro [3.5] nonan-7-yl) methyl) -2, 3-dihydro-4H-pyrido [3,2-b ] [1,4] oxazine-4-carboxylic acid ester as a yellow oil 100mg. LC-MS ESI m/z=701.4 [ m+h ] ⁺.

Step 2: see intermediate 3-INTB, procedure 2. 70mg of the product are obtained as a pale yellow oil. LC-MS ESI m/z=601.4 [ m+h ] ⁺.

Intermediate 49-INTB: preparation of methyl (S) -3- (3-bromo-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (2- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-6-yl) butanoate

Step 1: see intermediate 5-INTB step 3, wherein 49-INTA is prepared as 30-INTA. Column chromatography of the crude product (DCM/meoh=10/1) afforded 60mg as a yellow oil. LC-MS: ESI m/z 607.2[ M+H ] ⁺.

Intermediate 50-INTB: preparation of methyl (S) -3- (3- (tert-butyl) -5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (2- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-6-yl) butanoate

Step 1: see intermediate 5-INTB step 3, wherein 50-INTA is prepared as 30-INTA. Column chromatography of the crude product (DCM/meoh=10/1) afforded 60mg as a yellow oil. LC-MS: ESI m/z, 585.4[ M+H ] ⁺.

Intermediate 51-INTB: preparation of methyl (3S) -3- (3-bromo-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [4.4] nonan-2-yl) butanoate

Step 1: see intermediate 5-INTB step 3, wherein 51-INTA is prepared as 32-INTA. The crude product was chromatographed (DCM/MeOH=10/1) to give 50mg of the product as a yellow oil. LC-MS: ESI m/z 621.2[ M+H ] ⁺.

Intermediate 52-INTB: preparation of methyl (3S) -3- (3- (tert-butyl) -5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [4.4] nonan-2-yl) butanoate

Step 1: see intermediate 5-INTB step 3, wherein 52-INTA is prepared as 32-INTA. The crude product was purified by normal phase silica gel column chromatography (DCM/methanol=9/1) to give 140mg of the product as a yellow oil. LC-MS ESI m/z=599.6 [ m+h ] ⁺.

Intermediate 53-INTB: preparation of methyl (S) -3- (3-bromo-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (2- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonan-7-yl) butanoate

Step 1: see intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (DCM/meoh=10/1) to give 50mg of the product as a yellow oil. LC-MS: ESI m/z 621.2[ M+H ] ⁺.

Intermediate 54-INTB: preparation of methyl (S) -3- (3- (tert-butyl) -5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (2- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonan-7-yl) butanoate

Step 1: see intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (DCM/methanol=9/1) to give 50mg of the product as a yellow oil. . LC-MS: ESI m/z 599.4[ M+H ] ⁺.

Intermediate 62-INTB: preparation of methyl 2- (7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [4.4] nonan-2-yl) acetate

Step 1: tert-butyl 2, 7-diazaspiro [4.4] nonane-2-carboxylate (100 mg, 441.9. Mu. Mol) was dissolved in DCM (5 ml), then TEA (184.5. Mu.l, 1.3 mol) and methyl chloroacetate (62 mg, 574.4. Mu. Mol) was added sequentially and reacted at room temperature for 2 hours. The reaction solution was dried by spinning, washed with water (10 ml), extracted with ethyl acetate (10 ml), and the organic phase was dried, and the product tert-butyl 7- (2-methoxy-2-carbonylethyl) -2, 7-diazaspiro [4.4] nonane-2-carboxylate 130mg was obtained as a yellow oil by spinning. LC-MS ESI m/z=299.0 [ m+h ] ⁺.

Step 2: see intermediate 14-INB step 2. The product methyl 2- (2, 7-diazaspiro [4.4] nonan-2-yl) acetate was obtained as a yellow oil 90mg. LC-MS ESI m/z=199.2 [ m+h ] ⁺.

Step 3: see intermediate 5-INTB, procedure 3. The crude product was prepared by high performance liquid chromatography to give 100mg of a white solid product. LC-MS ESI m/z=345.5 [ m+h ] ⁺.

Intermediate 63-INTB: preparation of methyl (S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (9- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -1-oxa-4, 9-diazaspiro [5.5] undec-4-yl) butanoate

Step 1: see intermediate 3-INTB, procedure 3. Purifying the crude product by normal phase silica gel column chromatography (100% petroleum ether-ethyl acetate=2/1) to obtain yellow oily product tert-butyl (E) -4- (4-methoxy-4-carbonyl-but-2-alkene-1-yl) -1-oxa-4, 9-diazaspiro [5.5] undecane-9-carboxylic acid ester 400mg.¹H NMR(400MHz,CDCl3)δ6.83(m,1H)5.93(br d,J＝15.77Hz,1H)3.55-3.77(m,8H)3.06(m,2H)2.99(m,2H)2.31-2.40(m,2H)2.16(s,2H)1.87(m,2H)1.30-1.43(m,12H).

Step 2: see intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (100% petroleum ether-ethyl acetate=1/2) to give 420mg of tert-butyl (S) -4- (2- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4-methoxy-4-carbonylbutyl) -1-oxa-4, 9-diazaspiro [5.5] undecane-9-carboxylate as a yellow oily product. LC-MS ESI m/z=527.7 [ m+h ] ⁺.

Step 3: see intermediate 3-INTB, procedure 2. The product methyl (S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (1-oxa-4, 9-diazaspiro [5.5] undec-4-yl) butanoate (63-INTA) was obtained as a pale yellow oil in 500mg. LC-MS ESI m/z=427.6 [ m+h ] ⁺.

Step 4: see intermediate 5-INTB, procedure 3. The crude product was purified by normal phase silica gel column chromatography (100% dcm-methanol=4/1) to give 200mg of the product as a yellow oil. LC-MS ESI m/z=573.7 [ m+h ] ⁺.

Intermediate 64-INTB: preparation of methyl (S) -4- (7- ((3H-imidazo [4,5-b ] pyridin-5-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) butanoate

Step 1: see intermediate 5-INTB, procedure 3. The crude product was purified by normal phase silica gel column chromatography (100% dcm-methanol=4/1) to give tert-butyl (S) -5- ((2- (2- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4-methoxy-4-carbonylbutyl) -2, 7-diazaspiro [3.5] nonan-7-yl) methyl) -3H-imidazo [4,5-b ] pyridine-3-carboxylate as a yellow oil 30mg. LC-MS: ESI m/z 628.7[ M+H ] ⁺.

Step 2: see intermediate 3-INTB, procedure 2. Concentration gave 30mg of the product as a pale yellow oil. LC-MS: ESI m/z 528.6[ M+H ] ⁺.

Intermediate 65-INTB: preparation of methyl (S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) oxo) -2-azaspiro [3.5] nonan-2-yl) butanoate

Step 1: 7-hydroxy-2-azaspiro [3.5] nonane-2-carboxylic acid tert-butyl ester (200 mg,0.8 mmol), 1, 8-naphthyridin-2-ol (1 eq.) and triphenylphosphine (1.2 eq.) were dissolved in dry THF (20 ml) in this order. Di-tert-butyl azodicarbonate (1.2 eq.) was added dropwise to the solution under nitrogen at 0deg.C, and the reaction was stirred for 1 hour. The reaction solution was concentrated and the crude product was purified by normal phase silica gel column chromatography (PE/ea=4/1) to give the product tert-butyl 7- ((1, 8-naphthyridin-2-yl) oxo) -2-azaspiro [3.5] nonane-2-carboxylate as a yellow oil 160mg. LC-MS: ESI m/z 370.2[ M+H ] ⁺.

Step 2: see intermediate J, procedure 2. The product tert-butyl 7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) oxo) -2-azaspiro [3.5] nonane-2-carboxylate was obtained as a white solid, 50mg. LC-MS: ESI m/z 374.2[ M+H ] ⁺.

Step 3: see intermediate 14-INTB, procedure 2. The product 7- ((2-azaspiro [3.5] nonan-7-yl) oxo) -1,2,3, 4-tetrahydro-1, 8-naphthyridine was obtained as a white solid, 40mg. LC-MS: ESI m/z 274.2[ M+H ] ⁺.

Step 4: see intermediate 3-INTB, procedure 3. Purification of the crude product by normal phase silica gel column chromatography (PE/ea=2/1) afforded the product methyl (E) -4- (7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) oxo) -2-azaspiro [3.5] nonan-2-yl) but-2-enoate (65-INTA) as a yellow oil in an amount of 50mg. LC-MS: ESI m/z 372.2[ M+H ] ⁺.

Step 5: see intermediate 1-INTB step 4. The crude product was prepared by high performance liquid chromatography to yield 26mg of a yellow solid product. LC-MS: ESI m/z 544.3[ M+H ] ⁺.

Intermediate 66-INTB: preparation of methyl (S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- ((7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -7-azaspiro [3.5] nonan-2-yl) amino) butanoate

Step 1: see intermediate 3-INTB, procedure 3. Purification of the crude product by normal phase silica gel column chromatography (DCM/meoh=10/1) gives tert-butyl (E) -2- ((4-methoxy-4-carbonylbut-2-en-1-yl) amino) -7-azaspiro [3.5] nonane-7-carboxylate as a colourless oil 1.9g.LC-MS:ESI m/z＝339.5[M+H]⁺;¹H NMR(400MHz,CDCl3)δ＝6.98(td,J＝5.6,15.7Hz,1H),5.97(br d,J＝15.7Hz,1H),3.74(s,3H),3.37-3.23(m,7H),2.25-2.08(m,2H),1.52-1.42(m,15H).

Step 2: see intermediate 14-INTB, procedure 2. The yellow solid product methyl (E) -4- ((7-azaspiro [3.5] nonan-2-yl) amino) but-2-enoate 640mg was obtained.

Step 3: see intermediate 5-INTB, procedure 3. The crude product was prepared by high performance liquid chromatography to give 250mg of the brown solid product methyl (E) -4- ((7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -7-azaspiro [3.5] nonan-2-yl) amino) but-2-enoate (66-INTA). LC-MS ESI m/z=385.5 [ m+h ] ⁺.

Step 4: see intermediate 1-INTB step 4. The crude product is prepared into brown solid products by high performance liquid chromatography 50mg.LC-MS:ESI m/z＝557.4[M+H]⁺;¹H NMR(400MHz,METHANOL-d₄)δ＝7.58-7.50(m,1H),7.44-7.34(m,3H),7.25(d,J＝7.2Hz,1H),6.53(d,J＝7.2Hz,1H),6.09(s,1H),3.91(s,2H),3.71-3.62(m,1H),3.59(s,3H),3.56-3.48(m,1H),3.40(br t,J＝5.3Hz,2H),3.20(br d,J＝7.3Hz,2H),3.08-2.92(m,4H),2.88(br dd,J＝6.5,16.5Hz,1H),2.79-2.70(m,3H),2.28(br d,J＝14.4Hz,8H),1.98-1.78(m,8H).

Intermediate 67-INTB: preparation of methyl (S) -4- (8, 8-difluoro-2- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] oct-6-yl) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) butanoate

Step 1: see intermediate 3-INTB, procedure 3. Purifying the crude product by normal phase silica gel column chromatography (100% petroleum ether-ethyl acetate=2/1) to obtain yellow oily product tert-butyl (E) -8, 8-difluoro-6- (4-methoxy-4-carbonyl-but-2-ene-1-yl) -2, 6-diazaspiro [3.4] octane-2-carboxylic acid ester 100mg.¹H NMR(400MHz,CDCl3)δ6.88(m,1H)6.00(d,J＝15.65Hz,1H)4.19(d,J＝9.29Hz,2H)3.76(s,3H)3.70(d,J＝9.17Hz,2H)3.25(d,J＝5.14Hz,2H)2.92-3.09(m,4H)1.45(s,9H).

Step 2: see intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (100% petroleum ether-ethyl acetate=1/1) to give the pale yellow oily product tert-butyl (S) -6- (2- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4-methoxy-4-carbonylbutyl) -8, 8-difluoro-2, 6-diazaspiro [3.4] octane-2-carboxylate 120mg. LC-MS ESI m/z=519.5 [ m+h ] ⁺.

Step 3: see intermediate 14-INTB, procedure 2. 110mg of methyl (S) -4- (8, 8-difluoro-2, 6-diazaspiro [3.4] oct-6-yl) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) butanoate (67-INTA) was obtained as a pale yellow oily product. LC-MS ESI m/z=419.4 [ m+h ] ⁺.

Step 4: see intermediate 5-INTB, procedure 3. The crude product was purified by normal phase silica gel column chromatography (100% dcm-methanol=4/1) to give 120mg of the product as a yellow oil. LC-MS ESI m/z=565.5 [ m+h ] ⁺.

Intermediate 68-INTB: preparation of methyl (3S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (8-fluoro-2- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-6-yl) butanoate

The preparation method and the steps are the same as those of 67-INTB by taking tert-butyl 8-fluoro-2, 6-diazaspiro [3.4] octane-2-carboxylate as a starting material. 110mg of methyl (3S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (8-fluoro-2, 6-diazaspiro [3.4] octane-6-yl) butanoate (68-INTA) was obtained as a pale yellow oily intermediate. LC-MS ESI m/z=401.2 [ m+h ] ⁺. 120mg of the product was obtained as a yellow oil. LC-MS ESI m/z=547.3 [ m+h ] ⁺.

Intermediate 69-INTB: preparation of methyl (S) -4- (8, 8-difluoro-6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] oct-2-yl) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) butanoate

The preparation method and the steps are the same as those of 67-INTB by taking tert-butyl 8, 8-difluoro-2, 6-diazaspiro [3.4] octane-6-carboxylate as a starting material. 110mg of methyl (S) -4- (8, 8-difluoro-2, 6-diazaspiro [3.4] oct-2-yl) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) butanoic acid ester (69-INTA) was obtained as a pale yellow oily intermediate. LC-MS ESI m/z=419.2 [ m+h ] ⁺. 120mg of the product was obtained as a yellow oil. LC-MS ESI m/z=565.3 [ m+h ] ⁺.

Intermediate 70-INTB: preparation of methyl (3S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (8-fluoro-6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-2-yl) butanoate

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The preparation method and the steps are the same as those of 67-INTB by taking tert-butyl 8-fluoro-2, 6-diazaspiro [3.4] octane-6-carboxylate as a starting material. 110mg of methyl (3S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (8-fluoro-2, 6-diazaspiro [3.4] octane-2-yl) butanoate (70-INTA) was obtained as a pale yellow oily intermediate. LC-MS ESI m/z=401.2 [ m+h ] ⁺. 120mg of the product was obtained as a yellow oil. LC-MS ESI m/z=547.3 [ m+h ] ⁺.

Intermediate 71-INTB: preparation of methyl (S) -4- (5, 5-difluoro-7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) butanoate

Step 1: see intermediate 5-INTB, procedure 3. Purification of the crude product by normal phase silica gel column chromatography (100% dcm-methanol=7/3) afforded tert-butyl 5, 5-difluoro-7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonane-2-carboxylate as a yellow oil, 390mg. LC-MS ESI m/z=409.5 [ m+h ] ⁺.

Step 2: see intermediate 3-INTB, procedure 2. This gave the product 7- ((5, 5-difluoro-2, 7-diazaspiro [3.5] nonan-7-yl) methyl) -1,2,3, 4-tetrahydro-1, 8-naphthyridine as a pale yellow oil, 500mg.

Step 3: see intermediate 3-INTB, procedure 3. Purifying the crude product by normal phase silica gel column chromatography (100% petroleum ether-ethyl acetate=0/1) to obtain pale yellow oily product methyl (E) -4- (5, 5-difluoro-7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) but-2-enoate (71-INTA)60mg.¹H NMR(400MHz,CDCl3)δ8.41(br s,1H)7.32(br d,J＝7.21Hz,1H)6.83(dt,J＝15.80,5.49Hz,1H)6.61(d,J＝7.21Hz,1H)5.97(br d,J＝15.77Hz,1H)3.75(s,3H)3.64(s,2H)3.49(br s,2H)3.27-3.38(m,6H)2.75(br t,J＝6.17Hz,2H)2.63(br t,J＝10.94Hz,2H)2.55(br s,2H)2.13(br d,J＝4.77Hz,2H)1.89-1.97(m,2H).

Step 4: see intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (100% dcm-methanol=85/15) to give 70mg of the product as a pale yellow oil. LC-MS ESI m/z=579.6 [ m+h ] ⁺.

Intermediate 72-INTB: preparation of methyl (3S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (5-fluoro-7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) butanoate

Step 1: see procedure for the preparation of intermediate 1-INTB step 4, wherein 72-INTA is prepared as 71-INTA. The crude product was purified by normal phase silica gel column chromatography (100% dcm-methanol=85/15) to give 120mg of the product as a pale yellow oil. LC-MS ESI m/z=561.3 [ m+h ] ⁺.

Intermediate 73-INTB: preparation of methyl (S) -4- (5, 5-difluoro-2- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonan-7-yl) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) butanoate

The preparation method and the steps are the same as those of 67-INTB by taking tert-butyl 5, 5-difluoro-2, 7-diazaspiro [3.5] nonane-2-carboxylate as a starting material. 100mg of methyl (S) -4- (5, 5-difluoro-2, 7-diazaspiro [3.5] nonan-7-yl) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) butanoic acid ester (73-INTA) was obtained as a pale yellow oily intermediate. LC-MS ESI m/z=433.5 [ m+h ] ⁺. 85mg of the product are obtained as a yellow oil. LC-MS ESI m/z=579.7 [ m+h ] ⁺.

Intermediate 74-INTB: preparation of methyl (3S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (5-fluoro-2- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonan-7-yl) butanoate

The preparation method and the steps are the same as those of 67-INTB by taking tert-butyl 5-fluoro-2, 7-diazaspiro [3.5] nonane-2-carboxylate as a starting material. 100mg of methyl (3S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (5-fluoro-2, 7-diazaspiro [3.5] nonan-7-yl) butanoate (74-INTA) was obtained as a pale yellow oily intermediate. LC-MS ESI m/z=415.2 [ m+h ] ⁺. 85mg of the product are obtained as a yellow oil. LC-MS ESI m/z=561.3 [ m+h ] ⁺.

Intermediate 75-INTB: preparation of methyl (S) -3- (3-bromophenyl) -4- (7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) butanoate

Step 1: see intermediate 1-INTB step 4. The crude product was purified by high performance liquid chromatography to give 110mg of the product as a yellow oil. LC-MS ESI m/z=527.4 [ m+h ] ⁺.

Intermediate 76-INTB: preparation of methyl (S) -3- (3-bromo-5- (tert-butyl) phenyl) -4- (7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) butanoate

Step 1: see intermediate 1-INTB step 4. The crude product was purified by high performance liquid chromatography to give 210mg of tert-butyl (S) -2- (2- (3-bromo-5- (tert-butyl) phenyl) -4-methoxy-4-carbonylbutyl) -2, 7-diazaspiro [3.5] nonane-7-carboxylate as a colourless oil. LC-MS ESI m/z=537.5 [ m+h ] ⁺.

Step 2: see intermediate 14-INTB, procedure 2. The reaction mixture was concentrated directly to give 75mg of methyl (S) -3- (3-bromo-5- (tert-butyl) phenyl) -4- (2, 7-diazaspiro [3.5] nonan-2-yl) butanoate (76-INTA) as a yellow oily product.

Step 3: see intermediate 5-INTB, procedure 3. The crude product was purified by normal phase silica gel column chromatography (DCM/meoh=10/1) to give 70mg of the product as a yellow oil. LC-MS ESI m/z=583.6 [ m+h ] ⁺.

Intermediate 77-INTB: preparation of methyl (S) -3- (5-bromo-2-fluorophenyl) -4- (7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) butanoate

The preparation method and the steps are the same as those of 76-INTB by taking tert-butyl (E) -2- (4-methoxy-4-carbonylbut-2-en-1-yl) -2, 7-diazaspiro [3.5] nonane-7-carboxylate and (5-bromo-2-fluorophenyl) boric acid as starting materials. This gave 290mg of methyl (S) -3- (5-bromo-2-fluorophenyl) -4- (2, 7-diazaspiro [3.5] nonan-2-yl) butanoate (77-INTA) as a pale yellow oily intermediate. LC-MS ESI m/z=401.1 [ m+h ] ⁺. 150mg of a colourless oily product are obtained. LC-MS ESI m/z=547.2 [ m+h ] ⁺.

Intermediate 78-INTB: preparation of methyl (S) -3- (isoquinolin-7-yl) -4- (7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) butanoate

Step 1: see intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (DCM/meoh=10/1) to give 40mg of the product as a yellow oil. LC-MS ESI m/z=500.6 [ m+h ] ⁺.

Intermediate 79-INTB: preparation of methyl (S) -3- (3- (2-methoxyethoxy) phenyl) -4- (7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) butanoate

Step 1: see intermediate 1-INTB step 4. The crude product was purified by high performance liquid chromatography to give 180mg of the yellow solid product tert-butyl (S) -2- (4-methoxy-2- (3- (2-methoxyethoxy) phenyl) -4-carbonylbutyl) -2, 7-diazaspiro [3.5] nonane-7-carboxylate. LC-MS ESI m/z=478.7 [ m+h ] ⁺.

Step 2: see intermediate 3-INTB, procedure 2. Crude methyl (S) -3- (3- (2-methoxyethoxy) phenyl) -4- (2, 7-diazaspiro [3.5] nonan-2-yl) butanoate (79-INTA) 180mg is obtained. LC-MS ESI m/z=378.3 [ m+h ] ⁺.

Step 3: see intermediate 5-INTB, procedure 3. Purifying the crude product by high performance liquid chromatography to obtain yellow oily product 90mg.LC-MS:ESI m/z＝523.5[M+H]⁺;¹H NMR(400MHz,METHANOL-d₄)8.42(s,2H),7.34-7.24(m,2H),6.94-6.85(m,3H),6.59-6.52(m,1H),5.28-5.05(m,4H),4.17-4.11(m,2H),3.78-3.75(m,2H),3.72(s,2H),3.61(s,3H),3.57-3.53(m,4H),3.44(s,3H),3.44-3.42(m,1H),2.82-2.61(m,8H),1.97-1.87(m,6H).

Intermediate 80-INTB: preparation of methyl (S) -3- (5- (3, 5-dimethyl-1H-pyrazol-1-yl) -2-fluorophenyl) -4- (6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-2-yl) butanoate

Step 1: see intermediate 1-ITNB, step 4. Purification of the crude product by normal phase silica gel column chromatography (DCM/meoh=10/1) afforded tert-butyl (S) -2- (2- (5-bromo-2-fluorophenyl) -4-methoxy-4-carbonylbutyl) -2, 6-diazaspiro [3.4] octane-6-carboxylate as a yellow oil 480mg. LC-MS ESI m/z=485.4 [ m+h ] ⁺.

Step 2: tert-butyl (S) -2- (2- (5-bromo-2-fluorophenyl) -4-methoxy-4-carbonylbutyl) -2, 6-diazaspiro [3.4] octane-6-carboxylate (380 mg,0.8 mmol), (5-bromo-2-fluorophenyl) boronic acid (90.3 mg,0.9 mmol), palladium catalyst (66.9 mg,0.1 mmol) and cesium carbonate (765.2 mg,2.4 mmol) were dissolved in dioxane (10 ml), replaced with nitrogen three times, heated to 120℃under nitrogen and stirred for 16 hours. The reaction solution was concentrated and the crude product was purified by normal phase silica gel column chromatography (DCM/meoh=10/1) to give 200mg of tert-butyl (S) -2- (2- (5- (3, 5-dimethyl-1H-pyrazol-1-yl) -2-fluorophenyl) -4-methoxy-4-carbonylbutyl) -2, 6-diazaspiro [3.4] octane-6-carboxylate as a yellow oily product. LC-MS ESI m/z=501.5 [ m+h ] ⁺.

Step 3: see intermediate 3-INTB, procedure 2. The crude product was purified by high performance liquid chromatography to give 60mg of methyl (S) -3- (5- (3, 5-dimethyl-1H-pyrazol-1-yl) -2-fluorophenyl) -4- (2, 6-diazaspiro [3.4] octane-2-yl) butanoate (80-INTA) as a yellow oily product. LC-MS ESI m/z=401.5 [ m+h ] ⁺.

Step 4: see intermediate 5-INTB, procedure 3. The crude product was purified by high performance liquid chromatography to give 40mg of the product as a yellow oil. LC-MS ESI m/z=547.4 [ m+h ] ⁺.

Intermediate 81-INTB: preparation of methyl (S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) -5-fluorophenyl) -4- (6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-2-yl) butanoate

The preparation method and the steps are the same as those of the preparation of 80-INTB by taking (3-bromo-5-fluorophenyl) boric acid as a starting material. To give methyl (S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) -5-fluorophenyl) -4- (2, 6-diazaspiro [3.4] octane-2-yl) butanoate (81-INTA) as a pale yellow oily intermediate 60mg. LC-MS ESI m/z=401.2 [ m+h ] ⁺. 30mg of the product was obtained as a yellow oil. LC-MS ESI m/z=547.5 [ m+h ] ⁺.

Intermediate 82-INTB: preparation of methyl (S) -3- (3-bromo-5- (tert-butyl) phenyl) -4- (6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] oct-2-yl) butanoate

Tert-butyl (E) -2- (4-methoxy-4-carbonylbut-2-en-1-yl) -2, 6-diazaspiro [3.4] octane-6-carboxylate is used as starting material, and the preparation method and steps are the same as those of 76-INTB. 110mg of methyl (S) -3- (3-bromo-5- (tert-butyl) phenyl) -4- (2, 6-diazaspiro [3.4] oct-2-yl) butanoate (82-INTA) is obtained as a pale yellow oily intermediate. LC-MS ESI m/z=423.2 [ m+h ] ⁺. 120mg of the product was obtained as a yellow oil. LC-MS ESI m/z=569.5 [ m+h ] ⁺.

Intermediate 83-INTB: preparation of methyl (S) -3- (3- (1H-pyrrol-2-yl) phenyl) -4- (6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] oct-2-yl) butanoate

Step 1: see intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (DCM/meoh=100/0 to 20/1) to give benzyl (S) -2- (2- (3- (1- (tert-butoxycarbonyl) -1H-pyrrol-2-yl) phenyl) -4-methoxy-4-carbonylbutyl) -2, 6-diazaspiro [3.4] octane-6-carboxylate as a yellow solid 370mg. LC-MS ESI m/z=587.8 [ m+h ] ⁺.

Step 2: see intermediate J, procedure 2. To give 137mg of tert-butyl (S) -2- (3- (4-methoxy-4-carbonyl-1- (2, 6-diazaspiro [3.4] oct-2-yl) butan-2-yl) phenyl) -1H-pyrrole-1-carboxylate (83-INTA) as a colourless oil. LC-MS ESI m/z=454.5 [ m+h ] ⁺.

Step 3: see intermediate 5-INTB, procedure 3. The crude product was prepared by high performance liquid chromatography to give the colorless oily product tert-butyl (S) -2- (3- (4-methoxy-4-carbonyl-1- (6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] oct-2-yl) butan-2-yl) phenyl) -1H-pyrrole-1-carboxylate 80mg. LC-MS ESI m/z=600.5 [ m+h ] ⁺.

Step 4: tert-butyl (S) -2- (3- (4-methoxy-4-carbonyl-1- (6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] oct-2-yl) butan-2-yl) phenyl) -1H-pyrrole-1-carboxylate (40 mg,68 μmol) was dissolved in 5ml dcm, trifluoroacetic acid (2 mg) was added at room temperature and stirred at room temperature for 1 hour. The crude product after concentration of the reaction solution was subjected to high performance liquid chromatography to obtain 50mg of a brown solid product. LC-MS ESI m/z=500.3 [ m+h ] ⁺.

Intermediate 84-INTB: preparation of methyl (S) -3- (3-cyclopropylphenyl) -4- (6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-2-yl) butanoate

The preparation method and the steps are the same as those of 76-INTB by taking tert-butyl (E) -2- (4-methoxy-4-carbonylbut-2-en-1-yl) -2, 6-diazaspiro [3.4] octane-6-carboxylate and (3-cyclopropylphenyl) boric acid as starting materials. 160mg of methyl (S) -3- (3-cyclopropylphenyl) -4- (2, 6-diazaspiro [3.4] octane-2-yl) butanoate (84-INTA) is obtained as a pale yellow oily intermediate. LC-MS ESI m/z=329.5 [ m+h ] ⁺. 90mg of the product was obtained as a yellow oil. LC-MS ESI m/z=475.6 [ m+h ] ⁺.

Intermediate 85-INTB: preparation of methyl (S) -3- (5-bromo-2-fluorophenyl) -4- (6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-2-yl) butanoate

The preparation method and the steps are the same as those of 76-INTB by taking tert-butyl (E) -2- (4-methoxy-4-carbonylbut-2-en-1-yl) -2, 6-diazaspiro [3.4] octane-6-carboxylate and (5-bromo-2-fluorophenyl) boric acid as starting materials. 160mg of methyl (S) -3- (5-bromo-2-fluorophenyl) -4- (2, 6-diazaspiro [3.4] octane-2-yl) butanoate (85-INTA) is obtained as a pale yellow oily intermediate. LC-MS ESI m/z=385.1 [ m+h ] ⁺. 110mg of the product was obtained as a yellow oil. LC-MS ESI m/z=531.5 [ m+h ] ⁺.

Intermediate 86-INTB: preparation of methyl (S) -3- (3-cyclopropyl-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-2-yl) butanoate

Step 1: see intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (DCM: methanol=100:0-20:1) to give benzyl (S) -2- (2- (3-bromo-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4-methoxy-4-carbonylbutyl) -2, 6-diazaspiro [3.4] octane-6-carboxylate as a brown oil 400mg. LC-MS ESI m/z=595.4 [ m+h ] ⁺.

Step 2: benzyl (S) -2- (2- (3-bromo-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4-methoxy-4-carbonylbutyl) -2, 6-diazaspiro [3.4] octane-6-carboxylate (400 mg,0.7 mmol), cyclopropylboronic acid (87 mg,1 mmol), cesium carbonate (547 mg,1.7 mmol) and palladium catalyst (49 mg,0.1 mmol) were dissolved in dioxane (10 ml) in this order. Heated to 100 ℃ under nitrogen and stirred for 3 hours. The reaction solution was directly spin-dried and purified by normal phase silica gel column chromatography (DCM/meoh=100/0 to 10/1) to give the product benzyl (S) -2- (2- (3-cyclopropyl-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4-methoxy-4-carbonylbutyl) -2, 6-diazaspiro [3.4] octane-6-carboxylate 373mg as a brown oil. LC-MS ESI m/z=557.5 [ m+h ] ⁺.

Step 3: see intermediate J, procedure 2. The product methyl (S) -3- (3-cyclopropyl-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (2, 6-diazaspiro [3.4] octane-2-yl) butanoate (86-INTA) was obtained as a brown oil in 270mg. LC-MS ESI m/z=423.5 [ m+h ] ⁺.

Step 4: see intermediate 1-INTB step 3. Purification of the crude product by normal phase silica gel column chromatography (DCM (1% ammonia)/methanol=100/0-10/1) afforded 130mg as a brown oil. LC-MS ESI m/z=569.4 [ m+h ] ⁺.

Intermediate 87-INTB: preparation of methyl (S) -3- (3- (5-methyl-1H-imidazol-2-yl) phenyl) -4- (6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-2-yl) butanoate

Benzyl (E) -2- (4-methoxy-4-carbonyl but-2-alkene-1-yl) -2, 6-diazaspiro [3.4] octane-6-carboxylic acid ester and an intermediate L are used as starting materials, and the preparation method and the steps are the same as those of 83-INTB. To give 150mg of methyl (S) -3- (3- (5-methyl-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-imidazol-2-yl) phenyl) -4- (2, 6-diazaspiro [3.4] oct-2-yl) butanoate as a pale yellow oily intermediate. LC-MS ESI m/z=499.5 [ m+h ] ⁺. 100mg of the product was obtained as a yellow oil. LC-MS ESI m/z=515.4 [ m+h ] ⁺.

Intermediate 88-INTB: preparation of methyl (S) -3- (3- (1H-imidazol-2-yl) phenyl) -4- (6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] oct-2-yl) butanoate

Benzyl (E) -2- (4-methoxy-4-carbonyl but-2-alkene-1-yl) -2, 6-diazaspiro [3.4] octane-6-carboxylic acid ester and an intermediate M are used as starting materials, and the preparation method and the steps are the same as those of 83-INTB. The intermediate methyl (S) -4- (2, 6-diazaspiro [3.4] oct-2-yl) -3- (3- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-imidazol-2-yl) phenyl) butanoate (88-INTA) was obtained as a pale yellow oil in 350mg. LC-MS ESI m/z=485.4 [ m+h ] ⁺. 300mg of the product was obtained as a yellow oil. LC-MS ESI m/z=501.4 [ m+h ] ⁺.

Intermediate 89-INTB: preparation of methyl (S) -3- (5- (tert-butyl) -2-fluorophenyl) -4- (6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-2-yl) butanoate

Step 1: see intermediate 1-INTB step 4. Purification of the crude product by normal phase silica gel column chromatography (DCM/meoh=100/0-8/1) afforded the product methyl (E) -4- (6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] oct-2-yl) but-2-enoate (89-INTA) as a colourless oil 400mg. LC-MS ESI m/z=357.3 [ m+h ] ⁺.

Step 2: see intermediate 1-INTB step 4. The crude product is prepared into colorless oily product by high performance liquid chromatography 21mg.LC-MS:ESI m/z＝509.6[M+H]⁺.¹H NMR(400MHz,METHANOL-d₄)δ＝7.36-7.22(m,3H),6.99(br t,J＝9.5Hz,1H),6.52(d,J＝7.2Hz,1H),3.94(s,2H),3.73-3.46(m,9H),3.44-3.39(m,2H),3.27(s,2H),3.16-3.07(m,3H),2.83(br dd,J＝6.8,15.8Hz,1H),2.75(br t,J＝6.1Hz,2H),2.67(br dd,J＝8.0,16.0Hz,1H),2.31-2.14(m,2H),1.96-1.83(m,2H),1.39-1.26(m,9H).

Intermediate 90-INTB: preparation of methyl (S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (6- (2- (5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) ethyl) -2, 6-diazaspiro [3.4] octane-2-yl) butanoate

Step 1: see intermediate 20-INTB, procedure 1. Purification of the crude product by normal phase silica gel column chromatography (DCM/meoh=10/1) afforded tert-butyl (S) -7- (2- (2- (2- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4-methoxy-4-carbonylbutyl) -2, 6-diazaspiro [3.4] oct-6-yl) ethyl) -3, 4-dihydro-1, 8-naphthyridine-1 (2H) -carboxylate as a yellow oil 120mg. LC-MS ESI m/z=643.8 [ m+h ] ⁺.

Step 2: see intermediate 20-INTB, procedure 2. The crude product was purified by high performance liquid chromatography to give 50mg of a yellow solid product. LC-MS ESI m/z=543.5 [ m+h ] ⁺.

Intermediate 91-INTB: preparation of methyl (S) -3- (3- (tert-butyl) -5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (6- (2- (5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) ethyl) -2, 6-diazaspiro [3.4] octane-2-yl) butanoate

The intermediate 43-INTA is taken as a starting material, and the preparation method and the steps are the same as those of the preparation of 90-INTB. 400mg of the product are obtained as a yellow oil. LC-MS ESI m/z=599.6 [ m+h ] ⁺.

Intermediate 92-INTB: preparation of methyl (S) -3- (3-bromo-5- (tert-butyl) phenyl) -4- (6- (2- (5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) ethyl) -2, 6-diazaspiro [3.4] oct-2-yl) butanoate

The intermediate 82-INTA is taken as a starting material, and the preparation method and the steps are the same as those of the preparation of 90-INTB. 350mg of the product are obtained as a yellow oil. LC-MS ESI m/z=583.4 [ m+h ] ⁺.

Intermediate 93-INTB: preparation of methyl (S) -3- (5-bromo-2-fluorophenyl) -4- (6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.3] heptan-2-yl) butanoate

The preparation method and the steps are the same with 89-INTB preparation by taking methyl (E) -4- (2, 6-diazaspiro [3.3] heptane-2-yl) but-2-enoate as a starting material. The intermediate methyl (E) -4- (6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.3] heptane-2-yl) but-2-enoate (93-INTA)220mgmg.LC-MS:ESI m/z＝343.4[M+H]⁺;¹H NMR(400MHz,CDCl3)δ7.16(d,J＝7.21Hz,1H)6.81(m,1H)6.49(d,J＝7.34Hz,1H)5.92(br d,J＝15.77Hz,1H)5.75(br s,1H)3.74(s,3H)3.62(s,2H)3.58(s,4H)3.42(br s,2H)3.34(s,4H)3.16(br d,J＝4.52Hz,2H)2.72(br t,J＝6.11Hz,2H)1.91(m,2H). was obtained as a yellow oil to give 55mg of the product as a yellow oil. LC-MS ESI m/z=517.1 [ m+h ] ⁺.

Intermediate 94-INTB: preparation of methyl (3S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (1- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -1, 7-diazaspiro [4.4] nonan-7-yl) butanoate

Step 1: see intermediate 3-INTB, procedure 3. Purifying the crude product by normal phase silica gel column chromatography (100% petroleum ether-ethyl acetate=1/2) to obtain yellow oily product tert-butyl (E) -7- (4-methoxy-4-carbonyl-but-2-en-1-yl) -1, 7-diazaspiro [4.4] nonane-1-carboxylic acid ester 190mg.¹H NMR(400MHz,CDCl3)δ6.90(m,1H)5.92(br d,J＝15.65Hz,1H)3.67(s,3H)3.24-3.49(m,2H)3.12-3.23(m,2H)2.85(m,1H)2.57-2.66(m,1H)2.45-2.54(m,2H)2.06(m,1H)1.57-1.84(m,5H)1.36-1.45(s,9H).

Step 2: see intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (EA) to give tert-butyl 7- ((S) -2- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4-methoxy-4-carbonylbutyl) -1, 7-diazaspiro [4.4] nonane-1-carboxylate as a yellow oil, 140mg. LC-MS ESI m/z=497.6 [ m+h ] ⁺.

Step 3: see intermediate 3-INTB, procedure 2. The product methyl (3S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (1, 7-diazaspiro [4.4] nonan-7-yl) butanoate (94-INTA) was obtained as a pale yellow oil in 160mg. LC-MS ESI m/z=397.3 [ m+h ] ⁺.

Step 4: see intermediate 5-INTB, procedure 3. The crude product was purified by normal phase silica gel column chromatography (100% dcm-methanol=4/1) to give 200mg of the product as a yellow oil. LC-MS ESI m/z=543.6 [ m+h ] ⁺.

Intermediate 95-INTB: preparation of methyl (S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (7- (((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) amino) methyl) -3, 4-dihydroisoquinolin-2 (1H) -yl) butanoate

Step 1: pyridine-2, 6-diamine (5 g,45.8 mmol) and 1, 3-tetramethoxypropane (7.5 g,45.8 mmol) were dissolved in phosphoric acid (50 ml), and the mixture was heated to 70℃under nitrogen and stirred for 2 hours. The ph=10 of the reaction was adjusted with 5M aqueous sodium hydroxide at 0 ℃, the filter cake was filtered and washed with DCM (50 ml x 3), the resulting solution was further extracted with DCM (200 ml x 3), the organic phase was washed with saturated brine (100 ml x 2), dried over Na ₂SO₄, filtered and spun dry to give the crude product. The crude product was purified by normal phase silica gel column chromatography (DCM/meoh=10/1) to give 300mg of 1, 8-naphthyridin-2-amine as a red solid. LC-MS ESI m/z=146.3 [ m+h ] ⁺.

Step 2: see methods for the preparation of intermediate A-2 to give 290mg of crude 5,6,7, 8-tetrahydro-1, 8-naphthyridin-2-amine. LC-MS ESI m/z=151.4 [ m+h ] ⁺.

Step 3: see intermediate 3-INTB, procedure 2. 2.3g of crude 7-bromo-1, 2,3, 4-tetrahydroisoquinoline was obtained. LC-MS ESI m/z=215.1 [ m+h ] ⁺.

Step 4: see intermediate 6-INTB, procedure 1. Purification of the crude product by normal phase silica gel column chromatography (PE/ea=1/1) afforded the product methyl (E) -4- (7-bromo-3, 4-dihydroisoquinolin-2 (1H) -yl) but-2-enoate as a yellow oil 630mg. LC-MS ESI m/z=312.3 [ m+h ] ⁺.

Step 5: see intermediate 1-INTB step 4. Purification of the crude product by normal phase silica gel column chromatography (PE/ea=1/1) afforded the product methyl (S) -4- (7-bromo-3, 4-dihydroisoquinolin-2 (1H) -yl) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) butanoic acid ester as a yellow oil, 600mg. LC-MS ESI m/z=481.6 [ m+h ] ⁺.

Step 6: referring to the preparation of intermediate D-4, the crude product was purified by normal phase silica gel column chromatography (PE/ea=1/1) to give 340mg of methyl (S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (7-vinyl-3, 4-dihydroisoquinolin-2 (1H) -yl) butanoate as a yellow oil. LC-MS: ES m/z=452.4 [ m+na ] ⁺.

Step 7: referring to the preparation method of intermediate D-6, the crude product is purified by high performance liquid chromatography to obtain 70mg of methyl (S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (7-formyl-3, 4-dihydroisoquinolin-2 (1H) -yl) butanoate (95-INTA) as a yellow oily product. LC-MS ESI m/z=432.0 [ m+h ] ⁺.

Step 8: see intermediate 1-INTB step 3. The crude product was purified by high performance liquid chromatography to give a yellow solid product 10mg. LC-MS ESI m/z=565.4 [ m+h ] ⁺.

Intermediate 96-INTB: preparation of methyl (S) -4- (5, 5-difluoro-7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) -3- (5- (3, 5-dimethyl-1H-pyrazol-1-yl) -2-fluorophenyl) butanoate

The intermediate tert-butyl (E) -5, 5-difluoro-2- (4-methoxy-4-carbonyl-but-2-en-1-yl) -2, 7-diazaspiro [3.5] nonane-7-carboxylate is obtained by using tert-butyl 5, 5-difluoro-2, 7-diazaspiro [3.5] nonane-7-carboxylate as a starting material according to the preparation method of the intermediate 3-INTB step 3, and the subsequent preparation method and steps of the intermediate are the same as those of 80-INTB. To give the product methyl (S) -4- (5, 5-difluoro-2, 7-diazaspiro [3.5] nonan-2-yl) -3- (5- (3, 5-dimethyl-1H-pyrazol-1-yl) -2-fluorophenyl) butanoic acid ester (96-INTA) as a yellow oil (60 mg). LC-MS ESI m/z=451.2 [ m+h ] ⁺. 40mg of the product was obtained as a yellow oil. LC-MS ESI m/z=597.3 [ m+h ] ⁺.

Intermediate 97-INTB: preparation of methyl (S) -3- (3- (tert-butyl) -5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (5, 5-difluoro-7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) butanoate

Step 1: see intermediate 1-INTB step 4. The crude product was purified by high performance liquid chromatography to give a yellow solid product 10mg. LC-MS ESI m/z=635.4 [ m+h ] ⁺.

Intermediate 98-INTB: preparation of methyl (S) -3- (3-bromo-5- (tert-butyl) phenyl) -4- (5, 5-difluoro-7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) butanoate

Step 1: see intermediate 1-INTB step 4. The crude product was purified by high performance liquid chromatography to give a yellow solid product 10mg. LC-MS ESI m/z=605.2 [ m+h ] ⁺.

Intermediate 99-INTB: preparation of methyl (S) -3- (3-bromophenyl) -4- (5, 5-difluoro-7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) butanoate

Step 1: see intermediate 1-INTB step 4. The crude product was purified by high performance liquid chromatography to give a yellow solid product 10mg. LC-MS ESI m/z=563.2 [ m+h ] ⁺.

Intermediate 100-INTB: preparation of methyl (S) -3- (5-bromo-2-fluorophenyl) -4- (5, 5-difluoro-7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) butanoate

Step 1: see intermediate 1-INTB step 4. The crude product was purified by high performance liquid chromatography to give a yellow solid product 10mg. LC-MS ESI m/z=581.2 [ m+h ] ⁺.

Preparation of methyl (S) -4- (5, 5-difluoro-7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) -3- (3- (2-methoxyethoxy) phenyl) butanoate as intermediate 101-INTB

Step 1: see intermediate 1-INTB step 4. The crude product was purified by high performance liquid chromatography to give a yellow solid product 10mg. LC-MS ESI m/z=559.3 [ m+h ] ⁺.

Intermediate 102-INTB: preparation of methyl (S) -4- (5, 5-difluoro-7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) -3- (5- (3, 5-dimethyl-1H-pyrazol-1-yl) pyridin-3-yl) butanoate

Step 1: see intermediate 1-INTB step 4. The crude product was purified by high performance liquid chromatography to give 50mg of the yellow solid product methyl (S) -3- (5-bromopyridin-3-yl) -4- (5, 5-difluoro-7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) butanoate. LC-MS ESI m/z=564.2 [ m+h ] ⁺.

Step 2: see intermediate 44-INTB, procedure 2. The crude product was purified by high performance liquid chromatography to give a yellow solid product 15mg. LC-MS ESI m/z=580.3 [ m+h ] ⁺.

Intermediate 103-INTB: preparation of methyl (S) -4- (5, 5-difluoro-7- (2- (5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) ethyl) -2, 7-diazaspiro [3.5] nonan-2-yl) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) butanoate

Step 1: see intermediate 20-INTB, procedure 1. The product tert-butyl 7- (2- (2- (tert-butoxycarbonyl) -5, 5-difluoro-2, 7-diazaspiro [3.5] nonan-7-yl) ethyl) -3, 4-dihydro-1, 8-naphthyridine-1 (2H) -carboxylate was obtained as a yellow oil. LC-MS ESI m/z=523.3 [ m+h ] ⁺.

Step 2: see intermediate 3-INTB, procedure 2. The product 7- (2- (5, 5-difluoro-2, 7-diazaspiro [3.5] nonan-7-yl) ethyl) -1,2,3, 4-tetrahydro-1, 8-naphthyridine was obtained as a yellow oil. LC-MS ESI m/z=323.3 [ m+h ] ⁺.

Step 3: see intermediate 3-INTB, procedure 3. The product methyl (E) -4- (5, 5-difluoro-7- (2- (5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) ethyl) -2, 7-diazaspiro [3.5] nonan-2-yl) but-2-enoate (103-INTA) was obtained as a yellow oil. LC-MS ESI m/z=421.2 [ m+h ] ⁺.

Step 4: see intermediate 1-INTB step 4. A pale yellow solid product was obtained. LC-MS ESI m/z=593.3 [ m+h ] ⁺.

Intermediate 104-INTB: preparation of methyl (3S) -3- (3- (tert-butyl) -5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (5-fluoro-7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) butanoate

Step 1: see intermediate 1-INTB step 4. The crude product was purified by high performance liquid chromatography to give 50mg of a yellow solid product. LC-MS ESI m/z=617.4 [ m+h ] ⁺.

Intermediate 105-INTB: preparation of methyl (3S) -3- (3-bromo-5- (tert-butyl) phenyl) -4- (5-fluoro-7- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 7-diazaspiro [3.5] nonan-2-yl) butanoate

Step 1: see intermediate 1-INTB step 4. The crude product was purified by high performance liquid chromatography to give 50mg of a yellow solid product. LC-MS ESI m/z=601.2 [ m+h ] ⁺.

Intermediate 106-INTB: preparation of methyl (3S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (5-fluoro-7- (2- (5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) ethyl) -2, 7-diazaspiro [3.5] nonan-2-yl) butanoate

The preparation method and the steps are the same as those of 103-INTB by taking tert-butyl 5-fluoro-2, 7-diazaspiro [3.5] nonane-2-carboxylate as a starting material. To give the yellow oily product methyl (E) -4- (5-fluoro-7- (2- (5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) ethyl) -2, 7-diazaspiro [3.5] nonan-2-yl) but-2-enoate (106-INTA) 60mg. LC-MS ESI m/z=403.2 [ m+h ] ⁺. 40mg of the product was obtained as a yellow oil. LC-MS ESI m/z=575.3 [ m+h ] ⁺.

Intermediate 107-INTB: preparation of methyl (S) -4- (8, 8-difluoro-6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] oct-2-yl) -3- (5- (3, 5-dimethyl-1H-pyrazol-1-yl) -2-fluorophenyl) butanoate

Preparation of intermediate 107-INTA: the preparation method and the steps are the same as those of 103-INTA by taking tert-butyl 8, 8-difluoro-2, 6-diazaspiro [3.4] octane-2-carboxylate as a starting material.

Step 1: see intermediate 1-INTB step 4. The crude product was purified by high performance liquid chromatography to give 50mg of the yellow solid product methyl (S) -3- (5-bromo-2-fluorophenyl) -4- (8, 8-difluoro-6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-2-yl) butyrate. LC-MS ESI m/z=567.2 [ m+h ] ⁺.

Step 2: see intermediate 80-INTB, procedure 2. The crude product was purified by normal phase silica gel column chromatography (DCM/meoh=10/1) to give 20mg of the product as a yellow oil. LC-MS ESI m/z=583.3 [ m+h ] ⁺.

Intermediate 108-INTB: preparation of methyl (S) -3- (3- (tert-butyl) -5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (8, 8-difluoro-6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-2-yl) butanoate

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Step 1: see intermediate 1-INTB step 4. The crude product was purified by high performance liquid chromatography to give 50mg of a yellow solid product. LC-MS ESI m/z=621.4 [ m+h ] ⁺.

Intermediate 109-INTB: preparation of methyl (S) -3- (3-bromo-5- (tert-butyl) phenyl) -4- (8, 8-difluoro-6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-2-yl) butanoate

Step 1: see intermediate 1-INTB step 4. The crude product was purified by high performance liquid chromatography to give 50mg of a yellow solid product. LC-MS ESI m/z=605.4 [ m+h ] ⁺.

Intermediate 110-INTB: preparation of methyl (S) -4- (8, 8-difluoro-6- (2- (5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) ethyl) -2, 6-diazaspiro [3.4] octane-2-yl) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) butanoate

The preparation method and the steps are the same as those of 103-INTB by taking tert-butyl 8, 8-difluoro-2, 6-diazaspiro [3.4] octane-2-carboxylate as a starting material. To give 60mg of methyl (E) -4- (8, 8-difluoro-6- (2- (5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) ethyl) -2, 6-diazaspiro [3.4] oct-2-yl) but-2-enoate (110-INTA) as a yellow oil. LC-MS ESI m/z=407.2 [ m+h ] ⁺. 40mg of the product was obtained as a yellow oil. LC-MS ESI m/z=579.3 [ m+h ] ⁺.

Intermediate 111-INTB: preparation of methyl (3S) -3- (3- (tert-butyl) -5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (8-fluoro-6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-2-yl) butanoate

The preparation method and the steps are the same as those of 67-INTB by taking tert-butyl 8-fluoro-2, 6-diazaspiro [3.4] octane-6-carboxylate as a starting material. 100mg of methyl (3S) -3- (3- (tert-butyl) -5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (8-fluoro-2, 6-diazaspiro [3.4] octane-2-yl) butanoate (111-INTA) was obtained as a pale yellow oily intermediate. LC-MS ESI m/z=457.3 [ m+h ] ⁺. 85mg of the product are obtained as a yellow oil. LC-MS ESI m/z=603.4 [ m+h ] ⁺.

Intermediate 112-INTB: preparation of methyl (3S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (8-fluoro-6- (2- (5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) ethyl) -2, 6-diazaspiro [3.4] octane-2-yl) butanoate

Step 1: see intermediate 1-INTB preparation step 4. The crude product was purified by high performance liquid chromatography to give 50mg of a yellow solid product. LC-MS ESI m/z=561.3 [ m+h ] ⁺.

Intermediate 113-INTB: preparation of methyl (S) -3- (3- (tert-butyl) -5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (8, 8-difluoro-2- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-6-yl) butanoate

The preparation method and the steps are the same with the preparation of 76-INTB by taking tert-butyl (E) -8, 8-difluoro-6- (4-methoxy-4-carbonylbut-2-en-1-yl) -2, 6-diazaspiro [3.4] octane-2-carboxylate and intermediate I as starting materials. This gave 290mg of methyl (S) -3- (3- (tert-butyl) -5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (8, 8-difluoro-2, 6-diazaspiro [3.4] oct-6-yl) butanoate (113-INTA) as a pale yellow oily intermediate. LC-MS ESI m/z=475.3 [ m+h ] ⁺. 150mg of a yellow oily product was obtained. LC-MS ESI m/z=621.4 [ m+h ] ⁺.

Intermediate 114-INTB: preparation of methyl (S) -4- (8, 8-difluoro-2- (2- (5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) ethyl) -2, 6-diazaspiro [3.4] octane-6-yl) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) butanoate

The preparation method and the steps are the same as those of 103-INTB by taking tert-butyl 8, 8-difluoro-2, 6-diazaspiro [3.4] octane-6-carboxylate as a starting material. To give 60mg of methyl (E) -4- (8, 8-difluoro-2- (2- (5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) ethyl) -2, 6-diazaspiro [3.4] oct-6-yl) but-2-enoate (114-INTA) as a yellow oil. 40mg of the product was obtained as a yellow oil. LC-MS ESI m/z=579.3 [ m+h ] ⁺.

Intermediate 115-INTB: preparation of methyl (3S) -3- (3- (tert-butyl) -5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (8-fluoro-2- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-6-yl) butanoate

The preparation method and the steps are the same as those of 67-INTB by taking tert-butyl 8-fluoro-2, 6-diazaspiro [3.4] octane-2-carboxylate as a starting material. 110mg of the intermediate methyl (3S) -3- (3- (tert-butyl) -5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (8-fluoro-2, 6-diazaspiro [3.4] oct-6-yl) butanoate (115-INTA) was obtained as a pale yellow oil. LC-MS ESI m/z=603.4 [ m+h ] ⁺.

Intermediate 116-INTB: preparation of methyl (3S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (8-fluoro-2- (2- (5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) ethyl) -2, 6-diazaspiro [3.4] octane-6-yl) butanoate

The preparation method and the steps are the same as those of 103-INTB by taking tert-butyl 8-fluoro-2, 6-diazaspiro [3.4] octane-6-carboxylate as a starting material. To give 60mg of methyl (E) -4- (8-fluoro-2- (2- (5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) ethyl) -2, 6-diazaspiro [3.4] oct-6-yl) but-2-enoate (116-INTA) as a yellow oil. LC-MS ESI m/z=561.3 [ m+h ] ⁺.

Intermediate 117-INTB: preparation of methyl (S) -3- (3-cyclopropyl-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (8, 8-difluoro-6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-2-yl) butanoate

Step 1: the preparation method of step 4, see intermediate 1-INTB, uses tert-butyl (E) -8, 8-difluoro-2- (4-methoxy-4-carbonylbut-2-en-1-yl) -2, 6-diazaspiro [3.4] octane-6-carboxylate as starting material. The crude product was purified by flash column chromatography to give intermediate tert-butyl (S) -2- (2- (3-cyclopropyl-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4-methoxy-4-carbonylbutyl) -8, 8-difluoro-2, 6-diazaspiro [3.4] octane-6-carboxylate 30mg. LC-MS ESI m/z=559 [ m+h ] +.

Step 2: see intermediate 3-INTB, procedure 2. The crude 30mg was used directly in the next step. LC-MS ESI m/z=459 [ m+h ] ⁺.

Step 3: methyl (S) -3- (3-cyclopropyl-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (8, 8-difluoro-2, 6-diazaspiro [3.4] octane-2-yl) butanoate (30 mg,0.1 mmol) was dissolved in DCM (1.5 ml), A-3 (16 mg,0.1 mmol), octadecyl trimethyl ammonium bromide (STAB, 42mg,0.2 mmol) and one drop of acetic acid were added in this order to the solution, and stirring was carried out at room temperature for 2 hours. LCMS monitored completion of the reaction, water was added, DCM extracted and the organic phase dried over Na ₂SO₄ to give 29mg of product. LC-MS ESI m/z=605 [ m+h ] ⁺.

Intermediate 118-INTB: preparation of methyl (S) -3- (3-cyclobutyl-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (8, 8-difluoro-6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-2-yl) butanoate

See preparation of 117-INTB. LC-MS ESI m/z=619.4 [ m+h ] ⁺.

Intermediate 119-INTB: preparation of methyl (S) -3- (3-cyclopentyl-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (8, 8-difluoro-6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-2-yl) butanoate

See preparation of 117-INTB. LC-MS ESI m/z=633 [ m+h ] ⁺.

Intermediate 120-INTB: preparation of methyl (S) -3- (3-cyclohexyl-5- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (8, 8-difluoro-6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-2-yl) butanoate

See preparation of 117-INTB. LC-MS ESI m/z=633.7 [ m+h ] ⁺.

Intermediate 121-INTB: preparation of methyl (S) -3- (5- (tert-butyl) - [1,1' -biphenyl ] -3-yl) -4- (8, 8-difluoro-6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-2-yl) butanoate

See preparation of 117-INTB. LC-MS ESI m/z=603 [ m+h ] ⁺.

Intermediate 122-INTB: preparation of methyl (S) -3- (3, 5-di-tert-butylphenyl) -4- (8, 8-difluoro-6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-2-yl) butanoate

See preparation of 117-INTB. LC-MS ESI m/z=583 [ m+h ] ⁺.

Synthesis of examples 1 to 14, examples 19 to 21, examples 23 to 25, examples 29 to 54 and examples 62 to 66

Summary of the synthesis method: the ester intermediate of the example was dissolved in a solvent of methanol and water (volume ratio 5:1) and lithium hydroxide (5 equivalents) was added at room temperature. After stirring at room temperature for about 16 hours, LCMS detects complete reaction of the raw materials, concentrates the reaction mixture, and prepares (Column:Kromasil C18 150*30mm*5um;Condition:water(0.2％ammonium hydroxide)-ACN;Begin B:5;End B:100;Gradient Time(min):20;100％B Hold Time(Time):5;Flow Rate(ml/min):20;Detection wavelength:220nm and 254nm) purification of the obtained crude product by high performance liquid chromatography.

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Example 2: preparation of (S) -3- (3, 5-dimethyl-1H-pyrazol-1-yl) phenyl) -4- (6- ((5, 6,7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl) -2, 6-diazaspiro [3.4] octane-2-yl) butanoic acid (Compound 2)

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Intermediate 2-INTB (29 mg,0.1 mmol) was dissolved in 4N HCl/dioxane (1 mL) under nitrogen at room temperature and reacted for 2h. LCMS shows that the reaction is finished, the reaction solution is concentrated, and the crude product is purified by Prep-HPLC to obtain a white solid product 10mg.LC-MS:ESI m/z:515.3[M+H]⁺;¹HNMR(400MHz,CD₃OD)δ7.48(t,J＝8.0Hz,1H),7.36-7.30(m,4H),6.53(d,J＝7.2Hz,1H),6.07(s,1H),3.85(d,J＝10.8Hz,6H),3.44-3.40(m,3H),3.38-3.30(m,2H),3.19(s,2H),3.01(t,J＝6.8Hz,2H),2.77-2.70(m,3H),2.63-2.58(m,1H),2.27(s,3H),2.25(s,3H),2.27-2.23(m,2H),1.91-1.85(m,2H).

Synthesis of examples 15 to 18, example 22, examples 26 to 28 and examples 55 to 61

Summary of the synthesis method: sodium hydroxide is dissolved in 50% hydroxylamine water solution, and the temperature is reduced to 0 ℃ under the protection of nitrogen. After that, the ester intermediate was dissolved in methanol/THF (volume ratio 1:1) and slowly added dropwise to the above reaction solution, and stirring was continued at room temperature for 2 hours. LCMS detects complete reaction of the starting materials, concentrates the reaction mixture, and purifies the crude product by high performance liquid chromatography (Column:Kromasil C18 150*30mm*5um;Condition:water(0.2％ammonium hydroxide)-ACN;Begin B:5;End B:100;Gradient Time(min):20;100％B Hold Time(Time):5;Flow Rate(ml/min):20;Detection wavelength:220nm and 254nm) to obtain the product.

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Effect example 1: binding assays of Compounds with αvβ1, αvβ6 and αvβ8 proteins

1.1 Principle of fluorescence anisotropy test (Fluorescence Anisotropy)

The principle is based on the principle that the fluorescence polarization values in the horizontal direction and the vertical direction are calculated for relevant analysis by detecting the change of molecular weight before and after interaction of small molecules marked by fluorescein and other molecules. If the binding equilibrium between the fluorescent-labeled small molecule and the large molecule is established, the fluorescent-labeled small molecule moves slowly when excited, and the measured fluorescence polarization value increases. If the binding between the fluorescent-labeled small molecule and the large molecule is replaced by other ligands, the rotation or turnover speed of the fluorescent-labeled small molecule and the large molecule in a free state can be increased, the emitted light is depolarized relative to the excitation light plane, and the measured polarized light value is reduced, so that the fluorescence anisotropy of the sample can be calculated.

1.2 Experimental methods

40Ul of experimental reaction system is provided with multiple holes, and the alpha V beta 6 buffer solution is as follows: 50mM HEPES PH7.4,150mM Nacl,0.5mM CHAPS,0.4mM MgCl2, αvβ1 and αvβ8 buffers were: 50mM HEPES PH7.4,150mM Nacl,0.5mM CHAPS,0.1mM MnCl2. The working concentration of the fluorogenic substrate was 1nM, and the working concentrations of αvβ6 (recombinant human integrin produced by R & D systems, cat# 3817-AV) and αvβ8 (recombinant human integrin produced by R & D systems, cat# 4135-AV) were 8nM; the working concentration of alpha V beta 1 (product number 6579-AVB) is 12.5nM; the initial concentration of the compound was 1uM and 100nM, the final concentration of DMSO was two thousandths, and the Anisotropy values were determined after mixing all the components in 384 well plates (burning cat# CLS 3575) at room temperature (about 24 ℃) for 2 hours, the test instrument was a BioTek brand SYNERGY neo2 microplate reader, the specification was 4815 nM, and the emision was 530nM. Wells with buffer only were used as a blank for system readings. The average value of the compound wells is taken to calculate the inhibition rate, and the IC ₅₀ value of the compound with the inhibition rate of more than 50% under the condition of 100nM is measured.

PLN-74809 and GSK-3008348 were chosen as control compounds for this experiment. PLN-74809 (CAS: 2376257-44-0) and GSK-3008348 (CAS: 1629249-33-7) are two very potential integrin ligand molecules. The experimental treatment method is the same as that described above.

1.3 Calculation method

Inhibition ratio = (C-F)/(C-B) 100%

Wherein, C is Anisotropy value of complete combination of fluorogenic substrate and protein, F is Anisotropy value under corresponding concentration of compound, and B is Anisotropy background value of fluorogenic substrate. The corresponding IC ₅₀ values (Table 2) were calculated by S-curve with compound concentrations and corresponding inhibition values (Table 1). Each example was divided into three steps according to IC ₅₀ values, a: IC ₅₀<100nM;b：IC₅₀＝100-1000nM;c：IC₅₀ >1000nM. The a-grade example has excellent inhibitory activity on the target subtype; the embodiment of the b grade has stronger inhibition activity; the inhibitory activity of the c-stage examples is general.

The subtype selectivity calculation and evaluation method is as follows:

Fold=IC ₅₀ (subtype 1)/IC ₅₀ (subtype 2)

The examples are divided into three steps according to the Fold ratio, A: fold >4.0; b: fold = 1.0-4.0; c: fold <1.0. The a-stage example has strong selectivity for subtype 2 over subtype 1; gear B embodiment has medium selectivity; gear C is not selective.

1.4 Experimental results

TABLE 1 evaluation of the Effect (inhibition ratio) of the Compounds of the invention on the inhibition Activity of αvβ1, αvβ6 and αvβ8 subtypes

Note a: the inhibition rate is more than or equal to 50 percent; b: inhibition rate <50%; N/A indicates that no detection was made at this concentration

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TABLE 2 evaluation of the Selective Effect of the Compounds of the invention on αvβ1, αvβ6 and αvβ8 subtype inhibitory Activity (IC ₅₀ value Range) of the Compounds on αvβ1, αvβ8, respectively, on αvβ6

And (b) pouring a: IC50<100nM; b: i50=100-1000 nM; c: IC50>1000nM; a: fold >4.0, indicating that the compounds have high selectivity for a certain subtype; b: fold = 1.0-4.0, indicating that the compound is moderately selective for a certain subtype; c: fold <1.0, indicating that the compound is not selective for a subtype; N/A indicates that no detection is made;

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As shown in tables 1 and 2, the examples show different inhibitory effects on one, two or three of the αvβ1, αvβ6 and αvβ8 subtypes, respectively, and in particular show excellent inhibitory effect and selectivity on the αvβ6 single subtype and on the αvβ1 and αvβ6 two subtypes, such as examples 43, 44, 71, 72, 96-103 (type aaaCA) and the like, on both αvβ1 and αvβ6 and on αvβ8, and the selectivity is far superior to that of the positive reference PLN-74809. Examples 1, 5, 49-50, 52-54, 75, 77-78, etc. are excellent in inhibitory activity and specific selectivity for αvβ6. At the same time, compounds having inhibitory effects on all of αvβ1, αvβ6 and αvβ8 and not having selectivity were also selected, and were comparable to the activity and selectivity of positive reference GSK3008348, as in examples 82, 92, 113, 115, etc. Thus, the compounds of the present invention have excellent inhibitory activity and selectivity against integrin αvβ1, αvβ6 and αvβ8 subtypes, which are advantageous over the same type of molecules.

Effect example 2: degradation promoting effect of compound on fibrin alpha-SMA, fibronectin

2.1 Purpose of experiment

Fibroblasts (Fb) are the most predominant effector cells in fibrotic diseases, and when activated, undergo functional and phenotypic changes, transform into myofibroblasts (Myofibroblast, MFb), have contractile potential and strong collagen synthesis capacity, and are the main cellular processes for inducing fibrotic lesions. The phenotype of this cell activation is characterized by the expression of alpha smooth muscle actin (alpha-Smooth muscle actin, alpha-SMA) and Fibronectin (FN), so that the inhibition of fibroblast activation by integrin ligands can be verified by the variation of the expression levels of these two proteins.

2.2 Experimental materials and methods

Cell lines: human fetal lung fibroblasts (HFL-1); TGF- β: r & D company; culture medium: hyclone company F12K medium; serum: gibco company; fibrinectin antibody: abcam corporation, cat No.: ab45688; alpha-SMA antibody: abcam corporation, cat No.: ab124964.

HFL-1 cells were inoculated in F12K medium containing 10% FBS for routine culture. The different compounds are treated by inoculating 6-hole plates according to 1X 106 cells/hole, culturing for 24 hours under normal culture conditions, changing into serum-free culture medium, pretreating with excipient DMSO and different concentrations of compounds (3 concentrations of each compound and 3 compound holes of each concentration) for 2 hours, adding 10ng/ml of TGF-beta recombinant protein for 24 hours, and recovering cells after treatment for protein immunoblotting (Western blotting, WB) of the fibrinectin and alpha-SMA detection.

Pirfenidone (Pirfenidone) and GSK-3008348 were selected as control compounds for this experiment. Pirfenidone (CAS: 53179-13-8) is a widely used anti-fibrotic drug, and GSK-3008348 (CAS: 1629249-33-7) is a very potential integrin ligand molecule developed by the company Gelanin Smith (GSK Plc.). The concentration of pirfenidone drug is 0.5mg/mL, the concentration of GSK-3008348 drug is the same as that of the tested compound (3 concentrations, 3 compound wells for each concentration), and the treatment method is the same.

2.3WB Experimental procedure

Protein extraction: cells were washed with PBS, 100ul RIPA lysate was added to each well, scraped off with a cell scraper and collected in an EP tube, ice-bath for 30min, after sufficient lysis, 13000g was centrifuged for 10min and the supernatant was placed in a fresh EP tube. Protein quantification was performed according to BCA protein concentration assay kit (enhancement) instructions. According to the quantitative result, the sample is diluted to make the protein concentration consistent. Adding loading buffer solution, mixing, heating in boiling water bath for 3-5 min, denaturing protein thoroughly, cooling to room temperature, and loading.

And (3) glue preparation: injecting separating gel, reserving 1/5 space, and filling with deionized water. Standing at room temperature for 1 hour, pouring deionized water, sucking the deionized water by using water-absorbing filter paper, and filling the deionized water with concentrated glue; then quickly inserting a comb, taking care of removing bubbles by using the residual concentrated glue, and standing at room temperature for 30 min.

Sample addition/electrophoresis/transfer: the total protein loading of each sample was 25ug and was applied to the sample wells using a loading gun. Electrophoresis: 70V runs to the upper layer of the separation gel at the lower layer of the concentrated gel, and 120V runs out. Transferring: firstly, soaking a 0.45um PVDF film, filter paper and sponge in a transfer buffer solution, and marking the PVDF film; after electrophoresis, selecting proper glue; and (3) preparing a sandwich of the transfer film, placing the sandwich on a rack, filling with an electrotransfer buffer solution, and carrying out constant current 230mA for 120min.

Antigen blocking and antibody incubation: membranes were washed with PBS, western-blocking solution (5% non-mill) was added and incubated for 1h at room temperature with shaking. Incubation resistance: adding primary antibody alpha-SMA, fibronectin and internal reference Actin respectively, and standing overnight at 4deg.C; washing the primary antibody, adding TBST, and washing 3 times at room temperature for 5min each time. Secondary antibody incubation: the corresponding secondary antibody was added and incubated with shaking at room temperature for 1h. The secondary antibody was washed, TBST was added, and the mixture was subjected to shaking table washing at room temperature for 4 times, each for 6min. Finally, the fluorescent imager detects, and the optical density value of the protein band is quantitatively read. And comparing the expression conditions of the target proteins by taking the optical density value of the actin strip as an internal reference.

Because the molecular weight of the alpha-SMA is similar to that of the internal reference protein, an antibody stripping method (Antibody Stripping) is adopted in the experiment, firstly, the alpha-SMA protein is detected, then, a stripping buffer solution is used for removing the alpha-SMA primary antibody, and then, the Actin protein is detected. The incubation method for both antibodies was as above.

2.4 Experimental results:

Examples 1,2, 7, 15, 25 have significant degradation promotion of fibrin α -SMA, fibronectin (as shown in figure 1). The expression level of alpha-SMA protein is obviously reduced in examples 2, 7, 15 and 25, the alpha-SMA protein has excellent degradation promotion effect on the fibrotic protein alpha-SMA, is obviously superior to positive reference pirfenidone and GSK-3008348, and shows excellent anti-fibrosis effect; examples 1,2, 7 and 25 significantly reduced the expression level of the fibrinectin protein, and had excellent degradation promoting effect on the fibrinectin protein, which was also superior to positive reference pirfenidone and GSK-3008348. Examples 2, 7 and 25 all have excellent degradation promotion effect on two proteins of alpha-SMA and fibrauretin, and the effect is obviously superior to that of positive reference GSK-3008348 and pirfenidone. The ligand of the integrins alpha v beta 1, 6 and 8 has obvious inhibition effect on the secretion of alpha-SMA protein, the fibrinectin protein and the activation of the fibroblasts, and has potential of becoming an anti-fibrosis drug.

Effect example 3: liver microsomal metabolic stability of Compounds in humans, rats, mice, dogs, monkeys

3.1 Preparation of solutions

1Mg of test compound (substrate) was weighed, dissolved in DMSO to 10mM, and diluted to 10. Mu.M in 0.6% acetonitrile-water solution. A liver microsome assay system was formulated in which the NADPH generating system, including only glucose-6-phosphate, NADP+, glucose-6-phosphate dehydrogenase, was prepared as shown in Table 3 below.

TABLE 3 liver microsome assay system (total volume 200. Mu.L)

Reaction time 0, 10, 20, 30min

3.2 Sample handling

Substrate, liver microsomes, mgCl ₂, potassium phosphate buffer and water are added into a 96-well plate and mixed uniformly by gentle vortex. The microsomal mixture was pre-incubated in a 37℃water bath for 5min, and the reaction was initiated by addition of NADPH-generating system. After incubation for 0, 10, 20, 30min, 40. Mu.L of the reaction mixture was mixed with 100. Mu.L of acetonitrile (with internal standard 100 ng/mL) and the reaction was stopped. After the incubation, the mixture was centrifuged at 4600rpm for 10min at 4 ℃. 60 μl of the supernatant was diluted 1:1 with water and 5 μl of the sample was injected for LC-MS/MS analysis.

3.3 Chromatographic conditions

A Waters XBiridge C18 column (50 mm. Times.2.1 mm,5 μm) was used and the sample was taken in an amount of 5. Mu.L. The flow rate is 1mL/min, and the running time is 1.5min. Mobile phase a was water containing 0.1% formic acid and B was acetonitrile containing 0.1% formic acid, and the gradient elution procedure is as shown in table 4 below:

TABLE 4 gradient elution procedure

3.4 Mass Spectrometry Condition

High performance liquid chromatography tandem mass spectrometry (LC-MS/MS), model API 4500 (AB Sciex). Eluted compounds were detected using electrospray ionization (ESI) conditions, multiple Reaction Monitoring (MRM) mode. The dry gas temperature was 450 ℃, the pressure was 50psi, and the atomizing gas pressure was 20psi.

3.5 Conclusion of experiments

The metabolic stability results are shown in table 5 below.

Half-life of compounds of Table 5 tested in 5 species liver microsomes

As shown in Table 5, the compounds of the present invention were metabolically stable in human, mouse, rat, canine, and monkey liver microsomes, wherein examples 1,2, 6, 72, and 73 had half-lives in human liver microsomes superior to positive reference PLN-74809, examples 2, 6, 73, and 117 had half-lives in canine liver microsomes superior to PLN-74809, and other compounds of the present invention also had similar physicochemical properties.

Effect example 4: pharmacokinetics of Compounds in rats

4.1 Experimental method:

4.1.1 preparation of solutions and animal experiments

Male SD rats weighing about 200g were purchased from Shanghai Sipuler-BiKai laboratory animals Co., ltd, 3 per group. Each test compound was tested in two groups of tests, oral administration (PO) and intravenous Injection (IV).

The injection dose was 1mg/kg, and the test compound was prepared as a 0.1mg/mL solution using the same solvent, and tail vein injection was performed at a volume of 10 mL/kg. Blood was collected 5, 15, 30, 60, 120, 240, 480, 720, 1440min after administration, and the blood concentration was measured.

Oral doses of 10mg/kg, the test compound was first prepared as a 1mg/mL solution in solvent (DMA: solutol-HS-15: saline=10:10:80) and the stomach was irrigated at a volume of 10 mL/kg. Blood was collected at 15, 30, 60, 120, 240, 480, 720, 1440min (minutes) after administration, and the blood concentration was measured.

1Mg of test compound was precisely weighed and dissolved in a stock solution of 100. Mu.g/mL in methanol/water (MeOH: H2O=4:1, v/v). And diluting a proper amount of stock solution with a methanol/water solution to obtain serial standard solutions with mass concentrations of 20, 40, 100, 200, 400 and 1000ng/mL,2, 4, 10, 20, 40 and 100 mug/mL respectively. In addition, QC solutions with mass concentrations of 2 and 5 mug/mL are prepared for standby.

Taking 2.5 mu L of standard solution and QC solution with each concentration, dissolving in 47.5 mu L of mouse blank plasma, transferring into a 96-well plate, and preparing into standard plasma samples with mass concentrations of 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000 and 5000ng/mL and QC plasma samples with mass concentrations of 100 and 250ng/mL for later use.

4.1.2 Plasma sample treatment

The blood was collected at a volume of about 200. Mu.L each time, stored in EDTA-2K anticoagulation test tubes, and centrifuged at 5500rpm for 10min to separate plasma. 20. Mu.L of each time point test plasma sample, standard plasma sample, QC plasma sample were taken, mixed with 100. Mu.L of acetonitrile (100 ng/mL with internal standard), shaken at room temperature for 10min, and centrifuged at 3700rpm for 18min at 4 ℃. 60 μl of the supernatant was diluted 1:1 with water, shaken at room temperature for 10min, and then 5 μl of the sample was injected for LC-MS/MS analysis.

4.2. Sample measurement method

4.2.1 Chromatographic conditions

The chromatographic conditions were the same as those of effect example 3.

4.2.2 Mass Spectrometry conditions

High performance liquid chromatography tandem mass spectrometry (LC-MS/MS), model API 4500 (AB Sciex). Eluted compounds were detected using electrospray ionization (ESI) conditions, multiple Reaction Monitoring (MRM) mode. The ion source temperature was 450 ℃, the spray gas pressure and the auxiliary heating gas pressure were 50psi, the curtain gas pressure was 20psi, and the collision cell exit voltage was 13.0V. The declustering voltages of the internal standard and the tested compound are 79, 130 and 135eV respectively, and the collision energies are 19, 24 and 41eV respectively.

4.3 Conclusion of experiments

The pharmacokinetic results are shown in table 6 below.

TABLE 6 pharmacokinetic parameters of examples 71 and 72 in rats

As shown in table 6, the end elimination half-lives of the rats of examples 71 and 72 were 1.5 and 0.4 hours, respectively, after intravenous administration, and the injection AUC _INF was 4644h nm and 6511h nm, respectively. After gastric administration, the elimination half-life of the two ends is 3.0 and 0.7 hours respectively, the peak reaching time is 0.5 and 0.4 hours respectively, the peak reaching concentration is 4632 and 15946nM respectively, the oral AUC _INF is 12994h nM and 31406h nM respectively, the absorption of the rat is good, the oral bioavailability is 28% and 48% respectively, and the result is obviously better than the oral bioavailability of positive reference PLN-74809 (the actual measurement data of the rat is only 2%). Other compounds of the invention also have similar physicochemical properties, have excellent pharmaceutical potential and excellent in vivo metabolic characteristics.

Effect example 5: effect of compound on bleomycin-induced Idiopathic Pulmonary Fibrosis (IPF) in rats

5.1 Experimental method:

On Day 1 (Day 1), 3mg/kg bleomycin (purchased from japan chemical Co., ltd.) was administered to rats (SD) under anesthesia via an endotracheal tube, and IPF lesions were induced in the left lung, and a rat unilateral pulmonary fibrosis model was established. Continuous dosing was performed once daily on days 2 to 15. All rats were euthanized the next Day after the last dose (Day 16), and whole lungs were harvested after heart perfusion and fixed in 10% formalin. Paraffin embedding was then performed and precision sectioning was performed using a microtome (RM 2235, lycra LEICA). The tissue sections were subsequently H & E stained and Masson Trichrome stained to assess the pathological changes in inflammatory cell infiltration and fibrosis changes due to collagen deposition, respectively.

The experiments were divided into five groups, namely, a sham-operated group (G-1), a model group (G-2), a Nidamib control group (G-3), a low dose group (G-4) and a high dose group (G-5) of example 71, as shown in Table 7 below.

Table 7 experimental grouping and dosing schedules

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Experimental detection metrics included animal body weight and histopathological scores. Animal body weights were observed and recorded 1 time a day and the body weight gain was calculated. The histopathological scoring criteria were as follows:

In the H & E stained sections, 5 fields of view were randomly dispersed in and around the lesion area, and semi-quantitative scoring was performed on the terminal bronchioles and concomitant micro-pulmonary artery lesions and inflammatory changes, respectively. And 4 indexes of bronchial injury, bronchitis, arteriole injury and arteriole inflammation are evaluated together, wherein each index is 0-3 minutes, the total score is 0-12 minutes, wherein 0 is no pathological change, and 12 is the most serious pathological change.

In Masson Trichrome-stained sections, 10 fields of 1mm ² area size were randomly selected within the lesion area and semi-quantitatively scored by pathologists under double-blind conditions according to the Ashcroft scoring system. Ashcroft scores total 0-8, wherein 0 is no fibrosis lesions and 8 is complete fibrosis.

5.2 Data analysis method:

Data were collated in Office Excel and GRAPHPAD PRISM software and data were expressed as mean±sem (standard error). Analyzing by using One-way ANOVA and T-test, wherein the analysis method selects Tukey for comparing difference significance test between groups; when comparing the two groups, the T-test is used for two-tail detection between the two groups, and when p <0.05, the two groups have significant difference.

5.3 Discussion of experimental results

5.3.1 Body weight and body weight changes

The bleomycin-induced lung injury model, the rat body weight gradually decreases to the lowest point 3-4 days after surgery and then gradually recovers, which is the normal performance of the model. The rats in the low and high dose groups of example 71 recovered significantly faster body weight (fig. 2) over 14 days of continuous administration, with a higher rate of body weight gain than the model group and significantly better than the nilamide group (fig. 3), indicating that example 71 helped improve overall animal status, helped rats recovered body weight faster, and no significant side effects were seen in pulmonary fibrosis lesions.

5.3.2 Example 71 can reduce bleomycin-induced left lung tissue bronchial, arteriolar lesions and inflammatory infiltrates.

H & E stained sections were observed under a microscope and compared for the extent of left lung bronchi, pulmonary small vessels, perialveolar lesions and inflammatory cell infiltration. As can be seen from fig. 4, the low dose group (D) and the high dose group (E) of example 71 still have a phenomenon that part of inflammatory cells invade the inside of the tissue compared with the model group (B), but the degree is already reduced, which is comparable to that of the nilamide group (C). From the analysis of fig. 5, it was also found that the injury score of both dose groups of example 71 was significantly reduced (p < 0.001) compared to the model group, comparable to the reduction of the positive reference nilamide group (p < 0.001).

As can be seen from fig. 6, the low dose group (D) and the high dose group (E) of example 71 were able to reduce the degree of inflammation of the lung tissue at the edge of the lesion, which is significantly superior to the model group (B), wherein the high dose group (E) of example 71 reduced the degree of inflammation better than the low dose group and the positive reference nilamide group (C). From the analysis of fig. 7, it was also found that the ratio of the injury score to the model group was significantly reduced for both dose groups of example 71, wherein the injury score was significantly reduced for the high dose group (p < 0.001), which was far superior to the low dose group (p < 0.01) and the positive reference nilb group (p < 0.01), which was comparable to the nilb group.

As shown in fig. 8, the high dose group (E) of example 71 had the least alveolar wall thickening, and a slight alveolar wall thickening was observed at the arrow, and the pathological change was significantly lower than that of the model group (B), and also lower than that of the low dose group (D) of example 71 and that of the nilamide group (C), and the low dose group of example 71 was comparable to that of the nilamide group. This shows that the high dose group of example 71 has an advantage over the positive reference nildanib group and the efficacy is more pronounced.

5.3.3 Example 71 can alleviate bleomycin-induced alterations in pulmonary fibrosis

The extent of deposition of fibrotic collagen in lung tissue was observed under a microscope by Masson staining the sections, and fibrosis scoring was performed. As shown in fig. 9, the degree of fibrosis in the low dose group (D) of example 71 and the high dose group (E) of example 71 was significantly reduced compared to the model group (B), and the degree of reduction in both dose groups of example 71 was superior to that of the nilamide group (C). From the lung fibrosis scores of fig. 10, it was also found that the low dose group of example 71 had significantly reduced fibrosis scores (p < 0.001), the high dose group of example 71 had significantly reduced fibrosis scores (p < 0.001), and both were superior to the positive reference nilb group fibrosis scores (p < 0.05) as compared to the model group.

Fig. 11 shows the results of statistics of fibrosis light and severe duty cycles in the sham operation group, model group, nilamide cloth group, example 71 low dose group, and example 71 high dose group, respectively. Wherein a fibrosis score of less than or equal to 3 is considered as mild; a score of greater than or equal to 4 is considered to be severe. As shown, the low dose group severe duty cycle of example 71 was significantly reduced (p < 0.001) compared to the model group, and the high dose group severe duty cycle of example 71 was significantly reduced (p < 0.001). Compared with the positive reference nilamide group, the high-dose group and the low-dose group of the example 71 have the weight ratio which is obviously lower than that of the nilamide group, and the high-dose group of the example 71 has the weight ratio which is lower than that of the low-dose group and is obviously lower than that of the nilamide group. This indicates that more than 80% of the rats in the high dose group of example 71 are mild and the efficacy is extremely pronounced. Example 71 has a significant anti-pulmonary fibrosis effect as illustrated by figures 9, 10 and 11.

FIG. 12 shows the degree of collagen deposition in alveolar tissue of rats of each group in Masson staining. As shown, the collagen deposition was the least in the low dose group (D) of example 71 and the high dose group (E) of example 71, to a significantly lower extent than in the model group (B) and the nilamide group (C). As can also be seen from fig. 13, the collagen deposition area was significantly reduced in the two dose groups (p < 0.001) compared to the model group in example 71, and the reduction was significantly better than that in the nilamide group (p < 0.05). Example 71 the reduction in collagen deposition area was more pronounced in the high dose group, indicating that example 71 was equally effective in reducing lung collagen deposition.

Effect example 6: effect of Compounds on DDC-induced Primary Sclerosing Cholangitis (PSC) in mice

6.1 Experimental methods

On day 1, mice (C57 BL/6J) were randomized according to body weight. The normal diet in all cages except the normal control group, which provided normal diet and drinking water for 3 weeks (Day 2-Day 22), was changed to an additive containing 0.1% ddc (3, 5-diethoxycarbide-1, 4-dihydro-2, 4, 6-trimethylpyridine) from Day 2 to induce Primary Sclerosing Cholangitis (PSC) in mice. The corresponding drugs were given in different groups daily for 3 consecutive weeks (Day 2-Day 22) from Day 2. The next Day after the last dose (Day 23), mice were bled, blood samples were centrifuged at 4℃and 1500 Xg, serum was isolated and stored frozen at-80 ℃. Mice were euthanized after blood collection, intact livers were isolated and weighed, then 1/2 liver lobules were excised, fixed in 10% formalin (formaldehyde) solution for 48h, and subsequently pathologically examined.

The experimental detection indexes comprise animal weight, liver weight, serum liver function indexes and histopathological scores. Animal body weights were observed and recorded 1 time per day and percent change in body weight was compared. The levels of glutamic pyruvic transaminase (ALT), alkaline phosphatase (ALP), glutamic oxaloacetic transaminase (AST), total Bile Acid (TBA), total Bilirubin (TBIL) and the like in the blood sample of the mouse were measured using an automatic biochemical analyzer.

6.2 Discussion of experimental results

After DDC ingestion in mice, liver injury is gradually aggravated, and obvious biliary cirrhosis symptoms appear. All mice fed DDC-containing feed had a different degree of weight loss and became more severe over time. The significant increase in liver weight/body weight ratio occurred in the model group, and example 71 had a significant therapeutic effect compared to the model group, indicating that example 71 has excellent anti-hepatic fibrosis, anti-cholangiocarcinoma, and anti-inflammatory effects.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims

1. A class of bicyclic derivatives of formula I, as shown in formula I, and racemates, stereoisomers, tautomers, isotopic labels, nitroxides, solvates, polymorphs, metabolites, esters, prodrugs or pharmaceutically acceptable salts thereof:

wherein,

x is an oxygen atom or a nitrogen atom;

Is a single bond or a double bond;

n is 0,1, 2 or 3;

2. The bicyclic derivative of formula I according to claim 1,Is a substituted or unsubstituted spirocyclic ring, as shown in formula Ia:

A is a ring selected from the group consisting of: a substituted or unsubstituted quaternary heteroalkyl ring, a substituted or unsubstituted five-membered heteroalkyl ring, a substituted or unsubstituted six-membered heteroalkyl ring, a substituted or unsubstituted seven-membered heteroalkyl ring, a substituted or unsubstituted eight-membered heteroalkyl ring;

3. A bicyclic derivative according to any one of claims 1 or 2, wherein the heteroalkanes in the a and B rings each independently comprise 1-3 (preferably 1,2, 3) nitrogen atoms.

4. A bicyclic derivative as claimed in claim 3 wherein Y ₁ is linked to the a ring through a nitrogen atom in the heteroalkyl ring; the B ring is attached to Y ₂ through a nitrogen atom in the heteroalkyl ring.

5. The bicyclic derivative of claim 1 wherein R ₁ is substituted or unsubstitutedWherein the C ring is a C6-C10 aromatic ring or a 5-8 membered heteroaromatic ring, and the D ring is a substituted or unsubstituted 5-8 membered heteroaromatic ring or a substituted or unsubstituted 5-8 membered heteroalkane ring.

6. The bicyclic derivative of claim 1 wherein R ₂ is a hydrogen atom or-L1-L2; wherein, -L1-is- (substituted or unsubstituted phenyl) -, and L2 is a substituted or unsubstituted 5-6 membered nitrogen containing heteroaryl.

7. The bicyclic derivative of claim 1, wherein said bicyclic derivative is selected from the group consisting of:

8. A pharmaceutical composition, said pharmaceutical composition comprising:

(a) A therapeutically effective amount of a bicyclic derivative of any one of claims 1-7, and racemates, stereoisomers, tautomers, isotopic labels, nitroxides, solvates, polymorphs, metabolites, esters, prodrugs or pharmaceutically acceptable salts thereof; and

(B) A pharmaceutically acceptable carrier.

9. Use of a bicyclic derivative of formula (I) as defined in any one of claims 1 to 7, as well as its racemate, stereoisomer, tautomer, isotopic label, nitroxide, solvate, polymorph, metabolite, ester, prodrug or a pharmaceutically acceptable salt thereof, for the preparation of a pharmaceutical composition for the treatment or prophylaxis of a disease, disorder or condition associated with αvβ1, αvβ6 and αvβ8 integrin activity or expression.

10. The use according to claim 9, wherein the disease, disorder or condition associated with αvβ1, αvβ6 and αvβ8 integrin activity or expression level is selected from the group consisting of: autoimmune diseases, fibrotic diseases, inflammatory Bowel Disease (IBD), relapsing Multiple Sclerosis (RMS), progressive Multifocal Leukoencephalopathy (PML), ulcerative Colitis (UC), crohn's Disease (CD), chronic viral hepatitis B and hepatitis C, non-alcoholic fatty liver (NAFLD), age-related macular degeneration, diabetic retinopathy, retinal vascular disease, osteoporosis and cell proliferative diseases,

Preferably the fibrotic disease is selected from the group consisting of: pulmonary fibrosis, idiopathic pulmonary fibrosis, non-specific interstitial pneumonia (NSIP), conventional interstitial lung disease (UIP), radiation-induced pulmonary fibrosis, familial pulmonary fibrosis, airway pulmonary fibrosis, chronic Obstructive Pulmonary Disease (COPD), interstitial lung disease, liver fibrosis, chronic kidney disease, kidney fibrosis, skin fibrosis, systemic sclerosis, or a combination thereof,

The cell proliferative disorder is cancer selected from the group consisting of: breast cancer, cervical cancer, endometrial cancer, ovarian cancer, colon cancer, rectal cancer, pancreatic cancer, liver cancer, lung cancer, non-small cell lung cancer, brain metastases of lung cancer, oral squamous cell carcinoma, head cancer, neck cancer, head and neck squamous cell carcinoma, oral or nasal mucosa cancer, laryngeal cancer, renal cell carcinoma, ovarian cancer, spleen cancer, small intestine cancer, large intestine cancer, stomach cancer, esophageal cancer, lung squamous cell carcinoma, bile duct cancer, gall bladder cancer, melanoma, urothelial cancer, genitourinary tract cancer, genital cancer, prostate cancer, testicular cancer, bladder cancer, blood cancer, skin cancer, bone marrow cancer, brain cancer, central nervous system cancer, muscle tissue cancer, thyroid cancer, or a combination thereof.

11. A bicyclic derivative, and racemates, stereoisomers, tautomers, isotopic labels, nitroxides, solvates, polymorphs, metabolites, esters, prodrugs or pharmaceutically acceptable salts thereof, wherein said bicyclic derivative has the structure shown in the following formulas II-a, II-b, II-c:

wherein,

Each R _3a ^' is independently C1-C6 alkyl;

Is a single bond or a double bond;

each n is independently 0,1, 2 or 3;