CN115066417A

CN115066417A - Factor XIa inhibitors

Info

Publication number: CN115066417A
Application number: CN202080091567.8A
Authority: CN
Inventors: 安东; 刘煜; 骆庆合; 张斌; 苗帅; 伍广生; 卢凯
Original assignee: Shanghai Jingxin Biological Medical Co ltd; Zhejiang Jingxin Pharmaceutical Co Ltd
Current assignee: Shanghai Jingxin Biological Medical Co ltd; Zhejiang Jingxin Pharmaceutical Co Ltd
Priority date: 2019-12-31
Filing date: 2020-12-30
Publication date: 2022-09-16
Also published as: WO2021136390A1

Abstract

Factor XIa inhibitors of formula I, pharmaceutical compositions comprising the same, methods for their preparation, and their use in the preparation of medicaments for the prevention or treatment of thrombosis and embolism associated diseases.

Description

Factor XIa inhibitors

Technical Field

The invention relates to a novel coagulation factor XIa inhibitor, a pharmaceutical composition containing the same, a preparation method thereof and application thereof in preparing medicines for preventing or treating diseases related to thrombus and embolism.

Background

Cardiovascular and cerebrovascular diseases are common diseases seriously threatening human beings, have the characteristics of high morbidity, high disability rate and high mortality, are ranked first among all lethal factors, and are regarded as the first killers of the human beings. The third national cause of death survey published by the original Ministry of health of China shows that the cerebrovascular disease accounts for 22.45 percent of the total number of deaths. Each year in China, 250 million patients with stroke occur, and 130 million people die of stroke every year. The incidence of thrombotic diseases among cardiovascular and cerebrovascular diseases is the highest, and the number of thrombotic diseases is increasing in recent years, which is a hot and difficult problem in modern medical research.

Thrombotic diseases (thrombosis diseases), abbreviated as thrombosis, are diseases caused by abnormal blood clots formed in blood vessels in circulating blood during the survival of human beings and animals, and the diseases cause serious influences on organisms during the attack, such as blood vessel obstruction, embolism, heart valve deformation, extensive bleeding and the like. The mechanism of thrombus formation is very complex.

Studies report that Venous Thromboembolism (VTE) has now become the first leading cause of death in the population, a major health problem worldwide. The disease of abnormal coagulation of blood in human Venous lumens due to various factors to cause blood backflow disorder is called Deep Venous Thrombosis (DVT), after thrombus falls off, Pulmonary Embolism (PE) is formed along with the flow of blood to the lung, and if the thrombus does not fall off, the vein is blocked for a long time or the function of the valve is destroyed after the thrombus is recanalized, the corresponding symptom of Venous hypertension is called Post-Thrombotic Syndrome (PTS). Both DVT and PE are collectively called VTE.

Thrombotic diseases widely affect human health, and antithrombotic drugs are always hot spots in the pharmaceutical industry. The traditional anticoagulant drugs such as warfarin, heparin and Low Molecular Weight Heparin (LMWH) and new drugs on the market in recent years such as FXa inhibitors (rivaroxaban, apixaban and the like) and thrombin inhibitors (dabigatran etexilate, hirudin and the like) can effectively reduce the formation of venous thrombosis, and are widely used for clinically treating venous thrombosis and adult venous thrombosis in the bone joint replacement surgery process, reducing the risk of Pulmonary Embolism (PE) caused after acute DVT, and the risk of stroke and systemic embolism of some high-risk atrial fibrillation people (with heart failure, hypertension, age more than or equal to 75 years, diabetes history, stroke history and the like). Has good effect on reducing thrombosis, but all the medicines also have common defects. These drugs may cause bleeding complications, and bring about a great deal of serious examination for patients and doctors. To solve this problem, FXIa-targeted inhibitors have become the subject of major antithrombotic drug development and research in domestic and foreign companies.

Compounds like BMS262084, BMS724296, BMS962212, which are developed in succession by BMS companies, have entered clinical research. BAY1213790 developed by Bayer corporation, ONO8610539 developed by Xiaoye pharmacy, and other compounds are also under preclinical study. These clinical trials or preclinical compounds are surmised to have specific properties, such as BMS962212 clinical dosage forms being intravenous liquid formulations suggesting either difficult absorption through the stomach or unacceptable first pass effects. BMS262084 and BMS724296 are terminated after clinical phase I experiments, which indicates that the drug effect of the human body is possibly poor. The research states of BAY1213790 and ONO8610539 are not updated later, which indicates that the research progress is slow, and shows that although the target binding rate of the compounds is good, the anticoagulation effect on human whole blood is probably not good; there may also be some off-target effects that may cause other unacceptable side effects. It is known that new drug research is in the early stage, and the information is rarely disclosed, and the above presumption is only made by the experience of new drug research.

Given all published in vitro and in vivo information, Factor XIa is an attractive target for the treatment and prevention of thromboembolic disorders. Coagulation Factor XIa inhibitors may be a new, effective, potential, safer treatment for thrombotic diseases in humans.

Summary of The Invention

In one aspect, the present invention provides a novel class of factor XIa inhibitors. The compound of the invention can specifically act on blood coagulation Factor (Factor) XIa, and can remarkably prolong the coagulation time of human whole blood under the condition of very low concentration. The classical rat arteriovenous thrombosis test (AVST) proves that the compound has remarkable in-vivo antithrombotic effect. Therefore, the compounds of the present invention have greater clinical potential than the compounds mentioned in the published reports. The compounds of the invention are compounds of formula I or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, metabolite, or prodrug thereof:

wherein

A is selected from phenyl and cyclohexyl;

R ₁ each independently selected from halogen, C _1-6 Alkyl, tetrazolyl and triazolyl, said C _1-6 Alkyl, tetrazolyl or triazolyl are each optionally substituted with one or more substituents selected from amino and halo;

n is 0,1, 2 or 3;

R ₂ and R ₃ Each independently selected from H and C _1-6 An alkyl group;

m is 0 or 1;

R ₄ is H;

R ₅ is optionally substituted by one or more R ₈ Substituted phenyl or C _1-6 An alkyl group;

or R ₄ 、R ₅ And the atoms to which they are attached together form a 5-6 membered azacycloalkyl group optionally fused to a benzene ring, said 5-6 membered azacycloalkyl group or benzene ring fused thereto being optionally substituted by one or more R ₈ Substitution;

R ₈ each independently selected from H, -NR _a R _b Halogen, halogen,

5-6 membered cycloalkyl and phenyl;

R _a selected from H and C _1-6 An alkyl group;

R _b is- (CO) _p -(CH ₂ ) _q -R ₉ ；

p is 0 or 1;

q is 1 or 2;

R ₉ is selected from C _1-6 Alkoxy and-NR _c R _d ；

R _c And R _d Each independently selected from H and C _1-6 An alkyl group;

R ₆ is selected from H and C _1-6 An alkyl group;

R ₇ is optionally substituted by one or more R ₁₀ Substituted phenyl or benzyl;

R ₁₀ each independently selected from- (CH) ₂ ) _r -C(O) _s -R ₁₁ C optionally substituted by one or more halogens _1-6 Alkoxy, tetrazolyl, halo, -NHCOOC _1-6 Alkyl and-SO ₂ NHCOC _1-6 An alkyl group;

r is 0 or 1;

s is 1 or 2;

R ₁₁ selected from H, -NHSO ₂ R ₁₂ And optionally substituted with R ₁₃ Substituted C _1-6 An alkyl group;

R ₁₂ is selected from C _1-6 Alkyl radical, C _3-6 Cycloalkyl and phenyl;

R ₁₃ is selected from-OCOOR ₁₄ And

R ₁₄ is selected from C _1-6 Alkyl and C _3-6 A cycloalkyl group;

or R ₆ 、R ₇ And the N atom to which they are attached together form a 5-6 membered azacycloalkyl group,

wherein when R is ₈ Is composed of

And R is ₁₀ Is- (CH) ₂ ) _r -C(O) _s -R ₁₁ When R is ₆ Is C _1-6 An alkyl group, a carboxyl group,

wherein the wavy line

Indicates the point of attachment of the group to the rest of the molecule.

In another aspect, the invention provides a pharmaceutical composition comprising a prophylactically or therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, metabolite, or prodrug thereof, and one or more pharmaceutically acceptable carriers.

In another aspect, the present invention provides a compound of the present invention, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, metabolite or prodrug thereof, for use in the prevention or treatment of thrombosis and embolism associated disease.

In another aspect, the present invention provides the use of a compound of the present invention, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, metabolite or prodrug thereof, for the manufacture of a medicament for the prevention or treatment of thrombosis and embolism associated diseases.

In another aspect, the present invention provides a method for the prevention or treatment of thrombosis and embolism associated disorder, comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, metabolite or prodrug thereof, or a pharmaceutical composition of the present invention.

In another aspect, the invention provides a process for preparing a compound of the invention, according to scheme 1,2,3 or 4 below:

route 1

Route 2

Route 3

Route 4

Wherein R is ¹ 、R ² 、R ³ 、R ⁴ 、R ⁵ 、R ⁶ 、R ⁷ 、R ⁸ M and n are as defined above; and is

PG is a protecting group for amino group (e.g., t-butoxycarbonyl or 9-fluorenylmethoxycarbonyl).

Drawings

Figure 1 shows the extent of thrombus reduction and inhibition in rat AVST experiments for the compound of example 3.

Figure 2 shows the thrombus-inhibiting effect of the compound of example 3 in rat AVST experiments.

Detailed Description

Definition of

Unless defined otherwise below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. Reference to the techniques used herein is intended to refer to those techniques commonly understood in the art, including those variations of or alternatives to those techniques that would be apparent to those skilled in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.

As used herein, the terms "comprises," "comprising," "has," "containing," or "involving," and other variations thereof herein, are inclusive or open-ended and do not exclude additional unrecited elements or method steps.

The term "alkyl" as used herein is defined as a straight or branched chain saturated aliphatic hydrocarbon group. For example, as used herein, the term "C _1-6 Alkyl "refers to a straight or branched chain group having 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, etc.).

As used herein, the term "cycloalkyl" refers to a saturated, non-aromatic, monocyclic or polycyclic (such as bicyclic) hydrocarbon ring. For example, the term "C _3-6 Cycloalkyl "refers to a saturated non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon ring (e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl) having 3 to 6 ring carbon atoms. The term "C _5-6 Cycloalkyl "refers to a saturated non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon ring (e.g., cyclopentyl or cyclohexyl) having 5 to 6 ring carbon atoms.

As used herein, the term "alkoxy" means a group having an oxygen atom inserted at any reasonable position in the alkyl group (as defined above). For example, the term "C _1-6 Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, and,T-butoxy, pentyloxy, hexyloxy, and the like.

As used herein, the term "halo" or "halogen" group is defined to include fluorine, chlorine, bromine, or iodine.

The term "substituted" means that one or more (e.g., 1,2,3, or 4) hydrogens on the designated atom is replaced with a selection from the indicated group, provided that the designated atom's normal valency at the present time is not exceeded and the substitution results in a stable compound. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.

If a substituent is described as "optionally substituted", the substituent may be (1) unsubstituted or (2) substituted. If a carbon of a substituent is described as being optionally substituted with one or more of the list of substituents, one or more hydrogens on the carbon (to the extent of any hydrogens present) may be replaced individually and/or together with an independently selected substituent or not. If the nitrogen of a substituent is described as being optionally substituted with one or more of the list of substituents, then one or more hydrogens on the nitrogen (to the extent any hydrogen present) may each be replaced with an independently selected substituent or not.

If a substituent is described as "independently selected from" a group of groups, each substituent is selected independently of the other. Thus, each substituent may be the same as or different from another (other) substituent.

As used herein, the term "one or more" means 1 or more than 1, such as 2,3,4, 5, 6, 7, 8, 9 or 10, under reasonable conditions.

Unless indicated, as used herein, the point of attachment of a substituent may be from any suitable position of the substituent.

When a bond to a substituent is shown across the bond connecting two atoms in a ring, then such substituent may be bonded to any ring-forming atom in the substitutable ring.

The term "stereoisomer" denotes an isomer formed as a result of at least one asymmetric center. In compounds having one or more (e.g., 1,2,3, or 4) asymmetric centers, they can result in racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. Certain molecules may also exist as geometric isomers (cis/trans). Similarly, the compounds of the invention may exist as mixtures of two or more structurally different forms (commonly referred to as tautomers) in rapid equilibrium. Representative examples of tautomers include keto-enol tautomers, phenol-keto tautomers, nitroso-oxime tautomers, imine-enamine tautomers, and the like. It is understood that the scope of this application encompasses all such isomers or mixtures thereof in any ratio (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

The present invention encompasses all possible crystalline forms or polymorphs of the compounds of the present invention, which may be single polymorphs or mixtures of more than one polymorph in any ratio.

It will also be appreciated that certain compounds of the invention may be present in free form for use in therapy or, where appropriate, in the form of a pharmaceutically acceptable derivative thereof. In the present invention, pharmaceutically acceptable derivatives include, but are not limited to: pharmaceutically acceptable salts, solvates, metabolites or prodrugs thereof, which upon administration to a patient in need thereof are capable of providing, directly or indirectly, a compound of the present invention or a metabolite or residue thereof. Thus, when reference is made herein to "a compound of the invention," it is also intended to encompass the various derivative forms of the compounds described above.

Pharmaceutically acceptable salts of the compounds of the present invention include acid addition salts and base addition salts thereof. Suitable acid addition salts are formed from acids which form pharmaceutically acceptable salts. Suitable base addition salts are formed from bases which form pharmaceutically acceptable salts. For a review of suitable Salts, see Stahl and Wermuth, "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of the present invention are known to those skilled in the art.

The compounds of the invention may be present in the form of solvates, preferably hydrates, wherein the compounds of the invention comprise as structural element of the crystal lattice of the compound a polar solvent, such as in particular water, methanol or ethanol. The amount of polar solvent, particularly water, may be present in stoichiometric or non-stoichiometric proportions.

Those skilled in the art will appreciate that not all nitrogen-containing heterocycles can form N-oxides because nitrogen requires an available lone pair for oxidation to an oxide; one skilled in the art will recognize nitrogen-containing heterocycles that are capable of forming N-oxides. Those skilled in the art will also recognize that tertiary amines are capable of forming N-oxides. Synthetic methods for preparing N-oxides of heterocycles and tertiary amines are well known to those skilled in the art and include oxidation of heterocycles and tertiary amines with peroxy acids such as peracetic and m-chloroperbenzoic acid (MCPBA), hydrogen peroxide, alkyl hydroperoxides such as t-butyl hydroperoxide, sodium perborate, and dioxiranes (dioxiranes) such as dimethyldioxirane. These methods for preparing N-oxides have been widely described and reviewed in the literature, see for example: T.L.Gilchrist, Comprehensive Organic Synthesis, vol.7, pp 748-; a.r.katitzky and a.j.boulton, eds., Academic Press; and G.W.H.Cheeseman and E.S.G.Werstuk, Advances in Heterocyclic Chemistry, vol.22, pp 390-.

Also included within the scope of the present invention are metabolites of the compounds of the present invention, i.e., substances formed in vivo upon administration of the compounds of the present invention. Such products may result, for example, from oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc. of the administered compound. Accordingly, the present invention includes metabolites of the compounds of the present invention, including compounds made by the process of contacting the compounds of the present invention with a mammal for a time sufficient to produce a metabolite thereof.

The present invention further includes within its scope prodrugs of the compounds of the present invention which are certain derivatives of the compounds of the present invention which may themselves have little or no pharmacological activity which, when administered into or onto the body, may be converted to the compounds of the present invention having the desired activity by, for example, hydrolytic cleavage. Typically such prodrugs will be functional derivatives of the compounds which are readily convertible in vivo into the desired therapeutically active compound. Further information on the use of prodrugs can be found in "Pro-drugs as Novel Delivery Systems", volume 14, ACS Symposium Series (T.Higuchi and V.Stella) and "Bioreversible Carriers in Drug Design," Pergamon Press,1987(E.B.Roche editions, American Pharmaceutical Association). Prodrugs of the invention may be prepared, for example, by substituting certain moieties known to those skilled in the art as "pro-moieties" (e.g., "Design of Prodrugs", described in Bundgaard (Elsevier, 1985)) for appropriate functional groups present in compounds of the invention.

The invention also encompasses compounds of the invention containing a protecting group. In any process for preparing the compounds of the present invention, it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned, thereby forming a chemically protected form of the compounds of the present invention. This can be achieved by conventional protecting Groups, for example, as described in Protective Groups in Organic Chemistry, ed.j.f.w.mcomie, Plenum Press, 1973; and T.W.Greene & P.G.M.Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons,1991, which are incorporated herein by reference. The protecting group may be removed at a suitable subsequent stage using methods known in the art.

The term "about" means within. + -. 10%, preferably within. + -. 5%, more preferably within. + -. 2% of the stated value.

Compound (I)

It is an object of the present invention to provide a compound of formula I or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, metabolite or prodrug thereof:

wherein

A is selected from phenyl and cyclohexyl;

n is 0,1, 2 or 3;

R ₂ and R ₃ Each independently selected from H and C _1-6 An alkyl group;

m is 0 or 1;

R ₄ is H;

or R ₄ 、R ₅ And the atoms to which they are attached together form a 5-6 membered azacycloalkyl optionally fused to a phenyl ring, said 5-6 membered azacycloalkyl or phenyl ring fused thereto being optionally substituted by one or more R ₈ Substitution;

R ₈ each independently selected from H, -NR _a R _b Halogen, halogen,

5-6 membered cycloalkyl and phenyl;

R _a is selected from H and C _1-6 An alkyl group;

R _b is- (CO) _p -(CH ₂ ) _q -R ₉ ；

p is 0 or 1;

q is 1 or 2;

R ₉ is selected from C _1-6 Alkoxy and-NR _c R _d ；

R _c And R _d Each independently selected from H and C _1-6 An alkyl group;

R ₆ selected from H, C _1-6 Alkyl and C _3-6 A cycloalkyl group;

r is 0 or 1;

s is 1 or 2;

R ₁₂ is selected from C _1-6 Alkyl radical, C _3-6 Cycloalkyl and phenyl;

R ₁₃ is selected from-OCOOR ₁₄ And

R ₁₄ is selected from C _1-6 Alkyl and C _3-6 A cycloalkyl group;

wherein when R is ₈ Is composed of

wherein the waveThread

Indicates the point of attachment of the group to the rest of the molecule.

In accordance with some embodiments of the present invention,

selected from:

wherein the wavy line

Indicates the point of attachment of the group to the rest of the molecule.

According to some embodiments of the invention, R ₂ And R ₃ Is H.

In accordance with some embodiments of the present invention,

R ₄ is H;

R ₅ is optionally substituted by R ₈ Substituted phenyl;

R ₈ each independently selected from H, -NR _a R _b Halogen, halogen,

5-6 membered cycloalkyl and phenyl;

R _a selected from H and C _1-6 An alkyl group;

R _b is- (CO) _p -(CH ₂ ) _q -R ₉ ；

p is 0 or 1;

q is 1 or 2;

R ₉ is selected from C _1-6 Alkoxy and-NR _c R _d (ii) a And is

R _c And R _d Each independently selected from H and C _1-6 An alkyl group, a carboxyl group,

wherein the wavy line

Indicates the point of attachment of the group to the rest of the molecule.

In accordance with some embodiments of the present invention,

R ₄ is H;

R ₅ is optionally substituted by R ₈ A substituted phenyl group;

R ₈ is-NR _a R _b ；

R _a Is H;

R _b is- (CO) _p -(CH ₂ ) _q -R ₉ ；

p is 1;

q is 1;

R ₉ is-NR _c R _d (ii) a And is

R _c And R _d Each independently selected from C _1-6 An alkyl group.

In accordance with some embodiments of the present invention,

R ₄ 、R ₅ and the atoms to which they are attached together form a 5-6 membered azacycloalkyl optionally fused to a phenyl ring, said 5-6 membered azacycloalkyl or phenyl ring fused thereto being optionally substituted by R ₈ Substitution;

R ₈ each independently selected from H, -NR _a R _b Halogen, halogen,

5-6 membered cycloalkyl and phenyl;

R _a is selected from H and C _1-6 An alkyl group;

R _b is- (CO) _p -(CH ₂ ) _q -R ₉ ；

p is 0 or 1;

q is 1 or 2;

R ₉ is selected from C _1-6 Alkoxy and-NR _c R _d (ii) a And is

wherein the wavy line

Indicates the point of attachment of the group to the rest of the molecule.

In accordance with some embodiments of the present invention,

R ₄ 、R ₅ and the atoms to which they are attached together form a 1,2,3, 4-tetrahydroisoquinolinyl or pyrrolidinyl group, said 1,2,3, 4-tetrahydroisoquinolinyl or pyrrolidinyl group optionally substituted with R ₈ Substitution;

R ₈ each independently selected from H, -NR _a R _b Halogen, halogen,

5-6 membered cycloalkyl and phenyl;

R _a selected from H and C _1-6 An alkyl group;

R _b is- (CO) _p -(CH ₂ ) _q -R ₉ ；

p is 0 or 1;

q is 1 or 2;

R ₉ is selected from C _1-6 Alkoxy and-NR _c R _d (ii) a And is

wherein the wavy line

Indicates the point of attachment of the group to the rest of the molecule.

In accordance with some embodiments of the present invention,

R ₄ 、R ₅ and the atoms to which they are attached together form

R ₈ Selected from H, -NR _a R _b Halogen and

R _a selected from H and C _1-6 An alkyl group;

R _b is- (CO) _p -(CH ₂ ) _q -R ₉ ；

p is 0 or 1;

q is 1 or 2;

R ₉ is selected from C _1-6 Alkoxy and-NR _c R _d (ii) a And is

wherein the wavy line

Indicates the point of attachment of the group to the rest of the molecule.

In accordance with some embodiments of the present invention,

R ₄ 、R ₅ and the atoms to which they are attached together form

And is

R ₈ Selected from H, -NHCOCH ₂ OCH ₃ 、-NHCOCH ₂ N(CH ₃ ) ₂ 、-NH(CH ₂ ) ₂ N(CH ₃ ) ₂ 、-N(CH ₃ )COCH ₂ OCH ₃ 、-N(CH ₃ )COCH ₂ N(CH ₃ ) ₂ Br and

wherein the wavy line

Indicates the point of attachment of the group to the rest of the molecule.

In accordance with some embodiments of the present invention,

R ₄ 、R ₅ and atoms to which they are attached together form

And is

R ₈ Selected from the group consisting of cyclohexyl and phenyl,

wherein the wavy line

Indicates the point of attachment of the group to the rest of the molecule.

In accordance with some embodiments of the present invention,

R ₆ selected from H, C _1-6 Alkyl and C _3-6 A cycloalkyl group;

R ₇ is optionally substituted by one or two R ₁₀ Substituted phenyl or benzyl;

r is 0 or 1;

s is 1 or 2;

R ₁₂ is selected from C _1-6 Alkyl radical, C _3-6 Cycloalkyl and phenyl;

R ₁₃ is selected from-OCOOR ₁₄ And

and is

R ₁₄ Is selected from C _1-6 Alkyl and C _3-6 A cycloalkyl group,

wherein the wavy line

Indicates the point of attachment of the group to the rest of the molecule.

In accordance with some embodiments of the present invention,

R ₆ selected from H, C _1-6 Alkyl and C _3-6 A cycloalkyl group;

R ₇ is optionally substituted by one or two R ₁₀ Substituted phenyl or benzyl; and is

R ₁₀ Each independently selected from-COOH, -COOCH ₃ 、-COOCH ₂ CH ₃ 、-COOC(CH ₃ ) ₃ 、-COOCH ₂ OCOOCH ₂ CH ₃ 、-COOCH ₂ OCOOCH(CH ₃ ) ₂ 、-COOCH(CH ₃ )OCOOCH ₂ CH ₃ 、-COOCH(CH ₃ )OCOOCH(CH ₃ ) ₂ 、

-CONHSO ₂ CH ₃ 、

-CH ₂ COOH、-OCH ₃ 、-OCHF ₂ 、

-Cl、-F、-NHCOOCH ₃ and-SO ₂ NHCOCH ₃ ，

Wherein the wavy line

Denotes the attachment of a group to the rest of the moleculeAnd (4) point.

According to some embodiments of the invention, R ₆ 、R ₇ And the N atom to which it is attached together form a pyrrolidinyl group.

The present invention encompasses compounds of formula I obtained by any combination of the above preferred groups.

According to some embodiments of the invention, the compounds of the invention have the structure of formula II:

wherein R is ¹ 、R ² 、R ³ 、R ⁶ 、R ⁷ 、R ⁸ M and n are as defined above.

According to some embodiments of the invention, the compounds of the invention have the structure of formula II-1:

wherein R is ⁶ 、R ⁷ And R ⁸ As defined above.

According to some embodiments of the invention, the compounds of the invention have the structure of formula II-2:

wherein R is ⁶ 、R ⁷ And R ⁸ As defined above.

According to some embodiments of the invention, the compounds of the invention have the structure of formula II-3:

wherein R is ⁶ 、R ⁷ And R ⁸ As defined above.

According to some embodiments of the invention, the compound of the invention has the structure of formula II-4:

wherein R is ⁶ 、R ⁷ And R ⁸ As defined above.

According to some embodiments of the invention, the compounds of the invention have the structure of formula II-5:

wherein R is ⁶ 、R ⁷ And R ⁸ As defined above.

According to some embodiments of the invention, the compounds of the invention have the structure of formula III:

According to some embodiments of the invention, the compounds of the invention have the structure of formula III-1:

wherein R is ⁶ 、R ⁷ And R ⁸ As defined above.

According to some embodiments of the invention, the compounds of the invention have the structure of formula III-2:

wherein R is ⁶ 、R ⁷ And R ⁸ As defined above.

According to some embodiments of the invention, the compounds of the invention have the structure of formula IV:

According to some embodiments of the invention, the compounds of the invention have the structure of formula IV-1:

wherein R is ⁶ 、R ⁷ And R ⁸ As defined above.

According to some embodiments of the invention, the compounds of the invention have the structure of formula IV-2:

wherein R is ⁶ 、R ⁷ And R ⁸ As defined above.

According to some embodiments of the invention, the compounds of the invention have the structure of formula IV-3:

wherein R is ⁶ 、R ⁷ And R ⁸ As defined above.

Any combination of the above preferred groups can be made in keeping with the general knowledge in the art to arrive at various preferred embodiments of the present invention.

According to some embodiments of the invention, the compound of the invention is selected from:

preparation method

Another object of the present invention is to provide a process for the preparation of the compounds of the present invention, which is carried out according to scheme 1,2,3 or 4 below:

route 1

Route 2

Route 3

Route 4

Pharmaceutical composition

Another object of the present invention is to provide a pharmaceutical composition comprising a prophylactically or therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, metabolite or prodrug thereof, and one or more pharmaceutically acceptable carriers.

By "pharmaceutically acceptable carrier" in the context of the present invention is meant a diluent, adjuvant, excipient, or vehicle that is administered together with a therapeutic agent and which is, within the scope of sound medical judgment, suitable for contact with the tissues of humans and/or other animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio.

Water is an exemplary carrier when the pharmaceutical composition is administered intravenously. Physiological saline and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition may also optionally contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. Examples of suitable pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1990).

The pharmaceutical compositions of the present invention may act systemically and/or locally. For this purpose, they may be administered by a suitable route, for example by injection (e.g. intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular injection, including instillation) or transdermally; or by oral, buccal, nasal, transmucosal, topical, in the form of ophthalmic preparations or by inhalation.

For these routes of administration, the pharmaceutical compositions of the present invention may be administered in suitable dosage forms. Such dosage forms include, but are not limited to, tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups, and the like.

The amount or amount of a compound of the invention in a pharmaceutical composition may be from about 0.001 to 1000mg, suitably 0.01 to 800mg, preferably 0.05 to 500mg, more preferably 0.1 to 350mg, especially 0.5 to 100 mg.

In some embodiments, the present invention provides a method of making a pharmaceutical composition of the present invention, the method comprising combining a compound of the present invention, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, metabolite, or prodrug thereof, with one or more pharmaceutically acceptable carriers.

Methods of treatment and uses

It is another object of the present invention to provide a compound of the present invention or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, metabolite or prodrug thereof, for use in the prevention or treatment of thrombosis and embolism associated diseases.

Another object of the present invention is to provide a use of the compound of the present invention or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, metabolite or prodrug thereof for the preparation of a medicament for the prophylaxis or treatment of thrombosis and embolism associated diseases.

Another object of the present invention is to provide a method for preventing or treating thrombosis and embolism associated diseases, which comprises administering to a subject in need thereof a prophylactically or therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, metabolite or prodrug thereof, or a pharmaceutical composition of the present invention.

According to some embodiments of the invention, the thrombosis or embolism associated disorder is atherosclerotic disease, myocardial infarction, stroke, ischemic cerebral thrombosis, peripheral arterial disease, peripheral arterial occlusive disease, pulmonary embolism, deep vein thrombosis, acute coronary syndrome, or thrombosis following coronary intervention.

The dosing regimen may be adjusted to provide the best desired response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is noted that dosage values may vary with the type and severity of the condition being alleviated, and may include single or multiple doses. It is further understood that for any particular individual, the specific dosage regimen will be adjusted over time according to the individual need and the professional judgment of the person administering the composition or supervising the administration of the composition.

The amount of a compound of the invention administered will depend on the subject being treated, the severity of the disorder or condition, the rate of administration, the disposition of the compound, and the judgment of the prescribing physician. Generally, an effective dose is from about 0.0001 to about 50mg per kg body weight per day, e.g., from about 0.01 to about 10 mg/kg/day (single or divided administration). For a 70kg human, this may amount to about 0.007 mg/day to about 3500 mg/day, e.g., about 0.7 mg/day to about 700 mg/day. In some cases, dosage levels not higher than the lower limit of the aforesaid range may be sufficient, while in other cases still larger doses may be employed without causing any harmful side effects, provided that the larger dose is first divided into several smaller doses to be administered throughout the day.

As used herein, unless otherwise specified, the term "treating" or "treatment" means reversing, alleviating, inhibiting the progression of, or preventing such a disorder or condition, or one or more symptoms of such a disorder or condition, to which such term applies.

As used herein, "individual" includes a human or non-human animal. Exemplary human individuals include human individuals (referred to as patients) having a disease (e.g., a disease described herein) or normal individuals. "non-human animals" in the context of the present invention include all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock and/or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

Examples

In order to make the objects and technical solutions of the present invention clearer, embodiments of the present invention will be described in detail below with reference to examples. It will be understood by those skilled in the art that the following examples are illustrative of the present invention only and should not be taken as limiting the scope of the invention. The examples, in which the specific conditions are not specified, were conducted under the conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are commercially available, and are not indicated by manufacturers.

In the conventional syntheses as well as in the examples and intermediate syntheses, the meanings of the abbreviations are shown in the following table.

Abbreviations	Means of
Pd ₂ dba ₃	Tris (dibenzylacetone) dipalladium
BINAP	1,1 '-binaphthyl-2, 2' -bis (diphenylphosphines)
DIPEA	N, N-diisopropylethylamine
HATU	2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate
DMF	N, N-dimethylformamide
XPhos	2-dicyclohexylphosphine-2 ',4',6' -triisopropylbiphenyl
EA	Ethyl acetate
THF	Tetrahydrofuran (THF)

DCM	Methylene dichloride
TFA	Trifluoroacetic acid
Boc	Tert-butyloxycarbonyl radical
T3P	2,4, 6-tripropyl-1, 3,5,2,4, 6-trioxatriphosphoric acid-2, 4, 6-trioxides
Py	Pyridine compound
CbzCl	Chloroformic acid benzyl ester
DCC	N, N-dicyclohexylcarbodiimide
CMPI	2-chloro-1-methylpyridine iodide
DMAP	Dimethylaminopyridine
RT	At room temperature
LCMS	Liquid chromatography-mass spectrometry
DMSO-d ₆	Hexahydro-deuterated dimethyl sulfoxide
NMR	Nuclear magnetic resonance
MS	Mass spectrometry
s	Single peak (singlet)
d	Double peak (doublet)
t	Triple peak (triplet)
q	Quartet (quartz)
dd	Double two peak (double)
m	Multiplet (multiplet)et)
br	Broad peak (broad)
J	Coupling constant
Hz	Hertz's scale

Example 1: (E) preparation of (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2-methoxyacetylamino) -1,2,3, 4-tetrahydroisoquinoline-1-formylamino) benzoic acid

The first step is as follows: preparation of 5- (2-methoxyacetylamino) -3, 4-dihydroisoquinoline-2 (1H) -carboxylic acid tert-butyl ester (1-3)

2-Methoxyacetyl chloride (1-2,164. mu.L, 1.8mmol) was added dropwise to a dichloromethane solution (5mL) containing tert-butyl 5-amino-3, 4-dihydroisoquinoline-2 (1H) -carboxylate (1-1,372mg, 1.5mmol) under ice-cooling, and the mixture was stirred at room temperature overnight. Adding 20mL of water into the reaction solution for quenching, extracting by dichloromethane, drying the organic phase, filtering and concentrating to obtain a pale yellow solid 5- (2-methoxy acetamido) -3, 4-dihydroisoquinoline-2 (1H) -tert-butyl formate 480mg, which is directly used for the next reaction.

The second step: preparation of 2-methoxy-N- (1,2,3, 4-tetrahydroisoquinolin-5-yl) acetamide (1-4)

5- (2-Methoxyacetamido) -3, 4-dihydroisoquinoline-2 (1H) -carboxylic acid tert-butyl ester (1-3,360mg) was dissolved in dichloromethane (5mL), trifluoroacetic acid (3mL) was added thereto, and the mixture was stirred at room temperature for 2 hours, concentrated, and the residue was basified with 1mol/L aqueous sodium hydroxide solution, extracted with dichloromethane, dried, filtered and concentrated to give 2-methoxy-N- (1,2,3, 4-tetrahydroquinolin-5-yl) acetamide (230mg) as a yellow oil.

The third step: preparation of N- (3, 4-dihydroisoquinolin-5-yl) -2-methoxyacetamide (1-5)

2-methoxy-N- (1,2,3, 4-tetrahydroquinolin-5-yl) acetamide (1-4,230mg) was dissolved in methylene chloride (5mL), and manganese dioxide (918mg) was added and the mixture was stirred at room temperature overnight. And (5) filtering. The filtrate was concentrated, and the residue was purified by preparative thin layer chromatography to give 133mg of N- (3, 4-dihydroisoquinolin-5-yl) -2-methoxyacetamide as a yellow oil.

The fourth step: (E) preparation of tert-butyl (1-8) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2-methoxyacetylamino) -1,2,3, 4-tetrahydroisoquinoline-1-formamido) benzoate

N- (3, 4-dihydroisoquinolin-5-yl) -2-methoxyacetamide (1-5,133mg), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (1-7,172mg), 4-tert-butyl isocyanobenzoate (1-6,137mg) are dissolved in ethanol (5mL), the reaction solution is heated to 85 ℃ and stirred overnight, and the mixture is cooled to room temperature and purified by column chromatography to obtain white solid (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2-methoxyacetamido) -1, tert-butyl 2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (147mg, yield 35%).

The fifth step: (E) preparation of (01) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2-methoxyacetylamino) -1,2,3, 4-tetrahydroisoquinoline-1-formamido) benzoic acid

(E) Tert-butyl (1-8,80mg) 4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2-methoxyacetylamino) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate was dissolved in dichloromethane (4mL), trifluoroacetic acid (2mL) was added thereto, and the mixture was stirred at room temperature for 1.5 hours, followed by concentration and purification of the residue to give (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2-methoxyacetylamino) -1 as a white solid, 2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid (46 mg).

¹ HNMR(DMSO-d6 400MHz)δ10.79(s,1H),9.87(s,1H),9.48(s,1H),7.96(t,1H,J＝8.0Hz),7.86(d,2H,J＝8.8Hz),7.68-7.65(m,3H),7.49(d,1H，J＝7.2Hz),7.31(d,1H，J＝6.8Hz),7.26(t,1H,J＝7.8Hz),7.04(dd,2H,J＝15.8,52.2Hz),5.85(s,1H),4.11-4.05(m,1H),4.04(s，2H)，3.69-3.63(m,1H),3.42(s，3H)，3.00-2.91(m,1H),2.81-2.75(m,1H)。

MS m/z(ESI):634[M+H]+

Example 2

The compound obtained in example 1 was separated by preparative liquid chromatography to give the compound of example 2.

MS m/z(ESI):634[M+H]+

Example 3

The compound obtained in example 1 was separated by preparative liquid chromatography to give the compound of example 3.

MS m/z(ESI):634[M+H]+

Example 4: (E) preparation of (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2- (dimethylamino) acetamido) -1,2,3, 4-tetrahydroisoquinoline-1-formamido) benzoic acid

The first step is as follows: preparation of 5- (2- (dimethylamino) acetamido) -3, 4-dihydroisoquinoline-2 (1H) -carboxylic acid tert-butyl ester (4-2)

5-amino-3, 4-dihydroisoquinoline-2 (1H) -carboxylic acid tert-butyl ester (1-1, 373mg, 1.5mmol), dimethylglycine (4-1, 206mg, 2mmol) was dissolved in ethyl acetate (8mL), pyridine (0.365mL), T3P (50% ethyl acetate solution, 1.8mL) were added, and the mixture was stirred at room temperature overnight. Water was added to the reaction solution to quench, followed by extraction with ethyl acetate, drying of the organic phase, filtration, concentration and purification by column chromatography to obtain 394mg of 5- (2- (dimethylamino) acetamido) -3, 4-dihydroisoquinoline-2 (1H) -carboxylic acid tert-butyl ester as a white solid, in 79% yield.

The second step is that: preparation of 2- (dimethylamino) -N- (1,2,3, 4-tetrahydroisoquinolin-5-yl) acetamide (4-3)

5- (2- (two methyl amino) acetyl amino) -3, 4-two hydrogen isoquinoline-2 (1H) -formic acid tert-butyl ester (4-2, 394mg) dissolved in dichloromethane (10mL), added HCl/1, 4-dioxane solution (4mol/L, 10mL), at room temperature stirring overnight, concentration, residues added 1mol/L sodium hydroxide aqueous solution for alkalization, dichloromethane extraction, organic phase drying, filtering and concentration, white solid 2- (two methyl amino) -N- (1,2,3, 4-four hydrogen isoquinoline-5-yl) acetamide (270 mg).

The third step: preparation of N- (3, 4-dihydroisoquinolin-5-yl) -2- (dimethylamino) acetamide (4-4)

2- (dimethylamino) -N- (1,2,3, 4-tetrahydroisoquinolin-5-yl) acetamide (4-3, 320mg) was dissolved in methylene chloride (30mL), and manganese dioxide (2.38g) was added to stir at room temperature overnight. And (5) filtering. The filtrate was concentrated, and the residue was purified by preparative thin layer chromatography to give 213mg of N- (3, 4-dihydroisoquinolin-5-yl) -2- (dimethylamino) acetamide as a white solid.

The fourth step: (E) preparation of tert-butyl (4-5) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2- (dimethylamino) acetamido) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate

N- (3, 4-dihydroisoquinolin-5-yl) -2- (dimethylamino) acetamide (4-4, 85mg), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (1-7,119mg), and tert-butyl 4-isocyanobenzoate (1-6, 87mg) are dissolved in ethanol (5mL), the reaction solution is heated to 70 ℃, stirred overnight, cooled to room temperature and purified by column chromatography to obtain yellow solid (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2- (dimethylamino) acetamido) -1, tert-butyl 2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (118mg, yield 46%).

The fifth step: (E) preparation of (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2- (dimethylamino) acetamido) -1,2,3, 4-tetrahydroisoquinoline-1-formamido) benzoic acid (04)

(E) Tert-butyl (4-5,118mg) 4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2- (dimethylamino) acetamido) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate was dissolved in dichloromethane (5mL), trifluoroacetic acid (3mL) was added thereto, and the mixture was stirred at room temperature for 2 hours, followed by concentration and purification of the residue to give (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2- (dimethylamino) acetamido) -1 as a white solid, 2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid (50 mg).

¹ HNMR(DMSO-d6 400MHz)δ10.78(s,1H),9.87(s,1H),9.51(s,1H),7.96(t,1H,J＝8.2Hz),7.86(d,2H,J＝8.4Hz),7.66(d,4H，J＝8.8Hz),7.45(d,1H，J＝8.0Hz),7.25(t,1H,J＝8.0Hz),7.05(dd,2H,J＝15.8,67Hz),5.85(s,1H),4.15-4.07(m,1H),3.76-3.69(m,1H),3.11(s，2H)，3.05-2.93(m,1H),2.85-2.81(m,1H)，2.34(s，6H)。

MS m/z(ESI):647[M+H]+

Example 5

The compound obtained in example 4 was separated by preparative liquid chromatography to give the compound of example 5.

MS m/z(ESI):647[M+H]+

Example 6

The compound obtained in example 4 was separated by preparative liquid chromatography to give the compound of example 6.

MS m/z(ESI):647[M+H]+

Example 7: (E) preparation of (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- ((2- (dimethylamino) ethyl) amino) -1,2,3, 4-tetrahydroisoquinoline-1-formylamino) benzoic acid

The first step is as follows: n is a radical of ¹ - (isoquinolin-5-yl) -N ² ,N ² Preparation of (E) -dimethylethane-1, 2-diamine (7-3)

Under a nitrogen atmosphere, N-dimethylethylenediamine (7-2,66mg,0.75mmol), 5-bromoisoquinoline (7-1,104mg, 0.5mmol), Pd ₂ dba ₃ (91mg, 0.1mmol), BINAP (93mg, 0.15mmol), cesium carbonate (244mg, 0.75mmol) in 1, 4-dioxane (3mL) was heated to 100 deg.C and stirred overnight. Filtering and concentrating the reaction solution, and purifying the residue by column chromatography to obtain white solid N ¹ - (isoquinolin-5-yl) -N ² ,N ² 54mg of (E) -dimethylethane-1, 2-diamine, yield 50%.

The second step is that: n is a radical of ¹ ,N ¹ -dimethyl-N ² Preparation of- (1,2,3, 4-tetrahydroisoquinolin-5-yl) ethane-1, 2-diamine (7-4)

N ¹ - (isoquinolin-5-yl) -N ² ,N ² -dimethylethane-1, 2-diamine (7-3,54mg) was dissolved in methanol (3mL), platinum dioxide (10mg) was added, stirred overnight at room temperature under hydrogen atmosphere, filtered, and concentrated to give N as a white solid ¹ ,N ¹ -dimethyl-N ² - (1,2,3, 4-tetrahydroisoquinolin-5-yl) ethane-1, 2-diamine (50 mg).

The third step: n is a radical of ¹ - (3, 4-dihydroisoquinolin-5-yl) -N ² ,N ² Preparation of (E) -dimethylethane-1, 2-diamine (7-5)

N ¹ ,N ¹ -dimethyl-N ² - (1,2,3, 4-tetrahydroisoquinolin-5-yl) ethane-1, 2-diamine (7-4,50mg) was dissolved in methylene chloride (3mL), and manganese dioxide (200mg) was added thereto, followed by stirring at room temperature overnight. And (5) filtering. Concentrating the filtrate to obtain white solid N ¹ - (3, 4-dihydroisoquinolin-5-yl) -N ² ,N ² -dimethylethane-1, 2-diamine 45 mg.

The fourth step: (E) preparation of tert-butyl (7-6) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- ((2- (dimethylamino) ethyl) amino) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate

N ¹ - (3, 4-dihydroisoquinolin-5-yl) -N ² ,N ² -dimethylethane-1, 2-diamine (7-5,45mg), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (1-7,59mg),tert-butyl 4-isocyanobenzoate (1-6,46mg) was dissolved in ethanol (3mL), the reaction solution was warmed to 70 ℃ and stirred overnight, cooled to room temperature, and purified by column chromatography to give yellow solid tert-butyl (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- ((2- (dimethylamino) ethyl) amino) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (62mg, 43% yield).

The fifth step: (E) preparation of (07) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- ((2- (dimethylamino) ethyl) amino) -1,2,3, 4-tetrahydroisoquinoline-1-formylamino) benzoic acid

(E) Tert-butyl (7-6,62mg) 4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- ((2- (dimethylamino) ethyl) amino) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate was dissolved in dichloromethane (3mL), trifluoroacetic acid (2mL) was added, and stirring was carried out at room temperature for 2 hours, followed by concentration and purification of the residue to give (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- ((2- (dimethylamino) ethyl) amino) -1 as a white solid, preparation of 2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid (30 mg).

¹ HNMR(DMSO-d6 400MHz)δ10.63(s,1H),9.90(s,1H),7.96(t,1H,J＝8.2Hz),7.83-7.81(m,2H),7.66(d,1H，J＝8.8Hz),7.57-7.55(m,2H),7.36(d,1H,J＝7.6Hz),7.19(t,1H,J＝7.8Hz),7.03(d,1H,J＝7.6Hz),7.02(dd,2H,J＝15.8,61Hz),5.76(s,1H),5.26(s,1H),4.11-4.07(m,1H),3.76-3.69(m,1H),3.05-2.98(m,1H),2.94(t,2H,J＝7.0Hz)，2.81(t,2H,J＝7.0Hz)，2.43-2.38(m,1H)，2.14(s，6H)。

MS m/z(ESI):633[M+H]+

Example 8

The compound obtained in example 7 was separated by preparative liquid chromatography to give the compound of example 8.

MS m/z(ESI):633[M+H]+

Example 9

The compound obtained in example 7 was separated by preparative liquid chromatography to give the compound of example 9.

MS m/z(ESI):633[M+H]+

Example 10: (E) preparation of (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2-methoxy-N-methylacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-formamido) benzoic acid

The first step is as follows: preparation of 5- (2-methoxy-N-methylacetamido) -3, 4-dihydroisoquinoline-2 (1H) -carboxylic acid tert-butyl ester (10-1)

A tetrahydrofuran solution (6mL) of tert-butyl 5- (2-methoxyacetylamino) -3, 4-dihydroisoquinoline-2 (1H) -carboxylate (1-3,231mg, 0.72mmol) was added dropwise to a suspension of NaH (60mg, 1.5mmol) in tetrahydrofuran (2mL), and the mixture was stirred at room temperature for 0.5H. Methyl iodide (88. mu.L, 1.4mmol) was added dropwise to the reaction solution, and the mixture was stirred at room temperature overnight. Adding water into the reaction solution to quench, extracting with dichloromethane, drying the organic phase, filtering, concentrating, purifying the residue by column chromatography to obtain light yellow oily 5- (2-methoxy-N-methylacetamido) -3, 4-dihydroisoquinoline-2 (1H) -tert-butyl formate 240 mg.

The second step is that: preparation of 2-methoxy-N-methyl-N- (1,2,3, 4-tetrahydroisoquinolin-5-yl) acetamide (10-2)

5- (2-methoxy-N-methyl acetyl amino) -3, 4-two hydrogen isoquinoline-2 (1H) -formic acid tert-butyl ester (10-1,870mg) dissolved in dichloromethane (5mL), added HCl/1, 4-two oxygen six ring (4mol/L, 10mL), room temperature stirring for 3 hours, concentration, residues added 10% sodium hydroxide methanol solution for alkalization, filtration and concentration, yellow oily matter 2-methoxy-N-methyl-N- (1,2,3, 4-four hydrogen isoquinoline-5-yl) acetamide (650 mg).

The third step: preparation of N- (3, 4-dihydroisoquinolin-5-yl) -2-methoxy-N-methylacetamide (10-3)

2-methoxy-N-methyl-N- (1,2,3, 4-tetrahydroisoquinolin-5-yl) acetamide (10-2,650mg) was dissolved in methylene chloride (60mL), and manganese dioxide (4.70g) was added thereto, followed by stirring at room temperature overnight. And (5) filtering. The filtrate was concentrated, and the residue was purified by column chromatography to give N- (3, 4-dihydroisoquinolin-5-yl) -2-methoxy-N-methylacetamide as a yellow oil (590 mg).

The fourth step: (E) preparation of tert-butyl (10-4) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2-methoxy-N-methylacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-formylamino) benzoate

N- (3, 4-dihydroisoquinolin-5-yl) -2-methoxy-N-methylacetamide (10-3, 151mg), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (1-7, 193mg), and tert-butyl 4-isocyanobenzoate (1-6, 146mg) are dissolved in ethanol (15mL), the reaction solution is heated to 70 ℃ and stirred for 40 hours, and the mixture is cooled to room temperature and purified by column chromatography to obtain white solid (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2-methoxyacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid tert-butyl ester (171 mg).

The fifth step: (E) preparation of (10) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2-methoxy-N-methylacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-formamido) benzoic acid

(E) Tert-butyl (72mg) 4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2-methoxyacetylamino) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate was dissolved in dichloromethane (2mL), trifluoroacetic acid (2mL) was added thereto, and the mixture was stirred at room temperature for 2 hours, followed by concentration and purification of the residue to give (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2-methoxyacetylamino) -1 as a white solid, 2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid (45 mg).

¹ HNMR(DMSO-d6 400MHz)δ10.86(s,1H),9.87(s,1H),7.94(t,1H,J＝8.0Hz),7.87(d,2H，J＝7.2Hz),7.73-7.67(m,3H),7.36(d,1H,J＝7.6Hz),7.30(d,1H,J＝7.6Hz),7.16(t,1H,J＝7.8Hz),7.04(d,1H,J＝7.6Hz),7.02(dd,2H,J＝15.6,62.4Hz),5.89(d,1H,J＝8.8Hz),4.18-4.09(m,1H),3.71-3.65(m,1H),3.17(s，2H)，3.11(s，2H)，3.07(s，3H)，3.05-2.98(m,1H)，2.67-2.62(m,1H).

MS m/z(ESI):648[M+H]+

Example 11

The compound obtained in example 10 was separated by preparative liquid chromatography to give the compound of example 11.

MS m/z(ESI):648[M+H]+

Example 12

The compound obtained in example 10 was separated by preparative liquid chromatography to give the compound of example 12.

MS m/z(ESI):648[M+H]+

Example 13: (E) preparation of (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2- (dimethylamino) -N-methylacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-formylamino) benzoic acid

The first step is as follows: preparation of 5- (methylamino) -3, 4-dihydroisoquinoline-1, 2(1H) -dicarboxylic acid-2-tert-butyl ester-1-methyl ester (13-3)

5-bromo-3, 4-dihydroisoquinoline-1, 2(1H) -dicarboxylic acid-2-tert-butyl ester-1-methyl ester (13-1,1.017g, 2.75mmol), methylamine hydrochloride (13-2,362mg, 5.5mmol), Pd (OAc) under nitrogen atmosphere ₂ (61mg, 0.27mmol), XPhos (265mg, 0.55mmol), cesium carbonate (3.63g, 11mmol) in 1, 4-dioxane (18mL), sealed and heated to 105 deg.C with stirring overnight. The reaction solution was filtered and concentrated, and the residue was purified by column chromatography to give 5- (methylamino) -3, 4-dihydroisoquinoline-1, 2(1H) -dicarboxylic acid-2-tert-butyl ester-1-methyl ester as a pale yellow solid (860 mg, 97.7% yield).

The second step is that: preparation of 5- (2- (dimethylamino) -N-methylacetamido) -3, 4-dihydroisoquinoline-1, 2(1H) -dicarboxylic acid-2-tert-butyl ester-1-methyl ester (13-4)

5- (methylamino) -3, 4-dihydroisoquinoline-1, 2(1H) -dicarboxylic acid-2-tert-butyl ester-1-methyl ester (13-3,431mg, 1.35mmol), dimethylglycine (181mg) was dissolved in ethyl acetate (10mL), pyridine (326. mu.L), T3P (50% ethyl acetate solution, 1.6mL) were added, and the mixture was stirred at room temperature overnight. Adding water into the reaction solution to quench, extracting with ethyl acetate, drying the organic phase, filtering, concentrating and purifying by column chromatography to obtain white solid 5- (2- (dimethylamino) -N-methylacetamido) -3, 4-dihydroisoquinoline-1, 2(1H) -dicarboxylic acid-2-tert-butyl ester-1-methyl ester 540 mg.

The third step: preparation of 2- (tert-butoxycarbonyl) -5- (2- (dimethylamino) -N-methylacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid (13-5)

5- (2- (two methyl amino) -N-methyl acetyl amino) -3, 4-two hydrogen isoquinoline-1, 2(1H) -two acid-2-tert-butyl ester-1-methyl ester (13-4,540mg) dissolved in tetrahydrofuran (10mL), added lithium hydroxide aqueous solution (2mol/L, 10mL), methanol 3mL, room temperature stirring for 2 hours, organic solvent concentration, with 1mol/L hydrochloric acid to adjust pH to 6, dichloromethane extraction, organic phase drying filter concentration, white solid 2- (tert-butyl oxycarbonyl) -5- (2- (two methyl amino) -N-methyl acetyl amino) -1,2,3, 4-four hydrogen isoquinoline-1-carboxylic acid (470 mg).

The fourth step: synthesis of tert-butyl 1- ((4- (tert-butoxycarbonyl) phenyl) carbamoyl) -5- (2- (dimethylamino) -N-methylacetamido) -3, 4-dihydroisoquinoline-2 (1H) -carboxylate (13-7)

2- (tert-Butoxycarbonyl) -5- (2- (dimethylamino) -N-methylacetamino) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid (13-5,390mg, 1mmol), tert-butyl 4-aminobenzoate (13-6,201mg, 1.04mmol), HATU (402mg, 1.05mmol) dissolved in DMF/dichloromethane (1/1, 6mL), DIPEA (0.8mL) added and stirred at room temperature for 2 hours, water was added to quench the reaction, and the mixture was extracted with dichloromethane, dried, filtered, concentrated and purified by column chromatography to give 1- ((4- (tert-butoxycarbonyl) phenyl) carbamoyl) -5- (2- (dimethylamino) -N-methylacetamido) -3, 4-dihydroisoquinoline-2 (1H) -carboxylic acid tert-butyl ester as a white solid (560 mg).

The fifth step: preparation of tert-butyl 4- (5- (2- (dimethylamino) -N-methylacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate hydrochloride (13-8)

1- ((4- (tert-Butoxycarbonyl) phenyl) carbamoyl) -5- (2- (dimethylamino) -N-methylacetamido) -3, 4-dihydroisoquinoline-2 (1H) -carboxylic acid tert-butyl ester (13-7,560mg) was dissolved in tetrahydrofuran (30mL), ethyl acetate hydrochloride solution (3mol/L, 30mL) was added, stirred at room temperature for 3 hours, concentrated to give a crude product (600mg) which was used directly in the next reaction.

And a sixth step: (E) preparation of tert-butyl (13-9) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2- (dimethylamino) -N-methylacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate

4- (5- (2- (dimethylamino) -N-methylacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid tert-butyl ester hydrochloride (600mg), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (1-7,300mg), HATU (402mg, 1.05mmol) dissolved in DMF/dichloromethane (1/1, 10mL), DIPEA (3mL) added, stirred at room temperature for 2 hours, added with water to quench the reaction, dichloromethane extracted, filtered and concentrated by organic phase drying, and purified by column chromatography to obtain white solid (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) ) Tert-butyl 5- (2- (dimethylamino) -N-methylacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (680 mg).

The seventh step: (E) preparation of (13) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2- (dimethylamino) -N-methylacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid

(E) Tert-butyl (13-9,680mg) of (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2- (dimethylamino) -N-methylacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate was dissolved in dichloromethane (24mL), trifluoroacetic acid (8mL) was added thereto, and the mixture was stirred at room temperature for 2 hours, followed by concentration and purification of the residue to give (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2- (dimethylamino) -N-methylacetamido) as a white solid Amino) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid (548 mg).

¹ HNMR(DMSO-d6 400MHz)δ10.93(s,1H),9.88(s,1H),7.96(t,1H,J＝8.0Hz),7.88(d,2H,J＝8.4Hz),7.76-7.66(m,4H),7.40-7.36(m,1H),7.30-7.27(m,1H),7.03(dd,2H,J＝16.2,55Hz),5.91(d,1H,J＝8.8Hz),4.18-4.05(m,1H),3.69-3.58(m,1H),3.09(s，3H)，2.98-2.87(m,3H),2.76-2.71(m,1H)，2.22(s，3H)，2.14(s，3H)。

MS m/z(ESI):661[M+H]+

Example 14

The compound obtained in example 13 was separated by preparative liquid chromatography to give the compound of example 14.

MS m/z(ESI):661[M+H]+

Example 15

The compound obtained in example 13 was separated by preparative liquid chromatography to give the compound of example 15.

MS m/z(ESI):661[M+H]+

Example 16: (E) preparation of (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2- (dimethylamino) -N-methylacetamido) -N-methyl-1, 2,3, 4-tetrahydroisoquinoline-1-formamido) benzoic acid

The first step is as follows: preparation of tert-butyl 1- ((4- (tert-butoxycarbonyl) phenyl) methylcarbamoyl) -5- (2- (dimethylamino) -N-methylacetamido) -3, 4-dihydroisoquinoline-2 (1H) -carboxylate (16-2)

2- (tert-Butoxycarbonyl) -5- (2- (dimethylamino) -N-methylacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid (390mg, 1mmol), tert-butyl 4-methylaminobenzoate (210mg, 1.04mmol), HATU (402mg, 1.05mmol) dissolved in DMF/dichloromethane (1/1, 6mL), DIPEA (0.8mL) added and stirred at room temperature for 2 hours, water was added to quench the reaction, and the mixture was extracted with dichloromethane, dried, filtered, concentrated and purified by column chromatography to give 1- ((4- (tert-butoxycarbonyl) phenyl) methylcarbamoyl) -5- (2- (dimethylamino) -N-methylacetamido) -3, 4-dihydroisoquinoline-2 (1H) -carboxylic acid tert-butyl ester as a white solid (560 mg).

The second step is that: preparation of tert-butyl 4- (5- (2- (dimethylamino) -N-methylacetamido) -N-methyl-1, 2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate hydrochloride (16-3)

1- ((4- (tert-butoxycarbonyl) phenyl) methylcarbamoyl) -5- (2- (dimethylamino) -N-methylacetamido) -3, 4-dihydroisoquinoline-2 (1H) -carboxylic acid tert-butyl ester (560mg) was dissolved in tetrahydrofuran (30mL), ethyl acetate hydrochloride solution (3mol/L, 30mL) was added, and the mixture was stirred at room temperature for 3 hours and concentrated to give crude 4- (5- (2- (dimethylamino) -N-methylacetamido) -N-methyl-1, 2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid tert-butyl ester hydrochloride (600mg) which was used directly in the next reaction.

The third step: (E) preparation of tert-butyl (16-4) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2- (dimethylamino) -N-methylacetamido) -N-methyl-1, 2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate

4- (5- (2- (dimethylamino) -N-methylacetamino) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid tert-butyl ester hydrochloride (600mg), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (300mg), HATU (402mg, 1.05mmol) was dissolved in DMF/dichloromethane (1/1, 10mL), DIPEA (3mL) was added, the mixture was stirred at room temperature for 2 hours, water was added to the mixture to quench the reaction, dichloromethane was extracted, the organic phase was filtered, concentrated and then purified by column chromatography to obtain (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) compound as a white solid Tert-butyl 5- (2- (dimethylamino) -N-methylacetamido) -N-methyl-1, 2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (650 mg).

The fourth step: (E) preparation of (16) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2- (dimethylamino) -N-methylacetamido) -N-methyl-1, 2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid

(E) Tert-butyl (4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2- (dimethylamino) -N-methylacetamido) -N-methyl-1, 2,3, 4-tetrahydroisoquinoline-1-formamido) benzoate (100mg) was dissolved in dichloromethane (5mL), trifluoroacetic acid (3mL) was added, stirring was carried out at room temperature for 2 hours, concentration was carried out, and the residue was purified to obtain (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2- (dimethylamino) -N-substituted white solid Methylacetylamino) -N-methyl-1, 2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid (54 mg).

¹ HNMR(DMSO-d6 400MHz)δ9.88(s,1H),7.96(t,1H,J＝8.0Hz),7.88(d,2H,J＝8.4Hz),7.76-7.66(m,4H),7.40-7.36(m,1H),7.30-7.27(m,1H),7.03(dd,2H,J＝16.2,55Hz),5.91(d,1H,J＝8.8Hz),4.18-4.05(m,1H),3.69-3.58(m,1H),3.42(s,3H),3.09(s，3H)，2.98-2.87(m,3H),2.76-2.71(m,1H)，2.22(s，3H)，2.14(s，3H)。

MS m/z(ESI):675[M+H]+

Example 17

The compound obtained in example 16 was separated by preparative liquid chromatography to give the compound of example 17.

MS m/z(ESI):675[M+H]+

Example 18

The compound obtained in example 16 was separated by preparative liquid chromatography to give the compound of example 18.

MS m/z(ESI):675[M+H]+

Example 19: (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2-methoxy-N-methylacetamido) -N-methyl-1, 2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid

The first step is as follows: preparation of 5- (2-methoxy-N-methylacetamido) -3, 4-dihydroisoquinoline-dicarboxylic acid-2-tert-butyl ester-1-methyl ester (19-1)

Triethylamine (0.56mL) was added to a solution of 5- (methylamino) -3, 4-dihydroisoquinoline-1, 2(1H) -dicarboxylic acid-2-tert-butyl ester-1-methyl ester (400mg) in methylene chloride (20mL), 2-methoxyacetyl chloride (0.14mL) was added dropwise, and the reaction mixture was stirred at room temperature for 2 hours. Adding water to quench the reaction, separating the organic layer, drying, filtering, concentrating, and purifying the residue by column chromatography to obtain white solid 5- (2-methoxy-N-methylacetamido) -3, 4-dihydroisoquinoline-dicarboxylic acid-2-tert-butyl ester-1-methyl ester 450 mg.

The second step is that: preparation of 2- (tert-butoxycarbonyl) -5- (2-methoxy-N-methylacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid (19-2)

Adding aqueous solution of lithium hydroxide (2mol/L, 0.27mL) and 0.5mL of methanol to a tetrahydrofuran (2mL) solution of 5- (2-methoxy-N-methylacetamido) -3, 4-dihydroisoquinoline-dicarboxylic acid-2-tert-butyl ester-1-methyl ester (103mg), stirring at room temperature for 1.5 hours, adding 1mol/L hydrochloric acid to pH 5, adding 5mL of saturated saline, extracting with dichloromethane, drying and concentrating to obtain a white solid, 2- (tert-butoxycarbonyl) -5- (2-methoxy-N-methylacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid, 100 mg.

The third step: preparation of tert-butyl 1- ((4- (tert-butoxycarbonyl) phenyl) methylcarbamoyl) -5- (2- (methoxy) -N-methylacetamido) -3, 4-dihydroisoquinoline-2 (1H) -carboxylate (19-3)

2- (tert-Butoxycarbonyl) -5- (2- (methoxy) -N-methylacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid (100mg), tert-butyl 4-methylaminobenzoate (55mg), HATU (102mg) was dissolved in DMF/dichloromethane (1/1, 3mL), DIPEA (0.4mL) was added, and the mixture was stirred at room temperature for 2 hours, water was added to quench the reaction, and the mixture was extracted with dichloromethane, dried, filtered, concentrated and purified by column chromatography to give 1- ((4- (tert-butoxycarbonyl) phenyl) methylcarbamoyl) -5- (2- (methyloxy) -N-methylacetamido) -3, 4-dihydroisoquinoline-2 (1H) -carboxylic acid tert-butyl ester as a white solid (120 mg).

The fourth step: preparation of tert-butyl 4- (5- (2- (methoxy) -N-methylacetamido) -N-methyl-1, 2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate hydrochloride (19-4)

1- ((4- (tert-butoxycarbonyl) phenyl) methylcarbamoyl) -5- (2- (methoxy) -N-methylacetamido) -3, 4-dihydroisoquinoline-2 (1H) -carboxylic acid tert-butyl ester (120mg) was dissolved in tetrahydrofuran (3mL), ethyl acetate hydrochloride solution (3mol/L, 3mL) was added, stirred at room temperature for 3 hours, and concentrated to give crude 4- (5- (2- (methoxy) -N-methylacetamido) -N-methyl-1, 2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid tert-butyl ester hydrochloride (130mg) which was used directly in the next step.

The fifth step: (E) preparation of tert-butyl (19-5) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2- (methoxy) -N-methylacetamido) -N-methyl-1, 2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate

4- (5- (2- (methoxy) -N-methylacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid tert-butyl ester hydrochloride (120mg), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (67mg), HATU (91mg) dissolved in DMF/dichloromethane (1/1, 5mL), DIPEA (0.5mL) added, stirred at room temperature for 2 hours, added with water to quench the reaction, dichloromethane extracted, filtered, concentrated by organic phase, and purified by column chromatography to obtain (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (E) -5- (2- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (5-tetrahydroisoquinoline, 5- (1H-tetrazol-1-yl) phenyl) acrylic acid as a white solid Tert-butyl 2- (methoxy) -N-methylacetamido) -N-methyl-1, 2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (100 mg).

And a sixth step: (E) preparation of (19) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2- (methoxy) -N-methylacetamido) -N-methyl-1, 2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid

(E) Tert-butyl (4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2- (methoxy) -N-methylacetamido) -N-methyl-1, 2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (100mg) was dissolved in dichloromethane (5mL), trifluoroacetic acid (3mL) was added thereto, and stirred at room temperature for 2 hours, followed by concentration and purification of the residue to give (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2- (methoxy) -N-methylethyl) acryloyl) -4- (2- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5- (2- (methoxy) -N-methylethyl) as a white solid Amido) -N-methyl-1, 2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid (45 mg).

¹ HNMR(DMSO-d6 400MHz)δ9.87(s,1H),7.94(t,1H,J＝8.0Hz),7.87(d,2H，J＝7.2Hz),7.73-7.67(m,3H),7.36(d,1H,J＝7.6Hz),7.30(d,1H,J＝7.6Hz),7.16(t,1H,J＝7.8Hz),7.04(d,1H,J＝7.6Hz),7.02(dd,2H,J＝15.6,62.4Hz),5.89(d,1H,J＝8.8Hz),4.18-4.09(m,1H),3.71-3.65(m,1H),3.41(s,1H),3.17(s，2H)，3.11(s，2H)，3.07(s，3H)，3.05-2.98(m,1H)，2.67-2.62(m,1H).

MS m/z(ESI):662[M+H]+

Example 20

The compound obtained in example 19 was separated by preparative liquid chromatography to give the compound of example 20.

MS m/z(ESI):662[M+H]+

Example 21

The compound obtained in example 19 was separated by preparative liquid chromatography to give the compound of example 21.

MS m/z(ESI):662[M+H]+

Example 22: (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -N-methyl-5- (4-methyl-2-oxopiperazin-1-yl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid

The first step is as follows: preparation of 5- (4-methyl-2-oxopiperazin-1-yl) -3, 4-dihydroisoquinoline-1, 2(1H) -dicarboxylic acid-2-tert-butyl ester-1-methyl ester (22-2)

In a nitrogen atmosphere, 5-bromo-3, 4-dihydroisoquinoline-1, 2(1H) -dicarboxylic acid-2-tert-butyl ester-1-methyl ester (2.53g), 4-methylpiperazinone (1.017g), CuI (1.065g), phenanthroline (135mg) and potassium carbonate (2.84g) are added in dimethyl sulfoxide (11mL), heated to 130 ℃, and stirred overnight. Adding 3mol/L ammonia water into the reaction liquid, extracting by dichloromethane, drying, filtering, concentrating and purifying by column chromatography to obtain light yellow solid 5- (methylamino) -3, 4-dihydroisoquinoline-1, 2(1H) -dicarboxylic acid-2-tert-butyl ester-1-methyl ester 1.98 g.

The second step is that: preparation of 2- (tert-Butoxycarbonyl) -5- (4-methyl-2-oxopiperazin-1-yl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid (22-3) 5- (4-methyl-2-oxopiperazin-1-yl) -3, 4-dihydroisoquinoline-1, 2(1H) -dicarboxylic acid-2-tert-butyl ester-1-methyl ester (260mg) was dissolved in tetrahydrofuran (3mL), an aqueous solution of lithium hydroxide (1mol/L, 1.3mL) and methanol (2mL) were added thereto, and the mixture was stirred at room temperature for 4 hours, concentrated in an organic solvent, adjusted to pH 5 with 1mol/L hydrochloric acid, extracted with dichloromethane, filtered and concentrated to obtain 2- (tert-butyloxycarbonyl) -5- (4-methyl-2-oxol as a white solid Piperazin-1-yl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid (133 mg).

The third step: preparation of tert-butyl 1- ((4- (tert-butoxycarbonyl) phenyl) methylcarbamoyl) -5- (4-methyl-2-oxopiperazin-1-yl) -3, 4-dihydroisoquinoline-2 (1H) -carboxylate (22-4)

2- (tert-Butoxycarbonyl) -5- (4-methyl-2-oxopiperazin-1-yl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid (133mg), tert-butyl 4-aminobenzoate (85mg), HATU (156mg) was dissolved in DMF/dichloromethane (1/1, 6mL), DIPEA (0.2mL) was added, the mixture was stirred overnight at room temperature, quenched with water, extracted with dichloromethane, the organic phase was dried, filtered, concentrated and purified by column chromatography to give 1- ((4- (tert-butoxycarbonyl) phenyl) methylcarbamoyl) -5- (4-methyl-2-oxopiperazin-1-yl) -3, 4-dihydroisoquinoline-2 (1H) -carboxylic acid tert-butyl ester as a white solid (150 mg).

The fourth step: preparation of tert-butyl 4- (N-methyl-5- (4-methyl-2-oxopiperazin-1-yl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate hydrochloride (22-5)

1- ((4- (tert-butoxycarbonyl) phenyl) methylcarbamoyl) -5- (4-methyl-2-oxopiperazin-1-yl) -3, 4-dihydroisoquinoline-2 (1H) -carboxylic acid tert-butyl ester (150mg) was dissolved in tetrahydrofuran (3mL), ethyl acetate hydrochloride solution (3mol/L, 3mL) was added, and the mixture was stirred at room temperature for 3 hours and concentrated to give crude 4- (N-methyl-5- (4-methyl-2-oxopiperazin-1-yl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid tert-butyl ester hydrochloride (160mg) which was used directly in the next reaction.

The fifth step: (E) preparation of tert-butyl (22-6) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5-N-methyl- (4-methyl-2-oxopiperazin-1-yl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate

Tert-butyl 4- (N-methyl-5- (4-methyl-2-oxopiperazin-1-yl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate hydrochloride (160mg), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (84mg), HATU (119mg) was dissolved in DMF/dichloromethane (1/1, 5mL), DIPEA (0.3mL) was added, and the mixture was stirred at room temperature for 2 hours, adding water to quench the reaction, extracting with dichloromethane, drying the organic phase, filtering, concentrating, and purifying by column chromatography to obtain white solid (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazole-1).

-yl) phenyl) acryloyl) -5-N-methyl- (4-methyl-2-oxopiperazin-1-yl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid tert-butyl ester (145 mg).

And a sixth step: (E) preparation of (4) - (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -N-methyl-5- (4-methyl-2-oxopiperazin-1-yl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid (22)

(E) Tert-butyl (145mg) of (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5-N-methyl- (4-methyl-2-oxopiperazin-1-yl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate was dissolved in dichloromethane (4mL), trifluoroacetic acid (3mL) was added thereto, and the mixture was stirred at room temperature for 3 hours, followed by concentration and purification of the residue to give (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -N-methyl-5- (4-methyl-5-carboxylic acid as a white solid -2-oxopiperazin-1-yl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid (48 mg).

¹ HNMR(DMSO-d6 400MHz)δ9.70(s，1H),7.87(d,2H，J＝8.2Hz),7.87-7.83(m,1H),7.68(d，2H，J＝8.8Hz),7.68-7.62(m,1H),7.58(dd,1H，J＝1.0，8.8Hz,),7.35(t,1H，J＝7.8Hz),7.24(d,1H，J＝7.2Hz),6.98(dd,2H,J＝15.6,62.4Hz),5.89(s,1H),4.07-3.99(m,2H),3.98-3.85(m,4H),3.77-3.69(m,2H),3.42(s,3H),2.87(s,3H),2.66-2.59(m,2H)。

MS m/z(ESI):673[M+H]+

Example 23

The compound obtained in example 22 was separated by preparative liquid chromatography to give the compound of example 23.

MS m/z(ESI):673[M+H]+

Example 24

The compound obtained in example 22 was separated by preparative liquid chromatography to give the compound of example 24.

MS m/z(ESI):673[M+H]+

Example 25: (E) preparation of (E) -4- (5-bromo-2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -N-methyl-1, 2,3, 4-tetrahydroisoquinoline-1-formylamino) benzoic acid

The first step is as follows: preparation of 5-bromo-2- (tert-butoxycarbonyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid (25-1)

5-bromo-3, 4-dihydroisoquinoline-1, 2(1H) -dicarboxylic acid-2-tert-butyl ester-1-methyl ester (370mg) is dissolved in tetrahydrofuran (5mL), aqueous lithium hydroxide (1mol/L, 1.5mL) and 2mL of methanol are added, the mixture is stirred at room temperature for 4 hours, the organic solvent is concentrated, pH is adjusted to 5 with 1mol/L hydrochloric acid, dichloromethane is used for extraction, and the organic phase is dried, filtered and concentrated to obtain a white solid, 5-bromo-2- (tert-butoxycarbonyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid (253 mg).

The second step is that: preparation of 5-bromo-1- ((4- (tert-butoxycarbonyl) phenyl) methylcarbamoyl) -3, 4-dihydroisoquinoline-2 (1H) -carboxylic acid tert-butyl ester (25-2)

5-bromo-2- (tert-butoxycarbonyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid (253mg), tert-butyl 4-aminobenzoate (162mg), HATU (300mg) was dissolved in DMF/dichloromethane (1/1, 10mL), DIPEA (0.8mL) was added, the mixture was stirred at room temperature overnight, quenched with water, extracted with dichloromethane, dried, filtered, concentrated and purified by column chromatography to give 5-bromo-1- ((4- (tert-butoxycarbonyl) phenyl) methylcarbamoyl) -3, 4-dihydroisoquinoline-2 (1H) -carboxylic acid tert-butyl ester as a white solid (298 mg).

The third step: preparation of tert-butyl 4- (5-bromo-N-methyl-1, 2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate hydrochloride (25-3)

5-bromo-1- ((4- (tert-butoxycarbonyl) phenyl) methylcarbamoyl) -3, 4-dihydroisoquinoline-2 (1H) -carboxylic acid tert-butyl ester (298mg) is dissolved in tetrahydrofuran (5mL), ethyl acetate hydrochloride solution (3mol/L, 5mL) is added, stirring is carried out at room temperature for 3 hours, and crude product 4- (5-bromo-N-methyl-1, 2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid tert-butyl ester hydrochloride (300mg) is obtained by concentration and directly used for the next reaction.

The fourth step: (E) preparation of tert-butyl (25-4) -4- (5-bromo-2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -N-methyl-1, 2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate

4- (5-bromo-N-methyl-1, 2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid tert-butyl ester hydrochloride (300mg), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (176mg), HATU (249mg) dissolved in DMF/dichloromethane (1/1, 6mL), DIPEA (0.6mL) added, stirred at room temperature for 2 hours, quenched with water, dichloromethane extracted, dried, filtered, concentrated and purified by column chromatography to obtain (E) -4- (5-bromo-2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -N-methyl-1 as a white solid, tert-butyl 2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (300 mg).

The fifth step: (E) preparation of (e) -4- (5-bromo-2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -N-methyl-1, 2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid (25)

(E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -5-N-methyl-bromo-1, 2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid tert-butyl ester (139mg) was dissolved in dichloromethane (4mL), trifluoroacetic acid (3mL) was added thereto, stirred at room temperature for 3 hours, concentrated, and the residue was purified to give (E) -4- (5-bromo-2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -N-methyl-1 as a white solid, 2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid (48 mg).

¹ HNMR(DMSO-d6 400MHz)δ9.87(s,1H),7.96(t,1H,J＝8.2Hz),7.87(d,2H,J＝8.4Hz),7.67-7.64(m,4H),7.62-7.59(m,2H),7.25(t,1H,J＝7.6Hz),7.05(dd,2H,J＝15.6,75.6Hz),5.88(s,1H),4.16-4.13(m,1H),3.81-3.77(m,1H),3.45(s，3H)，3.10-3.07(m,1H),3.01-2.98(m,1H)。

MS m/z(ESI):638，640[M+H]+

Example 26

The compound obtained in example 25 was separated by preparative liquid chromatography to give the compound of example 26.

MS m/z(ESI):638，640[M+H]+

Example 27

The compound obtained in example 25 was separated by preparative liquid chromatography to give the compound of example 27.

MS m/z(ESI):638，640[M+H]+

Example 28: (E) preparation of (E) -N- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinolin-5-yl) -2-methoxy-N-methylacetamide

2-methoxy-N-methyl-N- (1,2,3, 4-tetrahydroisoquinolin-5-yl) acetamide (10-2, 70mg), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (1-7, 100mg) was dissolved in ethyl acetate (7mL), pyridine (75. mu.L), T3P (50% ethyl acetate solution, 0.36mL) was added, and the mixture was stirred at 60 ℃ overnight. Adding water into the reaction solution for quenching, extracting by dichloromethane, drying, filtering, concentrating and purifying by column chromatography to obtain white solid (E) -N- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazole-1-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-5-yl) -2-methoxy-N-methylacetamide 124mg, wherein the yield is 86%.

¹ HNMR(DMSO-d6 400MHz)δ9.86(s,1H),7.95(t,1H,J＝8.2Hz),7.66(dd,1H,J＝1.2，8.0Hz),7.33-7.29(m,2H),7.22-7.19(m,1H),7.00(dd,2H,J＝15.8,46.2Hz),4.79-4.64(m,2H),3.83-3.67(m,2H),3.49-3.35(m,2H),3.16(s，2H)，3.15(s，2H)，3.06(s，2H)，3.04(s，1H)，2.67-2.62(m,2H).

MS m/z(ESI):485[M+H]+

Example 29: (E) preparation of (E) -N- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -1- (pyrrolidine-1-carbonyl) -1,2,3, 4-tetrahydroisoquinoline-5-yl) -2-methoxy-N-methylacetamide

The first step is as follows: preparation of 5- (2-methoxy-N-methylacetamido) -1- (pyrrolidine-1-carbonyl) -3, 4-dihydroisoquinoline-2 (1H) -carboxylic acid tert-butyl ester (29-1)

2- (tert-Butoxycarbonyl) -5- (2-methoxy-N-methylacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid (19-2,100mg), pyrrolidine (0.045mL) was dissolved in ethyl acetate (4mL), pyridine (65. mu.L), T3P (50% ethyl acetate solution, 0.32mL) were added, and stirring was carried out overnight at 60 ℃. Adding water into the reaction solution for quenching, extracting by dichloromethane, drying, filtering, concentrating and purifying by column chromatography to obtain white solid 5- (2-methoxy-N-methylacetamido) -1- (pyrrolidine-1-carbonyl) -3, 4-dihydroisoquinoline-2 (1H) -tert-butyl formate 49 mg.

The second step is that: preparation of 2-methoxy-N-methyl-N- (1- (pyrrolidine-1-carbonyl) -1,2,3, 4-tetrahydroisoquinolin-5-yl) acetamide (29-2)

5- (2-methoxy-N-methyl acetyl amino) -1- (pyrrolidine-1-carbonyl) -3, 4-two hydrogen isoquinoline-2 (1H) -formic acid tert butyl ester (29-1,49mg) dissolved in dichloromethane (3mL), added with hydrochloric acid 1, 4-dioxane solution (4mol/L, 3mL), room temperature stirring for 2 hours, spin-dry to obtain light yellow solid 2-methoxy-N-methyl-N- (1- (pyrrolidine-1-carbonyl) -1,2,3, 4-four hydrogen isoquinoline-5-yl) acetamide 50 mg.

The third step: (E) preparation of (E) -N- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -1- (pyrrolidine-1-carbonyl) -1,2,3, 4-tetrahydroisoquinolin-5-yl) -2-methoxy-N-methylacetamide (29)

2-methoxy-N-methyl-N- (1- (pyrrolidine-1-carbonyl) -1,2,3, 4-tetrahydroisoquinolin-5-yl) acetamide (29-2,50mg), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (1-7,50mg) was dissolved in ethyl acetate (3mL), pyridine (35. mu.L), T3P (50% ethyl acetate solution, 0.17mL) was added, and stirring was carried out at 60 ℃ overnight. Water is added into the reaction liquid for quenching, dichloromethane is used for extraction, organic phase is dried, filtered, concentrated and purified by column chromatography to obtain white solid (E) -N- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazole-1-yl) phenyl) acryloyl) -1- (pyrrolidine-1-carbonyl) -1,2,3, 4-tetrahydroisoquinoline-5-yl) -2-methoxy-N-methylacetamide 45mg (the yield of the two steps is 68.2%).

¹ HNMR(DMSO-d6 400MHz)δ9.86(s,1H),7.95(t,1H,J＝8.2Hz),7.66(d,1H,J＝8.4Hz),7.33-7.27(m,3H),7.10(dd,1H,J＝5.6,15.6Hz),6.96(dd,1H,J＝1.8,15.8Hz),6.01(d，1H，J＝10.8Hz)，4.09-3.85(m,2H),3.80-3.74(m,2H),3.73-3.38(m,1H),3.48-3.40(m,1H),3.29-3.18(m,2H),3.16(d，3H，J＝1.2Hz)，3.07(d，3H，J＝8.8Hz)，2.99-2.79(m,1H)，2.72-2.60(m,1H)，2.04-1.88(m,2H)，1.84-1.76(m,2H)。

MS m/z(ESI):582[M+H]+

Example 30: (S, E) -4- (2- (3- (3-chloro-2-fluoro-6 (1H-tetrazol-1-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-formamido) benzoic acid

The first step is as follows: preparation of methyl 2- (benzyloxycarbonyl) - (S) -5-bromo-3, 4-dihydroisoquinoline-1, 2(1H) -carboxylate (30-2)

(S) -5-bromo-3, 4-dihydroisoquinoline-1, 2(1H) -carboxylic acid methyl ester hydrochloride (30-1,1g,3.26mmol), sodium bicarbonate (959mg,11.42mmol) were added to a mixed system of dioxane (10mL) and water (5mL) and stirred uniformly, benzyl chloroformate (612mg, 3.59mmol) was added and the reaction was stirred at room temperature for 4 hours, and the reaction was checked by LCMS to be complete. Adding 50mL of water, extracting with ethyl acetate, collecting an organic phase, washing, drying, concentrating, and purifying by column chromatography to obtain pure 2- (benzyloxycarbonyl) - (S) -5-bromo-3, 4-dihydroisoquinoline-1, 2(1H) -methyl formate (1.297g, yield 98%).

MS m/z(ESI):404,406[M+H]+

The second step is that: preparation of 2- (benzyloxycarbonyl) - (S) -5-bromo-3, 4-dihydroisoquinoline-1, 2(1H) -carboxylic acid (30-3)

Methyl 2- (benzyloxycarbonyl) - (S) -5-bromo-3, 4-dihydroisoquinoline-1, 2(1H) -carboxylate (30-2,1.297g,3.21mmol) was added to a mixed solvent of tetrahydrofuran (20mL) and water (20mL) and stirred well, then lithium hydroxide monohydrate (404mg,9.62mmol) was added thereto, and the reaction was stirred for 2 hours and was determined to be complete by LCMS. Adding 50mL of water, extracting with ethyl acetate, collecting an organic phase, washing, drying and concentrating to obtain a crude product of 2- (benzyloxycarbonyl) - (S) -5-bromo-3, 4-dihydroisoquinoline-1, 2(1H) -methyl formate (1.197g, yield 96%), and directly putting into the next step.

MS m/z(ESI):390,392[M+H]+

The third step: preparation of benzyl (S) -5-bromo-1- ((4- (tert-butoxycarbonyl) phenyl) carbamoyl) -3, 4-dihydroisoquinoline-2 (1H) -carboxylate (30-4)

Methyl 2- (benzyloxycarbonyl) - (S) -5-bromo-3, 4-dihydroisoquinoline-1, 2(1H) -carboxylate (30-3,1.197g,3.07mmol), tert-butyl 4-aminobenzoate (652mg,3.37mmol), HATU (1.4g,3.68mmol) and diisopropylethylamine (1.01mL,6.13mmol) were added to DCM (30mL) and stirred for 2H, LCMS checked for completion of the reaction. Spin-dry the solvent, add 100mL water, extract with ethyl acetate, collect the organic phase and wash, dry, concentrate, purify by column chromatography to get pure benzyl (S) -5-bromo-1- ((4- (tert-butoxycarbonyl) phenyl) carbamoyl) -3, 4-dihydroisoquinoline-2 (1H) -formate (1.55g, 89% yield).

MS m/z(ESI):563,565[M-H]-

The fourth step: preparation of tert-butyl (S) -4- (1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (30-5)

Benzyl (S) -5-bromo-1- ((4- (tert-butoxycarbonyl) phenyl) carbamoyl) -3, 4-dihydroisoquinoline-2 (1H) -carboxylate (30-4,200mg, 354. mu. mol) was added to methanol (50mL), Pd-C (100mg) was added, hydrogen was purged three times from the reaction system, the reaction was overnight under a hydrogen atmosphere, and the reaction was detected to be complete by LCMS. Filtration and concentration of the filtrate gave a pure product of tert-butyl (S) -4- (1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (125mg, yield 100%).

MS m/z(ESI):353[M+H]+

The fifth step: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6 (1H-tetrazol-1-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-formamido) benzoic acid tert-butyl ester (30-6)

Tert-butyl (S) -4- (1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (125mg, 355. mu. mol), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (105mg, 390. mu. mol), HATU (162mg, 426. mu. mol) and diisopropylethylamine (176. mu.l, 1.06mmol) were added to DCM (4mL) and stirred for a uniform reaction for 2 hours, and LCMS detected reaction completion. Spin-drying solvent, adding 20mL of water, extracting with ethyl acetate, collecting organic phase, washing, drying, concentrating, and purifying by column chromatography to obtain pure tert-butyl (S, E) -4- (2- (3- (3-chloro-2-fluoro-6 (1H-tetrazol-1-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-formylamino) benzoate (193mg, yield 90%).

MS m/z(ESI):410[M-192]+

And a sixth step: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6 (1H-tetrazol-1-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-formamido) benzoic acid (30)

Tert-butyl (S, E) -4- (2- (3- (3-chloro-2-fluoro-6 (1H-tetrazol-5-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (173mg, 287. mu. mol) was added to DCM (10mL), trifluoroacetic acid (5mL) was added, the reaction was stirred for 2 hours, and LCMS was performed to detect completion of the reaction. Spin-dry the solvent, prepare thin-layer chromatography and purify to obtain pure (S, E) -4- (2- (3- (3-chloro-2-fluoro-6 (1H-tetrazol-1-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-formylamino) benzoic acid (55mg, yield 35%).

MS m/z(ESI):545[M-1]-

Example 31: (S, E) -4- (2- (3- (5-chloro-2- (4-chloro-1H-1, 2, 3-triazole-1-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-formamido) benzoic acid

The first step is as follows: preparation of (S, E) -4- (2- (3- (5-chloro-2- (4-chloro-1H-1, 2, 3-triazole-1-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-formamido) tert-butyl benzoate (31-1)

Tert-butyl (S) -4- (1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (30-5,187mg, 531. mu. mol), (E) -3- (5-chloro-2- (4-chloro-1H-1, 2, 3-triazol-1-yl) phenyl) acrylic acid (31-2, 166mg, 584. mu. mol), HATU (242mg, 637. mu. mol) and diisopropylethylamine (263. mu.l, 1.59mmol) were added to DCM (5mL) and stirred for a uniform reaction for 2 hours, and the reaction was determined to be complete by LCMS. Spin-drying solvent, adding 20mL of water, extracting with ethyl acetate, collecting organic phase, washing, drying, concentrating, and purifying by column chromatography to obtain pure product (S, E) -4- (2- (3- (5-chloro-2- (4-chloro-1H-1, 2, 3-triazole-1-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-formamido) benzoic acid tert-butyl ester (270mg, yield 82%).

MS m/z(ESI):425[M-192]+

The second step is that: preparation of (S, E) -4- (2- (3- (5-chloro-2- (4-chloro-1H-1, 2, 3-triazole-1-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-formamido) benzoic acid (31)

Tert-butyl (S, E) -4- (2- (3- (5-chloro-2- (4-chloro-1H-1, 2, 3-triazol-1-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (31-1,270mg, 437. mu. mol) was added to DCM (10mL), trifluoroacetic acid (5mL) was added thereto, the reaction was stirred for 2 hours, and the reaction was detected to be complete by LCMS. The solvent was dried by spinning, and purified by thin layer chromatography to obtain pure (S, E) -4- (2- (3- (5-chloro-2- (4-chloro-1H-1, 2, 3-triazol-1-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid (50mg, yield 20%).

¹ HNMR(DMSO-d6 400MHz)δ12.67(brs，1H)，10.79(s,1H),8.87(s，1H)，8.42(d，1H，1.8Hz)，7.86(d,2H,J＝8.8Hz),7.72(dd,1H,J＝1.8，8.6Hz),7.68(d,2H,J＝7.6Hz),7.60(d,1H,J＝7.2Hz),7.63(t,1H,J＝4.4Hz),7.30-7.25(m,3H),7.29(dd,2H,J＝15.8,205.2Hz),5.82(s,1H),4.37-4.31(m,1H),3.91-3.85(m,1H),3.23-3.16(m,1H),2.95-2.89(m,1H)。

MS m/z(ESI):560[M-1]-

Example 32: (S, E) -4- (2- (3- (3-chloro-6- (4-chloro-1H-1, 2, 3-triazole-1-yl) -2-fluorophenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-formamido) benzoic acid

The first step is as follows: preparation of (S, E) -4- (2- (3- (3-chloro-6- (4-chloro-1H-1, 2, 3-triazole-1-yl) -2-fluorophenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-formamido) tert-butyl benzoate (32-1)

Tert-butyl (S) -4- (1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (30-5,187mg, 531. mu. mol), (E) -3- (3-chloro-6- (4-chloro-1H-1, 2, 3-triazol-1-yl) -2-fluorophenyl) acrylic acid (176mg, 584. mu. mol), HATU (242mg, 637. mu. mol) and diisopropylethylamine (263. mu.l, 1.59mmol) were added to DCM (5mL) and stirred for a uniform reaction for 2 hours, and the reaction was determined to be complete by LCMS. And (3) drying the solvent, adding 20mL of water, extracting with ethyl acetate, collecting an organic phase, washing, drying, concentrating, and purifying by column chromatography to obtain a pure product of tert-butyl (S, E) -4- (2- (3- (3-chloro-6- (4-chloro-1H-1, 2, 3-triazole-1-yl) -2-fluorophenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-formylamino) benzoate (242mg, yield 72%).

MS m/z(ESI):443[M-192]+

The second step is that: preparation of (S, E) -4- (2- (3- (3-chloro-6- (4-chloro-1H-1, 2, 3-triazole-1-yl) -2-fluorophenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-formamido) benzoic acid (32)

Tert-butyl (S, E) -4- (2- (3- (3-chloro-6- (4-chloro-1H-1, 2, 3-triazol-1-yl) -2-fluorophenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (32-1,242mg, 380. mu. mol) was added to DCM (10mL), trifluoroacetic acid (5mL) was added, the reaction was stirred for 2 hours, and the reaction was detected by LCMS to be complete. The solvent was dried by spinning, and purified by preparative thin layer chromatography to give pure (S, E) -4- (2- (3- (3-chloro-6- (4-chloro-1H-1, 2, 3-triazol-1-yl) -2-fluorophenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid (50mg, yield 23%).

¹ HNMR(DMSO-d6,400MHz)δ12.70(brs,1H),10.79(s,1H),8.89(s,1H),7.92(t,1H,J＝8.2Hz),7.86(d,2H,J＝8.8Hz),7.67(d,2H,J＝8.8Hz),7.64-7.60(m,2H),7.27-7.24(m,4H),7.05(dd,2H,J＝15.6,71.6Hz),5.81(s,1H),4.14-4.10(m,1H),3.69-3.63(m,1H),3.23-3.16(m,1H),2.91-2.85(m,1H)。

MS m/z(ESI):578[M-1]-

Example 33: (S, E) -4- (2- (3- (3-chloro-2-fluoro-6 (1H-tetrazol-1-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid tert-butyl ester

The title compound was prepared according to the fifth step of the synthesis of example 30.

¹ HNMR(DMSO-d6,400MHz)δ10.86(s,1H),9.93(s,1H),8.01(t,1H,J＝8.2Hz),7.89(d,2H,J＝8.8Hz),7.76-7.67(m,4H),7.33-7.30(m,3H),7.10(dd,2H,J＝16.0,65.6Hz),5.87(s,1H),4.21-4.18(m,1H),3.74-3.69(m,1H),3.28-3.21(m,1H),2.96-2.90(m,1H)，1.58(s，9H)。

MS m/z(ESI):603[M+H]+。

Example 34: (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid ((isopropoxycarbonyl) oxy) methyl ester

Adding (S, E) -4- (2- (3- (3-chloro-2-fluoro-6 (1H-tetrazol-1-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-formamido) benzoic acid (30,50mg,91 mu mol), potassium carbonate (38mg,274 mu mol) and potassium iodide (15mg,91 mu mol) into DMF (2mL), pumping nitrogen, adding chloromethyl isopropyl carbonate (21mg,137 mu mol) in nitrogen atmosphere, stirring uniformly, reacting at 60 ℃ for 16 hours, and detecting the reaction completion by LCMS. Spin-dry solvent, add 10mL water, ethyl acetate extraction, collect organic phase washing, drying, concentration, preparation thin layer chromatography purification to get pure (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazole-1-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-formyl amino) benzoic acid ((isopropoxycarbonyl) oxy) methyl ester 35 mg.

MS m/z(ESI):663[M+1]+

Example 35: (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid 1- ((ethoxycarbonyl) oxy) ethyl ester

Adding (S, E) -4- (2- (3- (3-chloro-2-fluoro-6 (1H-tetrazol-1-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-formamido) benzoic acid (30,50mg,91 mu mol), potassium carbonate (38mg,274 mu mol) and potassium iodide (15mg,91 mu mol) into DMF (2mL), replacing nitrogen, adding 1-chloroethyl ethyl carbonate (21mg,137 mu mol) under nitrogen atmosphere, stirring uniformly, reacting at 60 ℃ for 16 hours, and detecting the reaction completion by LCMS. Spin-dry the solvent, add 10mL water, ethyl acetate extraction, collect organic phase washing, drying, concentrate, prepare thin layer chromatography purification to get pure (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazole-1-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-formyl amino) benzoic acid ((isopropyl carbonyl) oxy) methyl ester 45 mg.

MS m/z(ESI):663[M+1]+

Example 36: 4- ((S) -2- ((E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid 1- ((isopropoxycarbonyl) oxy) ethyl ester

Adding (S, E) -4- (2- (3- (3-chloro-2-fluoro-6 (1H-tetrazol-1-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-formamido) benzoic acid (30,50mg,91 mu mol), potassium carbonate (38mg,274 mu mol) and potassium iodide (15mg,91 mu mol) into DMF (2mL), changing nitrogen, adding 1-chloroethyl isopropyl carbonate (21mg,137 mu mol) under nitrogen atmosphere, stirring uniformly, reacting at 60 ℃ for 16 hours, and detecting the reaction completion by LCMS. And (3) drying the solvent by spinning, adding 10mL of water, extracting by ethyl acetate, collecting an organic phase, washing, drying, concentrating, and preparing a pure product of 1- ((isopropyloxycarbonyl) oxy) ethyl 4- ((S) -2- ((E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-formamido) benzoate (40mg, yield 66%).

¹ HNMR(DMSO-d6,400MHz)δ10.89(s,1H),9.87(s,1H),7.96(t,1H,J＝8.2Hz),7.90(d,2H,J＝8.8Hz),7.73(d,2H,8.8Hz),7.66(dd,1H,J＝1.2,8.8Hz),7.63-7.61(m,1H),7.27-7.24(m,3H),7.04(dd,2H,J＝15.8,66.4Hz),6.84(q,1H,J＝5.2Hz),5.81(s,1H),4.15(q,2H,J＝7.2Hz),4.14-4.12(m,1H),3.68-3.62(m,1H),3.22-3.15(m,1H),2.91-2.84(m,1H),1.56(d,3H,J＝4.2Hz),1.20(t-d,3H,J＝0.8,7.6Hz)。

MS m/z(ESI):529[M-133]+

Example 37: (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid 1- (((cyclohexyloxy) carbonyl) oxy) ethyl ester

Adding (S, E) -4- (2- (3- (3-chloro-2-fluoro-6 (1H-tetrazol-1-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-formamido) benzoic acid (30,50mg,91 mu mol), potassium carbonate (38mg,274 mu mol) and potassium iodide (15mg,91 mu mol) into DMF (2mL), replacing nitrogen, adding 1-chloroethyl cyclohexyl carbonate (23mg,137 mu mol) in nitrogen atmosphere, stirring uniformly, reacting at 60 ℃ for 16 hours, and detecting the reaction completion by LCMS. And (3) drying the solvent by spinning, adding 10mL of water, extracting by ethyl acetate, collecting an organic phase, washing, drying, concentrating, preparing a thin layer chromatography, and purifying to obtain a pure product of 1- (((cyclohexyloxy) carbonyl) oxy) ethyl 4- ((S) -2- ((E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-formamido) benzoate (43 mg).

MS m/z(ESI):717[M+1]+

Example 38: (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid (5-methyl-2-oxo-1, 3-dioxol-4-yl) methyl ester

Adding (S, E) -4- (2- (3- (3-chloro-2-fluoro-6 (1H-tetrazol-1-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-formamido) benzoic acid (30,50mg,91 mu mol), potassium carbonate (38mg,274 mu mol) and potassium iodide (15mg,91 mu mol) into DMF (2mL), pumping nitrogen, adding 4-chloromethyl-5-methyl-2-dioxole (20mg,137 mu mol) under nitrogen atmosphere, stirring uniformly, reacting at 60 ℃ for 16 hours, and detecting complete reaction by LCMS. Spin-dry solvent, add 10mL water, ethyl acetate extraction, collect organic phase washing, drying, concentrate, prepare thin layer chromatography purification to get pure (E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazole-1-yl) phenyl) acryloyl) -1,2,3, 4-tetrahydroisoquinoline-1-formamido) benzoic acid (5-methyl-2-oxo-1, 3-dioxol-4-yl) methyl ester (33 mg).

MS m/z(ESI):659[M+1]+

Example 39: 4- ((S) -2- ((1R,4R) -4- (aminomethyl) cyclohexane-1-carbonyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid

The first step is as follows: preparation of tert-butyl 4- ((S) -2- ((1R,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carbonyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (39-2)

Tert-butyl (S) -4- (1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (30-5,178mg), and (1r,4r) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carboxylate (39-1, 129mg) were dissolved in ethyl acetate (3mL), DIPEA (0.59mL) and T3P (50% ethyl acetate solution, 0.59mL) were added, and the mixture was stirred at 55 ℃ overnight. Water was added to the reaction solution to quench, followed by extraction with ethyl acetate, drying of the organic phase, filtration, concentration and purification by column chromatography to give tert-butyl 4- ((S) -2- ((1r,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carbonyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (39-2) as a white solid (165 mg).

The second step is that: preparation of 4- ((S) -2- ((1R,4R) -4- (aminomethyl) cyclohexane-1-carbonyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid (39)

Tert-butyl 4- ((S) -2- ((1r,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carbonyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (39-2) (67mg) was dissolved in methylene chloride (1mL), and trifluoroacetic acid (1mL) was added to stir at room temperature for 2 hours, followed by concentration and purification by preparative thin layer chromatography to give 21mg of 4- ((S) -2- ((1r,4r) -4- (aminomethyl) cyclohexane-1-carbonyl) -1,2,3, 4-tetrahydroquinoline-1-carboxamido) benzoic acid as a white solid.

MS m/z(ESI):436[M+1]+

Example 40: 4- ((S) -2- ((1R,4R) -4- (aminomethyl) cyclohexane-1-carbonyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid ethyl ester

The first step is as follows: preparation of benzyl (S) -5-bromo-1- ((4- (ethoxycarbonyl) phenyl) carbamoyl) -3, 4-dihydroisoquinoline-2 (1H) -carboxylate (40-1)

Methyl 2- (benzyloxycarbonyl) - (S) -5-bromo-3, 4-dihydroisoquinoline-1, 2(1H) -carboxylate (30-3,1.197g,3.07mmol), ethyl 4-aminobenzoate (652mg,3.37mmol), HATU (1.4g,3.68mmol) and diisopropylethylamine (1.01mL,6.13mmol) were added to DCM (30mL) and stirred for 2H, and the reaction was determined to be complete by LCMS. Spin-dry the solvent, add 100mL water, extract with ethyl acetate, collect the organic phase and wash, dry, concentrate, purify by column chromatography to get pure benzyl (S) -5-bromo-1- ((4- (ethoxycarbonyl) phenyl) carbamoyl) -3, 4-dihydroisoquinoline-2 (1H) -formate (1.55g, 89% yield).

The second step is that: preparation of ethyl (S) -4- (1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (40-2)

Benzyl (S) -5-bromo-1- ((4- (ethoxycarbonyl) phenyl) carbamoyl) -3, 4-dihydroisoquinoline-2 (1H) -formate (40-1,200mg, 354. mu. mol) is added into methanol (50mL), then Pd-C (100mg) is added, hydrogen is pumped out from the reaction system for three times, the reaction is carried out overnight under hydrogen atmosphere, and the reaction is completely detected by LCMS. Filtration and concentration of the filtrate gave a pure product of ethyl (S) -4- (1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (125mg, yield 100%).

The third step: preparation of ethyl 4- ((S) -2- ((1R,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carbonyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (40-3)

Ethyl (S) -4- (1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (40-2,125mg), ethyl (1r,4r) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carboxylate (39-1, 99mg) was dissolved in ethyl acetate (3mL), DIPEA (0.4mL) and T3P (50% ethyl acetate solution, 0.4mL) were added, and the mixture was stirred at 55 ℃ overnight. Water was added to the reaction solution to quench, followed by extraction with ethyl acetate, organic phase was dried, filtered, concentrated and purified by column chromatography to obtain 159mg of ethyl 4- ((S) -2- ((1R,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carbonyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate as a white solid.

The fourth step: preparation of ethyl 4- ((S) -2- ((1R,4R) -4- (aminomethyl) cyclohexane-1-carbonyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (40)

Ethyl 4- ((S) -2- ((1R,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carbonyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (40-3, 159mg) was added to DCM (5mL), trifluoroacetic acid (3mL) was added, the reaction was stirred for 2 hours, and completion of the reaction was detected by LCMS. Solvent was dried by spinning, and purified by thin layer chromatography to obtain ethyl 4- ((S) -2- ((1R,4R) -4- (aminomethyl) cyclohexane-1-carbonyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate 75 mg.

MS m/z(ESI):464[M+1]+

Example 41: 4- ((S) -2- ((1R,4R) -4- (aminomethyl) cyclohexane-1-carbonyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid methyl ester

The first step is as follows: preparation of benzyl (S) -5-bromo-1- ((4- (methoxycarbonyl) phenyl) carbamoyl) -3, 4-dihydroisoquinoline-2 (1H) -formate (41-1)

Methyl 2- (benzyloxycarbonyl) - (S) -5-bromo-3, 4-dihydroisoquinoline-1, 2(1H) -carboxylate (30-3,389mg,1mmol), methyl 4-aminobenzoate (151mg,1mmol), HATU (0.382g,1mmol) and diisopropylethylamine (0.5mL) were added to DCM (10mL) and the reaction was stirred for 2 hours at room temperature and was checked for completion by LCMS. Spin-drying solvent, adding 50mL water, extracting with ethyl acetate, collecting organic phase, washing, drying, concentrating, purifying by column chromatography to obtain pure benzyl (S) -5-bromo-1- ((4- (methoxycarbonyl) phenyl) carbamoyl) -3, 4-dihydroisoquinoline-2 (1H) -formate (420mg, yield 80%).

The second step is that: preparation of methyl (S) -4- (1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (41-2)

Benzyl (S) -5-bromo-1- ((4- (methoxycarbonyl) phenyl) carbamoyl) -3, 4-dihydroisoquinoline-2 (1H) -formate (41-1,210mg) was added to methanol (10mL), Pd-C (20mg) was then added, the reaction system was purged with hydrogen three times, reacted overnight under a hydrogen atmosphere, and LCMS detected that the reaction was complete. Filtration and concentration of the filtrate gave a pure product of methyl (S) -4- (1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (124 mg).

The third step: preparation of methyl 4- ((S) -2- ((1R,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carbonyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (41-3)

Ethyl (S) -4- (1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (41-2,124mg), ethyl (1r,4r) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carboxylate (39-1, 103mg) was dissolved in ethyl acetate (3mL), DIPEA (0.4mL) and T3P (50% ethyl acetate solution, 0.4mL) were added, and the mixture was stirred at 55 ℃ overnight. Water was added to the reaction solution to quench, followed by extraction with ethyl acetate, drying of the organic phase, filtration, concentration and purification by column chromatography to give 150mg of methyl 4- ((S) -2- ((1R,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carbonyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate as a white solid.

The fourth step: preparation of methyl 4- ((S) -2- ((1R,4R) -4- (aminomethyl) cyclohexane-1-carbonyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (41)

Ethyl 4- ((S) -2- ((1R,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carbonyl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (41-3, 150mg) was added to DCM (5mL), trifluoroacetic acid (3mL) was added, the reaction was stirred for 2 hours, and completion of the reaction was detected by LCMS. Solvent is dried by spinning, and the methyl 4- ((S) -2- ((1R,4R) -4- (aminomethyl) cyclohexane-1-carbonyl) -1,2,3, 4-tetrahydroisoquinoline-1-formamido) benzoate is obtained by purifying the solvent by Thin Layer Chromatography (TLC).

MS m/z(ESI):450[M+1]+

Example 42: 4- (2- ((1R,4R) -4- (aminomethyl) cyclohexane-1-carbonyl) -5- (2- (dimethylamino) -N-methylacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid

The first step is as follows: preparation of tert-butyl 4- (2- ((1R,4R) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carbonyl) -5- (2- (dimethylamino) -N-methylacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (42-1)

Tert-butyl 4- (5- (2- (dimethylamino) -N-methylacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (13-8,80mg), 4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carboxylic acid (39-1, 51mg), HATU (79mg) and diisopropylethylamine (0.1mL) were added to dichloromethane (3mL) and the mixture was stirred at room temperature for 2 hours. Adding water, extracting with dichloromethane, collecting organic phase, washing, drying, concentrating, purifying by column chromatography to obtain tert-butyl 4- (2- ((1r,4r) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carbonyl) -5- (2- (dimethylamino) -N-methylacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-formylamino) benzoate (102 mg).

The second step is that: preparation of 4- (2- ((1R,4R) -4- (aminomethyl) cyclohexane-1-carbonyl) -5- (2- (dimethylamino) -N-methylacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoic acid (42)

Tert-butyl 4- (2- ((1R,4R) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carbonyl) -5- (2- (dimethylamino) -N-methylacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (42-1, 102mg) was added to methylene chloride (5mL), trifluoroacetic acid (3mL) was added thereto, the reaction was stirred for 2.5 hours, and the reaction was completed by LCMS. The solvent was dried by spinning, and purified by preparative thin layer chromatography to give methyl 4- ((S) -2- ((1R,4R) -4- (aminomethyl) cyclohexane-1-carbonyl) -1,2,3, 4-tetrahydroquinoline-1-carboxamido) benzoate as a pale yellow solid (85 mg).

MS m/z(ESI):550[M+1]+

Example 43: preparation of ethyl 4- (2- ((1R,4R) -4- (aminomethyl) cyclohexane-1-carbonyl) -5- (2- (dimethylamino) -N-methylacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (43)

The first step is as follows: synthesis of ethyl 1- ((4- (tert-butoxycarbonyl) phenyl) carbamoyl) -5- (2- (dimethylamino) -N-methylacetamido) -3, 4-dihydroisoquinoline-2 (1H) -carboxylate (43-1)

2- (tert-Butoxycarbonyl) -5- (2- (dimethylamino) -N-methylacetamino) -1,2,3, 4-tetrahydroisoquinoline-1-carboxylic acid (13-5,390mg, 1mmol), ethyl 4-aminobenzoate (13-6,165mg, 1.04mmol), HATU (402mg, 1.05mmol) dissolved in DMF/dichloromethane (1/1, 6mL), DIPEA (0.8mL) added and stirred at room temperature for 2 hours, water was added to quench the reaction, and the mixture was extracted with dichloromethane, dried, filtered, concentrated and purified by column chromatography to give ethyl 1- ((4- (tert-butoxycarbonyl) phenyl) carbamoyl) -5- (2- (dimethylamino) -N-methylacetamido) -3, 4-dihydroisoquinoline-2 (1H) -carboxylate (520mg) as a white solid.

The second step is that: preparation of ethyl 4- (5- (2- (dimethylamino) -N-methylacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate hydrochloride (43-2)

Ethyl 1- ((4- (tert-butoxycarbonyl) phenyl) carbamoyl) -5- (2- (dimethylamino) -N-methylacetamido) -3, 4-dihydroisoquinoline-2 (1H) -carboxylate (43-1,530mg) was dissolved in tetrahydrofuran (10mL), and ethyl acetate hydrochloride solution (3mol/L, 10mL) was added thereto, followed by stirring at room temperature for 3 hours and concentration to give a crude product (600mg) which was used directly in the next reaction.

The third step: preparation of ethyl 4- (2- ((1R,4R) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carbonyl) -5- (2- (dimethylamino) -N-methylacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (43-3)

Ethyl 4- (5- (2- (dimethylamino) -N-methylacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate hydrochloride (43-2,80mg), (1r,4r) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carboxylic acid (39-1, 51mg), HATU (79mg) and diisopropylethylamine (0.1mL) were added to dichloromethane (3mL) and the mixture was stirred at room temperature for reaction for 2 hours. Adding water, extracting with dichloromethane, collecting organic phase, washing, drying, concentrating, purifying by column chromatography to obtain ethyl 4- (2- ((1R,4R) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carbonyl) -5- (2- (dimethylamino) -N-methylacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-formylamino) benzoate (120 mg).

The second step is that: preparation of ethyl 4- (2- ((1R,4R) -4- (aminomethyl) cyclohexane-1-carbonyl) -5- (2- (dimethylamino) -N-methylacetamido) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (43)

Tert-butyl 4- (2- ((1r,4r) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carbonyl) -5- (2- (dimethylamino) -N-methylacetamino) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamido) benzoate (42-1, 120mg) was added to methylene chloride (5mL), trifluoroacetic acid (3mL) was added thereto, and the reaction was stirred for 2.5 hours and checked for completion by LCMS. The solvent was dried by spinning and purified by preparative thin layer chromatography to give 78mg of methyl 4- ((S) -2- ((1r,4r) -4- (aminomethyl) cyclohexane-1-carbonyl) -1,2,3, 4-tetrahydroquinoline-1-carboxamido) benzoate as a pale yellow solid.

MS m/z(ESI):577[M+1]+

Example 44: (E) preparation of- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -N- (4-difluoromethoxyphenyl) -5- (4-methyl-2-oxopiperazin-1-yl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamide

The first step is as follows: preparation of 1- (isoquinolin-5-yl) -4-methyloxopiperazin-2-one (44-1)

Under a nitrogen atmosphere, 5-bromoisoquinoline (7-1, 1.04g), 4-methylpiperazinone (22-1, 798mg), CuI (762mg), phenanthroline (101mg) and potassium carbonate (2.26g) are placed in dimethyl sulfoxide (8mL), heated to 130 ℃ and stirred overnight. Adding 3mol/L ammonia water into the reaction solution, extracting by dichloromethane, drying, filtering, concentrating and purifying by column chromatography to obtain light yellow solid 1- (isoquinoline-5-yl) -4-methyl oxo-piperazine-2-ketone 0.84 g.

The second step is that: preparation of 4-methyl-1- (1,2,3, 4-tetrahydroisoquinolin-5-yl) piperidin-2-one (44-2)

1- (isoquinoline-5-yl) -4-methyloxopiperazin-2-one (44-1, 590mg) was dissolved in ethanol (20mL), and platinum dioxide (102mg) was added thereto under 2MPa hydrogen atmosphere, followed by stirring at room temperature overnight, filtration and concentration to give 4-methyl-1- (1,2,3, 4-tetrahydroisoquinolin-5-yl) piperidin-2-one (503mg) as a yellow oil.

The third step: preparation of 1- (3, 4-dihydroisoquinolin-5-yl) -4-methylpiperidin-2-one (44-3)

4-methyl-1- (1,2,3, 4-tetrahydroisoquinolin-5-yl) piperidin-2-one (44-2, 503mg) was dissolved in methylene chloride (15mL), and manganese dioxide (1.42g) was added thereto, followed by stirring at room temperature overnight. And (5) filtering. The filtrate was concentrated to give 1- (3, 4-dihydroisoquinolin-5-yl) -4-methylpiperidin-2-one 450mg as a yellow oil.

The fourth step: (E) preparation of- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -N- (4-difluoromethoxyphenyl) -5- (4-methyl-2-oxopiperazin-1-yl) -1,2,3, 4-tetrahydroisoquinoline-1-carboxamide (44)

In a closed tube reactor, 1- (3, 4-dihydroisoquinolin-5-yl) -4-methylpiperidin-2-one (44-3, 100mg), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (1-7, 110mg), and 1- (difluoromethoxy) -4-isocyanobenzene (44-4, 69mg) were dissolved in methanol (3mL), and the reaction solution was heated to 65 ℃ and stirred for 48 hours. And (5) performing column chromatography purification to cool to room temperature to obtain white solid (E) - (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazole-1-yl) phenyl) acryloyl) -N- (4-difluoromethoxyphenyl) -5 (4-methyl 2-oxo-piperazin-1-yl) -1,2,3, 4-tetrahydroisoquinoline-1-formamide 75 mg.

1HNMR(DMSO-d6 400MHz)δ10.89-10.81(m，1H)，9.87(s，1H),7.96(t,1H，J＝8.0Hz),7.87(d,2H，J＝7.2Hz),7.73-7.67(m,4H),7.38(t，1H，J＝7.6Hz),7.30(d,1H，J＝7.6Hz),7.05-7.04(m，1H)，7.04(dd,2H,J＝15.6,62.4Hz),5.89(d,1H，J＝9.6Hz),4.07-3.99(m,2H),3.98-3.85(m,4H),3.77-3.69(m,2H),3.09(s,3H),2.66-2.59(m,2H)。

MS m/z(ESI):681[M+H]+

Preparation of intermediate 1- (difluoromethoxy) -4-isocyanobenzene (44-4)

The first step is as follows: preparation of N- (4- (difluoromethoxy) phenyl) carboxamide (44-6)

4-Difluoromethoxyaniline (44-5, 1.60g), DMAP (255mg), N-methylmorpholine (2.2mL), and formic acid (1.5mL) were added to dichloromethane (25mL) and stirred at room temperature overnight. Formic acid (1.0mL) was added and the mixture was refluxed for 5 hours. Cooled to room temperature, washed once with 1mol/L dilute hydrochloric acid, the organic layer separated, dried, filtered and concentrated to obtain 2.16g of brown oily N- (4- (difluoromethoxy) phenyl) formamide.

The second step is that: preparation of 1- (difluoromethoxy) -4-isocyanobenzene

N- (4- (difluoromethoxy) phenyl) carboxamide (44-6, 1.87g) was dissolved in methylene chloride (10mL), triethylamine (5mL) was added, and phosphorus oxychloride (1.4mL) was added dropwise and stirred at room temperature for 2 hours. Water was added to the reaction mixture to quench, followed by extraction with dichloromethane, and the organic layer was dried, filtered, concentrated and purified by column chromatography to give 850mg of 1- (difluoromethoxy) -4-isocyanobenzene as a yellow oil.

Example 45: 4- ((2S,4R) -1- ((1R,4S) -4- (aminomethyl) cyclohexyl-1-carbonyl) -4-cyclohexylpyrrolidine-2-carboxamido) benzoic acid

The first step is as follows: preparation of (2S,4R) -1-2-methyl-4- (p-toluenesulfonic acid) pyrrolidine-1, 2-dicarboxylic acid tert-butyl ester (45-2)

(2S,4R) -1-2-methyl-4-hydroxypyrrolidine-1, 2-dicarboxylic acid tert-butyl ester (45-1,3g,12.23mmol) and triethylamine (5.1mL,36.69mmol) were added to DCM (200mL), stirred well, p-toluenesulfonyl chloride (3.5g,18.35mmol) was added dropwise, reacted at room temperature for 4 hours, LCMS checked for reaction completion. The solvent was dried, 200mL of water was added, extraction was performed with ethyl acetate, the organic phase was collected and washed, dried, concentrated, and purified by column chromatography to give pure (2S,4R) -1-2-methyl-4- (p-toluenesulfonic acid) pyrrolidine-1, 2-dicarboxylic acid tert-butyl ester (3.5g, yield 72%).

MS m/z(ESI):400[M+H]+

The second step is that: preparation of (2S,4R) -1-tert-butoxycarbonyl-4- (p-toluenesulfonic acid) pyrrolidine-2-carboxylic acid (45-3)

(2S,4R) -1-2-methyl-4- (p-toluenesulfonic acid) pyrrolidine-1, 2-dicarboxylic acid tert-butyl ester (45-2,1.4g,3.5mmol) was added to a mixed solvent of THF (20mL) and water (20mL), and lithium hydroxide monohydrate (441mg,10.51mmol) was added thereto, followed by stirring for 2 hours and completion of the reaction was detected by LCMS. 50mL of water is added, ethyl acetate is used for extraction, an organic phase is collected and washed, dried and concentrated to obtain a crude product (2S,4R) -1-tert-butoxycarbonyl-4- (p-toluenesulfonic acid) pyrrolidine-2-carboxylic acid (1g, yield 74%) which is directly put into the next step.

MS m/z(ESI):386[M+H]+

The third step: preparation of (2S,4R) -1-tert-butoxycarbonyl-4-cyclohexylpyrrolidine-2-carboxylic acid (45-4)

(2S,4R) -1-tert-Butoxycarbonyl-4- (p-toluenesulfonic acid) pyrrolidine-2-carboxylic acid (45-3, 1g,2.59mmol), lithium methoxide (99mg,2.59mmol), cuprous iodide (99mg, 519. mu. mol) and tetramethylethylenediamine (121mg,1.04mmol) were added to THF (50mL), nitrogen was purged three times, cyclohexyl formatter (4.86g,25.94mmol) was added dropwise at 0 ℃ and reacted for 16 hours at 0 ℃. Dropwise adding water to quench the reaction until no air bubbles are generated, spin-drying the solvent, adding 50mL of water, adjusting the pH value to 3 with 1M hydrochloric acid, extracting with ethyl acetate, collecting an organic phase, washing, drying and concentrating to obtain a crude product (2S,4R) -1-tert-butoxycarbonyl-4-cyclohexylpyrrolidine-2-carboxylic acid (600mg, yield 78%), and directly adding the crude product to the next step.

MS m/z(ESI):298[M+H]+

The fourth step: preparation of (2S,4R) -2- ((4- (tert-butoxycarbonyl) phenyl) carbamoyl) -4-cyclohexylpyrrolidine-1-carboxylic acid tert-butyl ester (45-5)

(2S,4R) -1-tert-Butoxycarbonyl-4-cyclohexylpyrrolidine-2-carboxylic acid (45-4, 600mg,2.02mmol), tert-butyl 4-aminobenzoate (429mg,2.22mmol), HATU (921mg,2.42mmol) were added to DCM (20mL), stirred well, DIPEA (1mL,6.05mmol) was added, the reaction was allowed to react for 2 hours, and LCMS checked for completion. The solvent was dried, 30mL of water was added, extraction was performed with ethyl acetate, the organic phase was collected and washed, dried, concentrated, and purified by column chromatography to obtain pure (2S,4R) -2- ((4- (tert-butoxycarbonyl) phenyl) carbamoyl) -4-cyclohexylpyrrolidine-1-carboxylic acid tert-butyl ester (150mg, yield 16%).

MS m/z(ESI):473[M+H]+

The fifth step: preparation of tert-butyl 4- ((2S,4R) -4-cyclohexylpyrrolidine-2-carboxamido) benzoate (45-6)

(2S,4R) -2- ((4- (tert-butoxycarbonyl) phenyl) carbamoyl) -4-cyclohexylpyrrolidine-1-carboxylic acid tert-butyl ester (45-5, 150mg, 254. mu. mol) was added to a solution of ethyl hydrogen chloride in ethyl acetate (10mL) and reacted for 2 hours, and the reaction was checked by LCMS to be complete. The solvent was dried by evaporation to give pure tert-butyl 4- ((2S,4R) -4-cyclohexylpyrrolidine-2-carboxamido) benzoate (95mg, yield 100%).

MS m/z(ESI):373[M+H]+

And a sixth step: preparation of tert-butyl 4- ((2S,4R) -1- ((1R,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexyl-1-carbonyl) -4-cyclohexylpyrrolidine-2-carboxamido) benzoate (45-7)

Tert-butyl 4- ((2S,4R) -4-cyclohexylpyrrolidine-2-carboxamido) benzoate (45-6, 45mg, 121. mu. mol), (1R,4R) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexyl-1-carboxylate (34mg, 133. mu. mol), HATU (55mg, 145. mu. mol) and isopropyldiethylamine (45mg, 362. mu. mol) were added to DCM (3mL) and reacted for 2 hours with LCMS to check completion. The solvent was dried by evaporation, 10mL of water was added, ethyl acetate was extracted, the organic phase was collected and washed, dried, concentrated, and purified by column chromatography to give pure tert-butyl 4- ((2S,4R) -1- ((1R,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexyl-1-carbonyl) -4-cyclohexylpyrrolidine-2-carboxamido) benzoate (60mg, yield 81%).

MS m/z(ESI):612[M+H]+

The seventh step: preparation of 4- ((2S,4R) -1- ((1R,4S) -4- (aminomethyl) cyclohexyl-1-carbonyl) -4-cyclohexylpyrrolidine-2-carboxamido) benzoic acid (45)

Tert-butyl 4- ((2S,4R) -1- ((1R,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexyl-1-carbonyl) -4-cyclohexylpyrrolidine-2-carboxamido) benzoate (45-7, 60mg, 98. mu. mol) was added to a mixed solvent of trifluoroacetic acid (2mL) and DCM (4mL) and reacted for 1 hour with LCMS to check completion of the reaction. The solvent was dried by evaporation to give 4- ((2S,4R) -1- ((1R,4S) -4- (aminomethyl) cyclohexyl-1-carbonyl) -4-cyclohexylpyrrolidine-2-carboxamido) benzoic acid as a product (39mg, 87% yield).

MS m/z(ESI):456[M+H]+

Example 46: 4- ((2S,4R) -1- (4- (aminomethyl) cyclohexyl-1-carbonyl) -4-phenylpyrrolidine-2-carboxamido) benzoic acid trifluoroacetate

The first step is as follows: preparation of (2S,4R) -2- ((4- (tert-butoxycarbonyl) phenyl) carbamoyl) -4-phenylpyrrolidine-1-carboxylic acid tert-butyl ester (46-2)

(2S,4R) -1- (tert-butyloxycarbonyl) -4-phenylpyrrolidine-2-carboxylic acid (46-1,200mg, 686. mu. mol), tert-butyl 4-aminobenzoate (146mg, 755. mu. mol), HATU (313mg, 824. mu. mol) were added to DCM (10mL), stirred well, DIPEA (0.34mL,2.06mmol) was added, the reaction was allowed to react for 2 hours, and LCMS checked for completion. The solvent was dried, 20mL of water was added, extraction was performed with ethyl acetate, the organic phase was collected and washed, dried, concentrated, and purified by column chromatography to give pure (2S,4R) -2- ((4- (tert-butoxycarbonyl) phenyl) carbamoyl) -4-phenylpyrrolidine-1-carboxylic acid tert-butyl ester (320mg, yield 99%).

MS m/z(ESI):467[M+H]+

The second step is that: preparation of tert-butyl 4- ((2S,4R) -4-phenylpyrrolidine-2-carboxamido) benzoate (46-3)

(2S,4R) -2- ((4- (tert-butoxycarbonyl) phenyl) carbamoyl) -4-phenylpyrrolidine-1-carboxylic acid tert-butyl ester (46-2,320mg, 686. mu. mol) was added to a solution of ethyl hydrogen chloride in ethyl acetate (10mL) and reacted for 2 hours, and the reaction was checked for completion by LCMS. The solvent was dried by evaporation to give pure tert-butyl 4- ((2S,4R) -4-phenylpyrrolidine-2-carboxamido) benzoate (250mg, 99% yield).

MS m/z(ESI):367[M+H]+

The third step: preparation of tert-butyl 4- ((2S,4R) -1- (4- (aminomethyl) cyclohexyl-1-carbonyl) -4-phenylpyrrolidine-2-carboxamido) benzoate (46-4)

Tert-butyl 4- ((2S,4R) -4-phenylpyrrolidine-2-carboxamido) benzoate (46-3,100mg, 273. mu. mol), (1R,4R) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexyl-1-carboxylate (77mg, 300. mu. mol), HATU (125mg, 327. mu. mol) and isopropyldiethylamine (106mg, 817. mu. mol) were added to DCM (5mL) and reacted for 2 hours with LCMS to check completion. The solvent was dried, 10mL of water was added, extraction was performed with ethyl acetate, the organic phase was collected and washed, dried, concentrated, and purified by column chromatography to give pure tert-butyl 4- ((2S,4R) -1- (4- (aminomethyl) cyclohexyl-1-carbonyl) -4-phenylpyrrolidine-2-carboxamido) benzoate (120mg, yield 73%).

MS m/z(ESI):606[M+H]+

The fourth step: preparation of 4- ((2S,4R) -1- (4- (aminomethyl) cyclohexyl-1-carbonyl) -4-phenylpyrrolidine-2-carboxamido) benzoic acid trifluoroacetate (46)

Tert-butyl 4- ((2S,4R) -1- (4- (aminomethyl) cyclohexyl-1-carbonyl) -4-phenylpyrrolidine-2-carboxamido) benzoate (46-4,120mg, 198. mu. mol) was added to a mixed solvent of trifluoroacetic acid (2mL) and DCM (4mL) for 1 hour and the reaction was complete by LCMS. The solvent was dried by evaporation to give the product, 4- ((2S,4R) -1- (4- (aminomethyl) cyclohexyl-1-carbonyl) -4-phenylpyrrolidine-2-carboxamido) benzoic acid trifluoroacetate (80mg, 90% yield).

1HNMR(DMSO-d6 400MHz)δ12.69(s，1H)，10.34(s,1H),7.90(d,2H,J＝8.8Hz),7.73(d,2H,J＝8.8Hz),7.72(brs，3H)，7.36-7.33(m，4H),7.29-7.25(m,1H),4.51-4.47(m,1H),4.25-4.16(m,1H),3.52-3.47(m,2H),2.69-2.64(m,3H),1.99-1.96(m,1H),1.86-1.78(m,4H),1.58-1.47(m,1H),1.36-1.24(m,2H),1.05-0.96(m,2H)。

MS m/z(ESI):450[M+H]+

Example 47: preparation of 4- ((2S,4R) -1- ((E) -3- (3-chloro-2-fluoro-6- (4H-1,2, 3-tetrazol-4-yl) phenyl) acryloyl) -4-cyclohexylpyrrolidine-2-formamido) benzoic acid

The first step is as follows: preparation of tert-butyl 4- ((2S,4R) -1- ((E) -3- (3-chloro-2-fluoro-6- (4H-1,2, 3-tetrazol-4-yl) phenyl) acryloyl) -4-cyclohexylpyrrolidine-2-formylamino) benzoate (47-1)

Tert-butyl 4- ((2S,4R) -4-cyclohexylpyrrolidine-2-carboxamido) benzoate (45mg, 121. mu. mol), (E) -3- (3-chloro-2-fluoro-6- (1 hydro-tetrazol-1-yl) phenyl) acrylic acid (36mg, 133. mu. mol), HATU (55mg, 145. mu. mol) and isopropyldiethylamine (47mg, 362. mu. mol) were added to DCM (3mL) and reacted for 2 hours with LCMS to check completion. And (3) drying the solvent by spinning, adding 10mL of water, extracting by ethyl acetate, collecting an organic phase, washing, drying, concentrating, and purifying by column chromatography to obtain a pure product of tert-butyl 4- ((2S,4R) -1- ((E) -3- (3-chloro-2-fluoro-6- (4H-1,2, 3-tetrazol-4-yl) phenyl) acryloyl) -4-cyclohexylpyrrolidine-2-formylamino) benzoate (55mg, yield 73%).

MS m/z(ESI):622[M+H]+

The second step is that: preparation of 4- ((2S,4R) -1- ((E) -3- (3-chloro-2-fluoro-6- (4H-1,2, 3-tetrazol-4-yl) phenyl) acryloyl) -4-cyclohexylpyrrolidine-2-formylamino) benzoic acid (47)

Tert-butyl 4- ((2S,4R) -1- ((E) -3- (3-chloro-2-fluoro-6- (4H-1,2, 3-tetrazol-4-yl) phenyl) acryloyl) -4-cyclohexylpyrrolidine-2-carboxamido) benzoate (55mg, 88. mu. mol) was added to a mixed solvent of trifluoroacetic acid (2mL) and DCM (4mL) and reacted for 1 hour, and the reaction was detected to be complete by LCMS. The solvent was dried by evaporation to give 4- ((2S,4R) -1- ((E) -3- (3-chloro-2-fluoro-6- (4H-1,2, 3-tetrazol-4-yl) phenyl) acryloyl) -4-cyclohexylpyrrolidine-2-carboxamido) benzoic acid (28mg, 56% yield).

MS m/z(ESI):566[M+H]+

Example 48: 4- ((2S,4R) -1- ((E) -3- (3-chloro-2-fluoro-6- (4H-1,2, 3-tetrazol-4-yl) phenyl) acryloyl) -4-phenylpyrrolidine-2-formamido) benzoic acid

The first step is as follows: preparation of tert-butyl 4- ((2S,4R) -1- ((E) -3- (3-chloro-2-fluoro-6- (4H-1,2, 3-tetrazol-4-yl) phenyl) acryloyl) -4-phenylpyrrolidine-2-formylamino) benzoate (48-1)

Tert-butyl 4- ((2S,4R) -4-phenylpyrrolidine-2-carboxamido) benzoate (46-3,100mg, 273. mu. mol), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (81mg, 300. mu. mol), HATU (125mg, 327. mu. mol) and isopropyldiethylamine (106mg, 817. mu. mol) were added to DCM (5mL) and reacted for 2 hours with LCMS to detect completion. And (3) drying the solvent by spinning, adding 10mL of water, extracting by ethyl acetate, collecting an organic phase, washing, drying, concentrating, and purifying by column chromatography to obtain a pure product of tert-butyl 4- ((2S,4R) -1- ((E) -3- (3-chloro-2-fluoro-6- (4H-1,2, 3-tetrazol-4-yl) phenyl) acryloyl) -4-phenylpyrrolidine-2-formylamino) benzoate (110mg, yield 65%).

MS m/z(ESI):616[M+H]+

The second step is that: preparation of 4- ((2S,4R) -1- ((E) -3- (3-chloro-2-fluoro-6- (4H-1,2, 3-tetrazol-4-yl) phenyl) acryloyl) -4-phenylpyrrolidine-2-formamido) benzoic acid (48)

Tert-butyl 4- ((2S,4R) -1- ((E) -3- (3-chloro-2-fluoro-6- (4H-1,2, 3-tetrazol-4-yl) phenyl) acryloyl) -4-phenylpyrrolidine-2-carboxamido) benzoate (48-1,110mg, 179. mu. mol) was added to a mixed solvent of trifluoroacetic acid (2mL) and DCM (4mL) and reacted for 1 hour with LCMS detection of reaction completion. The solvent was dried by evaporation to give 4- ((2S,4R) -1- ((E) -3- (3-chloro-2-fluoro-6- (4H-1,2, 3-tetrazol-4-yl) phenyl) acryloyl) -4-phenylpyrrolidine-2-carboxamido) benzoic acid (71mg, 71% yield).

MS m/z(ESI):560[M+H]+

Example 49: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) benzoic acid

The first step is as follows: preparation of tert-butyl (S) -4- (2- (((9H-fluoren-ylmethoxy) carbonyl) amino) -2-phenylacetamido) benzoate (49-2)

Fmoc-phenylglycine (49-1,934mg, 2.5mmol), 13-6(486mg, 2.5mmol), HATU (1.16g, 3mmol) was dissolved in dichloromethane (15mL), DIPEA (1.3mL) was added, the mixture was stirred overnight at room temperature, water was added to quench the reaction, dichloromethane was extracted, the organic phase was filtered and concentrated, and the mixture was purified by column chromatography to give (S) -tert-butyl 4- (2- (((9H-fluoren-ylmethoxy) carbonyl) amino) -2-phenylacetamido) benzoate as a white solid in 1.29g, 94% yield.

The second step is that: preparation of tert-butyl (S) -4- (2-amino-2-phenylacetamido) benzoate (49-3)

Tert-butyl (S) -4- (2- (((9H-fluoren-ylmethoxy) carbonyl) amino) -2-phenylacetamido) benzoate (49-2,548mg) was dissolved in methylene chloride (6mL), dimethylamine (3mL) was added, the mixture was stirred at room temperature for 2 hours, and the reaction mixture was concentrated and purified by column chromatography to give (S) -4- (2-amino-2-phenylacetamido) benzoate (49-3,290mg) as a yellow oil.

The third step: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) benzoic acid tert-butyl ester (58)

(E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (1-7,270mg), tert-butyl (S) -4- (2-amino-2-phenylacetamido) benzoate (49-3,290mg), HATU (380mg) dissolved in dichloromethane (5mL), DIPEA (0.5mL) is stirred at room temperature overnight, water is added for quenching reaction, dichloromethane is used for extraction, organic phase is dried, filtered and concentrated, and then column chromatography is carried out for purification to obtain pale yellow solid (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) benzoic acid tert-butyl ester (58)510 mg.

The fourth step: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) benzoic acid (49)

(S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) benzoic acid tert-butyl ester (58,135mg) was dissolved in dichloromethane (4mL), trifluoroacetic acid (1.5mL) was added, stirred at room temperature for 2 hours, concentrated and purified by preparative thin layer chromatography to give (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) benzoic acid as a white solid (49,60 mg).

¹ HNMR(DMSO-d6 400MHz)δ12.63(brs，1H)，10.70(s,1H),9.86(s,1H),9.22(d,1H,J＝7.6Hz),7.93(t,1H,J＝8.2Hz),7.87(d,2H,J＝8.8Hz),7.68(d,2H,J＝8.8Hz),7.63(dd,1H,J＝1.2，8.8Hz),7.50(d,2H,J＝7.2Hz),7.40(t,2H,J＝7.4Hz),7.35-7.32(m,1H),6.90(dd,2H,J＝16.2,67.4Hz),5.71(d,1H,J＝7.6Hz)。

MS m/z(ESI):521[M+H]+

Example 50: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -N-methyl-2-phenylacetamido) benzoic acid

The first step is as follows: preparation of tert-butyl (S) -4- (2- (((9H-fluoren-ylmethoxy) carbonyl) amino) -N-methyl-2-phenylacetamido) benzoate (50-1)

Fmoc-phenylglycine (49-1,798mg, 2.1mmol), 16-1(414mg, 2.0mmol) was dissolved in pyridine (10mL), phosphorus oxychloride (0.25mL) was added dropwise in an ice bath, the mixture was stirred at room temperature for 1.5 hours, 1N diluted hydrochloric acid was added to quench the reaction, and the precipitated solid was collected by filtration and purified by column chromatography to give (S) -tert-butyl 4- (2- (((9H-fluoren-ylmethoxy) carbonyl) amino) -N-methyl-2-phenylacetamido) benzoate (50-1)1.04g as a white solid.

The second step is that: preparation of tert-butyl (S) -4- (2-amino-N-methyl-2-phenylacetamido) benzoate (50-2)

Tert-butyl (S) -4- (2- (((9H-fluoren-ylmethoxy) carbonyl) amino) -N-methyl-2-phenylacetamido) benzoate (50-1,1.04g) was dissolved in methylene chloride (21mL), dimethylamine (7mL) was added, the mixture was stirred at room temperature for 4 hours, and the reaction mixture was concentrated and purified by column chromatography to give tert-butyl (S) -4- (2-amino-N-methyl-2-phenylacetamido) benzoate (50-2,593mg) as a yellow oil.

The third step: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -N-methyl-2-phenylacetamido) benzoic acid tert-butyl ester (50-3)

(E) Dissolving (1-7,112mg) of (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid, (S) -4- (2-amino-N-methyl-2-phenylacetamido) (50-2) in pyridine (3mL), dropwise adding phosphorus oxychloride (0.048mL) under ice bath, stirring at room temperature for 4 hours, adding 1N diluted hydrochloric acid to quench reaction, extracting with dichloromethane, drying, filtering and concentrating the organic phase, and purifying by column chromatography to obtain a pale yellow solid (S, E) -4- (2- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -N-methyl-2-phenylacetamido) tert-butyl benzoate (50-3)125 mg.

The fourth step: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -N-methyl-2-phenylacetamido) benzoic acid (50)

(S, E) -tert-butyl 4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -N-methyl-2-phenylacetamido) benzoate (50-3,125mg) was dissolved in dichloromethane (5mL), trifluoroacetic acid (5mL) was added thereto, and the mixture was stirred at room temperature for 1.5 hours, after concentration, the mixture was purified by preparative thin layer chromatography to give (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -N-methyl-2-phenylacetamido) benzoic acid as a white solid (50,57 mg).

¹ HNMR(DMSO-d6 400MHz):12.63(brs，1H)，9.86(s,1H),9.12(d,1H,J＝7.6Hz),7.93(t,1H,J＝8.2Hz),7.87(d,2H,J＝8.8Hz),7.68(d,2H,J＝8.8Hz),7.63(dd,1H,J＝1.2，8.8Hz),7.50(d,2H,J＝7.2Hz),7.40(t,2H,J＝7.4Hz),7.35-7.32(m，1H),6.90(dd,2H,J＝16.2,67.4Hz),5.71(d,1H,J＝7.6Hz)，3.40(s,3H)。

MS m/z(ESI):535[M+H]+

Example 51: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -N-methyl-2-phenylacetamido) methyl benzoate

The first step is as follows: preparation of methyl (S) -4- (2- (((9H-fluoren-ylmethoxy) carbonyl) amino) -N-methyl-2-phenylacetamido) benzoate (51-2)

Fmoc-phenylglycine (49-1,374mg, 1mmol), methyl N-methylaminobenzoate (51-1, 175mg, 1.06mmol) was dissolved in ethyl acetate (7mL), DIPEA (0.5mL), T3P (50% ethyl acetate solution, 0.71mL) was added, and stirring was carried out overnight at 55 ℃. Water was added to the reaction mixture to quench, followed by extraction with methylene chloride, drying, filtration, concentration and purification by column chromatography to give (S) -methyl 4- (2- (((9H-fluoren-ylmethoxy) carbonyl) amino) -N-methyl-2-phenylacetamido) benzoate (51-2) as a white solid (406 mg, 78% yield).

The second step is that: preparation of methyl (S) -4- (2-amino-N-methyl-2-phenylacetamido) benzoate (51-3)

Methyl (S) -4- (2- (((9H-fluoren-ylmethoxy) carbonyl) amino) -N-methyl-2-phenylacetamido) benzoate (51-2,406mg) was dissolved in methylene chloride (9mL), dimethylamine (5mL) was added, the mixture was stirred at room temperature for 2.5 hours, and the reaction mixture was concentrated and purified by column chromatography to give methyl (S) -4- (2-amino-N-methyl-2-phenylacetamido) benzoate (51-3,230mg) as a white wax.

The third step: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -N-methyl-2-phenylacetamido) benzoic acid methyl ester (51)

(E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (1-7,119mg), methyl (S) -4- (2-amino-N-methyl-2-phenylacetamido) benzoate (51-3,98mg), HATU (172mg) dissolved in DMF (4mL), DIPEA (0.185mL) was added, the mixture was stirred at room temperature overnight, water was added to quench the reaction, and the precipitated solid was collected by filtration and purified by column chromatography to give (S, E) -methyl 4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -N-methyl-2-phenylacetamido) benzoate (51) as a white solid (121 mg).

¹ HNMR(DMSO-d6 400MHz)δ9.85(s,1H),9.07(d,1H,J＝7.6Hz),7.96(d,2H,J＝8.0Hz),7.92(t,1H,J＝8.2Hz),7.63(dd,1H,J＝1.2，8.8Hz),7.33-7.29(m,5H),7.01(brs,2H),6.84(dd,2H,J＝16,50Hz),5.54(s,1H)，3.87(s,3H)，3.20(s,3H)。

MS m/z(ESI):549[M+H]+

Example 52: preparation of (S, E) -3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) -N- (2- (methyl (phenyl) amino) -2-oxo-1-phenylethyl) acrylamide

The first step is as follows: preparation of (9H-fluoren-9-yl) methyl (S) - (2- (methyl (phenyl) amino) -2-oxo-1-phenylethyl) carbamate (52-2)

Fmoc-phenylglycine (560mg, 1.5mmol), N-methylaniline (201mg, 1.9mmol), DCC (380mg, 1.8mmol), DMAP (22mg, 0.18mmol) were dissolved in dichloromethane/DMF (12mL/1mL) and stirred at room temperature overnight. Water was added to the reaction mixture to quench, followed by filtration, extraction with methylene chloride, drying of the organic phase, filtration, concentration and purification by column chromatography to give 500mg of (9H-fluoren-9-yl) methyl (S) - (2- (methyl (phenyl) amino) -2-oxo-1-phenylethyl) carbamate as a white solid in a yield of 72%.

The second step is that: preparation of (S) -2-amino-N-methyl-N, 2-diphenylacetamide (52-3)

(9H-fluoren-9-yl) methyl (S) - (2- (methyl (phenyl) amino) -2-oxo-1-phenylethyl) carbamate (240mg) was dissolved in methylene chloride (9mL), dimethylamine (5mL) was added, the mixture was stirred at room temperature for 3 hours, and the reaction mixture was concentrated and purified by column chromatography to give (S) -2-amino-N-methyl-N, 2-di phenylacetamide (130mg) as a white solid.

The third step: preparation of (S, E) -3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) -N- (2- (methyl (phenyl) amino) -2-oxo-1-phenylethyl) acrylamide (52)

(E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (180mg), (S) -4- (2-amino-N-methyl-N, 2-diphenylacetamide (130mg), HATU (285mg) dissolved in DMF (4mL), DIPEA (0.3mL) was added, the mixture was stirred at room temperature overnight, water was added to quench the reaction, and the precipitated solid was collected by filtration and purified by column chromatography to give 163mg of (S, E) -3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) -N- (2- (methyl (phenyl) amino) -2-oxo-1-phenylethyl) acrylamide as a pale yellow solid.

¹ HNMR(DMSO-d6 400MHz)δ9.85(s,1H),9.04(d,1H,J＝7.2Hz),7.92(t,1H,J＝8.2Hz),7.62(dd,1H,J＝1.4，8.6Hz),7.41-7.38(m,3H),7.28-7.26(m,3H),7.14-7.12(m,2H),6.96-6.93(m,2H),6.84(dd,2H,J＝16.2,62.6Hz),5.51(d,1H，J＝7.6Hz)，3.16(s,3H)。

MS m/z(ESI):491[M+H]+

Example 53: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) methyl benzoate

The first step is as follows: preparation of methyl (S) -4- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -2-phenylacetamido) benzoate (53-1)

Fmoc-phenylglycine (378mg, 1mmol), methyl 4-aminobenzoate (151mg,1mmol), HATU (453mg, 1.2mmol) were dissolved in DMF (5mL), DIPEA (0.5mL) was added, and stirring was continued overnight at room temperature. Water was added to the reaction solution to quench, and methyl (S) -4- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -2-phenylacetamido) benzoate was collected by filtration as a pale yellow solid (500mg) and used as it was in the next reaction.

The second step is that: preparation of methyl (S) -4- (2-amino-2-phenylacetamido) benzoate (53-2)

Methyl (S) -4- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -2-phenylacetamido) benzoic acid (500mg) was dissolved in methylene chloride (14mL), dimethylamine (6mL) was added thereto, the mixture was stirred at room temperature for 2 hours, and the reaction mixture was concentrated and purified by column chromatography to give methyl (S) -4- (2-amino-2-phenylacetamido) benzoate (189mg) as a white solid.

The third step: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) methyl benzoate (53)

(E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (93mg), methyl (S) -4- (2-amino-2-phenylacetamido) benzoate (81mg), HATU (129mg) were dissolved in DMF (2.5mL), DIPEA (0.14mL) was added, stirred overnight at room temperature, quenched with water, and the precipitated solid was collected by filtration and purified by column chromatography to give methyl (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) benzoate as a pale yellow solid (136 mg).

¹ HNMR(DMSO-d6 400MHz)δ10.77(s，1H)，9.86(s,1H),9.23(d,1H,J＝7.6Hz),7.93(t,1H,J＝8.4Hz),7.91(dd,2H,J＝1.8，7.0Hz),7.72(dd,2H,J＝2.0，7.2Hz),7.63(dd,1H,J＝1.2，8.8Hz),7.51(d,2H,J＝8.4Hz),7.41(t,2H,J＝7.4Hz),7.36-7.32(m,1H),6.91(dd,2H,J＝16.2,67.8Hz),5.71(d,1H，J＝7.6Hz)，3.81(s,3H)。

MS m/z(ESI):535[M+H]+

Example 54: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) -N- (methylsulfonyl) benzamide

The first step is as follows: preparation of N- (methylsulfonyl) -4-nitrobenzamide (54-3)

P-nitrobenzoic acid (2g,11.97mmol), 2-chloro-1-methylpyridine iodide (3.67g,14.36mmol), DMAP (73mg, 598. mu. mol), and triethylamine (5mL,35.90mmol) were added to DCM (40mL) and stirred well before methanesulfonamide (2.28g,23.93mmol) was added and the reaction was complete by LCMS. Spin-drying solvent, adding 50mL water, extracting with ethyl acetate, collecting organic phase, washing, drying, concentrating, and purifying by column chromatography to obtain pure N- (methylsulfonyl) -4-nitrobenzamide (1.092g, yield 37%).

MS m/z(ESI):243[M-H]-

The second step is that: preparation of N- (methylsulfonyl) -4-aminobenzamide (54-4)

Adding N- (methylsulfonyl) -4-nitrobenzamide (1.092g,4.47mmol) into methanol (100mL), adding Pd-C (500mg), changing hydrogen for three times, reacting under hydrogen atmosphere overnight, and detecting the reaction completion by LCMS. Filtration and concentration of the filtrate gave pure N- (methylsulfonyl) -4-aminobenzamide (904mg, 94% yield).

MS m/z(ESI):215[M+H]+

The third step: preparation of tert-butyl (S) - (2- ((4- ((methylsulfonyl) carbamoyl) phenyl) amino) -2-oxo-1-phenylethyl) carbamate (54-5)

Boc-phenylglycine (251mg, 1mmol), 4-amino-N- (methylsulfonyl) benzamide (214mg, 1mmol), HATU (456mg, 1.2mmol) were dissolved in DMF (4mL), DIPEA (0.5mL) was added and the mixture was stirred at room temperature overnight. Water was added to the reaction mixture to quench, followed by extraction with dichloromethane, and the organic layer was dried, filtered, concentrated and purified by column chromatography to give 440mg of (S) - (2- ((4- ((methanesulfonyl) carbamoyl) phenyl) amino) -2-oxo-1-phenylethyl) carbamic acid tert-butyl ester as a white solid.

The fourth step: preparation of (S) -4- (2-amino-2-phenylacetylamino) -N- (methylsulfonyl) benzamide (54-6)

(S) -tert-butyl (2- ((4- ((methylsulfonyl) carbamoyl) phenyl) amino) -2-oxo-1-phenylethyl) carbamate (400mg) was dissolved in methanol (1.5mL), a hydrochloric acid methanol solution (5mL) was added, the mixture was stirred at room temperature for 3 hours, the reaction mixture was concentrated, the mixture was dissolved in a 2mol/L aqueous sodium hydroxide solution, extracted with dichloromethane, and the organic layer was dried, filtered, concentrated and purified by column chromatography to give (S) -4- (2-amino-2-phenylacetamido) -N- (methylsulfonyl) benzamide (300mg) as a white solid.

The fifth step: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) -N- (methylsulfonyl) benzamide (54)

(E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (30mg), (S) -4- (2-amino-2-phenylacetylamino) -N- (methylsulfonyl) benzamide (30mg), HATU (40mg) was dissolved in DMF (2mL), DIPEA (0.055mL) was added, the mixture was stirred at room temperature overnight, and the reaction was quenched with water, and the precipitated solid was collected by filtration and purified by column chromatography to give (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) -N- (methylsulfonyl) benzamide 13mg as a white solid.

¹ HNMR(DMSO-d6 400MHz)δ10.75(s，1H)，9.83(s,1H),9.22(d,1H,J＝7.6Hz),7.95(t,1H,J＝8.5Hz),7.90(dd,2H,J＝1.8，7.1Hz),7.77(dd,2H,J＝2.0，7.2Hz),7.60(dd,1H,J＝1.18，8.6Hz),7.55(d,2H,J＝8.4Hz),7.41(t,2H,J＝7.6Hz),7.38-7.29(m,1H),6.89(dd,2H,J＝16.2,67.8Hz),5.71(d,1H，J＝8.2Hz)，4.11(s,3H)。

MS m/z(ESI):598[M+H]+

Example 55: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) -N- (benzenesulfonyl) benzamide

The first step is as follows: preparation of N- (phenylsulfonyl) -4-nitrobenzamide (55-1)

P-nitrobenzoic acid (1g,5.98mmol), 2-chloro-1-methylpyridinium iodide (1.834g,7.18mmol), DMAP (37mg, 299. mu. mol), and triethylamine (2.5mL,17.95mmol) were added to DCM (20mL) and stirred until homogeneous, followed by benzenesulfonamide (1.88g,11.97mmol), reaction was carried out for 1 hour, and LCMS checked for completion. Spin-drying solvent, adding 20mL water, extracting with ethyl acetate, collecting organic phase, washing, drying, concentrating, and purifying by column chromatography to obtain pure N- (benzenesulfonyl) -4-nitrobenzamide (1.5g, yield 82%).

MS m/z(ESI):305[M-H]-

The second step: preparation of N- (phenylsulfonyl) -4-aminobenzamide (55-2)

Adding N- (benzenesulfonyl) -4-nitrobenzamide (1.5g,4.90mmol) into methanol (100mL), adding Pd-C (750mg), replacing hydrogen for three times, reacting under a hydrogen atmosphere overnight, and detecting the reaction completion by LCMS. After filtration, the filtrate was concentrated to give a pure 4-amino-N- (phenylsulfonyl) benzamide (1.3g, yield 96%).

MS m/z(ESI):277[M+H]+

The third step: preparation of tert-butyl (S) - (2-oxo-1-phenyl-2- ((4- ((benzenesulfonyl) carbamoyl) phenyl) amino) ethyl) carbamate (55-3)

Boc-phenylglycine (251mg, 1mmol), 4-amino-N- (phenylsulfonyl) benzamide (276mg, 1mmol), HATU (456mg, 1.2mmol) were dissolved in DMF (4mL), DIPEA (0.5mL) was added, and the mixture was stirred at room temperature overnight. Water was added to the reaction mixture to quench, followed by extraction with dichloromethane, and the organic layer was dried, filtered, concentrated and purified by column chromatography to give 432mg of tert-butyl (S) - (2-oxo-1-phenyl-2- ((4- ((benzenesulfonyl) carbamoyl) phenyl) amino) ethyl) carbamate as a white solid.

The fourth step: preparation of (S) -4- (2-amino-2-phenylacetylamino) -N- (benzenesulfonyl) benzamide (55-4)

Tert-butyl (S) - (2-oxo-1-phenyl-2- ((4- ((benzenesulfonyl) carbamoyl) phenyl) amino) ethyl) carbamate (255mg) was dissolved in methanol (1mL), a hydrochloric acid methanol solution (5mL) was added, the mixture was stirred at room temperature for 3 hours, the reaction solution was concentrated, and the residue was slurried with ethanol/diethyl ether ═ 1/10 to give (S) -4- (2-amino-2-phenylacetamido) -N- (benzenesulfonyl) benzamide (180mg) as a white solid.

The fifth step: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) -N- (benzenesulfonyl) benzamide (55)

(E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (115mg), (S) -4- (2-amino-2-phenylacetamido) -N- (benzenesulfonyl) benzamide (180mg), HATU (190mg) was dissolved in DMF (4mL), DIPEA (0.5mL) was added, stirred overnight at room temperature, the reaction was quenched with water, the precipitated solid was collected by filtration and slurried with ethyl acetate/petroleum ether ═ 1/4 to give (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) -N- (benzenesulfonyl) benzamide 265mg as a white solid.

¹ HNMR(DMSO-d6 400MHz)δ10.55(s,1H),9.87(s,1H),9.23(d,1H,J＝7.6Hz),7.93(t,1H,J＝8.2Hz),7.83-7.79(m,4H),7.62(dd,1H,J＝1.2,8.8Hz),7.53-7.50(m,4H),7.40-7.36(m,5H),7.33-7.29(m,1H),6.91(dd,2H,J＝16,80Hz),5.72(d,1H,J＝8.0Hz)。

MS m/z(ESI):660[M+H]+

Example 56: preparation of (S, E) -N- (2- ((4- (2H-tetrazol-5-yl) phenyl) amino) -2-oxo-1-phenylethyl) -3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamide

The first step is as follows: preparation of (S) - (2- ((4- (2H-tetrazol-5-yl) phenyl) amino) -2-oxo-1-phenylethyl) carbamic acid tert-butyl ester (56-2)

Boc-phenylglycine (251mg, 1mmol), 4- (2H-tetrazol-5-yl) aniline (161mg, 1mmol), HATU (456mg, 1.2mmol) were dissolved in DMF (4mL), DIPEA (0.5mL) was added and the mixture was stirred at room temperature overnight. Water was added to the reaction solution to quench, dichloromethane was then extracted, the organic layer was dried, filtered and concentrated, and the residue was slurried with dichloromethane/diethyl ether (1/4) to give 410mg of (S) - (2- ((4- (2H-tetrazol-5-yl) phenyl) amino) -2-oxo-1-phenylethyl) carbamic acid tert-butyl ester as a white solid.

The second step is that: preparation of (S) -N- (4- (2H-tetrazole-5-yl) phenyl) -2-amino-2-phenyl acetamide hydrochloride (56-3)

(S) - (2- ((4- (2H-tetrazol-5-yl) phenyl) amino) -2-oxo-1-phenylethyl) carbamic acid tert-butyl ester (240mg) was dissolved in tetrahydrofuran (5mL), a hydrochloric acid methanol solution (8mL) was added, stirring was carried out at room temperature for 3 hours, and the reaction solution was concentrated to obtain (S) -N- (4- (2H-tetrazol-5-yl) phenyl) -2-amino-2-phenylacetamide hydrochloride as a white solid (157 mg).

The third step: preparation of (S, E) -N- (2- ((4- (2H-tetrazol-5-yl) phenyl) amino) -2-oxo-1-phenylethyl) -3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamide (56)

(E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (165mg), (S) -N- (4- (2H-tetrazol-5-yl) phenyl) -2-amino-2-phenylacetamide hydrochloride (157mg), HATU (304mg) was dissolved in DMF (5mL), DIPEA (1mL) was added, stirred overnight at room temperature, quenched with water, the precipitated solid was collected by filtration, dissolved in methanol (2mL), diethyl ether (20mL) was added, and the precipitated solid was collected by filtration to give (S, E) -N- (2- ((4- (2H-tetrazol-5-yl) phenyl) amino) -2-oxo-1-phenylethyl) -3- (3-chloroethyl) as a white solid 66mg of (E) -2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamide.

¹ HNMR(DMSO-d6 400MHz)δ10.51(s，1H)，9.87(s,1H),9.24(d,1H,J＝8.0Hz),7.93(t,1H,J＝8.6 Hz),7.90(dd,2H,J＝2.0，6.8Hz),7.63(dd,2H,J＝1.8，7.0Hz),7.61(d,1H,J＝6.8Hz),7.52(d,2H,J＝7.2Hz),7.41(t,2H,J＝7.4Hz),7.34-7.30(m，1H)，6.93(dd,2H,J＝15.8,79.8Hz),5.75(d,1H，J＝7.6Hz)。

MS m/z(ESI):545[M+H]+

Example 57: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) ethyl benzoate

The first step is as follows: (S) -4- (2- ((tert-butoxycarbonyl) amino) -2-phenylacetylamino) benzoic acid (57-1) ethyl ester

Boc-phenylglycine (278mg, 1.5mmol), ethyl 4-aminobenzoate (250mg, 1.5mmol), HATU (690mg, 1.8mmol) were dissolved in DMF (4mL), DIPEA (0.78mL) was added, and the mixture was stirred at room temperature overnight. Water was added to the reaction mixture to quench it, and the precipitated pale yellow solid was collected by filtration to give 556mg of (S) -4- (2- ((tert-butoxycarbonyl) amino) -2-phenylacetylamino) benzoic acid ethyl ester in 93% yield.

The second step is that: preparation of ethyl (S) -4- (2-amino-2-phenylacetylamino) benzoate hydrochloride (57-2)

Ethyl (S) -4- (2- ((tert-butoxycarbonyl) amino) -2-phenylacetamido) benzoate (166mg) was dissolved in methanol (1mL), a hydrochloric acid methanol solution (4mol/L, 5mL) was added thereto, and the mixture was stirred at room temperature for 3 hours, and the reaction mixture was concentrated to give (S) -4- (2-amino-2-phenylacetamido) benzoate ethyl hydrochloride (147mg) as a yellow solid.

The third step: preparation of (S, E) -ethyl 4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) benzoate (57)

(E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (130mg), ethyl (S) -4- (2-amino-2-phenylacetamido) benzoate hydrochloride (147mg), HATU (230mg) dissolved in DMF (5mL), DIPEA (0.7mL) was added, the mixture was stirred at room temperature overnight, water was added to quench the reaction, and the precipitated solid was collected by filtration and purified by preparative thin layer chromatography to give 145mg of ethyl (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) benzoate as a pale yellow solid.

¹ HNMR(DMSO-d6 400MHz)δ10.73(s，1H)，9.86(s,1H),9.23(d,1H,J＝7.6Hz),7.93(t,1H,J＝8.0Hz),7.91(d,2H,J＝8.8Hz),7.71(d,2H,J＝9.2Hz),7.63(dd,1H,J＝1.2，8.8Hz),7.51(d,2H,J＝8.8Hz),7.40(t,2H,J＝7.4Hz),7.36-7.32(m,1H),6.90(dd,2H,J＝16,66.4Hz),5.70(d,1H，J＝7.6Hz)，4.27(q，2H，J＝7.2Hz)，1.30(t,3H，J＝7.0Hz)。

MS m/z(ESI):549[M+H]+

Example 58: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) benzoic acid tert-butyl ester

The title compound was prepared according to the third reaction step of example 49.

MS m/z(ESI):577[M+H]+

Example 59: 1- ((ethoxycarbonyl) oxy) ethyl 4- ((S) -2- ((E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -2-phenylacetamido) benzoate

Adding (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -2-phenylacetamido) benzoic acid (49, 27.5mg,53 mu mol), potassium carbonate (22mg,158 mu mol) and potassium iodide (9mg,53 mu mol) into DMF (1mL), pumping nitrogen, adding 1-chloroethyl ethyl carbonate (12mg,79 mu mol) under nitrogen atmosphere, stirring uniformly, reacting at 60 ℃ for 16 hours, and detecting the reaction completion by LCMS. Spin-drying the solvent, adding 10mL of water, extracting with ethyl acetate, collecting the organic phase, washing, drying, concentrating, and purifying by preparative thin-layer chromatography to obtain a pure product of 1- ((ethoxycarbonyl) oxy) ethyl 4- ((S) -2- ((E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl) -2-phenylacetamido) benzoate (24mg, yield 71%).

MS m/z(ESI):503[M-133]+

Example 60: preparation of (S, E) -2-chloro-4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) benzoic acid

The first step is as follows: preparation of tert-butyl 2-chloro-4-nitrobenzoate (60-2)

2-chloro-4-nitrobenzoyl chloride (1.11g, 5mmol) was dissolved in tetrahydrofuran (10mL), and a solution of lithium tert-butoxide in tetrahydrofuran (2.2mol/L, 3.4mL) was added dropwise with cooling in an ice bath and stirred at room temperature overnight. The reaction mixture was concentrated, and then saturated aqueous sodium hydrogencarbonate solution was added to conduct extraction with ethyl acetate, and the organic layer was separated, dried, filtered and concentrated to obtain 1.012g (yield: 78%) of tert-butyl 2-chloro-4-nitrobenzoate as a yellow solid.

The second step is that: preparation of tert-butyl 4-amino-2-chlorobenzoate (60-3)

Tert-butyl 2-chloro-4-nitrobenzoate (1.01g) was dissolved in methanol (20mL), reduced iron powder (1.21g) and glacial acetic acid (6.2mL) were added, the mixture was refluxed for three hours, filtered, the filtrate was concentrated, and the residue was purified by column chromatography to give tert-butyl 4-amino-2-chlorobenzoate 871mg as a white solid.

The third step: preparation of tert-butyl (S) -4- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -2-phenylacetamido) -2-chlorobenzoate (60-4)

Fmoc-phenylglycine (374mg, 1mmol), tert-butyl 4-amino-2-chlorobenzoate (228mg, 1mmol), HATU (461mg, 1.2mmol) were dissolved in dichloromethane (5mL), DIPEA (0.5mL) was added, and the mixture was stirred at room temperature overnight. Water was added to the reaction solution to quench, methylene chloride was used for extraction, the organic layer was separated, dried and filtered, and the residue was purified by column chromatography to give 440mg of (S) -4- (2- (((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -2-phenylacetylamino) -2-chlorobenzoic acid tert-butyl ester as a white solid in 75.5% yield.

The fourth step: preparation of (S) -4- (2-amino-2-phenylacetylamino) -2-chlorobenzoic acid tert-butyl ester (60-5)

Tert-butyl (S) -4- (2- (((9H-fluorenylmethoxy) carbonyl) amino) -2-phenylacetamido) -2-chlorobenzoate (250mg) was dissolved in methylene chloride (6mL), dimethylamine (3mL) was added, and the mixture was stirred at room temperature for 2 hours, and the reaction mixture was concentrated and purified by column chromatography to give tert-butyl (S) -4- (2-amino-2-phenylacetamido) -2-chlorobenzoate (129mg) as a yellow oil.

The fifth step: preparation of (S, E) -2-chloro-4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) benzoic acid (60-6)

(E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (115mg), tert-butyl (S) -4- (2-amino-2-phenylacetamido) -2-chlorobenzoate (129mg) was dissolved in ethyl acetate/DMF (3mL/1mL), DIPEA (0.4mL), T3P (50% ethyl acetate solution, 0.425mL) was added and stirred overnight at 55 ℃. Water is added into the reaction liquid for quenching, ethyl acetate is used for extraction, organic phase is dried, filtered and concentrated, and then white solid tert-butyl (S, E) -2-chloro-4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazole-2-yl) phenyl) acrylamido) -2-phenylacetamido) benzoate is obtained through purification by column chromatography.

And a sixth step: preparation of (S, E) -2-chloro-4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) benzoic acid (60)

(S, E) -tert-butyl 2-chloro-4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) benzoate (159mg) was dissolved in dichloromethane (5mL), trifluoroacetic acid (4mL) was added thereto, stirred at room temperature for 2 hours, concentrated and purified by preparative thin layer chromatography to give (S, E) -2-chloro-4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) benzoic acid as a white solid (100 mg).

¹ HNMR(DMSO-d6 400MHz)δ13.10(brs，1H)，10.79(s,1H),9.86(s,1H),9.24(d,1H,J＝7.2Hz),7.93(t,1H,J＝8.2Hz),7.86(d,1H,J＝2.0Hz),7.80(d,1H,J＝8.4Hz),7.63(dd,1H,J＝1.0，8.6Hz),7.52(dd,1H,J＝2.0，8.8Hz),7.49(d,2H,J＝7.2Hz),7.40(t,2H,J＝7.4Hz),7.36-7.33(m,1H),6.90(dd,2H,J＝16.2,61Hz),5.66(d,1H,J＝7.2Hz)。

MS m/z(ESI):555[M+H]+

Example 61: preparation of (S, E) -3-chloro-4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) benzoic acid

The first step is as follows: preparation of tert-butyl 4-amino-3-chlorobenzoate (61-1)

Tert-butyl 4-aminobenzoate (966mg, 5mmol) was dissolved in isopropanol/acetonitrile (12mL/12mL), heated to 60 deg.C, NCS (732mg, 5.5mmol/L) was added, and the mixture was stirred at 80 deg.C for one hour. The reaction solution was cooled and concentrated, and the residue was dissolved in methylene chloride, washed with a 1mol/L aqueous solution of sodium hydroxide and a saturated common salt solution, and the organic layer was separated, dried, filtered and concentrated to give 1.1g of 4-amino-3-chlorobenzoic acid tert-butyl ester as a brown solid.

The second step is that: preparation of (S, E) -3-chloro-4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) benzoic acid tert-butyl ester (61-2)

Fmoc-phenylglycine (374mg, 1mmol), tert-butyl 4-amino-3-chlorobenzoate (230mg, 1mmol) was dissolved in ethyl acetate (5mL), DIPEA (0.5mL), T3P (50% ethyl acetate solution, 1.2mL) was added, and the mixture was stirred at 55 ℃ overnight. Water is added into the reaction liquid for quenching, ethyl acetate is used for extraction, organic phase is dried, filtered and concentrated, and then the mixture is purified by column chromatography to obtain 553mg of beige solid tert-butyl (S, E) -3-chloro-4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) benzoate with the yield of 95 percent.

The third step: preparation of tert-butyl (S) -4- (2-amino-2-phenylacetylamino) -3-chlorobenzoate (61-3)

Tert-butyl (S) -4- (2- (((9H-fluorenylmethoxy) carbonyl) amino) -2-phenylacetamido) -3-chlorobenzoate (250mg) was dissolved in methylene chloride (6mL), dimethylamine (3mL) was added, and the mixture was stirred at room temperature for 2 hours, and the reaction mixture was concentrated and purified by column chromatography to give tert-butyl (S) -4- (2-amino-2-phenylacetamido) -3-chlorobenzoate (111mg) as a yellow oil.

The fourth step: preparation of (S, E) -3-chloro-4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) benzoic acid tert-butyl ester (61-4)

(E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (100mg), tert-butyl (S) -4- (2-amino-2-phenylacetamido) -2-chlorobenzoate (111mg) was dissolved in ethyl acetate/DMF (3mL/1mL), DIPEA (0.4mL), T3P (50% ethyl acetate solution, 0.365mL) was added, and the mixture was stirred overnight at 55 ℃. Adding water into the reaction solution for quenching, extracting by ethyl acetate, drying, filtering and concentrating the organic phase, and purifying by column chromatography to obtain 130mg of white solid (S, E) -3-chloro-4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) benzoic acid tert-butyl ester.

The fifth step: preparation of (S, E) -3-chloro-4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) benzoic acid (61)

(S, E) -3-chloro-4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) benzoic acid tert-butyl ester (130mg) was dissolved in dichloromethane (5mL), trifluoroacetic acid (3mL) was added, stirred at room temperature for 2 hours, concentrated and purified by preparative thin layer chromatography to give (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) benzoic acid as a white solid (79 mg).

¹ HNMR(DMSO-d6 400MHz)δ13.18(brs，1H)，10.09(s,1H),9.86(s,1H),9.22(d,1H,J＝7.6Hz),7.93(t,1H,J＝8.2Hz),7.92(d,1H,J＝1.2Hz),7.89-7.86(m,2H),7.63(dd,1H,J＝1.4，8.6Hz),7.54(d,2H,J＝7.2Hz),7.40(t,2H,J＝7.4Hz),7.36-7.33(m,1H),6.92(dd,2H,J＝16,60.4Hz),5.95(d,1H,J＝7.6Hz)。

MS m/z(ESI):555[M+H]+

Example 62: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) -2-fluorobenzoic acid

The first step is as follows: preparation of 2-fluoro-4-nitrobenzoyl chloride (62-2)

2-fluoro-4-nitrobenzoic acid (920mg) was dissolved in phosphorus oxychloride (10mL) and refluxed for 1.5 hours. The reaction liquid is cooled and concentrated to obtain a gray oily 2-fluoro-4-nitrobenzoyl chloride crude product which is directly used for the next reaction.

The second step is that: preparation of tert-butyl 2-fluoro-4-nitrobenzoate (62-3)

To tert-butanol (10mL) was added an n-butyllithium-n-hexane solution (2.5mol/L, 2.3mL), and the mixture was stirred at room temperature for 20 minutes. The crude 2-fluoro-4-nitrobenzoyl chloride was dissolved in tetrahydrofuran (4mL), added dropwise to the reaction mixture, and stirred at room temperature overnight. The reaction solution was concentrated, water was added, extraction was carried out with ethyl acetate, the organic layer was separated, dried, filtered and concentrated, and the residue was purified by column chromatography to give 2-fluoro-4-nitrobenzoic acid tert-butyl ester (435mg) as a white solid.

The third step: preparation of tert-butyl 4-amino-2-fluorobenzoate (62-4)

Tert-butyl 2-fluoro-4-nitrobenzoate (435mg) was dissolved in ethanol (8mL), 10% Pd/C (120mg) and ammonium formate (760mg) were added, the mixture was stirred at room temperature for 2 hours, filtered, the filtrate was concentrated, the residue was extracted with water and methylene chloride, and the organic layer was separated, dried, filtered and concentrated to give 367mg of tert-butyl 4-amino-2-fluorobenzoate as a white solid.

The fourth step: preparation of tert-butyl (S) -4- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -2-phenylacetylamino) -2-fluorobenzoate (62-5)

Fmoc-phenylglycine (380mg, 1mmol), tert-butyl 4-amino-3-chlorobenzoate (213mg, 1mmol) HATU (466mg, 1.2mmol) was dissolved in dichloromethane (5mL), DIPEA (0.5mL) was added, and the mixture was stirred at room temperature overnight. Water was added to the reaction solution to quench, dichloromethane was used for extraction, the organic layer was separated, dried and filtered, and the residue was purified by column chromatography to give 456mg of (S) -4- (2- ((((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -2-phenylacetylamino) -2-fluorobenzoic acid tert-butyl ester as a white solid in 81% yield.

The fifth step: preparation of tert-butyl (S) -4- (2-amino-2-phenylacetylamino) -2-fluorobenzoate (62-6)

(S) -tert-butyl 4- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -2-phenylacetamido) -2-fluorobenzoate (226mg) was dissolved in methylene chloride (6mL), dimethylamine (3mL) was added, stirring was performed at room temperature for 2 hours, and the reaction mixture was concentrated and purified by column chromatography to give tert-butyl (S) -4- (2-amino-2-phenylacetamido) -2-fluorobenzoate (100mg) as a yellow oil.

And a sixth step: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) -2-fluorobenzoic acid tert-butyl ester (62-7)

(E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (94mg), tert-butyl (S) -4- (2-amino-2-phenylacetamido) -2-fluorobenzoate (100mg) was dissolved in ethyl acetate (5mL), DIPEA (0.2mL), T3P (50% ethyl acetate solution, 0.34mL) was added, and the mixture was stirred at 55 ℃ overnight. Water is added into the reaction liquid for quenching, ethyl acetate is used for extraction, organic phase is dried, filtered and concentrated, and then white solid (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) -2-fluorobenzoic acid tert-butyl ester 95mg is obtained after column chromatography purification.

The seventh step: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) -2-fluorobenzoic acid (62)

(S, E) -tert-butyl 4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) -2-fluorobenzoate (95mg) was dissolved in dichloromethane (5mL), trifluoroacetic acid (3mL) was added, stirred at room temperature for 3 hours, concentrated and purified by preparative thin layer chromatography to give (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) -2-fluorobenzoic acid (48mg) as a white solid.

¹ HNMR(DMSO-d6 400MHz)δ12.90(brs,1H),10.87(s,1H),9.86(s,1H),9.24(d,1H,J＝7.2Hz),7.94(t,1H,J＝8.2Hz),7.83(t,1H,J＝8.4Hz),7.66-7.63(m,2H),7.49(d,2H,J＝7.2Hz),7.41(t,2H,J＝7.4Hz),7.37-7.32(m,2H),6.90(dd,2H,J＝16.2,60.6Hz),5.67(d,1H,J＝7.2Hz)。

MS m/z(ESI):539[M+H]+

Example 63: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) -3-fluorobenzoic acid

The first step is as follows: preparation of 3-fluoro-4-nitrobenzoyl chloride (63-2)

3-fluoro-4-nitrobenzoic acid (920mg) was dissolved in phosphorus oxychloride (10mL) and refluxed for 1.5 hours. The reaction liquid is cooled and concentrated to obtain a gray oily 2-fluoro-4-nitrobenzoyl chloride crude product which is directly used for the next reaction.

The second step is that: preparation of tert-butyl 3-fluoro-4-nitrobenzoate (63-3)

To tert-butanol (10mL) was added an n-butyllithium-n-hexane solution (2.5mol/L, 2.3mL), and the mixture was stirred at room temperature for 20 minutes. The crude 2-fluoro-4-nitrobenzoyl chloride was dissolved in tetrahydrofuran (4mL), added dropwise to the reaction mixture, and stirred at room temperature overnight. The reaction solution was concentrated, water was added, extraction was carried out with ethyl acetate, the organic layer was separated, dried, filtered and concentrated, and the residue was purified by column chromatography to give 3-fluoro-4-nitrobenzoic acid tert-butyl ester (450mg) as a white solid.

The third step: preparation of tert-butyl 4-amino-3-fluorobenzoate (63-4)

3-fluoro-4-nitrobenzoic acid tert-butyl ester (450mg) was dissolved in ethanol (8mL), 10% Pd/C (120mg) and ammonium formate (760mg) were added, stirred at room temperature for 2 hours, filtered, the filtrate was concentrated, the residue was added with water, extracted with dichloromethane, the organic layer was separated, dried, filtered and concentrated to give 380mg of white solid tert-butyl 4-amino-2-fluorobenzoate.

The fourth step: preparation of tert-butyl (S) -4- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -2-phenylacetylamino) -3-fluorobenzoate (63-5)

Fmoc-phenylglycine (373mg, 1mmol), tert-butyl 4-amino-3-fluorobenzoate (211mg, 1mmol), HATU (461mg, 1.2mmol) were dissolved in dichloromethane (5mL), DIPEA (0.5mL) was added, and the mixture was stirred at room temperature overnight. Water was added to the reaction solution to quench, methylene chloride was used for extraction, the organic layer was separated, dried and filtered, and the residue was purified by column chromatography to give (S) -4- (2- (((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -2-phenylacetylamino) -3-fluorobenzoic acid tert-butyl ester as a white solid (456 mg).

The fifth step: preparation of tert-butyl (S) -4- (2-amino-2-phenylacetylamino) -3-fluorobenzoate (63-6)

(S) -tert-butyl 4- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -2-phenylacetylamino) -3-fluorobenzoate (230mg) was dissolved in methylene chloride (6mL), dimethylamine (3mL) was added, the mixture was stirred at room temperature for 2 hours, and the reaction mixture was concentrated and purified by column chromatography to give tert-butyl (S) -4- (2-amino-2-phenylacetylamino) -3-fluorobenzoate (100mg) as a yellow oil.

And a sixth step: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) -3-fluorobenzoic acid tert-butyl ester (63-7)

(E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (97mg), tert-butyl (S) -4- (2-amino-2-phenylacetamido) -2-fluorobenzoate (100mg) was dissolved in ethyl acetate (5mL), DIPEA (0.2mL), T3P (50% ethyl acetate solution, 0.34mL) was added, and the mixture was stirred at 55 ℃ overnight. Adding water into the reaction solution for quenching, extracting by ethyl acetate, drying organic phase, filtering, concentrating, and purifying by column chromatography to obtain white solid (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazole-2-yl) phenyl) acrylamido) -2-phenylacetamido) -3-fluorobenzoic acid tert-butyl ester 105 mg.

The seventh step: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetylamino) -3-fluorobenzoic acid (63)

(S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) -3-fluorobenzoic acid tert-butyl ester (105mg) was dissolved in dichloromethane (5mL), trifluoroacetic acid (3mL) was added, stirred at room temperature for 3 hours, concentrated and purified by preparative thin layer chromatography to give (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) -3-fluorobenzoic acid (45mg) as a white solid.

¹ HNMR(DMSO-d6 400MHz)δ13.11(brs,1H),10.46(s,1H),9.86(s,1H),9.19(d,1H,J＝7.6Hz),8.07(t,1H,J＝8.0Hz),7.93(t,1H,J＝8.2Hz),7.72(d,1H,J＝8.4Hz),7.68(dd,1H,J＝1.2,11.8Hz),7.63(dd,1H,J＝1.2,8.8Hz),7.51(d,2H,J＝6.8Hz),7.40(t,2H,J＝7.4Hz),7.36-7.32(m,1H),6.90(dd,2H,J＝16,64.8Hz),5.94(d,1H,J＝7.2Hz)。

MS m/z(ESI):539[M+H]+

Example 64: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetylamino) -2-methoxybenzoic acid

The first step is as follows: preparation of tert-butyl 2-methoxy-4-nitrobenzoate (64-2)

2-methoxy-4-nitrobenzoic acid (500mg, 2.54mmol) was dissolved in toluene (15mL), heated to 80 deg.C, N-dimethylformamide di-tert-butyl acetal (1.2mL) was added and stirred at 80 deg.C for 3 hours. N, N-dimethylformamide di-tert-butyl acetal (1.2mL) was added and stirred at 80 ℃ overnight. The reaction solution was cooled, washed with a saturated aqueous solution of sodium hydrogencarbonate once, and the organic layer was separated, dried, filtered and concentrated to give 400mg of 2-methoxy-4-nitrobenzoic acid tert-butyl ester as a brown oily substance.

The second step is that: preparation of tert-butyl 4-amino-2-methoxybenzoate (64-3)

Tert-butyl 2-methoxy-4-nitrobenzoate (400mg) was dissolved in ethanol (10mL), 10% Pd/C (130mg) and ammonium formate (650mg) were added, the mixture was stirred at room temperature for 3 hours, filtered, the filtrate was concentrated, the residue was added with water, extracted with dichloromethane, the organic layer was separated, dried, filtered and concentrated to give 336mg of tert-butyl 4-amino-2-methoxybenzoate as a white solid.

The third step: preparation of tert-butyl (S) -4- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -2- (3-methoxyphenyl) acetylamino) benzoate (64-4)

Fmoc-phenylglycine (377mg, 1mmol), tert-butyl 4-amino-2-methoxybenzoate (224mg, 1mmol) was dissolved in ethyl acetate (5mL), DIPEA (0.5mL), T3P (50% ethyl acetate solution, 1.2mL) were added, and the mixture was stirred overnight at 55 ℃. Water was added to the reaction solution to quench, ethyl acetate was used for extraction, the organic layer was separated, dried and filtered, and the residue was purified by column chromatography to give 516mg of (S) -4- (2- (((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -2- (3-methoxyphenyl) acetylamino) benzoic acid tert-butyl ester as a yellow solid.

The fourth step: preparation of (S) -4- (2-amino-2-phenylacetylamino) -2-methoxybenzoic acid tert-butyl ester (64-5)

Tert-butyl (S) -4- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -2- (3-methoxyphenyl) acetylamino) benzoate (258mg) was dissolved in methylene chloride (5mL), dimethylamine (3mL) was added, the mixture was stirred at room temperature for 2 hours, and the reaction mixture was concentrated and purified by column chromatography to give tert-butyl (S) -4- (2-amino-2-phenylacetamido) -2-methoxybenzoate (139mg) as a yellow oil.

The fifth step: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetylamino) -2-methoxybenzoic acid tert-butyl ester (64-6)

(E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (125mg), (S) -4- (2-amino-2-phenylacetamido) -2-fluorobenzoic acid tert-butyl ester (139mg), HATU (150mg) was dissolved in DMF (3mL), DIPEA (0.2mL) was added, and stirring was carried out overnight at room temperature. Water was added to the reaction mixture to quench, and the precipitated solid was collected by filtration and purified by column chromatography to give 212mg of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetylamino) -2-methoxybenzoic acid tert-butyl ester as a pale yellow solid.

And a sixth step: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetylamino) -2-methoxybenzoic acid (64)

(S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) -2-methoxybenzoic acid tert-butyl ester 150mg) was dissolved in methylene chloride/tetrahydrofuran (4mL/1mL), trifluoroacetic acid (3mL) was added thereto, and the mixture was stirred at room temperature for 2 hours, after concentration, the mixture was purified by preparative thin layer chromatography to give (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) -2-methoxybenzoic acid as a white solid (105 mg).

¹ HNMR (DMSO-d 6400 MHz) δ 12.23(brs,1H),10.66(s,1H),9.86(s,1H),9.22(d,1H, J ═ 7.2Hz),7.94(t,1H, J ═ 8.2Hz),7.66(d,1H, J ═ 8.4Hz),7.64(dd,1H, J ═ 1.2, 8.8Hz),7.5(E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid 46(m,3H),7.41(t,2H, J ═ 7.2Hz),7.37-7.32(m,1H),7.19(dd,1H, J ═ 1.8,8.6Hz), 6.91(dd,2H, 68, J ═ 2H, J ═ 16.78, 3H, 3.78 (d, 3H, J ═ 8.6.78 Hz).

MS m/z(ESI):533[M-18]+

Example 65: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -2- (3- (2- (dimethylamino) acetamido) phenyl) acetamido) benzoic acid

The first step is as follows: preparation of 2-amino-2- (3-nitrophenyl) acetic acid (65-2)

L-phenylglycine (20g,132mmol) was added to a mixed solvent of fuming nitric acid (20mL) and concentrated sulfuric acid (20mL) at 0 ℃ and the reaction was stirred for 1 hour and then allowed to react at room temperature for 30 minutes. Pouring the reaction solution into ice water (400mL), adding 4M ammonia water to adjust the pH value to 6, separating out light yellow solid in the water, filtering, washing a filter cake with a small amount of ice water and methanol in sequence, and drying the filter cake to obtain a product 2-amino-2- (3-nitrophenyl) acetic acid (10g, the yield is 39%).

The second step is that: preparation of (S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -2- (3-nitrophenyl) acetic acid (65-3)

2-amino-2- (3-nitrophenyl) acetic acid (10g,51mmol) was added to 1, 4-dioxane (200mL) and water (100mL), stirred at 0 ℃ for 5 minutes, and an aqueous solution (100mL) of sodium carbonate (18.91g,178mmol) was added and stirred well. Fmoc-OSu was dissolved in 1, 4-dioxane (100mL), and the mixture was added dropwise to the reaction system and reacted at room temperature for 16 hours. LCMS check reaction complete. The 1, 4-dioxane is removed by spinning, ethyl acetate is used for extraction, an organic phase is collected and washed, dried, concentrated and purified by column chromatography to obtain a pure product (S) -2- ((((9H-fluorene-9-yl) methoxyl) carbonyl) amino) -2- (3-nitrophenyl) acetic acid (18g, the yield is 84%).

MS m/z(ESI):419[M+H]+

The third step: preparation of tert-butyl (S) -4- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -2- (3-nitrophenyl) acetamido) benzoate (65-4)

(S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -2- (3-nitrophenyl) acetic acid (5g,11.95mmol), tert-butyl 4-aminobenzoate (2.42g,12.55mmol), HATU (5.45g,14.34mmol) and diisopropylethylamine (3.95mL,23.9mmol) were added to DCM (100mL) and stirred for 2H, LCMS checked for reaction completion. The solvent was dried by spinning, 100mL of water was added, extraction was performed with ethyl acetate, the organic phase was collected and washed, dried, concentrated, and purified by column chromatography to obtain a pure product of tert-butyl (S) -4- (2- (((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -2- (3-nitrophenyl) acetylamino) benzoate (6g, yield 85%).

MS m/z(ESI):370[M-Fmoc-H]-

The fourth step: preparation of tert-butyl (S) -4- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -2- (3-aminophenyl) acetamido) benzoate (65-5)

Tert-butyl (S) -4- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -2- (3-nitrophenyl) acetamido) benzoate (500mg, 842. mu. mol) was added to methanol (50mL), Raney Ni (250mg) was added, reaction was carried out under hydrogen atmosphere for 2 hours, Raney Ni was filtered off, and the filtrate was dried by evaporation to give tert-butyl (S) -4- (2- (((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -2- (3-aminophenyl) acetamido) benzoate (350mg, yield 72%).

MS m/z(ESI):564[M+H]+

The fifth step: preparation of tert-butyl (S) -4- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -2- (3- (2- (dimethylamino) acetamido) phenyl) acetamido) benzoate (65-6)

Tert-butyl (S) -4- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -2- (3-aminophenyl) acetamido) benzoate (350mg, 621. mu. mol), dimethylaminoacetic acid (77mg, 745. mu. mol), HATU (354mg, 931. mu. mol) and diisopropylethylamine (256. mu.l, 1.55mmol) were added to DCM (20mL) and stirred for a homogeneous reaction for 2 hours, and the reaction was checked for completion by LCMS. The solvent was dried by spinning, 20mL of water was added, extraction was performed with ethyl acetate, the organic phase was collected, washed, dried, and concentrated to obtain crude tert-butyl (S) -4- (2- (((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -2- (3- (2- (dimethylamino) acetylamino) phenyl) acetylamino) benzoate (400mg, yield 99%) which was directly charged into the next step.

MS m/z(ESI):649[M+H]+

And a sixth step: preparation of tert-butyl (S) -4- (2-amino-2- (3- (2- (dimethylamino) acetylamino) phenyl) acetylamino) benzoate (65-7)

Tert-butyl (S) -4- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -2- (3- (2- (dimethylamino) acetylamino) phenyl) acetylamino) benzoate (400mg, 617. mu. mol) was added to a mixed solvent of diethylamine (10mL) and DCM (20mL), and the mixture was stirred for reaction for 2 hours, and the solvent was evaporated and purified by column chromatography to give tert-butyl (S) -4- (2-amino-2- (3- (2- (dimethylamino) acetylamino) phenyl) acetylamino) benzoate (222mg, yield 84%).

MS m/z(ESI):427[M+H]+

The seventh step: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -2- (3- (2- (dimethylamino) acetamido) phenyl) acetamido) benzoic acid tert-butyl ester (65-8)

Tert-butyl (S) -4- (2-amino-2- (3- (2- (dimethylamino) acetamido) phenyl) acetamido) benzoate (74mg, 174. mu. mol), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (51mg, 191. mu. mol), HATU (79mg, 208. mu. mol) and diisopropylethylamine (86. mu.l, 521. mu. mol) were added to DCM (5mL) and stirred for a uniform reaction for 2 hours, and the reaction was checked to be complete by LCMS. The solvent was dried by evaporation, 20mL of water was added, extraction was performed with ethyl acetate, the organic phase was collected and washed, dried, concentrated, and purified by column chromatography to give pure tert-butyl (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -2- (3- (2- (dimethylamino) acetylamino) phenyl) acetylamino) benzoate (87mg, 74% yield).

MS m/z(ESI):677[M+H]+

The eighth step: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -2- (3- (2- (dimethylamino) acetamido) phenyl) acetamido) benzoic acid (65)

Tert-butyl (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -2- (3- (2- (dimethylamino) acetamido) phenyl) acetamido) benzoate (87mg, 128. mu. mol) was added to DCM (8mL), trifluoroacetic acid (4mL) was added, the reaction was stirred for 2 hours and was checked for completion by LCMS. The solvent was dried by spinning, and purified by preparative thin layer chromatography to give pure (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -2- (3- (2- (dimethylamino) acetylamino) phenyl) acetylamino) benzoic acid (40mg, 50% yield).

¹ HNMR(DMSO-d6 400MHz)δ10.76(s,1H),10.63(s,1H),9.86(d,1H，J＝1.2Hz),9.79(brs,1H), 9.24(d,1H,J＝7.2Hz),7.94(t,1H,J＝8.2Hz),7.90(d,2H,J＝8.8Hz),7.72-7.63(m,3H),7.59(t,1H,J＝6.8Hz),7.48(d,1H,J＝8.4Hz),7.40(t,1H,J＝8.0Hz),7.25(d,1H，J＝7.6Hz),6.91(dd,2H,J＝16,67.6Hz),5.72(d,1H，J＝7.6Hz),4.11(s，2H)，2.87(s,6H)。

MS m/z(ESI):621[M+H]+

Example 66: (S) -4- (2- (4- (aminomethyl) benzoylamino) -2- (3- (2- (dimethylamino) acetylamino) phenyl) acetylamino) benzoic acid

The first step is as follows: preparation of tert-butyl (S) -4- (2- (4- (aminomethyl) benzoylamino) -2- (3- (2- (dimethylamino) acetylamino) phenyl) acetylamino) benzoate (66-1)

Tert-butyl (S) -4- (2-amino-2- (3- (2- (dimethylamino) acetamido) phenyl) acetamido) benzoate (74mg, 174. mu. mol), 4- (((tert-butoxycarbonyl) amino) methyl) benzoic acid (48mg, 191. mu. mol), HATU (79mg, 208. mu. mol) and diisopropylethylamine (86. mu.l, 521. mu. mol) were added to DCM (5mL) and stirred for a uniform reaction for 2 hours, and the reaction was checked by LCMS for completion. The solvent was dried by evaporation, 20mL of water was added, extraction was performed with ethyl acetate, the organic phase was collected and washed, dried, concentrated, and purified by column chromatography to give pure tert-butyl (S) -4- (2- (4- (aminomethyl) benzoylamino) -2- (3- (2- (dimethylamino) acetylamino) phenyl) acetylamino) benzoate (72mg, yield 63%).

MS m/z(ESI):660[M+H]+

The second step is that: preparation of (S) -4- (2- (4- (aminomethyl) benzoylamino) -2- (3- (2- (dimethylamino) acetylamino) phenyl) acetylamino) benzoic acid (66)

Tert-butyl (S) -4- (2- (4- (aminomethyl) benzoylamino) -2- (3- (2- (dimethylamino) acetylamino) phenyl) acetylamino) benzoate (72mg, 109. mu. mol) was added to DCM (8mL), trifluoroacetic acid (4mL) was added, the reaction was stirred for 2h and checked for completion by LCMS. The solvent was dried by evaporation, and purified by preparative thin layer chromatography to give pure (S) -4- (2- (4- (aminomethyl) benzoylamino) -2- (3- (2- (dimethylamino) acetylamino) phenyl) acetylamino) benzoic acid (30mg, yield 55%).

MS m/z(ESI):504[M+H]+

Example 67: preparation of 4- ((S) -2- ((1R,4S) -4- (aminomethyl) cyclohexyl-1-carboxamido) -2- (3- (2- (dimethylamino) acetylamino) phenyl) acetylamino) benzoic acid

The first step is as follows: preparation of tert-butyl 4- ((S) -2- ((1R,4S) -4- (aminomethyl) cyclohexyl-1-carboxamido) -2- (3- (2- (dimethylamino) acetylamino) phenyl) acetylamino) benzoate (67-1)

Tert-butyl (S) -4- (2-amino-2- (3- (2- (dimethylamino) acetamido) phenyl) acetamido) benzoate (74mg, 174. mu. mol), (1r,4r) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexyl-1-carboxylate (49mg, 191. mu. mol), HATU (79mg, 208. mu. mol) and diisopropylethylamine (86. mu.l, 521. mu. mol) were added to DCM (5mL) and stirred for a uniform reaction for 2 hours, and the reaction was checked for completion by LCMS. The solvent was dried by evaporation, 20mL of water was added, extraction was performed with ethyl acetate, the organic phase was collected and washed, dried, concentrated, and purified by column chromatography to give pure tert-butyl 4- ((S) -2- ((1R,4S) -4- (aminomethyl) cyclohexyl-1-carboxamido) -2- (3- (2- (dimethylamino) acetylamino) phenyl) acetylamino) benzoate (102mg, yield 88%).

MS m/z(ESI):666[M+H]+

The second step is that: preparation of 4- ((S) -2- ((1R,4S) -4- (aminomethyl) cyclohexyl-1-carboxamido) -2- (3- (2- (dimethylamino) acetylamino) phenyl) acetylamino) benzoic acid (67)

Tert-butyl 4- ((S) -2- ((1R,4S) -4- (aminomethyl) cyclohexyl-1-carboxamido) -2- (3- (2- (dimethylamino) acetylamino) phenyl) acetylamino) benzoate (102mg,153 μmol) was added to DCM (8mL), trifluoroacetic acid (4mL) was added, the reaction was stirred for 2 hours and LCMS checked for completion. The solvent was dried by evaporation and purified by preparative thin layer chromatography to give pure 4- ((S) -2- ((1R,4S) -4- (aminomethyl) cyclohexyl-1-carboxamido) -2- (3- (2- (dimethylamino) acetylamino) phenyl) acetylamino) benzoic acid (40mg, yield 51%).

MS m/z(ESI):510[M+H]+

Example 68: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) -N- (cyclopropylsulfonyl) benzamide

The first step is as follows: preparation of N- (cyclopropylsulfonyl) -4-nitrobenzamide (68-2)

P-nitrobenzoic acid (2g,11.97mmol), 2-chloro-1-methylpyridine iodide (3.67g,14.36mmol), DMAP (73mg, 598. mu. mol), and triethylamine (5mL,35.90mmol) were added to DCM (40mL) and stirred well before cyclopropyl sulfonamide (2.28g,23.93mmol) was added and the reaction was complete by LCMS. The solvent is dried by spinning, 50mL water is added, ethyl acetate is used for extraction, the organic phase is collected and washed, dried, concentrated and purified by column chromatography to obtain the pure product N- (cyclopropylsulfonyl) -4-nitrobenzamide (1.092g, yield 37%).

The second step is that: preparation of N- (cyclopropylsulfonyl) -4-aminobenzamide (68-3)

Adding N- (cyclopropylsulfonyl) -4-nitrobenzamide (1.092g,4.47mmol) into methanol (100mL), adding Pd-C (500mg), replacing hydrogen by the reaction system for three times, reacting under a hydrogen atmosphere overnight, and detecting the completion of the reaction by LCMS. Filtration and concentration of the filtrate gave pure N- (cyclopropylsulfonyl) -4-aminobenzamide (904mg, 94% yield).

The third step: preparation of tert-butyl (S) - (2- ((4- ((cyclopropylsulfonyl) carbamoyl) phenyl) amino) -2-oxo-1-phenylethyl) carbamate (68-4)

Boc-phenylglycine (251mg, 1mmol), 4-amino-N- (cyclopropylsulfonyl) benzamide (214mg, 1mmol), HATU (456mg, 1.2mmol) were dissolved in DMF (4mL), DIPEA (0.5mL) was added and the mixture was stirred at room temperature overnight. Water was added to the reaction mixture to quench, followed by extraction with dichloromethane, and the organic layer was dried, filtered, concentrated and purified by column chromatography to give 440mg of (S) - (2- ((4- ((cyclopropylsulfonyl) carbamoyl) phenyl) amino) -2-oxo-1-phenylethyl) carbamic acid tert-butyl ester as a white solid.

The fourth step: preparation of (S) -4- (2-amino-2-phenylacetylamino) -N- (cyclopropylsulfonyl) benzamide (68-5)

Tert-butyl (S) - (2- ((4- ((cyclopropylsulfonyl) carbamoyl) phenyl) amino) -2-oxo-1-phenylethyl) carbamate (400mg) was dissolved in methanol (1.5mL), a methanol solution (5mL) of hydrochloric acid was added, the mixture was stirred at room temperature for 3 hours, the reaction mixture was concentrated, the mixture was dissolved in a 2mol/L aqueous sodium hydroxide solution, extracted with dichloromethane, and the organic layer was dried, filtered, concentrated and purified by column chromatography to give (S) -4- (2-amino-2-phenylacetamido) -N- (cyclopropylsulfonyl) benzamide (300mg) as a white solid.

The fifth step: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) -N- (cyclopropylsulfonyl) benzamide (68)

(E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (30mg), (S) -4- (2-amino-2-phenylacetylamino) -N- (cyclopropylsulfonyl) benzamide (30mg), HATU (40mg) was dissolved in DMF (2mL), DIPEA (0.055mL) was added, the mixture was stirred at room temperature overnight, and the reaction was quenched with water, and the precipitated solid was collected by filtration and purified by column chromatography to give (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -2-phenylacetamido) -N- (cyclopropylsulfonyl) benzamide 13mg as a white solid.

MS m/z(ESI):624[M+H]+

Example 69: preparation of (S, E) -N- (2- ((4- (N-acetylsulfamoyl) phenyl) amino) -2-oxo-1-phenylethyl) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamide

The first step is as follows: preparation of tert-butyl (S) - (2- ((4- (N-acetylsulfamoyl) phenyl) amino) -2-oxo-1-phenylethyl) carbamate (69-2)

Boc-phenylglycine (503mg, 2mmol), sulfacetamide (430mg, 2mmol), HATU (912mg, 2.4mmol) was dissolved in dichloromethane (10mL), DIPEA (1mL) was added and stirred at room temperature overnight. Water was added to the reaction mixture to quench, followed by extraction with dichloromethane, and the organic layer was dried, filtered, concentrated and purified by column chromatography to give 847mg of (S) - (2- ((4- (N-acetylsulfamoyl) phenyl) amino) -2-oxo-1-phenylethyl) carbamic acid tert-butyl ester as a white solid.

The second step is that: preparation of (S) -N- (4- (N-acetylsulfamoyl) phenyl) -2-amino-2-phenylacetamide (69-3)

(S) -tert-butyl (2- ((4- (N-acetylsulfamoyl) phenyl) amino) -2-oxo-1-phenylethyl) carbamate (180mg) was dissolved in methanol (1mL), a methanol solution (4mL) of hydrochloric acid was added, the mixture was stirred at room temperature for 3 hours, and the reaction mixture was concentrated to give (S) -N- (4- (N-acetylsulfamoyl) phenyl) -2-amino-2-phenylacetamide (197mg) as a white solid.

The third step: preparation of (S, E) -N- (2- ((4- (N-acetylsulfamoyl) phenyl) amino) -2-oxo-1-phenylethyl) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamide (69)

(E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (100mg), (S) -N- (4- (N-acetylsulfamoyl) phenyl) -2-amino-2-phenylacetamide (197mg), HATU (155mg) was dissolved in DMF (2mL), DIPEA (0.2mL) was added, stirred overnight at room temperature, quenched with water, and the precipitated solid was collected by filtration and purified by column chromatography to give (S, E) -N- (2- ((4- (N-acetylsulfamoyl) phenyl) amino) -2-oxo-1-phenylethyl) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) propan-ol as a white solid Enamide 53 mg.

¹ HNMR(DMSO-d6 400MHz)δ11.95(s,1H),10.82(s,1H),9.86(s,1H),9.23(d,1H,J＝7.6Hz),7.93(t,1H,J＝8.2Hz),7.82(d,2H,J＝9.2Hz),7.76(d,2H,J＝8.8Hz),7.63(dd,1H,J＝1.2,8.8Hz),7.49(d,2H,J＝6.8Hz),7.40(t,2H,J＝7.0Hz),7.36-7.30(m,1H),6.90(dd,2H,J＝16,64.4Hz),5.69(d,1H,J＝7.6Hz),1.87(s,3H)。

MS m/z(ESI):598[M+H]+

Example 70: preparation of (S, E) - (4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -2-phenylacetamido) phenyl) methyl carbamate

The first step is as follows: preparation of methyl 4-tert-butoxycarbonylaminophenylcarbamate (70-2)

Tert-butyl (4-aminophenyl) carbamate (1.04g) and triethylamine (2.1mL) were dissolved in methylene chloride (20mL), and methyl chloroformate (0.426mL) was added thereto under cooling in an ice bath and stirred at room temperature overnight. Water was added thereto and the mixture was quenched, extracted with methylene chloride, and the organic layer was dried, filtered and concentrated to give methyl 4-t-butoxycarbonylaminophenylcarbamate (1.11g) as a brown solid.

The second step is that: preparation of methyl (4-aminophenyl) carbamate (70-3)

Methyl 4-tert-Butoxycarbonylaminophenylcarbamate (1.11g) was dissolved in methylene chloride (30mL), and trifluoroacetic acid (15mL) was added thereto and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated, and a saturated aqueous sodium hydrogencarbonate solution was added to the residue to conduct extraction with methylene chloride, and the organic layer was dried, filtered, concentrated and purified by column chromatography to give a yellow oily substance, which was allowed to solidify by standing to give methyl (4-aminophenyl) carbamate (347mg) as a yellow solid.

The third step: preparation of (9H-fluoren-9-yl) methyl (S) - (2- ((4- ((methoxycarbonyl) amino) phenyl) amino) -2-oxo-1-phenylethyl) carbamate (70-4)

Fmoc-phenylglycine (423mg), (4-aminophenyl) carbamic acid methyl ester (186mg), HATU (510mg) was dissolved in DMF (4mL), DIPEA (0.55mL) was added, and the mixture was stirred at room temperature overnight. Water was added to the reaction solution to quench, and the precipitated solid was collected by filtration and slurried with ethanol to give (9H-fluoren-9-yl) methyl (S) - (2- ((4- ((methoxycarbonyl) amino) phenyl) amino) -2-oxo-1-phenylethyl) carbamate as a white solid (508 mg).

The fourth step: preparation of methyl (S) - (4- (2-amino-2-phenylacetylamino) phenyl) carbamate (70-5)

(9H-Fluoren-9-yl) methyl (S) - (2- ((4- ((methoxycarbonyl) amino) phenyl) amino) -2-oxo-1-phenylethyl) carbamate (264mg) was dissolved in methylene chloride/tetrahydrofuran (10mL/10mL), diethylamine (5mL) was added, stirring was performed at room temperature for 3 hours, the reaction solution was concentrated, and the residue was purified by column chromatography to give methyl (S) - (4- (2-amino-2-phenylacetamido) phenyl) carbamate (158mg) as a pale yellow solid.

The fifth step: preparation of (S, E) - (4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -2-phenylacetamido) phenyl) methyl carbamate (70)

(E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (150mg), (S) - (4- (2-amino-2-phenylacetamido) phenyl) carbamic acid methyl ester (158mg), HATU (230mg) was dissolved in DMF (4mL), DIPEA (0.28mL) was added, the mixture was stirred at room temperature overnight, quenched with water, and the precipitated solid was collected by filtration and slurried with ethanol/ethyl acetate (1/1) to give 130mg of methyl (S, E) - (4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -2-phenylacetamido) phenyl) carbamate as a white solid.

¹ HNMR (DMSO-d 6400 MHz) δ 10.39(s,1H),9.86(s,1H),9.58(s,1H),9.21(d,1H, J ═ 7.6Hz),7.95(t,1H, J ═ 8.0Hz),7.63(dd,1H, J ═ 1.4, 8.6Hz),7.5(E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid 47(m,4H),7.39-7.35(m,4H),7.33-7.29(m,1H),6.91(dd,2H, J ═ 16.0,80.0Hz),5.70(d,1H, J ═ 8.0Hz), 3.64(s, 3H).

MS m/z(ESI):550[M+H]+

Example 71: (S, E) -2- (4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -N-methyl-2-phenylacetamido) phenyl) acetic acid

The first step is as follows: preparation of tert-butyl (S) -2- (4- (2- (((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -N-methyl-2-phenylacetamido) phenyl) acetate (71-2)

Fmoc-phenylglycine (49-1,374mg, 1mmol), tert-butyl 2- (4- (methylamino) phenyl) acetate (216mg, 1mmol) was dissolved in ethyl acetate (7mL), DIPEA (0.5mL), T3P (50% in ethyl acetate, 0.71mL) was added, and stirring was carried out overnight at 55 ℃. Water was added to the reaction mixture to quench, and the reaction mixture was extracted with ethyl acetate, dried, filtered, concentrated and purified by column chromatography to give 460mg of (S) -tert-butyl 2- (4- (2- (((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -N-methyl-2-phenylacetamido) phenyl) acetate as a pale yellow solid, which was obtained in 79% yield.

The second step is that: preparation of tert-butyl (S) -2- (4- (2-amino-N-methyl-2-phenylacetamido) phenyl) acetate (71-3)

Tert-butyl (S) -2- (4- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -N-methyl-2-phenylacetamido) phenyl) acetate (460mg) was dissolved in dichloromethane (8mL), diethylamine (5mL) was added, stirring was carried out at room temperature for 3 hours, the reaction mixture was concentrated, and the residue was purified by column chromatography to give tert-butyl (S) -2- (4- (2-amino-N-methyl-2-phenylacetamido) phenyl) acetate (278mg) as a pale yellow solid.

The third step: preparation of (S, E) -tert-butyl 2- (4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -N-methyl-2-phenylacetamido) phenyl) acetate (71-4)

(E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (127mg), (S) -tert-butyl 2- (4- (2-amino-N-methyl-2-phenylacetamido) phenyl) acetate (139mg), HATU (194mg) dissolved in DMF (4mL), DIPEA (0.19mL) was added, the mixture was stirred at room temperature overnight, water was added to quench the reaction, and the precipitated solid was collected by filtration and purified by column chromatography to give 209mg of tert-butyl (S, E) -2- (4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -N-methyl-2-phenylacetamido) phenyl) acetate as a white solid.

The fourth step: preparation of (S, E) -2- (4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -N-methyl-2-phenylacetamido) phenyl) acetic acid (71)

(S, E) -tert-butyl 2- (4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -N-methyl-2-phenylacetamido) phenyl) acetate (160mg) was dissolved in dichloromethane (9mL), trifluoroacetic acid (4mL) was added thereto, and the mixture was stirred at room temperature for 1.5 hours, after concentration, the mixture was purified by preparative thin layer chromatography to give (S, E) -2- (4- (2- (3- (3-chloro-2-fluoro-6- (2H-tetrazol-2-yl) phenyl) acrylamido) -N-methyl-2-phenylacetamido) phenyl) acetic acid as a white solid (75 mg).

¹ HNMR(DMSO-d6 400MHz)δ12.39(brs，1H)，9.85(s,1H),9.04(d,1H,J＝7.6Hz),7.92(t,1H,J＝8.2Hz),7.62(dd,1H,J＝1.0，8.6Hz),7.29-7.26(m,5H),7.05(d,2H,J＝7.2Hz),6.96(d,2H,J＝8.0Hz),6.84(dd,2H,J＝16.2,65.0Hz),5.51(d,1H,J＝7.2Hz),3.61(s，2H),3.15(s，3H)。

MS m/z(ESI):549[M+H]+

Example 72: preparation of 4- ((S) -2- ((1R,4S) -4- (aminomethyl) cyclohexyl-1-carboxamido) -2- (3- (dimethylamino) phenyl) acetamido) benzoic acid

The first step is as follows: preparation of (S) -2- (3-bromobenzene) -2- (tert-butoxycarbonylamino) acetic acid (72-2)

3-bromo-L-phenylalanine (2.07g,8.49mmol) was added to a mixed solvent of dichloromethane (60ml) and water (40ml), triethylamine (3ml,21.22mmol) was added thereto, and the mixture was stirred well, followed by addition of Boc ₂ O (2.1ml,9.34mmol), reaction for 4 hours, LCMS to check reaction completion. Dichloromethane was spin-dried, 1M hydrochloric acid was added to adjust PH to 5, ethyl acetate was extracted, the organic phase was collected and washed, dried, concentrated, and slurried with petroleum ether to obtain pure (S) -2- (3-bromobenzene) -2- (tert-butoxycarbonylamino) acetic acid (2.5g, yield 86%).

MS m/z(ESI):345[M+H] ⁺

The second step is that: preparation of tert-butyl (S) -4- (2- (3-bromobenzene) -2- (tert-butoxycarbonylamino) acetylamino) benzoate (72-3)

(S) -2- (3-bromobenzene) -2- (tert-butoxycarbonylamino) acetic acid (2.5g,7.26mmol), tert-butyl 4-aminobenzoate (1.54g,7.99mmol), HATU (3.31g,8.72mmol) and diisopropylethylamine (2.4ml,14.53mmol) were added to dichloromethane (75ml) and stirred for 2 hours and LCMS checked for completion. The solvent was dried, 100ml of water was added, extraction was performed with ethyl acetate, the organic phase was collected and washed, dried, concentrated, and purified by column chromatography to obtain pure (S) -tert-butyl 4- (2- (3-bromobenzene) -2- (tert-butoxycarbonylamino) acetylamino) benzoate (3.75g, yield 99%).

MS m/z(ESI):419,421[M-99] ⁺

The third step: preparation of tert-butyl (S) -4- (2- (tert-butoxycarbonylamino) -2- (3- (methylamino) phenyl) acetylamino) benzoate (72-4)

Tert-butyl (S) -4- (2- (3-bromobenzene) -2- (tert-butoxycarbonylamino) acetylamino) benzoate (3.75g,7.22mmol), methylamine hydrochloride (1.22g,18.05mmol), Pd2(dba)3(661mg, 722. mu. mol), XPhos (688mg,1.44mmol) and Cs2CO3(10.58g,32.49mmol) were added to 1, 4-dioxane (100ml), nitrogen was purged, the reaction was carried out at 110 ℃ for 16 hours, and the reaction was complete by LCMS. The solvent was dried by evaporation, 100ml of water was added, extraction was performed with ethyl acetate, the organic phase was collected and washed, dried, concentrated, and purified by column chromatography to obtain pure tert-butyl (S) -4- (2- (tert-butoxycarbonylamino) -2- (3- (methylamino) phenyl) acetylamino) benzoate (1.067g, yield 31%).

MS m/z(ESI):468[M-1] ^-

The fourth step: preparation of tert-butyl (S) -4- (2- (tert-butoxycarbonylamino) -2- (3- (dimethylamino) phenyl) acetylamino) benzoate (72-5)

Tert-butyl (S) -4- (2- (tert-butoxycarbonylamino) -2- (3- (methylamino) phenyl) acetylamino) benzoate (300mg, 639. mu. mol) was added to methanol (10ml), paraformaldehyde (38mg,1.28mmol) and sodium cyanoborohydride (120mg,1.92mmol) were added, and the mixture was stirred for 2 hours and LCMS showed completion of the reaction. The solvent was dried, 100ml of water was added, extraction was performed with ethyl acetate, the collected organic phase was washed, dried, concentrated, and purified by column chromatography to give pure tert-butyl (S) -4- (2- (tert-butoxycarbonylamino) -2- (3- (dimethylamino) phenyl) acetylamino) benzoate (203mg, yield 66%).

MS m/z(ESI):484[M+1]+

The fifth step: preparation of tert-butyl (S) -4- (2-amino-2- (3- (dimethylamino) phenyl) acetylamino) benzoate (72-6)

Tert-butyl (S) -4- (2- (tert-butoxycarbonylamino) -2- (3- (dimethylamino) phenyl) acetylamino) benzoate (203mg, 420. mu. mol) was added to a solution of ethyl hydrogen chloride in ethyl acetate (10ml), and the reaction was completed by LCMS. The solvent was dried by evaporation, and purified by column chromatography to give pure (S) -4- (2-amino-2- (3- (dimethylamino) phenyl) acetylamino) benzoic acid tert-butyl ester (67mg, 42% yield).

MS m/z(ESI):384[M+H]+

And a sixth step: preparation of tert-butyl 4- ((S) -2- ((1R,4S) -4- (aminomethyl) cyclohexyl-1-carboxamido) -2- (3- (dimethylamino) phenyl) acetylamino) benzoate (72-7)

Tert-butyl (S) -4- (2-amino-2- (3- (dimethylamino) phenyl) acetylamino) benzoate (67mg, 139. mu. mol), (1r,4r) -4- (((tert-oxycarbonyl) amino) methyl) cyclohexyl-1-carboxylate (39mg, 152. mu. mol), HATU (63mg, 166. mu. mol) and isopropyldiethylamine (54mg, 416. mu. mol) were added to dichloromethane (3ml) and reacted for 2 hours with LCMS to check completion. The solvent was dried by evaporation, 10ml of water was added, extraction was performed with ethyl acetate, the organic phase was collected and washed, dried, concentrated, and purified by column chromatography to give pure tert-butyl 4- ((S) -2- ((1r,4S) -4- (aminomethyl) cyclohexyl-1-carboxamido) -2- (3- (dimethylamino) phenyl) acetylamino) benzoate (55mg, yield 64%).

MS m/z(ESI):623[M+H]+

The seventh step: preparation of 4- ((S) -2- ((1r,4S) -4- (aminomethyl) cyclohexyl-1-carboxamido) -2- (3- (dimethylamino) phenyl) acetylamino) benzoic acid (72)

Tert-butyl 4- ((S) -2- ((1r,4S) -4- (aminomethyl) cyclohexyl-1-carboxamido) -2- (3- (dimethylamino) phenyl) acetylamino) benzoate (55mg, 88. mu. mol) was added to a mixed solvent of trifluoroacetic acid (2ml) and dichloromethane (4ml), and the reaction was carried out for 1 hour and was determined to be complete by LCMS. The solvent was dried by evaporation to give 4- ((S) -2- ((1r,4S) -4- (aminomethyl) cyclohexyl-1-carboxamido) -2- (3- (dimethylamino) phenyl) acetylamino) benzoic acid as a product (40mg, 97% yield).

MS m/z(ESI):467[M+H]+

Example 73: preparation of 4- ((S) -2- ((1R,4S) -4- (aminomethyl) cyclohexyl-1-carboxamido) -2- (4- (dimethylamino) phenyl) acetamido) benzoic acid

The first step is as follows: preparation of tert-butyl (S) -4- (2- (4-bromobenzene) -2- (tert-butoxycarbonylamino) acetylamino) benzoate (73-2)

(S) -2- (4-bromobenzene) -2- (tert-butoxycarbonylamino) acetic acid (2g,5.81mmol), tert-butyl 4-aminobenzoate (1.24g,6.39mmol), HATU (2.65g,6.97mmol) and diisopropylethylamine (1.92ml,11.62mmol) were added to dichloromethane (100ml) and stirred well for 2 hours and LCMS checked for completion of the reaction. The solvent was dried, 100ml of water was added, extraction was performed with ethyl acetate, the organic phase was collected and washed, dried, concentrated, and purified by column chromatography to obtain pure (S) -tert-butyl 4- (2- (4-bromobenzene) -2- (tert-butoxycarbonylamino) acetylamino) benzoate (2.5g, yield 83%).

MS m/z(ESI):419,421[M-99]+

The second step is that: preparation of tert-butyl (S) -4- (2- (tert-butoxycarbonylamino) -2- (4- (methylamino) phenyl) acetylamino) benzoate (73-3)

Tert-butyl (S) -4- (2- (4-bromobenzene) -2- (tert-butoxycarbonylamino) acetylamino) benzoate (1.64g,3.16mmol), methylamine hydrochloride (533mg,7.89mmol), Pd2(dba)3(29mg, 32. mu. mol), XPhos (62mg, 126. mu. mol) and Cs2CO3(4.58g,14.05mmol) were added to tetrahydrofuran (20ml), nitrogen was purged, and the reaction was completed by LCMS after 16 hours at 110 ℃. The solvent was dried, 50ml of water was added, extraction was performed with ethyl acetate, the collected organic phase was washed, dried, concentrated, and purified by column chromatography to give pure (S) -tert-butyl 4- (2- (tert-butoxycarbonylamino) -2- (4- (methylamino) phenyl) acetylamino) benzoate (230mg, yield 16%).

MS m/z(ESI):468[M-1]-

The third step: preparation of tert-butyl (S) -4- (2- (tert-butoxycarbonylamino) -2- (4- (dimethylamino) phenyl) acetylamino) benzoate (73-4)

Tert-butyl (S) -4- (2- (tert-butoxycarbonylamino) -2- (4- (methylamino) phenyl) acetylamino) benzoate (230mg, 490. mu. mol) was added to methanol (10ml), paraformaldehyde (29mg, 980. mu. mol) and sodium cyanoborohydride (92mg,1.47mmol) were added, and the reaction was stirred for 2 hours and checked for completion by LCMS. The solvent was dried, 500ml of water was added, extraction was performed with ethyl acetate, the collected organic phase was washed, dried, concentrated, and purified by column chromatography to give a pure product of tert-butyl (S) -4- (2- (tert-butoxycarbonylamino) -2- (4- (dimethylamino) phenyl) acetylamino) benzoate (143mg, yield 60%).

MS m/z(ESI):484[M+1]+

The fourth step: preparation of tert-butyl (S) -4- (2-amino-2- (4- (dimethylamino) phenyl) acetylamino) benzoate (73-5)

Tert-butyl (S) -4- (2- (tert-butoxycarbonylamino) -2- (4- (dimethylamino) phenyl) acetylamino) benzoate (143mg, 296. mu. mol) was added to a solution of ethyl hydrogen chloride in ethyl acetate (10ml), and the reaction was allowed to proceed for 3 hours and was checked by LCMS for completion. The solvent was dried by evaporation, and purified by column chromatography to give pure (S) -tert-butyl 4- (2-amino-2- (4- (dimethylamino) phenyl) acetylamino) benzoate (98mg, yield 86%).

MS m/z(ESI):384[M+H]+

The fifth step: preparation of tert-butyl 4- ((S) -2- ((1R,4S) -4- (aminomethyl) cyclohexyl-1-carboxamido) -2- (4- (dimethylamino) phenyl) acetylamino) benzoate (73-6)

Tert-butyl (S) -4- (2-amino-2- (4- (dimethylamino) phenyl) acetamido) benzoate (98mg, 203. mu. mol), (1r,4r) -4- (((tert-oxycarbonyl) amino) methyl) cyclohexyl-1-carboxylate (57mg, 223. mu. mol), HATU (92mg, 243. mu. mol) and isopropyldiethylamine (100mg, 208. mu. mol) were added to dichloromethane (5ml) and reacted for 2 hours with LCMS to check completion. The solvent was dried by evaporation, 10ml of water was added, extraction was performed with ethyl acetate, the organic phase was collected and washed, dried, concentrated, and purified by column chromatography to give pure tert-butyl 4- ((S) -2- ((1r,4S) -4- (aminomethyl) cyclohexyl-1-carboxamido) -2- (4- (dimethylamino) phenyl) acetylamino) benzoate (101mg, yield 80%).

MS m/z(ESI):623[M+H]+

And a sixth step: preparation of 4- ((S) -2- ((1R,4S) -4- (aminomethyl) cyclohexyl-1-carboxamido) -2- (4- (dimethylamino) phenyl) acetamido) benzoic acid (73)

Tert-butyl 4- ((S) -2- ((1R,4S) -4- (aminomethyl) cyclohexyl-1-carboxamido) -2- (4- (dimethylamino) phenyl) acetylamino) benzoate (101mg, 162. mu. mol) was added to a mixed solvent of trifluoroacetic acid (2ml) and dichloromethane (4ml), and the reaction was allowed to react for 1 hour and was determined to be complete by LCMS. The solvent was dried by evaporation to give 4- ((S) -2- ((1r,4S) -4- (aminomethyl) cyclohexyl-1-carboxamido) -2- (4- (dimethylamino) phenyl) acetylamino) benzoic acid as a product (70mg, 93% yield).

MS m/z(ESI):467[M+H]+

Example 74: preparation of 4- ((S) -2- ((1R,4S) -4- (aminomethyl) cyclohexyl-1-carboxamido) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) -N-ethylacetamido) -benzoic acid

The first step is as follows: preparation of (S) -2- (3-bromophenyl) -2- (tert-butoxycarbonylamino) acetic acid (74-2)

3-bromo-phenylglycine (10g,40.97mmol) and sodium bicarbonate (10.32g,123mmol) were added to a mixed solvent of dichloromethane (300ml) and water (300ml), stirred well, then Boc2O (10.36ml,45mmol) was added, the reaction was allowed to react for 4 hours, and LCMS checked for completion. Dichloromethane was spin-dried, 1M hydrochloric acid was added to adjust PH to 5, ethyl acetate was extracted, the organic phase was collected and washed, dried, concentrated, and slurried with petroleum ether to obtain pure (S) -2- (3-bromophenyl) -2- (tert-butoxycarbonylamino) acetic acid (14.1g, 99% yield).

The second step is that: preparation of benzyl (S) -2- (3-bromophenyl) -2- (tert-butoxycarbonylamino) acetate (74-3)

(S) -3- (3-bromophenyl) -2- (tert-butoxycarbonylamino) acetic acid (14.1g,10.96mmol) and cesium carbonate (10.94g,33.59mmol) were added to DMF (100ml), benzyl bromide (5.11ml,43mmol) was added, the reaction was allowed to react for 3 hours, and the reaction was checked for completion by LCMS. Adding 500ml of water, extracting with ethyl acetate, collecting an organic phase, washing with water, drying, concentrating, and purifying by column chromatography to obtain a pure product (S) -benzyl 2- (3-bromophenyl) -2- (tert-butoxycarbonylamino) acetate (16g, yield 90%).

The third step: preparation of benzyl (S) -2- (3- (methylamino) phenyl) -2- (tert-butoxycarbonylamino) acetate (74-4)

Benzyl (S) -2- (3-bromophenyl) -2- (tert-butoxycarbonylamino) acetate (2g,4.6mmol), methylamine hydrochloride (777mg,11.51mmol), Pd2(dba)3(42mg, 46. mu. mol), Brettphos (90mg, 184. mu. mol) and Cs2CO3(3g,9.21mmol) were added to tetrahydrofuran (20ml), nitrogen was purged, and the reaction was completed by LCMS at 110 ℃ for 16 hours. The solvent was dried by evaporation, 100ml of water was added, extracted with ethyl acetate, and the organic phase was washed, dried, concentrated, and purified by column chromatography to give pure (S) -benzyl 2- (3- (methylamino) phenyl) -2- (tert-butoxycarbonylamino) acetate (565mg, yield 32%).

The fourth step: preparation of benzyl (S) -2- (tert-butoxycarbonylamino) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) acetate (74-5)

Benzyl (S) -2- (3- (methylamino) phenyl) -2- (tert-butoxycarbonylamino) acetate (565mg,1.47mmol), dimethylaminoacetic acid (182mg,1.76mmol), HATU (838mg,2.2mmol) and diisopropylethylamine (607. mu.l, 3.67mmol) were added to dichloromethane (10ml) and stirred uniformly for 16 hours, and the reaction was checked for completion by LCMS. The solvent was removed by evaporation, 20ml of water was added, extraction was performed with ethyl acetate, the organic phase was collected and washed, dried, concentrated, and purified by column chromatography to give pure benzyl (S) -2- (tert-butoxycarbonylamino) -2- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) acetate (550mg, yield 80%).

The fifth step: preparation of (S) -2- (tert-butoxycarbonylamino) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) acetic acid (74-6)

Benzyl (S) -2- (tert-butoxycarbonylamino) -2- (3- (2- (dimethylamino) -N-methylacetamino) phenyl) acetate (550mg,1.17mmol) was added to methanol (50ml), palladium on charcoal (250mg) was added, the reaction was carried out for 2 hours under a hydrogen atmosphere, the palladium on charcoal was filtered off, and the filtrate was spin-dried to give (S) -2- (tert-butoxycarbonylamino) -2- (3- (2- (dimethylamino) -N-methylacetamino) phenyl) acetic acid (366mg, yield 82%).

And a sixth step: preparation of (S) -4- (2- (tert-butoxycarbonylamino) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) -N-ethylacetamido) -benzoic acid tert-butyl ester (74-7)

(S) -2- (tert-Butoxycarbonylamino) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) acetic acid (183mg, 482. mu. mol), tert-butyl 4-acetamidobenzoate (138mg, 530. mu. mol), HATU (220mg, 579. mu. mol) and diisopropylethylamine (159. mu.l, 965. mu. mol) were added to dichloromethane (5ml) and stirred uniformly for 2 hours, and the reaction was checked to be complete by LCMS. The solvent was dried, 100ml of water was added, extraction was performed with ethyl acetate, the collected organic phase was washed, dried, concentrated, and purified by column chromatography to give pure (S) -4- (2- (tert-butoxycarbonylamino) -2- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) -N-ethylacetamido) -benzoic acid tert-butyl ester (121mg, yield 40%).

The seventh step: preparation of (S) -4- (2-amino-3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) -N-ethylacetamido) -benzoic acid tert-butyl ester (74-8)

(S) -4- (2- (tert-butoxycarbonylamino) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) -N-ethylacetamido) -benzoic acid tert-butyl ester (121mg, 195. mu. mol) was added to a hydrogen chloride ethyl acetate solution (10ml) and reacted for 2 hours, and the reaction was completed by LCMS. The solvent was dried by spinning and purified by column chromatography to give pure (S) -4- (2-amino-2- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) -N-ethylacetamido) -benzoic acid tert-butyl ester (50mg, yield 49%).

Eighth step: preparation of 4- ((S) -2- ((1R,4S) -4- (tert-Butoxycarbonylaminomethyl) cyclohexyl-1-carboxamido) -3- (3- (2- (dimethylamino) -N-methylacetyl) phenyl) -N-ethylacetoylamino) -benzoic acid tert-butyl ester (74-9)

(S) -4- (2-amino-2- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) -N-ethylacetamido) -benzoic acid tert-butyl ester (50mg, 96. mu. mol), (1R,4R) -4- (((tert-oxycarbonyl) amino) methyl) cyclohexyl-1-carboxylic acid (27mg, 105. mu. mol), HATU (44mg, 105. mu. mol) and isopropyldiethylamine (48mg, 288. mu. mol) were added to dichloromethane (2ml) and reacted for 2 hours, and LCMS checked that the reaction was complete. The solvent was evaporated, 5ml of water was added, and extraction was performed with ethyl acetate, and the organic phase was collected and washed, dried, concentrated, and purified by column chromatography to give pure 4- ((S) -2- ((1R,4S) -4- (tert-butoxycarbonylaminomethyl) cyclohexyl-1-carboxamido) -3- (3- (2- (dimethylamino) -N-methylacetyl) phenyl) -N-ethylacetamido) -benzoic acid tert-butyl ester (45mg, yield 62%).

The ninth step: preparation of 4- ((S) -2- ((1R,4S) -4- (aminomethyl) cyclohexyl-1-carboxamido) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) -N-ethylacetamido) -benzoic acid (74)

4- ((S) -2- ((1R,4S) -4- (tert-Butoxycarbonylaminomethyl) cyclohexyl-1-carboxamido) -3- (3- (2- (dimethylamino) -N-methylacetyl) phenyl) -N-ethylacetamido) -benzoic acid tert-butyl ester (45mg, 59. mu. mol) was added to a mixed solvent of trifluoroacetic acid (2ml) and dichloromethane (4ml), reacted for 1 hour, and the reaction was checked for completion by LCMS. The solvent was dried by evaporation to give the product 4- ((S) -2- ((1R,4S) -4- (aminomethyl) cyclohexyl-1-carboxamido) -3- (3- (2- (dimethylamino) -N-methylacetyl) phenyl) -N-ethylacetamido) -benzoic acid (23mg, 64% yield).

MS m/z(ESI):552[M+H]+

Synthesis of intermediate tert-butyl 4-ethylanthranilate (74-M2)

Tert-butyl 4-aminobenzoate (13-6, 1.16g) was dissolved in methanol (15mL), and acetaldehyde tetrahydrofuran solution (5mol/L, 1.2mL) was added thereto, and sodium cyanoborohydride (560mg) was added in portions, followed by stirring at room temperature for 2 hours. The solvent was concentrated, and the residue was purified by column chromatography to give tert-butyl 4-ethylanthranilate (340 mg).

EXAMPLE 75 preparation of 4- ((S) -2- ((1R,4S) -4- (aminomethyl) cyclohexyl-1-carboxamido) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) -N-isopropylacetamido) -benzoic acid

The first step is as follows: preparation of (S) -4- (2- (tert-Butoxycarbonylamino) -3- (3- (2- (dimethylamino) -N-methylacetoacetamino) phenyl) -N-isopropylacetoacetamino) -benzoic acid tert-butyl ester (75-2)

(S) -2- (tert-butoxycarbonylamino) -3- (3- (2- (dimethylamino) -N-methylacetamino) phenyl) acetic acid (183mg, 482. mu. mol), tert-butyl 4-isopropylanthranilate (138mg, 530. mu. mol), HATU (220mg, 579. mu. mol) and diisopropylethylamine (159. mu.l, 965. mu. mol) were added to dichloromethane (5ml) and stirred uniformly for 2 hours, and the reaction was checked to be complete by LCMS. The solvent was dried, 100ml of water was added, extraction was performed with ethyl acetate, the collected organic phase was washed, dried, concentrated, and purified by column chromatography to give pure (S) -tert-butyl 4- (2- (tert-butoxycarbonylamino) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) -N-isopropylacetamido) -benzoate (121mg, yield 40%).

The second step is that: preparation of (S) -4- (2-amino-3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) -N-isopropylacetamido) -benzoic acid tert-butyl ester (75-3)

(S) -4- (2- (tert-butoxycarbonylamino) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) -N-isopropylacetamido) -benzoic acid tert-butyl ester (121mg, 195. mu. mol) was added to a hydrogen chloride ethyl acetate solution (10ml), reacted for 2 hours, and the reaction was completed by LCMS. The solvent was dried by evaporation, and purified by column chromatography to give pure (S) -4- (2-amino-3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) -N-isopropylacetamido) -benzoic acid tert-butyl ester (50mg, yield 49%).

The third step: preparation of 4- ((S) -2- ((1R,4S) -4- (tert-Butoxycarbonylaminomethyl) cyclohexyl-1-carboxamido) -3- (3- (2- (dimethylamino) -N-methylacetyl) phenyl) -N-isopropylacetamido) -benzoic acid tert-butyl ester (75-4)

(S) -4- (2-amino-3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) -N-isopropylacetamido) -benzoic acid tert-butyl ester (50mg, 96. mu. mol), (1R,4R) -4- (((tert-oxycarbonyl) amino) methyl) cyclohexyl-1-carboxylic acid (27mg, 105. mu. mol), HATU (44mg, 105. mu. mol) and isopropyldiethylamine (48mg, 288. mu. mol) were added to dichloromethane (2ml) and reacted for 2 hours with LCMS to check completion of the reaction. The solvent was dried, 5ml of water was added, extracted with ethyl acetate, the organic phase was collected and washed, dried, concentrated and purified by column chromatography to give pure 4- ((S) -2- ((1R,4S) -4- (tert-butoxycarbonylaminomethyl) cyclohexyl-1-carboxamido) -3- (3- (2- (dimethylamino) -N-methylacetyl) phenyl) -N-isopropylacetamido) -benzoic acid tert-butyl ester (45mg, yield 62%).

The fourth step: preparation of 4- ((S) -2- ((1R,4S) -4- (aminomethyl) cyclohexyl-1-carboxamido) -3- (3- (2- (dimethylamino) -N-methylacetyl) phenyl) -N-isopropylacetamido) -benzoic acid (75)

4- ((S) -2- ((1r,4S) -4- (tert-Butoxycarbonylaminomethyl) cyclohexyl-1-carboxamido) -3- (3- (2- (dimethylamino) -N-methylacetyl) phenyl) -N-isopropylacetamido) -benzoic acid tert-butyl ester (45mg, 59. mu. mol) was added to a mixed solvent of trifluoroacetic acid (2ml) and dichloromethane (4ml) and reacted for 1 hour, and the reaction was checked for completion by LCMS. The solvent was dried by evaporation to give the product 4- ((S) -2- ((1R,4S) -4- (aminomethyl) cyclohexyl-1-carboxamido) -3- (3- (2- (dimethylamino) -N-methylacetyl) phenyl) -N-isopropylacetamido) -benzoic acid (23mg, 64% yield).

MS m/z(ESI):566[M+H]+

Preparation of intermediate t-butyl isopropylanthranilate (75-M2)

Tert-butyl 4-aminobenzoate (13-6, 1.0g) was dissolved in methanol (15mL), acetone (1.5mL) was added, and sodium cyanoborohydride (448mg) was added in portions, and stirred at room temperature for 2 hours. The solvent was concentrated, and the residue was purified by column chromatography to give tert-butyl 4-isopropylanthranilate (700 mg).

Example 76: 4- ((S) -2- ((E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (1, 1-thiomorpholine) -4-oxobutanamido)) benzoic acid

The first step is as follows: preparation of tert-butyl (S) -2- ((((9H-fluoren-ylmethoxy) carbonyl) amino) -4- (1, 1-thiomorpholine) -4-oxobutyrate (76-2)

Aspartic acid (40mg), thiomorpholine-1, 1-dioxide (15mg), HATU (62mg) and diisopropylethylamine (0.30mL) were added to dichloromethane (3mL) and the reaction stirred for 2h, LCMS checked for completion. Adding water, extracting with dichloromethane, collecting organic phase, washing, drying, concentrating, and purifying by column chromatography to obtain tert-butyl (S) -2- ((((9H-fluoren-ylmethoxy) carbonyl) amino) -4- (1, 1-sulfur dioxide morpholine) -4-oxobutyrate (61 mg).

The second step is that: preparation of (S) -2- ((((9H-fluoren-ylmethoxy) carbonyl) amino) -4- (1, 1-thiomorpholine sulfide) -4-oxobutanoic acid (76-3)

Tert-butyl (S) -2- ((((9H-fluoren-ylmethoxy) carbonyl) amino) -4- (1, 1-thiomorpholine dioxide) -4-oxobutyrate (61mg) was added to dichloromethane (2mL), trifluoroacetic acid (1mL) was added, the reaction was stirred for 2 hours, LCMS detected complete reaction, solvent was spun off, and preparative thin layer chromatography purification gave (S) -2- (((((9H-fluoren-ylmethoxy) carbonyl) amino) -4- (1, 1-thiomorpholine dioxide) -4-oxobutyric acid (65 mg).

The third step: preparation of tert-butyl (S) -4- (2- ((((9H-fluoren-ylmethoxy) carbonyl) amino) -4- (1, 1-thiomorpholine sulfide) -4-oxobutylamine) benzoate (76-4)

(S) -2- ((((9H-fluoren-ylmethoxy) carbonyl) amino) -4- (1, 1-thiomorpholine sulfide) -4-oxobutanoic acid (65mg), tert-butyl 4-aminobenzoate (13-6, 44mg), HATU (93mg) and diisopropylethylamine (0.10mL) were added to dichloromethane (3mL) and stirred to react uniformly for 2 hours, and (3) adding water, extracting by using dichloromethane, collecting an organic phase, washing, drying, concentrating, and purifying by column chromatography to obtain (S) -4- (2- ((((9H-fluoren-ylmethoxy) carbonyl) amino) -4- (1, 1-sulfur dioxide morpholine) -4-oxobutylamine) tert-butyl benzoate (59 mg).

The fourth step: preparation of tert-butyl (S) -4- (2-amino-4- (1, 1-thiomorpholine-4-oxobutylamine) benzoate (76-5)

Tert-butyl (S) -4- (2- ((((9H-fluoren-ylmethoxy) carbonyl) amino) -4- (1, 1-thiomorpholine) -4-oxobutylamine) benzoate (59mg) was dissolved in methylene chloride (3mL), diethylamine (2mL) was added, the mixture was stirred at room temperature for 3 hours, the reaction mixture was concentrated, and the residue was purified by preparative thin-layer chromatography to give tert-butyl (S) -4- (2-amino-4- (1, 1-thiomorpholine) -4-oxobutylamine) benzoate (24 mg).

The fifth step: preparation of tert-butyl 4- ((S) -2- ((E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (1, 1-thiomorpholine) -4-oxobutyramido)) benzoate (76-6)

Tert-butyl (S) -4- (2-amino-4- (1, 1-thiomorpholine-dioxide) -4-oxobutylamine) benzoate (24mg), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (14mg), HATU (23mg) and diisopropylethylamine (0.1mL) were added to dichloromethane (2mL) and stirred uniformly for 2 hours, and the reaction was detected to be complete by LCMS. Adding water, extracting with dichloromethane, collecting organic phase, washing, drying, concentrating, and purifying by column chromatography to obtain tert-butyl 4- ((S) -2- ((E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (1, 1-sulfur dioxide morpholine) -4-oxobutyrylamino)) benzoate (25 mg).

And a sixth step: preparation of 4- ((S) -2- ((E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (1, 1-thiomorpholine) -4-oxobutyrylamino)) benzoic acid (76)

Tert-butyl 4- ((S) -2- ((E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (1, 1-thiomorpholine) -4-oxobutylamine)) benzoate (25mg) was added to dichloromethane (2mL), trifluoroacetic acid (1mL) was added, the reaction was stirred for 2 hours and LCMS checked for completion. The solvent was dried by spinning and purified by preparative thin layer chromatography to give 4- ((S) -2- ((E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (1, 1-thiomorpholine dioxide) -4-oxobutyramido)) benzoic acid (23 mg).

¹ HNMR(DMSO-d6 400MHz)δ12.56(brs,1H),10.43(s,1H),9.86(s,1H),8.78(d,1H,J＝5.4Hz),7.96-7.88(m,3H),7.71(d,2H,J＝8.4Hz),7.64(d,1H,J＝8.8Hz),6.82(d,1H，J＝15.6Hz),6.75(d,1H,J＝16.4Hz),4.87(q,1H,J＝6.0Hz),3.88-3.84(m，5H)，3.10-3.03(m，2H)，3.00-2.85(m,3H).

MS m/z(ESI):620[M+H]+

Example 77

Preparation of 1- ((isopropoxycarbonyl) oxy) ethyl 4- ((S) -2- ((E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (1, 1-thiomorpholine-dioxide) -4-oxobutyramido)) benzoate

4- ((S) -2- ((E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (1, 1-thiomorpholine dioxide) -4-oxobutyrylamino)) benzoic acid (23mg), potassium carbonate (16mg) and potassium iodide (6.5mg) were added to DMF (2mL), nitrogen was purged, isopropyl 1-chloroethylcarbonate (12.9mg) was added under nitrogen atmosphere, stirred uniformly, reacted at 60 ℃ for 16 hours, and LCMS detected reaction completion. Adding 10mL of water, extracting with ethyl acetate, collecting the organic phase, washing, drying, concentrating, and purifying by preparative thin layer chromatography to obtain 1- ((isopropoxycarbonyl) oxy) ethyl 4- ((S) -2- ((E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (1, 1-thiomorpholine-dioxide) -4-oxobutyrylamino)) benzoate (19mg)

MS m/z(ESI):750[M+H]+

¹ HNMR(DMSO-d6 400MHz)δ10.55(s,1H),9.86(s,1H),8.77(d,1H,J＝7.6Hz),7.96-7.92(m,3H),7.79(d,2H,J＝8.8Hz),7.64(dd,1H,J＝1.2,8.8Hz),6.85(dd,1H，J＝5.2,10.8Hz),6.83(d,1H，J＝16Hz),6.75(d,1H,J＝16.2Hz),4.87(q,1H,J＝6.9Hz),4.82-4.76(m,1H),3.92-3.81(m，4H)，3.27-3.23(m,2H),3.10-3.03(m，2H)，3.01-2.82(m,2H),1.57(d,3H,J＝5.4Hz),1.23(s,6H,J＝6.4Hz).

Example 78

Preparation of 4- ((S) -2- ((1R,4S) -4- (aminomethyl) cyclohexane-1-carboxamido) -4- (1, 1-thiomorpholine dioxide) -4-oxobutanoylamino) benzoic acid

The first step is as follows: preparation of tert-butyl 4- ((S) -2- ((1R,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carboxamido) -4- (1, 1-thiomorpholine) -4-oxobutanoylamino) benzoate (78-2)

Tert-butyl (S) -4- (2-amino-4- (1, 1-thiomorpholine-dioxide) -4-oxobutanoylamino) benzoate (180mg), (1r,4r) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carboxylate 39-1, 110mg), HATU (161mg), and diisopropylethylamine (0.23mL) were added to dichloromethane (5mL) and stirred for uniform reaction for 2 hours. Adding water, extracting with dichloromethane, collecting organic phase, washing, drying, concentrating, and purifying by column chromatography to obtain tert-butyl 4- ((S) -2- ((1R,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-formamido) -4- (1, 1-sulfur dioxide morpholine) -4-oxobutyrylamino) benzoate (180 mg).

The second step is that: preparation of 4- ((S) -2- ((1R,4S) -4- (aminomethyl) cyclohexane-1-carboxamido) -4- (1, 1-thiomorpholine) -4-oxobutanoylamino) benzoic acid (78)

Tert-butyl 4- ((S) -2- ((1R,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carboxamido) -4- (1, 1-thiomorpholine) -4-oxobutyrylamino) benzoate (180mg) was added to dichloromethane (2mL), trifluoroacetic acid (1mL) was added, the reaction was stirred for 2 hours, and the reaction was complete by LCMS. The solvent was dried by evaporation and purified by preparative thin layer chromatography to give 4- ((S) -2- ((1R,4S) -4- (aminomethyl) cyclohexane-1-carboxamido) -4- (1, 1-thiomorpholine) -4-oxobutyrylamino) benzoic acid (100 mg).

MS m/z(ESI):509[M+H]+

Biological evaluation

Experimental example 1: determination of the biological Activity of the Compounds of the invention as inhibitors of coagulation factor X1a

Test materials

Enzyme: human Factor Xa (Haematologic, cat # HCXIA-0160)

Substrate: PyroGlu-Pro-Arg-pNA (GL-China, cat # 695760)

Buffer solution: 50mM Tris-HCl, pH 7.5, 0.01% Tween-20,150mM NaCl,5mM CaCl ₂ And 0.01% BSA.

Test procedure

1 Xbuffer (50mm Tris-HCl, pH 7.5, 0.01% Tween-20,150mm NaCl,5mm CaCl2 and 0.01% BSA) was prepared. Compounds were diluted with DMSO. All test compounds were diluted to 10 concentrations of 100-fold solution using a 3-fold serial dilution method. The final starting concentration was 1. mu.M test compound. Using an automated liquid handler, 200nL of compound was transferred to 384 well plates. FXIa enzyme was diluted to 2X (0.5nM) concentration with buffer, 10. mu.L of FXIa was added to each well, followed by 10. mu.L of buffer. Incubate for 15 minutes at room temperature. 2 Xsubstrate solution (pyroGlu-Pro-Arg-pNA) was prepared in buffer, and 10. mu.L of diluted substrate solution was added to each well to start the reaction. OD was read dynamically at 405nM using an EnVision microplate reader.

Data processing:

the absorbance was curve-fitted with Prism and the inhibition per well was calculated to obtain IC ₅₀ 。

And (4) conclusion: the compound has obvious inhibitory activity on blood coagulation factor Xla.

Experimental example 2: determination of human blood anticoagulation in vitro

1. Test materials

aPTT reagent (Mindray, cat # 1904005)

CaCl ₂ (Mindray, cat # 1903021A)

Plasma: human blood is collected in an anticoagulant vacuum tube containing sodium citrate solution (the volume ratio is 1:9), and the blood is immediately centrifuged for 15min at 5000 Xg after blood collection, and the blood plasma is respectively separated and immediately used for aPTT detection.

2. Instrument for measuring the position of a moving object

Centrifuge (manufacturer: Eppendorf, model: 5810)

Full-automatic blood coagulation analyzer (manufacturer: Mindray, model: C3510)

Sonic pipetting system (manufacturer: Labcyte, model:

550)

3. test procedure

The compounds to be tested are prepared into 100% DMSO solutions with corresponding concentrations of 100 x according to the final test concentrations. 5uL of the corresponding 100 XDMSO solution was taken for testing and added to 495uL of plasma samples. The negative control was 5uL of 100% DMSO solution added to 495uL of plasma samples. And (3) sending the sample to be tested into a full-automatic coagulation analyzer for aPTT testing, and obtaining corresponding reading values.

4. Data processing

The values for compound concentration were taken as the X-axis and the corresponding aPTT readings as the Y-axis, curves were fitted using the analytical software GraphPad Prism 5 and calculated EC2.0X.

Compound numbering	Inhibiting platelet aggregation (EC2.0X, um)
Example 3	1.85
Example 9	2.51
Example 24	1.57
Example 30	4.15
Example 46	3.38
Example 54	3.50
Examples63	2.61
Example 66	3.22
Example 68	2.89
Example 72	5.35
Example 74	3.82

And (4) conclusion: the compound of the invention has obvious inhibitory activity on human blood coagulation.

Experimental example 3: rat arteriovenous thrombus experiment

1. Test materials

250 plus 350g Wistar rat, polyethylene hose, No. 3 operation silk thread, test drug

2. Test procedure

After all animals were dosed (compound of example 3), they were anesthetized by intraperitoneal injection of 1% sodium pentobarbital, rats were placed in supine position, the neck was dehaired and skin sterilised, the neck was opened by sterile surgical instruments, the trachea was isolated, the right common carotid artery and the left external jugular vein were isolated, 6cm of # 3 surgical silk was cut and placed in a three-stage polyethylene tube, the end of the silk was exposed 1cm from the junction near the arterial end, the length of the silk contacting the blood was 5cm, the polyethylene tube was filled with physiological saline, and a carotid artery and vein bypass circuit with clamped artery was established. After a certain time of administration, the artery clamp is opened, the surgical silk thread is taken out quickly after timing for 15min and weighed (wet weight), and then the surgical silk thread is placed in an oven at 60 ℃ for 24h and weighed (dry weight).

3. Data processing

And (5) recording the wet weight and the dry weight of the thrombus, and calculating the thrombus inhibition rate. Experimental data were calculated and statistically processed using Prism software. The experimental results are shown in fig. 1 and fig. 2.

And (4) conclusion: the compound of the invention has obvious inhibition effect on thrombosis in animal bodies.

Pharmacodynamic experiment results show that the compound has obvious effect of inhibiting the activity of the blood coagulation factor XIa in vitro and has obvious effect of resisting thrombosis in vivo.

Claims

A compound of formula I or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, metabolite, or prodrug thereof:

wherein

A is selected from phenyl and cyclohexyl;

R ₁ each independently selected from halogen, C _1-6 Alkyl, tetrazolyl and triazolyl, said C _1-6 Alkyl, tetrazolyl or triazolyl are each optionally substituted with one or more substituents selected from amino and halo;

n is 0,1, 2 or 3;

R ₂ and R ₃ Each independently selected from H and C _1-6 An alkyl group;

m is 0 or 1;

R ₄ is H;

R ₅ is optionally substituted by one or more R ₈ Substituted phenyl or C _1-6 An alkyl group;

or R ₄ 、R ₅ And the atoms to which they are attached together form a 5-6 membered azacycloalkyl optionally fused to a phenyl ring, said 5-6 membered azacycloalkyl or phenyl ring fused thereto being optionally substituted by one or more R ₈ Substitution;

R ₈ each independently selected from H, -NR _a R _b Halogen, halogen,
5-6 membered cycloalkyl and phenyl;

R _a is selected from H and C _1-6 An alkyl group;

R _b is- (CO) _p -(CH ₂ ) _q -R ₉ ；

p is 0 or 1;

q is 1 or 2;

R ₉ is selected from C _1-6 Alkoxy and-NR _c R _d ；

R _c And R _d Each independently selected from H and C _1-6 An alkyl group;

R ₆ selected from H, C _1-6 Alkyl and C _3-6 A cycloalkyl group;

R ₇ is optionally substituted by one or more R ₁₀ Substituted phenyl or benzyl;

R ₁₀ each independently selected from- (CH) ₂ ) _r -C(O) _s -R ₁₁ C optionally substituted by one or more halogens _1-6 Alkoxy, tetrazolyl, halo, -NHCOOC _1-6 Alkyl and-SO ₂ NHCOC _1-6 An alkyl group;

r is 0 or 1;

s is 1 or 2;

R ₁₁ selected from H, -NHSO ₂ R ₁₂ And optionally substituted with R ₁₃ Substituted C _1-6 An alkyl group;

R ₁₂ is selected from C _1-6 Alkyl radical, C _3-6 Cycloalkyl and phenyl;

R ₁₃ is selected from-OCOOR ₁₄ And

R ₁₄ is selected from C _1-6 Alkyl and C _3-6 A cycloalkyl group;

or R ₆ 、R ₇ And the N atom to which they are attached together form a 5-6 membered azacycloalkyl group,

wherein when R is ₈ Is composed of
And R is ₁₀ Is- (CH) ₂ ) _r -C(O) _s -R ₁₁ When R is ₆ Is C _1-6 An alkyl group, a carboxyl group,

wherein the wavy line
Indicates the point of attachment of the group to the rest of the molecule.
A compound of claim 1, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, metabolite, or prodrug thereof,

wherein,

selected from:

wherein the wavy line
Indicates the point of attachment of the group to the rest of the molecule.
A compound of claim 1 or 2, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, metabolite or prodrug thereof,

wherein,

R ₂ and R ₃ Is H.
A compound of any one of claims 1-3, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, metabolite, or prodrug thereof,

wherein,

R ₄ is H; and is

R ₅ Is optionally substituted by R ₈ A substituted phenyl group.
A compound of claim 4, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, metabolite, or prodrug thereof,

wherein,

R ₈ is-NR _a R _b ；

R _a Is H;

R _b is- (CO) _p -(CH ₂ ) _q -R ₉ ；

p is 1;

q is 1;

R ₉ is-NR _c R _d (ii) a And is

R _c And R _d Each independently selected from C _1-6 An alkyl group.
A compound of any one of claims 1-3, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, metabolite, or prodrug thereof,

wherein,

R ₄ 、R ₅ and the atoms to which they are attached together form a 5-6 membered azacycloalkyl optionally fused to a phenyl ring, said 5-6 membered azacycloalkyl or phenyl ring fused thereto being optionally substituted by R ₈ And (4) substitution.
A compound of claim 6, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, metabolite, or prodrug thereof,

wherein,

R ₄ 、R ₅ and the atoms to which they are attached together form a 1,2,3, 4-tetrahydroisoquinolinyl or pyrrolidinyl group, said 1,2,3, 4-tetrahydroisoquinolinyl or pyrrolidinyl group optionally substituted with R ₈ And (4) substitution.
A compound of claim 7, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, metabolite, or prodrug thereof,

wherein,

R ₄ 、R ₅ and the atoms to which they are attached together form
And is

R ₈ Selected from H, -NR _a R _b Halogen and

wherein the wavy line
Indicates the point of attachment of the group to the rest of the molecule.
The compound of claim 8, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, metabolite, or prodrug thereof,

wherein R is ₈ Selected from H, -NHCOCH ₂ OCH ₃ 、-NHCOCH ₂ N(CH ₃ ) ₂ 、-NH(CH ₂ ) ₂ N(CH ₃ ) ₂ 、-N(CH ₃ )COCH ₂ OCH ₃ 、-N(CH ₃ )COCH ₂ N(CH ₃ ) ₂ Br and

wherein the wavy line
Indicates the point of attachment of the group to the rest of the molecule.
A compound of claim 7, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, metabolite, or prodrug thereof,

wherein,

R ₄ 、R ₅ and atoms to which they are attached together form
And is

R ₈ Selected from the group consisting of cyclohexyl and phenyl,

wherein the wavy line
Indicates the point of attachment of the group to the rest of the molecule.
A compound of any one of claims 1-10, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, metabolite, or prodrug thereof,

wherein,

R ₆ is selected from H and C _1-6 An alkyl group; and is

R ₇ Is optionally substituted by one or two R ₁₀ Substituted phenyl or benzyl.
The compound of claim 11, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, metabolite, or prodrug thereof,

wherein,

R ₁₀ each independently selected from-COOH, -COOCH ₃ 、-COOCH ₂ CH ₃ 、-COOC(CH ₃ ) ₃ 、-COOCH ₂ OCOOCH ₂ CH ₃ 、-COOCH ₂ OCOOCH(CH ₃ ) ₂ 、-COOCH(CH ₃ )OCOOCH ₂ CH ₃ 、-COOCH(CH ₃ )OCOOCH(CH ₃ ) ₂ 、
-CONHSO ₂ CH ₃ 、

-CH ₂ COOH、-OCH ₃ 、-OCHF ₂ 、
-Cl、-F、-NHCOOCH ₃ and-SO ₂ NHCOCH ₃ ，

Wherein the wavy line
Indicates the point of attachment of the group to the rest of the molecule.
A compound of any one of claims 1-10, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, metabolite, or prodrug thereof,

wherein,

R ₆ 、R ₇ and the N atom to which it is attached, together form a pyrrolidinyl group.
The compound of claim 1, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, metabolite, or prodrug thereof, wherein said compound is selected from the group consisting of:
a pharmaceutical composition comprising a prophylactically or therapeutically effective amount of a compound according to any one of claims 1-14, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, metabolite, or prodrug thereof, and one or more pharmaceutically acceptable carriers.
Use of a compound of any one of claims 1-14, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, metabolite, or prodrug thereof, for the manufacture of a medicament for the prevention or treatment of thrombosis and embolism associated disorder.
The use of claim 16, wherein the thrombosis or embolism associated disorder is atherosclerotic disease, myocardial infarction, stroke, ischemic cerebral thrombosis, peripheral arterial disease, peripheral arterial occlusive disease, pulmonary embolism, deep vein thrombosis, acute coronary syndrome, or thrombosis following coronary intervention.
A process for the preparation of a compound according to any one of claims 1 to 14, according to scheme 1,2,3 or 4 below:

route 1

Route 2

Route 3

Route 4

Wherein R is ¹ 、R ² 、R ³ 、R ⁴ 、R ⁵ 、R ⁶ 、R ⁷ 、R ⁸ M and n are as defined in any one of claims 1 to 14; and PG is a protecting group for amino group, preferably t-butoxycarbonyl or 9-fluorenylmethoxycarbonyl.