CN113943259A - Amino acid derivative, preparation method and application thereof - Google Patents

Amino acid derivative, preparation method and application thereof Download PDF

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CN113943259A
CN113943259A CN202010692426.3A CN202010692426A CN113943259A CN 113943259 A CN113943259 A CN 113943259A CN 202010692426 A CN202010692426 A CN 202010692426A CN 113943259 A CN113943259 A CN 113943259A
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phenyl
amino
butyl
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王雪菊
戴静芳
吴少平
张俊清
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Xi'an Hepeng Biomedical Co ltd
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Abstract

The invention discloses an amino acid derivative with a blood coagulation Factor (Factor) XIa inhibition effect and application thereof. The invention provides a compound shown as a formula I or a pharmaceutically acceptable salt, a stereoisomer, a tautomer, a polymorph, a solvate, an N-oxide, a metabolite or a prodrug thereof, and in vitro multiple biological test results show that the compound can generate obvious inhibitory activity effect on blood coagulation Factor (Factor) XIa at an extremely low concentration, and some preferred compounds can obviously prolong the coagulation time of human whole blood under the condition of low concentration, namely 1.5 times or 2.0 times of coagulation time. All reach the practical standard and have optimistic patent medicine prospect. The compounds can be used as inhibitors of the blood coagulation factor XIa, pharmaceutical compositions containing the same, preparation methods of the compounds and applications of the compounds in preparation of medicines for preventing or treating diseases related to thrombus and vascular embolism.

Description

Amino acid derivative, preparation method and application thereof
Technical Field
The invention relates to a novel amino acid derivative which can be used as a coagulation factor XIa inhibitor, a pharmaceutical composition containing the same, a preparation method of the pharmaceutical composition and application of the pharmaceutical composition in preparation of medicines for preventing or treating diseases related to thrombus and vascular embolism.
Background
Thrombus is a pathological product, and is a solid mass formed by coagulation of blood or coagulation of some tangible components in blood in heart and blood vessel. After the thrombus is formed, part or all of the thrombus falls off, and the thrombus blocks a blood vessel along with the blood flow to form an embolism. Thrombosis or thromboembolism causes a range of cardiovascular and cerebrovascular diseases, and has become a leading cause of morbidity and mortality worldwide. More than 2600 million people die of various thrombotic diseases every year worldwide, and the incidence rate of the thrombotic diseases also rises year by year in our country. According to the third national cause of death survey published by the ministry of health of China, 22.5 percent of the death people die from cardiovascular and cerebrovascular diseases. Under the severe situation, the development of cheap, safe and effective antithrombotic drugs has very important significance for human health.
The classical coagulation cascade plays a key role in understanding the formation of venous thrombosis. The Factor XI (FXI) at the upstream of the reaction path is activated into Factor XIa (FXIa), can catalyze the Factor IX (FIX) into Factor IXa (FIXa), continues to be combined with the Factor VIIIa (FVIIIa), and jointly generates the Factor Xa (FXa) efficiently and lowly. Antagonizes the activity of Factor Xa (FXa), realizes the clinical effect of preventing blood coagulation, and is the action target of the sand plate drugs represented by rivaroxaban. If the activity of the Factor Xa (FXa) is maintained, then the Factor II (FII) can be activated into thrombin (Factor IIa, FIIa) through the synergistic action of the Factor Va (FVa), finally fibrin is formed, and thrombus plaques with different sizes are gathered. FXI is essential for the maintenance of the intrinsic pathway and plays a key role in the amplification of the coagulation cascade. In the coagulation cascade, thrombin (Factor IIa, FIIa) can feedback to activate FXI, which in turn drives the massive production of thrombin, thereby amplifying the coagulation cascade.
Meanwhile, clinical meta-studies have found that these anticoagulants cause bleeding risks in different degrees and in different proportions. Bleeding complications may be caused by both traditional anticoagulant drugs, such as warfarin, heparin, Low Molecular Weight Heparin (LMWH), and recently marketed new drugs, such as coagulation factor FXIa inhibitors (rivaroxaban, apixaban, etc.) and thrombin inhibitors (dabigatran etexilate, hirudin, etc.). Therefore, the research and development of new antithrombotic drugs with little hemorrhagic side effect has important value.
The coagulation factor FXI (Coag. mu. translation factor XI), also known as Plasma thromboplastin antecedent, is a serine hydrolase, an inactive state, synthesized in the liver and having a molecular weight of 160 KD. The coagulation factor FXI normally exists in the dimeric form (hobodimer) and is activated to convert to the coagulation factor FXI α. The conversion process comprises the following steps: in the endogenous coagulation process, when blood comes into contact with a negatively charged foreign surface, prothrombin (FII) first binds to the foreign surface, and is activated to thrombin (FII α), and then the coagulation factor FXI is activated to the coagulation factor FXI α, thereby initiating the intrinsic coagulation pathway. In the coagulation cascade described above, the initiation of the endogenous coagulation pathway and the formation of the coagulation factor FXI α are important for causing blood clot formation, as the coagulation factor FXI α is located near the origin of the intrinsic coagulation pathway. Recent studies have shown that increased levels of the coagulation factor FXI are associated with venous thrombosis and myocardial infarction in men and increase the chances of cerebrovascular and coronary artery disease. It is thus concluded that inhibition of FXI α may effectively inhibit thrombosis and not cause significant bleeding.
At present, about 23 candidate compounds are researched aiming at FXI/FXI alpha targets, no medicine is available on the market, and the most advanced is in phase II clinical research. By 10/05/2020, 6 candidate compounds are under development in clinical phase II, of which 3 are small molecule chemical drugs and 3 are biological drugs. There were 4 additional candidate compounds in phase I clinical development, totaling 10 in clinical research. Among the small molecule inhibitors, SHR-2285 and ONO-5450598 do not disclose any reported structure, and the following are the varieties of the disclosed structure in clinical research.
Figure BDA0002589780190000021
In summary, from the trend of drug development, there are at least many groups of small molecule compounds on which drug-forming studies are being conducted, but clinical data based on drug effects are not yet fully published, and no candidate compound has yet been developed in clinical stage III. Therefore, how to develop a new structural coagulation factor FXI alpha inhibitor which is suitable for clinical needs and has good pharmaceutical parameters remains a great challenge facing the field at present.
According to the invention, a series of organic small molecule structure-activity relationship researches are creatively carried out by deeply analyzing the molecular structure of a target protein, and a series of related compounds and in vitro multiple biological test results show that the compound can generate obvious inhibitory activity effect on blood coagulation Factor (Factor) XIa at an extremely low concentration, wherein some preferable compounds can obviously prolong the coagulation time of human whole blood under the condition of low concentration, namely 1.5 times or 2.0 times of coagulation prolonging time. All reach the practical standard and have optimistic patent medicine prospect. The classical rat arteriovenous thrombosis test (AVST) demonstrated that the compound of example 17 has significant in vivo antithrombotic effects. Therefore, the compounds of the present invention have greater clinical potential than the compounds mentioned in the background of the invention in this field.
Disclosure of Invention
The present invention provides a novel class of factor XIa inhibitors. The compound is a compound of formula I or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, metabolite, or prodrug thereof:
Figure BDA0002589780190000031
wherein
A is selected from phenyl and cyclohexyl;
R1each independently selected from halogen, C1-6Alkyl, tetrazolyl and triazolyl, said C1-6Alkyl, tetrazolyl or triazolyl are each optionally substituted with one or more substituents selected from amino and halo;
n is 0, 1, 2 or 3;
R2and R3Each independently selected from H and C1-6An alkyl group;
m is 0 or 1;
R4is H;
R6selected from H and C1-6An alkyl group;
R7is H or optionally substituted by one or more R10Substituted phenyl, benzyl, naphthyl, indenyl, indole, benzimidazole, cyclohexyl;
R10each independently selected from- (CH)2)r-C(O)s-R11；
r is 0 or 1;
s is 1 or 2;
R11selected from H, -CH2R12And optionally substituted with R13Substituted C1-6An oxoalkyl group;
R12is selected from C1-6Alkyl radical, C3-6Cycloalkyl and phenyl;
R13is selected from-OCOOR14And
Figure BDA0002589780190000032
R14is selected from C1-6Alkyl and C3-6A cycloalkyl group;
R5is optionally substituted by one or more R8Substituted phenyl or Cp alkyl;
p is 0, 1, 2, 3, 4, 5 or 6;
R8is-NR in ortho-or meta-or para-position in the phenyl substitution15R16；
R15Is H or CH 3；
R16is-C (O) -R17；
R17Is CH3、-CH2-N(CH3)2or-CH2-OCH3；
R8May also be-C (O) R substituted at Cp alkyl18；
R18May have any one of the following structures
Figure BDA0002589780190000033
Figure BDA0002589780190000041
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.
The present invention also provides a process for preparing a compound of the invention, comprising the steps of:
Figure BDA0002589780190000042
wherein R is1、R2、R3、R4、R5、R6、R7、R8M and n are as defined above; and PG is a protecting group for carboxyl group.
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 "C1-6 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 "C3-6 cycloalkyl" refers to a saturated non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon ring having 3 to 6 ring-forming carbon atoms (e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl). The term "C5-6 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-forming 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, representative examples of the term "C1-6 alkoxy" include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-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 with …," 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 of a substituent is shown through a bond connecting two atoms in a ring, then such substituent may be bonded to any ring 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 are capable of forming N-oxides, since the available lone pair is required for oxidation of the nitrogen to the 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 h. 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:
according to some embodiments of the invention, the compounds of the invention have the structure of formula I:
Figure BDA0002589780190000071
wherein R is1、R2、R3、R6、R7、R8M and n are as defined above.
In accordance with some embodiments of the present invention,
Figure BDA0002589780190000072
is selected from
Figure BDA0002589780190000073
Figure BDA0002589780190000074
Wherein the wavy line
Figure BDA0002589780190000076
Indicates the point of attachment of the group to the rest of the molecule.
R19Can be H or CH3；
R20Can be-CO2R21；
R21Is C1To C6Linear alkanes of (1).
According to some embodiments of the invention, the compounds of the invention have the structure of formula II:
Figure BDA0002589780190000075
wherein R is1、R2、R3、R6、R7、R8M and n are as defined above.
B is any one of the following structures,
Figure BDA0002589780190000081
Figure BDA0002589780190000082
R22each independently selected from-COOH, -COOCH3、-COOCH2CH3、-COOC(CH3)3、-COOCH2OCOOCH2CH3、-COOCH2OCOOCH(CH3)2、-COOCH(CH3)OCOOCH2CH3、-COOCH(CH3)OCOOCH(CH3)2、
Figure BDA0002589780190000083
-CONHSO2CH3、
Figure BDA0002589780190000084
-CH2COOH、-OCH3、-OCHF2、
Figure BDA0002589780190000085
-Cl、-F、-NHCOOCH3and-SO2NHCOCH3，
Wherein the wavy line
Figure BDA0002589780190000086
Indicates the point of attachment of the group to the rest of the molecule.
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.
Pharmaceutical composition
It is another object of the present invention 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.
It is another object of the present invention 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.).
The above preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention without departing from the common general knowledge in the art.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows: the results of in vitro multiple biological tests of a series of compounds related to the invention show that the compound can generate obvious inhibitory activity effect on blood coagulation Factor (Factor) XIa at extremely low concentration, and some of the compounds can obviously prolong the coagulation time of human whole blood under the condition of low concentration, namely the coagulation time is 1.5 times or 2.0 times. All meet the practical standard. The classical rat arteriovenous thrombosis test (AVST) verifies that the compound of example 3 has a significant in vivo antithrombotic effect. Therefore, the compound of the invention has larger clinical potential.
Drawings
FIG. 1 shows the results of the drug effects of the compounds of examples 5, 17 and 20 on inhibition of thrombosis in rat arteriovenous thrombosis model
Detailed Description
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 not indicated by the manufacturer, and are all conventional products commercially available.
In the conventional syntheses as well as in the examples and intermediate syntheses, the meanings of the abbreviations are as shown in Table 1 below.
Figure BDA0002589780190000101
Figure BDA0002589780190000111
TABLE 1 Alphabet abbreviation meaning Table
Example 1: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (methyl (2- (benzenesulfonyl) ethyl) amino) -4-oxobutanamido) benzoic acid
Figure BDA0002589780190000112
The first step is as follows: preparation of tert-butyl (S) -4- (4- (benzyloxy) -2- ((tert-butoxycarbonyl) amino) -4-oxobutanamido) benzoate
N-tert-Butoxycarbonyl-L-aspartic acid-4-benzyl ester (6.0g, 18.6mmol), tert-butyl 4-aminobenzoate (3.94g, 20.4mmol), EDCI (4.27g, 22.27mmol), HOBT (3.01g, 22.27mmol), triethylamine (5.62g, 55.7mmol) were dissolved in dichloromethane (120mL) and stirred at room temperature overnight. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give tert-butyl (S) -4- (4- (benzyloxy) -2- ((tert-butoxycarbonyl) amino) -4-oxobutanamido) benzoate (9.10g, yield 98%) as a white solid.
The second step is that: preparation of (S) -3- ((tert-butoxycarbonyl) amino) -4- ((4- (tert-butoxycarbonyl) phenyl) amino) -4-oxobutanoic acid tert-butyl (S) -4- (4- (benzyloxy) -2- ((tert-butoxycarbonyl) amino) -4-oxobutanoylamino) benzoate (9.10g, 18.3mmol) was dissolved in methanol (100mL), Pd/C (0.9g, 10% Pd, 50% wet) was added, and the mixture was stirred at room temperature overnight under a hydrogen atmosphere, filtered and concentrated to give (S) -3- ((tert-butoxycarbonyl) amino) -4- ((4- (tert-butoxycarbonyl) phenyl) amino) -4-oxobutanoic acid (7.16g, 96% yield).
The third step: preparation of N-methyl-2- (phenylsulfonyl) ethyl-1-amine
In a closed tube reactor, phenylvinylsulfone (166mg, 1mmol), methylamine hydrochloride (140mg, 2mmol) were dissolved in methanol (3mL), triethylamine (0.42mL, 3mmol) was added, and the mixture was stirred at 30 ℃ overnight, followed by concentration of the reaction mixture and purification by column chromatography to obtain N-methyl-2- (phenylsulfonyl) ethyl-1-amine (171mg, yield 86%) as a pale yellow solid.
The fourth step: (S) -4- (2- ((tert-butoxycarbonyl) amino) -4- (methyl (2- (benzenesulfonyl) ethyl) amino) -4-oxobutanamide)
Preparation of tert-butyl benzoate
N-methyl-2- (benzenesulfonyl) ethyl-1-amine (100mg, 0.50mmol), (S) -3- ((tert-butoxycarbonyl) amino) -4- ((4- (tert-butoxycarbonyl) phenyl) amino) -4-oxobutanoic acid (200mg, 0.49mmol), T3P (50% ethyl acetate solution, 0.59mL), DIPEA (0.59mL) was dissolved in ethyl acetate/DMF (4mL/1mL), heated to 55 deg.C, and stirred overnight. The organic layer was dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to give tert-butyl (S) -4- (2- ((tert-butoxycarbonyl) amino) -4- (methyl (2- (benzenesulfonyl) ethyl) amino) -4-oxobutanamido) benzoate (184mg, yield 64%) as a white solid.
The fifth step: tert-butyl (S) -4- (2-amino-4- (methyl (2- (phenylsulfonyl) ethyl) amino) -4-oxobutanamido) benzoate
Preparation of acid salts
Tert-butyl (S) -4- (2- ((tert-butoxycarbonyl) amino) -4- (methyl (2- (benzenesulfonyl) ethyl) amino) -4-oxobutanamido) benzoate (177mg, 0.3mmol) was dissolved in tetrahydrofuran (3mL) in ice, and an HCl ethyl acetate solution (3mol/L, 10mL) was added thereto, followed by stirring at 0-5 ℃ for 7 hours and concentration to give tert-butyl (S) -4- (2-amino-4- (methyl (2- (benzenesulfonyl) ethyl) amino) -4-oxobutanamido) benzoate hydrochloride (160mg) as a white solid.
And a sixth step: preparation of tert-butyl (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (methyl (2- (benzenesulfonyl) ethyl) amino) -4-oxobutanamido) benzoate
Tert-butyl (S) -4- (2-amino-4- (methyl (2- (benzenesulfonyl) ethyl) amino) -4-oxobutanamido) benzoate hydrochloride (160mg, ca. 0.3mmol), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (103mg, 0.38mol), T3P (50% ethyl acetate solution, 0.36mL), DIPEA (1.0mL) was dissolved in ethyl acetate/DMF (5mL/1mL), heated to 55 ℃ and stirred overnight. The organic layer was washed once with water, dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give tert-butyl (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (methyl (2- (benzenesulfonyl) ethyl) amino) -4-oxobutyramido) benzoate (78mg, yield 35%) as a white solid.
The seventh step: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (methyl (2- (benzenesulfonyl) ethyl) amino) -4-oxobutanamido) benzoic acid
(S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (methyl (2- (benzenesulfonyl) ethyl) amino) -4-oxobutanamido) benzoic acid tert-butyl ester (78mg, 0.105mmol) was added to HCl 1, 4-dioxane solution (4mol/L, 3mL), stirred at room temperature for 3 hours, the reaction mixture was concentrated, and the residue was purified by reverse phase column chromatography to give (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (methyl (2- (benzenesulfonyl) ethyl) amino) -4-oxobutanamido) benzamide Acid (56mg, 78% yield) as a white solid.
1HNMR(400MHz,DMSO-d6,ppm)δ12.73(brs,1H),10.44(d,J＝8.4Hz,1H),9.88(d,J＝4.0Hz,1H),8.75(d,J＝7.6Hz,1H),7.97-7.89(m,5H),7.76-7.72(m,3H),7.68-7.63(m,3H),6.86-6.77(m,2H),4.84-4.78(m,1H),3.78-3.74(m,1H),3.65-3.61(m,1H),3.56-3.47(m,2H),2.90(s,2H),2.73-2.61(m,3H).
MS m/z(ESI):684[M+1]+。
Example 2: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (4-methylpiperazin-1-yl) -4-oxobutanamido) benzoic acid
Figure BDA0002589780190000131
The first step is as follows: preparation of tert-butyl (S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4- (4-methylpiperazin-1-yl) -4-oxobutyrate
(((9H-fluoren-9-yl) methoxy) carbonyl) -L-aspartic acid (411mg, 1mmol), 1-methylpiperazine (0.11mL, 1mmol), HATU (402mg, 1.1mmol), DIPEA (0.5mL) were dissolved in dichloromethane (10mL) and stirred at room temperature for 3 hours. After the organic layer was washed with water once, dried over anhydrous sodium sulfate, filtered and concentrated, the residue was purified by column chromatography to give tert-butyl (S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4- (4-methylpiperazin-1-yl) -4-oxobutyrate (490mg, yield 100%) as a white solid.
The second step is that: preparation of (S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4- (4-methylpiperazin-1-yl) -4-oxobutanoic acid
Tert-butyl (S) -2- (((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4- (4-methylpiperazin-1-yl) -4-oxobutanoate (490mg, 1mmol) was dissolved in dichloromethane (10mL), trifluoroacetic acid (5mL) was added, stirring was performed at room temperature for 2 hours, and the reaction solution was concentrated to give (S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4- (4-methylpiperazin-1-yl) -4-oxobutanoic acid (430mg, yield 99%) as a white solid.
The third step: preparation of tert-butyl (S) -4- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4- (4-methylpiperazin-1-yl) -4-oxobutanamido) benzoate
(S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4- (4-methylpiperazin-1-yl) -4-oxobutanoic acid (430mg, 1mmol), tert-butyl 4-aminobenzoate (256mg, 1.3mmol), HATU (503mg, 1.3mmol), DIPEA (2mL) was dissolved in dichloromethane (10mL) and stirred at room temperature overnight. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give tert-butyl (S) -4- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4- (4-methylpiperazin-1-yl) -4-oxobutanoylamino) benzoate (478mg, yield 78%) as a white solid.
The fourth step: preparation of tert-butyl (S) -4- (2-amino-4- (4-methylpiperazin-1-yl) -4-oxobutanamido) benzoate
Tert-butyl (S) -4- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4- (4-methylpiperazin-1-yl) -4-oxobutanamido) benzoate (478mg, 0.78mmol) was dissolved in dichloromethane (8mL), diethylamine (5mL) was added, stirred at room temperature for 1 hour, the reaction was concentrated, and the residue was purified by column chromatography to give tert-butyl (S) -4- (2-amino-4- (4-methylpiperazin-1-yl) -4-oxobutanamido) benzoate (250mg, yield 82%) as a pale yellow solid.
The fifth step: preparation of tert-butyl (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (4-methylpiperazin-1-yl) -4-oxobutanamido) benzoate
Tert-butyl (S) -4- (2-amino-4- (4-methylpiperazin-1-yl) -4-oxobutanamido) benzoate (250mg, 0.64mmol), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (207mg, 0.77mmol), HATU (293mg, 0.77mmol), DIPEA (0.6mL) were dissolved in dichloromethane (10mL) and stirred at room temperature overnight. After the organic layer was washed once with water, and dried over anhydrous sodium sulfate, filtered and concentrated, the residue was purified by column chromatography to give tert-butyl (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (4-methylpiperazin-1-yl) -4-oxobutanamido) benzoate (328mg, yield 80%) as a white solid.
And a sixth step: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (4-methylpiperazin-1-yl) -4-oxobutanamido) benzoic acid
(S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (4-methylpiperazin-1-yl) -4-oxobutanamido) benzoic acid tert-butyl ester (100mg, 0.16mmol) was dissolved in dichloromethane (4mL), trifluoroacetic acid (2mL) was added thereto, the mixture was stirred at room temperature for 2 hours, the reaction mixture was concentrated, and the residue was purified by column chromatography to give (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (4-methylpiperazin-1-yl) -4-oxobutanamido) benzoic acid (51mg, yield 55%) white solid.
1HNMR(400MHz,DMSO-d6,ppm)δ10.36(s,1H),9.87(s,1H),8.95(d,J＝8.0Hz,1H),7.95(t,J＝8.0Hz,1H),7.88(d,J＝8.4Hz,2H),7.68-7.64(m,3H),6.84(d,J＝16Hz,1H),6.70(d,J＝16Hz,1H),5.24-5.19(m,1H),3.33-3.30(m,8H),3.20-3.09(m,1H),3.04-2.96(m,1H),2.76-2.67(m,3H).
MS m/z(ESI):585[M+H]+。
Example 3: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-oxo-4- (piperidin-1-yl) butanamido) benzoic acid
Figure BDA0002589780190000151
The first step is as follows: preparation of (S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4-oxo-4- (piperidin-1-yl) butanoic acid
(((9H-fluoren-9-yl) methoxy) carbonyl) -L-aspartic acid (411mg, 1mmol), piperidine (0.105mL, 1.1mmol), HATU (402mg, 1.1mmol), DIPEA (0.5mL) were dissolved in dichloromethane (10mL) and stirred at room temperature for 3 hours. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give (S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4-oxo-4- (piperidin-1-yl) butyric acid (480mg, yield 100%) as a white solid.
The second step is that: preparation of tert-butyl (S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4-oxo-4- (piperidin-1-yl) butyrate
(S) -2- (((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4-oxo-4- (piperidin-1-yl) butanoic acid (480mg, 1mmol) was dissolved in dichloromethane (10mL), trifluoroacetic acid (5mL) was added, stirring was performed at room temperature for 2 hours, and the reaction solution was concentrated to give tert-butyl (S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4-oxo-4- (piperidin-1-yl) butanoate (420mg, yield 99%) as a white solid.
The third step: preparation of tert-butyl (S) -4- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4-oxo-4- (piperidin-1-yl) butanamido) benzoate
(S) -tert-butyl 2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4-oxo-4- (piperidin-1-yl) butyrate (420mg, 1mmol), tert-butyl 4-aminobenzoate (256mg, 1.3mmol), HATU (503mg, 1.3mmol), DIPEA (2mL) was dissolved in dichloromethane (10mL) and stirred at room temperature overnight. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give tert-butyl (S) -4- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4-oxo-4- (piperidin-1-yl) butanamido) benzoate (515mg, yield 86%) as a white solid.
The fourth step: preparation of tert-butyl (S) -4- (2-amino-4-oxo-4- (piperidin-1-yl) butanamido) benzoate
Tert-butyl (S) -4- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4-oxo-4- (piperidin-1-yl) butanamido) benzoate (515mg, 0.86mmol) was dissolved in dichloromethane (8mL), diethylamine (5mL) was added, stirring was performed at room temperature for 1 hour, the reaction solution was concentrated, and the residue was purified by column chromatography to give tert-butyl (S) -4- (2-amino-4-oxo-4- (piperidin-1-yl) butanamido) benzoate (243mg, yield 75%) as a pale yellow solid.
The fifth step: preparation of tert-butyl (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-oxo-4- (piperidin-1-yl) butanamido) benzoate
Tert-butyl (S) -4- (2-amino-4-oxo-4- (piperidin-1-yl) butanamido) benzoate (243mg, 0.65mmol), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (210mg, 0.78mmol), HATU (296mg, 0.78mmol), DIPEA (0.6mL) were dissolved in dichloromethane (10mL) and stirred at room temperature overnight. After washing with water once, the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give tert-butyl (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-oxo-4- (piperidin-1-yl) butanamido) benzoate (340mg, yield 84%) as a white solid.
And a sixth step: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-oxo-4- (piperidin-1-yl) butanamido) benzoic acid
(S, E) -tert-butyl 4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-oxo-4- (piperidin-1-yl) butanamido) benzoate (200mg, 0.32mmol) was dissolved in methylene chloride (4mL), trifluoroacetic acid (2.5mL) was added thereto, the mixture was stirred at room temperature for 2 hours, the reaction mixture was concentrated, and the residue was purified by column chromatography to give (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-oxo-4- (piperidin-1-yl) butanamido) benzoic acid (124mg, yield 68%) of white solid.
1HNMR(400MHz,DMSO-d6,ppm)δ10.42(s,1H),9.86(s,1H),8.76(d,J＝7.6Hz,1H),7.93(t,J＝8.4Hz,1H),7.88(d,J＝8.8Hz,2H),7.73(d,J＝9.2Hz,1H),7.64(dd,J＝1.2,8.6Hz,1H),6.82(d,J＝16Hz,1H),6.77(d,J＝16.4Hz,1H),4.87-4.82(m,1H),3.45-3.39(m,4H),2.86-2.81(m,1H),2.76-2.70(m,1H),1.58-1.54(m,2H),1.50-1.46(m,2H),1.45-1.35(m,2H).
MS m/z(ESI):570[M+H]+。
Example 4: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-morpholin-4-oxobutanamido) benzoic acid
Figure BDA0002589780190000161
The first step is as follows: preparation of tert-butyl (S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4-morpholine-4-oxobutyrate
((9H-fluoren-9-yl) methoxy) carbonyl) -L-aspartic acid (418mg, 1mmol), morpholine (0.095mL, 1.1mmol), HATU (412mg, 1.1mmol), DIPEA (1mL) were dissolved in dichloromethane (10mL) and stirred at room temperature for 2H. Water was added thereto and the organic layer was washed once, dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give tert-butyl (S) -2- (((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4-morpholine-4-oxobutanoate (480mg, yield 100%) as a white solid.
The second step is that: preparation of (S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4-morpholin-4-oxobutanoic acid
Tert-butyl (S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4-morpholine-4-oxobutanoate (480mg, 1mmol) was dissolved in dichloromethane (6mL), trifluoroacetic acid (3mL) was added, stirring was carried out at room temperature for 2.5 hours, and the reaction solution was concentrated to give (S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4-morpholine-4-oxobutanoic acid (420mg, yield 99%) as a white solid.
The third step: preparation of tert-butyl (S) -4- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4-morpholin-4-oxobutanoylamino) benzoate (S) -2- ((((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4-morpholin-4-oxobutanoic acid (420mg, 1mmol), tert-butyl 4-aminobenzoate (256mg, 1.3mmol), HATU (503mg, 1.3mmol), DIPEA (2mL) was dissolved in dichloromethane (10mL) and stirred at room temperature overnight. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give tert-butyl (S) -4- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4-morpholine-4-oxobutanamido) benzoate (527mg, yield 88%) as a white solid.
The fourth step: preparation of tert-butyl (S) -4- (2-amino-4-morpholine-4-oxobutanamido) benzoate
Tert-butyl (S) -4- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4-morpholine-4-oxobutanamido) benzoate (527mg, 0.88mmol) was dissolved in dichloromethane (8mL), diethylamine (6mL) was added, stirring was carried out at room temperature for 1 hour, the reaction solution was concentrated, and the residue was purified by column chromatography to give tert-butyl (S) -4- (2-amino-4-morpholine-4-oxobutanamido) benzoate (330mg, yield 99%) as a yellow solid.
The fifth step: preparation of tert-butyl (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-morpholine-4-oxobutanamido) benzoate
Tert-butyl (S) -4- (2-amino-4-morpholin-4-oxobutanamido) benzoate (330mg, 0.875mmol), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (283mg, 1.05mmol), HATU (399mg, 1.05mmol), DIPEA (1mL) were dissolved in dichloromethane (12mL) and stirred at room temperature for 4H. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give tert-butyl (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-morpholin-4-oxobutanamido) benzoate (528mg, 96% yield) as a yellow solid.
And a sixth step: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-morpholin-4-oxobutanamido) benzoic acid
(S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-morpholin-4-oxobutanamido) benzoic acid tert-butyl ester (200mg, 0.32mmol) was dissolved in dichloromethane (5mL), trifluoroacetic acid (3mL) was added, stirring was carried out at room temperature for 2 hours, the reaction was concentrated, and the residue was purified by column chromatography to give (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-morpholin-4-oxobutanamido) benzoic acid (95mg, yield 52%), a white solid.
1HNMR(400MHz,DMSO-d6,ppm)δ10.47(s,1H),9.87(s,1H),8.80(d,J＝7.2Hz,1H),7.93(t,J＝8.0Hz,1H),7.88(d,J＝9.2Hz,2H),7.73(d,J＝8.8Hz,2H),7.63(dd,J＝1.2,8.4Hz,1H),6.82(d,J＝16Hz,1H),6.77(d,J＝16Hz,1H),4.85(q,J＝6.8Hz,1H),3.60-3.52(m,4H),3.48-3.41(m,4H),2.89-2.83(m,1H),2.79-2.73(m,1H).
MS m/z(ESI):572[M+H]+。
Example 5: preparation of (S, E) -4- (4- (4-acetylpiperazin-1-yl) -2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-oxobutanamido) benzoic acid
Figure BDA0002589780190000181
The first step is as follows: preparation of tert-butyl (S) -4- (4- (4-acetylpiperazin-1-yl) -2- ((tert-butoxycarbonyl) amino) -4-oxobutanamido) benzoate
(S) -3- ((tert-Butoxycarbonyl) amino) -4- ((4- (tert-butyloxycarbonyl) phenyl) amino) -4-oxobutanoic acid (203mg, 0.5mmol), 1-acetylpiperazine (0.125mL, 1.0mmol), T3P (50% ethyl acetate solution, 0.59mL), DIPEA (0.5mL) dissolved in ethyl acetate (6mL), heated to 50 ℃ and stirred overnight. The organic layer was dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to give tert-butyl (S) -4- (4- (4-acetylpiperazin-1-yl) -2- ((tert-butoxycarbonyl) amino) -4-oxobutanamido) benzoate (244mg, yield 94%) as a white solid.
The second step is that: preparation of tert-butyl (S) -4- (4- (4-acetylpiperazin-1-yl) -2-amino-4-oxobutanoylamino) benzoate
Tert-butyl (S) -4- (4- (4-acetylpiperazin-1-yl) -2- ((tert-butoxycarbonyl) amino) -4-oxobutanamido) benzoate (160mg, 0.31mmol) was dissolved in tetrahydrofuran (2mL) in an ice-water bath, an HCl ethyl acetate solution (3mol/L, 5mL) was added, the reaction mixture was stirred for 5 hours in an ice-water bath, the reaction mixture was concentrated, and the residue was purified by reverse phase column chromatography to give tert-butyl (S) -4- (4- (4-acetylpiperazin-1-yl) -2-amino-4-oxobutanamido) benzoate (52mg, yield 45%) as a white solid.
The third step: preparation of tert-butyl (S, E) -4- (4- (4-acetylpiperazin-1-yl) -2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-oxobutanamido) benzoate
Tert-butyl (S) -4- (4- (4-acetylpiperazin-1-yl) -2-amino-4-oxobutanamido) benzoate (52mg, 0.125mmol), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (37mg, 0.138mmol), T3P (50% ethyl acetate solution, 0.147mL), DIPEA (0.1mL) was dissolved in ethyl acetate (6mL), heated to 50 ℃ and stirred overnight. The organic layer was washed once with water, dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by reverse phase column chromatography to give tert-butyl (S, E) -4- (4- (4-acetylpiperazin-1-yl) -2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-oxobutyramido) benzoate (52mg, yield 62%) as a white solid.
The fourth step: preparation of (S, E) -4- (4- (4-acetylpiperazin-1-yl) -2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-oxobutanamido) benzoic acid
Tert-butyl (S, E) -4- (4- (4-acetylpiperazin-1-yl) -2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-oxobutanamido) benzoate (52mg, 0.078mmol) was dissolved in methylene chloride (2.5mL), trifluoroacetic acid (0.5mL) was added thereto, the mixture was stirred at room temperature for 3 hours, the reaction mixture was concentrated, and the residue was purified by reverse phase column chromatography to give (S, E) -4- (4- (4-acetylpiperazin-1-yl) -2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-oxobutanamido) benzoic acid (40mg, yield 84%) yellow solid.
1HNMR(400MHz,DMSO-d6,ppm)δ10.48(s,1H),9.88(s,1H),8.81(d,J＝7.2Hz,1H),7.95(t,J＝8.4Hz,1H),7.89(d,J＝9.2Hz,2H),7.74(d,J＝8.8Hz,2H),7.66(dd,J＝1.0,9.0Hz,1H),6.83(d,J＝22.4Hz,1H),6.77(d,J＝22.4Hz,1H),4.87(q,J＝6.8Hz,1H),3.50-3.41(m,8H),2.92-2.87(m,1H),2.82-2.77(m,1H),2.03(d,J＝5.2Hz,3H).
MS m/z(ESI):613[M+H]+。
Example 6: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (4- (dimethylamino) piperidin-1-yl) -4-oxobutanamido) benzoic acid
Figure BDA0002589780190000191
The first step is as follows: preparation of tert-butyl (S) -4- (2- ((tert-butoxycarbonyl) amino) -4- (4- (dimethylamino) piperidin-1-yl) -4-oxobutanamido) benzoate
(S) -3- ((tert-Butoxycarbonyl) amino) -4- ((4- (tert-butyloxycarbonyl) phenyl) amino) -4-oxobutanoic acid (205mg, 0.5mmol), N, N-dimethylpiperidin-4-amine (0.117mL, 1.0mmol), T3P (50% ethyl acetate solution, 0.59mL), DIPEA (0.5mL) were dissolved in ethyl acetate (6mL), heated to 50 ℃ and stirred overnight. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give tert-butyl (S) -4- (2- ((tert-butoxycarbonyl) amino) -4- (4- (dimethylamino) piperidin-1-yl) -4-oxobutanamido) benzoate (260mg, yield 100%) as a white solid.
The second step is that: preparation of tert-butyl (S) -4- (2-amino-4- (4- (dimethylamino) piperidin-1-yl) -4-oxobutanamido) benzoate
Tert-butyl (S) -4- (2- ((tert-butoxycarbonyl) amino) -4- (4- (dimethylamino) piperidin-1-yl) -4-oxobutanamido) benzoate (260mg, 0.5mmol) was dissolved in tetrahydrofuran (2mL) in an ice-water bath, an HCl ethyl acetate solution (3mol/L, 5mL) was added, the mixture was stirred in an ice-water bath for 5 hours, the reaction mixture was concentrated, and the residue was purified by reverse phase column chromatography to give tert-butyl (S) -4- (2-amino-4- (4- (dimethylamino) piperidin-1-yl) -4-oxobutanamido) benzoate (99mg, yield 47%) as a white solid.
The third step: preparation of tert-butyl (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (4- (dimethylamino) piperidin-1-yl) -4-oxobutanamido) benzoate
Tert-butyl (S) -4- (2-amino-4- (4- (dimethylamino) piperidin-1-yl) -4-oxobutanamido) benzoate (99mg, 0.237mmol), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (67mg, 0.25mmol), T3P (50% ethyl acetate solution, 0.28mL), DIPEA (0.2mL) was dissolved in ethyl acetate (6mL), heated to 50 ℃ and stirred overnight. The organic layer was washed once with water, dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by reverse phase column chromatography to give tert-butyl (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (4- (dimethylamino) piperidin-1-yl) -4-oxobutanamido) benzoate (39mg, 25% yield) as a white solid.
The fourth step: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (4- (dimethylamino) piperidin-1-yl) -4-oxobutanamido) benzoic acid
(S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (4- (dimethylamino) piperidin-1-yl) -4-oxobutanamido) benzoic acid tert-butyl ester (39mg, 0.058mmol) was dissolved in methylene chloride (2.5mL), trifluoroacetic acid (0.5mL) was added thereto, the mixture was stirred at room temperature for 3 hours, the reaction mixture was concentrated, and the residue was purified by reverse phase column chromatography to give (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (4- (dimethylamino) piperidin-1-yl) -4-oxobutanamido) Benzoic acid (20mg, 56% yield) as a yellow solid.
1HNMR(400MHz,DMSO-d6,ppm)δ12.64(brs,1H),10.46(s,1H),9.87(s,1H),8.83-8.77(m,1H),7.94(t,J＝8.2Hz,1H),7.89(d,J＝8.4Hz,2H),7.74(d,J＝8.4Hz,2H),7.65(d,J＝8.4Hz,1H),6.82(d,J＝16Hz,1H),6.76(d,J＝16Hz,1H),4.90-4.83(m,1H),4.49-4.44(m,1H),4.04-3.99(m,1H),3.01-2.74(m,4H),2.69(s,6H),2.01-1.97(m,3H),1.58-1.53(m,1H),1.35-1.31(m,1H).
MS m/z(ESI):613[M+H]+。
Example 7: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -5-morpholine-5-oxopentanamido) benzoic acid
Figure BDA0002589780190000211
The first step is as follows: preparation of tert-butyl (S) -4- (5- (benzyloxy) -2- ((tert-butoxycarbonyl) amino) -5-oxopentanamido) benzoate
N-tert-Butoxycarbonyl-L-glutamic acid 5-benzyl ester (6.0g, 17.8mmol), tert-butyl 4-aminobenzoate (3.78g, 19.6mmol), EDCI (4.09g, 21.34mmol), HOBT (2.88g, 21.34mmol), triethylamine (5.39g, 53.4mmol) were dissolved in dichloromethane (120mL) and stirred at room temperature overnight. Water was added thereto and the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give tert-butyl (S) -4- (5- (benzyloxy) -2- ((tert-butoxycarbonyl) amino) -5-oxopentanamido) benzoate (9.01g, yield 99%) as a white solid.
The second step is that: preparation of (S) -4- ((tert-butoxycarbonyl) amino) -5- ((4- (tert-butoxycarbonyl) phenyl) amino) -5-oxopentanoic acid
Tert-butyl (S) -4- (5- (benzyloxy) -2- ((tert-butoxycarbonyl) amino) -5-oxopentanamido) benzoate (9.01g, 17.6mmol) was dissolved in methanol (100mL), 10% Pd/C (0.9g, 10% Pd, 50% wet) was added, stirred overnight at room temperature under a hydrogen atmosphere, filtered and concentrated to give (S) -4- ((tert-butoxycarbonyl) amino) -5- ((4- (tert-butoxycarbonyl) phenyl) amino) -5-oxopentanoic acid (7.13g, 96% yield).
The third step: preparation of tert-butyl (S) -4- (2- ((tert-butoxycarbonyl) amino) -5-morpholine-5-oxopentanamido) benzoate
(S) -4- ((tert-Butoxycarbonyl) amino) -5- ((4- (tert-butyloxycarbonyl) phenyl) amino) -5-oxopentanoic acid (218mg, 0.5mmol), morpholine (0.088mL, 1.0mmol), T3P (50% ethyl acetate solution, 0.60mL), DIPEA (0.6mL) dissolved in ethyl acetate (5mL), heated to 50 ℃ and stirred overnight. The organic layer was dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to give tert-butyl (S) -4- (2- ((tert-butoxycarbonyl) amino) -5-morpholine-5-oxopentanamido) benzoate (244mg, yield 96%) as a yellow solid.
The fourth step: preparation of tert-butyl (S) -4- (2- ((tert-butoxycarbonyl) amino) -5-morpholine-5-oxopentanamido) benzoate
Tert-butyl (S) -4- (2- ((tert-butoxycarbonyl) amino) -5-morpholine-5-oxopentanamido) benzoate (244mg, 0.48mmol) was dissolved in tetrahydrofuran (3mL) in an ice-water bath, an HCl ethyl acetate solution (3mol/L, 5mL) was added, the mixture was stirred in an ice-water bath for 6 hours, the reaction mixture was concentrated, and the residue was purified by reverse phase column chromatography to give tert-butyl (S) -4- (2- ((tert-butoxycarbonyl) amino) -5-morpholine-5-oxopentanamido) benzoate (123mg, 65% yield) as a yellow solid.
The fifth step: preparation of tert-butyl (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -5-morpholine-5-oxopentanamido) benzoate
Tert-butyl (S) -4- (2- ((tert-butoxycarbonyl) amino) -5-morpholin-5-oxopentanamido) benzoate (123mg, 0.31mmol), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (90mg, 0.335mmol), T3P (50% in ethyl acetate, 0.37mL), DIPEA (0.3mL) dissolved in ethyl acetate (6mL), heated to 50 ℃ and stirred overnight. Water was added thereto and the organic layer was washed once, dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by reverse phase column chromatography to give tert-butyl (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -5-morpholin-5-oxopentanamido) benzoate (133mg, yield 65.8%) as a white solid.
And a sixth step: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -5-morpholine-5-oxopentanamido) benzoic acid
(S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -5-morpholine-5-oxopentanamido) benzoic acid tert-butyl ester (133mg, 0.21mmol) was dissolved in tetrahydrofuran (2mL), HCl 1, 4-dioxane solution (3mL) was added, the mixture was stirred at room temperature for 5 hours, the reaction mixture was concentrated, and the residue was purified by reverse phase column chromatography to give (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -5-morpholine-5-oxopentanamido) benzoic acid (58mg, yield 47%) yellow solid.
1HNMR(400MHz,DMSO-d6,ppm)δ12.68(s,1H),10.49(s,1H),9.88(s,1H),8.80(d,J＝7.2Hz,1H),7.96(t,J＝8.4Hz,1H),7.91(d,J＝8.8Hz,2H),7.72(d,J＝8.8Hz,2H),7.66(dd,J＝1.2,8.4Hz,1H),6.84(d,J＝18Hz,1H),6.80(d,J＝18Hz,1H),4.87(q,J＝7.1Hz,1H),3.55-3.52(m,4H),3.43-3.40(m,4H),2.42-2.37(m,2H),2.03-2.00(m,1H),1.94-1.84(m,1H).
MS m/z(ESI):586[M+H]+。
Example 8: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -5-oxo-5- (piperidin-1-yl) pentanamido) benzoic acid
Figure BDA0002589780190000221
The first step is as follows: preparation of tert-butyl (S) -4- (2- ((tert-butoxycarbonyl) amino) -5-oxo-5- (piperidin-1-yl) pentanamido) benzoate
(S) -4- ((tert-Butoxycarbonyl) amino) -5- ((4- (tert-butyloxycarbonyl) phenyl) amino) -5-oxopentanoic acid (213mg, 0.5mmol), piperidine (0.1mL, 1.0mmol), T3P (50% ethyl acetate solution, 0.59mL), DIPEA (0.59mL) dissolved in ethyl acetate (6mL), heated to 50 ℃ and stirred overnight. The organic layer was dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to give tert-butyl (S) -4- (2- ((tert-butoxycarbonyl) amino) -5-oxo-5- (piperidin-1-yl) pentanamide) benzoate (240mg, yield 98%) as a yellow solid.
The second step is that: preparation of tert-butyl (S) -4- (2-amino-5-oxo-5- (piperidin-1-yl) pentanamido) benzoate
Tert-butyl (S) -4- (2- ((tert-butoxycarbonyl) amino) -5-oxo-5- (piperidin-1-yl) pentanamide) benzoate (240mg, 0.48mmol) was dissolved in tetrahydrofuran (3mL) in an ice-water bath, an HCl ethyl acetate solution (3mol/L, 5mL) was added, the mixture was stirred in an ice-water bath for 6 hours, the reaction mixture was concentrated, and the residue was purified by reverse phase column chromatography to give tert-butyl (S) -4- (2-amino-5-oxo-5- (piperidin-1-yl) pentanamide) benzoate (143mg, yield 73%) as a white solid.
The third step: preparation of tert-butyl (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -5-oxo-5- (piperidin-1-yl) pentanamido) benzoate
Tert-butyl (S) -4- (2-amino-5-oxo-5- (piperidin-1-yl) pentanamido) benzoate (143mg, 0.37mmol), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (108mg, 0.40mmol), T3P (50% ethyl acetate solution, 0.435mL), DIPEA (0.4mL) was dissolved in ethyl acetate (5mL), heated to 50 deg.C and stirred overnight. After washing with water once, the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give tert-butyl (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -5-oxo-5- (piperidin-1-yl) pentanamido) benzoate (115mg, yield 49%) as a pale yellow solid.
The fourth step: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -5-oxo-5- (piperidin-1-yl) pentanamido) benzoic acid
(S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -5-oxo-5- (piperidin-1-yl) pentanamide) benzoic acid tert-butyl ester (115mg, 0.18mmol) was dissolved in tetrahydrofuran (2mL), HCl 1, 4-dioxane solution (3mL) was added thereto, the mixture was stirred at room temperature for 5 hours, the reaction mixture was concentrated, and the residue was purified by reverse phase column chromatography to give (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -5-oxo-5- (piperidin-1-yl) pentanamide) benzoic acid (55mg, yield 52%) white solid.
1HNMR(400MHz,DMSO-d6,ppm)δ12.69(s,1H),10.45(s,1H),9.88(s,1H),8.77(d,J＝7.6Hz,1H),7.96(t,J＝8.2Hz,1H),7.91(d,J＝9.2Hz,2H),7.72(d,J＝8.8Hz,2H),7.66(dd,J＝1.0,8.6Hz,1H),6.85(d,J＝18.8Hz,1H),6.81(d,J＝18.8Hz,1H),4.50(q,J＝7.1Hz,1H),3.41(t,J＝5.4Hz,2H),3.30(m,2H),2.38(t,J＝8.0Hz,2H),2.04-1.95(m,1H),1.93-1.87(m,1H),1.57-1.54(m,2H),1.51-1.44(m,2H),1.40-1.36(m,2H).
MS m/z(ESI):584[M+H]+。
Example 9: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -5- (4-methylpiperazin-1-yl) -5-oxopentanamido) benzoic acid
Figure BDA0002589780190000241
The first step is as follows: preparation of tert-butyl (S) -4- (2- ((tert-butoxycarbonyl) amino) -5- (4-methylpiperazin-1-yl) -5-oxopentanamido) benzoate
(S) -4- ((tert-butoxycarbonyl) amino) -5- ((4- (tert-butoxycarbonyl) phenyl) amino) -5-oxopentanoic acid (216mg, 0.51mmol), 1-methylpiperazine (0.11mL, 1.0mmol), T3P (50% ethyl acetate solution, 0.60mL), DIPEA (0.6mL) dissolved in ethyl acetate (5mL), heated to 50 ℃ and stirred overnight. The organic layer was dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to give tert-butyl (S) -4- (2- ((tert-butoxycarbonyl) amino) -5- (4-methylpiperazin-1-yl) -5-oxopentanamido) benzoate (250mg, yield 99%) as a pale yellow solid.
The second step is that: preparation of tert-butyl (S) -4- (2-amino-5- (4-methylpiperazin-1-yl) -5-oxopentanamido) benzoate
Tert-butyl (S) -4- (2- ((tert-butoxycarbonyl) amino) -5- (4-methylpiperazin-1-yl) -5-oxopentanamido) benzoate (250mg, 0.5mmol) was dissolved in tetrahydrofuran (2mL) in an ice-water bath, an HCl ethyl acetate solution (3mol/L, 6mL) was added, the mixture was stirred in an ice-water bath for 6 hours, the reaction mixture was concentrated, and the residue was purified by reverse phase column chromatography to give tert-butyl (S) -4- (2-amino-5- (4-methylpiperazin-1-yl) -5-oxopentanamido) benzoate (113mg, yield 56%) as a white solid.
The third step: preparation of tert-butyl (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -5- (4-methylpiperazin-1-yl) -5-oxopentanamido) benzoate
Tert-butyl (S) -4- (2-amino-5- (4-methylpiperazin-1-yl) -5-oxopentanamido) benzoate (113mg, 0.28mmol), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (83mg, 0.31mmol), T3P (50% in ethyl acetate, 0.33mL), DIPEA (0.3mL) was dissolved in ethyl acetate (5mL), heated to 50 ℃ and stirred overnight. After the organic layer was washed once with water, and dried over anhydrous sodium sulfate, filtered and concentrated, the residue was purified by column chromatography to give tert-butyl (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -5- (4-methylpiperazin-1-yl) -5-oxopentanamido) benzoate (89mg, yield 49%) as a pale yellow solid.
The fourth step: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -5- (4-methylpiperazin-1-yl) -5-oxopentanamido) benzoic acid
(S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -5- (4-methylpiperazin-1-yl) -5-oxopentanamido) benzoic acid tert-butyl ester (89mg, 0.136mmol) was dissolved in acetonitrile (2mL), HCl 1, 4-dioxane solution (4mol/L, 3mL) was added thereto, the mixture was stirred at room temperature for 3 hours, the reaction mixture was concentrated, and the residue was purified by reverse phase column chromatography to give (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -5- (4-methylpiperazin-1-yl) -5-oxopentanamido) Pentamido) benzoic acid (45mg, 55% yield) as a white solid.
1HNMR(400MHz,DMSO-d6,ppm)δ10.51(s,1H),9.88(s,1H),8.81(d,J＝8.0Hz,1H),7.96(t,J＝8.2Hz,1H),7.91(d,J＝8.8Hz,2H),7.73(d,J＝8.8Hz,2H),7.66(dd,J＝1.2,8.8Hz,1H),6.85(d,J＝20Hz,1H),6.81(d,J＝20Hz,1H),4.51(q,J＝7.6Hz,1H),3.53-3.49(m,4H),2.69-2.65(m,4H),2.46-2.41(m,4H),2.25-2.16(m,1H),2.14-2.06(m,1H),2.01-1.92(m,1H).
MS m/z(ESI):599[M+H]+。
Example 10: preparation of (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-oxo-4- (piperidin-1-yl) butanamido) -1H-indole-2-carboxylic acid
Figure BDA0002589780190000251
The first step is as follows: preparation of (S) -5- (2- ((tert-butoxycarbonyl) amino) -4-oxo-4- (piperidin-1-yl) butanamido) -1H-indole-2-carboxylic acid tert-butyl ester
(S) -4- ((2- (tert-Butoxycarbonyl) -1H-indol-5-yl) amino) -3- ((tert-butyloxycarbonyl) amino) -4-oxobutanoic acid (225mg, 0.5mmol), piperidine (0.1mL, 1.0mmol), T3P (50% ethyl acetate solution, 0.59mL), DIPEA (0.6mL) was dissolved in ethyl acetate (5mL), heated to 50 ℃ and stirred overnight. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give (S) -tert-butyl 5- (2- ((tert-butoxycarbonyl) amino) -4-oxo-4- (piperidin-1-yl) butanamido) -1H-indole-2-carboxylate (250mg, yield 99%) as a pale yellow solid.
The second step is that: preparation of (S) -5- (2- ((tert-butoxycarbonyl) amino) -4-oxo-4- (piperidin-1-yl) butanamido) -1H-indole-2-carboxylic acid tert-butyl ester
Tert-butyl (S) -5- (2- ((tert-butoxycarbonyl) amino) -4-oxo-4- (piperidin-1-yl) butanamido) -1H-indole-2-carboxylate (250mg, 0.5mmol) was dissolved in tetrahydrofuran (3mL) in an ice-water bath, an HCl ethyl acetate solution (3mol/L, 6mL) was added, the mixture was stirred for 8 hours in an ice-water bath, the reaction mixture was concentrated, and the residue was purified by reverse phase column chromatography to give (S) -5- (2- ((tert-butoxycarbonyl) amino) -4-oxo-4- (piperidin-1-yl) butanamido) -1H-indole-2-carboxylate (75mg, yield 36%) as a yellow solid.
The third step: preparation of (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-oxo-4- (piperidin-1-yl) butanamido) -1H-indole-2-carboxylic acid tert-butyl ester
(S) -tert-butyl 5- (2- ((tert-butoxycarbonyl) amino) -4-oxo-4- (piperidin-1-yl) butanamido) -1H-indole-2-carboxylate (75mg, 0.184mmol), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (54mg, 0.20mmol), T3P (50% in ethyl acetate, 0.22mL), DIPEA (0.2mL) was dissolved in ethyl acetate (5mL), heated to 50 ℃ and stirred overnight. The organic layer was washed once with water, dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give tert-butyl (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-oxo-4- (piperidin-1-yl) butanamido) -1H-indole-2-carboxylate (86mg, yield 70%) as a white solid.
The fourth step: preparation of (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-oxo-4- (piperidin-1-yl) butanamido) -1H-indole-2-carboxylic acid
(S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-oxo-4- (piperidin-1-yl) butanamido) -1H-indole-2-carboxylic acid tert-butyl ester (86mg, 0.13mmol) was dissolved in methylene chloride (2mL), trifluoroacetic acid (3mL) was added thereto, the mixture was stirred at room temperature for 5 hours, the reaction mixture was concentrated, and the residue was purified by reverse phase column chromatography to give (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-oxo-4- (piperidin-1-yl) butanamido) -1H Indole-2-carboxylic acid (42mg, 53% yield) as a white solid.
1HNMR(400MHz,DMSO-d6,ppm)δ11.51(brs,1H),9.99(s,1H),9.93(s,1H),9.92(s,1H),8.78(d,J＝7.6Hz,1H),8.01(s,1H),7.99(t,J＝8.2Hz,1H),7.70(d,J＝8.4Hz,1H),7.37(s,2H),6.96-6.92(m,1H),6.88-6.84(m,2H),4.93-4.88(m,1H),3.53-3.44(m,4H),2.95-.86(m,1H),2.81-2.73(m,1H),1.66-1.59(m,2H),1.58-1.50(m,2H),1.46-1.41(m,2H).
MS m/z(ESI):609[M+H]+。
Preparation of intermediate (S) -4- ((2- (tert-butyloxycarbonyl) -1H-indol-5-yl) amino) -3- ((tert-butyloxycarbonyl) amino) -4-oxobutanoic acid (10-1)
Figure BDA0002589780190000261
The first step is as follows: preparation of (S) -5- (4- (benzyloxy) -2- ((tert-butoxycarbonyl) amino) -4-oxobutanamido) -1H-indole-2-carboxylic acid tert-butyl ester
4-benzyl N-tert-butoxycarbonyl-L-aspartate (6.0g, 18.6mmol), tert-butyl 5-amino-1H-indole-2-carboxylate (4.3g, 18.6mmol), T3P (50% in ethyl acetate, 14.7g), DIPEA (7.2g) were dissolved in ethyl acetate (250mL), heated to 50 ℃ and stirred overnight. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give tert-butyl (S) -5- (4- (benzyloxy) -2- ((tert-butoxycarbonyl) amino) -4-oxobutanamide) -1H-indole-2-carboxylate (9.98g, yield 99%) as a white solid.
The second step is that: preparation of (S) -4- ((2- (tert-butoxycarbonyl) -1H-indol-5-yl) amino) -3- ((tert-butoxycarbonyl) amino) -4-oxobutanoic acid
(S) -tert-butyl 5- (4- (benzyloxy) -2- ((tert-butoxycarbonyl) amino) -4-oxobutanamido) -1H-indole-2-carboxylate (9.98g, 18.6mmol) was dissolved in methanol (200mL), 10% Pd/C (0.5g) was added, and the mixture was stirred under hydrogen atmosphere at room temperature overnight, filtered and concentrated to give (S) -4- ((2- (tert-butoxycarbonyl) -1H-indol-5-yl) amino) -3- ((tert-butoxycarbonyl) amino) -4-oxobutanoic acid (8.01g, 96% yield).
Preparation of intermediate 5-aminoindole-2-carboxylic acid tert-butyl ester (10-5)
Figure BDA0002589780190000271
The first step is as follows: 5-Nitroindole-2-carboxylic acid (7.0g, 34mmol) was dissolved in tetrahydrofuran (140mL), thionyl chloride (7mL, 96.5mmol) and DMF (0.1mL) were added, heated under reflux for 3 hours, cooled to room temperature, and concentrated to give 5-nitroindole-2-carbonyl chloride, which was used directly in the next reaction.
The second step is that: 5-Nitroindole-2-carbonyl chloride was dissolved in tetrahydrofuran (100mL), and a potassium tert-butoxide tetrahydrofuran solution (1mol/L, 75mL) was added dropwise to the solution, followed by stirring at room temperature for 1 hour. Water was added to quench the reaction, 1N hydrochloric acid was added to adjust the pH to neutral, and ethyl acetate extraction was carried out. The organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give tert-butyl 5-nitroindole-2-carboxylate (7.57g, yield 85%).
The third step: tert-butyl 5-nitroindole-2-carboxylate (7.57g, 28.9mmol) was dissolved in tetrahydrofuran (150mL), Pd/C (0.7g, 10% Pd, 50% wet) was added, and the mixture was stirred under hydrogen at room temperature overnight. Filtration and concentration gave tert-butyl 5-aminoindole-2-carboxylate (6.51g, 97% yield).
Example 11: preparation of (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (4-methylpiperazin-1-yl) -4-oxobutanamido) -1H-indole-2-carboxylic acid
Figure BDA0002589780190000272
The first step is as follows: preparation of (S) -5- (2- ((tert-butoxycarbonyl) amino) -4- (4-methylpiperazin-1-yl) -4-oxobutanamido) -1H-indole-2-carboxylic acid tert-butyl ester
(S) -4- ((2- (tert-butoxycarbonyl) -1H-indol-5-yl) amino) -3- ((tert-butoxycarbonyl) amino) -4-oxobutanoic acid (446mg, 1mmol), 1-methylpiperazine (0.225mL, 2.0mmol), T3P (50% ethyl acetate solution, 1.06mL), DIPEA (1mL) dissolved in ethyl acetate (10mL), heated to 50 ℃ and stirred overnight. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give (S) -tert-butyl 5- (2- ((tert-butoxycarbonyl) amino) -4- (4-methylpiperazin-1-yl) -4-oxobutanamido) -1H-indole-2-carboxylate (530mg, yield 100%) as a pale yellow solid.
The second step is that: preparation of (S) -5- (2-amino-4- (4-methylpiperazin-1-yl) -4-oxobutanamido) -1H-indole-2-carboxylic acid tert-butyl ester
Tert-butyl (S) -5- (2- ((tert-butoxycarbonyl) amino) -4- (4-methylpiperazin-1-yl) -4-oxobutanamido) -1H-indole-2-carboxylate (530mg, 1.0mmol) was dissolved in tetrahydrofuran (5mL) in an ice-water bath, an HCl ethyl acetate solution (3mol/L, 10mL) was added, the mixture was stirred in an ice-water bath for 8 hours, the reaction mixture was concentrated, and the residue was purified by reverse phase column chromatography to give (S) -5- (2-amino-4- (4-methylpiperazin-1-yl) -4-oxobutanamido) -1H-indole-2-carboxylate (191mg, yield 44%) as a yellow solid.
The third step: preparation of (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (4-methylpiperazin-1-yl) -4-oxobutanamido) -1H-indole-2-carboxylic acid tert-butyl ester
Tert-butyl (S) -5- (2-amino-4- (4-methylpiperazin-1-yl) -4-oxobutanamido) -1H-indole-2-carboxylate (191mg, 0.44mmol), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (122mg, 0.45mmol), T3P (50% ethyl acetate solution, 0.545mL), DIPEA (0.5mL) was dissolved in ethyl acetate (9mL), heated to 50 ℃ and stirred overnight. The organic layer was washed once with water, dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give tert-butyl (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (4-methylpiperazin-1-yl) -4-oxobutanamido) -1H-indole-2-carboxylate (100mg, yield 33%) as a white solid.
The fourth step: preparation of (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (4-methylpiperazin-1-yl) -4-oxobutanamido) -1H-indole-2-carboxylic acid
(S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (4-methylpiperazin-1-yl) -4-oxobutanamido) -1H-indole-2-carboxylic acid tert-butyl ester (100mg, 0.147mmol) was dissolved in methylene chloride (5mL), trifluoroacetic acid (3mL) was added thereto, the mixture was stirred at room temperature for 5 hours, the reaction mixture was concentrated, and the residue was purified by reverse phase column chromatography to give (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (4-methylpiperazin-1-yl) -4-oxo Butyrylamino) -1H-indole-2-carboxylic acid (45mg, yield 49%) as a white solid.
1HNMR(400MHz,DMSO-d6,ppm)δ11.70(brs,1H),10.03(s,1H),9.86(s,1H),8.76(d,J＝7.2Hz,1H),8.02(s,1H),7.95(t,J＝8.0Hz,1H),7.66(dd,J＝1.0,8.6Hz,1H),7.37(s,2H),7.04(d,J＝2.0Hz,1H),6.85-6.77(m,2H),4.89-4.83(m,1H),3.55-3.49(m,8H),2.89-2.85(m,1H),2.79-2.73(m,1H),2.46-2.35(m,3H).
MS m/z(ESI):624[M+H]+。
Example 12: preparation of (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-morpholine-4-oxobutanamido) -1H-indole-2-carboxylic acid
Figure BDA0002589780190000291
The first step is as follows: preparation of (S) -5- (2- ((tert-butoxycarbonyl) amino) -4-morpholine-4-oxobutanamido) -1H-indole-2-carboxylic acid tert-butyl ester
(S) -4- ((2- (tert-Butoxycarbonyl) -1H-indol-5-yl) amino) -3- ((tert-butyloxycarbonyl) amino) -4-oxobutanoic acid (223mg, 0.5mmol), morpholine (0.088mL, 1mmol), T3P (50% ethyl acetate solution, 0.59mL), DIPEA (0.5mL) dissolved in ethyl acetate (10mL), heated to 50 ℃ and stirred overnight. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give (S) -tert-butyl 5- (2- ((tert-butoxycarbonyl) amino) -4-morpholine-4-oxobutanamide) -1H-indole-2-carboxylate (230mg, yield 90%) as a pale yellow solid.
The second step is that: preparation of (S) -5- (2-amino-4-morpholine-4-oxobutanamido) -1H-indole-2-carboxylic acid tert-butyl ester
Tert-butyl (S) -5- (2- ((tert-butoxycarbonyl) amino) -4-morpholine-4-oxobutanamide) -1H-indole-2-carboxylate (230mg, 0.45mmol) was dissolved in tetrahydrofuran (2.5mL) in an ice-water bath, an HCl ethyl acetate solution (3mol/L, 9mL) was added, the mixture was stirred in an ice-water bath for 8 hours, the reaction mixture was concentrated, and the residue was purified by reverse phase column chromatography to give tert-butyl (S) -5- (2-amino-4-morpholine-4-oxobutanamide) -1H-indole-2-carboxylate (135mg, yield 64%) as a yellow solid.
The third step: preparation of (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-morpholine-4-oxobutanamido) -1H-indole-2-carboxylic acid tert-butyl ester
(S) -tert-butyl 5- (2-amino-4-morpholin-4-oxobutanamido) -1H-indole-2-carboxylate (135mg, 0.325mmol), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (91mg, 0.34mmol), T3P (50% in ethyl acetate, 0.385mL), DIPEA (0.3mL) dissolved in ethyl acetate (9mL), heated to 50 ℃ and stirred overnight. After washing with water once, the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give tert-butyl (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-morpholine-4-oxobutyramido) -1H-indole-2-carboxylate (85mg, yield 39%) as a white solid.
The fourth step: preparation of (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-morpholine-4-oxobutanamido) -1H-indole-2-carboxylic acid
(S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-morpholine-4-oxobutanamide) -1H-indole-2-carboxylic acid tert-butyl ester (85mg, 0.128mmol) was dissolved in dichloromethane (2mL), trifluoroacetic acid (2mL) was added thereto, the mixture was stirred at room temperature for 5 hours, the reaction mixture was concentrated, and the residue was purified by reverse phase column chromatography to give (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-morpholine-4-oxobutanamide) -1H-indole-2-carboxylic acid (40mg, yield 51%) white solid.
1HNMR(400MHz,DMSO-d6,ppm)δ11.52(s,1H),9.98(s,1H),9.87(s,1H),8.74(d,J＝7.2Hz,1H),7.98(s,1H),7.93(t,J＝8.2Hz,1H),7.63(d,J＝8.4Hz,1H),7.33(s,2H),6.95(s,1H),6.81(s,2H),4.86(q,J＝7.2Hz,1H),3.57-3.55(m,2H),3.53-3.50(m,2H),3.48-3.46(m,2H),3.43-3.41(m,2H),2.91-2.82(m,1H),2.77-2.67(m,1H).
MS m/z(ESI):611[M+H]+。
Example 13: preparation of (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-oxo-4- (piperidin-1-yl) butanamido) -1H-benzo [ d ] imidazole-2-carboxylic acid
Figure BDA0002589780190000301
The first step is as follows: preparation of di-tert-butyl (S) -5- (2- ((tert-butoxycarbonyl) amino) -4-oxo-4- (piperidin-1-yl) butanamido) -1H-benzo [ d ] imidazole-1, 2-dicarboxylate
(S) -4- ((1, 2-bis (tert-butoxycarbonyl) -1H-benzo [ d ] imidazol-5-yl) amino) -3- ((tert-butoxycarbonyl) amino) -4-oxobutanoic acid (225mg, 0.41mmol), piperidine (0.1mL, 1.0mmol), T3P (50% ethyl acetate solution, 0.59mL), DIPEA (0.6mL) was dissolved in ethyl acetate (5mL), heated to 50 ℃ and stirred overnight. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give di-tert-butyl (S) -5- (2- ((tert-butoxycarbonyl) amino) -4-oxo-4- (piperidin-1-yl) butanamido) -1H-benzo [ d ] imidazole-1, 2-dicarboxylate (250mg, yield 99%) as a pale yellow solid.
The second step is that: preparation of (S) -5- (2-amino-4-oxo-4- (piperidin-1-yl) butanamido) -1H-benzo [ d ] imidazole-2-carboxylic acid tert-butyl ester
Di-tert-butyl (S) -5- (2- ((tert-butoxycarbonyl) amino) -4-oxo-4- (piperidin-1-yl) butanamido) -1H-benzo [ d ] imidazole-1, 2-dicarboxylate (250mg, 0.4mmol) was dissolved in tetrahydrofuran (2.5mL) in an ice-water bath, an HCl ethyl acetate solution (3mol/L, 6mL) was added, the mixture was stirred in an ice-water bath for 8 hours, the reaction mixture was concentrated, and the residue was purified by reverse phase column chromatography to give tert-butyl (S) -5- (2-amino-4-oxo-4- (piperidin-1-yl) butanamido) -1H-benzo [ d ] imidazole-2-carboxylate (135mg, yield 79%) as a white solid.
The third step: preparation of (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-oxo-4- (piperidin-1-yl) butanamido) -1H-benzo [ d ] imidazole-2-carboxylic acid tert-butyl ester
Tert-butyl (S) -5- (2-amino-4-oxo-4- (piperidin-1-yl) butanamido) -1H-benzo [ d ] imidazole-2-carboxylate (135mg, 0.325mmol), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylate (91mg, 0.34mmol), T3P (50% in ethyl acetate, 0.385mL), DIPEA (0.3mL) dissolved in ethyl acetate (5mL), heated to 50 ℃ and stirred overnight. The organic layer was washed once with water, dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give tert-butyl (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-oxo-4- (piperidin-1-yl) butanamido) -1H-benzo [ d ] imidazole-2-carboxylate (85mg, yield 39%) as a white solid.
The fourth step: preparation of (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-oxo-4- (piperidin-1-yl) butanamido) -1H-benzo [ d ] imidazole-2-carboxylic acid
(S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-oxo-4- (piperidin-1-yl) butanamido) -1H-benzo [ d ] imidazole-2-carboxylic acid tert-butyl ester (85mg, 0.13mmol) was dissolved in dichloromethane (2mL), trifluoroacetic acid (3mL) was added thereto, the mixture was stirred at room temperature for 5 hours, the reaction mixture was concentrated, and the residue was purified by reverse phase column chromatography to give (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-oxo-4- (piperidin-1-yl) butanamide) -1H-benzo [ d ] imidazole-2-carboxylic acid (40mg, 51% yield) as a white solid.
1HNMR(400MHz,DMSO-d6,ppm)δ10.13(s,1H),9.99(s,1H),9.77(s,1H),8.63(d,J＝7.2Hz,1H),8.05(s,1H),7.95(d,J＝1.2Hz,1H),7.84(t,J＝8.4Hz,1H),7.54(d,J＝8.4Hz,1H),7.42-7.18(m,1H),6.73-6.66(m,2H),4.77-4.72(m,1H),3.29-3.27(m,4H),2.78-2.71(m,1H),2.66-2.57(m,1H),1.48-1.44(m,2H),1.42-1.36(m,2H),1.35-1.25(m,2H).
MS m/z(ESI):610[M+H]+。
Preparation of intermediate (S) -4- ((1, 2-di (tert-butoxycarbonyl) -1H-benzo [ d ] imidazol-5-yl) amino) -3- ((tert-butoxycarbonyl) amino) -4-oxobutanoic acid (13-1)
Figure BDA0002589780190000311
The first step is as follows: preparation of di-tert-butyl (S) -5- (4- (benzyloxy) -2- ((tert-butoxycarbonyl) amino) -4-oxobutanamido) -1H-benzo [ d ] imidazole-1, 2-dicarboxylate
4-benzyl N-tert-butoxycarbonyl-L-aspartate (2.68g, 8.3mmol), di-tert-butyl 5-amino-1H-benzo [ d ] imidazole-1, 2-dicarboxylate (2.76g, 8.3mmol), T3P (50% ethyl acetate solution, 6.61g), DIPEA (3.21g) were dissolved in ethyl acetate (50mL), heated to 50 ℃ and stirred overnight. Water was added to wash once, the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give di-tert-butyl (S) -5- (4- (benzyloxy) -2- ((tert-butoxycarbonyl) amino) -4-oxobutanamido) -1H-benzo [ d ] imidazole-1, 2-dicarboxylate (5.20g, yield 98%) as a white solid.
The second step is that: preparation of (S) -4- ((1, 2-di (tert-butoxycarbonyl) -1H-benzo [ d ] imidazol-5-yl) amino) -3- ((tert-butoxycarbonyl) amino) -4-oxobutanoic acid
Di-tert-butyl (S) -5- (4- (benzyloxy) -2- ((tert-butoxycarbonyl) amino) -4-oxobutanamido) -1H-benzo [ d ] imidazole-1, 2-dicarboxylate (5.20g, 8.1mmol) was dissolved in methanol (200mL), 10% Pd/C (0.5g) was added, stirring overnight at room temperature under a hydrogen atmosphere, and concentration by filtration gave (S) -4- ((1, 2-bis (tert-butoxycarbonyl) -1H-benzo [ d ] imidazol-5-yl) amino) -3- ((tert-butoxycarbonyl) amino) -4-oxobutanoic acid (4.20g, 94% yield).
Preparation of intermediate 5-amino-1H-benzo [ d ] imidazole-1, 2-dicarboxylic acid di-tert-butyl ester (13-5)
Figure BDA0002589780190000321
The first step is as follows: methyl 2,2, 2-trichloroacetimidate (7.91g, 44.8mmol) was added dropwise to a solution of 4-nitrophthalenediamine (6.168g, 40.3mmol) dissolved in acetic acid (83mL) under ice-bath, stirred for 5 hours, and the precipitated solid was collected by filtration to give 5-nitro-2- (trichloromethyl) -1H-benzo [ d ] imidazole (5.80g, yield 51.4%) as a white solid.
The second step is that: 5-Nitro-2- (trichloromethyl) -1H-benzo [ d ] imidazole (550mg, 1.96mmol) was added to a 1N aqueous NaOH solution (50mL) and stirred at room temperature for 4 hours. And (5) filtering. The filtrate was adjusted to pH 4 with 3N hydrochloric acid, and the precipitated solid was collected by filtration to give 5-nitro-1H-benzo [ d ] imidazole-2-carboxylic acid (377mg, yield 92%)
The third step: 5-Nitro-1H-benzo [ d ] imidazole-2-carboxylic acid (1.82g, 8.75mmol) and DMAP (2.135g, 17.5mmol) were dissolved in methylene chloride (100mL), and a solution of Boc2O (7.63g, 35mmol) in methylene chloride (50mL) was added dropwise thereto, followed by stirring at room temperature overnight. The reaction mixture was washed once with 1N diluted hydrochloric acid, saturated aqueous sodium bicarbonate solution and saturated brine, the organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give di-tert-butyl 5-nitro-1H-benzo [ d ] imidazole-1, 2-dicarboxylate (1.95g, yield 62%).
The fourth step: di-tert-butyl 5-nitro-1H-benzo [ d ] imidazole-1, 2-dicarboxylate (800mg, 2.2mmol) was dissolved in methanol/tetrahydrofuran (10mL/5 mL). Raney Ni (80mg) was added thereto, and the mixture was stirred overnight under a hydrogen atmosphere, filtered and concentrated to give di-tert-butyl 5-amino-1H-benzo [ d ] imidazole-1, 2-dicarboxylate (719mg, yield 98%).
Example 14: preparation of (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-morpholine-4-oxobutanamido) -1H-benzo [ d ] imidazole-2-carboxylic acid
Figure BDA0002589780190000322
The first step is as follows: preparation of (S) -5- (2- ((tert-butoxycarbonyl) amino) -4-morpholine-4-oxobutanamido) -1H-benzo [ d ] imidazole-2-carboxylic acid tert-butyl ester
(S) -4- ((1, 2-bis (tert-butoxycarbonyl) -1H-benzo [ d ] imidazol-5-yl) amino) -3- ((tert-butoxycarbonyl) amino) -4-oxobutanoic acid (550mg, 1mmol), morpholine (0.20mL, 2.0mmol), T3P (50% ethyl acetate solution, 1mL), DIPEA (1mL) was dissolved in ethyl acetate (8mL), heated to 50 ℃ and stirred overnight. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give tert-butyl (S) -5- (2- ((tert-butoxycarbonyl) amino) -4-morpholine-4-oxobutanamide) -1H-benzo [ d ] imidazole-2-carboxylate (520mg, yield 100%) as a pale yellow solid.
The second step is that: preparation of (S) -5- (2-amino-4-morpholine-4-oxobutanamido) -1H-benzo [ d ] imidazole-2-carboxylic acid tert-butyl ester
Tert-butyl (S) -5- (2- ((tert-butoxycarbonyl) amino) -4-morpholine-4-oxobutanamide) -1H-benzo [ d ] imidazole-2-carboxylate (520mg, 1.0mmol) was dissolved in tetrahydrofuran (5mL) in an ice-water bath, an HCl ethyl acetate solution (3mol/L, 10mL) was added, the mixture was stirred for 8 hours in an ice-water bath, the reaction mixture was concentrated, and the residue was purified by reverse phase column chromatography to give tert-butyl (S) -5- (2-amino-4-morpholine-4-oxobutanamide) -1H-benzo [ d ] imidazole-2-carboxylate (218mg, yield 52%) as a white solid.
The third step: preparation of (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-morpholine-4-oxobutanamido) -1H-benzo [ d ] imidazole-2-carboxylic acid tert-butyl ester
(S) -tert-butyl 5- (2-amino-4-morpholin-4-oxobutanamido) -1H-benzo [ d ] imidazole-2-carboxylate (218mg, 0.52mmol), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (141mg, 0.52mmol), T3P (50% ethyl acetate solution, 0.62mL), DIPEA (0.6mL) dissolved in ethyl acetate (10mL), heated to 50 ℃ and stirred overnight. The organic layer was washed once with water, dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give tert-butyl (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-morpholin-4-oxobutanamido) -1H-benzo [ d ] imidazole-2-carboxylate (165mg, yield 48%) as a white solid.
The fourth step: preparation of (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-morpholine-4-oxobutanamido) -1H-benzo [ d ] imidazole-2-carboxylic acid
(S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-morpholin-4-oxobutanamido) -1H-benzo [ d ] imidazole-2-carboxylic acid tert-butyl ester (81mg, 0.121mmol) was dissolved in dichloromethane (2mL), trifluoroacetic acid (1mL) was added thereto, the mixture was stirred at room temperature for 2 hours, the reaction mixture was concentrated, and the residue was purified by reverse phase column chromatography to give (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4-morpholin-4-oxobutanamido) -1H-benzo [ d ] imidazole-2- Formic acid (55mg, 74% yield) as a white solid.
1HNMR(400MHz,DMSO-d6,ppm)δ10.49(s,1H),10.35(s,1H),9.88(s,1H),9.27(s,1H),8.79(t,J＝7.4Hz,1H),8.19(d,J＝1.2Hz,1H),7.98-.29(m,1H),7.65(dd,J＝1.2,8.2Hz,1H),7.63-7.58(m,1H),7.46(dd,J＝2.0,8.8Hz,1H),6.82(t,J＝16.6Hz,2H),4.90-4.85(m,1H),3.58(brs,2H),3.53(brs,2H),3.47(brs,2H),3.43(brs,2H),2.90-2.84(m,1H),2.79-2.70(m,1H).
MS m/z(ESI):612[M+H]+。
Example 15: preparation of (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (4-methylpiperazin-1-yl) -4-oxobutanamido) -1H-benzo [ d ] imidazole-2-carboxylic acid
Figure BDA0002589780190000341
The first step is as follows: preparation of tert-butyl (S) -5- (2- ((tert-butoxycarbonyl) amino) -4- (4-methylpiperazin-1-yl) -4-oxobutanamido) -1H-benzo [ d ] imidazole-2-carboxylate
(S) -4- ((1, 2-bis (tert-butoxycarbonyl) -1H-benzo [ d ] imidazol-5-yl) amino) -3- ((tert-butoxycarbonyl) amino) -4-oxobutanoic acid (547mg, 1mmol), 1-methylpiperazine (0.20mL, 2.0mmol), T3P (50% ethyl acetate solution, 1mL), DIPEA (1mL) was dissolved in ethyl acetate (8mL), heated to 50 ℃ and stirred overnight. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give tert-butyl (S) -5- (2- ((tert-butoxycarbonyl) amino) -4- (4-methylpiperazin-1-yl) -4-oxobutanamido) -1H-benzo [ d ] imidazole-2-carboxylate (530mg, yield 100%) as a pale yellow solid.
The second step is that: preparation of (S) -5- (2-amino-4- (4-methylpiperazin-1-yl) -4-oxobutanamido) -1H-benzo [ d ] imidazole-2-carboxylic acid tert-butyl ester
Tert-butyl (S) -5- (2- ((tert-butoxycarbonyl) amino) -4- (4-methylpiperazin-1-yl) -4-oxobutanamido) -1H-benzo [ d ] imidazole-2-carboxylate (530mg, 1.0mmol) was dissolved in tetrahydrofuran (5mL) in an ice-water bath, an HCl ethyl acetate solution (3mol/L, 10mL) was added, the mixture was stirred in an ice-water bath for 8 hours, the reaction mixture was concentrated, and the residue was purified by reverse phase column chromatography to give tert-butyl (S) -5- (2-amino-4- (4-methylpiperazin-1-yl) -4-oxobutanamido) -1H-benzo [ d ] imidazole-2-carboxylate (198mg, yield 46%), a white solid.
The third step: preparation of (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (4-methylpiperazin-1-yl) -4-oxobutanamido) -1H-benzo [ d ] imidazole-2-carboxylic acid tert-butyl ester
Tert-butyl (S) -5- (2-amino-4- (4-methylpiperazin-1-yl) -4-oxobutanamido) -1H-benzo [ d ] imidazole-2-carboxylate (198mg, 0.46mmol), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (125mg, 0.47mmol), T3P (50% in ethyl acetate, 0.545mL), DIPEA (0.5mL) dissolved in ethyl acetate (8mL) was heated to 50 ℃ and stirred overnight. The organic layer was washed once with water, dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give tert-butyl (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (4-methylpiperazin-1-yl) -4-oxobutanamido) -1H-benzo [ d ] imidazole-2-carboxylate (51mg, yield 16%) as a white solid.
The fourth step: preparation of (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (4-methylpiperazin-1-yl) -4-oxobutanamido) -1H-benzo [ d ] imidazole-2-carboxylic acid
(S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (4-methylpiperazin-1-yl) -4-oxobutanamido) -1H-benzo [ d ] imidazole-2-carboxylic acid tert-butyl ester (51mg, 0.075mmol) was dissolved in dichloromethane (5mL), trifluoroacetic acid (4mL) was added thereto, the mixture was stirred at room temperature for 5 hours, the reaction mixture was concentrated, and the residue was purified by reverse phase column chromatography to give (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (4-methylpiperazin-1-yl) - 4-Oxobutylamino) -1H-benzo [ d ] imidazole-2-carboxylic acid (35mg, 75% yield) as a white solid.
1HNMR(400MHz,DMSO-d6,ppm)δ10.39(s,1H),9.87(s,1H),9.80(d,J＝7.6Hz,1H),8.21(s,1H),7.94(t,J＝8.2Hz,1H),7.64(d,J＝8.8Hz,1H),7.61(d,J＝8.8Hz,1H),7.44(d,J＝8.0Hz,1H),6.83(d,J＝16Hz,1H),6.78(d,J＝16Hz,1H),4.88(q,J＝6.8Hz,1H),3.77(t,J＝5.0Hz,3H),3.11(t,J＝4.8Hz,3H),3.08-3.00(m,2H),2.96-2.89(m,1H),2.82-2.76(m,1H),2.70(s,3H).
MS m/z(ESI):625[M+H]+。
Example 16: preparation of 5- ((S) -2- ((1r,4S) -4-cyanocyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propanamido) -1H-benzo [ d ] imidazole-2-carboxylic acid
Figure BDA0002589780190000351
The first step is as follows: preparation of di-tert-butyl (S) -5- (2- ((tert-butoxycarbonyl) amino) -3- (3- (N-methylacetamido) phenyl) propanamido) -1H-benzo [ d ] imidazole-1, 2-dicarboxylate
(S) -2- ((tert-butoxycarbonyl) amino) -3- (3- (N-methylacetamido) phenyl) propionic acid (111mg, 0.33mmol), di-tert-butyl 5-amino-1H-benzo [ d ] imidazole-1, 2-dicarboxylate (100mg, 0.30mmol), HATU (126mg, 0.33mmol), DIPEA (0.16mL) was added to dichloromethane (3mL), stirred at room temperature for 2 hours, washed once with water, the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give (S) -5- (2- ((tert-butoxycarbonyl) amino) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-1, 2-dicarboxylic acid di-tert-butyl ester (165mg, yield 77%).
The second step is that: preparation of (S) -5- (2-amino-3- (3- (N-methylacetamido) phenyl) propanamido) -1H-benzo [ d ] imidazole-2-carboxylic acid tert-butyl ester
Di-tert-butyl (S) -5- (2- ((tert-butoxycarbonyl) amino) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-1, 2-dicarboxylate (133mg, 0.204mmol) was dissolved in tetrahydrofuran (3mL) under ice-bath, an HCl ethyl acetate solution (3mol/L, 5mL) was added, stirring was carried out in an ice-water bath for 5 hours, the reaction was concentrated, and the residue was purified by reverse phase column chromatography to give tert-butyl (S) -5- (2-amino-3- (3- (N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-2-carboxylate (70mg, yield 76%).
The third step: preparation of tert-butyl 5- ((S) -2- ((1r,4S) -4-cyanocyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propanamido) -1H-benzo [ d ] imidazole-2-carboxylate
(S) -tert-butyl 5- (2-amino-3- (3- (N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-2-carboxylate (70mg, 0.155mmol), (1r,4r) -4-cyanocyclohexane-1-carboxylate (25mg, 0.16mmol), HATU (80mg, 0.21mmol), DIPEA (0.1mL) were added to dichloromethane (3mL), stirred at room temperature for 2 hours, washed once with water, the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give 5- ((S) -2- ((1r,4S) -4-cyanocyclohexane-1-carboxamide) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-2- Tert-butyl formate (52mg, 57% yield).
The fourth step: preparation of 5- ((S) -2- ((1r,4S) -4-cyanocyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propanamido) -1H-benzo [ d ] imidazole-2-carboxylic acid
Tert-butyl 5- ((S) -2- ((1r,4S) -4-cyanocyclohexane-1-carboxamide) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-2-carboxylate (52mg, 0.089mmol) was dissolved in dichloromethane (3mL), trifluoroacetic acid (2mL) was added, stirring at room temperature for 2 hours, concentration and purification by reverse phase column chromatography to give 5- ((S) -2- ((1r,4S) -4-cyanocyclohexane-1-carboxamide) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-2-carboxylic acid (35mg, yield 74%) white solid.
1HNMR(400MHz,DMSO-d6,ppm)δ11.57(s,1H),9.96(s,1H),8.15(d,J＝8.0Hz,1H),7.98(s,1H),7.62-7.53(m,2H),7.29-7.27(m,2H),7.25(s,1H),7.17(d,J＝8.0Hz,1H),7.11(s,1H),4.75-4.69(m,1H),3.10(s,3H),2.66-2.61(m,2H),2.39-2.07(m,2H),2.27-2.17(m,2H),2.01-1.93(m,5H),1.87-1.78(m,2H),1.60-1.30(m,3H).
MS m/z(ESI):531[M+H]+
Preparation of intermediate (S) -2- ((tert-butoxycarbonyl) amino) -3- (3- (N-methylacetamido) phenyl) propionic acid (16-1)
Figure BDA0002589780190000361
The first step is as follows: preparation of (S) -3- (3-bromophenyl) -2- ((tert-butoxycarbonyl) amino) propionic acid
L-3-Bromophenylalanine (15g, 61.5mmol) was added to 1, 4-dioxane/water (1/1, 200mL), di-tert-butyl dicarbonate (15.75mL, 68.6mmol) and sodium bicarbonate (15.75g, 187.5mmol) were added, and the mixture was stirred at room temperature for 4 hours. Under cooling in an ice bath, the aqueous phase was adjusted to pH 3 with 2N dilute hydrochloric acid, 1, 4-dioxane was distilled off under reduced pressure, the residue was extracted with ethyl acetate, the extracts were combined, washed with saturated brine to neutrality, dried over anhydrous magnesium sulfate, and ethyl acetate was distilled off under reduced pressure to give (S) -3- (3-bromophenyl) -2- ((tert-butoxycarbonyl) amino) propionic acid (19g, yield 90%) as a white solid.
The second step is that: preparation of benzyl (S) -3- (3-bromophenyl) -2- ((tert-butoxycarbonyl) amino) propionate
(S) -3- (3-bromophenyl) -2- ((tert-butoxycarbonyl) amino) propionic acid (19g, 55.2mmol) was dissolved in DMF (100mL), cesium carbonate (15g, 46mmol) was added, benzyl bromide (7mL, 58.9mmol) was added dropwise, and the mixture was stirred at room temperature for 4 hours. The reaction was quenched with water, extracted with ethyl acetate, and the combined extracts were washed with saturated brine, dried over anhydrous magnesium sulfate, evaporated under reduced pressure to remove ethyl acetate, and the residue was slurried with n-hexane to give benzyl (S) -3- (3-bromophenyl) -2- ((tert-butoxycarbonyl) amino) propionate (18.1g, yield 76%) as a white solid.
The third step: preparation of benzyl (S) -2- ((tert-butoxycarbonyl) amino) -3- (3- (methylamino) phenyl) propionate
In a sealed tube, benzyl (S) -3- (3-bromophenyl) -2- ((tert-butoxycarbonyl) amino) propionate (11.7g, 27mmol), methylamine hydrochloride (8.21g, 122mmol), Pd2(dba)3(250mg, 2.7mmol), Brettphos (580mg, 10.8mmol), and cesium carbonate (39.6g, 122mmol) were added to anhydrous tetrahydrofuran (100mL) under a nitrogen atmosphere and reacted overnight at 110 ℃. Cooled to room temperature, filtered, concentrated, and the residue purified by column chromatography to give benzyl (S) -2- ((tert-butoxycarbonyl) amino) -3- (3- (methylamino) phenyl) propionate (7.3g, 70% yield).
The fourth step: preparation of benzyl (S) -2- ((tert-butoxycarbonyl) amino) -3- (3- (N-methylacetamido) phenyl) propionate
Benzyl (S) -2- ((tert-butoxycarbonyl) amino) -3- (3- (methylamino) phenyl) propionate (2.0g, 5.3mmol) was dissolved in dichloromethane (40mL) and triethylamine (1.05g, 10.42mmol) and acetic anhydride (850mg, 8.33mmol) were added. After stirring at room temperature for 4 hours, the reaction was quenched with water, and the organic layer was dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to give benzyl (S) -2- ((tert-butoxycarbonyl) amino) -3- (3- (N-methylacetamido) phenyl) propionate (2.0g, yield 90%).
The fifth step: preparation of (S) -2- ((tert-butoxycarbonyl) amino) -3- (3- (N-methylacetamido) phenyl) propionic acid
Benzyl (S) -2- ((tert-butoxycarbonyl) amino) -3- (3- (N-methylacetamido) phenyl) propionate (2.0g, 4.7mmol) was dissolved in methanol (20mL), Pd/C (0.9g, 10% Pd, 50% wet) was added, stirred at room temperature under a hydrogen atmosphere for 2 hours, filtered and concentrated to give (S) -2- ((tert-butoxycarbonyl) amino) -3- (3- (N-methylacetamido) phenyl) propanoic acid (1.50g, 95% yield).
Preparation of intermediate (1r,4r) -4-cyanocyclohexane-1-carboxylic acid (16-4)
Figure BDA0002589780190000371
The first step is as follows: preparation of (1r,4r) -4-formamidocyclohexane-1-methyl formate
(1r,4r) -4- (methoxycarbonyl) cyclohexane-1-carboxylic acid (1.86g, 10mmol) was dissolved in methylene chloride (20mL), and thionyl chloride (3mL) was added thereto, and the mixture was stirred at room temperature for 2 hours and concentrated under reduced pressure. The residue was dissolved in methyl tert-butyl ether (20mL), concentrated aqueous ammonia was slowly added dropwise in an ice bath to precipitate a large amount of white solid, which was filtered, dissolved in dichloromethane, dried over anhydrous magnesium sulfate, filtered and concentrated to give methyl (1r,4r) -4-carboxamidocyclohexane-1-carboxylate (1.55g, 84% yield).
The second step is that: preparation of methyl (1r,4r) -4-cyanocyclohexane-1-carboxylate
Methyl (1r,4r) -4-carboxamidocyclohexane-1-carboxylate (300mg, 1.62mmol) was dissolved in DMF (5mL) and phosphorus oxychloride (0.5mL) was added dropwise with cooling in an ice bath. After the addition, the temperature was raised to room temperature, stirring was continued for 2 hours, water was added to the mixture, the mixture was extracted with ethyl acetate, the organic phase was dried over anhydrous magnesium sulfate, ethyl acetate was evaporated under reduced pressure, and the residue was purified by column chromatography to give methyl (1r,4r) -4-cyanocyclohexane-1-carboxylate (250mg, yield 92%).
The third step: preparation of (1r,4r) -4-cyanocyclohexane-1-carboxylic acid
(1r,4r) -4-cyanocyclohexane-1-carboxylic acid methyl ester (250mg, 1.49mmol) was dissolved in water/tetrahydrofuran/methanol (2mL/2mL/2mL), and lithium hydroxide monohydrate (83mg, 1.92mmol) was added and stirred at room temperature for 2 hours. The organic solvent was removed under reduced pressure, a small amount of water was added, 1N diluted hydrochloric acid was adjusted to pH3, ethyl acetate was extracted, the organic phase was dried over anhydrous magnesium sulfate, and ethyl acetate was distilled off under reduced pressure to give (1r,4r) -4-cyanocyclohexane-1-carboxylic acid (200mg, yield 88%).
Example 17: preparation of 5- ((S) -2- ((1r,4S) -4- ((dimethylamino) methyl) cyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propanamido) -1H-benzo [ d ] imidazole-2-carboxylic acid
Figure BDA0002589780190000381
The first step is as follows: preparation of tert-butyl 5- ((S) -2- ((1r,4S) -4- ((dimethylamino) methyl) cyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propanamido) -1H-benzo [ d ] imidazole-2-carboxylate
(S) -tert-butyl 5- (2-amino-3- (3- (N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-2-carboxylate (67mg, 0.15mmol), (1r,4r) -4- ((dimethylamino) methyl) cyclohexane-1-carboxylate (30mg, 0.16mmol), HATU (80mg, 0.21mmol), DIPEA (0.1mL) were added to dichloromethane (3mL), stirred at room temperature for 2 hours, washed once with water, the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give 5- ((S) -2- ((1r,4S) -4- ((dimethylamino) methyl) cyclohexane-1-carboxamide) -3- (3- (N-methylacetamido) phenyl) propionamido) -1 Tert-butyl H-benzo [ d ] imidazole-2-carboxylate (25mg, 27% yield).
The second step is that: preparation of 5- ((S) -2- ((1r,4S) -4- ((dimethylamino) methyl) cyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propanamido) -1H-benzo [ d ] imidazole-2-carboxylic acid
Tert-butyl 5- ((S) -2- ((1r,4S) -4- ((dimethylamino) methyl) cyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propanamido) -1H-benzo [ d ] imidazole-2-carboxylate (25mg, 0.040mmol) was dissolved in dichloromethane (3mL), trifluoroacetic acid (1mL) was added, stirring was carried out at room temperature for 3 hours, concentration was carried out, and purification was carried out by reverse phase column chromatography to give 5- ((S) -2- ((1r,4S) -4- ((dimethylamino) methyl) cyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propanamido) -1H-benzo [ d ] imidazole-2-carboxylic acid (18mg, yield 79%) of white solid.
1HNMR(400MHz,DMSO-d6,ppm)δ11.57(s,1H),9.96(s,1H),8.15(d,J＝8.0Hz,1H),7.98(s,1H),7.62-7.53(m,2H),7.29-7.27(m,2H),7.25(s,1H),7.17(d,J＝8.0Hz,1H),7.11(s,1H),4.83-4.71(m,1H),3.81-3.71(m,2H),3.18-3.13(m,4H),3.10(s,3H),2.75(s,6H),2.15(t,J＝10.8Hz,1H),1.73-1.51(m,4H),1.33-1.18(m,3H),0.93-0.89(m,2H).
MS m/z(ESI):563[M+H]+
Preparation of intermediate (1r,4r) -4- ((dimethylamino) methyl) cyclohexane-1-carboxylic acid (17-1)
Figure BDA0002589780190000382
The first step is as follows: preparation of benzyl (1r,4r) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carboxylate
(1r,4r) -4- (((tert-Butoxycarbonyl) amino) methyl) cyclohexane-1-carboxylic acid (0.415g, 1.61mmol) was dissolved in DMF (10mL), cesium carbonate (1.059g, 3.25mmol) was added, benzyl bromide (304mg, 1.78mmol) was added dropwise, and the mixture was stirred at room temperature for 4 hours. The reaction was quenched with water, extracted with ethyl acetate, and the combined extracts were washed with saturated brine, dried over anhydrous magnesium sulfate, and ethyl acetate was evaporated under reduced pressure, and the residue was purified by column chromatography to give benzyl (1r,4r) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carboxylate (0.516g, yield 92%) as a white solid.
The second step is that: preparation of benzyl (1r,4r) -4- (aminomethyl) cyclohexane-1-carboxylate hydrochloride
Benzyl (1r,4r) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carboxylate (1.76g, 5.07mmol) was dissolved in HCl ethyl acetate (3mol/L, 20mL), stirred at room temperature to precipitate a white solid, which was filtered and dried to give benzyl (1r,4r) -4- (aminomethyl) cyclohexane-1-carboxylate hydrochloride (1.20g, 83% yield).
The third step: preparation of benzyl (1r,4r) -4- ((dimethylamino) methyl) cyclohexane-1-carboxylate
(1r,4r) -4- (aminomethyl) cyclohexane-1-carboxylic acid benzyl ester hydrochloride (1.20g, 4.24mmol) was dissolved in methanol (20mL), and paraformaldehyde (382mg) and sodium cyanoborohydride (320mg) were added and stirred at room temperature overnight. Saturated aqueous sodium bicarbonate solution was added, extracted with ethyl acetate, the organic phase was dried over anhydrous magnesium sulfate, the ethyl acetate was evaporated under reduced pressure, and the residue was purified by column chromatography to give benzyl (1r,4r) -4- ((dimethylamino) methyl) cyclohexane-1-carboxylate (820mg, yield 70%).
The fourth step: preparation of (1r,4r) -4- ((dimethylamino) methyl) cyclohexane-1-carboxylic acid
Benzyl (1r,4r) -4- ((dimethylamino) methyl) cyclohexane-1-carboxylate (500mg, 1.82mmol) was dissolved in methanol (10mL), Pd/C (50mg, 10% Pd, 50% wet) was added, stirred at room temperature under a hydrogen atmosphere for 2 hours, filtered and concentrated to give (1r,4r) -4- ((dimethylamino) methyl) cyclohexane-1-carboxylate (330mg, 97% yield).
Example 18: preparation of 5- (2- (4- (aminomethyl) cyclohexane-1-carboxamido) -3- (3- (2-methoxy-N-methylacetamido) phenyl) propanamido) -1H-benzo [ d ] imidazole-2-carboxylic acid
Figure BDA0002589780190000391
The first step is as follows: preparation of benzyl (S) -2- ((tert-butoxycarbonyl) amino) -3- (3- (2-methoxy-N-methylacetamido) phenyl) propionate
Benzyl (S) -2- ((tert-butoxycarbonyl) amino) -3- (3- (methylamino) phenyl) propionate (384mg, 1mmol), triethylamine (0.5mL, 3.6mmol) were dissolved in dichloromethane, and 2-methoxyacetyl chloride (0.11mL, 1.1mmol) was added dropwise and stirred at room temperature for 1.2 hours. The reaction solution was washed once with water, and the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to give benzyl (S) -2- ((tert-butoxycarbonyl) amino) -3- (3- (2-methoxy-N-methylacetamido) phenyl) propionate (460mg, crude yield 100%) as a yellow oil.
The second step is that: preparation of benzyl (S) -2-amino-3- (3- (2-methoxy-N-methylacetamido) phenyl) propionate hydrochloride
Benzyl (S) -2- ((tert-butoxycarbonyl) amino) -3- (3- (2-methoxy-N-methylacetamido) phenyl) propionate (460mg, 1mmol) was dissolved in ethyl acetate, HCl/1, 4-dioxane solution (4mol/L, 4mL) was added, and the mixture was stirred at room temperature for 2 hours. Concentration gave 400mg of (S) -benzyl 2-amino-3- (3- (2-methoxy-N-methylacetamido) phenyl) propionate hydrochloride in 100% crude yield as a yellow solid.
The third step: preparation of (S) -1- (benzyloxy) -3- (3- (2-methoxy-N-methylacetamido) phenyl) -1-oxopropyl-2-yl (1r,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carboxy
(S) -benzyl 2-amino-3- (3- (2-methoxy-N-methylacetamido) phenyl) propionate hydrochloride (400mg, 1mmol), trans-4- (tert-butoxycarbonylaminomethyl) cyclohexanecarboxylic acid (258mg, 1mmol), HATU (436mg, 1.1mmol), DIPEA (2mL) was added to DMF/dichloromethane (3mL/3mL), stirred overnight at room temperature, washed once with water, the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, the residue was purified by column chromatography to give (S) -1- (benzyloxy) -3- (3- (2-methoxy-N-methylacetamido) phenyl) -1-oxopropyl-2-yl (1r,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carboxy 420mg, yield 70.6% as pale yellow solid.
The fourth step: preparation of (S) -2- (((1r,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carbonyl) oxo) -3- (3- (2-methoxy-N-methylacetamido) phenyl) propanoic acid
(S) -1- (benzyloxy) -3- (3- (2-methoxy-N-methylacetamido) phenyl) -1-oxopropyl-2-yl (1r,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carboxy (420mg, 0.71mmol) was dissolved in methanol (5mL), Pd/C (40mg, 10% Pd, 50% wet) was added, and the mixture was stirred overnight under a hydrogen atmosphere, filtered, and concentrated to give (S) -2- (((1r,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carbonyl) oxo) -3- (3- (2-methoxy-N-methylacetamido) phenyl) propionic acid (340mg, yield 95%) as a pale yellow solid.
The fifth step: preparation of di-tert-butyl 5- ((S) -2- (((1r,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carbonyl) oxo) -3- (3- (2-methoxy-N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-1, 2-dicarboxylate
(S) -2- (((1r,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carbonyl) oxo) -3- (3- (2-methoxy-N-methylacetamido) phenyl) propionic acid (252mg, 0.5mmol), di-tert-butyl 5-amino-1H-benzo [ d ] imidazole-1, 2-dicarboxylate (167mg, 0.5mmol), T3P (50% ethyl acetate solution, 0.59mL), DIPEA (0.59mL) was dissolved in ethyl acetate (10mL), heated to 55 ℃ and stirred overnight. The organic layer was washed once with water, dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give di-tert-butyl 5- ((S) -2- (((1r,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carbonyl) oxo) -3- (3- (2-methoxy-N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-1, 2-dicarboxylate (160mg, yield 38.9%) as a pale yellow solid.
And a sixth step: preparation of 5- ((S) -2- (((1r,4S) -4- (aminomethyl) cyclohexane-1-carbonyl) oxo) -3- (3- (2-methoxy-N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-2-carboxylic acid
Dissolving di-tert-butyl 5- ((S) -2- (((1r,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carbonyl) oxo) -3- (3- (2-methoxy-N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-1, 2-dicarboxylate (160mg, 0.19mmol) in dichloromethane (3mL), adding trifluoroacetic acid (2mL), stirring at room temperature for 2 hours, concentrating, and purifying by column chromatography to obtain 5- ((S) -2- (((1r,4S) -4- (aminomethyl) cyclohexane-1-carbonyl) oxo) -3- (3- (2-methoxy-N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] d Imidazole-2-carboxylic acid (57mg, 51% yield) as a white solid.
1HNMR(400MHz,DMSO-d6,ppm)δ10.51(s,1H),10.32(s,1H),9.41(s,1H),8.34-8.15(m,2H),7.69(s,2H),7.63(d,J＝8.8Hz,1H),7.42(dd,J＝1.6,9.0Hz,1H),7.36(t,J＝7.6Hz,1H),7.29(d,J＝7.2Hz,1H),7.24(s,1H),7.17(d,J＝7.6Hz,1H),4.78-4.72(m,1H),3.15(s,2H),3.11(s,3H),3.08(s,3H),2.93-2.69(m,1H),2.68-2.62(m,2H),2.13-2.10(m,1H),1.77-1.71(m,3H),1.59-1.43(m,3H),1.30-1.16(m,2H),0.95-0.88(m,2H).
MS m/z(ESI):565[M+H]+
Example 19: preparation of 5- ((S) -2- ((1r,4S) -4-cyanocyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propanamido) -1H-indole-2-carboxylic acid
Figure BDA0002589780190000411
The first step is as follows: preparation of (S) -2-amino-3- (3- (N-methylacetamido) phenyl) propionic acid benzyl ester hydrochloride
(S) -benzyl 2- ((tert-butoxycarbonyl) amino) -3- (3- (N-methylacetamido) phenyl) propionate (130mg, 0.305mmol) was dissolved in tetrahydrofuran (3mL), an HCl ethyl acetate solution (3mol/L, 5mL) was added, stirring was carried out at room temperature for 3 hours, and the reaction solution was concentrated to give (S) -benzyl 2-amino-3- (3- (N-methylacetamido) phenyl) propionate hydrochloride (111mg) which was used directly in the next reaction.
The second step is that: preparation of benzyl (S) -2- ((1r,4S) -4-cyanocyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propionate
Benzyl (S) -2-amino-3- (3- (N-methylacetamido) phenyl) propionate hydrochloride (111mg, 0.305mmol), (1r,4r) -4-cyanocyclohexane-1-carboxylate (51mg, 0.33mmol), HATU (127mg, 0.33mmol), DIPEA (0.16mL) was added to dichloromethane (3mL), stirred at room temperature for 2 hours, washed once with water, the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give benzyl (S) -2- ((1r,4S) -4-cyanocyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propionate (120mg, 85% yield).
The third step: preparation of (S) -2- ((1r,4S) -4-cyanocyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propionic acid
Benzyl (S) -2- ((1r,4S) -4-cyanocyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propionate (120mg, 0.26mmol) was dissolved in methanol (3mL), Pd/C (15mg, 10% Pd, 50% wet) was added, stirred at room temperature under a hydrogen atmosphere for 4 hours, filtered and concentrated to give (S) -2- ((1r,4S) -4-cyanocyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propanoate (95mg, 98% yield).
The fourth step: preparation of tert-butyl 5- ((S) -2- ((1r,4S) -4-cyanocyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propanamido) -1H-indole-2-carboxylate
(S) -2- ((1r,4S) -4-cyanocyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propionic acid (95mg, 0.256mmol), tert-butyl 5-amino-1H-indole-2-carboxylate (60mg, 0.26mmol), HATU (108mg, 0.28mmol), DIPEA (0.14mL) were added to dichloromethane (3mL), stirred at room temperature for 2 hours, washed once with water, the organic layer was dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to give tert-butyl 5- ((S) -2- ((1r,4S) -4-cyanocyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-indole-2-carboxylate (50mg, yield 33%).
The fifth step: preparation of 5- ((S) -2- ((1r,4S) -4-cyanocyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propanamido) -1H-indole-2-carboxylic acid
Tert-butyl 5- ((S) -2- ((1r,4S) -4-cyanocyclohexane-1-carboxamide) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-indole-2-carboxylate (50mg, 0.085mmol) was dissolved in dichloromethane (5mL), trifluoroacetic acid (2.5mL) was added, stirring was carried out at room temperature for 3 hours, the reaction solution was concentrated, and the residue was purified by ether beating to give 5- ((S) -2- ((1r,4S) -4-cyanocyclohexane-1-carboxamide) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-indole-2-carboxylic acid (32mg, yield 71%).
1HNMR(400MHz,DMSO-d6,ppm)δ11.44(s,1H),9.95(s,1H),8.14(d,J＝7.6Hz,1H),7.94(s,1H),7.45-7.14(m,6H),6.92(s,1H),4.73-4.71(m,1H),3.10(s,3H),2.66-2.61(m,2H),2.39-2.07(m,2H),2.27-2.17(m,2H),2.01-1.93(m,5H),1.87-1.78(m,2H),1.60-1.30(m,3H).
MS m/z(ESI):530[M+H]+
Example 20: 5- ((S) -2- ((1r,4S) -4-cyanocyclohexane-1-carboxamido) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propanamido) -1H-indole-2-carboxylic acid
Figure BDA0002589780190000421
The first step is as follows: preparation of benzyl (S) -2- ((tert-butoxycarbonyl) amino) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propionate
Benzyl (S) -2- ((tert-butoxycarbonyl) amino) -3- (3- (methylamino) phenyl) propionate (565mg, 1.47mmol), N, N-dimethylglycine (182mg, 1.77mmol), HATU (838mg, 2.2mmol), DIPEA (0.61mL) was dissolved in dichloromethane (10mL) and stirred at room temperature for 1.5 h. The organic layer was dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to give benzyl (S) -2- ((tert-butoxycarbonyl) amino) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propionate (670mg, yield 97%).
The second step is that: preparation of (S) -2- ((tert-butoxycarbonyl) amino) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propanoic acid
Benzyl (S) -2- ((tert-butoxycarbonyl) amino) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propionate (1g, 2.1mmol) was dissolved in methanol (10mL), Pd/C (0.1g, 10% Pd, 50% wet) was added, stirred at room temperature for 2 hours under a hydrogen atmosphere, filtered and concentrated to give (S) -2- ((tert-butoxycarbonyl) amino) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propanoic acid (768mg, 95% yield).
The third step: preparation of (S) -5- (2- ((tert-butoxycarbonyl) amino) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propanamido) -1H-indole-2-carboxylic acid tert-butyl ester
(S) -2- ((tert-butoxycarbonyl) amino) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propionic acid (379mg, 1mmol), tert-butyl 5-amino-1H-indole-2-carboxylate (232mg, 1mmol), HATU (418mg, 1.2mmol), DIPEA (0.49mL) was added to dichloromethane (3mL), stirred at room temperature for 2 hours, washed once with water, the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give (S) -tert-butyl 5- (2- ((tert-butoxycarbonyl) amino) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propionamido) -1H-indole-2-carboxylate (520mg, yield 88%).
The fourth step: preparation of (S) -5- (2-amino-3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propanamido) -1H-indole-2-carboxylic acid tert-butyl ester
Tert-butyl (S) -5- (2- ((tert-butoxycarbonyl) amino) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propionamido) -1H-indole-2-carboxylate (76mg, 0.128mmol) was dissolved in tetrahydrofuran (3mL) under ice-bath, HCl ethyl acetate solution (3mol/L, 5mL) was added, stirring was performed in ice-water bath for 5 hours, the reaction mixture was concentrated, and the residue was purified by reverse phase column chromatography to give tert-butyl (S) -5- (2-amino-3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propionamido) -1H-indole-2-carboxylate (60mg, yield 95%).
The fifth step: preparation of tert-butyl 5- ((S) -2- ((1r,4S) -4-cyanocyclohexane-1-carboxamido) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propanamido) -1H-indole-2-carboxylate
(S) -5- (2-amino-3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propanamido) -1H-indole-2-carboxylic acid tert-butyl ester (250mg, 0.507mmol), (1r,4r) -4-cyanocyclohexane-1-carboxylic acid (80mg, 0.52mmol), HATU (199mg, 0.52mmol), DIPEA (0.26mL) was added to dichloromethane (3mL), stirred at room temperature for 2 hours, washed once with water, the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give 5- ((S) -2- ((1r,4S) -4-cyanocyclohexane-1-carboxamido) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propanamido) ) -1H-indole-2-carboxylic acid tert-butyl ester (80mg, 26% yield).
And a sixth step: preparation of 5- ((S) -2- ((1r,4S) -4-cyanocyclohexane-1-carboxamido) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propanamido) -1H-indole-2-carboxylic acid
Tert-butyl 5- ((S) -2- ((1r,4S) -4-cyanocyclohexane-1-carboxamido) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propionamido) -1H-indole-2-carboxylate (80mg, 0.13mmol) was dissolved in dichloromethane (5mL), trifluoroacetic acid (2.5mL) was added, stirring was carried out at room temperature for 3 hours, the reaction solution was concentrated, and the residue was purified by ether beating to give 5- ((S) -2- ((1r,4S) -4-cyanocyclohexane-1-carboxamido) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propionamido) -1H-indole-2-carboxylic acid (70mg, yield 95%).
1HNMR(400MHz,DMSO-d6,ppm)δ12.85(brs,1H),11.71(s,1H),9.98(s,1H),9.62(s,1H),8.41(s,1H),8.19(d,J＝8.8Hz,1H),7.97(d,J＝1.2Hz,1H),7.44-7.23(m,5H),7.04(d,J＝1.6Hz,1H),4.81-4.75(m,1H),3.18(s,3H),3.17-3.09(m,2H),2.94-2.81(m,2H),2.73(s,6H),2.69-2.56(m,2H),2.27-2.17(m,1H),2.02-1.85(m,3H),1.71-1.68(m,1H),1.58-1.32(m,3H)
MS m/z(ESI):573[M+H]+
Example 21: preparation of 5- ((S) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) -2- ((1r,4S) -4- ((dimethylamino) methyl) cyclohexane-1-carboxamido) propionamido) -1H-indole-2-carboxylic acid
Figure BDA0002589780190000441
The first step is as follows: preparation of tert-butyl 5- ((S) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) -2- ((1r,4S) -4- ((dimethylamino) methyl) cyclohexane-1-carboxamido) propionamido) -1H-indole-2-carboxylate
(S) -tert-butyl 5- (2-amino-3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propionamido) -1H-indole-2-carboxylate (99mg, 0.2mmol), (1r,4r) -4- ((dimethylamino) methyl) cyclohexane-1-carboxylate (37mg, 0.2mmol), HATU (80mg, 0.21mmol), DIPEA (0.1mL) was added to dichloromethane (3mL), stirred at room temperature for 2 hours, washed once with water, the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give 5- ((S) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) -2- ((1r,4S) -4- ((dimethylamino) methyl) cyclohexane-1-carboxamido) propionamido) -1H-indole-2-carboxylic acid tert-butyl ester (55mg, 42% yield).
The second step is that: preparation of 5- ((S) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) -2- ((1r,4S) -4- ((dimethylamino) methyl) cyclohexane-1-carboxamido) propionamido) -1H-indole-2-carboxylic acid
Tert-butyl 5- ((S) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) -2- ((1r,4S) -4- ((dimethylamino) methyl) cyclohexane-1-carboxamido) propionamido) -1H-indole-2-carboxylate (55mg, 0.083mmol) was dissolved in dichloromethane (3mL), trifluoroacetic acid (1mL) was added, stirring was carried out at room temperature for 3 hours, concentration was carried out, and purification was carried out by reverse phase column chromatography to give 5- ((S) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) -2- ((1r,4S) -4- ((dimethylamino) methyl) cyclohexane-1-carboxamido) propionamido) -1H-indole-2-carboxylate Formic acid (38mg, 75% yield).
1HNMR(400MHz,DMSO-d6,ppm)δ11.71(s,1H),10.36(brs,1H),9.98(s,1H),8.16(d,J＝8.8Hz,1H),7.97(s,1H),7.72-7.23(m,6H),7.05(s,1H),5.31(s,1H),4.83-4.71(m,1H),3.81-3.71(m,2H),3.18-3.13(m,4H),2.75(s,6H),2.71(s,6H),2.15(t,J＝11.8Hz,1H),1.73-1.51(m,4H),1.33-1.18(m,3H),0.93-0.89(m,2H).
MS m/z(ESI):605[M+H]+。
Example 22: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (4-methylpiperazin-1-yl) -4-oxobutanamido) benzoic acid
Figure BDA0002589780190000442
The first step is as follows: preparation of di-tert-butyl (S) -5- (2- ((tert-butoxycarbonyl) amino) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propanamido) -1H-benzo [ d ] imidazole-1, 2-dicarboxylate
(S) -2- ((tert-Butoxycarbonyl) amino) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propionic acid (50mg, 0.132mmol), di-tert-butyl 5-amino-1H-benzo [ d ] imidazole-1, 2-dicarboxylate (44mg, 0.132mmol), T3P (50% ethyl acetate solution, 105mg, 0.165mmol), DIPEA (52mg, 0.40mmol) were dissolved in ethyl acetate (5mL), heated to 50 ℃ and stirred overnight. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give di-tert-butyl (S) -5- (2- ((tert-butoxycarbonyl) amino) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-1, 2-dicarboxylate (75mg, yield 82%).
The second step is that: preparation of (S) -5- (2-amino-3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propanamido) -1H-benzo [ d ] imidazole-2-carboxylic acid tert-butyl ester
Di-tert-butyl (S) -5- (2- ((tert-butoxycarbonyl) amino) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-1, 2-dicarboxylate (75mg, 0.108mmol) was dissolved in tetrahydrofuran (2mL) in ice bath, an HCl ethyl acetate solution (5mL) was added, stirring was carried out for 7 hours in ice bath, the reaction mixture was concentrated, and the residue was purified by reverse phase column chromatography to give tert-butyl (S) -5- (2-amino-3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-2-carboxylate (42 mg), yield 80%) white solid.
The third step: preparation of tert-butyl 5- ((S) -2- ((1r,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carboxamido) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propanamido) -1H-benzo [ d ] imidazole-2-carboxylate
Tert-butyl (S) -5- (2-amino-3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-2-carboxylate (42mg, 0.085mmol), (1r,4r) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carboxylate (22mg, 0.085mmol), HATU (35mg, 0.092mmol), DIPEA (0.05mL) were dissolved in dichloromethane (5mL) and stirred at room temperature for 3 hours. The organic layer was washed once with water, dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give tert-butyl 5- ((S) -2- ((1r,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carboxamido) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-2-carboxylate (41mg, yield 65%) as a white solid.
The fourth step: preparation of 5- ((S) -2- ((1r,4S) -4- (aminomethyl) cyclohexane-1-carboxamido) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propanamido) -1H-benzo [ d ] imidazole-2-carboxylic acid
5- ((S) -2- ((1r,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carboxamido) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propanamido) -1H-benzo [ d ] imidazole-2-carboxylic acid tert-butyl ester (41mg, 0.056mmol) was dissolved in dichloromethane (4mL), trifluoroacetic acid (2mL) was added, stirring was carried out at room temperature for 2 hours, the reaction solution was concentrated, and the residue was purified by column chromatography to give 5- ((S) -2- ((1r,4S) -4- (aminomethyl) cyclohexane-1-carboxamido) -3- (3- (2- (dimethylamino) -N-methylacetamido) phenyl) propanamido) -1 H-benzo [ d ] imidazole-2-carboxylic acid (20mg, 62% yield) as a white solid.
1HNMR(400MHz,DMSO-d6,ppm)δ10.29(s,1H),10.11(s,1H),8.20-8.16(m,1H),8.09-8.02(m,1H),7.79(s,2H),7.61-7.51(m,1H),7.42-7.21(m,5H),4.83-4.72(m,1H),3.87-3.56(m,2H),3.39(q,J＝6.8Hz,1H),3.16-3.08(m,3H),2.94-2.88(m,1H),2.63(s,6H),2.14(t,J＝12Hz,1H),1.78-1.71(m,3H),1.16-1.07(m,6H),0.98-0.89(m,2H).
MS m/z(ESI):578[M+H]+。
Example 23: preparation of 5- ((S) -2- ((1r,4S) -4- (aminomethyl) cyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propanamido) -1H-indole-2-carboxylic acid
Figure BDA0002589780190000461
The first step is as follows: preparation of (S) -5- (2- ((tert-butoxycarbonyl) amino) -3- (3- (N-methylacetamido) phenyl) propanamido) -1H-indole-2-carboxylic acid tert-butyl ester
(S) -2- ((tert-butoxycarbonyl) amino) -3- (3- (N-methylacetamido) phenyl) propionic acid (100mg, 0.297mmol), tert-butyl 5-amino-1H-indole-2-carboxylate (70mg, 0.30mmol), T3P (50% ethyl acetate solution, 0.425mL), DIPEA (0.4mL) was dissolved in ethyl acetate (5mL), heated to 50 ℃ and stirred overnight. The organic layer was dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to give (S) -tert-butyl 5- (2- ((tert-butoxycarbonyl) amino) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-indole-2-carboxylate (135mg, yield 83%).
The second step is that: preparation of (S) -5- (2-amino-3- (3- (N-methylacetamido) phenyl) propanamido) -1H-indole-2-carboxylic acid tert-butyl ester
Tert-butyl (S) -5- (2- ((tert-butoxycarbonyl) amino) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-indole-2-carboxylate (135mg, 0.245mmol) was dissolved in tetrahydrofuran (2mL) under ice bath, an HCl ethyl acetate solution (5mL) was added, the mixture was stirred under ice bath for 7 hours, the reaction was concentrated, and the residue was purified by reverse phase column chromatography to give (S) -5- (2-amino-3- (3- (N-methylacetamido) phenyl) propionamido) -1H-indole-2-carboxylate (65mg, yield 59%) as a white solid.
The third step: preparation of 5- ((S) -2- ((1r,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-indole-2-carboxylic acid tert-butyl ester
(S) -5- (2-amino-3- (3- (N-methylacetamido) phenyl) propionamido) -1H-indole-2-carboxylic acid tert-butyl ester (65mg, 0.144mmol), (1r,4r) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carboxylic acid (39mg, 0.151mmol), HATU (58mg, 0.153mmol), DIPEA (0.08mL) was dissolved in dichloromethane (5mL) and stirred at room temperature for 3 hours. The organic layer was washed once with water, dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give tert-butyl 5- ((S) -2- ((1r,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-indole-2-carboxylate (55mg, yield 55%) as a white solid.
The fourth step: preparation of 5- ((S) -2- ((1r,4S) -4- (aminomethyl) cyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propanamido) -1H-indole-2-carboxylic acid
Tert-butyl 5- ((S) -2- ((1r,4S) -4- (((tert-butoxycarbonyl) amino) methyl) cyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-indole-2-carboxylate (55mg, 0.079mmol) was dissolved in dichloromethane (4mL), trifluoroacetic acid (2mL) was added, stirring was carried out at room temperature for 2 hours, the reaction solution was concentrated, and the residue was purified by column chromatography to give 5- ((S) -2- ((1r,4S) -4- (aminomethyl) cyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-indole-2-carboxylic acid (35mg, yield 82%) of white solid.
1HNMR(400MHz,DMSO-d6,ppm)δ12.61(brs,1H),11.69(s,1H),9.99(s,1H),8.09(d,J＝8.4Hz,1H),7.99(s,1H),7.65(s,2H),7.38-7.34(m,2H),7.32-7.27(m,2H),7.23(s,1H),7.15(d,J＝8.0Hz,1H),7.04(d,J＝1.2Hz,1H),4.76-4.70(m,1H),3.10(s,3H),2.92-2.87(m,1H),2.64(d,J＝6.8Hz,2H),2.13(t,J＝11.8Hz,1H),1.78-1.70(m,6H),1.55-1.44(m,2H),1.27-1.07(m,3H),0.94-0.87(m,2H).
MS m/z(ESI):534[M+H]+。
Example 24: preparation of (S) -5- (2- (cyclohexanecarboxamido) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-indole-2-carboxylic acid
Figure BDA0002589780190000471
The first step is as follows: preparation of (S) -5- (2- (cyclohexanecarboxamido) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-indole-2-carboxylic acid tert-butyl ester
(S) -5- (2-amino-3- (3- (N-methylacetamido) phenyl) propionamido) -1H-indole-2-carboxylic acid tert-butyl ester (65mg, 0.144mmol), cyclohexanecarboxylic acid (19mg, 0.151mmol), HATU (58mg, 0.153mmol), DIPEA (0.08mL) was dissolved in dichloromethane (5mL) and stirred at room temperature for 3 hours. Water was added thereto and the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give tert-butyl (S) -5- (2- (cyclohexanecarboxamido) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-indole-2-carboxylate (62mg, yield 77%).
The second step is that: preparation of (S) -5- (2- (cyclohexanecarboxamido) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-indole-2-carboxylic acid
(S) -5- (2- (cyclohexanecarboxamido) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-indole-2-carboxylic acid tert-butyl ester (62mg, 0.11mmol) was dissolved in dichloromethane (5mL), trifluoroacetic acid (2.5mL) was added, stirring was performed at room temperature for 2.5 hours, the reaction solution was concentrated, and the residue was purified by reverse phase column chromatography to give (S) -5- (2- (cyclohexanecarboxamido) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-indole-2-carboxylic acid (31mg, yield 55%) as a white solid.
1HNMR(400MHz,DMSO-d6,ppm)δ11.57(s,1H),9.96(s,1H),8.03(d,J＝8.0Hz,1H),7.96(s,1H),7.36-7.34(m,2H),7.29-7.27(m,2H),7.23(s,1H),7.14(d,J＝8.0Hz,1H),6.97(s,1H),4.75-4.69(m,1H),3.10(s,3H),2.93-2.87(m,1H),2.18-2.13(m,1H),1.69-1.56(m,7H),1.46-1.44(m,1H),1.26-1.09(m,6H).
MS m/z(ESI):505[M+H]+。
Example 25: preparation of (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (1, 1-thiomorpholine) -4-oxobutylamine) pentanoic acid
Figure BDA0002589780190000481
The first step is as follows: preparation of tert-butyl 5- (benzyloxycarbonylamino) valerate
5- (benzyloxycarbonylamino) pentanoic acid (1.01g, 4mmol) was dissolved in dichloromethane (10mL), and DCC (2.48g, 12mmol), tert-butanol (1.76g, 24mmol), DMAP (105mg, 0.8mmol) were added and stirred at room temperature for 3.5 hours. Filtration, concentration of the filtrate, addition of 20mL of diethyl ether, re-filtration, concentration of the filtrate, and purification of the residue by column chromatography gave tert-butyl 5- (benzyloxycarbonylamino) pentanoate (950mg, yield 41%) as a pale yellow oil.
The second step is that: preparation of tert-butyl 5-Aminopentanoate
Tert-butyl 5- (benzyloxycarbonylamino) valerate (950mg, 3.1mmol) was dissolved in methanol (20mL), Pd/C (0.9g, 10% Pd, 50% wet) was added, the mixture was stirred overnight at room temperature under a hydrogen atmosphere, and the mixture was concentrated by filtration to give tert-butyl 5-aminopentanoate (520mg, 97% yield).
The third step: preparation of tert-butyl (S) -5- (2- ((((9H-fluoren-9-ylmethoxy) carbonyl) amino) -4- (1, 1-thiomorpholine) -4-oxobutylamine) pentanoate
(S) -2- ((((9H-fluoren-9-ylmethoxy) carbonyl) amino) -4- (1, 1-thiomorpholine dioxide) -4-oxobutanoic acid (95mg, 0.2mmol), tert-butyl 5-aminopentanoate (52mg, 0.3mmol), HATU (93mg, 0.24mmol) and diisopropylethylamine (0.10mL) were added to dichloromethane (3mL) and stirred at room temperature for 2 hours, water was added, dichloromethane was extracted, the organic phase was collected and washed, dried, concentrated, and purified by column chromatography to give tert-butyl (S) -5- (2- ((((9H-fluoren-9-ylmethoxy) carbonyl) amino) -4- (1, 1-thiomorpholine-dioxide) -4-oxobutylamine) pentanoate (105mg, 84% yield).
The fourth step: preparation of tert-butyl (S) -5- (2-amino-4- (1, 1-thiomorpholine) -4-oxobutylamine) valerate
Tert-butyl (S) -5- (2- ((((9H-fluoren-9-ylmethoxy) carbonyl) amino) -4- (1, 1-thiomorpholine) -4-oxobutylamine) valerate (105mg, 0.167mmol) was dissolved in dichloromethane (3mL), diethylamine (2mL) was added, stirring was performed at room temperature for 2 hours, the reaction mixture was concentrated, and the residue was purified by column chromatography to give tert-butyl (S) -5- (2-amino-4- (1, 1-thiomorpholine) -4-oxobutylamine) valerate (55mg, 81% yield).
The fifth step: preparation of (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamide) -4- (1, 1-thiomorpholine-dioxide) -4-oxobutylamine) tert-butyl valerate
Tert-butyl (S) -5- (2-amino-4- (1, 1-thiomorpholine-dioxide) -4-oxobutylamine) valerate (55mg, 0.135mmol), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (37mg, 0.135mmol), HATU (57mg, 0.15mmol) and diisopropylethylamine (0.1mL) were added to dichloromethane (4mL) and stirred for a uniform reaction for 2 hours. Water was added and extracted with dichloromethane, and the organic phase was collected, washed, dried, concentrated and purified by column chromatography to give tert-butyl (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (1, 1-thiomorpholin) -4-oxobutylamine) pentanoate (75mg, 84% yield).
And a sixth step: preparation of (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (1, 1-thiomorpholine) -4-oxobutylamine) pentanoic acid
Tert-butyl (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (1, 1-thiomorpholine) -4-oxobutylamine) pentanoate (75mg, 0.114mmol) was added to dichloromethane (2mL), trifluoroacetic acid (1mL) was added, and the mixture was stirred at room temperature for 2 hours. Concentration, thin layer chromatography purification of the residue to obtain (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamide) -4- (1, 1-thiomorpholine dioxide) -4-oxobutylamine) pentanoic acid (45mg, 66% yield) as a white solid.
1HNMR(400MHz,DMSO-d6,ppm)δ11.97(s,1H),9.86(s,1H),8.57(d,J＝8.0Hz,1H),7.98-7.91(m,2H),7.64(d,J＝8.4Hz,1H),6.80(d,J＝16.0Hz,1H),6.75(d,J＝16.0Hz,1H),4.65(q,J＝7.2Hz,1H),3.86-3.83(m,4H),3.28-3.23(m,2H),3.06-2.99(m,4H),2.83-2.71(m,2H),2.20(t,J＝7.2Hz,2H),1.49-1.37(m,4H).
MS m/z(ESI):600[M+H]+。
Preparation of intermediate (S) -2- ((((9H-fluoren-9-ylmethoxy) carbonyl) amino) -4- (1, 1-thiomorpholine) -4-oxobutanoic acid (25-3)
Figure BDA0002589780190000491
The first step is as follows: preparation of tert-butyl (S) -2- ((((9H-fluoren-9-ylmethoxy) carbonyl) amino) -4- (1, 1-thiomorpholine) -4-oxobutyrate
N- [ (9H-fluoren-9-ylmethoxy) carbonyl ] -L-aspartic acid 1-tert-butyl ester (4.1g, 10mmol), thiomorpholine-1, 1-dioxide (1.5g, 11mmol), HATU (4.56g, 12mmol) and diisopropylethylamine (5mL) were added to dichloromethane (50mL) and stirred at room temperature for 2 hours. Water was added and extraction was carried out with methylene chloride, and the organic phase was collected and washed, dried, concentrated, and purified by column chromatography to give tert-butyl (S) -2- (((((9H-fluoren-9-ylmethoxy) carbonyl) amino) -4- (1, 1-thiomorpholine dioxide) -4-oxobutyrate (5.0g, yield 95%).
The second step is that: preparation of (S) -2- ((((9H-fluoren-9-ylmethoxy) carbonyl) amino) -4- (1, 1-thiomorpholine-dioxide) -4-oxobutanoic acid
Tert-butyl (S) -2- ((((9H-fluoren-9-ylmethoxy) carbonyl) amino) -4- (1, 1-thiomorpholine dioxide) -4-oxobutanoate (5.0g, 9.45mmol) was added to methylene chloride (50mL), trifluoroacetic acid (20mL) was added, and the mixture was stirred at room temperature for 2 hours, concentrated, and purified by column chromatography to give (S) -2- ((((9H-fluoren-9-ylmethoxy) carbonyl) amino) -4- (1, 1-thiomorpholine dioxide) -4-oxobutanoic acid (4.2g, 94% yield).
Example 26: (E) preparation of (E) -5- ((S) -2- ((E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (1, 1-thiomorpholine) -4-oxobutylamine) 2-pentenoic acid
Figure BDA0002589780190000501
The first step is as follows: preparation of tert-butyl (S) -2-amino-4- (1, 1-thiomorpholine-dioxide) -4-oxobutyrate
Tert-butyl (S) -2- ((((9H-fluoren-9-ylmethoxy) carbonyl) amino) -4- (1, 1-thiomorpholine) -4-oxobutyrate (529mg, 1mmol) was added to methylene chloride (5mL), diethylamine (3mL) was added, and the mixture was stirred at room temperature for 2 hours, concentrated, and purified by column chromatography to give tert-butyl (S) -2-amino-4- (1, 1-thiomorpholine) -4-oxobutyrate (214mg, 70% yield).
The second step is that: preparation of (S, E) - (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (1, 1-thiomorpholine) -4-oxobutanoic acid tert-butyl ester
Tert-butyl (S) -2-amino-4- (1, 1-thiomorpholine dioxide) -4-oxobutyrate (153mg, 0.5mmol), (E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylic acid (134mg, 0.5mmol), HATU (209mg, 0.55mmol) and diisopropylethylamine (0.25mL) were added to dichloromethane (5mL) and the reaction 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 (S, E) - (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamide) -4- (1, 1-sulfur dioxide morpholine) -4-oxobutyrate (220mg, 79% yield).
The third step: preparation of (S, E) - (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamide) -4- (1, 1-thiomorpholine dioxide) -4-oxobutanoic acid
Tert-butyl (S, E) - (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (1, 1-thiomorpholine dioxide) -4-oxobutanoate (220mg, 0.39mmol) was added to dichloromethane (5mL), trifluoroacetic acid (2mL) was added thereto, and the mixture was stirred at room temperature for 2 hours, concentrated, and purified by column chromatography to give (S, E) - (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (1, 1-thiomorpholine-dioxide) -4-oxobutanoic acid (182mg, yield 92%).
The fourth step: (E) preparation of tert-butyl (E) -5- ((S) -2- ((E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (1, 1-thiomorpholine) 4-oxobutylamine) 2-pentenoate
Adding (S, E) - (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamide) -4- (1, 1-sulfur dioxide morpholine) -4-oxobutyric acid (182mg, 0.36mmol), (E) -5-amino-2-pentenoic acid tert-butyl ester (93mg, 0.55mmol), HATU (152mg, 0.4mmol) and diisopropylethylamine (0.18mL) into dichloromethane (5mL), stirring at room temperature for reaction for 2 hours, adding water, extracting with dichloromethane, collecting organic phase, washing, drying, concentrating, and purifying by column chromatography to obtain (E) -5- ((S) -2- ((E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acryloyl Amino) -4- (1, 1-thiomorpholine-dioxide) -4-oxobutylamine) tert-butyl 2-pentenoate (107mg, 45% yield).
The fifth step: (E) preparation of (E) -5- ((S) -2- ((E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (1, 1-thiomorpholine) -4-oxobutylamine) 2-pentenoic acid
To dichloromethane (4mL) was added (E) -5- ((S) -2- ((E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (1, 1-thiomorpholine-dioxide) -4-oxobutylamine) tert-butyl 2-pentenoate (107mg, 0.16mmol), and trifluoroacetic acid (2mL) was added and stirred at room temperature for 3 hours. Concentrating, purifying the residue by reversed-phase preparative column chromatography to obtain (E) -5- ((S) -2- ((E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamide) -4- (1, 1-sulfur dioxide morpholine) -4-oxobutylamine) 2-pentenoic acid (55mg, yield of 56%) and a white solid.
MS m/z(ESI):598[M+H]+。
Preparation of intermediate (E) -5-amino-2-pentenoic acid tert-butyl ester (26-4)
Figure BDA0002589780190000511
The first step is as follows: preparation of 3- (FMOC-amino) -1-propanal
3- (FMOC-amino) -1-propanol (1.025g, 3.45mmol) was dissolved in ethyl acetate (25mL) and IBX (5.78g, 10.4mmol) was added. Heated to 80 ℃ and stirred vigorously for 4 hours. Cooled to room temperature, filtered and concentrated to give crude 3- (FMOC-amino) -1-propanal (ca. 1.26g) which was used directly in the next reaction.
The second step is that: (E) preparation of tert-butyl (E) -5- ((((9H-fluoren-9-ylmethoxy) carbonyl) amino) 2-pentenoate
Crude 3- (FMOC-amino) -1-propanal (ca. 1.26g, ca. 3.45mmol) was dissolved in dichloromethane (20mL), and (tert-butoxycarbonylmethylene) triphenylphosphine (1.56g, 4.1mmol) was added and stirred at room temperature overnight. Concentration and column chromatography purification of the residue gave tert-butyl (E) -5- ((((9H-fluoren-9-ylmethoxy) carbonyl) amino) 2-pentenoate (1.18g, 87% yield).
The third step: (E) preparation of tert-butyl (5-amino-2-pentenoate)
(E) -tert-butyl 5- ((((9H-fluoren-9-ylmethoxy) carbonyl) amino) 2-pentenoate (787mg, 2mmol) was dissolved in dichloromethane (10mL), tetrabutylammonium fluoride trihydrate (1.89g, 6mmol) was added, stirring was carried out at room temperature for 5 hours, concentration was carried out, and the residue was purified by column chromatography to give tert-butyl (E) -5-amino-2-pentenoate (212mg, yield 62%).
Example 27: (E) preparation of (E) -4- ((S) -2- ((E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamide) -4- (1, 1-thiomorpholine) -4-oxobutylamine) 2-butenoic acid
Figure BDA0002589780190000521
The experimental procedures and reaction conditions were the same as in example 26.
MS m/z(ESI):584[M+1]+。
Example 28: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (methyl (2- (methylsulfonyl) ethyl) amino) -4-oxobutanamido) benzoic acid
Figure BDA0002589780190000522
The experimental procedures and reaction conditions were the same as in example 1.
1HNMR(400MHz,DMSO-d6,ppm)δ12.63(brs,1H),10.40(s,1H),9.88(s,1H),8.85(d,J＝8.0Hz,1H),7.93(t,J＝8.4Hz,2H),7.88(d,J＝8.8Hz,2H),7.73(d,J＝9.2Hz,1H),7.64(dd,J＝1.2,8.6Hz,1H),6.82(d,J＝16Hz,1H),6.77(d,J＝16.4Hz,1H),4.85-4.80(m,1H),3.78-3.74(m,1H),3.65-3.61(m,1H),3.56-3.47(m,2H),2.94(s,3H),2.86-2.83(m,1H),2.80(s,3H),2.76-2.70(m,1H).
MS m/z(ESI):622[M+1]+。
Example 29: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -5- (1, 1-thiomorpholine) -5-oxopentanamido) benzoic acid
Figure BDA0002589780190000523
The experimental procedures and reaction conditions were the same as in example 7.
1HNMR(400MHz,DMSO-d6,ppm)δ12.58(s,1H),10.43(s,1H),9.87(s,1H),8.80(d,J＝7.2Hz,1H),7.96(t,J＝8.4Hz,1H),7.91(d,J＝8.8Hz,2H),7.72(d,J＝8.8Hz,2H),7.65(dd,J＝1.2,8.4Hz,1H),6.84(d,J＝16Hz,1H),6.80(d,J＝16Hz,1H),4.87(q,J＝6.8Hz,1H),3.88-3.84(m,4H),3.10-3.03(m,4H),2.42-2.37(m,2H),2.03-2.00(m,1H),1.94-1.84(m,1H).
MSm/z(ESI):634[M+H]+。
Example 30: (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -5- (4-methylpiperazin-1-yl) -5-oxopentanamido) -1H-indole-2-carboxylic acid
Figure BDA0002589780190000531
The experimental procedures and reaction conditions were the same as in example 10.
1HNMR(400MHz,DMSO-d6,ppm)δ11.70(brs,1H),10.03(s,1H),9.86(s,1H),8.76(d,J＝7.2Hz,1H),8.02(s,1H),7.95(t,J＝8.0Hz,1H),7.66(dd,J＝1.0,8.6Hz,1H),7.37(s,2H),7.04(d,J＝2.0Hz,1H),6.85-6.77(m,2H),4.89-4.83(m,1H),3.55-3.49(m,8H),2.46-.35(m,3H),2.25-2.16(m,2H),2.14-2.06(m,1H),2.00-1.85(m,1H).
MS m/z(ESI):638[M+H]+。
Example 31: (S, E) -5- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -5- (4-dimethylaminopiperidin-1-yl) -5-oxopentanamido) -1H-indole-2-carboxylic acid
Figure BDA0002589780190000532
The experimental procedures and reaction conditions were the same as in example 10.
1HNMR(400MHz,DMSO-d6,ppm)δ11.72(brs,1H),10.13(s,1H),9.86(s,1H),8.76(d,J＝7.2Hz,1H),8.02(s,1H),7.95(t,J＝8.0Hz,1H),7.66(dd,J＝1.0,8.6Hz,1H),7.37(s,2H),7.04(d,J＝2.0Hz,1H),6.85-6.77(m,2H),4.89-4.83(m,1H),4.04-3.99(m,1H),3.01-2.74(m,3H),2.69(s,6H),2.01-1.97(m,3H),2.25-2.16(m,2H),2.14-2.06(m,1H),2.00-1.85(m,1H),1.58-1.53(m,1H),1.35-1.31(m,1H).
MS m/z(ESI):666[M+H]+。
Example 32: preparation of 5- ((S) -3- (3- (N-methylacetamido) phenyl) -2- ((1r,4S) -4- (tetrahydropyrrole-1-ylmethyl) cyclohexane-1-carboxamido) propanamido) -1H-benzo [ d ] imidazole-2-carboxylic acid
Figure BDA0002589780190000541
The experimental procedures and reaction conditions were the same as in example 16.
1HNMR(400MHz,DMSO-d6,ppm)δ11.57(s,1H),9.96(s,1H),8.15(d,J＝8.0Hz,1H),7.98(s,1H),7.62-7.53(m,2H),7.29-7.27(m,2H),7.25(s,1H),7.17(d,J＝8.0Hz,1H),7.11(s,1H),4.83-4.71(m,1H),3.81-3.71(m,2H),3.18-3.13(m,4H),3.10(s,3H),2.55-2.47(m,4H),2.15(t,J＝10.8Hz,1H),1.73-1.51(m,8H),1.33-1.18(m,3H),0.93-0.89(m,2H).
MS m/z(ESI):589[M+H]+
Example 33: preparation of 5- ((S) -2- ((1r,4S) -4- (((butoxycarbonyl) amino) methyl) cyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-2-carboxylic acid-2-ethoxy-2-oxoethyl ester
Figure BDA0002589780190000542
The first step is as follows: preparation of (1r,4r) -4- (((butoxycarbonyl) amino) methyl) cyclohexane-1-carboxylic acid
Tranexamic acid (1.26g,8mmol) was dissolved in 8% aqueous NaOH (4ml,8 mmol). Under ice-bath, butyl chloroformate (1.20g,8.8mmol) was added dropwise. Sodium carbonate (466mg, 4.4mmol) was slowly added and stirred at room temperature for 5 hours. 1N diluted hydrochloric acid (8mL) was added, and the resulting solid was collected by filtration to give (1r,4r) -4- (((butoxycarbonyl) amino) methyl) cyclohexane-1-carboxylic acid (1.67g, yield 81%).
The second step is that: preparation of tert-butyl 5- ((S) -2- ((1r,4S) -4- (((butoxycarbonyl) amino) methyl) cyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-2-carboxylate
(S) -tert-butyl 5- (2-amino-3- (3- (N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-2-carboxylate (70mg, 0.155mmol), (1r,4r) -4- (((butoxycarbonyl) amino) methyl) cyclohexane-1-carboxylate (41mg, 0.16mmol), HATU (80mg, 0.21mmol), DIPEA (0.1mL) was added to dichloromethane (3mL), stirred at room temperature for 2 hours, washed once with water, the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give 5- ((S) -2- ((1r,4S) -4-cyanocyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-benzo [d] Imidazole-2-carboxylic acid tert-butyl ester (70mg, 65% yield).
The third step: preparation of 5- ((S) -2- ((1r,4S) -4- (((butoxycarbonyl) amino) methyl) cyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-2-carboxylic acid
Tert-butyl 5- ((S) -2- ((1r,4S) -4- (((butoxycarbonyl) amino) methyl) cyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propanamido) -1H-benzo [ d ] imidazole-2-carboxylate (70mg, 0.101mmol) was dissolved in dichloromethane (3mL), trifluoroacetic acid (2mL) was added, stirring at room temperature for 2 hours, concentration and purification by reverse phase column chromatography to give 5- ((S) -2- ((1r,4S) -4- (((butoxycarbonyl) amino) methyl) cyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propanamido) -1H-benzo [ d ] imidazole-2-carboxylic acid (50mg, yield 78%), white solid.
The fourth step: preparation of 5- ((S) -2- ((1r,4S) -4- (((butoxycarbonyl) amino) methyl) cyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-2-carboxylic acid-2-ethoxy-2-oxoethyl ester
5- ((S) -2- ((1r,4S) -4- (((butoxycarbonyl) amino) methyl) cyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propanamido) -1H-benzo [ d ] imidazole-2-carboxylic acid (50mg, 0.0787mmol) was dissolved in DMF (2mL), ethyl chloroacetate (14mg,0.11mmol), potassium carbonate (44mg, 0.32mmol) were added and stirred at 40 ℃ for 4H. The reaction solution was poured into ice water, and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, ethyl acetate was evaporated under reduced pressure, and the residue was purified by column chromatography to give 2-ethoxy-2-oxoethyl 5- ((S) -2- ((1r,4S) -4- (((butoxycarbonyl) amino) methyl) cyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-2-carboxylate (48mg, yield 85%).
1HNMR(400MHz,DMSO-d6,ppm)δ9.96(s,1H),8.15(d,J＝8.0Hz,1H),7.98(s,1H),7.62-7.53(m,2H),7.29-7.27(m,2H),7.25(s,1H),7.17(d,J＝8.0Hz,1H),7.11(s,1H),5.21(s,2H),4.83-4.71(m,1H),4.21(q,J＝7.2Hz,2H),3.90(t,J＝7.2Hz,2H),3.81-3.71(m,2H),3.10(s,3H),2.88-2.82(m,2H),2.45-2.27(m,5H),1.73-1.51(m,8H),1.38-1.28(m,4H),1.23(t,J＝7.2Hz,3H),0.93(t,J＝7.2Hz,3H).
MS m/z(ESI):721[M+H]+。
Example 34: preparation of 1-isopropoxy-1- ((isopropoxycarbonyl) oxo) ethyl 5- ((S) -3- (3- (N-methylacetamido) phenyl) -2- ((1r,4S) -4- (piperidin-1-ylmethyl) cyclohexane-1-carboxamido) propanamido) -1H-benzo [ d ] imidazole-2-carboxylate
Figure BDA0002589780190000551
The experimental procedures and reaction conditions were the same as in example 33.
1HNMR(400MHz,DMSO-d6,ppm)δ11.55(s,1H),9.96(s,1H),8.15(d,J＝8.0Hz,1H),7.96-7.92(m,2H),7.62-7.53(m,2H),7.29-7.27(m,2H),7.25(s,1H),7.17(d,J＝8.0Hz,1H),7.11(s,1H),4.83-4.71(m,2H),3.81-3.71(m,2H),3.10(s,3H),2.55-2.47(m,5H),2.15-2.08(m,5H),1.73-1.61(m,8H),1.57(d,J＝5.4Hz,3H),1.33-1.25(m,6H),1.23(d,J＝6.4Hz,6H).
MS m/z(ESI):717[M+H]+
Example 35: preparation of methyl 5- ((S) -3- (3- (N-methylacetamido) phenyl) -2- ((1r,4S) -4- (morpholin-1-ylmethyl) cyclohexane-1-carboxamido) propionamido) -1H-benzo [ d ] imidazole-2-carboxylate
Figure BDA0002589780190000561
The first step is as follows: preparation of methyl (S) -5- (2- ((tert-butoxycarbonyl) amino) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-2-carboxylate
(S) -2- ((tert-butoxycarbonyl) amino) -3- (3- (N-methylacetamido) phenyl) propionic acid (101mg, 0.30mmol), methyl 5-amino-1H-benzo [ d ] imidazole-2-carboxylate (57mg, 0.30mmol), HATU (126mg, 0.33mmol), DIPEA (0.16mL) was added to dichloromethane (3mL), stirred at room temperature for 2 hours, the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give methyl (S) -5- (2- ((tert-butoxycarbonyl) amino) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-2-carboxylate (127mg, yield 83%).
The second step is that: preparation of (S) -5- (2-amino-3- (3- (N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-2-carboxylic acid methyl ester hydrochloride
(S) -methyl 5- (2- ((tert-butoxycarbonyl) amino) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-2-carboxylate (127mg, 0.25mmol) was dissolved in tetrahydrofuran (3mL), an HCl ethyl acetate solution (3mol/L, 5mL) was added, stirring was performed at room temperature for 5 hours, and the reaction was concentrated to give (S) -methyl 5- (2-amino-3- (3- (N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-2-carboxylate hydrochloride (110mg, 99% yield).
The third step: preparation of methyl 5- ((S) -3- (3- (N-methylacetamido) phenyl) -2- ((1r,4S) -4- (morpholin-1-ylmethyl) cyclohexane-1-carboxamido) propionamido) -1H-benzo [ d ] imidazole-2-carboxylate
(S) -methyl 5- (2-amino-3- (3- (N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-2-carboxylate hydrochloride (110mg, 0.25mmol), (1r,4r) -4- (morpholinomethyl) cyclohexane-1-carboxylate (57mg, 0.25mmol), HATU (105mg, 0.28mmol), DIPEA (0.15mL) were added to dichloromethane (3mL), stirred at room temperature for 2 hours, washed once with water, the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give 5- ((S) -3- (3- (N-methylacetamido) phenyl) -2- ((1r,4S) -4- (morpholin-1-ylmethyl) cyclohexane-1-carboxamido) propionamido) - Methyl 1H-benzo [ d ] imidazole-2-carboxylate (88mg, 57% yield).
1HNMR(400MHz,DMSO-d6,ppm)δ9.86(s,1H),8.25(d,J＝8.0Hz,1H),7.98(s,1H),7.62-7.53(m,2H),7.29-7.27(m,2H),7.25(s,1H),7.17(d,J＝8.0Hz,1H),7.11(s,1H),4.83-4.71(m,1H),4.10(s,3H),3.81-3.71(m,2H),3.48-3.43(m,4H),3.12(s,3H),2.55-2.47(m,4H),2.39-2.28(m,8H),1.73-1.51(m,7H).
MS m/z(ESI):619[M+H]+
Example 36: preparation of (S) -5- (3- (3- (N-methylacetamido) phenyl) -2- (4- (morpholinomethyl) benzamido) propionamido) -1H-indole-2-carboxylic acid
Figure BDA0002589780190000571
The experimental procedures and reaction conditions were the same as in example 19.
1HNMR(400MHz,DMSO-d6,ppm)δ11.55(s,1H),9.95(s,1H),8.85(s,1H),8.14(s,1H),7.94d,J＝7.6Hz,2H),7.45-7.39(m,4H),7.29-7.14(m,4H),7.00-6.92(m,2H),4.73-4.71(m,1H),3.60(s,2H),3.55-3.50(m,4H),3.10(s,3H),2.66-2.61(m,2H),2.39-2.29(m,4H),2.01-1.93(m,3H).
MS m/z(ESI):598[M+H]+
Example 37: preparation of (S) -5- (2- (2-chloro-4- (morpholinomethyl) benzamido) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-indole-2-carboxylic acid
Figure BDA0002589780190000572
The experimental procedures and reaction conditions were the same as in example 19.
1HNMR(400MHz,DMSO-d6,ppm)δ11.55(s,1H),9.95(s,1H),8.95(s,1H),8.14(d,J＝7.6Hz,1H),7.94-7.92(m,2H),7.45-7.39(m,4H),7.29-7.14(m,4H),6.92(s,1H),4.73-4.71(m,1H),3.60(s,2H),3.55-3.50(m,4H),3.10(s,3H),2.66-2.61(m,2H),2.39-2.29(m,4H),2.01-1.93(m,3H).
MS m/z(ESI):632[M+H]+
Example 38: preparation of 5- ((S) -2- ((1r,4S) -4- (N, N-dimethylcarbamimidoyl) cyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-benzo [ d ] imidazole-2-carboxylic acid
Figure BDA0002589780190000581
The experimental procedures and reaction conditions were the same as in example 16.
1HNMR(400MHz,DMSO-d6,ppm)δ11.25(s,1H),9.96(s,1H),9.62(s,1H),8.15(d,J＝8.0Hz,1H),7.98(s,1H),7.62-7.53(m,2H),7.29-7.27(m,2H),7.25(s,1H),7.17(d,J＝8.0Hz,1H),7.11(s,1H),4.83-4.71(m,1H),3.81-3.71(m,2H),3.10(s,3H),3.00(s,6H),2.36-2.26(m,2H),2.15(s,3H),1.73-1.51(m,4H),1.33-1.18(m,4H).
MS m/z(ESI):576[M+H]+
Example 39: preparation of (1S,4r) -4- ((S) -2- ((E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamide) -4- (1, 1-thiomorpholine dioxide) -4-oxobutanamide) cyclohexane-1-carboxylic acid
Figure BDA0002589780190000582
The experimental procedures and reaction conditions were the same as in example 26.
1HNMR(400MHz,DMSO-d6,ppm)δ10.57(s,1H),9.86(s,1H),8.57(d,J＝8.0Hz,1H),7.98-7.91(m,2H),7.64(d,J＝8.4Hz,1H),6.80(d,J＝16.0Hz,1H),6.75(d,J＝16.0Hz,1H),4.75(q,J＝7.2Hz,1H),3.86-3.83(m,4H),3.28-3.23(m,2H),3.10-3.06(m,2H),2.83-2.71(m,2H),2.31-2.10(m,2H),1.75-1.68(m,2H),1.49-1.37(m,6H).
MS m/z(ESI):626[M+H]+。
Example 40: preparation of (1S,4r) -4- (((S) -2- ((E) -3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -4- (1, 1-thiomorpholine dioxide) -4-oxobutanamide) methyl) cyclohexane-1-carboxylic acid
Figure BDA0002589780190000591
The experimental procedures and reaction conditions were the same as in example 26. 1HNMR (400MHz, DMSO-d6, ppm) δ 10.67(s,1H),9.86(s,1H),8.57(d, J ═ 8.0Hz,1H),7.98-7.91(m,2H),7.64(d, J ═ 8.4Hz,1H),6.80(d, J ═ 16.0Hz,1H),6.75(d, J ═ 16.0Hz,1H),4.65(q, J ═ 7.2Hz,1H),3.86-3.83(m,4H),3.28-3.23(m,2H),3.15-3.10(m,4H),2.83-2.71(m,2H),2.31-2.10(m,2H),1.75-1.68(m,2H), 1.49-1.6H).
MS m/z(ESI):640[M+H]+。
Example 41: preparation of 5- ((S) -2- ((1r,4S) -4- ((dimethylamino) methyl) cyclohexane-1-carboxamido) -3- (3- (N-methylacetamido) phenyl) propionamido) -1H-indole-2-carboxylic acid
Figure BDA0002589780190000592
The experimental procedures and reaction conditions were the same as in example 23.
1HNMR(400MHz,DMSO-d6,ppm)δ12.61(brs,1H),11.69(s,1H),9.99(s,1H),8.09(d,J＝8.4Hz,1H),7.99(s,1H),7.65(s,2H),7.38-7.34(m,2H),7.32-7.27(m,2H),7.23(s,1H),7.15(d,J＝8.0Hz,1H),7.04(d,J＝1.2Hz,1H),4.83-4.71(m,1H),3.81-3.71(m,2H),3.18-3.13(m,4H),3.10(s,3H),2.75(s,6H),1.73-1.51(m,4H),1.33-1.18(m,3H),0.93-0.89(m,2H).
MS m/z(ESI):562[M+H]+。
Example 42: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -5-morpholine-5-oxopentanamido) -2-naphthoic acid
Figure BDA0002589780190000593
The experimental procedures and reaction conditions were the same as in example 7.
1HNMR(400MHz,DMSO-d6,ppm)δ12.68(s,1H),10.49(s,1H),9.88(s,1H),8.80(d,J＝7.2Hz,1H),8.25(s,1H),8.02(s,1H),7.96(t,J＝8.4Hz,1H),7.66(dd,J＝1.2,8.4Hz,1H),7.44(t,J＝7.2Hz,2H),7.36(t,J＝7.2Hz,2H),6.84(d,J＝18Hz,1H),6.80(d,J＝18Hz,1H),4.87(q,J＝7.1Hz,1H),3.55-3.52(m,4H),3.43-3.40(m,4H),2.42-2.37(m,2H),2.03-2.00(m,1H),1.94-1.84(m,1H).
MS m/z(ESI):636[M+H]+。
Example 43: preparation of (S, E) -4- (2- (3- (3-chloro-2-fluoro-6- (1H-tetrazol-1-yl) phenyl) acrylamido) -5-morpholine-5-oxopentanamido) -1H-indene-2-carboxylic acid
Figure BDA0002589780190000601
The experimental procedures and reaction conditions were the same as in example 7.
1HNMR(400MHz,DMSO-d6,ppm)δ11.44(s,1H),10.49(s,1H),9.88(s,1H),8.83(d,J＝7.2Hz,1H),8.11(d,J＝7.6Hz,1H),7.96(t,J＝8.4Hz,1H),7.66(dd,J＝1.2,8.4Hz,1H),7.56(s,1H),7.40(d,J＝7.6Hz,1H),6.84(d,J＝18Hz,1H),6.80(d,J＝18Hz,1H),4.87(q,J＝7.1Hz,1H),3.59(s,2H).3.55-3.52(m,4H),3.43-3.40(m,4H),2.42-2.37(m,2H),2.03-.00(m,1H),1.94-1.84(m,1H).
MS m/z(ESI):624[M+H]+。
Biological evaluation
In vitro experimental example 1: determination of the biological Activity of the Compounds of the invention as inhibitors of coagulation factor X1a
Reagents and materials:
50mM HEPES；pH7.4；145mM NaCl；5mM KCl；0.01％Tween20
factor XIa protease 1. mu.L of factor XIa stock solution (5.1mg/mL) was diluted to 100. mu.g/mL with the reaction solution and dispensed into 2. mu.L bottles and stored at-80 ℃. Factor XIa-specific substrate (S-2366, pyroGlu-Pro-Arg-pNA. HCl,): the mixture was made up to 10mM in DMSO, and 66. mu.L of each tube was dispensed, and stored at-20 ℃.
The test process comprises the following steps:
test compounds dissolved in 100% DMSO were formulated at 10mM in 100% DMSO, diluted 100. mu.M in DMSO and serially diluted three-fold down to 14 concentrations (all 100X) at final system concentrations of 1.0. mu.M, 0.33. mu.M, 0.11. mu.M, 0.037. mu.M, 0.012. mu.M, 0.0041. mu.M, 0.0014. mu.M, 0.00046. mu.M, 0.00015. mu.M, 0.000051. mu.M. Add 0.2. mu.l of compound per well in 384-well plates, replace the blank with DMSO, centrifuge at 1500rpm for 1min, bring the compound to the bottom and all samples for duplicate wells.
Mu.l of the XIa enzyme solution prepared at 0.1nM using the reaction solution was added to each well, 10. mu.l of buffer was added to the blank wells instead, centrifuged, the enzyme solution was removed to the bottom, and incubated at 37 ℃ for 15 minutes.
Mu.l of 660. mu.M substrate prepared from the reaction mixture was added to each well, centrifuged, the substrate solution was drained to the bottom, and the OD 405 was measured every 3min by kinetic method for 1 h.
Data processing:
the software Gen5 outputs maxV for each kinetic curve, expressed as Mean + -SD.
Calculating the drug inhibition rate
Figure BDA0002589780190000611
Wherein max group is positive control, DMSO is used to replace drug; min group was negative control, DMSO was used instead of drug, and the reaction solution was used instead of XIa enzyme solution. Four-parameter curve fitting was performed on inhibition and log concentration values using Graphpad Prism, with the minimum value being 0 and the maximum value being 100, and IC50 was calculated.
In vitro experimental example 2: determination of anticoagulant activity in human whole blood liquid
Reagents and materials:
plasma: blood from 4 healthy women was collected in blood collection tubes containing 3.8% sodium citrate anticoagulant. Centrifuging at 3000rpm at room temperature for 10min, collecting plasma, mixing with 10 human plasma, and storing at 4 deg.C for 2 hr. Activated Partial Thromboplastin Time (APTT) assay kit (coagulation method) includes APTT reagent and calcium oxide solution. LG-PAPER-I type platelet aggregation coagulation factor analyzer, centrifuge, and vortex mixer.
The test process comprises the following steps:
mixing the blood plasma. Test compounds were diluted in 10mM in 100% DMSO in plasma to 1000, 100, 30, 10, 5, 2.5, 1, 0.3, 0.1 μ M. The blank control group was diluted with plasma in 100% DMSO at the corresponding ratio. All samples were tested in duplicate wells.
APTT reagent, CaCl2 reagent, plasma (containing test substance/DMSO) were placed in the appropriate locations in the coagulometer. Wherein the APTT reagent and the blood plasma are pre-warmed for 3min, the CaCl2 is pre-warmed for 15min (the temperature reaches 37 +/-1 ℃) so as to be fully dissolved, and the mixture is shaken and uniformly mixed before use.
When in test, firstly adding magnetic beads, taking 50 mu l of blood plasma to be tested and adding into a test cup, then adding 50 mu l of the prewarmed APTT reagent, placing in a prewarming channel at 37 ℃ for 5min, then adding 250 mu l of prewarmed CaCl, immediately automatically testing and recording the result in unit second(s)
Data processing:
the ratio of APTT to DMSO control was calculated for each compound, expressed as Mean + -SD, and linear fit to the corresponding concentration using Excel to calculate EC1.5×I.e. the concentration of compound corresponding to 1.5 times the APTT of the blank.
TABLE 2 inhibitory Activity and anti-blood clotting Activity of the Compounds of the examples on FXIad
Figure BDA0002589780190000612
Figure BDA0002589780190000621
Figure BDA0002589780190000631
Table 2 shows the inhibitory activity of the example compounds against the coagulation factor FXIa and the hysteresis effect on human blood coagulation.
In vivo experimental example 3: rat arteriovenous thrombus experiment
Reagents and materials: intravenous infusion needle, Polyethylene tube (i.d. ═ 0.58mm), No. 3 surgical silk thread, CMC-NA, PEG 300, DMAC, 5% glucose solution, microinjection pump, ultrasonic cleaner, cell disruption instrument, electronic balance
Test animals: feeding the seeds in an SPF environment at the temperature of 20-26 ℃, the relative humidity of 30-70% and the light dark illumination for 12:12 h; free drinking water and food intake. Wistar rat (SPF grade, male, 240-
Test procedure
Purchasing test animals, and performing adaptive breeding for 5 days; dividing the animal body weight into 7 groups, and dividing each group into 8 animals; animals were fasted and not water-fed for 16 hours before the start of the experiment; three-section polyethylene pipe specification: the length of the two end pipes is 3.5 cm. The middle section tube is a 0.8 venous transfusion needle hose, and the length is 12.5 cm. Three sections of tube-in-tube wire, No. 3 surgical wire.
After fasting is finished, all animals in a gavage administration group (PO solvent control, rivaroxaban and example compound) are subjected to intraperitoneal injection of 1% sodium pentobarbital (30mg/kg and 3mL/kg) for anesthesia after administration, rats are subjected to cervical incision by using a sterile surgical instrument, the trachea is separated, the right common carotid artery and the left external jugular vein are separated, 3 surgical silk threads are cut and placed in a three-section polyethylene tube, the length of the silk thread contacting blood is made to be a fixed length, the polyethylene tube is filled with physiological saline, and a carotid artery bypass circuit and a venous bypass circuit clamped by artery clamps are established. After a period of administration (example compound), the arterial clamp was opened. All animals are penetrated for 15min, then the silk thread is taken out quickly, and the wet weight of the thrombus (the wet weight of the thrombus is the total wet weight of the silk thread-net wet weight of the silk thread) is weighed and calculated; after drying in an oven at 60 ℃ for 24h, the dried thrombus was weighed and the dry thrombus weight was calculated (dry thrombus weight ═ total dry silk thread weight-dry net silk thread weight).
The group was administered intravenously (iv bolus + iv infusion), 1% sodium pentobarbital (30mg/kg) was intraperitoneally injected for anesthesia, and the rats were placed in the supine position, and the iv infusion administration was started immediately after the iv bolus administration. The sterilized surgical instrument for the anesthetized animals cuts the neck, separates the trachea, separates the right common carotid artery and the left external jugular vein, cuts the No. 3 surgical silk thread, places the surgical silk thread in a three-section polyethylene tube, enables the length of the silk thread contacting blood to be 6cm, fills the polyethylene tube with normal saline, and establishes a carotid artery and vein bypass loop clamped and closed by an artery clamp. After Iv infusion is administered for a period of time, opening an artery clamp, timing for 15min (continuously administering the drug during the period), quickly taking out the surgical silk thread, weighing, and calculating the wet weight of the thrombus (the wet weight of the thrombus is the total wet weight of the silk thread-net weight of the silk thread); after oven at 60 ℃ for 24h, the weight was measured, and the dry weight of the thrombus was calculated (dry weight of thrombus: total dry weight of silk thread-net weight of silk thread).
The wet weight (g), dry weight (g), wet weight inhibition (%) and dry weight inhibition (%) of the thrombus were calculated for each group, respectively.
Data processing: experimental data were calculated and statistically processed using Microsoft Office Excel 2010 software. Data are expressed as Mean ± standard error (Mean ± s.e) unless otherwise stated, and comparisons between groups were analyzed using t-test. A P value less than 0.05 considered the difference to be statistically significant.
And (4) test conclusion: the compound of the invention has obvious inhibition effect on thrombosis in animal bodies.
Animal pharmacodynamic experiment results show that the compound has obvious in-vivo antithrombotic effect.

Claims (7)

1.一种如式I所示的化合物或其药学上可接受的盐、立体异构体、互变异构体、多晶型物、溶剂合物、N-氧化物、代谢物或前药：1. a compound as shown in formula I or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, metabolite or prodrug thereof :
Figure FDA0002589780060000011
Figure FDA0002589780060000011
 其中in A选自苯基和环己基；A is selected from phenyl and cyclohexyl; R1各自独立地选自卤素、C1-6烷基、四唑基和三唑基，所述C1-6烷基、四唑基或三唑基各自任选地被一或多个选自氨基和卤素的取代基取代；R 1 are each independently selected from halogen, C 1-6 alkyl , tetrazolyl, and triazolyl, each of which is optionally selected by one or more Substituted from amino and halogen substituents; n为0、1、2或3；n is 0, 1, 2 or 3; R2和R3各自独立地选自H和C1-6烷基；R 2 and R 3 are each independently selected from H and C 1-6 alkyl; m为0或1；m is 0 or 1; R4为H；R 4 is H; R6选自H和C1-6烷基；R 6 is selected from H and C 1-6 alkyl; R7为H或任选地被一或多个R10取代的苯基、苄基、萘基，茚基、吲哚、苯并咪唑、环己基； R7 is H or phenyl, benzyl, naphthyl, indenyl, indole, benzimidazole, cyclohexyl optionally substituted with one or more R10; R10各自独立地选自-(CH2)r-C(O)s-R11；Each R 10 is independently selected from -(CH 2 )rC(O)sR 11 ; r为0或1；r is 0 or 1; s为1或2；s is 1 or 2; R11选自H、-CH2R12和任选地被R13取代的C1-6氧代烷基；R 11 is selected from H, -CH 2 R 12 and C 1-6 oxoalkyl optionally substituted by R 13 ; R12选自C1-6烷基、C3-6环烷基和苯基；R 12 is selected from C 1-6 alkyl, C 3-6 cycloalkyl and phenyl; R13选自-OCOOR14和
Figure FDA0002589780060000012
R 13 is selected from -OCOOR 14 and
Figure FDA0002589780060000012
 R14选自C1-6烷基和C3-6环烷基；R 14 is selected from C 1-6 alkyl and C 3-6 cycloalkyl; R5为任选地被一或多个R8取代的苯基或Cp烷基；R5 is phenyl or Cp alkyl optionally substituted with one or more R8 ; p为0、1、2、3、4、5或6；p is 0, 1, 2, 3, 4, 5, or 6; R8为在苯基取代位置为邻位或间位或对位的-NR15R16；R 8 is -NR 15 R 16 which is ortho or meta or para at the phenyl substitution position; R15为H或CH3；R 15 is H or CH 3 ; R16为-C(O)-R17；R 16 is -C(O)-R 17 ; R17为CH3、-CH2-N(CH3)2或-CH2-OCH3；R 17 is CH 3 , -CH 2 -N(CH 3 ) 2 or -CH 2 -OCH 3 ; R8也可为在Cp烷基取代的-C(O)R18；R 8 can also be -C(O)R 18 substituted at Cp alkyl; R18可以为如下结构中的任意一种
Figure FDA0002589780060000021
Figure FDA0002589780060000022
R 18 can be any one of the following structures
Figure FDA0002589780060000021
Figure FDA0002589780060000022
 2.如权利要求1所述的如式I所示的化合物或其药学上可接受的盐、立体异构体、互变异构体、多晶型物、溶剂合物、N-氧化物、代谢物或前药，其特征在于，2. the compound shown in formula I as claimed in claim 1 or its pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, A metabolite or prodrug, characterized in that,
Figure FDA0002589780060000023
选自
Figure FDA0002589780060000024
Figure FDA0002589780060000025
Figure FDA0002589780060000023
selected from
Figure FDA0002589780060000024
Figure FDA0002589780060000025
 其中，R19可为H或CH3；Wherein, R 19 can be H or CH 3 ; R20可为-CO2R21；R 20 can be -CO 2 R 21 ; R21为C1至C6的直链烷烃。R 21 is a C 1 to C 6 straight chain alkane. 3.如权利要求2所述的如式I所示的化合物或其药学上可接受的盐、立体异构体、互变异构体、多晶型物、溶剂合物、N-氧化物、代谢物或前药，其特征在于，所述的如式I所示的化合物为如下式II所示的结构：3. the compound shown in formula I as claimed in claim 2 or its pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, Metabolite or prodrug, characterized in that the compound shown in formula I is the structure shown in the following formula II:
Figure FDA0002589780060000026
Figure FDA0002589780060000026
 其中，R1、R2、R3、R6、R7、R8、m和n如权利要求1或2所定义。wherein R 1 , R 2 , R 3 , R 6 , R 7 , R 8 , m and n are as defined in claim 1 or 2 . 4.如权利要求3所述的如式I所示的化合物或其药学上可接受的盐、立体异构体、互变异构体、多晶型物、溶剂合物、N-氧化物、代谢物或前药，其特征在于，4. the compound shown in formula I as claimed in claim 3 or its pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, A metabolite or prodrug, characterized in that, B为如下结构中的任意一种，
Figure FDA0002589780060000027
Figure FDA0002589780060000028
B is any one of the following structures,
Figure FDA0002589780060000027
Figure FDA0002589780060000028
 和/或，R22各自独立地选自-COOH、-COOCH3、-COOCH2CH3、-COOC(CH3)3、-COOCH2OCOOCH2CH3、-COOCH2OCOOCH(CH3)2、-COOCH(CH3)OCOOCH2CH3、-COOCH(CH3)OCOOCH(CH3)2、
Figure FDA0002589780060000031
-CONHSO2CH3、
Figure FDA0002589780060000032
Figure FDA0002589780060000033
-CH2COOH、-OCH3、-OCHF2、
Figure FDA0002589780060000034
-Cl、-F、-NHCOOCH3和-SO2NHCOCH3。and/or, each R 22 is 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 ,
Figure FDA0002589780060000031
-CONHSO 2 CH 3 ,
Figure FDA0002589780060000032
Figure FDA0002589780060000033
-CH 2 COOH, -OCH 3 , -OCHF 2 ,
Figure FDA0002589780060000034
-Cl, -F, -NHCOOCH 3 and -SO 2 NHCOCH 3 . 5.一种如权利要求1-4中任一项所述的如式I所示的化合物的制备方法，其特征在于，其包括以下步骤：5. the preparation method of the compound shown in the formula I as described in any one of claim 1-4, is characterized in that, it comprises the following steps:
Figure FDA0002589780060000035
Figure FDA0002589780060000035
 其中，R1、R2、R3、R4、R5、R6、R7、R8、m和n如权利要求1-4中任一项所定义；且PG为羧基的保护基。wherein, R 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 1-4; and PG is a protecting group for carboxyl group. 6.一种药物组合物，其包含预防或治疗有效量的如权利要求1-4中任一项所述的化合物或其药学上可接受的盐、立体异构体、互变异构体、多晶型物、溶剂合物、N-氧化物、代谢物或前药，以及一种或多种药学上可接受的载体。6. A pharmaceutical composition comprising a prophylactically or therapeutically effective amount of a compound according to any one of claims 1-4 or a pharmaceutically acceptable salt, stereoisomer, tautomer, A polymorph, solvate, N-oxide, metabolite or prodrug, and one or more pharmaceutically acceptable carriers. 7.一种如权利要求1-4中任一项所述的化合物或其药学上可接受的盐、立体异构体、互变异构体、多晶型物、溶剂合物、N-氧化物、代谢物或前药在制备用于预防或治疗血栓和栓塞相关疾病的药物中的用途。7. A compound as claimed in any one of claims 1-4 or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxidation thereof Use of a compound, metabolite or prodrug in the preparation of a medicament for the prevention or treatment of thrombosis and thrombosis-related diseases.
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