CN102924567A - Peptide compound and preparation method and use of peptide compound - Google Patents

Abstract

The present invention discloses a class of novel peptide compounds represented by formula (I), its pharmaceutically acceptable salts, pharmaceutically acceptable solvates, derivatives, isomers, crystal forms and pharmaceutically acceptable precursors drug. The present invention also discloses the preparation method of the compound of formula (I). The invention also discloses the application of the peptide compound in the preparation of medicines for treating and preventing thrombin-mediated and thrombin-related diseases.

Description

A class of peptide compounds, its preparation method and use

This application is a divisional application of the patent application with the application number 200810201891.1, the application date is October 28, 2008, and the invention title is "a class of peptide compounds, their preparation method and application".

technical field

The present invention relates to a novel competitive inhibitor with inhibitory effect on trypsin-like serine proteases, especially thrombin, a method for synthesizing the competitive inhibitor and its use in the preparation of medicines. In particular, the present invention relates to peptide compounds having antithrombotic activity.

Background technique

The mortality rate of cardiovascular and cerebrovascular diseases has been ranked second in the world, among which thromboembolism is the main cause of high rate of morbidity and death of cardiovascular and cerebrovascular diseases. Especially with the changes in people's lifestyles and the increasing aging of the population, the incidence of such diseases shows a continuous upward trend. This makes it extremely urgent to explore and study drugs that can effectively prevent and treat such diseases, which is of great significance in both clinical application and basic research.

Traditional anticoagulant drugs currently used in clinical practice, such as heparin, warfarin, and hirudin, have serious defects in efficacy and safety, which greatly restrict their application. For example, heparin must be injected, and has no effect on thrombin in blood clots, and can cause thrombocytopenia symptoms; warfarin is the only oral drug, but since there is no clear drug target, individual anticoagulant responses vary greatly. There are many influencing factors, and coagulation function monitoring is required. Therefore, it is very important to research and develop artificially synthesized new anticoagulant drugs with oral activity.

Blood coagulation is the result of a complex series of enzyme actions, the key step of which is the activation of prothrombin to generate thrombin. Thrombin is a trypsin-like serine protease whose main function is to hydrolyze fibrinogen to form insoluble fibrous aggregates. Plays a key role in the blood coagulation cascade. Therefore, inhibiting the activity of thrombin can block the formation of thrombus. Due to its clear target and different targets from traditional anticoagulant drugs, direct thrombin inhibitors (DTI) are expected to overcome the application limitations of traditional anticoagulant drugs. Clearly, a potent, selective, and orally bioavailable thrombin inhibitor with convenient therapeutic control of thrombus progression represents an attractive target.

In the past thirty years, many major advances have been made in the study of artificially synthesized thrombin inhibitors as anticoagulant activity, and a large number of highly active and highly selective small molecule thrombin inhibitors have been reported. D-Phe-Pro-Arg-H and Me-D-Phe-Pro-Arg-H tripeptide aldehyde thrombin inhibitors are reported as in US4346078. Recently, D-Phe-Pro-agmatine and its derivatives were described as thrombin inhibitors in US4346078 and WO9311152. Later, a tripeptide inhibitor was reported in WO9429336 and WO9523609, in which 4-amidinobenzylamine was incorporated at the P1 position to replace agmatine and the like.

In US4101653, Okamoto etc. disclosed that N-tosyl arginine methyl ester (TAMA) was used as thrombin substrate, designed and synthesized a series of arginine derivatives, and found that argatroban (argatroban) has good inhibition of thrombin activity. After pharmacological clinical research, it was approved by the FDA as an injection in 2001. Argatroban is a reversible competitive thrombin inhibitor that binds to the active site of thrombin to play a role and is highly selective for thrombin. It is clinically used to treat peripheral thrombosis and acute stroke, and can also be used for heparin-induced thrombocytopenia and thrombotic syndrome.

Similarly, according to the structure of TAMA, the researchers of Hoffmann La Roche designed and synthesized a series of benzamidine compounds with weak basicity, and further structural modification found that the thrombin inhibitory activity of NAPAP was twice as strong as that of argatroban (J Med Chem , 1994, 37, 3889). A series of compounds that change NAPAP’s benzamidine to piperidine formamidine also have higher thrombin-inhibiting activity. Among them, napsagatran synthesized by Hoffmann-La Roche has the strongest activity and has an effect on blood fibrinase. It has been used in phase II clinical trials research, but the half-life is short and the oral bioavailability is poor.

Astra company discloses a class of benzamidine analogs in WO9429336, wherein melagatran (melagatran) has strong and selective inhibition of thrombin activity, no obvious bleeding problem can be safely used for deep vein thrombosis (DVT), but oral The bioavailability is low, and its double prodrug ximelagatran (ximelagatran) was further synthesized, which was launched in 2004. It was the first oral anticoagulant drug in more than 60 years after warfarin, but it was terminated in February 2006 after severe liver damage was found in clinical practice.

In addition, WO9311152, WO9715190, and US5510369 reported a class of thrombin inhibitors with a unique D-diphenylglycine at the P3 position. It is reported that these compounds have higher antithrombin activity (J Med Chem, 1997, 40, 830) compared with the corresponding D-phenylglycine analogs, and some of them have better oral bioavailability ( J Med Chem, 1997, 40, 3687; J Med Chem, 1997, 40, 3726).

Only a few of the reported compounds have suitable in vivo pharmacokinetic and pharmacodynamic properties. So far, the study of thrombin inhibitors is still one of the most intensely competitive research topics in the field of medicinal chemistry.

Contents of the invention

Aiming at the technical problems in the above-mentioned prior art, we have conducted sufficient research and discovered a class of novel peptide compounds with good thrombin inhibitory activity.

The first object of the present invention is to provide a novel peptide compound represented by formula (I), its pharmaceutically acceptable salt, pharmaceutically acceptable solvate, derivative, isomer, crystal form and pharmaceutical Acceptable prodrugs:

in,

n stands for 1~3;

A stands for Lys, Arg or Harg;

R ₁ represents aryl, 5-6 membered heterocycle or benzo saturated or unsaturated heterocycle;

B is for

in,

R ₂ represents H, C1～4 alkyl;

R ₃ represents C4～8 saturated or unsaturated cycloalkane;

Z represents H, carboxyl, methyl carboxyl, carbamoyl or aminoacetyl;

m stands for 0-3.

The aryl is substituted or unsubstituted phenyl or naphthyl, wherein the substituent is hydroxyl, carboxyl, halogen, alkoxy, amino, alkylamino, dialkylamino, cyano, ester, trifluoromethyl or A C1-4 alkyl group bearing the above groups.

The 5-6 membered heterocycle is a substituted or unsubstituted structural unit represented by formula (II):

Wherein the substituent is hydroxyl, carboxyl, halogen, alkoxy, amino, alkylamino, dialkylamino, cyano, ester, trifluoromethyl or C1-4 alkyl with the above groups.

The benzo saturated or unsaturated heterocycle is a substituted or unsubstituted structural unit represented by formula (III):

Wherein the substituent is hydroxyl, carboxyl, halogen, alkoxy, amino, alkylamino, dialkylamino, cyano, ester, trifluoromethyl or C1-4 alkyl with the above groups.

Preferred specific compounds of the invention include, but are not limited to: N-benzyl-L-lysyl-L-proline, N-benzyl-L-lysyl-D-proline, N-phenylethyl Base-L-lysyl-L-proline, N-phenylethyl-L-lysyl-D-proline, N-phenylpropyl-L-lysyl-L-proline, N-phenylpropyl-L-lysyl-D-proline, N-p-chlorobenzyl-L-lysyl-L-proline, N-p-chlorobenzyl-L-lysyl- D-proline, N-p-fluorobenzyl-L-lysyl-L-proline, N-p-fluorobenzyl-L-lysyl-D-proline, N-1-naphthyl -L-lysyl-L-proline, N-1-naphthyl-L-lysyl-D-proline, N-2-naphthyl-L-lysyl-L-proline , N-2-naphthyl-L-lysyl-D-proline, N-benzyl-L-lysyl-pyrrolidine, N-benzyl-L-lysyl-piperidine, N- Benzyl-L-lysyl-cyclohexylamine, N-benzyl-L-lysyl-cyclohexylamine, N-benzyl-L-lysyl-L-homoproline, N-benzyl -L-lysyl-D-homoproline, N-benzyl-L-lysyl-(R-3-formyl)piperidine, N-benzyl-L-lysyl-(S- 3-formyl)piperidine, N-benzyl-L-arginyl-D-proline or N-benzyl-L-homoarginyl-D-proline.

The present invention also includes its pharmaceutically acceptable salts, pharmaceutically acceptable solvates, derivatives, isomers, crystal forms and pharmaceutically acceptable prodrugs.

Unless otherwise stated, terms and abbreviations disclosed herein have their standard meanings.

Pharmaceutically acceptable salts of compounds of formula (I) include salts derived from pharmaceutically acceptable inorganic and organic acids. Examples of suitable acids include sulfuric acid, hydrochloric acid, hydrobromic acid, nitric acid, acetic acid, phosphoric acid, maleic acid, fumaric acid, citric acid, perchloric acid, p-toluenesulfonic acid, tartaric acid, formic acid, benzoic acid, malonic acid, Citric acid, methanesulfonic acid, benzenesulfonic acid, lactic acid, mandelic acid.

The compounds of the present invention can form hydrates and solvates with appropriate solvents. Preferred solvents for preparing solvate forms include water and alcohols, and preferred alcohols are methanol and ethanol. Other suitable solvents can be selected according to the size of solvent molecules. Solvates or hydrates are formed during recrystallization or during salt formation.

Since the compound of formula (I) of the present invention has one or more chiral carbon atoms in its structure, it can also exist as racemates, diastereomeric mixtures and pure enantiomers, and all these isomers belong to the present invention. within the scope of the invention.

The present invention also relates to pharmaceutically acceptable prodrugs of the compounds of general formula which, when administered, are metabolized to the active compounds. Suitable prodrugs are, for example, N-alkoxycarbonyl-protected derivatives or ester-protected derivatives of carboxylic acids of the general formula.

When the compounds are solids, the compounds and salts of the invention may exist in different crystalline or polymorphic forms, all within the scope of the invention and the specific formulae.

The second object of the present invention is to provide a preparation method of the peptide compound of formula (I), which includes the step of preparing the final product from an amino acid whose nitrogen position is protected by a protecting group. The method can be prepared by similar standard chemical reactions known in the prior art (the compound of the present invention can be prepared according to the following general processes), and the starting materials, reagents, techniques and methods used in the route are all well known, And are well known and understood by anyone of ordinary skill in the art.

The preparation method is described in detail as follows:

Route 1:

Route one provides the general synthetic method of wherein A is the formula (I) compound of lysyl, wherein n, _R The definition as above, B represents

Z represents carboxyl, methyl carboxyl,

m stands for 0~3,

R ₄ and R ₅ refer to suitable amino protecting groups, among which tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), fluorenyloxycarbonyl (Fmoc),

X refers to a suitable carboxylic acid protecting group for the Z group, where hydrogenolysis-labile esters such as benzyl ester (Bn), base-labile esters such as methyl ester (Me) or acid-labile esters such as tert-butyl are preferred base ester (tBu).

In route one, at first the C-terminal of compound 1 is coupled with the N-terminal of compound 2 to obtain compound 3, and the compound 3 removes the carboxylic acid protecting group X and the amino protecting group R at the Nα-terminal respectively to obtain intermediate ₅ , and then The group R ₁ -(CH ₂ ) _n is introduced into the Nα-terminus of compound 5, and finally the amino protecting group R ₅ on the side chain N is removed to obtain the desired compound.

Route two:

Route two provides the general synthetic method of wherein A is the formula (I) compound of lysyl, wherein n, _R The definition as above, B represents

Wherein _R represents H, C1～4 alkyl,

R ₃ represents C4～8 saturated or unsaturated cycloalkane,

Z represents H, carbamoyl, aminoacetyl,

m stands for 0~3,

R ₄ and R ₅ refer to suitable amino protecting groups, among which tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) and fluorenyloxycarbonyl (Fmoc) are preferred.

In route 2, firstly, the C-terminal of compound 1 and the N-terminal of B are coupled to obtain compound 4, which removes the amino protecting group R4 at the Nα-terminal to obtain intermediate 5, and then the group R1-(CH ₂ ) _n The Nα-terminal of compound 5 was introduced, and finally the amino protecting group R ₅ on the side chain N was removed to obtain the desired compound.

Line three:

Route 3 provides a general synthesis method for compounds of formula (I) wherein A is arginyl or homoarginyl, wherein n, R ₁ , are as defined above, and p represents 1-2.

B is for

Z represents carboxyl, methyl carboxyl,

m stands for 0~3,

R ₆ and R ₇ refer to suitable amino protecting groups, wherein preferred tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz),

X refers to a suitable carboxylic acid protecting group for the Z group, where hydrogenolysis-labile esters such as benzyl ester (Bn), base-labile esters such as methyl ester (Me) or acid-labile esters such as tert-butyl are preferred base ester (tBu).

In route three, at first the C-terminal of compound 7 is coupled with the N-terminal of compound 2 to obtain compound 8, and the compound ₈ removes the carboxylic acid protecting group X and the amino protecting group R at the Nα-terminal respectively to obtain intermediate 10, and then The group R1-(CH ₂ ) _n was introduced into the Nα-terminus of compound 10, and finally the amino protecting group R ₇ on the side chain N was removed to obtain the desired compound.

Line four:

Route 4 provides a general synthesis method for compounds of formula (I) wherein A is arginyl or homoarginyl, wherein n, R ₁ are as defined above, and p represents 1-2.

B is for

Wherein R ₂ represents H, C 1～4 alkyl,

R ₃ represents C4～8 saturated or unsaturated cycloalkane,

Z represents H, carbamoyl, aminoacetyl,

m stands for 0~3,

R ₆ and R ₇ refer to suitable amino protecting groups, among which tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) and fluorenyloxycarbonyl (Fmoc) are preferred.

In Route 4, the C-terminal of compound 7 and the N-terminal of B are firstly coupled to obtain compound 9, which removes the amino protecting group R ₆ at the Nα-terminal to obtain intermediate 10, and then the group R1-(CH ₂ ) _n was introduced into the Nα-terminus of compound 10, and finally the amino protecting group R ₇ on the side chain N was removed to obtain the desired compound.

Among the above four synthetic routes, amide coupling adopts standard peptide coupling method to carry out under inert gas such as nitrogen, such as azidation method, mixed anhydride method, carbodiimide (dicyclohexylcarbodiimide DCC, diisopropyl Carbodiimide EDC) method, active ester method, carbonyldiimidazole method, phosphorus reagents such as BOP-C1 method. Some of these methods (especially the carbodiimide method) can be improved by adding 1-hydroxybenzotriazole HOBt. If necessary, the coupling reaction needs to be carried out in the presence of an acid-binding agent. Examples of suitable acid-binding agents are organic tertiary amines such as diisopropylethylamine, triethylamine, trimethylamine, pyridine, N-methylmorpholine etc., N-methylmorpholine or diisopropylethylamine are particularly preferred as acid-binding agents.

The above reaction is carried out in the presence of anhydrous solvent, and the solvent examples that can be selected include: methylene chloride, tetrahydrofuran, ether, acetonitrile, ethylene dichloride, ethyl acetate, N,N-dimethylformamide, dimethylmethylene sulfone. The reaction temperature is generally not critical, and it is preferable to react at 0 to 30°C for 2 to 24 hours.

The method for removing the amino protecting group is carried out by a conventional method, such as hydrolysis in the presence of an acid (for example: organic acids such as trifluoroacetic acid, benzenesulfonic acid, formic acid, etc., or inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, etc.) or in the presence of Alkalis (for example: hydroxides, hydrides, carbonates or bicarbonates of alkali metals or alkaline earth metals, such as sodium hydroxide, potassium hydroxide, sodium hydride, potassium carbonate, sodium bicarbonate, etc., or organic bases such as di Hydrolysis in the presence of isopropylethylamine, triethylamine, piperidine, etc.), or reduction by hydrogenolysis with hydrogen in the presence of a catalyst (such as metal catalysts such as palladium, platinum, nickel, etc.). Suitable methods can be found in Protecting Groups in Organic Synthesis, Third Edition, T.W. Green and Peter G.M. Wuts (1999), Publisher: John Wiley & Sons, Inc.

In general, the reaction can be carried out in the presence of a solvent that has no adverse effect on the reaction. Examples of solvents that can be selected include: dichloromethane, alcohols such as methanol, ethanol, etc., tetrahydrofuran, dioxane, acetone, acetic acid, acetic acid ethyl ester. The reaction temperature is usually not critical, and is preferably 0 to 40°C.

The removal of the carboxylic acid protecting group is carried out by conventional methods, for example in the presence of a base (e.g. hydroxide, hydride, carbonate or bicarbonate of an alkali or alkaline earth metal, such as sodium hydroxide, potassium hydroxide, sodium hydride , potassium carbonate, sodium bicarbonate, etc., or organic bases such as diisopropylethylamine, triethylamine, etc.) in the presence of hydrolysis, or in the presence of catalysts (such as palladium, platinum, nickel and other metal catalysts) by using hydrogen restoration. Suitable methods can be found in Protecting Groups in Organic Synthesis, Third Edition, T.W. Green and Peter G.M. Wuts (1999), Publisher: John Wiley & Sons, Inc.

Generally, the reaction can be carried out in the presence of a solvent that has no adverse effect on the reaction. Examples of solvents that can be selected include: water, alcohols such as methanol, ethanol, etc., tetrahydrofuran, dioxane, and acetone. The reaction temperature is usually not critical, and is preferably 0 to 30°C.

Reductive amination and nucleophilic substitution can be used for alkylation on N. Such as using nucleophilic substitution method. Suitable nucleophilic substitution agents include, but are not limited to: alkyl bromides, alkyl iodides, alkyl chlorides, alkyl sulfonates, alkyl benzene sulfonates, alkyl p-toluene sulfonates, alkyl methane sulfonates Or contain acid sulfate. Reagent for methylsulfate. Preference is given to alkyl iodides or alkyl p-homenesylates. The nucleophilic substitution reaction needs to be carried out in the presence of an acid-binding agent, and a suitable acid-binding agent can be an inorganic base (hydroxide, hydride, carbonate bicarbonate, hydrogen phosphate of an alkali metal or an alkaline earth metal, such as hydroxide Sodium, sodium hydride, potassium carbonate, dipotassium hydrogen phosphate, etc.), or organic bases such as diisopropylethylamine, triethylamine, piperidine, etc. Examples are organic tertiary amines such as diisopropylethylamine, triethylamine , trimethylamine, pyridine, N-methylmorpholine, etc., particularly preferably dipotassium hydrogen phosphate or diisopropylethylamine as the acid-binding agent. Examples of solvents that can be used include tetrahydrofuran, dioxane, acetonitrile, acetone, N,N-dimethylformamide and the like. The reaction temperature is preferably 30 to 80°C.

Such as using the reductive amination method. The reactants and the corresponding aldehyde can be treated with a suitable reducing agent in a suitable solvent. Suitable reducing agents are well known in the art and include, but are not limited to, lithium tri-tert-butoxide, potassium borohydride, sodium borohydride, sodium triacetoxyborohydride, Raney nickel, lithium triethylborohydride, where Preference is given to potassium borohydride or sodium triacetoxyborohydride. Suitable solvents for the reductive amination reaction are known in the art, such as methanol, ethanol, tetrahydrofuran, dichloroethane, acetonitrile, and mixed solvents. The reaction temperature is preferably 0-50°C.

The product of each step can be purified by methods known in the art such as column chromatography and recrystallization.

General formula 1, 2 and 7 compounds as starting material, some are commercially available (Sigma-Aldrich company), some are known (Chem Pharm Bull, 1999,47,1489 and Synth Commun, 2000, 30, 2525), which can be prepared by methods described in the literature. The intermediates obtained by the method for preparing the compound of formula (I) according to the present invention are all novel compounds, except Lys-Pro, thus included in the scope of the present invention.

The third object of the present invention is to provide the application of the peptide compound represented by formula (I) in the preparation of thrombin inhibitors and the application in the preparation of medicines for the treatment and prevention of thrombin-mediated and thrombin-related diseases .

The compounds of the present invention are useful in the treatment and prevention of thrombin-mediated and thrombin-associated diseases. Such diseases include, but are not limited to: venous thrombosis and pulmonary embolism, arterial thrombosis such as myocardial ischemia, myocardial infarction, unstable angina, stroke due to thrombosis, and peripheral arteriogenesis; atherosclerotic diseases such as coronary artery disease, Cerebral arterial disease and peripheral arterial disease.

The peptide compounds of the present invention are also useful as anticoagulants in extracorporeal blood lines.

Additionally, such compounds are expected to be useful in the prevention and treatment of myocardial infarction together with thrombolytic agents. Furthermore, such compounds are expected to be useful in preventing re-thrombosis following microsurgery. Such compounds are expected to be useful in anticoagulant therapy for hemodialysis and disseminated intravascular coagulation. It can also be used for in vitro preservation of blood, plasma and other blood products.

The compounds of the present invention are contemplated to be administered orally or parenterally, eg by intravenous infusion, intramuscular injection, or subcutaneous injection. The exact amount at which the compounds administered according to the invention achieve a therapeutic or prophylactic effect will depend upon the particular circumstances of the case, including the form of administration, the rate of administration and the condition being treated. Typical oral daily doses to be effective are between about 0.01 mg/kg and about 1000 mg/kg; typical parenteral daily doses are between about 0.001 mg/kg and about 100 mg/kg. The mode of administration can be different, such as a single dose per day, or multiple doses such as 3-5 times per day may also be suitable. It is to be understood that the dosage and mode of administration may be routinely adjusted as necessary according to the age and weight of the patient and the severity of the disease being treated. The exact dosage and route of administration should be decided by the attending physician.

The fourth object of the present invention is to provide a pharmaceutical composition comprising the peptide compound represented by formula (I) and a pharmaceutically acceptable carrier.

The compounds of the invention may be administered in the form of pharmaceutical compositions. For example, for oral administration, the compound is prepared in the form of capsules or tablets, which may contain excipients such as lubricants, binders, and disintegrants. For injectable use, the compound is dissolved in a pharmaceutically acceptable vehicle such as sterile, pyrogen-free water, physiological saline.

Dosage forms may be solid, semi-solid or liquid preparations prepared by techniques known per se. The active ingredients in such compositions comprise from 0.1% to 99.9% by weight of the formula. Moreover, the carrier, diluent or excipient used in the composition is compatible with the active ingredient and is harmless to the recipient.

Abbreviations appearing in this application are as follows:

Boc: tert-butoxycarbonyl

Cbz: benzyloxycarbonyl

DCHA: Dicyclohexylamine

Lys: Lysyl

Pro: Prolyl

Hpro: high prolyl

Arg: Arginyl

Harg: high arginyl

Fmoc: 9-fluorenyloxycarbonyl

EDCI: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride

HOBt: 1-Hydroxybenzotriazole

TFA: Trifluoroacetic acid

AcOH: acetic acid

HBr: N,N'-dimethylformamide.

activity assay

The in vitro thrombin-inhibiting activity of the compounds of the present invention can be determined by the chromogenic substrate method. In the experiment, Ac-FVR-AMC was used as a chromogenic substrate to measure its activity of inhibiting human α-thrombin. And calculate its _IC50 value.

Detailed ways

The following examples will describe in detail the specific synthetic methods described in Routes 1 and 2, and synthesized preferred compounds of the present invention, but those skilled in the art understand that the chemical reactions can be slightly modified to prepare many other thrombin inhibitors of the present invention agent. For example, non-exemplified compounds of the present invention can be successfully synthesized by modifications apparent to those skilled in the art. These examples are for illustration only and do not limit the scope of the invention in any way.

Detection method:

The nuclear magnetic resonance instrument adopts the Varian INOVA-400 nuclear magnetic resonance instrument, with tetramethyl silicon as the internal standard, and the unit of chemical shift () is ppm. Thin-layer chromatography (TLC, using HSG-F254 high-performance thin-layer chromatography silica gel prefabricated plates, manufactured by Yantai Zhifu Huangwu Silica Gel Development and Experimental Factory) and HPLC were used to detect the reaction and product purity. For color development, use iodine vapor or irradiate under 254A and 310A ultraviolet lamps or 1% ethanol solution of ninhydrin. The reagents used were of analytical grade unless otherwise specified, and the anhydrous solvents and reagents were treated according to conventional methods. Melting points were determined with a micro melting point apparatus using an uncalibrated thermometer.

HPLC: Waters 1525; Detector: Waters 2487; Chromatographic column: Phenomenex C18 (4.6×250mm, 5μm); Detection wavelength: 220nm and 254nm; Column temperature: 40°C; Flow rate: 1.0ml/min;

Mobile phase 1: A: 0.1% trifluoroacetic acid in water, B: acetonitrile

Mobile phase 2: A: triethylamine phosphate buffer at pH 2.0, B: acetonitrile

Mobile phase 3: A: Phosphate buffer at pH 8.0, B: Acetonitrile

Method 1: Mobile phase A from 75% to 25%, gradient elution for 10 minutes

Method 2: Mobile phase A from 95% to 30%, gradient elution for 20 minutes

Embodiment 1: Synthesis of N-benzyl-L-lysyl-D-proline dihydrobromide (compound 1)

a) Synthesis of [L-Lys(Cbz)] ₂ Cu

A solution of L-lysine hydrochloride (18.2g), sodium bicarbonate (16.8g), copper sulfate pentahydrate (12.5g) in water (200ml) was stirred for 1h. Cool in an ice bath to 0°C, slowly add benzyloxycarbonyl chloride (14.3ml) dropwise, and adjust to pH 8.5 with 2mol/L sodium hydroxide solution, keep the pH value constant for more than 3 hours after the reaction, filter, and wash the obtained precipitate with water To neutrality, washed with acetone, quantitatively obtained blue solid. Proceed directly to the next reaction.

b) Synthesis of Boc-L-Lys(Cbz)OH·DCHA

[L-Lys(Cbz)] ₂ Cu (27.7g) was suspended in acetone (100ml) and 10% sodium carbonate (100ml) aqueous solution, and 8-hydroxyquinoline (14.95g) was added under vigorous stirring. After 2 hours, it was cooled to below 10°C in an ice bath, and a solution of di-tert-butyl dicarbonate (28 g) in acetone (60 ml) was slowly added dropwise, and the reaction was allowed to rise to room temperature naturally for 8 hours. Filter, wash the filter cake with water, concentrate the filtrate under reduced pressure to remove acetone, and extract with ethyl acetate until the organic layer is colorless. The aqueous phase was adjusted to pH 4 with saturated potassium hydrogensulfate solution and extracted with dichloromethane. The obtained organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a light yellow oil, which was dissolved in ethyl acetate (60ml), and DCHA (18ml) and n-hexane (100ml) were added under stirring, gradually A solid was precipitated and filtered, and the crude product was recrystallized from isopropanol/cyclohexane to obtain a white solid (46g, 82%), mp: 103-105°C, content 99% (HPLC, mobile phase 1, method 1). Rf=0.6 Developing agent dichloromethane/methanol=15/1 Color development: UV, iodine and 1% ninhydrin solution 1H NMR (DMSO) ppm: 1.43 (9H, s), 1.45-1.52 (2H, m), 1.58-1.72(2H, m), 1.82(2H, m), 3.14-1.18(2H, m), 4.26(1H, m), 5.09(2H, s), 7.27-7.34(5H, m)

c) synthesis of D-proline methyl ester hydrochloride

Dissolve D-proline (23g) in methanol (200ml), cool in an ice bath to below 0°C, slowly add thionyl chloride (36ml) dropwise for 1 hour, and react at 40°C for 5 hours. The solvent was concentrated under reduced pressure at 50°C to obtain a light yellow oil, and the crude product was recrystallized from isopropanol/isopropyl ether to obtain a white solid (29 g, 90%).

Rf=0.4 developing agent ethyl acetate/methanol/acetic acid=10/4/1 color development: iodine and 1% ninhydrin solution 1H NMR (DMSO) ppm: 4.43 (1H, t), 3.75 (3H, s), 3.20(3H,m), 2.25(1H,m), 1.95(3H,m)

d) Synthesis of Boc-L-Lys(Cbz)-D-Pro-OCH ₃

Dissolve Boc-L-Lys(Cbz)-OH·DCHA (7.3g) in 5% potassium bisulfate (40ml) solution and dichloromethane (40ml), stir for 0.5h, separate the layers, and wash the aqueous phase with dichloromethane Extracted with methane, combined the organic phases, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a light yellow oil.

Dissolve the freed Boc-L-Lys(Cbz)-OH, proline methyl ester hydrochloride (2.4g), N-methylmorpholine (3.37g) with anhydrous dichloromethane (50ml), nitrogen After protection, after cooling to 0°C, HOBt (2.1g) and EDCI (3g) were added, stirred at 0°C for 20min, then naturally rose to room temperature, and reacted for 8 hours. The organic layer was washed successively with 5% potassium bisulfate solution, saturated sodium bicarbonate solution and saturated saline solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 5.5 g of crude product, which was purified by column chromatography to obtain a colorless oil (5.3 g , yield: 83.7%).

Rf=0.7 developing agent ethyl acetate color development: ultraviolet and iodine

MS: 514 (M+Na+)

1H NMR (CDCl ₃ ) ppm: 1.41 (9H, s), 1.44-1.57 (4H, m), 1.58-1.72 (2H, m), 1.78 (2H, m), 1.82 (2H, m), 1.93-2.03 (2H, m), 2.2 (2H, m), 3.19-3.22 (2H, m), 3.60 (3H, s), 3.65-3.74 (2H, m), 4.43 (1H, m), 4.51-4.54 (1H , m), 5.08 (2H, s), 7.25-7.34 (6H, m)

e) Synthesis of Boc-L-Lys(Cbz)-D-Pro-OH

Dissolve Boc-L-Lys(Cbz)-D-Pro-OCH ₃ (5.3g) in methanol (50ml), cool to 0°C, add sodium hydroxide (1.7g), and react for 2 hours with 5% HCl solution Adjust to pH 4, concentrate under reduced pressure to remove the methanol solvent, and then extract with ethyl acetate. The resulting organic phase was dried and concentrated under reduced pressure to obtain a colorless thick product (4.8 g, 94%).

Rf=0.5 developer ethyl acetate color development: UV and 1% ninhydrin solution

MS: 476 (M-)

1H NMR (CDCl ₃ ) ppm: 1.51 (9H, s), 1.4-1.55 (2H, m), 1.59-1.67 (2H, m), 1.73-1.77 (2H, m), 1.97-2.10 (2H, m) , 2.16(2H, m), 3.57-3.63(2H, m), 3.74-3.76(2H, m), 4.46(1H, m), 4.54-4.59(1H, m), 5.09(2H, s), 7.27 -7.35(5H,m)

f) Synthesis of L-Lys(Cbz)-D-Pro-OH·TFA

Boc-L-Lys(Cbz)-D-Pro-OH (4.8g) was dissolved in dichloromethane (40ml), cooled in an ice bath to below 0°C, TFA (20ml) was added dropwise, reacted at 0°C for 1 hour, and Concentrate under reduced pressure, add diethyl ether to wash, and obtain an oily substance, pour off diethyl ether, and drain to obtain a white viscous solid (4.58 g, 92%).

Rf = 0.5 developing agent dichloromethane/methanol = 5/1 color development: ultraviolet and 1% ninhydrin solution

MS: 376 (M-)

g) Synthesis of N-benzyl-L-lysyl-D-proline dihydrobromide

Dissolve L-Lys(Cbz)-D-Pro-OH·TFA (491mg) in methanol (5ml), cool to 0°C, add triethylamine (0.21ml), benzaldehyde (121mg), stir for 3 hours, divide KBH4 (84 mg) was added in batches and reacted for 3 hours. Purified by flash column, washed with diethyl ether, poured diethyl ether, and dried to obtain a white thick substance.

The thick product obtained in the previous step and anisole (0.5ml) were dissolved in 36% HBr acetic acid solution (4ml), reacted at room temperature, HPLC detected that the reaction was complete, added diethyl ether (20ml) to the reaction solution, placed in the refrigerator overnight, and the solid was precipitated. Filter (300 mg, 61%).

MS: 334(M+1)

Embodiment 2: Synthesis of N-phenethyl-L-lysyl-D-proline dihydrobromide (compound 2)

Dissolve L-Lys(Cbz)-D-Pro-OH TFA (737mg) obtained in Example 1 in methanol (10ml), cool to 0°C, add triethylamine (277mg), phenylacetaldehyde (207mg) , stirred for 0.5 hours, added sodium triacetoxyborohydride (600 mg) in batches, reacted for 3 hours, concentrated under reduced pressure to remove the solvent, and purified by column chromatography to obtain a colorless thick substance.

Dissolve the thick product obtained in the previous step and anisole (0.5ml) in 36% HBr acetic acid solution (3ml), react at room temperature, HPLC detects that the reaction is complete, add ether (20ml) to the reaction solution, place it in the refrigerator overnight, and precipitate a solid , filtered (50mg, 6.6%)

MS: 348(M+1)

Embodiment 3: Synthesis of N-phenylpropyl-L-lysyl-D-proline dihydrobromide (compound 3)

Dissolve L-Lys(Cbz)-D-Pro-OH TFA (737mg) obtained in Example 1 in methanol (10ml), cool to 0°C, add triethylamine (277mg), phenylpropanal (211mg) , stirred for 0.5 hours, added sodium triacetoxyborohydride (600 mg) in batches, reacted for 3 hours, concentrated under reduced pressure to remove the solvent, and purified by column chromatography to obtain a colorless thick substance.

Dissolve the thick product obtained in the previous step and anisole (0.5ml) in 36% HBr acetic acid solution (3ml), react at room temperature, HPLC detects that the reaction is complete, add ether (20ml) to the reaction solution, place it in the refrigerator overnight, and precipitate a solid , filtered (167mg, 21%)

MS: 362(M+1)

Embodiment 4: Synthesis of N-1-naphthyl-L-lysyl-D-proline dihydrobromide (compound 4)

Dissolve L-Lys(Cbz)-D-Pro-OH·TFA (491mg) obtained in Example 1 in methanol (5ml), cool to 0°C, add triethylamine (0.21ml), 1-naphthaldehyde (175mg ), stirred for 3 hours, added KBH ₄ (83 mg) in batches, and reacted for 3 hours. Purified by flash column, washed with diethyl ether, poured diethyl ether, and dried to obtain a white thick substance.

The thick product obtained in the previous step and anisole (0.5ml) were dissolved in 36% HBr acetic acid solution (4ml), reacted at room temperature, HPLC detected that the reaction was complete, added diethyl ether (20ml) to the reaction solution, placed in the refrigerator overnight, and the solid was precipitated. Filtration (261 mg, 47.8%).

MS: 384(M+1)

Embodiment 5: Synthesis of N-2-naphthyl-L-lysyl-D-proline dihydrobromide (compound 5)

Dissolve L-Lys(Cbz)-D-Pro-OH·TFA (491mg) obtained in Example 1 in methanol (5ml), cool to 0°C, add triethylamine (0.21ml), 2-naphthaldehyde (175mg ), stirred for 3 hours, added KBH ₄ (80 mg) in batches, and reacted for 3 hours. Purified by flash column, washed with diethyl ether, poured diethyl ether, and dried to obtain a white thick substance.

The thick product obtained in the previous step and anisole (0.5ml) were dissolved in 36% HBr acetic acid solution (4ml), reacted at room temperature, HPLC detected that the reaction was complete, added diethyl ether (20ml) to the reaction solution, placed in the refrigerator overnight, and the solid was precipitated. Filtration (243mg, 44.6%).

MS: 384(M+1)

Embodiment 6: Synthesis of N-p-chlorobenzyl-L-lysyl-D-proline dihydrobromide (compound 6)

Dissolve L-Lys(Cbz)-D-Pro-OH·TFA (491mg) obtained in Example 1 in methanol (5ml), cool to 0°C, add triethylamine (0.21ml), p-chlorobenzaldehyde (145mg ), stirred for 3 hours, added KBH ₄ (91 mg) in batches, and reacted for 3 hours. Purified by flash column, washed with diethyl ether, poured diethyl ether, and dried to obtain a white thick substance.

The thick product obtained in the previous step and anisole (0.5ml) were dissolved in 36% HBr acetic acid solution (4ml), reacted at room temperature, HPLC detected that the reaction was complete, added diethyl ether (20ml) to the reaction solution, placed in the refrigerator overnight, and the solid was precipitated. Filtration (209mg, 39.4%).

MS: 369(M+1)

Embodiment 7: Synthesis of N-p-fluorobenzyl-L-lysyl-D-proline dihydrobromide (compound 7)

Dissolve L-Lys(Cbz)-D-Pro-OH·TFA (491mg) obtained in Example 1 in methanol (5ml), cool to 0°C, add triethylamine (0.21ml), p-fluorobenzaldehyde (132mg ), stirred for 3 hours, added KBH ₄ (87 mg) in batches, and reacted for 3 hours. Purified by flash column, washed with diethyl ether, poured diethyl ether, and dried to obtain a white thick substance.

The thick product obtained in the previous step and anisole (0.5ml) were dissolved in 36% HBr acetic acid solution (4ml), reacted at room temperature, HPLC detected that the reaction was complete, added diethyl ether (20ml) to the reaction solution, placed in the refrigerator overnight, and the solid was precipitated. Filter (215 mg, 42%).

MS: 352(M+1)

Embodiment 8: Synthesis of N-benzyl-L-lysyl-L-proline dihydrobromide (compound 8)

a) Synthesis of L-proline methyl ester hydrochloride

Dissolve L-proline (19.49g) in methanol (150ml), cool in an ice bath to below 0°C, slowly add thionyl chloride (38ml) dropwise for 1 hour, and react at 40°C for 5 hours. The solvent was concentrated under reduced pressure at 50°C to obtain a light yellow oil, and the crude product was recrystallized from isopropanol/isopropyl ether to obtain a white solid (25 g, 92%).

Rf=0.4 developing agent ethyl acetate/methanol/acetic acid=10/4/1 color development: iodine and 1% ninhydrin solution

b) Synthesis of Boc-L-Lys(Cbz)-L-Pro-OCH ₃

Dissolve Boc-L-Lys(Cbz)OH·DCHA (14g) in 5% potassium bisulfate (70ml) solution and dichloromethane (60ml), stir for 0.5h, separate layers, and extract the aqueous phase with dichloromethane , the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a light yellow oil.

Dissolve the freed Boc-L-Lys(Cbz)OH, L-proline methyl ester hydrochloride (4.8g), N-methylmorpholine (6.6g) with anhydrous dichloromethane (100ml), Under nitrogen protection, after cooling to 0°C, HOBt (4.2g) and EDCI (6g) were added, stirred at 0°C for 20min, then naturally rose to room temperature, and reacted for 8 hours. The organic layer was washed successively with 5% potassium bisulfate solution, saturated sodium bicarbonate solution and saturated saline solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography to obtain a colorless oil (10 g, yield : 79%).

Rf=0.8 developer ethyl acetate color development: ultraviolet and iodine

MS: 514 (M+Na ⁺ )

c) Synthesis of Boc-L-Lys(Cbz)-L-Pro-OH

Dissolve Boc-L-Lys(Cbz)-L-Pro-OCH ₃ (2g) in methanol (25ml), cool to 0°C, add sodium hydroxide (1.1g), react for 2 hours and adjust with 5% HCl solution To pH 4, concentrated under reduced pressure to remove the methanol solvent, then extracted with ethyl acetate, the resulting organic phase was dried and concentrated under reduced pressure to obtain a colorless thick product (1.9 g, 92%).

Rf=0.5 developer ethyl acetate color development: UV and 1% ninhydrin solution

MS: 476 (M-)

d) Synthesis of L-Lys(Cbz)-L-Pro-OH·TFA

Boc-L-Lys(Cbz)-L-Pro-OH (2.4g) was dissolved in dichloromethane (15ml), cooled in an ice bath to below 0°C, TFA (5ml) was added dropwise, reacted at 0°C for 1 hour, and Concentrate under reduced pressure, add diethyl ether to wash, and obtain an oily substance, pour off diethyl ether, and drain to obtain a white viscous solid (2.3 g, 92%).

Rf = 0.5 developing agent dichloromethane/methanol = 5/1 color development: ultraviolet and 1% ninhydrin solution

MS: 376 (M-)

e) Synthesis of N-benzyl-L-lysyl-L-proline dihydrobromide

Dissolve L-Lys(Cbz)-L-Pro-OH·TFA (491mg) in methanol (5ml), cool to 0°C, add triethylamine (0.21ml), benzaldehyde (121mg), stir for 3 hours, divide KBH4 (86 mg) was added in batches and reacted for 3 hours. Purified by flash column, washed with diethyl ether, poured diethyl ether, and dried to obtain a white thick substance.

The thick product obtained in the previous step and anisole (0.5ml) were dissolved in 36% HBr acetic acid solution (4ml), reacted at room temperature, HPLC detected that the reaction was complete, added diethyl ether (20ml) to the reaction solution, placed in the refrigerator overnight, and the solid was precipitated. Filter (154mg, 31%).

MS: 334(M+1)

Embodiment 9: Synthesis of N-phenethyl-L-lysyl-L-proline dihydrobromide (compound 9)

Dissolve L-Lys(Cbz)-L-Pro-OH TFA (737mg) obtained in Example 8 in methanol (10ml), cool to 0°C, add triethylamine (267mg), phenylacetaldehyde (210mg) , stirred for 0.5 hours, added sodium triacetoxyborohydride (592 mg) in batches, reacted for 3 hours, concentrated under reduced pressure to remove the solvent, and purified by column chromatography to obtain a colorless thick substance.

Dissolve the thick product obtained in the previous step and anisole (0.Sml) in 36% HBr acetic acid solution (3ml), react at room temperature, HPLC detects that the reaction is complete, add ether (20ml) to the reaction solution, place it in the refrigerator overnight, and precipitate Solid, filtered (61 mg, 8%)

MS: 348(M+1)

Embodiment 10: Synthesis of N-phenylpropyl-L-lysyl-L-proline dihydrobromide (compound 10)

Dissolve L-Lys(Cbz)-L-Pro-OH TFA (737mg) obtained in Example 8 in methanol (10ml), cool to 0°C, add triethylamine (274mg), phenylpropanal (220mg) , stirred for 0.5 hours, added sodium triacetoxyborohydride (611 mg) in batches, reacted for 3 hours, concentrated under reduced pressure to remove the solvent, and purified by column chromatography to obtain a colorless thick substance.

Dissolve the thick product obtained in the previous step and anisole (0.5ml) in 36% HBr acetic acid solution (3ml), react at room temperature, HPLC detects that the reaction is complete, add ether (20ml) to the reaction solution, place it in the refrigerator overnight, and precipitate a solid , filtered (184mg, 23%)

MS: 362(M+1)

Embodiment 11: Synthesis of N-1-naphthyl-L-lysyl-L-proline dihydrobromide (compound 11)

Dissolve L-Lys(Cbz)-L-Pro-OH·TFA (491mg) obtained in Example 8 in methanol (5ml), cool to 0°C, add triethylamine (0.21ml), 1-naphthaldehyde (177mg ), stirred for 3 hours, added KBH ₄ (89 mg) in batches, and reacted for 3 hours. Purified by flash column, washed with diethyl ether, poured diethyl ether, and dried to obtain a white thick substance.

The thick product obtained in the previous step and anisole (0.5ml) were dissolved in 36% HBr acetic acid solution (4ml), reacted at room temperature, HPLC detected that the reaction was complete, added diethyl ether (20ml) to the reaction solution, placed in the refrigerator overnight, and the solid was precipitated. Filtration (249mg, 45.6%).

MS: 384(M+1)

Example 12: Synthesis of N-2-naphthyl-L-lysyl-L-proline dihydrobromide (compound 12)

Dissolve L-Lys(Cbz)-L-Pro-OH·TFA (491mg) obtained in Example 8 in methanol (5ml), cool to 0°C, add triethylamine (0.21ml), 2-naphthaldehyde (181mg ), stirred for 3 hours, added KBH ₄ (95 mg) in batches, and reacted for 3 hours. Purified by flash column, washed with diethyl ether, poured diethyl ether, and dried to obtain a white thick substance.

The thick product obtained in the previous step and anisole (0.5ml) were dissolved in 36% HBr acetic acid solution (4ml), reacted at room temperature, HPLC detected that the reaction was complete, added diethyl ether (20ml) to the reaction solution, placed in the refrigerator overnight, and the solid was precipitated. Filter (211 mg, 40%).

MS: 384(M+1)

Example 13: Synthesis of N-p-chlorobenzyl-L-lysyl-L-proline dihydrobromide (compound 13)

Dissolve L-Lys(Cbz)-L-Pro-OH·TFA (491mg) obtained in Example 8 in methanol (5ml), cool to 0°C, add triethylamine (0.21ml), p-chlorobenzaldehyde (150mg ), stirred for 3 hours, added KBH ₄ (88 mg) in batches, and reacted for 3 hours. Purified by flash column, washed with diethyl ether, poured diethyl ether, and dried to obtain a white thick substance.

The thick product obtained in the previous step and anisole (0.5ml) were dissolved in 36% HBr acetic acid solution (4ml), reacted at room temperature, HPLC detected that the reaction was complete, added diethyl ether (20ml) to the reaction solution, placed in the refrigerator overnight, and the solid was precipitated. Filter (186 mg, 35%).

MS: 369(M+1)

Example 14: Synthesis of N-p-fluorobenzyl-L-lysyl-D-proline dihydrobromide (compound 14)

Dissolve L-Lys(Cbz)-L-Pro-OH·TFA (491mg) obtained in Example 8 in methanol (5ml), cool to 0°C, add triethylamine (0.21ml), p-fluorobenzaldehyde (143mg ), stirred for 3 hours, added KBH ₄ (97 mg) in batches, and reacted for 3 hours. Purified by flash column, washed with diethyl ether, poured diethyl ether, and dried to obtain a white thick substance.

The thick product obtained in the previous step and anisole (0.5ml) were dissolved in 36% HBr acetic acid solution (4ml), reacted at room temperature, HPLC detected that the reaction was complete, added diethyl ether (20ml) to the reaction solution, placed in the refrigerator overnight, and the solid was precipitated. Filter (224mg, 43.5%).

MS: 352(M+1)

Example 15: Synthesis of N-benzyl-L-lysyl-pyrrolidine dihydrobromide (compound 15)

a) Synthesis of Boc-L-Lys(Cbz)-pyrrolidine

Dissolve Boc-L-Lys(Cbz)OH·DCHA (1.9g) in 5% potassium bisulfate (15ml) solution and dichloromethane (15ml), stir for 0.5h, separate the layers, and wash the aqueous phase with two Extract with methyl chloride, combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain a light yellow oil.

Dissolve the freed Boc-L-Lys(Cbz)OH, pyrrolidine (367mg), N-methylmorpholine (719mg) in anhydrous dichloromethane (20ml), protect with nitrogen, cool to 0°C, add HOBt (811mg), EDCI (1.15g), stirred at 0°C for 20min, naturally warmed to room temperature, and reacted for 8 hours. The organic layer was washed successively with 5% potassium bisulfate solution, saturated sodium bicarbonate solution and saturated saline solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography to obtain a colorless oil (1.817 g, recovered rate: 83.8%).

Rf=0.8 developer ethyl acetate color development: ultraviolet and iodine

1H NMR: (CDCl ₃ )ppm: 1.44 (9H, s), 1.36-1.40 (2H, m), 1.47-1.62 (2H, m), 1.70-1.73 (2H, m), 1.85-1.90 (2H, m ), 1.94-1.99 (2H, m), 3.08-3.10 (2H, m), 3.38-3.44 (2H, m), 3.49-3.3.53 (2H, m), 4.45-4.47 (1H, m), 5.09 (2H, s), 7.26-7.35 (5H, m)

b) Synthesis of L-Lys(Cbz)-pyrrolidine·TFA

Dissolve Boc-L-Lys(Cbz)-pyrrolidine (867mg) in dichloromethane (10ml), cool in an ice bath to below 0°C, add TFA (8ml) dropwise, react at 0°C for 1h, and place the reaction solution at 25°C Concentrate under reduced pressure, add diethyl ether and wash to obtain an oily substance, pour off diethyl ether, and dry it to obtain a white viscous solid (850 mg, 98%).

Rf = 0.5 developing agent dichloromethane/methanol = 5/1 color development: ultraviolet and 1% ninhydrin solution

1H NMR: (CDCl ₃ )ppm: 1.34-1.38 (2H, m), 1.56-1.63 (2H, m), 1.69-1.73 (2H, m), 1.85-1.88 (4H, m), 2.99-3.18 (2H , m), 3.38-3.44 (4H, m), 3.61-3.63 (1H, m), 5.09 (2H, s), 7.26-7.35 (5H, m)

MS: 318(M+1)

c) Synthesis of N-benzyl-L-Lys--pyrrolidine·2HBr

Dissolve L-Lys(Cbz)-pyrrolidine·TFA (530mg) in methanol (10ml), cool to 0°C, add triethylamine (110mg) dropwise, add benzaldehyde (120mg), stir for 0.5h, and batch KBH ₄ (101 mg) was added to react for about 4 hours. HPLC detected that the reaction was complete, concentrated under reduced pressure to remove most of the solvent, and purified by column chromatography to obtain a white thick substance.

The thick product obtained in the previous step and anisole (0.5ml) were dissolved in 36% HBr/acetic acid solution (6ml), HPLC detected that the reaction was complete, ether (20ml) was added to the reaction solution, and the refrigerator was placed overnight to precipitate a solid. Filtration gave a white solid (264mg, 58%).

MS: 290(M+1)

Embodiment 16: Synthesis of N-benzyl-L-lysyl-piperidine dihydrobromide (compound 16)

a) Synthesis of Boc-L-Lys(Cbz)-piperidine

Dissolve Boc-L-Lys(Cbz)OH·DCHA (1.99g) in 5% potassium bisulfate (15ml) solution and dichloromethane (15ml), stir for 0.5h, separate the layers, and wash the aqueous phase with two Extract with methyl chloride, combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain a light yellow oil.

Dissolve the freed Boc-L-Lys(Cbz)OH, piperidine (434mg), N-methylmorpholine (720mg) in anhydrous dichloromethane (20ml), protect with nitrogen, cool to 0°C, add HOBt (834mg), EDCI (1.2g), stirred at 0°C for 20min, naturally warmed to room temperature, and reacted for 8 hours. The organic layer was washed successively with 5% potassium bisulfate solution, saturated sodium bicarbonate solution and saturated saline solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography to obtain a colorless oil (2 g, yield : 89%).

Rf=0.8 developer ethyl acetate color development: ultraviolet and iodine

1H NMR: (CDCl ₃ )ppm: 1.43 (9H, s), 1.31-1.35 (2H, m), 1.56-1.62 (8H, m), 1.73-1.75 (2H, m), 3.08-3.11 (2H, m ), 3.49-3.54 (2H, m), 4.45-4.47 (1H, m), 5.05 (2H, s), 7.33-7.45 (5H, m)

b) Synthesis of L-Lys(Cbz)-pyrrolidine·TFA

Dissolve Boc-L-Lys(Cbz)-piperidine (895mg) in dichloromethane (10ml), cool in an ice bath to below 0°C, add TFA (8ml) dropwise, react at 0°C for 1h, and place the reaction solution at 25°C Concentrate under reduced pressure, add diethyl ether to wash to obtain an oily substance, pour off diethyl ether, and dry it to obtain a white viscous solid (884 mg, 98%).

Rf = 0.5 developing agent dichloromethane/methanol = 5/1 color development: ultraviolet and 1% ninhydrin solution

1H NMR: (CDCl ₃ )ppm: 1.29-1.35 (2H, m), 1.56-1.63 (8H, m), 1.81-1.84 (2H, m), 3.09-3.13 (2H, m), 3.39-3.45 (2H , m), 3.58-3.64 (1H, m), 5.06 (2H, s), 7.33-7.45 (5H, m)

MS: 348(M+1)

c) Synthesis of N-benzyl-L-Lys-piperidine·2HBr

Dissolve L-Lys(Cbz)-piperidine·TFA (890mg) in methanol (10ml), cool to 0°C, add triethylamine (303mg) dropwise, add benzaldehyde (243mg), stir for 0.5h, batchwise KBH ₄ (106 mg) was added to react for about 4 hours. HPLC detected that the reaction was complete, concentrated under reduced pressure to remove most of the solvent, and purified by column chromatography to obtain a white thick substance.

The thick matter obtained in the previous step and anisole (0.65ml) were dissolved in 36% HBr/acetic acid solution (10ml), HPLC detected that the reaction was complete, ether (20ml) was added to the reaction solution, and the refrigerator was placed overnight to precipitate a solid. Filtration gave a white solid (455mg, 49%).

MS: 304(M+1)

Example 17: Synthesis of N-benzyl-L-lysyl-cyclohexylamine dihydrobromide (compound 17)

a) Synthesis of Boc-L-Lys(Cbz)-cyclohexylamine

Dissolve Boc-L-Lys(Cbz)OH·DCHA (1.9g) in 5% potassium bisulfate (15ml) solution and dichloromethane (15ml), stir for 0.5h, separate the layers, and wash the aqueous phase with two Extract with methyl chloride, combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain a light yellow oil.

Dissolve the freed Boc-L-Lys(Cbz)OH, cyclohexylamine (513mg), and N-methylmorpholine (719mg) in anhydrous dichloromethane (20ml), protect with nitrogen, and cool to 0°C. Added HOBt (817mg) and EDCI (1.15g), stirred at 0°C for 20min, naturally rose to room temperature, and reacted for 8 hours. The organic layer was washed successively with 5% potassium bisulfate solution, saturated sodium bicarbonate solution and saturated saline solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography to obtain a colorless oil (1.929 g, recovered rate: 81.6%).

Rf=0.5 developing agent ethyl acetate/petroleum ether=1/1 color development: ultraviolet and iodine

MS: 476(M+1)

b) Synthesis of L-Lys(Cbz)-cyclohexylamine·TFA

Dissolve Boc-L-Lys(Cbz)-cyclohexylamine (922mg) in dichloromethane (10ml), cool in an ice bath to below 0°C, add TFA (8ml) dropwise, and react at 0°C for 1h. Concentrate under reduced pressure at ℃, add diethyl ether to wash to obtain an oily substance, pour off diethyl ether, and dry it to obtain a white viscous solid (866 mg, 94%). Rf=0.5 developing agent dichloromethane/methanol=5/1 color development: ultraviolet and 1% ninhydrin solution MS: 362 (M+1)

c) Synthesis of N-benzyl-L-Lys-cyclohexylamine·2HBr

Dissolve L-Lys(Cbz)-cyclohexylamine·TFA (464mg) in methanol (5ml), cool to 0°C, add triethylamine (146mg) dropwise, add benzaldehyde (120mg), stir for 0.5h, divide KBH ₄ (67 mg) was added in batches to react for about 4 hours. HPLC detected the completion of the reaction, concentrated under reduced pressure to remove most of the solvent, and purified by column chromatography to obtain a white thick substance.

The thick matter obtained in the previous step and anisole (0.5ml) were dissolved in 36% HBr/acetic acid solution (4ml), HPLC detected that the reaction was complete, ether (20ml) was added to the reaction solution, and the refrigerator was placed overnight to precipitate a solid. Filtration gave a white solid (218mg, 45%).

MS: 318(M+1)

Example 18: Synthesis of N-benzyl-L-lysyl-cycloheptylamine dihydrobromide (compound 18)

a) Synthesis of Boc-L-Lys(Cbz)-Cycloheptylamine

Dissolve Boc-L-Lys(Cbz)OH·DCHA (1.9g) in 5% potassium bisulfate (15ml) solution and dichloromethane (15ml), stir for 0.5h, separate the layers, and wash the aqueous phase with two Extract with methyl chloride, combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain a light yellow oil.

Dissolve the freed Boc-L-Lys(Cbz)OH, cyclohexylamine (590mg), and N-methylmorpholine (719mg) in anhydrous dichloromethane (20ml), protect with nitrogen, and cool to 0°C. Added HOBt (814mg) and EDCI (1.15g), stirred at 0°C for 20min, naturally rose to room temperature, and reacted for 8 hours. The organic layer was washed successively with 5% potassium bisulfate solution, saturated sodium bicarbonate solution and saturated saline solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography to obtain a colorless oil (1.845 g, recovered rate: 75.4%).

Rf=0.55 developing agent ethyl acetate/petroleum ether=1/1 color development: ultraviolet and iodine

MS: 490(M+1)

b) Synthesis of L-Lys(Cbz)-Cycloheptylamine·TFA

Dissolve Boc-L-Lys(Cbz)-cyclohexylamine (950mg) in dichloromethane (10ml), cool in an ice bath to below 0°C, add TFA (8ml) dropwise, react at 0°C for 1h, and place the reaction solution at 25 Concentrate under reduced pressure at ℃, add diethyl ether to wash to obtain an oily substance, pour off diethyl ether, and dry it to obtain a white viscous solid (879 mg, 93%). Rf = 0.5 developing agent dichloromethane/methanol = 5/1 color development: ultraviolet and 1% ninhydrin solution

MS: 376(M+1)

c) Synthesis of N-Benzyl-L-Lys-Cycloheptylamine·2HBr

Dissolve L-Lys(Cbz)-cyclohexylamine·TFA (476mg) in methanol (5ml), cool to 0°C, add triethylamine (146mg) dropwise, add benzaldehyde (120mg), stir for 0.5h, divide KBH ₄ (67 mg) was added in batches to react for about 4 hours. HPLC detected the completion of the reaction, concentrated under reduced pressure to remove most of the solvent, and purified by column chromatography to obtain a white thick substance.

The thick matter obtained in the previous step and anisole (0.5ml) were dissolved in 36% HBr/acetic acid solution (4ml), HPLC detected that the reaction was complete, ether (20ml) was added to the reaction solution, and the refrigerator was placed overnight to precipitate a solid. Filtration gave a white solid (197 mg, 40%).

MS: 332(M+1)

Example 19: Synthesis of N-benzyl-L-lysyl-L-homoproline dihydrobromide (compound 19)

a) Synthesis of Boc-L-Lys(Cbz)-L-Hpro-OCH ₃

Dissolve Boc-L-Lys(Cbz)OH·DCHA (5.6g) in 5% potassium bisulfate (60ml) solution and dichloromethane (50ml), stir for 0.5h, separate the layers, and wash the water phase with dichloromethane After extraction, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a light yellow oil.

Dissolve the freed Boc-L-Lys(Cbz)OH, L-homoproline methyl ester hydrochloride (2g), diisopropylethylamine (3.2g) with anhydrous dichloromethane (50ml), Under nitrogen protection, after cooling to 0°C, HOBt (1.35g) and EDCI (2.3g) were added, stirred at 0°C for 20min, then naturally raised to room temperature, and reacted for 8 hours. The organic layer was washed successively with 5% potassium bisulfate solution, saturated sodium bicarbonate solution and saturated saline solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography to obtain a colorless oil (1.4 g, harvested rate: 27.7%).

Rf=0.8 developer ethyl acetate color development: ultraviolet and iodine

MS: 506(M+1)

b) Synthesis of B oc-L-Lys(Cbz)-L-Hpro-OH

Dissolve Boc-L-Lys(Cbz)-L-Hpro-OCH ₃ (1.4g) in methanol (10ml), cool to 0°C, add sodium hydroxide (400mg), react for 2 hours and adjust with 5% HCl solution To pH 4, concentrated under reduced pressure to remove the methanol solvent, then extracted with ethyl acetate, the obtained organic phase was dried, concentrated under reduced pressure to obtain a colorless thick product (800mg, 59%).

Rf=0.6 developing agent ethyl acetate color development: ultraviolet and 1% ninhydrin solution

MS: 492 (M-)

c) Synthesis of L-Lys(Cbz)-L-Hpro-OH·TFA

Dissolve Boc-L-Lys(Cbz)-L-Hpro-OH (800mg) in dichloromethane (15ml), cool in an ice bath to below 0°C, add TFA (5ml) dropwise, react at 0°C for 1 hour, and decompress Concentrate, add diethyl ether and wash to obtain an oily substance, pour off diethyl ether, and suck dry to obtain a white viscous solid (609 mg, 77%).

Rf = 0.5 developing agent dichloromethane/methanol = 5/1 color development: ultraviolet and 1% ninhydrin solution

MS: 392 (M-)

d) Synthesis of Nα-Benzyl-L-Lys-L-Hpro-OH·2HBr

Dissolve L-Lys(Cbz)-L-Hpro-OH·TFA (500mg) in methanol (8ml), cool to 0°C, add diisopropylethylamine (193mg), benzaldehyde (127mg), and stir for 3 hours , added KBH4 (76mg) in batches, and reacted for 3 hours. Purified by flash column, washed with diethyl ether, poured diethyl ether, and dried to obtain a white thick substance.

The thick product obtained in the previous step and anisole (0.5ml) were dissolved in 36% HBr acetic acid solution (4ml), reacted at room temperature, HPLC detected that the reaction was complete, added diethyl ether (20ml) to the reaction solution, placed in the refrigerator overnight, and the solid was precipitated. Filter (159 mg, 31%).

MS: 348(M+1)

Example 20: Synthesis of N-benzyl-L-lysyl-D-homoproline dihydrobromide (compound 20)

a) Synthesis of Boc-L-Lys(Cbz)-D-Hpro-OCH ₃

Dissolve Boc-L-Lys(Cbz)OH·DCHA (5.6g) in 5% potassium bisulfate (60ml) solution and dichloromethane (50ml), stir for 0.5h, separate the layers, and wash the water phase with dichloromethane After extraction, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a light yellow oil. Dissolve the freed Boc-L-Lys(Cbz)OH, D-homoproline methyl ester hydrochloride (2g), diisopropylethylamine (3.2g) with anhydrous dichloromethane (50ml), Under nitrogen protection, after cooling to 0°C, HOBt (1.35g) and EDCI (2.3g) were added, stirred at 0°C for 20min, then naturally raised to room temperature, and reacted for 8 hours. The organic layer was washed successively with 5% potassium bisulfate solution, saturated sodium bicarbonate solution and saturated saline solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography to obtain a colorless oil (1.6 g, harvested rate: 29%).

Rf=0.8 developer ethyl acetate color development: ultraviolet and iodine

MS: 506(M+1)

b) Synthesis of Boc-L-Lys(Cbz)-D-Hpro-OH

Dissolve Boc-L-Lys(Cbz)-D-Hpro-OCH ₃ (1.6g) in methanol (10ml), cool to 0°C, add sodium hydroxide (420mg), react for 2 hours and adjust with 5% HCl solution To pH 4, concentrated under reduced pressure to remove the methanol solvent, then extracted with ethyl acetate, the obtained organic phase was dried, concentrated under reduced pressure to obtain a colorless thick product (938mg, 69%).

Rf=0.6 developing agent ethyl acetate color development: ultraviolet and 1% ninhydrin solution

MS: 492 (M-)

c) Synthesis of L-Lys(Cbz)-D-Hpro-OH·TFA

Dissolve Boc-L-Lys(Cbz)-D-Hpro-OH (800mg) in dichloromethane (15ml), cool in an ice bath to below 0°C, add TFA (5ml) dropwise, react at 0°C for 1 hour, and decompress Concentrate, add diethyl ether and wash to obtain an oily substance, pour off diethyl ether, and suck dry to obtain a white viscous solid (711mg, 89%).

Rf = 0.5 developing agent dichloromethane/methanol = 5/1 color development: ultraviolet and 1% ninhydrin solution

MS: 392 (M-)

d) Synthesis of Nα-Benzyl-L-Lys-D-Hpro-OH·2HBr

Dissolve L-Lys(Cbz)-D-Hpro-OH·TFA (500mg) in methanol (8ml), cool to 0°C, add diisopropylethylamine (195mg), benzaldehyde (121mg), and stir for 3 hours , added KBH4 (76mg) in batches, and reacted for 3 hours. Purified by flash column, washed with diethyl ether, poured diethyl ether, and dried to obtain a white thick substance.

The thick product obtained in the previous step and anisole (0.5ml) were dissolved in 36% HBr acetic acid solution (4ml), reacted at room temperature, HPLC detected that the reaction was complete, added diethyl ether (20ml) to the reaction solution, placed in the refrigerator overnight, and the solid was precipitated. Filter (182mg, 36%).

MS: 348(M+1)

Example 21: Synthesis of N-benzyl-L-lysyl-(R-3-formyl)piperidine dihydrobromide (compound 21)

a) Synthesis of Boc-L-Lys(Cbz)-(R-3-formyl)piperidine ethyl ester

Dissolve Boc-L-Lys(Cbz)OH·DCHA (1.9g) in 5% potassium bisulfate (20ml) solution and dichloromethane (20ml), stir for 0.5h, separate the layers, and wash the aqueous phase with dichloromethane After extraction, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a light yellow oil.

Dissolve the freed Boc-L-Lys(Cbz)OH, R-3-piperidinecarboxylate ethyl ester hydrochloride (1g), diisopropylethylamine (1.5g) with anhydrous dichloromethane (20ml) , nitrogen protection, after cooling to 0°C, add HOBt (700mg), EDCI (1.1g), stir at 0°C for 20min, naturally rise to room temperature, react for 8 hours. The organic layer was washed successively with 5% potassium bisulfate solution, saturated sodium bicarbonate solution and saturated saline solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography to obtain a colorless oil (1.729 g, recovered rate: 67%).

Rf=0.8 developer ethyl acetate color development: ultraviolet and iodine

MS: 520(M+1)

b) Synthesis of Boc-L-Lys(Cbz)-(R-3-formyl)piperidine

Dissolve Boc-L-Lys(Cbz)-(R-3-formyl)piperidine ethyl ester (1g) in methanol (10ml), cool to 0°C, add sodium hydroxide (400mg), react for 2 hours and use The 5% HCl solution was adjusted to pH 4, concentrated under reduced pressure to remove the methanol solvent, and then extracted with ethyl acetate. The resulting organic phase was dried and concentrated under reduced pressure to obtain a colorless thick product (968mg, 98%).

Rf=0.6 developing agent ethyl acetate color development: ultraviolet and 1% ninhydrin solution

MS: 492 (M-)

c) Synthesis of L-Lys(Cbz)-(R-3-formyl)piperidine·TFA

Dissolve Boc-L-Lys(Cbz)-(R-3-formyl)piperidine (563mg) in dichloromethane (15ml), cool in an ice bath to below 0°C, add TFA (5ml) dropwise, and react at 0°C Concentrate under reduced pressure for 1 hour, add diethyl ether to wash to obtain an oily substance, pour off diethyl ether, and dry it to obtain a white sticky solid (507 mg, 90%).

Rf = 0.5 developing agent dichloromethane/methanol = 5/1 color development: ultraviolet and 1% ninhydrin solution

MS: 392 (M-)

d) Synthesis of N-benzyl-L-lysyl-(R-3-formyl)piperidine dihydrobromide

Dissolve L-Lys(Cbz)-(R-3-formyl)piperidine·TFA (507mg) in methanol (8ml), cool to 0°C, add diisopropylethylamine (195mg), benzaldehyde (121mg ), stirred for 3 hours, added KBH4 (76mg) in batches, and reacted for 3 hours. Purified by flash column, washed with diethyl ether, poured diethyl ether, and dried to obtain a white thick substance.

The thick product obtained in the previous step and anisole (0.5ml) were dissolved in 36% HBr acetic acid solution (4ml), reacted at room temperature, HPLC detected that the reaction was complete, added diethyl ether (20ml) to the reaction solution, placed in the refrigerator overnight, and the solid was precipitated. Filtration (241 mg, 47%).

MS: 348(M+1)

Example 22: Synthesis of N-benzyl-L-lysyl-(S-3-formyl)piperidine dihydrobromide (compound 22)

a) Synthesis of Boc-L-Lys(Cbz)-(S-3-formyl)piperidine ethyl ester

Dissolve Boc-L-Lys(Cbz)OH·DCHA (1.9g) in 5% potassium bisulfate (20ml) solution and dichloromethane (20ml), stir for 0.5h, separate the layers, and wash the aqueous phase with dichloromethane After extraction, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a light yellow oil.

Dissolve the freed Boc-L-Lys(Cbz)OH, S-3-piperidinecarboxylate ethyl ester hydrochloride (1g), diisopropylethylamine (1.5g) with anhydrous dichloromethane (20ml) , nitrogen protection, after cooling to 0°C, add HOBt (700mg), EDCI (1.1g), stir at 0°C for 20min, naturally rise to room temperature, react for 8 hours. The organic layer was washed successively with 5% potassium bisulfate solution, saturated sodium bicarbonate solution and saturated saline solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography to obtain a colorless oil (1.826 g, recovered rate: 70%).

Rf=0.8 developer ethyl acetate color development: ultraviolet and iodine

MS: 520(M+1)

b) Synthesis of Boc-L-Lys(Cbz)-(S-3-formyl)piperidine

Dissolve Boc-L-Lys(Cbz)-(S-3-formyl)piperidine ethyl ester (1g) in methanol (10ml), cool to 0°C, add sodium hydroxide (400mg), react for 2 hours and use The 5% HCl solution was adjusted to pH 4, concentrated under reduced pressure to remove the methanol solvent, and then extracted with ethyl acetate. The resulting organic phase was dried and concentrated under reduced pressure to obtain a colorless thick product (968mg, 98%).

Rf=0.6 developing agent ethyl acetate color development: ultraviolet and 1% ninhydrin solution

MS: 492 (M-)

c) Synthesis of L-Lys(Cbz)-(S-3-formyl)piperidine·TFA

Dissolve Boc-L-Lys(Cbz)-(S-3-formyl)piperidine (551mg) in dichloromethane (15ml), cool in an ice bath to below 0°C, add TFA (5ml) dropwise, and react at 0°C Concentrate under reduced pressure for 1 hour, add diethyl ether to wash, and obtain an oily substance, pour off diethyl ether, and dry it to obtain a white sticky solid (500 mg, 91%).

Rf = 0.5 developing agent dichloromethane/methanol = 5/1 color development: ultraviolet and 1% ninhydrin solution

MS: 392 (M-)

d) Synthesis of N-benzyl-L-lysyl-(S-3-formyl)piperidine dihydrobromide

Dissolve L-Lys(Cbz)-(S-3-formyl)piperidine·TFA (500mg) in methanol (8ml), cool to 0°C, add diisopropylethylamine (195mg), benzaldehyde (122mg ), stirred for 3 hours, added KBH4 (86mg) in batches, and reacted for 3 hours. Purified by flash column, washed with diethyl ether, poured diethyl ether, and dried to obtain a white thick substance.

The thick product obtained in the previous step and anisole (0.5ml) were dissolved in 36% HBr acetic acid solution (4ml), reacted at room temperature, HPLC detected that the reaction was complete, added diethyl ether (20ml) to the reaction solution, placed in the refrigerator overnight, and the solid was precipitated. Filter (235 mg, 46%).

MS: 348(M+1)

Example 23: Synthesis of N-benzyl-L-arginyl-D-proline dihydrobromide (compound 23)

a) Synthesis of Boc-L-Arg(Cbz) ₂ -D-Pro-OCH ₃

Boc-L-Arg(Cbz) ₂ -OH (2.7g), D-proline methyl ester hydrochloride (833mg), diisopropylethylamine (1.5g) were mixed with anhydrous dichloromethane (30ml) Dissolved, protected by nitrogen, cooled to 0°C, added HOBt (690mg), EDCI (1.1g), stirred at 0°C for 20min, naturally rose to room temperature, and reacted for 8 hours. The organic layer was washed successively with 5% potassium bisulfate solution, saturated sodium bicarbonate solution and saturated saline solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography to obtain a colorless oil (2.1 g, harvested rate: 65%).

Rf=0.5 developing agent ethyl acetate color development: ultraviolet and iodine

MS: 654(M+1)

b) Synthesis of Boc-L-Arg(Cbz) ₂ -D-Pro-OH

Dissolve Boc-L-Arg(Cbz) ₂ -D-Pro-OCH ₃ (1g) in methanol (8ml), cool to 0°C, add sodium hydroxide (300mg), react for 2 hours and adjust with 5% HCl solution To pH 4, concentrated under reduced pressure to remove the methanol solvent, then extracted with ethyl acetate, the obtained organic phase was dried, concentrated under reduced pressure to obtain a colorless thick product (896 mg, 92%).

Rf=0.4 developing agent dichloromethane/methanol=40/1 color development: ultraviolet and 1% ninhydrin solution

MS: 641(M-)

c) Synthesis of L-Arg(Cbz) ₂ -D-Pro-OH·TFA

Boc-L-Arg(Cbz) ₂ -D-Pro-OH (700mg) was dissolved in dichloromethane (15ml), cooled in an ice bath to below 0°C, TFA (5ml) was added dropwise, reacted at 0°C for 1 hour, and Concentrate under reduced pressure, add diethyl ether to wash to obtain an oily substance, pour off diethyl ether, and dry it to obtain a white viscous solid (637 mg, 91%).

Rf=0.2 developing agent dichloromethane/methanol=5/1 color development: ultraviolet and 1% ninhydrin solution

MS: 540 (M-)

d) Synthesis of N-benzyl-L-arginyl-D-proline dihydrobromide

Dissolve L-Arg(Cbz) ₂ -D-Pro-OH·TFA (637mg) in methanol (8ml), cool to 0°C, add diisopropylethylamine (190mg), benzaldehyde (121mg), and stir for 3 Hours, KBH4 (82mg) was added in portions, and the reaction was carried out for 3 hours. Purified by flash column, washed with diethyl ether, poured diethyl ether, and dried to obtain a white thick substance.

The thick product obtained in the previous step and anisole (0.5ml) were dissolved in 36% HBr acetic acid solution (4ml), reacted at room temperature, HPLC detected that the reaction was complete, added diethyl ether (20ml) to the reaction solution, placed in the refrigerator overnight, and the solid was precipitated. Filter (268mg, 51%).

MS: 362(M+1)

Example 24: Synthesis of N-benzyl-L-homoarginyl-D-proline dihydrobromide (compound 24)

a) Synthesis of Boc-L-Harg(Cbz) ₂ -D-Pro-OCH ₃

Boc-L-Harg (Cbz) ₂ -OH (2.7g), D-proline methyl ester hydrochloride (828mg), diisopropylethylamine (1.4g) were mixed with anhydrous dichloromethane (30ml) Dissolved, protected by nitrogen, cooled to 0°C, added HOBt (700mg), EDCI (1.15g), stirred at 0°C for 20min, naturally rose to room temperature, and reacted for 8 hours. The organic layer was washed successively with 5% potassium bisulfate solution, saturated sodium bicarbonate solution and saturated saline solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography to obtain a colorless oil (2.5 g, harvested rate: 75%).

Rf=0.6 developing agent ethyl acetate color development: ultraviolet and iodine

MS: 669(M+1)

b) Synthesis of Boc-L-Harg(Cbz) ₂ -D-Pro-OH

Dissolve Boc-L-Harg(Cbz) ₂ -D-Pro-OCH ₃ (1g) in methanol (8ml), cool to 0°C, add sodium hydroxide (300mg), react for 2 hours and adjust with 5% HCl solution To pH 4, concentrated under reduced pressure to remove the methanol solvent, then extracted with ethyl acetate, the obtained organic phase was dried, concentrated under reduced pressure to obtain a colorless thick product (860mg, 88%).

Rf=0.4 developing agent dichloromethane/methanol=40/1 color development: ultraviolet and 1% ninhydrin solution

MS: 655(M-)

c) Synthesis of L-Harg(Cbz) ₂ -D-Pro-OH·TFA

Dissolve Boc-L-Harg(Cbz) ₂ -D-Pro-OH (700mg) in dichloromethane (15mD, cool in an ice bath to below 0°C, add TFA (5ml) dropwise, react at 0°C for 1 hour, and depressurize Concentrate, add diethyl ether and wash to obtain an oily substance, pour off diethyl ether, and suck dry to obtain a white viscous solid (610 mg, 87%).

Rf=0.2 developing agent dichloromethane/methanol=5/1 color development: ultraviolet and 1% ninhydrin solution

MS: 554(M-)

d) Synthesis of N-benzyl-L-homoarginyl-D-proline dihydrobromide

Dissolve L-Harg(Cbz) ₂ -D-Pro-OH·TFA (610mg) in methanol (8ml), cool to 0°C, add diisopropylethylamine (185mg), benzaldehyde (115mg), and stir for 3 Hours, KBH4 (78 mg) was added in portions, and the reaction was carried out for 3 hours. Purified by flash column, washed with diethyl ether, poured diethyl ether, and dried to obtain a white thick substance.

The thick product obtained in the previous step and anisole (0.5ml) were dissolved in 36% HBr acetic acid solution (4ml), reacted at room temperature, HPLC detected that the reaction was complete, added diethyl ether (20ml) to the reaction solution, placed in the refrigerator overnight, and the solid was precipitated. Filtration (246 mg, 49%).

84 mg of compound 24 were prepared from Boc-L-Harg(Cbz) ₂ -OH by methods substantially equivalent to those described in Example 1-d to Example-g.

MS: 376(M+1)

Example 25: Determination of Antithrombin Activity

In the experiment, the sample to be tested was added to the enzyme reaction system containing 5.4 μg/ml freeze-dried human thrombin national standard purified from human blood and incubated for 30 minutes, then the specific substrate Ac -FVR-AMC (5μM), using the microplate detector Envision (PerkinElmer), at room temperature, dynamically detect the change of RFU (relative fluorescence unit, relative fluorescence intensity unit) within 10 minutes, and the enzyme kinetics is linear at the beginning of the reaction The slope of the chemical curve is the enzyme reaction initial velocity (RFU/sec).

Calculate the inhibition rate (%Inhibition) of the sample to the enzyme activity according to formula 1,

$<span\; class="notranslate">\; \%</span>\mathrm{<span\; class="notranslate">\; Inhibition</span>}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; v</span>}}_{\mathrm{<span\; class="notranslate">\; DMSO</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; v</span>}}_{\mathrm{<span\; class="notranslate">\; Sample</span>}}}{{\mathrm{<span\; class="notranslate">\; v</span>}}_{\mathrm{<span\; class="notranslate">\; D.</span>}\mathrm{<span\; class="notranslate">\; MSO</span>}}}<span\; class="notranslate">\; \&times;</span>\mathrm{<span\; class="notranslate">\; 100</span>}<span\; class="notranslate">\; \%</span>$

Formula 1

Where v _Sample represents the initial velocity of the drug-dosing group, and _vDMSO represents the initial velocity of the DMSO group (that is, the non-medicine group). The IC ₅₀ value (the concentration of the drug when the enzyme activity is inhibited by 50%) is calculated by nonlinear fitting of the inhibition rate (%Inhibition) to the logarithmic value X of the sample concentration by formula 2.

$<span\; class="notranslate">\; \%</span>\mathrm{<span\; class="notranslate">\; Inhibition</span>}<span\; class="notranslate">\; =</span>\frac{\mathrm{<span\; class="notranslate">\; 100</span>}}{{\mathrm{<span\; class="notranslate">\; 1</span>}<span\; class="notranslate">\; +</span>\mathrm{<span\; class="notranslate">\; 10</span>}}^{<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; LogIC</span>}\mathrm{<span\; class="notranslate">\; 50</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; x</span>}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; *</span>\mathrm{<span\; class="notranslate">\; h</span>}}}$

Formula 2

where h represents the Hill coefficient

In the table below, the _IC50 values of the compounds of the invention are listed

It should be noted that when n=1, A=Lys or Arg, the compound with a six-membered ring structure in the B part has a stronger inhibitory activity on thrombin, especially when A is Arg, the intensity of thrombin inhibition is obviously improved.

The following formulation examples are illustrative only and are not intended to limit the scope of the invention in any way. The active ingredient refers to a compound of general formula (I) or a pharmaceutically acceptable salt or solvate thereof.

Embodiment 26: the preparation of injection

prescription:

Appropriate dilute acids or bases or buffer salts may be added to adjust the pH to the most stable state, and antioxidants or metal chelating agents may also be included. The solution is sterilized by filtration and, under aseptic conditions, filled into sterile ampoules.

Example 27: Preparation of Tablets

prescription:

The active ingredient is thoroughly mixed with the excipients, sieved, and compressed on a tablet machine.

Embodiment 28: the preparation of hard capsule

prescription:

The active ingredient is sieved, mixed with the excipients, and, using suitable equipment, the mixture is filled into hard gelatin capsules.

Example 29: Preparation of Suspension

prescription:

The active ingredient is sieved, mixed with sodium carboxymethylcellulose and syrup to form a homogeneous paste, the pigment, benzoic acid is diluted with a part of purified water, added to the paste with stirring, and then enough water to create the required volume.

Although the invention has been described with specific examples, it should be understood that it can be further modified. This application will include any change, use, or follow-up of the invention which generally departs from and includes departures from the disclosure but which are known or conventional in the field of the invention. And the same applies to the above-mentioned essential features, and is included in the scope of the appended claims.

Claims (11)

1. A peptide compound represented by formula (I) or a pharmaceutically acceptable salt thereof:

in, n stands for 1~3; A stands for Arg or Harg; R ₁ represents an aryl group; B is for

in, Z represents H, carboxyl, methyl carboxyl, carbamoyl or aminoacetyl; m stands for 1 or 2. 2. The peptide compound according to claim 1, wherein the aryl group is substituted or unsubstituted phenyl or naphthyl, wherein the substituents are hydroxyl, carboxyl, halogen, alkoxy, amino, alkylamino , dialkylamino group, cyano group, ester group, trifluoromethyl group or C1~4 alkyl group with the above groups. 3. The peptide compound according to claim 1, characterized in that, the peptide compound is the following compounds: N-benzyl-L-lysyl-L-proline, N-benzyl-L-lysine Aminoacyl-D-proline, N-phenylethyl-L-lysyl-L-proline, N-phenylethyl-L-lysyl-D-proline, N-phenylpropyl -L-lysyl-L-proline, N-phenylpropyl-L-lysyl-D-proline, N-p-chlorobenzyl-L-lysyl-L-proline, N-p-chlorobenzyl-L-lysyl-D-proline, N-p-fluorobenzyl-L-lysyl-L-proline, N-p-fluorobenzyl-L-lysyl -D-proline, N-1-naphthyl-L-lysyl-L-proline, N-1-naphthyl-L-lysyl-D-proline, N-2-naphthalene Base-L-lysyl-L-proline, N-2-naphthyl-L-lysyl-D-proline, N-benzyl-L-lysyl-pyrrolidine, N-benzyl Base-L-lysyl-piperidine, N-benzyl-L-lysyl-cyclohexylamine, N-benzyl-L-lysyl-cyclohexylamine, N-benzyl-L-lysine Acyl-L-homoproline, N-benzyl-L-lysyl-D-homoproline, N-benzyl-L-lysyl-(R-3-formyl)piperidine, N -Benzyl-L-lysyl-(S-3-formyl)piperidine, N-benzyl-L-arginyl-D-proline or N-benzyl-L-N-benzyl-L- Homoarginyl-D-proline. 4. The preparation method of the peptide compound according to any one of claims 1-3, characterized in that the method comprises the step of preparing the final product from an amino acid whose nitrogen position is protected by a protecting group as a raw material. 5. The preparation method according to claim 4, wherein the protecting group is selected from tert-butoxycarbonyl, benzyloxycarbonyl or fluorenyloxycarbonyl. 6. Use of the peptide compound according to any one of claims 1-3 in the preparation of thrombin inhibitors. 7. Use of the peptide compound according to any one of claims 1-3 in the preparation of medicines for treating and preventing thrombin-mediated and thrombin-related diseases. 8. The application according to claim 7, characterized in that the disease is venous thrombosis and pulmonary embolism, arterial thrombosis, stroke and peripheral arterial formation caused by thrombosis, atherosclerotic disease, cerebral arteriopathy or Peripheral Artery Disease. 9. Use of the peptide compound according to any one of claims 1-3 as an anticoagulant in extracorporeal blood lines. 10. The pharmaceutical composition, characterized in that it comprises the peptide compound according to any one of claims 1-3 and a pharmaceutically acceptable carrier. 11. The pharmaceutical composition according to claim 10, characterized in that it is tablet, injection, suspension or capsule.