CN102924567B - Peptide compound and preparation method and use of peptide compound - Google Patents

Abstract

The invention discloses a novel peptide compound shown as a formula (I), pharmaceutically acceptable salts, pharmaceutically acceptable solvates, derivatives, isomers, crystal forms and pharmaceutically acceptable pro-drugs of the peptide compound. The invention also discloses a preparation method of the compound shown as the formula (I). The invention further discloses application of the peptide compound in preparation of drugs for treating and preventing thrombin mediated diseases and diseases related to thrombin.

Description

Peptide compound, Preparation Method And The Use

The application is the divisional application of the patent application that application number is 200810201891.1, the applying date is on October 28th, 2008, denomination of invention is " peptide compound, Preparation Method And The Use ".

Technical field

The present invention relates to the method for a class to the inhibited novel competitive inhibitor of Trypsin serine protease, particularly zymoplasm, synthetic this competitive inhibitor and in the purposes of preparing in medicine.Specifically, the present invention relates to have the peptide compounds of anti-thrombosis activity.

Background technology

The mortality ratio of cardiovascular and cerebrovascular diseases occupy second always in the world, and wherein thromboembolism is to cause the morbidity of height ratio cardiovascular and cerebrovascular diseases and main causes of death.Especially along with variation and the aging population degree of people life style are increasingly sharpened, the sickness rate of this class disease presents the trend that continues rising.This makes to explore and the medicine of this type of disease of the effective control of research seems particularly urgent, aspect clinical application or fundamental research, is all being significant.

Traditional anticoagulant of current clinical use, as heparin, warfarin and r-hirudin etc. exist major defect aspect efficacy and saferry, is very limited its application.As heparin must be injected use, and to the zymoplasm in blood clot without effect, and can cause thrombopenia symptom; Warfarin is unique oral pharmaceutical, but owing to there is no clear and definite drug targets, individual anticoagulant response differs greatly, and influence factor is many, need carry out coagulation function monitoring.Therefore the Syncumar medicine with Orally active of researching and developing synthetic is very important.

Blood coagulation is the result of the serial enzymes effect of a complexity, and wherein a crucial step is that prothrombin activation generates zymoplasm.Zymoplasm is a kind of serine protease of tryptase, and Main Function is the insoluble fibrous solidifying compound of the former generation of hydrolysis of fibrin.In blood coagulation cascade reaction, play a crucial role.Therefore, the activity of Trombin inhibiting, just can block the formation of thrombus.Because its target spot is clear and definite and different from the action target spot of traditional anticoagulant, direct thrombin inhibitor (DTI) is expected to overcome the application limitation of traditional anticoagulation.Clearly, convenient treatment control, the effective as selective to thrombus stroke and the thrombin inhibitors with oral biological effect have represented the target of a very attractive.

Three ten years in the past, obtain many great progress using the thrombin inhibitors of synthetic as anticoagulant active research, and the small molecules thrombin inhibitors of a large amount of high reactivities and highly selective is reported.As reported D-Phe-Pro-Arg-H and Me-D-Phe-Pro-Arg-H aldehydic tripeptide Thrombin-like enzyme inhibitor in US4346078.Recently, D-Phe-Pro-agmatine and derivative thereof have been described in US4346078 and WO9311152 as thrombin inhibitors.Afterwards, in WO9429336 and WO9523609, report again a kind of tripeptides type inhibitor, mixed 4-amidino benzylamines in P1 position to replace agmatine etc.

In US4101653; Okamoto etc. disclose taking N-p-toluenesulfonyl arginine methyl esters (TAMA) as zymoplasm substrate; serial arginine derivative has been synthesized in design, finds that argatroban (argatroban) has good anticoagulant enzymic activity.Go on the market as injection in calendar year 2001 FDA approval through pharmacology clinical study, argatroban is a kind of reversible competitive thrombin inhibitors, is combined and plays a role with the reactive site of zymoplasm, and zymoplasm is had to high selectivity.Clinical periphery thrombus disease and the acute apoplexy of being used for the treatment of, also can be used for thrombopenia and thrombus syndrome that heparin brings out.

Equally, the structure that the investigator of Hoffmann La Roche company presses TAMA, the weak benzenyl amidine compounds of design synthesizing series alkalescence, further structure of modification is found strong one times (the J Med Chem of anticoagulant activity ratio argatroban of NAPAP, 1994,37,3889).The series compound that the benzenyl amidine of NAPAP is changed into piperidines carbonamidine also has higher anticoagulant enzymic activity, wherein the synthetic napsagatran activity of Hoffmann-La Roche company is the strongest, the former proteolytic enzyme of fibrin is also had to effect, once carried out II phase clinical study, but the transformation period is short, oral administration biaavailability is poor.

Astra company discloses a class benzenyl amidine analogue at WO9429336, wherein Melagatran (melagatran) has by force and selective inhibition of coagulation enzymic activity, without obviously bleeding problems can be safely for degree of depth phlebothrombosis (DVT), but oral administration biaavailability is low, the uncommon Melagatran (ximelagatran) of further synthetic its bi precursor medicine, in listing in 2004.First oral anticoagulation medicine in more than 60 year after warfarin, but listing after at clinical middle discovery severe liver injury, stop in February, 2006.

In addition, in WO9311152, WO9715190, US5510369, reported class thrombin inhibitors with unique D-diphenyl glycine on P3 position.It is reported that this compounds is compared with corresponding D-PG analogue, antithrombin activity higher (J Med Chem, 1997,40,830), some compounds wherein have good oral bioavailability rate (J Med Chem, 1997,40,3687; J Med Chem, 1997,40,3726).

In the compound of having reported, only have minority to there is pharmacokinetics and pharmacodynamic properties in applicable body.Up to now, the research of thrombin inhibitors remains in current pharmaceutical chemistry field one of the fiercest research topic of competition.

Summary of the invention

The technical problem existing for above-mentioned prior art, we have carried out sufficient research, have found that a class has the novel peptide compounds of good thrombin-inhibiting activity.

First object of the present invention is to provide the peptide compounds shown in the formula (I) of a class novelty, its pharmacy acceptable salt, pharmaceutically acceptable solvate, derivative, isomer, crystal formation and the acceptable prodrug of pharmacy:

Wherein,

N represents 1～3;

A represents Lys, Arg or Harg;

R ₁represent aryl, 5-6 unit's heterocycle or benzo is saturated or unsaturated heterocycle;

B representative

Wherein,

R ₂represent H, C1～4 alkyl;

R ₃represent the saturated or unsaturated naphthenic hydrocarbon in C4～8;

Z represents H, carboxyl, methyl carboxyl, formamyl or glycyl;

M represents 0～3.

Described aryl is for replacing or unsubstituted phenyl or naphthyl, and wherein substituting group is the alkyl of hydroxyl, carboxyl, halogen, alkoxyl group, amino, alkylamino, dialkylamino, cyano group, ester group, trifluoromethyl or C1～4 with above-mentioned group.

The first heterocycle of described 5-6 is the structural unit replacing or unsubstituted formula (II) represents:

Wherein substituting group is hydroxyl, carboxyl, halogen, alkoxyl group, amino, alkylamino, dialkylamino, cyano group, ester group, trifluoromethyl or C1～4 alkyl with above-mentioned group.

The structural unit that described benzo is saturated or unsaturated heterocycle is replacement or unsubstituted formula (III) representative:

Wherein substituting group is hydroxyl, carboxyl, halogen, alkoxyl group, amino, alkylamino, dialkylamino, cyano group, ester group, trifluoromethyl or C1～4 alkyl with above-mentioned group.

The preferred particular compound of the present invention includes, but are not limited to: N-benzyl-L-lysyl-L-PROLINE, N-benzyl-L-lysyl-D-PROLINE, N-styroyl-L-lysyl-L-PROLINE, N-styroyl-L-lysyl-D-PROLINE, N-hydrocinnamyl-L-lysyl-L-PROLINE, N-hydrocinnamyl-L-lysyl-D-PROLINE, N-p-chlorobenzyl-L-lysyl-L-PROLINE, N-p-chlorobenzyl-L-lysyl-D-PROLINE, N-is to luorobenzyl-L-lysyl-L-PROLINE, N-is to luorobenzyl-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-tetramethyleneimine, N-benzyl-L-lysyl-piperidines, N-benzyl-L-lysyl-hexahydroaniline, N-benzyl-L-lysyl-cycloheptylamine, the high proline(Pro) of N-benzyl-L-lysyl-L-, the high proline(Pro) of N-benzyl-L-lysyl-D-, N-benzyl-L-lysyl-(R-3-formyloxy) piperidines, N-benzyl-L-lysyl-(S-3-formyloxy) piperidines, N-benzyl-L-arginyl-D-PROLINE or the high arginyl-D-PROLINE of N-benzyl-L-.

The present invention also comprises its pharmacy acceptable salt, pharmaceutically acceptable solvate, derivative, isomer, crystal formation and the acceptable prodrug of pharmacy.

Unless otherwise indicated, in the present invention, disclosed term has the implication of their standards with abridging.

Formula (I) compound pharmacy acceptable salt comprises and comes from pharmaceutically acceptable inorganic and organic acid salt.Suitably the example of acid comprises sulfuric acid, hydrochloric acid, Hydrogen bromide, nitric acid, acetic acid, phosphoric acid, toxilic acid, fumaric acid, citric acid, perchloric acid, tosic acid, tartrate, formic acid, phenylformic acid, propanedioic acid, Citric Acid, methylsulfonic acid, Phenylsulfonic acid, lactic acid, amygdalic acid.

Compound energy of the present invention and appropriate solvent generate hydrate and solvate.Comprise water, alcohols for the preparation of the good solvent of solvate form, alcohol is methyl alcohol and ethanol preferably.Other suitable solvents can be selected according to the size of solvent molecule.Solvate or hydrate form in recrystallization process or in salt generative process.

In structure due to formula of the present invention (I) compound, have one or more chiral carbon atoms, therefore it also can racemoid, non-enantiomer mixture and pure enantiomorph exist, within all these isomer all belong to the scope of the invention.

The invention still further relates to the acceptable prodrug of general formula compound pharmacy, it can be metabolized to active compound after being applied.Suitable prodrug is the derivative of N-alkoxy carbonyl protection of for example described general formula or the derivative that carboxylic acid is protected by ester.

In the time that compound is solid, the crystal that compound of the present invention and salt can be different or polymorphous form exist, all scopes that all belong to the present invention and specific formula.

Second object of the present invention is to provide the preparation method of formula (I) peptide compounds, and the method comprises that the amino acid protected by protecting group taking nitrogen position makes the step of final product as raw material.The method can be prepared (compound of the present invention can be prepared according to following process roughly) with similar standard chemical reaction well known in the prior art, in route, starting raw material used, reagent, technology and method are all well-known, and for any those of ordinary skill in this area be all know with understand.

Described preparation method describes in detail as follows:

Route one:

It is the general synthetic method of formula (I) compound of lysyl that route one provides wherein A, wherein n, R ₁definition as above, B representative

Z representation carboxy, methyl carboxyl,

M represents 0～3,

R ₄and R ₅refer to suitable amino protecting group, wherein preferred tertiary butoxy carbonyl (Boc), carbobenzoxy-(Cbz) (Cbz), fluorenylmethoxycarbonyl (Fmoc),

X refers to the carboxylic acid protecting group of suitable Z group, wherein preferably the unsettled ester of hydrogenolysis if benzyl ester (Bn), alkali labile ester are if methyl ester (Me) or sour unsettled ester are as tertiary butyl ester (tBu).

In route one, first the N-end coupling of the C-of compound 1 end and compound 2 makes compound 3, and this compound 3 is removed respectively the amino protecting group R of carboxylic acid protecting group X and N α-end ₄obtain intermediate 5, then by radicals R ₁-(CH ₂) _nn α-end of introducing compound 5, finally removes the amino protecting group R on side chain N ₅make required compound.

Route two:

It is the general synthetic method of formula (I) compound of lysyl that route two provides wherein A, wherein n, R ₁definition as above, B representative

Wherein R ₂represent H, C1～4 alkyl,

R ₃represent the saturated or unsaturated naphthenic hydrocarbon in C4～8,

Z represents H, formamyl, glycyl,

M represents 0～3,

R ₄and R ₅refer to suitable amino protecting group, wherein preferred tertiary butoxy carbonyl (Boc), carbobenzoxy-(Cbz) (Cbz), fluorenylmethoxycarbonyl (Fmoc).

In route two, first the N-end coupling of the C-of compound 1 end and B makes compound 4, and the amino protecting group R4 that this compound is removed N α-end obtains intermediate 5, then by radicals R 1-(CH ₂) _nn α-end of introducing compound 5, finally removes the amino protecting group R on side chain N ₅make required compound.

Circuit three:

It is the general synthetic method of formula (I) compound of arginyl or high arginyl that route three provides wherein A, wherein n, R ₁, definition as above, p represents 1～2.

B representative

Z representation carboxy, methyl carboxyl,

M represents 0～3,

R ₆and R ₇refer to suitable amino protecting group, wherein preferred tertiary butoxy carbonyl (Boc), carbobenzoxy-(Cbz) (Cbz),

X refers to the carboxylic acid protecting group of suitable Z group, wherein preferably the unsettled ester of hydrogenolysis if benzyl ester (Bn), alkali labile ester are if methyl ester (Me) or sour unsettled ester are as tertiary butyl ester (tBu).

In route three, first the N-end coupling of the C-of compound 7 end and compound 2 makes compound 8, and this compound 8 is removed respectively the amino protecting group R of carboxylic acid protecting group X and N α-end ₆obtain intermediate 10, then by radicals R 1-(CH ₂) _nn α-end of introducing compound 10, finally removes the amino protecting group R on side chain N ₇make required compound.

Circuit four:

It is the general synthetic method of formula (I) compound of arginyl or high arginyl that route four provides wherein A, wherein n, R ₁definition as above, p represents 1～2.

B representative

Wherein R ₂represent H, C 1～4 alkyl,

R ₃represent the saturated or unsaturated naphthenic hydrocarbon in C4～8,

Z represents H, formamyl, glycyl,

M represents 0～3,

R ₆and R ₇refer to suitable amino protecting group, wherein preferred tertiary butoxy carbonyl (Boc), carbobenzoxy-(Cbz) (Cbz), fluorenylmethoxycarbonyl (Fmoc).

In route four, first the N-end coupling of the C-of compound 7 end and B makes compound 9, and this compound is removed the amino protecting group R of N α-end ₆obtain intermediate 10, then by radicals R 1-(CH ₂) _nn α-end of introducing compound 10, finally removes the amino protecting group R on side chain N ₇make required compound.

In above-mentioned four kinds of synthetic routes, acid amides coupling adopt standard peptide couling process at rare gas element as carried out under nitrogen, as azide method, mixed acid anhydride method, carbodiimide (dicyclohexyl carbodiimide DCC, di-isopropyl carbodiimide EDC) method, active ester method, carbonyl dimidazoles method, phosphorus reagent is as BOP-C1 method.Some method in these methods (especially carbodiimide method) can be by adding I-hydroxybenzotriazole HOBt to be modified.If necessary, in coupled reaction, need under existing, acid binding agent carry out, the example of suitable acid binding agent be trimethylamine as diisopropylethylamine, triethylamine, Trimethylamine 99, pyridine, N-methylmorpholine etc., particularly preferably N-methylmorpholine or diisopropylethylamine are as acid binding agent.

Above-mentioned reaction is carried out under anhydrous solvent exists, and the examples of solvents that can select comprises: methylene dichloride, tetrahydrofuran (THF), ether, acetonitrile, ethylene dichloride, ethyl acetate, DMF, dimethyl sulfoxide (DMSO).Temperature of reaction is conventionally inessential, preferably reacts 2～24 hours at 0～30 DEG C.

Removing the method for amino protecting group is undertaken by ordinary method, for example in acid (for example: organic acid is as trifluoroacetic acid, Phenylsulfonic acid, formic acid etc., or mineral acid example hydrochloric acid, sulfuric acid, Hydrogen bromide etc.) hydrolysis or at alkali (for example: the oxyhydroxide of basic metal or alkaline-earth metal under existing, hydride, carbonate or supercarbonate, as sodium hydroxide, potassium hydroxide, sodium hydride, salt of wormwood, sodium bicarbonate etc., or organic bases is as diisopropylethylamine, triethylamine, piperidines etc.) exist under hydrolysis, or at catalyzer (as palladium, platinum, the metal catalysts such as nickel) exist under by the hydrogenolysis reducing carrying out with hydrogen.Proper method can be referring to " blocking group in organic synthesis ", the third edition, T.W.Green and Peter G..M.Wuts (1999), publisher: John Wiley & Sons, Inc.

Generally, this reaction can be carried out under the solvent that reaction is had no adverse effect exists, and can cross the examples of solvents of selecting and comprise: methylene dichloride, alcohol are as methyl alcohol, ethanol etc., tetrahydrofuran (THF), dioxane, acetone, acetic acid, ethyl acetate.Temperature of reaction is conventionally inessential, preferably 0～40 DEG C.

Removing carboxylic acid protecting group's method is undertaken by ordinary method; for example at alkali (for example: oxyhydroxide, hydride, carbonate or the supercarbonate of basic metal or alkaline-earth metal; as sodium hydroxide, potassium hydroxide, sodium hydride, salt of wormwood, sodium bicarbonate etc.; or organic bases is as diisopropylethylamine, triethylamine etc.) there is lower hydrolysis, or under catalyzer (as metal catalysts such as palladium, platinum, nickel) existence by the reduction of carrying out with hydrogen.Proper method can be referring to " blocking group in organic synthesis ", the third edition, T.W.Green and Peter G.M.Wuts (1999), publisher: John Wiley & Sons, Inc.

Generally, this reaction can be carried out under the solvent that reaction is had no adverse effect exists, and the examples of solvents that can select comprises: water, alcohol are as methyl alcohol, ethanol etc., tetrahydrofuran (THF), dioxane, acetone.Temperature of reaction is conventionally inessential, preferably 0～30 DEG C.

The upper alkylated reaction of N can adopt reduction amination method and nucleophilic substitution method.As use nucleophilic substitution method.Suitable nucleophilic substitution deed includes but not limited to: alkyl bromide, alkyl iodide, alkyl chloride, alkyl sulfonic ester, benzene sulfonamide acid esters, alkyl p-toluenesulfonic esters, alkyl methanesulfonates or contain bisulphate root.The reagent of methylsulfate.Preferred alkyl iodine or alkyl are to family's benzene sulfonate.Nucleophilic substitution reaction need to carry out under acid binding agent exists, suitable acid binding agent can be mineral alkali (oxyhydroxide of basic metal or alkaline-earth metal, hydride, carbonate carbonic acid hydrogen salt, hydrophosphate, as sodium hydroxide,, sodium hydride, salt of wormwood, dipotassium hydrogen phosphate etc.), or organic bases as the examples such as diisopropylethylamine, triethylamine, piperidines be trimethylamine as diisopropylethylamine, triethylamine, Trimethylamine 99, pyridine, N-methylmorpholine etc., particularly preferably dipotassium hydrogen phosphate or diisopropylethylamine are as acid binding agent.The examples of solvents that can select comprises: tetrahydrofuran (THF), dioxane, acetonitrile, acetone, DMF etc.Preferably 30～80 DEG C of temperature of reaction.

As use reduction amination method.Can by reactant and corresponding aldehyde in appropriate solvent with suitable reductive agent processing.Suitable reductive agent is well known in the art, includes but not limited to three-tert.-butoxy lithium, POTASSIUM BOROHYDRIDE, sodium borohydride, sodium triacetoxy borohydride, Raney's nickel, lithium triethylborohydride, wherein preferred POTASSIUM BOROHYDRIDE or sodium triacetoxy borohydride.The appropriate solvent that carries out reductive amination process is known in the art, as methyl alcohol, ethanol, tetrahydrofuran (THF), ethylene dichloride, acetonitrile and mixed solvent etc.Preferably 0～50 DEG C of temperature of reaction.

The product of each step can be by means known in the art as column chromatography and recrystallization method purifying.

As general formula 1,2 and 7 compounds of initial substance, some is business available (Sigma-Aldrich company), some (Chem Pharm Bull that is known on document, 1999,47,1489 and Synth Commun, 2000,30,2525), can be prepared by method described in document.The intermediate obtaining according to the method for preparation formula of the present invention (I) compound is novel compound, except Lys-Pro, thereby is included in the scope of the present invention.

The 3rd object of the present invention is to provide the peptide compounds shown in formula (I) preparing application in thrombin inhibitors and in preparation treatment and the application preventing in medicine thrombin-mediated and relevant with zymoplasm disease.

Compound of the present invention can be used for treatment and prevents thrombin-mediated and the disease relevant with zymoplasm.Described disease includes but not limited to: venous thrombosis and pulmonary infarction, the artery thrombosis apoplexy that for example myocardial ischemia, myocardial infarction, unstable angina, thrombosis cause and tip arteriogenesis; Atheromatosis is as coronary artery disease, cerebral arteries disease and tip arteriopathy.

Peptide compounds of the present invention also can be used as the antithrombotics in extracorporeal blood pipeline.

In addition, this compounds expection can be used from prevention with thrombolytic agent one and treatment myocardial infarction forms.In addition, this compounds expection can be used for preventing again forming thrombus after microsurgery.This compounds is having expected utility aspect the anticoagulation therapy of hemodialysis and disseminating property intravascular coagulation.And can be used for the in vitro conservation of blood, blood plasma and other blood products.

Compound of the present invention expection can per os or parenteral route as intravenous infusion, intramuscularly or subcutaneous injection administration.Obtain the concrete metering for the treatment of and preventive effect according to the compound of administration of the present invention, depend on the particular case of this case, comprise the form of administration, medicine-feeding rate and the illness for the treatment of.Produce the oral per daily dose of typical case of effectiveness between about 0.01mg/kg and about 1000mg/kg; Typical parenterai administration per daily dose is between about 0.001mg/kg and about 100mg/kg.Give the mode of dosage can be different, as can every day single dose, or every day, 3～5 such multiple dosess may be also suitable.Need be clear that, can, according to the severity of patient's age and body weight and the disease for the treatment of, do necessary routine adjustment to dosage and administering mode.Dosage and approach should have attending doctor to determine accurately.

The 4th object of the present invention is to provide a kind of pharmaceutical composition, shown in this pharmaceutical composition contained (I) peptide compounds and pharmaceutically acceptable carrier.

The compounds of this invention can pharmaceutical composition mode administration.Such as for oral administration, compound is made to capsule or tablet form, wherein may contain the vehicle such as lubricant, binding agent, disintegrating agent.In order to inject use, compound be dissolved in a kind of pharmaceutically acceptable solvent as aseptic, without heat source water, in physiological saline.

Formulation can be solid, semisolid or the liquid preparation of being made up of techniques known in themselves.In such composition, activeconstituents accounts for formulation weight 0.1% to 99.9%.And carrier, thinner or vehicle and activeconstituents used in composition are compatible, and harmless to received object.

The abbreviation occurring in the application is as follows:

Boc: tertbutyloxycarbonyl

Cbz: carbobenzoxy-(Cbz)

DCHA: dicyclohexyl amine

Lys: lysyl

Pro: prolyl

Hpro: high prolyl

Arg: arginyl

Harg: high arginyl

Fmoc:9-fluorenylmethoxycarbonyl

EDCI:1-ethyl-3-(3-dimethylaminopropyl) carbon imide hydrochloride

HOBt:1-hydroxybenzotriazole

TFA: trifluoroacetic acid

AcOH: acetic acid

HBr:N, N '-dimethyl formamide.

Determination of activity

The vitro inhibition thrombin activity of the compounds of this invention can be measured by chromophoric substrate method.In experiment, adopt Ac-FVR-AMC as chromogenic substrate, measure the activity that it suppresses human body α-zymoplasm.And calculate its IC ₅₀value.

Embodiment

The following example is the concrete synthetic method illustrating in detail described in route one, two, and synthesized preferred compound of the present invention, can slightly change, to prepare a lot of other thrombin inhibitorss of the present invention but technician understands described chemical reaction.For example, by the apparent improvement of those skilled in the art, can successfully synthesize the compound of non-example in the present invention.These embodiment only, for explanation, instead of limit the scope of the invention by any way.

Detection method:

Nuclear magnetic resonance analyser adopts Varian INOVA-400 type nuclear magnetic resonance analyser, and taking tetramethylsilane as interior mark, the unit of chemical shift () is ppm.Thin-layer chromatography (TLC, uses HSG-F254 High Performance Thin Layer Chromatography silica gel precoated plate, the yellow business of Yantai Zhifu silica gel development experiments factory system) and HPLC are for detection of reaction and product purity.Colour developing adopts iodine vapor or irradiates or the ethanolic soln of 1% triketohydrindene hydrate under 254A and 310A ultraviolet lamp.Agents useful for same removes specified otherwise and is analytical pure, and anhydrous solvent and reagent are processed according to a conventional method.Fusing point is measured with micro melting point apparatus, and thermometer used is not calibrated.

HPLC:Waters 1525; Detector: Waters 2487; Chromatographic column: Phenomenex C18 (4.6 × 250mm, 5 μ are m); Detect wavelength: 220nm and 254nm; Column temperature: 40 DEG C; Flow velocity: 1.0ml/min;

The trifluoroacetic acid aqueous solution of moving phase 1:A:0.1%, B: acetonitrile

The phosphoric acid triethylamine damping fluid of moving phase 2:A:pH2.0, B: acetonitrile

The phosphate buffered saline buffer of moving phase 3:A:pH8.0, B: acetonitrile

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

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

Embodiment 1: synthetic N-benzyl-L-lysyl-D-PROLINE two hydrobromates (compound 1)

A) synthetic [L-Lys (Cbz)] ₂cu

By water (200ml) the solution stirring 1h of L lysine HCL (18.2g), sodium bicarbonate (16.8g), cupric sulfate pentahydrate (12.5g).Ice bath is chilled to 0 DEG C, slowly drips benzene methoxy dicarbonyl chloride (14.3ml), and is adjusted to pH 8.5 with 2mol/L sodium hydroxide solution, question response pH value is held constant at more than 3 hours, filters, and gained precipitation washes with water to neutrality, with washing with acetone, quantitatively obtain blue solid.Directly carry out the next step.

B) synthetic Boc-L-Lys (Cbz) OHDCHA

By [L-Lys (Cbz)] ₂cu (27.7g) is suspended in acetone (100ml) and 10% sodium carbonate (100ml) aqueous solution, adds oxine (14.95g) under vigorous stirring.After 2 hours, ice bath is cooled to below 10 DEG C, slowly drips acetone (60ml) solution of tert-Butyl dicarbonate (28g), naturally rises to room temperature, reacts 8 hours.Filter, filter cake washes with water, and filtrate decompression is concentrated removes acetone, is extracted with ethyl acetate to organic layer colourless.Water is adjusted to pH 4 by potassium hydrogen sulfate saturated solution, uses dichloromethane extraction.The saturated common salt water washing of gained organic phase, anhydrous sodium sulfate drying, filters, concentrating under reduced pressure obtains light yellow oil, adds ethyl acetate (60ml) and dissolves, and adds DCHA (18ml) and normal hexane (100ml) under stirring, separate out gradually solid, filter, Virahol for crude product/hexanaphthene recrystallization obtains white solid (46g, 82%), mp:103～105 DEG C, content 99% (HPLC, moving phase 1, method 1).Rf=0.6 developping agent methylene chloride/methanol=15/1 colour developing: ultraviolet, iodine and 1% triketohydrindene hydrate liquid 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) synthetic D-PROLINE methyl ester hydrochloride

D-PROLINE (23g) is dissolved in to methyl alcohol (200ml), and ice bath was cooled to below 0 DEG C, slowly drips sulfur oxychloride (36ml), and 1h drips, in 40 DEG C of reactions 5 hours.At 50 DEG C, concentrating under reduced pressure removes desolventizing and obtains light yellow oil, and Virahol for crude product/isopropyl ether recrystallization obtains white solid (29g, 90%).

Rf=0.4 developping agent ethyl acetate/methanol/acetic acid=10/4/1 colour developing: iodine and 1% triketohydrindene hydrate liquid 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) synthetic Boc-L-Lys (Cbz)-D-Pro-OCH ₃

Boc-L-Lys (Cbz)-OHDCHA (7.3g) is dissolved in 5% sal enixum (40ml) solution and methylene dichloride (40ml), stir 0.5h, layering, water is used dichloromethane extraction again, merge organic phase, use saturated common salt water washing, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure obtains light yellow oil.

Boc-L-Lys (Cbz)-OH after free, proline methyl ester hydrochloride (2.4g), N-methylmorpholine (3.37g) are dissolved with anhydrous methylene chloride (50ml); nitrogen protection; be cooled to after 0 DEG C; add HOBt (2.1g); EDCI (3g); at 0 DEG C, stir 20min, naturally rise to room temperature, react 8 hours.Use 5% potassium hydrogen sulfate solution, saturated sodium bicarbonate solution and the saturated common salt aqueous solution wash organic layer successively, and anhydrous sodium sulfate drying filters, and concentrating under reduced pressure obtains crude product 5.5g, and column chromatography purification obtains colorless oil (5.3g, yield: 83.7%).

Rf=0.7 developping agent ethyl acetate colour developing: 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) synthetic Boc-L-Lys (Cbz)-D-Pro-OH

By Boc-L-Lys (Cbz)-D-Pro-OCH ₃(5.3g) be dissolved in methyl alcohol (50ml), be cooled to 0 DEG C, add sodium hydroxide (1.7g), react and be adjusted to pH 4 with 5%HCl solution after 2 hours, concentrating under reduced pressure is removed methanol solvate, then is extracted with ethyl acetate, and gained organic phase is dry, concentrating under reduced pressure obtains colourless thick liquid (4.8g, 94%).

Rf=0.5 developping agent ethyl acetate colour developing: ultraviolet and 1% triketohydrindene hydrate liquid

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) synthetic L-Lys (Cbz)-D-Pro-OHTFA

Boc-L-Lys (Cbz)-D-Pro-OH (4.8g) is dissolved in to methylene dichloride (40ml), ice bath is cooled to below 0 DEG C, drip TFA (20ml), 0 DEG C is reacted 1 hour, and concentrating under reduced pressure adds ether washing, obtain oily matter, the ether that inclines, drains and can obtain white sticky solid (4.58g, 92%).

Rf=0.5 developping agent methylene chloride/methanol=5/1 colour developing: ultraviolet and 1% triketohydrindene hydrate liquid

MS：376(M-)

G) synthetic N-benzyl-L-lysyl-D-PROLINE two hydrobromates

L-Lys (Cbz)-D-Pro-OHTFA (491mg) is dissolved in to methyl alcohol (5ml), be cooled to 0 DEG C, add triethylamine (0.21ml), phenyl aldehyde (121mg), stir 3 hours, add KBH4 (84mg) in batches, react 3 hours.Flash column purification, then with ether washing, topple over ether, drain to obtain white thick liquid.

Upper step gained thick liquid and methyl-phenoxide (0.5ml) are dissolved in to 36%HBr acetic acid solution (4ml), room temperature reaction, HPLC detection reaction is complete, add ether (20ml) to reaction solution, place refrigerator overnight, separate out solid, filter (300mg, 61%).

MS：334(M+1)

Embodiment 2: synthetic N-styroyl-L-lysyl-D-PROLINE two hydrobromates (compound 2)

The L-Lys of embodiment 1 gained (Cbz)-D-Pro-OHTFA (737mg) is dissolved in methyl alcohol (10ml), be cooled to 0 DEG C, add triethylamine (277mg), phenylacetic aldehyde (207mg), stir 0.5 hour, add sodium triacetoxy borohydride (600mg) in batches, react 3 hours, concentrating under reduced pressure is except desolventizing, and column chromatography purification obtains colourless thick liquid.

Upper step gained thick liquid and methyl-phenoxide (0.5ml) are dissolved in to 36%HBr acetic acid solution (3ml), room temperature reaction, HPLC detection reaction is complete, in reaction solution, add ether (20ml), place refrigerator overnight, separate out solid, filter (50mg, 6.6%)

MS：348(M+1)

Embodiment 3: synthetic N-hydrocinnamyl-L-lysyl-D-PROLINE two hydrobromates (compound 3)

The L-Lys of embodiment 1 gained (Cbz)-D-Pro-OHTFA (737mg) is dissolved in methyl alcohol (10ml), be cooled to 0 DEG C, add triethylamine (277mg), phenylpropyl aldehyde (211mg), stir 0.5 hour, add sodium triacetoxy borohydride (600mg) in batches, react 3 hours, concentrating under reduced pressure is except desolventizing, and column chromatography purification obtains colourless thick liquid.

Upper step gained thick liquid and methyl-phenoxide (0.5ml) are dissolved in to 36%HBr acetic acid solution (3ml), room temperature reaction, HPLC detection reaction is complete, in reaction solution, add ether (20ml), place refrigerator overnight, separate out solid, filter (167mg, 21%)

MS：362(M+1)

Embodiment 4: synthetic N-1-naphthyl-L-lysyl-D-PROLINE two hydrobromates (compound 4)

The L-Lys of embodiment 1 gained (Cbz)-D-Pro-OHTFA (491mg) is dissolved in to methyl alcohol (5ml), be cooled to 0 DEG C, add triethylamine (0.21ml), 1-naphthaldehyde (175mg), stir 3 hours, add KBH in batches ₄(83mg), reaction 3 hours.Flash column purification, then with ether washing, topple over ether, drain to obtain white thick liquid.

Upper step gained thick liquid and methyl-phenoxide (0.5ml) are dissolved in to 36%HBr acetic acid solution (4ml), room temperature reaction, HPLC detection reaction is complete, add ether (20ml) to reaction solution, place refrigerator overnight, separate out solid, filter (261mg, 47.8%).

MS：384(M+1)

Embodiment 5: synthetic N-2-naphthyl-L-lysyl-D-PROLINE two hydrobromates (compound 5)

The L-Lys of embodiment 1 gained (Cbz)-D-Pro-OHTFA (491mg) is dissolved in to methyl alcohol (5ml), be cooled to 0 DEG C, add triethylamine (0.21ml), 2-naphthaldehyde (175mg), stir 3 hours, add KBH in batches ₄(80mg), reaction 3 hours.Flash column purification, then with ether washing, topple over ether, drain to obtain white thick liquid.

Upper step gained thick liquid and methyl-phenoxide (0.5ml) are dissolved in to 36%HBr acetic acid solution (4ml), room temperature reaction, HPLC detection reaction is complete, add ether (20ml) to reaction solution, place refrigerator overnight, separate out solid, filter (243mg, 44.6%).

MS：384(M+1)

Embodiment 6: synthetic N-p-chlorobenzyl-L-lysyl-D-PROLINE two hydrobromates (compound 6)

The L-Lys of embodiment 1 gained (Cbz)-D-Pro-OHTFA (491mg) is dissolved in to methyl alcohol (5ml), be cooled to 0 DEG C, add triethylamine (0.21ml), 4-chloro-benzaldehyde (145mg), stir 3 hours, add KBH in batches ₄(91mg), reaction 3 hours.Flash column purification, then with ether washing, topple over ether, drain to obtain white thick liquid.

Upper step gained thick liquid and methyl-phenoxide (0.5ml) are dissolved in to 36%HBr acetic acid solution (4ml), room temperature reaction, HPLC detection reaction is complete, add ether (20ml) to reaction solution, place refrigerator overnight, separate out solid, filter (209mg, 39.4%).

MS：369(M+1)

Embodiment 7: synthetic N-is to luorobenzyl-L-lysyl-D-PROLINE two hydrobromates (compound 7)

The L-Lys of embodiment 1 gained (Cbz)-D-Pro-OHTFA (491mg) is dissolved in to methyl alcohol (5ml), be cooled to 0 DEG C, add triethylamine (0.21ml), p-Fluorobenzenecarboxaldehyde (132mg), stir 3 hours, add KBH in batches ₄(87mg), reaction 3 hours.Flash column purification, then with ether washing, topple over ether, drain to obtain white thick liquid.

Upper step gained thick liquid and methyl-phenoxide (0.5ml) are dissolved in to 36%HBr acetic acid solution (4ml), room temperature reaction, HPLC detection reaction is complete, add ether (20ml) to reaction solution, place refrigerator overnight, separate out solid, filter (215mg, 42%).

MS：352(M+1)

Embodiment 8: synthetic N-benzyl-L-lysyl-L-PROLINE two hydrobromates (compound 8)

A) synthetic L-PROLINE methyl ester hydrochloride

L-PROLINE (19.49g) is dissolved in to methyl alcohol (150ml), and ice bath was cooled to below 0 DEG C, slowly drips sulfur oxychloride (38ml), and 1h drips, in 40 DEG C of reactions 5 hours.At 50 DEG C, concentrating under reduced pressure removes desolventizing and obtains light yellow oil, and Virahol for crude product/isopropyl ether recrystallization obtains white solid (25g, 92%).

Rf=0.4 developping agent ethyl acetate/methanol/acetic acid=10/4/1 colour developing: iodine and 1% triketohydrindene hydrate liquid

B) synthetic Boc-L-Lys (Cbz)-L-Pro-OCH ₃

Boc-L-Lys (Cbz) OHDCHA (14g) is dissolved in 5% sal enixum (70ml) solution and methylene dichloride (60ml), stir 0.5h, layering, water is used dichloromethane extraction again, merge organic phase, use saturated common salt water washing, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure obtains light yellow oil.

Boc-L-Lys (Cbz) OH after free, L-PROLINE methyl ester hydrochloride (4.8g), N-methylmorpholine (6.6g) are dissolved with anhydrous methylene chloride (100ml); nitrogen protection; be cooled to after 0 DEG C; add HOBt (4.2g); EDCI (6g); at 0 DEG C, stir 20min, naturally rise to room temperature, react 8 hours.Use 5% potassium hydrogen sulfate solution, saturated sodium bicarbonate solution and the saturated common salt aqueous solution wash organic layer successively, and anhydrous sodium sulfate drying filters, and concentrating under reduced pressure obtains crude product, and column chromatography purification obtains colorless oil (10g, yield: 79%).

Rf=0.8 developping agent ethyl acetate colour developing: ultraviolet and iodine

MS：514(M+Na ⁺)

C) synthetic Boc-L-Lys (Cbz)-L-Pro-OH

By Boc-L-Lys (Cbz)-L-Pro-OCH ₃(2g) be dissolved in methyl alcohol (25ml), be cooled to 0 DEG C, add sodium hydroxide (1.1g), react and be adjusted to pH 4 with 5%HCl solution after 2 hours, concentrating under reduced pressure is removed methanol solvate, then is extracted with ethyl acetate, and gained organic phase is dry, concentrating under reduced pressure obtains colourless thick liquid (1.9g, 92%).

Rf=0.5 developping agent ethyl acetate colour developing: ultraviolet and 1% triketohydrindene hydrate liquid

MS：476(M-)

D) synthetic L-Lys (Cbz)-L-Pro-OHTFA

Boc-L-Lys (Cbz)-L-Pro-OH (2.4g) is dissolved in to methylene dichloride (15ml), ice bath is cooled to below 0 DEG C, drip TFA (5ml), 0 DEG C is reacted 1 hour, and concentrating under reduced pressure adds ether washing, obtain oily matter, the ether that inclines, drains and can obtain white sticky solid (2.3g, 92%).

Rf=0.5 developping agent methylene chloride/methanol=5/1 colour developing: ultraviolet and 1% triketohydrindene hydrate liquid

MS：376(M-)

E) synthetic N-benzyl-L-lysyl-L-PROLINE two hydrobromates

L-Lys (Cbz)-L-Pro-OHTFA (491mg) is dissolved in to methyl alcohol (5ml), be cooled to 0 DEG C, add triethylamine (0.21ml), phenyl aldehyde (121mg), stir 3 hours, add KBH4 (86mg) in batches, react 3 hours.Flash column purification, then with ether washing, topple over ether, drain to obtain white thick liquid.

Upper step gained thick liquid and methyl-phenoxide (0.5ml) are dissolved in to 36%HBr acetic acid solution (4ml), room temperature reaction, HPLC detection reaction is complete, add ether (20ml) to reaction solution, place refrigerator overnight, separate out solid, filter (154mg, 31%).

MS：334(M+1)

Embodiment 9: synthetic N-styroyl-L-lysyl-L-PROLINE two hydrobromates (compound 9)

The L-Lys of embodiment 8 gained (Cbz)-L-Pro-OHTFA (737mg) is dissolved in methyl alcohol (10ml), be cooled to 0 DEG C, add triethylamine (267mg), phenylacetic aldehyde (210mg), stir 0.5 hour, add sodium triacetoxy borohydride (592mg) in batches, react 3 hours, concentrating under reduced pressure is except desolventizing, and column chromatography purification obtains colourless thick liquid.

Upper step gained thick liquid and methyl-phenoxide (0.Sml) are dissolved in to 36%HBr acetic acid solution (3ml), room temperature reaction, HPLC detection reaction is complete, in reaction solution, add ether (20ml), place refrigerator overnight, separate out solid, filter (61mg, 8%)

MS：348(M+1)

Embodiment 10: synthetic N-hydrocinnamyl-L-lysyl-L-PROLINE two hydrobromates (compound 10)

The L-Lys of embodiment 8 gained (Cbz)-L-Pro-OHTFA (737mg) is dissolved in methyl alcohol (10ml), be cooled to 0 DEG C, add triethylamine (274mg), phenylpropyl aldehyde (220mg), stir 0.5 hour, add sodium triacetoxy borohydride (611mg) in batches, react 3 hours, concentrating under reduced pressure is except desolventizing, and column chromatography purification obtains colourless thick liquid.

Upper step gained thick liquid and methyl-phenoxide (0.5ml) are dissolved in to 36%HBr acetic acid solution (3ml), room temperature reaction, HPLC detection reaction is complete, in reaction solution, add ether (20ml), place refrigerator overnight, separate out solid, filter (184mg, 23%)

MS：362(M+1)

Embodiment 11: synthetic N-1-naphthyl-L-lysyl-L-PROLINE two hydrobromates (compound 11)

The L-Lys of embodiment 8 gained (Cbz)-L-Pro-OHTFA (491mg) is dissolved in to methyl alcohol (5ml), be cooled to 0 DEG C, add triethylamine (0.21ml), 1-naphthaldehyde (177mg), stir 3 hours, add KBH in batches ₄(89mg), reaction 3 hours.Flash column purification, then with ether washing, topple over ether, drain to obtain white thick liquid.

Upper step gained thick liquid and methyl-phenoxide (0.5ml) are dissolved in to 36%HBr acetic acid solution (4ml), room temperature reaction, HPLC detection reaction is complete, add ether (20ml) to reaction solution, place refrigerator overnight, separate out solid, filter (249mg, 45.6%).

MS：384(M+1)

Embodiment 12: synthetic N-2-naphthyl-L-lysyl-L-PROLINE two hydrobromates (compound 12)

The L-Lys of embodiment 8 gained (Cbz)-L-Pro-OHTFA (491mg) is dissolved in to methyl alcohol (5ml), be cooled to 0 DEG C, add triethylamine (0.21ml), 2-naphthaldehyde (181mg), stir 3 hours, add KBH in batches ₄(95mg), reaction 3 hours.Flash column purification, then with ether washing, topple over ether, drain to obtain white thick liquid.

Upper step gained thick liquid and methyl-phenoxide (0.5ml) are dissolved in to 36%HBr acetic acid solution (4ml), room temperature reaction, HPLC detection reaction is complete, add ether (20ml) to reaction solution, place refrigerator overnight, separate out solid, filter (211mg, 40%).

MS：384(M+1)

Embodiment 13: synthetic N-p-chlorobenzyl-L-lysyl-L-PROLINE two hydrobromates (compound 13)

The L-Lys of embodiment 8 gained (Cbz)-L-Pro-OHTFA (491mg) is dissolved in to methyl alcohol (5ml), be cooled to 0 DEG C, add triethylamine (0.21ml), 4-chloro-benzaldehyde (150mg), stir 3 hours, add KBH in batches ₄(88mg), reaction 3 hours.Flash column purification, then with ether washing, topple over ether, drain to obtain white thick liquid.

Upper step gained thick liquid and methyl-phenoxide (0.5ml) are dissolved in to 36%HBr acetic acid solution (4ml), room temperature reaction, HPLC detection reaction is complete, add ether (20ml) to reaction solution, place refrigerator overnight, separate out solid, filter (186mg, 35%).

MS：369(M+1)

Embodiment 14: synthetic N-is to luorobenzyl-L-lysyl-D-PROLINE two hydrobromates (compound 14)

The L-Lys of embodiment 8 gained (Cbz)-L-Pro-OHTFA (491mg) is dissolved in to methyl alcohol (5ml), be cooled to 0 DEG C, add triethylamine (0.21ml), p-Fluorobenzenecarboxaldehyde (143mg), stir 3 hours, add KBH in batches ₄(97mg), reaction 3 hours.Flash column purification, then with ether washing, topple over ether, drain to obtain white thick liquid.

Upper step gained thick liquid and methyl-phenoxide (0.5ml) are dissolved in to 36%HBr acetic acid solution (4ml), room temperature reaction, HPLC detection reaction is complete, add ether (20ml) to reaction solution, place refrigerator overnight, separate out solid, filter (224mg, 43.5%).

MS：352(M+1)

Embodiment 15: synthetic N-benzyl-L-lysyl-tetramethyleneimine two hydrobromates (compound 15)

A) synthetic Boc-L-Lys (Cbz)-tetramethyleneimine

Boc-L-Lys (Cbz) OHDCHA (1.9g) is dissolved in 5% sal enixum (15ml) solution and methylene dichloride (15ml), stir 0.5h, layering, water is used dichloromethane extraction again, merge organic phase, use saturated common salt water washing, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure obtains light yellow oil.

Boc-L-Lys (Cbz) OH after free, tetramethyleneimine (367mg), N-methylmorpholine (719mg) are dissolved with anhydrous methylene chloride (20ml); nitrogen protection; be cooled to after 0 DEG C; add HOBt (811mg), EDCI (1.15g); at 0 DEG C, stir 20min; naturally rise to room temperature, react 8 hours.Use 5% potassium hydrogen sulfate solution, saturated sodium bicarbonate solution and the saturated common salt aqueous solution wash organic layer successively, and anhydrous sodium sulfate drying filters, and concentrating under reduced pressure obtains crude product, and column chromatography purification obtains colorless oil (1.817g, yield: 83.8%).

Rf=0.8 developping agent ethyl acetate colour developing: 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) synthetic L-Lys (Cbz)-tetramethyleneimine TFA

Boc-L-Lys (Cbz)-tetramethyleneimine (867mg) is dissolved in to methylene dichloride (10ml), ice bath is cooled to below 0 DEG C, drip TFA (8ml), 0 DEG C of reaction 1h, by reaction solution concentrating under reduced pressure at 25 DEG C, adds ether washing, obtain oily matter, the ether that inclines, drains and can obtain white sticky solid (850mg, 98%).

Rf=0.5 developping agent methylene chloride/methanol=5/1 colour developing: ultraviolet and 1% triketohydrindene hydrate liquid

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) synthetic N-benzyl-L-Lys--tetramethyleneimine 2HBr

L-Lys (Cbz)-tetramethyleneimine TFA (530mg) is dissolved in methyl alcohol (10ml), is cooled to 0 DEG C, be added dropwise to triethylamine (110mg), add phenyl aldehyde (120mg), stir 0.5h, add KBH in batches ₄(101mg) react about 4h, HPLC detection reaction is complete, and concentrating under reduced pressure is removed most of solvent, and column chromatography purification obtains white thick liquid.

Upper step gained thick liquid and methyl-phenoxide (0.5ml) are dissolved in 36%HBr/ acetic acid solution (6ml), HPLC detection reaction is complete, in reaction solution, add ether (20ml), place refrigerator overnight, separate out solid, filtration obtains white solid (264mg, 58%).

MS：290(M+1)

Embodiment 16: synthetic N-benzyl-L-lysyl-piperidines two hydrobromates (compound 16)

A) synthetic Boc-L-Lys (Cbz)-piperidines

Boc-L-Lys (Cbz) OHDCHA (1.99g) is dissolved in 5% sal enixum (15ml) solution and methylene dichloride (15ml), stir 0.5h, layering, water is used dichloromethane extraction again, merge organic phase, use saturated common salt water washing, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure obtains light yellow oil.

Boc-L-Lys (Cbz) OH after free, piperidines (434mg), N-methylmorpholine (720mg) are dissolved with anhydrous methylene chloride (20ml); nitrogen protection; be cooled to after 0 DEG C; add HOBt (834mg), EDCI (1.2g); at 0 DEG C, stir 20min; naturally rise to room temperature, react 8 hours.Use 5% potassium hydrogen sulfate solution, saturated sodium bicarbonate solution and the saturated common salt aqueous solution wash organic layer successively, and anhydrous sodium sulfate drying filters, and concentrating under reduced pressure obtains crude product, and column chromatography purification obtains colorless oil (2g, yield: 89%).

Rf=0.8 developping agent ethyl acetate colour developing: 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) synthetic L-Lys (Cbz)-tetramethyleneimine TFA

Boc-L-Lys (Cbz)-piperidines (895mg) is dissolved in to methylene dichloride (10ml), ice bath is cooled to below 0 DEG C, drip TFA (8ml), 0 DEG C of reaction 1h, by reaction solution concentrating under reduced pressure at 25 DEG C, adds ether washing, obtain oily matter, the ether that inclines, drains and can obtain white sticky solid (884mg, 98%).

Rf=0.5 developping agent methylene chloride/methanol=5/1 colour developing: ultraviolet and 1% triketohydrindene hydrate liquid

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) synthetic N-benzyl-L-Lys-piperidines 2HBr

L-Lys (Cbz)-piperidines TFA (890mg) is dissolved in methyl alcohol (10ml), is cooled to 0 DEG C, be added dropwise to triethylamine (303mg), add phenyl aldehyde (243mg), stir 0.5h, add KBH in batches ₄(106mg) react about 4h, HPLC detection reaction is complete, and concentrating under reduced pressure is removed most of solvent, and column chromatography purification obtains white thick liquid.

Upper step gained thick liquid and methyl-phenoxide (0.65ml) are dissolved in 36%HBr/ acetic acid solution (10ml), HPLC detection reaction is complete, in reaction solution, add ether (20ml), place refrigerator overnight, separate out solid, filtration obtains white solid (455mg, 49%).

MS：304(M+1)

Embodiment 17: synthetic N-benzyl-L-lysyl-hexahydroaniline two hydrobromates (compound 17)

A) synthetic Boc-L-Lys (Cbz)-hexahydroaniline

Boc-L-Lys (Cbz) OHDCHA (1.9g) is dissolved in 5% sal enixum (15ml) solution and methylene dichloride (15ml), stir 0.5h, layering, water is used dichloromethane extraction again, merge organic phase, use saturated common salt water washing, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure obtains light yellow oil.

Boc-L-Lys (Cbz) OH after free, hexahydroaniline (513mg), N-methylmorpholine (719mg) are dissolved with anhydrous methylene chloride (20ml); nitrogen protection; be cooled to after 0 DEG C; add HOBt (817mg), EDCI (1.15g); at 0 DEG C, stir 20min; naturally rise to room temperature, react 8 hours.Use 5% potassium hydrogen sulfate solution, saturated sodium bicarbonate solution and the saturated common salt aqueous solution wash organic layer successively, and anhydrous sodium sulfate drying filters, and concentrating under reduced pressure obtains crude product, and column chromatography purification obtains colorless oil (1.929g, yield: 81.6%).

Rf=0.5 developping agent ethyl acetate/petroleum ether=1/1 colour developing: ultraviolet and iodine

MS：476(M+1)

B) synthetic L-Lys (Cbz)-hexahydroaniline TFA

Boc-L-Lys (Cbz)-hexahydroaniline (922mg) is dissolved in to methylene dichloride (10ml), ice bath is cooled to below 0 DEG C, drip TFA (8ml), 0 DEG C of reaction 1h, by reaction solution concentrating under reduced pressure at 25 DEG C, adds ether washing, obtain oily matter, the ether that inclines, drains and can obtain white sticky solid (866mg, 94%).Rf=0.5 developping agent methylene chloride/methanol=5/1 colour developing: ultraviolet and 1% triketohydrindene hydrate liquid MS:362 (M+1)

C) synthetic N-benzyl-L-Lys-hexahydroaniline 2HBr

L-Lys (Cbz)-hexahydroaniline TFA (464mg) is dissolved in methyl alcohol (5ml), is cooled to 0 DEG C, be added dropwise to triethylamine (146mg), add phenyl aldehyde (120mg), stir 0.5h, add KBH in batches ₄(67mg) react about 4h, HPLC detection reaction is complete, and concentrating under reduced pressure is removed most of solvent, and column chromatography purification obtains white thick liquid.

Upper step gained thick liquid and methyl-phenoxide (0.5ml) are dissolved in 36%HBr/ acetic acid solution (4ml), HPLC detection reaction is complete, in reaction solution, add ether (20ml), place refrigerator overnight, separate out solid, filtration obtains white solid (218mg, 45%).

MS：318(M+1)

Embodiment 18: synthetic N-benzyl-L-lysyl-cycloheptylamine two hydrobromates (compound 18)

A) synthetic Boc-L-Lys (Cbz)-cycloheptylamine

Boc-L-Lys (Cbz) OHDCHA (1.9g) is dissolved in 5% sal enixum (15ml) solution and methylene dichloride (15ml), stir 0.5h, layering, water is used dichloromethane extraction again, merge organic phase, use saturated common salt water washing, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure obtains light yellow oil.

Boc-L-Lys (Cbz) OH after free, hexahydroaniline (590mg), N-methylmorpholine (719mg) are dissolved with anhydrous methylene chloride (20ml); nitrogen protection; be cooled to after 0 DEG C; add HOBt (814mg), EDCI (1.15g); at 0 DEG C, stir 20min; naturally rise to room temperature, react 8 hours.Use 5% potassium hydrogen sulfate solution, saturated sodium bicarbonate solution and the saturated common salt aqueous solution wash organic layer successively, and anhydrous sodium sulfate drying filters, and concentrating under reduced pressure obtains crude product, and column chromatography purification obtains colorless oil (1.845g, yield: 75.4%).

Rf=0.55 developping agent ethyl acetate/petroleum ether=1/1 colour developing: ultraviolet and iodine

MS：490(M+1)

B) synthetic L-Lys (Cbz)-cycloheptylamine TFA

Boc-L-Lys (Cbz)-hexahydroaniline (950mg) is dissolved in to methylene dichloride (10ml), ice bath is cooled to below 0 DEG C, drip TFA (8ml), 0 DEG C of reaction 1h, by reaction solution concentrating under reduced pressure at 25 DEG C, adds ether washing, obtain oily matter, the ether that inclines, drains and can obtain white sticky solid (879mg, 93%).Rf=0.5 developping agent methylene chloride/methanol=5/1 colour developing: ultraviolet and 1% triketohydrindene hydrate liquid

MS：376(M+1)

C) synthetic N-benzyl-L-Lys-cycloheptylamine 2HBr

L-Lys (Cbz)-hexahydroaniline TFA (476mg) is dissolved in methyl alcohol (5ml), is cooled to 0 DEG C, be added dropwise to triethylamine (146mg), add phenyl aldehyde (120mg), stir 0.5h, add KBH in batches ₄(67mg) react about 4h, HPLC detection reaction is complete, and concentrating under reduced pressure is removed most of solvent, and column chromatography purification obtains white thick liquid.

Upper step gained thick liquid and methyl-phenoxide (0.5ml) are dissolved in 36%HBr/ acetic acid solution (4ml), HPLC detection reaction is complete, in reaction solution, add ether (20ml), place refrigerator overnight, separate out solid, filtration obtains white solid (197mg, 40%).

MS：332(M+1)

Embodiment 19: synthetic high proline(Pro) two hydrobromates of N-benzyl-L-lysyl-L-(compound 19)

A) synthetic Boc-L-Lys (Cbz)-L-Hpro-OCH ₃

Boc-L-Lys (Cbz) OHDCHA (5.6g) is dissolved in 5% sal enixum (60ml) solution and methylene dichloride (50ml), stir 0.5h, layering, water is used dichloromethane extraction again, merge organic phase, use saturated common salt water washing, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure obtains light yellow oil.

Boc-L-Lys (Cbz) OH after free, the high proline methyl ester hydrochloride of L-(2g), diisopropylethylamine (3.2g) are dissolved with anhydrous methylene chloride (50ml); nitrogen protection; be cooled to after 0 DEG C; add HOBt (1.35g); EDCI (2.3g); at 0 DEG C, stir 20min, naturally rise to room temperature, react 8 hours.Use 5% potassium hydrogen sulfate solution, saturated sodium bicarbonate solution and the saturated common salt aqueous solution wash organic layer successively, and anhydrous sodium sulfate drying filters, and concentrating under reduced pressure obtains crude product, and column chromatography purification obtains colorless oil (1.4g, yield: 27.7%).

Rf=0.8 developping agent ethyl acetate colour developing: ultraviolet and iodine

MS：506(M+1)

B) synthetic B oc-L-Lys (Cbz)-L-Hpro-OH

By Boc-L-Lys (Cbz)-L-Hpro-OCH ₃(1.4g) be dissolved in methyl alcohol (10ml), be cooled to 0 DEG C, add sodium hydroxide (400mg), react and be adjusted to pH 4 with 5%HCl solution after 2 hours, concentrating under reduced pressure is removed methanol solvate, then is extracted with ethyl acetate, and gained organic phase is dry, concentrating under reduced pressure obtains colourless thick liquid (800mg, 59%).

Rf=0.6 developping agent ethyl acetate colour developing: ultraviolet and 1% triketohydrindene hydrate liquid

MS：492(M-)

C) synthetic L-Lys (Cbz)-L-Hpro-OHTFA

Boc-L-Lys (Cbz)-L-Hpro-OH (800mg) is dissolved in to methylene dichloride (15ml), ice bath is cooled to below 0 DEG C, drip TFA (5ml), 0 DEG C is reacted 1 hour, and concentrating under reduced pressure adds ether washing, obtain oily matter, the ether that inclines, drains and can obtain white sticky solid (609mg, 77%).

Rf=0.5 developping agent methylene chloride/methanol=5/1 colour developing: ultraviolet and 1% triketohydrindene hydrate liquid

MS：392(M-)

D) synthetic N α-phenmethyl-L-Lys-L-Hpro-OH2HBr

L-Lys (Cbz)-L-Hpro-OHTFA (500mg) is dissolved in to methyl alcohol (8ml), be cooled to 0 DEG C, add diisopropylethylamine (193mg), phenyl aldehyde (127mg), stir 3 hours, add KBH4 (76mg) in batches, react 3 hours.Flash column purification, then with ether washing, topple over ether, drain to obtain white thick liquid.

Upper step gained thick liquid and methyl-phenoxide (0.5ml) are dissolved in to 36%HBr acetic acid solution (4ml), room temperature reaction, HPLC detection reaction is complete, add ether (20ml) to reaction solution, place refrigerator overnight, separate out solid, filter (159mg, 31%).

MS：348(M+1)

Embodiment 20: synthetic high proline(Pro) two hydrobromates of N-benzyl-L-lysyl-D-(compound 20)

A) synthetic Boc-L-Lys (Cbz)-D-Hpro-OCH ₃

Boc-L-Lys (Cbz) OHDCHA (5.6g) is dissolved in 5% sal enixum (60ml) solution and methylene dichloride (50ml), stir 0.5h, layering, water is used dichloromethane extraction again, merge organic phase, use saturated common salt water washing, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure obtains light yellow oil.Boc-L-Lys (Cbz) OH after free, the high proline methyl ester hydrochloride of D-(2g), diisopropylethylamine (3.2g) are dissolved with anhydrous methylene chloride (50ml); nitrogen protection; be cooled to after 0 DEG C; add HOBt (1.35g); EDCI (2.3g); at 0 DEG C, stir 20min, naturally rise to room temperature, react 8 hours.Use 5% potassium hydrogen sulfate solution, saturated sodium bicarbonate solution and the saturated common salt aqueous solution wash organic layer successively, and anhydrous sodium sulfate drying filters, and concentrating under reduced pressure obtains crude product, and column chromatography purification obtains colorless oil (1.6g, yield: 29%).

Rf=0.8 developping agent ethyl acetate colour developing: ultraviolet and iodine

MS：506(M+1)

B) synthetic Boc-L-Lys (Cbz)-D-Hpro-OH

By Boc-L-Lys (Cbz)-D-Hpro-OCH ₃(1.6g) be dissolved in methyl alcohol (10ml), be cooled to 0 DEG C, add sodium hydroxide (420mg), react and be adjusted to pH 4 with 5%HCl solution after 2 hours, concentrating under reduced pressure is removed methanol solvate, then is extracted with ethyl acetate, and gained organic phase is dry, concentrating under reduced pressure obtains colourless thick liquid (938mg, 69%).

Rf=0.6 developping agent ethyl acetate colour developing: ultraviolet and 1% triketohydrindene hydrate liquid

MS：492(M-)

C) synthetic L-Lys (Cbz)-D-Hpro-OHTFA

Boc-L-Lys (Cbz)-D-Hpro-OH (800mg) is dissolved in to methylene dichloride (15ml), ice bath is cooled to below 0 DEG C, drip TFA (5ml), 0 DEG C is reacted 1 hour, and concentrating under reduced pressure adds ether washing, obtain oily matter, the ether that inclines, drains and can obtain white sticky solid (711mg, 89%).

Rf=0.5 developping agent methylene chloride/methanol=5/1 colour developing: ultraviolet and 1% triketohydrindene hydrate liquid

MS：392(M-)

D) synthetic N α-phenmethyl-L-Lys-D-Hpro-OH2HBr

L-Lys (Cbz)-D-Hpro-OHTFA (500mg) is dissolved in to methyl alcohol (8ml), be cooled to 0 DEG C, add diisopropylethylamine (195mg), phenyl aldehyde (121mg), stir 3 hours, add KBH4 (76mg) in batches, react 3 hours.Flash column purification, then with ether washing, topple over ether, drain to obtain white thick liquid.

Upper step gained thick liquid and methyl-phenoxide (0.5ml) are dissolved in to 36%HBr acetic acid solution (4ml), room temperature reaction, HPLC detection reaction is complete, add ether (20ml) to reaction solution, place refrigerator overnight, separate out solid, filter (182mg, 36%).

MS：348(M+1)

Embodiment 21: synthetic N-benzyl-L-lysyl-(R-3-formyloxy) piperidines two hydrobromates (compound 21)

A) synthetic Boc-L-Lys (Cbz)-(R-3-formyloxy) piperidines ethyl ester

Boc-L-Lys (Cbz) OHDCHA (1.9g) is dissolved in 5% sal enixum (20ml) solution and methylene dichloride (20ml), stir 0.5h, layering, water is used dichloromethane extraction again, merge organic phase, use saturated common salt water washing, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure obtains light yellow oil.

Boc-L-Lys (Cbz) OH after free, R-3-piperidine ethyl formate hydrochloride (1g), diisopropylethylamine (1.5g) are dissolved with anhydrous methylene chloride (20ml); nitrogen protection; be cooled to after 0 DEG C; add HOBt (700mg); EDCI (1.1g); at 0 DEG C, stir 20min, naturally rise to room temperature, react 8 hours.Use 5% potassium hydrogen sulfate solution, saturated sodium bicarbonate solution and the saturated common salt aqueous solution wash organic layer successively, and anhydrous sodium sulfate drying filters, and concentrating under reduced pressure obtains crude product, and column chromatography purification obtains colorless oil (1.729g, yield: 67%).

Rf=0.8 developping agent ethyl acetate colour developing: ultraviolet and iodine

MS：520(M+1)

B) synthetic Boc-L-Lys (Cbz)-(R-3-formyloxy) piperidines

Boc-L-Lys (Cbz)-(R-3-formyloxy) piperidines ethyl ester (1g) is dissolved in to methyl alcohol (10ml), be cooled to 0 DEG C, add sodium hydroxide (400mg), react and be adjusted to pH 4 with 5%HCl solution after 2 hours, concentrating under reduced pressure is removed methanol solvate, then is extracted with ethyl acetate, and gained organic phase is dry, concentrating under reduced pressure obtains colourless thick liquid (968mg, 98%).

Rf=0.6 developping agent ethyl acetate colour developing: ultraviolet and 1% triketohydrindene hydrate liquid

MS：492(M-)

C) synthetic L-Lys (Cbz)-(R-3-formyloxy) piperidines TFA

Boc-L-Lys (Cbz)-(R-3-formyloxy) piperidines (563mg) is dissolved in to methylene dichloride (15ml), ice bath is cooled to below 0 DEG C, drip TFA (5ml), 0 DEG C is reacted 1 hour, and concentrating under reduced pressure adds ether washing, obtain oily matter, the ether that inclines, drains and can obtain white sticky solid (507mg, 90%).

Rf=0.5 developping agent methylene chloride/methanol=5/1 colour developing: ultraviolet and 1% triketohydrindene hydrate liquid

MS：392(M-)

D) synthetic N-benzyl-L-lysyl-(R-3-formyloxy) piperidines two hydrobromates

L-Lys (Cbz)-(R-3-formyloxy) piperidines TFA (507mg) is dissolved in to methyl alcohol (8ml), be cooled to 0 DEG C, add diisopropylethylamine (195mg), phenyl aldehyde (121mg), stir 3 hours, add KBH4 (76mg) in batches, react 3 hours.Flash column purification, then with ether washing, topple over ether, drain to obtain white thick liquid.

Upper step gained thick liquid and methyl-phenoxide (0.5ml) are dissolved in to 36%HBr acetic acid solution (4ml), room temperature reaction, HPLC detection reaction is complete, add ether (20ml) to reaction solution, place refrigerator overnight, separate out solid, filter (241mg, 47%).

MS：348(M+1)

Embodiment 22: synthetic N-benzyl-L-lysyl-(S-3-formyloxy) piperidines two hydrobromates (compound 22)

A) synthetic Boc-L-Lys (Cbz)-(S-3-formyloxy) piperidines ethyl ester

Boc-L-Lys (Cbz) OHDCHA (1.9g) is dissolved in 5% sal enixum (20ml) solution and methylene dichloride (20ml), stir 0.5h, layering, water is used dichloromethane extraction again, merge organic phase, use saturated common salt water washing, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure obtains light yellow oil.

Boc-L-Lys (Cbz) OH after free, S-3-piperidine ethyl formate hydrochloride (1g), diisopropylethylamine (1.5g) are dissolved with anhydrous methylene chloride (20ml); nitrogen protection; be cooled to after 0 DEG C; add HOBt (700mg); EDCI (1.1g); at 0 DEG C, stir 20min, naturally rise to room temperature, react 8 hours.Use 5% potassium hydrogen sulfate solution, saturated sodium bicarbonate solution and the saturated common salt aqueous solution wash organic layer successively, and anhydrous sodium sulfate drying filters, and concentrating under reduced pressure obtains crude product, and column chromatography purification obtains colorless oil (1.826g, yield: 70%).

Rf=0.8 developping agent ethyl acetate colour developing: ultraviolet and iodine

MS：520(M+1)

B) synthetic Boc-L-Lys (Cbz)-(S-3-formyloxy) piperidines

Boc-L-Lys (Cbz)-(S-3-formyloxy) piperidines ethyl ester (1g) is dissolved in to methyl alcohol (10ml), be cooled to 0 DEG C, add sodium hydroxide (400mg), react and be adjusted to pH 4 with 5%HCl solution after 2 hours, concentrating under reduced pressure is removed methanol solvate, then is extracted with ethyl acetate, and gained organic phase is dry, concentrating under reduced pressure obtains colourless thick liquid (968mg, 98%).

Rf=0.6 developping agent ethyl acetate colour developing: ultraviolet and 1% triketohydrindene hydrate liquid

MS：492(M-)

C) synthetic L-Lys (Cbz)-(S-3-formyloxy) piperidines TFA

Boc-L-Lys (Cbz)-(S-3-formyloxy) piperidines (551mg) is dissolved in to methylene dichloride (15ml), ice bath is cooled to below 0 DEG C, drip TFA (5ml), 0 DEG C is reacted 1 hour, and concentrating under reduced pressure adds ether washing, obtain oily matter, the ether that inclines, drains and can obtain white sticky solid (500mg, 91%).

Rf=0.5 developping agent methylene chloride/methanol=5/1 colour developing: ultraviolet and 1% triketohydrindene hydrate liquid

MS：392(M-)

D) synthetic N-benzyl-L-lysyl-(S-3-formyloxy) piperidines two hydrobromates

L-Lys (Cbz)-(S-3-formyloxy) piperidines TFA (500mg) is dissolved in to methyl alcohol (8ml), be cooled to 0 DEG C, add diisopropylethylamine (195mg), phenyl aldehyde (122mg), stir 3 hours, add KBH4 (86mg) in batches, react 3 hours.Flash column purification, then with ether washing, topple over ether, drain to obtain white thick liquid.

Upper step gained thick liquid and methyl-phenoxide (0.5ml) are dissolved in to 36%HBr acetic acid solution (4ml), room temperature reaction, HPLC detection reaction is complete, add ether (20ml) to reaction solution, place refrigerator overnight, separate out solid, filter (235mg, 46%).

MS：348(M+1)

Embodiment 23: synthetic N-benzyl-L-arginyl-D-PROLINE two hydrobromates (compound 23)

A) synthetic Boc-L-Arg (Cbz) ₂-D-Pro-OCH ₃

By Boc-L-Arg (Cbz) ₂-OH (2.7g), D-PROLINE methyl ester hydrochloride (833mg), diisopropylethylamine (1.5g) dissolve with anhydrous methylene chloride (30ml); nitrogen protection; be cooled to after 0 DEG C; add HOBt (690mg); EDCI (1.1g); at 0 DEG C, stir 20min, naturally rise to room temperature, react 8 hours.Use 5% potassium hydrogen sulfate solution, saturated sodium bicarbonate solution and the saturated common salt aqueous solution wash organic layer successively, and anhydrous sodium sulfate drying filters, and concentrating under reduced pressure obtains crude product, and column chromatography purification obtains colorless oil (2.1g, yield: 65%).

Rf=0.5 developping agent ethyl acetate colour developing: ultraviolet and iodine

MS：654(M+1)

B) synthetic Boc-L-Arg (Cbz) ₂-D-Pro-OH

By Boc-L-Arg (Cbz) ₂-D-Pro-OCH ₃(1g) be dissolved in methyl alcohol (8ml), be cooled to 0 DEG C, add sodium hydroxide (300mg), react and be adjusted to pH 4 with 5%HCl solution after 2 hours, concentrating under reduced pressure is removed methanol solvate, then is extracted with ethyl acetate, and gained organic phase is dry, concentrating under reduced pressure obtains colourless thick liquid (896mg, 92%).

Rf=0.4 developping agent methylene chloride/methanol=40/1 colour developing: ultraviolet and 1% triketohydrindene hydrate liquid

MS：641(M-)

C) synthetic L-Arg (Cbz) ₂-D-Pro-OHTFA

By Boc-L-Arg (Cbz) ₂-D-Pro-OH (700mg) is dissolved in methylene dichloride (15ml), ice bath is cooled to below 0 DEG C, drip TFA (5ml), 0 DEG C is reacted 1 hour, and concentrating under reduced pressure adds ether washing, obtain oily matter, the ether that inclines, drains and can obtain white sticky solid (637mg, 91%).

Rf=0.2 developping agent methylene chloride/methanol=5/1 colour developing: ultraviolet and 1% triketohydrindene hydrate liquid

MS：540(M-)

D) synthetic N-benzyl-L-arginyl-D-PROLINE two hydrobromates

By L-Arg (Cbz) ₂-D-Pro-OHTFA (637mg) is dissolved in methyl alcohol (8ml), is cooled to 0 DEG C, adds diisopropylethylamine (190mg), phenyl aldehyde (121mg), stir 3 hours, add KBH4 (82mg) in batches, react 3 hours.Flash column purification, then with ether washing, topple over ether, drain to obtain white thick liquid.

Upper step gained thick liquid and methyl-phenoxide (0.5ml) are dissolved in to 36%HBr acetic acid solution (4ml), room temperature reaction, HPLC detection reaction is complete, add ether (20ml) to reaction solution, place refrigerator overnight, separate out solid, filter (268mg, 51%).

MS：362(M+1)

Embodiment 24: synthetic high arginyl-D-PROLINE two hydrobromates of N-benzyl-L-(compound 24)

A) synthetic Boc-L-Harg (Cbz) ₂-D-Pro-OCH ₃

By Boc-L-Harg (Cbz) ₂-OH (2.7g), D-PROLINE methyl ester hydrochloride (828mg), diisopropylethylamine (1.4g) dissolve with anhydrous methylene chloride (30ml); nitrogen protection; be cooled to after 0 DEG C; add HOBt (700mg); EDCI (1.15g); at 0 DEG C, stir 20min, naturally rise to room temperature, react 8 hours.Use 5% potassium hydrogen sulfate solution, saturated sodium bicarbonate solution and the saturated common salt aqueous solution wash organic layer successively, and anhydrous sodium sulfate drying filters, and concentrating under reduced pressure obtains crude product, and column chromatography purification obtains colorless oil (2.5g, yield: 75%).

Rf=0.6 developping agent ethyl acetate colour developing: ultraviolet and iodine

MS：669(M+1)

B) synthetic Boc-L-Harg (Cbz) ₂-D-Pro-OH

By Boc-L-Harg (Cbz) ₂-D-Pro-OCH ₃(1g) be dissolved in methyl alcohol (8ml), be cooled to 0 DEG C, add sodium hydroxide (300mg), react and be adjusted to pH 4 with 5%HCl solution after 2 hours, concentrating under reduced pressure is removed methanol solvate, then is extracted with ethyl acetate, and gained organic phase is dry, concentrating under reduced pressure obtains colourless thick liquid (860mg, 88%).

Rf=0.4 developping agent methylene chloride/methanol=40/1 colour developing: ultraviolet and 1% triketohydrindene hydrate liquid

MS：655(M-)

C) synthetic L-Harg (Cbz) ₂-D-Pro-OHTFA

By Boc-L-Harg (Cbz) ₂-D-Pro-OH (700mg) is dissolved in methylene dichloride (15mD, ice bath is cooled to below 0 DEG C, drip TFA (5ml), 0 DEG C is reacted 1 hour, and concentrating under reduced pressure adds ether washing, obtain oily matter, the ether that inclines, drains and can obtain white sticky solid (610mg, 87%).

Rf=0.2 developping agent methylene chloride/methanol=5/1 colour developing: ultraviolet and 1% triketohydrindene hydrate liquid

MS：554(M-)

D) synthetic high arginyl-D-PROLINE two hydrobromates of N-benzyl-L-

By L-Harg (Cbz) ₂-D-Pro-OHTFA (610mg) is dissolved in methyl alcohol (8ml), is cooled to 0 DEG C, adds diisopropylethylamine (185mg), phenyl aldehyde (115mg), stir 3 hours, add KBH4 (78mg) in batches, react 3 hours.Flash column purification, then with ether washing, topple over ether, drain to obtain white thick liquid.

Upper step gained thick liquid and methyl-phenoxide (0.5ml) are dissolved in to 36%HBr acetic acid solution (4ml), room temperature reaction, HPLC detection reaction is complete, add ether (20ml) to reaction solution, place refrigerator overnight, separate out solid, filter (246mg, 49%).

By those methods that are substantially equivalent to described in embodiment 1-d to embodiment-g, from Boc-L-Harg (Cbz) ₂-OH has prepared 84mg compound 24.

MS：376(M+1)

Embodiment 25: antithrombin activity is measured

In experiment, sample to be measured is joined in the enzyme reaction system of the freeze dried human zymoplasm national standard that obtains of purifying from human blood that contains 5.4 μ g/ml and hatch after 30 minutes and add specific substrate A c-FVR-AMC (5 μ M), use microwell plate detector Envision (PerkinElmer), at room temperature, RFU (relative fluorescence unit in detection of dynamic 10 minutes, relative intensity of fluorescence unit) variation, the slope that the reaction initial stage is linear enzymatic kinetic curve is enzyme reaction initial velocity (RFU/sec).

Inhibiting rate (%Inhibition) according to formula 1 calculation sample to enzymic activity,

$\%\mathrm{Inhibition}=\frac{v_{\mathrm{DMSO}}-v_{\mathrm{Sample}}}{v_{D\mathrm{MSO}}}\×100\%$

Formula 1

Wherein v _samplerepresent the initial velocity of dosing group, v _dMSOrepresent the initial velocity of DMSO group (i.e. not dosing group).IC ₅₀value (concentration of medicine when enzymic activity suppressed 50%) is that inhibiting rate (%Inhibition) is carried out Nonlinear Quasi and calculated by formula 2 the logarithmic value X of sample concentration.

$\%\mathrm{Inhibition}=\frac{100}{{1+10}^{(\mathrm{LogIC}50-X)*h}}$

Formula 2

Wherein h represents Hill coefficient

In following table, list the IC of the compounds of this invention ₅₀value

Be noted that and work as n=1, A=Lys or Arg, B part be that the activity of compound Trombin inhibiting of six-membered ring structure is stronger, especially in the time that A is Arg, has obviously improved the intensity about zymoplasm inhibition.

Following formulation examples just illustrates, and limits the scope of the invention by any way unintentionally.Active ingredient refers to compound or its pharmaceutically acceptable salt or the solvate of general formula (I).

Embodiment 26: the preparation of injection

Prescription:

Suitable diluted acid or alkali or buffering salt be can add, by extremely pH regulator steady state, antioxidant or metal chelator also can be comprised.Solution, by filtration sterilization, under aseptic condition, is filled in aseptic ampulla.

Embodiment 27: the preparation of tablet

Prescription:

Active ingredient is fully mixed with vehicle, sieve, compressing tablet on tabletting machine.

Embodiment 28: the preparation of hard capsule

Prescription:

Active ingredient is sieved, with mixed with excipients, with suitable equipment, mixture is filled in hard gelatin capsule.

Embodiment 29: the preparation of suspensoid

Prescription:

Active ingredient is sieved, mix with Xylo-Mucine and syrup, form a kind of evenly paste, pigment, phenylformic acid dilute by a part of purified water, under agitation add in paste, then add enough water to produce needed volume.

Although the present invention is illustrated with specific examples, but still should be appreciated that it can further be improved the application and will comprise any variation, purposes, or follow the present invention generally away from and comprise and deviate from the disclosure but belong to the known or conventional application in field of the present invention, and be used in equally said essential characteristic above, and be included within the scope of appended claims.

Claims (11)

1. the peptide compounds shown in formula (I) or its pharmacy acceptable salt:

Wherein,

N represents 1～2;

A represents Arg or Harg;

R ₁represent aryl;

B representative

Wherein,

Z representation carboxy, methyl carboxyl, formamyl or glycyl;

M represents 1 or 2;

Described aryl is for replacing or unsubstituted phenyl or naphthyl, and wherein substituting group is carboxyl, halogen, alkoxyl group, amino, alkylamino, dialkylamino, cyano group, ester group or trifluoromethyl.

2. peptide compounds according to claim 1, is characterized in that, described peptide compounds is N-benzyl-L-arginyl-D-PROLINE or the high arginyl-D-PROLINE of N-benzyl-L-.

3. the preparation method of the peptide compounds described in claim 1 or 2, is characterized in that, the method comprises that the amino acid protected by protecting group taking nitrogen position makes the step of final product as raw material.

4. preparation method according to claim 3, is characterized in that, described protecting group is selected from tertbutyloxycarbonyl, carbobenzoxy-(Cbz) or fluorenylmethoxycarbonyl.

5. the peptide compounds described in claim 1 or 2 is in the application of preparing in thrombin inhibitors.

6. the peptide compounds described in claim 1 or 2 is in preparation treatment and the application preventing in the medicine of the disease relevant with zymoplasm.

7. application according to claim 6, is characterized in that, the disease that the described disease relevant with zymoplasm is thrombin-mediated.

8. application according to claim 6, is characterized in that, described disease is apoplexy and tip arteriogenesis, atheromatosis, cerebral arteries disease or the tip arteriopathy that venous thrombosis and pulmonary infarction, artery thrombosis, thrombosis cause.

Peptide compounds described in claim 1 or 2 in vitro in transducer potector as the application of antithrombotics.

10. pharmaceutical composition, is characterized in that, it comprises peptide compounds and pharmaceutically acceptable carrier described in claim 1 or 2.

11. pharmaceutical compositions according to claim 10, is characterized in that, it is tablet, injection, suspensoid or capsule.