CN101724016B

CN101724016B - Peptide compound and preparation method and application thereof

Info

Publication number: CN101724016B
Application number: CN2008102018911A
Authority: CN
Inventors: 李敏; 黄雨; 袁哲东; 俞雄; 朱雪焱
Original assignee: Shanghai Institute of Pharmaceutical Industry
Current assignee: Li Min
Priority date: 2008-10-28
Filing date: 2008-10-28
Publication date: 2013-09-25
Anticipated expiration: 2028-10-28
Also published as: CN101724016A

Abstract

The invention discloses a novel peptide compound as shown by the formula (I), and a pharmaceutically acceptable salt, a pharmaceutically acceptable solvate, a derivative, an isomer, a crystal form and a pharmaceutically acceptable precursor medicament of the peptide compound. The invention also discloses a preparation method for the compound of the formula (I). The invention also discloses application of the peptide compound in preparing medicaments for treating and preventing thrombin-mediated and thrombin-related diseases.

Description

Peptide compound, Preparation Method And The Use

Technical field

The present invention relates to a class to the method for the competitive inhibitor of the inhibited novelty of Trypsin enzyme serine protease, particularly zymoplasm, synthetic this competitive inhibitor and the purposes in the preparation medicine thereof.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 make to explore and research effectively medicine of this type of disease of control seem particularly urgent, still be all to be significant aspect the fundamental research in clinical application.

There are major defect in traditional anticoagulant such as heparin, warfarin and the r-hirudin etc. of present clinical use aspect curative effect and the security, its application is very limited.Must inject use as heparin, and the zymoplasm in the blood clot is not had effect, and can cause the thrombopenia symptom; Warfarin is unique oral pharmaceutical, but owing to there is not clear and definite drug targets, individual anticoagulant response differs greatly, and influence factor is many, need carry out the 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 a wherein crucial step is that the thrombogen activation generates zymoplasm.Zymoplasm is a kind of serine protease of tryptase, and main effect is the insoluble fibrous compound that coagulates of the former generation of hydrolysis of fibrin.In the blood coagulation cascade reaction, play a crucial role.Therefore, the activity of Trombin inhibiting just can be blocked the formation of thrombus.Because its target spot is clear and definite and different with the action target spot of traditional anticoagulant, directly thrombin inhibitors (DTI) is expected to overcome the application limitation of traditional anti-freezing medicine.Clearly, convenient treatment control, the effective as selective of thrombus stroke and thrombin inhibitors with oral biological drug effect have been represented the target of a very attractive.

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

In US4101653, it is the zymoplasm substrate that Okamoto etc. disclose with N-p-toluenesulfonyl arginine methyl esters (TAMA), and serial arginine derivative has been synthesized in design, finds that argatroban (argatroban) has the good restraining thrombin activity.Through the pharmacology clinical study in calendar year 2001 FDA approval as the injection listing, argatroban is a kind of reversible competitive thrombin inhibitors, is combined with the reactive site of zymoplasm and plays a role, and zymoplasm is had 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 more 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 the 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 also there is effect, once carried out II phase clinical study, but the transformation period is short, and oral administration biaavailability is poor.

Astra company discloses a class benzenyl amidine analogue at WO9429336, wherein Melagatran (melagatran) has strong and the selective inhibition of coagulation enzymic activity, do not have obvious bleeding problems and can be used for degree of depth phlebothrombosis (DVT) safely, but oral administration biaavailability is low, the uncommon Melagatran (ximelagatran) of further synthetic its bi precursor medicine was in listing in 2004.Be first oral anticoagulation medicine in more than 60 year behind the warfarin, but severe liver injury is found in the listing back in clinical, stop in February, 2006.

In addition, reported among WO9311152, WO9715190, the US5510369 that a class has the thrombin inhibitors of unique D-diphenyl glycine in the P3 position.It is reported that this compounds compares with corresponding D-phenylglycocoll analogue, antithrombin activity higher (J Med Chem, 1997,40,830), some compounds wherein have oral bioavailability rate (J Med Chem, 1997,40,3687 preferably; J Med Chem, 1997,40,3726).

Have only minority to have pharmacokinetics and pharmacodynamic properties in the suitable body in the compound of having reported.Up to now, the research of thrombin inhibitors remains and competes one of the fiercest research topic in the present pharmaceutical chemistry field.

Summary of the invention

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

First purpose of the present invention provides 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;

The B representative

Or

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

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

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

Saturated or the unsaturated heterocycle of described benzo is for replacing or the structural unit of unsubstituted formula (III) representative:

The preferred particular compound of the present invention includes, but are not limited to: N-benzyl-L-lysyl-L-proline(Pro), N-benzyl-L-lysyl-D-proline(Pro), N-styroyl-L-lysyl-L-proline(Pro), N-styroyl-L-lysyl-D-proline(Pro), N-hydrocinnamyl-L-lysyl-L-proline(Pro), N-hydrocinnamyl-L-lysyl-D-proline(Pro), N-p-chlorobenzyl-L-lysyl-L-proline(Pro), N-p-chlorobenzyl-L-lysyl-D-proline(Pro), the luorobenzyl of N--L-lysyl-L-proline(Pro), the luorobenzyl of N--L-lysyl-D-proline(Pro), N-1-naphthyl-L-lysyl-L-proline(Pro), N-1-naphthyl-L-lysyl-D-proline(Pro), N-2-naphthyl-L-lysyl-L-proline(Pro), N-2-naphthyl-L-lysyl-D-proline(Pro), N-benzyl-L-lysyl-tetramethyleneimine, N-benzyl-L-lysyl-piperidines, N-benzyl-L-lysyl-hexahydroaniline, N-benzyl-L-lysyl-cycloheptylamine, N-benzyl-high the proline(Pro) of L-lysyl-L-, N-benzyl-high the proline(Pro) of 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(Pro) or the high arginyl of N-benzyl-L--D-proline(Pro).

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, disclosed term has the implication of their standards with abridging among the present invention.

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.For the preparation of the solvate form preferably solvent comprise water, alcohols, 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 the salt generative process.

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

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

When compound was solid, the crystal that compound of the present invention and salt can be different or polymorphous form existed, and all belong to the scope of the present invention and specific formula.

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

Described preparation method is described in detail as follows:

Route one:

Route one provides wherein that A is the general synthetic method of formula (I) compound of lysyl, n wherein, 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 the preferred unsettled ester of hydrogenolysis such as benzyl ester (Bn), alkali labile ester such as methyl ester (Me) or sour unsettled ester such as tertiary butyl ester (tBu).

In route one, at first the N-end coupling of the C-of compound 1 end and compound 2 makes compound 3, and this compound 3 is removed the amino protecting group R of carboxylic acid protecting group X and N α-end respectively ₄Obtain intermediate 5, again with radicals R ₁-(CH ₂) n introduces the N α-end of compound 5, removes the amino protecting group R on the side chain N at last ₅Make required compound.

Route two:

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

Or

R wherein 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, at 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, again with radicals R 1-(CH ₂) n introduces the N α-end of compound 5, removes the amino protecting group R on the side chain N at last ₅Make required compound.

Circuit three:

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

The 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),

In route three, at first the N-end coupling of the C-of compound 7 end and compound 2 makes compound 8, and this compound 8 is removed the amino protecting group R of carboxylic acid protecting group X and N α-end respectively ₆Obtain intermediate 10, again with radicals R 1-(CH ₂) n introduces the N α-end of compound 10, removes the amino protecting group R on the side chain N at last ₇Make required compound.

Circuit four:

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

The B representative

Or

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

In route four, at 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, again radicals R 1-(CH2) n is introduced the N α-end of compound 10, remove the amino protecting group R on the side chain N at last ₇Make required compound.

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

Above-mentioned being reflected under the anhydrous solvent existence carried out, and the examples of solvents that can select for use comprises: methylene dichloride, tetrahydrofuran (THF), ether, acetonitrile, ethylene dichloride, ethyl acetate, N, dinethylformamide, dimethyl sulfoxide (DMSO).Temperature of reaction is inessential usually, and preferred 0～30 ℃ was reacted 2～24 hours down.

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

Generally speaking, this reaction can be carried out in the presence of the solvent that reaction is had no adverse effect, and can cross the examples of solvents of selecting for use and comprise: methylene dichloride, alcohol are as methyl alcohol, ethanol etc., tetrahydrofuran (THF), dioxane, acetone, acetic acid, ethyl acetate.Temperature of reaction is inessential usually, preferred 0～40 ℃.

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

Generally speaking, this reaction can be carried out in the presence of the solvent that reaction is had no adverse effect, and the examples of solvents that can select for use comprises: water, alcohol are as methyl alcohol, ethanol etc., tetrahydrofuran (THF), dioxane, acetone.Temperature of reaction is inessential usually, preferred 0～30 ℃.

The last alkylated reaction of N can adopt reduction amination method and nucleophilic substitution method.As use the 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 the bisulphate root.The reagent of methylsulfate.Preferred alkyl iodine or alkyl are to tame benzene sulfonate.Nucleophilic substitution reaction need carry out in the presence of acid binding agent, 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 examples such as organic bases such as diisopropylethylamine, triethylamine, piperidines are trimethylamine such as diisopropylethylamine, triethylamine, Trimethylamine 99, pyridine, N-methylmorpholine etc., and special preferably phosphoric acid hydrogen dipotassium or diisopropylethylamine are as acid binding agent.The examples of solvents that can select for use comprises: tetrahydrofuran (THF), dioxane, acetonitrile, acetone, N, dinethylformamide etc.Preferred 30～80 ℃ of temperature of reaction.

As use the reduction amination method.Reactant and corresponding aldehyde can be handled with suitable reductive agent in appropriate solvent.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.Preferred 0～50 ℃ of temperature of reaction.

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

As general formula 1,2 and 7 compounds of initial substance, some is that commerce can get (Sigma-Aldrich company), some be known on the document (Chem Pharm Bull, 1999,47,1489 and Synth Commun, 2000,30,2525), can be by the described method preparation of document.The intermediate that obtains 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 purpose of the present invention provide the peptide compounds shown in the formula (I) preparation in the thrombin inhibitors application and the preparation treatment and prevent thrombin-mediated and with the medicine of thrombin-related diseases in application.

Compound of the present invention can be used for treating and prevent thrombin-mediated and and thrombin-related diseases.Described disease includes but not limited to: venous thrombosis and pulmonary infarction, the artery thrombosis for example apoplexy that causes of myocardial ischemia, myocardial infarction, unstable angina, thrombosis and tip artery form; Atheromatosis such as coronary artery disease, cerebral arteries disease and tip arteriopathy.

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

In addition, this compounds expection can be used from prevention with thrombolytic agent one and the treatment myocardial infarction forms.In addition, this compounds expection can be used for preventing forming thrombus again after the 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.

But compound expection per os of the present invention or parenteral route such as intravenous infusion, intramuscularly or subcutaneous injection administration.According to the compound acquisition treatment of administration of the present invention and the concrete metering of preventive effect, depend on the particular case of this case, comprise form, medicine-feeding rate and the illness for the treatment of of administration.Produce the typical oral per daily dose 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, perhaps every day, 3～5 such multiple dosess also may be suitable.Need be clear that, can do necessary routine adjustment to dosage and administering mode according to the severity of patient's age and body weight and the disease for the treatment of.Dosage and approach should have the attending doctor to determine accurately.

The 4th purpose of the present invention provides a kind of pharmaceutical composition, this pharmaceutical composition comprise shown in the formula (I) peptide compounds and pharmaceutically acceptable carrier.

The compounds of this invention can pharmaceutical composition the mode administration.Such as for oral administration, compound is made capsule or tablet form, wherein may contain vehicle such as lubricant, binding agent, disintegrating agent.In order to inject use, compound is dissolved in a kind of pharmaceutically acceptable solvent such as aseptic, no heat source water, in the physiological saline.

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

The abbreviation that occurs among 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

The Fmoc:9-fluorenylmethoxycarbonyl

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

The 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 the 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 will specify route one, two described concrete synthetic methods in detail, and synthesize preferred compound of the present invention, can change slightly but the technician understands described chemical reaction, to prepare a lot of other thrombin inhibitorss of the present invention.For example, by the apparent improvement of those skilled in the art, can successfully synthesize the compound of non-example among the present invention.These embodiment only are used for explanation, rather than limit the scope of the invention by any way.

Detection method:

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

HPLC:Waters1525; Detector: Waters2487; Chromatographic column: PhenomenexC18 (4.6 * 250mm, 5 μ m); Detect wavelength: 220nm and 254nm; Column temperature: 40 ℃; 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(Pro) two hydrobromates (compound 1)

A) synthetic [L-Lys (Cbz)] ₂Cu

Water (200ml) solution stirring 1h with L lysine HCL (18.2g), sodium bicarbonate (16.8g), cupric sulfate pentahydrate (12.5g).Ice bath is chilled to 0 ℃, slowly drips benzene methoxy dicarbonyl chloride (14.3ml), and is adjusted to pH8.5 with the 2mol/L sodium hydroxide solution, question response pH value was held constant at more than 3 hours, filtered, and the gained precipitation washes with water to neutrality, use washing with acetone, quantitatively obtain blue solid.Directly carry out the next step.

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

With [L-Lys (Cbz)] ₂Cu (27.7g) is suspended in acetone (100ml) and 10% yellow soda ash (100ml) aqueous solution, adds oxine (14.95g) under the vigorous stirring.Ice bath is cooled to below 10 ℃ after 2 hours, slowly drips acetone (60ml) solution of tert-Butyl dicarbonate (28g), rises to room temperature naturally, reacts 8 hours.Filter, filter cake washes with water, and filtrate decompression concentrates and removes acetone, and is colourless to organic layer with ethyl acetate extraction.Water is adjusted to pH4 with 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 gets light yellow oil, adds ethyl acetate (60ml) dissolving, stirs to add DCHA (18ml) and normal hexane (100ml) down, separate out solid gradually, filter, crude product gets white solid (46g, 82%) with Virahol/hexanaphthene recrystallization, mp:103～105 ℃, content 99% (HPLC, moving phase 1, method 1).Rf=0.6 developping agent methylene chloride=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), and 1.58-1.72 (2H, m), 1.82 (2H, m), 3.14-1.18 (2H, m), 4.26 (1H, m), 5.09 (2H, s), and 7.27-7.34 (5H, m)

C) synthetic D-proline methyl ester hydrochloride

D-proline(Pro) (23g) is dissolved in methyl alcohol (200ml), and ice bath is cooled to below 0 ℃, slow dripping thionyl chloride (36ml), and 1h dripped, in 40 ℃ of reactions 5 hours.Obtain light yellow oil 50 ℃ of following concentrating under reduced pressure desolventizings, crude product gets white solid (29g, 90%) with Virahol/isopropyl ether recrystallization.

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 B oc-L-Lys (Cbz)-D-Pro-OCH ₃

Boc-L-Lys (Cbz)-OHDCHA (7.3g) is dissolved in 5% sal enixum (40ml) solution and the methylene dichloride (40ml), stir 0.5h, layering, water is used dichloromethane extraction again, merge organic phase, use the saturated common salt water washing, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure gets 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; after being cooled to 0 ℃; add HOBt (2.1g); EDCI (3g); stir 20min down at 0 ℃, rise to room temperature naturally, reacted 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 gets 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

With Boc-L-Lys (Cbz)-D-Pro-OCH ₃(5.3g) be dissolved in methyl alcohol (50ml), be cooled to 0 ℃, add sodium hydroxide (1.7g), react and be adjusted to pH4 with 5%HCl solution after 2 hours, concentrating under reduced pressure is removed methanol solvate, uses ethyl acetate extraction again, gained organic phase drying, concentrating under reduced pressure obtain 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 methylene dichloride (40ml), and ice bath is cooled to below 0 ℃, drips TFA (20ml), and 0 ℃ was reacted 1 hour, concentrating under reduced pressure adds the ether washing, gets oily matter, the ether that inclines is drained and can be got white sticky solid (4.58g, 92%).

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

MS：376(M-)

G) synthetic N-benzyl-L-lysyl-D-proline(Pro) two hydrobromates

L-Lys (Cbz)-D-Pro-OHTFA (491mg) is dissolved in methyl alcohol (5ml), is cooled to 0 ℃, add triethylamine (0.21ml), phenyl aldehyde (121mg) stirred 3 hours, added KBH4 (84mg) in batches, reacted 3 hours.The flash column purification with ether washing, is toppled over ether again, drain white thick liquid.

To go up step gained thick liquid and methyl-phenoxide (0.5ml) and be dissolved in 36%HBr acetic acid solution (4ml), room temperature reaction, the HPLC detection reaction finishes, and adds ether (20ml) to reaction solution, places refrigerator overnight, separates out solid, filters (300mg, 61%).

MS：334(M+1)

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

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

To go up step gained thick liquid and methyl-phenoxide (0.5ml) and be dissolved in 36%HBr acetic acid solution (3ml), room temperature reaction, the HPLC detection reaction finishes, and adds ether (20ml) in reaction solution, places refrigerator overnight, separates out solid, filters (50mg, 6.6%)

MS：348(M+1)

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

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

To go up step gained thick liquid and methyl-phenoxide (0.5ml) and be dissolved in 36%HBr acetic acid solution (3ml), room temperature reaction, the HPLC detection reaction finishes, and adds ether (20ml) in reaction solution, places refrigerator overnight, separates out solid, filters (167mg, 21%)

MS：362(M+1)

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

L-Lys (the Cbz)-D-Pro-OHTFA (491mg) of embodiment 1 gained is dissolved in methyl alcohol (5ml), is cooled to 0 ℃, add triethylamine (0.21ml), 1-naphthaldehyde (175mg) stirred 3 hours, added KBH in batches ₄(83mg), reaction is 3 hours.The flash column purification with ether washing, is toppled over ether again, drain white thick liquid.

To go up step gained thick liquid and methyl-phenoxide (0.5ml) and be dissolved in 36%HBr acetic acid solution (4ml), room temperature reaction, the HPLC detection reaction finishes, and adds ether (20ml) to reaction solution, places refrigerator overnight, separates out solid, filters (261mg, 47.8%).

MS：384(M+1)

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

L-Lys (the Cbz)-D-Pro-OHTFA (491mg) of embodiment 1 gained is dissolved in methyl alcohol (5ml), is cooled to 0 ℃, add triethylamine (0.21ml), 2-naphthaldehyde (175mg) stirred 3 hours, added KBH in batches ₄(80mg), reaction is 3 hours.The flash column purification with ether washing, is toppled over ether again, drain white thick liquid.

To go up step gained thick liquid and methyl-phenoxide (0.5ml) and be dissolved in 36%HBr acetic acid solution (4ml), room temperature reaction, the HPLC detection reaction finishes, and adds ether (20ml) to reaction solution, places refrigerator overnight, separates out solid, filters (243mg, 44.6%).

MS：384(M+1)

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

L-Lys (the Cbz)-D-Pro-OHTFA (491mg) of embodiment 1 gained is dissolved in methyl alcohol (5ml), is cooled to 0 ℃, add triethylamine (0.21ml), 4-chloro-benzaldehyde (145mg) stirred 3 hours, added KBH in batches ₄(91mg), reaction is 3 hours.The flash column purification with ether washing, is toppled over ether again, drain white thick liquid.

To go up step gained thick liquid and methyl-phenoxide (0.5ml) and be dissolved in 36%HBr acetic acid solution (4ml), room temperature reaction, the HPLC detection reaction finishes, and adds ether (20ml) to reaction solution, places refrigerator overnight, separates out solid, filters (209mg, 39.4%).

MS：369(M+1)

Embodiment 7: the synthetic luorobenzyl of N--L-lysyl-D-proline(Pro) two hydrobromates (compound 7)

L-Lys (the Cbz)-D-Pro-OHTFA (491mg) of embodiment 1 gained is dissolved in methyl alcohol (5ml), is cooled to 0 ℃, add triethylamine (0.21ml), p-Fluorobenzenecarboxaldehyde (132mg) stirred 3 hours, added KBH in batches ₄(87mg), reaction is 3 hours.The flash column purification with ether washing, is toppled over ether again, drain white thick liquid.

To go up step gained thick liquid and methyl-phenoxide (0.5ml) and be dissolved in 36%HBr acetic acid solution (4ml), room temperature reaction, the HPLC detection reaction finishes, and adds ether (20ml) to reaction solution, places refrigerator overnight, separates out solid, filters (215mg, 42%).

MS：352(M+1)

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

A) synthetic L-proline methyl ester hydrochloride

L-proline(Pro) (19.49g) is dissolved in methyl alcohol (150ml), and ice bath is cooled to below 0 ℃, slow dripping thionyl chloride (38ml), and 1h dripped, in 40 ℃ of reactions 5 hours.Obtain light yellow oil 50 ℃ of following concentrating under reduced pressure desolventizings, crude product gets white solid (25g, 92%) with Virahol/isopropyl ether recrystallization.

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 the methylene dichloride (60ml), stir 0.5h, layering, water is used dichloromethane extraction again, merge organic phase, use the saturated common salt water washing, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure gets 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; after being cooled to 0 ℃; add HOBt (4.2g); EDCI (6g); stir 20min down at 0 ℃, rise to room temperature naturally, reacted 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 gets 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

With Boc-L-Lys (Cbz)-L-Pro-OCH ₃(2g) be dissolved in methyl alcohol (25ml), be cooled to 0 ℃, add sodium hydroxide (1.1g), react and be adjusted to pH4 with 5%HCl solution after 2 hours, concentrating under reduced pressure is removed methanol solvate, uses ethyl acetate extraction again, gained organic phase drying, concentrating under reduced pressure obtain colourless thick liquid (1.9g, 92%).

MS：476(M-)

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

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

MS：376(M-)

E) synthetic N-benzyl-L-lysyl-L-proline(Pro) two hydrobromates

L-Lys (Cbz)-L-Pro-OHTFA (491mg) is dissolved in methyl alcohol (5ml), is cooled to 0 ℃, add triethylamine (0.21ml), phenyl aldehyde (121mg) stirred 3 hours, added KBH4 (86mg) in batches, reacted 3 hours.The flash column purification with ether washing, is toppled over ether again, drain white thick liquid.

To go up step gained thick liquid and methyl-phenoxide (0.5ml) and be dissolved in 36%HBr acetic acid solution (4ml), room temperature reaction, the HPLC detection reaction finishes, and adds ether (20ml) to reaction solution, places refrigerator overnight, separates out solid, filters (154mg, 31%).

MS：334(M+1)

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

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

To go up step gained thick liquid and methyl-phenoxide (0.5ml) and be dissolved in 36%HBr acetic acid solution (3ml), room temperature reaction, the HPLC detection reaction finishes, and adds ether (20ml) in reaction solution, places refrigerator overnight, separates out solid, filters (61mg, 8%)

MS：348(M+1)

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

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

To go up step gained thick liquid and methyl-phenoxide (0.5ml) and be dissolved in 36%HBr acetic acid solution (3ml), room temperature reaction, the HPLC detection reaction finishes, and adds ether (20ml) in reaction solution, places refrigerator overnight, separates out solid, filters (184mg, 23%)

MS：362(M+1)

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

L-Lys (the Cbz)-L-Pro-OHTFA (491mg) of embodiment 8 gained is dissolved in methyl alcohol (5ml), is cooled to 0 ℃, add triethylamine (0.21ml), 1-naphthaldehyde (177mg) stirred 3 hours, added KBH in batches ₄(89mg), reaction is 3 hours.The flash column purification with ether washing, is toppled over ether again, drain white thick liquid.

To go up step gained thick liquid and methyl-phenoxide (0.5ml) and be dissolved in 36%HBr acetic acid solution (4ml), room temperature reaction, the HPLC detection reaction finishes, and adds ether (20ml) to reaction solution, places refrigerator overnight, separates out solid, filters (249mg, 45.6%).

MS：384(M+1)

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

L-Lys (the Cbz)-L-Pro-OHTFA (491mg) of embodiment 8 gained is dissolved in methyl alcohol (5ml), is cooled to 0 ℃, add triethylamine (0.21ml), 2-naphthaldehyde (181mg) stirred 3 hours, added KBH in batches ₄(95mg), reaction is 3 hours.The flash column purification with ether washing, is toppled over ether again, drain white thick liquid.

To go up step gained thick liquid and methyl-phenoxide (0.5ml) and be dissolved in 36%HBr acetic acid solution (4ml), room temperature reaction, the HPLC detection reaction finishes, and adds ether (20ml) to reaction solution, places refrigerator overnight, separates out solid, filters (211mg, 40%).

MS：384(M+1)

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

L-Lys (the Cbz)-L-Pro-OHTFA (491mg) of embodiment 8 gained is dissolved in methyl alcohol (5ml), is cooled to 0 ℃, add triethylamine (0.21ml), 4-chloro-benzaldehyde (150mg) stirred 3 hours, added KBH in batches ₄(88mg), reaction is 3 hours.The flash column purification with ether washing, is toppled over ether again, drain white thick liquid.

To go up step gained thick liquid and methyl-phenoxide (0.5ml) and be dissolved in 36%HBr acetic acid solution (4ml), room temperature reaction, the HPLC detection reaction finishes, and adds ether (20ml) to reaction solution, places refrigerator overnight, separates out solid, filters (186mg, 35%).

MS：369(M+1)

Embodiment 14: the synthetic luorobenzyl of N--L-lysyl-D-proline(Pro) two hydrobromates (compound 14)

L-Lys (the Cbz)-L-Pro-OHTFA (491mg) of embodiment 8 gained is dissolved in methyl alcohol (5ml), is cooled to 0 ℃, add triethylamine (0.21ml), p-Fluorobenzenecarboxaldehyde (143mg) stirred 3 hours, added KBH in batches ₄(97mg), reaction is 3 hours.The flash column purification with ether washing, is toppled over ether again, drain white thick liquid.

To go up step gained thick liquid and methyl-phenoxide (0.5ml) and be dissolved in 36%HBr acetic acid solution (4ml), room temperature reaction, the HPLC detection reaction finishes, and adds ether (20ml) to reaction solution, places refrigerator overnight, separates out solid, filters (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 the methylene dichloride (15ml), stir 0.5h, layering, water is used dichloromethane extraction again, merge organic phase, use the saturated common salt water washing, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure gets light yellow oil.

Boc-L-Lys (Cbz) OH after free, tetramethyleneimine (367mg), N-methylmorpholine (719mg) are dissolved with anhydrous methylene chloride (20ml); nitrogen protection; after being cooled to 0 ℃; add HOBt (811mg), EDCI (1.15g); stir 20min down at 0 ℃; naturally rise to room temperature, reacted 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 gets colorless oil (1.817g, yield: 83.8%).

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 methylene dichloride (10ml), ice bath is cooled to below 0 ℃, drip TFA (8ml), 0 ℃ of reaction 1h at 25 ℃ of following concentrating under reduced pressure, adds the ether washing with reaction solution, get oily matter, the ether that inclines is drained and can be got white sticky solid (850mg, 98%).

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 the methyl alcohol (10ml), is cooled to 0 ℃, be added dropwise to triethylamine (110mg), add phenyl aldehyde (120mg), stir 0.5h, add KBH in batches ₄(101mg) the about 4h of reaction, the HPLC detection reaction finishes, and concentrating under reduced pressure is removed most of solvent, and column chromatography purification obtains white thick liquid.

To go up step gained thick liquid and methyl-phenoxide (0.5ml) and be dissolved in the 36%HBr/ acetic acid solution (6ml), the HPLC detection reaction finishes, and adds ether (20ml) in reaction solution, places refrigerator overnight, separates out solid, filters and 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 the methylene dichloride (15ml), stir 0.5h, layering, water is used dichloromethane extraction again, merge organic phase, use the saturated common salt water washing, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure gets light yellow oil.

Boc-L-Lys (Cbz) OH after free, piperidines (434mg), N-methylmorpholine (720mg) are dissolved with anhydrous methylene chloride (20ml); nitrogen protection; after being cooled to 0 ℃; add HOBt (834mg), EDCI (1.2g); stir 20min down at 0 ℃; naturally rise to room temperature, reacted 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 gets colorless oil (2g, yield: 89%).

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 methylene dichloride (10ml), ice bath is cooled to below 0 ℃, drip TFA (8ml), 0 ℃ of reaction 1h at 25 ℃ of following concentrating under reduced pressure, adds the ether washing with reaction solution, get oily matter, the ether that inclines is drained and can be got white sticky solid (884mg, 98%).

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 the methyl alcohol (10ml), is cooled to 0 ℃, be added dropwise to triethylamine (303mg), add phenyl aldehyde (243mg), stir 0.5h, add KBH in batches ₄(106mg) the about 4h of reaction, the HPLC detection reaction finishes, and concentrating under reduced pressure is removed most of solvent, and column chromatography purification obtains white thick liquid.

To go up step gained thick liquid and methyl-phenoxide (0.65ml) and be dissolved in the 36%HBr/ acetic acid solution (10ml), the HPLC detection reaction finishes, and adds ether (20ml) in reaction solution, places refrigerator overnight, separates out solid, filters and 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) OH after free, hexahydroaniline (513mg), N-methylmorpholine (719mg) are dissolved with anhydrous methylene chloride (20ml); nitrogen protection; after being cooled to 0 ℃; add HOBt (817mg), EDCI (1.15g); stir 20min down at 0 ℃; naturally rise to room temperature, reacted 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 gets 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 methylene dichloride (10ml), ice bath is cooled to below 0 ℃, drip TFA (8ml), 0 ℃ of reaction 1h at 25 ℃ of following concentrating under reduced pressure, adds the ether washing with reaction solution, get oily matter, the ether that inclines is drained and can be got white sticky solid (866mg, 94%).

MS：362(M+1)

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

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

To go up step gained thick liquid and methyl-phenoxide (0.5ml) and be dissolved in the 36%HBr/ acetic acid solution (4ml), the HPLC detection reaction finishes, and adds ether (20ml) in reaction solution, places refrigerator overnight, separates out solid, filters and 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) OH after free, hexahydroaniline (590mg), N-methylmorpholine (719mg) are dissolved with anhydrous methylene chloride (20ml); nitrogen protection; after being cooled to 0 ℃; add HOBt (814mg), EDCI (1.15g); stir 20min down at 0 ℃; naturally rise to room temperature, reacted 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 gets 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 methylene dichloride (10ml), ice bath is cooled to below 0 ℃, drip TFA (8ml), 0 ℃ of reaction 1h at 25 ℃ of following concentrating under reduced pressure, adds the ether washing with reaction solution, get oily matter, the ether that inclines is drained and can be got white sticky solid (879mg, 93%).

MS：376(M+1)

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

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

To go up step gained thick liquid and methyl-phenoxide (0.5ml) and be dissolved in the 36%HBr/ acetic acid solution (4ml), the HPLC detection reaction finishes, and adds ether (20ml) in reaction solution, places refrigerator overnight, separates out solid, filters and obtains white solid (197mg, 40%).

MS：332(M+1)

Embodiment 19: synthetic N-benzyl-high proline(Pro) two hydrobromates of 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 the methylene dichloride (50ml), stir 0.5h, layering, water is used dichloromethane extraction again, merge organic phase, use the saturated common salt water washing, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure gets 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; after being cooled to 0 ℃; add HOBt (1.35g); EDCI (2.3g); stir 20min down at 0 ℃, rise to room temperature naturally, reacted 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 gets colorless oil (1.4g, yield: 27.7%).

MS：506(M+1)

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

With Boc-L-Lys (Cbz)-L-Hpro-OCH ₃(1.4g) be dissolved in methyl alcohol (10ml), be cooled to 0 ℃, add sodium hydroxide (400mg), react and be adjusted to pH4 with 5%HCl solution after 2 hours, concentrating under reduced pressure is removed methanol solvate, uses ethyl acetate extraction again, gained organic phase drying, concentrating under reduced pressure obtain 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 methylene dichloride (15ml), and ice bath is cooled to below 0 ℃, drips TFA (5ml), and 0 ℃ was reacted 1 hour, concentrating under reduced pressure adds the ether washing, gets oily matter, the ether that inclines is drained and can be got white sticky solid (609mg, 77%).

MS：392(M-)

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

L-Lys (Cbz)-L-Hpro-OHTFA (500mg) is dissolved in methyl alcohol (8ml), is cooled to 0 ℃, add diisopropylethylamine (193mg), phenyl aldehyde (127mg) stirred 3 hours, added KBH4 (76mg) in batches, reacted 3 hours.The flash column purification with ether washing, is toppled over ether again, drain white thick liquid.

To go up step gained thick liquid and methyl-phenoxide (0.5ml) and be dissolved in 36%HBr acetic acid solution (4ml), room temperature reaction, the HPLC detection reaction finishes, and adds ether (20ml) to reaction solution, places refrigerator overnight, separates out solid, filters (159mg, 31%).

MS：348(M+1)

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

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

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; after being cooled to 0 ℃; add HOBt (1.35g); EDCI (2.3g); stir 20min down at 0 ℃, rise to room temperature naturally, reacted 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 gets colorless oil (1.6g, yield: 29%).

MS：506(M+1)

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

With Boc-L-Lys (Cbz)-D-Hpro-OCH ₃(1.6g) be dissolved in methyl alcohol (10ml), be cooled to 0 ℃, add sodium hydroxide (420mg), react and be adjusted to pH4 with 5%HCl solution after 2 hours, concentrating under reduced pressure is removed methanol solvate, uses ethyl acetate extraction again, gained organic phase drying, concentrating under reduced pressure obtain colourless thick liquid (938mg, 69%).

MS：492(M-)

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

Boc-L-Lys (Cbz)-D-Hpro-OH (800mg) is dissolved in methylene dichloride (15ml), and ice bath is cooled to below 0 ℃, drips TFA (5ml), and 0 ℃ was reacted 1 hour, concentrating under reduced pressure adds the ether washing, gets oily matter, the ether that inclines is drained and can be got white sticky solid (711mg, 89%).Rf=0.5 developping agent methylene chloride=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 methyl alcohol (8ml), is cooled to 0 ℃, add diisopropylethylamine (195mg), phenyl aldehyde (121mg) stirred 3 hours, added KBH4 (76mg) in batches, reacted 3 hours.The flash column purification with ether washing, is toppled over ether again, drain white thick liquid.

To go up step gained thick liquid and methyl-phenoxide (0.5ml) and be dissolved in 36%HBr acetic acid solution (4ml), room temperature reaction, the HPLC detection reaction finishes, and adds ether (20ml) to reaction solution, places refrigerator overnight, separates out solid, filters (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 the methylene dichloride (20ml), stir 0.5h, layering, water is used dichloromethane extraction again, merge organic phase, use the saturated common salt water washing, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure gets 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; after being cooled to 0 ℃; add HOBt (700mg); EDCI (1.1g); stir 20min down at 0 ℃, rise to room temperature naturally, reacted 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 gets colorless oil (1.729g, yield: 67%).

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 methyl alcohol (10ml), be cooled to 0 ℃, add sodium hydroxide (400mg), react and be adjusted to pH4 with 5%HCl solution after 2 hours, concentrating under reduced pressure is removed methanol solvate, uses ethyl acetate extraction again, gained organic phase drying, concentrating under reduced pressure obtains colourless thick liquid (968mg, 98%).

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 methylene dichloride (15ml), ice bath is cooled to below 0 ℃, drip TFA (5ml), 0 ℃ was reacted 1 hour, and concentrating under reduced pressure adds the ether washing, get oily matter, the ether that inclines is drained and can be got white sticky solid (507mg, 90%).

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 methyl alcohol (8ml), is cooled to 0 ℃, add diisopropylethylamine (195mg), phenyl aldehyde (121mg) stirred 3 hours, added KBH4 (76mg) in batches, reacted 3 hours.The flash column purification with ether washing, is toppled over ether again, drain white thick liquid.

To go up step gained thick liquid and methyl-phenoxide (0.5ml) and be dissolved in 36%HBr acetic acid solution (4ml), room temperature reaction, the HPLC detection reaction finishes, and adds ether (20ml) to reaction solution, places refrigerator overnight, separates out solid, filters (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 the methylene dichloride (20ml), stir 0.5h, layering, water is used dichloromethane extraction again, merge organic phase, use the saturated common salt water washing, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure gets 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; after being cooled to 0 ℃; add HOBt (700mg); EDCI (1.1g); stir 20min down at 0 ℃, rise to room temperature naturally, reacted 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 gets colorless oil (1.826g, yield: 70%).

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 methyl alcohol (10ml), be cooled to 0 ℃, add sodium hydroxide (400mg), react and be adjusted to pH4 with 5%HCl solution after 2 hours, concentrating under reduced pressure is removed methanol solvate, uses ethyl acetate extraction again, gained organic phase drying, concentrating under reduced pressure obtains colourless thick liquid (968mg, 98%).

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 methylene dichloride (15ml), ice bath is cooled to below 0 ℃, drip TFA (5ml), 0 ℃ was reacted 1 hour, and concentrating under reduced pressure adds the ether washing, get oily matter, the ether that inclines is drained and can be got white sticky solid (500mg, 91%).

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 methyl alcohol (8ml), is cooled to 0 ℃, add diisopropylethylamine (195mg), phenyl aldehyde (122mg) stirred 3 hours, added KBH4 (86mg) in batches, reacted 3 hours.The flash column purification with ether washing, is toppled over ether again, drain white thick liquid.

To go up step gained thick liquid and methyl-phenoxide (0.5ml) and be dissolved in 36%HBr acetic acid solution (4ml), room temperature reaction, the HPLC detection reaction finishes, and adds ether (20ml) to reaction solution, places refrigerator overnight, separates out solid, filters (235mg, 46%).

MS：348(M+1)

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

A) synthetic Boc-L-Arg (Cbz) 2-D-Pro-OCH3

With Boc-L-Arg (Cbz) ₂-OH (2.7g), D-proline methyl ester hydrochloride (833mg), diisopropylethylamine (1.5g) be with anhydrous methylene chloride (30ml) dissolving, nitrogen protection, be cooled to 0 ℃ after; add HOBt (690mg), EDCI (1.1g) stirs 20min down at 0 ℃; naturally rise to room temperature, reacted 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 gets 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

With Boc-L-Arg (Cbz) ₂-D-Pro-OCH ₃(1g) be dissolved in methyl alcohol (8ml), be cooled to 0 ℃, add sodium hydroxide (300mg), react and be adjusted to pH4 with 5%HCl solution after 2 hours, concentrating under reduced pressure is removed methanol solvate, uses ethyl acetate extraction again, gained organic phase drying, concentrating under reduced pressure obtain colourless thick liquid (896mg, 92%).

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

MS：641(M-)

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

With Boc-L-Arg (Cbz) ₂-D-Pro-OH (700mg) is dissolved in methylene dichloride (15ml), and ice bath is cooled to below 0 ℃, drips TFA (5ml), and 0 ℃ was reacted 1 hour, and concentrating under reduced pressure adds the ether washing, gets oily matter, and the ether that inclines is drained and can be got white sticky solid (637mg, 91%).

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

MS：540(M-)

D) synthetic N-benzyl-L-arginyl-D-proline(Pro) two hydrobromates

With L-Arg (Cbz) ₂-D-Pro-OHTFA (637mg) is dissolved in methyl alcohol (8ml), is cooled to 0 ℃, adds diisopropylethylamine (190mg), and phenyl aldehyde (121mg) stirred 3 hours, added KBH4 (82mg) in batches, reacted 3 hours.The flash column purification with ether washing, is toppled over ether again, drain white thick liquid.

To go up step gained thick liquid and methyl-phenoxide (0.5ml) and be dissolved in 36%HBr acetic acid solution (4ml), room temperature reaction, the HPLC detection reaction finishes, and adds ether (20ml) to reaction solution, places refrigerator overnight, separates out solid, filters (268mg, 51%).

MS：362(M+1)

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

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

With Boc-L-Harg (Cbz) ₂-OH (2.7g), D-proline methyl ester hydrochloride (828mg), diisopropylethylamine (1.4g) be with anhydrous methylene chloride (30ml) dissolving, nitrogen protection, be cooled to 0 ℃ after; add HOBt (700mg), EDCI (1.15g) stirs 20min down at 0 ℃; naturally rise to room temperature, reacted 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 gets 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

With Boc-L-Harg (Cbz) ₂-D-Pro-OCH ₃(1g) be dissolved in methyl alcohol (8ml), be cooled to 0 ℃, add sodium hydroxide (300mg), react and be adjusted to pH4 with 5%HCl solution after 2 hours, concentrating under reduced pressure is removed methanol solvate, uses ethyl acetate extraction again, gained organic phase drying, concentrating under reduced pressure obtain colourless thick liquid (860mg, 88%).

MS：655(M-)

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

With Boc-L-Harg (Cbz) ₂-D-Pro-OH (700mg) is dissolved in methylene dichloride (15ml), and ice bath is cooled to below 0 ℃, drips TFA (5ml), and 0 ℃ was reacted 1 hour, and concentrating under reduced pressure adds the ether washing, gets oily matter, and the ether that inclines is drained and can be got white sticky solid (610mg, 87%).

MS：554(M-)

D) the synthetic high arginyl of N-benzyl-L--D-proline(Pro) two hydrobromates

With L-Harg (Cbz) ₂-D-Pro-OHTFA (610mg) is dissolved in methyl alcohol (8ml), is cooled to 0 ℃, adds diisopropylethylamine (185mg), and phenyl aldehyde (115mg) stirred 3 hours, added KBH4 (78mg) in batches, reacted 3 hours.The flash column purification with ether washing, is toppled over ether again, drain white thick liquid.

To go up step gained thick liquid and methyl-phenoxide (0.5ml) and be dissolved in 36%HBr acetic acid solution (4ml), room temperature reaction, the HPLC detection reaction finishes, and adds ether (20ml) to reaction solution, places refrigerator overnight, separates out solid, filters (246mg, 49%).

With being equivalent to described those methods of embodiment 1-d to embodiment-g basically, 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 joined hatch adding specific substrate A c-FVR-AMC (5 μ M) after 30 minutes in the enzyme reaction system that contains the freeze dried human zymoplasm national standard product that purifying of 5.4 μ g/ml obtain from human blood, use microwell plate detector Envision (PerkinElmer), at room temperature, RFU (relative fluorescence unit in the detection of dynamic 10 minutes, relative intensity of fluorescence unit) variation, the reaction initial stage is linear enzyme kinetics slope of a curve and is enzyme reaction initial velocity (RFU/sec).

According to the inhibiting rate (%Inhibition) of formula 1 calculation sample to enzymic activity,

% Inhibition = \frac{v_{DMSO} - v_{Sample}}{v_{DMSO}} \times 100 %

Formula 1

V wherein _Samp/eThe initial velocity of expression dosing group, v _DMSOThe initial velocity of expression DMSO group (i.e. not dosing group).IC ₅₀To be inhibiting rate (%Inhibition) carry out non-linear fitting to the logarithmic value X of sample concentration by formula 2 to value (enzymic activity is suppressed 50% o'clock drug concentrations) calculates.

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

Formula 2

Wherein h represents the Hill coefficient

In following table, listed the IC of The compounds of this invention ₅₀Value

Be noted that and work as n=1 that A=Lys or Arg, B partly are that the activity of compound Trombin inhibiting of six-membered ring structure is stronger, have especially obviously improved the intensity that suppresses about zymoplasm when A is Arg.

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 can be added, with the extremely steady state of pH regulator, also antioxidant or metal chelator can be comprised.Solution by filtration sterilization, under aseptic condition, is filled in the aseptic peace bottle.

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, and with mixed with excipients, the equipment with suitable is filled in mixture in the hard gelatin capsule.

Embodiment 29: the preparation of suspensoid

Prescription:

Active ingredient is sieved, mix with Xylo-Mucine and syrup, form a kind of even paste, pigment, phenylformic acid dilute with a part of purified water, under agitation add in the paste, add enough water then 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 the said essential characteristic in front equally, and be included in the appended claims scope.

Claims

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

Wherein,

N represents 1～3;

A represents Lys;

R ₁Represent aryl; Described aryl is for replacing or unsubstituted phenyl or naphthyl, and wherein substituting group is hydroxyl, carboxyl, halogen, alkoxyl group, cyano group, ester group, trifluoromethyl or with the alkyl of C1～4 of above-mentioned group;

The B representative

Wherein,

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

M represents 1 or 2.

2. peptide compounds according to claim 1, it is characterized in that described peptide compounds is following compound: N-benzyl-L-lysyl-L-proline(Pro), N-benzyl-L-lysyl-D-proline(Pro), N-styroyl-L-lysyl-L-proline(Pro), N-styroyl-L-lysyl-D-proline(Pro), N-hydrocinnamyl-L-lysyl-L-proline(Pro), N-hydrocinnamyl-L-lysyl-D-proline(Pro), N-p-chlorobenzyl-L-lysyl-L-proline(Pro), N-p-chlorobenzyl-L-lysyl-D-proline(Pro), the luorobenzyl of N--L-lysyl-L-proline(Pro), the luorobenzyl of N--L-lysyl-D-proline(Pro), N-1-naphthyl-L-lysyl-L-proline(Pro), N-1-naphthyl-L-lysyl-D-proline(Pro), N-2-naphthyl-L-lysyl-L-proline(Pro), N-2-naphthyl-L-lysyl-D-proline(Pro), N-benzyl-L-lysyl-tetramethyleneimine, N-benzyl-L-lysyl-piperidines, N-benzyl-L-lysyl-hexahydroaniline, N-benzyl-L-lysyl-cycloheptylamine, N-benzyl-high the proline(Pro) of L-lysyl-L-, N-benzyl-high the proline(Pro) of 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(Pro) or the high arginyl of N-benzyl-L--D-proline(Pro).

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

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

5. the application of the arbitrary described peptide compounds of claim 1-2 in the preparation thrombin inhibitors.

6. the arbitrary described peptide compounds of claim 1-2 is in preparation treatment with prevent application in the medicine with thrombin-related diseases.

7. the described application of claim 6 is characterized in that, described disease is the disease of thrombin-mediated.

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

The arbitrary described peptide compounds of claim 1-2 in the extracorporeal blood pipeline as the application of antithrombotics.

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

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