CN1275979C - Carboline carboxylic acid contaiing P6A associated sequence and synthesis and medical application thereof - Google Patents

Abstract

The present invention relates to a protective intermediate compound of P6A correlated series, a compound of a deprotection product, corresponding pseudo peptide generated by coupling and deprotecting the compound of a deprotection product and the carboxyl of carboline carboxylic acid, and corresponding pseudopeptide generated by coupling and deprotecting the compound for a deprotection resultant and the secondary amino groups of the carboline carboxylic acid. The present invention also relates to preparation methods and application in medicine, such as application as thrombolytic medicines. The compounds have strong thrombolysis activity.

Description

The carboline carboxylate that contains the P6A correlated series, they synthetic and in medically application

Technical field

The present invention relates to the low-molecular-weight peptide class of class product, relate in particular to P6A related peptides sequence, it intends peptide, its preparation method and the application in the preparation medicine.

Background technology

Vessel embolism, for example myocardial infarction and cerebral apoplexy are the highest diseases of mortality ratio in the cardiovascular and cerebrovascular diseases.Seeking thrombolytic agent safely and effectively, is one of focus of cardiovascular and cerebrovascular diseases drug research.The inventor is first guide structure with fibrin degradation product (FDP) P6A (ARPAK), carries out structural modification by liquid phase is synthetic, has obtained a series of oligopeptides (contriver: Peng Shiqi, Zhao Ming, Wang Chao, the pivot Tang Dynasty, Wang Yinye; Denomination of invention: cardiovascular active polypeptide and their the synthetic and application in medical science; Granted publication CN 1055479C; Granted publication day: on August 16th, 2000).These oligopeptides have clear and definite thrombus dissolving activity, become outstanding thrombus dissolving oligopeptides elder generation guide structure.When the metabolism of research P6A and analogue, the inventor uses LC/MS tracking P6A and analogue in the intravital variation of mouse, finds that P6A is Arg-Pro-Ala-Lys-OH, Pro-Ala-Lys-OH, Ala-Arg-Pro-Ala-OH and four kinds of meta-bolitess of Ala-Arg-Pro-OH at the mouse vivo degradation.The contriver has prepared this four kinds of meta-bolitess with liquid phase synthesizing method, their application prospect (Peng Shiqi on the rat suppository model, have been confirmed as thrombolytic agent, Zhao Ming, Wang Chao, Xu Youxuan, Wu Yanfen, the meta-bolites of P6A and the application in medical science thereof, number of patent application 01136780.6, Intellectual Property Right Bureau of the RPC).P6A and analogue all have the inherent room for improvement as the meta-bolites of thrombus dissolving oligopeptides elder generation's guide structure and P6A as thrombus dissolving oligopeptides elder generation guide structure, be the albumen of existence in the body and the difficulty that polypeptide causes for their detection, and technology and financial difficulties that the LC/MS detection causes all have much room for improvement.If have a kind of existing antithrombotic acitivity the structure of uv-absorbing character to be incorporated in the first guide structure of above-mentioned thrombus dissolving oligopeptides again, can overcome and detect the difficulty that causes in the body.Reports such as Yao Xinsheng are separated to carboline carboxylate from the Onion head.Think that carboline carboxylate has outstanding antiplatelet aggregative activity (Yao Xinsheng etc., Chinese pharmaceutical chemistry magazine, 1995,5,134).The inventor adopts chemical process to synthesize carboline carboxylate, confirms that carboline carboxylate has outstanding antiplatelet aggregative activity really.The outstanding uv-absorbing character that the indole ring parent nucleus of carboline carboxylate brings and have outstanding platelet aggregation inhibitory activity as edible natural product has the potential application prospect.Peptide was intended in preparation among the present invention was incorporated into P6A and correlated series to carboline carboxylate as pharmacophore.The plan peptide that makes has comprised the thrombolysis activity of former peptide sequence, has comprised the platelet aggregation inhibitory activity of carboline carboxylate, and has the ultraviolet detectability.Described ultraviolet detectability makes the pharmacokinetic of this comparison peptide become simple and direct.

Summary of the invention

The object of the invention provides a class by the plan peptide of being made up of P6A correlated series polypeptide and carboline carboxylate, and this plan peptide removes has thrombolysis activity, also has the platelet aggregation inhibitory activity of carboline carboxylate and has the ultraviolet detectability.

The present invention adopts the quadrature strategy to adopt liquid phase process progressively to connect peptide from the L-amino acid of Boc protection by route shown in Figure 1, makes general formula (1) protection oligopeptides intermediate.AA wherein ₁And AA ₂Common is hydrogen atom, or AA ₁Be A, G, Q, K, N, R, AA ₂Be Arg (Tos) residue.

Boc-AA ₁-AA ₂-Pro-Ala-Lys (ClZ)-OBzl general formula (1)

The present invention adopts the quadrature strategy to adopt liquid phase process progressively to connect peptide from the L-amino acid of Boc protection by route shown in Figure 2, makes the protection oligopeptides intermediate of general formula (2).Boc-AA wherein ₃OH is OBzl or AA jointly ₃Be the Ala residue.

Boc-Ala-Arg (Tos)-Pro-AA ₃-OBzl general formula (2)

Compound deprotection with general formula (1) under the effect of HCl gets the compound of general formula (4); Compound deprotection with general formula (1) under the effect of NaOH gets the compound of general formula (5).AA wherein ₁And AA ₂Common is hydrogen atom, or AA ₁Be A, G, Q, K, N or R, AA ₂Be Arg (Tos) residue.

HClAA ₁-AA ₂-Pro-Ala-Lys (ClZ)-Obzl general formula (4)

Boc-AA ₁-AA ₂-Pro-Ala-Lys (ClZ) OH general formula (5)

As shown in Figure 4, with the carboxyl condensation of the compound and the N-Boc-carboline carboxylate of general formula (4), condensation product gets the compound of general formula (6) through the HF deprotection.AA ₁' and AA ₂' be hydrogen atom jointly; Or AA ₁' be A, G, Q, K, R, N, AA ₂' be the Arg residue.

As shown in Figure 5, with the amino condensation of the compound and the carboline carboxylate benzyl ester of general formula (5), condensation product gets the compound of general formula (7) through the HF deprotection.AA ₁' and AA ₂' be hydrogen atom jointly; Or AA ₁' be A, G, Q, K, R, N, AA ₂' be the Arg residue.

Compound deprotection with general formula (2) under the effect of HCl gets the compound of general formula (9); Compound deprotection with general formula (2) under the effect of NaOH gets the compound of general formula (10).AA wherein ₃Be Ala residue or AA ₃Disappearance.

HClAla-Arg (Tos)-Pro-AA ₃-OBzl general formula (9)

Boc-Ala-Arg (Tos)-Pro-AA ₃OH general formula (10)

As shown in Figure 7, with the carboxyl condensation of the compound and the N-Boc-carboline carboxylate of general formula (9), condensation product gets the compound of general formula (11) through the HF deprotection.AA wherein ₃Be Ala residue or AA ₃Disappearance.

As shown in Figure 8, with the amino condensation of the compound and the carboline carboxylate benzyl ester of general formula (10), condensation product gets the compound of general formula (12) through the HF deprotection.AA wherein ₃Be Ala residue or AA ₃Disappearance.

Plan peptide of the present invention removes the thrombolysis activity that has comprised former peptide sequence, has also comprised the platelet aggregation inhibitory activity of carboline carboxylate, and has had the ultraviolet detectability.So plan peptide of the present invention can be used to prepare the medicine for the treatment of thrombotic diseases, its ultraviolet detectability can make the pharmacokinetic of this comparison peptide become more simple and direct in addition.

Description of drawings

Fig. 1 is the synthetic route of P6A correlated series.

Fig. 2 is the synthetic route of P6A meta-bolites.

Fig. 3 is that the protection polypeptide of the relevant series of P6A takes off Boc, saponification, and deprotection.

Fig. 4 is the protection polypeptide of general formula (4) and the coupling of N-Boc-carboline carboxylate, deprotection.

Fig. 5 is the protection polypeptide of general formula (5) and the coupling of carboline carboxylate benzyl ester, deprotection.

Fig. 6 is that the protection polypeptide of P6A meta-bolites takes off Boc, saponification, and deprotection.

Fig. 7 is the protection polypeptide of general formula (9) and the coupling of N-Boc-carboline carboxylate, deprotection.

Fig. 8 is the protection polypeptide of general formula (10) and the coupling of carboline carboxylate benzyl ester, deprotection.

Plan peptide of the present invention has also comprised the platelet aggregation inhibitory activity of carboline carboxylic acid, and has had ultraviolet detection property except the thrombolysis activity that has comprised former peptide sequence. So plan peptide of the present invention can be for the preparation of the medicine for the treatment of thrombotic diseases, its ultraviolet detection property can make the pharmacokinetic of this class plan peptide become more simple and direct in addition.

The present invention adopts rat arteriovenous shut model to estimate general formula (3), (6), (7), (8), the thrombus dissolving activity of the compound of (11) and (12). The result shows that plan peptide provided by the invention not only has outstanding thrombolytic effect, and this kind thrombolytic effect has clear and definite structure dependence.

Embodiment

In order further to set forth the present invention, provide a series of embodiment below.These embodiment are illustrative fully, and they only are used for the present invention is specifically described, and not should be understood to limitation of the present invention.

In chemical embodiment, the protection amino acid of use is the L configuration, and intermediate and degree of purity of production confirm that with TLC FAB-MS measures with the ZAB-MS type High Resolution G C/MS/DS combined instrument that Britain VG company produces.Electrospray ionization mass spectrum is measured by the ES-5989X type mass spectrograph that U.S. Hewlett-Packard Corporation produces.Optically-active is measured with the polarimeter (Polartronic D type) that Schmidt+Haensch company produces.Fusing point is measured with micro-fusing point instrument, and thermometer is not proofreaied and correct.Silica gel for chromatography is produced by Haiyang Chemical Plant, Qingdao.Sephadex is available from Sigma company.

The preparation of embodiment 1.Boc-Lys (ClZ)-OBzl

160mg (0.491mmol) Cs ₂CO ₃Be dissolved in distilled water, join in the ethanol solution of 400mg (0.966mmol) Boc-Lys (ClZ)-OH, removal of solvent under reduced pressure behind the 40min, residue is put moisture eliminator and is spent the night, get white solid, this solid is dissolved in dry DMF, drips 0.112ml (0.98mmol) bromobenzyl, behind 50 ℃ of stirring 16hr, filtering CsBr precipitation, filtrate decompression is concentrated into dried, residue with acetic acid ethyl dissolution after, successively with saturated sodium bicarbonate aqueous solution, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate.The organic layer anhydrous sodium sulfate drying of telling filters, and filtrate gets title compound 480mg (98.5%) at 37 ℃ of following concentrating under reduced pressure, is colorless oil.

The preparation of embodiment 2.HClLys (ClZ)-OBzl

100mg (0.2mmol) Boc-Lys (ClZ)-OBzl is dissolved in 3ml 4mol/L anhydrous hydrogen chloride/ethyl acetate solution, stirring at room, and the adularescent precipitation generates behind the 20min, and TLC behind the 2hr (chloroform/methanol, 40/1) shows that raw material point disappears.Reaction mixture is concentrating under reduced pressure at room temperature, the residue acetic acid ethyl dissolution, and at room temperature concentrate, so repeatedly for several times, up to Ex-all free hydrogenchloride.Residue grinds with anhydrous diethyl ether, takes out ether, white solid, this solid soaks with sherwood oil (60-90 ℃), the sherwood oil that inclines, drain after for several times so repeatedly title compound, be directly used in next step reaction.

The preparation of embodiment 3.Boc-Ala-Lys (ClZ)-OBzl

38mg (0.2mmol) Boc-Ala-OH is dissolved among the anhydrous THF of 3ml, and ice bath adds 30mg (0.22mmol) N-hydroxy benzo triazole and 45mg (0.22mmol) DCC down.Stand-by behind the mixing 33min.88mg (0.2mmol) HCILys (ClZ)-OBzl is dissolved in the anhydrous THF of 5ml, drip N-methylmorpholine and make pH8-9, the mixture that adding obtains above is behind 0 ℃ of reaction 2hr, room temperature reaction 4hr, TLC (chloroform/methanol, 30/1) shows that raw material point disappears stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution is successively with saturated sodium bicarbonate aqueous solution, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate.The organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain title compound 112mg (98%), and mp94-96 ℃, FAB-MS (m/e) 575[M+H] ⁺, 475[M-Boc] ⁺

The preparation of embodiment 4.HClAla-Lys (ClZ)-OBzl

115mg (0.2mmol) Boc-Ala-Lys (ClZ)-OBzl is dissolved in 4ml 4mol/L anhydrous chlorides of rase chlorine/ethyl acetate solution, stirring at room, and TLC behind the 2hr (chloroform/methanol, 30/1) shows that raw material point disappears.Reaction mixture is concentrating under reduced pressure at room temperature, and residue also at room temperature concentrates with acetic acid ethyl dissolution, so repeatedly for several times, and up to Ex-all free hydrogenchloride.Residue grinds with anhydrous diethyl ether, gets title compound, is directly used in next step reaction.

The preparation of embodiment 5.Boc-Pro-Ala-Lys (ClZ)-OBzl

280mg (1.3mmol) Boc-Pro-OH is dissolved among the anhydrous THF of 30ml, and ice bath adds 170mg (1.26mmol) N-hydroxy benzo triazole and 270mg (1.31mmol) DCC down, and is stand-by behind the mixing 30min.657mg (1.28mmol) HCI.Ala-Lys (ClZ)-OBzl is dissolved in the anhydrous THF of 5ml, drip N-methylmorpholine and make pH value of solution 8-9, the mixture that adding obtains above, behind 0 ℃ of reaction 2hr, room temperature reaction 6hr, TLC (chloroform/methanol, 20/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, successively with saturated sodium bicarbonate aqueous solution, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain mp86-88 ℃ of title compound 134mg (98%), FAB-MS (m/e) 674[M+H] ⁺

The preparation of embodiment 6.HClPro-Ala-Lys (ClZ)-OBzl

128mg (0.19mmol) Boc-Pro-Ala-Lys (ClZ)-OBzl is dissolved in 5ml anhydrous hydrogen chloride/ethyl acetate solution, stirring at room, and TLC behind the 5hr (chloroform/methanol, 20/1) shows that raw material point disappears.Reaction mixture is concentrating under reduced pressure at room temperature, and residue also at room temperature concentrates with acetic acid ethyl dissolution, and for several times, up to Ex-all free hydrogenchloride, residue grinds with anhydrous diethyl ether so repeatedly, gets title compound, is directly used in next step reaction.

The preparation of embodiment 7.Boc-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl

45mg (0.074mmol) HCI Pro-Ala-Lys (ClZ)-OBzl is dissolved in the anhydrous THF of 5ml, 0 ℃ drips N-methylmorpholine down and makes pH value of solution 8-9, remain on 0 ℃, in reaction solution, add 33mg (0.077mmol) Boc-Arg (Tos)-OH successively, the anhydrous THF solution of 10mg (0.074mmol) N-hydroxy benzo triazole and 16mg (0.077mmol) DCC, behind 0 ℃ of reaction 2hr, change room temperature reaction 5hr over to, TLC (chloroform/methanol, 15/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, use saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain mp68-70 ℃ of title compound 70mg (96%), FAB-MS (m/e) 984[M+H] ⁺

The preparation of embodiment 8.HClArg (Tos)-Pro-Ala-Lys (ClZ)-OBzl

128mg (0.13mmol) Boc-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is dissolved in 5ml anhydrous hydrogen chloride/ethyl acetate solution, stirring at room, and TLC behind the 5hr (chloroform/methanol, 15/1) shows that raw material point disappears.Reaction mixture is concentrating under reduced pressure at room temperature, and residue also at room temperature concentrates with acetic acid ethyl dissolution, so repeatedly for several times, and up to Ex-all free hydrogenchloride.Residue grinds with anhydrous diethyl ether, gets title compound, is directly used in next step reaction.

Preparation 320mg (1.69mmol) Boc-Ala-OH of embodiment 9.Boc-Ala-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is dissolved among the anhydrous THF of 40ml, ice bath adds 220mg (1.63mmol) N-hydroxy benzo triazole and 350mg (1.70mmol) DCC down, and is stand-by behind the mixing 30min.1490mg (1.63mmol) HCI.Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is dissolved in the anhydrous THF of 25ml, drip N-methylmorpholine and make pH value of solution 8-9, the mixture that adding obtains above, behind 0 ℃ of reaction 2hr, room temperature reaction 16hr, TLC (chloroform/methanol, 10/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, successively with saturated sodium bicarbonate aqueous solution, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain the title compound crude product, through 200-300 order silica gel in atmosphere pressure column chromatography (developping agent CHCl ₃/ CH ₃OH, 30/1) separation and purification, get mp96-98 ℃ of white blister powder 1620mg (94%), FAB-MS (m/e) 1076.7[M+Na] ⁺, 1093.4[M+K] ⁺, 954.9[M-Boc] ⁺

The preparation of embodiment 10.Boc-Gly-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl

200mg (1.14mmol) Boc-Gly-OH is dissolved among the anhydrous THF of 40ml, and ice bath adds 150mg (1.11mmol) N-hydroxy benzo triazole and 235mg (1.14mmol) DCC down, and is stand-by behind the mixing 30min.930mg (1.01mmol) HCI.Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is dissolved in the anhydrous THF of 15ml, drip N-methylmorpholine and make pH value of solution 8-9, the mixture that adding obtains above, behind 0 ℃ of reaction 2hr, room temperature reaction 12hr, TLC (chloroform/methanol, 10/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, successively with saturated sodium bicarbonate aqueous solution, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain the title compound crude product, through 200-300 order silica gel in atmosphere pressure column chromatography (developping agent CHCl ₃/ CH ₃OH, 30/1) separation and purification, get white blister powder 770mg (73%), mp82-84 ℃, FAB-MS (m/e) 1038.6[M+H] ⁺, 1061.7[M+Na] ⁺

The preparation of embodiment 11.Boc-Lys (ClZ)-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl

340mg (0.82mmol) Boc-Lys (ClZ)-OH is dissolved among the anhydrous THF of 40ml, and ice bath adds 110mg (0.82mmol) N-hydroxy benzo triazole and 170mg (0.82mmol) DCC down, and is stand-by behind the mixing 30min.746mg (0.81mmol) HCl.Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is dissolved in the anhydrous THF of 50ml, drip N-methylmorpholine and make pH value of solution 8-9, the mixture that adding obtains above, behind 0 ℃ of reaction 2hr, room temperature reaction 16hr, TLC (chloroform/methanol, 10/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, successively with saturated sodium bicarbonate aqueous solution, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain the title compound crude product, through 200-300 order silica gel in atmosphere pressure column chromatography (developping agent CHCl ₃/ CH ₃OH, 30/1) separation and purification, get white blister powder 850mg (81%), mp78-88 ℃, FAB-MS (m/e) 1111.3[M+H] ⁺, 1132.5[M+Na] ⁺, 1010.0[M-Boc] ⁺

The preparation of embodiment 12.Boc-Gln-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl

467mg (0.51mmol) HCl.Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is dissolved in the 5ml dry DMF, drip N-methylmorpholine and make pH value of solution 8-9, add Boc-Gln-ONp195mg (0.53mmol), room temperature reaction 96hr, TLC (chloroform/methanol, 9/1) show the basic disappearance of raw material point, stopped reaction, room temperature dries up solvent, and residue dissolves with chloroform, directly through 200-300 order silica gel in atmosphere pressure column chromatography (developping agent CHCl ₃/ CH ₃OH, 10/1) separation and purification, get white blister powder 450mg (80%), mp80-82 ℃, FAB-MS (m/e) 1111.3[M+H] ⁺, 1132.5[M+Na] ⁺, 1010.0[M-Boc] ⁺

The preparation of embodiment 13.Boc-Asn-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl

1160mg (1.26mmol) HCI.Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is dissolved in the 50ml dry DMF, drip N-methylmorpholine and make pH value of solution 8-9, add Boc-Asn-ONp500mg (1.41mmol), room temperature reaction 96hr, TLC (chloroform/methanol, 9/1) show the basic disappearance of raw material point, stopped reaction, room temperature dries up solvent, and residue dissolves with chloroform, directly through 200-300 order silica gel in atmosphere pressure column chromatography (developping agent CHCl ₃/ CH ₃OH, 15/1) separation and purification, get white blister powder 800mg (57%), mp86-88 ℃, FAB-MS (m/e) 1097[M+H] ⁺

The preparation of embodiment 14.Boc-Arg (Tos)-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl

1636mg (1.78mmol) HCl Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is dissolved in the anhydrous THF of 5ml, 0 ℃ drips N-methylmorpholine down and makes pH value of solution 8-9, remain on 0 ℃, in reaction solution, add 760mg (1.77mmol) Boc-Arg (Tos)-OH successively, the anhydrous THF solution of 240mg (1.78mmol) N-hydroxy benzo triazole and 370mg (1.80mmol) DCC, behind 0 ℃ of reaction 2hr, change room temperature reaction 5hr over to, TLC (chloroform/methanol, 10/1) shows the basic disappearance of raw material point, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution is used saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate.The organic layer anhydrous magnesium sulfate drying of telling filters, and filtrate is evaporated to dried at 37 ℃, obtain the title compound crude product, through 200-300 order silica gel in atmosphere pressure column chromatography (developping agent CHCl ₃/ CH ₃OH, 30/1) separation and purification, obtain title compound 1330mg (58%), mp90-92 ℃, FAB-MS (m/e) 1293[M+H] ⁺

The preparation of embodiment 15.H-Ala-Arg-Pro-Ala-Lys-OH

250mg (0.237mmol) Boc-Ala-Arg (Tos)-Pro-Ala-Lys (ClZ) OBzl is with after the 1ml methyl-phenoxide mixes, feed the 2ml anhydrous HF, 0 ℃ of reaction 2hr, HF is removed in decompression, feed the 2ml anhydrous HF once more, 0 ℃ of reaction 1hr, HF is removed in decompression, residue solidifies with anhydrous diethyl ether, the SephadexG-10 desalination of the solids behind the suction filtration, and distilled water is moving phase, triketohydrindene hydrate detects collects the aqueous solution, get title compound 100mg (77.8%), mp224-6 ℃, FAB-MS (m/e) 542[M+H after the lyophilize] ⁺

The preparation of embodiment 16.H-Gly-Arg-Pro-Ala-Lys-OH

300mg (0.289mmol) Boc-Gly-Arg (Tos)-Pro-Ala-Lys (ClZ) OBzl is with after the 1ml methyl-phenoxide mixes, feed the 2ml anhydrous HF, 0 ℃ of reaction 2hr, HF is removed in decompression, feed anhydrous HF 2ml once more, 0 ℃ of reaction 1hr, HF is removed in decompression, residue solidifies with anhydrous diethyl ether, the SephadexG-10 desalination of the solids behind the suction filtration, and distilled water is moving phase, triketohydrindene hydrate detects collects the aqueous solution, get title compound 150mg (98%), mp168-170 ℃, FAB-MS (m/e) 528[M+H after the lyophilize] ⁺

The preparation of embodiment 17.H-Lys-Arg-Pro-Ala-Lys-OH

290mg (0.227mmol) Boc-Lys (ClZ)-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is with after the 1ml methyl-phenoxide mixes, feed the 2ml anhydrous HF, 0 ℃ of reaction 2hr, HF is removed in decompression, feed anhydrous HF 2ml once more, 0 ℃ of reaction 1hr, HF is removed in decompression, residue solidifies with anhydrous diethyl ether, the SephadexG-10 desalination of the solids behind the suction filtration, and distilled water is moving phase, triketohydrindene hydrate detects collects the aqueous solution, get title compound 109mg (80%), mp168-170 ℃, FAB-MS (m/e) 599[M+H after the lyophilize] ⁺

The preparation of embodiment 18.H-Gln-Arg-Pro-Ala-Lys-OH

250mg (0.225mmol) Boc-Gln-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is with after the 1ml methyl-phenoxide mixes, feed the 2ml anhydrous HF, 0 ℃ of reaction 2hr, HF is removed in decompression, feed anhydrous HF 2ml once more, 0 ℃ of reaction 1hr, HF is removed in decompression, residue solidifies with anhydrous diethyl ether, the SephadexG-10 desalination of the solids behind the suction filtration, and distilled water is moving phase, triketohydrindene hydrate detects collects the aqueous solution, get title compound 130mg (96%), mp180-182 ℃, FAB-MS (m/e) 600[M+H after the lyophilize] ⁺

The preparation of embodiment 19.H-Asn-Arg-Pro-Ala-Lys-OH

102mg (0.1mmol) Boc-Asn-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is with after the 1ml methyl-phenoxide mixes, feed the 2ml anhydrous HF, 0 ℃ of reaction 2hr, HF is removed in decompression, feed anhydrous HF 2ml once more, 0 ℃ of reaction 1hr, HF is removed in decompression, residue solidifies with anhydrous diethyl ether, the SephadexG-10 desalination of the solids behind the suction filtration, and distilled water is moving phase, triketohydrindene hydrate detects collects the aqueous solution, get title compound 43mg (80%), mp168-170 ℃, FAB-MS (m/e) 585[M+H after the lyophilize] ⁺

The preparation of embodiment 20.H-Arg-Arg-Pro-Ala-Lys-OH

280mg (0.188mmol) Boc-Arg (Tos)-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is with after the 1ml methyl-phenoxide mixes, feed the 2ml anhydrous HF, 0 ℃ of reaction 2hr, HF is removed in decompression, feed anhydrous HF 2ml once more, 0 ℃ of reaction 1hr, HF is removed in decompression, residue solidifies with anhydrous diethyl ether, the Sephadex GIO desalination of the solids behind the suction filtration, and distilled water is moving phase, triketohydrindene hydrate detects collects the aqueous solution, get title compound 100mg (85%), mp168-170 ℃, FAB-MS (m/e) 627[M+H after the lyophilize] ⁺

The preparation of embodiment 21.H-Arg-Pro-Ala-Lys-OH

280mg (0.285mmol) Boc-Arg (Tos)-Pro-Ala-Lys (ClZ) OBzl feeds the 2ml anhydrous HF with after the 1ml methyl-phenoxide mixes, 0 ℃ of reaction 2hr, HF is removed in decompression, feeds anhydrous HF 2ml once more, 0 ℃ of reaction 1hr, HF is removed in decompression, residue solidifies with anhydrous diethyl ether, solids Sephadex G-10 desalination, and distilled water is moving phase, triketohydrindene hydrate detects collects the aqueous solution, get title compound 100mg (75%), mp158-160 ℃, FAB-MS (m/e) 472[M+H after the lyophilize] ⁺

The preparation of embodiment 22.H-Pro-Ala-Lys-OH

280mg (0.417mmol) Boc-Pro-Ala-Lys (ClZ)-OBzl is with after the 1ml methyl-phenoxide mixes, feed the 2ml anhydrous HF, 0 ℃ of reaction 0.5hr, HF is removed in decompression, and residue solidifies with anhydrous diethyl ether, the Sephadex G-10 desalination of solids behind the suction filtration, distilled water is moving phase, and triketohydrindene hydrate detects collects the aqueous solution, gets title compound 110mg (84%) after the lyophilize, mp150-152 ℃, FAB-MS (m/e) 315[M+H] ⁺

The preparation of embodiment 23.Boc-Ala-Arg (Tos)-Pro-Ala-Lys (ClZ)-OH

1000mg (0.95mmol) Boc-Ala-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is dissolved in the 20ml methyl alcohol, ice bath drips 2mol/L NaOH 10ml down, TLC (chloroform/methanol behind the 30min, 10/1) detecting raw material point disappears, add an amount of 2mol/L HCl neutralization reaction liquid to pH7, concentrating under reduced pressure boils off methyl alcohol, residue adds an amount of 2mol/LHCl makes pH1-2, in a large amount of distilled water of impouring, have a large amount of white precipitates to generate, suction filtration, filter cake are with distilled water wash for several times, put in the moisture eliminator and spend the night, get title compound 880mg (91%).

The preparation of embodiment 24.Boc-Gly-Arg (Tos)-Pro-Ala-Lys (ClZ)-OH

230mg (0.221mmol) Boc-Gly-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is dissolved in the 6ml methyl alcohol, ice bath drips 2mol/L NaOH 2.5ml down, TLC (chloroform/methanol behind the 30min, 10/1) detecting raw material point disappears, add an amount of 2mol/L HCl neutralization reaction liquid to pH7, concentrating under reduced pressure boils off methyl alcohol, residue adds an amount of 2mol/L HCl makes pH1-2, in a large amount of distilled water of impouring, have a large amount of white precipitates to generate, suction filtration, filter cake are with distilled water wash for several times, put in the moisture eliminator and spend the night, get title compound 180mg (86%).

The preparation of embodiment 25.Boc-Lys (ClZ)-Arg (Tos)-Pro-Ala-Lys (ClZ)-OH

250mg (0196mmol) Boc-Lys (ClZ)-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is dissolved in the 5ml methyl alcohol, ice bath drips 2mol/L NaOH 2.0ml down, TLC (chloroform/methanol behind the 30min, 10/1) detecting raw material point disappears, add an amount of 2mol/L HCl neutralization reaction liquid to pH7, concentrating under reduced pressure boils off methyl alcohol, residue adds an amount of 2mol/L HCl makes pH1-2, in a large amount of distilled water of impouring, have a large amount of white precipitates to generate, suction filtration, filter cake are with distilled water wash for several times, put in the moisture eliminator and spend the night, get title compound 200mg (86%).

The preparation of embodiment 26.Boc-Gln-Arg (Tos)-Pro-Ala-Lys (ClZ)-OH

770mg (0.694mmol) Boc-Gln-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is dissolved in the 18ml methyl alcohol, ice bath drips 2mol/L NaOH 7.0ml down, TLC (chloroform/methanol behind the 30min, 10/1) detecting raw material point disappears, add an amount of 2mol/L HCl neutralization reaction liquid to pH7, concentrating under reduced pressure boils off methyl alcohol, residue adds an amount of 2mol/L HCl makes pH1-2, in a large amount of distilled water of impouring, have a large amount of white precipitates to generate, suction filtration, filter cake are with distilled water wash for several times, put in the moisture eliminator and spend the night, get title compound 610mg (86%).

The preparation of embodiment 27.Boc-Asn-Arg (Tos)-Pro-Ala-Lys (ClZ)-OH

300mg (0.210mmol) Boc-Asn-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is dissolved in the 5ml methyl alcohol, ice bath drips 2mol/L NaOH 2.0ml down, TLC (chloroform/methanol behind the 30min, 10/1) detecting raw material point disappears, add an amount of 2mol/L HCl neutralization reaction liquid to pH7, concentrating under reduced pressure boils off methyl alcohol, residue adds an amount of 2mol/L HCl makes pH1-2, in a large amount of distilled water of impouring, have a large amount of white precipitates to generate, suction filtration, filter cake are with distilled water wash for several times, put in the moisture eliminator and spend the night, get title compound 230mg (83%).

The preparation of embodiment 28.Boc-Arg (Tos)-Arg (Tos)-Pro-Ala-Lys (ClZ)-OH

600mg (0.464mmol) Boc-Arg (Tos)-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is dissolved in the 10ml methyl alcohol, ice bath drips 2mol/L NaOH 4.0ml down, TLC (chloroform/methanol behind the 30min, 10/1) detecting raw material point disappears, add an amount of 2mol/L HCl neutralization reaction liquid to pH7, concentrating under reduced pressure boils off methyl alcohol, residue adds an amount of 2mol/L HCl makes pH1-2, in a large amount of distilled water of impouring, have a large amount of white precipitates to generate, suction filtration, filter cake are with distilled water wash for several times, put in the moisture eliminator and spend the night, get title compound 420mg (76%).

The preparation of embodiment 29.Boc-Arg (Tos)-Pro-Ala-Lys (ClZ)-OH

500mg (0.51mmol) Boc-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is dissolved in the 5ml methyl alcohol, ice bath drips 2mol/L NaOH 5ml down, TLC (chloroform/methanol behind the 30min, 15/1) detecting raw material point disappears, add an amount of 2mol/L HCl neutralization reaction liquid to pH7, concentrating under reduced pressure boils off methyl alcohol, residue adds an amount of 2mol/L HCl makes pH1-2, in a large amount of distilled water of impouring, has a large amount of white precipitates to generate, suction filtration, filter cake is put in the moisture eliminator and is spent the night with distilled water wash for several times, gets title compound 380mg (84%), mp108-110 ℃, TOF-MS (m/e) 893.2[M+H] ⁺, 915.5[M+Na] ⁺, 937.2[M+2Na] ⁺

The preparation of embodiment 30.Boc-Pro-Ala-Lys (ClZ)-OH

135mg (0.2mmol) Boc-Pro-Ala-Lys (ClZ)-OBzl is dissolved in the 5ml methyl alcohol, ice bath drips 2mol/L NaOH 2.0ml down, TLC (chloroform/methanol behind the 30min, 20/1) detecting raw material point disappears, add an amount of 2mol/L HCl neutralization reaction liquid to pH7, concentrating under reduced pressure boils off methyl alcohol, residue adds an amount of 2mol/L HCl makes pH1-2, in a large amount of distilled water of impouring, have a large amount of white precipitates to generate, suction filtration, filter cake are with distilled water wash for several times, put in the moisture eliminator and spend the night, get title compound 100mg (86%).

The preparation of embodiment 31.Boc-Pro-OH

(1) Boc-N ₃Method

800mg (6.96mmol) proline(Pro) is suspended in the 12ml dry DMF, adds the 1.8ml triethylamine, and ice bath stirs down, adds 1.5mlBoc-N in 30 minutes ₃, the solid dissolving, the solution clarification, stirring at room 3 days, TLC (chloroform/methanol, 30/1) shows that the raw material primitive reaction finishes, and stopped reaction dries up solvent, and residue is with the 50ml acetic acid ethyl dissolution, successively with 5%KHSO ₄, saturated sodium-chloride water solution washes, the ester layer filters with anhydrous magnesium sulfate drying, when steaming near doing, its strong solution is positioned over crystallization in the refrigerator, the 1200mg white crystals, yield 80%.

(2) (Boc) ₂The O method

800mg (6.96mmol) proline(Pro) is suspended among the anhydrous THF of 20ml, adds the 2.5ml triethylamine, adds 1625mg (7.45mmol) under the room temperature (Boc) ₂O, stirred overnight at room temperature, TLC (chloroform/methanol, 30/1) shows that the raw material primitive reaction finishes, stopped reaction, solvent evaporated, residue is with the 50ml acetic acid ethyl dissolution, successively with 5%KHSO ₄, saturated sodium-chloride water solution is washed, and the ester layer is with anhydrous magnesium sulfate drying, and filtration when steaming near doing, is positioned over crystallization in the refrigerator with its strong solution, gets the 1350mg white crystals, yield 90%.

The preparation of embodiment 32.Boc-Pro-OBzl

33mg (0.10mmol) Cs ₂CO ₃Be dissolved in distilled water, join in the ethanol solution of 43mg (0.20mmol) Boc-Pro-OH, removal of solvent under reduced pressure behind the 40min, residue is put moisture eliminator and is spent the night, get white solid, this solid is dissolved in dry DMF, drips 34mg (0.2mmol) bromobenzyl, after 50 ℃ of constant temperature stir 16hr, filtering CsBr precipitation, filtrate decompression is concentrated into dried, residue with acetic acid ethyl dissolution after, successively with saturated sodium bicarbonate aqueous solution, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate.The organic layer anhydrous sodium sulfate drying of telling filters, and filtrate is at 37 ℃ of following concentrating under reduced pressure, and residue grinds with sherwood oil, gets title compound 54mg (88%), is colorless oil.

The preparation of embodiment 33.HCl-Pro-OBzl

61mg (0.2mmol) Boc-Pro-OBzl is dissolved in 3ml 4mol/L anhydrous hydrogen chloride/ethyl acetate solution, stirring at room, and the adularescent precipitation generates behind the 20min, and TLC behind the 2hr (chloroform/methanol, 30/1) shows that raw material point disappears.Reaction mixture is concentrating under reduced pressure at room temperature, the residue acetic acid ethyl dissolution, and at room temperature concentrate, so repeatedly for several times, up to Ex-all free hydrogenchloride.Residue grinds with anhydrous diethyl ether, takes out ether, white solid, this solid soaks with sherwood oil (60-90 ℃), the sherwood oil that inclines, drain after for several times so repeatedly title compound, be directly used in next step reaction.

The preparation of embodiment 34.Boc-Arg (Tos)-Pro-OBzl

18mg (0.075mmol) HCI Pro-OBzl is dissolved in the anhydrous THF of 5ml, 0 ℃ drips N-methylmorpholine down and makes pH value of solution 8-9, remain on 0 ℃, in reaction solution, add 33mg (0.077mmol) Boc-Arg (Tos)-OH successively, the anhydrous THF solution of 10mg (0.074mmol) N-hydroxy benzo triazole and 16mg (0.077mmol) DCC, behind 0 ℃ of reaction 2hr, change room temperature reaction 5hr over to, TLC (chloroform/methanol, 30/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, use saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain title compound 37mg (80%), and mp60-62 ℃, FAB-MS (m/e) 617[M+H] ⁺

The preparation of embodiment 35.HClArg (Tos)-Pro-OBzl

123mg (0.2mmol) Boc-Arg (Tos)-Pro-OBzl is dissolved in 3ml 4mol/L anhydrous hydrogen chloride/ethyl acetate solution, stirring at room, and the adularescent precipitation generates behind the 20min, and TLC behind the 2hr (chloroform/methanol, 20/1) shows that raw material point disappears.Reaction mixture is concentrating under reduced pressure at room temperature, the residue acetic acid ethyl dissolution, and at room temperature concentrate, so repeatedly for several times, up to Ex-all free hydrogenchloride.Residue grinds with anhydrous diethyl ether, gets title compound, is directly used in next step reaction.

The preparation of embodiment 36.Boc-Ala-Arg (Tos)-Pro-OBzl

280mg (1.3mmol) Boc-Ala-OH is dissolved among the anhydrous THF of 30ml, and ice bath adds 170mg (1.26mmol) N-hydroxy benzo triazole and 270mg (1.31mmol) DCC down, and is stand-by behind the mixing 30min.793mg (1.44mmol) HCI.Arg (Tos)-Pro-OBzl is dissolved in the anhydrous THF of 5ml, drip N-methylmorpholine and make pH value of solution 8-9, the mixture that adding obtains above, behind 0 ℃ of reaction 2hr, room temperature reaction 6hr, TLC (chloroform/methanol, 20/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, successively with saturated sodium bicarbonate aqueous solution, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain the title compound crude product, through 200-300 order silica gel in atmosphere pressure column chromatography (developping agent CHCl ₃/ CH ₃OH, 30/1) separation and purification, obtain title compound 550mg (55%), mp86-88 ℃, FAB-MS (m/e) 685[M+H] ⁺, 724[M+K] ⁺

The preparation of embodiment 37.HClAla-Arg (Tos)-Pro-OBzl

137mg (0.2mmol) Boc-Ala-Arg (Tos)-Pro-OBzl is dissolved in 3ml 4mol/L anhydrous hydrogen chloride/ethyl acetate solution, stirring at room, and the adularescent precipitation generates behind the 20min, and TLC behind the 2hr (chloroform/methanol, 20/1) shows that raw material point disappears.Reaction mixture is concentrating under reduced pressure at room temperature, the residue acetic acid ethyl dissolution, and at room temperature concentrate, so repeatedly for several times, up to Ex-all free hydrogenchloride.Residue grinds with anhydrous diethyl ether, gets title compound, is directly used in next step reaction.

The preparation of embodiment 38.Boc-Ala-Arg (Tos)-Pro-OH

550mg (0.80mmol) Boc-Ala-Arg (Tos)-Pro-OBzl is dissolved in the 10ml methyl alcohol, ice bath drips 2mol/LNaOH 5ml down, TLC (chloroform/methanol behind the 30min, 20/1) detecting raw material point disappears, add an amount of 2mol/L HCl neutralization reaction liquid to pH7, concentrating under reduced pressure boils off methyl alcohol, residue adds an amount of 2mol/LHCl makes pH1-2, in a large amount of distilled water of impouring, have a large amount of white precipitates to generate, suction filtration, filter cake are with distilled water wash for several times, put in the moisture eliminator and spend the night, get title compound 130mg (27%).

The preparation of embodiment 39.H-Ala-Arg-Pro-OH

105mg (0.153mmol) Boc-Ala-Arg (Tos)-Pro-OBzl feeds the 2ml anhydrous HF with after the 1ml methyl-phenoxide mixes, 0 ℃ of reaction 2hr, HF is removed in decompression, feeds anhydrous HF 2ml once more, 0 ℃ of reaction 1hr, HF is removed in decompression, residue solidifies with anhydrous diethyl ether, the Sephadex G-10 desalination of the solids behind the suction filtration, and distilled water is moving phase, triketohydrindene hydrate detects collects the aqueous solution, get title compound 10mg (19%), mp150-2 ℃, FAB-MS (m/e) 344[M+H after the lyophilize] ⁺

The preparation of embodiment 40.HClAla-OBzl

1070mg (0.012mol) L-Ala joins 25ml benzylalcohol and the 5g Tripyrophosphoric acid gets in the homogeneous solution, reaction solution is poured into reaction solution in the 200ml water that contains the 10ml concentrated hydrochloric acid then 90-95 ℃ of reaction 4 hours, adds ether, divide water-yielding stratum, the ether layer merges water with 2% salt pickling 3 times, adds solid yellow soda ash and makes PH be about 10, add the jolting of 100ml ether, tell the ether layer, behind anhydrous sodium sulfate drying, filter, in filtrate, feed anhydrous hydrogen chloride gas, have a large amount of white column precipitations to generate, filter, precipitation is washed with anhydrous diethyl ether, dry, get 1500mg (58%), mp140-142 ℃, 140 ℃ in document.

The preparation of embodiment 41.Boc-Pro-Ala-OBzl

500mg (2.326mmol) HCI Ala-OBzl is dissolved in the anhydrous THF of 50ml, 0 ℃ drips N-methylmorpholine down and makes pH value of solution 8-9, remain on 0 ℃, in reaction solution, add 500mg (2.326mmol) Boc-Pro-OH successively, the anhydrous THF solution of 300mg (2.222mmol) N-hydroxy benzo triazole and 480mg (2.331mmol) DCC, behind 0 ℃ of reaction 2hr, change room temperature reaction 5hr over to, TLC (chloroform/methanol, 30/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, use saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain mp100-101 ℃ of title compound 782mg (89%), FAB-MS (m/e) 377[M+H] ⁺

The preparation of embodiment 42.HClPro-Ala-OBzl

75mg (0.2mmol) Boc-Pro-Ala-OBzl is dissolved in 3ml 4mol/L anhydrous hydrogen chloride/ethyl acetate solution, stirring at room, and the adularescent precipitation generates behind the 20min, and TLC behind the 2hr (chloroform/methanol, 30/1) shows that raw material point disappears.Reaction mixture is concentrating under reduced pressure at room temperature, the residue acetic acid ethyl dissolution, and at room temperature concentrate, so repeatedly for several times, up to Ex-all free hydrogenchloride.Residue grinds with anhydrous diethyl ether, gets title compound, is directly used in next step reaction.

The preparation of embodiment 43.Boc-Arg (Tos)-Pro-Ala-OBzl

72mg (0.231mmol) HCl.Pro-Ala-OBzl is dissolved in the anhydrous THF of 50ml, 0 ℃ drips N-methylmorpholine down and makes pH value of solution 8-9, remain on 0 ℃, in reaction solution, add 100mg (0.234mmol) Boc-Arg (Tos)-OH, the anhydrous THF solution of 31mg (0.230mmol) N-hydroxy benzo triazole and 48mg (0.233mmol) DCC, behind 0 ℃ of reaction 2hr, change room temperature reaction 5hr over to, TLC (chloroform/methanol, 20/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, use saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain mp70-71.5 ℃ of title compound 120mg (75.6%), FAB-MS (m/e) 688[M+H] ⁺

The preparation of embodiment 44.HClArg (Tos)-Pro-Ala-OBzl

137mg (0.2mmol) Boc-Arg (Tos)-Pro-Ala-OBzl is dissolved in 3ml 4mol/L anhydrous hydrogen chloride/ethyl acetate solution, stirring at room, and the adularescent precipitation generates behind the 20min, and TLC behind the 2hr (chloroform/methanol, 20/1) shows that raw material point disappears.Reaction mixture is concentrating under reduced pressure at room temperature, the residue acetic acid ethyl dissolution, and at room temperature concentrate, so repeatedly for several times, up to Ex-all free hydrogenchloride.Residue grinds with anhydrous diethyl ether, gets title compound, is directly used in next step reaction.

The preparation of embodiment 45.Boc-Ala-Arg (Tos)-Pro-Ala-OBzl

45mg (0.072mmol) HClArg (Tos)-Pro-Ala-OBzl is dissolved in the anhydrous THF of 50ml, 0 ℃ drips N-methylmorpholine down and makes pH value of solution 8-9, remain on 0 ℃, in reaction solution, add 15mg (0.079mmol) Boc-Ala-OH successively, the anhydrous THF solution of 10mg (0.074mmol) N-hydroxy benzo triazole and 16mg (0.077mmol) DCC, behind 0 ℃ of reaction 2hr, change room temperature reaction 5hr over to, TLC (chloroform/methanol, 15/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, use saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain the title compound crude product, through 200-300 order silica gel in atmosphere pressure column chromatography (developping agent CHCl ₃/ CH ₃OH, 30/1) separation and purification, obtain mp80-82 ℃ of title compound 30mg (54%), FAB-MS (m/e) 758[M+H] ⁺

The preparation of embodiment 46.HClAla-Arg (Tos)-Pro-Ala-OBzl

151mg (0.2mmol) Boc-Ala-Arg (Tos)-Pro-Ala-OBzl is dissolved in 3ml 4mol/L anhydrous hydrogen chloride/ethyl acetate solution, stirring at room, and the adularescent precipitation generates behind the 20min, and TLC behind the 2hr (chloroform/methanol, 15/1) shows that raw material point disappears.Reaction mixture is concentrating under reduced pressure at room temperature, the residue acetic acid ethyl dissolution, and at room temperature concentrate, so repeatedly for several times, up to Ex-all free hydrogenchloride.Residue grinds with anhydrous diethyl ether, gets title compound, is directly used in next step reaction.

The preparation of embodiment 47.Boc-Ala-Arg (Tos)-Pro-Ala-OH

550mg (0.726mmol) Boc-Ala-Arg (Tos)-Pro-Ala-OBzl is dissolved in the 20ml methyl alcohol, ice bath drips 2mol/LNaOH 8ml down, TLC (chloroform/methanol behind the 30min, 15/1) detecting raw material point disappears, add an amount of 2mol/L HCl neutralization reaction liquid to pH7, concentrating under reduced pressure boils off methyl alcohol, residue adds an amount of 2mol/L HCl makes pH1-2, in a large amount of distilled water of impouring, have a large amount of white precipitates to generate, suction filtration, filter cake are with distilled water wash for several times, put in the moisture eliminator and spend the night, get title compound 330mg (67%).

The preparation of embodiment 48.H-Ala-Arg-Pro-Ala-OH

250mg (0.330mmol) Boc-Ala-Arg (Tos)-Pro-Ala-OBzl feeds the 2ml anhydrous HF with after the 1ml methyl-phenoxide mixes, 0 ℃ of reaction 2hr, HF is removed in decompression, feeds anhydrous HF 2ml once more, 0 ℃ of reaction 1hr, HF is removed in decompression, residue solidifies with anhydrous diethyl ether, the Sephadex G-10 desalination of the solids behind the suction filtration, and distilled water is moving phase, triketohydrindene hydrate detects collects the aqueous solution, get title compound 80mg (58%), mp157-159 ℃, FAB-MS (m/e) 415[M+H after the lyophilize] ⁺

Embodiment 49.2,3,4,9-Tetrahydro-1H-Pyrido[3,4-b]-preparation of Indole-3-Carboxylic Acid

1g (5mmol) tryptophane joins and contains the dense H of 0.5ml ₂SO ₄80ml water in, stir after 10 minutes, add 8ml formaldehyde (36-38%), promptly separate out a large amount of solids again after the reaction solution clarification, behind the stirring at room 2hr, transfer solution pH value 6-7 with strong aqua, refrigerator is placed and is spent the night, suction filtration gets yellow solid, it is dissolved in 250ml 50% gets in the ethanol, be heated to 50 ℃, drip strong aqua and make the solution clarification, add the 0.4g activated carbon decolorizing, filter, filtrate concentrating separated out a large amount of solids, and suction filtration gets off-white powder 6.0g, after concentrating, mother liquor gets 3.0g light yellow solid, total recovery 85%.

Embodiment 50.2-N-tert-Butyloxycarbonyl-3,4,9-Trihydro-1H-Pyri-do[3,4-b]-preparation of Indole-3-Carboxylic Acid

0.9g (4.17mmol) compound (49) is suspended in and contains 1.25g (Boc) ₂In the 15ml anhydrous DMF solution of O and 1.5ml triethylamine, 45 ℃ of following reaction 4hr, compound 49 dissolve gradually in the reaction process, TLC (chloroform/methanol, 20/1) shows to react and finishes, solvent evaporated, residue with acetic acid ethyl dissolution after, use 5%KHSO successively ₄, saturated NaCl washes, and the organic layer anhydrous sodium sulfate drying filters, evaporate to dryness, the chloroform recrystallization gets the 1g title compound, yield 76%, TLC (chloroform/methanol, 20/1) is consistent with standard substance, and mp168-171 ℃, document 165-170 ℃.

Embodiment 51.2,3,4,9-Tetrahydro-1H-Pyrido[3,4-b]-preparation of Indole-3-Carboxylic AcidBenzyl Ester

280mg (0.86mmol) Cs ₂CO ₃Be dissolved in distilled water, join in the ethanol solution of 540mg (1.71mmol) compound (50), removal of solvent under reduced pressure behind the 40min, residue is put moisture eliminator and is spent the night, get white solid, this solid is dissolved in dry DMF, drips 0.2ml (1.75mmol) bromobenzyl, after 50 ℃ of constant temperature stir 16hr, filtering CsBr precipitation, filtrate decompression is concentrated into dried, residue with acetic acid ethyl dissolution after, use saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous sodium sulfate drying of telling, filter, filtrate stops to concentrate when being evaporated to the 10ml volume under 37 ℃, adds 20ml 4mol/L anhydrous hydrogen chloride one ethyl acetate solution in reaction solution, stirring at room, have the brown precipitation to generate behind the 30min, TLC (chloroform/methanol, 20/1) shows that raw material point disappears.Reaction mixture at room temperature is evaporated to dried, the residue acetic acid ethyl dissolution, be washed till alkalescence with saturated sodium bicarbonate aqueous solution, wash with saturated sodium-chloride water solution again, tell organic layer and use anhydrous sodium sulfate drying, filter, evaporate to dryness gets near-white solid 400mg (76%), TLC (chloroform/methanol, 20/1) is consistent with standard substance.

The preparation of embodiment 52.Boc-Pro-Ala-Lys (ClZ)-KL-OBzl

320mg (1.046mmol) compound (51) is dissolved in the anhydrous THF of 25ml, in reaction solution, add 600mg (1.031mmol) compound 30 successively under 0 ℃, the anhydrous THF solution of 140mg (1.037mmol) N-hydroxy benzo triazole and 220mg (1.068mmol) DCC, behind 0 ℃ of reaction 2hr, change room temperature reaction 5hr over to, TLC (chloroform/methanol, 15/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, use saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain title compound 890mg (98%), and mp68-70 ℃, FAB-MS (m/e) 872[M+H] ⁺

The preparation of embodiment 53.Boc-KL-Pro-Ala-Lys (ClZ)-OBzl

390mg (0.670mmol) HCI Pro-Ala-Lys (ClZ)-OBzl is dissolved in the anhydrous THF of 15ml, 0 ℃ drips N-methylmorpholine down and makes pH value of solution 8-9, remain on 0 ℃, in reaction solution, add 210mg (0.686mmol) Boc-KL-OH successively, the anhydrous THF solution of 100mg (0.149mmol) N-hydroxy benzo triazole and 150mg (0.728mmol) DCC, behind 0 ℃ of reaction 2hr, change room temperature reaction 5hr over to, TLC (chloroform/methanol, 15/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, use saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain mp76-78 ℃ of title compound 520mg (89%), FAB-MS (m/e) 870[M+H] ⁺

The preparation of embodiment 54.Boc-Ala-Arg (Tos)-Pro-KL-OBzl

320mg (1.046mmol) compound (51) is dissolved in the anhydrous THF of 25ml, in reaction solution, add 130mg (0.218mmol) compound 38 successively under 0 ℃, the anhydrous THF solution of 30mg (0.222mmol) N-hydroxy benzo triazole and 45mg (0.218mmol) DCC, behind 0 ℃ of reaction 2hr, change room temperature reaction 5hr over to, TLC (chloroform/methanol, 15/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, use saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain mp70-72 ℃ of title compound 140mg (73%), FAB-MS (m/e) 886[M+H] ⁺

The preparation of embodiment 55.Boc-KL-Ala-Arg (Tos)-Pro-OBzl

208mg (0.335mmol) HCI Ala-Arg (Tos)-Pro-OBzl is dissolved in the anhydrous THF of 15ml, 0 ℃ drips N-methylmorpholine down and makes pH value of solution 8-9, remain on 0 ℃, in reaction solution, add 110mg (0.348mmol) Boc-KL-OH successively, the anhydrous THF solution of 45mg (0.333mmol) N-hydroxy benzo triazole and 72mg (0.350mmol) DCC, behind 0 ℃ of reaction 2hr, change room temperature reaction 5hr over to, TLC (chloroform/methanol, 15/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, use saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain mp76-78 ℃ of title compound 150mg (51%), FAB-MS (m/e) 883[M+H] ⁺

The preparation of embodiment 56.Boc-Arg (Tos)-Pro-Ala-Lys (ClZ)-KL-OBzl

100mg (.326mmol) compound (51) is dissolved in the anhydrous THF of 25ml, in reaction solution, add 290mg (0.338mmol) compound 29 successively under 0 ℃, the anhydrous THF solution of 40mg (0.296mmol) N-hydroxy benzo triazole and 70mg (.340mmol) DCC, behind 0 ℃ of reaction 2hr, change room temperature reaction 5hr over to, TLC (chloroform/methanol, 10/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, use saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain mp114-6 ℃ of title compound 340mg (89%), FAB-MS (m/e) 884[M+H] ⁺

The preparation of embodiment 57.Boc-KL-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl

450mg (0.490mmol) HCI Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is dissolved in the anhydrous THF of 15ml, 0 ℃ drips N-methylmorpholine down and makes pH value of solution 8-9, remain on 0 ℃, in reaction solution, add 180mg (0.570mmol) Boc-KL-OH successively, the anhydrous THF solution of 70mg (0.518mmol) N-hydroxy benzo triazole and 120mg (0.582mmol) DCC, behind 0 ℃ of reaction 2hr, change room temperature reaction 5hr over to, TLC (chloroform/methanol, 10/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, use saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain title compound 500mg (83%), and is white solid, and mp94-96 ℃, FAB-MS (m/e) 1182[M+H] ⁺, 1214[M+CH ₃OH] ⁺

The preparation of embodiment 58.Boc-Ala-Arg (Tos)-Pro-Ala-KL-OBzl

160mg (0.522mmol) compound (51) is dissolved in the anhydrous THF of 25ml, in reaction solution, add 330mg (0.495mmol) compound 47 successively under 0 ℃, the anhydrous THF solution of 70mg (0.518mmol) N-hydroxy benzo triazole and 110mg (534mmol) DCC, behind 0 ℃ of reaction 2hr, change room temperature reaction 5hr over to, TLC (chloroform/methanol, 10/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, use saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain mp104-6 ℃ of title compound 200mg (42%), FAB-MS (m/e) 1183[M+H] ⁺

The preparation of embodiment 59.Boc-KL-Ala-Arg (Tos)-Pro-Ala-OBzl

250mg (0.361mmol) HCI Ala-Arg (Tos)-Pro-Ala-OBzl is dissolved in the anhydrous THF of 15ml, 0 ℃ drips N-methylmorpholine down and makes pH value of solution 8-9, remain on 0 ℃, in reaction solution, add 120mg (0.392mmol) Boc-KL-OH successively, the anhydrous THF solution of 50mg (0.370mmol) N-hydroxy benzo triazole and 80mg (0.388mmol) DCC, behind 0 ℃ of reaction 2hr, change room temperature reaction 5hr over to, TLC (chloroform/methanol, 10/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, use saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain mp98-100 ℃ of title compound 160mg (45%), FAB-MS (m/e) 957[M+H] ⁺

The preparation of embodiment 60.HClAla-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl

100mg (0.080mmol) Boc-Ala-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is dissolved in 5ml anhydrous hydrogen chloride/ethyl acetate solution, stirring at room, and TLC behind the 5hr (chloroform/methanol, 10/1) shows that raw material point disappears.Reaction mixture is concentrating under reduced pressure at room temperature, and residue also at room temperature concentrates with acetic acid ethyl dissolution, so repeatedly for several times, and up to Ex-all free hydrogenchloride.Residue grinds with anhydrous diethyl ether, gets title compound, is directly used in next step reaction.

The preparation of embodiment 61.HClGly-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl

100mg (0.081mmol) Boc-Gly-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is dissolved in 5ml anhydrous hydrogen chloride/ethyl acetate solution, stirring at room, and TLC behind the 5hr (chloroform/methanol, 10/1) shows that raw material point disappears.Reaction mixture is concentrating under reduced pressure at room temperature, and residue also at room temperature concentrates with acetic acid ethyl dissolution, so repeatedly for several times, and up to Ex-all free hydrogenchloride.Residue grinds with anhydrous diethyl ether, gets title compound, is directly used in next step reaction.

The preparation of embodiment 62.HClLys (ClZ)-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl

100mg (0.068mmol) Boc-Lys (ClZ)-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is dissolved in 5ml anhydrous hydrogen chloride/ethyl acetate solution, stirring at room, and TLC behind the 5hr (chloroform/methanol, 10/1) shows that raw material point disappears.Reaction mixture is concentrating under reduced pressure at room temperature, and residue also at room temperature concentrates with acetic acid ethyl dissolution, so repeatedly for several times, and up to Ex-all free hydrogenchloride.Residue grinds with anhydrous diethyl ether, gets title compound, is directly used in next step reaction.

The preparation of embodiment 63.HClGln-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl

100mg (0.076mmol) Boc-Gln-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is dissolved in 5ml anhydrous hydrogen chloride/ethyl acetate solution, stirring at room, and TLC behind the 5hr (chloroform/methanol, 10/1) shows that raw material point disappears.Reaction mixture is concentrating under reduced pressure at room temperature, and residue also at room temperature concentrates with acetic acid ethyl dissolution, so repeatedly for several times, and up to Ex-all free hydrogenchloride.Residue grinds with anhydrous diethyl ether, gets title compound, is directly used in next step reaction.

The preparation of embodiment 64.HClAsn-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl

100mg (0.077mmol) Boc-Asn-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is dissolved in 5ml anhydrous hydrogen chloride/ethyl acetate solution, stirring at room, and TLC behind the 5hr (chloroform/methanol, 10/1) shows that raw material point disappears.Reaction mixture is concentrating under reduced pressure at room temperature, and residue also at room temperature concentrates with acetic acid ethyl dissolution, so repeatedly for several times, and up to Ex-all free hydrogenchloride.Residue grinds with anhydrous diethyl ether, gets title compound, is directly used in next step reaction.

The preparation of embodiment 65.HClArg (Tos)-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl

100mg (0.067mmol) Boc-Arg (Tos)-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is dissolved in 5ml anhydrous hydrogen chloride/ethyl acetate solution, stirring at room, and TLC behind the 5hr (chloroform/methanol, 10/1) shows that raw material point disappears.Reaction mixture is concentrating under reduced pressure at room temperature, and residue also at room temperature concentrates with acetic acid ethyl dissolution, so repeatedly for several times, and up to Ex-all free hydrogenchloride.Residue grinds with anhydrous diethyl ether, gets title compound, is directly used in next step reaction.

The preparation of embodiment 66.Boc-Ala-Arg (Tos)-Pro-Ala-Lys (ClZ)-KL-OBzl

280mg (0.915mmol) compound (51) is dissolved in the anhydrous THF of 25ml, in reaction solution, add 860mg (0.893mmol) compound 23 successively under 0 ℃, the anhydrous THF solution of 120mg (0.889mmol) N-hydroxy benzo triazole and 190mg (0.922mmol) DCC, behind 0 ℃ of reaction 2hr, change room temperature reaction 5hr over to, TLC (chloroform/methanol, 10/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, use saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain mp109-110 ℃ of title compound 850mg (76%), FAB-MS (m/e) 1253.9[M+H] ⁺, 1277.3[M+Na] ⁺

The preparation of embodiment 67.Boc-KL-Ala-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl

280mg (0.283mmol) HCI Ala-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is dissolved in the anhydrous THF of 15ml, 0 ℃ drips N-methylmorpholine down and makes pH value of solution 8-9, remain on 0 ℃, in reaction solution, add 100mg (0.316mmol) Boc-KL-OH successively, the anhydrous THF solution of 43mg (0.318mmol) N-hydroxy benzo triazole and 65mg (0.315mmol) DCC, behind 0 ℃ of reaction 2hr, change room temperature reaction 5hr over to, TLC (chloroform/methanol, 10/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, use saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain mp129-31 ℃ of title compound 300mg (84.7%), FAB-MS (m/e) 1252.1[M+H] ⁺, 1274.0[M+Na] ⁺, 1152.7[M-Boc] ⁺

The preparation of embodiment 68.Boc-Gly-Arg (Tos)-Pro-Ala-Lys (ClZ)-KL-OBzl

60mg (0.196mmol) compound (51) is dissolved in the anhydrous THF of 25ml, in reaction solution, add 180mg (0.190mmol) compound 24 successively under 0 ℃, the anhydrous THF solution of 25mg (0.185mmol) N-hydroxy benzo triazole and 40mg (0.194mmol) DCC, behind 0 ℃ of reaction 2hr, change room temperature reaction 5hr over to, TLC (chloroform/methanol, 10/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, use saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain mp118-9 ℃ of title compound 220mg (93%), FAB-MS (m/e) 1239.4[M+H] ⁺, 1275.7[M+K] ⁺

The preparation of embodiment 69.Boc-KL-Gly-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl

300mg (0.307mmol) HCI Gly-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is dissolved in the anhydrous THF of 15ml, 0 ℃ drips N-methylmorpholine down and makes pH value of solution 8-9, remain on 0 ℃, in reaction solution, add 115mg (0.364mmol) Boc-KL-OH successively, the anhydrous THF solution of 50mg (0.370mmol) N-hydroxy benzo triazole and 75mg (0.364mmol) DCC, behind 0 ℃ of reaction 2hr, change room temperature reaction 5hr over to, TLC (chloroform/methanol, 10/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, use saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain mp114-6 ℃ of title compound 270mg (72%), FAB-MS (m/e) 1239.1[M+H] ⁺, 1261.4[M+Na] ⁺, 1137.9[M-Boc] ⁺, 1373.9[M+Cs] ⁺

The preparation of embodiment 70.Boc-Lys (ClZ)-Arg (Tos)-Pro-Ala-Lys (ClZ)-KL-OBzl

53mg (0.173mmol) compound (51) is dissolved in the anhydrous THF of 25ml, in reaction solution, add 200mg (0.1683mmol) compound 25 successively under 0 ℃, the anhydrous THF solution of 23mg (0.170mmol) N-hydroxy benzo triazole and 36mg (0.175mmol) DCC, behind 0 ℃ of reaction 2hr, change room temperature reaction 5hr over to, TLC (chloroform/methanol, 10/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, use saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain mp94-6 ℃ of title compound 200mg (80%), FAB-MS (m/e) [M+H] ⁺1477.

The preparation of embodiment 71.Boc-KL-Lys (ClZ)-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl

330mg (0.271mmol) HCI Lys (ClZ)-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is dissolved in the anhydrous THF of 15ml, 0 ℃ drips N-methylmorpholine down and makes pH value of solution 8-9, remain on 0 ℃, in reaction solution, add 90mg (0.285mmol) Boc-KL-OH successively, the anhydrous THF solution of 40mg (0.296mmol) N-hydroxy benzo triazole and 60mg (0.291mmol) DCC, behind 0 ℃ of reaction 2hr, change room temperature reaction 5hr over to, TLC (chloroform/methanol, 10/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, use saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain mp86-88 ℃ of title compound 320mg (79%), FAB-MS (m/e) 1477[M+H] ⁺

The preparation of embodiment 72.Boc-Gln-Arg (Tos)-Pro-Ala-Lys (ClZ)-KL-OBzl

180mg (0.570mmol) compound (51) is dissolved in the anhydrous THF of 25ml, in reaction solution, add 540mg (0.529mmol) compound 26 successively under 0 ℃, the anhydrous THF solution of 70mg (0.52mmol) N-hydroxy benzo triazole and 120mg (0.582mmol) DCC, behind 0 ℃ of reaction 2hr, change room temperature reaction 5hr over to, TLC (chloroform/methanol, 10/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, use saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain mp118-120 ℃ of title compound 550mg (79%), FAB-MS (m/e) 1311.4[M+H] u, 1333.4[M+Na] ⁺, 1209.1[M-Boc] ⁺

The preparation of embodiment 73.Boc-KL-Gln-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl

440mg (0.421mmol) HCI Gln-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is dissolved in the 35ml dry DMF, 0 ℃ drips N-methylmorpholine down and makes pH value of solution 8-9, remain on 0 ℃, in reaction solution, add 140mg (0.443mmol) Boc-KL-OH successively, the anhydrous THF solution of 60mg (0.444mmol) N-hydroxy benzo triazole and 90mg (0.437mmol) DCC, behind 0 ℃ of reaction 2hr, change room temperature reaction 5hr over to, TLC (chloroform/methanol, 10/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, use saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain mp126-8 ℃ of title compound 250mg (45%), FAB-MS (m/e) 1309.3[M+H] ⁺, 1209.3[M-Boc] ⁺

The preparation of embodiment 74.Boc-Asn-Arg (Tos)-Pro-Ala-Lys (ClZ)-KL-OBzl

13mg (42.48umol) compound (51) is dissolved in the anhydrous THF of 10ml, in reaction solution, add 40mg (39.76umol) compound 27 successively under 0 ℃, the anhydrous DMF solution of 6mg (44.44mol) N-hydroxy benzo triazole and 9mg (43.69umol) DCC, behind 0 ℃ of reaction 2hr, change room temperature reaction 5hr over to, TLC (chloroform/methanol, 10/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, use saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain mp115-7 ℃ of title compound 40mg (77%), FAB-MS (m/e) 1553[M+8CH ₃OH+H] ⁺

The preparation of embodiment 75.Boc-KL-Asn-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl

94mg (0.091mmol) HCI Asn-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is dissolved in the 15ml dry DMF, 0 ℃ drips N-methylmorpholine down and makes pH value of solution 8-9, remain on 0 ℃, in reaction solution, add 30mg (0.095mmol) Boc-KL-OH successively, the anhydrous THF solution of 20mg (0.148mmol) N-hydroxy benzo triazole and 25mg (0.121mmol) DCC, behind 0 ℃ of reaction 2hr, change room temperature reaction 5hr over to, TLC (chloroform/methanol, 10/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, use saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain mp123-126 ℃ of title compound 50mg (42%), FAB-MS (m/e) 1351[M+C4H9] ⁺

The preparation of embodiment 76.Boc-Arg (Tos)-Arg (Tos)-Pro-Ala-Lys (ClZ)-KL-OBzl

120mg (0.392mmol) compound (51) is dissolved in the anhydrous THF of 35ml, in reaction solution, add 420mg (0.349mmol) compound 28 successively under 0 ℃, the anhydrous THF solution of 50mg (0.370mmol) N-hydroxy benzo triazole and 80mg (0.388mmol) DCC, behind 0 ℃ of reaction 2hr, change room temperature reaction 5hr over to, TLC (chloroform/methanol, 10/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, use saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain mp96-99 ℃ of title compound 230mg (44%), FAB-MS (m/e) 1491[M+H] ⁺

The preparation of embodiment 77.Boc-KL-Arg (Tos)-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl

665mg (0.541mmol) HCI Arg (Tos)-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is dissolved in the anhydrous THF of 15ml, 0 ℃ drips N-methylmorpholine down and makes pH value of solution 8-9, remain on 0 ℃, in reaction solution, add 180mg (0.570mmol) Boc-KL-OH successively, the anhydrous THF solution of 80mg (0.592mmol) N-hydroxy benzo triazole and 120mg (0.582mmol) DCC, behind 0 ℃ of reaction 2hr, change room temperature reaction 5hr over to, TLC (chloroform/methanol, 10/1) shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, use saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, obtain title compound 350mg (43%), and mp120-122 ℃, FAB-MS (m/e) 1492[M+H] ⁺

The preparation of embodiment 78.H-Pro-Ala-Lys-KL-OH

500mg (0.330mmol) Boc-Pro-Ala-Lys (ClZ)-KL-OBzl is with after the 1ml methyl-phenoxide mixes, feed the 2ml anhydrous HF, 0 ℃ of reaction 0.5hr, HF is removed in decompression, and residue solidifies with anhydrous diethyl ether, the Sephadex G-10 desalination of solids behind the suction filtration, distilled water is moving phase, and triketohydrindene hydrate detects collects the title compound aqueous solution, gets title compound 250mg (85%) after the lyophilize, mp 130-134 ℃, FAB-MS (m/e) 669[M+H] ⁺

The preparation of embodiment 79.H-KL-Pro-Ala-Lys

200mg (0.230mmol) Boc-KL-Pro-Ala-Lys (ClZ)-OBzl is with after the 1ml methyl-phenoxide mixes, feed the 3ml anhydrous HF, 0 ℃ of reaction 0.5hr, HF is removed in decompression, and residue solidifies with anhydrous diethyl ether, the Sephadex G-10 desalination of solids behind the suction filtration, distilled water is moving phase, and triketohydrindene hydrate detects collects the aqueous solution, gets title compound 70mg (67%) after the lyophilize, mp 186-188 ℃, FAB-MS (m/e) 513[M+H] ⁺

The preparation of embodiment 80.H-Ala-Arg-Pro-KL-OH

120mg (0.230mmol) Boc-Ala-Arg (Tos)-Pro-KL-OBzl is with after the 1ml methyl-phenoxide mixes, feed the 2ml anhydrous HF, 0 ℃ of reaction 0.5hr, HF is removed in decompression, and residue solidifies with anhydrous diethyl ether, the Sephadex G-10 desalination of solids behind the suction filtration, distilled water is moving phase, and triketohydrindene hydrate detects collects the title compound aqueous solution, gets title compound 250mg (85%) after the lyophilize, mp 130-134 ℃, FAB-MS (m/e) 669[M+H] ⁺

The preparation of embodiment 81.H-KL-Ala-Arg-Pro

120mg (0.230mmol) Boc-KL-Ala-Arg (Tos)-Pro-Obzl is with after the 1ml methyl-phenoxide mixes, feed the 3ml anhydrous HF, 0 ℃ of reaction 0.5hr, HF is removed in decompression, and residue solidifies with anhydrous diethyl ether, the Sephadex G-10 desalination of solids behind the suction filtration, distilled water is moving phase, and triketohydrindene hydrate detects collects the aqueous solution, gets title compound 40mg (55%) after the lyophilize, mp 186-188 ℃, FAB-MS (m/e) 541[M+H] ⁺

The preparation of embodiment 82.H-Arg-Pro-Ala-Lys-KL-OH

220mg (0.186mmol) Boc-Arg (Tos)-Pro-Ala-Lys (ClZ)-KL-OBzl is with after the 1ml methyl-phenoxide mixes, feed the 3ml anhydrous HF, 0 ℃ of reaction 2hr, HF is removed in decompression, feed anhydrous HF 2ml once more, 0 ℃ of reaction 1hr, HF is removed in decompression, residue solidifies with anhydrous diethyl ether, the SephadexG-10 desalination of the solids behind the suction filtration, and distilled water is moving phase, triketohydrindene hydrate detects collects the aqueous solution, get title compound 60mg (48%), mp114-116 ℃, FAB-MS (m/e) 667[M+H after the lyophilize] ⁺

The preparation of embodiment 83.H-KL-Arg-Pro-Ala-Lys-OH

440mg (0.373mmol) Boc-KL-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl feeds the 1ml anhydrous HF with after the 1.5ml methyl-phenoxide mixes, 0 ℃ of reaction 2hr, HF is removed in decompression, feed anhydrous HF 3ml once more, 0 ℃ of reaction 1hr, HF is removed in decompression, residue solidifies with anhydrous diethyl ether, the SephadexG-10 desalination of solids behind the suction filtration, distilled water are moving phase, and triketohydrindene hydrate detects collects the aqueous solution, get title compound 240mg (96%), mp240-245 ℃ of decomposition after the lyophilize.FAB-MS(m/e)669[M+H] ⁺。

The preparation of embodiment 84.H-Ala-Arg-Pro-Ala-KL-OH

220mg (0.186mmol) Boc-Ala-Arg (Tos)-Pro-Ala-KL-OBzl is with after the 1ml methyl-phenoxide mixes, feed the 3ml anhydrous HF, 0 ℃ of reaction 2hr, HF is removed in decompression, feed anhydrous HF 2ml once more, 0 ℃ of reaction 1hr, HF is removed in decompression, residue solidifies with anhydrous diethyl ether, the SephadexG-10 desalination of the solids behind the suction filtration, and distilled water is moving phase, triketohydrindene hydrate detects collects the aqueous solution, get title compound 60mg (48%), mp114-116 ℃, FAB-MS (m/e) 612[M+H after the lyophilize] ⁺

The preparation of embodiment 85.H-KL-Ala-Arg-Pro-Ala-OH

440mg (0.373mmol) Boc-KL-Ala-Arg (Tos)-Pro-Ala-OBzl feeds the 1ml anhydrous HF with after the 1.5ml methyl-phenoxide mixes, 0 ℃ of reaction 2hr, HF is removed in decompression, feed anhydrous HF 3ml once more, 0 ℃ of reaction 1hr, HF is removed in decompression, residue solidifies with anhydrous diethyl ether, the SephadexG-10 desalination of solids behind the suction filtration, distilled water are moving phase, and triketohydrindene hydrate detects collects the aqueous solution, get title compound 240mg (96%), mp240-245 ℃ of decomposition after the lyophilize.FAB-MS(m/e)612[M+H] ⁺。

The preparation of embodiment 86.H-Ala-Arg-Pro-Ala-Lys-KL-OH

100mg (0.080mmol) Boc-Ala-Arg (Tos)-Pro-Ala-Lys (ClZ)-KL-OBzl is with after the 0.5ml methyl-phenoxide mixes, feed the 1ml anhydrous HF, 0 ℃ of reaction 2hr, HF is removed in decompression, feed anhydrous HF 2ml once more, 0 ℃ of reaction 1hr, HF is removed in decompression, residue solidifies with anhydrous diethyl ether, the Sephadex G-10 desalination of the solids behind the suction filtration, and distilled water is moving phase, triketohydrindene hydrate detects collects the aqueous solution, get title compound 50mg (84.6%), mp236-239 ℃ of decomposition, ESI-MS (m/e) 741[M+H after the lyophilize] ⁺

The preparation of embodiment 87.H-KL-Ala-Arg-Pro-Ala-Lys-OH

100mg (0.080mmol) Boc-KL-Ala-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is with after the 1ml methyl-phenoxide mixes, feed the 0.5ml anhydrous HF, 0 ℃ of reaction 2hr, HF is removed in decompression, feed anhydrous HF 2ml once more, 0 ℃ of reaction 1hr, HF is removed in decompression, residue solidifies with anhydrous diethyl ether, the Sephadex G-10 desalination of the solids behind the suction filtration, and distilled water is moving phase, triketohydrindene hydrate detects collects the aqueous solution, get title compound 45mg (76%), mp220-224 ℃ of decomposition, FAB-MS (m/e) 740[M+H after the lyophilize] ⁺

The preparation of embodiment 88.H-Gly-Arg-Pro-Ala-Lys-KL-OH

100mg (0.081mmol) Boc-Gly-Arg (Tos)-Pro-Ala-Lys-KL-OBzl is with after the 0.5ml methyl-phenoxide mixes, feed the 1ml anhydrous HF, 0 ℃ of reaction 2hr, HF is removed in decompression, feed anhydrous HF 2ml once more, 0 ℃ of reaction 1hr, HF is removed in decompression, residue solidifies with anhydrous diethyl ether, the SephadexG-10 desalination of the solids behind the suction filtration, and distilled water is moving phase, triketohydrindene hydrate detects collects the aqueous solution, get title compound 50mg (85%), mp214-218 ℃, FAB-MS (m/e) 726[M+H after the lyophilize] ⁺

The preparation of embodiment 89.H-KL-Gly-Arg-Pro-Ala-Lys-OH

250mg (0.330mmol) Boc-KL-Gly-Arg (Tos)-Pro-Ala-Lys-OBzl is with after the 1ml methyl-phenoxide mixes, feed the 2ml anhydrous HF, 0 ℃ of reaction 2hr, HF is removed in decompression, feed anhydrous HF 2ml once more, 0 ℃ of reaction 1hr, HF is removed in decompression, residue solidifies with anhydrous diethyl ether, the SephadexG-10 desalination of the solids behind the suction filtration, and distilled water is moving phase, triketohydrindene hydrate detects collects the aqueous solution, get title compound 45mg (75%), mp210-213 ℃, FAB-MS (m/e) 726[M+H after the lyophilize] ⁺

The preparation of embodiment 90.H-Lys-Arg-Pro-Ala-Lys-KL-OH

185mg (0.125mmol) Boc-Lys (ClZ)-Arg (Tos)-Pro-Ala-Lys (ClZ)-KL-OBzl is with after the 0.5ml methyl-phenoxide mixes, feed the 2ml anhydrous HF, 0 ℃ of reaction 2hr, HF is removed in decompression, feed anhydrous HF 2ml once more, 0 ℃ of reaction 1hr, HF is removed in decompression, residue solidifies with anhydrous diethyl ether, the Sephadex G-10 desalination of the solids behind the suction filtration, and distilled water is moving phase, triketohydrindene hydrate detects collects the aqueous solution, get title compound 90mg (87%), mp250-260 ℃, FAB-MS (m/e) 797[M+H after the lyophilize] ⁺

The preparation of embodiment 91.H-KL-Lys-Arg-Pro-Ala-Lys-OH

290mg (0.196mmol) Boc-KL-Lys (ClZ)-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is with after the 1ml methyl-phenoxide mixes, feed the 2ml anhydrous HF, 0 ℃ of reaction 2hr, HF is removed in decompression, feed anhydrous HF 2ml once more, 0 ℃ of reaction 1hr, HF is removed in decompression, residue solidifies with anhydrous diethyl ether, the Sephadex G-10 desalination of the solids behind the suction filtration, and distilled water is moving phase, triketohydrindene hydrate detects collects the aqueous solution, get title compound 130mg (83%), mp206-208 ℃, FAB-MS (m/e) 797[M+H after the lyophilize] ⁺

The preparation of embodiment 92.H-Gln-Arg-Pro-Ala-Lys-KL-OH

280mg (0.200mmol) Boc-Gln-Arg (Tos)-Pro-Ala-Lys (ClZ)-KL-OBzl is with after the 1ml methyl-phenoxide mixes, feed the 3ml anhydrous HF, 0 ℃ of reaction 2hr, HF is removed in decompression, feed anhydrous HF 2ml once more, 0 ℃ of reaction 1hr, HF is removed in decompression, residue solidifies with anhydrous diethyl ether, the Sephadex G-10 desalination of the solids behind the suction filtration, and distilled water is moving phase, triketohydrindene hydrate detects collects the aqueous solution, get title compound 130mg (81%), mp230-232 ℃ of decomposition, FAB-MS (m/e) 798[M+H after the lyophilize] ⁺

The preparation of embodiment 93.H-KL-Gln-Arg-Pro-Ala-Lys-OH

250mg (0.330mmol) Boc-KL-Gln-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is with after the 1ml methyl-phenoxide mixes, feed the 2ml anhydrous HF, 0 ℃ of reaction 2hr, HF is removed in decompression, feed anhydrous HF 2ml once more, 0 ℃ of reaction 1hr, HF is removed in decompression, residue solidifies with anhydrous diethyl ether, the Sephadex G-10 desalination of the solids behind the suction filtration, and distilled water is moving phase, triketohydrindene hydrate detects collects the aqueous solution, get title compound 80mg (58%), mp218-220 ℃, FAB-MS (m/e) 797[M+H after the lyophilize] ⁺

The preparation of embodiment 94H-Asn-Arg-Pro-Ala-Lys-KL-OH

200mg (0.182mmol) Boc-Asn-Arg (Tos)-Pro-Ala-Lys (ClZ)-KL-OBzl is with after the 1ml methyl-phenoxide mixes, feed the 2ml anhydrous HF, 0 ℃ of reaction 2hr, HF is removed in decompression, feed anhydrous HF 2ml once more, 0 ℃ of reaction 1hr, HF is removed in decompression, residue solidifies with anhydrous diethyl ether, the Sephadex G-10 desalination of the solids behind the suction filtration, and distilled water is moving phase, triketohydrindene hydrate detects collects the aqueous solution, get title compound 70mg (51%), mp198-200 ℃, FAB-MS (m/e) 782[M+H after the lyophilize] ⁺

The preparation of embodiment 95H-KL-Asn-Arg-Pro-Ala-Lys-OH

250mg (0.330mmol) Boc-KL-Asn-Arg (Tos)-Pro-Ala-Lys-OBzl is with after the 1ml methyl-phenoxide mixes, feed the 2ml anhydrous HF, 0 ℃ of reaction 2hr, HF is removed in decompression, feed anhydrous HF 2ml once more, 0 ℃ of reaction 1hr, HF is removed in decompression, residue solidifies with anhydrous diethyl ether, the SephadexG-10 desalination of the solids behind the suction filtration, and distilled water is moving phase, triketohydrindene hydrate detects collects the aqueous solution, get title compound 80mg (58%), mp211-214 ℃, FAB-MS (m/e) 784[M+H after the lyophilize] ⁺

The preparation of embodiment 96H-Arg-Arg-Pro-Ala-Lys-KL-OH

200mg (0.134mmol) Boc-Arg (Tos)-Arg (Tos)-Pro-Ala-Lys (ClZ)-KL-OBzl is with after the 0.5ml methyl-phenoxide mixes, feed the 2ml anhydrous HF, 0 ℃ of reaction 2hr, HF is removed in decompression, feed anhydrous HF 2ml once more, 0 ℃ of reaction 1hr, HF is removed in decompression, residue solidifies with anhydrous diethyl ether, the Sephadex G-10 desalination of the solids behind the suction filtration, and distilled water is moving phase, triketohydrindene hydrate detects collects the aqueous solution, get title compound 80mg (72%), mp208-212 ℃ of decomposition, FAB-MS (m/e) 825[M+H after the lyophilize] ⁺

The preparation of embodiment 97H-KL-Arg-Arg-Pro-Ala-Lys-OH

340mg (0.228mmol) Boc-KL-Arg (Tos)-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is with after the 1ml methyl-phenoxide mixes, feed the 2ml anhydrous HF, 0 ℃ of reaction 2hr, HF is removed in decompression, feed anhydrous HF 2ml once more, 0 ℃ of reaction 1hr, HF is removed in decompression, residue solidifies with anhydrous diethyl ether, the Sephadex G-10 desalination of the solids behind the suction filtration, and distilled water is moving phase, triketohydrindene hydrate detects collects the aqueous solution, get title compound 130mg (69%), mp238-240 ℃ of decomposition, FAB-MS (m/e) 825[M+H after the lyophilize] ⁺

The preparation of embodiment 98Boc-Ala-Arg (Tos)-Pro-Ala-Lys (ClZ)-KL-OH

550mg (0.439mmol) Boc-Ala-Arg (Tos)-Pro-Ala-Lys (ClZ)-OBzl is dissolved in the 10ml methyl alcohol, ice bath drips 2mol/L NaOH 50ml down, TLC (chloroform/methanol behind the 30min, 10/1) detecting raw material point disappears, add an amount of 2mol/L HCl neutralization reaction liquid to pH7, concentrating under reduced pressure boils off methyl alcohol, residue adds an amount of 2mol/L HCl makes pH1-2, in a large amount of distilled water of impouring, there are a large amount of white precipitates to generate suction filtration, filter cake with distilled water wash for several times, put in the moisture eliminator and spend the night, get title compound 450mg (88%), FAB-MS (m/e) 1201.2[M+K] ⁺

The preparation of embodiment 99HClAla-Arg-Pro-Ala-Lys (ClZ)-KL-OH

100mg (0.086mmol) Boc-Ala-Arg (Tos)-Pro-Ala-Lys (ClZ)-KL-OH is dissolved in 5ml anhydrous hydrogen chloride/ethyl acetate solution, stirring at room, and TLC behind the 5hr (chloroform/methanol, 5/1) shows that raw material point disappears.Reaction mixture is concentrating under reduced pressure at room temperature, and residue also at room temperature concentrates with acetic acid ethyl dissolution, so repeatedly for several times, and up to Ex-all free hydrogenchloride.Residue grinds with anhydrous diethyl ether, gets title compound, is directly used in next step reaction.

The preparation of embodiment 100.Cyclo (Ala-Arg (Tos)-Pro-Ala-Lys (ClZ))

100mg compound H CL-Ala-Arg (Tos)-Pro-Ala-Lys (ClZ)-OH is dissolved in the 2ml dry DMF, 0 ℃ adds 15mg (0.111mmol) N-hydroxy benzo triazole and 20mg (0.097mmol) DCC down, behind 0 ℃ of reaction 5hr, TLC shows that raw material point disappears, add dry DMF 200ml, 0 ℃ drips N-methylmorpholine down and makes pH value of solution 8-9,0 ℃ drips N-methylmorpholine down and makes pH value of solution 8-9, change room temperature reaction 36hr over to, TLC (chloroform/methanol, 10/1) shows that raw material point disappears, stopped reaction dries up solvent, the residue acetic acid ethyl dissolution, use saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride water solution, the washing of 5% aqueous potassium hydrogen sulfate, the organic layer anhydrous magnesium sulfate drying of telling.Filter, filtrate is evaporated to dried at 37 ℃, and residue is walked big plate separation and obtained title compound 10mg (10%), ESI-MS (m/e) 1045[M+H] ⁺, 1061[M+H ₂O] ⁺

The thrombolysis activity of embodiment 101. general formulas (3), (6), (7)

101.1 the preparation of thrombus

The Glass tubing of past vertical fixing (long 15mm, internal diameter 2.5mm, external diameter 5.0mm, the pipe end, seal with plug) the middle 0.1ml of injection citron acidifying rat artery blood, the multiple calcium of this blood does not coagulate.Toward the interior thrombus standing bolt that inserts a stainless steel material rapidly of pipe.This thrombus standing bolt diameter is the Stainless Steel Wire coiled of 0.2mm, and the long 12mm of spiral part contains 15 bung flanges, and the diameter of bung flange is 1.0mm, and the holder handle links to each other with spiral, and long 7.0mm is the question mark type.After the blood coagulation 15 minutes, open the plug of Glass tubing bottom,, from Glass tubing, take out fixedly spiral of the thrombus that wrapped up by thrombus carefully, accurately weigh (mass range is 74-84mg) with the fixing fixing holder handle of spiral of thrombus of tweezers.

101.2 the preparation of bypass duct

Bypass duct constitutes by 3 sections, and the stage casing is a polyethylene rubber tube, long 60.0mm, internal diameter 3.5mm, two ends are identical polyethylene tube, long 100.0mm, internal diameter 1.0mm, external diameter 2.0mm, one end of this pipe is strained to point pipe (being used to insert rat carotid artery or vein), and external diameter is 1.0mm, the outer cover one segment length 7.0mm of the other end, the polyethylene tube of external diameter 3.5mm is used to insert in the polyethylene tube in stage casing.The equal silanization of the inwall of 3 sections pipes.The thrombus standing bolt that the thrombus of accurately weighing wraps up is put into the stage casing polyethylene tube, and the two ends of sebific duct link to each other with the sleeve end of firm two polyethylene tubes describing respectively.With syringe by sharp pipe end with filling with heparin-saline solution (50IU/kg body weight) in the pipe, standby.

101.3 rat neck arteriovenous shut intubate model

(male, 250～300g) anaesthetize by 80mg/kg dosage abdominal injection Sodital sodium solution the Wistar rat.Undergo surgery after the anesthetized rat dorsal position is fixing: separate tracheae, separate right common carotid artery and left external jugular vein.On the left external jugular vein that exposes, cut an angle carefully, the sharp pipe of the bypass duct for preparing is above inserted the proximal part of left external jugular vein opening by angle, simultaneously away from the holder handle of bypass tube stage casing thrombus standing bolt.Push the heparin-saline (50Iu/kg) of accurate amount with syringe by the sharp pipe of the other end, this moment, syringe was not withdrawn polyethylene tube.Proximal part in right common carotid artery stops blooding with bulldog clamp, right common carotid artery is cut an angle carefully nearby at the calcarine artery folder.Extract syringe from the tip of polyethylene tube, the tip of polyethylene tube is inserted the proximal part of artery angle.The two ends of bypass duct all use 4 trumpeter's art sutures and arteriovenous to fix.Open bulldog clamp, make blood flow flow to vein from artery by bypass duct.So, constituted rat arteriovenous shut Thrombolysis Model.

101.4 the thrombolytic effect of the compound of general formula (3) (6) (7)

With scalp acupuncture the normal saline solution (10umol/kg) of the compound of the normal saline solution (20000IU/kg) of physiological saline (3ml/kg), urokinase, general formula (3), (6), (7) is thrust away from the fixing nearly vein place of spiral of thrombus by the stage casing of bypass tube, slowly be injected into (about 6 minutes) in the blood.Make the compound of physiological saline (blank), urokinase (positive control) and general formula (3), (6), (7) cross blood circulation, the sequential action of pressing vein-heart-artery is to thrombus.Pick up counting from start injection contrast liquid or drug solution, 1.5 after hour from bypass tube the removal of thromboses standing bolt, accurately weigh, obtain thrombus standing bolt in every rat bypass duct and enter of poor quality before and after the bypass duct, add up the thrombus quality difference (x ± S) of each treated animal, and carry out the t check, data are listed table 1 in.

The thrombolytic effect of the compound of table 1. general formula (3), (6), (7)

Group	Dosage (dosage/kg)	The thrombus loss of weight (x ± S)
			NS UK P6A GP6A QP6A dAKL dKLA dGKL dKLG dQKL dKLQ dPAKKL dPAKKL dPAKKL dKLPAK	3ml 20000IU 5.4mg 5.3mg 6.0mg 7.4mg 7.4mg 7.2mg 7.2mg 8.0mg 8.0mg 5.1mg 2.5mg 0.5mg 5.1mg	15.31±3.57 24.10±3.54 18.84±3.18 1)2) 25.90±2.05 1) 21.28±3.46 1)2) 26.43±3.84 1) 16.13±7.83 2) 24.02±5.21 1) 19.7±3.71 2) 22.88±9.21 1) 17.27±3.57 2) 26.40±4.21 1)3) 22.31±3.66 1) 18.22±5.84 2)3) 20.80±3.96 2)

1) compares P＜0.05 with NS; 2) compare P＜0.05 with UK; 3) compare P＜0.05, N=9 with dPAKKL2.5mg/mg

The result shows that the thrombolysis activity of dPAKKL has dose-dependently.The 10umol/kg group, the thrombolysis activity and the dosage of 5umol/kg group and 1umol/kg group have linear dependence.The P6A group, GP6A group and QP6A group thrombolysis activity are the structure dependency.DXKL (X=Ala, Gly, Gln, PAK) group is active suitable with UK, corresponding dKLX (X=PAK, Ala, Gly, Gln) group does not have thrombolysis activity.

Claims (4)

1, a kind of by P6A correlated series polypeptide and carboline carboxylate with the plan peptide that covalent linkage connects and composes, it is characterized in that having the structure of following general formula (7):

AA wherein ₁' and AA ₂' be hydrogen atom jointly; Or AA ₁' be A, G or Q, AA ₂' be the Arg residue.

2, the method for preparing the described plan peptide of claim 1 comprises following raw material: Boc-AA ₁-AA ₂-Pro-Ala-Lys (ClZ) OH, with the amino condensation of carboline carboxylate benzyl ester, condensation product gets the compound of general formula (7) through the HF deprotection, in the above-mentioned raw material, AA ₁And AA ₂Common is hydrogen atom; Or AA ₁Be A, G or Q, AA ₂Be Arg (Tos) residue.

3, a kind of thrombolytic agent that contains the described plan peptide of claim 1.

4, the application of the plan peptide of claim 1 in the medicine of preparation treatment thrombotic diseases.

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