CN101318993B - Hybridized peptide with 3S-tetrahydrochysene-beta-carboline-3-carboxylic acid as connecting arm, preparation method and application thereof - Google Patents
Hybridized peptide with 3S-tetrahydrochysene-beta-carboline-3-carboxylic acid as connecting arm, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a general formula I compound and a general formula II compound having thrombus dissolving activity, a preparation method thereof and an application thereof as thrombus dissolving agent. The invention adopts a rat carotid-jugular vein bypass intubation thrombus model to evaluate the thrombus dissolving activity of the general formula I compound and the general formula II compound respectively. Animal experimental results show that: both the general formula I compound and the general formula II compound have excellent thrombus dissolving activity and can be applied to clinical practice as a thrombus dissolving agent.
Description
Technical field
The present invention relates to a kind of hybridization peptide, relating in particular to 3S-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is hybridization peptide, its preparation method of connecting arm, and the application that the invention still further relates to this hybridization peptide conduct and use thrombolytic agent belongs to field of medicaments.
Background technology
Medicine ubiquity systemic bleedings such as streptokinase, urokinase and the rt-PA tendency and the immunogenic response of the treatment acute thrombus embolism class diseases of present clinical use, seeking safely and effectively, thrombolytic agent is one of focus of new drug research.The contriver 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 number: CN1055479C; 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 contriver 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.The contriver notices that carboline carboxylate has outstanding antiplatelet aggregative activity (Yao Xinsheng etc., China's pharmaceutical chemistry magazine, 1995,5,134), the indole ring parent nucleus of carboline carboxylate has outstanding uv-absorbing character and has the potential application prospect as edible natural product simultaneously.The contriver adopts chemical process to synthesize carboline carboxylate, and carboline carboxylate is incorporated into preparation plan peptide in P6A and the correlated series as pharmacophore.The plan peptide that makes has comprised the thrombolysis activity of PAK sequence peptide, has also comprised the platelet aggregation inhibitory activity of carboline carboxylate, and has ultraviolet detectability (contriver: Peng Shiqi, Zhao Ming, Wang Chao, Wu Yanfen; Denomination of invention: contain the carboline carboxylate of P6A correlated series, their the synthetic and application in medical science; Granted publication number: CN1373139; Granted publication day: on October 9th, 2002).
Platelet aggregation plays an important role in thrombosis.Normal thrombocyte is activated under the inducing of thrombogen, adenosine diphosphate (ADP) or collagen, discharges arachidonic acid (AA).AA generates endoperoxide (PGH2) through cyclooxygenase catalysis.PGH2 generates thromboxane A2 (TXA2) under the effect of isomerase, cause platelet aggregation and adhesion.Behind the platelet activation, glycoprotein iib/iiia (GP IIb/IIIa) acceptor on surface exposes, and occurred conformation changes, and combines with Fibrinogen, causes thrombosis.In research GPIIb/IIIa acceptor and the scleroproein bonded process, find that the RGD sequence that exists among the scleroproein is mediation it self and GP IIb/IIIa bonded pass key sequence (Samanen J., et al., J.Med.Chem., 1991,34,3114).The contriver adopts liquid phase method to synthesize RGDS, RGDV and RGDF, and confirm that they can suppress thrombosis (what contain the RGD tetrapeptide synthesizes and function Acta Pharmaceutica Sinica, 1997,32,271 for Zhao Ming, Peng Shiqi).The contriver once reported, contained the tetrapeptide of RGD, for example RGDS, can be thrombolytic agent, for example urokinase is transported to the thrombosis position, has clear and definite target (Ling Shichang, Zhao Ming, Peng Shiqi, the liposome that RGDS modifies is as the research of pharmaceutical carrier targeting thrombolysis, journal of Beijing Medical University, 1994,26,174).
Summary of the invention
One of the object of the invention provide have thrombolytic agent active with 3S-tetrahydrochysene-β-Ka Lin-3-carboxylic acid be connecting arm the hybridization peptide.
One of the object of the invention is achieved through the following technical solutions:
The compound of following general formula I or general formula I I:
Wherein, AA
1Be selected from Ser, Val or Phe residue; Work as AA
3During for the Arg residue, AA
2Be selected from Ala, Gly or Gln residue, or AA
2Do not exist; Perhaps AA
2And AA
3All do not exist.
Two of the object of the invention provides a kind of method for preparing general formula I or general formula I I compound
A kind of method for preparing above-mentioned compound of Formula I comprises:
(1) prepares following three kinds of tetrapeptide: Arg-Gly-Asp-Ser-OH, Arg-Gly-Asp-Val-OH, Arg-Gly-Asp-Phe-OH respectively according to ordinary method; The N-end and the side chain of above-mentioned three kinds of tetrapeptides are protected with protecting group;
(2) prepare 3S-tetrahydrochysene-β-Ka Lin-3-carboxylic acid according to ordinary method, benzylization prepares 3S-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate then;
(3) prepare following 5 kinds of small peptide: Gly-Arg-Pro-Ala-Lys-OH, Gln-Arg-Pro-Ala-Lys-OH, Ala-Arg-Pro-Ala-Lys-OH, Arg-Pro-Ala-Lys-OH, the Pro-Ala-Lys-OH of C-end and side chain protected respectively according to ordinary method;
(4) according to ordinary method respectively with on Arg-Gly-Asp-Ser-OH, the Arg-Gly-Asp-Val-OH of N-end and side chain protected or 2 nitrogen that Arg-Gly-Asp-Phe-OH is connected to 3S-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate, slough carbobenzoxy; Respectively Gly-Arg-Pro-Ala-Lys-OH, Gln-Arg-Pro-Ala-Lys-OH, Ala-Arg-Pro-Ala-Lys-OH, Arg-Pro-Ala-Lys-OH or the Pro-Ala-Lys-OH of C-end and side chain protected are connected on the 3-carboxyl of 3S-tetrahydrochysene-β-Ka Lin-3-carboxylic acid, slough all protecting groups, obtain compound of Formula I.
A kind of method for preparing above-mentioned general formula I I compound comprises:
(1) prepares following three kinds of tetrapeptide: Arg-Gly-Asp-Ser-OH, Arg-Gly-Asp-Val-OH, Arg-Gly-Asp-Phe-OH respectively according to ordinary method; The C-end and the side chain of above-mentioned three kinds of tetrapeptides are protected with protecting group;
(2) prepare 3S-tetrahydrochysene-β-Ka Lin-3-carboxylic acid according to ordinary method, benzylization prepares 3S-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate then;
(3) prepare following 5 kinds of small peptide: Gly-Arg-Pro-Ala-Lys-OH, Gln-Arg-Pro-Ala-Lys-OH, Ala-Arg-Pro-Ala-Lys-OH, Arg-Pro-Ala-Lys-OH, the Pro-Ala-Lys-OH of N-end and side chain protected respectively according to ordinary method;
(4) respectively Gly-Arg-Pro-Ala-Lys-OH, Gln-Arg-Pro-Ala-Lys-OH, Ala-Arg-Pro-Ala-Lys-OH, Arg-Pro-Ala-Lys-OH or the Pro-Ala-Lys-OH of N-end and side chain protected is connected to earlier on 2 nitrogen of 3S-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate, sloughs carbobenzoxy according to ordinary method; Respectively C-end and side chain protected Arg-Gly-Asp-Ser-OH, Arg-Gly-Asp-Val-OH or Arg-Gly-Asp-Phe-OH are connected on the 3-position carboxyl of 3S-tetrahydrochysene-β-Ka Lin-3-carboxylic acid, slough all protecting groups again, obtain general formula I I compound.
Among the above-mentioned preparation method, described 3S-tetrahydrochysene-β-Ka Lin-3-carboxylic acid can be prepared by L-tryptophane and formaldehyde condensation.
The present invention adopts the thrombus dissolving activity of having estimated compound of Formula I (Ia-o) and general formula I I compound (IIa-o) on the rat neck arteriovenous shut intubate thrombus model respectively, experimentation on animals is the result show, compound of Formula I of the present invention (Ia-o) and general formula I I compound (IIa-o) all have outstanding thrombus dissolving activity, can be used as thrombus dissolving clinically and use.
Description of drawings
The synthetic route chart of Fig. 1 compound of Formula I; Wherein, AA
1Be selected from Ser, Val or Phe; Work as AA
3During for Arg, AA
2Be selected from Ala, Gly or Gln, or AA
2Do not exist; Perhaps AA
2And AA
3All do not exist.
The synthetic route chart of Fig. 2 general formula I I compound; Wherein, AA
1Be selected from Ser, Val or Phe; Work as AA
3During for Arg, AA
2Be selected from Ala, Gly or Gln, or AA
2Do not exist; Perhaps AA
2And AA
3All do not exist.
Embodiment
The invention will be further described below by embodiment.Should be pointed out that these embodiment only are illustrations of the present invention, should not be construed as limitation of the present invention.
The preparation of embodiment 1Boc-Pro-Ala-Lys (Z)-OBz
1) preparation Boc-Ala-Lys (Z)-Obzl
473mg (2.5mmol) Boc-Ala-OH is dissolved in 10ml anhydrous tetrahydro furan (THF), adds anhydrous tetrahydro furan (THF) solution of 10ml338mg (2.5mmol) N-hydroxybenzotriazole (HOBt) and 619mg (3mmol) dicyclohexyl carbonyl diimine (DCC) under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
936mg (2.3mmol) HClLys (Z)-OBzl and 232mg (2.3mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 6ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl3:MeOH=20:1) shows that HClLys (Z)-OBzl disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.204g (97%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate.Be white solid.
2) preparation HClAla-Lys (Z)-OBzl
TLC (developping agent CHCl will be stirred 1 hour under the mixture condition of ice bath of 1.354g (2.5mmol) Boc-Ala-Lys (Z)-OBzl and 15ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=20:1) show that Boc-Ala-Lys (Z)-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
3) preparation Boc-Pro-Ala-Lys (Z)-OBzl
538mg (2.5mmol) Boc-Pro-OH is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution of 10ml338mg (2.5mmol) HOBt and 619mg (3mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
1.099g (2.3mmol) HClAla-Lys (Z)-OBzl and 232mg (2.3mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 6ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=20:1) show that HClAla-Lys (Z)-OBzl disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 2.847g (98%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate.Be colorless solid.
Embodiment 2Boc-Arg (NO
2The preparation of)-Pro-Ala-Lys (Z)-OBzl
1) preparation HClPro-Ala-Lys (Z)-OBzl
TLC (developping agent CHCl will be stirred 1 hour under the mixture condition of ice bath of 1.596g (2.5mmol) Boc-Pro-Ala-Lys (Z)-OBzl and 15ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=20:1) show that Boc-Pro-Ala-Lys (Z)-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
2) preparation Boc-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
With 798mg (2.5mmol) Boc-Arg (NO
2)-OH is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution of 10ml338mg (2.5mmol) HOBt and 619mg (3mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
1.322g (2.3mmol) HClPro-Ala-Lys (Z)-OBzl and 232mg (2.3mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 6ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl3:MeOH=10:1) shows that HClPro-Ala-Lys (Z)-OBzl disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.642g (85%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate.Be colorless solid.Mp87~88℃[α]
D=-13.0(C=0.8,CHCl
3);ESI-MS(m/e)864[M+Na]
+。
Embodiment 3Boc-Ala-Arg (NO
2The preparation of)-Pro-Ala-Lys (Z)-OBzl
1) preparation HClArg (NO
2)-Pro-Ala-Lys (Z)-OBzl
With 2.099g (2.5mmol) Boc-Arg (NO
2Stirred 1 hour TLC (developping agent CHCl under the mixture condition of ice bath of)-Pro-Ala-Lys (Z)-OBzl and 15ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show Boc-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
2) Boc-Ala-Arg (NO
2The preparation of)-Pro-Ala-Lys (Z)-OBzl
473mg (2.5mmol) Boc-Ala-OH is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution that 10ml contains 338mg (2.5mmol) HOBt and 619mg (3mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
With 1.784g (2.3mmol) HClArg (NO
2)-Pro-Ala-Lys (Z)-OBzl and 232mg (2.3mmol) N-methylmorpholine are miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 6ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show HClArg (NO
2)-Pro-Ala-Lys (Z)-OBzl disappears.Reaction mixture is evaporated to dried, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 1.884g (90%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate.Be colorless solid.Mp97~98℃[α]
D=-23.0(C=0.8,CHCl
3)ESI-MS(m/e)911[M+H]
+。
Embodiment 4Boc-Gly-Arg (NO
2The preparation of)-Pro-Ala-Lys (Z)-OBzl
438mg (2.5mmol) Boc-Gly-OH is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution that 10ml contains 338mg (2.5mmol) HOBt and 619mg (3mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
With 1.784g (2.3mmol) HClArg (NO
2)-Pro-Ala-Lys (Z)-OBzl and 232mg (2.3mmol) N-methylmorpholine are miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 6ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show HClArg (NO
2)-Pro-Ala-Lys (Z)-OBzl disappears.Reaction mixture is evaporated to dried, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 1.855g (90%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate.Be colorless solid.Mp91~92℃[α]
D=-14.0(C=0.8,CHCl
3);ESI-MS(m/e)897[M+H]
+。
Embodiment 5Boc-Gln-Arg (NO
2The preparation of)-Pro-Ala-Lys (Z)-OBzl
615mg (2.5mmol) Boc-Gln-OH is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution that 10ml contains 338mg (2.5mmol) HOBt and 619mg (3mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
With 1.784g (2.3mmol) HClArg (NO
2)-Pro-Ala-Lys (Z)-OBzl and 232mg (2.3mmol) N-methylmorpholine are miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 6ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show HClArg (NO
2)-Pro-Ala-Lys (Z)-OBzl disappears.Reaction mixture is evaporated to dried, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 2.002g (90%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate.Be colorless solid.Mp83~85℃[α]
D=-18.0(C=0.8,CHCl
3);ESI-MS(m/e)968[M+H]
+。
Embodiment 6Boc-Arg (NO
2The preparation of)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl
1) preparation Boc-Ser (Bzl)-OBzl
738mg (2.5mmol) Boc-Ser (Bzl)-OH is dissolved in the 10ml dehydrated alcohol, adds 407mg (1.25mmol) Cs
2CO
3, room temperature reaction 15 minutes, TLC show that raw material point disappears.Concentrating under reduced pressure removes and desolvates, and gets corresponding cesium salt.The cesium salt that obtains is dissolved in the 2ml dry DMF earlier, adds 0.3ml (2.5mmol) bromobenzyl more inward.Reaction mixture stirs 16h, TLC (developping agent CHCl in 50-55 ℃
3: MeOH=20:1) cesium salt of demonstration Boc-Ser (Bzl)-OH disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling obtains 874mg (90.7%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless oil.
2) preparation HClSer (Bzl)-OBzl
TLC (developping agent CHCl will be stirred 1 hour under the mixture condition of ice bath of 963mg (2.5mmol) Boc-Ser (Bzl)-OBzl and 10ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=20:1) show that Boc-Ser (Bzl)-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
3) preparation Boc-Asp (OBzl)-Ser (Bzl)-OBzl
808mg (2.5mmol) Boc-Asp (OBzl)-OH is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution that 10ml contains 338mg (2.5mmol) HOBt and 619mg (3mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
740mg (2.3mmol) HClSer (Bzl)-OBzl and 232mg (2.3mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 6ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=20:1) show that Boc-Asp (OBzl)-OH disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.29g (95%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless oil.
4) preparation HClAsp (OBzl)-Ser (Bzl)-OBzl
TLC (developping agent CHCl will be stirred 1 hour under the mixture condition of ice bath of 1.477g (2.5mmol) Boc-Asp (OBzl)-Ser (Bzl)-OBzl and 10ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=20:1) show that Boc-Asp (OBzl)-Ser (Bzl)-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
5) preparation Boc-Gly-Asp (OBzl)-Ser (Bzl)-OBzl
438mg (2.5mmol) Boc-Gly-OH is dissolved in the anhydrous THF of 6ml, adds the anhydrous THF solution that 10ml contains 338mg (2.5mmol) HOBt and 619mg (3mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
1.212g (2.3mmol) HClAsp (OBzl)-Ser (Bzl)-OBzl and 232mg (2.3mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 6ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show that HClAsp (OBzl)-Ser (Bzl)-OBzl disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.461g (98.1%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.
6) preparation HClGly-Asp (OBzl)-Ser (Bzl)-OBzl
TLC (developping agent CHCl will be stirred 1 hour under the mixture condition of ice bath of 1.619g (2.5mmol) Boc-Gly-Asp (OBzl)-Ser (Bzl)-OBzl and 15ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show that Boc-Gly-Asp (OBzl)-Ser (Bzl)-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
7) preparation Boc-Arg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl
With 798mg (2.5mmol) Boc-Arg (NO
2)-OH is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution that 10ml contains 338mg (2.5mmol) HOBt and 619mg (3mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
1.343g (2.3mmol) HClGly-Asp (OBzl)-Ser (Bzl)-OBzl and 232mg (2.3mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 6ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show that HClGly-Asp (OBzl)-Ser (Bzl)-OBzl disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.461g (98.1%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.
Embodiment 7Boc-Arg (NO
2The preparation of)-Gly-Asp (OBzl)-Phe-OBzl
1) preparation HClPhe-OBzl
The suspension of 10.85g (66mmol) Phe-OH, 74.6ml phenylcarbinol and 14.93g polyphosphoric acid is stirred 8h in 90-95 ℃, make to become clarification.Reaction mixture is poured in 600ml (5%) the sulfuric acid liquid, used extracted with diethyl ether 5 times, divide water-yielding stratum.Ether layer is with 2% salt acid elution 3 times, the water NaHCO of merging
3Solid is regulated pH9.Water extracted with diethyl ether 3 times (100ml * 3), the ether layer of merging anhydrous Na 2SO
4Dry.Filter, in filtrate, led to hydrogen chloride gas about 20 minutes.Muddy ether layer filters, and filter collects 5.641g (30%) title compound.
2) preparation Boc-Asp (OBzl)-Phe-OBzl
808mg (2.5mmol) Boc-Asp (OBzl)-OH is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution that 10ml contains 338mg (2.5mmol) HOBt and 619mg (3mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
668mg (2.3mmol) HClPhe-OBzl and 232mg (2.3mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 6ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=20:1) show that HClPhe-OBzl disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.461g (98.1%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.
3) preparation HClAsp (OBzl)-Phe-OBzl
TLC (developping agent CHCl will be stirred 1 hour under the mixture condition of ice bath of 1.398g (2.5mmol) Boc-Asp (OBzl)-Phe-OBzl and 15ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=20:1) show that Boc-Asp (OBzl)-Phe-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
4) preparation Boc-Gly-Asp (OBzl)-Phe-OBzl
438mg (2.5mmol) Boc-Gly-OH is dissolved in the anhydrous THF of 5ml, adds the anhydrous THF solution that 10ml contains 338mg (2.5mmol) HOBt and 619mg (3mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
1.141g (2.3mmol) HClAsp (OBzl)-Phe-OBzl and 232mg (2.3mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show that HClAsp (OBzl)-Phe-OBzl disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.29g (91%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.
5) preparation HClGly-Asp (OBzl)-Phe-OBzl
TLC (developping agent CHCl will be stirred 1 hour under the mixture condition of ice bath of 1.541g (2.5mmol) Boc-Gly-Asp (OBzl)-Phe-OBzl and 15ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show that Boc-Gly-Asp (OBzl)-Phe-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
6) preparation Boc-Arg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl
With 798mg (2.5mmol) Boc Arg (NO
2)-OH is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution that 10ml contains 338mg (2.5mmol) HOBt and 619mg (3.0mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
1.272g (2.3mmol) HClGly-Asp (OBzl)-Phe-OBzl and 232mg (2.3mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show that HClGly-Asp (OBzl)-Phe-OBzl disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.29g (91%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.
Embodiment 8Boc-Arg (NO
2The preparation of)-Gly-Asp (OBzl)-Val-OBzl
1) preparation HClVal-OBzl
The suspension of 5.855g (50mmol) H-Val-OH, 55ml phenylcarbinol and 11.5g polyphosphoric acid is stirred 8h in 90-95 ℃, make to become clarification.Reaction mixture is poured in 300ml (5%) the sulfuric acid liquid, used extracted with diethyl ether 5 times, divide water-yielding stratum.Ether layer is with 2% salt acid elution 3 times, the water NaHCO of merging
3Solid is regulated pH9.Water extracted with diethyl ether 3 times (100ml * 3), the anhydrous MgSO of the ether layer of merging
4Dry.Filter, in filtrate, led to HCl gas about 20 minutes.Muddy ether layer is overanxious, and filter collects 3.034g (25%) title compound.
2) preparation Boc-Asp (OBzl)-Val-OBzl
808mg (2.5mmol) Boc-Asp (OBzl)-OH is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution that 10ml contains 338mg (2.5mmol) HOBt and 619mg (3.0mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
558mg (2.3mmol) HClVal-OBzl and 232mg (2.3mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=20:1) show that HClVal-OBzl disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.29g (96%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.
3) preparation HClAsp (OBzl)-Val-OBzl
TLC (developping agent CHCl will be stirred 1 hour under the mixture condition of ice bath of 1.278g (2.5mmol) Boc-Asp (OBzl)-Val-OBzl and 10ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=20:1) show that Boc-Asp (OBzl)-Val-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
4) preparation Boc-Gly-Asp (OBzl)-Val-OBzl
438mg (2.5mmol) Boc-Gly-OH is dissolved in the anhydrous THF of 6ml, adds the anhydrous THF solution that 10ml contains 338mg (2.5mmol) HOBt and 619mg (3.0mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
1.03g (2.3mmol) HClAsp (OBzl)-Val-OBzl and 232mg (2.3mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show that HClAsp (OBzl)-Val-OBzl disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.242g (95%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.
5) preparation HClGly-Asp (OBzl)-Val-OBzl
TLC (developping agent CHCl will be stirred 1 hour under the mixture condition of ice bath of 1.421g (2.5mmol) Boc-Gly-Asp (OBzl)-Val-OBzl and 10ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show that Boc-Gly-Asp (OBzl)-Val-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
6) preparation Boc-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl
With 798mg (2.5mmol) Boc-Arg (NO
2)-OH is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution that 10ml contains 338mg (2.5mmol) HOBt and 619mg (3.0mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
1.162g (2.3mmol) HClGly-Asp (OBzl)-Val-OBzl and 232mg (2.3mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show that HClGly-Asp (OBzl)-Val-OBzl disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.523g (86%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.
Embodiment 9 preparation 3S-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid
The H of 25ml1N
2SO
4, add 5g (24.5mmol) tryptophane in the stirring, add 75ml water again, the formaldehyde solution of 4ml (45.6mmol) 38%, reaction solution becomes clarification, separates out a large amount of solids about 5Min, react after 4 hours, be added dropwise to the 8ml strong aqua, transfer PH near neutral, standing over night, the a small amount of cold wash of suction filtration, filter cake is drained, dryly again must obtain 5.1g (96%) title compound, be buff powder.
Embodiment 10 preparation 3S-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate
1) the Tetrahydrocarboline benzyl carboxylate of preparation phos-phate forms
With 25.00g (115.7mmol) 3S-2,3,4, the suspension of 9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid, 150ml phenylcarbinol and 25g polyphosphoric acid makes to become clarification in 90-95 ℃ of stirring 10h, and reaction solution is a Vandyke brown.Be cooled to normal temperature, reaction mixture poured in the mixed solution of 200mlH2O and 300ml ether, machinery is powerful to be stirred 5 hours.Separate out a large amount of yellow powder shape solids, leave standstill hypsokinesis and go out supernatant liquid, filter yellow particle shape solid.Gained solid water is washed by rubbing with the hands repeatedly, until obtaining thin yellow powder shape solid.Filter, the phos-phate forms that gets carboline carboxylate benzyl ester is directly used in next step reaction.
2) preparation free Tetrahydrocarboline benzyl carboxylate
The previous step products therefrom is suspended in the mixed solution of 250ml and 100ml water, under the condition of ice bath, it is transparent to drip triethylamine solution no insoluble particle and reaction solution to the reaction solution.After the sustained reaction 2 hours, separatory is got ethyl acetate layer, adds saturated sodium bicarbonate aqueous solution and gives a baby a bath on the third day after its birth time, tells ethyl acetate layer with anhydrous sodium sulphate solid drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, recrystallization gets 19g (74%) title compound, is the yellowish white powder.
The preparation of embodiment 11Boc-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl)-OBzl
1) preparation Boc-Ser (Bzl)-OBzl
738mg (2.5mmol) Boc-Ser (Bzl)-OH is dissolved in the 10ml dehydrated alcohol, adds 4ml407mg (1.25mmol) Cs
2CO
3The aqueous solution, room temperature reaction 15 minutes, TLC show that Boc-Ser (Bzl)-OH disappears.Concentrating under reduced pressure removes and desolvates, and gets corresponding cesium salt.The cesium salt that obtains is dissolved in the 2ml dry DMF earlier, adds 0.3ml (2.5mmol) bromobenzyl more inward.Reaction mixture stirs 16h, TLC (developping agent CHCl in 50-55 ℃
3: MeOH=20:1) cesium salt of demonstration Boc-Ser (Bzl)-OH disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling obtains 874mg (90.7%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless oil.
2) preparation HClSer (Bzl)-OBzl
TLC (developping agent CHCl will be stirred 1 hour under the mixture condition of ice bath of 963mg (2.5mmol) Boc-Ser (Bzl)-OBzl and 10ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=20:1) show that Boc-Ser (Bzl)-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
3) preparation Boc-Asp (OcHex)-Ser (Bzl)-OBzl
789mg (2.5mmol) Boc-Asp (OcHex)-OH is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution that 10ml contains 338mg (2.5mmol) HOBt and 619mg (3mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
740mg (2.3mmol) HClSer (Bzl)-OBzl and 232mg (2.3mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 6ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=20:1) show that Boc-Asp (OcHex)-OH disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution collection successively.The ethyl acetate layer of telling gets 1.27g (95%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless oil.
4) preparation HClAsp (OcHex)-Ser (Bzl)-OBzl
TLC (developping agent CHCl will be stirred 1 hour under the mixture condition of ice bath of 1.457g (2.5mmol) Boc-Asp (OcHex)-Ser (Bzl)-OBzl and 10ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=20:1) show that Boc-Asp (OcHex)-Ser (Bzl)-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
5) preparation Boc-Gly-Asp (OcHex)-Ser (Bzl)-OBzl
438mg (2.5mmol) Boc-Gly-OH is dissolved in the anhydrous THF of 6ml, adds the anhydrous THF solution of 10ml338mg (2.5mmol) HOBt and 619mg (3mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
1.194g (2.3mmol) HClAsp (OBzl)-Ser (Bzl)-OBzl and 232mg (2.3mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 6ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show that HClAsp (OcHex)-Ser (Bzl)-OBzl disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.443g (98.1%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp96~97℃;[α]
D=-32(C=0.2,CHCl
3);ESI-MS(m/e)641[M+H]
+。
6) preparation HClGly-Asp (OcHex)-Ser (Bzl)-OBzl
TLC (developping agent CHCl will be stirred 1 hour under the mixture condition of ice bath of 1.599g (2.5mmol) Boc-Gly-Asp (OcHex)-Ser (Bzl)-OBzl and 15ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show that Boc-Gly-Asp (OcHex)-Ser (Bzl)-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
7) preparation Boc-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl)-OBzl
1.071g (2.5mmol) Boc-Arg (Tos)-OH is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution that 10ml contains 338mg (2.5mmol) HOBt and 619mg (3mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
1.325g (2.3mmol) HClGly-Asp (OcHex)-Ser (Bzl)-OBzl and 232mg (2.3mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 6ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show that HClGly-Asp (OcHex)-Ser (Bzl)-OBzl disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 2.144g (98.1%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp101~102℃[α]
D=-34.0(C=0.4,CHCl
3);ESI-MS(m/e)951[M+H]
+。
The preparation of embodiment 12Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe-OBzl
1) preparation HClPhe-OBzl
The suspension of 10.85g (66mmol) Phe-OH, 74.6ml phenylcarbinol and 14.93g polyphosphoric acid is stirred 8h in 90-95 ℃, make to become clarification.Reaction mixture is poured in 600ml (5%) the sulfuric acid liquid, used extracted with diethyl ether 5 times, divide water-yielding stratum.Ether layer is with 2% salt acid elution 3 times, the water NaHCO of merging
3Solid is regulated pH9.Water extracted with diethyl ether 3 times (100ml * 3), the ether layer anhydrous Na of merging
2SO
4Dry.Filter, in filtrate, led to HCl gas about 20 minutes.Muddy ether layer filters, and filter collects 5.641g (30%) title compound.
2) preparation Boc-Asp (OcHex)-Phe-OBzl
789mg (2.5mmol) Boc-Asp (OcHex)-OH is dissolved in the anhydrous THF of 10ml, adds anhydrous tetrahydro furan (THF) solution that 10ml contains 338mg (2.5mmol) HOBt and 619mg (3mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
668mg (2.3mmol) HClPhe-OBzl and 232mg (2.3mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 6ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=20:1) show that HClPhe-OBzl disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.242g (98.1%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp114~115℃[α]
D=-22.0(C=0.2,CHCl
3);ESI-MS(m/e)553[M+H]
+。
3) preparation HClAsp (OcHex)-Phe-OBzl
TLC (developping agent CHCl will be stirred 1 hour under the mixture condition of ice bath of 1.381g (2.5mmol) Boc-Asp (OcHex)-Phe-OBzl and 15ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=20:1) show that Boc-Asp (OcHex)-Phe-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
4) preparation Boc-Gly-Asp (OcHex)-Phe-OBzl
438mg (2.5mmol) Boc-Gly-OH is dissolved in the anhydrous THF of 5ml, adds the anhydrous THF solution that 10ml contains 338mg (2.5mmol) HOBt and 619mg (3mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
1.124g (2.3mmol) HClAsp (OcHex)-Phe-OBzl and 232mg (2.3mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show that HClAsp (OcHex)-Phe-OBzl disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.275g (91%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp109~111℃,[α]
D=-32(C=0.2,CHCl
3)ESI-MS(m/e)632[M+Na]
+。
5) preparation HClGly-Asp (OcHex)-Phe-OBzl
TLC (developping agent CHCl will be stirred 1 hour under the mixture condition of ice bath of 1.524g (2.5mmol) Boc-Gly-Asp (OcHex)-Phe-OBzl and 15ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show that Boc-Gly-Asp (OcHex)-Phe-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
6) preparation Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe-OBzl
1.071g (2.5mmol) Boc Arg (Tos)-OH is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution that 10ml contains 338mg (2.5mmol) HOBt and 619mg (3.0mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
1.256g (2.3mmol) HClGly-Asp (OcHex)-Phe-OBzl and 232mg (2.3mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show that HClGly-Asp (OcHex)-Phe-OBzl disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.925g (91%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp98.3~99.2℃,[α]
D=-46(C=0.4,CHCl
3)ESI-MS(m/e)943[M+Na]
+。
The preparation of embodiment 13Boc-Arg (Tos)-Gly-Asp (OcHex)-Val-OBzl
1) preparation HClVal-OBzl
The suspension of 5.855g (50mmol) H-Val-OH, 55ml phenylcarbinol and 11.5g polyphosphoric acid is stirred 8h in 90-95 ℃, make to become clarification.Reaction mixture is poured in 300ml (5%) the sulfuric acid liquid, used extracted with diethyl ether 5 times, divide water-yielding stratum.Ether layer is with 2% salt acid elution 3 times, the water NaHCO of merging
3Solid is regulated pH9.Water extracted with diethyl ether 3 times (100ml * 3), the anhydrous MgSO of the ether layer of merging
4Dry.Filter, in filtrate, led to hydrogenchloride 1 gas about 20 minutes.Muddy ether layer is overanxious, and filter collects 3.034g (25%) title compound.
2) preparation Boc-Asp (OcHex)-Val-OBzl
789mg (2.5mmol) Boc-Asp (OcHex)-OH is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution that 10ml contains 338mg (2.5mmol) HOBt and 619mg (3.0mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
558mg (2.3mmol) HClVal-OBzl and 232mg (2.3mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=20:1) show that HClVal-OBzl disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.11g (96%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is oily compound.Mp107~108℃;[α]
D=-36.0(C=0.2,CHCl
3);ESI-MS(m/e)505[M+H]
+。
3) preparation HClAsp (OcHex)-Val-OBzl
TLC (developping agent CHCl will be stirred 1 hour under the mixture condition of ice bath of 1.261g (2.5mmol) Boc-Asp (OcHex)-Val-OBzl and 10ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=20:1) show that Boc-Asp (OcHex)-Val-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
4) preparation Boc-Gly-Asp (OcHex)-Val-OBzl
438mg (2.5mmol) Boc-Gly-OH is dissolved in the anhydrous THF of 6ml, adds the anhydrous THF solution that 10ml contains 338mg (2.5mmol) HOBt and 619mg (3.0mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
1.014g (2.3mmol) HClAsp (OBzl)-Val-OBzl and 232mg (2.3mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show that HClAsp (OBzl)-Val-OBzl disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.242g (95%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp109~110℃;[α]
D=-19.0(C=0.2,CHCl
3);ESI-MS(m/e)584[M+Na]
+。
5) preparation HClGly-Asp (OcHex)-Val-OBzl
TLC (developping agent CHCl will be stirred 1 hour under the mixture condition of ice bath of 1.404g (2.5mmol) Boc-Gly-Asp (OcHex)-Val-OBzl and 10ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show that Boc-Gly-Asp (OcHex)-Val-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
6) preparation Boc-Arg (Tos)-Gly-Asp (OcHex)-Val-OBzl
1.071g (2.5mmol) Boc-Arg (Tos)-OH is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution that 10ml contains 338mg (2.5mmol) HOBt and 619mg (3.0mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
1.145g (2.3mmol) HClGly-Asp (OcHex)-Val-OBzl and 232mg (2.3mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show that HClGly-Asp (OcHex)-Val-OBzl disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.725g (86%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp97~98℃;[α]
D=-24.0(C=0.4,CHCl
3);ESI-MS(m/e)896[M+H+Na]
+。
Embodiment 14 preparation 3S-2-(Arg-Gly-Asp-Ser)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid
1) preparation Boc-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl)-OH
Earlier 2.376g (2.5mmol) Boc-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl)-OBzl is dissolved in the mixed solution of 20ml methyl alcohol, add 300mgPd/C (5%) again, the air in the reaction flask is discharged in decompression, feed hydrogen exchange, after replacing 5 times repeatedly, logical hydrogen stirring at room 3 hours, TLC (developping agent CHCl
3: MeOH=10:1+3 drips Glacial acetic acid) show that Boc-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl)-OBzl disappears.Stopped reaction, filtering Pd/C, filtrate decompression concentrates to remove and desolvates, and gets 2.108g (98%) title compound, is colorless solid.Mp108.1~109.0℃;[α]
D=-1.8685(C=0.2,CHCl
3)ESI-MS(m/e)861[M+H]
+。
2) preparation 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate
2.151g (2.5mmol) Boc-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl)-OH is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution that 10ml contains 338mg (2.5mmol) HOBt and 619mg (3.0mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
612mg (2.0mmol) 3S-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate is dissolved in the anhydrous THF of 10ml, miscible with top stand-by active ester solution then.The reaction mixture room temperature reaction that obtains 12 hours.TLC (developping agent CHCl
3: MeOH=10:1) show that 3S-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.88g (82%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp104.1~105.5℃;[α]
D=-48.0(C=1,CH
3OH);ESI-MS(m/e)1148[M+H]
+。
3) preparation 3S-2-(Arg-Gly-Asp-Ser)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid
With 200mg (0.26mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl)]-2,3,4, it is wetting that 9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate adds the 1ml methyl-phenoxide earlier, add 1ml trifluoracetic acid (TFA) dissolved compound under the condition of ice bath, in addition 3ml TFA and 1ml trifluoromethanesulfonic acid (TFMSA) are mixed the back adding, ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadex G-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 131mg (72%) title compound, is white solid.Mp109.1~110.8℃;[α]
D=-87.0(C=1,H
2O);ESI-MS(m/e)632[M+H]
+。
Embodiment 15 preparation 3S-2-(Arg-Gly-Asp-Ser)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Ala-Arg-Pro-Ala-Lys-OH (Ia)
1) preparation 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is earlier with 2.773g (2.41mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate is dissolved in 20ml methyl alcohol, add 300mgPd/C (5%) again, the air in the reaction flask is discharged in decompression, feeds hydrogen exchange, replace 5 times repeatedly after, logical hydrogen stirring at room 48 hours, TLC (developping agent CHCl
3: the MeOH=10:1+3d Glacial acetic acid) show the most of disappearance of raw material.Stopped reaction, filtering Pd/C, filtrate decompression concentrates to remove and desolvates, and getting 1.81g (71%) title compound is colorless solid.Mp101.9~102.2℃;[α]
D=-27.0(C=1,CH
3OH);ESI-MS(m/e)1058[M+H]
+。
4) preparation HClAla-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl
TLC (developping agent CHCl will be stirred 1 hour under the mixture condition of ice bath of 2.276g (2.5mmol) Boc-Ala-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl and 30ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show that Boc-Ala-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
2) preparation 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Ala-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl
With 300mg (0.31mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the anhydrous THF of 5ml, adds the anhydrous THF solution of 3ml42mg (0.31mmol) HOBt and 64mg (0.31mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
263g (0.31mmol) HClAla-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl and 31mg (0.31mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid disappears.Reaction mixture is evaporated to, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 247mg (45%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp147.1~148.2℃;[α]
D=-127.0(C=1,CH
3OH);ESI-MS(m/e)1761[M+H]
+。
3) preparation 3S-2-(Arg-Gly-Asp-Ser)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Ala-Arg-Pro-Ala-Lys-OH
With 200mg (0.26mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Ala-Arg (NO
2It is wetting that)-Pro-Ala-Lys (Z)-OBzl adds the 1ml methyl-phenoxide earlier, adds 1ml TFA dissolved compound under the condition of ice bath, in addition 3ml TFA and 1mlTFMSA mixing back added ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadexG-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 131mg (72%) title compound, is colorless solid.Mp204.9~206.1℃;[α]
D=-58.7(C=1,H
2O);ESI-MS(m/e)1156[M+H]
+。
Embodiment 16 preparation 3S-2-(Arg-Gly-Asp-Ser)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Gly-Arg-Pro-Ala-Lys-OH (Ib)
1) preparation HClGly-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl
TLC (developping agent CHCl will be stirred 1 hour under the mixture condition of ice bath of 2.241g (2.5mmol) Boc-Gly-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl and 30ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show that Boc-Gly-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
2) preparation 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Gly-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
With 350mg (0.36mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the anhydrous THF of 5ml, adds the anhydrous THF solution that 3ml contains 48mg (0.36mmol) HOBt and 74mg (0.36mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
300mg (0.36mmol) HClGly-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl and 36mg (0.36mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show that HClGly-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl disappears.Reaction mixture is evaporated to dried, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 322mg (51%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp175.8~177.6℃;[α]
D=-134.0(C=1,CH
3OH);ESI-MS(m/e)1747[M+H]
+。
3) preparation 3S-2-(Arg-Gly-Asp-Ser)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Gly-Arg-Pro-Ala-Lys-OH
With 200mg (0.26mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Gly-Arg (NO
2It is wetting that)-Pro-Ala-Lys (Z)-OBzl adds the 1ml methyl-phenoxide earlier, adds 1ml TFA dissolved compound under the condition of ice bath, in addition 3ml TFA and 1ml TFMSA mixing back added ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadex G-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 124mg (64%) title compound, is colorless solid.Mp204.9~206.1℃;[α]
D=-59.3(C=1,H
2O);ESI-MS(m/e)1142[M+H]
+。
Embodiment 17 preparation 3S-2-(Arg-Gly-Asp-Ser)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Gln-Arg-Pro-Ala-Lys-OH (Ic)
1) preparation HClGln-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl
TLC (developping agent CHCl will be stirred 1 hour under the mixture condition of ice bath of 2.418g (2.5mmol) Boc-Gln-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl and 30ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show that Boc-Gln-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
2) preparation 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
With 350mg (0.36mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the anhydrous THF of 5ml, adds the anhydrous THF solution that 3ml contains 48mg (0.36mmol) HOBt and 74mg (0.36mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
312mg (0.36mmol) HClGln-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl and 36mg (0.36mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show that HClGln-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl disappears.Reaction mixture is evaporated to dried, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 212mg (51%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp185.8~187.6℃;[α]
D=-52.0(C=1.0,CH
3OH);ESI-MS(m/e)1818[M+H]
+。
3) preparation 3S-2-(Arg-Gly-Asp-Ser)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Gln-Arg-Pro-Ala-Lys-OH
With 250mg (0.14mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Gln-Arg (NO
2It is wetting that)-Pro-Ala-Lys (Z)-OBzl adds the 1ml methyl-phenoxide earlier, adds 1ml TFA dissolved compound under the condition of ice bath, in addition 3ml TFA and 1ml TFMSA mixing back added ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadex G-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 171mg (62%) title compound, is colorless solid.Mp224.9~226.1℃;[α]
D=-157.7(C=1,H
2O);ESI-MS(m/e)1213[M+H]
+。
Embodiment 18 preparation 3S-2-(Arg-Gly-Asp-Ser)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Pro-Ala-Lys-OH (Id)
1) preparation HClArg (NO2)-Pro-Ala-Lys (Z)-OBzl
TLC (developping agent CHCl will be stirred 1 hour under the mixture condition of ice bath of 2.098g (2.5mmol) Boc-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl and 30ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show that Boc-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
2) preparation 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
With 350mg (0.36mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the anhydrous THF of 5ml, adds the anhydrous THF solution that 3ml contains 48mg (0.36mmol) HOBt and 74mg (0.36mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
179mg (0.36mmol) HClArg (NO2)-Pro-Ala-Lys (Z)-OBzl and 36mg (0.36mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show that HClArg (NO2)-Pro-Ala-Lys (Z)-OBzl disappears.Reaction mixture is evaporated to dried, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 127mg (61%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp205.8~207.6℃;[α]
D=-76.0(C=1,CH
3OH);ESI-MS(m/e)1690[M+H]
+。
3) preparation 3S-2-(Arg-Gly-Asp-Ser)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Pro-Ala-Lys-OH
With 250mg (0.16mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2It is wetting that)-Pro-Ala-Lys (Z)-OBzl adds the 1ml methyl-phenoxide earlier, adds 1ml TFA dissolved compound under the condition of ice bath, in addition 3ml TFA and 1mlTFMSA mixing back added ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadexG-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 101mg (74%) title compound, is colorless solid.Mp211.9~212.1℃;[α]
D=-129.3(C=1,H
2O);ESI-MS(m/e)1085[M+H]
+。
Embodiment 19 preparation 3S-2-(Arg-Gly-Asp-Ser)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Pro-Ala-Lys-OH (Ie)
1) preparation HClPro-Ala-Lys (Z)-OBzl
To stir 1 hour under the mixture condition of ice bath of 1.595g (2.5mmol) Boc-Pro-Ala-Lys (Z)-OBzl and 20ml hydrogenchloride-acetic acid ethyl fluid (4N), TLC (developping agent CHCl3:MeOH=10:1) shows that Boc-Pro-Ala-Lys (Z)-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
2) preparation 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Pro-Ala-Lys (Z)-OBzl
With 350mg (0.36mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the anhydrous THF of 5ml, adds the anhydrous THF solution that 3ml contains 48mg (0.36mmol) HOBt and 74mg (0.36mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
238mg (0.36mmol) HClPro-Ala-Lys (Z)-OBzl and 36mg (0.36mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show that HClPro-Ala-Lys (Z)-OBzl disappears.Reaction mixture is evaporated to dried, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 152mg (59%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp185.8~187.6℃;[α]
D=-45.0(C=1,CH3OH);ESI-MS(m/e)1488[M+H]
+。
3) preparation 3S-2-(Arg-Gly-Asp-Ser)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Pro-Ala-Lys-OH
With 250mg (0.17mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl)]-2; 3; 4; it is wetting that 9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Pro-Ala-Lys (Z)-OBzl adds the 1ml methyl-phenoxide earlier; add 1ml TFA dissolved compound under the condition of ice bath; in addition 3mlTFA and 1ml TFMSA are mixed the back adding, ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadex G-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 144mg (78%) title compound, is colorless solid.Mp224.9~226.1℃;[α]
D=-63.7(C=1,H
2O);ESI-MS(m/e)929[M+H]
+。
Embodiment 20 preparation 3S-2-[Arg-Gly-Asp-Val]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid
1) preparation Boc-Arg (Tos)-Gly-Asp (OcHex)-Val-OH
Earlier 2.180g (2.5mmol) Boc-Arg (Tos)-Gly-Asp (OcHex)-Val-OBzl is dissolved in 10ml methyl alcohol, adds 300mgPd/C (5%) again, the air in the reaction flask is discharged in decompression, feed hydrogen exchange, after replacing 5 times repeatedly, logical hydrogen stirring at room 4 hours, TLC (developping agent CHCl
3: MeOH=10:1+3 drips Glacial acetic acid) show that Boc-Arg (Tos)-Gly-Asp (OcHex)-Val-OBzl disappears.Stopped reaction, filtering Pd/C, filtrate decompression concentrates to remove and desolvates, and gets 1.916g (98%) title compound, is white solid.
2) preparation 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Val]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate
1.955g (2.5mmol) Boc-Arg (Tos)-Gly-Asp (OcHex)-Val-OH is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution that 10ml contains 338mg (2.5mmol) HOBt and 619mg (3.0mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
612mg (2.0mmol) 3S-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate is dissolved in the anhydrous THF of 10ml, miscible with top stand-by active ester solution then.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl3:MeOH=10:1) shows that 3S-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.69g (79%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is white solid.Mp102.5~103.1℃;[α]
D=-111(C=1,CH
3OH);ESI-MS(m/e)1070[M+H]
+。
3) preparation 3S-2-[Arg-Gly-Asp-Val]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid
With 300mg (0.28mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Val]-2,3,4, it is wetting that 9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate adds the 1ml methyl-phenoxide earlier, add the 1mlTFA dissolved compound under the condition of ice bath, in addition 3ml TFA and 1ml TFMSA are mixed the back adding, ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadex G-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 133mg (74%) title compound, is white solid.Mp134.9~136.1℃;[α]
D=-78(C=1,H
2O);ESI-MS(m/e)644[M+H]
+。
Embodiment 21 preparation 3S-2-(Arg-Gly-Asp-Val)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Ala-Arg-Pro-Ala-Lys-OH (If)
1) preparation 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Val]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid
Earlier with 2.577g (2.4mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Val]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate is dissolved in 10ml methyl alcohol, adds 300mgPd/C (5%) again, and the air in the reaction flask is discharged in decompression, feed hydrogen exchange, after replacing 5 times repeatedly, logical hydrogen stirring at room 4 hours, TLC (developping agent CHCl
3: MeOH=10:1+3 drips Glacial acetic acid) show that raw material partly disappears.Stopped reaction, filtering Pd/C, filtrate decompression concentrates to remove and desolvates, and gets 2.02g (86%) title compound, is colorless solid.Mp157.9~160.7℃;[α]
D=-46(C=1,CH
3OH);ESI-MS(m/e)981[M+H]
+。
2) preparation HClAla-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl
TLC (developping agent CHCl will be stirred 1 hour under the mixture condition of ice bath of 2.276g (2.5mmol) Boc-Ala-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl and 30ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show that Boc-Ala-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
3) preparation 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Val]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
With 1.1g (1.12mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Val]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution that 10ml contains 152mg (1.12mmol) HOBt and 232mg (1.12mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
890mg (1.07mmol) HClAla-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl and 107mg (1.07mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show that HClAla-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl disappears.Reaction mixture is evaporated to dried, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 1.52g (78%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is white solid.Mp139.3~140.5℃;[α]
D=-166(C=1,CH
3OH);ESI-MS(m/e)1773[M+H]
+。
4) preparation 3S-2-(Arg-Gly-Asp-Val)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Ala-Arg-Pro-Ala-Lys-OH
With 300mg (0.28mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Val]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Ala-Arg (NO
2It is wetting that)-Pro-Ala-Lys (Z)-OBzl adds the 1ml methyl-phenoxide earlier, adds 1ml TFA dissolved compound under the condition of ice bath, in addition 3ml TFA and 1mlTFMSA mixing back added ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadexG-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 133mg (74%) title compound, is colorless solid.Mp229.3~231.1℃;[α]
D=-52.7(C=1,H
2O);ESI-MS(m/e)1167[M+H]
+。
Embodiment 22 preparation 3S-2-(Arg-Gly-Asp-Val)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Gly-Arg-Pro-Ala-Lys-OH (Ig)
1) preparation HClGly-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl
TLC (developping agent CHCl will be stirred 1 hour under the mixture condition of ice bath of 958mg (1.07mmol) Boc-Gly-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl and 30ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show that Boc-Gly-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
2) preparation 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Val]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Gly-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl
With 1.1g (1.12mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Val]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution that 10ml contains 152mg (1.12mmol) HOBt and 232mg (1.12mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
890mg (1.07mmol) HClGly-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl and 107mg (1.07mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show that HClGly-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl disappears.Reaction mixture is evaporated to dried, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 1.52g (78%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp123.5~125.1℃;[α]
D=-178.3(C=1,CH
3OH);ESI-MS(m/e)1758[M+H]
+。
3) preparation 3S-2-(Arg-Gly-Asp-Val)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Gly-Arg-Pro-Ala-Lys-OH
With 250mg (0.14mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Val]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Gly-Arg (NO
2It is wetting that)-Pro-Ala-Lys (Z)-OBzl adds the 1ml methyl-phenoxide earlier, adds 1ml TFA dissolved compound under the condition of ice bath, in addition 3ml TFA and 1mlTFMSA mixing back added ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadexG-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 102mg (71%) title compound, is colorless solid.Mp175.8~177.3℃;[α]
D=-118(C=1,H
2O);ESI-MS(m/e)1154[M+H]
+。
Embodiment 23 preparation 3S-2-(Arg-Gly-Asp-Val)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Gln-Arg-Pro-Ala-Lys-OH (Ih)
1) preparation HClGln-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl
To stir 1 hour under the mixture condition of ice bath of 921mg (0.95mmol) Boc-Gln-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl and 15ml hydrogenchloride-acetic acid ethyl fluid (4N), TLC (developping agent CHCl3:MeOH=10:1) shows that Boc-Gln-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
2) preparation 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Val]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
With 980mg (1.0mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Val]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution that 10ml contains 136mg (1.0mmol) HOBt and 207mg (1.0mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
861mg (0.95mmol) HClGln-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl and 95mg (0.95mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show that HClGln-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl disappears.Reaction mixture is evaporated to dried, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 1.16g (67%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is white solid.Mp129.2~130.9℃;[α]
D=-243.7(C=1,CH
3OH);ESI-MS(m/e)1829[M+H]
+。
3) preparation 3S-2-(Arg-Gly-Asp-Val)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Gln-Arg-Pro-Ala-Lys-OH
With 250mg (0.14mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Val]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Gln-Arg (NO
2It is wetting that)-Pro-Ala-Lys (Z)-OBzl adds the 1ml methyl-phenoxide earlier, adds 1ml TFA dissolved compound under the condition of ice bath, in addition 3ml TFA and 1mlTFMSA mixing back added ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadexG-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 102mg (71%) title compound, is colorless solid.Mp216.6~218.1℃;[α]
D=-126.3(C=1,H2O);ESI-MS(m/e)1224[M+H]
+。
Embodiment 24 preparation 3S-2-(Arg-Gly-Asp-Val)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Pro-Ala-Lys-OH (Ii)
1) preparation HClArg (NO2)-Pro-Ala-Lys (Z)-OBzl
TLC (developping agent CHCl will be stirred 1 hour under the mixture condition of ice bath of 2.098g (2.5mmol) Boc-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl and 30ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show that Boc-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
2) preparation 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Val]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
With 350mg (0.36mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Val]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the anhydrous THF of 8ml, adds the anhydrous THF solution that 5ml contains 48mg (0.36mmol) HOBt and 74mg (0.36mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
291mg (0.38mmol) HClArg (NO2)-Pro-Ala-Lys (Z)-OBzl and 38mg (0.38mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Val]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid disappears.Reaction mixture is evaporated to dried, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 343mg (56%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp162.3~163.0C;[α]
D=-112(C=1,CH
3OH);ESI-MS(m/e)1724[M+H+Na]
+。
3) preparation [3S-2-(Arg-Gly-Asp-Val)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-acyl group]-Arg-Pro-Ala-Lys-OH
With 250mg (0.14mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Val]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2It is wetting that)-Pro-Ala-Lys (Z)-OBzl adds the 1ml methyl-phenoxide earlier, adds 1ml TFA dissolved compound under the condition of ice bath, in addition 3ml TFA and 1ml TFMSA mixing back added ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadex G-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 102mg (71%) title compound, is white solid.Mp184.9~186.1℃;[α]
D=-28.3(C=1,H
2O);ESI-MS(m/e)1097[M+H]
+。
Embodiment 25 preparation 3S-2-(Arg-Gly-Asp-Val)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Pro-Ala-Lys-OH (Ij)
1) preparation HClPro-Ala-Lys (Z)-OBzl
TLC (developping agent CHCl will be stirred 1 hour under the mixture condition of ice bath of 1.595g (2.5mmol) Boc-Pro-Ala-Lys (Z)-OBzl and 20ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show that Boc-Pro-Ala-Lys (Z)-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
2) preparation 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Val]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Pro-Ala-Lys (Z)-OBzl
With 350mg (0.36mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Val]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the anhydrous THF of 8ml, adds the anhydrous THF solution that 5ml contains 48mg (0.36mmol) HOBt and 74mg (0.36mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
216mg (0.38mmol) HClPro-Ala-Lys (Z)-OBzl and 38mg (0.38mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Val]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid disappears.Reaction mixture is evaporated to dried, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 275mg (51%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp124.0~125.5C;[α]
D=-133(C=1,CH
3OH);ESI-MS(m/e)1500[M+H]
+。3) preparation [3S-2-(Arg-Gly-Asp-Val)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-acyl group]-Pro-Ala-Lys-OH
With 250mg (0.17mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Val]-2; 3; 4; 9-tetrahydrochysene-β-Ka Lin-3-acyl group }-adding 1ml methyl-phenoxide is wetting earlier for Pro-Ala-Lys (Z)-OBzl; add 1ml trifluoracetic acid (TFA) dissolved compound under the condition of ice bath; in addition 3ml trifluoracetic acid (TFA) and 1ml trifluoromethanesulfonic acid (TFMSA) are mixed the back adding, ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadex G-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 132mg (81%) title compound, is white solid.Mp177.9~179.1℃[α]
D-21.7(C=1,H
2O)ESI-MS(m/e)941[M+H]
+963[M+Na]
+964[M+H+Na]
+。
Embodiment 26 preparation 3S-2-(Arg-Gly-As-Phe)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid
1) preparation Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe-OH
Earlier 2.300g (2.5mmol) Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe-OBzl is dissolved in 10ml methyl alcohol, adds 300mgPd/C (5%) again, the air in the reaction flask is discharged in decompression, feed hydrogen exchange, after replacing 5 times repeatedly, logical hydrogen stirring at room 6 hours, TLC (developping agent CHCl
3: MeOH=10:1+3 drips Glacial acetic acid) show that Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe-OBzl disappears.Stopped reaction, filtering Pd/C, filtrate decompression concentrates to remove and desolvates, and gets 2.033g (98%) title compound, is colorless solid.Mp118.9~119.6℃;[α]
D=-15.0(C=1,CH
3OH);ESI-MS(m/e)831[M+H]
+。
2) preparation 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate
2.075g (2.5mmol) Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe-OH is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution that 10ml contains 338mg (2.5mmol) HOBt and 619mg (3.0mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
612mg (2.0mmol) 3S-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate is dissolved in the anhydrous THF of 10ml, miscible with top stand-by active ester solution then.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show that 3S-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.76g (79%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp105.2~106.9℃;[α]
D=-25.0(C=1.0,CH
3OH);ESI-MS(m/e)1118[M+H]
+。
3) preparation 3S-2-(Arg-Gly-As-Phe)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid
With 250mg (0.22mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe]-2,3,4, it is wetting that 9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate adds the 1ml methyl-phenoxide earlier, add the 1mlTFA dissolved compound under the condition of ice bath, in addition 3ml TFA and 1ml TFMSA are mixed the back adding, ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadex G-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 100mg (66%) title compound, is colorless solid.Mp154.9~156.3℃;[α]
D=-36.3(C=1.0,H
2O);ESI-MS(m/e)692[M+H]
+。
Embodiment 27 preparation 3S-2-(Arg-Gly-Asp-Phe)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Ala-Arg-Pro-Ala-Lys-OH (Ik)
1) preparation 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid
Earlier with 2.697g (2.41mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate is dissolved in 10ml methyl alcohol, adds 300mgPd/C (5%) again, and the air in the reaction flask is discharged in decompression, feed hydrogen exchange, after replacing 5 times repeatedly, logical hydrogen stirring at room 48 hours, TLC (developping agent CHCl
3: MeOH=10:1+3 drips Glacial acetic acid) show that raw material partly disappears.Stopped reaction, filtering Pd/C, filtrate decompression concentrates to remove and desolvates, and gets 1.83g (74%) title compound, is colorless solid.Mp128.5~129.8℃;[α]
D=-36.3(C=1.0,CH
3OH);ESI-MS(m/e)1028[M+H]
+。
2) preparation HClAla-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl
TLC (developping agent CHCl will be stirred 1 hour under the mixture condition of ice bath of 2.276g (2.5mmol) Boc-Ala-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl and 30ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show that Boc-Ala-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
3) preparation 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Ala-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
With 500mg (0.49mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the anhydrous THF of 8ml, adds the anhydrous THF solution that 8ml contains 66mg (0.49mmol) HOBt and 101mg (0.49mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
433mg (0.51mmol) HClAla-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl and 51mg (0.51mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid disappears.Reaction mixture is evaporated to dried, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 452mg (51%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is white solid.Mp104.1~105.6℃;[α]
D=-178.0(C=1.0,CH
3OH);ESI-MS(m/e)1821[M+H]
+。
4) preparation 3S-2-(Arg-Gly-Asp-Phe)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Ala-Arg-Pro-Ala-Lys-OH
With 250mg (0.16mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Ala-Arg (NO
2It is wetting that)-Pro-Ala-Lys (Z)-OBzl adds the 1ml methyl-phenoxide earlier, adds 1ml TFA dissolved compound under the condition of ice bath, in addition 3ml TFA and 1mlTFMSA mixing back added ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadexG-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 151mg (78%) title compound, is colorless solid.Mp120~123℃;[α]
D=-103(C=1.0,H
2O);ESI-MS(m/e)1214[M]
+。
Embodiment 28 preparation 3S-2-(Arg-Gly-Asp-Phe)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Gly-Arg-Pro-Ala-Lys-OH (Il)
1) preparation HClGly-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl
TLC (developping agent CHCl will be stirred 1 hour under the mixture condition of ice bath of 2.241g (2.5mmol) Boc-Gly-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl and 30ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show that Boc-Gly-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
2) preparation 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Gly-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl
With 500mg (0.49mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the anhydrous THF of 8ml, adds the anhydrous THF solution that 8ml contains 66mg (0.49mmol) HOBt and 101mg (0.49mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
424mg (0.51mmol) HClGly-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl and 51mg (0.51mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid disappears.Reaction mixture is evaporated to dried, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 369mg (46%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is white solid.Mp136~137℃;[α]
D=-60.4(C=1.0,CH
3OH);ESI-MS(m/e)1806[M+H]
+。
3) preparation 3S-2-(Arg-Gly-Asp-Phe)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Gly-Arg-Pro-Ala-Lys-OH
With 250mg (0.16mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Gly-Arg (NO
2It is wetting that)-Pro-Ala-Lys (Z)-OBzl adds the 1ml methyl-phenoxide earlier, adds 1ml TFA dissolved compound under the condition of ice bath, in addition 3ml TFA and 1mlTFMSA mixing back added ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadexG-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 102mg (62%) title compound, is colorless solid.Mp146.1~147.3℃;[α]
D=-116.3(C=1.0,H
2O);ESI-MS(m/e)1201[M+H]
+。
Embodiment 29 preparation 3S-2-(Arg-Gly-Asp-Phe)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Gln-Arg-Pro-Ala-Lys-OH (Im)
1) preparation HClGln-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl
TLC (developping agent CHCl will be stirred 1 hour under the mixture condition of ice bath of 2.418g (2.5mmol) Boc-Gln-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl and 30ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show that Boc-Gln-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
5) preparation 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Gln-Arg (NO
2)-Pro-Ala-Lys (Z)-OBzl
With 500mg (0.49mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the anhydrous THF of 8ml, adds the anhydrous THF solution that 8ml contains 66mg (0.49mmol) HOBt and 101mg (0.49mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
461mg (0.51mmol) HClGln-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl and 51mg (0.51mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid disappears.Reaction mixture is evaporated to dried, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 395mg (43%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is white solid.Mp104~106℃;[α]
D=-213.0(C=1.0,CH
3OH);ESI-MS(m/e)1876[M+H]
+。
3) preparation 3S-2-(Arg-Gly-Asp-Phe)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Gln-Arg-Pro-Ala-Lys-OH
With 250mg (0.16mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Gln-Arg (NO
2It is wetting that)-Pro-Ala-Lys (Z)-OBzl adds the 1ml methyl-phenoxide earlier, adds 1ml TFA dissolved compound under the condition of ice bath, in addition 3ml TFA and 1mlTFMSA mixing back added ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadexG-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 94mg (46%) title compound, is white solid.Mp117~118℃;[α]
D=-116.7(C=1.0,H
2O);ESI-MS(m/e)1272[M+H]
+。
Embodiment 30 preparation 3S-2-(Arg-Gly-Asp-Phe)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Pro-Ala-Lys-OH (In)
1) preparation HClArg (NO2)-Pro-Ala-Lys (Z)-OBzl
TLC (developping agent CHCl will be stirred 1 hour under the mixture condition of ice bath of 2.098g (2.5mmol) Boc-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl and 30ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show that Boc-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
5) preparation 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl
With 500mg (0.49mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the anhydrous THF of 8ml, adds the anhydrous THF solution that 8ml contains 66mg (0.49mmol) HOBt and 101mg (0.49mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
396mg (0.51mmol) HClArg (NO2)-Pro-Ala-Lys (Z)-OBzl and 51mg (0.51mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid disappears.Reaction mixture is evaporated to dried, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 522mg (61%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is white solid.Mp117.1~119.6℃;[α]
D=-137.1(C=1.0,CH
3OH);ESI-MS(m/e)1772[M+H+Na]
+。
3) preparation 3S-2-(Arg-Gly-Asp-Phe)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Pro-Ala-Lys-OH
With 300mg (0.17mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2It is wetting that)-Pro-Ala-Lys (Z)-OBzl adds the 1ml methyl-phenoxide earlier, adds 1ml TFA dissolved compound under the condition of ice bath, in addition 3ml TFA and 1ml TFMSA mixing back added ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadex G-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 178mg (87%) title compound, is white solid.Mp156~157℃;[α]
D=-105.0(C=1.0,H
2O);ESI-MS(m/e)1144[M+H]
+。
Embodiment 31 preparation 3S-2-(Arg-Gly-Asp-Phe)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Pro-Ala-Lys-OH (Io)
1) preparation HClPro-Ala-Lys (Z)-OBzl
TLC (developping agent CHCl will be stirred 1 hour under the mixture condition of ice bath of 1.595g (2.5mmol) Boc-Pro-Ala-Lys (Z)-OBzl and 20ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show that Boc-Pro-Ala-Lys (Z)-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
5) preparation 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Pro-Ala-Lys (Z)-OBzl
With 500mg (0.49mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the anhydrous THF of 8ml, adds the anhydrous THF solution that 8ml contains 66mg (0.49mmol) HOBt and 101mg (0.49mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
293mg (0.51mmol) HClPro-Ala-Lys (Z)-OBzl and 51mg (0.51mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid disappears.Reaction mixture is evaporated to dried, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 478mg (63%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp100.8~101.9C;[α]
D=-157.3(C=1.0,CH
3OH);ESI-MS(m/e)1548[M+H]
+。
6) preparation 3S-2-(Arg-Gly-Asp-Phe)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Pro-Ala-Lys-OH
With 300mg (0.18mmol) 3S-2-[Boc-Arg (Tos)-Gly-Asp (OcHex)-Phe]-2; 3; 4; it is wetting that 9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Pro-Ala-Lys (Z)-OBzl adds the 1ml methyl-phenoxide earlier; add 1ml TFA dissolved compound under the condition of ice bath; in addition 3ml TFA and 1ml TFMSA are mixed the back adding, ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadex G-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 175mg (84%) title compound, is colorless solid.Mp193.1~194.8℃;[α]
D=-112.0(C=1.0,H
2O);ESI-MS(m/e)988[M+H]
+。
Embodiment 32 preparation 3S-2-(Ala-Arg-Pro-Ala-Lys)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Ser-OH (IIa)
1) preparation Boc-Ala-ONP
5g (26.5mmol) Boc-Ala-OH and 3.86g (30mmol) 4-nitrophenol (HONP) is miscible with the anhydrous THF of 20ml earlier, under condition of ice bath, add the anhydrous THF solution of 20ml5.73g (30mmol) DCC then.Reaction was reacted 6 hours at ambient temperature.TLC (developping agent CHCl
3: MeOH=20:1) show that Boc-Ala-OH disappears.Filtration under diminished pressure, filtrate decompression is concentrated into dried, adds a large amount of sherwood oils and wears away repeatedly for several times, gets 8.1g (93%) title compound.Be the pale yellow powder solid.
2) preparation Boc-Ala-Arg-OH
2g (11.4mmol) HClArg-OH and 1g (11.4mmol) sodium bicarbonate solid is earlier miscible in 10ml water, in addition 5.2g (16.7mmol) Boc-Ala-ONP fully is dissolved in the 20ml dioxane, two solution are mixed the 10min that bleeds that reduces pressure, normal-temperature reaction 10 hours.TLC (developping agent propyl carbinol: H
2O:HAc=4:1:1) show that HClArg-OH disappears.Dioxane is revolved in decompression, and residual water solution adds the ethyl acetate collection to be washed four times, and aqueous layer adds the saturated sodium pyrosulfate aqueous solution and regulates pH=2, and cold wind dries up.Add the dehydrated alcohol collection and carry three times, merge ethanol liquid, the decompression rotation concentrates.Get 3.0g (75%) title compound.Be colorless solid.
3) preparation Boc-Pro-Ala-Lys (Z)-OH
1.595g (2.5mmol) Boc-Pro-Ala-Lys (Z)-OBzl is dissolved among the 7ml MeOH, and ice bath drips 20ml1N NaOH down, reaction 1h.TLC (developping agent CHCl
3: MeOH=10:1+3 drips Glacial acetic acid) show that Boc-Pro-Ala-Lys (Z)-OBzl disappears.In reaction solution, add KHSO
4(s) and small amount of H
2O transfers pH=2, rotation steams MeOH, ethyl acetate extraction four times of the residual aqueous solution, combined ethyl acetate solution, and wash twice with saturated sodium-chloride water solution, the ethyl acetate layer of telling gets 1.233g (90%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colourless powder.
4) preparation 3S-2-[Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate
1.37g (2.5mmol) Boc-Pro-Ala-Lys (Z)-OH is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution that 10ml contains 338mg (2.5mmol) HOBt and 619mg (3.0mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
704mg (2.3mmol) 3S-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate and 232mg (2.3mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 10 hours.TLC (developping agent CHCl
3: MeOH=10:1) show that 3S-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.686g (72%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp85~87℃;[α]
D=-76.0(C=1.0,CH
3OH);ESI-MS(m/e)837[M+H]
+。
5) preparation 3S-2-[Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate hydrochloride
With 1.62g (1.94mmol) 3S-2-[Pro-Ala-Lys (Z)]-2,3,4, stirred 1 hour TLC (developping agent CHCl under the mixture condition of ice bath of 9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate and 15ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show 3S-2-[Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
6) preparation 3S-2-[Boc-Ala-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate
With the anhydrous N of 703mg (2.03mmol) Boc-Ala-Arg-OH10ml, dinethylformamide (DMF) adds the anhydrous THF solution of 10ml275mg (2.03mmol) HOBt and 420mg (2.03mmol) DCC inward under the ice bath.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
With 1.5g (1.94mmol) 3S-2-[Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate hydrochloride and 19.4mg (1.94mmol) N-methylmorpholine are miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show 3S-2-[Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate hydrochloride disappears.Reaction mixture cold wind dries up, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 1.46g (73%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp97~99℃;[α]
D=-118.0(C=1.0,CH
3OH);ESI-MS(m/e)1065[M+H]
+。
5) preparation 3S-2-[Boc-Ala-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid
With 1.3g (2.5mmol) 3S-2-[Boc-Ala-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate is dissolved among the 15ml MeOH, drips 30ml2N NaOH under the condition of ice bath, reaction 3h.TLC (developping agent CHCl
3: MeOH=10:1+3 drips Glacial acetic acid) demonstration 3S-2-[Boc-Ala-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate disappears.In reaction solution, add KHSO
4(s) and small amount of H
2O transfers pH=7, and rotation steams MeOH, and the residual aqueous solution adds KHSO
4(s) regulate pH=2, cold wind dries up, and with dehydrated alcohol extraction four times, merges ethanolic soln, and 37 ℃ of concentrating under reduced pressure get 900mg (67%) title compound, are pale yellow powder.Mp102~104℃;[α]
D=-109.0(C=1.0,CH
3OH);ESI-MS(m/e)974[M+H]
+。
8) preparation HClArg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl
With 165mg (0.19mmol) Boc-Arg (NO
2The mixture of)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl and 8ml hydrogenchloride-acetic acid ethyl fluid (4N) stirred 1 hour under condition of ice bath, TLC (developping agent CHCl
3: MeOH=10:1) show Boc-Arg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
9) preparation 3S-2-[Boc-Ala-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl
With 180mg (0.18mmol) 3S-2-[Boc-Ala-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the 5ml dry DMF, adds the anhydrous THF solution that 3ml contains 26mg (0.18mmol) HOBt and 40mg (0.18mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
With 152mg (0.19mmol) HClArg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl and 19.1mg (0.19mmol) N-methylmorpholine are miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 5ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show HClArg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl disappears.Reaction mixture cold wind dries up, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 167mg (57%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp119~120℃;[α]
D=-154.7(C=1.0,CH
3OH);ESI-MS(m/e)1705[M+H]
+。
10) preparation 3S-2-(Ala-Arg-Pro-Ala-Lys)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Ser-OH
With 200mg (0.12mmol) 3S-2-[Boc-Ala-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2It is wetting that)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl adds the 1ml methyl-phenoxide earlier, adds 1ml TFA dissolved compound under the condition of ice bath, in addition 3ml TFA and 1ml TFMSA mixing back added ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadex G-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 115mg (82%) title compound, is colorless solid.Mp168~169℃;[α]
D=-45.9(C=1.0,H
2O);ESI-MS(m/e)1156[M+H]
+。
Embodiment 33 preparation 3S-2-(Gly-Arg-Pro-Ala-Lys)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Ser-OH (IIb)
1) preparation Boc-Gly-ONP
With 5g (28.5mmol) Boc-Gly-OH and 4.17g (30mmol) HONP) miscible with the anhydrous THF of 30ml earlier, under condition of ice bath, add anhydrous (the THF solution that 30ml contains 6.18g (30mmol) DCC then.Reaction was reacted 6 hours at ambient temperature.TLC (developping agent CHCl
3: MeOH=20:1) show that Boc-Gly-OH disappears.Filtration under diminished pressure, filtrate decompression concentrates, and adds a large amount of sherwood oils and wears away repeatedly for several times, gets 8.0g (95%) title compound.Be the pale yellow powder solid.
2) preparation Boc-Gly-Arg-OH
3.3g (18.9mmol) HClArg-OH and 1.7g (20mmol) sodium bicarbonate solid is earlier miscible in 20ml water, in addition 8.0g (27.1mmol) Boc-Gly-ONP fully is dissolved in the 40ml dioxane, two solution are mixed the 10min that bleeds that reduces pressure, normal-temperature reaction 10 hours.TLC (developping agent propyl carbinol: H
2O:HAc=4:1:1) show that HClArg-OH disappears.Dioxane is revolved in decompression, and residual water solution adds the ethyl acetate collection to be washed four times, and aqueous layer adds saturated aqueous potassium hydrogen sulfate and regulates p=2, and cold wind dries up.Add the dehydrated alcohol collection and carry three times, merge ethanol liquid, the decompression rotation concentrates.Get 5.9g (92%) title compound.Be white solid.
3) preparation Boc-Pro-Ala-Lys (Z)-OH
1.595g (2.5mmol) Boc-Pro-Ala-Lys (Z)-OBzl is dissolved among the 7ml eOH, and ice bath drips 20ml1N aOH down, reaction 1h.TLC (developping agent CHCl
3: MeOH=10:1+3 drips acetic acid) show that Boc-Pro-Ala-Lys (Z)-OBzl disappears.In reaction solution, add KHSO
4(s) and small amount of H
2O transfers pH=2, rotation steams MeOH, ethyl acetate extraction four times of the residual aqueous solution, combined ethyl acetate solution, and wash twice with saturated sodium-chloride water solution, the ethyl acetate layer of telling gets 1.233g (90%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is no powder.Mp79~81℃;[α]
D=34.0(C=1.0,CH
3OH);ESI-MS(m/e)549[M+H]
+。
4) preparation 3S-2-[Boc-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate
1.37g (2.5mmol) Boc-Pro-Ala-Lys (Z)-OH is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution that 10ml contains 338mg (2.5mmol) HOBt and 619mg (3.0mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
704mg (2.3mmol) 3S-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate and 232mg (2.3mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 10 hours.TLC (developping agent CHCl
3: MeOH=10:1) show that H-Klss-oBzl disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.56g (73%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.
5) preparation 3S-2-[Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate hydrochloride
With 1.62g (1.94mmol) 3S-2-[Boc-Pro-Ala-Lys (Z)]-2,3,4, stirred 1 hour TLC (developping agent CHCl under the mixture condition of ice bath of 9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate and 15ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show that raw material disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
6) preparation 3S-2-[Boc-Gly-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate
With 675mg (2.04mmol) Boc-Gly-Arg-OH10ml dry DMF, add the anhydrous THF solution that 10ml contains 275mg (2.03mmol) HOBt and 420mg (2.03mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
With 1.5g (1.94mmol) 3S-2-[Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate hydrochloride and 19.4mg (1.94mmol) N-methylmorpholine are miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show 3S-2-[Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate hydrochloride disappears.Reaction mixture cold wind dries up, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 1.46g (73%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp97~99℃;[α]
D=-73.0(C=1.0,CH
3OH);ESI-MS(m/e)1051[M+H]
+。
7) preparation 3S-2-[Boc-Gly-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid
With 1.3g (2.5mmol) 3S-2-[Boc-Gly-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate is dissolved among the 15ml MeOH, drips 30ml2N NaOH under the condition of ice bath, reaction 3h.TLC (developping agent CHCl
3: MeOH=10:1+3 drips Glacial acetic acid) show that raw material disappears.In reaction solution, add KHSO
4(s) and small amount of H
2O transfers pH=7, and rotation steams MeOH, and the residual aqueous solution adds KHSO
4(s) regulate pH=2, cold wind dries up, and with dehydrated alcohol extraction four times, merges ethanolic soln, and 37 ℃ of concentrating under reduced pressure get 900mg (67%) title compound, are pale yellow powder.Mp95~97℃;[α]
D=-113.0(C=1.0,CH
3OH);ESI-MS(m/e)962[M+H]
+。
8) preparation HClArg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl
The mixture of 165mg (0.19mmol) Boc-Arg (NO2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl and 8ml hydrogenchloride-acetic acid ethyl fluid (4N) was stirred 1 hour TLC (developping agent CHCl under condition of ice bath
3: MeOH=10:1) show Boc-Arg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
9) preparation 3S-2-[Boc-Gly-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl
With 1.0g (1.04mmol) 3S-2-[Boc-Gly-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the 10ml dry DMF, adds the anhydrous THF solution that 5ml contains 144mg (1.04mmol) HOBt and 220mg (1.04mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
With 860mg (1.10mmol) HClArg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl and 110mg (1.11mmol) N-methylmorpholine are miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 15ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show 3S-2-[Boc-Gly-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid 1 disappears.Reaction mixture cold wind dries up, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 759mg (41%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp110~112℃;[α]
D=-158.7(C=1.0,CH
3OH);ESI-MS(m/e)1691[M+H]
+。
10) preparation 3S-2-(Gly-Arg-Pro-Ala-Lys)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Ser-OH
With 480mg (0.28mmol) 3S-2-[Boc-Gly-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2It is wetting that)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl adds the 1ml methyl-phenoxide earlier, adds 1ml TFA dissolved compound under the condition of ice bath, in addition 3ml TFA and 1ml TFMSA mixing back added ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadex G-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 263mg (80%) title compound, is colorless solid.Mp144~146℃;[α]
D=-55.7(C=1.0,H
2O);ESI-MS(m/e)1142[M+H]
+。
Embodiment 34 preparation 3S-2-(Gln-Arg-Pro-Ala-Lys)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Ser-OH (IIc)
1) preparation Boc-Gln-ONP
2.5g (10.2mmol) Boc-Gln-OH and 1.48g (10.6mmol) HONP is miscible with the anhydrous THF of 15ml earlier, under condition of ice bath, add the anhydrous THF solution that 10ml contains 2.2g (10.6mmol) DCC then.Reaction was reacted 6 hours at ambient temperature.TLC (developping agent CHCl
3: MeOH=20:1) show that Boc-Gln-OH disappears.Filtration under diminished pressure, filtrate decompression concentrates, and adds a large amount of sherwood oils and wears away repeatedly for several times, gets 3.54g (95%) title compound.Be the colourless powder solid.
2) preparation Boc-Gln-Arg-OH
710mg (4.08mmol) HClArg-OH and 480mg (4.10mmol) sodium bicarbonate solid is earlier miscible in 8ml water, in addition 2.0g (5.44mmol) Boc-Gln-ONP fully is dissolved in the 10ml dioxane, two solution are mixed the 10min that bleeds that reduces pressure, normal-temperature reaction 10 hours.TLC (developping agent propyl carbinol: H
2O:HAc=4:1:1) show that HClArg-OH disappears.Dioxane is revolved in decompression, and residual water solution adds the ethyl acetate collection to be washed four times, and aqueous layer adds saturated aqueous potassium hydrogen sulfate and regulates pH=2, and cold wind dries up.Add the dehydrated alcohol collection and carry three times, merge ethanol liquid, the decompression rotation concentrates.Get 1.62g (97%) title compound.Be colorless solid.
3) preparation Boc-Pro-Ala-Lys (Z)-OH
1.595g (2.5mmol) Boc-Pro-Ala-Lys (Z)-OBzl is dissolved among the 7ml MeOH, and ice bath drips 20ml1N NaOH down, reaction 1h.TLC (developping agent CHCl
3: MeOH=10:1+3 drips Glacial acetic acid) show that Boc-Pro-Ala-Lys (Z)-OBzl disappears.In reaction solution, add KHSO4 (s) and small amount of H
2O transfers pH=2, rotation steams MeOH, ethyl acetate extraction four times of the residual aqueous solution, combined ethyl acetate solution, and wash twice with saturated sodium-chloride water solution, the ethyl acetate layer of telling gets 1.233g (90%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colourless powder.Mp79~81℃;[α]
D=-46.0(C=1.0,CH
3OH);ESI-MS(m/e)549[M+H]
+。
4) preparation 3S-2-[Boc-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate
1.37g (2.5mmol) Boc-Pro-Ala-Lys (Z)-OH is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution that 10ml contains 338mg (2.5mmol) HOBt and 619mg (3.0mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
704mg (2.3mmol) 3S-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate and 232mg (2.3mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 10 hours.TLC (developping agent CHCl
3: MeOH=10:1) show that 3S-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.56g (83%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.
5) preparation 3S-2-[Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate hydrochloride
With 1.62g (1.94mmol) 3S-2-[Boc-Pro-Ala-Lys (Z)]-2,3,4, stirred 1 hour TLC (developping agent CHCl under the mixture condition of ice bath of 9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate and 15ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show that raw material 1 disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
6) preparation 3S-2-[Boc-Gln-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate
With 936mg (2.33mmol) Boc-Gln-Arg-OH10ml dry DMF, add the anhydrous THF solution that 10ml contains 314mg (2.33mmol) HOBt and 480mg (2.33mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
With 1.5g (1.94mmol) 3S-2-[Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate hydrochloride and 19.4mg (1.94mmol) N-methylmorpholine are miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show 3S-2-[Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate hydrochloride disappears.Reaction mixture cold wind dries up, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 1.46g (73%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp107~109℃;[α]
D=-214.0(C=1.0,CH
3OH);ESI-MS(m/e)1121[M+H]
+。
7) preparation 3S-2-[Boc-Gln-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid
With 1.3g (2.41mmol) 3S-2-[Boc-Gln-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate is dissolved among the 15ml MeOH, drips 30ml2N NaOH under the condition of ice bath, reaction 3h.TLC (developping agent CHCl
3: MeOH=10:1+3 drips Glacial acetic acid) show that raw material disappears.In reaction solution, add KHSO
4(s) and small amount of H 2O transfer pH=7, rotation steams MeOH, the residual aqueous solution adds KHSO
4(s) regulate pH=2, cold wind dries up, and with dehydrated alcohol extraction four times, merges ethanolic soln, and 37 ℃ of concentrating under reduced pressure get 852mg (59%) title compound, are pale yellow powder.Mp115~117℃;[α]
D=-311.7(C=1.0,CH
3OH);ESI-MS(m/e)1032[M+H]
+。
8) preparation HClArg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl
With 165mg (0.19mmol) Boc-Arg (NO
2The mixture of)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl and 8ml hydrogenchloride-acetic acid ethyl fluid (4N) stirred 1 hour under condition of ice bath, TLC (developping agent CHCl)
3: MeOH=10:1) show Boc-Arg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
9) preparation 3S-2-[Boc-Gln-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl
With 370mg (0.36mmol) 3S-2-[Boc-Gln-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the 10ml dry DMF, adds the anhydrous THF solution of 5ml50mg (0.36mmol) HOBt and 78mg (0.36mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
With 296mg (0.38mmol) HClArg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl and 3.8mg (0.38mmol) N-methylmorpholine are miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 15ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show 3S-2-[Boc-Gln-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid disappears.Reaction mixture cold wind dries up, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 272mg (43%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp140~142℃;[α]
D=-182.0(C=1.0,CH
3OH);ESI-MS(m/e)1761[M+H]
+。
10) preparation 3S-2-(Gln-Arg-Pro-Ala-Lys)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Ser-OH
With 250mg (0.14mmol) 3S-2-[Boc-Gln-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2It is wetting that)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl adds the 1ml methyl-phenoxide earlier, adds 1ml TFA dissolved compound under the condition of ice bath, in addition 3ml TFA and 1ml TFMSA mixing back added ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadex G-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 124mg (73%) title compound, is colorless solid.Mp212~214℃;[α]
D=-31.3(C=1.0,H
2O);ESI-MS(m/e)1213[M+H]
+。
Embodiment 35 preparation 3S-2-(Arg-Pro-Ala-Lys)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Ser-OH (IId)
1) preparation Boc-Arg (NO2)-Pro-Ala-Lys (Z)-OH
2.098g (2.5mmol) Boc-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl is dissolved among the 15mlMeOH, and ice bath drips 1NNaOH30ml down, reaction 1h.TLC (developping agent CHCl
3: MeOH=10:1+3 drips Glacial acetic acid) show that Boc-Arg (NO2)-Pro-Ala-Lys (Z)-OBzl disappears.In reaction solution, add KHSO4 (s) and small amount of H 2O and transfer PH=2, rotation steams MeOH, ethyl acetate extraction four times of the residual aqueous solution, combined ethyl acetate solution, and wash twice with saturated sodium-chloride water solution, the ethyl acetate layer of telling gets 1.686g (90%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colourless powder.Mp98.2~99.7℃;[α]
D=-42.0(C=1.0,CH
3OH);ESI-MS(m/e)750[M+H]
+。
2) preparation 3S-2-[Boc-Arg (NO2)-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate
1.3g (1.74mmol) Boc-Arg (NO2)-Pro-Ala-Lys (Z)-OH is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution that 10ml contains 239mg (1.74mmol) HOBt and 365mg (1.74mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
425mg (1.39mmol) 3S-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate is dissolved in the anhydrous THF of 10ml, miscible with top stand-by active ester solution then.The reaction mixture room temperature reaction that obtains 12 hours.TLC (developping agent CHCl
3: MeOH=10:1) show that 3S-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.11g (76%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp99.8~100.7℃;[α]
D=-382.0(C=1.0,CH
3OH);ESI-MS(m/e)1038[M+H]
+。
3) preparation 3S-2-[Boc-Arg (NO2)-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid
With 1.11g (2.5mmol) 3S-2-[Boc-Arg (NO2)-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate is dissolved among the 15ml MeOH, and ice bath drips 30ml2N NaOH down, reaction 3h.TLC (developping agent CHCl
3: MeOH=10:1+3 drips Glacial acetic acid) demonstration 3S-2-[Boc-Arg (NO2)-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate disappears.In reaction solution, add KHSO
4(s) and small amount of H 2O transfer pH=7, rotation steams MeOH, the residual aqueous solution adds KHSO4 (s) and regulates pH=2, with ethyl acetate extraction four times, combined ethyl acetate solution, and wash twice with saturated sodium-chloride water solution, the ethyl acetate layer of telling is with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, get 1.1g (69%) title compound, be colourless powder.Mp105.1~106.6℃;[α]
D=-311.0(C=1.0,CH
3OH);ESI-MS(m/e)948[M+H]
+。
4) preparation HClArg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl
With 854mg (1.01mmol) Boc-Arg (NO
2Stirred 1 hour TLC (developping agent CHCl under the mixture condition of ice bath of)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl and 10ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show Boc-Arg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
5) preparation 3S-2-[Boc-Arg (NO2)-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl
With 1g (1.01mmol) 3S-2-[Boc-Arg (NO2)-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the anhydrous THF of 6ml, adds the anhydrous THF solution that 6ml contains 143mg (1.01mmol) HOBt and 218mg (1.01mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
With 790mg (1.01mmol) HClArg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl and 10.2mg (1.01mmol) N-methylmorpholine are miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl3:MeOH=10:1) shows HClArg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl disappears.Reaction mixture is evaporated to dried, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 1.01g (61%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp108.3~19.1℃;[α]
D=-213.0(C=1.0,CH
3OH);ESI-MS(m/e)1701[M+H+Na]
+。
6) preparation 3S-2-(Arg-Pro-Ala-Lys)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Ser-OH
With 400mg (0.24mmol) 3S-2-[Boc-Arg (NO2)-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2It is wetting that)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl adds the 1ml methyl-phenoxide earlier, adds 1ml TFA dissolved compound under the condition of ice bath, in addition 3ml TFA and 1ml TFMSA mixing back added ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadex G-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 184mg (69%) title compound, is colorless solid.Mp152.7~153.9℃;[α]
D=-78.3(C=1.0,H
2O);ESI-MS(m/e)1085[M+H]
+。
Embodiment 36 preparation 3S-2-(Pro-Ala-Lys)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Ser-OH (IIe)
1) preparation Boc-Pro-Ala-Lys (Z)-OH
1.595g (2.5mmol) Boc-Pro-Ala-Lys (Z)-OBzl is dissolved among the 7mlMeOH, and ice bath drips 1NNaOH20ml down, reaction 1h.TLC (developping agent CHCl
3: MeOH=10:1+3 drips Glacial acetic acid) show that Boc-Pro-Ala-Lys (Z)-OBzl disappears.In reaction solution, add KHSO
4(s) and small amount of H
2O transfers pH=2, rotation steams MeOH, ethyl acetate extraction four times of the residual aqueous solution, combined ethyl acetate solution, and wash twice with saturated sodium-chloride water solution, the ethyl acetate layer of telling gets 1.233g (90%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colourless powder.
2) preparation 3S-2-[Boc-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate
1.37g (2.5mmol) Boc-Pro-Ala-Lys (Z)-OH is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution that 10ml contains 338mg (2.5mmol) HOBt and 619mg (3.0mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
704mg (2.3mmol) 3S-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate and 232mg (2.3mmol) N-methylmorpholine is miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 10 hours.TLC (developping agent CHCl
3: MeOH=10:1) show that 3S-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate disappears.Reaction mixture is evaporated to dried, residue acetic acid ethyl dissolution, filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer of telling gets 1.686g (72%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp93.7~95.3℃;[α]
D=-228(C=1,CH
3OH);ESI-MS(m/e)837[M+H]
+。
3) preparation 3S-2-[Boc-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid
With 1.8g (2.2mmol) 3S-2-[Boc-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate is dissolved among the 10ml MeOH, and ice bath drips 30ml2N NaOH down, reaction 2h.TLC (developping agent CHCl
3: MeOH=10:1+3 drips Glacial acetic acid) show that raw material disappears.In reaction solution, add KHSO
4(s) and small amount of H
2O transfers PH=7, and rotation steams MeOH, and the residual aqueous solution adds KHSO
4(s) regulate pH=2, use ethyl acetate extraction four times, combined ethyl acetate solution, and wash twice with saturated sodium-chloride water solution, the ethyl acetate layer of telling gets 1.1g (69%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colourless powder.Mp95.1~96.6℃;[α]
D=-222(C=1,CH
3OH);ESI-MS(m/e)837[M+H]
+。
4) preparation HClArg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl
With 360mg (0.42mmol) Boc-Arg (NO
2The mixture of)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl and 5ml hydrogenchloride-acetic acid ethyl fluid (4N) stirred 1 hour under condition of ice bath, TLC (developping agent CHCl
3: MeOH=10:1) show Boc-Arg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
6) preparation 3S-2-[Boc-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl
With 333mg (0.45mmol) 3S-2-[Boc-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the anhydrous THF of 4ml, adds the anhydrous THF solution that 3ml contains 60mg (0.45mmol) HOBt and 92mg (0.45mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
With 330mg (0.42mmol) HClArg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl and 43mg (0.42mmol) N-methylmorpholine are miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 4ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show HClArg (NO
2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl disappears.Reaction mixture is evaporated to dried, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 320mg (51%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp105.2~107.1℃;[α]
D=-333(C=1,CH
3OH);ESI-MS(m/e)1469[M+H]
+。
7) preparation 3S-2-(Pro-Ala-Lys)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Ser-OH
With 300mg (0.21mmol) 3S-2-[Boc-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-acyl group-Arg (NO
2It is wetting that)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl adds the 1ml methyl-phenoxide earlier, adds 1ml TFA dissolved compound under the condition of ice bath, in addition 3ml TFA and 1ml TFMSA mixing back added ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadex G-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 81mg (43%) title compound, is colorless solid.Mp134.9~135.8℃;[α]
D=-81(C=1,H
2O);ESI-MS(m/e)928[M+H]
+。
Embodiment 37 preparation 3S-2-(Ala-Arg-Pro-Ala-Lys)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Val-OH (IIf)
1) preparation HClArg (NO
2)-Gly-Asp (OBzl)-Val-OBzl
With 654mg (0.85mmol) Boc-Arg (NO
2Stirred 1 hour TLC (developping agent CHCl under the mixture condition of ice bath of)-Gly-Asp (OBzl)-Val-OBzl and 10ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show Boc-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
2) preparation 3S-2-[Boc-Ala-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl
With 869g (0.89mmol) 3S-2-[Boc-Ala-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the 10ml dry DMF, adds the anhydrous THF solution that 6ml contains 121mg (0.89mmol) HOBt and 184mg (0.89mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
With 600mg (0.85mmol) HClArg (NO
2)-Gly-Asp (OBzl)-Val-OBzl and 8.6mg (0.85mmol) N-methylmorpholine are miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show HClArg (NO
2)-Gly-Asp (OBzl)-Val-OBzl disappears.Reaction mixture cold wind dries up, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 728mg (52%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is white solid.Mp133~134℃;[α]
D=-235.7(C=1.0,CH
3OH)ESI-MS(m/e)1627[M+H]
+。
3) preparation 3S-2-(Ala-Arg-Pro-Ala-Lys)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Val-OH
With 200mg (0.12mmol) 3S-2-[Boc-Ala-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-acyl group-Arg (NO
2It is wetting that)-Gly-Asp (OBzl)-Val-OBzl adds the 1ml methyl-phenoxide earlier, adds 1ml TFA dissolved compound under the condition of ice bath, in addition 3ml TFA and 1ml TFMSA mixing back added ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadex G-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 104mg (77%) title compound, is white solid.Mp221~223℃;[α]
D=-17.7(C=1.0,H
2O);ESI-MS(m/e)1168[M+H]
+。
Embodiment 38 preparation 3S-2-(Gly-Arg-Pro-Ala-Lys)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Val-OH (IIg)
1) preparation HClArg (NO
2)-Gly-Asp (OBzl)-Val-OBzl
With 654mg (0.85mmol) Boc-Arg (NO
2Stirred 1 hour TLC (developping agent CHCl under the mixture condition of ice bath of)-Gly-Asp (OBzl)-Val-OBzl and 10ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show Boc-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
2) preparation 3S-2-[Boc-Gly-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl
With 400g (0.42mmol) 3S-2-[Boc-Gly-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the 8ml dry DMF, adds the anhydrous THF solution that 6ml contains 56mg (0.42mmol) HOBt and 91mg (0.42mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
With 310mg (0.44mmol) HClArg (NO
2)-Gly-Asp (OBzl)-Val-OBzl and 4.4mg (0.44mmol) N-methylmorpholine are miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show 3S-2-[Boc-Gly-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid disappears.Reaction mixture cold wind dries up, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 443mg (66%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp120~122℃;[α]
D=-167.7(C=1.0,CH
3OH);ESI-MS(m/e)1611[M+H]
+。
3) preparation 3S-2-(Gly-Arg-Pro-Ala-Lys)-2,3,4; 9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Val-OH is with 260mg (0.16mmol) 3S-2-[Boc-Gly-Arg-Pro-Ala-Lys (Z)]-2; 3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2It is wetting that)-Gly-Asp (OBzl)-Val-OBzl adds the 1ml methyl-phenoxide earlier, adds 1ml TFA dissolved compound under the condition of ice bath, in addition 3ml TFA and 1ml TFMSA mixing back added ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadex G-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 143mg (77%) title compound, is colorless solid.Mp170~172℃;[α]
D=-79.7(C=1.0,H
2O);ESI-MS(m/e)1154[M+H]
+。
Embodiment 39 preparation 3S-2-(Gln-Arg-Pro-Ala-Lys)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Val-OH (IIh)
1) preparation HClArg (NO
2)-Gly-Asp (OBzl)-Val-OBzl
With 654mg (0.85mmol) Boc-Arg (NO
2Stirred 1 hour TLC (developping agent CHCl under the mixture condition of ice bath of)-Gly-Asp (OBzl)-Val-OBzl and 8ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show Boc-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
2) preparation 3S-2-[Boc-Gln-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl
With 300g (0.29mmol) 3S-2-[Boc-Gln-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the 8ml dry DMF, adds the anhydrous THF solution that 6ml contains 41mg (0.29mmol) HOBt and 63mg (0.29mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
With 216mg (0.31mmol) HClArg (NO
2)-Gly-Asp (OBzl)-Val-OBzl and 3.1mg (0.31mmol) N-methylmorpholine are miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 8ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show 3S-2-[Boc-Gln-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid disappears.Reaction mixture cold wind dries up, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 298mg (61%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp110~112℃;[α]
D=-132.0(C=1.0,CH
3OH);ESI-MS(m/e)1683[M+H]
+。
3) preparation 3S-2-(Gln-Arg-Pro-Ala-Lys)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Val-OH
With 130mg (0.08mmol) 3S-2-[Boc-Gln-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2It is wetting that)-Gly-Asp (OBzl)-Val-OBzl adds the 1ml methyl-phenoxide earlier, adds 1ml TFA dissolved compound under the condition of ice bath, in addition 3ml TFA and 1ml TFMSA mixing back added ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadex G-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 54mg (57%) title compound, is white solid.Mp127~129℃;[α]
D=-52.3(C=1.0,H
2O);ESI-MS(m/e)1154[M+H]
+。
Embodiment 40 preparation 3S-2-(Arg-Pro-Ala-Lys)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Val-OH (IIi)
1) preparation HClArg (NO
2)-Gly-Asp (OBzl)-Val-OBzl
With 770mg (0.22mmol) Boc-Arg (NO
2Stirred 1 hour TLC (developping agent CHCl under the mixture condition of ice bath of)-Gly-Asp (OBzl)-Val-OBzl and 8ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show Boc-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
2) preparation 3S-2-[Boc-Arg (NO2)-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl
With 200mg (0.21mmol) 3S-2-[Boc-Arg (NO2)-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the anhydrous THF of 5ml, adds the anhydrous THF solution that 3ml contains 29mg (0.21mmol) HOBt and 45mg (0.21mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
With 157mg (0.23mmol) HClArg (NO
2)-Gly-Asp (OBzl)-Val-OBzl and 23.2mg (0.23mmol) N-methylmorpholine are miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 5ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show HClArg (NO
2)-Gly-Asp (OBzl)-Val-OBzl disappears.The reaction mixture concentrating under reduced pressure as for, residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 213mg (63%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp110~112℃;[α]
D=-143.0(C=1.0,CH
3OH);ESI-MS(m/e)1622[M+H+Na]
+。
3) preparation 3S-2-(Arg-Pro-Ala-Lys)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Val-OH
With 200mg (0.15mmol) 3S-2-[Boc-Arg (NO2)-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2It is wetting that)-Gly-Asp (OBzl)-Val-OBzl adds the 1ml methyl-phenoxide earlier, adds 1ml TFA dissolved compound under the condition of ice bath, in addition 3ml TFA and 1mlTFMSA mixing back added ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadexG-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 107mg (78%) title compound, is white solid.Mp189~191℃;[α]
D=-64.0(C=1.0,H
2O);ESI-MS(m/e)1097[M+H]
+。
Embodiment 41 preparation 3S-2-(Pro-Ala-Lys)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Val-OH (IIj)
1) preparation HClArg (NO
2)-Gly-Asp (OBzl)-Val-OBzl
With 1.767g (2.5mmol) Boc-Arg (NO
2Stirred 1 hour TLC (developping agent CHCl under the mixture condition of ice bath of)-Gly-Asp (OBzl)-Val-OBzl and 20ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show Boc-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
2) preparation 3S-2-[Boc-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl
With 300mg (0.40mmol) 3S-2-[Boc-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the anhydrous THF of 5ml, adds the anhydrous THF solution of 4ml54mg (0.40mmol) HOBt and 83mg (0.40mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
With 271mg (0.38mmol) HClArg (NO
2)-Gly-Asp (OBzl)-Val-OBzl and 38mg (0.38mmol) N-methylmorpholine are miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 4ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show HClArg (NO
2)-Gly-Asp (OBzl)-Val-OBzl disappears.Reaction mixture is evaporated to dried, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 260mg (48%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp112.3~113.9℃;[α]
D=-183.0(C=1.0,CH
3OH);ESI-MS(m/e)1400[M+H]
+。
6) preparation 3S-2-(Pro-Ala-Lys)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Val-OH
With 250mg (0.18mmol) 3S-2-[Boc-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2It is wetting that)-Gly-Asp (OBzl)-Val-OBzl adds the 1ml methyl-phenoxide earlier, adds 1ml TFA dissolved compound under the condition of ice bath, in addition 3ml TFA and 1ml TFMSA mixing back added ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadex G-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 119mg (71%) title compound, is white solid.Mp238.2~239.7℃;[α]
D=-39(C=1.0,H
2O);ESI-MS(m/e)940[M+H]
+。
Embodiment 42 preparation 3S-2-(Ala-Arg-Pro-Ala-Lys)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Phe-OH (IIk)
1) preparation HClArg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl
With 1.887g (2.5mmol) Boc-Arg (NO
2Stirred 1 hour TLC (developping agent CHCl under the mixture condition of ice bath of)-Gly-Asp (OBzl)-Phe-OBzl and 20ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show Boc-Arg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
9) preparation 3S-2-[Boc-Ala-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl
With 1.08g (1.11mmol) 3S-2-[Boc-Ala-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the 10ml dry DMF, adds the anhydrous THF solution that 6ml contains 151mg (1.11mmol) HOBt and 229mg (1.11mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
With 800mg (1.06mmol) HClArg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl and 10.6mg (1.06mmol) N-methylmorpholine are miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show HClArg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl disappears.Reaction mixture cold wind dries up, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 833mg (49%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is white solid.Mp131~134℃;[α]
D=-376.7(C=1.0,CH
3OH)ESI-MS(m/e)1675[M+H]
+。
10) preparation 3S-2-(Ala-Arg-Pro-Ala-Lys)-2,3,4; 9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Phe-OH is with 350mg (0.21mmol) 3S-2-[Boc-Ala-Arg-Pro-Ala-Lys (Z)]-2; 3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2It is wetting that)-Gly-Asp (OBzl)-Phe-OBzl adds the 1ml methyl-phenoxide earlier, adds 1ml TFA dissolved compound under the condition of ice bath, in addition 3ml TFA and 1ml TFMSA mixing back added ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadex G-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 244mg (87%) title compound, is colorless solid.Mp179~180℃;[α]
D=-61.3(C=1.0,H
2O);ESI-MS(m/e)1216[M+H]
+。
Embodiment 43 preparation 3S-2-(Gly-Arg-Pro-Ala-Lys)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Phe-OH (IIl)
1) preparation HClArg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl
With 1.887g (2.5mmol) Boc-Arg (NO
2Stirred 1 hour TLC (developping agent CHCl under the mixture condition of ice bath of)-Gly-Asp (OBzl)-Phe-OBzl and 20ml hydrogenchloride-acetic acid ethyl fluid (4N)
3: MeOH=10:1) show Boc-Arg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
2) preparation 3S-2-[Boc-Gly-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl
With 1.0g (1.04mmol) 3S-2-[Boc-Gly-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the 10ml dry DMF, adds the anhydrous THF solution that 6ml contains 141mg (1.04mmol) HOBt and 214mg (1.04mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
With 750mg (0.99mmol) HClArg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl and 9.9mg (0.99mmol) N-methylmorpholine are miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show HClArg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl disappears.Reaction mixture cold wind dries up, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 624mg (39%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is white solid.Mp140~141℃;[α]
D=-224.3(C=1.0,CH
3OH)ESI-MS(m/e)1661[M+H]
+。
3) preparation 3S-2-(Gly-Arg-Pro-Ala-Lys)-2,3,4; 9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Phe-OH is with 400mg (0.21mmol) 3S-2-[Boc-Gly-Arg-Pro-Ala-Lys (Z)]-2; 3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2It is wetting that)-Gly-Asp (OBzl)-Phe-OBzl adds the 1ml methyl-phenoxide earlier, adds 1ml TFA dissolved compound under the condition of ice bath, in addition 3ml TFA and 1ml TFMSA mixing back added ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadex G-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 246mg (73%) title compound, is white solid.Mp190~192℃;[α]
D=-23.3(C=1.0,H
2O);ESI-MS(m/e)1202[M+H]
+
Embodiment 44 preparation 3S-2-(Gln-Arg-Pro-Ala-Lys)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Phe-OH (IIm)
1) preparation HClArg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl
With 288mg (0.35mmol) Boc-Arg (NO
2Stirred 1 hour under the mixture condition of ice bath of)-Gly-Asp (OBzl)-Phe-OBzl and 8ml hydrogenchloride-acetic acid ethyl fluid (4N), TLC (developping agent CHCl3:MeOH=10:1) shows Boc-Arg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
2) preparation 3S-2-[Boc-Gln-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl
With 370mg (0.36mmol) 3S-2-[Boc-Gln-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the 8ml dry DMF, adds the anhydrous THF solution that 3ml contains 49mg (0.36mmol) HOBt and 74mg (0.36mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
With 266mg (0.35mmol) HClArg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl and 3.5mg (0.35mmol) N-methylmorpholine are miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 8ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show HClArg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl disappears.Reaction mixture cold wind dries up, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 447mg (74%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp102~105℃;[α]
D=-157.0(C=1.0,CH
3OH);ESI-MS(m/e)1732[M+H]
+。
3) preparation 3S-2-(Gln-Arg-Pro-Ala-Lys)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Phe-OH
With 400mg (0.20mmol) 3S-2-[Boc-Gln-Arg-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2It is wetting that)-Gly-Asp (OBzl)-Phe-OBzl adds the 1ml methyl-phenoxide earlier, adds 1ml TFA dissolved compound under the condition of ice bath, in addition 3ml TFA and 1ml TFMSA mixing back added ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadex G-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 143mg (46%) title compound, is colorless solid.Mp121~122℃;[α]
D=-51.0(C=1.0,H
2O);ESI-MS(m/e)1273[M+H]
+。
Embodiment 45 preparation 3S-2-(Arg-Pro-Ala-Lys)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Phe-OH (IIn)
1) preparation HClArg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl
With 818mg (0.22mmol) Boc-Arg (NO
2The mixture ice bath of)-Gly-Asp (OBzl)-Phe-OBzl and 8ml hydrogenchloride-acetic acid ethyl fluid (4N) stirred 1 hour, TLC (developping agent CHCl
3: MeOH=10:1) show Boc-Arg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
2) preparation 3S-2-[Boc-Arg (NO2)-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl
With 200mg (0.21mmol) 3S-2-[Boc-Arg (NO2)-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the anhydrous THF of 5ml, adds the anhydrous THF solution of 3ml29mg (0.21mmol) HOBt and 45mg (0.21mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
With 167mg (0.22mmol) HClArg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl and 22mg (0.22mmol) N-methylmorpholine are miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 8ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show HClArg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl disappears.Reaction mixture is evaporated to dried, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The chloroform layer of telling gets 295mg (84%) title compound with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, is colorless solid.Mp121.3~123.2℃;[α]
D=-173.0(C=1.0,CH
3OH);ESI-MS(m/e)1648[M+H]
+。
6) preparation 3S-2-(Arg-Pro-Ala-Lys)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Phe-OH
With 250mg (0.15mmol) 3S-2-[Boc-Arg (NO2)-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2It is wetting that)-Gly-Asp (OBzl)-Val-OBzl adds the 1ml methyl-phenoxide earlier, adds 1ml TFA dissolved compound under the condition of ice bath, in addition 3ml TFA and 1mlTFMSA mixing back added ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadexG-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 101mg (59%) title compound, is colorless solid.Mp169~170℃;[α]
D=-41.3(C=1.0,H
2O);ESI-MS(m/e)1145[M+H]
+。
Embodiment 46 preparation 3S-2-(Pro-Ala-Lys)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Phe-OH (IIo)
4) preparation HClArg (NO
2) GlyAsp (OBzl)-Phe-OBzl
With 910mg (1.1mmol) Boc-Arg (NO
2The mixture of)-Gly-Asp (OBzl)-Phe-OBzl and 10ml hydrogenchloride-acetic acid ethyl fluid (4N) stirred 1 hour under condition of ice bath, TLC (developping agent CHCl
3: MeOH=10:1) show Boc-Arg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl disappears.With water pump reaction solution is drained, added anhydrous diethyl ether, with water pump reaction solution is drained once more, repeat 5 times.Residue is washed with the anhydrous diethyl ether bubble, and plastic spatula is worn away, and anhydrous diethyl ether is inclined to 5 times repeatedly.The title compound that obtains is directly used in the next step.
5) preparation 3S-2-[Boc-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl
With 872mg (1.2mmol) 3S-2-[Boc-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is dissolved in the anhydrous THF of 10ml, adds the anhydrous THF solution that 10ml contains 158mg (1.2mmol) HOBt and 241mg (1.2mmol) DCC under the ice bath inward.The reaction mixture ice bath stirred 20 minutes, got corresponding active ester solution, and is stand-by.
With 840mg (1.1mmol) HClArg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl and 111mg (1.1mmol) N-methylmorpholine are miscible, miscible with top stand-by active ester solution then with the anhydrous THF of 10ml earlier.The reaction mixture room temperature reaction that obtains 24 hours.TLC (developping agent CHCl
3: MeOH=10:1) show HClArg (NO
2)-Gly-Asp (OBzl)-Phe-OBzl disappears.Reaction mixture is evaporated to dried, and residue dissolves with chloroform, the filtering insolubles.Filtrate is washed with 5% aqueous potassium hydrogen sulfate, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.(the eluent chloroform: methyl alcohol=20:1) get 780mg (48%) title compound is colorless solid to the chloroform layer of telling with anhydrous sodium sulfate drying, filtration, 37 ℃ of concentrating under reduced pressure of filtrate, column chromatography.Mp106.1~108.5℃;[α]
D=-243.0(C=1.0,CH
3OH);ESI-MS(m/e)1447[M+H]
+。
06) preparation 3S-2-(Pro-Ala-Lys)-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-acyl group]-Arg-Gly-Asp-Phe-OH
With 350mg (0.24mmol) 3S-2-[Boc-Pro-Ala-Lys (Z)]-2,3,4,9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg (NO
2It is wetting that)-Gly-Asp (OBzl)-Phe-OBzl adds the 1ml methyl-phenoxide earlier, adds 1ml TFA dissolved compound under the condition of ice bath, in addition 3ml TFA and 1ml TFMSA mixing back added ice bath reaction 1.5 hours.Disposable adding anhydrous diethyl ether 50ml promptly separates out a large amount of cotton-shaped solids.Leave standstill, inclining supernatant liquid, and repeatable operation three times is filtered, filter cake sephadex G-10 post desalination, and moving phase is 5% aqueous acetic acid, and triketohydrindene hydrate detects collects the aqueous solution, and lyophilize gets 123mg (51%) title compound, is colorless solid.Mp208.4~210.5℃;[α]
D=-44(C=1.0,H
2O);ESI-MS(m/e)989[M+H]
+。
Test example 1 The compounds of this invention thrombus dissolving activity is measured
1) laboratory animal
The SD male rat, body weight 180-220g (available from laboratory animal portion of Beijing Medical University, credit number is the moving word 01-3056 of doctor).
2) experiment material and apparatus
Polyethylene tube (two kinds of external diameter 1.6mm and 1.3mm), medical silicone tube (internal diameter 3.5mm), Glass tubing (long 15mm, internal diameter 2.5mm, external diameter 5.0mm), heparin (50IU/ml), urethanum (urethane).
3) preparation thrombus
The SD male rat is anaesthetized by 1200mg/kg dosage abdominal injection urethane solution.The anesthetized rat dorsal position is fixed, and separates right common carotid artery, in proximal part folder bulldog clamp, proximal part and distal end penetrate surgical thread respectively, the surgical thread of distal end are clamped with mosquito forceps in fur, in the distal end intubate, unclamp bulldog clamp, emit about 1ml arterial blood, be contained in the 1ml sub warhead.In the Glass tubing (the pipe end seals with plug) of vertical fixing, inject 0.1ml rat artery blood, insert the thrombus standing bolt of a stainless steel material in the past pipe rapidly.This thrombus fixedly spiral 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.Behind the blood coagulation 15min, open the plug of Glass tubing bottom,, from Glass tubing, take out fixedly spiral of the thrombus that wrapped up by thrombus carefully with the fixing fixing holder handle of spiral of thrombus of tweezers.Accurately weigh.
4) rat neck arteriovenous shut intubate
Rat neck arteriovenous shut intubate constitutes by 3 sections, and the stage casing is the medical silica-gel flexible pipe, long 60.0mm, internal diameter 3.5mm; Two ends are identical polyethylene tube, pipe range 100.0mm, internal diameter 1.0mm, one end of this pipe of external diameter 2.0mm pulls into point pipe (being used to insert rat carotid artery or vein), external diameter is about 1.0mm, the outer cover one segment length 7.0mm of the other end, external diameter are the equal silanization of inwall of 3 sections pipes of polyethylene tube (overstriking is used to insert in the medical silica-gel flexible pipe in stage casing) of 3.5mm.With the thrombus of thrombus parcel fixedly spiral put into the stage casing polyethylene rubber tube, the two ends of sebific duct are nested with two poly butt ends that add respectively.With syringe by sharp pipe end with filling with heparin-saline solution (50IU/kg) in the pipe, standby.
The left external jugular vein that separates rat, proximal part and distal end penetrate surgical thread respectively, 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, fix with No. 4 surgical thread, simultaneously away from the fixing holder handle of spiral of the interior thrombus in bypass tube stage casing (containing fixedly spiral of the thrombus of accurately weighing).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, clamped flexible pipe between syringe and the polyethylene tube with mosquito forceps.Proximal part in right common carotid artery stops blooding with bulldog clamp, right common carotid artery is being cut an angle carefully nearby from bulldog clamp.Extract syringe from the tip of polyethylene tube, the proximal part of artery angle is inserted in the tip of polyethylene tube, fix with No. 4 surgical thread.Form the closed circulation system of rat arteriovenous and bypass intubate.
5) administration
With scalp acupuncture with physiological saline (3ml/kg), the normal saline solution of urokinase (dosage is 20000IU/kg), respectively that the embodiment of the invention is prepared Ia, Ib, Ic, Id, Ie, If, Ig, Ih, Ii, Ij, Ik, Il, Im, In, Io, IIa, IIb, IIc, IId, IIe, IIf, IIg, IIh, IIi, IIj, IIk, IIl, IIm, IIn, the stage casing of the normal saline solution of IIo (dosage is 10nmol/kg) by bypass tube (containing fixedly spiral of the thrombus of accurately weighing), thrust away from the fixing nearly vein place of spiral of thrombus, open bulldog clamp, make blood flow flow to vein from artery by bypass duct, this is a rat arteriovenous shut Thrombolysis Model, slowly the liquid in the syringe is injected in the blood, make physiological saline (blank group), urokinase (positive controls) or Ia-o or II a-o (treatment group) are by blood circulation, and the sequential action of pressing vein-heart-artery is to thrombus.Timing during from start injection, behind the 1h from bypass duct the fixing spiral of removal of thromboses, accurately weigh.Ask fixedly of poor quality before and after the spiral administration of thrombus in every rat bypass duct, and thrombus quality difference of each group of statistics (X ± SD), and do the t check.
5) thrombus quality difference is listed table 1 in as a result.
The rat suppository loss of weight (X ± sD mg) that table 1 The compounds of this invention Ia-o and II a-o treatment cause
Compound | The thrombus loss of weight | Group | The thrombus loss of weight |
NS | 6.70±1.33 | UK | 24.50±1.62 b |
Ia | 15.65±2.0 a | IIa | 13.15±1.63 a |
Ib | 16.43±1.11 b | IIb | 13.16±1.24 a |
Ic | 19.09±1.21 b | IIc | 16.15±2.05 b |
Id | 15.03±1.08 b | IId | 13.71±1.45 a |
Ie | 18.12±0.74 b | IIe | 14.48±1.33 a |
If | 14.91±1.30 a | IIf | 19.10±2.18 b |
Ig | 13.17±1.82 a | IIg | 17.14±1.53 b |
Ih | 15.13±1.83 a | IIh | 13.21±1.92 a |
Ii | 17.05±1.49 b | IIi | 15.42±1.81 a |
Ij | 15.96±2.06 a | IIj | 15.00±2.08 a |
Ik | 16.66±1.57 a | IIk | 16.38±1.75 a |
Il | 14.15±1.19 a | IIl | 14.28±0.82 a |
Im | 15.88±1.31 a | IIm | 16.00±1.08 b |
In | 14.90±1.52 a | IIn | 16.08±1.55 b |
Io | 13.42±1.46 a | IIo | 14.43±1.69 a |
N=10; Physiological saline dosage: 3ml/kg; Urokinase dosage: 20000IU/kg; Ia-o dosage 10nmol/kg; IIa-o dosage 10 μ mol/kg.A) compare p<0.01 with NS; B) compare p<0.001 with NS.
Test example 2 is measured compound 3S-2-(Ala-Arg-Pro-Ala-Lys)-2,3,4 according to the method for test example 1, and 9-tetrahydrochysene-β-Ka Lin-3-formyl radical-Arg-Gly-Asp-Val-OH (IIf) is 1 * 10
-8Mol/kg, 1 * 10
-9Mol/kg and 1 * 10
-10Thrombus dissolving activity under three kinds of dosage of mol/kg the results are shown in Table 2.
Table 2 Compound I If dose-effect relationship data
Dosage | 1×10 -8mol/kg | 1×10 -9mol/kg | 1×10 -10mol/kg |
The thrombus loss of weight | 19.10±2.18 a | 13.47±1.01 b | 6.87±0.65 |
Annotate: n=10; A) with 1 * 10
-9Mol/kg dosage group is compared, p<0.01; B=and 1 * 10
-9Mol/kg dosage group is compared, p<0.01.
Claims (5)
2. method for preparing the described compound of Formula I of claim 1 comprises:
(1) prepares following three kinds of tetrapeptide: Arg-Gly-Asp-Ser-OH, Arg-Gly-Asp-Val-OH, Arg-Gly-Asp-Phe-OH respectively according to ordinary method; The N-end and the side chain of above-mentioned three kinds of tetrapeptides are protected with protecting group respectively;
(2) prepare 3S-tetrahydrochysene-β-Ka Lin-3-carboxylic acid according to ordinary method, benzylization prepares 3S-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate then;
(3) prepare following 5 kinds of small peptide: Gly-Arg-Pro-Ala-Lys-OH, Gln-Arg-Pro-Ala-Lys-OH, Ala-Arg-Pro-Ala-Lys-OH, Arg-Pro-Ala-Lys-OH, the Pro-Ala-Lys-OH of C-end and side chain protected respectively according to ordinary method;
(4) according to ordinary method respectively with on Arg-Gly-Asp-Ser-OH, the Arg-Gly-Asp-Val-OH of N-end and side chain protected or 2 nitrogen that Arg-Gly-Asp-Phe-OH is connected to 3S-tetrahydrochysene-β-Ka Lin-3-benzyl carboxylate, slough carbobenzoxy; Respectively Gly-Arg-Pro-Ala-Lys-OH, Gln-Arg-Pro-Ala-Lys-OH, Ala-Arg-Pro-Ala-Lys-OH, Arg-Pro-Ala-Lys-OH or the Pro-Ala-Lys-OH of C-end and side chain protected are connected on the 3-carboxyl of 3S-tetrahydrochysene-β-Ka Lin-3-carboxylic acid, slough all protecting groups, obtain compound of Formula I.
3. according to the described preparation method of claim 2, it is characterized in that: described 3S-tetrahydrochysene-β-Ka Lin-3-carboxylic acid is prepared by L-tryptophane and formaldehyde condensation.
4. pharmaceutical composition for the treatment of thrombus dissolving is made up of the described compound of Formula I of the claim 1 of significant quantity and pharmaceutically acceptable carrier or auxiliary material.
5. the described compound of Formula I of claim 1 is in the purposes of preparation in the thrombolytic agent.
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CN102796167B (en) * | 2011-05-26 | 2014-01-08 | 首都医科大学 | (S)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-formyl-L-prolyl-L-alanyl-L-amino acid, and preparation method and application thereof |
CN105884905A (en) | 2012-09-05 | 2016-08-24 | 上海晟顺生物科技有限公司 | Novel compound with thrombolytic, free radical scavenging and thrombus targeted functions, and preparation method and purposes thereof |
CN105218623A (en) * | 2014-06-10 | 2016-01-06 | 首都医科大学 | 1-(ethylamino acid benzyl ester)-β-carboline-3-benzyl carboxylate, nanostructure, preparation, active and application |
CN105153275A (en) * | 2014-06-10 | 2015-12-16 | 首都医科大学 | 1-(ethyl amino acid-OBzl)-beta-carboline-3-carboxylic acid benzyl ester, preparation, activity, nanostructure and application |
CN105061283B (en) * | 2015-08-09 | 2017-11-24 | 浙江大学 | The preparation method of amino-acid benzyl ester hydrochloride |
CN109265373A (en) * | 2018-11-03 | 2019-01-25 | 郑州安图生物工程股份有限公司 | A kind of amino acids substrate and its preparation method and application |
CN112898378B (en) * | 2019-12-02 | 2022-04-22 | 首都医科大学 | Dioxohexa-ring modified tetrahydrocarboline-3-formyl-The-HGE, preparation thereof, antithrombotic activity thereof and application thereof |
CN116804040B (en) * | 2023-05-26 | 2024-08-06 | 首都医科大学 | Carboline oral small molecule anti-inflammatory medicament and preparation method and application thereof |
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CN1373139A (en) * | 2002-01-28 | 2002-10-09 | 广州白云山制药股份有限公司广州白云山制药总厂 | Carboline carboxylic acid contaiing P6A associated sequence and synthesis and medical application thereof |
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