CN1978462A - Imidazoline modified amino acid, and its synthesizing method and use for polypeptide marking - Google Patents

Imidazoline modified amino acid, and its synthesizing method and use for polypeptide marking Download PDF

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CN1978462A
CN1978462A CN 200510105291 CN200510105291A CN1978462A CN 1978462 A CN1978462 A CN 1978462A CN 200510105291 CN200510105291 CN 200510105291 CN 200510105291 A CN200510105291 A CN 200510105291A CN 1978462 A CN1978462 A CN 1978462A
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dioxy
tetrahydroglyoxaline
tetramethyl
boc
asp
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CN1978462B (en
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彭师奇
赵明
张建伟
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Capital Medical University
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Abstract

This invention discloses a series of new amino acids modified by imidazoline and its synthesis method and application for polypeptide fixed-point marking. This invention prepares a series of new amino acids that contain 2-substitution-1,3-dioxy-imidazoline through linking 2-substitution-1,3-dioxy-imidazoline to alkaline side chain or alpha- amino of L-Lys or L-Arg, or linking to acidic side chain oralpha-oxatyl of L-Asp or L-Glu. The invented amino acids that are modified by imidazoline have following characteristics. 1) It has satisfactory chemical stability, can tolerance various kinds of reaction condition in process of polypeptide synthesis, can maintain nature of free radical or removing remove being not invariable, also can carry out fixed-point ESR marking like normal L- amino acid in the process of polypeptide synthesis according to random place of polypeptide or protein where need to be inserted; 2) It has satisfactory biocompatibility. Polypeptide that is obtained by amino acid fixed-point marking in this invention is compared with polypeptide obtained by N end marking in present technique, the physico-chemical property and biological activity of them is completely coincident.

Description

Imidazoline modified amino acid, its synthetic method and the application in polypeptide marker
Technical field
The amino acid that the present invention relates to modify relates in particular to imidazoline modified amino acid, and its synthetic method and the application of amino acid in polypeptide fixed point mark that should modify belong to chemical field.
Background technology
1970, nitronyl nitroxide (the 2-replacement-1 of having synthesized general formula 1 first, 3-dioxy base tetrahydroglyoxaline) (Edwin F.Ullman, Call L and Jeanne H.Osiecki, Stable freeradicals:New Imino, Amidino and Carbamoyl nitroxides, J.Org.Chem., 1970,25,3623-3631).In generation nineteen ninety, the effect of NO in living organism is familiar with gradually.Nitronyl Nitroxide begins to be subjected to extensive concern owing to can the specificity reaction take place and have paramagnetism with NO.
The structure of general formula 1.Nitronyl nitroxide (2-replaces-1,3-dioxy base tetrahydroglyoxaline).R=alkyl in the formula, aryl, heteroaryl etc.
1993, nitronyl nitroxide and NO react 2-replacement-1-oxygen base imidazolines product (the Joseph J that generates to have adopted the The Technique of Electron Paramagnetic Resonance detection by quantitative, Kalyanaraman B, HydeJS.Trapping of nitric oxide by nitronyl nitroxides:an electron spinresonance investigation. Biochem Biophys Res Commun 1993; 192:926-934).The ESR of Nitronyl nitroxide spectrum is split branch (Fig. 1) in typical 2: 2: 1 five.The ESR of the two generation-1-oxygen base tetrahydroglyoxalines of reaction product 2-composes to be and split branch (Fig. 2) in typical 5: 2 seven.
The distinct difference of ESR spectrum makes nitronyl nitroxide become the single-minded trapping agent of NO before and after the reactive behavior of Nitronyl nitroxide and NO and the reaction, and as the strong instrument of studying the NO function.Nitronyl nitroxide has play a part crucial for disclosing NO dual-use function as courier's factor and effector in biosystem really.For example, Maeda H etc. utilizes nitronylnitroxide to disclose mediation (the Maeda H of NO in the noumenal tumour blood penetration, NoguchiY, Sato K, Akaike T.Enhanced vascular permeability in solid tumoris mediated by nitric oxide and inhibited by both new nitric oxidescavenger and nitric oxide synthase inhibitor.Jpn J Cancer Res1994; 85:331-334), for treatment for cancer provides new thinking (Wartenberg M, Schallenberg M, Hescheler J, Sauer H.Reactive oxygenspecies-mediated regulation of eNOS and iNOS expression inmulticellular prostate tumor spheroids.Int J Cancer 2003; 104:274-282).It is microbic activity molecule (Maeda H such as scavenger cell kill bacteria that Maeda H etc. also utilize nitronyl nitroxide to disclose NO, Akaike T, Yoshida M, Sato K, Noguchi Y.A new nitric oxide scavenger, imidazolineoxyl N-oxidederivative, and its effects in pathophysiology and microbiology.Curr Top Microbiol Immunol 1995; 196:37-50).
The characteristic ESR spectrum that the free radical character of Nitronyl nitroxide self is brought, and the high sensitivity of ESR mensuration make such spectrum mark (ESR mark) become valuable.The ESR mark is owing to neither resemble and can cause radiocontamination the isotopic labeling, also do not resemble difficulty the fluorescent mark, thereby growing into one of most important labeling technique of biological study.
Nitronyl nitroxide makes itself have important use to be worth in medical science to the single-minded capture ability of NO.For example aryl class nitronyl nitroxide can effectively treat endotoxin shock, protect cardiovascular and renal function (Yoshida M, Akaike T, Wada Y, et al.Therapeutic effects of imidazolineoxyl N-oxide against endotoxinshock through its direct nitric oxide-scavenging activity.BiochemBiophys Res Commun 1994; 202:923-930).For example nitronyl nitroxide and the coupling of antithrombotic polypeptide again, the pseudo-peptide that generates has antithrombotic and NO removes dual-use function (JunlingLiu, Ming Zhao, Chao Wang, and Shiqi Peng, Synthesis andBioactivities of Nitronyl Nitroxide and RGD ContainingPseudopeptides, Bioorganic﹠amp; Medicinal Chemistry Letters, 2003,13,4065-4069).For example nitronyl nitroxide and the coupling of thrombus dissolving polypeptide again, the pseudo-peptide that generates has thrombus dissolving and NO removes dual-use function (Ming Zhao, Junling Liu, Chao Wang, LiliWang, Hu Liu, Shiqi Peng, Synthesis and Biological Activity ofNitronyl Nitroxide Containing Peptides, J Med.Chem.2005,48,4285-4292).
At present, still there is tangible limitation in the ESR mark of protein and polypeptide.The nitronyl nitroxide mark of bibliographical information all is a random labelling.Random labeled product complexity when protein and polypeptide exist alkalescence or acid side-chain (this situation is almost inevitable).The inventor discloses the new nitronyl nitroxide of a class in early-stage Study, these new nitronyl nitroxide are chemically enough stable, can position the ESR mark to protein and polypeptide, but these are used for the N end that specifically labelled nitronyl nitroxide can only be connected to protein and polypeptide.So present protein and polypeptide ESR labeling technique have obviously been limited to biology and study of pharmacy, the ESR of development protein and polypeptide fixed point labeling technique has clear and definite scientific meaning and using value.
Summary of the invention
The present invention's technical problem at first to be solved is to overcome the deficiencies in the prior art, provide a class new through imidazoline modified amino acid, this amino acid can be inserted into the optional position of polypeptide or protein chain as required as normal L-amino acid in polypeptide is synthetic, realize fixed point ESR mark.
The present invention's technical problem at first to be solved realizes by following technological approaches:
One class new through imidazoline modified amino acid, have the structure of following general formula (I)-(VI):
In the general formula (I), R 1Be carbobenzoxy-(Cbz) or hydrogen atom; In the general formula (II), R 2During for tertbutyloxycarbonyl, R 3Be hydrogen atom; R 2During for hydrogen atom, R 3Be methyl; In the general formula (III), R 4During for tertbutyloxycarbonyl, R 5Be methyl or hydrogen atom; R 4During for hydrogen atom, R 5Be methyl; In the general formula (IV), R 6Be methyl or hydrogen atom; In the logical formula V, work as R 7During for tertbutyloxycarbonyl, R 8Be hydrogen atom; R 7Be hydrogen atom, R 8Be methyl or hydrogen atom; In the general formula (VI), R 9During for tertbutyloxycarbonyl, R 10Be hydrogen atom; R 9During for hydrogen atom, R 10Be methyl or hydrogen atom.
In addition, the present invention also provides the key intermediate of preparation general formula (I) compound: N ω-carbobenzoxy-(Cbz)-N α-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl]-Lys-OBzl; The key intermediate of the logical formula V compound of preparation: Boc-Asp (NHCH 2CH 2Br)-OH; Boc-Asp (NHCH 2CH 2Br)-OCH 3Boc-Asp[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-OCH 3The key intermediate of preparation general formula (VI) compound: Boc-Glu (NHCH 2CH 2Br)-OH and Boc-Glu (NHCH 2CH 2Br)-OCH 3
The present invention by nitronyl nitroxide is guided to L-Lys and or the basic side chain or alpha-amino group of L-Arg on, nitronyl nitroxide is guided on the acid side-chain or α-carboxyl of L-Asp or L-Glu, prepare the above-mentioned general formula compound that contains nitronyl nitroxide.These compounds that contain nitronyl nitroxide have good chemical stability, can tolerate the various reaction conditionss of polypeptide in synthetic, keep free radical and remove the character of free radical constant, can also in polypeptide is synthetic, be inserted into the optional position of polypeptide or protein chain as normal L-Lys, L-Arg, L-Asp and L-Glu as required, realize fixed point ESR mark.The compounds of this invention also has excellent biological compatibility, the nitronyl nitroxide polypeptide that contains that contains nitronyl nitroxide polypeptide and the acquisition of available technology adopting N end mark that fixed point ESR mark of the present invention is obtained compares analysis, and physico-chemical property of the two and biological activity are in full accord.
Certainly, can prepare the salt of The compounds of this invention, these salt are included in the present invention.
The acid salt of The compounds of this invention is preferably pharmaceutically acceptable, with the suitable nontoxic salt of acid (for example hydrochloric acid, acetic acid) formation.Except pharmaceutically acceptable salt, other salt is also included among the present invention.
The invention still further relates to a kind of method for preparing general formula (I)-(VI) compound, comprise by synthetic 4-(1,3-dioxy-4,4,5, the 5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenylacetic acid of art methods; 2-is replaced-1,3-dioxy base tetrahydroglyoxaline is guided on the basic side chain or d-amino of L-Lys or L-Arg according to ordinary method, or with the derivative Boc-Asp (NHCH of L-Asp or L-Glu 2CH 2Br)-OH, Boc-Asp (NHCH 2CH 2Br)-OCH 3, Boc-Glu (NHCH 2CH 2Br)-OH or Boc-Glu (NHCH 2CH 2Br)-OCH 3Implement oxygen-alkylated reaction, promptly get general formula compound of the present invention.
In addition, the present invention also provides the novel polypeptide that adopts above-mentioned general formula (I)-(VI) compound fixed point mark, comprises that the N end contains the PAK (Pro-Ala-[N of the L-Lys of nitronyl nitroxide mark ω-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group ethanoyl] Lys-OH], N end contains the RGDV (N of the L-Arg of nitronyl nitroxide mark G-[4-(1; 3-dioxy-4; 4; 5; 5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group ethanoyl] Arg-Gly-Asp-Val-OH), ([4-(1 for Arg-Gly-[β-aspartoyl for 3 RGDS that contain the L-Asp of nitronyl nitroxide mark; 3-dioxy-4; 4; 5; 5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group third amino] Asp-Ser-OH] and N end contain the L-Glu of nitronyl nitroxide mark gsh (γ-[4-(and 1,3-dioxy-4,4; 5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group third amino] Glu-Cys-Gly-OH).By synthesizing of these representative peptides, illustrate that L-Lys, L-Arg, L-Asp and the L-Glu of the nitronylnitroxide of containing of the present invention has a wide range of applications in polypeptide marker.The structure of these novel polypeptides is as follows:
Figure A20051010529100111
The present invention also provides the intermediate of synthetic above-mentioned novel polypeptide, that is: synthetic Pro-Ala-[N ωThe intermediate of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group ethanoyl] Lys-OH: Boc-Ala-N ω-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Lys-OCH 3Ala-N ω-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Lys-OCH 3Boc-Pro-Ala-N ω-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Lys-OCH 3Boc-Pro-Ala-N ω-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Lys-OH.
Synthetic N GThe key intermediate of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group ethanoyl] Arg-Gly-Asp-Val-OH: N α-Boc-N G-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl] Arg-Gly-Ome; N α-Boc-N G-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino] Arg-Gly-OH; N α-Boc-N G-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino] Arg-Gly-Asp (OBzl)-Val-OMe; N α-Boc-N G-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino] Arg-Gly-Asp-Val-OH.
Synthetic Arg-Gly-[β-aspartoyl [4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group third amino] and the key intermediate of Asp-Ser-OH: Boc-Asp-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Ser-OCH 3Asp-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Ser-OCH 3Boc-Gly-Asp-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Ser-OCH 3Gly-Asp-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Ser-OCH 3Boc-Arg-Gly-Asp-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Ser-OCH 3Boc-Arg-Gly-Asp-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Ser-OH.
Synthetic γ-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group third amino] and the key intermediate of Glu-Cys-Gly-OH: Boc-Glu-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Cys-OMe; Boc-Glu-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Cys-OH; Boc-Glu-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Cys-Gly-OMe; Boc-Glu-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Cys-Gly-OH.
The L-Arg that the present invention adopts alpha-amino group nitronyl nitroxide to modify has synthesized RGDS, RGDV and the RGDF that the N end contains the L-Arg of nitronyl nitroxide mark.The RGDS that contains nitronylnitroxide, RGDV and RGDF that the mark of the N end of the new synthetic polypeptide of these the present invention and available technology adopting limitation is obtained carry out the comparison of chemistry and biology aspect, found that; The physico-chemical property and the biological activity of two class polypeptide are in full accord, illustrate to adopt L-Lys, L-Arg, L-Asp and the L-Glu labeling polypeptide of the nitronyl of containing nitroxide provided by the invention can guarantee accurate aspect chemistry and biology two.The structure of the polypeptide that contains nitronyl nitroxide that the mark of the N end of employing limitation of the prior art obtains is as follows:
Figure A20051010529100131
Contain 1 with what the present invention prepared, 3-dioxy-4,4,5, Lys, Arg, Asp, Glu, Arg-Gly-Asp-Ser-OH, Arg-Gly-Asp-Val-OH and the Glu-Cys-Gly-OH of 5-tetramethyl--2-tetrahydroglyoxaline measure on electron spin resonanceapparatus, and these compounds all show the feature ESR spectrum (Fig. 3) of two NO free radicals.This phenomenon explanation The compounds of this invention has good chemical resistance, is used for the character that the polypeptide building-up reactions can keep two NO free radicals.
Contain 1 with what the present invention prepared, 3-dioxy-4,4,5, the aqueous solution of Lys, Arg, Asp, Glu, Arg-Gly-Asp-Ser-OH, Arg-Gly-Asp-Val-OH and the Glu-Cys-Gly-OH of 5-tetramethyl--2-tetrahydroglyoxaline all is converted into single NO free radical product after feeding NO, these products with on electron spin resonanceapparatus, measure, all show the feature ESR spectrum (Fig. 4) of single NO free radical.This phenomenon explanation The compounds of this invention has good chemical resistance, is used for the polypeptide building-up reactions and can keeps NO to remove activity.
By following compound with the present invention preparation:
N α-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group ethanoyl] Arg-Gly-Asp-Ser-OH, N α-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group ethanoyl] Arg-Gly-Asp-Val-OH and N α-[4-(1; 3-dioxy-4; 4; 5; 5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group ethanoyl] Arg-Gly-Asp-Phe-OH and of the prior art 4-(1; 3-dioxy-4; 4; 5; 5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenylium is incorporated into Arg (Tos)-Gly-Asp (OcHex)-Ser-OBzl; on the α amino of the N of Arg (Tos)-Gly-Asp (OcHex)-Val-OBzl and Arg (Tos)-Gly-Asp (OcHex)-Phe-OBzl end Arg residue again the product that generates of deprotection carry out antithrombotic acitivity simultaneously relatively and estimate, the result shows that the antithrombotic acitivity of two class products is in par.
Another technical problem to be solved by this invention provides a kind of medicinal compositions, this medicinal compositions treat by the present invention effective dose general formula (I)-(VI) arbitrary compound or form by any polypeptide and the pharmaceutically acceptable carrier of this compound institute mark, be about to pharmaceutically acceptable consumption general formula (I)-(VI) arbitrary compound or by the polypeptide of this compound institute mark with after pharmaceutically acceptable carrier or thinner cooperate, by the formulation method of this area routine it is prepared into any one appropriate drug composition.
Usually said composition is suitable for oral administration and drug administration by injection, also is fit to other medication.
Said composition can be liquid preparation forms such as tablet, capsule, pulvis, granule, lozenge, suppository, or oral liquid.According to different medications, pharmaceutical composition of the present invention can contain 0.1%-99% weight, arbitrary compound of the general formula of the present invention (I-VI) of preferred 10-60% weight or arbitrary polypeptide of this compound institute mark.
Description of drawings
Fig. 1 is the ESR spectrum of Nitronyl nitroxide;
Fig. 2 is the ESR spectrum of the 2-replacement-1-oxygen base tetrahydroglyoxaline of nitronyl nitroxide and NO reaction generation;
Fig. 3 contains 1 for of the present invention, and 3-dioxy-4,4,5, Lys, Arg, Asp, Glu, Arg-Gly-Asp-Ser-OH, Arg-Gly-Asp-Val-OH and the Glu-Cys-Gly-OH of 5-tetramethyl--2-tetrahydroglyoxaline provide identical feature ESR spectrum;
Fig. 4 contains 1 for of the present invention, 3-dioxy-4,4,5, Lys, Arg, Asp, Glu, Arg-Gly-Asp-Ser-OH, Arg-Gly-Asp-Val-OH and the Glu-Cys-Gly-OH of 5-tetramethyl--2-tetrahydroglyoxaline provides identical feature ESR spectrum with the product that the NO reaction generates.
Breviary is explained: DCU (dicyclohexylurea (DCU)); DCC (dicyclohexyl imide); THF (tetrahydrofuran (THF)); Me (methyl); Bzl (benzyl); Ach (vagusstoff); NS (heparin-saline); BOC (tertbutyloxycarbonyl); DMF (dimethyl formamide); UK (urea kinases); NE (norepinephrine); CHex (cyclohexyl); SNP (Sodium Nitroprusside); Tos (p-toluenesulfonyl).
Further describe beneficial effect of the present invention by the following examples, it should be understood that these embodiment only are used for the purpose of illustration, never limit protection scope of the present invention.
Embodiment
The synthetic route of 4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenylium.
[intermediate preparation embodiment 1] 2,3-dimethyl-2, the preparation of 3-dinitro-propane (compound 2)
In 130ml NaOH (6mol/1) aqueous solution, add 69.0g (0.78mol) 2-nitropropane (1).The solution that obtains stirred in ice bath 5 minutes, slowly dripped 20ml (0.38mol) Br then 2, last 1 hour.Add 240ml ethanol in the reaction mixture then and in 90 ℃ of water-baths, refluxed 3 hours, during flaky precipitate appears.Reaction mixture is poured in the 800ml frozen water while hot, treated that ice dissolves the back decompress filter and collects plate crystal.Get 63.0g (92%) title compound after the drying.
[intermediate preparation embodiment 2] 2,3-dimethyl-2, the preparation of 3-dihydroxy aminobutane (compound 3)
By 10.0g (56.82mmol) 2,3-dimethyl-2,3-dinitro-propane (2), 5.6g NH 4(50% suspension that constitutes adds the 22.9g zinc powder at ice bath and under stirring, and lasts 3 hours for Cl and 112ml ethanolic soln.The reaction mixture stirring at room is 3 hours then.The reaction mixture decompress filter, filter cake is with 50% aqueous ethanolic solution repetitive scrubbing.Merge washings and filtrate, transfer pH2, be evaporated to muddy with concentrated hydrochloric acid.Slurry that obtains and an amount of K 2CO 3Mixed evenly to PH10, place apparatus,Soxhlet's CHCl then 3Extracted 6 hours.Extracting solution concentrating under reduced pressure, residue sherwood oil crystallization obtains 4.0g (47.57%) title compound, is colourless plate crystal, Mp157-159 ℃.
[intermediate preparation embodiment 3] 1,3-dihydroxyl-2-(4 '-hydroxy phenyl)-4,4,5, the preparation of 5-tetramethyl-imidazolidine (compound 4)
By 990mg (8.107mmol) p-Hydroxybenzaldehyde, 1.196g (8.078mmol) 2,3-dimethyl-2, the solution stirring at room that 3-dihydroxy aminobutane (3) and 4.8ml methyl alcohol constitute 6 hours, TLC shows that raw material point disappears, the reaction mixture decompress filter, obtain 1.47g (72%) title compound, be colourless crystallization, ESI-MS (m/e)=2 36[M] +Rf 0.64 (CHCl 3/ CH 3OH, 10: 1), mp:168-169; IR (KBr, cm -1): 3340,1600,1450; NMR (CDCl 3) δ=1.14 (s, 4-CH 3), 4.78 (s, CH), 7.31-7.51 (m, 5-ArH), 7.71 (2-OH).Ultimate analysis: C 13H 20N 2O 2Calculated value is: C, 66.07; H, 8.53; N, 11.85.
[intermediate preparation embodiment 4] 1,3-dioxy-2-(4 '-hydroxy phenyl)-4,4,5, the preparation of 5-tetramethyl--2-tetrahydroglyoxaline (compound 5)
Toward 1.47g (5.83mmol) 1,3-dihydroxyl-2-(4 '-hydroxy phenyl)-4,4,5, add 8.14g PbO in the solution of 5-tetramethyl-imidazolidine and 81ml methyl alcohol 2(34.40mmol).Behind the stirring at room 0.5hr, TLC shows that raw material point disappears.Reaction mixture filters, and filtrate decompression concentrates, and residue separates (petrol ether/ethyl acetate, 2: 1) with silica gel column chromatography.The fraction concentrating under reduced pressure of collecting that contains product obtains 1.06g (73%) title compound, is blue look solid.R f=0.54 (CHCl 3/ CH 3OH, 20: 1); Mp84-85 ℃; ESI-MS (m/e)=233[M] +IR (KBr, cm -1): 3325,1610,1500,1450,765; Ultimate analysis: C 13H 17N 2O 2Calculated value is: C, 66.93; H, 7.34; N, 12.01.
[intermediate preparation embodiment 5] 1,3-dioxy-2-(4-ethoxycarbonyl p-methoxy-phenyl)-4,4,5, the preparation of 5-tetramethyl--2-tetrahydroglyoxaline (compound 6)
By 500mg (2mmol) 1,3-dioxy-2-(4 '-hydroxy phenyl)-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline (5), 0.6ml bromoethyl acetate, 405mg sodium ethylate, and the solution that the 6ml anhydrous tetrahydro furan constitutes stirred the disappearance of TLC demonstration raw material point 5 hours in 60 ℃.The reaction mixture concentrating under reduced pressure, residue separates (petrol ether/ethyl acetate, 2: 1) with silica gel column chromatography.The fraction that contains required component is evaporated to dried, obtains 650mg (97%) title compound, Mp107-109 ℃; IR (cm -1, KBr): 2830,1750,1610,1370,840; ESI-MS (m/e)=336[M] +
The preparation of [intermediate preparation embodiment 6] 4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy acetic acid (compound 7)
By 100mg (0.2985mmol) 1,3-dioxy-2-(4-ethoxycarbonyl p-methoxy-phenyl)-4,4,5, the solution that 5-tetramethyl--2-tetrahydroglyoxaline (6) and 3ml methyl alcohol constitute mixes with 20 NaOH aqueous solution (2mol/l), stirring at room 30 minutes, the disappearance of TLC demonstration raw material point.The reaction mixture concentrating under reduced pressure, residue and 5ml saturated aqueous common salt mix the back and transfer pH5 with hydrochloric acid (2mol/l).The weakly acidic solution CHCl that obtains 3(3ml * 3) extraction.Merge organic phase, concentrating under reduced pressure obtains 92mg (100%) title compound, is blue look solid.Mp55-157 ℃, ESI-MS (m/e)=307[M] +IR (cm -1, KBr): 2330-2502,1760,1605,1490,1450,1260,830; Ultimate analysis: C 15H 19N 2O 5Calculated value is: C, 68.62; H, 6.23; N, 9.12; Measured value is C, 68.51; H, 6.08; N, 9.27.
Figure A20051010529100171
N ω-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group ethanoyl] naphthalene propylhomoserin methyl esters, N ω-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group ethanoyl] naphthalene propylhomoserin, α-Boc-N ω-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group ethanoyl] naphthalene propylhomoserin, N ω-carbobenzoxy-(Cbz)-N α-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group ethanoyl] naphthalene ammonia acid benzyl ester, N ω-carbobenzoxy-(Cbz)-N α-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group ethanoyl] naphthalene propylhomoserin and N αThe synthetic route of-[4-(1,3-4,4,5,5 tetramethyls-2-tetrahydroglyoxaline-2-yl) phenoxy group ethanoyl] naphthalene propylhomoserin.
[embodiment 1] N ωThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl]-Lys-OMe (compound 8)
By 203mg (0.66mmol) 4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) solution of phenylacetic acid, 130mg (0.66mmol) HClLys-OMe and 20ml anhydrous tetrahydro furan formation drips N-methylmorpholine down earlier in ice bath and transfers pH9, drips the solution of 168mg (0.82mmol) DCC and 12ml anhydrous tetrahydro furan then.Reaction mixture stirs 2 hours, stirred overnight at room temperature, the disappearance of TLC demonstration raw material point in 0 ℃.Reaction mixture filters, and filtrate decompression is concentrated into dried, the residue acetic acid ethyl dissolution.The solution that obtains is used 5%KHSO successively 4The aqueous solution is washed, saturated NaHCO 3The aqueous solution is washed, the saturated NaCl aqueous solution is washed.Tell ethyl acetate layer, anhydrous Na 2SO 4Drying is filtered, and filtrate decompression is concentrated into dried, obtains 196mg (83%) title compound, is blue look solid.IR(cm -1，KBr)：3431、1708、1658、1605、1490、1450、1260、830；ESI-MS(m/e)＝449[M] +；[α] 20 D=143.8(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 22H 33N 4O 6Calculated value is: C, 58.78; H, 7.40; N, 12.46; Measured value is C, 58.77; H, 7.26; N, 12.62.
[embodiment 2] N ωThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl]-Lys-OH (compound 9)
By 200mg (0.223mmol) N ω-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenyl acetyl]-Lys-OMe (8) and 10ml methyl alcohol constitute with stirring at room after 0 ℃ of the 2ml NaOH aqueous solution (1mol/l) mixes 30 minutes, TLC shows raw material point disappearance.The reaction mixture concentrating under reduced pressure, residue and 20ml saturated aqueous common salt are mixed, transfer pH5 with 2mol/l hydrochloric acid.The weakly acidic solution that obtains ethyl acetate extraction (6ml * 3).Obtain 180mg (93%) title compound, be blue look solid.IR(cm -1，KBr)：3422、2341-2520、1746、1668、1605、1490、1450、1260、830；ESI-MS(m/e)＝435[M] +。[α] 20 D＝-241.8(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 21H 31N 4O 6Calculated value is: C, 57.92; H, 7.17; N, 12.87; Measured value is C, 57.76; H, 7.33; N, 12.45.
[embodiment 3] N α-Boc-N ωThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl]-Lys-OH (compound 10)
200mg (0.46mmol) N ωSolution, 120mg (0.552mmol) Boc that-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenyl acetyl]-Lys-OH, the 15ml 1NNaOH aqueous solution constitute 2The solution of O and 15ml dioxane is regulated pH8.6 with the 2NNaOH aqueous solution under ice bath stirs, sustained reaction 12 hours, and TLC shows that raw material point disappears substantially.That reaction mixture is evaporated to is dried, residue with less water dissolving back with saturated aqueous potassium hydrogen sulfate adjusting pH2.0, use ethyl acetate extraction.Ethyl acetate layer with saturated nacl aqueous solution be washed till that neutrality, anhydrous sodium sulfate drying are spent the night, filtration, filtrate decompression be concentrated into dried, residue and solidify in ethyl acetate-sherwood oil.Obtain 218mg (91%) title compound, be blue look solid.IR(cm -1，KBr)：3435、1750、1662、1603、1496、1452、1392、1274、836；ESI-MS(m/e)＝519[M] +。[α] 20 D＝-155.2(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 26H 39N 4O 7Calculated value is: C, 60.10; H, 7.57; N, 10.78; Measured value is C, 60.27; H, 7.39; N, 10.62.
[embodiment 4] N ω-carbobenzoxy-(Cbz)-N αThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl]-Lys-OBzl (compound 11)
200mg (0.65mmol) 4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) solution of phenylacetic acid, 320mg (0.78mmol) Lys (Z)-OBzl, 90mg (0.65mmol) HOBt and 10ml anhydrous tetrahydro furan formation drips N-methylmorpholine down earlier in ice bath and transfers pH9, drips the solution of 160mg (0.78mmol) DCC and 10ml anhydrous tetrahydro furan formation then.Reaction mixture stirs 2 hours, stirred overnight at room temperature for 0 ℃, and TLC shows that raw material point disappears.Reaction mixture filters, filtrate decompression is concentrated into dried, residue acetic acid ethyl dissolution, and the solution that obtains is used 5%KHSO successively 4The aqueous solution is washed and saturated NaHCO 3The aqueous solution is washed, the saturated NaCl aqueous solution is washed.Tell ethyl acetate layer, anhydrous Na 2SO 4Drying, filtration, filtrate decompression are concentrated into the dried 280mg of obtaining (65%) title compound, are blue look solid.IR(cm -1，KBr)：3430、1757、1669、1600、1487、1450、1270、821；ESI-MS(m/e)＝659[M] +；[α] 20 D＝-183.9(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 36H 43N 4O 8Calculated value is: C, 65.54; H, 6.57; N, 8.49; Measured value is C, 65.71; H, 6.75; N, 8.64.
[embodiment 5] N ω-carbobenzoxy-(Cbz)-N αThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl]-Lys-OH (compound 12)
Under the ice bath with 180mg (0.27mmol) N ω-carbobenzoxy-(Cbz)-N α-[4-(1,3-dioxy-4,4; 5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenyl acetyl]-Lys-OBzl is dissolved in the 10ml methyl alcohol, and the solution that obtains adds the 3.5ml 1mol/lNaOH aqueous solution; reaction mixture stirs 30min for 0 ℃, and TLC shows that raw material point disappears.Mixing solutions, stirred 1 hour, TLC shows raw material point disappearance.That reaction solution is evaporated to is dried, residue is transferred pH5 with 2mol/l hydrochloric acid, and the weakly acidic solution that obtains is with ethyl acetate extraction (20ml * 3).Obtain 110mg (90%) title compound, be blue look solid.IR(cm -1，KBr)：3434、2365-2603、1752、1663、1605、1482、1455、1273、834；ESI-MS(m/e)＝435[M] +；[α] 20 D＝-92.9(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 29H 37N 4O 8Calculated value is: C, 61.15; H, 6.55; N, 9.84; Measured value is C, 61.02; H, 6.73; N, 9.66.
[embodiment 6] N αThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl]-Lys-OH (compound 13)
Under the ice bath with 180mg (0.02731mmol) N ω-carbobenzoxy-(Cbz)-N α-[4-(1; 3-dioxy-4,4,5; 5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl]-Lys-OH mixed, stirs 1 hour with the mixing solutions of 2.5ml trifluoroacetic acid/trifluoromethanesulfonic acid (4: 1) with the solution of 2.5ml trifluoroacetic acid, and TLC shows raw material point disappearance.Reaction mixture is evaporated to dried, and residue is with the 15ml acetic acid ethyl dissolution and be evaporated to dried.This operation repeatedly 3 times, so that residual trifluoroacetic acid and the trifluoromethanesulfonic acid of Ex-all, residue obtains 110mg (90%) title compound at last with the 4ml ether dissolution and be evaporated to driedly, is blue look solid.IR(cm -1，KBr)：3423、2360-2598、1750、1660、1602、1480、1450、1275、830；ESI(m/e)＝435[M] +；[α] 20 D＝-183.9(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 21H 31N 4O 6Calculated value is: C, 57.92; H, 7.17; N, 12.87; Measured value is C, 58.11; H, 7.33; N, 12.69.
Figure A20051010529100201
Boc-N G-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group ethanoyl] arginine, N G-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group ethanoyl] arginine methyl esters and N αThe arginic synthetic route of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group ethanoyl].
[embodiment 7] N α-Boc-N GThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl]-Arg-OMe (compound 14)
In the solution of 258mg (0.90mmol) Boc-Arg-OMe and 4ml anhydrous tetrahydro furan, add 143mg (2.39mmol, 40%) sodium hydride.Suspension that obtains and 100mg (0.30mmol) 1,3-dioxy-2-(4-ethoxycarbonyl p-methoxy-phenyl)-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline mixes with the solution of 3ml anhydrous tetrahydro furan.The reaction mixture stirred overnight at room temperature, TLC shows that raw material point disappears.Reaction mixture filters, filtrate decompression is concentrated into dried, residue acetic acid ethyl dissolution, and the solution that obtains is used 5%KHSO successively 4The aqueous solution is washed, saturated NaHCO 3The aqueous solution is washed, the saturated NaCl aqueous solution is washed.The ethyl acetate layer anhydrous Na of telling 2SO 4That drying, filtration, filtrate decompression are concentrated into is dried, residue separates (chloroform/methanol, 5: 1) with silica gel column chromatography, obtains 150mg (87%) title compound, is blue look solid.IR(cm -1，KBr)：3409、1762、1668、1601、1483、1454、1395、1370、1272、833；ESI(m/e)＝577[M] +；[α] 20 D＝70.2(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 27H 41N 4O 8Calculated value is: C, 56.14; H, 7.15; N, 14.55; Measured value is C, 56.28; H, 7.32; N, 14.39.
[embodiment 8] N α-Boc-N GThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl]-Arg-OH (compound 15)
Toward 58mg (0.10mmol) N-Boc-N GAdd the 3mlNaOH aqueous solution (1mol/l) in the solution of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenyl acetyl]-Arg-OMe and 6ml methyl alcohol.Reaction mixture stirred 1 hour for 0 ℃, and TLC shows that raw material point disappears.The reaction mixture concentrating under reduced pressure, residue mixes with the 2ml saturated aqueous common salt, transfers pH5 with 2mol/l hydrochloric acid.The weakly acidic solution that obtains ethyl acetate extraction (6ml * 3).Ethyl acetate layer is evaporated to the dried 43mg of obtaining (73%) title compound, is blue look solid.IR(cm -1，KBr)：3414、2366-2590、1752、1665、1606、1482、1454、1395、1272、833；ESI-MS(m/e)＝563[M] +；[α] 20 D＝58.1(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 26H 39N 4O 8Calculated value is: C, 55.41; H, 6.97; N, 14.91; Measured value is C, 55.24; H, 6.79; N, 15.10.
[embodiment 9] N GThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl]-Arg-OMe (compound 16)
Under the ice bath with 110mg (0.19mmol) N-Boc-N G-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenyl acetyl]-Arg-OMe is dissolved in the 15ml ethyl acetate.Add 2.5ml hydrogenchloride/ethyl acetate solution (4mol/l), stirring 20 minutes in the solution that obtains, TLC shows that raw material point disappears.Reaction solution is evaporated to dried, and residue is evaporated to after with the 10ml acetic acid ethyl dissolution dried again.This operation 3 times repeatedly is so that the residual hydrogenchloride of Ex-all.Residue obtains 90mg (98%) title compound again with the 10ml ether dissolution and be evaporated to driedly, is blue look solid.IR(cm -1，KBr)：3402、1760、1664、1605、1480、1452、1370、1270、830；ESI(m/e)＝477[M] +；ESI-MS(m/e)＝477[M] +；[α] 20 D＝-206.9(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 22H 33N 6O 6Calculated value is: C, 55.33; H, 6.97; N, 17.60; Measured value is C, 55.51; H, 6.60; N, 17.78.
[embodiment 10] N αThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl]-Arg-OMe (compound 17)
In the solution of 168mg (0.90mmol) H-Arg-OMe and 8ml anhydrous tetrahydro furan, add the 2.4ml N-methylmorpholine, transfer pH9.Suspension that obtains and 100mg (0.30mmol) 1,3-dioxy-2-(4-ethoxycarbonyl p-methoxy-phenyl)-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline mixes with the solution of 3ml anhydrous tetrahydro furan.The reaction mixture stirred overnight at room temperature, TLC shows that raw material point disappears.Reaction mixture filters, filtrate decompression is concentrated into dried, residue acetic acid ethyl dissolution, and the solution that obtains is used 5%KHSO successively 4The aqueous solution is washed, saturated NaHCO 3The aqueous solution is washed, the saturated NaCl aqueous solution is washed.The ethyl acetate layer anhydrous Na of telling 2SO 4That drying, filtration, filtrate decompression are concentrated into is dried, residue separates (chloroform/methanol, 5: 1) with silica gel column chromatography, obtains 150mg (87%) title compound, is blue look solid.IR(cm -1，KBr)：3345、3239、1766、1662、1604、1485、1455、1396、1373、1275、836；ESI(m/e)＝491[M] +；[α] 20 D＝-159.2(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 23H 35N 6O 6Calculated value is: C, 56.20; H, 7.18; N, 17.10; Measured value is C, 56.05; H, 7.02; N, 17.27.
[embodiment 11] N αThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl]-Arg-OH (compound 18)
Toward 49mg (0.10mmol) N αAdd the 3ml NaOH aqueous solution (1mol/l) in the solution of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenyl acetyl]-Arg-OMe and 6ml methyl alcohol.Reaction mixture stirred 1 hour for 0 ℃, and TLC shows that raw material point disappears.The reaction mixture concentrating under reduced pressure, residue mixes with the 2ml saturated aqueous common salt, transfers pH5 with 2mol/l hydrochloric acid.The weakly acidic solution that obtains ethyl acetate extraction (6ml * 3).Ethyl acetate layer is evaporated to the dried 43mg of obtaining (73%) title compound, is blue look solid.IR(cm -1，KBr)：3389、2362-2594、1754、1662、1604、1477、1452、1274、835；ESI-MS(m/e)＝477[M] +；[α] 20 D＝-125.6(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 22H 33N 6O 6Calculated value is: C, 55.33; H, 6.97; N, 17.60; Measured value is C, 55.52; H, 6.78; N, 17.44.
Boc-β-aspartoyl [4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group third amino] and aspartic acid and β-aspartoyl [4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group third amino] synthetic route of aspartic acid.
[intermediate preparation embodiment 7] Boc-Asp (NHCH 2CH 2Br)-preparation of OH (compound 19)
Ice bath is down toward 500mg (2.1mmol) Boc-Asp-OH, 435mg (2.1mmol) BrCH 2CH 2NH 2Drip the 1.4ml N-methylmorpholine in the solution of HBr and 30ml anhydrous tetrahydro furan, transfer pH9.The solution that the solution that obtains and 440mg (2.1mmol) DCC and 30ml anhydrous tetrahydro furan constitute mixes, 0 ℃ stir 2 hours, stirred overnight at room temperature, TLC and show the disappearance of raw material point.Reaction mixes and adds 10%KHSO 4The aqueous solution to pH2-3, filtration, filtrate decompression concentrates, residue adds ethyl acetate, leaches the colorless solid of generation, obtains 580mg (81%) title compound.IR(cm -1，KBr)：3326、2360-2594、1750、1625、1482、1454、1395、1272、833；ESI-MS(m/e)＝338[M] +。[α] 20 D=-33.4 ultimate analysis: C 11H 19N 2O 5Calculated value is: C, 38.95; H, 5.65; N, 8.26; Measured value is C, 39.13; H, 5.82; N, 8.42.
[embodiment 12] Boc-Asp[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-preparation of OH (compound 21)
By 226mg (0.91mmol) 1,3-dioxy-2-(4 '-hydroxy phenyl)-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline, 243mg (0.72mmol) Boc-Asp (NHCH 2CH 2Br)-and solution that OH, 240mg sodium ethylate and 6ml anhydrous tetrahydro furan constitute stirred 2 hours in 60 ℃, and TLC shows raw material point disappearance.Reacting liquid filtering, filtrate decompression concentrates.Residue adds 10%KHSO 4The aqueous solution is also used ethyl acetate extraction.The organic phase that merges is washed with the saturated NaCl aqueous solution.Tell ethyl acetate layer and use anhydrous Na 2SO 4That drying, filtration, filtrate decompression are concentrated into is dried, residue separates (petrol ether/ethyl acetate, 2: 1) with silica gel column chromatography, obtains 221mg (48%) title compound, is blue look solid.IR(cm -1，KBr)：3406、2342-2587、1750、1662、1603、1489、1450、1393、1270、830；ESI-MS(m/e)＝523[M] +；[α] 20 D＝-42.7(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 25H 39N 4O 8Calculated value is: C, 57.35; H, 7.51; N, 10.70; Measured value is C, 57.21; H, 7.69; N, 10.52.
[embodiment 13] Asp[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-preparation of OH (compound 22)
Under the ice bath with 200mg (0.39mmol) Boc-Asp[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-OH is dissolved in the 5ml ethyl acetate, the solution that obtains and 3ml hydrogenchloride/ethyl acetate solution (4mol/l), stirring 40 minutes, TLC shows that raw material point disappears.Reaction solution is evaporated to dried, and residue is evaporated to after with the 10ml acetic acid ethyl dissolution dried again.This operation 3 times repeatedly is so that the residual hydrogenchloride of Ex-all.Residue obtains 150mg (94%) title compound again with the 10ml ether dissolution and be evaporated to driedly, is blue look solid.IR(cm -1，KBr)：3416、2340-2581、1753、1660、1605、1484、1452、1272、834；ESI-MS(m/e)＝423[M] +；[α] 20 D＝-157.1(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 20H 31N 4O 6Calculated value is: C, 56.72; H, 7.38; N, 13.23; Measured value is C, 56.90; H, 7.56; N, 13.07.
Boc-β-aspartoyl [4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group third amino] and aspartic acid methyl esters and β-aspartoyl [4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group third amino] synthetic route of aspartic acid methyl esters.
[intermediate preparation embodiment 8] Boc-Asp (NHCH 2CH 2Br)-OCH 3The preparation of (compound 20)
Ice bath is down toward 517mg (2.1mmol) Boc-Asp-OCH3,435mg (2.1mmol) BrCH 2CH 2NH 2Drip the 1.4ml N-methylmorpholine in the solution of HBr and 30ml anhydrous tetrahydro furan, transfer pH9.The solution that the solution that obtains and 440mg (2.1mmol) DCC and 30ml anhydrous tetrahydro furan constitute mixes, 0 ℃ stir 2 hours, stirred overnight at room temperature, TLC and show the disappearance of raw material point.Reaction mixes and adds 10%KHSO 4The aqueous solution to pH2-3, filtration, filtrate decompression concentrates, residue adds ethyl acetate, leaches the colorless solid of generation, obtains 580mg (85%) title compound.IR(cm -1，KBr)：3335、1754、1622、1480、1450、1395、1370、1274、835；ESI-MS(m/e)＝352[M] +。[α] 20 D=-42.5 ultimate analysis: C 12H 21N 2O 5Calculated value is: C, 40.81; H, 5.99; N, 7.93; Measured value is C, 41.00; H, 5.81; N, 7.78.
[embodiment 14] Boc-Asp[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-OCH 3The preparation of (compound 23)
By 226mg (0.91mmol) 1,3-dioxy-2-(4 '-hydroxy phenyl)-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline, 253mg (0.72mmol) Boc-Asp (NHCH 2CH 2Br)-OCH 3, the solution that constitutes of 240mg sodium ethylate and 6ml anhydrous tetrahydro furan stirred 2 hours in 60 ℃, TLC shows raw material point disappearance.Reacting liquid filtering, filtrate decompression concentrates.Residue adds 10%KHSO 4The aqueous solution is also used ethyl acetate extraction.The organic phase that merges is washed with the saturated NaCl aqueous solution.Tell ethyl acetate layer and use anhydrous Na 2SO 4That drying, filtration, filtrate decompression are concentrated into is dried, residue separates (petrol ether/ethyl acetate, 2: 1) with silica gel column chromatography, obtains 264mg (54%) title compound, is blue look solid.IR(cm -1，KBr)：3406、1755、1660、1601、1454、1395、1370、1274、833；ESI-MS(m/e)＝537[M] +；[α] 20 D=？(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 26H 41N 4O 8Calculated value is: C, 58.08; H, 7.69; N, 10.42; Measured value is C, 58.22; H, 7.85; N, 10.27.
[embodiment 15] Asp[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-OCH 3The preparation of (compound 24)
Under the ice bath with 209mg (0.39mmol) Boc-Asp[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-OCH 3Be dissolved in the 5ml ethyl acetate, the solution that obtains and 3ml hydrogenchloride/ethyl acetate solution (4mol/1), stirring 40 minutes, TLC shows that raw material point disappears.Reaction solution is evaporated to dried, and residue is evaporated to after with the 10ml acetic acid ethyl dissolution dried again.This operation 3 times repeatedly is so that the residual hydrogenchloride of Ex-all.Residue obtains 150mg (94%) title compound again with the 10ml ether dissolution and be evaporated to driedly, is blue look solid.IR(cm -1，KBr)：3403、1756、1662、1603、1480、1450、1276、830；ESI-MS(m/e)＝437[M] +；[α] 20 D=？(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 21H 33N 4O 6Calculated value is: C, 57.65; H, 7.60; N, 12.81; Measured value is C, 57.79; H, 7.41; N, 12.62.
The Boc-gamma-glutamyl [4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group third amino] and L-glutamic acid and gamma-glutamyl [4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group third amino] synthetic route of L-glutamic acid.
[intermediate preparation embodiment 9] Boc-Glu (NHCH 2CH 2Br)-preparation of OH (compound 25)
Ice bath is down toward 100mg (0.40mmol) HO-Glu (Boc)-OH, 83mg (0.40mmol) BrCH 2CH 2NH 2.HBr with in the solution of 10ml anhydrous tetrahydro furan drip N-methylmorpholine, transfer pH9.The solution that obtains mixes with the solution that 83mg (0.40mmol) DCC and 10ml anhydrous tetrahydro furan constitute, 0 ℃ of stirring 2 hours, stirred overnight at room temperature, and TLC shows that raw material point disappears.Reaction mixture 10%KHSO 4The aqueous solution is transferred that pH2-3, filtration, filtrate decompression concentrate, residue adds ethyl acetate, is leached the colorless solid of generation, obtains 54mg (38%) title compound, is blue look solid.IR(cm -1，KBr)：3326、2364-2597、1755、1625、1480、1453、1393、1270、830；ESI(m/e)＝352[M] +；IR(neat)：3326，1625cm -1；[α] 20 D＝-107.8(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 12H 21N 2O 5Calculated value is: C, 40.81; H, 5.99; N, 7.93; Measured value is C, 41.00; H, 6.19; N, 7.75.
[embodiment 16] Boc-Glu[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-preparation of OH (compound 27)
By 150mg (0.6mmol) 1,3-dioxy-2-(4 '-hydroxy phenyl)-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline, 253mg (0.72mmol) Boc-Glu (NHCH 2CH 2Br)-and solution that OH, 210mg sodium ethylate and 15ml anhydrous tetrahydro furan constitute stirred 2 hours in 60 ℃, and TLC shows raw material point disappearance.Reaction mixture filters, filtrate decompression concentrates.Residue adds KHSO 4Use ethyl acetate extraction after the aqueous solution (10%) dissolving.The organic phase that merges is washed with the saturated NaCl aqueous solution.The ethyl acetate layer anhydrous Na of telling 2SO 4That drying, filtration, filtrate decompression are concentrated into is dried, residue separates (petrol ether/ethyl acetate, 2: 1) with silica gel column chromatography, obtains 150mg (48%) title compound, is blue look solid.IR(cm -1，KBr)：3320、3252、2348-2590、1750、1662、1602、1486、1455、1270、836；ESI-MS(m/e)＝537[M] +；[α] 20 D＝-77.1(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 26H 41N 4O 8Calculated value is: C, 58.08; H, 7.69; N, 10.42; Measured value is C, 58.22; H, 7.86; N, 10.23.
[embodiment 17] Glu[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-preparation of OH (compound 28)
Under the ice bath with 200mg (0.40mmol) Boc-Glu[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-OH is dissolved in the 15ml ethyl acetate, add 2.5ml hydrogenchloride/ethyl acetate solution (4mol/l) then and stirred the disappearance of TLC demonstration raw material point 20 minutes.Reaction solution is evaporated to dried, and residue is evaporated to after with the 10ml acetic acid ethyl dissolution dried again.This operation 3 times repeatedly is so that the residual hydrogenchloride of Ex-all.Residue obtains 160mg (100%) title compound again with the 10ml ether dissolution and be evaporated to driedly, is blue look solid.IR(cm -1，KBr)：3366、2306-2570、1750、1662、1603、1486、1450、1270、836；ESI-MS(m/e)＝437[M] +，[α] 20 D=-116.5(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 21H 33N 4O 6Calculated value is: C, 57.65; H, 7.60; N, 12.81; Measured value is C, 57.47; H, 7.44; N, 12.98.
Figure A20051010529100271
The Boc-gamma-glutamyl [4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group third amino] the glutamic acid methyl ester gamma-glutamyl [4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group third amino] synthetic route of glutamic acid methyl ester.
[intermediate preparation embodiment 10] Boc-Glu (NHCH 2CH 2Br)-OCH 3The preparation of (compound 26)
Ice bath is down toward 108mg (0.40mmol) HO-Glu (Boc)-OH, 83mg (0.40mmol) BrCH 2CH 2NH 2.HBr with in the solution of 10ml anhydrous tetrahydro furan drip N-methylmorpholine, transfer pH9.The solution that obtains mixes with the solution that 83mg (0.40mmol) DCC and 10ml anhydrous tetrahydro furan constitute, 0 ℃ of stirring 2 hours, stirred overnight at room temperature, and TLC shows that raw material point disappears.Reaction mixture 10%KHSO 4The aqueous solution is transferred that pH2-3, filtration, filtrate decompression concentrate, residue adds ethyl acetate, is leached the colorless solid of generation, obtains 81mg (55%) title compound, is blue look solid.IR(cm -1，KBr)：3328、1750、1626、1483、1450、1395、1275、833；ESI(m/e)＝367[M] +；IR(neat)：3326，1625cm -1；[α] 20 D＝-80.6(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 13H 23N 2O 5Calculated value is: C, 42.52; H, 6.31; N, 7.63; Measured value is C, 42.70; H, 6.16; N, 7.79.
[embodiment 18] Boc-Glu[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-OCH 3The preparation of (compound 29)
By 150mg (0.6mmol) 1,3-dioxy-2-(4 '-hydroxy phenyl)-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline, 264mg (0.72mmol) Boc-Glu (NHCH 2CH 2Br)-OCH 3, the solution that constitutes of 210mg sodium ethylate and 15ml anhydrous tetrahydro furan stirred 2 hours in 60 ℃, TLC shows raw material point disappearance.Reaction mixture filters, filtrate decompression concentrates.Residue adds KHSO 4Use ethyl acetate extraction after the aqueous solution (10%) dissolving.The organic phase that merges is washed with the saturated NaCl aqueous solution.The ethyl acetate layer anhydrous Na of telling 2SO 4That drying, filtration, filtrate decompression are concentrated into is dried, residue separates (petrol ether/ethyl acetate, 2: 1) with silica gel column chromatography, obtains 150mg (48%) title compound, is blue look solid.IR(cm -1，KBr)：3329、3254、1752、1665、1604、1482、1450、1396、1371、1273、834；ESI-MS(m/e)＝551[M] +；[α] 20 D=？(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 27H 43N 4O 8Calculated value is: C, 58.78; H, 7.86; N, 10.16; Measured value is C, 58.95; H, 7.69; N, 10.25.
[embodiment 19] Glu[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-OCH 3The preparation of (compound 30)
Under the ice bath with 220mg (0.40mmol) Boc-Glu[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-OCH 3Be dissolved in the 15ml ethyl acetate, add 2.5ml hydrogenchloride/ethyl acetate solution (4mol/l) then and stirred the disappearance of TLC demonstration raw material point 20 minutes.Reaction solution is evaporated to dried, and residue is evaporated to after with the 10ml acetic acid ethyl dissolution dried again.This operation 3 times repeatedly is so that the residual hydrogenchloride of Ex-all.Residue obtains 162mg (98%) title compound again with the 10ml ether dissolution and be evaporated to driedly, is blue look solid.IR(cm -1，KBr)：3326、3252、1755、1661、1602、1485、1452、1371、1275、832；ESI-MS(m/e)＝451[M] +，[α] 20 D＝？(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 22H 35N 4O 6Calculated value is: C, 58.52; H, 7.81; N, 12.41; Measured value is C, 58.38; H, 7.64; N, 12.59.
Pro-Ala-[N ωThe synthetic route of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group ethanoyl] Lys-OH.
[embodiment 20] Boc-Ala-N ω-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Lys-OCH 3The preparation of (compound 31)
Ice bath is down toward 166mg (0.37mmol) N ω-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Lys-OCH 3, drip N-methylmorpholine in the solution that constitutes of 77mg (0.41mmol) Boc-Ala-OH, 50mg (0.37mmol) HOBT and 8ml anhydrous tetrahydro furan and transfer pH9.The solution that obtains mixes with the solution that 85mg (0.41mmol) DCC and 8ml anhydrous tetrahydro furan constitute, 0 ℃ of stirring 2 hours, stirred overnight at room temperature, and TLC shows that raw material point disappears.Reaction mixture filters, filtrate decompression is concentrated into dried, residue and uses 5%KHSO successively with acetic acid ethyl dissolution, the solution that obtains 4The aqueous solution is washed, saturated NaHCO 3The aqueous solution is washed, the saturated NaCl aqueous solution is washed.The ethyl acetate layer anhydrous Na of telling 2SO 4That drying, filtration, filtrate decompression are concentrated into is dried, residue separates (petrol ether/ethyl acetate, 2: 1) with silica gel column chromatography, obtains 196mg (85%) title compound, is blue look solid.IR(cm -1，KBr)：3344、3251、1753、1662、1605、1480、1455、1395、1370、1274、832；ESI-MS(m/e)＝620[M] +，[α] 20 D＝-132.0(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 30H 46N 5O 9Calculated value is: C, 58.05; H, 7.47; N, 11.28; Measured value is C, 58.22; H, 7.66; N, 11.43.
[embodiment 21] Ala-N ω-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Lys-OCH 3The preparation of (compound 32)
Under the ice bath with 196mg (0.38mmol) Boc-Ala-N ω-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Lys-OCH 3Be dissolved in the 10ml ethyl acetate, add 2.5ml hydrogenchloride/ethyl acetate solution (4mol/l) then and stirred the disappearance of TLC demonstration raw material point 12 minutes.Reaction solution is evaporated to dried, and residue is evaporated to after with the 10ml acetic acid ethyl dissolution dried again.This operation 3 times repeatedly is so that the residual hydrogenchloride of Ex-all.Residue obtains 164mg (93%) title compound again with the 10ml ether dissolution and be evaporated to driedly, is blue look solid.IR(cm -1，KBr)：3340、3252、1750、1664、1603、1482、1454、1372、1276、830；ESI-MS(m/e)＝520[M] +；[α] 20 D＝+112.2(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 25H 38N 5O 7Calculated value is: C, 57.68; H, 7.36; N, 13.45; Measured value is C, 57.49; H, 7.19; N, 13.62.
[embodiment 22] Boc-Pro-Ala-N ω-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Lys-OCH 3The preparation of (compound 33)
Ice bath is down toward 164mg (0.30mmol) Ala-N ω-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Lys-OCH 3, drip N-methylmorpholine in the solution that constitutes of 75mg (0.35mmol) Boc-Pro-OH, 43mg (0.32mmol) HOBT and 8ml anhydrous tetrahydro furan and transfer pH9.The solution that obtains mixes with the solution that 72mg (0.35mmol) DCC and 8ml anhydrous tetrahydro furan constitute, 0 ℃ of stirring 2 hours, stirred overnight at room temperature, and TLC shows that raw material point disappears.Reaction mixture filters, filtrate decompression is concentrated into dried, residue and uses 5%KHSO successively with acetic acid ethyl dissolution, the solution that obtains 4The aqueous solution is washed, saturated NaHCO 3The aqueous solution is washed, the saturated NaCl aqueous solution is washed.The ethyl acetate layer anhydrous Na of telling 2SO 4That drying, filtration, filtrate decompression are concentrated into is dried, residue separates (petrol ether/ethyl acetate, 2: 1), obtains 170mg (80.5%) title compound with silica gel column chromatography, is blue look solid.IR(cm -1，KBr)：3360、3250、1754、1662、1605、1480、1452、1395、1370、1272、834；ESI-MS(m/e)＝717[M] +；[α] 20 D＝+169.4(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 35H 53N 6O 10Calculated value is: C, 58.56; H, 7.44; N, 11.71; Measured value is C, 58.38; H, 7.62; N, 11.90.
[embodiment 23] Boc-Pro-Ala-N ωThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Lys-OH (compound 34)
By 250mg (0.35mmol) Boc-Pro-Ala-N ω-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Lys-OCH 3The solution and 2mlNaOH (1mol/l) aqueous solution that constitute with 15ml methyl alcohol stirred 30 minutes for mixed back 0 ℃, and TLC shows that raw material point disappears.The reaction mixture concentrating under reduced pressure, residue is transferred pH5 with 10ml saturated common salt water dissolution and with hydrochloric acid (2mol/l).The weakly acidic solution that obtains is with ethyl acetate extraction (3ml * 3), isolating ethyl acetate layer anhydrous Na 2SO 4Drying, filtration, filtrate decompression are concentrated into dried, obtain 230mg (94%) title compound, are blue look solid.IR(cm -1，KBr)：3334、3250、2316-2572、1755、1660、1605、1484、1453、1275、834；ESI-MS(m/e)＝703[M] +；[α] 20 D＝-136.0(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 34H 51N 6O 10Calculated value is: C, 58.02; H, 7.30; N, 11.94; Measured value is C, 58.21; H, 7.49; N, 11.77.
[embodiment 24] Pro-Ala-N ωThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Lys-OH (compound 35)
Under the ice bath with 200mg (0.29mmol) Boc-Pro-Ala-N ωThe solution of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Lys-OH and 10ml ethyl acetate mixes with 3ml hydrogenchloride/ethyl acetate solution (4mol/l) and stirred 60 minutes, and TLC shows that raw material point disappears.Reaction solution is evaporated to dried, and residue is evaporated to after with the 10ml acetic acid ethyl dissolution dried again.This operation 3 times repeatedly is so that the residual hydrogenchloride of Ex-all.Residue obtains 170mg (99%) title compound again with the 10ml ether dissolution and be evaporated to driedly, is blue look solid.IR(cm -1，KBr)：3320、3239、2312-2574、1751、1662、1603、1483、1455、1270、832；ESI-MS(m/e)＝603[M] +；[α] 20 D＝-166.5(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 29H 43N 6O 8Calculated value is: C, 57.70; H, 7.18; N, 13.92; Measured value is C, 57.53; H, 7.00; N, 13.74.
N GThe synthetic route of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group ethanoyl] Arg-Gly-Asp-Val-OH.
[embodiment 25] N α-Boc-N GThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl] Arg-Gly-OMe (compound 36)
Ice bath is down toward 380mg (0.68mmol) N-Boc-N ω-[4-(1; 3-dioxy-4,4,5; 5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl] drip N-methylmorpholine in the solution that constitutes of Arg-OH, 85mg (0.68mmol) HClGlyOMe, 90mg (0.68mmol) HOBT and 15ml anhydrous tetrahydro furan, transfer pH9.The solution that obtains mixes with the solution of 180mg (0.87mmol) DCC and 3ml anhydrous tetrahydro furan, 0 ℃ of stirring 2 hours, stirred overnight at room temperature, and TLC shows that raw material point disappears.Reaction mixture filters, filtrate decompression is concentrated into dried, residue acetic acid ethyl dissolution, and the solution that obtains is used 5%KHSO successively 4The aqueous solution is washed, saturated NaHCO 3The aqueous solution is washed, the saturated NaCl aqueous solution is washed.The ethyl acetate layer anhydrous Na of telling 2SO 4That drying, filtration, filtrate decompression are concentrated into is dried, residue separates (petrol ether/ethyl acetate, 2: 1) with silica gel column chromatography, obtains 420mg (98%) title compound, is blue look solid.IR(cm -1，KBr)：3339、3253、1752、1664、1603、1482、1455、1396、1372、1275、830；ESI-MS(m/e)＝634[M] +；[α] 20 D＝-48.3(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 29H 44N 7O 9Calculated value is: C, 54.88; H, 6.99; N, 15.45; Measured value is C, 54.70; H, 7.17; N, 15.62.
[embodiment 26] .N α-Boc-N GThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino] Arg-Gly-OH (compound 37)
By 250mg (0.35mmol) N-Boc-N ωSolution and 2mlNaOH (1mol/l) aqueous solution that-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino] Arg-Gly-OMe and 15ml methyl alcohol constitute, reaction mixture stirred 1 hour for 0 ℃, and TLC shows that raw material point disappears.The reaction mixture concentrating under reduced pressure, residue is transferred pH5 with 10ml saturated common salt water dissolution and with hydrochloric acid (2mol/l).The weakly acidic solution that obtains is with ethyl acetate extraction (10ml * 3), isolating ethyl acetate layer anhydrous Na 2SO 4Drying, filtration, filtrate decompression are concentrated into dried, obtain 210mg (95%) title compound, are blue look solid.IR(cm -1，KBr)：3328、3233、2314-2577、1753、1665、1606、1485、1452、1274、835；ESI-MS(m/e)＝620[M] +；[α] 20 D＝-259.1(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 28H 42N 7O 9Calculated value is: C, 54.18; H, 6.82; N, 15.80; Measured value is C, 54.36; H, 7.02; N, 15.63.
[embodiment 27] N α-Boc-N GThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino] Arg-Gly-Asp (OBzl)-Val-OMe (compound 38)
Ice bath is down toward 270mg (0.44mmol) N-Boc-N ω-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino] drip N-methylmorpholine in the solution of Arg-Gly-OH, 150mg (0.44mmol) HClAsp (Bzl)-Val-OMe, 60mg (0.44mmol) HOBT and 10ml anhydrous tetrahydro furan, transfer pH9.The solution that obtains mixes with the solution that 100mg (0.49mmol) DCC and 5ml anhydrous tetrahydro furan constitute, 0 ℃ of stirring 2 hours, stirred overnight at room temperature, and TLC shows that raw material point disappears.Reaction mixture filters, filtrate decompression is concentrated into dried, residue acetic acid ethyl dissolution, and the solution that obtains is used 5%KHSO successively 4The aqueous solution is washed, saturated NaHCO 3The aqueous solution is washed, the saturated NaCl aqueous solution is washed.The ethyl acetate layer anhydrous Na of telling 2SO 4That drying, filtration, filtrate decompression are concentrated into is dried, residue separates (petrol ether/ethyl acetate, 2: 1) with silica gel column chromatography, obtains 240mg (59%) title compound, is blue look solid.IR(cm -1，KBr)：3329、3250、1750、1662、1606、1480、1452、1395、1370、1274、833；ESI-MS(m/e)＝924[M] +；[α] 20 D＝-70.9(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 44H 62N 9O 13Calculated value is: C, 57.13; H, 6.76; N, 13.63; Measured value is C, 57.30; H, 6.91; N, 13.45.
[embodiment 28] N α-Boc-N GThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino] Arg-Gly-Asp-Val-OH (compound 39)
By 240mg (0.26mmol) N-Boc-N ω-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino] solution and 5mlNaOH (1mol/l) aqueous solution that Arg-Gly-Asp (OBzl)-Val-OMe and 15ml methyl alcohol constitute, reaction mixture stirred 1 hour for 0 ℃, and TLC shows the disappearance of raw material point.The reaction mixture concentrating under reduced pressure, residue is transferred pH5 with 10ml saturated common salt water dissolution and with hydrochloric acid (2mol/l).The weakly acidic solution that obtains is with ethyl acetate extraction (10ml * 3), isolating ethyl acetate layer anhydrous Na 2SO 4Drying, filtration, filtrate decompression are concentrated into dried, obtain 200mg (97%) title compound, are blue look solid.IR(cm -1，KBr)：3332、3230、2325-2586、1755、1662、1604、1482、1455、1276、835；ESI-MS(m/e)＝834[M] +；[α] 20 D＝+13.8(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 37H 56N 9O 13Calculated value is: C, 52.23; H, 6.76; N, 15.10; Measured value is C, 52.40; H, 6.89; N, 15.28.
[embodiment 29] N GThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino] Arg-Gly-Asp-Val-OH (compound 40)
Under the ice bath with 200mg (0.26mmol) N-Boc-N ω-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino] solution of Arg-Gly-Asp-Val-OH and 10ml ethyl acetate mixes with 3ml hydrogenchloride/ethyl acetate solution (4mol/l) and stirred 30 minutes, and TLC shows raw material point disappearance.Reaction solution is evaporated to dried, and residue is evaporated to after with the 10ml acetic acid ethyl dissolution dried again.This operation 3 times repeatedly is so that the residual hydrogenchloride of Ex-all.Residue obtains 180mg (97%) title compound again with the 10ml ether dissolution and be evaporated to driedly, is blue look solid.IR(cm -1，KBr)：3329、3232、2318-2579、1753、1660、1605、1480、1453、1272、830；ESI-MS(m/e)＝734[M] +；[α] 20 D＝+11.5(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 32H 48N 9O 11Calculated value is: C, 52.31; H, 6.58; N, 17.16; Measured value is C, 52.49; H, 6.76; N, 17.36.
The synthetic route of Arg-Gly-[β-aspartoyl [4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group third amino] Asp-Ser-OH
[embodiment 30] Boc-Asp-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Ser-OCH 3The preparation of (compound 41)
Ice bath is down toward 209mg (0.40mmol) Boc-Asp-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-drip N-methylmorpholine in the solution of OH, 62mg (0.40mmol) HClSer-OMe, 54mg (0.40mmol) HOBt and 10ml anhydrous tetrahydro furan, transfer pH9.The solution that obtains mixes with the solution of 80mg (0.40mmol) DCC and 10ml anhydrous tetrahydro furan, 0 ℃ of stirring 2 hours, stirred overnight at room temperature, and TLC shows that raw material point disappears.Reaction mixture filters, filtrate decompression is concentrated into dried, residue acetic acid ethyl dissolution, and the solution that obtains is used 5%KHSO successively 4The aqueous solution is washed, saturated NaHCO 3The aqueous solution is washed, the saturated NaCl aqueous solution is washed, the ethyl acetate layer anhydrous Na of telling 2SO 4Drying is filtered, filtrate decompression is concentrated into dried, residue and separates (petrol ether/ethyl acetate, 2: 1), obtain 231mg (95%) title compound with silica gel column chromatography, is blue look solid.IR(cm -1，KBr)：3433、3330、3252、1753、1664、1604、1485、1451、1394、1372、1270、834；ESI-MS(m/e)＝608[M] +；[α] 20 D＝-100.4(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 28H 42N 5O 10Calculated value is: C, 52.31; H, 6.58; N, 17.16; Measured value is C, 52.50; H, 6.41; N, 17.33.
[embodiment 31] Asp-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Ser-OCH 3The preparation of (compound 42)
540mg under the ice bath (0.892mmol) Boc-Asp-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Ser-OCH 3The solution that constitutes with the 12ml ethyl acetate mixes with 2.5ml hydrogenchloride/ethyl acetate solution (4mol/l) and stirred 45 minutes, and TLC shows the disappearance of raw material point.Reaction solution is evaporated to dried, and residue is evaporated to after with the 10ml acetic acid ethyl dissolution dried again.This operation 3 times repeatedly is so that the residual hydrogenchloride of Ex-all.Residue obtains 450mg (98%) title compound again with the 10ml ether dissolution and be evaporated to driedly, is blue look solid.IR(cm -1，KBr)：3430、3335、3250、1756、1667、1605、1483、1456、1372、1274、832；ESI-MS(m/e)＝508[M] +；[α] 20 D＝-78.1(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 23H 34N 5O 8Calculated value is: C, 54.32; H, 6.74; N, 13.77; Measured value is C, 54.18; H, 6.57; N, 13.95.
[embodiment 32] Boc-Gly-Asp-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Ser-OCH 3The preparation of (compound 43)
Ice bath is down toward 225mg (0.45mmol) Asp-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Ser-OCH 3, 78mg (0.45mmol) Boc-Gly-OH, 61mg (0.45mmol) HOBT and 10ml anhydrous tetrahydro furan solution in drip N-methylmorpholine, transfer pH9.The solution that obtains mixes with the solution of 90mg (0.45mmol) DCC and 5ml anhydrous tetrahydro furan, 0 ℃ of stirring 2 hours, stirred overnight at room temperature, and TLC shows that raw material point disappears.Reaction mixture filters, filtrate decompression is concentrated into dried, residue acetic acid ethyl dissolution, and the solution that obtains is used 5%KHSO successively 4The aqueous solution is washed, saturated NaHCO 3The aqueous solution is washed, the saturated NaCl aqueous solution is washed.The ethyl acetate layer anhydrous Na of telling 2SO 4That drying, filtration, filtrate decompression are concentrated into is dried, residue separates (petrol ether/ethyl acetate, 2: 1) with silica gel column chromatography, obtains 36mg (61%) title compound, is blue look solid.IR(cm -1，KBr)：3430、3334、3250、1751、1662、1606、1483、1450、1395、1375、1272、830；ESI-MS(m/e)＝665[M] +；[α] 20 D＝-53.8(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 30H 45N 6O 11Calculated value is: C, 54.13; H, 6.81; N, 12.62; Measured value is C, 54.30; H, 7.00; N, 12.81.
[embodiment 33] Gly-Asp-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Ser-OCH 3The preparation of (compound 44)
360mg under the ice bath (0.54mmol) Boc-Gly-Asp-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Ser-OCH 3The solution that constitutes with the 10ml ethyl acetate mixes with 35ml hydrogenchloride/ethyl acetate solution (4mol/l) and stirred 35 minutes, and TLC shows the disappearance of raw material point.Reaction solution is evaporated to dried, and residue is evaporated to after with the 10ml acetic acid ethyl dissolution dried again.This operation 3 times repeatedly is so that the residual hydrogenchloride of Ex-all.Residue obtains 300mg (98%) title compound again with the 10ml ether dissolution and be evaporated to driedly, is blue look solid.ESI-MS(m/e)＝565[M] +；IR(cm -1，KBr)：3430、3334、3250、1751、1662、1606、1483、1450、1395、1375、1272、830；[α] 20 D＝-121.7(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 25H 37N 6O 9Calculated value is: C, 53.09; H, 6.59; N, 14.86; Measured value is C, 52.91; H, 6.41; N, 14.68.
[embodiment 34] Boc-Arg-Gly-Asp-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Ser-OCH 3The preparation of (compound 45)
Ice bath is down toward 360mg (0.64mmol) Gly-Asp-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Ser-OCH 3, drip N-methylmorpholine in 175mg (0.54mmol) Boc-Arg-OH, 73mg (0.54mmol) HOBt and the 15ml anhydrous tetrahydro furan, transfer pH9.The solution that obtains mixes with the solution of 108mg (0.54mmol) DCC and 5ml anhydrous tetrahydro furan, 0 ℃ of stirring 2 hours, stirred overnight at room temperature, and TLC shows that raw material point disappears.Reaction mixture filters, filtrate decompression is concentrated into dried, residue acetic acid ethyl dissolution, and the solution that obtains is used 5%KHSO successively 4The aqueous solution is washed, saturated NaHCO 3The ethyl acetate layer anhydrous Na that the aqueous solution is washed, the saturated NaCl aqueous solution is washed, told 2SO 4That drying, filtration, filtrate decompression are concentrated into is dried, residue separates (petrol ether/ethyl acetate, 2: 1) with silica gel column chromatography, obtains 244mg (55%) title compound, is blue look solid.IR(cm -1，KBr)：3429、3330、3252、1752、1664、1603、1481、1452、1396、1372、1275、833；ESI-MS(m/e)＝821[M] +；[α] 20 D＝-148.4(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 36H 57N 10O 12Calculated value is: C, 52.61; H, 6.99; N, 17.04; Measured value is C, 52.42; H, 6.80; N, 17.22.
[embodiment 35] Boc-Arg-Gly-Asp-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-preparation of Ser-OH (compound 46)
By 170mg (0.21mmol) Boc-Arg-Gly-Asp-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Ser-OCH 3With solution and 3mlNaOH (1mol/l) aqueous solution that 10ml methyl alcohol constitutes, reaction mixture stirred 1 hour for 0 ℃, and TLC shows that raw material point disappears.The reaction mixture concentrating under reduced pressure, residue is transferred pH5 with 10ml saturated common salt water dissolution and with hydrochloric acid (2mol/l).The weakly acidic solution that obtains is with ethyl acetate extraction (15ml * 3), isolating ethyl acetate layer anhydrous Na 2SO 4Drying, filtration, filtrate decompression are concentrated into dried, obtain 142mg (84%) title compound, are blue look solid.IR(cm -1，KBr)：3329、3232、2318-2579、1753、1660、1605、1480、1453、1272、830；ESI-MS(m/e)＝807[M] +；[α] 20 D＝-171.3(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 35H 55N 10O 12Calculated value is: C, 52.03; H, 6.86; N, 17.34; Measured value is C, 51.86; H, 6.70; N, 17.17.
[embodiment 36] Arg-Gly-Asp-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-preparation of Ser-OH (compound 47)
140mg under the ice bath (0.89mmol) Boc-Arg-Gly-Asp-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-solution that Ser-OH and 10ml ethyl acetate constitute mixes with 3.5ml hydrogenchloride/ethyl acetate solution (4mol/l) and stirred 15 minutes, and TLC shows the disappearance of raw material point.Reaction solution is evaporated to dried, and residue is evaporated to after with the 10ml acetic acid ethyl dissolution dried again.This operation 3 times repeatedly is so that the residual hydrogenchloride of Ex-all.Residue obtains 120mg (98%) title compound again with the 10ml ether dissolution and be evaporated to driedly, is blue look solid.IR(cm -1，KBr)：3332、3239、2325-2584、1750、1664、1602、1483、1455、1274、835；ESI-MS(m/e)＝707[M] +；[α] 20 D＝-138.5(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 30H 47N 10O 10Calculated value is: C, 50.91; H, 6.69; N, 19.79; Measured value is C, 51.10; H, 6.51; N, 19.98.
The synthetic route of γ-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group third amino] Glu-Cys-Gly-OH.
[embodiment 37] Boc-Glu-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-preparation of Cys-OMe (compound 48)
Ice bath is down toward 200mg (0.38mmol) Boc-Glu-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-drip N-methylmorpholine in the solution of OH, 72mg (0.42mmol) HClCys-OMe, 50mg (0.38mmol) HOBT and 10ml anhydrous tetrahydro furan, transfer pH9.The solution that obtains mixes with the solution of 90mg (0.42mmol) DCC and 5ml anhydrous tetrahydro furan, 0 ℃ of stirring 2 hours, stirred overnight at room temperature, and TLC shows that raw material point disappears.Reaction mixture filters, filtrate decompression is concentrated into dried, residue acetic acid ethyl dissolution, and the solution that obtains is used 5%KHSO successively 4The aqueous solution is washed, saturated NaHCO 3The ethyl acetate layer anhydrous Na that the aqueous solution is washed, the saturated NaCl aqueous solution is washed, told 2SO 4That drying, filtration, filtrate decompression are concentrated into is dried, residue separates (petrol ether/ethyl acetate, 2: 1) with silica gel column chromatography, obtains 230mg (95%) title compound, is blue look solid.IR(cm -1，KBr)：3430、3326、3245、1750、1661、1607、1482、1454、1395、1370、1272、835；ESI-MS(m/e)＝638[M] +；[α] 20 D＝-91.0(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 29H 44N 5O 9The S calculated value is: C, 54.53; H, 6.94; N, 10.96; Measured value is C, 54.70; H, 7.13; N, 10.78.
[embodiment 38] Boc-Glu-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-preparation of Cys-OH (compound 49)
By 230mg (0.37mmol) Boc-Glu-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-solution and 5ml NaOH (1mol/l) aqueous solution that Cys-OMe and 15ml methyl alcohol constitute, reaction mixture stirred 1 hour for 0 ℃, and TLC shows that raw material point disappears.The reaction mixture concentrating under reduced pressure, residue is transferred pH5 with 10ml saturated common salt water dissolution and with hydrochloric acid (2mol/l).The weakly acidic solution that obtains is with ethyl acetate extraction (3ml * 3), isolating ethyl acetate layer anhydrous Na 2SO 4Drying, filtration, filtrate decompression are concentrated into dried, obtain 170mg (70%) title compound, are blue look solid.IR(cm -1，KBr)：3336、3232、2329-2588、1753、1662、1605、1480、1453、1272、833；ESI-MS(m/e)＝624[M] +；[α] 20 D＝-115.7(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 28H 42N 5O 9The S calculated value is: C, 53.83; H, 6.78; N, 11.21; Measured value is C, 53.66; H, 6.96; N, 11.40.
[embodiment 39] Boc-Glu-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-preparation of Cys-Gly-OMe (compound 50)
Ice bath is down toward 160mg (0.25mmol) Boc-Glu-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-drip N-methylmorpholine in the solution of Cys-OH, 40mg (0.31mmol) HClGly-OMe, 40mg (0.30mmol) HOBt and 10ml anhydrous tetrahydro furan, transfer pH9.The solution that obtains mixes with the solution of 62mg (0.29mmol) DCC and 10ml anhydrous tetrahydro furan, 0 ℃ of stirring 2 hours, stirred overnight at room temperature, and TLC shows that raw material point disappears.Reaction mixture filters, filtrate decompression is concentrated into dried, residue acetic acid ethyl dissolution, and the solution that obtains is used 5%KHSO successively 4The aqueous solution is washed, saturated NaHCO 3The ethyl acetate layer anhydrous Na that the aqueous solution is washed, the saturated NaCl aqueous solution is washed, told 2SO 4That drying, filtration, filtrate decompression are concentrated into is dried, residue separates (petrol ether/ethyl acetate, 2: 1) with silica gel column chromatography, obtains 156mg (90%) title compound, is blue look solid.IR(cm -1，KBr)：3427、3320、3242、1755、1663、1604、1476、1455、1396、1372、1275、834；ESI-MS(m/e)＝695[M] +；[α] 20 D＝+161.9(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 31H 47N 6O 10The S calculated value is: C, 53.51; H, 6.81; N, 12.08; Measured value is C, 53.69; H, 6.98; N, 11.84.
[embodiment 40] Boc-Glu-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-preparation of Cys-Gly-OH (compound 51)
By 120mg (0.16mmol) Boc-Glu-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-solution and 5ml NaOH (1mol/l) aqueous solution that Cys-Gly-OMe and 15ml methyl alcohol constitute, reaction mixture stirred 2 hours for 0 ℃, and TLC shows that raw material point disappears.The reaction mixture concentrating under reduced pressure, residue is transferred pH5 with 2ml saturated common salt water dissolution and with hydrochloric acid (2mol/l).The weakly acidic solution that obtains is with ethyl acetate extraction (3ml * 3), isolating ethyl acetate layer anhydrous Na 2SO 4Drying, filtration, filtrate decompression are concentrated into dried, obtain 60mg (51%) title compound, are blue look solid.IR(cm -1，KBr)：3332、3228、2341-2583、1756、1660、1603、1485、1450、1274、835；ESI-MS(m/e)＝681[M] +；[α] 20 D＝-304.4(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 30H 45N 6O 10The S calculated value is: C, 52.85; H, 6.65; N, 12.33; Measured value is C, 52.67; H, 6.47; N, 12.51.
[embodiment 4] Glu-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-preparation of Cys-Gly-OH (compound 52)
120mg under the ice bath (0.18mmol) Boc-Glu-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-solution that Cys-Gly-OH and 10ml ethyl acetate constitute mixes with 2.5ml hydrogenchloride/ethyl acetate solution (4mol/l) and stirred 40 minutes, and TLC shows the disappearance of raw material point.Reaction solution is evaporated to dried, and residue is evaporated to after with the 10ml acetic acid ethyl dissolution dried again.This operation 3 times repeatedly is so that the residual hydrogenchloride of Ex-all.Residue obtains 80mg (78%) title compound again with the 10ml ether dissolution and be evaporated to driedly, is blue look solid.IR(cm -1，KBr)：3340、3235、2328-2582、1754、1666、1605、1480、1456、1277、836；ESI-MS(m/e)＝581[M] +；[α] 20 D＝+124.5(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 25H 37N 6O 8The S calculated value is: C, 51.62; H, 6.41; N, 14.45; Measured value is C, 51.44; H, 6.23; N, 14.63.
Figure A20051010529100411
N αThe synthetic route of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group ethanoyl] Arg-Gly-Asp-AA-OH, AA=Phe among the AA=Val, 72 and 75 among the AA=Ser, 71 and 74 in 70 and 73.
The preparation of [intermediate preparation embodiment 11] Boc-Ser (Bzl) OBzl (compound 53)
33mg (0.10mmol) Cs2CO3 is dissolved in distilled water, join in the ethanol solution of 59mg (0.20mmol) Boc-Ser (Bzl)-OH, removal of solvent under reduced pressure behind the 40min, residue places moisture eliminator to spend the night, the white solid that obtains is dissolved in dry DMF, drip 34mg (0.2mmol) bromobenzyl, after 50 ℃ of constant temperature stir 16hr, filtering CsBr precipitation, filtrate decompression is distilled to dried, the residue acetic acid ethyl dissolution is used saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride, the washing of 5% sulphur hydracid aqueous solutions of potassium, the organic layer anhydrous sodium sulfate drying of telling, filter, filtrate is at 37 ℃ of following concentrating under reduced pressure, and residue grinds with sherwood oil, obtains 76mg (99%) title compound.
The preparation of [intermediate preparation embodiment 12] HCl Ser (Bzl) OBzl (compound 54)
77mg (0.2mmol) Boc-Ser (Bzl) OBzl is dissolved in 6ml 4mol/L anhydrous hydrogen chloride-ethyl acetate solution, stirring at room, and TLC shows that raw material point disappears behind the 1hr.Reaction mixture is concentrating under reduced pressure at room temperature, and residue also at room temperature concentrates with acetic acid ethyl dissolution, so repeatedly for several times, and up to Ex-all free hydrogenchloride.Residue grinds with anhydrous diethyl ether, gets title compound, is directly used in next step reaction.
The preparation of [intermediate preparation embodiment 13] Boc-Asp (OcHex)-Ser (Bzl) OBzl (compound 55)
63mg (0.2mmol) Boc-Asp (OcHex)-OH is dissolved among the anhydrous THF of 3ml, ices molten 30mg (0.22mmol) N-hydroxy benzo triazole and 48mg (0.22mmol) DCC of adding down, and is stand-by behind the mixing 30min.64mg (0.2mmol) HCl Ser (Bzl) OBzl is dissolved in the anhydrous THF of 5ml, drip N-methylmorpholine and make pH value of solution 9, the mixture that adding obtains above, behind 0 ℃ of reaction 2hr, room temperature reaction 12hr, TLC shows that raw material point disappears stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution is used saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride, the washing of 5% sulphur hydracid aqueous solutions of potassium, the organic layer anhydrous sodium sulfate drying of telling, filter, filtrate is evaporated to dried under 37 ℃, obtains 114mg (98%) title compound, is colorless solid; [α] 20 D=15 (C=0.2, CHCl 3).
The preparation of [intermediate preparation embodiment 14] HCl Asp (OcHex)-Ser (Bzl) OBzl (compound 56)
116mg (0.2mmol) Boc-Asp (OcHex)-Ser (Bzl) OBzl is dissolved in 6ml 4mol/L anhydrous hydrogen chloride-ethyl acetate solution, stirring at room, and TLC shows that raw material point disappears behind the 1hr.Reaction mixture is concentrating under reduced pressure at room temperature, and residue also at room temperature concentrates with acetic acid ethyl dissolution, so repeatedly for several times, and up to Ex-all free hydrogenchloride.Residue grinds with anhydrous diethyl ether, gets title compound, is directly used in next step reaction.
The preparation of [intermediate preparation embodiment 15] Boc-Gly-Asp (OcHex)-Ser (Bzl) OBzl (compound 57)
35mg (0.2mmol) Boc-Gly-OH is dissolved among the anhydrous THF of 3ml, ices molten 30mg (0.22mmol) N-hydroxy benzo triazole and 48mg (0.22mmol) DCC of adding down, and is stand-by behind the mixing 30min.104mg (0.2mmol) HCl Asp (OcHex)-Ser (Bzl) OBzl is dissolved in the anhydrous THF of 5ml, drip N-methylmorpholine and make pH value of solution 8-9, the mixture that adding obtains above, behind 0 ℃ of reaction 2hr, room temperature reaction 12hr, TLC shows that raw material point disappears stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution is used saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride, the washing of 5% sulphur hydracid aqueous solutions of potassium, the organic layer anhydrous sodium sulfate drying of telling, filter, filtrate is evaporated to dried under 37 ℃, obtains 120mg (94%) title compound, is colorless solid.[α] 20 D＝-3(C2，CHCl 3)。
The preparation of [intermediate preparation embodiment 16] HClGly-Asp (OcHex)-Ser (Bzl) OBzl (compound 58)
128mg (0.2mmol) Boc-Gly-Asp (OcHex)-Ser (Bzl) OBzl is dissolved in 6ml 4mol/L anhydrous hydrogen chloride-ethyl acetate solution, stirring at room, and TLC shows that raw material point disappears behind the 1hr.Reaction mixture is concentrating under reduced pressure at room temperature, and residue also at room temperature concentrates with acetic acid ethyl dissolution, so repeatedly for several times, and up to Ex-all free hydrogenchloride.Residue grinds with anhydrous diethyl ether, gets title compound, is directly used in next step reaction.
The preparation of [intermediate preparation embodiment 17] HClValOBzl (compound 59)
61mg (0.2mmol) Boc-ValOBzl is dissolved in 6ml 4mol/L anhydrous hydrogen chloride-ethyl acetate solution, stirring at room, and TLC shows that raw material point disappears behind the 1hr.Reaction mixture is concentrating under reduced pressure at room temperature, and residue also at room temperature concentrates with acetic acid ethyl dissolution, so repeatedly for several times, and up to Ex-all free hydrogenchloride.Residue grinds with anhydrous diethyl ether, gets title compound, is directly used in next step reaction.
The preparation of [intermediate preparation embodiment 18] Boc-Asp (OcHex)-ValOBzl (compound 60)
63mg (0.2mmol) Boc-Asp (OcHex)-OH is dissolved among the anhydrous THF of 3ml, ices molten 30mg (0.22mmol) N-hydroxy benzo triazole and 48mg (0.22mmol) DCC of adding down, and is stand-by behind the mixing 30min.49mg (0.2mmol) HCl ValOBzl is dissolved in the anhydrous THF of 5ml, drip N-methylmorpholine and make pH value of solution 8-9, the mixture that adding obtains above, behind 0 ℃ of reaction 2hr, room temperature reaction 12hr, TLC shows that raw material point disappears stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution is used saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride, the washing of 5% sulphur hydracid aqueous solutions of potassium, the organic layer anhydrous sodium sulfate drying of telling, filter, filtrate is evaporated to dried under 37 ℃, obtains 99mg (98%) title compound, is colorless solid.[α] 20 D=13 (C2, CHCl 3) preparation of [intermediate preparation embodiment 19] HCl Asp (OcHex)-ValOBzl (compound 61)
101mg (0.2mmol) Boc-Asp (OcHex)-ValOBzl is dissolved in 6ml 4mol/L anhydrous hydrogen chloride-ethyl acetate solution, stirring at room, and TLC shows that raw material point disappears behind the 1hr.Reaction mixture is concentrating under reduced pressure at room temperature, and residue also at room temperature concentrates with acetic acid ethyl dissolution, so repeatedly for several times, and up to Ex-all free hydrogenchloride.Residue grinds with anhydrous diethyl ether, gets title compound, is directly used in next step reaction.
The preparation of [intermediate preparation embodiment 20] Boc-Gly-Asp (OcHex)-ValOBzl (compound 62)
35mg (0.2mmol) Boc-Gly-OH is dissolved among the anhydrous THF of 3ml, ices molten 30mg (0.22mmol) N-hydroxy benzo triazole and 48mg (0.22mmol) DCC of adding down, and is stand-by behind the mixing 30min.88mg (0.2mmol) HCl Asp (OcHex)-ValOBzl is dissolved in the anhydrous THF of 5ml, drip N-methylmorpholine and make pH value of solution 8-9, the mixture that adding obtains above, behind 0 ℃ of reaction 2hr, room temperature reaction 12hr, TLC shows that raw material point disappears stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution is used saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride, the washing of 5% sulphur hydracid aqueous solutions of potassium, the organic layer anhydrous sodium sulfate drying of telling, filter, filtrate is evaporated to dried under 37 ℃, obtains 107mg (95%) title compound, is colorless solid.[α] 20 D=7(C2，CHCl 3)
The preparation of [intermediate preparation embodiment 21] HClGly-Asp (OcHex)-ValOBzl (compound 63)
112mg (0.2mmol) Boc-Gly-Asp (OcHex)-ValOBzl is dissolved in 6ml 4mol/L anhydrous hydrogen chloride-ethyl acetate solution, stirring at room, and TLC shows that raw material point disappears behind the 1hr.Reaction mixture is concentrating under reduced pressure at room temperature, and residue also at room temperature concentrates with acetic acid ethyl dissolution, so repeatedly for several times, and up to Ex-all free hydrogenchloride.Residue grinds with anhydrous diethyl ether, gets title compound, is directly used in next step reaction.
The preparation of [intermediate preparation embodiment 22] Boc-PheOBzl (compound 64)
33mg (0.10mmol) Cs2CO3 is dissolved in distilled water, join in the ethanol solution of 53mg (0.20mmol) Boc-Phe-OH, removal of solvent under reduced pressure behind the 40min, residue places moisture eliminator to spend the night, the white solid that obtains is dissolved in dry DMF, drip 34mg (0.2mmol) bromobenzyl, after 50 ℃ of constant temperature stir 16hr, filtering CsBr precipitation, filtrate decompression is distilled to dried, and the residue acetic acid ethyl dissolution is used saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride, the washing of 5% sulphur hydracid aqueous solutions of potassium, the organic layer anhydrous sodium sulfate drying of telling, filter, filtrate is at 37 ℃ of following concentrating under reduced pressure, and residue grinds with sherwood oil, obtain 70mg (99%) title compound, be colorless solid.
The preparation of [intermediate preparation embodiment 23] HClPheOBzl (compound 65)
71mg (0.2mmol) Boc-PheOBzl is dissolved in 6ml 4mol/L anhydrous hydrogen chloride-ethyl acetate solution, stirring at room, and TLC shows that raw material point disappears behind the 1hr.Reaction mixture is concentrating under reduced pressure at room temperature, and residue also at room temperature concentrates with acetic acid ethyl dissolution, so repeatedly for several times, and up to Ex-all free hydrogenchloride.Residue grinds with anhydrous diethyl ether, gets title compound, is directly used in next step reaction.
The preparation of [intermediate preparation embodiment 24] Boc-Asp (OcHex)-PheOBzl (compound 66)
63mg (0.2mmol) Boc-Asp (OcHex)-OH is dissolved among the anhydrous THF of 3ml, ices molten 30mg (0.22mmol) N-hydroxy benzo triazole and 48mg (0.22mmol) DCC of adding down, and is stand-by behind the mixing 30min.58mg (0.2mmol) HCl PheOBzl is dissolved in the anhydrous THF of 5ml, drip N-methylmorpholine and make pH value of solution 8-9, the mixture that adding obtains above, behind 0 ℃ of reaction 2hr, room temperature reaction 12hr, TLC shows that raw material point disappears stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution is used saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride, the washing of 5% sulphur hydracid aqueous solutions of potassium, the organic layer anhydrous sodium sulfate drying of telling, filter, filtrate is evaporated to dried under 37 ℃, obtains 109mg (98%) title compound, is colorless solid.[α] 20 D＝12(C2，CHCl 3)
The preparation of [intermediate preparation embodiment 25] HClAsp (OcHex)-PheOBzl (compound 67)
Under the ice bath 110mg (0.2mmol) Boc-Asp (OcHex)-PheOBzl is dissolved in 6ml 4mol/L anhydrous hydrogen chloride-ethyl acetate solution, stirring at room, TLC shows that raw material point disappears behind the 1hr.Reaction solution is evaporated to dried, and residue is evaporated to after with the 10ml acetic acid ethyl dissolution dried again.This operation 3 times repeatedly is so that the residual hydrogenchloride of Ex-all.Residue again with the 10ml ether dissolution and be evaporated to dried, title compound, be directly used in next step reaction.
The preparation of [intermediate preparation embodiment 26] Boc-Gly-Asp (OcHex)-PheOBzl (compound 68)
35mg (0.2mmol) Boc-Gly-OH is dissolved among the anhydrous THF of 3ml, ices molten 30mg (0.22mmol) N-hydroxy benzo triazole and 48mg (0.22mmol) DCC of adding down, and is stand-by behind the mixing 30min.98mg (0.2mmol) HCl Asp (OcHex)-PheOBzl is dissolved in the anhydrous THF of 5ml, drip N-methylmorpholine and make pH value of solution 8-9, the mixture that adding obtains above, behind 0 ℃ of reaction 2hr, room temperature reaction 12hr, TLC shows that raw material point disappears stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution is used saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride, the washing of 5% sulphur hydracid aqueous solutions of potassium, the organic layer anhydrous sodium sulfate drying of telling, filter, filtrate is evaporated to dried under 37 ℃, obtains 121mg (97%) title compound, is colorless solid.[α] 20 D＝-2(C2，CHCl 3)
The preparation of [intermediate preparation embodiment 27] HClGly-Asp (OcHex)-PheOBzl (compound 69)
Under the ice bath 122mg (0.2mmol) Boc-Gly-Asp (OcHex)-PheOBzl is dissolved in 6ml4mol/L anhydrous hydrogen chloride-ethyl acetate solution, stirring at room, TLC shows that raw material point disappears behind the 1hr.Reaction solution is evaporated to dried, and residue is evaporated to after with the 10ml acetic acid ethyl dissolution dried again.This operation 3 times repeatedly is so that the residual hydrogenchloride of Ex-all.Residue again with the 10ml ether dissolution and be evaporated to dried, title compound, be directly used in next step reaction.
[embodiment 42] N αThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl] Arg-Gly-Asp (OcHex)-Ser (Bzl)-OBzl (compound 70)
95mg (0.20mmol) N α-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino] Arg-OH is dissolved in the anhydrous THF of 4ml, ices molten 30mg (0.22mmol) N-hydroxy benzo triazole and 48mg (0.22mmol) DCC of adding down, and is stand-by behind the mixing 30min.115mg (0.2mmol) HClGly-Asp (OcHex)-Ser (Bzl) OBzl is dissolved in the anhydrous THF of 5ml, drip N-methylmorpholine and make pH value of solution 8-9, the mixture that adding obtains above, behind 0 ℃ of reaction 2h, room temperature reaction 12h, TLC shows that raw material point disappears stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution is used saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride, the washing of 5% sulphur hydracid aqueous solutions of potassium, the organic layer anhydrous sodium sulfate drying of telling, filter, filtrate is evaporated to dried under 37 ℃, gets title compound 191mg (97%) title compound, is blue look solid.IR(cm -1，KBr)：3341、3332、3240、1750、1663、1603、1485、1456、1276、834；ESI-MS(m/e)＝985[M] +；[α] 20 D＝17.8(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 50H 66N 9O 12Calculated value is: C, 60.96; H, 6.75; N, 12.80; Measured value is C, 61.15; H, 6.56; N, 12.62.
[embodiment 43] N αThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl] Arg-Gly-Asp (OcHex)-Val-OBzl (compound 71)
95mg (0.20mmol) N α-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino] Arg-OH is dissolved in the anhydrous THF of 4ml, ices molten 30mg (0.22mmol) N-hydroxy benzo triazole and 48mg (0.22mmol) DCC of adding down, and is stand-by behind the mixing 30min.100mg (0.2mmol) HClGly-Asp (OcHex)-Val (Bzl) OBzl is dissolved in the anhydrous THF of 5ml, drip N-methylmorpholine and make pH value of solution 8-9, the mixture that adding obtains above, behind 0 ℃ of reaction 2h, room temperature reaction 12h, TLC shows that raw material point disappears stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution is used saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride, the washing of 5% sulphur hydracid aqueous solutions of potassium, the organic layer anhydrous sodium sulfate drying of telling, filter, filtrate is evaporated to dried under 37 ℃, gets title compound 171mg (94%) title compound, is blue look solid.IR(cm -1，KBr)：3338、3330、3242、1753、1662、1604、1486、1452、1277、836；ESI-MS(m/e)＝907[M] +；[α] 20 D＝19.5(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 45H 64N 9O 11Calculated value is: C, 59.59; H, 7.11; N, 13.90; Measured value is C, 59.39.15; H, 7.27; N, 13.72.
[embodiment 44] N αThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl] Arg-Gly-Asp (OcHex)-Phe-OBzl (compound 72)
91mg (0.20mmol) N α-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino] Arg-OH is dissolved in the anhydrous THF of 4ml, ices molten 30mg (0.22mmol) N-hydroxy benzo triazole and 48mg (0.22mmol) DCC of adding down, and is stand-by behind the mixing 30min.109mg (0.2mmol) HClGly-Asp (OcHex)-Ser (Bzl) OBzl is dissolved in the anhydrous THF of 5ml, drip N-methylmorpholine and make pH value of solution 8-9, the mixture that adding obtains above, behind 0 ℃ of reaction 2h, room temperature reaction 12h, TLC shows that raw material point disappears stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution is used saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride, the washing of 5% sulphur hydracid aqueous solutions of potassium, the organic layer anhydrous sodium sulfate drying of telling, filter, filtrate is evaporated to dried under 37 ℃, gets title compound 191mg (97%) title compound, is blue look solid.IR(cm -1，KBr)：3339、3330、3244、1752、1661、1605、1486、1452、1274、832；ESI-MS(m/e)＝954[M] +；[α] 20 D=-13(C＝0.2，CHCl 3∶CH 3OH，10∶1)。Ultimate analysis: C 49H 64N 9O 11Calculated value is: C, 61.62; H, 6.75; N, 13.20; Measured value is C, 61.44; H, 6.92; N, 13.37.
[embodiment 45] N αThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl] Arg-Gly-Asp-Ser-OH (compound 73)
99mg (0.1mmol) N α-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl]-Gly-Asp (OcHex)-Phe-OBzl mixes with 1ml methyl-phenoxide and 2ml anhydrous HF, HF is removed in 0 ℃ of reaction 1 hour, decompression.Feed 6ml HF, 0 ℃ of reaction 1 hour, decompression once more and remove HF.Residue solidifies with anhydrous diethyl ether, and the colorless solid thing that obtains changes blue look into after placing and spending the night in ether.Blue look solid SephadexG10 purifying is used the distilled water wash-out.Collect blue colour band, lyophilize, obtain 55mg (76%) title compound, be blue look solid.IR(cm -1，KBr)：3337、3326、3235、2327-2588、1755、1662、1604、1483、1454、1276、833；ESI-MS(m/e)＝722[M] +；[α] 20 D＝-40.0(C＝1.0，H 2O)。Ultimate analysis: C 30H 44N 9O 12Calculated value is: C, 49.86; H, 6.14; N, 17.44; Measured value is C, 49.67; H, 6.31; N, 17.30.
[embodiment 46] N αThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl] Arg-Gly-Asp-Val-OH (compound 74)
95mg (0.1mmol) N α-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl] Arg-Gly-Asp (OcHex)-Val-OBzl mixes with 1ml methyl-phenoxide and 2ml anhydrous HF, HF is removed in 0 ℃ of reaction 1 hour, decompression.Feed 6ml HF, 0 ℃ of reaction 1 hour, decompression once more and remove HF.Residue solidifies with anhydrous diethyl ether, and the colorless solid thing that obtains changes blue look into after placing and spending the night in ether.Blue look solid SephadexG10 purifying is used the distilled water wash-out.Collect blue colour band, lyophilize, get 56mg (76%) title compound, be blue look solid.IR(cm -1，KBr)：3341、3329、3233、2329-2589、1753、1664、1605、1480、1456、1274、836；ESI-MS(m/e)＝734[M] +；[α] 20 D=17.0(C＝1.0，H 2O)。Ultimate analysis: C 32H 48N 9O 11Calculated value is: C, 52.31; H, 6.58; N, 17.16; Measured value is C, 52.50; H, 6.42; N, 17.35.
[embodiment 47] N αThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl]-Gly-Asp-Phe-OH (compound 75)
95mg (0.1mmol) N α-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl] Arg-Gly-Asp (OcHex)-Phe-OBzl mixes with 1ml methyl-phenoxide and 2ml anhydrous HF, HF is removed in 0 ℃ of reaction 1 hour, decompression.Feed 6ml HF, 0 ℃ of reaction 1 hour, decompression once more and remove HF.Residue solidifies with anhydrous diethyl ether, and the colorless solid thing that obtains changes blue look into after placing and spending the night in ether.Blue look solid SephadexG10 purifying is used the distilled water wash-out.Collect blue colour band, lyophilize, get 60mg (80%) title compound, be blue look solid.IR(cm -1，KBr)：3339、3327、3235、2327-2586、1754、1662、1605、1482、1457、1276、834；ESI-MS(m/e)＝782[M] +；[α] 20 D＝20(C＝1.0，H 2O)。Ultimate analysis: C 36H 48N 9O 11Calculated value is: C, 55.23; H, 6.18; N, 16.10; Measured value is C, 55.42; H, 6.35; N, 16.28.
The preparation of [embodiment 48] .Boc-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl) OBzl (compound 76)
86mg (0.20mmol) Boc-Arg (Tos)-OH is dissolved in the anhydrous THF of 4ml, ices molten 30mg (0.22mmol) HOBt and 48mg (0.22mmol) DCC of adding down, stirs 30min.115mg (0.2mmol) HClGly-Asp (OcHex)-Ser (Bzl) OBzl is dissolved in the anhydrous THF of 5ml, drip N-methylmorpholine and make pH value of solution 9, add Boc-Arg (the Tos)-OH mixture that obtains, 0 ℃ of reaction 2hr, room temperature reaction 12hr, TLC show that raw material point disappears.Stopped reaction, filtering DCU, filtrate are evaporated to dried, residue acetic acid ethyl dissolution at 37 ℃.Ethyl acetate solution successively with saturated sodium bicarbonate aqueous solution wash, saturated sodium-chloride is washed, 5% sulphur hydracid aqueous solutions of potassium is washed.The organic layer of telling, anhydrous sodium sulfate drying, filtration, filtrate are evaporated to dried under 37 ℃, obtain 133mg (70%) title compound, are colorless solid.IR(cm -1，KBr)：3342、3325、3239、1766、1665、1605、1600、1590、1482、1457、1395、1370、1274、832；ESI-MS(m/e)＝949[M] +；[α] 20 D=-3.0(C＝2.0，CHCl 3)。Ultimate analysis: C 47H 63N 7O 12Calculated value is: C, 59.41; H, 6.68; N, 10.32; Measured value is C, 59.59; H, 6.80; N, 10.15.
The preparation of [embodiment 49] .HClArg (Tos)-Gly-Asp (OcHex)-Ser (Bzl) OBzl (compound 77)
Under the ice bath 189mg (0.2mmol) Boc-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl) OBzl is dissolved in 6ml4mol/L anhydrous hydrogen chloride-ethyl acetate solution, stirring at room, TLC shows that raw material point disappears behind the 1hr.Reaction solution is evaporated to dried, and residue is evaporated to after with the 10ml acetic acid ethyl dissolution dried again.This operation 3 times repeatedly is so that the residual hydrogenchloride of Ex-all.Residue again with the 10ml ether dissolution and be evaporated to dried, title compound, be directly used in next step reaction.
[embodiment 50] .N αThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl]-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl)-OBzl (compound 78)
61mg (0.2mmol) 4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy acetic acid is dissolved among the anhydrous THF of 3ml, and ice bath adds 30mg (0.22mmol) N-hydroxy benzo triazole and 48mg (0.22mmol) DCC down, and is stand-by behind the mixing 30min.177mg (0.2mmol) HClArg (Tos)-Gly-Asp (OcHex)-Ser (Bzl) Obzl is dissolved in the anhydrous THF of 5ml, drip N-methylmorpholine and make pH value of solution 8-9, the mixture that adding obtains above, behind 0 ℃ of reaction 2hr, room temperature reaction 12hr, TLC shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution is used saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride, the washing of 5% sulphur hydracid aqueous solutions of potassium, the organic layer anhydrous sodium sulfate drying of telling, filter, filtrate is evaporated to dried under 37 ℃, and silica gel dodges formula column chromatography (chloroform: methyl alcohol=10: 1), obtain 207mg (91%) title compound, be blue look solid.Mp76-78℃；IR(cm -1，KBr)：3338、3322、3250、1763、1662、1602、1591、1480、1455、1270、830；ESI-MS(m/e)＝1138[M] +；[α] 20 D＝50(C＝1.0，CH 3OH)。Ultimate analysis: C 57H 72N 9O 14The S calculated value is: C, 60.09; H, 6.37; N, 11.06; Measured value is C, 60.27; H, 6.52; N, 11.28.
The preparation of [embodiment 51] .Boc-Arg (Tos)-Gly-Asp (OcHex)-Val-OBzl (compound 79)
86mg (0.2mmol) Boc-Arg (Tos)-OH is dissolved in the anhydrous THF of 4ml, ices molten 30mg (0.22mmol) N-hydroxy benzo triazole and 48mg (0.22mmol) DCC of adding down and mixes, and stirs 30min.99mg (0.2mmol) HClGly-Asp (OcHex)-Va1OBzl is dissolved in the anhydrous THF of 5ml, drip N-methylmorpholine and make pH value of solution 9, the Boc-Arg that adding obtains above (Tos)-OH mixture, 0 ℃ of reaction 2hr, room temperature reaction 12hr, TLC show that raw material point disappears.Stopped reaction, filtering DCU, filtrate are evaporated to dried, residue acetic acid ethyl dissolution at 37 ℃.Ethyl acetate solution successively with saturated sodium bicarbonate aqueous solution wash, saturated sodium-chloride water solution is washed, 5% sulphur hydracid aqueous solutions of potassium is washed.The organic layer of telling is evaporated to dried under 37 ℃ with anhydrous sodium sulfate drying, filtration, filtrate, obtain 132mg (76%) title compound, is colorless solid.Mp79-81℃；IR(cm -1，KBr)：3340、3322、3236、1761、1660、1602、1601、1593、1480、1455、1396、1372、1276、830；ESI-MS(m/e)＝871[M] +；[α] 20 D=-6(C＝2.0，CHCl 3)。Ultimate analysis: C 42H 61N 7O 11The S calculated value is: C, 57.8 5; H, 7.05; N, 11.24; Measured value is C, 58.03; H, 7.22; N, 11.38.
The preparation of [embodiment 52] .HClArg (Tos)-Gly-Asp (OcHex)-Val-OBzl (compound 80)
Under the ice bath 174mg (0.2mmol) Boc-Arg (Tos)-Gly-Asp (OcHex)-ValOBzl is dissolved in 6ml 4mol/L anhydrous hydrogen chloride-ethyl acetate solution, stirring at room, TLC shows that raw material point disappears behind the 1hr.Reaction solution is evaporated to dried, and residue is evaporated to after with the 10ml acetic acid ethyl dissolution dried again.This operation 3 times repeatedly is so that the residual hydrogenchloride of Ex-all.Residue again with the 10ml ether dissolution and be evaporated to dried, title compound, be directly used in next step reaction.
[embodiment 53] .N αThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl]-Arg (Tos)-Gly-Asp (OcHex)-Val-OBzl (compound 81)
61mg (0.2mmol) 4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy acetic acid is dissolved among the anhydrous THF of 3ml, and ice bath adds 30mg (0.22mmol) N-hydroxy benzo triazole and 48mg (0.22mmol) DCC down and mixes, and stirs 30min.161mg (0.2mmol) HClArg (Tos)-Gly-Asp (OcHex)-ValOBzl is dissolved in the anhydrous THF of 5ml, drip N-methylmorpholine and make pH value of solution 9, the 4-(1 that adding obtains above, 3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) mixture of phenoxy acetic acid, 0 ℃ of reaction 2hr, room temperature reaction 12hr, TLC show that raw material point disappears.Stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, successively with saturated sodium bicarbonate aqueous solution wash, saturated sodium-chloride water solution is washed, 5% sulphur hydracid aqueous solutions of potassium is washed.The organic layer of telling is evaporated to dried, silica gel sudden strain of a muscle formula column chromatography (chloroform: methyl alcohol=10: 1), obtain 190mg (90%) title compound, be blue look solid with anhydrous sodium sulfate drying, filtration, filtrate under 37 ℃.Mp92-94℃；IR(cm -1，KBr)：3338、3325、3234、1760、1664、1606、1600、1590、1483、1450、1275、832；ESI-MS(m/e)＝1060[M] +；[α] 20 D＝55(C＝1.0，CH 3OH)。Ultimate analysis: C 52H 70N 9O 13The S calculated value is: C, 58.85; H, 6.65; N, 11.88; Measured value is C, 58.69; H, 6.83; N, 11.69.
The preparation of [embodiment 54] .Boc-Arg (Tos)-Gly-Asp (OcHex)-PheOBzl (compound 82)
86mg (0.2mmol) Boc-Arg (Tos)-OH is dissolved among the anhydrous THF of 4ml, ices molten 30mg (0.22mmol) N-hydroxy benzo triazole and 48mg (0.22mmol) DCC of adding down, and is stand-by behind the mixing 30min.109mg (0.2mmol) HCl Gly-Asp (OcHex)-PheOBzl is dissolved in the anhydrous THF of 5ml, drip N-methylmorpholine and make pH value of solution 8-9, the mixture that adding obtains above, behind 0 ℃ of reaction 2hr, room temperature reaction 12hr, TLC shows that raw material point disappears, stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, use saturated sodium bicarbonate aqueous solution successively, saturated sodium-chloride, the washing of 5% sulphur hydracid aqueous solutions of potassium, the organic layer anhydrous sodium sulfate drying of telling filters, filtrate is evaporated to dried under 37 ℃, gets mp79-81 ℃ of title compound 110mg (60%) [α] 20 D-8 (C2, CHCl 3)
The preparation of [embodiment 55] .HClArg (Tos)-Gly-Asp (OcHex)-PheOBzl (compound 83)
184mg (0.2mmol) Boc-Arg (Tos)-Gly-Asp (OcHex)-PheOBzl is dissolved in 6ml4mol/L anhydrous hydrogen chloride-ethyl acetate solution, stirring at room, and TLC shows that raw material point disappears behind the 1hr.Reaction mixture is concentrating under reduced pressure at room temperature, and residue also at room temperature concentrates with acetic acid ethyl dissolution, so repeatedly for several times, and up to Ex-all free hydrogenchloride.Residue grinds with anhydrous diethyl ether, gets title compound, is directly used in next step reaction.
[embodiment 56] .N αThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl]-Arg (Tos)-Gly-Asp (OcHex)-Phe-OBzl (compound 84)
61mg (0.2mmol) 4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy acetic acid is dissolved among the anhydrous THF of 3ml, and ice bath adds 30mg (0.22mmol) N-hydroxy benzo triazole and 48mg (0.22mmol) DCC down and mixes, and stirs 30min.171mg (0.2mmol) HClArg (Tos)-Gly-Asp (OcHex)-PheObzl is dissolved in the anhydrous THF of 5ml, drip N-methylmorpholine and make pH value of solution 9, the 4-(1 that adding obtains above, 3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) mixture of phenoxy acetic acid, 0 ℃ of reaction 2hr, room temperature reaction 12hr, TLC show that raw material point disappears.Stopped reaction, filtering DCU, filtrate is evaporated to dried at 37 ℃, the residue acetic acid ethyl dissolution, successively with saturated sodium bicarbonate aqueous solution wash, saturated sodium-chloride water solution is washed, 5% sulphur hydracid aqueous solutions of potassium is washed.The organic layer of telling is evaporated to dried, silica gel sudden strain of a muscle formula column chromatography (chloroform: methyl alcohol=10: 1), obtain 200mg (90%) title compound, be blue look solid with anhydrous sodium sulfate drying, filtration, filtrate under 37 ℃.Mp108-110 ℃; IR (cm -1, KBr): 3336,3322,3230,1764,1662,1604,1601,1593,1480,1452,1273,830; ESI-MS (m/e)=1108[M] +[α] 20 D=-38 (C=1.0, CH 3OH). ultimate analysis: C 56H 70N 9O 13The S calculated value is: C, 60.63; H, 6.36; N, 11.36; Measured value is C, 60.80; H, 6.17; N, 11.18.
[embodiment 57] .N αThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl]-Arg-Gly-Asp-SerOH (compound 85)
140mg (0.1mmol) N α-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl]-Arg (Tos)-Gly-Asp (OcHex)-Ser (Bzl)-OBzl mixes with 1ml methyl-phenoxide and 2ml anhydrous HF, HF is removed in 0 ℃ of reaction 1 hour, decompression.Feed 6ml HF, 0 ℃ of reaction 1 hour, decompression once more and remove HF.Residue solidifies with anhydrous diethyl ether, and the colorless solid thing that obtains changes blue look into after placing and spending the night in ether.Blue look solid SephadexG10 purifying is used the distilled water wash-out.Collect blue colour band, lyophilize, obtain 60mg (73%) title compound, be blue look solid.Mp154 ℃ of decomposition, IR (cm -1, KBr): 3335,3328,3233,2324-2585,1753,1662,1604,1480,1456,1274,830; [α] 20 D=-40 (C=1.0, H 2O), ESI-MS (m/e)=722[M] +Ultimate analysis: C 30H 44N 9O 12Calculated value is: C, 49.86; H, 6.14; N, 17.44; Measured value is C, 50.01; H, 5.97; N, 17.63.
[embodiment 58] .N αThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl]-Arg-Gly-Asp-Val-OH (compound 86)
106mg (0.1mmol) N α-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl]-Arg (Tos)-Gly-Asp (OcHex)-Val-OBzl is with after 1ml methyl-phenoxide and 2ml anhydrous HF are mixed, and HF is removed in 0 ℃ of reaction 1 hour, decompression.Feed 6ml HF, 0 ℃ of reaction 1 hour, decompression once more and remove HF.Residue solidifies with anhydrous diethyl ether, and the colorless solid thing that obtains changes blue look into after placing and spending the night in ether.Blue look solid SephadexG10 purifying is used the distilled water wash-out.Collect blue colour band, lyophilize, obtain 50mg (70%) title chemical combination, be blue look solid.Mp160 ℃ of decomposition, IR (cm -1, KBr): 3341,3329,3233,2329-2589,1753,1664,1605,1480,1456,1274,836; [α] 20 D=17 (C=1.0, H 2O), ESI-MS (m/e)=734[M] +Ultimate analysis: C 32H 48N 9O 11Calculated value is: C, 52.31; H, 6.58; N, 17.16; Measured value is C, 52.47; H, 6.74; N, 17.29.
[embodiment 59] N αThe preparation of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl]-Arg-Gly-Asp-PheOH (compound 87)
111mg (0.1mmol) N α-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl]-Arg (Tos)-Gly-Asp (OcHex)-Phe-OBzl is with after 1ml methyl-phenoxide and 2ml anhydrous HF are mixed, 0 ℃ of reaction 1 hour, and HF is removed in decompression.Feed 6ml HF, 0 ℃ of reaction 1 hour, decompression once more and remove HF.Residue solidifies with anhydrous diethyl ether, and the colorless solid thing that obtains changes blue look into after placing and spending the night in ether.Blue look solid SephadexG10 purifying is used the distilled water wash-out.Collect blue colour band, lyophilize, obtain 60mg (80%) title compound, be blue look solid.Mp158 ℃ of decomposition, IR (cm -1, KBr): 3337,3325,3233,2329-2588,1756,1661,1603,1484,1455,1273,831; [α] 20 D=20 (C=1.0, H 2O), ESI-MS (m/e)=782[M] +Ultimate analysis: C 36H 48N 9O 11Calculated value is: C, 55.23; H, 6.18; N, 16.10; Measured value is C, 55.42; H, 6.00; N, 16.28.
The determination of activity of [test example 1] The compounds of this invention protection cell Green Tea Extract damage
One, test compound: the following compound that the embodiment of the invention is prepared: compound 9, compound 12, compound 13, compound 15, compound 16, compound 18, compound 21, compound 22, compound 23, compound 24, compound 28, compound 35, compound 40, compound 47, compound 52, compound 73, compound 74, compound 75.
(DMSO content is lower than 0.5%, V: V) hydrotropy through φ 0.22 μ m filtering with microporous membrane degerming, is distributed into small packages-85 ℃ refrigerator and preserves in the DMEM serum-free medium solution of above-claimed cpd with DMSO (dimethyl sulfoxide (DMSO)).Thaw during experiment, be diluted to different concns with serum-free medium.
Two, test materials and reagent:
Sodium Nitroprusside (Sodium Nitroprusside, SNP), MTT (tetramethyl-azo azoles salt), poly-l-lysine; Sodium.alpha.-ketopropionate is available from Sigma company; High sugared Dulbecco ' s modified Eagle ' smedium (DMEM) substratum, foetal calf serum is available from Gibco BRL company; Horse serum is available from HyClone company; Ferrous sulfate, hydrogen peroxide is available from Beijing reagent company.
The PC12 cell is a rat suprarenal gland pheochromocytoma clone strain.
Complete culture solution: high sugared DMEM nutrient solution includes 10% horse serum, 5% foetal calf serum, 1.0mM Sodium.alpha.-ketopropionate, 100UmL -1Penicillin, 100gmL -1Streptomycin sulphate, pH are about 7.2.
The pastille nutrient solution: respectively with different compounds with the 0.5%DMSO hydrotropy, be made into concentration and be respectively 12.5 μ M, 25 μ M, 50 μ M, the high sugared DMEM nutrient solution of 100,200 μ M contains the 1.0mM Sodium.alpha.-ketopropionate, 100UmL -1Penicillin, 100gmL -1Streptomycin sulphate, pH are about 7.2, do not contain serum.
Three, test method
The PC12 cell is at 37 ℃, relative humidity 100%, 5%CO 2Under-95% air conditions, cultivate in the sugared DMEM complete culture solution of height, changed a not good liquor in 2-3 days, passed a generation in 5-6 days.Digest 1min with 0.02%EDTA when going down to posterity, the piping and druming cell dispersion becomes single cell suspension, and centrifugal (800rpm 5min), through the PBS washing once, again with the complete culture solution suspension cell, is inoculated in bag by the culturing bottle of poly-l-lysine.Get be in logarithmic phase cell with 2 * 10 4The density in/hole goes down to posterity to be inoculated in and wraps in advance by in 96 well culture plates of poly-l-lysine, cultivates and carries out following three tests in 24 hours later on respectively:
1, nitrogen protoxide is removed determination of activity
Cell inoculation is in 96 orifice plates after 24 hours, and the reject original fluid adds the pastille nutrient solution that contains different sorts and different concns test compound, is placed on 37 ℃, relative humidity 100%, 5%CO 2-95% air conditions is cultivated 40min down to adapt to pharmaceutical environment, adds the serum-free medium 10 μ l that contain SNP (Sodium Nitroprusside) then, makes final concentration reach 0.5mM, acts on after 12 hours, carries out cytoactive and measures.Experiment is provided with and does not add the blank group of damage, singly add the model group of damage, and has not only added medicine but also added the experimental group of damage, establishes 4 multiple holes for every group and makes parallel sample.Singly add the medicine contrast of medicine and the blank of DMSO and the experiment proved that with the normal blank of cultivating there is not significant difference, so medicine and DMSO ignore to the influence of experiment.
2, hydrogen peroxide is removed determination of activity
Method and nitric oxide production experimental model basically identical, damage condition are 400 μ M H 2O 2Act on 12 hours
3, hydroxy radical qiao is removed determination of activity
Method and nitric oxide production experimental model basically identical, damage condition are 80 μ M H 2O 2/ 30 μ MFe (II) act on 12 hours.
Adopt mtt assay to measure cell activity, concrete grammar is as follows:
The ultimate principle of mtt assay is yellow soluble substrate MTT[3-(4,5)-two methyl-2-thiazole-(2,5)-and phenylbenzene bromination tetrazole indigo plant] can be reduced to insoluble blue particle (formazan) by succinodehydrogenase in the plastosome, because the activity of the cell of damaged or the succinodehydrogenase of dead cell reduces or forfeiture, can be according to the relative vigor of how much estimating cell that generates formazan.
During concrete operations cell to be measured is added 2.5mg/ml MTT solution, 10 μ l/ holes make final concentration reach 0.5mg/mL, put into CO 2Incubator, 5%CO 2, hatched 4 hours for 37 ℃, remove MTT solution, add DMSO 100 μ l/ holes, horizontal shaking table shakes up, and detects the growing amount of blue first a ceremonial jade-ladle, used in libation after 20 minutes with full-automatic microplate reader, and the detection wavelength is 570/630nm.
The calculating of cell protective effect:
The protective effect of medicine pair cell is calculated according to following formula: Effect%=(A Drug-A Model)/(A Control-A Model) * 100.Carry out nonlinear fitting by drug concentrations and protection efficient and do the measuring effect curve, obtain the medium effective concentration EC of every kind of compound 50Value.
Four, test-results
Test-results sees Table 1.
Table 1 The compounds of this invention free radical scavenging activity test-results
EC 50(μ M) value
NO H 2O 2· ·OH
Compound 9 42.31±3.04 48.62±3.31 58.22±3.11
Compound 12 42.66±2.18 49.89±2.98 59.00±2.99
Compound 13 38.99±2.78 47.82±2.93 55.63±3.00
Compound 15 41.77±2.55 49.62±2.80 58.06±2.62
Compound 16 37.84±3.06 46.98±2.91 54.90±3.04
Compound 18 92.30±2.05 31.64±2.20 100.08±2.17
Compound 21 90.56±2.64 29.85±2.66 98.77±3.06
Compound 22 89.90±2.78 29.33±2.85 97.72.±3.22
Compound 23 90.60±2.79 30.07±2.88 99.11±3.09
Compound 24 92.88±2.95 32.07±2.69 100.92±3.12
Compound 28 93.00±2.90 32.41±2.94 101.76±2.99
Compound 35 93.61±3.00 33.36±2.93 102.85±2.92
Compound 40 88.99±3.22 28.83±3.11 99.00±3.03
Compound 47 92.86±2.92 32.83±2.87 101.78±3.20
Compound 52 93.01±3.00 32.04±3.19 101.95±3.24
Compound 73 92.92±3.05 31.99±3.10 101.09±3.31
Compound 74 93.34±3.11 32.62±3.18 102.11±2.96
Compound 75 93.62±3.40 33.01±3.22 103.01±3.60
n＝6
Test-results shows that The compounds of this invention has good free radical scavenging activity.
[test example 2] The compounds of this invention is removed the activity test of NO
One, test materials
1, for the reagent thing: the following compound that the embodiment of the invention is prepared: compound 9, compound 12, compound 13, compound 15, compound 16, compound 18, compound 21, compound 22, compound 23, compound 24, compound 28, compound 35, compound 40, compound 47, compound 52, compound 73, compound 74, compound 75.
2, laboratory animal: body weight 250-300g, male Wistar rat is available from Department Of Medicine, Peking University laboratory animal portion.
Two, test method and result
Body weight 250-300g male Wistar rat is divided into blank group and test group at random.Fasting is 12 hours before the art, freely drinks water, and dislocation of cervical vertebra causes death, and opens chest and wins thoracic aorta rapidly, peels off the reticular tissue that adheres to, and blood vessel is cut into the long arterial ring of 3-5mm, places in the 15ml perfusion bath, and bath fills and passes to 95%O 2=5%CO 237 ℃ of constant temperature Krebs-Henseleit (KH) liquid 15ml of gas, fixedly the hook of arterial ring connects tonotransducer, traces the vasomotion curve on dual-trace recorder, and chart speed is 1mm/min.The adjustment resting tension is 1.0g, and giving final concentration behind the balance 30min is 1 * 10 -9Mol/L NE (norepinephrine) shrinks to play to swash in advance artery and acts on, flush away NE, and behind the balance 30min, giving final concentration is 1 * 10 -9The NE of mol/L waits to shrink tension force continually and steadily after the platform level, and the blank group adds dehydrated alcohol 15 μ l, and it is 10 that test group adds that final concentration is respectively -4, 10 -5, 10 -6Mol/L for reagent thing 15 μ l (being dissolved in dehydrated alcohol), treat steadily after, (final concentration is 10 to add 1.5 μ l Ach -6Mol/L).Medicine is removed the ability of NO and is recently represented (n=6) to suppress Ach diastole percentage, and test-results sees Table 2.
Table 2. representative tetrahydroglyoxaline-amino acid of the present invention and tetrahydroglyoxaline-RGD peptide suppresses the Ach diastolic blood vessel activity
Percent inhibition
Figure A20051010529100581
Compound
100μmol 10μmol 1μmol
NS 1.64-1.4
Compound 9 98.3 ± 4.8 74.5 ± 5.2 27.8 ± 5.6
Compound 12 99.2 ± 4.5 73.1 ± 5.1 26.7 ± 5.2
Compound 13 98.6 ± 4.8 72.3 ± 5.0 25.4 ± 5.1
Compound 15 97.9 ± 4.2 72.7 ± 4.9 28.0 ± 5.5
Compound 16 98.9 ± 4.9 74.0 ± 5.7 28.7 ± 5.4
Compound 18 95.9 ± 5.3 70.7 ± 5.5 24.8 ± 5.6
Compound 21 95.0 ± 5.5 71.2 ± 5.0 24.1 ± 5.3
Compound 22 96.1 ± 4.9 72.4 ± 5.3 25.7 ± 5.6
Compound 23 99.1 ± 5.6 73.9 ± 5.4 26.6 ± 5.5
Compound 24 96.6 ± 5.4 72.8 ± 5.3 26.2 ± 5.6
Compound 28 95.9 ± 4.6 71.3 ± 5.0 24.0 ± 4.9
Compound 35 95.9 ± 5.0 70.8 ± 5.5 25.4 ± 5.6
Compound 40 95.5 ± 5.2 70.1 ± 5.3 24.1 ± 5.0
Compound 47 947 ± 5.1 70.8 ± 5.0 24.0 ± 5.4
Compound 52 96.1 ± 4.9 72.1 ± 5.3 25.2 ± 5.1
Compound 73 96.2 ± 4.9 72.1 ± 5.3 24.9 ± 5.3
Compound 74 96.0 ± 5.2 72.4 ± 5.2 26.0 ± 5.8
Compound 75 95.8 ± 5.0 71.6 ± 5.1 26.3 ± 5.5
n=6
Test-results shows that The compounds of this invention has good nitrogen protoxide and removes active.
The antithrombotic acitivity of [test example 3] The compounds of this invention
One, test materials
1, test compound: compound 73, compound 74, compound 75 that the embodiment of the invention is prepared; Positive control medicine: Asprin (Asprine); Negative control medicine: heparin-saline (NS).
2, laboratory animal: body weight 250-300g male wistar rat, available from Department Of Medicine, Peking University laboratory animal portion.
Two, test method and result
Male Wistar rat, abdominal injection Sodital sodium solution is anaesthetized, isolate right common carotid artery and left external jugular vein, put into the long silk thread of 6cm of weighing in advance in the stage casing of polyethylene tube, (50IU/kg) is full of polyethylene tube with heparin-saline, one end is inserted left external jugular vein, with syringe with physiological saline (3ml/kg), the normal saline solution of acetylsalicylic acid (20mgl/kg), the normal saline solution of test compound slowly injects polyethylene tube from the other end, original heparin has been pushed in the right external jugular vein in the pipe at this moment, and major part is a test compound solution in the pipe, then the injectable drug end is inserted right common carotid artery.Blood flows to left external jugular vein from right common carotid artery through polyethylene tube, and middle Herba Clinopodii behind the 15min takes out silk thread and weighs, and gross weight deducts silk thread weight and is wet weight of thrombus.Wet thrombus placed for 1 week in moisture eliminator after, measure the thrombus dry weight.The average of the thrombus weight of each group of statistics and standard deviation (X ± SE), and do the t check, the thrombus weight value that blank group, positive controls and different compounds give sees Table 3.
Table 3. tetrahydroglyoxaline of the present invention-RGD peptide suppresses the thrombosis activity
Dosage Wet weight of thrombus (mg) Thrombus dry weight (mg)
NS Asprine compound 73 compounds 74 compounds 75 3ml/kg 20mg/kg 5μmol/kg 5μmol/kg 5μmol/kg 39.5±5.4 23.3±3.0 a 25.0±2.8 a，b 26.1±3.6 a，b 27.2±3.4 a，b 6.8±1.9 3.0±1.4 a 3.2±1.7 a，b 3.6±1.1 a，b 3.5±1.5 a，b
N=10 a) compares p＜0.05 with NS; B) compare p＞0.05 with Asprine
The test-results explanation, The compounds of this invention 73, compound 74 and compound 75 have good antithrombotic acitivity.
The thrombus dissolving activity activity test of [test example 4] tetrahydroglyoxaline-PAK of the present invention
One, 1, test compound: the compound 35 that the embodiment of the invention is prepared; 2, control compound: PAK (Pro-Ala-Lys); UK (urea kinases), NS (physiological saline).
Two, test method and result:
The preparation of thrombus: in the Glass tubing of vertical fixing (external diameter 5.0mm, the pipe end seals with Capsule for long 15mm, internal diameter 2.5mm), fill with the rat artery fresh blood, the thrombus standing bolt of an acupuncture needle material of insertion rapidly in the pipe.This thrombus fixedly spiral diameter is acupuncture needle coiled on No. 4 syringe needles 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 shape.After the blood coagulation 15 minutes, the careful Capsule of removing the Glass tubing bottom, carefully be stained with the position and separate gently thrombus and glass mouth of pipe place are sticking with tweezers,, from Glass tubing, take out fixedly spiral of the thrombus that wrapped up by thrombus carefully then with the fixing fixing holder handle of spiral of thrombus of tweezers.Accurately weigh.
The preparation of bypass duct: bypass duct constitutes by three sections, and the stage casing is a polyethylene rubber tube, long 60.0mm, and internal diameter 3.5mm, two ends are the polyethylene tube of identical material, pipe range 100.0mm, internal diameter 1.0mm, external diameter 2.0mm.One end of this pipe pulls into point pipe (being used to insert rat carotid artery or vein), and external diameter is 1.0mm, the outer cover one segment length 7.0mm of the other end, and external diameter is the polyethylene tube (overstriking of 3.5mm; Be used for being connected with the polyethylene rubber tube in stage casing), the inwall of three sections pipes is all used 2% silicone oil diethyl ether solution silanization.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 fill with in the pipe heparin-saline solution (50IU/kg, 30mg/90ml) standby.
Rat neck arteriovenous shut Thrombolysis Model: the Wistar rat (male, 180g -320g) available from Department Of Medicine, Peking University laboratory animal portion (credit number: the moving word 01-3056 of doctor).Anaesthetize by 80mg/kg dosage abdominal injection Sodital sodium solution.Undergo surgery after the anesthetized rat dorsal position is fixing: separate tracheae, separate right common carotid artery, proximal part in right common carotid artery is clamped blood vessel with bulldog clamp, cut an angle carefully at distal end as far as possible, insert the polyethylene tube (internal diameter 1.0mm, external diameter 2.0mm) that one one end pulls into sharp pipe, from pipe, emit about 1ml arterial blood after, again clamp artery with bulldog clamp, prepare thrombus by 1.1 described methods immediately.In the preparation thrombus, separate the total vein of left neck.After treating that thrombus performs and performing bypass duct by 1.2 described methods, on the total vein proximal part of left neck that exposes, cut an angle carefully, the sharp pipe of the bypass duct for preparing is above inserted the proximal part of the total vein opening of left neck by angle, fix with 4 trumpeter's art sutures and vein, in the bypass duct stage casing thrombus fixedly the holder handle of spiral point to vein end.After pushing the heparin-saline solution (50IU/kg) of accurate amount with the sharp pipe of syringe by the bypass duct the other end, clamp vein with bulldog clamp at proximal part, take off syringe from the tip of polyethylene tube, take off the bloodletting pipe from artery, the proximal part of artery angle is inserted in the tip of polyethylene tube, fix, open bulldog clamp with 4 trumpeter's art sutures and artery, make blood flow flow to vein by bypass duct from artery, this is a rat neck arteriovenous shut Thrombolysis Model.
Thrombolysis experiment: adopt preface to be used to the method administration.Model manipulation is the same, in the end open before the bulldog clamp, with scalp acupuncture with physiological saline (blank), urokinase normal saline solution (positive control), the normal saline solution of testing compound is by the stage casing of bypass tube, thrust away from the fixing nearly vein place of spiral of thrombus, after opening bulldog clamp, slowly medicine is injected in the blood in 3 minutes, the sequential action of pressing vein-heart one artery is to thrombus.Timing immediately during from start injection, after 90 minutes from bypass duct the fixing spiral of removal of thromboses, accurately weigh, obtain the fixedly weight difference before and after the spiral administration of thrombus in every rat bypass duct, the difference average of the thrombus weight of each group of statistics and standard deviation (X ± S), and do the t check.
Test-results sees Table 4.
Table 4. tetrahydroglyoxaline of the present invention-PAK thrombus dissolving activity
Compound Dosage Thrombus loss of weight (mg)
NS 3ml/kg 15.31±3.57
UK 2000IU/kg 24.10±3.54
PAK 10umol/kg 24.63±4.44
Compound 35 10umol/kg 26.20±4.21
n＝10。
Test-results shows that The compounds of this invention 35 has good thrombus dissolving activity.

Claims (10)

1. the compound of following general formula (I)-(VI):
Wherein, in the general formula (I), R 1Be carbobenzoxy-(Cbz) or hydrogen atom; In the general formula (II), R 2During for tertbutyloxycarbonyl, R 3Be hydrogen atom; R 2During for hydrogen atom, R 3Be methyl; In the general formula (III), R 4During for tertbutyloxycarbonyl, R 5Be methyl or hydrogen atom; R 4During for hydrogen atom, R 5Be methyl; In the general formula (IV), R 6Be methyl or hydrogen atom; In the logical formula V, work as R 7During for tertbutyloxycarbonyl, R 8Be hydrogen atom; R 7Be hydrogen atom, R 8Be methyl or hydrogen atom; In the general formula (VI), R 9During for tertbutyloxycarbonyl, R 10Be hydrogen atom; R 9During for hydrogen atom, R 10Be methyl or hydrogen atom.
2. the intermediate of claim 1 formula of (I) compound: N ω-carbobenzoxy-(Cbz)-N α-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl]-Lys-OBzl; The key intermediate of claim 1 formula of (V) compound: Boc-Asp (NHCH 2CH 2Br)-OH; Boc-Asp (NHCH 2CH 2Br)-OCH 3Boc-Asp[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-OCH 3The key intermediate of preparation claim 1 formula of (VI) compound: Boc-Glu (NHCH 2CH 2Br)-OH, Boc-Glu (NHCH 2CH 2Br)-OCH 3
3. a method for preparing general formula (I)-(VI) compound of claim 1 comprises; 2-is replaced-1,3-dioxy base tetrahydroglyoxaline is guided on the basic side chain or alpha-amino group of L-Lys or L-Arg according to ordinary method, or with the derivative Boc-Asp (NHCH of L-Asp or L-Glu 2CH 2Br)-OH, Boc-Asp (NHCH 2CH 2Br)-OCH 3, Boc-Glu (NHCH 2CH 2Br)-OH, Boc-Glu (NHCH 2CH 2Br)-OCH 3Implement oxygen-alkylated reaction, promptly get general formula compound of the present invention.
4. by the following polypeptide of the compound of claim 1 fixed point mark: Pro-Ala-[N ω-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group ethanoyl] Lys-OH; N G-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group ethanoyl] Arg-Gly-Asp-Val-OH; Arg-Gly-[β-aspartoyl [4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group third amino] Asp-Ser-OH; γ-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group third amino] Glu-Cys-Gly-OH; The structural formula of above-mentioned 4 peptide species is as follows respectively:
Figure A2005101052910003C1
5. Pro-Ala-[N in the claim 4 ωThe intermediate of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group ethanoyl] Lys-OH:
Boc-Ala-N ω-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Lys-OCH 3Ala-N ω-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Lys-OCH 3Boc-Pro-Ala-N ω-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Lys-OCH 3Boc-Pro-Ala-N ω-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Lys-OH;
N in the claim 4 GThe intermediate of-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group ethanoyl] Arg-Gly-Asp-Val-OH: N α-Boc-N G-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethanoyl] Arg-Gly-Ome; N α-Boc-N G-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino] Arg-Gly-OH; N α-Boc-N G-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino] Arg-Gly-Asp (OBzl)-Val-OMe; N α-Boc-N G-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino] Arg-Gly-Asp-Val-OH;
Arg-Gly-[β-aspartoyl in the claim 4 [4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group third amino] and the intermediate of Asp-Ser-OH: Boc-Asp-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Ser-OCH 3Asp-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Ser-OCH 3Boc-Gly-Asp-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Ser-OCH 3Gly-Asp-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Ser-OCH 3Boc-Arg-Gly-Asp-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Ser-OCH 3Boc-Arg-Gly-Asp-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Ser-OH;
γ in the claim 4-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) phenoxy group third amino] and the intermediate of Glu-Cys-Gly-OH: Boc-Glu-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Cys-OMe; Boc-Glu-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Cys-OH; Boc-Glu-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Cys-Gly-OMe; Boc-Glu-[4-(1,3-dioxy-4,4,5,5-tetramethyl--2-tetrahydroglyoxaline-2-yl) benzene oxygen ethylamino]-Cys-Gly-OH.
6. the application of arbitrary compound of claim 1 formula of (I)-(VI) in synthetic polypeptide.
7. the application of arbitrary compound of claim 1 formula of (I)-(VI) in preparation removing nitrogen protoxide, hydrogen peroxide or hydroxy radical qiao medicine.
8. arbitrary polypeptide of claim 6 is preparing the purposes of removing in the nitrogen protoxide medicine.
9. a medicinal compositions is characterized in that upward the arbitrary compound and the pharmaceutically acceptable carrier of claim 1 general formula (I)-(VI) of effective dose are formed by treatment.
10. a medicinal compositions is characterized in that upward the arbitrary polypeptide and the pharmaceutically acceptable carrier of the claim 4 of effective dose are formed by treatment.
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JP2015529209A (en) * 2012-09-05 2015-10-05 シャンハイ ルーモサ セラピューティクス カンパニー リミテッド Novel compounds having both thrombolysis, free radical scavenging and thrombus targeting functions, and production methods and uses thereof

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