CN1919865A - Bioactivity peptide, preparing process and application thereof - Google Patents

Abstract

A biologically active peptide and its preparation method and application. During preparation, tripeptide alcohol is prepared first, then dipeptide acid is prepared, and then tripeptide alcohol and dipeptide acid are prepared in 2,4,6-trichlorobenzoyl chloride and N, Under the catalysis of N-dimethyl-4-methylaminopyridine, the esterification coupling reaction is carried out to obtain the linear pentapeptide N-tert-butoxycarbonyl-2-thiazolealanyl-(R)-3-aminopentanoyl-milk Acyl-N-methylvalyl-N-methylphenylalanine tert-butyl ester; finally remove the tert-butoxycarbonyl protecting group and tert-butyl ester protecting group of the linear pentapeptide with trifluoroacetic acid, and cyclize to obtain The compounds of the present invention are biologically active peptides. The invention can provide sufficient raw materials and effective synthetic routes for the research of antineoplastic drugs, the preparation method is simple, the conditions are easy to control, and has certain versatility. compound.

Description

A kind of bioactive peptide and its preparation method and application

technical field

The invention relates to a biologically active peptide, its preparation method and its application in anti-tumor. The biologically active peptide is a five-membered macrocyclic ester peptide with anti-tumor activity from the ocean.

Background technique

Most of marine cyclic peptides come from sponges, sea squirts, seaweeds and sea hares, etc. Their novel structures and remarkable biological activities have aroused great interest of synthetic chemists and medicinal chemists. However, marine cyclic peptides are rich in N-methyl amino acids, amino acids containing thiazole (line)/oxazole (line) hybrids and other highly variable amino acids, which brings great troubles to synthetic chemists.

Obyanamide is a novel cyclic ester peptide isolated from the marine cyanobacterium Lyngbyaconfervoides by Moore's group (Williams, PG; Yoshida, WY; Moore, RE; Paul, VJJNat.Prod.2002, 65, 29-31.). Its IC ₅₀ values for KB and LoVo cells are 0.58 and 3.14 mg/mL, respectively. Initially, the structure of the active peptide was deduced as shown in the figure below, and the stereoabsolute configuration is (3S, 10S, 13S, 23S, 29S).

However, with the in-depth study of the active substance, the inventors found that the above deduced structure is not correct, and the correct stereoabsolute configuration of the biologically active peptide is (3R, 10S, 13S, 23S, 29S). Therefore, it is very important to synthesize bioactive peptides with correct structure to understand and conduct the study of the structure-activity relationship of active peptide drugs. In addition, due to the small amount of natural products, it is difficult to purify, separate, and identify them, which greatly limits the progress of research on the activity of marine active peptides. Among them, chemical synthesis is an alternative method, so it is necessary to develop an effective method for preparing active peptides.

Contents of the invention

The purpose of the present invention is to provide a biologically active peptide and its effective preparation method and application, which can provide sufficient raw materials and effective synthesis routes for the research of antitumor drugs.

A biologically active peptide, characterized in that its molecular formula is C ₃₀ H ₄₁ N ₅ O ₆ S, its stereoabsolute configuration is (3R, 10S, 13S, 23S, 29S), and its structural formula is:

The preparation method of the above-mentioned biologically active peptide is characterized in that first tripeptide alcohol is prepared: N-benzyloxycarbonyl L-phenylalanine is nitrogen-methylated under alkaline conditions to obtain the methylated product N-(benzyloxy Carbonyl)-N-methylphenylalanine, N-(benzyloxycarbonyl)-N-methylphenylalanine reacts with tert-butanol, and the resulting N-(benzyloxycarbonyl)-N-methylphenylalanine Tert-butyl amino acid is catalyzed by palladium preparation to remove the benzyloxycarbonyl protecting group, and then condensed with N-(benzyloxycarbonyl)-N-methylvaline with a condensing agent to obtain the dipeptide fragment N-(benzyloxycarbonyl) -N-methylvalyl-N-methylphenylalanine tert-butyl ester, the dipeptide fragment is catalyzed by palladium to remove the benzyloxycarbonyl protecting group, and then use a condensing agent to react with (S)-2-benzyl Condensation of oxylactic acid to obtain the tripeptide fragment (2S, 12S, 18S)-O-(benzyl) lactoyl-N-methylvalyl-N-methylphenylalanine tert-butyl ester, the tripeptide fragment Catalyzed hydrogenolysis with palladium preparations to remove the benzyl ether protecting group to obtain tripeptide alcohol (2S, 12S, 18S)-lactyl-N-methylvalyl-N-methylphenylalanine tert-butyl ester; and then prepare dipeptide Acid: The amino group of (R)-2-aminobutyric acid is protected by tert-butoxycarbonyl, and the obtained (R)-2-(N-tert-butoxycarbonyl)aminobutyric acid is subjected to Arndt-Estel diazotization and Wulff rearrangement to obtain (R)-3-(N-tert-butoxycarbonyl)aminovaleric acid methyl ester, followed by removal of the tert-butoxycarbonyl protecting group and 2-[1-(tert-butoxycarbonylamino)ethyl Base] thiazole-4-carboxylic acid is condensed to obtain the corresponding dipeptide acid methyl ester, and the dipeptide acid methyl ester is hydrolyzed in lye to obtain dipeptide acid N-tert-butoxycarbonyl-2-thiazolealanyl-(R)- 3-aminovaleric acid; then the above-mentioned tripeptide alcohol and dipeptide acid are subjected to esterification coupling reaction under the catalysis of 2,4,6-trichlorobenzoyl chloride and N,N-dimethyl-4-methylaminopyridine , to obtain the linear pentapeptide N-tert-butoxycarbonyl-2-thiazolealanyl-(R)-3-aminopentanoyl-lactyl-N-methylvalyl-N-methylphenylalanine tert Butyl ester; finally remove the tert-butoxycarbonyl protecting group and tert-butyl ester protecting group of the linear pentapeptide with trifluoroacetic acid, and cyclize to obtain the biologically active peptide of the compound of the present invention.

The present invention provides an effective preparation method and application of biologically active peptides, which can provide sufficient raw materials and effective synthesis routes for the research of antineoplastic drugs, and comprehensively use various effective condensing agents and Arndt-AI Siter diazotization and Wulff rearrangement reactions, etc., effectively shorten the synthetic route, the preparation method is simple, the conditions are easy to control, and has certain versatility, and can be used to synthesize other peptides with similar structures to the biologically active peptides of the present invention. Peptide compounds provide a variety of compounds with different structures for drug screening and further study of structure-activity relationship.

Description of drawings

Accompanying drawing 1 is the difference diagram of the chemical shift of Obyanamide and the carbon spectrum of the natural product with the three-dimensional configuration (3S, 10S, 13S, 23S, 29S).

Accompanying drawing 2 is the difference diagram of the carbon spectrum chemical shift between Obyanamide 1a and the natural product whose stereo configuration is (3R, 10S, 13S, 23S, 29S).

Detailed ways

The preparation method of bioactive peptide Obyanamide 1a of the present invention comprises the following steps:

1. The preparation of tripeptide alcohol (2S, 12S, 18S)-lactyl-N-methylvalyl-N-methylphenylalanine tert-butyl ester (HO-Lac-MeVal-MePhe-OtBu), which The specific steps are as follows:

The benzyloxycarbonyl (Z) phenylalanine is first subjected to nitrogen methylation under basic conditions. During operation, dissolve 2.99g (10.0mmol) benzyloxycarbonylphenylalanine (Z-Phe-OH), 5.0mL (80.0mmol) CH ₃ I in 30mL anhydrous tetrahydrofuran (THF) and cool down to 0°C, stirring vigorously Next, add 1.20 g (30.0 mmol) NaH (60% by weight) in batches, let it rise to room temperature naturally after the addition, and continue to stir for 24 h. Thin layer chromatography (TLC) showed that after the reaction was completed, a little ice water was added dropwise to destroy excess NaH, and then THF was distilled off under reduced pressure. The residue was dissolved in 200 mL of distilled water, and washed three times with 20 mL of n-hexane. The aqueous layer was adjusted to pH = 1 with hydrochloric acid, and extracted five times with ethyl acetate, each time with a volume of 40 mL of ethyl acetate. The extracts were combined and washed three times with 10% (weight percent) Na ₂ S ₂ O ₃ aqueous solution, 20 mL each time, and then washed once with 20 mL of saturated brine, and the ethyl acetate organic layer was concentrated to obtain N-(benzyloxycarbonyl) -N-methylphenylalanine (Z-MePhe-OH) 2.87g, yield 91.7%.

Z-MePhe-OH was then esterified with tert-butanol. During operation, 3.13g (10.0mmol) Z-MePhe-OH was dissolved in 60mL of dichloromethane, 28mL of tert-butanol was added under stirring and the temperature was lowered to 0°C. Add 2.88g (15.0mmol) 1-[3-dimethylamino]propyl-3-ethylcarbodiimide hydrochloride [EDC·HCl, 1-[3-(dimethylamino)propyl]-3- ethylcarbodiimide hydrochloride] and 0.61 g (5.0 mmol) of N,N-dimethyl-4-methylaminopyridine (DMAP). After 2h, it was allowed to rise to room temperature naturally and stirring was continued for 20h. The residue after concentration under reduced pressure was dissolved in 250 mL ethyl acetate, washed three times with 10% citric acid aqueous solution, 25 mL each time, then washed three times with 5% _NaHCO3 aqueous solution, 25 mL each time, and washed three times with saturated brine, 25mL each time. The ethyl acetate organic layer was dried and concentrated for silica gel column chromatography, and eluted with ethyl acetate-petroleum ether (the volume ratio of the two was 1:8) to obtain N-(benzyloxycarbonyl)-N-methylbenzenepropane Amino acid tert-butyl ester (Z-MePhe-O ^t Bu) 3.14g, yield 85.1%.

Then palladium preparations are used to catalyze hydrogenolysis to remove the benzyloxycarbonyl protecting group, and the resulting hydrogenolysis product is condensed with N-(benzyloxycarbonyl)-N-methylvaline to obtain dipeptide fragments. During operation, 739 mg (2.0 mmol) of Z-MePhe-O ^t Bu was dissolved in 3 mL of ethyl acetate, and 0.2 mmol of palladium-carbon was added for hydrogenolysis at normal temperature and pressure. TLC showed that the reaction was complete in 2 hours. Palladium-carbon was removed by filtration with celite, and the filter cake was washed three times with ethyl acetate, 5 mL each time. The filtrates were combined and concentrated, and after vacuum drying for 4 hours, the concentrate was dissolved in 5 mL of dichloromethane, and then condensed with N-(benzyloxycarbonyl)-N-methylvaline (Z-MeVal-OH), that is, 531 mg (2.0 After adding mmol) Z-MeVal-OH to the above dichloromethane solution and cooling down to 0°C, 460 mg (2.4 mmol) of EDC·HCl and 327 mg (2.4 mmol) of 1-hydroxybenzotriazole (HOAt) were added in sequence. After reacting at 0° C. for 2 h, it was allowed to warm to room temperature naturally and stirring was continued for 18 h. Concentrate under reduced pressure to remove dichloromethane, dissolve the residue in 100 mL ethyl acetate, wash with 10% citric acid aqueous solution three times, each 25 mL, then wash with 5% NaHCO ₃ aqueous solution three times, each 25 mL, and then wash with saturated saline Wash three times, each 25mL. The ethyl acetate organic layer was dried and concentrated for silica gel column chromatography, and eluted with ethyl acetate-petroleum ether (the volume ratio of the two was 1:8) to obtain the dipeptide fragment as a colorless oily substance N-(benzyloxycarbonyl )-N-methylvalyl-N-methylphenylalanine tert-butyl ester (Z-MeVal-MePhe-O ^t Bu) 750 mg, yield 77.7%.

The obtained dipeptide fragments were catalyzed by hydrogenolysis to remove the benzyloxycarbonyl protecting group, and then condensed with (S)-2-benzyloxylactic acid to obtain tripeptide fragments. During operation, 965 mg (2.0 mmol) of Z-MeVal-MePhe-O ^t Bu was dissolved in 5 mL of ethyl acetate and hydrogenolyzed with 10% (weight percent, the same below) palladium-carbon for 2 h. Palladium carbon was removed by filtration with celite, and the filter cake was rinsed with ethyl acetate three times, 5 mL each time. After the obtained filtrate was concentrated and vacuum-dried for 4 h, the concentrate was dissolved in 5 mL of dichloromethane, 360 mg (2.0 mmol) of (S)-2-benzyloxylactic acid (BnO-Lac-OH) was added and the temperature was lowered to 0°C. 460 mg (2.4 mmol) of EDC·HCl and 327 mg (2.4 mmol) of HOAt were added in sequence. After reacting at 0° C. for 2 h, the reaction system was allowed to rise to room temperature naturally, and stirring was continued for 12 h. After TLC showed that the reaction was finished, the residue was dissolved in 100 mL ethyl acetate and washed three times with 10% aqueous citric acid, 25 mL each, then three times with 5% _NaHCO3 aqueous solution, 25 mL each, and then washed three times with saturated brine, 25mL each time. The ethyl acetate organic layer was dried and concentrated for silica gel column chromatography, and ethyl acetate-petroleum ether (the volume ratio of the two was 1:5) was used to obtain a colorless oil (2S, 12S, 18S)-O-(benzyl ) tert-butyl lactoyl-N-methylvalyl-N-methylphenylalanine (BnO-Lac-MeVal-MePhe-O ^t Bu) 950 mg, yield 93.0%.

Catalyzed hydrogenolysis again to remove the benzyl ether protecting group to obtain tripeptide alcohol. During operation, 709 mg of BnO-Lac-MeVal-MePhe-O ^t Bu was dissolved in 5 mL of ethyl acetate, and 0.01 mmol of palladium-carbon was added to carry out hydrogenolysis at normal temperature and pressure for 4 hours, and then the reaction was completed. Palladium-carbon was removed by diatomaceous earth filtration and the filter cake was rinsed with ethyl acetate 3 times, 5 mL each time, and the resulting filtrate was concentrated and vacuum-dried to obtain a white waxy solid tripeptide alcohol (2S, 12S, 18S)- Lactyl-N-methylvalyl-N-methylphenylalanine tert-butyl ester 572 mg, yield 98.0%. The structure of tripeptide alcohol is characterized as follows: the migration value is R _f =0.39 (1 ethyl acetate: sherwood oil); the specific rotation is ${\left[a\right]}_{\mathrm{D.}}^{20}=$ $-103.8$ (c=1.0, CH ₃ OH); ¹ H NMR (600MHz, CDCl ₃ ) d: 0.75(d, J=6.8Hz, 3H, H-14), 0.94(d, J=6.8 Hz, 3H, H-15), 1.05(d, J=6.4Hz, 3H, H-19), 1.47(s, 9H, C(CH ₃ ) ₃ ), 2.27-2.31(m, 1H, H-13 ), 2.33 (s, 3H, N-CH ₃ ), 2.79 (s, 3H, N-CH ₃ ), 2.39-2.97 (m, 1H, H-3a), 3.39 (dd, J=4.6, 15.1Hz, 1H, H-3b), 3.63 (br, 1H, Lac OH), 4.27-4.31 (m, 1H, -18), 4.99 (d, J=10.5Hz, 1H, H-12), 5.52 (dd, J =4.1, 11.9Hz, 1H, H-2), 7.16-7.33 (m, 5H, Ar H); the carbon nuclear magnetic resonance spectrum data is ¹³ C NMR (CDCl ₃ )d: 17.8 (C-14), 19.6 (C -15), 20.9(C-19), 27.9(C(CH ₃ ) ₃ ), 29.0(C-13), 29.5(N-CH ₃ ), 31.4(N-CH ₃ ), 34.2(C-3) , 57.8(C-2), 59.0(C-12), 64.3(C-18), 82.0(C(CH ₃ ) ₃ ), 126.6(C-7), 128.8and 129.4(C-5, C-6, C-8 and C-9), 137.1 (C-4), 169.5, 169.6 and 175.3 (C=0×3); mass spectrum data is ESI-MS (m/z) calcd.M=420.3, [MH] ^- = 419.3, found 421.0, 420.0.

2. Preparation of dipeptide methyl ester N-tert-butoxycarbonyl-2-thiazolealanyl-(R)-3-aminovaleric acid methyl ester [Boc-Ala(Thz)-(R)-Apa-OMe] , the reaction steps are as follows:

Dissolve 451 mg (1.5 mmol) N-tert-butoxycarbonyl-2-thiazole alanyl ethyl ester [Boc-Ala(Thz)-OEt] in a mixture of 4 mL THF, 1 mL MeOH and 2 mL H ₂ O and cool to 0°C. 126 mg (3.0 mmol) of lithium hydroxide monohydrate was added to the above solution, and the ice bath was removed after 20 min. After TLC showed that the starting material disappeared, the solution was concentrated under reduced pressure and diluted with 40 mL of saturated brine. The resulting aqueous solution was adjusted to pH=2~3 with hydrochloric acid, and then extracted five times with ethyl acetate, 20 mL each time. The extracts were combined, concentrated and vacuum-dried to obtain 2-[1-(tert-butoxycarbonylamino)ethyl]thiazole-4-carboxylic acid (also known as N-tert-butoxycarbonyl-2-thiazolealanine, Boc- Ala(Thz)-OH) spare.

Then use (R)-2-aminobutyric acid as a raw material, protect the amino group with tert-butoxycarbonyl, and then carry out Arndt-Eistert (Arndt-Eistert) diazotization and Wolff (Wolff) rearrangement to obtain (R)-3-aminovaleric acid methyl ester, removing the benzyloxycarbonyl protecting group, and condensing with Boc-Ala(Thz)-OH to obtain the corresponding dipeptide acid methyl ester. During operation, dissolve 347mg (1.5mmol) (R)-3-(N-tert-butoxycarbonyl)aminovaleric acid methyl ester [Boc-(R)-Apa-OMe] in 1.5mL dichloromethane, cool to 0°C Then, 1.5 mL of trifluoroacetic acid (TFA) was slowly dropped thereinto. After 15 min, the ice bath was removed and allowed to rise to room temperature naturally. After 1.5 h TLC showed disappearance of starting material. It was concentrated under reduced pressure at low temperature, and the resulting residue was dissolved in 2 mL of dichloromethane and concentrated again. This was done 3 times and dried under high vacuum for 4 h, then dissolved in 5 mL of dichloromethane. After adding 2-[1-(tert-butoxycarbonylamino)ethyl]thiazole-4-carboxylic acid (Boc-Ala(Thz)-OH), the solution was cooled to 0°C. 288 mg (1.5 mmol) EDC·HCl, 204 mg (1.5 mmol) HOAt, 524 mL (3.0 mmol) diisopropylethylamine (DIPEA) were added sequentially. The reaction solution was kept at 0°C, and after 2 hours, the ice bath was removed to allow it to rise to room temperature naturally and stirring was continued for 6 hours. TLC shows that after the reaction finishes, dilute the reaction solution with 80mL ethyl acetate and wash three times with 10% citric acid aqueous solution, each 25mL, then use 5%NaHCO _The aqueous solution washes three times, each 25mL, then washes three times with saturated brine, each time 25mL each time. Dry and concentrate the ethyl acetate organic layer for silica gel column chromatography, and use ethyl acetate-petroleum ether (the volume ratio of the two is 1:5) to obtain a colorless oily substance dipeptide methyl ester N-tert-butoxycarbonyl-2 - Thiazolealanyl-R-3-aminovaleric acid methyl ester [Boc-Ala(Thz)-(R)-Apa-OMe] 566 mg, yield 98.1%. The structural characterization of dipeptide methyl ester is as follows: the migration value is R _f =0.30 (EtOAc-petroleum ether=1:1); the specific rotation is [a] ²⁹ _D =-24.5 (c0.28, MeOH); NMR Proton spectrum data is ¹ H NMR (600MHz, CDCl ₃ )d 0.98(t, J=7.3Hz, 3H, H-5), 1.47(s, 9H, ^tBu ), 1.62(d, J=6.6Hz, 3H , H-11), 1.67-1.73 (m, 2H, H-4), 2.64 (dd, J=16.1, 5.9Hz, 1H, H-2a), 2.67 (dd, J=15.8, 5.5Hz, 1H, H-2b), 3.70(s, 3H, _CO2CH3 ), 4.32-4.38(m, 1H, H- ₃ ), 5.08(br, 1H, H-10), 5.14(br, 1H, NH), 7.64 (d, J=8.8Hz, 1H, NH), 8.00 (s, 1H, H-8); C NMR spectrum data is ¹³ C NMR (CDCl ₃ )d10.7 (C-5), 21.6 (C -11), 27.3(C-4), 28.3((CH ₃ ) ₃ COC=O), 38.4(C-2), 47.5(C-3), 48.7(C-10), 51.7(CO ₂ CH ₃ ), 80.3((CH ₃ ) ₃ COC=O), 123.0(C-8), 149.9(C-7), 155.0, 160.6, 171.9 and 174.0(4quat.C); high-resolution mass spectrometry data is HRESIMScalcd for C ₁₇ H ₂₇ N ₃ O ₅ SNa[M+Na] ⁺ 408.1569, found 408.1586.

3. The preparation of bioactive peptide Obyanamide 1a, the specific steps are as follows:

88 mg (0.23 mmol) of dipeptide acid methyl ester Boc-Ala(Thz)-(R)-Apa-OMe was demethylated and dried in vacuo to obtain dipeptide acid, which was then dissolved in 2 mL of anhydrous tetrahydrofuran. Under stirring, 156 mL of DIPEA and 105 mL of 2,4,6-trichlorobenzoyl chloride were sequentially injected. After 5 h of reaction, TLC showed that the raw materials disappeared, THF was evaporated under reduced pressure under the protection of argon, the residue was quickly dissolved in 2 mL of anhydrous toluene, and 84 mg (0.20 mmol) of tripeptide alcohol HO-Lac-MeVal-MePhe-O ^t was added successively Bu, 110mg DMAP, after 4 hours of reaction, TLC showed that the reaction was complete. The reaction solution was diluted with 20 mL ethyl acetate and washed three times with 10% citric acid aqueous solution, 2 mL each time, then washed three times with 5% NaHCO ₃ aqueous solution, 2 mL each time, and washed three times with saturated brine, 2 mL each time. The organic layer containing ethyl acetate was dried and concentrated for silica gel column chromatography, and ethyl acetate-petroleum ether (the volume ratio of the two was 1:5) was used to obtain a white foamy solid linear pentapeptide N-tert-butoxycarbonyl- 2-Thiazolylalanyl-R-3-aminovaleryl-lactyl-N-methylvalyl-N-methylphenylalanine tert-butyl ester [Boc-Ala(Thz)-(R)-Apa -Lac-MeVal-MePhe-O ^t Bu] 146 mg, yield 94.2%. The structural characterization of the linear pentapeptide is as follows: the migration value is R _f =0.11 (EtOAc-petroleum ether=3:2); the specific rotation is [a] ²⁷ _D =-96.6 (c 0.15, MeOH), and the H NMR spectrum The data is ¹ H NMR (CDCl ₃ , one main rotamer)d 0.77(d, J=6.5Hz, 3H, H-14), 0.91(d, J=6.2Hz, 3H, H-15), 0.95(t , J=7.4Hz, 3H, H-24), 1.15(d, J=6.6Hz, 3H, H-19), 1.46(s, 9H, ^tBu ), 1.47(s, 9H, Boc), 1.49( d like, overlapped, 3H, H-30), 1.56-1.65 (m, 2H, H-23), 2.23-2.28 (m, 1H, H-13), 2.44 (s, 3H, N-CH ₃ ), 2.64-2.76(dd and dd like, overlapped, 2H, H-21a and H-21b), 2.79(s, 3H, N-CH ₃ ), 2.90-2.95(m, 1H, H-3a), 3.38(dd , J=15.0, 4.4Hz, 1H, H-3b), 4.33-4.38(m, 1H, H-22), 4.95(d, .J=10.6Hz, 1H, H-12), 5.05-5.11(m , overlapped, 2H, H-29 and H-18), 5.53 (dd, J=12.0, 4.0Hz, 1H, H-2), 7.17-7.28 (m, 5H, Ar H), 7.82 (d, J= 8.8Hz, 1H, 22-NH), 8.00(s, 1H, H-27); the carbon nuclear magnetic resonance spectrum data is ¹³ C NMR (CDCl ₃ , one main rotamer)d 10.7(C-24), 16.1(C -19), 17.8(C-15), 19.7(C-14), 21.5(C-30), 26.8(C-13), 27.2(C-23), 28.0((CH ₃ ) ₃ C), 28.3 ((CH ₃ ) ₃ COC=O), 29.2(N-CH ₃ ), 31.4(N-CH ₃ ), 34.3(C-3), 38.3(C-21), 47.6(C-22), 48.7( C-29), 57.6 (C-12), 58.3 (C-2), 67.0 (C-18), 80.3 ((CH ₃ ) ₃ COC=O), 81.9 ((CH ₃ ) ₃ COC=O), 122.9(C-27), 126.6(C-7), 128.4 and 128.8(C-5, C-6, C-8 and C-9), 137.2(C-4), 150.1(C-26), 154.9 , 160.6, 169.6, 170.0, 170.8 (quat.C); high-resolution mass spectrometry data is HRESIMScalcd for C ₃₉ H ₅₉ N ₅ O ₉ SNa[M+Na] ⁺ 796.3931, found 796.3962.

Then remove the protecting group tert-butoxycarbonyl and tert-butyl ester protecting group with trifluoroacetic acid and cyclize to obtain the biologically active peptide of the compound of the present invention. During specific operation, 65.0 mg (0.084 mmol) of linear pentapeptide was dissolved in 0.5 mL of dichloromethane and the temperature was lowered to 0° C., and 0.5 mL of TFA was slowly added thereto. After 20 min, the ice bath was removed and stirring was continued at room temperature for 4 h. TLC showed that after the reaction was completed, the reaction solution was concentrated to dryness under reduced pressure at low temperature. The residue was dissolved in 1 mL of dichloromethane and concentrated again. This was done three times, and the concentrate was dried under high vacuum for 4 h, then dissolved in 170 mL of anhydrous THF (concentration of the concentrate was 5×10 ^-4 mol/L) and cooled to 0°C. Add 96 mg of O-(7-azobenzotriazole-1-O)-N,N,N',N'-tetramethyluronium hexaphosphate [HATU, 2-(azobenzotriazolyl-1-oxy) -N,N,N',N'-tetramethyluronium hexafluoroborate] and 117mL DIPEA. After 4h, the ice bath was removed to allow it to return to room temperature naturally and stirring was continued for 3d. Concentrate under reduced pressure to remove THF, and dissolve the residue in 20 mL of ethyl acetate, wash the reaction solution three times with 10% aqueous citric acid, 2 mL each time, then wash three times with 5% _NaHCO3 aqueous solution, 2 mL each time, and then Wash three times with saturated saline, 2 mL each time. The organic layer containing ethyl acetate was dried and concentrated for silica gel column chromatography, eluted with chloroform-methanol (the volume ratio of the two was initially 150:1 and then gradually increased to 60:1), and then purified with SephadexLH-20, ( The mobile phase is chloroform-methanol, the volume ratio of the two is 1:1) The purified white solid is the compound bioactive peptide of the present invention, also known as Obyanamide 1a 29.3mg, the yield is 58.1%. The structure of the biologically active peptide Obyanamide 1a is characterized as follows: the migration value is R _f =0.45 (CHCl ₃ -MeOH=20:1); the specific rotation is [a] ²⁷ _D =+22.2 (c 0.07, MeOH); nuclear magnetic resonance The hydrogen spectrum data is ¹ H NMR (CDCl ₃ )d 0.50(d, J=6.4Hz, 3H, H-25), 0.86(d, J=6.9Hz, 3H, H-26), 1.07(t, J= 7.3Hz, 3H, H-5), 1.24(d, J=6.8Hz, 3H, H-30), 1.43(d, J=6.9Hz, 3H, H-11), 1.61-1.69(m, 2H, H-4), 2.28-2.34(m, 1H, H-24), 2.41(dd, J=11.9, 2.8Hz, 1H, H-2a), 2.79(dd, J=11.9, 5.0Hz, 1H, H -2b), 2.90(dd, J=13.7, 6.4Hz, 1H, H-14a), 3.13(s, 3H, H-27), 3.14(s, 3H, H-21), 3.28(dd, J= 13.7, 8.2Hz, 1H, H-14b), 4.40-4.46(m, 1H, H-3), 5.04-5.07(m, overlapped, 1H, H-10), 5.07(d, J=10.1Hz, 1H , H-23), 5.20(q, J=6.8Hz, 1H, H-29), 5.45(t like, J=8.2, 6.9Hz, 1H, H-13), 7.08(t, J=7.3Hz, 1H, H-18), 7.17 (d, J=7.8Hz, 1H, H-16 and H-20), 7.19-7.22 (dd like, 2H, H-17 and H-19), 7.94 (d, J =6.0Hz, 1H, 10-NH), 8.02(s, 1H, H-8), 9.14(d, J=10.1Hz, 1H, 3-NH); the carbon nuclear magnetic resonance spectrum data is ¹³ C NMR (CDCl ₃ )d 11.2(C-5), 15.8(C-30), 18.5(C-25 and C-26), 24.1(C-11), 25.9(C-4), 27.4(C-24), 29.0( C-21), 29.8(C-27), 37.0(C-14), 38.8(C-2), 47.3(C-3), 48.0(C-10), 57.8(C-23), 60.7(C -13), 67.5(C-29), 123.1(C-8), 127.0(C-18), 128.7(C-16 and C-20), 129.1(C-17 and C-19), 136.3(C -15), 148.6(C-7), 160.4(C-6), 168.0(C-12), 169.5(C-9), 169.9(C-22), 170.3(C-1), 172.9(C- 28); high-resolution mass spectrometry data is HRESIMS calcd for C ₃₀ H ₄₂ N ₅ O ₆ S[M+H] ⁺ 600.2856, found 600.2875.

The palladium preparation for catalytic hydrogenolysis described in the present invention is palladium-carbon or palladium hydroxide. The condensing agent used for condensation with N-(benzyloxycarbonyl)-N-methylvaline is N, N-dicyclohexyl imine, 1-[3-dimethylamino]-propyl-3- Ethylcarbodiimide hydrochloride or 7-azabenzoxazol-1-yl-oxy(tris-(dimethylamino)phosphine)hexafluorophosphate. The lye obtained by hydrolyzing the dipeptide acid in the lye is an aqueous solution of sodium hydroxide or lithium hydroxide, and the molar ratio of the dipeptide fragment to the alkali is in the range of 1:1 to 1:2. The molar ratio of the tripeptide alcohol and dipeptide acid in the esterification coupling reaction is 1:1.2-1:5.0.

The structure of bioactive peptide Obyanamide 1a of the present invention is shown in the following formula:

In order to further confirm that the stereoabsolute configuration of the bioactive peptide synthesized by the present invention is (3R, 10S, 13S, 23S, 29S), the inventor also synthesized a stereoabsolute configuration of (3S, 10S, 13S, 23S, 29S ) Obyanamide cyclic peptide, the preparation method of the two is the same, only the latter adopts (S)-2-aminobutyric acid as raw material in the process of synthesizing dipeptide acid, and the bioactive peptide Obyanamide 1a of the present invention adopts (R) -2-aminobutyric acid as raw material. The structural characterization of the synthesized Obyanamide cyclic peptide is as follows:

The migration value is R _f = 0.46 (CHCl ₃ : MeOH = 20: 1); the specific rotation is [a] _D ²⁸ = -96.3 (c = 0.06, MeOH); the proton nuclear magnetic resonance spectrum data is ¹ H NMR (600MHz, CDCl ₃ )d: 0.59(d, J=6.6Hz, 3H, H-25), 0.88(d, J=6.6Hz, 3H, H-26), 1.02(t, J=7.3Hz, 3H, H- 5), 1.46(d, J=6.6Hz, 3H, H-11), 1.50(d, J=6.6Hz, 3H, H-30), 1.69-1.77(m, 1H, H-4a), 1.94- 2.01(m, 1H, H-4b), 2.26-2.31(m, 1H, H-24), 2.74(dd, J=4.4, 16.5Hz, 1H, H-2a), 2.80-2.84(m, 2H, H-2b and H-14a, overlapped), 3.08(s, 3H, H-21), 3.21(s, 3H, H-27), 3.44(dd, J=8.8, 13.6Hz, 1H, H-14b ), 4.10-4.15 (m, 1H, H-3), 5.02 (d, J=10.6Hz, 1H, H-23), 5.16 (dd, J=5.8, 8.8Hz, 1H, H-13), 5.31 -5.35(m, 1H, H-10), 5.42(q, J=7.0Hz, 1H, H-29), 7.15-7.25(m, 5H, Ar H), 7.96(s, 1H, H-8) , 8.00(d, J=7.7Hz, 1H, 10-NH), 8.82(d, J=9.1Hz, 1H, 3-NH); the carbon nuclear magnetic resonance spectrum data is ¹³ C NMR (150MHz, CDCl ₃ )d: 11.4(C-5), 16.2(C-30), 18.7(C-26), 18.9(C-25), 23.3(C-11), 27.57(C-24), 27.61(C-4), 29.0 (C-21), 30.1(C-27), 36.4(C-14), 37.7(C-2), 46.6(C-10), 47.8(C-3), 57.2(C-23), 60.9( C-13), 67.4(C-29), 122.3(C-8), 127.0(C-18), 128.7(C-17 and C-19), 129.4(C-16 and C-20), 136.3( C-15), 150.4(C-7), 160.4(C-6), 167.2(C-12), 168.9(C-9), 170.0(C-1), 170.4(C-22), 171.9(C -28); high-resolution mass spectrometry data is HRESIMS calcd.[M+Na] ⁺ : 622.2675.found: 622.2665.

Compare the spectrum data of the synthetic Obyanamide cyclic peptide with the carbon spectrum of the biologically active peptide Obyanamide1a of the present invention and the natural product, as shown in the accompanying drawings, wherein Figure 1 is (3S, 10S, 13S, 23S, 29S) Obyanamide cyclic peptide Figure 2 shows the difference between (3R, 10S, 13S, 23S, 29S) Obyanamide 1a and the chemical shift of the natural product carbon spectrum, and the standard value is the chemical shift value of the natural product. It can be seen from the accompanying drawing 1 that the difference between Obyanamide and the carbon spectrum chemical shift value of the natural product is not large, and it can be seen from the accompanying drawing 2 that the difference between Obyanamide 1a and the carbon spectrum chemical shift value of the natural product is almost the same, thus showing the true nature of the natural product Stereoabsolute configuration should be (3R, 10S, 13S, 23S, 29S), which is the biologically active peptide Obyanamide 1a of the present invention.

In vitro screening test for antitumor biological activity of bioactive peptide Obyanamide 1a

The screening method used tetrazolium salt (MTT) reduction method and sulforhodamine B (SRB) protein staining method, and the tumor cell lines used included LOVO (colorectal cancer cell line), KB (squamous cell line) and HL-60 (leukemia cell line), the action time is 72h. The inhibition rate of the biologically active peptide Obyanamide 1a of the present invention on the above-mentioned tumor cell lines is shown in the table below. Each table shows the relationship between the concentration of the biologically active peptide of the present invention and the growth inhibition rate of each tumor cell line.

Inhibitory rate of LOVO tumor cell growth Concentration M ^{10-4 10-5 10-6 10-7 10-8} Inhibition rate% 100 50.0 53.3 0.4 0

Inhibitory rate of KB tumor cell growth Concentration M ^{10-4 10-5 10-6 10-7 10-8} Inhibition rate% 96.8 80.0 21.0 1.5 19.1

Inhibitory rate of HL-60* tumor cell growth Concentration M ^{10-4 10-5 10-6 10-7 10-8} Inhibition rate% 100 100 41.8 6.1 6.7

The inhibitory rate IC ₅₀ values of the biologically active peptide Obyanamide 1a of the present invention to each tumor cell line are 6.8mM (LOVO), 6.7mM (KB), 19.0mM (HL-60*), all have strong antitumor active.

Claims (7)

1, a kind of biologically active peptides, the molecular formula that it is characterized in that it is C ₃₀H ₄₁N ₅O ₆S, three-dimensional absolute configuration be (3R, 10S, 13S, 23S, 29S), structural formula is:

2, the preparation method of the described biologically active peptides of claim 1, it is characterized in that at first preparing tripeptides alcohol: make N-carbobenzoxy-(Cbz) L-phenylalanine under alkaline condition, carry out nitrogen and methylate and obtain methylate N-(carbobenzoxy-(Cbz))-N-methylbenzene L-Ala, N-(carbobenzoxy-(Cbz))-N-methylbenzene L-Ala and trimethyl carbinol reaction, the N-of gained (carbobenzoxy-(Cbz))-N-methylbenzene L-Ala tert-butyl ester is removed the carbobenzoxy-(Cbz) protecting group with palladium preparation catalytic hydrogenolysis, obtain two peptide fragment N-(carbobenzoxy-(Cbz))-N-methyl valyl-N-methylbenzene L-Ala tert-butyl ester with N-(carbobenzoxy-(Cbz))-N-methylvaline with the condensing agent condensation subsequently, this two peptide fragment is removed the carbobenzoxy-(Cbz) protecting group with palladium preparation catalytic hydrogenolysis, use then condensing agent with (S)-condensation of 2-benzyloxy lactic acid obtains tripeptide fragment (2S, 12S, 18S)-O-(benzyl) lactoyl-N-methyl valyl-N-methylbenzene L-Ala tert-butyl ester, this tripeptide fragment is removed the benzyl oxide protecting group with palladium preparation catalytic hydrogenolysis get tripeptides alcohol (2S, 12S, 18S)-lactoyl-N-methyl valyl-N-methylbenzene L-Ala tert-butyl ester; Refabrication dipeptides acid: with the amino of tertbutyloxycarbonyl protection (R)-2-aminobutyric acid, with gained (R)-2-(N-tertbutyloxycarbonyl) aminobutyric acid carry out the extra heavy nitrogenize of Arndt-Ai Si and Wu Erfu reset (R)-3-(N-tertbutyloxycarbonyl) aminovaleric acid methyl esters, subsequent removal tertbutyloxycarbonyl protecting group and with 2-[1-(t-butoxycarbonyl amino) ethyl] thiazole-4-formic acid condensation gets corresponding dipeptides acid methyl esters, this dipeptides acid methyl esters of hydrolysis obtains dipeptides acid N-tertbutyloxycarbonyl-2-thiazole alanyl-(R)-3-aminovaleric acid in alkali lye; Then with above-mentioned tripeptides alcohol and dipeptides acid 2,4,6-trichloro-benzoyl chloride and N, carry out the esterification coupling reaction under N-dimethyl-4-methylamino pyridine catalysis, obtain the amino valeryl-lactoyl of line style pentapeptide N-tertbutyloxycarbonyl-2-thiazole alanyl-(R)-3--N-methyl valyl-N-methylbenzene L-Ala tert-butyl ester; Remove the tertbutyloxycarbonyl protecting group and the tert-butyl ester protecting group of linear pentapeptide at last with trifluoroacetic acid, and cyclization obtains compound biologically active peptides of the present invention.

3, as the preparation method of biologically active peptides as described in the claim 2, it is characterized in that described palladium preparation is palladium-carbon or palladium hydroxide.

4, the preparation method of biologically active peptides as claimed in claim 2, it is characterized in that described and condensing agent that N-(carbobenzoxy-(Cbz))-condensation of N-methylvaline is used are N, N-dicyclohexyl imines, 1-[3-dimethylamino]-propyl group-3-ethyl carbon imide hydrochloride or 7-azepine benzothiazole-1-base-oxygen (three-(dimethylin) phosphines) hexafluorophosphate.

5, as the preparation method of biologically active peptides as described in the claim 2, it is characterized in that the described alkali lye that hydrolysis obtains dipeptides acid in alkali lye is the aqueous solution of sodium hydroxide or lithium hydroxide, the molar ratio range of two peptide fragment and alkali is 1: 1～1: 2.

6,, it is characterized in that the mol ratio when described tripeptides alcohol carries out the esterification coupling reaction with dipeptides acid is 1: 1.2～1: 5.0 as the preparation method of biologically active peptides as described in the claim 2.

7, the application of the described biologically active peptides of claim 1 in the preparation antitumor drug.

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