CN114315963A - LSH series cyclic pentapeptide ester and synthetic method and application thereof - Google Patents
LSH series cyclic pentapeptide ester and synthetic method and application thereof Download PDFInfo
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- CN114315963A CN114315963A CN202111527415.0A CN202111527415A CN114315963A CN 114315963 A CN114315963 A CN 114315963A CN 202111527415 A CN202111527415 A CN 202111527415A CN 114315963 A CN114315963 A CN 114315963A
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Images
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-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention provides an LSH series cyclic pentapeptide ester and a synthesis method and application thereof, belonging to the field of pharmacy, wherein the LSH series cyclic pentapeptide ester is prepared by constructing a peptide chain by a plurality of amino acid derivatives through a 3+2 strategy, then cyclizing, obtaining cyclic peptide with carbon-carbon unsaturated bonds through elimination reaction under alkaline condition, and then performing acylation reaction; the LSH series cyclic pentapeptide ester is used for treating melanoma/lung cancer/colon cancer/gastric cancer/cervical cancer. The LSH series cyclic pentapeptide ester is an LSH series novel cyclic peptide, the structure of the cyclic peptide comprises chiral 2, 4-dimethylheptanoyl side chain and cis-form unsaturated amino acid, and the cyclic peptide has good antitumor activity, and the activity depends on the chirality of the side chain and the configuration of the amino acid in the cyclic peptide ester; the LSH series cyclic pentapeptide ester has higher activity on melanoma cells, lung cancer cells, colon cancer cells, stomach cancer cells and cervical cancer cells.
Description
Technical Field
The invention relates to a cyclic pentapeptide ester, in particular to an LSH series cyclic pentapeptide ester and a synthesis method and application thereof.
Background
The cyclic peptide ester and cyclic peptide are peptide compounds with special structures, are mostly present in marine organisms, biological fermentation liquor, grains and plants, and mostly have stronger physiological and pharmacological activities, wherein the antitumor activity is particularly outstanding. Because free carboxyl and amino groups do not exist in the structure, the peptide has better stability than the chain peptide, especially in organisms, and therefore has very high medicinal value. In addition, the structural characteristics provide a valuable molecular model for drug design and provide a reference lead compound library for the research and development of new drugs. In recent years, a number of polypeptide drugs have entered clinical trials or have been approved for marketing. Because different amino acids can form different cyclic peptides, and different cyclic peptides have different activities or even no activity, how to obtain a cyclic peptide structure with stronger antitumor activity is a difficult point of research.
Disclosure of Invention
Based on the physicochemical characteristics of the cyclic peptide ester, the invention provides an LSH series cyclic pentapeptide ester and a synthesis method and application thereof.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a cyclic pentapeptide ester of the LSH series, the chemical structural formula of which is as follows:
wherein:
R1and R2Each independently hydrogen or methyl;
R3is independently hydrogen, alkyl, halogen, hydroxyl, amino/amino, hydrocarbyloxy or siloxy;
R4and R5Each independently isopropyl, isobutyl, sec-butyl or benzyl.
Further, the raw material for preparing the LSH series cyclic pentapeptide ester comprises a plurality of amino acid derivatives;
the configuration of the amino acid derivatives is independent L type or D type.
Further, the plurality of amino acid derivatives are independent aliphatic amino acid derivatives or arylpropyl amino acid derivatives, and the plurality of amino acid derivatives are not identical.
Further, the aliphatic amino acid derivative is a threonine derivative, a leucine derivative, an isoleucine derivative or a valine derivative;
wherein the threonine derivative is threonine with a nitrogen end protected by tert-butyloxycarbonyl and a carbon end protected by allyl;
the leucine derivative is leucine with nitrogen end protected by tert-butyloxycarbonyl and carbon end protected by allyl;
the isoleucine derivative is isoleucine with the nitrogen end protected by tert-butyloxycarbonyl and the carbon end protected by allyl;
the valine derivative is valine with a nitrogen end protected by tert-butyloxycarbonyl and a carbon end protected by allyl;
the aryl alanine amino acid derivative is a phenylalanine derivative, a 4-substituted phenylalanine derivative or a tyrosine derivative;
wherein the phenylalanine derivative is phenylalanine with a nitrogen end protected by tert-butyloxycarbonyl group and a carbon end protected by allyl group;
the substituted phenylalanine derivative is 4-substituted phenylalanine with a nitrogen end protected by tert-butyloxycarbonyl and a carbon end protected by allyl;
the tyrosine derivative is tyrosine with nitrogen end protected by tert-butyloxycarbonyl and carbon end protected by allyl.
Further, R1And R2When both are methyl, R1And R2The configuration of the chiral carbon is independent R type or S type.
Further, the chemical structural formula of the LSH series cyclic pentapeptide ester is LSH-1-LSH-16, and a biological electron isosteric cyclic peptide derivative prepared from a hydroxy substituted phenylalanine derivative;
wherein, the specific structural formulas of LSH-1 to LSH-16 are as follows:
wherein, the bioisostere cyclopeptide derivative comprises corresponding compounds of which the hydroxyl group is substituted by alkyl, amino (amino) and halogen.
A method for synthesizing the LSH series cyclic pentapeptide ester comprises the steps of constructing a peptide chain by taking a plurality of amino acid derivatives through a 3+2 strategy, cyclizing, obtaining cyclic peptide with carbon-carbon unsaturated bonds through elimination reaction under an alkaline condition, and then carrying out acylation reaction to obtain the LSH series cyclic pentapeptide ester.
Further, the synthesis method comprises the following specific steps which are carried out in sequence:
1) synthesis of intermediate T-1
Taking initial raw materials S-1 and Boc-Thr (T-Bu) -OH, and carrying out condensation reaction under the action of a condensing agent and a catalyst to obtain an intermediate T-1, wherein the specific chemical reaction formula is as follows:
wherein, the Boc-Thr (t-Bu) -OH has a structural formula:
the solvent of the condensation reaction is a polar solvent;
the polar solvent is dichloromethane;
the condensing agent is 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDCI);
the catalyst is 4-dimethylamino pyridine (DMAP);
the temperature of the condensation reaction is room temperature;
after the condensation reaction is finished, washing the obtained reaction solution by potassium carbonate aqueous solution, water, hydrochloric acid and saturated salt solution, drying and concentrating to obtain an intermediate T-1;
2) synthesis of intermediate T-2:
after the intermediate T-1 is subjected to Boc protecting group removal by trifluoroacetic acid, the intermediate T-1 is reacted with Boc-Tyr (R) under the action of a peptide coupling reagent and an organic base3) And (3) carrying out condensation reaction on-OH again to obtain an intermediate T-2, wherein the specific chemical reaction formula is as follows:
wherein, Boc-Tyr (R)3) -OH has the formula:
R3is independently hydrogen, alkyl, halogen, hydroxyl, amino/amino, hydrocarbyloxy or siloxy;
the reaction solvent for removing the Boc protecting group is dichloromethane, and the temperature is room temperature;
after the reaction for removing the Boc protecting group is finished and before the condensation reaction, the solvent, trifluoroacetic acid and generated acetic acid in the reaction system are also required to be removed by concentration;
the peptide coupling reagent is benzotriazole-N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HBTU);
the organic base is N, N-Diisopropylethylamine (DIPEA);
the solvent of the condensation reaction is dichloromethane, and the temperature is room temperature;
after the condensation reaction is finished, the condensation is needed, the obtained remainder is added with water, and then is extracted by ethyl acetate, the phase separation is carried out, the ethyl acetate phase is subjected to 5 wt% of K2CO3Washing the aqueous solution, water, 2 wt% hydrochloric acid aqueous solution and saturated salt solution for several times, drying, filtering, concentrating and purifying to obtain an intermediate T-2;
3) synthesis of intermediate T-3:
after the intermediate T-2 is subjected to Boc protecting group removal by trifluoroacetic acid, the intermediate T-2 is subjected to condensation reaction with the raw material S-2 again under the action of a peptide coupling reagent and organic base to obtain an intermediate T-3, wherein the specific chemical reaction formula is as follows:
wherein R is4And R5Each independently isopropyl, isobutyl, sec-butyl or benzyl;
the reaction solvent for removing the Boc protecting group is dichloromethane, and the temperature is room temperature;
after the reaction for removing the Boc protecting group is finished and before the condensation reaction, the solvent, trifluoroacetic acid and generated acetic acid in the reaction system are also required to be removed by concentration;
the peptide coupling reagent is benzotriazole-N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HBTU);
the organic base is N, N-Diisopropylethylamine (DIPEA);
the solvent of the condensation reaction is dichloromethane, and the temperature is room temperature;
after the condensation reaction is finished, the condensation is needed, the obtained remainder is added with water, and then is extracted by ethyl acetate, the phase separation is carried out, the ethyl acetate phase is subjected to 5 wt% of K2CO3Washing the aqueous solution, water, 2 wt% hydrochloric acid aqueous solution and saturated salt solution for several times, drying, filtering, concentrating and purifying to obtain an intermediate T-3;
4) synthesizing an intermediate T-4:
removing allyl from the intermediate T-3 under the catalytic action of N-methylmorpholine and tetrakis (triphenylphosphine) palladium, removing Boc protecting group by trifluoroacetic acid, and performing condensation reaction under the action of a peptide coupling reagent and organic base to obtain an intermediate T-4, wherein the specific chemical reaction formula is as follows:
the reaction solvent for removing allyl is chloroform, and the temperature is room temperature;
after the allyl removal reaction and before the Boc protection group removal reaction, the reaction mixture was concentrated, dissolved in ethyl acetate and then subjected to 15 wt% KHSO4Washing the aqueous solution and water for several times, drying, filtering and concentrating;
the reaction solvent for removing the Boc protecting group is dichloromethane, and the temperature is room temperature;
after the reaction for removing the Boc protecting group is finished and before the condensation reaction, the solvent, trifluoroacetic acid and produced acetic acid in the reaction system are also required to be removed by concentration;
the peptide coupling reagent is 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU);
the organic base is N, N-Diisopropylethylamine (DIPEA);
the solvent of the condensation reaction is dichloromethane, and the temperature is room temperature;
after the condensation reaction is finished, the condensation is needed, the obtained remainder is added with water, and then is extracted by ethyl acetate, the phase separation is carried out, the ethyl acetate phase is subjected to 5 wt% of K2CO3Washing the aqueous solution, water, 2 wt% hydrochloric acid aqueous solution and saturated salt solution for several times, drying, filtering, concentrating and purifying to obtain an intermediate T-4;
5) synthesizing an intermediate T-5:
the intermediate T-4 reacts with di-tert-butyl dicarbonate under the catalysis of 4-dimethylaminopyridine, then carries out elimination reaction under the action of nano potassium carbonate, and simultaneously removes Fmoc protecting groups to obtain an intermediate T-5, wherein the specific chemical reaction formula is as follows:
6) synthesis of LSH series Cyclic pentapeptide esters
Carrying out ester exchange reaction on a raw material S-3 and isobutyl chloroformate under the action of N-methylmorpholine at-15 to-8 ℃, adding an intermediate T-5, and carrying out acylation reaction to obtain the LSH series cyclic pentapeptide ester, wherein the specific chemical reaction formula is as follows:
wherein R is1And R2Each independently hydrogen or methyl;
and after the acylation reaction is finished, adding water into the obtained reaction solution for dilution, extracting by ethyl acetate, and drying, filtering, concentrating and purifying the obtained ethyl acetate phase to obtain the LSH series cyclic pentapeptide ester.
An application of the LSH series cyclic pentapeptide ester in preparing medicines for treating melanoma/lung cancer/colon cancer/gastric cancer/cervical cancer is provided.
Further, the inhibitory activity IC of the LSH series cyclic pentapeptide ester on melanoma cells/lung cancer cells/colon cancer cells/stomach cancer cells/cervical cancer cells50≥0.98μM。
The LSH series cyclic pentapeptide ester and the synthesis method and the application thereof have the beneficial effects that:
the LSH series cyclic pentapeptide ester is an LSH series novel cyclic peptide, the structure of the cyclic peptide comprises chiral dimethyl heptanoyl side chain and cis-form unsaturated amino acid, and the cyclic peptide has good anti-tumor activity, and the activity depends on the chirality of the side chain and the sequence and the configuration of the amino acid in the cyclic peptide ester; the LSH series cyclic pentapeptide ester has higher activity on melanoma cells, lung cancer cells, colon cancer cells, stomach cancer cells and cervical cancer cells.
Drawings
FIG. 1 is a NMR spectrum of LSH-2 prepared in example 2 of the present invention;
FIG. 2 is a NMR carbon spectrum of LSH-2 prepared in example 2 of the present invention;
FIG. 3 is a NMR spectrum of LSH-4 prepared in example 4 of the present invention;
FIG. 4 is a NMR carbon spectrum of LSH-4 prepared in example 4 of the present invention;
FIG. 5 is a NMR spectrum of LSH-10 prepared in example 10 of the present invention;
FIG. 6 is a NMR carbon spectrum of LSH-10 prepared in example 10 of the present invention;
FIG. 7 is a NMR spectrum of LSH-14 prepared in example 14 of the present invention;
FIG. 8 is a NMR carbon spectrum of LSH-14 prepared in example 14 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Example 1 Synthesis of LSH series Cyclopentapeptide ester
The embodiment is a synthesis method of LSH series cyclic pentapeptide ester (LSH-1), and the specific preparation process comprises the following steps in sequence:
1) synthesis of intermediate T-1 (threo-threo):
10mmol of the starting Fmoc-L-Thr-OAllyl (S-1 in the formula) and 11mmol of Boc-Thr (t-Bu) -OH were dissolved in 100mL of dichloromethane, 12mmol of EDCI and 12mmol of DMAP were added, and the condensation was carried out at room temperature with stirring for 24 hours, after completion of the reaction, the resulting reaction solutions were each treated with 5 wt% of K2CO3Washing with water solution, water, 2 wt% hydrochloric acid water solution and saturated saline solution for several times, drying with anhydrous magnesium sulfate, filtering, concentrating, and purifying to obtain intermediate T-1 (threo-threo), wherein the specific chemical reaction formula is as follows:
2) synthesis of intermediate T-2 (Caso-threo)
Adding 8mmol of intermediate T-1 (threo-threo) into 100mL of dichloromethane solution of trifluoroacetic acid with the concentration of 10 wt% for dissolving, stirring for 30 minutes at room temperature, removing Boc protecting group, removing solvent, trifluoroacetic acid and produced acetic acid in the reaction system through reduced pressure concentration, dissolving the obtained residue in 100mL of dichloromethane, adding 8.8mmol of Boc-D-Tyr-OH, 8.8mmol of HBTU and 16mmol of DIPEA, stirring overnight at room temperature, concentrating the obtained reaction solution, adding water into the concentrated residue, extracting with ethyl acetate, separating phases, and separating the ethyl acetate phase with 5 wt% of K2CO3Washing the aqueous solution, water, 2 wt% hydrochloric acid aqueous solution and saturated salt solution for several times, drying with anhydrous magnesium sulfate, filtering, concentrating and purifying to obtain an intermediate T-2 (casein-threo), wherein the specific chemical reaction formula is as follows:
wherein R is3Is OTBS.
3) Synthesis of intermediate T-3 (Liang-Val-Tyr-Su)
Adding 6mmol of intermediate T-2 (casein-threo) into 60mL of dichloromethane solution of trifluoroacetic acid with the concentration of 10 wt% for dissolving, stirring for 30 minutes at room temperature, removing Boc protecting groups, removing the solvent, the trifluoroacetic acid and the produced acetic acid in the reaction system through concentration under reduced pressure, dissolving the obtained residue in 50mL of dichloromethane, adding 6.6mmol of Boc-L-Leu-L-Val-OH (raw material S-2), 6.6mmol of HBTU and 12mmol of DIPEA, stirring overnight at room temperature, concentrating the obtained reaction liquid, adding water into the concentrated residue, extracting with ethyl acetate, separating phases, and subjecting the ethyl acetate phase to 5 wt% of K2CO3Washing the aqueous solution, water, 2 wt% hydrochloric acid aqueous solution and saturated salt solution for several times, drying by anhydrous magnesium sulfate, filtering, concentrating and purifying to obtain an intermediate T-3 (leucine-valine-casein-threo), wherein the specific chemical reaction formula is as follows:
wherein R is4Is isopropyl, R5Is an isobutyl group.
4) Synthesis of intermediate T-4 (Cyclic peptide Liang-Val-Tyr-Su)
4mmol of intermediate T-3 (leucine-valine-casein-threo) were dissolved in 20mL of chloroform, 24mmol of N-methylmorpholine and 1.6mmol of tetrakis (triphenylphosphine) palladium were added, the mixture was stirred at room temperature overnight, the reaction mixture was concentrated under reduced pressure, ethyl acetate was added to dissolve the residue, and 15 wt% of KHSO was added to dissolve the residue4Washing the aqueous solution and water for several times, drying the aqueous solution and water-free magnesium sulfate, filtering and concentrating the aqueous solution and water, adding 20mL of dichloromethane solution of trifluoroacetic acid with the concentration of 10 wt% into the obtained concentrate for dissolving, stirring the solution for 30 minutes at room temperature, removing Boc protecting groups, removing the solvent, the trifluoroacetic acid and the produced acetic acid in a reaction system through reduced pressure concentration, dissolving the obtained chain peptide product into 50mL dichloromethane, adding 8mmol HATU and 8mmol DIPEA, stirring the solution at room temperature overnight, concentrating the obtained reaction solution, adding water into the residue after concentration, extracting the residue with ethyl acetate, separating the phases, and adding 5 wt% of K into an ethyl acetate phase2CO3Washing the aqueous solution, water, 2 wt% hydrochloric acid aqueous solution and saturated salt solution for several times, drying by anhydrous magnesium sulfate, filtering, concentrating and purifying to obtain an intermediate T-4 (cyclopeptide leu-valine-casein-threo), wherein the specific chemical reaction formula is as follows:
5) synthesis of intermediate T-5 (Cyclic peptide Liang-Val-Tyr-threo-Su-elimination product)
2mmol of intermediate T-4 (cyclic peptide leucine-valine-casein-threo) were dissolved in 50mL of acetonitrile and 2.2mmol (Boc) were added2O and 0.2mmol DMAP, stirring for 2 hours at room temperature, removing Fmoc protecting group, and then directly adding 6mmol nano K into the reaction solution2CO3Stirring at room temperature for 5 hr, filtering the reaction solution, concentrating under reduced pressure, adding the residue into water, stirring, extracting with ethyl acetate, separating phases, and subjecting the organic phase to KHSO 1M4Aqueous, 1M NaHCO3Washing with aqueous solution, drying, filtering, concentrating and purifying to obtain intermediate T-5 (cyclopeptide leu-val-tyr-threo-elimination product), the specific chemical reaction formula is as follows:
6) synthesis of LSH series Cyclopentapeptide esters (LSH-1)
Dissolving 1.1mmol of 2S, 4S-dimethylheptanoic acid (S-3) and 1.1mmol of N-methylmorpholine in 10mL of tetrahydrofuran, cooling to-10 ℃, adding 1.1mmol of isobutyl chloroformate, stirring for 30 minutes, heating to 0 ℃, adding 1mmol of intermediate T-5 (cyclic peptide leucine-valine-casein-threo-elimination product), stirring overnight at room temperature, diluting the obtained reaction solution with water, extracting with ethyl acetate, drying the obtained ethyl acetate phase with anhydrous magnesium sulfate, filtering, concentrating and purifying to obtain the final product of LSH series cyclic pentapeptide ester (called LSH-1), wherein the total yield is 44%, and the specific chemical reaction formula is as follows:
wherein R is1And R2Each methyl in the S configuration.
Example 2-16 Synthesis of cyclic pentapeptide esters of the LSH series
Examples 2 to 16 are methods for synthesizing LSH-series cyclic pentapeptide esters, which have substantially the same steps as example 1, but differ only in the amount of raw materials and process parameters, as detailed in tables 1 to 2:
TABLE 1 summary of the process parameters of examples 2 to 8
TABLE 2 summary of the process parameters of examples 9-16
Wherein, the steps and the process parameters of other parts of the embodiments 2-16 are the same as those of the embodiment 1;
the specific structural formulas of LSH-1 to LSH-16 are as follows:
to save time for examination, only the nmr spectra and mass spectra of four compounds were selected for presentation, as follows:
the nuclear magnetic resonance hydrogen spectrum of the LSH-2 is1H NMR (500MHz, Acetone-d6) δ 8.50-8.46(1H, m), 8.42(1H, s), 8.19(1H, s), 7.91-7.87(1H, m), 7.38(1H, t, J ═ 7.0Hz), 7.22(2H, t, J ═ 8.5Hz), 6.76(2H, d, J ═ 8.0Hz), 6.61-6.55(1H, m), 5.22-5.19(1H, m), 4.95-4.56(1H, m), 4.43-4.32(1H, m), 4.22-4.20(1H, m), 3.81-3.77(1H, m), 3.35-3.32(1H, m), 3.30-3.25(1H, 3.03, 2.03-2H, 2.82 (1H, m), 1H, m), 3.77(1H, m), 3.35-3.32(1H, m), 3.30-3.25(1H, 3.94.0, 2H, 1 m), 1H, 6.70H, 1H, m), 1.59-1.54(2H, m), 1.42-1.39(2H, m), 1.35(3H, d, J ═ 6.0Hz), 1.20-1.14(2H, m), 1.06(2H, d, J ═ 7.0Hz), 1.05-1.01(1H, m), 0.99-0.94(9H, m), 0.89-0.86(2H, m), 0.83-0.78(6H, m), 0.75(3H, d, J ═ 7.0 Hz); the carbon spectrum is13C NMR(125MHz，Acetone-d6)δ177.25，175.33，171.37，170.42，170.38，156.86，134.61，134.28，131.00，130.82，128.57，116.04，74.91，71.61，60.67，58.36，57.46，57.09，55.56，54.90，42.56，41.13，39.94，39.29，36.92，31.04，28.79，25.33，23.19，21.97，20.61，20.25，19.77，19.15, 16.37; mass spectrum HRMS (ESI) m/z: [ M + H ]]+found for 700.4277，calcd for C37H58N5O8 700.4285。
The nuclear magnetic resonance hydrogen spectrum of LSH-4 is1H NMR (500MHz, Acetone-d6) δ 8.47(1H, d, J ═ 7.0Hz), 8.42(1H, s), 8.20-8.19(1H, m), 7.91-7.88(1H, m), 7.41-7.38(1H, m), 7.23-7.20(2H, m), 7.08(1H, d, J ═ 9.0Hz), 6.76(2H, d, J ═ 8.5Hz), 6.61-6.56(1H, m), 5.23-5.20(1H, m), 4.88(1H, dd, J ═ 3.0, 9.0Hz), 4.43-4.34(1H, m), 4.22-4.18(1H, m), 3.83-3.77(1H, m), 3.34(1H, 3.83, t ═ 3.3.3H, 3.47, 1H, 3.83, 3.5H, 1H, 3.5H, 3H, 3, 3.47 (1H, 3H, m), 2H, 3.5H, 3, 2H, 3.5H, 3H, 2H, 3H, 3H, 2H, 3H, 3H, 1H, 3H, 2H, m), 1.71(3H, d, J ═ 7.0Hz), 1.68-1.64(1H, m), 1.59-1.53(1H, m), 1.42-1.39(1H, m), 1.37(3H, d, J ═ 6.0Hz), 1.24-1.16(2H, m), 1.07(3H, d, J ═ 7.0Hz), o.99-o.94(10H, m), 0.89-0.86(1H, m), 0.83-0.81(4H, m), 0.80(3H, d, J ═ 4.0 Hz); the carbon spectrum is13C NMR (125MHz, Acetone-d6) δ 177.28, 175.36, 171.44, 170.45, 169.72, 165.46, 156.88, 134.44, 130.88, 130.82, 130.42, 128.57, 116.09, 116.00, 74.56, 71.61, 60.65, 57.36, 55.96, 55.00, 42.50, 41.09, 40.28, 39.19, 36.83, 31.19, 28.79, 25.34, 23.17, 22.00, 21.77, 20.57, 19.86, 19.77, 19.27, 16.57, 14.79, 14.51; mass spectrum HRMS (ESI) m/z: [ M + H ]]+found for 700.4288，calcd for C37H58N5O8700.4285。
The nuclear magnetic resonance hydrogen spectrum of LSH-10 is1H NMR(500MHz，Acetone-d6)δ8.46(1H，d，J＝7.0Hz)，8.42(1H，s)，8.20(1H，br)，7.89(1H，s)，7.40(1H，d，J＝7.5Hz)，7.22(2H，d，J＝8.5Hz)，7.06(1H，d，J＝9.0Hz)，6.76(2H，d，J＝8.5Hz)，6.58(1H，q，J＝7.0Hz)，5.35(1H，t，J＝4.5Hz)，5.23-5.21(1H，m)，4.88(1H，dd，J＝3.0，9.0Hz)，4.40-4.35(1H，m)，4.22-4.18(1H，m)，3.35(1H，t，J＝6.5Hz)，3.28(1H，dd，J＝3.0，14.0Hz)，3.00(1H，t，J＝13.0Hz)，2.67-2.62(1H，m)，2.48-2.41(1H，m)，2.15(1H，t，J＝70Hz), 1.92-1.85(1H, m), 1.71(3H, d, J ═ 7.0Hz), 1.68-1.64(1H, m), 1.59-1.54(2H, m), 1.37(3H, d, J ═ 6.0Hz), 1.23-1.17(2H, m), 1.06(3H, d, J ═ 7.0Hz), 1.04-1.01(1H, m), o.98(6H, t, J ═ 6.5Hz), o.95(3H, d, J ═ 6.5Hz), 0.92-0.87(4H, m), o.82(3H, t, J ═ 4.0Hz), 0.80(3H, d, J ═ 4.0 Hz); the carbon spectrum is13C NMR (125MHz, Acetone-d6) δ 177.29, 175.36, 171.46, 170.47, 169.78, 165.46, 156.90, 134.48, 130.88, 130.59, 128.59, 116.10, 74.56, 60.67, 57.34, 55.97, 55.03, 42.52, 41.12, 40.30, 39.23, 36.84, 31.21, 27.79, 25.35, 23.18, 22.02, 21.77, 20.58, 19.87, 19.77, 19.27, 16.57, 14.77, 14.51; mass spectrum HRMS (ESI) m/z: [ M + H ]]+found for 700.4280，calcd for C37H58N5O8 700.4285。
The nuclear magnetic resonance hydrogen spectrum of the LSH-14 is1H NMR (500MHz, Acetone-d6) δ 8.14(1H, d, J-25.0 Hz), 8.07(1H, s), 7.79(1H, s), 7.48(1H, s), 7.20-7.18(3H, m), 6.76(2H, d, J-8.0 Hz), 6.61(1H, q, J-7.0 Hz), 5.23(1H, s), 4.77(1H, s), 4.48(1H, s), 4.05(1H, s), 3.86(1H, s), 3.33(1H, dd, J-3.5, 14.0Hz), 3.00(1H, t, J-13.0 Hz), 2.70(2H, q, J-7.0), 2.03-2.01 (1H, t, J-13.0 Hz), 2.70(2H, q, J-7.0), 2.01-2H, 1.01(1H, 1H-19.31H, 1H-19.0 Hz), 1H-49H, 1.49H, 1H, 3.0Hz, 1H, 1.49H, 1H, 3.0Hz, d, J ═ 6.5Hz), 1.03(1H, s), 0.95(3H, d, J ═ 6.5Hz), 0.92(3H, d, J ═ 6.5Hz), 0.90-0.87(4H, m), 0.84(3H, t, J ═ 7.0Hz), 0.72(3H, d, J ═ 6.5Hz), 0.67(3H, d, J ═ 6.5 Hz); the carbon spectrum is13C NMR (125MHz, Acetone-d6) δ 173.96, 171.40, 170.40, 169.25, 165.10, 156.99, 133.93, 130.94, 130.10, 128.26, 116.04, 74.00, 62.02, 56.87, 56.12, 54.36, 42.67, 40.06, 39.49, 36.73, 32.63, 31.12, 25.72, 23.80, 21.31, 20.69, 20.31, 19.35, 19.10, 18.46, 16.72, 14.88, 14.56; mass spectrum HRMS (ESI) m/z: [ M + H ]]+found for 700.4282，calcd for C37H58N5O8 700.4285。
Example 17 antitumor Activity test
The test was performed based on sulforhodamine b (srb) method, and samples to be tested (LSH series cyclic pentapeptide esters prepared in examples 1 to 16) were prepared into DMSO solutions of different concentrations of 1000, 500, 250, 100, 50, 25, and 10 μ M, respectively.
Suspending the selected tumor cells in 10% fresh fetal calf serum PRMI culture solution (DMEM culture solution can also be used), digesting with pancreatin, counting cells, and fixing cell concentration to 5 × 104cells/mL to obtain tumor cell fluid;
adding tumor cell fluid (tumor cells are respectively tested by melanoma cell line SK-MEL-1, lung cancer cell line HePG-2 and colon cancer cell line HT-29) into 96-well plate, adding 90 μ L of tumor cell fluid into each well, wherein each well contains about 4500 tumor cells, adding 10 μ L of DMSO solutions with different concentrations into each well, transferring to 37 deg.C, relative humidity of 100% and CO content2Culturing for 48 hours in an incubator with mixed atmosphere at an air ratio of 5: 95; taking out 96-well plate, discarding solution, adding 100 μ L TCA with concentration of 10 wt%, fixing at 4 deg.C for 30 min, discarding solution again, and adding 100 μ L ddH into each well2Washing with water for three times, drying at room temperature for 6 hr, adding 100 μ L of Trisbase, dissolving, shaking for 5 min, measuring absorbance at 570nm (570nm is the absorbance of the solution related to the number of living cells) with microplate reader, measuring cell proliferation, and calculating the inhibition rate of tumor cells. Each set of experiments was repeated three times and the average was calculated; replacing tumor cells with normal cells 239-T to carry out the experiment, and calculating the average value of the cell inhibition rate; the specific results are shown in tables 3-4 (the values in tables 3-4 are average values, it should be noted that "-" in the tables means that the specific test is not carried out, since the related tumor inhibition rate is measured on similar compounds, and the repeated measurement is not carried out subsequently).
TABLE 3 inhibition rates (25. mu.M) of LSH 1-LSH 8 for 48h tumor cells
TABLE 4 inhibition rates (25. mu.M) of LSH 9-LSH 16 for 48h of tumor cells
As can be seen from tables 3-4, the LSH series cyclic pentapeptide ester of the invention shows good inhibitory activity on various tumor cells, and is particularly more prominent in melanoma cell line SK-MEL-1, lung cancer cell line HePG-2 and colon cancer cell line HT-29. The LSH series cyclic pentapeptide ester has extremely low toxicity to normal cells 239-T under the concentration of 25 mu M.
Selecting compound LSH-15 to act on HT-29 tumor cells, carrying out dose-effect relationship research according to the experimental method (specific results are shown in Table 5), measuring inhibition rates in tumor cell fluids with different concentrations, finding that the two have good correlation, and calculating half inhibition concentration by using the concentration and inhibition rate data in Table 5 to obtain IC50It was 0.98. mu.M.
TABLE 5 inhibition of HT-29 cells for 48h (25. mu.M) by different concentrations of LSH-15
The obtained other compounds also have good tumor cell inhibiting activity, and specific experiments are not listed again.
It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (10)
1. A cyclic pentapeptide ester of the LSH series, which is characterized in that the chemical structural formula of the cyclic pentapeptide ester of the LSH series is as follows:
wherein:
R1and R2Each independently hydrogen or methyl;
R3is independently hydrogen, alkyl, halogen, hydroxyl, amino/amino, hydrocarbyloxy or siloxy;
R4and R5Each independently isopropyl, isobutyl, sec-butyl or benzyl.
2. The LSH-series cyclic pentapeptide ester of claim 1, wherein the raw material from which said LSH-series cyclic pentapeptide ester is made comprises a plurality of amino acid derivatives;
the configuration of the amino acid derivatives is independent L type or D type.
3. The LSH serial cyclopentapeptide ester according to claim 2, wherein the plurality of amino acid derivatives are each independently aliphatic amino acid derivatives or arylpropyl amino acid derivatives.
4. The LSH series cyclic pentapeptide ester of claim 3,
the fatty amino acid derivative is threonine derivative, leucine derivative, isoleucine derivative or valine derivative;
the aryl alanine amino acid derivative is phenylalanine derivative, 4-substituted phenylalanine derivative or tyrosine derivative.
5. The LSH series cyclic pentapeptide ester of any of claims 1-4, wherein R is1And R2When both are methyl, R1And R2The configuration of the chiral carbon is independent R type or S type.
6. The LSH-series cyclic pentapeptide ester of any of claims 1 to 4, wherein the LSH-series cyclic pentapeptide ester has a chemical structure of LSH-1 to LSH-16, and a bioisostere cyclic peptide derivative made of a hydroxy-substituted phenylalanine derivative;
wherein, the specific structural formulas of LSH-1 to LSH-16 are as follows:
7. the method for synthesizing the LSH-series cyclic pentapeptide ester according to any one of claims 1 to 6, wherein the method comprises the steps of constructing a peptide chain from a plurality of amino acid derivatives by a 3+2 strategy, cyclizing, carrying out elimination reaction under an alkaline condition to obtain cyclic peptide with carbon-carbon unsaturated bonds, and carrying out acylation reaction to obtain the LSH-series cyclic pentapeptide ester.
8. The method for synthesizing cyclic pentapeptide esters of LSH series according to claim 7, wherein said method comprises the following steps performed in sequence:
1) synthesis of intermediate T-1:
taking initial raw materials S-1 and Boc-Thr (T-Bu) -OH, and carrying out condensation reaction under the action of a condensing agent and a catalyst to obtain an intermediate T-1, wherein the specific chemical reaction formula is as follows:
wherein, the Boc-Thr (t-Bu) -OH has a structural formula:
2) synthesis of intermediate T-2:
after the intermediate T-1 is subjected to Boc protecting group removal by trifluoroacetic acid, the intermediate T-1 is reacted with Boc-Tyr (R) under the action of a peptide coupling reagent and an organic base3) And (3) carrying out condensation reaction on-OH again to obtain an intermediate T-2, wherein the specific chemical reaction formula is as follows:
wherein, Boc-Tyr (R)3) -OH has the formula:
R3is independently hydrogen, halogen, hydroxyl, amino/amino, hydrocarbyloxy or silyloxy;
3) synthesis of intermediate T-3:
after the intermediate T-2 is subjected to Boc protecting group removal by trifluoroacetic acid, the intermediate T-2 is subjected to condensation reaction with the raw material S-2 again under the action of a peptide coupling reagent and organic base to obtain an intermediate T-3, wherein the specific chemical reaction formula is as follows:
wherein R is4And R5Each independently isopropyl, isobutyl, sec-butyl or benzyl;
4) synthesizing an intermediate T-4:
removing allyl from the intermediate T-3 under the catalytic action of N-methylmorpholine and tetrakis (triphenylphosphine) palladium, removing Boc protecting group by trifluoroacetic acid, and performing condensation reaction under the action of a peptide coupling reagent and organic base to obtain an intermediate T-4, wherein the specific chemical reaction formula is as follows:
5) synthesizing an intermediate T-5:
the intermediate T-4 reacts with di-tert-butyl dicarbonate under the catalysis of 4-dimethylaminopyridine, then carries out elimination reaction under the action of nano potassium carbonate, and simultaneously removes Fmoc protecting groups to obtain an intermediate T-5, wherein the specific chemical reaction formula is as follows:
6) synthesis of LSH series Cyclic pentapeptide esters
Carrying out ester exchange reaction on a raw material S-3 and isobutyl chloroformate under the action of N-methylmorpholine, then adding an intermediate T-5, and carrying out acylation reaction to obtain the LSH series cyclic pentapeptide ester, wherein the specific chemical reaction formula is as follows:
wherein R is1And R2Each independently hydrogen or methyl.
9. Use of the LSH series cyclic pentapeptide ester of any of claims 1-6 in the manufacture of a medicament for the treatment of melanoma/lung/colon/stomach/cervical cancer.
10. The use of the LSH-series cyclic pentapeptide ester according to claim 9, wherein the inhibitory activity IC of the LSH-series cyclic pentapeptide ester on melanoma cells/lung cancer cells/colon cancer cells/stomach cancer cells/cervical cancer cells50≥0.98μM。
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