CN113121648A - N-methyl cyclic pentapeptide compound and synthesis method and application thereof - Google Patents

Abstract

The invention discloses an N-methyl cyclic pentapeptide compound and a synthesis method and application thereof, wherein the N-methyl cyclic pentapeptide compound has a structure shown in a general formula (I):

wherein: ar is a fused ring aryl or substituted fused ring aryl; R1-R4 are each independently isopropyl, isobutyl, methyl; r '1-R' 5 are independent hydrogen and methyl respectively. N-methyl Ring of the inventionThe pentapeptide compound has good anti-tumor activity, particularly has higher activity on resisting gastric cancer cells, cervical cancer cells, renal clear cancer cells or pancreatic cancer cells, and the action way of the pentapeptide compound is to induce caspase-3 to make tumor cells die through caspase-9. These cyclic peptides exhibit good enzyme stability and have a long half-life.

Description

N-methyl cyclic pentapeptide compound and synthesis method and application thereof

Technical Field

The invention relates to a cyclic pentapeptide compound, in particular to an N-methyl cyclic pentapeptide compound containing condensed ring aryl alanine and having anti-tumor activity, a synthetic preparation method and application thereof.

Background

The cyclic peptide is a peptide compound with a special structure, most of the compounds have good physiological and pharmacological activities, wherein the anti-tumor activity is particularly outstanding, the N-methylated cyclic peptide is widely existed in natural products, the N-methylation of the cyclic peptide can greatly improve the molecular structure, and the cyclic peptide shows good biological activity. The cyclic peptide has different structures, properties and activities according to different amino acid arrangement sequences.

Disclosure of Invention

The invention aims to provide an N-methyl cyclic pentapeptide compound, wherein the N-methyl cyclic pentapeptide has good anti-tumor activity, particularly has higher activity on resisting gastric cancer cells, cervical cancer cells, renal clear cancer cells or pancreatic cancer cells, and has higher stability and longer half-life.

The invention also aims to provide a synthetic preparation method of the N-methyl cyclic pentapeptide, which has the advantages of less synthetic steps, higher product yield and lower cost.

To solve the above technical problems, the present invention provides an N-methylcyclopentapeptide compound of the general formula (i):

wherein:

ar is a fused ring aryl or substituted fused ring aryl;

R1-R4 are each independently isopropyl, isobutyl, methyl;

r '1-R' 5 are independent hydrogen and methyl respectively.

Wherein the fused ring aryl group comprises a naphthyl group, a substituted naphthyl group, a quinolyl group or a substituted quinolyl group;

the substituent in the substituted condensed ring aryl comprises halogen and methoxy.

Wherein the N-methylcyclopentapeptide compound is composed of five L-amino acids.

The five L-amino acids are connected into a ring structure by peptide bonds, and are selected from N-methyl amino acid, naphthylalanine or substituted naphthylalanine or condensed ring aryl alanine amino acid and aliphatic amino acid; and the five L-amino acids are independent of each other, are not completely identical and are arranged in a random order.

Wherein, among the five L-amino acids,

one L-amino acid is an N-methyl amino acid;

one L-amino acid is condensed ring aryl alanine or substituted condensed ring aryl alanine;

the other three L-amino acids are aliphatic amino acids or cyclic amino acids.

Wherein the fatty amino acid is L-leucine, L-valine, L-isoleucine or L-alanine.

Wherein the N-methyl amino acid is an N-methylated amino acid.

The N-methyl cyclic pentapeptide compound is one of the chemical formulas LX-1, LX-2, LX-3, LX-4, LX-5, LX-6, LX-7, LX-8, LX-9, LX-10, LX-11 or LX-12

The LX-3, namely the application of the leu-N-methyl-leu-valine-leu-2-naphthylalanine cyclic pentapeptide in preparing the anti-cancer drugs for enabling melanoma cells, gastric cancer cells, anti-cervical cancer cells, anti-renal clear cancer cells or anti-pancreatic cancer cells to be apoptotic through caspase-3 enzyme.

The invention provides a method for synthesizing an N-methylcyclopentapeptide compound, which comprises the following steps:

the method comprises the following steps: the tripeptide fragment is synthesized by the following chemical reaction formula:

step two: the dipeptide fragment is synthesized by the following chemical reaction formula:

step three: the synthesis and cyclization reaction of pentapeptide has the following chemical reaction formula:

wherein:

ar is a fused ring aryl or substituted fused ring aryl;

R1-R4 are each independently isopropyl, isobutyl, methyl;

r '1-R' 5 are independent hydrogen and methyl respectively.

Wherein the LX-1 is leucine-valine-N-methylleucine-2-naphthylpropanepentaceptide, and the synthesis steps are as follows:

step A, synthesizing N-methyl-leucine-2-naphthylalanine methyl ester dipeptide;

a1 adding a certain amount of 2-naphthylalanine methyl ester, Boc-N-methyl-leucine HATU, DIPEA, DCM and N, N-dimethylformamide into a container with calcium chloride, and stirring overnight at room temperature;

a2 concentrating the solution obtained in step A1 under reduced pressure, extracting with ethyl acetate, and combining the organic phases;

a3 extracting the organic phase with water, K₂CO₃Washing the solution, HCl solution and saturated NaCl solution for several times, concentrating, and purifying to obtain colorless viscous liquid;

a4, dissolving the colorless viscous liquid in dichloromethane and trifluoroacetic acid solution, stirring for 5-8 hours, and then removing dichloromethane and trifluoroacetic acid under reduced pressure to obtain a crude product of N-methyl-leucine-2-naphthylalanine methyl ester;

b, synthesizing Boc-bright-valine tripeptide;

b1 adding valine methyl ester, Boc-leucine, HBTU, DIPEA, DCM and DMF into a drying container, and stirring at room temperature overnight;

b2, concentrating the solution obtained in the step B1 under reduced pressure, extracting the concentrated solution with ethyl acetate, and combining organic phases;

b3 extracting the organic phase with water, K₂CO₃Washing the solution, an HCl solution and a saturated NaCl solution for several times respectively, drying, filtering and concentrating to obtain a product, adding DCM and TFA into the product, stirring and reacting for 5-7h at room temperature, and removing DCM and TFA under reduced pressure to obtain the bright-valine methyl trifluoroacetate;

b4, dissolving leucine-valine trifluoroacetate in DCM and DMF, adding Boc-leucine, HBTU and DIPEA, and stirring overnight;

b5 concentrating the reaction liquid from step B4, adding water to the product, extracting with ethyl acetate, and separating the organic phase with water and K₂CO₃Washing the solution, HCl solution and saturated NaCl solution for several times, drying, filtering, concentrating and purifying to obtain solid product;

b6 dissolving the solid product with THF, transferring to a container containing LiOH and water, stirring overnight at room temperature, removing THF under reduced pressure, adding water, adjusting pH of the solution to 3-4 with hydrochloric acid, extracting with ethyl acetate, mixing organic phases, drying overnight, filtering, and concentrating to obtain Boc-bright-valine tripeptide;

step C, synthesizing leucine-valine-N-methyl-leucine-2-naphthylalanine methyl ester pentapeptide;

c1: stirring N-methyl-leucine-2-naphthylalanine methyl ester, Boc-leucine-valine tripeptide, HATU, DIPEA, DCM and DMF at room temperature overnight;

c2: concentrating the reactant in the step C1 under reduced pressure, extracting with ethyl acetate for several times, combining organic phases, washing with water, K2CO3 solution, HCl solution and saturated NaCl solution for several times in sequence, drying, filtering, concentrating and purifying to obtain Boc-bright-valine-N-methyl-bright-2-naphthylalanine methyl ester;

c3: dissolving the obtained solid with THF, transferring into a container containing LiOH and water to form a solution, stirring overnight at room temperature, removing THF under reduced pressure after the reaction is finished, adding water into the residue, adjusting pH to 3-4 with dilute hydrochloric acid, extracting with ethyl acetate, combining organic phases, drying overnight, concentrating under reduced pressure to obtain Boc-leucine-valine-N-methyl-leucine-2-naphthylalanine pentapeptide, dissolving the Boc-leucine-valine-N-methyl-leucine-2-naphthylalanine pentapeptide into a solution of DCM and TFA, stirring at room temperature for reaction for 5-8h, and removing DCM and TFA under reduced pressure to obtain leucine-valine-N-methyl-leucine-2-naphthylalanine pentapeptide;

step D, synthesizing leucine-valine-N-methyl-leucine-2-naphthylalanine cyclic pentapeptide;

d1: adding leucine-valine-N-methyl-leucine-2-naphthylalanine pentapeptide into a container, dissolving in a solution formed by DMF, tetrahydrofuran and dichloromethane, stirring to dissolve, adding PyAOP HATU and diisopropylethylamine, and stirring at normal temperature for reacting for 65-75 h;

d2: decompressing and concentrating the reaction liquid in the step D1, adding water into the residual liquid, extracting with ethyl acetate, combining organic phases, and sequentially using water and K₂CO₃Washing the solution, hydrochloric acid solution, water and saturated NaCl solution for several times respectively, drying overnight, filtering, concentrating, and purifying to obtain the leucine-valine-N-methyl-leucine-2-naphthylalanine cyclic pentapeptide.

The N-methyl cyclic pentapeptide compound is applied to the preparation of medicines for treating transparent cancers of stomach, cervix, pancreas, melanin and kidney.

Compared with the prior art, the invention has the following beneficial effects:

the N-methyl cyclic pentapeptide compound has good anti-tumor activity, particularly has higher activity on resisting melanoma cells, gastric cancer cells, cervical cancer cells, renal clear cancer cells or pancreatic cancer cells, shows good enzyme stability, is not easy to degrade in vivo, and has longer half-life; the N-methylcyclopentapeptide of the invention has very little side effect on smooth muscle cells, lymphocytes and cardiac muscle cells; the inhibition effect of the N-methyl cyclic pentapeptide on the tumor cells is exerted through a way of inducing apoptosis; the synthetic preparation method of the N-methyl cyclic pentapeptide has the advantages of less synthetic steps, higher product yield and lower cost.

Drawings

FIG. 1 is an image of the Western-Blot detection result of the effect of LX-3 of the present invention on B16.

Detailed Description

The invention provides a novel anti-tumor N-methylcyclopentapeptide compound, which has a structure shown in a formula (I):

wherein:

ar is condensed ring aryl or substituted condensed ring aryl, the condensed ring aryl comprises naphthyl, substituted naphthyl, quinolyl or substituted quinolyl and the like, and the substituent in the substituted condensed ring aryl comprises halogen, methoxyl and the like;

R1-R4 are each independently isopropyl, isobutyl, methyl, or the like;

r '1 to R' 5 are each independently hydrogen, methyl, or the like.

The anti-tumor N-methylcyclopentapeptide compound is formed by connecting five L-amino acids with each other by peptide bonds to form a ring structure, wherein the five amino acids are selected from N-methyl amino acid, naphthylalanine or substituted naphthylalanine or fused ring aryl alanine amino acid and aliphatic amino acid; and the five amino acids are independent of each other, are not completely identical and are arranged in a random order.

Specifically, the antitumor N-methyl cyclic pentapeptide compound only contains one N-methyl amino acid, one condensed ring aryl alanine amino acid or substituted condensed ring aryl alanine amino acid in five L-amino acids, and the others are all aliphatic amino acids or cyclic amino acids. Wherein the N-methyl amino acid is N-methylated amino acid, and the fatty group amino acid is selected from L-leucine, L-valine, L-isoleucine, and L-alanine.

The representative compounds of the antitumor N-methyl cyclic pentapeptide compound of the present invention have the chemical formula LX-1, LX-2, LX-3, LX-4, LX-5, LX-6, LX-7, LX-8, LX-9, LX-10, LX-11 or LX-12.

The synthesis and preparation method of the anti-tumor N-methylcyclopentapeptide compound comprises the following steps:

the synthesis of the antitumor N-methyl cyclic pentapeptide compound is realized by adopting a liquid phase synthesis method, the strategy is a technology of protecting the nitrogen end of amino acid by Boc, the synthesis route is a 2+3 convergent or one-by-one connection method, HBTU or HATU or HCTU or PyBOP and the like are used as condensing agents for the growth of a peptide chain, and the synthesis method specifically comprises the following steps:

the method comprises the following steps: the tripeptide fragment is synthesized by the following chemical reaction formula:

step two: the dipeptide fragment is synthesized by the following chemical reaction formula:

step three: the synthesis and cyclization reaction of pentapeptide has the following chemical reaction formula:

the synthetic preparation method of the N-methyl cyclic pentapeptide has the advantages of less synthetic steps, higher product yield and lower cost.

The present invention will be described in further detail with reference to specific embodiments.

EXAMPLE 1 Synthesis of Liang-Val-N-methylLiang-2-naphthalenepropanac pentapeptide (LX-1)

1. Synthesis of N-methyl-leucine-2-naphthylalanine methyl ester dipeptide.

2mmol of methyl 2-naphthylalanine, 3mmol of Boc-N-methyl-leucine, 2.4mmol of HATU, 14mmol of DIPEA, 50mL of DCM and 5mL of N, N-dimethylformamide were placed in a 100mL single-neck flask equipped with a calcium chloride dry tube and stirred at room temperature overnight. Concentrated under reduced pressure, 300mL of water was added, extracted five times with 60mL of ethyl acetate, and the organic phases were combined. The organic phase was washed with water and 5% K₂CO₃And 2% HCl saturated NaCl, washed three times respectively, dried by anhydrous magnesium sulfate, filtered by suction, concentrated and purified by silica gel column chromatography to obtain colorless viscous liquid with the yield of 79%. Then, this liquid was dissolved in 12mL of methylene chloride and 8mL of trifluoroacetic acid, and stirred at ordinary temperature for 6 hours. DCM and TFA were removed under reduced pressure to give the crude product N-methyl-leu-2-naphthylalanine methyl ester.

2. Synthesis of the tripeptide Boc-light-valine.

In a 100mL single-neck flask equipped with a drying tube, 2mmol of valine methyl ester, 3mmol of Boc-leucine, 2.4mmol of HBTU, 14mmol of DIPEA, 50mL of LPCM and 5mL of DMF were added, and the mixture was stirred at room temperature overnight. Concentrated under reduced pressure, 300mL of water was added, extracted five times with 60mL of ethyl acetate, and the organic phases were combined. The organic phase was washed with water and 5% K₂CO₃Washed three times with 2% HCl saturated NaCl, dried over anhydrous magnesium sulfate, filtered, concentrated, and the residue added with 12mL DCM and 8mL TFA, stirred at room temperature for 5-7h (preferably 6 h). DCM and TFA were removed under reduced pressure to give the light-valinyl trifluoroacetate salt. This salt was dissolved in 50mL DCM and 5mL DMF, after which 3mmol Boc-leucine and 2.4mmol HBTU and 14mmol DIPEA were added and stirred overnight. The reaction mixture was concentrated, 300mL of water was added to the residue, and the mixture was extracted with ethyl acetateOrganic phase respectively contains water and 5% K₂CO₃The reaction mixture was washed 3 times with 2% HCl saturated NaCl, and dried over anhydrous magnesium sulfate overnight. Filtering, concentrating, purifying the product by silica gel column chromatography to obtain a solid product with the yield of 98%. The resulting solid was dissolved in 52.5mL THF and transferred to a flask containing a solution of 18mmol LiOH in 15mL water and stirred overnight at room temperature. THF was removed under reduced pressure, 60mL of water was added, the pH of the solution was adjusted to 3-4 with 2% hydrochloric acid, extraction was performed with ethyl acetate, and the organic phases were combined and dried over anhydrous magnesium sulfate overnight. Filtering and concentrating to obtain Boc-bright-valine tripeptide.

3. Synthesis of leucine-valine-N-methyl-leucine-2-naphthylalanine methyl ester pentapeptide.

2mmol of methyl N-methyl-leu-2-naphthylalanine, 3mmol of Boc-leu-valine tripeptide, 2.4mmol of HATU, 14mmol of DIPEA, 50mL of DCM and 5mL of DMF are stirred at room temperature overnight and the progress of the reaction is checked by TLC. Concentrated under reduced pressure, 300mL of water was added, extracted five times with 60mL of ethyl acetate, and the organic phases were combined. Sequentially using water and 5% K₂CO₃And 2% HCl saturated NaCl for three times respectively, drying the solution by anhydrous magnesium sulfate, filtering the solution, concentrating the solution, and purifying the solution by silica gel column chromatography to obtain Boc-bright-valine-N-methyl-bright-2-naphthylalanine methyl ester with the yield of 68%. The resulting solid was dissolved in 17.5mL THF and transferred to a flask containing a solution of 3mmol LiOH in 2.5mL water and stirred overnight at room temperature. The reaction was followed by TLC and after the reaction was complete THF was removed under reduced pressure. To the residue was added 60mL of water, extracted four times with dilute hydrochloric acid to pH 3-4 and 60mL of ethyl acetate, and the organic phases were combined and extracted with anhydrous Na₂SO₄Dry overnight. Concentrating under reduced pressure to obtain Boc-light-valine-N-methyl-light-2-naphthylalanine pentapeptide, dissolving in 6mL DCM and 4mL TFA solution, and stirring at room temperature for 5-8h (preferably 6 h). DCM and TFA were removed under reduced pressure to give leu-valine-N-methyl-leu-2-naphthylalanine pentapeptide.

4. Synthesis of leu-valine-N-methyl-leu-2-naphthylalanine cyclic pentapeptide.

0.5mmol of leu-valine-N-methyl-leu-2-naphthylalanine pentapeptide was added to a single-neck flask and dissolved in a solution of 60mL of DMF, 240mL of tetrahydrofuran and 240mL of dichloromethane. After stirring and dissolution, 3mmol PyAOP and 1mmol PyAOP were addedHATU, 3.5mmol diisopropylethylamine, and the reaction is stirred at normal temperature for 65-75h (preferably 72 h). After concentration under reduced pressure, 400mL of water was added to the residue, and the mixture was extracted five times with 90mL of ethyl acetate, and the organic phases were combined. Sequentially using water and 5% K₂CO₃The mixture was washed twice with 2% hydrochloric acid, water and saturated brine, and dried over anhydrous sodium sulfate overnight. Suction filtration, concentration and silica gel column chromatography purification are carried out to obtain the leucine-valine-N-methyl-leucine-2-naphthylalanine cyclic pentapeptide with the yield of 37 percent.

1H NMR(500MHz,CDCl₃)δ7.81(t,J＝4.5Hz,1H),7.78-7.74(m,2H),7.67(d,J＝9.0Hz,1H),7.63(s,1H),7.49-7.43(m,3H),7.34-7.32(dd,J＝8.5Hz,J＝1.5Hz,1H),7.04(d,J＝6.5Hz,1H),6.79(d,J＝7.5Hz,1H),4.77(d,J＝7.5Hz,1H),4.49(t,J＝8.5Hz,1H),4.28(d,J＝7.5Hz,1H),3.83-3.81(m,1H),3.40-3.37(dd,J＝9.0Hz,J＝6.0Hz,1H),3.34-3.25(m,2H),3.20(s,3H),2.12(brs,1H),2.03-1.88(m,3H),1.64-1.61(m,1H),1.57-1.49(m,3H),1.40-1.34(m,1H),1.28-1.25(m,1H),0.96-0.90(m,12H),0.85-0.79(m,12H)；13C NMR(125MHz,CDCl₃)δ173.62,173.54,172.61,171.30,171.01,133.78,133.55,132.64,128.74,128.03,127.77,127.53,127.14,126.43,125.99,58.14(2C),55.36,54.71,53.06,40.40,39.18,37.64,37.59,30.62(2C),25.52,25.26,24.79,22.90,22.80,22.56,22.25,22.07,21.82,19.53,18.38；HRMS(ESI,M+1)C₃₇H₅₆N₅O₅The calculated value was 650.4203, found 650.4221.

EXAMPLE 2 Synthesis of Liang-Leu-N-methyl-Val-Leu-2-Naphthylpropylenepentaceptide (LX-2)

1. Synthesis of Boc-light dipeptide.

2mmol of leucine methyl ester, 3mmol of Boc-leucine, 0.24mmol of HBTU, 14mmol of DIPEA, 50mL of DCM and 5mL of DMF were added to a 100mL single-neck flask containing a dry tube and stirred at room temperature overnight. Concentrated under reduced pressure, added with 300mL of water, extracted five times with 60mL of ethyl acetate, and the organic phases combined. The organic phase was washed with water and 5% K₂CO₃Washed with 2% HCl and saturated NaCl for three times, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by silica gel column chromatography to obtain a white solid with a yield of 97%. The resulting solid was dissolved in 52.5mL THF and transferred to a container containing 18mmol LiOH and 15mL waterThe solution of (4) was stirred at room temperature overnight. After the reaction was complete, THF was removed under reduced pressure. To the residue was added 60mL of water, extracted four times with dilute hydrochloric acid to pH 3-4 and 60mL of ethyl acetate, and the organic phases were combined and extracted with anhydrous Na₂SO₄Dry overnight. Vacuum concentrating to obtain crude Boc-bright dipeptide product.

2. And (3) synthesizing the tripeptide of N-methyl-valine-leucine-2-naphthylalanine methyl ester.

2mmol of methyl-2-naphthylalanine, 3mmol of Boc-leucine, 0.24mmol of HBTU, 14mmol of DIPEA, 50mL of DCM and 5mL of DMF were added to a 100mL single-neck flask containing a dry tube and stirred at room temperature overnight. Concentrated under reduced pressure, added with 300mL of water, extracted five times with 60mL of ethyl acetate, and the organic phases combined. The organic phase was washed with water and 5% K₂CO₃Washed with 2% HCl and saturated NaCl for three times, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by silica gel column chromatography to obtain a white solid with a yield of 78%. This was then dissolved in 12mL DCM and 8mL TFA and the reaction was stirred at RT for 6 h. DCM and TFA were removed under reduced pressure to give the methyl ester acetate of leucine-2-naphthylalanine. This salt was dissolved in 50mL THF and 5mL DMF, after which 3mmol Boc-N-methyl-valine and 2.4mmol HATU and 14mmol DIPEA were added and stirred overnight. The solvent was evaporated under reduced pressure, 300mL of water was added, extraction was performed five times with 60mL of ethyl acetate, and the organic phases were combined. The organic phase was washed with water and 5% K₂CO₃Washing with 2% HCl and saturated NaCl for three times, drying with anhydrous magnesium sulfate, vacuum filtering, concentrating, and purifying by silica gel column chromatography to obtain colorless viscous liquid with yield of 70%. This was then dissolved in 12mL DCM and 8mL TFA and the reaction was stirred at RT for 6 h. DCM and TFA were removed under reduced pressure to obtain crude N-methyl-valine-leucine-2-naphthylalanine methyl ester.

3. Synthesis of leu-N-methyl-valine-leu-2-naphthylalanine pentapeptide.

2mmol of N-methyl-valine-leucine-2-naphthylalanine methyl ester tripeptide, 3mmol of Boc-leucine-L-leucine dipeptide, 2.4mmol of HATU, 14mmol of DIPEA, 50mL of DCM and 5mL of DMF were added to a 100mL single-neck flask with a drying tube and stirred at room temperature overnight. The solvent was evaporated under reduced pressure, 300mL of water was added, extraction was performed five times with 60mL of ethyl acetate, and the organic phases were combined. The organic phase was washed with water and 5% K₂CO₃Washed three times with 2% HCl and saturated NaCl, and dried over anhydrous magnesium sulfate overnight. And (4) carrying out suction filtration, concentration and silica gel column chromatography purification to obtain a pure Boc-leucine-N-methyl-valine-leucine-2-naphthylalanine methyl ester pentapeptide. The resulting solid was dissolved in 17.5mL THF and transferred to a flask containing a solution of 3mmol LiOH in 2.5mL water and stirred overnight at room temperature. The reaction was followed by TLC and after the reaction was complete THF was removed under reduced pressure. To the residue was added 60mL of water, and the mixture was extracted four times with dilute hydrochloric acid to adjust pH to 3-4 and 60mL of ethyl acetate, and the organic phases were combined and dried over anhydrous magnesium sulfate overnight. Concentrating under reduced pressure to obtain Boc-light-N-methyl-valine-light-2-naphthylalanine pentapeptide, dissolving into a solution of 6mL DCM and 4mL TFA, and stirring at normal temperature for reacting for 6 h. DCM and TFA were removed under reduced pressure to give leu-valine-N-methyl-leu-2-naphthylalanine pentapeptide.

4. Synthesis of leu-N-methyl-valine-leu-2-naphthylalanine cyclic pentapeptide.

0.5mmol of leu-valine-N-methyl-leu-2-naphthylalanine pentapeptide was added to a 1000mL single-neck flask with a dry tube (syringe pump added dropwise), 3mmol of PyAOP, 1mmol of HATU, 3.5mmol of DIPEA, 240mL of DCM, 240mL of THF and 60mL of DMF were added, and stirring was carried out at room temperature for 72 h. The solvent was removed under reduced pressure, 400mL of water was added, extraction was performed five times with 90mL of ethyl acetate, and the organic phases were combined. The organic phase is sequentially treated with water and 5% K₂CO₃Washed three times with 2% HCl and saturated NaCl, and dried over anhydrous magnesium sulfate overnight. Suction filtering, concentrating, purifying by silica gel column chromatography to obtain leucine-N-methyl-valine-leucine-2-naphthylalanine cyclic pentapeptide with the yield of 35%.

1H NMR(500MHz,CDCl₃)δ8.12(brs,1H),7.81-7.72(m,4H),7.64(s,1H),7.48-7.45(m,3H),7.33-7.31(dd,J＝8.0Hz,J＝1.5Hz,1H),6.24(d,J＝7.0Hz,1H),4.83-4.79(dd,J＝16.0Hz,J＝7.0Hz,1H),4.51-4.47(dd,J＝16.5Hz,J＝7.0Hz,1H),4.35-4.29(m,1H),4.10-4.05(m,1H),3.49-3.44(m,1H),3.30-3.26(m,1H),3.18-3.14(m,1H),2.72(s,3H),2.38(d,J＝11.5Hz,1H),1.82-1.80(m,1H),1.76-1.67(m,4H),1.61-1.50(m,4H),0.95(t,J＝7.0Hz,7H),0.86-0.81(dd,J＝17.5Hz,J＝6.0Hz,6H),0.74(d,J＝5.5Hz,8H),0.69(d,J＝6.5Hz,3H)；13C NMR(125MHz,CDCl3)δ174.19,172.38,171.43,171.35,170.75,134.54,133.64,132.49,128.97,128.51,127.99,127.71,127.21,126.43,125.96,65.69,56.74,54.37.,51.90,48.74,41.24,40.32,40.06,35.25,30.70,26.57,26.50,25.03,24.94,24.80,23.17,22.86,22.80,22.06,21.05,19.77,19.14；HRMS(ESI,M+1)C₃₇H₅₆N₅O₅The calculated value was 650.4203, found 650.4266.

EXAMPLE 3 Synthesis of leucine-N-methyl-leucine-valine-leucine-2-naphthylalanine Cyclopentapeptide (LX-3)

1. And synthesizing the tripeptide of valine-leucine-2-naphthylalanine methyl ester.

2mmol of methyl 2-naphthylalanine, 3mmol of Boc-leucine, 0.24mmol of HBTU, 14mmol of DIPEA, 50mL of DCM and 5mL of DMF were placed in a 100mL single-neck flask equipped with a drying tube and stirred at room temperature overnight. The solvent was evaporated under reduced pressure, 300mL of water was added, extraction was performed five times with 60mL of ethyl acetate, and the organic phases were combined. The organic phase was washed with water and 5% K₂CO₃Washed three times with 2% HCl and saturated NaCl, and dried over anhydrous magnesium sulfate overnight. Suction filtration, concentration and silica gel column chromatography purification to obtain white solid with yield of 78%. This was then dissolved in 12mL DCM and 8mL TFA and the reaction was stirred at RT for 6 h. DCM and TFA were removed under reduced pressure to give the methyl ester acetate of leucine-2-naphthylalanine. This salt was dissolved in 50mL DCM and 5mL DMF, after which 3mmol Boc-valine and 2.4mmol HATU and 14mmol DIPEA were added and stirred overnight. The solvent was evaporated under reduced pressure, 300mL of water was added, extraction was performed five times with 60mL of ethyl acetate, and the organic phases were combined. The organic phase was washed with water and 5% K₂CO₃Washed with 2% HCl and saturated NaCl for three times, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by silica gel column chromatography to obtain a white solid with a yield of 75%. This was dissolved in 12mL DCM and 8mL TFA and the reaction was stirred at RT for 6 h. DCM and TFA were removed under reduced pressure to give the methyl valine-leucine-2-naphthylalanine tripeptide.

2. Synthesis of N-methyl-light-valine-light-2-naphthylalanine methyl ester tetrapeptide.

2mmol of valine-leucine-2-naphthylalanine methyl ester tripeptide, 3mmol of Boc-N-methyl-leucine, 2.4mmol of HATU, 14mmol of DIPEA, 50mL of DCM and 5mL of DMF were added to a 100mL single-neck flask with a drying tube and stirred at room temperature overnight. The solvent was evaporated under reduced pressure, 300mL of water was added, and the mixture was extracted five times with 60mL of ethyl acetate, and the mixture was combinedAnd (4) an organic phase. The organic phase was washed with water and 5% K₂CO₃Washed three times with 2% HCl and saturated NaCl, and dried over anhydrous magnesium sulfate overnight. Suction filtration, concentration and purification by silica gel column chromatography to obtain white solid with the yield of 70 percent. This was dissolved in 12mL DCM and 8mL TFA and the reaction was stirred at RT for 6 h. DCM and TFA were removed under reduced pressure to give N-methyl-leu-valine-leu-2-naphthylalanine methyl ester tetrapeptide.

3. And (3) synthesizing the leucine-N-methyl-leucine-valine-leucine-L-2-naphthylalanine pentapeptide.

N-methyl-leucine-valine-leucine-2-naphthylalanine methyl ester tetrapeptide, 3mmol Boc-L-leucine, 2.4mmol HATU, 14mmol DIPEA, 50mL DCM and 5mL DMF were added to a 100mL single-neck flask with a drying tube and stirred at room temperature overnight. The solvent was evaporated under reduced pressure, 300mL of water was added, extraction was performed five times with 60mL of ethyl acetate, and the organic phases were combined. The organic phase was washed with water and 5% K₂CO₃Washed three times with 2% HCl and saturated NaCl, and dried over anhydrous magnesium sulfate overnight. Suction filtration, concentration and purification by silica gel column chromatography to obtain white solid with the yield of 65 percent. The resulting solid was dissolved in 17.5mL THF and transferred to a flask containing a solution of 3mmol LiOH in 2.5mL water and stirred overnight at room temperature. The reaction was followed by TLC and after the reaction was complete THF was removed under reduced pressure. To the residue was added 60mL of water, and the mixture was extracted four times with dilute hydrochloric acid to adjust pH to 3-4 and 60mL of ethyl acetate, and the organic phases were combined and dried over anhydrous magnesium sulfate overnight. Concentrating under reduced pressure to obtain Boc-leucine-N-methyl-leucine-valine-leucine-2-naphthylalanine pentapeptide. 0.5mmol was dissolved in 6mL DCM and 4mL TFA and the reaction was stirred at RT for 6 h. DCM and TFA were removed under reduced pressure to give the leucine-N-methyl-leucine-valine-leucine-L-2-naphthylalanine pentapeptide.

4. Synthesis of leu-N-methyl-leu-valine-leu-2-naphthylalanine cyclic pentapeptide.

0.5mmol of leu-N-methyl-leu-valine-leu-2-naphthylalanine pentapeptide was added to a 1000mL single-neck flask with a dry tube (syringe pump added dropwise), 3mmol of PyAOP, 1mmol of HATU, 3.5mmol of DIPEA, 240mL of DCM, 240mL of THF and 60mL of DMF were added, and stirring was carried out at room temperature for 72 h. The solvent was removed under reduced pressure, 400mL of water was added, extraction was performed five times with 90mL of ethyl acetate, and the organic phases were combined. The organic phase was washed with water and 5% K₂CO₃2% HCl, saturated NaCl were washed three times each and dried over anhydrous magnesium sulfate overnight. Vacuum filtering, decompression eliminating solvent, silica gel column chromatography and purification to obtain leucine-N-methyl-leucine-valine-leucine-2-naphthylalanine cyclic pentapeptide in 36% yield.

1H NMR(500MHz,CDCl₃)δ8.18(d,J＝6.5Hz,1H),7.83-7.81(m,1H),7.78(d,J＝8.5Hz,1H),7.75-7.72(m,1H),7.67-7.63(m,3H),7.49-7.46(m,2H),7.32-7.30(dd,J＝8.5Hz,J＝1.5Hz,1H),6.41(d,J＝7.0Hz,1H),4.86-4.82(dd,J＝14.5Hz,J＝8.0Hz,1H),4.49-4.45(dd,J＝13.0Hz,J＝7.5Hz,1H),4.09(t,J＝9.0Hz,1H),3.93-3.89(m,1H),3.55-3.50(dd,J＝13.5Hz,J＝7.5Hz,1H),3.37(t,J＝8.0Hz,1H),3.32-3.28(dd,J＝14.0Hz,J＝5.5Hz,1H),3.00(s,3H),2.14-2.10(m,1H),2.07-2.01(m,1H),1.95-1.88(m,1H),1.82-1.80(m,1H),1.75-1.72(m,1H),1.62-1.53(m,3H),1.46-1.42(m,2H),0.95-0.88(m,18H),0.76(t,J＝6.0Hz,6H)；13C NMR(125MHz,CDCl₃)δ173.34,172.86,171.99,171.76,170.63,133.93,133.62,132.73,128.89,128.18,127.88,127.57,127.23,126.57,126.12,59.68,56.04,54.12,49.21,46.46,46.42,41.15,39.75,38.76,37.18.30.02,26.59,26.34,24.94,24.74,23.01,22.90,22.64,22.23,21.68,19.29,18.97；HRMS(ESI,M+1)C₃₇H₅₆N₅O₅The calculated value was 650.4203, found 650.4280.

Compound (LX-6) can be synthesized in 30% yield by substituting [2- (5-methoxynaphthyl) ] alanine for (2-naphthyl) alanine in example 3.

Compound (LX-7) can be synthesized in 26% yield by substituting (2-quinolyl) alanine for (2-naphthyl) alanine in example 3.

Compound (LX-8) can be synthesized in 28% yield by substituting tryptophan for (2-naphthyl) alanine in example 3.

Compound (LX-10) can be synthesized in 42% yield by substituting [2- (5-bromonaphthyl) ] alanine for (2-naphthyl) alanine in example 3.

EXAMPLE 4 Synthesis of Leu-Val-N-methylLeu-2-Naphenylalanine Cyclopentapeptide (LX-4)

1. Synthesis of leucine-valine-N-methylleucine-2-naphthylalanine pentapeptide.

After synthesis to give Boc-leu-valine-N-methyl-leu-naphthylalanine methyl ester, according to the procedure described for 3+2 in example 1, 0.5mmol thereof was dissolved in 3mL of tetrahydrofuran and transferred into a 100mL single-neck flask. While stirring, a solution of 3mmol of lithium hydroxide in 2.5mL of water and 17.5mL of tetrahydrofuran was added and stirred at room temperature overnight. Tetrahydrofuran was removed under reduced pressure, 60mL of water was added, the mixture was extracted four times with dilute hydrochloric acid to pH 3-4 and 60mL of ethyl acetate, and the organic phases were combined and dried over anhydrous magnesium sulfate overnight. After recovering the solvent under reduced pressure, the obtained residue was transferred to a 100mL single-neck flask, and 6mL of methylene chloride and 3mL of trifluoroacetic acid were added thereto and stirred at normal temperature for 6 hours. Concentrating, recovering dichloromethane to obtain leucine-valine-N-methyl-leucine-2-naphthylalanine pentapeptide, and directly using in next reaction without purification.

2. Synthesis of leu-valine-N-methylleu-2-naphthylalanine cyclic pentapeptide (LX-4).

In a 1000mL single-neck flask containing 3mmol PyAOP, 1mmol HATU, 3.5mmol (8mL) diisopropylethylamine, 240mL dichloromethane, 240mL tetrahydrofuran and 60mL N, N-dimethylformamide, 0.5mmol solution of the crude chain pentapeptide obtained in the above step in tetrahydrofuran was slowly added while stirring. The reaction was stirred at room temperature for 72h and the solvent was removed under reduced pressure. 400mL of water was added to the residue, extracted five times with 90mL of ethyl acetate, and the organic phases were combined. The organic phase was washed with 90mL of water, 75mL of 5% potassium carbonate solution, 75mL of 2% hydrochloric acid, 90mL of water, and 75mL of saturated brine in this order, and the organic phase was dried over anhydrous magnesium sulfate overnight. And (3) carrying out suction filtration, decompressing to remove the solvent, and purifying by silica gel column chromatography to obtain the target compound, namely the cyclopeptide LX-4 with the yield of 37%.

1H NMR(500MHz,CDCl₃)δ7.81(t,J＝4.5Hz,1H),7.78-7.74(m,2H),7.67(d,J＝9.0Hz,1H),7.63(s,1H),7.49-7.43(m,3H),7.34-7.32(dd,J＝8.5Hz,J＝1.5Hz,1H),7.04(d,J＝6.5Hz,1H),6.79(d,J＝7.5Hz,1H),4.77(d,J＝7.5Hz,1H),4.49(t,J＝8.5Hz,1H),4.28(d,J＝7.5Hz,1H),3.83-3.81(m,1H),3.40-3.37(dd,J＝9.0Hz,J＝6.0Hz,1H),3.34-3.25(m,2H),3.20(s,3H),2.12(brs,1H),2.03-1.88(m,3H),1.64-1.61(m,1H),1.57-1.49(m,3H),1.40-1.34(m,1H),1.28-1.25(m,1H),0.96-0.90(m,12H),0.85-0.79(m,12H)；13C NMR(125MHz,CDCl3)δ173.62,173.54,172.61,171.30,171.01,133.78,133.55,132.64,128.74,128.03,127.77,127.53,127.14,126.43,125.99,58.14(2C),55.36,54.71,53.06,40.40,39.18,37.64,37.59,30.62(2C),25.52,25.26,24.79,22.90,22.80,22.56,22.25,22.07,21.82,19.53, 18.38; HRMS (ESI, M +1) calculated value C₃₇H₅₆N₅O₅650.4203, found 650.4221.

EXAMPLE 5 Synthesis of Leu-Val-Leu-N-Methylnaphthylalanine Cyclopentapeptide (LX-5)

1. Synthesis of leucine-valine-leucine-N-methylnaphthylalanine pentapeptide.

After synthesis to Boc-light-valine-light-N-methylnaphthylalanine methyl ester according to the procedure described in example 1 for 3+2, 0.5mmol thereof was dissolved in 3mL of tetrahydrofuran and transferred into a 100mL single-neck flask. While stirring, a solution of 3mmol of lithium hydroxide in 2.5mL of water and 17.5mL of tetrahydrofuran was added and stirred at room temperature overnight. Tetrahydrofuran was removed under reduced pressure, 60mL of water was added, the mixture was extracted four times with dilute hydrochloric acid to pH 3-4 and 60mL of ethyl acetate, and the organic phases were combined and dried over anhydrous magnesium sulfate overnight. After recovering the solvent under reduced pressure, the obtained residue was transferred to a 100mL single-neck flask, and 6mL of methylene chloride and 3mL of trifluoroacetic acid were added thereto and stirred at normal temperature for 6 hours. Concentrating and recovering dichloromethane to obtain the leucine-valine-leucine-N-methyl naphthylalanine pentapeptide which can be directly used for the next reaction without purification.

2. Synthesis of leu-valine-leu-N-methylnaphthylalanine cyclic pentapeptide (LX-5).

In a 1000mL single-neck flask containing 3mmol PyAOP, 1mmol HATU, 3.5mmol (8mL) diisopropylethylamine, 240mL dichloromethane, 240mL tetrahydrofuran and 60mL N, N-dimethylformamide, 0.5mmol solution of the crude chain pentapeptide obtained in the above step in tetrahydrofuran was slowly added while stirring. The reaction was stirred at room temperature for 72h and the solvent was removed under reduced pressure. 400mL of water was added to the residue, extracted five times with 90mL of ethyl acetate, and the organic phases were combined. The organic phase was washed with 90mL of water, 75mL of 5% potassium carbonate solution, 75mL of 2% hydrochloric acid, 90mL of water, and 75mL of saturated brine in this order, and the organic phase was dried over anhydrous magnesium sulfate overnight. And (3) carrying out suction filtration, decompressing to remove the solvent, and purifying by silica gel column chromatography to obtain the target compound, namely the cyclopeptide LX-5 with the yield of 30%.

1H NMR(500MHz,MeOD)δ7.83-7.78(m,3H),7.70(s,1H),7.48-7.43(m,2H),7.40-7.38(dd,J＝8.5Hz,J＝1.5Hz,1H),4.67-4.64(dd,J＝11.0Hz,J＝4.0Hz,1H),4.58(brs,2H),4.53-4.50(m,1H),3.52-3.49(dd,J＝14.0Hz,J＝4.5Hz,1H),3.28(brs,1H),3.21-3.18(m,1H),3.15(s,3H),2.67-2.59(m,1H),1.79-1.73(m,1H),1.70-1.65(m,2H),1.63-1.55(m,2H),1.51-1.45(m,1H),1.40-1.27(m,3H),1.01-0.95(m,18H),0.77(d,J＝6.5Hz,3H),0.68(d,J＝7.0Hz,3H)；13C NMR(125MHz,MeOD)δ174.87,174.67,173.94,173.41(2C),136.41,134.94,133.96,129.39,128.87,128.66,128.55,127.92,127.22,126.78,67.52,66.40,57.23,54.09,49.00,41.38,40.75,39.65,37.68,37.51,29.22,26.31,26.25,26.00,23.63,23.02,22.93,22.65,22.58,21.50,20.24,19.80；HRMS(ESI,M+1)C₃₇H₅₆N₅O₅The calculated value was 650.4203, found 650.4236.

Compound (LX-9) can be synthesized by substituting N-methyl- (2-naphthyl) alanine in example 5 with N-methyl- [2- (5-methoxynaphthyl) ] alanine, in a yield of 24.6%.

EXAMPLE 6 Synthesis of Pro-Leu-Val-Leu-Naphenylalanine Cyclopentapeptide (LX-11)

1. Synthesis of pro-naphthylalanine methyl ester dipeptide.

2mmol of methyl L-2-naphthylalanine, 3mmol of Boc-L-proline, 2.4mmol of HATU, 14mmol of DIPEA, 50mL of DCM and 5mL of DMF were added to a 100mL single-neck flask equipped with a drying tube and stirred at room temperature overnight. The solvent was evaporated under reduced pressure, 300mL of water was added, extraction was performed five times with 60mL of ethyl acetate, and the organic phases were combined. The organic phase was washed with water and 5% K₂CO₃Washed three times with 2% HCl and saturated NaCl, and dried over anhydrous magnesium sulfate overnight. Filtering, concentrating, purifying by silica gel column chromatography to obtain white solid with yield of 80%. This was then dissolved in 12mL DCM and 8mL TFA and the reaction was stirred at RT for 6 h. DCM and TFA were removed under reduced pressure to give 2-naphthylalanine methyl ester dipeptide prolyl.

2. Synthesis of the tripeptide Boc-light-valine.

In a 100mL single-neck flask equipped with a drying tube, 2mmol of valine methyl ester, 3mmol of Boc-leucine, 2.4mmol of HBTU, 14mmol of DIPEA, 50mL of LPCM and 5mL of DMF were added, and the mixture was stirred at room temperature overnight. Concentrating under reduced pressure, adding 300mL of water, extracting with 60mL of ethyl acetate five times, and combining the organic extractsAnd (4) phase(s). The organic phase was washed with water and 5% K₂CO₃Washed three times with 2% HCl saturated NaCl each, dried over anhydrous magnesium sulfate, filtered, concentrated, and to the residue 12mL of DCM and 8mL of TFA were added and the reaction was stirred at RT for 6 h. DCM and TFA were removed under reduced pressure to give the light-valinyl trifluoroacetate salt. This salt was dissolved in 50mL DCM and 5mL DMF, after which 3mmol Boc-leucine and 2.4mmol HBTU and 14mmol DIPEA were added and stirred overnight. The reaction mixture was concentrated, 300mL of water was added to the residue, extraction was performed with ethyl acetate, and the organic phase was water and 5% K₂CO₃The reaction mixture was washed 3 times with 2% HCl-saturated NaCl, and dried over anhydrous magnesium sulfate overnight. Filtering, concentrating, purifying the product by silica gel column chromatography to obtain a solid product with the yield of 98%. The resulting solid was dissolved in 52.5mL THF and transferred to a flask containing a solution of 18mmol LiOH in 15mL water and stirred overnight at room temperature. THF was removed under reduced pressure, 60mL of water was added, the pH of the solution was adjusted to 3-4 with 2% hydrochloric acid, extraction was performed with ethyl acetate, and the organic phases were combined and dried over anhydrous magnesium sulfate overnight. Filtering and concentrating to obtain Boc-bright-valine tripeptide.

3. Synthesis of prolyl-valine-leucine-naphthylalanine pentapeptide.

In a 100mL single-neck flask equipped with a drying tube, 2mmol of pro-2-naphthylalanine methyl ester dipeptide, 3mmol of Boc-light-valine tripeptide, 2.4mmol of HBTU, 14mmol of DIPEA, 50mL of LPCM and 5mL of DMF were added, and the mixture was stirred at room temperature overnight. Concentrated under reduced pressure, 300mL of water was added, extracted five times with 60mL of ethyl acetate, and the organic phases were combined. The organic phase was washed with water and 5% K₂CO₃Washed three times with 2% HCl saturated NaCl, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by silica gel column chromatography to give a white solid in 69% yield. 0.5mmol of the resulting solid was dissolved in 17.5mL of THF and transferred to a flask containing a solution of 3mmol of LiOH in 2.5mL of water and stirred at room temperature overnight. The reaction was followed by TLC and after the reaction was complete THF was removed under reduced pressure. To the residue was added 60mL of water, and the mixture was extracted four times with dilute hydrochloric acid to adjust pH to 3-4 and 60mL of ethyl acetate, and the organic phases were combined and dried over anhydrous magnesium sulfate overnight. Concentrating under reduced pressure to obtain Boc-leucine-N-methyl-leucine-valine-leucine-2-naphthylalanine pentapeptide. Dissolve in 6mL DCM and 4mL TFA and stir the reaction at room temperature for 6 h. Removing under reduced pressureDCM and TFA to obtain Pro-Leu-Val-Leu-Naphenylalanine pentapeptide.

4. Synthesis of prolyl-valine-leucine-naphthylalanine cyclic pentapeptide.

0.5mmol of Pro-Leu-Val-Leu-Naphenylalanine pentapeptide was added to a 1000mL single vial with a dry tube (syringe pump drop wise), 3mmol of PyAOP, 1mmol of HATU, 3.5mmol of DIPEA, 240mL of DCM, 240mL of THF, and 60mL of DMF and stirred at room temperature for 72 h. The solvent was removed under reduced pressure, 400mL of water was added, extraction was performed five times with 90mL of ethyl acetate, and the organic phases were combined. The organic phase was washed with water and 5% K₂CO₃Washed three times with 2% HCl and saturated NaCl, and dried over anhydrous magnesium sulfate overnight. Vacuum filtering, removing solvent under reduced pressure, and purifying by silica gel column chromatography to obtain pro-leu-valine-leu-naphthylalanine cyclic pentapeptide with yield of 31%.

1H NMR(500MHz,DMSO)δ8.43(d,J＝9.0Hz,1H),7.87(d,J＝7.5Hz,1H),7.83-7.80(m,3H),7.57(s,1H),7.49-7.42(m,3H),7.29-7.27(dd,J＝8.5Hz,J＝1.5Hz,1H),7.06(d,J＝9.5Hz,1H),4.75-4.71(m,1H),4.44(d,J＝7.5Hz,1H),4.29-4.26(dd,J＝9.0Hz,J＝3.0Hz,1H),4.15(t,J＝9.5Hz,1H),3.73-3.68(m,1H),3.51-3.48(m,1H),3.41-3.37(m,1H),3.08-3.04(dd,J＝13.5Hz,J＝5.5Hz,1H),2.95-2.90(m,1H),1.93-1.83(m,2H),1.75-1.69(m,1H),1.60-1.55(m,1H),1.53-1.44(m,3H),1.39-1.34(m,1H),1.32-1.21(m,3H),0.89-0.83(m,13H),0.76-0.73(m,5H)；13C NMR(125MHz,DMSO)δ171.67,171.30,170.63,170.30,169.71,134.88,133.14,132.27,128.25,128.09,127.90(2C),127.62,126.42,125.93,61.28,55.38,55.09,54.73(2C),46.61,42.05,40.70(2C),32.50,31.55,24.77,24.68,23.39,23.09,22.59,22.26,21.17,18.90,18.53；HRMS(ESI,M+1)C₃₅H₅₀N₅O₅The calculated value was 620.3734, found 620.3820.

Compound (LX-12) can be synthesized in a similar manner with a yield of 32%.

Example 7 antitumor Activity test

The testing is carried out based on a sulforhodamine B (SRB) method, and the anti-tumor N-methylcyclopentapeptide compounds of the samples to be tested are respectively prepared into DMSO solutions with different concentrations of 1000, 500, 250, 100, 50, 10 mu mol/L and the like.

Suspending the selected tumor cells in10% fresh fetal bovine serum in PRMI or DMEM medium, trypsinized, and the cells counted, the cell concentration was adjusted to 5X 10⁴cells/mL, 96-well plates were loaded with 90. mu.L of cells per well, approximately 4500 cells per well, followed by 10. mu.L of different concentrations of drug solution per well, at 37 ℃, 100% relative humidity and CO content₂Ratio to air 5: and culturing for 48 hours in an incubator with 95 mixed atmospheres. Discarding the solution with 96-well plate, adding 100ul 10% TCA gently, and fixing at 4 deg.C for 30 min; discarding the solution, adding 100ul ddH per well₂Washing with water for three times, and drying at room temperature for 1h or more; adding 100ul of 0.4% SRB, and dyeing for 20min (shaking the plate); removing the dye solution, washing with 1% acetic acid for three times, and drying at room temperature for 6h or overnight; adding 100ul Trisbase to dissolve, shaking the plate for 5min, measuring the absorbance of the solution at 570nm relative to the number of living cells by using an enzyme-labeling instrument, and measuring the cell proliferation condition. Each set of experiments had a backup and the reported data is the average.

Under the condition of 0.05 mu mol/mL, the inhibition rate of the leucine-N-methyl-leucine-valine-leucine-2-naphthylalanine cyclic pentapeptide on mouse melanoma cell strains B16, gastric cancer cell strains BGC-823, cervical cancer cell strains Hela, breast cancer cell strains MDA-MB231, breast cancer cell strains MCF-7 and pancreatic cancer cell strains BXPC-3 is over 80 percent, and the leucine-N-methyl-valine-leucine-2-naphthylalanine cyclic pentapeptide has the effect of inducing apoptosis of mouse melanoma cell strains B16.

Experiments prove that the anti-tumor N-methyl cyclic pentapeptide compound shows good activity on various tumor cells. Particularly, the cell line is more prominent for mouse melanoma cell line B16, gastric cancer cell line BGC-823, cervical cancer cell line Hela, renal clear cancer cell 786-O, breast cancer cell line MDA-MB231, breast cancer cell line MCF-7 and pancreatic cancer cell line BXPC-3. And the antitumor N-methyl cyclic pentapeptide compound has extremely low toxicity to normal cells under the concentration of 50 mu M. The N-methylcyclopentapeptide of the invention has very little side effect on smooth muscle cells, lymphocytes and cardiac muscle cells; the inhibition of tumor cells by N-methyl cyclic pentapeptide is achieved by inducing apoptosis.

Table one: LX-1, LX-2, LX-3, LX-5, N-methyl cyclic pentapeptide compounds with antitumor activity IC50 (mu M)

EXAMPLE 8 Western-Blot the expression of Caspase-related proteins in B16 cells was examined for compound LX-3 (leucine-N-methyl-leucine-valine-leucine-2-naphthylalanine cyclic pentapeptide).

Referring to FIG. 1, the image of the Western-Blot assay of the effect of LX-3 on B16 shows that caspase-9 induces the expression of caspase-3, thereby acting on the apoptosis of tumor cells, and thus the inhibition of B16 by N-methylcyclopentapeptide including LX-3 acts through the pathway of caspase-9-induced caspase-3 to perform tumor cell apoptosis. In the practical application process, the N-methyl cyclic pentapeptide compound can be used as a candidate of an anti-tumor medicament to be applied by utilizing the effect of the N-methyl cyclic pentapeptide compound on caspase-9, and the caspase-9 is used as a medicament target to develop and prepare the application of the anti-tumor medicament.

Specifically, the following steps are required to obtain the experimental structure of fig. 1:

and uniformly mixing the protein sample and the loading buffer solution, boiling for 5min, cooling to room temperature, adding the mixture into a loading hole of a gel plate, wherein the loading amount is about 40-60 mu g, and filling into 12% of separation gel and 5% of concentrated gel. And applying 90V stabilized voltage electrophoresis for about 30min, and when the protein sample reaches the interface of the separation gel and the concentrated gel, changing to 120V stabilized voltage electrophoresis until the front edge of the bromophenol blue moves to the bottom of the gel for about 1.5 h. Taking out the gel, spreading the prepared PVDF film on the gel, respectively placing three layers of filter paper on two sides of the PVDF film and the gel, placing the gel on a negative electrode and the PVDF film on a positive electrode, placing the gel in an electric rotating instrument, and carrying out the steps of carrying out. The PVDF membrane was removed, placed in 5% BSA (bovine serum albumin) TTBS, and blocked at 37 ℃ for 1 hour. The PVDF membrane is then placed in a primary antibody diluted appropriately with TTBS and incubated overnight at 4 ℃ with slow shaking. After washing with TTBS for 10min X3 times, the cells were incubated in corresponding secondary antibodies diluted with TTBS (dilution ratio 1:500) at 37 ℃ for 2 h. TTBS washing 20min × 3 times, ECL luminescence, darkroom exposure, and scanning analysis by gel imaging system.

The N-methyl cyclic pentapeptide compound has no definite carbon end and nitrogen end in the structure, and particularly after N-methylation, the N-methyl cyclic pentapeptide compound is not easy to degrade in vivo, has a longer half-life period and shows good enzyme stability, so the N-methyl cyclic pentapeptide compound can be applied to the preparation of anti-gastric cancer drugs, anti-cervical cancer drugs, anti-renal clear cancer drugs or anti-pancreatic cancer drugs.

The above description is only a preferred embodiment of the present invention, and should not be taken as limiting the scope of the invention, therefore, the present invention shall not be limited by the above description, but should be construed as being limited only by the appended claims.

Claims (2)

1. An N-methyl cyclic pentapeptide compound with apoptosis promoting activity on melanoma cell, gastric cancer cell, cervical cancer cell, renal clear cancer cell or pancreatic cancer cell has one of chemical formulas LX-1, LX-2, LX-3 and LX-5

2. The N-methylcyclopentapeptide compound according to claim 1, for use in the preparation of a medicament for treating gastric, cervical, pancreatic, melanin, renal clear cancer.

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