Short peptide derivative as well as synthesis method and application thereof

Abstract

The invention relates to the technical field of synthesis of oligopeptide derivatives, and discloses an oligopeptide derivative as well as a synthesis method and application thereof. The oligopeptide derivative is formed by coupling a tripeptide composed of an amino acid sequence Val-Leu-Gly and an alkaloid hydroxycamptothecin. The synthesis method comprises the following steps: taking valine I as a raw material, and reacting with Fmoc-OSu under the catalysis of NaHCO to generate a compound shown in a formula III; then condensing with a compound shown as a formula II in a methanol system under the catalysis of DMTMM to obtain a compound shown as a formula IV; further performing catalytic deprotection through p-toluenesulfonic acid to generate a compound shown as a formula V; coupling with glycine methyl ester, and carrying out acid catalytic hydrolysis to obtain an intermediate shown as a formula VII; finally, coupling with hydroxycamptothecine under the catalysis of diphenyl phosphate azide to obtain a target product shown as a formula VIII. According to the method, efficient preparation of the oligopeptide-alkaloid derivative is realized by optimizing a liquid phase synthesis path and reaction parameters.

Description

Technical Field

[0001] The present invention relates to the technical field of short peptide derivative synthesis, and in particular to a short peptide derivative, a synthesis method and an application thereof. Background Art

[0002] In recent years, the research and development of anti-tumor drugs has gradually expanded towards targeting, low toxicity, and high bioavailability. Short peptide drugs have attracted widespread attention due to their excellent biocompatibility, modifiability, and potential targeting capabilities. However, short peptide compounds are susceptible to enzymatic degradation in the body, have poor stability, and are often limited in efficacy when used alone. To enhance their therapeutic potential, researchers often conjugate short peptides with other active molecules (such as alkaloids) to synergistically enhance anti-tumor activity and improve pharmacokinetic properties.

[0003] Hydroxycamptothecin, a topoisomerase I inhibitor, possesses significant anti-tumor activity, but its clinical application is limited by poor water solubility, high toxicity, and a lack of targeting. By conjugating hydroxycamptothecin to short peptides, the targeted delivery function of the peptide chain can enhance drug accumulation at tumor sites while also improving its solubility and bioavailability. However, existing short peptide-alkaloid conjugation technologies face challenges such as complex synthesis steps, low coupling efficiency, and numerous byproducts. Liquid-phase synthesis of peptide sequences and the selection of protecting group strategies are particularly in need of optimization. Summary of the Invention

[0004] The purpose of the present invention is to provide a short peptide derivative, a synthesis method and application thereof, so as to provide a short peptide derivative that shows significant potential in anti-tumor treatment, which is expected to become a candidate molecule for a new generation of tumor treatment drugs by enhancing tumor tissue targeting, reducing systemic toxicity and improving pharmacokinetic properties; another purpose of the present invention is to provide a synthesis method of a short peptide derivative; another purpose of the present invention is to provide an application of a short peptide derivative.

[0005] Technical solution: The present invention provides a method for synthesizing a short peptide derivative, which uses a liquid phase method to prepare a short peptide derivative comprising an amino acid sequence Val-Leu-Gly and an alkaloid hydroxycamptothecin. The method comprises the following steps:

[0006] (a) Using valine I as a raw material, the compound of formula (I) was obtained by reacting with Fmoc-OSu under the catalysis of NaHCO3;

[0007] (b) reacting the compound of formula II obtained in step (a) with the compound of formula III in a methanol system under the catalysis of DMTMM to obtain a compound of formula IV;

[0008] (c) reacting the compound of formula IV obtained in step (b) with p-toluenesulfonic acid to obtain a compound of formula V;

[0009] (d) reacting the compound of formula V obtained in step (c) with glycine methyl ester under the catalysis of DMTMM to obtain the compound of formula VI;

[0010] (e) reacting the compound of formula VI obtained in step (d) with p-toluenesulfonic acid to obtain a compound of formula VII;

[0011] (f) reacting the compound of formula VII obtained in step (e) with hydroxycamptothecin to obtain a compound of formula VIII;

[0012] (g) removing the Fmoc protection of the compound of formula VIII obtained in step (f) by tetrabutylammonium fluoride to obtain short peptide derivatives;

[0013] .

[0014] Preferably, the reaction conditions of step (a) are as follows: the solvent is 50% acetonitrile aqueous solution, the volume is 20 times the weight of the compound of formula I, the molar ratio of sodium bicarbonate to the compound of formula I is 2:1, and the molar ratio of Fmoc-OSu to the compound of formula I is 1.3:1, and the reaction is carried out at room temperature after the addition is completed;

[0015] Furthermore, after the reaction in step (a) is completed, the post-treatment method is as follows: acetonitrile is evaporated at 50° C. under reduced pressure, 2M hydrochloric acid is added to the residue to adjust the pH to 4-5, and the mixture is extracted twice with a mixed solution of dichloromethane / methanol with a volume ratio of 9 / 1, which is 5 / 6 times the volume of a 50% acetonitrile aqueous solution. The organic phases are combined, washed once with water, and concentrated to dryness under reduced pressure at 40° C. to obtain the compound of formula II.

[0016] Preferably, the reaction conditions of step (b) are as follows: the reaction solvent is methanol, the volume amount is 15 times the weight of the compound of formula II, the molar ratio of the compound of formula II to the compound of formula II is 1:1.01-1:1.02, the reaction temperature is 20-30°C, and the reaction time is 12h;

[0017] Furthermore, after the reaction in step (b) is completed, the post-treatment method is: concentration under reduced pressure, passing through a silica gel column chromatography with a weight 8 times the weight of the concentrate, and eluting with DCM / methanol = 30:1-20:1-10:1 to obtain a compound of formula IV.

[0018] Preferably, the reaction conditions of step (c) are: the reaction solvent is acetonitrile / water = 1 / 1 (V / V), the volume is 20 times the weight of the compound of formula III, p-toluenesulfonic acid and formula The molar ratio of the compounds is 1.4:1-1.5:1, the temperature is 40-50°C, and the reaction time is 6 hours;

[0019] Furthermore, after the reaction in step (c) is completed, the post-treatment method is as follows: concentrating under reduced pressure at 50° C. to remove acetonitrile, adding a mixed solution of dichloromethane / methanol = 9 / 1 with a volume ratio of 1 / 3 times the volume of the reaction system before concentration, extracting twice / time, combining the organic phases, washing once with water, and concentrating under reduced pressure at 40° C. to dryness to obtain the compound of formula V.

[0020] Preferably, the reaction conditions of step (d) are: the reaction solvent is methanol, the volume amount is 15 times the weight of the compound of formula V, glycine methyl ester and formula The molar ratio of the compounds was 1.01-1.02:1, the reaction temperature was 50°C, and the reaction time was 10h;

[0021] Furthermore, after the reaction in step (d) is completed, the post-treatment method is: concentration under reduced pressure, column chromatography with silica gel 8 times the weight of the concentrate, and elution with DCM / methanol = 30:1-20:1-10:1 to obtain the compound of formula VI.

[0022] Preferably, the reaction conditions of step (e) are: the reaction solvent is acetonitrile / water = 1 / 1 (V / V), the volume is 20 times the weight of the compound, p-toluenesulfonic acid and formula The molar ratio of the compounds is 1.4:1-1.5:1, the temperature is 40-50°C, and the reaction time is 6 hours;

[0023] Furthermore, after the reaction in step (e) is completed, the post-treatment method is as follows: concentrating under reduced pressure at 50° C. to remove acetonitrile, adding 100 ml / time of a mixed solution of dichloromethane / methanol = 9 / 1 with a volume ratio of 1 / 3 times the volume of the reaction system before concentration, and extracting twice, combining the organic phases, washing once with water, and concentrating to dryness under reduced pressure at 40° C. to obtain the compound of formula VII.

[0024] Preferably, the reaction conditions of step (f) are: the reaction solvent is DMF, and the volume dosage is 7 times the weight of the compound, the catalyst is diphenyl phosphoazide, the amount is 0.5 times the weight of the compound, hydroxycamptothecin and formula The molar ratio of the compounds was 1.1:1, the reaction temperature was 20°C, and the reaction time was 12 h;

[0025] Furthermore, after the reaction in step (e) is completed, the post-treatment method is as follows: water with a volume 6.25 times the volume of DMF is added to the reaction solution under stirring, stirred for 30 minutes, filtered, and the filter cake is slurried with water with a volume 3.75 times the volume of DMF for 30 minutes, filtered, and dried in vacuo at 80°C to obtain a compound of formula VIII.

[0026] Preferably, the reaction conditions of step (g) are: the reaction solvent is DMF, the volume dosage is 5 times the weight of the compound, the molar ratio of tetrabutylammonium fluoride to the compound of formula is 1.5:1-2:1, the reaction temperature is 25-30°C, and the reaction time is 4-6h;

[0027] Furthermore, after the reaction in step (f) is completed, the post-treatment method is as follows: adding water with a volume of 5.25 times the volume of DMF to the reaction solution under stirring, stirring for 30 minutes, filtering, beating the filter cake with water with a volume of 3.5 times the volume of DMF for 30 minutes, filtering, dissolving the filter cake in methanol, and sequentially passing it through D001 and D201 resin columns, with the column volume of D001 and D201 resin columns being 1 times the volume of the reaction solution, until the effluent pH is ≈ 7, eluting with 5 volumes of methanol with the column volume of the resin column, and then concentrating under reduced pressure to obtain the formula Short peptide derivatives.

[0028] The short peptide derivatives synthesized by the above method can be used to prepare anti-tumor drugs, especially for the treatment of gastric cancer. They are expected to show stronger anti-tumor activity in in vitro and in vivo experiments, providing a technical basis for the development of new targeted chemotherapy drugs.

[0029] Beneficial effects:

[0030] 1. Using DMTMM as a condensation agent significantly improves the coupling efficiency of amino acids and short peptides, reduces the generation of side reactions, and optimizes reaction conditions to ensure the high purity of intermediates and final products, avoiding the common impurity accumulation problem in traditional solid-phase synthesis.

[0031] 2. Use tetrabutylammonium fluoride to gently remove the Fmoc protecting group in DMF solvent, avoiding the degradation of short peptides or side chain side reactions that may be caused by traditional strong alkaline conditions, and effectively protecting the integrity of the short peptide sequence.

[0032] 3. The hydrophilicity of the short peptide chain can alleviate the hydrophobicity problem of hydroxycamptothecin and improve its bioavailability; targeted delivery reduces nonspecific damage to normal tissues and alleviates the toxic side effects of traditional chemotherapy drugs.

[0033] 4. The final product has the dual functions of inhibiting topoisomerase I (hydroxycamptothecin) and regulating the tumor microenvironment (short peptide targeting) through a synergistic mechanism. It is expected to show stronger anti-tumor activity in in vitro and in vivo experiments, providing a technical basis for the development of new targeted chemotherapy drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a comparison of the pathological sections of gastric cancer BGC-823 tumors treated with the short peptide derivative of the present invention and hydroxycamptothecin at the same concentration for 48 hours and the initial tumors stained with HE method;

[0035] Figure 2 Formula synthesized in Example 1 of the present invention Liquid phase spectrum;

[0036] Figure 3 The formula synthesized in Example 2 of the present invention is Liquid phase spectrum;

[0037] Figure 4 The formula synthesized in Example 3 of the present invention is Liquid phase spectrum;

[0038] Figure 5 Formula synthesized in Example 1 of the present invention X-ray powder diffraction pattern of

[0039] Figure 6 The compound of the formula synthesized in Example 1 of the present invention is 1 H-NMR spectrum.

[0040] Reference numerals:

[0041] A, blank control; B, commercially available hydroxycamptothecin group; C, short peptide derivative group of Example 1 of the present invention. DETAILED DESCRIPTION

[0042] In order to make the technical solution of the present invention clearer, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0043] Example 1

[0044] 5.86 g of valine was added to a reaction flask, and 60 ml of a 50% acetonitrile aqueous solution was added. 8.4 g of NaHCO3 and 21.93 g of Fmoc-Osu were added under stirring. The mixture was reacted at room temperature for 8 h. The acetonitrile was evaporated at 50 °C under reduced pressure. 2 M hydrochloric acid was added to the residue to adjust the pH to 4-5. The mixture was extracted twice with 50 ml of a mixed solution of dichloromethane / methanol with a volume ratio of 9 / 1. The organic phases were combined, washed once with water, and concentrated at 40 °C under reduced pressure to obtain the product. Compound 14.62g;

[0045] 13.58 g of the compound of formula II was added to a reaction flask, followed by 205 ml of methanol, 11.07 g of DMTMM, and 5.87 g of the compound of formula III. The mixture was reacted at 20° C. for 12 h, concentrated under reduced pressure, and eluted with silica gel (8 times the weight of the concentrate) using a DCM / methanol ratio of 30:1-20:1-10:1 to obtain 16.53 g of the compound of formula IV.

[0046] 16.33 g of the compound of formula IV was added to a reaction flask, followed by 326 ml of a 1 / 1 (v / v) mixture of acetonitrile and water. 9.32 g of p-toluenesulfonic acid was added with stirring and reacted at 40°C for 6 h. The acetonitrile was removed by concentration at 50°C under reduced pressure. The remaining liquid was extracted twice with 100 ml of a 9 / 1 (v / v) mixture of dichloromethane and methanol. The organic phases were combined, washed once with water, and concentrated at 40°C under reduced pressure to dryness to obtain 13.63 g of the compound of formula V.

[0047] 13.13 g of the compound of formula V was added to a reaction flask, and 196 ml of methanol was added. 8.3 g of DMTMM and 2.61 g of glycine methyl ester were added with stirring at 50° C. for 10 h. The mixture was concentrated under reduced pressure and passed through a silica gel column chromatography with a weight 8 times the weight of the concentrate, and eluted with DCM / methanol in a ratio of 30:1-20:1-10:1 to obtain 14.13 g of the compound of formula VI.

[0048] 13.61 g of the compound of formula VI was added to a reaction flask, followed by 272 ml of acetonitrile / water = 1 / 1 (v / v). 6.92 g of p-toluenesulfonic acid was added with stirring and the mixture was reacted at 40°C for 6 h. The acetonitrile was removed by concentration at 50°C under reduced pressure. The remaining liquid was extracted twice with 100 ml of a mixed solution of dichloromethane / methanol = 9 / 1 by volume. The organic phases were combined, washed once with water, and concentrated to dryness under reduced pressure at 40°C to obtain 11.44 g of the compound of formula VII.

[0049] 10.88 g of the compound of formula VII was added to a reaction flask, and 80 ml of DMF was added. 5.44 g of diphenylphosphoryl azide and 8.82 g of hydroxycamptothecin were added under stirring and reacted at 20°C for 12 h. 500 ml of water was added to the reaction solution under stirring, stirred for 30 min, filtered, and the filter cake was slurried with 300 ml of water for 30 min, filtered, and dried in vacuo at 80°C to obtain 17.14 g of the compound of formula VIII.

[0050] 17.12g of compound VIII was added to a reaction flask, 86ml of DMF was added, 7.84g of tetrabutylammonium fluoride was added under stirring, and the mixture was reacted at 25°C for 6h. 450ml of water was added to the reaction solution under stirring, and the mixture was stirred for 30min and filtered. The filter cake was slurried with 300ml of water for 30min and filtered. The filter cake was dissolved in methanol and passed through D001 and D201 resin columns in sequence. The column volume of D001 and D201 resin columns was 100ml. When the pH of the effluent was ≈7, 500ml of methanol was added and the mixture was concentrated under reduced pressure to obtain the compound VIII. Short peptide derivative 11.16g, formula The liquid phase spectrum is attached. Figure 2 ,Mode The X-ray powder diffraction pattern of Figure 5 ,Mode of 1 H-NMR spectrum is attached Figure 6 .

[0051] Figure 1 This is a microscopic comparison of the pathological sections of the gastric cancer BGC-823 tumor stained with HE after the short peptide derivative prepared in this example was treated with hydroxycamptothecin at the same concentration for 48 hours.

[0052] A is a blank control with no necrosis, B is a commercially available hydroxycamptothecin group with obvious necrosis, and C is a short peptide derivative group according to Example 1 of the present invention with complete necrosis.

[0053] Example 2

[0054] 5.86 g of valine was added to a reaction flask, and 60 ml of a 50% acetonitrile aqueous solution was added. 8.4 g of NaHCO 3 and 21.93 g of Fmoc-Osu were added with stirring. The mixture was reacted at room temperature for 8 h. The acetonitrile was evaporated at 50° C. under reduced pressure. The residue was added with 2 M hydrochloric acid to adjust the pH to 4-5. The mixture was extracted twice with 50 ml of a mixed solution of dichloromethane / methanol in a volume ratio of 9 / 1. The organic phases were combined, washed once with water, and concentrated to dryness under reduced pressure at 40° C. to obtain 14.48 g of the compound of formula:

[0055] 13.58 g of the compound of formula II was added to a reaction flask, followed by 205 ml of methanol, 11.07 g of DMTMM, and 5.9 g of the compound of formula III. The mixture was reacted at 25° C. for 12 h, concentrated under reduced pressure, and eluted with silica gel (8 times the weight of the concentrate) using a DCM / methanol ratio of 30:1-20:1-10:1 ratio to obtain 16.61 g of the compound of formula IV.

[0056] 16.33 g of the compound of formula IV was added to a reaction flask, followed by 326 ml of a 1 / 1 (v / v) mixture of acetonitrile and water. 9.72 g of p-toluenesulfonic acid was added with stirring and reacted at 45°C for 6 h. The acetonitrile was removed by concentration at 50°C under reduced pressure. The remaining liquid was extracted twice with 100 ml of a 9 / 1 (v / v) mixture of dichloromethane and methanol. The organic phases were combined, washed once with water, and concentrated at 40°C under reduced pressure to dryness to obtain 13.47 g of the compound of formula V.

[0057] 13.13 g of the compound of formula V was added to a reaction flask, and 196 ml of methanol was added. 8.3 g of DMTMM and 2.62 g of glycine methyl ester were added with stirring at 50° C. for 10 h. The mixture was concentrated under reduced pressure and passed through a silica gel column chromatography with a weight 8 times the weight of the concentrate, and eluted with DCM / methanol in a ratio of 30:1-20:1-10:1 to obtain 14.08 g of the compound of formula VI.

[0058] 13.61 g of the compound of formula VI was added to a reaction flask, followed by 272 ml of acetonitrile / water = 1 / 1 (v / v). 7.17 g of p-toluenesulfonic acid was added with stirring and the mixture was reacted at 45°C for 6 h. The acetonitrile was removed by concentration at 50°C under reduced pressure. The remaining liquid was extracted twice with 100 ml of a mixed solution of dichloromethane / methanol = 9 / 1 by volume. The organic phases were combined, washed once with water, and concentrated to dryness under reduced pressure at 40°C to obtain 11.51 g of the compound of formula VII.

[0059] 10.88 g of the compound of formula VII was added to a reaction flask, and 80 ml of DMF was added. 5.44 g of diphenylphosphoryl azide and 8.82 g of hydroxycamptothecin were added under stirring and reacted at 20°C for 12 h. 500 ml of water was added to the reaction solution under stirring, and the mixture was stirred for 30 min. The mixture was filtered, and the filter cake was slurried with 300 ml of water for 30 min, filtered, and dried in vacuo at 80°C to obtain 17.22 g of the compound of formula VIII.

[0060] 17.12g of compound VIII was added to a reaction flask, 86ml of DMF was added, 8.89g of tetrabutylammonium fluoride was added under stirring, and the mixture was reacted at 27°C for 5h. 450ml of water was added to the reaction solution under stirring, and the mixture was stirred for 30min and filtered. The filter cake was slurried with 300ml of water for 30min and filtered. The filter cake was dissolved in methanol and passed through D001 and D201 resin columns in sequence. The column volume of D001 and D201 resin columns was 100ml. When the pH of the effluent was ≈7, 500ml of methanol was added and the mixture was concentrated under reduced pressure to obtain the compound VIII. Short peptide derivative 11.02g, formula The liquid phase spectrum is attached. Figure 3 .

[0061] Example 3

[0062] 5.86 g of valine was added to the reaction flask, and 60 ml of 50% acetonitrile aqueous solution was added. 8.4 g of NaHCO3 and 21.93 g of Fmoc-Osu were added under stirring. The reaction was carried out at room temperature for 8 h. The acetonitrile was evaporated at 50 ° C under reduced pressure. 2 M hydrochloric acid was added to the residue to adjust the pH to 4-5. The residue was extracted twice with 50 ml of a mixed solution of dichloromethane / methanol with a volume ratio of 9 / 1. The organic phases were combined, washed once with water, and concentrated at 40 ° C under reduced pressure to obtain the formula Compound 14.33g;

[0063] 13.58 g of the compound of formula II was added to a reaction flask, followed by 205 ml of methanol, 11.07 g of DMTMM, and 5.92 g of the compound of formula III. The mixture was reacted at 30° C. for 12 h, concentrated under reduced pressure, and eluted with silica gel (8 times the weight of the concentrate) using a DCM / methanol ratio of 30:1-20:1-10:1 to obtain 16.49 g of the compound of formula IV.

[0064] 16.33 g of the compound of formula IV was added to a reaction flask, followed by 326 ml of a 1 / 1 (v / v) mixture of acetonitrile and water. 9.99 g of p-toluenesulfonic acid was added with stirring at 50°C for 6 h. The acetonitrile was removed by concentration at 50°C. The remaining liquid was extracted twice with 100 ml of a 9 / 1 (v / v) mixture of dichloromethane and methanol. The organic phases were combined, washed once with water, and concentrated at 40°C to dryness to obtain 13.67 g of the compound of formula V.

[0065] 13.13 g of the compound of formula V was added to a reaction flask, and 196 ml of methanol was added. 8.3 g of DMTMM and 2.63 g of glycine methyl ester were added with stirring at 50° C. for 10 h. The mixture was concentrated under reduced pressure and passed through a silica gel column chromatography with a weight 8 times the weight of the concentrate, and eluted with DCM / methanol in a ratio of 30:1-20:1-10:1 to obtain 13.96 g of the compound of formula VI.

[0066] 13.61 g of the compound of formula VI was added to a reaction flask, followed by 272 ml of acetonitrile / water = 1 / 1 (v / v). 7.41 g of p-toluenesulfonic acid was added with stirring and the mixture was reacted at 50°C for 6 h. The acetonitrile was removed by concentration at 50°C under reduced pressure. The remaining liquid was extracted twice with 100 ml of a mixed solution of dichloromethane / methanol = 9 / 1 by volume. The organic phases were combined, washed once with water, and concentrated to dryness under reduced pressure at 40°C to obtain 11.33 g of the compound of formula VII.

[0067] 10.88 g of the compound of formula VII was added to a reaction flask, and 80 ml of DMF was added. 5.44 g of diphenylphosphoryl azide and 8.82 g of hydroxycamptothecin were added under stirring and reacted at 20°C for 12 h. 500 ml of water was added to the reaction solution under stirring, and the mixture was stirred for 30 min. The mixture was filtered, and the filter cake was slurried with 300 ml of water for 30 min, filtered, and dried under vacuum at 80°C to obtain 17.2 g of the compound of formula VIII.

[0068] 17.12g of compound VIII was added to a reaction flask, 86ml of DMF was added, 10.46g of tetrabutylammonium fluoride was added under stirring and the mixture was reacted at 30°C for 4h, 450ml of water was added to the reaction solution under stirring, the mixture was stirred for 30min, filtered, the filter cake was slurried with 300ml of water for 30min, filtered, the filter cake was dissolved in methanol and passed through D001 and D201 resin columns in sequence, the column volume of D001 and D201 resin columns was 100ml, the effluent pH was ≈7, 500ml of methanol was eluted and concentrated under reduced pressure to obtain the compound VIII. Short peptide derivative 11.03g, formula The liquid phase spectrum is attached. Figure 4 .

[0069] The corresponding test data statistics of the short peptide derivatives prepared in Examples 1 to 3 are shown in Table 1:

[0070] Table 1 Statistical table of data of short peptide derivatives prepared in Example

[0071] Weight / g Theoretical amount / g Yield / % Liquid phase purity / % Example 1 11.16 12.67 88.08 99.938 Example 2 11.02 12.67 86.98 99.967 Example 3 11.03 12.67 87.06 99.949

[0072] From the data, the yield of short peptide derivative prepared by this method is stable, and the purity is also relatively stable, which shows that this synthesis method is relatively mature and has strong adaptability to process scale-up production.

[0073] Take the logarithmic growth period of gastric cancer BGC-823 cells, 0.25% trypsin digestion of adherent tumor cells, prepare cell suspension with RPMI 1640 culture solution containing 10% calf serum, count plate count is 5×10 4 Cells / ml. 100ul was inoculated in each well of 96-well culture plate, and the cells were cultured in a 37℃, 5% CO2 incubator for 24h to synchronize the cell growth. Different concentrations of drugs were given, and the final concentrations were 10ug

[0074] / ml,5ug / ml,2.5ug / ml,1.25ug / ml,0.625ug / ml. Each drug set 5 dose groups, and each group set 5-6 parallel holes. The positive control group was the commercially available hydroxyl camptothecin (purity 99%), and the final concentrations were 10ug / ml,5ug / ml,2.5ug / ml,1.25ug / ml,0.625ug ml. Each drug set 5 dose groups, and each group set 5-6 parallel holes. The blank control group was added with the same volume of culture solution as the drug. Incubate in a 37℃, 5% CO2 incubator for 24h, 48h, discard the culture solution, add 100 ul 0.5% MTT solution (RPMI 1640) to each well. 37℃ incubation for 4h, discard the supernatant, add DMSO 100ul to each well, shake in water bath for 20 minutes, and make Formazan particles fully dissolved. Use the microplate reader to measure the optical density value (OD) at reference wavelength 450 nm, detection wavelength 570 nm, and calculate the tumor inhibition rate of the drug. And use Logit method to calculate the half inhibition concentration (IC 50 ) value of the drug.

[0075] Tumor inhibition rate=(1- average OD value of drug group / average OD value of blank group)×100%.

[0076] Table 1 Comparison data of short peptide derivative obtained from examples and hydroxyl camptothecin on gastric cancer BGC-823

[0077] <![CDATA[折合成羟基喜树碱的半数抑制浓度IC 50 ]]> Converted to effective dose of hydroxycamptothecin mg / kg Tumor weight at 48h under the same effective dose of 2.5μg / ml Tumor inhibition rate at the same dose of 2.0μg / ml (%) Example 1 1.8933μg / ml 2.5 0.623±0.0272 54.24 Example 2 1.8933μg / ml 2.5 0.623±0.0274 54.22 Example 3 1.8931μg / ml 2.5 0.623±0.0269 54.28 Hydroxycamptothecin 2. 9042μg / ml 2.7 0.841±0.0465 39.92

[0078] Note: The formula for converting the short peptide derivative into hydroxycamptothecin is: converted hydroxycamptothecin = short peptide derivative ÷ 633.70 × 364.35, where 633.70 is the molecular weight of the short peptide derivative of the present invention, and 364.35 is the molecular weight of hydroxycamptothecin.

[0079] By comparing the short peptide derivatives prepared by the present invention with commercially available hydroxycamptothecin (purity 99%), it can be seen that when converted into hydroxycamptothecin, the short peptide derivatives prepared by the present invention have a half inhibitory concentration IC 50 The effective dose is better than that of hydroxycamptothecin, which also reflects that the short peptide derivatives prepared by the present invention have higher bioavailability and better targeting. Compared with hydroxycamptothecin, the short peptide derivatives of the present invention have better 48h tumor weight and tumor inhibition rate at the same effective dose than hydroxycamptothecin, and thus can be further used in the development of anti-tumor drugs.

[0080] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A method for synthesizing a short peptide derivative, characterized in that: The method uses a liquid phase method to prepare a short peptide derivative comprising an amino acid sequence Val-Leu-Gly and an alkaloid hydroxycamptothecin, and the method comprises the following steps: (a) Using valine I as a raw material, the compound of formula (I) was obtained by reacting with Fmoc-OSu under the catalysis of NaHCO3; (b) reacting the compound of formula II obtained in step (a) with the compound of formula III in a methanol system under the catalysis of DMTMM to obtain a compound of formula IV; (c) reacting the compound of formula IV obtained in step (b) with p-toluenesulfonic acid to obtain a compound of formula V; (d) reacting the compound of formula V obtained in step (c) with glycine methyl ester under the catalysis of DMTMM to obtain the compound of formula VI; (e) reacting the compound of formula VI obtained in step (d) with p-toluenesulfonic acid to obtain a compound of formula VII; (f) reacting the compound of formula VII obtained in step (e) with hydroxycamptothecin to obtain a compound of formula VIII; (g) removing the Fmoc protection of the compound of formula VIII obtained in step (f) by tetrabutylammonium fluoride to obtain short peptide derivatives; 。 2. The method for synthesizing a short peptide derivative according to claim 1, wherein: Step (a) reaction conditions are: the solvent is 50% acetonitrile aqueous solution, the volume is The molar ratio of sodium bicarbonate to the compound of formula I is 2:1, and the molar ratio of Fmoc-OSu to the compound of formula I is 1.3:

1. The reaction is carried out at room temperature.

3. The method for synthesizing a short peptide derivative according to claim 1, wherein: The reaction conditions of step (b) are as follows: the reaction solvent is methanol, the volume amount is 15 times the weight of the compound of formula II, The molar ratio of the compound to the compound of formula II is 1:1.01-1:1.02, the reaction temperature is 20-30° C., and the reaction time is 12 h.

4. The method for synthesizing a short peptide derivative according to claim 1, wherein: Step (c) reaction conditions are: the reaction solvent is acetonitrile / water = 1 / 1 (V / V), the volume is 20 times the weight of the compound, p-toluenesulfonic acid and formula The molar ratio of the compounds is 1.4:1-1.5:1, the temperature is 40-50°C, and the reaction time is 6 hours.

5. The method for synthesizing a short peptide derivative according to claim 1, wherein: The reaction conditions of step (d) are as follows: the reaction solvent is methanol, the volume amount is 15 times the weight of the compound of formula V, glycine methyl ester and formula The molar ratio of the compounds is 1.01-1.02:1, the reaction temperature is 50° C., and the reaction time is 10 h.

6. The method for synthesizing a short peptide derivative according to claim 1, wherein: The reaction conditions of step (e) are: the reaction solvent is acetonitrile / water = 1 / 1 (V / V), the volume is 20 times the weight of the compound, p-toluenesulfonic acid and formula The molar ratio of the compounds is 1.4:1-1.5:1, the temperature is 40-50°C, and the reaction time is 6 hours.

7. The method for synthesizing a short peptide derivative according to claim 1, wherein: The reaction conditions of step (f) are: the reaction solvent is DMF, the volume dosage is 7 times the weight of the compound, the catalyst is diphenyl phosphoazide, the amount is 0.5 times the weight of the compound, hydroxycamptothecin and formula The molar ratio of the compounds was 1.1:1, the reaction temperature was 20°C, and the reaction time was 12 h.

8. The method for synthesizing a short peptide derivative according to claim 1, wherein: The reaction conditions of step (g) are: the reaction solvent is DMF, the volume dosage is 5 times the weight of the compound, tetrabutylammonium fluoride and formula The molar ratio of the compounds is 1.5:1-2:1, the reaction temperature is 25-30°C, and the reaction time is 4-6h.

9. A short peptide derivative prepared according to the method for synthesizing a short peptide derivative according to any one of claims 1 to 8.

10. Use of the short peptide derivative according to claim 9 in the preparation of anti-tumor drugs.

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