CN112094322A

CN112094322A - His-Gly-Lys modified methotrexate, synthesis, antitumor activity and application thereof

Info

Publication number: CN112094322A
Application number: CN201910527891.9A
Authority: CN
Inventors: 赵明; 彭师奇; 梁梦
Original assignee: Capital Medical University
Current assignee: Capital Medical University
Priority date: 2019-06-18
Filing date: 2019-06-18
Publication date: 2020-12-18
Anticipated expiration: 2039-06-18
Also published as: CN112094322B

Abstract

The invention discloses a His-Gly-Lys modified methotrexate (in the formula, R)₁R when His-Gly-Lys₂Is OH, R₁R is OH₂Is His-Gly-Lys, and R₁And R₂His-Gly-Lys) and discloses a process for their preparation and their anti-tumor activity. Further discloses the advantages of no myelosuppressive toxicity, no hepatotoxicity, no nephrotoxicity and strong antitumor activity of the methotrexate. Thus, the invention discloses that they are prepared without myelosuppressive toxicityThe methotrexate medicine has no hepatotoxicity, no nephrotoxicity and strong antitumor activity.

Description

His-Gly-Lys modified methotrexate, synthesis, antitumor activity and application thereof

Technical Field

The invention relates to His-Gly-Lys modified methotrexate (in the formula, R)₁R when His-Gly-Lys₂Is OH, R₁Is OH whenR₂Is His-Gly-Lys, and R₁And R₂His-Gly-Lys) to their preparation, to their anti-tumor activity, to their advantages in reducing the risk of myelosuppressive toxicity associated with methotrexate, and to their advantages in reducing the risk of hepatorenal toxicity associated with methotrexate. The invention thus relates to their use in the preparation of medicaments which are devoid of bone marrow toxicity, hepatotoxicity, nephrotoxicity and which inhibit tumour growth. The invention belongs to the field of biological medicine.

Background

Cancer is a disease in which cells proliferate uncontrollably and abnormally and have an opportunity to spread and metastasize to other parts through the body's blood system or lymphatic system, and is a serious medical difficulty worldwide. According to the results of the malignant tumor morbidity and mortality data in each region of 2015 of China released by the national cancer center 2019 at the beginning, the leukemia is 10 first malignant tumors with the highest mortality. Methotrexate has been used clinically for over 70 years as one of the earliest drugs used to treat acute leukemia. Due to the non-specificity of methotrexate action, it attacks normal tissue cells, for example, cells of the blood system, causing myelosuppressive toxicity, mainly manifested as a decrease in the number of leukocytes, erythrocytes and platelets. For example, renal toxicity can be triggered by renal clearance. In addition, the large dose of methotrexate therapy causes problems of hepatotoxicity, oromucosal side effects and multiple drug resistance. In order to overcome the disadvantages, structural modification of methotrexate has been studied vigorously, but problems still remain. In the past years, through reasonable drug design and systematic experimental research, the inventor finds that the following general formula of His-Gly-Lys-methotrexate (wherein R is₁R when His-Gly-Lys₂Is OH, R₁R is OH₂Is His-Gly-Lys, and R₁And R₂And His-Gly-Lys) not only overcome the disadvantages of methotrexate, but also enhance the anti-tumor activity of methotrexate. Based on these findings, the inventors have proposed the present invention.

Disclosure of Invention

The first aspect of the present invention is to provide His-Gly-Lys modified methotrexate of the following general formula (wherein R is₁R when His-Gly-Lys₂Is OH, R₁R is OH₂Is His-Gly-Lys, and R₁And R₂And His-Gly-Lys).

In a second aspect, the invention provides a method for preparing His-Gly-Lys modified methotrexate, the method comprising: 1, adopting dicyclohexylcarbodiimide as a condensing agent and N-hydroxybenzotriazole as a catalyst to synthesize Fmoc-His (Trt) -Gly-Lys (Cbz) -OBzl in a liquid phase manner;

2, removing Fmoc to synthesize His (Trt) -Gly-Lys (Cbz) -OBzl;

3 adopting dicyclohexylcarbodiimide as a condensing agent and N-hydroxybenzotriazole as a catalyst, coupling methotrexate and His (Trt) -Gly-Lys (Cbz) -OBzl to generate the following general formula of His (Trt) -Gly-Lys (Cbz) -OBzl modified methotrexate, wherein R in the formula₁R for His (Trt) -Gly-Lys (Cbz) -OBzl₂' is OH, R₁' is OH, R₂' is His (Trt) -Gly-Lys (Cbz) -OBzl, and R₁' and R₂' both His (Trt) -Gly-Lys (Cbz) -OBzl;

4 removing the protecting group under acidic condition to generate His-Gly-Lys modified methotrexate (in the formula, R is₁R when His-Gly-Lys₂Is OH, R₁R is OH₂Is His-Gly-Lys, and R₁And R₂And His-Gly-Lys).

In a third aspect of the invention, the tumor growth inhibitory activity of His-Gly-Lys modified methotrexate of the general formula is evaluated.

A fourth aspect of the invention is the evaluation of hepatotoxicity of His-Gly-Lys modified methotrexate of the general formula.

A fifth aspect of the invention is the evaluation of nephrotoxicity of His-Gly-Lys modified methotrexate of the general formula.

A sixth aspect of the invention is the evaluation of myelosuppressive toxicity of His-Gly-Lys modified methotrexate of the general formula.

Drawings

FIG. 1 is a synthetic route for His-Gly-Lys modified methotrexate. Anhydrous tetrahydrofuran, dicyclohexylcarbodiimide, N-hydroxybenzotriazole, N-methylmorpholine; (ii) a solution of hydrogen chloride in ethyl acetate; (iii) a 20% piperidine in dichloromethane solution; (iv) anhydrous N, N-dimethylformamide, dicyclohexylcarbodiimide, N-hydroxybenzotriazole, N-methylmorpholine; (v) trifluoroacetic acid/trifluoromethanesulfonic acid.

Detailed Description

To further illustrate the invention, a series of examples are given below. These examples are purely illustrative and are intended to be a detailed description of the invention only and should not be taken as limiting the invention.

EXAMPLE 1 preparation of Boc-Gly-Lys (Cbz) -OBzl (1)

1.02g (5.81mmol) of Boc-Gly was dissolved in 60mL of anhydrous tetrahydrofuran to obtain solution No. 1. A solution of 1.43g (6.98mmol) dicyclohexylcarbodiimide and 0.79g (5.85mmol) N-hydroxybenzotriazole in dry tetrahydrofuran is added to solution No. 1 at 0 ℃ with stirring and stirred for 30 minutes. Then 2.35g (5.78mmol) of HCl Lys (Cbz) -OBzl was added, the pH of the reaction solution was adjusted to 9 with N-methylmorpholine, the ice bath was removed, and the reaction was terminated by TLC (dichloromethane/methanol ═ 30/1) after stirring well at room temperature for 17 hours. Filtration and concentration were carried out, and the residue was dissolved in 100mL of ethyl acetate, and the solution was washed with a saturated aqueous solution of sodium hydrogencarbonate (30 mL. times.3), saturated aqueous solution of sodium chloride (30 mL. times.3), 5% aqueous solution of potassium hydrogensulfate (30 mL. times.3), and saturated chlorine, respectivelyWashed with an aqueous solution of sodium chloride (30 mL. times.3), with an aqueous solution of saturated sodium bicarbonate (30 mL. times.3), and with an aqueous solution of saturated sodium chloride (30 mL. times.3). The ethyl acetate phase obtained was dried over anhydrous sodium sulfate for 12 hours, filtered and concentrated to give 3.05g (100%) of the title compound as a yellow oil. ESI-MS (M/e):528[ M + H]⁺。

EXAMPLE 2 preparation of Gly-Lys (Cbz) -OBzl (2)

1.75g (3.32mmol) of compound (1) was dissolved in anhydrous ethyl acetate. 20mL of a solution of hydrogen chloride in ethyl acetate (4M) was added with stirring at 0 ℃, and TLC (dichloromethane/methanol ═ 30/1) showed completion of the reaction after stirring for 9 hours. The reaction solution was repeatedly concentrated under reduced pressure in a warm water bath, and then the concentrate was dissolved in anhydrous ethyl acetate, and the reaction solution was repeatedly concentrated under reduced pressure (3 times), and the reaction product was repeatedly washed with anhydrous ethyl ether to obtain 1.40g (98%) of the objective compound as a yellow viscous oil. ESI-MS (M/e):428[ M + H]⁺。

EXAMPLE 3 preparation of Fmoc-His (Trt) -Gly-Lys (Cbz) -OBzl (3)

Using the method of example 1, 2.02g (65%) of the title compound were obtained as colorless powder from 1.87g (3.02mmol) of Fmoc-His (Trt) and 1.40g (3.02mmol) of the compound (2). ESI + MS (M/e):1029[ M + H]⁺,¹H NMR(300MHz,DMSO-d₆):/ppm＝8.20(t,J＝5.4Hz,1H),7.90(s,1H),7.88(s,1H),7.68-7.64(m,2H),7.43-7.20(m,27H),7.06-7.03(m,6H),6.73(s,1H),5.12(s,1H),5.07(s,2H),5.00-4.99(m,2H),4.30-4.12(m,4H),3.75-3.67(m,2H),2.97-2.89(m,3H),2.83-2.74(m,1H),1.68-1.55(m,2H),1.38-1.24(m,4H)。

EXAMPLE 4 preparation of His (Trt) -Gly-Lys (Cbz) -OBzl (4)

After dissolving 1.80g (1.75mmol) of compound (3) in 20mL of 20% piperidine dichloromethane solution and stirring at 0 ℃ for 6 hours, TLC (dichloromethane/methanol ═ 20/1) showed completion of the reaction and the reaction was terminated. Concentration in a 25 ℃ water bath afforded a white solid, which was repeatedly triturated with petroleum ether, again with dry ether for 3 times, and purified by reduced pressure silica gel column chromatography to give 0.83g (59%) of the title compound as a colorless powder. ESI + MS (M/e):807[ M + H]⁺，¹H NMR(300MHz,DMSO-d₆):/ppm＝8.69(d,J＝7.5Hz,1H),8.17(s,1H),7.40-7.24(m,21H),7.08-7.06(m,6H),6.68(s,1H),5.04(s,2H),4.99(s,2H),4.32-4.38(m,1H),3.79-3.65(m,2H),3.42-3.41(m,1H),2.92(d,J＝5.7Hz,2H),2.80-2.64(m,2H),1.66-1.56(m,2H),1.31-1.24(m,4H)。

EXAMPLE 5 preparation of His (Trt) -Gly-Lys (Cbz) -OBzl modified methotrexate (1a,1b,1c)

0.47g (1.03mmol) of methotrexate was dissolved in 40mL of anhydrous N, N-dimethylformamide to give solution No. 1. A solution of 0.26g (1.26mmol) of dicyclohexylcarbodiimide and 0.14g (1.14mmol) of N-hydroxybenzotriazole in anhydrous N, N-dimethylformamide was added to the solution No. 1 at 0 ℃ with stirring, and stirred for 30 minutes. 0.83g (1.03mmol) of compound (4) was added, the reaction solution pH was adjusted to 9 with N-methylmorpholine, the ice bath was removed, TLC (ethyl acetate/water/glacial acetic acid ═ 6/1/1) showed completion after stirring thoroughly at room temperature for 8 hours, the insoluble white solid dicyclohexylurea was removed by filtration, the filtrate was evaporated under reduced pressure to remove the solvent, and purified by preparative thin layer chromatography (ethyl acetate/water/glacial acetic acid ═ 6/1/1) to give 0.27g (21%) of compound 1a,0.66g (63.63%) of compound 1b and 0.12g (10%) of compound 1 c. Their structures are as follows:

1a is orange powder, ESI-MS (M/e):1241[ M-H [)]^-,¹H NMR(300MHz,DMSO-d₆):/ppm＝11.98(s,1H),8.53(s,1H),8.36-8.15(m,3H),7.69(m,2H),7.32(m,22H),7.02(s,7H),6.70-6.63(m,5H),5.05(s,2H),4.98(s,2H),4.75(s,2H),4.39(m,1H),4.27-4.25(m,2H),3.74-3.51(m,2H),3.17(s,3H),2.89-2.85(m,4H),2.37(m,1H),2.28(m,1H),1.60-1.48(m,4H),1.32-1.24(m,4H)；¹³C NMR(125MHz,DMSO-d₆):/ppm＝173.41,172.24,171.70,128.49,128.46,128.28,169.45,163.33,163.16,162.79,156.52,155.67,151.23,149.47,146.36,142.65,142.63,142.54,137.74,136.40,129.70,129.64,129.57,128.86,128.77,128.60,128.57,128.49,128.46,128.40,128.35,128.28,128.24,128.14,128.10,127.99,121.93,121.67,119.36,111.44,111.28,74.89,66.21,66.17,65.54,52.44,47.97,42.57,40.57,31.24,30.90,30.85,29.38,25.96,24.56。

1b is orangePowder, Q-TOF-MS (M/e):2031[ M + H]⁺,¹H NMR(300MHz,DMSO-d₆):/ppm＝8.52(d,J＝2.7Hz,1H),8.44-8.37(m,2H),8.29-8.18(m,3H),7.69-7.61(m,2H),7.31-7.26(m,41H),7.22-7.20(m,2H),7.04-7.02(m,13H),6.73-6.61(m,6H),5.05(m,4H),4.98(s,4H),4.74(s,2H),4.54-4.38(m,1H),4.25(m,2H),4.12(m,2H),3.75-3.54(m,4H),3.15(s,3H),2.90-2.74(m,8H),2.27-2.02(m,2H),1.63-1.41(m,6H),1.24-0.98(m,8H)；¹³C NMR(125MHz,DMSO-d₆):/ppm＝172.33,172.27,172.18,172.07,171.67,171.64,169.51,169.47,169.41,169.27,167.24,163.33,163.17,162.78,156.52,155.67,151.33.149.51,146.42,146.39,142.57,142.52,137.73,136.70,136.36,129.67,129.51,128.86,128.84,128.77,128.59,128.56,128.51,128.42,128.39,128.34,128.24,128.15,127.98,127.79,127.10,127.06,126.87,126.85,121.92,121.50,119.95,119.94,111.29,111.26,89.29,66.28,66.24,55.35,53.26,52.36,47.97,42.45,40.59,40.43,40.26,36.25,33.82,31.24,31.00,30.98,30.94,29.35,27.38,22.93,22.79。

1c is orange powder, ESI-MS (M/e):1243[ M + H]⁺,¹H NMR(300MHz,DMSO-d₆):/ppm＝8.56(s,1H),8.41(m,1H),7.82-7.80(m,2H),7.64(m,3H),7.32(m,21H),7.05-7.03(m,6H),6.82-6.80(d,J＝7.2Hz,2H),6.70(s,1H),6.60(s,2H),5.06(s,2H),4.98(s,2H),4.76(s,2H),4.40(m,1H),4.23-4.11(m,5H),3.68(m,2H),3.18(s,3H),2.15(m,2H),1.86(m,2H),1.63(m,2H),1.31-1.24(m,4H)；¹³C NMR(125MHz,DMSO-d₆):/ppm＝173.16,173.36,172.27,172.23,169.83,169.72,163.31,163.17,162.80,156.54,155.65,151.16,149.59,146.47,142.62,137.77,136.42,129.66,128.87,128.77,128.68,128.61,128.40,128.13,128.11,122.65,122.57,121.90,119.90,111.69,74.97,66.21,66.18,55.35,53.54,53.44,52.47,47.97,42.76,40.56,33.81,32.61,32.20,31.25,25.79,22.95。

EXAMPLE 6 preparation of His-Gly-Lys modified methotrexate α carboxy (2a) having the formula

0.12g (0.095mmol) of the resulting mixture was weighed out and dissolvedAnd slowly adding 2mL of trifluoroacetic acid into the compound 1a at 0 ℃ under stirring, then adding 0.6mL of trifluoromethanesulfonic acid, stirring for 40 minutes, and then pumping out volatile acid gas in the reaction bottle by using a circulating water type vacuum pump at 0 ℃ under stirring for 30 minutes. Then, 30mL of glacial ethyl ether was added to precipitate an orange-yellow insoluble substance, which was then allowed to stand, and the supernatant was discarded and repeated 3 times. Dissolving the reactant in small amount of water, regulating pH to 8 with dilute ammonia water, and treating with C₁₈Purifying by column chromatography, collecting eluate, prefreezing the collected solution at-80 deg.C, and lyophilizing to obtain 0.032g (44%) of compound 2a as orange powder. ESI-MS (M/e):775[ M-H ]]^-,¹H NMR(300MHz,DMSO-d₆):/ppm＝8.60(d,J＝3.3Hz,1H),8.45(s,1H),8.32-8.11(m,5H),7.96(s,1H),7.76-7.71(m,2H),7.64(s,1H),7.23(s,1H),7.16(s,1H),7.06(s,1H),6.83-6.81(m,2H),4.82(s,2H),4.52(m,1H),4.31(m,1H),4.20(m,1H),3.84-3.65(m,2H),3.23(s,3H),3.12-2.93(m,2H),2.75(m,2H),2.27-2.25(m,1H),2.20-2.13(m,1H),1.90-1.87(m,2H),1.73-1.69(m,1H),1.64-1.50(m,3H),1.34-1.18(m,2H)；¹³C NMR(125MHz,DMSO-d₆):/ppm＝174.55,174.43,173.84,172.47,172.34,170.94,169.35,169.23,167.19,163.17,160.81,151.43,149.43,148.43,148.32,134.51,129.57,122.45,122.20,121.42,119.89,117.32,111.56,55.32,53.75,53.57,52.47,52.10,42.46,42.46,42.34,30.95,30.78,27.93,26.94,22.69。

EXAMPLE 7 preparation of His-Gly-Lys modified methotrexate α, γ carboxy (2b)

0.18g (0.089mmol) of Compound 1b was weighed, and 2mL of trifluoroacetic acid and then 0.6mL of trifluoromethanesulfonic acid were added slowly with stirring at 0 ℃ and the reaction was terminated after stirring for 40 minutes. And (3) pumping volatile acid gas in the reaction bottle for 30 minutes by using a circulating water type vacuum pump at the temperature of 0 ℃ under stirring. Then adding glacial ethyl ether 30mL, precipitating orange insoluble substance, standing, removing supernatant, repeating for 3 times, dissolving reactant with small amount of water, adjusting pH to 8 with dilute ammonia water, and filtering with C₁₈Purifying by column chromatography, and collecting eluate. Pre-freezing at-80 deg.C, lyophilizing the sample with lyophilizer to obtain 0.036g (37%) of the productCompound 2b, an orange powder. ESI-MS (M/e):1099[ M + H]⁺,¹H NMR(300MHz,DMSO-d₆):/ppm＝8.57(s,1H),8.32-8.26(m,8H),8.10(d,J＝7.5Hz,2H),7.88(s,2H),7.75-7.64(m,7H),7.18-7.03(m,4H),6.81(d,J＝8.4Hz,2H),4.83(s,2H),4.53-4.51(m,2H),4.20(m,3H),3.83-3.66(m,4H),3.24(s,3H),3.03-2.87(m,4H),2.76(m,4H),2.16(m,2H),1.91-1.72(m,4H),1.60-1.49(m,6H),1.33(m,4H)；¹³C NMR(75MHz,DMSO-d₆):/ppm＝173.94,173.88,173.84,172.50,172.29,171.37,171.12,169.33,169.23,167.15,163.18,161.45,151.42,149.41,147.87,134.84,134.63,131.33,131.19,129.55,127.58,123.30,122.14,121.52,121.38,119.03,117.54,111.57,55.33,53.73,52.78,52.57,52.17,42.51,42.47,42.35,41.13,32.29,28.35,28.31,27.36,27.33,27.31,27.27,27.00,22.73。

EXAMPLE 8 preparation of Gamma carboxy (2c) methotrexate modified by His-Gly-Lys of the formula

0.11g (0.087mmol) of Compound 1c was weighed, and 1mL of trifluoroacetic acid and then 0.3mL of trifluoromethanesulfonic acid were added slowly with stirring at 0 ℃ and the reaction was terminated after stirring for 40 minutes. Removing volatile acid gas in the reaction bottle by using a circulating water type vacuum pump for 30 minutes at 0 ℃ under stirring, immediately adding 30mL of diethyl ether to separate out orange insoluble substances, standing, removing supernatant, and repeating for 3 times. Dissolving the reactant in small amount of water, regulating pH to 7-8 with dilute ammonia water, and treating with C₁₈Purifying by column chromatography, collecting eluate, pre-freezing the collected solution at-80 deg.C, and lyophilizing to obtain 0.017g (25%) of compound 2c as orange powder. ESI-MS (M/e):775[ M-H ]]^-,¹H NMR(500MHz,DMSO-d₆/D₂O＝10/1):/ppm＝8.59(s,1H),7.83(d,J＝10.5Hz,1H),7.69(d,J＝8.0Hz,2H),6.96(d,J＝3Hz,1H),6.84(d,J＝9.0Hz,2H),4.82(s,2H),4.42-4.40(m,1H),4.08(m,1H),3.81-3.77(m,1H),3.70-3.65(m,1H),3.22(s,3H),3.04-3.01(m,1H),2.89-2.88(m,1H),2.79-2.78(m,2H),2.21(m,2H),2.02-2.00(m,1H),1.89-1.85(m,1H),1.74-1.73(m,1H),1.62-1.53(m,3H),1.27(m,2H)；¹³C NMR(125MHz,DMSO-d₆/D₂O＝10/1):/ppm＝175.67,175.59,175.50,175.40,174.07,172.57,169.31,166.97,162.98,162.28,154.18,151.45,149.62,147.84,134.99,132.42,129.06,121.86,121.21,117.39,111.78,55.33,54.45,54.37,53.66,52.96,43.00,42.95,31.15,29.05,27.27,27.22,22.86。

EXPERIMENTAL EXAMPLE 1 Activity of Compounds 2a-c to inhibit the growth of S180 fibrosarcoma

Experimental animals: SPF grade ICR mice, male, weighing 18-22g, were purchased from Peking Wintonlifys animal research technologies, Inc.

Experimental grouping and dosing: compounds 2a-c were administered at a dose of 0.29. mu. mol/kg/day; methotrexate was given as a positive control, 2.9 μmol/kg/day; the negative control group was given physiological saline. All groups were dosed by intraperitoneal injection at a volume of 10 mL/kg/day body weight.

And (3) experimental operation: collecting SPF-grade S180 ascites tumor ICR mice (the tumor source is S180 sarcoma cells purchased from animal experiment center of Beijing university department of medicine), dislocating cervical vertebrae, killing, opening abdominal cavity of mice with surgical scissors, sucking S180 tumor liquid in abdominal water into EP tube with injector, centrifuging (1000rpm,10 min), discarding supernatant, washing with cooled normal saline for multiple times, removing cell debris, floating blood, etc., adding cooled normal saline for resuspension, and mixing with 0.4% trypan blue solution at a ratio of 1: 9. In which dead cells are stained in a distinct blue color, while live cells are not stained, and the number of live cells is observed and counted under a microscope using a cell counting plate (cell survival rate)>90%) of the cell suspension to a viable cell density of 1.5X 10⁷The series of operations are completed as soon as possible, the cells are kept at a lower temperature as possible so as to preserve the cell viability, and the cell viability calculation method is shown in formula (1).

Before inoculation, a receptor mouse needs to be rested and adapted to the environment for one day, when the inoculation is carried out, the right armpit of the mouse is wiped and disinfected by a cotton ball containing 75% alcohol, and the right armpit of the mouse is inoculated with 0.2mL of subcutaneous tumor liquid by a 1mL syringe, and an S180 fibrosarcoma model is established. On the 7 th day of the inoculation, mung bean-sized solid tumors can be seen in most of mice under the right axilla, and the mice are randomly grouped and are subjected to intraperitoneal injection continuously for 10 days according to the administration dose. On day 11 of administration, mice were sacrificed by dislocation of cervical vertebrae, the right axillary solid tumor sites of the mice were fixed with forceps, then the skin portions were cut off with surgical scissors to expose the tumor tissues, the tumor tissues were taken out by blunt separation along the gaps between the tumor and the skin and the muscles, the weights were weighed, the tumor inhibition rates of the respective groups were calculated, and formula (2) was calculated.

The results are shown in Table 1, and the data are examined by t. It can be seen that when the administered dose of the compounds 2a-c is reduced to 1/10 of the administered dose of methotrexate, good in vivo anti-tumor growth activity is still exhibited, indicating that the present invention has excellent technical effects.

TABLE 1 Activity of Compounds 2a-c to inhibit the growth of S180 fibrosarcoma

a) P <0.01 compared to saline group; b) p <0.01 compared to saline group, P <0.05 compared to methotrexate group; n is 10.

EXAMPLE 2 Effect of Compounds 2a-c on liver function in mice

Glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase are important biochemical indexes reflecting liver injury. In order to examine the potential influence of the compounds 2a-c on the liver function of tumor mice, the present invention measured the concentrations of glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase in the serum of S180 mice of experimental example 1 using a fully automatic biochemical analyzer. The data are shown in Table 2, and the data are subjected to t test. The effect of compounds 2a-c on serum glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase concentrations in S180 mice at a dose of 0.29 μmol/kg/day did not show a significant increase compared to normal saline, with the glutamic-oxalacetic transaminase levels in mice in group 2b being reduced but shifted towards the normal serum glutamic-oxalacetic transaminase concentration range in mice. The influence of methotrexate on the serum glutamic-pyruvic transaminase concentration of the S180 mice is statistically different and is increased compared with the normal saline group, and the methotrexate has hepatotoxicity. Therefore, the compounds 2a-c reduce the hepatotoxicity brought by methotrexate, and the compounds 2a-c have remarkable technical advantages.

TABLE 2 Effect of Compounds 2a-c on serum glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase concentrations in S180 mice

a) P <0.05 compared to saline group; b) p >0.05 compared to saline group; n is 6.

EXAMPLE 3 Effect of Compounds 2a-c on mouse Kidney function

Creatinine is an important biochemical indicator reflecting renal injury. To examine the potential effect of compounds 2a-c on renal function in tumor mice, the present inventors determined creatinine concentration in serum of S180 mice of Experimental example 1 using a fully automated biochemical analyzer. The data are shown in Table 3, and the data are subjected to t test. The data show that the effect of compounds 2a-c on serum creatinine concentration in S180 mice at the 0.29 μmol/kg/day dose is not different from that of normal saline, and thus, the compound 2a-c treatment does not impair the renal function of S180 mice. In contrast, methotrexate affects serum creatinine concentration in S180 mice at a dose of 2.9. mu. mol/kg/day differently from normal saline, i.e., administration of methotrexate causes renal injury. As can be seen, the compounds 2a-c reduce the renal toxicity brought by methotrexate, and the compounds 2a-c of the invention have significant technical advantages.

TABLE 3 Effect of Compounds 2a-c on S180 mouse creatinine concentration

EXPERIMENTAL EXAMPLE 4 bone marrow toxicity of Compounds 2a-c to S180 mice

Methotrexate, when used clinically as an antitumor drug, causes myelosuppressive reaction due to poor selectivity, mainly resulting in reduction of peripheral blood platelets, leukocytes, erythrocytes and neutrophils, so that myelosuppressive toxicity of the drug can be evaluated by counting peripheral blood cells of S180 mice of experimental example 1. The results are shown in Table 4, and the data are tested by t test. The data show that the effect of compounds 2a-c on red blood cell, white blood cell, platelet and neutrophil counts in the blood of S180 mice at a dose of 0.29 μmol/kg/day is not different compared to normal saline, and thus, compound 2a-c treatment is not myelotoxic to S180 mice. In contrast, methotrexate affects the blood counts of erythrocytes, leukocytes, platelets and neutrophils in S180 mice at a dose of 2.9. mu. mol/kg/day, as compared to normal saline, and thus methotrexate is known to be myelotoxic to S180 mice. Compounds 2a-c significantly reduced the peripheral blood cell-reducing toxicity caused by methotrexate, i.e., reduced the myelosuppressive toxicity of methotrexate. It can be seen that compounds 2a-c of the present invention have significant technical advantages.

TABLE 4 Effect of Compounds 2a-c on mouse erythrocyte, leukocyte, platelet and neutrophil counts

a) P <0.01 compared to saline group; b) p <0.05 compared to saline group; c) p >0.05 compared to saline group, P <0.01 compared to methotrexate group; d) p >0.05 compared to saline group, P <0.05 compared to methotrexate group; n is 6.

Claims

1. His-Gly-Lys modified methotrexate with the following general formula,

in the formula R₁R when His-Gly-Lys₂Is OH, R₁R is OH₂Is His-Gly-Lys, and R₁And R₂And His-Gly-Lys.

2. A method of making His-Gly-Lys modified methotrexate according to claim 1, comprising:

2.1 adopting dicyclohexylcarbodiimide as a condensing agent and N-hydroxybenzotriazole as a catalyst to synthesize Fmoc-His (Trt) -Gly-Lys (Cbz) -OBzl in a liquid phase;

2.2 removing Fmoc to synthesize His (Trt) -Gly-Lys (Cbz) -OBzl;

2.3 coupling methotrexate with His (Trt) -Gly-Lys (Cbz) -OBzl using dicyclohexylcarbodiimide as condensing agent and N-hydroxybenzotriazole as catalyst to produce His (Trt) -Gly-Lys (Cbz) -OBzl modified methotrexate of the general formula₁R for His (Trt) -Gly-Lys (Cbz) -OBzl₂' is OH, R₁' is OH, R₂' is His (Trt) -Gly-Lys (Cbz) -OBzl, and R₁' and R₂' both His (Trt) -Gly-Lys (Cbz) -OBzl;

2.4 deprotection under acidic conditions produces His-Gly-Lys modified methotrexate according to claim 1.

3. Use of the His-Gly-Lys modified methotrexate of claim 1 in the preparation of an anti-tumor drug.