CN112094320B - His-Gly-Glu modified methotrexate, synthesis, antitumor activity and application thereof - Google Patents
His-Gly-Glu modified methotrexate, synthesis, antitumor activity and application thereof Download PDFInfo
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Abstract
The invention discloses a His-Gly-Glu modified methotrexate (in the formula, R) 1 R is His-Gly-Glu 2 Is OH, R 1 R is OH 2 Is His-Gly-Glu, and R 1 And R 2 His-Gly-Glu) and discloses a preparation method thereof and discloses anti-tumor activity thereof. Further discloses the advantages of no myelosuppressive toxicity, no hepatotoxicity, no nephrotoxicity and strong antitumor activity of the methotrexate. Thus, the present invention discloses their use in the preparation of medicaments without myelosuppressive toxicity, hepatotoxicity, nephrotoxicity and resistanceThe application of the methotrexate medicine with strong tumor activity.
Description
Technical Field
The invention relates to His-Gly-Glu modified methotrexate, a preparation method thereof, antitumor activity thereof, the advantage of reducing the risk of bone marrow suppression toxicity brought by the methotrexate, and the advantage of reducing the risk of hepatorenal toxicity brought by the 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 malignant tumor morbidity and mortality data in regions of 2015 in China released by the national cancer center 2019 at the beginning, the leukemia is ten top 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. The inventor discovers that the following general formula of His-Gly-Glu-methotrexate (wherein R is R) is obtained by modifying methotrexate with His-Gly-Glu through reasonable drug design and systematic experimental research in the last years 1 R is His-Gly-Glu 2 Is OH, R 1 R is OH 2 Is His-Gly-Glu, and R 1 And R 2 And His-Gly-Glu) 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-Glu modified methotrexate of the following general formula (wherein R is 1 R is His-Gly-Glu 2 Is OH, R 1 R is OH 2 Is His-Gly-Glu, and R 1 And R 2 And also His-Gly-Glu).
In a second aspect, the present invention provides a method for preparing His-Gly-Glu modified methotrexate, comprising:
1. adopting dicyclohexylcarbodiimide as a condensing agent and N-hydroxybenzotriazole as a catalyst to synthesize Fmoc-His (Trt) -Gly-Glu (OBzl) -OBzl in a liquid phase manner;
2. removing Fmoc to synthesize His (Trt) -Gly-Glu (OBzl) -OBzl;
3. coupling methotrexate with His (Trt) -Gly-Glu (OBzl) -OBzl by using dicyclohexylcarbodiimide as a condensing agent and N-hydroxybenzotriazole as a catalyst to generate the His (Trt) -Gly-Glu (OBzl) -OBzl modified methotrexate with the following general formula, wherein R 1 R when' is His (Trt) -Gly-Glu (OBzl) -OBzl 2 ' is OH, R 1 ' is OH, R 2 ' is His (Trt) -Gly-Glu (OBzl) -OBzl, and R 1 ' and R 2 ' both His (Trt) -Gly-Glu (OBzl) -OBzl;
4 removing protecting group under acidic condition to generate His-Gly-Glu modified methotrexate (in the formula, R) with the following general formula 1 R is His-Gly-Glu 2 Is OH, R 1 R is OH 2 Is His-Gly-Glu, and R 1 And R 2 And also His-Gly-Glu).
A third aspect of the present invention is the evaluation of the tumor growth inhibitory activity of His-Gly-Glu modified methotrexate of the general formula.
A fourth aspect of the present invention is the evaluation of hepatotoxicity of His-Gly-Glu modified methotrexate of the general formula.
A fifth aspect of the invention is the evaluation of nephrotoxicity of His-Gly-Glu modified methotrexate of the general formula.
A sixth aspect of the present invention is the evaluation of myelosuppressive toxicity of His-Gly-Glu modified methotrexate of the general formula.
Drawings
FIG. 1 is a synthetic route for His-Gly-Glu 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-Glu (OBzl) -OBzl (1)
1.00g (5.72mmol) of Boc-Gly was dissolved in 60mL of anhydrous tetrahydrofuran to obtain solution No. 1. A solution of 1.41g (6.84mmol) dicyclohexylcarbodiimide and 0.77g (5.71mmol) N-hydroxybenzotriazole in dry tetrahydrofuran was added to solution No. 1 and stirred for 30 minutes. 1.90g (5.22mmol) of HCl. Glu (OBzl) -OBzl was added, the pH of the reaction solution was adjusted to 9 with N-methylmorpholine, the ice bath was removed, and after stirring well at room temperature for 19 hours, the reaction was completed as shown by TLC (dichloromethane/methanol. RTM. 30/1), and the reaction was terminated. The reaction solution was filtered off white solid dicyclohexylurea, concentrated and the residue was taken up in 100mL of acetic acidThe ethyl ester was dissolved, and the solution was washed with a saturated aqueous solution of sodium hydrogencarbonate (30 mL. times.3), a saturated aqueous solution of sodium chloride (30 mL. times.3), a 5% aqueous solution of potassium hydrogensulfate (30 mL. times.3), a saturated aqueous solution of sodium chloride (30 mL. times.3), a saturated aqueous solution of sodium hydrogencarbonate (30 mL. times.3) and a saturated aqueous solution of sodium chloride (30 mL. times.3), respectively, in this order, and the obtained ethyl acetate phase was dried over anhydrous sodium sulfate for 12 hours, filtered and concentrated to obtain 2.53g (100%) of the title compound as a yellow oily substance. ESI-MS (M/e):485[ M + H] + 。
EXAMPLE 2 preparation of Gly-Glu (OBzl) -OBzl (2)
1.12g (2.32mmol) of compound (1) was dissolved in anhydrous ethyl acetate. 20mL of hydrogen chloride in ethyl acetate (4M) was added with stirring at 0 ℃ and the reaction was completed by TLC (dichloromethane/methanol 30/1) after stirring well for 9 hours. The reaction solution was repeatedly concentrated under reduced pressure in a warm water bath, and then the reaction product was dissolved in anhydrous ethyl acetate, concentrated (3 times), and then repeatedly washed with anhydrous ethyl ether to obtain 0.89g (100%) of the objective compound as a yellow viscous oil. ESI-MS (M/e):385[ M + H] + 。
EXAMPLE 3 preparation of Fmoc-His (Trt) -Gly-Glu (OBzl) -OBzl (3)
Using the method of example 1, 1.50g (67%) of the title compound were obtained as colorless powder from 1.40g (2.26mmol) of Fmoc-His (Trt) and 0.89g (2.32mmol) of the compound (2). ESI-MS (M/e):986[ M + H] + ,1H NMR(300MHz,DMSO-d 6 ):δ/ppm＝8.44(d,J＝7.5Hz,1H),8.34-8.28(m,1H),7.89(d,J＝7.5Hz,2H),7.68(m,2H),7.36-7.28(m,25H),7.06-7.03(m,6H),6.73(s,1H),5.07(s,2H),5.03(s,2H),4.39-4.38(m,1H),4.28-4.14(m,4H),3.78-3.67(m,2H),2.89-2.73(m,2H),2.43-2.38(m,2H),2.06-2.00(m,1H),1.89-1.80(m,1H)。
Example 4 preparation of His (Trt) -Gly-Glu (OBzl) -OBzl (4)
0.72g (0.73mmol) of compound (3) was dissolved in 10mL of 20% piperidine in dichloromethane at 0 ℃ with stirring, and after stirring for 5 hours, TLC (dichloromethane/methanol ═ 30/1) showed completion of the reaction, and the reaction was terminated. Removing solvent under reduced pressure in 25 deg.C water bath to obtain white solid, repeatedly washing the reactant with petroleum ether, washing with anhydrous diethyl ether for 3 times, and purifying by reduced pressure silica gel column chromatography to obtain 0.39g (68%) of target compoundA colorless powder. ESI + MS (M/e):764[ M + H] + , 1 H NMR(300MHz,DMSO-d 6 ):δ/ppm＝8.81(d,J＝8.1Hz,1H),8.20(s,1H),7.37-7.29(m,20H),7.08-7.06(m,6H),6.68(s,1H),5.06(s,2H),5.03(s,2H),4.45-4.44(m,2H),3.73-3.69(m,2H),3.42-3.41(m,1H),2.74-2.67(m,2H),2.42(m,2H),2.03-1.99(m,1H),1.85-1.80(m,1H)。
Example 5 preparation of His (Trt) -Gly-Glu (OBzl) -OBzl modified methotrexate (1a,1b,1c)
0.54g (1.19mmol) of methotrexate was dissolved in 50mL of anhydrous N, N-dimethylformamide to give solution No. 1, and a solution of 0.29g (1.41mmol) of dicyclohexylcarbodiimide and 0.17g (1.26mmol) of N-hydroxybenzotriazole in anhydrous N, N-dimethylformamide was added to solution No. 1 at 0 ℃ with stirring and stirred for 30 minutes. 0.92g (1.21mmol) 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, concentrated and purified by preparative thin layer chromatography (ethyl acetate/water/glacial acetic acid ═ 6/1/1) to give 0.28g (19%) of compound 1a,0.45g (38%) of compound 1b and 0.18g (12%) of compound 1c, respectively. Their structures are as follows:
1a is orange powder, ESI-MS (M/e):1198.86[ M + H] - ； 1 H NMR(300MHz,DMSO-d 6 ):δ/ppm＝8.53-8.51(m,1H),8.43(m,1H),8.23(s,1H),8.16-8.13(d,J＝7.8Hz,1H),7.95(s,1H),7.72(m,3H),7.33-7.26(m,21H),7.01-6.95(m,6H),6.85(s,1H),6.68-6.63(m,5H),5.05(s,2H),5.01(s,2H),4.70(s,2H),4.35(m,3H),3.71(m,2H),3.12(s,3H),2.89(m,2H),2.44-2.35(m,2H),2.00-1.53(m,6H)； 13 C NMR(125MHz,DMSO-d 6 ):δ/ppm＝172.54,172.46,171.81,171.70,171.68,169.55,169.53,163.33,163.15,162.80,155.67,151.26,149.53,149.50,146.40,146.37,142.57,142.50,136.59,136.51,136.59,136.51,136.32,129.70,129.64,129.64,129.54,128.83,128.80,128.60,128.58,128.41,128.37,128.34,128.24,128.19,128.09,128.07,127.99,127.11,121.93,121.66,121.57,111.43,111.28,74.94,66.36,65.89,55.35,55.32,53.90,51.46,51.52,51.48,31.24,30.11,30.01,26.40,25.80。
1b is an orange powder, Q-TOF-MS (M/e):1943[ M + H] - ； 1 H NMR(300MHz,DMSO-d 6 ):δ/ppm＝8.63-8.60(m,1H),8.51-8.46(m,2H),8.28-8.25(m,3H),7.96(s,2H),7.68-7.65(m,3H),7.39-7.30(m,39H),7.03-6.98(m,13H),6.73-6.66(m,5H),5.02(s,8H),4.74(s,2H),4.38(m,4H),4.27(m,1H),3.79-3.53(m,2H),3.15(s,3H),2.82-2.73(m,4H),2.38-2.28(m,4H),2.09-1.87(m,8H)； 13 C NMR(125MHz,DMSO-d 6 ):δ/ppm＝172.72,172.47,172.45,172.41,172.34,172.15,172.01,171.86,171.78,171.76,171.69,169.65,169.55,169.45,163.33,163.17,162.78,157.16,155.68,151.41,151.33,149.50,149.50,146.40,146.36,142.59,138.35,136.55,137.51,137.01,136.55,136.53,136.29,136.28,129.67,129.52,128.86,128.84,128.58,128.51,128.43,128.38,128.33,128.28,128.25,128.15,128.10,127.98,127.10,126.87,121.93,121.44,121.17,119.94,119.44,111.27,75.03,74.97,66.41,66.38,65.91,55.35,54.23,53.69,53.30,51.48,47.97,42.61,33.82,31.24,30.09,30.07,30.00,26.48,16.43,25.80。
1c is orange powder, ESI-MS (M/e):1198.76[ M + H] - ； 1 H NMR(300MHz,DMSO-d 6 ):δ/ppm＝8.56(s,1H),7.63(m,4H),7.36-7.30(m,21H),7.05-7.02(m,6H),6.80(d,J＝7.8Hz,21H),6.69(s,1H),6.62(s,2H),5.05(s,2H),5.02(s,2H),4.76(s,2H),4.37(m,3H),3.99(m,2H),3.18(s,3H),2.89(m,2H),2.39(m,2H),1.80(m,6H)； 13 C NMR(125MHz,DMSO-d 6 ):δ/ppm＝173.12,172.30,171.93,171.92,169.95,169.83,163.33,163.18,162.78,157.17,155.67,151.17,151.14,149.58,146.45,142.61,136.60,136.35,129.68,128.87,128.82,128.73,128.61,128.48,128.41,128.34,128.22,128.12,128.11,127.98,126.87,122.66,122.58,121.91,119.98,49.06,47.97,42.92,32.60,30.09,26.40,26.35。
EXAMPLE 6 preparation of His-Gly-Glu modified methotrexate α carboxy (2a) of formula
0.14g (0.12mmol) of Compound 1a was weighed, and 1.5mL of trifluoroacetic acid and 0.5mL of trifluoromethanesulfonic acid were added slowly with stirring at 0 ℃ to react for 40 minutes, followed by termination of the reaction. 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 8 with dilute ammonia water, and treating with C 18 Purifying by column chromatography, and collecting eluate. The pool was prefreezed at-80 ℃ and the sample was freeze-dried by a freeze-dryer to give 0.016g (17%) of compound 2a as an orange powder. ESI-MS (M/e):778[ M + H ]] + ,1H NMR(300MHz,DMSO-d 6 ):δ/ppm＝8.57(s,1H),8.38(d,J＝6.9Hz,1H),8.27(m,1H),8.22-8.20(m,1H),7.97(d,J＝7.5Hz,1H),7.74(d,J＝8.4Hz,2H),7.45(d,J＝7.5Hz,2H),6.84-6.82(m,3H),6.66(s,1H),4.80(s,2H),4.41-4.26(m,3H),3.85-3.59(m,2H),3.22(s,3H),2.98-2.93(m,2H),2.27-2.19(m,4H),1.98-1.82(m,4H)； 13 C NMR(125MHz,DMSO-d 6 /D 2 O＝10/1):δ/ppm＝176.02,173.07,172.87,171.95,171.74,169.46,168.27,168.11,162.99,161.98,153.74,151.67,149.60,148.05,134.56,131.81,131.42,129.45,121.87,120.54,117.50,111.72,55.04,53.79,53.24,52.92,42.58,42.50,31.17,30.97,27.95,27.35,26.58。
EXAMPLE 7 preparation of His-Gly-Glu modified methotrexate α, γ carboxy (2b) of formula
0.14g (0.12mmol) of Compound 1b was weighed, and 1.5mL of trifluoroacetic acid and then 0.5mL 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 5mL of water, adjusting the pH of the solution to 8 with dilute ammonia water, and filtering the solution through a filter C 18 Purifying by column chromatography, and collecting eluate. Pre-freezing the collected liquid at-80 deg.C, and freeze dryingThe sample was lyophilized on a desiccator to give 0.014g (11%) of compound 2b as an orange powder. ESI-MS (M/e) 1101.87[ M + H ]] + ； 1 H NMR(300MHz,DMSO-d 6 ):δ/ppm＝8.55(s,1H),8.40-8.31(m,4H),8.25(d,J＝6.3Hz,1H),8.10-8.03(m,2H),7.76-7.71(m,4H),7.66(s,1H),7.56(s,1H),0.92(d,J＝8.1Hz,2H),6.83-6.80(m,3H),4.80(s,2H),4.45-4.43(m,1H),4.24(m,4H),3.85-3.51(m,4H),3.22(s,3H),2.94-2.73(m,4H),2.26(m,4H),2.17-2.15(m,2H),1.99-1.95(m,3H),1.83-1.80(m,3H)； 13 C NMR(75MHz,DMSO-d 6 ):δ/ppm＝174.56,174.47,173.90,173.79,172.49,172.23,172.09,171.73,169.30,169.26,167.13,163.16,163.06,155.20,151.41,149.56,146.68,135.10,129.53,121.92,121.58,119.67,117.74,117.63,111.57,55.38,55.31,53.73,53.39,53.31,51.94,42.72,42.64,32.28,30.73,29.45,29.43,29.30,27.06,27.00。
EXAMPLE 8 preparation of His-Gly-Glu modified methotrexate Gamma carboxy (2c) having the structural formula
0.10g (0.088mmol) of compound 1c was weighed, and 1mL of trifluoroacetic acid was added slowly and then 0.3mL of trifluoromethanesulfonic acid was added slowly at 0 ℃ with stirring, and the reaction was terminated after 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 8 with dilute ammonia water, and treating with C 18 Purifying by column chromatography, and collecting eluate. The pool was pre-frozen at-80 ℃ and the sample was freeze dried by a freeze dryer to give 0.010g (15%) of compound 2c as an orange powder. ESI-MS (M/e) 776.92[ M + H ]] - ； 1 H NMR(300MHz,DMSO-d 6 ):δ/ppm＝8.56(s,1H),8.27-8.25(m,3H),7.96(m,1H),7.74-7.72(m,3H),7.58-7.56(m,1H),7.48(s,1H),6.82(m,3H),6.68(s,2H),4.79(s,2H),4.42-4.40(m,1H),4.28-4.26(m,2H),3.69-3.64(m,2H),3.21(s,3H),2.91(m,1H),2.83(m,1H),2.26-2.24(m,4H),2.05-1.76(m,4H)； 13 C NMR(75MHz,DMSO-d 6 ):δ/ppm＝174.38,174.19,173.69,172.23,171.84,169.38,166.73,163.18,163.09,155.27,151.38,149.60,146.66,146.61,135.08,133.30,129.37,121.90,121.73,117.60,111.58,55.33,53.35,52.58,51.69,42.58,42.55,32.39,30.57,29.57,29.53,27.07,26.90。
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 mouse (S180 sarcoma cell as tumor source, purchased from animal experiment center of Beijing university department of medicine), dislocation of cervical vertebra, killing, opening abdominal cavity with surgical scissors, sucking S180 tumor liquid in abdominal water into EP tube with injector, centrifuging (1000rpm,10 min), removing supernatant, washing with cooled normal saline for several times, removing cell debris, floating blood, etc., adding cooled normal saline, resuspending, mixing with 0.4% trypan blue solution and cell suspension at ratio of 1:9, wherein dead cells are dyed into blue, live cells are not dyed, observing under microscope with cell counting plate, and counting number of live cells (cell survival rate)>90%) of the cell suspension to a viable cell density of 1.5X 10 7 The 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 compound 2a,2b still showed good in vivo anti-tumor growth activity when the administered dose was reduced to 1/10 of the administered dose of methotrexate, while 2c did not have in vivo tumor growth inhibitory activity at the administered dose of 0.29 μmol/kg/day. The invention has excellent technical effect.
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 was not statistically different compared to normal saline. The influence of methotrexate on the content of glutamic-pyruvic transaminase in serum of mice is statistically different from that of a normal saline group, and the mice are increased and have 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.
Experimental example 3 Effect of Compounds 2a-c on mouse Kidney function
Creatinine is an important biochemical indicator reflecting renal injury. In order to examine the potential effect of compounds 2a-c on the renal function of tumor mice, the serum creatinine concentration of S180 mice of Experimental example 1 was measured using a fully automatic 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 that of normal saline. 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
a) P <0.05 compared to saline group; b) p >0.05 compared to saline group; n is 6.
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 at the dose of 0.29 μmol/kg/day, compounds 2a-2c do not differ from normal saline in red blood cell, white blood cell, platelet and neutrophil counts in the blood of S180 mice, and thus, compound 2a-2c 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 with a differential decrease from normal saline, indicating that methotrexate is myelotoxic in 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 (2)
1. The application of the His-Gly-Glu modified methotrexate with the following formula in preparing the medicine for inhibiting S180 fibrosarcoma,
in the formula R 1 R is His-Gly-Glu 2 Is OH, and R 1 And R 2 And also is His-Gly-Glu.
2. The use of the compound of claim 1, wherein the His-Gly-Glu modified methotrexate is administered in a manner that comprises the steps of:
(1) adopting dicyclohexylcarbodiimide as a condensing agent and N-hydroxybenzotriazole as a catalyst to synthesize Fmoc-His (Trt) -Gly-Glu (OBzl) -OBzl in a liquid phase manner;
(2) removing Fmoc to synthesize His (Trt) -Gly-Glu (OBzl) -OBzl;
(3) coupling methotrexate with His (Trt) -Gly-Glu (OBzl) -OBzl by using dicyclohexylcarbodiimide as a condensing agent and N-hydroxybenzotriazole as a catalyst to generate the His (Trt) -Gly-Glu (OBzl) -OBzl modified methotrexate with the following general formula, wherein R 1 R when' is His (Trt) -Gly-Glu (OBzl) -OBzl 2 ' is OH, R 1 ' is OH, R 2 ' is His (Trt) -Gly-Glu (OBzl) -OBzl, and R 1 ' and R 2 ' both His (Trt) -Gly-Glu (OBzl) -OBzl;
(4) deprotecting under acidic conditions to produce His-Gly-Glu modified methotrexate according to claim 1.
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