CN112110924B

CN112110924B - 6-S,9-N- (diacetyl-Lys-OBzl-mercapto) purine, synthesis, activity and application thereof

Info

Publication number: CN112110924B
Application number: CN201910542916.2A
Authority: CN
Inventors: 赵明; 王玉记; 樊琦
Original assignee: Capital Medical University
Current assignee: Capital Medical University
Priority date: 2019-06-21
Filing date: 2019-06-21
Publication date: 2023-01-13
Anticipated expiration: 2039-06-21
Also published as: CN112110924A

Abstract

The invention discloses 6-S,9-N- (diacetyl-Lys-OBzl-sulfhydryl) purine with a formula, a preparation method thereof and antitumor activity thereof. Therefore, the invention discloses the application of the compound in preparing anti-tumor drugs.

Description

6-S,9-N- (diacetyl-Lys-OBzl-mercapto) purine, synthesis, activity and application thereof

Technical Field

The invention relates to 6-S,9-N- (diacetyl-Lys-OBzl-sulfhydryl) purine, a preparation method thereof and antitumor activity thereof. Therefore, the invention relates to the application of the compound in preparing antitumor drugs. The invention belongs to the field of biological medicine.

Background

6-mercaptopurine is a common medicine for clinically treating acute lymphoblastic leukemia of children. However, it is also used to treat chorioepithelial carcinoma. However, some toxic side effects limit the clinical use of 6-mercaptopurine. For example, 6-mercaptopurine has a more severe myelosuppressive effect, a shorter half-life, and a higher dose. Although there have been numerous studies over the past decades attempting to overcome these disadvantages of 6-mercaptopurine, there has been no apparent success. The inventor discovers that CH can be used through years of exploration ₂ CO-Lys-OBzl modifies 6-SH and 9-N of 6-mercaptopurine, and can eliminate the bone marrow suppression effect of 6-mercaptopurine, prolong half life and reduce dosage. Based on these findings, the inventors have proposed the present invention.

Disclosure of Invention

The first aspect of the present invention is to provide 6-S,9-N- (diacetyl-Lys-OBzl-mercapto) purine.

A second aspect of the present invention provides a process for the preparation of 6-S,9-N- (diacetyl-Lys-OBzl-mercapto) purine, which process comprises:

1. synthesizing 6-S, 9-N-diacetyl-O-ethylmercaptopurine;

2. synthesizing 6-S, 9-N-dicarboxymethylmercaptopurine;

3. condensing 6-S,9-N- (dicarboxymethylmercapto) purine and Lys-OBzl by a liquid phase method using dicyclohexylcarbodiimide as a condensing agent and 1-hydroxybenzotriazole as a catalyst to prepare 6-S, 9-N-diacetyl-Lys (Boc) -OBzl-mercaptopurine;

4. deprotecting 6-S, 9-N-diacetyl-Lys (Boc) -OBzl-mercaptopurine to produce 6-S, 9-N-diacetyl-Lys-OBzl-mercaptopurine;

the third aspect of the present invention is to evaluate the antitumor effect of 6-S,9-N- (diacetyl-Lys-OBzl-mercapto) purine.

Drawings

FIG. 1. Scheme for the synthesis of 6-S,9-N- (diacetyl-Lys-OBzl-mercapto) purine i) ethyl bromoacetate, dimethylformamide, K ₂ CO ₃ ,65℃；ii)CH ₃ OH,2N NaOH aqueous solution; iii) Dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, N-methylmorpholine, dimethylformamide; iv) 4N HCl/EA solution, 0 ℃.

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 6-S,9-N- (diacetyl-O-ethylmercapto) purine (1)

50mL of N, N-dimethylformamide was added to 3.001g (19.70 mmol) of 6-mercaptopurine, and the mixture was stirred at 65 ℃ until the 6-mercaptopurine was completely dissolved, so that the solution was yellow, clear and transparent. 5.400g (39.50 mmol) of potassium carbonate as a catalyst was added, activation was carried out for 30min, and 6.60mL (59.20 mmol) of ethyl bromoacetate was added, followed by reaction at 65 ℃. TLC (petroleum ether/ethyl acetate = 1/1) after 48h showed 6-mercaptopurine disappearance. The reaction solution was filtered, the filtrate was concentrated under reduced pressure, and the resulting yellow oil was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 1/1) to obtain 1.500g (23%) of the title compound as a colorless solid. ESI-MS (m/e) 325[ 2 ], [ M ] +H] ⁺ ； ¹ H-NMR(300MHz,DMSO-d ₆ )δ/ppm＝8.692(s,1H),8.489(s,1H),5.214(s,2H),4.260(s,2H),4.215～4.098(dq,J ₁ ＝6.9Hz,J ₂ ＝7.2Hz,4H),1.238～1.165(dt,J ₁ ＝7.2Hz,J ₂ ＝11.0Hz,6H)； ¹³ C-NMR(75MHz,DMSO-d ₆ ):δ/ppm＝167.93,167.55,166.54,150.30,147.67,137.81,132.98,61.00,60.64,53.03,31.11,14.16。

EXAMPLE 2 preparation of 6-S,9-N- (dicarboxymethylmercapto) purine (2)

0.170g (0.52 mmol) of 6-S,9-N- (diacetyl-O-ethylmercapto) purine (1) was completely dissolved in 5mL of methanol, and the solution was clear and transparent. The reaction was monitored for completion by TLC (petroleum ether/ethyl acetate = 1/2) after adjusting the reaction mixture pH to 13 with 2N NaOH aqueous solution at 0 ℃, stirring for 4h at 0 ℃. Saturated KHSO is used for reaction liquid ₄ The aqueous solution was adjusted to pH 7, concentrated under reduced pressure to precipitate a colorless salt solid, and 5mL of water was added to completely dissolve the solid. The solution is saturated with KHSO at 0 deg.C ₄ The aqueous solution was adjusted to pH 2, extracted with ethyl acetate (20 mL. Times.3), the ethyl acetate layers were combined and washed with saturated NaCl (15 mL. Times.3), and the resulting ethyl acetate phase was dried over anhydrous sodium sulfate for 12 hours. Filtration and concentration of the filtrate under reduced pressure gave 0.138g (98%) of the title compound as a colorless solid. ESI-MS (m/e) 267[ m ] H] ⁺ ； ¹ H-NMR(300MHz,DMSO-d ₆ )δ/ppm＝13.011(s,2H),8.501(s,1H),8.078(s,1H),4.799(s,2H),4.026(s,2H)； ¹³ C-NMR(75MHz,DMSO-d ₆ ):δ/ppm＝174.90,171.01,159.64,158.52,151.60,140.33,122.48,54.85,32.01。

EXAMPLE 3 preparation of 6-S,9-N- [ diacetyl-Lys (Boc) -OBzl-mercapto ] purine (3 a)

0.300g (1.12 mmol) of 6-S,9-N- (dicarboxymethylmercapto) purine (2) was dissolved in 10mL of anhydrous N, N-dimethylformamide, 0.605g (2.24 mmol) of 1-hydroxybenzotriazole was added thereto at 0 ℃ and stirred for 10min, then 0.924g (4.48 mmol) of dicyclohexylcarbodiimide was added thereto and stirred for 30min. 1.890g (4.48 mmol) Lys (Boc) -OBzl was added to the reaction solution at 0 deg.C, the pH of the reaction mixture was adjusted to 9 with N-methylmorpholine, and the reaction was monitored by TLC (dichloromethane/methanol = 20/1) after stirring at room temperature for 12h. The reaction solution was filtered, the filtrate was concentrated under reduced pressure, the residue was dissolved in 50mL of ethyl acetate, the insoluble material was filtered off, and the filtrate was taken with saturated NaHCO ₃ Aqueous solution washing (30 mL. Times.3), saturated aqueous NaCl solution washing (30 mL. Times.3), 5% ₄ Washed with an aqueous solution (30 mL. Times.3) and saturatedAqueous NaCl (30 mL. Times.3) and saturated NaHCO ₃ The ethyl acetate phase was dried over anhydrous sodium sulfate for 12 hours, after washing with an aqueous solution (30 mL. Times.3) and with a saturated aqueous NaCl solution (30 mL. Times.3). Filtration and concentration of the filtrate under reduced pressure gave a yellow oil which was purified by silica gel column chromatography (dichloromethane/methanol = 40/1) to give 0.808g (80%) of the title compound. ESI-MS (m/e) 904[ M-H ], [ M/e ]] ^- ； ¹ H-NMR(300MHz,DMSO-d ₆ ):δ/ppm＝8.883～8.855(d,J＝8.4Hz,1H),8.677～8.651(d,J＝7.8Hz,1H),8.634(s,1H),8.416(s,1H),7.340(s,10H),6.754(m,2H),5.114(m,4H),5.040(m,2H),4.297(m,2H),4.184(m,2H),2.875(m,4H),1.715～1.580(m,6H),1.495～1.160(m,28H)； ¹³ C-NMR(75MHz,DMSO-d ₆ ):δ/ppm＝172.14,172.00,167.92,166.68,158.77,156.03,151.69,149.23,146.16,136.37,136.29,130.60,128.85,128.51,128.44,128.26,128.18,77.83,66.48,66.36,52.93,52.80,45.43,32.21,31.17,31.06,29.84,29.50,28.73,25.99,23.00。

EXAMPLE 4 preparation of 6-S,9-N- (diacetyl-Lys-OBzl-mercapto) purine (4 a)

To 0.800g (0.88 mmol) of compound 3a was added a solution of 8mL of 4N hydrogen chloride in ethyl acetate at 0 ℃, and completion of the reaction was monitored by TLC (dichloromethane/methanol = 20/1) after stirring for 1 h. Pumping out the reaction solution, adding 15mL of dry ethyl acetate, pumping out, and repeating for three times; 15mL of anhydrous ether was added, and the mixture was dried by suction and repeated 3 times. The resulting pale yellow solid was dissolved in 1mL of distilled water and RP-C was used ₁₈ Column chromatography purification (40% methanol in water) and fraction freeze-drying. The resulting colorless solids were combined to give a total of 0.498g (72%) of the title compound. FT-MS (m/e) 705.32[ 2 ], [ M + H ]] ⁺ ； ¹ H-NMR(300MHz,DMSO-d ₆ ):δ/ppm＝9.023～8.997(d,J＝7.8Hz,1H),8.773～8.748(d,J＝7.5Hz,1H),8.664(s,1H),8.449(s,1H),7.390～7.310(m,10H),5.124(m,4H),5.072(s,2H),4.355～4.254(m,2H),4.202(s,2H),2.744～2.708(m,4H),1.785～1.655(m,4H),1.616～1.538(m,4H),1.487～1.406(m,4H)； ¹³ C-NMR(75MHz,DMSO-d ₆ ):δ/ppm＝172.03,171.89,168.00,166.76,158.75,151.72,149.23,146.20,136.33,136.26,130.60,128.89,128.56,128.50,128.29,128.21,66.57,66.45,52.69,52.58,45.49,38.93,32.17,30.79,26.83,22.59。

Example 5 evaluation of the antitumor Effect of 4a

6-mercaptopurine and sodium carboxymethylcellulose were both purchased from national chemical group, inc. Male mice (20 ± 2 g) of SPF grade ICR strain were purchased from laboratory animal technology ltd, viton, beijing. The experiment used a transplanted mouse sarcoma model of S180.

The dose of compound 4a was 16. Mu. Mol/kg/day, the dose of positive control 6-mercaptopurine was 164. Mu. Mol/kg/day, and the negative control was CMCNa.

The tumor source for modeling the transplanted mouse S180 sarcoma model is S180 mouse sarcoma cells, which are purchased from animal experiment centers of department of medicine of Beijing university and are subjected to self-passage preservation according to a suspension cell culture method. A male ICR mouse with the SPF level of S180 ascites tumor after one week of passage is taken, the neck is cut off after the anesthesia of a proper amount of ether, the mouse is soaked in 75% alcohol for sterilization for 1min, the abdominal cavity is cut open, ascites S180 tumor liquid is taken, the centrifugation is carried out for 1000r/min multiplied by 10min, the supernatant is discarded, the residues are washed by a small amount of cooled normal saline to remove non-cell fragments, tissues and floating blood, the cell activity is calibrated by a MUSE flow cytometer, and the result shows that the cell activity reaches 94.67%. Viable cell count, density 4X 10 ⁷ One per mL. The living cells were suspended in cooled physiological saline to a cell density of 2X 10 ⁷ one/mL, used for inoculation as soon as possible. During inoculation, the mouse is fixed by the left hand, a 1mL syringe is held by the right hand to pierce the oxter of the right side of the mouse for about 2mm to be subcutaneous, a small cavity is separated in a gentle blunt manner, and 0.2mL of living cell suspension is injected.

The mice are observed every day after inoculation, when most mice can see mung bean particle size solid tumors (about 5 days of inoculation) in the armpits, the mice are grouped, 11 mice in each group are continuously administrated for 10 days, the mice in each group are weighed until 11 days, the mice are anesthetized by ether, the cervical vertebra is dislocated and killed, the growth positions of the solid tumors in the armpits of the mice are fixed, scissors are taken to cut the skin, the tumor bodies are fully exposed, and sarcomas are taken out and weighed along the skin and the upper limbs are separated in a blunt manner. The brain, heart, liver, spleen and kidney were dissected out in sequence and weighed, and the index of each organ was calculated.

The in-situ tumor weight of the data is represented by a mean value +/-SD g, the SD value is subjected to variance analysis through SPSS software, the homogeneity of the variance is detected, and statistical comparison among groups is performed by adopting t-test detection.

As a result, compound 4a exhibited antitumor activity, and 4a had antitumor activity comparable to that of 6-mercaptopurine at a dose reduced by 9-fold compared to that of 6-mercaptopurine. This is the unexpected technical effect of this case.

S180 antitumor Activity of Compound 4a of Table 1

Compound (I)	Dosage (mu mol/kg/day)	Tumor weight (mean + -SD g)
			CMCNa	-	2.34±0.67
6-mercaptopurine	164	1.10±0.33
			4a	16	1.25±0.60 ^a

a) P <0.05 to CMCNa, p >0.05 to 6-mercaptopurine; n =11.

Example 6 evaluation of myelosuppressive toxicity of 4a

The method comprises the steps of taking 20 mu L of eyeball blood before mice are sacrificed for 10 days after continuous administration treatment, placing the eyeball blood into a 0.5mL special EDTA blood collection tube, tightly covering the tube opening, shaking up and down uniformly, and detecting by using a full-automatic three-classification blood cell analyzer within 4 h. 4a is 6-S,9-N- (diacetyl-Lys-OBzl-mercapto) purine and control is CMCNa.

Results table 2 shows that the white blood cell count and platelet count of the 6-mercaptopurine group mice were significantly lower than those of the CMCNa group mice, and the red blood cell count did not significantly decrease. This indicates that myelosuppressive toxicity caused by 6-mercaptopurine is mainly manifested by inhibition of leukocytes and platelets. The white blood cell number and the platelet number of the 4a group of mice are obviously higher than those of the 6-mercaptopurine group of mice, and are very close to those of the CMCNa group of mice, namely, the acetyl-Lys-OBzl modified 6-S,9-N can reduce the bone marrow suppression side effect of the 6-mercaptopurine. This is the unexpected technical effect of this scheme.

TABLE 2 results of myelosuppressive toxicity assay in group 4a mice

Compound (I)	Number of leukocytes (. Times.10) ⁹ /L)	Platelet count (. Times.10) ¹¹ /L)	Number of erythrocytes (. Times.10) ¹² /L)
				CMCNa	18.39	13.75	6.54
6-mercaptopurine	10.06	7.46	6.25
				4a	16.94	13.11	6.77

Example 7 evaluation of plasma half-life of 4a

Under the anesthesia state, the plasma of the mouse is taken out, centrifuged at 3000r/min for 10min, and serum is taken out. 6-mercaptopurine and 4a were dissolved in 1mL of serum to give a concentration of 1mg/mL. Incubating 100 μ L of the sample at 37 deg.C, sampling at 0min,10min,30min,1h,2h, etc., precipitating protein with methanol, concentrating to obtain sample with the same concentration, and measuring the ultraviolet absorption of 4a with ultraviolet spectrophotometer.

The absorbance value at 0min is taken as A ₀ Calculating the content of the compound at different time points according to the formula, wherein the content is = A ₀ and/A. The data are shown in Table 3. The results show that the half-life of 4a in mouse plasma is 59.5min, which is significantly longer than the half-life of 6-mercaptopurine in mouse plasma (19.2 min). This is the unexpected technical effect of this case.

Table 3 half-life of 4a in mouse plasma

Compound (I)	Half-life period (min)
		6-mercaptopurine	19.2
4a	59.5

Claims

1. The structural formula of the compound is 6-S, 9-N-diacetyl-Lys-OBzl-mercaptopurine,

2. a process for the preparation of 6-S, 9-N-diacetyl-Lys-OBzl-mercaptopurine according to claim 1 which comprises:

2.1. synthesizing 6-S, 9-N-diacetyl-O-ethylmercaptopurine;

2.2. synthesizing 6-S, 9-N-dicarboxymethylmercaptopurine;

2.3. condensing 6-S,9-N- (dicarboxymethylmercapto) purine and Lys (Boc) -OBzl by a liquid phase method using dicyclohexylcarbodiimide as a condensing agent and 1-hydroxybenzotriazole as a catalyst to prepare 6-S, 9-N-diacetyl-Lys (Boc) -OBzl-mercaptopurine;

2.4. 6-S, 9-N-diacetyl-Lys-OBzl-mercaptopurine was deprotected to produce 6-S, 9-N-diacetyl-Lys-OBzl-mercaptopurine.

3. Use of 6-S, 9-N-diacetyl-Lys-OBzl-mercaptopurine according to claim 1 for the preparation of an anti-tumour medicament.