A nitrogen mustard derivative and preparation method thereof

Abstract

The invention discloses a novel nitrogen mustard derivative and a preparation method thereof. The method comprises: firstly synthesizing an amine raw material by using the hydrochloride of N,N-di(2-chloroethyl)-1,4-phenylenediamine, anhydrous DCM and triethylamine, reacting the amine raw material with triphosgene at room temperature, adding an anhydrous DCM solution of an aniline raw material under ice bath condition after confirming that the N,N-di(2-chloroethyl)-1,4-phenylenediamine is converted into an intermediate by TCL board, reacting at room temperature under nitrogen protection, stopping the reaction after confirming that the intermediate is completely converted by TCL board; adding water to the reaction solution to remove the remaining triethylamine, extracting with anhydrous DCM, collecting and merging organic layers, washing with saturated NaCl, drying with anhydrous Na2SO4, concentrating, purifying on a silica gel column, and recrystallizing to obtain the novel nitrogen mustard derivative. The present invention uses nitrogen mustard as the parent structure to design and synthesize 5 new nitrogen mustard derivatives 5c-5e, 5g-5h, which show broad-spectrum anti-tumor proliferation effects on CCRF-CEM, K562 and HL-60 cells, and have relatively low cytotoxicity to normal human liver cells L02.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical chemistry, and more specifically, relates to a novel nitrogen mustard derivative and a preparation method thereof. Background Art

[0002] The use of drugs is a key factor in chemotherapy. In the past two decades, although the research on cancer chemotherapy drugs has been accelerating, the selectivity of cancer drugs has always been the key point in evaluating their anti-cancer efficacy. Most anti-cancer drugs have good therapeutic effects, but due to their poor selectivity, these drugs have strong side effects on the healthy tissues and organs of patients. Although there are many drugs with novel structures and good therapeutic effects that are currently in clinical evaluation at different stages, these candidate drugs still have many shortcomings. Therefore, the development of anti-cancer drugs with less toxic side effects or even no toxic side effects has always been the goal of countless researchers.

[0003] Nitrogen mustard drugs are clinical first-line alkylating agents, which are highly toxic to cells. They mainly inhibit cell proliferation by cross-linking between or within the DNA in the cell nucleus to exert their biological activity. They are first used in the treatment of malignant lymphoma. At present, the widely used nitrogen mustard chemotherapy drugs in the clinic include chlorambucil, melphalan, cyclophosphamide, etc. However, the alkylation effect of nitrogen mustard drugs is non-specific, which requires a higher dose to make the actual plasma concentration of the drug reaching the site of action reach the required effective therapeutic amount, thereby invisibly increasing the toxicity of the patient's metabolic system, and in the process of use, due to the increase in the dosage, the formation of drug resistance in the body is accelerated. Chlorambucil (CLB) is clinically used for the treatment of diseases such as chronic lymphocytic leukemia, ovarian cancer and breast cancer. Because chlorambucil has no selectivity for cancer cells, this leads to strong side effects such as hepatotoxicity, ototoxicity, nephrotoxicity, etc. while exerting anti-cancer effects, which seriously affects the clinical application of the drug. The study of its anti-cancer mechanism found that it mainly binds to the N7 position of guanine or adenine or the N3 position of adenine in the DNA double helix chain in the cell, causing intra-chain or inter-chain cross-linking of the double helix chain, and then stimulates the corresponding stress response in the cell, thereby affecting the regulation of multiple related signal pathways of the intracellular cell cycle, DNA damage, energy metabolism or apoptosis. Generally speaking, the carcinogenic activity of alkylating agents is closely related to their anti-cancer activity. Generally, alkylating agents with strong anti-cancer activity also have relatively active carcinogenic activity. Therefore, in order to obtain nitrogen mustard drugs with better therapeutic effects and less toxic side effects, they need to be structurally modified. Summary of the invention

[0004] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.

[0005] In order to achieve these purposes and other advantages of the present invention, a novel nitrogen mustard derivative is provided, and the chemical structure of the novel nitrogen mustard derivative is as follows:

[0006]

[0007] Where R is:

[0008]

[0009] The preparation method of the novel nitrogen mustard derivative as described above comprises the following steps:

[0010] Step 1. Preparation: Clean and fully dry the instruments used;

[0011] Step 2, feed reaction: N, N-di(2-chloroethyl)-1, 4-phenylenediamine hydrochloride and anhydrous DCM are mixed, stirred and dissolved at room temperature to form a suspension, triethylamine is added dropwise thereto, and the reaction is completed when the suspension is clarified to obtain an amine raw material solution; triphosgene is dissolved in anhydrous DCM at room temperature, and the amine raw material solution is added dropwise thereto under ice bath conditions, and then the ice bath is removed, and the reaction is carried out at room temperature for 30 to 50 minutes. After the TCL plate confirms that N, N-di(2-chloroethyl)-1, 4-phenylenediamine is converted into an intermediate, an anhydrous DCM solution of an aniline raw material is added under ice bath conditions, and the reaction is carried out at room temperature for 10 to 14 hours under nitrogen protection. After the TCL plate confirms that the conversion of the intermediate is complete, the reaction is stopped;

[0012] Step 3, post-treatment: add water to the reaction solution obtained in step 2 to remove the remaining triethylamine, extract with anhydrous DCM 2 to 4 times, collect and combine the organic layers, wash with saturated NaCl 2 to 4 times, and then wash with anhydrous Na 2 SO 4 After drying for 1 to 2 hours and concentration, the product was purified on a silica gel column and recrystallized to obtain a novel nitrogen mustard derivative.

[0013] Preferably, in the step 2, the molar volume ratio of the hydrochloride of N,N-di(2-chloroethyl)-1,4-phenylenediamine to anhydrous DCM is 0.3-0.4 mmol:2-4 mL; the molar ratio of the hydrochloride of N,N-di(2-chloroethyl)-1,4-phenylenediamine to triethylamine is 1:1.2-1.8.

[0014] Preferably, in step 2, the molar volume ratio of triphosgene to anhydrous DCM is 0.1-0.2 mmol:2-5 mL; the molar ratio of triphosgene to N,N-di(2-chloroethyl)-1,4-phenylenediamine hydrochloride is 0.3-0.5:1.

[0015] Preferably, in step 2, the aniline raw material is one of 3-fluoro-5-methylaniline, 3,5-difluoro-4-methoxyaniline, 3-fluoro-5-methoxyaniline, 3,5-dimethoxyaniline and 3,5-dimethylaniline.

[0016] Preferably, in the step 2, in the anhydrous DCM solution of the aniline raw material, the molar volume ratio of the aniline raw material to anhydrous DCM is 0.35-0.45 mmol:4-6 mL; and the molar ratio of the aniline raw material to the hydrochloride of N,N-di(2-chloroethyl)-1,4-phenylenediamine is 1-1.5:1.

[0017] Preferably, in step 2, the synthesis method of the hydrochloride of N,N-di(2-chloroethyl)-1,4-phenylenediamine comprises:

[0018] Step 1, synthesis of compound 2:

[0019] S11. Preparation: Clean and fully dry the instruments used;

[0020] S12, feed reaction: weigh compound 1 and diethanolamine, mix them, and then reflux them in an oil bath at 120° C. for 2 h under nitrogen protection. After the conversion of the raw materials is confirmed by TCL plate, stop the reaction;

[0021] S13, post-treatment: add ice water to cool the reaction solution, observe the precipitation of flocculent precipitate, stir the reaction bottle at room temperature for 30 minutes, observe the precipitation of yellow precipitate, stop stirring and let the reaction solution stand until the precipitate is completely settled, filter to obtain the crude product, collect the crude product, package it, and put it in a vacuum drying oven to dry for 12 hours to obtain compound 2;

[0022] Step 2, synthesis of compound 3:

[0023] S21. Preparation: Clean and fully dry the instruments used;

[0024] S22, feeding reaction: dissolve compound 2 in anhydrous DCM at room temperature, then drip triethylamine at a uniform rate, and then drip a solution of thionyl chloride in anhydrous DCM within 1 hour. After the dripping is completed, heat to 40°C in an oil bath under nitrogen protection and reflux for 4 hours. After the conversion of the raw materials is confirmed by TCL plate, stop the reaction;

[0025] S23, post-treatment: dilute the reaction solution with anhydrous DCM, and then use saturated NaHCO 3 , saturated NaCl, and the reaction solution was washed three times. The organic layers were collected and combined with anhydrous Na 2 SO 4 Dry for 1 hour, concentrate under reduced pressure to obtain a crude product, add ethanol to the crude product and heat to dissolve, place in a refrigerator to precipitate yellow needle-shaped crystals of pure product, namely compound 3;

[0026] Step 3: Synthesis of the hydrochloride of N,N-di(2-chloroethyl)-1,4-phenylenediamine:

[0027] S31. Preparation: Clean and fully dry the instruments used;

[0028] S32, feed reaction: Compound 3 and 10-14 mol / L hydrochloric acid were mixed respectively, stirred at room temperature to dissolve, and then stannous chloride dihydrate was added. The mixture was refluxed in an oil bath at 100°C for 5 h under nitrogen protection. The reaction was stopped when the reaction solution turned colorless and transparent.

[0029] S33, post-treatment: add ice water to the reaction solution to precipitate flocculent precipitate, filter, add water to dissolve the precipitate, adjust the pH to 7.5-8.0 with ammonia water, extract three times with ethyl acetate, collect and combine the organic layer, wash the organic layer three times with saturated NaCl, anhydrous Na 2 SO 4 After drying for 1 hour, the dried solution was concentrated under reduced pressure and the concentrated solution was poured into an instrument passing hydrochloric acid gas to precipitate the product. After confirming that all the products in the reaction solution were precipitated by TCL plate, the gas introduction can be stopped and the precipitated crude product was placed in a vacuum drying oven for 12 hours to obtain the hydrochloride of N,N-di(2-chloroethyl)-1,4-phenylenediamine.

[0030] Preferably, the compound 1 is:

[0031] Preferably, in the step 1, the molar volume ratio of compound 1, diethanolamine and ice water is 1 mmol:4-6 mmol:80-120 mL;

[0032] In the step 2, in S21, the molar volume ratio of compound 2, anhydrous DCM, triethylamine and dithionyl chloride is 10-12 mmol:50-70 mL:1 mL:2 mL; in the anhydrous DCM solution of dithionyl chloride, the volume ratio of dithionyl chloride and anhydrous DCM is 1:4-6; in S22, the volume molar ratio of anhydrous DCM and compound 2 in S21 is 15-25 mL:10-12 mmol;

[0033] In the step 3, the molar volume ratio of compound 3, hydrochloric acid, stannous chloride dihydrate, ice water and water is 9-10 mmol: 35-45 mmol: 55-60 mmol: 40-60 mL: 20-40 mL.

[0034] Application of a novel nitrogen mustard derivative prepared by the preparation method described above in the preparation of anticancer drugs.

[0035] Preferably, the use of the novel nitrogen mustard derivative in the preparation of anticancer drugs comprises: firstly, the materials used are crushed separately, passed through an 80-mesh sieve, and set aside; then, the novel nitrogen mustard derivative, microcrystalline cellulose, 40-60% of ethyl cellulose, 40-60% of cross-linked polyvinylpyrrolidone and Tween-80 are mixed, and then ethanol is added for granulation, and after supercritical carbon dioxide drying treatment, the remaining cross-linked polyvinylpyrrolidone, ethyl cellulose and magnesium stearate are added, the mixture is ball-milled, and pressed into tablets to obtain anticancer drug tablets.

[0036] Preferably, the proportions of the components in the tablet are: 25-65wt% of the novel nitrogen mustard derivative, 15-30wt% of microcrystalline cellulose, 5-10wt% of ethyl cellulose, 5-15wt% of cross-linked polyvinylpyrrolidone, 2-6wt% of Tween-80, and 0.5-5wt% of magnesium stearate.

[0037] Preferably, the specific method of the supercritical carbon dioxide drying treatment is: treating at 1-10MPa and 20-40°C for 1-2h, and then removing carbon dioxide at a pressure relief rate of 0.05-1MPa / s; the specific method of the ball milling is: adding the mixture to a vacuum ball mill, the ball mill lining is agate, the grinding balls are 2mm, 4mm, and 8mm agate balls configured in a mass ratio of 0.5-1.5:1-3:2-4, the grinding ball filling amount is 40-80%, the ball-to-material ratio is 6-10:1, the vacuum degree is 50-150Pa, the ball mill speed is 150-300rpm, and an intermittent ball milling method is adopted, that is, grinding for 30-60min, pausing for 15-30min, and circulating 3 times.

[0038] The present invention has at least the following beneficial effects: the present invention uses nitrogen mustard as the parent structure, designs and synthesizes 5 novel nitrogen mustard derivatives 5c-5e, 5g-5h, among which 5c-5e exhibits a broad-spectrum anti-tumor proliferation effect on CCRF-CEM, K562, HL-60 cells, IC 50 The values ​​ranged from 14.12 to 0.822 μM. In particular, the three novel nitrogen mustard derivatives 5c to 5e had excellent antiproliferative effects on HL-60 cells, with IC 50 The values ​​were 0.8823~1.109μM; among them, 5e in HL-60 cells, IC 50 The value is 0.8823 μM, and its inhibitory activity on HL-60 cells is much better than that of chlorambucil (34.392 μM), and its cytotoxicity to normal human liver cells L02 is relatively small.

[0039] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the synthesis route of the hydrochloride of N,N-di(2-chloroethyl)-1,4-phenylenediamine in Example 1 of the present invention;

[0041] Figure 2 The synthetic routes are the novel nitrogen mustard derivatives 5c-5e, 5g-5h of Examples 2-6 of the present invention and the nitrogen mustard derivatives 5a, 5b, 5f of Comparative Examples 1-3. DETAILED DESCRIPTION

[0042] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0043] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0044] Pretreatment of reagents used in the following implementation:

[0045] (1) Anhydrous dichloromethane (DCM): Place dry calcium hydride solid in a reflux apparatus, then add dichloromethane, heat and reflux for 2 h, collect the fraction at the boiling point and then turn off the instrument. The collected fraction can be used after cooling to room temperature, and finally turn off the condensation water.

[0046] (2) Ethyl acetate treatment: Add a certain amount of ethyl acetate into a normal pressure distillation apparatus and heat for distillation. When the temperature reaches about 70°C, the solvent begins to boil. Collect the required amount of solvent by cutting off the head and tail, and it can be used after natural cooling.

[0047] Example 1

[0048] The synthesis method of the hydrochloride of N,N-di(2-chloroethyl)-1,4-phenylenediamine, the synthesis route is as follows Figure 1 As shown, including:

[0049] Step 1, synthesis of compound 2:

[0050] S11. Preparation: Clean and fully dry the glassware required for the experiment;

[0051] S12, feed reaction: according to the reaction feed ratio, compound 1 (5 g; 35.5 mmol) and diethanolamine (5 eq; 18.652 g; 177.5 mmol) were weighed and put into a reaction bottle, and then refluxed in an oil bath at 120° C. for 2 h under nitrogen protection. After the conversion of the raw materials was confirmed by TCL plate, the reaction was stopped;

[0052] S13, post-treatment: add 100mL ice water to cool the reaction solution, observe the precipitation of flocculent precipitate, stir the reaction bottle at room temperature for 30min, observe the precipitation of yellow precipitate, stop stirring and let the reaction solution stand until the precipitate is completely settled, filter to obtain a crude product, collect the crude product, package it, and put it into a vacuum drying oven to dry for 12h to obtain compound 2; wherein, the developing agent (EA:PE=1:5) for monitoring the reaction is used to obtain 5.8g of yellow solid, with a yield of 72%. 1 H-NMR (600MHz, DMSO) δ6.82(d,J=7.6Hz,2H),8.02(d,J=9.5Hz,2H),4.87(t,J=5.2Hz,2H),3.59(dt,J=9.7,5.3Hz,8H);

[0053] Step 2, synthesis of compound 3:

[0054] S21, preparation: same as the synthesis of compound 2;

[0055] S22, feeding reaction: according to the reaction feeding ratio, compound 2 (2.50 g; 11.05 mmol) was added to the reaction bottle, and compound 2 was dissolved with anhydrous DCM (60 mL) at room temperature, and triethylamine (1 mL) was added dropwise at a uniform rate, and then thionyl chloride (2 mL) was diluted with anhydrous DCM (10 mL), and the diluted thionyl chloride solution was added dropwise to the reaction bottle within 1 h through a constant pressure dropping funnel. After the addition was completed, the temperature was raised to 40 ° C. and the oil bath was refluxed for 4 h. The reaction process required nitrogen protection. After the TCL plate confirmed that the conversion of the raw materials was complete, the reaction was stopped;

[0056] S23, post-treatment: dilute the reaction solution with anhydrous DCM (20 mL), and then use saturated NaHCO 3 , saturated NaCl, and the reaction solution was washed three times. The organic layers were collected and combined with anhydrous Na 2 SO 4 Dry for 1 hour, concentrate under reduced pressure to obtain a crude product, add an appropriate amount of ethanol to the crude product and heat to dissolve, place it in a refrigerator to precipitate a pure yellow needle-shaped crystal, namely compound 3; wherein, the developing agent for monitoring the reaction (EA:PE=1:1) was used to obtain 2.27 g of yellow needle-shaped crystals, with a yield of 73%. 1 H-NMR (600MHz, DMSO) δ6.93(d,J=9.5Hz,2H),8.07(d,J=9.5Hz,2H),3.90(t,J=6.8Hz,4H),3.81(t,J=6.7Hz,4H);

[0057] Step 3, Synthesis of the hydrochloride of N,N-di(2-chloroethyl)-1,4-phenylenediamine (Compound 4):

[0058] S31, preparation: same as the synthesis of compound 2;

[0059] S32, feed reaction: according to the reaction feed ratio, compound 3 (2.5 g, 9.5 mmol) and 12 mol / L hydrochloric acid (40 mmol) were respectively taken into a reaction bottle, stirred at room temperature to dissolve, and then stannous chloride dihydrate (12.8 g, 57 mmol) was added. The mixture was refluxed in an oil bath at 100°C under nitrogen protection for 5 h. When the reaction solution turned colorless and transparent, the reaction was stopped;

[0060] S33, post-treatment: add 50 mL of ice water to the reaction solution to precipitate flocculent precipitate, filter, add water (30 mL) to dissolve the precipitate, adjust the pH to 8.0 with ammonia water, extract three times with treated ethyl acetate, collect and combine the organic layers, wash the organic layers three times with saturated NaCl, and wash with anhydrous Na 2 SO 4 After drying for 1 hour, the dried solution was concentrated to 120 mL under reduced pressure, and the concentrated solution was poured into an instrument passing hydrochloric acid gas to precipitate the product. After the TCL plate confirmed that all the products in the reaction solution were precipitated, the gas introduction could be stopped. The whole process lasted for about 40 minutes. The precipitated crude product was placed in a vacuum drying oven for 12 hours to obtain the hydrochloride of N, N-di(2-chloroethyl)-1, 4-phenylenediamine, i.e., the hydrochloride of compound 4; wherein, the developing agent (EA:PE=2:1) ​​for monitoring the reaction was used to obtain 2.02 g of a light gray solid with a yield of 73.5%. 1 H-NMR (600MHz, DMSO) δ6.52 (d, J = 8.7 Hz, 2H), 6.59 (d, J = 8.8 Hz, 2H), 3.62 (t, J = 6.9 Hz, 4H), 4.68 (s, 2H), 3.51 (t, J = 6.9 Hz, 4H).

[0061] Example 2

[0062] A method for preparing a novel nitrogen mustard derivative comprises the following steps:

[0063] Step 1. Preparation: Clean and fully dry the glassware required for the experiment;

[0064] Step 2, feeding reaction: according to the reaction feeding ratio, the hydrochloride of compound 4 (100 mg; 0.327 mmol) and anhydrous DCM (3 mL) were taken in a reaction bottle, stirred and dissolved at room temperature to form a suspension, triethylamine (1.5 eq; 0.490 mmol) was added dropwise, and the reaction was completed when the suspension was clarified to obtain an amine raw material solution; another reaction bottle was taken, triphosgene (0.35 eq; 0.114 mmol) was added, and then anhydrous DCM (3 mL) was added to dissolve the triphosgene at room temperature, and the amine raw material solution was dropped into the reaction solution under ice bath conditions, and then the ice bath was removed, and the reaction was carried out at room temperature for 40 min. After the TCL plate confirmed that compound 4 was converted into an intermediate, a solution of 3-fluoro-5-methylaniline (1.2 eq; 0.392 mmol) in anhydrous DCM (5 mL) was quickly added to the reaction solution under ice bath conditions, and the reaction was carried out at room temperature for 12 h under nitrogen protection. After the TCL plate confirmed that the conversion of the intermediate was complete, the reaction was stopped;

[0065] Step 3, post-treatment: add water to the reaction solution to remove the remaining triethylamine, extract with anhydrous DCM three times, collect and combine the organic layers, wash three times with saturated NaCl, and 2 SO 4 After drying for 1 h and concentration, the product was purified by silica gel column and recrystallized to obtain a novel nitrogen mustard derivative 5c, the chemical structure of which is as follows:

[0066]

[0067] Among them, there are many by-products in the product, which are not easy to observe, so two development systems (CH 3 OH:DCM=1:7;EA:PE=1:3), a white solid was obtained, 1 H NMR (500MHz, DMSO-d6) δ8.66(s,1H),8.38(s,1H),7.26(t,J=7.8Hz,3H),6.92(s,1H),6.71(d,J=8.7Hz,2H),6.5 8(dd,J=9.6,2.1Hz,1H),5.76(d,J=1.5Hz,1H),3.69(dt,J=10.7,5.3Hz,9H),3.32(d,J=1.9Hz,1H),2.27(s,3H).

[0068] Example 3

[0069] A method for preparing a novel nitrogen mustard derivative: 3,5-difluoro-4-methoxyaniline (1.2 eq; 0.392 mmol) was used to replace 3-fluoro-5-methylaniline in Example 2, and the remaining steps were the same as in Example 2 to obtain a novel nitrogen mustard derivative 5d, whose chemical structure is as follows:

[0070]

[0071] Among them, there are many by-products in the product, which are not easy to observe, so two development systems (CH 3 OH:DCM=1:6;EA:PE=1:2), a white solid was obtained, 1 H NMR(500MHz,DMSO-d6)δ8.76(s,1H),8.44(s,1H),7.29-7.24(m,2H),7.21(d ,J=10.7Hz,2H),6.74-6.67(m,2H),3.83(s,3H),3.70(p,J=5.2,4.5Hz,8H).

[0072] Example 4

[0073] A method for preparing a novel nitrogen mustard derivative: 3-fluoro-5-methoxyaniline (1.2 eq; 0.392 mmol) was used to replace 3-fluoro-5-methylaniline in Example 2, and the remaining steps were the same as in Example 2 to obtain a novel nitrogen mustard derivative 5e, whose chemical structure is as follows:

[0074]

[0075] Among them, there are many by-products in the product, which are not easy to observe, so two development systems (CH 3 OH:DCM=1:6;EA:PE=1:2), a white solid was obtained, 1 H NMR (500MHz, DMSO-d6) δ8.72(s,1H),8.37(s,1H),7.27(d,J=8.6Hz,2H),6.96(dt,J=11.4, 2.1Hz, 1H), 6.86-6.65 (m, 3H), 6.39 (dt, J = 11.0, 2.3Hz, 1H), 3.69 (dt, J = 10.7, 5.3Hz, 8H).

[0076] Example 5

[0077] A method for preparing a novel nitrogen mustard derivative: 3,5-dimethoxyaniline (1.2 eq; 0.392 mmol) was used to replace 3-fluoro-5-methylaniline in Example 2, and the remaining steps were the same as in Example 2 to obtain 5 g of a novel nitrogen mustard derivative, the chemical structure of which is as follows:

[0078]

[0079] Example 6

[0080] A method for preparing a novel nitrogen mustard derivative: 3,5-dimethylaniline (1.2 eq; 0.392 mmol) was used to replace 3-fluoro-5-methylaniline in Example 2, and the remaining steps were the same as in Example 2 to obtain a novel nitrogen mustard derivative 5h, whose chemical structure is as follows:

[0081]

[0082] Example 7

[0083] The application of a novel nitrogen mustard derivative in the preparation of an anticancer drug comprises: firstly, crushing the materials used respectively, passing through an 80-mesh sieve, and setting aside; mixing 100 g of the novel nitrogen mustard derivative 5e prepared in Example 4, 40 g of microcrystalline cellulose, 8 g of ethyl cellulose, 10 g of cross-linked polyvinylpyrrolidone, and 8 g of Tween-80, and then adding an appropriate amount of ethanol for granulation; after supercritical carbon dioxide drying, adding 15 g of cross-linked polyvinylpyrrolidone, 8 g of ethyl cellulose, and 2.5 g of magnesium stearate, mixing and ball milling, and pressing into tablets to obtain 1000 anticancer drug tablets, each tablet containing 100 mg of the novel nitrogen mustard derivative 5e; type nitrogen mustard derivative 5e; wherein the specific method of supercritical carbon dioxide drying is: drying at 5MPa and 30°C for 1h, and then removing carbon dioxide at a pressure relief rate of 0.1MPa / s; the specific method of ball milling is: adding the mixture into a vacuum ball mill, the ball mill lining is agate, the grinding balls are 2mm, 4mm, and 8mm agate balls configured in a mass ratio of 1:2:3, the grinding ball filling amount is 60%, the ball-to-material ratio is 10:1, the vacuum degree is 100Pa, the ball mill speed is 200rpm, and an intermittent ball milling method is adopted, that is, grinding for 30min, pausing for 15min, and repeating 3 times.

[0084] Comparative Example 1

[0085] A method for preparing a nitrogen mustard derivative: 3,4,5-trifluoroaniline (1.2 eq; 0.392 mmol) was used to replace 3-fluoro-5-methylaniline in Example 2, and the remaining steps were the same as in Example 2 to obtain a nitrogen mustard derivative 5a, whose chemical structure is as follows:

[0086]

[0087] Among them, there are many by-products in the product, which are difficult to observe, so the TCL plate needs two development systems (CH 3 OH:DCM=1:6;EA:PE=1:2), a white solid was obtained, 1H NMR (500MHz, Chloroform-d) δ7.64 (s, 2H), 7.19 (s, 2H), 7.09 (d, J = 8.6Hz, 2H), 6.59 (d, J = 8.4Hz, 2H), 3.65 (t, J = 7.0Hz, 4H), 3.54 (t, J = 6.9Hz, 4H).

[0088] Comparative Example 2

[0089] A method for preparing a nitrogen mustard derivative: 3,5-difluoroaniline (1.2 eq; 0.392 mmol) was used to replace 3-fluoro-5-methylaniline in Example 2, and the remaining steps were the same as in Example 2 to obtain a nitrogen mustard derivative 5b, whose chemical structure is as follows:

[0090]

[0091] Among them, there are many by-products in the product, which are not easy to observe, so two development systems (CH 3 OH:DCM=1:6;EA:PE=1:2), a white solid was obtained, 1 H NMR (500MHz, DMSO-d6) δ8.94(s,1H),8.49(s,1H),7.40-7.06(m,4H),6.81-6.61(m,3H),3.85-3.58(m,8H).

[0092] Comparative Example 3

[0093] A method for preparing a nitrogen mustard derivative: 2-fluoroaniline (1.2 eq; 0.392 mmol) was used to replace 3-fluoro-5-methylaniline in Example 2, and the remaining steps were the same as in Example 2 to obtain a nitrogen mustard derivative 5f, whose chemical structure is as follows:

[0094]

[0095] Among them, there are many by-products in the product, which are not easy to observe, so two developing systems (CH3OH:DCM=1:8;EA:PE=1:5) are required to apply TCL plate during the reaction to obtain a white solid. 1 H NMR(500MHz,DMSO-d6)δ8.75(s,1H),8.38(s,1H),8.15(td,J=8.3,1.7Hz,1H),7.28(d,J=9.1Hz,2H), 7.24-7.18(m,1H),7.11(t,J=7.8Hz,1H),6.97(t,J=10.4Hz,1H),6.72(d,J=9.1Hz,2H),3.31(s,8H).

[0096] The synthetic routes of the novel nitrogen mustard derivatives 5c-5e, 5g-5h of Examples 2-6 and the nitrogen mustard derivatives 5a, 5b, 5f of Comparative Examples 1-3 are as follows: Figure 2 shown.

[0097] Comparative Example 4

[0098] In this comparative example, vacuum drying at 30°C for 1 h was used instead of supercritical carbon dioxide drying, and the remaining steps were the same as in Example 7.

[0099] Comparative Example 5

[0100] In this comparative example, intermittent ball milling was replaced by direct ball milling for 90 min, and the remaining steps were the same as those in Example 7.

[0101] Anti-tumor activity test:

[0102] The cell lines used in the following experiments: human acute lymphoblastic leukemia cell line CCRF-CEM, human promyelocytic acute leukemia cells HL-60, human chronic myeloid leukemia cell line K562, and human normal liver cells L02 were all purchased from Shanghai Lanbao Biotechnology Co., Ltd.

[0103] 1. Reagent preparation

[0104] RPMI-1640 complete medium: Prepared at a ratio of RPMI1640 medium: FBS (fetal bovine serum): PS (penicillin-streptomycin) = 89:10:1 and stored in a refrigerator at 4°C;

[0105] IMDM complete medium: Prepared at a ratio of IMDM (containing glutamate): FBS (fetal bovine serum): PS (penicillin-streptomycin) = 79:20:1 and stored in a 4°C refrigerator;

[0106] DMEM complete medium: Prepared at a ratio of DMEM high glucose medium: FBS (fetal bovine serum): PS (penicillin-streptomycin) = 89:10:1 and stored in a refrigerator at 4°C;

[0107] DMEM maintenance medium: prepared at a ratio of DMEM high glucose medium: FBS (fetal bovine serum): PS (penicillin-streptomycin) = 96.5:2.5:1, and stored in a refrigerator at 4°C;

[0108] IMDM maintenance medium: Prepared at a ratio of IMDM (containing glutamate): FBS (fetal bovine serum): PS (penicillin-streptomycin) = 94:5:1 and stored in a 4°C refrigerator;

[0109] RPMI-1640 maintenance medium: prepared at a ratio of RPMI1640 medium: FBS (fetal bovine serum): PS (penicillin-streptomycin) = 96.5:2.5:1, and stored in a refrigerator at 4°C;

[0110] MTT working solution: weigh 500 mg of tetramethylthiazolium blue (MTT) and pour it into a sterilized glass bottle. Dissolve it in 100 mL of sterile phosphate buffer (PBS), and then filter the solution through a 0.22 μm sterile filter membrane to obtain a 5 mg / mL MTT working solution. To prevent the MTT working solution from becoming ineffective due to exposure to light and repeated freezing, dispense the prepared working solution into 2 mL EP tubes and store it in a -20°C refrigerator in the dark.

[0111] Triple lysis buffer: weigh 10 g of SDS (sodium dodecyl sulfate) and pour it into a sterilized glass bottle with a scale. Then use a pipette to draw 0.1 mL of HCl (10 M) and 5 mL of isobutanol into the glass bottle. 2 O to 100 mL, and then filter the solution through a 0.22 μm sterile filter membrane, sterilize the filtered solution with high-pressure steam, and store the solution at room temperature.

[0112] 2. Cell recovery

[0113] Preparation: prepare a 37°C water bath, prepare the required complete culture medium, disinfect the workbench half an hour in advance, and place the required items on the workbench after disinfection;

[0114] Thawing: Take the cells out of the frozen state and thaw quickly in a water bath. Shake the cryotubes constantly during the whole process to ensure that the cryosol is completely dissolved.

[0115] Centrifugation: After the thawed cryotubes are disinfected with alcohol, the cells in the tubes are quickly centrifuged (1000 rpm, 5 min). During the centrifugation, 4 mL of complete culture medium is added to a new culture dish (60 mm × 15 mm);

[0116] Prepare cell suspension: After centrifugation, collect cells, mix the cell pellet at the bottom of the tube with culture medium, and then use a pipette to transfer the mixed cell suspension into the culture dish. Make sure to mix the cells evenly.

[0117] Culture: Place the sterilized culture dish in an incubator at 37°C and 5% CO 2 After 24 h of culture, the cell recovery status was observed, and the subsequent experimental content was determined based on the cell status and cell growth density.

[0118] 3. Cell culture

[0119] Human acute lymphoblastic leukemia cell line CCRF-CEM and human chronic myeloid leukemia cells K562 were cultured in RPMI-1640 complete medium;

[0120] Human promyelocytic acute leukemia cells HL-60 were cultured in IMDM complete medium;

[0121] Human normal hepatocytes L02 were cultured in DMED complete medium;

[0122] The above cell lines were cultured in a sterile incubator (37°C, 5% CO 2 ).

[0123] 4. Cell passaging

[0124] Preparation: To confirm whether the cells can be used for subculture experiments, it is necessary to observe the cell growth status and growth density under a microscope. When the cell growth density reaches the subculture requirement, that is, 85-95% of the culture dish, the cells can be subcultured;

[0125] Digestion of adherent cells (L02 cell line): discard the old culture medium in the culture dish, and then use sterile phosphate buffer to gently wash away the cells with poor growth and dead cells, repeat the washing twice, after washing, add 2mL of trypsin digestion solution, shake the culture dish in all directions to make the digestion solution evenly permeate the entire bottom of the culture dish, generally digest for 2 minutes, and stop digestion when the cell protrusions can be observed to become round;

[0126] Passaging: discard the digestion solution, add 2 mL of complete culture medium, use the culture medium to slowly blow off the cells at the bottom of the dish, and then centrifuge the culture medium containing cells. After centrifugation, mix the cell pellet with the culture medium, and divide the cell suspension into three equal parts and transfer them into the culture dish to complete the passaging;

[0127] Suspension cells (CCRF-CEM, HL-60, K562 cell lines): The steps are the same as for adherent cells except that phosphate buffer washing and trypsin digestion are not required.

[0128] 5. MTT colorimetric assay to detect cell inhibition rate

[0129] Adherent cells (L02 cell line):

[0130] Inoculation: Logarithmic phase cells with good growth status are the inoculation objects. When the cells reach the subculture density under the microscope, they can be used for cell inoculation. Aspirate and discard the old culture medium, slowly wash the cells twice with phosphate buffer to wash away the cells with poor growth status, and then digest the cells and centrifuge them in the same way as subculture. After centrifugation, mix the cell pellet with maintenance culture medium, and add the cell suspension to the wells in the 96-well plate, 100 μL per well. To prevent edge effect, add the same volume of sterile phosphate buffer around the wells where the cell suspension is added. Try to keep the cell concentration of all wells consistent during the inoculation process;

[0131] Dosing: dissolve the drug to be used in dimethyl sulfoxide to prepare a 5 mM solution for standby use at low temperature; use maintenance medium to dilute the drug to 0.04, 0.40, 2.00, 10.00, 20.00, and 40.00 μM; take out the cells after 24 hours of culture to observe whether the cell state has reached the drug-adding state. After confirming the state, discard the old culture medium and add drug solutions of various concentrations (each concentration is repeated 3 times). The blank control group is only added with maintenance medium and cultured for 72 hours;

[0132] Detection: Take out the 96-well plate, discard the old culture medium, add 100 μL MTT working solution to each well, put it into the incubator and continue to culture for 4 hours, discard the MTT working solution, add 100 μL dimethyl sulfoxide to each well to dissolve for 10 minutes, and then use a microplate reader (ReadMax1900) to perform absorbance test (test procedure: shake for 1 minute; measure absorbance at 490nm); cell inhibition rate calculation formula (%) = (1-OD 实验组 / OD 对照组 )×100%, and the experimental data were analyzed using GraphPad Prism 8 software;

[0133] Suspension cells:

[0134] Plating: For suspension cells, the washing and digestion steps of adherent cells were omitted. The drug concentrations were diluted to 0.04, 0.40, 2.00, 10.00, 20.00, and 40.00 μM with a uniform cell suspension, and then inoculated into a 96-well plate, 90 μL per well (each concentration was repeated 3 times). The rest of the operations were the same as for adherent cells.

[0135] Detection: After 72 hours, add 20 μL of MTT working solution directly to each well and incubate for 4 hours. After the incubation is completed, add 100 μL of triple lysis solution to each well and continue incubating in the incubator for 10 hours. Finally, use an ELISA reader to perform absorbance test (test procedure: shake for 1 minute; measure absorbance at 570 nm). The data analysis method is the same as that of adherent cells.

[0136] The inhibitory activities of the novel nitrogen mustard derivatives 5c-5e prepared in Examples 2-4 of the present invention and the nitrogen mustard derivatives 5a, 5b, 5f of Comparative Examples 1-3 on CCRF-CEM, K562, HL-60 and L02 were detected by MTT colorimetry. Chlorambucil (CLB) was used as a positive control. The IC values ​​of these cell lines were 50 The values ​​are shown in Table 1. 50 The values ​​represent the drug concentration at which the cell growth rate was detected to decrease by 50% after incubating the cells under the action of the drug for 72 h.

[0137] As shown in Table 1, compared with CLB, the inhibitory activity of 5a-5f on the three leukemia cells was significantly enhanced, and the inhibitory activity of 5c, 5d, and 5e in Examples 2-4 was better than that of 5a, 5b, and 5f in Comparative Examples 1-3. 5c-5e showed a broad spectrum of anti-tumor proliferation effects on CCRF-CEM, K562, and HL-60 cells, IC 50 The values ​​ranged from 14.12 to 0.822 μM. More importantly, these three novel nitrogen mustard derivatives had excellent antiproliferative effects on HL-60 cells, with IC 50 The values ​​were 0.8823~1.109μM, among which 5e in HL-60 cells, IC 50 The value is 0.8823 μM, indicating that its inhibitory activity on cells is far superior to the positive control CLB (34.392 μM) and 5a, 5b, 5f (1.580-12.07 μM), and its cytotoxicity to normal human liver cells L02 is relatively small. These two indicators show that 5e is worthy of further study.

[0138] Furthermore, by comparing the IC of 5e to human normal liver cells L02 50 The value was divided by the IC value of HL-60 for leukemia cells. 50 The selectivity index (SI) results were obtained by quantifying the selectivity of 5e. As shown in Table 2, the minimum selectivity index of 5e was 48.64, the selectivity indexes of 5a, 5b, and 5f were 3.31, 10.15, and 25.32, respectively, and the selectivity index of the positive control CLB was 3.08, indicating that the selectivity of 5e for the HL-60 cell line was higher than that of 5a, 5b, 5f, and CLB.

[0139] Structure-activity relationship (SAR) analysis is a very important step in the development of new drugs, which is conducive to the design of compound structures with better biological activity. According to Table 1, the structure-activity relationship analysis of the three new nitrogen mustard derivatives of the present invention and the three nitrogen mustard derivatives of Comparative Examples 1 to 3 found that: 5a to 5f with the introduction of diaryl urea structure had significantly enhanced the inhibitory activity against three leukemia cells; in the study of the inhibitory activity of the HL-60 cell line, it was found that increasing the number of fluorine atoms (compounds 5a, 5b, 5f and 5d, 5e) slightly reduced the activity; at the same time, it was found that the introduction of methoxy (5e) had better inhibitory activity than the introduction of methyl (5c) in the same substitution position, which may be due to the methoxy group increasing the water solubility of the compound.

[0140] In summary, when there are substituents on the benzene ring, the hybrids containing electron-donating groups (methoxy, methyl) at the ortho, meta, and para positions of the benzene ring have better inhibitory activity against the three leukemia cells than the hybrids containing only electron-withdrawing groups (such as fluorine groups), and increasing the number of fluorine groups cannot increase the inhibitory activity of the compounds against the three leukemia cells.

[0141] Table 1

[0142] <![CDATA[IC 50 Values(μM)]]> CCRF-CEM K562 HL-60 L02 5c 1.831 14.12 1.109 12.56 5d 1.539 13.85 0.8914 5.628 5e 1.875 5.701 0.8223 >40 5a 3.043 38.66 12.07 >40 5b 1.734 22.57 1.703 17.28 5f 4.699 19.02 1.580 >40 CLB 12.025 >100 34.392 105.8

[0143] Table 2

[0144]

[0145]

[0146] The friability and hardness of the anticancer drug tablets prepared in Example 7, Comparative Example 4 and Comparative Example 5 were tested:

[0147] Indentation hardness: Test the initial hardness of the tablet and the hardness after 24 hours of storage, according to the Rockwell hardness (HR). It is generally believed that an indentation hardness greater than 20N has no effect on transportation, storage and subsequent use;

[0148] Friability: It is carried out in accordance with the tablet friability test method 0923 of the third volume of the "Chinese Pharmacopoeia". The particles need to be tumbled 100 times in the friability tester. The ratio of the lost weight to the original weight is the friability. If the friability exceeds 1%, it is unqualified.

[0149] As shown in Table 3, it can be seen that compared with Comparative Examples 4 to 5, the drugs prepared by the method of the present invention have high mixing uniformity and are easy to form, and the obtained tablets have smooth surface, low friability and high hardness. Especially after being placed for 24 hours, the indentation hardness of Comparative Example 4 is significantly reduced, which may be due to the fact that the moisture in the tablets is not completely dried, and the moisture is reduced during the placement process, resulting in a decrease in the hardness of the tablets.

[0150] Table 3

[0151]

[0152] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A nitrogen mustard derivative, characterized in that The chemical structure of the nitrogen mustard derivative is: 。 2. A method for preparing a nitrogen mustard derivative as claimed in claim 1, characterized in that: The following steps are involved: Step 1. Preparation: Clean and fully dry the instruments used; Step 2, feed reaction: N, N-di(2-chloroethyl)-1, 4-phenylenediamine hydrochloride and anhydrous DCM are mixed, stirred and dissolved at room temperature to form a suspension, triethylamine is added dropwise thereto, and the reaction is completed when the suspension is clarified to obtain an amine raw material solution; triphosgene is dissolved in anhydrous DCM at room temperature, and the amine raw material solution is added dropwise thereto under ice bath conditions, and then the ice bath is removed, and the reaction is carried out at room temperature for 30 to 50 min. After the conversion of N, N-di(2-chloroethyl)-1, 4-phenylenediamine into an intermediate is confirmed by TCL board, an anhydrous DCM solution of aniline raw material is added under ice bath conditions, and the reaction is carried out at room temperature for 10 to 14 h under nitrogen protection. After the conversion of the intermediate is confirmed by TCL board, the reaction is stopped; Step 3, post-treatment: add water to the reaction solution obtained in step 2 to remove the remaining triethylamine, extract with anhydrous DCM 2 to 4 times, collect and combine the organic layers, wash with saturated NaCl 2 to 4 times, dry with anhydrous Na2SO4 for 1 to 2 h, concentrate, purify on a silica gel column, and recrystallize to obtain a nitrogen mustard derivative; In the step 2, the aniline raw material is 3-fluoro-5-methoxyaniline; in the anhydrous DCM solution of the aniline raw material, the molar volume ratio of the aniline raw material to anhydrous DCM is 0.35-0.45 mmol: 4-6 mL; the molar volume ratio of the aniline raw material to the hydrochloride of N,N-di(2-chloroethyl)-1,4-phenylenediamine is 1-1.5: 1; the molar volume ratio of the hydrochloride of N,N-di(2-chloroethyl)-1,4-phenylenediamine and anhydrous DCM is 0.3-0.4 mmol: 2-4 mL; the molar volume ratio of the hydrochloride of N,N-di(2-chloroethyl)-1,4-phenylenediamine and triethylamine is 1: 1.2-1.

8.

3. The method for preparing a nitrogen mustard derivative according to claim 2, characterized in that: In the step 2, the molar volume ratio of triphosgene to anhydrous DCM is 0.1-0.2 mmol: 2-5 mL.

4. The method for preparing a nitrogen mustard derivative according to claim 2, characterized in that: In the step 2, the molar ratio of triphosgene to the hydrochloride of N,N-di(2-chloroethyl)-1,4-phenylenediamine is 0.3-0.5:

1.

5. Use of the nitrogen mustard derivative according to claim 1 in the preparation of anticancer drugs.

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