CN102532192B

CN102532192B - Acetaminophen-cyclophosphamide anti-tumor drug and preparation method thereof

Info

Publication number: CN102532192B
Application number: CN201110434041.8A
Authority: CN
Inventors: 宋杨; 宋尔群; 李效尧
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2011-12-22
Filing date: 2011-12-22
Publication date: 2014-10-15
Anticipated expiration: 2031-12-22
Also published as: CN102532192A

Abstract

The invention provides a chlormethine compound with double activity function groups and application of the chlormethine compound serving as an anti-tumor drug. A structure of the compound is shown in the specification. In-vitro anti-tumor experiments prove that the compound has good inhibiting effect on liver cancer cells of the human body. In-vitro deoxyribose nucleic acid (DNA) crosslinking experiments prove that the compound has good crosslinking effects on plasmid DNA. Mouse analgesia experiments prove that the compound has good analgesia effects. The chlormethine compound is simple in synthesizing steps and good in anti-tumor effects, possibly simulates the analgesia effects of acetaminophen in vivo, is an anti-tumor drug with double functions, and has great medical value.

Description

A kind of acetaminophen-cyclophosphamide anti-tumor drug and preparation method thereof

Technical field

Field of medicaments involved in the present invention, a kind of antineoplastic compound of paracetamol-Glyciphosphoramide derivative.

Background technology

Mustargen is the earliest for clinical and obtain the alkylating agent series antineoplastic medicament of outstanding curative effect, has highly active character.Because chlorine atom in chloroethyl is easily sloughed formation carbonium ion, more further there is annulation in molecule, the ethyleneimine ion that height of formation is active.Mustargen can react with macromolecular nucleophilic group in other bodies, as the amino of the carboxyl of protein, amino, sulfydryl, nucleic acid and hydroxyl, phosphate radical etc., carries out alkylation modification.Mustargen easily with the 7th nitrogen covalent attachment of guanine, the cross bracing that produces the interior different bases of same chain of cross bracing in the two strands of DNA or DNA.

Mustargen is cell cycle nonspecific agent (CCNSA), and poor selectivity during as antitumor drug, exists severe side effect, has affected clinical use.So the nitrogen mustards antitumor drug of design low toxicity becomes the focus of scientific research.On nitrogen-atoms, introduce aromatic group and form aromatic chlorethazine medicine, due to lone-pair electron and the phenyl ring conjugation of nitrogen-atoms, weakened the alkalescence of nitrogen-atoms, more difficult formation ring-type ethylenimine, plays a role by losing chlorine atom formation carbonium ion intermediate.

Tumour patient is accompanied by violent, lasting pain conventionally.Cancer pain is mainly the hypertrophy due to cancerous tissue, and pressuring nerve or nerve ending cause.In addition, cancerous tissue infection, ulcer, necrosis etc. also all can cause pain.The control of patient's pain is contributed to improve the quality of life of tumour patient and extend lifetime.

To the control of tumour patient pain, mainly use analgesic agent and the auxiliary medicines such as anxiety, anti-melancholy that use to improve other symptoms of cancer, Postoperation effect.Wherein Nonopioids is widely used acetamido phenol sheet.

Summary of the invention

The object of the invention is to invent a kind of aromatic chlorethazine medicine, its molecule contains paracetamol structure, and it can effectively reduce toxicity, in antineoplastic while, produces the similar analgesic activity of paracetamol, increases the scope of application.

Acetyl aminophenol-Glyciphosphoramide derivant structure that the present invention proposes is as follows:

Its constitutional features is that Glyciphosphoramide is in conjunction with 1-2 paracetamol.

The preparation method of described compound comprises two-step reaction,

The first step: adopt two Dichloroethyl amine hydrochlorates (1) and POCl ₃(2) reaction, prepares Glyciphosphoramide (3);

Second step: adopt Glyciphosphoramide (3) to react with paracetamol (4), prepare acetyl aminophenol-Glyciphosphoramide;

Detailed process is as follows:

Medicine synthetic method of the present invention is simple, is applicable in enormous quantities generation.

The present invention further proposes to protect the purposes of above-claimed cpd in preparing antitumor drug; especially the application in the medicine of preparation treatment leukemia, lung cancer, liver cancer, mammary cancer, ovarian cancer, cervical cancer, prostate cancer, and the application in preparation treatment cancer patients analgesic.

By anticancer experiment in vitro, find, the compounds of this invention has good restraining effect to human liver cancer cell, by the crosslinked experiment of external DNA, finds that it has good crosslinked action to plasmid DNA, finds that by mouse analgesic experiment it has good analgesic effect.Synthesis step of the present invention is simple, and antitumous effect is good, and may simulate in vivo the analgesic activity of paracetamol, is a kind of bifunctional antitumor drug that has, and has great pharmaceutical use.

Can be synthetic easily by chemical way.It demonstrates good anti-tumor activity and analgesic effect.

Accompanying drawing explanation

Fig. 1 is the gel electrophoresis figure of the crosslinked plasmid pBR322 of acetyl aminophenol-Glyciphosphoramide.

Embodiment

The invention will be further described by the following examples.

Embodiment 1: compound 5 synthetic

Step 1: the preparation of compound 3

The new POCl steaming of two Dichloroethyl amine hydrochlorates (1) (20g, 0.112mol) and 52mL (0.60mol) ₃(2) be placed in the round-bottomed flask of 200mL, 110 ℃ of backflow 20h are to the two Dichloroethyl amine hydrochlorates disappearances of solid.Excessive POCl is removed in decompression ₃the solid residue of gained adds acetic acid ethyl dissolution, crosses and filters out insoluble substance, concentrated ethyl acetate filtrate, filtrate acetone: sherwood oil (v/v=1: 5) recrystallization, obtain clear crystal, Glyciphosphoramide dichloro 3 is total to 18.0g, yield 61%, product structure is through fusing point, IR, NMR, MS and ultimate analysis are determined.

Step 2: the preparation of compound 5

Paracetamol (4) (151mg, 1mmol) be dissolved in 20mL methyl alcohol, in 0 ℃, add triethylamine (185mL, 1.34mmol) to stir, the Glyciphosphoramide dichloro (3) (0.13mg, 0.5mmol) that is dissolved in 10mL methyl alcohol is added to reaction system.In room temperature reaction 48h, methyl alcohol is removed in decompression, obtains solid residue.Add again ethyl acetate again to dissolve, filter and collect filtrate, concentrated filtrate.Crude product methyl alcohol: methylene dichloride (v/v=1: 10) cross silicagel column separation, obtain 100mg compound 5 (productive rate 20.5%).Product structure is through fusing point, IR, and NMR, MS and ultimate analysis are determined.

Embodiment 2: anticancer experiment in vitro

Select compound 5 of the present invention to test its cytotoxicity to cell strain HepG-2, A549 and HL-60, adopt mtt assay.

1, compound 5 is configured to different concns with RPMI-1640 nutrient solution respectively, 4 ℃ of preservations.

2, cancer cells is incubated at containing 10% calf serum, in the RPMI-1640 nutrient solution of 100kU/L penicillin and 100mg/L Streptomycin sulphate, is placed on 5%CO ₂constant temperature culture in saturated humidity cell culture incubator.

The cancer cells of 3, taking the logarithm vegetative period, adds 5.0 * 10 in 96 orifice plates ⁴individual/mL cell suspension 100 μ L.

4, blank and endoxan (CP) are set simultaneously, 5 FU 5 fluorouracil (5-Fu) positive control is empty, blank group of nutrient solution that adds same volume, and positive controls adds 4 μ mol/L, and each concentration is established eight parallel holes.

5, continue to cultivate after 48h, every hole adds MTT (5g/L, 20 μ L), continues to cultivate 5h.

6, nutrient solution (as far as possible completely) in sucking-off hole, adds DMSO liquid (100 μ L/ hole), puts on 37 ℃ of vibrators and shakes up 15min, and crystal is fully dissolved.

7, use microplate reader under wavelength 490nm, survey OD value.

From table-1, can find out, compound 5 has good anti-tumor activity.

Table-1: medicine (5) anti-tumor activity test result (mtt assay)

Embodiment 3: medicine and DNA are crosslinked

Select compound 5 of the present invention to detect its external crosslinking activity to plasmid pBR322DNA, adopt alkaline gel electrophoresis method.

1, prepare linear pBR322DNA.Prepare the mixture (totally 200 μ L) of following system: the pBR322DNA of superhelix type (10 μ L; 10 μ g); restriction enzyme EcoR I (4 μ L; 40-80u); 10 times of EcoR I buffered soln (20 μ L); acetylize BSA (20 μ L, 1mg/mL), intermediate water (146 μ L).Said mixture is cultivated to 3 hours in 37 ℃ of constant temperature water tanks, then add sodium-acetate (20 μ L, 3M) and ice ethanol (440 μ L) ,-20 ℃ of standing over night.Take out mixture, with 16,000 revs/min centrifugal 15 minutes, pour out ethanol, be inverted several minutes to vapor away residual ethanol.Add 30 μ L intermediate waters standby.Concentration approximately 0.25 μ g/ μ L.

2, in the EP of 0.2mL pipe, add linearizing pBR322DNA 2 μ L (0.25 μ g/ μ L), then add a certain amount of compound 5 solution, add the TE damping fluid of pH=8, regulate cumulative volume to 10 μ L, solution is mixed.

3, EP pipe is placed in 37 ℃ of constant temperature water tanks, hatches 50 minutes.

4,, after hatching, in each EP pipe, add 1 μ L sample loading buffer.

5, sample is placed on 0.9% sepharose, 40V voltage, under 120mA electric current, electrophoresis is approximately 2 hours, to the about 5cm of bromjophenol blue band migration, stops electrophoresis.Then gel is proceeded to solution (1M Tris pH=7/1.5MNaCl) inner in and 30min.

6, alkaline glue being placed in to EB staining fluid dyes 20 minutes.

7, take out, use clear water rinsing, imaging.

By the crosslinked experiment of figure-1 linear pBR322DNA, visible compound 5 has the ability of certain crosslinked DNA, judges that thus its mechanism that causes cytotoxicity and anti-tumor activity may be DNA crosslinked action.

Embodiment 4: medicine analgesic experiment

Get female body weight 18-22g female mice, by hot plate pain threshold detector induced pain (55.0 ± 0.5) ℃, the mouse of usining licks metapedes as pain indicator reaction.Before experiment, select in advance eligible, take and be no more than 60s latent period as qualified.Qualified mouse is divided into 5 groups at random, 10 every group: blank group (giving the physiological saline of same volume), paracetamol group (80mg/kg), the large, medium and small dosage group of compound 5 (40mg/kg, 80mg/kg and 160mg/kg).After tail vein injection administration, mouse is dropped in hot-plate instrument, recorded after mouse is dropped into hot-plate instrument to the time (s) that occurs licking metapedes, as the threshold of pain of this mouse.

Record after administration 30 and threshold of pain during 60min, the relatively difference of the threshold of pain between each group.

From table-2, can find out that compound 5 has good analgesic activity.

Table-2: the impact of compound 5 on the effect of mouse hot-plate analgesia

* compare with physiological saline group, equal p < 0.05 is organized in each metering of paracetamol group and medicine.

Experimental results show that medicine of the present invention has obvious restraining effect to tumor cell line, can be for the preparation of the medicine for the treatment of malignant cancer; Meanwhile, this medicine can effectively improve the pain threshold values of mouse, at antineoplastic analgesic activity of bringing into play simultaneously, increases the scope of application.

Claims

1. an anti-tumor compounds, has following structural formula, and its constitutional features is Glyciphosphoramide in conjunction with 2 paracetamol:

2. the purposes of compound as claimed in claim 1 in preparing antitumor drug.

3. the application of compound as claimed in claim 1 in the medicine of preparation treatment leukemia, lung cancer, liver cancer, mammary cancer, ovarian cancer, cervical cancer, prostate cancer.

4. the application of compound as claimed in claim 1 in preparation treatment cancer patients analgesic.