CN103351383B - 5-fluorouracil nitroxyl-free-radical anti-tumor drug - Google Patents

5-fluorouracil nitroxyl-free-radical anti-tumor drug Download PDF

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CN103351383B
CN103351383B CN201310321949.7A CN201310321949A CN103351383B CN 103351383 B CN103351383 B CN 103351383B CN 201310321949 A CN201310321949 A CN 201310321949A CN 103351383 B CN103351383 B CN 103351383B
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compound
fluorouracil
cancer
cell
follows
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CN103351383A (en
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贾艳艳
孙晓莉
文爱东
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Fourth Military Medical University FMMU
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Fourth Military Medical University FMMU
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Abstract

The invention discloses a compound expressed by a structural formula (I), wherein X can be NO2, OH, OCH3, halogens or H; Y can be oxygen or does not exist. The anti-tumor drug has the advantages as follows: the synthetic method of the compound is simple and convenient; each molecule of the compound contains a 5-fluorouracil structural unit and a nitroxyl free radical structural unit; the proliferation of the tumor cells of liver cancer, gastric cancer, breast cancer and the like can be obviously inhibited; besides, the anti-tumor drug has a synergistic effect.

Description

5 FU 5 fluorouracil nitroxyl radical anti-tumor medicaments
Technical field
The invention provides the novel 5 FU 5 fluorouracil nitroxyl radical anti-tumor medicaments of a class, belong to medical art.
Background technology
Malignant tumour is a class common disease of serious threat human health, has become the second largest factor of death in China's malignant tumour, and the annual whole world about has 7,000,000 people to die from cancer, accounts for 1/4th of total death toll.Chemotherapy is comparatively effective to malignant tumour, is the essential therapeutic arsenals of middle and terminal cancer patient and the methods for the treatment of that generally uses, in the treatment of cancer, plays irreplaceable effect.
At present, common clinical medicine mainly contains endoxan (CTX), methotrexate (MTX), Ro 2-9757, Zorubicin (ADM), pidorubicin (EPI), taxol, Docetaxel, platinum medicine etc.Though these medicines can reach the effect of certain Therapeutic cancer, but great majority can cause Neutrophilic granulocytopenia, gi tract syndromes and surrounding nerve toxic, to tumour cell and Normocellular selectivity poor, to kill or while inhibition tumor cell, also major injury normal cell, and directly affect the heart, liver, kidney and neural system.In addition, there is the problem such as poorly water-soluble and resistance toward contact in them.
Drug combination shows significant advantage in curative effect, but efficient also clinical less than 40%(single the efficient of medicine of combined chemotherapy is only 10-20%), and combined chemotherapy toxic side effect increases greatly, significantly limit the clinical application effect of chemotherapeutics.Increase dosage and administration time can improve the anti-tumor activity of medicine significantly, certainly will have a strong impact on healthy tissues, even cause " chemotherapy is dead ", thus cause chemotherapy to interrupt.Therefore, find effective cancer therapy drug and method is the important topic that world medicine faces, exploitation be efficient, the new drug of low toxicity, novel treatment malignant tumour, there is great theory significance and application prospect.
Summary of the invention
The object of the present invention is to provide a class contain can high-efficient cleaning except harmful free radicals " nitroxyl free radical " structural unit and there is the novel 5 FU 5 fluorouracil nitroxyl free radical compounds of 5 FU 5 fluorouracil structural unit of anti-tumor activity.
Another object of the present invention is the preparation method providing above-mentioned 5 FU 5 fluorouracil nitroxyl free radical compounds;
A further object of the invention is to provide the application of above-mentioned 5 FU 5 fluorouracil nitroxyl free radical compounds in preparation treatment antitumor drug, and described knurl medicine is cancer of the stomach, liver cancer, mammary cancer.
Implementation procedure of the present invention is as follows:
Compound shown in structural formula (I),
Wherein, X is selected from NO 2, OH, OCH 3, halogen or H, Y be oxygen or do not exist.
The synthetic method of compound shown in structural formula (I), synthetic route is as follows:
。
Synthetic route can also be as follows:
。
The synthetic method of compound shown in structural formula (I), synthetic route is as follows:
。
Synthetic route can also be as follows:
Above-mentioned condensing agent is 1,3-dicyclohexylcarbodiimide (DCC), 1,3-DIC (DIC) or 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC).
The application of compound shown in structural formula (I) in the antitumor monomer medicine of preparation or pharmaceutical composition, described medicine is tablet, capsule, powder, pill, granule, emulsion or injection.
Nitroxyl free radical is a kind of free radical with unique antioxidant property of discovered in recent years.Research confirms, activity in vivo oxygenate kind (ROS) increase and the change of cellular anti-oxidant enzyme can induced tumor, " principle of hybridization " of the present invention's foundation " antioxidant compound chain " theory and medicinal design, the nitroxyl free radical structural unit of harmful free radicals can be removed and there is the 5 FU 5 fluorouracil combination of highly effective antineoplastic activity by high-efficient cleaning, build the new type antineoplastic medicine structure of series structure novelty, complex function, anti-oxidant, the antitumor synergy of the two can be given full play to.5 FU 5 fluorouracil nitroxyl free radical compounds of the present invention has very high anti-tumor activity, proved by inside and outside pharmacodynamics test, such medicine has obvious restraining effect to liver cancer, cancer of the stomach, Cells Proliferation of Human Breast Cancer, have good anti-tumor activity, and resistance is better.
Accompanying drawing explanation
Fig. 1 is the inhibited proliferation of compound 1 pair of SMMC-7721 cell;
Fig. 2 is that Tunel method surveys cell in-situ apoptosis picture;
Fig. 3 is the anti-cancer of the stomach MKN45 cell proliferation experiment of compound 2 ~ 5;
Fig. 4 is the anti-MDA-MB-231 cell proliferation experiment of compound 6 ~ 10.
Embodiment
Specifically, can synthesize according to the inventive method and obtain following structural compounds.
Method 1:
Method 2:
According to similar route of synthesis, can also synthesize and obtain following representative configurations:
The compound of general formula (I) synthesizes as follows, changes substituting group and can obtain series derivates.
According to similar route of synthesis, can also synthesize and obtain following representative configurations:
embodiment 1
By 3.5 g(62.5 mmo1) KOH and 40 mL water are placed in there-necked flask, stirring and dissolving.Add 2.0 g (15.4 mmo1) 5 FU 5 fluorouracil, be warming up to 60 DEG C.Drip containing 2.3 g (21.6 mmol) the chloroacetic aqueous solution, TLC detects, and being cooled to room temperature after completion of the reaction, is 5 by concentrated hydrochloric acid adjust ph, elimination precipitates, adjust pH to 2, refrigerated overnight, suction filtration, throw out distilled water (20 mL × 3) washing 3 times, dry white crystal intermediate, weigh 2.46 g, productive rate 81%.
At N 2under protection; 1.5 g (12 mmo1) p-Hydroxybenzaldehyde, 0.1 g (5.8 mmol) anhydrous tosic acid, 15 mL pyridines are added in there-necked flask; 0 DEG C is stirred 0.5 h; add the DMF solution of 7 mL containing 2.5 g (12 mmol) DCC; under ice bath, drip the 10 mL dry pyridine solution containing step intermediate on 1.9 g.Finish, stirring at room temperature 36 h, add 10 mL Glacial acetic acid, stir 1 h, ice bath hold over night.Filter, with pyridine washing (10 mL × 2) washing, merge organic phase, stir in lower impouring frozen water, separate out white solid precipitation.Filter, distilled water wash, dry, Gossypol recrystallized from chloroform 2 times, obtains white solid product , drydry 1.4 g that weigh, productive rate 72.9%.
By 1.00 g(4.0 mmol) on walk solid product, 0.84 g(5.6 mmol) two azanol is placed in 100 mL single necked round bottom flask, adds 30 mL dissolve with methanol, stirred at ambient temperature reacts, and decompression is spin-dried for solvent, obtains faint yellow solid.Under ice-water bath, with 40 mL CH 2cl 2middle dissolved solids (being not easily long placed in), drips NaIO 4saturated aqueous solution, stop after reacting 10 min, the anhydrous MgSO of organic phase 4drying, spends the night, and Rotary Evaporators is except desolventizing, and column chromatography purification obtains blue solid target product 1, weigh 1.33g, productive rate 88.5%.HRMS (m/z): 420.323, [M+H] +; G=2.0096, | a n|=7.83G ultimate analysis: Found:C, 54.41; H, 4.81; N, 13.36 Calc. for C 19h 20fN 4o 6, C, 54.32; H, 4.96; N, 13.44%.
embodiment 2
By 3.5 g(62.5 mmo1) KOH and 40 mL water are placed in there-necked flask, stirring and dissolving.Add 2.0 g(15.4 mmo1) 5 FU 5 fluorouracil, be warming up to 60 DEG C.Drip containing 2.3 g (21.6 mmol) the chloroacetic aqueous solution, TLC detects, and being cooled to room temperature after completion of the reaction, is 5 by concentrated hydrochloric acid adjust ph, elimination precipitates, adjust pH to 2, refrigerated overnight, suction filtration, throw out distilled water (20 mL × 3) washing 3 times, dry white crystal intermediate, weigh 2.46 g, productive rate 81%.
At N 2under protection; by 1.75 g(12 mmo1) 3-fluoro-4-hydroxy benzaldehyde, 0.1 g (5.8 mmol) anhydrous tosic acid, 15 mL pyridines add in there-necked flask; 0 DEG C is stirred 0.5 h; add the DMF solution of 7 mL containing 2.5 g (12 mmol) DCC; under ice bath, drip the 10 mL dry pyridine solution containing step intermediate on 1.9 g.Finish, stirring at room temperature 36 h, add 10 mL Glacial acetic acid, stir 1 h, ice bath hold over night.Filter, with pyridine washing (10 mL × 2) washing, merge organic phase, stir in lower impouring frozen water, separate out white solid precipitation.Filter, distilled water wash, dry, Gossypol recrystallized from chloroform 2 times, obtains white solid product , drydry 3.0 g that weigh, productive rate 78.9%.
By 1.00 g(4.0 mmol) on walk solid product, 0.84 g(5.6 mmol) two azanol is placed in 100 mL single necked round bottom flask, adds 30 mL dissolve with methanol, stirred at ambient temperature reacts, and decompression is spin-dried for solvent, obtains faint yellow solid.Under ice-water bath, with 40 mL CH 2cl 2middle dissolved solids (being not easily long placed in), drips NaIO 4saturated aqueous solution, stop after reacting 10 min, the anhydrous MgSO of organic phase 4drying, spends the night, and Rotary Evaporators is except desolventizing, and column chromatography purification obtains target product 4, weigh 1.33g, productive rate 88.5%.HRMS (m/z): 437.22，[M+H] +；g=2.0045，|a N| =7.69G。Ultimate analysis: Found:C, 52.18; H, 4.38; N, 12.81 Calc. for C 19h 19f 2n 4o 6, C, 52.23; H, 4.40; N, 12.75%.
embodiment 3
By 2.0 g(15.4 mmo1) 5 FU 5 fluorouracil, 3.5 g(62.5 mmo1) KOH and 2.3 g(15.4 mmo1) terephthalaldehydic acid and 40 mL water are placed in there-necked flask, stirring reaction.TLC detects, and suction filtration after completion of the reaction, organic phase column chromatography purification obtains white crystal intermediate, and weigh 3.5 g, productive rate 87%.
By 1.00 g(4.0 mmol) on walk solid product, 0.84 g(5.6 mmol) two azanol is placed in 100 mL single necked round bottom flask, adds 30 mL dissolve with methanol, stirred at ambient temperature reacts, and decompression is spin-dried for solvent, obtains faint yellow solid.Under ice-water bath, with 40 mL CH 2cl 2middle dissolved solids (being not easily long placed in), drips NaIO 4saturated aqueous solution, stop after reacting 10 min, the anhydrous MgSO of organic phase 4drying, spends the night, and Rotary Evaporators is except desolventizing, and column chromatography purification obtains target product 6, weigh 1.02g, productive rate 65.8%.HRMS (m/z): [M+H] + 389.10；g=2.0067，|a N| =7.90G。Ultimate analysis: Found:C, 55.53; H, 4.66; N, 14.39 Calc. for C 18h 18fN 4o 5, C, 55.61; H, 4.62; N, 14.47%.
embodiment 4
By 2.0 g(15.4 mmo1) 5 FU 5 fluorouracil, 3.5 g(62.5 mmo1) KOH and 3.0 g(15.4 mmo1) 4 formyl radical-2 nitrobenzoic acids and 40 mL water are placed in there-necked flask, stirring reaction.TLC detects, and suction filtration after completion of the reaction, organic phase column chromatography purification obtains white crystal intermediate, and weigh 4.1 g, productive rate 86%.
By 1.23g(4.0 mmol) on walk solid product, 0.84 g(5.6 mmol) two azanol is placed in 100 mL single necked round bottom flask, adds 30 mL dissolve with methanol, stirred at ambient temperature reacts, and decompression is spin-dried for solvent, obtains faint yellow solid.Under ice-water bath, with 40 mL CH 2cl 2middle dissolved solids (being not easily long placed in), drips NaIO 4saturated aqueous solution, stop after reacting 10 min, the anhydrous MgSO of organic phase 4drying, spends the night, and Rotary Evaporators is except desolventizing, and column chromatography purification obtains target product 8, weigh 1.20g, productive rate 70.5%.HRMS (m/z): [M+H] + 434.25；g=2.0071，|a N| =7.85G。Ultimate analysis: Found:C, 49.77; H, 3.94; N, 16.12 Calc. for C 18h 17fN 5o 7, C, 49.68; H, 3.96; N, 16.50%.
embodiment 5: the antitumor pharmacodynamic experiment of compound 1
1. in vitro tests
(1) experimental technique
Liver cancer cell SMMC-7721 (this school physiopathology teaching and research room) cell is inoculated in the RPMI RPMI-1640 containing 100 mL/L newborn calf serums, puts 50 mL/L CO 2in incubator, 37 DEG C of cultivations, the cell of logarithmic phase is all selected in all experiments.The cell adjusting density is joined 96 well culture plates, every hole 6000-10000 cell, liquid is changed after cultivating 24 h, the medicine adding different concns (is diluted to different final concentrations with 1640 complete culture solutions containing 100 mL/L calf serums: 10 μ g/mL, 20 μ g/mL, 40 μ g/mL, 80 μ g/mL, 160 μ g/mL), every concentration all makes 6 parallel holes, separately establish blank well and control wells, during drug effect, with the growing state of inverted phase contrast microscope observation of cell, after acting on 24 h, every hole adds the MTT 20 μ L of 5 g/L, cultivate 4 h, abandon supernatant liquor, add DMSO about 100 μ L/ hole, vibrate 10 min, purple crystal thing is fully dissolved, the absorbance A in each hole is measured by microplate reader 570nm.
(2) test-results
MTT result display (Fig. 1), compound 1concentration be 20 μ g/mL, 40 μ g/mL, 80 μ g/mL, have obvious antiproliferative effect effect to SMMC-7721 cell during 160 μ g/mL.
From table 1, compound 1all restraining effect is had to different tumour cells, compared with Docetaxel, compound 1particularly remarkable to the growth inhibition effect of breast cancer cell MDA-MB-231 and stomach cancer cell MKN-45, MKN-28, and the murder by poisoning of normal cell GES is affected lower than Docetaxel.
2. experiment in vivo
(1) experimental technique
Logarithmic phase human breast carcinoma MDA-MB-231 cell, adjustment cell concn is 1-2 × 10 7cells/mL, it is subcutaneous that No. 6 needle injection are inoculated in nude mice femoribus internus, every only inoculation 0.2 mL.Inoculate 30 nude mices, lotus knurl success after two weeks, tumor average volume 122 ±11 mm 3in time, is used for testing.
The detection of tumor control rate
Select the nude mice 24 that tumor size difference is little, random packet, often organize 3, be divided into blank group, 1group (40 mg/kg), Docetaxel positive controls (40 mg/kg).Adopt knurl body local injection method, medication 1 time in every two days, each medication 50 μ L experimental period is two weeks.After experiment terminates, put to death all nude mices with cervical dislocation, dissect rapidly and win knurl block, measurement volumes.
TUNEL method in situ detection apoptosis
TUNEL in situ detection apoptosis kit (purchased from BD company) is adopted to detect apoptosis situation.
(2) experimental result
The detection of tumor control rate
Experimental result shows, compound 1, Docetaxel positive controls all shows the effect of comparatively obvious Tumor suppression growth.When the 12nd day, gross tumor volume difference is obvious.
As seen from Table 3, compound 1experimental group has good restraining effect to tumour, with in Docetaxel, low dose group there was no significant difference ( p>0.05).This experiment proves 1can increase by Tumor suppression preferably in animal level.
TUNEL method in situ detection cell apoptosis assay the results are shown in Figure 2.
Analyze from In situ cell apoptosis rate detected result, compound 1experimental group and Docetaxel positive controls all can cell death inducings, have compared with blank group significant difference ( p<0.05).
embodiment 6: the anti-cancer of the stomach MKN45 cell proliferation experiment of compound 2 ~ 5
Test working method with embodiment 5, experimental result as shown in Figure 3.In Fig. 3, data declaration compound 2 ~ 5(concentration is 40 μ g/mL) all there is certain restraining effect to people cancer of the stomach MKN45 cell proliferation.
embodiment 7: the anti-MDA-MB-231 cell proliferation experiment of compound 6 ~ 10
Examination on experimental operation is with embodiment 5, and experimental result as shown in Figure 4.Data declaration compound in Fig. 4 6~ 10(concentration is 40 μ g/mL) all has certain restraining effect to MDA-MB-231 cell proliferation.
embodiment 8: compound 1 anti-MDA-MB-231 cell proliferation experiment and existing nitroxyl free radical results contrast
By the IC of the anti-cancer of the stomach MDA-MB-231 cell proliferation experiment of upper table 50be worth visible, novel 5 FU 5 fluorouracil nitroxyl free radical of the present invention has and has better antitumor activity than the medicine of existing synthesis.

Claims (8)

1. the compound shown in structural formula (I),
Wherein, X is selected from NO 2, OH, OCH 3, halogen or H, Y be oxygen or do not exist.
2. the synthetic method of compound described in claim 1, is characterized in that synthetic route is as follows:
。
3. the synthetic method of compound according to claim 1, is characterized in that synthetic route is as follows:
。
4. the synthetic method of compound described in claim 1, is characterized in that synthetic route is as follows:
。
5. the synthetic method of compound according to claim 1, is characterized in that synthetic route is as follows:
。
6. according to the synthetic method of one of any described compound of claim 2 to 5, it is characterized in that: described condensing agent is 1,3-dicyclohexylcarbodiimide, 1,3-DIC or 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide.
7. the application of compound described in claim 1 in the antitumor monomer medicine of preparation or pharmaceutical composition.
8. apply according to claim 7, it is characterized in that described medicine is tablet, capsule, powder, pill, granule, emulsion or injection.
CN201310321949.7A 2013-07-29 2013-07-29 5-fluorouracil nitroxyl-free-radical anti-tumor drug Expired - Fee Related CN103351383B (en)

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