CN103626776A - Pyrazolo[1,5-alpha] pyrimidine nitrogen mustard derivatives, preparation methods of pyrazolo[1,5-alpha] pyrimidine nitrogen mustard derivatives, and application of pyrazolo[1,5-alpha] pyrimidine nitrogen mustard derivatives in oncotherapy - Google Patents
Pyrazolo[1,5-alpha] pyrimidine nitrogen mustard derivatives, preparation methods of pyrazolo[1,5-alpha] pyrimidine nitrogen mustard derivatives, and application of pyrazolo[1,5-alpha] pyrimidine nitrogen mustard derivatives in oncotherapy Download PDFInfo
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- C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
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Abstract
The invention relates to pyrazolo[1,5-alpha] pyrimidine nitrogen mustard derivatives or medical salts thereof, as well as application of the pyrazolo[1,5-alpha] pyrimidine nitrogen mustard derivatives or the medical salts thereof. The pyrazolo[1,5-alpha] pyrimidine nitrogen mustard derivatives and the medical salts thereof have the structure shown as the formula I. Pharmacological experiments show that the pyrazolo[1,5-alpha] pyrimidine nitrogen mustard derivatives and the medical salts thereof have inhibiting effects on the proliferation of various tumor cells. Moreover, the pyrazolo[1,5-alpha] pyrimidine nitrogen mustard derivatives are small in toxicity, have the advantages of selectivity on tumor cells, and are dual-functional anti-tumor drugs. Meanwhile, the pyrazolo[1,5-alpha] pyrimidine nitrogen mustard derivatives are easy to synthesize, and the overall yields are high. All the advantages show that the pyrazolo[1,5-alpha] pyrimidine nitrogen mustard derivatives have great potential of being anti-tumor drugs.
Description
Technical field
The present invention relates to new pyrazolo [1,5-a] pyrimidine nitrogen mustards derivative and preparation method thereof and as the application of anti-tumor medicine.
Background technology
Chlormethine series pharmaceuticals belongs to cytotoxic drug, is to be applied to the earliest clinical non-structure specificity anti-tumor biological alkylating agent.Since coming out, chlormethine series pharmaceuticals research and development are active, having brought into play vital role aspect treatment tumour, are at home and abroad widely used.But nitrogen mustards compound can produce many serious side reactions in inhibition and killing tumor cell, as nauseating, vomiting, bone marrow depression, alopecia etc.Therefore, how to reduce the untoward reaction that nitrogen mustards compound exists, improve therapeutic efficiency, reduce its toxicity and increase the study hotspot that the problems such as its selectivity become pharmaceutical field, attract and inspiring countless researchers to be devoted to the research and development of this type of medicine.Nitrogen mustards antineoplastic compound generally comprises alkylation part and carrier part.By can improve the pharmacokinetics character such as the absorption in vivo of this type of medicine, distribution to the modification of carrier part, thereby affect its anti-tumor activity.At present the research of nitrogen mustards compound is roughly concentrated on to three aspects: the one, by introduce sugar and derivative thereof in drug molecule, the 2nd, in drug molecule, introduce the acid of nitrogen base or little peptide, the 3rd, in drug molecule, introduce tumoraffin heterocycle.Wherein, most study be the 3rd aspect, introduce tumoraffin heterocycle the carrier part of nitrogen mustards compound carried out to structural modification, to reach, improve selectivity and fall hypotoxic object.
Cell cycle deopendent protein kinase (Cyclin-dependent kinases, CDKs) is one group of serine/threonine protein kitase, and its active accuracy controlling is to the propagation of cell and transcribe most important.By suppressing the activity of CDKs, just can reach the object of the proliferative diseases such as treatment cancer.CDK
2being a member in CDKs family, has been G
1phase and a very crucial cell cycle deopendent protein kinase that enters the S phase have important effect in the cell cycle.This enzyme not only can, with cyclin E (CyclinE) in conjunction with also activation, maintain G
1later stage pRb phosphorylation, makes cell pass through smoothly G
1phase enters the S phase, can also, with cyclin A (CyclinA) keying action in the initial phase of S of cell cycle, make the passivation of E2F transcription factor, and then cause the cell S phase to be stagnated or apoptosis, and likely act on the G of cell cycle
2phase is an important oncotherapy target spot of generally acknowledging at present.CDK
2crystalline structure in 1993, be confirmed, along with the continuous understanding of people to its protein structure, the competitive small molecules CDK of increasing ATP
2the studied report of inhibitor.In recent years to CDK
2have the micromolecular inhibitor of selectivity inhibition as Flavopiridol, R-roscovitine (CYC202), 7-hydroxyl staurosporine (UCN-01) and aminothiazole compounds (BMS-387032) etc. have entered clinical study, being expected to is providing more selection in the future for clinicist.
Pyrazolo [1,5-a] pyrimidines is the novel CDK of a class
2inhibitor is one of study hotspot of antitumor drug in recent years.The female ring of this compounds is by a pyrazole ring and a fused heterocycle that pyrimidine ring forms, hydrogen and CDK in its 7 bit amino
2in skeleton between the carbonylic oxygen atom of Leu83,1 nitrogen-atoms and the CDK of pyrazole ring
2in skeleton between the amino hydrogen atom of Leu83,2 hydrogen atoms and the CDK of pyrazole ring
2in skeleton, between the carbonylic oxygen atom of Glu81, all can form hydrogen bond.Due to CDK
2in tumour cell, there is overexpression, as CDK
2the pyrazolo of inhibitor [1,5-a] pyrimidines is, after carrier is connected with nitrogen mustards Compound Phase, to have it to become the Potential feasibility of specificity and targeting tumour.So far, about the nitrogen mustards compound that is carrier with pyrazolo [1,5-a] pyrimidines, as the research of antitumor drug, there is not yet report, this also makes this invention have higher novelty.
Summary of the invention
The invention provides new pyrazolo [1,5-a] pyrimidine nitrogen mustards derivative or its pharmacologically acceptable salt, it is characterized in that: one end has mustargen alkanisation group; The other end has also [1,5-a] pyrimidine of precursor structure 3-cyano pyrazole, its 5 substd R
1, be respectively methyl, chloromethyl, its 7 amidos are connected with substituent R by phenyl ring, and R is the mustargen group of 3-, 4-.They show less toxicity in and selectivity and body inhibited to the propagation of tumour cell, have the great potential that becomes antitumor drug.Structure is suc as formula I:
Wherein:
R
1be the heteroaryl that replaces of alkyl, haloalkyl, alkoxyl group, aryl, heteroaryl, alkyl, heterolipid cyclic group or-NR
4r
5;
R
4and R
5independently selected from hydrogen, alkyl, cycloalkyl, aryl or heteroaryl, or R
4and R
5form altogether a heteroaryl or heterolipid cyclic group;
R is N (CH
2cH
2cl)
2.
Above-claimed cpd or its pharmacologically acceptable salt, structure is suc as formula II:
Wherein:
R
1alkyl or haloalkyl;
R
2=N (CH
2cH
2cl)
2, R
3=H, or R
2=H, R
3=N (CH
2cH
2cl)
2.
Its concrete synthesis step comprises three parts, and route is as follows:
Pyrazolo [1,5-a] pyrimidine female ring synthetic route:
Lymphoquin synthetic route:
Target compound synthetic route:
The synthesis step of route A
1. ethoxy methylene the third two eyeballs is synthetic
The third two eyeball 9.9g (0.15mol), triethyl orthoformate 33.3g (0.23mol) and acetic anhydride 38.4g (0.38mol) are joined in 250mL single port flask.After back flow reaction 6h, cooling, add gac post-heating backflow 30min, heat filtering, with after hot ethanol washing leaching cake, filtrate is placed in refrigerator overnight, and suction filtration is dried to obtain faint yellow solid 15.4g, productive rate 84.3%.
Synthesizing of 2.3-amino-4-cyano pyrazole
Ethoxy methylene the third two eyeball 15g (0.12mol) at room temperature slowly join in 85% hydrazine hydrate 12mL (0.25mol), and heating in water bath for reaction 1h adds 10mL water in reaction system.In refrigerator, place and spend the night, suction filtration, washing filter cake, dries to obtain faint yellow solid 10.6g, productive rate 81.7%.
3.3-cyano group-5-(chlorine) methyl-7-hydroxypyrazoles is synthesizing of [1,5-a] pyrimidine also
3-amino-4-cyano pyrazole 4g (37mmol) and (chlorine) methyl aceto acetate (38mmol) are added in 33mL glacial acetic acid to back flow reaction 4h.Be cooled to after completion of the reaction room temperature, filter, with Glacial acetic acid and water wash filter, obtain yellow solid successively.By re-crystallizing in ethyl acetate, obtain sterling, productive rate 80-89%.
Synthesizing of 4.3-cyano group-5-(chlorine) methyl-7-chlorine pyrazolo [1,5-a] pyrimidine
In the four-necked bottle of 250mL, add 3-cyano group-5-(chlorine) methyl-7-hydroxypyrazoles also [1,5-a] after pyrimidine (9.95mmol) and pyridine 0.82mL (10.55mmol), slowly drip phosphorus oxychloride 4.05mL (43.2mmol), finish after being slowly heated to 85 ℃ and start to stir, in 120 ℃ of reaction 1h.After below being cooled to 60 ℃, add chloroform 150mL.Back flow reaction 1h.While being cooled to 0-5 ℃, add cold water 90mL, suction filtration is removed insolubles, separates organic phase, washes twice, each water 90mL.Revolve after chloroform is removed in steaming and obtain solid 3-cyano group-5-(chlorine) methyl-7-chlorine pyrazolo [1,5-a] pyrimidine, productive rate 81-87%.
The synthesis step of route B
1.N, N-bis-(2-hydroxyethyl)-4-N-methyl-p-nitroaniline synthetic
Get 4-chloronitrobenzene 10.34g (0.07mol), diethanolamine 17.56g (0.17mol) mixes in 250mL round-bottomed flask, and 120 ℃ are stirred 6h, pour into while hot in 90 ℃ of hot water, fully stir.Filtered while hot, standing rear filtrate is separated out solid, suction filtration, filter cake is with obtaining yellow solid 5.46g with ethyl alcohol recrystallization, productive rate 36.3% after ether washing.
2.N, N-bis-(2-hydroxyethyl)-3-N-methyl-p-nitroaniline synthetic
Get 3-N-methyl-p-nitroaniline 5.52g (0.04mol), add in the aqueous acetic acid of 80mL25%, under ice bath, drip oxyethane 10mL.Be warming up to room temperature, stir 24h, suction filtration, washes filter cake with water, obtains orange/yellow solid 8.02g, productive rate 88.7% after oven dry.
3.N, N-bis-(2-methanesulfonates ethyl) N-methyl-p-nitroaniline synthetic
Get N, N-bis-(2-hydroxyethyl) N-methyl-p-nitroaniline 2.26g (0.01mol), adds in the methylene dichloride that 30mL is dry, under ice bath, adds 3.32mL (0.025mol) triethylamine, after add methylsulfonyl chloride 1.93mL (0.025mol).TLC detects, and after 10min, reacts completely.Reaction solution is used saturated sodium bicarbonate solution successively, after saturated sodium-chloride water solution washing, and organic phase anhydrous sodium sulfate drying.After revolving desolventizing, obtain orange solids, productive rate 90-98%.
4.N, N-bis-(2-chloroethyl) N-methyl-p-nitroaniline synthetic
By N, N-bis-(2-methanesulfonates ethyl) N-methyl-p-nitroaniline 1.58g (4.98mmol) is dissolved in 3mL DMF, adds lithium chloride 0.42g (10mmol), is heated to 110 ℃.TLC detects, and after 10min, reacts completely, and in cooling backward reaction solution, adds 45mL water, dichloromethane extraction.Organic phase saturated aqueous common salt washed twice, anhydrous sodium sulfate drying.After revolving desolventizing, obtain yellow solid, productive rate 90-95%.
5.N, N-bis-(2-chloroethyl) phenylenediamine synthetic
Get N, N-bis-(2-chloroethyl) N-methyl-p-nitroaniline 1.04g (4mmol) is dissolved in 5mL methyl alcohol, adds 0.1g palladium carbon (10%) catalytic hydrogenation.TLC detects, and after 1h, reacts completely, and suction filtration is removed palladium carbon, directly carries out next step reaction after revolving rapidly desolventizing.
The synthesis step of route C
Synthesizing of 3-cyano group-5-(chlorine) methyl-7-(N, N-bis-(2-chloroethyl) amino phenyl amino) pyrazolo [1,5-a] pyrimidine
By the N of reduction gained, N-bis-(2-chloroethyl) phenylenediamine (2mmol) is dissolved in 10mL ethanol, adds 3-cyano group-5-(chlorine) methyl-7-chlorine pyrazolo [1,5-a] pyrimidine (2.2mmol), reflux 2.5-3h, TLC detection reaction is complete, has solid to separate out after standing.Suction filtration, obtains yellow solid product, productive rate 76-88%.
In preferred embodiments, the invention provides nitrogen mustard derivatives or its pharmacologically acceptable salt of the formula A with good antitumous effect:
Correspondingly, the preparation method of nitrogen mustard derivatives or its pharmacologically acceptable salt of formula A is provided, it is characterized in that following synthetic route: the pyrazolo [1 that the propane dinitrile of take replaces as the synthetic 7-chlorine of raw material, 5-a] pyrimidine female ring, take p-Nitrophenyl chloride as the synthetic Lymphoquin of raw material, by the method that two compounds of gained are refluxed in ethanol, obtain A.
In preferred embodiments, the invention provides nitrogen mustard derivatives or its pharmacologically acceptable salt of the formula B with good antitumous effect:
Correspondingly, the preparation method of nitrogen mustard derivatives or its pharmacologically acceptable salt of formula B is provided, the synthetic route that it is characterized in that compound: the pyrazolo [1 that the propane dinitrile of take replaces as the synthetic 7-chlorine of raw material, 5-a] pyrimidine female ring, take m-nitraniline as the synthetic Lymphoquin of raw material, by the method that two compounds of gained are refluxed in ethanol, obtain B.
In preferred embodiments, the invention provides nitrogen mustard derivatives or its pharmacologically acceptable salt of the formula C with good antitumous effect:
Correspondingly, the preparation method of nitrogen mustard derivatives or its pharmacologically acceptable salt of formula C is provided, it is characterized in that following synthetic route: the pyrazolo [1 that the propane dinitrile of take replaces as the synthetic 7-chlorine of raw material, 5-a] pyrimidine female ring, take p-Nitrophenyl chloride as the synthetic Lymphoquin of raw material, by the method that two compounds of gained are refluxed in ethanol, obtain C.
In preferred embodiments, the invention provides nitrogen mustard derivatives or its pharmacologically acceptable salt of the formula D with good antitumous effect:
Correspondingly, the preparation method of nitrogen mustard derivatives or its pharmacologically acceptable salt of formula D is provided, the synthetic route that it is characterized in that compound: the pyrazolo [1 that the propane dinitrile of take replaces as the synthetic 7-chlorine of raw material, 5-a] pyrimidine female ring, take m-nitraniline as the synthetic Lymphoquin of raw material, by the method that two compounds of gained are refluxed in ethanol, obtain D.
Advantage of the present invention:
1) in the present invention, the synthesis step productive rate of target compound is higher, is easy to make.
2) in the present invention, the test of the external activity of target compound shows that such compound has good anti-tumor activity and selectivity.
3) in the present invention, the toxicity in vivo of target compound is relatively little, is the not available feature of general nitrogen mustards compound.
Embodiment
By embodiment, can make the present invention be more clearly described below, but they should not be construed as and limit the scope of the invention, and be only its illustration and representative.
Biology embodiment
Embodiment A. the inhibition to five kinds of common tumour cells
Suppress cell proliferating determining method and adopt conventional mtt assay, this measuring method can be used for measuring the inhibition ability of different target compounds of the present invention to five kinds of cancer cell multiplications, utilize method well known in the art, can use similar measuring method to any cancer cells.Tumour cell in logarithmic phase (HepG2, SH-SY5Y, MCF-7, A549, DU145) is used to 0.25% trysinization, then use nutrient solution (DMEM+10%FBS or PRMI1640+10%FBS) that cell dilution is suspended into single cell suspension, adjusting cell density is 2.0 * 10
4individual/mL, every hole adds 100 μ L to be inoculated in 96 orifice plates, in 37 ℃, the incubator of saturated humidity, 5% carbonic acid gas, cultivate after 24h, the formula A-D compound that adds respectively 0.1 μ M, 1 μ M, 5 μ M, 10 μ M, 20 μ M, 30 μ M, 50 μ M, 100 μ M by experimental design, parallel 5 the multiple holes of each concentration, and experimental group and control group be set respectively, continue to hatch after 72h, in every hole, add 10 μ LMTT solution (5mg/mL), then hatch 4h at 37 ℃, then add 100 μ L cell pyrolysis liquid (10%SDS+0.1%NH in every hole
4cl), lucifuge overnight incubation.Inferior daily microplate reader is measured each hole at absorbancy (OD) value at 570nm place, reference wavelength 650nm.According to absorbance, calculate inhibitory rate of cell growth: inhibiting rate (100%)=[1-(experimental group OD average-blank group OD average)/(control group OD average-blank group OD average)] * 100%.Take drug level as X-coordinate, and cell inhibitory rate is ordinate zou, draws cell growth inhibition curve.Above-mentioned experiment repeats 3 times.By the Probit module in IBM SPSSTM Statistics20 statistical software, the probit Return Law, calculate IC
50value.
Following table 1 has shown wherein substituent R
1=methyl and R
2=mustargen, R
3the formula A of=H or R
2=H, R
3the formula B compound of=mustargen and wherein R
1=chloromethyl and R
2=mustargen, R
3the formula C of=H or R
2=H, R
3the IC of the formula D compound of=mustargen to five kinds of common cells
50:
Table 1:
As can be seen from the above table, this compounds has good inhibition to above five kinds of tumour cells.Wherein, formula C is best to the inhibition of liver cancer (HepG2), neuroblastoma (SH-SY5Y) and lung cancer (A549); Formula D is better to the inhibition of liver cancer (HepG2) and neuroblastoma (SH-SY5Y); Formula A is better to the inhibition of neuroblastoma (SH-SY5Y) and lung cancer (A549).
Embodiment B. acute toxicity test
For the result of embodiment A, to working as substituent R
1=chloromethyl, and R
2=mustargen, R
3the toxicity in vivo of the formula C compound of=hydrogen is measured.Subjects is Kunming small white mouse (18-22g, male and female half and half), and fasting 12h before test, can't help water.It is stand-by that the injection liquid that formula C is mixed with to different concns irradiates 15min under ultraviolet lamp.Get 10 of small white mouses, take 2 as one group (female half and half), divide 5 groups, a series of dosage that selective dose spacing is larger, the injection liquid of organizing the formula C of mouse injection different concns respectively obtains 0% and 100% lethal quantity scope to each.It is 10 (male and female half and half) that each dosage treated animal number is chosen in formal experiment, and body weight and sex stratified random are distributed, and completes tail vein injection administration after animal grouping and Rapid Dose Calculation.During administration, first therefrom dosage group starts, so that the reaction that can accept medicine from initial several treated animals judges that whether the dosage of two groups is suitable, otherwise can adjust at any time, make as far as possible the mortality ratio of animal upper and lower 50%, when mortality ratio is 0% or 100%, can not be for calculating.Give observation post administration small white mouse activity change situation and death toll, Continuous Observation 3 days.With weighting probit (Bliss method), calculate the Lethal Dose 50 (LD of small white mouse
50) and 95% fiducial limit, the results are shown in following table 2.
Table 2:
By the toxicity in vivo comparison with carmustine (45.2mg/kg), melphalan (32mg/kg) and camptothecine (57.3mg/kg), the toxicity in vivo of upper table data sheet Ming Dynasty style C compound is lower.
Synthetic example
Embodiment 1
What according to following steps, prepare is the substituent R of formula A
1=methyl, R
2=mustargen, R
3the compound of=hydrogen.
1.1 ethoxy methylenes, the third two eyeballs synthetic
The third two eyeball 9.9g (0.15mol), triethyl orthoformate 33.3g (0.23mol) and acetic anhydride 38.4g (0.38mol) are joined in 250mL single port flask.After back flow reaction 6h, cooling, add gac post-heating backflow 30min, heat filtering, with after hot ethanol washing leaching cake, filtrate is placed in refrigerator overnight, and suction filtration is dried to obtain faint yellow solid 15.4g, productive rate 84.3%.Gained spectrogram is consistent with bibliographical information.
Synthesizing of 1.2 3-amino-4-cyano pyrazoles
Ethoxy methylene the third two eyeball 15.0g (0.12mol) at room temperature slowly join in 85% hydrazine hydrate 12mL (0.25mol), and heating in water bath back flow reaction 1h adds 10mL water in reaction system.In refrigerator, place and spend the night, suction filtration, washing filter cake, dries to obtain faint yellow solid 10.6g, productive rate 81.7%.Gained spectrogram is consistent with bibliographical information.
1.3 3-cyano group-5-methyl-7-hydroxypyrazoles are synthesizing of [1,5-a] pyrimidine also
3-amino-4-cyano pyrazole 4.0g (37mmol) and methyl aceto acetate (38mmol) are added in 33mL glacial acetic acid to back flow reaction 4h.Be cooled to after completion of the reaction room temperature, filter, use successively Glacial acetic acid and water wash filter cake each 3 times, obtain yellow solid.By re-crystallizing in ethyl acetate, obtain sterling, productive rate 80.8%.Gained spectrogram is consistent with bibliographical information.
Synthesizing of 1.4 3-cyano group-5-methyl-7-chlorine pyrazolo [1,5-a] pyrimidines
In the four-necked bottle of 250mL, add 3-cyano group-5-methyl-7-hydroxypyrazoles also [1,5-a] after pyrimidine (9.95mmol) and pyridine 0.82mL (10.55mmol), slowly drip phosphorus oxychloride 4.05mL (43.2mmol), finish after being slowly heated to 85 ℃ and start to stir, in 120 ℃ of reaction 1h.After below being cooled to 60 ℃, add chloroform 150mL.Back flow reaction 1h.While being cooled to 0-5 ℃, add cold water 90mL, suction filtration is removed insolubles, separates organic phase, washes twice, each water 90mL.Revolve after chloroform is removed in steaming and obtain yellow solid, productive rate 81.7%.Gained spectrogram is consistent with bibliographical information.
1.5 N, N-bis-(2-hydroxyethyl)-4-N-methyl-p-nitroaniline synthetic
Get 4-chloronitrobenzene 10.3g (0.07mol), diethanolamine 17.5g (0.17mol), in 250mL round-bottomed flask, mixes, and 120 ℃ are stirred 6h, pour into while hot in 90 ℃ of hot water, fully stir.Filtered while hot, standing rear filtrate is separated out solid, suction filtration, filter cake is with obtaining yellow solid 5.5g with ethyl alcohol recrystallization, productive rate 36.3% after ether washing.Gained spectrogram is consistent with bibliographical information.
1.6 N, N-bis-(2-methanesulfonates ethyl)-4-N-methyl-p-nitroaniline synthetic
Get N, N-bis-(2-hydroxyethyl) N-methyl-p-nitroaniline 2.3g (0.01mol), adds in the methylene dichloride that 30mL is dry, under ice bath, adds 3.3mL triethylamine, after add methylsulfonyl chloride 1.9mL (0.025mol).TLC detects, and after 10min, reacts completely.Reaction solution is used saturated sodium bicarbonate solution successively, after saturated sodium-chloride water solution washing, and organic phase anhydrous sodium sulfate drying.After revolving desolventizing, obtain orange solids, productive rate 90.6%.Gained spectrogram is consistent with bibliographical information.
1.7 N, N-bis-(2-chloroethyl)-4-N-methyl-p-nitroaniline synthetic
By N, N-bis-(2-methanesulfonates ethyl) N-methyl-p-nitroaniline 1.6g (4.98mmol) is dissolved in 3mL DMF, adds lithium chloride 0.4g (10mmol), is heated to 110 ℃.TLC detects, and after 10min, reacts completely, and in cooling backward reaction solution, adds 45mL water, dichloromethane extraction.Organic phase saturated aqueous common salt washed twice, anhydrous sodium sulfate drying.After revolving desolventizing, obtain yellow solid.Productive rate 90.3%.Gained spectrogram is consistent with bibliographical information.
1.8 N, N-bis-(2-chloroethyl) Isosorbide-5-Nitrae-phenylenediamine synthetic
Get N, N-bis-(2-chloroethyl) N-methyl-p-nitroaniline 1.0g (4mmol) is dissolved in 5mL methyl alcohol, adds 0.1g palladium carbon (10%) catalytic hydrogenation.TLC detects, and after 1h, reacts completely, and suction filtration is removed palladium carbon, directly carries out next step reaction after revolving rapidly desolventizing.
Synthesizing of 1.9 3-cyano group-5-methyl-7-(4-N, N-bis-(2-chloroethyl) amino phenyl amino) pyrazolo [1,5-a] pyrimidine
By the N of reduction gained, N-bis-(2-chloroethyl)-Isosorbide-5-Nitrae-phenylenediamine (2mmol) is dissolved in 10mL ethanol, add 3-cyano group-5-methyl-7-chlorine pyrazolo [1,5-a] pyrimidine (2.2mmol), reflux 2.5h, TLC detection reaction is complete, has solid to separate out after standing.Suction filtration, obtains yellow solid product, productive rate 87.8%.
1H?NMR(400MHz，CDCl
3)δ8.24(s，1H，-CH)，7.82(s，1H，-NH)，7.23(d，J=8.88Hz，2H，ArH)，6.78(d，J=8.88Hz，2H，ArH)，6.13(s，1H，-CH)，3.79(t，J＝6.76Hz，4H，-CH
2CH
2Cl)，3.68(t，J=6.60Hz，4H，-CH
2CH
2Cl)，2.52(s，3H，-CH
3).
13C?NMR(100MHz，CDCl
3)δ163.73，150.61，146.37，146.32，145.77，127.06，124.90，113.65，112.99，89.21，80.59，53.56，40.33，25.25.MS(ESI
+)m／z：389.3(M+H
+).IR(KBr?pellet，cm
-1)：3352，2222，1616，1589，1519，1421，1184，918，808，719.
Embodiment 2
What according to following steps, prepare is the substituent R of formula B
1=methyl, R
2=hydrogen, R
3the compound of=mustargen.
2.1 N, N-bis-(2-hydroxyethyl)-3-N-methyl-p-nitroaniline synthetic
Get 3-N-methyl-p-nitroaniline 5.52g (0.04mol), add in the aqueous acetic acid of 80mL25%, under ice bath, drip oxyethane 10mL.Be warming up to room temperature, stir 72h, suction filtration, water washing filter cake, dries to obtain yellowish-orange solid 8.02g, productive rate 88.5%.Gained spectrogram is consistent with bibliographical information.
2.2 N, N-bis-(2-methanesulfonates ethyl)-3-N-methyl-p-nitroaniline synthetic
Get N, N-bis-(2-hydroxyethyl)-3-N-methyl-p-nitroaniline 2.26g (0.01mol), adds in the methylene dichloride that 30mL is dry, under ice bath, adds 3.32mL (0.025mol) triethylamine, after add methylsulfonyl chloride 1.93mL (0.025mol).TLC detects, and after 10min, reacts completely.Reaction solution is used saturated sodium bicarbonate solution successively, after saturated sodium-chloride water solution washing, and organic phase anhydrous sodium sulfate drying.After revolving desolventizing, obtain orange solids, productive rate 90.8%.Gained spectrogram is consistent with bibliographical information.
2.3 N, N-bis-(2-chloroethyl)-3-N-methyl-p-nitroaniline synthetic
Get N, N-bis-(2-methanesulfonates ethyl)-3-N-methyl-p-nitroaniline is dissolved in 1.58g (4.98mmol) in 3mL DMF, adds lithium chloride 0.42g (10mmol), is heated to 110 ℃.TLC detects, and after 10min, reacts completely, and in cooling backward reaction solution, adds 45mL water, dichloromethane extraction.Organic phase saturated aqueous common salt washed twice, anhydrous sodium sulfate drying.After revolving desolventizing, obtain yellow solid, productive rate 91.3%.Gained spectrogram is consistent with bibliographical information.
2.4 N, N-bis-(2-chloroethyl) 1,3-phenylenediamine synthetic
Get N, N-bis-(2-chloroethyl)-3-N-methyl-p-nitroaniline 1.04g (4mmol) is dissolved in 5mL methyl alcohol, adds 0.1g palladium carbon (10%) catalytic hydrogenation.TLC detects, and after 1h, reacts completely, and suction filtration is removed palladium carbon, directly carries out next step reaction after revolving rapidly desolventizing.
Synthesizing of 2.5 3-cyano group-5-methyl-7-(3-N, N-bis-(2-chloroethyl) amino phenyl amino) pyrazolo [1,5-a] pyrimidine
By the N of reduction gained, N-bis-(2-chloroethyl) 1,3-phenylenediamine (2mmol) is dissolved in 10mL ethanol, add 3-cyano group-5-methyl-7-chlorine pyrazolo [1,5-a] pyrimidine (2.2mmol), reflux 2.5h, TLC detection reaction is complete, has solid to separate out after standing.Suction filtration, obtains yellow solid product.Productive rate 76.6%.
1H?NMR(400MHz，CDCl
3)δ8.25(s，1H，-CH)，7.98(s，1H，-NH)，7.36(t，J=8.12Hz，1H，ArH)，6.77(d，J=7.76Hz，1H，ArH)，6.66(d，J=8.44Hz，1H，ArH)，6.62(s，1H，-CH)，6.41(s，1H，-ArH)，3.78(t，J=6.60Hz，4H，-CH
2CH
2Cl)，3.67(t，J=6.52Hz，4H，-CH
2CH
2Cl)，2.56(s，3H，-CH
3).
13C?NMR(100MHz，CDCl
3)δ163.90，150.57，147.85，146.31，145.21，137.01，131.16，113.53，112.79，110.67，107.44，89.76，80.90，53.37，40.29，25.35.MS(ESI
+)m／z：389.3(M+H
+).IR(KBr?pellet，cm
-1)：3356，2224，1605，1570，1537，1500，1418，1280，1168，997，748.
Embodiment 3
What according to following steps, prepare is the substituent R of formula C
1=chloromethyl, R
2=mustargen, R
3the compound of=hydrogen.
3.1 3-cyano group-5-chloromethyl-7-hydroxypyrazoles are synthesizing of [1,5-a] pyrimidine also
3-amino-4-cyano pyrazole 4g (37mmol) and chloroacetyl acetacetic ester (38mmol) are added in 33ml glacial acetic acid to back flow reaction 4h.Be cooled to after completion of the reaction room temperature, filter, use successively Glacial acetic acid and water wash filter cake each 3 times, obtain yellow solid.By re-crystallizing in ethyl acetate, obtain sterling, productive rate 82.3%.Gained spectrogram is consistent with bibliographical information.
Synthesizing of 3.2 3-cyano group-5-chloromethyl-7-chlorine pyrazolo [1,5-a] pyrimidines
In the four-necked bottle of 250mL, add 3-cyano group-5-chloromethyl-7-hydroxypyrazoles also [1,5-a] after pyrimidine (9.95mmol) and pyridine 0.82mL (10.55mmol), slowly drip phosphorus oxychloride 4.05mL (43.2mmol), finish after being slowly heated to 85 ℃ and start to stir, in 120 ℃ of reaction 1h.After below being cooled to 60 ℃, add chloroform 150mL.Back flow reaction 1h.While being cooled to 0-5 ℃, add cold water 90ml, suction filtration is removed insolubles, separates organic phase, washes twice, each water 90mL.Revolve after chloroform is removed in steaming and obtain yellow solid, productive rate 81.2%.Gained spectrogram is consistent with bibliographical information.
Synthesizing of 3.3 3-cyano group-5-chloromethyl-7-(4-N, N-bis-(2-chloroethyl) amino phenyl amino) pyrazolo [1,5-a] pyrimidine
By the N of reduction gained, N-bis-(2-chloroethyl) Isosorbide-5-Nitrae-phenylenediamine (2mmol) is dissolved in 10mL ethanol, add 3-cyano group-5-chloromethyl-7-chlorine pyrazolo [1,5-a] pyrimidine (2.2mmol), reflux 3h, TLC detection reaction is complete, has solid to separate out after standing.Suction filtration, obtains yellow solid product, productive rate 76.9%.
1H?NMR(400MHz，CDCl
3)δ8.30(s，1H，-CH)，7.99(s，1H，-NH)，7.25(d，J=8.76Hz，2H，ArH)，6.79(d，J=8.76Hz，2H，ArH)，6.50(s，1H，-CH)，4.59(s，2H，-CH
2Cl)，3.80(t，J=6.68Hz，4H，-CH
2CH
2Cl)，3.69(t，J=6.64Hz，4H，-CH
2CH2Cl).
13C?NMR(100MHz，CDCl
3)δ161.03，150.08，147.13，146.82，145.92，126.86，124.37，113.22，112.94，88.05，81.75，53.51，46.19，40.26.MS(ESI
+)m／z：423.3(M+H
+).IR(KBr?pellet，cm
-1)：3337，2225，1710，1616，1589，1520，1416，1205，823，729.
Embodiment 4
What according to following steps, prepare is the substituent R of formula C
1=chloromethyl, R
2=hydrogen, R
3the compound of=mustargen.
Synthesizing of 3-cyano group-5-chloromethyl-7-(3-N, N-bis-(2-chloroethyl) amino phenyl amino) pyrazolo [1,5-a] pyrimidine
By the N of reduction gained, N-bis-(2-chloroethyl) 1,3-phenylenediamine (2mmol) is dissolved in 10mL ethanol, add 3-cyano group-5-chloromethyl-7-chlorine pyrazolo [1,5-a] pyrimidine (2.2mmol), reflux 3h, TLC detection reaction is complete, has solid to separate out after standing.Suction filtration, obtains yellow solid product.Productive rate 78.7%.
1H?NMR(400MHz，CDCl
3)δ8.32(s，1H，-CH)，8.14(s，1H，-NH)，7.38(t，J=8.16Hz，1H，ArH)，6.62(s，1H，-CH)，6.78(d，J=8.48Hz，1H，ArH)，6.69(d，J＝8.44Hz，1H，ArH)，6.64(s，1H，-ArH)，4.62(s，3H，-CH
3)，3.78(t，J=6.60Hz，4H，-CH
2CH
2Cl)，3.67(t，J=6.52Hz，4H，-CH
2CH
2Cl).
13C?NMR(100MHz，CDCl
3)δ161.23，150.03，147.90，146.82，146.10，136.61，131.34，113.08，112.68，110.99，107.23，88.55，82.06，53.41，46.21，40.28.MS(ESI
+)m／z：423.3(M+H
+).IR(KBr?pellet，cm
-1)：3319，2227，1605，1570，1535，1500，1417，1283，1168，995，735.
Claims (16)
1. for pyrazolo [1,5-a] pyrimidine nitrogen mustard derivatives or its pharmacologically acceptable salt of the formula I structure of oncotherapy:
Wherein:
R
1be the heteroaryl that replaces of alkyl, haloalkyl, alkoxyl group, aryl, heteroaryl, alkyl, heterolipid cyclic group or-NR
4r
5;
R
4and R
5independently selected from hydrogen, alkyl, cycloalkyl, aryl or heteroaryl, or R
4and R
5form altogether a heteroaryl or heterolipid cyclic group;
R is N (CH
2cH
2cl)
2.
2. compound or pharmaceutically acceptable salt thereof according to claim 1, wherein said pyrazolo [1,5-a] pyrimidine nitrogen mustard derivatives has as shown in the formula II:
Wherein:
R
1alkyl or haloalkyl;
R
2=N (CH
2cH
2cl)
2, R
3=H, or R
2=H, R
3=N (CH
2cH
2cl)
2.
3. compound or pharmaceutically acceptable salt thereof according to claim 2, wherein said pyrazolo [1,5-a] pyrimidine nitrogen mustard derivatives has following formula III:
R wherein
2=N (CH
2cH
2cl)
2, R
3=H, or R
2=H, R
3=N (CH
2cH
2cl)
2.
4. compound or pharmaceutically acceptable salt thereof according to claim 2, wherein said pyrazolo [1,5-a] pyrimidine nitrogen mustard derivatives has as shown in the formula IV:
R wherein
2=N (CH
2cH
2cl)
2, R
3=H, or R
2=H, R
3=N (CH
2cH
2cl)
2.
5. the preparation method of pyrazolo claimed in claim 2 [1,5-a] pyrimidine nitrogen mustard derivatives or its pharmacologically acceptable salt, is characterized in that synthetic route comprises following three parts:
Pyrazolo [1,5-a] pyrimidine female ring synthetic route:
Lymphoquin synthetic route:
Target compound synthetic route:
6. compound or pharmaceutically acceptable salt thereof according to claim 3, wherein said pyrazolo [1,5-a] pyrimidine nitrogen mustard derivatives has as shown in the formula A:
7. compound or pharmaceutically acceptable salt thereof according to claim 3, wherein said pyrazolo [1,5-a] pyrimidine nitrogen mustard derivatives has as shown in the formula B:
8. compound or pharmaceutically acceptable salt thereof according to claim 4, wherein said pyrazolo [1,5-a] pyrimidine nitrogen mustard derivatives has as shown in the formula C:
9. compound or pharmaceutically acceptable salt thereof according to claim 4, wherein said pyrazolo [1,5-a] pyrimidine nitrogen mustard derivatives has as shown in the formula D:
10. pyrazolo [1 claimed in claim 6,5-a] preparation method of pyrimidine nitrogen mustard derivatives or its pharmacologically acceptable salt, it is characterized in that following synthetic route: the pyrazolo [1 that the propane dinitrile of take replaces as the synthetic 7-chlorine of raw material, 5-a] pyrimidine female ring, take p-Nitrophenyl chloride as the synthetic Lymphoquin of raw material, by the method that two compounds of gained are refluxed in ethanol, obtain A.
11. pyrazolos [1 claimed in claim 7,5-a] preparation method of pyrimidine nitrogen mustard derivatives or its pharmacologically acceptable salt, it is characterized in that following synthetic route: the pyrazolo [1 that the propane dinitrile of take replaces as the synthetic 7-chlorine of raw material, 5-a] pyrimidine female ring, take m-nitraniline as the synthetic Lymphoquin of raw material, by the method that two compounds of gained are refluxed in ethanol, obtain B.
12. pyrazolos [1 claimed in claim 8,5-a] preparation method of pyrimidine nitrogen mustard derivatives or its pharmacologically acceptable salt, it is characterized in that following synthetic route: the pyrazolo [1 that the propane dinitrile of take replaces as the synthetic 7-chlorine of raw material, 5-a] pyrimidine female ring, take p-Nitrophenyl chloride as the synthetic Lymphoquin of raw material, by the method that two compounds of gained are refluxed in ethanol, obtain C.
13. pyrazolos [1 claimed in claim 9,5-a] preparation method of pyrimidine nitrogen mustard derivatives or its pharmacologically acceptable salt, it is characterized in that following synthetic route: the pyrazolo [1 that the propane dinitrile of take replaces as the synthetic 7-chlorine of raw material, 5-a] pyrimidine female ring, take m-nitraniline as the synthetic Lymphoquin of raw material, by the method that two compounds of gained are refluxed in ethanol, obtain D.
14. 1 kinds of pharmaceutical compositions, comprise in claim 1-4 and 6-9 the pyrazolo described in any one [1,5-a] pyrimidine nitrogen mustard derivatives or its pharmacologically acceptable salt and comprise pharmaceutically acceptable diluent or carrier.
Pyrazolo [1,5-a] pyrimidine nitrogen mustard derivatives in 15. claim 1-4 and 6-9 described in any one or the application of its pharmacologically acceptable salt in preparing anti-tumor medicine.
Application described in 16. claims 15, wherein said cancer is HepG2, SH-SY5Y, MCF-7, A549 and DU145.
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| CN1780840A (en) * | 2003-02-28 | 2006-05-31 | 帝人制药株式会社 | Pyrazolo [1, 5-a ] pyrimidine derivatives |
| CN101039945A (en) * | 2004-08-13 | 2007-09-19 | 帝人制药株式会社 | Pyrazolo [1, 5-a ] pyrimidine derivatives |
| CN101054380A (en) * | 2006-04-13 | 2007-10-17 | 沈阳药科大学 | Pyrazolopyrimidine derivative used as cell cycle dependency protein kinase inhibito |
| CN101119998A (en) * | 2004-12-21 | 2008-02-06 | 先灵公司 | Pyrazolo [1,5-a] pyrimidine adenosine a2a receptor antagonists |
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| CN101039945A (en) * | 2004-08-13 | 2007-09-19 | 帝人制药株式会社 | Pyrazolo [1, 5-a ] pyrimidine derivatives |
| CN101119998A (en) * | 2004-12-21 | 2008-02-06 | 先灵公司 | Pyrazolo [1,5-a] pyrimidine adenosine a2a receptor antagonists |
| CN101054380A (en) * | 2006-04-13 | 2007-10-17 | 沈阳药科大学 | Pyrazolopyrimidine derivative used as cell cycle dependency protein kinase inhibito |
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