CN1970552A - Substituted[1,3,5] triazine compound, its preparing process and its application - Google Patents
Substituted[1,3,5] triazine compound, its preparing process and its application Download PDFInfo
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Abstract
The invention discloses a chemical acceptable salt and solvent complex or hydrate and composition and application with substituted [1,3,5] triazine compound, which is characterized by the following: displaying obvious EGFR enzyme inhibiting activity and tumour-proof activity in a series of sieving experiment.
Description
Technical field
The present invention relates to pharmaceutical chemistry and pharmacotherapeutics field, be specifically related to replace [1,3,5] compound in triazine class and its production and application, and the pharmaceutical composition that comprises this replacement [1,3,5] compound in triazine class.
Background technology
The pathogenic process of numerous disease all has the participation of inflammatory mediator, some diseases associated with inflammation (as rheumatoid arthritis, rheumatic fever, osteoarthritis), asthma, chronic obstructive lung illness, wound, burn, endotoxin shock, alzheimer's disease, even heart failure etc. all has the participation of inflammatory mediator.Consider the complicacy and the diversity of medium, single medium is not the physiological whole reasons of the disorderly pathology of inflammation.Therefore, can disturb the compound of a plurality of media or enzymic activity may be more influential to inflammation simultaneously than the compound of single-activity.In the multiple inflammatory mediator, (Arachidonic Acid, meta-bolites prostaglandin(PG) (PGs) AA) and leukotriene (LTs) are two important inflammatory mediators to arachidonic acid.The metabolism of AA has following two approach: (1) cyclooxygenase (COX) pathways metabolism, and promptly AA is transformed into PGs through the series reaction metabolism under the catalysis of COX; (2) lipoxygenase (5-LOX) pathways metabolism, i.e. AA metabolism under the catalysis of 5-LOX is transformed into LTs.Therefore, block of the generation of these two pathways metabolisms of AA, thereby reach the purpose for the treatment of or weakening inflammation with regard to energy inflammation-inhibiting medium PGs and LTs
Wherein, article one pathways metabolism comprises following series reaction: membrane phospholipid is via phospholipase A
2(PLA
2) producing free arachidonic acid (AA), AA is converted into PGG through the effect of cyclooxygenase (COX-1 and COX-2)
2(PG
2), its peroxidase activity then is converted into PGH with its product
2(PGH
2), tissue-specific isozyme is with PGH
2Metabolism is other form or the thromboxane A of prostaglandin(PG)
2(TxA
2).Simultaneously, AA metabolism under the effect of 5-lipoxygenase is leukotriene (LTs).
For the treatment of inflammation, early stage people use glucocorticosteroid AID (SAID) more, easily cause complication such as adrenal cortex function decline but take this class medicine for a long time.Nineteen fifty-two, Phenylbutazone was used for clinically, proposed this notion of NSAID (non-steroidal anti-inflammatory drug) (NSAID) in the world first.Emerge large quantities of NSAID with anti-inflammatory and analgesic effect in the period of twenty or thirty subsequently, as the at present still indomethacin of widespread use clinically, Ibuprofen BP/EP etc., such medicine occupies first place in world's medicine output always.Yet prolonged application NSAID can cause the particularly infringement of stomach mucous membrane of gi tract, shows as petechial hemorrhage, the shallow table mucosal erosion of diffusivity, local dark ulcer, profuse bleeding even perforation.
1991, a new direction was opened up in the research that is found to be NSAID of COX-2.Studies show that: COX-1 is Normocellular constitutive protein at normal tissue expression; And COX-2 is the form of inducing of enzyme, mainly at inflammatory cell, as expressing in the endotheliocyte after the tissue injury, scavenger cell, sliding liquid fiber cell, dendritic cell, chondrocyte and the scleroblast.COX-2 has similar active combining site to COX-1 to AA or NSAID; COX-2 can be induced by the multiple factor in the inflammation tissue, and its level will rise by the PGE at the position that causes inflammation with 8～10 times speed play
2, PGI
2And PGE
1Content increase, promoted inflammatory reaction and tissue injury.1994, J.R.Vane etc. pointed out that NSAID comes from its inhibition to COX-2 to effective therapeutic action of inflammation, and untoward reaction belongs to the inhibition to COX-1.Therefore, since the nineties, seeking the COX-2 selective depressant has become the important channel of finding the novel anti-inflammatory medicine.But, the potential safety hazard that the COX-2 selective depressant exists aspect cardiovascular.
Studies show that, have certain balance restricting relation in two pathways metabolisms of AA, promptly when the activity of COX was suppressed, the increased activity of 5-LOX made more AA enter the 5-LOX pathways metabolism and produces LTs.Equally, when the activity of 5-LOX is suppressed, then has more AA to enter the COX pathways metabolism and make PG generate increase, the result makes inflammation increase the weight of.Therefore, a simple inhibition wherein pathways metabolism will cause that all a large amount of AA enters another pathways metabolism, thereby cause inflammation to further develop.Classical NSAIDs such as Asprin, indomethacin, diclofenac etc. all are the COX selective depressants; generally inoperative to the 5-LOX metabolism; they not only influence PGs synthetic that gastric mucosa is had provide protection; and since single inhibition COX-2 so that cause the LOX metabolic activity to increase; cause the AA metabolic imbalance; promote the synthetic increase of LTs material, thereby promote leukocyte chemotaxis to assemble, increase vascular permeability.So need to propose a comprehensive and little antiphlogiston of toxic side effect of effect, reply COX and 5-LOX produce restraining effect simultaneously.Thereby design COX-2/5-LOX double inhibitor is the primary study direction of domestic and international medical worker's in recent years anti-inflammatory drug to reach the purpose of collaborative anti-inflammatory.
Recently discover NSAIDs particularly optionally cox 2 inhibitor (as celecoxib etc.) as synergism and other chemotherapeutic agent molecule acting in conjunction, can treat the rectum cancer.With respect to the research of cox 2 inhibitor on the rectum cancer, optionally 5-LOX inhibitor and the research of COX-2/5-LOX double inhibitor on the rectum cancer to lack many.Yet COX-2 selective depressant or COX-2/5-LOX double inhibitor, especially will more and more receive publicity in the control of carcinoma of the colon and rectum in the oncotherapy field as antitumor drug and/or its ancillary drug, and will become the focus of research from now on.
In recent years, " area of computer aided medicinal design " (Computer-Aided Drug Design, CADD) important method and the instrument of modern medicines research and development have been become, with the particularly circulation of area of computer aided combinatorial chemical library design adding new drug research of area of computer aided medicinal design, can shorten the cycle of new drug research, save research and development cost, improve the hit rate of new medicament screen." combinatorial chemistry " (Combinatorial Chemistry) is a new technology that occurs in pharmaceutical chemistry and the synthetic chemistry in recent years, can produce a large amount of molecular structures rapidly to carry out high flux screening (High Throughput Screening)." structure biology " also enters the applied research stage from original fundamental research, one of them main application fields is to study the interaction of medicine and target protein on molecule and atomic structure level, measure the crystalline structure of medicine-protein complex, for the design of new compound and the structure of modification of lead compound provide useful structural information.
Summary of the invention
The present inventor discloses the synthesis preparation method of piperazine compound in triazine class and has contained the pharmaceutical composition of this compounds in Chinese patent application CN 200310109187.0, found that the piperazine compound in triazine class with brand new has good combination to the COX-2 enzyme, dissociation constant reaches 10
-6And mouse carrageen colloidality foot swelling model there is good anti-inflammatory action.And, according to COX-1, (wherein COX-1 and COX-2 have crystalline structure to the three-dimensional structure of COX-2 and 5-LOX, the three-dimensional structure of 5-LOX makes up with homologous protein mould construction method), integrated use area of computer aided SARS drug design, combinatorial chemistry, molecular biology and structure biology method, further seek lead compound with the effect of COX-2/5-LOX double inhibitor, and carry out composition optimizes at its pharmacological action, thereby find on the basis of above-mentioned patented invention: (1) some piperazine compound in triazine class all has good keying action to COX-2 enzyme and 5-LOX enzyme, and dissociation constant reaches 10
-6To N-substituted aryl-N '-substituted aryl-6-wherein (piperazine-1-replaces)-[1,3,5]-triazine-2, the 4-diamine compounds has carried out various pharmacological testings, finds that this compounds causes on the scorching model of swollen model, mouse ear caused by dimethylbenzene xylene, rat assist agent arthritis (prevention primary affection) model and rat assist agent arthritis (prevention secondary affection) model at the rat paw carrageenin good anti-inflammatory action is all arranged; And in Ames experiment and micronuclei in mice experiment, show security preferably, gi tract are had no side effect.(2) some replacements [1,3,5] compound in triazine class has certain inhibition activity to people's colon-cancer cell strain HT-29 and HCT-116, finds that simultaneously the morpholine compound in triazine class of similar structures but has potent inhibition active to people's colon-cancer cell strain HT-29 and HCT-116, a few compounds IC
50Reach the nM level; It is active that the morpholine compound in triazine class has also showed EGF-R ELISA (EGFR) inhibition preferably in addition; The compound performance of accepting the cell chromosome teratogenic test is good, does not all have teratogenecity, thereby has finished the present invention.
Therefore, the purpose of this invention is to provide a kind of replacement [1,3,5] compound in triazine class that COX-2/5-LOX is had double inhibitor effect and the effect of EGFR enzyme inhibitors.
A further object of the present invention provides the preparation method of above-claimed cpd.
Still a further object of the present invention provides the application of above-claimed cpd.
A further object of the present invention provides the pharmaceutical composition that comprises above-claimed cpd.
A further object of the present invention provides the application of aforementioned pharmaceutical compositions.
According to technical scheme of the present invention, the invention provides a kind of replacement [1,3,5] compound in triazine class, its pharmacy acceptable salt and solvate or hydrate with structure shown in the formula (I):
Wherein,
R
1, R
2, R
3And R
4Be hydrogen, C independently of one another
1-C
8Straight or branched alkyl, replacement or unsubstituted aryl, aralkyl, C
1-C
4Alkaryl, aroyl, wherein aryl is selected from phenyl, naphthyl and xenyl, and substituting group is that individual being selected from of 1-4 comprises halogen, C
1-C
6Straight or branched alkyl, cyano group, nitro, amino, hydroxyl, methylol, trifluoromethyl, trifluoromethoxy, carboxyl, C
1-C
4Alkoxyl group, sulfydryl, C
1-C
4Alkylthio, C
1-C
4Alkyl carbonyl, C
1-C
4The group of the group of carbalkoxy, alkylsulfonyl; And
R
5Structural formula be
R wherein
6And R
7Comprise hydrogen, halogen, C for being selected from independently of one another
1-C
4Straight or branched alkyl, cyano group, nitro, amino, hydroxyl, methylol, trifluoromethyl, trifluoromethoxy, carboxyl, C
1-C
4Alkoxyl group, sulfydryl, C
1-C
4Alkylthio, C
1-C
4The group of the group of acyl group and alkylsulfonyl; Y is CH
2, O, S or RN, wherein R is hydrogen, C
1-C
4Straight or branched alkyl, hydroxyl, C
1-C
4Hydroxyalkyl, carboxyl, C
1-C
4Alkyl carbonyl, C
1-C
4The group of carbalkoxy, alkylsulfonyl; M, n are 0,1,2 or 3 independently of one another, perhaps, and R
5Be selected from hydroxyl, amino, substituted-amino, C
1-C
6Alkyl, C
1-C
6The alkane hydroxyl ,-C (O) R
8,-(CH
2)
xR
8,-CH
2CH=CHR
8,-C (O) OR
8Or-S (O)
2R
8, wherein, x is 0,1,2 or 3; R
8Be hydrogen, hydroxyl, aryl, hetero-aromatic ring base, heterolipid cyclic group or C
2-C
6Thiazolinyl.
The present invention also provides the method for preparing above-claimed cpd, and this method may further comprise the steps: react compound shown in the general formula (II) under 0-50 ℃ of temperature in basic solvent (1) with replacement amine,
Wherein, R
xBe hydrogen, halogen, hydroxyl, sulfydryl, C
1-C
4Alkylamine or C
1-C
4Aminoalkyl; R
yAnd R
zBe respectively hydrogen, halogen, C
1-C
4Aminoalkyl or C
1-C
4The halo alkyl, thus compound shown in the general formula (III) obtained:
Wherein, R
1, R
2, R
xAnd R
yIdentical with definition among general formula (I), (II);
(2) with compound shown in the general formula (III) in basic solvent under 20-100 ℃ of temperature with replace reactions such as amine, substituted aromatic amines, substituted aryl alkanamine, alcohol or mercaptan, obtain compound shown in the general formula (IV):
Wherein, R
1, R
2, R
3, R
4Identical with definition in the general formula (I), R
xIdentical with definition in the general formula (II);
(3) under 0-120 ℃, in basic solvent, with compound shown in the general formula (IV) with replace aliphatic cyclic amine, substituted aryl amine, substituted aryl alkanamine, alcohol or mercaptan and react, obtain compound shown in the formula (I);
(4) as required, be carried out to reactant salt, or form solvate or hydrate by this area ordinary method.
The method according to this invention, wherein, basic solvent described in step (1) and the step (2) is the alkaline solution with the inert solvent preparation, described alkali is selected from organic bases that comprises pyridine, triethylamine, 4-dimethylamino pyridine, diisopropylethylamine and the group that comprises the mineral alkali of yellow soda ash, salt of wormwood, sodium hydroxide, potassium hydroxide, and described inert solvent comprises tetrahydrofuran (THF), ether (Et for one or more are selected from
2O), the solvent of the group of dimethyl formamide, glycol dimethyl ether, ethylene glycol diethyl ether, dioxane.
The method according to this invention, wherein, basic solvent described in the step (3) is that described polar organic solvent is selected from the solvent of the group that comprises dimethyl formamide, dimethyl sulfoxide (DMSO), dioxane, ethanol, methyl alcohol, acetone, ethyl acetate and tetrahydrofuran (THF) for one or more with the alkaline solution of polar organic solvent preparation; Described alkali is selected from the organic bases that comprises pyridine, triethylamine, 4-dimethylamino pyridine (DMAP), diisopropylethylamine for one or more and comprises the alkali of group of the mineral alkali of yellow soda ash, salt of wormwood, sodium hydroxide, potassium hydroxide.
According to replacement [1 of the present invention, 3,5] compound in triazine class or its pharmacy acceptable salt or their solvate or hydrate can be used as cyclooxygenase-2 inhibitors, 5-lipoxygenase inhibitor, cyclooxygenase and 5-lipoxygenase double inhibitor and EGFR enzyme inhibitors.
According to replacement [1 of the present invention, 3,5] compound in triazine class or its pharmacy acceptable salt or their solvate or hydrate can also be used to prevent and/or treat inflammatory disease, can also be used to prevent and/or treat and/or auxiliary for treating cancer, intestinal cancer especially.
It is a kind of with the pharmaceutical composition of compound shown in the formula (I) as active ingredient that the present invention also provides, described pharmaceutical composition comprise the treatment significant quantity suc as formula the replacement [1 shown in (I), 3,5] compound in triazine class or its pharmacy acceptable salt or their solvate or hydrate and at least a pharmaceutically acceptable carrier.Described pharmaceutically acceptable carrier comprises partial glycerol ester mixture, the water of ion-exchanger, aluminum oxide, aluminum stearate, Yelkin TTS, serum protein, buffer substance such as phosphoric acid salt, glycerine, Sorbic Acid, potassium sorbate, saturated vegetable fatty acid, salt or ionogen, Sodium phosphate dibasic, potassium hydrogen phosphate, sodium-chlor, zinc salt, colloided silica, Magnesium Trisilicate, polyvinylpyrrolidone, cellulosic material, polyoxyethylene glycol, Xylo-Mucine, polyacrylic ester, beeswax and lanolin, but be not limited to this.
Aforementioned pharmaceutical compositions can be used to prevent and/or treat diseases associated with inflammation, and prevents and/or treats and/or adjuvant therapy of tumors disease, intestinal cancer especially.
The pharmaceutical composition of The compounds of this invention can be used by following any-mode: oral, spraying sucks, rectal application, nasal cavity applied medicine, the cheek medication, local application, non-enterally administer, as in subcutaneous, vein, intramuscular, intraperitoneal, the sheath, in the ventricle, in the breastbone and intracranial injection or input, or by the medication of a kind of outer planting reservoir.Preferred oral or intramuscular injection when wherein treating inflammation, intraperitoneal or intravenous administration mode.
It may be noted that in addition, the using dosage of The compounds of this invention and using method depend on all multifactor, comprise activity intensity, Time of Administration, metabolic rate, the severity of illness and diagnosis and treatment doctor's the subjective judgement of patient's age, body weight, sex, natural health situation, nutritional status, compound.For example, recommended doses is beginning 5mg～10mg/kg every day, and maintenance dose can reduce to every order 3mg/kg.Capsule: 0.25g/ grain.Injection liquid 0.25g/5ml.Oral liquid: 5g/50ml.
Replace [1 shown in the formula of the present invention (I), 3,5] compound in triazine class or its pharmacy acceptable salt all show significant anti-inflammatory activity in a series of researchs of COX-2 and 5-LOX enzyme level and integral animal level (carrageenin inductive mouse pedal swelling model, carrageenin inductive rat paw edema model, dimethylbenzene inductive Mice Auricle inflammatory model, former of rat assist agent arthritis, secondary affection model).Ames experiment and micronuclei in mice experiment there are security preferably, have no side effect, be used for interior therapeutic and have biocompatibility GI.Pharmaceutical composition of the present invention can be used to prevent and/or treat inflammatory disease, and good curative effect is arranged.
Replace [1 shown in the formula of the present invention (I), 3,5] compound in triazine class or its pharmacy acceptable salt show significant EGFR enzyme inhibition activity and anti-tumor activity in a series of shaker tests of EGFR enzyme level and tumor line cell levels (people's intestinal cancer HT-29 and HCT-116).Therefore, pharmaceutical composition of the present invention can also be used for preventing and/or treating of tumor disease and/or assisting therapy, and good curative effect is arranged.
Description of drawings
Fig. 1 is for replacing the binding kinetics curve of [1,3,5] compound in triazine class and cyclooxygenase (COX-1 and COX-2).Used instrument is BIACORE3000, and analysis software is the Kinetic Analysis among the ApplicationWizard.Cyclooxygenase 2 proteopexies are on the CM5 chip during mensuration.Wherein, Fig. 1-1 and 1-2, Fig. 1-3 and 1-4, Fig. 1-5 and 1-6, Fig. 1-7 and 1-8 and Fig. 1-9 and Fig. 1-10 represent positive compound, compd B, compd B 52, compd B 58 and compound 61 and COX-2 and COX-1 bonded KD value respectively.
Fig. 2 shows the binding kinetics curve of replacement [1,3,5] compound in triazine class and 5-lipoxygenase (5-LOX).Used instrument is BIACORE3000, and analysis software is the Kinetic Analysis among the Application Wizard.The proteopexy of 5-lipoxygenase is on the CM5 chip during mensuration.Wherein, Fig. 2-1 and Fig. 2-2 represents positive compound ETYA and compound B-26 2 and 5-LOX bonded K respectively
D
Fig. 3 respectively organizes the photo of section situation for the gastrointestinal side effect laboratory report.The wherein negative contrast of Fig. 3-1, Fig. 3-2 is an indomethacin, and Fig. 3-3 is Compound C elecoxib, and Fig. 3-4 is a compound B-26 2, and Fig. 3-5 is a compound B-26 8.
Embodiment
Further use the preparation of embodiment formula (I) compound below, but these embodiment never are any limitation of the invention.
Nuclear magnetic resonance spectrum is measured on Bruker AM-400, and mass spectrum carries out on MAT-95 type mass spectrograph.Ultimate analysis is finished by analyzer room, institute of materia medica, Chinese Academy of Sciences Shanghai.Fusing point is measured on electric heating melting point tube or b type melting point tube, and thermometer is not calibrated; It is that 0.8% Xylo-Mucine distilled water solution fully stirs evenly bed board afterwards that thin layer chromatography (tlc) (TLC) adopts silica GF254 (Haiyang Chemical Plant, Qingdao's productions) and concentration, dry, preservation is standby in moisture eliminator after 100～110 oC activate 1～2 hour, ultraviolet lamp (λ: 254nm) colour developing; Column chromatography adopts 200 orders～300 order column chromatography silica gels (Haiyang Chemical Plant, Qingdao's production).
Embodiment 1 aniline list replaces the synthetic of three polychlorostyrene piperazines (4,6-two chloro-N-phenyl-[1,3,5]-triazine-2-amine)
In the 50ml eggplant-shape bottle, 4.726g three polychlorostyrene piperazines are dissolved in 20ml 1, the 4-dioxane stirs the aqueous solution that slowly splashes into 2.337ml aniline and 1.03gNaOH down, stirring reaction 6 hours; Add suction filtration after the frozen water, wash title compound 3.259g, productive rate 53%.Mp 136～138℃。
In the 50ml eggplant-shape bottle, 0.421g three polychlorostyrene piperazines are dissolved in 5ml 1,4-dioxane, the aqueous solution of adding 0.42ml aniline and 0.2gNaOH under the stirring at room; Spend the night at 20-100 ℃ of stirring reaction.Suction filtration, wash pale solid (crude product), after the drying title compound be 0.515g, productive rate 76% (slightly).Mp 197℃。
Embodiment 3 N, N '-phenylbenzene-6-(piperazine-1-replace)-[1,3,5]-triazine-2,4-diamines (compd B) synthetic
In the 100ml eggplant-shape bottle, the 2.915g piperazine hexahydrate is dissolved in butanone, add 1.117g aniline two and replace three polychlorostyrene piperazines, under-40-40 ℃ and alkaline condition, stir 5 hours (or reflux 2～3 hours).Stopped reaction is separated out white precipitate, suction filtration behind the adding cold water.Dried white crystal is got title compound 0.702g through column chromatography for separation, productive rate 54%.Mp 189～192oC。
1HNMR(400Mz，CDCl
3，TMS)δ(ppm)：2.40(br-s，1H)；2.91(t，J＝4.92Hz，4H)；3.83(t，J＝4.92Hz，4H)；7.04(t，J＝7.3 8Hz，2H)；7.10(br-s，2H)；7.30(t，J＝7.91Hz，4H)；7.55(t，J＝7.70Hz，4H).LREI(m/z)：347(M+)，291(50)，279(100)，220，144，119，92，77，56。
In the 50ml eggplant-shape bottle, 0.400g is replaced three polychlorostyrene piperazines to the aniline list is dissolved in 15ml acetone, under the stirring at room with constant pressure funnel toward wherein slowly splashing into the 10ml acetone soln of 0.248g to methylsulfonyl aniline; Add the 10ml aqueous solution of 0.080g sodium hydroxide after dripping off again, stirring at room is the system muddiness after 2 hours, and back flow reaction is spent the night, and separates out white precipitate, and suction filtration gets crude product, column chromatography (EA/PE=1: 1; Rf=0.26) purify pure product title compound 0.170g, productive rate 27%.
Embodiment 5 N-are to methylsulfonyl phenyl-N '-phenyl-6-(piperazine-1-replace)-[1,3,5]-triazine-2,4-diamines (compd B 51) synthetic
In the 50ml eggplant-shape bottle, 0.250g (1.29mmol) piperazine hexahydrate is dissolved in about 20ml acetone, wherein slowly splash into 0.160g (0.426mmol) 6-chloro-N-with constant pressure funnel is past under the stirring at room to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-and triazine-2, the 8ml acetone soln of 4-diamines; Drip off the back and continued stirring at room 3～4 hours, the pressure reducing and steaming proper amount of acetone stirs adding capacity cold water down, separates out white precipitate, and suction filtration is washed to neutrality repeatedly; After the gained crude product drying column chromatography purify title compound 0.102g, productive rate 56%.Mp 166～170℃。
1HNMR(400Mz，CD
3OD，TMS)δ(ppm)：7.95(d，J＝8.8Hz，2H)；7.83(d，J＝8.8Hz，2H)；7.65(d，J＝8.8Hz，2H)；7.30(t，J＝7.8Hz，2H)；7.02(t，J＝7.6Hz 1H)；3.85(t，J＝5.2Hz，4H)；3.10(s，3H)；2.85(t，J＝5.2Hz，4H)。LREI(m/z)：425(M+)，357(100)。
Synthesizing of embodiment 6 4-(4-anilino-6-(piperazine-1-replaces)-[1,3,5]-triazine-2-amino) benzsulfamide (compd B 52)
Except replace 6-chloro-N-with 4-(6-chloro-4-anilino-[1,3,5]-triazine-2-amino) benzsulfamide to methylsulfonyl phenyl-N '-phenyl-[1; 3,5]-triazine-2, outside the 4-diamines; press embodiment 5 operations, obtain title compound white powder solid, productive rate 83%.Mp 209～211℃。
1HNMR(400Mz，CD
3OD，TMS)δ(ppm)：2.88(t，J＝5.12Hz，4H)；3.84(t，J＝5.12Hz，4H)；7.01(t，J＝7.42Hz，1H)；7.29(t，J＝7.42Hz，2H)；7.65(d，J＝8.66Hz，2H)；7.78(d，J＝8.93Hz，2H)；7.88(d，J＝8.93Hz，2H)。
13CNMR(400Mz，DMSO-d6)δ(ppm)：164.5，164，143.5，140，136，128.5，126，122，120.5，119.5，46，44。LREI(m/z)：426(M
+)，384，370，358(100)，299，144，119，92，77，56。HREI: calculated value is (with C
19H
22N
8O
2The S meter) 426.1597; Measured value 426.1584.IR(cm
-1)：3374.9，2950.6，1577.5，1533.2，1500.4，1417.4，1317.2，1269.0，1228.5，1155.2，1099.2，879.4，835.0，804.2，694.3，586.3，540.0。
Embodiment 7 N-phenyl-6-(piperazine-1-replace)-N '-to Trifluoromethoxyphen-l-[1,3,5]-triazine-2,4-diamines (compd B 53) synthetic
6-chloro-N-phenyl-N '-to Trifluoromethoxyphen-l-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1 except using; 3,5]-triazine-2, outside the 4-diamines; press embodiment 5 operations, obtain title compound white powder solid, productive rate 65%.Mp 89～91℃。
1HNMR(400Mz，DMCO-d6，TMS)δ(ppm)：7.90(d，J＝9.07Hz，2H)；7.73(d，J＝7.97Hz，2H)；7.28(t，J＝7.97Hz，2H)；7.23(t，J＝8.80Hz，2H)；6.98(t，J＝7.43Hz，1H)；4.01(t，J＝5.2Hz，4H)；3.15(t，J＝5.2Hz，4H)。
Embodiment 8 N-are to bromophenyl-N '-phenyl-6-(piperazine-1-replace)-[1,3,5]-triazine-2,4-diamines (compd B 54) synthetic
Except using 6-chloro-N-to bromophenyl-N '-phenyl-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2, outside the 4-diamines, presses embodiment 5 operations, obtains title compound white powder solid.Mp 220～222℃。
1HNMR(400Mz，DMSO-d6，TMS)δ(ppm)：7.74(m，4H)；7.43(d，J＝9.0Hz，2H)；7.28(t，J＝8.1Hz，2H)；6.96(t，J＝7.2Hz，1H)；3.69(t，J＝4.8Hz，4H)；2.72(t，J＝4.8Hz，4H)。
Embodiment 9 N-phenyl-6-(piperazine-1-replace)-N '-p-methylphenyl-[1,3,5]-triazine-2,4-diamines (compd B 55) synthetic
Except using 6-chloro-N-phenyl-N '-p-methylphenyl-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2, outside the 4-diamines, obtains title compound white powder solid by embodiment 5 operations.Mp 197～199℃。
1HNMR(400Mz，CDCl
3，TMS)δ(ppm)：7.58(d，J＝Hz，2H)；7.44(d，J＝Hz，2H)；7.32(t，J＝Hz，2H)；7.14(d，J＝Hz，2H)；7.02(t，J＝Hz 1H)；3.82(t，J＝5.2Hz，4H)；2.92(t，J＝5.2Hz，4H)；2.32(s，3H)。
Embodiment 10 N-rubigan-N '-phenyl-6-(piperazine-1-replace)-[1,3,5]-triazine-2,4-diamines (compd B 56) synthetic
Except using 6-chloro-N-rubigan-N '-phenyl-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2, outside the 4-diamines, presses embodiment 5 operations, obtains title compound white powder solid.Mp 208～211℃。
1HNMR(400Mz，CDCl
3，TMS)δ(ppm)：7.55(d，J＝Hz，2H)；7.51(d，J＝Hz，2H)；7.32(t，J＝Hz，2H)；7.26(d，J＝Hz，2H)；7.06(t，J＝Hz 1H)；3.80(t，J＝5.2Hz，4H)；2.92(t，J＝5.2Hz，4H)。
Fluorophenyl-N '-phenyl-6-between embodiment 11 N-(piperazine-1-replace)-[1,3,5]-triazine-2,4-diamines (compd B 57) synthetic
Except using fluorophenyl-N '-phenyl-[1,3,5]-triazine-2 between 6-chloro-N-, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2, outside the 4-diamines, presses embodiment 5 operations, obtains title compound white powder solid.Mp 86～93 ℃。
1HNMR(400Mz，DMCO-d6，TMS)δ(ppm)：7.75(d，J＝Hz，2H)；7.43(dd，J＝Hz，2H)；7.28(t，J＝Hz，2H)；7.28(s，1H)；6.98(t，J＝Hz 1H)；6.71(td，J＝Hz，1H)；3.79(t，J＝5.2Hz，4H)2.81(t，J＝5.2Hz，4H)。
Embodiment 12 N-p-methoxyphenyl-N '-phenyl-6-(piperazine-1-replace)-[1,3,5]-triazine-2,4-diamines (compd B 58) synthetic
Except using 6-chloro-N-p-methoxyphenyl-N '-phenyl-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2, outside the 4-diamines, presses embodiment 5 operations, obtains title compound white powder solid.Mp 198～199℃。
1HNMR(400Mz，CDCl
3，TMS)δ(ppm)：2.91(t，J＝5.0Hz，4H)；3.80(t，J＝5.0Hz，4H)；6.86(d，J＝Hz，2H)；6.94(br-s，1H)；7.02(t，J＝Hz，1H)；7.02(s，1H)；7.29(t，J＝Hz，2H)；7.43(d，J＝5.0Hz，2H)；7.55(d，2H)。
13CNMR(400Mz，DMSO-d6)δ(ppm)：54.0，55.9，65.2(-OCH3)，123.9，130.0，132.0，138.1，143.5，150.2，164.4，174.0，174.8。LREI(m/z)：377(M
+)，321，309(100)，250，149，119，92，77。HREI: calculated value is (with C
20H
23N
7The O meter) 377.1964; Measured value 377.1952.IR (cm
-1): 3322.8,2950.6,2856.1,1608.4,158 1.4,1538.9,1494.6,1436.7,1376.9,1359.6,1272.8,1236.2,1027.9,806.1,750.2. analytical calculation value is (with C
20H
23N
7The O meter) (%): C, 63.64; H, 6.14; N, 25.98.Measured value: C, 63.71; H, 6.17; N, 25.53.
Bromophenyl-N '-phenyl-6-between embodiment 13 N-(piperazine-1-replace)-[1,3,5]-triazine-2,4-diamines (compd B 59) synthetic
Except using bromophenyl-N '-phenyl-[1,3,5]-triazine-2 between 6-chloro-N-, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2, outside the 4-diamines, presses embodiment 5 operations, obtains title compound white powder solid.Mp 89～92℃。
1HNMR(400Mz，CDCl
3，TMS)δ(ppm)：7.98(s，1H)；7.54(dd，J＝8.6&1.0Hz，2H)；7.30(m，3H)；7.14(m，4H)；7.05(t，J＝7.6Hz，1H)；3.80(t，J＝5.2Hz，4H)2.90(t，J＝5.2Hz，4H)。
Except using 6-chloro-N-p-nitrophenyl-N '-phenyl-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2, outside the 4-diamines, presses embodiment 5 operations, obtains title compound white powder solid.Mp 246～247℃。
1HNMR(400Mz，DMCO-d6，TMS)δ(ppm)：2.81(t，J＝5.2Hz，4H)；3.80(t，J＝5.2Hz，4H)；7.01(t，J＝7.4Hz，1H)；7.30(t，J＝8.0Hz，2H)；7.75(d，J＝8.0Hz，2H)；8.08(d，J＝9.2Hz，1H)；8.18(d，J＝9.2Hz，2H)。
Embodiment 15 N-(3, the 4-dimethoxy) phenyl-N '-phenyl-6-(piperazine-1-replace)-[1,3,5]-triazine-2,4-diamines (compound B-26 1) synthetic
Except using 6-chloro-N-(3, the 4-dimethoxy) phenyl-N '-phenyl-[1,3,5]-triazine-2; the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2; outside the 4-diamines, press embodiment 5 operations, obtain title compound white powder solid.Mp 97～100℃。
1HNMR(400Mz，CDCl
3 TMS)δ(ppm)：2.80(t，J＝5.1Hz，4H)；3.79(t，J＝5.1Hz，4H)；3.79(s，6H)；6.86(d，J＝8.7Hz，1H)；6.95(t，J＝8.7Hz，1H)；7.15(d，J＝8.9Hz，1H)；7.25(t，J＝7.4Hz，2H)；7.52(s，1H)；7.78(d，J＝7.7Hz，2H)。LREI(m/z)：407(M
+)，351，339(100)，323，280，144，119，92，77，56。
Embodiment 16 N-(2,3-dihydrobenzo [1,4] two evil-6-replace)-N '-phenyl-6-(piperazine-1-replaces)-[1,3,5]-triazine-2,4-diamines (compound B-26 2) synthetic
Except using 6-chloro-N-(2,3-dihydrobenzo [1,4] two evil-6-replace)-N '-phenyl-[1,3; 5]-and triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N ' phenyl-[1,3,5]-triazine-2; outside the 4-diamines, press embodiment 5 operations, obtain title compound white powder solid.Mp 105～109℃。
1HNMR(400Mz，DMCO-d6，TMS)δ(ppm)：2.79(t，J＝4.9Hz，4H)；3.75(t，J＝4.8Hz，4H)；4.25(m，4H)；6.74(d，J＝8.8Hz，1H)；6.95(t，J＝7.3Hz，1H)；7.10(dd，J＝8.8 and 2.6Hz，1H)；7.25(t，J＝7.4Hz，2H)；7.43(s，1H)；7.78(d，J＝7.7Hz，2H)。
Synthesizing of embodiment 17 4-(4-P-nethoxyaniline base-6-(piperazine-1-replaces)-[1,3,5]-triazine-2-amino) benzsulfamide (compound B-26 3)
Except replace 6-chloro-N-with 4-(6-chloro-4-P-nethoxyaniline base-[1,3,5]-triazine-2-amino) benzsulfamide to methylsulfonyl phenyl-N '-phenyl-[1; 3,5]-triazine-2, outside the 4-diamines; press embodiment 5 operations, obtain title compound white powder solid.Mp 251～254℃。
1HNMR(400Mz，DMSO-d6，TMS)δ(ppm)：(not split clear)2.75(s，4H)；3.70(s，4H)；3.75(s，3H)；6.89(d，J＝6.8Hz，2H)；7.12(s，1H)；7.60(d，J＝9.4Hz，2H)；7.70(d，J＝8.8 Hz，2H)；7.92(d，J＝8.5Hz，2H)。
Embodiment 18 N-are to ethoxyl phenenyl-N '-phenyl-6-(piperazine-1-replace)-[1,3,5]-triazine-2,4-diamines (compound B-26 4) synthetic
Except using 6-chloro-N-to ethoxyl phenenyl-N '-phenyl-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2, outside the 4-diamines, presses embodiment 5 operations, obtains title compound white powder solid.Mp 185～187℃。
1HNMR(400Mz，DMSO-d6，TMS)δ(ppm)：1.38(t，J＝7.0Hz，3H)；2.80(t，J＝5.0Hz，4H)；3.78(t，J＝5.0Hz，4H)；4.02(q，J＝6.8Hz，2H)；6.85(d，J＝6.8Hz，2H)；6.95(t，J＝7.2Hz，1H)；7.26(t，J＝7.6Hz，2H)；7.64(d，J＝7.2Hz，2H)；7.79(d，J＝7.6Hz，2H)。
Embodiment 19 N-(3, the 4-dimethyl) phenyl-N '-phenyl-6-(piperazine 1-replacement)-[1,3,5]-triazine-2,4-diamines (compound B-26 5) synthetic
Except using 6-chloro-N-(3, the 4-dimethyl) phenyl-N '-phenyl-[1,3,5]-triazine-2; the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2; outside the 4-diamines, press embodiment 5 operations, obtain title compound white powder solid.Mp 185～187℃。
1HNMR(400Mz，DMSO-d6，TMS)δ(ppm)：2.19(s，3H)；2.21(s，3H)；3.75(s，3H)；2.79(t，J＝5.2Hz，4H)；3.75(t，＝4.8Hz，4H)；6.95(t，1H)；7.01(d，J＝8.0Hz，1H)；7.25(t，2H)；7.45(d，1H)；7.58(s，1H)；7.78(d，J＝8.4Hz，2H)。
Embodiment 20 N, N '-di-p-methoxy phenyl-6-(piperazine-1-replace)-[1,3,5]-triazine-2,4-diamines (compound B-26 6) synthetic
Except using 6-chloro-N, N '-di-p-methoxy phenyl-[1,3,5]-triazine-2,4-diamines replace 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2, outside the 4-diamines, press embodiment 5 operations, obtain title compound white powder solid.Mp 168～170℃。
1HNMR(400Mz，CDCl
3，TMS)δ(ppm)：2.90(t，J＝5.0Hz，4H)；3.78(t，J＝5.0Hz，4H)；3.80(s，6H)；6.65(br-s，2H)；6.85(d，J＝6.8Hz，4H)；7.43(d，J＝6.8 Hz，4H)。
Embodiment 21 N-o-methoxyphenyl-N '-phenyl-6-(piperazine-1-replace)-[1,3,5]-triazine-2,4-diamines (compound B-26 7) synthetic
Except using 6-chloro-N-o-methoxyphenyl-N '-phenyl-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2, outside the 4-diamines, presses embodiment 5 operations, obtains title compound white powder solid.Mp 89～92℃。
1HNMR(400Mz，CDCl
3，TMS)δ(ppm)：2.92(t，J＝5.2Hz，4H)；3.83(t，J＝5.2Hz，4H)；3.88(s，3H)；6.88(m，2H)；6.97(m，2H)；7.06(t，J＝7.2Hz，1H)；7.33(t，J＝7.0Hz，2H)；7.40(s，1H)；7.58(d，J＝8.8Hz，2H)；8.42(m，1H)。
Embodiment 22 N-are to methylthio group phenyl-N '-phenyl-6-(piperazine-1-replace)-[1,3,5]-triazine-2,4-diamines (compound B-26 8) synthetic
Except using 6-chloro-N-to methylthio group phenyl-N '-phenyl-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2, outside the 4-diamines, presses embodiment 5 operations, obtains title compound white powder solid.Mp 90～94℃。
1HNMR(400Mz，CDCl
3，TMS)δ(ppm)：2.44(s，3H)；2.92(t，J＝5.0Hz，4H)；3.80(t，J＝5.2Hz，4H)；6.93(Br-s，2H)；7.05(t，J＝7.2Hz，1H)；7.25(d，J＝8.8Hz，2H)；7.31(t，J＝8.8&7.2Hz，2H)；7.50(d，J＝6.6Hz，2H)；7.55(d，J＝8.4Hz，2H)。
Embodiment 23 N-benzyl-N '-phenyl-6-(piperazine-1-replace)-[1,3,5]-triazine-2,4-diamines (compd B 70) synthetic
Except using N-benzyl-6-chloro-N '-phenyl-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2, outside the 4-diamines, presses embodiment 5 operations, obtains title compound white powder solid.Mp 72～75 ℃。
1HNMR(400Mz，CDCl
3，TMS)δ(ppm)：7.53(dd，J＝8.4&0.8Hz，2H)；7.30(m，7H)；6.95(t，J＝7.2Hz，1H)；6.90(s，1H)；5.30(s，1H)；4.60(d，J＝6Hz，2H)；3.78(s，4H)；2.85(t，J＝5.Hz，4H)。
Embodiment 24 N-(3, the 4-dichloro) phenyl-N '-phenyl-6-(piperazine-1-replace)-[1,3,5]-triazine-2,4-diamines (compd B 75) synthetic
Except using 6-chloro-N-(3, the 4-dichloro) phenyl-N '-phenyl-[1,3,5]-triazine-2; the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2; outside the 4-diamines, press embodiment 5 operations, obtain title compound white powder solid.Mp 107～109℃。
1HNMR(400Mz，CDCl
3，TMS)δ(ppm)：7.94(d，J＝2.4Hz，1H)；7.54(d，J＝7.6Hz，2H)；7.34(t，J＝8Hz，3H)；7.23(dd，J＝8.4&2.4Hz，1H)；7.07(t，J＝7.2Hz，1H)；6.92(s，1H)；6.90(s，1H)；3.82(t，J＝4.8Hz，4H)；2.94(t，J＝4.8Hz，4H)。
Embodiment 25 N-are to fluorophenyl-N '-phenyl-6-(piperazine-1-replace)-[1,3,5]-triazine-2,4-diamines (compd B 76) synthetic
Except using 6-chloro-N-to fluorophenyl-N '-phenyl-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2, outside the 4-diamines, presses embodiment 5 operations, obtains title compound white powder solid.Mp 191～192℃。
1HNMR(400Mz，CDCl
3，TMS)δ(ppm)：7.55(dd，J＝8.8&1.2Hz，2H)；7.49(m，2H)；7.31(t，J＝8.4Hz，2H)；7.02(m，3H)；6.87(s，1H)；6.83(s，1H)；3.81(t，J＝5.2Hz，4H)；2.92(t，J＝5.2Hz，4H)。
Embodiment 26 N-m-methoxyphenyl-N '-phenyl-6-(piperazine-1-replace)-[1,3,5]-triazine-2,4-diamines (compd B 77) synthetic
Except using 6-chloro-N-m-methoxyphenyl-N '-phenyl-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2, outside the 4-diamines, presses embodiment 5 operations, obtains title compound white powder solid.Mp85～88℃。
1HNMR(400Mz，CDCl
3，TMS)δ(ppm)：7.56(d，J＝7.6Hz，2H)；7.34(m，3H)；7.20(t，J＝8.0Hz，1H)；7.03(t，J＝6.8Hz，2H)；6.92(s，2H)；6.60(dd，J＝8.4&2.8Hz，1H)；3.83(t，J＝5.2Hz，4H)；3.80(s，3H)；2.93(t，J＝5.2Hz，4H)。
Embodiment 27 N, N '-dibenzyl-6-(piperazine-1-replace)-[1,3,5]-triazine-2,4-diamines (compound B-26 9) synthetic
Except using N, N '-dibenzyl-6-chloro-[1,3,5]-triazine-2,4-diamines replace 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2, outside the 4-diamines, press embodiment 5 operations, obtain the colourless thick solid of title compound.
1HNMR(400Mz，CDCl
3，TMS)δ(ppm)：7.18-7.28(m，10H)；4.40(s，4H)；3.58(t，J＝4.4Hz，4H)；2.62(t，J＝4.4Hz，4H)。
Embodiment 28 N, N '-phenylbenzene-6-(4-p-toluenesulfonyl piperazine-1-replace)-[1,3,5]-triazine-2,4-diamines (compd B 81) synthetic
With 200mg N, N '-phenylbenzene-6-(piperazine-1-replaces)-[1,3,5]-and triazine-2, the 4-diamines is dissolved in the 10ml anhydrous pyridine, ice bath stirs down, add Tosyl chloride 120mg in batches, add an amount of frozen water toward system after 3 hours, stir a quarter after suction filtration get the yellow-green colour solid crude product, purify through column chromatography, obtain pure product white powder 260mg.Yield 90%.Mp 214～218℃。
1HNMR(400Mz，DMCO-d6，TMS)δ(ppm)：7.72(d，J＝7.6Hz，2H)；7.65(d，J＝7.6Hz，2H)；7.43(d，J＝8Hz，2H)；7.25(t，J＝7.6Hz，4H)；6.95(t，J＝7.6Hz，2H)；3.90(t，J＝5.2Hz，4H)；2.93(t，J＝5.2Hz，4H)；2.38(s，3H)。LREI(m/z)：501(M+)，346(100)。
Embodiment 29 N-p-methoxyphenyl-N '-phenyl-6-(4-p-toluenesulfonyl piperazine-1-replace)-[1,3,5]-triazine-2,4-diamines (compd B 82) synthetic
Except using N-p-methoxyphenyl-N '-phenyl-6-(piperazine-1-replaces)-[1,3,5]-triazine-2, the 4-diamines replaces N, N '-phenylbenzene-6-(piperazine-1-replaces)-[1,3,5]-triazine-2, outside the 4-diamines, press embodiment 28 operations, obtain title compound white powder solid.Mp 114～117℃。
1HNMR(400Mz，CDCl
3，TMS)δ(ppm)：7.62(d，J＝8.4Hz，2H)；7.47(d，J＝7.6Hz，2H)；7.36(d，J＝8.8Hz，2H)；7.30(m，4H)；7.02(t，J＝7.6Hz，1H)；6.84(d，J＝6.8Hz，2H)；3.91(t，J＝5.2Hz，4H)；3.80(s，3H)；3.01(t，J＝5.2Hz，4H)；2.40(s，3H)。
Except replacing the Tosyl chloride with methylsulfonyl chloride, press embodiment 29 operations, obtain title compound white powder solid.Mp 212～214℃。
1HNMR(400Mz，CDCl
3，TMS)δ(ppm)：7.52(d，J＝8.4Hz，2H)；7.42(d，J＝6.8Hz，2H)；7.31(t，J＝7.6Hz，2H)；7.05(t，J＝7.6Hz，1H)；6.95(s，1H)；6.87(d，J＝6.8Hz，2H)；6.85(s，1H)；3.95(t，J＝4.8Hz，4H)；3.80(s，3H)；3.25(t，J＝4.8Hz，4H)；2.77(s，3H)。
Embodiment 31 N, N '-phenylbenzene-6-(4-methylsulfonyl piperazine-1-replace)-[1,3,5]-triazine-2,4-diamines (compd B 84) synthetic
Except replacing the Tosyl chloride with methylsulfonyl chloride, press embodiment 28 operations, obtain title compound.Mp 216～218℃。
1HNMR(400Mz，CDCl
3，TMS)δ(ppm)：7.55(d，J＝7.6Hz，4H)；7.33(t，J＝7.6Hz，4H)；7.07(t，J＝7.6Hz，2H)；6.95(s，2H)；3.97(t，J＝4.8Hz，4H)；3.27(t，J＝4.8Hz，4H)；2.80(s，3H)。
Embodiment 32 4-[4-aniline-6-(4-methylsulfonyl piperazine-1-replace)-[1,3,5]-triazine-2-amino] benzsulfamide (compd B 85) synthetic
Except replacing N with 4-(4-anilino-6-(piperazine-1-replaces)-[1,3,5]-triazine-2-amino) benzsulfamide, N '-phenylbenzene-6-(piperazine-1-replaces)-[1,3,5]-triazine-2, outside the 4-diamines, press embodiment 30 operations, obtain title compound white powder solid.Mp 247～252℃。
1HNMR(400Mz，DMSO-d6，TMS)δ(ppm)：9.60(s，1H)；9.33(s，1H)；7.92(d，J＝9.2Hz，2H)；7.72(d，J＝9.2Hz，4H)；7.31(t，J＝8.0Hz，2H)；7.16(s，2H)；7.02(t，J＝7.6Hz，1H)；3.91(t，J＝5.2Hz，4H)；3.22(t，J＝5.2Hz，4H)；2.91(s，3H)。
Embodiment 33 4-[4-anilino-6-(4-ethanoyl piperazine-1-replace)-[1,3,5]-triazine-2-amino] benzsulfamide (compound B-11 96) synthetic
Except using 4-(4-anilino-6-(piperazine-1-replaces)-[1,3,5]-and triazine-2-amino) benzsulfamide replacement N, N '-phenylbenzene-6-(piperazine-1-replaces)-[1,3,5]-triazine-2, the 4-diamines, Acetyl Chloride 98Min. replaces outside the Tosyl chloride, presses embodiment 28 operations, obtains title compound white powder solid.
1HNMR(300Mz，DMSO-d6，TMS)δ(ppm)：7.93(d，J＝8.7Hz，2H)；7.69-7.24(m，4H)；7.31(t，J＝7.5Hz，2H)；7.00(t，J＝7.5Hz，1H)；3.73-3.82(m，4H)；3.54(m，4H)；2.06(s，3H)。
Except using 4-(4-anilino-6-(piperazine-1-replaces)-[1,3,5]-and triazine-2-amino) benzsulfamide replacement N, N '-phenylbenzene-6-(piperazine-1-replaces)-[1,3,5]-triazine-2, the 4-diamines, ethyl bromoacetate replaces outside the Tosyl chloride, presses embodiment 28 operations, obtains title compound white powder solid.
1HNMR(300Mz，DMSO-d6，TMS)δ(ppm)：7.93(d，J＝8.1Hz，2H)；7.69-7.74(m，4H)；7.31(t，J＝7.8Hz，2H)；7.01(t，J＝7.2Hz，1H)；4.10(q，J＝7.2Hz，2H)；3.79(m，4H)；2.59(m，4H)；1.20(t，J＝7.2Hz，3H)。
Embodiment 35 N-rubigan-6-(morpholine-4-replace)-N '-phenyl-[1,3,5]-triazine-2,4-diamines (Compound C 151) synthetic
Except using 6-chloro-N-rubigan-N '-phenyl-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1; 3,5]-and triazine-2, the 4-diamines; outside the morpholino TEPA piperazine, press embodiment 5 operations, obtain title compound white powder solid.Mp 226℃。
1HNMR(300Mz，CDCl
3，TMS)δ(ppm)：7.55(d，J＝7.8Hz，2H)；7.50(d，J＝6.6Hz，2H)；7.33(t，J＝7.8Hz，2H)；7.27(dd，J＝1.5&7.2Hz，2H)；7.08(t，J＝7.2Hz1H)；7.02(Br-s，2H)；3.74～3.84(m，8H)。
Embodiment 36 N-o-methoxyphenyl-6-(morpholine-4-replace)-N '-phenyl-[1,3,5]-triazine-2,4-diamines (Compound C 152) synthetic
Except using 6-chloro-N-o-methoxyphenyl-N '-phenyl-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1; 3,5]-and triazine-2, the 4-diamines; outside the morpholino TEPA piperazine, press embodiment 5 operations, obtain title compound white powder solid.Mp 162～163℃。
1HNMR(300Mz，DMSO-d6，TMS)δ(ppm)：8.13(Br-s，1H)；7.74(Br-s，1H)；7.70(d，J＝8.1Hz，2H)；7.26(t，J＝8.1Hz，2H)；7.05(d，J＝3.6Hz，2H)；6.92-6.98(m，2H)；3.86(s，3H)；3.73(m，4H)；3.66(m，4H)。
Embodiment 37 N-m-methoxyphenyl-6-(morpholine-4-replace)-N '-phenyl-[1,3,5]-triazine-2,4-diamines (Compound C 153) synthetic
Except using 6-chloro-N-m-methoxyphenyl-N '-phenyl-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1; 3,5]-and triazine-2, the 4-diamines; outside the morpholino TEPA piperazine, press embodiment 5 operations, obtain title compound white powder solid.Mp 164℃。
1HNMR(300Mz，DMSO-d6，TMS)δ(ppm)：7.75(d，J＝7.5Hz，2H)；7.43(s，1H)；7.27(m，3H)；7.17(t，J＝8.1Hz，1H)；6.96(t，J＝7.5Hz，1H)；6.55(d，J＝7.5Hz，1H)；3.65～3.75(m，11H)。
Embodiment 38 N, N '-di-p-methoxy phenyl-6-(morpholine-4-replace)-[1,3,5]-triazine-2,4-diamines (Compound C 154) synthetic
Except using 6-chloro-N, N '-di-p-methoxy phenyl-[1,3,5]-triazine-2; the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2,4-diamines; outside the morpholino TEPA piperazine, press embodiment 5 operations, obtain title compound white powder solid.Mp 192～193℃。
1HNMR(300Mz，CDCl
3，TMS)δ(ppm)：7.42(d，J＝8.7Hz，4H)；6.86(d，J＝8.7Hz，4H)；6.78(s，2H)；3.72～3.80(m，14H)。
Embodiment 39 6-(morpholine-4-replace)-N-phenyl-N '-to Trifluoromethoxyphen-l-[1,3,5]-triazine-2,4-diamines (Compound C 155) synthetic
6-chloro-N-phenyl-N '-to Trifluoromethoxyphen-l-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1 except using; 3,5]-and triazine-2, the 4-diamines; outside the morpholino TEPA piperazine, press embodiment 5 operations, obtain title compound white powder solid.Mp178～180℃。
1HNMR(300Mz，DMSO-d6，TMS)δ(ppm)：7.85(d，J＝9.0Hz，2H)；7.72(d，J＝7.8Hz，2H)；7.28(m，4H)；6.97(t，J＝7.5Hz，1H)；3.64～3.85(m，8H)。
Embodiment 40 N-are to bromophenyl-6-(morpholine-4-replace)-N '-phenyl-[1,3,5]-triazine-2,4-diamines (Compound C 156) synthetic
Except with N-to bromophenyl-6-chloro-N '-phenyl-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1; 3,5]-and triazine-2, the 4-diamines; outside the morpholino TEPA piperazine, press embodiment 5 operations, obtain title compound white powder solid.Mp 227～228℃。
1HNMR(300Mz，DMSO-d6，TMS)δ(ppm)：7.73(m，4H)；7.43(d，J＝9.0Hz，2H)；7.29(t，J＝7.8Hz，2H)；6.98(t，J＝7.5Hz，1H)；3.65～3.74(m，8H)。
Embodiment 41 N-are to ethoxyl phenenyl-6-(morpholine-4-replace)-N '-phenyl-[1,3,5]-triazine-2,4-diamines (Compound C 157) synthetic
Except using 6-chloro-N-to ethoxyl phenenyl-N '-phenyl-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1; 3,5]-and triazine-2, the 4-diamines; outside the morpholino TEPA piperazine, press embodiment 5 operations, obtain title compound white powder solid.Mp 174～175℃。
1HNMR(300Mz，DMSO-d6，TMS)δ(ppm)：7.74(d，J＝7.8Hz，2H)；7.59(d，J＝9.0Hz，2H)；7.27(t，J＝7.8Hz，2H)；6.95(t，J＝7.2Hz，1H)；6.85(d，J＝9.0Hz，2H)；3.98(q，J＝6.9Hz，2H)；3.64～3.73(m，8H)；1.31(t，J＝6.9Hz，3H)。
Synthesizing of embodiment 42 4-(6-(morpholine-4-replaces)-4-anilino-[1,3,5]-triazine-2-amino) benzamide (Compound C 158)
Except replace 6-chloro-N-with 4-(6-chloro-4-anilino-[1,3,5]-triazine-2-amino) benzamide to methylsulfonyl phenyl-N '-phenyl-[1; 3,5]-and triazine-2, the 4-diamines; outside the morpholino TEPA piperazine, press embodiment 5 operations, obtain title compound white powder solid.Mp 222～223℃。
1HNMR(300Mz，DMSO-d6，TMS)δ(ppm)：7.78～7.85(m，5H)；7.73(d，J＝8.1Hz，2H)；7.30(t，J＝8.1Hz，2H)；7.18(s，1H)；6.99(t，J＝7.5Hz，1H)；3.65～3.76(m，8H)。
Embodiment 43 N-are to fluorophenyl-N '-p-methoxyphenyl-6-(morpholine-4-replace)-[1,3,5]-triazine-2,4-diamines (Compound C 159) synthetic
Except using 6-chloro-N-to fluorophenyl-N '-p-methoxyphenyl-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1; 3,5]-and triazine-2, the 4-diamines; outside the morpholino TEPA piperazine, press embodiment 5 operations, obtain title compound white powder solid.Mp 200～201℃。
1HNMR(300Mz，CDCl
3，TMS)δ(ppm)：7.47(m，2H)；7.41(d，J＝9.0Hz，2H)；6.70(t，J＝8.7Hz，2H)；6.87(d，J＝9.3Hz，2H)；6.82(s，1H)；6.77(s，1H)；3.71～3.81(m，8H)。
Embodiment 44 N-are to fluorophenyl-6-(morpholine-4-replace)-N '-to Trifluoromethoxyphen-l-[1,3,5]-triazine-2,4-diamines (Compound C 160) synthetic
Except with 6-chloro-N-to fluorophenyl-N '-to Trifluoromethoxyphen-l-[1; 3; 5]-triazine-2; the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2; the 4-diamines; outside the morpholino TEPA piperazine, press embodiment 5 operations, obtain title compound white powder solid.Mp198～199℃。
1HNMR(300Mz，CDCl
3，TMS)δ(ppm)：7.55(d，J＝9.3Hz，2H)；7.47(m，2H)；7.15(d，J＝8.7Hz，2H)；7.01(t，J＝9.0Hz，2H)；6.88(s，1H)；6.80(s，1H)；3.73～3.82(m，8H)。
Embodiment 45 N-are to fluorophenyl-6-(morpholine-4-replace)-N '-phenyl-[1,3,5]-triazine-2,4-diamines (Compound C 161) synthetic
Except using 6-chloro-N-to fluorophenyl-N '-phenyl-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1; 3,5]-and triazine-2, the 4-diamines; outside the morpholino TEPA piperazine, press embodiment 5 operations, obtain title compound white powder solid.Mp 210～211℃。
1HNMR(300Mz，CDCl
3，TMS)δ(ppm)：7.54(dd，J＝1.2&8.7Hz，2H)；7.48(m，2H)；7.31(t，J＝8.4Hz，2H)；6.97～7.08(m，3H)；6.96(s，1H)；6.91(s，1H)；3.73～3.82(m，8H)。
Synthesizing of embodiment 46 4-(4-P-nethoxyaniline base-6-(morpholine-4-replaces)-[1,3,5]-triazine-2-amino) benzsulfamide (Compound C 162)
Except replace 6-chloro-N-with 4-(6-chloro-4-P-nethoxyaniline base-[1,3,5]-triazine-2-amino) benzsulfamide to methylsulfonyl phenyl-N '-phenyl-[1; 3,5]-and triazine-2, the 4-diamines; outside the morpholino TEPA piperazine, press embodiment 5 operations, obtain title compound white powder solid.Mp 257～258℃。
1HNMR(300Mz，DMSO-d6，TMS)δ(ppm)：7.91(d，J＝7.2Hz，2H)；7.68(d，J＝8.7Hz，2H)；7.57(d，J＝8.7Hz，2H)；6.88(t，J＝7.2Hz，2H)；3.73(m，7H)；3.65(m，4H)。
Embodiment 47 N-p-methoxyphenyl-N '-(2-(morpholine-4-replace) ethyl)-N "-phenyl-[1,3,5]-triazine-2,4,6-triamine (Compound C 163) synthetic
Except using 6-chloro-N-p-methoxyphenyl-N '-phenyl-[1; 3; 5]-triazine-2; the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2; the 4-diamines; 2-(morpholine-4-replaces) ethamine replaces outside the piperazine, presses embodiment 5 operations, obtains title compound white powder solid.Mp 147～148℃。
1HNMR(300Mz，CDCl
3，TMS)δ(ppm)：7.57(d，J＝7.2Hz，2H)；7.45(d，J＝8.7Hz，2H)；7.30(t，J＝8.7Hz，2H)；7.04(t，J＝7.2Hz，1H)；6.89(Br-s，1H)；6.87(d，J＝9.0Hz，2H)；6.85(Br-s，1H)；5.54(t，J＝7.2Hz，1H)；3.81(s，3H)；3.71(t，J＝4.8Hz，4H)；3.51(q，J＝5.7Hz，2H)；2.56(t，J＝5.4Hz，2H)；2.47(t，J＝4.5Hz，4H)。LREI(m/z)：421(M
+)，309(100)。
Embodiment 48 N-(2-(morpholine-4-replace) ethyl)-N ', N "-phenylbenzene-[1,3,5]-triazine-2,4,6-triamine (Compound C 164) synthetic
Except using 6-chloro-N, N '-phenylbenzene-[1,3,5]-triazine-2; the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2,4-diamines; 2-(morpholine-4-replaces) ethamine replaces outside the piperazine, presses embodiment 5 operations, obtains title compound white powder solid.Mp 167～168℃。
1HNMR(300Mz，CDCl
3，TMS)δ(ppm)：7.59(d，J＝7.2Hz，4H)；7.34(t，J＝7.5Hz，4H)；7.07(t，J＝7.2Hz，2H)；6.96(s，2H)；5.58(t，J＝6.0Hz，1H)；3.74(t，J＝4.8Hz，4H)；3.55(q，J＝5.7Hz，2H)；2.59(t，J＝6.0Hz，2H)；2.50(t，J＝4.8Hz，4H)。LREI(m/z)：391(M
+)，279(100)。
Embodiment 49 N-benzyl-N '-(2-(morpholine-4-replace) ethyl)-N "-phenyl-[1,3,5]-triazine-2,4,6-triamine (Compound C 165) synthetic
Except using N-benzyl-6-chloro-N '-phenyl-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1; 3,5]-and triazine-2, the 4-diamines; 2-(morpholine-4-replaces) ethamine replaces outside the piperazine, presses embodiment 5 operations, obtains title compound thickness oily matter.
1HNMR(300Mz，CDCl
3，TMS)δ(ppm)：7.54(m，2H)；7.04～7.34(m，7H)；7.01(t，J＝7.2Hz，1H)；6.87(Br-s，1H)；5.44(t，J＝7.2Hz，1H)；5.38(Br-s，1H)；4.62(d，J＝5.7Hz，2H)；3.71(t，J＝4.8Hz，4H)；3.49(q，J＝5.7Hz，2H)；2.54(t，J＝6.0Hz，2H)；2.46(t，J＝4.8Hz，4H)。
Embodiment 50 N-benzyl-6-(morpholine-4-replace)-N '-phenyl-[1,3,5]-triazine-2,4-diamines (Compound C 166) synthetic
Except using N-benzyl-6-chloro-N '-phenyl-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2, and the 4-diamines outside the morpholino TEPA piperazine, is pressed embodiment 5 operations, obtains the title compound white solid.Mp 121～123℃。
1HNMR(300Mz，CDCl
3，TMS)δ(ppm)：7.53(d，J＝8.7Hz，2H)；7.25～7.34(m，7H)；7.01(t，J＝7.2Hz，1H)；6.87(s，1H)；5.31(Br-s，1H)；4.61(d，J＝5.7Hz，2H)；3.69～3.77(m，8H)。
Embodiment 51 N-benzyl-6-(morpholine-4-replace)-N '-p-methylphenyl-[1,3,5]-triazine-2,4-diamines (Compound C 167) synthetic
Except using N-benzyl-6-chloro-N '-p-methylphenyl-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1; 3,5]-and triazine-2, the 4-diamines; outside the morpholino TEPA piperazine, press embodiment 5 operations, obtain title compound white sticky solid.
1HNMR(300Mz，DMSO-d6，TMS)δ(ppm)：7.73(d，J＝7.8Hz，1H)；7.63(d，J＝8.1Hz，1H)；7.22(m，4H)；7.11(d，J＝8.1Hz，1H)；6.89(t，J＝7.2Hz，1H)；4.42(d，J＝13.2Hz，2H)；3.60～3.67(m，8H)；2.25(s，3H)。
Synthesizing of embodiment 52 4-(4-benzamido group-6-(morpholine-4-replaces)-[1,3,5]-triazine-2-amino) benzsulfamide (Compound C 168)
Except replace 6-chloro-N-with 4-(4-benzamido group-6-chloro-[1,3,5]-triazine-2-amino) benzsulfamide to methylsulfonyl phenyl-N '-phenyl-[1; 3,5]-and triazine-2, the 4-diamines; outside the morpholino TEPA piperazine, press embodiment 5 operations, obtain title compound white sticky solid.Mp 205～206℃。
1HNMR(300Mz，DMSO-d6，TMS)δ(ppm)：7.89(d，J＝8.7Hz，1H)；7.75(d，J＝8.7Hz，1H)；7.63(m，2H)；7.17～731(m，5H)；4.46(d，J＝12Hz，2H)；3.59～3.66(m，8H)。
Embodiment 53 N-benzyl-N '-p-methoxyphenyl-6-(morpholine-4-replace)-[1,3,5]-triazine-2,4-diamines (Compound C 169) synthetic
Except using N-benzyl-6-chloro-N '-p-methoxyphenyl-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1; 3,5]-and triazine-2, the 4-diamines; outside the morpholino TEPA piperazine, press embodiment 5 operations, obtain the title compound white solid.Mp 152～153℃。
1HNMR(300Mz，CDCl
3，TMS)δ(ppm)：7.40(d，J＝9.0Hz，2H)；7.26～7.33(m，5H)；6.83(d，J＝9.0Hz，2H)；6.75(Br-s，1H)；5.27(Br-s，1H)；4.59(d，J＝5.7Hz，2H)；3.68～3.78(m，11H)。
Embodiment 54 N-benzyl-N '-to fluorophenyl-6-(morpholine-4-replace)-[1,3,5]-triazine-2,4-diamines (Compound C 170) synthetic
N-benzyl-6-chloro-N '-to fluorophenyl-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1 except using; 3,5]-and triazine-2, the 4-diamines; outside the morpholino TEPA piperazine, press embodiment 5 operations, obtain the title compound white solid.Mp 143～144℃。
1HNMR(300Mz，CDCl
3，TMS)δ(ppm)：7.45(m，2H)；7.25～7.33(m，5H)；6.96(t，J＝8.7Hz，2H)；6.78(s，1H)；5.28(t，J＝5.7Hz，1H)；4.59(d，J＝6.0Hz，2H)；3.69～3.76(m，8H)。
Embodiment 55 N, N '-dibenzyl-6-(morpholine-4-replace)-[1,3,5]-triazine-2,4-diamines (Compound C 176) synthetic
Except using N, N '-dibenzyl-6-chloro-[1,3,5]-triazine-2,4-diamines replace 6-chloro-N-to methylsulfonyl phenyl-N ' phenyl-[1,3,5]-triazine-2, and the 4-diamines outside the morpholino TEPA piperazine, is pressed embodiment 5 operations, obtains the title compound white solid.Mp 134～136℃。
1HNMR(300Mz，CDCl
3，TMS)δ(ppm)：7.23～7.3 1(m，10H)；5.1 7(Br-s，2H)；4.57(d，J＝5.7Hz，4H)；3.66～3.73(m，8H)。LREI(m/z)：376(M
+)，73(100)。
Embodiment 56 2-[4,6-hexichol amido-[1,3,5]-triazine-2-amino] ethanol (Compound C 177) synthetic
Except using 6-chloro-N, N '-phenylbenzene-[1,3,5]-triazine-2; the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2,4-diamines; the 2-monoethanolamine replaces outside the piperazine, presses embodiment 5 operations, obtains the title compound white solid.Mp 182～183℃。
1HNMR(300Mz，DMSO-d6，TMS)δ(ppm)：7.80(d，J＝8.1Hz，4H)；7.25(t，J＝7.8Hz，4H)；6.94(t，J＝7.5Hz，3H)；3.54(t，J＝6.0Hz，2H)；3.38(q，J＝5.7Hz，2H)。LREI(m/z)：322(M
+)，304(100)。
Embodiment 57 2-[4-P-nethoxyaniline base-6-anilinos-[1,3,5]-triazine-2-amino] ethanol (Compound C 179) synthetic
Except using 6-chloro-N-p-methoxyphenyl-N '-phenyl-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1; 3,5]-and triazine-2, the 4-diamines; the 2-monoethanolamine replaces outside the piperazine, presses embodiment 5 operations, obtains the title compound white solid.Mp 173～174℃。
1HNMR(300Mz，CDCl
3，TMS)δ(ppm)：7.55(d，J＝8.7Hz，2H)；7.42(d，J＝9.0Hz，2H)；7.31(t，J＝7.5Hz，2H)；7.06(t，J＝7.5Hz，1H)；6.86(d，J＝7.8Hz，2H)；6.79(s，1H)；6.70(s，1H)；5.45(t，J＝5.4Hz，1H)；3.78～3.82(m，5H)；3.58(q，J＝5.1Hz，2H)。LREI(m/z)：352(M
+)，321(100)。
Embodiment 58 2-[4-benzamido group-6-anilino-[1,3,5]-triazine-2-amino] ethanol (Compound C 180) synthetic
Except using N-benzyl-6-chloro-N '-phenyl-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1; 3,5]-and triazine-2, the 4-diamines; the 2-monoethanolamine replaces outside the piperazine, presses embodiment 5 operations, obtains the thick solid of title compound.
1HNMR(300Mz，CDCl
3，TMS)δ(ppm)：7.49(d，J＝8.4Hz，2H)；7.22～7.31(m，7H)；7.01(t，J＝7.5Hz，1H)；4.56(d，J＝6.0Hz，2H)；3.73(m，2H)；3.52(m，2H)。LREI(m/z)：336(M
+)，305(100)。
Embodiment 59 4,6-two (morpholine-4-replace)-N-phenyl-[1,3,5]-triazine-2-amine (Compound C 171) synthetic
Except with 4,6-two chloro-N-phenyl-[1,3,5]-triazine-2-amine replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2, and the 4-diamines outside the morpholino TEPA piperazine, press embodiment 5 and operated, and obtains the title compound white solid.Mp 168～170℃。
1HNMR(300Mz，CDCl
3，TMS)δ(ppm)；7.55(d，J＝10.4Hz，2H)；7.30(t，J＝9.6Hz，2H)；7.02(t，J＝10.0Hz，1H)；6.74(s，1H)；3.78(m，8H)；3.73(m，8H)。
Embodiment 60 4-[4,6-two (morpholine-4-replace)-[1,3,5]-triazine-2-amino] benzsulfamide (Compound C 172) synthetic
Except using 4-[4,6-two chloro-[1,3,5]-triazine-2-amino] benzsulfamide replacement 6-chloro-N-is to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2, and the 4-diamines outside the morpholino TEPA piperazine, is pressed embodiment 5 operations, obtains the title compound white solid.Mp is more than 300 ℃.
1HNMR(300Mz，DMSO-d6，TMS)δ(ppm)：7.84(d，J＝9.0Hz，2H)；7.71(d，J＝8.7Hz，2H)；3.71(m，8H)；3.64(m，8H)。
Embodiment 61 N-p-methoxyphenyls-4,6-two (morpholine-4-replace)-[1,3,5]-triazine-2-amine (Compound C 173) synthetic
Except with 4,6-two chloro-N-p-methoxyphenyls-[1,3,5]-triazine-2-amine replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2, and the 4-diamines outside the morpholino TEPA piperazine, press embodiment 5 and operated, and obtains the title compound white solid.Mp 206～207℃。
1HNMR(300Mz，DMSO-d6，TMS)δ(ppm)：7.55(d，J＝9.0Hz，2H)；6.84(d，J＝6.6Hz，2H)；3.60～3.70(m，19H)。
Embodiment 62 N-are to fluorophenyl-4,6-two (morpholine-4-replaces)-[1,3,5]-triazine-2-amine (Compound C 174) synthetic
Except with 4,6-two chloro-N-replace 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2 to fluorophenyl-[1,3,5]-triazine-2-amine, and the 4-diamines outside the morpholino TEPA piperazine, press embodiment 5 and operated, and obtains the title compound white solid.Mp 199～200℃。
1HNMR(300Mz，CDCl
3，TMS)δ(ppm)：7.48(m，2H)；7.01(t，J＝8.4Hz，2H)；3.71～3.81(m，16H)。
Embodiment 63 N-benzyls-4, the synthetic of 6-two (morpholine-4-replaces)-[1,3,5]-triazine-2-amine (Compound C 175) is synthetic
Except with 4,6-two chloro-N-benzyls-[1,3,5]-triazine-2-amine replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2, and the 4-diamines outside the morpholino TEPA piperazine, press embodiment 5 and operated, and obtains the title compound white solid.Mp 160～161℃。
1HNMR(300Mz，CDCl
3，TMS)δ(ppm)：7.36(t，J＝6.6Hz，1H)；7.17～7.29(m，5H)；4.40(d，J＝6.3Hz，2H)；3.55～3.61(m，16H)。
Synthesizing of embodiment 64 4-chloro-6-(morpholine-4-replaces)-N-phenyl-[1,3,5]-triazine-2-amine (Compound C 181)
Except replacing the methylsulfonyl aniline with morpholino, press embodiment 4 operations, obtain the title compound white solid.Mp 198～199℃。
1HNMR(300Mz，CDCl
3，TMS)δ(ppm)：7.53(d，J＝8.1Hz，2H)；7.37(t，J＝8.1Hz，2H)；7.26(s，1H)；7.14(t，J＝7.5Hz，1H)；3.87(m，4H)；3.77(m，4H)。LREI(m/z)：291(M
+，100)。
Embodiment 65 4-[4-chloro-6-(morpholine-4-replace)-[1,3,5]-triazine-2-amino] benzsulfamide (Compound C 182) synthetic
Except using 4-[4,6-two chloro-[1,3,5]-triazine-2-amino] benzsulfamide replacement 4,6-two chloro-N-phenyl-[1,3,5]-triazine-2-amine, morpholino is pressed embodiment 4 operations for to outside the methylsulfonyl aniline, obtains the title compound white solid.Mp is more than 300 ℃.
1HNMR(300Mz，DMSO-d6，TMS)δ(ppm)：7.55～7.83(m，4H)；3.64～3.77(m，8H)。
Synthesizing of embodiment 66 4-chloro-N-p-methoxyphenyl-6-(morpholine-4-replaces)-[1,3,5]-triazine-2-amine (Compound C 183)
Except with 4,6-two chloro-N-p-methoxyphenyls-[1,3,5]-triazine-2-amine replaces 4,6-two chloro-N-phenyl-[1,3,5]-triazine-2-amine, and morpholino press embodiment 4 and is operated for to outside the methylsulfonyl aniline, obtains the title compound white solid.Mp 164～165℃。
1HNMR(300Mz，CDCl
3，TMS)δ(ppm)：7.39(d，J＝9.0Hz，2H)；7.10(s，1H)；6.90(d，J＝8.7Hz，2H)；3.72～3.85(m，11H)。
Embodiment 67 4-chloro-N-are synthetic to fluorophenyl-6-(morpholine-4-replaces)-[1,3,5]-triazine-2-amine (Compound C 184)
Except with 4,6-two chloro-N-replace 4 to fluorophenyl-[1,3,5]-triazine-2-amine, 6-two chloro-N-phenyl-[1,3,5]-triazine-2-amine, and morpholino press embodiment 4 and is operated for to outside the methylsulfonyl aniline, obtains the title compound white solid.Mp 234～235℃。
1HNMR(300Mz，CDCl
3，TMS)δ(ppm)：7.42～7.47(m，2H)；7.04(t，J＝8.7Hz，2H)；3.72～3.86(m，8H)。
Synthesizing of embodiment 68 N-benzyl-4-chloro-6-(morpholine-4-replaces)-[1,3,5]-triazine-2-amine (Compound C 185)
Except using N-benzyl-4,6-two chloro-[1,3,5]-triazine-2-amine replaces 4,6-two chloro-N-phenyl-[1,3,5]-triazine-2-amine, and morpholino is pressed embodiment 4 operations for to outside the methylsulfonyl aniline, obtains the title compound white solid.Mp 169～170℃。
1HNMR(300Mz，DMSO-d6，TMS)δ(ppm)：7.23～7.33(m，5H)；4.45(d，J＝6.3Hz，2H)；3.56～3.67(m，8H)。
Embodiment 69 2-chloro-4,6-two (morpholine-4-replace)-[1,3,5]-triazine (Compound C 188) synthetic
Except replacing the aniline with morpholino, press embodiment 2 operations, obtain the title compound white solid.Mp 175℃。
1HNMR(300Mz，CDCl
3，TMS)δ(ppm)：3.70～3.78(m，16H)。
Embodiment 70 2-methoxyl groups-4,6-two (morpholine-4-replace)-[1,3,5]-triazine (Compound C 186) synthetic
Except using 2-chloro-4,6-two (morpholine-4-replaces)-[1,3,5]-triazine replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1; 3,5]-and triazine-2, the 4-diamines, sodium methylate replaces piperazine; methyl alcohol replaces outside the acetone, presses embodiment 5 operations, obtains the title compound white solid.Mp 161～162℃。
1HNMR(300Mz，CDCl
3，TMS)δ(ppm)：3.90(s，3H)；3.70～3.82(m，16H)。
Synthesizing of embodiment 71 N-benzyl-4-methoxyl group-6-(morpholine-4-replaces)-[1,3,5]-triazine-2-amine (Compound C 187)
Except replacing 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3 with N-benzyl-4-chloro-6-(morpholine-4-replaces)-[1,3,5]-triazine-2-amine; 5]-and triazine-2, the 4-diamines, sodium methylate replaces piperazine; methyl alcohol replaces outside the acetone, presses embodiment 5 operations, obtains the title compound white solid.Mp174～175℃。
1HNMR(300Mz，CDCl
3，TMS)δ(ppm)：7.27～7.35(m，5H)；4.61(s，2H)；3.67～3.90(m，11H)。LREI(m/z)：301(M
+，100)。
Embodiment 72 2, and 4,6-three (morpholine-4-replace)-[1,3,5]-triazine (Compound C 189) synthetic
Except replacing the aniline with morpholino, press embodiment 2 operations, obtain the title compound white solid.273 ℃ of volatilizations of Mp.
1HNMR(300Mz，CDCl
3，TMS)δ(ppm)：3.69～3.78(m，24H)。
Synthesizing of embodiment 73 4-methoxyl group-N-p-methoxyphenyl-6-(morpholine-4-replaces)-[1,3,5]-triazine-2-amine (Compound C 190)
Except using 4-chloro-N-p-methoxyphenyl-6-(morpholine-4-replaces)-[1; 3; 5]-triazine-2-amine replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1; 3,5]-and triazine-2, the 4-diamines; sodium methylate replaces piperazine; methyl alcohol replaces outside the acetone, presses embodiment 5 operations, obtains the title compound white solid.Mp 164～165℃。
1HNMR(300Mz，CDCl
3，TMS)δ(ppm)：7.44(d，J＝9.0Hz，2H)；6.87(d，J＝9.0Hz，2H)；3.91(s，3H)；3.81(m，7H)；3.72(m，4H)。
Embodiment 74 2-[4-(4,6-di-p-methoxy anilino-[1,3,5]-triazine-2-replaces) piperazine-1-replaces] ethanol (Compound C 191) synthetic
Except using 6-chloro-N; N '-di-p-methoxy phenyl-[1; 3,5]-and triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1; 3; 5]-and triazine-2, the 4-diamines, 2-(piperazine-1-replaces) ethanol replaces outside the piperazine; press embodiment 5 operations, obtain title compound white powder solid.Mp 172～173℃。
1HNMR(300Mz，CDCl
3，TMS)δ(ppm)：7.44(d，J＝9.3Hz，4H)；6.86(d，J＝9.0Hz，4H)；3.84(m，10H)；3.68(t，J＝6.8Hz，2H)；2.54～2.61(m，6H)。
Embodiment 75 2-[4-(4-P-nethoxyaniline base-6-anilino-[1,3,5]-triazine-2-replace) piperazine-1-replaces] ethanol (Compound C 192) synthetic
Except using 6-chloro-N-p-methoxyphenyl-N-phenyl-[1; 3; 5]-triazine-2; the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2; the 4-diamines; 2-(piperazine-1-replaces) ethanol replaces outside the piperazine, presses embodiment 5 operations, obtains title compound thickness oily matter.
1HNMR(300Mz，CDCl
3，TMS)δ(ppm)：7.54(d，J＝8.7Hz，2H)；7.44(d，J＝6.6Hz，2H)；7.30(t，J＝8.1Hz，2H)；7.03(t，J＝7.8Hz，1H)；7.02(s，1H)；6.93(s，1H)；6.86(d，J＝7.2Hz，2H)；3.87(t，J＝5.1Hz，2H)；3.80(s，3H)；3.69(t，J＝5.7Hz，2H)；3.00(s，2H)；2.58～2.64(m，6H)。
Embodiment 76 2-[4-(4,6-hexichol amido-[1,3,5]-triazine-2-replaces) piperazine-1-replaces] ethanol (Compound C 193) synthetic
Except using 6-chloro-N, N '-phenylbenzene-[1,3,5]-triazine-2; the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2,4-diamines; 2-(piperazine-1-replaces) ethanol replaces outside the piperazine, presses embodiment 5 operations, obtains title compound white powder solid.Mp 151～152℃。
1HNMR(300Mz，CDCl
3，TMS)δ(ppm)：7.57(d，J＝9.0Hz，4H)；7.33(t，J＝8.4Hz，4H)；7.06(t，J＝7.2Hz，2H)；6.98(s，2H)；3.88(t，J＝5.1Hz，4H)；3.69(t，J＝7.2Hz，2H)；2.90(s，1H)；2.56～2.63(m，6H)。
Embodiment 77 6-(morpholine-4-replace)-N, N '-phenylbenzene-[1,3,5]-triazine-2,4-diamines (Compound C 71) synthetic
Except using 6-chloro-N, N '-phenylbenzene-[1,3,5]-triazine-2; the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N ' phenyl-[1,3,5]-triazine-2,4-diamines; outside the morpholino TEPA piperazine, press embodiment 5 operations, obtain title compound white powder solid.Mp 195～197℃。
1HNMR(400Mz，CDCl
3，TMS)δ(ppm)：7.55(d，J＝7.8Hz，4H)；7.32(t，J＝6.8Hz，4H)；7.05(t，J＝7.8Hz，2H)；6.88(s，2H)；3.82(t，J＝4.8Hz，4H)；3.75(t，J＝4.8Hz，4H)。LREI(m/z)：348(M
+，100％)。
Embodiment 78 N-p-methoxyphenyl-6-(morpholine-4-replace)-N '-phenyl-[1,3,5]-triazine-2,4-diamines (Compound C 72) synthetic
Except using N-p-methoxyphenyl-6-chloro-N '-phenyl-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1; 3,5]-and triazine-2, the 4-diamines; outside the morpholino TEPA piperazine, press embodiment 5 operations, obtain title compound white powder solid.Mp 198～200℃。
1HNMR(400Mz，CDCl
3，TMS)δ(ppm)：7.55(d，J＝7.6Hz，2H)；7.45(d，J＝7.2Hz，2H)；7.30(t，J＝7.6Hz，2H)；7.05(t，J＝7.6Hz，1H)；6.91(s，1H)；6.88(d，J＝6.8Hz，2H)；6.81(s，1H)；3.81(m，7H)；3.75(t，J＝4.8Hz，4H)。LREI(m/z)：378(M
+，100％)。
Embodiment 79 N-(3, the 4-dimethoxy) phenyl-6-(morpholine-4-replace)-N '-phenyl--[1,3,5]-triazine-2,4-diamines (Compound C 73) synthetic
Except using 6-chloro-N-(3, the 4-dimethoxy) phenyl-N '-phenyl-[1,3,5]-triazine-2; the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1,3,5]-triazine-2,4-diamines; outside the morpholino TEPA piperazine, press embodiment 5 operations, obtain title compound white powder solid.Mp132～135℃。
1HNMR(400Mz，CDCl
3 TMS)δ(ppm)：7.53(dd，J＝7.6&0.8Hz，2H)；7.30(t，J＝7.6Hz，2H)；7.23(s，1H)；7.04(t，J＝7.2Hz，1H)；6.95(dd，J＝8.4&2.4Hz，2H)；6.91(s，1H)；6.82(d，J＝8.4Hz，1H)；3.87(s，3H)；3.82(m，7H)；3.73(t，J＝5.2Hz，4H)。LREI(m/z)：408(M
+，100％)。
Synthesizing of embodiment 80 4-(4-anilino-6-(morpholine-4-replaces)-[1,3,5]-triazine-2-amino) benzsulfamide (Compound C 74)
Except replace 6-chloro-N-with 4-(6-chloro-4-anilino-[1,3,5]-triazine-2-amino) benzsulfamide to methylsulfonyl phenyl-N '-phenyl-[1,3; 5]-and triazine-2, the 4-diamines is outside the morpholino TEPA piperazine; press embodiment 5 operations, obtain title compound white powder solid, productive rate 92%.Mp245～248℃。
1HNMR(400Mz，DMCO-d6，TMS)δ(ppm)：7.95(d，J＝8.8Hz，2H)；7.78(d，J＝8.8Hz，2H)；7.74(d，J＝8.8Hz，2H)；7.29(t，J＝7.6Hz，2H)；7.00(t，J＝7.6Hz，1H)；3.79(t，J＝4.8Hz，1H)；3.68(t，J＝4.8Hz，4H)。LREI(m/z)：427(M
+，100％)。
Embodiment 81 N-are to methylthio group phenyl-6-(morpholine-4-replace)-N '-p-methylphenyl-[1,3,5]-triazine-2,4-diamines (Compound C 19) synthetic
Except using 6-chloro-N-to methylthio group phenyl-N '-p-methylphenyl-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1; 3,5]-and triazine-2, the 4-diamines; outside the morpholino TEPA piperazine, press embodiment 5 operations, obtain title compound white powder solid.
1HNMR(300Mz，DMSO-d6，TMS)δ(ppm)：7.70(d，J＝8.1Hz，2H)；7.59(d，J＝7.5Hz，2H)；7.20(d，J＝8.7Hz，2H)；7.07(d，J＝8.4Hz，2H)；3.64～3.73(m，8H)；2.44(s，3H)；2.25(s，3H)。LREI(m/z)：408(M
+，100)。
Embodiment 82 6-(morpholine-4-replace)-N-phenyl-N '-(pyridine-4-replaces)-[1,3,5]-triazine-2,4-diamines (Compound C 20) synthetic
Except using 6-chloro-N-phenyl-N '-(pyridine-4-replaces)-[1,3,5]-triazine-2, the 4-diamines replaces 6-chloro-N-to methylsulfonyl phenyl-N '-phenyl-[1; 3,5]-and triazine-2, the 4-diamines; outside the morpholino TEPA piperazine, press embodiment 5 operations, obtain the title compound pulverulent solids.
1HNMR(300Mz，DMSO-d6，TMS)δ(ppm)：8.48(d，J＝8.7Hz，2H)；7.10(d，J＝8.1Hz，2H)；7.01(d，J＝7.8Hz，2H)；6.68(t，J＝7.5Hz，2H)；6.50(d，J＝7.8Hz，2H)；3.67(m，4H)；2.92(m，4H)。LREI(m/z)：349(M
+，100)。
Test example 1
Test 1, utilize surface plasma resonance (Surface Plasmon Resonance, SPR) biosensor technique Biacore 3000 researchs replacement [1 prepared in accordance with the present invention, 3,5] interaction (Fig. 1) of compound in triazine class and cyclooxygenase (COX-1 and COX-2)
Experimental technique:
(Sweden) (Amersham) at room temperature finishes the experiment that combines of compound and cyclooxygenase (COX-1 and COX-2) for BIACORE AB, Uppsala to adopt BIACORE3000.Chip and buffering solution are as follows: the CM5 chip, and EDC, NHS, thanomin, HBS-EP is (available from BIACORE AB company (Uppsala, Sweden)).
Operation steps: the piperazine compound in triazine class is dissolved with DMSO, be diluted to respective concentration (0.625,1.25,2.5,5.0 and 10.0 μ M) with HBS-EP, the content of DMSO is 0.4%.The albumen of purifying is connected on the chip by the amino coupled method.Dynamic analysis Wizard with BIACORE 3000 carries out dynamic experiment, carries out the Collection and analysis of data.
According to 1:1 Langmuir binding model analytical data, the series of results that obtains is as shown in table 1.
Table 1, piperazine compound in triazine class and cyclooxygenase (COX-1 and COX-2) bonded kinetics is normal
Number is analyzed
| Compound | COX-2 K D(M) | COX-1 K D(M) |
| Celecoxib B51 B52 B53 B55 B56 B57 B58 | 8.7×10 -5 8.9×10 -5 5.3×10 -5 8.6×10 -5 8.1×10 -6 8.7×10 -7 1.9×10 -5 3.4×10 -6 | 1.7×10 -4 8.2×10 -4 8.4×10 -4 7.2×10 -4 8.6×10 -4 6.7×10 -4 5.4×10 -4 1.2×10 -5 |
| B59 B60 B61 B62 B63 B64 B65 B66 B67 B68 B70 B71 B72 B73 B74 B75 B81 B82 B83 B84 B | NO NO 9.2×10 -5 9.4×10 -5 9.3×10 -5 8.6×10 -5 7.5×10 -6 7.8×10 -5 8.9×10 -5 4.1×10 -6 8.1×10 -4 9.2×10 -4 9.8×10 -4 6.7×10 -5 3.49×10 -5 2.97×10 -5 2.39×10 -6 5.4×10 -6 7.66×10 -6 1.85×10 -5 6.4×10 -5 | NO NO 7.8×10 -5 4.21×10 -4 4.11×10 -4 8.7×10 -4 4.3×10 -4 9.4×10 -4 9.1×10 -4 7.4×10 -5 5.5×10 -5 1.1×10 -3 8.7×10 -3 7.3×10 -3 3.1×10 -3 2.4×10 -3 1.25×10 -5 2.27×10 -5 1.35×10 -5 5.6×10 -3 5.6×10 -4 |
Sample source: test compounds is by institute of materia medica, Shanghai drug discovery and the preparation of compound experiment chamber, design centre (DDDC), and positive compound Celecoxib and ETYA (eicosa-5,8,11-tetraynoic acid) purchase from Calbiochem company.The 5-LOX enzyme, DDDC biology laboratory eukaryotic system is expressed preparation.
Plant and instrument: Biacore S51 instrument, Sweden Biacore company.
Experiment condition: temperature: 25 ℃; Enzyme dissolving damping fluid: PBS.Compound concentration (Fig. 2): positive compound ETYA is followed successively by 3.125e-6; 6.25e-6; 12.5e-6; 25e-6; 50e-6M; Compd B 52 is followed successively by 1.6807e-6,2.401e-6,3.43e-6,4.9e-6,7.0e-6,10.0e-6M.
Experimental result:
1.Celecoxib: native system fail to detect Celecoxib can with the information of 5-LOX enzyme effect.(not seeing that pertinent literature report Celecoxib has the 5-LOX enzyme inhibition activity).
2.ETYA and compound B-26 2 shows that with the test that combines of 5-lipoxygenase its dissociation constant is respectively: ETYA:K
D=3.4 * 10
-6M; B62:K
D=5.08 * 10
-6The M (see figure 2).The result of ETYA conforms to the bibliographical information value.
Test example 2 isolated rat neutrophil leucocytes discharge the restraining effect test of leukotriene B4
Experiment reagent: II type glycogen (Sigma-Aldrich Co, 10K154), indomethacin (Sigma-Aldrich Co, 061K1368), A23187 (Sigma-Aldrich Co), L-halfcystine (Kangda Amino-acid Factory of Shanghai, lot number: 20030601), the zileuton bulk drug (Beijing Zhaoyan New Drug Research Center, purity is 99.8%, lot number is 20020902).LTB
4EIA measures test kit (Cayman Chemical Company, lot number are 121237).
Experimental animal: the SD rat, the cleaning level, male and female are regardless of, body weight 200 ± 20g (commercially available getting).
Laboratory apparatus: microplate reader (Thermo, Multiskan spectrum), constant water bath box etc.
Experimental technique and data analysis:
The preparation of 1 rat leukocyte suspension
Get normal rat, 0.2% glycogen 20mL/kg carries out abdominal injection (ip), femoral artery sacrificed by exsanguination rat behind the 16h, every rat carries out peritoneal lavage with Hanks balanced salt damping fluid 10mL, collects the intraperitoneal irrigating solution, the centrifugal 10min of 2000r/min, sedimentation cell adds freezing distilled water 5mL lysed erythrocyte, add isopyknic 1.8% sodium chloride solution behind the 1min immediately, the centrifugal 5min of 2000r/min, sedimentation cell suspend with Hanks balanced salt damping fluid and wash 2 times.Trypan blue is got rid of dyeing, cytoactive＞95%, and Wright-Giemsa dyeing, morphological observation neutrophil leucocyte ratio＞80%, all the other are monocyte.
2 leukotriene Bs
4Generation
With Hanks balanced salt damping fluid the cell of above-mentioned collection is adjusted to 5 * 10
6/ mL, press the 0.5mL packing, hatch 10min for 37 ℃, add L-halfcystine (10mM) successively, indomethacin (1mg/L) and each concentration test-compound (50,5,0.5 μ M), 37 ℃ hatch 30min after, add calcium ion carrier A 23187 (5 μ M), 37 ℃ continue to hatch 30min after, immediately in centrifugal 5 minutes of 4 ℃, 14000r/min, supernatant be stored in-70 ℃ standby.Solvent final concentration in the reaction system≤0.21%.
3 leukotriene Bs
4Mensuration
According to the test kit explanation, after the damping fluid dilution of cell extract with commercial EIA test kit, add 96 hole enzyme plates, each sample is established two multiple holes, and twice, 4 ℃ of overnight incubation of duplicate detection, add developer next day, detect absorbancy behind the lucifuge reaction 90min in 412nm place, and according to LTB in the typical curve conversion test sample of standard substance foundation
4Content.
4 statistical analysis
Each handles LTB in the sample
4Concentration represent that with mean ± SEM compound produces LTB to neutrophil leucocyte
4The calculation formula of inhibiting rate be:
Inhibiting rate=(solvent pipe concentration-sample hose concentration)/solvent pipe concentration * 100%
According to this formula, draw the different concns compound neutrophil leucocyte is produced LTB
4Inhibiting rate.
5 results
5.1 the foundation of typical curve
The logarithm regression equation of setting up according to the concentration gradient of standard substance is: Y=-1.0064ln (x)+3.6998, R
2=0.9823.
5.2 compound produces LTB to neutrophil leucocyte
4Inhibition
After in the neutrophil leucocyte Incubating Solution, adding the compound of different concns, to the LTB under the calcium ion carrier A 23187 stimulation
4Generate the restraining effect (table 2) that produces in various degree.
Table 2: calcium ion carrier A 23187 stimulates isolated rat neutrophil leucocyte (5 * 10 down
6/ mL) LTB
4Burst size and compound different concns restraining effect (mean ± SEM)
| Compound | LTB 4Concentration (ng/mL) | Inhibiting rate (%) | |
| Solvent B B62 B55 B68 B61 | 0.5μM 5μM 50μM 0.5μM 5μM 50μM 0.5μM 5μM 50μM 0.5μM 5μM 50μM 0.5μM 5μM 50μM | 362.24±87.49 278.83±34.74 201.36±37.91 97.41±21.74 248.07±44.06 54.45±35.63 33.04±28.31 359.78±56.58 310.90±71.63 301.82±0.00 305.40±80.31 239.32±15.42 186.24±37.65 361.30±77.18 306.35±142.89 286.51±47.24 | 1.56 43.94 72.88 30.95 84.84 90.80 0.00 13.46 15.99 14.99 33.38 48.16 0.00 14.72 20.25 |
| Zileuton | 0.5μM 5μM 50μM | 170.93±4.73 47.25±17.98 40.20±35.47 | 52.42 86.85 88.81 |
As seen from Table 2, the test compounds B of institute, B55, B62, B61, B68 discharges LTB to the isolated rat neutrophil leucocyte
4Restraining effect is in various degree arranged, but inhibition strength is inferior to positive control Zileuton, two compounds that restraining effect is stronger are B62, B68.
Further test routine method and tested compound B-26 2, B68, C72 and positive control Zileuton isolated rat neutrophil leucocyte (5 * 10 under the calcium ion carrier A 23187 stimulation with this
6/ ml) discharge LTB
4IC
50Value is respectively 1.52 μ M, 31.12 μ M, 11.78 μ M and 0.86 μ M.
Test example 3 anti-inflammatory drugs screening pharmacodynamic experiment ()
Laboratory animal: male and healthy mouse, body weight 22-25g.
Experimental technique and observation index: experiment mice foot sole of the foot carrageenin causes swollen method
Get body weight 22-25g male and healthy mouse random packet, 10 every group.Negative if (CMC sodium, sodium carboxymethyl cellulose solution) and positive (indomethacin, 5mg/kg) control group.Be dissolved in the CMC solution by the reagent thing, drug dosage is 50mg/kg.Yu Zhiyan administration in preceding 30 minutes (abdominal injection or filling stomach).Then mouse is fixed in the mouse frame, stretching hind leg is injected 0.2% carrageenin (Carrageenin), 20 μ l with No. 4 syringes to the mouse foot sole of the foot.Cause scorching back 4 hours and put to death mouse, along hind leg joint difference clip left and right sides hind leg, relatively the difference of administration group, control group and positive drug control group is analyzed by statistics and is obtained mean value, SD, P value respectively and suppress percentage (seeing Table 3).
Table 3
| Compound | Dosage | Route of administration a | Inhibiting rate (%) | SD | The |
| Indomethacin | |||||
| 10 | |
40 | 12.63 | 3.83E-05 |
| celecoxib B B51 B52 B53 B54 B55 B56 B57 B58 B59 B60 B61 B62 B63 B64 B65 B66 B67 B68 B70 B75 B76 B77 | 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 | ip ip ip ip ip ip ip ip ip po ip ip ip ip ip ip ip ip ip ip ip ip ip ip | 59 52 24 24 38 29 26 37 26 24 28 30 53 36 22 23 44 49 42 51 27 35 24 13 | 8.25 19 10.6 8.1 11 13 17.8 11.3 15.3 11 13 14 10.47 10.27 5.31 11.24 8.9 10 9 10.7 16.06 11.65 13.5 13.5 | 5.37E-08 3.30E-05 1.65E-02 1.70E-04 9.00E-07 1.90E-04 2.80E-03 2.22E-05 1.81E-02 6.50E-07 3.00E-02 3.00E-02 2.63E-07 3.0E-03 2.50E-03 6.60E-03 1.55E-04 7.00E-04 7.50E-06 5.51E-04 3.60E-03 6.13E-05 2.40E-03 6.14E-03 |
| B81 B82 B83 B84 B85 | 50 50 50 50 50 | ip ip ip ip ip | 35 13 0 30 27 | 11.4 9.8 14.7 10 13.6 | 2.00E-04 17.9E-02 9.80E-01 7.00E-03 4.50E-02 |
aIp: abdominal injection or filling stomach; Po: oral administration.
Find out from above experimental result, have in 28 compounds that screened 25 to mouse foot sole of the foot carrageenin inhibitory rate of intumesce all greater than 20%, have better antiphlogistic effects; Wherein the inhibiting rate of compd B and B68 is greater than 50%, and antiphlogistic effects is remarkable, therefore based on this model, has done further screening at these two compounds by various dose, the results are shown in Table 4.
Table 4
| Compound | Dosage | Route of administration | Inhibiting rate (%) | SD | The p value |
| B | 6.25 12.5 25 50 12.5 25 50 | ip po | 29 32 29 52 15 19 26 | 7.17 5.2 11.37 19 19.97 11.72 9.92 | 8.8E-03 3.7E-03 2.1E-02 3.3E-05 1.5E-01 1.4E-02 1E-03 |
| |
6 12.5 25 50 | |
10 21 33 48 | 16.72 19.64 9.28 15.10 | 7.6E-02 6.9E-03 1.70E-05 1.03E-06 |
aIp: abdominal injection or filling stomach; Po: oral administration.
The Orally active of compound B-26 8 obviously is better than compd B on this model as can be seen from Table 3.
Test example 4 anti-inflammatory drugs screening pharmacodynamic experiment (two)
Laboratory animal: male Wister rat, body weight 150-180g; Male mice, body weight 26-30g.
Experimental technique and observation index:
Test 1. rat paw carrageenins and cause swollen method
Get body weight 150-180g male Wister rat random packet, 10 every group.Negative if (the CMC aqueous solution) and positive (indomethacin 3.6mg/kg) control group.Be dissolved in the CMC solution by the reagent thing, 15 kinds of compound amount are 15mg/kg.Preceding 60 minutes gastric infusions of Yu Zhiyan.Give the left back sufficient plantar aponeurosis of rat injection 1% carrageenin 0.1ml down then, cause scorching back 3 hours and measure the ankle joint girth.
Difference with joint, left and right sides girth is the swelling level index, carries out the t check, the significance of comparative group differences.
Test the scorching method of 2. mouse ear caused by dimethylbenzene xylene
Get body weight 26-30g male mice, random packet, 10 every group.Negative if (the CMC aqueous solution) and positive (indomethacin 7.2mg/kg) control group.Be dissolved in the CMC solution by the reagent thing, (B68) consumption is 15mg/kg to 3 kinds of compounds for B61, B62.Preceding 60 minutes gastric infusions of Yu Zhiyan.Then dimethylbenzene 0.05ml is dripped in the mouse auris dextra, left ear compares.After 2 hours the dislocation of mouse cervical vertebra is caused death, cut two ears, lay circular auricle at the same position of left and right ear respectively, weigh with punch tool along the auricle baseline.
Difference with left and right sides auricle weight is the swelling level index, carries out the t check, the significance of comparative group differences.
Test 3. rat assist agent arthritis (prevention primary affection)
Get body weight 180 ± 20g male SD rat, random packet, 10 every group.Negative if (the CMC aqueous solution) and positive (indomethacin 0.3mg/kg) control group.Be dissolved in the CMC solution by the reagent thing, the heavy dose of 30mg/kg of B62, middle dosage 10mg/kg, low dose of 3mg/kg, the heavy dose of 100mg/kg of B68, middle dosage 50mg/kg, low dose of 25mg/kg.Preceding 60 minutes gastric infusions of Yu Zhiyan.
Left back sufficient sole of the foot intradermal injection Freund ' the s Freund's complete adjuvant of every mouse (FCA) 0.1ml causes inflammation.Cause the volume of sufficient pawl about measurement in scorching back 18 hours.With about the difference of sufficient pawl volume be the swelling level index, carry out the t check, the significance of comparative group differences.
Experimental result:
Test 1. rat paw carrageenins and cause swollen method
The rat paw carrageenin causes swollen swelling degree and the inhibiting rate of The compounds of this invention the results are shown in Table 5～7.
Table 5 rat paw carrageenin causes swollen method experimental result
| Medicine | The swelling degree | Inhibiting rate |
| Negative control Indomethacin Celecoxib B62 B54 B53 B64 B81 B61 B68 | 6.10±1.45 3.90±1.02 3.90±0.97 ** 4.00±1.13 ** 5.00±1.53 5.70±0.89 5.75±1.80 6.25±1.01 6.80±2.14 8.35±1.63 | 36.07％ 36.07％ 34.43％ 18.03％ 6.56％ 5.74％ -2.46％ -11.48％ -36.89％ |
| B | 4.2±2.39 * | 31.15％ |
*P＜0.05
*Each group of p＜0.01 compares (t check) with negative control group.
Above experimental result shows positive control drug and B62, and Celecoxib, B all have significant antiphlogistic effects.Further the effect of compd B and the anti-carrageenin inductive of compound B-26 2 large, medium and small dosage groups rat paw edema is measured, be the results are shown in Table 5 and table 6.
The influence of table 6 compd B on Carrageenan inductive rat paw edema
| Medicine | n | The swelling degree | Inhibiting rate |
| Negative control positive control 1mg/kg group 3mg/kg group 10mg/kg group 30mg/ |
10 10 10 10 10 10 | 7.30±2.18 3.90±1.02 ** 4.20±1.30 ** 4.45±1.89 ** 4.70±0.95 ** 4.90±1.85 * | 46.58％ 42.47％ 39.04％ 35.62％ 32.88％ |
*P＜0.05
*Each group of p＜0.01 compares (t check) with negative control group.
The influence of table 7 compound B-26 2 on Carrageenan inductive rat paw edemas
| Medicine | n | The swelling degree | Inhibiting rate |
| Dosage (10mg/kg) heavy dose of (30mg/kg) in negative control positive control low dose of (1mg/kg) low dose of (3mg/kg) | 10 10 10 10 10 10 | 7.30±2.18 3.90±1.02 7.70±1.27 4.80±2.26 * 4.05±2.72 ** 6.05±2.19 | 46.58％ -5.48％ 34.25％ 44.52％ 17.12％ |
*P＜0.05
*Each group of p＜0.01 compares (t check) with negative control group.
Test the scorching method of 2. mouse ear caused by dimethylbenzene xylene
The swelling degree of mouse ear caused by dimethylbenzene xylene inflammation and the inhibiting rate of The compounds of this invention the results are shown in Table 8～10.
The scorching method experimental result of table 8 mouse ear caused by dimethylbenzene xylene
| Medicine | The swelling degree | Inhibiting rate |
| Negative control positive control B62 B61 B68 | 17.80±4.24 8.90±4.09 ** 9.40±4.60 ** 18.80±4.18 13.30±5.23 * | 50.00％ 47.19％ -5.62％ 25.28％ |
*P＜0.05
*Each group of p＜0.01 compares (t check) with negative control group.
Above experimental result shows positive control drug and B62, and B68 all has significant antiphlogistic effects.Further the effect of compd B and the anti-dimethylbenzene inductive of compound B-26 2 large, medium and small dosage groups Mice Auricle inflammation is measured, be the results are shown in Table 9 and table 10.
The influence of table 9 compd B p-Xylol inductive Mice Auricle inflammation
| Medicine | n | Swelling long-range degree | Inhibiting rate |
| Negative control positive control 1mg/kg group 3mg/kg group 10mg/ |
10 10 10 10 10 | 17.80±4.24 8.90±4.09 ** 10.50± 3.75 ** 11.80± 4.87 ** 12.70±4.85 * | 50.00％ 41.01％ 33.71％ 28.65％ |
| The 30mg/ |
10 | 8.40±4.95 ** | 52.81％ |
*P＜0.05
*Each group of p＜0.01 compares (t check) with negative control group.
The influence of table 10 compound B-26 2 p-Xylol inductive Mice Auricle inflammation
| Medicine | n | The swelling degree | Inhibiting rate |
| Dosage (10mg/kg) heavy dose of (30mg/kg) in negative control positive control low dose of (1mg/kg) low dose of (3mg/kg) | 10 10 10 10 10 10 | 17.80±4.24 8.90±4.09 ** 6.40±4.06 ** 9.50±4.77 ** 5.80±4.64 ** 6.10±4.41 ** | 50.00％ 64.04％ 46.63％ 67.42％ 65.73％ |
*P＜0.05
*Each group of p＜0.01 compares (t check) with negative control group.
Test 3. rat assist agent arthritis (prevention primary affection)
The compounds of this invention Chinese People's Anti-Japanese Military and Political College mouse adjuvant-induced arthritis the results are shown in Table 10.
The influence of 2 pairs of rat assist agent arthritis primary affections of table 11 compound B-26
| Medicine | n | The swelling degree | Inhibiting rate |
| Negative control positive control low dose, dosage (3mg/kg), (10mg/kg) heavy dose, (30mg/kg) | 10 10 10 10 10 | 0.95±0.16 0.50±0.14 ** 0.56±0.27 ** 0.45±0.14 ** 0.53±0.16 ** | 47.36％ 40.80％ 52.96％ 43.66％ |
*P＜0.05
*Each group of p＜0.01 compares (t check) with negative control group.
The test of test example 5 anti-inflammatory compound gastrointestinal side effects
Experiment purpose: estimate its gastric injury character with the oral four kinds of compounds of fasting rat.
Laboratory animal: male SD rat
Be subjected to the reagent thing: indomethacin, Celecoxib, B62, B68
Experimental technique and observation index:
Get body weight 200-220g male SD rat at random, 10 every group.If feminine gender and positive controls.Gastric infusion, once a day, continuous 4 days, during this in to food, water do not add control.Taking-up stomach and small intestine along little curved a kerf of vertically doing, cleaned stomach and small intestine with flowing water, with forefinger stomach were opened, and checked stomach degree of impairment and record with sacrifice of animal in 24 hours after the last administration.There are a place or many places damages (blutpunkte, erosion, ulcer or perforation) promptly to think positive.Use formalin fixed then, sections observation.Calculating is tried the per-cent of gastric injury animal in the treated animal.Comprehensive each result makes comparisons.
Be dissolved in the 0.08%CMC solution by the reagent thing, positive control drug indomethacin 3.6mg/kg, Celecoxib, B62, B68 are 30mg/kg, and negative control group is given 0.08%CMC solution.The photo that the gastrointestinal side effect laboratory report is respectively organized the section situation as shown in Figure 3.
Experimental result: respectively be subjected to the reagent thing to cause that the rat stomach injury situation sees Table 1
Table 12 visual inspection rat stomach injury recorder
| Be subjected to the reagent thing | Degree of impairment | Positive rate | |||
| The blutpunkte | Rotten to the corn | Ulcer | Perforation | ||
| Negative control group | Do not have | Do not have | Do not have | Do not have | 0％ |
| Indomethacin | Have, serious | Do not have | Do not have | Do not have | 100％ |
| Celecoxib | Do not have | Do not have | Have | Do not have | 20％ |
| B62 | Have, slight | Do not have | Do not have | Do not have | 20％ |
| B68 | Do not have | Do not have | Do not have | Do not have | 0％ |
From last table as seen, Celecoxib, B62, B68 gastrointestinal side effect symptom are lighter than indomethacin, and wherein the demonstration of B68 does not have gastrointestinal side effect.
Test example 6 compd Bs and B62 Ames test
Experiment purpose: observing B and B62 has not modificator gene sudden change to the in vitro tests of mouse Salmonellas.
Compound method:
Be made into the B and the B62 of different concns before the experiment with DMSO, make each dosage add the soup of equal volume.
Reference substance:
Negative control: DMSO
Positive control: (divide no S
9Metabolic system and through S
9Metabolic system)
-S
9System:
TA97:9-aminoacridine is produced by Sigma Chemical Company lnc
TA98:2-Nitroflucrene is produced by Aldrich Chemical Company lnc
TA100:Methyl methanesulfonate is produced by Sigma Chemical Company lnc
TA102:Mitomycin C is produced by KYOWA HAKKO KOGYO CO.LTD.
TA1535:Sodium azide is produced by Mcrck
+ S
9System:
TA97, TA98, TA100:2-aminofluorene is produced by Sigma Chemical Company lnc
TA102:1,8-dihydroxyanthraquinone is produced by Aldrich Chemical Company lnc
TA1535: Cyclophosphamide for injection is produced by Hualian Pharmaceutical Co., Ltd., Shanghai
Test strain:
The strain of Salmonella typhimurium Histidine nutrient defect mutation.
TA97, TA98, TA100, TA102 and TA1535 (commercially available getting).Liquid nitrogen is preserved bacterial classification.
Hereditary property is identified:
The evaluation content comprises: histidine auxotroph, lipopolysaccharides barrier defective (rfa), ultraviolet repair deficiency (except the Δ uvrB, TA102), and the R-factor.TA97, TA98 and TA100 have the pKM101 plasmid, and anti-penbritin effect is arranged, and TA102 has pKM101 and pAQ1 plasmid, and anti-penbritin and tsiklomitsin effect are arranged.Above-mentioned accreditation person, the satisfactory bacterial strain of spontaneous mutation number increases bacterium, as the mutagenesis experimental strain.
Dosage is selected:
Adding S
9Select for use 5000,1000,500,50,5 μ g/ wares to survey compd B in the experimental system 5 bacterial strains are carried out toxicity prediction.The toxicity assessment standard: the first, observation bacterial growth background is compared as if thinning or disappearance with negative control group then be the toxicity performance.The second, compare the parameter that returns of the average every ware of counting with negative control, obviously descend or be and have dose-dependently then to show if return parameter for toxicity.
The metabolism activation agent:
Induce preparation rat liver S with Aroclor 1254
9Sprague-Dawley rat with about 200 grams of body weight, abdominal injection Aroclor 1254 (Nihon Pharmaceutical Co., Ltd.) 500mg/kg put to death on the 5th day, and aseptic condition is flushing and taking-up liver down, use 4 ℃ of 0.15MKCl lavations immediately, the ratio in the 3ml/g weight in wet base adds 0.15M KCl again.4 ℃ of homogenate, 9000 * g is centrifugal, gets its supernatant liquor and is S
9Preserve in the liquid nitrogen.Before experiment is used, refrigerated S9 is slowly melted, all use freshly prepd S at every turn
9Mix.
Experimental technique:
Observing time: cultivate after 72 hours, count for 37 ℃.
Experiment: with the soup of DMSO preparation different concns.+ S
9System's dosage is 5000,1000,500,50,5 μ g/ wares.-S
9System's dosage is 500,50,5,0.5,0.05 μ g/ ware.
The dressing plate infiltration method is used for surveying the direct effect of medicine without metabolism activation, and its test top layer is:
2.0ml top layer
0.1ml soup
0.1ml bacterium liquid
0.5ml phosphoric acid buffer
Pre-cultivation then is used to survey the mutagenesis of drug metabolism activation, and it tests consisting of of top layer:
2.0ml top layer
0.1ml soup
0.1ml bacterium liquid
0.5ml S
9Mixed solution
The soup of being surveyed, bacterium liquid, S
9Mixed solution after 25 fens 35 ℃ of jolting temperature are incubated, experimentizes by the dressing plate infiltration method earlier again.Each dosage group is established 3 wares, counts each bacterial strain without drug metabolism activation or through drug metabolism activation system (S
9Or+S
9) return to become colony number X ± SD.
Effectively standard is accepted in experiment:
1. high density must reach the standard that meets governing principle, or reaches the concentration that toxicity allows.
2. positive (feminine gender) contrast must drop on or near conforming within the scope of this breadboard historical background data or with documents and materials.
3. do not answer the pollution of immingling technology problem, excessive toxicity.
The evaluation of result standard:
1. the reverse mutation value of all test group is judged to feminine gender less than 2 times of negative control group.
2. the reverse mutation value of any test group is greater than 4 times of negative control group, and the conclusion of trial test and master trip conforms to, and shellfish is judged to the positive.
3. as if between 2～4 times of the negative contrasts of test group maximum effect group, then do further to analyze.
A) after the Bonterron of one-sided Dunnett ' the s check of data-switching and multiple comparisons proofreaied and correct, still nonsignificance (p≤0.05) then can be judged to feminine gender.
B) if the statistics significant difference should compare with historical negative control data 98% higher limit, if less than still being judged to feminine gender.Greater than the time then can be judged to the positive with reference to dose relationship and biological significance.Then need repeat when being difficult to determine.
Result and evaluation
1. test compound B and B62 maximum concentration meet the governing principle requirement, and negative, positive reverse mutation rate all meets this breadboard background information, and is pollution-free in the experiment.It is effective that this experiment is used for estimating.
2. surveying Salmonellas reverse mutation result with the dressing plate infiltration method shows:
1. compd B is at-S
9When concentration is 500 μ g/ wares in the experimental system, TA97, TA98, TA102 bacterial strain there is bacteriostatic action.And when dosage is 50,5,0.5,0.05 μ g/ ware, do not have bacteriostatic action, there is not mutagenesis yet.The results are shown in Table 13.
2. compd B is at+S
9During 5000,1000 μ g/ wares, TA97, TA98, TA100, TA102, TA1535,5 bacterial strains there is bacteriostatic action in the experimental system.When dosage is 500 μ g/ wares, TA97, TA98, TA102,3 bacterial strains there is bacteriostatic action.50,5 μ g/ wares do not have the antibacterial mutagenesis of yet not having.The results are shown in Table 14.
3. compound B-26 2 is at-S
9With+S
9When concentration is 5000 μ g/ wares in the experimental system, TA97, TA98, TA100, TA102,4 bacterial strains there is bacteriostatic action.And work as dosage is that 1000,500,50,5 μ g/ wares do not have bacteriostatic action.The growth of unrestraint bacteria colony count did not have the mutagenesis of appearance yet when TA1535 bacterial strain dosage was 5000,1000,500,50,5 μ g/ wares.The results are shown in Table 15 and table 16.
To sum up: B and B62 are under this experiment condition, and no mutagenesis does not have mutagenesis.
Table 13.B is without S
9After the metabolic system effect
Mutagenesis testing to Salmonella typhimurium
| Dosage μ m/ ware | Bacterium colony and time change colony number | ||||
| TA97 | TA98 | TA100 | TA102 | TA1535 | |
| DMSO 0.05 | 111±10 98±6 | 22±2.6 20±3.2 | 132±17 121±5 | 251±26 249±25 | 10±2.1 13±1.2 |
| 0.5 5 50 500 positive * | 93 ± 14 97 ± 7 103 ± 8 antibacterial 1141 ± 86 | 18 ± 3.2 20 ± 0.6 20 ± 3.1 antibacterial 1200 ± 2 08 | 115±5 121±11 120±13 112±7 2123±1 90 | 240 ± 25 259 ± 8 252 ± 21 antibacterial 1971 ± 1 30 | 8±1.2 12±3.6 13±2.9 10±1.2 1079± 72 |
*TA97, TA98, TA100:2-aminofiuorene (50 μ g/ ware)
TA102:1,8-dihydroxyanthraquinone (50 μ g/ ware)
TA1535:cyclophosphamide 200 μ g/ wares
Table 14.B is through S
9The metabolic system effect
Mutagenesis to Salmonella typhimurium
| Dosage μ g/ ware | Bacterium colony and time change colony number | ||||
| TA97 | TA98 | TA100 | TA102 | TA1535 | |
| DMSO 5 50 500 1000 5000 | 123 ± 12 135 ± 23 130 ± 7 is antibacterial antibacterial | 35 ± 3.0 31 ± 4.7 31 ± 1.2 is antibacterial antibacterial | 147 ± 18 134 ± 9 137 ± 9 166 ± 40 is antibacterial | 301 ± 6 269 ± 20 295 ± 9 is antibacterial antibacterial | 19 ± 5.0 14 ± 4.0 17 ± 3.5 17 ± 4.0 is antibacterial |
| Positive * | 1341±163 | 2052±178 | 1413±161 | 1158±156 | 319±19 |
*TA97:9-aminoacridine (50 μ g/ ware)
TA98:2-Nitroflucrene (20 μ g/ ware)
TA100:Methy methanesulfonate (1 μ l/ ware)
TA102:Mitomycin C (0.5 μ g/ ware)
TA1535:Sodium azide (4 μ g/ ware)
Table 15.B62 is without S
9The metabolic system effect
Mutagenesis to Samonella typhimurium
| Dosage μ g/ ware | Bacterium colony and time change colony number | ||||
| TA97 | TA98 | TA100 | TA102 | TA1535 | |
| DMSO 5 50 500 1,000 5000 positives * | 135 ± 7 112 ± 13 127 ± 15 135 ± 16 99 ± 11 antibacterial 1141 ± 86 | 30 ± 8.5 31 ± 7.3 31 ± 6.2 29 ± 3.0 31 ± 3.8 antibacterial 1200 ± 208 | 140 ± 8 122 ± 9 135 ± 25 132 ± 14 135 ± 18 antibacterial 2123 ± 190 | 307 ± 8 227 ± 32 235 ± 10 214 ± 4 248 ± 35 antibacterial 1971 ± 130 | 19±2.0 24±3.6 20±1.0 18±1.0 16±2.0 10±4.5 1079±72 |
*TA97:9-aminoacridine (50 μ g/ ware)
TA98:2-Nitroflucrene (20 μ g/ ware)
TA100:Methyl methanesulfonate (1 μ l/ ware)
TA102:Muitomycin C (0.5 μ g/ ware)
TA1535:Sodium azide (4 μ g/ ware)
Table 16.B62 is through S
9After the metabolic system effect
Mutagenesis testing to Samonella typhimurium
| Dosage μ g/ ware | Bacterium colony and time change colony number | ||||
| TA97 | TA98 | TA100 | TA102 | TA1535 | |
| DMSO 5 50 500 1,000 5000 positives * | 118 ± 21 124 ± 21 119 ± 12 108 ± 19 117 ± 11 antibacterial 1341 ± 163 | 35 ± 6.6 29 ± 5.3 35 ± 8.7 36 ± 3.6 40 ± 3.6 antibacterial 2052 ± 178 | 135 ± 14 116 ± 10 120 ± 14 124 ± 9 132 ± 14 antibacterial 1413 ± 161 | 237 ± 57 239 ± 12 231 ± 7 218 ± 24 222 ± 15 antibacterial 1158 ± 156 | 7±1.5 7±1.0 10±3.2 17±4.4 15±3.5 10±3.5 319±19 |
*TA97, TA98, TA100:2-aminofiuorene (50 μ g/ ware)
TA102:1,8-dihydroxyanthraquinone (50 μ g/ ware)
TA1535:Cyclophosphamide 200 μ g/ wares
Test example 7 compound B-26s 2 and B68 mouse bone marrow cells micronucleus test
Test objective:
Whether test b 62 and B68 cause the chromosomal damage of mouse polychromatic erythrocytes to the whole administration of mouse.
Reference substance
Solvent control: 0.5% Xylo-Mucine.
Positive reference substance: endoxan (Hualian Pharmaceutical Co., Ltd., Shanghai's product).
Animal
ICR kind mouse 90 (♀ 45, and ♂ 45) provides conformity certification by Chinese Academy of Sciences's Shanghai Experimental Animal Center: laboratory animal certification of fitness credit number SCXK (Shanghai) 2002-0010.Mouse was raised in institute of materia medica, Chinese Academy of Sciences Shanghai Animal House adaptability through three days, and body weight is 18～22 grams during administration, and by the body weight random packet, trial-product 14 (7 of ♀, 7 of ♂) is a dosage group, 10 every group of control groups (5 of ♀, 5 of ♂).Feed is available from the joint western pul of China and Britain-Bi Kai laboratory animal company limited.Freely fetch water, raising temperature is 23 ± 2 ℃, and humidity is 60 ± 10%.
Dosage
1.B62 dosage is selected test
Select that dosage is 2000,1000,500,250,5 dosage groups of 125mg/kg, 14 every group, male and female half and half, continuous 2 days, every day, 1 gastric infusion write down the animal dead situation of observing, and death (table 1) does not appear in mouse after the administration of high dosage 2000mg/kg group as a result.
By table as seen, mouse LD50 is greater than 2000mg/kg.
B62 dosage is selected test card
| Dosage (mg/kg) | Number of animals (n) | Animal dead number (n) |
| 125 250 500 1000 2000 | 14 14 14 14 14 | 0 0 0 0 0 |
2.B68 dosage is selected test
Select that dosage is 2000,1000,500,250,5 dosage groups of 125mg/kg, 14 every group, male and female half and half, continuous 2 days, every day 1 gastric infusion, the animal dead situation observed in record, experimental result sees the following form 1.
By table as seen, mouse LD50 is about 1000mg/kg.
B68 dosage is selected test card
| Dosage (mg/kg) | Number of animals (n) | Animal dead number (n) |
| 125 250 500 1000 2000 | 14 14 14 14 14 | 0 0 3 7 8 |
Test dose
This experiment mice is irritated stomach B62 maximum dose level and is elected 2000mg/kg as, and other establishes 1000,3 dosage groups of 500mg/kg and 1 solvent control group, 1 positive controls.
This experiment mice is irritated stomach B68 maximum dose level and is elected 500mg/kg as, and other establishes 250,125mg/kg totally 3 dosage
Agent distance: successively decrease for 0.5 times with maximum dose level.
Administration number of times: once a day, continuous 2 days.
Administration volume: 0.2ml/10g.
Route of administration: irritate stomach.
Experimental technique
1. medication:
Press the technical requirements of ICH, twice or repeatedly give and tried thing a time point sampling analysis can selected between 12～24h after the last administration.Mouse stomach B62:2000,1000,500mg/kg/day and negative control group successive administration 2 days; B68:500,250,125mg/kg/day and negative control group successive administration 2 days.Positive controls once abdominal cavity injection (60mg/kg), 24h sampling after the last administration.
2. various dose influence that micronucleus is formed:
24h behind 3 dosage fillings of B62:2000,1000,500mg/kg stomach, the sampling microscopy;
24h behind 3 dosage fillings of B68:500,250,125 mg/kg stomach, the sampling microscopy.
Preparation of specimen
After animal dislocation is put to death, take out the bilateral femur, with the flushing of deactivation calf serum, centrifugal, cell dispersion smear, dry and through methyl alcohol fixedly Giemsa dye, microscopy.
The microscopy method
Every smear is 2000 good dispersions of observed and recorded at least, the polychromatic erythrocyte of complete form (PCE) and have the PCE (MNPCE) of micronucleus, the red corpuscle that writes down PCE simultaneously and just dying (NCE), both sums are more than 1000, and obtain the ratio of PCE/NCE, to observe medicine whether the effect of the medullary cell of inhibition is arranged.
Data analysis
Efficiency test is accepted standard
1. the used dosage of mouse must reach the standard that meets governing principle.Maximum dose level is as if no serious Signs or serious bone marrow toxicity, and maximum dose level must be greater than 1/2 LD
50
2. positive must drop on the micronuclear rates of the caused mouse polychromatic erythrocytes of negative control product or near conforming within the scope of the historical background data of this testing laboratory or with documents and materials.
3. administration group PCE/NCE ratio does not have tangible medullary cell toxic action in suitable scope.
Judgement criteria
1. it is similar to the negative control group micronuclear rates that all dosage groups are tried the micronuclear rates of thing, or be no more than 2 times, then can be judged to feminine gender.
2. the increase of any one group of micronuclear rates that is tried thing and brought out surpasses 4 times of negative control group and then is judged to the positive.
3. then done further statistical study if try between 2～4 times of the negative contrasts of thing maximum effect group.
A. after the Bonferrom of the one-sided Dunnett ' s check of data-switching and multiple comparisons proofreaied and correct, still nonsignificance (p≤0.05) then can be judged to feminine gender.
B. if the statistics significant difference should compare with historical negative control data 95% higher limit, if less than still being judged to feminine gender.Greater than the time then will be with reference to dose relationship, micronuclear rates increases with dosage, the statistics significant difference then be judged to the positive.
The result
1. this tests used dosage, meets the governing principle requirement.Administration group and control group PCE/NCE ratio, no significant difference shows that filling stomach B62 and B68:24h do not see restraining effect to medullary cell.The negative control result is in the scope of this breadboard historical background data.The positive reference substance endoxan causes that micronuclear rates obviously increases.So it is suitable that this experiment is used for the evaluation of mutagenesis micronuclear rates.
2. continuous 2 days of mouse is irritated stomach B62, and sampling surveys 2000,1000 on 24h time point after the last administration, 3 dosage groups of 500mg/kg/day micronuclear rates is similar to negative control group, and no significant difference the results are shown in table 17.
3. mouse is irritated stomach B68 continuous 2 times, and sampling surveys 125,250 on 24h time point after the last administration, 3 dosage groups of 500mg/kg/day micronuclear rates is similar to negative control group, and no significant difference the results are shown in table 18.
Table 17. is irritated stomach B62 24h the ICR mouse bone marrow cells is had a liking for polychromatophilia
The influence that red corpuscle (PCE) micronucleus forms
| Medicine | Dosage (mg/kg) | Number of animals (only) | Microscopy PCE number | MNPCE | MNPCE/PCE x±SD‰ | PCE/NCE x±SD |
| CMC B62 endoxan | 500 1000 2000 60 | 10 10 10 10 10 | 20271 20238 20171 20216 20278 | 33 40 54 61 1073 | 1.63±0.84 1.98±1.06 2.68±0.84 3.02±0.96 52.96±19.87 | 1.18±0.24 1.01±0.20 0.95±0.12 1.04±0.13 1.07±0.19 |
Table 18. is irritated stomach B68 24h the ICR mouse bone marrow cells is had a liking for polychromatophilia
The influence that red corpuscle (PCE) micronucleus forms
| Medicine | Dosage (mg/kg) | Number of animals (only) | Microscopy PCE number | MNPCE | MNPCE/PCE x±SD‰ | PCE/NCE x±SD |
| CMC B68 endoxan | 125 250 500 60 | 10 10 10 10 10 | 20271 20213 20258 20213 20278 | 33 46 48 46 1073 | 1.63±0.84 2.28±0.94 2.37±1.70 2.27±1.09 52.96±19.87 | 1.18±0.24 1.08±0.25 1.03±0.18 1.13±0.52 1.07±0.19 |
To sum up can get conclusion: B62 and B68 and under this experiment condition, on the mouse bone marrow cells micronucleus test, not increase the micronucleus rate of formation.
Test example 8 compd Bs bring out cultivates the mammalian cell chromosomal aberration test
Experiment purpose
With the chromosome aberration is index, and observing the compd B in vitro tests has the damage that does not cause the CHL cell chromosome.
Compound method:
Tried thing and added a small amount of DMSO, added RPMI-1640 again.Make each dosage group nutrient solution volume identical.
Reference substance:
Positive control: silk splits the plain C of enzyme (Japanese Kyowa company product) as (S
9) experiment
Reference substance.
Endoxan (Shanghai the 12 pharmaceutical products) is as metabolism activation system reference substance.
Negative control: RPMI-1640
Cell
The CHL cell is as the target cell of test B to the karyomit(e) influence.The CHL cell is introduced by the institute for drug control, Shanghai, and mycoplasma is checked negative.
Substratum
RPMI1640 (GIBCO product) cell adds 15% calf serum and places 37 ℃, 5%CO
2Incubator is made monolayer cell and is cultivated.
Dosage
Measure IC
50Value is 1.82 μ g/ml, and as maximum dose level two-fold dilution successively, ultimate density is three dosage groups of 1.82,0.91,0.455 μ g/ml.
The metabolism activation agent
Induce preparation rat liver S with Aroclor 1254
9The Sprague-Dawley rat of about 200 grams of body weight, ip Aroclor 1254 (Nihon Pharmaceutical Co., Ltd.) 500mg/kg, put to death in the 5th day, aseptic condition is flushing and taking-up liver down, use 4 ℃ of 0.15M KCl lavation immediately, add 4 ℃ of 0.15M KCl homogenate in 3ml/g weight in wet base ratio again, 9000 * g is centrifugal, gets its supernatant liquor and both has been S
9On Salmonellas TA97, check S in addition
9Biological activity.
Drug treating time
Non-metabolism activation experiment medicine and cell directly act on 24h.
The metabolism activation experiment is to renew bright nutrient solution behind the 6h to continue to be cultured to 24h.
Preparation of specimen's time
Non-metabolism activation group and metabolism activation group 24h harvested cell are made sample.
Experiment
1.IC
50Measure
Inoculation about 1 * 10 in the 25ml culturing bottle
5Cell is cultivated through 24h, adds test article, and making ultimate density is 6 concentration of 31.25,15.6,7.8,3.9,1.95,0.98 μ g/ml.Continue to be cultured to 48h.Discard nutrient solution, after digestion, beat even cell, and counting, record the IC that is tried thing B with the Logit method
50Value is 1.82 μ g/ml.
2. chromosomal aberration test
Inoculation CHL cell, every bottle contains cell 1 * 10 approximately
5Add behind 37 ℃ of cultivation 24h and tried thing solution, make ultimate density in nutrient solution: be 1.82,0.91,0.455 μ g/ml.Each three dosage groups and negative control group, positive controls.Non-metabolism activation what 24h collecting cell.The metabolism activation group adds 0.1ml S again except adding soup
9Mixed solution is equally also surveyed 3 dosage groups and negative, positive controls.Renew bright nutrient solution after cultivating 6h, continue to be cultured to the 24h collecting cell.
3. Chromosome Preparation
3h adds colchicine (Colchicine) 0.2 μ g/ml before the collecting cell, and is again through trypsin treatment, centrifugal, supernatant liquor inclines.After the effect of 0.075M KCl hypotonic medium, at methyl alcohol: fixing in Glacial acetic acid (3: the 1) stationary liquid.Peek is dripped hanging drop on the cleaning glass sheet, Giemsa dyeing, microscopy.
4. microscopy
Microscopy under 100 times of oily mirrors.Each dosage group observation phase cell in 100 mid-terms.Press IshidateM.Jr
*Standard judgment experiment result.
5. the check of experiment validity
1. maximum concentration should meet the requirement of China's governing principle.Can reach the maximum concentration that solubleness or toxicity allow.
2. the negative control value should be in this laboratory in the past within the scope of negative control background information 99% fiducial limit.
3. the influence of necessary no any technical problem.As pollute, cytotoxicity is too high, or is not suitable for pH etc.
6. judgement criteria
Propose to such an extent that standard is estimated the result by Ishidate, wherein crack and polyploid are included.
| Negative (-): aberration rate＜4.9% |
| Suspicious (±): aberration rate 5～9.9% |
| Positive (+): |
| Than strong positive (++): aberration rate 20～49.9% |
| Strong positive (+++): aberration rate 〉=50% |
Negative: if the aberration rate of all test group all 4.9% with the next feminine gender that is judged to.
Positive: as, and to have the dependency of dosage maybe can repetition person then can be judged to positive findings if test group has at least one group to reach positive criteria.The result does not affirm that the person then answers revision test.
Experimental result
(+S
9) group: the compd B ultimate density is that 1.82,0.91,0.455 μ g/ml and negative control group chromosome aberrations rate are respectively: 2%, 4% and 3%.In the experiment, high, medium and low dosage group chromosome aberration rate＜5% is judged to feminine gender.It is 13% positive (seeing Table 19) that positive drug endoxan 24h brings out aberration rate.
Table 19 compd B is to CHL cell chromosome aberration rate (+S
9Group)
| Medicine | Dosage (μ g/ml) | Time (h) | ------distortion type *-------- | Aberration rate (%) | Evaluation ** | |||||||||
| b | f | t | p | q | e | d | r | l | tr | |||||
| Nutrient solution | 24 | 2 | 2 | 3 | - | |||||||||
| B | 1.82 0.91 0.455 | 24 24 24 | 3 1 | 2 1 2 | 2 4 3 | - - - | ||||||||
| Endoxan | 50 | 24 | 2 | 2 | 5 | 4 | 13 | + | ||||||
Annotate: positive control: endoxan is used as+S
9System experimentation
*Distortion type: b-fracture; The p-polyploid; T-three width of cloth bodies; The q-quadriradiatus; A-is damaged; The e-exchange; The f-fragmentation; The d-dicentromere; The g-crack; The r-ring-type; L-loses: the tr-transposition
*Judgement criteria: (-):＜5%; (±): 5-9%; (+): 10-19%; (++): 20-49%; (+++): 〉=50%;
N kind shape appears in a cell chromosome distortion, and aberration rate is calculated as 1 time
The 9 mouse stomach B68 acute toxicity tests of test example
Experiment purpose
Observation is tried thing and is once irritated behind the stomach acute toxic reaction and death condition to mouse produced.
Compound method:
To be tried thing and be mixed with respective concentration, each dosage group administration volume is equated with 0.5%CMC-Na, now with the current.
Animal
60 of Kunming mouses (♀ 30, and ♂ 30) provide conformity certification by Chinese Academy of Sciences's Shanghai Experimental Animal Center: No. the 005th, the moving pipe of middle section.Raise through all adaptability.Feed is available from by Shanghai Shi Lin Science and Technology Ltd..Freely fetch water 23 ± 2 ℃ of raising temperatures.Body weight: body weight is the 18-22 gram during administration.Sex: male and female half and half.Every treated animal number: by the body weight random packet, be a dosage group with per 10 during experiment.
Dosage
Dosage is provided with: by pre-experimental result just, maximum dose level is decided to be 4082mg/kg, and the ratio row with 0.7 successively decrease, and is 686,980,1400,2000,2857,4082mg/kg totally 6 dosage groups; Agent distance: 0.7; Every animals received capacity: irritate body of stomach and amass: the 0.2ml/g body weight,
Route of administration
Gastric infusion of mouse.
Method
Mouse is respectively divided 6 groups at random by body weight, and 10 every group, every group of mouse body weight distributes similar.Promptly observe animal each side response situation after the administration, dead animal is dissected, and checks internal organ, the death toll of record animal every day.
Observation index
Observation period: 14 days
Toxic reaction: situations such as observation and inspection mouse outward appearance, behavior, feed, ight soil, dead animal becomes celestial.When experiment finished in the 15th day, survival mice was dissected, organ diseases such as the visual control heart, lung, liver,spleen,kidney.
The result
1 toxic reaction and the cause of death
Behind the mouse stomach about 30 minutes appearance activity minimizing, paroxysmal tremble, lurch, behind the medicine about 1 hour beginning dead.Most of death occurred in 4 hours.Toxic reaction and dosage are proportional.The mouse internal organs visual control that becomes celestial shows no obvious abnormalities.Survival mice was all dissected on the 15th day, and the visual control internal organ are not seen obvious pathology.Death condition is listed in the table below.
Irritate the tabulation of stomach B68 dead mouse situation
| Tried agent amount (mg/kg) | Number of animals (n) | 4b | Death condition | Add up to (n) | |||||||||||||||
| 1 | 2 | D3 | D4 | D5 | D6 | D7 D14 | |||||||||||||
| ♀ | ♂ | ♀ | ♂ | ♀ | ♂ | ♀ | ♂ | ♀ | ♂ | ♀ | ♂ | ♀ | ♂ | ♀ | ♂ | ♀ ♂ | |||
| 686 980 1400 2000 2857 4082 | 5 5 5 5 5 5 | 5 5 5 5 5 5 | 0 1 2 0 1 4 | 0 0 1 1 1 2 | 0 0 0 2 2 0 | 0 0 0 1 1 2 | 0 0 0 0 0 0 | 0 0 0 0 1 0 | 0 0 0 0 0 0 | 0 0 0 0 0 0 | 0 0 0 0 0 0 | 0 0 0 0 0 0 | 0 0 0 0 0 0 | 0 0 0 0 0 0 | 0 0 0 0 0 0 | 0 0 0 0 0 0 | 0 0 0 0 0 0 | 0 0 0 0 0 0 | 0 1 3 4 6 8 |
2 mouse dose-response numerical tabulars
| Tried thing (mg/kg) | Log10 dose (x) | Number of animals (only) | Dead animal number (only) | Mortality ratio (%) | Probit (Y) | LD50 and 95% fiducial limit (mg/kg) |
| 686 980 1400 2000 2857 4082 | 2.84 2.99 3.15 3.30 3.46 3.61 | 10 10 10 10 10 10 | 0 1 3 4 6 8 | 0 10 30 40 60 80 | 0 3.72 4.48 4.75 5.25 5.84 | 2315 (1790～2993) |
12.3 LD
50Value and statistical method
Calculate LD with the Bliss method
50As follows:
Mouse LD
50=2315mg/kg; The 95% credible 1790～2993mg/kg that is limited to.
Brief summary
Behind the mouse stomach about 30 minutes appearance activity minimizing, paroxysmal tremble, lurch, behind the medicine about 1 hour beginning dead.Most of death occurred in 4 hours.Toxic reaction and dosage are proportional.The survivor all dissected on the 15th day, and the visual control internal organ are not seen obvious pathology.Mortality ratio is tried to achieve LD with the Bliss method
50For: 2315mg/kg; The 95% credible 1790～2993mg/kg that is limited to.
Test example 10 cell levels test compounds EGFR suppress active
Experiment purpose:
From 27 kinds of tyrosine kinase inhibitor newtype drugs of new synthetic, filter out a kind of cell growth inhibiting efficient height (effective concentration is low), specificity at EGFR tyrosine kinase pathway, the stable treatment polycystic kidney new drug of effect, prepare animal experiment.
Experiment material:
Cell: SPCA1 human lung carcinoma cell; Stimulating factor: hEGF: human epidermal growth factor (Rand D Catalog:236-EG); Substratum: DMEM/F12 1: 1 (GIBCO); Calf serum (Hangzhou folium ilicis chinensis); DMSO; MTT (5g/l)
Experimental technique: MTT
Experimental procedure:
One, new drug configuration
Be made into the DMSO solution of 10mM/L according to molecular weight;-20 degree are preserved;
Two, at cell levels medicine is carried out rough sizing, find out and suppress the medicine that cel l proliferation is the strongest, effective concentration is minimum.
1, first day, in 96 orifice plates, cell density was 1 * 10 with the DMEM/F12 nutrient solution inoculating cell that contains 10% serum
4Individual/hole;
2, second day, the DMEM/F12 synchronization of usefulness serum-free 24 hours;
3, the 3rd day, abandon original nutrient solution, dosing; Each concentration is respectively established 3 multiple holes; Every hole adds 100ul; Drug intervention 48 hours; Get the mother liquor of 10mM/L and do dilution in 1: 1000 (DMEM/F12 that uses serum-free is as solvent),, make doubling dilution, establish 7 drug levels altogether as maximum concentration; That is: 10um, 5um, 2.5um, 1.25um, 0.625um, 0.3125um, 0.156um, 0um (negative control);
4, the 5th day, every hole added 10ul MTT, surveyed the OD value after 4 hours in the 492nm place;
5, interpretation of result;
Proliferation inhibition rate with negative control group is 0, and all the other each concentration are compared with it, draw inhibiting rate; Calculating the inhibiting rate formula is:
(control group OD value-experimental group OD value)/control group OD value * 100%
Three, in order to filter out the new drug of specificity at the EGFR tyrosine kinase pathway, this experiment has been set up and has been added the hEGF stimulating group, medicine after the scalping is carried out fine screening, find out suppress that cel l proliferation is the strongest, activity is minimum, the novel tyrosine kinase inhibitor of high specificity.
1, first day, in 96 orifice plates, cell density was 1 * 10 with the DMEM/F12 nutrient solution inoculating cell that contains 10% serum
4Individual/hole;
2, second day, the DMEM/F12 synchronization of usefulness serum-free 24 hours;
3, the 3rd day, abandon original nutrient solution, dosing; Each concentration is respectively established 3 multiple holes; Every hole adds 100ul; Drug intervention 48 hours; Be divided into hEGF stimulating group and no hEGF stimulating group;
No hEGF stimulating group:
Get the mother liquor of 10mM/L and do dilution in 1: 1000 (DMEM/F12 that uses serum-free is as solvent),, make doubling dilution, establish 7 drug levels altogether as maximum concentration; That is: 10um, 5um, 2.5um, 1.25um, 0.625um, 0.3125um, 0.156um, 0um (negative control);
Add the hEGF stimulating group:
The same, with the DMEM/F12 of serum-free contain 10ng/ml hEGF as solvent, medicine is done as above dilution, negative control is the DMEM/F12 that contains the serum-free of 10ng/ml hEGF.
4, the 5th day, every hole added 10ul MTT, surveyed the OD value after 4 hours in the 490nm place;
5, interpretation of result;
Proliferation inhibition rate with negative control group is 0, and all the other each concentration are compared with it, draw inhibiting rate; Calculating the inhibiting rate formula is:
(control group OD value-experimental group OD value)/control group OD value * 100%
Experimental result: see Table 20 and table 21
Table 20 Compound C 151～C154 cell levels test EGFR suppresses active result
| Concentration (M) Compd. | 0μm | 0.01μm | 0.06μm | 0.32μm | 1.6μm | 8μm | 40μm | 200μm |
| C151 C152 C153 C154 | 0 0 0 0 | -10.9％ -15.5％ -14.6％ -0.15％ | -5.83％ -14.6％ -21.5％ -12.0％ | -16.7％ -18.5％ -20.7％ -19.1％ | -3.57％ -9.63％ -0.26％ 0.45％ | 9.62％ 4.25％ 5.87％ 36.1％ | 37.9％ 17.2％ 58.9％ 76.1％ | 61.3％ 57.4％ 79.1％ 69.5％ |
Table 21 Compound C 151, C153﹠amp; C154 secondary cell horizontal checkout EGFR suppresses active knot
Really
| Concentration (M) Compd. | 0μm | 0.75μm | 1.5μm | 3μm | 6μm | 12μm | 25μm | 50μm |
| C151 C153 C154 | 0 0 0 | -5.77％ 9.63％ 13.4％ | 7.49％ 21.0％ 21.9％ | 18.2％ 30.8％ 24.7％ | 19.7％ 32.9％ 33.7％ | 26.9％ 48.0％ 64.5％ | 46.6 46.3％ 77.0％ | 59.4％ 56.8％ 81.4％ |
Test example 11 anti-tumor biological body outer screening tests
Experiment 1:
Screening method: sulphonyl rhodamine B (sulforhodamine B, SRB) protein staining method
Cell strain: HCT-116 people's intestinal cancer
Action time: 72
*
Result evaluation: invalid: 10
-5Mol/L＜85%;
The weak effect: 10
-5Mol/L 〉=85% or 10
-6Mol/L＞50%;
Potent: 10
-6Mol/L 〉=85% or 10
-7Mol/L＞50%.
Table 22HCT-116 people intestinal cancer body outer screening test result (part)
Inhibiting rate (%) to growth of tumour cell
| Concentration (M) Compd. | HCT- | Estimate | |||||
| 10 -4 | 10 -5 | 10 -6 | 10 -7 | 10 -8 | |||
| B B51 B53 B54 B55 B56 B57 B59 B60 B62 | 97.8 96.7 77.6 92.2 94.0 94.1 95.6 89.8 95.3 86.7 | 98.8 48.7 97.9 98.8 98.2 98.6 98.4 97.8 97.8 98.6 | 23.2 0 82.7 42.2 30.3 54.0 40.2 54.3 30.7 5.6 | 0 0 0 6.6 3.6 10.0 0 2.1 0 0 | 0 0 0 3.5 2.9 10.6 0 4.6 0 3.3 | The weak effect of the weak effect weak effect of the weak effect of the weak effect of the weak effect of the weak effect of the weak effect of invalid weak effect | |
| B64 B65 B66 B67 B68 B75 B77 B85 C20 | 95.2 92.5 97.5 96.4 95.2 80.7 96.4 91.5 70.0 | 98.0 98.2 98.7 98.8 99.5 98.4 96.8 97.5 71.1 | 11.5 8.4 9.0 2.2 79.5 49.6 4.5 0 74.3 | 0 0 0 0 0 18.0 7.4 0 79.1 | 0 17.8 0 0 0 25.3 0 0 78.4 | The weak effect of the weak effect of the weak effect weak effect of the weak effect of the weak effect of invalid weak effect is potent |
| C151 C152 C153 C154 C71 C72 C74 C158 | 58.3 77.7 97.8 92.0 58.0 96.7 74.0 97.6 | 81.3 81.4 84.7 96.2 51.7 78.3 53.3 85.5 | 64.6 72.5 72.9 72.1 70.3 54.1 68.4 63.6 | 42.6 52.9 60.1 63.9 19.6 13.8 16.7 39.1 | 52.3 40.8 53.4 46.0 11.4 43.3 23.6 0 | The weak effect of the weak effect of the weak effect of potent weak effect |
Experiment 2:
Screening method: sulphonyl rhodamine B (sulforhodamine B, SRB) protein staining method
Cell strain: HT-29 people's intestinal cancer
Action time: 72
*
Result evaluation: invalid: 10
-5Mol/L＜85%;
The weak effect: 10
-5Mol/L 〉=85% or 10
-6Mol/L＞50%;
Potent: 10
-6Mol/L 〉=85% or 10
-7Mol/L＞50%.
Table 23HT-29 people intestinal cancer body outer screening test result (part)
Inhibiting rate (%) to growth of tumour cell
| Concentration (M) Compd. | HT-29 | |
||||
| 10 -4 | 10 -5 | 10 -6 | 10 -7 | 10 -8 | ||
| B53 B54 B56 B59 B60 B65 B67 B70 B75 B76 B84 B85 C19 C20 C151 C152 C153 C154 | 59.7 77.9 81.0 68.1 85.5 78.1 98.8 98.2 79.1 68.8 86.2 60.5 56.0 90.2 80.5 | 86.5 88.4 91.8 88.7 88.9 91.8 100 100 93.9 92.1 86.6 89.0 46.7 54.1 71.2 100 76.4 80.4 | 42.7 0 14.9 42.8 0 76.6 55.6 70.8 46.8 0 0 0 69.1 61.3 85.0 91.6 51.3 48.8 | 0 0 0 2.1 0 0 0 5.5 39.0 0 0 0 17.1 19.4 12.5 97.2 5.5 39.0 | 0 0 0.9 9.6 0 0 0 0 4.4 0 0 0 15.3 0 0 37.8 16.7 33.6 | The weak effect of the weak effect of the weak effect of the weak effect of the weak effect of the weak effect of the weak effect of the weak effect of the weak effect of the weak effect of the weak effect of the weak effect of the weak effect of weak effect is potent invalid |
| C71 C72 C73 C74 C155 C156 C157 C158 C159 C160 C161 C162 C163 C166 | 82.4 92.6 92.4 84.7 90.0 91.2 85.9 93.4 74.6 77.5 87.7 82.0 91.6 93.1 | 87.1 89.3 87.4 80.6 87.3 89.7 87.5 87.8 82.3 85.5 90.0 83.2 74.6 90.5 | 88.0 67.5 91.5 87.8 77.2 94.1 81.5 81.2 69.3 40.8 56.5 71.1 23.5 0 | 78.4 41.6 27.5 66.6 0 54.3 40.8 72.0 15.4 0 0 44.3 0 0 | 23.3 36.0 24.9 43.9 20.4 28.3 6.8 58.5 12.5 0 0 20.0 0 0 | The weak effect of the potent weak effect of the potent weak effect potent weak effect weak effect of the weak effect of the weak effect of the weak effect of potent weak effect |
The possibility of utilizing on industry
The preparation method of replacement of the present invention [1,3,5] compound in triazine class has that reaction condition gentleness, abundant raw material are easy to get, operation and the advantage such as post processing is simple.
Replacement [1 of the present invention, 3,5] compound in triazine class is at computer virtual screening and cyclooxygenase and 5-lipoxygenase (COX-1, COX-2 and 5-LOX) confirm all that in conjunction with pharmacological testing in experiment and animal body this compounds is the double inhibitor of cyclooxygenase and 5-lipoxygenase (COX-2 and 5-LOX), Experimental Inflammation there is preventive and therapeutic action preferably, and Ames experiment and micronuclei in mice experiment are had security preferably, GI side effect is obviously weakened.
On the interactional model of biosensor technique Biacore 3000 research replacement of the present invention [1,3,5] compound in triazine class and cyclooxygenase and 5-lipoxygenase (COX-1, COX-2 and 5-LOX), observe many compounds 10-7-10
-5In the M concentration range, this model all there is effect.
Compound of the present invention is to mouse Irish moss colloidality foot swelling model, the rat paw carrageenan causes swollen model, the scorching model of mouse ear caused by dimethylbenzene xylene, rat assist agent arthritis (prevention primary affection) model, rat assist agent arthritis (prevention secondary affection) model has good antiinflammatory action.
Toxicity of compound of the present invention is very low. Ames experiment and micronuclei in mice experiment there are is security preferably, intestines and stomach are had no side effect.
Replacement of the present invention [1,3,5] compound in triazine class has demonstrated and has suppressed preferably active in the body outer screening test of antitumor cell strain HCT-116 people's intestinal cancer and HT-29 people's intestinal cancer, many compounds show as potent inhibitor.
Compound of the present invention has shown also that in the experiment of EGFR cell tests the inhibition of μ M level is active.
Therefore, compound of the present invention can be used for preparing the medicine that prevents and/or treats diseases associated with inflammation; Compound of the present invention also can be used for preventing and/or treating and/or the medicine of adjuvant therapy of tumors disease.
Claims (16)
1, a kind of replacement [1,3,5] compound in triazine class, its pharmacy acceptable salt and solvate or hydrate with structure shown in the formula (I):
Wherein,
R
1, R
2, R
3And R
4Be hydrogen, C independently of one another
1-C
8Straight or branched alkyl, replacement or unsubstituted aryl, aralkyl, C
1-C
4Alkaryl, aroyl, wherein aryl is selected from phenyl, naphthyl and xenyl, and substituting group is that individual being selected from of 1-4 comprises halogen, C
1-C
6Straight or branched alkyl, cyano group, nitro, amino, hydroxyl, methylol, trifluoromethyl, trifluoromethoxy, carboxyl, C
1-C
4Alkoxyl group, sulfydryl, C
1-C
4Alkylthio, C
1-C
4Alkyl carbonyl, C
1-C
4The group of the group of carbalkoxy, alkylsulfonyl; And
R
5Structural formula be
, R wherein
6And R
7Comprise hydrogen, halogen, C for being selected from independently of one another
1-C
4Straight or branched alkyl, cyano group, nitro, amino, hydroxyl, methylol, trifluoromethyl, trifluoromethoxy, carboxyl, C
1-C
4Alkoxyl group, sulfydryl, C
1-C
4Alkylthio, C
1-C
4The group of the group of acyl group and alkylsulfonyl; Y is CH
2, O, S or RN, wherein R is hydrogen, C
1-C
4Straight or branched alkyl, hydroxyl, C
1-C
4Hydroxyalkyl, carboxyl, C
1-C
4Alkyl carbonyl, C
1-C
4The group of carbalkoxy, alkylsulfonyl; M, n are 0,1,2 or 3 independently of one another,
Perhaps, R
5Be selected from hydroxyl, amino, substituted-amino, C
1-C
6Alkyl, C
1-C
6The alkane hydroxyl ,-C (O) R
8,-(CH
2)
xR
8,-CH
2CH=CHR
8,-C (O) OR
8Or-S (O)
2R
8, wherein, x is 0,1,2 or 3; R
8Be hydrogen, hydroxyl, aryl, hetero-aromatic ring base, heterolipid cyclic group or C
2-C
6Thiazolinyl.
2, according to replacement [1,3,5] compound in triazine class, its pharmacy acceptable salt and the solvate or the hydrate of claim 1, it is characterized in that described replacement [1,3,5] compound in triazine class:
3, replace the preparation method of [1,3,5] compound in triazine class or its pharmacy acceptable salt or their solvate or hydrate shown in the described general formula of claim 1 (I), it is characterized in that may further comprise the steps:
(1) compound shown in the general formula (II) is reacted with replacement amine under 0-50 ℃ of temperature in basic solvent,
Wherein, R
xBe hydrogen, halogen, hydroxyl, sulfydryl, C
1-C
4Alkylamine or C
1-C
4Aminoalkyl; R
yAnd R
zBe respectively hydrogen, halogen, C
1-C
4Aminoalkyl or C
1-C
4The halo alkyl, thus compound shown in the general formula (III) obtained:
Wherein, R
1, R
2, R
xAnd R
yIdentical with definition among general formula (I), (II);
(2) with compound shown in the general formula (III) in basic solvent under 20-100 ℃ of temperature with replace reactions such as amine, substituted aromatic amines, substituted aryl alkanamine, alcohol or mercaptan, obtain compound shown in the general formula (IV):
Wherein, R
1, R
2, R
3, R
4Identical with definition in the general formula (I), R
xIdentical with definition in the general formula (II);
(3) under 0-120 ℃, in basic solvent, with compound shown in the general formula (IV) with replace aliphatic cyclic amine, substituted aryl amine, substituted aryl alkanamine, alcohol or mercaptan and react, obtain compound shown in the formula (I);
(4) as required, be carried out to reactant salt, or form solvate or hydrate by this area ordinary method.
4, preparation method as claimed in claim 3, wherein the basic solvent described in step (1) and the step (2) is that described alkali is selected from organic bases that comprises pyridine, triethylamine, 4-dimethylamino pyridine, diisopropylethylamine and the group that comprises the mineral alkali of yellow soda ash, salt of wormwood, sodium hydroxide, potassium hydroxide with the alkaline solution of inert solvent preparation.
5, preparation method as claimed in claim 4, wherein said inert solvent is selected from the solvent of the group that comprises tetrahydrofuran (THF), ether, dimethyl formamide, glycol dimethyl ether, ethylene glycol diethyl ether, dioxane for one or more.
6, preparation method as claimed in claim 3, wherein the basic solvent described in the step (3) is that described polar organic solvent is selected from the solvent of the group that comprises dimethyl formamide, dimethyl sulfoxide (DMSO), dioxane, ethanol, methyl alcohol, acetone, ethyl acetate and tetrahydrofuran (THF) for one or more with the alkaline solution of polar organic solvent preparation; Described alkali is selected from the organic bases that comprises pyridine, triethylamine, 4-dimethylamino pyridine (DMAP), diisopropylethylamine for one or more and comprises the alkali of group of the mineral alkali of yellow soda ash, salt of wormwood, sodium hydroxide, potassium hydroxide.
7, the described replacement of claim 1 [1,3,5] compound in triazine class or its pharmacy acceptable salt or their solvate or hydrate are as the application of cyclooxygenase-2 inhibitors.
8, the described replacement of claim 1 [1,3,5] compound in triazine class or its pharmacy acceptable salt or their solvate or hydrate are as the application of 5-lipoxygenase inhibitor.
9, the described replacement of claim 1 [1,3,5] compound in triazine class or its pharmacy acceptable salt or their solvate or hydrate are as the application of cyclooxygenase and 5-lipoxygenase double inhibitor.
10, the described replacement of claim 1 [1,3,5] compound in triazine class or its pharmacy acceptable salt or their solvate or hydrate prevent and/or treat the application on the medicine of inflammatory disease in preparation.
11, the described replacement of claim 1 [1,3,5] compound in triazine class or its pharmacy acceptable salt or their solvate or hydrate are as the EGFR application of enzyme inhibitors.
12, the described replacement of claim 1 [1,3,5] compound in triazine class or its pharmacy acceptable salt or their solvate or hydrate are used to prevent and/or treat and/or the application of auxiliary for treating cancer.
13, a kind of pharmaceutical composition, it is characterized in that, described pharmaceutical composition comprise the treatment significant quantity suc as formula [1,3, the 5] compound in triazine class of the replacement shown in (I) or its pharmacy acceptable salt or their solvate or hydrate and at least a pharmaceutically acceptable carrier.
14, pharmaceutical composition as claimed in claim 13, it is characterized in that, described pharmaceutically acceptable carrier comprises partial glycerol ester mixture, the water of ion-exchanger, aluminum oxide, aluminum stearate, Yelkin TTS, serum protein, buffer substance such as phosphoric acid salt, glycerine, Sorbic Acid, potassium sorbate, saturated vegetable fatty acid, salt or ionogen, Sodium phosphate dibasic, potassium hydrogen phosphate, sodium-chlor, zinc salt, colloided silica, Magnesium Trisilicate, polyvinylpyrrolidone, cellulosic material, polyoxyethylene glycol, Xylo-Mucine, polyacrylic ester, beeswax and lanolin.
15, pharmaceutical composition as claimed in claim 13 is used to prevent and/or treat the application of diseases associated with inflammation.
16, pharmaceutical composition as claimed in claim 13 is used to prevent and/or treat and/or the application of adjuvant therapy of tumors disease.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610057786 CN1970552A (en) | 2005-11-25 | 2006-02-27 | Substituted[1,3,5] triazine compound, its preparing process and its application |
| PCT/CN2006/003428 WO2007095812A1 (en) | 2006-02-27 | 2006-12-15 | Substituted [1,3,5] triazine compounds, their processes for preparation and uses thereof |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200510115078 | 2005-11-25 | ||
| CN200510115078.9 | 2005-11-25 | ||
| CN 200610057786 CN1970552A (en) | 2005-11-25 | 2006-02-27 | Substituted[1,3,5] triazine compound, its preparing process and its application |
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| Publication Number | Publication Date |
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| CN1970552A true CN1970552A (en) | 2007-05-30 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200610057786 Pending CN1970552A (en) | 2005-11-25 | 2006-02-27 | Substituted[1,3,5] triazine compound, its preparing process and its application |
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| CN (1) | CN1970552A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102573483A (en) * | 2009-06-08 | 2012-07-11 | 加利福尼亚资本权益有限责任公司 | Triazine derivatives and their therapeutical applications |
| CN102675234A (en) * | 2012-05-07 | 2012-09-19 | 陕西师范大学 | Synthetic method of sym-triazine derivative |
| CN102887867A (en) * | 2011-07-21 | 2013-01-23 | 中国科学院上海药物研究所 | Triazine compound and preparation method and application of triazine compound |
| CN110357863A (en) * | 2019-08-27 | 2019-10-22 | 药雅科技(上海)有限公司 | Double aromatic ring derivative egf inhibitors of a kind of triazine and preparation method thereof and purposes |
| CN111662242A (en) * | 2019-03-08 | 2020-09-15 | 中国药科大学 | Heterocyclic IDH mutant inhibitor, preparation method and application thereof |
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2006
- 2006-02-27 CN CN 200610057786 patent/CN1970552A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102573483A (en) * | 2009-06-08 | 2012-07-11 | 加利福尼亚资本权益有限责任公司 | Triazine derivatives and their therapeutical applications |
| CN102887867A (en) * | 2011-07-21 | 2013-01-23 | 中国科学院上海药物研究所 | Triazine compound and preparation method and application of triazine compound |
| CN102887867B (en) * | 2011-07-21 | 2015-04-15 | 中国科学院上海药物研究所 | Triazine compound and preparation method and application of triazine compound |
| CN102675234A (en) * | 2012-05-07 | 2012-09-19 | 陕西师范大学 | Synthetic method of sym-triazine derivative |
| CN102675234B (en) * | 2012-05-07 | 2015-01-07 | 陕西师范大学 | Synthetic method of sym-triazine derivative |
| CN111662242A (en) * | 2019-03-08 | 2020-09-15 | 中国药科大学 | Heterocyclic IDH mutant inhibitor, preparation method and application thereof |
| CN111662242B (en) * | 2019-03-08 | 2023-10-24 | 中国药科大学 | Heterocyclic IDH mutant inhibitor, preparation method and application thereof |
| CN110357863A (en) * | 2019-08-27 | 2019-10-22 | 药雅科技(上海)有限公司 | Double aromatic ring derivative egf inhibitors of a kind of triazine and preparation method thereof and purposes |
| CN110357863B (en) * | 2019-08-27 | 2023-04-14 | 药雅科技(上海)有限公司 | Triazine double aromatic ring derivative epidermal growth factor inhibitor and preparation method and application thereof |
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