CN101323629A - 4-{6-[5-(2-chlorinde-6- methylaniline formyl)-thiazole-2-amido]-2-methyl pyrimidine-4}-piperazine-1- diethyl methylphosphate - Google Patents
4-{6-[5-(2-chlorinde-6- methylaniline formyl)-thiazole-2-amido]-2-methyl pyrimidine-4}-piperazine-1- diethyl methylphosphate Download PDFInfo
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Abstract
The invention relates to a chemical compound of 4-(6-(5-(2-chlorine-6-methylaniline formoxyl)-thiazole-2-amido)-2-methylpyrimidine-4)-piperazine-1-methyl triethyl phosphate shown in general formula I, salts thereof for medical purposes, hydrates and solvates thereof, polycrystals and eutectic crystals thereof, precursors or derivatives thereof with the same biological function, a preparation method thereof, a composition containing one or more chemical compounds and the application of the chemical compound in the treatment of diseases relating to protein tyrosine kinase, such as immune disorder and oncology diseases.
Description
Technical field
The present invention relates to the 4-{6-[5-shown in the formula I (2-chloro-6-methylaniline formyl)-thiazole-2-amino]-2-methylpyrimidine-4}-piperazine-1-methyl acid phosphate diethyl ester; its pharmacologically acceptable salt; its hydrate and solvate; its polycrystalline and eutectic; the precursor of its same biological function or derivative; and preparation method thereof, contain one or more these compound compositions and this compounds purposes aspect treatment and protein tyrosine kinase diseases associated such as immune disorder and tumor disease.
Background technology
In recent years, because the Study on Molecular Mechanism of tumor development has obtained breakthrough, the specific molecular biosciences target spot that the research steering of new drug works in the nosetiology of cancer and pathologic process (Science, 1993,260 (5110), 918-919).(Science，1995，267(5205)，1782-1787)。Studies show that, oncogene more than 80% and proto-oncogene are present in people's the cancer proteins encoded Tyrosylprotein kinase (PTK), the generation of human various cancers is relevant with the abnormal cells signal conduction that comes from protein tyrosine kinase with development, an increase that principal feature is a tyrosine kinase activity of malignant cell.In addition, the overexpression of normal former carcinogenic Tyrosylprotein kinase also can cause proliferative disease.Verified in the laboratory: by the undue expression or the various receptor tyrosine kinases that make a variation, increase its activity, normal cell can be converted to cancer cells, and the degree of pernicious transformation is closely related with tyrosine kinase activity; And antibody by utilizing acceptor or special kinase inhibitor reduce that kinase activity can make canceration reverse (Drugs, 2000,59 (4): 753) again in the acceptor.Therefore, suppress tyrosine kinase activity, block the new way that its activatory signal conducting path becomes the control tumour.
Protein tyrosine kinase (PTK) is a kind of enzyme, except comprising receptor tyrosine kinase (RPTK): as the member (for example HER1 and HER2) of epidermal growth factor kinase family, outside Thr6 PDGF BB (PDGF) and the kinases (Tie-2 and KDR) that in vascularization, works, also comprise nonreceptor tyrosine kinase Syk, the member of JAK and Src family (Sro for example, Fyn, Lyn, Lck and Blk) (see " the src family of the tyrosine protein kinase in the hematopoiesis signal transduction ", the United States Federal's experimental biology meeting federation's magazine (FASEB J.), 1992,6,3403-3409; " Role in Plant Signal Transduction " with acceptor of tyrosine kinase activity, cell (Cell), 1990,61,203-212; " the Janus protein tyrosine kinase in the conduction of hematopoietic cytokine signal ", immunology investigation literary composition volume (Sem.Immun01.), 1995,7,247-254).
The activity of PTK not only all demonstrates enhancing in many pernicious and non-neoplasm diseases, and PTK plays a crucial role in immune cell regulate and control.Therefore, ptk inhibitor can be influential to many kinds of tumours and amynologic disease.Can be by optionally suppressing certain acceptor or non-acceptor PTK, for example Lck perhaps owing to the homology between all kinds of PTK, utilizes inhibitor to suppress more than one PTK, thereby these illnesss is eased.
A kind of PTK of particularly important is the Lck that finds in the T cell, and it is relevant with the phosphorylation of key protein substrate in the T cell. it is that conduction of productivity antigen receptor signal and cell activation are needed.When not having Lck active, TXi Baoshouti (TCR) zeta chain is not by phosphorylation, and kinases ZAP-70 is not activated, and can not take place for the activation of the vital Ca particle of T cell activation passage, therefore, the inhibitor of Lck can be used for treating the cell-mediated disease of T.For example, the chronic disease that the T cell plays an important role, the acute disease that plays an important role therein as rheumatoid arthritis, multiple sclerosis disease and lupus and known T cell, for example acute transplant rejection and delayed hypersensitivity.
Though it has been found that many small molecules tyrosine kinase inhibitors, as WO9903854, WO2004005281, WO0062778, WO2005013983 has made very big contribution to this area, but for improving anticancer and immune disorder medicine, research is still being continued in this area.
Summary of the invention
The object of the present invention is to provide a kind of new tyrosine kinase inhibitor 4-{6-[5-(2-chloro-6-methylaniline formyl)-thiazole-2-amino]-2-methylpyrimidine-4}-piperazine-1-methyl acid phosphate diethyl ester; its pharmacologically acceptable salt; its solvate, its prodrug, its polycrystalline or eutectic.
Another object of the present invention is to provide a kind of 4-{6-[5-of preparation (2-chloro-6-methylaniline formyl)-thiazole-2-amino]-method of 2-methylpyrimidine-4}-piperazine-1-methyl acid phosphate diethyl ester.
A further object of the present invention is to provide a kind of pharmaceutical composition that contains one or more this compounds.
Another purpose of the present invention be to provide this compounds anticancer and immune and with the medicine of Tyrosylprotein kinase diseases related in purposes.
In order to finish the present invention's purpose, can adopt following technical scheme:
The present invention relates to have 4-{6-[5-(the 2-chloro-6-methylaniline formyl)-thiazole-2-amino of following formula I structure]-2-methylpyrimidine-4}-piperazine-1-methyl acid phosphate diethyl ester:
Or its pharmacologically acceptable salt, hydrate and solvate, its polycrystalline and eutectic, the precursor of its same biological function or derivative.
The invention also discloses the method for preparing The compounds of this invention, comprise the steps:
(a) pyrimidine derivatives links to each other with phosphate derivative earlier,
(b) and then with thiazole derivative link to each other,
(c) link to each other with 2-chloro-6-methyl phenylamino again, finally obtain the described compound of formula I;
In addition, starting raw material and intermediate in the above-mentioned reaction obtain easily, or can be easy to the ordinary method in the organic synthesis synthesize to those skilled in the art.
Wherein, piperazine in the step (a)-1-methyl acid phosphate diethyl ester and 2-methyl-4, the 6-dichloro pyrimidine links to each other; It is anhydrous 1 to be reflected at diisopropyl ethamine and solvent, carries out under the condition that the 4-dioxane exists; Obtain product 4-(6-chloro-2-methylpyrimidine-4) piperazine-1-methyl acid phosphate diethyl ester.
4-(6-chloro-2-methylpyrimidine-4) piperazine-1-methyl acid phosphate diethyl ester and 2-amino-5-ethoxycarbonyl thiazole carry out under the condition of solvent dry DMF and sodium hydride existence in the step (b), obtain product 2-{6-[4-(diethoxy phosphoryl methyl) piperazine-1]-2-methylpyrimidine-4-amino }-thiazole-5-carboxylic acid ethyl ester.
2-{6-[4-(diethoxy phosphoryl methyl) piperazine-1 in the step (c)]-2-methylpyrimidine-4-amino }-thiazole-5-carboxylic acid ethyl ester is under the condition that solvent methanol exists, slough ester group with sodium hydroxide, obtain 2-{6-[4-(diethoxy phosphoryl methyl) piperazine-1]-2-methylpyrimidine-4-amino }-thiazole-5-carboxylic acid;
The 2-{6-[4-that obtains (diethoxy phosphoryl methyl) piperazine-1]-2-methylpyrimidine-4-amino }-thiazole-5-carboxylic acid and 2-chloro-6-methyl phenylamino, reaction obtains product 4-{6-[5-(2-chloro-6-methylaniline formyl)-thiazole-2-amino under the condition that triethylamine, solvent dry DMF, diethyl phosphorocyanidate (DECP) exist]-2-methylpyrimidine-4}-piperazine-1-methyl acid phosphate diethyl ester.
The described 4-{6-[5-of formula I (2-chloro-6-methylaniline formyl)-thiazole-2-amino]-2-methylpyrimidine-4}-piperazine-1-methyl acid phosphate diethyl ester can solvate or the form of non-solvent compound exist, utilize different solvents to carry out crystallization and may obtain different solvates.The described pharmacy acceptable salt of formula I comprises different acid salt, as following mineral acid or organic acid acid salt: hydrochloric acid, Hydrogen bromide, phosphoric acid, sulfuric acid, methylsulfonic acid, tosic acid, trifluoroacetic acid, matrimony vine acid, toxilic acid, tartrate, fumaric acid, citric acid, lactic acid.The described pharmacy acceptable salt of I also comprises Different Alkali metal-salt (lithium, sodium, sylvite), alkaline earth salt (calcium, magnesium salts) and ammonium salt and the salt of physiologically acceptable cationic organic bases can be provided, as methylamine, dimethylamine, Trimethylamine 99, piperidines, the salt of morpholine and three (2-hydroxyethyl) amine.All these salt within the scope of the present invention all can adopt the ordinary method preparation.At described 4-{6-[5-(2-chloro-6-methylaniline formyl)-thiazole-2-amino]-preparation process of 2-methylpyrimidine-4}-piperazine-1-diethyl phosphoric acid and solvate thereof and its salt in, polycrystalline or eutectic may appear in different crystallization conditions.
The invention still further relates to the pharmaceutical composition of The compounds of this invention as active ingredient.This pharmaceutical composition can be according to method preparation well known in the art.Can be by the pharmaceutically acceptable solid of The compounds of this invention and one or more or liquid excipient and/or assistant agent being combined, make any formulation that is suitable for human or animal's use.The content of The compounds of this invention in its pharmaceutical composition is generally 0.1-95 weight %.
The compounds of this invention or contain its pharmaceutical composition can the unit dosage form administration, route of administration can be enteron aisle or non-enteron aisle, as oral, intravenous injection, intramuscular injection, subcutaneous injection, nasal cavity, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum etc.
Form of administration can be liquid dosage form, solid dosage or semisolid dosage form.Liquid dosage form can be solution (comprising true solution and colloidal solution), emulsion (comprising o/w type, w/o type and emulsion), suspensoid, injection (comprising aqueous injection, powder injection and transfusion), eye drops, nasal drop, lotion and liniment etc.; Solid dosage can be tablet (comprising ordinary tablet, enteric coated tablet, lozenge, dispersible tablet, chewable tablet, effervescent tablet, orally disintegrating tablet), capsule (comprising hard capsule, soft capsule, enteric coated capsule), granule, powder, micropill, dripping pill, suppository, film, paster, the agent of gas (powder) mist, sprays etc.; Semisolid dosage form can be ointment, gelifying agent, paste etc.
The compounds of this invention can be made ordinary preparation, also make is sustained release preparation, controlled release preparation, targeting preparation and various particulate delivery system.
For The compounds of this invention is made tablet, can be extensive use of various vehicle well known in the art, comprise thinner, tamanori, wetting agent, disintegrating agent, lubricant, glidant.Thinner can be starch, dextrin, sucrose, glucose, lactose, N.F,USP MANNITOL, sorbyl alcohol, Xylitol, Microcrystalline Cellulose, calcium sulfate, secondary calcium phosphate, lime carbonate etc.; Wetting agent can be water, ethanol, Virahol etc.; Tackiness agent can be starch slurry, dextrin, syrup, honey, glucose solution, Microcrystalline Cellulose, mucialga of arabic gummy, gelatine size, Xylo-Mucine, methylcellulose gum, Vltra tears, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyoxyethylene glycol etc.; Disintegrating agent can be dry starch, Microcrystalline Cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, croscarmellose sodium, sodium starch glycolate, sodium bicarbonate and Citric Acid, polyoxyethylene sorbitol fatty acid ester, sodium laurylsulfonate etc.; Lubricant and glidant can be talcum powder, silicon-dioxide, stearate, tartrate, whiteruss, polyoxyethylene glycol etc.
Tablet further can also be made coating tablet, for example sugar coated tablet, thin membrane coated tablet, ECT, or double-layer tablets and multilayer tablet.
For capsule is made in the administration unit, the effective constituent The compounds of this invention can be mixed with thinner, glidant, mixture is directly placed hard capsule or soft capsule.Also the effective constituent The compounds of this invention particle or micropill be can be made with thinner, tamanori, disintegrating agent earlier, hard capsule or soft capsule placed again.Each thinner, tamanori, wetting agent, disintegrating agent, the glidant kind that are used to prepare the The compounds of this invention tablet also can be used for preparing the capsule of The compounds of this invention.
For The compounds of this invention is made injection, can water, ethanol, Virahol, propylene glycol or their mixture as solvent and add an amount of this area solubilizing agent commonly used, solubility promoter, pH and adjust agent, osmotic pressure regulator.Solubilizing agent or solubility promoter can be poloxamer, Yelkin TTS, hydroxypropyl-beta-cyclodextrin etc.; PH adjustment agent can be phosphoric acid salt, acetate, hydrochloric acid, sodium hydroxide etc.; Osmotic pressure regulator can be sodium-chlor, N.F,USP MANNITOL, glucose, phosphoric acid salt, acetate etc.As prepare lyophilized injectable powder, also can add N.F,USP MANNITOL, glucose etc. as propping agent.
In addition, as needs, also can in pharmaceutical preparation, add tinting material, sanitas, spices, correctives or other additive.
For reaching the medication purpose, strengthen result of treatment, medicine of the present invention or pharmaceutical composition can be with any known medication administrations.
The dosage of The compounds of this invention pharmaceutical composition is according to character and the severity that will prevent or treat disease, the individual instances of patient or animal, and route of administration and formulation etc. can have large-scale variation.In general, the suitable dose scope of the every day of The compounds of this invention is the 0.001-150mg/Kg body weight, is preferably the 0.01-100mg/Kg body weight.Above-mentioned dosage can a dose unit or is divided into several dose unit administrations, and this depends on doctor's clinical experience and comprises the dosage regimen of using other treatment means.
Compound of the present invention or composition can be taken separately, or merge use with other treatment medicine or symptomatic drugs.When compound of the present invention and other medicine existence synergy, should adjust its dosage according to practical situation.
The compounds of this invention is tyrosine kinase inhibitor or its precursor, not only suppresses non-receptor protein tyrosine kinase, as the Src family kinase, and is used for kinds of tumors and amynologic disease; And the inhibition receptor protein tyrosine kinase, as HER1 and HER2, therefore can be used for treating proliferative disease, as psoriasis and cancer.These compounds suppress the ability of HER1 and other receptor kinase, also make it can be used as anti-angiogenic formation agent and are used for the treatment of such as illnesss such as cancer and diabetic retinopathies.In addition, the activation of T cell has been blocked in the inhibition of Lck in the Src family, and this compounds can be used for the cell-mediated disease of T.Because this compounds can block the activation of epidermic cell PTK, thereby limit the surface expression that brings out neutrophilia bonded adhesion molecule, and suppressed neutrophilia and activate necessary PTK, thereby be used for the treatment of ischemic disorders and reperfusion injury.With vascular smooth muscle cells migration and increment diseases associated therapeutic action is arranged in addition, especially anti-tumor activity is obvious.The compounds of this invention has high bioavailability, can be used for the treatment of multiple human malignancies, comprises leukemia, neurospongioma, incidence cancer, nonsmall-cell lung cancer, carcinoma of the pancreas, colorectal cancer, bladder cancer and mammary cancer, ovarian cancer, squamous cell carcinoma etc.
Description of drawings
Fig. 1. the effect of 5 pairs of K562 apoptosis-inducings of embodiment
Fig. 2. the effect of 5 pairs of KR/1.6 apoptosis-inducings of embodiment
Fig. 3. the effect of 5 pairs of KR/3.0 apoptosis-inducings of embodiment
5 couples of Bcr-Abl of Fig. 4 embodiment, the restraining effect of c-src and Lyn protein expression level
Embodiment
Below with reference to embodiment invention is described further, but does not limit the scope of the invention.
Determining instrument: NMR (Nuclear Magnetic Resonance) spectrum Vaariaan Mercury 300 type nuclear magnetic resonance analyser.Mass spectrum ZAD-2F and VG300 mass spectrograph.
Embodiment 1.4-(diethoxy phosphoryl methyl)-piperazine-1-benzyl carboxylate
Place 250 ml flasks reflux to reacting completely for 120 milliliters 1 milliliter in the formaldehyde and the benzene of 2.2 gram (10 mmole) piperazine-1-benzyl carboxylates and 1.38 gram (10 mmole) diethyl phosphites and 37%; cooling; revolve and desolvate; add acetic acid ethyl dissolution, sodium hydrogen carbonate solution washing, washing; salt is washed; anhydrous sodium sulfate drying revolves and desolvates, and the short column separation obtains product 4-(diethoxy phosphoryl methyl)-piperazine-1-benzyl carboxylate.
1HNMR (300MHz, D-acetone), δ (ppm): 7.36 (m, 5H, ArH), 5.10 (s, 2H, ArH), 4.08 (m, 4H, 2OCH
2), 3.45 (t, 4H, 2NCH
2), 2.60 (t, 4H, 2NCH
2), 2.78 (s, 1H, CH
2), 2.74 (s, 1H, CH
2), 1.26 (t, 6H, 2CH
3) .FABMS:(M+1)
+=371.
Embodiment 2. piperazines-1-methyl acid phosphate diethyl ester
370 milligrams of (1 mmole) 4-(diethoxy phosphoryl methyl)-piperazine-1-benzyl carboxylate is dissolved in 25 milliliters of tetrahydrofuran (THF)/ethanol (1: 1) mixed solvent; add palladium carbon (10%; 180 milligrams); 40-50 ℃ of normal pressure hydrogenation spends the night; the complete after-filtration of hydrogenation, revolving desolvates obtains product piperazine-1-methyl acid phosphate diethyl ester.
1HNMR (300MHz, D-acetone), δ (ppm): 4.09 (m, 4H, 2OCH
2), 2.85 (t, 4H, 2NCH
2), 2.64 (t, 4H, 2NCH
2), 2.74 (s, 1H, CH
2), 2.70 (s, 1H, CH
2), 1.27 (t, 6H, 2CH
3) .FABMS:(M+1)
+=237.
Embodiment 3.4-(6-chloro-2-methylpyrimidine-4-yl)-piperazine-1-methyl acid phosphate diethyl ester
With 236 milligrams of (1 mmole) piperazine-1-methyl acid phosphate diethyl esters and 243 milligrams of (1.5 mmole) 2-methyl-4, the 6-dichloro pyrimidine is dissolved in 20 milliliters anhydrous 1, in the 4-dioxane, add 130 milligrams of (1 mmole) diisopropyl ethamine, reflux is stirred to raw material and disappears, revolve and desolvate, add acetic acid ethyl dissolution, the sodium hydrogen carbonate solution washing, washing, anhydrous sodium sulfate drying revolves the short column separation of desolvating and obtains product liquid 4-(6-chloro-2-methylpyrimidine-4-yl)-piperazine-1-methyl acid phosphate diethyl ester.
1H NMR (300MHz, CDCl
3), δ (ppm) 6.30 (s, 1H, ArH), 4.12 (m, 4H, 2OCH
2), 3.64 (t, 4H, 2NCH
2), 2.83 (s, 1H, CH
2), 2.79 (s, 1H, CH
2), 2.70 (t, 4H, 2NCH
2), 2.45 (s, 3H, CH
3), 1.31 (t, 6H, 2CH
3). FABMS:(M+1)
+=363.
Embodiment 4.2-{6-[4-(diethoxy phosphoryl methyl) piperazine-1]-2-methylpyrimidine-4-amino }-thiazole-5-carboxylic acid ethyl ester (compound 4)
362 milligrams of (1 mmole) 4-(6-chloro-2-methylpyrimidine-4-yl)-piperazine-1-methyl acid phosphate diethyl ester and 172 milligrams of (1 mmole) 2-amino-5-ethoxycarbonyl thiazoles are dissolved in 20 milliliters of dry DMF; stir and add 100 milligrams of (2.5 mmole) sodium hydrides (60%) down; stirring at room to raw material disappears; in the ice-cold sodium hydrogen carbonate solution of cooling back impouring; ethyl acetate extraction; washing; salt is washed; anhydrous sodium sulfate drying, the short column that desolvates separation obtains product 2-{6-[4-(diethoxy phosphoryl methyl) piperazine-1]-2-methylpyrimidine-4-amino }-thiazole-5-carboxylic acid ethyl ester.
1H NMR (300MHz, DMSO-D6), δ (ppm) 11.62 (S, 1H, NH), 8.02 (S, 1H, ArH), 6.04 (s, 1H, ArH), 4.25 (q, 2H, OCH
2), 4.05 (m, 4H, 2OCH
2), 3.68 (t, 4H, 2NCH
2), 2.84 (S, 1H, CH
2), 2.80 (S, 1H, CH
2), 2.62 (m, 4H, 2NCH
2), 2.40 (s, 3H, CH
3), 1.25 (m, 9H, 3CH
3).
FABMS：(M+1)
+＝499.
Embodiment 5.4-{6-[5-(2-chloro-6-methylaniline formyl)-thiazole-2-amino]-2-methylpyrimidine-4}-piperazine-1-methyl acid phosphate diethyl ester (compound 5)
With 498 milligrams of (1 mmole) 2-{6-[4-(diethoxy phosphoryl methyl) piperazine-1]-2-methylpyrimidine-4-amino }-thiazole-5-carboxylic acid ethyl ester is dissolved in 10 ml methanol; add 10 milliliters in 2.5N sodium hydroxide; stirring at room to raw material disappears; methyl alcohol is revolved in decompression; ethyl acetate extraction; the water layer ice-water bath drips concentrated hydrochloric acid and is neutralized to a large amount of precipitations of generation; leave standstill suction filtration after half an hour; the washing solid, drying obtains product 2-{6-[4-(diethoxy phosphoryl methyl) piperazine-1]-2-methylpyrimidine-4-amino }-thiazole-5-carboxylic acid.
With 470 milligrams of (1 Bo mole) 2-{6-[4-(diethoxy phosphoryl methyl) piperazine-1]-2-methylpyrimidine-4-amino }-thiazole-5-carboxylic acid and 126 milligrams of (1 mmole) 2-chloro-6-methyl phenylaminos and 152 milligrams of (1.5 mmole) triethylamines are dissolved in 15 milliliters of dry DMF; stir and drip 326 milligrams of (2 mmole) diethyl phosphorocyanidates (DECP) down; be heated to 60-70 ℃ gradually; the stirring raw material disappears; revolve desolvate after; add dehydrated alcohol; separate out white solid, filter and obtain product 4-{6-[5-(2-chloro-6-methylaniline formyl)-thiazole-2-amino]-2-methylpyrimidine-4}-piperazine-1-methyl acid phosphate diethyl ester.
1H NMR (300MHz, DMSO-D6), δ (ppm) 11.46 (S, 1H, NH), 9.86 (S, 1H, NH), 8.20 (S, 1H, ArH), 7.38 (m, 1H, ArH), 7.26 (m, 2H, ArH), 6.04 (S, 1H, ArH), 4.03 (m, 4H, 2OCH
2), 3.50 (m, 4H, 2NCH
2), 2.85 (S, 1H, PCH
2), 2.81 (S, 1H, PCH
2), 2.62 (m, 6H, 3NCH
2), 2.39 (s, 3H, CH
3), 2.22 (s, 3H, CH
3), 1.23 (t, 6H, 2CH
3).
31P-NMR (200MHz, DMSO-d
6+ D
2O+KH
2PO
4) δ (ppm) 21.78 (S) .FABMS:(M+1)
+=594.
Pharmacologically active
External activity is estimated:
1. to the restraining effect of growth of tumour cell
1 cell cultures
Test used K562 cell and the K562 cell of anti-Imatinib in containing 5%CO
237 ℃ of incubators in cultivate and go down to posterity, cultivate with the RPMI RPMI-1640 that contains 10% foetal calf serum and 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates, go down to posterity weekly twice.
2 mtt assay are measured the tumour cell survival rate
Being mixed with concentration behind the cell centrifugation with logarithmic phase is 1 * 10
5The cell suspension of cell/ml, be inoculated in 96 orifice plates by 10000/hole, every hole adds 100 μ l, add the fresh culture that contains different concns medicine and coordinative solvent contrast, every hole adds 100 μ l (DMSO final concentration＜0.1%), establish three parallel holes for every group, in 37 ℃ cultivate 3 days after, every hole adds the serum free medium of the freshly prepared 5mg/ml of the containing MTT of 20 μ l, continues to cultivate 4 hours, and is centrifugal, abandon supernatant, every hole adds 200 μ l DMSO dissolving MTT first hairpin precipitation, with the microoscillator mixing that vibrates, with MK3 type microplate reader at reference wavelength 450nm, detect under the wavelength 570nm condition and measure optical density value (OD), the tumour cell of handling with solvent control is a control group, calculates the inhibiting rate of medicine to tumour cell with following formula, and calculates IC50 by middle efficacious prescriptions journey:
Result: embodiment 5 compounds are to the growth-inhibiting effect of kinds of tumor cells system
3 flow cytometry analysis apoptosis and cell cycles
Collecting cell removes supernatant after centrifugal, and PBS washs once.Cell is flicked, and every pipe adds the about 2ml of 70% ethanol to be fixed, and 4 ℃ are spent the night.Next day 1, centrifugal 5 minutes of 000rpm removes ethanol, PBS washing 2 times, 1, centrifugal 5 minutes of 000rpm, repeated centrifugation washed twice.Abandon supernatant, keep about 0.1ml liquid.Add RNAase 50 μ l (final concentration is 60 μ g/ml), vortex, 37 ℃ of incubations 30 minutes.Add PI (propidium iodide) (final concentration is 50 μ g/ml) and to 0.5ml, shake up, 4 ℃ of lucifuges act on 60 minutes.Through nylon net filter, on flow cytometer, measure each phase dna content of cell and carry out cell cycle analysis.
1) to the influence of K562 cell cycle
Sample effect after 24 hours, adopts the influence of flow cytometer test sample cell cycle distribution in the K562 cell.
The influence of 5 pairs of K562 cell cycle distribution of table 2. embodiment.
2) to the effect of K562 apoptosis-inducing
After 48 hours, flow cytometer detects and shows that the apoptotic cell ratio obviously increases sample effect in the K562 cell.2.5nmol/L, 5nmol/L, 10nmol/L embodiment 5 induce the ratio that produces apoptosis to be respectively 92.1%, 95.6%, 97.0%; And dasatinib10nmol/L institute inductive apoptosis rate is 95.2%.(seeing figure .1)
3) to the influence of KR/1.6 cell cycle
Sample effect after 24 hours, adopts the influence of flow cytometer test sample cell cycle distribution in the KR/1.6 cell.The result shows: 5 pairs of KR/1.6 cells of embodiment have tangible G
1The effect of phase retardance.
The influence of 5 pairs of KR/1.6 cell cycle distribution of table 3. embodiment.
4) to the effect of KR/1.6 apoptosis-inducing
The flow cytometer detected result shows that after 48 hours, the apoptotic cell ratio obviously increases sample effect in the KR/1.6 cell.2.5nmol/L, 5nmol/L, 10nmol/L embodiment 5 induce the ratio that produces apoptosis to be respectively 23.8%, 84.2%, 87.2%; And dasatinib10nmol/L institute inductive apoptosis rate is 84.8%.(seeing figure .2)
5) to the influence of KR/3.0 cell cycle
Sample effect after 24 hours, adopts the influence of flow cytometer test sample cell cycle distribution in the KR/1.6 cell.The result shows: 5 pairs of KR/3.0 cells of embodiment have tangible G
1The effect (seeing Table 4) of phase retardance.
The influence of 5 pairs of KR/3.0 cell cycle distribution of table 4. embodiment.
6) to the effect of KR/3.0 apoptosis-inducing
The flow cytometer detected result shows that after 48 hours, the apoptotic cell ratio obviously increases sample effect in the KR/3.0 cell.2.5nmol/L, 5nmol/L, 10nmol/L embodiment 5 induce the ratio that produces apoptosis to be respectively 16.0%, 36.6%, 58.8%; And dasatinib10nmol/L institute inductive apoptosis rate is 58.8%.(see figure 3) 7) 5 couples of Bcr-Abl of embodiment, the restraining effect (see figure 4) of c-src and Lyn protein expression level
4. the foundation of leukemia mouse model
The SCID mouse, female, age in 6-8 week, body weight 20-25g, Chinese Academy of Medical Sciences's zooscopy provides.Continuous 2 days of abdominal cavity injection endoxan (CTX) 2mg/ only, inject once every day, the vegetative period K562 cell of taking the logarithm in the 3rd day is used for transplanting, with the disposable injection 1 * 10 of tail vein
7Individual/mouse, the SCID mouse number of each tail vein injection is identical.
5. animal grouping
The injection cell is after 20 days, and animal skin is wrinkling, and is dispirited few moving, loses weight.At this moment begin oral administration.Be divided into five groups, 4 every group, control group, the oral equivalent physiological saline of leukemia model mice.The Dasatinib group is pressed the 30mg/kg body weight, and embodiment 5 presses 18mg/kg, 36mg/kg body weight oral administration, successive administration 20 days, the lifetime of then observing mouse.
The influence of 5 couples of human chronic myelogenous leukemia K562 of embodiment cell NOD/SCID heteroplastic transplantation model mice spleen index
*P＜0.05,
*P＜0.01,
* *P＜0.001 is compared with the normal control group; #P＜0.05, ##＜0.01, ###＜0.001 is compared with model group
The influence of 5 couples of human chronic myelogenous leukemia K562 of embodiment cell NOD/SCID heteroplastic transplantation model mice peripheral white blood cell
*P＜0.05,
*P＜0.01,
* *P＜0.001 is compared with the normal control group; #P＜0.05, ##＜0.01, ###＜0.001 is compared with model group.
Claims (10)
1, the 4-{6-[5-shown in the formula I (2-chloro-6-methylaniline formyl)-thiazole-2-amino]-2-methylpyrimidine-4}-piperazine-1-methyl acid phosphate diethyl ester compounds; its pharmacologically acceptable salt; its hydrate and solvate, its polycrystalline and eutectic, the precursor of its same biological function or derivative
According to the compound of claim 1, it is characterized in that 2, described pharmacologically acceptable salt comprises: hydrochloride, hydrobromate, phosphoric acid salt, vitriol, mesylate, tosilate, trifluoroacetate, matrimony vine hydrochlorate, maleate, tartrate, fumarate, Citrate trianion, lactic acid salt, lithium salts, sodium salt, sylvite, calcium salt, magnesium salts, ammonium salt, methylamine salt, dimethylamine salt, front three amine salt, piperidinium salt, alkylbenzyldimethylasaltsum saltsum and three (2-hydroxyethyl) amine salt.
3, the method for the described compound of preparation claim 1 comprises the steps:
(a) pyrimidine derivatives links to each other with phosphate derivative earlier,
(b) and then with thiazole derivative link to each other,
(c) link to each other with 2-chloro-6-methyl phenylamino again, finally obtain the described compound of formula I;
4, according to the preparation method of claim 3, it is characterized in that, piperazine in the step (a)-1-methyl acid phosphate diethyl ester and 2-methyl-4, the 6-dichloro pyrimidine links to each other; It is anhydrous 1 to be reflected at diisopropyl ethamine and solvent, carries out under the condition that the 4-dioxane exists; Obtain product 4-(6-chloro-2-methylpyrimidine-4) piperazine-1-methyl acid phosphate diethyl ester.
5, according to the preparation method of claim 3; it is characterized in that; 4-(6-chloro-2-methylpyrimidine-4) piperazine-1-methyl acid phosphate diethyl ester and 2-amino-5-ethoxycarbonyl thiazole carry out under the condition of solvent dry DMF and sodium hydride existence in the step (b), obtain product 2-{6-[4-(diethoxy phosphoryl methyl) piperazine-1]-2-methylpyrimidine-4-amino }-thiazole-5-carboxylic acid ethyl ester.
6, according to the preparation method of claim 3, it is characterized in that, 2-{6-[4-(diethoxy phosphoryl methyl) piperazine-1 in the step (c)]-2-methylpyrimidine-4-amino }-thiazole-5-carboxylic acid ethyl ester is under the condition that solvent methanol exists, slough ester group with sodium hydroxide, obtain 2-{6-[4-(diethoxy phosphoryl methyl) piperazine-1]-2-methylpyrimidine-4-amino }-thiazole-5-carboxylic acid;
The 2-{6-[4-that obtains (diethoxy phosphoryl methyl) piperazine-1]-2-methylpyrimidine-4-amino }-thiazole-5-carboxylic acid and 2-chloro-6-methyl phenylamino, reaction obtains product 4-{6-[5-(2-chloro-6-methylaniline formyl)-thiazole-2-amino under the condition that triethylamine, solvent dry DMF, diethyl phosphorocyanidate (DECP) exist]-2-methylpyrimidine-4}-piperazine-1-methyl acid phosphate diethyl ester.
7, a kind of composition of medicine is characterized in that, contains the compound and the technology of pharmaceutics acceptable carrier of claim 1.
8, the application of the compound of claim 1 in the medicine of preparation prevention and treatment and protein tyrosine kinase diseases associated.
According to the application of claim, it is characterized in that 9, described and protein tyrosine kinase diseases associated is immune disorder, tumor disease.
According to the application of claim 9, it is characterized in that 10, described tumor disease is a leukemia, neurospongioma, incidence cancer, nonsmall-cell lung cancer, carcinoma of the pancreas, colorectal cancer, bladder cancer and mammary cancer, ovarian cancer, squamous cell carcinoma.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2008100081978A CN101323629B (en) | 2007-02-16 | 2008-02-18 | 4-{6-[5-(2-chlorinde-6- methylaniline formyl)-thiazole-2-amido]-2-methyl pyrimidine-4}-piperazine-1- diethyl methylphosphate |
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| CN2008100081978A CN101323629B (en) | 2007-02-16 | 2008-02-18 | 4-{6-[5-(2-chlorinde-6- methylaniline formyl)-thiazole-2-amido]-2-methyl pyrimidine-4}-piperazine-1- diethyl methylphosphate |
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| CN2008100081978A Active CN101323629B (en) | 2007-02-16 | 2008-02-18 | 4-{6-[5-(2-chlorinde-6- methylaniline formyl)-thiazole-2-amido]-2-methyl pyrimidine-4}-piperazine-1- diethyl methylphosphate |
| CN2008100081963A Expired - Fee Related CN101265274B (en) | 2007-02-16 | 2008-02-18 | Pyrimidinthiazolamine derivatives, and preparation method, medicament composition and use thereof |
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| CN101265274B (en) * | 2007-02-16 | 2013-09-04 | 中国医学科学院药物研究所 | Pyrimidinthiazolamine derivatives, and preparation method, medicament composition and use thereof |
| CN112057451A (en) * | 2014-12-09 | 2020-12-11 | 正大天晴药业集团股份有限公司 | Quinoline derivatives for treating non-small cell lung cancer |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101812060B (en) * | 2010-02-02 | 2011-08-17 | 南京卡文迪许生物工程技术有限公司 | Simple novel method for preparing high-purity Sprycel, and intermediate compound |
| CN102786517B (en) * | 2011-05-18 | 2017-06-06 | 中国医学科学院药物研究所 | The pyrimidine thiazole amines derivative of GK and PPAR double excitation activity |
| CN103130740A (en) * | 2011-11-29 | 2013-06-05 | 长江大学 | Amino acid derivatives of seedvax and application for being used as bactericides thereof |
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| CO4940418A1 (en) * | 1997-07-18 | 2000-07-24 | Novartis Ag | MODIFICATION OF A CRYSTAL OF A DERIVATIVE OF N-PHENYL-2-PIRIMIDINAMINE, PROCESSES FOR ITS MANUFACTURE AND USE |
| EP3222619A1 (en) * | 1999-04-15 | 2017-09-27 | Bristol-Myers Squibb Holdings Ireland | Cyclic protein tyrosine kinase inhibitors |
| GB0126902D0 (en) * | 2001-11-08 | 2002-01-02 | Novartis Ag | Organic compounds |
| US20050009891A1 (en) * | 2003-07-09 | 2005-01-13 | Lee Francis Y. | Combination of SRC Kinase inhibitors and chemotherapeutic agents for the treatment of proliferative diseases |
| CN101265275B (en) * | 2007-02-16 | 2012-09-05 | 中国医学科学院药物研究所 | Diethyl 4-{6-[5-(2-chloro-6-methylanilinoformacyl)-thiazolyl-2-amino]-2-methylpyrimidin-4}-piperazin-1-phosphate |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101265274B (en) * | 2007-02-16 | 2013-09-04 | 中国医学科学院药物研究所 | Pyrimidinthiazolamine derivatives, and preparation method, medicament composition and use thereof |
| CN112057451A (en) * | 2014-12-09 | 2020-12-11 | 正大天晴药业集团股份有限公司 | Quinoline derivatives for treating non-small cell lung cancer |
| CN112057452A (en) * | 2014-12-09 | 2020-12-11 | 正大天晴药业集团股份有限公司 | Quinoline derivatives for treating non-small cell lung cancer |
Also Published As
| Publication number | Publication date |
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| CN101323629B (en) | 2011-08-17 |
| CN101265275A (en) | 2008-09-17 |
| CN101265274A (en) | 2008-09-17 |
| CN101265274B (en) | 2013-09-04 |
| CN101265275B (en) | 2012-09-05 |
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