CN103202843B - Application of pyrimidine derivative in preparation of drugs capable of prevention and/or treatment and/or adjuvant therapy of cancers - Google Patents
Application of pyrimidine derivative in preparation of drugs capable of prevention and/or treatment and/or adjuvant therapy of cancers Download PDFInfo
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- 0 *c(cc1)ccc1N Chemical compound *c(cc1)ccc1N 0.000 description 1
- BGVNTIGSCSPASF-UHFFFAOYSA-N Cc1cc(Nc2cc(N(CC3)CCN3O)nc(Sc(cc3)ccc3NC(C3CC3)=O)n2)n[nH]1 Chemical compound Cc1cc(Nc2cc(N(CC3)CCN3O)nc(Sc(cc3)ccc3NC(C3CC3)=O)n2)n[nH]1 BGVNTIGSCSPASF-UHFFFAOYSA-N 0.000 description 1
Abstract
The invention discloses application of a pyrimidine derivative in preparation of drugs capable of prevention and/or treatment and/or adjuvant therapy of cancers. The pyrimidine derivative comprises a pyrimidine compound having a structural formula (I), a pharmaceutically acceptable salt thereof, a solvate, a polymorphic substance, a tautomer and a prodrug or a composition containing the pyrimidine compound. The structural formula (I) is described in the specification. According to the invention, the pyrimidine derivative with novel Aurora kinase inhibition activity is formed through different C-N couplings and introduction of different pharmacophores respectively at positions 2, 4 and 6 of a pyrimidine ring, and the pyrimidine derivative can be used for preparation of drugs capable of prevention and/or treatment and/or adjuvant therapy of cancers.
Description
Technical field
The present invention relates to field of medicaments, be specifically related to a kind of pyrimidine derivatives preparation prevent and/or treat and/or auxiliary for treating cancer medicine in purposes.
Background technology
Aurora A is the serine/threonine kinase that a class has important regulating and controlling effect in cell growth cycle, participates in regulating spindle formation, centrosome maturation, chromosome differentiation and cytokinesis process, has pivotal role to the genomic stability of maintenance.Research finds, the overexpression of Aurora A easily causes cell mitogen abnormal, closely related with the formation of tumor.Aurora A overexpression in many cancerous cell (as pulmonary carcinoma, breast carcinoma, rectal cancer, thyroid carcinoma, cancer of pancreas), suppresses the activity of Aurora A can cause the gathering of tumor cell polyploid, promotes apoptosis, blocks cell proliferation.Be that the research and development that target spot carries out antitumor drug are also more and more subject to people's attention with Aurora A.Aurora A inhibitor belongs to anti-tumor drugs targeting, compared with other non-specific cell cytotoxic drug, will have larger advantage.
According to the difference of aminoacid sequence, mankind's Aurora A is divided into AuroraA, AuroraB and AuroraC tri-kinds of hypotypes.These three kinds of hypotypes have very conservative C-and hold catalytic domain and N-to hold variable region, namely have the ATP-binding site (C-holds catalytic domain) of very high homology, but have larger difference on the amino acid length and sequence of N-end.
Wherein Aurora A is positioned at the centrosome and mitotic spindle two ends that copy, regulates the maturation of centrosome, the assembling of spindle and mitotic start-up course.Aurora A can the regulatory factor Cdc25b of phosphorylation cell periodic protein B 1-Cdk1, regulates the start-up course of cell mitogen after activation Cdc25b.AuroraA is also by participating in adjustment three kinds of centrosome protein, i.e. centrosome protein (centroso minute, CNN) acid coiled-coil protein (trans-for minute gacidiccoiled-coil protein, is transformed, TACC) and spindle deficiency albumen (spindle defective-2, SPD-2) ensure the maturation of centrosome in a fission process.
After scientist in 1998 confirms that the generation of tumor and the overexpression of Aurora A have substantial connection, academia and pharmaceuticals industry circle are caused using the research boom of Aurora A as antitumor drug action target spot.The application of the deep and computer modeling technique studied along with Aurora A crystal structure, it is found that the Aurora A inhibitor of great majority synthesis belongs to ATP (adenosine triphosphate) competitive inhibitors of kinases, the kinase whose ATP-binding site of competition binding, cut off the direct energy source of kinases, thus suppress it active.
Research finds, the purine ring in ATP structure can be incorporated into the hydrophobic pocket of Aurora A structure, and forms hydrogen bond with the amino acid residue of bonding pad.ATP binding pocket is made up of following region: kinases hinge region, hydrophobic pocket land, phosphate ditch buried district, solvent can and district and ribose moieties.
Aurora A family ATP-binding site has very high homology, and this makes the selectivity of inhibitors of kinases become a very large challenge.The Aurora A inhibitor of many reports still can embody good selectivity, one of them major reason is inhibitor except by except ATP-binding site and kinase interactions, also act on the region near ATP binding pocket, comprise the hydrophobic pocket at kinases rear portion, the difference of all these zone of action amino acid residues determines inhibitors of kinases optionally difference jointly.The basic structure feature of the inhibitors of kinases reported is: the heterocyclic system with a plane is with competition binding kinase whose ATP pocket and simulate adenine and kinase whose interaction; Also " Donor-Acceptor-Donor " binding mode is formed by hydrogen bond action between inhibitor and kinases hinge region; The functional group of inhibitor often can enter kinase phosphorylation salt binding region or selective binding pocket.From structure, the Aurora A inhibitor of synthesis is at present mainly pyrimidine lopps, pyrrolo-pyrazole class, indoles, quinazoline ditosylate salt, benzo nitrogen
miazines and other Structure type compound.
Pyrimidines is the Aurora A inhibitor reported the earliest, U.S.'s Vertex Standard (Vertex) company (US7361492B2, WO03092607A2) reported first in 2003 AuroraA kinases and pyrimidine compound N-(3-cyclopropyl-1H-pyrazoles-5-base)-2-phenylquinazoline-4-amine compound crystal structure.Point out in patent, two atom N on this complex pyrazole ring and kinase whose hinge region form hydrogen bond action, in kinase whose glycine rich region, have π-π to interact between kinases and the phenyl ring of this complex.The achievement in research of Vertex company is that follow-up appropriate design Aurora A inhibitor is laid a good foundation.In patent WO03092607A2, structural formula of compound is as follows:
Vertex company and Merck (Merck) company have developed jointly first Aurora A inhibitor VX-680 (having another name called MK-0457) (WO2004000833A1).This molecular structure is Y shape, and the pyrimidine ring at center is positioned at kinase whose hydrophobic region, and has hydrophobic interaction between kinase amino acid residue; Arm containing amino-pyrazol stretches into kinase whose hinge area, and nitrogen-atoms and the hinge region amino acid residue of pyrazole ring have hydrogen bond action; Arm containing phenyl stretches in kinase active site pocket, and the cyclopropyl of phenyl end and the kinase whose F275 residue of Aurora A have hydrophobic interaction; Piperazine sidechain then by the active region of enzyme be stretched over enzyme solvent can and district.Research finds, VX-680 has good inhibitory action to AuroraA, B and C, IC
50value is respectively 0.6,18 and 4.6nM, can suppress the propagation of numerous tumor cells such as colorectal carcinoma, breast carcinoma, carcinoma of prostate, cancer of pancreas, melanoma, tumor colli, leukemia.2006, VX-680 entered the clinical II phase and studies, and was mainly used in treating obstinate chronic lymphocytic leukemia and acute lymphatic leukemia.Research finds the QTc of patient can be caused to extend risk by this medicine, and (QTc is the time difference between T ripple and Q ripple that electrocardiogram corrects, QTc prolongation easily causes arrhythmia), in November, 2007, Merck terminated II clinical trial phase (the Expert Opin.Investing Drugs.2009 of VX-680,18,379).
Although there is the compound inhibited to Aurora A that some are similar to VX-680 in prior art, but still need that further research has the inhibiting compound of Aurora A more, be suitable for prevent and/or treat and/or the needs of medicine of auxiliary for treating cancer so that produce more.
Summary of the invention
The object of the invention be to provide a kind of pyrimidine derivatives preparation prevent and/or treat and/or auxiliary for treating cancer medicine in purposes, a kind of pyrimidine derivatives with new construction is made into for preventing and/or treating and/or the medicine of auxiliary for treating cancer.
For this reason, provide in the present invention a kind of pyrimidine derivatives preparation prevent and/or treat and/or adjuvant therapy of tumors medicine in purposes, pyrimidine derivatives comprise there is structural formula (I) pyrimidine compound, its pharmaceutically acceptable salt, solvate, polymorph, tautomer, prodrug, or the compositions containing pyrimidine compound, structural formula (I) is as follows:
Preferably, in such use, synthesize the above-mentioned method with the pyrimidine compound of structural formula (I) to comprise the following steps: the compd A and 1-methyl piperazine with structural formula (1) are dissolved into 1 according to mol ratio 1: 8 ~ 12, in 4-dioxane, 100 ~ 140 DEG C, microwave reaction 30 ~ 60 minutes, reaction gained mixture is spin-dried for solvent, must have the pyrimidine compound of structural formula (I), wherein, in formula (1), X is halogen.
Preferably, in such use, synthesize above-mentioned have be spin-dried for solvent process in the method for the pyrimidine compound of structural formula (I) after comprise step with reverse phase silica gel column purification further, with the step of reverse phase silica gel column purification be with volume ratio be 80: 20 ~ 40: 60 water and methanol mixed solvent carry out eluting, collect the pyrimidine compound with structural formula (I) of purification.
Preferably, in such use, the method of synthesis above-claimed cpd A is as follows: compd B and the methyl p-aminobenzoate will with structural formula (2), p-methyl benzenesulfonic acid is dissolved in n-butyl alcohol according to mol ratio 1: 1 ~ 1.2: 0.8 ~ 1, back flow reaction 10 ~ 12h at 130 ~ 150 DEG C, be cooled to room temperature after reaction, add saturated NaHCO
3solution regulates pH to 7 ~ 8, makes to be extracted with ethyl acetate 3 ~ 5 times, organic layer saturated common salt water washing 3 ~ 5 times, then uses anhydrous Na
2sO
4drying, is spin-dried for solvent and namely obtains compd A, and wherein, in formula (2), X is halogen.
Preferably, in such use, synthesize the step comprising further after being spin-dried for solvent process in the method for the above-mentioned compd A had in structural formula (1) and use purification by column chromatography, with the step of purification by column chromatography be with volume ratio be 3: 1-1: 2 petroleum ether and ethyl acetate mixed solvent carry out eluting, collect the compd A of purification.
Preferably, in such use, the method for synthesizing the above-mentioned compd B had in structural formula (2) is as follows: by 2, and 4,6-tri-halogenated pyrimidine and 3-amino-5-methylpyrazole are dissolved in dehydrated alcohol according to mol ratio 1: 1 ~ 2, add triethylamine, 0 DEG C of stirring reaction 8 ~ 16h, after reaction stops, water is added in reaction system, sucking filtration after precipitation white solid, adopts frozen water and ice methanol wash successively, obtains having the compd B in structural formula (2) after drying.
Preferably, in such use, synthesizing 2,4,6-tri-halogenated pyrimidines in the method for the above-mentioned compd B had in structural formula (2) is 2,4,6-trichloropyrimidine.
Preferably, in such use, above-mentioned tumor is the tumor because the unconventionality expression of Aurora A causes.
Preferably, in such use, above-mentioned tumor is nasopharyngeal carcinoma, breast carcinoma and neoplastic hematologic disorder.
Simultaneously, additionally provide a kind of purposes of pyrimidine derivatives vitro inhibition cancer cell growth in the present invention, pyrimidine derivatives comprise there is structural formula (I) pyrimidine compound, its pharmaceutically acceptable salt, solvate, polymorph, tautomer, prodrug, or the compositions containing pyrimidine compound, structural formula (I) is as follows:
Pass through in 2,4,6-positions of pyrimidine ring in the present invention, carry out different C-N couplings respectively, introduce different pharmacophore, form the new pyrimidine derivatives with Aurora kinase inhibitory activity, this pyrimidine derivatives can be prepared into and can prevent and/or treat and/or the medicine of auxiliary for treating cancer.
Except object described above, feature and advantage, the present invention also has other object, feature and advantage.Below with reference to figure, the present invention is further detailed explanation.
Accompanying drawing explanation
Accompanying drawing form this description a part, for understanding the present invention further, accompanying drawing shows the preferred embodiments of the present invention, and be used for principle of the present invention is described together with description.In figure:
Fig. 1 shows the proton nmr spectra spectrogram of the pyrimidine compound according to the present invention with structural formula (I);
Fig. 2 shows the carbon-13 nmr spectra spectrogram of the pyrimidine compound according to the present invention with structural formula (I);
Fig. 3 shows has the pyrimidine compound (AKI602) of structural formula (I) to AuroraA kinase inhibition Comparative result figure according to the present invention;
What Fig. 4 showed variable concentrations has the pyrimidine compound (AKI602) of structural formula (I) for the kinase whose inhibitory action figure of AuroraA in Leukemia cells NB4 according to the present invention;
The pyrimidine compound (AKI602) having structural formula (I) according to the present invention that Fig. 5 a shows variable concentrations suppresses leukaemia HL-60 growth result figure;
The pyrimidine compound (AKI602) having structural formula (I) according to the present invention that Fig. 5 b shows variable concentrations suppresses the growth result figure of leukaemia KG1a;
Fig. 6 a shows the cell cycle arrest result figure of pyrimidine compound (AKI602) for Leukemia cells NB4 according to the present invention with structural formula (I) of variable concentrations;
Fig. 6 b shows the cell cycle arrest result figure of pyrimidine compound (AKI602) for leukaemia HL-60 according to the present invention with structural formula (I) of variable concentrations;
Fig. 6 c shows the cell cycle arrest result figure of pyrimidine compound (AKI602) for leukaemia U937 according to the present invention with structural formula (I) of variable concentrations;
The pyrimidine compound (AKI602) having structural formula (I) according to the present invention that Fig. 7 a shows variable concentrations impels the result figure of Leukemia cells NB4 generation apoptosis;
The pyrimidine compound (AKI602) having structural formula (I) according to the present invention that Fig. 7 b shows variable concentrations impels leukaemia HL-60 that the result figure of apoptosis occurs; And
The pyrimidine compound (AKI602) having structural formula (I) according to the present invention that Fig. 7 c shows variable concentrations impels leukaemia U937 that the result figure of apoptosis occurs.
Detailed description of the invention
Below in conjunction with the accompanying drawing in the embodiment of the present invention, technical scheme in embodiments of the invention is described in detail, but following embodiment and accompanying drawing are only understand the present invention, and the present invention can not be limited, the multitude of different ways that the present invention can be defined by the claims and cover is implemented.
In one embodiment of the invention, provide a kind of pyrimidine derivatives preparation prevent and/or treat and/or auxiliary for treating cancer medicine in purposes, this pyrimidine derivatives comprise there is structural formula (I) pyrimidine compound, its pharmaceutically acceptable salt, solvate, polymorph, tautomer, prodrug, or the compositions containing pyrimidine compound, structural formula (I) is as follows:
The pyrimidine compound that the present invention has structural formula (I) is 4-[4-(5-methyl isophthalic acid H-pyrazole-3-yl) amino-6-(4-methylpiperazine-1-yl) pyrimidine-2-base] Methyl anthranilate, chemical formula is C21H26N8O2, and relative molecular weight is: 422.2.This compound is by 2,4, the 6-positions at pyrimidine ring, carry out different C-N couplings respectively, introduce different pharmacophore, form the new pyrimidine derivatives with Aurora kinase inhibitory activity, this pyrimidine derivatives can be made into and can prevent and/or treat and/or the medicine of auxiliary for treating cancer.
In one embodiment of the invention, provide one to prepare the above-mentioned method with the pyrimidine compound of structural formula (I) and comprise the following steps: the compd A and 1-methyl piperazine with structural formula (1) are dissolved into 1 according to mol ratio 1: 8 ~ 12, in 4-dioxane, 100 ~ 140 DEG C, microwave reaction 30 ~ 60 minutes, reaction gained mixture is spin-dried for solvent, must have the pyrimidine compound of structural formula (I), in its Chinese style (2), X is halogen.
Preferably, synthesize above-mentioned have be spin-dried for solvent process in the method for the pyrimidine compound of structural formula (I) after comprise step with reverse phase silica gel column purification further, with the step of reverse phase silica gel column purification be with volume ratio be 80: 20 ~ 40: 60 water and methanol mixed solvent carry out eluting, collect the pyrimidine compound with structural formula (I) of purification.Add the step with reverse phase silica gel column purification in the method, the increase of this step is conducive to the purity with the pyrimidine compound of structural formula (I) synthesized by raising, is conducive to later stage pharmaceutical applications.
In one embodiment of the invention, the method of the compd A that synthesis has in structural formula (1) comprises and will have compd B and the methyl p-aminobenzoate of structural formula (2), p-methyl benzenesulfonic acid is dissolved in n-butyl alcohol according to mol ratio 1: 1 ~ 1.2: 0.8 ~ 1, back flow reaction 10 ~ 12h at 130 ~ 150 DEG C, be cooled to room temperature after reaction, add saturated NaHCO
3solution regulates pH to 7 ~ 8, makes to be extracted with ethyl acetate 3 ~ 5 times, organic layer saturated common salt water washing 3 ~ 5 times, then uses anhydrous Na
2sO
4drying, is spin-dried for solvent and namely obtains the compd A with structural formula (1), and in its Chinese style (2), X is halogen.
Preferably, synthesize after being spin-dried for solvent process in the method for the compd A had in structural formula (1) and comprise the step using purification by column chromatography further, be with 200 ~ 300 object silica gel for chromatography by the step of purification by column chromatography, with volume ratio be 3: 1-1: 2 petroleum ether and ethyl acetate mixed solvent carry out eluting, collect the compd A had in structural formula (1) of purification.Add the step of purification by column chromatography in the method, the increase of this step is conducive to obtaining the higher compd A of purity, and then the by-product being conducive to reducing when follow-up synthesis has a pyrimidine compound of structural formula (I), and then improve the purity with the pyrimidine compound of structural formula (I), be conducive to later stage pharmaceutical applications.
In one embodiment of the invention, the method of the compd B that synthesis has in structural formula (2) comprises 2,4,6-tri-halogenated pyrimidine and 3-amino-5-methylpyrazole are dissolved in dehydrated alcohol according to mol ratio 1: 1 ~ 2, add triethylamine, 0 DEG C of stirring reaction 8 ~ 16h, reaction adds water after stopping in reaction system, sucking filtration after precipitation white solid, adopt frozen water and ice methanol wash successively, after drying, obtain that there is the compd B in structural formula (2).Wherein, 2,4,6-tri-halogenated pyrimidine is preferably 2,4,6-trichloropyrimidine.
According to foregoing, to have the reaction scheme of the synthetic reaction of the pyrimidine compound of structural formula (I) as follows for one in the present invention:
In one embodiment of the present invention, tumor is the tumor because the unconventionality expression of Aurora A causes.Be preferably nasopharyngeal carcinoma, breast carcinoma and neoplastic hematologic disorder.
Simultaneously, in one embodiment of the invention, provide a kind of pyrimidine derivatives in the purposes for suppressing cancer cell growth in vitro, this pyrimidine derivatives comprise there is structure above (I) pyrimidine compound, its pharmaceutically acceptable salt, solvate, polymorph, tautomer, prodrug, or the compositions containing pyrimidine compound.
Exemplary of the present invention will be described in detail below.But these embodiments only for the purpose of illustration, are not intended to limit the scope of the invention.
As used herein, if for providing concrete restriction, term of the present invention has following implication.
Halogen comprises fluorine, chlorine, bromine and iodine.
Treats effective dose and refers to when giving the mammal needing such treatment, is enough to the amount of the general formula compound of effectively treatment.Treatment effective dose changes depending on the given activity of healing potion used, age of patient, physiological situation, the existence of Other diseases state and nutriture.In addition, the other medicines treatment that patient may just accept will affect the determination of the treatment effective dose of the healing potion that will give.
Compound of the present invention also comprises all isotopic atoms, no matter is at intermediate or last compound.Isotopic atom comprises and has identical atomic number, but different quality number.Such as, the isotope of hydrogen comprises tritium and deuterium.
Compound of the present invention also comprises pharmaceutically acceptable salt.Pharmaceutically acceptable salt refers to the form the basic group conversion salify in parent compound.Pharmaceutically acceptable salt include but not limited to, the inorganic or organic acid salt of basic group such as amine (ammonia) base.Pharmaceutically acceptable salt of the present invention can be synthesized by parent compound, and the acid of the basic group namely in parent compound and 1-4 equivalent is reacted in a solvent system.Suitable salt is set forth in Re minute gton
pharmaceuticalSciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p.1418 with Journal ofPharmaceutical Science, 66, in 2 (1977).
Pharmaceutically acceptable acid-addition salts can be synthesized by inorganic and organic acid.Hydrochloric acid, hydrobromic acid, sulphuric acid, nitric acid, phosphoric acid etc. are comprised by the mineral acid of derived acids addition salts.Acetic acid, propanoic acid, glycolic, acetone acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethyl sulfonic acid, p-methyl benzenesulfonic acid, salicylic acid, benzenesulfonic acid etc. are comprised by the organic acid of derived acids addition salts.The mineral acid of derived acids addition salts and organic acid are especially selected from hydrochloric acid, phosphoric acid, sulphuric acid, nitric acid, perchloric acid, hydrobromic acid, acetic acid, benzoic acid and p-methyl benzenesulfonic acid.
As used herein, pharmaceutically acceptable carrier comprise any and whole solvents, disperse medium, coating, antibacterium and antifungal medicine, etc. blend absorption delay agent etc.Such medium and medicament are used for pharmaceutically active substances and are well known in the art.Unless any conventional media or medicament incompatible with active component, its application in therapeutic combination is expected.The active component supplemented also can be incorporated in compositions.
Said composition is preferably formulated into unit dosage forms.Term unit dosage forms refers to the physical discrete unit being suitable for use as and giving human experimenter and other mammiferous single doses, and per unit contains the predetermined amount and relevant suitable pharmaceutical excipient (as tablet, capsule, ampoule) that calculate to produce the effective active substance of required treatment.The compound of general formula (I) is effective and usually gives active drug amount in dosage range widely.Preferably, for oral administration, each dosage unit comprises general formula (I) compound of 10mg to 2g, be more preferably 10 to 700mg, and for parenteral, be preferably general formula (I) compound of 10 to 700mg, more preferably from about 50 to 200mg.But, should understand, the amount of actual general formula (I) compound given is determined by doctor according to relevant situation, comprise the disease that will treat, the route of administration selected, the pragmatize compound given and its relative activity, age of each patient, body weight and reaction, the seriousness etc. of patients symptomatic.
In order to synthesis of solid compositions is as tablet, main active component carried out mixing to form solid preformulation composition with drug excipient (or carrier), it comprises the homogeneous mixture of compound of the present invention.In time claiming these preformulation composition to be uniform, it refers to that active component is dispersed in whole compositions, so that compositions easily can be subdivided into identical effective unit dosage forms as tablet, pill and capsule.
Tablet of the present invention or pill applied or otherwise can be had to provide a kind of the dosage form extending effect beneficial by compound, or protection tablet or pill are from the effect of acid condition in stomach.Such as, tablet or pill can comprise interior dosage and external dose composition, and the latter has the form of the crust on the former.Can separate two kinds of compositions with enteric layer, wherein enteric layer is used for stoping disintegrate under one's belt and composition is complete in allowing enters duodenum or be delayed by release.Various material may be used for such enteric layer or coating, and above-mentioned material comprises many polymer acids and polymer acid and such material as the mixture of Lac, hexadecanol and cellulose acetate.
Solution in pharmaceutically acceptable aqueous solvent or organic solvent or its mixture and suspension is included in for the compositions of inhalation or insufflation, and powder.Liquid or solid compositions can comprise as suitable pharmaceutical excipient above.Preferably, these compositionss are given to obtain local or systemic effect by oral or nasal respiratory route.The compositions in the acceptable solvent of preferred pharmacy can be atomized by using noble gas.Directly can suck atomized soln from atomising device, or atomising device can be connected to face shield account shape thing or intermittent positive pressure breathing machine.Can by the device sending dosage form in a suitable manner, preferred oral or nose approach, give solution, suspensoid or powder composite.
Compound of the present invention and pharmaceutically acceptable salt also comprise the form of solvate or hydrate.In general, the form of solvate or hydrate is equal to form that is non-solvated or non-hydrated, and contains within the scope of the invention.Likely there is polycrystal or unbodied form in some compound in the present invention.Generally speaking, all physical form have equal purposes, and contain within the scope of the invention.
The present invention also comprises the prodrug of compound.Prodrug is a pharmacological agents (medicine), is derived by parent drug.Once enter in body, prodrug is just become parent drug by metabolic transformation.Prodrug synthesizes by replacing one or more functional groups of parent drug, and its substituted radical will be degraded and discharge parent compound in vivo.Synthesis and the use of prodrug can at T.Higuchi and V.Stella, Pro-drugs as Novel Delivery Systems, Vol.14of the A.C.S.SymposiumSeries, with Bioreversible Carriers in Drug Design, ed.Edward B.Roche, American PharmaceuticalAssociation and Pergamon Press, finds in 1987.
The present invention also provides the pharmaceutical composition comprising general formula (I) compound or the acceptable salt of its pharmacy or its prodrug and the pharmaceutically acceptable carrier of at least one.Pharmaceutical composition of the present invention can be oral, and injection is injected, and spraying sucks, skin external, rectum use, nasal cavity use, vagina use, abdominal cavity use, or use by implanting the approach such as reservoir or transdermal patch.
Beneficial effect of the present invention is proved further below with reference to specific embodiment.
Embodiment 1
Synthesis compd B:
Take 2,4,6-trichloropyrimidine (1.83g, 10mmol), 3-amino-5-methylpyrazole (0.97g, 10mmol) is dissolved in 100mL dehydrated alcohol, adds 1.4mL triethylamine, stirring reaction 12h at 0 DEG C.After stopped reaction, in reaction solution, add 150mL water, separate out a large amount of white solids immediately, sucking filtration, and use 50mL frozen water and 20mL ice methanol wash successively, after drying, obtain white solid B (1.82g, 75%).
Product analysis: the nuclear magnetic data of the white solid B obtained is
1h NMR (400MHz, DMSO-d
6) δ: 12.21 (s, 1H), 10.63 (s, 1H), 7.74and6.76 (m, 1H), 6.38,5.78 (m, 1H), 2.21 (s, 3H);
13c NMR (100MHz, DMSO-d
6) δ: 161.1,159.7,158.4,147.1,139.0,102.6,95.2,10.4ppm.White solid B obtained as can be seen here has structure in structural formula (2).
Synthesis compd A:
Weigh Compound B (244mg, 1mmol), methyl p-aminobenzoate (181mg, 1.2mmol), p-methyl benzenesulfonic acid (152mg, 0.8mmol), is dissolved in 10mL n-butyl alcohol, back flow reaction 12h at 130 DEG C, is cooled to room temperature, adds the saturated NaHCO of 2.5mL
3solution regulates pH=7.5, make to be extracted with ethyl acetate (200mL?), saturated aqueous common salt (50mL?) washing organic layer, then use anhydrous Na
2sO
4drying, is spin-dried for solvent, purification by column chromatography (petroleum ether: ethyl acetate=3: 1-1: 2) obtain white solid A (233mg, 65%).
Product analysis: the nuclear magnetic data of the white solid A obtained is
1h NMR (400MHz, DMSO-d
6) δ: 12.09 (s, 1H), 9.92 (s, 2H), 7.87 (t, J=8.3Hz, 4H), 6.25 (s, 2H), 3.80 (s, 3H), 2.23 (s, 3H);
13c NMR (100MHz, DMSO-d
6) δ: 166.0,160.8,158.7,144.9,130.0,122.0,118.2,96.3,51.7,10.8ppm.White solid A obtained as can be seen here has structure in structural formula (2).
Synthesis has the pyrimidine compound of structural formula (I)
Weigh Compound A (179mg, 0.5mmol) and 1-methyl piperazine (0.55mL, 5mmol) are dissolved in the Isosorbide-5-Nitrae-dioxane of 4mL, and at 140 DEG C, microwave reaction 30 minutes, is spin-dried for solvent, use reverse phase silica gel column purification (H
2o: methanol=80: 20-40: 60), obtains white solid (180mg, 85%).
Product analysis: the nuclear magnetic data of the white solid obtained is
1h NMR (400MHz, DMSO-d
6) δ: 11.84 (s, 1H), 9.12 (s, 1H), 8.94 (s, 1H), 7.91 (s, 2H), 7.84 (s, 2H), 6.19 (s, 1H), 6.11 (s, 1H), 3.80 (s, 3H), 3.49 (s, 4H), 2.39 (s, 4H), 2.22 (s, 6H);
13c NMR (100MHz, DMSO-d
6) δ: 166.1,163.3,160.6,158.3,149.2,146.1,138.1,130.0,120.5,117.3,95.3,77.7, the white solid high resolution mass spectrum data that 54.2,51.5,45.7,43.8,10.7ppm. obtains are HRMS (ESI-TOF): m/z calcd. for C
21h
25n
8o
2[M-H]
-: 421.2100; Found:421.2103.As can be seen here obtain the pyrimidine compound with structural formula (I).
Embodiment 2
Synthesis compd B:
Take 2,4,6-trichloropyrimidine (1.83g, 10mmol), 3-amino-5-methylpyrazole (1.94g, 20mmol) is dissolved in 100mL dehydrated alcohol, adds 1.4mL triethylamine, stirring reaction 16h at 0 DEG C.After stopped reaction, in reaction solution, add 150mL water, separate out a large amount of white solids immediately, sucking filtration, and use 50mL frozen water and 20mL ice methanol wash successively, after drying, obtain white solid B (1.82g, 70%).
Synthesis compd A:
Weigh Compound B (244mg, 1mmol), methyl p-aminobenzoate (150.8mg, 1mmol), p-methyl benzenesulfonic acid (190mg, 1mmol), is dissolved in 10mL n-butyl alcohol, back flow reaction 10h at 150 DEG C, is cooled to room temperature, adds the saturated NaHCO of 2.5mL
3solution is adjusted to pH=7, make to be extracted with ethyl acetate (200mL?), saturated aqueous common salt (50mL?) washing organic layer, then use anhydrous Na
2sO
4drying, is spin-dried for solvent, purification by column chromatography (petroleum ether: ethyl acetate=3: 1-1: 2) obtain white solid A (233mg, 63%).
Synthesis has the pyrimidine compound of structural formula (I)
Weigh Compound A (179mg, 0.5mmol) and 1-methyl piperazine (0.44mL, 4mmol) are dissolved in the Isosorbide-5-Nitrae-dioxane of 4mL, and at 140 DEG C, microwave reaction 60 minutes, is spin-dried for solvent, use reverse phase silica gel column purification (H
2o: methanol=80: 20-40: 60), obtains white solid (180mg, 73%), this white solid through nuclear-magnetism, infrared detection analysis for having the pyrimidine compound of structural formula (I).
Can both obtain by method in embodiment 1 and embodiment 2 pyrimidine compound that the application has structural formula (I), wherein adopt method in embodiment 1 to obtain compound yield higher.
One: the pyrimidine compound (AKI602) with structural formula (I) that embodiment 1 is synthesized is to AuroraA kinase inhibition assay.
AuroraA kinase inhibition assay (K-LI SA
tMauroraActivity Kit, Calbiochem) main experimental methods: the first step, in 96 orifice plates, successively add 10 masurium reaction solutions, 10 masurium AuroraA kinases, 10 masurium substrates, 10 masurium testing compound solutions, 10 masurium ATP solution, mix latter 30 DEG C and hatch 30 minutes; Second step, adds 10 masurium kinase reaction stop baths in each orifice plate; 3rd step, adds 100 masurium phosphoric acid-histone H 3 antibody, hatches 60 minutes at 25 DEG C in each orifice plate; 4th step, adds 100 masurium HRP-antibody chelating agent solutions, hatches 60 minutes at 25 DEG C in each orifice plate; 5th step, adds 100 masurium tmb substrates (developer), hatches the 10 minutes: six step at 25 DEG C in each orifice plate, add 100 masurium ELISA stop baths, with the reading of enzyme-linked immunosorbent assay instrument record 450nm in each orifice plate.Not add the solvent blank of medicine as negative control, take VX-680 as positive control.
According to relation: after adding compound, the ability of tyrosine phosphorylation substrate take VX-680 as reference.
Experimental result: as shown in Figure 3, the pyrimidine compound synthesized by the present invention with structural formula (I) has AuroraA kinase inhibitory activity, and the pyrimidine compound with structural formula (I) is better than VX-680 to AuroraA kinase inhibitory effect.
Two, the pyrimidine compound (AKI602) with structural formula (I) that embodiment 1 is synthesized is tested the kinase whose inhibit activities of Aurora-A in cell.
Experiment object: Leukemia cells NB4 cell.
Experimental technique: by NB4 cell culture in 6 orifice plates, adopt the pyrimidine compound process NB4 cell with structural formula (I) that the embodiment 1 of variable concentrations is synthesized, 24 h before harvest cells, adopt Western blot to detect the expression (T288) of phosphorylation Aurora-A.GAPDH is as internal reference.Concrete steps are collect the cell through drug treating, abundant cell lysis; Leave the heart 15 minutes with 4 DEG C 14000, get supernatant.Determining the protein quantity adopts Bradford albuminimetry, gets protein sample 40 μ g, to carry out after SDS-PAGE electrophoresis by protein delivery to NC film, after carry out antibody incubation and chemiluminescence development.
Experimental result: as shown in Figure 4, the pyrimidine compound energy dose dependent synthesized by the present invention with structural formula (I) reduces the expression of the phosphorylation Aurora-A (T288 site) in NB4 cell, therefore this compound can suppress the kinase whose activity of AuroraA, shows that this compound has targeting specific.
Three, the pyrimidine compound (AKI602) with structural formula (I) of embodiment 1 synthesis is to the experiment suppressing leukemic cell growth.
Experiment object: leukaemia HL-60 and leukaemia KG1a.
Experimental technique: by HL-60, KG1a cell with 10
4the density of/100 μ l is incubated in 96 orifice plates, the pyrimidine compound process with structural formula (I) adopting the embodiment 1 of variable concentrations to synthesize adds 10 μ l WST-8 (Dojindo) after 24 hours, 37 Fei cultivate after 4 hours and detect OD value (cells growth activity) with 450nm.
Experimental result: as shown in Figure 5 a, the growth of pyrimidine compound to leukaemia HL-60 synthesized by the present invention with structural formula (I) has obvious inhibitory action, and along with having the increase of pyrimidine compound content of structural formula (I), its inhibition is better, best when the pyrimidine compound content particularly with structural formula (I) is 1 ~ 10 μM.As shown in Figure 5 b, the growth of pyrimidine compound to leukaemia KG1a synthesized by the present invention with structural formula (I) has obvious inhibitory action, and along with having the increase of pyrimidine compound content of structural formula (I), its inhibition is better, best when the pyrimidine compound content particularly with structural formula (I) is 1 ~ 5 μM.
Four, the cell cycle arrest experiment of the pyrimidine compound (AKI602) with structural formula (I) of embodiment 1 synthesis.
Experiment object: Leukemia cells NB4, leukaemia HL-60 and leukaemia U937.
Cell cycle arrest experimental technique: by the synthesized pyrimidine compound process leukaemia with structural formula (I), 24 h before harvest cells, dye with 50 μ g/mL PI after 70% ethanol is fixing, adopt flow cytometer to carry out cell cycle detection.Each compound 6 concentration, are respectively 0 Jian, 1 Jian, 5 Jian, 10 Jian, 15 Jian, 20 Jian, judge compound inhibit activities by the retardance rate of cell G2/M phase.
Experimental result: as shown in Fig. 6 a, 6b, 6c, in leukaemia HL-60, U937, NB4, the synthesized pyrimidine compound with structural formula (I) all can obviously cause G2/M cell cycle arrest at 1-20 μM, and there is polyploid phenomenon, show that the pyrimidine compound with structural formula (I) has an impact by suppressing Aurora-A and then cell cycle.
Five, the pyrimidine compound (AKI602) with structural formula (I) that embodiment 1 is synthesized impels leukaemia that the experiment of apoptosis occurs.
Experiment object: Leukemia cells NB4, leukaemia HL-60 and leukaemia U937.
Experimental technique: having in the pyrimidine compound of structural formula (I) synthesized by variable concentrations is processed leukaemia, 24 h before harvest cells, adopts Western blot to detect the expression of Cleaved PARP.GAPDH is as internal reference.Concrete steps are collect the cell through drug treating, abundant cell lysis; Leave the heart 15 minutes with 4 DEG C 14000, get supernatant.Determining the protein quantity adopts Bradford albuminimetry.Get protein sample 40 μ g, to carry out after SDS-PAGE electrophoresis by protein delivery to NC film, after carry out antibody incubation and chemiluminescence development.
Experimental result: as shown in Fig. 7 a, 7b, 7c, the pyrimidine compound with structural formula (I) synthesized by the present invention has the significantly apoptotic effect of promotion, and the expression showing as Cleaved PARP increases progressively with compound effects increasing concen-trations.The expression of obvious Cleaved PARP is there is in HL-60 and NB4 cell when activity is 1 μM; The expression of obvious Cleaved PARP is there is in U937 when activity is 5 μMs.The effect showing this compound killing tumor cell is relevant to activation apoptotic signal path.
These are only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. pyrimidine derivatives preparation prevent and/or treat and/or adjuvant therapy of tumors medicine in a purposes, it is characterized in that, described pyrimidine derivatives for having the pyrimidine compound of structural formula (I) or its pharmaceutically acceptable salt,
Described structural formula (I) is as follows:
2. purposes according to claim 1, is characterized in that, the method described in synthesis with the pyrimidine compound of structural formula (I) comprises the following steps:
The compd A and 1-methyl piperazine with structural formula (1) are dissolved into 1 according to mol ratio 1:8 ~ 12, in 4-dioxane, 100 ~ 140 DEG C, microwave reaction 30 ~ 60 minutes, reaction gained mixture is spin-dried for solvent, has the pyrimidine compound of structural formula (I) described in obtaining;
Wherein, in formula (1), X is halogen.
3. purposes according to claim 2, it is characterized in that, there is described in synthesis the step comprising further after being spin-dried for solvent process in the method for the pyrimidine compound of structural formula (I) and use reverse phase silica gel column purification, be be that the water of 80:20 ~ 40:60 and methanol mixed solvent carry out eluting by volume ratio by the step of reverse phase silica gel column purification, collect the pyrimidine compound described in purification with structural formula (I).
4. purposes according to claim 3, is characterized in that, the method for synthesizing described compd A is as follows:
Compd B and the methyl p-aminobenzoate of structural formula (2) will be had, p-methyl benzenesulfonic acid is dissolved in n-butyl alcohol according to mol ratio 1:1 ~ 1.2:0.8 ~ 1, at 130 ~ 150 DEG C, back flow reaction 10 ~ 12h, is cooled to room temperature after reaction, adds saturated NaHCO
3solution regulates pH to 7 ~ 8, makes to be extracted with ethyl acetate 3 ~ 5 times, organic layer saturated common salt water washing 3 ~ 5 times, then uses anhydrous Na
2sO
4drying, is spin-dried for solvent and namely obtains described compd A,
Wherein, in formula (2), X is halogen.
5. purposes according to claim 4, it is characterized in that, synthesize the step comprising further after being spin-dried for solvent process in the method for described compd A and use purification by column chromatography, be be that the petroleum ether of 3:1-1:2 and ethyl acetate mixed solvent carry out eluting by volume ratio by the step of purification by column chromatography, collect the described compd A of purification.
6. purposes according to claim 4, is characterized in that, the method for synthesizing described compd B is as follows:
By 2,4,6-tri-halogenated pyrimidine and 3-amino-5-methylpyrazole are dissolved in dehydrated alcohol according to mol ratio 1:1 ~ 2, add triethylamine, 0 DEG C of stirring reaction 8 ~ 16h, after reaction stops, water is added in reaction system, sucking filtration after precipitation white solid, adopts frozen water and ice methanol wash successively, obtains described compd B after drying.
7. purposes according to claim 6, is characterized in that, described in the method for synthesizing described compd B, 2,4,6-tri-halogenated pyrimidines are 2,4,6-trichloropyrimidine.
8. the purposes described in any one of claim 1 to 7, is characterized in that, described tumor is the tumor because the unconventionality expression of Aurora A causes.
9. purposes according to claim 8, is characterized in that, described tumor is nasopharyngeal carcinoma, breast carcinoma and neoplastic hematologic disorder.
10. a purposes for pyrimidine derivatives vitro inhibition cancer cell growth, is characterized in that, described pyrimidine derivatives for having the pyrimidine compound of structural formula (I) or its pharmaceutically acceptable salt,
Described structural formula (I) is as follows:
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