CN101054380B - Pyrazolopyrimidine derivative used as cell cycle dependency protein kinase inhibitor - Google Patents
Pyrazolopyrimidine derivative used as cell cycle dependency protein kinase inhibitor Download PDFInfo
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Abstract
The present invention discloses pyrazolopyrimidine derivative as cell cycle dependent protein kinase inhibitor, and its optical isomer and pharmaceutically acceptable salt, hydrate. The constitutional formula is indicated as formula I, which can be prepared for medicament for treating cancer, in which the substituting group X,Y,Q1,Q2 are separately selected from hydro-, C1-C10 alkyl, C3-C7 cyclane, C2-C10 alkenyl and C2-C10 alkynyl and can be replaced by 1-3 same or different R4. Derivative of formula I can be used for preparing cell cycle dependent protein kinase inhibitor and also be used in preparing medicine for treatment or prophylaxis of cancer.
Description
Technical field:
The present invention relates to the pyrazolo [1 shown in the general formula I, 5-a] pyridine derivatives, and optically active body and its pharmaceutically acceptable salt, hydrate, with be the pharmaceutical composition of activeconstituents with this derivative, and at preparation cell cycle dependent protein kinase inhibitor and be used for the treatment of and/or prevent purposes in the medicine of various cancers.
Background technology:
Cancer is called malignant tumour again, is a class common disease of serious threat human health, and the mortality ratio of cancer still rising, also lacks effective medicine to common solid tumor at present.By some link kill cancer cell of interference cell fission process, its action target spot there is no essential distinction to existing chemotherapeutics in cancer cells and normal cell, in kill cancer cell, also can produce toxic side effect mostly.
Cell cycle is meant that cycling cell finishes the whole process that is experienced to next mitotic division end from a mitotic division.Whether cell cycle is moved and is controlled by accurate cell cycle regulating mechanism.The positivity regulatory factor that this machine-processed core is the cell cycle and cell cycle negativity regulatory factor coordinative role in the cell cycle by to corresponding substrate phosphorylation, are being ordered about cell and are being finished the cell cycle.Cell positivity regulatory factor is a histone kinase, and (cyclin-dependent-kinase CDKs), has found CDK1-77 at present, and is relatively stable in their content in the whole cell cycle to be called the cell cycle deopendent protein kinase.The negativity regulatory factor of cell cycle then is blocking-up cell cycle, the arrestin that suppresses cytodifferentiation, is called the cell cycle dependent protein kinase inhibitor.
Cell cycle and multiple human disease-related wherein the most important thing is to have confidential relation with cancer.The major cause of cancer is the unusual of the unrestricted differentiation of cell that causes after the cell cycle imbalance and propagation, thereby jeopardizes the general safety of body.Dedifferenting with malignancy of cell is often closely related with cell cycle regulatory factors.The overexpression of positivity cell cycle regulatory factors can cause that cell fission is out of control, causes cancer to form.The abnormal expression of CDK2-Cyclin E is very typical feature in hysteroma, mammary cancer and lung cancer.
The active regulation and control of CDKs are up-and-coming antitumous effect target spots, have at present the CDKs inhibitor of a lot of novelties, particularly CDK2 inhibitor be in clinical before or clinical study in, be proved to be better curative effect.
The inventor is on the basis of reference, and a series of imidazo [1,5-a] pyridine derivatives has been synthesized in design, through the anti tumor activity in vitro screening, shows to have anti-tumor activity.
Summary of the invention:
The derivative of the general formula I that the present invention relates to be defined as follows, and optically active body and its pharmaceutically acceptable salt, hydrate,
Wherein
Q
1And Q
2Identical or different, be independently selected from hydrogen, C respectively
1-C
10Alkyl, C
3-C
7Cycloalkyl, C
2-C
10Thiazolinyl and C
2-C
10Alkynyl can be by 1～3 identical or different R
4The optional replacement;
Or Q
1And Q
2Form 5-10 unit heterocyclic radical with the nitrogen-atoms that is connected with them, described heterocyclic radical can contain 1-4 heteroatoms that is selected from N, O and S, chooses wantonly to comprise 1 or 2 carbon-carbon double bond or three key, and described heterocyclic radical can be by 1～3 identical or different R
4The optional replacement;
Perhaps Q
1And Q
2One of them is H, and another is-(CH
2)
m-Ar
1
When X be direct key ,-O-(CH
2)
pIn-time, Y is
When X is
The time, Y is
Or-OR
3
When X is
The time, Y is Ar
2
Ar
1Be C
6-C
10Aryl, 5-10 unit heteroaryl, wherein, described heteroaryl can contain 1-4 heteroatoms that is selected from N, O or S, and Ar
1Can be by 1-3 identical or different R
5The optional replacement;
Ar
2Be C
6-C
10Aryl, 5-10 unit heteroaryl, wherein, described heteroaryl contains 1-4 heteroatoms that is selected from N, O or S, and Ar
2Can be by 1-3 identical or different R
6The optional replacement;
R
1And R
2Identical or different, be independently selected from hydrogen, C respectively
1-C
10Alkyl, C
3-C
7Cycloalkyl, C
2-C
10Thiazolinyl and C
2-C
10Alkynyl, C
6-C
10Aryl, C
6-C
10Aryl C
1-C
4Alkyl, 5-10 unit heterocyclic radical, the heterocyclic radical C of 5-10 unit
1-C
4Alkyl, 5-10 unit heteroaryl, the heteroaryl C of 5-10 unit
1-C
4Alkyl can be by 1～3 identical or different R
7The optional replacement; Described cycloalkyl can comprise 1～2 carbon-carbon double bond or three key, and described heterocyclic radical and heteroaryl contain 1-4 heteroatoms that is selected from N, O or S; R
1And R
2Can be by 1～3 identical or different R
7The optional replacement;
Or R
1And R
2Form 5-10 unit's heterocyclic radical or 5-10 unit heteroaryl with the nitrogen-atoms that is connected with them, described heterocyclic radical and heteroaryl can contain 1-4 heteroatoms that is selected from N, O and S, optional comprise 1 or 2 carbon-carbon double bond or three key, can be by 1～3 identical or different R
7The optional replacement;
R
3Be hydrogen, C
1-C
10Alkyl, C
3-C
7Cycloalkyl, C
6-C
10Aryl, 5-10 unit heterocyclic radical, 5-10 unit heteroaryl can be by 1～3 identical or different R
8The optional replacement;
R
4, R
7, R
8Be independently selected from hydroxyl, halogen, nitro, cyano group, trifluoromethyl, trifluoromethoxy, C
1-C
4Alkyl, C
1-C
4Alkoxy methyl, N, N-two C
1-C
4Alkylamino, C
3-C
7Cycloalkyl, described cycloalkyl can comprise 1～2 carbon-carbon double bond or three key;
R
5, R
6Be independently selected from C
1-C
4Alkyl, C
1-C
4Alkoxyl group, hydroxyl, optional by hydroxyl, amino or halogenated C
1-C
4Alkyl or C
1-C
4Alkoxyl group, free, salifiable, esterification with amidated carboxyl, halo, C
1-C
4Alkyl acyl, nitro, cyano group, amino, C
1-C
6Alkylamidoalkyl or coverlet or two (C
1-C
4Alkyl) amido of Qu Daiing, C
1-C
4Alkoxy methyl, formamyl, N-C
1-C
4Alkyl-carbamoyl, N, N-two C
1-C
4Alkyl-carbamoyl, amino-sulfonyl, N-C
1-C
4Alkyl amino sulfonyl, N, N-two C
1-C
4Alkyl amino sulfonyl, C
1-C
3Alkylenedioxy group;
M is the integer between 0～4;
P, q are the integer between 1～4;
R is the integer between 0～2.
The preferred Q of the present invention
1And Q
2One of them is H, and another is-(CH
2)
m-Ar
1The general formula I derivative, and optically active body and its pharmaceutically acceptable salt, hydrate.
The present invention goes back the following general formula I derivative of preferred definition, and optically active body and its pharmaceutically acceptable salt, hydrate,
Wherein
Q
1And Q
2One of them is H, and another is-(CH
2)
m-Ar
1
When X be direct key ,-O-(CH
2)
pIn-time, Y is
When X is
The time, Y is
Or-OR
3
When X is
The time, Y is Ar
2
Ar
1For phenyl or by 1-3 identical or different R
5The optional phenyl that replaces;
Ar
2For phenyl or by 1-3 identical or different R
6The optional phenyl that replaces, perhaps for containing 1-4 heteroatomic 5-6 ring heteroaryl that is selected from N, O or S, described heteroaryl can be by 1-3 identical or different R
6The optional replacement;
R
1And R
2Identical or different, be independently selected from hydrogen, C respectively
1-C
10Alkyl, C
3-C
7Cycloalkyl, C
6-C
10Aryl, C
6-C
10Aryl C
1-C
4Alkyl, 5-10 unit heterocyclic radical, the heterocyclic radical C of 5-10 unit
1-C
4Alkyl, 5-10 unit heteroaryl, the heteroaryl C of 5-10 unit
1-C
4Alkyl can be by 1～3 identical or different R
7The optional replacement; Described cycloalkyl can comprise 1～2 carbon-carbon double bond or three key, and described heterocyclic radical and heteroaryl contain 1-4 heteroatoms that is selected from N, O or S; R
1And R
2Can be by 1～3 identical or different R
7The optional replacement;
Or R
1And R
2Form 5-10 unit's heterocyclic radical or 5-10 unit heteroaryl with the nitrogen-atoms that is connected with them, described heterocyclic radical and heteroaryl can contain 1-4 heteroatoms that is selected from N, O and S, optional comprise 1 or 2 carbon-carbon double bond or three key, can be by 1～3 identical or different R
7The optional replacement;
R
3Be hydrogen, C
1-C
10Alkyl, C
3-C
7Cycloalkyl can be by 1～3 identical or different R
8The optional replacement;
R
7, R
8Be independently selected from hydroxyl, halogen, nitro, cyano group, trifluoromethyl, trifluoromethoxy, C
1-C
4Alkyl, C
1-C
4Alkoxy methyl, C
3-C
7Cycloalkyl, N, N-two C
1-C
4Alkylamino, described cycloalkyl can comprise 1～2 carbon-carbon double bond or three key;
R
5, R
6Be independently selected from C
1-C
4Alkyl, C
1-C
4Alkoxyl group, hydroxyl, optional by hydroxyl, amino or halogenated C
1-C
4Alkyl or C
1-C
4Alkoxyl group, free, salifiable, esterification with amidated carboxyl, halo, C
1-C
4Alkyl acyl, nitro, cyano group, amino, C
1-C
6Alkylamidoalkyl or coverlet or two (C
1-C
4Alkyl) amido of Qu Daiing, C
1-C
4Alkoxy methyl, formamyl, N-C
1-C
4Alkyl-carbamoyl, N, N-two C
1-C
4Alkyl-carbamoyl, amino-sulfonyl, N-C
1-C
4Alkyl amino sulfonyl, N, N-two C
1-C
4Alkyl amino sulfonyl, C
1-C
3Alkylenedioxy group;
M is the integer between 0～4;
P, q are the integer between 1～4;
R is the integer between 0～2.
The general formula I derivative that the special preferred definition of the present invention is following, and optically active body and its pharmaceutically acceptable salt, hydrate,
Wherein
Q
1And Q
2One of them is H, and another is a phenyl or by 1-3 identical or different R
5The optional phenyl that replaces;
When X be direct key ,-O-(CH
2)
pIn-time, Y is
When X is
The time, Y is
Or-OR
3
R
1And R
2Identical or different, be independently selected from hydrogen, C respectively
1-C
6Alkyl, C
3-C
6Cycloalkyl, C
3-C
6Methyl cycloalkyl, C
3-C
6The cycloalkyl ethyl, phenyl, phenmethyl, styroyl, furfuryl, furans ethyl, picolyl, pyridine ethyl, tetrahydrobenzene methyl, tetrahydrobenzene ethyl, cyclopentenes methyl, cyclopentenes ethyl;
Or R
1And R
2Form 1-pyrrolidyl, 4-morpholinyl, piperidino, 1-piperazinyl, 4-methylpiperazine base, 1-imidazolyl, 2-methyl isophthalic acid-imidazolyl, 1-triazol radical, 1-tetrazole base with the nitrogen-atoms that is connected with them;
R
3Be hydrogen, C
1-C
10Alkyl, C
3-C
7Cycloalkyl;
R
5Be independently selected from C
1-C
4Alkyl, C
1-C
4Alkoxyl group, hydroxyl, optional by hydroxyl, amino or halogenated C
1-C
4Alkyl or C
1-C
4Alkoxyl group, free, salifiable, esterification with amidated carboxyl, halo, C
1-C
4Alkyl acyl, nitro, cyano group, amino, C
1-C
6Alkylamidoalkyl or coverlet or two (C
1-C
4Alkyl) amido of Qu Daiing; C
1-C
4Alkoxy methyl, formamyl, N-C
1-C
4Alkyl-carbamoyl, N, N-two C
1-C
4Alkyl-carbamoyl, amino-sulfonyl, N-C
1-C
4Alkyl amino sulfonyl, N, N-two C
1-C
4Alkyl amino sulfonyl, C
1-C
3Alkylenedioxy group;
P, q are the integer between 1～4.
The general formula I derivative that the also special preferred definition of the present invention is following, and optically active body and its pharmaceutically acceptable salt, hydrate,
Wherein
Q
1And Q
2One of them is H, and another is a phenyl or by 1-3 identical or different R
5The optional phenyl that replaces;
When X be direct key ,-O-(CH
2)
pIn-time, Y is
When X is
The time, Y is
Or-OR
3
R
1And R
2Identical or different, be independently selected from hydrogen, C respectively
1-C
6Alkyl, C
3-C
6Cycloalkyl, C
3-C
6Methyl cycloalkyl, C
3-C
6Cycloalkyl ethyl, phenyl, phenmethyl, styroyl, furfuryl, furans ethyl, picolyl, pyridine ethyl, tetrahydrobenzene methyl, tetrahydrobenzene ethyl, cyclopentenes methyl, cyclopentenes ethyl;
Or R
1And R
2Form 1-pyrrolidyl, 4-morpholinyl, piperidino, 1-piperazinyl, 4-methylpiperazine base, 1-imidazolyl, 2-methyl isophthalic acid-imidazolyl, 1-triazol radical, 1-tetrazole base with the nitrogen-atoms that is connected with them;
R
3Be hydrogen, C
1-C
4Alkyl, C
3-C
7Cycloalkyl;
R
5Be independently selected from C
1-C
4Alkyl, C
1-C
4Alkoxyl group, hydroxyl, optional by hydroxyl, amino or halogenated C
1-C
4Alkyl or C
1-C
4Alkoxyl group, free, salifiable, esterification with amidated carboxyl, halo, C
1-C
4Alkyl acyl, nitro, cyano group, amino, C
1-C
6Alkylamidoalkyl or coverlet or two (C
1-C
4Alkyl) amido of Qu Daiing, C
1-C
4Alkoxy methyl, formamyl, N-C
1-C
4Alkyl-carbamoyl, N, N-two C
1-C
4Alkyl-carbamoyl, amino-sulfonyl, N-C
1-C
4Alkyl amino sulfonyl, N, N-two C
1-C
4Alkyl amino sulfonyl, C
1-C
3Alkylenedioxy group;
P is 1 or 2;
Q is the integer between 1～4.
The general formula I derivative that the also special preferred definition of the present invention is following, and optically active body and its pharmaceutically acceptable salt, hydrate,
Wherein
Q
1And Q
2One of them is H, and another is a phenyl or by 1-3 identical or different R
5The optional phenyl that replaces;
When X be direct key ,-O-(CH
2)
pIn-time, Y is
When X is
The time, Y is
Or-OR
3
R
1And R
2Identical or different, be independently selected from hydrogen, C respectively
1-C
6Alkyl, C
3-C
6Cycloalkyl, C
3-C
6Methyl cycloalkyl, C
3-C
6Cycloalkyl ethyl, phenyl, phenmethyl, styroyl, furfuryl, furans ethyl, picolyl, pyridine ethyl, tetrahydrobenzene methyl, tetrahydrobenzene ethyl, cyclopentenes methyl, cyclopentenes ethyl;
Or R
1And R
2Form 1-pyrrolidyl, 4-morpholinyl, piperidino, 1-piperazinyl, 4-methylpiperazine base, 1-imidazolyl, 2-methyl isophthalic acid-imidazolyl, 1-triazol radical, 1-tetrazole base with the nitrogen-atoms that is connected with them;
R
3Be hydrogen, C
1-C
4Alkyl, C
3-C
7Cycloalkyl;
R
5Be selected from fluorine, chlorine, bromine, trifluoromethyl, trifluoromethoxy, nitro, cyano group, amino, methylene radical dioxy base, amino-sulfonyl, formamyl, two (C
1-C
4Alkyl) amido of Qu Daiing.
P is 1 or 2;
Q is the integer between 1～4.
And according to some usual methods in field under the present invention, the pyrazolo of following formula I of the present invention [1,5-a] pyridine derivatives can generate its pharmaceutically acceptable salt with acid.Acid can comprise mineral acid or organic acid, and the salt that forms with following acid is particularly preferred: hydrochloric acid, Hydrogen bromide, sulfuric acid, phosphoric acid, methylsulfonic acid, ethyl sulfonic acid, toluenesulphonic acids, Phenylsulfonic acid, naphthalene disulfonic acid, acetate, propionic acid, lactic acid, trifluoroacetic acid, toxilic acid, citric acid, fumaric acid, oxalic acid, Tartaric acid, Phenylsulfonic acid, phenylformic acid or tosic acid etc.
Derivative of the present invention can exist with stereoisomer form, and these stereoisomeric forms in any ratio can be enantiomorph or diastereomer.The present invention had both related to enantiomorph or diastereomer, also related to their mixtures separately, as diastereomer, can racemic form be separated into stereomeric one-component according to self known method.
In addition, the present invention also comprises the prodrug of derivative of the present invention.According to the present invention, prodrug is the derivative of general formula I, they self may have more weak active or even do not have activity, but after administration, (for example by metabolism, solvolysis or other mode) is converted to corresponding biologically active form under physiological condition.
Unless otherwise noted, term used herein " halo " is meant fluorine, chlorine, bromine or iodine generation; " alkyl " is meant the alkyl of straight or branched; " alkylidene group " is meant the alkylidene group of straight or branched; " cycloalkyl " is meant and replaces or unsubstituted cycloalkyl; Heteroaryl comprises the heteroatoms that contains one or more O of being selected from, N and S, wherein the ring-type system of each heteroaryl can be monocycle or polycyclic, the ring-type system is an aromaticity, can enumerate for example imidazolyl, pyridyl, pyrimidyl, pyrazolyl, (1,2,3)-and (1,2,4)-triazolyl, pyrazinyl, tetrazyl, furyl, thienyl, isoxazolyl, oxazolyl, pyrazolyl, pyrryl, thiazolyl, benzothienyl, benzofuryl, benzimidazolyl-, benzothiazolyl, indyl, quinolyl etc.; Saturated heterocyclyl comprises the heteroatoms that contains one or more O of being selected from, N and S, and the ring-type system can be monocycle or polycyclic, can enumerate for example pyrrolidyl, morpholinyl, piperazinyl, piperidyl, pyrazolidyl, imidazolidyl and thiazolinyl etc.
Specific derivatives of the present invention can have asymmetric center, and therefore the form with different enantiomorphs and diastereomer exists.The present invention relates to all optically active isomers, raceme of derivative of the present invention and composition thereof." raceme " is meant the mixture of a pair of enantiomer that contains equivalent.
The present invention includes pharmaceutical composition, said composition contains pyrazolo [1, the 5-a] pyridine derivatives of following formula I, and optically active body and its pharmaceutically acceptable salt, hydrate or as activeconstituents, and pharmaceutically acceptable excipient.Described pharmaceutically acceptable excipient is meant any thinner, auxiliary and/or carrier that can be used for pharmaceutical field.Derivative of the present invention can be used in combination with other activeconstituentss, as long as they do not produce other disadvantageous effect, for example anaphylaxis.
Pharmaceutical composition of the present invention can be mixed with some kinds of formulations, wherein contains some vehicle commonly used in the pharmaceutical field; For example, oral preparations (as tablet, capsule, solution or suspension); Injectable preparation (as injectable solution or suspension, or injectable dried powder, adding water for injection before injection can use immediately); Topical formulations (for example ointment or solution).
The carrier that is used for pharmaceutical composition of the present invention is the available common type of pharmaceutical field, comprising: the tackiness agent that oral preparations is used, lubricant, disintegrating agent, solubility promoter, thinner, stablizer, suspension agent, non-pigment, correctives etc.; The sanitas that injectable formulation is used, solubilizing agent, stablizer etc.; The matrix that topical formulations is used, thinner, lubricant, sanitas etc.Pharmaceutical preparation can oral administration or parenteral mode (for example intravenously, subcutaneous, intraperitoneal or part) administration, if some drugs is unsettled under the stomach condition, it can be mixed with enteric coated tablets.
Pass through antitumor activity in vitro, we find that The compounds of this invention has anti-tumor activity, therefore The compounds of this invention can be used to prepare the medicine that treats and/or prevents various cancers, as the cancer of mammary gland, lung, colon, rectum, stomach, prostate gland, bladder, uterus, pancreas and ovary.
Derivative according to the present invention can be used as activeconstituents and is used for preparation and treats and/or prevents various cancers, the present invention also provides treatment or prevents the method for above-mentioned disease, comprise suffer from or easily suffer from this sick patient significant quantity according to derivative of the present invention.The clinical dosage that the pyrazolo of following formula I [1,5-a] pyridine derivatives is used for the patient must rely on the main body of being treated, administration concrete approach, treat severity of disease and is changed, and optimal dose is definite by the concrete patient's of treatment doctor.
Active compound of the present invention can be used as unique cancer therapy drug and uses, and perhaps can unite use with one or more other antitumor drugs.Combination therapy realizes by each being treated component while, order or separating administration.
The compounds of this invention and preparation method thereof is further illustrated and illustrated to embodiment that hereinafter provides and preparation example.The scope that should be appreciated that following embodiment and preparation example also limits the scope of the invention never in any form.In the following embodiments, except as otherwise noted, otherwise the molecule with a chiral centre exists with the form of racemic mixture.Except as otherwise noted, otherwise the molecule with two or more chiral centres is a racemic mixture as diastereomer exists.Independent enantiomorph/diastereomer can obtain by method known to those skilled in the art.
Below synthetic route the preparation of formula I derivative of the present invention has been described, all raw materials all are the method preparation known by the method for describing in these synoptic diagram, by the organic chemistry filed those of ordinary skill or commercially available.All final derivative of the present invention all is to prepare by the method for describing in these synoptic diagram or by method similar with it, and these methods are that the organic chemistry filed those of ordinary skill is known.Whole variable factors of using in these synoptic diagram are as hereinafter definition or as the definition in the claim.
Route 1
According to formula I derivative of the present invention, in route 1, X be direct key ,-O-(CH
2)
p-,
-S-, other each substituting group such as summary of the invention part definition.
When X is-SO-, or-SO
2In-time, promptly the method according to route 1 at first makes formula II compound,, adopt suitable oxygenant (Sodium peroxoborate, dioxygen employing sodium wolframate and hydrogen peroxide are adopted in single oxidation) to carry out oxidizing reaction then and make target product (route 2).
Route 2
Embodiment:
Embodiment is intended to set forth rather than limit the scope of the invention.The proton nmr spectra of derivative is measured with Bruker ARX-300, and mass spectrum is measured with Agilent1100LC/MSD; Agents useful for same is analytical pure or chemical pure.
Embodiment 1:3-cyano group-5-[(4-methyl isophthalic acid-piperazinyl) methyl]-the 7-[(4-Trifluoromethoxyphen-l) the also preparation of [1,5-a] pyrimidine of amidopyrazole
Steps A: the preparation of ethoxymethylidene base propane dinitrile
132g (2.0mol) propane dinitrile and 440g (4.32mol) diacetyl oxide are added in the three-necked bottle of 1L, are heated to 110 ℃ of reactions after 45 minutes, drip 304g (2.1mol) triethyl orthoformate, use 40 minutes approximately.This reaction is thermopositive reaction, and the speed that control drips makes temperature remain on 108-120 ℃.Begin air distillation after the dropping triethyl orthoformate finishes, receive cut with being furnished with the container that stirs and the mixed solution of 68mL ammoniacal liquor (1.0mol) and 132mL water is housed, temperature surpasses 115 ℃ in reaction vessel, uses 1.5 hours approximately.Decompression (〉 100mm gradually then), continue distillation 1 hour temperature to the reaction vessel and be raised to 125 ℃.Distilling the initial stage, receiving bottle has the ammonium salt solid wall built-up, and liquid is divided into two-phase.Along with acetic acid amount in the distillate increases relatively, ammonium salt dissolves gradually, and distillation final stage liquid also becomes homogeneous phase.
The preparation of step B 3-amino-4-cyano pyrazole
In the 1L three-necked bottle, ethoxymethylidene base propane dinitrile is heated to 65 ℃, is dripping the 400mL Virahol in half an hour approximately then.In the dropping process, temperature will remain 65 ℃ to avoid crystallization to solidify, and the temperature of mixture also should not surpass 65 ℃, because Virahol and ethoxymethylidene base propane dinitrile mix the meeting heat release, the too high meeting of temperature causes product yield low.Make solution be cooled to 10 ℃ after dropwising, drip 112g (2.24mol) hydrazine hydrate again, bathe cooling outside and dripped about 2.5 hours down, obtain soup compound.Be heated to 20 ℃ of reactions 1 hour,,, steam Virahol 60 ℃-63 ℃ decompressions again after the mixture heating up to 65 ℃ then 35 ℃ of reactions 1 hour.Follow per 20 minutes and drip a 50mL water, Dropwise 35 0mL water needs 2.5 hours altogether, obtains the 440mL distillate at last.The liquid that obtains dark red filters and obtains beige solid 0 ℃-5 ℃ cooling 2 hours, cleans with 150mL cold water, and then stirs into pulpous state with 400mL cold water and refilter the cleaning of 150mL cold water.Vacuum-drying obtains the 134.5g solid, two step yields 62%.
Step C 3-cyano group-5-chloromethyl-7-hydroxypyrazoles is the preparation of [1,5-a] pyrimidine also
In the 250mL three-necked bottle, use the 100mL glacial acetic acid as solvent, add 10.8g (0.1mol) 3-amino-4-cyano group-pyrazoles and 23g (0.15mol) chloroacetyl acetacetic ester, the initial stage raw material does not dissolve, and is heated to 80 ℃ of left and right sides raw materials and dissolves substantially, back flow reaction 4 hours, there is solid to generate, with the reaction solution cool to room temperature, filters after reaction finishes, clean with Glacial acetic acid, seasoning obtains milk yellow solid 19.7g, yield 95%.
The preparation of step D 3-cyano group-5-chloromethyl-7-chlorine pyrazolo [1,5-a] pyrimidine
In having churned mechanically 1L three-necked bottle, add 3-cyano group-5-chloromethyl-7-chlorine pyrazolo [1,5-a] pyrimidine 82g (0.36mol) and 34g (0.43mol) pyridine, add 90g (0.59mol) POCl again
3, producing a large amount of smog, the raw material cure hard slowly heats with airbath, and when temperature reached 85 ℃, mixture was molten state, used mechanical stirring, 120 ℃ of reactions 1 hour, finished when reaction solution becomes shiny black soup compound reaction.When reaction solution is cooled to 60 ℃, adding the 500mL chloroform stirred 1 hour, add 300mL cold water after being cooled to 0-5 ℃, cross and filter out a small amount of insoluble solids, separatory cleans chloroform layer until neutrality with 300mL cold water again, chloroform is taken off in underpressure distillation, seasoning obtains khaki color solid 70g, yield 78%.
Step e 3-cyano group-5-chloromethyl-7-[(4-Trifluoromethoxyphen-l) amido | azoles is the preparation of [1,5-a] pyrimidine also
Make solvent with the 50mL Virahol, in the 100L three-necked bottle, add 8g (0.035mol) 3-cyano group-5-chloromethyl-7-chlorine pyrazolo [1,5-a] the 3-chloro-4-fluoroaniline of pyrimidine and 7.9g (0.045mol), be heated to 60 ℃, the beginning raw material does not dissolve, along with the rising raw material of temperature dissolves gradually, reaction has solid to generate after half hour, and afterreaction finished in 2 hours.The reaction solution cool to room temperature filters, and solid cleans twice with cold Virahol, and seasoning obtains white solid 10.7g, yield 84%.
Step F 3-cyano group-5-[(4-methyl isophthalic acid-piperazinyl) methyl]-the 7-[(4-Trifluoromethoxyphen-l) amido] preparation of pyrazolo [1,5-a] pyrimidine
With 3-cyano group-5-chloromethyl-7-[(4-Trifluoromethoxyphen-l) amido] pyrazolo [1,5-a] pyrimidine 1g (0.0027mol) and 0.8g (0.006mol) K
2CO
3And 0.6g (0.006mol) N methyl piperazine joins in the 50mL three-necked bottle that 20mL DMF is housed together, be heated to 50 ℃ in the oil bath, afterreaction finished in 1 hour, and cooling is poured reaction solution in the 250mL beaker that 100mL is housed into, the adularescent solid is separated out, filter with B, clean three times seasoning then again with 10mL cold water, obtain solid 1g, yield 85%.
MS：(M+H)432.
1H-NMR(DMSO，δ(ppm))：2.13(s，3H)，2.26(t，4H)，2.49(t，4H)，3.53(s，2H)，6.62(s，1H)，7.50(d，2H)，7.58(d，2H)，8.73(s，1H)，10.53(s，1H).
According to the preparation method of embodiment 1, select suitable raw material, make embodiment 2-embodiment 28 compounds.
Embodiment 2 3-cyano group-5-[(1-Pyrrolidine base) methyl]-the 7-[(4-trifluoromethyl) amido] preparation of pyrazolo [1,5-a] pyrimidine
MS：(M+H)387.
1H-NMR(DMSO，δ(ppm))：1.69(3，4H)，2.50(t，4H)，3.68(s，2H)，6.74(s，1H)，7.65(d，2H)，7.82(d，2H)，8.73(s，1H)，10.67(s，1H).
Embodiment 3 3-cyano group-5-[(N, N-dimethyl amido) methyl]-the 7-[(4-Trifluoromethoxyphen-l) amido] preparation of pyrazolo [1,5-a] pyrimidine
MS：(M+H)377.
1H-NMR(DMSO，δ(ppm))：2.18(s，6H)，3.45(s，2H)，6.66(s，1H)，7.50(d，2H)，7.50(d，2H)，8.60(s，1H)，10.53(s，1H).
Embodiment 4 3-cyano group-5-[(N-methyl-N-ethyl amido) methyl]-the 7-[(3-chloro-phenyl-) amido] preparation of pyrazolo [1,5-a] pyrimidine oxalate
MS：(M+H)341.
1H-NMR(DMSO，δ(ppm))：1.17(t，3H)，2.50(s，3H)，2.94(q，2H)，4.15(s，2H)，6.75(s，1H)，7.41(d，1H)，7.47(d，1H)，7.54(d，1H)，7.57(s，1H)，8.82(s，1H)，10.47(s，1H).
Embodiment 5 3-cyano group-5-[(1-Pyrrolidine) methyl]-the 7-[(2-fluorophenyl) amido] preparation of pyrazolo [1,5-a] pyrimidine oxalate
MS：(M+H)337.
1H-NMR(DMSO，δ(ppm))：1.90(3，4H)，3.09(t，4H)，4.27(s，2H)，6.28(s，1H)，7.36(d，1H)，7.45(1H)，7.48(1H)，7.53(1H)，8.83(s，1H)，10.57(s，1H).
Embodiment 6 3-cyano group-5-[(1-piperidyl) methyl]-7-[[3, two (trifluoromethyl) phenyl of 5-] amido] preparation of pyrazolo [1,5-a] pyrimidine
MS：(M+H)469.
1H-NMR(DMSO，δ(ppm))：1.36(m，2H)，1.42(m，4H)，2.36(t，4H)，3.27(s，2H)，6.22(s，1H)，7.48(s，1H)，7.56(s，2H)，8.21(s，1H).
Embodiment 7 3-cyano group-5-[(1-piperidyl) methyl]-the 7-[(4-trifluoromethyl) amido] preparation of pyrazolo [1,5-a] pyrimidine oxalate
MS：(M+H)351.
1H-NMR(DMSO，δ(ppm))：1.45(m，2H)，1.59(3，4H)，2.82(t，4H)，3.99(s，2H)，6.33(s，1H)，7.37(t，1H)，7.50(m，3H)，8.81(s，1H)，10.70(s，1H).
Embodiment 8 3-cyano group-5-[(N-methyl-N-propyl group amido) methyl]-the 7-[(3-fluorophenyl) amido] preparation of pyrazolo [1,5-a] pyrimidine oxalate
MS：(M+H)339.
1H-NMR(DMSO，δ(ppm))：0.88(t，3H)，162(q，2H)2.60(s，3H)，2.83(q，2H)，4.13(s，2H)，6.81(s，1H)，7.16(t，1H)，7.35(m，2H)，7.55(q，1H)，8.82(s，1H)，10.47(s，1H).
Embodiment 9 3-cyano group-5-[[N-methyl-N-(2-hydroxyethyl) amido] methyl]-the 7-[(3-fluorophenyl) amido] preparation of pyrazolo [1,5-a] pyrimidine
MS：(M+H)341.
H
1-NMR(DMSO，δ(ppm))：2.25(s，3H)，2.49(t，2H)，3.47(t，2H)，3.61(s，2H)，4.20(s，1H)，6.81(s，1H)，7.16(t，1H)，7.35(m，2H)，7.53(q，1H)，8.74(s，1H)，10.51(s，1H).
Embodiment 10 3-cyano group-5-[(1-Pyrrolidine base) methyl]-the 7-[(4-p-methoxy-phenyl) methylamino] preparation of pyrazolo [1,5-a] pyrimidine
MS：(M+H)363
1H-NMR(DMSO，δ(ppm))：1.75(3，4H)，2.66(t，4H)，3.71(s，3H)，3.86(s，2H)，4.57(d，2H)，6.52(s，1H)，6.90(d，2H)，7.35(d，2H)，8.66(s，1H)，9.18(t，1H).
Embodiment 11 3-cyano group-5-[(4-methyl isophthalic acid-piperazinyl) methyl]-the 7-[(4-p-methoxy-phenyl) methylamino] preparation of pyrazolo [1,5-a] pyrimidine
MS：(M+H)392.
1H-NMR(DMSO，δ(ppm))：2.16(s，3H)，2.24(t，4H)，2.32(t，4H)，3.46(s，2H)，3.72(s，3H)，4.57(d，2H)6.38(s，1H)，6.90(d，2H)，7.30(d，2H)，8.63(s，1H)，9.12(t，1H).
Embodiment 12 3-cyano group-5-[(N, N-dimethyl amido) methyl]-the 7-[(4-p-methoxy-phenyl) methylamino] preparation of pyrazolo [1,5-a] pyrimidine
MS：(M+H)337，(2M+Na
+)695.
1H-NMR(DMSO，δ(ppm))：2.15(s，6H)，3.43(s，2H)，3.71(s，3H)，4.57(d，2H)，6.43(s，1H)，6.90(d，2H)，7.32(d，2H)，8.63(s，1H)，9.04(t，1H).
Embodiment 13 3-cyano group-5-[[N-methyl-N-(2-hydroxyethyl) amido] methyl]-the 7-[(4-p-methoxy-phenyl) methylamino] preparation of pyrazolo [1,5-a] pyrimidine
MS：(M+H)367.
1H-NMR(DMSO，δ(ppm))：2.20(s，3H)，2.46(t，2H)，3.48(m，2H)，3.56(s，2H)，3.71(s，3H)，4.25(t，1H)，4.55(d，2H)，6.59(s，1H)，6.90(d，2H)，7.32(d，2H)，8.62(s，1H)，9.04(t，1H).
Embodiment 14 3-cyano group-5-[[2-(1-cyclohexenyl) ethylamino-] methyl]-the 7-[(3-trifluoromethyl) amido] preparation of pyrazolo [1,5-a] pyrimidine oxalate
MS：(M+H)441.
Embodiment 153-cyano group-5-[2-(4-morpholinyl) ethylamino-] methyl-7-[(4-trifluoromethyl) amido] preparation of pyrazolo [1,5-a] pyrimidine oxalate
MS：(M+H)446.
Embodiment 163-cyano group-5-[[3-(2-methyl isophthalic acid-piperidyl) Propylamino] methyl]-the 7-[(4-trifluoromethyl) amido] preparation of pyrazolo [1,5-a] pyrimidine
MS：(M+H)472.
Embodiment 173-cyano group-5-[(2-furyl methylamino) methyl]-the 7-[(4-trifluoromethyl) amido] preparation of pyrazolo [1,5-a] pyrimidine
MS：(M+H)413.
Embodiment 183-cyano group-5-(amylamine ylmethyl)-7-[(4-trifluoromethyl) amido] preparation of pyrazolo [1,5-a] pyrimidine
MS：(M+H)403
Embodiment 193-cyano group-5-[(3-isopropoxy Propylamino) methyl]-the 7-[(4-fluorophenyl) amido] preparation of pyrazolo [1,5-a] pyrimidine
MS：(M+H)383.
Embodiment 203-cyano group-5-[[3-(4-morpholinyl) Propylamino] methyl]-7-[(3, the 5-difluorophenyl) amido] preparation of pyrazolo [1,5-a] pyrimidine
MS：(M+H)428.
Embodiment 21 3-cyano group-5-[[(1-phenmethyl-4-piperidyl) methyl amido)]-the 7-[(3-trifluoromethyl) amido] preparation of pyrazolo [1,5-a] pyrimidine
MS：(M+H)506.
Embodiment 223-cyano group-5-[((2-furfuryl) methyl amido)]-the 7-[(3-trifluoromethyl) amido] preparation of pyrazolo [1,5-a] pyrimidine oxalate
MS：(M+H)413.
Embodiment 233-cyano group-5-[[(2-(1-cyclohexenyl) ethylamino-] methyl]-the 7-[(3-trifluoromethyl) amido] preparation of pyrazolo [1,5-a] pyrimidine oxalate
MS：(M+H)441.
Embodiment 243-cyano group-5-[[3-(methylphenylamine base) Propylamino] methyl]-the 7-[(4-trifluoromethyl) amido] preparation of pyrazolo [1,5-a] pyrimidine oxalate
MS：(M+H)480.
Embodiment 253-cyano group-5-[[(4-cyclohexyl amido) butylamine base] methyl]-the 7-[(4-fluorophenyl) amido] preparation of pyrazolo [1,5-a] pyrimidine
MS：(M+H)436.
Embodiment 263-cyano group-5-[(3-butoxy propyl amine base) methyl]-7-[(3, the 5-difluorophenyl) amido] preparation of pyrazolo [1,5-a] pyrimidine
MS：(M+H)415.
Embodiment 273-cyano group-5-[[(3-pyridyl) methylamino] methyl]-the 7-[(3-trifluoromethyl) amido] preparation of pyrazolo [1,5-a] pyrimidine
MS：(M+H)424.
Embodiment 283-cyano group-5-[(cyclohexyl methyl amido) methyl]-the 7-[(3-trifluoromethyl) amido] preparation of pyrazolo [1,5-a] pyrimidine oxalate
MS：(M+H)429.
Embodiment 293-cyano group-5-[[2-(4-morpholinyl) oxyethyl group] methyl]-the 7-[(3-trifluoromethyl) amido] pyrazolo [1,5-a] pyrimidine
Preparation method steps A～E according to embodiment 2 compounds prepares 3-cyano group-5-chloromethyl-7-(3-trifluoromethylbenzene amido) pyrazolo [1,5-a] pyrimidine, makes target product then in accordance with the following methods.
2-(4-morpholinyl) ethanol of exsiccant 20mL DMF and 0.6g (0.0062mol) is joined in the 50mL three-necked bottle, add 60% NaH0.3g (0.0062mol) then, stir, heat release and have bubble and produce.Be warmed up to 35 ℃, restir half an hour add 3-cyano group-5-chloromethyl-7-(3-trifluoromethylbenzene amido) pyrazolo [1,5-a] pyrimidine 1g (0.00284mol), keep 2 hours afterreactions of this temperature to finish.Reaction solution is poured in the 250mL beaker that 150mL water is housed, had solid to produce, leave standstill half a day, filter, clean with 20mL water, seasoning obtains white solid 1.1g, yield 86%.
MS：(M+H).
H
1-NMR(DMSO，δ(ppm))：2.32(t，4H)，2.46(t，2H)，3.43(t，4H)，3.61(t，2H)，4.54(s，1H)，6.58(s，1H)，7.16(t，2H)，7.35(t，1H)，7.53(s，1H)，8.77(s，1H)，10.71(s，1H).
Preparation method according to embodiment 29 makes embodiment 30～36 compounds.
Embodiment 30 3-cyano group-5-[[2-(4-morpholinyl) oxyethyl group] methyl]-7-[(3, the 4-dichlorophenyl) amido] preparation of pyrazolo [1,5-a] pyrimidine oxalate
MS：(M+H)447.
1H-NMR(DMSO，δ(ppm))：2.89(t，4H)，2.99(t，2H)，3.66(t，4H)，3.76(t，2H)，4.59(s，1H)，6.60(s，1H)，7.55(dd，1H)，7.77(m，2H)，8.78(s，1H)，10.51(s，1H).
Embodiment 31 3-cyano group-5-[[2-(N, N-diethyl amido) oxyethyl group] methyl]-the 7-[(2-fluorophenyl) amido] preparation of pyrazolo [1,5-a] pyrimidine oxalate
MS：(M+Na)383.
1H-NMR(DMSO，δ(ppm))：1.11(m，6H)，3.10(q，4H)，3.27(t，2H)，3.82(t，2H)，4.60(s，1H)，6.31(d，1H)，7.53(m，4H)，8.79(s，1H)，10.51(s，1H).
Embodiment 32 3-cyano group-5-[[2-(piperidino) oxyethyl groups] methyl]-the 7-[(3-chloro-phenyl-) amido] preparation of pyrazolo [1,5-a] pyrimidine oxalate
MS：(M+H)411.
1H-NMR(DMSO，δ(ppm))：1.49(m，2H)，1.68(m，4H)，3.11(t，4H)，3.22(t，2H)，3.84(t，4H，)，4.60(s，1H)6.57(s，1H)，7.55(dd，1H，)，7.77(m，2H)，8.78(s，1H)，10.51(s，1H).
Embodiment 33 3-cyano group-5-[2-[(N, N-diethyl amido) oxyethyl group] methyl]-7-[(3-fluoro-4-bromophenyl) amido] preparation of pyrazolo [1,5-a] pyrimidine oxalate
MS：(M+H)461.
1H-NMR(DMSO，δ(ppm))：1.11(m，6H)，3.11(q，4H)，3.28(t，2H)，3.83(t，2H)，4.62(s，1H)，6.64(s，1H)，7.30(d，1H)，7.53(d，1H)，7.78(m，4H)，8.79(s，1H)，10.36(s，1H).
Embodiment 343-cyano group-5-[[2-(1-Pyrrolidine base) oxyethyl group] methyl]-7-[(3,4-methylenedioxyphenyl base) amido] preparation of pyrazolo [1,5-a] pyrimidine oxalate
MS：(M+H)407.
Embodiment 35 3-cyano group-5-[[2-(4-morpholinyl) oxyethyl group] methyl]-7-[(3,4-methylenedioxyphenyl base) amido] preparation of pyrazolo [1,5-a] pyrimidine oxalate
MS：(M+H)423.
Embodiment 363-cyano group-5-[(3-octyloxy Propylamino) methyl]-the 7-[(4-fluorophenyl) amido] preparation of pyrazolo [1,5-a] pyrimidine
MS：(M+H)453.
Embodiment 37 3-cyano group-5-(4-hydroxyl) benzene thiomethyl-7-[(3,5-difluorophenyl) amido] preparation of pyrazolo [1,5-a] pyrimidine
1.2g (0.0094mol) thiophenol and 0.3g (0.007mol) NaOH are equipped with in the 30mL alcoholic acid 50mL three-necked bottle in the room temperature adding, stir half an hour, add 3-cyano group-5-chloromethyl-7-(3 again, 5-difluoroaniline base) pyrazolo [1,5-a] pyrimidine 1.5g reaction, afterreaction finished in 4 hours, reaction solution was poured in the 250mL beaker that 150mL water is housed, separate out white solid, left standstill 5 hours, and filtered, clean with 20mL water, obtain the 1.9g light yellow solid, yield 98%.
MS：(M+H)410.
1H-NMR(DMSO，δ(ppm))：4.12(s，2H)，6.60(s，1H)，6.66(d，2H)，7.15(m，3H)，7.23(d，2H)，8.77(s，1H)，9.58(s，1H)，10.71(s，1H).
Preparation method according to embodiment 37 makes embodiment 38～39 compounds.
Embodiment 38 3-cyano group-5-[(4-hydroxy phenyl) thiomethyl]-7-[(3-fluoro-4-bromophenyl) amido] preparation of pyrazolo [1,5-a] pyrimidine
MS：(M+Na)492.
1H-NMR(DMSO，δ(ppm))：4.07(s，2H)，6.39(s，1H)，6.70(d，2H)，7.02(dd，1H)，7.22(d，2H)，7.35(dd，1H)，7.75(t，1H)，8.75(s，1H)，9.66(s，1H)，10.58(s，1H).
Embodiment 39 3-cyano group-5-[(4-hydroxy phenyl) thiomethyl]-the 7-[(4-Trifluoromethoxyphen-l) amido] preparation of pyrazolo [1,5-a] pyrimidine
MS：(M+H)480.
1H-NMR(DMSO，δ(ppm))：4.04(s，2H)，6.16(s，1H)，6.74(d，2H)，7.22(d，2H)，7.32(d，2H)，7.44(d，2H)，8.74(s，1H)，9.72(s，1H)，10.52(s，1H).
Embodiment 40 3-cyano group-5-[(4-hydroxy phenyl) sulfinyl methyl]-7-[(3, the 5-difluorophenyl) amido] preparation of pyrazolo [1,5-a] pyrimidine
With 3-cyano group-5-[(4-hydroxy phenyl) thiomethyl]-7-[(3, the 5-difluorophenyl) amido] pyrazolo [1,5-a] pyrimidine 0.4g (0.00098mol) is as raw material, join in the three-necked bottle of the 100mL that the 30mL Glacial acetic acid is housed, add 0.16g (0.00102mol) Sodium peroxoborate again, raw material does not dissolve after being warmed up to 50 ℃, along with the carrying out of reaction, raw material dissolves gradually, solution clarification after 4 hours, reaction finishes, join in the 250mL beaker that 150mL water is housed, separate out solid, leave standstill filtration, obtain 0.3g white solid, yield 72% with the cleaning of 20mL water.
MS：(M+Na
+)448.
H
1-NMR(DMSO，δ(ppm))：4.32(d，2H)，6.66(s，1H)，6.88(d，2H)，7.17(m，3H)，7.48(d，2H)，8.79(s，1H)，10.08(s，1H)，10.72(s，1H).
Embodiment 41 3-cyano group-5-[(4-hydroxy phenyl) sulfonymethyl]-7-[(3, the 5-difluorophenyl) amido] preparation of pyrazolo [1,5-a] pyrimidine
Join in the three-necked bottle of the 100mL that 30mL methyl alcohol is housed as raw material with 3-cyano group-5-(4-hydroxyl) benzene thiomethyl-7-(3,5-difluoroaniline base) pyrazolo [1,5-a] pyrimidine 0.4g (0.00098mol), add 30% H
2O
21.1g (0.00098mol) with the 0.08g sodium wolframate, the stirring at room material dissolution is separated out gradually along with reaction has solid, afterreaction finished in 6 hours, filtered, and used washed with methanol, obtained white solid 0.4g, yield 92%.
MS：(M+H)442.
1H-NMR(DMSO，δ(ppm))：4.79(s，2H)，6.68(s，1H)，6.87(d，2H)，7.22(m，3H)，7.59(d，2H)，8.79(s，1H)，10.57(s，1H)，10.78(s，1H).
Preparation method according to embodiment 41 makes embodiment 42 compounds.
Embodiment 423-cyano group-5-[(4-hydroxy phenyl) sulfonymethyl]-the 7-[(4-Trifluoromethoxyphen-l) amido] preparation of pyrazolo [1,5-a] pyrimidine
MS：(M+H)490.
1H-NMR(DMSO，δ(ppm))：4.74(s，2H)，6.27(s，1H)，6.90(d，2H)，7.37(d，2H)，7.47(d，2H)，7.60(d，2H)，8.78(s，1H)，10.66(s，1H).
The pharmacological research of product of the present invention
The anti tumor activity in vitro test
1) cell recovery
The careful cell (frozen pipe) that takes out all melts rapidly in 37～40 ℃ of water-baths from liquid nitrogen, makes cell cross 0～5 ℃ of very easily impaired temperature range rapidly.Put into centrifuge tube with liquid-transfering gun sucking-off cell under aseptic condition, centrifugal 3min under 1300r/min adds nutrient solution after the abandoning supernatant gently, and piping and druming mixing cell is put into CO2gas incubator in the immigration culturing bottle and cultivated, and changes liquid behind the 4h once.
2) passage
Need cultivating behind the cell recovery goes down to posterity treats that it can experimentize after stable for 2-3 time, goes down to posterity to stick with cell to be as the criterion bottom the culturing bottle at every turn.
3) cell buried plate
When sticking the culturing bottle bottom, the cell growth it is digested from the culturing bottle bottom with trypsin solution (0.25%).Cell dissociation buffer poured into then add nutrient solution in the centrifuge tube to stop digestion.With centrifuge tube centrifugal 3min under 1300r/min, add the 5mL nutrient solution gently after the abandoning supernatant, piping and druming mixing cell is drawn in the 10uL cell suspension adding cell counting count board and is counted, and adjusting cell concn is 10
4Individual/hole.Removing the A1 hole in 96 orifice plates is that blank well does not add the extracellular, and all the other all add the 100uL cell suspension.96 orifice plates are put into incubator cultivate 24h.
4) cell dosing
Earlier with 50 μ l DMSO dissolved substances.Then add an amount of nutrient solution, make medicine dissolution become the 2mg/mL soup.In 24 orifice plates, medicine dissolution is become 16,8 then, 4,2,1 μ g/mL.Each concentration adds 3 holes, and wherein two row, two row cell growing ways are affected by environment bigger on every side, only uses as blank cell hole.96 orifice plates are put into incubator cultivate 24h.
5) mtt assay measurement result
Will be in 96 orifice plates band medicine nutrient solution discard,, in every hole, add after MTT (0.5mg/mL) 100uL puts into incubator 4h cell flushing twice with PBS, discard MTT (tetrazole) solution, add DMSO100uL.Vibration is fully dissolved survivaling cell and MTT reaction product Jia Za on the magnetic force vibrator, puts into the microplate reader measurement result.Can obtain medicine IC by the Bliss method
50Value.
6) the inhibition oral carcinoma of compound and fibrosarcoma activity the results are shown in Table 1.
Table 1
| The embodiment sequence number | IC 50KB (cancer cell of oral cavity) (ug/mL) | IC 50HT-1080 (fibrosarcoma) (ug/mL) |
| Embodiment 1 | 7 | 5 |
| Embodiment 2 | 10 | 1l |
| Embodiment 5 | 8 | 6 |
| Embodiment 6 | 7 | 12 |
| Embodiment 12 | 8 | 14 |
| Embodiment 15 | 10 | 8 |
| Embodiment 18 | 11 | 14 |
| Embodiment 19 | 14 | 14 |
| Embodiment 20 | 2 | 8 |
| Embodiment 27 | 4 | 11 |
| Embodiment 29 | 6 | 7 |
| Embodiment 30 | 10 | 12 |
| Embodiment 31 | 4 | 14 |
| Embodiment 32 | 8 | 11 |
| Embodiment 33 | 9 | 8 |
| Cis-platinum | 9 | 7 |
Claims (8)
1. the compound of general formula I and pharmaceutically acceptable salt,
Wherein
Q
1And Q
2Identical or different, be independently selected from hydrogen, C respectively
1-C
10Alkyl, C
3-C
7Cycloalkyl, C
2-C
10Thiazolinyl and C
2-C
10Alkynyl can be by 1～3 identical or different R
4The optional replacement;
Or Q
1And Q
2Form 5-10 unit heterocyclic radical with the nitrogen-atoms that is connected with them, described heterocyclic radical contains 1-4 heteroatoms that is selected from N, O and S, chooses wantonly to comprise 1 or 2 carbon-carbon double bond or three key, and described heterocyclic radical can be by 1～3 identical or different R
4The optional replacement;
Perhaps Q
1And Q
2One of them is H, and another is-(CH
2)
m-Ar
1
When X be direct key ,-O-(CH
2)
pIn-time, Y is
When X is
The time, Y is
Or-OR
3
When X is
The time, Y is Ar
2
Ar
1Be C
6-C
10Aryl, 5-10 unit heteroaryl, wherein, described heteroaryl contains 1-4 heteroatoms that is selected from N, O or S, and Ar
1Can be by 1-3 identical or different R
5The optional replacement;
Ar
2Be C
6-C
10Aryl, 5-10 unit heteroaryl, wherein, described heteroaryl contains 1-4 heteroatoms that is selected from N, O or S, and Ar
2Can be by 1-3 identical or different R
6The optional replacement;
R
1And R
2Identical or different, be independently selected from hydrogen, C respectively
1-C
10Alkyl, C
3-C
7Cycloalkyl, C
2-C
10Thiazolinyl, C
2-C
10Alkynyl, C
6-C
10Aryl, C
6-C
10Aryl, C
1-C
4Alkyl, 5-10 unit heterocyclic radical, the heterocyclic radical C of 5-10 unit
1-C
4Alkyl, 5-10 unit heteroaryl, the heteroaryl C of 5-10 unit
1-C
4Alkyl can be by 1～3 identical or different R
7The optional replacement; Described cycloalkyl can comprise 1～2 carbon-carbon double bond or three key, and described heterocyclic radical and heteroaryl contain 1-4 heteroatoms that is selected from N, O or S; R
1And R
2Can be by 1～3 identical or different R
7The optional replacement;
Or R
1And R
2Form 5-10 unit's heterocyclic radical or 5-10 unit heteroaryl with the nitrogen-atoms that is connected with them, described heterocyclic radical and heteroaryl contain 1-4 heteroatoms that is selected from N, O and S, choose wantonly to comprise 1 or 2 carbon-carbon double bond or three key, can be by 1～3 identical or different R
7The optional replacement;
R
3Be hydrogen, C
1-C
10Alkyl, C
3-C
7Cycloalkyl, C
6-C
10Aryl;
R
4, R
7, R
8Be independently selected from hydroxyl, halogen, nitro, cyano group, trifluoromethyl, trifluoromethoxy, C
1-C
4Alkyl, C
1-C
4Alkoxy methyl, N, N-two C
1-C
4Alkylamino, C
3-C
7Cycloalkyl, described cycloalkyl can comprise 1～2 carbon-carbon double bond or three key;
R
5, R
6Be independently selected from C
1-C
4Alkyl, C
1-C
4Alkoxyl group, hydroxyl, optional by hydroxyl, amino or halogenated C
1-C
4Alkyl or C
1-C
4Alkoxyl group, carboxyl, halo, C
1-C
4Alkyl acyl, nitro, cyano group, amino, C
1-C
6Alkylamidoalkyl, coverlet or two (C
1-C
4Alkyl) amido of Qu Daiing, C
1-C
4Alkoxy methyl, formamyl, N-C
1-C
4Alkyl-carbamoyl, N, N-two C
1-C
4Alkyl-carbamoyl, amino-sulfonyl, N-C
1-C
4Alkyl amino sulfonyl, N, N-two C
1-C
4Alkyl amino sulfonyl, C
1-C
3Alkylenedioxy group;
M is the integer between 0～4;
P, q are the integer between 1～4;
R is the integer between 0～2.
2. the compound of Formula I of claim 1 and pharmaceutically acceptable salt thereof,
Wherein
Q
1And Q
2One of them is H, and another is-(CH
2)
m-Ar
1
3. the compound of Formula I of claim 1 and pharmaceutically acceptable salt thereof,
Wherein
Q
1And Q
2One of them is H, and another is-(CH
2)
m-Ar
1
Ar
1For phenyl or by 1-3 identical or different R
5The optional phenyl that replaces;
Ar
2For phenyl or by 1-3 identical or different R
6The optional phenyl that replaces, perhaps for containing 1-4 heteroatomic 5-6 ring heteroaryl that is selected from N, O or S, described heteroaryl can be by 1-3 identical or different R
6The optional replacement;
R
1And R
2Identical or different, be independently selected from hydrogen, C respectively
1-C
10Alkyl, C
3-C
7Cycloalkyl, C
6-C
10Aryl, C
6-C
10Aryl C
1-C
4Alkyl, 5-10 unit heterocyclic radical, the heterocyclic radical C of 5-10 unit
1-C
4Alkyl, 5-10 unit heteroaryl, the heteroaryl C of 5-10 unit
1-C
4Alkyl, can be by 1～3 identical or different R
7The optional replacement; Described cycloalkyl can comprise 1～2 carbon-carbon double bond or three key, and described heterocyclic radical and heteroaryl contain 1-4 heteroatoms that is selected from N, O or S; R
1And R
2Can be by 1～3 identical or different R
7The optional replacement;
Or R
1And R
2Form 5-10 unit's heterocyclic radical or 5-10 unit heteroaryl with the nitrogen-atoms that is connected with them, described heterocyclic radical and heteroaryl contain 1-4 heteroatoms that is selected from N, O and S, choose wantonly to comprise 1 or 2 carbon-carbon double bond or three key, can be by 1～3 identical or different R
7The optional replacement;
R
3Be hydrogen, C
1-C
10Alkyl, C
3-C
7Cycloalkyl can be by 1～3 identical or different R
8The optional replacement;
R
7, R
8Be independently selected from hydroxyl, halogen, nitro, cyano group, trifluoromethyl, trifluoromethoxy, C
1-C
4Alkyl, C
1-C
4Alkoxy methyl, N, N-two C
1-C
4Alkylamino, C
3-C
7Cycloalkyl, described cycloalkyl can comprise 1～2 carbon-carbon double bond or three key;
R
5, R
6Be independently selected from C
1-C
4Alkyl, C
1-C
4Alkoxyl group, hydroxyl, optional by hydroxyl, amino or halogenated C
1-C
4Alkyl or C
1-C
4Alkoxyl group, carboxyl, halo, C
1-C
4Alkyl acyl, nitro, cyano group, amino, C
1-C
6Alkylamidoalkyl or coverlet or two (C
1-C
4Alkyl) amido of Qu Daiing; C
1-C
4Alkoxy methyl, formamyl, N-C
1-C
4Alkyl-carbamoyl, N, N-two C
1-C
4Alkyl-carbamoyl, amino-sulfonyl, N-C
1-C
4Alkyl amino sulfonyl, N, N-two C
1-C
4Alkyl amino sulfonyl, C
1-C
3Alkylenedioxy group;
M is the integer between 0～4;
P, q are the integer between 1～4;
R is the integer between 0～2.
4. the compound of Formula I of claim 1 and pharmaceutically acceptable salt,
Wherein
Q
1And Q
2One of them is H, and another is a phenyl or by 1-3 identical or different R
5The optional phenyl that replaces;
When X be direct key ,-O-(CH
2)
pIn-time, Y is
When X is
The time, Y is
Or-OR
3
R
1And R
2Identical or different, be independently selected from hydrogen, C respectively
1-C
6Alkyl, C
3-C
6Cycloalkyl, C
3-C
6Methyl cycloalkyl, C
3-C
6Cycloalkyl ethyl, phenyl, phenmethyl, styroyl, furfuryl, furans ethyl, picolyl, pyridine ethyl, tetrahydrobenzene methyl, tetrahydrobenzene ethyl, cyclopentenes methyl, cyclopentenes ethyl;
Or R
1And R
2Form 1-pyrrolidyl, 4-morpholinyl, piperidino, 1-piperazinyl, 4-methylpiperazine base, 1-imidazolyl, 2-methyl isophthalic acid-imidazolyl, 1-triazol radical, 1-tetrazole base with the nitrogen-atoms that is connected with them;
R
3Be hydrogen, C
1-C
10Alkyl, C
3-C
7Cycloalkyl;
R
5Be independently selected from C
1-C
4Alkyl, C
1-C
4Alkoxyl group, hydroxyl, optional by hydroxyl, amino or halogenated C
1-C
4Alkyl or C
1-C
4Alkoxyl group, carboxyl, halo, C
1-C
4Alkyl acyl, nitro, cyano group, amino, C
1-C
6Alkylamidoalkyl or coverlet or two (C
1-C
4Alkyl) amido of Qu Daiing; C
1-C
4Alkoxy methyl, formamyl, N-C
1-C
4Alkyl-carbamoyl, N, N-two C
1-C
4Alkyl-carbamoyl, amino-sulfonyl, N-C
1-C
4Alkyl amino sulfonyl, N, N-two C
1-C
4Alkyl amino sulfonyl, C
1-C
3Alkylenedioxy group;
P, q are the integer between 1～4.
5. the compound of Formula I of claim 1 and pharmaceutically acceptable salt thereof,
Wherein
Q
1And Q
2One of them is H, and another is a phenyl or by 1-3 identical or different R
5The optional phenyl that replaces;
When X be direct key ,-O-(CH
2)
pIn-time, Y is
When X is
The time, Y is
Or-OR
3
R
1And R
2Identical or different, be independently selected from hydrogen, C respectively
1-C
6Alkyl, C
3-C
6Cycloalkyl, C
3-C
6Methyl cycloalkyl, C
3-C
6Cycloalkyl ethyl, phenyl, phenmethyl, styroyl, furfuryl, furans ethyl, picolyl, pyridine ethyl, tetrahydrobenzene methyl, tetrahydrobenzene ethyl, cyclopentenes methyl, cyclopentenes ethyl;
Or R
1And R
2Form 1-pyrrolidyl, 4-morpholinyl, piperidino, 1-piperazinyl, 4-methylpiperazine base, 1-imidazolyl, 2-methyl isophthalic acid-imidazolyl, 1-triazol radical, 1-tetrazole base with the nitrogen-atoms that is connected with them;
R
3Be hydrogen, C
1-C
4Alkyl, C
3-C
7Cycloalkyl;
R
5Be independently selected from C
1-C
4Alkyl, C
1-C
4Alkoxyl group, hydroxyl, optional by hydroxyl, amino or halogenated C
1-C
4Alkyl or C
1-C
4Alkoxyl group, carboxyl, halo, C
1-C
4Alkyl acyl, nitro, cyano group, amino, C
1-C
6Alkylamidoalkyl or coverlet or two (C
1-C
4Alkyl) amido of Qu Daiing, C
1-C
4Alkoxy methyl, formamyl, N-C
1-C
4Alkyl-carbamoyl, N, N-two C
1-C
4Alkyl-carbamoyl, amino-sulfonyl, N-C
1-C
4Alkyl amino sulfonyl, N, N-two C
1-C
4Alkyl amino sulfonyl, C
1-C
3Alkylenedioxy group;
P is 1 or 2;
Q is the integer between 1～4.
6. the compound of Formula I of claim 1 and pharmaceutically acceptable salt thereof,
Wherein
Q
1And Q
2One of them is H, and another is a phenyl or by 1-3 identical or different R
5The optional phenyl that replaces;
When X be direct key ,-O-(CH
2)
pIn-time, Y is
When X is
The time, Y is
Or-OR
3
R
1And R
2Identical or different, be independently selected from hydrogen, C respectively
1-C
6Alkyl, C
3-C
6Cycloalkyl, C
3-C
6Methyl cycloalkyl, C
3-C
6Cycloalkyl ethyl, phenyl, phenmethyl, styroyl, furfuryl, furans ethyl, picolyl, pyridine ethyl, tetrahydrobenzene methyl, tetrahydrobenzene ethyl, cyclopentenes methyl, cyclopentenes ethyl;
Or R
1And R
2Form 1-pyrrolidyl, 4-morpholinyl, piperidino, 1-piperazinyl, 4-methylpiperazine base, 1-imidazolyl, 2-methyl isophthalic acid-imidazolyl, 1-triazol radical, 1-tetrazole base with the nitrogen-atoms that is connected with them;
R
3Be hydrogen, C
1-C
4Alkyl, C
3-C
7Cycloalkyl;
R
5Be selected from fluorine, chlorine, bromine, trifluoromethyl, trifluoromethoxy, nitro, cyano group, amino, methylene radical dioxy base, amino-sulfonyl, formamyl, two (C
1-C
4Alkyl) amido of Qu Daiing;
P is 1 or 2;
Q is the integer between 1～4.
7. medicinal compositions comprises any one compound and the pharmaceutically acceptable salt thereof as the claim 1-6 of activeconstituents, and pharmaceutically acceptable excipient.
8. each compound or its pharmaceutically acceptable salt of claim 1-6 treats and/or prevents application in oral carcinoma and the fibrosarcoma medicine in preparation.
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| MX2012007684A (en) | 2009-12-30 | 2012-10-05 | Avila Therapeutics Inc | Ligand-directed covalent modification of protein. |
| US8969363B2 (en) | 2011-07-19 | 2015-03-03 | Infinity Pharmaceuticals, Inc. | Heterocyclic compounds and uses thereof |
| CN102690270B (en) * | 2012-05-28 | 2014-02-05 | 苏州科捷生物医药有限公司 | Pyrimidine compound and purpose thereof |
| CN103626776B (en) * | 2013-12-05 | 2017-02-15 | 北京师范大学 | Pyrazolo[1,5-alpha] pyrimidine nitrogen mustard derivatives, preparation methods of pyrazolo[1,5-alpha] pyrimidine nitrogen mustard derivatives, and application of pyrazolo[1,5-alpha] pyrimidine nitrogen mustard derivatives in oncotherapy |
| CN108484613B (en) * | 2018-05-22 | 2020-07-07 | 江南大学 | Pyrazolo [1,5-a ] pyrimidine compound and application thereof |
| CN116761609A (en) * | 2021-01-22 | 2023-09-15 | 上海海雁医药科技有限公司 | Substituted pyrazolo [1,5-a ] pyrimidin-7-amine derivatives, compositions thereof and pharmaceutical uses thereof |
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Non-Patent Citations (4)
| Title |
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| Douglas SW,Martin J P ,J ustin FB , et al ..Structure-guided design of pyrazolo [ 1 , 5a ] pyrimidines as inhibitors of human cyclin-dependent kinase 2.《Bioorg.Med. Chem. Lett.》.2005,第15卷(第4期),863 - 867. |
| Douglas SW,Martin J P,J ustin FB, et al ..Structure-guided design of pyrazolo [ 1, 5a ] pyrimidines as inhibitors of human cyclin-dependent kinase 2.《Bioorg.Med. Chem. Lett.》.2005,第15卷(第4期),863- 867. * |
| Thomas N,Roland KR..Synthesis and antifungal properties of certain 7-alkylaminopyrazolo [ 1, 5a ] pyrimidines.《J. Med. Chem.》.1977,第20卷(第2期),296-299. * |
| ThomasN Roland KR..Synthesis and antifungal properties of certain 7-alkylaminopyrazolo [ 1 |
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