CN101245051B - 2,4-di-substituted amido-6-substituted-[1,3,5]triazine or miazines compound, preparation method, pharmaceutical combination and use of the same - Google Patents
2,4-di-substituted amido-6-substituted-[1,3,5]triazine or miazines compound, preparation method, pharmaceutical combination and use of the same Download PDFInfo
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- CN101245051B CN101245051B CN2006101700247A CN200610170024A CN101245051B CN 101245051 B CN101245051 B CN 101245051B CN 2006101700247 A CN2006101700247 A CN 2006101700247A CN 200610170024 A CN200610170024 A CN 200610170024A CN 101245051 B CN101245051 B CN 101245051B
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Abstract
The invention relates to a novel 2, 4-disubstituted amino-6-substituted-(1, 3, 5) triazine or a 1, 3-pyrimidine derivative, the preparation method, a drug combination and the pharmacological usage; the structure general formula is shown as the formula (I), wherein, the definitions of R1, R2, R3, R4, R5, R6, A, B, X, Y and Z are described in an instruction. The compounds and a HIV-1 integrase havehigh binding activity and can effectively inhibit the binding of the integrase with a substrate in a substrate competition test. Therefore, the compounds are stronger HIV-1 integrase inhibitors, which are expected to be developed into new anti-HIV drugs.
Description
Technical field
The present invention relates to pharmaceutical chemistry and pharmacotherapeutics field, be specifically related to 2,4-disubstituted amido-6-replacement-[1,3,5] triazine or 1,3-pyrimidines, its preparation method, the pharmaceutical composition that contains this compounds and purposes.
Background technology
From 1981 since the U.S. finds the first acquired immune deficiency syndrome (AIDS) AIDS (acquiredimmunodeficiency syndrome, acquired immunodeficiency disease disease) patient, acquired immune deficiency syndrome (AIDS) spreads fast in more than 180 countries and regions in the whole world.Present global AIDS patient and virus carrier's sum have reached more than 4,200 ten thousand, and this is state-owned nearly 1,000,000 in wherein, occupies the 2nd in the Asia, occupies the 14th in the whole world.And the China's AIDS case load has become an increasingly serious public safety problem increasing fast with 30～40% speed every year on average in recent years.
HIV virus is as the pathogenic agent of AIDS, and it infects behind human body in vivo reproduction process needs intergrase (integrase, participation IN) just can be finished.IN can finish the process that the HIV double chain DNA molecule is integrated into host chromosome.IN has the endonuclease activity and the chain transfer activity of 3 ' cutting in vivo.IN cuts two bases at the double-stranded two ends of the linear flush end DNA of HIV by 3 '-5 ' direction earlier, forms the recessed end of CA-OH-3 ', cuts the breach of a 5bp on the dna double chain of host cell; The recessed end of the CA-OH-3 ' of HIV dna double chain combines with the form of phosphodiester bond with the 5bp of host cell DNA chain otch under the effect of IN then, forms to integrate intermediate; In cell, remove viral DNA 5 ' terminal unpaired two bases again under the mechanism of action of some enzyme, repair the breach of virus and host cell DNA chain.
The protein of the molecular weight 32kD that IN is made up of 288 amino acid, it structurally is divided into three parts: N petiolarea, core area and C petiolarea.The N petiolarea is made up of 1～50 amino acids, is a conservative HHCC Zinc finger domain, has the viral DNA recognition function, promotes the function of IN four dimerizations and enhancing catalytic activity in addition in addition.Core area is made up of 52～210 amino acids, is the most important position of IN catalysis.It comprises three acidic amino acid residues (Asp64, Asp116 and Glu152), is arranged in the DD35E type, is very conservative structure, is the site of interior ribozyme and nucleotidyltransferase.This position and one or two divalent cation (Mg
2+Or Mn
2+) in conjunction with being essential to catalytic activity.The C petiolarea is the zone of conservative property minimum among the IN, is made up of 213～288 amino acids.It comprises nuclear localization signal, can be nonspecific in conjunction with DNA, also participate in the multimerization of IN.The crystalline structure in three districts of IN reaches and the compound crystal structure of various inhibitor all obtains, but the crystalline structure of IN total length does not still solve.
Because IN plays indispensable vital role in the HIV virus replication, and IN do not have the functional analogue of IN in human body cell, thereby IN is the desirable target of AIDS treatment.Seeking new IN inhibitor, in conjunction with hiv reverse transcriptase and proteinase inhibitor drug combination, can improve antiviral activity, reduce toxic side effect and reduce resistance, is treatment plan highly significant.
Up to the present the inhibitor of the IN of Fa Xianing comprises: DNA wedding agent, oligonucleotide and nucleoside analog, sulfuric ester analogue, polypeptide and hydroxyl substituted aromatics etc.Follow the binding site of IN can be divided into four classes according to these inhibitor.
1 nucleosides binding site
The nucleoside compound that is used as the research of IN inhibitor the earliest is AZT and derivative AZTMP thereof.The single phosphoric acid of AZT, bis phosphoric acid and triphosphoric acid all have anti-IN chain transfer activity.They are by disturbing IN to realize with combining of DNA to the inhibition of IN.The K156 of IN, K159 and K160 are the binding sites of monokaryon glycosides, and wherein the K159 of high conservative is IN and DNA bonded key.Find dinucleotide and different dinucleotide subsequently, also have the oligonucleotide compounds that the activity that suppresses IN is also arranged.
2 metal ion binding sites
Divalent-metal ion (the Mg of polyhydroxy fragrant compound and IN
2+Or Mn
2+) thereby chelating suppresses the activity of IN.This has and is in the news at most in the inhibiting compound of IN, lignanolide class, coumarin monomeric or dimer class, vinylbenzene quinoline is arranged and contain volution class anthraquinone class of pyrocatechol etc.This compounds has the good restraining effect to IN in external test, but too big in the cell cultures toxic.
3LTR (long terminal repetition) dna sequence dna binding site
This class inhibitor has been reported at present dna double spiral minor groove binding (as spindle mycin and distamycin), DNA intercalator (as Dx) and topoisomerase enzyme inhibitor (as the many anthraquinones of rice).
4 other sites
Merck ﹠ Co., Inc. had found the two kinds of diketone acid inhibitor that can contain IN in 2000, and this is that a unique so far class selectively acting is in the inhibitor of IN.This compounds is to the selective restraining effect of second step of integrating remark.In addition other inhibitor all may have a plurality of action sites simultaneously.
Although to the research in surplus the inhibitor of IN existing ten year, the IN inhibitor did not have the medicine listing at present, there only have minority to enter to be clinical.Trace it to its cause, mainly contain the restriction of two aspects: the one, the restriction of activity determination method: can't determine based on the mensuration of cell cultures whether compound directly acts on IN, and the screening of molecular level has too many false positive; The 2nd, the crystalline structure of the crystalline structure of the restriction of structure biology aspect: IN and DNA substrate complex thereof all do not have to determine, and integrates in this complicated biological process and also have many mechanism not understand.
At present the treatment of AIDS disease is mainly adopted the strategy of drug combination, promptly adopt hiv reverse transcriptase and proteinase inhibitor drug combination, but the toxic side effect situation of resistance that the HIV virus mutation causes in the therapeutic process and reversed transcriptive enzyme and proteinase inhibitor is serious.Therefore, seek and find that the HIV-1 integrase inhibitor becomes the new important theme of AIDS treatment.From natural product, find the new inhibitor of HIV-1 intergrase, have great ground-breaking meaning for the treatment of effective HIV-1 integrase inhibitor of further design synthesizing new and AIDS.
Summary of the invention
An object of the present invention is to provide and had the novel 2 of anti HIV-1 virus effect, 4-disubstituted amido-6-replacement-[1,3,5] triazine or 1, the 3-pyrimidines, its general structure is suc as formula shown in (I).
Another object of the present invention provides with 2,4,6-three chloro-[1,3,5] triazine or 1, and the 3-pyrimidine is 2 of a raw material synthesizing new, 4-disubstituted amido-6-replacement-[1,3,5] triazine or 1, the method for 3-pyrimidines.
A further object of the present invention provides and comprises 2,4-disubstituted amido-6-replacement-[1,3,5] triazine or 1, the pharmaceutical composition of the application of the anti HIV-1 virus aspect of 3-pyrimidines.
Compound involved in the present invention can be used as the micromolecular inhibitor of HIV-1 intergrase, and the HIV double chain DNA molecule that participates in by blocking-up IN is integrated into the process of host chromosome, thereby plays the effect that suppresses virus replication.Therefore, be expected to develop and become new anti HIV-1 virus medicine.
The invention provides and have 2 of following general formula (I) structure, 4-disubstituted amido-6-replacement-[1,3,5] triazine or 1,3-pyrimidines or its pharmacy acceptable salt:
Wherein:
X is selected from C or N.
Y is selected from O or NH.
A is selected from CH, CH
2, N or NH.
B is selected from CH, CH
2, N or NH.
Z represents the group of atom or atom.Wherein, described atom is carbon atom or nitrogen-atoms, and the group of described atom is alkoxyl group, sulfydryl, the acyl group of C1～C4 and the carbon atom that group replaced of aromatic base Ar of the straight or branched alkyl of the alkyl, the C1～C6 that are selected from hydrogen atom, halogen, halo C1～C3, cyano group, nitro, amino, hydroxyl, methylol, trifluoromethyl, trifluoromethoxy, carboxyl, C1～C4.
N is selected from 0 to 3 integer.
Dotted line is represented singly-bound or two key.
R
1Be selected from hydrogen, C
1～C
6Saturated or the unsaturated alkyl of straight or branched, C
3～C
7Cyclic hydrocarbon radical, alkyloyl RCO, aromaticacyl radical ArCO, alkane alkylsulfonyl RSO
2, fragrant alkylsulfonyl ArSO
2, benzyl, aromatic base Ar, 5～7 membered aromatic heterocycles (contain 1～3 heteroatoms that is selected from oxygen, sulphur, nitrogen, can and close by phenyl and aromatic heterocycle, or by one or more halogen, C of being selected from
1～C
6Straight or branched alkyl, cyano group, nitro, amino, hydroxyl, methylol, trifluoromethyl, trifluoromethoxy, carboxyl, C
1～C
4Alkoxyl group, sulfydryl, C
1～C
4The group of acyl group, aromatic base Ar replaces).
R
2Be selected from hydrogen, halogen, C
1～C
6Straight or branched alkyl, C
1～C
4Straight or branched alkoxyl group, cyano group, trifluoromethyl, trifluoromethoxy, R
1The Y-group.
R
3, R
4, R
5And R
6Be selected from hydrogen, C independently of one another
1～C
6Saturated or the unsaturated alkyl of straight or branched and alkoxyl group, C
3～C
7Cyclic hydrocarbon radical, benzyl, aromatic base Ar, 5～7 membered aromatic heterocycles (contain 1～3 heteroatoms that is selected from oxygen, sulphur, nitrogen, can and close by phenyl and aromatic heterocycle, or by one or more halogen, C of being selected from
1～C
6Straight or branched alkyl, cyano group, nitro, amino, hydroxyl, methylol, trifluoromethyl, trifluoromethoxy, carboxyl, C
1～C
4Alkoxyl group, sulfydryl, C
1～C
4The group of acyl group, aromatic base Ar replaces).
Aromatic base Ar can be a phenyl, (substituting group can be 1～4 and be selected from halogen, C substituted-phenyl
1～C
6Straight or branched alkyl, cyano group, nitro, amino, hydroxyl, methylol, trifluoromethyl, trifluoromethoxy, carboxyl, C
1～C
4Alkoxyl group, sulfydryl, C
1～C
4The group of acyl group), naphthyl, xenyl.
" pharmacy acceptable salt " described in this specification sheets can be enumerated and propionic acid particularly, oxalic acid, propanedioic acid, succsinic acid, fumaric acid, toxilic acid, lactic acid, oxysuccinic acid, tartrate, citric acid, Deng organic acid and aspartic acid, acidic amino acids such as L-glutamic acid form behind the ester salt that forms with mineral alkali again, as sodium, potassium, calcium, aluminium salt and ammonium salt, or the salt that forms with organic bases, as methylamine salt, ethylamine salt, ethanolamine salt etc., or and Methionin, arginine, basic aminoacidss such as ornithine form the hydrochloric acid behind the ester, Hydrogen bromide, hydrofluoric acid, sulfuric acid, nitric acid, the salt of mineral acids such as phosphoric acid, or and formic acid, acetate, picric acid, methylsulfonic acid, organic acid salt such as ethyl sulfonic acid.
A preferred embodiment of formula of the present invention (I) compound is following 2, and 4-disubstituted amido-6-replaces-[1,3,5] compound in triazine class or its pharmacy acceptable salt:
Wherein, X is N; Y is O;
A, B, Z, n, R
1, R
2, R
3, R
4, R
5And R
6Definition as mentioned above.
Another preferred embodiment of formula of the present invention (I) compound is following 2, and 4-disubstituted amido-6-replaces-[1,3,5] compound in triazine class or its pharmacy acceptable salt:
Wherein, X is N; Y is NH;
A, B, Z, n, R
1, R
2, R
3, R
4, R
5And R
6Definition as mentioned above.
Another preferred embodiment of formula of the present invention (I) compound is following 2, and 4-disubstituted amido-6-replaces-1,3-pyrimidines or its pharmacy acceptable salt:
Wherein, X is C; Y is O;
A, B, Z, n, R
1, R
2, R
3, R
4, R
5And R
6Definition as mentioned above.
The 4th preferred embodiment of formula of the present invention (I) compound is following 2, and 4-disubstituted amido-6-replaces-1,3-pyrimidines or its pharmacy acceptable salt:
Wherein, X is C; Y is NH;
A, B, Z, n, R
1, R
2, R
3, R
4, R
5And R
6Definition as mentioned above.
The 5th preferred embodiment of formula of the present invention (I) compound is following 2,4-disubstituted amido-6-replacement-[1,3,5] triazine or 1, and 3-pyrimidines or its pharmacy acceptable salt:
Wherein, A is C; B is N;
X, Y, Z, n, R
1, R
2, R
3, R
4, R
5And R
6Definition as mentioned above.
The 6th preferred embodiment of formula of the present invention (I) compound is following 2,4-disubstituted amido-6-replacement-[1,3,5] triazine or 1, and 3-pyrimidines or its pharmacy acceptable salt:
Wherein, A is CH; B is CH;
X, Y, Z, n, R
1, R
2, R
3, R
4, R
5And R
6Definition as mentioned above.
The 7th preferred embodiment of formula of the present invention (I) compound is following 2,4-disubstituted amido-6-replacement-[1,3,5] triazine or 1, and 3-pyrimidines or its pharmacy acceptable salt:
Wherein, A is CH; B is N;
X, Y, Z, n, R
1, R
2, R
3, R
4, R
5And R
6Definition as mentioned above.
The 8th preferred embodiment of formula of the present invention (I) compound is following 2,4-disubstituted amido-6-replacement-[1,3,5] triazine or 1, and 3-pyrimidines or its pharmacy acceptable salt:
Wherein, A is N; B is CH;
X, Y, Z, n, R
1, R
2, R
3, R
4, R
5And R
6Definition as mentioned above.
The 9th preferred embodiment of formula of the present invention (I) compound is following 2,4-disubstituted amido-6-replacement-[1,3,5] triazine or 1, and 3-pyrimidines or its pharmacy acceptable salt:
Wherein, A is N; B is N;
X, Y, Z, n, R
1, R
2, R
3, R
4, R
5And R
6Definition as mentioned above.
The tenth preferred embodiment of formula of the present invention (I) compound is following 2,4-disubstituted amido-6-replacement-[1,3,5] triazine or 1, and 3-pyrimidines or its pharmacy acceptable salt:
Wherein, Z is NH;
X, Y, A, B, n, R
1, R
2, R
3, R
4, R
5And R
6Definition as mentioned above.
Concrete preferred embodiment of the present invention is selected from following compounds:
4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-and the hydrazono-methyl]-1, the 3-dihydroxy-benzene,
2-chloroformic acid 3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
Phenylformic acid 3-benzoyloxy-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
2-acetoxy-benzoic acid 3-(2-acetoxyl group benzoyloxy)-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
4-pivaloyl aminobenzoic acid 3-(4-pivaloyl oxygen base benzoyloxy)-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
2 hydroxybenzoic acid 3-(2-(2-hydroxybenzoyl) oxygen base)-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
2-benzaminic acid 3-(2-aminobenzoic acyl-oxygen base)-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
2-acetylamino benzoic acid 3-(2-kharophen benzoyloxy)-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
N, N-dimethylamino formic acid 3-(N, N-formyl oxygen dimethylamino)-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
N-methylamino-formic acid 3-(N-methylamino-methanoyl)-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
Succinic Acid 3-(3-hydroxyl formyl radical propionyloxy)-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
Phenylformic acid 3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
4-{N '-[4-(3-the bromo-benzyloxy-)-rare base of 2-methoxyl group-benzyl]-diazanyl }-6-morpholine-4-base-[1,3,5]-triazine-2-yl }-aniline,
3-bromo-benzoic acid 3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
4-trifluoromethylbenzoic acid 3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
4-toluene sulfonic acide acid 3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
3-bromo-benzoic acid 2-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
3-bromo-benzoic acid 2-oxyethyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
3-bromo-benzoic acid 3-methoxyl group-4-{[4-phenylamino-6-(4-sulfamyl phenylamino)-[1,3,5] triazine-2-yl]-the hydrazine methene }-phenyl ester,
4-chloro-benzoic acid 3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
4-bromo-benzoic acid 3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
4-chloro-benzoic acid 3-hydroxyl-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
2,6-dichlorobenzoic acid 3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazono-methyl]-phenol,
4-methoxybenzoic acid 3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
3-bromo-benzoic acid 4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
3-bromo-benzoic acid 4-[(4,6-two (4-anisole amino)-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
N-{4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-and the hydrazine methene] phenyl }-the 3-brombenzamide,
N '-(2, the 4-dihydroxy phenyl)-N-(4-morpholine-6-anilino-1,3,5-triazines-2-yl) first miaow,
Phenylformic acid 3-methoxyl group-4-((4-morpholine-6-anilino-1,3,5-triazines-2-base is amino) methene amido) phenyl ester,
4-(2-(4-morpholine-6-anilino-1,3,5-triazines-2-yl) acetal amino)-1, the 3-dihydroxy-benzene,
Phenylformic acid 3-methoxyl group-4-(2-(4-morpholine-6-anilino-1,3,5-triazines-2-yl) acetal amino) phenyl ester,
4-(2-(4-morpholine-6-anilino-1,3,5-triazines-2-base is amino) vinyl)-1, the 3-dihydroxy-benzene,
Phenylformic acid 3-methoxyl group-4-((4-morpholine-6-anilino-1,3,5-triazines-2-base is amino) vinyl) phenyl ester,
4-(3-(4-morpholine-6-anilino-1,3,5-triazines-2-yl) third-1-thiazolinyl)-1, the 3-dihydroxy-benzene,
Phenylformic acid 3-methoxyl group-4-(2-(4-morpholine-6-anilino-1,3,5-triazines-2-yl) third-1-thiazolinyl) phenyl ester,
4-(((4-morpholine-6-anilino-1,3,5-triazines-2-yl) methyl-imino) methyl)-1, the 3-dihydroxy-benzene, or
Phenylformic acid 3-methoxyl group-4-(((4-morpholine-6-(anilino)-1,3,5-triazines-2-yl) methyl-imino) methyl) phenyl ester.
The I that the formula Compound I of the invention provides comprises
A, I
B, I
C, I
D, and I
EThe preparation method of compounds and intermediate II thereof, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX.R wherein
1, R
2, R
3, R
4, R
5, R
6With the definition of Y as mentioned above.
I
AThe synthetic of compounds is with 2,4, and 6-three chloro-[1,3,5] triazine is a raw material, replaces through monoamine respectively, and two ammonia replace, and hydrazine replaces, and Y-replaces and condensation reaction makes.Synthesis strategy is embodied in the graphic scheme 1.
Scheme 12,4-disubstituted amido-6-replaces diazanyl-[1,3,5] compound in triazine class I
APreparation
I
BThe synthetic of compounds is with 2,4,6-three chloro-1, and the 3-pyrimidine is a raw material, replaces through monoamine respectively, (reaction conditions is than I in two ammonia replacements
AHarsh), hydrazine replaces, and Y-replaces and condensation reaction makes.Synthesis strategy is embodied in the graphic scheme 2.
Scheme 22,4-disubstituted amido-6-replaces diazanyl-1,3-pyrimidines I
BPreparation
I
CCompounds synthetic is to be raw material with N-Boc-2-(4,6-two chloro-[1,3,5] triazine-2 base) methylamine, replaces through monoamine respectively, and two ammonia replace, and take off the Boc-protecting group, and Y-replaces and condensation reaction makes.Synthesis strategy is embodied in the graphic scheme 3.
Scheme 32,4-disubstituted amido-6-replacement-[1,3,5] compound in triazine class I
CPreparation
I
DThe synthetic of compounds is with 2-Boc-amino-4, and 6-two chloro-[1,3,5] triazine is a raw material, replaces through monoamine respectively, and two ammonia replace, and take off the Boc-protecting group, and Y-replaces and condensation reaction makes.Synthesis strategy is embodied in the graphic scheme 4.
Scheme 42,4-disubstituted amido-6-replacement-[1,3,5] compound in triazine class I
DPreparation
I
EThe synthetic of compounds is that 6-two chloro-1,3,5-triazines are raw material with 2-((1,3-dimercapto-2-yl) methyl)-4, replaces through monoamine respectively, and two ammonia replace, and take off 1,3-dimercapto protecting group, and Y-replaces and condensation reaction makes.Synthesis strategy is embodied in the graphic scheme 5.
Scheme 52,4-disubstituted amido-6-replacement-[1,3,5] compound in triazine class I
EPreparation
Preparation formula I
A2 of expression, the method for 4-disubstituted amido-6-replacement-[1,3,5] compound in triazine class, this method may further comprise the steps:
1) 4,6-two chloro-[1,3,5] triazine-2-base-N-substitutional amine-group compound II's is synthetic:
To 2,4, add the replacement amine and the alkali of equivalent in the acetone soln of 6-three chloro-[1,3,5] triazine, ice bath stirred 2-48 hour; In the big water gaging of reaction solution impouring, stirred 30 minutes, separate out a large amount of solids, suction filtration, washing, the dry product II that gets;
Synthesis strategy is as follows:
2) 2-replacement-6-chloro-N
4-replacement-[1,3,5] triazine-diamine compounds III's is synthetic:
To 4, add the replacement amine and the alkali of equivalent in the dioxane solution of 6-two chloro-[1,3,5] triazine-2-base-N-substitutional amine-group compound II, ice bath stirred 2～48 hours; In the big water gaging of reaction solution impouring, stirred 30 minutes, separate out a large amount of solids, suction filtration, washing, the dry product III that gets;
Synthesis strategy is as follows:
3) 2,4,6-three replacement-[1,3,5] triazine-diamine compounds IV's is synthetic:
With 2-replacement-6-chloro-N
4-replacement-[1,3,5] triazine-diamine compounds III and excessive hydrazine hydrate reaction reflux and stirred 3～24 hours, are chilled to room temperature, suction filtration, and drying gets product IV;
Synthesis strategy is as follows:
4) Y
4-replacement-R
2Replace the synthetic of compounds V:
Under alkaline condition, 20～70 ℃ of temperature controls stirred 5～24 hours with aldehyde or the amine of protecting and excessive carboxylic acid halides/alkane halogen, and in the big water gaging of reaction solution impouring, organic solvent extraction is used pickling, alkali cleaning, washing successively, and drying is filtered, and removes solvent under reduced pressure and gets product V;
Synthesis strategy is as follows:
5) Compound I
ASynthetic:
Compound IV and V refluxed in alcohol 2～24 hours, and the small amount of acid catalyzed reaction is chilled to room temperature, suction filtration, and drying gets product I
A
Synthesis strategy is as follows:
Used alkali is mineral alkali or organic bases in the above steps, and described mineral alkali is selected from yellow soda ash, salt of wormwood, sodium hydroxide and the sodium bicarbonate, and described organic bases is selected from triethylamine, diethylamine and the pyridine; Step 1) or 3) acetone or dioxane solvent used in are interchangeable, also replaceable one-tenth methyl alcohol, THF, propyl alcohol or Virahol; Used acid is Glacial acetic acid, hydrochloric acid, sulfuric acid or phosphoric acid in the step 5).
Preparation formula I
C2 of expression, the method for 4-disubstituted amido-6-replacement-[1,3,5] compound in triazine class is characterized in that this method may further comprise the steps:
1) N-Boc-2-(6-chloro-N
4-replacement-[1,3,5] triazine-2 base) methylamines IX's is synthetic:
Add the replacement amine and the alkali of equivalent in the dioxane solution of N-Boc-2-(4,6-two chloro-[1,3,5] triazine-2 base) methylamine, ice bath stirred 2～48 hours; In the big water gaging of reaction solution impouring, stirred 30 minutes, separate out a large amount of solids, suction filtration, washing, the dry product IX that gets;
Synthesis strategy is as follows:
2) 2,4,6-three replacement-[1,3,5] triazine-diamine compounds XI's is synthetic:
With N-Boc-2-(6-chloro-N
4-replacement-[1,3,5] triazine-2 base) methylamines IX and excessive replacement amine reaction reflux and stirred 3～24 hours, are chilled to room temperature, suction filtration, and drying gets product X, and X gets product X I through sloughing Boc again;
Synthesis strategy is as follows:
3) Y
4-replacement-R
2Replace the synthetic of compounds V:
Under alkaline condition, 20～70 ℃ of temperature controls stirred 5～24 hours with aldehyde or the amine of protecting and excessive carboxylic acid halides/alkane halogen, and in the big water gaging of reaction solution impouring, organic solvent extraction is used pickling, alkali cleaning, washing successively, and drying is filtered, and removes solvent under reduced pressure and gets product V;
Synthesis strategy is as follows:
4) Compound I
CSynthetic:
Compounds X I and V refluxed in alcohol 2～24 hours, and the small amount of acid catalyzed reaction is chilled to room temperature, suction filtration, and drying gets product I
C
Synthesis strategy is as follows:
Used alkali is mineral alkali or organic bases in the above steps, and described mineral alkali is selected from yellow soda ash, salt of wormwood, sodium hydroxide and the sodium bicarbonate, and described organic bases is selected from triethylamine, diethylamine and the pyridine; Used acid is Glacial acetic acid, hydrochloric acid, sulfuric acid or phosphoric acid in the step 4).
At preparation formula I
AOr formula I
C2 of expression, in the method for 4-disubstituted amido-6-replacement-[1,3,5] compound in triazine class, each goes on foot products therefrom and can purify respectively, obtains pure products, and wherein the method for Ti Chuning comprises column chromatography or recrystallization.
Another aspect of the present invention relates to the pharmaceutical composition that prevents and/or treats the HIV virus infection, described pharmaceutical composition contains shown in the formula (I) 2,4-disubstituted amido-6-replaces-[1,3,5] triazine or 1,3-pyrimidines or its pharmacy acceptable salt and at least a pharmaceutically acceptable carrier, it can be used for interior therapeutic and has biocompatibility.Described pharmaceutical composition can be prepared into various forms according to different way of administration.Pharmaceutical composition of the present invention can be used to prevent and/or treat the HIV virus infection.
Medicinal compositions provided by the present invention can be a various ways, as tablet, capsule, powder, syrup, solution shape, suspension and aerosol etc., and may reside in suitable solid or liquid support or the diluent and the suitable disinfector injecting or instil of being used for.This medicinal compositions also can comprise odorant agent, flavouring agent etc., its ideal ratio is, formula (I) compound accounts for gross weight than more than 65% as activeconstituents, rest part is for accounting for gross weight than 0.5～40%, or more preferably 1～20%, or be preferably 1～10% pharmaceutically acceptable carrier, diluent or solution or salts solution.
The compound of aforesaid structural formula (I) can comprise humans and animals to the clinical use of Mammals, can through port, the route of administration of nose, skin, lung or gi tract etc.The best is preferably oral.Best preferred per daily dose is 0.01～200mg/kg body weight, disposable taking, or 0.01～100mg/kg body weight part vic.Which kind of instructions of taking that don't work, individual's optimal dose should be decided according to concrete treatment.Generally be from low dose, increase dosage gradually until find optimal dosage.
Formula (I) compound is integrated into the process of host chromosome by the HIV double chain DNA molecule that blocking-up IN participates in, thereby plays the effect that suppresses virus replication.Be used for the treatment of HIV virus infection.
Description of drawings
Fig. 1 is for detecting the proteic electrophorogram of purifying;
Fig. 2 is IA-3 and the dynamic analysis of HIV-1 intergrase;
Fig. 3 is the mensuration of the IC50 value of IA-3.
Embodiment
In following embodiment, will further illustrate the present invention.These embodiment only are used to illustrate the present invention, but do not limit the present invention in any way.All parameters among the embodiment and remaining explanation unless otherwise indicated, all are the explanation foundation with the quality.
Embodiment 1
4,6-two chloro-[1,3,5] triazine-2-base aniline (II-1)
With 2.0 grams 2,4,6-three chloro-[1,3,5] triazine is dissolved in 12 milliliters of dioxane, under the ice bath cooling and stirring, slowly drips the acetone soln (/ 2 milliliters of 1 grams) of aniline.After dropwising, disposable adding 1.16 gram yellow soda ash continue ice bath and stir after 5 hours, are warming up to stirring at room 2 hours.In reaction solution, drip in 150 ml waters, stirred 30 minutes, separate out a large amount of solids, suction filtration, washing, dry that light yellow solid product 2.48 restrains yield 95.4%.Mp?132-133℃(lit.133-135℃)。MS-EI?240(M+，100％)。
Embodiment 2
(4-chloro-6-morpholine-4-base-[1,3,5] triazine-2-yl) aniline (III-1)
1.0 gram II-1 are dissolved in 6 milliliters of dioxane, under the ice bath cooling and stirring, slowly drip the acetone soln (/ 1 milliliter of 0.36 gram) of morpholine.After dropwising, disposable adding 0.438 gram yellow soda ash continues ice bath and stirs after 2 hours, is warming up to stirring at room 1 hour.In reaction solution, drip in 70 ml waters, stirred 30 minutes, separate out a large amount of solids, suction filtration, washing, dry that light yellow solid product 1.06 restrains yield 88.0%.Mp?193-195℃(lit.198.5-199.5℃)。1H-NMR(400Hz，CDCl3)δ：3.75(4H，m)，3.85(4H，m)，7.14(1H，t)，7.18(1H，br)，7.38(2H，t)，7.52(2H，t)。
Embodiment 3
(4-diazanyl-6-morpholine-4-base-[1,3,5] triazine-2-yl) aniline (IV-1)
0.5 gram III-1 is put in 15 milliliter 85% the hydrazine hydrate, refluxes and stirred 2 hours, be chilled to room temperature, pumping rate, a small amount of dehydrated alcohol is washed, off-white color solid 0.4 gram, yield 81.6%.Mp?170-171℃；MS-EI?287(M+，100％)。
Phenylformic acid-3-methoxyl group-4-formyl radical-phenyl ester (V-1)
0.2 gram 2-methoxyl group-4-hydroxyl-phenyl aldehyde is dissolved in 5 milliliters of anhydrous pyridines, under the stirring at room, drips 0.36 gram Benzoyl chloride, finished stirring at room 12 hours, in reaction solution impouring 50 ml waters, ethyl acetate extracts (30mlx3), merges organic layer, dilute acid solution is given a baby a bath on the third day after its birth inferior, aqueous sodium carbonate is washed once, and anhydrous magnesium sulfate drying filters, remove solvent under reduced pressure and get light yellow solid 0.16 gram, yield 48.5%.1H-NMR(400Hz，CDCl3)δ：3.95(3H，s)，6.93(2H，m)，7.56(2H，t)，7.67(1H，d)，7.94(1H，d)，8.22(2H，d)，10.44(1H，s)。
4-(3-bromo-benzyloxy-)-2-methoxyl group-phenyl aldehyde (V-2)
With 0.88 gram 3-bromobenzyl chlorine, 0.5 gram 4-hydroxyl-2-methoxybenzaldehyde and 0.59 gram salt of wormwood are put in 10 milliliters of acetone, reflux and stir 12 hours, be chilled to room temperature, add 50 ml waters, ethyl acetate extracts, column chromatography for separation gets white solid 0.46 gram, Mp 101-103 ℃, yield 43.6%.
1H-NMR(400Hz，CDCl
3)δ：3.90(3H，s)，5.10(2H，s)，6.54(2H，s)，7.30(1H，t)，7.36(1H，d)，7.50(1H，d)，7.61(1H，s)，7.83(1H，d)，10.30(1H，s)。MS-EI?320(M
+)，169(100％)。
Phenylformic acid 3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester (I
A-1)
128 milligrams of V-1 and 144 milligrams of IV-1 are put in 25 milliliters of dehydrated alcohols, add 2 Glacial acetic acid, stirring and refluxing 2 hours is chilled to room temperature, pumping rate, and small amount of ethanol is washed, and gets white solid I
A-1145 milligrams, Mp 211-213 ℃, yield 55.3%.
1H-NMR(，400Hz，DMSO-d6)δ：3.66(4H，m)，3.75(4H，m)，3.85(3H，s)，7.00(2H，m)，7.09(1H，s)，7.30(2H，t)，7.65(2H，t)，7.74-7.85(3H，m)，7.95(1H，d)，8.16(2H，d)，8.46(1H，s)。MS-EI?525(M
+)，271(100％)。
(4-{N '-[4-(3-the bromo-benzyloxy-)-rare base of 2-methoxyl group-benzyl]-diazanyl }-6-morpholine-4-base-[1,3,5]-triazine-2-yl)-aniline (I
A-2)
V-1 is replaced to V-2, and all the other desired raw materials, reagent and preparation method get white solid I with embodiment 6
A-2187 milligrams, Mp 185-186 ℃, yield 63.4%.
1H-NMR(400Hz，DMSO-d6)δ：3.67(4H，m)，3.75(4H，m)，3.84(3H，s)，5.19(2H，s)，6.73(2H，s)，6.98(1H，t)，7.30(2H，t)，7.41(1H，t)，7.51(1H，d)，7.58(1H，d)，7.70(1H，s)，7.78-7.79(3H，m)，8.39(1H，s)。MS-EI?589(M
+)，271(100％)。
3-bromo-benzoic acid 3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester (I
A-3)
Benzoyl chloride is replaced to the 3-bromo-benzoyl chloride, and all the other desired raw materials, reagent and preparation method get white solid I with embodiment 4 and 6
A-3270 milligrams, Mp 175-177 ℃, yield 89.4%.
1H-NMR(400Hz，DMSO-d6)δ：3.66(4H，m)，3.74(4H，m)，3.84(3H，s)，6.92-7.01(2H，m)，7.12(1H，s)，7.29(2H，t)，7.62(1H，t)，7.82(2H，br)，7.99(2H，m)，8.15(1H，d)，8.26(1H，s)，8.45(1H，s)。MS-EI?603(M
+)，271(100％)。
4-trifluoromethylbenzoic acid 3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester (I
A-4)
Benzoyl chloride is replaced to the 4-trifluoromethyl benzoyl chloride, and all the other desired raw materials, reagent and preparation method get white solid I with embodiment 4 and 6
A-4135 milligrams, Mp 234-236 ℃, yield 67.2%.
1H-NMR(400Hz，DMSO-d6)δ：3.65(4H，m)，3.75(4H，m)，3.85(3H，s)，6.92-7.03(2H，m)，7.12(1H，s)，7.29(2H，t)，7.82(2H，br)，7.95(1H，d)，8.01(2H，d)，8.36(2H，d)，8.46(1H，s)。MS-EI?593(M
+)，271(100％)。
4-toluene sulfonic acide acid 3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester (I
A-5)
Benzoyl chloride is replaced to 4-Methyl benzenesulfonyl chlorine, and all the other desired raw materials, reagent and preparation method get white solid I with embodiment 4 and 6
A-5 240 milligrams, Mp 239-241 ℃, yield 83.6%.
1H-NMR(400Hz，DMSO-d6)δ：2.41(3H，s)，3.65(4H，m)，3.75(7H，m)，6.64-6.70(2H，m)，6.97(1H，t)，7.28(2H，t)，7.50(2H，d)，7.79(5H，m)，8.35(1H，s)。MS-EI?575(M
+)，271(100％)。
Embodiment 11
3-bromo-benzoic acid 2-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester (I
A-6)
2-methoxyl group-4-hydroxy benzaldehyde is replaced to 3-methoxyl group-4-hydroxy benzaldehyde, and all the other desired raw materials, reagent and preparation method get white solid I with embodiment 4 and 6
A-6173 milligrams, Mp 142-144 ℃, yield 79.0%.
1H-NMR(400Hz，DMSO-d6)δ：3.66(4H，m)，3.75(4H，m)，3.84(3H，s)，6.97(1H，t)，7.29(2H，t)，7.33(2H，s)，7.48(1H，s)，7.62(1H，t)，7.81(2H，br)，7.99(1H，d)，8.15(2H，d)，8.22(1H，s)。MS-EI?603(M
+)，271(100％)。
Embodiment 12
3-bromo-benzoic acid 2-oxyethyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester (I
A-7)
2-methoxyl group-4-hydroxy benzaldehyde is replaced to vanirom, and all the other desired raw materials, reagent and preparation method get light yellow solid I with embodiment 4 and 6
A-7240 milligrams, Mp 118-119 ℃, yield 77.7%.
1H-NMR(400Hz，DMSO-d6)δ：1.28(3H，t)，3.66(4H，m)，3.75(4H，m)，4.14(2H，q)，6.98(1H，t)，7.29(2H，t)，7.33(2H，s)，7.47(1H，s)，7.62(1H，t)，7.81(2H，br)，7.99(1H，d)，8.14(2H，d)，8.23(1H，s)。MS-EI?617(M
+)，271(100％)。
Embodiment 13
3-bromo-benzoic acid 3-methoxyl group-4-{[4-phenylamino-6-(4-sulfamyl phenylamino)-[1,3,5] triazine-2-yl]-the hydrazine methene }-phenyl ester (I
A-8)
With aniline, morpholine and Benzoyl chloride replace to sulfanilamide (SN) respectively, aniline and 3-bromo-benzoyl chloride, and all the other desired raw materials, reagent and preparation method get off-white color solid I with embodiment 1-4 and 6
A-8157 milligrams, Mp 218-220 ℃, yield 76.2%.
1H-NMR(400Hz，DMSO-d6)δ：3.87(3H，s)，7.05(2H，m)，7.15(1H，d)，7.35(2H，t)，7.63(1H，t)，7.73(2H，d)，7.85(2H，br)，7.99(2H，m)，8.06(2H，br)，8.16(1H，d)，8.27(1H，t)，8.54(1H，s)。MS-ESI?689[M+H]
+。
Embodiment 14
4-chloro-benzoic acid 3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester (I
A-9)
Benzoyl chloride is replaced to the 4-chloro-benzoyl chloride, and all the other desired raw materials, reagent and preparation method get white solid I with embodiment 4 and 6
A-9250 milligrams, Mp 211-212 ℃, yield 86.6%.
1H-NMR(400Hz，DMSO-d6)δ：3.66(4H，m)，3.74(4H，m)，3.85(3H，s)，7.00(2H，m)，7.10(1H，s)，7.29(2H，t)，7.71(2H，d)，7.81(2H，br)，7.94(1H，d)，8.16(2H，d)，8.45(1H，s)。MS-EI?559(M
+)，271(100％)。
4-bromo-benzoic acid 3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester (I
A-10).
Benzoyl chloride is replaced to the 3-bromo-benzoyl chloride, and all the other desired raw materials, reagent and preparation method get 4-bromo-benzoic acid 3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl with embodiment 4 and 6)-the hydrazine methene]-phenyl ester.
Embodiment 16
4-methoxybenzoic acid 3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester (I
A-11)
Benzoyl chloride is replaced to the 4-methoxy benzoyl chloride, and all the other desired raw materials, reagent and preparation method get 4-methoxybenzoic acid 3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl with embodiment 4 and 6)-the hydrazine methene]-phenyl ester.
Embodiment 17
2-chloro-benzoic acid 3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester (I
A-12)
Benzoyl chloride is replaced to the 2-chloro-benzoyl chloride, and all the other desired raw materials, reagent and preparation method get 2-chloro-benzoic acid 3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl with embodiment 4 and 6)-the hydrazine methene]-phenyl ester.
Embodiment 18
2,6-dichlorobenzoic acid 3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester (I
A-13)
Benzoyl chloride is replaced to 2, the 6-dichlorobenzoyl chloride, all the other desired raw materials, reagent and preparation method get 2,6-dichlorobenzoic acid 3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl with embodiment 4 and 6)-the hydrazine methene]-phenyl ester.
Embodiment 19
3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenol (I
A-14)
V-1 is replaced to 2-methoxyl group-4-hydroxyl-phenyl aldehyde, and all the other desired raw materials, reagent and preparation method get 3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl with embodiment 6)-the hydrazine methene]-phenol.
3-bromo-benzoic acid 4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester (I
A-15)
2-methoxyl group-4-hydroxyl-phenyl aldehyde is replaced to 4-hydroxyl-phenyl aldehyde, with Benzoyl chloride replace to the 3-bromo-benzoyl chloride with all the other desired raw materials, reagent and preparation method with embodiment 4 and 6, get 3-bromo-benzoic acid 4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester.
Embodiment 21
4-chloro-benzoic acid 3-hydroxyl-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester (I
A-16)
2-methoxyl group-4-hydroxyl-phenyl aldehyde is replaced to 2,4-dihydroxyl-phenyl aldehyde, with Benzoyl chloride replace to the 4-chloro-benzoyl chloride with all the other desired raw materials, reagent and preparation method with embodiment 4 and 6, get 4-chloro-benzoic acid 3-hydroxyl-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester.
Embodiment 22
4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-and the hydrazono-methyl]-1,3-dihydroxy-benzene (I
A-17)
V-1 is replaced to 2,4-dihydroxyl-phenyl aldehyde, all the other desired raw materials, reagent and preparation method get 4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl with embodiment 6)-the hydrazono-methyl]-1, the 3-dihydroxy-benzene.1H-NMR(300Hz，d6-DMSO)δ3.64(m，4H)，3.74(m，4H)，6.34(d，s，2H)，6.96(m，1H)，7.16(d，1H，J＝8.1Hz)，7.27(m，2H)，7.75(br，2H)，8.17(s，1H)。MS-EI?m/z：407(M
+)。HR?EI-MS：Calcd.forC
20H
21N
7O
3：407.1706；Found：407.1715。
Embodiment 23
3-bromo-benzoic acid 4-[(4,6-two (4-anisole amino)-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester (I
A-18)
With aniline, morpholine and Benzoyl chloride replace to 4-anisidine and 3-bromo-benzoyl chloride respectively, and all the other desired raw materials, reagent and preparation method are with embodiment 1-4 and 6, get 3-bromo-benzoic acid 4-[(4,6-two (4-anisole amino)-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester.
Embodiment 24
2-acetoxy-benzoic acid 3-(2-acetoxyl group benzoyloxy)-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester (I
A-19)
The acetylsalicylic acid of 99mg (0.55mmol) is suspended in the exsiccant CH of 5mL
2Cl
2, ice bath adds the oxalyl chloride of the new steaming of 55 μ L (0.61mmol) down, adds a DMF catalyzed reaction, changes stirring at room 4-5h behind the 10min; Then, ice bath directly adds (the I of 100mg down
A-17) (0.25mmol), stir 10min after, slowly add TEA and be weakly alkaline to solution, continue reaction 1h, the CH of adding 20mL in the solution
2Cl
2, washing is (10ml) once, and saturated NaCl washes once (10ml), anhydrous Na
2SO
4Drying is drained, and obtains the 185mg crude product, and the chromatographic column purifying obtains 165mg 2-acetoxy-benzoic acid 3-(2-acetoxyl group benzoyloxy)-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester (I
A-19), yield 91.7%, Mp 99-101 ℃.
1H-NMR(300Hz，CDCl
3)δ2.29(s，3H)，2.33(s，3H)，3.74(m，4H)，3.83(m，4H)，7.05(t，1H，J＝7.5Hz)，7.13(m，2H)，7.20(m，3H)，7.32(t，2H，J＝7.5Hz，)，7.42(q，2H，J＝7.8Hz)，7.59-7.71(m，4H)，7.91(s，1H)，8.23(t，2H，J＝7.8Hz)；MS-EI?m/z：731(M
+)。
4-pivaloyl aminobenzoic acid 3-(4-pivaloyl oxygen base benzoyloxy)-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester (I
A-20)
Acetylsalicylic acid is replaced to 4-pivaloyl aminobenzoic acid, all the other desired raw materials, reagent and preparation method are with embodiment 24, get 176mg 4-pivaloyl aminobenzoic acid 3-(4-pivaloyl oxygen base benzoyloxy)-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester (I
A-20), yield 96.2%.
1H-NMR(300Hz，CDCl
3)δ1.36(s，18H)，3.72(m，4H)，3.80(m，4H)，7.04(t，1H，J＝7.8Hz)，7.15-7.32(m，9H)，7.35(m，2H)，7.98(s，1H)，8.18-8.26(m，4H)；MS-EIm/z：815(M
+)。
Embodiment 26
Phenylformic acid 3-benzoyloxy-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester (I
A-21)
Acetylsalicylic acid is replaced to phenylformic acid, and all the other desired raw materials, reagent and preparation method get phenylformic acid 3-benzoyloxy-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl with embodiment 24)-the hydrazine methene]-phenyl ester.
Embodiment 27
2 hydroxybenzoic acid 3-(2-(2-hydroxybenzoyl) oxygen base)-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester (I
A-22)
Acetylsalicylic acid is replaced to 2 hydroxybenzoic acid, all the other desired raw materials, reagent and preparation method are with embodiment 24, get phenylformic acid 2 hydroxybenzoic acid 3-(2-(2-hydroxybenzoyl) oxygen base)-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester.
Embodiment 28
2-benzaminic acid 3-(2-aminobenzoic acyl-oxygen base)-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester (I
A-23)
Acetylsalicylic acid is replaced to the 2-benzaminic acid, all the other desired raw materials, reagent and preparation method are with embodiment 24, get phenylformic acid 2-benzaminic acid 3-(2-aminobenzoic acyl-oxygen base)-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester.
Embodiment 29
2-acetylamino benzoic acid 3-(2-kharophen benzoyloxy)-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester (I
A-24)
Acetylsalicylic acid is replaced to the 2-acetylamino benzoic acid, all the other desired raw materials, reagent and preparation method are with embodiment 24, get 2-acetylamino benzoic acid 3-(2-kharophen benzoyloxy)-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester.
Embodiment 30
(2,6-dichloro pyrimidine-4-yl)-(4-p-methoxy-phenyl) amine (VI-1)
With 2,4,6-trichloropyrimidine 2.36 grams, 4-anisidine 3.17 grams and yellow soda ash 1.36 grams are put in 20 milliliters of ethanol, reflux and stir 2 hours.Be chilled to room temperature, add 70 ml waters, pumping rate obtains to such an extent that thick product gets white solid 2.4 grams, Mp158-160 ℃ (lit.160-162 ℃), yield 69.4% with 75% aqueous ethanolic solution recrystallization.
1H-NMR(400Hz，CDCl
3)δ：3.84(3H，s)，6.37(1H，s)，6.98(2H，d)，7.26(2H，d)，7.37(1H，br)。
Embodiment 31
6-chloro-N
4-(4-p-methoxy-phenyl)-N
2-phenyl-pyrimidine-2,4-diamines (VII-1)
With the VI-11.2 gram, aniline 3.17 grams and yellow soda ash 1.2 grams are put in 20 milliliters of dioxane, reflux and stir 48 hours.Be chilled to room temperature, add 70 ml waters, dichloromethane extraction, drying concentrates, and the resistates column chromatography for separation gets off-white color solid phase prod 0.8 gram, and Mp 102-105 ℃, yield 55.2%.MS-EI?326(M
+)，269(100％)。
Embodiment 32
6-diazanyl-N
4-(4-p-methoxy-phenyl)-N
2-phenyl-pyrimidine-2,4-diamines (VIII-1)
VII-20.3 is restrained in the hydrazine hydrate of putting into 10 milliliter 85%, and stirring and refluxing 2 hours is chilled to room temperature, and pumping rate gets off-white color solid product 0.23 gram.Mp 173-174 ℃, yield 77.7%.MS-EI?322(M
+，100％)。
Embodiment 33
3-bromo-benzoic acid 3-methoxyl group-4-{[6-(4-anisole amido)-2-anilino-pyrimidine-4-yl]-the hydrazine methene }-phenyl ester (I
B-1)
With Benzoyl chloride, IV-1 replaces to the 3-bromo-benzoyl chloride respectively, VIII-1, and all the other desired raw materials, reagent and preparation method get off-white color solid I with embodiment 4 and 6
B-1105 milligrams, Mp 187-188 ℃, yield 55.0%.
1H-NMR(400Hz，DMSO-d6)δ：3.74(3H，s)，3.87(3H，s)，6.04(1H，s)，6.92(3H，m)，7.02(1H，d)，7.14(1H，s)，7.27(2H，t)，7.57-7.64(3H，m)，7.83(2H，d)，7.91(1H，d)，8.00(1H，d)，8.17(1H，d)，8.28(1H，s)，8.42(1H，s)。MS-EI?602(M
+)，270(100％)。
Embodiment 34
N-Boc-2-(4-anilino-6-chloro-[1,3,5] triazine-2 base) methylamine (IX-1)
With 2,4,6-three chloro-[1,3,5] triazine replaces to N-Boc-2-(4,6-two chloro-[1,3,5] triazine-2 base) methylamine, and all the other desired raw materials, reagent and preparation method get N-Boc-2-(4-anilino-6-chloro-[1,3,5] triazine-2 base) methylamine with embodiment 1.
Embodiment 35
N-Boc-2-(4-anilino-6-morpholine-[1,3,5] triazine-2 base) methylamine (X-1)
With 4,6-two chloro-[1,3,5] triazine-2-base aniline replaces to N-Boc-2-(4-anilino-6-chloro-[1,3,5] methylamine triazine-2 base), all the other desired raw materials, reagent and preparation method are with embodiment 2, get N-Boc-2-(4-anilino-6-morpholine-[1,3,5] triazine-2 base) methylamine.
Embodiment 36
2-(4-anilino-6-morpholine-[1,3,5] triazine-2 base) methylamine (XI-1)
With 0.5 gram N-Boc-2-(4-anilino-6-morpholine-[1,3,5] triazine-2 base) methylamine is dissolved in 20 milliliters of 20% trifluoroacetic acids/dry methylene chloride solution, after the normal-temperature reaction 2 hours, solid is collected in underpressure distillation, washing repeatedly, get 2-(4-anilino-6-morpholine-[1,3,5] triazine-2 base) methylamine.
Embodiment 37
Phenylformic acid 3-methoxyl group-4-(((4-morpholine-6-(anilino)-1,3,5-triazines-2-yl) methyl-imino) methyl) benzene fat (I
C-1)
With (4-diazanyl-6-morpholine-4-base-[1,3,5] triazine-2-yl) aniline replaces to 2-(4-anilino-6-morpholine-[1,3,5] methylamine triazine-2 base), all the other desired raw materials, reagent and preparation method get phenylformic acid 3-methoxyl group-4-(((4-morpholine-6-(anilino)-1 with embodiment 6,3,5-triazine-2-yl) methyl-imino) methyl) benzene fat.
Embodiment 38
2-Boc-amino-4-anilino-6-chloro-[1,3,5] triazines (XII-1)
With 2,4,6-three chloro-[1,3,5] triazine replaces to 2-Boc-amino-4, and 6-two chloro-[1,3,5] triazine, all the other desired raw materials, reagent and preparation method get 2-Boc-amino-4-anilino-6-chloro-[1,3,5] triazine with embodiment 1.
Embodiment 39
2-Boc-amino-4-anilino-6-morpholine-[1,3,5] triazines (XIII-1)
With 4,6-two chloro-[1,3,5] triazine-2-base aniline replaces to 2-Boc-amino-4-anilino-6-chloro-[1,3,5] triazine, and all the other desired raw materials, reagent and preparation method get 2-Boc-amino-4-anilino-6-morpholine-[1,3,5] triazine with embodiment 2.
Embodiment 40
2-amino-4-anilino-6-morpholine-[1,3,5] triazines (XIV-1)
N-Boc-2-(4-anilino-6-morpholine-[1,3,5] triazine-2 base) methylamine is replaced to 2-Boc-amino-4-anilino-6-morpholine-[1,3,5] triazine, all the other desired raw materials, reagent and preparation method are with embodiment 36, get 2-amino-4-anilino-6-morpholine-[1,3,5] triazine.
Embodiment 41
Phenylformic acid-3-methoxyl group-4-(N-Boc amino)-phenyl ester (XV-1)
2-methoxyl group-4-hydroxyl-phenyl aldehyde is replaced to 2-methoxyl group-4-hydroxy-n-Boc-aniline, and all the other desired raw materials, reagent and preparation method get phenylformic acid-3-methoxyl group-4-(N-Boc amino)-phenyl ester with embodiment 4.
Embodiment 42
Phenylformic acid-3-methoxyl group-4-amino-phenyl ester (XVI-1)
With N-Boc-2-(4-anilino-6-morpholine-[1,3,5] triazine-2 base) methylamine replaces to phenylformic acid-3-methoxyl group-4-(N-Boc amino)-phenyl ester, and all the other desired raw materials, reagent and preparation method get phenylformic acid-3-methoxyl group-4-amino-phenyl ester with embodiment 36.
Embodiment 43
Phenylformic acid 3-methoxyl group-4-((4-morpholine-6-anilino-1,3,5-triazines-2-base is amino) methene amido) phenyl ester (ID-1)
100 milligrams of IXV-1 and 1 equivalent XVI-1 are dissolved in an amount of tetrahydrofuran (THF), add monovalent formaldehyde, stirring at room, underpressure distillation after reaction is finished, column chromatography gets compound phenylformic acid 3-methoxyl group-4-((4-morpholine-6-anilino-1,3,5-triazines-2-base is amino) methene amido) phenyl ester.
Embodiment 44
2-((1,3-dimercapto-2-yl) methyl)-4-anilino-6-chloro-1,3,5-triazines (XVII-1)
With 2,4,6-three chloro-[1,3,5] triazine replaces to 2-((1,3-dimercapto-2-yl) methyl)-4,6-two chloro-1,3,5-triazines, all the other desired raw materials, reagent and preparation method are with embodiment 1, get 2-((1,3-dimercapto-2-yl) methyl)-4-anilino-6-chloro-1,3,5-triazines.
Embodiment 45
2-((1,3-dimercapto-2-yl) methyl)-4-anilino-6-morpholine-1,3,5-triazines (XVIII-1)
With 4,6-two chloro-[1,3,5] triazine-2-base aniline replaces to 2-((1,3-dimercapto-2-yl) methyl)-4-anilino-6-chloro-1,3, the 5-triazine, all the other desired raw materials, reagent and preparation method are with embodiment 2, get 2-((1,3-dimercapto-2-yl) methyl)-4-anilino-6-morpholine-1,3,5-triazines.
Embodiment 46
2-aldehyde-base-4-anilino-6-morpholine-1,3,5-triazines (XIX-1)
With 0.5 gram 2-((1,3-dimercapto-2-yl) methyl)-4-anilino-6-morpholine-1,3, the 5-triazine is dissolved in the proper amount of acetone, adds the cupric chloride of equivalent and the cupric oxide of equivalent, refluxes three hours, column chromatography gets product 2-aldehyde-base-4-anilino-6-morpholine-1,3,5-triazines.
Embodiment 47
Phenylformic acid 3-methoxyl group-4-(2-(4-morpholine-6-anilino-1,3,5-triazines-2-yl) acetal amino) phenyl ester (I
E-1)
With (4-diazanyl-6-morpholine-4-base-[1; 3; 5] triazine-2-yl) aniline and phenylformic acid-3-methoxyl group-4-formyl radical-phenyl ester replaces to 2-aldehyde-base-4-anilino-6-morpholine-1 respectively; 3; 5-triazine and phenylformic acid-3-methoxyl group-4-amino-phenyl ester, all the other desired raw materials, reagent and preparation method get phenylformic acid 3-methoxyl group-4-(2-(4-morpholine-6-anilino-1 with embodiment 6; 3,5-triazine-2-yl) acetal amino) phenyl ester.
Embodiment 48
The HIV-1 intergrase of GST mark (IN, the structure of fusion rotein plasmid pGEX-4T-1-IN (F185K) integrase):
(1) experimental technique:
1) with the dna fragmentation (gene order according to AF040373 (GENEBANK)) of round pcr from pUC18-IN plasmid acquisition IN.
Primer: FW:5 '-ata tgg atc ctt ttta gat gga ata gat-3 '
RV：5′-ata?tct?cga?gct?aat?cct?cat?cct?g-3′
94 ℃ of sex change 5min begin circulation then: 94 ℃ of 45s, and 55 ℃ of 45s, 72 ℃ of 1min30s repeat circulation, 72 ℃ of 10min then 29 times.
Adopt 1% agarose gel electrophoresis evaluation PCR product and reclaim the PCR fragment.
2) with IN PCR fragment cloning to carrier pGEX-4T-1: IN PCR product and pGEX-4T-1 carry out endonuclease reaction with BamHI and XhoI respectively; Reclaim test kit recovery enzyme with glue and cut product; Do ligation with the T4DNA ligase enzyme; The connection product is transformed into DH5 α competent cell and is coated on the flat board that contains penbritin (100mg/l); Cut and check order by enzyme and identify correct clone.
3) phenylalanine with 185 of HIV-1 intergrases is mutated into Methionin: this step adopts the point mutation test kit of Invitrogen company.
Mutant primer: FW:5 '-ggcagtattcatccacaataagaaaagaaaaggggggattgg-3 '
RV：5’-ccaatccccccttttcttttcttattgtggatgaatactgcc-3’
Determine to take place the cloned plasmids pGEX-4T-1-IN (F185K) of correct sudden change by checking order.
(2) experimental result
1) agarose gel electrophoresis evaluation enzyme is cut the result: two restriction enzyme sites of BamHI and XhoI are arranged on the multiple clone site of pGEX-4T-1 carrier, IN is cloned on the pGEX-4T-1 by these two sites, and correct enzyme is cut the result and cut out the about 900bp of fragment of IN itself and the about 4900bp of fragment of carrier;
2) sequencing result shows that clone's sequence is identical with AF040373 (GENEBANK), only in the 553-555 position of sequence by original " ttt " sport " aaa ", the amino acid that corresponds to 185 is mutated into Methionin by phenylalanine.
Embodiment 49
The expression and purification of the HIV-1 intergrase (GST-IN) of GST mark:
(1) experimental technique
1) plasmid pGEX-4T-1-IN (F185K) is changed among the expression strain BL21 (DE3), select the correct 37 ℃ of overnight shakings that are cloned in and be incubated at 10ml ammonia benzyl LB substratum (yeast extract 5g/L, Tryptones 10g/L and NaCl 10g/L, penbritin concentration is 100mg/L) in, be transferred in the fresh ammonia benzyl LB substratum by 1: 100, shaking culture is to OD
600When value is between the 0.6-0.8, adds IPTG and be 0.2mM and culture temperature is reduced to 25 ℃ of inducible proteins expressed 5-7 hour to final concentration.5, the centrifugal 10min of 000rpm collects bacterium, washes recentrifuge behind the bacterium with the PBS damping fluid is outstanding, and places-70 ℃ of refrigerators preservations stand-by the bacterium of collecting.
2) stand-by thalline hangs with 20ml PBS, the ultrasonication bacterium, and 15, get the cleer and peaceful good Glutathione Sepharose of PBS balance that used behind the centrifugal 30min of 000rpm
TMThe affine resin of 4B is at 4 ℃ of mixing 4hrs, pass pillar after, with 100-150ml PBS flush away foreign protein, use the IN of 50mM GSH wash-out GST mark at last.
3) specification sheets that provides according to Amersham Biosciences company of the protein solution of wash-out utilizes the further separation and purification of Hiload 16/60Superdex75 molecular sieve prepacked column, used solution is 10mM HEPES and 150mM NaCl, collects albumen and detects purity with SDS-PAGE.
(2) experimental result
8%SDS-PAGE also detects the albumen of purifying with coomassie brilliant blue staining: 282 amino acid of HIV-1 intergrase total length, add that size is the GST mark of 27kD, and whole fusion rotein size is about 60kD.As shown in Figure 1, left side swimming lane is marker, and the right then is the good albumen HIV-1 intergrase of purifying, can be clearly seen that the band at about 60kD place on the SDS-PAGE glue after the dyeing, judges that purity is greater than 95%.
Embodiment 50
Utilize biosensor Biacore 3000 to detect I
A-3 is active with combining of HIV-1 intergrase:
(1) experimental technique
1) HIV-1 intergrase that purifying is the good program of utilizing Biacore3000 to carry with the method for amino coupled is coupled on the CM5 chip.The Biacore3000 system running environment is HBS-EP damping fluid (3mM EDTA and 0.01%P20, pH 7.4 for 10mM HEPES, 150mM NaCl).
2) with the I of different concns
A-3 are made into HBS-EP damping fluid (the 10mM HEPES that contains 0.1%DMSO, 150mM NaCl, 3mM EDTA and 0.01%P20, pH 7.4) do dynamic analysis, draw the equilibrium dissociation constant K of this compound and HIV-1 intergrase with Biacore evaluation 3.2 software analysis
D
(2) experimental result
I
A-3 show the active K of the very high combination of HIV-1 intergrase
D=0.38 μ M (Fig. 2)
Embodiment 51
With the IC of biosensor assay PLK275 to the HIV-1 intergrase
50Value:
(1) experimental technique
1) substrate analogue with the HIV-1 intergrase is coupled on the biosensor SA chip:
Synthesize the substrate similar DNA according to the terminal substrate sequences Design of the viral DNA of HIV-1 intergrase, and add vitamin H (biotin) mark at 5 ' end.Sequence: biotin-5 '-GTGTGGAAAATCTCTAGGTGT-3 '.Utilize vitamin H and Streptomycin sulphate bonded principle to be coupled to the SA chip the biotin labeled DNA of synthetic band.
2) biosensor assay I
AThe IC of-3 pairs of HIV-1 intergrases
50Value:
The HIV-1 intergrase that purifying is good and the I of different concns
A-3 hatch after 1 hour on ice and to do dynamic analysis, because the compound competition in conjunction with the HIV-1 intergrase, makes the HIV-1 intergrase that the binding ability of substrate DNA analogue on the chip is descended, show as the decline of RU value.According to the RU value and the concentration mapping of the different concns correspondence of compound, draw the IC of compound with software Origin analysis
50Value.
(2) experimental result
Can see increase along with compound concentration by Fig. 3, the HIV-1 intergrase descends to the RU value of SA chip.According to the RU value and the concentration mapping of the different concns correspondence of compound, analyze with Origin and to draw Compound I
A-3 IC
50Value is 0.97 μ M.
12 external anti-HIV-1 activity of compound such as embodiment 52Xs2
1. material and method
(1) medicine and compound
Testing sample Xs2, Xs19, Xs32, Xs77, XsF2, XsF9, Xs274, Xs275, Xs276, Xs277, Xs278, Xs279 is provided by professor Shen Xu of Shanghai Pharmaceutical Inst., Chinese Academy of Sciences.Positive control compound Zidovodine (3 '-Azido-3 '-deoxythymidine, AZT) available from Sigma company.12 samples all are dissolved among the DMSO, and stock solution concentration is 25mg/ml, and condition of storage is :-20 ℃; AZT is dissolved in the RPMI-1640 perfect medium, 0.22 μ m membrane filtration degerming ,-20 ℃ of preservations after the packing.
(2) reagent and solution a. reagent
HEPES (N-2 (2-Hydroxyothyl) piperazine-N '-(2-ethanesufonic acid), MTT (3, (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), DMF (N, N '-Dimethyl formamine), penicillin (Penicillin), Vetstrep (Streptomycin sulfate), glutamine (Glutamine) are all available from Sigma company; (2-ME 2-Mercaptoethanol) is Bio-Rad company product to 2 mercapto ethanol.RPMI-1640 and newborn calf serum are Gibco company product.
B. substratum
The RPMI-1640 perfect medium contains 10% deactivation newborn calf serum, 2mM L-glutaminate, 10Mm HEPES, 50 μ M 2 mercapto ethanols, 100,000IU penicillin, 100 μ g/ml Streptomycin sulphates.
(3) cell and virus
The human T lymphocyte is C8166, MT4 cell and HIV-1 experiment strain HIV-1
IIIBBy Britain Medical Research Council, AIDS Reagent Project is so kind as to give.All cells and virus are all cultivated with the RPMI-1640 perfect medium that contains 10% calf serum.Prepare HIV-1 according to a conventional method
IIIB, titration also calculates viral TCID
50After the packing of virus stock solution, put-70 ℃ of preservations.The frozen according to a conventional method and recovery of cell and virus.
(4) HIV-1 infectious titration
HIV-1
IIIBPress Johnson﹠amp; The described method improvement of Byington carries out titration, is summarized as follows: the HIV-1 stock solution is done 4 times of dilutions on 96 orifice plates, and 10 gradients, 6 repeating holes of every gradient are provided with control wells 6 holes simultaneously.Every hole adds C8166 cell 50 μ l (4 * 10
5/ ml), every hole final volume is 200 μ l.37 ℃, 5%CO
2Cultivate.Added fresh RPMI-1640 perfect medium 100 μ l on the 3rd day, under inverted microscope, observed HIV-1 inductive cytopathic effect (Cytopathic effect in every hole on the 7th day, CPE), whether there is the formation of synplasm (Syncytium) to determine with every hole; Press Reed﹠amp; The Muench method is calculated the TCID of virus
50(50%Tissue culture infection dose).
(5) compound is to the toxicity test of C8166 cell
4 * 10
5/ ml C8166 cell suspension 100ul and different testing compound solution mixing establish three repeating holes.The control wells that does not contain compound, 37 ℃, 5%CO are set simultaneously
2Cultivated 3 days, and adopted the MTT colorimetry to detect cytotoxicity.The ELx800 microplate reader is measured the OD value, and the mensuration wavelength is 595nm, and reference wavelength is 630nm.Calculate CC
50Value (50%Cytotoxicconcentration), the compound concentration during promptly to 50% normal T lymphocyte series C8166 toxigenicity.
(6) compound H IV-1
IIIBThe inhibition experiment of cytopathogenic effect (CPE)
With 8 * 10
5/ ml C8166 cell 50ul/ hole is inoculated on the 96 porocyte culture plates that contain 100 μ l/ hole gradient doubling dilution compounds, adds the HIV-1 of 50 μ l then
IIIBThe dilution supernatant, 1300TCID
50/ hole.If 3 repeating holes.The normal cell control wells that does not contain compound is set simultaneously.The positive medicine contrast of AZT.37 ℃, 5%CO
2Cultivated 3 days, (100 *) count plasmodial formation under the inverted microscope.EC
50(50%Effective concentration) forms 50% o'clock compound concentration for suppressing synplasm.
(7) compound infects the provide protection experiment of MT4 cell to HIV-1
With 8 * 10
5/ ml MT4 cell 50ul/ hole is inoculated on the 96 porocyte culture plates that contain 100 μ l/ hole doubling dilution compounds, and half hole of culture plate adds the HIV-1 of 50 μ l
IIIBDilution (M.O.I.=006), second half hole adds 50 μ l substratum.2 repeating holes of each concentration gradient are provided with control wells and the blank hole that does not contain compound, 37 ℃, 5%CO simultaneously
2Cultivate, 100 μ l fresh cultures were added in every hole in the 3rd day, adopted the MTT colorimetry to detect cell survival rate in the 5th day or the 6th day.The ELx800ELISA instrument is measured the OD value, and the mensuration wavelength is 595nm, and reference wavelength is 630nm.Calculate compound to Normocellular toxicity with to HIV-1 with formula
IIIBThe provide protection of cells infected.
(8) calculation formula
Draw dose response curve according to experimental result, press Reed﹠amp; The Muench method calculates the 50% effective concentration (EC that compound suppresses virus
50), 50% cell growth inhibiting concentration (CC
50) and the active therapeutic index of anti-HIV-1 (Therapeutic index TI) is: TI=CC
50/ EC
50
1, cell growth survival rate (%)=experimental port OD value/control wells OD value * 100
2, the cytopathogenic inhibiting rate of HIV-1 (%)=(1-experimental port synplasm number/control wells synplasm number) * 100
3, the protection ratio of cells infected (%)=(experimental port OD value-positive control hole OD value)/(negative control hole OD value-positive control hole OD value) * 100
2. result
(1) compound is the toxic action of C8166 cell to the human T lymphocyte
Table 1 compound is to the experimental data of the toxic action of C8166 cell
(2) compound is to HIV-1
IIIBInduce the cytopathic restraining effect of C8166
Table 2. compound is to HIV-1
IIIBInduce the cytopathic restraining effect experimental data of C8166
(3) compound is to HIV-1
IIIBInfect MT
4The provide protection of cell
Table 3. compound is to experiment strain HIV-1
IIIBInfect MT
4The experimental data of cytoprotection
3. test-results gathers
Table 4: the active summary sheet as a result of compound vitro cytotoxicity and anti-HIV-1:
Compound shown in the present and HIV-1 intergrase have very high combination activity, and can effectively suppress the combination of intergrase to substrate in the substrate competition test.The present inventor also finds, the compound that embodiment 8 (IA-3) is disclosed is in external anti HIV-1 virus experiment, toxicity to the C8166 cell is less, CC50 is greater than 200 μ g/ml, the EC50 that induces the C8166 cell to form the synplasm inhibition to HIV-1 is 1.13-2.41 μ g/ml, therapeutic index TI is 177.46～73.63, has further confirmed the effect of this compounds anti HIV-1 virus.
Industrial applicibility
Of the present invention 2,4-disubstituted amido-6-replacement-[1,3,5] triazine or 1, advantage such as the preparation method of 3-pyrimidines has that reaction conditions gentleness, abundant raw material are easy to get, operation and aftertreatment are simple.
Of the present invention 2,4-disubstituted amido-6-replacement-[1,3,5] triazine or 1, the 3-pyrimidines the computer virtual screening and with the binding constant of HIV-1 intergrase, and in the substrate competition test, can effectively suppress the combination of intergrase to substrate.Verified the mechanism of action that anti HIV-1 virus infects.
Toxicity of compound of the present invention is very low.
Therefore, compound of the present invention can be used for preparing the medicine of treatment HIV virus infection.
Claims (8)
1.2 4-disubstituted amido-6-replacement-[1,3,5] compound in triazine class or its salt that is become with pharmaceutically acceptable bronsted lowry acids and bases bronsted lowry is specially:
4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-and the hydrazono-methyl]-1, the 3-dihydroxy-benzene,
2-acetoxy-benzoic acid 3-(2-acetoxyl group benzoyloxy)-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
4-pivaloyl aminobenzoic acid 3-(4-pivaloyl oxygen base benzoyloxy)-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
Phenylformic acid 3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
(4-{N '-[4-(3-the bromo-benzyloxy-)-rare base of 2-methoxyl group-benzyl]-diazanyl }-6-morpholine-4-base-[1,3,5]-triazine-2-yl)-aniline,
3-bromo-benzoic acid 3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
4-trifluoromethylbenzoic acid 3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
4-toluene sulfonic acide acid 3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
3-bromo-benzoic acid 2-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester,
3-bromo-benzoic acid 2-oxyethyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester, or
4-chloro-benzoic acid 3-methoxyl group-4-[(4-morpholine-4-base-6-phenylamino-[1,3,5] triazine-2-yl)-the hydrazine methene]-phenyl ester.
2. a medicinal compositions is characterized in that, it is described 2 that this medicinal compositions comprises claim 1, and 4-disubstituted amido-6-replaces-[1,3,5] compound in triazine class and vehicle, carrier or thinner.
3. medicinal compositions as claimed in claim 2 comprises the described compound of 0.05mg～200mg in the unitary dose of its pharmaceutical formulation.
4. medicinal compositions as claimed in claim 2 comprises the described compound of 0.1mg～100mg in the unitary dose of its pharmaceutical formulation.
5. medicinal compositions as claimed in claim 2, its route of administration are oral, nasal cavity suction, Transdermal absorption, pulmonary administration or parenteral drug administration by injection.
6. medicinal compositions as claimed in claim 2 is characterized in that: when it is used for the treatment of, described medicinal compositions is used in combination with other anti HIV-1 virus infection medicine.
7. medicinal compositions according to claim 2 is characterized in that: described medicinal compositions exists with the composition forms of anti HIV-1 virus infection medicine with other, optionally, exists with the unit dosage form that separates when drug packages.
8. claim 1 is described 2, and 4-disubstituted amido-6-replacement-[1,3,5] compound in triazine class or the medicinal compositions that comprises the described compound of claim 1 prevent and/or treat application in the medicine of HIV virus infection and relative disease thereof in preparation.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2006101700247A CN101245051B (en) | 2005-12-22 | 2006-12-22 | 2,4-di-substituted amido-6-substituted-[1,3,5]triazine or miazines compound, preparation method, pharmaceutical combination and use of the same |
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| CN200510132243 | 2005-12-22 | ||
| CN200510132243.1 | 2005-12-22 | ||
| CN2006101700247A CN101245051B (en) | 2005-12-22 | 2006-12-22 | 2,4-di-substituted amido-6-substituted-[1,3,5]triazine or miazines compound, preparation method, pharmaceutical combination and use of the same |
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| CN101245051A CN101245051A (en) | 2008-08-20 |
| CN101245051B true CN101245051B (en) | 2011-04-20 |
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| WO (1) | WO2007071199A1 (en) |
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| WO2011039735A2 (en) * | 2009-10-02 | 2011-04-07 | Consiglio Nazionale Delle Ricerche | Compounds with ddx3 inhibitory activity and uses thereof |
| CA2803687C (en) * | 2010-07-20 | 2019-04-30 | Vestaron Corporation | Insecticidal triazines and pyrimidines |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999036410A1 (en) * | 1998-01-13 | 1999-07-22 | Scriptgen Pharmaceuticals, Inc. | Triazine antiviral compounds |
| CN1295566A (en) * | 1998-03-27 | 2001-05-16 | 詹森药业有限公司 | Trisubstituted 1,3,5-triazine derivatives for treatment by HIV infections |
| WO2003062392A2 (en) * | 2002-01-18 | 2003-07-31 | Ceretek Llc | Methods of treating conditions associated with an edg receptor |
| GB2397301A (en) * | 2003-01-14 | 2004-07-21 | Novo Pharmaceuticals Ltd De | Substituted 1,3,5-triazine derivatives |
| US20040167132A1 (en) * | 2003-01-16 | 2004-08-26 | Geetha Shankar | Methods of treating conditions associted with an Edg-2 receptor |
| CN1668305A (en) * | 2002-05-13 | 2005-09-14 | 泰博特克药品有限公司 | Microbicidal pyrimidine or triazine for preventing sexual HIV transmission |
-
2006
- 2006-12-22 WO PCT/CN2006/003546 patent/WO2007071199A1/en active Application Filing
- 2006-12-22 CN CN2006101700247A patent/CN101245051B/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999036410A1 (en) * | 1998-01-13 | 1999-07-22 | Scriptgen Pharmaceuticals, Inc. | Triazine antiviral compounds |
| CN1295566A (en) * | 1998-03-27 | 2001-05-16 | 詹森药业有限公司 | Trisubstituted 1,3,5-triazine derivatives for treatment by HIV infections |
| WO2003062392A2 (en) * | 2002-01-18 | 2003-07-31 | Ceretek Llc | Methods of treating conditions associated with an edg receptor |
| CN1668305A (en) * | 2002-05-13 | 2005-09-14 | 泰博特克药品有限公司 | Microbicidal pyrimidine or triazine for preventing sexual HIV transmission |
| GB2397301A (en) * | 2003-01-14 | 2004-07-21 | Novo Pharmaceuticals Ltd De | Substituted 1,3,5-triazine derivatives |
| US20040167132A1 (en) * | 2003-01-16 | 2004-08-26 | Geetha Shankar | Methods of treating conditions associted with an Edg-2 receptor |
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|---|---|
| WO2007071199A1 (en) | 2007-06-28 |
| CN101245051A (en) | 2008-08-20 |
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