CN101438288A - Regulation of phosphoryl transferase activity of glutamine synthetase - Google Patents

Regulation of phosphoryl transferase activity of glutamine synthetase Download PDF

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CN101438288A
CN101438288A CNA2006800545929A CN200680054592A CN101438288A CN 101438288 A CN101438288 A CN 101438288A CN A2006800545929 A CNA2006800545929 A CN A2006800545929A CN 200680054592 A CN200680054592 A CN 200680054592A CN 101438288 A CN101438288 A CN 101438288A
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atp
adenylylation
alkyl
heteroaryl
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科林·彼得·凯尼恩
林登·凯里·奥德菲尔德
克里斯蒂安·韦南德·范德-韦斯特许仁
阿曼达·路易丝·鲁索
克里斯托弗·约翰·帕金森
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CSIR Corp
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Abstract

The present invention provides a method of screening and designing compounds that are inhibitors of glutamine synthetase, including adenylylated glutamine synthetase. The invention also provides compounds and compositions for treating, preventing and/or ameliorating bacterial infections, including mycobacterium tuberculosis.

Description

The adjusting of the phosphoryl transferase activity of glutamine synthelase
Technical field
The present invention relates to be used for the phosphoryl transferase activity of regulatory enzyme, comprise material and method by the phosphoryl transferase activity of phosphine formic acid (carboxyphosphate) intermediate mediation.For example, be provided for regulating the material and the method for activity of glutamine synthetase, comprise the material and the method in the phosphoryl transferase site that is used to regulate the adenylylation glutamine synthelase.
Background technology
Glutamine synthelase (GS, EC 6.3.1.2) is to participate in nitrogen metabolism and the reversible core enzyme that is converted into L-glutaminate, ADP and inorganic phosphate of catalysis L-glutamic acid, ATP and ammonia.This reaction is regulated by gamma-glutamyl phosphoric acid intermediate.There are three kinds of multi-form glutamine synthelase: GSI, GSII and GSIII.The enzyme of GSI form only is present in bacterium (eubacteria) and the ancient bacterium (archeobacteria).GSII is present in eukaryotic and some soil bacteria, and only finds the GSIII gene in the minority bacterial classification.
Two important GSI subphylums are arranged: GSI-α and GSI-β.In Thermophilic Bacteria, low G+C gram-positive bacteria and wide ancient bacterium (comprising methanogen class, Halophiles class and some thermophilic mushrooms), find GSI-α gene, and in all other bacteriums, find GSI-β gene.GSI-β enzyme is by adenylylation/go the adenylylation cascade to be conditioned, and contains non-existent 25 aminoacid insertion sequences in the GSI-alpha form.Bacterium with GSI-β gene comprises: Bacterium diphtheriae, gonococcus, Escherichia coli, salmonella typhimurium, salmonella typhi, Klebsiella Pneumoniae, serratia marcescens, proteus vulgaris, Shigella dysenteriae, comma bacillus, pseudomonas aeruginosa, Bacillus foecalis alkaligenes, helicobacter pylori, haemophilus influenzae, Bordetella pertussis, the special bacterium of bronchitis Boulder, Neisseria meningitidis, Brucella melitensis, Much's bacillus, Mycobacterium leprae, Tyreponema pallidum, leptospira interrogans, actinomyces israelii, Nocardia asteroides, Thiobacillus ferrooxidans, Azospirillum brasilense, anabena, Fremyella diplosiphon and streptomyces coelicolor.The bacterium of GSI-α subphylum comprises Bacillus cercus, bacillus subtilis, Bacillus anthracis, streptococcus pneumonia, streptococcus pyogenes, staphylococcus aureus, clostridium botulinum, clostridium tetani and C.perfringens.
In the Escherichia coli with GSI-β gene and other bacterium, regulate the GS activity by the adenylylation between 1 and 12 the GS subunits.The adenylylation site of all protokaryon bacteriums (being equivalent to the Tyr397 in the Escherichia coli) all shows high conservative, and the utilizability of the nitrogen energy and the carbon energy is relevant in adenylylation degree and the nutrient culture media.Have in the cell that glutamine synthelase results from excess nitrogen and the carbon existence of limiting the quantity of is grown down of 10-12 adenylylation subunit.The adenylylation of GS also changed enzyme to bivalent metal ion from Mg 2+To Mn 2+Specificity.
The destruction of the society and economy that flourishing and underdeveloped countries is brought in view of bacterial infection and the growth at full speed of bacteria antibiotic endurance strain, being used for the treatment of, preventing and improve the microbiotic of the new kind of bacterial infection can be of great use.
Summary of the invention
Unless specify in addition, all GS total number of atnino acid that this paper identifies are meant the residue of Escherichia coli (E.coli) GS.If provide the homology between the bacterium GS peptide sequence, those skilled in the art just can utilize methods such as comparison of homology for example or molecular simulation to measure the corresponding residue that GS paid close attention to of other kind.
This instructions is based on following discovery, and promptly in the process that forms gamma-glutamyl phosphoric acid intermediate, the GS of adenylylation form utilizes and comprises (Mn 2+) 3(HCO 3 -) 12Unique reaction mechanism of ATP compound is transferred to γ-phosphate of ATP the γ-carboxylate of glutamic acid.Therefore, can utilize about this compound, this compound binding site and the information of the relevant phosphine formic acid intermediate phosphoryl transfer mechanism of proposition compound of designing target adenylylation GSI-β enzyme on GS.It is active and therefore be used for the treatment of, prevent or improve bacterial infection that this compound can be used for suppressing adenylylation GS.Because by adenylylation/go adenylylation cascade scalable bacterium GSI-β enzyme; but can not regulate mammiferous GSII enzyme; therefore adenylylation GS there is inhibiting effect but to going adenylylation GS not have or only have minimum inhibiting compound to can be used for optionally suppressing the growth of GSI-β bacterial cell, the negative effect to mammalian cell simultaneously also is minimized.Therefore provide computer assistant methods with design experiment inhibitor compound and the method that is used for the inhibition activity of shaker test molecule in external and the body.This paper also is provided for suppressing the GS activity, comprises adenylylation and removes the compound and the composition of adenylylation GS activity; Be used to suppress or prevent the compound and the composition of bacterial growth in external and the body; And the compound and the composition that are used for the treatment of, prevent or improve the mammal bacterial infection.
Therefore; in one embodiment; this paper provides the computer-aid method of test inhibitor of the phosphoryl transferase site activity of a kind of generation adenylylation glutamine synthelase (GS) polypeptide; described method is used the program mode computing machine that comprises processor and input equipment, and described method comprises:
(a) comprise the data of the phosphoryl transferase site structure of GS polypeptide to the input of described input equipment;
(b) using described processor will test inhibitor molecules is docked in the described phosphoryl transferase site; And
(c), determine whether described test inhibitor molecules for example suppresses the activity in described phosphoryl transferase site by the formation that suppresses phosphine formic acid intermediate based on described butt joint.This method can further comprise (Mn 2+) 3(HCO 3 -) 12One or more structural motifs (structuralmotifs) of ATP compound are docked in the described phosphoryl transferase site.This method can comprise based on described butt joint, determines whether described test inhibitor molecules suppresses described (Mn 2+) 3(HCO 3 -) 12One or more structural motifs of ATP compound combine with described phosphoryl transferase site, perhaps suppress the formation of phosphine formic acid intermediate.
In some embodiment, this method can comprise design by the definite test inhibitor of step (c) with the active and described test inhibitor of in-vitro evaluation that suppresses described phosphoryl transferase site inhibition activity to adenylylation glutamine synthelase polypeptide.In-vitro evaluation can comprise the analytic approach of using energy measurement ATP hydrolysis, ADP formation, glutamic acid utilization or glutamine to form.
In some embodiment; this method can comprise further that the described test inhibitor of in-vitro evaluation is to going the inhibition activity of adenylylation glutamine synthelase polypeptide; active to estimate described test inhibitor to the specificity inhibition of described adenylylation glutamine synthelase polypeptide; and/or generate described test inhibitor and estimate the inhibition activity of described test inhibitor the bacterial growth that contains GSI-β glutamine synthetase gene; for example, this bacterium is selected from Bacterium diphtheriae; gonococcus; Escherichia coli; salmonella typhimurium; salmonella typhi; Klebsiella Pneumoniae; serratia marcescens; proteus vulgaris; Shigella dysenteriae; comma bacillus; pseudomonas aeruginosa; Bacillus foecalis alkaligenes; helicobacter pylori; haemophilus influenzae; Bordetella pertussis; the special bacterium of bronchitis Boulder; Neisseria meningitidis; Brucella melitensis; Much's bacillus; Mycobacterium leprae; Tyreponema pallidum; leptospira interrogans; actinomyces israelii; Nocardia asteroides; Thiobacillus ferrooxidans; Azospirillum brasilense; anabena; Fremyella diplosiphon and streptomyces coelicolor.
In some embodiment, this method comprises estimates described test inhibitor to eukaryotic, for example the inhibition activity of mammalian cell growth.
On the other hand, provide a kind of method that produces the compound of the phosphoryl transferase site activity that suppresses adenylylation glutamine synthelase polypeptide, said method comprising the steps of:
(a) provide the three-dimensional structure of glutamine synthelase polypeptide; And
(b) based on described three-dimensional structure, design can suppress described phosphoryl transferase site and (Mn 2+) 3(HCO 3 -) 12Interactional test compound between one or more structural motifs of ATP compound.The three-dimensional structure of described glutamine synthelase polypeptide comprises the (Mn that is attached to described phosphoryl transferase site 2+) 3(HCO 3 -) 12One or more structural motifs of ATP compound.
In another embodiment, provide a kind of method that produces the test compound of the phosphoryl transferase site activity that suppresses adenylylation glutamine synthelase polypeptide, described method comprises:
(a) (Mn is provided 2+) 3(HCO 3 -) 12The three-dimensional structure of ATP compound; And
(b) based on described three-dimensional structure, design has and is similar to (Mn 2+) 3(HCO 3 -) 12The test compound of one or more structural motifs of ATP composite structure.
On the other hand, provide a kind of body outer screening test compound to determine whether described test compound suppresses the method for the phosphoryl transferase site activity of adenylylation glutamine synthelase polypeptide, and described method comprises:
(a) under the effective condition of formation, adenylylation glutamine synthelase polypeptide is contacted with test compound to phosphoryl transferase activity and/or phosphine formic acid intermediate; And
(b) determine with respect to also not with the activity of the contacted adenylylation glutamine synthelase polypeptide of described test compound, whether the activity of the phosphoryl transferase of described adenylylation glutamine synthelase polypeptide decreases.This method can further comprise determines whether the described activity of the phosphoryl transferase of adenylylation glutamine synthelase polypeptide of going decreases with respect to also there not being the activity of removing adenylylation glutamine synthelase polypeptide contacted with described test compound.
A kind of in-vitro method that is used to suppress the phosphoryl transferase site activity of adenylylation GS polypeptide also is provided, and described method comprises to be made adenylylation GS polypeptide and contains the compound compositions that formula I as herein described, II, III, IV, V, VI or VII represent and contact.
A kind of in-vitro method that is used to suppress contain the bacterial growth of GSI-β gene also is provided, and wherein said method comprises to be made described bacterium and contains the compound compositions that formula I as herein described, II, III, IV, V, VI or VII represent and contact.
On the other hand, provide a kind of and be used for the treatment of, prevention or relevant with mammiferous bacterial infection or relevant with mammiferous bacterial infection one or more symptoms of improvement or the method for obstacle with bacterial infection danger, wherein said bacterial infection causes by the bacterium that contains GSI-β gene, and described method comprises and gives described mammal with containing the compound compositions that formula I as herein described, II, III, IV, V, VI or VII represent.
Method in a kind of body of the phosphoryl transferase site activity that is used to suppress adenylylation glutamine synthelase polypeptide also is provided, and described method comprises:
(a) will contain the mammal that compound compositions that formula I as herein described, II, III, IV, V, VI or VII represent is subjected to bacterial infection, wherein said bacterial infection is caused by the bacterium that contains GSI-β gene.
This paper also provides the compound of for example representing suc as formula I, II, III, IV, V, VI or VII, simultaneously the pharmaceutical composition that its pharmaceutically acceptable salt and derivant also is provided and contains these compounds.Also be provided for treating, preventing or improve the purposes of the compound or the pharmaceutical composition of mammal bacterial infection.The specific compound that for example has the specific identification compound number of the middle use of same procedure also is provided.
Unless otherwise defined, all technical terms used herein and scientific terminology and general technical staff of the technical field of the invention the same meaning generally understood.If any conflict, presents comprises that definition will determine protection domain.Although also can be used for implementing the present invention or the present invention being tested following preferable methods and the material put down in writing with method described herein and materials similar or the method that is equal to and material.All publications, patented claim, patent and other list of references all are incorporated herein with integral body.Material disclosed herein, method and embodiment only are used for explanation and the intent of the present invention without limits.
From following explanation, accompanying drawing and claims, will obviously find out other features and advantages of the present invention, and for example suppress the activity of adenylylation GS and be used for the treatment of the method for bacterial infection thus.
Description of drawings
Fig. 1 has described the catalytic mechanism of proposition in the phosphoryl transferase site of adenylylation GS.
Lowercase is meant step proposed below:
A. the cooperation of carbon dioxide huge legendary turtle is used;
B. electric charge displacement;
C. the molecule inner proton shifts;
D. metal formization;
E. the formation of carbamyl phosphate;
F. phosphoryl shifts; And
G. collapse.
Embodiment
In other was found, the inventor had found than at Mg 2+The enzyme that goes the adenylylation form under existing is at Mn 2+There is a kind of new glutamine synthelase ATP-phosphoryl transfer reaction mechanism in the GS enzyme of the adenylylation form under existing.This reaction mechanism shows need exist carbonate and Mn 2+, with ATP with (Mn 2+) 3(HCO 3 -) 12The form of ATP compound plays a role.Further illustrate as following, the reaction of supposing comprises the N of ATP 7Carboxylated, form to allow C 8The HCO of proton by associating 3 -The cationic imide of deprotonation (immonium species), and follow formation Mn 2+Cabbeen, and carry out N subsequently 7Carboxylate forms phosphine formic acid intermediate to the attack of γ-phosphoric acid, impels then by some important amino acid side chain of GS (that is, His269 and His271) to make phosphoryl transfer to glutamic acid.Referring to Fig. 1.
Therefore, this paper proposes, based on (Mn 2+) 3(HCO 3 -) 12The structure of ATP compound or the ingredient of its structure and can design specific adenylylation GS inhibitor based on the analysis of their binding sites in adenylylation GS, and propose these inhibitor and make enzyme be attached to (Mn 2+) 3(HCO 3 -) 12ATP-binding site is to suppress bacterial enzyme; Or inhibition (Mn 2+) 3(HCO 3 -) 12ATP is in the combination of binding site; Or the formation of inhibition phosphine formic acid intermediate. Definition
Except as otherwise noted, term " GS polypeptide " and " glutamine synthelase polypeptide " are used interchangeably in this article, for example are meant the bacterium GSI polypeptide from GSI-α or GSI-β bacterium.Except as otherwise noted, this term comprises the polypeptide and the fragment thereof of total length.In addition, as those skilled in the art will generally acknowledge, GS can be used as ten dimers in the body, the GS avtive spot can be made up of the amino acid that comes from an above monomer.Therefore, this term also comprises the polymer of polymer (for example dimer, tripolymer, the tetramer, pentamer, six aggressiveness, heptamer, eight aggressiveness, nine aggressiveness, ten aggressiveness, 11 aggressiveness and ten dimers), the GS fragment of total length GS, as one or more residues of one or more GS avtive spot parts (one group of residue for example, described residue may be in the primary sequence of GS adjacency not, but form at least a portion of GS avtive spot) and the polymer of these combinations.
" polypeptide " and " albumen " is used interchangeably, and is meant no matter length or posttranslational modification, amino acid whose any peptides connection chain (peptide-linked chain).
Term " separation " can refer to not have homologue that nature exists or the polypeptide of isolated or purified from for example following tissue of the composition that accompanies naturally with it or body fluid, and described organizing for example is pancreas, liver, spleen, ovary, testis, muscle, joint tissue, nerve fiber, gastrointestinal tissue or tumor tissues (for example breast cancer or colon cancer tissue); Described body fluid for example is blood, serum or urine, or from bacterium or fungus culture medium the polypeptide of isolated or purified.Usually, when the polypeptide of at least 70% dry weight does not contain the albumen that is associated naturally with it and other organic molecule that exists naturally, think that polypeptide is " separation ".
Preferably, polypeptide formulations is the polypeptide of at least 80%, more preferably at least 90% and most preferably at least 99% dry weight.Because the polypeptide of chemosynthesis itself is from separating the composition of coexistence naturally with it, so synthetic polypeptide is " separation ".
The polypeptide that separates can be for example by from natural origin (for example from tissue) extraction, by coded polypeptide recombinant nucleic acid expression or obtain by chemosynthesis.Because must not contain the composition that accompanies naturally with it, therefore the polypeptide that produces in the cell system different with producing the polypeptide source naturally is " separation ".By any suitable method, for example column chromatography, polyacrylamide gel electrophoresis or HPLC analyze the degree that can measure isolated or purified.
Before detecting, can modify for example adenylylation, phosphorylation or glycosylation by methods known in the art and method described herein to any polypeptide.
" pharmaceutically acceptable derivates " of compound used herein comprises salt, ester, enol ether, enol ester, acetal, ketal, ortho esters, hemiacetal, hemiketal, acid, alkali, solvate, hydrate or its pro-drug.Use is used for these known methods of deriving, and those skilled in the art can prepare these derivants at an easy rate.The compound that generates is given the animal or human and do not have substantial toxic action, and this compound has pharmaceutical active or is pro-drug.
Pharmaceutically acceptable salt comprises amine salt, N for example, N '-dibenzyl-ethylenediamin, chloroprocanine, choline, ammonia, diethanolamine and other hydroxyalkyl amine, ethylenediamine, N-methylglucosamine, procaine, N-benzyl-1-phenylethylamine, 1-be right-benzyl chloride base-2-pyrrolidine-1 '-ylmethyl-benzimidazole, diethylamine and other alkyl amine, piperazine and three (methylol) aminomethane, but be not limited thereto; Alkali metal salt, for example lithium salts, sylvite and sodium salt, but be not limited thereto; Alkali salt, for example barium salt, calcium salt and magnesium salts, but be not limited thereto; Transition metal salt, zinc salt for example, but be not limited thereto; With other slaine, for example dibastic sodium phosphate and disodium hydrogen phosphate, but be not limited thereto; Also include but not limited to nitrate, borate, mesylate, benzene sulfonate, toluene sulfonate, inorganic acid salt (for example hydrochloride, hydrobromate, hydriodate and sulfate, but be not limited thereto); And acylate, for example acetate, trifluoroacetate, maleate, oxalates, lactate, malate, tartrate, citrate, benzoate, salicylate, ascorbate, succinate, butyrate, valerate and fumarate, but be not limited thereto.Pharmaceutically acceptable ester comprises Arrcostab, alkenyl esters, alkynyl ester, aryl ester, heteroaryl ester, aralkyl ester, heteroarylalkyl ester, cycloalkyl ester and the heterocyclic ester of acidic group, but be not limited thereto, wherein said acidic group comprises carboxylic acid, phosphoric acid, hypophosphorous acid, sulfonic acid, sulfinic acid and boric acid, but is not limited thereto.Pharmaceutically acceptable enol ether comprises the derivant of formula C=C (OR) expression, and wherein R is hydrogen, alkyl, thiazolinyl, alkynyl, aryl, heteroaryl, aralkyl, heteroarylalkyl, naphthenic base or heterocyclic radical, but is not limited thereto.Pharmaceutically acceptable enol ester comprises the derivant of formula C=C (OC (O) R), and wherein, R is hydrogen, alkyl, thiazolinyl, alkynyl, aryl, heteroaryl, aralkyl, heteroarylalkyl, naphthenic base or heterocyclic radical, but is not limited thereto.Pharmaceutically acceptable solvate and hydrate are the compound of one or more solvents or hydrone and compound, or the compound of about 10 or about 2 of 1-, 1-of about 100 or 1-of 1-about 3 or about 4 solvents of 1-or hydrone and compound.
Treatment used herein mean make by any way bacterial infection for example one or more symptoms of m tuberculosis infection be improved or other take place and advantageously change.Treatment also comprises any medicinal usage of composition as herein described, for example be used for the treatment of mammal (for example people) suspect be subjected to bacterial infection or with the purposes of disease, obstacle or the imbalance of bacterial infection implication.
Used hereinly be improved and be meant and anyly be attributable to that composition gives or give relevant alleviating with composition by giving symptom that specific compound or pharmaceutical composition make particular obstacle, no matter this alleviate be lasting or interim, continue or of short duration.
IC used herein 50Be meant in the method for measuring response, the inhibition of peak response reached amount, concentration or the dosage of 50% special test compound.
Term Ki used herein represents the dissociation constant of enzyme/inhibitor complexes.Ki is irrelevant with the substrate that is suppressed the agent test in theory.User's formula K i=IC 50* K m/ (S+K m) by IC 50But calculating K i, wherein, S is a concentration of substrate, K mBe that reaction velocity is the concentration of substrate (when not having inhibitor) of maximal value one half.Be used to suppress specific substrates (fixing K m) the K of inhibitor iIt is constant.EC used herein 50Be meant the drug concentration when reaching 50% inhibition, and CC 50Be meant the drug concentration when reaching 50% toxicity.
Pro-drug used herein is to give in body, by one or more steps or process by metabolism or by other mode be converted into this compound biologically, pharmaceutically or the activity form on the acology.For generating pro-drug, reactive compound is pharmaceutically modified so that this reactive compound is regenerated by metabolic process.Can design pro-drug to change its metabolic stability or drug transport characteristic, cover spinoff or toxicity, improve the medicine taste or to change other characteristic or the character of medicine.In a single day those skilled in the art know the compound with pharmaceutical active, the pro-drug that can design this compound by the knowledge of interior medicine dynamics process and drug metabolism is (referring to for example Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, 388-392).
It should be understood that compound provided herein can contain chiral center.This chiral center is (R) or (S) configuration, perhaps can be its potpourri.Therefore, compound provided herein can be enantiomer-pure or spatial isomerism or diastereoisomeric potpourri.For amino acid residue, these residues can be L-type or D-type.Naturally the configuration of the amino acid residue of Cun Zaiing L-type normally.When not specifying, residue then is the L-type.Term " amino acid " is meant a-amino acid as used herein, is racemic or D-form or L-configuration.The preceding symbol " d " of amino acid title (for example dAla, dSer, dVal etc.) is meant amino acid whose D-isomeride.Symbol " dl " before the amino acid title is meant amino acid L-isomeride and D-mixture of isomers.Epimerization may take place in chiral center in vivo that it should be understood that compound provided herein.Similarly, those skilled in the art will recognize that the compound for epimerization takes place in vivo gives (R) type compound and is equal to and gives (S) type compound.
Used herein about compound " quite pure " to such an extent as to be meant that enough homogeneous are when using those skilled in the art estimate the standard method of analysis that purity is used, for example thin layer chromatography (TLC), gel electrophoresis, high performance liquid chromatography (HPLC) and/or mass spectroscopy (MS) are when measuring, show the impurity do not contain easy detection, to such an extent as to or enough purely be further purified the physical property that can not detect material and the change of chemical property (for example enzymatic activity and biologically active).Those skilled in the art become known for compound is carried out purifying to produce the chemically method of quite pure compound.Yet chemically quite pure compound can be the potpourri of steric isomer.In this case, be further purified the specific activity that can increase compound.
" alkyl " used herein, " thiazolinyl " and " alkynyl " are meant it can is the carbochain of straight or branched.Exemplary alkyl, thiazolinyl and the alkynyl of this paper comprises methyl, ethyl, propyl group, isopropyl, isobutyl, normal-butyl, sec-butyl, the tert-butyl group, isopentyl, neopentyl, tertiary pentyl, isohesyl, allyl (propenyl) and propargyl (propinyl), but is not limited thereto.
" naphthenic base " used herein is meant saturated monocycle or polycyclic system, contains 3-10 carbon atom in some embodiments, contains 3-6 carbon atom in other embodiments.The member ring systems of naphthenic base can comprise one or two or more rings, links together with condensed ring, bridged ring or volution ways of connecting.Example comprises cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl, but is not limited thereto.
" aryl " used herein is meant aromatic series monocycle or the many cyclic groups that contains 6-19 carbon atom.Aryl comprises for example unsubstituted or fluorenyl that replaces, unsubstituted or the phenyl and the naphthyl unsubstituted or that replace that replace, but is not limited thereto.
" heteroaryl " used herein is meant monocycle or Ppolynuclear aromatic member ring systems, in some embodiments, be about 5-15 unit ring, wherein the one or more atoms in the member ring systems are heteroatoms, in one embodiment, the atom of the 1-4 in the member ring systems is a heteroatoms, and described heteroatoms is other atoms of elements beyond the carbon, comprise nitrogen, oxygen or sulphur, but be not limited thereto.Randomly, heteroaryl can be fused to phenyl ring.Heteroaryl comprises furyl, imidazole radicals, pyrimidine radicals, tetrazole radical, thienyl, pyridine radicals, pyrrole radicals, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, quinolyl and isoquinolyl, but is not limited thereto.
" heterocyclic radical " used herein is meant the non-aromatic member ring systems of monocycle or many rings, be 3-10 unit ring in one embodiment, be 4-7 unit ring in another embodiment, be 5-6 unit ring in another embodiment, wherein the one or more atoms in the member ring systems are heteroatoms, in some embodiments, 1-3 in a member ring systems atom is a heteroatoms, described heteroatoms is other atoms of elements beyond the carbon, comprises nitrogen, oxygen or sulphur, but is not limited thereto.
" halogen " used herein, " halogen " or " halogenide " are meant F, Cl, Br or I.
Pseudohalide used herein or plan halogen are that its behavior is similar to halid group substantially.Using of this compound with handle can be identical with halogenide.Pseudohalide comprises prussiate, cyanate, thiocyanate, selenium cyanate, trifluoromethoxy and azide, but is not limited thereto.
" alkylhalide group " used herein is meant the alkyl that one or more hydrogen atoms are substituted by halogen.
" carboxyl " used herein is meant bivalent radical-C (O) O-.
" amino carbonyl " used herein is meant-C (O) NH 2
" aminoalkyl " used herein is meant-RNH 2, wherein R is an alkyl.
" alkoxy " used herein and " alkylthio group " are meant RO-and RS-, and wherein R is an alkyl.
" aryloxy group " used herein and " arylthio " are meant RO-and RS-, and wherein R is an aryl.
" amide group " used herein is meant divalent group-C (O) NH-.
" hydrazides " used herein is meant divalent group-C (O) NHNH-.
Given substituent number is uncertain (for example alkylhalide group) arbitrarily, may have one or more substituting groups.For example, " alkylhalide group " can comprise one or more identical or different halogens.
Except as otherwise noted, the abbreviation of any protecting group used herein, amino acid and other compound all meets their usual usage, generally acknowledged abbreviation or the IUPAC-IUB council regulation (referring to (1972) Biochem.11:942-944) for biological chemical name.
The method for designing of the inhibitor in phosphoryl transferase activity site
This paper is provided for the method for compound that design was attached to and/or suppressed the phosphoryl transferase site of GS polypeptide (particularly adenylylation GS polypeptide, more especially from the adenylylation GS polypeptide of GSI-β bacterium), comprises computer-based method.As described herein, the inventor supposes (Mn 2+) 3(HCO 3 -) 12The ATP compound is attached to the phosphoryl transfer activity site of adenylylation GS, thereby therefore produces unique reaction mechanism of γ-phosphoric acid of ATP being transferred to γ-carboxylate formation gamma-glutamyl phosphoric acid intermediate of glutamic acid.By analyzing (Mn 2+) 3(HCO 3 -) 12Interaction between ATP compound and the phosphoryl transfer activity site; one skilled in the art will recognize that how to use standard molecule simulation or other technology to identify polypeptide, intend peptide and micromolecule, thereby this polypeptide, plan peptide and micromolecule will suppress the activity of phosphoryl transferase in conjunction with the phosphoryl transferase site of adenylylation GS polypeptide.For example; micromolecule can directly be used for suppressing assumed response mechanism (for example preventing the formation of phosphine formic acid intermediate) with some amino acid in the phosphoryl transferase site; perhaps can be in the allosteric site effect, this allosteric site does not promptly participate in phosphoryl transferase activity directly but compound combines molecular domains that meeting causes (for example changing by the inducing molecule conformation) active inhibition with it.
" molecular simulation " is meant the quantitative and/or qualitative analysis based on the 26S Proteasome Structure and Function of the physical action of three-dimensional structure Information And Action model.This comprises conventional molecular dynamics and energy minimization model, interactive computer graphical model, decorating molecule mechanical model, geometric distance and based on the restricted model of other structure based on numeral.Usually use a computer and carry out molecular simulation and can use known method further to optimize.
Design specificity (for example with high affinity) is attached to the phosphoryl transferase site; the method in the phosphoryl transferase site of adenylylation GS polypeptide (for example adenylylation GSI-beta polypeptides) for example; usually also be based on computing machine, and relate to the computing machine that use has the program that can produce atomic model.Use X ray crystallization data computing machine program to be particularly useful for these compounds of design.For example, program (for example RasMol) can be used for producing the three-dimensional model of following material, for example phosphoryl transferase site or the (Mn of adenylylation GS, adenylylation GS 2+) 3(HCO 3 -) 12The ATP compound.Computer program, for example INSIGHT (Accelrys, Burlington, MA), Auto-Dock (Accelrys) and Discovery Studio 1.5 (Accelrys) allow further to handle and possess the ability of introducing new construction.
Can use for example computer hardware or software or the composite design compound of the two.Yet, preferably implement design with one or more computer programs of carrying out on one or more program mode computing machines, each program mode computing machine includes processor and at least one input equipment.Computerized optimization also comprises data-storage system (comprising impermanency and nonvolatil storer and/or memory element) and at least one output device.Service routine code input data realize above-mentioned functions and produce output information.In known manner output information is applied on one or more output devices.Computing machine for example can be personal computer, microcomputer or the workstation of conventional design.
Thereby preferably carrying out each program with high-caliber process programming language or object-oriented programming language exchanges with computer system.Yet, if need, can assembly language or machine language come executive routine.Under any circumstance, described language can be compiling type language or interpreted languages.
Preferably each computer program storage is used on the storage medium or equipment (for example ROM or disk) general or that special-purpose programmable calculator promptly can read.When program was read by computing machine, the effect of this computer program was that setting and operational computations machine are carried out program as herein described.Also can implement method of the present invention, realize function as herein described thereby the described storage medium that wherein so is provided with makes computing machine operate in specific and default mode by the computer-readable recording medium that computer program is set.
For example, in the method for design experiment compound, need the step of computing machine to comprise:
(a) for example import data by keyboard, disk or tape to input equipment, described data (for example atomic coordinates) limit three-dimensional (3-D) structure of first molecule or the compound that are attached to second molecule or compound.Described first molecule or compound for example be GS polypeptide, adenylylation GS polypeptide, GS polypeptide or adenylylation GS polypeptide fragment, constitute one group of residue in GS polypeptide or adenylylation GS polypeptide active site (for example phosphoryl transferase site), any in them can comprise ATP, ADP, glutamine or the glutamic acid molecule of one or more combinations; Described second molecule or compound for example are ATP, ADP, (Mn 2+) 3(HCO 3 -) 12The part of ATP compound or this compound; And
(b) use processor to measure and participate in being attached to first molecule of second molecule or compound or the 3-D structure (for example atomic model) in the site on the compound.
From the information that mode thus obtains, those skilled in the art for example can be on some residue and in some way (for example hydrogen bond action, electrostatic interaction and/or Van der Waals interact) design and make inhibition compound with suitable 3-D structure (for example peptide, non-peptide micromolecule, plan peptide and fit (for example aptamer)).For example, those skilled in the art can design and the interactive inhibition compound of some residue of first molecule.It should be noted, although original GS3-D structure from a kind, those skilled in the art can be by standard method, for example the corresponding residue that GS paid close attention to of other kind is determined in homology comparison or molecular simulation.
In addition, if obtain to be used for the 3-D data (for example, X-ray crystallization data) that the computing machine of candidate compound can be used, in conjunction with above-mentioned computer based step (a) and (b), can carry out one or more following computer based steps:
(c) for example by keyboard, disk or tape data (for example atomic coordinates) to input equipment input qualification candidate compound three-dimensional (3-D) structure;
(d) use processor to measure the 3-D structure (for example atomic model) of described candidate compound;
(e) use described processor to determine whether candidate compound is attached to the site on first molecule or the compound, or suppress the formation of phosphine formic acid intermediate, or prevent for example (Mn 2+) 3(HCO 3 -) 12The combination of ATP compound; And
(f) identify that candidate compound is to suppress to interact between first and second molecules or the compound or suppress the formation of phosphine formic acid intermediate or prevent for example (Mn 2+) 3(HCO 3 -) 12The compound of ATP compound combination.
This method can comprise other step, promptly exports the model of the 3-D structure of this compound to output device.In addition, the 3-D data of candidate compound can be compared with the Computer Database of the 3-D structure of for example storing in data-storage system.
For example, in some embodiment, the computer-aid method of the test inhibitor of the phosphoryl transferase site activity of generation adenylylation GS polypeptide can comprise:
(a) to input equipment input data, described data comprise the (Mn in the phosphoryl transferase site that is attached to the GS polypeptide 2+) 3(HCO 3 -) 12The structure of one or more structural motifs of ATP compound (structured data that for example, comprises the one group of residue that constitutes the phosphoryl transferase site);
(b) using processor will test inhibitor molecules is docked in the described phosphoryl transferase site; And
(c) based on described butt joint, whether the confirmed test inhibitor molecules suppresses the activity in phosphoryl transferase site.
Use other based on structure Design/analogue technique (referring to for example Jackson (1997) Seminars in Oncology 24:L164-172; With people (1996) J.Med.Chem.39:904-917 such as Jones), also can design compound of the present invention alternately by the structural information of compound described herein.Also can by the simulation that for example uses a computer identify candidate compound with compound of the present invention and polypeptide is accredited as spatially and optionally (promptly with high affinity) match with the phosphoryl transferase site.
Then, the cell inhibition method of the standard of being familiar with those skilled in the art or acellular enzyme inhibition method or enzyme inhibition method detect the candidate compound of above-mentioned evaluation.This paper has described exemplary method.
Can measure the 3-D structure of biomacromolecule (for example GS, adenylylation GS, nucleic acid, carbohydrates and lipid) by the data that obtain by the whole bag of tricks.These methods that have been used to estimate albumen 3-D structure most effectively comprise: (a) X ray Laue method; (b) nuclear magnetic resonance (NMR) spectroscopic methodology; (c) analysis of the physical distance constraint that forms between definite site on the big molecule, for example chemical crosslinking in the molecule between the residue on the albumen (international patent application no PCT/US00/14667 for example, its disclosure is incorporated herein by reference with integral body), and (d) based on the molecule simulation method of the primary structure knowledge of concern albumen, the program that for example uses a computer for example MONSSTER ( MOdeling Of NEw STructures from SEcondary and TErtiary REstraints) homology modeling technique, thread algorithm or the structural modeling that starts anew of (referring to for example international patent application no PCT/US99/11913, its disclosure is incorporated herein by reference with integral body).Also can use other Molecular Simulation Technique according to the invention (people (1990) J.Med.Chem.33:883-894 such as Cohen for example; People such as Navia (1992) Current Opinions in StructuralBiology, 2,202-210, its disclosure is incorporated herein by reference with integral body).The data that all these methods produce all can be subjected to the guidance of Computer Analysis.Also can be used for the inventive method but other spectroscopic methodology of relevant biomolecule atomic level structural details is not provided at present, comprise circular dichroism spectrometry, fluorescence chromatography and ultraviolet absorption spectroscopy.Preferred analytical approach is the X ray Laue method.Below this method and NMR spectroscopic methodology are described.
The X ray Laue method
The X ray Laue method is in the crystal of interest region, by centering on nuclear electron cloud, based on the X-ray diffraction of characteristic wavelength.This technology use the crystal of the biomacromolecule (but these biomacromolecules usually comprise solvent composition, co-factor, substrate or other part) of purifying to measure to constitute the macromolecular atom of particular organisms near atomic resolution.The condition precedent that discloses big molecule 3-D structure by the X ray Laue method is the orderly crystal of the strong diffraction X ray of energy.This method with a branch of x-ray bombardment to many same moleculars in order, the array that repeats so that X ray by this array can recover the pattern diffraction of individual molecule structure.Orderly crystal, for example the orderly crystal of globulin molecule be have irregular surface, big, coccoid or oval thing.Between individual molecule, crystal contains macropore.These account for crystal volume duct over half usually and are filled by unordered solvent molecule, and protein molecular only contacts with each other at place, minority zonule.A protein structure reason identical usually in the crystal why that Here it is with protein structure in the solution.
GS is by crystallization repeatedly, for example from the GS (Almassy, people such as R.J. (1986) Nature (London) 323:304-309, Liaw, people such as S-H. (1993) Proc.Natl.Acad.Sci. (USA) 90:4996-5000) of salmonella typhimurium; The GS (Liaw, S-H and D.Eisenberg (1994) J.Biol.Chem.33:675-681) that in avtive spot, has glycocoll, alanine and serine; In the avtive spot of 5 structures, have AMPPNP, glutamic acid, L-methionine-S-sulfoxide acid imide, glutamine and ADP GS (Liaw, S-H., Jun, G. and D.Eisenberg (1993) Protein Science, 2:470-471); And the GS (Liaw, S-H., Jun, G. and D.Eisenberg (1994) Biochemistry33:11184-11188) that in avtive spot, has ATP.Below describe or those skilled in the art have known the method that GS or GS fragment comprise the GS of adenylylation that obtains.The formation of crystal depends on some different parameters, comprises the character of pH, temperature, biomacromolecule concentration, solvent and precipitation agent and interpolation ion or the protein ligands that exists.Need many conventional crystallization experiments to be used for the combination of all these parameters with screening, the combination of described parameter can obtain being suitable for the crystal of X-ray diffraction analysis.The crystallization automation can make a large amount of repeatability work of crystallization experiment realize operating automatically and obtain quickening (referring to for example U.S. Patent number 5,790,421).
The polypeptide crystallization occurs in peptide concentration and surpasses in the solution of its maxima solubility (that is, polypeptide solution is oversaturated).This solution can restore balance by reducing peptide concentration, and described reduction peptide concentration is the precipitation by polypeptide crystal preferably.Usually, induce polypeptide crystallization from supersaturated solution by interactional reagent between interpolation change polypeptide surface charge or interference polypeptide and the body water to promote to cause the association of crystallization.
Crystallization is carried out between 4 ℃ to 20 ℃ usually.Usually use known material as " precipitation agent ", by around peptide molecule, being formed on disadvantageous precipitation depletion layer on the energy, reduce the solubleness of polypeptide in strong solution (Weber (1991) Advances in Protein Chemistry, 41:1-36).Except that precipitation agent, also add other material sometimes to the polypeptide crystallization solution.These materials comprise the buffering agent of regulator solution pH and reduce the salt of polypeptide solubleness.Known in the art have various precipitation agents, and comprise following listed: ethanol, 3-ethyl-2,4 pentanediol and many polyethylene glycols be polyglycol (PEG) for example.Precipitation solution can comprise for example 13-24%PEG 4000,5-41% ammonium sulfate and 1.0-1.5M sodium chloride, and pH is in the 5-7.5 scope.Other adjuvant can comprise 0.1MHepes, 2-4% butanols, 0.1M or 20mM sodium acetate, 50-70mM citric acid, 120-130mM sodium phosphate, 1mM ethylenediamine tetraacetic acid (EDTA) and 1mM dithiothreitol (DTT) (DTT).These reagent are prepared in damping fluid, and dropwise add in the crystallization damping fluid with various combinations.
Normally used polypeptide method for crystallising comprises following technology: batch (-type) crystallization, hanging drop type crystallization, crystal seed cause formula crystallization and dialysis.In above-mentioned every kind of method, importantly by keeping promoting continuous crystallisation behind the solution supersaturation formation nucleus.In the batch (-type) method for crystallising, polypeptide and precipitant mix to be to reach supersaturation, with seal of vessel and shelve until crystal and occur.In dialysis process, polypeptide is retained in the sealing dialysis membrane, described sealing dialysis membrane is placed the solution that contains precipitation agent.The whole machine balancing of film has increased the concentration of polypeptide and precipitation agent, makes polypeptide reach the supersaturation level thus.
Preferred hanging drop type crystallization technique (McPherson (1976) J.Biol.Chem., 251:6300-6306) in, precipitation agent added to generating initial mixtures of polypeptides in the polypeptide strong solution.The concentration of polypeptide and precipitation agent should be and makes polypeptide non-crystallizable under this original form.This potpourri of one droplet is placed on the microslide, described microslide is inverted and is hung on the liquid storage tank top of second solution.Then with system sealing.Usually, second solution contains precipitation agent or other dewatering agent of higher concentration.The difference of precipitation agent concentration makes the vapour pressure of this protein solution than the second solution height.Seal because contain the system of two kinds of solution, so equilibrium establishment, water is transferred to second solution by mixtures of polypeptides.This balance increases the concentration of polypeptide in the polypeptide solution and precipitation agent.In the critical concentration of polypeptide and precipitation agent, can form the crystal of polypeptide.
The other method of crystallization forms the site with nucleus and introduces the polypeptide strong solution.Usually, preparation polypeptide strong solution and the polypeptide crystal seed introduced this solution.If the concentration of polypeptide and any precipitation agent is suitable, crystal seed can provide nucleus to form the site, than megacryst formation around this nucleus forms site.
In addition, the other method of crystallization is the electrocrystallization method, in the method, utilizes the dipole moment (referring to U.S. Patent number 5,597,457) of the protein macromolecule of auto arrangement (self-align) in the contiguous Helmholtz layer of electrode.
Some albumen may be resisted crystallization.Yet those skilled in the art can use several technological guide crystallizations.For example, the flexible polypeptide portion except that deproteinized amino or c-terminus can promote to generate the crystalline protein sample.For example trypsase, chymotrypsin or subtilopeptidase A are handled albumen to remove these parts can to use Protocols in Molecular Biology or use proteinase.
In diffraction experiment, obtain narrow and parallel X ray light beam and shine on the crystal to produce diffracted beam by x-ray source.The incident main beam all causes damage to big molecule and solvent molecule.Therefore, make crystal cooling (for example being cooled to-220 ℃ to-50 ℃) to prolong its life-span.Main beam must be from a plurality of direction shooting crystal to produce all possible point diffraction, and therefore, at experimental session, crystal rotates in light beam.Be recorded in point diffraction on the film or by the electronic detectors record.The film of exposure must be carried out digitizing and quantification in scanning device, and electronic detectors are directly imported computing machine with its detected signal.The electronics regions detecting device significantly reduces to be gathered and measures the required time of diffraction data.The every bundle diffraction light that is write down with the point on the film uses three kinds of character to define: amplitude, measure according to the intensity of point; Wavelength is set according to x-ray source; And phase place, its loss in the X ray experiment.In order to measure the atom site that causes diffracted beam, all diffracted beams all need all these three kinds of character.A kind of mode of measuring phase place is called multiple isomorphous replace-ment (MIR), and it need introduce external source X ray scatterer heavy atoms such as (for example) metallic atoms the structure cell of crystal.About the more detailed description of MIR, referring to U.S. Patent number 6,093,573 the 15th hurdle.
Atomic coordinates is meant that described data are that the pattern that is obtained by the homogeneous X-ray diffraction of the atom (scattering center) of the biomacromolecule of being paid close attention to of crystal form obtains by relating to the Cartesian coordinates (x, y and z position) that the synthetic mathematical equation of data Fourier obtains.Diffraction data is used to calculate the electron-density map of repetitive (structure cell) in the crystal.Electron-density map is used for the position (atomic coordinates) of interior each atom of structure cell of definite crystal.Because belong to atomic coordinates absolute value can by prolong x, y and/or z axle together or respectively rotation and/or translation change, but keep the relative space relation between the atom constant, so the spatial relationship between the absolute value representation atom of atomic coordinates.Therefore, biomacromolecule (for example albumen), the absolute atomic coordinates value of one group can by rotate and/or translational adjustment for one group of another sample analysis formerly measured value conform to, can think that the atomic coordinates of this biomacromolecule is with identical by the atomic coordinates of this another sample acquisition.
By U.S. Patent number 6,093,573 and international application no PCT/US99/18441, PCT/US99/11913 and PCT/US00/03745 can obtain the further details of relevant X ray Laue method.
The NMR spectroscopic methodology
The macromolecular monocrystalline that the X ray Laue method need be paid close attention to, but the NMR measurement is but carried out near the solution under the physiological condition.Yet the structure that the structure that NMR obtains does not have crystal to obtain is detailed like that.
Though the use of NMR spectroscopic methodology still is limited to the 3-D structure of the relatively little molecule of elaboration (for example albumen of 100-150 amino acid residue) as of late, but comprise the feasible bigger molecule of analysis that this method expanded to of latest developments that the molecule of being paid close attention to is carried out isotope labeling and transverse relaxation-optimization spectrometry (TROSY), for example the 110kDa molecular weight protein (Wider (2000) BioTechniques, 29:1278-1294).
NMR uses radio-frequency radiation to check the environment that is come the magnetic atom nuclear in the homogeneous magnetic field of pulse by particular radio-frequency.This impulse disturbances has the nuclear magnetisation of those atoms of nonzero spin nuclear.When the system recovery balance, the detected transient time-domain signal.The Fourier of this transient signal is changed to frequency domain acquisition one-dimensional NMR spectrum.The chemical shift of various active nucleus is represented at the peak of these spectrum.The chemical shift of atom is decided by its local electronic environment.Two dimension NMR experiment can provide the information about various atom degrees of closeness in structure and three dimensions.By carrying out (being three-dimensional or four-dimensional sometimes) the NMR experiment of some two dimensions and using gained information can measure protein structure as the constraint in the serial protein folding simulation.
More information about the NMR spectroscopic methodology, comprising that the raw data that the NMR experiment is obtained about how is used to measure the detailed description of big molecule 3-D structure can be referring to following document: ProteinNMR Spectroscopy, Principles and Practice, people such as J.Cavanagh, AcademicPress, San Diego, 1996; People such as Gronenborn (1990) Anal.Chem.62 (1): 2-15; And Wider (2000), the same.
Use the aforementioned calculation machine,, can use any available method to make up the 3-D model in the GS zone of being paid close attention to by data and/or the NMR data of X ray crystallization figure.Can make up this model by the analysis data point of input equipment input computing machine with by the processor that uses the known software bag, described software package is CATALYST (Accelrys), INSIGHT (Accelrys) and CeriusII, HKL, MOSFILM, XDS, CCP4, SHARP, PHASES, HEAVY, XPLOR, TNT, NMRCOMPASS, NMRPIPE, DIANA, NMRDRAW, FELIX, VNMR, MADIGRAS, QUANTA, BUSTER, SOLVE, O, FRODO or CHAIN for example.Pass through computer output equipment, use available system, can make by the models show of these data construct, described available system is Silicon Graphics, Evans and Sutherland, SUN, Hewlett Packard, Apple Macintosh, DEC, IBM or Compaq for example.
Design compound of the present invention
In case having set up, any in the use said method be attached to (GS phosphoryl transferase site for example, the GS polypeptide zone of paying close attention to; comprise adenylylation GS phosphoryl transferase site) the 3-D structure of compound, just can prepare and have and the essentially identical 3-D structure of the institute's compounds identified compound of (or comprising the essentially identical territory of structure).In this article, " have essentially identical 3-D structure " and be meant and have the hydrogen bond that is similar to institute's compounds identified and the compound of hydrophobic character.In some cases, has near the zone that the compound with the essentially identical 3-D structure of institute's compounds identified can comprise heterocyclic system and show hydrophobic property the hydrogen bond zone, although the zone of this hydrophobic property can have some hydrogen bond property.This compounds can include but not limited to spatially to give the substituting group of spatial volume, and this space can be sealed manganese and the phosphoric acid skeleton of carbonate coordination, for example (Mn with institute's authenticating compound feature in addition 2+) 3(HCO 3 -) 12ATP.
Possess above-mentioned 3-D structured data and know the chemical constitution (for example, being amino acid sequence) of interest region, one skilled in the art will recognize that how to make compound with above-mentioned character for albumen.These methods comprise chemical synthesis, are recombination method (with reference to above) for albumen.For example, can use suitably to be placed in the compound territory of compound or compound is constrained to suitable 3-D structure with the cysteine residues that forms disulfide bond.In addition, itself be polypeptide or comprising in the compound of polypeptide domain, one skilled in the art will recognize which kind of amino acid described compound comprises and which kind of sequence to comprise these amino acid with in order to produce alpha-helix, beta structure or corner or the bending in the polypeptide backbone for example.
Though computer-based method is optional, it can be used for designing compound of the present invention.The computer program that is fit to comprises: InsightII (Accelrys), CATALYST (Accelrys), LUDI (Accelrys., San Diego, CA), Aladdin (Daylight Chemical InformationSystems, Irvine, CA); With people (1985) J.Med.Chem.36 (20): 2921-2928 such as LEGEND[Nishibata].
The The compounds of this invention that itself is peptide also comprises said structure, but just can use in vivo after modified, promptly by being beneficial to related polypeptide survival in vivo at amino and/or c-terminus interpolation blocking agent.Peptide end is easily by the situation of proteasome degradation of great use before cell is taken in for those for this.Relevant or the incoherent peptide sequence that this blocking agent can include but not limited to add, these sequences can be connected on the amino and/or c-terminus residue of the peptide that will give.This can finish by chemical mode in the process of synthetic peptide, and perhaps the method for being familiar with by those of ordinary skills uses recombinant DNA technology to finish.
Perhaps, blocking agent (for example pyroglutamic acid) or other molecule known in the art can be connected to aminoterminal residue and/or c-terminus residue, perhaps available different piece is come the amino of substituted-amino end or the carboxyl of c-terminus.In addition, before giving, peptide compounds covalently or non-covalently can be coupled to pharmaceutically acceptable " carrier " albumen.
What paid close attention in addition is, based on the amino acid sequence of peptide compounds of the present invention and the plan peptide compounds that designs.Intend peptide compounds and be having synthetic compound with the essentially identical three-dimensional conformation of the three-dimensional conformation of selected peptide (i.e. " peptide motif ").Intend peptide compounds and can have the bells and whistles that strengthens effectiveness in its body, for example increase the cell permeability and prolong biological half-life.
Intend peptide and have the skeleton of the non-peptide of part or the skeleton of complete non-peptide usually, but have with its based on peptide in the identical side group of side group of the amino acid residue that exists.In the plan peptide that makes up antiprotease, the normally useful peptide bond substituent of a few class chemical bond known in the art, these chemical bonds be ester bond, thioester bond, thioamides key, contrary acid amides (retroamide) key, reducing carbonyl key, dimethylene key and ketone methylene (ketomethylene) key for example.
As (Mn 2+) 3(HCO 3 -) 12The analog of ATP compound and/or the micromolecular compound that can be attached to the phosphoryl transferase site acquire a special sense.Micromolecular out of Memory of this Special Category and the synthetic method for preparing these molecules below are provided.
Screening is analyzed
Below also be provided for the in-vitro method of authenticating compound, described compound suppresses the GS activity, comprises adenylylation GS activity, for example the phosphoryl transferase activity of adenylylation GS polypeptide.
The specificity that adenylylation GS suppresses is based on the following fact: promptly the structure of the structure of adenylylation GS and the reaction mechanism of removing adenylylation GS, avtive spot and reaction intermediate is different.In the method for screening compounds, GS polypeptide (comprising adenylylation GS polypeptide) is shifting under the effective particular analysis condition and can contact with test compound adenylylation GS polypeptide generation phosphoryl, and the compound that is screened suppresses (Mn 2+) 3(HCO 3 -) 12ATP compound or its part are attached to the GS polypeptide by phosphine formic acid intermediate, for example the phosphoryl transferase site of adenylylation GS polypeptide, particularly adenylylation GS polypeptide.
The analytic approach of estimating adenylylation GS activity is different with the analytic approach of removing adenylylation GS usually.Can be at pH6.3 and HCO 3 -: Mn 2+: the concentration ratio of ATP is to analyze adenylylation GS under the condition of 12:3:1, and at pH7.2 and Mg 2+: the concentration ratio of ATP is to analyze under the condition of 1:1 to remove adenylylation GS.The canonical analysis condition of adenylylation GS is 20mM imidazole buffer (pH 6.3), 1mM ATP, 3mM MnCl 2, 12mM NaHCO 3, 4mM NH 4Cl and 2mM sodium glutamate; And the canonical analysis condition of removing adenylylation GS is 20mM imidazole buffer (pH 7.2), 1mMATP, 1mM MgCl 2, 12mM NaCl, 4mM NH 4Cl and 2mM sodium glutamate.Above-mentioned analysis can be carried out under 37 ℃.
Can under test compound existence and non-existent condition, measure the hydrolysis of ATP, generation and/or the utilization of glutamic acid and the generation of glutamine of ADP.For example, in one embodiment, a kind of body outer screening test compound is to determine that whether it suppresses the method for the phosphoryl transferase site activity of adenylylation GS polypeptide, comprising:
(a) under effective condition, adenylylation GS polypeptide is contacted with test compound to phosphoryl transferase activity; And
(b) determine with respect to also not with the activity of the contacted adenylylation GS polypeptide of test compound, whether the phosphoryl transferase activity of described adenylylation GS polypeptide decreases.Can regulate the phosphoryl transferase site activity by phosphine formic acid intermediate.Any inhibition activity can be compared with the inhibition that test compound suppresses to go adenylylation GS polypeptide to be obtained.
Compound and pharmaceutical composition
This paper also provides compound, for example, and the compound that pharmaceutical composition as herein described comprised and/or be used for the compound of methods described herein.As shown in this paper and following examples; shift the structure and mechanism information in site based on the phosphoryl of GS, design and prepare some class pyrimidines and class purine molecule and relevant analog to estimate their inhibiting effect to adenylylation GS phosphoryl transferase site activity.For example, considered some analogs, around 6 yuan of rings, had the various combination of space characteristics, hydrogen bond network and pole diagram based on adenine.These analogs comprise 5,6 dicyclic compounds that condense, 6,6 dicyclic compounds that condense, 6,6 dicyclic compounds and the adenine analog with metal-complexing ability.This compound can be used for suppressing adenylylation GS activity; Be used to suppress to have the growth of the bacterium (comprising Much's bacillus) of GSI-β gene; And be used for the treatment of, prevent or improve to be subjected to, may to be subjected to or suspect the mammal that is subjected to bacterial infection (for example having infected Much's bacillus).
Therefore, in some embodiment, the compound that is used for method as herein described or is included in composition described herein can be suc as formula I:
Figure A200680054592D00381
Compound or its pharmaceutically acceptable salt or the derivant of expression, wherein:
R 1Be hydrogen, halogen, OR 5Or NR 6R 7
R 2Be hydrogen, halogen or NR 7R 8
R 3Be hydrogen, halogen or NR 6R 7
R 4Be SR 5, NR 6R 7Or H;
R 5Be H, replacement or unsubstituted C1-C20 alkyl, alkenyl or alkynyl, wherein said alkyl, alkenyl or alkynyl can be straight chain, side chain or ring-type; Or replace or unsubstituted aryl or heteroaryl; And
R 6, R 7And R 8Each is independently selected from H; Acyl group; Hydroxyl; And replace or unsubstituted alkyl, naphthenic base, aryl or heteroaryl; Or R 6And R 7Can form together replacement or unsubstituted naphthenic base, heteroaryl or heterocyclic radical; Or NR 7R 8Can be the form of N=O,
Wherein 1-3 substituting group allows on the part of any replacement, and wherein substituting group can be independently selected from alkyl, thiazolinyl, alkynyl, aryl, heteroaryl, halogen, carboxylate, acid amides, NR 7R 8, OR 5, ketone group, SH and SO 3H.
In some embodiment, R 6And/or R 7And/or R 8Can be the alkyl or cycloalkyl that replaces, for example, the naphthenic base that hydroxyl replaces, for example carbohydrates part.
In some embodiment, R 1Be chlorine.
In some embodiment, R 2Be NR 7R 8
In some embodiment, R 4Be H.
In some embodiment, R 1Be NR 6R 7, R wherein 6Be H, R 7Be methyl, benzyl, 2-hydroxyethyl, 4-bromophenyl or 2-pyridine radicals.
In some embodiment, R 2Be nitroso-, amino, bromine, aminoalkyl or ammonia aryl, for example benzyl amino.
In some embodiment, R 3It is chlorine, dimethylamino, pyrrolidinyl, morpholinyl or 2-(pyrrolidine-1-yl) carboxylate.
In one embodiment, R 4Be H, R 1Be NR 6R 7, R wherein 6Be H, R 7Be benzyl, R 2Be nitroso-, R 3Be chlorine.In another embodiment, R 4Be H, R 1Be NR 6R 7, R wherein 6Be H, R 7Be the 2-hydroxyethyl, R 2Be amino, R 3It is pyrrolidinyl.As following further as described in, compound 97,105 and 111 also is the embodiment of formula I.
The compound that can use standard chemical synthetic method preparation to represent suc as formula I, shown in following examples like that.
In some cases, can be suc as formula II:
Compound or its pharmaceutically acceptable salt or the derivant of expression, wherein:
R 1Be hydrogen, halogen, OR 5Or NR 6R 7
R 4Be hydrogen, SR 5, NR 6R 7Or OR 5
R 5Be H, alkyl, the alkenyl or alkynyl of replacement or unsubstituted C1-C20, wherein said alkyl, alkenyl or alkynyl can be straight chain, side chain or ring-type; Replace or unsubstituted aryl or heteroaryl;
R 6, R 7And R 8Each is independently selected from H; Acyl group; Hydroxyl; And replace or unsubstituted alkyl, naphthenic base, aryl or heteroaryl; Or R 6And R 7Can form together replacement or unsubstituted naphthenic base, heteroaryl or heterocyclic radical; Or NR 7R 8It can be the form of N=O;
R 9Be H, halogen or replacement or unsubstituted alkyl, aryl, heterocyclic radical, heteroaryl, OR 5Or NR 6R 7
X and Y can be N or CH independently; And
Wherein 1-3 substituting group allows on the part of any replacement, and wherein substituting group can be independently selected from alkyl, thiazolinyl, alkynyl, aryl, heteroaryl, halogen, carboxylate, acid amides, NR 7R 8, OR 5, ketone group, SH and SO 3H.
In some embodiment, R 1Be OH or NH 2
In some embodiment, R 4Be H, OH or NH 2
In some embodiment, R 6Be H or alkyl.
In some embodiment, R 9Be alkyl, thiazolinyl, alkynyl or the aryl that replaces.In some embodiment, R 9It is the amino alkyl that replaces.
In one embodiment, R 4Be H, R 6Be benzyl, R 9Be H and R 1Be NR 6R 7, R wherein 6Be methyl and R 7It is methyl.In another embodiment, R 4Be NH 2, R 1Be OH, R 6Be H, R 9It is phenyl.Below further the compound 81 of record be the example of the compound represented suc as formula II.
Those of ordinary skills use standard synthetic method and/or the described scheme of following embodiment can prepare the compound of representing suc as formula II.In some embodiment, for example can obtain the compound represented suc as formula II by formula I compound deriving by the method for substitution that is fit to and closed loop method.
Compound for example is used for the compound of methods described herein, can also be as formula III
Figure A200680054592D00401
Compound or its pharmaceutically acceptable salt or the derivant of expression, wherein:
R 1Be hydrogen, halogen, OR 5Or NR 6R 7
R 4Be hydrogen, SR 5, NR 6R 7Or OR 5
R 5Be H, replacement or unsubstituted C1-C20 alkyl, alkenyl or alkynyl, wherein said alkyl, alkenyl or alkynyl can be straight chain, side chain or ring-type; Replace or unsubstituted aryl or heteroaryl;
R 6And R 7Each is independently selected from H; Acyl group, hydroxyl; And replace or unsubstituted alkyl, naphthenic base, aryl or heteroaryl; Or R 6And R 7Can form together replacement or unsubstituted naphthenic base, heteroaryl or heterocyclic radical;
X, Y can be CH or N independently;
Wherein 1-3 substituting group allows on the part of any replacement, and wherein substituting group can be independently selected from alkyl, thiazolinyl, alkynyl, aryl, heteroaryl, halogen, carboxylate, acid amides, NR 7R 8, OR 5, ketone group, SH and SO 3H.
In some embodiment, R 1Be OH or H.
In some embodiment, R 4Be H, OH or NH 2
In some embodiment, R 6Be OH or H.
In some embodiment, R 7It is the alkyl of H or replacement.
Use the known standard synthetic method of those of ordinary skills can prepare the compound that formula III is represented.In some cases, the compound that formula I can be represented is converted into the formula III compound, for example shown in following examples like that.
In some embodiment, compound can be suc as formula IV
Figure A200680054592D00411
Compound or its pharmaceutically acceptable salt or the derivant of expression, wherein:
R 11Be hydrogen or replacement or unsubstituted C1-C20 alkyl, alkenyl or alkynyl, wherein said alkyl, alkenyl or alkynyl can be straight chain, side chain or ring-type; Replace or unsubstituted aryl or heteroaryl; The R that allows in any replacement of 1-3 substituting group wherein 11On the part, wherein substituting group can be independently selected from alkyl, thiazolinyl, alkynyl, aryl, heteroaryl, halogen, carboxylate, acid amides, NR 7R 8, OR 5, ketone group, SH and SO 3H; And
R 12The alkyl or alkenyl that is unsubstituted or replaces or aryl unsubstituted or that replace, wherein R 12Substituting group can be selected from NH 2, OH, COOH, CHO, NCHO, CONH 2, halogen, OR 5, CO 2R 5And NR 6R 7, wherein:
R 5Be H, replacement or unsubstituted C1-C20 alkyl, alkenyl or alkynyl, wherein said alkyl, alkenyl or alkynyl can be straight chain, side chain or ring-type; Replace or unsubstituted aryl or heteroaryl; And
R 6And R 7Each is independently selected from H; Acyl group, hydroxyl; And replace or unsubstituted alkyl, naphthenic base, aryl or heteroaryl; Or R 6And R 7Can form together replacement or unsubstituted naphthenic base, heteroaryl or heterocyclic radical; And
Wherein 1-3 substituting group allows independently at R 5, R 6Or R 7On the part that replaces, wherein substituting group can be independently selected from alkyl, thiazolinyl, alkynyl, aryl, heteroaryl, halogen, carboxylate, acid amides, NR 7R 8, OR 5, ketone group, SH and SO 3H.
In some embodiment, R 11Be alkyl or H.
In some embodiment, R 12Be aryl or thiazolinyl unsubstituted or that replace, described aryl or thiazolinyl for example have 1-10 C atom.
Use the known standard synthetic method of those of ordinary skills can prepare the compound of representing suc as formula IV, for example shown in following examples like that.
In some embodiment, compound can be suc as formula V:
Figure A200680054592D00421
Compound or its pharmaceutically acceptable salt or the derivant of expression, wherein:
R 1Be hydrogen, halogen, OR 5Or NR 6R 7
R 5Be H, replacement or unsubstituted C1-C20 alkyl, alkenyl or alkynyl, wherein said alkyl, alkenyl or alkynyl can be straight chain, side chain or ring-type; Replace or unsubstituted aryl or heteroaryl;
R 6And R 7Each is independently selected from H; Acyl group, hydroxyl; And replace or unsubstituted alkyl, naphthenic base, aryl or heteroaryl; Or R 6And R 7Can form together replacement or unsubstituted naphthenic base, heteroaryl or heterocyclic radical;
R 13Be independently replace or unsubstituted alkyl, aryl, heteroaryl or naphthenic base;
R 14Be H or NHR 15, R wherein 15Be independently replace or unsubstituted alkyl, aryl, heteroaryl or naphthenic base;
X and Y can be N or CH independently; And
Wherein 1-3 substituting group allows on the part of any replacement, and wherein substituting group can be independently selected from alkyl, thiazolinyl, alkynyl, aryl, heteroaryl, halogen, carboxylate, acid amides, NR 7R 8, OR 5, ketone group, SH and SO 3H.
In some embodiment, R 1Be H.
In some embodiment, R 13Be that replace or unsubstituted aryl.
In some embodiment, R 14Be replace or unsubstituted aryl, alkyl or cycloalkyl.
In some embodiment, X and Y all are CH.
As following further as described in, compound 117 is examples suc as formula the V compound.
Use the known standard synthetic method of those of ordinary skills can prepare the compound of representing suc as formula V.In some cases, shown in for example following embodiment like that, use heterocyclic aromatic amine, aldehyde and isocyanide, can prepare the compound of representing suc as formula V by 3 component coupling reactions.
In some embodiment, compound can be suc as formula VI
Figure A200680054592D00431
Compound or its pharmaceutically acceptable salt or the derivant of expression, wherein:
R 13Be hydrogen or replacement or unsubstituted C1-C20 alkyl, alkenyl or alkynyl, wherein said alkyl, alkenyl or alkynyl can be straight chain, side chain or ring-type; Replace or unsubstituted aryl or heteroaryl; The R that allows in any replacement of 1-3 substituting group wherein 13On the part, wherein substituting group can be independently selected from alkyl, thiazolinyl, alkynyl, aryl, heteroaryl, halogen, carboxylate, acid amides, NR 7R 8, OR 5, ketone group, SH and SO 3H; And
R 12The alkyl or alkenyl that is unsubstituted or replaces or aryl unsubstituted or that replace, wherein R 12Substituting group can be selected from NH 2, OH, COOH, CHO, NCHO, CONH 2, halogen, OR 5, CO 2R 5And NR 6R 7, wherein:
R 5Be H, replacement or unsubstituted C1-C20 alkyl, alkenyl or alkynyl, wherein said alkyl, alkenyl or alkynyl can be straight chain, side chain or ring-type; Replace or unsubstituted aryl or heteroaryl; And
R 6And R 7Each is independently selected from H; Acyl group, hydroxyl; And replace or unsubstituted alkyl, naphthenic base, aryl or heteroaryl; Or R 6And R 7Can form together replacement or unsubstituted naphthenic base, heteroaryl or heterocyclic radical; And
Wherein 1-3 substituting group allows independently at R 5, R 6Or R 7On the part that replaces, wherein substituting group can be independently selected from alkyl, thiazolinyl, alkynyl, aryl, heteroaryl, halogen, carboxylate, acid amides, NR 7R 8, OR 5, ketone group, SH and SO 3H.
In some embodiment, R 13Be H.
In some embodiment, R 12Be replace or unsubstituted aryl or thiazolinyl.
Use the known standard synthetic method of those of ordinary skills can prepare the compound of representing suc as formula VI.In some embodiment, shown in following examples like that, the Hydrolyze method by being fit to for example can obtain the compound represented suc as formula VI by formula IV compound deriving.
Compound for example is used for the compound of methods described herein, can also be suc as formula VII
Compound or its pharmaceutically acceptable salt or the derivant of expression, wherein:
R 6And R 7Each is independently selected from H; Acyl group, hydroxyl; And replace or unsubstituted alkyl, naphthenic base, aryl or heteroaryl; Or R 6And R 7Can form together replacement or unsubstituted naphthenic base, heteroaryl or heterocyclic radical; And
R 14Be H; Acyl group, replacement or unsubstituted alkyl, naphthenic base, aryl or heteroaryl;
Wherein 1-3 substituting group allows on the part of any replacement, and wherein substituting group can be independently selected from alkyl, thiazolinyl, alkynyl, aryl, heteroaryl, halogen, carboxylate, acid amides, NR 7R 8, OR 5, ketone group, SH and SO 3H.
In some embodiment, R 6Be H.
In some embodiment, R 14Be H.
Use the known standard synthetic method of those of ordinary skills can prepare the compound of representing suc as formula VII, for example shown in following examples like that.
The preparation of compound
The compound that is used for composition provided herein and method can obtain maybe can prepare by method well known to those skilled in the art or by method shown in this article by being purchased (for example Sigma, Aldrich, Riedel de Hah, Merck and Acros).Those skilled in the art change by the routine of these methods, use the raw material that is fit to can prepare all compounds used herein.
The preparation of pharmaceutical composition
Pharmaceutical composition provided herein contains the compound that one or more this paper provided and the pharmaceutically acceptable carrier for the treatment of effective dose, described compound is used for the treatment of, prevention or improvement and the bacterial infection (bacterium that for example contains GSI-β gene, Much's bacillus for example) one or more relevant symptoms are subjected to obstacle, illness or the imbalance of bacterial infection with bacterial infection implication or suspection.The pharmaceutical carrier that is suitable for giving compound provided herein comprises the specific any carrier that gives mode that is suitable for well known by persons skilled in the art.
In addition, the composition that described compound can be mixed with as single pharmacy activity component or with the combination of other active component.For example, described compound can prepare or with known antimicrobial compound, antiinflammation compound, steroid and/or the combination of antivirotic class.
This paper provides and contains one or more compound compositions.In one embodiment, described compound is mixed with suitable pharmaceutical preparation, the solution, supensoid agent, tablet, Disket, pill, capsule, pulvis, sustained release agent or the elixir that for example are used for oral administration, or be used for the sterile solution or the suspension form of parenteral and transdermal patch and Foradil Aerolizer formoterol fumarate.In one embodiment, use technology well known in the art and technology, above-claimed cpd is mixed with pharmaceutical composition (referring to for example Ansel Introduction to Pharmaceutical Dosage Forms, the 4th edition, 1985,126).
In these compositions, one or more compounds of effective concentration or its pharmaceutically acceptable derivates are mixed with suitable pharmaceutical carrier.As mentioned above, before preparation, can be corresponding salt, ester, enol ether or enol ester, acetal, ketal, ortho esters, hemiacetal, hemiketal, acid, alkali, solvate, hydrate or pro-drug with compound deriving.In the composition compound concentrations be operational throughput after giving to treatment, prevent or to improve one or more bacterial infection symptoms effective.
In one embodiment, composition being formulated as single dose gives.Be compositions formulated, with the compound of weight fraction with effective concentration dissolving, suspendible, be scattered in the selected carrier or and be mixed in the selected carrier in other mode, the illness of being treated is eased or one or more symptoms improve.
Reactive compound is included in the pharmaceutically acceptable carrier with the consumption of abundance, thereby the patient of treatment is brought into play the beneficial effect of treatment under the condition of no unfavorable spinoff.Can pass through (ex vivo) system and interior this compound of system testing of body in vitro system, the junctor, determine to treat effective concentration empirically, infer the dosage that is used for the people then thus.
The concentration of reactive compound depends on absorptivity, inactivation rate and the excretion rate of reactive compound, physicochemical characteristic, dosage arrangement (dosage schedule), administered dose and the other factors well known by persons skilled in the art of compound in the pharmaceutical composition.
The haplotype preparation (dosage unit forms) of preparation medicine, every haplotype preparation is contained have an appointment (0.01mg, 0.1mg or 1mg) to the active component of (500mg, 1000mg or 2000mg) or the combination of basis approximately, in one embodiment, be the about 500mg of about 10mg-.
Can disposablely give active component, maybe can be divided into a plurality of low doses and in the time interval, give.Be understandable that accurate dose and treatment cycle and the obstacle of being treated have substantial connection, and can use known experimental technique or by in the body or the in vitro test data infer and carry out determining empirically.It should be noted that concentration and dose value also can be with waiting that the order of severity that alleviates illness changes.It should also be understood that for any specific experimenter; according to individual demand and give composition or the personage's that the supervision group compound gives professional judgement; should adjust specific dosage in time, and the concentration range that this paper proposes is scope and enforcement exemplary and that be not intended to limit composition required for protection.
For the situation of compound exhibits solubleness deficiency, can use the method for dissolved compound.This method is well known by persons skilled in the art, comprises the use cosolvent, for example dimethyl sulfoxide (DMSO) (DMSO); Use surfactant, for example
Figure A200680054592D00471
Or be dissolved in sodium bicarbonate aqueous solution, but be not limited thereto.Also can use the derivant of compound, for example the pro-drug of compound preparation drug composition effective.
After compound or the interpolation, the gained potpourri can be solution, suspension, emulsion etc.The form of gained potpourri depends on some factors, comprises desired mode that gives and the solubleness of compound in selected carrier or excipient.Effective concentration is enough to improve the illness of disease symptoms, obstacle or treatment, and can determine this effective concentration empirically.
Can give described pharmaceutical composition to humans and animals according to the form of haplotype preparation, described haplotype formulation example is as the tablet that contains an amount of compound or its pharmaceutically acceptable derivates, capsule, pill, pulvis, granule, the aseptic outer solution of stomach and intestine or supensoid agent and oral solutions or supensoid agent and oil-in-water emulsions.In one embodiment, the compound and the derivant thereof of preparing and giving pharmaceutically to have therapeutic activity with single dose form (unit-dosage forms) or multiple dose form (multiple-dosage form).As known in the art, single dose form used herein is meant the physics discrete unit that is suitable for the humans and animals experimenter and packs separately.Each single dose form contains the compound with therapeutic activity of scheduled volume, in conjunction with needed pharmaceutical carrier, excipient or thinning agent, is enough to produce desirable result of treatment.The example of single dose form comprises the tablet or the capsule of ampoule and syringe and independent packing.Can partly or give single dose form exponentially.The multiple dose form is a plurality of identical single dose form that is packaged in the single container, thereby gives with the multiple dose form of separating.The example of multiple dose form comprises bottle, the bottle of tablet or capsule or the bottle of adorning with pint or gallon.Therefore, the multiple dose form is a plurality of single dose forms of not separating in packing.
Pharmaceutically the fluid composition that can give can be for example by dissolving in carrier, disperse or with other mode mix as defined above reactive compound and optionally pharmaceutic adjuvant prepare, form solution or supensoid agent thus, described carrier is water, salt solution, D/W, glycerine, ethylene glycol, ethanol etc. for example.If need, desire the administered agents composition and also can contain a spot of nontoxic auxiliary agent, for example wetting agent, emulsifying agent, solubilizer, pH buffering agent etc., for example acetate, sodium citrate, cyclodextrine derivatives, sorbitan mono-laurate, triethanolamine, sodium acetate, triethanol amine oleate and other this type of reagent.
The current methods for preparing this formulation will be for maybe being known to those skilled in the art; For example, referring to Remington ' s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., the 15 edition, 1975.
Can prepare formulation or composition with the active component that contains 0.005%-100% of non-toxic carrier balance.These method for compositions of the known preparation of those skilled in the art.The composition of expection can contain the active component of 0.001%-100%, perhaps in one embodiment for containing the active component of 0.1-95%.
1. the composition that is used for orally give
Oral Pharmaceutical dosage forms is solid formulation, gel or liquid agent.Solid dosage forms is tablet, capsule, granule and loose powder agent (bulk powders).The oral tablet type comprises the lozenge chewed and the tablet of compression, and described lozenge and tablet can be enteric, sugar-coat or film-coated.Capsule can be hard gelatine capsule or soft gelatine capsule, and granule and pulvis can non-effervesce form or effervesce form and other composition well known by persons skilled in the art combine and provide.
A. the solid composite that is used for orally give
In some embodiments, preparation is a solid dosage forms, in one embodiment, is capsule or tablet.Tablet, pill, capsule, lozenge etc. can contain the compound of one or more following compositions or similar quality: bonding agent; Lubricant; Thinning agent; Glidant; Disintegrant; Colorant; Sweetener; Flavoring additives; Wetting agent; Emetic coating agent (emetic coating); And film coating agent.The bonding agent example comprises microcrystalline cellulose, tragacanth, glucose solution, mucialga of arabic gummy, gel solution, molasses, polyvinylpyrrolidone, polyvidone, polyvinylpolypyrrolidone, sucrose and gelatinized corn starch.Lubricant comprises talcum, starch, dolomol or calcium stearate, lycopod and stearic acid.Thinning agent comprises for example lactose, sucrose, starch, porcelain earth, salt, sweet mellow wine and Dicalcium Phosphate.Glidant comprises cataloid, but is not limited thereto.Disintegrant comprises Ac-Di-Sol, sodium carboxymethyl starch, alginic acid, cornstarch, potato starch, bentonitic clay, methylcellulose, agar and carboxymethyl cellulose.Colorant comprises any water-soluble FD of approval of certification for example and C dyestuff and composition thereof; And the water-insoluble FD and the C dyestuff that are suspended in hydrated alumina.Sweetener comprises sucrose, lactose, sweet mellow wine and artificial sweetening agent for example asccharin and many spray drying flavoring additivess.Flavoring additives comprises from plant (for example fruit) intermixture of the compound of the natural flavouring that extracts and the synthetic joyful sense of generation, for example peppermint oil and cresotinic acid, but be not limited thereto.Wetting agent comprises propylene glycolmonostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether.Emetic coating agent comprises fatty acid, fat, wax, lac, contains ammonia lac and CAP.Film coating comprises hydroxyethyl cellulose, sodium carboxymethyl cellulose, Macrogol 4000 and CAP.
Compound or its pharmaceutically acceptable derivates form with composition can be provided to protect it to be subjected to the infringement of sour environment in the stomach.For example, composition can be formulated as the form of enteric coating, make its under one's belt being kept perfectly property and in intestines release of active compounds.Can also be in conjunction with antiacid or other this constituents compositions formulated.
When the haplotype preparation was capsule, except that the material of the above-mentioned type, it also can contain for example fat oil of liquid-carrier.In addition, the haplotype preparation can contain various other materials of the dosage unit physical form that changes, for example sugar-coat agent and other enteric agents.The component that compound also can be used as elixir, supensoid agent, syrup, wafer, spray (sprinkle), chewing gum etc. gives.Except that reactive compound, syrup can contain as the sucrose of sweetener and some antiseptic, dyes and dyestuffs and flavoring additives.
Active substance also can mix with other active substance that does not destroy desired effect, or mixes with the material that replenishes desired effect.Active component is compound as herein described or its pharmaceutically acceptable derivates.Can comprise the active component of higher concentration up to about 98 weight %.
In all embodiments, just as is known to the person skilled in the art, can carry out dressing to change or to keep the dissolubility of active component to tablet and capsule.Therefore, for example can use the digested dressing (for example phenyl salicytate, wax and CAP) of conventional enteric that they are carried out dressing.
B. the fluid composition that is used for orally give
Liquid oral dosage form comprises by non-effervescent and reproducing and the aqueous solution, emulsion fluid, supensoid agent, solution and/or the suspension that come and reproduced and the effervescent formulation that comes by effervescent.Aqueous solution comprises for example elixir and syrup.Emulsion fluid is oil-in-water or Water-In-Oil.
Elixir is limpid, as to have sweet taste water alcohol formulations.The pharmaceutically acceptable carrier that is used for elixir comprises solvent.Syrup is the concentrated aqueous solution of sugar (for example sucrose), and can contain antiseptic.Emulsion fluid is diphasic system, and wherein a kind of liquid is distributed in whole another liquid with the bead form.The pharmaceutically acceptable carrier that is used for emulsion fluid is on-aqueous liquid, emulsifying agent and antiseptic.Supensoid agent uses pharmaceutically acceptable supensoid agent and antiseptic.Thereby be used for the pharmaceutically acceptable material that non-effervescent reproduces to liquid oral dosage form and comprise thinning agent, sweetener and wetting agent.Thereby be used for the pharmaceutically acceptable material that effervescent reproduces to liquid oral dosage form and comprise organic acid and carbon dioxide source.All above-mentioned formulations are all used colorant and flavoring additives.
Solvent comprises glycerine, sorbierite, ethanol and syrup.Examples of preservatives comprises glycerine, methyl p-hydroxybenzoate and propylparaben, benzoic acid, Sodium Benzoate and alcohol.The example that is used for the on-aqueous liquid of emulsion fluid comprises mineral oil and cottonseed oil.The example of emulsifying agent comprises gelatin, gum arabic, tragacanth, bentonitic clay and surfactant (for example octadecanoic acid ester of polyethylene glycol).Supensoid agent comprises sodium carboxymethyl cellulose, pectin, tragacanth, aluminium-magnesium silicate (Veegum) and gum arabic.Sweetener comprises sucrose, syrup, glycerine and artificial sweetening agent (for example asccharin).Wetting agent comprises propylene glycolmonostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether.Organic acid comprises citric acid and tartrate.Carbon dioxide source comprises sodium bicarbonate and sodium carbonate.Colorant comprises the water-soluble FD of any approval of certification and C dyestuff and composition thereof.Flavoring additives comprises the intermixture of the compound of the natural flavouring that extracts from plant (for example fruit) and the synthetic joyful sense of generation.
For solid dosage forms, for example solution or the suspension in carbonic allyl ester, vegetable oil or the triglyceride enclosed in the gelatine capsule in one embodiment.In U.S. Patent No. 4,328, these solution and preparation and encapsulated are disclosed in 245,4,409,239 and 4,410,545.For liquid dosage form, the solution in the polyglycol for example, can use capacity pharmaceutically acceptable liquid-carrier (for example water) thus dilution easily measure being used to and give.
Perhaps, by dissolving or dispersed activity compound or salt in vegetable oil, ethylene glycol, triglyceride, propylene glycol ester (for example carbonic allyl ester) and other examples of such carriers, and in hard gelatine capsule shell or soft gelatine capsule shell, enclose these solution or suspension, can prepare liquid or semisolid oral formulations.At U.S. Patent No. RE28, set forth other the useful preparation that comprises them in 819 and 4,358,603.In brief, said preparation includes but not limited to contain those preparations of the compound (the single aklylene glycol or the poly alkylene glycol of dialkyl groupization) that this paper provides, include but not limited to 1, the 2-dimethoxymethane, diethylene glycol dimethyl ether, triglyme, tetraethylene glycol dimethyl ether, polyglycol-350-dimethyl ether, polyglycol-550-dimethyl ether, polyglycol-750-dimethyl ether, wherein 350,550 and 750 are meant the approximate mean molecular weight of polyglycol, said preparation also comprises one or more antioxidants, for example Yoshinox BHT (BHT), butylated hydroxyanisol (BHA), n-propyl gallate, vitamin E, p-dihydroxy-benzene, Hydroxycoumarin, monoethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbierite, phosphoric acid, thio-2 acid and ester thereof and dithiocarbamate.
Other preparation comprises the aqueous alcoholic liquid that contains pharmaceutically acceptable acetal, but is not limited thereto.The alcohol that is used for these preparations is any pharmaceutically acceptable water-miscible solvent with one or more hydroxyls, comprises propylene glycol and ethanol, but is not limited thereto.Acetal includes but not limited to two low-carbon alkyl acetal, for example acetaldehyde diethyl acetals of low-carbon alkyl aldehyde.
2. injection-type formulation soln and emulsion fluid
This paper is also contained stomach and intestine and is given outward, in one embodiment, it is characterized by subcutaneous, intramuscular or intravenous injection.Injection can be prepared as regular dosage form, is prepared as liquid solution agent or supensoid agent, was suitable for the solution in the liquid or the solid dosage forms of supensoid agent before injection, or be prepared as emulsion fluid.Injection, solution and emulsion also contain one or more excipient.Suitable excipient is water, salt solution, dextrose, glycerine or ethanol for example.In addition, if need, desire the administered agents composition and also can contain a spot of nontoxic auxiliary agent, for example wetting agent or emulsifying agent, pH buffering agent, stabilizing agent, solubilizer and other this type of reagent, for example sodium acetate, sorbitan mono-laurate, triethanol amine oleate and cyclodextrin.
The implantation of slow release or lasting delivery systme also contained in this paper, makes dosage remain on stable level (referring to for example U.S. Patent No. 3,710,795).In brief, make compound provided herein be scattered in following solid interior matrix, described internal matrix is polymethylmethacrylate for example, poly-n-butyl methacrylate, plasticised polyvinyl chloride or not plasticized polyvinyl chloride, plasticising nylon, the plasticising polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, tygon, vinyl-vinyl acetate copolymer, silicon rubber, dimethyl silicone polymer, the silicon-carbon acid ester copolymer, hydrophilic polymer is the hydrogel of acrylate and methacrylate for example, collagen, the polyvinyl acetate of crosslinked polyvinyl alcohol (PVA) and crosslinked partial hydrolysis, it is surrounded by the outer polymer film, described polymkeric substance is tygon for example, polypropylene, ethylene/propene copolymer, the ethylene/ethyl acrylate multipolymer, ethylene, silicon rubber, dimethyl silicone polymer, chloroprene rubber, haloflex, Polyvinylchloride, the multipolymer of vinyl chloride and vinyl acetate, 1, the 1-dichloroethylene, ethylene-propylene, the ionomer polyethylene terephthalate, butyl rubber-epichlorohydrin rubber, the ethylene/vinyl alcohol copolymer, Ethylene/vinyl acetate/vinyl alcohol terpolymer and ethylene/vinyl ethoxy-ethanol multipolymer, described outer polymer film is insoluble to body fluid.In the rate of release controlled step, compound spread outside polymer film.The percentage of the reactive compound that the outer composition of these stomach and intestine contains depends on its specific character strongly, and compound activity and experimenter's needs.
The stomach and intestine of composition comprise that outward intravenous, subcutaneous and intramuscular give.Be used for that preparation that stomach and intestine give comprises the sterile solution preparing to be used to inject, prepared with the solubility goods (for example freeze dried powder comprises hypodermic tablet) of the aseptic drying of solvent outward before using, the aseptic supensoid agent preparing to be used to inject, before using, prepare insoluble goods and aseptic emulsion fluid with the aseptic drying of mixed with excipients.Solution can be aqueous solution or non-aqueous solution.
If intravenous gives, suitable carrier comprises physiological saline or phosphate buffered saline (PBS) (PBS) and contains thickening agent and the solution of solubilizer that described thickening agent and solubilizer is glucose, polyglycol and polypropylene glycol and composition thereof for example.
The pharmaceutically acceptable carrier that is used for parenteral formulation comprises water-based excipient, non-aqueous excipient, antiseptic, isotonic agent, damping fluid, antioxidant, local anesthetic, suspendible and spreading agent, emulsifying agent, sequestering agent or sequestrant and other pharmaceutically acceptable material.
The example of water-based excipient comprise sodium chloride injection, Lin Ge (Ringers) parenteral solution, etc. open dextrose parenteral solution, sterilized water parenteral solution, dextrose and lactic acid ringer's injection.The outer excipient of non-aqueous stomach and intestine comprises fixedly oil, cottonseed oil, corn oil, sesame oil and the peanut oil of plant origin.Must antiseptic be joined in the parenteral formulation that is packaged in the multi-dose container with the concentration that suppresses bacterium or inhibition fungi, this antiseptic comprises phenol or cresols, mercurial, phenmethylol, methaform, methyl p-hydroxybenzoate and propylparaben, thimerosal, benzalkonium chloride and benzethonium chloride.Isotonic agent comprises sodium chloride and dextrose.Buffering agent comprises phosphate and citrate.Antioxidant comprises niter cake.Local anesthetic comprises procaine hydrochloride.Suspendible and spreading agent comprise sodium carboxymethyl cellulose, hydroxypropyl methylcellulose and polyvinylpyrrolidone.Emulsifying agent comprise polyoxyethylene sorbitan monoleate (
Figure A200680054592D00521
80).The sequestering agent or the sequestrant of metallic ion comprise EDTA.Pharmaceutical carrier also comprises ethanol, polyglycol and the propylene glycol that is used for the water miscibility excipient; And the NaOH, hydrochloric acid, citric acid or the lactic acid that are used to regulate pH.
The concentration of pharmaceutically active compound is regulated, thereby make injection can provide effective dose to produce desirable drug effect.As known in the art, definite dosage depends on age, body weight and the illness of patient or animal.
Come the parenteral formulation of packing unit metering with ampoule, bottle or band needle injection.As known in the art with put into practice like that, it should be aseptic being used for all preparations that stomach and intestine give outward.
As an example, to contain the aseptic aqueous solution of reactive compound be the pattern that effectively gives for intravenous or endoarterial infusion.Another embodiment is that injection contains the aseptic aqueous solution of active substance or oily solution or supensoid agent to produce desirable drug effect when needing.
Be designed for the injection-type preparation that topical administration and whole body give.In one embodiment, with the treatment effective dose be mixed with tissue for treatment contain concentration at least about 0.1%w/w to about 90%w/w or higher, in some embodiments, the tissue for treating contains the reactive compound of concentration greater than 1%w/w.
Compound can the micro mist form or other form suspendible that is fit to exist, perhaps can derive to generate the better activated product of dissolubility or to generate pro-drug.The form of gained potpourri depends on some factors, comprises the pattern that gives and the dissolubility of compound in selected carrier or excipient of expectation.Effective concentration is enough to improve the symptom of illness and can determine by rule of thumb.
3. freeze dried powder
The freeze dried powder in addition that this paper paid close attention to can be dissolved as it solution, emulsion fluid and other potpourri and be used to give.Also can and be formulated as solid or gel with its dissolving.
By being dissolved in The suitable solvent, compound provided herein or its pharmaceutically acceptable derivates prepare aseptic freeze-dried pulvis.Described solvent can contain excipient, and described excipient improves other pharmacology component of pulvis or the stability of reproducing solution that is prepared by pulvis.Spendable excipient comprises dextrose, sorbierite, fructose, corn syrup, xylitol, glycerine, glucose, sucrose or other suitable reagent, but is not limited thereto.Described solvent also can contain damping fluid, and for example other this type of buffer solution known to citrate buffer solution, sodium phosphate or buffer solution of potassium phosphate or those skilled in the art in one embodiment, contains pH and is bordering on neutral buffer solution.Subsequently, carrying out the aseptic filtration of solution under the standard conditions known to those skilled in the art, is freeze-drying then, obtains desirable preparation.In one embodiment, gained solution branch is installed to be used for freeze-drying in the bottle.Each bottle contains the compound of single dose or multiple dose.Can be under suitable condition, for example about 4 ℃ store freeze dried powder to room temperature.
Use water for injection to dissolve above-mentioned freeze dried powder, obtain the preparation that stomach and intestine give usefulness outward.For realizing dissolving, freeze dried powder is added in sterilized water or other the suitable carrier.Accurate consumption depends on selected compound.Can determine this consumption by rule of thumb.
4. topical administration
Preparation is used for the part potpourri that topical administration and whole body give as previously mentioned.The gained potpourri can be solution, supensoid agent, emulsion fluid etc., and can be mixed with creme, gel, ointment, emulsion, solution, elixir, lotion, supensoid agent, tincture, patch, foaming agent, aerosol, lavage solution, spray, suppository, bandage, skin patch or be suitable for any other preparation of topical administration.
Compound or its pharmaceutically acceptable derivates can be mixed with aerosol and be used for local the use, for example by sucking (referring to for example U.S. Patent No. 4,044,126,4,414,209 and 4,364,923, it has been described and has been used for the treatment of the especially aerosol that is used to carry steroids of asthma of inflammatory disease).Be used for these preparations that respiratory tract gives can aerosol or the form of solution be used for sprayer or be used for sucking with the form of fine pulvis, use separately or with inert carrier for example lactose be used in combination.In this case, in one embodiment, the diameter of preparation granules is less than 50 microns, and in one embodiment, the diameter of preparation granules is less than 10 microns.
Compound preparation can be used for part or local the use, for example be applied topically to skin and mucous membrane (for example intraocular mucous membrane) and be used for eyes, or be used in the brain pond or in the backbone with gel, creme and lotion form.The topical administration expection is used for through the skin conveying and is used to give eyes or mucous membrane or is used for sucking treatment.Also can give nose solution, described nose only contains the pharmaceutically acceptable excipient composition of reactive compound or reactive compound and other with solution.
This class solution can be expected that especially the solution that is used for eye is formulated as 0.01%～10% isotonic solution with the salt that is fit to, pH is about 5～7.
5. be used for the composition that other gives approach
This paper is also contained other and is given approach, for example comprise iontophoresis equipment and electrophoresis equipment through the skin patch, and rectum gives.
What comprise iontophoresis equipment and electrophoresis equipment is well known to those skilled in the art through the skin patch.For example, U.S. Patent No. 6,267,983,6,261,595,6,256,533,6,167,301,6,024,975,6,010,715,5,985,317,5,983,134,5,948,433 and 5,860,957 disclose this patch.
For example, being used for rectum administered agents formulation is rectal suppository, capsule and the tablet with whole body effect.Rectal suppository used herein is meant the solid formulation that is used to insert rectum, and this solid formulation melts under body temperature or be softening, discharges one or more pharmacological components or therapeutic active component.The pharmaceutically acceptable material that is used for rectal suppository is for improving matrix or the carrier and the reagent of fusing point.The example of this matrix comprises the suitable potpourri of cocoa butter (cupu oil), glycerine-gelatin, carbowax (Carbowax) (polyglycol) and glycerine monofatty ester, fatty acid diglyceride and fatty acid triglycercide.Can use the combination of different substrates.The reagent that improves the suppository fusing point comprises spermaceti and wax.Can be equipped with rectal suppository by compression method or molding legal system.In one embodiment, rectal suppository weight is about 2～3 grams.
Use pharmaceutically acceptable material identical and identical method preparation to be used for tablet and the capsule that rectum gives with the orally give preparation.
6. target preparation
Also compound provided herein or its pharmaceutically acceptable derivates can be mixed with targeting preparation, other position of particular organization, acceptor or health of the experimenter that described preparation target is to be treated.Those skilled in the art have known many targeted approach.All targeted approach that are used for instant combination contained in this paper.About the non-limitative example of targeted approach referring to for example U.S. Patent No. 6,316,652,6,274,552,6,271,359,6,253,872,6,139,865,6,131,570,6,120,751,6,071,495,6,060,082,, 6,048,736,6,039,975,6,004,534,5,985,307,5,972,366,5,900,252,5,840,674,5,759,542 and 5,709,874.
In one embodiment, the liposome supensoid agent comprises that the liposome (for example liposome of target tumor) of target tissue also can be suitable for use as pharmaceutically acceptable carrier.Can prepare these preparations according to method known to those skilled in the art.For example can be according to U.S. Patent No. 4,522,811 described such Liposomal formulations that prepare.In brief, phosphatidylserine (mol ratio 7:3) in egg yolk lecithin in the flask and the brain can be prepared for example multilamellar liposome (MLV) of liposome by drying.The solution that is dissolved in the compound provided herein in the phosphate buffered saline (PBS) (PBS) of no bivalent cation is added flask and vibration is dispersed until lipid film.Washing gained microvesicle, to remove non-encapsulated compound, centrifugal balling-up is resuspended among the PBS then.
7. goods
Compound or pharmaceutically acceptable derivates can be packaged into goods (for example kit), described goods contain wrappage, are positioned at compound provided herein or its pharmaceutically acceptable derivates and the label of wrappage, and described label shows compound or composition or its pharmaceutically acceptable derivates is used for the treatment of, prevention or improvement comprise one or more symptoms or the obstacle that m tuberculosis infection is relevant with bacterial infection.
Goods provided herein contain wrappage.Those skilled in the art has known and has been used to pack the pharmaceutical packing material.Referring to for example U.S. Patent number 5,323,907,5,052,558 and 5,033,252.The drug packages examples of material comprises blister package (blister packs), bottle, pipe, inhalator, pump, bag, bottle, container, syringe and is suitable for the mode that gives of selected prescription and hope and any wrappage of therapeutic modality, but is not limited thereto.
8. sustained release agent
The present invention also provides sustained release preparation, with compound with high cyclical level (10 -9With 10 -4Between the M) be transported to desirable target.The sustained release preparation of this level carries out systemic circulation in patient's body, or in one embodiment, rests on and for example benumb the position.
Be understandable that as desired, the compound level remains unchanged in certain period, those skilled in the art can easily measure this level.Slowly-releasing mode by conveying equipment well known to those skilled in the art can prepare this sustained release preparation and/or timing releasing agent, and for example U.S. Patent No. 3,845, and 770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 4,710,384; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556 and 5,733,566 described preparations are incorporated herein by reference the disclosed content of these documents.Use following material these pharmaceutical compositions can be used for slowly or lastingly discharging of one or more reactive compounds, these materials are hydroxypropyl methylcellulose, other polymer substrate, gel, permeable membrane, osmosis system, multiple coatings, microsome, liposome, microsphere etc. for example.Can select the suitable sustained release preparation known to those skilled in the art at an easy rate, comprise preparation as herein described, use with pharmaceutical composition provided herein.Therefore, the haplotype preparation of the orally give that is suitable for slowly-releasing contained in this paper, for example tablet, capsule, soft capsule, Caplet, pulvis etc., but be not limited thereto.
In one embodiment, sustained release preparation contains reactive compound, for example microcrystalline cellulose, maltodextrin, ethyl cellulose and dolomol, but be not limited thereto.As mentioned above, all the known encapsulating methods with disclosed compound property compatibility contained in this paper.By using slow insoluble polymer may pharmaceutical composition particle provided herein or particle are carried out the dressing of different-thickness or seal sustained release preparation by microencapsulation.In one embodiment, use the coating material of different-thickness (for example about 1 micron～200 microns) to seal sustained release preparation, make pharmaceutical composition dissolving in about 48～72 hours after giving mammal.In another embodiment, described coating material is the adjuvant that can be used for food through approval.
In another embodiment, sustained release preparation is to be pressed into the stromatolysis device that tablet is made by delaying the insoluble polymer may carrier with medicine.In one embodiment, as U.S. Patent No. 4,710,384 and 5,354,556 is disclosed, and dressing particle size is about 0.1～300 micron, and these document integral body are incorporated herein by reference.Each particle exists with the micromatrix form, and active component is evenly distributed in the whole polymkeric substance.
For example in U.S. Patent No. 4, those disclosed sustained release preparation in 710,384, it is incorporated herein by reference with integral body, this sustained release preparation contains high relatively number percent in dressing plastifier is disclosed in the document, thereby so that the essence breakage in the enough elasticity prevention compressing tablet process to be provided.The concrete consumption of plastifier is different, depends on the character of dressing and used certain plasticizers.By detecting the release characteristic of made tablet, can easily determine plasticizer dosage by rule of thumb.If drug is too fast, then use the plastifier of more amount.Release characteristic also is the function of coating thickness.When the plasticizer consumption that uses was quite big, the slow-release capability of dressing weakened.Therefore, can increase the increase of coating thickness a little with the compensation plasticizer consumption.Usually, in embodiment, the plasticizer dosage of existence is about 15～30% of slow-release material in the dressing, in one embodiment, be 20～25%, and the dressing consumption is 10～25% of an active substance weight, in another embodiment, the dressing consumption is 15～20% of an active substance weight.The pharmaceutically acceptable plastifier of any routine can be added in the dressing.
Compound provided herein can be mixed with sustained release preparation and/or regularly release agent.All slow releasing pharmaceutical goods all can reach and improve curative effect of medication to surpass the purpose of its non-slowly-releasing homologue.Desirable situation is, in medical treatment, uses the characteristics of the sustained release preparation of optimal design to be to use minimum drug substance to cure or controls illness.The advantage of sustained release preparation can comprise: 1) composition is active prolongs 2) minimizing of medicament access times, and 3) raising of patient's compliance.In addition, sustained release preparation also can be used for influencing drug treating time or other performance, and for example the haemoconcentration of composition can influence the generation of drug side-effect thus.
Sustained release preparation provided herein is designed to the therapeutic combination consumption that initial release can produce desired curative effect fast, then gradually and other consumption of sustained-release composition, makes the curative effect that in long-time, can keep this level.In order to keep this level stable in vivo, must from preparation, discharge therapeutic combination with certain speed, so that can compensate composition by internal metabolism and secretion.
Can be by for example pH, temperature, enzyme, water, other physiological condition or the compound slow release that comes the stimulating activity composition of different inducements.
The orally give preparation suitably can be mixed with can the sustained release reactive compound.In one embodiment, compound is formulated as the sustained release pulvis of the disperse particles that can be easy to be mixed with liquid form.The slowly-releasing pulvis comprises the particle that contains active component, and can choose wantonly and comprise the excipient with at least a nontoxic polymer.
Pulvis can disperse or be suspended in liquid phase carrier and can keep its sustained releasing character during use.These spreading agents or supensoid agent have chemical stability and rate of dissolution stability.Pulvis can contain the excipient that comprises polymkeric substance, and this polymkeric substance can be soluble, insoluble, permeable, impermeable or biodegradable.This polymkeric substance can be polymkeric substance or multipolymer.This polymkeric substance can be natural or synthetic polymkeric substance.Natural polymer comprises polypeptide (for example zein), polysaccharide (for example cellulose) and alginic acid.Representational synthetic polymer comprises U.S. Patent No. 5,354, the 3rd hurdle, capable those that put down in writing of 33-45 in 556 (document is incorporated herein by reference with integral body), but be not limited thereto.Particularly suitable polymkeric substance comprises U.S. Patent No. 5,354, those that the 3rd hurdle the 46th row-Di 4 hurdle eighth rows are put down in writing in 556 (document is incorporated herein by reference with integral body), but be not limited thereto.
Can prepare and be used for the slow releasing composition provided herein that stomach and intestine give outward, for example by intramuscular injection or be implanted to hypodermis and give with different body cavitys and transdermal device.In one embodiment, the intramuscular injection agent is formulated as aqueous suspension or oil suspension.In aqueous suspension, reduction of reactive compound solubleness or rate of dissolution reduce partly to cause slow releasing function in the recombination process.Oil suspension and solution have adopted similar approach, wherein measure the rate of release of reactive compound by reactive compound is assigned to aqueous medium on every side from oil.Have only the molten and reactive compound that have desirable distribution character of oil to be only suitable.The oil that can be used for intramuscular injection comprises sesame oil, olive oil, arachis oil, corn oil, apricot kernel oil, soybean oil, cottonseed oil and castor oil, but is not limited thereto.
Can in the time period of a couple of days to several years, the very ripe drug conveying form of slowly-releasing be to be implanted into the drug-carrying polymer device at subcutaneous or different body cavitys.The polymeric material that is used to implant must be a bio-compatible and nontoxic, and it comprises hydrogel, silicone, tygon, vinyl-vinyl acetate copolymer or biodegradable polymkeric substance, but is not limited thereto.
The activity rating of compound
Provided herein as the GS activity (activity of adenylylation GS for example; the phosphoryl transferase activity that comprises the phosphine formic acid intermediate mediation of adenylylation GS) activity of the compound of inhibitor; and/or, all can measure or estimate by standard analytical process as the activity of the compound of treatment, prevention or improvement one or more symptoms, illness or the obstacle relevant with bacterial infection (for example m tuberculosis infection).Can use enzyme to suppress method (for example gamma glutamyltransferase method, ATP Hydrolyze method, ADP form and glutamic acid utilization and glutamine forming method), the growth inhibited of bacterium and cytoprotection, survival and the cytotoxicity analysis of cell, all these are well known to those of ordinary skill in the art and/or as mentioned below.
The using method of compound and composition
Use compound as herein described and composition can implement external or the interior method of body.The external application of The compounds of this invention can be used for the basic scientific research of GS reaction mechanism for example or is used for the treatment of, prevents, weakens or suppress bacterial contamination or infection or be used to suppress the GS phosphoryl transferase activity.This compound also can be used as interior therapeutic reagent with the treatment bacterial infection, comprises the infection of pathogen or opportunist.Especially, this compound can be used as treatment reagent to treat by the bacterial infection of GSI-β or by the bacterial infection of GSI-α, and described GSI-β bacterium is Bacterium diphtheriae for example, gonococcus, Escherichia coli, salmonella typhimurium, salmonella typhi, Klebsiella Pneumoniae, serratia marcescens, proteus vulgaris, Shigella dysenteriae, comma bacillus, pseudomonas aeruginosa, Bacillus foecalis alkaligenes, helicobacter pylori, haemophilus influenzae, Bordetella pertussis, the special bacterium of bronchitis Boulder, Neisseria meningitidis, Brucella melitensis, Much's bacillus, Mycobacterium leprae, Tyreponema pallidum, leptospira interrogans, actinomyces israelii, Nocardia asteroides, Thiobacillus ferrooxidans, Azospirillum brasilense, anabena, Fremyella diplosiphon and streptomyces coelicolor.This compound can be used for preventing and/or treating and relates to microorganism in the cell infectious factor of time multiplexed cell system (promptly), for example disease of bacterium (for example Mycobacterium tuberculosis) in the cell.
The inventive method can be used for the species of wide region, for example mammal for example monkey, horse, ox, pig, sheep, goat, dog, cat, rabbit, cavy, hamster, rat and mouse of people, non-human primates for example.Because the GSI-β enzyme of bacterium is regulated by adenylylation/go adenylylation cascade; and mammiferous GSII enzyme is not like this; and some inhibitor of this paper is supposed the optionally GS of target adenylylation, and minimally has a negative impact to mammalian cell so this compound and composition can be used for optionally suppressing bacterial cell growth.
In a kind of body in the method, with compound as herein described or pharmaceutical composition (the represented compound of formula I, II, III, IV, V, VI or VII for example; Or those materials as being set forth among the embodiment) give the experimenter, for example mammal is for example suspected the mammal that is subjected to bacterial infection or is being subjected to bacterial infection.Usually, The compounds of this invention is suspended in the pharmaceutically acceptable carrier (for example physiological saline), and oral administration gives transdermal administration or carry out in intravenous, subcutaneous, the muscle, in the peritonaeum, in the rectum, in the leaf sheath, in the nose, in the stomach, in the tracheae or pulmonary injection (or infusion).They can be delivered directly to the suitable tissue that is subjected to affect.
The used inhibition compound and the dosage of assistant medicament depend on the selection of the approach of giving; The character of preparation; The character of patient disease; Experimenter's build, body weight, surface area, age and sex; The other medicines that give; And attending doctor's judgement.The dosage that is fit to is generally 0.0001-100.0mg/kg.Consider the kind and the various different efficient that give approach of available compound and assistant medicament, estimate that required dosage can great changes have taken place.For example, estimate orally give than intravenous injection give need be bigger dosage.The variation of these dosage levels can use the usual manner of the standard experience that is used to optimize well known in the art to regulate.Can one-pack type or the form of multi-form (for example 2 times, 3 times, 4 times, 6 times, 8 times, 10 times, 20 times, 50 times, 100 times, 150 times or more times) give compound and/or assistant medicament.
Embodiment
Embodiment 1Mn (HCO 3 ) 2 Illustrating of the catalytic mechanism of-ATP compound and structure
Theoretical background
Use the conformation (vide infra) of adenosine 5'-triphosphate (ATP) in the nuclear magnetic relaxation technical measurement manganese compound.By Solomon-Bleombergen equation (Bloembergen, N. (1957) J.Chem.Phys.27:(2), 572-596; Mildvan, A.S. and Eagle, J.L. (1972) Methods Enzymol.26:654-682 and Mildvan, A.S. and Cohn, M. (1970) Adv.Enzymol.33:1-70) dipole term calculate Mn 2+To the distance of ATP core, this equation is as follows:
1 T 1 M = 2 15 S ( S + 1 ) γ I 2 g 2 β 2 r 6 [ 3 τ c 1 + ω I 2 τ c 2 + 7 τ c 1 + ω s 2 τ c 2 ] + 2 3 S ( S + 1 ) A 2 h 2 [ τ e 1 + ω s 2 τ e 2 ]
1 T 21 M = 1 15 S ( S + 1 ) γ I 2 g 2 β 2 r 6 [ 4 τ c + 3 τ c 1 + ω I 2 τ c 2 + 13 τ c 1 + ω s 2 τ c 2 ] + 1 3 S ( S + 1 ) A 2 h 2 [ τ e + τ e 1 + ω s 2 τ e 2 ]
Wherein:
S=electron spin quantum number is (for Mn 2+Be 5/2)
γ I=nuclear magnetogyric ratio (for 1H is 2.675 * 10 4Arcsecond-1 Gauss -1)
" g " factor of g=electronics is (for Mn 2+Be 2)
β=Bohr magneton (8.795 * 10 6Arcsecond -1Gauss -1)
τ c=dipole correlation time is (for Mn-(H 2O) 6For
Figure A200680054592D0061153404QIETU
3.0 * 10 -11)
ω I=nuclear resounce frequency ( 1H is 6.28 * 10 in 23.487 Gausses 8)
ω S=electron spin resonance frequency ( 1H is 4.13 * 10 in 23.487 Gausses 11)
The h=hyperfine coupling constant
The mean distance of r=electronics-nucleon (centimetre)
T 1The longitudinal relaxation time of=proton
T 2The T2 of=proton
T e=electron spin correlation time
P=paramagnetic ion and ligand concentration ratio
Calculate paramagnetic to longitudinal relaxation speed and transverse relaxation speed 1/T by the contribution that deducts anti-magnetic 1pAnd 1/T 2pContribution:
1 T 1 p = 1 T 1 ( obs ) - 1 T 1 ( o ) = pq T 1 M
Wherein, 1/T 1 (obs)Be the relaxation rate when having paramagnet, 1/T 1 (o)It is the relaxation rate when not having paramagnet.Paramagnetic contribution 1/T to relaxation rate 1pRelaxation rate 1/T with first coordination shell 1MRelevant, relational expression is as follows:
1 T 1 p = 1 T 1 ( obs ) - 1 T 1 ( o ) = pq τ m + T 1 M + 1 T 1 ( os )
Wherein, 1/T 1 (os)Be to the contribution of the skin of relaxation rate, p is a concentration of paramagnetic ions and the ratio of ligand concentration, and q is the part number in the coordination shell.The q value is obtained by the relaxation rate of water, and how many hydrones illustrate has replaced by the part coordination on the paramagnetic ion.Nucleon is at the residence time τ of paramagnetic ion first coordination shell MConsider combining form and the exchange rate between the combining form not.By multiplying each other, with 1/T with p 1pAnd 1/T 2pCarry out the normalization of concentration.In conjunction with the relaxation time T of first coordination shell of ATP magnetic nuclear 1MAt quick exchange limit place and pqT 1pEquate.
In order to explain the structure and the kinetic property of paramagnetic metal-organic molecule compound, must measure q, r and τ cValue (or limit).Correlation time τ cCharacterized the rate process of regulating dipolar interaction, it is defined by following formula:
1 τ c = 1 τ r + 1 τ s + 1 τ m
To correlation time contributive parameter be: the timeconstant that internuclear ion-the nucleon radius vector rotatablely moves r, electron spin relaxation time τ sWith the residence time τ of nucleon at paramagnetic ion first coordination shell m1/ τ mValue is combining form and the ligand exchange speed between the combining form not.By rapid rate process or τ r, τ sOr τ mIn the shortest time measure τ correlation time cNeed these times of estimation could calculate 1/T c
In 25-46 ℃ of temperature range, obtain Mn (HCO 3 -) 2-ATP and MnCl 2The ATP proton H of-ATP compound 8, H 2, H 1And H 2The T of O 1And T 2Relaxation time.Measure the T of each proton then 1MAnd T 2MValue and relaxation rate 1/T 1pAnd 1/T 2p200,300,400 and 500MHz under measure proton H 8, H 2, H 1And H 2The T of O 1And T 2Relaxation time is to the dependence of frequency.
Then temperature and frequency are used to resolve different correlation times to 1/pT 1pAnd 1/pT 2pThe contribution of value.
With respect to hydrate (Mn (H 2O) 6 2+) relaxation rate, the increase of relaxation rate is expected.In hydrate, rotational time τ rDecision τ cIf at the residence time of first coordination shell τ mDecision longitudinal relaxation speed 1/T 1P, τ then mT 1M, because T 2M≤ T 1M, τ mNecessarily also determine 1/T 2P, 1/T 1P
Figure A200680054592D0062161745QIETU
1/T 2PBecause τ mReduce with the temperature increase, so 1/T 1PAnd 1/T 2PNecessarily increase with temperature.Because τ mDo not rely on frequency, 1/T 1PAnd 1/T 2PDo not rely on frequency, so when changing NMR frequency (ω I 2) time, T 1PFigure should show T 1PTo frequency dependence.If T 1PDo not rely on frequency (ω I) 2, τ then mBe factor of determination.At T 1To ω I 2Figure in, slope and ratio of distance are τ c 2, therefore could calculate τ cIf relaxation rate reduces with the increase of temperature, then T 1MAnd T 2M(outer relaxation) decision 1/T 1PAnd 1/T 2PExcept that depending on temperature, also depend on frequency if observe relaxation rate, then except that chemical exchange, rate process is to T 1PMake remarkable contribution.
Work as 1/T 1PFigure to temperature is timing, and reason may be following 3 kinds of possibilities:
1. chemical exchange speed 1/ τ MTo such an extent as to enough slowly determine 1/T 1P
2. if quick exchange and τ take place sDecision T 1M, τ under this condition sHas positive temperature coefficient (PTC).
3. if τ c10 -8To such an extent as to second ω I 2τ c 21, f (τ c) be 1/ τ cFunction, and work as τ cWhen shortening with the temperature increase, f (τ c) increase.
Following equation has provided the correlation time τ that characterizes the scalar interaction that transmits by chemical bond rather than by the space e:
1 τ e = 1 τ s + 1 τ m
Under most conditions, τ sLess than τ m, at high temperature, the τ of some ion mMay be less than τ s
τ cCan be by T 1To ω I 2The ratio (=τ of the slope of figure and intercept c 2) obtain.If τ cLess than τ sLower limit, then quick exchange can not take place, and τ sCan not decision T 1MUnder this condition, τ cMay not have positive temperature coefficient (PTC).
If at 1/T 1pPositive temperature coefficient region, find that the NMR frequency is to 1/T 1pDo not have influence, then Zhu Dao relaxation mechanism is attributable to ligand exchange speed 1/T M
Therefore, use 1/T 1pTemperature and the combination of judging which rate process or rate process of frequency dependence and EPR spectrum ( τR, τ sOr τ m) be the reason that causes the nuclear spin relaxation.Therefore, by Mn 2+The EPR spectrum of compound can obtain electron spin relaxation time τ sLower limit.
The disassociation of supercarbonate and in the presence of bivalent metal ion to the influence of ATP
The disassociation of bicarbonate ion in solution depends on pH.At Mn 2+And Mg 2+Exist and measure HCO down 3 -Disassociation is to the influence of GS activity.This has physiology importance, because with regard to the activity of GS, depends on the adenylylation state of enzyme, and GS has two optimal pHs, i.e. pH 6.3 and pH 7.3.Based on the dissociation theory of classics, when pH 6.3, bicarbonate radical and CO 2Ratio with 50:50 exists, and at pH 7.3, then almost only with HCO 3 -There is CO 3 2-The amount of form can be ignored.In the time of between pH 5.0 and pH 8.0, supercarbonate is NH for example 4HCO 3Or NaHCO 3Ionic dissociation equilibrium as follows:
Figure A200680054592D00641
In the polyprotonic acid aqueous solution,, be difficult to conductivity measurement is provided simple an explanation because ionic dissociation equilibrium is overlapping.In the pH scope that this research is paid close attention to, promptly pH 5.5-8.0 supposes that very important material has only NH 4 +, HCO 3-and CO 2CO 3 2-Role can be ignored or not have in assumed response mechanism.
The equilibrium constant of dissociation reaction obtains by following formula:
K 1 = [ NH 4 + ] [ HCO 3 - ] NH 4 H CO 3 F 1
Yet, depend on the pH of solution, set up second balance:
K 2 = [ NH 3 ] [ CO 2 ] HCO 3 - F 2
Wherein, F iThe merchant of expression vivacity:
F 1 = f NH 4 + f HCO 3 - f NH 4 HCO 3
F 2 = f NH 3 f CO 2 f HCO 3 -
By the Henderson-Hasselbach equation, determine the concentration of the different material that exists in the solution by pH value of solution and total dissociation degree α:
Figure A200680054592D00647
Yet, under pH5.5-8.0 and concentration 1mM-5mM condition, non-dissociated NH 4HCO 3Amount can be ignored.Usually
Figure A200680054592D00648
[NH 4HCO 3]＝c(1-α)
Figure A200680054592D00651
Yet, for NH 4HCO 3, suppose under low consistency conditions:
[ HCO 3 - ] [ NH 4 HC O 3 ] = 10 ( pH - pK a )
Figure A200680054592D00653
[CO 2]＝c(1-α)
Figure A200680054592D00654
Therefore,
[ HCO 3 - ] [ CO 2 ] = 10 ( pH - p K a )
When pH was increased to pH 8.0 by pH 5.5, the electrical conductivity of solution in this pH horizontal extent should be indicated HCO in the solution 3 -Concentration, and by CO in the solution 2The difference of concentration is indicated.In the presence of 10mM imidazoles HCl buffer solution, the relative concentration difference.Therefore, determine and containing NH 4HCO 3Solution in, the existence of imidazoles influences HCO with which kind of degree 3 -Dissociate into CO 2Importantly should be understood that at (Mn 2+) 3(HCO 3 -) 12The ATP compound exists down, and the relative concentration of above-mentioned every kind of material influences the function of adenylylation GS.Can also think HCO 3 -And CO 2In the reaction that adenylylation GS catalysis is regulated, all play a role.The pK of imidazoles HCl in the time of 25 ℃ aBe 6.92.Containing NH 4HCO 3Solution in, imidazole salts is as HCO 3 -Counter ion counterionsl gegenions.Under low pH condition, NH 4HCO 3Be tending towards disassociation and form soluble CO 2And NH 3
Figure A200680054592D00656
Imidazoles HCl dissociating in aqueous solution:
Figure A200680054592D00657
Contain 1mM NH 4HCO 310mM imidazoles HCl solution under higher pH, imidazoles excessive in the solution can become HCO 3 -Counter ion counterionsl gegenions, generate and can measure the other ionic species of its existence by the conductivity analysis.
Figure A200680054592D00661
Considering that observed molar conductivity A is the contribution summation that constitutes the ion j of electrolytic solution under the different pH levels, λ iIt is the conductivity of each ion.
Λ＝α(λ j[NH 4 +]+λ j[HCO 3 -]+λ j[Imi +])
It is 10mM imidazoles and 1mM NH that the conductivity analysis is used to be determined at concentration ratio 4HCO 3Imidazole buffer solution in, but quantitative measurement NH whether still 4HCO 3Disassociation.In pH value scope, add 1mM NH 4HCO 3Thereby the ultimate density that makes imidazoles is a 10mM preparation imidazoles HCl solution.Proof-the summary of catalytic mechanism
(Mn 2+) 3(HCO 3 -) 12The structure analysis of ATP compound and the described (Mn of molecular simulation explanation 2+) 3(HCO 3 -) 12The ATP compound plays a part very important in reaction mechanism.Two kinds of methods are used to prove this reaction mechanism.Rite-directed mutagenesis (SDM) is used for proving the effect of the main amino acid of enzyme active sites in the phosphoryl transfer process.SDM occurs in His269, His271, Glu207 and Arg356.As previously mentioned, all amino acid residue numberings are corresponding to colibacillary GS residue; Use known technology, for example molecular simulation or homology comparison, those skilled in the art can be by the corresponding residue among other species mensuration GS.Can measure then each mutant enzyme gamma glutamyltransferase enzymatic activity and compare with the enzymatic activity of the gamma glutamyltransferase of mutant enzyme not.For obtaining to go fully the enzyme of adenylylation, carry out SDM in GS adenylylation site, just make Tyr397 sport Val397.As Senior, P.J. (1975) J.Bact.123:(2), 407-418 outline like that, at nitrogen excessive and carbon is limited the quantity of or nitrogen is limited the quantity of and the excessive condition of carbon under, also can obtain complete adenylylation or remove the GS of adenylylation by the Continuous Cultivation Escherichia coli.Adenylylation GS is preferably at Mn fully 2+Play a role when existing, and go adenylylation GS preferably at Mg 2+Play a role when existing.
The second method that is used for proved response mechanism is to use NMR spectroscopic methodology proof to use Mg in the phosphoryl transfer reaction of ATP catalysis 2+Or Mn 2+As the producible function difference of bivalent metal ion.These are reflected under the no enzyme condition and take place.Proton N MR relaxation data declaration is for Mn 2+, this bivalent metal ion may be close to the adenine ring, and initial hypothesis thinks that this may play a role to catalytic process.Find under certain conditions the C of ATP subsequently 8The proton mutability, a kind of mechanism that soluble this situation takes place is Mn 2+Whether be attached to C 8Form metal carbene.Someone proposes can take place by coordination HCO in the process of enzyme bound substrates 3 -The N that causes 7Possible carboxylation or protonated, at N 7The place brings out cationic imide.This cationic imide further impels Mn 2+With C 8Thereby between form supposition combination form metal carbene.Someone proposes the HCO of electric charge (effectively proton) by coordination 3 -Be displaced to N 7Identical result can pass through N 7Carboxylation regulate.
Metal carbene is in case formation, then N 7Carboxyl can in the phosphoryl transfer process, play a role, form carboxyl-phosphoric acid, follow the fracture of metal carbene simultaneously.In the enzymatic process, phosphoryl is transferred to glutamic acid by the side chain of amino acid residue His269 and His271, forms gamma-glutamyl phosphoric acid, and Glu207 and Arg356 also play a role in catalytic process.For proving variable C 8-H is at D 2Prepare Mg-ATP, Mn (HCO among the O 3) 2-ATP and MnCl 2-ATP compound, and pass through 1H NMR monitors C 8The existence of-H.At D 2Entire reaction takes place among the O, because if C 8The proton mutability, C in course of reaction 8Proton 1H NMR displacement meeting disappears, because this proton and body exchange mutually of ATP can take place, promptly this proton is entered D by exchange 2Among the O.In course of reaction, also monitor the hydrolysis of ATP.ATP by in the glutamine synthelase analytic approach, using C8 deuterium generation and measure the catalysis isotope effect that causes, also provable C 8The effect of proton in reaction.Find that the catalysis isotope effect only occurs in by using Mn 2+The reaction regulated of adenylylation glutamine synthelase in, and do not occur in by using Mg 2+Go in the reaction that the adenylylation glutamine synthelase regulates.
Proof-the material of catalytic mechanism and method
Mn (HCO 3) 2-ATP and MnCl 2The preparation of-ATP compound
With Na 2ATP is dissolved in water, makes its concentration be about 80mM.Make solution pass through the Dowex 50 WX2 strong cation-exchanging resins of acid then, from ATP, remove Na +Ion.The 50mM HCl that makes 3 times of bed volumes uses the H of 5 times of bed volumes then by post 2O washes post, and Dowex 50WX2 resin is converted into acid.Collect to merge all samples that contains acid-ATP, and with etc. the MnCO of volumetric molar concentration 3, Mg (OH) 23MgCO 33H 2O reaction or and MnCl 2Mix.Stirring contains Mg (OH) 23MgCO 33H 2O and MnCO 3Solution until all MnCO 3Or Mg (OH) 23MgCO 33H 2O is dissolved.Use NaHCO then 3With Mn (HCO 3 -The pH regulator of)-ATP solution is to pH 6.3-7.0.Regulate the Mg of ATP with NaOH 2+And MnCl 2The pH of compound.Prepare Na by the pH that regulates 80mM solution with NaOH to pH 7.0 4ATP.Exist (data not shown) with electrospray ionization mass spectrum proof compound.Measure Mn with ICP 2+Correct stoichiometry (data not shown).Measure Mn (HCO with single metering method (monometrically) 3 -) 2HCO in the-ATP compound 3 -Roughly stoichiometry.
ATP concentration is to Na 4ATP, Mn (HCO 3 -) 2-ATP, Mg-ATP and MnCl 2The influence that the NMR of-ATP compound analyzes
For measuring to ATP's 1The pile up effect of H NMR is measured Na 4ATP, Mn (HCO 3 -) 2-ATP, Mg-ATP and MnCl 2The ATP concentration of-ATP is to H 8, H 2, H 1And H 2The T of O proton 1The influence in relaxation time.With Na 410 of ATP or Mg-ATP concentration -3Concentration, with Mn (HCO 3 -) 2-ATP and MnCl 2-ATP adds Na 4ATP and Mg-ATP.Concentration range with 5-120mM adds ATP.
Na 4ATP, Mn (HCO 3 -) 2-ATP, Mg-ATP and MnCl 2The NMR of-ATP compound analyzes
Measure Mn (HCO 3 -) 2-ATP compound and MnCl 2-ATP compound is to H 8, H 2, H 1And H 2Vertical T of O proton 1With horizontal T 2The influence in relaxation time.With Mn (HCO 3 -) 2-ATP compound and MnCl 2-ATP compound adds 60mM Na 4In ATP or the Mg-ATP solution, make its concentration become 60 μ M.To contain Mn (HCO 3 -) 2-ATP or MnCl 2The 60mMNa of-ATP compound 4The freeze-drying of ATP solution stores down at-20 ℃, and as required, by being dissolved in D 2The preparation solution of O.There is and do not exist Mn (HCO 3 -) 2-ATP or MnCl 2Under the condition of-ATP, use Varian UNITY plus 400MHz nuclear magnetic resonance analyser to Na 4Thereby ATP or Mg-ATP compound carry out nuclear magnetic resonance experiment obtains T 1And T 2Relaxation time.Mn (HCO 3 -) 2-ATP compound is based on the Na that joins in the solution 2HCO 3, MnCO 3, ATP relative concentration.In temperature range, measure the relaxation time.
Under 200MHz, 300MHz, 400MHz and 500MHz, measure the NMR frequency to T 1And T 2The influence in relaxation time.The equipment that uses is Varion Gemini200/2000 (200MHz), Varion Unity Inova 400 (400MHz), Br ü ker ARX 300 (300MHz) and Advance500 (500MHz).
PH is to NH in the imidazole buffer 4HCO 3The influence of disassociation
Measure pH to NH in the imidazole buffer 4HCO 3The influence of disassociation.The NH that is dissolved in imidazoles HCl damping fluid (10mM) in the preparation pH horizontal extent 4HCO 3(1mM) solution.The only 10mM imidazoles HCl and the 10mM imidazoles HCl that also measure under the different pH levels add 1mMNH 4HCO 3Conductivity.Prepare all solution with the water that than conductivity is 18 μ S.Use Jenway 4150 conductivity meters to measure then and compare conductivity.Therefore, the difference between the ratio conductivity of two of gained associations comprises the contribution of the ratio conductivity of the ion j that constitutes electrolyte solution.Molar conductivity Λ under the viewed different pH level is the summation of single molar conductivity.
Λ＝α(λ j[NH 4 +]+λ j[HCO 3 -]+λ j[Imi +])
ATP hydrolysis rate and Na 4ATP, Mn (HCO 3 -) 2-ATP, Mg-ATP and MnCl 2C-H in the-ATP compound 8Deuterium generation
The NMR spectroscopic methodology is used to prove the C of ATP under certain conditions 8Proton is variable and only at Mn 2+Be attached to C 8Shi Fasheng.For proving variable C 8-H, preparation is dissolved in D 2The Mg-ATP of O, Mn (HCO 3 -) 2-ATP and MnCl 2-ATP compound, and pass through 1H NMR monitors C 8The existence of-H over time.At D 2Thereby carry out entire reaction among the O and measure Mn 2+Or Mg 2+To C 8The proton deuterium is for the influence of speed, because with this proton and D 2In the course of reaction that is exchanged for the result of O, C 8Proton 1H NMR displacement disappears.Therefore ATP is at C 8The position is by deuterium generation.Also can be determined at the hydrolysis rate of ATP in the course of reaction.Use H 2This reaction also can take place as solvent in O.Respond and occur in the imidazole buffer of pH 6.3 or pH 7.3.The NaHCO of use experimentize at imidazole concentration during for 20mM 3, MnCl 2And MgCl 2Concentration is NaHCO 3Between 0-12mM, change MnCl 2And MgCl 2Between 0-4mM, change.In particular time interval, get 150 μ L samples, and use D 2O or H 2O is diluted to 730 μ L.Adding EDTA (20 μ L) is 2.0mM to its concentration, and centrifugal sample is to remove bivalent metal ion behind the 10min.By 1H NMR analytic sample is measured ADP concentration by HPLC then.
M 2+Concentration is to adenylylation and the influence of going adenylylation GS activity
At certain ATP concentration range and certain M 2+In the ATP ratio ranges, measure Mn 2+And Mg 2+Concentration is to adenylylation and the influence of going adenylylation GS activity.The ATP concentration of using is 200 μ M, 400 μ M, 600 μ M, 800 μ M and 1000 μ M.In each used ATP concentration, add MnCl 2Or MgCl 2It to its concentration 1,2,3 or 4 times of ATP concentration.Other component of analytic approach is 20mM imidazoles HCl, 12mM NaHCO 3, 4mM NH 4Cl and 4mML-glutamic acid and 4mM NH 4Cl.Use the analytic approach of adenylylation glutamine synthelase 6.3 times at pH, and make the analytic approach that spends the adenylylation glutamine synthelase 7.2 times at pH.With NaHCO 3As the compound of last adding, prepared fresh analytical solution immediately before use, and adding the fashionable pH that regulates immediately.
HCO 3 -Concentration is to adenylylation and the influence of going adenylylation GS activity
At certain Mn 2+Measure HCO in the concentration range 3 -Concentration is to the influence of adenylylation GS activity.Analyte comprises 20mM imidazoles HCl, 4mM NH 4Cl, 600 μ M ATP, 1.8mM MnCl 2With 4mM L-glutamic acid, and carry out this analysis 6.3 times at pH.NaHCO 3Concentration is at 1-12 μ mole NaHCO 3Change between/μ mole the ATP.Can not make to spend adenylylation GS and analyze, because when the high carbon acid salinity, Mg 2+Precipitate.
The ATP in C-8 position deuterium generation and 13C HCO 3 -Influence to adenylylation GS specific activity
By under 60 ℃ with 20mM Na 2ATP joins and is dissolved in D 2In the 50mM triethanolamine of O, make Na 2The ATP deuterium was for 96 hours.Store down with the sample freeze-drying and at-20 ℃ then, when needs, it is dissolved in the water.Use Luna C then 18Reversed-phase column (250mm * 25.2mm), is 7.0ml/min with ammonium acetate as moving phase, flow velocity, the ATP in deuterium generation is carried out purifying.Collect sample and freeze-drying then to remove moving phase.Check then gained ATP in the C-8 position deuterium generation whether fully.
The concentration of ATP that is determined at C-8 position deuterium generation is to the influence of adenylylation GS activity.Analyte comprises 20mM imidazoles HCl, 12mM NaHCO 3, 4mM L-glutamic acid and 4mM NH 4Cl analyzes at pH 6.3.Can not make to spend adenylylation GS and analyze, because ATP is in the deuterium specific activity not influence (data not shown) of generation to removing adenylylation GS of C-8 position.
In deuterium generation with also can not measure Na in the presence of the ATP in deuterium generation 13HCO 3Influence to the glutamine synthelase specific activity.Analyte comprises 20mM imidazoles HCl, 12mM NaHCO 3(or Na 13HCO 3), 4mM L-glutamic acid and 4mM NH 4Cl, and carry out for 6.3 times at pH.Under 800mM ATP, analyze, relatively the ATP in C-8 position deuterium generation and the ATP of natural abundance.Bacterial isolates and nutrient culture media
Table 1 has been listed used bacterial isolates and carrier.In the 38%m/v glycerite of all bacterial isolates low-temperature preservations under-70 ℃.Except as otherwise noted, all culture of Escherichia coli are remained on LM nutrient culture media (5g/l NaCl, 10g/l yeast extract, 10g/l tryptone; PH7.2) on.If need, then add the agar that concentration is 15g/l.For pAlter-1, with 50 μ g/ml ampicillins or 12.5 μ g/ml tetracycline supplementing culture mediums, and for pBluescript II SK +, replenish with 100 μ g/ml ampicillins.
The M9 nutrient culture media that contains trace salt by use is realized the growth of mutant strain on minimal medium.Trace salt prepares in 0.1N HCl, and this trace salt comprises following (being expressed as every liter of solution contains): 3.5g FeSO 47H 2O, 0.5g MnSO 4H 2O, 0.11g Na 2B 4O 710H 2O, 0.13gNa 2MoO 42H 2O, 1.1g ZnSO 4, 0.1g CuSO 45H 2O and FeCl 36H 2O.Before the use, trace salt diluted in isopyknic 0.1N NaOH and add with the concentration of 20mL/L M9 nutrient culture media.When needs, be 50 μ gmL with concentration respectively with ampicillin and tetracycline antibiotic -1With 12.5 μ gmL -1Add in the nutrient culture media.
Table 1
Figure A200680054592D00711
The separation of glnA gene and the structure of carrier
Use QiaPrep Spin Miniprep Kit TM(Qiagen) or by alkaline lysis (Sambrook, J., Fritsch, E.F. and T.Maniatis (1989) In:Molecular Cloning:A Laboratory Manual., second edition Cold Spring Habor Laboratory LaboratoryPress) the small-scale DNA isolation.Using Qiagen Midi DNA separating kit (Qiagen) to finish extensive DNA separates.Use illustrates that available from the restriction enzyme of Amersham Biosciences and according to manufacturer carrying out the DNA enzyme cuts.Alkaline phosphatase is from AmershamBiosciences.The T4 dna ligase uses from Promega and according to the scheme that this enzyme provides.The agarose that is used for the DNA electrophoresis is a molecular biology grade.
Taq polymerase (rTaq) is from TaKaRa Bio Inc. and be used for general screening.The Taq polymerase (ExTaq) of high fidelity is also from TaKaRa Bio Inc. and be used for amplification and be used for cloned genes.
Use standard step (Sambrook, J., Fritsch, E.F. and T.Maniatis (1989) In:Molecular Cloning:A Laboratory Manual., second edition Cold Spring HaborLaboratory Press) carry out restriction enzyme digestion and agarose gel electrophoresis.1kb dna ladder shape band is used for all electrophoresis.
According to the scheme that each kit provides, use from the external sudden change kit of the Altered Sites of Promega company, or carry out rite-directed mutagenesis from the QuikChange XL rite-directed mutagenesis kit of Stratagene.
All chemicals are AG or molecular biology grade, and need not to be further purified and can use.Except as otherwise noted, all chemicals are from Merck or Sigma.
The clone of Escherichia coli glutamine synthetase gene
Design of primers is arrived the Escherichia coli glnA gene order that obtains by Genbank (accession number X05173).Design of primers is become at 5 ' end to have NsiI restriction site (shown in the boldface type).Below show this primer:
5 ' primer: 5 '-GATATGCATCCGTCAAATGCG-S ' (SEQ ID NO:1)
3 ' primer: 5 '-GCGATGCATAAAGTTTCCACGG-3 ' (SEQ ID NO:2)
Use the DNA of pGLn6 to carry out PCR as template and above-mentioned primer.The PCR potpourri contains 1 μ l plasmid DNA (50ng), every kind of primer of 5 μ l (2.5pmol/ μ l), 4 μ l 2.5mM dNTP, contains 20mM MgCl 25 μ l 10X damping fluids and the Taq polymerase (2.5 units) of the high fidelity of 0.5 μ l.
Carry out PCR as follows, promptly earlier to template DNA at 95 ℃ of pre-sex change 5min down, subsequently 95 ℃ of following sex change 5min, 55 ℃ down annealing 1min and 72 ℃ extend 2min down, carry out 30 such circulations altogether.In the last extension step of carrying out 10min under 72 ℃ also adition process.Carry out the fragment length that agarose gel electrophoresis produces with checking PCR.Use high-purity PCR purification kit (Roche Diagnostics) purified pcr product, and use NsiI that this product is carried out enzyme and cut.
For make up using Altered Sites system to carry out the template of SDM, with PstI to the pAlter-1 linearization and before connection dephosphorylation.With embolus: carrier is than being that 3:1 connects embolus and carrier.
According to manufacturer's explanation, use Bio-Rad gene pulse instrument the coupled reaction product to be transformed among the Escherichia coli JM 109 by electroporation.On the LM agar medium that is supplemented with 12.5 μ g/ml tetracyclines, 80 μ g/ml X-Gal and 1mM IPTG, screen transformant.
Subsequently by use the alkaline lysis DNA isolation, enzyme is cut the row agarose gel electrophoresis of going forward side by side and is screened by transforming the dull and stereotyped several white colonies that obtain of going up then.Check order to confirm existing and sequence of Escherichia coli glnA gene.Except using M13/F and M13/R universal primer, also design the inner primer of gene specific by known gene order.Table 2 shows these primers.
The sequence specific primers that table 2. couple clone's Escherichia coli glnA gene checks order and uses
Primer Primer sequence
GlnSeq1 5’-GCTGAACACGTACTGACGATG-3’(SEQID NO：3)
GlnSeq2 5’-GTGGGAACCGGAGATAGATGATC-3’(SEQ ID NO：4)
GlnSeq3 5’-CGATGTTCGGTGATAACGGCTC-3’(SEQID NO：5)
GlnSeq4 5’-CGTACTTCGATACGACGTGCTTTC-3’(SEQ ID NO：6)
For using QuikChange TMSystem constructing is used for the template of SDM, by pAlter construct subclone glutamine synthetase gene as the SacI-HindIII fragment.The band that will contain the glnA gene with knife blade scales off from gel, thereby pushes Eppendorf pipe pulverizing agarose by the syringe of 2ml.(balance is to pH 8.0) adds this pipe with 1ml phenol, then with suspension vortex 1min.Descend freezing samples 30min at least at-70 ℃.Sample one thaws, just with its in the Eppendorf microcentrifuge with the centrifugal 15min of 13 000rpm.Water is moved to clean pipe, and use phenol then: chloroform: isoamylol (25:24:1) extracts once, then uses chloroform: isoamylol (24:1) extracts once.With ethanol with DNA precipitation and be resuspended in the TE damping fluid.Then with this fragment with embolus: carrier is than being that 3:1 is connected to pBluescript II SK +In, carry out enzyme with SacI and HindIII again and cut.This coupled reaction product is transformed among the Escherichia coli XL1-Blue by electroporation, and on the LM agar plate that contains 100 μ g/ml ampicillins, carries out flat board and cultivate.By the alkaline lysis isolated plasmid dna, use the BamHI enzyme to cut DNA and use agarose gel electrophoresis to analyze these fragments subsequently and screen single transformant bacterium colony (single transformant colonies).
Rite-directed mutagenesis (SDM)
Use Qiagen Midi Prep kit to separate the DNA of the wild type glnA gene among the pAlter-1 by e. coli jm109.Table 3 has been listed and has been used this system to carry out the designed oligonucleotides of glnA gene mutation.Be building up in the SDM oligonucleotides introducing restriction site be beneficial to the to suddenly change silent mutation of elementary screening.These are also shown in the table 3.
Table 3. uses Altered Sites TMThe sudden change of the Escherichia coli glnA gene that system carries out.The sudden change that changes amino acid residue illustrates with boldface type, lines out below the restriction site.
Sudden change Oligonucleotide sequence Restriction site
Y397V 5’-TGGACAAAAACCTG GTCGACCTGCCGCCAG-3’(SEQ ID NO：7) SalI
Sudden change is selected screen mutation list bacterium colony after taking place, and DNA isolation from incubated overnight liquid is passed through in described screening, and uses the enzyme of the specific sudden change of being screened to carry out restriction analysis.The expression of mutator
Cut by using SacI and HindIII to carry out enzyme, separate mutator by the pAlter-1 clone body.The material that enzyme is scaled off carries out agarose gel electrophoresis with carrier of separating and embolus band then.As mentioned above, the band that will contain gene scales off and uses phenol extraction DNA from gel.
Cut pBluescript II SK with SacI and HindIII enzyme +, then with embolus: carrier is that 3:1 is connected to each mutator in the carrier.The coupled reaction product is transformed among the glutamine synthelase auxotrophy strain Escherichia coli YMC11 by electroporation, and on the LM agar medium that is supplemented with 50 μ g/ml ampicillins, selects transformant.
By using the PCR of M13/F and M13/R universal primer, to transforming the single bacterium colony enforcement screening step that obtains on the flat board.In this step, single bacterium colony is resuspended in the 20 μ l distilled water.This bacterium colony suspension of 1 μ l is added in the PCR reaction system, and described PCR reaction system contains every kind of primer of 2.5 μ l (2.5pmol/ μ l), 2 μ l 2.5mM dNTP, 2.5 μ l 10X damping fluids, 2 μ l 25mMMgCl 2With 0.1 μ l Taq polymerase (0.5u).Carry out the PCR circulation as mentioned above.Subsequently, separate the PCR product, and select positive transformant based on correct stripe size by agarose gel electrophoresis.Positive control and negative control are introduced the result that this step obtains with checking.
QuikChange TMSudden change
Use Qiagen MidiPrep kit is separated the DNA (pBluescript construct) of selected template from Escherichia coli XL1-Blue.Table 4 has been listed and has been used this system to carry out the designed oligonucleotides of SDM.Because this is based on the system of PCR, so two kinds of oligonucleotides of each reaction needed (justice with antisense).
The sudden change of the Escherichia coli glnA gene that table 4. use QuikChange system carries out.The sudden change that changes amino acid residue illustrates with boldface type, lines out below the restriction site.
Sudden change Oligonucleotide sequence Restriction site
E207T 5’-CAGATGGG ACTAGTGGTTACTGCCCAT-CACCACGAAGTAG-3’(SEQ ID NO：8)5’-CTACTTCGTGGTGATGGGCAGTAACC- ACTAGTCC-CATCTG-3’(SEQ ID NO：9) SpeI
H269N 5’-TTCGGTGATAA CGGATCCGGTATGAAT-TGCCACATGTCTCTGTC-3’(SEQ IDNO：10)5’-GACAGAGACATGTGGCAATTCATACC- GGATCCGTTATCACCGAA-3’(SEQ IDNO：11) BamHI
H271N 5’-GATAACGGCTCCG GCATGCACTGCAAT-ATGTCTCTGTCTAAAAACG-3’(SEQ IDNO：12)5’-CGTTTTTAGACAGAGACATATTGCAGT- GCATGCCGGAGCCGTTATC-3’(SEQ IDNO：13) SphI
R355Q 5’-GAAAGCACGTCAAATCGAAGTACGTTTCCC AGATCCG-3’(SEQ ID NO：14)5’-CGGATC TGGGAAACGTACTTCGATTTGACGTGCTTTC-3’(SEQ ID NO：15) Remove the BamHI site
Sudden change is selected screen mutation list bacterium colony after taking place, and DNA isolation from incubated overnight liquid is passed through in described screening, and uses the enzyme of the specific sudden change of being screened to carry out restriction analysis.Use QuikChange TMSystem is transformed into the positive mutating strain that obtains and is used among the Escherichia coli YMC11 expressing.
The affirmation of sudden change
Check order to confirm each sudden change by external agency.For this purpose, design forward and reverse gene-specific primer by known gene order.These are shown in Table 5.
Table 5. is used for confirming the employed sequence specific primers that exists of the various sudden changes of Escherichia coli glnA gene
Figure A200680054592D00761
The result
The clone of Escherichia coli glutamine synthetase gene
With Escherichia coli wild type glnA gene magnification is the 2.1kb fragment, and code length is 471 amino acid whose albumen.With embolus: carrier is that the glnA gene of the pcr amplification of the 3:1 2124bp that will contain NsiI flank restriction site is connected among the SDM carrier pAlter-1 that the PstI enzyme cuts.DNA isolation and use BamHI and EcoRI carries out restriction analysis from some transformant.According to the known array of gene and carrier, with BamHI construct (having this glnA gene at required 5 correct ' end to 3 ' end orientation) is carried out restriction enzyme digestion, can produce the fragment of 6012bp and 1797bp.Identified correct construct by this way, and called after pGln12.
Use QuikChange for making up TMThe wild type template that system suddenlys change scales off the fragment as SacI-HindIII with the glnA gene from pGln12, and with embolus: carrier is that 3:1 is connected to the pBluescript II SK that cuts through similar enzyme +In.The coupled reaction product is transformed among the Escherichia coli XL1-Blue, and on the LM agar medium that is supplemented with 100 μ g/ml ampicillins, 80 μ g/ml X-Gal and 1mM IPTG, carries out flat board and cultivate.In order to identify positive subclone strain, DNA isolation also limits it restriction analysis with SacI, HindIII and BamHI from some white colonies.Identify correct construct by this way and with its called after pBSK-ECgln.Before carrying out any SDM, two clone body are carried out the integrality of sequential analysis with the checking wild type gene.
Rite-directed mutagenesis (SDM)
Altered Sites TMSystem
Carry out rite-directed mutagenesis according to such scheme.Use is carried out enzyme to the special enzyme that suddenlys change to the DNA that separates from mutant and is cut, and carries out size fractionation (size-fractionated) to confirm to exist sudden change.
Under any circumstance, use identical enzyme that wild type construct (pGln12) is carried out enzyme and cut contrast as a comparison.The sudden change such as the table 6 that have separately the restriction site introduced and desired fragment length are listed.
Table 6. uses Altered Sites system to introduce the tabulation of the sudden change among the pGln12, demonstrates desired restriction fragment
Figure A200680054592D00771
Owing to obtain desired dna fragmentation length, agarose gel electrophoresis confirms to exist sudden change.The expression of mutator
With mutator (from pAlter) subclone to pBluescript II SK +In, and be transformed among the Escherichia coli YMC11, to promote the research of protein purification and enzyme.Confirm that by the PCR screening subclone gene is present in the carrier.After testing, the transformant bacterium colony that contains sudden change glnA gene is the 2170bp band on the Ago-Gel.Comprise the negative control of being made up of the carrier that is transformed among the Escherichia coli YMC11 in the PCR screening, and this negative control is presented on the gel as band, this band has the size of carrier multiple clone site.The PCR screening also comprises the also positive control of the pBSK-ECgln DNA in Escherichia coli YMC11.It is presented on the gel as band, with any positive the big or small identical of subclone body of suddenling change.Use then and the special restriction enzyme that suddenlys change is carried out enzyme to the DNA from single subclone body of each mutant that identifies by this way cut, to confirm to exist specific mutations.
The tabulation of the restriction fragment that table 7. sudden change glnA subclone body is desired
Figure A200680054592D00781
Because the dna fragmentation length that obtains expecting, agarose gel electrophoresis confirm to exist in the subclone body sudden change.
The QuikChange sudden change
Scheme uses the QuikChange system to carry out SDM as described above.DNA isolation from possible mutant, use carry out enzyme to the special enzyme that suddenlys change to DNA to be cut, and carries out size fractionation to confirm to exist sudden change.Use identical enzyme that enzyme is carried out in the contrast of being made up of the parental generation template and cut contrast as a comparison.The restriction site and the sudden change such as the table 8 of desired fragment length that have separately are listed.PBSK-ECgln is used for E207T, H269N, H271N and ER355Q sudden change as template.In pBSK-Y397V, produce double-mutant E207T Y397V, H269NY397V, H271N Y397V and R355Q Y397V.
Table 8. uses the QuikChange system to be incorporated into the tabulation of the sudden change in each template, shows desired restriction fragment
Owing to obtain desired dna fragmentation length, agarose gel electrophoresis confirms to exist sudden change.Enzyme is analyzed
Use the enzymatic activity of measuring the GS gamma glutamyltransferase as described standard methods of people (1970) Adv Enzyme Reg:8:99-118 such as Stadtman E.R..Prove the homogeney of enzyme with polyacrylamide gel electrophoresis (PAGE).Measure the generating rate of glutamine and ADP by high pressure lipuid chromatography (HPLC) (HPLC) and measure GS forward reaction speed.The GS forward reaction comprises (unless otherwise defined): 11mM (NH 4) HPO 4, 1.0mM glutamic acid and 1.0mM M 2+-ATP compound (is Na 2Mn (HCO 3) 2-ATP, Na 2Mg-ATP or Na 2MnCl 2-ATP).Being reflected at pH 6.3 or pH carries out for 7.2 times.
The purifying of Escherichia coli glutamine synthelase
M9 nutrient culture media (6g/l Na in the modification that is supplemented with 70mM L-glutamic acid, 5mM L-glutaminate and 100 μ g/ml ampicillins 2HPO 4, 3g/l KH 2PO 4, 0.5g/l NaCl) in, cultivate all recombinant precursors be used to separate GS.At 37 ℃, rotating speed is to all nutrient solution shaken cultivation 48 hours under the 220rpm.Centrifugal under 4 ℃, 10000rpm, collecting cell from nutrient culture media.Use these living beings then immediately or descend storage when needs use at-20 ℃.In addition; by the wild type glutamine synthelase (from Escherichia coli pBSK-ECgln) that the living beings purifying obtains adenylylation and goes two kinds of forms of adenylylation to exist, described living beings are from obtaining as the described Continuous Cultivation of Senior P.J. (1975) J.Bact:123 (2): 407-418.
Under the condition that the excessive and carbon of nitrogen is limited the quantity of, generate the adenylylation enzyme, and nitrogen limit the quantity of and the excessive condition of carbon under generate and remove the adenylylation enzyme.Centrifugal 10min collects the cell that obtains under 4 ℃, 10000rpm, and-20 ℃ down storage when needs use.
The method that is used for the purifying glutamine synthelase is developed by reported method (Shapiro and Stadtman (1970) Methods Enzymol.17A:910-922.).
To be resuspended in the resuspended buffering agent A of 10ml or (RBA) (10mM imidazoles-HCl, 2mM beta-mercaptoethanol, 10mM MnCl from the living beings of 1L culture 24H 2O; PH 7.0) in.With cell at 50% dutycycle (duty cycle), 6 ℃ ultrasonic 10min down.With this ultrasonic solution centrifugal 10min under 10 000rpm, and keep supernatant.Add streptomycin sulphate (10%w/v 10%), and stir suspension 10min down at 4 ℃.Centrifugal 10min and keep supernatant under 10 000rpm then.Regulate supernatant pH to 5.15 with sulfuric acid.Stir this potpourri 15min, centrifugal 10min under 13 000rpm then down at 4 ℃.Keep supernatant once more.Add saturated ammonium sulfate (volume 30%) and regulate pH to 4.6 with sulfuric acid.Stir suspension 15min, centrifugal 10min under 13 000rpm then down at 4 ℃.The precipitation that obtains is resuspended in 2-5ml RBA and regulates pH to 5.7 with sulfuric acid.4 ℃ stir down that these suspensions spend the night so that glutamine synthelase be able to resuspended, centrifugal 10min under 13 000rpm then.Keep supernatant and with suspension pH regulator to 7.0.
Use AKTA Explorer (Amersham Biosciences) to utilize affinity chromatography that wild-type enzyme is further purified.Use length to realize separating with 5 ' AMP agarose 4b resin (Amersham Biosciences) as the HR10/10 post of 10mm with internal diameter as 10cm.To preparative column, described preparative column is with 10mM imidazoles (pH 7.0), 150mM NaCl and 10mM MnCl with partially purified glutamine synthelase preparation (being about 2ml) application of sample 24H 2O comes balance.At the 40ml NaCl of whole linear gradient, use 2.5mM ADP that the glutamine synthelase of combination is eluted from post, and collect the 1ml component from 150mM to 500mM.Collect then and merge the component that contains pure glutamine synthelase and use the RBA dialysed overnight.
According to standard scheme, the aliquot to each albumen suspension on the 7.5%SDS-PAGE gel is carried out electrophoresis.Use Lowry Protein Detection method to measure protein concentration, described concentration is used to measure the specific activity of all enzymes.
The rite-directed mutagenesis of Escherichia coli glutamine synthelase (GS)
GS sequential analysis and protein molecular simulation
Use
Figure A200680054592D00811
(Lynnon Biosoft, Quebec Canada) carry out the comparison of protein sequence homology.(MSI Inc., San Diego USA) are used for the protein molecular simulation that all use Silicon Graphics Octane processor to Accelrys Inc. molecular simulation software.
Proof-the result of catalytic mechanism and discussion
The ATP structure analysis
ATP concentration is to Na 4ATP, Mn (HCO 3 -) 2-ATP, Mg-ATP and MnCl 2The influence that the NMR of-ATP compound analyzes
For measuring in the solution ATP to ATP's 1The pile up effect of H NMR is at Mn (HCO 3 -) 2-ATP or MnCl 2-ATP exists down, measures ATP concentration (Na 4ATP or Mg-ATP) to H 8, H 2, H 1And H 2The T of O proton 1The influence in relaxation time.With Na 410 of ATP or Mg-ATP concentration -3Concentration, with Mn (HCO 3 -) 2-ATP and MnCl 2-ATP adds Na 4ATP and Mg-ATP.Concentration range with 5-120mM adds ATP.Along with ATP concentration is increased to 60mmol, 1/pT 1pIncrease with concentration is linear increase.Can determine to measure T by these data 1And T 2All analyses of relaxation rate should be at the Na of 60mmol 4The MnCl of ATP and Mg-ATP and 60 μ mol 2-ATP or Mn (HCO 3 -) 2Carry out during the ATP concentration of-ATP.
Temperature and frequency are to pT 1p -1And pT 2p -1The influence of relaxation rate
Use Varian UNITY plus 400MHz nuclear magnetic resonance analyser to obtain Mn (HCO 3) 2-ATP compound, MnCl 2-ATP compound and Na 4ATP is at the T of 400MHz 1And T 2Relaxation rate.In certain temperature range, experimentize, and draw H 8, H 2, H 1And H 2The pT of O proton 1P -1And pT 2P -1Relaxation rate is to the figure of 1000/K.According to definition, if relaxation rate reduces with the increase of temperature, T then 1MAnd T 2MOr outer relaxation decision pT 1p -1And pT 2p -1When energy of activation greater than 4kcal/mole and T 1P7/6 T 2PThe time, can get rid of Mn (HCO 3) 2-ATP compound and MnCl 2Proton H in the-ATP compound 2And H 1Outer relaxation.Mn (HCO 3) 2-ATP compound and MnCl 2The H of-ATP 8The proton behavior shows different.
Between 25 ℃-35 ℃ (1000/K is between 3.20 and 3.35), MnCl 2The H of-ATP compound 8The T of proton 1P -1Figure to 1000/K does not show temperature effect.For Mn (HCO 3) 2-ATP compound, T 1P -1H to 1000/K 8Figure increases between 25 ℃-35 ℃, descends subsequently.Explanation to this increase is, if ligand exchange decision relaxation rate, then 1/ τ M(combination and not the ligand exchange speed between the combining form) decision relaxation rate T IP -1And T 2P -1If τ MDecision T 1P, τ then MT 1M, and because T 2M≤ T 1M, τ then MNecessarily also determine T 2P, the result is T 1P -1
Figure A200680054592D0082145827QIETU
T 2P -1Work as T 2P -1/ T 1P -1Significantly less than 1 o'clock, then situation was different.Work as τ MWhen reducing with the temperature increase, T 1P -1And T 2P -1Necessarily increase with temperature.Because at T 1P -1Or T 2P -1Among the figure to frequency, τ MDo not rely on frequency, so T 1P -1And T 2P -1Should not rely on frequency yet.If observed relaxation rate depends on frequency, except that chemical exchange, rate process is to T 1PMake remarkable contribution.Mn (HCO 3 -) 2-ATP compound and MnCl 2The T of-ATP 1PAnd T 2PThe mensuration of frequency has been shown dependence to frequency.Thereby the linear regression of data is used for the ratio of slope calculations and intercept calculates τ c 2Therefore, the rate process except that chemical exchange is to T 1PMake remarkable contribution.
Mn (HCO 3) 2-ATP compound and MnCl 2Proton H among the-ATP 2And H 1All demonstrate frequency dependence and relaxation rate T 1P -1Reduce with the temperature increase.Because the chemical exchange rate has positive temperature coefficient (PTC), the negative dependence of temperature coefficient illustrates 1/ τ MCan not become the speed limit process.So T 1MDecision T 1P -1, and T 1MConversely by τ r, τ sOr τ MDecision.Common τ rAnd τ mReduce (1/ τ with the temperature rising rWith 1/ τ mIncrease with the temperature rising), however Mn 2+The τ of compound sBut may increase or reduce.
Because find H 8The T of proton 1P -1Figure to temperature is shown as the decline then of rising earlier, and T 1MShow positive frequency dependence, so can measure temperature to Mn (HCO 3) 2-ATP compound and MnCl 2The influence (data not shown) in the electron spin relaxation time of-ATP compound.The order of magnitude of finding the electron spin relaxation time is 1.5 * 10 -9Second.If τ cLess than τ s, quick exchange does not then take place, and can not be by τ sDecision T 1MTable 9 has been listed the T by each proton 1PThe correlation time that frequency dependence and electron spin relaxation time calculate relatively.Because T 1P -1Be frequency τ cFunction, so can calculate q and r by equation 1 and equation 3.Someone thinks, understands the molecular dynamics of these ATP compounds in used temperature range, and when temperature is tending towards 35 ℃, Mn (HCO 3) 2Manganese in the-ATP compound arrives the C of next-door neighbour ATP 8The position of carbon.This situation is confirmed by the interatomic disance that uses the Solomon-Bloemgergen Equation for Calculating.Because there is the C that is obtained down in bicarbonate radical 8The proton behavioral data is different from the data of chlorion under existing, so the existence that also shows bicarbonate radical plays an important role to the structure of ATP compound.This is for pT 2p -1Especially correct.Under the situation that bicarbonate radical exists, C 8-H interatomic disance is also very near.T 2MAspect the dipolar interaction that value is transmitted at the scalar interaction that transmits by chemical bond with by the space significant contribution is arranged all.T 1MHas only the dipole contribution.When between very little or observed nucleon of hyperfine constant A and the paramagnetic particle during no chemical bond, T 1MAnd T 2MAlmost equal.For C 8Proton, Mn (HCO 3) 2The T of-ATP compound 2P -1/ T 1P -1Value is significantly less than MnCl 2The T of-ATP compound 2P -1/ T 1P -1Value.Someone proposes this is Mn (HCO 3) 2The Mn of-ATP compound 2+The tight result who acts on of π-track with the adenine ring.
By the data of interatomic disance as can be known, under all probe temperatures, show Mn 2+And HCO 3 -Existence to Mn 2+Near C 8Proton plays a significant role.
Table 9. temperature is to Mn (HCO 3) 2-ATP compound and MnCl 2The influence of dipole correlation time of-ATP compound.
Table 10. temperature is to Mn (HCO 3) 2-ATP compound and MnCl 2The Mn of-ATP compound 2+The influence of the interatomic disance of-proton.
Figure A200680054592D00841
Mn (HCO 3) 2The structure of-ATP compound is different from the structure of MgATP compound basically; Particularly metallic ion coordination is to C 8On the carbon.This structure is used for using the Accelrys software kit to carry out designing program based on the rational drug of part.
[Mn 2+], [Mg 2+] and [HCO 3 -] to the C of ATP 8The influence of deuterium generation and ATP hydrolysis
At pH 6.3, the ATP hydrolysis rate depends on Mn 2+Concentration and existence.Na only 2ATP, NaHCO 3And Na 2ATP, NaHCO 3, MgCl 2And Na 2ATP all shows identical hydrolysis rate.Containing MgCl 2And NaHCO 3Solution in, magnesium carbonate precipitation causes experiment to close on error in data takes place when finishing.Add Mn 2+Demonstrate and to significantly improve hydrolysis rate.Contain 2 molecule NaHCO 3With 2 molecule MnCl 2The hydrolysis rate of solution be higher than and contain 1 molecule NaHCO 3With 2 molecule MnCl 2The hydrolysis rate of solution, so NaHCO 3Concentration also demonstrates and plays a role.
Under pH6.3 and 7.3 and 37 ℃, in 336 hours, measure NaHCO 3, MnCl 2, MgCl 2And D 2O is to the influence of ATP hydrolysis rate.Hydrolysis rate is calculated by the regretional analysis of data (table 11 and table 12).In 336 hours, data are by 5 data points of each concentration, each data point are established 4 samples and calculated.With D 2O is that solvent reacts K and U under each pH.
Increasing exponentially of imidazole concentration significantly improves hydrolysis rate effectively.When pH 6.3, Mn 2+Concentration is bigger than the influence when the pH 7.3 to the influence of hydrolysis rate difference.Because by OH -The hydrolysis that causes, when pH 7.3, the hydrolysis rate when not having metallic ion is higher than the hydrolysis rate when pH 6.3, yet when higher pH, HCO 3 -And Mn 2+Influence all reduce to some extent.When having 3 manganese ions, compare reaction rate, then Mn 2+Influence clearly.There is Mg in pH 7.3 times 2+The time hydrolysis rate raising do not have HCO 6.3 times greater than pH 3 -The time hydrolysis rate raising.HCO 3 -Existence also improved pH and had Mg 7.3 times 2+The time hydrolysis rate.Because MgHCO 3There is Mg in precipitation 6.3 times so can not determine pH 2+The time HCO 3 -Influence to hydrolysis rate.D 2The existence of O has improved the hydrolysis rate under the pH 6.3.
NaHCO 3Increasing exponentially of concentration improved at each equal Mn 2+Deuterium under the concentration is for speed.Under the low-carbonate concentration, the ATP hydrolysis rate obviously depends on Mn 2+Concentration.Therefore, deuterium is for depending on carbonate concentration.At high NaHCO 3Under the concentration, the ATP hydrolysis rate shows and does not rely on Mn 2+Concentration; Yet at Mn 2+With the concentration ratio of ATP less than 3:1, i.e. 2Mn 2+: ATP and 1Mn 2+: during ATP, hydrolysis rate and deuterium then are non-linear for speed.Mn 2+Obviously has influence with the concentration ratio of ATP.
Table 11.NaHCO 3, MnCl 2And MgCl 2Concentration and D 2O is to the influence of ATP hydrolysis rate.Analyze A-L and comprise 10mM imidazoles and 1mM ATP, comprise 20mM imidazoles and 1mM ATP and analyze M-U.Under pH 6.3 and 37 ℃, analyze.Reaction K and U comprise the D as solvent 2O.The reaction carried out 336 hours and these data by 5 data point calculation.In each data point, each sample carries out 4 analyses.Owing to there is Mg 2+And carbonate, so reaction J precipitates.Used NaHCO 3, MnCl 2Or MgCl 2Concentration is as follows.
[NaHCO 3] [MnCl 2] [MgCl 2] ADP speed average SD RMS mM mM mM (μ M.hr -1)
A 0 0 0.561 13.58 0.974B 3 0 0.545 12.03 0.966C 3 1 0.449 3.78 0.988D 1 2 0.923 5.01 0.997E 2 2 0.990 3.27 0.994
F 3 2 0.951 4.58 0.998G 0 2 0.881 6.52 0.991
H 3 3 2.161 20.93 0.959I 0 2 0.399 3.28 0.975J 3 2 -0.019 61.85 0.027
K 3 2 1.125 3.78 1.000L 0 2 1.112 2.86 1.000 [NaHCO 3] [MnCl 2] deuterium is for speed RMS ADP speed RMS mM mM (μ M.hr -1) (μM.hr -1)M 0 0 1.452 0.876 1.071 0.796N 12 0 1.353 0.979 0.830 0.826O 6 1 1.670 0.906 1.486 0.837P 6 2 1.586 0.437 1.233 0.973Q 6 3 1.760 0.955 1.424 0.991R 12 1 1.889 0.957 1.320 0.929S 12 2 1.727 0.966 1.333 0.991T 12 3 2.001 0.970 1.497 0.968U 12 4 2.587 0.766 1.666 0.981
Table 12.NaHCO 3, MnCl 2, MgCl 2And D 2O is to the influence of ATP hydrolysis rate.Analysis comprises 10mM imidazoles and 1mM ATP, and analyzes under pH 7.3 and 37 ℃.Reaction K and L comprise the D as solvent 2O.The reaction carried out 336 hours and these data by 5 data point calculation.In each data point, each sample carries out 4 analyses.Owing to there is Mg 2+And carbonate, so reaction J precipitates.
[NaHCO 3] [MnCl 2] [MgCl 2] speed average SD RMS mM mM mM (μ M.hr -1)
A 0 0 0.661 9.64 0.991B 3 0 0.550 11.77 0.971C 3 1 0.609 2.06 0.943D 1 2 0.955 5.33 0.999E 2 2 0.961 5.30 1.000
F 3 2 0.968 4.66 0.999G 0 2 0.962 1.30 1.000
H 3 3 1.307 7.92 0.999I 0 2 0.499 11.02 0.952J 3 2 0.595 4.38 0.913
K 3 2 1.014 1.46 1.000L 0 2 1.100 8.56 1.000
Obtain the GS enzyme for Mn 2+And HCO 3 -The data of demand (vide infra) are measured [Mn under higher concentration 2+] and [HCO 3 -] to ATP hydrolysis rate and ATP at C 8The influence in-H deuterium generation.In stoichiometry is 6HCO 3 -: 3Mn 2+: during 1ATP, the hydrolysis rate ratio is higher when not having metallic ion; Yet find that hydrolysis and deuterium generation is not linear all.In stoichiometry is 12HCO 3 -: 3Mn 2+: during 1ATP, find that hydrolysis rate and deuterium are linearity for speed.In stoichiometry is 12HCO 3 -: 1Mn 2+: during 1ATP, find that once more data are not linear.Shown in GS enzyme data, the someone thinks to demonstrate and has formed stabilized complex Mn 2+3 (HCO 3 -) 12-ATP.In adenylylation GS zymetology, also find this situation (vide infra).There is not HCO 3 -Or Mn 2+The time, deuterium is for speed and hydrolysis rate and only have HCO 3 -Shi Xiangtong.Pass through H 2O or by relating to the CO of coordination 2Another mechanism the hydrolysis of ATP can take place.That can expect is the CO of coordination 2At N 7Form the carboxylation intermediate, this carboxylation intermediate attack γ-phosphoric acid then, thus its hydrolysis is formed phosphine formic acid.
Compare 12HCO 3 -And 3Mn 2+With 6HCO 3 -And 3Mn 2+Under deuterium for speed explanation deuterium for speed at 12HCO 3 -Higher when existing.Hydrolysis rate and deuterium are all unequal under two kinds of conditions for speed, and the deuterium under two kinds of conditions generation is all fast than hydrolysis.Mn 2+And HCO 3 -In deuterium generation and hydrolysis, all play a role, yet the rate dependent that obtains to equate is in obtaining hydrolysis and deuterium for the Mn that all needs 2+And HCO 3The accurate stereochemistry of-coordination.Someone proposes, and the main side chain that exists by glutamine synthelase helps forming Mn 2+And HCO 3 -The accurate stereochemistry of coordination.
PH is to NH in the imidazole buffer 4HCO 3The influence of disassociation
Measure pH to containing 1mM NH 4HCO 310mM imidazoles HCl and the influence of the ratio conductivity of 10mM imidazoles HCl.Be used to calculate NH than conductivity difference between two data sets 4HCO 3In the contribution of the molar conductivity of different pH levels disassociation, and measure the HCO of imidazoles to causing by pH to conductivity 3 -To CO 2The influence that transforms.Then with its with compare by any the theoretical molar conductivity that calculates in the following formula:
Λ＝α(λ j[NH 4 +]+λ j[HCO 3 -]+λ j[Imi +])
Perhaps
Λ＝α(λ j[NH 4+]+λ j[HCO 3 -]
Based on NH 4HCO 3Dissociate into NH 4 +And HCO 3 -Or NH 4 +, HCO 3 -And imidazol ion, and the imidazol ion that should dissociate and exist can be to NH 4CO 3Dissociate into HCO 3 -And CO 2Have a negative impact.This calculates the HCO that obtains based on by the Henderson-Hasselbach equation 3 -Concentration, described equation are used to obtain pH to [HCO 3 -] and [CO 2] the theory influence of relative scale.Also need to consider NH 4 +And HCO 3 -Molar conductivity and by NH 4 +Form imidazol ion.
Figure A200680054592D00871
Λ pH = ( λ NH 4 + [ HCO 3 - ] N H 4 + + λ HCO 3 - [ HCO 3 - ] NH 4 + + λ HCO 3 - [ HCO 3 - ] Im i + + λ Im i + j [ HCO 3 - ] Im i + )
Wherein, Λ PHMolar conductivity under=each specific pH
The ratio conductivity of each ion of λ j=
Figure A200680054592D00873
Figure A200680054592D00874
When only supposing with NH 4 +As HCO 3 -Counter ion counterionsl gegenions when calculating, gross data and experimental data are unequal.As hypothesis HCO 3 -Counter ion counterionsl gegenions be NH 4 +During with two kinds of imidazol ions, experimental data equates with gross data.By these data, pass through NH 4 +The pK of concentration and imidazoles HCl aCan indicate the disassociation of imidazol ion, the most a high proportion of imidazoles counter ion counterionsl gegenions occur when hanging down the pH value.By the NH in the solution 4HCO 3Concentration rather than imidazoles HCl concentration are indicated the ratio of the imidazol ion of formation.Therefore, in the presence of imidazoles, NH 4HCO 3Disassociation defer to theoretical disassociation really, and when pH is reduced to pH 6.3 by pH 7.2, the CO in the solution 2Concentration increases.This to the glutamine synthelase reaction mechanism that proposed highly significant because when pH6.3, under the enzyme analysis condition, HCO can occur 3 -And CO 2Volumetric molar concentration equate.In assumed response mechanism, there is the people to propose, when glutamine synthelase exists with complete adenylylation form, CO 2In the phosphoryl transfer process, play indispensable effect.
Mn 2+Or Mg 2+Concentration and ATP concentration are to adenylylation and the influence of going adenylylation GS activity
In the ATP concentration range that is detected, [Mn 2+] influence that forms the adenylylation GS specific activity of measuring by ADP is increased to optimum 3Mn 2+/ ATP.The NaHCO that exists 3Concentration is carried out all analyses during for 12mmole.When measuring the specific activity of adenylylation GS, demonstrate with [Mg by the formation of ADP 2+] the active not increase of increase.When comparing, can see similar trend with the adenylylation GS specific activity of measuring by the formation of glutamine.At [Mn 2+] exist down, the activity of adenylylation GS is increased to optimal value 3Mn in the ATP concentration range that is detected 2+/ ATP, and at [Mg 2+] exist down, the GS activity does not increase in the ATP concentration range that is detected.Use the specific activity data of adenylylation GS, and hypothesis forms stable ATP and Mn at each ATP concentration place that analyzes based on ADP 2+Compound, in this compound, to corresponding 3 manganese ions of each ATP.For example, when 200 μ M ATP are added 200 μ M MnCl 2Form " stable " Mn 3-ATP compound is 66.7 μ M Mn 3During-ATP.[Mn 2+ 3-ATP] influence that forms the adenylylation GS specific activity of measuring by ADP is illustrated that the RMS deviation that in fact forms " stable " compound and data is about 0.96.Can find out that also the gained curve is S-shaped, demonstrate and have concertedness (data not shown).
Use each [Mn 2+] [ATP] data Eadie-Hofstee figure of calculating v/s show that data are non-linear.Use [Mn 3-ATP] the compound Eadie-Hofstee figure that calculates v/s shows that also data are non-linear, yet when and use [Mn 2+] calculating the figure of v/s, this video data is concentrated together.Data do not have linearity also to illustrate and have positive cooperativity.As the Mn that adds 1 equivalent and 2 equivalents to each ATP 2+The time, Mn 2+Other rate constant that-ATP compound forms makes and is used for calculating [Mn 2+-ATP] data become complicated.As the Mn that adds 3 equivalents to each ATP 2+The time, the differential of the Eadie-Hofstee curve that the activity data that use obtains is determined calculates the K in the v/s value scope m(table 13).Can find out by these data, along with Mn in the solution 3The raising of-ATP concentration, enzyme is to Mn 3The affinity of-ATP increases, thereby also demonstrates positive cooperativity.
The K of the adenylylation glutamine synthelase that table 13. is calculated by the rate of curve of the active figure of Eadie-Hofstee mThe Mn that adds 3 equivalents to each ATP 2+
[Mn 3-ATP]μM K m(μM)
200 942
400 702
600 479
800 261
1000 126
Because data presentation the Mn of enzyme 3Positive homotroplc cooperativlty between the-ATP-binding site is so use Hill equation drawing data.
log ( v o v max - v o ) = n log [ s ] + log K b
Discovery is 2.0 by the Hill coefficient (h) of the slope acquisition of Hill figure, shows that 2 enzyme subunits interact.Use Mg 2+As the counter ion counterionsl gegenions of ATP, not from the correlativity of the activity influence of Hill graph discovery adenylylation GS.Use Molecular Simulation Technique,, show that in fact the result who carries out the enzyme adenylylation by two subunits positive coorperativity may take place by the manual adenylylation of T397 residue is estimated the GS crystal structure.Positive coorperativity can be by being the diagonal angle between two subunits (for example subunit A and H) two AMP residues of adenylylation glutamine synthelase take place.Positive coorperativity also can only take place between 2 avtive spot subunits, and this can be confirmed by the data of Hill figure.
Do not find [Mg 2+] be increased to 4 pairs of adenylylation GS specific activities of measuring by forming of ADP that go with the ratio of [ATP] by 1 and produce material impact.The specific activity of measuring by the formation of ADP that removes adenylylation GS shows that also activity is not with [Mn 2+] increase and increase, yet, [Mn 2+] the specific activity that removes adenylylation GS under existing is but significantly less than Mg 2+There is measured activity down.When with the formation by glutamine measure go adenylylation GS specific activity to compare the time, can see similar trend.When drawing [Mg 2+] with [ATP] variable concentrations than under [ATP] to the figure of the influence of going adenylylation GS activity represented based on the specific activity of ADP and based on the specific activity of glutamine the time, also find similar trend.At 1[Mg 2+] with the concentration ratio of [ATP] under, find departs from linear, this may be because 1[Mg 2+] equilibrium constant out of true that forms with [ATP] is 1.
[ATP] is at [Mn 2+] with [ATP] variable concentrations than down to going showing to increase based on the active of ADP with based on the influence of the specific activity of the activity of glutamine and actively significantly not increasing of adenylylation GS with [ATP]; Yet, under these two kinds of situations, with [Mn 2+] increase with [ATP] concentration ratio, activity all reduces.The adenylylation degree of used enzyme is about 8%.Under this adenylylation degree, the allosteric interaction of subunit may be not obvious.
Bicarbonate concentration is to the influence of adenylylation GS specific activity
Because the activity of GS adenylylation and degree and intracellular metabolic flux are related, therefore measure [NaHCO 3] to the influence of adenylylation GS specific activity.In ATP concentration is 600 μ M and Mn 2+Concentration is that the condition of 1800 μ M gets off to measure specific activity.At 12NaHCO 3Under the concentration ratio of 1ATP, active obvious optimal value has appearred.Therefore, the Mn-ATP compound optimal value of adenylylation GS activity demonstrates and comprises 12HCO 3 -: 3Mn 2+: 1ATP.At ratio is 8NaHCO 3/ ATP reaches less than under this ratio, by ATP the active reaction efficiency of the conversion of glutamine activity recently being measured with glutamic acid of ADP is shown that it is subjected to appreciable impact, because reaction efficiency drops to 65% by 100%.At ratio is 8NaHCO 3During/ATP, bicarbonate radical also makes specific activity increase by five times.
At C 8The ATP in position deuterium generation and 13C HCO 3 -Influence to adenylylation GS specific activity
Be determined at C 8The ATP concentration in position deuterium generation is to the influence of adenylylation GS activity, and with the ATP that uses deuterium not generation under the same conditions the influence of adenylylation GS activity compared.ATP is at C 8The result in position deuterium generation finds significant catalysis isotope effect.Find out obviously that by these data ATP is at C 8The deuterium of position is for the catalytic activity of appreciable impact adenylylation glutamine synthelase.ATP is at C 8The deuterium of position is for the increase that has caused forming by ADP the enzymatic activity of speed measurement.Yet, by transformation efficiency show glutamine to form efficient lower.There is hypothesis to think, at (Mn 2+) 3(HCO 3 -) 12-ATP exists the phosphoryl transfer reaction of the adenylylation glutamine synthelase that carries out down to regulate by metal carbene.Referring to Fig. 1.The first step of reaction is with N 7Thereby protonated or carboxylation is at N 7Form cationic imide.Also may be by being coordinated in a Mn 2+CO on the ion 2Make and N 7Carboxylation.In case close the cationic imide that forms ATP by the carbon dioxide huge legendary turtle, then the carboxylate of coordination will be assisted C 8Deprotonation, generation can be in conjunction with Mn 2+The carbenoid material.Mn 2+Combination depend on life-span (Fig. 1) of carbenoid material IV.The life-span of this material may depend on the dissociation rate of the carbamyl-hydrogen bond that forms in molecule inner proton transfer step.Therefore, it is stable with the deuterium instead of hydrogen material IV to be obtained, and has promoted the formation of material V, allows phosphoryl to transfer on the contiguous carbamyl residue.The fact is that the adenylylation enzyme is best in pH 6.3 performance functions, at this pH, NaHCO 3The HCO of volumetric molar concentration such as dissociate into 3 -And CO 2, this may be at the HCO that promotes by existing 3 -And CO 2Play an important role in the catalytic step of regulating.At this moment, but γ-phosphoric acid of carboxyl attack ATP forms phosphine formic acid, thereby makes the δ-carboxyl phosphorylated of glutamic acid form gamma-glutamyl phosphoric acid then.
In order to prove HCO 3 -Effect in the reaction of being regulated is measured 13C HCO 3 -Influence; Referring to table 14.In each data point, clearly the glutamine synthelase of two kinds of heavy isotope forms is all influential to the specific activity of adenylylation glutamine synthelase.Because it is right 13C HCO 3 -The catalysis isotope effect fairly obvious, so increase when very remarkable when active, carbonate must be brought into play the effect that exceeds the solute effect in reaction.
Table 14. 13C HCO 3With 12C HCO 3And deuterium generation and not the ATP in deuterium generation to the influence of the specific activity of adenylylation glutamine synthelase
Figure A200680054592D00911
Someone thinks CO 2(or its hydrated form) participated in the phosphoryl transfer process from mechanism, therefore can reasonably propose, and the phosphoryl transfer rate should be limited by the kinetic isotope effect that is produced by used form of carbon dioxide.Possible situation is that the speed that the carbon dioxide huge legendary turtle that causes material II to generate is closed is very slow, and balance may be to moving to left-the dissociation rate height.Therefore, the gathering of compound is determined by speed.So, use heavy carbon dioxide isotope will cause positive reaction and reversed reaction all to lag behind.If subsequently pass through carbamyl at C 8Deprotonation (and subsequent reaction) very fast, then heavy isotope will prolong this material life-span-increase the possibility of the further reaction in the cascade that can carry out.In fact, when using carbon dioxide source 13When C variant check glutamine formation and ATP consume, can be observed this situation.
Rate effect is the performance that from mechanism participate in of carbon dioxide (or equivalent of hydration) in the phosphoryl transfer mechanism.
HCO 3 -There is the proof of the carbamate intermediate of ATP down
Be dissolved in D 2The concentration of O is the KH of 20mM 2HPO 4/ K 2HPO 4Experimentize used ATP, NaHCO in the damping fluid 3, MnCl 2And MgCl 2Concentration change, ATP is at 0-1mM, NaHCO 3At 0-12mM, MnCl 2And MgCl 2In the 0-3mM scope, change.In order to prove the N of adenine ring 7Effect, also substitute ATP and carry out response analysis by adding adenosine or tubercidin.In tubercidin, substitute N with carbon atom 7With sodium dithionate (Na 2S 2O 2) to add sample to concentration be 4mM and pD is adjusted to pD 6.3.After 48 hours, adding EDTA is 4.0mM to its concentration, and centrifugal 10min removes bivalent metal ion with sample.Obtain then 1H NMR spectrogram, because if form the carbamate intermediate, then this intermediate will be reduced to the formic acid that can identify by NMR.
HCO 3 -There is the proof of the carbamate intermediate of ATP down
There is HCO in the assumed response potpourri 3 -, ATP is at Na 2S 2O 2Exist and generate formic acid down.Someone proposes this formic acid is by at position N 7The carbamate intermediate that forms generates.By Powers and Meister (1976) Proc.Natl.Acad.Sci.73:(9), 3020-3024) this reduction can take place in described similar mechanism, yet, be to pass through Na in this case 2S 2O 2Rather than KBH 4Reduce.
ATP is in conjunction with CO 2The evaluation of ATP-carbamate intermediate
Prove by the following the ATP cationic imide is induced at N 7Carbamate, promptly by at Na 2S 2O 2Make ATP, HCO under existing 3 -, Mn 2+Or Mg 2+Reaction, thus show that the carbamate intermediate is reduced formation formic acid.All analyses all are being dissolved in D 2Carry out in the 20mM phosphate buffer of the pD 6.3 of O, and all reagent all are dissolved in D 2Prepare among the O.Table 15 has been listed the analysis of being carried out.Reaction mixture comprises that concentration is the Na of 4mM 2S 2O 4Adding methyl alcohol is that 2mM is as internal standard compound until its concentration.Add Na 2S 2O 4The time, with 1M DCl pD is adjusted to pD 6.3.Reaction was carried out 48 hours, and at this moment adding USB EDTA, to make the ultimate density of EDTA be 4mM.Thereby with sample centrifugal Mn that removes under 10000 * g 2+, obtain NMR 1The H spectrogram.Measure the formic acid concn of generation with respect to the relative displacement intensity of methyl alcohol internal standard compound by formic acid.Analyze: (1) 1mM ATP, 3mM MnCl 2, 12mM NaHCO 3With 4mM Na 2S 2O 4(2) 0mM ATP, 0mM MnCl 2, 12mM NaHCO 3With 4mM Na 2S 2O 4(3) 1mMATP, 0mM MnCl 2, 12mM NaHCO 3With 4mM Na 2S 2O 4(4) 0mM ATP, 3mMMnCl 2, 12mM NaHCO 3With 4mM Na 2S 2O 4(5) 1mM ATP, 3mM MnCl 2, 12mM NaCl and 4mM Na 2S 2O 4(6) 1mM ATP, 3mM MgCl 2, 12mM NaHCO 3With 4mM Na 2S 2O 4(7) 1mM adenosine, 3mM MnCl 2, 12mM NaHCO 3And 4mMNa 2S 2O 4(8) 1mM tubercidin, 3mM MnCl 2, 12mM NaHCO 3And 4mMNa 2S 2O 4(9) contain the reactant 1 of the 2mM formic acid of adding; And (10) 2mM formic acid standard items.
Table 15. analysis condition.Analyzing in 5, using 12mM NaCl to substitute NaHCO 3Analyzing in 6, use 3mM MgCl 2Substitute MnCl 2Analyzing in 7, using the 1mM adenosine to substitute ATP; And analyzing in 8, use the 1mM tubercidin to substitute ATP.
[ATP]mM [MnCl 2]mM [NaHCO 3] [formic acid] mM
1 1 3 12 0.15
2 0 0 12 0
3 1 0 12 0
4 0 3 12 0
5 1 3 0 0
6 1 0 12 0
7 0 3 12 0
8 0 3 12 0
By being reduced to the provable certain carbamate intermediate that formed of formic acid (the NMR displacement is arranged when the 8.55ppm).The formation of formic acid depends on bicarbonate radical, ATP and Mn 2+Existence.Analysis 7 and analysis 8 comprise adenosine and tubercidin respectively.Set up these two analyses with 7 pairs of proof nitrogen positions form carbamate with and be reduced to the necessity of formic acid.Can find out Mn by these data 2+Be coordinated on the polyphosphoric acid of ATP be react necessary.When using Mg 2+Substitute Mn 2+The time, can find out to have formed a spot of formic acid.
Embodiment 2 molecular simulations and rational drug design
The analysis of GS crystal structure
By Brookhaven albumen database (accession number 1f52.pdb) obtain from the colibacillary crystal structure that removes adenylylation GS (Gill, H.S. and D.Eisenburg (1994) Biochemistry, 40:1903-1912).Because described albumen is all had the ADP residue in crystallization and each avtive spot, so use this structure.Thereby produce the GS polypeptide tetramer by this structure and form single avtive spot.(the Mn of " sealing " form that proposes in the ATP structure analysis 2+) 3(HCO 3 -) 12-ATP compound is based on 1The Mn of H NMR data 2+Vicinity and Mn 2+Coordinative Chemistry require with at C 8Deuterium played a role in generation.Use InsightII (Accelrys) software building (Mn 2+) 3(HCO 3 -) 12-ATP structure is also carried out energy minimization.The model configuration that produces have be positioned on the phosphoric acid afterbody and under 2 Mn 2+And the 3rd Mn of contiguous adenine ring coordination 2+Use Accelrys software with (Mn then 2+) 3(HCO 3 -) 12The adenine ring of-ATP compound is added on the adenine ring of ADP in the avtive spot, thereby this structure is inserted avtive spot.Assembly is carried out energy minimization, thereby and the evaluation amino acid side chain relevant with ATP can carry out rite-directed mutagenesis to these residues so that can illustrate their effects in the catalysis that glutamine synthelase is regulated.Amino acid residue through identifying is from Glu129, Glu207, His269, His271, Arg224 and Arg355 and the Lys47 ' of adjacent subunit.
The rite-directed mutagenesis of Escherichia coli GS and enzyme analysis
To on Glu207, His269, His271 and Arg355, carrying out rite-directed mutagenesis from colibacillary GS.When separating from each clone body and purifying GS and when using the specific activity of gamma glutamyltransferase assay enzyme of GS, find that these sudden changes have destroyed the functional of enzyme.These amino acid residues show in the GS avtive spot and play a significant role, and have hypothesis to think that His269 and His271 residue play a role in phosphoryl shifts.When based on ADP and glutamine formation use HPLC measurement enzymatic activity, found similar results.
Use two kinds of enzyme assays to estimate activity of glutamine synthetase.First kind of used analytic approach gamma glutamyltransferase analytic approach measured " contrary " reaction as the glutamyl transferase activity.In this reversed reaction, in the presence of ADP, arsenate and manganese or magnesium, azanol and glutamine reaction generate gamma-glutamyl hydroximic acid and free ammonia (Shapiro and Stadtman (1970) Methods inEnzymol.17A:910-922).This has formed the basis of activity of glutamine synthetase analytic approach.Under the correct pH that measures enzyme isoelectric point gained, the adenylylation of glutamine synthelase is identical with the transferase active that goes two kinds of forms of adenylylation.Yet the enzyme of these two kinds of forms can obtain distinguishing, because when isoelectric point, the glutamine synthelase of adenylylation is by 60mM Mg fully 2+Suppress fully, go the enzyme of adenylylation then unaffected (Bender R A, K A Janssen, A D Resnick, M Blumberg, F Foor and B Magasanik, (1977) Journal of Bacteriology 129:1001-1009).Therefore, can based on they under pH7.15, Mn 2+Or Mg 2+Its active difference when existing makes a distinction the activity of two kinds of form enzymes.Measure activity of glutamine synthetase in two kinds of different analysis of mixtures: a kind of contains Mn, and second kind contains Mn and Mg.All reagent prepare in imidazole buffer (pH 7.0).In cumulative volume 600 μ l, implement two kinds of analytic approachs.As shown in table 16 below, set up the Mn analytic approach, and shown in table 17, set up combined analytical method.
Table 16. is based on Mn 2+The analysis of mixtures of glutamyl transferase analytic approach
Component Ultimate density in analytic approach
L-glutaminate 15mM
NaADP 0.4mM
Natrium arsenicum 30mM
MnCl 2·4H 2O 0.3mM
Azanol 60mM
Table 17. is based on Mn 2+And Mg 2+The analysis of mixtures of glutamyl transferase analytic approach
Component Ultimate density in analytic approach
L-glutaminate 15mM
NaADP 0.4mM
Natrium arsenicum 30mM
MnCl 2·4H 2O 0.3mM
MgCl 2·7H 2O 60mM
Azanol 60mM
With with Mn 2+Reacting phase mode together prepares blank reactant, but substitutes ADP and arsenate solution with isopyknic water.Under 37 ℃, make analysis of mixtures balance 5min, add 50 μ l enzyme preparations then and begin reaction.Make this reaction carry out 30min, add 900 μ l Stop Mix (1MFeCl then 36H 2O, 0.2M trichloroacetic acid and 7.1%v/v HCl) stop this reaction.Next, with sample in the Eppendorf microcentrifuge under 13000rpm centrifugal 2min and under 540nm, measure absorbance removing any precipitation that may form.All results are expressed as with the μ mole glutamy hydroxamic acid specific activity that compound/min/mg albumen is represented.Consider the number of subunit, by removing the active and total gamma glutamyltransferase activity (Mn of adenylylation gamma glutamyltransferase 2+Reaction) the adenylylation degree of recently calculating.
In addition, use HPLC to estimate the conversion ratio that is converted into glutamine and ADP by ATP, glutamic acid and ammonia.This is called " just " reaction or " biosynthesizing " reaction, and use two kinds of different analytic approachs to analyze: a kind of is to measure in the presence of ATP, glutamine synthelase turns to the ability of glutamine with glutamic acid rotating, and second kind is determined in the identical analysis of mixtures ATP to the conversion of ADP and AMP.Set up this analysis to measure the positive reaction of glutamine synthelase.Analytic approach is measured at MnHCO 3The amount of the glutamine that forms by L-glutamic acid under-ATP (basis of first kind of reaction) and MgATP (basis of second kind of reaction) exist, and measure ATP, the ADP of formation and the amount of AMP.Same analysis mixed solution carries out with 2 kinds of HPLC methods, a kind ofly is used for glutamic acid/glutamine analysis, and another kind is used for ATP/ADP/AMP and analyzes.Shown in table 18, set up this analytic approach.
Table 18. is used for the HPLC analytic approach to measure the analysis component that glutamic acid and ATP are converted into the conversion ratio of glutamine and ADP.
Figure A200680054592D00951
Carry out Mn 6.3 times at pH 2+Analytic approach is carried out Mg 7.3 times at pH 2+Analytic approach.The volume of all enzyme preparations with 50 μ l added in the analysis of mixtures.The interpolation of enzyme has started reaction, makes its reaction 1 hour then.Add 6 μ l, 50% trichloroacetic acid solution and stop this reaction.Then each analyte is all assigned to 4 HPLC bottles (150 μ l/ bottle) and analyzed, on Agilent 100HPLC equipment, use Phenomenex Luna 5 μ C18 posts that wherein two are carried out glutamic acid and glutamine analysis and ATP/ADP and analyze.All analyze parallel carrying out three times.
Can be found out by these data: H271 is connected on the enzyme of adenylylation form, because when comprising two sudden change Y397V, generate when removing the enzyme of adenylylation form fully, all activity will be lost.Therefore there is the people to propose His271 and in the phosphoryl transfer reaction of the supposition of adenylylation form enzyme, brings into play key effect.Histidine 269 demonstrations play a significant role, but its influence elaboration is clear and definite inadequately.
The glutamyl transferase analysis result of the mutant of table 19. Escherichia coli WT and structure.The WT enzyme is meant the bacterial strain of growing in the M9 nutrient culture media of modifying, WT (AD) and WT (DD) are meant the adenylylation enzyme that Continuous Cultivation produces respectively and remove the adenylylation enzyme.
Enzyme Total enzyme activity:4.5mM MnCl 2(μ mole/min/mg albumen) Go the activity of adenylylation enzyme:4.5mM MnCl 2+60mM MgCl 2(μ mole/min/mg albumen) The percentage of adenylylation
WT 91.5 20.2 78
WT(AD) 84.9 21.1 75
WT(DD) 55.8 56.1 0
Y397V 64.5 5.1 92
E207T 24.1 10.6 56
E207T Y397V 45.1 10.4 77
H269N 0.2 0.0 Na
H269N Y397V 2.5 0.1 96
H271N 39.3 10.2 74
H271N Y397V 0.0 0.0 -
R355Q 30.3 6.1 80
R355Q Y397V 34.8 12.3 65
Table 20. shows the glutamic acid of use HPLC mensuration and the analysis result that ATP is converted into the conversion ratio of glutamine and ADP.The WT enzyme is meant the bacterial strain of growing in the M9 nutrient culture media of modifying, and WT (AD) and WT (DD) are meant the adenylylation that produces and remove the adenylylation enzyme in Continuous Cultivation.Institute's indicating value is represented the different mean value of analyzing three times, and wherein the deviation that should be worth is less than 5%.
Enzyme Specific activity based on glutamine: Mn 2+Analytic approach (μ mole/min/mg albumen) Specific activity based on ADP: Mn 2+Analytic approach (μ mole/min/mg albumen) Percentage conversion Mn 2+Analytic approach Specific activity based on glutamine: Mg 2+Analytic approach (μ mole/min/mg albumen) Specific activity based on ADP: Mg 2+Analytic approach (μ mole/min/mg albumen) Percentage conversion Mg 2+Analytic approach
WT 4.31 4.37 98 4.26 4.21 100
WT(AD) 3.76 3.65 100 3.02 3.12 97
WT(DD) 4.98 4.62 100 14.54 13.92 100
Y397V 2.81 4.95 57 0.12 0.23 50
MnHCO 3 The ATP combination
E207T 1.72 2.34 73 0.15 0.26 60
E207TY397V 2.28 3.69 62 0.26 0.33 80
H269N 0.45 0.47 94 0.10 0.06 100
H269NY397V 0.26 0.33 79 0.18 0.21 87
H271N 3.05 4.28 71 4.36 6.22 70
H271NY397V 0.00 0.09 0 0.18 0.81 22
R355Q 5.83 6.53 89 4.26 4.88 87
R355QY397V 4.01 4.91 82 2.88 4.07 71
The data that participate in from mechanism
The ratio and the concentration of ATP and enzyme during given pH 6.3 are defined as major parameter with the manganese content and carbon dioxide (or its hydrated form) content of system.Investigated and had enzyme and do not having the influence of these parameters under the condition of enzyme.Document is verified under the condition that does not have enzyme, aspect ATP generation ADP, and the quantitative effect relevant with the change of important parameter, promptly major parameter is to C 8Deuterium is combined with influence (wherein, at the D as the body solvent substrate 2Checked the influence of this parameter among the O).Under the condition that enzyme exists, can be with the check of phosphoryl transfer efficiency (can regard that the generation by ADP forms the possibility of glutamine as) probe as mechanism.
Under the condition that does not have enzyme, the speed that ATP is hydrolyzed to ADP depends on the ratio of manganese ion and ATP to a great extent.On the contrary, ADP generating rate (under stable manganese level) is irrelevant with the stoichiometry of carbonate basically.The conclusion that this observation produces is that carbonate (or carbon dioxide) is very little to the effect of ATP hydrolysis.Because pH is a constant 6.3, the absolute concentration of hydroxide ion is a constant, therefore the unique determinative of ATP hydrolysis is a concentration of metal ions, and this concentration of metal ions determines the level near the metal the phosphoric acid skeleton-oxygen compound (may be that metal is incorporated into imidazoles) conversely in this example.When using D 2When O is hydrolyzed research as solvent substrate, observes deuterium and be attached to C 8Other result.Than the data relevant, observedly increase (O in the table 11 and R, P and S, Q and T item) for speed with carbonate concentration, and do not follow the raising of hydrolysis rate to the deuterium under stable manganese ion concentration with hydrolysis.This observation explanation deuterium is in conjunction with depending on carbonate.
When the glutamic acid of checking enzymatic and ATP to regulate in a similar manner when glutamine transforms, the participation on mechanism of these observationss becomes apparent.ADP shifts (generation glutamine) generation by hydrolysis (not generating glutamine) and phosphoryl now.Glutamine forms the difference of speed and ADP formation speed and has illustrated hydrolysis rate.Obviously, glutamine forms rate dependent in the stoichiometry of manganese ion and carbonate.Therefore the conclusion that draws is the main effect of carbonate performance in ADP generates, and wherein the generation of ADP has caused the successful transfer (in the background hydrolysis, not observing the effect of carbonate) of phosphoryl.This fact is also passed through 13C HCO 3 -Positive catalysis isotope effect to the activity of glutamine synthetase of adenylylation form is confirmed.Because when not having enzyme, at D 2C in the O matrix 8The deuterium of position is for following similar trend, and therefore hint probably participates in the transition state generation deuterium generation that phosphoryl successfully shifts by being similar to.This prompting is before being attached to enzyme active sites, and preliminary self aggregation to a certain degree takes place compound, and this is the reason that causes phosphorylated.
Because carbonate phosphoryl transfer mechanism has involving on the mechanism, therefore use 13C CO 2(or its hydrated form) observes kinetic isotope effect is rational.In fact, can be observed the influence that glutamine is formed.Yet, observe corresponding increase prompting that ATP consumes and be suitable under the condition that phosphoryl shifts, by observed similar mechanism when not having enzyme, enzymatic advances the activation of γ-phosphoryl residue.
In brief, the conclusion that can draw is that manganese ion concentration (until reaching optimal value) is the main cause of the γ that causes ATP-phosphoryl residue activation.Yet, cause the phosphoryl transfer mechanism and the carbonate implication of glutamine formation reason and relate to C 8The fracture of-H residue.When not needing carbonate (but enough manganese ions are arranged) on mechanism, enzyme can activate γ-phosphoryl residue, but lacks the ability of residue effectively being transferred to glutamic acid.
Discuss
A kind of Mn by supposition is proposed 2+ 3(HCO 3 -) 12The new reaction mechanism (Fig. 1) that-ATP compound is regulated.Someone proposes the HCO by coordination 3 -Carry out protonated or pass through N 7Carboxylation is at N 7The place induces cationic imide.Someone thinks reaction pH and HCO 3 -When pH6.3, dissociate into CO 2Be closely connected, then probably produce the carboxylation intermediate.By containing HCO 3 -Reactant in the resulting data of formic acid of catching hinted that also the carbamate intermediate has generated cationic imide.Huge legendary turtle is closed carbon dioxide (or its hydrated form for example bicarbonate radical) and is similar to Ashman in this way, L.K. and D.B.Keech, (1975) mode that J.Biol.Chem.250:14-21 put down in writing, this mode proposes in the relation between the ATP hydrolysis of studying biotin regulatory enzyme sheep kidney pyruvate carboxylase and carbon dioxide fixation, and wherein proposing phosphine formic acid is the transition intermediate.So, at N 7Place's formation cationic imide makes can be by the carbamic acid of formed deprotonation or the HCO of another coordination 3 -(deprotonation) makes C 8Deprotonation, the intermediate that is generated (IV) passes through M n 2+And C 8Between the supposition that forms in conjunction with being stablized.Formed material (V) causes that terminal phosphoryl residue is near carbamyl; and cause that this phosphoryl residue is closed by carbamyl residue huge legendary turtle; (the close of β-phosphoric acid of former ATP makes this step have reversibility to generate the carbamyl phosphoryl acid anhydride (VI) that activates; as people such as Kaziro (1962) J.Biol.Chem.237:(5), what 1460-1468) proposed in studying about the propiono carboxylase is such).The CO that carboxylation is required 2CO from coordination 2(or its hydrated form).PH 6.3 times, HCO 3 -With CO 2Ratio to be 50:50 most important to the peak optimization reaction speed that obtains the adenylylation glutamine synthelase.PH optimal value pH6.3 demonstration obtains CO easily for this process 2And HCO 3 -And these two kinds of materials all play a role in catalysis.So, the N of carboxylation 7γ-phosphoric acid hydrolysis by ATP can be used in the formation of phosphine formic acid.Then, in reaction, phosphoric acid forms gamma-glutamyl phosphoric acid by the His271 and the γ-carboxyl that may be displaced to glutamic acid by His269.Gamma-glutamyl phosphoric acid is subjected to nucleophilic attack in another mechanism then, forms glutamine.E207 and Arg355 residue play a role in stablizing phosphoryl transfer intermediate by hydrogen bond.At Mn 2+ 3(HCO 3 -) 12The possible coordination that takes place in-ATP the compound is as follows: 2 Mn 2+Ion be positioned on the phosphoric acid tail plane and under, a Mn 2+Ion coordination is to the adenine ring.
In a word, the catalytic mechanism that is proposed based on following some:
■When there being Mn 2+The time, show that the phosphoric acid chain of ATP is coordinated to Mn 2+, and Mn 2+Be coordinated to the C of adenine ring conversely again 8Carbon.
■When there being HCO 3 -There is Cl then again -The time, Mn 2+With C 8Proton draws closer together.
■When not having enzyme, the hydrolysis rate of ATP depends on Mn 2+And HCO 3 -Concentration and existence.
■When not having enzyme, show the hydrolysis rate of ATP with 12HCO 3 -: 3Mn 2+: the Mn that the ratio of 1ATP exists 2+And HCO 3 -Relevant.
■The used reaction mechanism of adenylylation ATP needs Mn 2+Be used for reaction, and go adenylylation ATP to need Mg 2+Be used for reaction.
■The adenylylation glutamine synthelase can use Mg in reaction 2+, be affected but the ATP hydrolysis forms the transformation efficiency of glutamine.
■Each ATP of adenylylation glutamine synthelase needs 3 Mn 2+Ion is to reach optimum active.
■The adenylylation glutamine synthelase needs HCO 3 -And CO 2To reach optimum active.
■PH is that 6.3 definite supercarbonates dissociate into HCO 3 -And CO 2
■The adenylylation glutamine synthelase uses Mn 2+, HCO 3 -With the ratio of ATP be 3:12:1.
■C 8Proton shows that the activity to the glutamine synthelase of adenylylation form plays catalytic action.
Synthetic and the evaluation of embodiment 3 test inhibitor
Preparation purine and pyrimidine analogue are also studied their effects to the GS phosphoryl transferase activity, comprise the effect to ATP hydrolysis, ADP formation, glutamine formation, gamma glutamyltransferase activity and transformation efficiency.
The purine in target phosphoryl transferase site and the synthetic reaction method of pyrimidine analogue
ATP mainly depends on arrangement and several hydrophobic interaction of hydrogen bond around the purine part of molecule with combining of GS avtive spot.Can use back one feature to increase any given micromolecular specificity, form because the hydrophobic region of known ATP-binding site is made up by independent amino acid sequence based on purine type skeleton.Synthetic these molecules with hydrophobic part will make micromolecule tightr with combining of enzyme-specific avtive spot, but repel all similar ATP binding pockets (except the family member who is closely connected most) to a great extent, described hydrophobic part stretches into these sites or pocket, thereby described site or pocket have appropriate space characteristics and optimum effect can take place with the amino acid residue in the pocket electronic characteristic.As long as hydrogen bond can with the main amino acid effect of ATP binding pocket, then this situation is applicable to the structure based on non-purine too.
In order to generate these compounds, studied several groups of universalization compounds, the compound of initial set includes that to have with the adjacent diamines of characteristic be aryl or the heteroaryl that ring replaces part.The heteroaryl system is all based on the pyrimidine skeleton.What it is contemplated that is, depends on used specific diamines, these systems can be converted into purine (II, X, Y=N), quinoxaline (III, X, Y=CH), pteridine (III, X, Y=N) or carbenoid (II, X, Y=N) material.Back one material has participated in the inhibition (E.Piers and J.Y.Roberge, Tetrahedron Letters, 1992,33,6923-6926 and list of references wherein) of ATP enzyme.Also comprise exist with the benzimidazole form, (Y=CH), although because there is not azo to replace, known these material biologically actives are (referring to for example V.Klime for II, X without any the system of hydrogen bond around the hexatomic ring
Figure A200680054592D0101181800QIETU
People such as ov á, Eur.J.Med Chem., 2002,37,409-418).
Figure A200680054592D01011
Also comprise other system in this research, comprise relevant imidazo [1,2-α] pyridine (V) and saturated pyrimidine structure (IV).These two kinds of systems all have required hydrogen bond pattern, and may have a large amount of substituting group variation that the design inhibitor needs, and making may be to suppress the mode combination that required phosphoryl shifts.
Figure A200680054592D01012
Based on (mixing) aryl-1, the molecule of 2-diamines
Be available commercially required diamines, for example 1,2-phenylenediamine 1 and 6-hydroxyl-2,4,5-Triaminopyrimidine 2, synthetic obtain (J.A.Van Allen, Org.Synth.Coll.Vol.VI, people such as N.Rabjohn (editor) perhaps as mentioned below, John Wiley and Sons, Inc. (New York), 1963, pp.245-246; W.R.Sherman and E.C.Taylor, the same, pp.247-249).
Figure A200680054592D01021
If use two hydrochloric acid malonamides directly synthetic 5 as raw material, can not generate neatly required 4,6-di-amino-pyrimidine 4 people J.Chem.Soc such as (, 1943,574) G.W.Kenner.4 another kind preparation (relating to the reaction of thiocarbamide and malononitrile) obtains 4,6-diamido-2-mercaptopyrimidine 3 (scheme 1) (A.Bendich, J.F.Tinker and G.B.Brown, J.Chem.Soc, 1948,3109).The Raney's nickel of mercaptan 3 (Raney nickel) reduction is proved to be to select preferably, and 4,6-di-amino-pyrimidine 4 obtains to separate (D.J.Brown, J.Soc.Chem.Ind, 1950,69,353) with good yield.
Figure A200680054592D01022
Scheme 1
Though existing lot of documents has been put down in writing the nitrosation of pyrimidine in acetate, reported that particularly 4 nitrosifying yield is very low, and need have mineral acid [(a) B.Lythgoe, A.R.Todd and A.Topham, J.Chem.Soc, 1944,315; (b) J.Baddiley, B.Lythgoe, D.McNeil and A.R.Todd, J.Chem.Soc, 1943,383].Though nitrosation is carried out smoothly, find nitroso-product 6 instability when separating under acid condition.6 of humidity is carried out the hyposulfite reduction immediately, generate unsettled triamine 5, yet, can be separated into stable hydrosulfate.
Synthesize 4 in a similar manner, at this moment 5-diamido-6-hydroxy pyrimidine 10 handles thiocarbamide to form mercaptan 7 (W.Traube with ethyl cyanoacetate, Ann.Chem., 1904,331,64), nitrous turns to stable nitrosothiols 9 then, hyposulfite are reduced to diamido mercaptan 8 (A.R.Pagano, W.M.Lajewski and R.A.Jones, J.Am.Chem.Soc, 1995,117,11669) and with Raney's nickel desulfurization in ammoniacal liquor obtain diamines 10.Directly generate 10 trial and unsuccessful (A.Laxer, D.T.Major, H.E.Gottlieb and B.Fischer, J.Org.Chem., 2001,66,5463) by the Raney's nickel reduction by mercaptan 9.Similarly, obtain 5 by handle urea (obtaining 6-amino-uracil 11) with ethyl cyanoacetate, 6-diamido uracil hydrochloride 13 carries out the reduction of nitrosation (obtaining 12) and hyposulfite and obtains diamines 13.The free alkali instability is oxidized to highly colored pyrimido pteridine easily.
During this period, use the 6-amino-1 that is available commercially, test obtains xanthine by the uracil that N-replaces thereby 3-dimethyl uracil 14 carries out ring-closure reaction, and purpose is applied to the N-benzyl derivative with identical method after being.Carry out nitrosation with sodium nitrite to 14 and obtain 15, use the hydrosulfurous acid sodium reduction then, obtain 5,6-diaminostilbene, 3-dimethyl uracil 16 (scheme 2).
Figure A200680054592D01031
Scheme 2
5, the bicyclic system that 6-condenses:
A) benzimidizole derivatives (II, X, Y=CH, R 1, R 4=H)
The benzimidazole of some N1 or C2 replacement is prepared into the adenine analog of the simplification that has nonpolar hexatomic ring.
Figure A200680054592D01032
The most direct synthetic method of the benzimidazole that C-2 replaces relates to phenylenediamine 1 and the carboxylic acid reaction (scheme 3) that is fit to.
Figure A200680054592D01041
Scheme 3
Under 150 ℃, in 85% phosphoric acid; two components (phenylenediamine and corresponding carboxylic acid) are condensed, the amido protecting that is fit to then, thus can separate and purifying compounds (except 5-bromine pentanoate derivant); obtain compound 17-20, all these at room temperature are solid.Product after the protection is not attempted being hydrolyzed.
Figure A200680054592D01042
For example, under 60-100 ℃, by sodium hydride and allyl bromide, bromoallylene that use is in the dry dimethyl formamide benzimidazole 21 is handled 18h, generate allyl benzo imidazoles 22 and obtain the benzimidazole (scheme 4) that N1-replaces (people Bioorg.Med.Chem.Letters such as K.-L.Yu, 2003,13,2141-2144).Use the similar scheme of two alkylations of methylene bromide and furfuryl alcohol to obtain glycation product 23 (A.Hol
Figure A200680054592D0104182141QIETU
Deng the people, J.Med.Chem., 1999,42,2064-2086; People such as A.Khalafi-Nezhad, Tetrahedron, 2002,58,10341-10344].
Scheme 4
B) purine derivative of Qu Daiing: (II, X, Y=N)
C-8 replaces:
Use the purine (scheme 5) of conventional method by diamines 2,5,10 and 8 replacements of 13 preparations.Use the Schotten-Baumann condition, uses the selected acid chloride that is purchased optionally to locate to carry out acidylate and obtain 5-amine pyrimidine 24 in that the strongest 5-of alkalescence is amino 2,5,10 and 13.In some cases, use the dicyandiamide solution of modification to realize this conversion.Use phosphorous oxychloride or sodium methoxide to carry out 2,10 and 13 acid amides cyclisation, form phenols (25, X=OH) or chloride (25, X=Cl) (G.B.Elion, E.Burgi and G.H.Hitchings, J.Am.Chem.Soc, 1951,73,5235-5239).By the acid amides of triamine 5 only with the methoxide cyclisation, obtain adenine (25, X=NH 2) (B.Lucas, N.Rosen and G.Chiosis, J.Comb.Chem., 2001,3,518-520).
Figure A200680054592D01051
Scheme 5
2,5,10 and 13 acidylate and corresponding cyclisation result are shown in table 21.
Figure A200680054592D01061
Figure A200680054592D01071
Another preparation method of various 8-substituted purins comprise by 2 with prepared in reaction carbamate 55 (the P.R Shildneck and the W.Windus of S-methyl-isourea, Org.Syn.Coll.Vol.II, A.H.Blatt (ed.), 1943, John Wiley and Sons (New York), 411) (scheme 6).
Figure A200680054592D01081
Scheme 6
Handle 5-nitroso--6-amino-1 by using benzylamine and concentrated hydrochloric acid aqueous solution, 3-dimethyl uracil 15 also can prepare xanthine 48 (scheme 7) (C.E.M ü ller, M.Thorand, R.Qurishi, M.Diekmann, K.A.Jacobson, W.L.Padgett and J.W.Daly, J.Med.Chem., 2002,45,3440).
Figure A200680054592D01082
Scheme 7
N-6 and N-9 replace
As everyone knows, 4, the nucleophilic displacement of fluorine of 56 pairs of nitrogen of 6-dichloro pyrimidine and oxygen affinity nuclear reagent is particularly responsive.The replacement of first chloro is easy to, and second chlorine then needs more mandatory condition (C.W.Whitehead and J.J.Traverso, J.Am.Chem.Soc., 1958,80,2185-2189; People such as M.H.Norman, J.Med.Chem., 2000,43,4288-4312).Yet pyrimidine ring can be activated.Can realize this activation by introducing electron withdraw group at C-5, this electron withdraw group for example is nitro or the nitroso-that replaces by parent's electricity.Permission replaces a chlorine substituent with the amine list, and nitrosation and the scheme that replaces second chlorine substituent subsequently can obtain as scheme 8 described Triaminopyrimidine precursors then.Reduction nitroso-, and then closed loop earlier can obtain desirable adenine derivative.
Figure A200680054592D01091
Scheme 8
Select various amine to replace chloro,, comprise alkyl amine, arylamine and heteroaryl amine to cover molecule as much as possible " scope ".Be used in combination a primary amine and a secondary amine to guarantee that product is single when cyclisation is purine compound.
Select following amine:
Primary amine:
Figure A200680054592D01092
Secondary amine:
Initial substitution reaction is included in the isopropyl alcohol of boiling and handles 56 (C.Temple, C.L.Kussner and J.A.Montgomery, J.Med.Chem., 1962,5,866-870 with the twice excess amine; People J.Med.Chem. such as M.Israel, 1964,7,792-799), perhaps, another kind is when having the triethylamine of monovalent, only uses the amine of monovalent can carry out this reaction (N.Baindur, N.Chadha and M.R.Player, J.Comb.Chem., 2003,5,653-659).In the crude mixture or separated product of monoamine product 57-61, all do not separate or detect disubstituted product.It is shown in table 22 to carry out mono-substituted result with primary amine to 56.Amazingly be, derive and yield that the pyrimidine 60 that comes generates is very low by phenylalanine methyl ester hydrochloride, may be because the ester hydrochloride intermediate that forms be considerably less.As expected, alkaline very weak arylamine needs mandatory condition.Yet, the 2-aminopyridine can not with 56 the reaction.
Be easy to take place the nitrosation of secondary amine 57-59, obtain the good product 62-64 of yield (people such as D.E.O ' Brien, J.Med.Chem., 1962,5,1085-1103).Perplexing is that phenylalanyl derivant 60 can not react under this condition.Similarly, prove under different condition, be difficult to activation anil 61 and carry out nitrosation (table 22).Then, fully activate under the condition of pyrimidine, handle nitroso-product 62-64 with secondary amine in the hypothesis nitrosation.The aromatic amine that will be difficult for reacting in this stage is introduced the trial of these compounds and is got nowhere.
Under the condition of sodium hydrosulfite and aqueous sulfuric acid existence, make the Triaminopyrimidine 72-78 (table 23) of nitroso compound 65-71 reduction to obtain replacing.Carry out the cyclisation of pyrimidine 72,73,75,76 and 78 by heating pyrimidine in the 1:1 of acetic anhydride and triethyl orthoformate potpourri, obtain the adenine 79-83 (C.Temple, C.L.Kussner and the J.A.Montgomery that replace respectively, J.Med.Chem., 1962,5,866-870).Yet, all with the pyrimidine 74 and 77 of hydroxyethyl chain replacement, obtained the composite mix of product, show that this composite mix contains the intermediate of desirable product and cyclization.
Figure A200680054592D01131
At first, to handle 56 with secondary amine, carry out the scheme identical then with aforementioned schemes with the similar mode of primary amine.The result is shown in table 24.
Table 24: under mandatory condition, make 56 with secondary amine reaction, carry out the nitrosation test and carry out replacement second time with primary amine
Figure A200680054592D01141
aObtain by condensing by 57
bObtain by condensing by 58
dObtain by condensing by 59
The nitrosation of four tertiary amine 84-87 is all failed, the not charged nitrosifying N-nitroso-intermediate that mechanism reaction needed secondary amine with formation reaction takes place, and nitrosation (J.H.Boyer takes place at the ortho position, The Chemistry of the Nitro and Nitroso Groups, H.Feuer (ed.), JohnWiley and Sons (New York), 1969, pp.223-225).Tertiary amine helps the direct nitrosation of contraposition, and this replaces parent's electricity can not carry out (R.G.Coombes, ComprehensiveOrganic Chemistry in these cases, D.Barton and W.D.Ollis (eds.), Pergamon Press (Oxford), 1979, pp.308-309).Therefore, use mandatory condition that primary amine is directly introduced product 84-87, can achieve success in varying degrees, sum up as table 24.Utilize these conditions, use pyrrolidine and dimethylamine also can successfully make the 60 direct aminations of phenylalanyl derivant, thereby obtain bisamination product 100 and 101 respectively.
In order to use 2-aminopyrazole derivatives 99, replacing, it is nitrated to obtain 102 to carry out.But can use the Raney's nickel reduction, therefore prove this unstable products and decomposition fast.
Use excessive a little bromine, in methylene chloride, carry out the bromination of diamines 89-96, obtain bromo pyrimi piperidine 103-110 (table 25).Use the anhydrous cuprous iodide in the excess amine to be used for inserting, bromide 105 and 108 is under the Ullmann amination condition, all are dissolved in the N that contains hypophosphite monohydrate potassium, N-dimethylethanolamine (chelating solvent) is as alkali (F.Y.Kwong and S.L.Buchwald, Org.Letters, 2003,5,793-796, J.P.Wolfe, S.Wagaw, J.-F.Marcoux and S.L.Buchwald, Ace.Chem.Res., 1998,31,805-818).Under inert atmosphere in 100 ℃ with mixture heated 18h.In the amine that this replacement is tested, have only benzylamine to succeed, obtain Triaminopyrimidine 111 and 112.
Imidazo [1,2-α] pyridine (V, X, Y=CH, R 1=H)
Because imidazopyridine, Imidazopyrazines and imidazopyrimidine are extensively treated the interesting biologically active that demonstrates on the kind, be subjected to pharmacy industry very big concern (A.R.Katritzky, Y.-J Xu and H.Tu, J Org.Chem., 2003,68,4935].Though have some route of synthesis can obtain imidazo [1,2-α] pyridine ring system, prevailing method relates to 2-aminopyridine and α-Lu Daitangjihuahewu (halocarbonyl compound) coupling.In initial research, react with phenacyl bromide and PBPB respectively by the 2-aminopyridine and to prepare imidazopyridine 113 and 114.
Figure A200680054592D01171
More method in common is to use three component coupling (3CC) [(a) C.Blackburn, B.Guan, P.Fleming, K.Shiosaki and S.Tsai, Tetrahedron Lett., 1998,39,3635 that relate to aldehyde, 2-aminopyridine and isocyanide condensation; (b) C.Blackburn, Tetrahedron Lett., 1998,39,5469; (c) C.Blackburn and B.Guan, Tetrahedron Lett., 2000,41,1495] (scheme 9).
Figure A200680054592D01172
Scheme 9
Although also use excessive glyoxalic acid (M.A.Lyon and T.S.Kercher, Org.Lett., 2004,6,4989) or polynite (R.S.Varma and D.Kumar, Tetrahedron Lett, 1999,40,7665), but in the presence of acid catalyst, often use trifluoromethayl sulfonic acid scandium (III) [(a) C.Blackburn, B.Guan, P.Fleming, K.Shiosaki and S.Tsai, Tetrahedron Lett., 1998,39,3635; (b) C.Blackburn, Tetrahedron Lett., 1998,39,5469; (c) C.Blackburn and B.Guan, Tetrahedron Lett., 2000,41,1495; (d) S.M.Ireland, H.Tye and M.Whittaker, Tetrahedron Lett., 2003,44,4369; (e) G.S.Mandair, M.Light, A.Russell, M.Hursthouse and M.Bradley, Tetrahedron Lett., 2002,43,4267] carry out the 3CC reaction.Acid catalyst has promoted the first step reaction among the 3CC, forms imines.Usually can use conventional heating or microwave reactor at room temperature to carry out the reaction of long-time (48h).
With zinc chloride or polynite K10 is catalyzer, prepares some imidazos [1,2-α] pyridine 115-131 by the 2-aminopyridine.These results are summarised in the table 26.
The 3CC reaction of table 26:2-aminopyridine
Figure A200680054592D01181
aPolynite K10 refluxes 5h
bZnCl 2, microwave, 2h
cPolynite K10, microwave, 2h
6, the bicyclic system that 6-condenses
Especially with glyoxal (obtaining 132-134) (H.B.Gillespie, F.Spano and S.Graff, J.Org.Chem., 1960,25,942-944; L.G.Fr
Figure A200680054592D0118182840QIETU
People J.Med.Chem. such as lich, 1999,42,4108-4121) with methyl-oxalacetic ester sodium salt (obtaining 135-137) [D.Farquhar and T.L.Loo, J.Med.Chem., 1972,15,567-568] be the dicarbapentaborane component, target is to form based on 1, one group 6 of 2-dicarbapentaborane system and diamines 1,2 and 13 condensations, the bicyclic system (table 27) that 6-condenses.Proof is difficult for carrying out based on 1 reaction unusually, mainly be because the contact between the reagent is very poor, and triamine 2 has obtained interesting tricyclic compound 133 in excessive glyoxal.
Table 27: use glyoxal or methyl-oxalacetic ester to form quinoxaline and pteridine
Figure A200680054592D01191
A obtains with the form of the potpourri of quinoxaline and imidic acid diester intermediate
6, the 6-bicyclic system
Pyrimidine derivatives for the non-aroma properties of the hydrogen bond network that obtains having extension, the preparation 3 of using Biginelli at first to put down in writing, three component reaction (J.C.Bussolari and the P.A.McDonnell of 4-dihydro-pyrimidin-2 (1H)-ketone, J.Org.Chem., 2000,65,6777 and list of references wherein).This multi-component reaction comprises the condensation reaction of the aromatic aldehyde, urea and the ethyl acetoacetate that are in the acid ethanol solution, generates the pyrimidone of highly functionalization.The amended Biginelli reaction that use is relevant with Bussolari and McDonnell is used to be in anhydrously 1, and the methyl-oxalacetic ester sodium salt that contains urea in the 2-ethylene dichloride, various aldehyde and trifluoroacetic acid obtain pyrimidone 138-141 (table 28).
Table 28: the cyclizative condensation of methyl-oxalacetic ester sodium salt and 138 hydrolysis
Figure A200680054592D01201
By handle 138 suspensions in the absolute ethyl alcohol with the potassium hydroxide solution in the absolute ethyl alcohol, the hydrolysis of 138 the ethyl ester of realizing that oxyhydroxide induces, the pyrimidone 142 that obtains dewatering.
Adenosine with metal-complexing ability
Transfer to the mechanism prediction and the structure prediction of the character of glutamic acid by ATP based on the phosphoryl of the glutamine synthelase of adenylylation form; someone proposes; by in this zone of possible inhibitor molecules, incorporating the metal binding site into, can very well simulate metal close to adenine C-8.Someone proposes, the substituting group that contains two or more contiguous heteroatomss (for example morpholino part) in this position, can bring into play the effect in coordination site simultaneously and second heteroatoms placed area of space, this area of space is similar to the zone that the near-ring shape phosphoric acid skeleton of ATP compound occupies.Then, be embedded in metallic ion in this compound can obtain water or carbon dioxide (with hydrated form) thus infer and finish coordination shell to be similar to the similar mode of parent ATP.
Can realize the synthetic of desired substance by following steps; that is: make adenosine 143 (bromine aqueous solution) bromination to obtain 8-bromine adenosine 144; use 2 then; the potpourri of 2-dimethoxy propane and acetone (equal-volume) and the toluenesulfonic acid of 5 equivalents as 2 ', 3 '-isopropylidene acetal 145 protects (scheme 10).In the presence of excessive morpholine, use microwave irradiation,, in the 4-diox solvent halogenide is replaced, thereby obtain 146 1.In tetrahydrofuran and concentrated hydrochloric acid, under room temperature, carry out deprotection generating desired substance 147, with preparative liquid chromatography handle 147 with obtain authentic sample (T.Sasaki, K.minamoto and H, Itoh, J.Org.Chem., 1978,43,2320-2325).
Scheme 10
Adopt similar scheme can introduce another substituting group, thereby change space characteristics and in conjunction with feature in the 8-position.
The material of synthetic schemes and method
All initiation materials all by commercially available and obtain the back from supplier and directly use, are not further purified.
Use Merck Kiesel gel 60 (particle size 0.040-0.063mm) to carry out column chromatography, use aluminium base silica gel plate 60 F of Merck 254Carry out thin-layer chromatography.
On Varian Gemini 200 nuclear magnetic resonance analyser, write down the NMR spectrogram in 200MHz.With ppm record chemical shift data, and with Hz record coupling constant.
On the Waters liquid chromatographic system, use Varian 9050 UV/VTS detecting devices in 254nm record HPLC data.Use the isocratic elution system at Phenomenex
Figure A200680054592D0121162746QIETU
Luna TMCarry out all separation on 5 μ C-18 (2) 150mm * 4.60mm posts.The methyl alcohol under the pH 4 of solvent for use shown in being and the potpourri of 25mM ammonium acetate aqueous buffer solution are 1cm at flow velocity 3Carry out wash-out under the/min.
Standard fabrication is to use organic solvent extraction, then with dried over mgso and carry out the vacuum distillation of solvent on Rotary Evaporators.
Record fusing point and not proofreading and correct on Reichert Hotplate.
4,6-di-amino-pyrimidine-2-mercaptan (3)
(1.8g 0.078mol) is dissolved in absolute ethyl alcohol (40cm to make sodium metal under nitrogen atmosphere 3).To its add thiocarbamide (6.0g, 0.079mol), and then add malononitrile (5.0g, 0.076mol).With gained heterogeneous body potpourri backflow heating 2h, make it be cooled to room temperature then.Add entry (120cm 3) dissolve fully to promote it, with glacial acetic acid this uniform homogeneous blend (to pH6.0) that neutralizes.Potpourri is cooled to 0 ℃ and filtration.With collected product vacuum drying to obtain 4,6-di-amino-pyrimidine-2-mercaptan 3 (7.80g, 69%).δ H(200MHz，d 6-DMSO)5.01(1H，s，H-6)，6.4-6.7，(4H，br s，NH 2)；mp>280℃。
4,6-Diaminopyrimidines thiamine hydrochloride (4)
With 4, (4.10g 0.029mol) is dissolved in 2M sodium hydrate aqueous solution (18cm to 6-di-amino-pyrimidine-2-mercaptan 3 3) in, and in ice bath, be cooled to 10 ℃.To keep temperature to be lower than 15 ℃ speed, dropwise add in the stirring aqueous hydrogen peroxide solution (3%, 62cm 3).After adding end (about 20min), further reaction stirred 30min under the condition that need not to cool off, during this period of time, reaction mixture becomes opaque, arrives pH 4.0 with the glacial acetic acid acidifying then.With the cooling of gained slurry, collect product by filtering, vacuum drying obtains 4, and 6-di-amino-pyrimidine-2-sulfinic acid (3.48g, 69%), fusing point are 164-170 ℃ (literature value is 168-170 ℃).Should acid (3.40g 0.020mol) is added in 5 ℃ down in the dry ethanol with the saturated mistakes of hydrogen chloride gas, at room temperature stirs gained potpourri 30min, forms thick slurry during this period.After forming this slurry, potpourri is cooled to 0 ℃ and stir 1h.With the product isolated by filtration,, obtain 2.78g (97%) 4,6-Diaminopyrimidines thiamine hydrochloride 4 with ether washing and vacuum drying.δ H(200MHz, d 6-DMSO) 5.60 (1H, s, H-5), 7.40-7.81 (4H, br s, NH 2), 8.20 (1H, s, H-2); Fusing point 194-196 ℃ (literature value is 196-198 ℃).
4, another preparation method of 6-di-amino-pyrimidine (4)
With 4, (24.85g 0.18mol) is suspended in 5% ammoniacal liquor (1.2L) 6-diamido-2-mercaptopyrimidine 3, and is heated to 85 ℃ to impel its dissolving.Raney's nickel (slurry that 50g is wet) a part in 10min is carefully added in the hot mixt.With gained potpourri backflow heating 1h.Thermal reaction mixture filtered and with hot water (200cm 3) washing leaching cake.Filtrate decompression is concentrated, obtain 4,6-di-amino-pyrimidine 4 (15.9g, 83%).δ H(200MHz, D 2O) 7.82 (1H, s, H-2) and 5.55 (1H, s, H-5).δ C(50MHz, d 6-DMSO) 159.6 (C-4 and C-6), 149.9 (C-2) and 81.2 (C-5).
4,5,6-Triaminopyrimidine hydrosulfate (5)
With 4, (15.90g 0.14mol) is suspended in aqueous hydrochloric acid solution (1M, 500cm to 6-di-amino-pyrimidine 4 3) and be cooled to 2 ℃.To keep temperature of reaction to be lower than 4 ℃ speed, with sodium nitrite (14.90g, aqueous solution (35cm 0.22mol) 3) dropwise add in the cooling solution and (carry out 30min with this speed).Make potpourri in 1h, be warming up to room temperature naturally.After this, add sodium bicarbonate for a part, green and brown color contamination compound is neutralized to pH7.0 with solid form.With the blue-green sedimentation and filtration that forms, but not exclusively dry.Unsettled nitroso compound is at water (220cm 3) in become slurry immediately, (52.80g 0.25mol) handles at room temperature to use a part of sodium hydrosulfite that adds.With 50% aqueous sulfuric acid (150cm 3, 1.4mol) handle yellow mixture and be heated to 80 ℃ and keep 3min, in ice bath, be cooled to room temperature then.With the sedimentation and filtration that forms, with ethanol water (30cm 3) washing and dry, obtain 4,5,6-Triaminopyrimidine hydrosulfate 5 (23.0g, 71%).δ H(200MHz, D 2O) 7.61 (1H, s, H-2); δ C(50MHz, d 6-DMSO) 148.1 (C-4 and C-6), 140.4 (C-2) and 105.9 (C-5).By preparing unhindered amina in the sodium hydrate aqueous solution that hydrosulfate is dissolved in minimum 2M heat.Unhindered amina precipitation in cooling procedure, and can from water, come out by recrystallization.
4-Amide-6-hydroxy-2--mercaptopyrimidine (7)
(4.60g 0.20mol) is dissolved in absolute ethyl alcohol (150cm under nitrogen atmosphere with sodium metal 3), and with ethyl cyanoacetate (22.0g, 0.19mol) and thiocarbamide (16.0g 0.21mol) handles.With the gained potpourri under nitrogen atmosphere, reflux the heating 2h.After this, potpourri is cooled to room temperature, forms white precipitate and filter this white precipitate.Filter cake is dissolved in water (150cm 3) and be acidified to pH 4.0 with 50% acetic acid aqueous solution.With formed sedimentation and filtration, obtain 4-Amide-6-hydroxy-2--mercaptopyrimidine 7 (28.0g, 100%) white solid.δ H(200MHz, D 2O) 5.06 (1H, s, H-5); δ C(50MHz, D 2O) 177.4 (C-6) a, 170.0 (C-2) a, 164.9 (C-4) aWith 82.1 (C-5).
4,5-diamido-6-hydroxyl-2-mercaptopyrimidine (8)
(17.60g is 0.10mol) at saturated sodium bicarbonate aqueous solution (400cm with 4-Amide-6-hydroxy-2--sulfydryl-5-nitroso-pyrimidine 9 3) in become slurry, and (42.7g 0.25mol) handles with a part of sodium hydrosulfite that adds in 10min.Light yellow potpourri is stirred 7h down at 5 ℃, use acetate (24cm then 3) handle.Beginning forms white precipitate gradually.With sedimentation and filtration, with ethanol water (30cm 3) washing and dry, obtain 4,5-diamido-6-hydroxyl-2-mercaptopyrimidine 8 (18.3g, 100%).δ C(50MHz, D 2O) 168.3 (C-6) a, 158.7 (C-2) a, 142.3 (C-4) aWith 103.1 (C-5).
4-Amide-6-hydroxy-2--sulfydryl-5-nitroso-pyrimidine (9)
(16.80g 0.12mol) is suspended in water (300cm with 4-Amide-6-hydroxy-2--mercaptopyrimidine 7 3) in and with acetate (60cm 3) handle.Be dissolved in water (35cm by dropwise adding 3) sodium nitrite (15.0g 0.22mol) handles this suspension.The orange mixture of gained at room temperature stirred spend the night.Behind the 16h, potpourri is filtered, successively water (20cm 3) and ethanol (20cm 3) washing leaching cake and dry, obtain 4-Amide-6-hydroxy-2--sulfydryl-brick-red solid of 5-nitroso-pyrimidine 9 (17.6g, 87%).Need not to characterize or be further purified, can in afterreaction, use this crude product.
4,5-diamido-6-hydroxy pyrimidine (10)
With 4, (16.20g 0.10mol) is dissolved in 5% ammoniacal liquor (440cm to 5-diamido-6-hydroxyl-2-mercaptopyrimidine 8 3) in, and in 5min, handle with a part of Raney's nickel that adds (slurry that 45.3g is wet).With gained potpourri backflow heating 1.5h.Thermal reaction mixture is filtered, filtrate decompression is concentrated, obtain 4,5-diamido-6-hydroxy pyrimidine 10 (11.5g, 88%).δ H(200MHz, D 2O) 7.54 (1H, s, H-2); δ C(50MHz, D 2O) 157.0 (C-6) a, 147.8 (C-4) a, 138.7 (C-2) and 111.1 (C-5).
5,6-diaminourea pyrimidine hydrochloride (13)
(3.9g is 0.17mol) with the absolute ethyl alcohol (100cm in many mouthfuls of round-bottomed flasks of fritter adding with sodium 3) in.After all sodium all disappears, to its add ethyl cyanoacetate (9.8g, 0.087mol) and urea (5.2g 0.087mol), and refluxes reactant and heats 4h.Be solid this moment on the reaction mixture certain degree, adds hot water (100cm to it 3) dissolve this material, and heat this potpourri 15min down at 80 ℃.With the careful neutralization reactant of glacial acetic acid, add glacial acetic acid (7.5cm in addition 3), and then adding is dissolved in water (7cm 3) in sodium nitrite (6.5g, 0.094mol).With this solution cooling, by removing by filter pink solid 12 and washing with frozen water.Substance transfer after this filtration is returned original flask and added warm water (40cm 3).Heat this slurry to 100 ℃ while stirring, add the nitroso compound disappearance of solid sodium hydrosulfite (about 20g) until redness.Add a few gram hyposulfite in addition and continue heating 15min.Make the potpourri cooling, filtration diamido uracil sulphite also washes with water.With concentrated hydrochloric acid (20cm 3) add in the sulphite in the conical flask agitating heating 1h on electric hot plate.Filter and fully wash, obtain 5,6-diamido uracil hydrochloride 13 tawny solids (7.53g, 49%) with acetone.δ C(50MHz, D 2O/NaOH) 96.0 (C-5), 158.0 (C-6), 159.3 (C-2) and 162.2 (C-4).
6-amino-1,3-dimethyl-5-nitroso-uracil (15)
With 6-amino-1, (5.0g 32mmol) is dissolved in 50% acetic acid aqueous solution (150cm to 3-dimethyl uracil 14 3) in.Be dissolved in water (20cm to its adding 3) in sodium nitrite (4.4g, 64mmol).Reactant almost becomes the brilliant violet look immediately, at room temperature stirs 1h.With the potpourri cooling and by filtering collecting precipitation, fully wash with cold water, obtain 6-amino-1,3-dimethyl-5-nitroso-uracil 15 brilliant violet look solids (5.8g, 98%).δ H(200MHz, d 6-DMSO) 3.26 (3H, s, CH 3N), 3.28 (3H, s, CH 3N), 9.05 and 12.97 (2H, 2 * br s, NH 2).
5,6-diaminostilbene, 3-dimethyl uracil hydrosulfite (16)
With 6-amino-1, (3.5g 19mmol) is suspended in the warm water 3-dimethyl-5-nitroso-uracil 15, and disappears until purple to its adding sodium hydrosulfite.In this stage, all substances are all in solution.Remove by evaporation and to anhydrate, with solid filtering and wash with water, obtain 5,6-diaminostilbene, 3-dimethyl uracil hydrosulfite 16 light yellow solids (2.2g, 46%) until obtaining slurry.δ H(200MHz .d 6-DMSO) 3.14 (3H, s, CH 3N), 3.30 (3H, s, CH 3N), 3.36 (2H, br s, NH 2) and 6.13 (2H, br s, NH 2); δ C(50MHz, d 6-DMSO) 28.3 and 30.5 (2 * CH 3), 96.7 (C-5), 145.6 (C-6), 150.5 (C-2) and 159.7 (C-4).
The conventional method of carboxylic acid and o-phenylenediamine (1) condensation
O-phenylenediamine 1 (1 equivalent) and required carboxylic acid (1.2 equivalent) are introduced not with the 2cm in the round-bottomed flask of condenser 3In 85% phosphoric acid.With this mixture heated to 150-200 ℃, minimum heating 2h, normally 18h obtains dark blue solution as time passes.Then hot mixt is decanted into 100cm 3In the unsaturated carbonate aqueous solutions of potassium, obtain pH greater than 7 blue solution.Constant is to add isopyknic ethyl acetate is not dissolved in water with extraction all organic components to it.Below provide each preparation method.
(2S)-2-(pyrrolidine-1-t-butyl formate (17) of 1H-benzimidazolyl-2 radicals-yl)
As treatment of o-phenylenediamine 1 as described in the above-mentioned conventional method (1.00g, 9.21mmol) and the L-proline (1.31g, 11.35mmol).The white granular pastel that the water simmer down to is contained red patch.With three parts of 10cm 3Cold water carefully dissolves this white paste, removes patch.Make it be dissolved in 3:1 (v/v) methyl alcohol: in the ethyl acetate, any solid of gained to be filtered and with the red foam of red solution simmer down to.This residue is dissolved in contain NaOH (1.36g, 33.88mmol) and di-tert-butyl dicarbonate (4.84g, water (25cm 22.19mmol) 3, 0.4M) in, at room temperature stir 18h.With ethyl acetate (2 * 25cm 3) extract, with organic layer drying (MgSO 4) and concentrate, obtain limpid jelly, with 3:10 (v/v) ethyl acetate: hexane grinds, and obtains thin white powder (2S)-2-(1H-benzimidazolyl-2 radicals-yl)-pyrrolidine-1-t-butyl formate 17 (obtaining 0.51g, 19% through the reaction of 2 steps).δ H(200MHz, CDCl 3) 7.49-7.68 (2H, m, aryl H-4 and H-7), 7.18-7.30 (2H, m, aryl H-5 and H-6), 5.09-5.18 (1H, br dm, pyrroles H-2, J 7.2), 3.36-3.57 (2H, br t, pyrroles H-5), 2.98-3.21 (1H, br s, pyrroles H-3a), 1.95-2.39 (3H, 2 * br m, pyrroles H-3a and H-4) and 1.53[9H, s, OC (CH 3) 3]; δ C(50MHz, CDCl 3) 156.8 (NCO), 155.1 (benzimidazole C-2), 122.7 (aryl C-4, C-5, C-6 and C-7), 115.5 (br s, season aryl C-3a and C-7a), 80.9 (pyrroles C-2), 54.9[OC (CH 3) 3], 47.6 (pyrroles C-5), 28.8[OC (CH 3) 3], 28.4 (pyrroles C-3) and 25.2 (pyrroles C-4).
The methyl of N-acetyl group-N-[(1-acetyl group-1H-benzimidazolyl-2 radicals-yl)] acetamide (18)
(1.68g is 22.08mmol) with o-phenylenediamine 1 (1.96g, 18.11mmol) 18h to handle glycocoll with above-mentioned conventional method.After neutralization and adding ethyl acetate, water layer is separated and simmer down to cream-coloured (beige) jelly.Under 160 ℃, with acetate (10cm 3) in 1:1 (v/v) acetic anhydride handle 0.31g jelly 18h.Solution is decanted into 100cm 3In the unsaturated carbonate aqueous solutions of potassium, with ethyl acetate (2 * 100cm 3) extract dry (MgSO 4) and the simmer down to jelly.In ethyl acetate/hexane, make its grinding, obtain white powder N-acetyl group-N-[(1-acetyl group-1H-benzimidazolyl-2 radicals-yl)-methyl] acetamide 18 (0.22g, 4%).δ H(200MHz, CDCl 3) 7.29-7.80,7.60-7.68 and 7.35-7.45 (4H, 3 * m, aryl H), 5.38 (2H, s, CH 2), 2.89 (3H, s, benzimidazole COCH 3), 2.52[6H, s, N (COCH 3) 2]; δ C(50MHz, CDCl 3) 173.4[N (COCH 3) 2], 169.6 (benzimidazole COCH 3), 152.8 (benzimidazole C-2), 143.1 (season aryl C-3a), 132.8 (season aryl C-7a), 125.1 and 124.8 (aryl C-4 and C-7), 121.4 (aryl C-6), 113.3 (aryl C-5), 45.9 (CH 2), 26.9 (benzimidazole COCH 3) and 26.6[N (COCH 3) 2].
The 2-{[(tertbutyloxycarbonyl) amino] methyl }-1H-benzimidazole-1-t-butyl formate (19)
Used part jelly (1.04g) in the preparation 18 is dissolved in and contains NaOH (4.51g) and di-tert-butyl dicarbonate (2.43g, water (50cm 11.14mmol) 3) in, at room temperature stir 18h.With ethyl acetate (2 * 25cm 3) extract, with organic layer drying (MgSO 4) and concentrate, obtain limpid jelly.(eluent is 1:10-1:5 (v/v) ethyl acetate: this product of purifying hexane) to carry out column chromatography, obtain light yellow oil, this grease is cured as beige solid through placing, the 2-{[(tertbutyloxycarbonyl) methyl amino) }-1H-benzimidazole-1-t-butyl formate 19 (1.20g, based on the raw material diamines is 19%), δ H(200MHz, CDCl 3) 7.92-7.98,7.65-7.74 and 7.26-7.39 (4H, 3 * m, aryl H), 5.85 (1H, br s, NH), 4.81 (2H, d, CH 2, J 5.4), 1.73[9H, s, benzimidazole OC (CH 3) 3] and 1.51[9H, s, OC (CH 3) 3].
2-(4-brombutyl)-1H-benzimidazole (20)
(1.00g, 9.27mmol) (2.15g 11.85mmol) places the round-bottomed flask of being furnished with condenser, simple agitating heating 3h under 150 ℃ with 5-bromine valeric acid with o-phenylenediamine 1.Gained purple solid is pulverized, used 10cm 3The ethyl acetate washing obtains purple powder 2-(4-brombutyl)-1H-benzimidazole 20 (1.28g, 73%) with precipitation.δ H(200MHz, CDCl 3) 7.61-7.68 and 7.18-7.32 (4H, aryl H), 6.26 (1H, br s, NH), 4.09 (2H, t ,=CCH 2, J 6.0), 3.11 (2H, t, CH 2OH, J 6.3) and 1.95-2.22[4H, m, (CH 2) 2CH 2Br].
The alkylating conventional method 21 of benzimidazole that alkali is regulated
Under nitrogen atmosphere, be in dry N with sodium hydride, the potpourri of benzimidazole 21 in the dinethylformamide (1 equivalent) and alkylating agent (1.1-1.3 equivalent).Then, solution is heated to 100 ℃ and lasting 18h.The gained brown solution is decanted in the ethyl acetate, uses the water of equivalent to extract three times.After dry and concentrated, (eluent is 1:10-1:2 (v/v) ethyl acetate: this residue of purifying hexane) by column chromatography.
1-allyl-1H-benzimidazole (22)
According to above-mentioned conventional method, (60% oil solution, 0.23g 5.66mmol) are in dry N, dinethylformamide (10cm with sodium hydride 3) in benzimidazole 21 (0.49g, 4.15mmol) with allyl bromide, bromoallylene (0.40cm 3, potpourri 4.62mmol).Obtain cream-coloured grease 1-allyl-1H-benzimidazole 22 (0.56g, 86%) by column chromatography.δ H(200MHz, CDCl 3) 7.92 (1H, br s, H-2), 7.77-7.89 and 7.24-7.45 (4H, 2 * m, aryl H), 6.02 (1H, ddt, CH 2CH=, J 17.0,10.0 and 5.8), 5.31 (1H, dq, CH=CH aH b, J 10.2 and 1.8), 5.21 (1H, dq, CH=CH aH b, J 17.0 and 1.6) and 4.79 (2H, NCH 2, J 5.4 and 1.8).
1-[(2-furans methoxyl) methyl]-1H-benzimidazole (23)
According to above-mentioned conventional method, (0.38g 9.25mmol) is in dry N, dinethylformamide (10cm with sodium hydride 3) in benzimidazole 21 (0.51g, 4.24mmol), methylene bromide (0.33cm 3, 4.6mmol) and furfuryl alcohol (0.40cm 3, 4.6mmol).Obtain yellow oil 1-[(2-furans methoxyl by column chromatography) methyl]-1H-benzimidazole 23 (0.39g, 40%).δ H(200MHz, CDCl 3) 8.00 (1H, s, benzimidazole H-2), 7.79-7.88 and 7.47-7.59 (2H, 2 * m, benzimidazole H-4 and H-7), 7.43-7.47 (1H, m, furyl H-5), 7.26-7.40 (2H, m, benzimidazole H-5 and H-6), (6.38 1H ,～dd, furyl H-4, J 3.2 and 1.8), (6.33 1H, br dd, furyl H-3, J 3.0 and 1.8), 5.57 (2H, s, NCH 2O) and 4.41 (2H, s, OCH 2Furyl).
The conventional method of preparation acidylate pyrimidine
N-(2,4-diamido-6-hydroxy pyrimidine-5-yl) benzamide (26)
With 2,4, (1.0g 4.2mmol) is dissolved in 2M sodium hydroxide solution (25cm to 5-triamido-6-hydroxy pyrimidine 2 3) in, with ice bath solution is cooled to 0 ℃.(0.6g 4.2mmol) adds in the solution with chlorobenzoyl chloride in 5min to use syringe then.The 15min that under 0 ℃, stirs the mixture, add in a similar manner another part chlorobenzoyl chloride (0.6g, 4.2mmol).In ice bath, stirred the mixture again 1 hour.From ice bath, remove reaction mixture then and be heated to room temperature.With glacial acetic acid solution is transferred to pH 5, this moment, product was by being precipitated out in the solution.Filter the light brown precipitation and with deionized water (3 * 10cm with Buchner funnel 3) washing.Dry cake 3h on funnel transfers to flask and vacuum drying then at first.N-(2,4-diamido-6-hydroxy pyrimidine-5-yl) benzamide 26 (0.78g, 56%).δ H(200MHz, D 2O) 7.79 (2H, d, J 6.6,2 * ArCH) and 7.35-7.58 (3H, m, 3 * ArCH).Can be directly used in the step of back.
Obtain following product in a similar manner:
N-(2,4-diamido-6-hydroxyphenyl) caprylamide (28)
(0.34g，35％)。δ H(200MHz, D 2O) 2.36 (J 7.7, COCH for 2H, t 2), 1.45-1.70 (2H, m, COCH 2CH 2), 1.10-1.40 (8H, m, 4 * CH 2) and 0.82 (3H, br s, CH 3).
N-(2,4-diamido-6-hydroxyphenyl)-2,2-dimethyl propylene acid amides (29)
(0.38g，43％)。δ H(200MHz，D 2O)1.24(9H，s，3×CH 3)。
N-(1-{[(2,4-diamido-6-hydroxy pyrimidine-5-yl) amino] carbonyl }-the 2-methyl-propyl) benzamide (30)
At room temperature, with chlorobenzoyl chloride (18.7cm 3, 0.16mol) processing is dissolved in water (150cm 3) in the L-valine (15.75g, 0.13mol) and potassium hydroxide (12.00g 0.21mol), and stirs 72h.With chloroform (200cm 3) extract, earlier with isopyknic 1M salt acid elution, use the saturated potassium carbonate solution washing more then.Dry (MgSO 4) and concentrate after, make beige solid be dissolved in chloroform (200cm 3).Under nitrogen atmosphere, with thionyl chloride (14.7cm 3, 0.21mol) handle this solution with three dimethyl formamides.With mixture heated 1h, the yellow substance of simmer down to viscosity then.Under 0 ℃, two parts of acid chlorides (every part of 4.01g) are added in 2h and be in 1M sodium hydrate aqueous solution (50cm 3) in 13 (2.00g 8.38mmol) in the solution, after this, regulates pH to being about 4 with glacial acetic acid.Beige solid 30 settles down from solution, filters then, and drying need not to characterize and can directly use.
The conventional method of the cyclisation of the acidylate pyrimidine that sodium methoxide is regulated
2-amino-8-phenyl-9H-purine-6-alcohol (31)
(0.3g, 1.2mmol) adding is dissolved in methyl alcohol (10%m/m, 5cm with N-(2,4-diamido-6-hydroxy pyrimidine-5-yl) benzamide 26 3) sodium methoxide in.With the gained potpourri in oil bath, reflux the heating 5h.Then reaction mixture is cooled to room temperature, adds entry (5cm 3), evaporate methyl alcohol with Rotary Evaporators.With glacial acetic acid obtained aqueous solution is acidified to pH 5, at this moment, solid is precipitated out from solution.With solid filtering and water (3 * 10cm 3) washing.On funnel, filtration cakes torrefaction is spent the night, obtain 2-amino-8-phenyl-9H-purine-6-alcohol 31 (0.78g, 60%) δ H(200MHz, D 2O) 7.96 (2H, d, J 7.0,2 * ArCH) and 7.28-7.46 (3H, m, 3 * ArCH).
Prepare following product in a similar manner:
2-amino-8-heptyl-9H-purine-6-alcohol (33)
(0.12g，46％)。δ H(200MHz, D 2O) 2.59 (2H, t, J7.7, ArCH 2), 1.52-1.70 (2H, m, ArCH 2CH 2), 1.08-1.35 (8H, m, 4 * CH 2) and 0.78 (3H, br s, CH 3).
2-amino-8-the tert-butyl group-9H-purine-6-alcohol (34)
(0.11g，53％)。δ H(200MHz，D 2O)1.30(9H，s，3×CH 3)。
The conventional method of the cyclisation of the acidylate pyrimidine that phosphorous oxychloride is regulated
6-chloro-8-phenyl-9H-purine-2-amine (35)
To be dissolved in phosphorous oxychloride (35cm 3) benzamide 26 (1.66g, 6.75mmol) the heating 18h that under nitrogen atmosphere, refluxes.Under vacuum, remove excessive solvent, trash ice is added residue, obtain the black suspension under the stirring.Solid is filtered, and make the filtrate alkalization with liquor ammoniae fortis.Form beige solid.With solid filtering, water, ethanol and washing with acetone, dry then, obtain 6-chloro-8-phenyl-9H-purine-2-amine 35 (0.39g, 24%).δ H(200MHz, d 6-DMSO) 8.02-7.95 and 7.62-7.38 (5H, 3 * m, aryl H), 7.12 (1H, br s, NH), 6.25 and 6.32 (2H, 2 * br s, NH 2); δ C(50MHz, d 6-DMSO) 161.2 (C-6), 156.4 (C-4), 154.2 (C-8), 153.4 (C-2), 130.13 (season aryl C), 129.13,128.7 and 125.9 (aryl CH) and 108.5 (C-5).
6-chloro-8-heptyl-9H-purine-2-amine (36)
According to above-mentioned conventional method, use POCl 3(35cm 3) (1.71g 6.38mmol), obtains 6-chloro-8-heptyl-9H-purine-2-amine 36 (0.19g, 11%) orange solids to handle acid amides 28.δ H(200MHz, d 6-DMSO) 6.39 (～1H, br s, NH), 1.12 (12H, br s, 6 * CH 2) and 0.83 (3H, br m, CH 3); δ C(50MHz, d 6-DMSO) 160.2 (C-6), 150.6 (C-8), 146.1 (C-4), 144.3 (C-2), 112.7 (C-5), 31.4,28.9,28.7,28.6,27.6 and 22.3 (6 * CH 2) and 13.8 (CH 3).
N-(4-amino-6-hydroxy pyrimidine-5-yl) benzamide (37)
According to the conventional method of above-mentioned acidylate, with chlorobenzoyl chloride (1.84cm 3, 15.85mmol) processing is dissolved in 2M NaOH aqueous solution (20cm 3) in diamines 10 (1.00g 7.93mmol), obtains N-(4-amino-6-hydroxy pyrimidine-5-yl) benzamide 37 (1.35g, 74%) yellow powder.Need not to characterize and directly to use.
N-(4-amino-6-hydroxy pyrimidine-5-yl) oleamide (38)
According to the conventional method of above-mentioned acidylate, with oleoyl chloride (5.24cm 3, 15.85mmol) processing is dissolved in 2M NaOH aqueous solution (20cm 3) in diamines 10 (1.00g 7.93mmol), obtains the brown jelly of N-(4-amino-6-hydroxy pyrimidine-5-yl) oleamide 38 (0.33g, 11%).Need not to characterize and directly to use.
N-(4-amino-6-hydroxy pyrimidine-5-yl) caprylamide (39)
According to the conventional method of above-mentioned acidylate, with caprylyl chloride (2.71cm 3, 15.88mmol) processing is dissolved in 2M NaOH aqueous solution (20cm 3) in diamines 10 (1.06g 8.43mmol), obtains N-(4-amino-6-hydroxy pyrimidine-5-yl) caprylamide 39 (0.16g, 8%) cream-coloured powder.Need not to characterize and directly to use.
8-heptyl-9H-purine-6-alcohol (40)
The above-mentioned conventional method of the closed loop of regulating according to sodium methoxide is with being dissolved in methyl alcohol (15cm 3) sodium methoxide (1.97g, (0.18g 0.72mmol), obtains 8-heptyl-9H-purine-6-alcohol 40 (0.13g, 77%) white powder 36.53mmol) to handle acid amides 39.δ H(200MHz, d 6-DMSO) 8.54 (～1H, br s, NH), 7.74 (～1H, br s, H-2), 6.02 (1H, br s, OH), 2.17-2.41,1.01-1.82 and 0.75-1.12[15H, 6 * m, (CH 2) 5CH 3]; δ C(50MHz, d 6-DMSO) 171.6 (C-6), 159.2 (C-8), 158.5 (C-4), 146.9 (C-2), 98.9 (C-5), 35.3,31.2,28.7,28.5,27.4,25.1 and 22.0 (6 * CH 2) and 13.9 (CH 3).
N-(6-amino-2,4-dihydroxy-pyrimidine-5-yl) benzamide (41)
Use the conventional method of above-mentioned acidylate, with 5, (0.50g 2.8mmol) is dissolved in 10% sodium hydrate aqueous solution (12cm to 6-diamido uracil hydrochloride 13 3) in, in ice bath, cool off, in 1.5h with two parts of chlorobenzoyl chloride (0.33cm 3, 2.8mmol) handle.Regulate pH to 7-8 with acetate, product is precipitated out from solution, collects and washes with water by filtering, and obtains N-(6-amino-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-yl) benzamide 41 yellow solids (0.68g, 99%).δ H(200MHz, d 6-DMSO) 10.37 (1H, s, NH), 10.19 (1H, s, NH), 8.79 (1H, s, NHC=O), 7.94-8.00 (2H, m, aryl H), 7.41-7.55 (3H, m, aryl H) and 6.10 (2H, br s, NH 2).
6-amino-5-{[(1E, 2E)-3-phenyl-2-allylidene] amino } pyrimidine-2,4-glycol (42)
With 5, (0.50g 2.8mmol) is dissolved in methyl alcohol (10cm to 6-diamido uracil hydrochloride 13 3), add cinnamic acid (0.35cm to it 3, reaction stirred 2h 2.8mmol) and at room temperature.Add entry, obtain viscous solid, grind this solid, collect by filtering then, obtain 6-amino-5-{[(1E with ether, 2E)-3-phenyl-2-allylidene] amino } pyrimidine-2,4-glycol 42 bright orange solids (0.64g, 89%).δ H(200MHz, d 6-DMSO) 10.92 (1H, s, NH), 10.76 (1H, s, NH), 9.44 and 9.69 (1H, 2 * d J 8, N=CH) and 7.10-7.80 (7H, m, aryl H, CH=CH); δ C(50MHz, d 6-DMSO) 159.9153.8,152.7 and 149.2 (C-2, C-4, C-5 and C-6), 136.3,130.5 and 131.9 (C=C and C=N), 129.8,129.4,129.2 and 128.3 (aryl C).
Xanthine (43)
With triethyl orthoformate (5cm 3), triethylamine (0.39cm 3, 2.8mmol) and N, dinethylformamide (2cm 3) heating 5 that refluxes, 6-diamido uracil hydrochloride 13 (0.50g, 2.8mmol) 19h.Solid matter is filtered and wash, obtain xanthine 43 beige solids (0.45g, 90%) with ether.δ H(200MHz, d 6-DMSO) 7.9 (1H, s, H-8); δ C(50MHz, d 6-DMSO) 156.2 (C-6), 152.1 (C-2), 149.5 (C-4), 141.2 (C-8) and 107.4 (C-5).
6-chloro-8-phenyl-9H-purine-2-alcohol (44)
(0.14g is 0.057mmol) at POCl for benzamide 41 with N-(6-amino-2,4-dihydroxy-pyrimidine-5-yl) 3(5cm 3) middle boiling reflux 4h.In fuming cupboard, remove excessive POCl3 by rotary evaporation, trash ice is added residue.By filtering collecting precipitation and washing with water, obtain 6-chloro-8-phenyl-9H-purine-2-alcohol 44 brown solids (0.17g, product add external phosphorus compound).δ H(200MHz, d 6-DMSO) 10.85 (1H, s, NH), 8.02-8.10 (2H, m, aryl H) and 7.38-7.60 (3H, m, aryl H); δ C(50MHz, d 6-DMSO) 156.0 (C-6), 152.0 (C-2), 150.6 and 150.2 (C-4 and C-8), 130.8,129.7,129.5 and 126.9 (aryl C) and 108.7 (C-5).
8-[(E)-the 2-styryl]-9H-purine-2,6-glycol (45)
With 6-amino-5-{[(1E, 2E)-3-phenyl-2-allylidene] amino } pyrimidine-2, (0.27g 1.1mmol) is dissolved in thionyl chloride (10cm to 4-glycol 42 3) and the heating 6.5h that refluxes, in course of reaction, carry out HPLC and analyze.After the raw material peak disappears, excessive thionyl chloride is removed in reactant cooling and decompression, is obtained crude product 8-[(E)-the 2-styryl]-9H-purine-2,6-glycol 45.δ C(50MHz, d 6-DMSO) 155.8 and 152.0 (C-6 and C-2), 150.0 (simultaneous C-8 and C-4), 136.1 and 135.6 (CH=CHPh and aryl C), 129.8,129.7 and 127.8 (aryl C), 116.4 (CH=CHPh) and 108.1 (C-5).
N-(6-amino-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-yl) benzamide (46)
With 5, the 6-diaminostilbene, (0.25g 0.99mmol) is suspended in 10% NaOH (4cm to 3-dimethyl uracil hydrosulfite 16 3) and in ice bath, be cooled to 0 ℃.Add chlorobenzoyl chloride (1 equivalent, 0.12cm to it 3), stir the chlorobenzoyl chloride that adds another part equivalent behind the 15min again.Making reactant be warming up to room temperature naturally and stir spends the night.Use the acetate acidified reaction mixture,, obtain N-(6-amino-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-yl) benzamide 46 yellow solids (0.10g, 37%) by filtering collecting precipitation and washing with water.δ H(200MHz, d 6-DMSO) 8.90 (1H, s, NHC=O), 7.95-8.02 (2H, m, aryl H), 7.42-7.55 (3H, m, aryl H), 3.35 (3H, s, CH 3N) and 3.15 (3H, s, CH 3N).
N-(6-amino-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-yl) caprylamide (47)
With 5, the 6-diaminostilbene, (0.25g 0.99mmol) is dissolved in pyridine (2cm to 3-dimethyl uracil hydrosulfite 16 3) and solution is cooled to 0 ℃.Add caprylyl chloride (1 equivalent, 0.17cm to it 3), make reactant be warming up to room temperature naturally and stir and spend the night.Add volume acetate seldom to it, reaction mixture distributes between ethyl acetate and water.Water layer removed and with twice of 1M salt acid elution organic layer.Use MgSO 4Dry organic layer also removes and desolvates, and obtains crude product N-(6-amino-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-yl) caprylamide 47 yellow solids (0.05g, 17%).δ H(200MHz, d 6-DMSO) 8.91 (1H, s, NHC=O), 8.24 and 6.47 (2H, 2 * s, NH 2), 3.31 (3H, s, CH 3N), 3.12 (3H, s, CH 3N), 2.14-2.30[2H, m, CH 3(CH 2) 5CH 2C=O], 1.40-1.61 and 1.17-1.38[10H, 2 * m, CH 3(CH 2) 5CH 2C=O] and 0.80-0.92[3H, m, CH 3(CH 2) 6].
1,3-dimethyl-8-phenyl-3,9-dihydro-1H-purine-2,6-diketone (48)
(0.10g 0.36mmol) is suspended in 2M NaOH (2cm with N-(6-amino-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-yl) benzamide 46 3) and methyl alcohol (1cm 3) in.With reactant boiling reflux 3h.All substances are all dissolved in course of reaction, then form white precipitate.Add entry (1cm with the reaction mixture cooling and to it 3), be acidified with acetic acid to pH 5 then.By filtering collecting precipitation and washing with water, obtain 1,3-dimethyl-8-phenyl-3,9-dihydro-1H-purine-2,6-diketone 48 white solids (0.06g, 65%).δ H(200MHz, d 6-DMSO) 8.12-8.19 (2H, m, aryl H), 7.48-7.57 (3H, m, aryl H), 7.28 (1H, s, NH), 3.52 (3H, s, CH 3N) and 3.28 (3H, s, CH 3N).
Another kind of preparation method:
Under 175 ℃, with 6-amino-1, (0.50g 2.7mmol) and uses concentrated hydrochloric acid (0.16g, the benzylamine (2cm that 4.3mmol) handled to 3-dimethyl-5-nitroso-uracil 15 3) heating 3h.Make the reactant cooling, make material curing and be suspended in ethanol (5cm 3).It is filtered and water (2cm 3) stir solids 2h.By solid collected by filtration and wash with water, obtain 48 white solids (0.12g, 17%).
1,3-dimethyl-8-heptyl-3,9-dihydro-1H-purine-2,6-diketone (49)
(0.29g is 0.99mmol) at 2M NaOH (3cm with N-(6-amino-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-yl) caprylamide 47 3) and methyl alcohol (1.5cm 3) middle boiling reflux 4h.After the cooling, add entry (1cm earlier to it 3) add several acetate again.The gained white precipitate is filtered, and water and washing with acetone, obtain 1,3-dimethyl-8-heptyl-3,9-dihydro-1H-purine-2,6-diketone 49 white solids (0.19g, 70%).δ H(200MHz, d 6-DMSO) 3.41 (3H, s, CH 3N), 3.22 (3H, s, CH 3N), 1.60-1.73 and 1.17-1.35[12H, 2 * m, CH 3(CH 2) 6] and 0.82-0.90[3H, m, CH 3(CH 2) 6].
4,5, the amidated conventional method of 6-Triaminopyrimidine:
N-(4,6-di-amino-pyrimidine-5-yl) benzamide (50)
Make 4,5, (0.36g 2.88mmol) becomes slurry to 6-Triaminopyrimidine 5 in tetrahydrofuran/water [1:1 (v/v), 30cm3], and (1.2 μ l 8.63mmol) handle with triethylamine.Potpourri is cooled to 0 ℃ in ice bath, dropwise adds to it and be dissolved in tetrahydrofuran (5cm 3) chlorobenzoyl chloride (0.33cm 3, 2.88mmol) in.After adding, spend the night after potpourri is warming up to room temperature naturally.Decompression is removed tetrahydrofuran and with the gained sedimentation and filtration, is obtained N-(4,6-di-amino-pyrimidine-5-yl) benzamide 50 (97mg, 15%).δ H(200MHz, d 6-DMSO) 9.20 (1H, br s, NH), 8.03 (2H, dd, J 8.2 and 1.8,2 * aryl CH), 7.82 (1H, s, H-2), 7.44-7.56 (3H, m, 3 * aryl CH), 6.23 (2H, br s, NH 2) and 6.04 (2H, s, NH 2); δ C(50MHz, d 6-DMSO) 166.3 (PhCONH), 160.5 (C-4 and C-6), 156.1 (C-2), 135.1 (aryl C), 131.8,128.9 and 128.6 (aryl CH) and 96.1 (C-5).ES-MS m/z 230.1 (M+H), 212.1 and 104.9.
Prepare following compound with above-mentioned conventional method:
N-(4,6-di-amino-pyrimidine-5-yl) oleamide (51)
(0.26g，17％)。δ H(200MHz, d 6-DMSO) 8.55 (1H, br s, NH), 7.76 (1H, s, H-2), 5.77 (4H, br s, 2 * NH 2), (CH=CH), 2.32 (J 7.5, NHCOCH for 2H, t for 2H, m for 5.25-5.38 2), 1.88-2.09 (4H, m, 2 * CH 2CH=C), 1.48-1.63 (2H, m, CH 2), 1.15-1.46 (20H, m, 10 * CH 2) and 0.86 (J 6.6, CH for 3H, t 3); δ C(50MHz, d 6-DMSO) 173.0 (CONH), 159.5 (C-4 and C-6), 154.4 (C-2), 130.3 (CH=CH), 96.2 (C-5), 35.9,32.0,29.8,29.6,29.3,27.3 and 25.4 (7 * CH 2), 22.8 (CH 2CH 3) and 14.6 (CH 3).
N-(4,6-di-amino-pyrimidine-5-yl) caprylamide (52)
(0.37g，36％)。δ H(200MHz, d 6-DMSO) 8.59 (1H, br s, NH), 7.82 (1H, s, H-2), 5.99 (4H, br s, 2 * NH 2), 2.33 (J 7.4, NHCOCH for 2H, t 2), 1.50-1.68 (2H, m, CH 2), 1.13-1.40 (8H, m, 4 * CH 2) and 0.84 (J 6.6, CH for 3H, t 3); δ C(50MHz, d 6-DMSO) 173.0 (CONH), 158.2 (C-4 and C-6), 151.8 (C-2), 95.8 (C-5), 36.0 (COCH 2), 31.9,29.6,29.3,25.4 and 22.8 (5 * CH 2) and 14.6 (CH 3); (ES) M/zM/z 252.2 (M+H), 234.2 and 126.0.
4,5 of sodium methoxide adjusting, the conventional method of the closed loop of 6-Triaminopyrimidine:
8-phenyl-9H-purine-6-amine (53)
To be dissolved in methyl alcohol (0.5cm 3) N-(4,6-di-amino-pyrimidine-5-yl) benzamide 50 (0.17g, 0.72mmol) add sodium methoxide (25%, 6cm 3) methanol solution in.With in nitrogen atmosphere, under this temperature, reflux heating and stir 16h of potpourri.Potpourri is cooled to room temperature, and regulates pH to 4 with the 1M aqueous hydrochloric acid solution.Form precipitation,, obtain 8-phenyl-9H-purine-6-amine 53 (0.12g, 77%) this sedimentation and filtration and dry.δ H(200MHz, d 6-DMSO) 9.13 (2H, br s, NH 2), 8.55 (1H, s, H-2), 8.20-8.25 (2H, m, 2 * aryl CH) and 7.60-7.63 (3H, m, 3 * aryl CH).δ C(50MHz, d 6-DMSO) 153.5 (C-6), 152.1 (C-4), 150.8 (C-8), 146.3 (C-2), 132.2 and 130.0 (each aryl CH), 128.7 (aryl C), 127.5 (aryl CH) and 110.1 (C-5) .ES-MSm/z, 212.2 (M+H).
8-heptyl-9H-purine-6-amine (54)
According to above-mentioned conventional method preparation, obtain (82mg, 65%).δ H(200MHz, d 6-DMSO) 8.87 (2H, br s, NH 2), 8.46 (H-2), 2.87 (J 7.6, CH for 2H, t for 1H, s 2(CH 2) 5CH 3), 1.70-1.87 (2H, m, CH 2), 1.17-1.23 (8H, m, 4 * CH 2) and 0.86 (J 6.6, CH for 3H, t 3); δ C(50MHz, d 6-DMSO) 157.7 (C-6), 151.3 (C-4), 150.2 (C-8), 145.4 (C-2), 110.0 (C-5), 31.8,29.3,29.2,29.0,27.7 and 22.8 (6 * CH 2) and 14.6 (CH 3).
2-amino-6-hydroxyl-9H-purine-8-aminocarbamic acid methyl esters (55)
To be dissolved in water (2cm 3) the S-methyl-isourea (0.97g, 3.48mmol) and methylchloroformate (0.36cm 3, potpourri 4.7mmol) is cooled to 0 ℃ in ice bath, and regulates pH to 8.0 by dropwise adding 25% sodium hydrate aqueous solution.After this 10min that stirs the mixture under this temperature, recalls to 5.0 with glacial acetic acid with pH.Add earlier to it then and be in water (2cm 3) in slurry 2,4,5-triamido-6-hydroxy pyrimidine 2 (1.00g, 4.18mmol), add again the solid sodium acetate (0.29g, 4.18mmol).Under 85 ℃, reaction mixture is heated 1.5h.After this, potpourri is cooled to room temperature, product is filtered and vacuum drying, obtain 2-amino-6-hydroxyl-9H-purine-8-aminocarbamic acid methyl esters 55 (0.90g, 96%).δ H(200MHz，D 2O)3.61(3H，s，CO 2CH 3)。
4, the conventional method (56) of 6-dichloro pyrimidine monoamineization
To be dissolved in the solution boiling reflux 1-5h of 56 in the isopropyl alcohol (1.0M) that contains amine (2.1 equivalent) or amine (1.2 equivalent) and the potpourri of triethylamine (1.2 equivalent), until exhausting all raw materials.Solvent is removed under vacuum, the gained stickum is distributed between water and ethyl acetate or methylene chloride.Go forward side by side column criterion preparation of extraction obtains pure product by column chromatography then.
6-chloro-N-methylpyrimidine-4-amine (57)
According to above-mentioned conventional method, with methylamine hydrochloride (5.06g, 74.97mmol) and triethylamine (10.3cm 3, 73.8mmol) processing is dissolved in isopropyl alcohol (168cm 3) in 56 (10.03g, 67.12mmol), be prepared and use 1:10 (v/v) ethyl acetate then: hexane carries out column chromatography as eluent to be separated, and obtains white crystalline solid 6-chloro-N-methylpyrimidine-4-amine 57 (7.26g, 75.2%, measuring its purity by HPLC is 99.9%).δ H(200MHz, CDCl 3) 8.33 (and 1H, s, H-2), 6.35 (1H, s, H-5), 5.72 (1H, br s, NH) and 2.94 (3H, d, NHMe, J5.2); δ C(50MHz, CDCl 3) 164.0 (C-6), 160.8 (C-4), 158.2 (C-2), 101.3 (br, C-5) and 28.2 (NHMe); t R3.10min (50% methyl alcohol: the 25mM ammonium acetate); (ES) m/z 116 (93), 119 (32), 144 (100, M +, C 5H 6 35ClN 3Requirement is 144) and 146 (42, M +.C 5H 6 37ClN 3Requirement is 146).
N-benzyl-6-chlorine pyrimidine-4-amine (58)
Use above-mentioned conventional method, with benzylamine (8.07cm 3, 73.84mmol) and triethylamine (10.3cm 3, 73.8mmol) processing is dissolved in isopropyl alcohol (168cm 3) in 56 (10.04g, 67.36mmol), be prepared and use 1:10 (v/v) ethyl acetate then: hexane carries out column chromatography as eluent to be separated, and obtains orange solids N-benzyl-6-chlorine pyrimidine-4-amine 58 (12.66g, 85.5%, measuring it by HPLC is 99.7%).δ H(200MHz, CDCl 3) 8.09 (1H, s, H-2), 7.19-7.43 (5H, m, aryl H), 6.42 (1H, br s, NH), 6.36 (1H, s, H-5) and 4.50 (2H, d, PhCH 2, J 5.4); δ C(50MHz, CDCl 3) 163.2 (C-6), 159.7 (C-4), 158.2 (C-2), 136.8 (season aryl C), 128.8,127.9 and 127.4 (aryl C), 101.9 (br, C-5) and 45.6 (PhCH 2); t R14.28min (50% methyl alcohol: the 25mM ammonium acetate); (ES) m/z141 (10), 201 (12), 220 (100, M +.C 11H 10 35ClN 3Requirement is 220), 221 (15) and 222 (30, M +.C 11H 10 37ClN 3Requirement is 222).
2-[(6-chlorine pyrimidine-4-yl) amino] ethanol (59)
Use above-mentioned conventional method, with monoethanolamine (4.25cm 3, 70.48mmol) and triethylamine (10.3cm 3, 73.8mmol) processing is dissolved in isopropyl alcohol (168cm 3) in 56 (10.08g, 67.64mmol), be prepared and use 1:10 (v/v) ethyl acetate then: hexane carries out column chromatography as eluent to be separated, and obtains beige solid 2-[(6-chlorine pyrimidine-4-yl) amino] ethanol 59 (9.19g, 78.3%, measuring its purity by HPLC is 98.9%).δ H(200MHz, CDCl 3+ d 6-DMSO) 8.22 (1H, s, H-2), 6.45 (1H, br s, NH), 6.35 (1H, s, H-5), 3.70 (2H, t, OCH 2, J5.0) with 3.41 (3H, br s, OH and NHCH 2); δ C(50MHz, CDCl 3+ d 6-DMSO) 163.8 (C-6), 158.4 (C-2 and C-4), 103.3 (br, C-5), 61.1 (OCH 2) and 44.2 (NHCH 2); t R2.40min (50% methyl alcohol: the 25mM ammonium acetate); (ES) m/z110 (64), 128 (100), 129 (43), 130 (32), 132 (17), 156 (100, M +-H 2O), 158 (36, M +-H 2O), 174 (68, M +.C 6H 8 35ClN 3It is 174 that O requires) and 176 (23, M +.C 6H 8 37ClN 3It is 176 that O requires).
(2R)-and 2-[(6-chlorine pyrimidine-4-yl) amino]-3-phenylpropionic acid methyl esters (60)
Use above-mentioned conventional method, with the L-phenylalanine methyl ester hydrochloride (by being dissolved in methyl alcohol (75cm 3) L-phenylalanine (12.32g, 74.57mmol) prepared fresh obtains, this methyl alcohol was handled with hydrogen chloride gas) and triethylamine (19.6cm 3, 0.14mol) handle (168cm that is dissolved in isopropyl alcohol 3) 56 (10.04g, 67.38mmol), be prepared and use 1:10 (v/v) ethyl acetate then: hexane carries out column chromatography as eluent, obtain orange (2R)-2-[(6-chlorine pyrimidine radicals-4-yl) amino]-3-phenylpropionic acid methyl esters 60 (2.08g, 11.0%, measuring its purity by HPLC is 99.5%).δ H(200MHz, CDCl 3) 8.35 (1H, s, H-2), 7.01-7.35 (5H, m, aryl H), 6.36 (1H, s, H-5), 5.79 (1H, br d, NH, J7.6), 4.68-5.21 (1H, br m, CHCO), 3.75 (3H, s, OCH 3) 3.24 (1H, dd, PhCH aH b, J 5.6 and 13.8) and 3.41 (1H, dd, PhCH aH b, J6.4 and 14.0); δ C(50MHz, CDCl 3) 172.0 (CO), 162.1 (C-6), 159.1 (C-4), 158.3 (C-2), 135.5 (season aryl C), 129.1,128.5 and 127.1 (aryl C), 103.9 (br, C-5), 54.6 (OCH 3), 52.3 (PhCH 2) and 37.8 (CHCO); t R(1.87min methyl alcohol); (ES) m/z 120 (13), 169 (17), 205 (21), 232 (100, M +-CO 2Me), 233 (28), 234 (73, M +-CO 2Me), 292 (45, M +.C 14H 14 35ClN 3O 2Requirement is 292) and 294 (18, M +.C 14H 14 37ClN 3O 2Requirement is 294).
N-(4-bromophenyl)-6-chlorine pyrimidine-4-amine (61)
Use above-mentioned conventional method, with the 4-bromaniline (12.10g, 69.50mmol) and triethylamine (10.3cm 3, 73.8mmol) processing is dissolved in isopropyl alcohol (168cm 3) 56 (9.98g, 67.01mmol).After the heating, add isopyknic water and ethyl acetate, the gained suspension is filtered, obtain cream-coloured powder N-(4-bromophenyl)-6-chlorine pyrimidine-4-amine 61 (12.73g 67.0%, measures its purity by HPLC〉99.9%) to it.δ H(200MHz, CDCl 3) 9.11 (and 1H, br s, H-2), 8.05 (1H, s, NH), 7.17 (aryl H, J 8.8 for 2H, d), 7.05 (2H, d, aryl H, J9.0) and 6.40 (1H, s, H-5); δ C(50MHz, CDCl 3+ d 6-DMSO) 169.4 (C-6), 161.9 (C-4), 158.1 (C-2), 137.6 (season aryl C), 131.7 and 122.7 (aryl C), 116.3 (season aryl C) and 104.6 (C-5); t R(2.15min methyl alcohol); (ES) m/z 204 (80), 205 (100, M +-Br), 206 (42), 207 (35), 210 (10), 213 (10), 259 (10), 284 (78, M +, C 10H7 79Br 35ClN 3Requirement is 284), 286 (100, M +.C 10H 7 81Br 35ClN 3Requirement is 286) and 288 (29).
6-chloro-N-methyl-5-nitroso-pyrimidine-4-amine (62)
According to above-mentioned conventional method, in 30min, will be dissolved in water (84cm 3) sodium nitrite (6.31g 91.38mmol) dropwise adds to handle and is dissolved in acetate (25cm 3) chloride 57 (7.26g, 50.58mmol).After 2h forms solid, solid is separated, obtain yellow powder 6-chloro-N-methyl-5-nitroso-pyrimidine-4-amine 62 (7.68g, 88%, measuring its purity by HPLC is 97.4%).δ H(200MHz, CDCl 3) 8.83 (and 1H, s, H-2), 8.06 (1H, s, NH) and 3.45 (3H, s, NHMe); δ C(50MHz, CDCl 3) 161.9 (C-4 and C-6), 158.4 (C-2), 107.9 (C-5) and 27.6 (NHMe); t R9.10min (50% methyl alcohol: the 25mM ammonium acetate); (ES) m/z 130 (18, C 4H 4 35ClN 3), 143 (39), 144 (100, M +-NO), 145 (21), 146 (33, M +-NO) and 173 (1, M +.C 5H 5 35ClN 4It is 173 that O requires).
N-benzyl-6-chloro-5-nitroso-pyrimidine-4-amine (63)
Use above-mentioned conventional method, in 30min, will be dissolved in water (95cm 3) sodium nitrite (7.11g 0.10mol) dropwise adds to handle and is dissolved in concentrated hydrochloric acid (28cm 3) chloride 58.Be settled out solid through 18h, this solid is separated, obtain oldlace powder N-benzyl-6-chloro-5-nitroso-pyrimidine-4-amine 63 (12.75g, 90.2%, measuring its purity by HPLC is 96.3%).δ H(200MHz, CDCl 3) 8.91 (1H, br s, H-2), 8.08 (1H, s, NH), 7.29 (5H, br s, aryl H) and 5.37 (2H, s, PhCH 2); δ C(50MHz, CDCl 3) 162.1 (C-4 and C-6), 158.4 (C-2), 134.2 (season aryl C), 128.6,128.3 and 127.9 (aryl C), 108.0 (C-5) and 43.6 (PhCH 2); t R(2.12min methyl alcohol); (ES) m/z106 (12), 218 (80, C 11H 9 35ClN 3), 219 (65), 220 (100, C 11H 9 37ClN 3), 221 (26) and 222 (23).There is not M +(C 11H 9 35ClN 4It is 249 that O requires).
2-[(6-chloro-5-nitroso-pyrimidine-4-yl) amino] ethanol (64)
Use above-mentioned conventional method, in 30min, will be dissolved in water (132cm 3) sodium nitrite (6.65g 96.32mol) dropwise adds to handle and is dissolved in acetate (26cm 3) chloride 59 (9.19g, 52.96mmol).In 18h, form solid, this solid is separated and use ethyl acetate extraction solution.Wash organic phase with the 2M aqueous sodium hydroxide washes, partial concentration is an orange.This grease separated out and merge, obtain light orange powder 2-[(6-chloro-5-nitroso-pyrimidine-4-yl with the solid that filters out) amino] ethanol 64 (8.54g, 79.6%, measuring its purity by HPLC is 86.0%).δ H(200MHz, CDCl 3) 8.86 (1H, br s, H-2), 8.05 (1H, s, NH), 4.36 (2H, t, OCH 2, J5.5), 3.73 (2H, t, NHCH 2, J5.6) and 2.25 (1H, br s, OH); δ C(50MHz, CDCl 3) 162.5 (C-6), 158.2 (C-2 and C-4), 108.1 (C-5), 59.6 (OCH 2) and 42.7 (NHCH 2); t R(4.52min methyl alcohol); (ES) m/z 130 (24), 142 (95), 143 (100, C 5H 6 35ClN 3), 144 (36), 145 (32), 156 (10, C 6H 7 35ClN 3), 172 (29), 174 (64, C 6H 8 37ClN 3O), 176 (16) and 203 (13, M +.C 6H 7 35ClN 4O 2Requirement is 203).
The nitrosifying conventional method of pyrimidine
Dropwise handle the pyrimidine solution that is dissolved in acetate or hydrochloric acid (2M) with the sodium nitrite that is dissolved in water (6.3M) (1.8 equivalent) solution.In adition process, produce brown gas, and pass formation solid precipitation in time.With solid filtering, wash with water and carry out suction dried.
N, N, N '-trimethyl-5-nitroso-pyrimidine-4,6-diamines (65)
Dropwise add dimethylamine (33% alcoholic solution, 1.74cm 3, 12.75mmol) handle and be dissolved in methylene chloride (2.9cm 3) pyrimidine 62 (0.97g, potpourri 5.65mmol) cause spontaneous heating.18h at room temperature stirs the mixture.Use 1:10-3:10 (v/v) ethyl acetate: hexane carries out the column chromatography purifying as eluent to resulting orange solution, obtains yellow solid N, N, N '-trimethyl-5-nitroso-pyrimidine-4,6-diamines 65 (0.60g, 59.0%).δ H(200MHz, CDCl 3) 8.52 (1H, s, H-2), 7.02 (1H, s, NH), 3.47 (3H, s, NCH 3) and 3.18[H, s, N (CH 3) 2]; δ C(50MHz, CDCl 3) 163.1 (C-6), 160.1 (C-4), 157.4 (C-2), 87.4 (C-5), 37.5[N (CH 3) 2] and 28.0 (NHCH 3).
N-benzyl-N ', N '-dimethyl-5-nitroso-pyrimidine-4,6-diamines (66)
Use is similar to 65 method, and (be dissolved in the alcohol, concentration is 33%, 1.00cm dropwise to add dimethylamine 3, 7.32mmol) handle and be dissolved in methylene chloride (1cm 3) pyrimidine 63 (0.51g, potpourri 2.04mmol) cause spontaneous heating.Potpourri is at room temperature stirred 2h.Use 1:10-3:10 (v/v) ethyl acetate: hexane is as eluent, resulting orange solution is carried out the column chromatography purifying, obtain light yellow solid 4-benzyl amino-6-dimethylamino-5-nitroso-pyrimidine 66 (0.48g, 90.7%, measuring its purity by HPLC is 99.6%).δ H(200MHz, CDCl 3) 8.57 (1H, s, H-2), 7.21-7.31 (5H, m, aryl H), 7.05 (1H, s, NH), 5.39 (2H, s, PhCH 2) and 3.18[6H, s, N (CH 3) 2]; δ C(50MHz, CDCl 3) 163.2 (C-6), 159.9 (C-4), 157.5 (C-2), 135.1 (season aryl C), 128.3,128.2 and 127.4 (aryl C), 87.6 (C-5), 43.7 (PhCH 2) and 37.5[N (CH 3) 2]; t R(2.17min methyl alcohol); (ES) m/z 105 (12), 123 (31), 151 (11), 199 (12), 227 (94), 228 (100, C 13H 16N 4), 229 (83) and 258 (1, MH +.C 13H 16N 5It is 258 that O requires).
2-{[6-(dimethylamino)-5-nitroso-pyrimidine-4-yl] amino } ethanol (67)
Use is similar to 65 method, and (be dissolved in the alcohol, concentration is 33%, 1.48cm dropwise to add dimethylamine 3, 10.86mmol) handle and be dissolved in methylene chloride (2.5cm 3) pyrimidine 64 (1.02g, potpourri 5.03mmol) cause spontaneous heating.Potpourri is at room temperature stirred 18h.Use 1:10-3:10 (v/v) ethyl acetate: hexane is as eluent, resulting orange solution is carried out the column chromatography purifying, obtain yellow solid 2-{[6-(dimethylamino)-5-nitroso-pyrimidine-4-yl] amino } ethanol 67 (0.76g, 72.0%, measuring its purity by HPLC is 99.5%).δ H(200MHz, CDCl 3) 8.48 (1H, s, H-2), 6.97 (1H, s, NH), 4.30 (2H, t, OCH 2, J4.9), 3.69 (2H, t, NHCH 2, J4.9) and 3.19[6H, s, N (CH 3) 2]; δ C(50MHz, CDCl 3) 163.1 (C-6), 159.9 (C-4), 157.0 (C-2), 88.0 (C-5), 60.3 (OCH 2), 44.1 (NHCH 2) and 37.5[N (CH 3) 2]; t R3.87min (50% methyl alcohol: the 25mM ammonium acetate); (ES) m/z 151 (100, C 7H 11N 4), 152 (14), 182 (10, C 8H 14N 4O) and 183 (25).There is not M +(C 8H 13N 5O 2Requirement is 211).
N-methyl-5-nitroso--6-pyrrolidine-1-yl pyrimidines-4-amine (68)
With pyrimidine 62 (0.97g, 5.61mmol) and pyrrolidine (1.16cm 3, pure mixture heated to 150 13.91mmol) ℃, and continue heating 1h.Use 1:10-3:10 (v/v) ethyl acetate: hexane is as eluent, resulting dark solution is carried out the column chromatography purifying, obtain yellow solid N-methyl-5-nitroso--6-pyrrolidine-1-yl pyrimidines-4-amine 68 (0.97g, 83.1%, measuring its purity by HPLC is 99.9%).δ H(200MHz, CDCl 3) 8.44 (1H, s, H-2), 6.80 (1H, s, NH), 3.48[4H, br s, N (CH 2) 2], 3.40 (3H, s, NHCH 3) and 1.78-2.13[4H, br m, (CH 2) 2]; δ C(50MHz, CDCl 3) 160.7 (C-6), 159.5 (C-4), 157.8 (C-2), 88.1 (C-5), 46.5[N (CH 2) 2], 27.8 (NHCH 3) and 25.1[(CH 2) 2]; t R11.57min (50% methyl alcohol: the 25mM ammonium acetate); (ES) m/z 123 (12), 149 (15), 150 (51), 177 (49, M +-NO), 178 (89, MH +-NO), 179 (100), 180 (10) and 208 (1, MH +.C 9H 13N 5It is 208 that O requires).
N-benzyl-5-nitroso--6-pyrrolidine-1-yl pyrimidines-4-amine (69)
Dropwise add pyrrolidine (0.34cm 3, 4.02mmol) processing is dissolved in methylene chloride (1cm 3) pyrimidine 63 (0.50g, potpourri 2.00mmol) cause spontaneous heating.Potpourri is at room temperature stirred 10min.Use 1:10-3:10 (v/v) ethyl acetate: hexane is as eluent, resulting brown solution is carried out the column chromatography purifying, obtain light yellow solid N-benzyl-5-nitroso--6-pyrrolidine-1-yl pyrimidines-4-amine 69 (0.53g, 94.1%, measuring its purity by HPLC is 96.6%).δ H(200MHz, CDCl 3) 8.56 (1H, s, H-2), 7.15-7.31 (5H, m, aryl H), 6.90 (1H, s, NH), 5.39 (2H, s, PhCH 2), 3.53[4H, br s, N (CH 2) 2] and 1.87-2.21[4H, br m, (CH 2) 2]; δ C(50MHz, CDCl 3) 160.9 (C-6), 159.4 (C-4), 157.7 (C-2), 135.1 (season aryl C), 128.3,128.1 and 127.3 (aryl C), 88.5 (C-5), 46.7[N (CH 2) 2], 43.6 (PhCH 2) and 25.2[(CH 2) 2]; t R(2.28min methyl alcohol); (ES) m/z 149 (17), 226 (22), 253 (78, M +-NO), 254 (100, MH +-NO), 255 (86), 256 (14) and 284 (1, MH +.C 15H 18N 5It is 284 that O requires).
2-[(5-nitroso--6-pyrrolidine-1-yl pyrimidines-4-yl) amino] ethanol (70)
Use is similar to 68 method, with pyrimidine 64 (0.96g, 4.76mmol) and pyrrolidine (0.98cm 3, pure mixture heated to 150 11.85mmol) ℃, and continue heating 1h.Use 1:10-3:10 (v/v) ethyl acetate: hexane is as eluent, resulting dark solution is carried out the column chromatography purifying, obtain beige solid 2-[(5-nitroso--6-pyrrolidine-1-yl pyrimidines-4-yl) amino] ethanol 70 (0.41g, 36.6%, it is 99.7% that HPLC measures its purity by HPLC).δ H(200MHz, CDCl 3) 8.49 (1H, s, H-2), 6.84 (1H, s, NH), 4.31 (2H ,～t, OCH 2, J4.8), 3.70 (2H ,～t, NHCH 2, J4.9), 3.11-3.85[4H, br m, N (CH 2) 2] and 1.82-2.23[4H, br m, (CH 2) 2]; δ C(50MHz, CDCl 3) 160.9 (C-6), 159.5 (C-4), 157.3 (C-2), 89.0 (C-5), 60.4 (OCH 2), 46.8[br, N (CH 2) 2], 44.1 (NHCH 2) and 25.3[br, (CH 2) 2]; t R6.43min (50% methyl alcohol: the 25mM ammonium acetate).
N-benzyl-6-morpholine-4-base-5-nitroso-pyrimidine-4-amine (71)
Use is similar to 66 method, and (1.23g 14.1mmol) handles and is dissolved in dichloromethane solution (10cm dropwise to add morpholine 3) pyrimidine 58 (1.46g, potpourri 5.87mmol) cause spontaneous heating.Potpourri at room temperature stirred spend the night.Pour the gained deep yellow solution into ethyl acetate (20cm 3) and use the equal-volume water washing.Solvent removed obtain viscous solid, in hexane, grind this solid.By filter collecting yellow solid N-benzyl-6-morpholine-4-base-5-nitroso-pyrimidine-4-amine 71[1.42g, 85%, at 1:1 (v/v) ethyl acetate: the R in the hexane fBe 0.43].δ H(200MHz, CDCl 3) 8.60 (1H, s, H-2), 7.26 (5H, br s, aryl H), 7.14 (1H, s, NH), 5.39 (2H, s, PhCH 2), 3.80 (4H, m, 2 * OCH 2) and 3.72 (4H, m, 2 * NCH 2); δ C(50MHz, CDCl 3) 163.5 (C-6), 161.0 (C-4), 157.5 (C-2), 135.0 (season aryl C), 128.7,128.5 and 127.7 (aryl C), 87.8 (C-5), 66.5 (2 * OCH 2), 45.0 (2 * NCH 2) and 43.5 (PhCH 2).
The conventional method of nitroso-reduction:
N 4-benzyl-6-pyrrolidine-1-yl pyrimidines-4,5-diamines (76)
(0.41g 1.45mmol) is suspended in water (1.45cm with N-benzyl-5-nitroso--6-pyrrolidine-1-yl pyrimidines-4-amine 69 3) in, (0.53g 3.04mmol) handles with a part of solid sodium hydrosulfite that adds.To its dropwise add aqueous sulfuric acid (50%v/v, 4.08g), under 130 ℃ with gained potpourri agitating heating 5min.After this, reaction mixture becomes colourless, cools off in ice.With the 2M sodium hydrate aqueous solution potpourri pH is transferred to pH〉10, and with methylene chloride (3 * 50cm 3) extract.Organic extract concentrating under reduced pressure with merging goes out crude product from the dichloromethane/hexane recrystallization, obtains N 4-benzyl-6-pyrrolidine-1-yl pyrimidines-4,5-diamines 76 (0.34g, 87%).δ H(200MHz, CDCl 3) 8.10 (1H, s, H-2), 7.25-7.35 (5H, m, aryl H), 5.35 (1H, br s, NH 2), 5.14 (1H, s, NH 2), 4.41 (2H, d, J5.8, CH 2Ph), 3.36 (4H, br s, 2 * CH 2N) and 1.90-1.96 (4H, m, 2 * CH 2CH 2N); δ C(50MHz, CDCl 3) 162.5 (C-4) a, 160.9 (C-6) a, 157.9 (C-2), 138.7 (season aryl C), 128.7,127.6 and 127.5 (aryl C), 80.9 (C-5), 46.4 (2 * CH 2N), 46.1 (CH 2Ph) and 25.5 (2 * CH 2CH 2N); (ES) m/z 255.2[(M-N)+and H], 228.2,163.1 and 138.0.
Use the following compound of this method preparation:
N 4, N 4, N 6-trimethyl pyrimidine-4,5,6-triamine (72)
(31mg，81％)。δ H(200MHz, CDCl 3) 8.16 (1H, s, H-2), 5.28 (1H, s, NH 2), 4.82 (1H, br s, NH 2), 3.09[6H, s, N (CH 3) 2] and 2.89[H, d, J 5.4, N (CH 3) 2].
N 6-benzyl-N 4, N 4-dimethyl pyrimidine-4,5,6-triamine (73)
(91.9mg，80％)。δ H(200MHz, CDCl 3) 8.16 (1H, s, H-2), 7.26-7.35 (5H, m, aryl H), 5.30 (1H, s, NH 2), 5.10 (1H, br s, NH 2), 4.45 (J 5.8, CH for 2H, d 2Ph), 3.01 (6H, s, 2 * NCH 3) and 1.78 (1H, br s, NH); δ C(50MHz, CDCl 3) 163.0 (C-4 and C-6), 157.6 (C-2), 138.7 (season aryl C), 128.8 and 127.5 (aryl C), 80.2 (C-5), 46.0 (CH 2Ph) and 37.4 (2 * NCH 3); (ES) m/z 229.2[(M-N)+and H], 202.1,137.1 and 138.0.
2-[5-amino-6-(dimethylamino) pyrimidine-4-yl] ethylaminoethanol (74)
(0.29g，54％)。δ H(200MHz, CDCl 3) 8.15 (1H, s, H-2), 5.36 (1H, s, NH 2), 5.00 (1H, br s, NH 2), 3.80 (J 4.9, CH for 2H, t 2OH), 3.42-3.50 (2H, m, CH 2NH), 3.05 (6H, s, 2 * NCH 3) and 2.87 (1H, brs, NH); δ C(50MHz, CDCl 3) 162.9 (C-4) a, 162.0 (C-6) a, 157.3 (C-2), 80.9 (C-5), 62.9 (CH 2OH), 44.8 (CH 2NH) and 37.5 (2 * NCH 3); (ES) m/z 183.1[(M-N)+and H], 165.1,139.0 and 110.9.
N 4-methyl-6-pyrrolidine-1-yl pyrimidines-4,5-diamines (75)
(0.66g，86％)。δ H(200MHz, CDCl 3) 8.11 (1H, s, H-2), 5.13 (1H, s, NH 2), 4.76 (1H, br s, NH 2), 3.36-3.48 (4H, m, 2 * CH 2N), 2.85 (J 5.4, NCH for 3H, d 3) and 1.94-2.00 (4H, m, 2 * CH 2CH 2N); δ C(50MHz, CDCl 3) 163.4 (C-4) a, 161.8 (C-6) a, 157.8 (C-2), 79.9 (C-5), 46.5 (2 * CH 2N), 28.7 (NCH 3) bWith 25.5 (2 * CH 2CH 2N) b(ES) m/z 179.1[(M-N)+and H], 137.0 and 120.9.
2-[(5-amino-6-pyrrolidine-1-yl pyrimidines-4-yl) amino] ethanol (77)
(0.23g，71％)。δ H(200MHz, CDCl 3) 8.12 (1H, s, H-2), 5.22 (1H, s, NH 2), 5.06 (1H, br s, NH 2), 3.79 (J 4.9, CH for 2H, t 2OH), 3.30-3.48 (7H, m, CH 2NH, 2 * CH 2N and NH) and 1.94-2.01 (4H, m, 2 * CH 2CH 2N); δ C(50MHz, CDCl 3) 162.6 (C-4) a, 160.7 (C-6) a, 157.5 (C-2), 81.4 (C-5), 62.5 (CH 2OH), 46.5 (2 * CH 2N), 44.7 (CH 2NH) and 25.5 (2 * CH 2CH 2N); (ES) m/z 209.1[(M-N)+and H], 191.1,182.1,165.1 and 138.0.
N 4-benzyl-6-morpholine-4-yl pyrimidines-4,5-diamines (78)
Use is similar to 76 method, and (1.35g 4.63mmol) is suspended in water (50cm with nitroso compound 71 3) in, (1.70g 9.74mmol) handles with a part of solid sodium hydrosulfite that adds.In 3min, with aqueous sulfuric acid (50%w/w 9.09g) dropwise adds, with the gained potpourri at 140 ℃ of following agitating heating 5min.After this, reaction mixture becomes colourless, makes it be cooled to 40 ℃.With the 2M sodium hydrate aqueous solution potpourri pH is transferred to pH〉10, with ethyl acetate (2 * 50cm 3) extraction gained solution.Organic extract concentrating under reduced pressure with merging goes out crude product from the dichloromethane/hexane recrystallization, obtains N 4-benzyl-6-morpholine-4-yl pyrimidines-4,5-diamines 87 (0.90g, 70%) white solid.δ H(200MHz, CDCl 3) 8.20 (1H, s, H-2), 7.35 (5H, br s, aryl H), 5.41 (1H, s, NH), 5.35 (br, NH 2), 4.48 (2H, d, J 5.8Hz, CH 2Ph), 3.75 (4H, m, 2 * CH 2O) and 3.51 (4H, m, 2 * NCH 2); δ C(50MHz, CDCl 3) 164.5 (C-6), 163.0 (C-4), 157.5 (C-2), 138.5 (season aryl C), 129.0,128.0 and 127.5 (aryl C), 81.5 (C-5), 66.5 (2 * OCH 2), 46.0 (PhCH 2) and 44.5 (2 * NCH 2).
The conventional method of Triaminopyrimidine 72-78 closed loop:
9-benzyl-6-pyrrolidine-1-base-9H-purine (82)
(39.6mg 0.15mmol) is suspended in the heating of the potpourri of acetic anhydride (5 mass equivalents, 143mg, 130 μ l) and triethyl orthoformate (5 mass equivalents, 143mg, 160 μ l) and stirring and refluxing with 5-amino-4-benzyl amino-6-(pyrrolidine-1-yl) pyrimidine 76.All raw materials all dissolve in heating process.Behind the backflow 4h,, excessive acetic acid acid anhydride and triethyl orthoformate decompression are removed the potpourri cooling.With thick residue purifying, obtain 9-benzyl-6-pyrrolidine-1-base-9H-purine 82 (32.8mg, 80%) by silica gel column chromatography (using ethyl acetate) as eluent.δ H(200MHz, CDCl 3) 8.45 (1H, s, H-2), 7.24-7.28 (5H, m, aryl H), 6.19 (1H, s, H-8), 5.11 (1H, s, CH 2Ph), 3.18-3.62 (4H, br s, 2 * CH 2N) and 1.90-2.06 (4H, m, 2 * CH 2CH 2N); δ C(50MHz, CDCl 3) 171.2 (C-6) a, 161.3 (C-4) a, 158.4 (C-8) a, 158.4 (C-2), 138.0 (season aryl C), 128.7,127.7 and 127.4 (aryl C), 98.1 (C-5), 50.5 (2 * CH 2N), 46.7 (CH 2Ph) and 25.5 (2 * CH 2CH 2N); (ES) m/z 291.2[M+NH 4 +], 255.2,205.1 and 166.1.
Use the following compound of this method preparation:
N, N, 9-trimethyl-9H-purine-6-amine (79)
(0.12g，100％)。δ H(200MHz, CDCl 3) 8.47 (1H, s, H-2), 6.50 (1H, s, H-8), 3.36 (3H, s, NCH 3) and 3.13[6H, s, N (CH 3) 2]; δ C(50MHz, CDCl 3) 171.3 (C-6) a, 163.5 (C-4) a, 161.7 (C-8), 157.8 (C-2), 96.1 (C-5), 37.6 (2 * NCH 3) and 35.0 (NCH 3).
9-benzyl-N, N ,-dimethyl-9H-purine-6-amine (80)
(46.0mg，88％)。δ H(200MHz, CDCl 3) 8.16 (1H, s, H-2), 7.26-7.35 (5H, m, aryl H), 6.33 (1H, s, H-8), 5.30 (1H, s, NH 2), 5.10 (1H, br s, NH 2), 4.45 (J 5.8, CH for 2H, d 2Ph), 3.01 (6H, s, 2 * NCH 3) and 1.78 (1H, br s, NH); (ES) m/z 229.2[(M-N)-and KH], 202.1,137.1 and 138.0.
9-methyl-6-pyrrolidine-1-base-9H-purine (81)
(47.1mg，67％)。δ H(200MHz, CDCl 3) 8.41 (1H, s, H-2), 6.29 (1H, s, H-8), 3.34-3.56 (4H, m, 2 * CH 2N), 3.31 (3H, s, NCH 3) and 1.92-2.04 (4H, m, 2 * CH 2CH 2N).δ C(50MHz, CDCl 3) 171.2 (C-6) a, 161.3 (C-4) a, 158.4 (C-8), 158.1 (C-2), 96.9 (C-5), 46.8 (2 * CH 2N), 35.0 (NCH 3) and 25.4 (2 * CH 2CH 2N).(ES) m/z 221.2[M+NH 4 +], 179.1 and 166.1.
9-benzyl-6-morpholine-4-base-9H-purine (83)
(59.7mg，68％)。δ H(200MHz, CDCl 3) 8.49 (1H, s, H-2), 7.21-7.35 (5H, m, aryl H), 6.66 (1H, s, H-8), 5.18 (2H, s, CH 2Ph), 3.74-3.80 (4H, m, 2 * CH 2O), 3.56-3.61 (4H, m, 2 * CH 2N) and 1.78 (1H, br s, NH); δ C(50MHz, CDCl 3) 171.6 (C-6) a, 163.5 (C-4) a, 161.35 (C-8), 158.0 (C-2), 137.9 (season aryl C), 128.8,127.5 and 127.4 (aryl C), 96.9 (C-5), 66.6 (2 * CH 2O), 50.4 (CH 2Ph) and 44.6 (2 * CH 2N); (ES) m/z221.2[M+NH 4 +], 312.1,268.9 and 91.1.
6-chloro-N, N-dimethyl pyrimidine-4-amine (84)
Be dissolved in isopropyl alcohol (100cm according to above-mentioned conventional method processing 3) in 56 (15.03g, 0.10mol), dimethylamine (60% aqueous solution, 9.20cm 3, 0.12mol) and triethylamine (17.07cm 3, 0.12mol) solution.By chromatogram (use 1:10-1:5 (v/v) ethyl acetate: hexane is as eluent), obtain beige crystals 6-chloro-N, (amount is 16.73g to N-dimethyl pyrimidine-4-amine 84; Measuring its purity by HPLC is 99.1%).δ H(200MHz, CDCl 3) 8.38 (1H, s, H-2), 6.39 (1H, s, H-5) and 3.14 (6H, s, NMe 2); δ C(50MHz, CDCl 3) 159.2 (C-6), 158.9 (C-4), 158.0 (C-2), 101.3 (C-5) and 37.8 (NMe 2); t R4.48min (50% methyl alcohol: the 25mM ammonium acetate); (TSP) m/z 158 (100, M +.C 6H 8 35ClN 3Requirement is 157.6) and 160 (35, C 6H 8 37ClN 3).
6-chloro-4-pyrrolidine-1-yl pyrimidines (85)
Be dissolved in isopropyl alcohol (100cm according to above-mentioned conventional method processing 3) in 56 (15.00g, 0.10mol), pyrrolidine (9.37cm 3, 0.112mol) and triethylamine (17.1cm 3, 0.12mol) solution.Obtain beige crystals 6-chloro-4-pyrrolidine-1-yl pyrimidines 85 (17.85g, 98.3% by chromatogram (use 1:10-1:5 (v/v) ethyl acetate: hexane is as eluent); Measuring its purity by HPLC is 98%).δ H(200MHz, CDCl 3) 8.36 (1H, s, H-2), 6.25 (1H, s, H-5), and 3.58 and 3.31[4H, 2 overlapping br s, N (CH 2) 2] and 2.12[4H, br s, (CH 2) 2]; δ C(50MHz, CDCl 3) 160.9 (C-6), 158.9 (C-4), 158.0 (C-2), 101.0 (C-5), 46.3[N (CH 2) 2] and 25.3[(CH 2) 2]; t R7.13min (50% methyl alcohol: the 25mM ammonium acetate); (TSP) m/z 184 (100, M +.C 8H 10 35ClN 3Requirement is 184) and 186 (28, M +.C 8H 10 37ClN 3).
1-(6-chlorine pyrimidine-4-yl) pyrrolidine-2-methyl formate (86)
Be dissolved in methyl alcohol (100cm with the hydrogen chloride gas processing 3) in the L-proline (14.26g, 0.12mol) 30min stir it 1h then.Then that solution concentration is extremely dry, need not to characterize and can use this methyl ester hydrochloride.Be dissolved in isopropyl alcohol (100cm according to above-mentioned conventional method processing 3) in above-mentioned methyl ester, 56 (15.01g, 0.10mol) and triethylamine (31.3cm 3, 0.23mol).Obtain viscosity orange 1-(6-chlorine pyrimidine-4-yl) pyrrolidine-2-methyl formate 86 (19.74g, 81% by chromatogram (use 1:5 (v/v) ethyl acetate: hexane is as eluent); Measuring its purity by HPLC is 89.3%).δ H(200MHz, CDCl 3) 8.39 (1H, s, H-2), 6.39 (1H, br s, H-5), 4.63 (1H, br s, COCH), 3.73 (3H, s, OMe), 3.81-3.28 (2H, 2 overlapping br s, NCH 2) and 2.41-1.95[4H, m, (CH 2) 2]; δ C(50MHz, CDCl 3) 172.8 (CO), 160.9 (C-6), 159.8 (C-4), 158.0 (C-2), 101.2 (C-5), 59.8 (OMe), 52.2 (COCH), 46.5 (NCH 2), 30.1 and 24.1[(CH 2) 2]; t R5.27min (50% methyl alcohol: the 25mM ammonium acetate); (TSP) m/z 242 (100, M +.C 10H 14 35ClN 3O 2Requirement is 241.7), 243 (10) and 244 (25, M +.C 10H 14 37ClN 3O 2).
4-(6-chlorine pyrimidine-4-yl) morpholine (87)
Be dissolved in isopropyl alcohol (100cm according to above-mentioned conventional method processing 3) in 56 (15.00g, 0.10mol) and morpholine (18.00g, 0.21mol).With the concentrated crystalline solid that obtains of extract.On Buchner funnel, wash this crystal with saturated sodium bicarbonate aqueous solution, remove with minimum ethyl acetate then and anhydrate, obtain beige crystals 4-(6-chlorine pyrimidine-4-yl) morpholine 87 (18.30g, 92%).δ H(200MHz, CDCl 3) 8.39 (1H, s, H-2), 6.48 (1H, s, H-5), 3.91-3.72[4H, m, O (CH 2) 2] and 3.70-3.41[4H, m, N (CH 2) 2]; δ C(50MHz, CDCl 3) 162.6 (C-6), 160.2 (C-4), 158.0 (C-2), 101.4 (C-5), 66.3[O (CH 2) 2] and 44.2[N (CH 2) 2].
N-methyl-6-pyrrolidine-1-yl pyrimidines-4-amine (89)
With chloride 85 (1.45g, 10.11mmol) and pyrrolidine (1.86cm 3, 22.24mmol) under 250 ℃, condense 1h.Continuation is at ethyl acetate (50cm 3) middle processing, water (50cm 3) washing, dry (MgSO 4), the brown jelly of simmer down to.By obtain light brown solid N-methyl-6-pyrrolidine-1-yl pyrimidines-4-amine 89 (0.63g, 35%, measuring its purity by HPLC is 99%) by column chromatography (1:9 (v/v) methyl alcohol: ethyl acetate is as eluent).δ H(200MHz, CDCl 3) 8.11 (1H, s, H-2), 5.13 (1H, s, H-5), 4.89 (1H, br s, NH), 3.52-3.28[4H, m, O (CH 2CH 2) 2N], 2.85 (3H, d, NHCH 3, J 5.2) and 2.09-1.81[4H, m, O (CH 2CH 2) 2N]; δ C(50MHz, CDCl 3) 162.8 and 160.7 (C-4 and C-6), 157.2 (C-2), 79.5 (C-5), 46.3[(CH 2CH 2) 2N], 28.5 (NHCH 3) and 25.3[(CH 2CH 2) 2N]; t R2.25min (50% methyl alcohol: the 25mM ammonium acetate).
N-methyl-6-morpholine-4-yl pyrimidines-4-amine (90)
With chloride 87 (1.45g, 10.11mmol) and morpholine (2.28cm 3, 22.23mmol) under 250 ℃, condense 1h.Continuation is at ethyl acetate (50cm 3) middle processing, water (50cm 3) washing, dry (MgSO 4), the brown jelly of simmer down to.Obtain yellow powder N-methyl-6-morpholine-4-yl pyrimidines-4-amine 90 (1.51g, 77%, measuring its purity by HPLC is 96%) by column chromatography (1:9 (v/v) methyl alcohol: ethyl acetate is as eluent).δ H(200MHz, CDCl 3) 8.14 (1H, s, H-2), 5.37 (1H, s, H-5), 5.05 (1H, br s, NH), 3.77[4H, t, O (CH 2CH 2) 2N, J 4.9], 3.54[4H, t, O (CH 2CH 2) 2N, J 4.9] and 2.87 (3H, d, NHCH 3, J 5.2); δ C(50MHz, CDCl 3) 163.7 and 163.1 (C-4 and C-6), 157.2 (C-2), 79.9 (C-5), 66.5[O (CH 2CH 2) 2N], 44.5[O (CH 2CH 2) 2N] and 28.4 (NHCH 3); t R2.11min (50% methyl alcohol: the 25mM ammonium acetate).
N-benzyl-6-pyrrolidine-1-yl pyrimidines-4-amine (91)
With chloride 85 (0.98g, 5.35mmol), benzylamine (1.24cm 3, 11.4mmol) and toluene (5cm 3) potpourri place glass tube, sealing is also at 130 ℃ of heating 18h down.In 30min, in solution, form one deck crystallization.After the cooling, use ethyl acetate from water, to extract solution, thereby and be prepared and obtain orange.Obtain beige solid N-benzyl-6-pyrrolidine-1-yl pyrimidines-4-amine 91 (0.62g, 46%, measuring its purity by HPLC is 97.5%) by column chromatography (ratio of ethyl acetate-methyl alcohol in ethyl acetate is that 1:10 (v/v) is as eluent).δ H(200MHz, CDCl 3) 8.15 (1H, s, H-2), 7.21-7.40 (5H, m, aryl H), 5.30 (1H, br t, NH), 5.18 (1H, s, H-5), 4.46 (2H, d, PhCH 2, J 5.6), 3.39[4H, br s, N (CH 2) 2] and 1.90-2.05[4H, m, (CH 2) 2]; δ C(50MHz, CDCl 3) 162.1 (C-6), 160.6 (C-4), 157.5 (C-2), 138.4 (season aryl C), 128.6,127.3 and 127.2 (aryl C), 80.6 (C-5), 46.2[N (CH 2) 2], 45.9 (PhCH 2) and 25.3[(CH 2) 2]; t R10.13min (50% methyl alcohol: the 25mM ammonium acetate); (ES) m/z 138 (38), 163 (51), 228 (22), 255 (100, MH +.C 16H 18N 4Requirement is 255) and 256 (27).
N-benzyl-1-[6-(benzyl amino) pyrimidine-4-yl] pyrrolidine-2-formamide (92)
Use is similar to 91 method, with chloride 86 (1.67g, 6.92mmol), benzylamine (2.17cm 3, 20.0mmol) and toluene (20cm 3) place in the glass tube sealing and heating 18h under 130 ℃.In 30min, in solution, form one deck crystallization.After the cooling, use ethyl acetate from water, to extract solution, thereby and be prepared and obtain orange.Obtain beige solid N-benzyl-1-[6-(benzyl amino) pyrimidine-4-yl by column chromatography (ratio of ethyl acetate-methyl alcohol in ethyl acetate is that 1:10 (v/v) is as eluent)] pyrrolidine-2-formamide 92 (1.73g, 62%).δ H(200MHz, CDCl 3) 8.12 (1H, s, H-2), and 7.15-7.44 (5H, m, aryl H), 5.30 (2H, br m, H-5 and NH), 4.53-4.66 (1H, m, NCHCO), 5.27-4.52 (4H, m, 2 * PhCH 2), 3.42-3.56 (1H ,～br dd, NCH aH b, J 6.8 and 9.4), 3.19-3.38 (1H ,～br q, NCH aH b, J 9.0), 2.38-2.52 and 1.94-2.20[4H, 2 * m, (CH 2) 2]; δ C(50MHz, CDCl 3) 172.2 (CO), 162.7 (C-6), 161.3 (C-4), 157.5 (C-2), 138.3 and 138.1 (2 * season aryl C), 128.7,128.5,127.5,127.4,127.3 and 127.2 (aryl C), 81.7 (C-5), 61.1 (NCHCO), 47.5 (PhCH 2), 45.8 (NCH 2), 43.3 (PhCH 2), 29.2 and 24.3[(CH 2) 2]; (ES) m/z 201 (19), 205 (13), 253 (100, C 11H 19N 5O 2), 254 (52), 281 (100, C 13H 23N 5O 2), 282 (22), 361 (18), 388 (80, MH +.C 23H 25N 5O 2Requirement is 388) and 389 (28).
N-benzyl-6-morpholine-4-yl pyrimidines-4-amine (93)
Under 250 ℃, with chloride 87 (2.29g, 10.44mmol) and morpholine (2.00cm 3, 23.00mmol) (1.28g 11.48mmol) condenses 1h under the existence at potassium tert-butoxide.Continuation is at ethyl acetate (50cm 3) middle processing, water (50cm 3) washing, dry (MgSO 4), the brown jelly of simmer down to.Obtain the yellow solid of viscosity by column chromatography (methyl alcohol: ethyl acetate is that 1:9 (v/v) is as eluent).It is dissolved in acetone and comes precipitated solid with hexane, obtain yellow powder N-benzyl-6-morpholine-4-base-pyrimidine-4-amine (93) (1.56g, 55%, measuring its purity by HPLC is 95%).δ H(200MHz, CDCl 3) 8.16 (1H, s, H-2), 7.48-7.05 (5H, m, aryl H), 5.39 (2H, br m, H-5 and NH), 4.46 (2H, d, PhCH 2, J 6.0), 3.74[4H, t, O (CH 2CH 2) 2N, J 4.9] and 3.48[4H, t, O (CH 2CH 2) 2N, J 4.9]; δ C(50MHz, CDCl 3) 162.9 and 162.8 (C-4 and C-6), 157.3 (C-2), 138.0 (season phenyl C), 128.8,127.5 and 127.2 (phenyl CH), 81.0 (C-5), 66.5[O (CH 2CH 2) 2N], 45.8 (PhCH 2) and 44.5[O (CH 2CH 2) 2N]; t R6.82min (50% methyl alcohol: the 25mM ammonium acetate).
2-[(6-pyrrolidine-1-yl pyrimidines-4-yl) amino] ethanol (94)
With chloride 85 (1.95g, 5.10mmol), monoethanolamine (1.89cm 3, 31.37mmol) and toluene (20cm 3) potpourri place in the glass tube, sealing is also at 130 ℃ of heating 18h down.As time passes, form brown oil at the pipe end.After the cooling, use ethyl acetate from water, to extract solution, thereby and be prepared and obtain orange.Obtain a meter brown solid 2-[(6-pyrrolidine-1-yl pyrimidines-4-yl by column chromatography (ratio of ethyl acetate-methyl alcohol in ethyl acetate is that 1:10 (v/v) is as eluent)) amino] ethanol 94 (1.10g, 50.6%, measuring its purity by HPLC is 94.1%).δ H(200MHz, CDCl 3) 8.07 (1H, s, H-2), 5.30 (1H, br t, NH), 5.18 (1H, s, H-5), 4.08 (1H, br s, OH), 3.78 (24H, t, CH 2OH, J 4.6), 3.22-3.49[6H, br m, N (CH 2) 2And CH 2OH] and 1.90-2.05[4H, m, (CH 2) 2]; δ C(50MHz, CDCl 3) 162.2 (C-6), 160.4 (C-4), 157.1 (C-2), 80.8 (C-5), 61.6 (CH 2OH), 46.3[N (CH 2) 2], 44.3 (CH 2NH) and 25.2[(CH 2) 2]; t R2.17min (50% methyl alcohol: the 25mM ammonium acetate); (ES) m/z 110 (15), 121 (13), 165 (15), 191 (85), 209 (100, MH +.C 11H 16N 4It is 209 that O requires) and 210 (22).
N-(2-hydroxyethyl)-1-{6-[(2-hydroxyethyl) amino] pyrimidine-4-yl } pyrrolidine-2-formamide (95)
Use is similar to 91 method, with chloride 86 (1.61g, 6.67mmol), monoethanolamine (1.21cm 3, 20.0mmol) and toluene (20cm 3) place in the glass tube sealing and heating 18h under 130 ℃.As time passes, form brown oil at the pipe end.After the cooling, use ethyl acetate from water, to extract solution, thereby and be prepared and obtain orange.Obtain brown oil N-(2-hydroxyethyl)-1-{6-[(2-hydroxyethyl by column chromatography (ratio of ethyl acetate-methyl alcohol in ethyl acetate is that 1:10 (v/v) is as eluent)) amino] pyrimidine-4-yl } pyrrolidine-2-formamide 95 (1.02g, 54%, measuring its purity by HPLC is 99.9%).δ H(200MHz, CDCl 3) 7.97 (1H, s, H-2), 7.43 (1H, br t, CONH, J 5.0), 5.90 (1H, br t, NH, J5.6), 5.27 (1H, s, H-5), 4.20-4.35 (1H, br m, NCHCO), 3.18-3.38 and 3.39-3.75[10H, 2 * m, 2 * NH (CH 2) 2OH and NCH 2] and 1.74-2.20[4H, m, (CH 2) 2]; δ C(50MHz, CDCl 3) 172.5 (CO), 162.1 (C-6), 160.1 (C-4), 156.4 (C-2), 81.3 (C-5), 60.4 (NCHCO), 60.2[2 * (CH 2) 2OH], 46.6 (NCH 2), 45.0[NH (CH 2) 2], 41.3[NH (CH 2) 2], 29.4 and 23.2[(CH 2) 2]; t R1.77min (50% methyl alcohol: the 25mM ammonium acetate); (ES) m/z 207 (10), 235 (52, C 11H 15N 4O 2), 269 (11), 296 (100, MH +.C 13H 21N 5O 3Requirement is 295) and 297 (19).
2-[(6-morpholine-4-yl pyrimidines-4-yl) amino] ethanol (96)
According to the method that is similar to 91, with chloride 59 (1.88g, 10.84mmol) and morpholine (2.08cm 3, 23.84mmol) heat 1h down at 250 ℃, use ethyl acetate (3 * 50cm then 3) from saturated sodium-chloride water solution (50cm 3) and methyl alcohol (10cm 3) potpourri in extract.Extract concentrated and by column chromatography (1:9-2:8 (v/v) methyl alcohol: ethyl acetate is as eluent) purifying.Obtain wax shape beige solid 2-[(6-morpholine-4-yl pyrimidines-4-yl) amino] ethanol (96) (1.82g, 75%).δ H(200MHz, CDCl 3) 7.96 (1H, s, H-2), 5.65 (1H, br m, NH), 5.35 (1H, s, H-5), 4.68 (1H, br s, OH), 3.59[6H, t and unconspicuous m, O (CH 2CH 2) 2N, J 4.8 and CH 2OH], 3.34[4H, t, O (CH 2CH 2) 2N, J 4.9] and 3.24 (2H, q, NHCH 2, J 5.0); δ C(50MHz, CDCl 3) 169.2 and 162.4 (C-4 and C-6), 156.8 (C-2), 81.2 (C-5), 66.1[O (CH 2CH 2) 2N], 61.0 (CH 2OH), 44.1[O (CH 2CH 2) 2N] and 43.8 (NHCH 2).
N-4-bromophenyl-6-pyrrolidine-1-yl pyrimidines-4-amine (97)
At room temperature, (3.52g, 31.37mmol) processing is dissolved in toluene (20cm in reaction tube with potassium tert-butoxide 3) chloride 85 (1.93g, 10.50mmol) and 4-bromaniline (2.73g, potpourri 15.87mmol).Carry out instantaneous heating, form dense thick brown precipitate.As 93 described, reaction tube is sealed and handles, obtain brown ceramic powder by column chromatography (ratio of ethyl acetate-methyl alcohol in ethyl acetate is that 1:10 (v/v) is as eluent) then.This powder is partially dissolved in small amount of acetone and precipitates with hexane.By the isolated by filtration solid, obtain cream-coloured powder N-4-bromophenyl-6-pyrrolidine-1-yl pyrimidines-4-amine 97 (1.24g, 37.1%, measuring its purity by HPLC is 98.2%).δ H(200MHz, CDCl 3+ d 6-DMSO) 8.15 (1H, s, H-2), 7.58 (1H, br s, NH), 7.31 (2H ,～d, aryl H, J 6.6), 7.20 (2H ,～d, aryl H, J 6.7), 5.55 (1H, s, H-5), 3.32[4H, br s, N (CH 2) 2] and 1.88-1.98[4H, m, (CH 2) 2]; δ C(50MHz, CDCl 3) 160.4 (C-6), 159.7 (C-4), 157.4 (C-2), 138.8 (season aryl C), 131.8 and 122.5 (aryl C), 115.2 (season aryl C), 82.8 (C-5), 46.1[N (CH 2) 2And 25.0[(CH 2) 2]; t R(2.23min methyl alcohol); (ES) m/z 319 (100, M +.C 15H 16 79BrN 4Requirement is 319), 320 (12), 321 (100, M +.C 15H 16 81BrN 4Requirement is 321) and 322 (13).
The 1-{6-[(4-bromophenyl) amino] pyrimidine-4-yl } pyrrolidine-2-carboxylic acid (98)
Use is similar to 97 method, and at room temperature, (2.26g, 20.14mmol) processing is dissolved in toluene (20cm in reaction tube with potassium tert-butoxide 3) chloride 86 (1.61g, 6.66mmol) and 4-bromaniline (1.75g, potpourri 10.18mmol).Carry out instantaneous heating, form dense thick brown precipitate.As 91 described, reaction tube is sealed and handles, obtain cream-coloured powder 1-{6-[(4-bromophenyl by column chromatography (ratio of ethyl acetate-methyl alcohol in ethyl acetate is that 1:10 (v/v) is as eluent) then) amino] pyrimidine-4-yl } pyrrolidine-2-carboxylic acid 98 (0.47g, 19.5%, measuring its purity by HPLC is 99.2%).δ H(200MHz, CDCl 3+ d 6-DMSO) 9.73 (1H, br s, NH), 7.67 (～1H, m, H-2), 7.46 (2H, m, aryl H), 7.28 (2H, m, aryl H), 5.07 (1H ,～d, H-5, J 3.2), and 4.23-4.61 (1H, br m, NCHCO), 3.18-3.39 and 3.39-3.62 (2H, 2 * br m, NCH 2) and 1.82-2.25[4H, m, (CH 2) 2]; δ C(50MHz, CDCl 3) 170.3 (CO), 161.6 (C-6), 159.6 (C-4), 147.2 (C-2), 137.0 (season aryl C), 130.3 and 120.4 (aryl C), 114.6 (season aryl C), 85.7 (C-5), 60.3 (NCHCO), 46.5 (NCH 2), 29.4 and 22.7[(CH 2) 2]; t R11.30min (50% methyl alcohol: the 25mM ammonium acetate); (ES) m/z 164 (88), 192 (100), 363 (87, M +.C 15H 15 79BrN 4O 2Requirement is 363), 364 (14), 365 (86, M +.C 15H 15 81BrN 4O 2Requirement is 365) and 366 (15).
N-pyridine-2-base-6-pyrrolidine-1-yl pyrimidines-4-amine (99)
Use is similar to 97 method, and at room temperature, (3.54g, 31.51mmol) processing is dissolved in toluene (20cm in reaction tube with potassium tert-butoxide 3) chloride 85 (1.91g, 10.40mmol) and 2-aminopyridine (1.51g, potpourri 16.07mmol).Carry out instantaneous heating, form dense thick brown precipitate.As 91 described, reaction tube is sealed and handles, obtain cream-coloured powder N-pyridine-2-base-6-pyrrolidine-1-yl pyrimidines-4-amine 99 (0.38g by column chromatography (ratio of ethyl acetate-methyl alcohol in ethyl acetate is that 1:10 (v/v) is as eluent) then, 15.3%, measuring its purity by HPLC is 94.0%).δ H(200MHz, CDCl 3) 8.35 (1H, s, H-2), 8.30 (1H, dd, pyridine H-6, J 2.0 and 7.0), 7.60 (1H ,～ddd, pyridine H-4, J 1.8,6.8 and 7.2), 7.32 (1H, d, pyridine H-3, J 8.2), 6.87 (1H, dd, pyridine H-5, J 6.0 and 7.2), 6.78 (1H, s, H-5), 3.52[4H, br s, N (CH 2) 2And 1.90-2.18[4H, m, (CH 2) 2]; δ C(50MHz, CDCl 3) 161.0 (C-6), 158.1 (C-4), 157.5 (C-2), 153.8 (pyridine C-2), 147.7 (pyridine C-6), 137.6 (pyridine C-4), 116.7 (pyridine C-3), 112.6 (pyridine C-5), 86.7 (C-5), 46.4[N (CH 2) 2] and 25.3[(CH 2) 2]; t R5.17min (50% methyl alcohol: the 25mM ammonium acetate); (ES) m/z138 (65), 146 (11), 171 (15), 242 (100, M +.C 13H 15N 5Requirement is 242) and 243 (31).N-(1-benzyl-2-oxo-2-pyrrolidine-1-base ethyl)-6-pyrrolidine-1-yl pyrimidines-4-amine (100)
According to the method that is similar to 97, at pyrrolidine (0.57cm 3, 6.89mmol) exist down, be dissolved in toluene (7cm with being sealed in the glass tube 3) chloride 60 (1.00g is 3.43mmol) in 130 ℃ of following heating 72h.After concentrating, separate (uses methyl alcohol: ethyl acetate as 2:8 (v/v) as eluent) by column chromatography after, obtain orange solids N-(the basic ethyl of 1-benzyl-2-oxo-2-pyrrolidine-1-)-6-pyrrolidine-1-yl pyrimidines-4-amine 100 (0.33g, 26%).δ H(200MHz, CDCl 3) 8.18 (1H, br s, H-2), 7.41-7.09 (5H, m, aryl H), 5.45 (1H, br d, NH, J 8.8), 5.25 (1H, s, H-5), 5.03 (1H, dt, NHCH, J 14.2 and 6.0), 3.62-3.19[3H, m, CONCH aH b(CH 2)-], 3.38[4H, t, N (CH 2) 2-, J 7.0], 3.13 (1H, dd, PhCH aH b, J 13.2 and 7.2), 3.02 (1H, dd, PhCH aH b, J 13.0 and 9.2), 2.80-2.42[1H, m, CONCH aH b(CH 2)-], 2.13-1.82 and 1.82-1.44[8h, 2 * m, 2 * N (CH 2CH 2) 2]; δ C(50MHz, CDCl 3) 170.6 (CO), 161.2 and 160.2 (C-4 and C-6), 157.5 (C-2), 137.0 (season phenyl C), 129.4,128.2 and 126.7 (phenyl CH), 82.9 (C-5), 53.8 (NHCH), 46.3 and 45.7[CON (CH 2) 2], 46.2[N (CH 2) 2], 39.7 (PhCH 2), 25.1 and 24.0[CON (CH 2CH 2) 2] and 25.3[N (CH 2CH 2) 2].
2-{[6-(dimethylamino) pyrimidine-4-yl] amino }-3-phenylpropionic acid methyl esters (101)
According to the method that is similar to 100, at dimethylamine (33% ethanolic solution, 3.0cm 3, 22.0mmol) exist down, be dissolved in toluene (7cm with being sealed in the glass tube 3) chloride 60 (0.98g is 3.37mmol) in 130 ℃ of following heating 72h.After concentrating, obtain orange solids 2-{[6-(dimethylamino) pyrimidine-4-yl by column chromatography (use methyl alcohol: ethyl acetate as 1:9-2:8 (v/v) as eluent)] amino }-3-phenylpropionic acid methyl esters 101 (0.21g, 17%).δ H(200MHz, CDCl 3) 8.20 (1H, br s, H-2), 7.38-7.11 (5H, m, aryl H), 5.47 (1H, br d, NH, J 8.4), 5.38 (1H, s, H-5), 5.31 (1H, dt, NHCH, J 8.2 and 6.0), 3.10 (1H, dd, PhCH aH b, J 13.2 and 5.8), 3.08 (3H, s, OCH 3), 3.02 (1H, unconspicuous dd, PhCH aH b, J 13.2 and 3.4), 2.08 and 2.73 (6H, 2 * s, 2 * CH 3); δ C(50MHz, CDCl 3) 172.3 (CO), 162.5 and 161.6 (C-4 and C-6), 157.3 (C-2), 136.9 (season phenyl C), 129.4,128.3 and 126.7 (phenyl CH), 82.5 (C-5), 51.4 (OCH 3), 39.8 (NHCH), 37.1 (PhCH 2), 36.9 and 35.6[N (CH 3) 2].
The conventional method that pyrimidine is nitrated
To be in quick stirring suspension in the conical flask, that be dissolved in the pyrimidine in the concentrated sulphuric acid (3-4 equivalent) and in ice-salt bath, be cooled to-10 ℃.Then nitric acid (65%, the 1-2 equivalent) is dropwise added in the potpourri, as time passes, obtain the glassy yellow blend, and become darkorange gradually.In 18h, this blend is warming up to room temperature naturally then.Then potpourri is decanted on the trash ice, and is prepared by filtering or extracting as required.
5-nitro-N-pyridine-2-base-6-pyrrolidine-1-yl pyrimidines-4-amine (102)
Use above-mentioned nitrated conventional method, 0 ℃ mix down pyrimidine 99 (0.28g, 1.16mmol), nitric acid (65%, 0.24cm 3, 2.4mmol) and the concentrated sulphuric acid (2.3cm 3), obtain yellowish orange solution.After the preparation, use ethyl acetate: hexane carries out column chromatography as 1:10 (v/v) to be separated, and obtains yellow solid 5-nitro-N-pyridine-2-base-6-pyrrolidine-1-yl pyrimidines-4-amine 102 (0.28g, 83.1%).δ H(200MHz, CDCl 3) 10.2 (1H, br s, NH), 8.42 (1H, dd, pyridine H-6, J 0.8 and 8.4), 8.35 (1H, dt, pyridine H-4, J 1.0 and 4.8), 8.23 (1H, s, H-2), 7.71 (1H, ddd, H-5, J 1.9,7.8 and 8.8), 7.04 (1H, dd, H-3, J 4.8 and 7.2), 3.47[4H, br s, N (CH 2) 2] and 1.96-2.05[4H, m, (CH 2) 2]; δ C(50MHz, CDCl 3) 156.9 (pyridine C-2), 153.5 (C-6), 151.5 (C-4), 148.2 (pyridine C-6), 137.8 (pyridine C-4), 119.6 (C-2), 115.8 (pyridine C-5), 109.7 (pyridine C-3), 80.2 (C-5), 50.0[N (CH 2) 2] and 25.2[br, (CH 2) 2]; (ES) m/z 213 (10), 241 (65), 242 (125), 287 (100, MH +.C 13H 15N 6O 2Requirement is 287) and 288 (18).
4, the conventional method of 6-di-amino-pyrimidine bromination
To be in pyrimidine solution under the room temperature, that be dissolved in methylene chloride and handle, obtain exotherm usually,, then follow the quick boiling of dichloromethane solution, obtain red solution to black if should operation too fast with bromine (1.5-2 equivalent).This potpourri is covered stopper place 18h, be decanted into then in the 1M sodium hydrate aqueous solution, and extract with methylene chloride.Carry out standard fabrication and separate, obtain the bromide of solid-state or oily with column chromatography.
5-bromo-N-methyl-6-pyrrolidine-1-yl pyrimidines-4-amine (103)
According to above-mentioned conventional method, with bromine (0.17cm 3, 3.33mmol) processing is dissolved in methylene chloride (7.0cm 3) in pyrimidine 89 (0.50g, 2.78mmol) solution.After column chromatography separation (ethyl acetate: hexane is that 1:4-1:1 (v/v) is as eluent), obtain beige solid 5-bromo-N-methyl-6-pyrrolidine-1-yl pyrimidines-4-amine 103 (0.54g, 75%; Measuring its purity by HPLC is 100%).δ H(200MHz, CDCl 3) 8.09 (1H, s, H-2), 5.31 (1H, br s, NH), 3.72[4H, t, (CH 2) 2N, J 6.7], 3.01 (3H, d, NHCH 3, J 4.8) and 2.02-1.75[4H, m, O (CH 2) 2-]; δ C(50MHz, CDCl 3) 160.0 and 158.1 (C-4 and C-6), 154.8 (C-2), 83.2 (C-5), 50.0[(CH 2CH 2) 2N], 28.7 (NHCH 3) and 25.7[(CH 2CH 2) 2N]; t R(2.15min methyl alcohol).
5-bromo-N-methyl-6-morpholine-4-yl pyrimidines-4-amine (104)
According to above-mentioned conventional method, with bromine (0.48cm 3, 9.34mmol) processing is dissolved in methylene chloride (19.5cm 3) in pyrimidine 90 (1.51g, 7.78mmol) solution.After separating (ethyl acetate: hexane is that 1:4-1:1 (v/v) is as eluent) by column chromatography, obtain beige solid 5-bromo-N-methyl-6-morpholine-4-yl pyrimidines-4-amine 104 (1.66g, 78%, measuring its purity by HPLC is 99%).δ H(200MHz, CDCl 3) 8.22 (1H, s, H-2), 5.39 (1H, br s, NH), 3.79[4H, t, O (CH 2CH 2) 2N, J 4.6], 3.44[4H, t, O (CH 2CH 2) 2N, J4.7] and 3.03 (3H, d, NHCH 3, J 5.0); δ C(50MHz, CDCl 3) 162.3 and 160.5 (C-4 and C-6), 157.3 (C-2), 90.6 (C-5), 66.8[O (CH 2CH 2) 2N], 49.1[O (CH 2CH 2) 2N] and 28.6 (NHCH 3); t R(1.97min methyl alcohol).
N-benzyl-5-bromo-6-pyrrolidine-1-yl pyrimidines-4-amine (105)
Use the conventional method of above-mentioned bromination, to being dissolved in methylene chloride (4.4cm 3) pyrimidine 91 (0.55g, 2.17mmol) and bromine (0.17cm 33.26mmol) solution handles, separate (ethyl acetate: hexane is 1:10-1:5 (v/v)) with column chromatography through preparation after, obtain orange N-benzyl-5-bromo-6-pyrrolidine-1-yl pyrimidines-4-amine 105 (0.59g, 81.8%, measuring its purity by HPLC is 92.4%).δ H(200MHz, CDCl 3) 8.12 (1H, s, H-2), 7.25-7.39 (5H, m, aryl H), 5.65 (1H, br m, NH), 4.72 (2H, d, PhCH 2, J 5.4), 3.72-3.82[4H, m, N (CH 2) 2] and 1.85-1.98[4H, m, (CH 2) 2]; δ C(50MHz, CDCl 3) 159.4 (C-6), 158.2 (C-4), 154.8 (C-2), 139.1 (season aryl C), 128.6,127.5 and 127.5 (aryl C), 83.1 (C-5), 50.1[N (CH 2) 2], 45.6 (PhCH 2) and 25.7[br, (CH 2) 2]; t R(2.38min methyl alcohol); (ES) m/z 241 (41), 243 (42), 333 (100, M +.C 15H 17 79BrN 4Requirement is 333), 334 (14), 335 (100, M +.C 15H 17 81BrN 4Requirement is 335) and 366 (15).
N-benzyl-1-[6-(benzyl amino)-5-bromo pyrimi piperidine-4-yl] pyrrolidine-2-formamide (106)
According to the conventional method of above-mentioned bromination, to being dissolved in methylene chloride (4.5cm 3) in product 92 (1.69g, 4.35mmol) and bromine (0.27cm 35.2mmol) handle, with ethyl acetate: to be 1:10-1:5 (v/v) carry out obtaining yellowish orange grease N-benzyl-1-[6-(benzyl amino)-5-bromo pyrimi piperidine-4-yl after column chromatography separates as eluent hexane] pyrrolidine-2-formamide 106 (1.52g, 75%).δ H(200MHz, CDCl 3) 8.01 (1H, s, H-2), 7.02-7.34 (10H, m, aryl H), 6.65 (1H, br t, CONH, J 5.8), 5.69 (1H, br t, aryl NH, J5.7), 4.88 (1H, dd, NCHCO, J 6.2 and 7.4), 4.63 (2H, d, PhCH 2, J5.8), 4.37 (2H, dd, PhCH 2, J 2.0 and 5.8), 4.02 (1H, ddd, NCH aH b, J 7.2,9.2 and 16.2), 3.68 (1H, ddd, NCH aH b, J 7.6,10.2 and 13.4) and 1.70-2.31[4H, m, (CH 2) 2]; δ C(50MHz, CDCl 3) 172.8 (CO), 159.6 (C-6), 158.9 (C-4), 154.8 (C-2), 138.6 and 138.3 (season aryl C), 128.5,128.4,127.3,127.2,127.2 and 127.1 (aryl C), 85.2 (C-5), 63.1 (NCHCO), 52.0 (NCH 2), 45.4 and 43.0 (2 * PhCH 2), 29.8 and 25.3[(CH 2) 2]; (ES) m/z 255 (11), 283 (10), 345 (100, C 16H 17 79BrN 4), 346 (15), 347 (98, C 16H 17 81BrN 4), 348 (16), 373 (58, C 16H 16 79BrN 5O), 375 (58, C 16H 16 81BrN 5O), 376 (12) and 388 (1, M +-Br).There is not M +(C 23H 24 79BrN 5It is 466 that O requires).
N-benzyl-5-bromo-6-morpholine-4-yl pyrimidines-4-amine (107)
According to above-mentioned conventional method, with bromine (0.31cm 3, 6.06mmol) processing is dissolved in methylene chloride (13.0cm 3) in pyrimidine 93 (1.37g, 5.05mmol).Through column chromatography (ethyl acetate: hexane is that 1:4-1:1 (v/v) is as eluent), obtain orange N-benzyl-5-bromo-6-morpholine-4-yl pyrimidines-4-amine 107 (1.07g, 61%, measuring its purity by HPLC is 99%).δ H(200MHz, CDCl 3) 8.23 (1H, s, H-2), 7.48-7.12 (5H, m, aryl H), 5.70 (1H, br m, NH), 4.71 (2H, d, PhCH 2, J5.8), 3.81[4H, t, O (CH 2CH 2) 2N, J 4.6] and 3.48[4H, t, O (CH 2CH 2) 2N, J 4.6]; δ C(50MHz, CDCl 3) 162.7 and 159.9 (C-4 and C-6), 155.4 (C-2), 138.5 (season phenyl C), 128.7,127.5 and 127.4 (phenyl CH), 90.4 (C-5), 66.8[O (CH 2CH 2) 2N], 49.1 (PhCH 2) and 45.5[O (CH 2CH 2) 2N]; t R(2.10min methyl alcohol).
2-[(5-bromo-6-pyrrolidine-1-yl pyrimidines-4-yl) amino] ethanol (108)
Use the conventional method of above-mentioned bromination, to being dissolved in methylene chloride (23cm 3) in pyrimidine 94 (0.96g, 4.96mmol) and bromine (0.26cm 35.1mmol) handle, the process preparation separates (ethyl acetate: hexane is that 1:10-1:5 (v/v) is as eluent) with column chromatography after, obtain yellow oil 2-[(5-bromo-6-pyrrolidine-1-yl pyrimidines-4-yl) amino] ethanol 108 (1.04g, 79.0%, measuring its purity by HPLC is 99%).δ H(200MHz, CDCl 3) 7.93 (1H, s, H-2), 5.74 (1H, br m, NH), 4.49 (1H, br s, OH), 3.42-3.76[8H, 2 * m, HO (CH 2) 2NH and N (CH 2) 2] and 1.82-1.97[4H, m, (CH 2) 2]; δ C(50MHz, CDCl 3) 159.7 (C-6), 158.0 (C-4), 154.1 (C-2), 82.6 (C-5), 62.9 (OCH 2), 49.9[N (CH 2) 2], 44.7 (CH 2NH) and 25.5[(CH 2) 2]; t R8.40min (50% methyl alcohol: the 25mM ammonium acetate); (ES) m/z 162 (12), 189 (79), 190 (18), 207 (27), 269 (47, M +-H 2O), 271 (48, M +-H 2O), 287 (100, M +.C 10H 15 79BrN 4It is 287 that O requires) and 289 (96, M +.C 10H 15 81BrN 4It is 289 that O requires).
N-(2-hydroxyethyl)-1-{6-[(2-hydroxyethyl) amino]-5-bromo pyrimi piperidine-4-yl } pyrrolidine-2-formamide (109)
Use the conventional method of above-mentioned bromination, to being dissolved in methylene chloride (23cm 3) in pyrimidine 95 (0.968g, 4.96mmol) and bromine (0.26cm 35.17mmol) handle, the process preparation separates (ethyl acetate: hexane is that 1:10-1:5 (v/v) is as eluent) with column chromatography after, obtain yellow oil N-(2-hydroxyethyl)-1-{6-[(2-hydroxyethyl) amino]-5-bromo pyrimi piperidine-4-yl } pyrrolidine-2-formamide 109 (1.04g, 79.0%).δ H(200MHz, CDCl 3) 7.93 (1H, s, H-2), 5.74 (1H, br m, NH), 4.49 (1H, br s, OH), 3.42-3.76[8H, 2 * m, HO (CH 2) 2NH and N (CH 2) 2] and 1.82-1.97[4H, m, (CH 2) 2]; δ C(50MHz, CDCl 3) 159.7 (C-6), 158.0 (C-4), 154.1 (C-2), 82.6 (C-5), 62.9 (OCH 2), 49.9[N (CH 2) 2], 44.7 (CH 2NH) and 25.5[(CH 2) 2].
2-[(5-bromo-6-morpholine-4-yl pyrimidines-4-yl) amino] ethanol (110)
According to above-mentioned conventional method, with bromine (0.43cm 3, 8.37mmol) processing is dissolved in methylene chloride (17.0cm 3) in pyrimidine 96 (1.57g, 6.98mmol).After separating (with ethyl acetate: hexane is 1:4 (v/v)-methyl alcohol: ethyl acetate is that 1:9 (v/v) is as eluent) by column chromatography, obtain beige solid 2-[(5-bromo-6-morpholine-4-yl pyrimidines-4-yl) amino] ethanol 110 (1.04g, 49%, measuring its purity by HPLC is 98%).δ H(200MHz, CDCl 3) 8.12 (1H, s, H-2), 5.81 (1H, br m, NH), 3.90-3.67 (7H, br t and unconspicuous m, OH, O (CH 2CH 2) 2N, J 4.0 and CH 2OH), 3.63 (2H ,～dt, NHCH 2, J 5.5 and 4.2) and 3.46[4H, t, O (CH 2CH 2) 2N, J 4.2]; δ C(50MHz, CDCl 3) 162.5 and 160.3 (C-4 and C-6), 154.9 (C-2), 90.1 (C-5), 66.8[O (CH 2CH 2) 2N], 62.8 (CH 2OH), 49.0[O (CH 2CH 2) 2N] and 44.7 (NHCH 2); t R3.87min (50% methyl alcohol: the 25mM ammonium acetate).
The conventional method of 5-bromo pyrimi piperidine catalytic aminoization
At room temperature, with stand-by 5-bromo pyrimi piperidine and required amine (4 equivalent), potassium phosphate hydrate (2 equivalent) and the N that is purchased, N-dimethylethanolamine (0.1M) places in the reaction tube.Purge the about 15min of potpourri with nitrogen stream then, after this, add anhydrous, white cuprous iodide (CuI, 1.1 equivalents), obtain green mixture usually to it.Reaction tube at the nitrogen atmosphere lower seal, is heated to 100 ℃ then, and continues heating 18h.Potpourri presents atropurpureus gradually with reaction.After the cooling, handle potpourri removing the copper residue with liquor ammoniae fortis, and extract with methylene chloride.Concentrate and with methyl alcohol: ethyl acetate be 1:10 (v/v) as eluent, if need, then use little ammonia to carry out column chromatography, obtain the product of purifying.
N 4, N 5-dibenzyl-6-pyrrolidine-1-yl pyrimidines-4,5-diamines (111)
Use above-mentioned conventional method, bromizate thing 105 (0.19g, 0.58mmol), benzylamine (0.26cm 3, 2.4mmol), potassium phosphate hydrate (0.29g, 1.28mmol), cuprous iodide (0.15g, 0.78mmol) and N, N-dimethylethanolamine (1cm 3) boiling together, produce brown solution.Be prepared and chromatographic resolution, obtain yellow solid N 4, N 5-dibenzyl-6-pyrrolidine-1-yl pyrimidines-4, and 5-diamines 111 (0.15g, 69%, measuring its purity by HPLC is 78.0%).δ H(200MHz, CDCl 3) 8.15 (1H, s, H-2), 7.12-7.45 (10H, m, aryl H), 5.17 and 5.42 (2H, br s and br m, 2 * NH), 4.44 (2H, d, PhCH 2, J 4.8), 3.89 (2H, br s, PhCH 2), 3.39[4H, br m, N (CH 2) 2] and 1.75-2.05[4H, m, (CH 2) 2]; δ C(50MHz, CDCl 3) 162.2 (C-6), 160.6 (C-4), 157.6 (C-2), 138.4 (season aryl C), 128.6,128.5,127.3,127.2,127.0 and 126.8 (aryl C), 80.6 (C-5), 46.2[N (CH 2) 2], 45.8 (2 * PhCH 2) and 25.2[br, (CH 2) 2]; t R9.78min (50% methyl alcohol: the 25mM ammonium acetate); (ES) m/z 138 (29), 163 (48, C 8H 12N 4), 228 (21), 255 (100, C 15H 18N 4) and 256 (25).There is not M +(C 22H 25N 5Requirement is 360).
2-{[5-(benzyl amino)-6-pyrrolidine-1-yl pyrimidines-4-yl] amino } ethanol (112)
Use above-mentioned conventional method, bromizate thing 108 (0.30g, 1.04mmol), benzylamine (0.47cm 3, 4.32mmol), potassium phosphate hydrate (0.59g, 2.57mmol), cuprous iodide (0.23g, 1.21mmol) and N, N-dimethylethanolamine (1.8cm 3) boiling together, produce purple brown solution.Be prepared and chromatographic resolution, obtain yellow solid 2-{[5-(benzyl amino)-6-pyrrolidine-1-yl pyrimidines-4-yl] amino } ethanol 112 (0.30g, 92.3%).δ H(200MHz, CDCl 3) 8.10 (1H, br s, H-2), 7.12-7.40 (5H, m, aryl H), 4.41 and 5.13 (4H, 2 * br s, 2 * NH and PhCH 2), 3.80 (1H, br s, OH), 3.54-3.68 (2H, m, OCH 2), 3.19-3.42[4H, br m, N (CH 2) 2], 2.05 (2H, s, NHCH 2) and 1.86-1.95[4H, m, (CH 2) 2]; δ C(50MHz, CDCl 3) 162.3 (C-6), 160.3 (C-4), 157.2 (C-2), 140.1 (season aryl C), 128.2,127.6 and 126.4 (aryl C), 80.8 (C-5), 61.1 (OCH 2), 55.3 (PhCH 2), 46.1[N (CH 2) 2], 44.1 (CH 2NH) and 25.1[(CH 2) 2].
The 2-phenylimidazole is [1,2-α] pyridine (113) also
(2.03g, 13.1mmol) (1.24g is 13.1mmol) at dimethyl formamide (25cm with the 2-aminopyridine with phenacyl chloride 3) in be heated to 120 ℃, and continue heating 5h.With the potpourri cooling, at room temperature stir and spend the night then.Pour potpourri into water (50cm 3) in, with ethyl acetate (2 * 50cm 3) extract.Solvent is removed from organic phase, generated the grease of blackout, this grease is carried out flash chromatography separate (with ethyl acetate: hexane is that 1:2-2:1 (v/v) is as eluent).The light brown crystalline material is separated (R f0.23 ethyl acetate: hexane is 1:1 (v/v)), be defined as title compound 113 (0.79g, 31%).δ H(CDCl 3, 200MHz) 8.13 (1H, d, H-5, J 6.6), 7.98 (2H, d, phenyl H, J 6.8), 7.87 (1H, s, H-3), 7.67 (1H, d, H-8, J 9.0), 7.43 (3H, m, phenyl H), 7.19 (1H, dd, H-7, J 9.0 and 7.8) and 6.79 (1H, dd, H-6, J 7.8 and 6.6).
2-(4-bromophenyl) imidazo [1,2-α] pyridine (114)
(2.44g, 10.9mmol) (1.03g is 10.9mmol) at dimethyl formamide (20cm with the 2-aminopyridine with 4-bromobenzene formyl methyl chloride 3) in be heated to 120 ℃, and continue heating 5h.With the potpourri cooling, at room temperature stir and spend the night then.Pour potpourri into water (40cm 3) in, with ethyl acetate (2 * 40cm 3) extract.Solvent is removed from organic phase, generated the grease of blackout, this grease is carried out flash chromatography separate (with ethyl acetate: hexane is that 2:3 (v/v) is as eluent).The yellow crystal material is separated (R f0.35 ethyl acetate: hexane is 1:1 (v/v)), be defined as title compound 114 (2.14g, 72%).δ H(CDCl 3, 200MHz) 8.15 (1H, d, H-5, J 6.7), 7.87 (1H, s, H-3), 7.85 (2H, d, phenyl H-3 and H-5, J 8.5), 7.66 (1H, d, H-8, J 9.1), 7.57 (2H, d, phenyl H-2 and H-6, J 8.5), 7.22 (1H, dd, H-7, J 9.1 and 7.1) and 6.80 (1H, dd, H-6, J 6.7 and 7.1).Use zinc chloride to prepare the conventional method of imidazo [1,2-α] pyridine
With the 2-aminopyridine (0.13mg 1.33mmol) is dissolved in the diox, with zinc chloride (5mol%, 0.07mmol), aldehyde (1.0 equivalents, 1.33mmol) and isocyanide (1.0 equivalents 1.328mmol) are handled and are sealed in the microwave reactor.Potpourri is shone 1.5h under (600W's) 40% power.After this, reaction mixture is concentrated, and handle crude mixture to obtain the product precipitation with ethyl acetate/hexane.
Use polynite K10 to prepare the conventional method of imidazo [1,2-α] pyridine
With the 2-aminopyridine (0.13mg 1.33mmol) is dissolved in the diox, with polynite K10 (1 mass equivalent, 0.13mg), aldehyde (1.0 equivalents, 1.33mmol) and isocyanide (1.0 equivalents 1.33mmol) are handled and are sealed in the microwave reactor.Potpourri is shone 1.5h under (600W's) 40% power.After this, reaction mixture is filtered and several times with methyl alcohol or washed with dichloromethane polynite.Filtrate decompression is concentrated and handle crude mixture to obtain the product precipitation with ethyl acetate/hexane.
Perhaps, can use conventional method agitating heating reaction mixture 5 hours.
Use a kind of or use routine in above-mentioned two kinds of methods to add the following compound of hot preparation:
N-(2,6 3,5-dimethylphenyl)-2-phenylimidazole is [1,2-α] pyridine-3-amine (115) also
(0.29g，72％)。δ H(200MHz, CDCl 3) 8.12 (2H, d, 2 * aryl H), 7.61 (2H, d, 2 * aryl H), 7.24-7.41 (3H, m, 3 * aryl H), 7.16 (1H, m, aryl H), 7.00 (J 7.6 for 2H, d, 2 * aryl H), 6.78-6.86 (2H, m, 2 * aryl H), (6.67-6.74 2H, m, 2 * aryl H), 5.43 (1H, br s is NH) with 2.02 (6H, s, 2 * CH 3); δ C(50MHz, CDCl 3) 141.5 (C-8) a, 140.5 (C-3) a133.8 (C-2), 130.1 (2 * xylyl C-3), 128.6 (2 * phenyl C-2), (127.8 phenyl C-4), (127.3 2 * phenyl C-3), 125.7 (C-4), 124.5 (C-5), (122.6 2 * xylyl C-2), (121.3 xylyl C-4), 117.8 (C-6), 112.5 (C-7) and 18.5 (2 * CH 3); (EI) m/z 313.2,220.1, and 194.0 and 79.1.
N-(2, the 6-xylyl)-2-(4-methoxyphenyl) imidazo [1,2-α] pyridine-3-amine (116)
(0.20g，48％)。δ H(200MHz, CDCl 3) 8.04 (J 8.4 for 2H, d, aryl H), 7.58 (J 1.0 for 2H, d, aryl H), 7.16-6.63 (7H, m, 7 * aryl H), 5.41 (1H, s, NH), 3.82 (3H, s, OCH 3) and 2.01 (6H, s, 2 * CH 3); δ C(50MHz, CDCl 3) 149.2 (pyridine radicals C-2 ' and C-6 '), 141.3 (C-8) a, 140.6 (aryl C) a138.5 (C-3), (137.5 pyridine radicals C-4 '), 130.1 (2 * xylyl C-3 '), 128.5 (2 * phenyl C-2), 125.4 (C-4), 124.3 (C-5), 122.4 (2 * xylyl C-2 '), 121.1 (xylyl C-4 '), 117.3 (C-6), 112.3 (C-7), 55.4 (OCH 3) and 18.7 (2 * CH 3); (EI) m/z 343.4,224.9, and 210.9,79.0 and 77.7.
The N-tert-butyl group-2-phenylimidazole is [1,2-α] pyridine-3-amine (117) also
(0.25g，60％)。δ H(200MHz, CDCl 3) 8.55 (1H, d, J9.2, aryl H), 8.15 (1H, d, J 6.6 aryl H), 7.47 (2H, br d, 2 * aryl H), 7.28 (1H, br t, aryl H), 6.89-7.04 (4H, m, 4 * aryl H) and 0.79 (9H, s, 3 * CH 3); δ C(50MHz, CDCl 3) 142.0 (C-8) a, 137.9 (C-3) a, 131.4 (C-2), 129.9 (2 * phenyl C-2), 127.7 (phenyl C-4), 127.4 (2 * phenyl C-3), 127.0 (C-4), 124.7 (phenyl C-1), 123.4 (C-5), 118.0 (C-6), 113.5 (C-7), 56.6[C (CH 3) 3] and 30.2[C (CH 3) 3]; (EI) m/z 265.1,208.1, and 181.0 and 78.1.
The N-tert-butyl group-2-(4-methoxyphenyl) imidazo [1,2-α] pyridine-3-amine (118)
(0.14g，28％)。δ H(200MHz, CDCl 3) 8.05 (1H, br m, aryl H), 7.61-7.70 (1H, br m, aryl H), 7.29 (2H, br d, J 7.2,2 * aryl H), 7.16 (1H, br t, J7.1, aryl H), 6.72-7.00 (4H, m, 4 * aryl H), 5.42 (1H, br s, NH), 3.828 (3H, s, OCH 3) and 2.01 (9H, s, 3 * CH 3); δ C(50MHz, DMSO) 159.2 (C-3), 158 (C-2), 148.20 (2 * phenyl C-2), (127.7 phenyl C-4), 137.40 (2 * phenyl C-3), 135.26 (C-4), 128.49 (phenyl C-1), 114.22 (C-5), 113.02 (C-6), 109.11 (C-7), 61.63[C (CH 3) 3] and 55.84 (OCH 3); (EI) m/z 295.0,238.9, and 237.8,210.9,212.2,94.0,77.9,66.9,55.6 and 50.7.
N-(2-morpholine-4-base ethyl)-2-phenylimidazole is [1,2-α] pyridine-3-amine (119) also
(0.12g，14％)。δ H(200MHz, CDCl 3) 8.11 (1H, d, J7.0, aryl H), 8.03 (2H, d, J 8.4,2 * aryl H), 7.56 (2H, d, J 9.2,2 * aryl H), 7.34-7.48 (3H, m, 3 * aryl H), (7.18 1H, br t, aryl H), 6.79 (1H, br t, aryl H), 4.08 (1H, br s, NH), 3.72 (4H, t, J 4.5,2 * CH 2O), 3.07-3.10 (2H, m, CH 2NH), 2.52-2.58 (2H, m, CH 2N) and 2.44 (4H, t, J 4.5,2 * CH 2N); δ C(50MHz, CDCl 3) 141.6 (C-8) a, 135.17 (C-3) a, 134.7 (C-2), 128.8 (2 * phenyl C-2), 127.5 (phenyl C-4), 127.3 (2 * phenyl C-3), 126.7 (phenyl C-1), 123.9 (C-4), 122.6 (C-5), 117.8 (C-6), 111.8 (C-7), 67.2 (2 * CH 2O), 58.5 (CH 2N), 53.9 (2 * CH 2N) and 44.4 (CH 2NH); (EI) m/z 322.1,220.0, and 99.9 and 78.1.
N-(2-morpholine-4-base ethyl)-2-(4-methoxyphenyl) imidazo [1,2-α] pyridine-3-amine (120)
(0.10g，20％)。δ H(200MHz, CDCl 3) 8.30 (J 6.6 for 1H, d, aryl H), 8.10 (2H, d, J 8.4,2 * aryl H), 7.60 (2H, d, J 9.2,2 * aryl H), 7.14-7.32 (3H, m, 3 * aryl H), 6.93 (J 8.6 for 2H, br t, aryl H), 4.14 (NH), 3.92 (J 8.4, CH for 3H, d for 1H, br s 3O), 3.76 (4H, t, J 4.1,2 * CH 2O), 3.07-3.10 (2H, m, CH 2NH), 2.52-2.58 (2H, m, CH 2N) and 2.44 (4H, t, J 4.0,2 * CH 2N); δ C(50MHz, CDCl 3) 133.1 (C-8) a, 128.5 (C-3) a, 127.5 (C-2), 122.0 (2 * phenyl C-2), 127.5 (phenyl C-4), 127.3 (2 * phenyl C-3), 126.7 (phenyl C-1), 123.9 (C-4), 122.6 (C-5), 118.9 (C-6), 116.4 (C-7), 71.9 (2 * CH 2O), 60.3 (CH 2N), 58.7 (2 * CH 2N), 49.1 (OCH 3) and 45.0 (CH 2NH); (EI) m/z 352.2,251.9, and 225.0,224.0,211.0,128.0,114.0,113.0,101.0,99.7,78.9,77.9,55.7 and 50.6.
N-cyclohexyl-2-phenylimidazole is [1,2-α] pyridine-3-amine (121) also
(0.60g，78％)。δ C(50MHz, CDCl 3) 141.7 (C-8) a, 136.7 (C-3) a, 134.6 (C-2), 129.6 (phenyl C-1), (128.7 2 * phenyl C-2), (127.5 phenyl C-4), 127.3 (2 * phenyl C-3), 124.1 (C-4), 122.9 (C-5), 117.6 (C-6), 111.8 (C-7), 57.2 (CHNH), (34.4 2 * cyclohexyl C-2 '), 26.0 (2 * cyclohexyl C-3 ') and 25.1 (cyclohexyl C-4 '); (EI) m/z 290.9,208.0, and 180.9 and 78.2.
N-(cyclohexyl)-2-(4-methoxyphenyl) imidazo [1,2-α] pyridine-3-amine (122)
(0.18g，42％)。δ H(200MHz, CDCl 3) 8.11 (J 6.8 for 2H, d, aryl H), 8.02 (J 8.6 for 2H, d, aryl H), 7.16-7.08 (1H, m, aryl H), 7.02-6.98 (J 8.6 for 2H, d, aryl H), 6.80-6.73 (1H, m, aryl H), 3.87 (3H, s, OCH 3) and 1.80-1.59 (10H, br m, 5 * cyclohexyl CH 2); δ C(50MHz, CDCl 3) 141.6 (C-8), 134.6 (C-2), 128.6 (2 * phenyl C-2), 127.3 (phenyl C-4), 124.3 (2 * phenyl C-3), 123.9 (C-4), 122.9 (C-5), 117.3 (C-6), 111.7 (C-7), 57.1 (CHNH), 55.5 (OCH 3), 34.4 (2 * cyclohexyl C-2 '), 26.0 (2 * cyclohexyl C-3 ') and 25.1 (cyclohexyl C-4 '); (EI) m/z 231.2,237.8, and 210.8 and 78.0.
N-(2, the 6-3,5-dimethylphenyl)-2-pyrimidin-3-yl imidazo [1,2-α] pyridine-3-amine (123)
(0.22g，54％)。δ H(200MHz, CDCl 3) 9.29 (J 1.6 for 1H, d, pyridine radicals H-2 '), 8.48 (1H, dd, J 5 and 1.6, pyridine radicals H-6 '), 8.33 (1H, dt, J 8 and 1.8, H-4), 7.59-7.69 (2H, m, 2 * aryl H), (7.15-7.30 2H, m, 2 * aryl H), 6.98 (2H, d, J 7.4,2 * xylyl H-3 '), 6.72-6.86 (2H, m, 2 * aryl H), 5.49 (1H, br s is NH) with 2.01 (6H, s, 2 * CH 3); δ C(50MHz, CDCl 3) 148.5 (pyridine radicals C-2 ' and C-6 '), 141.9 (C-8) a, 140.1 (aryl C) a, 134.7 (C-3), 134.5 (pyridine radicals C-4 '), (130.1 2 * xylyl C-3 '), 129.8 (C-2), 125.9 (C-4), 125.0 (C-5), (123.5 pyridine radicals C-5 '), (122.6 2 * xylyl C-2 '), 122.1 (pyridine radicals C-3 '), 121.8 (xylyl C-4 '), 117.8 (C-6), 112.9 (C-7) and 18.7 (2 * CH 3); (EI) m/z 314.1,221.0, and 194.9 and 78.0.
The N-tert-butyl group-2-pyridin-3-yl imidazo [1,2-α] pyridine-3-amine (124)
(0.21g，57％)。δ H(200MHz, CDCl 3) 9.23 (1H, d, J1.6, pyridine radicals H-2 '), 8.56 (1H, dd, J 5 and 1.6, pyridine radicals H-6 '), 8.31 (1H, dt, J 8 and 1.6, pyridine radicals H-4 '), 8.23 (1H, dt, J 7.0 and 1.2, H-4), 7.56 (1H, dd, J 9.0 and 1.1, H-7), 7.38 (1H, ddd, J8.0,5.0 and 1.0, pyridine radicals H-5 '), 7.17 (1H, ddd, J 9.0,7.0 and 1.2, H-6), 6.81 (1H, dt, J 7.0 and 1.1, H-5), 3.09 (1H, br s, NH) and 1.02 (9H, s, 3 * CH 3); δ C(50MHz, CDCl 3) 149.3 (pyridine radicals C-2 ') a, 148.5 (pyridine radicals C-6 ') a, 142.80 (C-8) a, 135.6 (C-3), 135.5 (pyridine radicals C-4 '), 131.6 (C-2), 124.7 (C-4), 123.7 (C-5), 123.6 (pyridine radicals C-5 '), 122.5 (pyridine radicals C-3 '), 117.8 (C-6), 111.9 (C-7), 55.7[C (CH 3) 3] and 30.7[C (CH 3) 3]; (EI) m/z 266.1,210.0, and 181.9 and 78.0.
N-(2-morpholine-4-base ethyl)-2-pyridin-3-yl imidazo [1,2-α] pyridine-3-amine (125)
(49.5mg，11％)。δ H(200MHz, CDCl 3) 9.30 (J 1.0 for 1H, d, pyridine radicals H-2 '), 8.56 (1H, dd, J 5.0 and 1.6, pyridine radicals H-6 '), 8.38 (1H, dt, J 7.9 and 1.6, pyridine radicals H-4 '), 8.16 (1H, d, J 6.8, H-4), 7.58 (1H, d, J9.4, H-7), 7.38 (1H, dd, J 7.9,5.0, pyridine radicals H-5 '), 7.18 (J 8.0 for 1H, br t, H-6), 6.84 (J 6.8 for 1H, br t, H-5), and 4.01 (1H, br s, NH), 3.76 (4H, t, J 4.6,2 * CH 2O), 3.14 (J 5.7, CH for 2H, br d 2NH), 2.60 (J 5.7, CH for 2H, t 2N) and 2.50 (4H, t, J 4.6,2 * CH 2N); δ C(50MHz, CDCl 3) 148.4 (pyridine radicals C-2 ') a, 148.3 (pyridine radicals C-6 ') a, 142.07 (C-8), 140.4 (C-2) a, 134.5 (pyridine radicals C-4 ') a, 130.7 (C-3) a, 127.1 (pyridine radicals C-3 '), 124.4 (C-4), 123.8 (C-5), 122.6 (pyridine radicals C-5 '), 118.0 (C-6), 112.2 (C-7), 67.1 (2 * CH 2O), 58.5 (CH 2N), 53.9 (2 * CH 2N) and 44.5 (CH 2NH); (EI) m/z 323.3,223.2, and 100.0 and 78.2.
N-(cyclohexyl)-2-pyridin-3-yl imidazo [1,2-α] pyridine-3-amine (126)
(0.23g，59％)。δ H(200MHz, CDCl 3) 9.33 (1H, dd, J 1.8 and 0.8, pyridine radicals H-2 '), 8.55 (1H, dd, J 4.7 and 1.8, pyridine radicals H-6 '), 8.41 (1H, dt, J 7.9 and 1.8, pyridine radicals H-4 '), 8.10 (1H, dt, J 6.8 and 1.1, H-4), 7.56 (1H, dt, J 9.1 and 1.1, H-7), 7.38 (J 7.9 for 1H, ddd, 4.7 with 0.8, pyridine radicals H-5 '), 7.17 (1H, ddd, J 9.1,6.8 and 1.1, H-6), 6.82 (1H, dt, J 6.8 and 1.1, H-5), 3.11 (1H, br s, NH), 2.86-3.08 (1H, m, CHNH), 1.46-1.92 and 1.00-1.40 (10H, 2 * m, 5 * cyclohexyl CH 2); δ C(50MHz, CDCl 3) 148.4 (pyridine radicals C-2 ') a, 148.3 (pyridine radicals C-6 ') a, 142.3 (C-8) a, 134.7 (pyridine radicals C-4 ') a, 134.6 (C-3) a, 130.9 (C-2), 125.6 (pyridine radicals C-3 '), 124.6 (C-4), 123.7 (C-5), 122.9 (pyridine radicals C-5 '), 117.8 (C-6), 112.1 (C-7), 57.2 (CHNH), 34.5 (2 * cyclohexyl C-2 '), 25.9 (2 * cyclohexyl C-3 ') and 25.1 (cyclohexyl C-4 '); (EI) m/z 292.1,209.0, and 181.9 and 78.0.
N-(2, the 6-3,5-dimethylphenyl)-2-(2-furyl) imidazo [1,2-α] pyridine-3-amine (127)
(99.4mg，25％)。δ H(200MHz, CDCl 3) 7.65 (J 7.0 for 1H, d, H-4), and 7.55 (1H, d, J 9.0, H-7), and 7.43 (1H, br s, furyl H-5 '), 7.06-7.18 (1H, m, H-6), 6.99 (2H, d, J 6.9,2 * xylyl H-3 '), 6.80-6.91 (H-5), 6.70 (J 6.9 for 1H, d for 1H, m, xylyl H-4 '), 6.59 (J 3.3 for 1H, d, furyl H-3 '), 6.42 (1H, dd, J 3.3 and 1.6, furyl H-4 ') and 2.03 (6H, s, 2 * CH 3).
The N-tert-butyl group-2-(2-furyl) imidazo [1,2-α] pyridine-3-amine (128)
(0.20g，60％)。δ H(200MHz, CDCl 3) 8.27 (J 6.8 for 1H, d, H-4), and 7.44-7.58 (2H, m, H-7 and furyl H-5 '), 7.06-7.22 (1H, m, H-6), (6.93 J 3.4 for 1H, d, furyl H-3 '), 6.78 (1H, t, J 6.8, H-5), 6.53 (1H, dd, J 3.4 and 1.6, furyl H-4 '), 3.54 (1H, br s, NH) and 1.18[9H, s, C (CH 3) 3].
N-(cyclohexyl)-2-(2-furyl) imidazo [1,2-α] pyridine-3-amine (129)
(0.27g，73％)。δ H(200MHz, CDCl 3) 8.07 (1H, dd, J 6.7 and 1.2, H-4), 7.53 (J 9.0 for 1H, d, H-7), 7.51 (J 1.6 for 1H, d, furyl H-5 '), 7.15 (1H, ddd, J 9.0,6.7 and 1.2, H-6), 6.91 (J 3.3 for 1H, d, furyl H-3 '), 6.79 (1H, td, J 6.7 and 1.0, H-5), 6.55 (1H, dd, J 3.3 and 1.6, furyl H-4 '), 3.62 (1H, br s, NH), 2.85-3.10 (1H, m, CHNH), 1.50-2.00 and 1.05-1.45 (10H, m, 5 * cyclohexyl CH 2).
4-{3-[(2, the 6-xylyl) amino] imidazo [1,2-α] pyridine-2-yl } benzene-1,3-glycol (130)
(0.11g，23％)。δ H(200MHz, CDCl 3) 8.04 (J 8.6 for 1H, d, H-4), and 7.69 (1H, d, J 7.0, H-6 '), 7.56 (J 9.4, H-7) for 1H, d, 7.15-7.28 (H-6), 7.02 (J 7.4 for 2H, d for 1H, m, 2 * xylyl H-3 '), 6.72-6.92 (2H, m, H-5 and xylyl H-4 '), 6.53 (1H, d, J 1.7, H-3 '), 6.33 (1H, dd, J 8.2 and 1.7, H-5 '), 5.36 (1H, br s is NH) with 2.04 (6H, s, 2 * CH 3).
4-(3-cyclohexyl amino) imidazo [1,2-α] pyridine-2-yl) benzene-1,3-glycol (131)
(0.13g，26％)。δ H(200MHz, CDCl 3) 8.04 (J 6.8 for 1H, d, H-4), and 7.76 (1H, d, J 8.0, H-6 '), 7.28-7.38 (1H, m, H-7), 6.96-7.10 (1H, m, H-5 '), 6.71 (1H, t, J 6.8, H-5), 6.21-6.38 (2H, m, H-6 and H-3 '), 3.51 (1H, br s, NH), 2.71-2.95 (1H, m, CHNH), 1.30-1.75 and 0.85-1.28 (10H, 2 * m, 5 * cyclohexyl CH 2).
The conventional method of diamines and glyoxal water solution condensation
With 1 of aequum, the 2-diamines places 3cm 3In the water, add 40% glyoxal water solution (with labelled amount) to it.Add potassium hydroxide (2.1-3.1 equivalent) (except that not needing to add the o-phenylenediamine 1 of alkali) then, diamines is dissolved immediately.At room temperature, potpourri is stirred at the appointed time, in this process, generate the solid precipitation.With glacial acetic acid pH is dropped to and to be about 5, collect solid by vacuum filtration.Water, ethyl acetate or washing with acetone, vacuum drying then.
2-amino-4-hydroxy pteridine (132)
According to above-mentioned conventional method, with glyoxal (0.68cm 3, 4.66mmol) and potassium hydroxide (0.52g 9.31mmol) handles 4-hydroxyl-2,5,6-Triaminopyrimidine sulfate 2 (1.06g, 4.43mmol).In the time adding glyoxal, a spot of brown solid settles down from solution.By filtering out this solid in the yellow solution, this yellow solution forms thin yellow mercury oxide gradually at 18h between the reaction period.After the acidifying, yellow powder is filtered, water and washing with acetone and dry vacuum obtain 2-amino-4-hydroxy pteridine 132 (0.49g, 67%).δ H(200MHz, D 2O/NaOD) 8.47 (1H, d, N=CH aCH b=N, J 2.4) and 8.27 (1H, d, N=CH aCH b=N, J 2.4); δ C(50MHz, D 2O/NaOD) 173.4 (C-4), 156.7 (C-2), 148.6 (C-7), 138.7 (C-8) and 129.6 (C-10).
8,9-dihydroxy-8,9-glyoxalidine be [1,2-α] pteridine-5 (7H)-ketone (133) also
According to above-mentioned conventional method, with glyoxal (2cm 3) and potassium hydroxide (0.11g 1.97mmol) handles 4-hydroxyl-2,5, and 6-Triaminopyrimidine sulfate 2 (0.23g, 0.99mmol).In the 18h course of reaction, pink solution exists always, obtains pink powder after the acidifying.Solid separates, and is dried and reuses same steps as, and yellow powder is filtered, and water and washing with acetone and vacuum drying obtain 8, and 9-dihydroxy-8,9-glyoxalidine be [1,2-α] pteridine-5 (7H)-ketone 133 (0.16g, 77%) also.δ H(200MHz, d 6-DMSO) 9.34 (1H, br s, tradable D 2O, NH), 8.72 (1H, d, N=CH 3CH b=N, J 1.8), 8.48 (1H, d, N=CH aCH b=N, J 2.0), 7.48[1H, d, tradable D 2O, N ArylHCH a(OH), J 6.8], 6.59[1H, d, tradable D 2O, NHCH b(OH), J 7.8], 5.58[1H, d, N ArylHCH a(OH), J 6.8] and 4.97[1H, d, NHCH b(OH), J 8.0]; δ C(50MHz, d 6-DMSO) 159.1 (C-5), 158.2 (C-6a), 155.2 (C-4a), 150.1 (C-3), 140.3 (C-2), 130.8 (C-9b), 85.5 (C-8) and 84.1 (C-9).
Pteridine-2,4 (1H, 3H)-diketone (134)
According to above-mentioned conventional method, with glyoxal (2cm 3) and potassium hydroxide (0.11g, 1.90mmol) processing 5,6-diamido uracil sulfate 13 (0.22g, 0.91mmol) 18h.Form yellow mercury oxide this moment.After it adds acetate, dense thick yellow mercury oxide is filtered, water and washing with acetone and vacuum drying, obtain yellow powder pteridine-2,4 (1H, 3H)-diketone 134 (89.0mg, 60%).δ H (200MHz, d 6-DMSO) 11.73 (2H, br s, NH), 8.64 (1H, d, N=CH aCH b=N, J 2.2) and 8.52 (1H, d, N=CH aCH b=N, J 2.2).
(2-amino-4,7-dihydroxy pteridine-6-yl) ethyl acetate (136)
With 6-hydroxyl-2,4, (0.25g 1.03mmol) places the glacial acetic acid (7cm that is preheating to 90 ℃ to 5-Triaminopyrimidine 2 3) in, add immediately afterwards the methyl-oxalacetic ester sodium salt (0.27g, 1.27mmol).The heating that then solution refluxed fast makes potpourri boiling 18h.Obtain the particulate suspension.After being cooled to 5 ℃,, wash with water and vacuum drying, obtain thin dark brown powder (2-amino-4,7-dihydroxy pteridine-6-yl) ethyl acetate 136 (0.22g, 79%) by filtering the solid of collecting precipitation.δ H(200MHz, d 6-DMSO) 7.01 (1H, br s, OH), 6.39 and 6.29 (1H, 2 * br s), 4.09 (2H, q, OCH 2CH 3, J 7.0), 3.61 (2H, s, CH 2CO) and 1.18 (3H, t, OCH 2CH 3, J 7.0); δ C(50MHz, d 6-DMSO) 170.2 (CO), 166.1 and 159.3 (C-4 and C-7), 157.5 (C-6), 155.6 (C-8a), 152.1 (C-4a), 146.0 (C-6), 61.0 (OCH 2CH 3), 40.1 (CH 2CO) and 14.8 (OCH 2CH 3).
(7-hydroxyl-2,4-dioxo-1,2,3,4-tetrahydropteridine-6-yl) ethyl acetate (137)
With 4, (0.31g 1.30mmol) places the glacial acetic acid (25cm that is preheating to 90 ℃ to 5-diamido uracil sulfate 13 3) in, add immediately afterwards the methyl-oxalacetic ester sodium salt (0.35g, 1.65mmol).The heating that then solution refluxed fast makes potpourri boiling 5h.Obtain the particulate suspension.After being cooled to 5 ℃,, use 10cm by filtering the solid of collecting precipitation 3Hot ethanol washing and vacuum drying obtain thin dark brown powder (7-hydroxyl-2,4-dioxo-1,2,3,4-tetrahydropteridine-6-yl) ethyl acetate 137 (0.21g, 71%).δ H(200MHz, d 6-DMSO) 11.23 (1H, br s, OH), 4.10 (2H, q, OCH 2CH 3, J 7.0), 3.72 (2H, s, CH 2CO) and 1.19 (3H, t, OCH 2CH 3, J 7.0); δ C(50MHz, d 6-DMSO) 170.2 (CO), 160.9 and 160.7 (C-2 and C-4), 150.8 (C-7), 148.2 (C-6), 61.1 (OCH 2CH 3), 39.3 (CH 2CO) and 14.8 (OCH 2CH 3).
2,4-dihydroxy-4,5-(diethoxy carbonyl)-6-(4-nitrobenzophenone)-3,4,5,6-tetrahydropyrimidine (138)
With trifluoroacetic acid (3.2cm 3, 41.54mmol) add be dissolved in anhydrous 1,2-ethylene dichloride (92cm 3) urea (4.17g, 99.37mmol) (7.84g is in potpourri 51.91mmol) with the 4-nitrobenzaldehyde.(95%, 10.23g 46.24mmol) adds in the reaction mixture, reactant is refluxed heat 23h with the methyl-oxalacetic ester sodium salt.Reaction mixture is cooled to room temperature, concentrates, obtain brown viscosity grease with final vacuum.With ethyl acetate (150cm 3) continuation processing residue and water (3 * 150cm 3) washing.With ethyl acetate (200cm 3) water-washing liquid that extract to merge.Merging organic extract also, vacuum is concentrated into the about 150cm of initial volume 3The gained potpourri that will contain insoluble precipitation is transferred in the conical flask, and the heating that refluxes is until all insoluble matter dissolvings.The hexane that adds capacity to hot solution produces precipitation to induce.Potpourri will be stirred be cooled to room temperature.By filtering collecting precipitation, obtain waxy solid, this solid becomes slurry in hexane.By filtering collecting precipitation and using hexane wash.With the filter cake drying under reduced pressure, weight is about 20.31g.With solid by ethyl acetate-hexanes mixtures recrystallization, obtain cream-coloured amorphous solid, it is carried out spectral analysis, find that it is 2,4-dihydroxy-4,5-(diethoxy carbonyl)-6-(4-nitrobenzophenone)-3,4,5, the non-enantiomer mixture of the 6:1.4:1 of 6-tetrahydropyrimidine 138 (11.61g, 66%) is (by the NMR spectroscopic methodology; Vide infra); δ H(50MHz; DMSO-d 6) 8.26-8.18 and 8.20 (3 * 2H, m and d, J are about 8.5,6 * aryl H), 7.71-7.57 (3 * 2H, m and d, J are about 8.5,6 * aryl H), 7.37,7.06,6.94,6.71,6.63 and 6.52 (6 * 1H, 6 * s, 6 * NH), 5.20 (J 5.2, H-6) for 1H, d, 4.95-4.89 and 4.92 (J 11.5, H-6) for 2 * 1H, 2 overlapping d, 4.28-4.04 (J is about 7.0,3 * OCH for 3 * 2H, 2 overlapping q 2CH 3), (J is about 7.0,3 * OCH to 4.00-4.62 for 3 * 2H, 2 overlapping q 2CH 3), 3.25-3.10 and 3.20 (J 11.5 for 3 * 1H, 3 overlapping d, H-5), 1.30-1.22,1.27 and 1.26 (J is about 7.1,2 * OCH for 2 * 3H, 2 overlapping t 2CH 3), 1.67 (J is about 7.2 for 3H, t, OCH 2CH 3), 1.02-0.93,0.98 and 0.96 (2 * 3H, 2 overlapping t, J7.0 and 7.1,2 * OCH 2CH 3), 0.747 (J 7.2, OCH for 3H, t 2CH 3); δ C(50MHz; DMSO-d 6) 169.8 (CO 2Et), 167.7 (CO 2Et), 154.2 (NHCONH), 148.7,147.8,130.5 and 124.0 (aryl C), 81.7 (C-4), 62.4 (C-5), 61.3 (C-6), 52.9 and 52.7 (OCH 2CH 3), 14.6 and 14.2 (OCH 2CH 3).
2,4-dihydroxy-4,5-(diethoxy carbonyl)-6-(3,5-dimethoxy-4 '-hydroxyphenyl)-3,4,5,6-tetrahydropyrimidine (139)
With trifluoroacetic acid (2.1cm 3, 27.26mmol) add be dissolved in anhydrous 1,2-ethylene dichloride (62cm 3) in urea (2.66g, 44.29mmol) and syringaldehyde (6.04g is in potpourri 32.48mmol).(95%, 6.53g 29.53mmol) adds in the reaction mixture, reactant is refluxed heat 19h with the methyl-oxalacetic ester sodium salt again.In heating process, brown insoluble viscosity grease obviously appears.Reaction mixture is cooled to room temperature, and solvent decant from be not dissolved in grease is come out.Wash the reaction mixture behind the decant with water, dry (MgSO 4) and vacuum concentrate, obtain thick brown oil, analyze by TLC, this grease mainly is made up of unreacted syringaldehyde.Water (50cm 3) and ethyl acetate (50cm 3) the brown viscosity grease of mixture process remnants, and vigorous stirring number minute is dissolved in water-ethyl acetate mixture fully until grease.Potpourri is transferred to separating funnel, in this process, is divided into three layers.The bottom water is separated mutually with the emulsification mutually from remaining ethyl acetate.This two-phase is transferred in the conical flask, and the hexane that adds capacity in this conical flask produces precipitation to induce.With slurry stirred for several minute, after this, by filtering collecting precipitation.Filter cake is carried out drying under reduced pressure, obtain 2,4-dihydroxy-4,5-(diethoxy carbonyl)-6-(3,5-dimethoxy-4 '-hydroxyphenyl)-3,4,5, the non-enantiomer mixture of the 6:1 of 6-tetrahydropyrimidine 139 (3.57g, 29%); δ H(50MHz; DMSO-d 6) 8.30 (PhOH), 7.81 (J 3.3, NH) for 1H, br d for 1H, br s, 7.46 (J 1.6 for 1H, br d, NH), and 6.73 (1H, s, NH), 6.82 (1H, s, NH), 6.58 (2 * 1H, s, aryl H), 6.54 (2 * 1H, s, aryl H), 6.44 (1H, br s, OH), 5.10 (J 3.0 for 1H, br d, H-6), and 4.70 (1H, d, J 11.5, H-6), 4.28-3.78 (4 * 2H, 4 overlapping q, OCH 2CH 3), 3.78 and 3.77 (2 * 3H, 2 * s, 2 * aryl OCH 3), 3.10 (1H, d, J11.5, H-5), 1.31-1.22,1.28 and 1.26 (2 * 3H, 2 overlapping t, J 7.0 and 7.2, OCH 2CH 3), 1.11 (J 7.0, OCH for 3H, t 2CH 3), 0.98 (J 7.2, OCH for 3H, t 2CH 3); δ C(50MHz; DMSO-d 6) 170.1 (CO 2Et), 168.3 (CO 2Et), 154.4 (NHCONH), 148.3,136.1,130.7 and 106.2 (aryl C), 81.7 (C-4), 62.3 (C-5), 60.9 (C-6), 56.8 (aryl OCH 3), 53.4 (OCH 2CH 3), 14.6 and 14.4 (OCH 2CH 3).
2,4-dihydroxy-4,5-(diethoxy carbonyl)-6-(4-bromophenyl)-3,4,5,6-tetrahydropyrimidine (140)
With trifluoroacetic acid (3.2cm 3, 41.54mmol) add be dissolved in anhydrous 1,2-ethylene dichloride (90cm 3) in urea (4.09g, 68.02mmol) (9.32g is in potpourri 49.88mmol) with the 4-bromobenzaldehyde.(95%, 10.03g 45.34mmol) adds in the reaction mixture, reactant is refluxed heat 41h with the methyl-oxalacetic ester sodium salt again.Make reaction mixture be cooled to room temperature, concentrate, obtain the viscosity orange with final vacuum.Under intense stirring condition, use ethyl acetate (100cm 3) and water (100cm 3) this residue of continuation processing.Potpourri is phase-splitting subsequently, water (2 * 100cm 3) the washing organic phase.With ethyl acetate (2 * 100cm 3) water-washing liquid that extract to merge.Organic extract is merged, and vacuum concentrates, and obtains thick orange solids, and this solid is dissolved in the ethyl acetate of boiling.Make solution be cooled to room temperature, add hexane then and produce precipitation to induce.The gained slurry is stirred 15min.After this, with sedimentation and filtration and use hexane wash.With the filter cake drying under reduced pressure, obtain 2,4-dihydroxy-4,5-(diethoxy carbonyl)-6-(4-bromophenyl)-3,4,5, the non-enantiomer mixture of the 4:1 of 6-tetrahydropyrimidine 140 (4.92g, 26%); δ H(50MHz; DMSO-d 6) 7.61-7.51 and 7.53 (J 8.4, aryl H for 4 * 1H, 2 overlapping m and d), 7.35-7.20 and 7.33 (4 * 1H, 2 overlapping m and d, J 8.4, aryl H), 7.09 (1H, s, NH), 6.91 (1H, br d, J 1.4, and NH), 6.58 (J 1.0 for 1H, br d, NH), 6.50 (1H, s, NH), 5.03 (1H, br d, J 4.5, H-6), 4.76 (1H, d, J 11.5, H-6), and 4.25-4.05 (2 * 2H, 2 overlapping q, OCH 2CH 3), 3.93-3.65 (2 * 2H, 2 overlapping q, OCH 2CH 3), 3.20-3.10 and 3.13 (2 * 1H, overlapping d and br dd, J 11.5 and 0.8,2 * H-5), 1.26 (J is about 7.1 for 3H, t, OCH 2CH 3) 1.16 (J 7.0, OCH for 3H, t 2CH 3), 0.97 (J is about 7.1 for 3H, t, OCH 2CH 3) and 0.78 (J is about 7.1 for 3H, t, OCH 2CH 3); δ C(50MHz; DMSO-d 6) 169.9 (CO 2Et), 167.8 (CO 2Et), 154.9 (NHCONH), 154.2 (NHCONH), 140.5,131.8,131.1,129.6 and 121.6 (aryl C), 81.7 and 80.3 (C-4), 62.4 (C-5), 61.1 and 60.5 (C-6), 53.0 and 52.8 (OCH 2CH 3), 14.6 and 14.4 (OCH 2CH 3).
2,4-dihydroxy-4,5-(diethoxy carbonyl)-6-(trans-2-styrene)-3,4,5,6-tetrahydropyrimidine (141)
With trifluoroacetic acid (3.2cm 3, 41.54mmol) add be dissolved in anhydrous 1,2-ethylene dichloride (91cm 3) in urea (4.10g, 68.31mmol) and trans-cinnamic aldehyde (6.62g is in potpourri 50.09mmol).(95%, 10.07g 45.54mmol) adds reaction mixture, will react the heating 22h that refluxes with the methyl-oxalacetic ester sodium salt again.Reaction mixture is cooled to room temperature, concentrates, obtain the viscosity orange with final vacuum.With ethyl acetate (150cm 3) continuation processing residue and water (2 * 100cm 3) washing.With ethyl acetate (2 * 150cm 3) water-washing liquid that extract to merge.Organic extract is merged, and dry and vacuum concentrates, and obtains thick orange viscosity grease, because from the trial of the direct crystal of ethyl acetate-hexanes mixtures and unsuccessful, so this grease is carried out purifying by column chromatography.Obtain thick wax shape orange solids, this solid recrystallization from the hexane of ethyl acetate and minimal volumes comes out, obtain white powder 2,4-dihydroxy-4,5-(diethoxy carbonyl)-6-(trans-2-styrene)-3,4,5, the non-enantiomer mixture of the 4:1 of 6-tetrahydropyrimidine 141 (1.82g, 11%); δ H(50MHz; DMSO-d 6) 7.50-7.25 (2 * 5H, m, aryl H), 6.96 (1H, br s, NH), 6.77 (1H, br s, NH), 6.63 (2 * 1H, d, J is about 16.0, PhCH=CH), 6.47 (1H, br s, NH), 6.18 (2 * 1H, dd, J are about 16.0 and 8.0, PhCH=CH), 4.64-4.56 (1H, m, H-6), 4.38 (1H, dd, J is about 11.0 and 8.0, H-6), and 4.24-4.07 (2 * 2H, 2 overlapping q, OCH 2CH 3), 4.07-3.90 (2 * 2H, 2 overlapping q, OCH 2CH 3), 6.05 (J is about 4.0 for 1H, br d, H-5), 2.97 (J is about 11.0 for 1H, d, H-5), 1.30-1.16,1.27 and 1.19 (J is about 7.1 and 7.0, OCH for 2 * 3H, 2 overlapping t 2CH 3), 1.11-1.07,1.07 and 1.06 (J 7.0, OCH for 2 * 3H, 2 overlapping t 2CH 3); δ C(50MHz; DMSO-d 6) 170.0 (CO 2Et), 168.2 (CO 2Et), 154.2 (NHCONH), 137.0,133.0,131.7,128.4 with 127.4 (aryl C), 129.3 (CH=CHPh), 128.9 (CH=CHPh), 127.1 (CH=CHPh), 127.0 (CH=CHPh), 81.6 (C-4), 62.3 (C-5), 61.1 (C-6), 51.4 and 51.2 (OCH 2CH 3), 14.6 and 14.5 (OCH 2CH 3).
6-(4-nitrobenzophenone)-2-oxo-1,2,3,4-tetrahydropyridine-4,5-dicarboxylic acid (142)
To be dissolved in absolute ethyl alcohol (2cm 3) potassium hydroxide (0.13g, 2.24mmol) solution adds and to be dissolved in absolute ethyl alcohol (3cm 3) diethoxy carbonyl-tetrahydropyrimidine 138 (0.22g is 0.56mmol) in the suspension.Reaction mixture refluxed is heated 10min.In this process, precipitation almost exists always.The burgundy reaction mixture is cooled to room temperature, in cooling procedure, with aqueous hydrochloric acid solution (10%) potpourri is acidified to pH and is about 1-2, it is bright orange that color is become.By filtering the simple collection product, with a few fraction absolute ethanol washings, drying under reduced pressure obtains 6-(4-nitrobenzophenone)-2-oxo-1,2,3 then, 4-tetrahydropyridine-4,5-dicarboxylic acid 142 (0.16g, 90%); δ H(50MHz; DMSO-d 6) 11.26 (2 * 1H, br s, 2 * CO 2H), 8.31 (J is about 8.5 for 2H, d, aryl H), 8.18 (1H, br s, NH), 7.99 (J is about 8.5 for 2H, d, aryl H), 5.95 (1H, s, H-6).
8-morpholine and adenosine (147)
With morpholine (2.0cm 3) add 8-bromine adenosine 145 (0.15g, 0.43mmol the) diox (5cm of isopropylidene protection 3) in the solution, gained solution is carried out microwave irradiation (under 50% power of 600W) 40min.The hydrobromate sedimentation and filtration of morpholine is removed, and solvent is removed from filtrate.With gained solution ether (2 * 5cm 3) grind the solid (0.12mg, 71%) of the paste (cream collared) of the 8-morpholino adenosine 146 of generation isopropylidene protection.δ H(200MHz, CDCl 3) 8.18 (1H, s, H-2), 5.90 (1H, d, H-1 ', J 5.6), 5.27 (1H, dd, H-2 ', J 5.6 and 5.4), 5.08 (1H, d, H-3 ', J 5.4), 4.42 (1H, s, H-4 '), 3.90 (2H, m, H-5 '), 3.78[4H, m, O (CH 2CH 2) 2N], 3.39[2H, m, O (CH 2CH aH b) 2N], 3.15[2H, m, O (CH 2CH aH b) N], 1.60 and 1.38[6H, 2 * s, C (CH 3) 2]; (EI) m/z 392 (C 17H 24N 6O 5Requirement is 392).Spend the night by in aqueous hydrochloric acid solution (2M, 5), stirring, with gained material (10mg) deprotection in tetrahydrofuran.At this moment, in the sample and after carry out HPLC and analyze, find not have raw material remnants.After solvent removed, thick 147 can be used for suppressing research.
The in-vitro evaluation of embodiment 4 inhibitor
By limit the quantity of (adenylylation form) at the excessive and carbon of nitrogen or nitrogen is limited the quantity of and the condition of carbon excessive (going the adenylylation form) under the Continuous Cultivation Escherichia coli, can generate adenylylation and remove the GS enzyme of adenylylation form.Use the enzyme of two kinds of forms of AMP-agarose affinity chromatography purifying, analyze to detect the inhibition of glutamine synthelase.Be hydrolyzed to ADP and form glutamine by ATP and measure enzymatic activity by glutamic acid.
Material and method
Generate the enzyme of two kinds of forms by the reorganization glutamine synthelase construct pBSK-ECgln of glutamine synthelase deficiency host strain Escherichia coli YMC11.Make biology growing by Continuous Cultivation; wherein for the adenylylation enzyme; under limiting the quantity of condition, cultivates in the excessive and carbon of nitrogen; and for removing the adenylylation enzyme; under the condition that nitrogen is limited the quantity of and carbon is excessive, cultivate, as document (Senior, P.J. (1975); J.Bact:123 (2), 407-418) described such.The fermentation of carrying out under the excessive and carbon of nitrogen is limited the quantity of condition, the initial growth speed of its growth is 0.07 hour -1, after through 5 retention times, growth rate was reduced to 0.01 hour -1Thereby, reach stable state.(N.J., USA) fermentation tank are that 1v/v/m and stir speed (S.S.) are to carry out all fermentations under the 600rpm at 37 ℃, pH 7.15, air velocity to use NewBrunswick 3000.Biomass collection, and preservation under 6 ℃ continuously, after this, with its centrifugal 20min and freezing down under 10,000 * g at-20 ℃.Stablize guaranteeing by measuring culture absorbance (600nm) and cell survival rate and continuing to monitor fermentation by the mensuration biomass concentration, wherein cell survival rate obtains by plate count agar is carried out plate count.During the fermentation, from fermentation system, take out single bacterium colony chorista at set intervals, and contain the integrality of the plasmid of GS gene by PCR screening and plasmid separate authentication.
Living beings are resuspended in resuspended buffering agent A or RBA (10mM imidazoles-HCl, 2mM beta-mercaptoethanol, 10mM MnCl 24H 2O; PH 7.0) in.With cell ultrasonic 10min under 50% dutycycle.With this ultrasonic solution centrifugal 10min under 12000 * g, and keep supernatant.Add streptomycin sulphate (10%w/v 10%), and stir suspension 10min down at 4 ℃.The supernatant of centrifugal 10min under 12000 * g, and reservation then.Regulate supernatant pH to 5.15 with sulfuric acid.Stir this potpourri 15min, centrifugal 10min under 20000 * g then down at 4 ℃.Keep supernatant once more.Add saturated ammonium sulfate (volume 30%) and regulate pH to 4.6 with sulfuric acid.Stir suspension 15min, centrifugal 10min under 20000 * g then down at 4 ℃.The precipitation that obtains is resuspended in RBA and regulates pH to 5.7 with sulfuric acid.4 ℃ stir down that these suspensions spend the night so that glutamine synthelase be able to resuspended, centrifugal 10min under 20000 * g then.Keep supernatant and with suspension pH regulator to 7.0.
Use AKTA Explorer (Amersham Biosciences), utilize affinity chromatography that the enzyme of two kinds of forms is further purified.Use length to realize separating with 5 ' AMP agarose resin as the HR10/10 post of 10mm with internal diameter as 10cm.To preparative column, described preparative column is with 10mM imidazoles (pH7.0), 150mM NaCl and 10mM MnCl with glutamine synthelase preparation (being about 2ml) application of sample 24H 2O comes balance.At the 40mlNaCl of whole linear gradient, use 2.5mM ADP that the glutamine synthelase of combination is eluted from post, and collect the 1ml component from 150mM to 500mM.Collect then and merge the component that contains pure glutamine synthelase and use the RBA dialysed overnight.
According to standard scheme, the aliquot to each albumen suspension on the 7.5%SDS-PAGE gel is carried out electrophoresis.Use Lowry Protein Detection method to measure protein concentration, described concentration is used to measure the specific activity of all enzymes.
The analytic approach that use comes from measuring enzyme positive reaction development is estimated the influence of every kind of inhibitor to activity of glutamine synthetase.In each case, every kind of inhibitor is all detected the adenylylation form of enzyme and goes the adenylylation form.All analyze parallel carrying out twice.In inhibitor concentration is to carry out primary dcreening operation under the 1mM.The analyte component is shown in table 29.
Table 29. analyte component
Figure A200680054592D01741
All inhibitor are prepared into the 10mM storing solution of dimethyl sulfoxide (DMSO).Under pH6.3, carry out the analysis of adenylylation enzyme, and the analysis of under pH7.2, removing the adenylylation enzyme.The volume of all enzyme preparations with 50 μ l added in the analysis of mixtures.The adding of enzyme has started reaction, then, for the adenylylation enzyme, can carry out 2.5 hours reaction, for removing the adenylylation enzyme, can carry out the reaction of 45min.Making its concentration by the adding trichloroacetic acid is that 0.5%m/v comes cessation reaction.Then each analyte is all assigned to the HPLC bottle, on Agilent 100 HPLC equipment, use Phenomenex Luna 5 μ C18 posts that wherein two are carried out glutamic acid and glutamine analysis, and other two carried out ADP and ATP analyzes.
The inhibition analysis of enzyme
Calculate the specific enzyme activity that every kind of inhibitor is analyzed with μ mole/min/mg albumen.Then every kind of inhibitor analysis is compared with the analysis (check analysis that does not promptly contain inhibitor) of untamed enzyme, calculated the reduction of enzymatic activity, report with percentage then.The active scope that reduces can be selected 80-100%, 60-80%, 30-60% and 0-30%.These results are shown in table 30.
The relative inhibition level that the inhibitor compound that table 30. detected produces adenylylation and the enzyme that goes the adenylylation form.
Figure A200680054592D01742
Figure A200680054592D01761
Based on the result shown in the table 30, select some inhibitor compounds further to detect.Table 31 has shown these inhibitor compounds and their relative inhibition level.
Table 31. is used for the further inhibitor compound of detection
Figure A200680054592D01771
Can select useful inhibitor based on the inhibition percentage of GS activity.With respect to removing adenylylation GS, the ADP of adenylylation GS or glutamine are formed active selection inhibition be particularly useful.In addition, with respect to glutamine, show that to ADP is synthetic more highly inhibited inhibitor may also be useful.
The inhibitor activity evaluation of embodiment 5 Much's bacillus and mammalian cell
Material and method
The BACTEC system is designed to be used to monitor the mycobacterium growth of slow growth species.Bacterium is grown, the radioactivity carbon dioxide of generation directly be directly proportional with mycobacterium growth speed (Siddiqi, S.H., BACTEC 460 TB system, Product and proceduremanual (1995)) on radioactivity matrix.Read value is expressed as growth index (GI).On the 7H9 mycobacterium nutrient culture media (Difco) that is rich in ADC (Biolabart C70), Much's bacillus reference strain H37Rv (ATCC 25618) is continued to cultivate under the stirring at 37 ℃.When the A600nm value is between the 0.4-0.6 time, the growth of logarithmic phase can be accepted.When the A600nm of culture value reaches (one McFarland) when being about 0.16 density, get 0.1ml and be inoculated in the Bactec bottle.Cultivate these primary cultures down at 37 ℃ and reach 500 (+/-50) until growth index.These primary cultures are used for inhibitor to be detected.
By having the aperture is that the 13mm organic solvent-resistant syringe filters in 0.22 micron hole (Millex-LG) is sterilized to the detection compound in the dimethyl sulfoxide (DMSO).GIA to undiluted sample detects.Will show that thereby active undiluted sample dilution mensuration in turn in aseptic DMSO continues the dilution gfactor of GIA.The inhibitor that detects is shown in table 32.
The inhibitor that detects during table 32.BACTEC analyzes
Compound Bacteriostat is/denys
Cinchonine Not
Beta, gamma-methylene adenosine-5 '-triphosphate Not
Thiamine hydrochloride Not
4,5,6-Triaminopyrimidine hydrosulfate (5) Be
6-amino-1,3-dimethyl-5-nitroso-uracil (15) Not
N-(4-amino-6-hydroxy pyrimidine-5-yl) benzamide (37) Not
6-amino-5-{[(1E, 2E)-3-phenyl-2-allylidene] amino } pyrimidine-2,4 (1H, 3H)-glycol (42) Be
9-methyl-6-pyrrolidine-1-base-9H-purine (81) Not
N-(2-hydroxyethyl)-1-{6-[(2-hydroxyethyl) amino] pyrimidine-4-yl } pyrrolidine-2-formamide (95) Not
N-(4-bromophenyl)-6-pyrrolidine-1-yl pyrimidines-4-amine (97) Be
N-benzyl-5-bromo-6-pyrrolidine-1-yl pyrimidines-4-amine (105) Be
N 4，N 5-dibenzyl-6-pyrrolidine-1-yl pyrimidines-4,5-diamines (111) Be
The N-tert-butyl group-2-phenylimidazole is [1,2-α] pyridine-3-amine (117) also Be
(2-amino-4,7-dihydroxy pteridine-6-yl) ethyl acetate (136) Not
0.1ml primary culture and inhibitor compound are added in the BACTEC bottle, cultivate bottle down at 37 ℃, and every once its growth of monitoring in 24 hours.Contrast includes inhibitor and does not have inhibitor and solvent is arranged and do not have the culture of solvent.Proceed the GI reading and reach 999 until the maximum GI value of contrast.
For the vitro cytotoxicity of test sample, use 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) analytic approach to Chinese hamster ovary (CHO) cell to mammal cell line.
Under every kind of situation, to the parallel detection of all samples three times.Use MTT-analytic approach cell growth and survival to carry out colorimetric analysis and compare with other used analytic approach (people such as Mosman, 1983 and people such as Rubinstein, 1990).Use tetrazolium salts MTT to measure all growths and chemosensitivity.
By the 10mM storing solution among the CSIR acquisition 100%DMSO.If dissolve bad then, and be stored under-20 ℃ until use with suspension form test sample.The all test specimens of screening under 50 μ M.Select this concentration not influence cell survival with used solvent strength in guaranteeing to analyze.High solvent concentration pair cell survival that cell contacted does not have the influence (data not shown) that can measure.All the experiment in, with emetine with comparing medicine.The initial concentration of emetine is 100 μ g/ml, with 10 times of dilution stepwise dilutions, obtains six concentration in complete medium, and least concentration is 0.001 μ g/ml.
By the GraphPad Prism non-linear dose-response curve Fitting Analysis of software v.4, obtain 50% inhibition concentration (IC of these samples by dose-response curve 50) value.
Result and discussion
In the presence of different inhibitor, carry out the Bactec growth response curve of Much's bacillus.By measuring the influence of inhibitor concentration, detect the LC of inhibitor then to Much's bacillus Bactec growth 50Concentration, those inhibitor that detected are to find to suppress the inhibitor of Much's bacillus.LC 50Concentration is shown in table 33.
The LC of table 33. Much's bacillus inhibitor 50Concentration (μ M)
Inhibitor LC 50
97 87.79
105 52.68
111 56.44
117 73.33
Detect the cytotoxicity of all inhibitor then to mammalian cell (for example Chinese hamster ovary celI).Be to begin to detect all samples under the 50 μ M in concentration earlier.By the gained result as can be known, all samples all shows there is not significant cytotoxicity (table 34) in this concentration to Chinese hamster ovary celI system.
The cell survival percentage of table 34. under 50 μ M
Compound Cell survival percentage
Cinchonine 92.5
Beta, gamma-methylene adenosine-5 '-triphosphoric acid 93.0
Thiamine hydrochloride 93.6
5 87.6
15 89.5
37 100.9
42 92.1
81 89.0
95 96.3
97 66.6
105 66.4
111 50.8
117 83.2
136 94.8
IC 50(μg/ml)
Emetine 0.06
Emetine 0.07
This paper has described some embodiments of the present invention.Yet, be understandable that, under the condition that does not depart from the present invention's spirit and protection domain, can make various modifications.Therefore, other embodiment also drops in the scope of following claims.

Claims (68)

1. the computer-aid method of the test inhibitor of a phosphoryl transferase site activity that produces adenylylation glutamine synthelase (GS) polypeptide, described method is used the program mode computing machine that comprises processor and input equipment, and described method comprises:
(a) comprise the data of the phosphoryl transferase site structure of GS polypeptide to the input of described input equipment;
(b) using described processor will test inhibitor molecules is docked in the described phosphoryl transferase site; And
(c), determine whether described test inhibitor molecules suppresses the activity in described phosphoryl transferase site based on described butt joint.
2. method according to claim 1 further comprises (Mn 2+) 3(HCO 3 -) 12One or more structural motifs of ATP compound are docked in the described phosphoryl transferase site.
3. method according to claim 2 further comprises based on described butt joint, determines whether described test inhibitor molecules suppresses described (Mn 2+) 3(HCO 3 -) 12One or more structural motifs of ATP compound and combining of described phosphoryl transferase site.
4. method according to claim 1, further comprise design by the definite test inhibitor of step (c) with the active and described test inhibitor of in-vitro evaluation that suppresses described phosphoryl transferase site inhibition activity to adenylylation glutamine synthelase polypeptide.
5. method according to claim 4, wherein said in-vitro evaluation comprise the analytic approach of using energy measurement ATP hydrolysis, ADP formation, glutamic acid utilization or glutamine to form.
6. method according to claim 4; further comprise the described test inhibitor of in-vitro evaluation to going the inhibition activity of adenylylation glutamine synthelase polypeptide, active to estimate described test inhibitor to the specificity inhibition of described adenylylation glutamine synthelase polypeptide.
7. method according to claim 1 further comprises generating described test inhibitor and estimating the inhibition activity of described test inhibitor to the bacterial growth that contains GSI-β glutamine synthetase gene.
8. method according to claim 7, wherein said bacterium is selected from Bacterium diphtheriae, gonococcus, Escherichia coli, salmonella typhimurium, salmonella typhi, Klebsiella Pneumoniae, serratia marcescens, proteus vulgaris, Shigella dysenteriae, comma bacillus, pseudomonas aeruginosa, Bacillus foecalis alkaligenes, helicobacter pylori, haemophilus influenzae, Bordetella pertussis, the special bacterium of bronchitis Boulder, Neisseria meningitidis, Brucella melitensis, Much's bacillus, Mycobacterium leprae, Tyreponema pallidum, leptospira interrogans, actinomyces israelii, Nocardia asteroides, Thiobacillus ferrooxidans, Azospirillum brasilense, anabena, Fremyella diplosiphon and streptomyces coelicolor.
9. method according to claim 7 further comprises and estimates the inhibition activity of described test inhibitor to the eukaryotic growth.
10. method according to claim 9, wherein said eukaryotic is a mammalian cell.
11. method according to claim 5, wherein said analytic approach are included in pH and are about under the condition of 6.0-6.5, and described adenylylation glutamine synthelase polypeptide is contacted with described test inhibitor.
12. a method that produces the compound of the phosphoryl transferase site activity that suppresses adenylylation glutamine synthelase polypeptide, described method comprises:
(a) provide the three-dimensional structure of glutamine synthelase polypeptide; And
(b) based on described three-dimensional structure, design can suppress described phosphoryl transferase site and (Mn 2+) 3(HCO 3 -) 12Interactional test compound between one or more structural motifs of ATP compound.
13. method according to claim 12, the three-dimensional structure of wherein said glutamine synthelase polypeptide comprises the (Mn that is attached to described phosphoryl transferase site 2+) 3(HCO 3 -) 12One or more structural motifs of ATP compound.
14., further comprise the test compound that generates described step (b) and the described test compound of in-vitro evaluation inhibition activity to adenylylation glutamine synthelase polypeptide according to claim 12 or 13 described methods.
15., further comprise the test compound that generates described step (b) and estimate the inhibition activity of described test compound to the bacterial growth that contains GSI-β glutamine synthetase gene according to claim 12 or 13 described methods.
16. method according to claim 15, wherein said bacterium is selected from Bacterium diphtheriae, gonococcus, Escherichia coli, salmonella typhimurium, salmonella typhi, Klebsiella Pneumoniae, serratia marcescens, proteus vulgaris, Shigella dysenteriae, comma bacillus, pseudomonas aeruginosa, Bacillus foecalis alkaligenes, helicobacter pylori, haemophilus influenzae, Bordetella pertussis, the special bacterium of bronchitis Boulder, Neisseria meningitidis, Brucella melitensis, Much's bacillus, Mycobacterium leprae, Tyreponema pallidum, leptospira interrogans, actinomyces israelii, Nocardia asteroides, Thiobacillus ferrooxidans, Azospirillum brasilense, anabena, Fremyella diplosiphon and streptomyces coelicolor.
17. method according to claim 15 further comprises and estimates the inhibition activity of described test compound to the eukaryotic growth.
18. method according to claim 17, wherein said eukaryotic is a mammalian cell.
19. a method that produces the test compound of the phosphoryl transferase site activity that suppresses adenylylation glutamine synthelase polypeptide, described method comprises:
(a) (Mn is provided 2+) 3(HCO 3 -) 12The three-dimensional structure of ATP compound; And
(b) based on described three-dimensional structure, design has and is similar to described (Mn 2+) 3(HCO 3 -) 12The test compound of one or more structural motifs of ATP composite structure.
20. a body outer screening test compound is to determine whether described test compound suppresses the method for the phosphoryl transferase site activity of adenylylation glutamine synthelase polypeptide, and described method comprises:
(a) under effective condition, adenylylation glutamine synthelase polypeptide is contacted with test compound to phosphoryl transferase activity; And
(b) determine with respect to also not with the activity of the contacted adenylylation glutamine synthelase polypeptide of described test compound, whether the activity of the phosphoryl transferase of described adenylylation glutamine synthelase polypeptide decreases.
21. method according to claim 20; further comprise and determine whether the described activity of the phosphoryl transferase of adenylylation glutamine synthelase polypeptide of going decreases with respect to also there not being the activity of removing adenylylation glutamine synthelase polypeptide contacted with described test compound.
22. method according to claim 20, the analytic approach of wherein using energy measurement ATP hydrolysis, ADP formation, glutamic acid utilization or glutamine to form is measured the activity of described phosphoryl transferase.
Make adenylylation GS polypeptide and contain the compound compositions that formula I as herein described, II, III, IV, V, VI or VII represent and contact 23. an in-vitro method that is used to suppress the phosphoryl transferase site activity of adenylylation GS polypeptide, described method comprise.
Make described bacterium and contain the compound compositions that formula I as herein described, II, III, IV, V, VI or VII represent and contact 24. an in-vitro method that is used to suppress contain the bacterial growth of GSI-β gene, wherein said method comprise.
25. one kind be used for the treatment of, one or more symptoms that prevention or improvement are relevant with the mammal bacterial infection or the method for obstacle, wherein said bacterial infection causes by the bacterium that contains GSI-β gene, and described method comprises and gives described mammal with containing the compound compositions that formula I as herein described, II, III, IV, V, VI or VII represent.
26. method in the body of a phosphoryl transferase site activity that is used to suppress adenylylation glutamine synthelase polypeptide, described method comprises:
(a) will contain the mammal that compound compositions that formula I as herein described, II, III, IV, V, VI or VII represent is subjected to bacterial infection, wherein said bacterial infection is caused by the bacterium that contains GSI-β gene.
27. the computer-aid method based on the test inhibitor of the phosphoryl transfer reaction of phosphine formic acid of design adenylylation glutamine synthelase (GS) polypeptide; described method is used the program mode computing machine that comprises processor and input equipment, and described method comprises:
(a) the householder method design experiment inhibitor molecules that uses a computer, described test inhibitor molecules contain and are similar to described (Mn 2+) 3(HCO 3 -) 12ATP compound or based on one or more structural motifs of the described transition state intermediate of the phosphoryl transfer reaction intermediate of phosphine formic acid; And
(b) the auxiliary docking concept that uses a computer determines whether designed test inhibitor molecules suppresses based on the active of the phosphoryl transferase of phosphine formic acid or do not interact with one or more amino acid of described adenylylation glutamine synthelase polypeptide.
28. method according to claim 27, wherein interacting with described one or more amino acid is to interact by one or more amino acid whose one or more hydrogen bonds in the avtive spot or Van der Waals.
29. method according to claim 27 further comprises and uses the analytic approach of energy measurement ATP hydrolysis, ADP formation, glutamic acid utilization or glutamine formation to come the inhibition activity of the designed test inhibitor molecules of in-vitro evaluation to adenylylation GS polypeptide.
30. according to each described method among the claim 23-36, wherein said composition contains the compound that is selected from compound 97,105,111 and 117.
31. according to each described method in claim 24 or 25, the bacterium of the wherein said GSI-of containing β gene is selected from Bacterium diphtheriae, gonococcus, Escherichia coli, salmonella typhimurium, salmonella typhi, Klebsiella Pneumoniae, serratia marcescens, proteus vulgaris, Shigella dysenteriae, comma bacillus, pseudomonas aeruginosa, Bacillus foecalis alkaligenes, helicobacter pylori, haemophilus influenzae, Bordetella pertussis, the special bacterium of bronchitis Boulder, Neisseria meningitidis, Brucella melitensis, Much's bacillus, Mycobacterium leprae, Tyreponema pallidum, leptospira interrogans, actinomyces israelii, Nocardia asteroides, Thiobacillus ferrooxidans, Azospirillum brasilense, anabena, Fremyelladiplosiphon and streptomyces coelicolor.
32. according to claim 24 or 25 described methods, wherein said composition causes the activity of mammal GSII polypeptide to reduce about 0-30%.
33. method according to claim 32, wherein said mammal is the people.
34. according to claim 24 or 25 described methods, wherein said composition causes the activity of adenylylation GSI-beta polypeptides to reduce about 0-30%.
35. according to claim 24 or 25 described methods, the bacterium of the wherein said GSI-of containing β gene is a Much's bacillus.
36. one kind suc as formula I
Figure A200680054592C00071
Compound or its pharmaceutically acceptable salt or the derivant of expression, wherein:
R 1Be hydrogen, halogen, OR 5Or NR 6R 7
R 2Be hydrogen, halogen or NR 7R 8
R 3Be hydrogen, halogen or NR 6R 7
R 4Be SR 5, NR 6R 7Or H;
R 5Be H, replacement or unsubstituted C1-C20 alkyl, alkenyl or alkynyl, wherein said alkyl, alkenyl or alkynyl can be straight chain, side chain or ring-type; Or replace or unsubstituted aryl or heteroaryl; And
R 6, R 7And R 8Each is independently selected from H; Acyl group; Hydroxyl; And replace or unsubstituted alkyl, naphthenic base, aryl or heteroaryl; Or R 6And R 7Can form together replacement or unsubstituted naphthenic base, heteroaryl or heterocyclic radical; Or NR 7R 8Can be the form of N=O,
Wherein 1-3 substituting group allows on the part of any replacement, and wherein substituting group can be independently selected from alkyl, thiazolinyl, alkynyl, aryl, heteroaryl, halogen, carboxylate, acid amides, NR 7R 8, OR 5, ketone group, SH and SO 3H.
37. compound according to claim 36, wherein R 6And/or R 7And/or R 8It can be the alkyl or cycloalkyl that replaces.
38. compound according to claim 36, wherein R 1Be chlorine.
39. compound according to claim 36, wherein R 1Be NR 6R 7, R wherein 6Be H and R 7Be methyl, benzyl, 2-hydroxyethyl, 4-bromophenyl or 2-pyridine radicals.
40. compound according to claim 36, wherein R 2Be nitroso-, amino, bromine, aminoalkyl or ammonia aryl.
41. compound according to claim 36, wherein R 3It is chlorine, dimethylamino, pyrrolidinyl, morpholinyl or 2-(pyrrolidine-1-yl) carboxylate.
42. one kind suc as formula II
Figure A200680054592C00091
Compound or its pharmaceutically acceptable salt or the derivant of expression, wherein:
R 1Be hydrogen, halogen, OR 5Or NR 6R 7
R 4Be hydrogen, SR 5, NR 6R 7Or OR 5
R 5Be H, alkyl, the alkenyl or alkynyl of replacement or unsubstituted C1-C20, wherein said alkyl, alkenyl or alkynyl can be straight chain, side chain or ring-type; Replace or unsubstituted aryl or heteroaryl;
R 6, R 7And R 8Each is independently selected from H; Acyl group; Hydroxyl; And replace or unsubstituted alkyl, naphthenic base, aryl or heteroaryl; Or R 6And R 7Can form together replacement or unsubstituted naphthenic base, heteroaryl or heterocyclic radical; Or NR 7R 8It can be the form of N=O;
R 9Be H, halogen or replacement or unsubstituted alkyl, aryl, heterocyclic radical, heteroaryl, OR 5Or NR 6R 7
X and Y can be N or CH independently; And
Wherein 1-3 substituting group allows on the part of any replacement, and wherein substituting group can be independently selected from alkyl, thiazolinyl, alkynyl, aryl, heteroaryl, halogen, carboxylate, acid amides, NR 7R 8, OR 5, ketone group, SH and SO 3H.
43. according to the described compound of claim 42, wherein R 1Be OH or NH 2
44. according to the described compound of claim 42, wherein R 4Be H, OH or NH 2
45. according to the described compound of claim 42, wherein R 9Be alkyl, thiazolinyl, alkynyl or the aryl that replaces.
46. one kind as formula III
Figure A200680054592C00101
Compound or its pharmaceutically acceptable salt or the derivant of expression, wherein:
R 1Be hydrogen, halogen, OR 5Or NR 6R 7
R 4Be hydrogen, SR 5, NR 6R 7Or OR 5
R 5Be H, replacement or unsubstituted C1-C20 alkyl, alkenyl or alkynyl, wherein said alkyl, alkenyl or alkynyl can be straight chain, side chain or ring-type; Replace or unsubstituted aryl or heteroaryl;
R 6And R 7Each is independently selected from H; Acyl group, hydroxyl; And replace or unsubstituted alkyl, naphthenic base, aryl or heteroaryl; Or R 6And R 7Can form together replacement or unsubstituted naphthenic base, heteroaryl or heterocyclic radical;
X, Y can be CH or N independently; And
Wherein 1-3 substituting group allows on the part of any replacement, and wherein substituting group can be independently selected from alkyl, thiazolinyl, alkynyl, aryl, heteroaryl, halogen, carboxylate, acid amides, NR 7R 8, OR 5, ketone group, SH and SO 3H.
47. according to the described compound of claim 46, wherein R1 is OH or H.
48. one kind suc as formula IV
Figure A200680054592C00102
Compound or its pharmaceutically acceptable salt or the derivant of expression, wherein:
R 11Be hydrogen or replacement or unsubstituted C1-C20 alkyl, alkenyl or alkynyl, wherein said alkyl, alkenyl or alkynyl can be straight chain, side chain or ring-type; Replace or unsubstituted aryl or heteroaryl; The R that allows in any replacement of 1-3 substituting group wherein 11On the part, wherein substituting group can be independently selected from alkyl, thiazolinyl, alkynyl, aryl, heteroaryl, halogen, carboxylate, acid amides, NR 7R 8, OR 5, ketone group, SH and SO 3H; And
R 12The alkyl or alkenyl that is unsubstituted or replaces or aryl unsubstituted or that replace, wherein R 12Substituting group can be selected from NH 2, OH, COOH, CHO, NCHO, CONH 2, halogen, OR 5, CO 2R 5And NR 6R 7, wherein:
R 5Be H, replacement or unsubstituted C1-C20 alkyl, alkenyl or alkynyl, wherein said alkyl, alkenyl or alkynyl can be straight chain, side chain or ring-type; Replace or unsubstituted aryl or heteroaryl; And
R 6And R 7Each is independently selected from H; Acyl group, hydroxyl; And replace or unsubstituted alkyl, naphthenic base, aryl or heteroaryl; Or R 6And R 7Can form together replacement or unsubstituted naphthenic base, heteroaryl or heterocyclic radical; And
Wherein 1-3 substituting group allows independently at R 5, R 6Or R 7On the part that replaces, wherein substituting group can be independently selected from alkyl, thiazolinyl, alkynyl, aryl, heteroaryl, halogen, carboxylate, acid amides, NR 7R 8, OR 5, ketone group, SH and SO 3H.
49. according to the described compound of claim 48, wherein R 11Be alkyl or H.
50. one kind suc as formula V
Compound or its pharmaceutically acceptable salt or the derivant of expression, wherein:
R 1Be hydrogen, halogen, OR 5Or NR 6R 7
R 5Be H, replacement or unsubstituted C1-C20 alkyl, alkenyl or alkynyl, wherein said alkyl, alkenyl or alkynyl can be straight chain, side chain or ring-type; Replace or unsubstituted aryl or heteroaryl;
R 6And R 7Each is independently selected from H; Acyl group, hydroxyl; And replace or unsubstituted alkyl, naphthenic base, aryl or heteroaryl; Or R 6And R 7Can form together replacement or unsubstituted naphthenic base, heteroaryl or heterocyclic radical;
R 13Be independently replace or unsubstituted alkyl, aryl, heteroaryl or naphthenic base;
R 14Be H or NHR 15, R wherein 15Be independently replace or unsubstituted alkyl, aryl, heteroaryl or naphthenic base;
X and Y can be N or CH independently; And
Wherein 1-3 substituting group allows on the part of any replacement, and wherein substituting group can be independently selected from alkyl, thiazolinyl, alkynyl, aryl, heteroaryl, halogen, carboxylate, acid amides, NR 7R 8, OR 5, ketone group, SH and SO 3H.
51. according to the described compound of claim 50, wherein R 13Be that replace or unsubstituted aryl.
52. according to the described compound of claim 50, wherein R 14Be replace or unsubstituted aryl, alkyl or cycloalkyl.
53. one kind suc as formula VI
Figure A200680054592C00121
Compound or its pharmaceutically acceptable salt or the derivant of expression, wherein:
R 13Be hydrogen or replacement or unsubstituted C1-C20 alkyl, alkenyl or alkynyl, wherein said alkyl, alkenyl or alkynyl can be straight chain, side chain or ring-type; Replace or unsubstituted aryl or heteroaryl; The R that allows in any replacement of 1-3 substituting group wherein 13On the part, wherein substituting group can be independently selected from alkyl, thiazolinyl, alkynyl, aryl, heteroaryl, halogen, carboxylate, acid amides, NR 7R 8, OR 5, ketone group, SH and SO 3H; And
R 12The alkyl or alkenyl that is unsubstituted or replaces or aryl unsubstituted or that replace, wherein R 12Substituting group can be selected from NH 2, OH, COOH, CHO, NCHO, CONH 2, halogen, OR 5, CO 2R 5And NR 6R 7, wherein:
R 5Be H, replacement or unsubstituted C1-C20 alkyl, alkenyl or alkynyl, wherein said alkyl, alkenyl or alkynyl can be straight chain, side chain or ring-type; Replace or unsubstituted aryl or heteroaryl; And
R 6And R 7Each is independently selected from H; Acyl group, hydroxyl; And replace or unsubstituted alkyl, naphthenic base, aryl or heteroaryl; Or R 6And R 7Can form together replacement or unsubstituted naphthenic base, heteroaryl or heterocyclic radical; And
Wherein 1-3 substituting group allows independently at R 5, R 6Or R 7On the part that replaces, wherein substituting group can be independently selected from alkyl, thiazolinyl, alkynyl, aryl, heteroaryl, halogen, carboxylate, acid amides, NR 7R 8, OR 5, ketone group, SH and SO 3H.
54. according to the described compound of claim 53, wherein R 12Be replace or unsubstituted aryl or thiazolinyl.
55. one kind suc as formula VII
Figure A200680054592C00131
Compound or its pharmaceutically acceptable salt or the derivant of expression, wherein:
R 6And R 7Each is independently selected from H; Acyl group, hydroxyl; And replace or unsubstituted alkyl, naphthenic base, aryl or heteroaryl; Or R 6And R 7Can form together replacement or unsubstituted naphthenic base, heteroaryl or heterocyclic radical; And
R 14Be H; Acyl group, replacement or unsubstituted alkyl, naphthenic base, aryl or heteroaryl;
Wherein 1-3 substituting group allows on the part of any replacement, and wherein substituting group can be independently selected from alkyl, thiazolinyl, alkynyl, aryl, heteroaryl, halogen, carboxylate, acid amides, NR 7R 8, OR 5, ketone group, SH and SO 3H.
56. a pharmaceutical composition, it comprises the compound of representing suc as formula I, II, III, IV, V, VI or VII with the combination of pharmaceutically acceptable carrier or thinning agent.
57. a compound of representing suc as formula I, II, III, IV, V, VI or VII is used for the treatment of, prevents or improves mammiferous bacterial infection.
58. a compound of representing suc as formula I, II, III, IV, V, VI or VII is used for the treatment of, prevents in preparation or improves application in the medicine of mammal bacterial infection.
59. a compound or its pharmaceutically acceptable salt or derivant, described compound is selected from the compound in the following table, and wherein said compound has the structure of the compound of described numbering:
Compound 2,5,8,10,18,17,19,20,21,23,35,36,37,39,40,41,42,45,48,50,52,53,54,55,57,58,60,61,62,63,64,66,67,68,69,70,73,74,75,76,77,80,81,82,83,88,89,90,91,93,94,95,96,97,98,99,100,102,104,105,108,110,111,113,114,115,117,121,124,132,134,136,137,138,139,140,141 and 142.
60. a pharmaceutical composition, it comprises according to the compound of claim 59 and pharmaceutically acceptable carrier or thinning agent.
61. be used for the treatment of, prevent in preparation or improve application in the medicine of mammal bacterial infection according to the compound of claim 59.
62. the compound according to claim 59 is used for the treatment of, prevents or improves mammiferous bacterial infection.
63. an in-vitro method that is used to suppress the phosphoryl transferase site activity of adenylylation GS polypeptide, described method comprises makes adenylylation GS polypeptide contact with the compound compositions that contains with good grounds claim 59.
64. an in-vitro method that is used to suppress contain the bacterial growth of GSI-β gene, described method comprises makes described bacterium contact with the compound compositions that contains with good grounds claim 59.
65. one kind be used for the treatment of, one or more symptoms that prevention or improvement are relevant with the mammal bacterial infection or the method for obstacle, wherein said bacterial infection is caused that by the bacterium that contains GSI-β gene described method comprises that the compound compositions that will contain with good grounds claim 59 gives described mammal.
66. method in the body of a phosphoryl transferase site activity that is used to suppress adenylylation glutamine synthelase polypeptide, described method comprises:
(a) compound compositions that will contain with good grounds claim 59 is subjected to the mammal of bacterial infection, and wherein said bacterial infection is caused by the bacterium that contains GSI-β gene.
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