CN101766602B - Application of Substituted Arylhydrazone Compounds as Tumor Necrosis Factor Inhibitor Drugs - Google Patents
Application of Substituted Arylhydrazone Compounds as Tumor Necrosis Factor Inhibitor Drugs Download PDFInfo
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Abstract
本发明涉及取代芳香基腙类化合物及其衍生物作为抗肿瘤坏死因子抑制剂在临床上的应用。该化合物在动物体内试验结果显示具有较低的细胞毒性，因此，该药物化合物有望作为抗TNF-a和TNF-β抑制剂在临床上推广应用，主要用于治疗一些TNF相关的免疫性疾病，如风湿性关节炎、炎性肠道疾病、糖尿病、败血病、银屑病、强直性脊柱炎以及包括HIV在内的其他一些传染性疾病，目前的TNF相关的研究进展还提示，该药物化合物还可用于临床血液及实体肿瘤(如骨髓增生异常综合征、骨髓纤维化症、急性骨髓细胞白血病以及急性/慢性移植物抗宿主反应、卵巢癌、肾细胞癌等疾病)的治疗。The invention relates to the clinical application of substituted aryl hydrazone compounds and their derivatives as anti-tumor necrosis factor inhibitors. The test results of the compound in animals show that it has low cytotoxicity. Therefore, the drug compound is expected to be used as an anti-TNF-α and TNF-β inhibitor in clinical practice, mainly for the treatment of some TNF-related immune diseases. Such as rheumatoid arthritis, inflammatory bowel disease, diabetes, sepsis, psoriasis, ankylosing spondylitis and some other infectious diseases including HIV, the current research progress related to TNF also suggests that the drug The compound can also be used in the treatment of clinical blood and solid tumors (such as myelodysplastic syndrome, myelofibrosis, acute myeloid leukemia and acute/chronic graft-versus-host reaction, ovarian cancer, renal cell carcinoma and other diseases).
Description
Technical field
The invention belongs to technical field of medicine synthesis, relate to substituted aromatic base hydrazone compounds, derivant and salt thereof as anti-tumor necrosis factor (tumor necrosis factor, TNF) TNF-a and the application clinically of TNF-beta inhibitor.
Background technology
Immune system is a most important system of defense in the human body, and normal immunne response is given the human body infection, discerns oneself and nonego, in time removes the ability of the cell of aging, pathological changes in the body.But immunoreaction abnormity also has diseases induced probability, and promptly physiological reaction is to the transformation of pathological reaction.Research shows: body can discharge a large amount of inflammatory factors in the antigenic immunoreactive process, like interleukin-11, interleukin 6, tumor necrosis factor etc., can make the local organization organ injury.Clinical common rheumatoid arthritis, inflammatory enteritis, chronic glomerulonephritis, systemic lupus erythematosus (sle) etc. are all relevant with the immune inflammation damage; Therefore we can say that immunoreation is the double-edged sword of life; How to utilize various means; Promoting its physiological reaction and suppress its pathological effect, go after profits and advoid disadvantages, is the vital task of immunization therapy.
(tumor necrosis factor TNF) mainly contains two types of TNF-α and TNF-β to tumor necrosis factor, and wherein TNF-α is mainly by monokaryon-macrophage secretion; TNF-β is mainly by the activated T lymphocytic emiocytosis.TNF-α is very similar with the biological action of TNF-β; All can stimulate the generation of IL-1 in vivo and in vitro; All can kill some tumor cell (cytolyticaction), or suppress proliferation function (cytostaticaction), this maybe be relevant with the homogeneity of the similarity of molecular structure and receptor.The native form of TNF-α and β performance biological effect is homologous trimer.TNF receptor (TNF-R) can be divided into amphitypy: I type TNF-R (55kDa, CD120a, 439 amino acid residues), and this receptor possibly play a major role on dissolved cell activity; II type TNF-R (75kDa, CD120b, 426 amino acid residues), this receptor maybe be relevant with the T cell proliferation with the signal transmission.The mechanism of TNF killing tumor cells is different from complement or perforin (perforin) killer cell, the TNF killer cell phenomenon of not boring a hole, and also the process of killing and wounding is relatively slow.TNF-R belongs to trk C (NGFR) superfamily, is present in multiple normal and tumor cell surface, and generally each cell receptor number has 10
3～10
4, the number of different cell surface TNFR and affinity and cell are also not parallel to the sensitivity of TNF.After TNF and the corresponding receptors bind signal transmission maybe with PKC (PKC), catalysis receptor protein phosphorylation is relevant.Handle obviously enhance TNF tumor cell killing activity of tumor cell (like l cell strain L929) with actinomycin D, ametycin, cycloheximide etc.
TNF α accounts for mastery reaction in the immunoloregulation function of TNF; And be a kind of cytokine of early carrying out clinical trial, upwards can trace back to the seventies in 20th century, but because its toxic and side effects is comparatively serious; And can not control, so fail to promote the use of clinically always.Along with in the last few years to the understanding of TNF progressively deeply; People generally believe that this cytokine is one " three sword swords "; The immunoregulation effect that promptly is low concentration TNF is a mastery reaction, and it can the activation neutrophilic granulocyte, stimulates mononuclear cell; Activating macrophage also can influence special and non-specific immunity through the adjusting of T, bone-marrow-derived lymphocyte; The cell toxicant toxicity of high concentration TNF becomes mastery reaction; It can kill and wound some tumor cell or make the in-vivo tumour tissue that hemorrhagic necrosis take place on the one hand; It is again a kind of stronger endogenous inflammatory mediator on the other hand, and excessive TNF can cause after tissue injury, severe infection property shock, autoimmune disease, the organ transplantation immunological rejection etc., causes body physiological function disorderly; Often symptom is dangerous, the treatment difficulty.Therefore, blocking-up TNF α can improve the symptom of these diseases significantly, and has become the Critical policies of the above-mentioned immune disease of treatment.At present, be used for anti-TNF alpha medicine for treatment thing in the world and mainly contain infliximab, CDP571; Etanercept, onercept, adalimumab (D2E7); CDP870 (seeing table 1); The data of just having delivered at present, this type biopharmaceutical macromolecular drug (overwhelming majority is the antibody of anti-TNF alpha) can have made marked progress about two weeks to rheumatoid arthritis, ankylosing spondylosis and chronic eczema property arthritic (80%) evident in efficacy and quick.Wherein, especially what deserves to be mentioned is infliximab, from be applied to by FDA approval in 1998 clinical since, become the choice drug of inflammatory diseasess such as treatment rheumatic arthritis and remarkable in economical benefits (seeing table 2).Also have great deal of research results to show that the anti-TNF alpha therapy successfully treated immune diseases such as Er Shi disease at the bottom of uveitis, vasculitis, the history, inflammatory bowel, dermatomyositis in addition, make specificity anti-TNF alpha preparation become one of the most potential immunosuppressant of current treatment autoimmune disease gradually.In the last few years, become a new research focus again around generation, the Research on development of TNF-a and tumor, existing result of study shows the generation of TNF-a and neonate tumour blood vessel, drug resistance and tumor and shifts relevant.Therefore, blocking-up TNF α can improve the symptom of above-mentioned immune disease significantly, can suppress generation, growth and the transfer of tumor again.At present; The inhibitor of TNF-a has been used to some blood of clinical treatment and entity tumor; Like myelodysplastic syndrome, myelofibrosis, acute myeloid leukemia and acute/chronic graft versus host reaction, ovarian cancer, renal cell carcinoma etc., and obtained result safely and effectively.
Mostly current TNF α preparation is biopharmaceutical macromolecular drug, and they have the characteristics that specificity is good, binding ability is strong on the one hand, but the one side less stable; In patient's body, be prone to degraded; And the biomacromolecule that the life-time service immunogenicity is strong will cause the immunologic tolerance of patient to it, and this is disadvantageous for the autoimmune disease patient especially, because this type patient need use immunosuppressive drug for a long time, repeatedly; Therefore; Explore new immunosuppressant research strategy and develop the small-molecule drug of targeting anti-TNF alpha, overcome the shortcoming of above-mentioned macromolecular drug, become domestic current clinical immunization therapy and be badly in need of one of difficult problem that solves.
Table 1.TNF-a inhibitor
12 kinds of biotech drugs that table 2.2006 year global marketing volume is the highest
Undoubtedly; Closely during the last ten years; Along with the arrival in functional genome's epoch and the high speed development of proteomics, and some the relevant new theorys and the emerge in multitude and the extensive use of new technique, especially multiple nuclear magnetic resonance, NMR (NMR) technology and area of computer aided drug design (computer-aided drug design; CADD) series connection is used; And the continual renovation of the continuous expansion of albumen database (PDB) and molecular simulation software, for solving an above-mentioned difficult problem opportunity is provided, and has made " order formula " design and screening micromolecule targeted drug become possibility.In simple terms, this newtype drug research and development strategy was divided into for six steps:
1. from the PDB library, search for the composite crystalline structure (TNF-β and the protein bound composite crystalline structure of its receptor P55) of target protein and its receptor or part; And related data downloaded to the computer graphical work station; And analyze both binding sites detailed structure character; Like the distribution in zones such as electrostatic interaction, hydrophobic interaction and hydrogen bonded, and the composition of amino acid residue;
2. on the computer graphical work station, all amino acid residues of above-mentioned receptor and part junction or partial peptide section are done as a wholely to peel off out; And the three dimensions conformation of keeping them is constant; Then on SGI02 (R12000) graphics workstation with the core texture of InsightII software analysis and definite land part or acceptor portion and influence key amino acid residue (the one 7 fragments of peptides (RKEMGQV in the 2nd loop ring of receptor P55 albumen second domain of its affinity; No.77-83)); Last is constant " anchor point " with above-mentioned key amino acid residue; Remove all hydrones, hetero atom etc., after the SYBYL simulation has belonged to the field of force, supply butt joint to use.Receptor or ligand molecular surface are generated by the DMS program; Then based on above-mentioned " anchor point ", the stereoscopic features in the binding cavity cave when the rigidity bead methods of filling different sizes with the SPHGEN program are described receptor and combined with part, the ball site of taking-up irrational distribution; Obtain the land negative-appearing image of being piled into by 32 balls; Represent the land characteristic, then, write input file dock.in; Confirm the position of each Parameter File, desired parameters when comprising the receptor structure data file handled well, avtive spot negative-appearing image data file, scoring grid data file and ligand structure data file and DOCK screening;
3. arrangement is collected in companies such as specs chemical compound micromolecule in kind storehouse, form two-dimentional SDF file.Utilize the spl programming language in the SYBYL6.7 of the Tripos company software kit to combine other auxiliary routines of studying voluntarily; Under Unix and linux system; The Specs chemical compound two bit data library files of collecting are automatically converted to the file of three-dimensional mol2 form; Comprising hydrogenation, add electric charge and the 500 step POWLL field of forces are energy-optimised; Operation DOCK simulation and screening under Linux then searched among the above-mentioned data base the possible best conformation of land negative-appearing image docking in the micromolecular compound and above-mentioned steps 2 with anchor point search (achor-first search) method, again according to the energy array data as a result of giving a mark.
4. select 500-1000 lower chemical compound of energy marking; Cluster analysis through the main functional group similarity is classified it according to framing structure, and final selection and purchase (or synthetic) 50-100 representative compound is done further biological activity determination behind the analysis-by-synthesis;
5. Determination of biological activity comprises activity experiment and animal vivo test on body outer cell line or the primary cell, as induces or suppress apoptosis, promotion or blocking-up cell migration, inhibition or promote angiogenesis and cell cycle influence etc.;
6. the micromolecular compound that filters out of analysis-by-synthesis is confirmed candidate's lead compound, in order to further further investigation later on.
This research promptly is based on biological macromolecule crystal structure, computer virtual screening and cytoactive triage techniques, the inhibitor of screening TNF-β and TNF-a from the storehouse in kind of existing micromolecular compound.Find first that finally substituted aromatic base hydrazone compounds of the present invention can directly combine with TNF-a; Can significantly suppress TNF-β and TNF-a to the L929 cytotoxicity; Can demonstrate effective pharmacologically active at vitro inhibition TNF-β and the inductive L929 apoptosis of TNF-a.The method of using in the research of this chemical compound and the research process thereof will be established theory and experiment basis for the chemicals of developing targeting TNF molecule from now on.In several years backward; Along with becoming better and approaching perfection day by day of above-mentioned serial connection technology; The fine structure character of a large amount of receptors and part junction will be decrypted; Also make the small-molecule drug of " order formula " accurate targeting part of development or receptor become possibility, this will provide unprecedented opportunities and challenge for the development of immunization therapy.Before the present invention, do not see that the aromatic radical hydrazone compounds is as directly combine the relevant report and the patent application of the inhibitor of the BA of inhibition TNF-β and TNF-a with TNF-β and TNF-a.
Summary of the invention
First purpose of the present invention has been to disclose the application aspect preparation tumor necrosis factor (TNF-a and TNF-β) inhibitor medicaments of substituted aromatic base hydrazone compounds, derivant and salt thereof.
Another object of the present invention is to disclose substituted aromatic base hydrazone compounds, derivant and salt thereof in the relevant immune disease of some TNF of preparation treatment, like rheumatic arthritis, inflammatory bowel disease, diabetes, septicemia, psoriasis, ankylosing spondylitis and the application of medicine aspect that comprises some other infectious disease of HIV.
A further object of the invention is to disclose substituted aromatic base hydrazone compounds, derivant and salt thereof at some neoplastic hematologic disorders of preparation treatment, like the application of the medicine aspect of myelodysplastic syndrome, myelofibrosis, acute medullary cell leukemia and acute/chronic graft versus host reaction.
Further object of the present invention is to disclose substituted aromatic base hydrazone compounds, derivant and salt thereof at some entity tumors of preparation treatment, like the application of the medicine aspect of diseases such as ovarian cancer, renal cell carcinoma.
Last purpose of the present invention is the pharmaceutical composition that is to be provided for treating autoimmune disease and tumor, and it comprises substituted aromatic base hydrazone compounds, derivant and salt thereof and one or more pharmaceutically acceptable carriers, excipient or diluent combined.
The invention provides following technical scheme:
Substituted aromatic base hydrazone compounds, derivant and salt thereof with following general structure C are as anti-tumor necrosis factor inhibitor application clinically
Wherein:
X
1Be phenyl, substituted phenyl, heterocycle, substituted heterocycle;
X
2Be phenyl, substituted phenyl, halogen, NO
2, H, OH, NH2, HSO3, straight or branched alkyl;
X
3Be phenyl, substituted phenyl;
X
1Or/and X
2Or/and X
3Can be to contain single or a plurality of substituent groups, and be in aromatic rings or heterocyclic para-position respectively or/and the ortho position or/and a position.
X of the present invention
1Or/and X
2Or/and X
3The substituent group that contains can be C
1-C
6Straight or branched alkyl, C
1-C
4Alkoxyl, C
1-C
4Perfluoroalkyl, C
1-C
4Ester group, substituted aryl, halogen, NO
2, H, OH, NH
2, HSO
3, CH
3S.
General formula C chemical compound pharmaceutically acceptable salt means: The compounds of this invention and mineral acid, organic acid salify, preferred especially salt is: hydrochlorate, hydrobromate, hydriodate, sulfate, disulfate, phosphate, acetate, propionate, butyrate, lactate, mesylate, tosilate, maleate, benzoate, succinate, tartrate, citrate, fumarate, taurate or the like.As described salt, they can also be the salt that forms with conventional alkali, for example alkali metal salt.
C of the present invention
1-C
6The straight or branched alkyl comprises methyl, ethyl, propyl group, isopropyl, butyl, isobutyl group, tert-butyl group or the like.Described C
1-C
3Alkyl or chlorine, fluorine replace C
1-C
3Alkyl can be methyl, ethyl, propyl group, chloroethyl, fluoro ethyl; C
1-C
4Alkoxyl or chlorine, fluorine list or disubstituted C
1-C
3Alkoxyl; C
1-C
6Alkoxyl can be methoxyl group, ethyoxyl, propoxyl group, butoxy, isobutoxy, chloroethoxy, chlorine propoxyl group, 1,1-dichloro propoxyl group, 1-fluoro-2-chlorine propoxyl group or the like.
The present invention is preferred described to have the chemical compound of pharmacologically active to be selected from following chemical compound.
Experiment numbers C-12
3-benzene-1-(4-benzene-1,3-thiazoles-2-yl)-1 hydrogen-pyrazoles-4,5-diketone-4-({ 4-chloro-3-Nitrobenzol } hydrazone) structural formula is following:
The present invention and experiment numbers C-12 have the chemical compound of identity function following:
3-benzene-1-(4-benzene-1,3-thiazoles-2-yl)-1 hydrogen-pyrazoles-4,5-diketone-4-({ 4-chloro-3-Nitrobenzol } hydrazone);
(trans)-4-(2-(4-(2,4-dichloro benzyloxy (base) benzylidene) diazanyl-N-(2, the 3-3,5-dimethylphenyl)-6-(the 4-methyl piperidine-1-yl)-1,3,5-triazines-2-amine;
(trans)-5-(6-(2-(2-benzyloxy-3,5-dibromobenzene methylene) diazanyl)-[1,2,5] oxadiazoles [3,4-b] pyrazine-5-base is amino)-1H-benzo [d] imidazoles-2 (3H)-ketone;
(trans)-2-(5-(benzoic acid of (2-(3-cyanic acid-4-methoxyl methyl-6-picoline-2-yl) inferior hydrazine) methyl) furan-2-yl);
(trans)-N '-((2-(3-bromo-benzyloxy-) naphthal-1-yl) methylene)-2-(3-chlorphenyl) propyl group hydrazine;
(trans)-2-(2-(4-methoxyl group)-3-(2-nitro benzyloxy) diazanyl)-4, and 6-two (piperidines-1-yl)-1,3,5-triazines;
(cis)-2-(2-((the inferior hydrazine of 2-(2-(1-(4-benzyl chloride base)-1H-benzo [d] imidazoles-2-sulfenyl) acetyl)) methyl) benzene oxygen) acetic acid.
2-hydroxyl-3,5-dinitrobenzal-dehyde [4-(benzamido group)-6-(dimethylamino)-1,3,5-triazines-2-yl] hydrazone
2-hydroxy-5-methyl oxygen benzaldehyde [4-aniline-6-(3,5-dimethyl pyrazole-1 hydrogen-pyrazoles-1-yl)-and 1,3,5-triazines benzamide-2-yl] hydrazone
The present invention further discloses the pharmaceutical composition of substituted aromatic base hydrazone compounds as tumor necrosis factor inhibitors, it contains substituted aromatic base hydrazone compounds and one or more pharmaceutically acceptable carriers, excipient or diluent.
The preparation of pharmaceutical compositions of chemical compound according to the invention is following:
Use standard and conventional technology make on The compounds of this invention and the galenic pharmacy acceptable solid or liquid-carrier combine, and make it at random with galenic pharmacy on acceptable adjuvant and excipient combine to be prepared into microgranule or microsphere.Solid dosage forms comprises tablet, discrete particles, capsule, slow releasing tablet, slow-release micro-pill or the like.Solid carrier can be at least a material, and it can serve as diluent, flavouring agent, solubilizing agent, lubricant, suspending agent, binding agent, disintegrating agent and coating agent.Inert solid carrier comprises magnesium phosphate, magnesium stearate, smoothers sugar, lactose, pectin, propylene glycol, polyoxyethylene sorbitan monoleate, dextrin, starch, gelatin, cellulose substances for example methylcellulose, microcrystalline Cellulose, low melt point paraffin, Polyethylene Glycol, mannitol, cocoa butter etc.Liquid dosage form comprises solvent, suspension for example injection, powder or the like.
The amount of the active ingredient that contains in pharmaceutical composition and the unit dosage form (The compounds of this invention) can be according to patient's the state of an illness, specific being applied of situation of diagnosis; The amount of used chemical compound or concentration are regulated in the scope of a broad; Usually, the weight range of reactive compound is 0.5%～90% (weight) of compositions.Another preferred range is 0.5%-70%.
The present invention is based on biological macromolecule crystal structure, computer virtual screening and cytoactive triage techniques, the inhibitor of screening TNF-β and TNF-a from the storehouse in kind of existing micromolecular compound.In the experiment; We are with the possible best conformation of nearly 100,000 micromolecular compounds and ligand binding domain negative-appearing image docking in anchor point search (achor-first search) method search database; The DOCK program can be automatically according to energy marking result according to array data from high to low; Through 1000 forward chemical compounds of analysis-by-synthesis marking, choose wherein representational 50 chemical compounds and carry out the BA experiment again.The chemical compound that contains the hydrazone structure in preceding 50 chemical compounds that will choose is below selected, and with corresponding energy marking tabulation 1.
Part contains the chemical structural formula and the energy marking value of the chemical compound of hydrazone structure in preceding 50 chemical compounds of table 1..
Experimental result shows: above-mentioned chemical compound in various degree can suppress TNF-β and TNF-a to the L929 cytotoxicity; Wherein substituted aromatic base-hydrazone micromolecular chemical compound (C-12) has obvious suppression TNF-β and TNF-a to the L929 cytotoxicity, demonstrates effective pharmacologically active.
Description of drawings:
Fig. 1. diagram TNF-α and TNF-β crystal structure on the SGI computer workstation.TNF-alpha molecule data are from 1TNF.pdb among the PDB; TNF-β from 1TNR.pdb;
Fig. 2 .TNF-β and the protein bound crystal structure of its receptor P55 (1TNR) and be used for the confirming of part template of molecular docking (docking).The protein bound crystal structure data of TNF-β and its receptor P55 are from 1TNR.pdb among the PDB.
965 interactional energy profiles of chemical compound behind Fig. 3 .Dock;
Fig. 4. chemical compound comprises Fig. 4-1, Fig. 4-2, Fig. 4-3, Fig. 4-4 to TNF-a and the beta mediated cytotoxic inhibitory action of L929 of TNF-.
Fig. 5 .C-12 chemical compound comprises Fig. 5-1,5-2 to the inhibition of the beta mediated cytotoxicity of TNF-a and TNF-.
Fig. 6 .C-12 chemical compound directly suppresses TNF-a and representes blank and negative control (quantum dot-labeled irrelevant albumen *) respectively with the combine A and the C of its receptor, and B, D and E are experimental group.
The cytotoxicity of Fig. 7 .C-12 chemical compound and to the inhibition of the apoptosis effect of TNF-a mediation; A, B, C and D are the inhibition result of C-12 chemical compound to the apoptosis effect of TNF-a mediation; E and F are the cytotoxicity result of this chemical compound.All be added with the 1ug/ml actinomycin D in all reaction systems.
Fig. 8. 9 fragments of peptides (No.75-83) and micromolecular compound C-12 binding pattern in the 2nd loop ring of graphical analysis TNF-β and its receptor P55 albumen second domain.The result shows formation 8 hydrogen bonds (A) between TNF-beta molecule and 9 fragments of peptides; Form 4 hydrogen bonds (B) with C-12.Hydrogen bond shows with blue lines; TNF-beta molecule part is represented with thin black line; Ligand molecular and micromolecular compound are represented with thick lines.
The specific embodiment
Below in conjunction with embodiment the present invention is done further explanation, it is indicative that embodiment is merely, and means that never it limits scope of the present invention by any way, and chemical compound of the present invention all adopts the conventional method preparation maybe can buy.In order to show the practicality of target micromolecular compound, further specify the effect of The compounds of this invention below through pharmacodynamic experiment.With typical C-No. 12 is example: and 3-benzene-1-(4-benzene-1,3-thiazoles-2-yl)-1 hydrogen-pyrazoles-4,5-diketone-4-({ 4-chloro-3-Nitrobenzol } hydrazone) further explains.
Test a .TNF-β and the protein bound crystal structure of its receptor P55 (1TNR) and be used for the confirming and the process of virtual screening candidate compound molecule of part template of molecular docking (docking).
Principle: proteic crystal composite construction can accurately reflect each proteic space conformation, the especially embedded structure of two kinds of albumen junctions when receptor and part combine under the naturalness, for accurately seeking proteic active site possibility is provided.The computer virtual screening just is based on a kind of drug screening pattern that proteic crystal structure development comes; In brief; Step is following: at first on the computer graphical work station, all amino acid residues of receptor and part junction are done as a wholely to peel off out; And the three dimensions conformation of keeping them is constant, then, confirms the core texture of land part or acceptor portion and influences the key amino acid residue of its affinity; Be that constant " anchor point " removed all hydrones, hetero atom etc. then with above-mentioned key amino acid residue; After SYBYL simulation has belonged to the field of force, the stereoscopic features in the binding cavity cave when the rigidity bead methods that reuse SPHGEN program is filled different sizes are described receptor and combined with part, the structured data in last cave, chamber based on above-mentioned acquisition; Carry out docking with the chemical micromolecule of three dimensional stress, to seek the candidate compound molecule.
Experimental technique:
1. the structure of three-dimensional Chemoinformatics structural database
Arrangement is collected in companies such as specs chemical compound micromolecule in kind storehouse, form two-dimentional SDF file.Utilize the spl programming language in the SYBYL6.7 of the Tripos company software kit to combine other auxiliary routines of studying voluntarily; Under Unix and linux system; The Specs chemical compound two bit data library files of collecting are automatically converted to the file of three-dimensional mol2 form, comprising hydrogenation, add electric charge and the 500 step POWLL field of forces are energy-optimised.
2. the selection of the part template of computer virtual screening
TNF-alpha molecule data are from 1TNF.pdb among the PDB; TNF-β and the protein bound composite crystalline structure of its receptor P55 are from 1TNR.pdb.Based on TNF-β and the protein bound crystal structure of its receptor P55, select on the INSIGHT II platform on the part P55 with the chimeric one section outstanding ring structure of TNF-α outer surface void structure as receptor templates.
3. the receptor structure data are prepared and the negative-appearing image generation
From Protein Data Bank (PDB) crystal structure data base, download tumor necrosis factor (TNF-β; LT) with its receptor TNFR1 (p55; Composite crystal data structure (1TNR.pdb) 55kDa); Remove all hydrones, hetero atom etc., after the SYBYL simulation has belonged to the field of force, supply butt joint to use.The acceptor molecule surface is generated by the DMS program; The stereoscopic features in the binding cavity cave when the bead methods of filling different sizes with the SPHGEN program then are described in receptor and combine with part; Take out the ball site of irrational distribution; Obtain the land negative-appearing image of being piled into, represent the land characteristic by 32 balls.
4. butt joint and scoring
Write input file dock.in; Confirm the position of each Parameter File; Desired parameters when comprising the receptor structure data file handled well, avtive spot negative-appearing image data file, scoring grid data file and ligand structure data file and DOCK screening, operation DOCK simulation and screening under Linux then.Search for the possible best conformation of part among the above-mentioned data base with anchor point search (achor-first search) method, according to energy marking array data as a result.
Experimental result: confirmed to be used for computer virtual screening TNF-α micromolecular inhibitor the part template.
The bonded crystal structure data of TNF-alpha molecule and part still fail to obtain so far; Therefore; This experiment is based on TNF-α and TNF-β and the protein bound crystal structure of its receptor P55; And TNF-a has highly similar space structure and identical receptor (see figure 1) with TNF-β, adopts computer-aided design to confirm to be used for the part template of molecular docking (docking).After the above-mentioned albumin crystal structure of comparative analysis, (RKEMGQV is No.77-83) as the part template (see figure 2) of docking to confirm one 7 fragments of peptides in the 2nd loop ring of receptor P55 albumen second domain.
Three dimensional structure based on above-mentioned part template; Specs (containing 90,000 micromolecule of having an appointment) three-dimensional data base is carried out obtaining behind the virtual screening chemical compound of 965 kinds of structural similarities with the docking method; From these 965 kinds of chemical compounds, select 300 kinds of minimum chemical compound (see figure 3)s of energy marking then; Cluster analysis through the main functional group similarity is divided into the principle that 48 big classes are followed quasi-medicated property and structure diversity then according to framing structure with it; Pick out representational chemical compound in total, obtain 50 chemical compounds from the purchase of Specs company at last and carry out screening active ingredients.
Experiment two. candidate compound is to the cytotoxic inhibitory action of TNF-β and TNF-a mediation.
Principle: TNF-α is mainly by monokaryon-macrophage secretion; TNF-β is mainly by the activated T lymphocytic emiocytosis, the generation that both all can inside and outsides all can stimulate IL-1.TNF-α is very similar with the biological action of TNF-β; In vivo, externally all can kill some tumor cell (cytolyticaction); Or suppressing proliferation function (cytostaticaction), this maybe be relevant with the homogeneity of the similarity of molecular structure and receptor.The mechanism of TNF killing tumor cells is compared different with complement or perforin (perforin) killer cell, the TNF killer cell phenomenon of not boring a hole, and also the process of killing and wounding is relatively slow.Handle tumor cell (like l cell strain L929) obviously enhance TNF-α and TNF-β tumor cell killing activity with actinomycin D, ametycin, cycloheximide etc.The antagonist that blocking-up TNF engages with its receptor can suppress the effect of above-mentioned TNF killer cell.
Experimental technique:
Getting the l cell that is in exponential phase is the L929 cell, join in 96 well culture plates with the density in 2 * 104/hole, and every hole 100ul RPMI1640 culture fluid (10% hyclone), and in 37 ℃, 5%CO
2Cultivated 24 hours in the incubator.In RPMI-1640, adding final concentration is 1ug/ml ActD and 1.0ng/mlTNF-a, joins in 96 orifice plates by the 100ul/ hole, adds candidate compound simultaneously, and establishes the variable concentrations hole.Put into incubator and cultivate after 20 hours, every hole adds 20ulMTT (5mg/ml) continuation effect 4 hours.Abandon supernatant, press the 100ul/ hole and add DMSO, concussion 1min makes fully dissolving of deposition, the OD value when on ELIASA, measuring wavelength 546nm.Calculate the cell toxicant suppression ratio according to following formula: [(ODactD+TNFa+compound-ODactD+TNFa)/(ODactD-ODactD+TNFa)] * 100%
Experimental result: the method with MTT detects the malicious inhibitory action (Fig. 4) of 50 chemical compound pair cells, establishes two compound concentrations of 20uM and 4uM and negative control group respectively.Wherein the C-12 chemical compound has the obvious suppression effect to TNF-a and the beta mediated cell toxicant of TNF-.
Testing three .C-12 chemical compounds inhibition TNF-β and TNF-a tests the L929 cytotoxicity.
Principle: the same
Experimental technique: the same
Experimental result: detect behind the C-12 compound effects 24h the Cytotoxic inhibition of TNF-a (Fig. 5) with the method for MTT.Establish 7 concentration and ineffective treatment compound (C-17) matched group respectively.As can be seen from the figure, the C-12 chemical compound is concentration dependent form to the inhibition of TNF-a (1.0ng/ml) and TNF-β (1.0ng/ml), and matched group does not have this inhibitory action, has significant difference [(P=0.0106) between the two; (P=0.0014)].(half-maximum inhibition concentration IC50) is respectively 10umol/L and 19.8umol/L to calculation of half inhibitory concentration.
Test the inhibitory action of four .C-12 chemical compounds to quantum dot-labeled TNF-a bind receptor.
Principle: quantum dot (Quantum Dots), be called inorganic nanocrystal again, have unique optics and electrical properties.Quantum dot can be used as fluorescent probe and is used for aspects such as biomarker, biological detection and bio-imaging.Compare with the organic fluorescence molecule; Quantum dot has the optical characteristics of many uniquenesses: its emission peak wavelength can be regulated by composition material and size; Its excitation wavelength scope is very wide; In addition, quantum dot has good photochemical stability, can tolerate stronger exciting light and longer light emissioning cycle.Quantum dot in the reaction system that coupling agent exists can with radical reactions such as amino in the protein molecular, hydroxyl, sulfydryl, thereby be tagged on the protein molecular, as the indication fluorescent labeling.The albumen that is labeled with can be on flow cytometer and fluorescence co-focusing microscope after cell combines distinctive detecting.
Experimental technique:
1.TNF-a coupling with EviTag Quantum Dots
Select for use PBS (0.01mol/L, pH7.2) solution is the coupling buffer of quantum dot, TNF-a, adds water-soluble quantum dot, TNF-a (about 100ug) and EDC coupling agent according to 1: 15: 4000 mol ratio, adjusts pH to 7.5 with NaOH, the room temperature jog is hatched 2h.Quantum dot behind the labelling-TNF-a complex after the high speed centrifugation separation and purification, dissolve the PBS that is dispersed in 1ml (0.01mol/L, pH7.2,0.5%BSA) in.
2. micromolecular compound suppresses the TNF-a and the receptors bind experiment of quantum dot (Quantum Dots) labelling
Get the l cell that is in exponential phase and be 6 * 105 in L929 cell and add in the streaming pipe, subsequent use with twice of PBS washed cell.3 different compounds concentration group are established in experiment, establish irrelevant labelled protein matched group and blank group simultaneously.In above-mentioned cell pipe, add the quantum dot-labeled TNF-a of 40ul during experiment in every pipe, and add irrelevant albumen and variable concentrations C-12 chemical compound simultaneously.Hatch 30min for 4 ℃.With cold PBS thorough washing cell 3 times, every thereafter pipe adds 400ulPBS, and mixing carries out flow cytometer immediately and detects.
Experimental result:
Can the emulative inhibition quantum dot-labeled TNF-a of micromolecular compound C-12 combines with the L929 cell surface receptor.Can find out that from the result C-12 chemical compound has obvious suppression to the TNF-a bind receptor, suppression ratio was respectively 25.4% and 10.3% when micromolecular compound concentration was 0.5mM and 0.25mM, was concentration dependent and concerned (see figure 6).
Test the cytotoxicity of five .C-12 chemical compounds and to the apoptotic inhibitory action of L929 of TNF-β and TNF-a mediation.
Principle: TNF-β and TNF-a through with its receptors bind, can mediate lethal effect to target cell.Apoptosis is one of basic mode of cell death, and it plays an important role at the aspect such as stable of the fetal development of body, tissue repair, interior environment.Annexin V is a kind of Ca
2+Dependency phospholipids incorporate albumen, can with apoptosis process in be turned to the outer Phosphatidylserine generation specificity of film and combine.With labelling the Annexin V of FITC as fluorescent probe, utilize flow cytometer or fluorescence microscope can detect apoptotic generation.Propidium iodide (PI) is-kind of nucleic acid dye, and it can not see through complete cell membrane, but at the cell and dead cell of apoptosis middle and advanced stage, and PI can permeate through cell membranes and make that nucleus is red to be dyed.Annexin V and PI coupling are used, and the cell that can apoptosis is early stage and the cell in late period and dead cell make a distinction.
Experimental technique: getting the l cell that is in exponential phase is the L929 cell, joins in 12 well culture plates with the density in 6 * 105/hole, and every hole 1ml RPMI1640 culture fluid (10% hyclone), and in 37 ℃, 5%CO
2Cultivated 24 hours in the incubator.In RPMI-1640, adding final concentration is 1ug/ml ActD and 5.0ng/mlTNF-a, joins in 96 orifice plates by the 1ml/ hole, adds target compound simultaneously, and establishes variable concentrations and do not have TNF-a, no chemical compound control wells, puts into incubator and cultivates 24 hours.Abandon supernatant, 0.25% trypsinization 3mins, with cold PBS washed cell 2 times, 1 * binding buffer is 1 * 106/ml with cell dilution.Get 100ul cell suspension (1 * 105) to 5ml streaming pipe, add 5ul Annexin V-FITC and 5ul PI, mixing, the room temperature lucifuge is placed 15mins.Add 400ul 1 * binding buffer in each streaming pipe, mixing carries out flow cytometer immediately and detects.
Experimental result:
TNF-a has mediated the link coupled apoptosis pathway of caspase through TNFR.With TNF-a effect L929 cell, add the C-12 chemical compound that concentration is respectively 50uM and 12.5uM simultaneously, cultivate 24h, and establish PBS, singly add micromolecular compound and irrelevant micromolecular compound matched group.The result shows: the C-12 chemical compound has the obvious suppression effect to the inductive apoptosis of TNF-a, and the apoptosis suppression ratio was 35.8% when compound concentration was 50uM; 12.5uM the time be 24.5%, be concentration dependence (see figure 7).Adding chemical compound matched group result separately can find out, the cell toxicant toxicity of C-12 chemical compound self pair cell is lower.
Experiment six. the micromolecular compound that the comparative analysis of molecule iconology filters out combines with the P55 receptor.
Principle: with method one.
Experimental technique: with method one.
Experimental result:
Based on Chimera V.1.0 the software analysis result show: P55 albumen No.75-83 peptide section forms a loop ring in the 1TNR crystal structure, embeds deeply in the cave, chamber of part, forms a stable composite structure through 8 hydrogen bonds between the two; The C-12 micromolecule can be deep in the cave, chamber of part; The fragrant ring of one of them benzene extends to center, cave, chamber; This chemical compound can form 5 hydrogen bonds with part; There wherein have three hydrogen bonds to form in position (Ser (No.82), Lys (No.84) and Pro (No.155)) and the above-mentioned mineral crystal complex to be identical, thereby can occupy the position that combines of receptor and part, blocks the (see figure 8) that combines of Tumor Necrosis Factor Receptors P55 and its native ligand.
The animal toxicity in vivo experimental result of data 1. substituted aromatic bases-hydrazone compounds (C-12).
Analysis indexes is that (Median Lethal Dose, LD50): behind (be generally for 2 weeks) within a certain period of time per os or the transdermal administration given the test agent, making animal subject generation POD is 50% dosage for LD50, half lethal dose.Accept the quality (mg/kg bw or g/kg bw) of given the test agent representes with per weight.
Minimum visible illeffects level (Lowest Observed Adverse Effect Level; LOAEL): under the experimental condition of regulation, given the test agent causes that harmful minimum poisoning dosage or concentration that changes takes place for laboratory animal form, function, growth promoter etc.
In sum, further prove through pharmacological evaluation and interpretation of result: substituted aromatic base hydrazone compounds of the present invention (C-12) can directly combine with TNF-a; The BA that can suppress TNF-β and TNF-a, and can demonstrate effective pharmacologically active at the inductive L929 apoptosis of external remarkable inhibition TNF-a; The characteristics that have low toxicity simultaneously; Therefore; Be expected to apply clinically as anti-TNF-a and TNF-beta inhibitor; Be mainly used in the relevant immune disease of some TNF of treatment, like rheumatic arthritis, inflammatory bowel disease, diabetes, septicemia, psoriasis, ankylosing spondylitis and some other infectious disease of comprising HIV, the progress that present TNF is relevant is also pointed out; This medical compounds also possibly be used for some blood of clinical treatment and entity tumor, like diseases such as myelodysplastic syndrome, myelofibrosis, acute medullary cell leukemia and acute/chronic graft versus host reaction, ovarian cancer, renal cell carcinomas.
FORMULATION EXAMPLE 1
Every preparation tablets that contains the 100mg active component:
With active component; Lactose, starch, microcrystalline Cellulose are crossed 100 mesh sieves; And abundant mixing, 2% hydroxyl methylcellulose aqueous solution joined in the above-mentioned mixed-powder mix, cross 20 mesh sieve system soft materials; Make wet granular in 45-55 ℃ of drying, carboxymethylstach sodium, magnesium stearate are joined tabletting in the above-mentioned dried particles.
FORMULATION EXAMPLE 2
It is following that every capsule contains the capsular preparation of 100mg active component:
FORMULATION EXAMPLE 3
The preparation of injection
Making pH value is that 7.0-7.5 filtration filter liquor concentration is 3 mg/ml, by 2 milliliters of packing of every peace bottle, promptly gets injection after the lyophilization.
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| CN1215407A (en) * | 1996-02-09 | 1999-04-28 | Basf公司 | Human antibodies that bind huma TNF 'alpha' |
| CN101343268B (en) * | 2008-08-20 | 2011-01-12 | 中国科学院上海有机化学研究所 | A kind of thiazole dipyrazolone compound and its application as Bcl-2 family protein antagonist |
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| CN1215407A (en) * | 1996-02-09 | 1999-04-28 | Basf公司 | Human antibodies that bind huma TNF 'alpha' |
| CN101343268B (en) * | 2008-08-20 | 2011-01-12 | 中国科学院上海有机化学研究所 | A kind of thiazole dipyrazolone compound and its application as Bcl-2 family protein antagonist |
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| 谭兵.抗肿瘤坏死因子-α治疗的研究进展.《国际内科学杂志》.2007,第34卷(第3期), * |
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