CN1846694B - Substituted aromatic radical biguanide compounds and the application of their medicine composition in preparing medicine for resisting malignant tumors - Google Patents
Substituted aromatic radical biguanide compounds and the application of their medicine composition in preparing medicine for resisting malignant tumors Download PDFInfo
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Abstract
The present invention relates to substituted aromatic radical biguanide compounds and the application of their medicine composition in preparing medicine for treating malignant tumors. The substituted aromatic radical biguanide compounds possess, extracorporeal and intracorporeal experiments shows, obvious tumor cell inhibiting effect and the features of high efficiency and low toxicity, so that the medicine compounds are expected to be applied in inhibiting tumor attack and metastasis, inhibiting angiogenesis, and treating human melanoma, glioma, lung cancer and other malignant tumors with high expression of integrin.
Description
Technical field
The present invention relates to use the method for series of substituted aromatic radical biguanide compound treatment malignant tumor, a kind of application of pharmaceutical composition aspect multiple malignant tumor such as the Humanmachine tumour that suppresses tumor invasion transfer and angiogenesis and treatment integrin alpha v beta 3 high expressed, glioma, pulmonary carcinoma, gastric cancer, carcinoma of prostate, ovarian cancer that has the substituted aromatic base biguanide compound of anti-tumor activity and contain this compounds of treating effective dose of saying so more specifically.
Background technology
Malignant tumor is one big type of disease of serious threat human health, and its paraplasm is that information unusual in the cell is controlled, and is closely related with the Showed Very Brisk and the inactivation of corresponding gene in the tumor cell.Traditional cancer therapy drug mainly is a cell toxicity medicament; Difference between the kinetics of their dependence tumor cells and normal cell growth, reparation, death is come killing tumor cell; Therefore this type medicine inevitably can influence normal cell; It is narrow to treat window clinically, has also limited the enforcement of multiple medicines combined chemotherapy.There are many cancer therapy drugs to molecular target to occur in recent years, this type medicine with strong points, effect is remarkable, just looks like that to hit target the same, therefore is called as " target agent ".Paul Ehrlich has just proposed the notion of targeted therapy (targeting therapy) before 100 years; Along with development of molecular biology and people to the going deep into of tumor mechanism research, obtained very fast development, the carcinogenic cause of disease of these medicine direct aggressions in recent years to the target agent of specific carcinogenic mechanism; Selectivity is strong; Clinical trial result is remarkable, and side effect is light, and multiple medicines is united the effect that use can strengthen traditional chemotherapeutics often.
Integrating plain (integrin) is one type of important cell surface molecule, belongs to cell surface receptor family, is made up of α and two kinds of subunits of β.The generation of integrating plain adhesion and numerous disease through mediated cell and cell, cell and extracellular matrix is relevant, and wherein integrin alpha v beta 3 (CD51/CD61) plays an important role in processes such as neoplasm metastasis, angiogenesis, inflammation, neointimal hyperplasia.Why integrin alpha v beta 3 becomes an efficacious therapy target spot; Be because its wide expression in normal cell not; Just high expressed in the smooth muscle cell of new vessels and endotheliocyte and the especially invasive tumor cell of multiple malignant tumor comprises Humanmachine tumour, glioma, pulmonary carcinoma, gastric cancer, carcinoma of prostate, ovarian cancer etc.
A lot of tumor patients are unavoidable after treatment recurrence to be occurred and extensively shifts, three adhesions that committed step is tumor cell and basement membrane that tumor invasion shifts, degradation of extracellular matrix, and cell migration.Special adhesion process of many stages is arranged in this process; The expression of some of them cell adhesion molecule; Especially the metastatic potential of integrin alpha v beta 3 and tumor cell is closely related; It through promoting tumor transfer and invade the malignant phenotype that the profit ability makes tumor and increase, can be used as a sign of the tumor cell of many invasion and attack.For example in one of the strongest tumor of transitivity human melanoma; Tumor cell to the invasive ability of many tissues and extracellular matrix to a great extent owing to the receptor-mediated effect of surface adhesion; Wherein, Integrin alpha v beta 3 has been brought into play important function in melanomatous transfer and propagation, it can through with the adhesion of its part, the mediation MC shifts to many tissues.It is and for example local that to invade profit be one of characteristics of glioblastoma; This is also closely related with the unconventionality expression of integrin alpha v beta 3; Invading the glioma cell of profit can send out along some structure that contains stromatin (like the medial surface of blood vessel wall basement membrane) through the effect of α v β 3.
The formation of new vessels can impel tumor to increase rapidly, and increases the risk of neoplasm metastasis.Integrin alpha v beta 3 is confirmed to be a sign of angiogenesis; It is seldom expressed in the normal blood vessels of resting stage, but under the perhaps certain growth factor-induced of tumor, integrin alpha v beta 3 obviously increases in the expression of new vessels endotheliocyte; Can be through promoting the new vessels endothelial cell migration; Suppress its apoptosis, and promote former with the little vessel growth of metastatic tumour and increase it and invade the profit ability, its malignant phenotype is increased.Can regulate the angiogenesis of tumor inducing to the inhibition of integrin alpha v beta 3, increase the apoptosis of the little vascular endothelial cell relevant with tumor, angiogenesis reduces, and can also regulate the activity of osteoclast, and the hypercalcemia that occurs together of control malignant tumor.
At present the inhibitor to integrin alpha v beta 3 mainly contains 37 kinds, wherein has two kinds carrying out II phase clinical experiment, is a kind ofly carrying out I phase clinical experiment.These inhibitor mainly are divided three classes: the first kind is a monoclonal antibody; Humanized monoclonal antibodies Vitaxin and its second filial generation antibody Vitaxin II comprising integrin alpha v beta 3; In its II phase clinical experiment, be used for studying to the melanoma of carcinoma of prostate, transfer and the colon cancer in late period and leiomyosarcoma patient's therapeutic activity.The humanized antibody CTNO-95 that also has Johnson & Johnson company in addition, clinical preceding experiment shows that it can suppress the growth of nude mice human melanin transplanted tumor, is carrying out multicenter I phase clinical experiment at present; Second type is polypeptide and polypeptide analogies; Comprise by the little molecule cyclic peptide analog EMD121974 of Merck company exploitation; Can the antagonism integrin alpha v beta 3 vascular endothelial cell and the interaction of extracellular matrix of mediation; Carry out the II clinical trial phase, be used to treat the research of tumor of head and neck, pulmonary carcinoma, cancer of pancreas, breast carcinoma, melanoma, tumor of kidney and colon cancer; The 3rd type is non-peptide type small molecular inhibitor, and what studying at present mainly is benzodiazepines, comprises SB223245 etc., can significantly suppress the cell adhesion of integrin alpha v beta 3 mediation.The peptide class is subject to the degraded of enzyme in vivo, and the half-life is short, develops the medicine of corresponding non-peptide type small molecular inhibitor, long action time in vivo not only, and can orally use, be one of development in future direction.Reported the crystal structure of integrin alpha v beta 3 extracellular region and ligand complex in April, 2002 on the Science; This research just is being based on this achievement in research; The means of utilizing the computer high flux screening to combine with Chemoinformatics; Receptor binding site to integrin alpha v beta 3 carries out database search, and screening is sought the new drug of diseases such as transfer of control tumor invasion and angiogenesis to the micromolecular inhibitor of integrin alpha v beta 3.
We find first; Substituted aromatic base biguanide compound of the present invention is aspect the medicine of multiple malignant tumor such as the Humanmachine tumour for preparing inhibition tumor invasion transfer and angiogenesis and treatment integrin alpha v beta 3 high expressed, ovarian cancer, glioma, pulmonary carcinoma, gastric cancer, carcinoma of prostate; Demonstrate effective pharmacologically active, open up a new way for seeking new effective antitumour medicine.
Before the present invention; The substituted aromatic base biguanide compound is once by as 5-hydroxytryptamine receptor 3 agonist; Can further stop the conduction of postsynaptic noxious stimulus through promoting the release of GABA, once be used for the research etc. that non-opium does not have addiction property analgesic.Brain Res.2003 Jul 18; 978 (1-2): 250-5; Pain.1997Jul; 71 (3): 237-47; Gen Pharmacol.1991; 22 (2): reported this pharmacological action of 5-hydroxytryptamine receptor agonist among the 247-51.But it is at anti-tumor aspect, and the application that especially suppresses tumor invasion transfer and angiogenesis aspect was not studied always.
Synthesizing referring to Cohn of substituted aryl biguanide compound of the present invention, J.Prakt.Chem. [2] 84,396 Jacobs, Jolles, Brit, pat.587,907 (1947 to ICI).Chemical compound of the present invention can have been bought from market or make through the method for reporting above-mentioned method or other document.
Summary of the invention
First purpose of the present invention has been to disclose the application of substituted aromatic base biguanide compound at the preparation medicine for resisting malignant tumors.
The present invention further discloses the application of substituted aromatic base biguanide compound aspect preparation inhibition tumor invasion diversion medicaments.
The present invention further discloses the application of substituted aromatic base biguanide compound aspect preparation inhibition neonate tumour blood vessel medicine.
The present invention further discloses the application of substituted aromatic base biguanide compound aspect the medicine of malignant tumor such as preparation treatment Humanmachine tumour, ovarian cancer, pulmonary carcinoma, gastric cancer, carcinoma of prostate, glioma again.
Second purpose of the present invention has been to provide the pharmaceutical composition that is used to treat malignant tumor, and it comprises as the chemical compound of the claim 1 of active component or 5 or 6 and one or more pharmaceutically acceptable carriers, excipient or diluent.
The invention provides as shown in the formula the application of the substituted aromatic base biguanide compound of I at the preparation medicine for resisting malignant tumors with anti-tumor activity:
Wherein:
R ' is H, phenyl, substituted phenyl, benzene alkyl, benzene thiazolinyl, benzene alkoxyl, phenoxy group, thiophenyl;
R is R
1Substituted phenyl, benzene alkyl, benzene thiazolinyl, benzene alkoxyl, phenoxy group, thiophenyl;
R
1For containing C
1-C
6Straight or branched alkyl, C
1-C
4Perfluoroalkyl, C
1-C
4Alkanoyl, C
1-C
4Ester group, aryl, halogen, NO
2, NH
2,-H ,-OR
2,-SR
2
R
2Be C
1-C
6Straight or branched alkyl, C
1-C
4Perfluoroalkyl, C
1-C
4Alkanoyl;
R or R ' can be single or a plurality of substituent groups, and are in para-position or an ortho position or a position on the aromatic ring respectively.
The present invention includes the pharmaceutically acceptable mineral acid and the acylate of I compounds, comprise hydrochlorate, phosphate, sulfate, acetate, maleate, citrate, benzene sulfonate, toluenesulfonate, fumarate, tartrate etc.
The present invention seeks the antitumor drug that suppresses tumor invasion transfer and angiogenesis with the means of computer ancillary drug design; We are to the three-dimensional structure database that contains 90,000 micromolecular compounds screening of giving a mark; (energy value is low more finally to obtain 1000 forward chemical compounds of score; Score is forward more), wherein the substituted aromatic base biguanide compound has all got into the scope of preceding 1000 chemical compounds, and representative chemical compound has:
| Chemical compound | Energy value |
| N-benzyl biguanide p-methyl benzenesulfonic acid salt (1) | -46.44 |
| N-{4-[(trifluoromethyl) sulfo-] phenyl } biguanide hydrochloride (2) | -54.06 |
| N-(3-chloro-phenoxy group) biguanide (3) | -34.19 |
| 3-biguanide-ethyl benzoate hydrochlorate (4) | -33.89 |
| N-(2-ethylphenyl) biguanide hydrochloride (5) | -36.9 |
| N-benzyl-N-phenyl biguanide p-methyl benzenesulfonic acid salt (6) | -42.76 |
| N-(3, the 4-Dichlorobenzene base) biguanide (7) | -47.07 |
| N-{3-[(methyl) sulfo-] phenyl } biguanide hydrochloride (8) | -43.35 |
| N-{2-[(methyl) sulfo-] phenyl } biguanide hydrochloride (9) | -45.48 |
| N-(2-chloro-5-trifluoromethyl) biguanide (10) | -47.24 |
| N-(2-ethoxyl phenenyl) biguanide (11) | -46.96 |
| N-(5-chloro-2-aminomethyl phenyl) biguanide (12) | -38.38 |
| N-{4-[(difluoromethyl) sulfo-] phenyl } biguanide hydrochloride (13) | -46.77 |
| N-(3-chloro-4-ethoxy benzyl) biguanide hydrochloride (14) | -48.86 |
| N-(3-chloro-4-methoxy-benzyl) biguanide hydrochloride (15) | -50.16 |
| N-(4-chloro-2-aminomethyl phenyl) biguanide hydrochloride (16) | -42.43 |
| N-(3-chloro-4-fluorophenyl) biguanide hydrochloride (17) | -44.85 |
| N-(4-Trifluoromethoxyphen-l) biguanide hydrochloride (18) | -38.95 |
| N-(3-nitrobenzophenone) biguanide (19) | -46.02 |
| N-(3-chloro-4-fluorobenzene methoxyl group) biguanide (20) | -39.89 |
| N-(2, the 4-Dichlorobenzene base) biguanide hydrochloride (21) | -47.70 |
| N-(2, the 3-Dichlorobenzene base) biguanide hydrochloride (22) | -46.73 |
| N-(4-fluorophenyl) biguanide hydrochloride (23) | -48.30 |
We have further carried out Determination of biological activity to these substituted aromatic base biguanide compounds, find that they all have inhibitory action in various degree to tumor cell adhesion, Invasion and Metastasis and neonate tumour blood vessel.
Description of drawings:
Fig. 1 is a substituted aromatic base biguanide compound structural formula;
Fig. 2 suppresses lab diagram for tumor cell adhesion;
Fig. 3 is cell injury healing lab diagram;
Fig. 4 forms lab diagram for the vascular endothelial cell tube chamber; Wherein, Fig. 4 A is for being the inductive blank group of the no factor, and Fig. 4 B is the positive controls of the bFGF of adding 100ng/ml, and Fig. 4 C is for add the processed group of 100ng/mlbFGF and 10 μ g/ml N-(3, the 4-Dichlorobenzene base) biguanide simultaneously.
Fig. 5 is the apoptosis lab diagram, wherein, adds N-(3, the 4-Dichlorobenzene base) biguanide of 0,5 μ g/ml and 10 μ g/ml among Fig. 5 A, Fig. 5 B, Fig. 5 C respectively.
At this,, provide the pharmacological experimental data of exemplary embodiments N-of the present invention (3, the 4-Dichlorobenzene base) biguanide below in order to show the practicality of target compound.
The experiment of pharmacological evaluation one tumor cell adhesion
Principle:
Corresponding part adheres to tumor cell in film surface receptor such as element and the extracellular matrix through integrating, and adhesion is the moving step of beginning that tumor cell invasion shifts, and helps normal structures such as tumor cell infringement basement membrane.This method is used to measure the influence of chemical compound to tumor cell and basement membrane components adhesive capacity.Select the human melanoma cell system of integrin alpha v beta 3 high expressed to be as subject cell; Select the ligands specific albumen vitronectin of integrin alpha v beta 3 to encapsulate 96 orifice plates; Add test-compound when adding cell; After hatching after a while, rinse out not adherent cell, adhere to the light absorption value that the cell quantity on the plate measures with the crystal violet method and reflect.
Experimental technique:
Experiment is divided into two groups: N-(3, the 4-Dichlorobenzene base) biguanide group and negative control chemical compound group.Establish six dosage of 50,25,12.5,6.25,3.13,1.56 μ g/ml altogether.Other establishes and adds isopyknic solvent in the blank, adheres to contrast, cell adhesion suppression ratio and half amount of suppression (IC50) in the experiment with computing group as cell 100%.
Add the every hole 100 μ l of 5 μ g/ml vitreous adhesion albumen (vitronectin) in (1) 96 orifice plate, 4 ℃ are spent the night.Control wells encapsulates with 5 μ g/ml bovine serum albumin (BSA), and every hole 100 μ l get rid of the influence of non-specific binding.
(2) sucking-off next day coating buffer adds the 1%BSA sealing, every hole 100 μ l, and incubated at room 1 hour, phosphate buffer (PBS) is washed twice.
(3) to contain 1%BSA, 1mM CaCl
2, 1mM MgCl
2, 1mM MnCl
2RPMI1640 serum-free medium suspension M21 human melanoma cell, every hole adds 8 * 10
4Cell, the experimental group adding receives the reagent thing, and the blank group adds solvent PBS as contrast, 37 ℃ of 5%CO
2Hatched in the incubator 1 hour.
(4) sucking-off culture medium, every hole add 200 μ l PBS, wash gently three times, to remove not adherent cell.
(5) discard PBS, every hole added 100 μ l, 10% glutaraldehyde fixing 30 minutes.
(6) thoroughly clean with deionized water wash to glutaraldehyde, put 37 ℃ of baking oven finish-dryings.
(7) the crystal violet pair cell of adding 0.1% dyes jolting 30 minutes.
(8) use distilled water wash, rinse well, put 37 ℃ of baking oven finish-dryings to unnecessary crystal violet.
(9) crystal violet of the acetic acid pair cell of adding 10% absorption is extracted.
On ELIASA, measure after (10) 1 hours, wavelength is 595nm.
Experimental result:
N-(3; The 4-Dichlorobenzene base) biguanide shows the tumor cell of integrin alpha v beta 3 high expressed and the adhesion experimental result of ligands specific vitronectin; N-(3; The 4-Dichlorobenzene base) biguanide can obviously suppress the adhesion of tumor cell and vitronectin, and is the dose-dependence (see figure 2), and the IC50 value is 10.61 ± 1.04 μ g/ml.Control compound does not almost have inhibitory action to the adhesion of tumor cell.
Pharmacological evaluation two cell injury healing experiment
Principle:
The wound healing experiment is used for studying the migration of number of different types cell, and in the research that is commonly used in metastasis and angiogenesis medicament.After the cell of monolayer culture was damaged or strikes off, the cell of damage zone will increase the secretion of somatomedin, propagation through cell and the migration damage of healing.The wound healing of cell monolayer can be observed in 3-24 hour.When carrying out the wound healing experiment, on the cell of monolayer growth, mark a band with rifle head or syringe needle usually, cell outstanding and migration to injury region in damage back is estimated the result in the distance of different time migration through the record cell.
Method:
Experiment is divided into the every hole of 24 orifice plates and at the bottom of plate, does three markings longitudinally.Get the good human melanoma cell of growth conditions, the adjustment cell concentration is 2 * 10
4/ ml is inoculated in 24 orifice plates, and every hole 500 μ l are cultured to 90% cell and converge, and are replaced by serum-free medium, spends the night in 37 ℃ of 5%CO2 incubators, uses diameter to be about standardized horizontal scratch of lancet point of 2.5mm.With aseptic PBS washing three times, remove the cell that comes off in the scratching process.Add N-(3, the 4-Dichlorobenzene base) biguanide of 2.5,5,10 μ g/ml, and establish the matched group that does not add medicine, establish three parallel holes for every group.The migration distance of scratch and markings infall cell after the following record of the inverted phase contrast microscope different time points.
Experimental result:
As shown in Figure 3, line damage is after 8 hours, and cellular control unit has moved 0.31mm, and under the N-(3, the 4-Dichlorobenzene base) of 2.5,5,10 μ g/ml biguanide effect, and 0.21,0.083 and 0mm, the migration suppression ratio is respectively 32.3%, 73.2% and 100%.After 18 hours, cellular control unit has moved 1.19mm, and the experimental group cell that has added 2.5,5,10 μ g/mlN-(3, the 4-Dichlorobenzene base) biguanide has moved 0.72,0.39 and 0.2mm respectively, and the migration suppression ratio is respectively 39.5%, 67.2% and 83.2%.Therefore, this chemical compound not only can obviously suppress tumor cell migration, can also play long-acting inhibitory action.
Pharmacological evaluation three vascular endothelial cell tube chambers form experiment
Principle:
The umbilical vein vascular endothelial cell can be under the effect of VEGF; In the gel rubber system that artificial Matrigel forms, form the blood capillary spline structure: the cell that is cobble appearance profile originally; Cell space elongates and has blastogenesis shape tubular structure to stretch out and make cell space irregular; Often interconnect with tubular structure between adjacent ameboid cell, prolonging in the cyton longitudinal axis or the overhanging tubular structure Chang Kejian has translucent cavity or crack to occur.Research at present thinks that endotheliocyte is similar to its angiogenic process in vivo in these changes of artificial Matrigel, thereby can be used to detect the combined influence of different drug to endotheliocyte angiogenesis ability.
Method:
The artificial Matrigel of ECM is spread 96 orifice plates into pre-cooling, every hole 60 μ l, 37 ℃ solidified 1 hour.Every hole adds 2 * 10
4Human umbilical vein endothelial cells (HUVEC); The basic fibroblast growth factor (bFGF) that adds 100ng/ml in the positive controls; Negative control group as contrast, adds the bFGF of 100ng/ml and the test-compound of 10 μ g/ml with PBS in the processed group, establish three parallel holes for every group.Hatch observed result after 6 hours under 37 ℃ of 5%CO2 conditions
Experimental result:
Human umbilical vein endothelial cells can form the tube chamber spline structure that is similar to new vessels on artificial Matrigel under the inducing of bFGF, see Fig. 4; Wherein, Fig. 4 A does not have the inductive endotheliocyte of the factor, and B is that the bFGF of 100ng/ml induces down the interconnected tube chamber spline structure that forms, and C is for having added 10 μ g/ml N-(3; The 4-Dichlorobenzene base) processed group of biguanide; Though also added the somatomedin bFGF of same amount in this group, because the anti-angiogenic rebirth effect of chemical compound can not form the tube chamber spline structure.
The two staining apoptosis experiments of pharmacological evaluation four Annexin V/PI
Principle:
Tumor cell can promote tumor existence through the adhesion of cell and extracellular matrix, if this adhesive attraction is suppressed, then cell death inducing rapidly is called as " anoikis " (anoikis).Apoptotic cell has anti-metachromia to the dyestuff PI that is used for the cytoactive evaluation, and non-viable non-apoptotic cell then can not.Cell membrane has the DNA of the non-viable non-apoptotic cell of damage by PI to dye the generation red fluorescence, and the apoptotic cell that cell membrane remains intact does not then have red fluorescence and produces.Therefore, apoptotic early stage PI not can dye and do not have the red fluorescence signal.Normal living cells similarly.On the scatterplot of bivariate flow cytometer, left lower quadrant shows living cells, is (FITC
-/ PI
-); Right upper quadrant is non-living cells, and promptly non-viable non-apoptotic cell is (FITC
+/ PI
+); And right lower quadrant is an apoptotic cell, manifests (FITC
+/ PI
-)
Method:
(1) the take the logarithm human melanoma cell of trophophase, the adjustment cell concentration is 5 * 10
5/ ml spreads into 6 porocyte culture plates, adds N-(3, the 4-Dichlorobenzene base) biguanide of different 0,5,10 μ g/ml respectively, hatches 24 hours under 37 ℃ of 5%CO2 conditions.
After (2) 24 hours, collecting cell, centrifugal 5 minutes of 4 ℃ of 1000rpm abandon supernatant.
(3) with the dilution proportion binding buffer liquid (binding buffer) of sterilized water by 1: 4
(4) concentration of using binding buffer to adjust cell to be measured is 5 * 10
5～1 * 10
6/ ml. gets the 1ml cell, and centrifugal 5 minutes of 4 ℃ of 1000rpm abandon supernatant.
(5) with 1ml PBS washing 1 time, centrifugal 5 minutes of 1000rpm.
(6) with 0.5ml binding buffer re-suspended cell, get 195 μ l cell suspension, add 5 μ l AnnexinV and 10 μ l PI, lucifuge is hatched 10 ~ 15min under the room temperature
(7) add 300 μ l Binding Buffer, go up machine (flow cytometer) immediately and detect: the flow cytometer excitation wavelength is used 488nm, uses a wavelength to detect FITC fluorescence as the passband filter of 515nm, and another wavelength detects PI greater than the filter of 560nm.
Experimental result:
The result shows; See that with untreated cell Fig. 5 (Fig. 5 A) compares; The N-of 5 μ g/ml (Fig. 5 B) and 10 μ g/ml (Fig. 5 C) (3, the 4-Dichlorobenzene base) biguanide was handled after 24 hours, thereby can both be through suppressing cell adhesion inducing cell " anoikis "; Apoptosis rate increases by 27.7% and 44.9% respectively, is dose dependent.
Preliminary study in the pharmacological evaluation five body constituents
Utilize the human melanoma cell of integrating plain high expressed to set up the experimental metastasis model of nude mice, in order to assessing compound antitumor action in vivo.
(1) foundation of the experimental metastasis model of nude mice
BALB/C nu/nu nude mouse, in 4～6 ages in week, body weight 18～22 grams available from Nat'l Pharmaceutical & Biological Products Control Institute's animal center, are raised under the qualified environment of SPF (specific pathogen free) level.Nude mouse is through 150cGy's
137The total irradiation of Cs-gamma-rays, random packet, every group of 3 animals.Use in 12～72h
Number injection needle slowly injects 2 * 10 through the tail vein
6The M21 cell.
(2) anti-tumor in vivo transfer activity experiment
Experiment is divided into negative control group and treatment group, and negative control group gives normal saline as contrast.The biguanide to N-(3, the 4-Dichlorobenzene base) is organized in treatment, is divided into two dosage of height.From inoculating back the 2nd day, administration every day 1 time, totally 20 times.
(3) observation of curative effect
Observe the general situation of nude mice during the administration, body weight of weighing in per two days.The 7th weekend, disconnected neck was put to death animal in the inoculation back.Separate after weighing and cut the nude mice lungs, use scales/electronic balance weighing, again that it is fixing with BouinShi liquid (saturated picric acid 75ml+40% formaldehyde 25ml+ glacial acetic acid 5ml), lung tissue is yellow behind the 48h, and the neoplasm metastasis kitchen range becomes the white protuberance.Observe and record lung MET number and size with anatomic microscope, and press the size fractionation of MET.The routine paraffin wax section, HE dyeing, pathological study.The result shows that N-(3, the 4-Dichlorobenzene base) biguanide can obviously suppress lung in vivo and shift nodular formation.The variation of treatment back nude mice body weight can reflect the toxic action of medicine indirectly; The animal of treatment group is compared with not treatment group; Not only do not alleviate, on the rise on the contrary, N-(3 is described; The 4-Dichlorobenzene base) biguanide does not have overt toxicity to animal, and can overcome not treatment group owing to shifting losing weight of causing.The PRELIMINARY RESULTS prompting; N-(3, the 4-Dichlorobenzene base) biguanide can be efficiently, the inhibition neoplasm metastasis of low toxicity, because N-(3; The 4-Dichlorobenzene base) biguanide can estimate that to the targeting of integrin alpha V β 3 it also possibly produce tangible antimetastatic activity to the tumor of other integrin alpha v beta 3 high expressed.
In sum; Vivo and vitro experiment through pharmacology can further prove: substituted aryl biguanides derivant of the present invention demonstrates the effect of transfer of obvious suppression tumor cell invasion and angiogenesis; Have characteristics efficient, low toxicity simultaneously, therefore be expected to using aspect the multiple malignant tumor medicines such as the Humanmachine tumour that suppresses tumor invasion transfer and angiogenesis and treatment integrin alpha v beta 3 high expressed, ovarian cancer, pulmonary carcinoma, gastric cancer, carcinoma of prostate, glioma.
The present invention is used to treat the pharmaceutical composition of malignant tumor, and it comprises as the substituted aromatic base biguanide compound of active component and one or more pharmaceutically acceptable carriers, excipient or diluent.
At this, contain the formed pharmaceutical composition of exemplary embodiments 7 effective ingredient of the present invention in order to show the practicality of target compound (I), to provide below.
Substituted aromatic base biguanides series compound of the present invention is normally taken with the form of pharmaceutical composition; Orally-ingestible or non-oral administration are perhaps with the oral or non-oral administration of chemical compound (like tablet, slow releasing preparation, capsule, injection, the solution) safety that forms with pharmaceutically acceptable carrier, excipient and other additive.When oral administration, compositions can be mixed with tablet, sugar-coat agent or capsule.For the preparation combination of oral medication can adopt lactose or starch to do carrier, gelatin, sodium carboxymethyl cellulose, methylcellulose, polyvinylpyrrolidone etc. are suitable bonding agent or become an agent.Can select starch or microcrystalline Cellulose for use as disintegrating agent, often with Pulvis Talci, santocedl, tristerin, calcium stearate or magnesium etc. are as suitable antiadhesives and lubricant.For example, can prepare tablet through the compacting wet granular.Active component and carrier and optionally with a disintegrate additive composition mixture; The aqueous solution of this mixture and binding agent; Alcohol property or aqueous alcohol property solution carry out granulating in suitable device; Dried particles adds other disintegrating agent subsequently, and lubricant and antiplastering aid are with this mixture tabletting.
Substituted aromatic base biguanides series compound of the present invention can the injection form administration, though dosage changes according to treatment target, administering mode, symptom and other factor.When parenterai administration, compositions of the present invention is made into ejection preparation.
Chemical compound of the present invention is effective in quite wide dosage range.The dosage of for example taking every day can be in the scope of the about 0.5mg-1200mg of per kilogram of body weight.In adult's treatment, dosage range once or is several times taken preferably at 1mg/kg--50mg/kg.The dosage of the actual chemical compound of taking should be decided according to relevant situation by the doctor; These situation comprise by the condition of therapist; The person's of choosing route of administration, age, body weight, patient are to the individual reaction of medicine; Order of severity of patient's symptom or the like, therefore above-mentioned dosage range is not to limit scope of the present invention by any way.
The specific embodiment
In order to explain enforcement of the present invention more fully, following FORMULATION EXAMPLE is provided.These embodiment explain rather than limit scope of the present invention.Preparation can adopt the active component of any chemical compound among the present invention.
Preparation 1
Every preparation tablets that contains the 10mg active component is following:
Consumption/sheet weight concentration (%)
Chemical compound (2) 100mg 10.0
Microcrystalline Cellulose 35mg 35.0
Starch 45mg 45.0
Polyvinylpyrrolidone 4mg 4.0
Carboxymethyl starch sodium salt 4.5mg 4.5
Magnesium stearate 0.5mg 0.5
Pulvis Talci 1mg 1.0
Amount to 100 100.0
With active component, starch and cellulose sieve, and fully mix; Polyvinylpyrrolidonesolution solution is mixed with above-mentioned powder, sieve, make wet granular in 50-60 ℃ of drying; With the carboxymethyl starch sodium salt, magnesium stearate and Pulvis Talci sieve in advance, join tabletting in the above-mentioned granule then.
Preparation 2
The preparation of injection
Chemical compound (5) 200mg
Mannitol 700mg
PEG3000 10mg
Distilled water 100ml
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.
Preparation 3
It is following that every capsule contains the capsular preparation of 100mg active component:
Consumption/capsule weight concentration (%)
Chemical compound (7) 100mg 30.0
Polyoxyethylene dehydration Pyrusussuriensis 0.05mg 0.02
The sugar alcohol monoleate
Starch 250mg 69.98
Amount to 350.05mg 100.00
Claims (8)
1. the application of substituted aromatic base biguanide compound aspect preparation inhibition tumor invasion diversion medicaments; It is selected from the following chemical compound one or more:
N-benzyl biguanide p-methyl benzenesulfonic acid salt;
N-{4-[(trifluoromethyl) sulfo-] phenyl } biguanide hydrochloride;
N-(3-chloro-phenoxy group) biguanide;
3-biguanide-ethyl benzoate hydrochlorate;
N-(2-ethylphenyl) biguanide hydrochloride;
N-benzyl-N-phenyl biguanide p-methyl benzenesulfonic acid salt;
N-(3, the 4-Dichlorobenzene base) biguanide;
N-{3-[(methyl) sulfo-] phenyl } biguanide hydrochloride;
N-{2-[(methyl) sulfo-] phenyl } biguanide hydrochloride;
N-(2-chloro-5-trifluoromethyl) biguanide;
N-(2-ethoxyl phenenyl) biguanide;
N-(5-chloro-2-aminomethyl phenyl) biguanide;
N-{4-[(difluoromethyl) sulfo-] phenyl } biguanide hydrochloride;
N-(3-chloro-4-ethoxy benzyl) biguanide hydrochloride;
N-(3-chloro-4-methoxy-benzyl) biguanide hydrochloride;
N-(4-chloro-2-aminomethyl phenyl) biguanide hydrochloride;
N-(3-chloro-4-fluorophenyl) biguanide hydrochloride;
N-(4-Trifluoromethoxyphen-l) biguanide hydrochloride;
N-(3-nitrobenzophenone) biguanide;
N-(3-chloro-4-fluorobenzene methoxyl group) biguanide;
N-(2, the 4-Dichlorobenzene base) biguanide hydrochloride;
N-(2, the 3-Dichlorobenzene base) biguanide hydrochloride;
N-(4-fluorophenyl) biguanide hydrochloride.
2. the described application of claim 1, it is selected from following chemical compound: N-benzyl biguanide p-methyl benzenesulfonic acid salt N-(2, the 4-Dichlorobenzene base) biguanide hydrochloride N-(2, the 3-Dichlorobenzene base) biguanide hydrochloride; N-(2-chloro-5-trifluoromethyl) biguanide; N-(5-chloro-2-aminomethyl phenyl) biguanide; N-{3-[(methyl) sulfo-] phenyl } biguanide hydrochloride N-{4-[(trifluoromethyl) sulfo-] phenyl } biguanide hydrochloride; N-(3-chloro-phenoxy group) biguanide; 3-biguanide-ethyl benzoate hydrochlorate; N-(2-ethylphenyl) biguanide hydrochloride; N-benzyl-N-phenyl biguanide p-methyl benzenesulfonic acid salt or N-(3, the 4-Dichlorobenzene base) biguanide.
3. the described application of claim 2, it is selected from following chemical compound: N-(2-ethylphenyl) biguanide hydrochloride N-(2, the 4-Dichlorobenzene base) biguanide hydrochloride; N-(2, the 3-Dichlorobenzene base) biguanide hydrochloride; N-(2-chloro-5-trifluoromethyl) biguanide; N-(5-chloro-2-aminomethyl phenyl) biguanide N-{3-[(methyl) sulfo-] phenyl } biguanide hydrochloride or N-(3, the 4-Dichlorobenzene base) biguanide.
4. each defined application of claim 1-3, the application of its chemical compound aspect the Humanmachine tumour for preparing treatment integrin alpha v beta 3 high expressed, ovarian cancer, pulmonary carcinoma, gastric cancer, carcinoma of prostate, neuroglia tumor medicine.
5. each defined application of claim 1-3, the application of its chemical compound aspect the medicine of preparation treatment Humanmachine tumour malignant tumor.
6. each defined application of claim 1-3, the application of its chemical compound aspect preparation inhibition neonate tumour blood vessel medicine.
7. each defined application of claim 1-3, the application of its N-(3, the 4-Dichlorobenzene base) biguanide compound aspect preparation inhibition tumor invasion diversion medicaments.
8. each defined application of claim 1-3, the application of its N-(3, the 4-Dichlorobenzene base) biguanide compound aspect the medicine of preparation treatment Humanmachine tumour malignant tumor.
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| US20130059916A1 (en) * | 2010-05-26 | 2013-03-07 | Stephane Rocchi | Biguanide compounds and its use for treating cancer |
| JP5927518B2 (en) * | 2011-08-08 | 2016-06-01 | イムノメット セラピューティクス インク | N1-cyclic amine-N5-substituted phenyl biguanide derivative, process for its preparation and pharmaceutical composition containing it |
| KR102216701B1 (en) | 2014-11-20 | 2021-02-18 | 이뮤노메트테라퓨틱스 인코포레이티드 | Biguanide compounds and use thereof |
| CN106748941A (en) * | 2016-11-28 | 2017-05-31 | 武汉福山生物科技有限公司 | Selenium-containing compound and application thereof |
| CN106986791A (en) * | 2017-05-11 | 2017-07-28 | 杜剑平 | It is a kind of to treat medical compounds of tumour and its preparation method and application |
| CN112574071A (en) * | 2019-09-30 | 2021-03-30 | 复旦大学 | Amphiphilic metformin derivative with biguanide group connected with fatty carbon chain and pharmaceutical application thereof |
| CN111138660B (en) * | 2019-12-20 | 2020-12-08 | 浙江大学 | Preparation method of fluorinated polyhexamethylene biguanide |
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