Meishadazole compounds and preparation method and application thereof
阅读说明：本技术 一组美沙达唑类化合物及其制备方法和应用 (Meishadazole compounds and preparation method and application thereof ) 是由 吴长生 李岳兰 李越中 于 2021-06-17 设计创作，主要内容包括：本发明公开了一组美沙达唑类化合物,是如式(Ⅰ)所示结构的化合物之一。该类化合物是由黏球菌(Myxococcus sp.)SDU36CCTCC NO：M 2021520经过培养和发酵,从其发酵液中分离获得的。实验证实：本发明所述美沙达唑类化合物能明显促进血管破损斑马鱼的血管的生成,同时还对血栓具有一定的缓解作用。提示本发明的化合物有望增益其在促进血管生成和抗血栓活性中的应用价值,为制备新一代心血管系统药物的开发和应用提供新的途径,在临床应用中具有较好的社会效益、经济价值和市场前景。(The invention discloses a group of mesartan compounds, which are one of compounds with a structure shown as a formula (I). The compound is prepared from mucococcus (Myxococcus sp.) SDU36CCTCC NO: m2021520 is obtained by culturing and fermenting, and separating from its fermentation liquid. The experiment proves that: the mesartan compound can obviously promote the generation of blood vessels of damaged blood vessels of zebra fish, and has a certain relieving effect on thrombus. The compound is expected to gain the application value of the compound in promoting angiogenesis and antithrombotic activity, provides a new way for the development and application of preparing a new generation of cardiovascular system medicaments, and has better social benefit, economic value and market prospect in clinical application.)
1. A group of mesartan azole compounds is characterized in that: the mesartan compound is one of compounds with a structure shown in a formula (I):
wherein:
。
2. The mesartan compound according to claim 1, characterized in that: the methoxadazole compound is named as methoxadazole A1The compound 1 of (1); or is named as the metxadazole A2Compound 2 of (1); or is named as the metxadazole B2Compound 3 of (1).
3. A process for the preparation of mesartan compounds according to claim 1 or 2, characterized in that: the mesartan compound is prepared from mucococcus (Myxococcus sp.) SDU36CCTCC NO: m2021520 was obtained from the fermentation product after liquid fermentation.
4. Use of mesartan compounds according to claim 1 for the preparation of medicaments having a pro-angiogenic and/or antithrombotic effect.
5. Use according to claim 4, characterized in that: the methoxadazole compound is named as methoxadazole A1The compound 1 of (1); or is named as the metxadazole A2Compound 2 of (1); or is named as the metxadazole B2Compound 3 of (1).
6. A angiogenesis promoting medicine containing a mesartan compound is characterized in that: the drug contains a therapeutically effective amount of the mesartan compound and a pharmaceutically acceptable carrier.
7. An antithrombotic effect medicine containing a mesartan compound, which is characterized in that: the drug contains a therapeutically effective amount of the mesartan compound and a pharmaceutically acceptable carrier.
8. A angiogenesis promoting pharmaceutical composition containing mesartan compounds is characterized in that: the pharmaceutical composition is an angiogenesis promoting pharmaceutical composition prepared from a therapeutically effective amount of a mesartan azole compound, a therapeutically effective amount of one or any combination of a danhong injection and a danshensu sodium.
9. An antithrombotic pharmaceutical composition containing a mesartan compound, which is characterized in that: the pharmaceutical composition is an antithrombotic pharmaceutical composition prepared from a therapeutically effective amount of a mesartan azole compound and a therapeutically effective amount of one or any combination of aspirin, clopidogrel, salvianolate, diltiazem hydrochloride, thromboxane and Xuesaitong.
Technical Field
The invention relates to a group of mesartan compounds, a fermentation separation preparation method of the mesartan compounds and application of the mesartan compounds in preparation of medicines for treating and preventing cardiovascular diseases. Belongs to the field of microbial technology, product and application technology.
Background
Slime bacteria are a group of gram-negative bacteria with complex cellular social behavior and multi-cellular developmental cycles, capable of gliding, forming morphologically specific fruiting bodies, sporulating and predating other bacteria, and secondlyThe secondary metabolite is abundant and has various biological activities, and is more and more concerned by scholars at home and abroad (J).F.J.Marcos-Torres,E.García-Bravo,A. J.P rez, front. Microbiol.2016,7, 781-781.). Myxobacteria are even a rich source of hybrid natural products, the most well-known congeneric Sorangium cellulosum (Sorangium cellulosum) synthetic series of NRPS-PKS hybrid epothilone epothilones, whose synthetic analogs ixabepilone (ixabepilone) and Youtellone (utidelone) were approved by the FDA and SFDA, respectively, for marketing in 2007 and 2021. Therefore, the slime bacteria have great synthesis potential and excavation prospect of novel active products.
The mesartan compounds are alkaloid new frameworks formed by the hybridization of fatty acid chains of isoxazole rings derived from mucobacteria and N-ribitol-5, 6-dimethyl benzimidazole. Isoxazole rings are quite rare in nature, and only 5 natural products containing isoxazole fragments are currently found from actinomycetes and fungi: cycloserine (M. -L.Svensson, S.Gatenbeck, Arch.Microbiol. 1982,131,129-131), acivicin and 4-hydroxyaacivicin (S.J.Gould, S.Ju, J.Am.Chem.Soc.1993, 114,10166-10172), muscarine (M.Onda, H.Fukushima, M.Akagawa, chem.pharm.Burm.1964, 12,751-751), amanithine (K.Bowden, A.C.Drysdale, G.A.Mogey, Nature 1965,206, 1359-1360). And the mesartan compound is the first discovery of an isoxazole ring in a mucobacteria. Even more remarkably, the presence of only vitamin B has been previously reported for dimethylbenzimidazole12This also highlights the novelty of the structure of mesartan. Through retrieval, various compounds related to the mesartan and fermentation, separation and preparation of the mesartan and application of the compounds in preparation of medicaments for treating and preventing cardiovascular diseases are not reported.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide a group of mesartan compounds, a preparation method thereof and application thereof in preparing medicines with angiogenesis promoting and/or antithrombotic effects.
The invention relates to a group of mesartan compounds, which are characterized in that: the mesartan compound is one of compounds with a structure shown in a formula (I):
wherein:
7' configuration
R group
Name of
Compound 1
S
R＝Me
Metronidazole A1
Compound 2
R
R＝Me
Metronidazole A2
Compound 3
R
R＝H
Metronidazole B2
Compound 4
S
R＝H
Metronidazole B1
The above-mentioned methoxadiazole compound is preferably named methoxadazole A1The compound 1 of (1); or is named as the metxadazole A2Compound 2 of (1); or is named as the metxadazole B2Compound 3 of (1).
The preparation method of the mesartan compound is characterized by comprising the following steps: the mesartan compound is prepared from mucococcus (Myxococcus sp.) SDU36CCTCC NO: m2021520 was obtained from the fermentation product after liquid fermentation.
The invention relates to application of a mesartan compound in preparation of drugs with angiogenesis promoting and/or antithrombotic effects.
Wherein: the methoxadazole compound is preferably named as methoxadazole A1The compound 1 of (1); or is named as the metxadazole A2Compound 2 of (1); or is named as the metxadazole B2Compound 3 of (1).
The experiment proves that: the mesartan compound can obviously promote the generation of blood vessels of zebra fish with damaged blood vessels, can repair the blood vessels damaged by PTK787 induction under the dosage of 10 mu M and 25 mu M, and has the repair capability equivalent to that of a danhong injection (positive control). At the same time, it was also confirmed that: the mesartan azole compound has a certain relieving effect on thrombus at the concentrations of 10 mu M and 25 mu M, and the treatment effects are 31.7 percent and 59.0 percent respectively (the positive control group is aspirin 71.1 percent); wherein is named as the metxadazole A1The compound (1) is most effective.
The invention further discloses a angiogenesis promoting medicine containing the mesartan compound, which is characterized in that: the drug contains a therapeutically effective amount of the mesartan compound and a pharmaceutically acceptable carrier.
The invention further discloses an antithrombotic drug containing the mesartan compound, which is characterized in that: the drug contains a therapeutically effective amount of the mesartan compound and a pharmaceutically acceptable carrier.
The invention further discloses an angiogenesis promoting pharmaceutical composition containing the mesartan compound, which is characterized in that: the pharmaceutical composition is an angiogenesis promoting pharmaceutical composition prepared from a therapeutically effective amount of a mesartan azole compound, a therapeutically effective amount of one or any combination of a danhong injection and a danshensu sodium.
The invention further discloses an antithrombotic pharmaceutical composition containing the mesartan compound, which is characterized in that: the pharmaceutical composition is an antithrombotic pharmaceutical composition prepared from a therapeutically effective amount of a mesartan azole compound and a therapeutically effective amount of one or any combination of aspirin, clopidogrel, salvianolate, diltiazem hydrochloride, thromboxane and Xuesaitong.
The pharmaceutically acceptable carrier refers to a conventional pharmaceutical carrier in the pharmaceutical field, such as: diluents, excipients such as water and the like, fillers such as starch, sucrose and the like, binders such as cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone; humectants such as glycerol; disintegrating agents such as agar, calcium carbonate and sodium bicarbonate; absorption enhancers such as quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorption carriers such as kaolin and bentonite; lubricants such as talc, calcium and magnesium stearate, and polyethylene glycol, and the like. Other adjuvants such as flavoring agent, sweetener, etc. can also be added into the composition.
The mesartan compound of the present invention is preferably administered in the form of a composition to a patient in need of treatment of the condition by oral, nasal inhalation or parenteral administration. For oral administration, it can be made into conventional solid preparations such as tablet, powder, granule, capsule, etc., liquid preparations such as aqueous or oil suspension, or other liquid preparations such as syrup, elixir, etc.; for parenteral administration, it can be formulated into solution for injection, aqueous or oily suspension, etc. Preferred forms are tablets, coated tablets, capsules, suppositories, nasal sprays and injections, particularly preferred formulations for delivery at specific sites in the intestinal tract.
The various dosage forms of the pharmaceutical compositions of the present invention may be prepared according to conventional manufacturing methods in the pharmaceutical art, for example by mixing the active ingredient with one or more carriers and then formulating the same into the desired dosage form.
The pharmaceutical composition of the present invention preferably contains 0.1% to 99.5% by weight (or volume) of the active ingredient, and most preferably 0.5% to 95% by weight (or volume) of the active ingredient.
The amount of the mesartan compound to be administered according to the present invention may vary depending on the route of administration, the age, body weight of the patient, the type and severity of the disease to be treated, etc., and the daily dose thereof may be 0.01 to 10mg/kg body weight, preferably 0.1 to 5mg/kg body weight. One or more administrations may be carried out.
The invention discloses a group of mesartan compounds, a preparation method thereof and application thereof in preparing medicaments with angiogenesis promoting and/or antithrombotic effects. Wherein the mesartan compound is a novel skeleton natural product of isoxazole-benzimidazole hybrid, and is also the first discovery of the isoxazole compound in mucobacteria. The experiment proves that: the mesartan compound can obviously promote the generation of blood vessels of zebra fish with damaged blood vessels, can repair the blood vessels damaged by PTK787 induction under the dosage of 10 mu M and 25 mu M, and has the repair capability equivalent to that of a danhong injection (positive control). At the same time, it was also confirmed that: the mesartan compound has a certain relieving effect on thrombus under the concentration of 10 mu M and 25 mu M, and the treatment effect is 31.7 percent and 59.0 percent respectively (aspirin 71.1 percent in a positive control group). The compound is prompted to promote angiogenesis of the transgenic zebra fish in vivo and has certain antithrombotic activity. The mesartan compound obtained by the invention is expected to gain the application value of the mesartan compound in promoting angiogenesis and antithrombotic activity, provides a new way for preparing a new generation of cardiovascular system medicaments, and generates better social benefit and economic value. The mesartan compound provided by the invention has wide clinical application and market prospect.
Drawings
FIG. 1: the evolutionary position of the strain SDU36 is at the evolutionary position in mucobacteriales.
FIG. 2: metronidazole A1/A2(1-2) Key HMBC and1H-1the H COSY signal.
FIG. 3: metronidazole B2(3) Key HMBC and1H-1the H COSY signal.
FIG. 4: metronidazole A1(1) Analysis of coupling constants of ribitol chains.
FIG. 5: vitamin B12 is degraded to N-ribityl-dimethylbenzimidazole.
FIG. 6: metronidazole A1/A2(1-2) steric configuration.
Wherein: the left of the figure is compound 1, and the right of the figure is compound 2.
FIG. 7: CP3 probability analysis.
FIG. 8: metronidazole A1/A2(1-2) analysis of intramolecular hydrogen bond interaction.
Wherein: (A) metronidazole A1/A2(1-2) AIM topology analysis chart. Within the circle is the critical point for the key chemical Bond (BCP). Metronidazole A1(1, left of the figure) has 5 hydrogen bonds, and mexadazole A2(2, right in the figure) has 4 hydrogen bonds. (B) Metronidazole A1/A2(1-2) structural schematic diagram. In mosatidazole A1In (1, left of the figure), three methylene groups in C-11', C-12' and C-13' highlighted by dotted lines can flexibly rotate without being bound; in mosatidazole A2In (2, the right part of the figure), three methylene groups in C-11', C-12' and C-13' are in a rigid ring formed by hydrogen bonds, and the single bond cannot rotate, so that two hydrogen atoms on the methylene groups are not chemically equivalent.
FIG. 9: metronidazole A2/B2And (2-3) comparison of ECD spectra.
FIG. 10: metronidazole A1/A2/B2(1-3) pairsThe zebra fish has the effects of promoting angiogenesis and resisting thrombosis.
Wherein: panels a and B show the pro-angiogenic activity of compounds: (A) zebrafish internode blood vessel (ISV) images. (B) quantitative analysis of the length of ISV in zebrafish. Panels C and D show the antithrombotic activity of the compounds: (C) red blood cell image in zebrafish heart, where black dots in the magnified region represent red blood cells in the heart. (D) Quantitative analysis of the staining intensity of erythrocytes in zebrafish hearts.
FIG. 11: metronidazole A1/A2/B2(1-3) the angiogenesis promoting and antithrombotic effects of the composition on zebrafish.
Wherein: panels a and B show the pro-angiogenic activity of compounds: (A) zebrafish internode blood vessel (ISV) images. (B) quantitative analysis of the length of ISV in zebrafish. Panels C and D show the antithrombotic activity of the compounds: (C) red blood cell image in zebrafish heart, where black dots in the magnified region represent red blood cells in the heart. (D) Quantitative analysis of the staining intensity of erythrocytes in zebrafish hearts.
FIG. 12: metronidazole A1Is/are as follows1H nuclear magnetic resonance spectroscopy.
FIG. 13: metronidazole A1Is/are as follows13C nuclear magnetic resonance spectroscopy (DEPTQ).
FIG. 14: metronidazole A1HSQC two-dimensional nuclear magnetic resonance spectrum of (1).
FIG. 15: metronidazole A1HMBC two-dimensional nuclear magnetic resonance spectroscopy.
FIG. 16: metronidazole A1Is/are as follows1H-1H COSY two-dimensional nuclear magnetic resonance spectrum.
FIG. 17: metronidazole A1High resolution mass spectrometry.
FIG. 18: metronidazole A2Is/are as follows1H nuclear magnetic resonance spectroscopy.
FIG. 19: metronidazole A2Is/are as follows13C nuclear magnetic resonance spectroscopy (DEPTQ).
FIG. 20: metronidazole A2HSQC two-dimensional nuclear magnetic resonance spectrum of (1).
FIG. 21: metronidazole A2HMBC two-dimensional nuclear magnetic resonance spectroscopy.
FIG. 22: metronidazole A2Is/are as follows1H-1H COSY two-dimensional nuclear magnetic resonance spectrum.
FIG. 23: metronidazole A2High resolution mass spectrometry.
FIG. 24: metronidazole B2Is/are as follows1H nuclear magnetic resonance spectroscopy.
FIG. 25: metronidazole B2Is/are as follows13C nuclear magnetic resonance spectroscopy (DEPTQ).
FIG. 26: metronidazole B2HSQC two-dimensional nuclear magnetic resonance spectrum of (1).
FIG. 27 is a schematic view showing: metronidazole B2HMBC two-dimensional nuclear magnetic resonance spectroscopy.
FIG. 28: metronidazole B2Is/are as follows1H-1H COSY two-dimensional nuclear magnetic resonance spectrum.
FIG. 29: metronidazole B2High resolution mass spectrometry.
FIG. 30: metronidazole A1According to the HECADE-HSQC two-dimensional nuclear magnetic resonance spectrum.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. The following examples are only preferred embodiments of the present invention, and it should be noted that the following descriptions are only for explaining the present invention and not for limiting the present invention in any form, and any simple modifications, equivalent changes and modifications made to the embodiments according to the technical spirit of the present invention are within the scope of the technical solution of the present invention.
In the following examples, materials, reagents and the like used were obtained commercially unless otherwise specified.
The mucococcus SDU36 is separated from a bottom mud sample of a cave lake of a cave measure in the Reparing county of the autonomous region of Tibet by a conventional method, is identified as a Myxococcus sp SDU36 by a microorganism national key laboratory of Shandong university, and is preserved in a China center for type culture Collection (Wuhan, Wuhan university, Wuhan, China) in 2021, 5 and 12 days, wherein the preservation center is numbered as follows: mucococcus (Myxococcus sp.) SDU36CCTCC NO: m2021520.
Example 1 obtaining of the StrainPreservation and separation and purification of methoxadiazole compound (with methoxadiazole A)1Mexadifen A2Metronidazole B2Example of the design reside in
1.1 isolation of the Strain
The separation method comprises the following steps: after the WCX medium is solidified, smearing colibacillus. And (3) uniformly scattering a fresh or air-dried sample (a soil sample of bottom soil of a lake and a cave in Rechengxian county in Tibet). After culturing to 3d, the plates were examined daily under a stereomicroscope for the presence of a fruiting body or a pellicle of slime bacteria. If so, carefully pick the top of the fruiting body or scrape the edges of the mycoderm, and transfer to new WCX medium plate with E.coli pad or VY/2 medium for purification. A yellow bacterium was obtained in the experiment and named SDU 36.
Wherein, the formulation of the WCX culture medium is as follows: CaCl2·2H2O0.1 g/100ml, agar 1.5g/100ml, pH 7.2. After sterilization cycloheximide was added to a final concentration of 25. mu.g/mL. The formula and the preparation method of the VY/2 culture medium are as follows: angel yeast powder 0.5g/100ml, MgSO4·7H2O 0.1g/100ml，CaCl20.07g/100ml, pH 7.2. VY/2 solid medium was supplemented with 1.5g/100ml agar.
The determination of physiological and biochemical reactions and the analysis of 16S rRNA gene sequence were carried out on the strain SDU36, and it was confirmed that the 16S rRNA gene nucleotide sequence of the strain SDU36 is shown in SEQ ID NO.1, and the strain was identified as a genus Myxococcus (FIG. 1).
The above-mentioned Myxococcus sp SDU36 shows negative gram stain, and vegetative cells of the strain are rod-shaped with two pointed ends and a size of 8-10 μm × 0.2-0.8 μm. The colony grows yellow, thin and expands to the edge, has a slide mark, and the myxospore is oval or elliptical. The strain SDU36 can adsorb congo red and produce catalase, cannot utilize urea, gelatin, starch, cellulose and sucrose, does not produce oxidase and cannot reduce nitrate.
The growth temperature of the above-mentioned mucococcus (Myxococcus sp.) SDU36 is 25-35 deg.C, and the optimum growth temperature is 30 deg.C.
The above-mentioned Myxococcus (Myxococcus sp.) SDU36 in CTT mediumAfter the growth and the shake culture for 48 hours, the density of the thalli reaches 2.23 plus or minus 0.05, and the pH value is 6.2 plus or minus 0.1. The formula of the CTT culture medium is as follows: casein peptone 1g/100ml, Tris-HCl (pH 7.6)10mM, PBS (pH 7.6)10mM, MgSO4·7H2O 0.1g/100ml。
The strain is preserved in China center for type culture Collection (address: Wuhan, Wuhan university, China) in 2021, 5 months and 12 days, and the preservation number is CCTCC NO: m2021520.
The research of the inventor shows that: mucococcus (Myxococcus sp.) SDU36CCTCC NO: m2021520 is a bacterium producing the mesartan compound. By using the liquid fermentation and LC-MS detection method of the strain, the inventor detects and separates the mexadazole compound, namely the mexadazole A, from the strain1Mexadifen A2Metronidazole B2。
1.2 Myxococcus (Myxococcus sp.) SDU36CCTCC NO: fermentation of M2021520 and extraction of Compounds
The mucococcus (Myxococcus sp.) SDU36CCTCC NO was picked from the solid WCX medium described above in the conventional manner: m2021520 cells were inoculated onto VY/2 plates. After 3-4 days of culture at 30 ℃, scraping thalli (10 mu L of the full ring of the inoculated bacteria), inoculating the thalli into a 100ml liquid VY/2 culture medium shake flask, culturing for 4-5 days at 30 ℃, and after reaching the logarithmic growth phase, transferring the thalli to a fresh VY/2 liquid culture medium by taking 10% as the inoculum size for expanding culture. Culturing at 30 deg.C and 200rpm for 7 days, filtering to remove thallus, adding 2g of adsorption resin HP-20 into each 100ml of culture solution, adsorbing at 30 deg.C and 200rpm overnight, and collecting resin to obtain maxadazole compounds.
For example: collecting adsorption resin HP-20 in 80L fermentation liquid, oven drying at 40 deg.C to remove excessive water, loading resin into glass chromatographic column, eluting resin with 20L methanol, and evaporating the extractive solution by rotary evaporator under reduced pressure at 45 deg.C to obtain crude extract (8.0g) containing mexadazole.
The formula and the preparation method of the VY/2 culture medium comprise the following steps: angel yeast powder 0.5g/100ml, MgSO4·7H2O 0.1g/100ml， CaCl20.07g/100ml, pH adjusted to 7.2 with sodium hydroxide. Wherein, VY/2 solid culture medium1.5% agar was added.
1.3 separation and purification of Maxamadazole compounds
The crude extract (8.0g) containing the mesartan azole compounds is further subjected to reversed-phase medium-low pressure column chromatography (instrument: Qi Pure C-810, equipped with FlashPure EcoRFlex C18 chromatographic column), gradient elution is carried out by a methanol-water system (flow rate is 12 ml/min; 10% -100%: 0-180 min; 100%: 30min), and finally 10 sub-fractions are obtained by merging according to TLC detection results. Then, the above mesartan compounds were detected in Fr 5 (50% elution fraction) and Fr 6 (60% elution fraction) by liquid chromatography.
Fr 5(300.0mg) is separated by Sephadex LH-20 gel column chromatography (filling 100g), eluted by methanol, and the eluate is manually received by 4 ml/bottle, and finally combined into 5 components according to TLC detection, and liquid quality detection shows that the component Fr 5-1 contains the mesartan azole compound. Eluting component Fr 5-1 (121.1mg) with reverse phase medium pressure liquid column chromatography, and gradient eluting with methanol-water system at flow rate of 5ml/min 10% -100% for 0-60 min 100% for 15 min. After impurities are removed, 2 mesartan azole compounds are obtained by high performance liquid chromatography purification, and the compounds are named as mesartan A1Compound 1 (t)R14.0min) and denominated methoxazole B)2Compound 3 (t)R12.4 min). The liquid chromatograph is of a Daian U3000 type; adopting Feinuomei Luna-C185 μm column (10X 250mm), ultraviolet detection wavelength of 210 nm, elution condition of 1.8ml/min flow rate, 70% methanol water.
Fr 6(146.3mg) is separated by Sephadex LH-20 gel column chromatography (filling 100g), eluted by methanol, and the eluate is manually received by 4 ml/bottle, and is combined into 10 components according to TLC detection, and liquid quality detection shows that the component Fr 6-4 contains the mesartan compound. Component Fr F-4(21.3mg) was purified by high performance liquid chromatography, eluting with 40% acetonitrile water at a flow rate of 1.8mL/min, at tRCollecting the product at 10.9min and naming as metxadazole A2Compound 2 of (1).
1.4 physicochemical properties and spectral data of Meishadazole compounds
Further, the enriched mexadazole compound is subjected to detection of high-resolution mass spectrum, nuclear magnetism, circular dichroism, optical rotation and infrared spectrum.
The instrument model is as follows: nuclear magnetic testing Bruker Avance III (600 MHz); HRESIMS test for LTQ-Orbitrap XL; testing a Chirascan by a UV/ECD test; optically active assay Anton Paar MCP 200; infrared test Nicolet iN 10 micro FTIR spectrometer.
The physical and chemical properties and spectrum data of the mesartan compound are as follows:
metronidazole A1(1) Light yellow oily matter;UV(MeOH)λmax(logε)279 (3.40),287(3.35)nm；ECD(MeOH):203(Δε+16.34),224(Δε+5.38)nm；IRνmax 3340,2929, 1679,1563,1470,1133cm-1；for 1H NMR and 13c NMR nuclear magnetic data, see table 1; HRESIMS at M/z546.3168 [ M]+(calcd for C29H44O7N3,546.3174).
Metronidazole A2(2) Yellow solid;UV(MeOH)λmax(logε)279(3.69), 287(3.64)nm；ECD(MeOH):203(Δε+5.73),218(Δε+5.40)nm；IRνmax 3397,2953,2925,1651, 1561,1403,1135cm-1；for 1H NMR and 13c NMR nuclear magnetic data, see table 1; HRESIMS at M/z546.3178 [ M]+(calcd for C29H44O7N3,546.3174).
Metronidazole B2(3) Yellow solid;UV(MeOH)λmax(logε)279(3.80), 286(3.75)nm；ECD(MeOH):202(Δε+7.50),216(Δε+4.09)；IRνmax 3351,2957,2930,1654, 1561,1403cm-1；for 1H NMR and 13c NMR nuclear magnetic data, see table 1; HRESIMS at M/z 532.3016[ M]+ (calcd for C28H42O7N3,532.3017).
EXAMPLE 2 structural characterization of Compound 1, Compound 2 and Compound 3
2.1 planar Structure characterization of Compound 1
The high resolution mass spectrum shows the excimer peak [ M + H ] of the compound 1]+m/z546.3168(calcd 546.3174), thereby inferring a molecular weight of 545 and a molecular formula C29H44N3O7The unsaturation degree was 10.
The infrared absorption spectra are respectively 3340cm-1And 1563cm-1The characteristic absorption bands of hydroxyl groups (associative) and strong N-O stretching vibrations are shown.
DEPTQ spectrum of Compound 1(13One of the C spectra) gives a 29 carbon signal: including 4 methyl groups, 9 methylene groups, 5 methine groups (three linked oxygen), 10 aromatic carbons and 1 carboxyl group (table 1).1H–1The H COSY and related HMBC signals show that the structure comprises 3 sets of spin coupling systems which are respectively: a fatty acid chain from C-1 'to C-7', a fatty acid chain from C-11 'to C-14', and a pentose alcohol chain from C-1 'to C-5' (FIG. 2). The hydrogen spectrum shows that the compound has 4 aromatic hydrogen signals including H-2 (delta)H9.91)、H-4(δH7.82)、H-7(δH7.86) and H-9' (delta)H6.74). Wherein at δCCharacteristic Low-field proton of methine C-2 at 141.4 [ H-2 (. delta.) ]H9.91)]Indicating that it is attached to a nitrogen atom. The inventors passed on key HMBC signals [ H-2/C-3a, C-7a ]]Deducing the presence of imidazole ring and based on HMBC signal [ H-4 (delta.)H7.82)/C-6、 C-7a、5-CH3And H-7 (delta)H7.86)/C-3a, C-5 and 6-CH3]The imidazole ring fragment was determined to be fused with an ortho-dimethylbenzene fragment to form 5, 6-dimethylbenzimidazole. In addition, another critical HMBC signal H2-1”(δH4.66 and 4.41)/C-3a, C-2 indicate that the pentitol side chain is attached to the N-3 of the above-mentioned 5, 6-dimethylbenzimidazole fragment. Similarly, in combination with the deduced fatty acid chain fragments, the inventors applied critical HMBC Signal [ H-9' (delta)H6.74)/C-10', C-11', H-7'/C-8', C-9 'and H-11'/C-9', C-10']And deducing a fourteen-element long-chain fatty acid. In order to meet the requirements of molecular formula and unsaturation degree of the structure and characteristic chemical shift, the fourteen-membered long-chain fatty acid C-8' -C-10 ' position must form isoxazole heterocycle, and C-14' must exist in the form of carboxylate (and benzimidazole 1-N position)+Internal salt formation). In conclusion, the inventors have reasonably determined the planar configuration of compound 1, which is a hybrid of N-ribityl-5, 6-dimethylbenzimidazole in the form of an inner salt with C-8 '-C-10' to form an isoxazole heterocyclic tetradecanoic fatty acid, containing a rare parent nucleus of isoxazole-benzimidazole.
Table 1: nuclear magnetic resonance data for Compounds 1-3 (hydrogen Spectrum 600 MHz; carbon Spectrum 150MHz)
2.2 determination of the planar configuration of Compound 2
The high resolution mass spectrum shows the excimer peak [ M + H ] of the compound 2]+m/z546.3178(calcd 546.3174), also 545 in molecular weight and C in molecular formula29H44N3O7。
The DEPTQ spectrum of compound 2 gave 29 signals: including 4 methyl groups, 9 methylene groups, 5 methine groups (three linked oxygen), 10 aromatic carbons and 1 carboxyl group (table 1). Compound 2 has the same molecular formula as compound 1 and the 1D and 2D nuclear magnetic data are very similar (table 1 and figure 2), thus the inventors speculate that compound 2 is a diastereomer of compound 1.
2.3 determination of Compound 3 planar configuration
The high resolution mass spectrum shows the excimer peak [ M + H ] of the compound 3]+m/z 532.3016(calcd 532.3017), its molecular weight is 531, and the molecular formula of the compound is C28H42N3O7One methylene CH less than in compounds 1 and 22(14Da)。
Further, its HSQC, HMBC and COSY signals confirmed the absence of heterobranching at C-1' for compound 3 (fig. 3), which is the only difference in planar configuration of compound 3 from compounds 1 and 2.
2.4 determination of the stereoregularity of the Compounds 1-3
The inventors have performed a configuration analysis of the side chain of pentitol of compound 1 based on coupling constants (J-based configuration analysis, JBCA, an empirical method for determining the relative configuration of chiral centers on carbon chains, G.Bifulco, P.Dambruos, L.Gomez-Paloma, R.Riccio, chem.Rev.2007,107,3744-3779.) to determine the relative configuration of three consecutive chiral centers at C2 "-C3" -C4 "in the 1-3 pentosyl fragment (FIG. 4). Of moderate intensity3JH2”-H3”(4.6Hz) showed rapid conformational interconversion between C2 "and C3". Coupling constant analysis based on 1, 2-secondary methyl system, small coupling value3JC4”-H2”And the value of the intermediate coupling3JC1”-H3”Indicating that C2 "-C3" is erythro. Likewise, large coupling values2JC3”-H4”And2JC4”-H3”c3 "-C4" was also shown to be erythro. In addition, in the same deuterated solvent (DMSO-d)6) Of the ribitol side chain moiety of riboflavin measured in13C chemical shift sum3JHHIt is more consistent (Y.Hu, K.Wang, J.B.MacMillan, org.Lett.2013,15, 390-393.). The absolute configuration of C2 '-C3' -C4 'in compounds 1-3 was assigned as 2' S,3 'S, 4' R, in conjunction with the biogenic hypothesis of the reductive opening of the α -D-ribosyl to ribitol (FIG. 5).
Since both compounds 1 and 2 are ribityl, we speculate that they are a pair of C-7' epimers caused by the opposite attack direction of the aza-Michelal addition reaction (FIG. 6). The opposite values of the beta (1 is positive and 2 is negative) confirm the inventors' guess. Further, the inventors found, through nuclear magnetic calculations and statistical results of CP 3: compound 1 is 7'S,2 "S, 3" S,4 "R (configuration a in nuclear magnetic calculations) and compound 2 is 7' R, 2" S,3 "S, 4" R (configuration a in nuclear magnetic calculations)The probability of configuration b) of (2) is 100% (FIG. 7). Thus, the stereoconfigurations of compounds 1 and 2 were identified as 7'S,2 "S, 3" S,4 "R and 7' R, 2" S,3 "S, 4" R. The chemical equivalence of methylene groups at C-11', C-12' and C-13' positions in the compound 1 and the compound 2 also provides important clues for configuration verification. In view of the structural characteristics of the compounds, the inventor believes that under the action of intramolecular hydrogen bonds, a stable rigid ring is formed in the structure of the compound 2, so that three methylene groups on the ring are fixed and the single bond is prevented from rotating to cause chemical inequivalence. To demonstrate this, the inventors analyzed the optimal conformation of compounds 1 and 2 using molecular atomic quantum topology theory (QTAIM), and showed that a key intramolecular hydrogen bond C was present in compound 29'-H···O＝C14'-O-A more stable rigid ring is promoted, and the three methylene groups on the ring are bound, resulting in chemical inequivalence (fig. 8). It was also confirmed that nuclear magnetic calculations concluded that the configurations of compounds 1 and 2 were correct. In conclusion, the inventors determined that the stereoconfigurations of compounds 1 and 2 are 7'R,2 "S, 3" S,4 "R and 7' S, 2" S,3 "S, 4" R, respectively, designated as mesartan a1And A2. The chemical inequivalence of the 3 methylene groups in the C-11', C-12', C-13' positions in Compound 3 indicates that its relative configuration is the same as that of Compound 2. In addition, the same negative values of the specific rotation values and the same trend of the ECD curves of the compound 3 and the compound 2 (FIG. 9) indicate that the absolute configurations of the compound 3 and the compound 2 are the same, and are also 7'R, 2' S,3 'S, 4' R and are named as mesalamine B2。
Table 2: boltzmann distribution of all conformations for NMR calculationsa
aBolded to the optimal conformation for each configuration
Table 3: actual chemical shifts of Compounds 1 and 2 and calculated chemical shifts of isomers a and b
TABLE 4 Properties of Hydrogen bond Critical points for Compounds 1 and 2
2.5 Quantum chemical computation method
2.5.1 conformational search
The structures of the isomers (a and b) were imported into the Multiwfn program to generate the xyz starting structure. Kinetic modeling of all conformations under GFN0-xTB generated a reasonable set of initial conformations by the procedure of xtb. Each frame of kinetics was batch optimized under GFN0-xTB by molplus call xtb, and was reordered to give a batch of conformations by isostat reordering (isostat module in molplus software), and each of the conformations generated above was batch optimized in the implicit solvent DMSO by GFN2-xTB by molplus call xtb, and reordered by isostat. And (3) calling Gaussian by Molclus, optimizing the conformation selected in the last step under vacuum, performing vibration analysis to obtain a more reliable structure and free energy heat correction value (B3LYP-D3 (BJ)/6-31G), calling an ORCA program to calculate single point energy (PWPB95-D3(BJ)/def2-QZVPP) of each conformation in a DMSO solvent, and reordering by isostat to finally obtain a batch of conformations with the lowest energy.
2.5.2 high precision Single Point energy calculation
Single point energy calculations were performed on the optimal conformation described above on the DFT (M06-2X/def2TZVP) scale using Gaussian (with DMSO as the solvent and IEFPCM as the solvent model) for analysis of hydrogen bonding interactions.
2.5.3 Nuclear magnetic Calculations
Compound 1 (7' S,2 "S, 3" S, 4) was calculated at mPW1PW91-SCRF/6-311+ G (2d, p) levels using the GIAO methodShielding constants for "R) (isomer a) and compound 2(7' R, 2" S,3 "S, 4" R) (isomer b). DMSO was used as solvent and IEFPCM was used as solvent model. Chemical shifts were calculated using a calibration method, by deltascaled＝(δcalc-formula of intercept)/slope to eliminate systematic errors.
2.6 Atom In Molecule (AIM) topology analysis of Compounds 1 and 2
Compounds 1 and 2 were subjected to AIM analysis by the AIMALL software package to investigate the nature of the intramolecular hydrogen bonds. The topological parameters at the hydrogen bond critical point BCP (p,and epsilon, etc.) are used to predict the nature of hydrogen bonding interactions. Quantitative analysis of the topological parameters was obtained by the software Multiwfn 3.8. AIM topological analysis graph is obtained through software VMD 1.9.4. The hydrogen bond binding energy E was calculated at the level of M06-2X/def2 TZVP.
EXAMPLE 3 test of the angiogenic Activity of Compounds 1-3
Healthy zebrafish larvae (line AB) and transgenic line Tg (fli1-EGFP) were provided by the institute of sciences, Shandong province. According to the Zebra fish handbook, they were placed in a closed flow system of tap water filtered with activated carbon and incubated at 28 ℃ for 10 hours in the dark and 14 hours in the light interval. The AB line wild zebra fish can be used for observing thrombus. The expression of Green Fluorescent Protein (GFP) exists in endothelial cells of the transgenic zebra fish Tg (fli1-EGFP), so that the transgenic zebra fish can be used for observing an angiogenesis experiment.
Reference is made to the literature for methods of angiogenic activity (M.Zhang, P.Li, F.Wang, S.Zhang, H.Li, Y.Zhang, X.Wang, K.Liu, X.Li, Food funct.2021,12, 2282-. The PTK 787-induced injury model of zebrafish internodal blood vessels (ISV) was used to assess the effect of compounds 1-3 on angiogenesis.
Collecting fertilized eggs of zebra fish of the same parent, placing in a 28 ℃ illumination incubator for 24h, demoulding embryos, randomly grouping, and placing in a 24-hole plate (10 eggs/hole, 2mL of culture solution per empty). The ISV injury model was first established by adding the VEGFR tyrosine kinase inhibitor PTK787 (final concentration 0.225. mu.g/mL) to the culture medium and the control group was treated with 0.1% by volume DMSO. After 3 hours of incubation, successfully molded zebrafish were randomly divided into 3 groups, treated with danhong injection (final concentration 9 μ L/mL) as a positive control group, treated with 0.1% by volume DMSO as an ISV injury model group, and treated with test compounds 1-3 at two final concentrations of 10 μ M and 25 μ M, respectively, as experimental groups. Next, zebrafish larvae were cultured for another 24 hours, photographed with a fluorescence microscope (Olympus IX53, japan), and the blood vessel length was calculated using Image Pro Plus 5.1. All treatments were performed in duplicate.
Concentration gradient experiments of angiogenesis promoting activity show that the ISV relative generation rate gradually increases with the increase of the medicine adding amount of the compound, and angiogenesis promoting activity is most remarkable at 10 mu M and 25 mu M and then gradually decreases. The 10. mu.M and 25. mu.M doses were determined to be preferred.
The results of the experiments show that the compounds 1-3 can repair ISV damaged by PTK787 induction under the dosage of 10 mu M and 25 mu M, and the repair capability of the compounds is equivalent to that of 9 mu L/mL danhong injection (positive control) (figure 10, A and B).
EXAMPLE 4 antithrombotic Activity testing of Compounds 1-3
Methods for testing antithrombotic activity are described in the literature (M.Zhang, P.Li, F.Wang, S.Zhang, H.Li, Y.Zhang, X.Wang, K.Liu, X.Li, Food funct.2021,12, 2282-2291.).
At 72 hours post fertilization, healthy zebrafish larvae (line AB) were selected and randomly plated in 24-well plates (10/well, 2mL per empty culture). Aspirin (final concentration 166. mu.M) was added to the positive control group, compounds 1-3 (test concentrations 10. mu.M and 25. mu.M) were added to the experimental group, and DMSO was added to the normal control group and the model control group at a volume ratio of 0.1%. After 6 hours of incubation in light conditions, the medium of all groups (except the normal control group) was replaced with fresh medium containing 80 μ M arachidonic acid (used to create the thrombus model). After further incubation for 1 hour, zebrafish were placed in a dark room and stained with 1mg/mL o-biphenylamine for 10 minutes.
The Staining Intensity (SI) of red blood cells in the heart of zebrafish was calculated by observing each zebrafish under a microscope (X100) and Image Pro Plus 5.1. All treatments were performed in duplicate.
The antithrombotic effect of the drug was evaluated according to the following formula:
therapeutic efficacy (%) ═ SIMedicine–SIModel (model))/(SIControl–SIModel (model))×100％。
The concentration gradient experiment of the antithrombotic shows that the red blood cells in the heart of the zebra fish are gradually increased along with the increase of the medicine adding amount of the compound, and the antithrombotic treatment effect is better when the red blood cells are 10 mu M and 25 mu M, but then the red blood cells are gradually reduced. The 10. mu.M and 25. mu.M doses were determined to be preferred.
The experimental results show that compared with the treatment rate of 71.1% of aspirin in a positive control group, the compound 1 has certain relieving effect on thrombus at the concentrations of 10 mu M and 25 mu M, and the treatment effect is 31.7% and 59.0% respectively (fig. 10, C and D). Likewise, other compounds should also have angiogenic and antithrombotic activity.
TABLE 5 angiogenic and antithrombotic effects of Compounds 1-3a
aThe relative ratio is the ratio of the length of the blood vessel of the model group to the length of the blood vessel of the dosing group
Example 5: metronidazole A-containing compound1Tablets with angiogenesis promoting and antithrombotic effects
The preparation method comprises the following steps: mexadifen A1Mixing with lactose and corn starch, moistening with water, sieving, drying, sieving, adding magnesium stearate, tabletting, each tablet weight is 240mg, and Metronidazole A1The content was 1 mg.
Example 6: metronidazole A-containing compound1Capsule with angiogenesis promoting and antithrombotic effects
The preparation method comprises the following steps: mexadifen A1Mixing with lactose and magnesium stearate, sieving, mixing in a suitable container, and encapsulating the mixture into hard gelatin capsules each weighing 200mg, and mexadazole A1The content was 1 mg.
Example 7: metronidazole A-containing compound1Injection with angiogenesis promoting and antithrombotic effects
The preparation method comprises the following steps: mexadifen A1And sodium chloride are dissolved in an appropriate amount of water for injection, and the resulting solution is filtered and filled into an ampoule under aseptic conditions.
Example 8:
similarly, the method and formulation described in examples 5-7 was followed with Metxadazole A2Or B2Respectively preparing tablets, capsules and injection with angiogenesis promoting and antithrombotic effects; wherein the mesartan A2Or B2The contents of (A) were all 1 mg.
Example 9: meishadazole compound (A)1、A2、B2) The angiogenesis promoting pharmaceutical composition
Mexadazole A was prepared according to the method described in example 7 and the related formulation1Or A2Or B2All prepared into injection with the medicine content of 0.5mg/mL, and the prepared mexadazole A1Or A2Or B2The injections were mixed with a commercially available Danhong injection (Danhong injection, DHI, national standard Z20026866, 2 mL/tube) at a volume ratio of 1: 9 mixing, filtering to obtain a solution, and filling the solution into an ampoule bottle under the aseptic condition to obtain the mexadazole A1Or A2Or B2A medicinal composition of the red sage root and safflower injection.
The experimental procedure of example 3 was followed for the above-mentioned mesartan1Composition (1+ DHI) of danhong injection and mexadazole A2Composition (2+ DHI) of danhong injection and mexadazole B2The angiogenesis promoting activity of the composition (3+ DHI) with Danhong injection was measured, and the results are shown in FIG. 11(A and B). The compositions 1+ DHI, 2+ DHI, 3+ DHI (final concentration 9 μ L/mL) were shown to be able to significantly repair the damage of internode blood vessels caused by PTK787, wherein: the repair effect of composition 2+ DHI was superior to that of the positive control group (9. mu.L/mL DHI).
Example 10: meishadazole compound (A)1、A2、B2) The antithrombotic pharmaceutical composition
Mexadazole A was prepared according to the method described in example 6 and the related formulation1Or A2Or B2Respectively mixing the powder with Aspirin (ASP), clopidogrel, salvianolic acid or diltiazem hydrochloride powder according to the weight ratio of 1: 9 mixing, mixing the mixture 1mg with 197mg lactose and 2mg magnesium stearate, sieving, mixing in a suitable container, and encapsulating the resulting mixture into hard gelatin capsules to obtain pharmaceutical composition capsules weighing 200mg per capsule.
The experimental procedure of example 4 was followed for the above-mentioned mesartan1Aspirin-and-aspirin combination (1+ ASP), and mexadazole A2Aspirin and composition (2+ ASP), methoxazole B2The antithrombotic activity of the aspirin-and-aspirin combination (3+ ASP) was measured, and the results are shown in FIG. 11(C and D). The composition 1+ ASP, 2+ ASP and 3+ ASP (final concentration 6mg/mL) can increase the number of erythrocytes in the heart to a certain extent and has potential antithrombotic effect.
Sequence listing
<110> Shandong university
<120> group of mesartan azole compounds, preparation method and application thereof
<141>2021-05-12
<160>1
<210>1
<211> 1525
<212> DNA
<213> Myxococcus sp
<221> Myxococcus sp (SDU 36CCTCC NO: M202152016S rRNA gene nucleotide sequence
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