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:

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

。

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.

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 t_RCollecting 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)λ_max(logε)279 (3.40),287(3.35)nm；ECD(MeOH):203(Δε+16.34),224(Δε+5.38)nm；IRν_max 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)λ_max(logε)279(3.69), 287(3.64)nm；ECD(MeOH):203(Δε+5.73),218(Δε+5.40)nm；IRν_max 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)λ_max(logε)279(3.80), 286(3.75)nm；ECD(MeOH):202(Δε+7.50),216(Δε+4.09)；IRν_max 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(δ_H7.82)、H-7(δ_H7.86) and H-9' (delta)_H6.74). Wherein at δ_CCharacteristic 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”(δ_H4.66 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 intensity³J_H2”-H3”(4.6Hz) showed rapid conformational interconversion between C2 "and C3". Coupling constant analysis based on 1, 2-secondary methyl system, small coupling value³J_C4”-H2”And the value of the intermediate coupling³J_C1”-H3”Indicating that C2 "-C3" is erythro. Likewise, large coupling values²J_C3”-H4”And²J_C4”-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 sum³J_HHIt 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 2_9'-H···O＝C_14'-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 calculations^a

^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 delta_scaled＝(δ_calc-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 (%) ═ SI_Medicine–SI_{Model (model)})/(SI_Control–SI_{Model (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 1-3^a

^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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