CN116535459A - Phenolic acid compound and synthesis method and application thereof - Google Patents

Phenolic acid compound and synthesis method and application thereof Download PDF

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Publication number
CN116535459A
CN116535459A CN202310388189.5A CN202310388189A CN116535459A CN 116535459 A CN116535459 A CN 116535459A CN 202310388189 A CN202310388189 A CN 202310388189A CN 116535459 A CN116535459 A CN 116535459A
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sepsis
acid compound
phenolic acid
resin
fmoc
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杨阳
梁琦
赵新锋
杨雯雯
薛承旭
陆晨曦
吴雪
雷王蕊
侯雨萱
张燕
唐冉
李嘉雯
刘延庆
徐晓玲
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NORTHWEST UNIVERSITY
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NORTHWEST UNIVERSITY
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/07—Optical isomers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The invention belongs to the technical field of medicine research, and particularly relates to a phenolic acid compound, a synthesis method and application thereof, in particular to a phenolic acid compound, a synthesis method thereof and application thereof in preparing medicines for resisting sepsis and treating/preventing myocardial injury induced by the same. The structural formula of the phenolic acid compound JY790 is shown as formula I. The research of the invention finds that: JY790 can increase survival rate of mice after sepsis injury, improve sepsis score and anal temperature, increase leukocyte and platelet count, decrease erythrocyte count, decrease lactate dehydrogenase and urea nitrogen levels, and alleviate sepsisA symptomatic bacterial infection; meanwhile, the JY790 is found to have the functions of improving the heart function and maintaining the normal morphology of myocardial tissue, and the JY790 can inhibit the elevation of molecules related to myocardial injury inflammation induced by sepsis, thereby playing the role of resisting sepsis and myocardial injury induced by the sepsis;

Description

Phenolic acid compound and synthesis method and application thereof
Technical Field
The invention belongs to the technical field of medicine research, and particularly relates to a phenolic acid compound, a synthesis method and application thereof, in particular to a phenolic acid compound, a synthesis method thereof and application thereof in preparing medicines for resisting sepsis and myocardial injury induced by sepsis.
Background
Sepsis refers to a life threatening organ dysfunction caused by a deregulation of the host's response to an infection. Exacerbation of sepsis can cause septic shock, disseminated intravascular coagulation, and impaired organ function, severely threatening the patient's life. The heart is one of the most severe organs of sepsis injury. Clinical studies have shown that mortality in sepsis patients with severe myocardial damage is as high as 50%. Early in sepsis, myocardial damage can accelerate the progression of sepsis. Sepsis-induced myocardial injury belongs to one type of infectious myocardial injury. Infectious myocardial injury refers to myocardial injury such as heart enlargement, heart failure, cardiogenic shock or abnormal heart rhythm occurring during viral infection or in recovery period, and typical symptoms are fatigue weakness, inappetence, nausea, vomiting, dyspnea, pale complexion and fever.
In the prior art, in the treatment of sepsis and myocardial injury caused by sepsis, other than basic inflammation (medicines such as antibiotics) response inhibition, there are adopted various adrenergic medicines such as dobutamine, norepinephrine, statin medicines, esmolol and the like. However, the antibiotic drugs in the drugs have strong drug resistance and are easy to cause secondary infection, while the adrenergic drugs are easy to cause side effects such as palpitation, arrhythmia and the like. Therefore, there is a lack of effective therapeutic drugs that are safe, nontoxic, and specific for sepsis and its induced myocardial damage.
Disclosure of Invention
In view of the above technical problems, the present invention provides the following technical solutions:
in a first aspect of the present invention, a phenolic acid compound is provided, and the structural formula of the phenolic acid compound is shown in formula I:
in a second aspect of the present invention, a method for synthesizing the phenolic acid compound is provided, comprising the following steps:
s1, swelling resin, mixing the resin with Fmoc-Leu-OH, and covalently connecting carboxyl of the Fmoc-Leu-OH to the resin to obtain an intermediate product A;
s2, mixing the intermediate product A with Fmoc-aminomethylbenzoic acid for acylation reaction, removing Fmoc protecting group from the obtained product, and then carrying out acylation reaction with Fmoc-3-cyclohexyl-L-alanine, and removing the Fmoc protecting group; then carrying out acylation reaction with Fmoc-cyclopropyl alanine, removing Fmoc protecting group, and collecting intermediate product B;
s2, mixing the intermediate product B obtained in the step S2 with ferulic acid for acylation reaction, removing Fmoc protecting groups, cutting the target compound from resin, precipitating, centrifuging and washing to obtain the phenolic acid compound.
Preferably, the resin is a 2-chlorotrimethylbenzene resin (i.e., a dichloro resin);
the acylation reaction is carried out under the action of N, N-diisopropylethylamine and O-benzotriazole-tetramethylurea hexafluorophosphate;
the Fmoc protecting group is removed by using a 20% piperidine solution with a volume fraction;
the cutting is carried out by mixing trifluoroacetic acid, anisole, 1, 2-ethanedithiol, phenol and water according to the volume ratio of 87.5:5:2.5:2.5:2.5 mixing and cutting as cutting fluid.
In a third aspect of the present invention, there is provided a use of the phenolic acid compound, the use comprising any one of the following:
(1) Use in the manufacture of an anti-sepsis medicament;
(2) Use in the manufacture of a medicament for the prevention/treatment of sepsis-induced myocardial damage.
Preferably, the therapeutically effective amount of the phenolic acid compound is 5 mg-20 mg/Kg.
In a fourth aspect of the invention, there is provided a medicament for use against sepsis comprising the phenolic acid compound.
In a fifth aspect of the present invention, there is provided a medicament for preventing/treating sepsis-induced myocardial damage, which comprises the phenolic acid compound.
Preferably, pharmaceutically acceptable excipients or carriers are also included.
Preferably, the dosage form of the medicament is an injection preparation.
Preferably, the injection preparation is an intravenous injection preparation or an intraperitoneal injection preparation.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a phenolic acid compound JY790, which is found by the research: JY790 can improve the survival rate of mice after sepsis injury, improve sepsis score and anal temperature, increase the number of white blood cells and platelets, reduce the number of red blood cells, reduce the level of lactate dehydrogenase and urea nitrogen, and alleviate sepsis bacterial infection; meanwhile, JY790 is found to have the functions of improving cardiac function and maintaining normal morphology of myocardial tissue, and JY790 can inhibit the elevation of molecules related to myocardial injury inflammation induced by sepsis, thereby exerting the functions of resisting sepsis and myocardial injury induced by the sepsis.
Drawings
FIG. 1 is a screening process of JY 790;
FIG. 2 is a graph showing enrichment index analysis of a library of screened DNA encoding compounds;
FIG. 3 is alpha 1 -molecular docking visualization of AR and JY790, a: JY790 structural formula; c: alpha 1 -AR protein structure; b: hydrogen bonding of the interaction between the two;
FIG. 4 is a modeling picture of the survival rate of mice;
FIG. 5 is a graph of mouse survival; in comparison with the group of CLPs, * P<0.05；
FIG. 6 is a functional modeling image of a mouse;
FIG. 7 is sepsis score results; in comparison with the group of CLPs, ** P<0.01， **** P<0.0001；
FIG. 8 is a graph of a statistical analysis of anal temperatures in mice; in comparison with the group of CLPs, ** P<0.01， **** P<0.0001；
FIG. 9 is a graph showing the effect of JY790 on the conventional indices of mouse blood after 8h of CLP injury; WBC, white blood cells; PLT, platelets; RBC, red blood cells; in comparison with the group of CLPs, * P<0.05， ** P<0.01， *** P<0.001；
FIG. 10 is a graph showing the effect of JY790 on various indices of mouse blood biochemistry after 8h of CLP injury; LDH, lactate dehydrogenase; BUN, urea nitrogen; in comparison with the group of CLPs, * P<0.05， ** P<0.01， **** P<0.0001；
FIG. 11 is a graph showing the effect of JY790 on bacterial infection in blood 8h after CLP injury;
FIG. 12 is an exemplary M-mode image of an echocardiogram short axis slice;
FIG. 13 is a graph of a statistical analysis of a portion of cardiac performance metrics; SV, stroke volume; CO, cardiac output; LVESV, left ventricular end systole volume; in comparison with the group of CLPs, *** P<0.001， **** P<0.0001；
FIG. 14 is a graph of a statistical analysis of a portion of cardiac performance metrics; LVEDV, left ventricular end diastole volume; LVPWs, left ventricular end-systole post wall thickness; LVPWd, left ventricular end-diastole post wall thickness; in comparison with the group of CLPs, ** P<0.01， *** P<0.001， **** P<0.0001；
FIG. 15 is a graph showing the effect of JY790 on myocardial tissue morphology after 8h of CLP injury;
FIG. 16 is a graph showing the effect of JY790 on the index related to myocardial inflammation after 8h of CLP injury.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
The cecal ligation perforation (Cecal Ligation and Puncture, CLP) model is a classical animal model of sepsis, and the invention adopts the CLP animal model as a study object, and the animal model can also cause acute myocardial injury, namely sepsis-induced myocardial injury.
The invention relates to infectious myocardial injury, in particular to myocarditis lesions caused by bacterial endocarditis, systemic or bacteremia caused by other organ infections.
The invention will now be further illustrated with reference to specific examples, which are given solely for the purpose of illustration and are not to be construed as limiting the invention.
The purity of JY790 used in the examples below was 95% or higher by HPLC. The animals are purchased from experimental animal centers of medical universities of air force army, and the reagents are purchased in the market. The experimental methods or related detection methods used in the examples below employ methods known in the art unless otherwise specified.
Example 1
JY790 binding to adrenergic receptor
Structurally modifying phenolic acid derivative with DNA encoding technology, synthesizing DNA encoding compound library containing phenolic acid derivative, and immobilizing adrenergic receptor alpha 1 (adrenergic receptor，α 1 -AR) chromatography to screen the library of compounds for access, and finally selecting the compounds according to the enrichment index and the in vitro molecular docking coefficient. The specific operation process is as follows:
(1) Construction of DNA coding Compound library:
(1) a DNA encoding library based on 20 Fmoc amino acids and 4 phenolic acids was constructed using conventional resolution and pooling procedures;
(2) repeating three cycles of acylation reaction and Fmoc protecting group removal reaction to obtain a DNA coding compound library containing 8000 compounds;
(3) substitution of 20 amino acids with 4 phenolic acids (caffeic acid, protocatechuic acid, gallic acid and ferulic acid) resulted in 32000 DNA encoding compounds (see figure 1).
(2) With alpha 1 -AR receptor chromatography column screening of phenolic acid active compounds
The PCR products were high throughput sequenced using a second generation sequencing technique, the DNA strand encoding the building block was significantly enriched (as in FIG. 2), and the z-score data normalization method disclosed in the document "Discovery of potent thrombin inhibitors from a protease-focused DNA-encoded chemical library" was usedLine analysis by comparison of alpha 1 The enrichment of AR (x-axis) and the enrichment of the blank (y-axis) were found to be the highest-enrichment factor (about 47) small molecule compound designated JY790.
(3) Molecular docking:
(1) alpha acquisition by alpha Fold 1 -AR's structure file, downloading α using uniprot (ID: P35348) 1 Three-dimensional structure of AR protein, followed by evaluation of alpha using the SAVES (https:// SAVES. Mbi. Ucla. Edu) website 1 -AR prediction effect;
(2) macromolecular alpha using AutoDockToolv1.5.7 software 1 -AR and small molecule ligand JY790.
(3) The molecular docking results were visualized using Discovery Studio.
The molecular docking results are shown in FIG. 3, which shows that alpha 1 -AR and JY790 have good binding capacity, and JY790 is a modification of phenolic acid.
The following describes the synthesis method of the compound JY790 provided by the invention in detail, and the synthesis process specifically comprises the following steps:
(1) Weighing 0.6g of 2-chlorotrimethylbenzene resin with the substitution degree of 1.0mmol/g, putting the resin into a reaction tube, adding 10ml of DCM, and oscillating for 30min; the solvent was filtered off with suction through a sand core, 3 times the molar amount of Fmoc-Leu-OH was added, a small amount of DMF was added for dissolution, 6 times the molar amount of DIPEA was added, and finally 3 times the molar amount of the condensing reagent HBTU was added and nitrogen was bubbled for 0.5h. Washing 3-4 times by using DMF, and taking a small amount of resin to detect ninhydrin, wherein the resin is colorless. 10ml of 20% piperidine/DMF solution was added for reaction for 5min, the solvent was filtered off, and 10ml of 20% piperidine/DMF solution was added for reaction for 15min. Washing with DMF for 5 times, and taking a small amount of resin to detect ninhydrin to be blue;
(2) To the resin obtained in (1) was added further 3-fold molar amount of Fmoc-aminomethylbenzoic acid, a small amount of DMF was added for dissolution, 6-fold molar amount of DIPEA was added, and finally 3-fold molar amount of condensing agent HBTU was added, and nitrogen was purged for 0.5h. Washing 3-4 times by using DMF, and taking a small amount of resin to detect ninhydrin, wherein the resin is colorless. 10ml of 20% piperidine/DMF solution was added for reaction for 5min, the solvent was filtered off, and 10ml of 20% piperidine/DMF solution was added for reaction for 15min. Washing with DMF for 5 times, and taking a small amount of resin to detect ninhydrin to be blue;
(3) To the resin obtained in (2) was added 3-fold molar amount of Fmoc-3-cyclohexyl-L-alanine, a small amount of DMF was added for dissolution, 6-fold molar amount of DIPEA was added, and finally 3-fold molar amount of condensing reagent HBTU was added, and nitrogen was purged for 0.5 hour. Washing 3-4 times by using DMF, and taking a small amount of resin to detect ninhydrin, wherein the resin is colorless. 10ml of 20% piperidine/DMF solution was added for reaction for 5min, the solvent was filtered off, and 10ml of 20% piperidine/DMF solution was added for reaction for 15min. Washing with DMF for 5 times, and taking a small amount of resin to detect ninhydrin to be blue;
(4) To the resin obtained in (3) was added 3-fold molar amount of Fmoc-cyclopropylalanine, a small amount of DMF was added for dissolution, 6-fold molar amount of DIPEA was added, and finally 3-fold molar amount of condensing reagent HBTU was added, and nitrogen was purged for 0.5h. Washing 3-4 times by using DMF, and taking a small amount of resin to detect ninhydrin, wherein the resin is colorless. 10ml of 20% piperidine/DMF solution was added for reaction for 5min, the solvent was filtered off, and 10ml of 20% piperidine/DMF solution was added for reaction for 15min. Washing with DMF for 5 times, and taking a small amount of resin to detect ninhydrin to be blue;
(5) Adding 3 times mole of ferulic acid into the resin obtained in the step (4), adding a small amount of DMF for dissolution, adding 6 times mole of DIPEA, and finally adding 3 times mole of condensing reagent HBTU, and blowing nitrogen for 0.5h. Washing 3-4 times by using DMF, and taking a small amount of resin to detect ninhydrin, wherein the resin is colorless. 10ml of 20% piperidine/DMF solution was added for reaction for 5min, the solvent was filtered off, and 10ml of 20% piperidine/DMF solution was added for reaction for 15min. The reaction mixture was washed 5 times with DMF and a small amount of resin was taken for ninhydrin detection as blue.
(6) Configuration of trifluoroacetic acid: anisole: 1, 2-ethanedithiol: phenol: water = 87.5:5:2.5:2.5:2.5 Adding the resin obtained in the step (5) into the cutting fluid in the volume ratio, and performing precipitation, centrifugation and washing to obtain JY790.
Example 2
JY790 can improve survival rate of mice after sepsis injury
1. Animal model construction for sepsis
A model of sepsis was constructed at the body level using CLP surgery. The mice were normally kept for molding on day 7. The specific operation steps are as follows:
mice were anesthetized with a small animal inhalation anesthesia system: the mice inhale 3% isoflurane (air flow 1L/min), and the isoflurane concentration during molding was 1.5% (air flow 1L/min) with continued anesthesia. The anesthesia degree monitoring standard is that withdrawal reflex of limbs disappears, and the mice are fixed and continuously inhaled with 2% of oxygen containing isoflurane to maintain anesthesia;
skin preparation in the middle region of the abdomen of a mouse is carried out by sterilizing the skin twice by 75% ethanol, cutting and separating the skin and subcutaneous tissues layer by layer along the middle line of the middle lower abdomen to form a white line, cutting rectus abdominus and peritoneum along the white line of the abdomen, wetting the two sides of the cut by 0.9% physiological saline, feeding the abdomen by using curved forceps, gently pulling out the feces near the ileocecum after finding the ileocecum, gently squeezing the feces towards the tail end of the cecum (avoiding air residues), ligating the cecum by using a No. 4 sterile operation suture at the position 2/3 on the connecting line between the tail end of the cecum and the ileocecum, penetrating the ligatured cecum (avoiding blood vessels) by using a 25G syringe needle at the midpoint between the ligature and the tail end of the cecum, gently squeezing the cecum after perforation (fig. 4), and allowing the content substances of the cecum at the visible ligature section to flow out along the puncture hole, taking the cecum together with all surrounding intestinal tubes, and gradually closing the peritoneum and skin layer by using a No. 4 sterile operation suture;
after the operation, all experimental mice are injected subcutaneously into the back of the patient immediately after the operation with physiological saline (10 ml/kg body weight) at 37 ℃ for liquid resuscitation, and the mice are put back into a squirrel cage after proper marking and wait for awakening.
The time counting is started after the CLP operation, the mice are observed every 1h, the death number of each group of mice in 72h is recorded, and the survival rate is counted and analyzed.
2. Grouping and administration
Grouping: the sepsis model mice were randomly divided into CLP group, low dose JY790+clp group (5 mg/kg), medium dose JY790+clp group (10 mg/kg) and high dose JY790+clp group (20 mg/kg), each group was 12. 12 mice were also selected as a Sham group, and the Sham group was constructed in the same manner as the CLP group except that no cecal ligation puncture was performed.
Administration: pretreatment (intraperitoneal injection) was performed on each group of mice 6 days before CLP molding, and Sham group, CLP group dimethyl sulfoxide (DMSO) was administered; low, medium, high JY790+CLP group: JY790 was dissolved in DMSO to prepare JY790 solutions at doses of 5mg/kg, 10mg/kg, and 20mg/kg, and administered 1 time every 2 days, 3 times total, to ensure the same time period and operation time for each administration.
Based on the survival rate results of each group, the JY790 appropriate concentration in this example was selected, and then the functional experiment was performed, and the obtained specimens were subsequently examined.
3. Results
The results of the mice survival curve are shown in figure 5. Compared to Sham group, mice had a survival rate of 41.67% (P < 0.05) within 72h after CLP treatment. Compared with the CLP group, the survival rate of mice treated with 5mg/kgJY790 is 25.00% (P < 0.05); the survival rate of the mice after 10mg/kgJY790 treatment is 91.67% (P < 0.05); the survival rate of mice treated with 20mg/kg JY790 was 58.33% (P < 0.05). The JY790 was suggested to increase survival rate after CLP in mice, and the optimal protection dose of JY790 was 10mg/kg (this optimal protection dose was used for subsequent functional assays).
Example 3
JY790 can improve sepsis score, mice anal temperature and blood routine change caused by sepsis
1. Sepsis model construction
The procedure was the same as in example 2 with the model construction procedure using ligation of the cecal end to 1/3 of the ileocecal valve linkage, as shown in FIG. 6.
2. Grouping and administration
Grouping: the sepsis model mice were randomly divided into CLP groups and jjjjjjjjjjjjjjjjjjj 790+clp groups (10 mg/kg), 6 each. 6 mice were also selected as a Sham group, and the Sham group was constructed in the same manner as the CLP group except that no cecal ligation puncture was performed.
3. Detection index
(1) Mice 8h after CLP surgery were scored according to the sepsis scoring table disclosed in literature "ShrumB, ananthaRV, xuSX, et al.
(2) Detection of changes in anal temperature in mice 8h after CLP surgery: after injury for 8 hours, the mice are fixed, the anus of the mice is cleaned up by cotton balls, a temperature detection probe is gently inserted into the anus, and the temperature is recorded after the data are stable.
(3) Detecting the change of routine indexes of the blood of the mice after 8 hours of CLP operation: after injury for 8 hours, adopting an eyeball-picking blood-taking method to take blood, and carrying out blood routine detection by using a full-automatic blood routine instrument.
4. Results
Sepsis scores were performed 8h after CLP treatment in mice and the results are shown in fig. 7, CLP group sepsis scores were significantly increased (P < 0.0001) compared to Sham group, and sepsis scores were significantly decreased (P < 0.01) after JY790 treatment.
The results of anal temperature measurements performed 8h after CLP treatment in mice are shown in fig. 8, where CLP group showed significantly lower anal temperature (P < 0.001) compared to Sham group and significantly higher anal temperature (P < 0.01) after JY790 treatment.
Changes in blood routine related indicators were detected 8h after CLP treatment in mice, and as shown in fig. 9, WBCs and PLTs were significantly reduced (P < 0.001) and RBCs were significantly increased (P < 0.001) compared to Sham group; compared with CLP group, after JY790 treatment, WBC and PLT were significantly increased (P < 0.001), RBC was significantly decreased (P < 0.01).
Example 4
JY790 can improve blood biochemical change caused by sepsis
Sepsis and its induced myocardial injury model were constructed as described in example 3 and pre-treated with JY790.
Detecting the change of various indexes of blood biochemistry of the mice after 8 hours of CLP operation: after injury for 8 hours, blood is taken by adopting an eyeball-taking blood-taking method, each group of whole blood is collected, the whole blood is centrifuged for 10 minutes at 3000rpm, serum is sucked, and then the whole blood is detected by using a full-automatic blood biochemical analyzer.
The results are shown in FIG. 10, and the levels of LDH and BUN in serum are significantly increased (P < 0.0001) after 8h of CLP injury compared with the Sham group; compared with the CLP group, the levels of LDH and BUN were significantly reduced (P < 0.0001) after JY790 treatment.
Example 5
JY790 can reduce bacterial infection caused by sepsis
1. Method of
Sepsis and its induced myocardial injury model were constructed as described in example 3 and pre-treated with JY790.
2. Detection index
Detection of changes in blood culture in mice 8h after CLP surgery: after injury for 8 hours, blood was collected from each group of whole blood using a heart blood collection method. The specific operation steps of blood culture are as follows:
(1) preparation of agar plates:
the autoclaved solid medium is poured into a dish, preferably to cover the entire bottom surface of the dish. And (5) placing the cover half buckle on an ultra-clean bench, standing and solidifying. After solidification, the dish was placed upside down.
(2) Blood collection sample preparation
Collecting blood of experimental animal, adding into anticoagulant tube, reversing upside down, and mixing blood with anticoagulant. If the blood culture cannot be immediately performed, the blood culture can be performed as soon as possible while the blood culture is being performed at 4 ℃.
(3) Blood coating
A6 cm or 9cm plate requires 50-100. Mu.l of blood. Adding blood onto agar plate, cooling the flame burnt coating rod, pushing the blood away by the coating rod, uniformly distributing on the whole agar plate surface, and burning and sterilizing the coating rod. Note that the labeling of each plate was done.
(4) Bacterial culture
After the homogenization, the blood-cultured agar plates were placed in an incubator at 37℃for cultivation for 24 hours or 48 hours.
(5) Result observation and image acquisition
After the blood culture is finished, the growth condition of bacteria on the agar plate is observed, and the bacteria are photographed and retained, and all images are arranged and analyzed.
3. Results
The results of blood culture are shown in FIG. 11, which shows a significant increase in bacterial numbers after CLP injury compared to Sham; the bacterial numbers were significantly reduced after JY790 treatment compared to the CLP group.
Example 6
JY790 can improve heart function injury caused by sepsis myocardial injury
1. Method of
Sepsis and its induced myocardial injury model were constructed as described in example 3 and pre-treated with JY790.
2. Detection index
Mice heart function 8h after CLP surgery were examined ultrasonically: the animals of each group are unhaired in the left chest area of the mice one day before ultrasonic detection, the mice are anesthetized by 2% isoflurane, the anesthetic gas flow is 1L/min, after isoflurane inhalation anesthesia, the animals are fixed on a thermostatic plate at 37 ℃, the left chest is fully exposed, a 30MHz probe is adopted, a standard left ventricular papillary muscle short axis section is selected, an M-mode heart ultrasonic section image is recorded, and the measurement indexes comprise: stroke volume, cardiac output, left ventricular end systole volume, left ventricular end diastole volume, left ventricular end systole back wall thickness, left ventricular end diastole back wall thickness, and the like.
During the detection, the following points should be noted, which may affect the details of the detection result: first, the anesthetic state cannot be too deep, otherwise the heart rate and contractile function of the mice are affected; secondly, the body position of the mouse is well arranged, the limbs of the mouse cannot be fixedly stretched too tightly, otherwise, the heart of the mouse is pressed, and the accuracy of heart function detection is finally affected; thirdly, the part of the mouse close to the heart is subjected to dehairing at least one day in advance, new hair is generated during detection when dehairing is too early, artifacts are generated during imaging, an ultrasonic result is affected, and the mouse is in a stress state to interfere with a heart function result when the dehairing is too late.
3. Results
The heart contractile function of the mice after 8 hours of CLP operation is detected by the ultrasonic method, and the results are shown in figures 12-14 (short axis ultrasonic results of left room), compared with Sham, SV, CO, LVESV, LVEDV of the heart of the mice after CLP injury is obviously reduced (P < 0.0001), LVPWs and LVPWD are obviously increased (P < 0.0001); compared with the CLP group, the cardiac function is obviously improved after JY790 treatment.
Example 7
JY790 can improve myocardial tissue injury caused by sepsis
1. Method of
Sepsis and its induced myocardial injury model were constructed as described in example 3 and pre-treated with JY790.
2. Detection index
The heart was taken and fixed in 4% paraformaldehyde for subsequent HE staining.
Organ tissue HE staining:
(1) and (3) paraffin embedding: placing the tissue into 4% paraformaldehyde, and fixing for at least 24 hours; paraffin embedding, slicing and dewaxing (soaking in 80%, 95%, 100% ethanol for 40min, soaking in 100% ethanol, 100% ethanol-xylene=1:1 mixed solution for 30min, dehydrating and transparentizing tissue, soaking in embedding machine for 3h, and finally embedding by dripping paraffin.
(2) Slicing: the thickness of the slice is set to be 5 mu m, the slice is stuck on a polylysine coated glass slide by a slice pulling method, and after the slice is baked for 1h at 70 ℃, the slice is baked for 5h at 60 ℃.
(3) Dyeing: soaking the slices in xylene for 10min, replacing xylene, soaking again for 10min, and sequentially soaking for 2min in order of 100%, 95%, 80% ethanol and deionized water for dewaxing to obtain dyeing; immersing the slice into hematoxylin dye solution for dyeing for 3min, and gently flushing for 5min by using tap water; immersing in 1% ethanol hydrochloride for 30s, decolorizing with 1% ammonia water for 3min, and gently washing with tap water for 3min; immersing in eosin dye solution for dyeing for 3min, and gently flushing for 3min by using tap water; soaking for 2min respectively according to the sequence of 70%, 80% ethanol 30s,95%, 100% gradient ethanol, xylene and xylene for dehydration transparent treatment; and (5) sealing the neutral resin.
3. Results
The results of HE staining of mouse myocardial tissue are shown in FIG. 15, and compared with Sram, the CLP has the advantages of disordered myocardial tissue structure, broken myocardial tissue fibers, increased interstitial edema and damaged cell integrity; compared with the CLP group, the JY790 treatment significantly reduced the myocardial morphology damage caused by the CLP.
Example 8
JY790 ameliorates CLP-induced myocardial injury by reducing the inflammatory response.
1. Method of
Sepsis and its induced myocardial injury model were constructed as described in example 3 and pre-treated with JY790.
2. Detection index
Immunohistochemical detection:
(1) paraffin embedding and slicing steps were the same as in example 7.
(2) Dyeing: sections were routinely dewaxed to water: according to kit requirements (purchased from wuneseir biotechnology limited): taking paraffin sections of heart tissues of each group of mice respectively, sequentially carrying out xylene for 2 times, 10 minutes each time, 100% ethanol for 2 times, and 10 minutes each time; 95%, 90%, 80%, 70% ethanol for 1 time each for 5min, and soaking in distilled water for 5min; antigen retrieval: repairing the sodium citrate buffer solution with microwave antigen for 20min, and washing with running water for 10min; blocking endogenous peroxidases: 3% hydrogen peroxide, and room temperature for 20min. Washing with PBS for 3 times, each time for 5min; closing: dripping 5% normal goat serum blocking solution, and incubating for 30min at room temperature; dripping primary antibody: excess serum was wiped off, primary antibody was added and incubated overnight at 4 ℃. Washing with PBS for 3 times, each time for 5min; dripping secondary antibody: dripping horseradish peroxidase HRP-labeled secondary antibody (1:5000, prepared by PBS), incubating for 1h in a 37 ℃ incubator, and washing with PBS for 3 times, each time for 5min; DAB color development: dripping DAB for 0.5-3min, controlling color development under a mirror, washing with running water for 10min, counterstaining with hematoxylin, differentiating with 1% hydrochloric acid alcohol, decolorizing with 1% ammonia water, dehydrating, transparency with xylene, and sealing with neutral resin.
(3) Observed under a microscope and photographed: and observing and photographing under a microscope, randomly finding out 20-30 non-overlapping fields of view from each slice, and taking a brown yellow particle deposition area of the tissue slice under a light microscope as a positive staining part.
3. Results
As shown in FIG. 16, the IHC staining results of the myocardial tissue of the mice show that Ly6g, ly6c and IL-6 expression are obviously increased after CLP injury compared with the Sham group; the expression of JY790 was significantly reduced after treatment compared to the CLP group.
While the invention has been described in terms of preferred embodiments, it is not intended to be limited thereto, but rather to enable any person skilled in the art to make various changes and modifications without departing from the spirit and scope of the present invention, which is therefore to be limited only by the appended claims.

Claims (10)

1. The phenolic acid compound is characterized in that the structural formula is shown as formula I:
2. a method for synthesizing the phenolic acid compound as claimed in claim 1, comprising the steps of:
s1, swelling resin, mixing the resin with Fmoc-Leu-OH, and covalently connecting carboxyl of the Fmoc-Leu-OH to the resin to obtain an intermediate product A;
s2, mixing the intermediate product A with Fmoc-aminomethylbenzoic acid for acylation reaction, removing Fmoc protecting group from the obtained product, and then carrying out acylation reaction with Fmoc-3-cyclohexyl-L-alanine, and removing the Fmoc protecting group; then carrying out acylation reaction with Fmoc-cyclopropyl alanine, removing Fmoc protecting group, and collecting intermediate product B;
s2, mixing the intermediate product B obtained in the step S2 with ferulic acid for acylation reaction, removing Fmoc protecting groups, cutting the target compound from resin, precipitating, centrifuging and washing to obtain the phenolic acid compound.
3. The synthesis method according to claim 2, wherein,
the resin is 2-chlorotrimethylbenzene resin;
the acylation reaction is carried out under the action of N, N-diisopropylethylamine and O-benzotriazole-tetramethylurea hexafluorophosphate;
the Fmoc protecting group is removed by using a 20% piperidine solution with a volume fraction;
the cutting is carried out by mixing trifluoroacetic acid, anisole, 1, 2-ethanedithiol, phenol and water according to the volume ratio of 87.5:5:2.5:2.5:2.5 mixing and cutting as cutting fluid.
4. Use of phenolic acids according to claim 1, characterized in that it comprises any one of the following:
(1) Use in the manufacture of an anti-sepsis medicament;
(2) Use in the manufacture of a medicament for the prevention/treatment of sepsis-induced myocardial damage.
5. The use according to claim 4, wherein the effective amount of phenolic acid compound is 5 mg-20 mg/Kg.
6. A medicament for the treatment of sepsis, characterized in that it comprises a phenolic acid compound according to claim 1.
7. A medicament for preventing/treating sepsis-induced myocardial damage, characterized in that it comprises the phenolic acid compound of claim 1.
8. The medicament according to claim 6 or 7, further comprising pharmaceutically acceptable excipients or carriers.
9. The medicament according to claim 8, wherein the dosage form of the medicament is an injectable formulation.
10. The medicament according to claim 9, wherein the injection preparation is an intravenous injection preparation or an intraperitoneal injection preparation.
CN202310388189.5A 2023-04-12 2023-04-12 Phenolic acid compound and synthesis method and application thereof Pending CN116535459A (en)

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