CN116350624B

CN116350624B - Application of compound in serving as or preparing SIRT1 receptor agonist

Info

Publication number: CN116350624B
Application number: CN202111616318.9A
Authority: CN
Inventors: 徐愿坚
Original assignee: Hangzhou Yijian Biomedical Technology Co ltd
Current assignee: Hangzhou Yijian Biomedical Technology Co ltd
Priority date: 2021-12-27
Filing date: 2021-12-27
Publication date: 2023-10-10
Anticipated expiration: 2041-12-27
Also published as: CN116350624A

Abstract

The invention belongs to the field of medicines or health products, and particularly relates to application of a compound in serving as or preparing an SIRT1 receptor agonist. The compound (shown in the formula I or the formula II) and enantiomer, diastereoisomer, salt, ester, prodrug, solvate or solvate of salt thereof can obviously reduce uric acid concentration when being used as or prepared into SIRT1 receptor agonists, and compared with the prior related commercial medicines, the uric acid reducing effect is better.

Description

Application of compound in serving as or preparing SIRT1 receptor agonist

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of a compound in serving as or preparing an SIRT1 receptor agonist.

Background

Uric acid is a metabolite of purine compounds digested in the human body, and is mainly excreted from the body through the kidneys. If uric acid production is increased (about 15%) or uric acid excretion is impaired (about 85%) in the human body, an increase in uric acid concentration in blood, namely Hyperuricemia (HUA), is caused. According to the national institute of Chinese hyperuricemia related diseases diagnosis and treatment multidisciplinary expert consensus issued by the China's China medical society in 2017, HUA is defined as that under normal purine diet, men with fasting blood uric acid level of No. two times a day are >420 mu mol/L (7 mg/dL), and women are >360 mu mol/L (6 mg/dL). The basis of this limit is that the saturated concentration of urate in blood is 420 mu mol/L, beyond which urate will deposit in joint synovium, bursa, cartilage and other tissues, causing gout and even causing serious consequences such as joint injury and kidney function injury.

Food such as seafood, meat products and beer is rich in purine compounds, so gout is also called "arthritis of rich people" in the period of lack of supplies, but with the remarkable improvement of the whole living standard of China in recent years, patients suffering from hyperuricemia are rapidly increased, and the gout is the second digestive metabolic disease only in diabetes. At present, the drug treatment for reducing uric acid is mainly divided into two types: one is a drug represented by allopurinol and febuxostat, which reduces uric acid synthesis by inhibiting xanthine oxidase activity; another drug represented by benzbromarone is a drug which can inhibit the reabsorption of tubular uric acid by inhibiting the tubular uric acid transporter-1 (URAT 1) to promote uric acid excretion. However, although the two medicines have better uric acid reducing effect, the two medicines have certain damage to kidney function, and patients with hyperuricemia are likely to suffer from kidney function diseases, so that development of novel uric acid reducing medicines with low toxic and side effects is urgently needed.

Disclosure of Invention

In view of the above, the invention aims to provide a natural SIRT1 agonist which can not only remarkably reduce serum uric acid, but also fundamentally prevent and treat gout; but also has the functions of inhibiting inflammation and relieving joint inflammation of patients who have developed gout.

The structural formula of the SIRT1 agonist is shown as formula I or formula II:

wherein R is ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ 、R ₆ Independently of each other, selected from hydrogen, hydroxy, methoxy, acetyl.

Preferably, the compound of formula I or formula II is selected from the group consisting of compounds of formula III, formula IV, formula V, formula VI, formula VII, formula VIII, formula IX, and formula X;

in particular, sirtuin 1 (Silent information regulator, SIRT 1) is one of the Sirtuin protein family members, and recent studies have found that SIRT1 agonists can prevent recurrent episodes of gouty arthritis by modulating metabolism to reduce hyperuricemia on the one hand, and can also prevent episodes of gouty arthritis by inhibiting inflammation on the other hand.

Specifically, herba lycopi (Eupatorium lindleyanum DC.) is a plant name of herba Eupatorii Lindleyani, a perennial herb, in Jiangsu region and land of China. The Chinese medicinal herb lindley eupatorium herb has bitter taste, calm nature, and liver and spleen meridian return, has the functions of resolving phlegm and relieving cough, clearing heat and detoxicating, promoting urination and reducing swelling and lowering blood pressure, and is used for treating common cold, cough with excessive phlegm, headache, tonsillitis, bacillary dysentery, hypertension and the like. Lindley eupatorium is commonly used for treating chronic tracheitis, bronchitis, hypertension and the like in clinic. The branch and leaf are used as medicine for relieving exterior syndrome, eliminating dampness, regulating middle warmer, and eliminating dampness, and can be used for treating cough due to fatigue, hematemesis, hemoptysis, stranguria with turbid urine, leucorrhea, and innominate swelling and pain. The modern pharmaceutical research shows that the active ingredients in the lycopus herb mainly comprise more than ten hundred compounds such as volatile oil, organic acids, triterpenes, sesquiterpenes, trace elements and the like. Among them, guaiane-type sesquiterpenoids are considered as a class of compounds with remarkable biological activity.

The invention aims to provide the use of any one of the compounds (formulae I to x) and enantiomers, diastereomers, salts, esters, prodrugs, solvates or solvates of salts thereof as or in the preparation of a SIRT1 receptor agonist.

The invention aims to provide the application of any one of the compounds (formula I-formula X) and enantiomers, diastereomers, salts, esters, prodrugs, solvates or solvates of salts thereof in the preparation of medicines for reducing uric acid.

Further, the medicament comprises an oral dosage form or an injection dosage form, wherein the oral dosage form comprises the following dosage forms: tablets, powders, granules, tea, capsules, soft capsules, oral liquid, pills, ointment and wine; the injection is injection.

The invention aims to provide the application of any one of the compounds (formula I-formula X) and enantiomers, diastereomers, salts, esters, prodrugs, solvates or solvates of salts thereof in preparing medicines for treating gout.

The invention aims to provide the use of any one of the compounds (formula I-formula X) and enantiomers, diastereomers, salts, esters, prodrugs, solvates or solvates of salts thereof as or in the manufacture of a medicament for inhibiting inflammation.

Specifically, the method for preparing the compound shown in the formula III or the formula IV comprises the following steps:

(1) Extracting. Collecting dried whole plant of herba Lycopi, pulverizing, reflux extracting with 90% methanol or ethanol, concentrating the extractive solution, removing wax with petroleum ether, adding water and chloroform or dichloromethane, extracting water layer with chloroform or dichloromethane for 2-3 times, concentrating chloroform or dichloromethane layer, and drying to obtain extract;

(2) Removing impurities. Separating the extract with AB-8 macroporous resin, eluting with ethanol water solvent, mixing the components between 40% -60% ethanol, concentrating, and drying to obtain hydrolysis raw material;

(3) And (5) hydrolyzing. Dissolving the hydrolysis raw material in 90% methanol or ethanol, adding concentrated sulfuric acid or concentrated hydrochloric acid or phosphoric acid, adjusting the pH value to be between 0.1 and 2.0, and hydrolyzing for 8 to 72 hours at the temperature of between 0 and 65 ℃ to obtain a hydrolysis crude product solution;

(4) And (5) separating and purifying. Separating the hydrolyzed crude product solution with AB-8 macroporous resin, eluting with ethanol water solvent, collecting 15% ethanol-eluted component and 25% ethanol-eluted component, concentrating and drying respectively. Separating and purifying the 15% ethanol eluting component by adopting a high-efficiency countercurrent chromatography ethyl acetate and water solvent system, using a water layer as a stationary phase and an ethyl acetate layer as a mobile phase, and collecting the eluent in a column volume period of 0.9-1.1 to obtain a compound shown in a formula III; separating and purifying the 25% ethanol eluting component by using high performance countercurrent chromatography ethyl acetate and water solvent system, wherein an ethyl acetate layer is used as a stationary phase, a water layer is used as a mobile phase, and collecting the eluent with a column volume period of 1.4-1.6 to obtain the compound shown in the formula IV.

In the invention, if the compound is used for preparing medicines, the compound is taken as an active substance, and then auxiliary materials for medicaments are added to prepare medicines.

The invention has the beneficial effects that

According to the embodiment, the compound and the enantiomer, diastereoisomer, salt, ester, prodrug, solvate or solvate of the salt thereof provided by the invention can obviously reduce the concentration of the uric acid when being used as or used for preparing the SIRT1 receptor agonist, and compared with the prior related commercial medicines, the uric acid reducing effect is better.

Drawings

FIG. 1 shows the results of various mouse model tests (administration concentration: 20 mg/kg).

Detailed Description

The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.

In the embodiment of the invention, allopurinol and benzbromarone come from commercial medicines.

In an embodiment of the present invention, an apparatus includes: API 4000 triple quadrupole LC-MS/MS Mass Spectrometry System (ABSCIEX Co., USA); LC-30AD ultra high performance liquid System (Shimadzu Corp.); R21G type high-speed refrigerated centrifuge (japanese Hitachi Co.); elix10 ultra pure water purification system (millpoer, usa); XS-105 parts per million precision electronic balance (METTLER toldo company, switzerland).

In an embodiment of the present invention, the reagent comprises: formic acid (Formic acid, sigma-Aldrich, batch No. 56302-10 ML) acetonitrile and methanol were all chromatographically pure (Merck, germany), and water was ultrapure water.

In the examples of the present invention, the chromatographic conditions in LC-MS/MS detection are shown in Table 1:

TABLE 1LC-MS/MS detection chromatography conditions

Chromatographic column	Kinetex HILIC column 2.1X100 mm,2.6 μm (S/N719247-4, USA)
		Mobile phase	Acetonitrile: 5mM ammonium acetate (0.1% formic acid)
Flow rate	0.25mL/min
		Column temperature	30℃
Sample injection volume	2μL
		Elution mode	Gradient elution (elution procedure see Table 2)

TABLE 2 LC-MS/MS detection gradient elution procedure

Time (min)	A (5 mM ammonium acetate (0.1% formic acid))	B (acetonitrile)
			0.1	98	2
3.5	98	2
			4	10	90
6.5	10	90
			7	98	2
10	98	2

In the embodiment of the invention, mass spectrum conditions in LC-MS/MS detection are as follows: using MRM mode, gas1:30psi, gas2:50psi, IS:5500v, tem:500 ℃, CAD:4psi, CUR:15Psi, ion pairs are shown in Table 3 below:

table 3 LC-Mass Spectrometry ion pair and other conditions in MS/MS detection

Sequence number

Ion pair

Name of the name

DP

CE

CXP

Scan mode

1

167.0/124.0

UA

80

20

30

Negative

In the embodiment of the invention, the preparation of the standard solution is as follows: accurately weighing 11.04mg of uric acid standard substance, placing into a 10mL volumetric flask, adding methanol water for dissolution, and fixing the volume to 10mL to obtain 1.104mg/mL of uric acid standard stock solution. And storing the mixture in a refrigerator at the temperature of 4 ℃ until the mixture is used.

In the embodiment of the invention, the blank serum sample treatment method comprises the following steps: precisely removing 50 mu L of blank serum sample, precisely adding 10 mu L of water, adding 400 mu L of methanol after vortex mixing for 30s, vortex mixing for 1min, centrifuging at 12000rpm for 15min, taking 100 mu L of supernatant, and carrying out sample injection analysis in a sample injection bottle at 2 mu L.

In the embodiment of the invention, the preparation and treatment of the drug-containing serum are as follows: 50uL of sample serum was precisely aspirated into a 1.5mL EP tube, 400 uL of methanol was added, vortexed and mixed well for 1min, centrifuged at 12000rpm for 15min, 100 uL of supernatant was taken in a sample bottle, and 2 uL of sample was analyzed.

In the embodiment of the invention, an SIRT1 receptor agonist, named as an "agonist A", is prepared, and the structural formula of the SIRT1 receptor agonist is as follows:

in the embodiment of the invention, an SIRT1 receptor agonist, named as an "agonist B", is prepared, and the structural formula of the SIRT1 receptor agonist is as follows:

in the embodiment of the invention, an SIRT1 receptor agonist, named as an "agonist C", is prepared, and the structural formula of the SIRT1 receptor agonist is as follows:

example 1

(1) Extracting by taking 800g of the whole plant of Eupatorium adenophorum, pulverizing, and reflux-extracting with 90% ethanol with volume 5 times; concentrating the extractive solution by rotary evaporation, and removing wax with petroleum ether; then adding 5 times of water and 3 times of dichloromethane into the rest part, separating and recovering a dichloromethane layer by using a separating funnel, extracting a water layer by using dichloromethane for 3 times, combining the collected dichloromethane solution, evaporating, concentrating and drying to obtain 69g of extract;

(2) Removing impurities, separating the extract with AB-8 macroporous resin, eluting with ethanol-water (100:0-0:100), mixing the components between 40% -60% ethanol, concentrating, and drying to obtain 14g hydrolysis raw material;

(3) Hydrolysis, namely dissolving the hydrolysis raw material in 90% methanol, then adjusting the pH value to 1.3 by using concentrated sulfuric acid, and hydrolyzing in a water bath kettle at the temperature of 60+/-1 ℃ for 60 hours to obtain a crude product solution;

(4) Separating and purifying by separating the crude product solution with AB-8 macroporous resin, eluting with ethanol-water (100:0-0:100), collecting 15% ethanol-eluted component and 25% ethanol-eluted component, concentrating and drying respectively; separating and purifying the 15% ethanol eluting component by adopting a high-efficiency countercurrent chromatography ethyl acetate and water solvent system, taking a water layer as a stationary phase and an ethyl acetate layer as a mobile phase, collecting eluent in a column volume period of 0.9-1.1, and freeze-drying to obtain 1.76g of agonist A with the purity of 96%; separating and purifying the 25% ethanol eluting component by using high performance countercurrent chromatography ethyl acetate and a water solvent system, using an ethyl acetate layer as a stationary phase and a water layer as a mobile phase, collecting eluent of 1.4-1.6 column periods, and freeze-drying to obtain 2.81g of agonist B with purity of 95%; the total yield of the two compounds agonist a, agonist B was 0.571%.

Example 2

(1) Extracting: taking 1500g of the whole plant of lycopus herb, crushing, and extracting with 90% ethanol with the volume of 3 times under reflux; concentrating the extractive solution by rotary evaporation, and removing wax with petroleum ether; then adding 4 times of water and 2 times of dichloromethane into the rest part, separating and recovering a dichloromethane layer by using a separating funnel, extracting a water layer by using dichloromethane for 3 times, combining the collected dichloromethane solution, evaporating, concentrating and drying to obtain 115g of extract;

(2) Removing impurities: separating the extract with AB-8 macroporous resin, eluting with ethanol-water (100:0-0:100), mixing the components between 40% -60% ethanol, concentrating, and drying to obtain 23g hydrolysis raw material;

(3) Hydrolysis: dissolving the hydrolysis raw material in 90% methanol, then regulating the pH value to 0.8 by phosphoric acid, and hydrolyzing in a water bath kettle at 58+/-1 ℃ for 72 hours to obtain a crude product solution;

(4) And (3) separating and purifying: separating the crude product solution with AB-8 macroporous resin, eluting with ethanol-water (100:0-0:100), collecting 15% ethanol-eluted component and 25% ethanol-eluted component, concentrating and drying respectively; separating and purifying the 15% ethanol eluting component by adopting a high-efficiency countercurrent chromatography ethyl acetate and water solvent system, taking an ethyl acetate layer as a stationary phase and a water layer as a mobile phase, collecting eluent in a column volume period of 0.9-1.1, and freeze-drying to obtain 3.27g of an agonist A with a purity of 97%; separating and purifying 25% ethanol eluting component by adopting high performance countercurrent chromatography ethyl acetate and water solvent system, collecting eluent of 1.4-1.6 column volume time periods by using ethyl acetate layer as stationary phase and water layer as mobile phase, and lyophilizing to obtain 4.39g of agonist B with purity of 96%; the total yield of both agonist a and agonist B compounds was 0.511%.

Example 3

(1) Extracting: taking 2000g of the whole plant of lycopus herb, crushing, and extracting with 90% ethanol with the volume of 3 times under reflux; concentrating the extractive solution by rotary evaporation, and removing wax with petroleum ether; then adding 4 times of water and 2 times of dichloromethane into the rest part, separating and recovering a dichloromethane layer by using a separating funnel, extracting a water layer by using dichloromethane for 3 times, combining the collected dichloromethane solution, evaporating, concentrating and drying to obtain 135g of extract;

(2) Removing impurities: separating the extract with AB-8 macroporous resin, eluting with ethanol-water (100:0-0:100), mixing the components between 40% -60% ethanol, concentrating, and drying to obtain 30g hydrolysis raw material;

(3) Hydrolysis: dissolving the hydrolysis raw material in 90% methanol, then adjusting the pH value to 0.8 by using concentrated hydrochloric acid, and hydrolyzing in a water bath kettle at a temperature of 50+/-1 ℃ for 24 hours to obtain a crude product solution;

(4) And (3) separating and purifying: separating the crude product solution with AB-8 macroporous resin, eluting with ethanol-water (100:0-0:100), collecting 20% ethanol-eluted component, concentrating, and drying; separating and purifying by adopting a high-efficiency countercurrent chromatography dichloromethane, water and methanol (1:1:1) solvent system, taking an upper phase as a stationary phase and a lower phase as a mobile phase, collecting eluent with the volume of 1.9-2.1 column periods, performing rotary evaporation concentration, and then performing freeze drying to obtain 6.18g of an agonist C with the purity of 98%; the total yield of the compound was 0.309%.

Example 4 Effect verification

(1) And selecting 70 qualified six-month-old mice, wherein the weight of each mouse is 20+/-3 g, and the mice are adaptively fed for 1 week under the environment of intermittent illumination every 12 hours at the temperature of 24+/-2 ℃ and the relative humidity of 55+/-5%. Thereafter, mice were randomly divided into 7 groups, model, blank, allopurinol, benzbromarone, agonist a, agonist B, agonist C.

(2) The concentration of each of allopurinol group, benzbromarone group, agonist A group, agonist B group and agonist C group was 20mg/kg, and the administration was continued for 14 days from day 15, 1 time per day. At day 8, the administration of 250mg/kg of potassium oxazinate and 300mg/kg of hypoxanthine was performed at a time of simultaneous intragastric administration, and hyperuricemia was induced. The blank was filled with 0.5% CMC-Na.

(3) Serum uric acid concentrations of blank experimental groups, allopurinol groups, benzbromarone groups, agonist A groups, agonist B groups and agonist C groups were detected by LC-MS/MS analysis, the results are shown in FIG. 1, and the experimental results show that:

the uric acid concentration of the agonist A is reduced from 11.01mg/L to 4.68mg/L, which is equivalent to the uric acid reducing effect of allopurinol, so that the prepared agonist A has good uric acid reducing effect.

The uric acid concentration of the agonist B is reduced to 4.37mg/L, which is equivalent to the uric acid reducing effect of the benzbromarone, which shows that the prepared agonist B can also effectively reduce uric acid.

The uric acid concentration of the agonist C is reduced by 4.81mg/L, and the uric acid reducing effect is slightly lower than that of allopurinol and benzbromarone, and the agonist C has obvious uric acid reducing effect although the agonist A and the agonist B are not the same.

Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.

Claims

1. The application of a compound serving as an SIRT1 receptor agonist in preparing a medicament for treating hyperuricemia is characterized in that the structural formula of the compound is shown as a formula III, a formula IV or a formula V,

2. the use according to claim 1, wherein the medicament comprises an oral dosage form or an injectable dosage form, the oral dosage form being of the following: tablets, powders, granules, capsules, oral liquid, pills and paste; the injection is injection.

3. The application of a compound serving as an SIRT1 receptor agonist in preparing a medicament for treating gout is characterized in that the structural formula of the compound is shown as formula III, IV or V,

4. the use according to claim 3, wherein the medicament comprises an oral dosage form or an injectable dosage form, the oral dosage form being of the following: tablets, powders, granules, capsules, oral liquid, pills and paste; the injection is injection.