CN115873057A - 26-carboxylic acid triterpene compound in sequoia, preparation method thereof and application thereof as acetyl coenzyme A carboxylase inhibitor - Google Patents

Abstract

The application discloses a 26-carboxylic acid triterpene compound in sequoia, a preparation method thereof and application of the 26-carboxylic acid triterpene compound as an acetyl coenzyme A carboxylase inhibitor, wherein the structural formula of the 26-carboxylic acid triterpene compound in the sequoia is one of the structures shown in the specification：

Description

26-carboxylic acid triterpene compound in sequoia, preparation method thereof and application thereof as acetyl coenzyme A carboxylase inhibitor

Technical Field

The application relates to the technical field of medicines, in particular to a 26-carboxylic acid triterpene compound in sequoia, a preparation method thereof and application thereof as an acetyl coenzyme A carboxylase inhibitor.

Background

Abnormal elevation of fatty acid synthesis (fayn), impaired fatty acid hyperoxidation (FAOxn), or disorders of fatty acid metabolism caused by both, resulting from abnormal fatty acid metabolism, are hallmarks of various metabolic abnormalities, including insulin antagonism, hepatic steatosis, dyslipidemia, obesity, metabolic syndrome (MetSyn), and nonalcoholic fatty liver disease (NAFLD), among others. These metabolic abnormalities may lead to the development of type 2 Diabetes (T2 DM), nonalcoholic steatohepatitis (NASH), and atherosclerotic vascular disease (Expert Opin thertargets, 2005,9,267-281.; JAMA,2001,285,2486-2497.; hepatology,2010,52, 774-788.; miller et al, circulation,2011,123,2292-2333.; neuropharmacology,2012,63,57-75.; diabetes Care,2014,37, S81-S90.). Altered fatty acid metabolism is also a hallmark of cancer and leads to abnormal and sustained cell proliferation of malignant cells (Baenke et al, dis Model Mech,2013,6, 1353-1363.; mounier et al, int J Oncol,2014, 45. Therefore, inhibition of fayn and/or stimulation of FAOxn may advantageously affect these diseases.

There are two known classes of acetyl-coa carboxylases, ACC1 and ACC2, in rodent and human body organs, which are encoded by different genes and share about 70% of the amino acids. ACC1 is acetyl-coa carboxylase 1, encoding a 265KD protein, located in liver and adipose tissue, while ACC2 is acetyl-coa carboxylase 2, encoding a 280KD protein, preferentially expressed in oxidized tissue, skeletal muscle and heart (Mao et al, proc.nato.acad.sci.usa,2003, 100. ACC1 is a key enzyme (rate-limiting enzyme) in the fatty acid synthesis process, requiring biotin as a coenzyme and relying on the energy provided by ATP to carboxylate acetyl-coa, which then catalyzes the production of malonyl-coa and thus regulates lipid metabolism (Ronnebaum et al, j. Biol. Chem.,2008,283, 14248-14256.). In the liver, malonyl-coa formed by ACC1 in the cytoplasm is mainly used in fayn and its related derivatives (Expert Opin Ther Targets,2005, 9.

In summary, ACC1 inhibitors have important implications for the treatment of metabolic syndrome with disorders of fatty acid anabolism (Veprik et al, commun. Biol.2022,5,238, chen et al, expert Opin Investig drugs.2019,28, 917-930.). Unfortunately, no ACC1 inhibitor is successfully marketed at present, and the ACC1 small-molecule inhibitors such as Gemcabene and the like which are researched at present are in the clinical third-stage and have optimistic prospects. GS-0976 significantly reduced insulin levels, increased insulin sensitivity, and reduced levels of triacylglycerols and cholesterol in the liver and blood (Loomba et al, gastroenterology,2018,155, 1463-1467.), while Gemcabene increased clearance of very low density lipoprotein cholesterol (VLDL) in the plasma and inhibited production of cholesterol and triglycerides in the liver (Srivastava et al, front Pharmacol,2018,9,471.). Other ACC1 inhibitors under investigation have been slow to develop due to low cell penetration, low affinity for ACC1, or poor specificity. Therefore, the search for the small-molecule ACC1 inhibitor which is efficient, high in selectivity and good in pharmacokinetic property is of great significance.

Natural products have a unique and important position for guidance and reference of new drug development and are an important source of new drug discovery (Atanasov et al, nat. Rev. Drug discov.2021,20,200-216, newman et al, j. Nat. Prod.2020,83,770-803, tiago et al, nat. Chem.2016,8, 531-541. The natural product is generated by long-time selection evolution through a natural rule, can be effectively combined with biological macromolecules, and can be considered as an elite compound library left after a biological system highly related to human protein is screened and eliminated for years. Meanwhile, the single target point medicine is often poor in curative effect, and the traditional Chinese medicine/botanical medicine and the components thereof have the characteristics of novel structure (chemical components), multiple target points and multiple ways, and have unique advantages in the process of treating complex diseases. Therefore, the method has important research value in searching and developing novel, efficient and low-toxic-side-effect ACC1 inhibitors from chemical components of natural sources (particularly plant sources).

Natural products with unique sources and structural features may be an antecedent in the intense competition in the current field of new drug development for diseases of dyslipidemic metabolism. The statistical analysis is carried out on the natural sources of the drug molecules, and the results show that: drug molecules derived from terrestrial plants are concentrated in some specific families (active natural products are derived from 792 families of 6763 families in nature, the proportion is only 11.7%), and marketed drugs and candidate chemicals in clinical phase are concentrated in 144 families, while most rare endangered plants are apparently in the genera of these plant families capable of producing drugs (Zhu et al, proc. Natl. Acad. Sci. U.S. A.2011,108, 12943-12948; mohamed et al, proc. Natl. Acad. Sci.2013,110: 16832-16837), which indicates that the novelty and drugability of natural products in rare endangered plants are much higher than the average level of general plants.

Significantly, pinaceae (Pinaceae) is the top 20 of the genera of the most potent drug molecule producing organisms (Zhu et al, proc.natl.acad.sci.u.s.a.2011,108, 12943-12948). The oil fir [ Keteleeria for tunei (Murr.) Carr ] belongs to the Pinaceae family of the genus of oil fir (Keteleeria), is recorded in 1992 in the book "Chinese plant Red book-rare endangered plant" and is classified as "gradually dangerous species". If a few oil fir plant samples (such as renewable branches and leaves) can be protectively collected to carry out systematic chemical composition research, scientific understanding, active protection and comprehensive development and utilization of the rare plant resource can be actively promoted, so that the rare plant resource can be continuously served for human beings.

Disclosure of Invention

The application provides a novel natural 26-carboxylic acid triterpenoid compound derived from branches and leaves of taxus chinensis, and the compound is found to have a good ACC1 inhibition effect in-vitro pharmacological activity screening, so that the compound has a treatment effect on hyperlipidemia, atherosclerosis, fatty liver, type II diabetes, cancer and other ACC1 mediated diseases, and has huge potential application in the pharmaceutical field. Based on the application, the application also provides a new application of the triterpenoids in the sequoia oleifera.

The application separates a series of 26-carboxylic acid triterpenoids (the structures are shown as formulas 1-4) with novel structures from methanol extracts of branches and leaves of taxus chinensis for the first time. Wherein compound 1 is a conventional lanostane-type triterpene, compound 2 is a methyl-rearranged lanostane-type triterpene, compound 3 is a cycloartene-type triterpene, compound 4 is a methyl-rearranged cycloartene-type triterpene, and the skeleton is very rare in nature. In addition, the side chains of compounds 1,2 and 4 all form one identical furan ring structural fragment, except for the C-26 carboxylation. Multiple pharmacological test researches show that the compounds have obvious ACC1 inhibiting activity.

26-carboxylic acid triterpenoid in the sequoia, wherein the structural formula of the 26-carboxylic acid triterpenoid is one of the following structures:

the application also provides application of the 26-carboxylic acid triterpene compound in preparing an Acetyl-CoA carboxylase 1 (Acetyl-CoA carboxylase 1, ACCC 1) inhibitor.

The application also provides application of the 26-carboxylic acid triterpene compound in preparing a medicament for preventing, delaying or treating diseases mediated by acetyl coenzyme A carboxylase 1.

Alternatively, the diseases mediated by acetyl-coa carboxylase 1 include hyperlipidemia, non-alcoholic fatty liver, type II diabetes, or obesity.

The application also provides an acetyl coenzyme A carboxylase 1 inhibitor which takes one or more of the 26-carboxylic acid triterpene compounds with the structures shown in formulas 1 to 4 as an active ingredient.

The triterpene compound of the application shows a strong inhibitory effect in an ACC1 inhibitory activity test.

The application also provides a pharmaceutical composition, which comprises one or more of 26-carboxylic acid triterpene compounds with the structures of formula 1-formula 4 in a therapeutically effective amount as an active ingredient.

The pharmaceutical composition adopts one or more of the triterpene compounds as raw materials, comprises one or more of the compounds as active ingredients in a therapeutically effective amount, and can further comprise pharmaceutically acceptable auxiliary materials, such as carriers, excipients, adjuvants or diluents and the like. The pharmaceutical composition can be used for preparing medicaments for preventing, delaying or treating diseases (particularly hyperlipidemia and related cardiovascular diseases) related to glycolipid disorder mediated by ACC1 or used as lead compounds of the medicaments.

Based on the advantages of the compounds in the aspects of novel chemical structure, remarkable biological activity and the like, the compounds have good development prospect and are expected to be developed into a therapeutic drug or lead compound with novel structure for ACC1 mediated diseases.

The compounds described herein can be isolated and purified from plants; can also be obtained by chemical synthesis methods well known to those skilled in the art.

Wherein, the method for separating and purifying the plant comprises the following steps:

air drying branches and leaves of Metasequoia fortunei (Murr.) Carr at room temperature, pulverizing, extracting with 90% methanol at room temperature for 5 times, concentrating the extractive solution, adding water, suspending, and extracting with petroleum ether, ethyl acetate and n-butanol respectively; concentrating the obtained ethyl acetate extract under reduced pressure, sequentially separating by silica gel, microporous resin (MCI), sephadex LH-20 gel and reversed-phase semi-preparative high performance liquid chromatography (semi-preparative RP-HPLC) to obtain the compounds shown in formulas 1-4.

In the above method, the methanol/water solution may be a methanol-water solution with a concentration of 70% or more, preferably a methanol-water solution with a volume fraction of 90%; the time for room-temperature extraction is not particularly limited, and can be 12 hours/time or more; the number of times of extraction may be one or more, preferably 3 or more, and more preferably 5.

Alternatively, the ethyl acetate extract was concentrated under reduced pressure and then eluted with a gradient of petroleum ether-ethyl acetate (15 → 1 → 10 → 5:1 → 3:1 → 1:1 → 0, v/v) by silica gel column chromatography, followed by ethyl acetate-methanol (5:1 → 1:1 → 0) to give 8 components (fr.1-fr.8);

for petroleum ether: ethyl acetate 5:1 eluate fraction fr.5 was subjected to MCI column chromatography and purified in water: gradient elution is carried out on methanol in a volume ratio of 50Liquid; for water: the elution components with the methanol volume ratio of 15 ₂ O,90:10,v/v；flow rate,3mL/min,t _R =16.0 min) to obtain compound 3; for water: the elution components with the methanol volume ratio of 20 ₂ O,90:10,v/v；flow rate,3mL/min,t _R =16.0 min) to give compound 1.

For petroleum ether: ethyl acetate 3:1 eluate fraction fr.7 was subjected to MCI column chromatography and purified in water: gradient elution is carried out on methanol in a volume ratio of 50 to 0; for water: the elution components with the methanol volume ratio of 20 ₂ O,90:10,v/v；flow rate,3mL/min,t _R =16.0 min) to give compound 4; for water: the elution components with the methanol volume ratio of 20 ₂ O,75:25,v/v；flow rate,3mL/min,t _R After =22.6 min) compound 2 was obtained.

Compared with the prior art, the application has at least one of the following remarkable advantages:

(1) The compounds are all novel 26-carboxylic acid triterpenoids which are firstly found and are derived from the natural world, wherein the compound 1 is a conventional lanostane-type triterpene, the compound 2 is a methyl-rearranged lanostane-type triterpene, the compound 3 is a cycloartene-type triterpene, and the compound 4 is a methyl-rearranged cycloartene-type triterpene. The side chains of compounds 1,2 and 4 all form one identical fragment of the furan ring structure.

(2) Meanwhile, the compound is found for the first time to have remarkable Acetyl-CoA carboxylase 1 (Acetyl-CoA carboxylase 1, ACC 1) inhibition activity through a plurality of in vitro activity tests, and can be applied to preparation of medicines for preventing, delaying or treating glycolipid metabolic disorders mediated by ACC1 and other related diseases. The application also can provide a lead compound for developing novel medicines for diseases related to glycolipid metabolic disturbance. The method has important application prospect for diseases related to the glycolipid metabolic disturbance, such as hyperlipidemia, non-alcoholic steatohepatitis, type II diabetes and the like, which are highly developed in modern people.

Drawings

FIG. 1 shows a scheme for preparing Compound 1 ¹ H NMR spectrum;

FIG. 2 shows a scheme for preparing Compound 1 ¹³ A C NMR spectrum;

FIG. 3 is a drawing of Compound 2 ¹ An H NMR spectrum;

FIG. 4 is a drawing of Compound 2 ¹³ A C NMR spectrum;

FIG. 5 shows the preparation of Compound 3 ¹ An H NMR spectrum;

FIG. 6 is a drawing of Compound 3 ¹³ A C NMR spectrum;

FIG. 7 is a drawing of Compound 4 ¹ An H NMR spectrum;

FIG. 8 is a drawing of Compound 4 ¹³ C NMR spectrum.

Detailed Description

The preparation steps and pharmacological test procedures of the compounds of the present application are further illustrated by the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present application, and that various modifications and changes may be made thereto by those skilled in the art.

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. The terminology used in the description of the present application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

The oil fir [ Ketleeria fortunei (Murr.) Carr ] belongs to the Pinaceae of the genus of oil fir (Ketleeria), the inventor protectively collects a small amount of samples of the Chinese eastern yellow fir plant (branches and leaves of the Chinese eastern yellow fir plant are easy to regenerate), extracts active substances of the Chinese eastern yellow fir plant and develops new application of the Chinese eastern yellow fir plant in pharmacy.

Taking branches and leaves of sequoia oleifera (Murr.) Carr, airing at room temperature, crushing, extracting with 90% methanol at room temperature, concentrating an extracting solution, adding water for suspension, and extracting with petroleum ether, ethyl acetate and n-butyl alcohol respectively; concentrating the obtained ethyl acetate extract under reduced pressure, sequentially separating by silica gel, microporous resin (MCI), sephadex LH-20 gel and reversed-phase semi-preparative high performance liquid chromatography (semi-preparative RP-HPLC) to obtain the compounds shown in formulas 1-4.

The following is illustrated by specific examples:

branches and leaves of Chinese fir (keteleria fortunei) are collected from Yunnan Kunming, dried in the shade, and pulverized into powder; specific optical rotation test was performed by Rudolf Autopol IV polarimeter at 25 ℃; hitachi U-2900E type ultraviolet spectrometer; thermo Scientific Nicolet Is5 FT-IR type infrared spectrometer; ECD spectra were measured by JASCO-810 CD spectrometer; ESI-MS was measured by Agilent model 1100 LC-MS instrument, HR-ESIMS was measured by AB Sciex TripleTOF model 5600 instrument; the silica gel used is produced by Qingdao ocean chemical company; the silica gel thin layer plate is produced by a tobacco platform Jiang You silica gel development company Limited, and the specification is GF254/0.25mm; MCI gel CHP20P is produced by Mitsubishi corporation of Japan, and the specification is 75-150 μm; sephadex LH-20 gel is produced by GE Healthcare Bio-Sciences, switzerland; semi-preparative HPLC was Shimadzu LC-20AT, equipped with SPD-M20A PDA detector and Waters Xbridge ODS and Cosmosil semi-preparative columns (250X 10mm,5 μ M); all the analytical pure reagents are produced by Shanghai national drug group chemical reagent company Limited; chromatographic grade solvents are produced by Shanghai Starfish high purity solvents, inc.; the deuterated reagent is produced by Sigma-Aldrich.

Example 1: preparation of the Compounds

Pulverizing dried branches and leaves of Metasequoia glyptostroboides (10.0 kg), extracting with 90% methanol (10L) at room temperature for 5 times, each for 24 hr, mixing extractive solutions, and concentrating under reduced pressure to obtain total extract 2.66kg. Dispersing the extract with 1L water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol with equal volume for three times respectively to obtain four components of petroleum ether, ethyl acetate, n-butanol and water. The ethyl acetate fraction amounted to 134.0g, and was eluted with a petroleum ether-ethyl acetate (15 → 10, 1 → 5:1 → 3:1 → 1:1 → 0, 100, v/v) gradient by silica gel column chromatography, followed by ethyl acetate-methanol (5:1 → 1:1 → 0), and the resulting lower column liquid was collected, subjected to color development by TLC thin layer chromatography, and the lower column liquids having the same spots were combined to give 8 fractions (fr.1 to fr.8).

For petroleum ether: ethyl acetate 5:1 eluate fraction (fr.5) was subjected to MCI column chromatography and purified with water: methanol volume ratio 50. For water: the elution components with the methanol volume ratio of 15 to 75 are sequentially subjected to Sephadex LH 20 gel chromatography (methanol elution) separation and reversed-phase semi-preparative high performance liquid chromatography (MeOH-H) separation ₂ O,90:10,v/v；flow rate,3mL/min,t _R =16.0 min) to obtain compound 3; for water: the elution components with the methanol volume ratio of 20 ₂ O,90:10,v/v；flow rate,3mL/min,t _R =16.0 min) to yield compound 1.

For petroleum ether: ethyl acetate 3:1 eluate fraction (fr.7) was subjected to MCI column chromatography and purified with water: gradient elution is carried out on methanol in a volume ratio of 50. For water: the elution components with the methanol volume ratio of 20 to 80 are sequentially subjected to Sephadex LH 20 gel chromatography (methanol elution) separation and reversed-phase semi-preparative high performance liquid chromatography (MeOH-H) separation ₂ O,90:10,v/v；flow rate,3mL/min,t _R =16.0 min) to give compound 4; for water: the elution components with the methanol volume ratio of 20 ₂ O,75:25,v/v；flow rate,3mL/min,t _R =22.6 min) to yield compound 2.

Compound 1, with the following nuclear magnetic and physicochemical data:

White powder；[α]25D–14.8(c 0.4,MeOH)；UV(MeOH)λ _max (logε)246(3.91)nm； ¹ H NMR(600MHz,pyridine-d ₆ )δ _H :3.66(1H,t,J＝3.0Hz,H-3),1.73(1H,dd,J＝11.0,6.8Hz,H-5),5.72(1H,m,H-7),2.37(1H,dd,J＝12.3,6.8Hz,H-9),0.98(3H,s,H-18),1.13(3H,s,H-19),0.89(3H,d,J＝6.5Hz,H-21),2.78(1H,dd,J＝14.8,3.6Hz,H-22a),2.30(1H,dd,J＝14.8,9.6Hz,H-22b),6.82(1H,s,H-24),8.47(1H,s,H-27),1.25(3H,s,H-28),0.99(3H,s,H-29),1.03(3H,s,H-30)； ¹³ C NMR(600MHz,pyridine-d ₆ )δ _c :30.5(C-1),26.9(C-2),75.2(C-3),37.9(C-4),43.0(C-5),28.9(C-6),122.2(C-7),149.0(C-8),49.1(C-9),36.0(C-10),23.2(C-11),23.5(C-12),43.8(C-13),53.0(C-14),33.7(C-15),35.2(C-16),53.6(C-17),23.8(C-18),24.8(C-19),36.5(C-20),18.7(C-21),34.7(C-22),157.3(C-23),107.3(C-24),122.1(C-25),165.8(C-26),146.7(C-27),29.6(C-28),23.7(C-29),30.8(C-30)；HRESIMS m/z 467.3165[M+H] ⁺ (calcd for C ₃₀ H ₄₅ O ₄ ,467.3167,Δ＝–0.3ppm).

¹ H NMR spectrum of compound 1in pyridine-d ₅ as shown in fig. 1; ¹³ C NMR spectrum of compound 1in pyridine-d ₅ as shown in fig. 2.

The nuclear magnetic and physicochemical data for compound 2 are as follows:

Colorless,amorphous powder；[α]25D+31.2(c 0.85,CHCl ₃ )；UV(MeOH)λ _max (logε)246(3.93)nm； ¹ H NMR(600MHz,CDCl ₃ )δ _H :4.68(1H,br s,H-3),1.61(1H,dd,J＝12.0,5.0Hz,H-5),5.59(1H,br s,H-7),5.12(1H,br s,H-15),0.94(3H,s,H-18),1.00(3H,s,H-19),0.83(3H,d,J＝6.8Hz,H-21),2.77(1H,dd,J＝14.3,3.2Hz,H-22a),2.28(1H,dd,J＝14.3,10.4Hz,H-22b),6.37(1H,s,H-24),7.98(1H,s,H-27),1.01(3H,s,H-28),0.95(3H,s,H-29),1.00(3H,s,H-30),2.08(3H,s,H-2′)； ¹³ C NMR(600MHz,CDCl ₃ )δ _c :27.3(C-1),22.3(C-2),78.2(C-3),35.7(C-4),38.2(C-5),21.9(C-6),120.0(C-7),136.1(C-8),52.3(C-9),34.0(C-10),32.9(C-11),25.6(C-12),51.2(C-13),152.2(C-14),114.5(C-15),44.6(C-16),50.2(C-17),15.9(C-18),21.7(C-19),37.0(C-20),15.1(C-21),30.9(C-22),157.5(C-23),104.9(C-24),118.3(C-25),167.2(C-26),146.9(C-27),27.8(C-28),22.6(C-29),23.2(C-30),170.1(C-1′),21.6(C-2′)；HRESIMS m/z 507.3189[M–H] ^- (calcd for C ₃₂ H ₄₃ O ₅ ,507.3189,Δ＝+0.1ppm).

¹ H NMR spectrum of compound 2in CDCl ₃ as shown in fig. 3; ¹³ C NMR spectrum of compound 2in CDCl ₃ as shown in fig. 4.

Compound 3, with the following nuclear magnetic and physicochemical data:

White powder；[α]25D+4.0(c 0.1,MeOH)；UV(MeOH)λ _max (logε)210(3.90)nm； ¹ H NMR(600MHz,CDCl ₃ )δ _H :3.49(1H,br s,H-3),1.84(1H,dd,J＝11.0,7.3Hz,H-5),5.23(1H,br s,H-16),1.06(3H,s,H-18),0.32(1H,d,J＝4.4Hz,H-19a),0.54(1H,d,J＝4.4Hz,H-19b),1.04(3H,d,J＝6.8Hz,H-21),6.89(1H,t,J＝7.0Hz,H-24),1.89(3H,s,H-27),0.98(3H,s,H-28),0.90(3H,s,H-29),0.94(3H,s,H-30)； ¹³ C NMR(600MHz,CDCl ₃ )δ _c :27.5(C-1),20.8(C-2),77.0(C-3),39.5(C-4),41.3(C-5),19.9(C-6),27.3(C-7),46.2(C-8),22.4(C-9),26.0(C-10),28.6(C-11),26.2(C-12),48.5(C-13),51.6(C-14),42.0(C-15),119.6(C-16),156.1(C-17),22.5(C-18),31.4(C-19),31.8(C-20),22.0(C-21),35.3(C-22),27.2(C-23),145.5(C-24),126.5(C-25),170.5(C-26),12.1(C-27),25.9(C-28),21.2(C-29),20.5(C-30)；HRESIMS m/z 453.3362[M–H] ^- (calcd for C ₃₀ H ₄₇ O ₃ ,453.3374,Δ＝–2.8ppm).

¹ H NMR spectrum of compound 3in CDCl ₃ as shown in fig. 5; ¹³ C NMR spectrum of compound 3 in CDCl ₃ as shown in fig. 6.

Compound 4, with the following nuclear magnetic and physicochemical data:

Colorless,amorphous powder；[α]25D+23.7(c 0.3,MeOH)；UV(MeOH)λ _max (logε)246(3.82)nm； ¹ H NMR(600MHz,CDCl ₃ )δ _H :3.50(1H,t,J＝3.2Hz,H-3),1.80(1H,dd,J＝12.1,4.4Hz,H-5),5.46(1H,dd,J＝8.0,2.5Hz,H-12),1.08(3H,s,H-18),0.20(1H,d,J＝4.2Hz,H-19a),0.49(1H,d,J＝4.2Hz,H-19b),0.83(3H,d,J＝6.8Hz,H-21),2.97(1H,dd,J＝14.2,1.6Hz,H-22a),2.70(1H,dd,J＝16.5,14.2Hz,H-22b),6.36(1H,s,H-24),7.97(1H,s,H-27),0.99(3H,s,H-28),0.89(3H,s,H-29),0.91(3H,s,H-30)； ¹³ C NMR(600MHz,CDCl ₃ )δ _c :28.3(C-1),26.3(C-2),77.1(C-3),39.2(C-4),41.1(C-5),19.7(C-6),25.5(C-7),50.2(C-8),28.3(C-9),26.8(C-10),31.4(C-11),115.8(C-12),159.3(C-13),46.5(C-14),37.6(C-15),32.9(C-16),49.1(C-17),25.6(C-18),27.8(C-19),38.1(C-20),14.9(C-21),30.8(C-22),158.4(C-23),105.6(C-24),118.8(C-25),167.1(C-26),147.4(C-27),26.0(C-28),21.4(C-29),17.7(C-30)；HRESIMS m/z 465.3004[M–H] ^- (calcd for C ₃₀ H ₄₁ O ₄ ,465.3010,Δ＝–1.3ppm).

¹ H NMR spectrum of compound 4 in CDCl ₃ as shown in fig. 7; ¹³ C NMR spectrum of compound 4in CDCl ₃ as shown in fig. 8.

Example 2: EXAMPLE 1 ACC1 inhibitory Activity test of the isolated Compound

ACC1 is located in the liver and adipocytes and is a key enzyme in fatty acid synthesis, requiring biotin as a coenzyme and catalyzing the production of malonyl-coa upon carboxylation of acetyl-coa depending on the energy provided by ATP to regulate lipid metabolism. This reaction is accompanied by the consumption of ATP, and therefore the ADP-Glo kinase assay reagent is used to detect the change in ATP, thereby mediating the inhibitory effect of the compound on ACC1 enzyme.

The concrete expression is as follows: the samples were dissolved in DMSO immediately before use to give appropriate concentrations, diluted 3-fold, subjected to 7-fold gradient, and triplicated wells, and 1. Mu.L of sample solution was added to a standard assay (40mM Tris, pH 8.0,10mM MgCl) ₂ 5mM DTT, ATP, coA, sodium citrate and ACC 1), incubated for 0.5 hours at room temperature. After that, 2.5. Mu.L of ADP-Glo in the kit was added and incubated at room temperature for 1 hour to consume the unreacted ATP as a substrate and terminate the reaction. Then adding a kinase detection reagent, and incubating for 0.5h. Converting ADP to ATP. The readings were then examined using an EnVision instrument. Meanwhile, a solvent control group, a positive control group and a blank control group which replace the compound to be detected with DMSO are arranged in the reaction, and each sample is provided with 3 multiple wells at each concentration. The final volume of the reaction was 12.5. Mu.L, the data were processed to plot the log of the concentration against the percentage of activity, then a fitted curve was calculated using non-linear regression, and IC was calculated using the software GraphPad Prism 5 formula log (inhibitor) vs. response- -Variable slope ₅₀ Value, weight of experimentTriplicates were performed and the results averaged over triplicates. The positive control was ND630, IC ₅₀ The value was 4.3. + -. 0.5nM.

TABLE 1 ACC1 inhibitory Activity data for 26-Carboxylic acid triterpenoids in Sequoia oleifera

The test results show that the four novel 26-carboxylic acid triterpene compounds in the patent show significant inhibitory activity on ACC1, and the compounds can be used for preparing medicaments for treating diseases related to glycolipid metabolic disorder or serving as lead compounds of the medicaments.

The above-mentioned embodiments only express several embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent application shall be subject to the appended claims.

Claims (10)

1. 26-carboxylic acid triterpene compounds in hemlock spruce are characterized in that the structural formula of the 26-carboxylic acid triterpene compounds is one of the following structures:

2. use of a triterpene 26-carboxylate compound as defined in claim 1 for the preparation of an acetyl-coa carboxylase 1 inhibitor.

3. Use of a 26-carboxylic acid triterpene compound according to claim 1 in the preparation of a medicament for preventing, delaying or treating a disease mediated by acetyl-coa carboxylase 1.

4. The use according to claim 3, wherein the diseases mediated by acetyl-CoA carboxylase 1 include hyperlipidemia, non-alcoholic fatty liver, type II diabetes, or obesity.

5. An acetyl-coa carboxylase 1 inhibitor comprising one or more of the 26-carboxylic acid triterpene compounds according to claim 1 as an active ingredient.

6. A pharmaceutical composition comprising a therapeutically effective amount of one or more selected from the 26-carboxylic acid triterpene compounds according to claim 1 as an active ingredient.

7. The pharmaceutical composition of claim 6, further comprising pharmaceutically acceptable pharmaceutical excipients.

8. A method for preparing 26-carboxylic acid triterpene compounds in the oil fir as claimed in claim 1, which comprises:

air drying branches and leaves of Metasequoia fortunei (Murr.) Carr at room temperature, pulverizing, extracting with methanol, concentrating the extractive solution, adding water, suspending, and sequentially extracting with petroleum ether, ethyl acetate and n-butanol; concentrating the obtained ethyl acetate extract under reduced pressure, sequentially separating by silica gel, microporous resin, sephadex LH-20 gel and reversed-phase semi-preparative high performance liquid chromatography to obtain the compounds shown in formulas 1-4.

9. The preparation method according to claim 9, wherein the ethyl acetate extract is concentrated under reduced pressure and then subjected to silica gel column chromatography with a gradient of 15;

for petroleum ether: ethyl acetate 5:1 eluate fraction fr.5 was chromatographed on MCI column and purified by water: gradient elution is carried out on methanol in a volume ratio of 50 to 0; for water: sequentially carrying out Sephadex LH 20 gel chromatographic separation and reversed-phase semi-preparative high performance liquid chromatographic separation on the elution components with the methanol volume ratio of 15; for water: and sequentially carrying out Sephadex LH 20 gel chromatographic separation and reversed-phase semi-preparative high performance liquid chromatographic separation on the elution components with the methanol volume ratio of 20.

For petroleum ether: ethyl acetate 3:1 eluate fraction fr.7 was chromatographed on MCI column and purified by water: gradient elution is carried out on methanol in a volume ratio of 50 to 0; for water: sequentially carrying out Sephadex LH 20 gel chromatographic separation and reversed-phase semi-preparative high performance liquid chromatographic separation on the elution components with the methanol volume ratio of 20; for water: sequentially carrying out Sephadex LH 20 gel chromatographic separation and reversed-phase semi-preparative high performance liquid chromatographic separation on the elution components with the methanol volume ratio of 20;

in the process of separating the compounds 1 to 4, methanol is adopted for elution in the step of Sephadex LH 20 gel chromatography separation; separating compound 1, compound 3 and compound 4 under MeOH-H conditions in the reverse phase semi-preparative HPLC ₂ O,90:10,v/v；flow rate,3mL/min,t _R =16.0min; separating compound 2 under MeOH-H in a reverse phase semi-preparative HPLC separation step ₂ O,75:25,v/v；flow rate,3mL/min,t _R ＝22.6min。

10. The method according to claim 9, wherein the methanol is 90% methanol; the extraction times are 3-5 times.

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