1. The intermolecular adduct of different terpenes in lancangchan fir shown as the following is characterized in that the intermolecular adduct of rearranged lanostan triterpenes and different terpenes is formed by Diels-Alder ([4+ 2]]-cycloaddition) reaction to give compounds of the new structure: skeleton (C) of rearranged lanospermane triterpenoid and abietane diterpenoid₃₀-C₂₀Formula 1 to formula 4) or a skeleton (C) which is combined with a laurane-type monoterpene₃₀-C₁₀The formula 5 to the formula 6),

2. the process for preparing intermolecular adducts of different terpenes in lancangchua, as claimed in claim 1, which comprises the steps of:

drying branches and leaves of Picea crassa (Pseudotsugaforrestii Craib) collected from the principle of Yunnan, pulverizing, extracting with 90% methanol at room temperature for 7 times, concentrating the extractive solution, adding water, suspending, and extracting with petroleum ether, ethyl acetate and n-butanol respectively; concentrating the ethyl acetate extract under reduced pressure, and separating by silica gel, microporous resin (MCI), Sephadex LH-20 gel and reverse phase semi-preparative high performance liquid chromatography (semi-preparative RP-HPLC) to obtain compounds shown in formulas 1-6.

3. Use of intermolecular adducts of different terpenoids in the lancangchua according to claim 1 for the preparation of drugs for the prevention, delay or treatment of glycolipid metabolic disorders and complications thereof, as well as other ACL mediated diseases.

4. The use as claimed in claim 3, wherein the disease associated with disturbance of glycolipid metabolism comprises: hyperlipidemia, nonalcoholic steatohepatitis, type 2 diabetes, and obesity.

5. A pharmaceutical composition comprising a therapeutically effective amount of one or more compounds selected from the compounds of claim 1 as an active ingredient, together with pharmaceutically acceptable excipients.

6. Use of a pharmaceutical composition according to claim 5 for the preparation of an ATP-citrate lyase (ACL) inhibitor.

Background

The prior art discloses that endogenous cholesterol synthesis is a key source of systemic circulating plasma cholesterol, and acetyl-CoA produced by the mitochondrial tricarboxylic acid cycle (TCA cycle) is a source substrate for endogenous cholesterol synthesis. Generally, acetyl-coa produced from the tricarboxylic acid cycle cannot directly permeate the mitochondrial membrane into the cytosol, but citrate is produced in mitochondria by the action of citrate synthase and passes through citrate transporters on the mitochondrial membrane into the cytoplasm, and is then hydrolyzed by ATP-citrate lyase (ACL) distributed on the endoplasmic reticulum into acetyl-coa and oxaloacetate (Beigneux et al, j.biol.chem.2004,279, 9557-9564); acetyl coenzyme A is taken as a substrate for the de novo synthesis of cholesterol, and the cholesterol is generated through 33 steps of reaction. Studies have disclosed that human ACL enzyme proteins are homotetramers composed of 4 identical 120kDa subunits, each polypeptide chain containing 1101 amino residues (Singh et al, J.biol.chem.1976,251, 5254-5250); it can be seen that ACL is closely related to the synthesis of fatty acids and cholesterol, and the change of expression thereof is closely related to hyperlipidemia (hyperlipidemia) and Cardiovascular Diseases (CVDs) caused thereby (burkey et al, curr. opin. lipiodol.2017, 28, 193-200; Pinkosky et al, Trends mol. med.2017,23, 1047-1063); meanwhile, abnormal metabolism of triglyceride also increases the risk factors of Nonalcoholic Fatty Liver (NAFLD) and Type II Diabetes Mellitus (T2 DM), which are related diseases of glycolipid metabolic disorders, so ACL can also be used as a potential treatment target of Nonalcoholic Fatty Liver and Type II Diabetes Mellitus (Armstrong et al, 2014,59, 1174. quadrature 1197; Cohen et al, Science 2011,332, 1519. quadrature 1523). It has also been shown that ACLs are closely associated with the development of Cancer, and that increased lipid synthesis provides the necessary lipids for cell growth and division, and is one of the important hallmarks of carcinogenesis, as well as an early event in tumorigenesis (Migita et al, Cancer Res.2008,68, 8547-) -8554. ACL is a major source of acetyl-coa (an important component of de novo fatty acid synthesis), and inhibition of its activity can significantly inhibit proliferation and induce apoptosis in tumor cells; accordingly, ACLs have been extensively studied in recent years as potential targets for Cancer (Granchi et al, Eur.J.Med.chem.2018,157, 1276-1291; Zaidi et al, Cancer Res.2012,72: 3709-.

In conclusion, ACL has become a hot spot for innovative drug research on glycolipid metabolic disorder diseases in recent years as a new drug target. With the wide application of high-throughput screening technology, a large variety of ACL small-molecule inhibitors have been discovered in succession, but no ACL inhibitors have been successfully marketed. Currently only the ACL small molecule inhibitor Bempedoic acid developed by american corporation Therapeutics (ESPR) enters the phase III clinical trial phase for the treatment of hypercholesterolemia and atherogenic cardiovascular disease; other ACL inhibitors have been limited in their research because of their low cell penetration, low affinity for ACL, and poor specificity. The search for the micromolecular ACL inhibitor which is efficient and high in selectivity and has good pharmacokinetic properties has important significance, and has wide application prospect in treatment of cardiovascular diseases, cancers and other diseases.

Natural products have a unique and important position for guidance and reference of new drug development, and are an important source for new drug discovery (Newman et al, J.Nat.Prod.2016,79: 629-661; 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 medicine with a single target point is often poor in curative effect, and the traditional Chinese medicine/botanical medicine and the components thereof have the characteristics of multi-target point and multi-path effects and have unique advantages in the process of treating complex diseases. Therefore, the research and development of novel, efficient and low-toxic-side-effect ACL inhibitors from chemical components of natural sources (particularly plant sources) has important research value. Natural products with unique sources and structural features may be a precursor in the intense competition in the current field of new drug development for diseases of dyslipidemic lipid metabolism.

The pseudocanna yellow fir (pseudotuotsuga forrestii Craib) belongs to the genus pseudonanus (Pinaceae) and is a special seed plant in China, namely evergreen arbor, distributed in the middle upper part to the middle part of the middle mountain in the middle south of the transected mountain range, recorded by the Chinese plant Redbud-rare endangered plant in 1992 and listed as a gradually dangerous seed. At present, the chemical components of the plant have not been reported, and if a few lancangchua plant samples can be protectively collected to carry out systematic chemical component research, scientific understanding can be actively promoted, and the rare plant resource can be actively protected and utilized to serve human beings.

Based on the current situation of the prior art, the inventor intends to provide intermolecular adducts of different terpenoids in the lancang yellow fir, and a preparation method and pharmaceutical application thereof.

Disclosure of Invention

The invention aims to provide intermolecular adducts of different terpenoids in the lancang-yellow fir, a preparation method and pharmaceutical application thereof based on the current situation of the prior art.

The invention separates a series of Diels-Alder adducts (with structures shown in formulas 1-6) among different terpenoids molecules with novel structures from methanol extracts of branches and leaves of the lancang yellow fir, and pharmacological tests show that the compounds have obvious ACL inhibitory activity, and the compounds can be used for preparing ATP-citric acid lyase inhibitors and treating hyperlipidemia, non-alcoholic steatohepatitis, type II diabetes, cancer and other ACL mediated diseases.

The invention provides a preparation method of Diels-Alder adducts among different terpenes,

the invention separates and identifies 6 rearranged lanospirane triterpenes from the plant of the genus Taxus of the family Pinaceae (Pseudotsuga forrestii Craib) and the different terpenes between the molecules are separated and identified through Diels-Alder [4+2] Alder]The compound with new skeleton produced by cycloaddition reaction has the structural feature of the combination of rearranged lanosporane type triterpene and abietane diterpene (C)₃₀-C₂₀) Or with laurane-type monoterpene (C)₃₀-C₁₀)。

More particularly, the present invention provides a Diels-Alder type adduct selected from:

the compounds of the present invention can be isolated and purified from plants, or synthesized by chemical methods well known to those skilled in the art.

The invention provides a preparation method of the Diels-Alder adduct, which comprises the following steps:

the compound is prepared from branches and leaves of lancang yellow fir (Pseudotsuga forrestii Craib) by an extraction and separation method, and comprises the following steps: soaking and extracting the dried and crushed branches and leaves of the lancang yellow fir with methanol/water solution at room temperature, concentrating the extracting solution under reduced pressure, recovering the solvent, and mixing to obtain an extract; dispersing the extract with water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol to obtain petroleum ether fraction, ethyl acetate fraction, n-butanol fraction and water soluble fraction; the ethyl acetate fraction is repeatedly separated and purified by silica gel, microporous resin (MCI), Sephadex LH-20 and reversed-phase semi-preparative high performance liquid chromatography (semi-preparative RP-HPLC) to obtain compounds 1-6.

In the above method, the methanol/water solution is a methanol-water solution with a concentration of 70% or more, preferably 90% methanol-water solution; extracting at room temperature for more than 12 hours; the extraction times are one or more, preferably 3 or more.

The Diels-Alder adduct of the invention shows stronger inhibition effect in ACL inhibition activity test, and the activity is equivalent to that of positive control BMS 303141.

The invention further provides the application of the compound or the composition in preparing a medicament for treating ACL-mediated diseases. Based on the advantages of the compounds in novel chemical structures and biological activity, the compounds have good development prospects and are expected to be therapeutic drugs or lead compounds with novel structures for ACL-mediated diseases.

The invention provides a pharmaceutical composition, which adopts one or more of the Diels-Alder adducts as raw materials, contains one or more of the compounds as active ingredients in effective treatment amount, and can further comprise pharmaceutically acceptable auxiliary materials, such as carriers, excipients, adjuvants or diluents. The medicine composition can be used for preparing medicines for preventing, delaying or treating glycolipid disorder related diseases mediated by ACL, particularly hyperlipidemia and related cardiovascular diseases, or used as lead compounds of the medicines.

The invention has the following remarkable advantages:

the compound is a novel compound separated from the nature, and is a Diels-Alder adduct (triterpene and diterpene or triterpene and monoterpene are combined) with a unique [4+2] -type cyclization structure; meanwhile, the compound has obvious ACL inhibition activity; has important application prospect for diseases related to glycolipid metabolic disturbance, such as hyperlipidemia, non-alcoholic steatohepatitis, type II diabetes and the like which are highly developed in modern people.

Detailed Description

The preparation steps and pharmacological experimental procedures of the compounds of the present invention are further illustrated by the following specific examples. It is to be understood that the following examples are illustrative only and are not intended to limit the scope of the invention, which is to be given numerous changes and modifications by one skilled in the art.

In the invention, branches and leaves of lancang yellow fir (Pseudotsuga forrestii Craib) are collected from Yunnan Dali, dried in the shade and crushed 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; the instrument used for the single crystal diffraction experiment was a Bruker D8 Venture diffractometer (gallium target); 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 Yangtze river friend silica gel development company Limited, and the specification is GF254/0.25 mm; MCI gel CHP20P is manufactured by Mitsubishi corporation of Japan, and has a specification of 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 Star high purity solvents, Inc.; the deuterated reagent is produced by Sigma-Aldrich.

Example 1 preparation of the Compounds of the invention

(1) Drying and pulverizing branches and leaves of Arcanna yellow fir (first batch: collected in 10 months in 2017, dry weight 13kg), extracting with 90% methanol (6L) solution at room temperature for 7 times, each for 24 hr;

(2) mixing the extractive solutions obtained in step (1), concentrating under reduced pressure to remove methanol to obtain extract 2.46kg (semi-dry), and dispersively soaking with 2L waterExtracting the paste by using petroleum ether, ethyl acetate and n-butanol which are equal in volume sequentially for three times, and concentrating the extraction liquid under reduced pressure to obtain 4 components of a petroleum ether part, an ethyl acetate part, an n-butanol part and water respectively, wherein 10 components are obtained after the crude extract (409g) of the ethyl acetate part is separated by silica gel column chromatography (gradient washing by petroleum ether-ethyl acetate, 30:1 → 0:1, v/v), and Fr.1-Fr.10; (3) separating the fraction Fr.5 of step (2) by MCI column chromatography with MeOH-H₂Gradient elution with O (60:40 → 70:30 → 80:20 → 90:10 → 100:0, v/v) gives 10 subfractions Fr.5A-Fr.5J, which are further purified by reverse phase HPLC after silica gel chromatography (XBridge column; mobile phase: MeOH/H)₂O,92:8, v/v; flow rate: 3 mL/min; column temperature 25 deg.C, detection at 205nm wavelength), collecting eluate for 13.0-15.0min, concentrating under reduced pressure, collecting eluate to obtain compound 6(3.5mg), eluting subfraction Fr.5I with pure methanol by gel column chromatography (SephadexLH-20), and further purifying by reverse phase HPLC (Xbridge column, 5 μm,250 × 10 mm; mobile phase: MeCN/H₂O,90:10, v/v; flow rate: 3 mL/min; column temperature 25 deg.C, detecting at 205nm wavelength), collecting eluate for 18.0-22.0min, concentrating under reduced pressure, and collecting eluate to obtain compound 5(3.9 mg);

(4) two batches of branches and leaves of blue canna yellow fir (second batch: collected 6 months in 2019, dry weight 15kg) collected from Yunan theory were dried and pulverized, and extracted with 90% methanol (6L) solution at room temperature for 7 times, each for 24 hours. Combining the extracting solutions, concentrating under reduced pressure to remove methanol to obtain 3.9kg (semi-dry) of extract, dispersing the extract with 3L of water, sequentially extracting for three times by using petroleum ether, ethyl acetate and n-butanol which are equal in volume respectively, and concentrating under reduced pressure to obtain 4 components of a petroleum ether part, an ethyl acetate part, an n-butanol part and water respectively, wherein 8 components are obtained after the crude extract of the ethyl acetate part is subjected to silica gel column chromatography (gradient washing with petroleum ether-ethyl acetate, 30:1 → 0:1, v/v) separation, and Fr.1-Fr.8;

(5) separating by HPLC-MS guidance, selecting component Fr.3(44g) in step (4) for separation by MCI column chromatography, MeOH-H₂Gradient elution with O (70:30 → 80:20 → 90:10 → 100:0, v/v) gives 7 subfractions Fr.3A to Fr.3G. Subfraction Fr.3F (2.7g) was eluted with pure methanol by gel column chromatography (Sephadex LH-20) and further purified by reverse phase HPLC (mobile phase: MeOH/H)₂O96:4, v/v; flow rate: 3 mL/min; column temperature 25 deg.C, detecting at 205nm wavelength), collecting eluate of 17.0-19.0min and 30.0-33.0min, respectively, concentrating under reduced pressure, and collecting eluate to obtain compound 1(100.0mg) and compound 3(100.0 mg);

(6) the fraction Fr.4(35g) from step (4) was isolated by MCI column chromatography in MeOH-H₂Gradient elution with O (70:30 → 80:20 → 90:10 → 100:0, v/v) gives 5 subfractions Fr.4A to Fr.4E; separation was guided by HPLC-MS and subfraction Fr.4C (6.6g) was selected for further purification by reverse phase HPLC (mobile phase: MeOH/H)₂O95:5, v/v; flow rate: 3 mL/min; column temperature 25 deg.C, detecting at 205nm wavelength), collecting eluate of 26.0-28.0min and 29.0-31.0min, respectively, concentrating under reduced pressure, and collecting eluate to obtain compound 2(8.0mg) and compound 4(10.0 mg).

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

a sheet-shaped single crystal, which is,+55.4(c0.27,MeOH)；UV(MeOH)λ_max(logε)206(3.57)nm；IR(KBr)v_max2952,2870,2649,1695,1469,1387,1273,1116,935,882,761cm^-1；¹H-NMR(CDCl₃,600MHz):δ1.62(1H,m,H-1a),1.96(1H,m,H-1b),2.41(1H,m,H-2a),2.65(1H,m,H-2b),1.57(1H,m,H-5),1.63(1H,m,H-6a),1.44(1H,m,H-6b),1.93(2H,m,H-7),2.13(1H,m,H-11a),2.12(1H,m,H-11b),1.40(2H,m,H-12),2.39(1H,m,H-15a),2.27(1H,m,H-15b),1.92(1H,m,H-16a),1.46(1H,m,H-16b),0.77(3H,s,H-18),1.11(3H,s,H-19),2.38(1H,m,H-20),0.74(3H,d,J＝6.5Hz,H-21),2.44(1H,m,H-22a),2.05(1H,m,H-22b),2.79(1H,br s,H-24),1.17(3H,s,H-26),1.09(6H,br s,H-29,30),4.72(1H,br s,H-30a),4.46(1H,br s,H-30b),0.76(1H,m,H-1'a),1.45(1H,m,H-1'b),1.56(1H,m,H-2'a),1.46(1H,m,H-2'b),1.61(1H,m,H-3'a),1.54(1H,m,H-3'b),1.69(1H,m,H-5'),1.58(1H,m,H-6'a),1.33(1H,m,H-6'b),1.59(2H,m,H-7'),1.61(1H,m,H-9'),2.12(1H,m,H-11'a),1.04(1H,m,H-11'b),2.76(1H,br s,H-12'),5.36(1H,s,H-14'),2.34(1H,m,H-15'),1.04(6H,br d,J＝6.5Hz,H-16’,17’),1.16(3H,s,H-18’),0.62(3H,s,H-20')；¹³C-NMR(CDCl₃,150MHz):δ35.8(C-1),34.6(C-2),216.7(C-3),47.4(C-4),51.4(C-5),20.9(C-6),26.0(C-7),136.1(C-8),147.9(C-9),35.9(C-10),26.8(C-11),30.9(C-12),67.9(C-13),154.3(C-14),27.0(C-15),38.1(C-16),48.1(C-17),18.3(C-18),18.8(C-19),36.3(C-20),15.4(C-21),44.8(C-22),213.8(C-23),62.2(C-24),49.8(C-25),18.5(C-26),184.2(C-27),21.2(C-28),26.3(C-29),103.7(C-30),38.2(C-1'),17.0(C-2'),37.3(C-3'),46.9(C-4'),48.4(C-5'),22.0(C-6'),30.9(C-7'),45.8(C-8'),49.0(C-9'),37.5(C-10'),20.3(C-11'),34.7(C-12'),149.4(C-13'),125.2(C-14'),32.3(C-15'),20.1(C-16'),20.2(C-17'),16.0(C-18'),186.6(C-19'),17.3(C-20')；ESIMS m/z 769[M+H]⁺；HRESIMS m/z 769.5392[M+H]⁺(calcd for C₅₀H₇₃O₆,769.5402,Δ＝+1.02ppm).

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

a white amorphous powder of a crystalline substance,+26.4(c0.35,MeOH)；UV(MeOH)λ_max(logε)206(3.67)nm；IR(KBr)v_max2950,2927,2870,1688,1464,1379,1277,1108,881,759cm^-1；¹H-NMR(CDCl₃,600MHz):δ1.71(1H,m,H-1a),1.89(1H,m,H-1b),2.49(1H,m,H-2a),2.61(1H,m,H-2b),1.66(1H,m,H-5),1.74(1H,m,H-6a),1.54(1H,m,H-6b),1.94(2H,m,H-7),2.16(1H,m,H-11a),2.05(1H,m,H-11b),1.41(1H,m,H-12a),1.36(1H,m,H-12b),2.38(1H,m,H-15a),2.30(1H,m,H-15b),1.83(1H,m,H-16a),1.47(1H,m,H-16b),0.84(3H,s,H-18),1.08(3H,s,H-19),2.37(1H,m,H-20),0.80(3H,d,J＝6.5Hz,H-21),2.72(1H,d,J＝17.8Hz,H-22a),2.06(1H,m,H-22b),3.35(1H,br s,H-24),0.83(3H,s,H-26),1.11(3H,s,H-29),1.08(3H,s,H-30),4.73(1H,br s,H-30a),4.48(1H,br s,H-30b),0.83(1H,m,H-1'a),1.39(1H,m,H-1'b),1.60(1H,m,H-2'a),1.45(1H,m,H-2'b),1.48(1H,m,H-3'a),1.45(1H,m,H-3'b),1.48(1H,m,H-5'),2.04(1H,m,H-6'a),1.38(1H,m,H-6'b),2.04(1H,m,H-7'a),1.38(1H,m,H-7'b),2.11(1H,m,H-9'),1.52(1H,m,H-11'a),1.08(1H,m,H-11'b),2.54(1H,m,H-12'),5.37(1H,s,H-14'),2.67(1H,m,H-15'),1.13(3H,d,J＝6.5Hz,H-16’),1.05(3H,d,J＝6.5Hz,H-17’),1.16(3H,s,H-18’),0.63(3H,s,H-20')；¹³C-NMR(CDCl₃,150MHz):δ35.9(C-1),34.4(C-2),218.1(C-3),47.2(C-4),51.0(C-5),21.1(C-6),26.4(C-7),136.6(C-8),147.5(C-9),35.9(C-10),26.7(C-11),31.6(C-12),68.0(C-13),155.3(C-14),27.2(C-15),38.0(C-16),48.8(C-17),19.2(C-18),18.5(C-19),33.4(C-20),16.0(C-21),44.6(C-22),210.9(C-23),59.0(C-24),55.8(C-25),16.5(C-26),183.2(C-27),21.1(C-28),26.5(C-29),103.9(C-30),38.0(C-1'),16.9(C-2'),35.9(C-3'),46.6(C-4'),49.3(C-5'),21.5(C-6'),32.0(C-7'),45.5(C-8'),47.6(C-9'),35.5(C-10'),29.5(C-11'),36.5(C-12'),148.2(C-13'),124.7(C-14'),32.2(C-15'),19.9(C-16'),21.1(C-17'),16.9(C-18'),185.2(C-19'),16.3(C-20')；ESIMS m/z 769[M+H]⁺；HRESIMS m/z 769.5406[M+H]⁺(calcd for C₅₀H₇₃O₆,769.5402,Δ＝+0.6ppm).

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

a white amorphous powder of a crystalline substance,+58.9(c0.27,MeOH)；UV(MeOH)λ_max(logε)206(3.67)nm；IR(KBr)v_max3070,2955,2937,2870,2651,1698,1462,1379,1276,1197,759cm^-1；¹H-NMR(CDCl₃,600MHz):δ1.71(1H,m,H-1a),1.94(1H,m,H-1b),2.49(1H,m,H-2a),2.67(1H,m,H-2b),1.68(1H,m,H-5),1.76(1H,m,H-6a),1.68(1H,m,H-6b),2.26(2H,m,H-7),2.05(2H,m,H-11),1.67(1H,m,H-12a),1.45(1H,m,H-12b),2.40(1H,m,H-15a),2.21(1H,m,H-15b),1.52(1H,m,H-16a),1.44(1H,m,H-16b),0.91(3H,s,H-18),1.12(3H,s,H-19),2.38(1H,m,H-20),0.87(3H,d,J＝6.5Hz,H-21),2.66(1H,m,H-22a),2.24(1H,m,H-22b),3.16(1H,d,J＝17.6Hz,H-24a),2.63(1H,d,J＝17.6Hz,H-24b),2.34(1H,m,H-26a),0.95(1H,m,H-26b),1.14(3H,s,H-28),1.09(3H,s,H-29),4.77(1H,br s,H-30a),4.53(1H,br s,H-30b),0.84(1H,m,H-1'a),1.43(1H,m,H-1'b),1.55(1H,m,H-2'a),1.46(1H,m,H-2'b),1.64(2H,m,H-3'),1.54(1H,m,H-5'),1.49(1H,m,H-6'a),1.29(1H,m,H-6'b),2.06(2H,m,H-7'),1.68(1H,m,H-9'),1.14(1H,m,H-11'a),1.33(1H,m,H-11'b),2.49(1H,m,H-12'),5.21(1H,s,H-14'),2.36(1H,m,H-15'),1.03(3H,d,J＝6.5Hz,H-16'),1.04(3H,d,J＝6.5Hz,H-17’),1.12(3H,s,H-18’),0.62(3H,s,H-20')；¹³C-NMR(CDCl₃,150MHz):δ35.6(C-1),34.5(C-2),217.4(C-3),47.3(C-4),51.0(C-5),20.7(C-6),26.7(C-7),136.2(C-8),147.9(C-9),36.0(C-10),26.3(C-11),32.1(C-12),68.2(C-13),155.7(C-14),27.2(C-15),37.8(C-16),49.4(C-17),18.7(C-18),19.0(C-19),34.0(C-20),16.3(C-21),45.2(C-22),209.1(C-23),50.7(C-24),50.8(C-25),40.0(C-26),181.4(C-27),21.0(C-28),26.5(C-29),104.1(C-30),38.4(C-1'),17.1(C-2'),36.9(C-3'),47.0(C-4'),49.1(C-5'),22.0(C-6'),32.8(C-7'),42.9(C-8'),49.3(C-9'),38.0(C-10'),27.5(C-11'),32.7(C-12'),149.4(C-13'),123.5(C-14'),32.7(C-15'),20.2(C-16'),20.3(C-17'),16.3(C-18'),185.6(C-19'),16.7(C-20')；ESIMS m/z 769[M+H]⁺；HRESIMS m/z 769.5403[M+H]⁺(calcd for C₅₀H₇₃O₆,769.5402,Δ＝-0.1ppm).

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

acicular single crystal (CHCl)₃),+13.9(c0.336,MeOH)；UV(MeOH)λ_max(logε)206(3.67)nm；IR(KBr)v_max2960,2922,2870,1693,1462,1382,1270,1110,928,878,754,686,569cm^-1；¹H-NMR(CDCl₃,600MHz):δ1.70(1H,m,H-1a),1.92(1H,m,H-1b),2.47(1H,m,H-2a),2.61(1H,m,H-2b),1.65(1H,m,H-5),1.68(1H,m,H-6a),1.56(1H,m,H-6b),2.11(1H,m,H-7a),1.95(1H,m,H-7b),2.14(1H,m,H-11a),1.71(1H,m,H-11b),2.04(1H,m,H-12a),1.44(1H,m,H-12b),2.42(1H,m,H-15a),2.34(1H,m,H-15b),1.53(1H,m,H-16a),1.44(1H,m,H-16b),0.83(3H,s,H-18),1.05(3H,s,H-19),2.31(1H,m,H-20),0.77(3H,d,J＝6.5Hz,H-21),2.55(1H,dd,J＝14.0,2.6Hz,H-22a),2.02(1H,m,H-22b),3.16(1H,d,J＝18.5Hz,H-24a),2.23(1H,d,J＝18.5Hz,H-24b),2.71(1H,dd,J＝17.6,14.0,3.4Hz,H-26a),0.82(1H,m,H-26b),1.12(3H,s,H-28),1.09(3H,s,H-29),4.75(1H,br s,H-30a),4.50(1H,br s,H-30b),0.83(1H,m,H-1'a),1.40(1H,m,H-1'b),1.51(1H,m,H-2'a),1.39(1H,m,H-2'b),1.64(2H,m,H-3'),1.57(1H,m,H-5'),1.48(1H,m,H-6'a),1.36(1H,m,H-6'b),1.81(1H,m,H-7'a),1.77(1H,m,H-7'b),1.62(1H,m,H-9'),1.10(1H,m,H-11'a),1.71(1H,m,H-11'b),2.46(1H,m,H-12'),5.31(1H,s,H-14'),2.31(1H,m,H-15'),1.02(3H,d,J＝6.5Hz,H-16’),1.00(3H,d,J＝6.5Hz,H-17’),1.15(3H,s,H-18’),0.60(3H,s,H-20')；¹³C-NMR(CDCl₃,150MHz):δ35.6(C-1),34.5(C-2),218.0(C-3),47.2(C-4),51.0(C-5),20.6(C-6),26.7(C-7),136.1(C-8),147.8(C-9),35.9(C-10),26.3(C-11),32.8(C-12),68.1(C-13),155.8(C-14),27.3(C-15),37.8(C-16),49.3(C-17),18.6(C-18),18.5(C-19),35.2(C-20),16.1(C-21),44.9(C-22),210.4(C-23),51.5(C-24),53.3(C-25),39.4(C-26),180.5(C-27),21.1(C-28),26.6(C-29),104.1(C-30),38.0(C-1'),17.1(C-2'),37.2(C-3'),46.5(C-4'),48.6(C-5'),22.0(C-6'),31.2(C-7'),43.3(C-8'),49.9(C-9'),37.8(C-10'),26.6(C-11'),33.1(C-12'),150.5(C-13'),124.3(C-14'),32.6(C-15'),20.5(C-16'),20.3(C-17'),16.7(C-18'),184.3(C-19'),16.6(C-20')；ESIMS m/z 769[M+H]⁺；HRESIMS m/z 769.5415[M+H]⁺(calcd for C₅₀H₇₃O₆,769.5402,Δ＝+1.7ppm)..

Compound 5, with the following nuclear magnetic and physicochemical data:

a white amorphous powder of a crystalline substance,+15.5(c0.39,MeOH)；UV(MeOH)λ_max(logε)206(3.48)nm；IR(KBr)v_max3369,2967,2935,2848,1706,1462,1382,1250,1122,1030,891cm^-1；¹H-NMR(CDCl₃,600MHz):δ1.70(1H,m,H-1a),1.91(1H,m,H-1b),2.49(1H,ddd,J＝16.2,7.5,3.4Hz,H-2a),2.58(1H,ddd,J＝16.2,10.5,7.5Hz,H-2b),1.69(1H,br d,overlapped,H-5),1.76(1H,dd,J＝13.0,6.0Hz,H-6a),1.58(1H,m,H-6b),2.00(1H,m,H-7a),2.08(1H,m,H-7b),2.08(1H,m,H-11a),2.00(1H,m,H-11b),1.91(1H,dd,J＝7.5,3.4Hz,H-12a),1.36(1H,dd,J＝12.2,7.5Hz,H-12b),2.40(1H,m,H-15a),2.34(1H,m,H-15b),1.56(1H,m,H-16a),1.49(1H,m,H-16b),0.90(3H,s,H-18),1.06(3H,s,H-19),2.44(1H,m,H-20),0.81(3H,d,J＝6.5Hz,H-21),2.65(1H,d,J＝16.0Hz,H-22a),2.38(1H,m,H-22b),3.04(1H,dd,J＝12.1,5.3Hz,H-24),1.23(3H,s,H-26),1.13(3H,s,H-28),1.07(3H,s,H-29),4.75(1H,br s,H-30a),4.49(1H,br s,H-30b),2.25(1H,m,H-1'a),1.92(1H,m,H-1'b),5.39(1H,br s,H-3'),2.38(1H,m,H-4'a),2.02(1H,m,H-4'b),2.09(1H,m,H-5'a),2.01(1H,m,H-5'b),2.26(2H,m,H-6'),5.04(1H,t,J＝6.7Hz,H-7'),1.68(3H,s,H-9'),1.60(3H,s,H-10')；¹³C-NMR(CDCl₃,150MHz):δ35.5(C-1),34.4(C-2),217.3(C-3),47.1(C-4),51.0(C-5),20.8(C-6),26.2(C-7),136.5(C-8),147.7(C-9),35.8(C-10),26.2(C-11),31.7(C-12),68.0(C-13),154.9(C-14),27.1(C-15),38.0(C-16),48.6(C-17),18.9(C-18,19),33.5(C-20),15.8(C-21),43.4(C-22),211.3(C-23),52.4(C-24),42.3(C-25),182.4(C-26),15.7(C-27),26.7(C-28),20.9(C-29),103.8(C-30),28.6(C-1'),135.1(C-2'),118.8(C-3'),37.6(C-4'),37.1(C-5'),26.7(C-6'),123.5(C-7'),132.0(C-8'),25.7(C-9'),17.7(C-10')；ESIMS m/z 625[M+Na]⁺；HRESIMS m/z 625.4205[M+Na]⁺(calcd for C₄₀H₅₈O₄Na,625.4227,Δ＝-3.5ppm).

compound 6, with the following nuclear magnetic and physicochemical data:

a white amorphous powder of a crystalline substance,+9.1(c0.15,MeOH)；UV(MeOH)λ_max(logε)206(3.84)nm；IR(KBr)v_max3449,2932,2878,1716,1462,1387,1295,1182,1078,1035cm^-1；¹H-NMR(CDCl₃,600MHz):δ1.28(1H,ddd,J＝14.8,14.6,3.6Hz,,H-1a),1.67(1H,m,H-1b),1.73(1H,m,H-2a),1.62(1H,m,H-2b),3.29(1H,dd,J＝12.1,4.4Hz,H-3),1.10(1H,br d,J＝11.4Hz,H-5),1.82(1H,m,H-6a),1.50(1H,m,H-6b),1.91(1H,m,H-7a),2.19(1H,m,H-7b),2.08(1H,m,H-11a),1.99(1H,m,H-11b),1.87(1H,m,H-12a),1.36(1H,m,H-12b),2.40(1H,m,H-15a),2.30(1H,m,H-15b),1.55(1H,m,H-16a),1.46(1H,m,H-16b),0.87(3H,s,H-18),0.96(3H,s,H-19),2.41(1H,m,H-20),0.79(3H,d,J＝5.8Hz,H-21),2.67(1H,br d,J＝15.2Hz,H-22a),2.38(1H,m,H-22b),3.03(1H,dd,J＝11.7,4.4Hz,H-24),1.22(3H,s,H-26),1.03(3H,s,H-28),0.82(3H,s,H-29),4.73(1H,br s,H-30a),4.49(1H,br s,H-30b),2.27(1H,dd,overlapped,H-1'a),1.92(1H,dd,overlapped,H-1'b),5.38(1H,br s,H-3'),2.39(1H,br d,overlapped,,H-4'a),2.04(1H,br d,overlapped,,H-4'b),2.08(1H,m,H-5'a),2.00(1H,m,H-5'b),2.24(2H,m,H-6'),5.06(1H,t,J＝6.8Hz,H-7'),1.68(3H,s,H-9'),1.60(3H,s,H-10')；¹³C-NMR(CDCl₃,150MHz):δ35.4(C-1),27.7(C-2),79.0(C-3),38.7(C-4),50.9(C-5),19.4(C-6),27.0(C-7),135.3(C-8),149.6(C-9),36.1(C-10),26.2(C-11),31.7(C-12),68.0(C-13),155.0(C-14),27.1(C-15),38.0(C-16),48.5(C-17),18.8(C-18),19.5(C-18),33.5(C-20),15.8(C-21),43.4(C-22),211.4(C-23),52.4(C-24),42.3(C-25),182.4(C-26),15.7(C-27),28.0(C-28),15.3(C-29),103.7(C-30),28.5(C-1'),135.2(C-2'),118.7(C-3'),37.6(C-4'),37.1(C-5'),26.3(C-6'),123.6(C-7'),131.9(C-8'),25.7(C-9'),17.7(C-10')；ESIMS m/z 627[M+Na]⁺；HRESIMS m/z 627.4356[M+Na]⁺(calcd for C₄₀H₆₀O₄Na,627.4384,Δ＝-4.5ppm).。

example 2: ACL inhibitory Activity test of Compounds prepared according to the present invention

The experimental method comprises the following steps: in the experiment, the ACL can catalyze and convert citric acid into acetyl coenzyme A, so that a precursor molecule of fatty acid synthesis, namely malonyl coenzyme A, is generated, and the reaction is accompanied with the consumption of ATP, so that ADP-Glo and a kinase detection kit can be used for detecting the change of ATP, and indirectly reacting a compound to inhibit the activity of the ACL enzyme; in the embodiment, the percentage inhibition rate of the activity of the ACL enzyme is investigated when the concentration of the compound selected by the primary screening is 20 mug/ml, and the test result shows that the compounds all have the enzyme activity inhibition effect;

further determination of IC₅₀The value: the samples were dissolved in DMSO just before use to make appropriate concentrations, 3-fold diluted, 7 dilutions, triplicate wells, 2. mu.L of sample solution was added to a standard viability assay system (40mM Tris, pH8.0,10mM MgCl)₂5mM DTT, ATP, CoA, sodium citrate and ACL), and incubating at 37 ℃ for 30 min; then, adding 25 mu of LADP-Glo reagent into the system, and incubating for 30min at room temperature to terminate the reaction and consume the rest ATP; adding a kinase detection reagent, incubating for 30min, reading a fluorescence signal by EnVision, and taking the slope of the first-order reaction of a kinetic curve as an activity index of the enzyme; the relative activity is plotted against the concentration of the compound, as shown by the formula v/v₀＝100/(1+b*[I]/IC₅₀) Fitting to obtain IC₅₀Value, experiment was repeated three timesAnd taking the average value of the three times; IC of positive control BMS303141₅₀The value was 0.20. + -. 0.04. mu.M.

Table 1 shows the data on the ACL inhibitory activity of Diels-Alder type terpene adducts in Lancang Yew.

Table 1.

The results are shown in Table 1 for ACL inhibitory activity data (IC) of Diels-Alder terpene adducts₅₀Value), all 6 compounds show significant inhibitory activity to ACL, which indicates that the compound of the invention can be used for preparing medicines for treating diseases related to glycolipid metabolic disturbance or used as a lead compound of the medicines.