CN115745790A - Eight-ring five-terpenoid compound in lancang yellow fir and preparation and application thereof - Google Patents
Eight-ring five-terpenoid compound in lancang yellow fir and preparation and application thereof Download PDFInfo
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Abstract
The application discloses aThe octacyclic five-terpene compound in the lantake-cangchamia fulvidraco has a structural formula shown in one of the following structures:
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to an octacyclo-pentaterpenoid compound in lantake yellow fir as well as preparation and application thereof.
Background
Abnormal elevation of fatty acid synthesis (fayn), impaired fatty acid hyperoxidation (FAOxn), or disorder of fatty acid metabolism caused by both of them due to abnormal fatty acid metabolism are markers 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 Ther Targets,2005, 9. Altered fatty acid metabolism is also a hallmark of cancer and leads to abnormal and sustained cell proliferation of malignant cells (Baenke F et al, dis Model Mech,2013,6 1353-1363, mounier C et al, int J Oncol,2014, 45. Therefore, inhibition of faayn and/or stimulation of FAOxn may have a beneficial effect on these diseases.
There are two known classes of acetyl-coa carboxylases, ACC1 and ACC2, encoded by different genes and sharing about 70% of the amino acids in rodent and human body organs. 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 J et al, proc.nat. Acad.sci.usa,2003,100, 7515-7520 abu-Elheiga L et al, J Biol Chem,1997, 10669-10677. ACC1 is a key enzyme in the fatty acid synthesis process (rate-limiting enzyme) and, after carboxylation of acetyl-coa, which requires biotin as a coenzyme and relies on the energy provided by ATP, catalyzes the production of malonyl-coa and thus regulates lipid metabolism (Ronnebaum et al, j.biol.chem.,2008, 283. 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 conclusion, ACC1 inhibitors are of great interest for the treatment of metabolic syndrome with disorders of fatty acid anabolism. Unfortunately, no ACC1 inhibitor is currently successfully marketed, and at the clinical stage three ACC1 small molecule inhibitors GS-0976 and Gemcabene, GS-0976 can significantly reduce insulin content, increase insulin sensitivity, reduce levels of triacylglycerols and cholesterol in liver and blood (Loomba et al, gastroenterology,2018,155, 1463-1467.), while Gemcabene can increase clearance of very low density lipoprotein cholesterol (VLDL) in plasma and inhibit production of cholesterol and triglycerides in liver (Srivastava RAK et al, front Pharmacol,2018,471. 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 are uniquely and important for guidance and reference of new drug discovery and are an important source of new drug discovery (Newman et al, j.nat. Prod.2020,83 770-803 tiago et al, nat. Chem.2016, 8. 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 for 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 (792 family of natural 6763 family, active natural products with a proportion of only 11.7%), and 144 families in which marketed drugs and candidate chemicals in clinical phase are concentrated, while most rare endangered plants are apparently in 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: 16832-16816837.), which indicates that the novel druggability of natural products in rare endangered plants is much higher than the average level of general plants.
Significantly, the 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 Lancese cang yellow fir (Pseudotsuga forrestii Craib) belongs to the genus Taxus (Pseudotsuga) of the family Pinaceae, and is a unique species in China. It is an evergreen arbor, distributed from the upper part of the middle mountain to the middle part of the high mountain in the middle south of the transected mountain, recorded in 1992 by the book "Chinese plant Honpi-rare endangered plant", listed as "gradual-dangerous seed". If a few lancang yellow fir plant samples (such as reproducible branches and leaves) can be protectively collected to carry out systematic chemical component 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 compound is rearranged spiro lanoline alkane type triterpene separated from the picea lanuginosa or an adduct of intermolecular Diels-Alder reaction type of conventional lanoline alkane type triterpene (dienophile) and abietanediterpene (diene), and has a remarkable ACC1 inhibition effect in vitro pharmacological activity screening, so that the compound can have a treatment effect on hyperlipidemia, non-alcoholic steatohepatitis, type II diabetes mellitus, cancer and other ACC1 mediated diseases, and has huge potential application in the pharmaceutical field.
The application separates and identifies 3 rearranged spiro lanoline triterpene from methanol extract of branches and leaves of lancang picea aspera for the first time or a compound with a new skeleton generated by Diels-Alder [4+2] cycloaddition reaction between molecules of conventional lanoline triterpene and abietane diterpene, and the structure is characterized in that: rearrangement spiro lanolin alkane type triterpene or conventional lanolin alkane type triterpene (dienophile) and abietane diterpene (diene) are combined (structure is shown in formula 1-formula 3), wherein formula 1 and formula 2 are skeleton of the combination of rearrangement spiro lanolin alkane type triterpene and abietane diterpene; the formula 3 is the skeleton of the combination of conventional lanoline alkane type triterpene and abietane diterpene. Multiple pharmacological test researches show that the compounds have obvious ACC1 inhibiting activity.
On one hand, the application provides the eight-ring pentaterpenoid compound in the lancangchan, and the structural formula of the eight-ring pentaterpenoid compound is one of the following structures:
on the other hand, the compounds have remarkable ACC1 inhibiting activity:
the application also provides application of the octacyclic pentaterpenoid in the lancang yellow fir in preparing Acetyl-CoA carboxylase 1 (Acetyl-CoA carboxylase 1, ACC 1) inhibitors.
The application also provides an application of the eight-ring pentaterpenoid in the lancang yellow fir 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 disease, type II diabetes or obesity.
The application also provides an acetyl coenzyme A carboxylase 1 inhibitor which takes one or more of the eight-ring pentaterpenoids in the lancang yellow fir with the structures shown as formulas 1 to 3 as an active ingredient.
The application also provides a pharmaceutical composition which comprises one or more of the eight-ring pentaterpenoids in the lancang yellow fir with the structure shown in formula 1-formula 3 in a therapeutically effective amount as an active ingredient.
The pharmaceutical composition of the present application uses one or more of the above-mentioned Diels-Alder adducts as raw material, contains one or more of the above-mentioned compounds as active ingredient in therapeutically effective amount, and may further comprise pharmaceutically acceptable excipients, such as carrier, excipient, adjuvant or diluent. The pharmaceutical composition can be used for preparing medicines for preventing, delaying or treating diseases (particularly hyperlipidemia and related cardiovascular diseases) related to glycolipid disturbance mediated by ACC1 or lead compounds of the medicines.
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 compound of the invention can be obtained by separating and purifying 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:
the compound is prepared from branches and leaves of bluebush (Pseudotsuga forrestii Craib) by a conventional extraction and separation method related in the field, and the steps are as follows: 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 part 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 1-3.
In the above method, the methanol/water solution may be a 70% or more methanol-water solution (v/v), preferably a 90% methanol-water solution (v/v); 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, for example 3 to 8; further preferably extracted 7 times.
Optionally, after concentrating the ethyl acetate extract under reduced pressure, performing silica gel column chromatography and gradient elution with petroleum ether-ethyl acetate according to a volume ratio of 30 → 1 → 0, and collecting eluent to obtain 8 components fr.1-fr.8;
an MCI column chromatography was performed on a 8 2 Performing O gradient elution, and dividing the mixture into 3 subfractions Fr.4A-Fr.4C after color development and combination according to the conditions of a mobile phase and a TLC spot plate;
and (4) for volume ratio of 80:20MeOH-H 2 Performing 100-200-mesh silica gel column chromatography on the O elution component Fr.4A, and performing gradient elution by using petroleum ether-ethyl acetate according to a volume ratio of 10;
and (4) for volume ratio of 80:20MeOH-H 2 The O elution component Fr.4B is subjected to semi-preparative HPLC segmentation to obtain a component Fr.4B1 (t R = 8-14 min) and Fr.4B2 (t) R = 15-30 min), wherein Fr.4B2 is t R Collecting the obtained eluent component within 15-30 min, and carrying out semi-preparative HPLC purification on Fr.4B2 again to obtain a compound 3;
the petroleum ether-ethyl acetate eluate fraction fr.6 in a volume ratio of 3 2 Performing O gradient elution to obtain 5 subfractions Fr.6A-Fr.6E; to volume ratio 90:10MeOH: h 2 Performing Sephadex LH-20 column chromatography on the O elution component Fr.6D, eluting with MeOH, and purifying by semi-preparative HPLC to obtain a compound 2;
alternatively, the conditions for semi-preparative HPLC purification when isolating compound 1 are: X-Bridge, mobile phase: meOH-H 2 O; 95; v/v; flow rate: 3mL/min; column temperature: detection was carried out at 25 ℃ and a wavelength of 205 nm.
Alternatively, the conditions for semi-preparative HPLC purification when isolating compound 2 are: X-Bridge, mobile phase: meOH-H 2 O, 85; flow rate: 3mL/min; column temperature: at 25 ℃ inDetection at a wavelength of 205 nm.
Alternatively, the conditions for semi-preparative HPLC purification when isolating compound 3 are: X-Bridge, mobile phase: meOH-H 2 O, 98; flow rate: 3mL/min; column temperature: detection was carried out at 25 ℃ and a wavelength of 205 nm.
The two types of adducts in the application show stronger inhibition effect in the ACC1 inhibitory activity test, and the activity is equivalent to that of the positive control ND 630.
The invention has the following remarkable advantages:
the compounds are all novel compounds which are firstly separated from the nature, and are Diels-Alder [4+2] -type adducts with unique cyclization structures (rearrangement spiro lanoline alkane type triterpenes or conventional lanoline alkane type triterpenes are dienophiles, and abietane diterpenes are dienes); meanwhile, the compounds are found to have remarkable ACC1 inhibition activity for the first time. The polypeptide has important application prospect on diseases related to glycolipid metabolic disturbance, such as hyperlipidemia, non-alcoholic steatohepatitis, type II diabetes and the like which are frequently developed in modern people.
Drawings
FIG. 1 is a HR-ESIMS plot for Compound 1;
FIG. 2 is a HR-ESIMS plot for Compound 2;
FIG. 3 is a HR-ESIMS graph for Compound 3.
Detailed Description
The technical solutions of the present application will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope 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. The terminology used herein in the description of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.
The application protectively collects a few samples of the Lanceolaria lanuginosa plant (such as reproducible branches and leaves) to carry out systematic chemical component research, extracts active substances of the Lanceolaria lanuginosa plant and develops new application of the Lanceolaria lanuginosa plant in pharmacy. The application separates a series of different terpenoid molecules (rearranged spiro lanoline alkane type triterpene or conventional lanoline alkane type triterpene and abietane diterpene) with novel structures from methanol extracts of branches and leaves of lancang picea aspera for the first time to obtain adducts (the structures are shown as formulas 1-3) obtained by a Diels-Alder addition mode. Multiple pharmacological test researches show that the compounds have obvious ACC1 inhibition activity.
One way of obtaining the compound: air drying and pulverizing branches and leaves of lancang yellow fir, soaking and extracting with methanol/water solution at room temperature, concentrating the extractive solution under reduced pressure, recovering solvent, and mixing to obtain extract. Dispersing the extract with water, and 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 part 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 1-3.
Branches and leaves of Lancebush yellow fir (Pseudotsuga forrestii Craib) are collected from Yunan Dali, dried in the shade, and pulverized into powder; specific rotation measurements were performed by a 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-810CD spectrometer; the instrument used in the single crystal diffraction experiment is 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 GF254/0.25mm; 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 Star high purity solvents, inc.; the deuterated reagent is produced by Sigma-Aldrich.
The following is a detailed description of specific examples:
preparation of the Compound of example 1
(1) After drying and pulverizing the branches and leaves of the blueberries of the Yunnan Spanish tree (collected in 6 months in 2019, 15kg in dry weight), extracting for 7 times 24 hours each time at room temperature by using 90% methanol (6L) solution. The extracts were combined and concentrated under reduced pressure to remove methanol to give 3.9kg of extract (semi-dry). Dispersing the extract with 3L of water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol with equal volume for three times, and concentrating the extractive solutions under reduced pressure to obtain 4 components, namely petroleum ether fraction, ethyl acetate fraction, n-butanol fraction and water. Wherein, 8 components are obtained after the crude extract of the ethyl acetate part is separated by silica gel column chromatography (petroleum ether-ethyl acetate gradient washing, 30.
(2) The component Fr.4 (35 g, obtained in step (1) via petroleum ether: ethyl acetate 8 2 After O-gradient elution (70 → 80. Fr.4A (6.6 g, in MeOH: H) 2 O-80: 20) was purified by column chromatography over silica gel (100-200 mesh, eluting with a petroleum ether-ethyl acetate gradient, 10: meOH-H 2 O, 95; flow rate: 3mL/min; column temperature: 25 ℃ at 205 nm) to give compound 1 (70.0 mg R ＝30.1min)。
Fr.4B (2 g, in MeOH: H) 2 O-80: 20) was fractionated by semi-preparative HPLC (X-Bridge, mobile phase: meOH-H 2 O, 95; flow rate: 3mL ofMin; column temperature: 25 ℃ at a wavelength of 205 nm) to give a fraction Fr.4B1 (t) R = 8-14 min) and Fr.4B2 (t) R = 15-30 min), fr.4b2 was again subjected to semi-preparative HPLC with chromatographic conditions (X-Bridge, mobile phase: meOH-H 2 O, 85; flow rate: 3mL/min; column temperature: 25 ℃ at 205 nm) to give compound 3 (4.5mg R ＝34.0min)。
(3) The fraction Fr.6 from step (1) (22 g, from step (1) eluting with petroleum ether: ethyl acetate 3) was selected for separation by MCI column chromatography with MeOH-H 2 The O gradient elution (70. Fr.6D (1.6 g, in MeOH: H) 2 O-90: 10) was purified by Sephadex LH-20 (MeOH) column chromatography followed by semi-preparative HPLC (X-Bridge, mobile phase: meOH-H 2 O, 98; flow rate: 3mL/min; column temperature: 25 ℃ at a wavelength of 205 nm) was purified to give compound 2 (9.8mg R ＝17.7min)。
The HR-ESIMS graph of the compound 1 is shown in a figure 1, and the nuclear magnetism and physicochemical data are as follows:
acicular single crystal (CHCl) 3 ),
(c 0.34,MeOH)；UV(MeOH)λ max (logε)206(3.67)nm；ECD(c 7.94×10 -5 M,MeCN)λ max (Δε)225(-1.20),252(-0.04),281(-0.14),343(+0.05)nm；IR(KBr)v max 2960,2922,2870,1693,1462,1382,1270,1110,928,878,754,686,569cm -1 ； 1 H-NMR(CDCl 3 ,600MHz):δ1.70(1H,m,H-1a),1.92(1H,m,H-1b),2.49(1H,ddd,J＝16.2,7.4,3.2Hz,H-2a),2.62(1H,ddd,J＝16.2,10.6,7.7Hz,H-2b),1.65(1H,br d,J＝10.4Hz,H-5),1.68(1H,m,H-6a),1.56(1H,m,H-6b),2.02(1H,m,H-7a),2.14(1H,m,H-7b),2.15(1H,m,H-11a),1.95(1H,m,H-11b),2.04(1H,m,H-12a),1.44(1H,m,H-12b),2.42(1H,ddd,J＝17.6,10.0,1.8 Hz,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＝6.5 Hz,H-21),2.55(1H,dd,J＝14.4,2.6 Hz,H-22a),2.02(1H,dd,overlapped,H-22b),3.16(1H,d,J＝18.5 Hz,H-24a),2.23(1H,d,J＝18.5 Hz,H-24b),2.71(1H,dd,J＝14.0,3.4 Hz,H-26a),0.82(1H,dd,overlapped,H-26b),1.09(3H,s,H-28),1.12(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(1H,m,H-3'a),1.57(1H,m,H-3'b),1.57(1H,br d,J＝11.2 Hz,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,dd,overlapped,H-9'),1.74(1H,m,H-11'a),1.11(1H,m,H-11'b),2.47(1H,br s,H-12'),5.32(1H,br s,H-14'),2.31(1H,m,H-15'),1.01(3H,d,J＝6.5 Hz,H-16’),1.00(3H,d,J＝6.5 Hz,H-17’),1.15(3H,s,H-19’),0.60(3H,s,H-20')； 13 C-NMR(CDCl 3 ,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),26.3(C-7),136.1(C-8),147.8(C-9),35.9(C-10),26.7(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),26.6(C-28),21.1(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'),43.4(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'),20.3(C-16'),20.5(C-17'),184.3(C-18'),16.7(C-19'),16.6(C-20')；ESIMS m/z 769[M+H] + ；HRESIMS m/z 769.5415[M+H] + (calcd for C 50 H 73 O 6 ,769.5402,Δ＝+1.7ppm).
The HR-ESIMS graph of compound 2 is shown in FIG. 2, and the nuclear magnetic and physicochemical data are as follows:
White powder,
(c 0.50,MeOH)；UV(MeOH)λ max (logε)206(3.59)nm；ECD(c 7.27×10 -5 M,MeCN)λ max (Δε)192(+7.68),225(-1.63),253(-0.05),283(-0.34)nm；IR(KBr)v max 2935,2873,1691,1467,1382,1372,1272,1207,1187,1025,913,883,731cm -1 ； 1 H-NMR(CDCl 3 ,600MHz):δ1.29(1H,ddd,J＝13.0,11.6,4.1Hz,H-1a),1.65(1H,m,H-1b),1.72(1H,m,H-2a),1.65(1H,m,H-2b),3.30(1H,dd,J＝10.8,4.5Hz,H-3),1.09(1H,br d,J＝10.8Hz,H-5),1.72(1H,m,H-6a),1.51(1H,m,H-6b),1.90(1H,m,H-7a),2.08(1H,m,H-7b),2.10(1H,m,H-11a),1.99(1H,m,H-11b),1.98(1H,m,H-12a),1.37(1H,m,H-12b),2.38(1H,br dd,J＝17.7,10.2Hz,H-15a),2.30(1H,m,H-15b),1.54(1H,m,H-16a),1.43(1H,m,H-16b),0.82(3H,s,H-18),0.96(3H,s,H-19),2.28(1H,m,H-20),0.76(3H,d,J＝6.5Hz,H-21),2.59(1H,br d,J＝14.3Hz,H-22a),1.98(1H,dd,overlapped,H-22b),3.14(1H,d,J＝18.4Hz,H-24a),2.20(1H,d,J＝18.4Hz,H-24b),2.68(1H,br d,J＝14.0Hz,H-26a),0.83(1H,dd,overlapped,H-26b),1.02(3H,s,H-28),0.84(3H,s,H-29),4.73(1H,br s,H-30a),4.50(1H,br s,H-30b),0.83(1H,m,H-1'a),1.39(1H,m,H-1'b),1.54(1H,m,H-2'a),1.40(1H,m,H-2'b),1.61(1H,m,H-3'a),1.60(1H,m,H-3'b),1.58(1H,br d,J＝12.0Hz,H-5'),1.33(1H,m,H-6'a),1.50(1H,m,H-6'b),1.79(1H,m,H-7'a),1.77(1H,m,H-7'b),1.64(1H,dd,overlapped,H-9'),1.73(1H,m,H-11'a),1.09(1H,br d,J＝11.8Hz,H-11'b),2.46(1H,br s,H-12'),5.31(1H,br s,H-14'),2.31(1H,m,H-15'),1.01(3H,d,J＝6.5Hz,H-16’),1.01(3H,d,J＝6.5Hz,H-17’),1.14(3H,s,H-19’),0.59(3H,s,H-20')； 13 C-NMR(CDCl 3 ,150MHz):δ35.4(C-1),27.5(C-2),79.2(C-3),38.7(C-4),50.7(C-5),19.2(C-6),26.7(C-7),134.9(C-8),149.6(C-9),36.0(C-10),26.4(C-11),32.5(C-12),68.1(C-13),155.8(C-14),27.2(C-15),38.0(C-16),49.1(C-17),18.6(C-18),19.2(C-19),35.1(C-20),16.0(C-21),44.7(C-22),210.6(C-23),51.4(C-24),53.3(C-25),39.4(C-26),180.6(C-27),28.0(C-28),15.5(C-29),103.9(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'),43.4(C-8'),49.8(C-9'),37.8(C-10'),26.5(C-11'),33.1(C-12'),150.4(C-13'),124.3(C-14'),32.6(C-15'),20.3(C-16'),20.5(C-17'),184.2(C-18'),16.7(C-19'),16.7(C-20')；HRESIMS m/z 793.5378[M+Na] + (calcd for C 50 H 74 O 6 Na,793.5378,Δ＝+0.1ppm).
the HR-ESIMS graph of compound 3 is shown in FIG. 3, and the nuclear magnetic and physicochemical data are as follows:
White powder,
(c 0.015,MeOH)；UV(MeOH)λ max (logε)204(3.98)nm；ECD(c 6.49×10 -5 M,MeCN)λ max (Δε)193(-4.19),214(+4.20),269(+0.11),298(+0.27)nm； 1 H-NMR(CDCl 3 ,600MHz):δ1.67(1H,m,H-1a),1.98(1H,m,H-1b),2.40(1H,ddd,J＝16.0,6.7,3.2Hz,H-2a),2.60(1H,ddd,J＝16.0,11.2,7.0Hz,H-2b),1.60(1H,dd,overlapped,H-5),1.66(1H,m,H-6a),1.59(1H,m,H-6b),2.08(2H,m,H-7),1.61(1H,m,H-11a),2.04(1H,m,H-11b),1.74(1H,m,H-12a),1.62(1H,m,H-12b),1.22(1H,m,H-15a),1.64(1H,m,H-15b),1.85(1H,m,H-16a),1.29(1H,m,H-16b),1.47(1H,m,H-17),0.73(3H,s,H-18),1.12(3H,s,H-19),1.95(1H,m,H-20),0.90(3H,d,J＝6.5Hz,H-21),2.33(1H,br d,J＝14.4Hz,H-22a),1.98(1H,dd,overlapped,H-22b),2.81(1H,br s,H-24),1.21(3H,s,H-26),1.10(3H,s,H-28),1.07(3H,s,H-29),0.87(3H,s,H-30),0.85(1H,m,H-1'a),1.43(1H,m,H-1'b),1.57(1H,m,H-2'a),1.46(1H,m,H-2'b),1.60(1H,m,H-3'a),1.65(1H,m,H-3'b),1.67(1H,dd,overlapped,H-5'),1.57(1H,m,H-6'a),1.28(1H,m,H-6'b),1.56(1H,m,H-7'a),1.67(1H,m,H-7'b),1.67(1H,dd,overlapped,H-9'),2.19(1H,br dd,J＝13.0,10.3Hz,H-11'a),1.00(1H,br d,J＝13.0Hz,,H-11'b),2.71(1H,br s,H-12'),5.38(1H,br s,H-14'),2.34(1H,sep t,J＝6.5Hz,H-15'),1.04(3H,d,J＝6.5Hz,H-16’),1.04(3H,d,J＝6.5Hz,H-17’),1.16(3H,s,H-19’),0.62(3H,s,H-20')； 13 C-NMR(CDCl 3 ,150MHz):δ36.0(C-1),34.6(C-2),217.8(C-3),47.4(C-4),51.2(C-5),19.4(C-6),26.3(C-7),135.1(C-8),133.2(C-9),36.9(C-10),21.0(C-11),30.8(C-12),44.5(C-13),50.2(C-14),30.7(C-15),28.4(C-16),50.0(C-17),15.8(C-18),18.7(C-19),34.3(C-20),19.9(C-21),51.5(C-22),213.6(C-23),59.7(C-24),50.3(C-25),17.7(C-26),183.7(C-27),26.1(C-28),21.3(C-29),24.2(C-30),37.9(C-1'),17.1(C-2'),37.0(C-3'),47.0(C-4'),49.0(C-5'),21.9(C-6'),30.8(C-7'),45.8(C-8'),48.1(C-9'),37.6(C-10'),20.9(C-11'),35.2(C-12'),148.6(C-13'),125.7(C-14'),32.3(C-15'),20.2(C-16'),20.2(C-17'),186.3(C-18'),16.2(C-19'),17.3(C-20')；HRESIMS m/z 793.5372[M+Na] + (calcd for C 50 H 74 O 6 Na,793.5378,Δ＝-0.7ppm).
activity measurement of the Compound of example 2
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, relying on the energy provided by ATP, to regulate lipid metabolism. This reaction is accompanied by the consumption of ATP, and therefore the change in ATP is detected using an ADP-Glo kinase detection reagent, whereby the inhibitory effect of the compound on ACC1 enzyme is indirectly reacted.
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 Values, experiments were repeated three times and results were averaged over three times. The positive control was ND630, IC 50 The value was 8.12. + -. 1.49nM.
TABLE 1 ACC1 inhibitory Activity data of pentacyclic terpenoid compounds in Cusuavecang Yew
ACC1 inhibitory Activity data (IC) of said Ocyclopentaterpenoid 50 Values) are shown in table 1. The test results show that all 3 compounds in the application show significant inhibitory activity on ACC1, and the compounds can be used for preparing medicaments for treating diseases related to glycolipid metabolic disorder or lead compounds of the medicaments.
In conclusion, the compound has obvious Acetyl-Coenzyme A carboxylase 1 (Acetyl-Coenzyme A carboxylase 1, ACC 1) inhibition activity through multiple in-vitro activity tests, and can be applied to preparation of medicines for preventing, delaying or treating ACC 1-mediated glycolipid metabolic disorders and other related diseases. The invention also provides a lead compound for developing novel medicaments for diseases related to glycolipid metabolic disturbance.
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 shall be subject to the appended claims.
Claims (10)
1. The eight-ring pentaterpenoid compound in the lancang yellow fir is characterized in that the structural formula is one of the following structures:
2. use of octacyclic pentaterpenoids in lancangchua as claimed in claim 1, for the preparation of acetyl-coa carboxylase 1 inhibitors.
3. Use of the octacyclic pentaterpenoids of lancangchua as claimed in claim 1 for the preparation of a medicament for the prevention, delay or treatment of diseases 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, characterized by comprising one or more of the octacyclic pentaterpenoids in the lanuginosa as claimed in claim 1 as an active ingredient.
6. A pharmaceutical composition characterized by comprising, as an active ingredient, a therapeutically effective amount of one or more selected from the group consisting of the octacyclic pentaterpenoids of lancang-fir as claimed in claim 1.
7. The pharmaceutical composition of claim 6, further comprising a pharmaceutically acceptable pharmaceutical excipient.
8. A process for preparing octacyclic pentaterpenoids in lantake-take fir as claimed in claim 1, which comprises:
drying branches and leaves of Lancese Canadensis yellow fir (Pseudotsuga forrestii Craib) at room temperature, pulverizing, extracting with 90% methanol at room temperature for several times, concentrating the extractive solution, adding water, suspending, and extracting with petroleum ether, ethyl acetate and n-butanol respectively; wherein the obtained ethyl acetate extract is subjected to vacuum concentration and then sequentially separated by the techniques of silica gel, microporous resin, sephadex LH-20 gel, reverse-phase semi-preparative high performance liquid chromatography and the like to respectively prepare the compounds shown in the formulas 1 to 3.
9. The preparation method according to claim 8, wherein the ethyl acetate extract is concentrated under reduced pressure, subjected to silica gel column chromatography and eluted with a petroleum ether-ethyl acetate gradient of a volume ratio of 30 → 1 → 0, and the eluate is collected to obtain 8 fractions fr.1 to fr.8;
an MCI column chromatography was performed on a 8 2 Performing O gradient elution, and dividing into 3 subcomponents Fr.4A-Fr.4C after color development and combination according to the conditions of a mobile phase and a TLC spot plate;
and (4) for volume ratio of 80:20MeOH-H 2 O elution component fr.4a was subjected to 100-200 mesh silica gel column chromatography and eluted with a 1Purifying the ester elution component by semi-preparative HPLC to obtain a compound 1;
and (4) volume ratio of 80:20MeOH-H 2 The O elution component Fr.4B is subjected to semi-preparative HPLC segmentation to obtain components Fr.4B1 and Fr.4B2, wherein the retention time t of Fr.4B2 for collection R Performing semi-preparative HPLC purification on Fr.4B2 again to obtain a compound 3 in eluent component of = 15-30 min;
the petroleum ether-ethyl acetate eluate fraction fr.6 in a volume ratio of 3 2 Performing O gradient elution to obtain 5 subfractions Fr.6A-Fr.6E; to volume ratio 90:10MeOH: h 2 Performing Sephadex LH-20 column chromatography on the O elution component Fr.6D, eluting with MeOH, and purifying by semi-preparative HPLC to obtain a compound 2;
10. the method according to claim 9,
the conditions for semi-preparative HPLC purification for isolation of compound 1 were: X-Bridge, mobile phase: meOH-H 2 O; 95; v/v; flow rate: 3mL/min; column temperature: detecting at 25 deg.C at 205nm wavelength;
the conditions for semi-preparative HPLC purification for isolation of compound 2 were: X-Bridge, mobile phase: meOH-H 2 O, 85; flow rate: 3mL/min; column temperature: detecting at 25 deg.C at 205nm wavelength;
the conditions for semi-preparative HPLC purification for isolation of compound 3 were: X-Bridge, mobile phase: meOH-H 2 O, 98; flow rate: 3mL/min; column temperature: and detecting at 25 ℃ at a wavelength of 205 nm.
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