CN112159378A - Gilmane type sesquiterpene lactone compound and preparation method and application thereof - Google Patents
Gilmane type sesquiterpene lactone compound and preparation method and application thereof Download PDFInfo
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- CN112159378A CN112159378A CN202010880632.7A CN202010880632A CN112159378A CN 112159378 A CN112159378 A CN 112159378A CN 202010880632 A CN202010880632 A CN 202010880632A CN 112159378 A CN112159378 A CN 112159378A
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- Prior art keywords
- germacrane
- type sesquiterpene
- preparation
- sesquiterpene lactone
- compound
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- 229930009674 sesquiterpene lactone Natural products 0.000 title claims abstract description 21
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- 238000002360 preparation method Methods 0.000 title claims abstract description 14
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- IBMAYSYTZAVZPY-QDMKHBRRSA-N germacrane Chemical compound CC(C)[C@H]1CC[C@@H](C)CCC[C@@H](C)CC1 IBMAYSYTZAVZPY-QDMKHBRRSA-N 0.000 claims abstract description 24
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- CBOIHMRHGLHBPB-UHFFFAOYSA-N hydroxymethyl Chemical compound O[CH2] CBOIHMRHGLHBPB-UHFFFAOYSA-N 0.000 claims abstract description 4
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D307/93—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems condensed with a ring other than six-membered
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Physical Education & Sports Medicine (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Rheumatology (AREA)
- Medicinal Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
Abstract
The invention discloses a germacrane type sesquiterpene lactone compound and a preparation method and application thereof. The germacrane type sesquiterpene lactone compound has a structural formula shown in formulas (I) to (IV):
wherein R is1Is H or OH; r2OH, OMearr or OEt; r3Is OAng, OH or OMeacr; r4Is OTig or OAng; r5Is CH2OH or CHO. The germacrane sesquiterpenoids provided by the invention have obvious effect of inhibiting differentiation on osteoclast induced by RANKL, most of the compounds have IC50 below 10.0 mu M, do not show cytotoxicity under 10 mu M, and can be applied to preparation of medicines for treating and/or preventingThe osteoporosis medicine has good application prospect.
Description
Technical Field
The invention relates to the technical field of compounds and medicines, and particularly relates to a germacrane type sesquiterpene lactone compound as well as a preparation method and application thereof.
Background
Osteoporosis (osteoporotis) is a systemic bone disease in which bone brittleness is increased due to decrease in bone density and bone quality caused by various reasons and destruction of bone microstructure, so that fracture is easily caused, and primary osteoporosis is most common among menopausal women and old people. Due to the aggravation of aging of the population, osteoporosis becomes a global public health problem, and the quality of life of the middle-aged and old people is seriously affected.
The pathogenesis of osteoporosis is complicated, but finally, the dynamic balance maintained between bone formation under the action of osteoblasts and osteoclast-mediated bone resorption is broken, so that the bone resorption effect is greater than that of bone formation, and the bone mass is lost to cause osteoporosis. The RANKL/RANK/OPG and Wnt/beta-catenin pathways are important pathways influencing bone metabolism (Dongbei, Sundao. osteoporosis treatment progresses from molecular mechanism to drug target [ J ]. the J. China osteoporosis and bone mineral disease journal, 2018,11(06):620 and 627), and at present, drugs aiming at osteoporosis regulate the balance between bone resorption and bone formation by acting on key targets of the pathways. Many drugs are still in clinical tests, safety evaluation and even basic research stages, most of the marketed drugs have single target points and narrow treatment ranges and have adverse reactions, and the search for high-efficiency and low-toxicity anti-osteoporosis drugs is urgently needed clinically.
Disclosure of Invention
The invention aims to overcome the defects or shortcomings of single target, narrow treatment range and adverse reaction of the current clinical anti-osteoporosis drugs, and provides a germacrane type sesquiterpene lactone compound. The germacrane sesquiterpenoids provided by the invention have obvious effect of inhibiting differentiation on osteoclast induced by RANKL, most of IC50 of the compounds are below 10.0 mu M, and do not show cytotoxicity under 10 mu M, so that the germacrane sesquiterpenoids can be applied to preparation of drugs for treating and/or preventing osteoporosis, and have good application prospect.
Another object of the present invention is to provide a process for producing the aforementioned germacane type sesquiterpene lactone compounds.
The invention also aims to provide the application of the germacrane type sesquiterpene lactone compound in preparing medicaments for preventing or treating osteoporosis.
Another object of the present invention is to provide a medicament for preventing and/or treating osteoporosis.
In order to achieve the above purpose of the present invention, the present invention provides the following technical solutions:
a gemma alkane type sesquiterpene lactone compound has structural formulas shown in formulas (I) to (IV):
wherein R is1Is H or OH; r2OH, OMeacr (methacryloyloxy) or OEt; r3OAng, OH or OMeacr (methacryloyloxy); r4Is OTig or OAng; r5Is CH2OH or CHO.
The traditional Chinese medicine herba siegesbeckiae is a commonly used traditional Chinese medicine in China, has the effects of expelling wind-damp, benefiting joints, clearing heat and removing toxicity, and is clinically used for treating rheumatic arthralgia, myasthenia of bones and muscles, soreness and weakness of waist and knees, quadriplegia, hemiplegia, rubella and eczema and the like. It is recorded in the first part of the 2015 edition of "Chinese pharmacopoeia".
The plants Siegesbeckiae Herba L, Siegesbeckiae glandularis Pubescens M, Siegesbeckiae Herba and Siegesbeckiae Herba Glaberescens M, all belonging to the genus Siegesbeckiae of the family Compositae, are plants from which Siegesbeckiae Herba is derived, and the pharmacological activities of the three plants are reported to be similar but different in chemical composition (Zhang Q R, Zhang Z F, SangW, et al. Herba Siegesbeckiae Pubescens is dicotyledonous plant of Siegesbeckiae of Compositae. Is distributed in the northeast and southwest of China. The herba siegesbeckiae contains Gimeranthan type, guaiane type sesquiterpene, pimarane type, and kaurane type diterpene. Modern pharmacological research reveals that the monomeric compound extracted from siegesbeckia plant has pharmacological effects of resisting inflammation, lowering blood pressure, suppressing immunity, resisting bacteria and malaria, etc.
In the study on siegesbeckia Pubescens Makino, the inventors of the present invention extracted several germacrane type sesquiterpene lactone compounds from siegesbeckia Pubescens Makino, and these germacrane type sesquiterpene lactone compounds have an obvious effect of inhibiting differentiation of osteoclasts induced by RANKL, and IC of most compounds50All are below 10.0. mu.M. In particular, IC of Compounds XX-1, XX-8, XX-9, and XX-10 against osteoblast differentiation Activity50The drug effect evaluation of the above 4 compounds shows that the compounds can block the downstream NF-kB and NFATc1 channels and remarkably inhibit osteoclast differentiation. And showed no cytotoxicity at 10. mu.M. The germacrane type sesquiterpenoids extracted from siegesbeckia Pubescens Makino can be applied to preparation of drugs for treating and/or preventing osteoporosis, and has good application prospect.
Preferably, said R is1Is H, R2Is OEt, R3Is OMeacr; or R4Is OAng; or R5Is CH2OH or CHO.
The preparation method of the germacrane type sesquiterpene lactone compound comprises the following steps:
s1: drying aerial part of siegesbeckia Pubescens Makino, pulverizing, leaching, and concentrating under reduced pressure to obtain crude extract;
s2: suspending the crude extract, extracting, concentrating under reduced pressure, eluting with chromatographic column or sephadex column, and separating by chromatography and high performance liquid chromatography to obtain the germacrane type sesquiterpene lactone compounds.
Preferably, the S1 is leached with an ethanol solution.
More preferably, the volume fraction of the ethanol solution in the S1 is 85-95%.
Preferably, the leaching times in the S1 are 3-4 times, and the time of single leaching is 2-5 days.
Preferably, the extraction in S2 is with ethyl acetate.
Preferably, the eluent eluted in S2 is a dichloromethane/methanol mixed solution; the gradient of elution was: the volume ratio of dichloromethane/methanol is 0 → 40 min: 1:0 → 100:1, 40 → 80 min: 100:1 → 50:1, 80 → 120 min: 50:1 → 20:1, 120 → 160 min: 20:1 → 10: 1.
Specifically, the germacrane type sesquiterpene lactone compound is prepared by the following steps:
crushing the dried overground part of siegesbeckia Pubescens Makino into small segments, and leaching the small segments with 85-95% ethanol water solution at room temperature for 3-4 times, wherein each time lasts for 2-5 days; concentrating the extracting solution under reduced pressure to obtain a crude extract; suspending the extract in water, extracting for 3-6 times by using ethyl acetate, concentrating under reduced pressure to obtain an ethyl acetate part, performing silica gel column chromatography with 200-300 meshes, and performing gradient elution by using dichloromethane/methanol; volume ratio of dichloromethane/methanol in gradient elution 0 → 40 min: 1:0 → 100:1, 40 → 80 min: 100:1 → 50:1, 80 → 120 min: 50:1 → 20:1, 120 → 160 min: 20:1 → 10: 1. And purifying the fractions obtained in the above steps sequentially by silica gel column chromatography, sephadex column chromatography and high performance liquid chromatography to obtain the final product.
The application of the germacrane type sesquiterpene lactone compound in preparing the medicine for preventing and/or treating osteoporosis is also within the protection scope of the invention.
A medicament for preventing and/or treating osteoporosis contains one or more of the above germacrane type sesquiterpenoids, pharmaceutically acceptable carriers or adjuvants; the mass fraction of the sesquiterpene compound and the pharmaceutically acceptable carrier or auxiliary material in the medicine is 0.1-99%.
The medicament for preventing and/or treating osteoporosis of the present invention can be prepared into various existing types of pharmaceutical preparations.
Preferably, the medicament is in the form of injection, tablet or capsule.
Compared with the prior art, the invention has the following advantages and effects:
the germacrane type sesquiterpene lactone compound provided by the invention has an obvious effect of inhibiting differentiation on RANKL-induced osteoclasts, and the IC of most compounds50All are below 10.0. mu.M. In particular, IC of Compounds XX-1, XX-8, XX-9, and XX-10 against osteoblast differentiation Activity50All below 1 μ M, and the drug effects of the above 4 compounds were evaluatedIt is shown that it can block the downstream NF-kB and NFATc1 pathways, and significantly inhibit osteoclast differentiation. And showed no cytotoxicity at 10. mu.M. The germacrane type sesquiterpenoids extracted from siegesbeckia Pubescens Makino can be applied to preparation of drugs for treating and/or preventing osteoporosis, and has good application prospect.
Drawings
FIG. 1 shows the structures XX-1 to XX-10 of the isolated compounds;
FIG. 2 shows that the compounds XX-1, XX-6 are primary1H-1H COSY
HMBC
And NOESY
Correlation
FIG. 3 is an ECD spectrum of compounds XX-1, XX-6;
FIG. 4 is a graph showing the results of compounds XX-1 to XX-10 in inhibiting RANKL-induced osteoclasts; a: measuring the toxicity of XX-1 to XX-10 to BMM cells; b, C: effects of XX-1 to XX-10 on osteoclast differentiation at different concentrations; d: TRAcP staining experimental result graphs of XX-1, XX-8, XX-9 and XX-10;
FIG. 5 is a graph showing the results of experiments with luciferase reporter genes for compounds XX-1, XX-4, XX-6, XX-8, XX-9 and XX-10.
Detailed Description
The present invention will be further explained with reference to the following examples and drawings, but the examples are not intended to limit the present invention in any manner. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the present invention are commercially available.
Example 1
XX-1 to XX-10 are prepared from siegesbeckia Pubescens Makino.
Drying aerial parts (5.0kg) of Siegesbeckiae herba, pulverizing into small pieces, and extracting with 95% ethanol water solution at room temperature for 3 times each for 3 days; concentrating the extracting solution under reduced pressure to obtain a crude extract; suspending the extract in water, extracting with ethyl acetate for 5 times, concentrating under reduced pressure to obtain ethyl acetate part, performing 200-300 mesh silica gel column chromatography, and performing gradient elution with dichloromethane/methanol. The volume ratio of dichloromethane/methanol in gradient elution is 0 → 40min in sequence: 1:0 → 100:1, 40 → 80 min: 100:1 → 50:1, 80 → 120 min: 50:1 → 20:1, 120 → 160 min: 20:1 → 10: 1. After thin-layer chromatography identification, the fractions are divided and concentrated into 5 fractions (A to E). And (3) carrying out reverse phase column chromatography on the fraction C (50g), and eluting with methanol/water (30-100%) to obtain a compound XX-4(300 mg). Fraction C3 was eluted through Sephadex column with methanol and divided into three fractions C3.1-C3.3, C3.2 were separated by HPLC to give compounds XX-1(30mg), XX-3(20mg), XX-5(5mg), XX-6(5 mg). C3.3 separation by high performance liquid chromatography gave compounds XX-7(8mg), XX-8(12mg), XX-9(10 mg). C4 separation by high performance liquid chromatography gave Compound XX-2(20mg), XX-10(30 mg). The structure of the compound is shown in figure 1.
The data of the physical and chemical properties of 10 monomeric compounds are as follows:
XX-1, colorless gum, C21H26O6，EI-MS m/z：397.13[M+Na]+；
+7(c 0.1，MeOH)；HRESIMS m/z 397.1623[M+Na]+(calcd for C21H26O6，397.1622)；UV(MeOH)λmax(log)195(0.32)，203(1.12)nm；IR(KBr)νmax 3372，2933，1714，1675，1459，1399，1031cm-1；1H NMR(400MHz，CDCl3)：9.44(d，J＝2.1Hz，H-14)，6.74(dd，J＝10.0，7.4Hz，H-1)，6.55(dd，J＝8.3，1.7Hz，H-8)，6.21(d，J＝3.5Hz，H-13a)，6.02(brs，H-5a)，5.83(d，J＝3.5Hz，H-13b)，5.55(t，J＝1.2Hz，H-5’)，5.02(t，J＝10.0Hz，H-6)，4.90(dd，J＝10.0，1.5Hz，H-5b)，3.65(dd，J＝8.3，2.2Hz，H-9)，3.37(dq，J＝9.2，7.0Hz，H-1’a)，3.06(dq，J＝9.2，7.0Hz，H-1’b)，2.67(m，H-2a)，2.59(dtd，J＝10.0，3.3，1.7Hz，H-7)，2.41(ddd，J＝12.0，5.9，2.3Hz，H-3a)，2.32(m，H-2b)，2.07(td，J＝12.0，2.3，H-3b)，1.91(brs，H3-15)，1.91(brs，H3-6’)，1.01(t，J＝7.0Hz，H3-2’).13C NMR(400MHz，CDCl3)：194.3(C-14)，169.6(C-12)，165.9(C-3')，156.0(C-1)，141.3(C-10)，137.1(C-4)，136.1(C-4’)，134.2(C-11)，127.3(C-5)，125.7(C-5)，122.3(C-13)，75.2(C-6)，70.1(C-8)，64.6(C-1’)，50.9(C-7)，36.9(C-3)，26.5(C-2)，18.5(C-6’)，17.0(C-15)，15.1(C-2’)。
XX-2, colorless gum, C20H24O7，EI-MS m/z：385.22[M+Na]+；1H NMR(400MHz，MeOD)：9.47(d，J＝2.1Hz，H-14)，6.79(dd，J＝10.2，7.4Hz，H-1)，6.58(dd，J＝8.4，1.6Hz，H-8)，6.19(overlap，H-3’a)，6.16(overlap，H-13a)，5.75(d，J＝3.1Hz，H-13b)，5.67(t，J＝1.6Hz，H-3’b)，5.36(t，J＝10.1Hz，H-6)，5.13(t，J＝10.6Hz，H-5)，4.49(dd，J＝13.1，1.1Hz，H-15a)，4.26(d，J＝13.1Hz，H-15b)，4.19(dd，J＝8.4，2.1Hz，H-9)，2.86(m，H-7)，2.82(m，H-3a)，2.68(m，H-2a)，2.56(m，H-2b)，2.05(td，J＝12.7，2.4Hz，H-3b)，1.98(s，Me-4’)；13C NMR(100MHz，MeOD)：197.1(C-14)，171.3(C-12)，167.9(C-1’)，157.5(C-1)，145.9(C-4)，142.4(C-10)，137.5(C-2’)，136.9(C-11)，129.3(C-5)，126.7(C-3’)，121.3(C-13)，75.7(C-6)，72.8(C-8)，69.8(C-9)，60.6(C-15)，52.3(C-7)，33.1(C-3)，27.8(C-2)，18.6(C-4’)。
XX-3, colorless gum, C19H22O6，EI-MS m/z：369.18[M+Na]+；1H NMR(500MHz，CDCl3)：9.42(s，H-14)，6.73(ddd，J＝9.5，7.5，1.5Hz，H-1)，6.35(d，J＝2.2Hz，H-13a)，6.12(s，H-3’a)，5.65(brs，H-13b)，5.61(brs，H-3’b)，5.35(d，J＝8.4Hz，H-8)，5.21(t，J＝10.0Hz，H-6)，5.12(d，J＝8.4Hz，H-9)，4.88(d，J＝10.0Hz，H-5)，2.95(q，J＝12.2Hz，H-2a)，2.62(m，H-2b)，2.46(m，H-3a)，2.42(m，H-7)，2.08(t，J＝12.2Hz，H-3b)，1.98(s，H3-15)，1.92(s，H3-4’)。13C NMR(125MHz，CDCl3):194.4(C-14)，169.8(C-12)，167.0(C-1’)，159.6(C-1)，141.6(C-10)，138.5(C-4)，136.0(C-11)，135.9(C-2’)，127.1(C-5)，127.0(C-3’)，120.6(C-13)，74.5(C-6)，73.5(C-9)，68.2(C-8)，51.6(C-7)，37.1(C-3)，27.1(C-2)，18.4(C-15)，17.2(C-4’)。
XX-4, colorless gum, C21H26O7，EI-MS m/z：413.17[M+Na]+；1H NMR(400MHz，CDCl3)：9.39(s，H-14)，6.74(dd，J＝10.1，7.4Hz，H-1)，6.46(d，J＝7.2，H-8)，6.13(d，J＝3.4Hz，H-13a)，6.03(s，H-3’a)，5.78(d，J＝3.4Hz，H-13b)，5.51(s，H-3’b)，5.19(t，J＝10.2Hz，H-6)，4.94(d，J＝10.2Hz，H-5)，4.38(d，J＝12.8Hz，H-15a)，4.28(d，J＝12.8Hz，H-15b)，3.90(dd，J＝8.3，2.1Hz，H-9)，3.28(m，H-5’a)，3.03(m，H-5’b)，2.75(m，H-2)，2.65(m，C-3a)，2.60(m，C-7)，2.51(m，C-2b)，1.94(m，H-3b)，1.86(s，H3-4’)，0.94(t，J＝7.0Hz，H3-6’).13C NMR(100MHz，CDCl3)：194.9(C-14)，169.8(C-12)，166.1(C-1’)，156.5(C-1)，141.2(C-10)，140.5(C-4)，135.9(C-2’)，134.2(C-11)，128.5(C-5)，125.9(C-3’)，122.1(C-13)，75.9(C-9)，74.0(C-6)，70.2(C-8)，64.4(C-5’)，60.5(C-15)，50.8(C-7)，32.6(C-3)，27.5(C-2)，18.3(C-4’)，14.9(C-6’)。
XX-5, colorless gum, C22H28O7，EI-MS m/z：427.20[M+Na]+；1H NMR(500MHz，CDCl3)：9.50(s，H-14)，6.76(t，J＝9.8Hz，H-1)，6.62(d，J＝8.4Hz，H-8)，6.27(d，J＝2.9Hz，H-13a)，6.04(q，J＝7.2Hz，H-3’)，5.90(s，H-13b)，5.19(t，J＝10.1Hz，H-6)，5.02(d，J＝10.6Hz，H-5)，4.46(d，J＝12.6Hz，H-15a)，4.35(dd，J＝12.9，4.1Hz，H-15b)，3.93(d，J＝8.3Hz，H-9)，3.37(dq，J＝9.0，7.0，H-6’a)，3.09(dq，J＝9.0，7.0，H-6’b)，2.84(dd，J＝12.5，5.6Hz，H-3a)，2.71(m，H-7)，2.64(d，J＝9.8Hz，H-2a)，2.58(dd，J＝14.4，12.5Hz，H-2b)，2.01(m，H-3b，1.95(d，J＝7.3Hz，H3-4’)，1.87(s，H3-5’)，1.05(t，J＝7.0Hz，H3-7’)。13C NMR(125MHz，CDCl3)：194.5(C-14)，169.6(C-12)，166.6(C-1’)，155.8(C-1)，141.6(C-4)，139.9(C-10)，138.2(C-3’)，134.0(C-11)，129.4(C-5)，127.5(C-2’)，122.8(C-13)，76.3(C-9)，74.0(C-6)，69.2(C-8)，64.6(C-6’)，60.9(C-15)，51.1(C-7)，32.7(C-3)，27.6(C-2)，20.7(C-5’)，15.8(C-7’)，15.1(C-4’)。
XX-6, colorless gum, C21H26O7，EI-MS m/z：427.20[M+Na]+；
+7(c 0.1，MeOH)；HRESIMS m/z 397.1623[M+Na]+(calcd for C21H26O6，397.1622)；UV(MeOH)λmax(log)195(0.32)，203(1.12)nm；IR(KBr)νmax 3372，2933，1714，1675，1459，1399，1031cm-1；1H NMR(400MHz，CDCl3)：6.23(d，J＝3.2Hz，H-13a)，6.09(dd，J＝9.3，1.9Hz，H-8)，6.03(brs，H-3’a)，5.76(t，J＝8.5Hz，H-1)，5.68(d，J＝3.2Hz，H-13b)，5.59(t，J＝1.5Hz，H-3’b)，5.44(d，J＝9.3Hz，H-9)，5.11(t，J＝11.0Hz，H-6)，5.04(d，J＝11.0Hz，H-5)，4.38(d，J＝12.5Hz，C-14a)，4.20(d，J＝12.5Hz，H-14b)，3.32(m，H-7)，2.42(m，H-2a)，2.29(m，C-2b)，2.00(d，J＝1.3Hz，H3-15)，1.92(s，H3-6’)，1.90(s，H3-4’)。13C NMR(100MHz，CDCl3)：169.8(C-12)，169.8(C-5’)，166.4(C-1’)，139.3(C-4)，136.3(C-10)，135.5(C-2’)，134.8(C-11)，134.5(C-1)，126.9(C-3’)，126.1(C-5)，121.4(C-13)，75.7(C-6)，72.8(C-9)，70.2(C-8)，64.2(C-14)，50.8(C-7)，37.9(C-3)，25.5(C-2)，21.0(C-6’)，18.4(C-4’)，17.0(C-15)。
XX-7, colorless glueForm C of20H28O6，EI-MS m/z：387.41[M+Na]+；1H NMR(400MHz，CDCl3)：6.79(m，H-3’)，6.26(d，J＝1.9Hz，H-13a)，5.68(m，H-13b)，5.33(m，H-8)，4.94(brs，H-6)，4.15(d，J＝4.3Hz，H-14)，3.44(dd，J＝5.4，5.3Hz，H2-15)，3.15(brs，H-7)，2.72(dd，J＝13.9，10.1Hz，H-9a)，2.59(dd，J＝13.9，5.4Hz，H-9b)，1.88(m，H-4)，1.78(d，J＝1.3Hz，H3-4’)，1.76(s，H3-5’)。13C NMR(100MHz，CDCl3)167.0(C-12)，167.4(C-1’)，139.0(C-3’)，136.9(C-11)，133.6(C-10)，131.3(C-1)，128.1(C-2’)，123.9(C-13)，79.2(C-6)，76.9(C-8)，68.1(C-15)，67.8(C-14)，42.2(C-7)，40.5(C-4)，31.0(C-5)，30.3(C-9)，26.6(C-2)，14.7(C-4’)，12.0(C-5’)。
XX-8, colorless gum, C19H26O6，EI-MS m/z：373.32[M+Na]+；1H NMR(400MHz，CDCl3)：6.28(d，J＝2.0Hz，H-13a)，6.04(s，H-3’)，5.69(d，H-13b)，5.67(m，H-1)，5.39(m，H-8)，4.93(ddd，J＝6.0，4.4，2.0Hz，H-7)，4.15(d，J＝4.3Hz，H2-14)，3.44(m，H2-15)，3.16(t，J＝2.2Hz，H-7)，2.74(dd，J＝13.9，10.2Hz，H-9a)，2.60(dd，J＝13.9，5.4Hz，H-9b)，2.30(m，H2-2)，2.02(m，H-5a)，1.95(dq,J＝7.3,1.6Hz,H3-5’),1.80(t,J＝1.5Hz,H3-4’)，1.55(ddd,J＝14.9,9.5,5.3Hz,H-5b)，1.19(m，H-3a)，1.14(m，H-3b)。13C NMR(100MHz，CDCl3)：170.0(C-12)，167.1(C-1’)，140.1(C-3’)，136.9(C-11)，133.5(C-10)，131.6(C-1)，127.3(C-2’)，123.9(C-13)，79.3(C-6)，76.4(C-8)，68.0(C-15)，67.8(C-14)，42.3(C-4)，40.6(C-5)，31.1(C-9)，30.4(C-3)，26.6(C-2)，20.5(C-5’)，15.9(C-4’)。
XX-9, colorless gum, C19H26O6，EI-MS m/z：373.32[M+Na]+；1H NMR(400MHz，CDCl3)：6.28(d，J＝2.0Hz，H-13)，6.04(t，J＝1.2Hz，H-3’a)，5.69(m，H-3)，5.57(t，J＝1.5Hz，H-6)，5.33(m，H-3’b)，4.93(ddd，J＝6.0，4.4，2.0Hz，H-8)，4.15(d，J＝4.3Hz，H2-15)，3.47(m，H-7)，3.45(m，H-14a)，3.16(t，J＝2.2Hz，H-14b)，2.74(dd，J＝13.9，10.2Hz，H-5a)，2.60(dd，J＝13.9，5.4Hz，H-5b)，2.30(m，H2-2)，2.06(m，H-9a)，2.02(m，H-1a)，1.95(m，H-10)，1.87(s，H3-4’)，1.55(m，H-9b)，1.20(m，H-1b)。13C NMR(100MHz，CDCl3)：169.9(C-12)，166.7(C-1’)，136.8(C-11)，135.7(C-2’)，133.4(C-4)，131.6(C-3)，126.9(C-3’)，124.0(C-13)，79.1(C-8)，77.4(C-6)，68.0(C-14)，67.9(C-15)，42.2(C-10)，40.5(C-9)，30.9(C-5)，30.3(C-1)，26.6(C-2)，18.3(C-4’)。
XX-10, colorless gum, C19H24O6，EI-MS m/z：371.18[M+Na]+；1H NMR(400MHz，CDCl3)：9.46(s，H-15)，6.72(t，J＝8.2Hz，H-3)，6.25(d，J＝2.1Hz，H-13a)，5.97(t，J＝1.2Hz，H-3’a)，5.67(d，J＝1.9Hz，H-13b)，5.62(s，H-3’b)，5.47(m，H-6)，4.76(brs，H-8)，3.44(m，H-14)，2.93(dd，J＝14.5，5.5Hz，H-5a)，2.78(brs，H-7)，2.65(m，H2-2)，2.64(m，H-5)，2.18(m，H-1a)，1.96(m，H-9a)，1.83(s，H3-4’)，1.41(m，H-9b)，1.38(m，H-1b)。13C NMR(100MHz，CDCl3)：194.6(C-15)，169.6(C-12)，166.2(C-1’)，157.5(C-3)，138.7(C-4)，135.6(C-2’)，126.6(C-3’)，124.5(C-13)，79.2(C-8)，75.1(C-6)，67.5(C-14)，42.1(C-10)，40.4(C-9)，30.2(C-1)，27.9(C-2)，27.4(C-5)，18.2(C-4’)。
Structural analysis of the novel compound:
XX-1, colorless gum. The high resolution mass spectrum shows that the ion peak is 397.1623[ M + Na [ ]]Indicate a molecular formula of C21H26O6The unsaturation degree was 9. Infrared Spectroscopy (IR) gave a hydroxyl group (3372 cm)-1) Aldehyde group (1714 cm)-1) And carbon-carbon double bonds (1675 cm)-1) Characteristic absorption peak of (1). The nuclear magnetic data of the compound is compared with the gemma type sesquiterpene lactone compound (1: (1) (R))10) E,4Z,6 α,8 β,9 α) -9-Ethoxy-6,15-dihydroxy-8- (2-methyacrylic) -14-oxomarccra-1 (10),4,11(13) -trieno-12,6-lactone (compound XX-4) are very similar, except that XX-1 lacks a hydroxyl group at C-15. This is represented by H in HMBC spectra3-15(H1.91) and C-3(C36.9) and C-5 (C: (C5)C127.3) to determine the planar structure thereof.
Relative configuration of Compound XX-1 by NMR1H and NOESY spectra, assuming that H-7 is in the beta orientation, then H-6: (H5.02)/H-7(H 2.59),H-7/H-8(H 6.55),H-8/H-9(H3.65) of the coupling constant (J)6,7＝10Hz,J7,8＝1.7Hz,J8,98.3Hz) shows H-6, H-8 and H-9 as α, β, α orientation, respectively, as also demonstrated in part by H-6/H-9 in the NOESY spectra. Furthermore H-1(H 6.74)/H-14(H9.44) and H-3(H 2.05)/H3-15(H1.91) NOESY correlation also determined the configuration of 1(10) E, 4E. The absolute configuration of compound 18 was confirmed by the method of calculating CD. Adopting a time-dependent density functional theory (TD-DFT) in quantum chemistry, using a B3LYP/6-31G (d, P) unit to carry out configuration optimization in air, selecting the PBE1PBE/6-31+ + G (2d,2P) unit level, and calculating the ECD of XX-1 by using methanol as a solvent. The calculated results and experimental ECD values were compared using SpecDis treatment transformations and the absolute configuration of XX-1 was determined to be 6S,7R,8R,9R by selecting the structure corresponding to the calculated value that more closely matched the experimental value. Thus, the structure of the compound XX-1 is determined as (6R,8S,9S) -9-Ethoxy-6-hydroxy-8-methyloxy-14-oxormacra-1 (10) E,4E,11(13) -triene-12, 6-lactone.
XX-6, colorless gum. The high resolution mass spectrum shows that the ion peak is 413.1570[ M + Na [ ]]+The molecular formula is shown as C21H26O7The unsaturation degree was 9. One-dimensional NMR spectrum shows that XX-6 has a structure similar to that of XX-1 and contains a germacrane type sesquiterpene lactone skeleton but has one more carbonyl group (C: (C) (C))C169.80 and a hydroxyl group (C)C64.21) lacks an aldehyde group. By HMBC spectrum H-9(H5.44) and C-1' ((II)C169.80) determination that the carbonyl group is at the 1' position, C-14(C64.21 and H-1 (C)H5.76) and H-9(H5.44) also confirmed a hydroxyl substitution on C-14. Due to the similarity of the one-dimensional NMR spectra of compounds XX-6 and XX-1 with the NOESY spectra, the relative configurations of the two are essentially the same, except that the NOESY of H-5/H-15 in XX-6 correlates with the 4Z configuration indicated. The absolute configuration of Compound XX-6 was confirmed by the same method for calculating its ECD as above. The absolute configuration of XX-6 is determined to be 6R, 7S, 8S, 9S. Thus, the structure of the compound XX-6 is determined as (1(10) E,4Z) -8S- (methacyloxy) -9S- (acetoyloxy) -6R,14-dihydroxy-germacra-1(10),4,11(13) -triene-12, 6-lactone.
Hydrocarbon remote correlation HMBC and hydrogen remote correlation COSY and NOESY of compounds XX-1, XX-6 are shown in FIG. 2, and ECD spectrum is shown in FIG. 3.
The structures of compounds XX-1 to XX-10 are shown in FIG. 1.
Example 2
And (3) determining the anti-osteoporosis activity of the compound.
First, cell culture
The assay used was shared to two cells: mouse primary Bone Marrow macrophages (Bone Marrow Macrophage, BMM) and RAW264.7 mononuclear macrophages.
(1) Isolated culture of BMM cells: taking 8-week-old female C57BL/6 mice, immersing the mice in 75% ethanol for sterilization for 10min after cervical vertebra dislocation, transferring the mice to a clean bench for dissecting and separating shin bones and thighbones of double hind limbs, cutting off redundant muscle tissues, and immersing the mice for 5min by using an alpha-MEM culture medium containing 1% double antibody. The joints at both ends of the bone were cut and placed in α -MEM complete medium containing 10% Fetal Bovine Serum (FBS) and 1% double antibody. The bone marrow cells were blown out with a 1mL syringe and filtered with a cell filter having a diameter of 40. mu.M. The cell suspension was transferred to a centrifuge tube and centrifuged at 1000rpm for 10min to remove the supernatant. The cells were resuspended in α -MEM containing 10% FBS and 25ng/mL macrophage colony stimulating factor (M-CSF) and plated onto 10cm diameter dishes. And after the cells adhere to the wall for 24 hours, removing the cells which do not adhere to the wall in the supernatant, blowing and falling off the cells at the bottom of the culture dish by using a new alpha-MEM complete culture medium, transferring the cells into a centrifuge tube, centrifuging at 1000rpm for 10min, collecting the BMM cells at the bottom of the centrifuge tube, and plating.
(2) Culture and passage of RAW264.7 cells: RAW264.7 cells were used for storage in this laboratory. After the cells were recovered, they were cultured in α -MEM medium containing 1% double antibody and 10% FBS. When the cells grow until the fusion degree is about 90%, passage is carried out, the original culture medium is sucked off, the cells are slightly blown down by the culture medium, and passage is carried out according to the ratio of 1: 4. All cells were exposed to 5% CO237 ℃ incubator.
Second, evaluation of cytotoxicity (CCK8 method)
(1) Adjusting the BMM cell suspension to 50000 cells/mL, inoculating the BMM cell suspension into a 96-well cell plate, wherein each well is 100 mu L, and the cell density is 5000 cells/well; after the cells are attached to the wall, the culture is continued for 12 h.
(2) Replacing the old culture medium with a drug-containing culture medium with a final compound concentration of 10 mu mol/L; 3 duplicate wells, control and zero wells (no cells) were set and incubation continued for 48 h.
(3) Discarding the old culture medium, and replacing with 100 μ L serum-free culture medium; the wells were protected from light, 10. mu.L of CCK-8 solution was added to each well, and incubated in an incubator for 2 hours.
(4) Taking out the culture plate, measuring absorbance value under the wavelength of 450nm, and calculating cell activity according to the absorbance of each hole, wherein the calculation method comprises the following steps:
third, osteoclast differentiation and TRAcP staining experiment
(1) Adjusting the BMM cell suspension to 50000 cells/mL, inoculating the BMM cell suspension into a 96-well cell plate, wherein each well is 100 mu L, and the cell density is 5000 cells/well; after the cells are attached to the wall, the culture is continued for 12 h.
(2) Preparing a compound to be tested by using a complete culture medium, and replacing an old culture medium with a medicine-containing culture medium with compound final concentrations of 0.03, 0.1, 0.3, 1, 3 and 10 mu mol/L; duplicate wells were set for 3, control and zero wells (no cells) were set. The model group and the administration group stimulate the differentiation of Osteoclast (OC) with RANKL of 50ng/mL, and the culture medium is replaced every other day until the model group is microscopically observed to form obvious OC after 4-5 days.
(3) TRAcP staining was performed.
a) Fixing: the medium in the 96-well plate was discarded, and 35. mu.L of 4% paraformaldehyde was added to each well, and the cells were fixed at room temperature for 2 hours.
b) Washing the plate: paraformaldehyde was discarded and 100. mu.L of ddH was used2O washes three times.
c) Dyeing: mu.L of TRAcP dye was added to each well and incubated at 37 ℃ for 1 hour.
d) And (4) observation: the number of mature osteoclasts was counted under a microscope at random 5 fields per well. Osteoclasts were identified as stained purple red under a light microscope and having a cell nucleus number of more than 3.
Fourth, luciferase reporter gene experiment
(1) Adjusting the density of Raw264.7 cell suspensions stably transfected by p-NF-kB-TA-Luc and p-NFAT-TA-Luc to 500000/mL respectively, and inoculating the cell suspensions in a 96-well cell plate, wherein each well is 100 mu L, and the cell density is 50000 per well; after the cells are attached to the wall, the culture is continued for 12 h.
(2) And (3) induction dosing: preparing a compound to be detected by using a complete culture medium, and replacing the old culture medium with a medicine-containing culture medium with the compound final concentration of 3 and 10 mu mol/L; 3 duplicate wells were set for each group, the control group was not treated with RANK, and the model and administered groups were stimulated with RANKL at 50ng/mL for 8 hours (for NF-. kappa.B transcriptional activity) and 12 hours (for NFATc1 transcriptional activity) on Raw264.7 cells.
(3) And (3) detection: according to
Single luciferase reporter gene detection system (Promega) instructions: a1 XPassive Lysis Buffer (PLB) cell lysate and a Luciferase Assay Reagent II (LARII) detection solution were prepared. The medium was discarded, and 1 Xpassive Lysis Buffer (20. mu.L/well) was added to the cell lysate and placed on a shaker and shaken for 20 min. 10 μ L of lysed sample was taken in an opaque 384-well white plate. Set the detection interval at 500ms to lyse the sample from the fluorescein reporter substrate 1: 1, 10 mu L of fluorescein reporter gene substrate is added and immediately placedAnd detecting the chemiluminescence value of the sample in a multifunctional microplate reader, wherein 8 samples are detected each time, and the time interval of each operation is unified. The fluorescence values obtained are the fluorescence generated after transcription and translation of NF-. kappa.B or NFAT vectors. And calculating the mean value according to the obtained numerical values, repeating the experiment for three times, and performing statistical analysis.
Fifth, statistical method
Data analysis was performed using GraphPad Prism 7.0, statistics expressed as mean ± Standard Deviation (SD), and differences between groups were compared using one-way analysis of variance (Bonferroni test). Statistical significance was considered when p <0.05 or < 0.01.
The inhibition rates of 10 compounds are shown in figure 4.
The compound shown in figure 1 has almost obvious differentiation inhibition activity on osteoclast BMM under 10 mu M, and has obvious inhibition effect on osteoclast BMM under 3 mu M. Further activity evaluation showed IC of most compounds50All are below 10.0. mu.M. In particular, IC of Compounds XX-1, XX-8, XX-9 and XX-10 against osteoblast differentiation Activity50All below 1 μ M, 0.51,0.80,0.50, and 0.83 μ M, respectively. Luciferase reporter gene experiments showed that compounds XX-1, XX-4, XX-6, XX-8, XX-9 were able to significantly inhibit NF- κ B and NFATc1 activation at a concentration of 10 μ M, as shown in figure 5, revealing that these compounds regulate osteoclast differentiation by inhibiting the transcriptional activity of NF- κ B and NFATc 1.
Example 3
The preparation of the compounds of the formulae (I), (II), (III) and (IV) is carried out as described in example 1, by adding water for injection and Tween 80, fine-filtering, filling and sterilizing to give injection.
Example 4
The compounds of the formulae (I), (II), (III) and (IV) are prepared as described in example 1, dissolved in sterile water for injection, stirred to dissolve and filtered through a sterile suction funnel. Then sterile fine filtering, subpackaging in ampoules, freeze-drying at low temperature, and aseptically sealing by melting to obtain powder for injection.
Example 5
The process of example 1 is followed to prepare the compounds of formulae (I), (II), (III) and (IV), which are mixed with excipients (e.g. starch slurry) in a ratio of 5:1 by weight, and the mixture is granulated and tabletted.
Example 6
The preparation of the compounds of formulae (I), (II), (III) and (IV) is carried out as described in example 1, and the capsules are prepared by adding the excipients in a ratio of 5:1 by weight to the excipients (e.g. polyethylene glycol 400).
Example 7
The preparation of the compounds of formulae (I), (II), (III) and (IV) is carried out as described in example 1, and the capsules are prepared by adding the excipients in a weight ratio of 3:1 to the excipients (e.g. Tween 80).
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A germacrane type sesquiterpene lactone compound is characterized by having a structural formula shown in formulas (I) to (IV):
wherein R is1Is H or OH; r2OH, OMearr or OEt; r3Is OAng, OH or OMeacr; r4Is OTig or OAng; r5Is CH2OH or CHO.
2. The method for preparing a germacrane type sesquiterpene lactone compound of claim 1, which comprises the steps of:
s1: drying aerial part of siegesbeckia Pubescens Makino, pulverizing, leaching, and concentrating under reduced pressure to obtain crude extract;
s2: suspending the crude extract, extracting, concentrating under reduced pressure, eluting with chromatographic column or sephadex column, and separating by chromatography and high performance liquid chromatography to obtain the germacrane type sesquiterpene lactone compounds.
3. The method according to claim 2, wherein the S1 is extracted by ethanol solution.
4. The method according to claim 3, wherein the volume fraction of the ethanol solution in S1 is 85-95%.
5. The preparation method according to claim 2, wherein the leaching in S1 is performed 3-4 times, and the time of a single leaching is 2-5 days.
6. The method according to claim 2, wherein the S2 is extracted with ethyl acetate.
7. The method according to claim 2, wherein the eluent eluted in S2 is a mixed solution of dichloromethane/methanol; the gradient of elution was: the volume ratio of dichloromethane/methanol is 0 → 40 min: 1:0 → 100:1, 40 → 80 min: 100:1 → 50:1, 80 → 120 min: 50:1 → 20:1, 120 → 160 min: 20:1 → 10: 1.
8. Use of the germacrane type sesquiterpene lactones according to claim 1 for the preparation of a medicament for the prevention and/or treatment of osteoporosis.
9. A medicament for preventing and/or treating osteoporosis, which comprises one or more of the germacrane type sesquiterpenoids according to claim 1, and pharmaceutically acceptable carriers or auxiliary materials; the mass fraction of the sesquiterpene compound and the pharmaceutically acceptable carrier or auxiliary material in the medicine is 0.1-99%.
10. The medicament of claim 9, wherein the medicament is in the form of an injection, a tablet or a capsule.
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| CN112876339A (en) * | 2021-01-29 | 2021-06-01 | 济南大学 | Preparation of sesquiterpenoids and application of sesquiterpenoids in synergism of fluconazole in resisting drug-resistant fungi |
| CN112876339B (en) * | 2021-01-29 | 2023-03-10 | 济南大学 | Preparation of sesquiterpenoids and application of sesquiterpenoids in synergism of fluconazole in resisting drug-resistant fungi |
| CN112876460A (en) * | 2021-02-05 | 2021-06-01 | 山西大学 | 7-diethylamino-3-acetyl coumarin derivative and synthetic method and application thereof |
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| CN115819409A (en) * | 2022-12-06 | 2023-03-21 | 江西省科学院应用化学研究所 | Gilmane type sesquiterpene lactone derivative and preparation method and application thereof |
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