CN112159378B - Gemmaxane type sesquiterpene lactone compound and preparation method and application thereof - Google Patents
Gemmaxane type sesquiterpene lactone compound and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a germacraneSesquiterpene lactone compounds and preparation method and application thereof. The germacrane sesquiterpene lactone compound has structural formulas shown in formulas (I) - (IV):wherein R is 1 Is H or OH; r is R 2 OH, OMeacr or OEt; r is R 3 Is OAng, OH or OMeacr; r is R 4 Is OTig or OAng; r is R 5 Is CH 2 OH or CHO. The germacrane sesquiterpenoids provided by the invention have obvious differentiation inhibition effect on RANKL-induced osteoclasts, the IC50 of most compounds is below 10.0 mu M, and the compounds do not show cytotoxicity at 10 mu M, so that the germacrane sesquiterpenoids can be applied to preparing medicines for treating and/or preventing osteoporosis, and have good application prospects.
Description
Technical Field
The invention relates to the technical field of compounds and medicines, in particular to a germacrane sesquiterpene lactone compound and a preparation method and application thereof.
Background
Osteoporosis (osteoporosis) is a systemic bone disease that is prone to fracture due to a decrease in bone density and bone mass, destruction of bone microstructure, and increased bone fragility, and is most common in menopausal women and the elderly. Due to the aggravation of aging population, osteoporosis has become a global public health problem, and the quality of life of middle-aged and elderly people is seriously affected.
The pathogenesis of osteoporosis is complex, but the dynamic balance maintained between bone formation under the action of osteoblasts and bone resorption mediated by osteoclasts is finally broken, so that the bone resorption is greater than the bone formation, and bone mass is lost to cause osteoporosis. RANKL/RANK/OPG and Wnt/β -catenin pathways are important pathways affecting bone metabolism (Dong Bingzi, sun Xiaofang. New developments in osteoporosis treatment from molecular mechanisms to drug targets [ J ]. Chinese osteoporosis and bone mineral diseases journal 2018,11 (06): 620-627), current drugs for osteoporosis regulate the balance between bone resorption and bone formation, mostly through key targets acting on these pathways. Many medicines are still in clinical trials and safety evaluation even basic research stages, most of the medicines on the market have single target points, narrow treatment range and adverse reactions, and the search for high-efficiency low-toxicity anti-osteoporosis medicines is needed in clinic.
Disclosure of Invention
The invention aims to overcome the defects or shortcomings of single target point, narrow treatment range and adverse reaction of the existing clinical anti-osteoporosis medicines, and provides a germacrane sesquiterpene lactone compound. The germacrane sesquiterpenoids provided by the invention have obvious differentiation inhibition effect on RANKL-induced osteoclasts, the IC50 of most compounds is below 10.0 mu M, and the compounds do not show cytotoxicity at 10 mu M, so that the germacrane sesquiterpenoids can be applied to preparing medicines for treating and/or preventing osteoporosis, and have good application prospects.
Another object of the present invention is to provide a process for producing the above-mentioned germamane sesquiterpene lactone compound.
The invention also aims to provide application of the germacrane sesquiterpene lactone compound in preparation of medicines for preventing or treating osteoporosis.
It is another object of the present invention to provide a medicament for preventing and/or treating osteoporosis.
In order to achieve the above object of the present invention, the present invention provides the following technical solutions:
a germacrane sesquiterpene lactone compound has a structural formula shown in formulas (I) - (IV):
wherein R is 1 Is H or OH; r is R 2 OH, OMeacr (methacryloyloxy) or OEt; r is R 3 OAng, OH or ome (methacryloyloxy); r is R 4 Is OTig or OAng; r is R 5 Is CH 2 OH or CHO.
The traditional Chinese medicine herba siegesbeckiae, which is a common traditional Chinese medicine in China, has the effects of dispelling wind-damp, benefiting joints, clearing heat and detoxicating, and is clinically used for treating rheumatalgia, muscle and bone weakness, soreness and weakness of waist and knees, quadriplegia, hemiplegia, rubella and wet sore and the like. Is recorded in the first part of the 2015 edition of Chinese pharmacopoeia.
Siegesbeckia orientalis Siegesbeckia orientalis l, siegesbeckia adenophora siegesbeckia pubescens m. And siegesbeckia pubescens Siegesbeckia glabrescens m. Are all used as source plants for the traditional Chinese herbs siegesbeckia orientalis, and the literature (Zhang Q R, zhong Z F, sang W, et al comparative comprehension on the anti-rheumatic Chinese herbal medicine Siegesbeckiae Herba: combined computational predictions and experimental investigations [ J ]. Journal ofEthnopharmacology,2018,228 ]) reports similar pharmacological activities but different chemical compositions. Siegesbeckiae herba is a plant of Siegesbeckiae herba of dicotyledonous Compositae. Distributed in northeast and southwest places of China. The materials contained in siegesbeckia orientalis mainly germacrane type, guaiane type sesquiterpene, pimarane type and kaurane type diterpene. Modern pharmacological research reveals that the monomeric compounds extracted from siegesbeckia plants have pharmacological effects of anti-inflammatory, antihypertensive, immunity-inhibiting, antibacterial, antimalarial and the like.
In the research of siegesbeckia orientalis, the inventor of the invention extracts a plurality of germacrane type sesquiterpene lactone compounds from the siegesbeckia orientalis, and the germacrane type sesquiterpene lactone compounds have obvious differentiation inhibition effect on RANKL-induced osteoclasts, and the IC of most compounds 50 Are all below 10.0 mu M. In particular, compounds XX-1, XX-8, XX-9, and XX-10 are IC's active against osteoclast differentiation 50 The efficacy evaluation of the above 4 compounds is below 1 mu M, which shows that the compounds can block the downstream NF- κB and NFATc1 channels and obviously inhibit osteoclast differentiation. And showed no cytotoxicity at 10 μm. The germacrane sesquiterpene compound extracted from siegesbeckia orientalis can be applied to preparation of medicines for treating and/or preventing osteoporosis, and has a good application prospect.
Preferably, said R 1 Is H, R 2 For OEt, R 3 Is OMeacr; or R is 4 OAng; or R is 5 Is CH 2 OH or CHO.
The preparation method of the germacrane sesquiterpene lactone compound comprises the following steps:
s1: drying aerial parts of siegesbeckia orientalis, pulverizing, leaching, 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 sesquiterpene lactone compound.
Preferably, the leaching in S1 is performed using an ethanol solution.
More preferably, the volume fraction of the ethanol solution in S1 is 85 to 95%.
Preferably, the number of leaches in S1 is 3-4, and the time of a single leaching is 2-5 days.
Preferably, ethyl acetate is used for extraction in S2.
Preferably, the eluent eluted in S2 is a dichloromethane/methanol mixed solution; the gradient of elution is: the volume ratio of dichloromethane/methanol is 0-40 min:1:0→100:1, 40→80min:100:1 to 50:1, 80 to 120min:50:1→20:1, 120→160min: 20:1.fwdarw.10:1.
Specifically, the germacrane sesquiterpene lactone compound is prepared through the following steps:
pulverizing dry aerial parts of siegesbeckia orientalis into small segments, leaching with 85-95% ethanol water solution for 3-4 times at room temperature for 2-5 days each time; concentrating the extractive solution under reduced pressure to obtain crude extract; suspending the extract in water, extracting with ethyl acetate for 3-6 times, concentrating under reduced pressure to obtain ethyl acetate part, subjecting to 200-300 mesh silica gel column chromatography, and gradient eluting with dichloromethane/methanol; the volume ratio of dichloromethane/methanol in the gradient elution is 0-40 min:1:0→100:1, 40→80min:100:1 to 50:1, 80 to 120min:50:1→20:1, 120→160min: 20:1.fwdarw.10:1. Purifying the fraction obtained in the steps by silica gel column chromatography, sephadex column chromatography and high performance liquid chromatography in sequence.
The application of the germacrane 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, wherein the medicament contains one or more of the germacrane sesquiterpenoids, pharmaceutically acceptable carriers or auxiliary materials; the mass fraction of the sesquiterpenoids, pharmaceutically acceptable carriers or auxiliary materials in the medicine is 0.1-99%.
The medicament for preventing and/or treating osteoporosis can be prepared into various existing medicament preparation types.
Preferably, the dosage form of the medicament is injection, tablet or capsule.
Compared with the prior art, the invention has the following advantages and effects:
the germacrane sesquiterpene lactone compound provided by the invention has obvious differentiation inhibition effect on RANKL-induced osteoclast, and most of compounds are IC 50 Are all below 10.0 mu M. In particular, compounds XX-1, XX-8, XX-9, and XX-10 are IC's active against osteoclast differentiation 50 The efficacy evaluation of the above 4 compounds is below 1 mu M, which shows that the compounds can block the downstream NF- κB and NFATc1 channels and obviously inhibit osteoclast differentiation. And showed no cytotoxicity at 10 μm. The germacrane sesquiterpene compound extracted from siegesbeckia orientalis can be applied to preparation of medicines for treating and/or preventing osteoporosis, and has a good application prospect.
Drawings
FIG. 1 shows the structures XX-1 to XX-10 of the compounds obtained by separation;
FIG. 2 is a main view of the compounds XX-1, XX-6 1 H- 1 HCOSY()，HMBC(/>) And NOESY (>) Correlation of
FIG. 3 is an ECD spectrum of compounds XX-1, XX-6;
FIG. 4 is a graph showing the results of inhibition of RANKL induced osteoclast by compounds XX-1 to XX-10; a: determining toxicity of XX-1 to XX-10 on BMM cells; b, C: effect of XX-1 to XX-10 on osteoclast differentiation at different concentrations; d: a TRAcP dyeing experimental result diagram of XX-1, XX-8, XX-9 and XX-10;
FIG. 5 is a graph showing the results of luciferase reporter experiments for compounds XX-1, XX-4, XX-6, XX-8, XX-9, XX-10.
Detailed Description
The present invention is further explained below with reference to examples and drawings, but the examples are not intended to limit the present invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
The reagents and materials used in the present invention are commercially available unless otherwise specified.
Example 1
XX-1 to XX-10 is prepared from siegesbeckia orientalis.
Drying aerial parts (5.0 kg) of siegesbeckia orientalis, pulverizing into small pieces, extracting with 95% ethanol water solution at room temperature for 3 times each for 3 days; concentrating the extractive solution under reduced pressure to obtain crude extract; suspending the extract in water, extracting with ethyl acetate for 5 times, concentrating under reduced pressure to obtain ethyl acetate part, subjecting to 200-300 mesh silica gel column chromatography, and eluting with dichloromethane/methanol according to gradient. The volume ratio of dichloromethane/methanol in the gradient elution is 0-40 min in sequence: 1:0→100:1, 40→80min:100:1 to 50:1, 80 to 120min:50:1→20:1, 120→160min: 20:1.fwdarw.10:1. After identification by thin layer chromatography, the fractions were separated and concentrated into 5 fractions (A to E). Fraction C (50 g) was subjected to reverse phase column chromatography, eluting with methanol/water (30% -100%), to give compound XX-4 (300 mg). Fraction C3 was eluted with methanol through a sephadex column and divided into three components C3.1 to C3.3, and C3.2 was separated by high performance liquid chromatography to give Compound XX-1 (30 mg), XX-3 (20 mg), XX-5 (5 mg), XX-6 (5 mg). C3.3 Compound XX-7 (8 mg), XX-8 (12 mg) and XX-9 (10 mg) were isolated by high performance liquid chromatography. C4 was separated by high performance liquid chromatography to give Compound XX-2 (20 mg), XX-10 (30 mg). The structure of the compound is shown in figure 1.
The physicochemical property data of the 10 monomer compounds are as follows:
XX-1, colorless gum, C 21 H 26 O 6 ，EI-MS m/z：397.13[M+Na] + ；+7(c 0.1，MeOH)；HRESIMS m/z397.1623[M+Na] + (calcdforC 21 H 26 O 6 ，397.1622)；UV(MeOH)λ max (logε)195(0.32)，203(1.12)nm；IR(KBr)ν max 3372，2933，1714，1675，1459，1399，1031cm -1 ； 1 HNMR(400MHz，CDCl 3 )δ：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，H 3 -15)，1.91(brs，H 3 -6’)，1.01(t，J＝7.0Hz，H 3 -2’). 13 CNMR(400MHz，CDCl 3 )δ：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, C 20 H 24 O 7 ，EI-MSm/z：385.22[M+Na] + ； 1 HNMR(400MHz，MeOD)δ：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’)； 13 CNMR(100MHz，MeOD)δ：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, C 19 H 22 O 6 ，EI-MS m/z：369.18[M+Na] + ； 1 HNMR(500MHz，CDCl 3 )：δ9.42(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，H 3 -15)，1.92(s，H 3 -4’)。 13 CNMR(125MHz，CDCl 3 ):δ194.4(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, C 21 H 26 O 7 ，EI-MS m/z：413.17[M+Na] + ； 1 HNMR(400MHz，CDCl 3 )δ：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，H 3 -4’)，0.94(t，J＝7.0Hz，H 3 -6’). 13 C NMR(100MHz，CDCl 3 )δ：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, C 22 H 28 O 7 ，EI-MS m/z：427.20[M+Na] + ； 1 HNMR(500MHz，CDCl 3 )δ：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，H 3 -4’)，1.87(s，H 3 -5’)，1.05(t，J＝7.0Hz，H 3 -7’)。 13 C NMR(125MHz，CDCl 3 )δ：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, C 21 H 26 O 7 ，EI-MS m/z：427.20[M+Na] + ；+7(c 0.1，MeOH)；HRESIMS m/z 397.1623[M+Na] + (calcd for C 21 H 26 O 6 ，397.1622)；UV(MeOH)λ max (logε)195(0.32)，203(1.12)nm；IR(KBr)ν max 3372，2933，1714，1675，1459，1399，1031cm -1 ； 1 HNMR(400MHz，CDCl 3 )δ：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，H 3 -15)，1.92(s，H 3 -6’)，1.90(s，H 3 -4’)。 13 C NMR(100MHz，CDCl 3 )δ：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 gum, C 20 H 28 O 6 ，EI-MS m/z：387.41[M+Na] + ； 1 HNMR(400MHz，CDCl 3 )δ：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，H 2 -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，H 3 -4’)，1.76(s，H 3 -5’)。 13 C NMR(100MHz，CDCl 3 )δ167.0(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, C 19 H 26 O 6 ，EI-MS m/z：373.32[M+Na] + ； 1 HNMR(400MHz，CDCl 3 )δ：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，H 2 -14)，3.44(m，H 2 -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，H 2 -2)，2.02(m，H-5a)，1.95(dq,J＝7.3,1.6Hz,H 3 -5’),1.80(t,J＝1.5Hz,H 3 -4’)，1.55(ddd,J＝14.9,9.5,5.3Hz,H-5b)，1.19(m，H-3a)，1.14(m，H-3b)。 13 C NMR(100MHz，CDCl 3 )δ：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, C 19 H 26 O 6 ，EI-MS m/z：373.32[M+Na] + ； 1 HNMR(400MHz，CDCl 3 )δ：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，H 2 -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，H 2 -2)，2.06(m，H-9a)，2.02(m，H-1a)，1.95(m，H-10)，1.87(s，H 3 -4’)，1.55(m，H-9b)，1.20(m，H-1b)。 13 C NMR(100MHz，CDCl 3 )δ：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, C 19 H 24 O 6 ，EI-MS m/z：371.18[M+Na] + ； 1 H NMR(400MHz，CDCl 3 )δ：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，H 2 -2)，2.64(m，H-5)，2.18(m，H-1a)，1.96(m，H-9a)，1.83(s，H 3 -4’)，1.41(m，H-9b)，1.38(m，H-1b)。 13 C NMR(100MHz，CDCl 3 )δ：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 resolution of the novel compounds:
XX-1, colorless gum. High resolution mass spectrum shows its ion peak 397.1623[ M+Na ]]++, hint molecular formula C 21 H 26 O 6 The unsaturation was 9. Infrared Spectroscopy (IR) gives hydroxy (3372 cm) -1 ) Aldehyde group (1714 cm) -1 ) And a carbon-carbon double bond (1675 cm) -1 ) Is characterized by an absorption peak. The nuclear magnetic data are very similar to those of the germamane sesquiterpene lactone compound (1 (10) E,4Z,6 alpha, 8 beta, 9 alpha) -9-Ethoxy-6, 15-dihydro-8- (2-methylacryloxy) -14-oxomacroa-1 (10), 4,11 (13) -trieno-12,6-lactone (compound XX-4), except that XX-1 lacks a hydroxyl group at C-15. This is explained by H in HMBC spectra 3 -15(δ H 1.91 And C-3 (delta) C 36.9 And C-5 (delta) C 127.3 To determine the planar structure thereof.
The relative configuration of Compound XX-1 is determined by NMR 1 H and NOESY spectra determined, assuming H-7 is beta oriented, then H-6 (delta H 5.02)/H-7(δ H 2.59),H-7/H-8(δ H 6.55),H-8/H-9(δ H 3.65 Coupling constant (J) 6,7 ＝10Hz,J 7,8 ＝1.7Hz,J 8,9 =8.3 Hz) shows that H-6,H-8 and H-9 are alpha, beta, alpha oriented, respectively, as also partially demonstrated by H-6/H-9 in the NOESY spectrum. In addition H-1 (delta) H 6.74)/H-14(δ H 9.44 And H-3 (delta) H 2.05)/H 3 -15(δ H 1.91 NOESY correlation of (1) (10) E, 4E). The absolute configuration of compound 18 was confirmed by the method of calculating CD. Using quantum chemistryThe time-density functional theory (TD-DFT) is adopted, a B3LYP/6-31G (d, p) unit is used for configuration optimization in the air, then the PBE1PBE/6-31++ G (2 d, 2P) unit level is selected, and the ECD of XX-1 is calculated by taking methanol as a solvent. The calculated and experimental ECD values were compared using SpecDis treatment transformation, and the structure corresponding to the calculated value that more closely matched the experimental value was selected to determine that XX-1 had an absolute configuration of 6S,7R,8R,9R. Thus, the structure of the compound XX-1 was determined to be (6R, 8S, 9S) -9-Ethoxy-6-hydroxy-8-metacryloxy-14-oxoermacra-1 (10) E,4E,11 (13) -triene-12,6-lactone.
XX-6, colorless gum. High resolution mass spectrum shows its ion peak 413.1570[ M+Na ]] + Prompt molecular formula C 21 H 26 O 7 The unsaturation was 9. One-dimensional NMR spectrum showed that XX-6 has a structure similar to XX-1 and contains one germamane type sesquiterpene lactone skeleton, but one more carbonyl group (delta) than XX-1 C 169.80 And a hydroxy group (delta) C 64.21 Lack one aldehyde group). By H-9 (delta) in HMBC spectra H 5.44 And C-1' (delta) C 169.80 Determination of the carbonyl group at the 1' position, C-14 (delta) C 64.21 And H-1 (delta) H 5.76 And H-9 (delta) H 5.44 Also corroborating the substitution of hydroxyl groups on C-14. Because of the degree of similarity of the one-dimensional NMR spectra of compounds XX-6 and XX-1 to the NOESY spectra, the relative configuration of the two is essentially the same, except for the 4Z configuration indicated by the NOESY correlation of H-5/H-15 in XX-6. The absolute configuration of compound XX-6 is confirmed by the same method as above for calculating its ECD. The absolute configuration of XX-6 was determined to be 6R,7S,8S,9S. The structure of compound XX-6 was thus defined as (1 (10) E, 4Z) -8S- (metacryloxy) -9S- (acetyloxy) -6R, 14-dihydro-germacra-1 (10), 4,11 (13) -triene-12,6-lactone.
Hydrocarbon remote related HMBC and hydrogen remote related COSY and NOESY related compounds XX-1, XX-6 are shown in fig. 2 and ecd spectra are shown in fig. 3.
The structures of the compounds XX-1 to XX-10 are shown in FIG. 1.
Example 2
The anti-osteoporosis activity of the compounds is measured.
1. Cell culture
The common use for this experiment was to two cells: mouse primary bone marrow macrophages (Bone Marrow Macrophage, BMM) and RAW264.7 mononuclear macrophages.
(1) Isolated culture of BMM cells: female C57BL/6 mice with the age of 8 weeks are taken, after dislocation of cervical vertebrae is killed, the mice are soaked in 75% ethanol for sterilization for 10min, then the mice are transferred to an ultra-clean workbench for dissecting and separating tibia and femur of double hind limbs, redundant muscle tissues are cut off, and alpha-MEM culture medium containing 1% double antibodies is used for soaking for 5min. The joints at both ends of the bone were cut out and placed in alpha-MEM complete medium containing 10% fetal bovine serum (fetal bovine serum, FBS) and 1% diabody. Bone marrow cells were blown out with a 1mL syringe and filtered through a cell filter with a diameter of 40 μm. The cell suspension was transferred to a centrifuge tube and centrifuged at 1000rpm for 10min, and the supernatant was removed. Cells were resuspended in alpha-MEM medium containing 10% FBS and 25ng/mL macrophage colony stimulating factor (macrophage colony-stimulating factor, M-CSF) and plated onto 10cm diameter dishes. After the cells were attached for 24 hours, the unattached cells in the supernatant were discarded, the cells at the bottom of the dish were blown off with new alpha-MEM complete medium, transferred to a centrifuge tube, centrifuged at 1000rpm for 10min, and BMM cells at the bottom of the centrifuge tube were collected and plated.
(2) Culture and passaging of RAW264.7 cells: RAW264.7 cells were stored for use in this laboratory. After resuscitating the cells, they were cultured in a-MEM medium containing 1% diabody and 10% FBS. When the cells grow to the fusion degree of about 90%, the cells are passaged, the original culture medium is sucked off, the cells are lightly blown down by the culture medium, and the passaged is carried out at the ratio of 1:4. All cells were exposed to 5% CO 2 Is a constant temperature incubator at 37 ℃.
2. Cytotoxicity evaluation (CCK 8 method)
(1) Adjusting BMM cell suspension to 50000/mL, inoculating into 96-well cell plates, wherein each well is 100 mu L, and the cell density is 5000/well; after the cells were attached, the culture was continued for 12 hours.
(2) Changing the old culture medium into a medicated culture medium with a final concentration of 10 mu mol/L; 3 duplicate wells were set, control and zeroing wells (no cells) were set and culture was continued for 48h.
(3) Discarding the old medium and replacing the old medium with 100 mu L of serum-free medium; in the dark, 10. Mu.L of CCK-8 solution was added to each well, and incubated in an incubator for 2 hours.
(4) The plates were removed, absorbance values were measured at a wavelength of 450nm, and cell viability was calculated from the absorbance of each well as follows:
3. osteoclast differentiation and TRAcP staining experiments
(1) Adjusting BMM cell suspension to 50000/mL, inoculating into 96-well cell plates, wherein each well is 100 mu L, and the cell density is 5000/well; after the cells were attached, the culture was continued for 12 hours.
(2) Preparing a compound to be tested by using a complete culture medium, and replacing the old culture medium with a drug-containing culture medium with the final concentration of the compound of 0.03, 0.1, 0.3, 1, 3 and 10 mu mol/L; 3 duplicate wells were set, control and zeroing wells (no cells) were set. Model and dosing groups Osteoclast (OC) differentiation was stimulated with RANKL at 50ng/mL, medium was changed every other day until after 4-5 days, and model differentiation was observed under the microscope to form distinct OC.
(3) TRAcP staining was performed.
a) Fixing: the medium in the 96-well plate was discarded, 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: the paraformaldehyde was discarded and 100. Mu.L of ddH was used 2 O was washed three times.
c) Dyeing: mu.L of TRAcP dye was added to each well and incubated at 37℃for 1 hour.
d) And (3) observation: the number of mature osteoclasts was counted by randomly selecting 5 fields/well under the microscope. The cells were stained mauve under an optical microscope and were considered osteoclasts with a nucleus number greater than 3.
4. Luciferase reporter assay
(1) The density of the stably transfected Raw264.7 cell suspensions of p-NF- κB-TA-Luc and p-NFAT-TA-Luc is adjusted to 500000/mL, and the cell suspensions are inoculated into 96-well cell plates, 100 μl per well and cell density of 50000 cells/well; after the cells were attached, the culture was continued for 12 hours.
(2) And (3) induction and dosing: preparing a compound to be tested by using a complete culture medium, and replacing the old culture medium with a drug-containing culture medium with the final concentration of the compound of 3 and 10 mu mol/L; 3 duplicate wells were placed in each group, control group was not treated with RANK, model group and dosing group stimulated raw264.7 cells with RANKL at 50ng/mL for 8 hours (assay of NF- κb transcriptional activity) and 12 hours (assay of NFATc1 transcriptional activity).
(3) And (3) detection: according to Dual-Shan Yingguang luciferase reporter gene detection system (Promega) instructions: 1X Passive Lysis Buffer (PLB) cell lysate and Luciferase Assay Reagent II (LAR II) assay were prepared. The medium was discarded, cell lysate 1X Passive Lysis Buffer (20. Mu.L/well) was added and the mixture was placed on a shaker for 20min. 10. Mu.L of the lysed sample was placed in an opaque 384 well whiteboard. The detection interval was set at 500ms to cleave the sample from the fluorescein reporter substrate 1:1, 10 mu L of fluorescein reporter gene substrate is added in proportion, and the mixture is immediately placed in a multifunctional enzyme-labeled instrument to detect the chemiluminescence values of the mixture, 8 samples are detected each time, and the time interval of each operation is unified. The resulting fluorescence value is that produced after transcription and translation of NF- κB or NFAT vectors. And calculating an average value according to the obtained values, repeating the experiment for three times, and carrying out statistical analysis.
5. Statistical method
Data analysis was performed using GraphPad Prism 7.0, statistical results were expressed as mean ± Standard Deviation (SD), and differences between groups were compared using one-way analysis of variance (Bonferroni test). A statistical significance is considered when p <0.05 or < 0.01.
The inhibition rates of the 10 compounds are shown in fig. 4.
The compound shown in FIG. 1 has a remarkable differentiation inhibition activity on the BMM of the osteoclast at 10 mu M and has a remarkable inhibition effect on the BMM of the osteoclast at 3 mu M. Further activity evaluation showed IC of most compounds 50 Are all below 10.0 mu M. In particular, compounds XX-1, XX-8, XX-9 and XX-10 are resistant to osteoclastsIC with cell differentiation activity 50 Are each below 1. Mu.M, 0.51,0.80,0.50, and 0.83. Mu.M, respectively. Luciferase reporter experiments showed that compounds XX-1, XX-4, XX-6, XX-8, XX-9 were able to significantly inhibit NF- κB and NFATc1 activation at a concentration of 10 μM, as shown in FIG. 5, revealing that these compounds modulate osteoclast differentiation by inhibiting NF- κB and NFATc1 transcriptional activity.
Example 3
The preparation of the injection solutions of the formulae (I), (II), (III), (IV) is carried out by the method of example 1, adding water for injection and Tween 80, fine filtering, packaging and sterilizing.
Example 4
The preparation of the compounds of the formula (I), (II), (III), (IV) is carried out as described in example 1, which is dissolved in sterile water for injection and stirred for dissolution and filtered with a sterile suction filter funnel. And then carrying out sterile fine filtration, subpackaging in ampoules, and carrying out low-temperature freeze drying and sterile sealing to obtain the powder injection.
Example 5
The process of example 1 is followed by the preparation of the formulations (I), (II), (III), (IV) by adding the excipient in a weight ratio of 5:1 to the excipient (e.g. starch slurry), granulating and tabletting.
Example 6
The process of example 1 is followed by the preparation of the formulations (I), (II), (III), (IV) by adding the excipient in a weight ratio of 5:1 to the excipient (e.g. polyethylene glycol 400) and making into capsules.
Example 7
The process of example 1 is followed by the preparation of the formulations (I), (II), (III), (IV) by adding the excipient in a weight ratio of 3:1 to the excipient (e.g. Tween 80) and making into capsules.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (8)
1. The application of the germacrane type sesquiterpene lactone compound in preparing a medicament for preventing and/or treating osteoporosis is characterized in that the germacrane type sesquiterpene lactone compound is a compound XX-1, a compound XX-4, a compound XX-5, a compound XX-6, a compound XX-7, a compound XX-8, a compound XX-9 or a compound XX-10:
。
2. the use according to claim 1, wherein the preparation method of the germacrane sesquiterpene lactone compound comprises the following steps:
s1: drying aerial parts of siegesbeckia orientalis, pulverizing, leaching, 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 sesquiterpene lactone compound.
3. Use according to claim 2, characterized in that S1 is leached with an ethanol solution.
4. The use according to claim 3, wherein the volume fraction of the ethanol solution in S1 is 85-95%.
5. The use according to claim 2, wherein the number of leaches in S1 is 3-4 and the time of a single leaching is 2-5 days.
6. Use according to claim 2, characterized in that in S2 extraction is performed with ethyl acetate.
7. Use according to claim 2, characterized in that the eluent eluted in S2 is a dichloromethane/methanol mixed solution; the gradient of elution is: the volume ratio of dichloromethane/methanol is 0-40 min:1:0→100:1, 40→80min:100:1 to 50:1, 80 to 120min:50:1→20:1, 120→160min: 20:1.fwdarw.10:1.
8. The use according to claim 1, wherein the medicament is in the form of an injection, a tablet or a capsule.
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