CN110498755A - Dihydro phenanthrene derivatives and its preparation method and application - Google Patents

Dihydro phenanthrene derivatives and its preparation method and application Download PDF

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CN110498755A
CN110498755A CN201810471340.0A CN201810471340A CN110498755A CN 110498755 A CN110498755 A CN 110498755A CN 201810471340 A CN201810471340 A CN 201810471340A CN 110498755 A CN110498755 A CN 110498755A
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mixed solvent
water
dihydro phenanthrene
methanol
phenanthrene derivatives
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李宁
陈刚
周地
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Shenyang Pharmaceutical University
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Abstract

The invention belongs to pharmaceutical technology fields, it is related to dihydro phenanthrene derivatives and its preparation method and application, more particularly to 7 new dihydro phenanthrene derivatives and preparation method thereof and the application in preparation prevention or treatment neurodegenerative disease drug field, the structure of the compound is as follows:

Description

Dihydro phenanthrene derivatives and its preparation method and application
Technical field
The invention belongs to pharmaceutical technology fields, and in particular in bletilla new dihydro phenanthrene derivatives and preparation method thereof and Using.
Background technique
Bletilla (Bletilla striata) is also known as Lian Jicao, Bai Gen, bletilla striata etc..It is mainly distributed on Guizhou, Sichuan, cloud South, Hunan, Hubei, Anhui etc. save.
Bletilla is as rare rare traditional Chinese medicine, bitter, sweet, puckery, cool in nature, return lung, stomach, Liver Channel, cure mainly hemoptysis, haematemesis, Traumatic hemorrhage, sore swollen toxin, chapped skin.It is apparent antitumor, anti-oxidant, anti-that modern pharmacology activity research shows that bletilla has Bacterium promotes the effects of wound healing, and chemical component is mainly bibenzyl, luxuriant and rich with fragrance class, dihydro phenanthrene class, joins luxuriant and rich with fragrance class, terpene, polysaccharide Deng.
Summary of the invention
The purpose of the present invention is to provide a series of dihydro phenanthrene derivatives and preparation method thereof and new medical usage.
New dihydro phenanthrene derivatives and its pharmaceutically acceptable salt, isomers or solvate provided by the invention, It has the following structure:
R1For hydrogen, hydroxyl, methoxyl group or pyrrolones ring；R2For hydroxyl, methoxyl group, para hydroxybenzene methyl or 4-O-glu- benzene Methyl；R3、R4、R5、R6、R7For hydrogen, hydroxyl or methoxyl group；
The invention particularly discloses following 7 particular compounds:
The present invention also provides the preparation methods of the new dihydro phenanthrene derivatives 1-7, and this method comprises the following steps:
(1) 70%~95% ethyl alcohol heating and refluxing extraction of the dry tuber of bletilla (Bletilla striata), recycling Extracting solution obtains crude extract；
(2) alcohol extracts obtained by step (1) are extracted after water dissolves with organic solvent, and the organic solvent is petroleum Ether, methylene chloride, ethyl acetate or n-butanol are successively extracted with the volume ratio 1:1 of water phase and organic phase, obtain opposed polarity Extract；
(3) gained acetic acid ethyl ester extract is separated through silica gel column chromatography in above-mentioned steps (2), with petroleum ether and acetic acid second Ester mixed solvent 100:10~1: 1, petroleum ether and acetone mixed solvent 100: 10~1: 1, chloroform and acetone mixed solvent 100: 1 ~100: 15, methylene chloride and acetone mixed solvent 100: 1~100: 15, chloroform and methanol mixed solvent 100:1~100:15, Methylene chloride and methanol mixed solvent 100:1~100:15 gradient elution；
(4) gained 100:1~100:25 flow point is mixed molten through ODS pillar layer separation in above-mentioned steps (3) with first alcohol and water Agent, or eluted using acetonitrile and water mixed solvent as eluent gradient；
(5) the middle gained first alcohol and water 3:7~9:1 of above-mentioned steps (4), acetonitrile and water 1:9~6:4 eluate are through preparative HPLC-UV is further separated, and with methanol and water mixed solvent 4:6~9:1, or with acetonitrile and water 3:7~6:4 mixed solvent is stream Dynamic phase gradient elution, obtains the racemic mixture of dihydro phenanthrene derivatives 3-5 and dihydro phenanthrene derivatives 1 and 2,6 and 7；
(6) racemic mixture of above-mentioned steps (5) obtained Dihydrophenanthrene is obtained through HPLC chiral resolution Compound 1 and 2,6 and 7.
The preparation method of the new dihydro phenanthrene derivatives 1-7 provided by the invention, extracting method described in step (1) To be heated to reflux ethyl alcohol extraction or heating ultrasonic extraction 1~3 time, the ethyl alcohol that solvent for use is 70%~95%, preferably 75%~ 95% ethyl alcohol.Medicinal material: the w/v of solvent is 1:5~1:25g/mL, preferably 1:10~1:15g/mL.
The preparation method of the new dihydro phenanthrene derivatives 1-7 provided by the invention, it is organic molten described in step (2) Agent extraction dissolves crude extract using water, according to volume ratio 1:1, respectively using petroleum ether, methylene chloride, ethyl acetate and just Butanol successively extracts 3-6 times, and preferably 5 times, the above organic solvent is recovered under reduced pressure.
The preparation method of the new dihydro phenanthrene derivatives 1-7 provided by the invention, eluting solvent described in step (3) The volume ratio of petroleum ether and ethyl acetate mixed solvent, petroleum ether and acetone mixed solvent is 100:10~1:1, preferably 10:1 ~10:4；Methylene chloride and acetone mixed solvent, chloroform and acetone mixed solvent, methylene chloride and methanol mixed solvent or chlorine The volume ratio of imitative and methanol mixed solvent is 100:1~100:15, preferably 100:1~100:8.
The preparation method of the new dihydro phenanthrene derivatives 1-7 provided by the invention, first alcohol and water described in step (4) The volume ratio of mixed solvent is 3:7~9:1, preferably 6:4~8:2；The volume ratio of acetonitrile and water mixed solvent is 1:9~6: 4, preferably 4:6~1:1.
The preparation method of the new dihydro phenanthrene derivatives 1-7 provided by the invention, first alcohol and water described in step (5) Mixed solvent, acetonitrile and water mixed solvent, the wherein volume ratio of methanol and water mixed solvent are as follows: 4:6~9:1, preferably 6:4~ 8:2；The volume ratio of acetonitrile and water mixed solvent is 3:7~6:4, preferably 4:6~1:1.
The preparation method of the new dihydro phenanthrene derivatives 1-7 provided by the invention, chiral chromatogram described in step (6) Column resolution solvent is n-hexane and dehydrated alcohol mixed solvent, and volume ratio is 70:30~95:5, preferably 75:25~80: 20。
The present invention induces BV2 microglia overactivity model with LPS, to the new dihydro phenanthrene derivatives being prepared The anti-neuroinflamation activity of 1-7 is evaluated.The results show that noval chemical compound 3 and 4 can be significant inhibit LPS induce mistake The release for spending the BV2 microglia NO of activation shows the anti-neuroinflamation activity of some strength, other compounds are to NO's Generation also shows certain inhibiting effect.Therefore, the new Dihydrophenanthrene prepared in the present invention can be in exploitation treatment mind Application in terms of through degenerative disease drug.
The present invention is provided for the first time using bletilla as raw material, preparation, the method for identifying 7 new dihydro phenanthrene derivatives, and is System has rated the activity in terms of its neuroprotection, illustrates its answering in terms of developing and treating neurodegenerative disease drug With.
Specific embodiment
The following examples will be further described the present invention, but not thereby limiting the invention.
Embodiment 1
(1) bletilla 900g is recovered under reduced pressure extracting solution and is obtained slightly with ethyl alcohol heating and refluxing extraction 3 times (dosage: 22.5L) of 75% Extract；
(2) ethanol extract obtained by step (1) is dissolved through water, successively with petroleum ether, methylene chloride, ethyl acetate and positive fourth Alcohol extraction, volume ratio 1:1, each solvent extraction 5 times obtain the extract at opposed polarity position；
(3) acetic acid ethyl ester extract in step (2), separates through silica gel column chromatography, successively mixed with petroleum ether and ethyl acetate Bonding solvent 100:3,100:8,100:10 elution；
(4) petroleum ether obtained in above-mentioned steps (3): ethyl acetate 100:8~100:10 flow point is through ODS chromatography, with 30: The mixed solvent gradient elution of 70,50:50,70:30,90:10 methanol-water；
(5) gained methanol-water (50:50~90:10) flow point is prepared through HPLC-UV chromatographic isolation in above-mentioned steps (4), 210nm detection, flow velocity 4mL/min, mobile phase is methanol: water=75:25 obtains the racemic mixture of dihydro phenanthrene class 1 and 2 (tR=40min) (yield 0.00010%)；Mobile phase is methanol: water=70:30, and the racemization for obtaining dihydro phenanthrene class 6 and 7 is mixed Close object (tR=50min) (yield 0.00010%)；The racemic mixture of compound 1 and 2 chiral pillar layer separation again, with N-hexane: ethyl alcohol (75:25) is that mobile phase affords 1 new (9.958min), and (yield is respectively 2 (11.304min) 0.00005%).The racemic mixture of compound 6 and 7 chiral pillar layer separation again, with n-hexane: ethyl alcohol (75:25) is stream Dynamic phase affords 6 new (8.958min), 7 (10.304min) (yield is respectively 0.00005%)
(6) middle gained methanol-water (70:30~90:10) flow point of above-mentioned steps (4) is through HPLC-UV chromatographic isolation, 210nm Detection, flow velocity 4mL/min obtain 3 (t of compound using the mixed solvent of 60:40 methanol-water as mobile phaseR=35min) it (receives 0.00013%) rate is.Fraction (50:50) HPLC-UV chromatographic isolation, 210nm detection, flow velocity 4mL/min, with 50:50 first Alcohol-water mixed solvent is mobile phase, obtains 4 (t of compoundR=35min) (yield 0.0001%) and 5 (t of compoundR= 25min) (yield 0.00014%).
Its structure is identified according to the physicochemical property of compound 1-7 and spectral data.
The Structural Identification data of compound 1 are as follows:
Purple powder (methanol).HR-ESI-MS provides quasi-molecular ion peak [M+H]+ M/z:326.1375 (calcd.326.1387for C19H20NO4), prompting its molecular formula is C19H19NO4。1HNMR(600MHz, CD3OD): δH7.88 (1H, d, J=8.4Hz, H-5), 6.59 (1H, dd, J=8.4,2.4Hz, H-6), 6.62 (1H, d, J= 2.4Hz, H-8) it is ABX Coupling System hydrogen signal on one group of phenyl ring, 6.46 (1H, s, H-3) are the hydrogen signal on phenyl ring；δH2.59-2.78 (4H, m, H-9,10) is Dihydrophenanthrene feature hydrogen signal；δH3.79(3H,s,4-OCH3) it is a first Oxygroup hydrogen signal, in addition there are also one group of hydrogen signals: δH5.36 (1H, m, H-5'), 2.58 (1H, m, H-3'a), 2.47 (1H, m, H-3'b), 2.28 (1H, m, H-4'a), 2.47 (1H, m, H-4'b) are 5 aliphatic hydrogen signals, include two groups of methylene hydrogen letters Number, it is one group of pyrrolidones hydrogen signal.13CNMR(150MHz,CD3OD 19 carbon signals: δ) are provided in spectrumC118.1 (C-1), 156.8 (C-2), 99.6 (C-3), 158.2 (C-4), 117.5 (C-4a), 130.4 (C-5), 126.0 (C-5a), 113.5 (C- 6), 156.2 (C-7), 114.4 (C-8), 140.6 (C-8a), 140.3 (C-10a) are 12 sp2Hydridization carbon signal；δC55.8(4- OCH3), 30.9 (C-9), 27.2 (C-10) they are fatty carbon signal；181.3 (C-2'), 32.3 (C-3'), 27.5 (C-4'), 53.2 (C-5') a penta azacyclo-pyrrolidones is formed.
The NMR of 1 compound 1 and 2 of table belongs to
All hydrogen carbon signals are belonged to using HSQC, and by HMBC spectrum that the position of substituent group is further true It is fixed.In HMBC spectrum, δH5.36 (H-5') and δC118.1 (C-1), 156.8 (C-2), 140.3 (C-10a) are long-range related, prompt Pyrrolidones is connected to C-1,3.79 (4-OCH3) to the long-range related prompt methoxyl group of 158.2 (C-4) C-4 are connected to, It is thus determined that the planar structure of the compound, absolute configuration further pass through with TDDFT (Time-dependent Density Functional Theory, density functional theory when Time Dependent) method carries out ECD and calculates to determine, first with 3.0 software of Discovery Studio, using Systematic search, energy is that 20kcal/mol carries out stable conformation.It finds excellent Gesture conformation carries out geometry optimization with Gaussian09 software.Calculated result equalizes (Boltzmann with Boltzmann method Averaged), it is since the fine difference of the selected preferential conformation calculated and practical conformation is made that map is not exactly the same At.The ECD map of spectrogram and (aR, the 5'S) that is calculated that experiment is measured is compared, the two spectrogram matches, table Negative cotton effect now is shown for the region 200-225nm and 250-325nm, shows positive cotton in the region 225-250nm Effect.It is thus determined that the absolute configuration of the compound is (aR, 5'S), it is retrieved as one and has no noval chemical compound reported in the literature, is ordered Entitled (aR, 5'S)-dusuanlansin F.
The Structural Identification data of compound 2 are as follows:
Purple powder (methanol).HR-ESI-MS provides quasi-molecular ion peak [M+H]+ M/z:326.1375 (calcd.326.1387for C19H20NO4), prompting its molecular formula is C19H19NO4.Compound 2 and 1 is one To enantiomter, hydrogen spectrum carbon modal data is shown in Table 1, and absolute configuration is further true compared with surveying ECD by calculating ECD It is fixed, compare with calculating (aS, 5'R), show as the region 200-225nm and 250-325nm positive cotton effect and The negative cotton effect in the region 225-250nm；Its cotton effect is with compound 1 completely on the contrary, determining that its absolute configuration is (aS, 5'R) is retrieved as one and has no noval chemical compound reported in the literature, be named as (aS, 5'R)-dusuanlansin F.
The Structural Identification data of compound 3 are as follows:
Yellow powder (MeOH).HR-ESI-MS provides quasi-molecular ion peak [M+H]+M/z:349.1451 (calcd.349.1434for C22H21O4), prompting its molecular formula is C22H20O4,1H NMR(400MHz,CD3OD low field area in) Hydrogen signal: δH6.52 (1H, s, H-4) be phenyl ring on hydrogen signal, 6.64 (2H, m, H-6,8), 8.05 (1H, d, J=8.8Hz, H-5 it) is coupled hydrogen signal for ABX on one group of phenyl ring, in addition there are also the hydrogen signals of one group of AA'BB' coupling: δH7.08 (2H, d, J= 8.4Hz, H-2', 6'), 6.63 (2H, d, J=8.4Hz, H-3', 5')；δH3.28(3H,s,1-OCH3) it is 1 methoxyl group hydrogen letter Number, 3.92 (2H, s, H-7') are the methylene hydrogen signal being connected with two phenyl ring, δH2.64 (4H, m, H-9,10) are dihydro phenanthrene 9,10 feature hydrogen signals of class compound.13CNMR(100MHz,CD3OD 22 carbon signals) are provided in spectrum, include 14 dihydro phenanthrenes Class parent nucleus carbon signal: δC158.1 (C-1), 121.8 (C-2), 155.5 (C-3), 111.6 (C-4), 120.3 (C-4a), 128.9 (C-5), 126.1 (C-5a), 114.3 (C-6), 156.5 (C-7), 115.2 (C-8), 140.6 (C-8a), 139.6 (C-10a), 30.9 (C-9), 31.0 (C-10), 6 phenyl ring carbon signals: 134.5 (C-1'), 130.4 (C-2', 6'), 115.7 (C-3', 5'), 156.0 (C-4'), high field region provide 1 methoxyl group carbon signal δC60.3(2-OCH3) and methylene rouge of 27.4 (C-7') Fat race carbon signal shows it for 2- (p-hydroxybenzyl) dihydrophenanthrene class compound.
The NMR of 2 compound 3 of table belongs to
The position to substituent group further will be composed by HMBC as shown in table by hydrocarbon signals assignment above by hsqc spectrum It is confirmed.In HMBC spectrum, 3.92 (2H, s, H-7') and 121.8 (C-2), 155.5 (C-3) and 158.1 (C-1) long-range phases It closes, shows that p-hydroxy benzyl segment is connected to C-2；δH3.28(3H,s,1-OCH3) and 158.1 (C-1) and δH 2.64 (4H, m, H-9,10) and δCThe long-range correlation of 158.1 (C-1), show the position of substitution of methoxyl group at C-1, therefore, The structure of the compound is determined, and is retrieved as one and has no noval chemical compound reported in the literature, is named as 2- (4- Hydroxybenzyl)-1-methoxy-9,10-dihydrophenanthrene-3,7-diol。
The Structural Identification data of compound 4 are as follows:
Yellow powder (MeOH).HR-ESI-MS provides quasi-molecular ion peak [M+NH4]+M/z:528.2246 (calcd.528.2234for C28H34NO9), prompting its molecular formula is C28H30O9。1H NMR(600MHz,CD3OD low field area in) Hydrogen signal: δH6.52 (1H, s, H-4) are the hydrogen signal on phenyl ring, 8.05 (1H, d, J=8.8Hz, H-5), 6.63 (2H, m, H- It 6,8) is ABX Coupling System hydrogen signal on one group of phenyl ring；δH3.28(3H,s,1-OCH3) be a methoxyl group hydrogen signal, 3.96 (2H, s, H-7') is a methylene hydrogen signal, and 2.63 (4H, s, H-9,10) are dihydro phenanthrene feature hydrogen signal, 4.83 (1H, d, J =7.2Hz, H-1 ") it is glucose end group hydrogen signal.13CNMR(150MHz,CD3OD 28 carbon signals: δ) are provided in spectrumC 158.1 (C-1), 121.5 (C-2), 155.6 (C-3), 111.6 (C-4), 120.3 (C-4a), 126.1 (C-5a), 128.9 (C- 5), 115.2 (C-6), 156.6 (C-7), 114.3 (C-8), 140.6 (C-8a), 139.8 (C-10a), 134.5 (C-1'), 130.5 (C-2', 6'), 117.4 (C-3', 5'), 157.1 (C-4') are 18 sp2Hydridization carbon signal；Other carbon signals: 102.5 (C-1 "), 75.0 (C-2 "), 78.0 (C-3 "), 71.4 (C-4 "), 78.1 (C-5 "), 62.5 (C-6 ") are one group of Glucose Carbon letter Number, δC60.3(1-OCH3), 31.2 (C-9), 31.4 (C-10) they are fatty carbon signal.
The NMR of 3 compound 4 of table belongs to
Further hydrocarbon signal is belonged to using hsqc spectrum, and further true to the position of substituent group by HMBC spectrum Card.δH3.28(1-OCH3) related to 158.1 (C-1's) prompt methoxyl group is connected to C-1,3.96 (H-7') and 121.5 (C- 2), the long-range related prompt p-hydroxybenzene of 134.5 (C-1') is connected to C-2, δH4.83 (H-1's ") and 157.1 (C-4') Long-range correlation shows that glucose is connected in C-4', is retrieved as one and has no noval chemical compound reported in the literature, is named as 2- (4- Hydroxybenzyl)-1-methoxy-9,10-dihydrophenanthrene-3,7-diol-4'-O-glucoside。
The Structural Identification data of compound 5 are as follows:
Yellow powder (MeOH).HR-ESI-MS provides quasi-molecular ion peak [M+H]+M/z:485.1957 (calcd.485.1959for C30H29O6), prompting its molecular formula is C30H28O6,1H NMR(600MHz,CD3OD low field area in) Hydrogen signal: δH6.56 (1H, s, H-3) are the hydrogen signal on phenyl ring, 6.58 (1H, d, J=2.4Hz, H-8), 8.04 (1H, d, J= 8.4Hz, H-5) and 6.62 (1H, dd, J=8.4,2.4Hz, H-6) be that ABX is coupled hydrogen signal on one group of phenyl ring, between one group of phenyl ring The hydrogen signal that position replaces: 6.93 (1H, t, J=7.8Hz, H-5 "), 6.49 (1H, dd, J=7.8,2.0Hz, H-4 "), 6.39 (2H, m, H-2 ", 6 "), one group of meta position are coupled hydrogen signal: 6.35 (2H, m, H-4', 6'), δH3.63(3H,s,3'-OCH3) and 3.89(3H,s,5'-OCH3) it is 2 methoxyl group hydrogen signals, 2.53 (4H, m) and 2.48 (2H, m), 2.27 (2H, m) are 8 rouge Fat race hydrogen signal is the feature hydrogen signal of dihydro phenanthrene and Bibenzyl compound, thus speculates that the compound should be a pair of horses going side by side of the two Close object.13CNMR(150MHz,CD3OD 30 carbon signals: δ) are provided in spectrumC116.3 (C-1), 158.9 (C-2), 100.6 (C- 3), 154.9 (C-4), 141.4 (C-4a), 130.3 (C-5), 140.7 (C-5a), 113.5 (C-6), 156.0 (C-7), 114.7 (C-8), 117.3 (C-8a), 126.6 (C-10a), 144.7 (C-1'), 117.3 (C-2'), 98.2 (C-4'), 158.2 (C- 5'), 109.2 (C-6'), 145.2 (C-1 "), 116.1 (C-2 "), 158.2 (C-3 "), 113.6 (C-4 "), 130.2 (C-5 "), 120.7 (C-6 ") are 24 sp2Hydridization carbon signal；High field region provides 2 methoxyl group carbon signal δC56.0(5'-OCH3) and 55.9 (3'-OCH3), 28.4 (C-9), 31.7 (C-10) and 37.7 (C- α '), 38.1 (C- α) are 4 aliphatic carbon signals.
The NMR of 4 compound 5 of table belongs to
It is as follows by hydrocarbon signals assignment of the hsqc spectrum to the compound, it is further composed by HMBC and its structure is carried out really Card.In HMBC spectrum, δH3.63(3'-OCH3) and δH3.89(5'-OCH3) respectively with δC160.3 (C-3') and 158.2 (C-5') Correlation and δH6.35 (H-4') and δC160.3 (C-3'), the correlation of 158.2 (C-5'), it is determined that two methoxyl groups take Subrogate and sets, δH2.48 (H-10) and δC116.3 (C-1), 2.53 (H- α ') and 117.3 (C-2'), 3.63 (3'-OCH3) with The long-range correlation of 117.3 (C-2') has determined the connection position of 9,10-dihydrophenanthrene and dihydrostilbene It is set to 1,2', one is retrieved as and has no noval chemical compound reported in the literature, be named as phochinenin M.
The Structural Identification data of compound 6 are as follows:
Brown powder (MeOH).HR-ESI-MS provides quasi-molecular ion peak [M-H]+ m/z∶419.1477(calcd.419.1500for C25H23O6), prompting its molecular formula is C25H24O6,1H NMR(600MHz, CD3OD low field area hydrogen signal in): δH6.31 (1H, d, J=2.0Hz, H-1) and 6.40 (1H, d, J=2.0Hz, H-3), one group pair Position coupling hydrogen signal: δH8.06 (1H, s, H-5), 6.67 (1H, s, H-8) and one group of ABX Coupling System hydrogen signal: δH6.77(1H, D, J=8.0Hz, H-5'), 6.83 (1H, dd, J=8.0,1.8Hz, H-6'), 6.95 (1H, d, J=1.8Hz, H-2')；δH3.81(3H,s,3'-OCH3), 3.82 (3H, s, 4-OCH3) it is two methoxyl group hydrogen signals, δH2.64-2.67(4H,m,H-9, It 10) is Bibenzyl compound methylene feature hydrogen signal, 5.49 (1H, d, J=5.4Hz, H-11), 3.47 (1H, m, H-12), 3.76 (1H, m, H-13a), 3.85 (1H, m, H-13b) are one group of five membered oxygen rings hydrogen signal；13CNMR(150MHz,CD3OD) in spectrum Provide 25 carbon signals: δC108.3 (C-1), 157.6 (C-2), 99.3 (C-3), 159.0 (C-4), 140.3 (C-4a), 125.8 (C-5), 142.0 (C-5a), 125.4 (C-6), 159.1 (C-7), 109.0 (C-8), 127.3 (C-8a), 116.9 (C- 10a), 135.4 (C-1'), 110.4 (C-2'), 149.5 (C-3'), 147.4 (C-4'), 116.1 (C-5'), 119.6 (C-6') For 18 sp2Hydridization carbon signal；δC56.4(3'-OCH3), 55.9 (4-OCH3) be two methoxyl group carbon signals, 31.6 (C-9), 31.8 (C-10) are fatty carbon signal, and 88.7 (C-11), 55.2 (C-12) are what phenyl ring was connected with dihydro phenanthrene with 65.6 (C-13) Five membered oxygen rings carbon signals.
The NMR of 5 compound 6 and 7 of table belongs to
Hydrocarbon signal is belonged to by hsqc spectrum, and structure is further confirmed by HMBC spectrum.In HMBC In spectrum, δH3.81(3'-OCH3), 6.95 (H-2') and 149.5 (C-3') and 3.82 (4-OCH3), 6.40 (H-3) and 159.0 (C-4) long-range correlation has determined the position of substitution of two methoxyl groups, 5.49 (H-11) and 159.1 (C-7), 135.4 (C-1'), 110.4 (C-2'), 3.47 (H-12) and 125.8 (C-6), 135.4 (C-1'), the long-range phase of 6.67 (H-8) and 31.6 (C-9) It closes, it is determined that the connection type and link position of five membered oxygen rings are at 6,7.In NOESY spectrum, it can be observed that δH 8.06(H- And δ 5)H 3.82(4-OCH3), 3.85 (H-13a) and 3.76 (H-13b) and δH6.67 (H-8) and δH 6.77(H-5')、 6.83 (H-6') are related, it is determined that the connection type and methylol of lactone ring five membered and dihydro phenanthrene and the link position of phenyl ring, separately Outside, 5.49 (H-11) it is related to 3.85 (H-13a) and 3.76 (H-13b) show C-11 and C-12 be it is trans-, determine the compound Relative configuration, the ECD spectrogram that absolute configuration further passes through actual measurement ECD spectrogram and (aS, 11S, the 12R) that calculates comes true Fixed, the two spectrogram matches, and shows be positive cotton effect and the area 205-250nm in the region 200-205nm and 250-310nm Domain shows the cotton effect that is negative, it is thus determined that its absolute configuration is (aS, 11S, 12R).It is retrieved as one and has no document report Noval chemical compound, be named as bletillin C1.
The Structural Identification data of compound 7 are as follows:
Brown powder (MeOH).HR-ESI-MS provides quasi-molecular ion peak [M-H]+ M/z:419.1477 (calcd.419.1500for C25H23O6), prompting its molecular formula is C25H24O6.Since compound 7 and 6 is A pair of of enantiomter, hydrogen spectrum carbon modal data are shown in Table 5, and absolute configuration further passes through actual measurement ECD spectrogram and calculating The ECD spectrogram of (aR, 11R, 12S) determines that the two spectrogram matches, show as in the region 200-205nm and 250-310nm Negative cotton effect and the positive cotton effect in the region 205-250nm, and with 6 spectrogram of compound completely on the contrary, by its absolute structure Type is determined as (aR, 11R, 12S).It is retrieved as one and has no noval chemical compound reported in the literature, be named as bletillin C2.
Embodiment 2
(1) bletilla 1000g is recovered under reduced pressure extracting solution and is obtained slightly with ethyl alcohol heating and refluxing extraction 2 times (dosage: 20L) of 80% Extract；
(2) ethanol extract obtained by step (1) is dissolved through water, successively with petroleum ether, methylene chloride, ethyl acetate and positive fourth Alcohol extraction, according to the volume ratio 1:1 of water phase and organic phase, each solvent extraction 3 times obtains the extract at opposed polarity position；
(3) acetic acid ethyl ester extract in step (2), separates through silica gel column chromatography, successively mixed with petroleum ether and ethyl acetate Bonding solvent 100:2,100:4,100:8,100:10 elution；
(4) petroleum ether obtained in above-mentioned steps (3): ethyl acetate 100:8~100:10 flow point is through ODS chromatography, with 30: The mixed solvent gradient elution of 70,50:50,60:40,70:30,90:10 methanol-water；
(5) gained methanol-water (50:50~60:40) flow point is prepared through HPLC-UV chromatographic isolation in above-mentioned steps (4), 210nm detection, flow velocity 4mL/min, mobile phase is methanol: water=75:25 obtains the racemic mixture of dihydro phenanthrene class 1 and 2 (tR=40min) (yield 0.00011%)；Mobile phase is methanol: water=75:25, and the racemization for obtaining dihydro phenanthrene class 6 and 7 is mixed Close object (tR=45min) (yield 0.00011%)；The racemic mixture of compound 1 and 2 chiral pillar layer separation again, with N-hexane: ethyl alcohol (75:25) is that mobile phase affords 1 new (9.958min), and (yield is respectively 2 (11.304min) 0.00005%).The racemic mixture of compound 6 and 7 chiral pillar layer separation again, with n-hexane: ethyl alcohol (77:23) is stream Dynamic phase affords 6 new (8.958min), 7 (10.304min) (yield is respectively 0.00005%)
(6) gained methanol-water (60:40) flow point is flowed through HPLC-UV chromatographic isolation, 210nm detection in above-mentioned steps (4) Speed obtains 3 (t of compound using the mixed solvent of 62:38 methanol-water as mobile phase for 4mL/minR=37min) (yield is 0.00012%).Fraction (50:50) HPLC-UV chromatographic isolation, 210nm detection, flow velocity 4mL/min, with 50:50 methanol-water Mixed solvent be mobile phase, obtain 4 (t of compoundR=38min) (yield 0.00011%) and 5 (t of compoundR=27min) (yield 0.00014%).
The Structural Identification method of dihydro phenanthrene derivatives 1-7 is shown in embodiment 1.
Embodiment 3
(1) bletilla 500g is with ethyl alcohol heating and refluxing extraction 3 times (dosage: 15L) of 85%, extracting solution is recovered under reduced pressure obtains and slightly mention Object；
(2) ethanol extract obtained by step (1) is dissolved through water, successively with petroleum ether, methylene chloride, ethyl acetate and positive fourth Alcohol extraction, the volume ratio 1:1 of water phase and organic phase, each solvent extraction 4 times obtain the extract at opposed polarity position；
(3) acetic acid ethyl ester extract in step (2), separates through silica gel column chromatography, is successively mixed with methylene chloride and acetone Solvent 100:2,100:4,100:6,100:8,100:10 elution；
(4) methylene chloride obtained in above-mentioned steps (3): acetone 100:6~100:8 flow point is through ODS chromatography, with 30:70, The mixed solvent gradient elution of 50:50,60:40,70:30,90:10 methanol-water；
(5) gained methanol-water (50:50~60:40) flow point is prepared through HPLC-UV chromatographic isolation in above-mentioned steps (4), 210nm detection, flow velocity 4mL/min, mobile phase is methanol: water=75:25 obtains the racemic mixture of dihydro phenanthrene class 1 and 2 (tR=42min) (yield 0.00010%)；Mobile phase is methanol: water=76:24, and the racemization for obtaining dihydro phenanthrene class 6 and 7 is mixed Close object (tR=45min) (yield 0.00011%)；The racemic mixture of compound 1 and 2 chiral pillar layer separation again, with N-hexane: ethyl alcohol (75:25) is that mobile phase affords 1 new (9.958min), and (yield is respectively 2 (11.304min) 0.00005%).The racemic mixture of compound 6 and 7 chiral pillar layer separation again, with n-hexane: ethyl alcohol (77:23) is stream Dynamic phase affords 6 new (8.908min), 7 (10.104min) (yield is respectively 0.00005%)
(6) gained methanol-water (60:40) flow point is flowed through HPLC-UV chromatographic isolation, 210nm detection in above-mentioned steps (4) Speed obtains 3 (t of compound using the mixed solvent of 62:38 methanol-water as mobile phase for 4mL/minR=35min) (yield is 0.00012%).Fraction (50:50) HPLC-UV chromatographic isolation, 210nm detection, flow velocity 4mL/min, with 53:47 methanol-water Mixed solvent be mobile phase, obtain 4 (t of compoundR=39min) (yield 0.00012%) and 5 (t of compoundR=27min) (yield 0.00011%).
The Structural Identification method of dihydro phenanthrene derivatives 1-7 is shown in embodiment 1.
Embodiment 4
(1) bletilla 1200g is recovered under reduced pressure extracting solution and is obtained slightly with ethyl alcohol heating and refluxing extraction 3 times (dosage: 15L) of 80% Extract；
(2) ethanol extract obtained by step (1) is dissolved through water, successively with petroleum ether, methylene chloride, ethyl acetate and positive fourth Alcohol extraction, water phase and organic phase volume ratio 1:1, each solvent extraction 5 times obtain the extract at opposed polarity position；
(3) acetic acid ethyl ester extract in step (2), separates through silica gel column chromatography, successively with chloroform and acetone mixed solvent 100:2,100:4,100:6,100:8 elution；
(4) chloroform obtained in above-mentioned steps (3): acetone 100:6~100:8 flow point is through ODS chromatography, with 30:70,50: The mixed solvent gradient elution of 50,60:40,70:30,90:10 methanol-water；
(5) gained methanol-water (50:50~60:40) flow point is prepared through HPLC-UV chromatographic isolation in above-mentioned steps (4), 210nm detection, flow velocity 4mL/min, mobile phase is methanol: water=75:25 obtains the racemic mixture of dihydro phenanthrene class 1 and 2 (tR=42min) (yield 0.00011%)；Mobile phase is methanol: water=76:24, and the racemization for obtaining dihydro phenanthrene class 6 and 7 is mixed Close object (tR=45min) (yield 0.00013%)；The racemic mixture of compound 1 and 2 chiral pillar layer separation again, with N-hexane: ethyl alcohol (76:24) is that mobile phase affords 1 new (9.403min), and (yield is respectively 2 (11.104min) 0.00005%).The racemic mixture of compound 6 and 7 chiral pillar layer separation again, with n-hexane: ethyl alcohol (77:23) is stream Dynamic phase affords 6 new (8.908min), 7 (10.104min) (yield is respectively 0.00006%)
(6) gained methanol-water (60:40) flow point is flowed through HPLC-UV chromatographic isolation, 210nm detection in above-mentioned steps (4) Speed obtains 3 (t of compound using the mixed solvent of 62:38 methanol-water as mobile phase for 4mL/minR=37min) (yield is 0.00011%).Fraction (50:50) HPLC-UV chromatographic isolation, 210nm detection, flow velocity 4mL/min, with 53:47 methanol-water Mixed solvent be mobile phase, obtain 4 (t of compoundR=39min) (yield 0.00013%) and 5 (t of compoundR=29min) (yield 0.00012%).
The Structural Identification method of dihydro phenanthrene derivatives 1-7 is shown in embodiment 1.
Embodiment 5
(1) bletilla 1200g is recovered under reduced pressure extracting solution and is obtained slightly with ethyl alcohol heating and refluxing extraction 3 times (dosage: 15L) of 80% Extract；
(2) ethanol extract obtained by step (1) is dissolved through water, successively with petroleum ether, methylene chloride, ethyl acetate and positive fourth Alcohol extraction, the volume ratio 1:1 of water phase and organic phase, each solvent extraction 5 times obtain the extract at opposed polarity position；
(3) acetic acid ethyl ester extract in step (2), separates through silica gel column chromatography, is successively mixed with methylene chloride and acetone Solvent 100:2,100:4,100:6,100:8 elution；
(4) methylene chloride obtained in above-mentioned steps (3): acetone 100:6~100:8 flow point is through ODS chromatography, with 30:70, The mixed solvent gradient elution of 50:50,60:40,70:30,90:10 methanol-water；
(5) gained methanol-water (50:50~60:40) flow point is prepared through HPLC-UV chromatographic isolation in above-mentioned steps (4), 210nm detection, flow velocity 4mL/min, mobile phase is methanol: water=75:25 obtains the racemic mixture of dihydro phenanthrene class 1 and 2 (tR=42min) (yield 0.00013%)；Mobile phase is methanol: water=76:24, and the racemization for obtaining dihydro phenanthrene class 6 and 7 is mixed Close object (tR=45min) (yield 0.00010%)；The racemic mixture of compound 1 and 2 chiral pillar layer separation again, with N-hexane: ethyl alcohol (76:24) is that mobile phase affords 1 new (9.403min), and (yield is respectively 2 (11.104min) 0.00006%).The racemic mixture of compound 6 and 7 chiral pillar layer separation again, with n-hexane: ethyl alcohol (77:23) is stream Dynamic phase affords 6 new (8.908min), 7 (10.104min) (yield is respectively 0.00005%)
(6) gained methanol-water (60:40) flow point is flowed through HPLC-UV chromatographic isolation, 210nm detection in above-mentioned steps (4) Speed obtains 3 (t of compound using the mixed solvent of 62:38 methanol-water as mobile phase for 4mL/minR=37min) (yield is 0.00013%).Fraction (50:50) HPLC-UV chromatographic isolation, 210nm detection, flow velocity 4mL/min, with 53:47 methanol-water Mixed solvent be mobile phase, obtain 4 (t of compoundR=39min) (yield 0.00014%) and 5 (t of compoundR=29min) (yield 0.00011%).The Structural Identification method of dihydro phenanthrene derivatives 1-7 is shown in embodiment 1.
Embodiment 6
(1) bletilla 1200g is recovered under reduced pressure extracting solution and is obtained slightly with ethyl alcohol heating and refluxing extraction 3 times (dosage: 15L) of 80% Extract；
(2) ethanol extract obtained by step (1) is dissolved through water, successively with petroleum ether, methylene chloride, ethyl acetate and positive fourth Alcohol extraction, the volume ratio 1:1 of water phase and organic phase, each solvent extraction 5 times obtain the extract at opposed polarity position；
(3) acetic acid ethyl ester extract in step (2), separates through silica gel column chromatography, successively with chloroform and methanol mixed solvent 100:2,100:4,100:6,100:7 elution；
(4) chloroform obtained in above-mentioned steps (3): methanol 100:6~100:8 flow point is through ODS chromatography, with 30:70,50: The mixed solvent gradient elution of 50,60:40,70:30,90:10 methanol-water；
(5) gained methanol-water (50:50~60:40) flow point is prepared through HPLC-UV chromatographic isolation in above-mentioned steps (4), 210nm detection, flow velocity 4mL/min, mobile phase is methanol: water=75:25 obtains the racemic mixture of dihydro phenanthrene class 1 and 2 (tR=42min) (yield 0.00011%)；Mobile phase is methanol: water=76:24, and the racemization for obtaining dihydro phenanthrene class 6 and 7 is mixed Close object (tR=45min) (yield 0.00011%)；The racemic mixture of compound 1 and 2 chiral pillar layer separation again, with N-hexane: ethyl alcohol (76:24) is that mobile phase affords 1 new (9.403min), and (yield is respectively 2 (11.104min) 0.00005%).The racemic mixture of compound 6 and 7 chiral pillar layer separation again, with n-hexane: ethyl alcohol (77:23) is stream Dynamic phase affords 6 new (8.908min), 7 (10.104min) (yield is respectively 0.00005%)
(6) gained methanol-water (60:40) flow point is flowed through HPLC-UV chromatographic isolation, 210nm detection in above-mentioned steps (4) Speed obtains 3 (t of compound using the mixed solvent of 62:38 methanol-water as mobile phase for 4mL/minR=37min) (yield is 0.00012%).Fraction (50:50) HPLC-UV chromatographic isolation, 210nm detection, flow velocity 4mL/min, with 53:47 methanol-water Mixed solvent be mobile phase, obtain 4 (t of compoundR=39min) (yield 0.00011%) and 5 (t of compoundR=29min) (yield 0.00014%).The Structural Identification method of dihydro phenanthrene derivatives 1-7 is shown in embodiment 1.
The anti-neuroinflamation active testing for the new dihydro phenanthrene derivatives 1-7 being prepared in 7 embodiment 1-6 of embodiment
(1) experimental principle: the chronic inflammatory reaction that Activated Microglia mediates is the generation of neurodegenerative disease, hair Important link during exhibition inhibits the activation of microglia to be likely to become a new target spot of drug discovery.LPS activation Microglia discharges NO, pro-inflammatory cytokine and active oxygen etc..This experiment activates the small colloid of BV2 by establishing external LPS It is anti-to evaluate new dihydro phenanthrene derivatives 1-7 to activate microglia release NO as index for the screening model of cell abnormal activation Scorching activity.
(2) experimental method:
1. the culture of mouse microglia system BV2
All glasswares used in cell culture and model foundation and metallic weapon (culture bottle, pipette, solution bottle Deng), pass through 121 DEG C of high pressure sterilization 30min, to completely remove the LPS of pollution.In being configured to based on DMEM culture medium Cell culture fluid containing 10% fetal calf serum and 50 μM of 2 mercapto ethanols.Microglia is with about 4 × 105Cells/ml's is dense Degree is in 5%CO2, secondary culture in 37 DEG C of culture bottles, until third day attached cell accounts for about culture bottle floor space 50-60%, with pancreas Enzymic digestion attached cell is passaged to another culture bottle.Using the BV2 after -80 DEG C of ultra low temperature freezer cryopreservation resuscitations as the first generation, choosing 3-8 is selected to be tested for BV2 cell.
2. method for preparation of drug
Test compound be it is powdered, dissolved with DMSO.It is made into mother liquor, concentration 50mM is stored in -20 DEG C.Face use When be diluted with DMEM culture solution, be successively diluted to 100 μM, 30 μM, 10 μM, 3 μM, 1 μM.DMSO final concentration < 1 ‰.
3. Griess method detection compound is to the inhibiting effect of LPS activation microglia
The BV2 microglia of logarithmic growth phase, with the fresh DMEM medium containing 5% fetal calf serum by cell density It is adjusted to 3 × 105Cells/ml is inoculated in 96 orifice plates, 100 μ l/well, in 37 DEG C, 5%CO2Incubator in culture.Cell Adhere-wall culture changes the fresh medium of serum-free into afterwards for 24 hours, while carrying out agent-feeding treatment.Every kind of compound set dosage 1,3,10, 30,100 μM and LPS collective effect.Blank control is set simultaneously.The final concentration of 100ng/ml of LPS in each administration group.After cell dosing After continuing culture for 24 hours, supernatant, NO in Griess colorimetric determination supernatant are collected2-Content.
4. influence of the mtt assay detection compound to microglia cell survival rate
The BV2 microglia of logarithmic growth phase culture, with the fresh DMEM medium containing 5% fetal calf serum by cell Density is adjusted to 3 × 105Cells/ml is inoculated in 96 orifice plates, 100 μ l/well, in 37 DEG C, 5%CO2Incubator in culture. Cell adhere-wall culture changes fresh medium into afterwards for 24 hours, while carrying out agent-feeding treatment.Every kind of compound set dosage 1,3,10,30, 100 μM and LPS collective effect.Blank control is set simultaneously.The final concentration of 100ng/ml of LPS in each administration group.Cell dosing is subsequent For 24 hours, MTT solution is then added in continuous culture into cell liquid, and 10 μ l/well are total at 37 DEG C by cell and 0.25mg/ml MTT With 3h is incubated for, culture solution is absorbed, the DMSO solution of 100 μ l is then added, measures its optical density OD value.Data processing utilizes enzyme It marks instrument corresponding software and carries out data processing, calculate the average value of 6 hole OD values of each sample, as follows using average value It calculates cell survival rate (cell viability, CV%).
Average value/blank control group OD value average value × 100% of cell survival rate %=sample sets OD value
5. statistical method
Whole data are tested analysis using SPSS (13.0) statistical package.As a result average value ± standard error table is used Show, evaluates globality difference, mean carries out homogeneity of variance analysis using One-Way ANOVA analytic approach between group, and combines Dunnett ' s test analysis method carries out comparison among groups.Multisample homogeneity test of variance is examined using Levene, works as p > 0.05, Variance be it is neat, using Dunnett ' s bilateral T examine multiple groups between mean difference, when p < 0.05, heterogeneity of variance, use Dunnett T3 examines the difference of mean between multiple groups.
⑥IC50Calculation method
The parameters such as each dosage and inhibiting rate nonlinear regression and fitting is calculated into IC50
(3) 3 experimental result: are shown in Table
The new dihydro phenanthrene derivatives 1-7 of table 6 inhibits Activated Microglia to act on experimental result
P < 0.001 * * Significance:*P < 0.05, * * P < 0.01, * is compared with LPS induction group；###P < 0.001 with it is right Is compared according to group
As a result it is found that the new Dihydrophenanthrene 3 and 4 (100 μM) being prepared in embodiment 1-6 can significantly press down The release of the BV2 microglia NO of the overactivity of LPS induction processed；1,2,5,6 and 7 is right in higher test concentrations (100 μM) The release of NO also has certain inhibiting effect.

Claims (10)

1. dihydro phenanthrene derivatives and its pharmaceutically acceptable salt, isomers or solvate, which is characterized in that have as follows General structure:
R1For hydrogen, hydroxyl, methoxyl group or pyrrolones ring；R2For hydroxyl, methoxyl group, para hydroxybenzene methyl or 4-O-glu- benzyl； R3、R4、R5、R6、R7For hydrogen, hydroxyl or methoxyl group.
2. following dihydro phenanthrene derivatives and its pharmaceutically acceptable salt, isomers or solvate, which is characterized in that its For with one or more of flowering structure,
3. according to the preparation method of dihydro phenanthrene derivatives and its pharmaceutically acceptable salt described in claim 2, feature exists In:
This method comprises the following steps:
(1) plant bletilla (Bletilla striata) ethonal extraction, recycling extracting solution obtain crude extract；
(2) crude extract obtained by step (1) is dissolved through water, and organic solvent extraction obtains the extract of opposed polarity；
(3) gained acetic acid ethyl ester extract is separated through silica gel column chromatography in above-mentioned steps (2), mixed with petroleum ether and ethyl acetate Bonding solvent, petroleum ether and acetone mixed solvent, chloroform and acetone mixed solvent, methylene chloride and acetone mixed solvent, chloroform and Methanol mixed solvent, methylene chloride and methanol mixed solvent gradient elution；
(4) 100: 1~100: 25 flow points of gained are through ODS pillar layer separation in above-mentioned steps (3), with methanol and water mixed solvent, Or it is eluted using acetonitrile and water mixed solvent as eluent gradient；
(5) in above-mentioned steps (4) gained first alcohol and water 3: 7~9: 1, acetonitrile and 1: 9~6: 4 eluate of water through HPLC-UV into one Step separation with methanol and water mixed solvent, or using acetonitrile and water mixed solvent as eluent gradient elution, obtains dihydro phenanthrene class and spreads out Biological 3-5 and the racemic mixture of dihydro phenanthrene derivatives 1 and 2,6 and 7；
(6) chemical combination that the racemic mixture of above-mentioned steps (5) obtained Dihydrophenanthrene is obtained through HPLC chiral resolution Object 1 and 2 and 6 and 7.
4. the preparation method of dihydro phenanthrene derivatives described in accordance with the claim 3, it is characterised in that: described in step (1) Extracting method is heating and refluxing extraction or heating ultrasonic extraction 1~3 time, and the volumetric concentration of ethyl alcohol is 70%~95%, bletilla: second The w/v of alcohol is 1: 5~1: 25g/mL；Organic solvent extractionprocess described in step (2), according to water phase and organic phase Volume ratio 1: 1, successively extracted 3-6 times using petroleum ether, methylene chloride, ethyl acetate and n-butanol respectively.
5. the preparation method of dihydro phenanthrene derivatives described in accordance with the claim 3, it is characterised in that: step is washed described in (3) The volume ratio of desolventizing petroleum ether and ethyl acetate mixed solvent, petroleum ether and acetone mixed solvent is 100: 10~1: 1, Methylene chloride and acetone mixed solvent, chloroform and acetone mixed solvent, methylene chloride and methanol mixed solvent or chloroform and methanol Mixed solvent volume ratio be 100: 1~100: 15.
6. the preparation method of dihydro phenanthrene derivatives described in accordance with the claim 3, it is characterised in that: flowed described in step (4) The volume ratio of dynamic phase methanol and water mixed solvent is 3: 7~9: 1, and the volume ratio of acetonitrile and water mixed solvent is 1: 9~6: 4。
7. the preparation method of dihydro phenanthrene derivatives described in accordance with the claim 3, it is characterised in that: first described in step (5) Pure and mild water mixed solvent, acetonitrile and water mixed solvent, the wherein volume ratio of methanol and water mixed solvent are as follows: 4: 6~9: 1, second The volume ratio of nitrile and water mixed solvent is 3: 7~6: 4.
8. the preparation method of dihydro phenanthrene derivatives described in accordance with the claim 3, it is characterised in that: hand described in step (6) Property to split the solvent that uses be n-hexane and dehydrated alcohol mixed solvent, volume ratio 70: 30~95: 5.
9. a kind of pharmaceutical composition includes dihydro phenanthrene derivatives of any of claims 1 or 2 and its pharmaceutically acceptable salt And pharmaceutically acceptable carrier.
10. dihydro phenanthrene derivatives of any of claims 1 or 2 and its pharmaceutically acceptable salt or as claimed in claim 9 Application of the pharmaceutical composition in preparation prevention or treatment neurodegenerative disease drug.
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