CN113683507A - Guaiane sesquiterpene derivative in regachia and application thereof - Google Patents
Guaiane sesquiterpene derivative in regachia and application thereof Download PDFInfo
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- 150000000176 guaiane derivatives Chemical class 0.000 title claims abstract description 27
- 238000002360 preparation method Methods 0.000 claims abstract description 15
- 206010029240 Neuritis Diseases 0.000 claims abstract description 14
- 239000003814 drug Substances 0.000 claims abstract description 12
- 238000004440 column chromatography Methods 0.000 claims abstract description 11
- 229940079593 drug Drugs 0.000 claims abstract description 10
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 7
- 101100313763 Arabidopsis thaliana TIM22-2 gene Proteins 0.000 claims abstract description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 36
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 23
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 22
- 229940125904 compound 1 Drugs 0.000 claims description 21
- 229940126214 compound 3 Drugs 0.000 claims description 13
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 12
- 229940125782 compound 2 Drugs 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 10
- 238000010828 elution Methods 0.000 claims description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- 238000011894 semi-preparative HPLC Methods 0.000 claims description 6
- 238000010898 silica gel chromatography Methods 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 4
- 239000004952 Polyamide Substances 0.000 claims description 3
- 241001646984 Thamnolia Species 0.000 claims description 3
- 239000003208 petroleum Substances 0.000 claims description 3
- 239000008194 pharmaceutical composition Substances 0.000 claims description 3
- 229920002647 polyamide Polymers 0.000 claims description 3
- 238000002953 preparative HPLC Methods 0.000 claims description 3
- 238000010992 reflux Methods 0.000 claims description 3
- 238000013375 chromatographic separation Methods 0.000 claims description 2
- 235000008216 herbs Nutrition 0.000 claims description 2
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims description 2
- 150000001875 compounds Chemical class 0.000 abstract description 33
- 230000000694 effects Effects 0.000 abstract description 8
- 241001534930 Thymelaeaceae Species 0.000 abstract description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 abstract 1
- 238000004128 high performance liquid chromatography Methods 0.000 abstract 1
- 239000000741 silica gel Substances 0.000 abstract 1
- 229910002027 silica gel Inorganic materials 0.000 abstract 1
- 229910052799 carbon Inorganic materials 0.000 description 14
- 238000001228 spectrum Methods 0.000 description 9
- 239000000126 substance Substances 0.000 description 9
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 8
- 241000934856 Daphne Species 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 8
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 7
- 238000005160 1H NMR spectroscopy Methods 0.000 description 7
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 6
- 238000004364 calculation method Methods 0.000 description 6
- 125000002837 carbocyclic group Chemical group 0.000 description 6
- 239000013078 crystal Substances 0.000 description 6
- 238000005481 NMR spectroscopy Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 5
- 238000000990 heteronuclear single quantum coherence spectrum Methods 0.000 description 5
- 210000000274 microglia Anatomy 0.000 description 5
- 229930004725 sesquiterpene Natural products 0.000 description 5
- 150000004354 sesquiterpene derivatives Chemical class 0.000 description 5
- 238000001026 1H--1H correlation spectroscopy Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000001052 heteronuclear multiple bond coherence spectrum Methods 0.000 description 4
- UNILWMWFPHPYOR-KXEYIPSPSA-M 1-[6-[2-[3-[3-[3-[2-[2-[3-[[2-[2-[[(2r)-1-[[2-[[(2r)-1-[3-[2-[2-[3-[[2-(2-amino-2-oxoethoxy)acetyl]amino]propoxy]ethoxy]ethoxy]propylamino]-3-hydroxy-1-oxopropan-2-yl]amino]-2-oxoethyl]amino]-3-[(2r)-2,3-di(hexadecanoyloxy)propyl]sulfanyl-1-oxopropan-2-yl Chemical compound O=C1C(SCCC(=O)NCCCOCCOCCOCCCNC(=O)COCC(=O)N[C@@H](CSC[C@@H](COC(=O)CCCCCCCCCCCCCCC)OC(=O)CCCCCCCCCCCCCCC)C(=O)NCC(=O)N[C@H](CO)C(=O)NCCCOCCOCCOCCCNC(=O)COCC(N)=O)CC(=O)N1CCNC(=O)CCCCCN\1C2=CC=C(S([O-])(=O)=O)C=C2CC/1=C/C=C/C=C/C1=[N+](CC)C2=CC=C(S([O-])(=O)=O)C=C2C1 UNILWMWFPHPYOR-KXEYIPSPSA-M 0.000 description 3
- 208000036110 Neuroinflammatory disease Diseases 0.000 description 3
- PNUZDKCDAWUEGK-CYZMBNFOSA-N Sitafloxacin Chemical compound C([C@H]1N)N(C=2C(=C3C(C(C(C(O)=O)=CN3[C@H]3[C@H](C3)F)=O)=CC=2F)Cl)CC11CC1 PNUZDKCDAWUEGK-CYZMBNFOSA-N 0.000 description 3
- KGNDCEVUMONOKF-UGPLYTSKSA-N benzyl n-[(2r)-1-[(2s,4r)-2-[[(2s)-6-amino-1-(1,3-benzoxazol-2-yl)-1,1-dihydroxyhexan-2-yl]carbamoyl]-4-[(4-methylphenyl)methoxy]pyrrolidin-1-yl]-1-oxo-4-phenylbutan-2-yl]carbamate Chemical compound C1=CC(C)=CC=C1CO[C@H]1CN(C(=O)[C@@H](CCC=2C=CC=CC=2)NC(=O)OCC=2C=CC=CC=2)[C@H](C(=O)N[C@@H](CCCCN)C(O)(O)C=2OC3=CC=CC=C3N=2)C1 KGNDCEVUMONOKF-UGPLYTSKSA-N 0.000 description 3
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- 238000002212 electronic circular dichroism spectrum Methods 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
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- 238000001819 mass spectrum Methods 0.000 description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 3
- 230000003959 neuroinflammation Effects 0.000 description 3
- 125000003003 spiro group Chemical group 0.000 description 3
- 208000024827 Alzheimer disease Diseases 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 2
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 2
- 210000003169 central nervous system Anatomy 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
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- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 2
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- SNVLJLYUUXKWOJ-UHFFFAOYSA-N methylidenecarbene Chemical group C=[C] SNVLJLYUUXKWOJ-UHFFFAOYSA-N 0.000 description 2
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- 230000003287 optical effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- ZWEHNKRNPOVVGH-UHFFFAOYSA-N 2-Butanone Chemical group CCC(C)=O ZWEHNKRNPOVVGH-UHFFFAOYSA-N 0.000 description 1
- IGRCWJPBLWGNPX-UHFFFAOYSA-N 3-(2-chlorophenyl)-n-(4-chlorophenyl)-n,5-dimethyl-1,2-oxazole-4-carboxamide Chemical compound C=1C=C(Cl)C=CC=1N(C)C(=O)C1=C(C)ON=C1C1=CC=CC=C1Cl IGRCWJPBLWGNPX-UHFFFAOYSA-N 0.000 description 1
- 206010008111 Cerebral haemorrhage Diseases 0.000 description 1
- 238000004057 DFT-B3LYP calculation Methods 0.000 description 1
- 241001310717 Daphne genkwa Species 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 208000030886 Traumatic Brain injury Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000002441 X-ray diffraction Methods 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
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- 230000005784 autoimmunity Effects 0.000 description 1
- 125000002619 bicyclic group Chemical group 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000012230 colorless oil Substances 0.000 description 1
- 238000005100 correlation spectroscopy Methods 0.000 description 1
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- 238000010586 diagram Methods 0.000 description 1
- SNRUBQQJIBEYMU-NJFSPNSNSA-N dodecane Chemical group CCCCCCCCCCC[14CH3] SNRUBQQJIBEYMU-NJFSPNSNSA-N 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 description 1
- -1 methylene hydrogen Chemical compound 0.000 description 1
- GRVDJDISBSALJP-UHFFFAOYSA-N methyloxidanyl Chemical compound [O]C GRVDJDISBSALJP-UHFFFAOYSA-N 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000002314 neuroinflammatory effect Effects 0.000 description 1
- 239000004090 neuroprotective agent Substances 0.000 description 1
- 230000000324 neuroprotective effect Effects 0.000 description 1
- BKIMMITUMNQMOS-UHFFFAOYSA-N nonane Chemical group CCCCCCCCC BKIMMITUMNQMOS-UHFFFAOYSA-N 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- AICOOMRHRUFYCM-ZRRPKQBOSA-N oxazine, 1 Chemical compound C([C@@H]1[C@H](C(C[C@]2(C)[C@@H]([C@H](C)N(C)C)[C@H](O)C[C@]21C)=O)CC1=CC2)C[C@H]1[C@@]1(C)[C@H]2N=C(C(C)C)OC1 AICOOMRHRUFYCM-ZRRPKQBOSA-N 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- LZPZPHGJDAGEJZ-AKAIJSEGSA-N regadenoson Chemical compound C1=C(C(=O)NC)C=NN1C1=NC(N)=C(N=CN2[C@H]3[C@@H]([C@H](O)[C@@H](CO)O3)O)C2=N1 LZPZPHGJDAGEJZ-AKAIJSEGSA-N 0.000 description 1
- 229960003614 regadenoson Drugs 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
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- 238000004611 spectroscopical analysis Methods 0.000 description 1
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- 239000006228 supernatant Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 230000009529 traumatic brain injury Effects 0.000 description 1
- 239000011715 vitamin B12 Substances 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
- C07C69/74—Esters of carboxylic acids having an esterified carboxyl group bound to a carbon atom of a ring other than a six-membered aromatic ring
- C07C69/757—Esters of carboxylic acids having an esterified carboxyl group bound to a carbon atom of a ring other than a six-membered aromatic ring having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
- C07C49/587—Unsaturated compounds containing a keto groups being part of a ring
- C07C49/703—Unsaturated compounds containing a keto groups being part of a ring containing hydroxy groups
- C07C49/743—Unsaturated compounds containing a keto groups being part of a ring containing hydroxy groups having unsaturation outside the rings, e.g. humulones, lupulones
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2602/00—Systems containing two condensed rings
- C07C2602/50—Spiro compounds
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2603/00—Systems containing at least three condensed rings
- C07C2603/56—Ring systems containing bridged rings
- C07C2603/86—Ring systems containing bridged rings containing four rings
Abstract
Guaiane sesquiterpene derivatives in regaining daphne and application thereof, belong to the field of medical science and technology, and relate to three guaiane sesquiterpene derivatives with novel structures extracted and separated from the regaining daphne of the family Thymelaeaceae. The compound is obtained by adopting chromatographic methods such as silica gel, ODS column chromatography, HP20 column chromatography, HPLC and the like. The invention also provides application of the three compounds in preparing anti-neuritis medicines. The preparation method is simple, the reproducibility is good, the purity of the obtained compound is high, and the obtained compound has good anti-neuritis activity on BV-2 cell line induced by LPS.
Description
Technical Field
The invention belongs to the technical field of medicines, relates to a method for preparing guaiane sesquiterpene derivatives from regaining senegal and application of the derivatives, and particularly relates to application of 3 guaiane sesquiterpene derivatives extracted and separated from regaining senegal in preparing anti-neuritis drugs.
Background
Neuroinflammation is a condition observed in the Central Nervous System (CNS) under the stimulation of infection, toxic metabolites, trauma or autoimmunity; the main characteristics of the disease are symmetric sensory movement and vegetative nerve dysfunction, and a plurality of common diseases are related to neuroinflammation, wherein the common diseases comprise Alzheimer Disease (AD), cerebral hemorrhage, traumatic brain injury and the like. The method for treating neuroinflammation mainly comprises supplementing vitamin B12Administration of some neuroprotective drugs, etc.
During the previous research on the chemical composition of Daphne genkwa sieb.et Zucc, some guaiane sesquiterpene derivatives with better neuroprotective activity were found, and then during the research on other Daphne plants, 3 guaiane sesquiterpene derivatives with neuritis resistance were found from Minjiang Daphne, one area from Wentang to Mao county, widely distributed in Sichuan province. The sesquiterpene derivative is prepared by extracting and extracting the regadeno regale, and has good activity of resisting neuritis. The compounds and the activities thereof related to the present invention have not been reported in patents or literatures so far.
Disclosure of Invention
The primary object of the present invention is to provide 3 guaiane sesquiterpene derivatives prepared from Min jiang daphne.
The invention also provides a preparation method of the 3 guaiane sesquiterpene derivatives and application of the guaiane sesquiterpene derivatives in preparation of anti-neuritis drugs.
The 3 guaiane sesquiterpene derivatives extracted and separated from daphne plant regadenoides of daphne of Thymelaeaceae have the following structures:
the method for extracting and separating the 3 guaiane sesquiterpene derivatives from the Thamnolia regale comprises the following steps:
(1) extracting dried whole herbs of regale daphne with ethanol, combining extracting solutions and concentrating to obtain an extract; sequentially extracting the obtained extract by using ethyl acetate and n-butanol, performing gradient elution on the ethyl acetate part and the n-butanol part by silica gel column chromatography respectively and dichloromethane/methanol according to the volume ratio of 100: 1-1: 1, and collecting 4 fractions Fr.A-Fr.D;
(2) subjecting the fraction Fr.A to polyamide column chromatography, eluting with ethanol water in a gradient manner, and collecting 2 fractions Fr.A 1-Fr.A 2;
(3) the fraction Fr.A1 is subjected to HP20 column chromatographic separation and ODS column separation sequentially, and is subjected to gradient elution by ethanol water, and 5 fractions Fr.A1-I, Fr.A1-II, Fr.A1-III, Fr.A1-IV and Fr.A1-V are collected;
(4) subjecting the fraction Fr.A1-I to silica gel column chromatography, performing gradient elution by petroleum ether/ethyl acetate according to the volume ratio of 100: 1-5: 1, subjecting the 2 nd fraction Fr.A1-I-2 obtained by elution to preparative HPLC, eluting by methanol/water to obtain 8 fractions Fr.A1-I-2-1-Fr.A1-I-2-8, subjecting the Fr.A1-I-2-3 to semi-preparative HPLC chromatography, eluting by acetonitrile/water, separating and purifying to obtain a compound 1, and subjecting the Fr.A1-I-2-7-5 to semi-preparative HPLC chromatography, eluting by methanol/water, and separating and purifying to obtain a compound 2 and a compound 3.
The above extraction method, wherein:
the ethanol in the step (1) is 70-80% industrial ethanol, and reflux extraction is carried out for 2-3 times.
The gradient concentration of the ethanol/water in the step (2) is as follows: 20% and 70%.
The gradient concentrations of the ethanol water separated by the HP20 column chromatography and the ODS column chromatography in the step (3) are respectively as follows: 20%, 50%, 90% and 40%, 50%, 60%, 70%, 80%, 90%.
The obtained compound is identified by a system structure, and the result is as follows, and the corresponding map is shown in figures 2-26:
daphenonid a (1): a colorless crystal (methanol),
HRESIMS gave the excimer peak [ M + H ]]+Peak m/z 291.1220(calcd for C)16H19O5291.1227), in combination1H-NMR、13C-NMR presumed to be of the formula C16H18O5The unsaturation was calculated to be 8. An absorption peak at a wavelength of 238nm, UV (MeOH). lambda.max(logε):238nm(0.81)。
1H-NMR(600MHz,CDCl3) Middle, deltaH6.16(1H, d, J ═ 1.5Hz) is an olefinic hydrogen proton signal, δH3.93(1H, s) is a hydroxyl signal, δH2.11(1H, brd, J. 5.3Hz) is a methine proton signal, δH3.63(3H, s) is the proton signal on the methoxy radical, δH2.02(3H,d,J＝1.4Hz)，δH1.23(3H, s) is the hydrogen signal on two methyl carbons. Combined with HSQC spectrum, pair13C-NMR(150MHz,CDCl3) Analysis of the spectra showed 16 carbon signals, of which there were four sp3Hybrid quaternary carbon signal (delta)C84.7,68.2,60.5, 59.0), four sp)2Hybrid quaternary carbon signal (delta)C212.6,198.0,171.0, 158.9), a double bond carbon signal (. delta.) with attached protonsC125.4), one methine carbon signal (δ)C53.5), three methylene carbon signals (. delta.))C38.6, 33.0, 32.2) and three methyl carbon signals (δ)C52.5, 22.7, 11.9, whereinC52.5 carbon-to-oxygen). Consider that there is one pairBond, three carbonyl groups and eight unsaturations, it is concluded from the above information that compound 1 may be a sesquiterpene possessing a tetracyclic system.
By analysis of the HMBC spectra, H-5 (. delta.)H2.11) and C-3 (. delta.)C212.6) correlation, H-2 (. delta.))H2.98,δH2.57) and C-5 (. delta.)C53.5) correlation and H3-15(δH1.23) and C-3 (. delta.)C212.6)，C-4(δC59.0) and C-5 (. delta.))C53.5) confirms the presence of the five-membered carbocyclic ring A consisting of C-1, C-2, C-3, C-4, C-5 and its presence of the ketone at C-3 and of the methyl group at C-4. H3-16(δH3.63) and C-13 (. delta.))C171.0) confirmed the presence of methoxyacyl groups. To further determine the planar structure of compound 1, we obtained a series of possible planar structures that fit the 2D nuclear magnetic data of compound 1 using a computer-aided structure analysis system (CASE), and then selected the most likely structure based on empirical NMR chemical shift predictions. "CASE results and key NOESY related and crystal structure of compound 1" as shown below, 4 structures were generated within 1.0s and based on13The average deviations of C were ranked. Compound 1 was determined to match the best structure according to the lowest standard deviation value. In the MCD diagram, methylene hydrogen (. delta.) at the C-12 positionH2.31, 1.82) associated with the presence of five quaternary carbons (C-1, C-3, C-4, C-7, C-11) and one tertiary carbon (C-5), determined the bridging of C-1 and C-7 together by C-11 and the linkage of methoxyacyl groups at the C-11 position, and the presence of a five-membered carbocyclic ring B consisting of C-1, C-4, C-5, C-12 and C-11. OH in position 7 (. delta.)H3.93) and C-6 (. delta.))C32.3)，C-8(δC198.0)，C-11(δC68.2) there is a correlation, indicating that the 7-OH is attached to C-7 and that a ketocarbonyl group is present at the C-8 position. Based on the above information, in combination1H-1The spin-coupled system shown in the H COSY spectra, illustrates the presence of a cage-like structure. Finally, it was concluded that the planar structure of Compound 1 is a tetracyclic [5.3.2.0 ]1,6.04,11]Novel caged sesquiterpenes with dodecane skeleton.
The relative configuration of Compound 1, H-5 (. delta.) is next determined by NOESY spectroscopyH2.11) and H-15 (. delta.))H1.23) NOE, indicating that H-5 is on the same side as H-15, ascribed to the α orientation; the above inference is further confirmed by the correlation of H-2 β/H-12 β and H-6 β/H-12 α. The relative configuration was assigned as 1R,4R,5R,7S,11S due to the steric hindrance of the 5/5/5/6 caged four-ring system and the rigid connection of the five-membered carbocyclic ring C and ring D. Then, the planar structure and relative configuration of compound 1 were further determined using NMR chemical shift calculations. To determine its absolute configuration, compound 1 was subjected to computational ECD at B3LYP/6-311+ + G (2d, p) levels, where the calculated ECD spectrum of (1R,4R,5R,7S,11S) -1 matched the experimental ECD spectrum of compound 1. After many attempts, single crystals of compound 1 were obtained in a mixed solvent of dichloromethane/methanol (1:1), and X-ray diffraction results confirmed assignment of absolute configuration and presumption of steric structure (fig. 10, CCDC 1876453, flip parameter ═ 0.04 (3)). After the database search of the scifinider, the compound 1 is an unreported new compound and is named as Daphnenoid A.
Daphnenoid B and Daphnenoid C (2-3): a colorless oil, compound 2 having an optical rotation value of
The optical rotation value of the compound 3 is
HRESIMS gave the excimer peak M/z273.1464[ M + Na ] of Compound 2]+(calcd for C15H22O3Na,273.1461), excimer peak M/z273.1462[ M + Na ] of Compound 3]+(calcd for C15H22O3Na, 273.1461). Bonding of1H-NMR、13C-NMR presumed that both of the compounds 2 and 3 have the formula C15H22O3The unsaturation was calculated to be 5. For compounds 2 and 313C-NMR(150MHz,CDCl3) And analysis of the HSQC spectra showed 15 carbon signals, including one sp3Hybridized quaternary carbon signal, three sp2A hybridized quaternary carbon signal, one protonated olefinic hydrogen carbon signal, three methine carbon signals, five methylene carbon signals, and two methyl carbon signals. In all the observed signals, one double bond and two carbonyl groups occupy three unsaturations, the remaining two unsaturations possibly constituting two carbocyclic skeletons, indicating, according to the above information, that compounds 2 and 3 are a pair of sesquiterpenes having the same bicyclic skeleton.
For HMBC spectral analysis, H2Correlation of-2 with C-1, C-4 and C-5 and H2The correlation of-3 with C-1 and C-5 indicates the presence of a five-membered carbocyclic ring A, and the presence of a ketocarbonyl group in the C-1 position and a methyl group in the C-4 position. H2The correlation of-13 with HMBC at C-7, C-11 and C-12 also indicates the presence of a terminal double bond. HMBC's with H-4 and C-6 and C-12, H-12 and C-1 and C-6 and H-6 and C-1 and C-12 illustrate bicyclic ring systems linked together by a spiro carbon atom, C-5. Assignment of Ring B is made difficult by the critical HMBC-related deletion of H-7 and the overlap of chemical shift values of H-8 and H-3, although H-6 α is associated with C-11, there is not enough evidence to justify the presence of the five-membered carbocyclic ring B. Furthermore, the peculiar 5/5 spiro sesquiterpene in nature has not been discovered, and even the 5/6 spiro system sesquiterpenes are rare in plants and marine microorganisms. To further confirm the structure of Ring B accurately, a computer-aided Structure analysis System (CASE) was used to generate all possible structures, with structures having 5/5 fused spiro ring systems having the lowest possible structure compared to other candidate structures13C mean deviation, is considered the most reasonable structure. This was confirmed by NMR chemical shift calculations. Finally, the plane structures of Daphnenoid B and Daphnenoid C (2-3) were determined and their spiro [4.4 ]]The nonane skeleton is also determined.
By analysis of the NOESY spectra of Compounds 2 and 3, H-6. beta. was compared with H-9 and H3Correlation of-15 demonstrates CH3-15 and H-7 are both in the alpha-orientation; the correlation of H-7 with H-12 was found in compound 2, indicating that H-7 is in the same side as H-12 in the alpha-orientation, whereas no correlation of H-7 with H-12 was found in compound 3, but a correlation of H-4 with H-12 was found, indicating that H-12 is in the beta-orientation in compound 3, indicating that compounds 2 and 123 is a diastereomer. In addition, to further confirm the diastereomeric differences of compounds 2 and 3, NMR chemical shifts were then calculated for the two candidate structures (A and B) in chloroform in a PCM solvent model at mPW1PW91/6-311+ G (d, p)// ω B97XD/6-31G (d) levels, first using R obtained based on quantum mechanical chemical shift analysis2Value to distinguish Compounds 2 and 3, but R2The difference in values was small and could not be used to confirm the relative configuration of H-12 for compounds 2 and 3; thereafter, MAE was usedΔΔδFrom the configuration of H-12, lowest MAEΔΔδThe values indicate that compound 2 best matches the combination of structure a and compound 3 best matches structure B; finally, using another highly reliable parameter for diastereomer structural assignment CP3, CP3 calculation showed that the 4R,5R,7S, 12R configuration (i.e. structure B) belongs to compound 3, and based on experiments and calculations1H and13the probability was determined to be 100% as a result of the chemical shift comparison. Accordingly, NOESY analysis, R2Value, MAEΔΔδAnd CP3 results were consistent, so the relative configurations of compounds 2 and 3 were 4R,5R,7S, 12S and 4R,5R,7S, 12R, respectively. But due to the flexibility of the 2-butanone moiety, no cultivation success was achieved for single crystals of compounds 2 and 3; and finally, comparing the experiment and the calculated ECD spectrogram to determine the absolute configurations of the compounds 2 and 3, wherein the absolute configurations of the compounds 2 and 3 are respectively determined as 4S,5S,7R,12R and 4S,5S,7R and 12S.
Upon database search by scifininder, compounds 2 and 3 were unreported new compounds, named daphenoid B (2) and daphenoid C (3).
Of compounds 1 to 31H NMR data and13c NMR data
HMBC and of compounds 1-21H-1H COSY correlation
CASE results for Compound 1 correlate with key NOESY and crystal structure
Key NOESY correlation of Compounds 2-3
The invention also provides a pharmaceutical composition, which comprises the guaiane sesquiterpene derivative, a pharmaceutically acceptable salt of the compound, a pharmaceutically acceptable excipient or/and a carrier.
The invention also provides application of the Thamnolia regale extract in a medicament for treating the neuritis.
The invention also provides application of the 3 guaiane sesquiterpene derivatives in medicines for treating neuritis.
The 3 guaiane sesquiterpene derivatives related to the invention were evaluated for their anti-neuritic activity. Results compounds 1-3 showed comparable LPS-induced anti-neuritic activity against BV-2 mouse microglia as the positive drug Dex. Therefore, the guaiane sesquiterpene derivative has the potential of clinically treating the anti-neuritis drugs.
The guaiane sesquiterpene derivatives have the advantages that the guaiane sesquiterpene derivatives are all novel compounds, are novel in structure, are all optically pure compounds with determined stereo configuration, have good anti-neuritis activity and have further development value.
Drawings
The structures of compounds 1-3 of FIG. 1;
figure 2 mass spectrum of compound 1;
FIG. 3 preparation of Compound 11H-NMR Spectroscopy (600MHz, CDCl)3)；
FIG. 4 preparation of Compound 113C-NMR Spectroscopy (150MHz, CDCl)3)；
FIG. 5 HSQC spectra (600MHz, CDCl) of Compound 13)；
FIG. 6 HMBC spectra (600MHz, CDCl) of Compound 13)；
FIG. 7 preparation of Compound 11H-1H COSY spectrum;
FIG. 8 NOESY spectrum (600MHz, CDCl) of Compound 13)；
FIG. 9 generation of a possible structure and MCD map of Compound 1 from the CASE system;
FIG. 10 comparison of Compound 1 calculation with Experimental ECD and single crystal plot;
figure 11 mass spectrum of compound 2;
FIG. 12 preparation of Compound 21H-NMR Spectroscopy (600MHz, CDCl)3)；
FIG. 13 preparation of Compound 213C-NMR Spectroscopy (150MHz, CDCl)3)；
FIG. 14 HSQC spectra (600MHz, CDCl) of Compound 23)；
FIG. 15 HMBC spectra (600MHz, CDCl) of Compound 23)；
FIG. 16 preparation of Compound 21H-1H COSY spectrum;
FIG. 17 NOESY spectrum (600MHz, CDCl) of Compound 23)；
Figure 18 mass spectrum of compound 3;
FIG. 19 preparation of Compound 31H-NMR Spectroscopy (600MHz, CDCl)3)；
FIG. 20 preparation of Compound 313C-NMR Spectroscopy (150MHz, CDCl)3)；
FIG. 21 HSQC spectra (600MHz, CDCl) of Compound 33)；
FIG. 22 HMBC spectra (600MHz, CDCl) of Compound 33)；
FIG. 23 preparation of Compound 31H-1H COSY spectrum;
FIG. 24 NOESY spectrum (600MHz, CDCl) of Compound 33)；
FIG. 25 experiments and calculations of Compounds 2 and 313Chemical potential of C NMRShifted CP3 computation and MAEΔΔδA parameter;
figure 26 experimental and calculated ECD spectra for compounds 2 and 3;
FIG. 27 protective activity of compounds 1-3 against LPS-induced neuroinflammatory injury of BV-2 mouse microglia; all data are expressed as means ± SD (three independent experiments); dex is a positive drug.
Detailed Description
The examples set out below are intended to assist the person skilled in the art in a better understanding of the invention, but do not limit it in any way.
Example 1: the preparation method of guaiane sesquiterpene derivatives 1-3 from regained daphne comprises the following steps:
(1) taking dry whole regadenoson plants (48kg) and carrying out reflux extraction for three times by 75% industrial ethanol, combining extracting solutions and concentrating to obtain an extract. The obtained extract is extracted by ethyl acetate and n-butanol successively. The ethyl acetate layer extract (600g) and the n-butanol layer extract (1.2kg) were subjected to silica gel column chromatography under reduced pressure, and gradient elution was performed with a methylene chloride/methanol system at a volume ratio of 100:1, 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, 1:1, and 4 fractions Fr A to Fr D were collected in total.
(2) The fraction fr.a (200g) was subjected to polyamide column chromatography and gradient elution (20%, 70%) with an ethanol water system, and 2 fractions fr.a1 to fr.a2 were collected in total.
(3) The fraction Fr.A1(90g) was first subjected to HP20 column chromatography eluting with an aqueous ethanol gradient (20%, 50%, 90%), and then subjected to ODS column chromatography eluting with an aqueous ethanol gradient (40%, 50%, 60%, 70%, 80%, 90%), and the fractions were collected to obtain 5 fractions Fr.A1-I, Fr.A1-II, Fr.A1-III, Fr.A1-IV, Fr.A1-V.
(4) Fraction fr. a1-I (12g) was subjected to silica gel column chromatography eluting with a petroleum ether/ethyl acetate gradient (volume ratio 100:1, 50:1, 30:1, 20:1, 10:1, 5: 1). The obtained 2 nd fraction Fr.A1-I-2 was subjected to preparative HPLC and eluted with methanol/water (volume ratio 40:60) to obtain 8 fractions (Fr.A1-I-2-1 to Fr.A1-I-2-8), Fr.A1-I-2-3 was subjected to semi-preparative HPLC chromatography and eluted with acetonitrile/water (volume ratio 20:80) to isolate and purify to obtain compound 1(12mg), and Fr.A1-I-2-7-5 was subjected to semi-preparative HPLC chromatography and eluted with methanol/water (volume ratio 25:75) to isolate and purify to obtain compound 2(1.2mg) and compound 3(2.2 mg). The structures of the obtained compounds 1 to 3 are shown in FIG. 1.
Example 2: and detecting the anti-neuritis activity of the obtained guaiane sesquiterpene derivatives 1-3.
BV2 mouse microglia were placed in 10% FBS-containing DMEM medium and incubated at 37 ℃ with 5% CO2Culturing in a saturated humidity incubator. Passage was performed when the cell confluence reached 80%.
The anti-neuritis activity of the guaiane sesquiterpene derivative 1-3 obtained from regained daphne is examined through NO content detection. Seeding BV2 microglia in 96-well plate with plate density of 6 × 104Each well is 100 μ L, 3 multiple wells are arranged, and the mixture is placed at 37 ℃ and 5% CO2Culturing in a saturated humidity incubator. After 12h, the drug (10. mu.M) was dosed. After 1h, BV2 microglia cells were stimulated by the addition of 10. mu.g/mL LPS. After 24 hours, the culture supernatant was collected, and the nitric oxide content in the supernatant was measured using an NO assay kit.
First, the Griess Reagent I and Reagent II are restored to room temperature, the culture supernatant is added into a 96-well plate according to 50 mu L of each well, then the Griess Reagent I and Reagent II are sequentially added, and the absorbance is measured at 540nm by adopting an enzyme-labeling instrument. All experiments were performed in parallel and repeated three times, blanks and positive controls were set up and analyzed using GraphPad Prism 6 software, with the results shown in fig. 27.
Claims (9)
1. A guaiane sesquiterpene derivative in regained jiang daphne is characterized by having the following structure:
2. the process for the preparation of guaiane sesquiterpene derivatives according to claim 1 comprising the steps of:
(1) extracting dried whole herbs of regale daphne with ethanol, combining extracting solutions and concentrating to obtain an extract; sequentially extracting the obtained extract by using ethyl acetate and n-butanol, performing gradient elution on the ethyl acetate part and the n-butanol part by silica gel column chromatography respectively and dichloromethane/methanol according to the volume ratio of 100: 1-1: 1, and collecting 4 fractions Fr.A-Fr.D;
(2) subjecting the fraction Fr.A to polyamide column chromatography, eluting with ethanol water in a gradient manner, and collecting 2 fractions Fr.A 1-Fr.A 2;
(3) the fraction Fr.A1 is subjected to HP20 column chromatographic separation and ODS column separation sequentially, and is subjected to gradient elution by ethanol water, and 5 fractions Fr.A1-I, Fr.A1-II, Fr.A1-III, Fr.A1-IV and Fr.A1-V are collected;
(4) subjecting the fraction Fr.A1-I to silica gel column chromatography, performing gradient elution by petroleum ether/ethyl acetate according to the volume ratio of 100: 1-5: 1, subjecting the 2 nd fraction Fr.A1-I-2 obtained by elution to preparative HPLC, eluting by methanol/water to obtain 8 fractions Fr.A1-I-2-1-Fr.A1-I-2-8, subjecting the Fr.A1-I-2-3 to semi-preparative HPLC chromatography, eluting by acetonitrile/water, separating and purifying to obtain a compound 1, and subjecting the Fr.A1-I-2-7-5 to semi-preparative HPLC chromatography, eluting by methanol/water, and separating and purifying to obtain a compound 2 and a compound 3.
3. The method according to claim 2, wherein the ethanol in step (1) is 70-80% industrial ethanol, and the reflux extraction is performed 2-3 times.
4. The method according to claim 2, wherein the gradient concentration of ethanol/water in the step (2) is: 20% and 70%.
5. The process according to claim 2, wherein the gradient concentrations of the ethanol water separated by the HP20 column chromatography and the ODS column chromatography in step (3) are as follows: 20%, 50%, 90% and 40%, 50%, 60%, 70%, 80%, 90%.
6. A pharmaceutical composition comprising the guaiane sesquiterpene derivatives of claim 1 in association with a pharmaceutically acceptable excipient or/and a carrier.
7. Use of the guaiane sesquiterpene derivatives according to claim 1 or the pharmaceutical composition according to claim 6 in the preparation of anti-neuritis drugs.
8. A regadenox extract comprising the guaiane sesquiterpene derivative of claim 1.
9. The application of the Thamnolia regale extract as claimed in claim 8, which is used for preparing the anti-neuritis medicine.
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| CN110305092A (en) * | 2019-04-11 | 2019-10-08 | 沈阳药科大学 | Guainane sequiterpene and its preparation and application |
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| CN110305092A (en) * | 2019-04-11 | 2019-10-08 | 沈阳药科大学 | Guainane sequiterpene and its preparation and application |
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| CN115215881A (en) * | 2022-07-29 | 2022-10-21 | 沈阳药科大学 | Guaiane type sesquiterpenoids prepared from Thorellan odorata, and preparation method and application thereof |
| CN115215881B (en) * | 2022-07-29 | 2023-11-03 | 沈阳药科大学 | Guaiane sesquiterpene compound prepared from daphne tanguticum, and preparation method and application thereof |
| CN115677520A (en) * | 2022-11-18 | 2023-02-03 | 扬州大学 | Diterpene compound and preparation method and application thereof |
| CN115677520B (en) * | 2022-11-18 | 2024-04-02 | 扬州大学 | Diterpene compound and preparation method and application thereof |
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