CN117945865A - Bibenzyl compound and preparation method and application thereof - Google Patents
Bibenzyl compound and preparation method and application thereof Download PDFInfo
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- CN117945865A CN117945865A CN202311748080.4A CN202311748080A CN117945865A CN 117945865 A CN117945865 A CN 117945865A CN 202311748080 A CN202311748080 A CN 202311748080A CN 117945865 A CN117945865 A CN 117945865A
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- -1 Bibenzyl compound Chemical class 0.000 title claims abstract description 122
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- 201000001441 melanoma Diseases 0.000 claims abstract description 24
- 239000003814 drug Substances 0.000 claims abstract description 19
- 238000000034 method Methods 0.000 claims abstract description 17
- 238000000926 separation method Methods 0.000 claims abstract description 14
- 230000008569 process Effects 0.000 claims abstract description 5
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 180
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 93
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 80
- 238000000605 extraction Methods 0.000 claims description 45
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 43
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 42
- 238000006243 chemical reaction Methods 0.000 claims description 41
- 241001678082 Dendrobium huoshanense Species 0.000 claims description 39
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 39
- 239000012046 mixed solvent Substances 0.000 claims description 38
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 36
- 238000010828 elution Methods 0.000 claims description 36
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 28
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 27
- 230000002829 reductive effect Effects 0.000 claims description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 26
- 239000012043 crude product Substances 0.000 claims description 25
- NOVNXQOBKQLFEX-UHFFFAOYSA-N 3,4-dimethoxy-5-phenylmethoxybenzaldehyde Chemical compound COC1=CC(C=O)=CC(OCC=2C=CC=CC=2)=C1OC NOVNXQOBKQLFEX-UHFFFAOYSA-N 0.000 claims description 24
- NVLTWXMZECWWPC-UHFFFAOYSA-N 3-hydroxy-4,5-dimethoxybenzaldehyde Chemical compound COC1=CC(C=O)=CC(O)=C1OC NVLTWXMZECWWPC-UHFFFAOYSA-N 0.000 claims description 24
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 24
- RIOQSEWOXXDEQQ-UHFFFAOYSA-N triphenylphosphine Chemical compound C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-N 0.000 claims description 24
- 238000010438 heat treatment Methods 0.000 claims description 23
- 238000004809 thin layer chromatography Methods 0.000 claims description 22
- 239000007787 solid Substances 0.000 claims description 21
- 238000000746 purification Methods 0.000 claims description 20
- 239000000843 powder Substances 0.000 claims description 19
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims description 18
- 239000003208 petroleum Substances 0.000 claims description 18
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 17
- PQVSTLUFSYVLTO-UHFFFAOYSA-N ethyl n-ethoxycarbonylcarbamate Chemical compound CCOC(=O)NC(=O)OCC PQVSTLUFSYVLTO-UHFFFAOYSA-N 0.000 claims description 17
- GLXDVVHUTZTUQK-UHFFFAOYSA-M lithium hydroxide monohydrate Substances [Li+].O.[OH-] GLXDVVHUTZTUQK-UHFFFAOYSA-M 0.000 claims description 17
- 229940040692 lithium hydroxide monohydrate Drugs 0.000 claims description 17
- 238000010898 silica gel chromatography Methods 0.000 claims description 17
- 239000000499 gel Substances 0.000 claims description 15
- 238000010992 reflux Methods 0.000 claims description 15
- 229910052757 nitrogen Inorganic materials 0.000 claims description 14
- 239000011541 reaction mixture Substances 0.000 claims description 14
- 238000012544 monitoring process Methods 0.000 claims description 13
- UKVWNSDFMYZNKC-UHFFFAOYSA-N 1-(dichloromethyl)-4-methoxybenzene Chemical compound COC1=CC=C(C(Cl)Cl)C=C1 UKVWNSDFMYZNKC-UHFFFAOYSA-N 0.000 claims description 12
- 125000004803 chlorobenzyl group Chemical group 0.000 claims description 12
- 239000003480 eluent Substances 0.000 claims description 12
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 claims description 12
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 12
- 238000002953 preparative HPLC Methods 0.000 claims description 11
- UKVIEHSSVKSQBA-UHFFFAOYSA-N methane;palladium Chemical compound C.[Pd] UKVIEHSSVKSQBA-UHFFFAOYSA-N 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 10
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 9
- 238000001514 detection method Methods 0.000 claims description 8
- 239000001257 hydrogen Substances 0.000 claims description 8
- 229910052739 hydrogen Inorganic materials 0.000 claims description 8
- XFYWJTQOVFWIEK-UHFFFAOYSA-N 1,2,3,4-tetrahydrophosphinine Chemical compound C1CPC=CC1 XFYWJTQOVFWIEK-UHFFFAOYSA-N 0.000 claims description 7
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 claims description 7
- 208000030381 cutaneous melanoma Diseases 0.000 claims description 7
- 238000005227 gel permeation chromatography Methods 0.000 claims description 7
- 201000003708 skin melanoma Diseases 0.000 claims description 7
- 238000003756 stirring Methods 0.000 claims description 7
- QWUWMCYKGHVNAV-UHFFFAOYSA-N 1,2-dihydrostilbene Chemical group C=1C=CC=CC=1CCC1=CC=CC=C1 QWUWMCYKGHVNAV-UHFFFAOYSA-N 0.000 claims description 6
- 229920005654 Sephadex Polymers 0.000 claims description 6
- 239000012507 Sephadex™ Substances 0.000 claims description 6
- 125000000051 benzyloxy group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])O* 0.000 claims description 6
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 8
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
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- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 238000005481 NMR spectroscopy Methods 0.000 description 4
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 4
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- FDKXTQMXEQVLRF-ZHACJKMWSA-N (E)-dacarbazine Chemical compound CN(C)\N=N\c1[nH]cnc1C(N)=O FDKXTQMXEQVLRF-ZHACJKMWSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 2
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 2
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- 229960003862 vemurafenib Drugs 0.000 description 2
- GPXBXXGIAQBQNI-UHFFFAOYSA-N vemurafenib Chemical compound CCCS(=O)(=O)NC1=CC=C(F)C(C(=O)C=2C3=CC(=CN=C3NC=2)C=2C=CC(Cl)=CC=2)=C1F GPXBXXGIAQBQNI-UHFFFAOYSA-N 0.000 description 2
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- 210000004185 liver Anatomy 0.000 description 1
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- DKPFODGZWDEEBT-QFIAKTPHSA-N taxane Chemical class C([C@]1(C)CCC[C@@H](C)[C@H]1C1)C[C@H]2[C@H](C)CC[C@@H]1C2(C)C DKPFODGZWDEEBT-QFIAKTPHSA-N 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to a bibenzyl compound, a preparation method and application thereof, and belongs to the technical field of antitumor activity research. The bibenzyl compound has a structural formula shown in formula (I); Formula (I). The in vitro experiments show that the dibenzyl compound can obviously inhibit the proliferation of melanoma cells of the skin of mice, and the IC 50 value is 4.938 mu mol/L; in vivo experiment results show that the dibenzyl compound can obviously inhibit the growth and metastasis of the melanoma of mice. The separation and preparation method of the bibenzyl compound has the characteristics of green and economical property, simple process, high yield and the like, can be used for researching and developing anti-melanoma medicaments, and has great application potential.
Description
Technical Field
The invention belongs to the technical field of anti-tumor activity research, and particularly relates to a bibenzyl compound, and a preparation method and application thereof.
Background
Melanoma is a tumor developed by malignant growth of melanocytes, has the characteristics of high metastatic and heterogeneous properties, can metastasize to any organ, accounts for more than 80% of all skin cancer deaths, and is the most serious type of skin cancer. Advanced morbidity, drug resistance and cancer cell metastasis are the main causes of high mortality of melanoma, with poor prognosis for patients with melanoma after metastasis and median survival of only 6 months.
Currently, chemotherapy drugs for treating middle and late (stage iii and stage iv) melanoma are mainly ipilimumab (ipilimumab), nivolumab (nivolumab), dacarbazine (Dacarbazine) and the like. However, the existing chemotherapeutics have low response rate to melanoma, and have the problems of strong toxic and side effects, drug resistance and the like. Therefore, the novel, low-toxicity and high-efficiency anti-tumor drug is developed, and has important significance for helping hand of 'healthy China'.
In the aspect of treating various diseases, the natural product shows unique curative effect and has the characteristics of toxicity reduction, synergy, low cost and low circulation. In recent years, the discovery of antitumor-active lead compounds represented by taxane compounds RP-56976 has attracted attention. From 1981 to 2019, 185 anticancer small molecule compounds were found worldwide, 156 (84%) of which are natural products. Therefore, the discovery of anticancer lead compounds with novel structures from natural products is a current research hotspot. The invention discovers a novel bibenzyl compound with anti-melanoma activity, and related reports are not yet seen at present.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provides a bibenzyl compound, and a preparation method and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a dibenzyl compound has a structural formula shown in formula (I);
the invention provides a natural preparation and separation method of the bibenzyl compound, which comprises the following steps:
Step (1): extracting stem of Dendrobium huoshanense with absolute ethanol, and concentrating the extractive solution under reduced pressure to obtain Dendrobium huoshanense extract;
step (2): dissolving the dendrobium huoshanense extract obtained in the step (1) with water, extracting with ethyl acetate, and concentrating an ethyl acetate phase under reduced pressure to obtain dendrobium huoshanense extract;
Step (3): separating the dendrobium huoshanense extract obtained in the step (2) through normal phase silica gel column chromatography, performing gradient elution, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, and combining the same parts to obtain 7 components Fr.1-Fr.7; the mobile phase adopted by gradient elution is a mixed solvent of dichloromethane and methanol;
Step (4): separating the component Fr.2 obtained in the step (3) by normal phase silica gel column chromatography, performing gradient elution, collecting gradient eluents of each gradient, concentrating, monitoring by TLC, and combining the same parts to obtain 6 subfractions 2A1-2A6; the mobile phase adopted by gradient elution is a mixed solvent of petroleum ether and ethyl acetate;
Step (5): and (3) carrying out Sephadex LH-20 Sephadex gel column and semi-preparative high performance liquid chromatography separation and purification on the subfraction 2A1 obtained in the step (4) to obtain the bibenzyl compound shown in the formula (I).
Further, preferably, in the step (1), the extraction mode is immersion extraction, ultrasonic extraction or reflux extraction; the extraction times are 1-5 times; the extraction time is 5-9 days each time;
In the step (2), the volume ratio of the ethyl acetate to the water is 0.5-4:1; extracting by adopting ultrasonic waves; the extraction times are 3-6 times; the extraction time is 0.5-2 hours each time, and the mixture is left standing overnight after extraction.
Further, it is preferable that in the step (3), the volume ratio of dichloromethane to methanol in the mixed solvent of dichloromethane and methanol is sequentially 100:0, 100:1, 80:1, 50:1, 30:1, 10:1, 5:1, 1:1, 0:1;
In the step (4), the volume ratio of petroleum ether to ethyl acetate is 100:1, 60:1, 30:1, 10:1 and 1:1 in sequence.
Further, preferably, in the step (5), the gel column adopted in the gel chromatography purification is Sephade x LH-20, and a mixed solvent of dichloromethane and methanol with the volume ratio of 1:1 is used as a mobile phase;
The chromatographic conditions for semi-preparative high performance liquid chromatography purification are:
The mobile phase is a mixed solvent of acetonitrile and water in a volume ratio of 1:1; the flow rate is 3mL/min; collecting the components with the peak starting time of 36 min; a C 18 reverse-phase chromatographic column is adopted; the specification is preferably: 4.6mm by 250mm,5 μm; column temperature: 30 ℃; sample size per sample: 20. Mu.L; the detection wavelength was 210nm using an ultraviolet detector.
The invention also provides a preparation method of the bibenzyl compound, which comprises the following steps:
S1: heating a mixture of 4-methoxychlorobenzyl chloride, triphenylphosphine and toluene under the protection of nitrogen, and carrying out reflux reaction to obtain white solid powder (chloro- (4-methoxybenzyl) triphenyl-lambda 5 -phosphane);
S2: adding chlorobenzyl and potassium carbonate into DMF containing 3, 4-dimethoxy-5-hydroxybenzaldehyde under the protection of nitrogen, and heating for reaction to obtain a (3- (benzyloxy) -4, 5-dimethoxy benzaldehyde) crude product;
S3: lithium hydroxide monohydrate is added into isopropanol solution of (chloro- (4-methoxybenzyl) triphenyl-lambda 5 -phosphane) at the temperature of 0 ℃ for reaction; then adding (3- (benzyloxy) -4, 5-dimethoxy benzaldehyde) crude product, and heating to react to obtain yellow oily (1- (benzyloxy) -2, 3-dimethoxy-5- (4-methoxy styryl) benzene);
S4: the obtained yellow oily matter (1- (benzyloxy) -2, 3-dimethoxy-5- (4-methoxystyryl) benzene) is dissolved in a mixed solvent of ethyl acetate and ethanol, hydrogen is introduced, then active palladium carbon is added, and stirring reaction is carried out at room temperature, so as to obtain the bibenzyl compound shown in the formula (I).
Further, it is preferable that in S1, reflux reaction is carried out at 100 to 140℃for 10 to 14 hours; the dosage ratio of 4-methoxychlorobenzyl chloride, triphenylphosphine and toluene was 1mmol:1-1.2mmol:1.5-2mL;
S2, heating at 70-90 ℃ for 5-7 hours; the dosage ratio of potassium carbonate, chlorobenzyl, 3, 4-dimethoxy-5-hydroxybenzaldehyde and DMF is 1mmol:1.9-2.1mmol:1.9-2.1 mmol: 2-2.5mL.
Further, it is preferable that in S3, the ratio of the amount of lithium hydroxide monohydrate, white solid powder (chloro- (4-methoxybenzyl) triphenyl-. Lamda. 5 -phosphane), isopropyl alcohol and (3- (benzyloxy) -4, 5-dimethoxybenzaldehyde) crude product is 1.5 to 1.7mmol:1.4-1.6mmol:4-5mL:1mmol; lithium hydroxide monohydrate is added into isopropanol solution of white solid powder (chlorine- (4-methoxybenzyl) triphenyl-lambda 5 -phosphane) to react for 14 to 16 minutes at the temperature of 0 ℃, then (3- (benzyloxy) -4, 5-dimethoxy benzaldehyde) crude product is added, and after the addition is finished, the reaction mixture is heated to 50 to 70 ℃ to react for 9 to 11 hours;
In S4, the dosage ratio of yellow oily matter (1- (benzyloxy) -2, 3-dimethoxy-5- (4-methoxystyryl) benzene), ethyl acetate, ethanol and active palladium on charcoal is 2.4-2.6mmol:19-22mL:9-11mL:0.19-0.21g; activated palladium carbon is 5% in concentration; the reaction is stirred at room temperature for 2.5 to 3.5 hours.
The invention also provides application of the bibenzyl compound in preparing medicines for treating and/or preventing melanoma.
Further, it is preferable that the melanoma is B16-F10 mouse skin melanoma.
In the step (1), the dendrobium huoshanense is extracted in a dipping extraction mode, an ultrasonic extraction mode or a reflux extraction mode, and dipping extraction is preferred; the number of extractions is 1-5, for example 3; the extraction time is 5-9 days, such as 7 days.
Preferably, the step (1) specifically comprises: removing She Baojing fresh herba Dendrobii, pulverizing to 0.8-1.2cm with pulverizer, soaking and extracting with 20L absolute ethanol at room temperature for 3 times, 7 days each time, filtering to obtain filtrate, and concentrating under reduced pressure to obtain herba Dendrobii extract.
In step (2) of the present invention, the volume ratio of ethyl acetate to water is 0.5-4:1, e.g. 1:1; performing extraction operation by adopting ultrasonic waves, and standing overnight after extraction; the extraction times are 3-6 times.
Preferably, in the step (2), the specific steps are: dissolving the extract obtained in the step (1) with water, adding an equal volume of ethyl acetate, performing ultrasonic treatment at room temperature for 0.5-2 hours, standing at room temperature for 8-12 hours, taking an ethyl acetate phase, and concentrating under reduced pressure; the extraction times are 3-6 times.
In the step (3), dry sample loading is preferably adopted in the normal phase silica gel column chromatographic separation process; preferably, the concrete method for dry loading of the extractum comprises the following steps: dissolving the extract with 10 times of ethyl acetate, adsorbing with 80-100 mesh silica gel with the same mass as the extract, and loading; the normal phase silica gel column is filled with 200-300 mesh normal phase silica gel. After each gradient elution until TLC plate was not shown, the next gradient elution was changed to give 7 total fr.1-fr.7 fractions.
In the step (4), the dry method is adopted for sample loading in the chromatographic separation process of the normal phase silica gel column; preferably, the specific method of dry loading is as follows: dissolving the component Fr.2 with 10 times of methanol, adsorbing with 80-100 mesh silica gel with the same mass as the component Fr.2, and loading; the normal phase silica gel column is filled with 200-300 mesh normal phase silica gel. After each gradient elution until the TLC plate showed no spots, the next gradient elution was changed to give 6 sub-fractions 2A1-2 A6.
In the step (5), the subfraction 2A1 is separated and purified by gel column chromatography Sephadex LH-20 and HPLC; the mobile phase of gel column chromatography is dichloromethane/methanol (1:1); the mobile phase of HPLC is acetonitrile/water (1:1), the flow rate is 3mL/min, and the components with the peak starting time of 36min are collected;
Preferably, in the case of gel chromatography purification, subfraction 2A1 is loaded after dissolution with 2 volumes of methanol, monitored by TLC and the same fractions combined; specifically: collection was performed using 10mL tubes, 5mL per tube, 60 tubes were collected in total, monitored by TLC, the same fractions were combined, 10-20 tubes were combined, concentrated under reduced pressure and purified by HPLC.
Preferably, the HPLC chromatographic condition is that the mobile phase is a mixed solvent of acetonitrile and water (volume ratio is 1:1), the flow rate is 3mL/min, the components with the peak starting time of 36min are collected, and an Agilent C18 reverse chromatographic column is adopted, wherein the specification is as follows: 4.6mm by 250mm,5 μm; column temperature: 30 ℃, the sample injection amount is equal to: 20. Mu.L, using an ultraviolet detector, the detection wavelength was 210nm.
In the invention S1, after the reaction, the reaction mixture is cooled to room temperature, solid is precipitated, the reaction mixture is filtered, and the reaction mixture is washed by toluene, acetone and n-hexane in sequence, so that white solid powder 1 is obtained.
In the invention S2, the reaction mixture is cooled to room temperature, poured into ice water, extracted three times by ethyl acetate, washed by saturated sodium chloride solution, dried by anhydrous sodium sulfate, and the solvent is removed under reduced pressure to obtain a crude product 2, and the crude product 2 is directly put into the next reaction without purification.
In the invention S3, thin layer chromatography is adopted to monitor the reaction in the reaction process. The reaction mixture was quenched with water and extracted 3 times with ethyl acetate. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and the crude product obtained by concentration under reduced pressure was purified by column chromatography on silica gel (petroleum ether: ethyl acetate=4:1) to give yellow oil 3.
In the invention S4, the reactant is filtered by diatomite, the filtrate is concentrated under reduced pressure, and the filtrate is purified by silica gel column chromatography (petroleum ether: ethyl acetate=5:1) to obtain colorless oily matter 4, namely the bibenzyl compound shown in formula (I), wherein nuclear magnetic hydrogen spectrum, carbon spectrum and high-resolution mass spectrum of the bibenzyl compound are consistent with natural products separated from dendrobium huoshanense. Activated palladium on charcoal was 5% strength (commercial product).
The bibenzyl provided by the invention can be used for preparing anti-melanoma medicines. The dibenzyl compound can effectively inhibit the growth and metastasis of the melanoma B16-F10 cells of the skin of the mice and tumors in vivo under the condition of low concentration, and has great application potential.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention provides a novel bibenzyl compound, and also provides an application of the bibenzyl compound in preparing a medicament for treating and/or preventing melanoma, wherein the bibenzyl compound can inhibit proliferation of skin melanoma cells of mice under the condition of low concentration, shows remarkable activity, and has an IC 50 value of 4.938 mu mol/L; in vivo experiments show that the dibenzyl compound can inhibit the growth and metastasis of melanoma of mice. Therefore, the dibenzyl compound has great application potential in development of anti-melanoma medicaments.
(2) The invention also provides a natural separation preparation method of the bibenzyl compound (I), which can rapidly, accurately and efficiently prepare the bibenzyl compound from dendrobium huoshanense. Dendrobium huoshanense is a perennial herb medicine and food homologous plant, and a novel compound bibenzyl compound (I) is extracted and separated from the dendrobium huoshanense, has good anti-melanoma activity, and provides a new idea for subsequent drug development.
(3) The invention further provides a chemical synthesis preparation method of the dibenzyl compound (I), which can be used for preparing a large amount of the compound rapidly and efficiently. It is difficult to obtain large amounts of natural products from natural sources of plant resources and chemical synthesis methods are employed to prepare materials that can be used as alternative sources to provide large amounts of products that meet the needs of subsequent research.
Drawings
FIG. 1 is a 1 H NMR chart of the dibenzyl compound (I) according to the invention;
FIG. 2 is a 13 C NMR chart of the dibenzyl compound (I) according to the present invention;
FIG. 3 is a 1 H NMR chart of the synthetic dibenzyl compound (I) according to the invention;
FIG. 4 is a 13 C NMR chart of the synthetic dibenzyl compound (I) according to the present invention;
FIG. 5 is a graph showing the activity of the dibenzyl compound (I) in inhibiting skin melanoma B16-F10 of mice; A-D are respectively a structure diagram of the dibenzyl compound (I), a cell proliferation experimental result diagram, an IC 50 diagram of the dibenzyl compound (I) on B16-F10 cells and a cell migration experimental result diagram;
FIG. 6 is a graph showing the growth and metastasis activity of the dibenzyl compound (I) in inhibiting subcutaneous B16-F10 xenograft tumor mice model. A-E are the mouse weight change map, the mouse tumor size map, the mouse spleen map, the mouse weight map and the mouse tumor weight map respectively.
Detailed Description
The present invention will be described in further detail with reference to examples.
It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The materials or equipment used are conventional products available from commercial sources, not identified to the manufacturer.
Example 1
A dibenzyl compound has a structural formula shown in formula (I);
The natural preparation and separation method of the bibenzyl compound comprises the following steps:
Step (1): extracting stem of Dendrobium huoshanense with absolute ethanol, and concentrating the extractive solution under reduced pressure to obtain Dendrobium huoshanense extract;
step (2): dissolving the dendrobium huoshanense extract obtained in the step (1) with water, extracting with ethyl acetate, and concentrating an ethyl acetate phase under reduced pressure to obtain dendrobium huoshanense extract;
Step (3): separating the dendrobium huoshanense extract obtained in the step (2) through normal phase silica gel column chromatography, performing gradient elution, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, and combining the same parts to obtain 7 components Fr.1-Fr.7; the mobile phase adopted by gradient elution is a mixed solvent of dichloromethane and methanol;
Step (4): separating the component Fr.2 obtained in the step (3) by normal phase silica gel column chromatography, performing gradient elution, collecting gradient eluents of each gradient, concentrating, monitoring by TLC, and combining the same parts to obtain 6 subfractions 2A1-2A6; the mobile phase adopted by gradient elution is a mixed solvent of petroleum ether and ethyl acetate;
Step (5): and (3) carrying out Sephadex LH-20 Sephadex gel column and semi-preparative high performance liquid chromatography separation and purification on the subfraction 2A1 obtained in the step (4) to obtain the bibenzyl compound shown in the formula (I).
Example 2
A dibenzyl compound has a structural formula shown in formula (I);
The natural preparation and separation method of the bibenzyl compound comprises the following steps:
Step (1): extracting stem of Dendrobium huoshanense with absolute ethanol, and concentrating the extractive solution under reduced pressure to obtain Dendrobium huoshanense extract;
step (2): dissolving the dendrobium huoshanense extract obtained in the step (1) with water, extracting with ethyl acetate, and concentrating an ethyl acetate phase under reduced pressure to obtain dendrobium huoshanense extract;
Step (3): separating the dendrobium huoshanense extract obtained in the step (2) through normal phase silica gel column chromatography, performing gradient elution, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, and combining the same parts to obtain 7 components Fr.1-Fr.7; the mobile phase adopted by gradient elution is a mixed solvent of dichloromethane and methanol;
Step (4): separating the component Fr.2 obtained in the step (3) by normal phase silica gel column chromatography, performing gradient elution, collecting gradient eluents of each gradient, concentrating, monitoring by TLC, and combining the same parts to obtain 6 subfractions 2A1-2A6; the mobile phase adopted by gradient elution is a mixed solvent of petroleum ether and ethyl acetate;
Step (5): and (3) carrying out Sephadex LH-20 Sephadex gel column and semi-preparative high performance liquid chromatography separation and purification on the subfraction 2A1 obtained in the step (4) to obtain the bibenzyl compound shown in the formula (I).
In the step (1), the extraction mode is immersion extraction; the extraction times are 1 time; the extraction time of each time is 5 days;
In the step (2), the volume ratio of ethyl acetate to water is 0.5:1; extracting by adopting ultrasonic waves; the extraction times are 3 times; the extraction time was 0.5 hour each, and the mixture was allowed to stand overnight after extraction.
In the step (3), the volume ratio of the dichloromethane to the methanol in the mixed solvent of the dichloromethane and the methanol is sequentially 100:0, 100:1, 80:1, 50:1, 30:1, 10:1, 5:1, 1:1 and 0:1;
In the step (4), the volume ratio of petroleum ether to ethyl acetate is 100:1, 60:1, 30:1, 10:1 and 1:1 in sequence.
In the step (5), a gel column adopted in the gel chromatography purification is Sephadex LH-20, and a mixed solvent of dichloromethane and methanol with the volume ratio of 1:1 is used as a mobile phase;
The chromatographic conditions for semi-preparative high performance liquid chromatography purification are:
The mobile phase is a mixed solvent of acetonitrile and water in a volume ratio of 1:1; the flow rate is 3mL/min; collecting the components with the peak starting time of 36 min; a C 18 reverse-phase chromatographic column is adopted; the specification is preferably: 4.6mm by 250mm,5 μm; column temperature: 30 ℃; sample size per sample: 20. Mu.L; the detection wavelength was 210nm using an ultraviolet detector.
Example 3
A dibenzyl compound has a structural formula shown in formula (I);
The natural preparation and separation method of the bibenzyl compound comprises the following steps:
Step (1): extracting stem of Dendrobium huoshanense with absolute ethanol, and concentrating the extractive solution under reduced pressure to obtain Dendrobium huoshanense extract;
step (2): dissolving the dendrobium huoshanense extract obtained in the step (1) with water, extracting with ethyl acetate, and concentrating an ethyl acetate phase under reduced pressure to obtain dendrobium huoshanense extract;
Step (3): separating the dendrobium huoshanense extract obtained in the step (2) through normal phase silica gel column chromatography, performing gradient elution, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, and combining the same parts to obtain 7 components Fr.1-Fr.7; the mobile phase adopted by gradient elution is a mixed solvent of dichloromethane and methanol;
Step (4): separating the component Fr.2 obtained in the step (3) by normal phase silica gel column chromatography, performing gradient elution, collecting gradient eluents of each gradient, concentrating, monitoring by TLC, and combining the same parts to obtain 6 subfractions 2A1-2A6; the mobile phase adopted by gradient elution is a mixed solvent of petroleum ether and ethyl acetate;
Step (5): and (3) carrying out Sephadex LH-20 Sephadex gel column and semi-preparative high performance liquid chromatography separation and purification on the subfraction 2A1 obtained in the step (4) to obtain the bibenzyl compound shown in the formula (I).
In the step (1), the extraction mode is ultrasonic extraction; the extraction times are 5 times; the extraction time of each time is 9 days;
in the step (2), the volume ratio of ethyl acetate to water is 4:1; extracting by adopting ultrasonic waves; the extraction times are 6 times; each extraction time was 2 hours and allowed to stand overnight after extraction.
In the step (3), the volume ratio of the dichloromethane to the methanol in the mixed solvent of the dichloromethane and the methanol is sequentially 100:0, 100:1, 80:1, 50:1, 30:1, 10:1, 5:1, 1:1 and 0:1;
In the step (4), the volume ratio of petroleum ether to ethyl acetate is 100:1, 60:1, 30:1, 10:1 and 1:1 in sequence.
In the step (5), a gel column adopted in the gel chromatography purification is Sephadex LH-20, and a mixed solvent of dichloromethane and methanol with the volume ratio of 1:1 is used as a mobile phase;
The chromatographic conditions for semi-preparative high performance liquid chromatography purification are:
The mobile phase is a mixed solvent of acetonitrile and water in a volume ratio of 1:1; the flow rate is 3mL/min; collecting the components with the peak starting time of 36 min; a C 18 reverse-phase chromatographic column is adopted; the specification is preferably: 4.6mm by 250mm,5 μm; column temperature: 30 ℃; sample size per sample: 20. Mu.L; the detection wavelength was 210nm using an ultraviolet detector.
Example 4
A dibenzyl compound has a structural formula shown in formula (I);
The natural preparation and separation method of the bibenzyl compound comprises the following steps:
Step (1): extracting stem of Dendrobium huoshanense with absolute ethanol, and concentrating the extractive solution under reduced pressure to obtain Dendrobium huoshanense extract;
step (2): dissolving the dendrobium huoshanense extract obtained in the step (1) with water, extracting with ethyl acetate, and concentrating an ethyl acetate phase under reduced pressure to obtain dendrobium huoshanense extract;
Step (3): separating the dendrobium huoshanense extract obtained in the step (2) through normal phase silica gel column chromatography, performing gradient elution, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, and combining the same parts to obtain 7 components Fr.1-Fr.7; the mobile phase adopted by gradient elution is a mixed solvent of dichloromethane and methanol;
Step (4): separating the component Fr.2 obtained in the step (3) by normal phase silica gel column chromatography, performing gradient elution, collecting gradient eluents of each gradient, concentrating, monitoring by TLC, and combining the same parts to obtain 6 subfractions 2A1-2A6; the mobile phase adopted by gradient elution is a mixed solvent of petroleum ether and ethyl acetate;
Step (5): and (3) carrying out Sephadex LH-20 Sephadex gel column and semi-preparative high performance liquid chromatography separation and purification on the subfraction 2A1 obtained in the step (4) to obtain the bibenzyl compound shown in the formula (I).
In the step (1), the extraction mode is reflux extraction; the extraction times are 3 times; the extraction time of each time is 7 days;
In the step (2), the volume ratio of the ethyl acetate to the water is 2:1; extracting by adopting ultrasonic waves; the extraction times are 4 times; each extraction time was 1 hour and allowed to stand overnight after extraction.
In the step (3), the volume ratio of the dichloromethane to the methanol in the mixed solvent of the dichloromethane and the methanol is sequentially 100:0, 100:1, 80:1, 50:1, 30:1, 10:1, 5:1, 1:1 and 0:1;
In the step (4), the volume ratio of petroleum ether to ethyl acetate is 100:1, 60:1, 30:1, 10:1 and 1:1 in sequence.
In the step (5), a gel column adopted in the gel chromatography purification is Sephadex LH-20, and a mixed solvent of dichloromethane and methanol with the volume ratio of 1:1 is used as a mobile phase;
The chromatographic conditions for semi-preparative high performance liquid chromatography purification are:
The mobile phase is a mixed solvent of acetonitrile and water in a volume ratio of 1:1; the flow rate is 3mL/min; collecting the components with the peak starting time of 36 min; a C 18 reverse-phase chromatographic column is adopted; the specification is preferably: 4.6mm by 250mm,5 μm; column temperature: 30 ℃; sample size per sample: 20. Mu.L; the detection wavelength was 210nm using an ultraviolet detector.
Example 5
A dibenzyl compound has a structural formula shown in formula (I);
The preparation method of the bibenzyl compound comprises the following steps:
S1: heating a mixture of 4-methoxychlorobenzyl chloride, triphenylphosphine and toluene under the protection of nitrogen, and carrying out reflux reaction to obtain white solid powder (chloro- (4-methoxybenzyl) triphenyl-lambda 5 -phosphane);
S2: adding chlorobenzyl and potassium carbonate into DMF containing 3, 4-dimethoxy-5-hydroxybenzaldehyde under the protection of nitrogen, and heating for reaction to obtain a (3- (benzyloxy) -4, 5-dimethoxy benzaldehyde) crude product;
S3: lithium hydroxide monohydrate is added into isopropanol solution of (chloro- (4-methoxybenzyl) triphenyl-lambda 5 -phosphane) at the temperature of 0 ℃ for reaction; then adding (3- (benzyloxy) -4, 5-dimethoxy benzaldehyde) crude product, and heating to react to obtain yellow oily (1- (benzyloxy) -2, 3-dimethoxy-5- (4-methoxy styryl) benzene);
S4: the obtained yellow oily matter (1- (benzyloxy) -2, 3-dimethoxy-5- (4-methoxystyryl) benzene) is dissolved in a mixed solvent of ethyl acetate and ethanol, hydrogen is introduced, then active palladium carbon is added, and stirring reaction is carried out at room temperature, so as to obtain the bibenzyl compound shown in the formula (I).
Example 6
A dibenzyl compound has a structural formula shown in formula (I);
The preparation method of the bibenzyl compound comprises the following steps:
S1: heating a mixture of 4-methoxychlorobenzyl chloride, triphenylphosphine and toluene under the protection of nitrogen, and carrying out reflux reaction to obtain white solid powder (chloro- (4-methoxybenzyl) triphenyl-lambda 5 -phosphane);
S2: adding chlorobenzyl and potassium carbonate into DMF containing 3, 4-dimethoxy-5-hydroxybenzaldehyde under the protection of nitrogen, and heating for reaction to obtain a (3- (benzyloxy) -4, 5-dimethoxy benzaldehyde) crude product;
S3: lithium hydroxide monohydrate is added into isopropanol solution of (chloro- (4-methoxybenzyl) triphenyl-lambda 5 -phosphane) at the temperature of 0 ℃ for reaction; then adding (3- (benzyloxy) -4, 5-dimethoxy benzaldehyde) crude product, and heating to react to obtain yellow oily (1- (benzyloxy) -2, 3-dimethoxy-5- (4-methoxy styryl) benzene);
S4: the obtained yellow oily matter (1- (benzyloxy) -2, 3-dimethoxy-5- (4-methoxystyryl) benzene) is dissolved in a mixed solvent of ethyl acetate and ethanol, hydrogen is introduced, then active palladium carbon is added, and stirring reaction is carried out at room temperature, so as to obtain the bibenzyl compound shown in the formula (I).
S1, carrying out reflux reaction for 10 hours at 100 ℃; the dosage ratio of 4-methoxychlorobenzyl chloride, triphenylphosphine and toluene was 1mmol:1mmol:1.5mL;
S2, heating at 70 ℃ for 5 hours; the dosage ratio of potassium carbonate, chlorobenzyl, 3, 4-dimethoxy-5-hydroxybenzaldehyde and DMF is 1mmol:1.9mmol:1.9mmol:2mL.
In S3, the ratio of the amount of lithium hydroxide monohydrate, white solid powder (chloro- (4-methoxybenzyl) triphenyl-. Lamda. 5 -phosphane), isopropyl alcohol and crude (3- (benzyloxy) -4, 5-dimethoxybenzaldehyde) was 1.5mmol:1.4mmol:4mL:1mmol; lithium hydroxide monohydrate is added into isopropanol solution of white solid powder (chlorine- (4-methoxybenzyl) triphenyl-lambda 5 -phosphane) to react for 14 minutes, and then (3- (benzyloxy) -4, 5-dimethoxy benzaldehyde) crude product is added, and after the addition is finished, the reaction mixture is heated to 50 ℃ to react for 9 hours;
in S4, the ratio of the amount of the yellow oily substance (1- (benzyloxy) -2, 3-dimethoxy-5- (4-methoxystyryl) benzene), ethyl acetate, ethanol and active palladium on charcoal is 2.4mmol:19mL:9mL:0.19g; activated palladium carbon is 5% in concentration; the reaction was stirred at room temperature for 2.5 hours.
Example 7
A dibenzyl compound has a structural formula shown in formula (I);
The preparation method of the bibenzyl compound comprises the following steps:
S1: heating a mixture of 4-methoxychlorobenzyl chloride, triphenylphosphine and toluene under the protection of nitrogen, and carrying out reflux reaction to obtain white solid powder (chloro- (4-methoxybenzyl) triphenyl-lambda 5 -phosphane);
S2: adding chlorobenzyl and potassium carbonate into DMF containing 3, 4-dimethoxy-5-hydroxybenzaldehyde under the protection of nitrogen, and heating for reaction to obtain a (3- (benzyloxy) -4, 5-dimethoxy benzaldehyde) crude product;
S3: lithium hydroxide monohydrate is added into isopropanol solution of (chloro- (4-methoxybenzyl) triphenyl-lambda 5 -phosphane) at the temperature of 0 ℃ for reaction; then adding (3- (benzyloxy) -4, 5-dimethoxy benzaldehyde) crude product, and heating to react to obtain yellow oily (1- (benzyloxy) -2, 3-dimethoxy-5- (4-methoxy styryl) benzene);
S4: the obtained yellow oily matter (1- (benzyloxy) -2, 3-dimethoxy-5- (4-methoxystyryl) benzene) is dissolved in a mixed solvent of ethyl acetate and ethanol, hydrogen is introduced, then active palladium carbon is added, and stirring reaction is carried out at room temperature, so as to obtain the bibenzyl compound shown in the formula (I).
S1, carrying out reflux reaction at 140 ℃ for 14 hours; the dosage ratio of 4-methoxychlorobenzyl chloride, triphenylphosphine and toluene was 1mmol:1.2mmol:2mL;
S2, heating at 90 ℃ for 7 hours; the dosage ratio of potassium carbonate, chlorobenzyl, 3, 4-dimethoxy-5-hydroxybenzaldehyde and DMF is 1mmol:2.1mmol:2.1mmol:2.5mL;
In S3, the ratio of the amount of lithium hydroxide monohydrate, white solid powder (chloro- (4-methoxybenzyl) triphenyl-. Lamda. 5 -phosphane), isopropyl alcohol and crude (3- (benzyloxy) -4, 5-dimethoxybenzaldehyde) was 1.7mmol:1.6mmol:5mL:1mmol; lithium hydroxide monohydrate is added into isopropanol solution of white solid powder (chlorine- (4-methoxybenzyl) triphenyl-lambda 5 -phosphane) to react for 16 minutes, and then (3- (benzyloxy) -4, 5-dimethoxy benzaldehyde) crude product is added, and after the addition is finished, the reaction mixture is heated to 70 ℃ to react for 11 hours;
in S4, the ratio of the amount of the yellow oily substance (1- (benzyloxy) -2, 3-dimethoxy-5- (4-methoxystyryl) benzene), ethyl acetate, ethanol and active palladium on charcoal is 2.6mmol:22mL:11mL:0.21g; activated palladium carbon is 5% in concentration; the reaction was stirred at room temperature for 3.5 hours.
Example 8
A dibenzyl compound has a structural formula shown in formula (I);
The preparation method of the bibenzyl compound comprises the following steps:
S1: heating a mixture of 4-methoxychlorobenzyl chloride, triphenylphosphine and toluene under the protection of nitrogen, and carrying out reflux reaction to obtain white solid powder (chloro- (4-methoxybenzyl) triphenyl-lambda 5 -phosphane);
S2: adding chlorobenzyl and potassium carbonate into DMF containing 3, 4-dimethoxy-5-hydroxybenzaldehyde under the protection of nitrogen, and heating for reaction to obtain a (3- (benzyloxy) -4, 5-dimethoxy benzaldehyde) crude product;
S3: lithium hydroxide monohydrate is added into isopropanol solution of (chloro- (4-methoxybenzyl) triphenyl-lambda 5 -phosphane) at the temperature of 0 ℃ for reaction; then adding (3- (benzyloxy) -4, 5-dimethoxy benzaldehyde) crude product, and heating to react to obtain yellow oily (1- (benzyloxy) -2, 3-dimethoxy-5- (4-methoxy styryl) benzene);
S4: the obtained yellow oily matter (1- (benzyloxy) -2, 3-dimethoxy-5- (4-methoxystyryl) benzene) is dissolved in a mixed solvent of ethyl acetate and ethanol, hydrogen is introduced, then active palladium carbon is added, and stirring reaction is carried out at room temperature, so as to obtain the bibenzyl compound shown in the formula (I).
S1, carrying out reflux reaction at 120 ℃ for 12 hours; the dosage ratio of 4-methoxychlorobenzyl chloride, triphenylphosphine and toluene was 1mmol:1.1mmol:1.7mL;
s2, heating at 80 ℃ for 6 hours; the dosage ratio of potassium carbonate, chlorobenzyl, 3, 4-dimethoxy-5-hydroxybenzaldehyde and DMF is 1mmol:2mmol:2mmol:2.2mL.
In S3, the ratio of the amount of lithium hydroxide monohydrate, white solid powder (chloro- (4-methoxybenzyl) triphenyl-. Lamda. 5 -phosphane), isopropyl alcohol and crude (3- (benzyloxy) -4, 5-dimethoxybenzaldehyde) was 1.6mmol:1.5mmol:4.5mL:1mmol; lithium hydroxide monohydrate is added into isopropanol solution of white solid powder (chlorine- (4-methoxybenzyl) triphenyl-lambda 5 -phosphane) to react for 15 minutes, and then (3- (benzyloxy) -4, 5-dimethoxy benzaldehyde) crude product is added, and after the addition is finished, the reaction mixture is heated to 60 ℃ to react for 10 hours;
In S4, the ratio of the amount of the yellow oily substance (1- (benzyloxy) -2, 3-dimethoxy-5- (4-methoxystyryl) benzene), ethyl acetate, ethanol and active palladium on charcoal is 2.5mmol:20mL:10mL:0.2g; activated palladium carbon is 5% in concentration; the reaction was stirred at room temperature for 3 hours.
Application example 1 preparation of bibenzyl Compound (I)
The preparation method of the dibenzyl compound (I) comprises the following steps:
Step (1): removing She Baojing from 20kg fresh herba Dendrobii sample, pulverizing to 0.8-1.2cm with pulverizer, soaking and extracting with 20L absolute ethanol at room temperature for 3 times, 7 days each time, filtering to obtain filtrate, and concentrating under reduced pressure to obtain herba Dendrobii extract;
Step (2): dissolving the dendrobium huoshanense extract obtained in the step (1) with water, extracting with ethyl acetate, wherein the volume ratio of water to ethyl acetate is 1:1, and concentrating an ethyl acetate phase under reduced pressure to obtain 967g of dendrobium huoshanense extract;
Step (3): dissolving the dendrobium huoshanense extract obtained in the step (2) by using ethyl acetate, adsorbing by using silica gel, mixing the sample, and performing rough-fraction gradient elution by using a forward silica gel chromatographic column to obtain components Fr.1-Fr.7, wherein a stationary phase of silica gel column chromatography is 200-300 meshes of forward silica gel, and a mobile phase is dichloromethane/methanol (volume ratio is 100:0-0:100) in sequence;
the method comprises the following steps: dissolving the extract with 10 times of ethyl acetate, adsorbing with 80-100 mesh silica gel with the same mass as the extract, and loading;
the mobile phase adopted by the gradient elution is a mixed solvent of dichloromethane and methanol in turn according to the elution sequence. The volume ratio of dichloromethane to methanol in the mixed solvent of dichloromethane and methanol is sequentially 100:0, 100:1, 80:1, 50:1, 30:1, 10:1, 5:1, 1:1 and 0:1, and after each gradient elution is carried out until a TLC (thin layer chromatography) plate shows no point, the next gradient elution is replaced, so that 7 components Fr.1-Fr.7 are obtained.
Step (4): subjecting the component Fr.2 to normal phase silica gel column chromatography, TLC monitoring, combining parts of the same components to obtain 6 subfractions (2A 1-2A 6), wherein the stationary phase of the silica gel column chromatography is normal phase silica gel of 200-300 meshes, and the mobile phase is petroleum ether/ethyl acetate (volume ratio 100:1-1:1);
The method comprises the following steps: dissolving the component Fr.2 with 10 times of methanol, adsorbing with 80-100 mesh silica gel with the same mass as the component Fr.2, and loading; the mobile phase adopted by the gradient elution is a mixed solvent of petroleum ether and ethyl acetate, the volume ratio of petroleum ether to ethyl acetate is sequentially 100:1, 60:1, 30:1, 10:1 and 1:1, and after each gradient elution is not displayed on a TLC (thin layer chromatography) plate, the next gradient elution is replaced to obtain 6 sub-components in total of 2A1-2A 6.
Step (5): separating and purifying 2A1 by gel column chromatography Sephadex LH-20 and HPLC; the mobile phase of the gel is methylene dichloride/methanol (volume ratio 1:1); the mobile phase of HPLC was acetonitrile/water (volume ratio 1:1), the flow rate was 3mL/min, and the fractions having a peak starting time of 36min were collected to give compound (I) (2.2 mg).
During gel chromatography purification, the subfraction 2A1 is dissolved by 2 times of methanol and is loaded; collection was performed using 10mL tubes, 5mL per tube, 60 tubes were collected in total, monitored by TLC, the same fractions were combined, 10-20 tubes were combined, concentrated under reduced pressure and purified by HPLC.
The HPLC chromatographic condition is that the mobile phase is a mixed solvent of acetonitrile and water (volume ratio is 1:1), the flow rate is 3mL/min, the components with the peak starting time of 36min are collected, and an Agilent C18 reverse chromatographic column is adopted, wherein the specification is as follows: 4.6mm by 250mm,5 μm; column temperature: 30 ℃, the sample injection amount is equal to: 20. Mu.L, using an ultraviolet detector, the detection wavelength was 210nm.
The structure of the obtained monomer compound (I) is determined by a nuclear magnetic resonance spectrum, and the properties and spectrum data of the obtained compound are as follows:
The structural formula of the dibenzyl compound (I) is shown as follows;
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Compound (i): 2,3-dimethoxy-5- (4-methoxyphenethyl) phenol, light yellow oily liquid, molecular formula of C 17H20O4 and molecular weight of 289.1433.
1H NMR(500MHz,CDCl3)δH:7.10(2H,d,J＝8.49Hz,H-2′,H-6′),6.83(2H,d,J＝8.51Hz,H-3′,H-5′),6.46(1H,s,H-6),6.24(1H,s,H-2),3.87(3H,s,H3-8),3.81(3H,s,H3-7),3.79(3H,s,H3-7′),2.82(2H,m,H2-8′),2.79(2H,m,H2-9);5.71(1H,s,OH).
13C NMR(150MHz,CDCl3)δC:158.0(C-4′),152.2(C-3),149.2(C-5),138.4(C-1),133.9(C-1′),133.7(C-4),129.5(C-2′),129.5(C-6′),113.9(C-3′),113.9(C-5′),107.9(C-6),104.5(C-2),61.1(C-8),55.9(C-7),55.4(C-7′),38.4(C-9),37.0(C-8′).
Application example 2 preparation of chemical Synthesis of bibenzyl Compound (I)
S1: a mixture of 4-methoxychlorobenzyl chloride (9.24 g,59.0 mmol), triphenylphosphine (17.0 g,64.8 mmol) and toluene (100 mL) was heated to 120deg.C under nitrogen and refluxed for 12 hours. After cooling to room temperature, a solid was precipitated, and the reaction solution was filtered, washed successively with toluene, acetone and n-hexane to give 1- (chloro- (4-methoxybenzyl) triphenyl-. Lamda. 5 -phosphane) (22.7 g, 92%) as a white solid powder.
S2: chlorobenzyl (0.696 g,5.50 mmol) and potassium carbonate (0.380 g,2.75 mmol) were added to a 25mL flask containing 3, 4-dimethoxy-5-hydroxybenzaldehyde (1.00 g,5.50 mmol) and DMF (6 mL) under nitrogen, heated to 80℃and reacted for 6 hours. The reaction mixture was cooled to room temperature, poured into ice water, extracted three times with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give crude product 2- (3- (benzyloxy) -4, 5-dimethoxybenzaldehyde) which was directly put into the next reaction without purification.
S3: lithium hydroxide monohydrate (0.369 g,8.80 mmol) was added to S1 to give a white solid powder (chloro- (4-methoxybenzyl) triphenyl-. Lambda. 5 -phosphane) (3.45 g,8.26 mmol) in isopropanol (25 mL) at 0deg.C and reacted for 15 min, followed by addition of S2 to give the crude product (3- (benzyloxy) -4, 5-dimethoxybenzaldehyde) (1.50 g,5.50 mmol). After the addition was completed, the reaction mixture was heated to 60℃and reacted for 10 hours (the reaction was monitored by thin layer chromatography). The reaction mixture was quenched with water and extracted 3 times with ethyl acetate. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude product which was purified by column chromatography over silica gel (petroleum ether: ethyl acetate=4:1) to give 3- (1- (benzyloxy) -2, 3-dimethoxy-5- (4-methoxystyryl) benzene) (2.06 g, 99%) as a yellow oil.
S4: the yellow oily substance (1- (benzyloxy) -2, 3-dimethoxy-5- (4-methoxystyryl) benzene) (2.06 g,5.46 mmol) obtained in S3 was dissolved in ethyl acetate (44 mL) and ethanol (22 mL), and hydrogen gas was introduced thereinto, followed by addition of 5% active palladium on charcoal (0.450 g). After stirring at room temperature for 3 hours, the reaction mixture was filtered through celite, the filtrate was concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether: ethyl acetate=5:1) to give a colorless oil (0.883 g, 56%), i.e., the dibenzyl compound of formula (i) whose nuclear magnetic hydrogen spectrum, carbon spectrum and high resolution mass spectrum were identical to those of the natural product isolated from dendrobium huoshanense.
Application example 3 Activity study of bibenzyl Compound (I) against cutaneous melanoma B16-F10 in mice
1. Material
1.1 Cells
Mouse cutaneous melanoma cells (B16-F10) were purchased from the national academy of sciences (Shanghai)
1.2 Materials
Fetal bovine serum was purchased from GBICO, DMEM medium, phosphate Buffered Saline (PBS), penicillin-streptomycin, 0.25% trypsin solution was purchased from Gibco (usa), and CCK8 kit was purchased from Abcam (usa).
1.3 Instruments
Carbon dioxide incubator (China hong Kong Likang biomedical technology Co., ltd.), desktop high-speed centrifuge (China Kunshan ultrasonic instruments Co., ltd.), enzyme-labeled instrument (Bio-TEK Co., U.S.A.).
2. Method of
2.1 Cell culture
B16-F10 cells were cultured in DMEM medium containing 10% fetal bovine serum by volume fraction, cultured in 5% CO 2 in 37℃incubator, digested with pancreatin and passaged at 1:3 ratio.
2.3CCK8 detection of cell proliferation
The inhibitory effect of compound (I) on B16-F10 cells was studied at different concentrations, and different groups of cells were cultured by adding compound (I) at concentrations of 30. Mu. Mol/L, 15. Mu. Mol/L, 10. Mu. Mol/L, and 5. Mu. Mol/L, respectively.
B16-F10 cells in the logarithmic growth phase were inoculated into 96-well plates at 100. Mu.L per well and cultured in an incubator (37 ℃ C., 5% CO 2) for 24 hours with the cell concentration adjusted to 5X 10 4/mL to 8X 10 4/mL. The 96-well plate was removed, the medium was discarded, 100. Mu.L of medium containing different concentrations of dibenzyl compounds (DMEM medium containing 10% fetal bovine serum by volume fraction) was added to each well of the experimental group, and 5 wells were set up, respectively. The culture was continued for 24 hours, the drug-containing medium was aspirated, washed 3 times with PBS, after the completion of the observation, 10. Mu.L of CCK8 with a volume fraction of 10% was added to each well, incubated at 37℃for 4 hours in the absence of light, absorbance (OD value) was measured at 450nm with a microplate reader, and cell viability was calculated.
2.4 Cell migration experiments
The inhibition effect of the positive drugs and the compound (I) on B16-F10 cell migration is studied, a control group (complete culture medium), a positive drug group (Vemurafenib, 3 mu mol/L) and a compound (I) group (5 mu mol/L) are arranged, and different groups of cells are respectively added with different drugs for culture.
B16-F10 cells in the logarithmic growth phase were inoculated into 6-well plates at a concentration of 6X 10 4 to 8X 10 4 cells/mL and 2mL per well, and cultured in an incubator (37 ℃ C., 5% CO 2) for 24 hours. The 6-well plate was removed, the medium was discarded, and the streaked cells were removed by washing 3 times with PBS using a tip of a 200. Mu.L pipette. 2mL of culture medium containing different concentrations of dibenzyl compounds (the culture medium adopts DMEM (medium with 1% fetal bovine serum) with volume fraction) is added into each hole of the experimental group, 3 compound holes are respectively arranged, and photographing recording is carried out under a 4-fold mirror for 0 hour for sampling. The culture was continued for 24 hours, the drug-containing medium was aspirated and removed, washed 3 times with PBS, after the observation was completed, fixed with 4% paraformaldehyde fixing solution for 5 minutes, then stained with 1% crystal violet staining solution, washed 3 times with PBS, the excess crystal violet staining solution was removed, and photographed under a 4-fold mirror for recording for 24 hours for sampling.
3 Results of experiments
(1) The results of cell proliferation experiments show that the dibenzyl compounds with the concentration of 30 mu mol/L, 15 mu mol/L, 10 mu mol/L and 5 mu mol/L have different degrees of inhibition on B16-F10 cells (figures 5B and 5C), so that the compound (I) has obvious inhibition activity on B16-F10 cells, and the IC 50 value is 4.938 mu mol/L.
(2) The results of the cell migration experiments showed that the intercellular spaces of the control group were significantly reduced, while the differences between the positive drug group and the dibenzyl compound groups at the respective concentrations were not significantly changed (fig. 5D). Thus, compound (I) significantly inhibits B16-F10 cell migration.
Conclusion of the experiment
The compound (I) can obviously inhibit proliferation of B16-F10 cells, and the IC 50 value is 4.938 mu mol/L. Therefore, the dibenzyl compound (I) has great potential in the application of anti-melanoma medicaments.
Application example 4 bibenzyl compound Compound (I) inhibition of growth and metastasis Activity studies in subcutaneous B16-F10 xenograft tumor mice model.
1 Material
1.1 Cells and animals
Mouse skin melanoma cells (B16-F10) were purchased from the national academy of sciences (Shanghai); mice (C57 BL/6, male, SPF grade, 18-20g,5-6 weeks old) were purchased from Liaoning Biotechnology Co., ltd
1.2 Materials
Fetal bovine serum was purchased from GBICO, DMEM medium, phosphate Buffered Saline (PBS), penicillin-streptomycin, 0.25% trypsin solution was purchased from Gibco (America)
1.3 Instruments
Carbon dioxide incubator (hong Kong Likang biomedical technology Co., ltd., china), desktop high-speed centrifuge (Kunshan ultrasonic instruments Co., ltd.).
2 Method
2.1 Experimental grouping
The inhibitory effect of different doses of compound (I) on the subcutaneous tumor-bearing model of melanoma of B16-F10 mice was studied, and different administration groups of mice were respectively perfused with 25mg/kg and 50mg/kg of bibenzyl compound, dissolved in physiological saline, and administered with vitamin Mo Feini (Vemurafenib, 50 mg/kg) in positive drug group, and the same dose of physiological saline solution was administered to control group and model group of mice. The control group mice are healthy mice without tumor, the administration group, the positive drug group and the model group mice are tumor model mice, and 6 mice are used in each group.
2.2 Cell culture
B16-F10 cells were cultured in DMEM medium containing 10% fetal bovine serum by volume fraction, cultured in 5% CO 2 in 37℃incubator, digested with pancreatin and passaged at 1:3 ratio.
2.3 Animal model establishment
The armpit of the right forelimb of the mouse is wiped with alcohol with the volume concentration of 75 percent, 1X 10 6 cells/cell suspension of melanoma B16-F10 of the mouse is taken by a 1mL syringe, and the cell suspension is inoculated into the armpit of the right forelimb of the mouse in a subcutaneous injection mode, so that a tumor model is established. When subcutaneous tumors were observed, mice were randomized into experimental groups and subsequently received different treatment regimens.
3 Results of experiments
After administration of positive drugs and different concentrations of dibenzyl compounds, the tumor growth rate was significantly reduced. Furthermore, the tumors of the treatment group were significantly inhibited compared to the model group (fig. 6B, 67E). And treatment with compound (i) did not affect the body weight of mice (fig. 6A, 6D), indicating low toxicity of compound (i). Furthermore, it can be clearly observed in fig. 6C that compound (i) significantly inhibited the metastasis of melanoma cells in liver tissue. Notably, the high dose group of compound (i) did not develop melanoma metastasis in the liver compared to the other groups.
Conclusion of the experiment
The dibenzyl compound can effectively inhibit growth and metastasis of subcutaneous B16-F10 xenograft tumors. Therefore, the dibenzyl compound has great potential in the application of anti-melanoma medicaments.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. A dibenzyl compound is characterized in that the structural formula is shown as a formula (I);
2. The method for the separation of dibenzyl compounds according to claim 1, characterized by comprising the steps of:
Step (1): extracting stem of Dendrobium huoshanense with absolute ethanol, and concentrating the extractive solution under reduced pressure to obtain Dendrobium huoshanense extract;
step (2): dissolving the dendrobium huoshanense extract obtained in the step (1) with water, extracting with ethyl acetate, and concentrating an ethyl acetate phase under reduced pressure to obtain dendrobium huoshanense extract;
Step (3): separating the dendrobium huoshanense extract obtained in the step (2) through normal phase silica gel column chromatography, performing gradient elution, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, and combining the same parts to obtain 7 components Fr.1-Fr.7; the mobile phase adopted by gradient elution is a mixed solvent of dichloromethane and methanol;
Step (4): separating the component Fr.2 obtained in the step (3) by normal phase silica gel column chromatography, performing gradient elution, collecting gradient eluents of each gradient, concentrating, monitoring by TLC, and combining the same parts to obtain 6 subfractions 2A1-2A6; the mobile phase adopted by gradient elution is a mixed solvent of petroleum ether and ethyl acetate;
Step (5): and (3) carrying out Sephadex LH-20 Sephadex gel column and semi-preparative high performance liquid chromatography separation and purification on the subfraction 2A1 obtained in the step (4) to obtain the bibenzyl compound shown in the formula (I).
3. The method for producing a dibenzyl compound according to claim 2, characterized in that, in step (1), the extraction is immersion extraction, ultrasonic extraction or reflux extraction; the extraction times are 1-5 times; the extraction time is 5-9 days each time;
In the step (2), the volume ratio of the ethyl acetate to the water is 0.5-4:1; extracting by adopting ultrasonic waves; the extraction times are 3-6 times; the extraction time is 0.5-2 hours each time, and the mixture is left standing overnight after extraction.
4. The preparation method of the dibenzyl compound according to claim 2, characterized in that in the step (3), the volume ratio of dichloromethane to methanol in the mixed solvent of dichloromethane and methanol is sequentially 100:0, 100:1, 80:1, 50:1, 30:1, 10:1, 5:1, 1:1, 0:1;
In the step (4), the volume ratio of petroleum ether to ethyl acetate is 100:1, 60:1, 30:1, 10:1 and 1:1 in sequence.
5. The method for preparing bibenzyl compound according to claim 2, wherein in step (5), the gel column used in the gel chromatography purification is Sephadex LH-20, and a mixed solvent of dichloromethane and methanol in a volume ratio of 1:1 is used as a mobile phase;
The chromatographic conditions for semi-preparative high performance liquid chromatography purification are:
The mobile phase is a mixed solvent of acetonitrile and water in a volume ratio of 1:1; the flow rate is 3mL/min; collecting the components with the peak starting time of 36 min; a C 18 reverse-phase chromatographic column is adopted; the specification is preferably: 4.6mm by 250mm,5 μm; column temperature: 30 ℃; sample size per sample: 20. Mu.L; the detection wavelength was 210nm using an ultraviolet detector.
6. The chemical synthesis preparation method of the dibenzyl compound as claimed in claim 1, which is characterized by comprising the following steps:
S1: heating a mixture of 4-methoxychlorobenzyl chloride, triphenylphosphine and toluene under the protection of nitrogen, and carrying out reflux reaction to obtain white solid powder (chloro- (4-methoxybenzyl) triphenyl-lambda 5 -phosphane);
S2: adding chlorobenzyl and potassium carbonate into DMF containing 3, 4-dimethoxy-5-hydroxybenzaldehyde under the protection of nitrogen, and heating for reaction to obtain a (3- (benzyloxy) -4, 5-dimethoxy benzaldehyde) crude product;
S3: lithium hydroxide monohydrate is added into isopropanol solution of (chloro- (4-methoxybenzyl) triphenyl-lambda 5 -phosphane) at the temperature of 0 ℃ for reaction; then adding (3- (benzyloxy) -4, 5-dimethoxy benzaldehyde) crude product, and heating to react to obtain yellow oily (1- (benzyloxy) -2, 3-dimethoxy-5- (4-methoxy styryl) benzene);
S4: the obtained yellow oily matter (1- (benzyloxy) -2, 3-dimethoxy-5- (4-methoxystyryl) benzene) is dissolved in a mixed solvent of ethyl acetate and ethanol, hydrogen is introduced, then active palladium carbon is added, and stirring reaction is carried out at room temperature, so as to obtain the bibenzyl compound shown in the formula (I).
7. The process for producing a dibenzyl compound according to claim 6, characterized in that, in S1, the reaction is carried out at 100 to 140℃for 10 to 14 hours under reflux; the dosage ratio of 4-methoxychlorobenzyl chloride, triphenylphosphine and toluene was 1mmol:1-1.2mmol:1.5-2mL;
S2, heating at 70-90 ℃ for 5-7 hours; the dosage ratio of potassium carbonate, chlorobenzyl, 3, 4-dimethoxy-5-hydroxybenzaldehyde and DMF is 1mmol:1.9-2.1mmol:1.9-2.1 mmol: 2-2.5mL.
8. The process for producing a dibenzyl compound according to claim 6, characterized in that in S3, lithium hydroxide monohydrate, white solid powder (chloro- (4-methoxybenzyl) triphenyl-. Lamda. 5 -phosphane), isopropyl alcohol and (3- (benzyloxy) -4, 5-dimethoxybenzaldehyde) crude product are used in an amount ratio of 1.5 to 1.7mmol:1.4-1.6mmol:4-5mL:1mmol; lithium hydroxide monohydrate is added into isopropanol solution of white solid powder (chlorine- (4-methoxybenzyl) triphenyl-lambda 5 -phosphane) to react for 14 to 16 minutes at the temperature of 0 ℃, then (3- (benzyloxy) -4, 5-dimethoxy benzaldehyde) crude product is added, and after the addition is finished, the reaction mixture is heated to 50 to 70 ℃ to react for 9 to 11 hours;
In S4, the dosage ratio of yellow oily matter (1- (benzyloxy) -2, 3-dimethoxy-5- (4-methoxystyryl) benzene), ethyl acetate, ethanol and active palladium on charcoal is 2.4-2.6mmol:19-22mL:9-11mL:0.19-0.21g; the reaction is stirred at room temperature for 2.5 to 3.5 hours.
9. Use of a dibenzyl compound according to claim 1 in the preparation of a medicament for the treatment and/or prevention of melanoma.
10. Use of a dibenzyl compound according to claim 9 for the preparation of a medicament for the treatment and/or prevention of melanoma, characterized in that said melanoma is B16-F10 mouse skin melanoma.
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