CN117430512A - Mould acid compound with anti-tumor activity and preparation method and application thereof - Google Patents
Mould acid compound with anti-tumor activity and preparation method and application thereof Download PDFInfo
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- CN117430512A CN117430512A CN202311296356.XA CN202311296356A CN117430512A CN 117430512 A CN117430512 A CN 117430512A CN 202311296356 A CN202311296356 A CN 202311296356A CN 117430512 A CN117430512 A CN 117430512A
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- acid compound
- mycolic acid
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 52
- 239000002253 acid Substances 0.000 title claims abstract description 43
- 230000000259 anti-tumor effect Effects 0.000 title claims abstract description 23
- 238000002360 preparation method Methods 0.000 title claims abstract description 7
- 239000000499 gel Substances 0.000 claims abstract description 9
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000000741 silica gel Substances 0.000 claims abstract description 6
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 6
- 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 abstract description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 36
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 36
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 30
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 27
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 26
- 238000004809 thin layer chromatography Methods 0.000 claims description 22
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 19
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 14
- 239000003208 petroleum Substances 0.000 claims description 13
- 238000000855 fermentation Methods 0.000 claims description 12
- 230000004151 fermentation Effects 0.000 claims description 12
- 238000004458 analytical method Methods 0.000 claims description 11
- 239000003795 chemical substances by application Substances 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 239000000287 crude extract Substances 0.000 claims description 8
- 239000003480 eluent Substances 0.000 claims description 8
- 238000003892 spreading Methods 0.000 claims description 8
- VDZOOKBUILJEDG-UHFFFAOYSA-M tetrabutylammonium hydroxide Chemical compound [OH-].CCCC[N+](CCCC)(CCCC)CCCC VDZOOKBUILJEDG-UHFFFAOYSA-M 0.000 claims description 8
- 238000010898 silica gel chromatography Methods 0.000 claims description 7
- 241000187563 Rhodococcus ruber Species 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 4
- 241000186046 Actinomyces Species 0.000 claims description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 3
- 244000061456 Solanum tuberosum Species 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 3
- 238000003287 bathing Methods 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 238000004440 column chromatography Methods 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 6
- 239000002246 antineoplastic agent Substances 0.000 claims 1
- 229940041181 antineoplastic drug Drugs 0.000 claims 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 6
- 239000003814 drug Substances 0.000 abstract description 6
- 238000004587 chromatography analysis Methods 0.000 abstract description 4
- 210000004881 tumor cell Anatomy 0.000 abstract description 4
- 201000011510 cancer Diseases 0.000 abstract description 3
- 238000000638 solvent extraction Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 7
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 6
- 239000000126 substance Substances 0.000 description 5
- 239000004215 Carbon black (E152) Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 229930195733 hydrocarbon Natural products 0.000 description 4
- 150000002430 hydrocarbons Chemical class 0.000 description 4
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 3
- 229910052740 iodine Inorganic materials 0.000 description 3
- 239000011630 iodine Substances 0.000 description 3
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 3
- XILIYVSXLSWUAI-UHFFFAOYSA-N 2-(diethylamino)ethyl n'-phenylcarbamimidothioate;dihydrobromide Chemical compound Br.Br.CCN(CC)CCSC(N)=NC1=CC=CC=C1 XILIYVSXLSWUAI-UHFFFAOYSA-N 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 125000004432 carbon atom Chemical group C* 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 2
- 229960001231 choline Drugs 0.000 description 2
- 238000005100 correlation spectroscopy Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 241000187654 Nocardia Species 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000013065 commercial product Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000005570 heteronuclear single quantum coherence Methods 0.000 description 1
- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 description 1
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- XOFYZVNMUHMLCC-ZPOLXVRWSA-N prednisone Chemical compound O=C1C=C[C@]2(C)[C@H]3C(=O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 XOFYZVNMUHMLCC-ZPOLXVRWSA-N 0.000 description 1
- 229960004618 prednisone Drugs 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
- C07C69/66—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
- C07C69/73—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids
- C07C69/732—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids of unsaturated hydroxy carboxylic acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/04—Actinomyces
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- 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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Abstract
The invention discloses a mycolic acid compound with anti-tumor activity, a preparation method and application thereof, wherein the molecular formula of the mycolic acid compound with anti-tumor activity is C 45 H 88 O 3， The structural formula is as follows:
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a mycolic acid compound with anti-tumor activity, and a preparation method and application thereof.
Background
The mycolic acid is a higher branched fatty acid of alpha-alkyl beta-hydroxy. Originally isolated from human tubercle bacillus by prednisone (R.J. Anderson) and the like. The chemical structure of mycolic acid is complex and is classified into 3 types according to the number of carbon atoms and groups: (1) choline type mycolic acid: r=ch3 (CH 2) 14, R' =c14h29; choline type mycoenoic acid: r=ch3 (CH 2) 5ch=ch (CH 2) 7, R' =c14h29; (2) the carbon number of mycolic acid from nocardia is 50; (3) the mycolic acid derived from Mycobacteria has 80 carbon atoms.
It is reported that mycolic acid has biological activities such as anti-tumor; the mycolic acid is tightly combined with the cell wall, and the polarity is smaller, and the currently adopted extraction and purification method is to degrade the cell wall firstly, generally adopt alkaline substances such as tetrabutyl ammonium hydroxide, sodium hydroxide, potassium hydroxide and the like for treatment, then adopt low-polarity organic solvents such as normal hexane, petroleum ether and the like for extraction, and then prepare the product by normal phase silica gel chromatography and thin layer chromatography. However, it has been reported that it is more difficult to purify structurally similar mycolic acids.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a mycolic acid compound with anti-tumor activity, and a preparation method and application thereof.
One of the technical schemes adopted for solving the technical problems is as follows:
mycolic acid compound with antitumor activity and molecular formula of C 45 H 88 O 3， The structural formula is as follows:
the second technical scheme adopted by the invention for solving the technical problems is as follows:
a method for preparing a mycolic acid compound having anti-tumor activity, the method comprising:
(1) Activating actinomyces ruber (Nocardia rubra) and then carrying out liquid submerged fermentation, wherein the formula of a culture medium for liquid submerged fermentation comprises the following components in parts by weight: 18-22% of potato, 1.5-2.5% of glucose and the balance of water, sterilizing for 30min at the temperature of 121 ℃ under the natural pH of 0.1Mpa, and culturing for 10-15 days in a constant temperature shaking table at the temperature of 25-28 ℃ and at the speed of 180-230 r/min;
(2) Separating mycelium from fermentation liquid by centrifugation after fermentation to obtain mycelium, adding tetrabutylammonium hydroxide, water-bathing at 50-100deg.C for 12-24 hr, extracting with equal volume of water and dichloromethane for 1-3 times, concentrating dichloromethane phase to obtain organic crude extract;
(3) Performing gel column chromatography on the organic crude extract obtained in the step (2), using methanol, acetone and chloroform as eluent, collecting the components, performing thin layer chromatography analysis, and combining according to the thin layer chromatography color development result to obtain an initial component containing a target compound;
(4) Subjecting the initial component containing the target compound obtained in step (3) to reverse phase silica gel column chromatography using methanol/acetone: eluting with water gradient (0-100% to 100-0%), collecting, concentrating, performing thin layer chromatography analysis, and combining according to thin layer chromatography color development result to obtain final component containing target compound;
(5) Subjecting the final component containing the target compound obtained in the step (4) to normal phase silica gel column chromatography, and using petroleum ether/dichloromethane/chloroform: the gradient eluent is acetone/ethyl acetate/methanol with the ratio of 500-10:10-1, the gradient eluent is collected in a fractional manner, thin layer chromatography analysis is carried out, and the mixture is combined according to the thin layer chromatography color development result to obtain the mycolic acid compound.
Further, in the step (3), during thin layer chromatography, the spreading agent is petroleum ether/n-hexane: acetone/ethyl acetate=10-30:1-5.
Further, in the step (4), in the case of thin layer chromatography, the layer-spreading agent is petroleum ether/n-hexane: acetone/ethyl acetate=10-30:1-5.
Further, in the step (5), in the case of thin layer chromatography, the layer-spreading agent is petroleum ether/n-hexane: acetone/ethyl acetate=10-30:1-5.
Further, in the step (3), the gel column is a Sephadex LH-20 gel column.
Further, in the step (4), the reverse phase silica gel column is SP-120-40/60-ODS-RPS reverse phase silica gel column.
The third technical scheme adopted by the invention for solving the technical problems is as follows:
an application of an anti-tumor mycolic acid compound in preparing an anti-tumor medicament.
Compared with the background technology, the technical proposal has the following advantages:
the invention adopts gel with higher carbon content (such as Sephadex LH-20) and reverse phase silica gel (such as SP-120-40/60-ODS-RPS), combines chromatographic techniques such as solvent extraction, normal phase chromatography and the like to obtain the mycolic acid compound with higher purity, can be used for resisting Bel-7405, hepG2 and huh7 tumor cells, and has great significance for developing medicaments for treating various malignant tumors; the technology of the invention can prepare a large amount of mycolic acid compounds, and is suitable for industrial application.
Drawings
The invention is further described below with reference to the drawings and examples.
FIG. 1 is a hydrogen spectrum of a mycolic acid compound having anti-tumor activity;
FIG. 2 is a carbon spectrum of a mycolic acid compound having anti-tumor activity.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The actinomycetes nocardia rubra used in the following examples was a commercial product bacterium.
Example 1: preparation of mycolic acid compound with antitumor activity
(1) Activating actinomyces ruber (Nocardia rubra) and then carrying out liquid submerged fermentation, wherein the formula of a culture medium for liquid submerged fermentation comprises the following components in parts by weight: 20% of potato, 2% of glucose and the balance of water, sterilizing for 30min at the temperature of 121 ℃ under the natural pH of 0.1Mpa, and culturing in a shaking table at the constant temperature of 200r/min at the temperature of 26 ℃ for 13 days;
(2) After fermentation, separating mycelium from fermentation liquid by centrifugation to obtain mycelium, adding tetrabutylammonium hydroxide, water-bathing at 80 ℃ for 16h, extracting with equal volume of water and dichloromethane for 3 times, concentrating dichloromethane phase to obtain organic crude extract;
(3) Performing gel column chromatography on the organic crude extract obtained in the step (2), controlling the flow rate to 15 s/drop by using methanol, acetone and chloroform as eluent, collecting the organic crude extract in a fractional manner, and performing thin layer chromatography analysis on 6ml of the organic crude extract in each fraction, wherein the layer expanding agent is petroleum ether/n-hexane: acetone/ethyl acetate=20:3, and observed with a color developer such as iodine and sulfuric acid, and combined according to Rf value and color development condition to obtain an initial component containing a target compound;
(4) Subjecting the initial component containing the target compound obtained in step (3) to reverse phase silica gel column chromatography using methanol/acetone: water gradient (0% -100%:100% -0%) elution, flow rate control 15mL/min, fractional collection, thin layer chromatography analysis after each concentration, layer spreading agent petroleum ether/n-hexane: acetone/ethyl acetate=20:3, and observed with a color developer such as iodine and sulfuric acid, and combined according to Rf value and color development condition to obtain a final component containing the target compound;
(5) Subjecting the final component containing the target compound obtained in the step (4) to normal phase silica gel column chromatography, and using petroleum ether/dichloromethane/chloroform: the acetone/ethyl acetate/methanol ratio is 250:5, the gradient eluent is collected in parts, 6ml of each part is collected, the thin layer chromatography analysis is carried out, and the spreading agent is petroleum ether/n-hexane: acetone/ethyl acetate=20:3, and the mycolic acid compound is obtained by combining the Rf value and the color development condition by observing with a color developer such as iodine and sulfuric acid.
Subjecting the obtained mycolic acid compound to nuclear magnetic resonance spectrum 1 H-NMR、 13 C-NMR、DEPT、HSQC、HMBC、 1 H－ 1 H COSY), the structural formula of the compound was identified as follows:
the mycolic acid compound is white and can be dissolved in chloroform and methanol. Analysis based on NMR spectrum data of mycolic acid compounds as shown in Table 1 1 H and 13 c NMR and DEPT data: the compounds are shown to contain 3 methyl groups, 30 methylene groups, 4 methine groups (2 of which are oxo, 2 of which are ethylenic carbons), 1 quaternary carbon (4 of which are carbo-yl, 1 of which is ethylenic carbon), and of formula C 45 H 88 O 3 . H-1 and C-2/C-3/C-1', H-2 and C-1/C-2'/C-3', H-2' and C-1'/C-1/C-2/C-3' are observed in HMBC experiments to be remotely related to hydrocarbon and from 1 H- 1 H-1 was related to H-2' hydrogen and C-1 (delta) observed in the H COSY experiments C 72.5)、C-2(δ C 35.9)、C-1'(δ C 176.4 And C-2' (delta) C 51.1 Chemical shift values, the mycolic acid characteristic structural fragments (C-1/2 and C-1 '/2') of the compound can be deduced. Also based on the observation from HMBC experiments that H-8/13 is related to C-10/11, H-9/12 is related to C-8/13, and H-10/11 is related to C-8/13 in hydrocarbon remoteness, and based on C-8/13 (delta C 27.6)、C-9/13(δ C 28.0 And C-10/11 (delta) C 130.2 Chemical shift values of (C-8) to C-13) of the compound. In addition, according to H 3 -1 'is remotely related to C-1' hydrocarbon, and the oxygen-linked methyl group (C-1 ') is presumed to be attached to C-1'. Based on the remaining hydrocarbon chemical shift values, a long chain alkyl group is presumed. By combining the above data, it can be speculated that the compound has the following structural formula:
table 1 NMR spectrum data (CDCL 3 ，500M)
Example 2: antitumor experimental analysis of mycolic acid compounds
Experimental method
(1) Culturing Bel-7405, hepG2, huh7 cells: bel-7405, hepG2, huh7 cells were cultured in DMEM complete medium (Hyclone) containing 1% diabody (mixed solution of green and streptomycin, gibco) and 10% FBS (Gibco), respectively, at 37℃and 5% CO 2 Maintaining saturated humidity for culture, and carrying out conventional passage when the cell density is about 80%; bel-7405, hepG2, huh7 cells were plated in 96-well plates at 5000 cells/well until the cells attached;
(2) and (3) performing drug treatment: 2.7mg of mycolic acid compound sample is added with 54ml of LDMSO solvent to prepare mother liquor with the concentration of 50 mug/mu L, 25 mug, 12.5 mug/mu L, 6.25 mug/mu L, 3.125 mug/mu L and 1.5625 mug/mu L respectively, and 1/mu L of mother liquor containing mycolic acid compound is added into each hole respectively, and 4 repeats are carried out, and DMSO solvent is used as a control group;
(3) after 48h of treatment, the old medium was discarded, CCK8 reagent (Biosharp) was added to detect cell viability, OD450 values were read over 2h, and IC was calculated 50 The detection results are shown in Table 2.
Inhibitory Activity of the compounds of Table 2 against 3 tumor cell lines
As shown in Table 2, the compounds have strong inhibitory activity on 3 tumor cell lines, can be used for resisting Bel-7405, hepG2 and huh7 cells, and have great significance in developing medicaments for treating various malignant tumors.
The foregoing description is only illustrative of the preferred embodiments of the present invention, and therefore should not be taken as limiting the scope of the invention, for all changes and modifications that come within the meaning and range of equivalency of the claims and specification are therefore intended to be embraced therein.
Claims (8)
1. A mycolic acid compound with antitumor activity is characterized in that the molecular formula is C 45 H 88 O 3， The structural formula is as follows:
2. the method for producing a mycolic acid compound having an antitumor activity according to claim 1, wherein the method comprises:
(1) Activating actinomyces ruber (Nocardia rubra) and then carrying out liquid submerged fermentation, wherein the formula of a culture medium for liquid submerged fermentation comprises the following components in parts by weight: 18-22% of potato, 1.5-2.5% of glucose and the balance of water, sterilizing for 30min at the temperature of 121 ℃ under the natural pH of 0.1Mpa, and culturing for 10-15 days in a constant temperature shaking table at the temperature of 25-28 ℃ and at the speed of 180-230 r/min;
(2) Separating mycelium from fermentation liquid by centrifugation after fermentation to obtain mycelium, adding tetrabutylammonium hydroxide, water-bathing at 50-100deg.C for 12-24 hr, extracting with equal volume of water and dichloromethane for 1-3 times, concentrating dichloromethane phase to obtain organic crude extract;
(3) Performing gel column chromatography on the organic crude extract obtained in the step (2), using methanol, acetone and chloroform as eluent, collecting the components, performing thin layer chromatography analysis, and combining according to the thin layer chromatography color development result to obtain an initial component containing a target compound;
(4) Subjecting the initial component containing the target compound obtained in step (3) to reverse phase silica gel column chromatography using methanol/acetone: eluting with water gradient (0-100% to 100-0%), collecting, concentrating, performing thin layer chromatography analysis, and combining according to thin layer chromatography color development result to obtain final component containing target compound;
(5) Subjecting the final component containing the target compound obtained in the step (4) to normal phase silica gel column chromatography, and using petroleum ether/dichloromethane/chloroform: the gradient eluent is acetone/ethyl acetate/methanol with the ratio of 500-10:10-1, the gradient eluent is collected in a fractional manner, thin layer chromatography analysis is carried out, and the mixture is combined according to the thin layer chromatography color development result to obtain the mycolic acid compound.
3. The method for producing a mycolic acid compound having an antitumor activity according to claim 2, characterized in that: in the step (3), during thin layer chromatography, the spreading agent is petroleum ether/n-hexane: acetone/ethyl acetate=10-30:1-5.
4. The method for producing a mycolic acid compound having an antitumor activity according to claim 2, characterized in that: in the step (4), during thin layer chromatography, the spreading agent is petroleum ether/n-hexane: acetone/ethyl acetate=10-30:1-5.
5. The method for producing a mycolic acid compound having an antitumor activity according to claim 2, characterized in that: in the step (5), during thin layer chromatography, the spreading agent is petroleum ether/n-hexane: acetone/ethyl acetate=10-30:1-5.
6. The method for producing a mycolic acid compound having an antitumor activity according to claim 2, characterized in that: in the step (3), the gel column is a Sephadex LH-20 gel column.
7. The method for producing a mycolic acid compound having an antitumor activity according to claim 2, characterized in that: in the step (4), the reverse phase silica gel column is SP-120-40/60-ODS-RPS reverse phase silica gel column.
8. The use of an antitumor mycolic acid compound according to claim 1 for the preparation of antitumor drugs.
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