CN102964321B - Nepetalactone fluorobenzoate, preparation technology and usage thereof - Google Patents
Nepetalactone fluorobenzoate, preparation technology and usage thereof Download PDFInfo
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- CN102964321B CN102964321B CN201210508922.4A CN201210508922A CN102964321B CN 102964321 B CN102964321 B CN 102964321B CN 201210508922 A CN201210508922 A CN 201210508922A CN 102964321 B CN102964321 B CN 102964321B
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- schizonepetolactone
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- OSTIHFXUTPZJQL-UHFFFAOYSA-N fluoro benzoate Chemical compound FOC(=O)C1=CC=CC=C1 OSTIHFXUTPZJQL-UHFFFAOYSA-N 0.000 title claims abstract description 45
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- ZDKZHVNKFOXMND-UHFFFAOYSA-N cis-Nepetalactone Natural products O=C1OC=C(C)C2C1C(C)CC2 ZDKZHVNKFOXMND-UHFFFAOYSA-N 0.000 title abstract 6
- ZDKZHVNKFOXMND-NBEYISGCSA-N cis-trans-nepetalactone Chemical compound O=C1OC=C(C)[C@@H]2[C@H]1[C@@H](C)CC2 ZDKZHVNKFOXMND-NBEYISGCSA-N 0.000 title abstract 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 25
- 239000003814 drug Substances 0.000 claims abstract description 10
- 241000712461 unidentified influenza virus Species 0.000 claims abstract description 7
- 238000006243 chemical reaction Methods 0.000 claims description 22
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 20
- 150000001875 compounds Chemical class 0.000 claims description 19
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 18
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 16
- 239000000741 silica gel Substances 0.000 claims description 16
- 229910002027 silica gel Inorganic materials 0.000 claims description 16
- 239000000126 substance Substances 0.000 claims description 14
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 12
- MXNBDFWNYRNIBH-UHFFFAOYSA-N 3-fluorobenzoic acid Chemical compound OC(=O)C1=CC=CC(F)=C1 MXNBDFWNYRNIBH-UHFFFAOYSA-N 0.000 claims description 10
- 229960000549 4-dimethylaminophenol Drugs 0.000 claims description 10
- 239000000463 material Substances 0.000 claims description 9
- 238000005352 clarification Methods 0.000 claims description 8
- 238000004440 column chromatography Methods 0.000 claims description 8
- 238000004821 distillation Methods 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 8
- 239000002904 solvent Substances 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 8
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 7
- -1 silica gel compound Chemical class 0.000 claims description 5
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 claims description 4
- 239000003208 petroleum Substances 0.000 claims description 2
- 239000000377 silicon dioxide Substances 0.000 claims description 2
- 238000000605 extraction Methods 0.000 abstract description 7
- 238000002844 melting Methods 0.000 abstract 1
- 230000008018 melting Effects 0.000 abstract 1
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Natural products C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 abstract 1
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 21
- 238000000034 method Methods 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 238000010790 dilution Methods 0.000 description 9
- 239000012895 dilution Substances 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- 241000700605 Viruses Species 0.000 description 8
- 230000003612 virological effect Effects 0.000 description 7
- 241000951376 Schizonepeta tenuifolia Species 0.000 description 6
- 239000003480 eluent Substances 0.000 description 6
- 239000003921 oil Substances 0.000 description 6
- 230000003647 oxidation Effects 0.000 description 6
- 238000007254 oxidation reaction Methods 0.000 description 6
- 238000001256 steam distillation Methods 0.000 description 6
- 239000000341 volatile oil Substances 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 5
- 230000021615 conjugation Effects 0.000 description 4
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 3
- 206010022000 influenza Diseases 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- NSTREUWFTAOOKS-UHFFFAOYSA-N 2-fluorobenzoic acid Chemical compound OC(=O)C1=CC=CC=C1F NSTREUWFTAOOKS-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000012930 cell culture fluid Substances 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- 230000032050 esterification Effects 0.000 description 2
- 238000005886 esterification reaction Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- XEVQXKKKAVVSMW-WRWORJQWSA-N loliolide Chemical compound C1[C@@H](O)CC(C)(C)C2=CC(=O)O[C@@]21C XEVQXKKKAVVSMW-WRWORJQWSA-N 0.000 description 2
- 238000004264 monolayer culture Methods 0.000 description 2
- 238000010606 normalization Methods 0.000 description 2
- 229940055695 pancreatin Drugs 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- XEVQXKKKAVVSMW-UHFFFAOYSA-N D-epiloliolide Natural products C1C(O)CC(C)(C)C2=CC(=O)OC21C XEVQXKKKAVVSMW-UHFFFAOYSA-N 0.000 description 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- IWUCXVSUMQZMFG-AFCXAGJDSA-N Ribavirin Chemical compound N1=C(C(=O)N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 IWUCXVSUMQZMFG-AFCXAGJDSA-N 0.000 description 1
- 241000951473 Schizonepeta Species 0.000 description 1
- 239000000730 antalgic agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000007541 cellular toxicity Effects 0.000 description 1
- 210000003837 chick embryo Anatomy 0.000 description 1
- 229940124579 cold medicine Drugs 0.000 description 1
- 125000000753 cycloalkyl group Chemical group 0.000 description 1
- 230000000120 cytopathologic effect Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- ROAYSRAUMPWBQX-UHFFFAOYSA-N ethanol;sulfuric acid Chemical compound CCO.OS(O)(=O)=O ROAYSRAUMPWBQX-UHFFFAOYSA-N 0.000 description 1
- 230000035931 haemagglutination Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- XWYLNCDCFDBLGT-HTRCEHHLSA-N loliolide Natural products CC1(C)C[C@H](O)C[C@H]2OC(=O)C=C12 XWYLNCDCFDBLGT-HTRCEHHLSA-N 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005497 microtitration Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229930003658 monoterpene Natural products 0.000 description 1
- 235000002577 monoterpenes Nutrition 0.000 description 1
- 231100000028 nontoxic concentration Toxicity 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- CFJYNSNXFXLKNS-UHFFFAOYSA-N p-menthane Chemical compound CC(C)C1CCC(C)CC1 CFJYNSNXFXLKNS-UHFFFAOYSA-N 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 229960000329 ribavirin Drugs 0.000 description 1
- HZCAHMRRMINHDJ-DBRKOABJSA-N ribavirin Natural products O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1N=CN=C1 HZCAHMRRMINHDJ-DBRKOABJSA-N 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 231100001274 therapeutic index Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
Abstract
The invention discloses a nepetalactone fluorobenzoate, a preparation technology and the usage of the nepetalactone fluorobenzoate. The nepetalactone fluorobenzoate is characterized by having the molecular formula of C17H17FO4, the melting point of 158-160 DEG C and the specific rotation of [alpha]D<20>=+13.2 degrees (c 1.0, CH3OH). The invention also discloses an extraction method of the nepetalactone fluorobenzoate and the application of the nepetalactone fluorobenzoate in the preparation of medicine for resisting influenza virus.
Description
Technical field
The present invention relates to a kind of Schizonepetolactone fluorobenzoate and preparation technology and purposes, belong to technical field of medicine.
Background technology
Schizonepetolactone (C
10h
14o
3) for by extracting a kind of Loliolide obtaining in Chinese medicinal materials schizonepeta, be colourless grain brilliant (ethyl acetate), mp:188~189 DEG C.TLC aobvious red (10% sulfuric acid ethanol), molecular formula is C
10h
14o
3, its structural formula is
Chinese patent CN01108186.4 has disclosed structure collection of illustrative plates of Schizonepetolactone and uses thereof, it can be used for preparing anti-cold medicine, anti-inflammation drugs, antibacterials, analgesic agent etc., Chinese patent CN201010217622.1 has disclosed the one of Schizonepetolactone and has improved preparation technology, and preparation manipulation simplification, yield raising, the production cost of Schizonepetolactone are reduced.But Schizonepetolactone in vitro infected by influenza reactive force a little less than, in order to obtain the medicine of stronger resisiting influenza virus effect, intend by structure of modification, there is the sick active Schizonepetolactone derivative of stronger In Vitro Anti influenza to obtain.But up to now, not yet there is report after fluorobenzoic acid esterification, to be obtained Schizonepetolactone fluorobenzoate by Schizonepetolactone.
Summary of the invention
The object of the present invention is to provide a kind of Schizonepetolactone fluorobenzoate and its preparation method and purposes as medicine of being obtained by Schizonepetolactone after fluorobenzoic acid esterification.
Technical scheme of the present invention is as following
Schizonepetolactone fluorobenzoate, is characterized in that molecular formula is C
17h
17fO
4, fusing point is 158-160 DEG C, its specific optical rotation is=+ 13.2 ° of (c 1.0, CH
3oH), its structural formula is as follows, the temporary called after Schizonepetolactone of inventor fluorobenzoate:
Schizonepetolactone fluorobenzoate preparation method of the present invention comprises the steps:
A. get Schizonepetolactone, m-fluorobenzoic acid and DMAP (english abbreviation is DMAP) are appropriate, these three kinds of material amount of substance ratios are 0.5~1.5:1.5~2.5:0.5~1.5, be placed in reaction vessel, add methylene dichloride, stirring is dissolved it completely, add N, N'-dicyclohexylcarbodiimide (DCC) or 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (english abbreviation is EDCI), these two kinds of material amount of substances are 1.5~2.5 times of Schizonepetolactone, in room temperature reaction, TLC checks reaction end, obtains reaction solution;
B. by A step gained reaction solution, drip methyl alcohol and make solution clarification, add silica gel, underpressure distillation solvent, to dry, obtains silica gel compound sample;
C. silica obtained B step compound sample dress post is carried out to column chromatography, the petroleum ether-ethyl acetate that is 4~7:1 with volume proportion carries out wash-out, stream part that collection contains Schizonepetolactone fluorobenzoate, reclaim under reduced pressure elutriant after merging, can obtain Schizonepetolactone fluorobenzoate white crystalline powder.
The preparation method of Schizonepetolactone fluorobenzoate in above-mentioned steps A, Schizonepetolactone, three kinds of material amount of substance ratios of m-fluorobenzoic acid and DMAP are 1:2:1; The N adding, N'-dicyclohexylcarbodiimide or 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride, these two kinds of material amount of substances are 2 times of Schizonepetolactone; The volume proportion of step C PetroChina Company Limited. ether-ethyl acetate is 5:1.
The In Vitro Anti influenza virus pharmacodynamics test of Schizonepetolactone fluorobenzoate shows, it has good anti-H
3n
2activity, therefore can be used for preparing anti-influenza virus medicament.
Schizonepetolactone fluorobenzoate of the present invention is a kind of new menthane type monoterpenes compound, and simple in structure, preparation method is easy to be inexpensive, and has stronger physiologically active.
Brief description of the drawings
Fig. 1 is the ultraviolet spectrogram of compound Schizonepetolactone fluorobenzoate, λ max (CH
3oH): 227.6nm, prompting compound has conjugation Absorption Characteristics.
Fig. 2 is the HPLC purity collection of illustrative plates of compound Schizonepetolactone fluorobenzoate, and it is 96.2% that normalization method calculates Schizonepetolactone parabromobenzoic acid ester purity.
Embodiment
Form by the following examples, Schizonepetolactone fluorobenzoate the present invention relates to and preparation method thereof is described in further detail, but this should be interpreted as to the scope of the above-mentioned theme of the present invention only limits to following example, all technology realizing based on foregoing of the present invention all belong to scope of the present invention.
Embodiment 1: take the Schizonepetolactone (method disclosing according to Chinese patent CN201010217622.1, adopt labiate schizonepeta Schizonepeta tenuifolia Briq. flower fringe to obtain through oxidation preparation with the commercially available Herba Schizonepetae volatile oil of extraction by steam distillation gained, purity is more than 98%) 18.2mg (0.10mmol), m-fluorobenzoic acid 35.1mg (0.25mmol, domestic AR level), DMAP 12.3mg (0.10mmol, domestic AR level), be placed in 50mL round-bottomed flask, add 4mL methylene dichloride (domestic AR level), stirring is dissolved it completely, add DCC 51.6mg (0.25mmol, domestic AR level), react in room temperature (20-25 DEG C), TLC checks reaction end.After reaction finishes, drip methyl alcohol (domestic AR level) and make solution clarification, add the about 500mg of triplication silica gel (Haiyang Chemical Plant, Qingdao produces, and column chromatography is used), underpressure distillation solvent is to dry.Take 5g silica gel dress post, eluent (sherwood oil (60-90 DEG C, domestic AR level): ethyl acetate (domestic AR level)=5:1) wash-out, when wash-out 40mL the left and right, TLC follows the tracks of can find target compound Schizonepetolactone fluorobenzoate, now collect elutriant, every 5mL collects once,, TLC follows the tracks of and finds target compound wash-out substantially completely when about 30mL the left and right, merge elutriant, reclaim under reduced pressure elutriant, can obtain Schizonepetolactone fluorobenzoate white crystalline powder, purity is more than 96%, yield 87%.
Embodiment 2:
The Schizonepetolactone fluorobenzoate structure elucidation that embodiment 1 prepares:
HR-ES IMS:m/z 305.1454[M+H]
+(calculated value C
17h
17fO
4for: 304.3123);
(c 1.0, CH
3oH).
IR (KBr compressing tablet, cm
-1): 3070(v
Ф-H); 2931,2848(saturated cyclic hydrocarbons v
cH); Ester carbonyl group v in 1704(
c=O); 1655(conjugation carbonyl v
c=O); 1606,1551,1505,1454(conjugation phenyl ring v
c=C); Between 899,795,691(phenyl ring, position replaces γ
Ф-H).
UV (CH
3oH) λ max:227.6nm, prompting compound has conjugation Absorption Characteristics.See accompanying drawing 1
H
1-NMR(300MHz,CDCl
3,ppm):δ1.04(d,3H,J=6.6Hz,6’-CH3),1.07(dd,1H,J=4.2,13.2Hz,5a-H),1.29(m,1H,4a-H),1.91(s,3H,3’-H),1.94-2.03(m,2H,5b-H,6-H),2.23(m,1H,4b-H),2.78-2.87(m,2H,7H),7.28-7.79(m,4H,Ar-H)。
13C-NMR(75MHz,CDCl
3,ppm):δ171.3(C-8),164.1(C-7’),162.6(C-3),160.8(C-1’),132.3,130.2(C-2’,C-6’),127.8,125.5(C-3’,C-5’),123.3(C-4’),116.7(C-7),104.5(C-4),45.1(C-5),34.5(C-1),28.9(C6),24.3(C-2),20.9(C-10),8.3(C-9)。
Schizonepetolactone fluorobenzoate purity: utilize HPLC method to detect, Schizonepetolactone fluorobenzoate peak area is 7802336, and impurity total peak area is 306852, adopting normalization method to calculate Schizonepetolactone fluorobenzoate purity is 96.2%.See accompanying drawing 2
Embodiment 3: take the Schizonepetolactone (method disclosing according to Chinese patent CN201010217622.1, adopt labiate schizonepeta Schizonepeta tenuifolia Briq. flower fringe to obtain through oxidation preparation with the commercially available Herba Schizonepetae volatile oil of extraction by steam distillation gained, purity is more than 98%) 18.2mg (0.10mmol), m-fluorobenzoic acid 35.1mg (0.25mmol, domestic AR level), DMAP 12.3mg (0.10mmol, domestic AR level), be placed in 50mL round-bottomed flask, add 4mL methylene dichloride (domestic AR level), stirring is dissolved it completely, add EDCI 46mg (0.24mmol, domestic AR level), react in room temperature (20-25 DEG C), TLC checks reaction end.After reaction finishes, drip methyl alcohol (domestic AR level) and make solution clarification, add the about 500mg of triplication silica gel (Haiyang Chemical Plant, Qingdao produces, and column chromatography is used), underpressure distillation solvent is to dry.Take 5g silica gel dress post, eluent (sherwood oil (60-90 DEG C, domestic AR level): ethyl acetate (domestic AR level)=5:1) wash-out, when wash-out 40mL the left and right, TLC follows the tracks of can find target compound Schizonepetolactone fluorobenzoate, now collect elutriant, every 5mL collects once,, TLC follows the tracks of and finds target compound wash-out substantially completely when about 30mL the left and right, merge elutriant, reclaim under reduced pressure elutriant, can obtain Schizonepetolactone fluorobenzoate white crystalline powder, purity is more than 97%, yield 84%.
Embodiment 4: take the Schizonepetolactone (method disclosing according to Chinese patent CN201010217622.1, adopt labiate schizonepeta Schizonepeta tenuifolia Briq. flower fringe to obtain through oxidation preparation with the commercially available Herba Schizonepetae volatile oil of extraction by steam distillation gained, purity is more than 98%) 24.3mg (0.15mmol), m-fluorobenzoic acid 44.8mg (0.32mmol, domestic AR level), DMAP18.5mg (0.15mmol, domestic AR level), be placed in 50mL round-bottomed flask, add 4mL methylene dichloride (domestic AR level), stirring is dissolved it completely, add DCC 66.1mg (0.32mmol, domestic AR level), react in room temperature (20-25 DEG C), TLC checks reaction end.After reaction finishes, drip methyl alcohol (domestic AR level) and make solution clarification, add the about 550mg of triplication silica gel (Haiyang Chemical Plant, Qingdao produces, and column chromatography is used), underpressure distillation solvent is to dry.Take 5g silica gel dress post, eluent (sherwood oil (60-90 DEG C, domestic AR level): ethyl acetate (domestic AR level)=6:1) wash-out, when wash-out 45mL the left and right, TLC follows the tracks of can find target compound Schizonepetolactone fluorobenzoate, now collect elutriant, every 5mL collects once,, TLC follows the tracks of and finds target compound wash-out substantially completely when about 35mL the left and right, merge elutriant, reclaim under reduced pressure elutriant, can obtain Schizonepetolactone fluorobenzoate white crystalline powder, purity is more than 97%, yield 88%.
Embodiment 5: take the Schizonepetolactone (method disclosing according to Chinese patent CN201010217622.1, adopt labiate schizonepeta Schizonepeta tenuifolia Briq. flower fringe to obtain through oxidation preparation with the commercially available Herba Schizonepetae volatile oil of extraction by steam distillation gained, purity is more than 98%) 24.3mg (0.15mmol), m-fluorobenzoic acid 44.8mg (0.32mmol, domestic AR level), DMAP 18.5mg (0.15mmol, domestic AR level), be placed in 50mL round-bottomed flask, add 4mL methylene dichloride (domestic AR level), stirring is dissolved it completely, add EDCI61.3mg (0.32mmol, domestic AR level), react in room temperature (20-25 DEG C), TLC checks reaction end.After reaction finishes, drip methyl alcohol (domestic AR level) and make solution clarification, add the about 550mg of triplication silica gel (Haiyang Chemical Plant, Qingdao produces, and column chromatography is used), underpressure distillation solvent is to dry.Take 5g silica gel dress post, eluent (sherwood oil (60-90 DEG C, domestic AR level): ethyl acetate (domestic AR level)=6:1) wash-out, when wash-out 45mL the left and right, TLC follows the tracks of can find target compound Schizonepetolactone fluorobenzoate, now collect elutriant, every 5mL collects once,, TLC follows the tracks of and finds target compound wash-out substantially completely when about 35mL the left and right, merge elutriant, reclaim under reduced pressure elutriant, can obtain Schizonepetolactone fluorobenzoate white crystalline powder, purity is more than 97%, yield 83%.
Embodiment 6: take the Schizonepetolactone (method disclosing according to Chinese patent CN201010217622.1, adopt labiate schizonepeta Schizonepeta tenuifolia Briq. flower fringe to obtain through oxidation preparation with the commercially available Herba Schizonepetae volatile oil of extraction by steam distillation gained, purity is more than 98%) 36.5mg (0.20mmol), m-fluorobenzoic acid 53.2mg (0.38mmol, domestic AR level), DMAP 24.5mg (0.20mmol, domestic AR level), be placed in 50mL round-bottomed flask, add 5mL methylene dichloride (domestic AR level), stirring is dissolved it completely, add DCC 78.5mg (0.38mmol, domestic AR level), react in room temperature (20-25 DEG C), TLC checks reaction end.After reaction finishes, drip methyl alcohol (domestic AR level) and make solution clarification, add the about 600mg of triplication silica gel (Haiyang Chemical Plant, Qingdao produces, and column chromatography is used), underpressure distillation solvent is to dry.Take 6g silica gel dress post, eluent (sherwood oil (60-90 DEG C, domestic AR level): ethyl acetate (domestic AR level)=7:1) wash-out, when wash-out 50mL the left and right, TLC follows the tracks of can find target compound Schizonepetolactone fluorobenzoate, now collect elutriant, every 5mL collects once,, TLC follows the tracks of and finds target compound wash-out substantially completely when about 40mL the left and right, merge elutriant, reclaim under reduced pressure elutriant, can obtain Schizonepetolactone fluorobenzoate white crystalline powder, purity is more than 97%, yield 86%.
Embodiment 7: take the Schizonepetolactone (method disclosing according to Chinese patent CN201010217622.1, adopt labiate schizonepeta Schizonepeta tenuifolia Briq. flower fringe to obtain through oxidation preparation with the commercially available Herba Schizonepetae volatile oil of extraction by steam distillation gained, purity is more than 98%) 36.5mg (0.20mmol), m-fluorobenzoic acid 53.2mg (0.38mmol, domestic AR level), DMAP 24.5mg (0.20mmol, domestic AR level), be placed in 50mL round-bottomed flask, add 5mL methylene dichloride (domestic AR level), stirring is dissolved it completely, add EDCI 72.8mg (0.38mmol, domestic AR level), react in room temperature (20-25 DEG C), TLC checks reaction end.After reaction finishes, drip methyl alcohol (domestic AR level) and make solution clarification, add the about 600mg of triplication silica gel (Haiyang Chemical Plant, Qingdao produces, and column chromatography is used), underpressure distillation solvent is to dry.Take 6g silica gel dress post, eluent (sherwood oil (60-90 DEG C, domestic AR level): ethyl acetate (domestic AR level)=7:1) wash-out, when wash-out 50mL the left and right, TLC follows the tracks of can find target compound Schizonepetolactone fluorobenzoate, now collect elutriant, every 5mL collects once,, TLC follows the tracks of and finds target compound wash-out substantially completely when about 40mL the left and right, merge elutriant, reclaim under reduced pressure elutriant, can obtain Schizonepetolactone fluorobenzoate white crystalline powder, purity is more than 97%, yield 85%.
The anti-H of embodiment 8 Schizonepetolactone fluorobenzoates
3n
2virus function
1 virus titer (TCID
50) mensuration
The preparation of 1.1 influenza viruses
Utilize chick embryo allantoic cavity inoculation method to prepare influenza virus, results allantoic fluid, measure viral existence by hemagglutination test (HA test), after packing-70 DEG C frozen.
The dilution of 1.2 viruses
Get the frozen viral allantoic fluid of a pipe, 1:10 dilution.
The virus liquid that the first round adds 146 μ L 1:10 to dilute, then does serial log10 dilution, makes it to become 10
-1, 10
-2, 10
-3... 10
-10.100 μ L virus liquids are contained in every hole, and each extent of dilution is inoculated a tandem totally 8 holes.
The preparation of 1.3MDCK cell and the mensuration of virus titer
Use mdck cell 1:10 was gone down to posterity in first 2 days, make it 70%~90% in blocks, discard cell culture fluid, wash cell once with 5mLEDTA-pancreatin, then discard.4mL-5mL EDTA pancreatin is covered to thin (162cm
2tissue Culture Flask) digest 10min~20min in 37 DEG C of incubators.In the time that cell starts to come off, add 5mL~10mL mdck cell nutrient solution, piping and druming cell dispersion also proceeds to centrifuge tube by cell, and the centrifugal 5min of 2000r/min, with PBS washing 2 times, to remove bovine serum.Cell suspension, in 1mL viral dilution liquid, is is fully blown and beaten to cell dispersion with suction pipe, add viral dilution liquid to 10mL, by cell counting count board counting cells quantity.Cell dilution is become to 1.5 × 10 with viral dilution liquid
5cell/mL, adds 100 μ L cells (1.5 × 10
4/ hole) in the good viral Microtitration plates of dilution.If two tandems are made in normal cell contrast, at 37 DEG C, 5%CO
2in incubator, cultivate, observe 7 days and record result.
Press Reed-Muench Liang Shi method and calculate TCID
50
2 sample cell toxicity tests
The preparation of 2.1 sample concentrations
Prepare sample by above-described embodiment 1 method, be first mixed with 20mg/mL with methyl-sulphoxide.Then using mdck cell maintenance medium (not containing the cell culture fluid of serum) to dilute is respectively 200 μ g/mL, 100 μ g/mL, 50 μ g/mL, 25 μ g/mL, 12.5 μ g/mL, 6.25 μ g/mL, 3.125 μ g/mL, 1.562 μ g/mL.
2.2 toxotest
Mdck cell is 37 DEG C of 96 well culture plate monolayer culture, and 5%CO2 cultivates 24 hours, inhales and abandons supernatant liquor, adds respectively different concns liquid, 4 holes, and every hole adds liquid 100 μ L.Through 72 hours observation of cell forms, calculate medicine and cause cell half toxicity T C
50.
3 cytopathic effect inhibition tests
Mdck cell is at 37 DEG C of 96 well culture plate monolayer culture, 5%CO
2cultivate 24 hours, inhale and abandon supernatant liquor, cell washs through diluent, and every hole adds washings 100 μ L, inhales and abandons washings, adds 30TCID
50virus liquid, 37 DEG C, 5%CO
2adsorb 2 hours, suck virus, add liquid under maximal non-toxic concentration, 4 holes, every hole dosing 100 μ L.Through 37 DEG C, 5%CO
2cultivate 72 hours, observe CPE(pathology), establish cell control group, virus control group, positive controls (ribavirin) and medicine group are observed CPE simultaneously, calculate IC
50(half effective inhibition concentration) and therapeutic index TI value.
4 results
Compound Schizonepetolactone fluorobenzoate shows good anti-H
3n
2activity, IC
50be 10.53 μ g/mL, TC
50be 29.33 μ g/mL, TI is 2.78.Under similarity condition, Schizonepetolactone does not show good anti-H
3n
2activity, therefore, Schizonepetolactone fluorobenzoate has the anti-H that is significantly better than Schizonepetolactone in vitro
3n
2active.In table 1.
Table 1 Schizonepetolactone fluorobenzoate In Vitro Anti H
3n
2active
Note: "-" represents that sample is at TC
50concentration lower time has no maybe will not be detected viral inhibition.
TI=TC
50/IC
50。
Claims (2)
1. the application of Schizonepetolactone fluorobenzoate in the anti-H3N2 influenza virus medicine of preparation, is characterized in that Schizonepetolactone fluorobenzoate, has following structure:
Preparation method comprises the steps:
A. get Schizonepetolactone, m-fluorobenzoic acid and DMAP are appropriate, and these three kinds of material amount of substance ratios are 0.5~1.5:1.5~2.5:0.5~1.5, are placed in reaction vessel, add methylene dichloride, stirring is dissolved it completely, adds N, N'-dicyclohexylcarbodiimide or 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride, these two kinds of material amount of substances are 1.5~2.5 times of Schizonepetolactone, in room temperature reaction, TLC checks reaction end, obtains reaction solution;
B. by A step gained reaction solution, drip methyl alcohol and make solution clarification, add silica gel, underpressure distillation solvent, to dry, obtains silica gel compound sample;
C. silica obtained B step compound sample dress post is carried out to column chromatography, the petroleum ether-ethyl acetate that is 4~7:1 with volume proportion carries out wash-out, stream part that collection contains Schizonepetolactone fluorobenzoate, reclaim under reduced pressure elutriant after merging, can obtain Schizonepetolactone fluorobenzoate white crystalline powder.
2. the application of Schizonepetolactone fluorobenzoate as claimed in claim 1 in the anti-H3N2 influenza virus medicine of preparation, is characterized in that Schizonepetolactone in preparation method's steps A, and three kinds of material amount of substance ratios of m-fluorobenzoic acid and DMAP are 1:2:1; The N adding, N'-dicyclohexylcarbodiimide or 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride, these two kinds of material amount of substances are 2 times of Schizonepetolactone; The volume proportion of step C PetroChina Company Limited. ether-ethyl acetate is 5:1.
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