CN104725367A - Tetrahydrobenzothiazole-7-ketoxime medicine as well as preparation method and application thereof - Google Patents
Tetrahydrobenzothiazole-7-ketoxime medicine as well as preparation method and application thereof Download PDFInfo
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- C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
- C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
- C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
Abstract
The invention relates to a tetrahydrobenzothiazole-7-ketoxime medicine. The structural formula of tetrahydrobenzothiazole-7-ketoxime medicine comprises a cis-isomer and a trans-isomer (shown in the specification). The tetrahydrobenzothiazole-7-ketoxime medicine further comprises pharmaceutically acceptable salts (HX) of the cis-isomer and the trans-isomer. A preparation method particularly comprises the following steps: carrying out reflux on 2-bromo-1,3-cyclohexanedione (a raw material 1) and thioacid amide by taking ethanol as a solvent for one night, so as to obtain 2-substituted tetrahydrobenzothiazole-7-one (an intermediate 2); carrying out reflux on the intermediate 2 and hydroxylamine hydrochloride in an absolute ethanol solvent in the presence of sodium glacial acetic acid, so as to obtain 2-substituted tetrahydrobenzothiazole-7-ketoxime (an intermediate 3); dissolving the intermediate 3 and propargyl bromide into anhydrous acetonitrile, carrying out stirring reaction in the presence of sodium hydride, so as to obtain 2-substituted tetrahydrobenzothiazole-7-ketoxime ether (an intermediate 4); carrying out 1,3-dipolar cycloaddition on the intermediate 4 and a nitrine intermediate by taking CuI as a catalyst, so as to obtain the tetrahydrobenzothiazole-7-ketoxime medicine. The preparation method can be applied to the preparation of anti-gastric-cancer medicines.
Description
Technical field
The present invention relates generally to a kind of biological activity of tetrahydro benzothiazol-7-oxime compound anti-tumor aspect, and it is synthesized, Structural Identification, active testing and analysis, finally determine that this compounds has certain In Vitro Anti cancer of the stomach active.
Background technology
Show according to the World Health Organization (WHO) research in " world's cancer report 2014 ", malignant tumour has become the subject matter of harm humans publilc health, and wherein lung cancer, mammary cancer and cancer of the stomach M & M is in the world come first three always.Since 20 century 70s, China's Cancer Mortality and mortality ratio present sustainable growth trend.Chen Wanqing the gathering and analyzing discovery China's tumour registration data and Third National coroner's inquest data of whole nation tumour Register, China's Cancer Mortality was obvious ascendant trend between nearly 20 years, particularly change in latter 10 years is especially remarkable, annual tumour new cases about 3,090,000 at present, dead 1,960,000, lung cancer, women with breast cancer, cancer of the stomach, liver cancer and the esophageal carcinoma are modal malignant tumours, account for the 1/4 (Chen Wanqing that tumor mortality accounts for whole cause of the death, Zheng Rongshou, Zeng Hongmei, Zou peasant, Zhang Siwei, He Jie .2011 China's Incidence and Study on mortality China tumour, 2015, 24, 1-10).In the face of so severe anticancer situation, study and develop the vital task that antineoplastic newtype drug is medical personal.
Benzothiazole compound is that a class has extensive bioactive nitrogenous, sulfurheterocyclic compound, and agriculture production can be used for antimycotic (as: 2-amino-4-chloro benzo thiazole), weeding (such as: mefenacet), plant-growth regulator (as 2-trichloromethyl benzo thiazole) etc.Such as Ke Hou Zhong etc. report on " fine-chemical intermediate " " synthesis of N-benzothiazole N '-tertiary butyl hydrazide kind compound and bioactivity research " (the 32nd volume in 2002,4th phase, 19-20 page), synthesize a series of benzothiazolyl acyl callosity compounds and carried out desinsection, sterilization biological activity test, having found that this compounds is by affecting and disturbing the normal physiological activity of insect to control the generation of disease and insect.Benzothiazole compound can be used for thiofide, uv-absorbing agent, liquid crystal display material, electroluminescent material and prominent light probe etc. at Material Field.Benzothiazole compound medically can be used for antibacterial (as cefotaxime sodium), antituberculosis, parasiticide, wind resistance diseases caused by dampness and the biological activity widely such as antiviral.Li Na etc. obtain 2-cyano group-3-(4-methyl benzo [d] thiazole-2-is amino)-3-phenyl acrylates compounds by chemosynthesis, find that it has therapeutic activity to cucumber mosaic virus and tobacco mosaic virus (TMV), inhibiting rate reaches 56.31% (Li Na, Wei Xue, Fan Huitao, Bi Liang, Li Xiangyang, Yang Song, Hu Deyu, the synthesis of Song Baoan .2-cyano group-3-(4-methyl benzo [d] thiazole-2-is amino)-3-phenyl acrylates compounds and antiviral activity, organic chemistry, the 31st volume the 8th phase 1300-1311 page in 2011).Sun Fang in master thesis to the key intermediate 2 for the treatment of Parkinson medicinal pramipexole, 6-diamino-4,5,6,7-tetrahydro benzothiazol has carried out chemosynthesis, (Changjiang University's master thesis, 2,6-diamino-4,5, the synthesising process research of 6,7-tetrahydro benzothiazol, 2012).The a series of benzothiazole compound of Li Jian brave design and synthesis, and screened their anti-tumor activity, find that 3-(4-(benzo [d] thiazol-2-y1)-1-phenyl-1H-pyrazol-3-y1) phenyl acetate (DPB-5) has higher cytotoxicity, its mechanism of action is AIF signal path cell death inducing (the Zhejiang University Ph.D. Dissertation mediated by ROS, the synthesis of benzothiazole antitumor drug, screening and the mechanism of action thereof, 2010).In sum, the derivative of benzothiazole has good biological activity, and along with the research and development of increasing benzothiazole antitumor drug, benzothiazole oneself through becoming an indispensable female ring in antitumor drug research.
The synthetic method of tetrahydro benzothiazol class heterocycle mainly comprises following two large classes: one is synthesize benzothiazole derivant by intermolecular reaction, the most frequently used raw material is o-amino thiophenol, more commonly itself and aldehyde, acyl chlorides, ester, carboxylic acid, acid anhydrides and nitrile etc. are acted at present and obtain object, mostly need in reaction process to use catalyzer, as polyphosphoric acid, polyphosphate // Vanadium Pentoxide in FLAKES, methylsulfonic acid and phosphorus trichloride etc.Two is by recirculation synthesis benzothiazole derivant in molecule, adopt thioamides or thiocarbamide and corresponding derivative as raw material, because thioamides or thiocarbamide can designed, designeds according to actual needs, therefore substituting group selectable range in product is larger, various structures, but shortcoming is reaction needs multistep to complete, and the reaction times is generally longer.Such as, Hou Yihua etc. have studied the condensation reaction of the amino tetrahydro benzothiazol of 2-and aromatic aldehyde, 5 N-tetrahydro benzothiazol group with imine moiety are synthesized, react with Thiovanic acid respectively again prepared 5 4-thiazolidone compound (Hou Yihua, silver is beautiful, Lu Jun, Yang Bingqin, the synthesis of Ma Huairang .N-tetrahydro benzothiazol imines and repercussion study, Northwest University's journal (natural science edition), the 29th volume the 1st phase 27-29 page in 1999).Li Kai with 4-Phthalimide hexalin for starting raw material, through Ca (ClO)
2the oxidation of/TEMPO/NaBr system, HBr/H
2o
2the important intermediate 2 of system bromo, Hantzsch cyclization, hydrolysis anti-Parkinson medicine body of Pramipexole dihydrochloride, 6-diamino-4,5,6,7-tetrahydro benzothiazol (Li Kai, outstanding .2, the 6-diamino-4,5 of ox, 6, the improvement in synthesis of 7-tetrahydro benzothiazol, Zhejiang chemical industry, the 44th volume the 9th phase 4-6 page in 2013).
Literature research shows, tetrahydro benzothiazol is the compound that a class has Chinese traditional medicine biology activity, in agricultural chemicals, medicine and Material Field important use, but utilizes this compounds to have no bibliographical information as anti-gastric cancer medicament purposes.This patent is intended to prepare tetrahydro benzothiazol-4-ketoxime ether compound by organic synthesis mode, develops its purposes as anti-gastric cancer medicament, verifies its anti-cancer of the stomach mechanism with pharmacological experiment and calculational chemistry mode.
Summary of the invention
The object of the present invention is to provide a kind of tetrahydro benzothiazol-7-ketoxime medicine, the structural formula of this medicine is:
This structural formula comprises cis-isomeride and the trans-isomer(ide) of tetrahydro benzothiazol-7-ketoxime;
This medicine also comprises pharmacy acceptable salt (HX), example hydrochloric acid salt, hydrobromate, phosphoric acid salt, oxalate or vitriol etc.
The invention also discloses a kind of tetrahydro benzothiazol-7-ketoximes derivatives medicine, the structural formula of this medicine is:
Wherein, the structural formula of this derivative comprises cis-isomeride and trans-isomer(ide), R
1for alkyl (as methyl, ethyl, n-propyl, normal-butyl, sec.-propyl, benzyl etc.), R
2for alkyl or aryl (as methyl, ethyl, benzyl, phenyl, rubigan etc.), such as
in any one.
More preferably R
1for CH
3-, R
2for
Medicine of the present invention also comprises the composition of the medicine containing tetrahydro benzothiazol-7-ketoxime, and said composition comprises any one of said medicine, and pharmaceutically acceptable auxiliary material or carrier.
The preparation method of above-mentioned middle tetrahydro benzothiazol-7-ketoxime medicine is as follows
Experiment material
Experiment reagent
Hydroresorcinol (analytical pure, Shanghai Jingchun Industrial Co., Ltd.), thioacetamide (analytical pure, Shanghai Jingchun Industrial Co., Ltd.), bromine simple substance (analytical pure, Tianjin recovery fine chemistry industry institute), oxammonium hydrochloride (analytical pure, Tianjin Kermel Chemical Reagent Co., Ltd.), sodium-acetate (analytical pure, Shanghai Jingchun Industrial Co., Ltd.), sherwood oil (boiling range 60-90 DEG C, Tianjin Heng Xing chemical reagent Manufacturing Co., Ltd), ethyl acetate (analytical pure, the Tianjin limited department of Ke Miou chemical reagent), acetonitrile (analytical pure, Tianjin Kermel Chemical Reagent Co., Ltd.), ethanol (analytical pure, Tianjin Kermel Chemical Reagent Co., Ltd.), anhydrous sodium sulphate (analytical pure, Chemical Reagent Co., Ltd., Sinopharm Group), pyridine (analytical pure, Chemical Reagent Co., Ltd., Sinopharm Group), sodium hydride (analytical pure, Tianjin Heng Xing chemical reagent Manufacturing Co., Ltd), cuprous iodide (analytical pure, Tianjin Heng Xing chemical reagent Manufacturing Co., Ltd), propargyl bromide (analytical pure, Tianjin Kermel Chemical Reagent Co., Ltd.), thin layer precoated plate (Qingdao is up to Chemical Co., Ltd.), silica gel G F-254 (Qingdao is up to Chemical Co., Ltd.).
Laboratory apparatus
ZXZ-4 type rotary-vane vaccum pump (Linhai City Tan Shi vacuum apparatus company limited), DZF-6020 type vacuum drying oven (Shanghai new talent medicine equipment Manufacturing Co., Ltd), SHB-IIIA circulating water type Multi-purpose vaccum pump (Shanghai Yu Kang Science & Teaching Instrument equipment company limited), the dull and stereotyped magnetic stirring apparatus (Zhengzhou Greatwall Scientific Industrial & Trading Co., Ltd.) of CL-4 type, EYELA SB-1100 Rotary Evaporators (Shanghai Ai Lang Instrument Ltd.), FA2104B analytical balance (Shanghai Yue Ping tech equipment company limited), the micro-melting point detector of XRC-1 type (tech factory of Sichuan University), DF-101S heat collecting type constant-temperature heating magnetic stirring apparatus (Ying Yu Yu Hua instrument plant of Gongyi City), GZX-9240MBE digital display air dry oven (Medical Equipment Plant of Shanghai Boxun Industrial Co., Ltd.), ZF-6 type ultraviolet analysis instrument for three purposed (Shanghai Jia Peng Science and Technology Ltd.)
Preparation process
Synthetic route
2-bromo-hydroresorcinol (raw material 1) and thioamides flow through night next time in the condition of ethanol as solvent, obtain 2-substituted-tetrahydro benzothiazole-7-ketone (intermediate 2); Intermediate 2 and oxammonium hydrochloride are refluxed in anhydrous ethanol solvent under the effect of glacial acetic acid sodium, obtains 2-substituted-tetrahydro benzothiazole-7-ketoxime (intermediate 3); Be dissolved in anhydrous acetonitrile by intermediate 3 and propargyl bromide, under the effect of sodium hydride, stirring reaction obtains 2-substituted-tetrahydro benzothiazole-7-ketoxime ether (intermediate 4); Intermediate 4 is made catalyzer and azide intermediate at CuI 1,3-Dipolar Cycloaddition occurs obtain tetrahydro benzothiazol-7-ketoxime class medicine (target product 5).Synthetic route is as follows:
Synthesis condition: (a) R
1c (S) NH
2, pyridine, 80 DEG C, 2 hours; (b) NH
2oH-HCl, pyridine, 110 DEG C, 2 hours; (c) BrCH
2c ≡ CH, K
2cO
3, CH
3cN, 80 DEG C, 1 hour; (d) R
2n
3, CuI, 20 DEG C, 10 minutes.
Specifically comprise the steps:
1) by 2-bromine hexanaphthene-1,3-diketone (raw material 1) and alkylthioamide R
1c (S) NH
2be dissolved in pyridine solution, under nitrogen protection, stir, mix, be heated to 70-90 DEG C, after reaction 1.5-3h, washing, vacuum, except pyridine, obtain product, by product dichloromethane extraction, anhydrous sodium sulfate drying, vacuum concentration, obtains 2-and replaces-5,6-dihydrobenzos [d] thiazole-7 (4H)-one (intermediate 2) after purifying;
2) under nitrogen protection, intermediate 2 is dissolved in the pyridine of drying, be heated to 100-120 DEG C, pour in ice-water mixture after being cooled to normal temperature after stirring 1.5-3h, obtain white depositions, filtering precipitate is also dry, be separated, obtain 2-and replace-5, the cis-trans-isomer mixture (intermediate 3) of 6-dihydrobenzo [d] thiazole-7 (4H)-one oxime, for white solid, separation and purification obtains trans-isomer(ide) (intermediate 3a) and cis-isomeride (intermediate 3b);
3) intermediate 3 is dissolved in anhydrous acetonitrile, add Anhydrous potassium carbonate and propargyl bromide, 0.5-1.5h is stirred at 70-90 DEG C, removing solvent acetonitrile, after product extracted into EtOAc, dry, vacuum concentration, obtain yellowish crude product, separation and purification obtains 2-substituted-tetrahydro benzothiazole-7-ketoxime ether (intermediate 4);
4) intermediate 4 and triazo-compound (R is got
2n
3) mix in mortar, add cuprous iodide, grind 10 minutes under room temperature until reactant completely consumed, reaction mixture water coolant is diluted, adds ethyl acetate and extract organism, through anhydrous sodium sulfate drying, obtain white solid after evaporate to dryness, be tetrahydro benzothiazol-7-ketoxime medicine.
In synthesis step, intermediate 2 can solvent be methyl alcohol, ethanol, toluene, pyridine, be wherein that the productive rate of solvent is the highest with pyridine; Intermediate 3 can solvent be toluene, benzene, tetrahydrofuran (THF), acetonitrile, dioxane etc., be wherein that the productive rate of solvent is the highest with acetonitrile; The extraction solvent of target product can be chloroform, ethyl acetate, methylene dichloride, ethanol, methyl alcohol etc., and the highest with the extraction efficiency of ethyl acetate, impurity is less, ethyl acetate.
Above-mentioned step 1) in, raw material 1 is 1:1-1.3 with the mol ratio of thioamides; Step 3) in, the mol ratio of intermediate 3, Anhydrous potassium carbonate, propargyl bromide is 1:1.2-2.5:1-1.5; Step 4) in, the mol ratio of intermediate 4, nitrine, cuprous iodide is: 10:8-15:0.8-1.5.
More preferably step 1) in, raw material 1 is 1:1 with the mol ratio of thioamides; Step 3) in, the mol ratio of intermediate 3, Anhydrous potassium carbonate, propargyl bromide is 1:2:12; Step 4) in, the mol ratio of intermediate 4, nitrine, cuprous iodide is: 10:10:1.
The invention also discloses a kind of preparation method of tetrahydro benzothiazol-7-ketoximes derivatives medicine, comprise the steps, a kind of preparation method of tetrahydro benzothiazol-7-ketoxime medicine, is characterized in that, comprise the steps:
1) by 2-bromine hexanaphthene-1,3-diketone (raw material 1) and alkylthioamide R
1c (S) NH
2be dissolved in pyridine solution, under nitrogen protection, stir, mix, be heated to 70-90 DEG C, after reaction 1.5-3h, washing, vacuum, except pyridine, obtain product, by product dichloromethane extraction, anhydrous sodium sulfate drying, vacuum concentration, obtains 2-and replaces-5,6-dihydrobenzos [d] thiazole-7 (4H)-one (intermediate 2) after purifying;
2) under nitrogen protection, intermediate 2 is dissolved in the pyridine of drying, be heated to 100-120 DEG C, pour in ice-water mixture after being cooled to normal temperature after stirring 1.5-3h, obtain white depositions, filtering precipitate is also dry, be separated, obtain 2-and replace-5, the cis-trans-isomer mixture (intermediate 3) of 6-dihydrobenzo [d] thiazole-7 (4H)-one oxime, for white solid, separation and purification obtains trans-isomer(ide) (intermediate 3a) and cis-isomeride (intermediate 3b);
3) intermediate 3 is dissolved in anhydrous acetonitrile, add Anhydrous potassium carbonate and propargyl bromide, 0.5-1.5h is stirred at 70-90 DEG C, removing solvent acetonitrile, after product extracted into EtOAc, dry, vacuum concentration, obtain yellowish crude product, separation and purification obtains 2-substituted-tetrahydro benzothiazole-7-ketoxime ether (intermediate 4);
4) intermediate 4 and nitrine (R is got
2n
3) mix in mortar, add cuprous iodide, 10 minutes are ground until reactant completely consumed under room temperature, reaction mixture water coolant is diluted, add ethyl acetate and extract organism, through anhydrous sodium sulfate drying, after evaporate to dryness, obtain white solid, be tetrahydro benzothiazol-7-ketoximes derivatives medicine.
Wherein, step 1) in, raw material 1 is 1:1-1.3 with the mol ratio of thioamides; Step 3) in, the mol ratio of intermediate 3, Anhydrous potassium carbonate, propargyl bromide is 1:1.2-2.5:1-1.5; Step 4) in, the mol ratio of intermediate 4, nitrine, cuprous iodide is: 10:8-15:0.8-1.5.
More preferably step 1) in, raw material 1 is 1:1 with the mol ratio of thioamides; Step 3) in, the mol ratio of intermediate 3, Anhydrous potassium carbonate, propargyl bromide is 1:2:12; Step 4) in, the mol ratio of intermediate 4, nitrine, cuprous iodide is: 10:10:1.
Adopt above-mentioned processing step, the present invention synthesizes further and obtains following tetrahydro benzothiazol-7-ketoximes derivatives medicine:
The structural analysis of compound is as follows:
Experiment reagent
Deuterochloroform-d (D atom content 99.8%, TMS content 0.03%V/V, 10*0.5mL/ box, ARMAR company of Switzerland); Trifluoroacetic acid aqueous solution (content 99.99%, 4L is bottled, German MILAK company); Distilled water (4.5L/ bucket, Watson Watsons company); Nuclear magnetic tube (5mm 100/pk 2ST500-8, Norell company of the U.S.).
Laboratory apparatus
Ultrashied 400MHz Plus nuclear magnetic resonance analyser (Bruker company of Switzerland), API 4000LC-MS/MS mass spectrograph (German Brooker dalton company)
Test process
Accurately take 10mg target compound, dissolve with 0.5mL deuterochloroform in nuclear magnetic tube, test its chemical structure by nuclear magnetic resonance analyser.
hydrogen nuclear magnetic resonance spectrogram, carbon spectrogram, electrospray ionization mass spectrum figure are as shown in Figure 1, 2, 3.
Nuclear magnetic data:
1h NMR (400MHz, CDCl
3) δ: 7.56 (s, 2H), 7.26 (d, J=5.6Hz, 4H), 5.54 (s, 4H), 5.30 (s, 4H), 2.93 (t, J=6.0Hz, 4H), 2.65 (s, 6H), 2.58 (t, J=6.4Hz, 4H), 2.04-1.98 (m, 4H).
13c NMR (400MHz, CDCl
3) δ: 170.1,159.2,149.1,145.0,135.2,128.5,123.0,118.5,67.2,53.5,29.2,27.9,22.6,18.7. interpretation of result: chemical displacement value is unimodal at the 6H at 2.65 places should be two methyl peaks, and the impact by the C=N double bond of side connection makes its shift value move to low field; In δ=2.93,2.58 places are all triplets, can infer the side carbon be attached thereto should have two hydrogen make it produce to split point, and δ=2.04-1.98 place is the multiplet of four hydrogen, structure expected structure according to compound can be inferred, has three CH in structure
2be connected, coincide with its expected structure.Four hydrogen at δ=7.26 place, coupling constant is 5.6Hz, is therefore benzene ring hydrogen, splits point identical, is all doublet, can infers on phenyl ring it is para-orientation; Two hydrogen at δ=7.56 place are unimodal, can judge it is the alkene hydrogen in triazole ring according to its chemical displacement value and proton peak; And δ=5.54,5.30 places are also all unimodal, should be two CH connected separately respectively
2, the CH that its chemical displacement value is all more general
2greatly, so its two ends connect respectively should be the heteroatomss such as N, O or the electron-withdrawing group such as phenyl ring, double bond.Be in carbon spectrum δ=29.2 of High-Field, 27.9,22.6,18.7 places are the CH not only not connected heteroatoms but also do not connected double bond
2, CH
3carbon, what δ=67.2,53.5 places were that side is connected is the carbon of heteroatoms and phenyl ring, and be subject to the effect of deshielding of electron-withdrawing group, chemical displacement value moves to low field; δ=170.1,159.2 are two quaternary carbons of C=N double bond; All the other carbon are carbon on olefinic carbon and phenyl ring.
Electrospray ionization mass spectrum map analysis: compound molecule formula C
30h
32n
10o
2s
2, molecular weight 628.77, mass spectrometric measurement result 651.85 (M+Na) is ion adduction peak, and add sodium peak with molecular weight and coincide, molecular weight is correct.
The invention also discloses described tetrahydro benzothiazol-7-ketoxime and composition, tetrahydro benzothiazol-7-ketoximes derivatives and composition thereof or the application of its salt in the anti-gastric cancer medicament of preparation treatment.
The external stomach cancer cell cytotoxic activity test of tetrahydro benzothiazol-7-ketoxime class medicine
Reagent and instrument
Experiment reagent
Laboratory apparatus
| Instrument title | Model | Manufacturer |
| Low speed centrifuge | TDZ4-WS | The ordinary instrument company limited in Changsha |
| Supercentrifuge | TGL-16C | Anting Scientific Instrument Factory, Shanghai |
| Electric heating constant temperature tank | DK-8A | The grand experimental installation company limited of upper Nereid |
| Fixed temperature and humidity incubator | HTC-100A | Instrument Ltd. is risen in Shanghai three |
| Vertical pressure steam sterilizer | LDZX-30KBS | Shenan Medical Appliances Factory, Shanghai |
| Computer-shape inverted microscope | XDS-113 | Shanghai Yu Guang Instrument Ltd. |
| Turbula shaker | Vortex-Genie2 | Ao Ran Science and Technology Ltd. of the U.S. |
| Enzyme-linked immunosorbent assay instrument | StatFax-2100 | U.S. Awareness |
| CO 2Incubator | SANYO GS company | |
| Automatic distilled water distiller | Shanghai Ya Rong biochemical equipment Instrument Ltd. | |
| Bechtop | Beijing Dong Lianhaer instrument manufacturing company limited | |
| Ultrasonic washing instrument | Ride the waves Xin Zhi biotech inc |
Experimental section
Mtt assay is adopted to test the anti tumor activity in vitro of compound to HGC-27 (Human Gastric carcinoma's cell), with antitumor drug paclitaxel (Paclitaxel) for positive control.
Inoculating cell step is as follows:
(1) with the monolayer cell that tryptic digestion converges, by cell harvesting in the substratum containing serum.
(2) eccentric cell suspension, makes cell deposition get off.Use substratum re-suspended cell, counting.
(3) by cell dilution to 2.5 × 10
3-50 × 10
3individual/mL, each titer plate can hold 20mL cell re-suspension liquid.
(4) add 200 μ L cell suspensions with in each hole of sample injector ten row in the middle of flat 96 orifice plates, every hole adds 0.5 × 10
3-10 × 10
3individual cell.
(5) 200 μ L substratum are added in 8 holes of the 1st row and the 12nd row.1st row read the blank of plate view.
(6) culture plate is put in plastic lunch box, incubation 1-3 days in 37 DEG C of moist environments, when waiting cell to enter exponential phase of growth, can medicine be added.Dosing:
(1) with substratum by cytotoxic drug 5 times of serial dilutions, altogether prepare 8 concentration.
(2) for the cell of adherent growth, the substratum that 2-11 arranges each hole is removed.
(3) in the 2nd row and the 11st 8 holes arranged, add the freshly prepared substratum of 200 μ L, these cells in contrast.
(4) in the cell of 3-10 row, cytotoxic drug is added.Each drug level only needs 4 holes, and such A-D is capable can be used for the first medicine, and E-H is capable is used for the second medicine.
(5) 200 μ L drug solutions are added to often organizing in 4 holes.
(6) culture plate is put back in plastics casing, be incubated to the time of determination.
Vegetative period:
(1) at the end of the drug effect phase, remove all containing the substratum in the hole of cell, add the substratum that 200 μ L are fresh.
(2) liquid is changed every day to 2-3 PDTs [population doubling time].
The estimation of survivaling cell number:
(1) in the growth end of term, in every hole, add the substratum that 200 μ L are fresh, in 1-11 row institute is porose, respectively adds 50 μ L MTT.
(2) culture plate is wrapped up with aluminium foil, incubation 4h in 37 DEG C of moist environments.
(3) discard the substratum in hole and MTT, in 1-11 row institute is porose, respectively adds 150 μ L DMSO, to dissolve residual MTT-formazan crystallization.
(4) culture plate is put shake on the oscillator, make reaction solution even.
(5) immediately at 492nm place record light absorption value.Read plate instrument with in the 1st row containing substratum and MTT but not celliferous each hole zeroing.Absorbance is detected at microplate reader 492nm place, and calculate inhibiting rate, calculation formula is as follows:
Inhibiting rate=(1-administration group absorbance/blank group absorbance) × 100%
The sample that when selecting 50 μ g/mL, inhibiting rate is greater than 50%, resets concentration and carries out dusting cover. and add in 96 orifice plates by testing sample with 1,2,4,8,16,32,64 μ g/mL concentration, cultivate 48 hours, detect inhibiting rate, adopt SPSS computed in software IC
50value.
Accompanying drawing explanation
Fig. 1 is
hydrogen nuclear magnetic resonance spectrogram.
Fig. 2 is
carbon-13 nmr spectra figure.
Fig. 3 is
electrospray ionization mass spectrum figure.
Embodiment
Embodiment 1
Synthetic route:
2-bromo-hydroresorcinol (raw material 1) and thioamides flow through night next time in the condition of ethanol as solvent, obtain 2-substituted-tetrahydro benzothiazole-7-ketone (intermediate 2); Intermediate 2 and oxammonium hydrochloride are refluxed in anhydrous ethanol solvent under the effect of glacial acetic acid sodium, obtains 2-substituted-tetrahydro benzothiazole-7-ketoxime (intermediate 3); Be dissolved in anhydrous acetonitrile by intermediate 3 and propargyl bromide, under the effect of sodium hydride, stirring reaction obtains 2-substituted-tetrahydro benzothiazole-7-ketoxime ether (intermediate 4); Intermediate 4 is made catalyzer and azide intermediate at CuI 1,3-Dipolar Cycloaddition occurs obtain tetrahydro benzothiazol-7-ketoxime class medicine (target product 5).Synthetic route is as follows:
Synthesis condition: (a) alkylthioamide R
1c (S) NH
2, pyridine, 80 DEG C, 2 hours; (b) NH
2oH-HCl, pyridine, 110 DEG C, 2 hours; (c) BrCH
2c ≡ CH, K
2cO
3, CH
3cN, 80 DEG C, 1 hour; (d) nitrine R
2n
3, CuI, 20 DEG C, 10 minutes.
Building-up process:
The preparation of intermediate 2: accurately take 3.80g with electronic balance, the 2-bromine hexanaphthene-1,3-diketone (1) of 20mmol and 20mmol alkylthioamide R
1c (S) NH
2in the pyridine solution of 50mL, be filled with nitrogen as protection gas, be uniformly mixed, be heated to 80 DEG C, react 2 hours.After having reacted, solvent is removed under vacuo, and by resistates 50mL saturated common salt water washing.Product methylene dichloride (3 × 30mL) is extracted.Organic extract liquid is merged, with anhydrous sodium sulfate drying, and concentrate under vacuo, obtain dark sticky oil thing, passed through flash column chromatography (eluent: ethyl acetate/petroleum ether=1:4, V/V), obtain 2.40g 2-and replace-5,6-dihydrobenzo [d] thiazole-7 (4H)-one (2) is yellow oil.
The preparation of intermediate 3: accurately take 10.0mmol 2-and replace-5; 6-dihydrobenzo [d] thiazole-7 (4H)-one (intermediate 2) is dissolved in the pyridine of 40mL drying; use the pyridinium dissolution of 10mL drying under nitrogen protection; be heated to 110 DEG C stir 2 hours; then cool and pour in 250mL ice-water mixture, obtaining white depositions.Filtering precipitate is also dry, and be separated by column chromatography, obtaining the cis-trans-isomer mixture that 1.80g 2-replaces-5,6-dihydrobenzos [d] thiazole-7 (4H)-one oxime, is white solid.0.81g trans-isomer(ide) (intermediate 3a) and 0.85g cis-isomeride (intermediate 3b) is obtained by column chromatographic isolation and purification.
The preparation of intermediate 4: accurately take 20mmol intermediate 3, is dissolved in 20mL anhydrous acetonitrile, adds 4.1g Anhydrous potassium carbonate (30mmol) and 2.4g propargyl bromide (20mmol), stirs 1 hour at 80 DEG C.Except desolventizing, product ethyl acetate (3 × 20mL) is extracted.Merged by organic extract liquid, drying also concentrates under vacuo, obtains yellowish crude product.Through flash column chromatography (normal hexane: ethyl acetate=3:1, V/V), obtain 4.1g intermediate 4.
The preparation of target product 5: accurately take 1.0mmol intermediate 4 and 1.0mmol nitrine R
2n
3mix in mortar, add 0.01mmol cuprous iodide, grind 10 minutes under room temperature until reactant completely consumed.By reaction mixture 10mL water coolant dilution, add ethyl acetate and extract organism, through anhydrous sodium sulfate drying, after evaporate to dryness, obtain white solid, be tetrahydro benzothiazol-7-ketoxime class medicine.
In the preparation of intermediate 2, pyridine solvent can also be any one in methyl alcohol, ethanol or toluene,
In the preparation of intermediate 4, acetonitrile solvent can also be any one in toluene, benzene, tetrahydrofuran (THF) or dioxane;
In the preparation of target product 5, acetate extractant can also be any one in chloroform, methylene dichloride, ethanol or methyl alcohol.
The experimental result of anti-gastric cancer medicament is as follows:
Cytotoxic activity evaluation result: tetrahydro benzothiazol-7-ketoxime class medicine has good anti-stomach cancer cell cytotoxic activity, with the anti-cancer of the stomach activity (IC of commercially available taxol
50: 4.16 μMs) compare, the half-inhibition concentration of compound 5j, 5k is low reaches less than 10 μMs, quite active with the anti-cancer of the stomach of taxol; The half-inhibition concentration of compound 5d, 5f and 5i, between 20 ~ 30 μMs, also has good anti-cancer of the stomach active.In sum, compound has deep anti-gastric cancer medicament Development volue.
Claims (10)
1. a tetrahydro benzothiazol-7-ketoxime medicine, is characterized in that the structural formula of this medicine is containing triazole methylene substituent:
This structural formula comprises cis-isomeride and the trans-isomer(ide) of tetrahydro benzothiazol-7-ketoxime, and its structural formula is:
This medicine also comprises above-mentioned cis-trans-isomer pharmacy acceptable salt (HX), acceptable salt comprise in hydrochloride, hydrobromate, phosphoric acid salt, oxalate or vitriol any one.
2. a tetrahydro benzothiazol-7-ketoximes derivatives medicine, is characterized in that, the structural formula of this medicine is:
Wherein, the structural formula of this derivative comprises cis-isomeride and trans-isomer(ide), R
1for alkyl, R
2for alkyl or aryl.
3. tetrahydro benzothiazol-7-ketoximes derivatives medicine according to claim 2, is characterized in that, R
1for any one in methyl, ethyl, n-propyl, normal-butyl, sec.-propyl or benzyl, R
2for
in any one.
4. tetrahydro benzothiazol-7-ketoximes derivatives medicine according to claim 2, is characterized in that, R
1for CH
3-, R
2for
5. the composition of medicine containing tetrahydro benzothiazol-7-ketoxime described in any one of claim 1-4, it is characterized in that, said composition comprises the medicine described in any one of Claims 1 to 4, and pharmaceutically acceptable auxiliary material or carrier.
6. a preparation method for tetrahydro benzothiazol-7-ketoxime or derivatives thereof medicine, is characterized in that, comprise the steps:
1) by 2-bromine hexanaphthene-1,3-diketone (raw material 1) and thioamides or R
1c (S) NH
2be dissolved in pyridine solvent, under nitrogen protection, stir, mix, be heated to 70-90 DEG C, after reaction 1.5-3h, washing, vacuum, except pyridine, obtain product, by product dichloromethane extraction, anhydrous sodium sulfate drying, vacuum concentration, obtains 2-and replaces-5,6-dihydrobenzos [d] thiazole-7 (4H)-one (intermediate 2) after purifying;
2) under nitrogen protection, intermediate 2 is dissolved in the pyridine of drying, be heated to 100-120 DEG C, pour in ice-water mixture after being cooled to normal temperature after stirring 1.5-3h, obtain white depositions, filtering precipitate is also dry, be separated, obtain 2-and replace-5, the cis-trans-isomer mixture (intermediate 3) of 6-dihydrobenzo [d] thiazole-7 (4H)-one oxime, for white solid, separation and purification obtains trans-isomer(ide) (intermediate 3a) and cis-isomeride (intermediate 3b);
3) intermediate 3 is dissolved in anhydrous acetonitrile solvent, add Anhydrous potassium carbonate and propargyl bromide, 0.5-1.5h is stirred at 70-90 DEG C, removing solvent acetonitrile, after product extracted into EtOAc, dry, vacuum concentration, obtain yellowish crude product, separation and purification obtains 2-substituted-tetrahydro benzothiazole-7-ketoxime ether (intermediate 4);
4) intermediate 4 and nitrine or R is got
2n
3mix in mortar, add cuprous iodide, 10 minutes are ground until reactant completely consumed under room temperature, reaction mixture water coolant is diluted, add extraction into ethyl acetate organism, through anhydrous sodium sulfate drying, after evaporate to dryness, obtain white solid, be tetrahydro benzothiazol-7-ketoxime medicine or tetrahydro benzothiazol-7-ketoximes derivatives medicine.
7. the preparation method of tetrahydro benzothiazol-7-ketoxime or derivatives thereof medicine according to claim 6, is characterized in that,
Step 1) in, pyridine solvent can also be any one in methyl alcohol, ethanol or toluene;
Step 3) in, acetonitrile solvent can also be any one in toluene, benzene, tetrahydrofuran (THF) or dioxane;
Step 4) in, acetate extractant can also be any one in chloroform, methylene dichloride, ethanol or methyl alcohol.
8. the preparation method of tetrahydro benzothiazol-7-ketoxime or derivatives thereof medicine according to claim 6, is characterized in that, step 1) in, raw material 1 is 1:1-1.3 with the mol ratio of thioamides;
Step 3) in, the mol ratio of intermediate 3, Anhydrous potassium carbonate, propargyl bromide is 1:1.2-2.5:1-1.5;
Step 4) in, the mol ratio of intermediate 4, nitrine, cuprous iodide is: 10:8-15:0.8-1.5.
9. the preparation method of tetrahydro benzothiazol-7-ketoxime or derivatives thereof medicine according to claim 6, is characterized in that,
Step 1) in, raw material 1 is 1:1 with the mol ratio of thioamides;
Step 3) in, the mol ratio of intermediate 3, Anhydrous potassium carbonate, propargyl bromide is 1:2:12;
Step 4) in, the mol ratio of intermediate 4, nitrine, cuprous iodide is: 10:10:1.
10. tetrahydro benzothiazol-7-the ketoxime described in any one of claim 1-9 and composition, tetrahydro benzothiazol-7-ketoximes derivatives and composition thereof or the application of its salt in the anti-gastric cancer medicament of preparation treatment.
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| CN107868063A (en) * | 2017-12-18 | 2018-04-03 | 泰山医学院 | A kind of acetone 9 oxime derivate of tetrahydro benzothiazol 2 and its preparation method and application |
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| US20090011975A1 (en) * | 2003-04-03 | 2009-01-08 | Semafore Pharmaceuticals Inc. | Targeted bone marrow protection agents |
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| US20090011975A1 (en) * | 2003-04-03 | 2009-01-08 | Semafore Pharmaceuticals Inc. | Targeted bone marrow protection agents |
| CN1590379A (en) * | 2003-07-28 | 2005-03-09 | 瑟维尔实验室 | New heterocyclic oxime compounds, preparing process and medical composition thereof |
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| CAO CHENG-QIAO ET AL.: "Synthesis and Crystal Structure of (Z)-2-Methyl-5,6-dihydrobenzo[d]thiazol-7(4H)-one O-Prop-2-yn-1-yl Oxime Derivatives", 《CHINESE J. STRUCT. CHEM.》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107868063A (en) * | 2017-12-18 | 2018-04-03 | 泰山医学院 | A kind of acetone 9 oxime derivate of tetrahydro benzothiazol 2 and its preparation method and application |
| CN107868063B (en) * | 2017-12-18 | 2021-02-12 | 泰山医学院 | Tetrahydrobenzothiazole-2-acetoxime derivative and preparation method and application thereof |
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