CN106146429A - The structure of isosulfocyanate precursor compound, prepare and apply - Google Patents
The structure of isosulfocyanate precursor compound, prepare and apply Download PDFInfo
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- CN106146429A CN106146429A CN201510191603.9A CN201510191603A CN106146429A CN 106146429 A CN106146429 A CN 106146429A CN 201510191603 A CN201510191603 A CN 201510191603A CN 106146429 A CN106146429 A CN 106146429A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
- C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
- C07D277/62—Benzothiazoles
- C07D277/68—Benzothiazoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 2
- C07D277/70—Sulfur atoms
- C07D277/74—Sulfur atoms substituted by carbon atoms
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C333/00—Derivatives of thiocarbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
- C07C333/14—Dithiocarbamic acids; Derivatives thereof
- C07C333/18—Esters of dithiocarbamic acids
- C07C333/20—Esters of dithiocarbamic acids having nitrogen atoms of dithiocarbamate groups bound to hydrogen atoms or to acyclic carbon atoms
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D257/00—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms
- C07D257/02—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms not condensed with other rings
- C07D257/04—Five-membered rings
Abstract
The invention provides the structure of isosulfocyanate precursor compound, the formula of described compound is:；Wherein R1For: H3C-、、C4-C6Alkyl, C1-C4Alkoxyl, C1-C6Alkenyl, C2-C10Alkynyl, C1-C4Alkanoyl, C1-C4Alkanoyloxy,、Or；R2For:；R3For: Cl, Br, alkyl, C1-C6Alkenyl, C2-C10Alkynyl, C1-C4Alkanoyl, C1-C4Alkanoyloxy；R4For: H, OH, CF3Or methoxyl group；X is: O, S or NH；N=0,1 or 2.Present invention also offers preparation and the application thereof of isosulfocyanate precursor compound, these compounds can slowly release reactive compound, and drug resistance is low, and toxic and side effects is little, may be used for prevention or treatment kinds cancer.
Description
Technical field
The present invention relates to medical compounds field, more particularly, to isosulfocyanate precursor compound structure, prepare and apply.
Background technology
In research process, find that some medical compounds structure has unstability, meanwhile, the most easy to store under air and normal temperature environment.And these existing cancer therapy drugs and are difficult to reach effective drug level in human body, and action time is short, also cannot give full play to anticancer efficacy.Therefore, Effect of Anti tumour medicine slow-released system becomes the emphasis of tumor pharmacother, and slow releasing pharmaceutical can strengthen the targeting to tumor cell or tissue, extends pharmaceutical release time simultaneously, reduces absorption rate, steady blood drug level, lowers toxic and side effects.It is contemplated that it is little to find a kind of Stability Analysis of Structures, toxic and side effects, targeting is strong, therapeutic effect is preferable and the prodrug that can slowly discharge.
CN101731258A discloses a kind of aromatic radical isothiocyanate and the compositions of allyl group isosulfocyanate, and its weight ratio is 1: 9～9: 1, and discloses above-mentioned composition application in preparation prevents and treats the pesticide fumigant of soil-borne pathogen, soil nematodes.
CN104173272A discloses the slow releasing preparation of a kind of isosulfocyanate compound or derivatives thereof, and specifically, described slow releasing preparation contains the isosulfocyanate compound or derivatives thereof of (a) therapeutically effective amount；(b) pharmaceutically acceptable carrier, it provides the form of a kind of compositions.CN101822663B discloses the isothiocyanate purposes at preparation preventing and treating drug-resistant tumor drug.CN101780066A discloses the isothiocyanate purposes at preparation preventing and treating tumor invasion and metabasis medicine.CN102631340A discloses a kind of antitumor drug containing isothiocyanate and application thereof.CN104072395A discloses isocyanate derivative has fragmentation effect to tumor cell.
But up to the present, there is no the prodrug of isosulfocyanate, it is desirable to improve to some extent in this field.
Summary of the invention
The invention provides a kind of Stability Analysis of Structures, toxic and side effects is little, targeting is strong, therapeutic effect is preferable and the precursor compound that can slowly discharge, and the formula of described compound is:
Wherein R1For:C4-C6Alkyl, C1-C4Alkoxyl, C1-C6Alkenyl, C2-C10Alkynyl, C1-C4Alkanoyl, C1-C4Alkanoyloxy, R2For:R3For: Cl, Br, alkyl, C1-C6Alkenyl, C2-C10Alkynyl, C1-C4Alkanoyl, C1-C4Alkanoyloxy；R4For: H, OH, CF3Or methoxyl group；X is: O, S or NH；N=0,1 or 2.
According to embodiments of the invention, it is provided that compound is for preventing or treating the application in terms of cancer.
In above-mentioned application, wherein, described cancer includes breast carcinoma, leukemia, proliferating epidermal cancer, hepatocarcinoma, cancer of pancreas, cerebral glioma, hepatocarcinoma, cutaneous melanoma, neuroblastoma, pulmonary carcinoma.
According to another embodiment of the present invention, it is provided that the preparation method of a kind of compound, described method includes: add 2-dimethylamino thioglycolic hydrochlorate and ethanol solution in a reservoir；It is subsequently added 5-[(4-isothiocyanate butyl) sulfur]-1-phenyl-1H-tetrazole；With alkaline solution, the pH value of reactant liquor being adjusted to 5-7, described reactant liquor is stirred overnight under inert gas shielding；In described reactant liquor, drip alkaline solution pH value is adjusted to alkalescence；Being spin-dried for by described reactant liquor, gained surplus materials is dissolved in dichloromethane, and washs, and gained organic facies is dried and is concentrated thus obtains the thick product of described compound；And the thick product of described compound is purified.
According to another embodiment of the present invention, it is provided that the preparation method of a kind of compound, described method includes: add 2-dimethylamino thioglycolic hydrochlorate and ethanol solution in a reservoir；It is subsequently added Sulforaphane；With alkaline solution, the pH value of reactant liquor is adjusted to 5-7, is stirred overnight under inert gas shielding；In described reactant liquor, drip alkaline solution pH value is adjusted to alkalescence；Being spin-dried for by described reactant liquor, gained surplus materials is dissolved in dichloromethane, and washs, and gained organic facies is dried and is concentrated thus obtains the thick product of described compound；And the thick product of described compound is purified.
According to another embodiment of the present invention, it is provided that the preparation method of a kind of compound, described method includes: be dissolved in dichloromethane by 5-((4-isothiocyanate butyl) sulfur)-1-phenyl-1H-tetrazole；It is subsequently adding dimethylamino naphthyridine and 2-(dimethylamino) ethyl-3-sulfydryl-2-methyl propyl ester；System is stirred at room temperature under inert gas shielding reacts；Rotation, except solvent, obtains the thick product of described compound；And the thick product of described compound is purified.
According to another embodiment of the present invention, it is provided that the preparation method of a kind of compound, described method includes: Sulforaphane is dissolved in dichloromethane；It is subsequently adding N, N-dimethylamino naphthyridine and 2-(dimethylamino) ethyl-3-sulfydryl-2-methyl propyl ester；System is stirred at room temperature under inert gas shielding reacts；Rotation, except solvent, obtains the thick product of described compound；And the thick product of described compound is purified.
According to another embodiment of the present invention, it is provided that the preparation method of a kind of compound, described method includes: 5-((4-isothiocyanate butyl) sulfur)-1-phenyl-1H-tetrazole is dissolved in dichloromethane；It is subsequently adding triethylamine and 2-(2-(2-(dimethylamino) ethyoxyl) ethyoxyl) ethyl mercaptan；System is stirred at room temperature under inert gas shielding reacts；Rotation, except solvent, obtains the thick product of described compound；And the thick product of described compound is purified.
According to another embodiment of the present invention, it is provided that the preparation method of a kind of compound, described method includes: be dissolved in dichloromethane by Sulforaphane；It is subsequently adding triethylamine and 2-(2-(2-(dimethylamino) ethyoxyl) ethyoxyl) ethyl mercaptan；System is stirred at room temperature under inert gas shielding reacts；Rotation, except solvent, obtains the thick product of described compound；And the thick product of described compound is purified.
According to another embodiment of the present invention, it is provided that the preparation method of a kind of compound, described method includes: be dissolved in ethanol by 2-dimethylamino thioglycolic hydrochlorate；It is subsequently adding (4-bromophenyl) (4-isothiocyanate butyl) thioether；By alkaline solution, the pH value of reactant liquor is adjusted to 5-7, described reactant liquor is stirred；Rotation is except solution, and gained surplus materials is dissolved in dichloromethane, organic facies is washed, is dried and concentrates, obtains the thick product of described compound；And the thick product of described compound is purified.
According to another embodiment of the present invention, it is provided that the preparation method of a kind of compound, described method includes: be dissolved in ethanol by 2-dimethylamino thioglycolic hydrochlorate；It is subsequently adding 2-((4-isothiocyanate butyl) sulfur) benzothiazole；By alkaline solution, the pH value of reactant liquor is adjusted to 5-7, described reactant liquor is stirred；Rotation is except solution, and gained surplus materials is dissolved in dichloromethane, organic facies is washed, is dried and concentrates, obtains the thick product of described compound；And the thick product of described compound is purified.
The compound structure that the present invention provides is stable, reactive compound can be slowly released, extend pharmaceutical release time, can effectively suppress survival and the growth of the tumor cells such as MCF-7, SUM-159, KG1a, HELA, HEPG2, PANC-1, SHG44, PLC/PRF5, A375, SH-SY5Y, NCI-H446, and drug resistance is low, toxic and side effects is little.It may be used for the cancer such as prevention or treatment breast carcinoma, leukemia, proliferating epidermal cancer, hepatocarcinoma, cancer of pancreas, cerebral glioma, hepatocarcinoma, cutaneous melanoma, neuroblastoma, pulmonary carcinoma.
Accompanying drawing explanation
Fig. 1 and Fig. 2 respectively illustrates compound 1c's and compound 1d1HNMR schemes.
Fig. 3 and Fig. 4 respectively illustrates compound 1e's1HNMR figure and13CNMR schemes.
Fig. 5 and Fig. 6 respectively illustrates compound 11HNMR figure and13CNMR schemes.
Fig. 7 and Fig. 8 respectively illustrates compound 21HNMR figure and13CNMR schemes.
Fig. 9 and Figure 10 respectively illustrates compound 3c's1HNMR figure and13CNMR schemes.
Figure 11 and Figure 12 respectively illustrates compound 3d's1HNMR figure and13CNMR schemes.
Figure 13 and Figure 14 respectively illustrates compound 31HNMR figure and13CNMR schemes.
Figure 15 and Figure 16 respectively illustrates compound 41HNMR figure and13CNMR schemes.
Figure 17 and Figure 18 respectively illustrates compound 5b's1HNMR figure and13CNMR schemes.
Figure 19 and Figure 20 respectively illustrates compound 5d's1HNMR figure and13CNMR schemes.
Figure 21 and Figure 22 respectively illustrates compound 5e's1HNMR figure and13CNMR schemes.
Figure 23 and Figure 24 respectively illustrates compound 51HNMR figure and13CNMR schemes.
Figure 25 and Figure 26 respectively illustrates compound 61HNMR figure and13CNMR schemes.
Figure 27 and Figure 28 respectively illustrates compound 71HNMR figure and13CNMR schemes.
Figure 29 and Figure 30 respectively illustrates compound 81HNMR figure and13CNMR schemes.
Figure 31 shows compound 1, compound 1e, the standard curve of carbamazepine.
Figure 32 shows the decomposition curve under compound 1 different condition in vitro.
Figure 33 shows the decomposition curve under compound 3 different condition in vitro.
Figure 34 shows the decomposition curve under compound 5 different condition in vitro.
Figure 35 shows the decomposition curve under compound 6 different condition in vitro.
Figure 36 shows the suppression of compound 1e, 1,3,5 pair MCF-7 cell.
Figure 37 shows the suppression of compound 2a, 4,6 pair MCF-7 cell.
Figure 38 shows the suppression of compound 1e, 1,3,5 pair sum159 cell.
Figure 39 shows the suppression of Figure 39 compound 2a, 4,6 pair Sum159 cell.
Figure 40 shows the suppression of compound 1e, 3,5 pair 293T cell.
Figure 41 shows the suppression of compound 2a, 4,6 pair 293T cell.
Figure 42 shows compound 2a, the suppression of 1e, 1,3 pair KG1a cell.
Figure 43 and Figure 44 shows the compound 1 suppression to Hela, HepG2, Panc-1, SHG44, PLC-PRF-5, A375, SH-SY5Y, NCI-H446 cell.
Detailed description of the invention
The following examples can make those skilled in the art that the present invention is more fully understood, but limits the present invention never in any form.
The activity form (I) of medicine and the formula of prodrug (II) thereof are as follows:
Wherein R1For:C4-C6Alkyl, C1-C4Alkoxyl, C1-C6Alkenyl, C2-C10Alkynyl, C1-C4Alkanoyl, C1-C4Alkanoyloxy,
R2For:
R3For: Cl, Br, alkyl, C1-C6Alkenyl, C2-C10Alkynyl, C1-C4Alkanoyl, C1-C4Alkanoyloxy；R4For: H, OH, CF3Or methoxyl group；
X is: O, S or NH；N=0,1,2.
Synthesis step and the collection of illustrative plates of various compound are described below.The building-up process of dimethylamino-(phenyl-1H-tetrazole) isothiocyanate conjugates (representing with compound 1) is as follows:
Below each step is progressively described.
The synthesis of 2-[4-((1-phenyl-1H-tetrazole-5-base) sulfur) butyl] isoindoline-1,3-diketone (compound 1c)
In dry two mouthfuls of round-bottomed flasks of 50mL, solid powdery Feldalat NM 0.82g (15.03mmol) is joined 20mL heavily steam in methanol solution, after solution stirring is clarified, add compound 1-phenyl-1H-tetrazole-5-mercaptan 2.68g (15.03mmol), after being stirred at room temperature 20 minutes, add compound N-(4-brombutyl) phthalimide 4.00g (14.18mmol).By TLC (petroleum ether: ethyl acetate=3:1) detection reaction, after question response terminates, adding 10mL ultra-pure water to reactant liquor, after continuing stirring 10 minutes, rotation is except methanol, separate out the thick product of a large amount of white solid, being washed twice with water, the drying of 45 DEG C of vacuum drying oven put into by gained solid, obtains white solid product, weight is 4.58g, and productivity is 85.0%.
1H-NMR(400MHz,CDCl3) δ 7.86-7.82 (m, 2H), 7.74-7.70 (m, 2H), 7.59-7.52 (m, 5H), 3.74 (t, J=6.8Hz, 2H), 3.45 (t, J=6.8Hz, 2H), 1.93-1.84 (m, 4H), compound 1c's1HNMR figure figure 1 illustrates.
The synthesis of 4-[(1-phenyl-1H-tetrazole-5-base) sulfur] butyl-1-amine (compound 1d)
In two mouthfuls of round-bottomed flasks of 500mL, add compound 2-[4-((1-phenyl-1H-tetrazole-5-base) sulfur) butyl] isoindoline-1,3-diketone 7.59g (20.0mmol) and 300mL ethanol solution, add 5mL hydrazine hydrate solution (about 100mmol) under room temperature, stir 4 hours under the conditions of 50 DEG C.By TLC (dichloromethane: methanol=10:1,0.5% ammonia) reaction of 1,2,3-indantrione monohydrate color developing detection, question response is filtered to remove white solid after being fully completed, gained solid washing with alcohol twice, is spin-dried for solvent through Rotary Evaporators after merging mother solution, obtain oily liquids, after silicagel column (dichloromethane: methanol=40:1,0.5% ammonia) removes most hydrazine hydrate, obtain pale yellow oily liquid body, weight is 4.76g, and productivity is 95.4%.
1H-NMR(400MHz,CDCl3) δ 7.56-7.51 (m, 5H), 3.40 (t, J=7.6Hz, 2H), 2.79 (t, J=6.8Hz, 2H), 2.67 (br, 2H), 1.94-1.86 (m, 2H), 1.68-1.61 (m, 2H), compound 1d's1HNMR figure figure 2 illustrates.
The synthesis of 5-[(4-isothiocyanate butyl) sulfur]-1-phenyl-1H-tetrazole (compound 1e)
In two mouthfuls of round-bottomed flasks of 250mL, add compound 4-[(1-phenyl-1H-tetrazole-5-base) sulfur] butyl-1-amine 1.15g (4.61mmol), 1M NaOH aqueous solution 7.7mL (7.65mmol) and 200mL dichloromethane solution, after reactant liquor is down to 0 DEG C of stirring 10 minutes in low-temp reaction is bathed, rapidly joining thiophosgene 0.80g (6.92mmol), reactant liquor moves to room temperature and continues stirring 2 hours after stirring 10 minutes.By TLC (petroleum ether: ethyl acetate=2:1) detection reaction, after question response is fully completed, adding saturated sodium-chloride water solution in solution, extract and separate organic facies, after saturated sodium-chloride water solution washs, organic facies anhydrous sodium sulfate is dried, rotation is except solvent, and gained faint yellow solid is through silica column purification (petroleum ether: ethyl acetate=10:1), and obtaining product is faint yellow solid, weight is 1.09g, and productivity is 81%.
1H-NMR(400MHz,CDCl3) δ 7.58-7.54 (m, 5H), 3.59 (t, J=6.4Hz, 2H), 3.43 (t, J=6.8Hz, 2H), 2.03-1.96 (m, 2H), 1.90-1.83 (m, 2H)；13C-NMR(100 MHz,CDCl3) δ 154.0,133.6,130.2,123.0,129.9,123.9,44.5,32.3,28.9,26.4, compound 1e's1HNMR figure and13CNMR figure illustrates the most in figs. 3 and 4.
The synthesis of dimethylamino-(phenyl-1H-tetrazole) isothiocyanate conjugates (compound 1)
In two mouthfuls of round-bottomed flasks of 5mL; add compound 2-dimethylamino thioglycolic hydrochlorate 100mg (0.706mmol) and 1mL95% ethanol; it is subsequently added compound 5-[(4-isothiocyanate butyl) sulfur]-1-phenyl-1H-tetrazole 171mg (0.588mmol); with the sodium hydrate aqueous solution of 1M, reactant liquor being adjusted to about pH6, reactant liquor is warming up to 40 DEG C under argon shield and is stirred overnight.By TLC (dichloromethane: methanol=10:1,0.5% ammonia) detection reaction.After question response is complete, the sodium hydrate aqueous solution dripping 1M in reactant liquor adjusts pH to alkalescence, being spin-dried for by reactant liquor, gained surplus materials is dissolved in dichloromethane, respectively washes twice through saturated sodium carbonate solution and saturated aqueous common salt, gained organic facies anhydrous sodium sulfate concentrates after drying, obtaining faint yellow oily crude product, with silica column purification (dichloromethane: methanol=150:1), obtaining product is oily thick liquid, weight is 27.3mg, and productivity is 20.0%.
1H-NMR(400MHz,CDCl3) δ 11.07 (br, 1H), 7.55-7.51 (m, 5H), 3.71-3.66 (m, 2H), 3.39 (t, J=7.2Hz, 2H), 3.01 (t, J=5.2Hz, 2H), (2.72 t, J=5.6Hz, 2H), 2.30 (s, 6H), 1.95-1.87 (m, 2H), 1.82-1.75 (m, 2H)；13C-NMR(100MHz,CDCl3) δ 196.5,154.3,133.5,130.2,129.8,123.8,60.9,46.2,45.2,34.1,32.7,27.2,26.7, compound 11HNMR figure and13CNMR figure illustrates the most in fig. 5 and fig..
The building-up process of 2-(dimethylamino) ethyl [4-(methylsulfinyl) butyl] dithio formate (compound 2) is as follows:
In two mouthfuls of round-bottomed flasks of 25mL; add compound 2-dimethylamino thioglycolic hydrochlorate 240mg (1.69mmol) and 10mL 95% ethanol solution; it is subsequently added compound Sulforaphane 330mg (1.86mmol); with the sodium hydrate aqueous solution of 1M, reactant liquor being adjusted to about pH6, reactant liquor is warming up to 40 DEG C under argon shield and stirs 12 hours.By TLC (dichloromethane: methanol=10:1,0.5% ammonia) detection reaction, find reaction the most not exclusively, increase in time and there will be more side reaction product.The sodium hydrate aqueous solution dripping 1M in reactant liquor adjusts pH to alkalescence, reactant liquor is spin-dried for, gained surplus materials is dissolved in dichloromethane, respectively washing twice through saturated sodium carbonate solution and saturated aqueous common salt, gained organic facies anhydrous sodium sulfate concentrates after drying, obtains faint yellow oily crude product, with silica column purification (dichloromethane: methanol=150:1), obtaining product is oily thick liquid, and weight is 56.9mg, and productivity is 11.9%.
1H-NMR(400MHz,CDCl3) δ 11.06 (br, 1H), 3.72-3.68 (m, 2H), 3.05 (t, J=5.2Hz, 2H), 2.76-2.72 (m, 4H), 2.57 (s, 3H), 2.34 (s, 6H), 1.90-1.80 (m, 4H)；13C-NMR(100MHz,CDCl3) δ 196.6,60.7,53.9,46.3,45.2,38.8,33.8,29.0,27.4,20.2, compound 21HNMR figure and13CNMR figure illustrates the most in figures 7 and 8.
The building-up process of 2-(dimethylamino) ethyl-3-sulfydryl-2-methyl-propyl-(4-((benzothiazole-2-base) sulfur) butyl) dithiocarbamate (compound 3) is as follows:
The synthesis of 2-(dimethylamino) ethyl-3-(ethanethioyl)-2 Methylpropionic acid ester (compound 3c)
In the two-mouth bottle of a 25mL, acetyl mercapto isopropylformic acid. 200mg (1.23mmol) is dissolved in 15mL dichloromethane, under the conditions of 0 DEG C, add N, N-dimethylamino naphthyridine 15mg (0.123mmol) and 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDCI) 284mg (1.48mmol), 0 DEG C is kept to stir 30 minutes, it is subsequently adding dimethylaminoethanol 121mg (1.36mmol), reaction system is naturally raised to room temperature and continues stirring reaction 6h, by TLC detection reaction (dichloromethane: methanol=20:1,0.5%NH3·H2O), after question response is complete, with Rotary Evaporators rotation except solvent, obtain crude product, through silica gel column chromatography purification (dichloromethane: methanol=60:1,0.5%NH3·H2O) obtaining yellow viscous liquid, weight is 250mg, and yield is 86.9%.
1H-NMR (400MHz, CDCl3) δ 4.22 (t, J=5.8Hz, 2H), 3.14 3.03 (m, 2H), 2.72 (td, J=7.3,6.2Hz, 1H), 2.62 (t, J=5.8Hz, 2H), 2.32 (s, 9H), 1.24 (d, J=7.1Hz, 3H)；13C-NMR (100MHz, CDCl3) δ 195.30,174.64,62.30,57.44,45.43,39.86,31.73,30.58,16.76, compound 3c's1HNMR figure and13CNMR figure illustrates the most in figure 9 and in figure 10.
The synthesis of 2-(dimethylamino) ethyl-3-(sulfydryl)-2 Methylpropionic acid ester (compound 3d)
In the two-mouth bottle of a 25mL, 2-(dimethylamino) ethyl-3-acetyl mercapto-2-methyl propyl ester 160mg (0.69mmol) is dissolved in 10mL methanol, add sodium bicarbonate 69.1mg (0.82mmol), reaction 7h is stirred at room temperature, by TLC detection reaction (dichloromethane: methanol=20:1,0.5%NH3·H2O), after question response is complete, with Rotary Evaporators rotation except solvent, gained grease is dissolved in 10mL dichloromethane, adds 10mL H2O, extraction, organic facies anhydrous sodium sulfate is dried, and filters, and with Rotary Evaporators rotation except solvent, obtains crude product, through silica gel column chromatography purification (dichloromethane: methanol=20:1,0.5%NH3·H2O) obtaining yellow viscous liquid, weight is 108mg, and productivity is 82.3%.
1H-NMR(400MHz,CDCl3) δ 4.29 4.18 (m, 2H), 2.81 2.62 (m, 3H), 2.59 (d, J=5.7Hz, 2H), 2.30 (s, 6H), 1.24 (d, J=6.8Hz, 3H)；13C-NMR(100MHz,CDCl3) δ 174.59,62.20,57.74,45.56,43.11,27.72,16.69, compound 3d's1HNMR figure and13CNMR schemes respectively shown in Figure 11 and Figure 12.
The synthesis of 2-(dimethylamino) ethyl-3-sulfydryl-2-methyl-propyl-(4-((benzothiazole-2-base) sulfur) butyl) dithiocarbamate (compound 3)
In the two of a 10mL in bottle; 5-((4-isothiocyanate butyl) sulfur)-1-phenyl-1H-tetrazole 100mg (0.34mmol) is dissolved in 3mL dichloromethane; add dimethylamino naphthyridine 13.5mg (0.11mmol) and 2-(dimethylamino) ethyl-3-sulfydryl-2-methyl propyl ester 47mg (0.25mmol); system is stirred at room temperature reaction 10h under argon shield; by TLC detection reaction (dichloromethane: methanol=20:1,0.5%NH3·H2O), after question response is complete, with Rotary Evaporators rotation except solvent, obtaining crude product, obtain clear yellow viscous solid through silica gel column chromatography purification (dichloromethane: methanol=50:1), weight is 105mg, and productivity is 88.7%.
1H-NMR(400MHz,CDCl3) δ 8.00 (s, 1H), 7.57 (s, 5H), 4.34 4.13 (m, 2H), 3.82 (dd, J=12.3,6.8Hz, 2H), 3.57 3.32 (m, 4H), 2.96 2.84 (m, 1H), 2.73 2.58 (m, 2H), 2.32 (d, J=12.8Hz, 6H), 1.98-1.91 (m, 2H), 1.89 1.78 (m, 2H), 1.35 1.17 (m, 2H)；13C-NMR(100MHz,CDCl3) δ 197.37,175.15,154.36,133.52,129.83,123.82,62.40,57.58,46.22,45.63,39.95,37.81,32.48,26.96,26.64,17.03, compound 31HNMR figure and13CNMR figure illustrates the most in figs. 13 and 14.
The building-up process of 2-(dimethylamino) ethyl-3-sulfydryl-2-methyl-propyl-(4-(methylsulfinyl) butyl) dithio formate (compound 4) is as follows:
In the two of a 10mL in bottle; Sulforaphane 60.3mg (0.34mmol) is dissolved in 5mL dichloromethane; add N; N-dimethylamino naphthyridine 13.5mg (0.11mmol) and 2-(dimethylamino) ethyl-3-sulfydryl-2-methyl propyl ester 47mg; (0.25mmol); system is stirred at room temperature reaction 10h under argon shield, by TLC detection reaction (dichloromethane: methanol=20:1,0.5%NH3·H2O), after question response is complete, with Rotary Evaporators rotation except solvent, obtaining crude product, obtain clear yellow viscous solid through silica gel column chromatography purification (dichloromethane: methanol=50:1), weight is 105mg, and productivity is 88.7%.
1H-NMR (400MHz, CDCl3) δ 8.00 (s, 1H), 7.57 (s, 5H), 4.34 4.13 (m, 2H), 3.82 (dd, J=12.3,6.8Hz, 2H), 3.57 3.32 (m, 4H), 2.96 2.84 (m, 1H), 2.73 2.58 (m, 2H), 2.32 (s, 6H), 1.98-1.91 (m, 2H), 1.89 1.78 (m, 2H), 1.35 1.17 (m, 2H)；13C-NMR(100MHz,CDCl3)δ197.37,175.15,154.36,133.52,129.83,123.82,62.40,57.58,46.22,45.63,39.95,37.81,32.48,
26.96,26.64,17.03, compound 41HNMR figure and13CNMR schemes respectively shown in Figure 15 and Figure 16.
The building-up process of 2-(2-(2-(dimethylamino) ethyoxyl) ethyoxyl) ethylmercapto group-(4-((benzothiazole-2-base) sulfur) butyl) dithiocarbamate (compound 5) is as follows:
The synthesis of 2-(2-(2-(dimethylamino) ethyoxyl) ethyoxyl) ethyl chloride (compound 5b)
In the two-mouth bottle of a 50mL, 2-(2-(2-(dimethylamino) ethyoxyl) ethyoxyl) ethanol 410mg (2.31mmol) is dissolved in 20mL chloroform, reaction system is placed in 0 DEG C, it is slowly added dropwise thionyl chloride 825.6mg (6.94mmol), drip complete, reaction system is heated to 70 DEG C of reaction 1h, by TLC detection reaction (dichloromethane: methanol=5:1,0.5%NH3·H2O), after question response is complete, adding 20mL saturated sodium carbonate solution, extraction, organic facies anhydrous sodium sulfate is dried, and filters, and filtrate is spin-dried for Rotary Evaporators, obtains combining red liquid, and weight is 430mg, and productivity is 95%.
1H-NMR (400MHz, CDCl3) δ 3.74 (t, J=5.9Hz, 2H), 3.67 (m, 2H), 3.64 3.57 (m, 6H), 2.53 (t, J=5.8Hz, 2H), 2.28 (s, 6H)；13C-NMR (100MHz, CDCl3) δ 71.44,70.77,70.47,69.51,58.92,45.95,42.77, compound 5b's1HNMR figure and13CNMR schemes respectively shown in Figure 17 and Figure 18.
S-(2-(2-(2-(dimethylamino) ethyoxyl) ethyoxyl) ethyl) acetyl thioester (compound 5d) synthesizes
In the two-mouth bottle of a 50mL, 2-(2-(2-(dimethylamino) ethyoxyl) ethyoxyl) ethyl chloride 430mg (2.2mmol) is dissolved in 25mL N, N-dimethylamino Methanamide, add thioacetic acid potassium 301mg (2.64mmol), reaction system is placed in 75 DEG C of reaction 12h, by TLC detection reaction (dichloromethane: methanol=5:1,0.5%NH3·H2O), after question response is complete, it is spin-dried for solvent with Rotary Evaporators, the red comprehensive color thick liquid of gained is dissolved in 20mL dichloromethane, adds 1M hydrochloric acid regulation pH=1, adds 10mL water, extraction, collect aqueous phase, add saturated sodium carbonate solution and adjust pH=10, add dichloromethane (3 × 20mL) extraction, merge organic facies, being dried with anhydrous sodium sulfate, filter, filtrate is spin-dried for Rotary Evaporators, obtain crude product, obtaining combining red liquid through silica gel column chromatography purification (dichloromethane: methanol=10:1), weight is 345mg, and productivity is 66.7%.
1H-NMR (400MHz, CDCl3) δ 3.72 (t, J=5.4Hz, 2H), 3.61 (s, 4H), 3.58 (t, J=6.5Hz, 2H), 3.07 (t, J=6.4Hz, 2H), 2.77 (t, J=5.3Hz, 2H), 2.48 (s, 6H), 2.32 (s, 3H)；13C-NMR (100MHz, CDCl3) δ 195.58,70.44,70.25,69.82,68.06,58.17,45.14,30.68,28.90, compound 5d's1HNMR figure and13CNMR schemes respectively shown in Figure 19 and Figure 20.
The synthesis of 2-(2-(2-(dimethylamino) ethyoxyl) ethyoxyl) ethyl mercaptan (compound 5e)
In the two-mouth bottle of a 25mL; S-(2-(2-(2-(dimethylamino) ethyoxyl) ethyoxyl) ethyl) acetyl thioester 250mg (1.06mmol) is dissolved in 10mL methanol; add hydrazine hydrate 271.3g (5.31mmol); reaction system is stirred at room temperature 3h under argon shield; by TLC detection reaction (dichloromethane: methanol=5:1,0.5%NH3·H2O), after question response is complete, it is spin-dried for solvent with Rotary Evaporators, adds 10mL saturated sodium carbonate solution and dichloromethane, extraction, organic facies anhydrous sodium sulfate is dried, filtering, filtrate is spin-dried for Rotary Evaporators, obtains crude product, through silica gel column chromatography purification (dichloromethane: methanol=10:1,0.5%NH3·H2O) obtaining combining red liquid, weight is 105mg, and productivity is 51.1%.
1H-NMR (400MHz, CDCl3) δ 3.60 (m, 8H), 2.69 (d, J=5.6Hz, 2H), 2.53 (t, J=5.8Hz, 2H), 2.28 (s, 6H), 1.60 (br, 1H)；13C-NMR (100MHz, CDCl3) δ 73.00,70.45,70.35,69.39,58.88,45.95,24.39, compound 5e's1HNMR figure and13CNMR schemes respectively shown in Figure 21 and Figure 22.
The synthesis of 2-(2-(2-(dimethylamino) ethyoxyl) ethyoxyl) ethylmercapto group-(4-((benzothiazole-2-base) sulfur) butyl) dithiocarbamate (compound 5)
In the two of a 10mL in bottle; 5-((4-isothiocyanate butyl) sulfur)-1-phenyl-1H-tetrazole 100mg (0.34mmol) is dissolved in 3mL dichloromethane; add triethylamine 11.6mg (0.11mmol) and 2-(2-(2-(dimethylamino) ethyoxyl) ethyoxyl) ethyl mercaptan 44mg (0.23mmol); system is stirred at room temperature reaction 10h under argon shield; by TLC detection reaction (dichloromethane: methanol=20:1,0.5%NH3·H2O), after question response is complete, with Rotary Evaporators rotation except solvent, obtaining crude product, obtain clear yellow viscous solid through silica gel column chromatography purification (dichloromethane: methanol=50:1), weight is 78mg, and productivity is 70.3%.
1H-NMR (400MHz, CDCl3) δ 8.51 (s, 1H), 7.57 (s, 5H), 3.77 (s, 4H), 3.69 3.64 (m, 2H), 3.61 (s, 4H), 3.41 (d, J=7.1Hz, 4H), 2.59 (t, J=5.7Hz, 2H), 2.33 (s, 6H), 1.95 (s, 2H), 1.81 (s, 2H)；13C-NMR (100MHz, CDCl3) δ 197.37,154.38,133.61,129.89,123.89,70.55,70.45,70.15,68.97,58.75,46.12,45.66,35.26,32.57,27.05,26.71, compound 51HNMR figure and13CNMR schemes respectively shown in Figure 23 and Figure 24.
The building-up process of 2-(2-(2-(dimethylamino) ethyoxyl) ethyoxyl) ethylmercapto group-(4-(methylsulfinyl) butyl) dithio formate (compound 6) is as follows:
In the two of a 25mL in bottle; Sulforaphane 280mg (1.58mmol) is dissolved in 15mL dichloromethane; add triethylamine 26.6mg (0.26mmol) and 2-(2-(2-(dimethylamino) ethyoxyl) ethyoxyl) ethyl mercaptan 254.4mg (1.32mmol); system is stirred at room temperature reaction 10h under argon shield; by TLC detection reaction (dichloromethane: methanol=20:1,0.5%NH3·H2O), question response completely after, with Rotary Evaporators rotation except solvent, obtain crude product, obtain clear yellow viscous solid through silica gel column chromatography purification (dichloromethane: methanol=50:1), weight be 250mg productivity be 64.5%.
1H-NMR (400MHz, CDCl3) δ 8.66 (s, 1H), 3.75 (t, J=5.8Hz, 4H), 3.69 3.58 (m, 6H), 3.37 (t, J=5.7Hz, 2H), 2.79 2.71 (m, 2H), 2.64 (t, J=5.7Hz, 2H), 2.58 (s, 3H), 2.35 (s, 6H), 1.87 (br, 4H)；13C-NMR (100MHz, CDCl3) δ 197.35,70.45,70.40,68.78,58.63,53.72,46.35,45.59,42.05,38.68,35.20,27.20,20.22, compound 61HNMR figure and13CNMR schemes respectively shown in Figure 25 and Figure 26.
The building-up process of 2-(dimethylamino) ethyl (4-((4-bromophenyl) sulfur) butyl) dithiocarbamate (compound 7) is as follows:
In a 5mL two-mouth bottle, 2-dimethylamino thioglycolic hydrochlorate 121.0mg (0.854mmol) is dissolved in 2mL 95% ethanol, add (4-bromophenyl) (4-isothiocyanate butyl) thioether 284.0mg (0.940mmol), add 1mol/L sodium hydroxide solution, reactant liquor is adjusted to PH6, and reactant liquor is warming up to 40 DEG C and stirs 12 hours.By TLC detection reaction (DCM:MeOH=10:1,0.5%NH3·H2O), rotation is except solution, gained surplus materials is dissolved in dichloromethane, organic facies is washed through a small amount of saturated sodium carbonate solution and saturated aqueous common salt, gained organic facies anhydrous sodium sulfate concentrates after drying, through silica column purification (DCM:MeOH=100:1), obtains oily thick liquid, weight is 67.9mg, and productivity is 19.5%.
1H-NMR (400MHz, CDCl3) δ 11.04 (bs, 1H), 7.37 (d, J=8.4Hz, 2H), 7.16 (d, J=8.4Hz, 2H), 3.66-3.61 (m, 2H), 2.98 (t, J=4.8Hz, 2H), 2.92 (t, J=7.2Hz, 2H), 2.73 (t, J=6.4Hz, 2H), 2.29 (s, 6H), 1.77-1.65 (m, 4H)；13C-NMR (100MHz, CDCl3) δ 196.3,135.5,132.0,131.9,131.0,130.8,119.8,61.1,46.5,45.2,34.2,33.3,27.3,26.4, compound 71HNMR figure and13CNMR schemes respectively shown in Figure 27 and Figure 28.
The building-up process of 2-(dimethylamino) ethyl (4-((benzothiazole-2-base) sulfur) butyl) dithiocarbamate (compound 8) is as follows:
In a 5mL two-mouth bottle, 2-dimethylamino thioglycolic hydrochlorate 91.85mg (0.648mmol) is dissolved in 2mL 95% ethanol, add 2-((4-isothiocyanate butyl) sulfur) benzothiazole 200.0mg (0.713mmol), add 1mol/L sodium hydroxide solution, reactant liquor is adjusted to PH6, and reactant liquor is warming up to 40 DEG C and stirs 12 hours.By TLC detection reaction (DCM:MeOH=10:1,0.5%NH3·H2O), rotation is except solution, gained surplus materials is dissolved in dichloromethane, organic facies is washed through a small amount of saturated sodium carbonate solution and saturated aqueous common salt, gained organic facies anhydrous sodium sulfate concentrates after drying, through silica column purification (DCM:MeOH=90:1), obtains oily thick liquid, weight is 64.4mg, and productivity is 25.7%.
1H-NMR (400MHz, CDCl3) δ 11.05 (bs, 1H), 7.84 (d, J=8.0Hz, 1H), 7.73 (d, J=8.0Hz, 1H), 7.39 (t, J=8.0Hz, 1H), 7.28 (d, J=7.6Hz, 1H), 3.73-3.68 (m, 2H), (3.39 t, J=6.8Hz, 2H), (2.99 t, J=5.2Hz, 2H), (2.71 t, J=5.6Hz, 2H), 2.27 (s, 6H), 1.94-1.87 (m, 4H), 1.85-1.77 (m, 4H)；13C-NMR (100MHz, CDCl3) δ 196.3,166.7,153.2,135.2,126.1,124.3,121.5,121.0,61.0,46.6,45.2,34.1,32.9,27.2,27.0, compound 81HNMR figure and13CNMR schemes respectively shown in Figure 29 and Figure 30.
Slow release experimental procedure and collection of illustrative plates
Vitro stability part
Present invention also offers precursor compound stability experiment scheme in vitro, being included in the slowly releasing effect in pH7.4, pH5.4, pH4.4, pH3.4 buffer and rat plasma, experiment uses internal mark method determination precursor compound concentration change in 37 DEG C of buffer of constant temperature and blood plasma.First, make the standard curve (see Figure 31) of compound 1, compound 1e and carbamazepine, thus confirm the linear relationship of compound peaks area and concentration.The slow release experimental program of precursor compound is specifically described in detail as a example by compound 5, and remaining is similar or identical.Specifically comprise the following steps that
Step 1): with acetonitrile as solvents precision the preparation compound 1 of 50umol/L, 100umol/L, 200umol/L, 300umol/L, 400umol/L, 500umol/L, 600umol/L, compound 1e, carbamazepine solution respectively, HPLC sample introduction, measure the peak area of variable concentrations solution, it is repeated 3 times, average, make the standard curve (see Figure 31) of compound 1, compound 1e and carbamazepine.
Step 2): with acetonitrile as solvents precision preparation compound 5 solution of 600umol/L, the compound 1e solution of 600umol/L, the carbamazepine solution of 300umol/L respectively.
Step 3): take compound 5 solution, compound 1e solution and carbamazepine solution that 100uL newly prepares respectively, add 300uL acetonitrile (CCompound 5 =100umol/L,C1e=100umol/L, CCarbamazepine=50umol/L), HPLC sample introduction, measure peak area and be respectively SCompound 5、S1e、SCarbamazepine, by calculated by peak area correction factor f, (fCompound 5=CCompound 5*SCarbamazepine/(CCarbamazepine*SCompound 5), f1e=C1e*SCarbamazepine/(CCarbamazepine*S1e))。
Step 4): take the compound 5 solution (600umol/L) that 1mL newly prepares, add 9mL buffer and (add the PBS of pH7.4 and pH5.4 the most respectively；PH4.4 and pH3.4 acetic acid/sodium acetate buffer), now solution concentration is CCompound 5=60umol/L.
Step 5): the buffer (60umol/L) of compound 5 is put in the thermostat water bath of 37 DEG C, isothermal holding, take this buffer of 500uL in design time point, add 100uL carbamazepine solution (300umol/L) (now CCompound 5=50umol/L, CCarbamazepine=50umol/L), HPLC sample introduction, measure compound 5, compound 1e, the peak area S of carbamazepineCompound 5、S1e、SCarbamazepine, calculated the concentration (C of compound 5 the most in the same time by correction factor with internal standard methodCompound 5=fCompound 5*CCarbamazepine*SCompound 5/SCarbamazepine, C1e=f1e*CCarbamazepine*S1e/SCarbamazepine), 24h, repetitive operation 3 times, average, make the elution profiles figure (see Figure 34) of compound 5 altogether
Step 6): take rat artery blood, add anticoagulant, centrifugal 15min (4 DEG C, 12000rpm), take upper plasma, be dispensed into small test tube ,-20 DEG C of freezen protective are standby.
Step 7): the blood plasma of freezen protective is taken out from refrigerator, puts and the most naturally thaw, the plasma sample of the compound 5 of accurate preparation 600umol/L, the compound 1e acetonitrile solution of 300umol/L and the carbamazepine acetonitrile solution of 300umol/L.
Step 8): precision measures the compound 5 plasma sample (600umol/L) that 100uL newly prepares respectively, 200uL compound 1e acetonitrile solution (300umol/L), 200uL carbamazepine acetonitrile solution (300umol/L), adds 700uL protein precipitant acetonitrile (now CCompound 5=50umol/L, C1e=50umol/L, CCarbamazepine=50umol/L), vortex 1min, centrifugal 10min (4 DEG C, 12000rpm), take the supernatant, filtering with microporous membrane, HPLC sample introduction, measure peak area and be respectively SCompound 5、S1e、SCarbamazepine, by calculated by peak area correction factor f, (fCompound 5=CCompound 5*SCarbamazepine/(CCarbamazepine*SCompound 5), f1e=C1e*SCarbamazepine/(CCarbamazepine*S1e))。
Step 9): the compound 5 plasma sample (600umol/L) newly prepared is put into isothermal holding in 37 DEG C of thermostat water baths, this plasma sample of 100uL is taken in design time point, add 200uL carbamazepine acetonitrile solution (300umol/L), add protein precipitant acetonitrile 900uL, vortex 1min, centrifugal 10min (4 DEG C, 12000rpm), take the supernatant, filtering with microporous membrane, HPLC sample introduction, measures the peak area S of compound 5, compound 1e, carbamazepineCompound 5、S1e、SCarbamazepine, calculated the concentration (C of compound 5 and compound 1e the most in the same time by correction factor with internal standard methodCompound 5=fCompound 5*CCarbamazepine*SCompound 5/SCarbamazepine, C1e=f1e*CCarbamazepine*S1e/SCarbamazepine), 24h, repetitive operation 3 times, average, make the decomposition curve figure (see Figure 34) of compound 5 altogether.
The chromatographic condition wherein used is as follows, and flow phase: water/acetonitrile=30/70 (wherein adds 2 ‰ trifluoroacetic acids, pH1.35) in water；Flow velocity: 1mL/min；Column temperature: 25 DEG C；Detection wavelength: 240nm；Sample size: 20uL.
Compound 5 is stable existence in the acetonitrile solution that pH is 5.4, along with the rising of pH is constantly decomposed into active component i.e. compound 1e；When pH is 7.4, in 7h, compound 5 and compound 1e reach poised state；Compound 1e is stable existence in acetonitrile dissolves；Compound 5 about 10h in blood plasma is completely decomposed into active component i.e. compound 1e, and compound 1e is almost metabolism completely in blood plasma in 10h.
Figure 32 shows the decomposition curve under compound 1 different condition in vitro.The method making decomposition curve is similar with compound 5, is not repeated at this.
Compound 1 in acid condition can stable existence, along with the rising of pH is constantly decomposed into active component i.e. compound 1e；In 10h, active component i.e. compound 1e it is completely decomposed into when pH is 7.4；Compound 1e is stable existence in acetonitrile dissolves；Compound 1 about 100min in blood plasma is completely decomposed into active component i.e. compound 1e, and compound 1e is almost metabolism completely in blood plasma in 6h.
Figure 33 shows the decomposition curve under compound 3 different condition in vitro.The method making decomposition curve is similar with compound 5, is not repeated at this.
Compound 3 is stable existence in the acetonitrile solution that pH is 3.4, along with the rising of pH, is constantly decomposed into active component i.e. compound 1e；It is decomposed into active component i.e. compound 1e and midbody compound 1e* when pH is 7.4, and in 7h, reaches balance；Compound 1e is stable existence in acetonitrile dissolves；In blood plasma, about 25h is completely decomposed into active component i.e. compound 1e and midbody compound 1e*, and midbody compound 1e* is the most constantly converted to compound 1e, forms a balance.The title of midbody compound 1e* and structure such as following formula:
2-methyl-3-(4-((benzothiazole-2-base) sulfur) butyl) dithiocarbamates propanoic acid
Figure 35 shows the decomposition curve under compound 6 different condition in vitro.The method making decomposition curve is similar with compound 1, is not repeated at this.
Compound 6 is stable existence in the acetonitrile solution that pH is 4.4, along with the rising of pH is constantly decomposed into active component i.e. compound 2a；When pH is 7.4, in 25h, compound 6 is not completely decomposed into compound 2a yet；Compound 2a is stable existence in acetonitrile dissolves；In blood plasma, about 25h is completely decomposed into active component i.e. compound 2a, and compound 2a is almost metabolism completely in blood plasma in 25h.
It is found through experiments, R2The slow release situation of the compound that structure is identical is similar, wherein, the slow release situation of compound 2, compound 7 and compound 8 is similar to compound 1, the slow release situation of compound 4 is similar to compound 3, and the description of the slow release situation of compound 2, compound 4, change compound 7 and compound 8 is not repeated at this.
Active testing and collection of illustrative plates thereof
Pharmacologically active part
The present invention uses MTT colorimetric method for determining cytoactive.
MTT colorimetry is a kind of method detecting cell survival and growth.Its Cleaning Principle is that the succinate dehydrogenase in living cells mitochondrion can make exogenous MTT be reduced to water-insoluble bluish violet crystallization first a ceremonial jade-ladle, used in libation (Formazan) and be deposited in cell, and dead cell is without this function.Dimethyl sulfoxide (DMSO) can dissolve the first a ceremonial jade-ladle, used in libation in cell, measures its absorbance value with enzyme-linked immunosorbent assay instrument at 490nm wavelength, can indirectly reflect living cells quantity.In the range of certain cell number, the amount that the crystallization of first a ceremonial jade-ladle, used in libation is formed is directly proportional to cell number.
The entitled 3-of chemistry (4,5-dimethylthiazole-2)-2 of MTT, 5-diphenyltetrazolium bromide bromide, trade name tetrazolium bromide, is the dyestuff of a kind of yellow color.
MTT powder is bought in Sigma company, and during use, phosphate buffer (PBS) is configured to the solution that concentration is 5mg/ml, with 0.22 μm membrane filtration to remove the antibacterial in solution, then keeps in Dark Place at 4 DEG C.Specifically as a example by the sum159 cell of compound 1, remaining compound carries out similar or identical process.
MTT colorimetric method for determining cytoactive includes following several step:
Step 1): dosing spreads 96 orifice plates with sum159 cell (purchased from gold amethyst bio tech ltd, Beijing) noon before that day.Collect the sum159 cell of exponential phase, adjust cell concentration after viable count to 2.5 × 104cells/mL.Inoculating cell in 96 orifice plates, every hole adds 100 μ L cell suspension bed boards, and final cell to be measured is 2500cells/ hole.Surrounding marginal pore not inoculating cell, only add 100 μ L cell culture mediums (cell culture medium used in this experiment is modified form RPMI-1640 (Hyclone) basal medium, adds the hyclone (Hyclone) of 10%).5%CO2, 37 DEG C of overnight incubation, so that cell is the most adherent.
Step 2): dosing in morning next day.First dilute medicine, prepare corresponding drug concentration gradient.The cell of 96 orifice plates completed to the previous day adds the medicine of the 100 corresponding concentration of μ L, being provided with 9 Concentraton gradient for sum159 cell in this experiment, system Chinese medicine final concentration gradient is: 300 μMs, 100 μMs, 33.3333 μMs, 11.1111 μMs, 3.7037 μMs, 1.2345 μMs, 0.4115 μM, 0.1371 μM, 0.04572 μM.Each concentration arranges 5 repetitions.The hole simultaneously arranging not dosing only inoculating cell is matched group, and the cell culture medium of 100 μ L is added in matched group not dosing；Arrange non-inoculating cell only to add the hole of culture medium and be set to blank well, also add 100 μ L cell culture mediums.5%CO2, 37 DEG C of incubators hatch X (X=24,48,72,96) hour.
Step 3): after X hour, every hole adds 20 μ L MTT solution (5mg/ml, MTT), continues to cultivate 4 hours.If medicine can react with MTT, can first be centrifuged and discard culture fluid afterwards, after carefully rinsing 2-3 time with PBS, add the culture fluid containing MTT.
Step 4): terminate after 4 hours cultivating, carefully suck liquid in hole.Every hole adds 150 μ L dimethyl sulfoxide, and 37 DEG C of incubators hatch 10 minutes.Enzyme-linked immunosorbent assay instrument MULTISKAN FC (Thermo scientific) is used to measure the light absorption value in each hole at 490nm, using blank well as zeroing hole during measurement.
Step 5): process data.Initially with following equation calculating suppression ratio:
Suppression ratio=1-dosing group OD value/matched group OD value
Then with Log C (drug level logarithm) as abscissa, suppression ratio is vertical coordinate, carry out probit weighted regression method (Bliss method) with data processing software SPSS software (IBM Corporation) and carry out data process, mapping, obtain IC50Value.
According to above-mentioned method of testing, record each compound at 48 hours suppression IC to MCF-7, SUM-159, KG1a and 297T cell50And IC90And the inhibitory action that compound 1 is to other various kinds of cell such as HELA, HEPG2, PANC-1, it is shown in Table 1, table 2 and Figure 36 to Figure 44, the active substance gone out with compound 2a and 1e for slow release the most respectively, for positive standard specimen:
Table 1----MCF-7, SUM-159, KG1a and 293T active testing result
The table 2----compound 1 active testing result to various cells
As described above, it is appreciated that, the compounds of this invention can slowly release reactive compound, it is thus possible to effectively suppress survival and the growth of the tumor cells such as MCF-7, SUM-159, KG1a, HELA, HEPG2, PANC-1, SHG44, PLC/PRF5, A375, SH-SY5Y, NCI-H446, and drug resistance is low, toxic and side effects is little.It may be used for the cancer such as prevention or treatment breast carcinoma, leukemia, proliferating epidermal cancer, hepatocarcinoma, cancer of pancreas, cerebral glioma, hepatocarcinoma, cutaneous melanoma, neuroblastoma, pulmonary carcinoma.
It will be understood by those skilled in the art that above example is only exemplary embodiment, without departing from the spirit and scope of the present invention, multiple change can be carried out, replace and change.
Claims (11)
1. a compound, the formula of described compound is:
Wherein R1For:C4-C6Alkyl, C1-C4Alkoxyl, C1-C6
Alkenyl, C2-C10Alkynyl, C1-C4Alkanoyl, C1-C4Alkanoyloxy,
R2For:
R3For: Cl, Br, alkyl, C1-C6Alkenyl, C2-C10Alkynyl, C1-C4Alkanoyl, C1-C4Alkane
Acyloxy；
R4For: H, OH, CF3Or methoxyl group；
X is: O, S or NH；
N=0,1 or 2.
Compound the most according to claim 1 is for preventing or treating the application in terms of cancer.
Application the most according to claim 2, wherein, described cancer include breast carcinoma, leukemia,
Proliferating epidermal cancer, hepatocarcinoma, cancer of pancreas, cerebral glioma, hepatocarcinoma, cutaneous melanoma, neural mother are carefully
Born of the same parents' tumor, pulmonary carcinoma.
4. a preparation method for compound, described method includes:
Add 2-dimethylamino thioglycolic hydrochlorate and ethanol solution in a reservoir；
It is subsequently added 5-[(4-isothiocyanate butyl) sulfur]-1-phenyl-1H-tetrazole；
With alkaline solution, the pH value of reactant liquor being adjusted to 5-7, described reactant liquor is under inert gas shielding
It is stirred overnight；
In described reactant liquor, drip alkaline solution pH value is adjusted to alkalescence；
Being spin-dried for by described reactant liquor, gained surplus materials is dissolved in dichloromethane, and washs, by institute
Organic facies is dried and concentrates thus obtains the thick product of described compound；And
The thick product of described compound is purified.
5. a preparation method for compound, described method includes:
Add 2-dimethylamino thioglycolic hydrochlorate and ethanol solution in a reservoir；
It is subsequently added Sulforaphane；
With alkaline solution, the pH value of reactant liquor is adjusted to 5-7, is stirred overnight under inert gas shielding；
In described reactant liquor, drip alkaline solution pH value is adjusted to alkalescence；
Being spin-dried for by described reactant liquor, gained surplus materials is dissolved in dichloromethane, and washs, by institute
Organic facies is dried and concentrates thus obtains the thick product of described compound；And
The thick product of described compound is purified.
6. a preparation method for compound, described method includes:
5-((4-isothiocyanate butyl) sulfur)-1-phenyl-1H-tetrazole is dissolved in dichloromethane；
It is subsequently adding dimethylamino naphthyridine and 2-(dimethylamino) ethyl-3-sulfydryl-2-methyl propyl ester；
System is stirred at room temperature under inert gas shielding reacts；
Rotation, except solvent, obtains the thick product of described compound；And
The thick product of described compound is purified.
7. a preparation method for compound, described method includes:
Sulforaphane is dissolved in dichloromethane；
It is subsequently adding N, N-dimethylamino naphthyridine and 2-(dimethylamino) ethyl-3-sulfydryl-2-methyl propyl ester；
System is stirred at room temperature under inert gas shielding reacts；
Rotation, except solvent, obtains the thick product of described compound；And
The thick product of described compound is purified.
8. a preparation method for compound, described method includes:
5-((4-isothiocyanate butyl) sulfur)-1-phenyl-1H-tetrazole is dissolved in dichloromethane；
It is subsequently adding triethylamine and 2-(2-(2-(dimethylamino) ethyoxyl) ethyoxyl) ethyl mercaptan；
System is stirred at room temperature under inert gas shielding reacts；
Rotation, except solvent, obtains the thick product of described compound；And
The thick product of described compound is purified.
9. a preparation method for compound, described method includes:
Sulforaphane is dissolved in dichloromethane；
It is subsequently adding triethylamine and 2-(2-(2-(dimethylamino) ethyoxyl) ethyoxyl) ethyl mercaptan；
System is stirred at room temperature under inert gas shielding reacts；
Rotation, except solvent, obtains the thick product of described compound；And
The thick product of described compound is purified.
10. a preparation method for compound, described method includes:
2-dimethylamino thioglycolic hydrochlorate is dissolved in ethanol；
It is subsequently adding (4-bromophenyl) (4-isothiocyanate butyl) thioether；
By alkaline solution, the pH value of reactant liquor is adjusted to 5-7, described reactant liquor is stirred；
Rotation is except solution, and gained surplus materials is dissolved in dichloromethane, organic facies is washed, is dried and concentrates,
Obtain the thick product of described compound；And
The thick product of described compound is purified.
The preparation method of 11. 1 kinds of compounds, described method includes:
2-dimethylamino thioglycolic hydrochlorate is dissolved in ethanol；
It is subsequently adding 2-((4-isothiocyanate butyl) sulfur) benzothiazole；
By alkaline solution, the pH value of reactant liquor is adjusted to 5-7, described reactant liquor is stirred；
Rotation is except solution, and gained surplus materials is dissolved in dichloromethane, organic facies is washed, is dried and concentrates,
Obtain the thick product of described compound；And
The thick product of described compound is purified.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510191603.9A CN106146429A (en) | 2015-04-21 | 2015-04-21 | The structure of isosulfocyanate precursor compound, prepare and apply |
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| CN108354920A (en) * | 2018-01-10 | 2018-08-03 | 天津国际生物医药联合研究院 | A kind of application of sulfoxide compound in treating inflammatory bowel disease |
| CN108383766A (en) * | 2018-03-28 | 2018-08-10 | 北京化工大学 | The preparation and its application in inhibiting the drug of cancer cell multiplication and/or treating cancer of dithiocarbamates compound |
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