CN103265478A - Mercaptonicotinic acid compounds and preparation method thereof - Google Patents
Mercaptonicotinic acid compounds and preparation method thereof Download PDFInfo
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- CN103265478A CN103265478A CN2013101880432A CN201310188043A CN103265478A CN 103265478 A CN103265478 A CN 103265478A CN 2013101880432 A CN2013101880432 A CN 2013101880432A CN 201310188043 A CN201310188043 A CN 201310188043A CN 103265478 A CN103265478 A CN 103265478A
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Abstract
The invention belongs to the technical field of medicine, and provides substituted mercaptonicotinic acid compounds and a preparation method thereof. The structural general formula of the compounds is shown as formula I described in the specification, and the compounds comprise a pharmaceutically acceptable salt, ester and solvate. A substituted aniline compound is used as a raw material and reacted with chloroacetyl chloride or bromoacetyl chloride to generate amide, and then the amide is reacted with a mercapto-substituted nicotinic acid or isonicotinic acid compound to obtain the mercaptonicotinic acid compounds. The invention further provides an application of the compounds in a medicament.
Description
Technical field
The invention belongs to medical technical field, be specifically related to class sulfydryl niacin compound serving and preparation method thereof.
Background technology
The fusobacterium bacteriotoxin comprises botulinum neurotoxin and tetanus toxin.Tetanus toxin is produced in anaerobic environment by Clostridium tetanus bacillus fusiformis, can cause tetanus clinically.Botulinum neurotoxin (Clostridium Botulinum Neurotoxins, be called for short botulinus toxin) be one group of protein (comprising the A-G type) that known virulence is the strongest, mainly be to be produced in anaerobic environment by the Clostridium Clostridium botulinum, its intravenous injection medium lethal dose is about 1ng/kg, sucks mld and is about 3ng/kg.It mainly is to take place by digesting the generation active toxin owing to the food that eats pollution, wound infection generation toxin enter the toxin precursor that reaches in the body in the baby intestinal that common botulinus toxin is poisoned.In addition, botulinum toxin type A has been widely used in the clinical treatment of cholinergic nerve and muscle dysfunction, and is applied to face beauty and smoothing wrinkles, during treatment is used might because of over administration, misuse abuse and (or) whole body sausage poisoning symptom appears in untoward reaction.Sausage poisoning is shorter latent period, and course of disease development is fast, and the state of an illness is more serious, the case fatality rate height.What wherein cause human poisoning mainly is A, B, E, F type, and Type B is the strongest to people's neurotoxicity.The medicine of botulinum toxin type B poisoning prevention and treatment is the toxinicide horse serum, can be effective to the poisoning patient more than 80%, but there is significant side effects in this type of medicine, serum sickness and anaphylaxis (Black RE appear in about 9% case of clinical report, et al.Am J Med, 1980,69:567-570), seriously limited the antitoxic widespread use of horse serum.For solving the defective that this type of medicine exists, become the Recent study focus at botulinus toxin light chain design micromolecular inhibitor, Roques BP etc. develops and a class high reactivity sulfur-bearing polypeptide compounds, the most highly active compound reaches 2.3nM to the Ki value of botulinum toxin type B light chain, but do not see below the active report of continuous integral animal (Anne C, et al.J Med Chem, 2003,46:4648-4656; Bioorg Med Chem, 2003,11:4655-4660; Blommaert A, et al.Bioorg Med Chem, 2004,12:3055-3062).Janda KD etc. find that in 2011 the L-chicoric acid can significantly suppress the botulinum toxin type B light chain, and this compound also has to a certain degree inhibition activity (Salzameda NT, et al.Chem Commun, 2011,47:1713 – 1715) to the A hypotype.
Tetanus has similar pathogenic course with sausage poisoning, be to be combined by the Sphingolipids,sialo of heavy chain C end on neuron membrane of toxin, sour environment makes its structural rearrangement, and impel heavy chain N end to enter in the film, while light chain unfolding, disulfide linkage is reduced the back and changes cell over to as the zine ion metalloenzyme, substrate protein (A in the catalytic pyrolysis one class born of the same parents, E type botulinum neurotoxin acts on SNAP SNAP-25, B, D, F, G type botulinum neurotoxin and tetanus toxin act on synaptic vesicle related membrane protein VAMP), thereby influence the transfer of vagusstoff, intervene the conduction of nerve impulse, and cause motorius paralysis or excited.If in conjunction with, change over to and carry out the inhibition of one or several link with 3 links of catalysis or check, can effectively suppress the neurotoxicity of toxin.Become in recent years research focus and design for target spot and develop catalysis class inhibitor with the toxin light chain with enzymic activity.External high flux screening plants such as Harry B, ocean tissue, fungi natural extract produce the material that suppresses to the fusobacterium toxin, have found 30 kinds of non-hypotype inhibitor, and wherein 5 kinds of extracts can suppress Type B, E BOTULINUM TOXIN TYPE A A simultaneously.Smith LA etc. has found that in 2009 quinolinol derivative CB7969312 is a kind of potential inhibitor, the Zn catalysis region of the big hydrophobic region pocket by being attached to botulinum toxin type A light chain avtive spot, effectively and botulinum toxin type A to the toxicity (Roxas-Duncan of N2a cell, V, et al.Agents Chemother.2009,53:3478-3486; Pang, Y.-P.et al.PLos One2009,4, e7730.).Janda KD etc. found in 2010 lomofungin can suppress the botulinum toxin type A light chain (Ki value 6.7 ± 0.7uM), demonstrate typical noncompetitive kinetics (Eubanks, L.M., et al.ACS Med.Chem.Lett.2010,1:268-272.).The compound contratoxin light chain of these reports has the vitro inhibition effect, and some compound also has the toxic effect of pressing down at whole toxin and animal model level, but activity still has room for promotion.In addition, it is active that the most compounds of report has the special inhibition of toxin type at present, and the intersection restraining effect is still waiting to improve.
Therefore be necessary researchdevelopment one class active compound, vitro inhibition and the antagonistic action that can have wider scope to fusobacterium neurotoxin (many types of botulinus toxin and tetanus toxin), and can reveal higher antitoxin activity in the whole animal level body, therefrom research and develop new medicine.
Summary of the invention
The object of the present invention is to provide a class sulfydryl niacin compound serving and pharmacy acceptable salt thereof, ester, solvate; Another object of the present invention provides the preparation method of this compounds and pharmacy acceptable salt thereof, ester, solvate; The 3rd purpose of the present invention provides this compounds and pharmacy acceptable salt thereof, ester, solvate as the application of medicine.
A first aspect of the present invention provides a class sulfydryl niacin compound serving and pharmacy acceptable salt thereof, ester, solvate, and this structural general formula is suc as formula shown in the I:
(formula I)
Among the formula I:
R
1, R
2, R
3, R
4Represent in the following radicals any one independently: hydrogen, hydroxyl, amino, halogen, cyano group, diazanyl, azido-, low-grade alkyl amino, low alkyl group, low-grade halogenated alkyl, cycloalkyl, lower alkoxy, elementary halogenated alkoxy, low-grade alkenyl, rudimentary cyano group alkyl, rudimentary 4-nitro alkyl, amide group, rudimentary amido alkyl, rudimentary diazanyl alkyl, rudimentary azido-alkyl, (CH
2)
mNR
7R
8, (CH
2)
mOR
9, (CH
2)
mSR
9, (CH
2)
mNR
7C (O) R
9, (CH
2)
mC (O) R
9, (CH
2)
mOC (O) R
9, O (CH
2)
mNR
7R
8, OC (O) NR
7, OC (O) (CH
2)
mOC (O) R
9
Perhaps, R
1And R
2Carbon atom on the phenyl ring that they connect forms 5 or 6 yuan of rings;
Perhaps, R
2And R
3Carbon atom on the phenyl ring that they connect forms 5 or 6 yuan of rings;
Perhaps, R
3And R
4Carbon atom on the phenyl ring that they connect forms 5 or 6 yuan of rings;
The unit that forms described 5 or 6 yuan of rings is selected from CH, CH
2, O, S or NR
7
R
5, R
6Represent in the following radicals any one independently: hydrogen, hydroxyl, amino, halogen, cyano group, diazanyl, azido-, low-grade alkyl amino, low alkyl group, low-grade halogenated alkyl, lower alkoxy, low-grade alkenyl and (CH
2)
mCOOH;
R
7, R
8, R
9Represent hydrogen, low alkyl group or cycloalkyl independently;
M is the integer between 0 to 3;
In this article, the term " rudimentary " relevant with alkyl and alkoxyl group refers to contain the straight or branched saturated fatty alkyl of 1 to 6 carbon atom, for example: methyl, ethyl, propyl group, sec.-propyl, butyl, the tertiary butyl, methoxyl group and oxyethyl group; The term relevant with alkenyl " rudimentary " refers to contain the group of 2 to 6 carbon atoms and one or more pairs of keys, for example: vinyl, allyl group, isoolefine propyl group, pentenyl, hexenyl, propenyl.The term cycloalkyl refers to contain the ring of 3 to 7 carbon, for example: cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.Term halogen refers to chlorine, bromine, iodine or fluorine.Group corresponding to term low-grade halogenated alkyl, rudimentary cyano group alkyl, rudimentary 4-nitro alkyl, rudimentary amido alkyl, rudimentary diazanyl alkyl, rudimentary azido-alkyl refers to respectively be replaced by one to three halogen, cyano group, nitro, amide group, diazanyl, azido-.Low-grade alkyl amino can contain one or two low alkyl group, for example represents NHCH
3, NHCH
2CH
3, N (CH
3)
2Or CH
3NCH
2CH
3
More excellent, among the formula I:
R
1, R
2, R
3, R
4Represent in the following radicals any one independently: hydrogen, hydroxyl, amino, halogen, low alkyl group, low-grade halogenated alkyl, lower alkoxy and elementary halogenated alkoxy;
R
5, R
6Expression independently: hydrogen or (CH
2)
mCOOH;
M is the integer between 0 to 3.
More excellent, compound shown in the formula I is selected from following compound:
2-(2-(5-chloro-2-anisole amido) ethanoyl sulfo-) naotin,
2-(2-(5-chloro-2-anisole amido) ethanoyl sulfo-) nicotinic acid,
2-(2-(5-chloro-2-anisole amido) ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(3-chloro-5-anisole amido)-2-ethanoyl sulfo-) naotin,
2-(2-(2-phenetole amido) ethanoyl sulfo-) nicotinic acid,
2-(2-(5-chloro-2-phenetole amido) ethanoyl sulfo-) nicotinic acid,
2-(2-(5-chloro-2-propoxy-anilino) ethanoyl sulfo-) nicotinic acid,
2-(2-(2-chloroanilino) ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(2,5-dibromobenzene amido) ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(4-chloroanilino) ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(2,5-dichlorobenzene amido) ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(4-bromobenzene amido) ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(5-fluoro-2-anisole amido) ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(5-methoxyl group-2-chloroanilino) ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(5-chloro-2-anisole amido) ethanoyl sulfo-) Yi Yansuan potassium,
2-(2-(2,5-dimethoxy benzene amido) ethanoyl sulfo-) nicotinic acid potassium,
2-(anilino-2-ethanoyl sulfo-) nicotinic acid,
2-(2-(5-fluoro-2-chloroanilino)-2-ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(4-fluoroanilino) ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(5-chloroanilino) ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(4-trifluoro-methoxyaniline base) ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(2,5-difluoroaniline base) ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(5-chloro-2-benzylaniline base) ethanoyl sulfo-) naotin,
2-(2-(2-trifluoro-methoxyaniline base) ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(3-trifluoro-methoxyaniline base) ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(5-trifluoro methoxy-2-bromobenzene amido) ethanoyl sulfo-) nicotinic acid potassium, or
2-(2-(5-fluoro-2-phenetole amido) ethanoyl sulfo-) nicotinic acid.
Compound pharmacy acceptable salt shown in the above-mentioned formula I is can be all kinds of salt that pyridine substitution in ring carboxyl becomes with metal ion or amino-complex.Described salt can be the salt that forms with mineral acid or the salt that forms with organic acid, and the salt that forms with mineral acid is hydrochloride, vitriol, phosphoric acid salt, diphosphate, hydrophosphate, hydrobromate or nitrate etc.; The salt that forms with organic acid be acetate, maleate, fumarate, tartrate, succinate, lactic acid salt, Citrate trianion, tosilate, salicylate, oxalate, palmitate or stearate etc.
The pharmaceutically acceptable ester of compound shown in the above-mentioned formula I refers in vivo ester that can hydrolysis and comprises the ester that can be broken down into its parent compound or salt in human body easily, for example C1-C6 alkyl ester etc.
The pharmaceutically acceptable solvate of compound shown in the above-mentioned formula I refers to and the pharmaceutically acceptable solvent solvation form that forms such as water, ethanol, DMSO for example.
A second aspect of the present invention has provided the preparation method of above-mentioned sulfydryl niacin compound serving and pharmacy acceptable salt thereof, ester, solvate, and the preparation of compound shown in its Chinese style I comprises the steps:
A) make the reaction of the compound shown in the formula II and chloroacetyl chloride or bromoacetyl chloride, obtain compound shown in the formula III;
(formula II) (formula III)
R in formula II, the formula III
1, R
2, R
3, R
4Definition cotype I;
B) make the reaction of compound shown in compound shown in the formula III and the formula IV, obtain compound shown in the formula I;
(formula IV)
R in the formula IV
5, R
6Definition cotype I.
A third aspect of the present invention has provided above-mentioned sulfydryl niacin compound serving and pharmacy acceptable salt thereof, ester, solvate as the application of medicine.
One of application provided by the present invention be the sulfydryl niacin compound serving that replaces shown in the formula I or its pharmacy acceptable salt, ester, solvate and their mixture, preparation preparation prevent and/or treat that botulinus toxin exposes and/or the poisoning medicine in application.
Two of application provided by the present invention is sulfydryl niacin compound serving or the application in preparation botulinus toxin endopeptidase inhibitor of its pharmacy acceptable salt, ester, solvate and their mixture that replace shown in the formula I.
To be the sulfydryl niacin compound serving that replaces shown in the formula I or its pharmacy acceptable salt, ester, solvate and their mixture prevent and/or treat application in the tetanus medicine in preparation in three of application provided by the present invention.
Four of application provided by the present invention is sulfydryl niacin compound serving or the application in preparation tetanus toxin endopeptidase inhibitor of its pharmacy acceptable salt, ester, solvate and their mixture that replace shown in the formula I.
Botulinus toxin described in the present invention comprises botulinum toxin type A, botulinum toxin type B, E BOTULINUM TOXIN TYPE A A, F BOTULINUM TOXIN TYPE A A.
Be preventing and/or treating medicine that botulinus toxin exposes and/or poison, preventing and/or treating tetanic medicine and also all belong to protection scope of the present invention of activeconstituents preparation with compound shown in the formula I or its pharmacy acceptable salt, ester, solvate or their mixture.
Described medicine can import body such as muscle, intracutaneous, subcutaneous, vein, mucosal tissue by the method for injection, injection, collunarium, eye drip, infiltration, absorption, physics or chemistry mediation; Or mixed by other materials or wrap up the back and import body.
When needing, in said medicine, can also add one or more pharmaceutically acceptable carriers.Described carrier comprises thinner, vehicle, weighting agent, tackiness agent, wetting agent, disintegrating agent, absorption enhancer, tensio-active agent, absorption carrier, lubricant of pharmaceutical field routine etc.
Described medicine can be made various ways such as injection liquid, suspension agent, pulvis, tablet, granule.The medicine of above-mentioned various formulations all can be according to the ordinary method preparation of pharmaceutical field.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.Should be understood that following examples are only for explanation the present invention but not for limiting scope of the present invention.
Experimental technique described in the following embodiment if no special instructions, is ordinary method; Described reagent and material if no special instructions, all can obtain from commercial channels.
Embodiment 1,2-(2-(5-chloro-2-anisole amido) ethanoyl sulfo-) naotin (formula I-1) is synthetic
(formula I-1)
3.14 gram (0.02mol) 2-methoxyl group-5-chloroanilines are dissolved in 30 milliliters of Glacial acetic acid, drip 2.5 gram (0.02mol) chloroacetyl chlorides, stirred overnight at room temperature.Then reaction solution is poured in 50 milliliters of frozen water, stirred 1 hour, separate out solid, filter, washing, the dry crude product that gets.
To go up the step product and 1.55 gram (0.01mol) 2-sulfydryl nicotinic acid join in 25 milliliters of THF and the water mixed solution (volume ratio 1:1), be cooled to 0 degree, add sodium hydroxide 1.6 grams, stirring reaction, be warming up to room temperature, stirring is spent the night, and has a large amount of solids to separate out, and filters, washing, drying obtains off-white color solid 3.55 grams, yield 86%.
The structural identification result is as follows:
1H-NMR(DMSO-d6):10.08(s,1H),8.48-8.50(m,1H),8.28(d,1H),8.14-8.16(m,1H),7.20-7.22(m,1H),7.02-7.04(m,1H),6.98(d,1H),3.79(s,2H),3.74(s,3H).EI-MS:m/z:353.2[M+H]
+.
Through identifying that products therefrom really is target compounds of formula I-1.
Embodiment 2,2-(2-(5-chloro-2-anisole amido) ethanoyl sulfo-) nicotinic acid (formula I-2) is synthetic
(formula I-2)
2-(2-(5-chloro-2-anisole amido)-2-ethanoyl sulfo-with embodiment 1 preparation) naotin places water, uses 1M hydrochloric acid adjust pH to 2 then, filter, and washing, drying obtains white solid, yield 97%.
The structural identification result is as follows: 1H-NMR (DMSO-d6): 13.56(s, 1H), 9.75 (s, 1H), and 8.69-8.70 (m, 1H), 8.29-8.30 (m, 1H), 8.19(d, 1H), and 7.35-7.37 (m, 1H), 7.01-7.08 (m, 2H), 4.02 (s, 2H), 3.79 (s, 3H) .EI-MS:m/z:375.4[M+H]+.
Through identifying that products therefrom really is target compounds of formula I-2.
Embodiment 3,2-(2-(5-chloro-2-anisole amido) ethanoyl sulfo-) nicotinic acid potassium (formula I-3) is synthetic
(formula I-3)
Restrain 2-methoxyl group-5-chloroanilines with 3.14 and be dissolved in 30 milliliters of Glacial acetic acid, drip 2.5 gram chloroacetyl chlorides, stirred overnight at room temperature is poured reaction solution in 50 milliliters of frozen water then, stirs 1 hour, separates out solid, filters washing, the dry crude product that gets.
To go up the step product and 1.55 gram 2-sulfydryl nicotinic acid join in 25 milliliters of THF and the water mixed solution (volume ratio 1:1), be cooled to 0 degree, add potassium hydroxide 2.0 grams, stirring reaction, be warming up to room temperature, stirring is spent the night, and has a large amount of solids to separate out, and filters, washing, vacuum-drying obtains off-white color solid 2.62 grams, yield 67%.
The structural identification result is as follows:
1H-NMR(DMSO-d6):9.93(s,1H),8.57-8.58(m,1H),8.24(d,1H),8.19-8.21(m,1H),7.26-7.28(m,1H),6.99-7.06(m,2H),3.89(s,2H),3.77(s,3H).EI-MS:m/z:391.4[M+H]
+.
Through identifying that products therefrom really is target compounds of formula I-3.
Synthesizing of embodiment 4,2-(2-(3-chloro-5-anisole amido) ethanoyl sulfo-) naotin (formula I-4)
(formula I-4)
With reference to embodiment 1, substitute 2-methoxyl group-5-chloroaniline, yield 77% with 3-methoxyl group-5-chloroaniline.
The structural identification result is as follows: 1H-NMR (DMSO-d6): 9.97 (s, 1H), 8.48-8.51 (m, 1H), 8.25 (d, 1H), 8.12-8.15 (m, 1H), 7.32 (s, 1H), 7.05 (s, 1H), 6.95 (s, 1H), 3.77 (s, 2H), 3.71 (s, 3H) .EI-MS:m/z:353.0[M+H]
+.
Through identifying that products therefrom really is target compounds of formula I-4.
Embodiment 5,2-(2-(2-phenetole amido) ethanoyl sulfo-) nicotinic acid (formula I-5) is synthetic
(formula I-5)
With reference to embodiment 1 and 2, substitute 2-methoxyl group-5-chloroaniline, yield 84% with the 2-phenetidine.
The structural identification result is as follows:
1H-NMR(DMSO-d6):13.63(s,1H),9.31(s,1H),8.66(d,1H),8.29(d,1H),8.10(d,1H),7.31-7.33(m,1H),6.95-6.99(m,2H),6.86-6.88(m,1H),3.97-4.02(m,4H),1.16(t,3H).EI-MS:m/z:333.3[M+H]
+.
Through identifying that products therefrom really is target compounds of formula I-5.
Embodiment 6,2-(2-(5-chloro-2-phenetole amido) ethanoyl sulfo-) nicotinic acid (formula I-6) is synthetic
(formula I-6)
With reference to example 1 and example 2, substitute 2-methoxyl group-5-chloroaniline, yield 82% with 2-oxyethyl group-5-chloroaniline.
The structural identification result is as follows: 1H-NMR (DMSO-d6): 13.60(s, 1H), 9.43 (s, 1H), and 8.64-8.66 (m, 1H), 8.28-8.30 (m, 1H), 8.18 (d, 1H), and 7.32-7.34 (m, 1H), 6.98-7.05 (m, 2H), 4.00-4.04 (m, 4H), 1.17 (t, 3H) .EI-MS:m/z:367.3[M+H]+.
Through identifying that products therefrom really is target compounds of formula I-6.
Embodiment 7,2-(2-(5-chloro-2-propoxy-anilino) ethanoyl sulfo-) nicotinic acid (formula I-7) is synthetic
(formula I-7)
With reference to embodiment 1 and 2, substitute 2-methoxyl group-5-chloroaniline, yield 80% with 2-propoxy--5-chloroaniline.
The structural identification result is as follows: 1H-NMR (DMSO-d6): 13.60 (s, 1H), 9.37 (s, 1H), 8.62-8.64 (m, 1H), 8.28-8.30 (m, 1H), 8.20 (d, 1H), 7.31-7.33 (m, 1H), 7.00-7.05 (m, 2H), 4.02 (s, 2H), 3.91 (t, 2H), 1.54-1.57 (m, 2H), 0.81 (t, 3H).EI-MS:m/z:381.4[M+H]
+.
Through identifying that products therefrom really is target compounds of formula I-7.
Embodiment 8,2-(2-(2-chloroanilino) ethanoyl sulfo-) nicotinic acid potassium (formula I-8) is synthetic
(formula I-8)
With reference to embodiment 3, substitute 2-methoxyl group-5-chloroaniline, yield 85% with the 2-chloroaniline.
The structural identification result is as follows: 1H-NMR (DMSO-d6): 9.83 (s, 1H), 8.41-8.43 (m, 1H), 8.05-8.10 (m, 2H), and 7.41-7.44 (m, 1H), 7.26-7.32 (m, 1H), 7.05-7.13 (m, 2H), 3.79 (s, 2H) .EI-MS:m/z:363.2[M+H]
+.
Through identifying that products therefrom really is target compounds of formula I-8.
Embodiment 9,2-(2-(2,5-dibromobenzene amido) ethanoyl sulfo-) nicotinic acid potassium (formula I-9) is synthetic
(formula I-9)
With reference to example 3, substitute 2-methoxyl group-5-chloroaniline, yield 87% with 2,5-dibromo aniline.
The structural identification result is as follows: 1H-NMR (DMSO-d6): 9.76 (s, 1H), 8.44-8.47 (m, 1H), 8.24(d, 1H), 8.05-8.08 (m, 1H), 7.54 (d, 1H), 7.08-7.22 (m, 2H), 3.79 (s, 2H) .EI-MS:m/z:385.1[M+H]
+.
Through identifying that products therefrom really is target compounds of formula I-9.
Embodiment 10,2-(2-(4-chloroanilino) ethanoyl sulfo-) nicotinic acid potassium (formula I-10) is synthetic
(formula I-10)
With reference to embodiment 3, substitute 2-methoxyl group-5-chloroaniline, yield 83% with the 4-chloroaniline.
The structural identification result is as follows: 1H-NMR (DMSO-d6): 10.38 (s, 1H), 8.30-8.33 (m, 1H), 8.00-8.03(m, 1H), 7.62(d, 2H), 7.31 (d, 2H), 7.01-7.05 (m, 1H), 3.79 (s, 2H) .EI-MS:m/z:362.6[M+H]
+.
Through identifying that products therefrom really is target compounds of formula I-10.
Embodiment 11,2-(2-(2,5-dichlorobenzene amido) ethanoyl sulfo-) nicotinic acid potassium (formula I-11) is synthetic
(formula I-11)
With reference to embodiment 3, substitute 2-methoxyl group-5-chloroaniline, yield 86% with 2,5-dichlorphenamide bulk powder.
The structural identification result is as follows: 1H-NMR (DMSO-d6): 9.97 (s, 1H), 8.42-8.44 (m, 1H), 8.21(m, 1H), 8.08(d, 1H), 7.47 (d, 1H), 7.10-7.17 (m, 2H), 3.82 (s, 2H) .EI-MS:m/z:396.1[M+H]
+.
Through identifying that products therefrom really is target compounds of formula I-11.
Embodiment 12,2-(2-(4-bromobenzene amido) ethanoyl sulfo-) nicotinic acid potassium (formula I-12) is synthetic
(formula I-12)
With reference to embodiment 3, substitute 2-methoxyl group-5-chloroaniline, yield 85% with the 4-bromaniline.
The structural identification result is as follows: 1H-NMR (DMSO-d6): 10.42 (s, 1H), 8.31-8.33 (m, 1H), 8.4(d, 1H), 7.57(d, 2H), 7.44 (d, 2H), 7.01-7.05 (m, 1H), 3.86 (s, 2H) .EI-MS:m/z:405.4[M+H]
+.
Through identifying that products therefrom really is target compounds of formula I-12.
Embodiment 13,2-(2-(5-fluoro-2-anisole amido) ethanoyl sulfo-) nicotinic acid potassium (formula I-13) is synthetic
(formula I-13)
With reference to embodiment 3, substitute 2-methoxyl group-5-chloroaniline, yield 82% with the 4-fluoroaniline.
The structural identification result is as follows: 1H-NMR (DMSO-d6): 9.97 (s, 1H), 8.55-8.57 (m, 1H), and 8.20-8.23 (d, 1H), 8.01-8.06 (m, 1H), 7.24-7.28 (m, 1H), and 6.94-6.99 (m, 1H), 6.78-6.84(m, 1H), 3.87 (s, 2H), 3.73 (s, 3H) .EI-MS:m/z:375.3[M+H]
+.
Through identifying that products therefrom really is target compounds of formula I-13.
Embodiment 14,2-(2-(5-methoxyl group-2-chloroanilino) ethanoyl sulfo-) nicotinic acid potassium (formula I-14) is synthetic
(formula I-14)
With reference to embodiment 3, substitute 2-methoxyl group-5-chloroaniline, yield 87% with 5-methoxyl group-2-chloroaniline.
The structural identification result is as follows: 1H-NMR (DMSO-d6): 9.78 (s, 1H), 8.41-8.44 (m, 1H), and 8.06-8.10 (m, 1H), 7.81 (d, 1H), 7.30-7.32 (m, 1H), and 7.09-7.13 (m, 1H), 6.65-6.69(m, 1H), 3.79 (s, 2H), 3.71 (s, 3H) .EI-MS:m/z:391.4[M+H]
+.
Through identifying that products therefrom really is target compounds of formula I-14.
Embodiment 15,2-(2-(5-chloro-2-anisole amido) ethanoyl sulfo-) Yi Yansuan potassium (formula I-15) is synthetic
(formula I-15)
With reference to embodiment 3, substitute 2-sulfydryl nicotinic acid, yield 79% with 2-sulfydryl Yi Yansuan.
The structural identification result is as follows: 1H-NMR (DMSO-d6): 10.12 (s, 1H), 8.44 (d, 1H), 8.23 (d, 1H), 7.61 (s, 1H), 7.48 (d, 1H), 6.99-7.09 (m, 2H), 4.00 (s, 2H), 3.79 (s, 3H) .EI-MS:m/z:391.2[M+H]
+.
Through identifying that products therefrom really is target compounds of formula I-15.
Embodiment 16,2-(2-(2,5-dimethoxy benzene amido) ethanoyl sulfo-) nicotinic acid potassium (formula I-16) is synthetic
(formula I-16)
With reference to embodiment 3, substitute 2-methoxyl group-5-chloroaniline, yield 87% with 2,5-dimethoxyaniline.
The structural identification result is as follows: 1H-NMR (DMSO-d6): 9.61 (s, 1H), 8.68-8.69 (m, 1H), 8.26-8.30 (m, 1H), 7.81 (d, 1H), 7.32-7.36 (m, 1H), 6.89 (d, 1H), 6.54-6.58(m, 1H), 3.98 (s, 2H), 3.70 (s, 3H), 3.69 (s, 3H) .EI-MS:m/z:387.2[M+H]
+.
Through identifying that products therefrom really is target compounds of formula I-16.
Embodiment 17,2-(anilino-2-ethanoyl sulfo-) nicotinic acid (formula I-17) is synthetic
(formula I-17)
With reference to embodiment 3, substitute 2-methoxyl group-5-chloroaniline, yield 89% with aniline.
The structural identification result is as follows: 1H-NMR (DMSO-d6): 13.5 (s, 1H), 10.20 (s, 1H), 8.58-8.60 (m, 1H), 8.23 (d, 1H), 7.57 (d, 2H), 7.22-7.31 (m, 3H), 7.00-7.08 (m, 1H), 4.02 (s, 2H) .EI-MS:m/z:389.3[M+H]
+.
Through identifying that products therefrom really is target compounds of formula I-17.
Embodiment 18,2-(2-(5-fluoro-2-chloroanilino)-2-ethanoyl sulfo-) nicotinic acid potassium (formula I-18) is synthetic
(formula I-18)
With reference to embodiment 3, substitute 2-methoxyl group-5-chloroaniline, yield 75% with 5-fluoro-2-chloroaniline.
The structural identification result is as follows: 1H-NMR (DMSO-d6): 9.98 (s, 1H), 8.43-8.45 (m, 1H), 8.01-8.11 (m, 2H), and 7.45-7.50 (m, 1H), 7.10-7.15 (m, 1H), 6.95-6.99 (m, 1H), 3.82 (s, 2H) .EI-MS:m/z:379.5[M+H]
+.
Through identifying that products therefrom really is target compounds of formula I-18.
Embodiment 19,2-(2-(4-fluoroanilino) ethanoyl sulfo-) nicotinic acid potassium (formula I-19) is synthetic
(formula I-19)
With reference to embodiment 3, substitute 2-methoxyl group-5-chloroaniline, yield 79% with the 4-fluoroaniline.
The structural identification result is as follows: 1H-NMR (DMSO-d6): 10.29 (s, 1H), 8.32-8.34 (m, 1H), 8.00-8.03 (m, 1H), 7.57-7.62 (m, 2H), 7.01-7.13 (m, 3H), 3.78 (s, 2H) .EI-MS:m/z:345.5[M+H]
+.
Through identifying that products therefrom really is target compounds of formula I-19.
Embodiment 20,2-(2-(5-chloroanilino) ethanoyl sulfo-) nicotinic acid potassium (formula I-20) is synthetic
(formula I-20)
With reference to embodiment 3, substitute 2-methoxyl group-5-chloroaniline, yield 82% with the 5-chloroaniline.
The structural identification result is as follows: 1H-NMR (DMSO-d6): 10.47 (s, 1H), 8.31-8.33 (m, 1H), 8.01-8.04 (m, 1H), 7.81(s, 1H), and 7.43-7.46 (m, 1H), 7.27-7.32 (m, 1H), 7.01-7.07 (m, 2H), 3.79 (s, 2H) .EI-MS:m/z:361.3[M+H]
+.
Through identifying that products therefrom really is target compounds of formula I-20.
Embodiment 21,2-(2-(4-trifluoro-methoxyaniline base) ethanoyl sulfo-) nicotinic acid potassium (formula I-21) is synthetic
(formula I-21)
With reference to embodiment 3, substitute 2-methoxyl group-5-chloroaniline, yield 80% with the 4-trifluoro-methoxyaniline.
The structural identification result is as follows: 1H-NMR (DMSO-d6): 10.45 (s, 1H), 8.31-8.33 (m, 1H), 8.00-8.03 (m, 1H), 7.69 (d, 2H), 7.28 (d, 2H), 7.01-7.05 (m, 1H), 3.79 (s, 2H) .EI-MS:m/z:411.8[M+H]
+.
Through identifying that products therefrom really is target compounds of formula I-21.
Embodiment 22,2-(2-(2,5-difluoroaniline base) ethanoyl sulfo-) nicotinic acid potassium (formula I-22) is synthetic
(formula I-22)
With reference to embodiment 3, substitute 2-methoxyl group-5-chloroaniline, yield 78% with 2,5-difluoroaniline.
The structural identification result is as follows: 1H-NMR (DMSO-d6): 10.50 (s, 1H), 8.37 (d, 1H), 7.94-8.08 (m, 2H), and 7.23-7.31 (m, 1H), 7.10-7.14 (m, 1H), 6.86-6.92 (m, 1H), 3.79 (s, 2H) .EI-MS:m/z:363.7[M+H]
+.
Through identifying that products therefrom really is target compounds of formula I-22.
Embodiment 23,2-(2-(5-chloro-2-benzyloxy-aniline base) ethanoyl sulfo-) naotin (formula I-23) is synthetic
(formula I-23)
With 3.47 gram 2-nitro-4-chlorophenols, 3.76 gram benzyl bromines, 5.52 gram Anhydrous potassium carbonates are put among 20 milliliters of DMF, and stirred overnight at room temperature is poured reaction solution in 100 milliliters of frozen water then, has a large amount of faint yellow solids to separate out.Filter after washing, the dry 2-benzyloxy-5-chloronitrobenzene that gets.The said products is joined in 40 milliliters of Glacial acetic acid, add 4.2 gram reduced iron powders, stirred overnight at room temperature, filter washing, merging filtrate, place 100 milliliters of there-necked flasks, drip 2.5 gram chloroacetyl chlorides, after dropwising, stirred overnight at room temperature, pour reaction solution into 200 milliliters of frozen water, separate out solid, filter, washing, dry that crude product is directly used in next step reaction.
To go up the step product and 1.55 gram 2-sulfydryl nicotinic acid join in 30 milliliters of THF and the water mixed solution (volume ratio 1:1), be cooled to 0 degree, add potassium hydroxide 2.24 grams, stirring reaction, be warming up to room temperature, stirring is spent the night, and has a large amount of solids to separate out, and filters, washing, drying obtains off-white color solid 4.29 grams, yield 92%.
The structural identification result is as follows: 1H-NMR (DMSO-d6): 9.93 (s, 1H), 8.29 (d, 1H), 8.02-8.08 (m, 2H), 7.25-7.35 (m, 5H), 6.94-7.04 (m, 3H), 5.16 (s, 2H), 3.77 (s, 2H) .EI-MS:m/z:467.3[M+H]
+.
Through identifying that products therefrom really is target compounds of formula I-23.
Embodiment 24,2-(2-(2-trifluoro-methoxyaniline base) ethanoyl sulfo-) nicotinic acid potassium (formula I-24) is synthetic
(formula I-24)
With reference to embodiment 3, substitute 2-methoxyl group-5-chloroaniline, yield 75% with the 2-trifluoro-methoxyaniline.
The structural identification result is as follows:
1H-NMR(DMSO-d6):9.98(s,1H),8.32-8.35(m,1H),8.05-8.14(m,2H),7.31-7.36(m,2H),7.08-7.19(m,2H),3.79(s,2H).EI-MS:m/z:411.5[M+H]
+.
Through identifying that products therefrom really is target compounds of formula I-24.
Embodiment 25,2-(2-(3-trifluoro-methoxyaniline base) ethanoyl sulfo-) nicotinic acid potassium (formula I-25) is synthetic
(formula I-25)
With reference to embodiment 3, substitute 2-methoxyl group-5-chloroaniline, yield 77% with the 3-trifluoro-methoxyaniline.
The structural identification result is as follows:
1H-NMR(DMSO-d6):10.60(s,1H),8.31-8.33(m,1H),8.02-8.05(m,1H),7.78(s,1H),7.37-7.51(m,2H),6.97-7.05(m,2H),3.82(s,2H).EI-MS:m/z:411.8[M+H]
+.
Through identifying that products therefrom really is target compounds of formula I-25.
Embodiment 26,2-(2-(5-trifluoro methoxy-2-bromobenzene amido) ethanoyl sulfo-) nicotinic acid potassium (formula I-26) is synthetic
(formula I-26)
With reference to embodiment 3, substitute 2-methoxyl group-5-chloroaniline, yield 81% with 5-trifluoro methoxy-2-bromaniline.
The structural identification result is as follows: 1H-NMR (DMSO-d6): 9.82 (s, 1H), 8.47-8.48 (m, 1H), 8.08-8.13 (m, 2H), 7.72 (d, 1H), 7.02-7.13 (m, 2H), 3.82 (s, 2H) .EI-MS:m/z:491.6[M+H]
+.
Through identifying that products therefrom really is target compounds of formula I-26.
Embodiment 27,2-(2-(5-fluoro-2-phenetole amido) ethanoyl sulfo-) nicotinic acid is synthetic
(formula I-27)
With reference to embodiment 2 and 3, substitute 2-methoxyl group-5-chloroaniline, yield 73% with 5-fluoro-2-phenetidine.
The structural identification result is as follows:
1H-NMR(DMSO-d6):13.60(s,1H),9.44(s,1H),8.63-8.65(m,1H),8.27-8.30(m,1H),7.96-8.00(m,1H),7.30-7.34(m,1H),6.94-6.99(m,1H),6.80-6.81(m,1H),3.94-4.03(m,4H),1.15(t,3H).EI-MS:m/z:351.4[M+H]
+.
Through identifying that products therefrom really is target compounds of formula I-27.
Embodiment 28, sulfydryl niacin compound serving are to the vitro inhibition effect of botulinum toxin type A endopeptidase activity
Adopt FRET probe method detection compound activity.The structure of A type FRET probe and expression and purification are referring to document (D.R.Ruge, F.M.Dunning, T.M.Piazza, B.E.Molles, M.Adler, F.N.Zeytin, W.C.Tucker, Detection of six serotypes of botulinum neurotoxin using fluorogenic reporters, J. Anal. Biochem. 2011,411:200 – 209.), the expression of botulinum toxin type A light chain (BoNT/A-LC) and purifying are referring to document (L.Li, B.R.Singh, High-Level expression, purification, and characterization of recombinant type A botulinum neurotoxin light chain, Protein Expr Purif.1999,17:339 – 344.).
At 100 μ l reaction solutions (50mM Hepes – NaOH pH7.4,10mM NaCl, 0.1%Tween20,5mM dithiothreitol, 10 μ M ZnCl
2) in, adding the BoNT/A-LC of final concentration 2nM simultaneously, the FRET probe (at botulinum toxin type A) of final concentration 300nM and the compound of 10 μ g mix back 37 ° of C and hatch.(BioTek USA) finishes, and 96 hole FluoroNunc blackboards are available from Denmark Nunc company by SynergyTM HT enzyme connection instrument for fluorescence detection of dynamic and analysis, testing conditions is that excitation wavelength is 420/50, emission wavelength is respectively 485/20 and 528/20, and pitch time, 2min detected duration 2h.Data are by Prism5.0(Graphpad Software) the software analysis processing, 528/485 pair of timing relationship mapping, 528/485 variation in the slope of a curve representation unit time is so the slope of any point is exactly the speed of reaction of this corresponding time on the curve.If the speed of reaction when not adding compound is V
0, the speed of reaction behind the adding compound is V
i, inhibiting rate (inhibition percentage ratio) i%=(1-V
i/ V
0) * 100%.
Active result is as shown in table 1 for FRET probe method detection compound vitro inhibition, compound has different restraining effect to the botulinum toxin type A endopeptidase activity, its inhibiting rate scope 2.7-78.0%, wherein the inhibition of embodiment 23 is remarkable, inhibiting rate can reach 78.0%, secondly be embodiment 4, inhibiting rate is 45.4%.
Table 1.FRET probe in detecting compound activity result
Embodiment 29, sulfydryl niacin compound serving are to the vitro inhibition effect of botulinum toxin type B endopeptidase activity
Adopt FRET probe method detection compound activity.The structure of Type B FRET probe and expression and purification are referring to document (D.R.Ruge, F.M.Dunning, T.M.Piazza, B.E.Molles, M.Adler, F.N.Zeytin, W.C.Tucker, Detection of six serotypes of botulinum neurotoxin using fluorogenic reporters, J.Anal.Biochem.2011,411:200 – 209.), the expression of botulinum toxin type B light chain (BoNT/B-LC) and purifying are referring to document (J.Gilsdorf, N.Gul, L.A.Smith, Expression, purification, and characterization of Clostridium botulinum type B light chain, Protein Expr Purif46 (2006) 256 – 267.).
At 100 μ l reaction solutions (50mM Hepes – NaOH pH7.4,10mM NaCl, 0.1%Tween20,5mM dithiothreitol, 10 μ M ZnCl
2) in, adding the BoNT/B-LC of final concentration 2nM simultaneously, the FRET probe (at botulinum toxin type B) of final concentration 300nM and the compound of 10 μ g mix back 37 ° of C and hatch.(BioTek USA) finishes, and 96 hole FluoroNunc blackboards are available from Denmark Nunc company by SynergyTM HT enzyme connection instrument for fluorescence detection of dynamic and analysis, testing conditions is that excitation wavelength is 420/50, emission wavelength is respectively 485/20 and 528/20, and pitch time, 2min detected duration 2h.Data are by Prism5.0(Graphpad Software) the software analysis processing, 528/485 pair of timing relationship mapping, 528/485 variation in the slope of a curve representation unit time is so the slope of any point is exactly the speed of reaction of this corresponding time on the curve.If the speed of reaction when not adding compound is V
0, the speed of reaction behind the adding compound is V
i, inhibiting rate (inhibition percentage ratio) i%=(1-V
i/ V
0) * 100%.
It is as shown in table 2 that the FRET probe method detects the active result of compound vitro inhibition of the present invention, compound of the present invention has different restraining effect to the botulinum toxin type B endopeptidase activity, its inhibiting rate scope 2.7-95.5%, wherein the compound inhibition of embodiment 4 is remarkable, inhibiting rate can reach 95.5%, secondly be the compound of embodiment 23, inhibiting rate is 55.7%.
The active result of table 2.FRET probe in detecting The compounds of this invention
Embodiment 30, sulfydryl niacin compound serving are to the vitro inhibition effect of E BOTULINUM TOXIN TYPE A A endopeptidase activity
Adopt FRET probe method detection compound activity.The structure of E type FRET probe and expression and purification are referring to document (D.R.Ruge, F.M.Dunning, T.M.Piazza, B.E.Molles, M.Adler, F.N.Zeytin, W.C.Tucker, Detection of six serotypes of botulinum neurotoxin using fluorogenic reporters, J.Anal.Biochem.2011,411:200 – 209.), the expression of E BOTULINUM TOXIN TYPE A A light chain (BoNT/E-LC) and purifying are referring to document (Rakhi Agarwal, Subramaniam Eswaramoorthy, Desigan Kumaran, Cloning, high level expression, purification, and crystallization of the full length Clostridium botulinum neurotoxin type E light chain, Protein Expr Purif.2004,34:95 – 102).
At 100 μ l reaction solutions (50mM Hepes – NaOH pH7.4,10mM NaCl, 0.1%Tween20,5mM dithiothreitol, 10 μ M ZnCl
2) in, adding the BoNT/E-LC of final concentration 2nM simultaneously, the compound of the FRET probe of final concentration 300nM (at the E BOTULINUM TOXIN TYPE A A) and 10 μ g mixes back 37 ° of C and hatches.(BioTek USA) finishes, and 96 hole FluoroNunc blackboards are available from Denmark Nunc company by SynergyTM HT enzyme connection instrument for fluorescence detection of dynamic and analysis, testing conditions is that excitation wavelength is 420/50, emission wavelength is respectively 485/20 and 528/20, and pitch time, 2min detected duration 2h.Data are by Prism5.0(Graphpad Software) the software analysis processing, 528/485 pair of timing relationship mapping, 528/485 variation in the slope of a curve representation unit time is so the slope of any point is exactly the speed of reaction of this corresponding time on the curve.If the speed of reaction when not adding compound is V
0, the speed of reaction behind the adding compound is V
i, inhibiting rate (inhibition percentage ratio) i%=(1-V
i/ V
0) * 100%.
Active result is as shown in table 3 for FRET probe method detection compound vitro inhibition, compound has different restraining effect to E BOTULINUM TOXIN TYPE A A endopeptidase activity, its inhibiting rate scope 2.1-50.7%, wherein the inhibition of embodiment 23 is remarkable, inhibiting rate can reach 50.7%, secondly be embodiment 4, inhibiting rate is 43.2%.
Table 3.FRET probe in detecting compound activity result
Embodiment 31, sulfydryl niacin compound serving are to the vitro inhibition effect of F BOTULINUM TOXIN TYPE A A endopeptidase activity
Adopt FRET probe method detection compound activity.The structure of F type FRET probe and expression and purification are referring to document (D.R.Ruge, F.M.Dunning, T.M.Piazza, B.E.Molles, M.Adler, F.N.Zeytin, W.C.Tucker, Detection of six serotypes of botulinum neurotoxin using fluorogenic reporters, J.Anal.Biochem.2011,411:200 – 209.), the purifying of F BOTULINUM TOXIN TYPE A A (BoNT/F) is referring to document (Yang K.H., Sugiyama H.Purification and Properties of Clostridium botulinum Type F Toxin, Appl.Microbiol.1975,29 (5): 598-603).
At 100 μ l reaction solutions (50mM Hepes – NaOH pH7.4,10mM NaCl, 0.1%Tween20,5mM dithiothreitol, 10 μ M ZnCl
2) in, adding the BoNT/F-LC of final concentration 2nM simultaneously, the compound of the FRET probe of final concentration 300nM (at the F BOTULINUM TOXIN TYPE A A) and 10 μ g mixes back 37 ° of C and hatches.(BioTek USA) finishes, and 96 hole FluoroNunc blackboards are available from Denmark Nunc company by SynergyTM HT enzyme connection instrument for fluorescence detection of dynamic and analysis, testing conditions is that excitation wavelength is 420/50, emission wavelength is respectively 485/20 and 528/20, and pitch time, 2min detected duration 2h.Data are by Prism5.0(Graphpad Software) the software analysis processing, 528/485 pair of timing relationship mapping, 528/485 variation in the slope of a curve representation unit time is so the slope of any point is exactly the speed of reaction of this corresponding time on the curve.If the speed of reaction when not adding compound is V
0, the speed of reaction behind the adding compound is V
i, inhibiting rate (inhibition percentage ratio) i%=(1-V
i/ V
0) * 100%.
Active result is as shown in table 4 for FRET probe method detection compound vitro inhibition, compound has different restraining effect to F BOTULINUM TOXIN TYPE A A endopeptidase activity, its inhibiting rate scope 3.0-48.6%, wherein the inhibition of embodiment 4 is remarkable, inhibiting rate can reach 48.6%, secondly be embodiment 23, inhibiting rate is 35.8%.
Table 4.FRET probe in detecting compound activity result
Embodiment 32, sulfydryl niacin compound serving are to the vitro inhibition effect of tetanus toxin endopeptidase activity
Adopt FRET probe method detection compound activity.The structure of tetanus toxin FRET probe and expression and purification are referring to document (D.R.Ruge, F.M.Dunning, T.M.Piazza, B.E.Molles, M.Adler, F.N.Zeytin, W.C.Tucker, Detection of six serotypes of botulinum neurotoxin using fluorogenic reporters, J.Anal.Biochem.2011,411:200 – 209.), the expression of tetanus toxin light chain (TeNt-LC) and purifying are referring to document (Fiorella Tonello, Rossella Pellizzari, Sebastiano Pasqualato, Recombinant and Truncated Tetanus Neurotoxin Light Chain:Cloning, Expression, Purification, and Proteolytic Activity, Protein Expr Purif.1999,15:221 – 227).
At 100 μ l reaction solutions (50mM Hepes – NaOH pH7.4,10mM NaCl, 0.1%Tween20,5mM dithiothreitol, 10 μ M ZnCl
2) in, adding the TET-LC of final concentration 2nM simultaneously, the FRET probe (at tetanus toxin) of final concentration 300nM and the compound of 20 μ g mix back 37 ° of C and hatch.(BioTek USA) finishes, and 96 hole FluoroNunc blackboards are available from Denmark Nunc company by SynergyTM HT enzyme connection instrument for fluorescence detection of dynamic and analysis, testing conditions is that excitation wavelength is 420/50, emission wavelength is respectively 485/20 and 528/20, and pitch time, 2min detected duration 2h.Data are by Prism5.0(Graphpad Software) the software analysis processing, 528/485 pair of timing relationship mapping, 528/485 variation in the slope of a curve representation unit time is so the slope of any point is exactly the speed of reaction of this corresponding time on the curve.If the speed of reaction when not adding compound is V
0, the speed of reaction behind the adding compound is V
i, inhibiting rate (inhibition percentage ratio) i%=(1-V
i/ V
0) * 100%.
Active result is as shown in table 5 for FRET probe method detection compound vitro inhibition, compound has different restraining effect to the tetanus toxin endopeptidase activity, its inhibiting rate scope 2.7-34.5%, wherein the inhibition of embodiment 4 is remarkable, inhibiting rate can reach 34.5%, secondly be embodiment 23, inhibiting rate is 30.3%.
Table 5.FRET probe in detecting compound activity result
The protection effect that embodiment 33, sulfydryl niacin compound serving are poisoned to the animal botulinum toxin type A
The extraction of botulinum toxin type A (BoNT/A) and authentication method are referring to document (C.J.Malizio, M.C.Goodnough, E.A.Johnson, Purification of Clostridium botulinum type A neurotoxin, Methods Mol Biol.2000,145:27-39.), Balb/c mouse 14-16g is available from Military Medical Science Institute's Experimental Animal Center.
The protectiveness experiment reference literature that botulinus toxin is poisoned is described (C.H.Hatheway; J.D.Snyder; J.E.Seals; T.A.Edell; G.E.Lewis; Jr.Antitoxin levels in botulism patients treated with trivalent equine botulism antitoxin to toxin types A; B; and E.Infect Dis1984; 150:145-151.) mouse sausage poisoning model method; concise and to the point method is as follows: be test animal with the Balb/C mouse; carry out random packet, 10/group, with the positive contrast of A type horse serum toxinicide; the different compounds of 200ug are sample to be checked (having the inhibiting part water soluble compounds of enzymic activity), respectively with 5 times of half lethal dose (5LD
50) botulinum toxin type A mix, 37 ° of C are hatched 30min, 500ul/ only, abdominal injection arranges placebo simultaneously.Continue to observe above 5 days, observation mouse sausage poisoning sign (as wasp waist occurring, fur stands upside down, shallow breathing, tetraplegia is until death), record mouse survival time is calculated survival rate.
The antitoxic action of compound in mouse sausage poisoning model is as shown in table 6; compound can suppress botulinum toxin type A in various degree to the lethal effect of animal subject; the protection ratio the highest (100%) of compound embodiment 4 and embodiment 23 wherein, secondly the protection ratio of embodiment 8, embodiment 15, embodiment 10, embodiment 3, embodiment 21, embodiment 14 is followed successively by 80%, 80%, 60%, 40%, 40%, 20% etc. from high to low.
The antitoxic action of table 6. compound in mouse sausage poisoning model
Annotate: placebo is solvent physiological saline in the table 6; Toxinicide is A type horse serum toxinicide (available from National Institute for Food and Drugs Control)
The protection effect that embodiment 34, sulfydryl niacin compound serving are poisoned to the animal botulinum toxin type B
The extraction of botulinum toxin type B (BoNT/B) and authentication method are referring to document (H.Arimitsu, K.Inoue, Y.Sakaguchi, J.Lee, Y.Fujinaga, T.Watanabe, T.Ohyama, R.Hirst, K.Oguma, Purification of fully activated Clostridium botulinum serotype B toxin for treatment of patients with dystonia, Infect Immun.71 (2003) 1599-1603.), Balb/c mouse 14-16g is available from Military Medical Science Institute's Experimental Animal Center.
The protectiveness experiment reference literature that botulinus toxin is poisoned is described (C.H.Hatheway; J.D.Snyder; J.E.Seals; T.A.Edell; G.E.Lewis; Jr.Antitoxin levels in botulism patients treated with trivalent equine botulism antitoxin to toxin types A; B; and E.Infect Dis1984; 150:145-151.) mouse sausage poisoning model method; method is as follows: be test animal with the Balb/C mouse; carry out random packet, 10/group, with the positive contrast of Type B horse serum toxinicide; the different compounds of 200ug are sample to be checked (having the inhibiting part water soluble compounds of enzymic activity), respectively with 5 times of half lethal dose (5LD
50) botulinum toxin type B mix, 37 ° of C are hatched 30min, 500ul/ only, abdominal injection arranges placebo simultaneously.Continue to observe above 5 days, observation mouse sausage poisoning sign (as wasp waist occurring, fur stands upside down, shallow breathing, tetraplegia is until death), record mouse survival time is calculated survival rate.
The antitoxic action of compound of the present invention in mouse sausage poisoning model is as shown in table 7; compound of the present invention can suppress botulinum toxin type B in various degree to the lethal effect of animal subject; compound embodiment 4 protection ratios the highest (100%) wherein; secondly; that have high protection ratio is compound embodiment 23, embodiment 24 and embodiment 12(80%), the protection ratio of compound embodiment 1, embodiment 3, embodiment 9, embodiment 18, embodiment 21, embodiment 26 is followed successively by 40%, 60%, 20%, 40%, 40%, 20% etc. in addition.
The antitoxic action of table 7. The compounds of this invention in mouse sausage poisoning model
Annotate: placebo is solvent physiological saline in the table 7; Toxinicide is Type B horse serum toxinicide (available from National Institute for Food and Drugs Control)
More than show and described ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; that describes in above-described embodiment and the specification sheets just illustrates principle of the present invention; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements all fall in the claimed scope of the invention.The claimed scope of the present invention is defined by appending claims and equivalent thereof.
Claims (8)
1. a class sulfydryl niacin compound serving and pharmacy acceptable salt thereof, ester, solvate, this structural general formula is suc as formula shown in the I:
(formula 1)
Among the formula I:
R
1, R
2, R
3, R
4Represent in the following radicals any one independently: hydrogen, hydroxyl, amino, halogen, cyano group, diazanyl, azido-, low-grade alkyl amino, low alkyl group, low-grade halogenated alkyl, cycloalkyl, lower alkoxy, elementary halogenated alkoxy, low-grade alkenyl, rudimentary cyano group alkyl, rudimentary 4-nitro alkyl, amide group, rudimentary amido alkyl, rudimentary diazanyl alkyl, rudimentary azido-alkyl, (CH
2)
mNR
7R
8, (CH
2)
mOR
9, (CH
2)
mSR
9, (CH
2)
mNR
7C (O) R
9, (CH
2)
mC (O) R
9, (CH
2)
mOC (O) R
9, O (CH
2)
mNR
7R
8, OC (O) NR
7, OC (O) (CH
2)
mOC (O) R
9
Perhaps, R
1And R
2Carbon atom on the phenyl ring that they connect forms 5 or 6 yuan of rings;
Perhaps, R
2And R
3Carbon atom on the phenyl ring that they connect forms 5 or 6 yuan of rings;
Perhaps, R
3And R
4Carbon atom on the phenyl ring that they connect forms 5 or 6 yuan of rings;
The unit that forms described 5 or 6 yuan of rings is selected from CH, CH
2, O, S or NR
7
R
5, R
6Represent in the following radicals any one independently: hydrogen, hydroxyl, amino, halogen, cyano group, diazanyl, azido-, low-grade alkyl amino, low alkyl group, low-grade halogenated alkyl, lower alkoxy, low-grade alkenyl and (CH
2)
mCOOH;
R
7, R
8, R
9Represent hydrogen, low alkyl group or cycloalkyl independently;
M is the integer between 0 to 3;
Described low alkyl group, lower alkoxy refer to contain the straight or branched saturated fatty alkyl of 1 to 6 carbon atom;
Described low-grade alkenyl refers to contain the group of 2 to 6 carbon atoms and at least one carbon-carbon double bond;
Described low-grade cycloalkyl refers to contain the ring of 3 to 7 carbon;
The amino of described low-grade alkyl amino for being replaced by one or two low alkyl group;
Described halogen refers to chlorine, bromine, iodine or fluorine;
The group of described low-grade halogenated alkyl, rudimentary cyano group alkyl, rudimentary 4-nitro alkyl, rudimentary amido alkyl, rudimentary diazanyl alkyl, rudimentary azido-alkyl refers to respectively be replaced by one to three halogen, cyano group, nitro, amide group, diazanyl, azido-.
2. a class sulfydryl niacin compound serving according to claim 1 and pharmacy acceptable salt thereof, ester, solvate is characterized in that, among the formula I,
R
1, R
2, R
3, R
4Represent in the following radicals any one independently: hydrogen, hydroxyl, amino, halogen, low alkyl group, low-grade halogenated alkyl, lower alkoxy, or elementary halogenated alkoxy;
R
5, R
6Expression independently: hydrogen or (CH
2)
mCOOH;
M is the integer between 0 to 3.
3. a class sulfydryl niacin compound serving according to claim 1 and pharmacy acceptable salt thereof, ester, solvate is characterized in that compound is selected from shown in the formula I:
2-(2-(5-chloro-2-anisole amido) ethanoyl sulfo-) naotin,
2-(2-(5-chloro-2-anisole amido) ethanoyl sulfo-) nicotinic acid,
2-(2-(5-chloro-2-anisole amido) ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(3-chloro-5-anisole amido)-2-ethanoyl sulfo-) naotin,
2-(2-(2-phenetole amido) ethanoyl sulfo-) nicotinic acid,
2-(2-(5-chloro-2-phenetole amido) ethanoyl sulfo-) nicotinic acid,
2-(2-(5-chloro-2-propoxy-anilino) ethanoyl sulfo-) nicotinic acid,
2-(2-(2-chloroanilino) ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(2,5-dibromobenzene amido) ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(4-chloroanilino) ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(2,5-dichlorobenzene amido) ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(4-bromobenzene amido) ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(5-fluoro-2-anisole amido) ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(5-methoxyl group-2-chloroanilino) ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(5-chloro-2-anisole amido) ethanoyl sulfo-) Yi Yansuan potassium,
2-(2-(2,5-dimethoxy benzene amido) ethanoyl sulfo-) nicotinic acid potassium,
2-(anilino-2-ethanoyl sulfo-) nicotinic acid,
2-(2-(5-fluoro-2-chloroanilino)-2-ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(4-fluoroanilino) ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(5-chloroanilino) ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(4-trifluoro-methoxyaniline base) ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(2,5-difluoroaniline base) ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(5-chloro-2-benzylaniline base) ethanoyl sulfo-) naotin,
2-(2-(2-trifluoro-methoxyaniline base) ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(3-trifluoro-methoxyaniline base) ethanoyl sulfo-) nicotinic acid potassium,
2-(2-(5-trifluoro methoxy-2-bromobenzene amido) ethanoyl sulfo-) nicotinic acid potassium, or
2-(2-(5-fluoro-2-phenetole amido) ethanoyl sulfo-) nicotinic acid.
4. according to claim 1,2 or 3 described class sulfydryl niacin compound servings and pharmacy acceptable salt thereof, ester, solvate, it is characterized in that described pharmacy acceptable salt is inorganic acid salt or organic acid salt.
5. a class sulfydryl niacin compound serving according to claim 4 and pharmacy acceptable salt thereof, ester, solvate is characterized in that, inorganic acid salt is hydrochloride, vitriol, phosphoric acid salt, diphosphate, hydrophosphate, hydrobromate or nitrate; Organic acid salt be acetate, maleate, fumarate, tartrate, succinate, lactic acid salt, Citrate trianion, tosilate, salicylate, oxalate, palmitate or stearate.
6. according to claim 1,2 or 3 described class sulfydryl niacin compound servings and pharmacy acceptable salt thereof, ester, solvate, it is characterized in that described pharmaceutically acceptable ester refers to the C1-C6 alkyl ester.
7. according to claim 1,2 or 3 described class sulfydryl niacin compound servings and pharmacy acceptable salt thereof, ester, solvate, it is characterized in that, described pharmaceutically acceptable solvate refers to the solvation form that forms with water, ethanol or DMSO.
8. the preparation method as claim 1,2 or 3 described sulfydryl niacin compound servings and pharmacy acceptable salt thereof, ester, solvate is characterized in that the preparation of compound shown in its Chinese style I comprises the steps:
A) make the reaction of the compound shown in the formula II and chloroacetyl chloride or bromoacetyl chloride, obtain compound shown in the formula III;
(formula II) (formula III)
R in formula II, the formula III
1, R
2, R
3, R
4Definition cotype I;
B) make the reaction of compound shown in compound shown in the formula III and the formula IV, obtain compound shown in the formula I;
(formula IV)
R in the formula IV
5, R
6Definition cotype I.
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| PCT/CN2014/077768 WO2014187291A1 (en) | 2013-05-20 | 2014-05-19 | Mercaptonicotinic acid compound and preparation method and use thereof |
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