CN102731459A - Scutellarin aglycone Mannich derivatives, and preparation method and application thereof - Google Patents
Scutellarin aglycone Mannich derivatives, and preparation method and application thereof Download PDFInfo
- Publication number
- CN102731459A CN102731459A CN2012101988298A CN201210198829A CN102731459A CN 102731459 A CN102731459 A CN 102731459A CN 2012101988298 A CN2012101988298 A CN 2012101988298A CN 201210198829 A CN201210198829 A CN 201210198829A CN 102731459 A CN102731459 A CN 102731459A
- Authority
- CN
- China
- Prior art keywords
- scutellarein
- mannich
- verivate
- scutellarin
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
The invention relates to the technical field of medicines and specifically to novel scutellarin aglycone Mannich derivatives, a preparation method thereof and application of the derivatives in preparation of medicines used for preventing and controlling thrombus. According to results of experiments, compared with scutellarin, the scutellarin aglycone Mannich derivatives provided in the invention have better dissolvability and better pharmacological activity in inhibiting oxidation, cell injury, blood coagulation and the like and are expected to be developed into a novel medicine used for preventing and treating diseases like myocardial infarction, senile dementia and cerebral infarction.
Description
Technical field
The present invention relates to medical technical study field, be specifically related to one type of novel Scutellarein Mannich verivate (I), and its preparation method and the application in control thrombus medicine.
Background technology
Scutellarin is a flavonoid compound in the short booth bitter fleabane of the composite family class plant Herba Erigerontis; Herba Erigerontis has another name called Herba Erigerontis; Another name oil lamp chrysanthemum, TUXIXIN, push up grass, root of Chinese wild ginger grass, eastern chrysanthemum etc.; Herba Erigerontis hardship cold in nature, little, Gan Wenxin have expelling cold and relieving exterior syndrome, dispel rheumatism, the effect of promoting blood circulation and removing blood stasis, clearing and activating the channels and collaterals, anti-inflammatory analgetic.Breviscarpine is the flavonoid active ingredient that from the natural phant Herba Erigerontis, extracts, and the mixture of breviscapine, scutellarin etc. is arranged, and is mainly scutellarin (content occupies more than 95%) (Chen Yiyue; Wang Shengtao, Ceng Wenshan, Zhu Yinghong; Fu Yongmei; The river great waves. Breviscarpine is to the relexation of rat aorta flesh ring. new Chinese medicine and clinical pharmacology .1994,5 (2), 15-19).Last century since the seventies, Breviscapine just was applied to clinical; Through nearly 30 years clinical application and wide deep pharmacological research; The curative effect that it is unique and the characteristics of safety and low toxicity have obtained social recognition; Modern medicines research proof, Breviscarpine has blood flow increasing, effects such as microcirculation improvement, vasodilation, reduction blood viscosity, reducing blood-fat, short fibrinolytic, antithrombotic, platelet aggregation-against; Its injection and tablet have become clinical common drug, at aspects such as treatment cardiovascular and cerebrovascular diseases, rheumatic arthritis and apoplexy sequelas significant curative effect are arranged.
Modern clinical research finds that bioavailability is lower in the Breviscarpine process of clinical application, is poorly soluble on the one hand, and the solubleness of bibliographical information Breviscarpine in water is merely 0.16mgmL
-1(Zhang Haiyan, flat its ability, Guo Jianxin, behaviour's cutting edge of a knife or a sword. Breviscarpine and Benexate Hydrochloride thereof are in pharmacokinetics in rats. Acta Pharmaceutica Sinica; 2005,40 (6), 563-567), secondly Breviscarpine is fat-soluble also very poor in the PBS of pH4.2 solution; LogP is-2.56 (behaviour's cutting edge of a knife or a sword, Guo Jianxin, flat its ability, Shao Yun; Liang Jing. synthetic, the physico-chemical property of scutellarin class prodrug and Study on degradation. Acta Pharmaceutica Sinica, 2006,41 (7), 595-602).Research is also found in addition, and the lower reason of Breviscarpine bioavailability is except poorly soluble, and beyond being difficult to absorb, also having an important reasons is that its staple scutellarin is easy to metabolism in vivo; No matter people such as Ge Qinghua discover oral or intravenously administrable, scutellarin metabolism elimination speed in animal body is fast, and the oral absolute bioavailability of Beagle dog is (0.40 ± 0.19) % (Ge Qinghua, Zhou Zhen only; Zhi Xiaojin, Ma Lili, Chen Xiuhua. Breviscarpine is in intravital pharmacokinetics of dog and absolute bioavailability research. Chinese Journal of Pharmaceuticals, 2003; 34 (12), 618-621), and the quiet notes of domesticated dog are eliminated the transformation period weak point, are (52 ± 29) min (Jiang Xuehua; Li Suhua, Lan Ke, Yang Junyi; Week is quiet. and Breviscarpine is in the intravital pharmacokinetics of domesticated dog. Acta Pharmaceutica Sinica .2003,38 (5), 371-373).People such as Feng Fang have studied the pharmacokinetic parameters behind the low dose of scutellarin dripping pill of the oral 60mg of human body; The discovery scutellarin is eliminated very fast in vivo; Be merely biological half-life (2.27 ± 0.58) min (Feng Fang, Shen Yulan. foundation and the pharmacokinetic studies of trace scutellarin SPE-HPLC/MS/MS in the human plasma. Chinese Pharmaceutical Journal .2006,41 (6); 457), scutellarin because of the low big limitations of its poorly soluble and bioavailability its clinical application.
More domestic scientific research personnel hope by means of exploitation Breviscarpine novel form, to improve its oral administration biaavailability or to prolong the transformation period in its body.In recent years, the invention disclosed patent was nearly 59, related to injection, liposome, phospholipid complex, oral cavity quick disintegrating slice, Sublingual tablet, sustained release pellet, controlled releasing penetrant pump, cyclodextrin inclusion compound, dripping pill, self-emulsifier etc.But at present the novel form of listing is few, and this shows, and change through formulation can not improve well that Breviscarpine is poorly soluble, absorption difference, problem that bioavailability is low.
Scutellarin is hydrolyzed the glucal acidic group in vivo, and forming Scutellarein is the major cause that limits its bioavailability.Occupy people such as Wen Zheng and measure scutellarin Plasma Concentration and clinical pharmacokinetics thereof, experimenter's oral administration 360mg scutellarin is 1,3,5,8h gets blood and survey scutellarin, but only when 5h, detect 20ngmL
-1And in blood plasma and urine, find a large amount of aglycons; Point out a large amount of scutellarins to be hydrolyzed to aglycon (LiuY, HuM.Absorption and metabolism of flavonoids in the Caco-2cell culture model and a perfused rat intestinal model.Drug Metab Dispos, 2002 in vivo; 30 (4), 370-377).Therefore; After introducing alkylamino radical through the Mannich reaction 8 of Scutellareins, on the one hand the alkylamino radical molecule have bigger water-soluble, thereby can solve the water-soluble too poor problem that is difficult to absorb of scutellarin; On the other hand because this compounds is difficult to the hydrolysis metabolism; Thereby can improve these compounds bioavailability in vivo, aspect cardiovascular and cerebrovascular diseases medicament research, will have very significant meaning.
Summary of the invention:
Goal of the invention: technical problem to be solved by this invention is; Overcome the poorly soluble deficiency of prior art scutellarin; With the scutellarin glycosides be raw material through the glycosides hydrolysis, thereby Mannich reaction synthesizing series has pharmaceutical use and solvability is good, bioavailability is high, untoward reaction is low, the Scutellarein Mannich verivate of drug safety, another object of the present invention provides the preparation method and its application in preparation control thrombus disease medicine of Scutellarein Mannich verivate.
Technical scheme: in order to realize above purpose, the logical formula I of Scutellarein Mannich verivate provided by the invention is following:
R wherein
1=R
2, all represent alkyl;
Perhaps R1 is a hydrogen, and R2 is morpholinyl, piperazinyl or substituted piperazinyl.
As preferred version, above-described Scutellarein Mannich verivate, described alkyl is methyl, ethyl, n-propyl, sec.-propyl, normal-butyl or isobutyl-.
As another preferred version, above-described Scutellarein Mannich verivate, the substituting group in the described substituted piperazinyl is methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, isobutyl-, n-pentyl or isopentyl.
The preparation method of Scutellarein Mannich verivate provided by the invention specifically may further comprise the steps:
A, to get scutellarin be raw material, and under the catalysis of the vitriol oil, heating hydrolysis in 95% ethanolic soln obtains Scutellarein, and is subsequent use;
B, get Scutellarein that step a obtains in methyl alcohol or ethanolic soln, with
Reaction obtains the described Scutellarein Mannich of claim 1 verivate (I), wherein R with formalin
1=R
2, all represent alkyl; Perhaps R1 is a hydrogen, and R2 is morpholinyl, piperazinyl or substituted piperazinyl.The structure of Scutellarein Mannich verivate (I) is following:
As preferred version; The preparation method of above-described Scutellarein Mannich verivate; The volumetric molar concentration of the used vitriol oil is 0.5 ~ 3mol/L among the step a, and scutellarin is 5g/100ml～10g/100ml with 95% consumption of ethanol ratio, and temperature of reaction is 110～120 ℃; Reaction times is 36～54 hours.
As preferred version; The preparation method of above-described Scutellarein Mannich verivate; The molar equivalent of used formaldehyde is 1:1～2 among the step b; The molar equivalent of
is 1:1～2, and temperature of reaction is 25 ~ 35 ℃
As preferred version; The salt of above-described Scutellarein Mannich verivate, said salt are that said Scutellarein Mannich verivate and alkali metal hydroxide, alkaline earth metal hydroxides, alkaline carbonate, alkaline earth metal carbonate, alkali metal hydrocarbonate or alkali metal bicarbonates prepared in reaction obtain.
The application of Scutellarein Mannich verivate provided by the invention in preparation control thrombotic diseases medicine, said thrombotic diseases is myocardial infarction, ischemia injury.
As further preferred version, Scutellarein Mannich derivative salt and pharmaceutically acceptable carrier are processed the medicine of tablet, capsule, granule, sprays, injection, micro-capsule, ointment or skin-permeable and control-released plaster formulation.
When Scutellarein Mannich derivative salt provided by the invention is processed tablet, Scutellarein Mannich derivative salt and carrier lactose or W-Gum, add magnesium stearate lubricant when needing, mix, compressing tablet is processed tablet then.When processing capsule, Scutellarein Mannich derivative salt provided by the invention mixes Scutellarein Mannich derivative salt and carrier lactose or W-Gum whole grain, the encapsulated then capsule of processing.When Scutellarein Mannich derivative salt provided by the invention is processed granule, mix compsn and thinner lactose or W-Gum, whole grain, drying is processed granule.When processing injection liquid to Scutellarein Mannich derivative salt provided by the invention, get Scutellarein Mannich derivative salt and add solubilizing agent, stir; 80 ℃ were heated 30 minutes; Filter, regulate pH value, be filtered to clear and bright with sintered glass funnel or other filter; Can was processed injection liquid in 30 minutes 100 to 115 ℃ of sterilizations.
Beneficial effect: Scutellarein Mannich verivate provided by the invention and prior art are at present than having the following advantages:
1, serial scutellarin aglycon Mannich verivate provided by the invention; Through connection ester dissolubility and the water-soluble functional group at Scutellarein Mannich derivatives selectively, the Scutellarein Mannich verivate solvability that obtains is good, can improve human body and take the artifact availability; It is active to strengthen the antithrombotic pharmacology; And untoward reaction is low, and medication is safer, and Scutellarein Mannich verivate can prepare salt with multiple alkali reaction; And can be made into multiple pharmaceutical dosage form, make things convenient for clinical application.
2, the preparation method of Scutellarein Mannich verivate provided by the invention; With the scutellarin glycosides is that raw material passes through the glycosides hydrolysis, thereby Mannich reaction synthesizing series has pharmaceutical use and solvability is good, the new compound of the Scutellarein Mannich verivate that bioavailability is high, preparation method provided by the invention; Workable; Production efficiency is high, and cost is low, and the finished product yield is high, purity is high.
Description of drawings
Fig. 1 is the structural representation of Scutellarein Mannich verivate of the present invention (I).
Fig. 2 is Scutellarein Mannich verivate of the present invention (I) preparing method's a reacting flow chart.
Embodiment
According to following embodiment, can understand the present invention better.Yet, those skilled in the art will readily understand that the described concrete material proportion of embodiment, processing condition and result thereof only are used to explain the present invention, and the present invention that should also can not limit in claims to be described in detail.
The preparation of embodiment 1 Scutellarein Mannich verivate
1, the preparation of Scutellarein (5,6,7-trihydroxy--2-(4-hydroxy phenyl)-4H-chromone)
(5g 10.82mmol) joins 3molL to get scutellarin
-190% vitriol oil ethanolic soln 50mL in, 120 ℃ with N
2Protective reaction 48h, reaction finishes postcooling, and reaction solution is poured in the water of 8 times of amounts, suction filtration, the filter cake washing is to neutrality, 50 ℃ of dryings.Filter cake with 80%, 50% ethanol repeatedly recrystallization get the Scutellarein bullion, separate through silica gel column chromatography again that (methylene dichloride: methyl alcohol=60:1) obtains Scutellarein 790mg.Yellow powder, productive rate 25.5%.m.p.160-162℃。IR(KBr,cm
-1):3430,2026,1661,1586,1491,1369,1247,1181.
1H?NMR(300MHz,DMSO-d
6)δ：12.79(s,1H,5-OH);10.44(s,1H,7-OH);10.30(s,1H,4′-OH);8.71(s,1H,6-OH);7.90-7.93(d,2H,J=8.8Hz,2′,6′-H);6.90-6.93(d,2H,J=8.8Hz,3′,5′-H);6.73(s,1H,8-H);6.78(s,1H,3-H)。ESI-MS:m/z285[M-H]
-。
2, Scutellarein Mannich response derivative is synthetic
2.1 get above-mentioned Scutellarein (100mg; 0.35mmol) 7 parts; Be dissolved in respectively in the 6.5ml methyl alcohol; 30 ℃ are stirred down 15min, treat that substrate dissolves fully after, get n n dimetylaniline, 0.35mmol diethylamine, the Diisopropylamine of 0.7mmol, the morphine quinoline of 0.7mmol, the N methyl piperazine of 0.7mmol, the N-ethyl piperazidine of 0.35mmol, 0.7mmol N-butyl piperazine that above 7 parts of Scutellareins add 0.7mmol respectively; Add formalin (0.027ml) after letting respectively, continue to keep magnetic agitation reaction 12h at 35 ℃ respectively.
Question response is got each reaction solution and is used silica gel mixed sample after finishing, respectively through column chromatography for separation, respectively with methylene dichloride: methyl alcohol=5:1 eluent, get Scutellarein Mannich response derivative I-1 to I-7 respectively, concrete product is following:
I-18-(N, N-dimethyl-)-methene amido-5,6,7,4 '-tetrahydroxy-2-phenyl-4H-1-chromene-4-ketone
The I-1 structure elucidation:
1H-NMR (DMSO, 500MHz) δ: 13.11 (s, 1H, C
5-OH), 10.25 (s, 1H, C
4 '-OH), 8.05 (s, 1H, C
6-OH), 7.95 (d, J=9Hz, 2H, C
2 'C
6 '-H), 6.93 (d, J=9Hz, 2H, C
3 'C
5 '-H), 6.96 (s, 1H, C
3-H), 4.13 (s, 2H ,-CH
2-), 2.56 (s, 6H, 2CH
3) .ESI-MS:m/z344 [M+H]
+
I-28-(N, N-diethylammonium)-methene amido-5,6,7,4 '-tetrahydroxy-2-phenyl-4H-1-chromene-4-ketone
The I-2 structure elucidation:
1H-NMR (DMSO, 500MHz) δ: 12.75 (s, 1H, C
5-OH), 7.91 (d, J=9Hz, 2H, C
2 'C
6 '-H), 6.94 (d, J=9Hz, 2H, C
3 'C
5 '-H), 6.68 (s, 1H, C
3-H), 4.28 (s, 2H ,-CH
2-), 2.902-2.974 (q, 2H ,-CH
2-), 1.170-1.218 (t, 3H ,-CH
3) .ESI-MS:m/z372 [M+H]
+
I-38-(N, N-diisopropylamino)-methene amido-5,6,7,4 '-tetrahydroxy-2-phenyl-4H-1-chromene-4-ketone
The I-3 structure elucidation:
1H NMR (DMSO, 500MHz) δ: 12.78 (s, 1H, C
5-OH), 7.95 (d, J=8.5Hz, 2H, C
2 'C
6 '-H), 6.93 (d, J=9Hz, 2H, C
3 'C
5 '-H), 6.88 (s, 1H, C
3-H), 4.28 (s, 2H ,-CH
2-), 2.30-2.34 (m, 2H, 2CH), 1.19-1.23 (d, 12H, 4CH
3) .ESI-MS:m/z 400 [M+H]
+
I48-(morphine quinoline base)-methene amido-5,6,7,4 '-tetrahydroxy-2-phenyl-4H-1-chromene-4-ketone
The I-4 structure elucidation:
1H NMR (DMSO, 500MHz) δ: 12.89 (s, 1H, C
5-OH), 10.29 (s, 1H, C
4 '-OH), 7.94 (d, J=9Hz, 2H, C
2 'C
6 '-H), 6.95 (d, J=9Hz, 2H, C
3 'C
5 '-H), 6.75 (s, 1H, C
3-H), 3.94 (s, 2H ,-CH
2-), 3.61 (t, 4H, 2CH
2) .ESI-MS:m/z387 [M+H]
+
I-58-(N methyl piperazine base)-methene amido-5,6,7,4 '-tetrahydroxy-2-phenyl-4H-1-chromene-4-ketone
The I-5 structure elucidation:
1H NMR (DMSO, 500MHz) δ: 12.86 (s, 1H, C
5-OH), 10.28 (s, 1H, C
4 '-OH), 7.92 (d, J=8.5Hz, 2H, C
2 'C
6 '-H), 6.93 (d, J=8.5Hz, 2H, C
3 'C
5 '-H), 6.73 (s, 1H, C
3-H), 3.95 (s, 2H ,-CH
2-), 2.62 (t, 4H, 2 * CH
3), 2.48 (t, 4H, 2 * CH
3), 1.22 (s, 3H ,-CH
3) .ESI-MS:m/z399 [M+H]
+
I-68-(N-ethyl piperazidine base)-methene amido-5,6,7,4 '-tetrahydroxy-2-phenyl-4H-1-chromene-4-ketone
The I-6 structure elucidation:
1H NMR (DMSO, 500MHz) δ: 12.83 (s, 1H, C
5-OH), 7.93 (d, J=8.7Hz, 2H, C
2 'C
6 '-H), 6.95 (d, J=8.7Hz, 2H, C
3 'C
5 '-H), 6.73 (s, 1H, C
3-H), 4.02 (s, 2H ,-CH
2-), 2.68 (s, 4H, 2CH
2), 2.42 (s, 4H, 2CH
2), 2.33-2.40 (q, 2H, CH
2), 0.97-1.02 (t, 3H, CH
3) .ESI-MS:m/z413 [M+H]
+
I-78-(N-butyl piperazinyl)-methene amido-5,6,7,4 '-tetrahydroxy-2-phenyl-4H-1-chromene-4-ketone
1H?NMR(DMSO,500MHz)δ：12.87(s,1H,C
5-OH),10.26(s,1H,C
4′-OH),7.93(d,J=8.5Hz,2H,C
2′C
6′-H),6.94(d,J=8.5Hz,2H,C
3′C
5′-H,),6.73(s,1H,C
3-H),4.01(s,2H,-CH
2-),2.63-2.67(m,4H,2CH
2),2.30-2.42(m,4H,2CH
2),1.38-1.41(t,2H,-CH
2-),1.23-1.31(m,4H,2CH
2),0.856-0.885(t,3H,CH
3).ESI-MS:m/z441[M+H]
+。
Embodiment 2 Scutellarein Mannich response derivative solvabilities are measured
Physico-chemical properties such as medicine dissolution property can influence the absorption and the bioavailability thereof of medicine; Scutellarin mainly absorbs in having weakly alkaline enteron aisle; But the interior metabolism easily of, body poor owing to its soluble ester dissolubility has influenced its bioavailability; In order to examine or check the solubleness of Scutellarein Mannich response derivative in weakly alkaline solution, adopt determined by ultraviolet spectrophotometry.
Get each 1mg of Scutellarein Mannich response derivative that scutellarin and embodiment 2 prepare,, add methanol constant volume then to the 100ml volumetric flask, promptly get the standardized solution of compound 10 μ g/ml with the small amount of methanol dissolving.
Respectively get reference substance standardized solution 3,4,5,6,7,8ml; Place the 25ml volumetric flask respectively; Add methyl alcohol to scale, compound concentration is 6 parts of the reference substance working solutions of 1.2 μ g/ml, 1.6 μ g/ml, 2.0 μ g/ml, 2.4 μ g/ml, 2.8 μ g/ml and 3.2 μ g/ml, with the absorbancy at UV spectrophotometric determination 333nm place; Say that then absorbancy and reference substance solution concentration carries out linear regression, obtain the typical curve equation.
Get testing compound and place the 10ml volumetric flask respectively, add 0.15mol/L NaHCO respectively
3Solution 2ml adds propyl carbinol 4ml respectively under 20 ℃, and is airtight; Vortex 10min carries out centrifugal treating, and rotating speed is 30000r/min; Draw upper strata (propyl carbinol) and lower aqueous solution respectively; It is the same to measure the optical density method, and through the absorbancy computerized compound concentration in each phase that obtains, concrete experimental result is as described in Table 1:
Table 1 Scutellarein Mannich response derivative is at 0.15mol/L NaHCO
3Solution and n-Octanol dissolubility data
Can find out that through table 1 scutellarin is at 0.15mol/LNaHCO
3Solubleness in the solution and in n-Octanol is respectively 2.9922mg/ml and 0.2138mg/ml, and the water-soluble and ester dissolubility of Scutellarein Mannich response derivative all has significantly raising than scutellarin.
The research of the external removing of embodiment 3 Scutellarein Mannich response derivatives DPPH free radical activity
Modern medicine points out, thus the generation that the 26S Proteasome Structure and Function of radical through the damaging cells film gives rise to diseases, like aging, tumour, inflammation and cerebral ischemia etc.(1,1-diphenyl-2-picrylhydrazyl) radical is the most commonly used to remove DPPH in the vitro detection antioxidant for clearing radical method.DPPH solution has absorption more by force at the 517nm place; When the inhibitor of hydrogen supply capacity exists; With DPPH coupling the DPPH purple is disappeared or weaken (reaction as follows); And the degree that absorbancy and radical are eliminated is linear, calculates free radical scavenging activity through the linear change that adds inhibitor front and back absorbancy.
If receive reagent can remove the DPPH radical, then point out this medicine to have the effective concentration that reduces radicals such as hydroxy radical qiao, alkane radical or peroxy radical in the body, interrupt the effect of lipid peroxidation chain reaction.Research thinks that it is to influence that the flavonoid compound resistance of oxidation mainly receives the substitute mode of phenolic hydroxyl group and number, and the phenolic hydroxyl group H institute of flavonoid compound is positively charged big more, accepts the easy more more by force and combined with radical of electronic capability.Form semiquinone formula radical structure after flavonoid compound and the free radical reaction, the number that increases phenolic hydroxyl group to a certain extent can improve the anti-oxidant activity of flavonoid compound.Ortho-phenolic hydroxyl can produce intramolecular hydrogen bond after forming semiquinone formula radical, and intramolecular hydrogen bond can obviously reduce maximum system energy makes semiquinone formula radical more stable, and it is many more to form intramolecular hydrogen bond, and the compound with oxidation resistance activity is high more.Research relatively Scutellarein-7-position glycosyl derivatives has great importance for control ischemic cerebrovascular disease and developing new drug to measured by esr technique.
Materials and methods
Take the microwell plate method, measure scutellarin and Scutellarein Mannich response derivative thereof clearance rate at the 517nm place, the antioxidation activity in vitro of comparative evaluation scutellarin and Scutellarein Mannich response derivative thereof the DPPH radical.
Experimental technique
Scutellarein Mannich response derivative and DPPH that embodiment 2 is prepared use anhydrous alcohol solution.(maximum concentration is 500 μ molL to be tried thing to 100 μ L respectively
-1) be added in 96 orifice plates, (ultimate density is 80 μ molL to add 100 μ LDPPH solution then
-1), jolting is even, and light absorption value is measured in lucifuge reaction 30 minutes at the 517nm place.Calculate free radical scavenging activity according to following formula:
A
0: the absorbancy after solvent and the DPPH effect; A
1: the absorbancy after Scutellarein Mannich response derivative and the DPPH effect; A
2: the absorbancy when not adding DPPH, it is as described in Table 2 specifically to test structure:
Table 2 Scutellarein Mannich response derivative anti-oxidant activity data
Can find out that from table 2 the anti-DPPH oxidation activity of some compound such as Scutellarein Mannich response derivative I-1, I-2 is better than scutellarin, its IC
50Value is respectively 26.46 μ M, 24.96 μ M, and anti-DPPH oxidation activity of other Scutellarein Mannich response derivative and scutellarin are quite active.The protection activity experiment of the external PC12 cellular oxidation of embodiment 4 Scutellarein Mannich response derivatives damage model
Experimental principle
Scutellarin pours into back injury protection mechanism of action again to cardiac-cerebral ischemia and it be not immediately clear.After having research to think the heart and brain tissues ischemia-reperfusion, the oxyradical in the tissue (ROS) is a large amount of to be produced, thereby causes the oxidative damage of cell.The PC12 cell of differentiation has the characteristic of typical neuroendocrine cell on form and function, be widely used in studying curative effect and the toxicological effect of neuronal cell death mechanism, the mechanism of action of NGFF, neural medication etc.H
2O
2Being one of staple of oxyradical (ROS), is a kind of cellular oxidation stress-induced agent commonly used, is widely used in the cell death inducing Study of model.
Discover that scutellarin is to hydrogen peroxide (H
2O
2) and the PC12 cellular oxidation of glutamate induction damage significant provide protection is arranged, can significantly improve PC12 cellular oxidation reducing power, suppress H
2O
2Inductive cell membrane phospholipid acyl Serine turns up, suppresses H
2O
2Inductive DNA oxidation scission, inhibition caspase-3 mRNA active, that promote the Bcl-2 gene express, reduce intracellular reactive oxyradical (ROS) and Ca
2+Concentration, stability line mitochondrial membrane potential etc.Research thinks that extensively scutellarin can improve the redox ability of neurocyte under the cerebral ischemia pathological state, prevents and treats the cerebral ischemia neural cell injury.In the activity rating system of scutellarin, be external model often, with H with the PC12 cell
2O
2With the oxidative damage of PC12 cytosis analog cell, the research scutellarin is intended the provide protection of ischemia injury to the PC12 cell.
Mtt assay is to utilize the desaturase relevant with NADP that exists in the viable cell plastosome can make ectogenic bromination tetrazole blue (MTT) reduction, generates into the bluish voilet crystallisate (Formazan) of insoluble and is deposited in the cell, and dead cell does not have this function.DMSO 99.8MIN. (DMSO) or three liquid (10%SDS-5% isopropylcarbinol-0.01mol/L HCl) the purple crystal thing in can dissolved cell is measured its light absorption value with enzyme-linked immunosorbent assay instrument in the 570nm wavelength and is reflected its viable cell quantity indirectly.
Working method: get the same generation PC12 cell, the digestion back is by 5 * 10
4ML
-1Be inoculated in 96 orifice plates, every hole 100 μ L put into incubator and continue to cultivate 24h.Experiment is divided into blank group and H
2O
2Damage model is established 5 multiple holes for every group.The blank group is the DMEM nutrient solution of serum-free.H
2O
2The damage model group is for choosing 400 μ molL
-1H
2O
2After 1 hour damage time, add 100 μ L Scutellarein Mannich response derivatives, investigate Scutellarein Mannich response derivative concentration and be respectively 400 μ molL
-1(height), 200 μ molL
-1(in), 100 μ molL
-1Protection to PC12 cellular oxidation damage model when (low) is active.
Mtt assay detects cell viability: add 20 μ L MTT (5mgmL
-1), put hatch 4h in the incubator after lucifuge add 150 μ L DMSO, jolting is 10 minutes gently, and crystallisate is fully dissolved.Detect absorbancy (OD value) with ELIASA at the 570nm place.
Scutellarein Mannich response derivative dissolves with DMSO, and nutrient solution is diluted to 2.5%, and the final concentration of Scutellarein Mannich response derivative is 100 μ molL
-1, scutellarin is as positive control; Data all with
Expression, with the t check, experimental result is seen table 3 between group.
The external H of table 3 Scutellarein Mannich response derivative
2O
2Induce the protection of PC12 cellular oxidation damage model active
Can find out that from above activity data Scutellarein Mannich response derivative is to H
2O
2Inductive PC12 cellular oxidation damage all have a good provide protection, show that cardiac-cerebral ischemia is had the better protecting effect.
Embodiment 6 Scutellarein Mannich response derivatives are to the influence of rat thrombin time
At present, the screening ordinary method of antithrombotic compound is to investigate the active of compound anticoagulant and to the influence of thrombin time, the present invention investigates each compound antithrombotic acitivity through measuring compound to the influence of thrombin time.
Concrete grammar: get the healthy male rabbit; The vetanarcol physiological salt soln auricular vein injecting anesthetic of 30mg/kg rabbit body weight; Operation separates carotid atery and gets blood, is collected in the plastic centrifuge tube 3.8% Sodium Citrate aqueous solution anti-freezing (blood and antithrombotics volume ratio are 9:1).The centrifugal 10min of 800r/min, and the preparation platelet rich plasma (Platelet-rich plasma, PRP), the centrifugal 10min of 3000r/min, the preparation platelet poor plasma (Platelet-poor plasma, PPP).
With the scutellarin glycosides as control group; The Scutellarein Mannich response derivative that the embodiment of the invention 2 prepares is as experimental group; Sample is dissolved in 80% ethanol, is made into the solution that starting point concentration is respectively 1.2mg/ml, 0.6mg/ml, 0.3mg/ml, 0.15mg/ml, 0.075mg/ml.
The mensuration of PT (prothrombin time):
Principle: thrombokinase and calcium ion mixture can make thrombogen change zymoplasm into, and zymoplasm makes Fibrinogen change fibrin clot into, and time that grumeleuse forms and the extrinsic soagulation factor content in the blood plasma are negative correlation.
Method: add solvent or given the test agent 10 μ L, PPP 50 μ L in the test cup, preparatory temperature 3min in 37 ℃ of preparatory temperature holes will test cup and change TCH test channel over to, add 37 ℃ of inductor PT reagent 100 μ L of temperature in advance, the time that record PPP solidifies.
The mensuration of APTT (activated partial thromboplastin time):
Principle: blood plasma to be measured adds the activated partial thromboplastin solution, and Fibrinogen changes scleroproein into, measures and solidifies the required time, is blood plasma activated partial thromboplastin time to be measured (APTT).If the intrinsic pathway defectiveness, setting time promptly prolongs, and is directly proportional with the degree of single-factor shortage.Equally also lack and be directly proportional with the accumulation of the required factor of intrinsic pathway.
Method: solvent or Mannich response derivative 10 μ L, adding PPP 50 μ L and warm in advance APTT reagent 50 μ L in the test cup; Preparatory temperature 5min in 37 ℃ of preparatory temperature holes; To test cup and change TCH test channel over to, add 37 ℃ of inductor CaCl2 reagent 50 μ L of temperature in advance, the time that record PPP solidifies.
The mensuration of TT (thrombin time):
Principle: blood plasma to be measured adds the thrombin solution of demarcating, and Fibrinogen changes scleroproein into, measures and solidifies the required time, is thrombin time of blood plasma to be measured (TT).
Method: add solvent or Mannich response derivative 10 μ L, PPP 50 μ L in the test cup, preparatory temperature 3min in 37 ℃ of preparatory temperature holes will test cup and change TCH test channel over to, add the inductor TT reagent 50 μ L of room temperature, the time that record PPP solidifies.
The mensuration of FIB (Fibrinogen):
Principle: the quantitatively determined Fibrinogen is the classical way that generally uses, and this method is after adding zymoplasm, to measure the aggegation time of diluting plasma.
Method: 1.: the preparation of typical curve: the definite value blood plasma after will redissolving is processed the diluting plasma of 1:5,1:10,1:15,1:20,1:30 respectively.Get each 200 μ L of diluting plasma of different concns, 37 ℃ of temperature 3 minutes in advance add FIB reagent 100 μ L then respectively, measure setting time, by the automatic formation curve of coagulo meter and preserve.2.: add solvent or Mannich response derivative 10 μ L, PPP 50 μ L in the test cup, preparatory temperature 3min in 37 ℃ of preparatory temperature holes will test cup and change TCH test channel over to, add zymoplasm (FIB) the 50 μ L of room temperature, time or concentration that record PPP solidifies.
More than all experimental datas represent with
; Mean relatively adopts the t check between group, and concrete experimental result is as shown in table 4.
Table 4 Scutellarein Mannich response derivative is to the situation that influences of thrombin time and platelet aggregation activity
Show from the result of above table 4; Scutellarein Mannich response derivative all has good anticoagulant effect; And compound I-1, I-2, I-3 are stronger than scutellarin with the I-6 anticoagulant active, and its TT, PT, APTT are longer than scutellarin, and FIB is lower than scutellarin.Scutellarein Mannich response derivative I-4, I-5, I-7 anticoagulant active and scutellarin are quite active.
Show by above table experimental result; Compare with the control group scutellarin; Scutellarein Mannich response derivative with formula I provided by the invention can more obviously improve water-soluble, ester dissolubility, and Scutellarein Mannich response derivative has demonstrated good DPPH anti-oxidant activity, PC12 cellular oxidation injury protection activity and anticoagulant active.And the present invention can prepare serial salt to Scutellarein Mannich response derivative and alkali reaction; Water-soluble with further improvement; Thereby be prepared into different dosage form, therefore Scutellarein Mannich response derivative provided by the invention and salt thereof are expected to be developed further into the medicine into thrombotic diseases such as treatment myocardial infarction, ischemia injury, apoplexy.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.
Claims (8)
1. Scutellarein Mannich verivate is characterized in that, they be have general formula (
I) shown in compound:
R wherein
1=R
2, all represent alkyl;
Perhaps R1 is a hydrogen, and R2 is morpholinyl, piperazinyl or substituted piperazinyl.
2. Scutellarein Mannich verivate according to claim 1 is characterized in that described alkyl is methyl, ethyl, n-propyl, sec.-propyl, normal-butyl or isobutyl-.
3. Scutellarein Mannich verivate according to claim 1 is characterized in that the substituting group in the described substituted piperazinyl is methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, isobutyl-, n-pentyl or isopentyl.
4. the preparation method of the described Scutellarein Mannich of claim 1 verivate is characterized in that, may further comprise the steps:
A, to get scutellarin be raw material, and under the catalysis of the vitriol oil, heating hydrolysis in 95% ethanolic soln obtains Scutellarein, and is subsequent use;
B, get Scutellarein that step a obtains in methyl alcohol or ethanolic soln, with
With formalin reaction obtain the described Scutellarein Mannich of claim 1 verivate (
I), R wherein
1=R
2, all represent alkyl; Perhaps R1 is a hydrogen, and R2 is morpholinyl, piperazinyl or substituted piperazinyl.
5. the preparation method of Scutellarein Mannich verivate according to claim 4; It is characterized in that; The volumetric molar concentration of the used vitriol oil is 0.5 ~ 3mol/L among the step a; Scutellarin is 5g/100ml～10g/100ml with 95% consumption of ethanol ratio, and temperature of reaction is 110～120 ℃; Reaction times is 36～54 hours.
6. the preparation method of Scutellarein Mannich verivate according to claim 4; It is characterized in that; The molar equivalent of used formaldehyde is 1:1～2 among the step b; The molar equivalent of
is 1:1～2, reaction
Temperature is 25 ~ 35 ℃, and reaction solvent is methyl alcohol, ethanol equal solvent, and the reaction times is 30min ~ 24 hour.
7. the salt of the described Scutellarein Mannich of claim 1 verivate; It is characterized in that said salt is that said Scutellarein Mannich verivate and alkali metal hydroxide, alkaline earth metal hydroxides, alkaline carbonate, alkaline earth metal carbonate, alkali metal hydrocarbonate or alkali metal bicarbonates prepared in reaction obtain.
8. the application of each described Scutellarein Mannich verivate of claim 1 to 3 in preparation control thrombotic diseases medicine, said thrombotic diseases is myocardial infarction, ischemia injury.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210198829.8A CN102731459B (en) | 2012-06-15 | 2012-06-15 | Scutellarin aglycone Mannich derivatives, and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210198829.8A CN102731459B (en) | 2012-06-15 | 2012-06-15 | Scutellarin aglycone Mannich derivatives, and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102731459A true CN102731459A (en) | 2012-10-17 |
| CN102731459B CN102731459B (en) | 2014-05-28 |
Family
ID=46987841
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210198829.8A Expired - Fee Related CN102731459B (en) | 2012-06-15 | 2012-06-15 | Scutellarin aglycone Mannich derivatives, and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102731459B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103113497A (en) * | 2013-01-24 | 2013-05-22 | 昆明理工大学 | Scutellarein aglycone prodrug and preparation method thereof |
| CN112094255A (en) * | 2020-08-19 | 2020-12-18 | 昆明龙津药业股份有限公司 | Scutellarin aglycone derivative, preparation method and application thereof |
| CN113549044A (en) * | 2021-07-23 | 2021-10-26 | 中国药科大学 | 8-azacyclo-substituted chromone derivative and preparation method and pharmaceutical application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07324037A (en) * | 1993-03-01 | 1995-12-12 | Nippon Getsutou Kk | Production of platelet coagulation inhibiting substance from shell flower |
| WO2000019998A1 (en) * | 1998-10-06 | 2000-04-13 | Kgk Synergize | Compositions and methods of inhibiting neoplastic diseases with compounds related to limocitrin and 5-desmethyl sinensetin |
| CN101337957A (en) * | 2008-08-22 | 2009-01-07 | 四川大学 | Scutellarin derivates, preparation method and application thereof |
| CN101824017A (en) * | 2010-05-20 | 2010-09-08 | 南京中医药大学 | Method for preparing scutellarin aglycone |
-
2012
- 2012-06-15 CN CN201210198829.8A patent/CN102731459B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07324037A (en) * | 1993-03-01 | 1995-12-12 | Nippon Getsutou Kk | Production of platelet coagulation inhibiting substance from shell flower |
| WO2000019998A1 (en) * | 1998-10-06 | 2000-04-13 | Kgk Synergize | Compositions and methods of inhibiting neoplastic diseases with compounds related to limocitrin and 5-desmethyl sinensetin |
| CN101337957A (en) * | 2008-08-22 | 2009-01-07 | 四川大学 | Scutellarin derivates, preparation method and application thereof |
| CN101824017A (en) * | 2010-05-20 | 2010-09-08 | 南京中医药大学 | Method for preparing scutellarin aglycone |
Non-Patent Citations (2)
| Title |
|---|
| YANG PING，等: "Synthesis and biological evaluation of 8-substituted and deglucuronidated scutellarin and baicalin analogues as antioxidant responsive element activators", 《SCIENCE CHINA: CHEMISTRY》, vol. 54, no. 10, 25 August 2011 (2011-08-25), pages 1573, XP019957785, DOI: doi:10.1007/s11426-011-4361-4 * |
| 葛庆华，等: "灯盏花素在犬体内的药动学和绝对生物利用度研究", 《中国医药工业杂志》, vol. 34, no. 12, 20 December 2003 (2003-12-20), pages 618 - 620 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103113497A (en) * | 2013-01-24 | 2013-05-22 | 昆明理工大学 | Scutellarein aglycone prodrug and preparation method thereof |
| CN112094255A (en) * | 2020-08-19 | 2020-12-18 | 昆明龙津药业股份有限公司 | Scutellarin aglycone derivative, preparation method and application thereof |
| CN112094255B (en) * | 2020-08-19 | 2023-05-09 | 昆明龙津药业股份有限公司 | Scutellarin aglycone derivative, and preparation method and application thereof |
| CN113549044A (en) * | 2021-07-23 | 2021-10-26 | 中国药科大学 | 8-azacyclo-substituted chromone derivative and preparation method and pharmaceutical application thereof |
| CN113549044B (en) * | 2021-07-23 | 2024-01-23 | 中国药科大学 | 8-azacyclo-substituted chromone derivative and preparation method and pharmaceutical application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102731459B (en) | 2014-05-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Sui et al. | Recent advances on synthesis and biological activities of aurones | |
| CN101891728B (en) | Scutellarein derivative as well as preparation method and application thereof | |
| CN100596299C (en) | Dehydrosilibinin diester derivatives, preparation method and use thereof | |
| CN104860993B (en) | A kind of chromocor compound prodrug and application thereof | |
| CN102702155B (en) | Scutellarin aglycone methylate product based on in-vivo metabolic mechanism as well as preparation method and application of scutellarin aglycone methylate product | |
| CN101967135A (en) | 4-aryl coumarin compound and preparation method and application thereof | |
| CN102731459B (en) | Scutellarin aglycone Mannich derivatives, and preparation method and application thereof | |
| CN104478897A (en) | Oxazino-scutellarin aglycone derivative as well as preparation method and application thereof | |
| CN106674176B (en) | 7-substituted-4-aryl coumarin compound and preparation method and application thereof | |
| CN110498784B (en) | Nobiletin derivatives or pharmaceutically acceptable salts thereof, and preparation method and application thereof | |
| CN1944448B (en) | Puerarin derivative and its medicinal use | |
| CN102321134B (en) | Compound, preparation method and application | |
| CN104910119A (en) | Coumarin substituted flavonoid derivatives, and preparation method and application thereof | |
| CN105541859B (en) | Dihydrofuran and chromanone derivatives and preparation method thereof and medical usage | |
| CN105198849A (en) | Scutellarein amine derivative and preparation method and application thereof | |
| Wang et al. | Synthesis of Ring A‐Modified Baicalein Derivatives | |
| Shen et al. | Efficient synthesis of 6-O-methyl-scutellarein from scutellarin via selective methylation | |
| KR20010070381A (en) | Anti-cancer agent containing naphthoquinone compound | |
| WO2022124994A1 (en) | Methods to synthesize flavonoid dimers and oligomers and the use thereof | |
| CN102690308A (en) | Scutellarin-7-glycosyl derivatives, and preparation method and application thereof | |
| CN101966175A (en) | Application of dehydroabietic silybin in preparing glycosidase inhibitors | |
| CN106928074A (en) | Isopropanolamine substituted beta elemene derivatives and its production and use | |
| CN106565734A (en) | Psoralen ester derivatives and applications thereof | |
| CN106543197A (en) | A kind of psoralen Schiff basess derivant and purposes | |
| CN108586426B (en) | Alkoxy biphenyl/chalcone hybrid compound, and preparation method and medical application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140528 Termination date: 20210615 |