Study On The Preparation Of 3β-[methyl-[2-(4-nitrophenoxy)ethyl] Amino]con-5-enine Hydrochloride And Its Pharmacodynamics Of Anti-Alzheimer’s Disease

Alzheimer’s disease（AD）is a progressive neurological degeneration of senile disease,which associated with memory loss,cognitive disorders and other disease.The social life,individuals living and emotional personality of older persons were also affacted by AD.Currently,the most effective drugs to treat AD are acetylcholinesterase inhibitors（AchEI）.Due to acetylcholinesterase inhibitors on mild to moderate AD effective,acetylcholinesterase inhibitors was considered as the first-line drug against disease.In the previous study,we found a potent acetylcholinesterase inhibitor named as（3β-[methyl-[2-（4-nitrophenoxy）ethyl] amino]con-5-enine）.However,the preparation process of this drug was complex and because of requires repeated column chromatography.Preparation of this drug requires for a long period of time,which dely pharmacodynamics and pharmacological studies.Therefore,In the present study,we optimizated preparation process of 3β-[methyl-[2-（4-nitrophenoxy）ethyl]amino]con-5-enine hydrochloride and evaluated the pharmacokinetics,pharmacodynamics and tissue distribution of 3β-[methyl-[2-（4-nitrophenoxy）ethyl]amino]con-5-enine in rats.The main contents and results were as follows:1.Preparation process optimization of 3β-[methyl-[2-（4-nitrophenoxy）ethyl]amino]con-5-enine hydrochloride（1）Optimization of extraction process of total alkaloids from Holarrhena antidysenteriacaSingle-factor design was employed to obtian four main process parameters.The selected variables were optimized by response surface analysis based on the extraction yield of total alkaloids.The results showed that the effect of the investigated factors on the total alkaloids extraction yield was in the order as: extraction temperature>ethanol concentration>extraction time> material-solvent ratio.The optimal extraction parameters were 85% ethanol,extraction at 80 ℃for 3 h with a material-solvent ratio 1:10 g/mL.The extraction yield of total alkaloids was（23.8±0.12）mg/g under these conditions.（2）Preparation process of 3β-[methyl-[2-（4-nitrophenoxy）ethyl]amino]con-5-enine hydrochlorideTotal alkaloids were isolated by using macroporous resin column chromatography which to yield lowly pure isoconessimine.A mixture of lowly pure isoconessimine and 2-Bromoethyl-（4-nitrophenyl）phenol ether in anhydrous acetonitrile was reflux reaction 16 h at 85°C.The crude product was chromatographed on a silica gel column to obtain 3β-[methyl-[2-（4-nitrophenoxy）ethyl]amino]con-5-enine（purity,98%）.The 3β-[methyl-[2-（4-nitrophenoxy）ethyl]amino]con-5-enine hydrochloride was obtained by acidizing 3β-[methyl-[2-（4-nitrophenoxy）ethyl]amino]con-5-enine with HCl.Compared with the previous preparation process,the current preparation process have advantages of simplicity of operater and short preparation period.2.Study on the method for the determination of 3β-[methyl-[2-（4-nitrophenoxy）ethyl]amino]con-5-enine in ratsA high performance liquid chromatography（HPLC）method was established for the determination of the content of 3β-[methyl-[2-（4-nitrophenoxy）ethyl]amino]con-5-enine hydrochloride in rats.The chromatographic conditions are as follows: chromatographic column: phenomenex C18,（5 g,110 A,150 * 4.6mm）;mobile phase: methanol: water: three ethylamine（70:30:3 v/v/v）;detection wavelength: 300nm;flow rate: 1.0mL/min.The precision and accuracy,recovery rate,dilution linearity and stability were also investigated.The results showed that the coefficient correlation of concentrations-area ranged from 0.9987 to 0.9996.The lowest limit of quantitation（LLOQ）was 0.08μg/mL.The relative standard deviation（RSD）of intra-day and inter-day were 0.69¹.05% and 0.78⁰.96%,which were less than 15%.The recovery rate was 87.95%⁹7.25%.The method gave high sensitivity,wide linearity,and specificity,which is suitable for the study of pharmacokinetics and distribution regularity of 3β-[methyl-[2-（4-nitrophenoxy）ethyl]amino]con-5-enine hydrochloride.4.Study on the pharmacokinetics and tissue distribution of 3β-[methyl-[2-（4-nitrophenoxy）ethyl]amino]con-5-enine hydrochlorideAfter the rats were orally administered of 3β-[methyl-[2-（4-nitrophenoxy）ethyl]amino] con-5-enine hydrochloride.the pharmacokinetic parameters were calculated by DAS3.0 software.The pharmacokinetic parameters was as followig:C_max:2.81±0.36μg/mL;T_max:2.48±0.12h;T_1/2:12.24±1.15h:AUC（0-t）:68.80±2.14μg/L.h;CL:0.71±0.21 L/h/kg;Vd : 12.42 ± 1.26 L/kg 。 The results showed that the concentration-time data and pharmacokineties parameters conformed to a 2-compartment model.3β-[methyl-[2-（4-nitrophenoxy）ethyl]amino]con-5-enine hydrochloride has a slowly clearance and large distribution volume in rats.Tissue distribution result showed the distribution of 3β-[methyl-[2-（4-nitrophenoxy）ethyl]amino]con-5-enine hydrochloride was rapid and wide.After orally administration 9 h,3β-[methyl-[2-（4-nitrophenoxy）ethyl]amino]con-5-enine were determined in the liver,small intestine,stomachand large intestine,which the major distribution tissues in rats.It was found that 3β-[methyl-[2-（4-nitrophenoxy）ethyl]amino]con-5-enine could be detected in brain,which suggested that 3β-[methyl-[2-（4-nitrophenoxy）ethyl]amino]con-5-enine could cross the blood-brain barrier.4.Study on pharmacodynamic of 3β-[methyl-[2-（4-nitrophenoxy）ethyl] amino] con-5-enine hydrochlorideIn this part,we established scopolamine-induced rats model of learning and memory impairment.The effects of 3β-[methyl-[2-（4-nitrophenoxy）ethyl]amino]con-5-enine hydrochloride on learning and memory disorder rats was evaluated by Morris water maze test.The results of decreasing escape latency,increasing searching for the security platform and effective coverage times showed that the 3β-[methyl-[2-（4-nitrophenoxy）ethyl]amino]con-5-enine hydrochloride can significantly improve the learning performances in scopolamine-induced learning and memory impairments in rats.