CN104356138A - Vasicine R-type optical isomer as well as preparation method and application thereof - Google Patents

Abstract

The invention discloses an R-type optical isomer of orchidine, a preparation method and application thereof. The R-type optical isomer of orchidine has the following chemical structural formula: The method is to prepare the ethanol-saturated solution by preparing the racemate of scallopine, adopt high-performance liquid chromatography, and use the mixed solution formed by ethanol and diethylamine at a volume ratio of 100:0.1 as the mobile phase, and use a chiral chromatographic column in the The column temperature is 25°C, the ultraviolet detection wavelength is 210nm, and the flow rate is 0.5mL/min for separation. The fractions in the chromatogram with a retention time in the range of 8.8 to 10.8 minutes are collected, concentrated and recrystallized. Experiments have proved that the pure R-type optical isomers of anthosine have stronger anti-AChE and anti-BChE activities than S-types, especially have significant anti-BChE activity, and can be used to prepare cholinesterase inhibitors and drugs for the treatment of dementia.

Description

An R-type optical isomer of orchidine and its preparation method and use

technical field

The present invention relates to an R-type optical isomer of oracine and its preparation method and application, and belongs to the technical field of natural medicines.

Background technique

Alzheimer's disease (AD for short), also known as senile dementia, is a chronic degenerative disease. Modern medical research has found that the neurobiological changes in the brain of AD patients are mainly the weakening of cholinergic function, the decrease of acetylcholine (Acetylcholine, ACh) level and activity in wide areas such as cerebral cortex and hippocampus, the degeneration of cholinergic neurons and fibrosis, necrotic or absent. Decreased activity of choline acetyltransferase (ChAT) and ACh content in the brain is recognized as an important feature of AD. The lack of ACh is a characteristic of AD. In clinical practice, various drugs are also tried to increase the level of ACh in the brain of patients to compensate for the loss of choline function, so as to achieve the purpose of treating AD. Currently the most mature and successful treatment for AD is the use of acetylcholinesterase (AcetylcholineEsterase, AChE for short) inhibitors. So far, the US FDA has approved four AChE inhibitors as therapeutic drugs for AD. These drugs include donepezil (Aricept ^™ ), rivastigmine (Rivastigmine, Exelon ^™ ), galantamine (Reminyl ^™ ) and tacrine (Tacrine, Cognex ^™ ).

AChE inhibitors increase the content of ACh by inhibiting the hydrolysis of ACh in the synaptic cleft, and finally achieve the purpose of treating AD. In the normal human brain, the ACh level in the brain is mainly regulated by AChE, butyrylcholinesterase (BChE) plays a very small role; while the activity of AChE in the brain of AD patients remains unchanged or decreases , but the activity of BChE increased. Therefore, the development of substances with both AChE and BChE inhibitory effects or the combined use of substances with AChE or BChE inhibitory effects is more effective for the treatment of AD (see literature: Greig N H, et al.Current Medical Research and Opinion, 2001, 17 (3): 159-165).

Dracine is a pyrrolo[2,1b] isolated from plants such as Adhatoda vasica Nees of the Acanthaceae family and Peganum harmala Linn of the Zygophyllaceae family. - quinazoline alkaloids. Although it has been reported in the literature that orchidine has inhibitory effects on acetylcholine and butyrylcholinesterase, there is no relevant report on the pharmacodynamic study of its optical isomers so far.

Contents of the invention

The object of the present invention is to provide an R-type optical isomer of oracine and its preparation method and application, so as to better utilize the medicinal value of oracine.

The R-type optical isomer of orchidine of the present invention has the following chemical structural formula:

The preparation method of the described rhizoid R-type optical isomer comprises the steps of:

a) dissolving the raceme of scallopedine with ethanol to make a saturated ethanol solution;

b) Inject the above-mentioned saturated ethanol solution into a high-performance liquid chromatograph, use a mixed solution formed by ethanol and diethylamine at a volume ratio of 100:0.1 as the mobile phase, use a chiral chromatographic column at a column temperature of 25°C, and detect by ultraviolet light Separation is carried out at a wavelength of 210nm and a flow rate of 0.5mL/min, and the fractions whose retention time is within the range of 8.8 to 10.8 minutes in the chromatogram are collected;

c) concentrating to dryness the solvent in the collected fractions to obtain the crude product;

d) Recrystallize the crude product with a mixed solvent formed by methanol and ethyl acetate at a volume ratio of 1:1, and then dry it to obtain the R-type optical isomer of orthosine.

As a preferred solution, the chiral chromatographic column is a Chiralpak AY-H chromatographic column with a column length of 150 mm, a column inner diameter of 4.6 mm, and a filler particle diameter of 5.0 μm.

As a preferred solution, the drying in step d) is vacuum drying at 35-45°C.

The R-type optical isomer of orchidine described in the present invention can be used to prepare cholinesterase inhibitors, and the cholinesterase inhibitors include acetylcholinesterase inhibitors or/and butyrylcholinesterase inhibitors agent.

The R-type optical isomer of orchidine described in the invention can be used to prepare medicine for treating senile dementia.

The medicine of the present invention can be used in various dosage forms for patients, such as various common oral solid preparations (tablets, capsules, granules, etc.), and other dosage forms such as sustained release and controlled release, and injections.

The R-type optical isomer of orchidine described in the present invention can be applied in the form of a free base or an addition salt formed with an acid, and the acid can be an inorganic acid, such as: sulfuric acid, hydrochloric acid, hydrobromic acid, Phosphoric acid, etc.; can also be organic acids, such as: acetic acid, oxalic acid, citric acid, gluconic acid, succinic acid, tartaric acid, p-toluenesulfonic acid, methanesulfonic acid, benzoic acid, lactic acid, maleic acid, etc.

Compared with the prior art, the present invention has the following beneficial effects:

The experiment proves that: the pure R-type optical isomer of oracine (abbreviated as R-type oracine) has a stronger The anti-AChE and anti-BChE activities, especially the significant anti-BChE activity, are 33.7 times of that of S-type schipin. The invention has important value for screening cholinesterase inhibitors with remarkable curative effect and good safety.

Description of drawings

Fig. 1 is the HPLC pattern of the raceme of orchidine used in the embodiment.

Fig. 2 is the HPLC spectrum of the S-type scallopine obtained by separation in the embodiment.

Fig. 3 is the HPLC spectrum of the R-type sucine obtained by separation in the embodiment.

Detailed ways

The present invention will be described in further detail and completely below in conjunction with embodiment; The experimental method that does not indicate specific condition in the following embodiment, usually according to routine condition or according to the condition suggested by the manufacturer.

Example 1: Separation and preparation of R-type orchidine and S-type orchidine from the racemate of orchidine

a) Dissolve 300mg of orbiline racemate in ethanol at 30°C to prepare a saturated ethanol solution (concentration: 50mg/mL);

b) Inject the above-mentioned ethanol saturated solution into a high-performance liquid chromatograph, use a mixed solution formed by ethanol and diethylamine in a volume ratio of 100:0.1 as the mobile phase, use a column length of 150 mm, a column inner diameter of 4.6 mm, and a filler The Chiralpak AY-H chiral chromatographic column with a particle diameter of 5.0 μm was separated under the conditions of a column temperature of 25°C, a mobile phase flow rate of 0.5mL/min, and a UV detection wavelength of 210nm. The retention times in the collected chromatograms were Fraction A within the range of 5.8 to 7.3 minutes and fraction B within the range of 8.8 to 10.8 minutes;

c) Concentrating to dryness the solvents in the collected Fraction A and Fraction B, respectively, to obtain the crude S-type and R-type rhizoids.

d) Recrystallize the crude S-type and R-type duckbilline with a mixed solvent of methanol and ethyl acetate at a volume ratio of 1:1, and then vacuum-dry them at 40°C until Constant weight, can be separated to obtain S-type orchidine and R-type orchidine.

Optical purity test: the HPLC purity of the isolated S-type and R-type antin are both >99%.

Fig. 1 is the HPLC spectrum of the racemate of orchidine used, Fig. 2 is the HPLC spectrum of the isolated S-type orchidine, and Fig. 3 is the HPLC spectrum of the isolated R-type orchidine.

Anticholinesterase activity test:

The purpose of the experiment: To investigate the effects of R-type orchidine, S-type orchidine, the mixture of R-type and S-type orchidine, and the racemate of orchidine on acetylcholinesterase (AChE) and butyrylcholine, respectively. Alkaline esterase (BChE) inhibitory activity comparison.

Experimental principle: Acetylcholine (ACh) or butyrylcholine (BCh) can be hydrolyzed under the action of AChE or BChE to generate acetic acid or butyric acid and choline, so the activity of AChE or BChE can be determined by measuring the amount of choline produced. When substances with AChE or BChE inhibitory activity are added, the production of choline will decrease, and the inhibition rate of the test substance can be calculated by measuring the production of choline.

Materials and Methods:

Tested substances: R-type orchidine, S-type orchidine, raceme of orchidine. Before the test, prepare a stock solution of the required concentration with methanol for future use.

Main reagents and materials: acetylcholinesterase (AChE) from Electrophorus electricus, butyrylcholinesterase (BChE) from horse serum, acetylcholine chloride (ACh), butyrylcholine chloride (BCh), chloride Choline (Ch), internal standard chlormequat (IS), and galantamine were all purchased from Sigma-Aldrich Company in the United States, and basil, chromatographic-grade methanol and acetonitrile were purchased from Fisher Co. in the United States, and chromatographic-grade formic acid was purchased from Ultrapure water was obtained from Tedia Inc. in the United States through Milli-Q Academic System, and other reagents were commercially available analytical grade.

Main instruments: Waters-ACQUITYTM UPLC system produced by Waters America, Micromass Quattro Premier XE produced by Waters UK, tandem triple quadrupole mass spectrometer, refrigerated centrifuge and micropipette produced by Eppendorf, Germany.

Reagent preparation:

Na ₂ HPO ₄ solution with a molar concentration of 1mol/L: Weigh 358.14g of dodecahydrate Na ₂ HPO ₄ and dissolve it in 1L of ultrapure water;

NaH ₂ PO ₄ solution with a molar concentration of 1mol/L: Weigh 156.01g of NaH ₂ PO ₄ dihydrate and dissolve it in 1L of ultrapure water;

Phosphate buffer solution with a molar concentration of 1mol/L and _a pH value of 7.6: take 84.5mL of a _Na2HPO4 solution with a molar concentration of 1mol/L and _15.5mL of a _NaH2PO4 solution with a molar concentration of 1mol/L and dilute to 1000mL is ready;

Phosphate buffer solution with a molar concentration of 20mmol/L and a pH value of 7.6: take 100mL of Na ₂ HPO ₄ solution with a molar concentration of 0.02mol/L and add a certain amount of NaH ₂ PO ₄ solution with a molar concentration of 0.02mol/L and mix them Adjust the pH value to 7.6;

Preparation of standard solution and quality control sample: Take appropriate amount of Ch and IS in a 25mL volumetric flask, add methanol to dissolve and prepare stock solutions with molar concentrations of 5.386mmol/L and 2.012mmol/L respectively, and use the initial mobile phase to pass through a Serial dilutions were used to obtain working solutions and quality control samples, and methanol was used to dilute to obtain IS working solutions with a molar concentration of 1.899 μmol/L. All solutions were stored at 4°C and placed at room temperature before use;

Preparation of sample solution: use phosphate buffer solution with a molar concentration of 20mmol/L and a pH value of 7.6 to prepare AChE and BChE mother solutions with a concentration of 3.470unit/mL and store them at -80°C; use a molar concentration of 20mmol/L, Phosphate buffer solution with a pH value of 7.6 was prepared to obtain ACh and BCh substrate solutions with a molar concentration of 11.011 μmol/L and 14.305 μmol/L respectively; an appropriate amount of the sample to be tested was prepared by dissolving an appropriate amount of dimethyl sulfoxide with a concentration of 0.2% by volume Obtain a mother solution with a molar concentration of 20mmol/L, and dilute it with a phosphate buffer solution with a molar concentration of 20mmol/L and a pH value of 7.6 to obtain a series of concentrations of the sample solutions to be tested, as shown in Table 1, wherein: RS is duckbill Anthocyanin racemate; S is S-type orchidine; R is R-type orchidine.

Table 1, the composition of the sample to be tested and the molar concentration of the sample solution to be tested

The composition of the sample to be tested Molar concentration of sample solution to be tested (μmol/L) Galantamine 5,2,1,0.5,0.2,0.1,0.05,0.02,0.01 RS 100, 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128 S 100, 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128 R 100, 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128 S:R=10:1 100, 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128 S:R=8:1 100, 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128 S:R=6:1 100, 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128 S:R=4:1 100, 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128 S:R=2:1 100, 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128 S:R=1:2 100, 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128 S:R=1:4 100, 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128 S:R=1:6 100, 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128 S:R=1:8 100, 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128 S:R=1:10 100, 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128

AChE and BChE inhibition test:

AChE inhibition test: first take 10 μL of the sample solution to be tested, then add 40 μL of AChE enzyme solution with a concentration of 0.0035 unit/mL to mix, leave it for 15 minutes, add 50 μL of ACh substrate solution with a molar concentration of 5.505 μmol/L, and react at 25 °C After 20 minutes, stop the reaction immediately with 300 μL of IS-ice acetonitrile solution at 0 °C containing a molar concentration of 1.899 μmol/L, centrifuge with a centrifuge at 15,000 rpm for 10 minutes, and take the supernatant for analysis.

BChE inhibition test: Take 10 μL of the sample solution to be tested, then add 40 μL of BChE enzyme solution with a concentration of 0.008 unit/mL to mix, let it stand for 15 minutes, add 50 μL of BCh substrate solution with a molar concentration of 7.152 μmol/L, and react at 25 °C for 20 Minutes later, stop the reaction with 300 μL of IS-ice acetonitrile solution at a temperature of 0 °C and a molar concentration of 1.899 μmol/L, centrifuge with a centrifuge at 15,000 rpm for 10 minutes, and take the supernatant for analysis.

Sample analysis:

Use the Waters-ACQUITYTM UPLC system system to separate, the chromatographic column uses the ACQUITY UPLC HSS T3 chromatographic column with a column length of 100mm, a column inner diameter of 2.1mm, and a filler particle diameter of 1.8μm. The column temperature is 40°C, and the mobile phase is 0.1vol% formic acid A mixed solution of aqueous solution and methanol with a volume ratio of 98:2, the flow rate of the mobile phase is 0.3mL/min, isocratic elution, and the injection volume is 5μL.

A Micromass Quattro Premier XE series triple quadrupole mass spectrometer combined with electrospray was used as the analytical detector, and the multiple reaction monitoring mode was adopted in the positive ion mode. The mass spectrometry parameters were: capillary voltage 3.00kV, extraction voltage 3.00V, ion source temperature 120°C , the desolvation temperature is 400°C, the desolvation gas is nitrogen with a flow rate of 550L/hour, the cone gas is nitrogen with a flow rate of 50L/hour and a purity of 99.9%, and the collision gas is argon with a purity of 99.999%. MassLynx 4.1 software complete.

data processing:

Calculate the inhibition rate of each sample to be tested to AChE and BChE by the results obtained by the determination in the following format:

Inhibition rate=(negative control sample-test sample)/negative control sample×100%.

The _IC50 values were obtained by nonlinear correlation analysis and fitting using Prism software, as shown in Table 2 for details.

Table 2, the inhibitory activity of test substance to AChE and BChE

From the results in Table 2, it can be seen that the inhibitory activity of the test substance on AChE and BChE is positively correlated with the proportion of R-type orchidine in the test substance, that is, the greater the proportion of R-type orchidine, the stronger the activity ; Pure R-type duckbilline has stronger anti-AChE and anti-BChE activities than S-type duckbilline, especially has significant anti-BChE activity, which is 33.7 times that of S-type duckbilline. R-type duckbilline can be used to prepare cholinesterase inhibitor medicines, and the cholinesterase inhibitor medicines can be acetylcholinesterase inhibitor medicines, butyrylcholinesterase inhibitor medicines, acetylcholinesterase inhibitor medicines, and acetylcholinesterase inhibitor medicines. drugs and butyrylcholinesterase inhibitors. Furthermore, the R-type anthalline can be used to prepare medicine for treating senile dementia, and has medicinal value.

Embodiment 2: the preparation of R type duckbilline tablet

a) Prepare 1000 prescriptions:

raw material Weight (g) R-type duckbilline 15.0 starch 122.0 Sodium carboxymethyl starch 7.5 Povidone K ₃₀ 2.5 talcum powder 3.0

b) Preparation method: Take 15.0g of R-type duckbilline and 122.0g of starch, pass through a 80-mesh sieve, add 7.5g of sodium carboxymethyl starch according to the internal and external addition method, mix well, add a volume ratio containing 2.5g of povidone K30 The ethanol solution with a concentration of 10% is used to make soft materials, and granules are made into granules with a 20-mesh sieve. After the wet granules are dried at 60-80°C, 3.0 g of talcum powder is added, mixed evenly, granulated through a 16-mesh sieve, and pressed into 1000 tablets, that is.

Embodiment 3: the preparation of R-type scallopine injection

a) Prescription:

raw material Weight (g) R-type duckbilline 10.0 lactic acid 1.0 Sodium chloride 18.0 Sulfite 2.0 0.1mol/L citric acid Adjust pH to 2.0~5.0 Water for Injection 2000mL

b) Preparation method: Weigh 10.0g of R-type duckbilline, 1.0g of lactic acid, and 18.0g of sodium chloride, place them in a preparation container, add 2/3 of the prescription amount of water for injection, stir and add 0.1M citric acid to make Dissolve the main drug and adjust the pH to 2.0~5.0, then add 2.0g of antioxidant sodium sulfite, add water for injection to 2000mL full amount, stir for about 30 minutes, add medicinal activated carbon for adsorption for 15~20 minutes, the amount of medicinal carbon used is 0.03% Left and right, filtered, aseptically filled and sterilized to obtain the injection.

To sum up: the R-type orchidine provided by the present invention has stronger AChE and BChE inhibitory activity than the S-type orchidine racemate, and can be formed in the form of free base or with acid Addition salt form and pharmaceutical auxiliary materials prepare various pharmaceutical dosage forms of cholinesterase inhibitors and treatment of senile dementia, such as various common oral solid preparations (tablets, capsules, granules, etc.), compared with the same kind in the prior art The medicine has the advantages of remarkable curative effect and small dosage, can greatly reduce the incidence of adverse reactions, and has significant medicinal value.

Finally, it is necessary to explain here that: the above content is only used to further describe the technical solution of the present invention in detail, and cannot be interpreted as limiting the protection scope of the present invention. Improvements and adjustments all belong to the protection scope of the present invention.

Claims (10)

1. a vasicine R type optical isomer, is characterized in that, has following chemical structural formula:

2. prepare a method for vasicine R type optical isomer according to claim 1, it is characterized in that, comprise the steps:

A) by vasicine raceme dissolve with ethanol, alcohol saturated solution is made;

B) above-mentioned alcohol saturated solution is injected high performance liquid chromatograph, the mixing solutions formed for 100:0.1 by volume with ethanol and diethylamine is for moving phase, use chiral chromatographic column column temperature be 25 DEG C, ultraviolet detection wavelength is 210nm, flow velocity for 0.5mL/ minute under be separated, collecting retention time in color atlas is the cut within the scope of 8.8 ~ 10.8 minutes;

C) solvent in concentrated dry collected cut, obtains crude product;

D) carry out drying after recrystallization process being carried out to crude product with the mixed solvent that methyl alcohol and ethyl acetate are formed for 1:1 by volume, obtain vasicine R type optical isomer.

3. method as claimed in claim 2, is characterized in that: described chiral chromatographic column selects the Chiralpak AY-H chromatographic column that column length is 150mm, column internal diameter is 4.6mm, filling agent particle diameter is 5.0 μm.

4. vasicine R type optical isomer according to claim 1 is for preparing the pharmaceutical use of anticholinesterase.

5. the anticholinesterase described in claim 4 comprises acetylcholinesterase depressant or/and butyrylcholinesterase inhibitor.

6. vasicine R type optical isomer according to claim 1 is for preparing the pharmaceutical use for the treatment of senile dementia.

7. the addition salt form that the vasicine R type optical isomer described in claim 4 or 6 is free alkali form or is formed with acid.

8. the acid described in claim 7 is mineral acid or organic acid.

9. the mineral acid described in claim 8 is sulfuric acid, hydrochloric acid, Hydrogen bromide or phosphoric acid.

10. the organic acid described in claim 8 is acetic acid, oxalic acid, citric acid, gluconic acid, succsinic acid, tartrate, tosic acid, methylsulfonic acid, phenylformic acid, lactic acid or toxilic acid.