CN107652233B - Preparation method and application of tacrine-sinapic acid heterozygote - Google Patents
Preparation method and application of tacrine-sinapic acid heterozygote Download PDFInfo
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- CN107652233B CN107652233B CN201711054725.9A CN201711054725A CN107652233B CN 107652233 B CN107652233 B CN 107652233B CN 201711054725 A CN201711054725 A CN 201711054725A CN 107652233 B CN107652233 B CN 107652233B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D219/00—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems
- C07D219/04—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the ring system
- C07D219/08—Nitrogen atoms
- C07D219/10—Nitrogen atoms attached in position 9
- C07D219/12—Amino-alkylamino radicals attached in position 9
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/473—Quinolines; Isoquinolines ortho- or peri-condensed with carbocyclic ring systems, e.g. acridines, phenanthridines
Abstract
The invention discloses a tacrine-sinapic acid hybrid, a medicinal composition containing the tacrine-sinapic acid hybrid, a preparation method and application. The tacrine-sinapic acid hybrid has the following structure:
Description
Technical Field
The invention belongs to the field of organic synthesis and medicines, and particularly relates to a tacrine-sinapic acid hybrid for treating Alzheimer disease, a medicinal composition containing the tacrine-sinapic acid hybrid, a preparation method and application, in particular to a tacrine-sinapic acid hybrid prepared by reacting amino tacrine with sinapic acid, and application of the tacrine-sinapic acid hybrid in preparing anti-Alzheimer medicines.
Background
Tacrine is the first drug approved by the FDA for the treatment of Alzheimer's Disease (AD) and belongs to the first generation of acetylcholinesterase inhibitors. However, its clinical use is limited due to severe hepatotoxicity. The research proves that the hepatotoxicity of tacrine is closely related to free primary amino group of tacrine, and the current research mostly adopts molecular fragments with different functions to conjugate tacrine, so that on one hand, the free primary amino group of tacrine is shielded, the hepatotoxicity of the whole molecule is reduced, on the other hand, new functions can be added, and the whole molecule has the characteristics of multifunctional and multi-target function[1-2]。
Fang et al[3-4]It is reported that tacrine-ferulic acid hybrid (I) has both acetylcholinesterase inhibitory activity and antioxidant activity, which is nearly 10 times that of tacrine. Chao et al[5]It was reported that tacrine-caffeic acid hybrid (II) has acetylcholinesterase inhibitory activity nearly 3 times that of tacrine, and inhibits amyloid A β aggregation and neuroprotective activity, Xie et al[6]Tacrine-vitamin E hybrid (iii) was reported to have approximately 11-fold higher acetylcholinesterase inhibitory activity than tacrine, no significant neurocytotoxicity (PC12) at 12.5 μ M, and significantly reduced acute hepatotoxicity. Chen et al[7]Silibinin-tacrine heterozygote (IV) is synthesized, the acetylcholinesterase inhibition capacity of the silybin-tacrine heterozygote is 0.29 times that of tacrine, but the toxicity of the whole molecule is obviously reduced compared with tacrine. The compound has no obvious neurocytotoxicity (HT22) at 50 mu M and shows a certain neurocyte protection effect;in addition, its hepatotoxicity is also significantly reduced.
The research shows that the conjugate of sinapic acid and tacrine, namely the target compound in the patent, has more excellent acetylcholinesterase inhibition activity than ferulic acid-tacrine (10 times of tacrine) or caffeic acid-tacrine (3 times of tacrine), and inhibits the acetylcholinesterase inhibition activity and the acetylcholinesterase inhibition activity as well as the acetylcholinesterase inhibition activity, the amyloid self-aggregation inhibition activity, the oxidation resistance, the neuroprotection and the hepatotoxicity, compared with the tacrine inhibition activity, namely the target compound in the patent, compared with the target compound in ferulic acid-tacrine (10 times of tacrine) or the caffeic acid-tacrine (3 times of tacrine), and inhibits the acetylcholinesterase inhibition activity and the amyloid A β aggregation and the neuroprotective activity, 1) the target product has the acetylcholinesterase inhibition activity of 15 times of tacrine and the amyloid A β aggregation and the neuroprotective activity in the experimental range of 12. mu. the survival rate of cells is 130. mu.25.25.25. the target product has no toxicity, and the growth of the target product has no toxicity to PC in the range of the cell growth of the cell2The injured PC12 cells have obvious protective effect, the neuroprotective activity is obvious dose-dependent (74.5%, 92.4%, 99.5% and 106.8%), the cell survival rate is obviously increased along with the increase of the concentration.3) the tacrine-sinapic acid hybrid of the invention has the activity of inhibiting the self-aggregation of the A β 42 protein, the inhibition rate of the self-aggregation of the A β 42 protein is correspondingly increased along with the increase of the concentration of the tacrine-sinapic acid hybrid in the concentration interval of 0.0064-100 mu M, the dose-effect relationship is S-shaped curve, the action mode of the enzyme-substrate is typical, the obvious effect is better than that of the control drug tacrine.4), the liver drug enzyme research (ALT/AST) reveals that the liver toxicity is greatly relieved and is close to a blank control groupThe composition is a high-efficiency and low-toxicity drug molecule for treating Alzheimer's disease.
Reference to the literature
[1]Wu W Y,Dai Y C,Li N G,et al.Novel multitarget-directed tacrinederivatives as potential candidates for the treatment of Alzheimer's disease[J].J Enzyme Inhib Med Chem,2017, 32(1):572-587.
[2]Guzior N,Wieckowska A,Panek D,et al.Recent development ofmultifunctional agents as potential drug candidates for the treatment ofAlzheimer's disease[J].Curr Med Chem, 2015,22(3):373-404.
[3]Fang L,Kraus B,Lehmann J,et al.Design and synthesis of tacrine-ferulic acid hybrids as multi-potent anti-Alzheimer drug candidates[J].BioorgMed Chem Lett,2008,18(9): 2905-2909.
[4] Tacrine-ferulic acid heterocomplex, its preparation method and its pharmaceutical composition [ P ] Chinese patent, 200810028558.5, 2008-06-05.
[5]Chao X,He X,Yang Y,et al.Design,synthesis and pharmacologicalevaluation of novel tacrine-caffeic acid hybrids as multi-targeted compoundsagainst Alzheimer's disease[J]. Bioorg Med Chem Lett,2012,22(20):6498-6502.
[6]Xie S S,Lan J S,Wang X B,et al.Multifunctional tacrine-troloxhybrids for the treatment of Alzheimer's disease with cholinergic,antioxidant,neuroprotective and hepatoprotective properties[J].Eur J MedChem,2015,93:42-50.
[7]Chen X1,Zenger K,Lupp A,et al.Tacrine-silibinin codrug showsneuro-and hepatoprotective effects in vitro and pro-cognitive andhepatoprotective effects in vivo[J].J Med Chem,55(11):5231-5342.
Disclosure of Invention
It is an object of the present invention to provide a tacrine-sinapic acid hybrid for use in the treatment of alzheimer's disease.
It is a second object of the invention to provide a method for preparing a tacrine-sinapic acid hybrid.
It is a third object of the present invention to provide a pharmaceutical composition comprising tacrine-sinapic acid hybrid or tautomer thereof or pharmaceutically acceptable salt thereof or prodrug thereof or solvate thereof as an active ingredient and a pharmaceutically acceptable carrier.
It is a fourth object of the present invention to provide a tacrine-sinapic acid hybrid and the use of pharmaceutical compositions thereof as anti-alzheimer agents.
The technical scheme of the invention is summarized as follows:
a tacrine-sinapic acid hybrid having the structure:
a method for preparing a tacrine-sinapic acid hybrid is characterized in that the hybrid is prepared according to the following steps of 1: 1 by reacting aminotaraxacum with sinapic acid catalyzed by EDCI and DMAP to obtain a tacrine-sinapic acid hybrid having the following structure:
the above method is preferably: 0.754g (3.36mmol) of sinapinic acid, 0.773g (4.03mmol) of EDCI, 0.123g of DMAP (1.01mmol) and 10mL of anhydrous N, N-dimethylformamide are added in turn in a round-bottomed flask; then, 1g (3.36mmol) of Aminotacrine was added, and the reaction was stirred at room temperature for 12 hours. The reaction was almost completed by TLC (dichloromethane: methanol 10: 1), the solvent was evaporated under reduced pressure, and the mixture was separated by silica gel column chromatography (eluent: dichloromethane: methanol 30: 1-10: 1) to obtain 1.091g of tacrine-sinapic acid hybrid (pale yellow oil) with a yield of 65%.
A pharmaceutical composition containing tacrine-sinapic acid heterozygote is prepared by mixing
The tacrine-sinapic acid heterozygote or the tautomer thereof or the medicinal salt thereof or the prodrug thereof or the solvate thereof is mixed with one or more pharmaceutically acceptable carriers, so that the amount of the tacrine-sinapic acid heterozygote accounts for 0.5 to 60 percent of the total weight of the pharmaceutical composition, and the tacrine-sinapic acid heterozygote is prepared into any clinically acceptable dosage form, including tablets, pills, capsules, granules, suspensions, aerosols, oral liquid, ointments, gels, patches, injections, infusion solutions, freeze-dried powders and sustained and controlled release preparations.
The pharmaceutically acceptable carrier includes conventional diluent (such as at least one of water for injection, microcrystalline cellulose, etc.), filler (such as at least one of mannitol, sucrose, lactose, polyethylene glycol, tween 80, sorbitol, menthol, liquid paraffin, vaseline, stearic acid, glyceryl monostearate, lanolin, mineral oil, DMSO, etc.), binder (such as at least one of carbomer, acacia, starch, cellulose, gelatin, polyvinylpyrrolidone, polyacrylamide, etc.), disintegrant (such as at least one of sodium carboxymethyl starch, croscarmellose sodium, hypromellose, low-substituted hydroxypropyl cellulose, etc.), lubricant (such as at least one of talc, magnesium stearate, calcium stearate, solid polyethylene glycol, lecithin, silicon dioxide, silica gel micropowder, etc.), humectant (such as at least one of propylene glycol, glycerol, ethanol, etc.), stabilizer (at least one of ethylene diamine tetraacetic acid, sodium thiosulfate, sodium metabisulfite, sodium sulfite, sodium bisulfite, ethanolamine, sodium bicarbonate, sodium acetate, nicotinamide, vitamin C and the like), osmotic pressure regulator (at least one of sodium chloride, glucose and the like), pH regulator (at least one of triethanolamine, sodium hydroxide, sodium citrate and the like), and preservative (at least one of chlorobutanol, nipagin ester, ethylparaben, benzalkonium bromide and the like). The auxiliary materials can be common dosage, and are mixed with the tacrine-sinapic acid heterozygote according to common proportion, and when the dosage of the tacrine-sinapic acid heterozygote is determined, the proportion of the medicinal auxiliary materials can be properly adjusted according to requirements.
The medicinal preparation contains 1-500mg of tacrine-sinapic acid heterozygote per unit dose, and the application amount can be adjusted according to various factors such as the application route, the age, the weight, the disease type and the severity of patients and the like, and can be properly changed according to the manifestations of clinical cases. The daily dose is generally 200-400mg/kg, preferably 250-350mg/kg, and most preferably 300-350 mg/kg.
Application of a tacrine-sinapic acid heterozygote and a pharmaceutical composition thereof in preparing anti-Alzheimer drugs.
The tacrine-sinapic acid heterozygote has strong inhibitory activity on acetylcholinesterase and butyrylcholinesterase, and the inhibition capacity of the tacrine-sinapic acid heterozygote is 15 times that of tacrine. The tacrine-sinapic acid heterozygote has a strong inhibiting effect on amyloid A beta self-aggregation, also has obvious antioxidant activity, has no obvious in vitro neurocytotoxicity and in vivo acute hepatotoxicity, shows obvious in vitro neurocyte protective activity, and can be used for treating Alzheimer's disease.
Drawings
FIG. 1 is a scheme for the synthesis of intermediate 1 of example 1 of the present invention.
FIG. 2 is a high resolution mass spectrum of intermediate 1 of example 1 of the present invention.
FIG. 3 is a scheme of synthesis of intermediate 2 of example 2 of the present invention.
FIG. 4 shows the synthesis of Aminotacrine in example 3 of the present invention.
FIG. 5 is a high resolution mass spectrum of Aminotacrine of example 3 of the present invention.
FIG. 6 shows the NMR spectrum of Aminotacrine in example 3 of the present invention.
FIG. 7 is a scheme showing the synthesis of tacrine-sinapic acid hybrid according to example 4 of the present invention.
FIG. 8 is a high resolution mass spectrum of a tacrine-sinapic acid hybrid of example 4 of the present invention.
FIG. 9 is a NMR spectrum of a tacrine-sinapic acid hybrid of example 4 of the present invention.
FIG. 10 is a carbon nuclear magnetic resonance spectrum of a tacrine-sinapic acid hybrid according to example 4 of the present invention.
FIG. 11 is a graph showing the inhibitory activity of tacrine-sinapic acid hybrid on amyloid A β self-aggregation in example 25 of the present invention.
FIG. 12 is an atomic force micrograph of the amyloid A β auto-aggregation inhibitory activity of tacrine-sinapic acid hybrid of example 26 of the present invention.
FIG. 13 is a graph of the neurotoxicity and neuroprotective effect of tacrine-sinapic acid hybrids on PC12 cells of example 28 of the present invention.
Detailed Description
The invention is further illustrated by the following examples, which are intended only for a better understanding of the invention and do not limit the scope of the invention:
example 1 Synthesis of intermediate 1
Anthranilic acid (50.0g, 364mmol) was weighed out and dissolved in 100ml toluene, cyclohexanone (45.3ml, 437mmol) was added, the reaction was refluxed at 110.6 ℃ for 2h, and about 6.8ml water was removed. The mixture was cooled to room temperature, needle crystals precipitated, further cooled to 0 ℃, filtered, and washed with toluene (50mL) and ethanol (50 mL). The filter cake was collected and dried in a vacuum oven to give 53.2g of intermediate 1 as white crystals in 67% yield (see FIG. 1 for the synthetic route and FIG. 2 for the characterization pattern).
Example 2 Synthesis of intermediate 2
The intermediate 1(67.0g, 309mmol) and phosphorus oxychloride (120ml, 1.29mol) were added sequentially to a three-necked flask and reacted at 85 ℃ under reflux for 2 h. Cooled to room temperature, cold 54% aqueous KOH (540g, 1000ml) was added dropwise with rapid stirring, and after the addition was complete, a precipitate (yellow solid) precipitated with a supernatant pH of 10 ± 1. Then adding CH2Cl2(1500ml) dissolve the solid and use CH2Cl2(3 × 1000ml) the aqueous layer was extracted, the organic phases combined, anhydrous MgSO4Drying, filtration and solvent recovery gave intermediate 2 as a yellow solid 60.0g with a yield of 90% (see FIG. 3 for the synthetic route).
Example 3 Synthesis of Aminotacrine
Adding intermediate 2(30.0g, 0.14mol), hexamethylenediamine (54ml, 0.42mol) and 1.5 gKI into a three-necked flask in turn, dissolving with 200ml n-amyl alcohol, refluxing at 140 ℃ for 18h, cooling the reaction solution to 0 ℃, adjusting the pH to 1.0 with 6M hydrochloric acid, standing for layering, adjusting the pH of the water layer to 10.0 with cold KOH solution, extracting with dichloromethane (3 × 200ml), combiningOrganic phase, washed three times with saturated NaCl solution, anhydrous Na2SO4Drying, filtering, and vacuum evaporating. Column chromatography purification (eluent: dichloromethane: methanol ═ 50: 1-10: 1-5: 1, 10ml triethylamine/1000 ml) gave 36.3g of amino tacrine as a pale yellow oily liquid in 88% yield (synthetic route shown in fig. 4, characterization shown in fig. 5-6).
Example 4 Synthesis of tacrine-sinapic acid hybrids
Sinapinic acid (0.754g, 3.36mmol), EDCI (0.773g, 4.03mmol), DMAP (0.123g, 1.01mmol) and 10mL of anhydrous N, N-dimethylformamide were added sequentially in a round bottom flask; then (1g, 3.36mmol) of amino tacrine is added, and the mixture is stirred and reacted for 12 hours at normal temperature. The reaction was essentially complete by TLC (dichloromethane: methanol 10: 1), the solvent was evaporated under reduced pressure and the mixture was separated on silica gel column (eluent: dichloromethane: methanol 30: 1-10: 1) to give 1.091g of tacrine-sinapic acid hybrid (pale yellow oil) in 65% yield (synthetic route see fig. 7, characterization see fig. 8-10).
EXAMPLE 5 tablets (Dry Process)
Taking 150g of the tacrine-sinapic acid heterozygote prepared in the example 4, adding 75g of hydroxypropyl cellulose, 75g of sodium carboxymethyl starch and 180g of starch, uniformly mixing by an equivalent incremental method, and then performing dry granulation; adding 20g of magnesium stearate, and uniformly mixing; and (4) pressing into tablets.
EXAMPLE 6 tablet (Wet process)
300g of the tautomer of tacrine-sinapic acid hybrid prepared in example 4 was taken, 500g of starch, 100g of magnesium stearate, 200g of carboxymethylcellulose, 2700g of ethanol (70% by volume), 2500g of microcrystalline cellulose and 3200g of corn starch were added, mixed thoroughly to prepare wet granules, dried at 60-70 ℃ for 2-4h and compressed into tablets. The excipient for tabletting comprises magnesium sulfate, corn starch, and pulvis Talci.
EXAMPLE 7 pellets
100g of the medicinal salt of the tacrine-sinapic acid heterozygote prepared in the embodiment 4 is taken, 900g of polyethylene glycol is added, the mixture is heated and melted at 90 ℃, the raw material and the auxiliary material are fully mixed and melted, the melt is dropped into the simethicone receiving liquid, and the mixture is naturally cooled to form the dropping pill, so that each dropping pill contains 100 mg of the tacrine-sinapic acid heterozygote and 200mg of the tacrine-sinapic acid heterozygote.
EXAMPLE 8 capsules
300g of the prodrug of tacrine-sinapic acid hybrid prepared in example 4 was taken, 20g of magnesium stearate, 200g of carboxymethyl cellulose and 480g of microcrystalline cellulose were added, mixed well with stirring to prepare wet granules, the prepared wet granules were directly dried at 60-70 ℃ for 2-4h, and then filled into empty capsule shells, each capsule containing 300mg of tacrine-sinapic acid hybrid.
Example 9 granules
600g of the solvate of tacrine-sinapic acid hybrid prepared in example 4 was added with 20g of magnesium stearate, 180g of carboxymethyl cellulose, and 200g of microcrystalline cellulose, mixed by stirring thoroughly, and granulated with a 12-14 mesh sieve. Drying at 60-70 deg.C for 2-4 hr to obtain granule containing 300mg of tacrine-sinapic acid hybrid.
Example 10 suspension
20g of the tacrine-sinapic acid hybrid prepared in example 4 was taken, and 420g of purified water, 390g of glycerol, 70g of sorbitol and 100g of propylene glycol were added to prepare a suspension uniformly dispersed in an aqueous carrier.
EXAMPLE 11 Aerosol preparation
1g of the tautomer of tacrine-sinapic acid hybrid prepared in example 4 was dissolved in propylene glycol, 0.5g of vitamin C and 0.2mL of chlorobutanol were added, and mixed to form a transparent solution, which was charged into a pressure vessel together with compressed nitrogen gas according to specifications.
EXAMPLE 12 Aerosol preparation
50g of the medicinal salt of the tacrine-sinapic acid hybrid prepared in example 4 is added with 200g of glycerol, 100g of propylene glycol, 1g of tween 80, 2g of menthol and 100g of pure water, stirred uniformly and filled into a container with a nozzle to obtain the tacrine-sinapic acid hybrid.
EXAMPLE 13 oral liquid
50g of the prodrug of tacrine-sinapic acid hybrid prepared in example 4 was added with 7.45kg of purified water and 2.5kg of edible sugar, stirred to dissolve, packaged and sterilized to obtain the composition.
EXAMPLE 14 ointment (o/w type emulsifier base)
100g of stearic acid, 70g of glycerin monostearate, 70g of white vaseline and 60g of liquid paraffin are taken and placed in a beaker, the beaker is heated to 80 ℃ in water bath and stirred to be melted, and 0.5g of Tween 80 and 600ml of distilled water are taken and placed in another small beaker, the beaker is heated to 80 ℃ in water bath and stirred uniformly. The aqueous phase was added to the oil phase in small streams at the same temperature and stirred continuously in a water bath to a milky semi-solid which was then stirred at room temperature to near condensation. 100g of the solvate of the tacrine-sinapic acid hybrid prepared in example 4 was placed on an ointment plate and in a mortar, and the prepared O/W type emulsifier matrix was added in portions and ground uniformly to obtain the final product.
EXAMPLE 15 gels
Dispersing 93410 g carbomer in distilled water, slowly swelling, adding 100g glycerol, and stirring to obtain transparent gel matrix. Dissolving Mentholum in ethanol, dissolving boric acid 10g in appropriate amount of water, mixing, and stirring. And (3) sprinkling 10g of tacrine-sinapic acid heterozygote prepared in the example 4 while stirring, adjusting the pH value to 4.5-5.5 by triethanolamine, adding water to 1000ml, stirring uniformly, and subpackaging to obtain the tacrine-sinapic acid heterozygote.
EXAMPLE 16 Patch
Weighing 4g of carbomer, propylene glycol, tween-80 and distilled water according to a weight ratio of 10: 20: 3: 12 heating to 60 deg.C and stirring for 20min, adding 1g of the tautomer of tacrine-sinapic acid hybrid prepared in example 4, and stirring; coating on the lining cloth in several times until reaching the required thickness, air drying or oven drying, and cutting according to the preparation specification.
EXAMPLE 17 injection
Dissolving 3g of vitamin C in 2800ml of water for injection, heating to 30-40 ℃, adding 90g of medicinal salt of the tacrine-sinapic acid heterozygote prepared in example 4, adding 18g of Tween-80, cooling to room temperature, adding 27 g of sodium chloride, adjusting the pH value to 7.0-7.5 by using a 5% sodium citrate solution, and fixing the volume to 3000ml by using the water for injection; subpackaging 3-5ml per ampoule according to the preparation specification, and sealing by fusing; sterilizing with flowing steam at 100 deg.C for 30 min.
EXAMPLE 18 injection
A solution of 10g of the prodrug of tacrine-sinapic acid hybrid prepared in example 4, 50g of glucose and 940g of water for injection was added dropwise in a volume ratio of 1: 1, regulating pH to 4.0, adding activated carbon 1g, boiling under stirring for 30min, filtering to remove activated carbon, filtering with 0.22 μm filter membrane to remove bacteria to obtain clear sterile solution, bottling, and sterilizing at 115 deg.C under hot pressure for 30 min.
EXAMPLE 19 infusion solution
Taking 10g of the solvate of the tacrine-sinapic acid hybrid prepared in the example 4, adding 4g of polyethylene glycol, 3g of lecithin, 6g of sodium acetate, 6g of sodium sulfite and 900g of sodium chloride, dissolving in water for injection, filtering, filling and sealing, sterilizing and packaging to obtain the tacrine-sinapic acid hybrid.
EXAMPLE 20 infusion solution
Taking 10g of the tacrine-sinapic acid heterozygote prepared in the example 4, adding 4g of polyethylene glycol, 4g of lecithin, 4g of sodium acetate, 8g of sodium sulfite and 5kg of glucose, dissolving in water for injection, filtering, filling and sealing, sterilizing and packaging to obtain the tacrine-sinapic acid heterozygote. Each preparation unit contains tacrine-sinapic acid hybrid 50mg, and can be used for intramuscular injection or intravenous injection, with dosage of 1-2 preparation units per time and 1-2 times daily.
EXAMPLE 21 lyophilized powder for injection
The tautomer 45g of tacrine-sinapic acid hybrid prepared in example 4, 40g of mannitol, was taken, 910 g of water for injection was added, stirred to dissolve, and added dropwise in a volume ratio of 1: 1, regulating the pH value to 6.2, adding 5g of activated carbon for injection, stirring for 20min at room temperature, filtering to remove the activated carbon, filtering with a 0.22 mu m filter membrane for sterilization to obtain a clear sterile liquid, filling into 5ml tube bottles with 3ml per bottle, and freeze-drying in a vacuum freeze-dryer to obtain the product.
Example 22 sustained-release tablet
300g of the pharmaceutical salt of tacrine-sinapic acid hybrid prepared in example 4 was added with 70g of hydroxypropyl methylcellulose, 250g of carboxymethyl cellulose, 370g of ethanol (70% by volume), 10g of magnesium stearate, etc., and mixed thoroughly to form wet granules, which were dried at 60-70 ℃ for 2-4h and compressed into tablets.
Example 23 sustained-release tablet
600g of the prodrug of tacrine-sinapic acid hybrid prepared in example 4 was added with 280g of hydroxypropylmethylcellulose, 30g of stearic acid, 80g of lactose, and 10g of magnesium stearate, mixed well, dry granulated, and tabletted, each tablet containing 300mg of tacrine-sinapic acid hybrid.
Example 24 in vitro evaluation of acetylcholinesterase and butyrylcholinesterase inhibitory activity of tacrine-sinapic acid hybrids prepared in example 4, comprising the steps of:
preparing an acetylcholinesterase solution: the solution is prepared into Tris-HCl buffer solution with 0.1% w/v bovine serum albumin to have the concentration of 0.22U/ml, and the solution is subpackaged into 5ml centrifuge tubes and stored at the temperature of minus 20 ℃ for standby. Preparation of butyrylcholinesterase solution: the solution is prepared into Tris-HC1 buffer solution with 0.1% w/v bovine serum albumin to have the concentration of 0.3U/ml, and the solution is subpackaged into 5ml centrifuge tubes and stored at the temperature of minus 20 ℃ for standby. Preparation of 5,5' -dithiobis (2-nitrobenzoic acid) solution: PBS (pH 7.4) was added to a concentration of 1.5mM, and the mixture was stored at 4 ℃ in the dark for further use. Preparing a thioacetyl choline iodide/thiobutyrylcholine iodide solution: the solution was prepared to a concentration of 30mM using PBS pH 7.4 and dispensed into 1ml centrifuge tubes and stored at-20 ℃ until use. Preparation of a solution of a test compound: DMSO was formulated into 0.01M concentration stock solution, diluted in PBS pH 7.4 to the corresponding concentration gradient.
Mu.l PBS was added to each well of the 96-well plate, 20. mu.l solutions of the test compounds at different concentrations were added, and 50. mu.l acetylcholinesterase/butyrylcholinesterase solutions were added to each well. After incubation for 5min at 37 ℃ in a shaker, 100. mu.l of 5,5' -dithiobis (2-nitrobenzoic acid) solution was added and incubated for 5min at 37 ℃ in a shaker away from light. Finally, 10 mul of substrate thioacetylcholine iodide/thiobutyrylcholine iodide solution is added, shaking table incubation is carried out at 37 ℃ for 3min, an enzyme-labeling instrument is used for measuring the absorbance (OD value) at 415nm, and each experiment is independently repeated for three times. And (3) calculating the inhibition rate of the test compound on acetylcholinesterase/butyrylcholinesterase, wherein the inhibition rate (%) is (OD1-OD2)/(OD1-OD 0). (OD 1: OD value of the test compound with no enzyme added, OD 0: OD value of blank with no enzyme added, OD 2: OD values of the test compound with enzyme added). Finally, calculating IC by using the inhibition rates corresponding to the compounds to be detected under different concentrations50The values, results are shown in Table 1.
Inhibitory Activity of the Compounds of Table 1 on Acetylcholinesterase and Butyrylcholinesterase (IC)50)
The results show that the tacrine-sinapic acid heterozygote has remarkable acetylcholinesterase and butyrylcholinesterase inhibiting activities, and the acetylcholinesterase inhibiting activity of the compound reaches the nmol level which is about 15 times that of tacrine.
Example 25 in vitro evaluation of the inhibitory activity of tacrine-sinapic acid hybrids against amyloid a β self-aggregation prepared in example 4, comprising the steps of:
pretreatment and solubilization of amyloid a β: dissolving Abeta lyophilized powder in appropriate amount of hexafluoroisopropanol, performing ultrasonic treatment for 5-10min to dissolve completely, and standing at room temperature overnight. Hexafluoroisopropanol was removed by vacuum drying at ambient temperature and the dried a β dry powder was stored at-20 ℃. The aged dry abeta powder was dissolved in DMSO at 2mM solubility as a stock solution and diluted to the desired solubility for use. Preparing a thioflavin T solution: taking 1mg of thioflavin T, adding 2ml of PBS solution (10mM, pH 7.4) into a 10ml centrifuge tube, dissolving by ultrasonic for 5min, and filtering through a 0.22 mu m filter membrane to obtain a thioflavin T stock solution. 1ml of thioflavin T stock solution was diluted to 10. mu.M, its UV absorbance at 416nm was measured, and the concentration was adjusted to 0.266. Preparation of a solution of a test compound: stock solutions were prepared in DMSO at 0.01M concentration and then diluted with PBS at pH 7.4 to the corresponding concentration gradient (0.0064,0.032,0.16,0.8,4,20,100 μ M).
Mu.l of prepared amyloid Abeta 42 solution (40. mu.M) was added to a 96-well plate, and 10. mu.l of test compounds of different concentrations were added simultaneously. The prepared mixed solution is incubated at 37 ℃ for 24h, and then 180. mu.l of 10. mu.M thioflavin T solution is added. Detecting by a full-wavelength multifunctional microplate reader, exciting the wavelength of 435nm, and reading the fluorescence intensity value of 485 nm. And (3) calculating the inhibition rate of the test compound, wherein the inhibition rate (%) is (F1-F2)/(F1-F0). (F1: adding A beta 42 and not adding the fluorescence value of the compound to be tested, F0: adding A beta 42 and the compound to be tested, the background fluorescence value of the thioflavin T solution, F2: adding A beta 42 and the fluorescence value of the compound to be tested), and finally, plotting the inhibition rates corresponding to the compound to be tested under different concentrations (see figure 11).
The results show that the tacrine-sinapic acid hybrid of the invention exhibits a certain activity of inhibiting the self-aggregation of the a β 42 protein. Observing a concentration-inhibition rate curve of the tacrine-sinapic acid hybrid, wherein the inhibition rate is correspondingly increased along with the increase of the concentration of the compound to be detected in a concentration range of 0.0064-100 mu M and presents a better S-shaped curve, and the activity of the tacrine-sinapic acid hybrid for inhibiting the aggregation of the Abeta 42 protein is better than that of a control drug tacrine at 20 mu M and 100 mu M.
Example 26 in vitro evaluation of the inhibitory activity of tacrine-sinapic acid hybrids prepared in example 4 on amyloid a β self-aggregation, comprising the steps of:
pretreatment and solubilization of amyloid a β: dissolving Abeta lyophilized powder in appropriate amount of hexafluoroisopropanol, performing ultrasonic treatment for 5-10min to dissolve completely, and standing at room temperature overnight. Hexafluoroisopropanol was removed by vacuum drying at ambient temperature and the dried a β dry powder was stored at-20 ℃.
Sample preparation: mu.l of the prepared amyloid Abeta 42 solution (2mM) was added to the 96-well plate, and 2.5. mu.l of the test compound was added simultaneously. Then, 45. mu.l of PBS (pH 7.4) was added. The prepared mixed solution is placed in a shaking table to shake for 12 hours and then is placed in a 37 ℃ for cultivation, and the cultivation time is recorded. Samples were prepared and observed 4 days after the culture. Test compounds were prepared in 2mM stock solution in DMSO. The blank control group was 2.5. mu.l of amyloid Abeta.42 solution and 2.5. mu.l of DMSO in 45. mu.l of PBS buffer, and the other treatment conditions were the same as those of the test sample. Sample detection: the mixture (1. mu.l) in the 96-well plate was diluted 100-fold, and 5. mu.l of the diluted solution was spotted on a previously prepared AFM mica plate, and allowed to stand overnight in a desiccator for drying. The atomic force microscope scans the sample in an intelligent mode with a force constant of 3N/m.
The results show (see fig. 12) that after 4 days of incubation, both the amyloid a β control group and the tacrine group showed a significant aggregation state, but no significant fibrillar aggregates were formed in the tacrine group. The aggregation of amyloid a β is significantly reduced after treatment with the tacrine-sinapic acid hybrid, and no significant lumpy or fibrillar aggregates are formed, suggesting that the tacrine-sinapic acid hybrid has a significant ability to inhibit the self-aggregation of amyloid a β.
Example 27 in vitro evaluation of antioxidant activity of tacrine-sinapic acid hybrids prepared from example 4, comprising the steps of:
preparing a 1, 1-diphenyl-2-trinitrophenylhydrazine solution: 1, 1-diphenyl-2-trinitrophenylhydrazine is dissolved by methanol to prepare a solution of 300 mu M, which is prepared at present and stored in a low-temperature sealing and dark place. Preparation of a solution of a test compound: the test compound was dissolved in methanol and diluted to the corresponding concentration gradient (five concentrations, 7.81-250. mu.M).
Taking 5 mul of compounds to be detected with different concentrations to a 96-well plate, adding 95 mul of 1, 1-diphenyl-2-trinitrophenylhydrazine solution, and incubating for 30min in a shaking table at 37 ℃ with a tin foil paper coated plate. The microplate reader measures the absorbance (OD value) at 517nm per well. Calculating the radical scavenging activity of the compound to be detected, wherein the scavenging rate is [ (-) ] [ 1- (A ]sample－Ablank)/Acontrol]×100％。(AcontrolAdding 1, 1-diphenyl-2-trinitrophenylhydrazine without adding the OD value of the compound to be tested, AblankThe OD value of the compound to be tested, A, is not added with 1, 1-diphenyl-2-trinitrophenylhydrazine sample1, 1-diphenyl-2-trinitrophenylhydrazine and the OD value of the compound to be tested) are added, and the experiment is independently repeated for three times, and the result is shown in a table 2.
TABLE 2 inhibitory Activity of the Compounds on 1, 1-Diphenyl-2-trinitrophenylhydrazine (IC)50)
The results show that tacrine-sinapic acid hybrids exhibit significant free radical scavenging activity (IC)50＝85.8±3.5μM)。
Example 28 in vitro evaluation of the neuroprotective activity of tacrine-sinapic acid hybrids prepared in example 4, comprising the steps of:
preparation of a solution of a test compound: the test compound DMSO was dissolved and diluted to the corresponding concentration gradient (1.25, 2.50, 5, 10. mu.M).
Culturing adrenal gland chromaffin tumor thin line of rat by taking complete culture mediumCell PC12, when the cell concentration reaches the appropriate amount, the original culture medium is removed, blank RPMI 1640 is added, starved cells 14 h.120. mu.l are inoculated in a 96-well plate coated with polylysine, 90% RPMI 1640, 10% fetal calf serum and 100U/ml double-antibody culture medium are cultured, the number of cells in each well is about 7 × 103. And adding 0.05 mu g/ml of NGF (nerve growth factor) to induce the cell to differentiate, and performing differentiation culture for 48 hours until the cell grows out the axon similar to the nerve cell. The final concentration was 1.25, 2.50, 5, 10 μ M and each concentration was plated in 4 wells in parallel, and equal volume of culture medium was added to the control. The compounds were dissolved in DMSO, diluted in medium, and the final concentration of DMSO was less than 0.1%. After 36h of drug action, PC12 cells were damaged by addition of cobalt chloride to a final concentration of 300. mu.M/L. After the cells were injured by molding for 12 hours, 20. mu.L (5mg/mL) of MTT was added, and the culture was continued for 4 hours. The supernatant was carefully removed to prevent the crystals formed from being drawn off. Add 100. mu.L DMSO into each well, put on a micro-oscillator to shake for 5min to fully dissolve, measure the absorbance of each well at 490nm, and derive the data. The nerve cytotoxicity test was performed as described above without damaging the PC12 cells with cobalt chloride. Data as mean ± standard deviation
It is shown that the SAS8.2 statistical software performs data statistics and one-way analysis of variance, and the results are shown in FIG. 13 for three independent replicates.
The results show that the neurotoxicity of tacrine is enhanced along with the increase of the concentration, and the cell activity at 5 mu M and 10 mu M is obviously reduced and reaches 82 percent and 76 percent respectively. In contrast, the tacrine-sinapic acid hybrid did not only cause damage to differentiated PC12 cells, but also had some growth-promoting effect, with a survival rate of 130% at a maximum concentration of 10. mu.M. CoCl2Injury resulted in a significant decrease in survival (51.2%) of normal NGF differentiated PC12 cells, and the antioxidant vitamin E (10. mu.M) significantly increased CoCl2The number of surviving PC12 cells damaged (101.3%). In addition, tacrine-sinapic acid hybrids and tacrine pairs CoCl2Fine lines of lesion PC12The cells have significant protective effects, especially the neuroprotective activity of tacrine-sinapic acid heterozygotes is significantly dose-dependent (74.5%, 92.4%, 99.5% and 106.8%), and the cell survival rate is significantly increased with increasing concentration.
Example 29 in vivo evaluation of hepatotoxicity of tacrine-sinapic acid hybrids prepared from example 4, comprising the steps of:
preparation of the medicine: weighing 1g of sodium carboxymethylcellulose by an analytical balance, dissolving in a beaker filled with 200ml of distilled water, heating in a water bath at 95 ℃, continuously stirring by a glass rod, performing ultrasonic treatment, and repeatedly stirring and performing ultrasonic treatment for several times until the sodium carboxymethylcellulose is completely dissolved. Standing, cooling and reserving for later use. Weighing a certain amount of tacrine medicine to prepare a liquid medicine of 1.5mg/ml, preparing a liquid medicine of 3.22mg/ml which is an equimolar amount by using a tacrine-sinapic acid heterozygote, and preparing the liquid medicine at present.
Mice were randomly grouped by weight after acclimation for one week, and the experiment was divided into 3 groups: blank Control (Control), Tacrine (Tacrine), drug (Tacrine-sinapic acid hybrid), 8 mice per group. Starving for twelve hours before administration to ensure water supply. Gavage (0.1 ml/10g of mice dosing standard), tacrine dosing dose 3mg/100g, equimolar tacrine-sinapic acid hybrid dosing in drug group, and equal volume of 0.5% sodium carboxymethylcellulose solution gavage in blank control group animals.
Orbital bleeds were taken at time points 8h, 24h, and 36h post-dose, respectively. Standing at 4 deg.C for 1h, centrifuging at 3000r for 15min, and collecting upper layer serum at-80 deg.C. The glutamate-pyruvate transaminase ALT/aspartate aminotransferase AST is then determined according to the kit instructions. Data as mean ± standard deviation
It is shown that data statistics and one-way anova were performed by SAS8.2 statistical software, and the results are shown in table 3.
Table 3 changes in mouse liver ALT/AST following oral administration of tacrine-sinapic acid hybrid (n-8,
)
note: p is less than or equal to 0.05, p is less than or equal to 0.01, compared with blank control group at the same time
The results show that oral administration of tacrine results in a significant increase in its AST/ALT index. And the medicinal tacrine-sinapic acid heterozygote obviously inhibits AST/ALT, and compared with the bulk drug tacrine, the liver injury toxicity of the medicinal tacrine-sinapic acid heterozygote is greatly relieved.
Claims (4)
1. A tacrine-sinapic acid hybrid characterized by the following structure:
2. a pharmaceutical composition containing tacrine-sinapic acid heterozygote is prepared by mixing the tacrine-sinapic acid heterozygote or medicinal salt thereof with one or more pharmaceutically acceptable carriers, so that the amount of the tacrine-sinapic acid heterozygote accounts for 0.5-60% of the total weight of the pharmaceutical composition, and the tacrine-sinapic acid heterozygote is prepared into clinically acceptable tablets, pills, capsules, granules, suspensions, aerosols, oral liquids, ointments, gels, patches, injections, freeze-dried powders and sustained and controlled release preparations, and has the following structure:
3. use of the tacrine-sinapic acid hybrid of claim 1 in the preparation of an anti-alzheimer's drug.
4. Use of a pharmaceutical composition comprising a tacrine-sinapic acid hybrid according to claim 2 for the manufacture of an anti-alzheimer's drug.
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| CN102617465A (en) * | 2011-11-14 | 2012-08-01 | 中山大学 | Tacrine-caffeic acid hetero-blends, their preparation method and medicinal compositions |
| CN106187891A (en) * | 2016-07-18 | 2016-12-07 | 南京中医药大学 | There is compound of anti-Alzheimer disease effect and preparation method and application |
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| CN102617465A (en) * | 2011-11-14 | 2012-08-01 | 中山大学 | Tacrine-caffeic acid hetero-blends, their preparation method and medicinal compositions |
| CN106187891A (en) * | 2016-07-18 | 2016-12-07 | 南京中医药大学 | There is compound of anti-Alzheimer disease effect and preparation method and application |
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