CN100503575C - Helicid hydrastine acetali mine its preparation method and its application - Google Patents

Abstract

This inventio relates to preparation method of helicid -huperzine methylal imine. Structural formula as (1) described. It also relates to it' s as drug compound of effective ingredient, as well as application in preparing restraining acetyl lipase activity medicine.

Description

Helicidum-huperzine aldimine imines, its preparation method and its application

Technical field: the present invention relates to novel helicidum-huperzine aldimine group with imine moiety, its preparation method is the pharmaceutical composition of activeconstituents with the compound, and its application in the medicine of preparation inhibition acetyl courage lipase activity.

Background technology: along with the increase year by year of world population mean lifetime, the sickness rate of senile dementia is also along with rapid rising, have 50%-70% to be Alzheimer (AD) in the senile dementia, Alzheimer's disease claims senile dementia early again, patient's brain function rate is gradually moved back, memory, abstract thinking ability occur and ability of language expression goes down, handicapped and other functional disorder, the system function that affects the nerves finally causes death.AD is a kind of central nervous system degenerative disease based on cognition of carrying out property and memory injury, estimates that global world Alzheimer number of the infected is up to more than 1,200 ten thousand.In this disease of developed country is to cause dead principal disease after cardiovascular diseases and tumour.The medicine of treatment AD mainly contains choline drugs thing, neurocyte metabolism Reinforcing agent, antioxidant, calcium ion antagonist, nerve growth factor, oestrogenic hormon, antiphlogiston, anti-beta amyloid medicine etc.Treating the successful method of this disease at present clinically is exactly by improving cholinergic neurotransmitter---the level of vagusstoff in the central nervous system, improving patient's AD symptom.Wherein at pass through suppress AChE the Degradation of vagusstoff improved the level of the vagusstoff in the brain, be the effective way of improving symptom.Effective clinically medicine such as tacrine (Cognex, tacrine), aricept (Aricept, donepezil), and Exelon (Exelon, rivastigmine), lycoremine (Reminyl, galantamine) and selagine (Hup A) be acetylcholinesterase depressant, their levels of acetylcholine in the brain that can effectively raise are improved people's cognitive function.And as the famous natural product selagine of acetylcholinesterase depressant, its drug effect has obtained clinical abundant affirmation, Shanghai Pharmaceutical Inst., Chinese Academy of Sciences synthesizes the derivative of a series of selagines on this basis, found the medicine " Xi Pulin " of a novel anti-senile dementia, entered for three phases at present in Europe clinical, and listing can be given the ratification after the expectation 2-3.Xi Pulin is as prodrug, and is suitable to AChE inhibitor activity and selagine, and selectivity is slightly high, but bioavailability obviously improves.

The report that the huperzine A derivative of helicidum is not arranged in the prior art up to now.

Summary of the invention: the huperzine A derivative that the object of the present invention is to provide a kind of helicidum is helicidum-huperzine aldimine group with imine moiety, its preparation method, with the compound is the pharmaceutical composition of activeconstituents, and its application in the medicine of preparation inhibition acetyl courage lipase activity.

In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:

Helicidum shown in structural formula (I)-huperzine aldimine imines.

The present invention provides the method for preparation formula (I) compound simultaneously, gets selagine, places the three footpath bottles that have prolong, adds absolute ethyl alcohol and stirring and dissolving; Other gets helicidum and is dissolved in the dehydrated alcohol, adds after triethylamine mixes, and slowly splashes in the three footpath bottles, and mixed solution magnetic agitation and 50 ℃-55 ℃ heating 1 hour is cooled to room temperature, and concentrating under reduced pressure gets final product through silica gel column chromatography.

The pharmaceutical composition that is used to suppress acetyl courage lipase activity of the present invention wherein contains the claim 1 helicidum-huperzine aldimine group with imine moiety and/or the pharmaceutically acceptable carrier for the treatment of significant quantity.

The present invention also provides the application of helicidum-huperzine aldimine group with imine moiety in preparation inhibition acetyl courage lipase activity medicine.

The proposition of technical solution of the present invention is the inspiration that is subjected to the Xi Pulin chemical structure, rely on structure and active characteristics to natural compounds, attempt to prepare the huperzine A derivative of helicidum, expection should have significant inhibiting activity of acetylcholinesterase,, and more widely to neural activity.Test-results meets the expection expectation.

When The compounds of this invention is used as medicine, can adds vehicle and directly use, or form compound preparation with other drug and use with suitable preparation.All the other are acceptable to humans and animals nontoxic and inert pharmaceutically acceptable carrier and/or vehicle on the pharmacology.Pharmaceutical carrier or vehicle are acceptable to the nontoxic and inert solid of humans and animals, semisolid and liquid diluent, filler and pharmaceutical preparation assistant agent on the pharmacology.Pharmaceutical composition of the present invention is used with the form of per weight dose.Medicine of the present invention can be through injection (quiet notes, intramuscular injection) and oral two kinds of form administrations.Usage quantity can be carried out one or many according to variations such as the type of route of administration, patient's age, body weight, the disease of being treated and severity and be used.Concerning the adult, 1～100mg every day is proper for dosage.

Natural product raw material and source thereof that The compounds of this invention helicidum-huperzine aldimine imines relates to:

1) helicidum (Hel)

Helicidum (Hilieidum, Helicid), chemical structure is 4-formylphenyl-beta-D-allopyranosid-(4-formylpheny-β-D-allopyranoside), being a kind of effective constituent that extraction separation obtains from Yunnan Province of China Proteaceae plant Fruit of Edible Ironweed (Helicia nilagirica Beed) fruit, is allose phenolic glycoside monomeric compound rarer in a kind of natural product.Prove through a large amount of pharmacology and clinical study: helicidum has pain relieving, sleeps peacefully and sedative effect, and is used to diseases such as neurasthenia, neurasthenia syndrome, insomnia and angioneurotic headache.

The extraction and purification process of helicidum: Fruit of Edible Ironweed crude drug powder, use cold water soak after a few hours, boiling 3 times, each 1 hour, filter merging filtrate, reduction vaporization becomes small volume, add 95% ethanol again and contain the alcohol amount greater than 75%, placed 5-6 hour, filter (residue washing with alcohol 2-3 time) to solution, after getting the stillness of night and washings recovery ethanol, leave standstill, separate out the off-white color crystal, coarse crystal 90% alcohol reflux, add 0.5% decolorizing with activated carbon, filtered while hot, filtrate left standstill 48 hours, separated out crystal, filter, drying, the pure product of helicidum of 0.4-0.5%, can be used as pharmaceutical raw material.It is at present domestic that tens of families pharmacy corporation is arranged is the medicine of effective constituent producing helicidum.

The sign of helicidum:

Helicidum is white, needle-shaped crystals or crystalline powder, odorless, mildly bitter flavor; Molecular formula C ₁₃H ₁₆O ₇, molecular weight 284.27;

Be dissolved in hot water, slightly soluble in water, the methyl alcohol, solubleness is atomic in the ethanol, and is insoluble substantially in ether, the chloroform;

Fusing point 190-198 ℃

UV?λmax：280nm

The NMR data of helicidum:

¹H?NMR?δ：7.85(1H，d，J＝9.2，2-H)，7.22(1H，d，J＝8.7，3-H)，7.22(1H，d，J＝8.7，5-H)，7.85(1H，d，J＝9.2，6-H)，9.85(1H，s，7-H)，5.38(1H，J＝7.8，1’-H)，3.70(1H，m，2’-H)，3.86(1H，m，3’-H)，3.62(1H，m，4’-H)，3.88(1H，m，5’-H)，4.15(1H，m)；3.86(1H，m)(6’-2H)

¹³C?NMR?δ：132.6(s，C-1)，132.8(d，C-2)，117.9(d，C-3)，164.3(s，C-4)，117.9(d，C-5)，132.8(d，C-6)，192.9(s，C-7)，99.8(d，C-1’)，72.1(d，C-1’)，73.0(d，C-3’)，68.8(d，C-4’)，76.0(d，C-5’)，63.0(t，C-6’).

Chemical structure:

Chemical name: 4-formylphenyl-beta-D-allopyranosid-

(4-formylpheny-β-D-allopyranoside)

2) selagine (HupA)

Selagine [(-) Huperzine A] is a kind of novel lycopodium alkaloid effective constituents monomer that the eighties in 20th century, Chinese scholar was found from traditional herbal medicine Herba Lycopodii serrati (Herba Lycopodii serrati) (Huperzia serrata); And in the nineties some scholars successfully develop become the treatment alzheimer's disease (Alzheimer ' s disease, natural drug AD), commodity are called " huperzine A ".Compare with similar AD medicine in the world, the chemical structure of selagine has specificity, has acetylcholinesterase in the inhibition brain of highly significant and strengthens the function of cholinergic neuron in the brain.Isotropic substance H3-selagine 183ug/ml intravenous injection was arrived mouse after 15 minutes, the autography experimental result shows: in periphery and each brain district radiopharmaceutical distribution is arranged all, after 3 hours, mainly be distributed in volume, top, cortex of temporal lobe, hippocampus, nucleus accumbens septi, zones such as fourth ventricle's basis pontis and adenohypophysis, more obvious.Show that selagine extremely is easy to enter maincenter, and selectivity is distributed in and learns, remember relevant nerve pathway zone, acetylcholinesterase in the brain is had higher selective inhibitory activity.

And the neuroprotective of selagine is also fairly obvious; studies show that: apoptosis and oxidative stress have participated in the pathologic process of AD and VD; the oxidative damage that free radical causes is considered to the final common path of different reason inducing neural degeneration; except AD and VD; atherosclerosis, cerebral ischemia, epilepsy, amyotrophy lateral sclerosis, the gloomy syndromes of Pa Shi gold etc. are all pointed out may stop these advancings of disease to anti-apoptotic and oxidative stress medicine.Selagine is by the raising antioxidase activity, has remarkable directed against amyloid-beta albumen (β-amyloid, A β) toxic action, can obviously improve the oxidative stress that the senile rat aging causes, and its antioxygenation helps to delay, stops the AD development.The vigor that selagine not only can improve superoxide dismutase (SOD) quickens the removing of interior free yl, reduces the damage that oxyradical causes.The prompting selagine is not only relevant with acetylcholine esterase inhibition activity, also improves the antioxidant radical function with it, to reduce the damage that free radical causes relevant.Illustrate that the effect of selagine anti-senile dementia is not only relevant with acetylcholine esterase inhibition activity, with improve the antioxidant radical function, to reduce the damage that free radical causes relevant.A β precursor protein APP (secretor type APP) is considered to have neuroprotective, can cause secretor type APP and PKC level to descend behind the injection A β in the rat tricorn.Selagine can obviously raise the PKC level, increases the expression of secretor type APP, and plays the neuro-protective effect, improves because study, the dysmnesia that A β causes.

Selagine has nootropic effect to adult, old monkey, to Scopolamine, shock by electricity, go back the cognitive disorder that hexanoyl imines, Sodium Nitrite or carbonic acid gas bring out, the dysmnesia that intracerebral injection cholinergic selectivity toxic agent AF64A brings out, brain space learning, the dysmnesia of bringing out after the injection A β 1-40 fragment in the tricorn, selagine all has the improvement effect.

Selagine be applied in 1994 clinical after, go down improving memory and the cognitive ability that optimum aging hypomnesis of person in middle and old age and presenile dementia cause, and improve teenager's memory and the school grade raising obtains positive effect.

The sign of selagine:

Selagine is white or little yellow crystalline powder, odorless, mildly bitter flavor; C ₁₅H ₁₈N ₂O, molecular weight 242.32

Be soluble in methyl alcohol, chloroform, be dissolved in ethanol, water insoluble; Fusing point 224-229 ℃; Specific rotatory power [α] _24.5 ^D-150.4 ° (c=0.498 methyl alcohol); UV λ max:231,313nm

The NMR data of selagine:

¹H?NMR?δ(400MHz，CDCl ₃)：6.41(1H，d，J＝9.4，2-H)，7.86(1H，d，J＝9.4，3-H)，2.71(2H，ddd，J _6α，6β＝16.8，J _6α，7＝1.16，J _6β，7＝0.52，6-H)，3.60(1H，brs，7-H)，5.39(1H，J＝4.6，8-H)，1.66(3H，d，J＝6.7，10-H)，5.47(3H，d，J＝6.7，11-H)，2.11(2H，s，14-H)，1.54(3H，s，16-H)，13.15(1H，s，NH).

¹³C?NMR?δ(100MHz，CDCl ₃)：165.4(s，C-1)，116.9(d，C-2)，140.2(d，C-3)，122.8(s，C-4)，142.5(s，C-5)，35.2(t，C-6)，32.8(d，C-7)，124.2(d，C-8)，12.3(q，C-10)，111.2(d，C-11)，143.1(s，C-12)，54.3(s，C-13)，49.1(t，C-14)，134.1(s，C-15)，22.6(q，C-16).

Chemical structure:

Chemical name: (5R, 9R, 11E)-5-amino-11-ethylidene-5,6,9,10-tetrahydrochysene-7-methyl-5,9-methylene ring cycloocta--2 (1H)-pyridone, (English: [5R-(5 α, 9 β, 11E)]-5-amino-11-ethylid-ene-5,6,9,10-tetrahydro-7-methyl-5,9-Methanocycloocta[b] pyridine-2 (1H)-one)

Embodiment: the following examples can make those skilled in the art more fully understand the present invention, but do not limit the present invention in any way.

Embodiment 1:

The preparation of helicidum-huperzine aldimine group with imine moiety:

Helicidum and huperzine aldimine prepared in reaction helicidum derivative (1), reaction formula:

HupA Hel compound 1

Get 48.4mg (0.2mmol) HupA and place the three footpath bottles that have prolong, add absolute ethyl alcohol and stirring and the dissolving of 8ml.Helicidum 71.1mg (0.25mmol) is dissolved in the dehydrated alcohol of 8ml, and after the triethylamine that adds 0.5ml mixes, slowly splashes in the three footpath bottles.Mixed solution magnetic agitation and heating, temperature maintenance are about 55 ℃, and 1 little back is detected by TLC, shows that reaction finishes.After reactant is cooled to room temperature, concentrating under reduced pressure, resistates silica gel column chromatography (CHCl ₃/ CH ₃OH 40:1 20:1), obtains product 1,80mg, yield 79.2%.White solid.

Prepare the helicidum derivative by schiff bases.The selagine of using in the experiment (95%) is opened up health Bioisystech Co., Ltd available from Changsha.All the other reagent are learned reagent company limited available from the lark waffle.

Embodiment 2:

Get 120mg (～0.5mmol) HupA places the three footpath bottles that have prolong, adds absolute ethyl alcohol and stirring and the dissolving of 25ml.Helicidum 200mg (～0.7mmol) be dissolved in the dehydrated alcohol of 25ml, and after the triethylamine that adds 1.3ml mixes, slowly splash in the three footpath bottles.Mixed solution magnetic agitation and heating, temperature maintenance are about 55 ℃, and 1 little back is detected by TLC, shows that reaction finishes.After reactant is cooled to room temperature, concentrating under reduced pressure, resistates silica gel column chromatography (CHCl ₃/ CH ₃OH 40:1 20:1), obtains product 1,210mg, yield 83.3%.White solid.

The sign of helicidum derivative 1:

White, needle-shaped crystals, mp.198-200 ℃. optically-active is than [α] ²⁰ _D:-2.42 (c=1.24, CHCl ₃);

¹H?NMR?δ(500MHz，CDCl ₃)：6.32(1H，d，J＝9.4，2-H)，7.22(1H，d，J＝9.4，3-H)，2.80(2H，ddd，J＝16.8，3.5，1，6-H)，3.68(1H，brs，7-H)，5.34(1H，d，J＝7.2，8-H)，1.65(3H，d，J＝6.5，10-H)，5.12(1H，q，J＝6.8，11-H)，(2H，d，J＝16.0，14-H)，1.28(3H，s，16-H)，7.81(1H，d，J＝8.6，2’-H)，7.17(1H，d，J＝8.6，3’-H)，7.81(1H，d，J＝8.6，6’-H)，8.43(1H，s，7’-H)，5.50(1H，s，1”-H)，3.69(1H，m，2”-H)，3.86(1H，m，3”-H)，3.61(1H，m，4”-H)，3.86(1H，overlap，5”-H)，4.14(1H，brs，6”-H)，3.89(1H，overlap，6”-H).

¹³C?NMR?δ(125MHz，CDCl ₃)：165.7(s，C-1)，118.0(d，C-2)，142.9(d，C-3)，122.7(s，C-4)，144.7(s，C-5)，36.0(t，C-6)，34.2(d，C-7)，125.3(d，C-8)，12.6(q，C-10)，115.6(d，C-11)，140.8(s，C-12)，65.7(s，C-13)，46.7(t，C-14)，135.5(s，C-15))，23.0(q，C-16)；131.4(s，C-1’)，131.1(d，C-2’)，117.8(d，C-3’)，161.9(s，C-4’)，117.8(d，C-5’)，131.1(d，C-6’)，163.8(d，C-7’)；99.7(d，C-1”)，72.1(d，C-2”)，73.0(d，C-3”)，68.7(d，C-4”)，75.8(d，C-5”)，62.9(t，C-6”).

(+)FAB-MS?m/z(％)：509(M+1，100)，347(M+1-All，78)，331(7)，243(14)，226(242-NH2，77)，211(13)，198(12)，122(19)

According to the reaction process of starting raw material, and the structure of above spectral data compound 1 can be inferred.

The chemical name of compound 1: because the systematic naming method complexity is pressed the natural product custom, name compound 1 is helicidum-huperzine aldimine imines.

Following example of formulations is that the method by embodiment 1 makes helicidum-huperzine aldimine imines earlier, pulvis, pelletizing press sheet, the capsule making injection liquid, powder injection, oral liquid routinely and add vehicle.

[example of formulations 1] tablet

Prepare tablet according to methods known in the art, every contains following compositions:

Formula (I) compound 50mg

Lactose 70mg

Magnesium Stearate 3mg

Polyvinylpyrrolidone 7mg

Add up to 130mg

If desired, tablet can carry out the film coating with Vltra tears, talcum and tinting material.

[example of formulations 2] capsule

Prepare capsule according to methods known in the art, contain following compositions in each capsule:

Formula (I) compound 50mg

Lactose 70mg

W-Gum 25mg

Magnesium Stearate 1mg

Polyvinylpyrrolidone 4mg

Add up to 150mg

[example of formulations 3] injection

Method by embodiment 1 makes formula of the present invention (I) compound earlier, prepares injection by methods known in the art, adds the injection water, smart filter, and injection liquid is made in the embedding sterilization.

Following test example can further specify the beneficial effect of helicidum of the present invention-huperzine aldimine imines and pharmaceutical composition thereof, and the test example has comprised pharmacodynamics test of the present invention etc.

Test example 1: the acetylcholinesterase of helicidum-huperzine aldimine imines suppresses experiment

Experimental principle:

Acetylcholinesterase (acetylcholinesterase, AChE) major physiological function is by quick hydrolysis neurotransmitter vagusstoff (ACh), end the pulse transmission in the cholinergic synapse, so AChE claims acetylcholine hydrolyzation enzyme (acetylcholine hydrolase) again ¹²

The product that acetylcholinesterase and acetylthiocholine iodide generate, with 5, the two nitrobenzoic acids of 5-two sulphur generate a kind of yellow substance, and this model utilizes this color reaction, measures the inhibition degree of testing sample to acetylcholinesterase.

Material:

Acetylcholinesterase (AChE), positive control tacrine (Tacrine) are purchased the company in SIGMA, and acetylthiocholine iodide is purchased the company in FLUKA, and the two nitrobenzoic acids of 5,5-two sulphur are purchased the company in ACROS, and other is homemade analytical pure.

Helicidum-huperzine aldimine imines is got the pears type pipe of 5 phosphate buffer solns to the inhibition determination of activity of acetylcholinesterase, the sample DMSO solution that adds different 1.0mM successively, make it use the DMSO polishing to the scale volume, concentration is controlled to be 50.0,25.0,12.5,6.25,3.13 (x10 ^-9M).Add a certain amount of 4mg/mL5 respectively, 5-dithio two (2-nitrobenzoic acid) and acetylcholinesterase solution (0.1mg/ml), and behind 37 ℃ of insulation 15min.Add a certain amount of 2mg/mL acetylthiocholine solution immediately, shake up the back and survey its A value (A at λ=412nm place _n).Reference 0.1M pH8.0 phosphate buffer soln.Not add the measured A of sample _ControlValue is as 100 unit of activity:

Relative enzyme activity=(A _n/ A _Control) * 100

With the relative vigor of enzyme inhibitor concentration is mapped then, try to achieve the IC of all cpds according to suppressing curve ₅₀Value (inhibitor concentration during inhibitory enzyme vigor 50%).Helicidum-huperzine aldimine imines is to the inhibition activity (table 1) of acetylcholinesterase.

The active result of the preliminary screening of table 1 compound 1

Compound	Inhibiting rate % (1 * 10 -3mol/l)	Inhibiting rate % (1 * 10 -4mol/l)	Inhibiting rate % (1 * 10 -5mol/l)
				1	99.09±0.34	93.26±0.76	93.41±2.67
HupA	99.675±0.16	85.7±1.14	81.93±3.40
				Hel	24.835±11.82
Tacrine	IC50： 1.15×10 -6mol/l

The positive contrast of tacrine, concrete concentration and inhibiting rate are:

Concentration 1 * 10 ^-3M, inhibiting rate 97.36%%; Concentration 1 * 10 ^-4M, inhibiting rate 94.46%; Concentration 1 * 10 ^-5M, inhibiting rate 90.93%; Concentration 1 * 10 ^-6M, inhibiting rate 71.91%; Concentration 1 * 10 ^-7M, inhibiting rate 17.25%; Concentration 1 * 10 ^-8M, inhibiting rate 0.

As seen, compound 1 is 1 * 10 ^-5Under the concentration of mol/L, the inhibiting rate of acetylcholinesterase is arrived 93.4%, and in same experiment under the same concentration, selagine (HupA) arrives 81.9% to the inhibiting rate of acetylcholinesterase, and helicidum (Hel) is 1 * 10 ^-3Under the concentration of mol/L, the inhibiting rate of acetylcholinesterase is only reached 24.8%.In view of the inhibition activity of 1 pair of acetylcholinesterase of compound is stronger, further repeated experiments has been measured IC50 (table 2):

The positive control p0407 stronger (acetylsalicylic acid Physostigmine) in the table than the tacrine activity.

Table 2 explanation compound helicidum-huperzine aldimine imines has the inhibition activity of stronger acetylcholinesterase.

The inhibition activity (IC50) of 1 pair of acetylcholinesterase of table 2 compound

Claims (4)

1, the helicidum shown in structural formula (I)-huperzine aldimine imines

2, the method for formula (I) compound of preparation claim 1 is got selagine, places the three footpath bottles that have prolong, adds absolute ethyl alcohol and stirring and dissolving; Other gets helicidum and is dissolved in the dehydrated alcohol, adds after triethylamine mixes, and slowly splashes in the three footpath bottles, and mixed solution magnetic agitation and 50 ℃-55 ℃ heating 1 hour is cooled to room temperature, and concentrating under reduced pressure gets final product through silica gel column chromatography.

3, be used to suppress the pharmaceutical composition of acetyl courage lipase activity, wherein contain the helicidum-huperzine aldimine group with imine moiety and/or the pharmaceutically acceptable carrier of the claim 1 for the treatment of significant quantity.

4, the application of the helicidum of claim 1-huperzine aldimine group with imine moiety in preparation inhibition acetyl courage lipase activity medicine.