CN105616400A

CN105616400A - Use of arctigenin carbamate derivatives in preparation of drug for treating Alzheimer disease

Info

Publication number: CN105616400A
Application number: CN201410619560.5A
Authority: CN
Inventors: 胡立宏; 沈旭; 雷敏; 朱志远; 刘军华; 陈静
Original assignee: Shanghai Institute of Materia Medica of CAS
Current assignee: Shanghai Institute of Materia Medica of CAS
Priority date: 2014-11-05
Filing date: 2014-11-05
Publication date: 2016-06-01

Abstract

The invention relates to a use of arctigenin carbamate derivatives with a general formula I in preparation of a drug for treating Alzheimer disease. The arctigenin carbamate derivatives can reduce beta-amyloid peptide (A beta) content and can be metabolized in vivo to form arctigenin (ATG) and thus the arctigenin carbamate derivatives can be used as drugs for treating Alzheimer disease.

Description

One class arctigenin carbamate derivatives is used for the purposes treating in the medicine of Alzheimer in preparation

Technical field

The present invention relates to the medical usage of a class arctigenin carbamate derivatives. Described carbamic acid arctigenin carbamate derivatives can reduce the content of beta amyloid peptide (A ��), and can metabolism be arctigenin (Arctigenin, ATG) in vivo, can as the medicine for the treatment of Alzheimer.

Background technology

Alzheimer (Alzheimer ' sdisease, AD) also known as alzheimer disease, it is a kind of progressive nerve retrograde affection, mainly suffers the neuronal death of irreversibility and the thing followed to include remembering and the intellectual damage of thinking of logic etc. Cause that the factor of AD is a lot, have gene and extragenic factor. There are about the AD of 5% is familial, and it causes mainly due to the sudden change of amyloid precursor protein (APP) and presenilin (PS) gene; But not the risk factor of the potential AD of familial includes the [HardyJ such as age, apolipoprotein E, vascular lesion and traumatic brain injury, SelkoeDJ.TheamyloidhypothesisofAlzheimer ' sdisease:progressandproblemsontheroadtotherapeutics.Scie nce, 2002; 297:353-356.]. Total course of disease of AD is 3��20 years, and making a definite diagnosis rear mean survival time is about 10 years. This disease to experience two kinds of death, is first that spirit is dead, dead followed by the human body. According to estimates, the expense that AD patient is looked after by the current whole world every year has reached more than 3,000 hundred million dollars (U.S., Alzheimer association adds up). Thus AD has become society and heavy family burden. Along with China steps into the society of the aged, the AD patient of China also increases year by year, and the medicine therefore finding treatment AD not only becomes a kind of malpractice insurance, more becomes a kind of social responsibility.

The marketed drug for the treatment of AD mainly includes following several: acetylcholinesterase (AChE) inhibitor donepezil (Aricept, defend material pharmacy), Rivastigmine (Exelon, Novartis), galantamine (Razadyne/Reminyl, Johnson & Johnson), tacrine (Cognex, Warner-Lambert), huperzine A (Huperizine, multiple China of Shanghai Fudan University Pharmaceutical) and NMDA (N-methyl-D-aspartate) receptor antagonist Memantine hydrochloride (Namenda/Axura, Merck). Current AD therapy is both for what disease symptoms carried out. They can only in 6 to 12 months by a small margin stable or improve human-subject test and functional symptoms, some can continue to play lentamente drug effect after this. But these medicines are all without stopping or from substantially slowing down disease process.

AD patient's postmortem shows that obvious atrophy occurs in its cerebral tissue, and brain cell occurs extensively dead, the particularly brain cell in ganglion basal district, and the visible pathological changes such as senile plaque, neurofibrillary tangle. The Main Tissues pathological characteristics of AD is: under cerebral cortex, Hippocampus, some cortex, core group has substantial amounts of senile plaque (senileplaque, SP) to be formed in corpus amygdaloideum, Basal forebrain nucleus and thalamus. Senile plaque is by the amyloid-beta (��-amyloidprotein that a large amount of threadinesss, molecular weight are 4kD; A ��) assemble form.

The neuro chemistry of research discovery AD and neuro pathology change all close contacting with A ��, and the main hypothesis of current AD pathomechanism is A beta hypothesis. This hypothesis is thought: the gathering of A �� is the initiation factor causing these a series of pathological changes such as the amylaceous precipitation formation of speckle, the entanglement of nerve fiber, neuronal inflammation, neuronal function forfeiture and dead and finally dull-witted formation; When A �� generation is too much or its removing reduces time, a�� protein can be assembled to become highly neurovirulent polymer; This polyprotein can cause the factor of the induced neuronal death such as the hyperphosphorylation of body generation oxidative stress, inflammation, Tau albumen. So the generation of A �� and gathering are the key links of the main pathogenesis of AD. Therefore, based on the A �� important function risen in AD pathogenesis, the anti-AD medicine of targeting A �� is considered as be hopeful most to play the medicine [YaminG delaying AD pathogenesis always, OnoK, InayathullahM, TeplowDB.Amyloidbeta-proteinassemblyasatherapeutictarget ofAlzheimer ' sdisease.CurrentPharmaceuticalDesign, 2008,14:3231-3246; KurzA, PerneczkyR.AmyloidclearanceasatreatmenttargetagainstAlzh eimer ' sdisease.JournalofAlzheimer'sdisease, 2011,24 [Suppl2]: 61-73.].

The adjustment factor that internal A �� removes is very many, and one of them important factor is exactly autophagy. Remove it is now recognized that autophagy can increase intracellular A ��, A �� can be swallowed in autophagy vesicle after autophagy is activated, then carry out merging with lysosome and make A �� be degraded, thus reaching the effect of A �� in scavenger cell. Thus strengthening autophagocytosis is considered as a kind of good strategy [YangDS treating these neurodegenerative diseases, StavridesP, MohanPS, etal.Therapeuticeffectsofremediatingautophagyfailureinam ousemodelofAlzheimerdiseasebyenhancinglysosomalproteolys is.Autophagy, 2011,7:788-789; BachmeierC, Beaulieu-AbdelahadD, MullanM, ParisD.Selectivedihydropyiridinecompoundsfacilitatethecl earanceofbeta-amyloidacrosstheblood-brainbarrier.Europea nJournalofPharmacology, 2011,659:124-129.].

Fructus Arctii ArctiumlappaL. is that Compositae Arctium lappa belongs to 2 years raw herbaceous plant. Fructus Arctii is its dry mature fruit, is the traditional Chinese medicine of China. ATG is the topmost active component of Fructus Arctii. By the screening on cellular level, inventor have found that ATG can suppress A �� to generate and can increase again its removing. Further Study on Molecular Mechanism finds, ATG can suppress A �� to generate by reducing BACE1 to express, and can strengthen the removing of A �� by starting autophagy simultaneously; AD transgenic models mice carries out 100 days continuous intraperitoneal injection ATG (3mg/kg/d), and in result of the test display AD mouse brain, albumen precipitation speckle significantly reduces, and memory injury is substantially recovered. The early-stage Study result of inventor has been published in [ZhuY, YanJM, JiangW, YaoXG, ChenJ, ChenL on JournalofNeuroscience; LiCJ, HuLH*, JiangHL*, ShenX*.Arctigenineffectivelyamelioratesmemoryimpairmenti nAlzheimer'sdiseasemodelmicetargetingboth ��-amyloidproductionandclearance.JournalofNeuroscience, 2013,33 (32): 13138-13149.]. In research process before, inventor has synthesized a series of aretigenin carbamate derivates, and has applied for Patents [inventor: Hu Lihong, Shen Xu, Zhu Zhiyuan, Lei Min, Chen Jing, Yan Jianming; Arctigenin carboxamide derivatives and preparation method thereof, comprise this derivant compositions, and application thereof; Application number: 201210186849.3]. But before the aretigenin carbamate derivates of patent report fat-soluble strong, poorly water-soluble, oral administration biaavailability are relatively low, and not easily crystallization causes purification difficult. In order to overcome the deficiency of above-claimed cpd, inventor has redesigned the aretigenin carbamate derivates replaced containing amino that a class is new, by becoming salt with acid, the stability of its water solublity, oral administration biaavailability, compound can be improved, to salt by recrystallization purifying, its purifying process can be greatly simplified.

Summary of the invention

By the Study on Molecular Mechanism suppressing A �� nucleus formation to arctigenin (ATG), inventor have found that ATG can suppress A �� to generate by reducing BACE1 to express, the removing of A �� can be strengthened simultaneously by starting autophagy. But ATG oral administration biaavailability low (F about 1%), poorly water-soluble (is about 0.01mg/mL). Then for the water solublity and the oral administration biaavailability that improve ATG, inventor designs, has synthesized a class new carbamic acid aretigenin esters derivative prodrug as ATG. On such compound structure, feature is, the nitrogen of carbamate all has a nitrogenous substituted radical, and this nitrogenous substituent group can with organic acid or inorganic acids. The water solublity of this compounds improves nearly 50-500 times than ATG, and biological activity is maintained or improves, and after this compounds becomes salt, it is easy to form solid, it is simple to the separation of compound, purification, also improve the stability of compound.

One aspect of the present invention provides the arctigenin carbamate derivatives shown in formula (I) below a class or the purposes that its pharmaceutically acceptable salt is in preparing the medicine for treating Alzheimer:

X is-(CH₂)_n-or arlydene; It is preferably-(CH₂)_n-or phenylene;

N is 1,2 or 3;

R₁��R₂And R₃It is each independently hydrogen; C₁-C₆Alkyl; C₃-C₈Cycloalkyl; Unsubstituted or by C₁-C₄Alkyl, C₁-C₄The aryl that substituent group in alkoxyl, halogen, nitro, cyano group and hydroxyl replaces; Unsubstituted or wherein aryl by C₁-C₄Alkyl, C₁-C₄The aryl C that substituent group in alkoxyl, halogen, nitro, cyano group and hydroxyl replaces₁-C₄Alkyl; Unsubstituted or wherein heteroaryl by C₁-C₄Alkyl, C₁-C₄The 5-7 unit heteroaryl C that substituent group in alkoxyl, halogen, nitro, cyano group and hydroxyl replaces₁-C₄Alkyl; Preferably, R₁��R₂And R₃It is each independently hydrogen, C₁-C₄Alkyl or C₃-C₆Cycloalkyl; It is highly preferred that R₂For hydrogen or methyl;

It is further preferred that X is phenylene,For N, the N-dimethylamino being connected with phenyl ring ortho position.

Or,

X and R₃, and and R₃The nitrogen-atoms being connected collectively forms unsubstituted or is selected from C₁-C₄Alkyl, halogen ,-NH₂4-8 member heterocyclic ring containing nitrogen with the substituent group replacement in hydroxyl, it is preferable that X and R₃, and and R₃The nitrogen-atoms being connected collectively forms unsubstituted or is selected from C₁-C₄Alkyl, halogen ,-NH₂The 4-8 member heterocyclic ring containing nitrogen containing a nitrogen-atoms with the substituent group replacement in hydroxyl, it is more preferred to, X and R₃, and and R₃The nitrogen-atoms being connected collectively forms piperidine ring;

Or

X and adjacent two nitrogen-atoms, R₃And R₁Collectively form unsubstituted or be selected from C₁-C₄Alkyl, halogen ,-NH₂4-8 member heterocyclic ring containing nitrogen with the substituent group replacement in hydroxyl, it is preferable that X and adjacent two nitrogen-atoms, R₃And R₁Collectively form unsubstituted or be selected from C₁-C₄Alkyl, halogen ,-NH₂The 4-8 member heterocyclic ring containing nitrogen containing two nitrogen-atoms with the substituent group replacement in hydroxyl; It is highly preferred that X and adjacent two nitrogen-atoms, R₃And R₁Collectively form piperazine ring.

In the present invention, term " aryl " refers to aromatic ring base, it will be preferred that carbon number is the aryl of 6-14, more preferably carbon number is the aryl of 6-10, such as phenyl, naphthyl, xenyl.

In the present invention, term " arlydene " is and is preferably the arlydene that carbon number is 6-14, more preferably carbon number is the arlydene of 6-10, such as phenylene, naphthylene, biphenylene.

In the present invention, term " 5-7 unit heteroaryl " refers to containing the heteroatomic aromatic ring base of at least one in O, N and S, it is preferred to furyl, pyrrole radicals, thienyl, pyrazolyl, imidazole radicals, pyridine radicals, pyrazinyl or azoles base.

In the present invention, term " C₁-C₆Alkyl " refer to the straight or branched alkyl with 1 to 6 carbon atom, include methyl, ethyl, propyl group, isopropyl, butyl, isobutyl group, sec-butyl, the tert-butyl group, amyl group, base etc. without limitation; Preferred methyl, ethyl, propyl group, butyl. Similarly, term " C₁-C₄Alkyl " refer to the straight or branched alkyl with 1 to 4 carbon atom, include methyl, ethyl, propyl group, isopropyl, butyl, isobutyl group etc. without limitation; Preferred methyl, ethyl, propyl group, butyl.

In the present invention, term " C₃-C₈Cycloalkyl " refer to the cyclic alkyl on ring with 3 to 8 carbon atoms, include cyclopropyl, cyclobutyl, cyclopenta, cyclohexyl or suberyl without limitation; Preferred cyclohexyl. Similarly, term " C₃-C₆Cycloalkyl " refer to the cyclic alkyl on ring with 3 to 6 carbon atoms, include cyclopropyl, cyclobutyl, cyclopenta or cyclohexyl without limitation; Preferred cyclohexyl.

In the present invention, term " 4-8 member heterocyclic ring containing nitrogen " refers to have at least one N heteroatomic 4-8 unit saturated heterocyclic on ring, includes without limitation: piperidine ring or piperazine ring.

In the present invention, term " pharmaceutically acceptable salt " refers to and the mineral acids such as phosphoric acid, sulphuric acid, hydrochloric acid, or the organic acid such as acetic acid, tartaric acid, citric acid, malic acid, or the salt that the acidic amino acid such as aspartic acid, glutamic acid is formed, or become, with above-mentioned acid, the salt formed again after ester or amide with inorganic base, such as sodium, potassium, calcium, aluminium salt and ammonium salt.

In the present invention, the arctigenin carbamate derivatives shown in described formula (I) is preferably selected from following compounds:

Arctigenin carbamate derivatives shown in formula of of the present invention (I) can adopt synthetic method as known in the art to prepare, or adopts following synthetic method to prepare:

Wherein, X, R₁, R₂And R₃Define same as described above;

ATG and 4-nitroxyl chloride phenyl formate generation esterification generates intermediate A, and then A obtains compound of formula I with corresponding amine generation substitution reaction.

Specifically, ATG is dissolved in solvent (such as, oxolane, dichloromethane, acetonitrile etc.) in and add N, N-diisopropyl ethyl amine (DIPEA), add 4-nitroxyl chloride phenyl formate, heat to the 6h that refluxes, after reaction terminates, reactant liquor saturated sodium carbonate is washed, and obtains the solution of intermediate A. N, N-diisopropyl ethyl amine (DIPEA) and corresponding amine is added in this solutionHeating to being back to reaction and terminate, reactant liquor saturated sodium carbonate washes, obtain formula 1 compound through column chromatography after dry and concentration.

Arctigenin carbamate derivatives shown in formula of of the present invention (I) or its pharmaceutically acceptable salt can deliver medicine to people, it is possible to oral, rectum, parenteral (intravenous, intramuscular or subcutaneous), topical (powder, ointment or drop).

Solid dosage forms for oral administration includes capsule, tablet, pill, powder and granule. In these solid dosage formss, reactive compound mixes with at least one conventional inert excipients (or carrier), such as sodium citrate or dicalcium phosphate, or mix with following compositions: (a) filler or bulking agent, such as, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binding agent, for instance, hydroxymethyl cellulose, alginate, gelatin, polyvinyl pyrrolidone, sucrose and arabic gum; (c) wetting agent, for instance, glycerol; (d) disintegrating agent, for instance, agar, calcium carbonate, potato starch or tapioca, alginic acid, some composition silicate and sodium carbonate; (e) retarding solvent, for instance paraffin; F () absorbs accelerator, for instance, quaternary ammonium compound; (g) wetting agent, for instance spermol and glyceryl monostearate; (h) adsorbent, for instance, Kaolin; (i) lubricant, for instance, Talcum, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulphate, or its mixture. In capsule, tablet and pill, dosage form also can comprise buffer agent.

Solid dosage forms such as tablet, sugar pill, capsule, pill and granule can adopt coating and shell material to prepare, such as casing and other material well known in the art. They can comprise opacifying agent, and, in this compositions, the release of reactive compound or compound can release in the part of certain in digestive tract in a delayed fashion. The example of adoptable embedding component is polymeric material and Wax. If desired, reactive compound also can with one or more formation microencapsulation form in above-mentioned excipient.

Liquid formulation for oral administration includes pharmaceutically acceptable emulsion, solution, suspension, syrup or tincture. Except active ingredient beyond the region of objective existence, liquid dosage form can comprise the conventional inert diluent adopted in this area, such as water or other solvent, solubilizing agent and emulsifying agent, example is known, the mixture etc. of ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3 butylene glycol, dimethylformamide and oil, particularly Oleum Gossypii semen, Oleum Arachidis hypogaeae semen, maize embryo oil, olive oil, Oleum Ricini and Oleum sesami or these materials.

Except these inert diluents, above-mentioned composition also can comprise auxiliary agent, such as wetting agent, emulsifying agent and suspending agent, sweeting agent, correctives and spice.

Except active ingredient beyond the region of objective existence, suspension can comprise suspending agent, for instance, the mixture etc. of ethoxylation isooctadecane alcohol, polyoxyethylene sorbitol and Isosorbide Dinitrate, microcrystalline Cellulose, aluminium methoxide and agar or these materials.

Compositions for parenteral injection can comprise physiologically acceptable sterile, aqueous or anhydrous solution, dispersion liquid, suspension or emulsion, and for being again dissolved into the sterilized powder of aseptic Injectable solution or dispersion liquid. Moisture and the nonaqueous carrier, diluent, solvent or the excipient that are suitable for include water, ethanol, polyhydric alcohol and suitable mixture thereof.

Dosage form for the compounds of this invention of topical includes ointment, powder, propellant and inhalant. Active component aseptically with physiologically acceptable carrier and any preservative, buffer agent, or if desired be likely to need propellant be mixed together.

Present invention also offers a kind of method treating Alzheimer, described method includes the arctigenin carbamate derivatives shown in the formula of of the present invention (I) to patient's drug treatment effective dose with these needs and one or more in its pharmaceutically acceptable salt.

Beneficial effect

Shown in the formula (I) of the present invention, arctigenin carbamate derivatives or its pharmaceutically acceptable salt have the effect of anti-AD. Whole animal test shows, this compounds has can substantially reverse the memory injury performance that transgenic mice occurs, therefore can as the medicine for the treatment of Alzheimer.

Accompanying drawing explanation

Fig. 1 is the chart that display ATG-A02 hydrochlorate alleviates the memory impairment of transgenic mice, the wherein transgenic mice of ATG-A02-HCl process in T-ATG-A02HCl:10mg/kg/ days, T-V: the transgenic mice that solvent processes, N-V: the nontransgenic mice that solvent processes.

Fig. 2 is the chart that display ATG-A02 hydrochlorate alleviates the memory impairment of transgenic mice, the wherein transgenic mice of ATG-A02-HCl process in T-ATG-A02HCl:10mg/kg/ days, T-V: the transgenic mice that solvent processes, N-V: the nontransgenic mice that solvent processes.

Detailed description of the invention

Below in conjunction with specific embodiment, the present invention is further elaborated, but the present invention is not limited to this.

In following preparation example,¹H-NMR VarianMercuryAMX300,400,500 type Instrument measuring. MS VGZAB-HS or VG-7070 type and Esquire3000plus-01005 measure. All solvents are before use all through re-distillation, and the anhydrous solvent used is all obtain by standard method dried. Unless otherwise indicated, being responded is all carry out under argon shield and follow the tracks of with TLC, all through saturated common salt washing and anhydrous magnesium sulfate dry run during post processing. The purification of product unless otherwise indicated all uses the column chromatography of silica gel, and the silica gel used is 200-300 order, GF₂₅₄Produce for Haiyang Chemical Plant, Qingdao or Yantai Yuan Bo silica gel company.

Preparation embodiment

Embodiment 1: the synthesis of compound ATG-A01

By p-nitrophenyl chloroformate ester (9.2mmol, 1.85g) stirring and dissolving in 60mLTHF, being subsequently adding DIPEA (1.6mL), reactant liquor occurs white solid at once. Add ATG (4mmol, 1.54g) under stirring, heat to back flow reaction 6h, TLC detection reaction until raw material A TG point disappears. After reaction terminates, repeatedly it is washed till water layer with saturated sodium carbonate to colourless, obtains the THF solution of the compound of structural formula A. Adding DIPEA (1.6mL), N in this solution, N-dimethyl-1��2-diaminoethane (12mmol, 1.0g) also heats to backflow, and TLC detection reaction is until raw material A point disappears. After reaction terminates, reactant liquor washes with water after once, is repeatedly washed till water layer with saturated sodium carbonate colourless, and THF layer is again with after saturated common salt washing once, and decompression lower removing solvent obtains yellow solid. Yellow solid obtains compound ATG-A01 through column chromatography for separation (petroleum ether/acetone=3:1), and yield is 54%.

¹HNMR(400MHz,CDCl₃) �� 6.97 (dd, J=8.1, 1.8Hz, 1H), 6.75 (d, J=8.1Hz, 1H), 6.73 (d, J=1.9Hz, 1H), 6.65 (dd, J=7.9, 2.0Hz, 1H), 6.53 (d, J=7.9Hz, 1H), 6.49 (d, J=2.0Hz, 1H), 4.13 (dd, J=6.3, 5.7Hz, 1H), 3.88 (t, J=5.7Hz, 1H), 3.84 (s, 3H), 3.82 (s, 3H), 3.76 (s, 3H), 3.64 (brs, 2H), 3.51 (brs, 2H), 2.95 (d, J=5.7Hz, 2H), 2.93 2.86 (m, 4H), 2.78 2.31 (m, 4H) .ESI-MS:m/z=487 ([M+H]⁺)��

Embodiment 2: the synthesis of compound ATG-A02

Outside replacing N, N-dimethyl-1��2-diaminoethane except with piperazine, prepare compound ATG-A02 in the same manner as example 1. Silica gel column chromatography eluent: petroleum ether/acetone=3:1, obtains the compound ATG-A02 of white powder, and yield is 53%.

¹HNMR(400MHz,CDCl₃) �� 8.61 (s, 1H), 8.56 (d, J=4.9Hz, 1H), 7.71 (d, J=7.9Hz, 1H), 7.30 (dd, J=7.9, 4.9Hz, 1H), 7.01 (d, J=8.0Hz, 1H), 6.77 (d, J=8.3Hz, 1H), 6.75 (d, J=2.0Hz, 1H), 6.65 (dd, J=8.0, 1.9Hz, 1H), 6.54 (dd, J=8.1, 2.0Hz, 1H), 6.50 (d, J=2.0Hz, 1H), 5.52 (t, J=6.2Hz, 1H), 4.47 (d, J=6.2Hz, 2H), 4.16 (dd, J=9.1, 7.2Hz, 1H), 3.89 (dd, J=9.1, 7.6Hz, 1H), 3.84 (s, 3H), 3.81 (s, 3H), 3.78 (s, 3H), 2.96 (d, J=5.7Hz, 2H), 2.70 2.41 (m, 4H), ESI-MS:m/z=485 ([M+H]⁺)��

Embodiment 3: the synthesis of compound ATG-A03

Outside replacing N, N-dimethyl-1��2-diaminoethane except with 1-methyl piperazine, prepare compound ATG-A03 in the same manner as example 1. Silica gel column chromatography eluent: petroleum ether/acetone=3:1, obtains the compound ATG-A03 of white powder, and yield is 52%.

¹HNMR(400MHz,CDCl₃) �� 6.97 (d, J=8.0Hz, 1H), 6.76 (d, J=8.1Hz, 1H), 6.73 (d, J=1.9Hz, 1H), 6.65 (dd, J=8.1, 1.9Hz, 1H), 6.53 (dd, J=8.1, 2.0Hz, 1H), 6.49 (d, J=2.0Hz, 1H), 4.14 (dd, J=9.1, 7.1Hz, 1H), 3.88 (dd, J=9.1, 7.0Hz, 1H), 3.85 (s, 3H), 3.82 (s, 3H), 3.76 (s, 3H), 3.70 (brs, 2H), 3.56 (brs, 2H), 2.96 (d, J=5.8Hz, 2H), 2.74 2.48 (m, 4H), 2.44 (t, J=5.1Hz, 4H), 2.33 (s, 3H), ESI-MS:m/z=499 ([M+H]⁺)��

Embodiment 4: the synthesis of compound ATG-A04

Outside replacing N, N-dimethyl-1��2-diaminoethane except with 4-amino piperidine, prepare compound ATG-A04 in the same manner as example 1. Silica gel column chromatography eluent: petroleum ether/acetone=3:1, obtains the compound ATG-A04 of white powder, and yield is 52%.

¹HNMR(400MHz,CDCl₃) �� 6.97 (d, J=8.0Hz, 1H), 6.76 (d, J=8.1Hz, 1H), 6.73 (d, J=1.8Hz, 1H), 6.65 (dd, J=8.0, 1.9Hz, 1H), 6.54 (dd, J=8.3, 2.0Hz, 1H), 6.50 (d, J=2.0Hz, 1H), 4.31 4.20 (m, 1H), 4.14 (dd, J=9.1, 6.8Hz, 2H), 3.91 3.86 (m, 1H), 3.85 (s, 3H), 3.83 (s, 3H), 3.77 (s, 3H), 3.18 2.85 (m, 6H), 2.75 2.40 (m, 4H), 1.97 1.81 (m, 2H), 1.46 1.28 (m, 2H), ESI-MS:m/z=499 ([M+H]⁺)��

Embodiment 5: the synthesis of compound ATG-A05

Outside replacing N, N-dimethyl-1��2-diaminoethane except with 2-(dimethylamino)-aniline, prepare compound ATG-A05 in the same manner as example 1. Silica gel column chromatography eluent: petroleum ether/acetone=3:1, obtains the compound ATG-A05 of white powder, and yield is 34%.

¹HNMR(400MHz,CDCl₃) �� 7.12 7.04 (m, 2H), 6.97 (dd, J=7.8, 1.7Hz, 1H), 6.82 (d, J=7.9Hz, 1H), 6.76 6.71 (m, 2H), 6.62 (d, J=1.9Hz, 1H), 6.59 (dd, J=7.9, 2.0Hz, 1H), 6.53 (dd, J=8.1, 2.0Hz, 1H), 6.45 (d, J=2.1Hz, 1H), 5.79 (s, 1H), 4.13 (dd, J=9.1, 7.1Hz, 1H), 3.87 (dd, J=9.1, 7.2Hz, 1H), 3.84 (s, 3H), 3.80 (s, 6H), 3.42 (s, 6H), 2.91 (d, J=5.7Hz, 2H), 2.65 2.57 (m, 4H), ESI-MS:m/z=535 ([M+H]⁺)��

Embodiment 6: the synthesis of compound ATG-A01 hydrochlorate

Being dissolved in ethanol by compound ATG-A01, dropping ethanol solution hydrochloride to pH value is 4-5, and removal of solvent under reduced pressure obtains compound ATG-A01 hydrochlorate again through ethyl alcohol recrystallization.

The hydrochlorate of other compounds all, all can react corresponding compound with ethanol solution hydrochloride by the method for embodiment 6 and be prepared.

The organic acid of the compound mentioned by the present invention and inorganic acid salt all can use method similar to Example 6 and be prepared with corresponding organic acid or inorganic acid reaction by described compound.

Test example

The water solublity of test example 1 arctigenin derivant

Synthesized arctigenin derivant hydrochloric acid salt dynamic solubility algoscopy is measured, i.e. precise 50mg compound, and dripped deionized water, until solid is completely dissolved, writing down the volume v of water, represent the dissolubility of compound with 50/v, unit is mg/mL.

The hydrochlorate of the arctigenin derivant of the designed synthesis of the present invention, its water solublity is at 0.5-5mg/mL, water solublity (0.01mg/mL) compared to ATG and the B-08 (compound that in Chinese patent application 201210186849.3, activity is the strongest, by reference the complete disclosure of Chinese patent application 201210186849.3 is incorporated into herein at this) water solublity (< 0.01mg/mL), improve nearly 50-500 times.

Table 1, arctigenin derivant hydrochloric acid salt dissolubility

Test example 2 cellular level test arctigenin derivant is to the A �� inhibitory action formed

The present invention tests arctigenin derivant to the A �� inhibitory action formed by enzyme-linked immunosorbent assay (ELISA) on the Chinese hamster ovary cell (Chinese hamster ovary celI) of high expressed amyloid-beta precursor protein (APP) and beta-secretase (BACE1), and the A �� contents level obtained directly reflects its compound activity level.

1, experimental principle

Adopt A �� content in ELISA method detection cell conditioned medium, its principle is: the A �� formed in cell can secrete to cell culture fluid, therefore white clear 96 orifice plate of A �� antibody there are to hatch culture fluid and coupling, another site antibodies being subsequently adding A �� is hatched simultaneously, by antigen and antibody specific identification combination principle, it is adsorbed on 96 orifice plates by simultaneously specific to A �� and antibody thereof; Then two anti-and chromogenic substrate (Tetramethylbenzidine of coupling horseradish peroxidase are used, TMB) hatch, the coloured product depth generated is directly proportional to A �� content, the absorbance (OD450) of bioassay standard pipe and sample tube respectively, calculates the content of A ��.

2, experiment material and method

1) A beta determination test kit is purchased from invitrogen company, and cell culture reagent is all purchased from Gibico company.

2) CHO-APP&BACE1 (proceeds to pcDNA3.1a-APP plasmid in Chinese hamster ovary celI, then passes through G418 antibiotic and carry out stable strain screening, obtain the stable strain of CHO-APP, continue to enter pSecTag2-BACE1 plasmid at CHO-APP transit cell, then pass through two kinds of antibiotic of zeocin and G418 and carry out stable strain screening, obtain the stable strain of CHO-APP&BACE1) cell cultivation: CHO-APP&BACE1 F12 culture fluid (10%FBS) is incubated in 24 orifice plates, when 70% cell density, it is separately added into various arctigenin derivant (20 ��Ms) (referring to table 1) hatches 24 hours, take out culture fluid 10, 000 is centrifuged 10 minutes, take supernatant, add protease inhibitor (cocktail), then pass through ELISA method and measure A �� content in supernatant.

3) cell conditioned medium standard dilution is carried out ten times of dilutions, then according to the provided method of test kit detects. Namely at room temperature 96 orifice plates of the supernatant after dilution, A �� antibody and coupling A �� another kind antibody are hatched 3 hours jointly, then carry out washing plate 4 times with the rinsing liquid provided in test kit, add coupling and have the two anti-incubated at room temperature 30 minutes of horseradish peroxidase, plate is washed five times again with rinsing liquid, it is subsequently adding nitrite ion in incubated at room 30 minutes, is eventually adding stopping of reaction liquid. With Bio-Rad microplate reader in 450nm place reading OD value.

3, experimental result

Result is as shown in table 2, and arctigenin carboxamide derivatives ATG-A01��ATG-A05 all has the effect suppressing A �� formation, and the physicochemical property of these compounds all significantly improves than ATG simultaneously.

Table 2, arctigenin and derivant thereof are to the A �� inhibitory activity formed

Compound	Suppression ratio (20 ��Ms)	Compound	Suppression ratio (20 ��Ms)
				DMSO	0	ATG	34.84%
ATG-A01-HCl	35.02%	ATG-A02-HCl	36.71%
				ATG-A03-HCl	36.40%	ATG-A04-HCl	37.30%
ATG-A05-HCl	73.50%

The pharmacokinetic of test example 3ATG-A02 hydrochlorate and B-08

Single dose intravenous (IV) and oral (PO) give SpragueDawley rat tested material ATG-A02 hydrochlorate 50mg/kg, gathering blood sample in different time points, LC/MS/MS measures the concentration of tested material in rat plasma after giving tested material and calculates relevant parameter. What detect in blood plasma after oral is mainly composed of ATG-A02 and ATG. Test result is in Table 3. Count its oral administration biaavailability (F) for 61.7% with ATG-A02, count its oral administration biaavailability (F) for 31.3% with ATG.

Table 3, rat single oral give ATG-A02 and the ATG main pharmacokinetic parameters after ATG-A02 hydrochlorate

AUC_(0-t)For area under the drug-time curve, refer to that plasma concentration curve is from 0 to the t area surrounded with time shaft.

AUC_(0-��)For area under the drug-time curve, refer to the entire area that time shaft is surrounded by plasma concentration curve.

MRT_(0-��)For average residence time, the meansigma methods of refer to drug molecule time of staying in vivo.

t_1/2zFor t1/2, refer to medicine reach distribution equilibrium after blood drug level reduce the time needed for 50%.

T_maxFor peak time, after referring to single medication, blood drug level reaches the time of peak value.

C_maxFor peak concentration of drug, the blood drug level peak occurred after showing medicine.

F is bioavailability, is absorbed into the relative quantity of systemic blood circulation medicine after referring to the outer administration of medicine intravascular.

Similarly, single dose intravenous (IV) and oral (PO) give SpragueDawley rat tested material B-0810mg/kg, gathering blood sample in different time points, LC/MS/MS measures the concentration of tested material in rat plasma after giving tested material and calculates relevant parameter. What detect in blood plasma after oral is mainly composed of B-08 and ATG. Count its oral administration biaavailability for 1.7% with B-08, count its oral administration biaavailability for 9.2% with ATG.

Test example 4ATG-A02 hydrochlorate reverses the effect of alzheimer disease transgenic models mouse memory power damage

The present invention evaluates compound ATG-A02 hydrochlorate to memory injury improvement result by APP/PS1 double transgenic Alzheimer's disease model mouse. Result shows that compound ATG-A02 hydrochlorate can substantially reverse the memory injury of Alzheimer's disease model mouse.

1, experimental principle

That this experiment adopts is APP/PS1 double transgenic Alzheimer's disease model mouse (B6C3 transgenic mice, APPswe, PS1dE9). This kind of transgenic mice energy high expressed is embedding and the presenilin 1 albumen (presenilin, PS1-dE9) of the 9th exon is deleted in the swedish mutant APP (Mo/HuAPP695swe) of Mus/people and people source. Can there is A �� deposition and can occur the infringement of spatial memory when 7 months when 6-7 month in this type of transgenic mice.

We adopt the Morris water maze laboratory (MorrisWaterMaze, MWM) of classics to evaluate the memory injury situation of Alzheimer's disease model mouse. Experiment is divided into two parts: experiment found by training experiment and platform. Search out plateau time length with training experiment small mouse and platform finds experiment small mouse spanning platform number of times for evaluating the index of mouse memory power situation.

2, experiment material and method

1) APP/PS1 double transgenic Alzheimer's disease model mouse buys the JacksonLaboratory company in the U.S., then breeding and qualification. By adopting, mice being cut tail, then PCR identifies that the gene order of the APP/PS1 of mice is to identify murine genes type. Adopt the mice not proceeding to the two gene after identifying as the Negative control mice in experiment in an experiment. Mice raises (SPF level environment, light and shade circulation in 12/12 hour, enough water and food, the constant temperature of 22 DEG C, the humidity of 60%) at the standard conditions.

2) Alzheimer's disease model mouse administration: when mice size in June, 20 transgenic mices are randomly divided into 2 groups of (transgenic group of solvents, transgenic ATG-A02 hydrochlorate-10mg/kg dosage group), 10 nontransgenic mice are as negative control group. Compound ATG-A02 hydrochlorate is directly dissolved in normal saline, adopts gastric infusion 100 days, then starts Behaviors survey detection (Morris water maze laboratory).

3) mice training experiment: mice carries out three training every day, continues six days. In training at three times, mice is placed in water towards pool wall at three quadrantal points removing platform place by respectively, looks for position of platform then to mice 90 seconds, and mice stops 20 seconds to help its memory position of platform on platform. During this period, record mice finds time and the incubation period of platform. At the 6th day, after three times are trained, mice carries out platform and finds experiment. In current experiment, diving and removed in undersurface platform, then allow mice continue to look for for 90 seconds platform in pond, record mice spanning platform number of times is as the evaluation index of its memory, and number of times more more shows that its memory is more good. All of zoopery operation all strictly observes " management of laboratory animal regulations ".

3, experimental result

Experimental result finds, it is considerably longer than nontransgenic mice (Fig. 1) incubation period of transgenic mice, and spanning platform number of times is considerably less than nontransgenic mice (Fig. 2), it was shown that substantially damage occurs in the memory of transgenic mice, it was shown that its Alzheimer-like Disease Model is correct; Give compound ATG-A02 hydrochlorate (10mg/kg/day,) transgenic mice be significantly shorter than the transgenic mice (Fig. 1) that solvent processes incubation period, and spanning platform number of times is significantly more than the transgenic mice (Fig. 2) that solvent processes, it was shown that compound ATG-A02 hydrochlorate can substantially reverse the memory injury performance that transgenic mice occurs; These results show that compound ATG-A02 hydrochlorate can play treatment Alzheimer effect very well.

Claims

1. the arctigenin carbamate derivatives shown in below formula (I), or its pharmaceutically acceptable salt is used for the purposes treating in the medicine of Alzheimer in preparation,

X is-(CH₂)_n-, or arlydene;

N is 1,2 or 3;

R₁��R₂And R₃It is each independently hydrogen; C₁-C₆Alkyl; C₃-C₈Cycloalkyl; Unsubstituted or by C₁-C₄Alkyl, C₁-C₄The aryl that substituent group in alkoxyl, halogen, nitro, cyano group and hydroxyl replaces; Unsubstituted or wherein aryl by C₁-C₄Alkyl, C₁-C₄The aryl C that substituent group in alkoxyl, halogen, nitro, cyano group and hydroxyl replaces₁-C₄Alkyl; Unsubstituted or wherein heteroaryl by C₁-C₄Alkyl, C₁-C₄The 5-7 unit heteroaryl C that substituent group in alkoxyl, halogen, nitro, cyano group and hydroxyl replaces₁-C₄Alkyl;

Or

X and R₃, and and R₃The nitrogen-atoms being connected collectively forms unsubstituted or is selected from C₁-C₄Alkyl, halogen ,-NH₂With in hydroxyl substituent group replace 4-8 member heterocyclic ring containing nitrogen, or

X and adjacent two nitrogen-atoms, R₃And R₁Collectively form unsubstituted or be selected from C₁-C₄Alkyl, halogen ,-NH₂4-8 member heterocyclic ring containing nitrogen with the substituent group replacement in hydroxyl.

2. purposes according to claim 1, wherein, X is-(CH₂)_n-, or phenylene.

3. purposes according to claim 2, wherein, X is phenylene,For N, the N-dimethylamino being connected with phenyl ring ortho position.

4. purposes according to claim 1, wherein, R₁��R₂And R₃It is each independently hydrogen, C₁-C₄Alkyl or C₃-C₆Cycloalkyl.

5. purposes according to claim 1, wherein, R₂For hydrogen or methyl.

6. purposes according to claim 1, wherein, X and R₃, and and R₃The nitrogen-atoms being connected collectively forms unsubstituted or is selected from C₁-C₄Alkyl, halogen ,-NH₂With the substituent group in hydroxyl replace containing the 4-8 member heterocyclic ring containing nitrogen of a nitrogen-atoms, or

X and adjacent two nitrogen-atoms, R₃And R₁Collectively form unsubstituted or be selected from C₁-C₄Alkyl, halogen ,-NH₂The 4-8 member heterocyclic ring containing nitrogen containing two nitrogen-atoms with the substituent group replacement in hydroxyl.

7. purposes according to claim 1, wherein, X and R₃, and and R₃The nitrogen-atoms being connected collectively forms piperidine ring, or

X and adjacent two nitrogen-atoms, R₃And R₁Collectively form piperazine ring.

8. purposes according to claim 1, wherein, described arctigenin carbamate derivatives is selected from following compounds: