A kind of substituted pyrrolidine carboxylic acid's ester type compound and its preparation method and applicationTechnical field
The present invention relates to technical field of pharmaceuticals, more particularly to a kind of substituted pyrrolidine carboxylic acid's ester type compoundAnd its preparation method and application.
Background technology
Asthma and COPD (COPD) are common diseases in tuberculosis.Asthma is particularly fertile by various kinds of cellThe chronic airway inflammation that maxicell, eosinophil and T lymphocytes participate in；Such a inflammation can cause repeatedly in susceptible personBreaking-out pant, shortness of breath, it is uncomfortable in chest and cough etc. symptom.The global people of asthma disease patient about 300,000,000, Chinese patients with asthma about 3000Ten thousand.In recent years, asthma prevalence rose year by year, and Beijing, Shanghai two places asthma prevalence rise 116.5% during the decade.When nineteen ninety, national pathogenesis of childhood asthma rate rises to 4.5%, or even send out in some developed countries 0.91% at presentSick rate can reach more than ten to three ten percent.COPD (COPD) common sympton is chronic cough, expectoration, is breathed heavilyIt is breath, uncomfortable in chest, had difficulty in breathing after activity, during diseases acute flare-ups or disease enters late period, severe stage, under patient's quiescent condition i.e.It is likely to occur expiratory dyspnea.COPD occupies the 4th of the global cause of death at present.Number of patients increases year by year, death rate rise, shapeInto serious social economical burden weight, it has also become influence the important public health problem of human health.I am more than 40 years old crowdCOPD illness rates are 9-14%.
Above-mentioned disease generally use bronchiectasis medicine is treated, and one kind is by β2Acceptor (adrenocepter) excitementAgent forms, such as salbutamol, Formoterol and salmeterol；It is another kind of be by m receptor (M-ChR) antagonist group into,Such as isopropyl support amine, tiotropium.
B-adrenergic receptor belongs to G- protein coupling receptor superfamilies, is divided into three hypotypes, i.e. β1-β3.AutoradiographTechnology confirms β2It is widely distributed in lungs, it is not only present on airway smooth muscle cells film, exists in other lung tissue cellsSuch as mucomembranous epithelial cell, vascular endothelial cell, II types alveolar epithelial cells and mast cell cell membrane.On smooth muscle cellDistribution density with air flue be classified and gradually increase, be the major receptors of expansion bronchus.β2The classical signals transduction of acceptorApproach is cAMP Dependents, or activates calcium by the non-dependent approach of cAMP and activate potassium-channel, makes smooth muscle cellInterior efflux of K+ ions, cell membrane potential are in super excited state, so as to diastole smooth muscle.
M receptor belongs to G- protein coupling receptor superfamilies together with beta-receptor, there is 5 i.e. M of hypotype1-M5, wherein only M1-M3AcceptorIt is distributed in the intrapulmonary of people and most animals.The M being distributed on smooth muscle cell membrane3The density of acceptor with the classification of air flue and byGradually reduce, its excitation energy shrinks smooth muscle.By suppressing or stimulating M3Acceptor can be controlled by fiber institute after parasympathetic ganglionThe smooth muscle contraction of organ and glandular secretion where the effector cell of domination.M3Receptor antagonist is that clinical treatment COPD is heldThe first-line drug of continuous patient with sympotoms.
Selective β2Receptor stimulating agent and M3Receptor antagonist is that current clinical practice is most wide, the bronchus solution of most speciesConvulsion agent, especially inhalant dosage form are widely used to controlling for bronchial astehma and COPD (COPD) acute attackTreat, can effectively alleviate the acute symptom of disease.Hughes A.D. et al., which are devised, a series of has β2Receptor stimulating agent and M3Receptor antagonist double pharmacological action drug molecule treats asthma and COPD, thus gives full play to the collaboration of two kinds of pharmacological actionsComplementary drug action, it is possible to obtain the mutual facilitation effect of " 1+1=3 ".Preliminary research shows that some dual pharmacology are madeIt is significantly larger than the exercising result of single-acting molecule with the drug effect of molecule, but and not all there is β2Receptor stimulating agent and M3AcceptorAntagonist double pharmacological action drug molecule all has the effect of this high-drug-effect.
Therefore it provides there is β while brand-new2Receptor agonist activity and M3The high-drug-effect chemical combination of receptor antagonist activityThing, more medicament selections can be provided for patient, reduce asthma and COPD treatment costs, and these compounds pass through two kindsIndependent action pattern provides bronchiectasis has single molecular medicine dynamic metabolism simultaneously, and this takes selectivity than simultaneouslyβ2Receptor stimulating agent and M3Receptor antagonist pharmaceuticals are more convenient.
The content of the invention
In view of this, it is an object of the invention to provide a kind of substituted pyrrolidine carboxylic acid's ester type compound and its preparation sideMethod and application so that the compound has β simultaneously2Receptor stimulating agent and M3Receptor antagonist double pharmacological action, and showHigher than the drug effect of single-acting molecule, can be applied in asthma and COPD treatment.
For achieving the above object, the present invention provides following technical scheme：
A kind of substituted pyrrolidine carboxylic acid's ester type compound of structure shown in formula 1：
Or its stereoisomer, geometric isomer, dynamic isomer, nitrogen oxides, solvate, metabolite, pharmacyUpper acceptable salt or its prodrug；
Wherein, R1Selected from hydrogen, C1-C10Straight chained alkyl, R2Selected from phenyl, C3-C10Cycloalkyl.
Preferably, R1For hydrogen, methyl or ethyl.Propyl group, fourth can also be selected from the embodiment of the present inventionBase, amyl group, hexyl, heptyl, octyl group, nonyl, decyl.
Preferably, R1For para-orientating group.
Preferably, R2For phenyl, cyclohexyl or pentamethylene base.It can also be selected in the embodiment of the present inventionFrom cyclopropane base, cyclobutane base, cycloheptyl alkyl, cyclooctane base, cyclononane base, cyclodecane base.
Preferably, the compound has the structure of one of：
In to the effect of m receptor subtype-selective and beta receptor subtype-selective Effect study, compound of the present inventionWith to M3The selectivity of receptor subtype and to β2Receptor subtype-selective.Also, in mouse test, chemical combination of the present inventionThe ED of thing anti-asthmatic action50There is β compared to existing2The ED of the salbutamol of receptor agonist activity50, it is its 5-10 times, while withWith M3The medicine glycopyrronium bromide of receptor antagonist activity is slightly good or suitable compared to activity, shows that compound of the present invention hasSplendid drug effect, it can be used for treating in the tuberculosis such as asthma and COPD.
Based on above-mentioned technique effect, the invention provides compound described in any one technical scheme of the present invention to control in preparationTreat the application in tuberculosis medicine.Preferably, the tuberculosis is asthma and COPD.
In addition, the present invention is based on application field, a kind of medical composition is additionally provided, the present invention comprising effective dose is anyCompound and pharmaceutically acceptable auxiliary material or active material described in one technical scheme, the pharmaceutically acceptable activityMaterial is the active compound or group for not producing antagonism with compound described in any one technical scheme of the present inventionCompound.
Preferably, the medical composition is oral formulations or spray-type, but this area can also be made simultaneouslyOther common formulations.
Meanwhile present invention also offers the preparation method of the compound, including：
With (4'- hydroxyl -3'- methylols) phenyl-(2- hydroxyl -4- bromos) butane substitution occurs for compound shown in formula 2 insteadAnswer compound shown in production 1；
Wherein, R1Selected from hydrogen, C1-C10Straight chained alkyl, R2Selected from phenyl, C3-C10Cycloalkyl.
The compound of formula 2 of the present invention and (4'- hydroxyl -3'- methylols) phenyl-(2- hydroxyl -4- bromos) butane are equalCan synthesize and obtain according to synthetic method that this area has been reported, pertinent literature referring to Antonio Mete, Keith Bowers,Eric Chevalier,David K.Donald,Helen Edwards,Katherine J.Escott,Rhonan Ford,Ken Grime, Ian Millichip, Barry Teobald, Vince Russell, Bioorganic＆MedicinalChemistry Letters 21(2011)7440–7446；
Anqiu Liu,Ling Huang,Zhiren Wang,Zonghua Luo,Fei Mao,Wenjun Shan,Jiaxing Xie, Kefang Lai, Xingshu Li, Bioorganic＆Medicinal Chemistry Letters 23(2013)1548–1552；
Preferably, the preparation method includes：
Compound shown in formula 2 is with excessive (4'- hydroxyl -3'- methylols) phenyl-(2- hydroxyl -4- bromos) butane in acetonitrileReaction is stirred at reflux in solvent, thin-layer chromatography detection reaction reaches balance, is cooled to room temperature, boils off solvent, residue acetic acidEthyl ester washs, and ethyl alcohol recrystallization, obtains compound shown in formula 1.
From above technical scheme, the invention provides a kind of substituted pyrrolidine carboxylic acid's ester type compound, it is oneKind brand-new has β2Receptor stimulating agent and M3Receptor antagonist double pharmacological action drug molecule, by the collaboration of two kinds of pharmacologyEffect, its drug effect are significantly larger than the exercising result of single-acting molecule, can apply to treat asthma and COPDEtc. in tuberculosis.
Embodiment
The invention discloses a kind of substituted pyrrolidine carboxylic acid's ester type compound and its preparation method and application, this area skillArt personnel can use for reference present disclosure, be suitably modified technological parameter realization.In particular, all similar replacements andChange apparent to those skilled in the art, they are considered as being included in the present invention.Chemical combination of the present inventionThing and its preparation method and application is described by preferred embodiment, and related personnel can substantially not depart from the present inventionMethod described herein and application are modified or suitably changed with combining in content, spirit and scope, to realize and applyThe technology of the present invention.
Raw material in compounds process for production thereof of the present invention can pass through commercially available acquisition.With reference to embodiment, enterOne step illustrates the present invention.
Embodiment 1：Preparation (the R of formula 1-A compounds1For hydrogen, R2For phenyl)
By 2.81g 2,2- diphenyl acetic acids (pyrrolidines -4-) ester and excessive (4'- hydroxyl -3'- methylols) phenyl-(2-Hydroxyl -4- bromos) butane is dissolved in 100mL acetonitriles, is stirred at reflux 5 hours, and thin-layer chromatography detection reaction reaches balance, cooling chamberTemperature, boils off solvent, and residue with Ethyl acetate washs 3 times, ethyl alcohol recrystallization, obtains target compound, obtain the compound as yellow of formula 1Solid 1.84g, yield 41%, consistent with expected structure after testing, appraising datum is as follows：
M.p.91.0~93.0 DEG C.MS(m/z):478(M+1)+。
1H-NMR(CDCl3,400MHz)δ:1.31-1.45 (m, 2H), 1.63-1.84 (m, 2H), 1.90-1.94 (m, 3H),2.11~2.24 (m, 4H), 2.30~2.35 (m, 2H), 4.01~4.05 (m, 1H), 4.52~4.56 (m, 1H), 4.72~4.75 (m, 2H), 4.88 (s, 1H), 6.52~6.67 (d, 1H), 6.85~6.98 (m, 2H), 7.06~7.18 (m, 10H).
Reaction equation：
Embodiment 2：Preparation (the R of formula 1-B compounds1To align substituent methyl, R2For phenyl)
In 1000mL three-necked flasks, by 2.95g 2- p-methylphenyl -2- phenylacetic acids (pyrrolidines -3-) esters and excess(4'- hydroxyl -3'- methylols) phenyl-(2- hydroxyl -4- bromos) butane is dissolved in 300mL acetonitriles, is stirred at reflux 5 hours, thin layerChromatography detection reaction reaches balance, cools down room temperature, boils off solvent, and residue with Ethyl acetate is washed 3 times, ethyl alcohol recrystallization, obtainedTo target compound, the compound as yellow solid 2.24g of formula 1, yield 46%, consistent with expected structure after testing, appraising datum are obtainedIt is as follows：
M.p.85.0~87.0 DEG C.MS(m/z):499(M+1)+。
1H-NMR(CDCl3,400MHz)δ:1.35-1.49 (m, 2H), 1.65-1.90 (m, 6H), 1.95-1.99 (m, 3H),2.15~2.29 (m, 4H), 2.32~2.39 (m, 2H), 2.46 (d, 3H), 4.03~4.09 (m, 1H), 4.48~4.52 (m,1H), 4.69~4.73 (m, 2H), 4.91 (s, 1H), 6.51~6.62 (d, 1H), 6.87~6.98 (m, 4H), 7.04 (d, 2H),7.09~7.18 (m, 5H).
Reaction equation：
Embodiment 3：Preparation (the R of formula 1-C compounds1For hydrogen, R2For cyclohexyl)
In 1000mL three-necked flasks, by the cyclohexyl-acetic acid of 2.87g 2- phenyl -2 (pyrrolidines -3-) ester and excessive (4'-Hydroxyl -3'- methylols) phenyl-(2- hydroxyl -4- bromos) butane is dissolved in 300mL acetonitriles, 5 hours are stirred at reflux, thin-layer chromatographyDetection reaction reaches balance, cools down room temperature, boils off solvent, and residue with Ethyl acetate washs 3 times, ethyl alcohol recrystallization, obtains meshCompound is marked, obtains the compound as yellow solid 2.35g of formula 1, yield 49%, consistent with expected structure after testing, appraising datum is as follows：
M.p.90.0~92.0 DEG C.MS(m/z):482(M+1)+。
1H-NMR(CDCl3,400MHz)δ:1.20-1.25 (m, 2H), 1.41-1.52 (m, 10H), 1.61-1.80 (m,6H), 1.84-1.97 (m, 3H), 2.15~2.21 (m, 4H), 2.24~2.35 (m, 2H), 2.54~2.58 (m, 1H), 4.00~4.05 (m, 1H), 4.44~4.50 (m, 1H), 4.78~4.81 (m, 2H), 4.81 (s, 1H), 6.48~6.86 (d, 3H), 7.22~7.58 (m, 5H).
Reaction equation：
Embodiment 4：Preparation (the R of formula 1-D compounds1To align methyl, R2For cyclohexyl)
In 1000mL three-necked flasks,
By 3.0g 2- p-methylphenyl -2- cyclohexyl-acetic acids (pyrrolidines -3-) esters and excessively (4'- hydroxyl -3'- methylols)Phenyl-(2- hydroxyl -4- bromos) butane is dissolved in 300mL acetonitriles, is stirred at reflux 5 hours, and thin-layer chromatography detection reaction reaches flatWeighing apparatus, room temperature is cooled down, boil off solvent, residue with Ethyl acetate washs 3 times, ethyl alcohol recrystallization, obtains target compound, obtain formula 1Compound as yellow solid 2.03g, yield 51%, consistent with expected structure after testing, appraising datum is as follows：
M.p.89.0~91.0 DEG C.MS(m/z):496(M+1)+。
1H-NMR(CDCl3,400MHz)δ:1.24-1.38 (m, 4H), 1.42-1.55 (m, 8H), 1.61-1.81 (m, 6H),1.83-1.93 (m, 3H), 2.13~2.34 (m, 6H), 2.0 (d, 3H), 2.55~2.59 (m, 1H), 4.03~4.08 (m, 1H),4.49~4.53 (m, 1H), 4.76~4.81 (m, 2H), 4.96 (s, 1H), 6.43~6.52 (d, 1H), 6.84~6.91 (m,2H), 6.96 (d, 2H), 7.5 (d, 2H).
Reaction equation：
Embodiment 5：Preparation (the R of formula 1-E compounds1For hydrogen, R2For pentamethylene base)
In 1000mL three-necked flasks, by 2.73g 2- phenyl -2- 2-Cyclopentylacetic acids (pyrrolidines -3-) esters and excessive (4'-Hydroxyl -3'- methylols) phenyl-(2- hydroxyl -4- bromos) butane is dissolved in 300mL acetonitriles, 5 hours are stirred at reflux, thin-layer chromatographyDetection reaction reaches balance, cools down room temperature, boils off solvent, and residue with Ethyl acetate washs 3 times, ethyl alcohol recrystallization, obtains meshCompound is marked, obtains the compound as yellow solid 2.19g of formula 1, yield 47%, consistent with expected structure after testing, appraising datum is as follows：
M.p.85.0~87.0 DEG C.MS(m/z):468(M+1)+。
1H-NMR(CDCl3,400MHz)δ:1.25-1.32 (m, 2H), 1.36-1.57 (m, 6H), 1.61-1.80 (m, 6H),1.81-1.90 (m, 3H), 2.12~2.21 (m, 4H), 2.24~2.32 (m, 2H), 2.61~2.72 (m, 1H), 4.11~4.15(m, 1H), 4.42~4.49 (m, 1H), 4.70~4.77 (m, 2H), 4.95 (s, 1H), 6.42~6.56 (d, 1H), 6.85~6.92 (m, 2H), 7.09~7.21 (m, 5H).
Reaction equation：
Embodiment 6：Preparation (the R of formula 1-F compounds1To align methyl, R2For pentamethylene base)
In 1000mL three-necked flasks, by 2.87g 2- p-methylphenyl -2- 2-Cyclopentylacetic acids (pyrrolidines -3-) esters and excess(4'- hydroxyl -3'- methylols) phenyl-(2- hydroxyl -4- bromos) butane is dissolved in 300mL acetonitriles, is stirred at reflux 5 hours, thin layerChromatography detection reaction reaches balance, cools down room temperature, boils off solvent, and residue with Ethyl acetate is washed 3 times, ethyl alcohol recrystallization, obtainedTo target compound, the compound as yellow solid 1.97g of formula 1, yield 47%, consistent with expected structure after testing, appraising datum are obtainedIt is as follows：
M.p.97.0~99.0 DEG C.MS(m/z):482(M+1)+。
1H-NMR(CDCl3,400MHz)δ:1.19-1.28 (m, 2H), 1.31-1.52 (m, 6H), 1.60-1.93 (m, 9H),2.11~2.32 (m, 6H), 2.44 (d, 3H), 2.65~2.71 (m, 1H), 4.14~4.17 (m, 1H), 4.44~4.47 (m,1H), 4.71~4.75 (m, 2H), 4.96 (s, 1H), 6.45~6.54 (d, 1H), 6.81~6.90 (m, 2H), 6.95 (d, 2H),7.09 (d, 2H).
Reaction equation：
Embodiment 7：To m receptor subtype-selective Effect study
Epicyte protein Competitive assays Binding experiment：
With [3H] NMS is tagged ligand, concentration is fixed as 1nM, 1ml reaction systems, and membrane protein concentration is fixed as 20-40 μG/ is managed, and non-specific pipe adds 10 μM of atropine, and the noval chemical compound for adding various concentrations is competition medicine, uses reaction bufferCumulative volume 1ml is added to, 25 DEG C of incubation 60min, 3ml ice-cold buffer terminating reactions is added, makes a collection of specimens, negative pressure leaching.BufferingLiquid is rinsed 3 times, and diaphragm is dodged in liquid and 3ml scintillation solutions are added in bottle in 80 DEG C of roasting films 2 hours, behind with step saturation experiments.InvestigateDifferent compounds and the binding ability of m receptor different subtype, the results are shown in Table 1.
Percentage bound=(testing tube-non-specific binding) cpm/ (total binding pipe-non-specific binding) cpm × 100%
Table 1, compound are to the binding ability (n=3) of m receptor different subtype
As can be seen from the data in the table, compound of the present invention can with Reverse transcriptase [3H] NMS and five acceptor AsiaType combines, and comparative compound and m receptor hypotype, compound is to M3The Ki values of receptor subtype are smaller than the Ki values of other hypotypes, toolHave to M3The selectivity of receptor subtype.
Embodiment 8：To beta receptor subtype-selective Effect study
(1) in lung film preparation thing (β2Based on acceptor) in competion experiment
Take the μ L of lung film preparation thing 100 and3H-DHA(2×10-9mol·L-1) in Tris buffer solutions with sample sets 1-A~1-F (final concentrations 10-10~10-4mol·L-1) or isoprel (final concentration 10-10~10-5mol·L-1) reacted, measureCompetition binding (CB).Simultaneously by method used in saturation experiments, parallel one group only adds 4 × 10-4mol·L-1Pu CailuoYour non-specific binding pipe, and one group of total binding pipe for being not added with Pu Cailuoer, react the μ L of cumulative volume 250, and above-mentioned reaction is answeredPipe.Operated after sample-adding identical with above-mentioned acceptor saturation experiments.The difference of activity between total binding pipe and non-specific binding pipeFor 100%, obtain corresponding in the presence of various concentrations test medicine3H-DHA and membrane receptor combination percentage.By that will tieThe half binding capacity IC of sample sets can be drawn to drug concentration after the mapping of logarithm-logit methods from curve by closing percentage50, lead toCross formula:Ki=IC50/(1+L/KD) it can then calculate the apparent dissociation constant K that medicine combines to acceptori, wherein KDBy above acceptorFull close is tried to achieve in experiment.
(2) in atrium film preparation thing (βlBased on acceptor) in competion experiment
Core the μ L of room film preparation thing 100 and3H-DHA(2×10-9mol·L-1) in Tris buffer solutions with sample sets 1-A~1-F (final concentrations 10-10~10-4mol·L-1) or isoprel (final concentration 10-10~10-5mol·L-1) carry out ibidReaction.Obtain corresponding in the presence of various concentrations sample sets3H-DHA and membrane receptor combination percentage.Draw competition bindingCurve.The half binding capacity IC of sample sets is calculated by upper method50.And the apparent dissociation constant K that medicine combines to acceptori。
It the results are shown in Table 2.
Table 2
Result of study shows：Sample sets and it can compete lung film (β with radioligand with isoprel2Based on acceptor)Receptor binding site in prepared product.Half binding capacity ICs of the formula 1-A~formula 1-F to acceptor50For 21.4~29.8nmol·L-1, apparent dissociation constant KiFor 17.2~21.2nmolL-1.Isoprel shows similar acceptorBinding characteristic, its IC50And KiRespectively 120.8nmolL-1, and 98.3nmolL-1, i.e. knot of the sample sets to lung membrane receptorConjunction ability is strong about 6 times compared with isoprel.
Sample sets and with isoprel can with radioligand compete atrium film (βlBased on acceptor) in prepared productReceptor binding site.Sample 1-A~1-F half binding capacities IC50For 119.2~142.3nmolL-1, apparent dissociation constant KiFor101.3~112.0nmolL-1.Isoprel shows similar receptor binding characteristics, its IC50And KiRespectively45.2nmol·L-1, and 34.1nmolL-1.That is about 3 times weak compared with isoprel to the binding ability of acceptor of sample sets.
To sum up, sample sets show stronger β2Receptor-selective.
Embodiment 9：Draw the protection and therapeutic action of asthma to cavy medicine
Baby guinea pig (150~250g of body weight), male and female are random.1d carries out drawing asthma experiment before experiment, selects asthmatic latent periodCavy less than 120s.Qualified cavy is randomly divided into 12 groups during each compound experiment, i.e., vehicle control group (gives w=1%CMC), salbutamol 1,3,9mg kg-1Group, glycopyrronium bromide 1,3,9mg kg-1Group and the compounds of this invention 0.0625,0.125、0.25、0.5、1.0mg kg-1Group, said medicine are dissolved in 1% (w) CMC.1h after administration, animal is placed in volumeFor in 4L spray tank, the mixed liquor 5s of 2% (w) acecoline and 0.1% (w) histamine is sprayed into constant flow rate.SprayingAfter stopping, observing the asthmatic latent period (to asthma attack, the time for tumble of twitching i.e. since spraying) of animal and twitchNumber of animals.6min is observed, non-tumble person's asthmatic latent period is calculated by 360s, and accounts for each test group number of animals with the animal that do not fallPercentage as effective percentage, calculate the ED of each medicine50。ED50It is as follows：
The compound of embodiment 1：0.12±0.05mg·kg-1；
The compound of embodiment 2：0.34±0.05mg·kg-1；
The compound of embodiment 3：0.27±0.05mg·kg-1；
The compound of embodiment 4：0.20±0.05mg·kg-1；
The compound of embodiment 5：0.36±0.05mg·kg-1；
The compound of embodiment 6：0.38±0.05mg·kg-1；
Salbutamol：2.01±0.22mg·kg-1,
Glycopyrronium bromide：0.21±0.22mg·kg-1,
From the above results, compound of the present invention and salbutamol all can significantly extend by acetylcholine and histamineMixed liquor spraying causes the incubation period that cavy pants, and the i.e. noval chemical compound of its anti-asthmatic action draws asthma when oral to cavy medicineProtective effect is strong about 5-10 times compared with salbutamol, slightly good or suitable with glycopyrronium bromide activity.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the artFor member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also shouldIt is considered as protection scope of the present invention.