CN102964279A - Kappa-opioid receptor agonists with peripheral analgesic effect - Google Patents
Kappa-opioid receptor agonists with peripheral analgesic effect Download PDFInfo
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- CN102964279A CN102964279A CN2012105437612A CN201210543761A CN102964279A CN 102964279 A CN102964279 A CN 102964279A CN 2012105437612 A CN2012105437612 A CN 2012105437612A CN 201210543761 A CN201210543761 A CN 201210543761A CN 102964279 A CN102964279 A CN 102964279A
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Abstract
The invention relates to kappa-opioid receptor agonists disclosed as Formula (I) or pharmaceutically acceptable salts thereof. The compounds has strong activity of in-vitro agonistic kappa-opioid receptors, the EC50 is on the nM level, and the EC50 of the compound with the strongest activity is 8.74+/-0.72nM; and the compounds can be used for peripheral analgesia. The analgesic test proves that the inhibitory rate and the compound concentration have a dosage dependency relationship.
Description
Technical field
The present invention relates to a class kappa opioid receptor agonist, can behind activated receptor, produce Peripheral Analgesic Effect.
Background technology
Kappa opioid receptor (κ-opioid receptor, OP2, KOR) belongs to a kind of hypotype in 7 transmembrane receptor superfamily members of G albumen coupling.Studies show that kappa opioid receptor is relevant with the secretion of pain sensation adjusting, diuretic properties, neuroendocrine thing.After activating, kappa opioid receptor can suppress adenylate cyclase (AC) activity by the adjusting of Gi albumen, reduce the level of cyclic amp (cAMP) in the cell, the protein phosphorylation of arrestin kinases A (PKA) and regulation and control thereof, the performance analgesic activity.
Kappa opioid receptor (κ-opioid receptor, OP2, KOR) belongs to a kind of hypotype in 7 transmembrane receptor superfamily members of G albumen coupling.Studies show that kappa opioid receptor is relevant with the secretion of pain sensation adjusting, diuretic properties, neuroendocrine thing.Can suppress adenylate cyclase (AC) activity by the adjusting of Gi albumen after kappa opioid receptor activates, reduce the level of cAMP in the cell, the protein phosphorylation of arrestin kinases A (PKA) and regulation and control thereof.Exist kappa opioid receptor in the Primary Sensory Neuron and on the periphery tip, in the situation of inflammation and nerve injury, the synthetic increase of kappa opioid receptor also is transported to nerve ending or damaged part, increase the expression of the kappa opioid receptor in the nerve fiber that conducts pain sensation impulsion, strengthen Peripheral Analgesic Effect.
Pain is that a kind of Physiological Psychology of complexity is movable, is one of modal symptom clinically.It comprises that noxious stimulation acts on the caused pain of body and feels, and body is to the pain reaction (somatic movement reaction and/or internal organ vegetative reaction often are attended by strong mood color) of noxious stimulation.The pain sensation can be used as a kind of warning that body comes to harm, and causes the protective reaction of body series of defence.But then, pain also has its limitation (when pain occurring such as cancer etc., coming too late) as reporting to the police.And some long-term having an intense pain has become a kind of insufferable torment to body.The perception of pain relates to simultaneously sense organ and emotion changes, and especially chronic pain often is accompanied by the decline of depression, anxiety and quality of life.Therefore, analgesia is the vital task that the medical personnel faces.Opioid drug plays an important role in the treatment of acute and chronic pain.The characteristics complete annotation in experimentation on animals and clinical investigation during although the sense organ in acute pain of this type of medicine is differentiated, their effects in chronic pain are further investigation not.At present, be applied to clinical periphery analgesic and be mainly the mu opioid receptor anodyne, such as fentanyl class medicine.There also do not have the kappa opioid receptor anodyne to be applied to be clinical.
In recent years, great majority studies show that the activation of kappa opioid receptor can change heart function and only have a few studies to show that kappa opioid receptor is relevant with pain.
Summary of the invention
The object of the present invention is to provide a kind of compound for the treatment of alzheimer's disease, this compound is the kappa opioid receptor agonist, and alzheimer's disease is had prevention or therapeutic action.Compound of the present invention comprises compound or its pharmacy acceptable salt that contains the chemical formula (I) for the treatment of significant quantity.The present invention adopts High Throughput Screening Assay, set up kappa opioid receptor agonist high flux screening model and be applied to the compound Large-scale Screening, searched out the kappa opioid receptor agonist with chemical formula (I) by screening a collection of compound from the purchase of CHEMDIV company and carrying out functional checking, simultaneously to its AD pharmacodynamics and Mechanism Study of being correlated with.
Technical scheme of the present invention is: stable transfection kappa opioid receptor in Chinese hamster ovary cancer cells (CHO), set up kappa opioid receptor agonist high flux screening model, carry out primary dcreening operation, multiple sieve, structure activity relationship is analyzed, carry out on this basis the integral animal pharmacodynamic experiment, obtain the drug candidate that a class has Peripheral Analgesic Effect.Concrete steps are as follows:
Step 1: the Chinese hamster ovary celI strain of setting up and cultivating the stable transfection kappa opioid receptor.
Step 2: the mensuration of typical curve and optimum cell number are determined.
Step 3: positive drug checking.
Step 4: adopt stable cell strain and the optimal detection condition groped carries out the high flux screening of kappa opioid receptor agonist to compound, obtain the compound of obvious dose-effect relationship.
Step 5: carry out the integral animal analgesic experiment, obtain the drug candidate that a class has Peripheral Analgesic Effect.
Description of drawings:
Fig. 1: typical curve and optimum cell number curve
Fig. 2: positive drug (kappa opioid receptor specific agonist: U50,488) amount effect relation curve
Fig. 3: compound amount effect relation curve
Fig. 4: compound 8 is on the impact of mouse Glacial acetic acid writhing test writhing number of times
Fig. 5: compound 8 is on the preclinical impact of mouse Glacial acetic acid writhing test
Embodiment
Below in conjunction with description of drawings the specific embodiment of the present invention
1. set up and cultivate the Chinese hamster ovary celI strain of stable transfection kappa opioid receptor.
Use box switching technology (the Recombinase-Mediated Cassette Exchange of recombinase-mediated, RMCE) make up in batches the g protein coupled receptor cell strain, and make its stable transfection kappa opioid receptor, cultivate the CHO-OP2 cell and make it reach the stable growth state.
2. the mensuration of typical curve and optimum cell number are determined.
For the exactness of confirmatory experiment system and can filter out reliable kappa opioid receptor agonist, when setting up model, need the bioassay standard curve and determine the optimum cell number.Treat CHO-OP2 Growth of Cells to 80% the time with the PBS peptic cell of 0.5mM EDTA, centrifugal removal supernatant liquid, at last with cell with stimulating damping fluid resuspended for definite optimum cell number.Use the LANCE of PerkinElmer company
TMCAMP 384Kit test kit, preparation standard cAMP gradient dilution liquid and adenylate cyclase activating agent (forskolin) gradient dilution liquid.The standard curve determination method: with the standardized solution 4 μ mol/L of cAMP (
The cAMP detection kit carries), be the working concentration of 4 times of final concentrations with stimulating the damping fluid stepwise dilution, each 5 μ l of cAMP and antibody-solutions add hatched in 384 orifice plates 45 minutes jointly, added 10 μ l stop buffers, hatched after one hour and detected; Optimum cell is counted detection method: be the working concentration of 2 times of final concentrations with the forskolin mother liquor of 5mM with stimulating the damping fluid stepwise dilution, forskolin adds with each 5 μ l of the antibody-solutions that contains cell hatched in 384 orifice plates 45 minutes jointly, add 10 μ l stop buffers, hatch after one hour and detect at EnVision microwell plate plate reading.According to forskolin amount effect curve window (property is made an uproar than S/B) and the curve cell concn close with the cAMP slope of standard curve as the optimum cell number.Typical curve and optimum cell number determine to see Fig. 1.
3. select kappa opioid receptor selective agonist U50, its EC is verified and calculated according to amount effect curve to the 488 pairs of high flux screening models of setting up
50
The positive drug measuring method: with the U50 of 23.8mM, 488 mother liquors are the working concentration of 4 times of final concentrations with stimulating the damping fluid stepwise dilution, and configuration can cause that optimum cell counts Ratio E
C9The forskolin diluent of 0 effect.Positive drug U50,4882.5 adding with the antibody-solutions 5 μ l that contain the optimum cell number, μ l jointly hatched in 384 orifice plates 15 minutes, again forskolin diluent 2.5 μ l are added and hatched in 384 orifice plates 30 minutes, add 10 μ l stop buffers and hatch again after one hour with the detection of EnVision microwell plate plate reading.The positive drug amount effect curve is seen Fig. 2.
4. under above-mentioned optimal detection condition, carry out the kappa opioid receptor agonist primary dcreening operation and multiple sieve.
Primary dcreening operation is with to sieve again process identical, primary dcreening operation only selects a concentration that 80,000 compounds are carried out preliminary screening, select again 8 concentration of primary dcreening operation compounds effective gradient dilution to carry out multiple sieve, process is as follows: with Janus full-automatic application of sample workstation diluted compounds and draw 5 μ l and transfer in 384 orifice plates, the antibody-solutions 5 μ l that will contain the optimum cell number with the Multidrop automatic sample adding instrument again add in 384 orifice plates, after both hatch 45 minutes jointly, the Multidrop automatic sample adding instrument adds 10 μ l stop buffers in 384 orifice plates, again hatched one hour, and detected with EnVision microwell plate plate reading.Multiple sieve obtains a series of compounds with kappa opioid receptor agonism, the EC of each compound
50Figure sees Fig. 3.
Sieve again experimental result
The compound that screening obtains can be summarized as according to the structure of parent nucleus a class, structural formula and EC
50As follows:
I:3,4-dimethoxy-N-(2-oxoethyl)-N-p-methylphenyl sulphonamide
The class active compound structure that the multiple sieve of table 1 obtains
The active compound EC that the multiple sieve of table 2 obtains
50
The animal ethology experimental result
Experimental technique: the mouse administration is injected Glacial acetic acid after half an hour, and writhing number of times and the latent period of 20min mouse the results are shown in Figure 4, Fig. 5 behind observation and the record injection Glacial acetic acid, calculate the analgesia percentage, carry out statistical procedures, with its significance of T test and judge.Analgesia percentage (%)=(the average writhing number of times of the control group-average writhing number of times of administration group)/average writhing number of times of control group
Table 3 compound 8 is on the impact of mouse Glacial acetic acid writhing test
Annotate: compare * P＜0.05, * * P＜0.01 with control group
Conclusion: the result of analgesic test shows that the analgesic effect of compound 8 when high density slightly is weaker than positive drug U50,488, and effect is better.There are dose-dependence in writhing inhibiting rate and concentration.
Claims (3)
1. following formula: compound or its acceptable salt pharmaceutically:
Wherein:
R represents aniline or aliphatic amide, wherein can be replaced by chlorine atom or low alkyl group on the phenyl ring of aniline, can connect alkyl or phenyl ring with 4-8 carbon atom on the aliphatic amide nitrogen-atoms.
The compound of the claimed formula (I) of claim 1 or its pharmaceutically acceptable salt for the preparation of the purposes of kappa opioid receptor agonist.
3. according to the purposes of claim 2, as the kappa opioid receptor agonist, be used for the treatment of peripheral pain.
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| CN109988086A (en) * | 2019-04-04 | 2019-07-09 | 三峡大学 | A kind of micromolecular inhibitor AZIN30 and its application in pharmacy |
Citations (4)
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| CN1111612A (en) * | 1993-12-09 | 1995-11-15 | 小野药品工业株式会社 | Naphthyloxyacetic acid derivatives |
| CN1200127A (en) * | 1995-08-18 | 1998-11-25 | 阿斯特拉公司 | Novel opioid peptides |
| CN1469862A (en) * | 2000-10-12 | 2004-01-21 | Ss制药株式会社 | 2,2-diphenyl butanamide derivatives and medicines containing the same |
| WO2009086303A2 (en) * | 2007-12-21 | 2009-07-09 | University Of Rochester | Method for altering the lifespan of eukaryotic organisms |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1111612A (en) * | 1993-12-09 | 1995-11-15 | 小野药品工业株式会社 | Naphthyloxyacetic acid derivatives |
| CN1200127A (en) * | 1995-08-18 | 1998-11-25 | 阿斯特拉公司 | Novel opioid peptides |
| CN1469862A (en) * | 2000-10-12 | 2004-01-21 | Ss制药株式会社 | 2,2-diphenyl butanamide derivatives and medicines containing the same |
| WO2009086303A2 (en) * | 2007-12-21 | 2009-07-09 | University Of Rochester | Method for altering the lifespan of eukaryotic organisms |
Non-Patent Citations (2)
| Title |
|---|
| HAMED AISSAOUI等: "N-Glycine-sulfonamides as potent dual orexin 1/orexin 2 receptor antagonists", 《BIOORGANIC & MEDICINAL CHEMISTRY LETTERS》 * |
| PATRICK MARKT等: "Discovery of Novel CB2 Receptor Ligands by a Pharmacophore-Based Virtual Screening Workflow", 《J. MED. CHEM.》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109988086A (en) * | 2019-04-04 | 2019-07-09 | 三峡大学 | A kind of micromolecular inhibitor AZIN30 and its application in pharmacy |
| CN109988086B (en) * | 2019-04-04 | 2021-06-18 | 三峡大学 | Small molecule inhibitor AZIN30 and application thereof in pharmacy |
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Address after: Lishui County of Nanjing City, Jiangsu province 211200 Yong Yang Town Natural Bridge Road No. 688 Branch Center Patentee after: China Pharmaceutical University Address before: No. 639 Jiangning longmian Road District of Nanjing City, Jiangsu province 211198 Patentee before: China Pharmaceutical University |
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