CN115340508A - Compound with D2 receptor agonism and NMDA receptor antagonism and application thereof - Google Patents
Compound with D2 receptor agonism and NMDA receptor antagonism and application thereof Download PDFInfo
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- CN115340508A CN115340508A CN202211007790.7A CN202211007790A CN115340508A CN 115340508 A CN115340508 A CN 115340508A CN 202211007790 A CN202211007790 A CN 202211007790A CN 115340508 A CN115340508 A CN 115340508A
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- C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
- C07D277/62—Benzothiazoles
- C07D277/68—Benzothiazoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 2
- C07D277/82—Nitrogen atoms
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- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
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Abstract
The invention discloses a compound with a structure shown as a formula I or a pharmaceutically acceptable salt thereof, wherein X is selected from CH 2 O; n is an integer selected from 1 to 6, and m is an integer selected from 1 to 6. The compounds of the present invention have at least NMDA receptor antagonism; particularly, when X is selected from O, the compound has D2 receptor agonism and NMDA receptor antagonism at the same time, the NMDA receptor antagonism does not influence the normal function, improves the motor symptoms of the PD model mouse and the cognitive impairment of the PD model mouse, and has better Parkinson disease treatment effect. The invention also discloses application of the compound or the pharmaceutically acceptable salt thereof in preparing a D2 receptor agonist and/or an NMDA receptor antagonist. The invention also discloses the application of the compound or the pharmaceutically acceptable salt thereof in preparing medicaments for preventing and treating Parkinson's diseaseThe use of (1).
Description
Technical Field
The invention belongs to the field of pharmacy, and relates to a novel Parkinson disease treatment drug with both a D2 receptor agonism effect and an NMDA receptor partial antagonism effect.
Background
Parkinson's Disease (PD) is the second leading neurodegenerative disease in the world, and 2% of people over the age of 65 have their health and quality of life threatened. The pathological features of PD are mainly dopamine neuron damage of the substantia nigra pars compacta of the midbrain, accompanied by motor symptoms such as resting tremor, rigidity, bradykinesia and the like and non-motor symptoms such as sleep disorder, cognitive disorder and the like.
Currently, the clinical treatment strategies for Parkinson's disease are mainly divided into protection of dopaminergic neurons and dopamine supplementation. Among them, levodopa, a dopamine-like drug, is associated with dyskinesia such as dyskinesia, and the like, with the progress of the disease condition and the increase of the dosage, and affects the therapeutic efficacy. Dopamine receptor agonists (mostly dopamine type ii (D2) receptor agonists) are accepted by more and more patients because they improve parkinson-like symptoms and have fewer adverse effects. Pramipexole (chemical name is (S) -2-amino-4, 5,6, 7-tetrahydro-6-propylamine-benzothiazole) is a D2 receptor agonist which is commonly used in clinic for treating PD, can excite D2 receptors and inhibit NO-GO effect of indirect pathway mediated by D2 neurons, thereby playing a role in relieving dyskinesia.
Clinical data show that up to 40 percent of PD patients are likely to suffer from cognitive dysfunction and dementia, and the probability of the dementia and the cognitive dysfunction is increased along with the progress of PD diseases, and NMDA receptor antagonists such as amantadine and memantine can improve the cognitive dysfunction of the PD patients accompanied with the dementia, can block abnormal glutamic acid signals in striatum of the PD patients, reduce neurotoxicity, play a role in neuroprotection and delay the progress of diseases to a certain extent.
In the process of onset of Parkinson's disease, there are many pathogenesis including environmental factors, oxidative stress, excitotoxicity, etc. Therefore, it is difficult to achieve satisfactory therapeutic effects only for one of the targets.
Disclosure of Invention
The invention aims to provide a multifunctional Parkinson treatment medicine.
The purpose of the invention is realized by the following technical scheme:
a compound having the structure shown in formula I:
wherein X is selected from CH 2 O; n is an integer selected from 1 to 6, and m is an integer selected from 1 to 6.
Preferably, X is selected from O; n is an integer selected from 1 to 6, and m is an integer selected from 1 to 6.
In particular, selected from the following compounds:
pharmaceutically acceptable salts are hydrochloride, hydrobromide, nitrate, perchlorate, phosphate, sulphate, formate, acetate, aconate, ascorbate, benzenesulphonate, benzoate, cinnamate, citrate, heptanoate, fumarate, glutamate, glycolate, lactate, maleate, malonate, mandelate, methanesulphonate, naphthalene-2-sulphonate, phthalate, salicylate, sorbate, stearate, succinate, tartrate or p-toluenesulphonate.
The compounds of the present invention have at least NMDA receptor antagonism; particularly, when X is selected from O, the compound has D2 receptor agonism and NMDA receptor antagonism, the NMDA receptor antagonism does not affect normal functions, the motor symptoms of a Parkinson disease model (MPTP model) mouse are improved, the motor and cognitive disorders of the MPTP model mouse are also improved, the damage of dopamine neurons in the substantia nigra pars compacta of the MPTP model mouse is relieved, and the mechanism of the compound is probably related to the fact that the compound can improve the abnormal excitability of NMDAR receptors and cholinergic neurons in striatum and improve the overall output function of the striatum. Therefore, the compound has better Parkinson disease treatment effect.
Therefore, the invention also aims to provide the application of the compound shown as the formula I or the pharmaceutically acceptable salt thereof in preparing D2 receptor agonist and/or NMDA receptor antagonist.
The invention also provides application of the compound shown as the formula I or pharmaceutically acceptable salts thereof in preparing medicaments for preventing and treating the Parkinson's disease.
Another object of the present invention is to provide a process for the preparation of compounds of formula I, which comprises the following steps:
the invention has the beneficial effects that:
the compound is a novel Parkinson disease treatment drug with multiple pharmacological mechanisms, has the D2 receptor agonism and NMDA receptor antagonism, can improve the movement and cognitive disorder of a PD model mouse, and has better treatment effect than a single-target drug.
Drawings
FIG. 1: the agonism effect of pramipexole, compounds ML-A, ML-B and ML-C on D2 receptors under different concentrations results; wherein, A: the upper picture is a fluorescence picture of striatum parts in a Chat-eGFP mouse brain slice, and the lower picture is a schematic diagram of recording cholinergic neurons by a glass electrode; b: representative diagrams of action potentials of cholinergic neurons before and after 50 mu M, 100 mu M and 200 mu M compound ML-B and 200 mu M pramipexole perfused brain slices; c: a statistics result diagram of action potential of cholinergic neuron before and after brain perfusion with 50 μ M, 100 μ M and 200 μ M compound ML-B; d: a statistical result graph of action potentials of cholinergic neurons before and after 200 mu M pramipexole perfused brain slices; e: comparing the percentage of action potential reduction of cholinergic neurons for 200 μ M compound ML-B to 200 μ M pramipexole under the same baseline conditions; f: representative diagrams of action potentials of cholinergic neurons before and after perfusion of a brain slice with 200 mu M of the compound ML-A and 200 mu M of the compound ML-C; g: a statistical result graph of action potentials of cholinergic neurons before and after perfusion of a 200 mu M compound ML-A to a brain slice; h: a statistical result graph of action potentials of cholinergic neurons before and after brain perfusion with 200 mu M compound ML-C;
FIG. 2: the antagonistic effect of pramipexole, compounds ML-A, ML-C and compounds ML-B on NMDA receptors under different concentrations results; wherein, A: recording a schematic representation of NMDAR-mediated EPSCs of striatal MSNs; b: the upper part is respectively a representation diagram of NMDAR mediated EPSC in MSNs before and after the perfusion of 50 mu M, 100 mu M and 200 mu M compounds ML-B to the brain slice, and the lower part is respectively a statistical result diagram; c: the upper part is a representation diagram of the EPSC mediated by the NMDAR in MSNs before and after the 200 mu M pramipexole perfusion brain slice, and the lower part is a statistical result diagram; d: the upper part is a representation diagram of the NMDAR mediated EPSC in MSNs before and after the 200 mu M compound ML-A perfusion brain slice, and the lower part is a statistical result diagram; e: the upper part is a representation diagram of the EPSC mediated by the NMDAR in MSNs before and after the 200 mu M pramipexole 200 mu M compound ML-C perfusion brain slice, and the lower part is a statistical result diagram;
FIG. 3: the time course plot of compound ML-B; wherein, A: time-of-drug profile of time-dependent concentration of compound ML-B in brain; b: time-of-drug plot of drug concentration in plasma as a function of time.
FIG. 4 is a schematic view of: HE staining pattern of paraffin sections of organs after one month of continuous administration (compound ML-B) to mice; wherein, A: organ maps of control mice, B: the organ images of mice in the administration group are heart, liver, spleen, lung and kidney from left to right in fig. 4A and fig. 4B.
FIG. 5 is a schematic view of: a bar rotating experiment result chart; wherein, A: schematic diagram of rod rotation experiment, B: and (4) a bar rotating experiment result graph.
FIG. 6: open field experimental result chart; wherein, A: each group of mice rotates the bar experiment trace chart; b: total distance of movement in the open field experiment of each group of mice; c: the movement speed of each group of mice in an open field experiment; d: percentage of immobility time in open field experiments for each group of mice.
FIG. 7 is a schematic view of: a TH staining pattern of the substantia nigra pars compacta; wherein, A: each group of mice substantia nigra pars compacta dopamine neuron tyrosine hydroxylase staining immunofluorescence map; b: TH staining statistical result chart of dopamine neuron in substantia nigra pars compacta of each group of mice.
FIG. 8: a new object identification experiment result graph; wherein, A: new object recognition experimental schematic, B: statistical results of the exploration behavior of different groups of mice for two identical objects, C: and (3) a statistical result graph of the exploration behaviors of the mice in different groups for two objects with different colors and shapes.
FIG. 9: elevated plus maze test result chart; wherein, A: elevated plus maze schematic, B: statistical results of different groups of mice.
FIG. 10: effect of compound ML-B on striatal cholinergic neuronal firing; a: recording striatal cholinergic neuron action potential pattern map, B: the action potential generated under different injection current intensities of each group of mice is represented as a graph, C: the statistical result graph of action potential generated under different injection current intensity of each group of mice.
FIG. 11: the effect of compound ML-B on NMDA receptor activity in striatal MSN; a: representative plot of NMDAR-mediated EPSCs in each group of mice given five different stimulation intensities, B: and (5) a statistical result graph.
FIG. 12: compound ML-B effects on striatal synaptic plasticity; a: recording a striatum field potential pattern diagram; b: normal group field potential baseline and fpsps post-administration high frequency stimulation record statistical result figures, C: model group field potential baseline and fpsps after high frequency stimulation recording statistical result graphs, D: statistical plots of field potential baseline in the group of doses and fEPSP recordings after high frequency stimulation.
Detailed Description
The following examples are presented to enable one of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way.
Example 1
1.1 Synthesis of 6-hydroxyhexyl p-toluenesulfonate
1, 6-hexanediol (30mmol, 3.54g), et 3 N (30 mmol) in dry CH with catalytic amount of DMAP (4-dimethylaminopyridine) 2 Cl 2 To (150 mL) was added TsCl (4-methylsulfonyl chloride, 33 mmol) slowly with stirring at 0 ℃ and the reaction was stirred at room temperature for 4 hours; after the reaction is finished, saturated NH is used 4 The reaction was quenched with 50mL of a Cl solution under vigorous stirring, the reaction mixture was extracted with ethyl acetate (3X 50 mL), the ethyl acetate extract was washed with saturated brine and then with anhydrous Na 2 SO 4 Drying, concentration, dilution with ethyl acetate/petroleum ether =1:1v/V to precipitate most of the bis-p-toluenesulfonate product, filtration, concentration of the filtrate, and purification by column chromatography on silica gel using PE: EA =2:1v/V as eluent gave the product (3.12 g, 38.2% yield), which was identified as 6-hydroxyhexyl-p-toluenesulfonate. 1 H NMR(400MHz,CDCl 3 )d:7.79(d,2H,J=8.36Hz),7.35(d,2H,J=8.0),4.03(t,2H,J=6.44Hz),3.60(t,2H,J=6.56Hz),2.45(s,3H),1.70-1.61(m,2H),1.55-1.48(m,2H),1.38-1.27(m,4H).
1.2, 6- (3, 5-dimethyladamantan-1-yl) amino-1-hexanol
6-hydroxyhexyl-p-toluenesulfonylAcid ester (11.5 mmmol), memantine hydrochloride (9.55 mmmol) and K 2 CO 3 (23.8 mmol) is dissolved in DMF (50 mL) and stirred for reaction for 48h at the temperature of 80 ℃ under the protection of nitrogen; then the solvent is evaporated to dryness, using CH 2 Cl 2 (3X 10 mL), the organic layer was concentrated and chromatographed on silica gel using CH 2 Cl 2 :CH 3 OH 33% ammonia =9 as eluent, 0.15v/V gave the product (1.63 g, 61.1% yield) identified as 6- (3, 5-dimethyladamantan-1-yl) amino-1-hexanol. 1 H NMR(400MHz,CDCl 3 )d:3.64(t,2H,J=6.50Hz),2.59(t,2H,J=7.16Hz),1.60-1.46(m,6H),1.39-1.25(m,12H),1.15-1.06(m,2H),0.84(s,6H).
1.3, 6- (N-Boc-N- (3, 5-dimethyladamantan-1-yl)) amino-1-hexanol
6- (3, 5-Dimethyladamantan-1-yl) amino-1-hexanol (5.73 mmol) was dissolved in 30mL of THF/H 2 To a solution of O (2/1, v/v), na was added 2 CO 3 (14.25 mmol) and (Boc) 2 O (di-tert-butyl dicarbonate, 8.59 mmol), stirring overnight at room temperature; evaporating solvent to dryness, dissolving in water, extracting with ethyl acetate, collecting ethyl acetate extractive solution, washing with saturated saline solution, and adding anhydrous Na 2 SO 4 Dried, concentrated and subjected to silica gel column chromatography using PE: EA =6:4v/V as eluent to give the product (0.95 g, 43.7% yield) which was identified as 6- (N-Boc-N- (3, 5-dimethyladamantan-1-yl)) amino-1-hexanol. 1 H NMR(400MHz,CDCl 3 )d:3.64(t,2H,J=6.48Hz),3.22(t,2H,J=7.88Hz),2.17-2.11(m,1H),1.78-1.68(m,4H),1.62-1.51(m,4H),1.50-1.43(m,11H),1.41-1.32(m,4H),1.30-1.21(m,4H),1.17-1.07(m,2H),0.84(s,6H).
1.4, 6- (N-Boc-N- (3, 5-dimethyladamantan-1-yl) amino) hexyl p-toluenesulfonate ester
0.93g (2.45 mmmol) of 6- (N-Boc-N- (3, 5-dimethyladamantan-1-yl)) amino-1-hexanol, et 3 N0.68 mL (4.90 mmol) with catalytic amount of DMAP in dry 50mL CH 2 Cl 2 Adding TsCl at the temperature of 0 ℃ under stirring, and stirring at room temperature for reaction for 3.5 hours; after the reaction is finished, saturated NH is used 4 The reaction was quenched with aqueous Cl solution, extracted with ethyl acetate (3X 50 mL), and the ethyl acetate extract was washed with saturated brine and then with anhydrous Na 2 SO 4 Drying, evaporation of solvent and silica gel column chromatography with PE: EA =9, 1v/V as eluent gave the product (0.717 g, 54.8% yield) identified as 6- (N-Boc-N- (3, 5-dimethyladamantan-1-yl) amino) hexyl p-toluenesulfonate. 1 H NMR(400MHz,CDCl 3 )d:7.78(d,2H,J=8.4Hz),7.34(d,2H,J=7.84Hz),4.02(t,2H,J=6.52Hz),3.17(t,2H,J=8.08Hz),2.45(s,3H),2.16-2.11(m,1H),1.92-1.88(m,2H),1.74-1.67(m,4H),1.67-1.60(m,2H),1.47-1.37(m,11H),1.37-1.30(m,4H),1.28-1.21(m,2H),1.21-1.15(m,2H),1.14-1.07(m,2H),0.84(s,6H).
1.5 tert-butyl (6- (((S) -2-amino-4, 5,6, 7-tetrahydrobenzo [ d ] thiazol-6-yl) (propyl) amino) hexyl) (3, 5-dimethyladamantan-1-yl) carbamate (Pu-C6-Me (Boc))
In a pressure resistant tube, 0.715g (1.34 mmol) of 6- (N-Boc-N- (3, 5-dimethyladamantan-1-yl) amino) hexyl p-toluenesulfonate, 0.232g (1.12 mmol) of pramipexole, et 3 Dissolving 0.31mL (2.24 mmol) of N in 12mL of MeCN (acetonitrile), replacing with nitrogen, and stirring at 80 ℃ for reaction for 48 hours; the solvent was evaporated to dryness and the residue was subjected to silica gel column chromatography with EA: meOH =20:1v/V as eluent to give the product (0.20 g, yield 31.3%) which was identified as (6- (((S) -2-amino-4, 5,6, 7-tetrahydrobenzo [ d ]]Thiazol-6-yl) (propyl) amino) hexyl) (3, 5-dimethyladamantan-1-yl) carbamic acid tert-butyl ester (noted Pu-C6-Me (Boc)). 1 H NMR(400MHz,CDCl 3 )d:4.92(br,s,1H),3.25-3.17(m,2H),3.03(br,s,1H),2.73-2.38(m,7H),2.17-2.10(m,2H),2.01-1.95(m,1H),1.95-1.89(m,2H),1.78-1.67(m,5H),1.52-1.40(m,14H),1.40-1.19(m,10H),1.18-1.07(m,2H),0.88(t,3H,J=7.32Hz),0.84(s,6H).
1.6、(6S)-N 6 - (6- ((3, 5-dimethyladamantan-1-yl) amino) hexyl) -N 6 -propyl-4, 5,6, 7-tetrahydrobenzo [ d ]]Thiazole-2, 6-diamine (Pu-C6-Me, compound ML-C)
2.6mL of 4M HCl dioxane solution was added dropwise to 0.2g (0.35 mmol) of Pu-6C-Me (Boc) at 0 deg.C, stirred at room temperature for 2h, the solvent was evaporated to dryness, purified by high performance liquid chromatography, lyophilized, and converted to the hydrochloride salt to give 0.14g of product in 84.8% yield. 1 H NMR(400MHz,DMSO-d 6 )d:10.94(s,1H),9.45(s,2H),9.04(s,2H),3.25-2.95(m,6H),2.92-2.75(m,3H),2.74-2.56(m,2H),2.43-2.33(m,1H),2.21-2.13(m,1H),2.03-1.88(m,1H),1.84-1.63(m,8H),1.61-1.50(m,4H),1.42-1.22(m,8H),1.19-1.07(m,2H),0.92(t,3H,J=7.24Hz),0.86(s,6H).Mass[M+H]found:473.7.
Example 2
2.1, 4-hydroxybutyl-p-toluenesulfonate ester
Starting from 1, 4-butanediol, the preparation was carried out as in example 1.1, replacing 1, 6-hexanediol by an equimolar amount of 1, 4-butanediol and was purified by silica gel column chromatography (eluent ethyl acetate: petroleum ether = 1V/V) to give the product (yield 26.9%), which was identified as 4-hydroxybutyl-p-toluenesulfonate. 1 H NMR(400MHz,CDCl 3 )d:7.79(d,2H,J=8.32Hz),7.35(d,2H,J=8.08),4.06(t,2H,J=6.32Hz),3.620(t,2H,J=6.24Hz),2.44(s,3H),1.80-1.72(m,2H),1.64-1.56(m,2H).
2.2, 4- (3, 5-Dimethyladamantan-1-yl) amino-1-butanol
4- (3, 5-Dimethyladamantan-1-yl) amino-1-butanol was prepared in 25.2% yield by the method of example 1.2, starting from 4-hydroxybutyl-p-toluenesulfonate and replacing 6- (3, 5-dimethyladamantan-1-yl) amino-1-hexanol with an equimolar amount of 4-hydroxybutyl-p-toluenesulfonate. 1 H NMR(400MHz,CDCl 3 )d:3.60(t,2H,J=5.08Hz),2.72(t,2H,J=5.86Hz),2.19-2.14(m,1H),1.79-1.71(m,2H),1.70-1.65(m,2H),1.65-1.61(m,2H),1.46-1.24(m,8H),1.14(s,2H),0.85(s,6H).
2.3, 4- (N-Boc-N- (3, 5-dimethyladamantan-1-yl)) amino-1-butanol
4- (N-Boc-N- (3, 5-dimethyladamantan-1-yl)) amino-1-butanol was prepared in 35% yield according to the procedure of example 1.3, starting from 4- (3, 5-dimethyladamantan-1-yl) amino-1-butanol, replacing 6- (3, 5-dimethyladamantan-1-yl) amino-1-hexanol with an equimolar amount of 4- (3, 5-dimethyladamantan-1-yl) amino-1-butanol. 1 H NMR(400MHz,CDCl 3 )d:3.71-3.63(m,2H),3.30-3.24(m,2H),2.18-2.11(m,1H),1.95-1.91(m,2H),1.76-1.71(m,4H),1.58-1.48(m,4H),1.46(s,9H),1.39-1.32(m,2H),1.29-1.23(m,2H),1.17-1.08(m,2H),0.85(s,6H).
2.4, 4- (N-Boc-N- (3, 5-dimethyladamantan-1-yl) amino) butyl p-toluenesulfonate ester
4- (N-Boc-N- (3, 5-dimethyladamantan-1-yl) amino) butyl p-toluenesulfonate was prepared in 43% yield by the method of example 1.4 starting from the product of example 2.3 by replacing 6- (N-Boc-N- (3, 5-dimethyladamantan-1-yl)) amino-1-hexanol with an equimolar amount of 4- (N-Boc-N- (3, 5-dimethyladamantan-1-yl)) amino-1-butanol. 1 H NMR(400MHz,CDCl 3 )d:7.79(d,2H,J=8.28Hz),7.34(d,2H,J=7.84Hz),4.03(t,2H,J=6.4Hz),3.23-3.17(m,2H),2.45(s,3H),2.16-2.10(m,1H),1.90-1.86(m,2H),1.74-1.65(m,4H),1.52-1.47(m,4H),1.42(s,9H),1.37-1.29(m,4H),1.14-1.09(m,2H),0.84(s,6H).
2.5 tert-butyl (4- (((S) -2-amino-4, 5,6, 7-tetrahydrobenzo [ d ] thiazol-6-yl) (propyl) amino) butyl) (3, 5-dimethyladamantan-1-yl) carbamate (Pu-C4-Me (Boc))
(4- (((S) -2-amino-4, 5,6, 7-tetrahydrobenzo [ d ] b-ydrobenzo [ d) was prepared in the same manner as in example 1.5, starting from the product of example 2.4 by replacing 6- (N-Boc-N- (3, 5-dimethyladamantan-1-yl) amino) hexyl p-toluenesulfonate with an equimolar amount of 4- (N-Boc-N- (3, 5-dimethyladamantan-1-yl) amino) butyl p-toluenesulfonate]Thiazol-6-yl) (propyl) amino) butyl) (3, 5-dimethyladamantan-1-yl) carbamic acid tert-butyl ester (noted Pu-C4-Me (Boc)) in 68.3% yield. 1 H NMR(400MHz,CDCl 3 )d:4.70(s,1H),3.28-3.19(m,2H),3.01(br,s,1H),2.75-2.37(m,7H),2.18-2.10(m,1H),2.01-1.87(m,3H),1.75-1.65(m,5H),1.50-1.40(m,14H),1.39-1.31(m,3H),1.28-1.22(m,4H),1.18-1.07(m,2H),0.88(t,3H,J=7.08Hz),0.85(s,6H).
2.6、(6S)-N 6 - (6- ((3, 5-dimethyladamantan-1-yl) amino) butyl) -N 6 -propyl-4, 5,6, 7-tetrahydrobenzo [ d ]]Thiazole-2, 6-diamine (Pu-C4-Me, compound ML-A)
Prepared by the method of example 1.6 starting from the product of example 2.5, replacing Pu-C6-Me (Boc) with an equimolar amount of Pu-C4-Me (Boc) to give (6S) -N6- (6- ((3, 5-dimethyladamantan-1-yl) amino) butyl) -N6-propyl-4, 5,6, 7-tetrahydrobenzo [ d]Thiazole-2, 6-diamine (Pu-C4-Me, compound ML-A), yield 88%. 1 H NMR(400MHz,DMSO-d 6 )d:10.96(s,1H),9.46(s,2H),9.13(s,2H),3.16-2.93(m,5H),2.89-2.77(m,3H),2.69-2.53(m,2H),2.39-2.30(m,1H),2.16-2.09(m,1H),1.97-1.79(m,3H),1.79-1.64(m,6H),1.58-1.46(m,4H),1.28-1.16(m,5H),1.15-1.02(m,2H),0.87(t,3H,J=7.12Hz),0.81(s,6H).Mass[M+H]found:445.7.
Example 3
3.1 (2-hydroxyethoxy) ethyl p-toluenesulfonate
Starting from diethylene glycol, 1, 6-hexanediol was replaced by an equimolar amount of diethylene glycol, prepared as in example 1.1 and purified by column chromatography on silica gel (eluent ethyl acetate: petroleum ether = 1V/V) to give the product in 35% yield, which was identified as (2-hydroxyethoxy) ethyl p-toluenesulfonate. 1 H NMR(400MHz,CDCl 3 )d:7.81(d,2H,J=8.12Hz),7.36(d,2H,J=8.24),4.22-4.18(m,2H),3.72-3.65(m,4H),3.56-3.52(m,2H).
3.2, 2- (2- (3, 5-Dimethyladamantan-1-yl) amino) -ethoxyethanol
Prepared as in example 1.2 starting from (2-hydroxyethoxy) ethyl p-toluenesulfonate with an equimolar amount of (2-hydroxyethoxy) ethyl p-toluenesulfonate replacing 6- (3, 5-dimethyladamantan-1-yl) amino-1-hexanol to give 2- (2- (3, 5-dimethyladamantan-1-yl) amino) -ethoxyethanol in 61.0% yield. 1 H NMR(400MHz,CDCl 3 )d:4.8(br,m,2H),3.85-3.80(m,2H),3.77-3.73(m,2H),3.71-3.66(m,2H),2.99-2.95(m,2H),2.21-2.16(m,1H),1.68-1.63(m,2H),1.49-1.37(m,4H),1.33-1.29(m,4H),1.18-1.06(m,2H),0.85(s,6H).
3.3, 2- (2- (N-Boc-N- (3, 5-dimethyladamantan-1-yl)) amino) ethoxy) ethanol
Prepared as in example 1.3 starting from the product of example 3.2 by replacing 6- (3, 5-dimethyladamantan-1-yl) amino-1-hexanol with an equimolar amount of 2- (2- (3, 5-dimethyladamantan-1-yl) amino) -ethoxyethanol to give 2- (2- (N-Boc-N- (3, 5-dimethyladamantan-1-yl)) amino) ethoxy) ethanol in 43% yield. 1 H NMR(400MHz,CDCl 3 )d:3.73(t,2H,J=4.48Hz),3.57(t,2H,J=4.36Hz),3.51-3.48(m,4H),2.18-2.11(m,1H),1.95-1.91(m,2H),1.73(s,4H),1.46(s,9H),1.38-1.31(m,2H),1.28-1.22(m,2H),1.14-1.10(m,2H),0.85(s,6H).
3.4, 2- (2- (N-Boc-N- (3, 5-dimethyladamantan-1-yl)) amino) ethoxy) ethyl p-toluenesulfonate
Prepared according to the method of example 1.4 starting from the product of example 3.3 by replacing 6- (N-Boc-N- (3, 5-dimethyladamantan-1-yl)) amino-1-hexanol with an equimolar amount of 2- (2- (N-Boc-N- (3, 5-dimethyladamantan-1-yl)) amino) ethoxy) ethanol to give 2- (2- (N-Boc-N- (3, 5-dimethyladamantan-1-yl)) amino) ethoxy) ethyl p-toluenesulfonate in 68.3% yield. 1 H NMR(400MHz,CDCl 3 )d:7.80(2H,d,J=8.70Hz),7.53(d,2H,J=7.84Hz),4.17-4.13(m,2H),3.65-3.61(m,2H),3.42-3.38(m,4H),2.45(s,3H),2.15-2.10(m,1H),1.90-1.87(m,2H),1.74-1.67(m,4H),1.44(s,9H),1.35-1.22(m,4H),1.14-1.06(m,2H),0.83(m,6H).
3.5 tert-butyl (2- (2- (((S) -2-amino-4, 5,6, 7-tetrahydrobenzo [ d ] thiazol-6-yl) (propyl) amino) ethoxy) ethyl) (3, 5-dimethyladamantan-1-yl) carbamate (Pu-3O-Me (Boc))
From example 3.4The product was prepared as in example 1.5, starting with an equimolar amount of 2- (2- (N-Boc-N- (3, 5-dimethyladamantan-1-yl)) amino) ethoxy) ethyl p-toluenesulfonate instead of 6- (N-Boc-N- (3, 5-dimethyladamantan-1-yl) amino) hexyl p-toluenesulfonate, to give (2- (2- (((S) -2-amino-4, 5,6, 7-tetrahydrobenzo [ d-b-is-c-2-amino-4, 5,6, 7-tetrahydrobenzo [ d ])]Thiazol-6-yl) (propyl) amino) ethoxy) ethyl) (3, 5-dimethyladamantan-1-yl) carbamic acid tert-butyl ester (noted Pu-3O-Me (Boc)) yield 31.6%. 1 H NMR(400MHz,CDCl 3 )d:4.80(s,2H),3.50-3.40(m,6H),3.08-2.97(m,1H),2.75-2.62(m,4H),2.60-2.45(m,4H),2.16-2.10(m,1H),2.02-1.95(m,1H),1.95-1.90(m,2H),1.77-1.68(m,5H),1.49-1.40(m,11H),1.37-1.31(m,2H),1.28-1.21(m,2H),1.16-1.06(m,2H),0.88(t,3H,J=7.28Hz),0.83(s,6H).
3.6、(6S)-N 6 - (2- (2- ((3, 5-dimethyladamantan-1-yl) amino) ethoxy) ethyl) -N 6 -propyl-4, 5,6, 7-tetrahydrobenzo [ d ]]Thiazole-2, 6-diamine (Pu-3O-Me, compound ML-B)
Preparation was carried out in the same manner as in example 1.6, starting from the product of example 3.5, replacing Pu-C6-Me (Boc) with an equimolar amount of Pu-3O-Me (Boc), to give (6S) -N6- (2- (2- ((3, 5-dimethyladamantan-1-yl) amino) ethoxy) ethyl) -N6-propyl-4, 5,6, 7-tetrahydrobenzo [ d ] benzo]Thiazole-2, 6-diamine (Pu-3O-Me, compound ML-B), yield 90%. 1 H NMR(400MHz,DMSO-d 6 )d:10.06-10.43(br,1H),9.48-9.14(m,4H),3.84-3.71(m,5H),3.57-3.27(m,6H),3.20-3.04(m,5H),2.75-2.65(m,1H),2.21-2.14(m,1H),1.92-1.76(m,4H),1.72-1.58(m,4H),1.33-1.26(m,4H),1.19-1.06(m,2H),0.92(t,3H,J=7.12Hz),0.85(s,6H).Mass[M+H]found:461.6
Example 4D2 receptor agonism assay
1. Reagent preparation
1.1 slicing solution rich in sucrose: 40mmol/L NaCl,4.5mmol/L KCl,1.25mmol/L NaH 2 PO 4 ,25mmol/L NaHCO 3 10mmol/L glucose, 148.5mmol/L sucrose, 1mmol/L ascorbic acidA blood acid salt, 3mmol/L sodium pyruvate, 3mmol/L inositol, 0.5mmol/L CaCl 2 ,7mmol/L MgCl 2 。
1.2 artificial cerebrospinal fluid: 125mmol/L NaCl,4.5mmol/L KCl,1.25mmol/L NaH 2 PO 4 ,2.5mmol/L CaCl 2 ,1.3mmol/L MgSO 4 ,25mmol/L NaHCO 3 15mmol/L glucose, 15mmol/L sucrose.
1.3 electrode internal solution for recording action potential: 123mmol/L potassium methanesulfonate, 8mmol/L NaCl,15mmol/L HEPES,0.2mmol/L EGTA,1mmol/L MgCl 2 ·H 2 O,2mmol/L MgATP,0.3mmol/L Na 3 GTP,7mmol/L Na 2 CrPi is used. The osmotic pressure was 290mOsm, and the pH was adjusted to 7.2 with KOH.
The slice liquid and the artificial cerebrospinal fluid are respectively used for incubation of the rat brain slice and the brain slice, and the electrode internal liquid is used for recording cell reaction.
2. Recording of cholinergic neuronal current-induced action potentials
Selecting a Chat-eGFP mouse, as shown in figure 1A, adopting a patch clamp current clamp whole cell mode, and observing for at least 5min before starting recording to ensure that cells are in a stable state. A GABA current blocking agent (BIC, 10 μ M) was added to the perfusate to clamp cholinergic neurons at-70 mV, and the number of current-induced action potentials was recorded within 300 ms. The effects of compounds ML-B, pramipexole, ML-A and ML-C on the number of action potentials were observed by separately perfusing compounds ML-B, pramipexole, ML-A and ML-C, each prepared from a physiological saline solution (0.9% NaCl solution).
3. Results of the experiment
The results are shown in FIG. 1, which shows that the compound ML-B has no D2 receptor agonism at a concentration of 50. Mu.M, the compound ML-B has D2 receptor agonism at a concentration of 100. Mu.M, the compound ML-B also has D2 receptor agonism at a concentration of 200. Mu.M, and the compound ML-B shows better dose correlation. When the pramipexole (Prami) concentration reaches 200 mu M, the D2 receptor agonism is achieved; the compounds ML-A and ML-C do not have D2 receptor agonism even when the concentration reaches 200. Mu.M.
Example 5 NMDA receptor antagonism assay
1. Reagent preparation
1.1 sucrose-rich slice: 40mmol/L NaCl,4.5mmol/L KCl,1.25mmol/L NaH 2 PO 4 ,25mmol/L NaHCO 3 10mmol/L glucose, 148.5mmol/L sucrose, 1mmol/L ascorbate, 3mmol/L sodium pyruvate, 3mmol/L inositol, 0.5mmol/L CaCl 2 ,7mmol/L MgCl 2 。
1.2 magnesium-free artificial cerebrospinal fluid: 125mmol/L NaCl,4.5mmol/L KCl,1.25mmol/L NaH 2 PO 4 ,2.5mmol/L CaCl 2 ,25mmol/L NaHCO 3 15mmol/L glucose, 15mmol/L sucrose.
1.3 electrode internal solution to record excitatory postsynaptic current: 119mmol/L CsMeSO 4 ,8mmol/L TEACl,15mmol/L HEPES,0.6mmol/L EGTA,0.3mmol/L Na 3 GTP,4mmol/L MgATP,5mmol/L QX-314.CL,7mmol/L Na 2 CrPO 4 . The osmotic pressure was 290mOsm, and the pH was adjusted to 7.2 with CsOH.
Recording of NMDA receptor-mediated excitatory postsynaptic currents
And (3) observing for at least 5min before recording by adopting a patch clamp voltage clamp whole cell recording mode, and ensuring that the cells are in a stable state. A GABA current blocking agent (BIC, 10 μ M) and an AMPA receptor blocking agent (CNQX, 10 μ M) are added into a perfusion solution, a stimulating electrode is placed, cells are clamped at-70 mV near the stimulating electrode, NMDA-mediated excitatory postsynaptic current is recorded, compounds ML-B, pramipexole, compounds ML-A and compounds ML-C with different concentrations prepared by normal saline are respectively perfused, and the influence of the compounds on NMDA-mediated excitatory postsynaptic current amplitude is observed.
3 results of the experiment
The results are shown in FIG. 2, which shows that compound ML-B has no NMDA receptor antagonism at 50. Mu.M, has NMDA receptor antagonism at 100. Mu.M, and also shows NMDA receptor antagonism at 200. Mu.M, and shows better dose-dependence. Pramipexole and ML-A showed no significant NMDA receptor antagonism at 200. Mu.M, while the compound ML-C at 200. Mu.M showed complete NMDA receptor antagonism.
The results of the D2 receptor agonistic effect test in example 4 show that the compound ML-B has both NMDA receptor antagonistic effect and D2 receptor agonistic effect.
EXAMPLE 6 measurement of the concentration of Compound ML-B in plasma and brain homogenates
1. Solution preparation
1.1 preparation of Standard solutions of Compounds: accurately weighing 2.0 mg of the compound ML-B in a 10mL volumetric flask, dissolving with methanol to a constant volume, preparing a standard solution with the concentration of 0.2mg/mL, and storing at 4 ℃.
1.2 preparation of internal standard: 4.15mg fluoxetine is accurately weighed into a 10mL volumetric flask, dissolved with methanol to a constant volume, and prepared into a standard solution with a concentration of 415 g/mL. Taking 0.1mL of standard solution in a 10mL volumetric flask, using methanol to fix the volume, and preparing the standard solution with the concentration of 4.15g/mL as an internal standard.
1.3 preparation of dosing solution: compound ML-B (4.8 mg) was weighed out accurately and dissolved in 19.2mL of physiological saline.
2. Detection conditions are as follows:
agilent 1200 series liquid phase System with Sunfire pretreatment C 18 Column (4.6 mm × 250 mm), mobile phase 0.1% formic acid water (mobile phase a) =20,v/V methanol (mobile phase B) =80, flow rate 0.25mL/min.
3. Plasma pretreatment
Placing 100 mu L of blank plasma in a 1.5mL centrifuge tube, adding 10 mu L of internal standard, adding 300 mu L of methanol-acetonitrile = 1V/V mixed solvent to precipitate protein, carrying out mixed rotation on a vortex instrument for 1min, centrifuging in a centrifuge for 10min at 12000r/min, and taking 10 mu L of supernatant for injection.
4. Drawing of Standard Curve
Taking blank plasma, preparing a standard blank plasma series sample with a compound standard solution, wherein the plasma concentration of the compound ML-B is 0.05, 0.1, 0.2, 1, 2.5 and 5 mu g/mL, treating the sample according to the item of 'plasma pretreatment', and measuring the sample according to the item of 'detection condition'. Recording chromatographic peak areas of the plasma sample and the internal standard, performing linear regression operation by taking the concentration of the compound ML-B as a horizontal ordinate and taking a peak area ratio (the ratio of peak areas of the compound and the internal standard) as a vertical ordinate to obtain a linear regression equation of y =1.2182x-0.1107,r 2 The value is 0.9943, and the result shows that the compound ML-B has a good linear relation with the peak area ratio in the range of 0.05-5 mu g/mL in the plasma.
5. Brain tissue pretreatment
The mice were sacrificed quickly, brain tissue was dissected out and rinsed with normal saline to remove blood stain on the tissue surface, the tissue surface was blotted with filter paper, weighed, added with a fixed amount of normal saline (1 g tissue added with 3mL normal saline) and made into homogenate with an electric homogenizer, and sonicated for 10min to remove air bubbles.
Placing 100 mu L of blank brain tissue into a 1.5mL centrifuge tube, adding 10 mu L of internal standard, adding 300 mu L of methanol acetonitrile = 1.
6. Drawing of brain homogenate standard curve
Taking a blank brain tissue, preparing a blank sample with the concentration of 10, 50, 100, 200, 400 and 600ng/g by using a compound standard solution, processing the blank sample according to the 'brain tissue pretreatment' term, measuring according to the 'detection condition' term, recording the chromatographic peak areas of a brain homogenate sample and an internal standard, performing linear regression operation by using the concentration of the compound as a horizontal coordinate and the peak area ratio as a vertical coordinate, and obtaining a linear regression equation of y =0.0035x +0.0191, r 2 The value is 0.9931, and the result shows that the compound ML-B has good linear relation with the peak area ratio in the range of 10-600 ng/g in the brain homogenate.
7. Abdominal cavity administration experiment of mouse
Mice of 8-10 weeks old and weighing 20-30g were divided at random into 2 mice per time point, and were intraperitoneally injected with compound ML-B (prepared from normal saline) at a dose of 2.5mg/kg. The mice are fasted for 12 hours without water supply, water is freely drunk in the test period, blood is taken from orbital venous plexus respectively for 5min, 10min, 30min, 60min, 90min, 180min and 240min after administration, plasma samples are placed in centrifugal tubes which are treated by heparin in advance, then the mice are killed, and brain tissues are dissected and taken out; and (3) centrifuging the plasma sample in a centrifuge at 12000r/min for 10min, processing the plasma sample according to the 'plasma pretreatment' term, processing the brain tissue according to the 'brain tissue pretreatment' term, analyzing the processed sample according to the analysis condition under the 'detection condition', substituting the peak area ratio of the compound ML-B and the internal standard peak area ratio into a corresponding standard curve to obtain the plasma drug concentration and the brain tissue drug concentration of the compound ML-B at each time point, drawing a line graph, and calculating corresponding pharmacokinetic parameters.
TABLE 1 pharmacokinetic parameters of Compound ML-B in plasma
t 1/2 (min) | 521.78 |
Tmax(min) | 5 |
Cmax(ng/mL) | 3268.57 |
The results are shown in Table 1 and FIG. 3, which indicate that compound ML-B can exert therapeutic effects through the blood-brain barrier.
Example 7HE staining experiment
The experimental steps are as follows: C57/B6 mice (8-10 weeks old, 20-30 g) are taken and randomly divided into a control group and an administration group; the administration group continuously injects compound ML-B (prepared into solution with the concentration of 0.025 percent by normal saline) into the abdominal cavity for one month, the dosage is 2.5mg/kg, and the dosage is converted into 0.1mL/10 g; the control group was injected with normal saline intraperitoneally continuously for one month, and the amount of normal saline was converted according to the body weight of the mouse. One month later, the heart, liver, spleen, lung and kidney of the mouse were collected after heart perfusion, and were soaked in 10% neutral formalin solution, paraffin sections and HE staining were performed, and then photographed.
The results of HE staining in the control group and the drug group are shown in fig. 4, the heart, liver, spleen, lung and kidney of the control group (fig. 4A) and the drug group (fig. 4B) have no obvious difference, and the drug has no obvious toxic reaction to the important organs of the mice.
Example 8
Establishment of mouse MPTP model
1. Animals: C57/B6 mice (8-10 weeks old, 20-30 g). The room temperature was maintained at 22 + -2 deg.C and light (12 h light cycle), and food and water were taken ad libitum.
2. Preparing a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine, MPTP) solution: the physiological saline is used as a solvent, an MPTP solution with the concentration of 0.6% is prepared, and the administration volume is 0.05mL/10g, namely the administration dose is 30mg/kg.
3. And (4) evaluation results: after MPTP injection, the mice developed symptoms such as salivation, arch back, tremor of limbs, etc.
4. The administration mode comprises the following steps: the mice were randomly divided into a model group (MPTP), an administration group, a positive drug group (Prami) and a normal group (Ctrl); injecting MPTP solution with the concentration of 0.6% into the abdominal cavity of a model group mouse, wherein the administration volume is 0.05mL/10g, namely the administration dosage is 30mg/kg, injecting physiological saline into the abdominal cavity after one hour, the administration volume is 0.05mL/10g, and continuously performing the same operation for five days; the mice in the administration group are injected with MPTP solution with the concentration of 0.6 percent in the abdominal cavity, the administration volume is 0.05mL/10g, namely the administration dose is 30mg/kg, and equal volume of ML-B solution with different concentrations (prepared by normal saline; the concentration of the compound ML-B is 0.05 percent, the administration dose is 2.5mg/kg; the concentration of the compound ML-B is 0.02 percent, the administration dose is 1.0mg/kg; the concentration of the compound ML-B is 0.008 percent, the administration dose is 0.4 mg/kg) is injected in the abdominal cavity after one hour, and the same operation is carried out continuously for five days; the positive medicine group mice are injected with MPTP solution with the concentration of 0.6% in the abdominal cavity, the administration volume is 0.05mL/10g, namely the administration dose is 30mg/kg, the equal volume of pramipexole solution (prepared by normal saline, the pramipexole concentration is 0.02%, and the administration dose is 1 mg/kg) is injected in the abdominal cavity after one hour, and the same operation is carried out continuously for five days; normal group mice were injected with normal saline twice a day, the two times of administration were separated by one hour, the volume of each administration was 0.05mL/10g, and the same procedure was performed for five consecutive days.
Rod rotation experiment
The experimental steps are as follows: the mice after molding and dosing were removed and placed on a rotarod apparatus as shown in fig. 5A. The rotating speed of the rod rotating instrument is set to be increased from 5rpm/min to 40rpm/min at a constant speed within 5min, the rod time of the mice is recorded, and the rod residence time of each group of mice is compared.
The results are shown in fig. 5B, which indicates that: the compound ML-B can prolong the rod residence time of an MPTP model mouse, and the compound ML-B shows better dose correlation; compared with the pramipexole serving as a positive drug, the compound ML-B has no obvious difference in the effect of improving the mice in a model group.
Open field experiment
The experimental steps are as follows: and (3) placing the mice after the model building and the drug administration are finished in an open field box (40 cm multiplied by 40 cm), and recording the total movement distance, the movement speed, the motionless time (the speed is less than 0.2 cm/s) percentage and the movement track of each group of mice within 5min by using behavioristics experiment software.
The experimental results are shown in fig. 6, and fig. 6A, 6B, 6C, and 6D are graphs of experimental trajectories, total movement distance, movement speed, and percentage of motionless time of each group of mice respectively, which show that: the compound ML-B can increase the total movement distance and the movement speed of an MPTP model mouse and reduce the immobility time percentage of the mouse; compared with the pramipexole serving as a positive drug, the compound ML-B has no obvious difference in the effect of improving the mice in a model group.
Immunofluorescence staining experiment for dopamine neuron in substantia nigra pars compacta
The experimental steps are as follows: after the behavioral investigation is finished, the heart of the mouse is perfused with 20mL of PBS solution and 40mL of 4% paraformaldehyde solution, then the head is broken and the brain is taken out, the mouse brain is placed in the 4% paraformaldehyde solution for fixing overnight, the next day is changed into 30% sucrose solution for sugar precipitation for two days, the frozen section of the substantia nigra compact part with the thickness of 20 mu m is carried out after embedding by an embedding medium, the material is pasted and then subjected to Tyrosine Hydroxylase (TH) immunofluorescence staining, after the mounting by adopting an anti-fluorescence quenching glycerol mounting medium containing dapi, the material is photographed under a 10-fold fluorescence microscope, and the number of TH staining positive cells is recorded.
The experimental result is shown in figure 7, the dark part in the fluorescence image is the background, the bright round dots are TH positive cells, namely dopamine neurons, and the area outlined by the dotted line is the mouse substantia nigra pars compacta. The more bright dots in the area framed by the fluorescence, the greater the number of dopamine neurons. The results show that: when the administration dose of the compound ML-B is 2.5mg/kg, the compound ML-B can relieve the damage of dopamine neurons in the density of the substantia nigra of an MPTP model mouse, reduce the speed of the decrease of the number of the dopamine neurons and play a role in neuroprotection; compared with pramipexole, the compound ML-B has no statistical difference.
Example 9
Experiment of new object recognition
The experimental steps are as follows: mice were randomly divided into a model group (MPTP), a drug administration group, a positive drug group (Prami), and a normal group (Ctrl). Injecting MPTP solution with the concentration of 0.6% into the abdominal cavity of a model group mouse, wherein the administration volume is 0.05mL/10g, namely the administration dose is 30mg/kg, injecting physiological saline into the abdominal cavity after one hour, the administration volume is 0.05mL/10g, and the same operation is carried out continuously for five days; the mice in the administration group are injected with MPTP solution with the concentration of 0.6 percent in the abdominal cavity, the administration volume is 0.05mL/10g, namely the administration dose is 30mg/kg, ML-B solution with the same volume (prepared by normal saline, the concentration of the compound ML-B is 0.05 percent, the administration dose is 2.5 mg/kg) is injected in the abdominal cavity after one hour, and the same operation is carried out continuously for five days; the positive medicine mice are injected with MPTP solution with the concentration of 0.6% in the abdominal cavity, the administration volume is 0.05mL/10g, namely the administration dose is 30mg/kg, the equal volume of the positive medicine pramipexole solution (prepared by normal saline, the pramipexole concentration is 0.02%, and the administration dose is 1 mg/kg) is injected in the abdominal cavity after one hour, and the same operation is carried out continuously for five days; normal group mice were injected with normal saline twice a day, the two times of administration were separated by one hour, the volume of each administration was 0.05mL/10g, and the same procedure was performed for five consecutive days.
The experiment of identifying new objects is generally divided into an adaptation period, a familiarity period and a testing period. At the acclimation period, mice were placed in a laboratory box (40 × 40 × 40 cm) and were made familiar with the environment. The following day of the acclimation period, the mice were subjected to the experiments of the familiarity period and the test period. As shown in fig. 8A, in the familiarity period, two identical objects were placed in the experimental box, and the exploration behavior of the mouse for the two objects within 10min was recorded; after 60min, the experiment is carried out in a test period, one of two identical objects is replaced by an object with different color and shape, the position is unchanged, the search time of the mouse for a Familiar object (Familiar) and a Novel object (Novel) within 5min is recorded, and the learning and memory ability of the mouse is evaluated by a Discrimination Index (DI).
The results are shown in FIGS. 8B and 8C, indicating that: compared with the mice in the model group, the time for the mice in the compound ML-B group to explore new objects is obviously increased, which shows that the compound ML-B improves the cognitive dysfunction of the mice in the MPTP model, and the pramipexole which is a positive drug has no obvious improvement effect on the cognitive dysfunction of the mice in the MPTP model.
Elevated plus maze experiment
The experimental steps are as follows: as shown in FIG. 9A, the elevated plus maze typically consists of two opposing open arms (open arm) and two opposing closed arms (closed arm), and the maze is 50cm high from the ground. And (3) installing a camera right above the maze, placing the mouse at the tail end of the open arm far from the central area, facing the tail end far from the central area, recording the time of the mouse from the open arm to the first time when the mouse enters the closed arm, and continuously measuring twice, wherein the interval time is 24h each time. The index for evaluating learning and memory of mice is the ratio of the time of two passes from the open arm to the closed arm (transfer latency, TL).
The results are shown in fig. 9B, which indicates that: compared with the mice in the model group, the time for the mice in the compound ML-B group to enter the closed arm from the open arm for the second time is obviously reduced, which shows that the compound ML-B improves the cognitive dysfunction of the mice in the MPTP model, and the pramipexole as a positive drug has no obvious improvement effect on the cognitive dysfunction of the mice in the MPTP model.
Example 10
Striatum cholinergic neuron spontaneous discharge and current-induced action potential recording experiment
1. Reagent preparation
1.1 sucrose-rich slice: 40mmol/L NaCl,4.5mmol/L KCl,1.25mmol/L NaH 2 PO 4 ,25mmol/L NaHCO 3 10mmol/L glucose, 148.5mmol/L sucrose, 1mmol/L ascorbate, 3mmol/L sodium pyruvate, 3mmol/L inositol, 0.5mmol/L CaCl 2 ,7mmol/L MgCl 2 。
1.2 artificial cerebrospinal fluid: 125mmol/L NaCl,4.5mmol/L KCl,1.25mmol/L NaH 2 PO 4 ,2.5mmol/L CaCl 2 ,1.3mmol/L MgSO 4 ,25mmol/L NaHCO 3 15mmol/L glucose, 15mmol/L sucrose.
1.3 electrode internal liquid for recording action potential and spontaneous discharge: 123mmol/L potassium methanesulfonate, 8mmol/L NaCl,15mmol/L HEPES,0.2mmol/L EGTA,1mmol/L MgCl 2 ·H 2 O,2mmol/L MgATP,0.3mmol/L Na 3 GTP,7mmol/L Na 2 CrPi is used as a reference. The osmotic pressure was 290mOsm, and the pH was adjusted to 7.2 with KOH.
2. Recording of cholinergic neuronal spontaneous discharge and current-induced action potentials
After the rod rotation experiment and the open field experiment are finished, the mice are divided into the following mice: control group (two intraperitoneal injections of normal saline), model group (one intraperitoneal injection of MPTP followed by one-hour intraperitoneal injection of an equal volume of normal saline), administration group (one intraperitoneal injection of MPTP followed by one-hour intraperitoneal injection of 2.5mg/kg of compound ML-B).
Referring to FIG. 10A, before starting recording, the cells are observed for at least 5min in the patch-clamp voltage-clamp cell attachment mode to ensure that the cells are in a stable state. Adding GABA receptor blocker (BIC, 10 μ M) into the perfusion liquid, clamping the cells at-65 mV, gradually injecting current in a current clamp mode, and recording the number of current-induced action potentials under different injected currents.
3. Results of the experiment
The results are shown in FIGS. 10B, 10C, indicating that: the compound ML-B can reduce the number of action potentials induced by the cholinergic neuron current of the striatum of a PD model mouse and reduce the excitability of the cholinergic neuron.
Example 11
Striatum medium spine neuron NMDA receptor activity detection experiment
1. Reagent preparation
1.1 slicing solution rich in sucrose: 40mmol/L NaCl,4.5mmol/L KCl,1.25mmol/L NaH 2 PO 4 ,25mmol/L NaHCO 3 10mmol/L glucose, 148.5mmol/L sucrose, 1mmol/L ascorbate, 3mmol/L sodium pyruvate, 3mmol/L inositol, 0.5mmol/L CaCl 2 ,7mmol/L MgCl 2 。
1.2 magnesium-free artificial cerebrospinal fluid: 125mmol/L NaCl,4.5mmol/L KCl,1.25mmol/L NaH 2 PO 4 ,2.5mmol/L CaCl 2 ,25mmol/L NaHCO 3 15mmol/L glucose, 15mmol/L sucrose.
1.3 electrode internal solution to record excitatory postsynaptic current: 119mmol/L CsMeSO 4 ,8mmol/L TEACl,15mmol/L HEPES,0.6mmol/L EGTA,0.3mmol/L Na 3 GTP,4mmol/L MgATP,5mmol/L QX-314.CL,7mmol/L Na 2 CrPO 4 . The osmotic pressure was 290mOsm, and the pH was adjusted to 7.2 with CsOH.
Registration of NMDA receptor-mediated excitatory postsynaptic and NMDA-induced currents
The mice are randomly divided into a model group (MPTP), an administration group, a positive medicine group (Prami) and a normal group (Ctrl), MPTP solution with the concentration of 0.6% is injected into the abdominal cavity of the mice in the model group, the administration volume is 0.05mL/10g, namely the administration dose is 30mg/kg, physiological saline is injected into the abdominal cavity after one hour, the administration volume is 0.05mL/10g, and the same operation is carried out continuously for five days; the mice in the administration group are injected with MPTP solution with the concentration of 0.6 percent in the abdominal cavity, the administration volume is 0.05mL/10g, namely the administration dosage is 30mg/kg, and after one hour, the ML-B solution with the same volume (prepared by normal saline, the concentration of the compound ML-B is 0.05 percent, and the administration dosage is 2.5 mg/kg) is injected in the abdominal cavity, and the same operation is carried out continuously for five days; the positive medicine group mouse is injected with MPTP solution with the concentration of 0.6% in the abdominal cavity, the administration volume is 0.05mL/10g, namely the administration dose is 30mg/kg, the equal volume of pramipexole solution (prepared by normal saline, the concentration of pramipexole is 0.02%, the administration dose is 1 mg/kg) is injected in the abdominal cavity after one hour, and the same operation is carried out continuously for five days; normal group mice were injected with normal saline twice a day, the two times of administration were separated by one hour, the volume of each administration was 0.05mL/10g, and the same procedure was performed for five consecutive days. After the rod rotating experiment and the open field experiment are finished, mouse brain slices of all groups are respectively taken, slice liquid and artificial cerebrospinal fluid are used for incubation of the mouse brain slices and the brain slices, and electrode internal liquid is used for recording cell reaction. And (3) observing for at least 5min before recording by adopting a patch clamp voltage clamp whole cell recording mode, and ensuring that the cells are in a relatively stable state. GABA current blocking agent (BIC, 10 μ M) and AMPA receptor blocking agent (CNQX, 10 μ M) are added into the perfusate, a stimulating electrode is placed, cells are clamped at-70 mV near the stimulating electrode, five stimulation intensities of 1V, 2V, 3V, 4V and 5V are sequentially given, and NMDAR mediated excitatory postsynaptic currents in a control group, a model group, an administration group and a positive medicine group are recorded.
3. Results of the experiment
The results are shown in FIG. 11, which shows: the amplitude of change of the NMDAR-mediated excitatory postsynaptic current recorded in the compound ML-B under the same stimulus intensity is smaller than that of the NMDAR-mediated excitatory postsynaptic current recorded in the PD model mouse under the same stimulus intensity, and the abnormal NMDAR excitatory synaptic transmission in the PD model mouse is reduced.
Example 12
Striatal field potential recording experiment
1. Reagent preparation
1.1 slicing solution rich in sucrose: 40mmol/L NaCl,4.5mmol/L KCl,1.25mmol/L NaH 2 PO 4 ,25mmol/L NaHCO 3 10mmol/L glucose, 148.5mmol/L sucrose, 1mmol/L ascorbate, 3mmol/L sodium pyruvate, 3mmol/L inositol, 0.5mmol/L CaCl 2 ,7mmol/L MgCl 2 。
1.2 artificial cerebrospinal fluid: 125mmol/L NaCl,4.5mmol/L KCl,1.25mmol/L NaH 2 PO 4 ,2.5mmol/L CaCl 2 ,1.3mmol/L MgSO 4 ,25mmol/L NaHCO 3 15mmol/L glucose, 15mmol/L sucrose.
1.3 electrode internal liquid for recording striatum field potential: 1mol/L NaCl.
2. Recording of striatal excitatory postsynaptic potentials
Mice were randomly divided into model group (MPTP), administration group and normal group (Ctrl); injecting MPTP solution (prepared by normal saline) with the concentration of 0.6% into the abdominal cavity of a model group mouse, wherein the administration volume is 0.05mL/10g, namely the administration dosage is 30mg/kg, injecting the normal saline into the abdominal cavity after one hour, and the administration volume is 0.05mL/10g, and continuously performing the same operation for five days; the mice in the administration group are injected with MPTP solution with the concentration of 0.6 percent in the abdominal cavity, the administration volume is 0.05mL/10g, namely the administration dose is 30mg/kg, ML-B solution with the same volume (prepared by normal saline, the concentration of the compound ML-B is 0.05 percent, the administration dose is 2.5 mg/kg) is injected in the abdominal cavity after one hour, and the same operation is carried out continuously for five days; normal group mice were injected with normal saline twice a day, the two times of administration were separated by one hour, the volume of each administration was 0.05mL/10g, and the same procedure was performed for five consecutive days. After the rod rotating experiment and the open field experiment are finished, mouse brain slices of all groups are respectively taken, slice liquid and artificial cerebrospinal fluid are used for incubation of the mouse brain slices and the brain slices, and electrode internal liquid is used for recording cell reaction. Brain patch clamp experiments were performed by adding a GABA receptor antagonist (BIC, 10 μ M) to the perfusate, recording in patch clamp current clamp mode, placing the stimulating electrode in the cortex, the glass recording electrode in the striatum, and recording the field excitatory postsynaptic potential of the cortex-striatum pathway (fEPSP). The stimulation intensity was first adjusted to bring the fEPSP within the appropriate range. After stable recording for 10min baseline, long-term inhibition (LTD) of fEPSP was induced by high frequency stimulation, and recording was continued for 45min after induction stimulation was completed. The amplitude of fEPSP after high frequency stimulation was compared to the baseline amplitude.
3. Results of the experiment
The results are shown in FIG. 12, which shows: in the MPTP model mice, the amplitude of fpsps in the cortical-striatal pathway did not change significantly upon high frequency stimulation, and long-term inhibition (LTD) could not be induced. The compound ML-B can restore the condition of synaptic plasticity impairment in an MPTP model mouse and improve the integral output function of striatum.
Claims (9)
2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein: x is selected from O; n is an integer selected from 1 to 6, and m is an integer selected from 1 to 6.
5. the compound according to any one of claims 1-4, or a pharmaceutically acceptable salt thereof, wherein: pharmaceutically acceptable salts are hydrochloride, hydrobromide, nitrate, perchlorate, phosphate, sulphate, formate, acetate, aconate, ascorbate, benzenesulphonate, benzoate, cinnamate, citrate, heptanoate, fumarate, glutamate, glycolate, lactate, maleate, malonate, mandelate, methanesulphonate, naphthalene-2-sulphonate, phthalate, salicylate, sorbate, stearate, succinate, tartrate or p-toluenesulphonate.
6. Use of a compound of any one of claims 1-3, or a pharmaceutically acceptable salt thereof, for the manufacture of a D2 receptor agonist and/or an NMDA receptor antagonist.
7. Use of a compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prevention and treatment of parkinson's disease.
8. Use of a compound of claim 4 or a pharmaceutically acceptable salt thereof for the preparation of a D2 receptor agonist and an NMDA receptor antagonist.
9. The use of a compound of claim 4, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prevention and treatment of parkinson's disease.
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张丽云 等: "普拉克索联合盐酸美金刚治疗老年帕金森病疗效及对相关蛋白影响", 《GUIZHOU MEDICINAL JOURNAL》, vol. 44, no. 12, pages 1960 - 1961 * |
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