CN115340508B - Compounds having both D2 receptor agonism and NMDA receptor antagonism and uses thereof - Google Patents

Compounds having both D2 receptor agonism and NMDA receptor antagonism and uses thereof Download PDF

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CN115340508B
CN115340508B CN202211007790.7A CN202211007790A CN115340508B CN 115340508 B CN115340508 B CN 115340508B CN 202211007790 A CN202211007790 A CN 202211007790A CN 115340508 B CN115340508 B CN 115340508B
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马腾飞
厉廷有
谢倩
秦亚娟
李嘉欣
胡梦溪
李子艺
李继阳
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Nanjing Medical University
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Abstract

The invention discloses a compound with a structure shown in a formula I or pharmaceutically acceptable salt thereof, wherein X is selected from CH 2 and O; n is an integer selected from 1 to 6, and m is an integer selected from 1 to 6. The compounds of the invention have at least NMDA receptor antagonism; especially when X is selected from O, the compound has both D2 receptor agonism and NMDA receptor antagonism, and the NMDA receptor antagonism does not affect normal functions, improves the motor symptoms of PD model mice, improves the cognitive dysfunction of PD model mice, and has better treatment effect on Parkinson's disease. The invention also discloses application of the compound or pharmaceutically acceptable salt thereof in preparing D2 receptor agonist and/or NMDA receptor antagonist. The invention also discloses application of the compound or pharmaceutically acceptable salt thereof in preparing medicines for preventing and treating parkinsonism.

Description

Compounds having both D2 receptor agonism and NMDA receptor antagonism and uses thereof
Technical Field
The invention belongs to the field of pharmacy, and relates to a novel parkinsonism treatment medicine with the functions of D2 receptor agonism and NMDA receptor partial antagonism.
Background
Parkinson's Disease (PD) is the second most neurodegenerative disease in the world, and 2% of the population aged 65 years and older are threatened by this. The pathological features of PD are mainly midbrain substantia nigra compact dopamine neuron injury, accompanied by motor symptoms such as resting tremor, rigidity, slow movement and the like, sleep disorder, cognitive disorder and other non-motor symptoms.
Currently, the clinical treatment strategies for parkinson's disease are mainly divided into protecting dopaminergic neurons and supplementing dopamine. Wherein, the quasi-dopamine drug levodopa can be generated along with dyskinesia such as catabolism with the progress of illness state and the increase of dosage, and the curative effect of treatment is affected. Dopamine receptor agonists, most of which are dopamine ii (D2) receptor agonists, are accepted by an increasing number of patients because they can ameliorate parkinson-like symptoms in the patient with fewer adverse effects. Pramipexole (chemical name of (S) -2-amino-4, 5,6, 7-tetrahydro-6-propylamine-benzothiazole) is a clinically common D2 receptor agonist for treating PD, and can excite the D2 receptor and inhibit the NO-GO effect of an indirect pathway mediated by D2 neurons, thereby playing a role in relieving dyskinesia.
Clinical data show that up to 40% of PD patients may develop cognitive dysfunction and dementia, and as the PD condition progresses, the probability of dementia and cognitive dysfunction increases, and NMDA receptor antagonists such as amantadine, memantine and the like can improve cognitive dysfunction in PD patients with dementia, block abnormal glutamate signals in the striatum of PD patients, reduce neurotoxicity, thereby playing a neuroprotective role and delaying the progression of the disease to a certain extent.
In the course of parkinson's disease, there are many pathogenesis including environmental factors, oxidative stress, excitotoxicity, etc. Therefore, it is difficult to achieve a satisfactory therapeutic effect only for one of the targets.
Disclosure of Invention
The invention aims to provide a multifunctional parkinsonism treatment medicine.
The invention aims at realizing the following technical scheme:
A compound having the structure shown in formula i:
wherein X is selected from CH 2 and 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.
Specifically, the compound is selected from the following compounds:
Pharmaceutically acceptable salts are hydrochloride, hydrobromide, nitrate, perchlorate, phosphate, sulfate, formate, acetate, aconate, ascorbate, benzenesulfonate, benzoate, cinnamate, citrate, heptanoate, fumarate, glutamate, glycolate, lactate, maleate, malonate, mandelate, methanesulfonate, naphthalene-2-sulfonate, phthalate, salicylate, sorbate, stearate, succinate, tartrate or p-toluenesulfonate.
The compounds of the invention have at least NMDA receptor antagonism; especially when X is selected from O, the compound has D2 receptor agonism and NMDA receptor antagonism, and the NMDA receptor antagonism does not affect normal functions, and improves the motor symptoms of a parkinsonism model (MPTP model) mouse, and simultaneously improves the movement and cognitive disorder of the MPTP model mouse, relieves the damage of dopamine neurons in the substantia nigra compacta of the MPTP model mouse, and the mechanism of the compound is possibly related to the fact that the compound can improve the abnormal excitability of NMDAR receptors and cholinergic neurons in the striatum and improve the overall output function of the striatum. The compound has better parkinsonism treatment effect.
It is therefore a further object of the present invention to provide the use of a compound of formula i or a pharmaceutically acceptable salt thereof for the preparation of D2 receptor agonists and/or NMDA receptor antagonists.
The invention also aims to provide application of the compound shown in the formula I or pharmaceutically acceptable salt thereof in preparing medicines for preventing and treating parkinsonism.
Another object of the present invention is to provide a process for the preparation of a compound of formula I, the synthetic route being as follows:
the invention has the beneficial effects that:
the compound disclosed by the invention is a novel parkinsonism treatment drug with multiple pharmacological mechanisms, has both D2 receptor agonism and NMDA receptor antagonism, can improve the movement and cognitive dysfunction of PD model mice, and has a better treatment effect than a single-target drug.
Drawings
Fig. 1: results of agonism of pramipexole, compound ML-A, ML-B, ML-C, at different concentrations at the D2 receptor; wherein A: the upper graph is a fluorescence graph of striatum parts in a Chat-eGFP mouse brain slice, and the lower graph is a schematic diagram of a cholinergic neuron recorded by a glass electrode; b: action potentials representative of cholinergic neurons before and after 50. Mu.M, 100. Mu.M, 200. Mu.M of compound ML-B, 200. Mu.M pramipexole perfused brain slices; c: action potential statistics graphs of 50. Mu.M, 100. Mu.M, 200. Mu.M compound ML-B perfusion brain slice pre-and post cholinergic neurons; d: action potential statistics of 200 mu M pramipexole perfusion brain slice front and back cholinergic neurons; e: comparing the percentage of action potential of 200 μm compound ML-B to 200 μm pramipexole to reduce cholinergic neurons at the same baseline conditions; f: action potential representative graphs of cholinergic neurons before and after perfusion of 200. Mu.M compound ML-A, 200. Mu.M compound ML-C into brain sheets G: action potential statistics of cholinergic neurons before and after 200 μm compound ML-a perfused brain slice H: action potential statistics of cholinergic neurons before and after 200 mu M compound ML-C perfusion brain slice;
Fig. 2: antagonism results of pramipexole, compound ML-A, ML-C, compound ML-B at different concentrations on NMDA receptors; wherein A: schematic of NMDAR-mediated EPSC recording striatal MSNs; b: NMDAR mediated EPSC representative graphs in MSNs before and after 50 μm, 100 μm, 200 μm compound ML-B perfusion brain sheets, respectively, in the upper part, and statistical result graphs, respectively; c: the upper part is a representation graph of NMDAR mediated EPSC in MSNs before and after the pramipexole perfusion brain slice with 200 mu M, and the lower part is a statistical result graph; d: the upper part is a representation of NMDAR mediated EPSC in MSNs before and after the compound ML-A perfusion brain slice of 200 mu M, and the lower part is a statistical result graph E: the upper part is a representation graph of NMDAR mediated EPSC in MSNs before and after 200 mu M pramipexole and 200 mu M compound ML-C perfusion brain slice, and the lower part is a statistical result graph;
fig. 3: time to drug profile for compound ML-B; wherein A: time plot of compound ML-B concentration in brain over time; b: time profile of drug concentration in plasma over time.
Fig. 4: organ paraffin section HE staining pattern after one month of continuous administration of mice (compound ML-B); wherein A: organ map of control mice, B: in the organ diagrams of mice in the administration group, in fig. 4A and 4B, the heart, liver, spleen, lung and kidney are sequentially arranged from left to right.
Fig. 5: a rotating rod experimental result diagram; wherein A: rotating rod experimental schematic diagram, B: and a rotating rod experimental result diagram.
Fig. 6: an open field experimental result diagram; wherein A: a rotating rod experimental track diagram of each group of mice; b: total distance moved in the open field experiments for each group of mice; c: the movement speed of each group of mice in the open field experiment; d: the percentage of immobility time in the open field experiments for each group of mice.
Fig. 7: a black compact TH staining chart; wherein A: immunofluorescence of dopamine neuron tyrosine hydroxylase staining of substantia nigra compact part of each group of mice; b: and (5) a drawing of TH staining statistical results of the dopamine neurons of the substantia nigra compact part of each group of mice.
Fig. 8: a new object identification experiment result diagram; wherein A: new object recognition experimental schematic diagram, B: exploratory behavior statistics graphs for two identical objects for different groups of mice, C: and a diagram of the search behavior statistics of mice of different groups on objects with different colors and shapes.
Fig. 9: an overhead cross maze experimental result diagram; wherein A: overhead cross maze schematic, B: statistics graphs of mice in different groups.
Fig. 10: effect of compound ML-B on striatal cholinergic neuron firing; a: record striatal cholinergic neuron action potential pattern, B: action potentials generated at different injection current intensities for each group of mice represent graphs, C: action potential statistics generated for each group of mice at different injection current intensities.
Fig. 11: effect of compound ML-B on NMDA receptor activity in striatal MSN; a: representative plot of NMDAR-mediated EPSC for each group of mice, B: and (5) counting a result graph.
Fig. 12: compound ML-B effects on striatal synaptic plasticity; a: recording a striatal field potential pattern diagram; b: statistics of the normal group field potential baseline and fEPSP after high frequency stimulation are recorded, C: model group field potential baseline and fEPSP record statistical results plot after high frequency stimulation, D: the dosing group field potential baseline and fEPSP after high frequency stimulation were used to record the statistical plots.
Detailed Description
The following examples will provide those skilled in the art with a more complete understanding of the invention, but are not intended to limit the invention in any way.
Example 1
Synthesis of 1, 6-hydroxyhexyl p-toluenesulfonate
1, 6-Hexanediol (30 mmol,3.54 g), et 3 N (30 mmol) and a catalytic amount of DMAP (4-dimethylaminopyridine) were dissolved in dry CH 2Cl2 (150 mL), tsCl (4-methylsulfonyl chloride, 33 mmol) was added slowly with stirring at 0deg.C, and the reaction was stirred at room temperature for 4h; after the reaction, the reaction was quenched with 50mL of saturated NH 4 Cl solution under vigorous stirring, the reaction solution was extracted with ethyl acetate (3×50 mL), the ethyl acetate extract was washed with saturated brine, dried over anhydrous Na 2SO4, concentrated, diluted with ethyl acetate: petroleum ether=1:1v/V, most of the di-p-toluenesulfonate product was precipitated, filtered, the filtrate was concentrated, and subjected to silica gel column chromatography, and purified with PE: ea=2:1v/V as eluent to give the product (3.12 g, yield 38.2%) identified as 6-hydroxyhexyl p-toluenesulfonate .1H NMR(400MHz,CDCl3)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-toluenesulfonate (11.5 mmmol), memantine hydrochloride (9.55 mmmol) and K 2CO3 (23.8 mmol) were dissolved in DMF (50 mL) and reacted under nitrogen at 80℃with stirring for 48h; the solvent was then evaporated off, extracted with CH 2Cl2 (3X 10 mL), the organic layer was taken and concentrated, and chromatographed on silica gel using CH 2Cl2:CH3 OH:33% ammonia=9:1:0.15V/V/V as eluent to give the product (1.63 g, 61.1% yield), identified as 6- (3, 5-dimethyladamantan-1-yl) amino-1-hexanol .1H NMR(400MHz,CDCl3)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 in30 mL of THF/H 2 O (2/1, v/v), na 2CO3 (14.25 mmol) and (Boc) 2 O (di-tert-butyl dicarbonate, 8.59 mmol) were added and stirred at room temperature overnight; the solvent was evaporated to dryness, dissolved in water and extracted with ethyl acetate, the ethyl acetate extract was collected, washed with saturated brine, dried over anhydrous Na 2SO4, 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), identified as 6- (N-Boc-N- (3, 5-dimethyladamantan-1-yl)) amino-1-hexanol .1H NMR(400MHz,CDCl3)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
0.93G (2.45 mmmol) of 6- (N-Boc-N- (3, 5-dimethyladamantan-1-yl)) amino-1-hexanol, 0.68mL (4.90 mmol) of Et 3 N and a catalytic amount of DMAP are dissolved in 50mL of CH 2Cl2, tsCl is added under stirring at 0 ℃ and the mixture is stirred at room temperature for 3.5h; after the completion of the reaction, the reaction was quenched with saturated aqueous NH 4 Cl, extracted with ethyl acetate (3×50 mL), the ethyl acetate extract was washed with saturated brine, dried over anhydrous Na 2SO4, and the solvent was evaporated to dryness, followed by column chromatography on silica gel using PE: ea=9:1v/V as eluent to give the product (0.717 g, 54.8% yield) identified as 6- (N-Boc-N- (3, 5-dimethyladamantan-1-yl) amino) hexyl p-toluenesulfonate .1H NMR(400MHz,CDCl3)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, 0.31mL (2.24 mmol) of Et 3 N were dissolved in 12mM MECN (acetonitrile), and after nitrogen substitution, the mixture was stirred at 80℃for 48 hours; the solvent was evaporated and the residue was subjected to silica gel column chromatography using EA: meoh=20:1V/V as eluent to give the product (0.20 g, 31.3% yield) identified as tert-butyl (6- (((S) -2-amino-4, 5,6, 7-tetrahydrobenzo [ d ] thiazol-6-yl) (propyl) amino) hexyl) (3, 5-dimethyladamantan-1-yl) carbamate (noted Pu-C6-Me(Boc)).1H NMR(400MHz,CDCl3)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)
Dripping 2.6mL of 4M HCl dioxane solution into 0.2g (0.35 mmol) of Pu-6C-Me (Boc) at 0 ℃, stirring for 2h at room temperature, evaporating the solvent, purifying by high performance liquid chromatography, lyophilizing, converting into hydrochloride to obtain 0.14g of product with yield 84.8％.1H NMR(400MHz,DMSO-d6)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
Prepared as in example 1.1 starting from 1, 4-butanediol, replacing 1, 6-hexanediol with an equimolar amount of 1, 4-butanediol, purifying by column chromatography on silica gel (eluent ethyl acetate: petroleum ether=1:1V/V) to give the product (yield 26.9%), identified as 4-hydroxybutyl-p-toluenesulfonate .1H NMR(400MHz,CDCl3)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
From 4-hydroxybutyl-p-toluenesulfonate, 6- (3, 5-dimethyladamantan-1-yl) amino-1-hexanol was replaced by an equimolar amount of 4-hydroxybutyl-p-toluenesulfonate, 4- (3, 5-dimethyladamantan-1-yl) amino-1-butanol was prepared in the same manner as in example 1.2, yield 25.2％.1H NMR(400MHz,CDCl3)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
From 4- (3, 5-Dimethyladamantan-1-yl) amino-1-butanol, 6- (3, 5-Dimethyladamantan-1-yl) amino-1-hexanol was replaced with an equimolar amount of 4- (3, 5-Dimethyladamantan-1-yl) amino-1-butanol, and 4- (N-Boc-N- (3, 5-Dimethyladamantan-1-yl)) amino-1-butanol was prepared in the same manner as in example 1.3, yield 35％.1H NMR(400MHz,CDCl3)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
From the product of example 2.3, 6- (N-Boc-N- (3, 5-dimethyladamantan-1-yl)) amino-1-hexanol was replaced with an equimolar amount of 4- (N-Boc-N- (3, 5-dimethyladamantan-1-yl)) amino-1-butanol, 4- (N-Boc-N- (3, 5-dimethyladamantan-1-yl) amino) butyl p-toluenesulfonate was prepared in the same manner as in example 1.4, yield 43％.1H NMR(400MHz,CDCl3)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))
From the product of example 2.4, 6- (N-Boc-N- (3, 5-dimethyladamantan-1-yl) amino) hexyl p-toluenesulfonate was replaced with an equimolar amount of 4- (N-Boc-N- (3, 5-dimethyladamantan-1-yl) amino) butyl p-toluenesulfonate, tert-butyl (3, 5-dimethyladamantan-1-yl) carbamate (denoted Pu-C4-Me (Boc)) was prepared as in example 1.5 in yield 68.3％.1H NMR(400MHz,CDCl3)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)
Starting from the product of example 2.5, pu-C6-Me (Boc) was replaced with an equimolar amount of Pu-C4-Me (Boc), prepared as in example 1.6 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％.1H NMR(400MHz,DMSO-d6)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
Prepared as in example 1.1 starting from ethylene glycol and substituting an equimolar amount of ethylene glycol for 1, 6-hexanediol, purified by column chromatography on silica gel (eluent ethyl acetate: petroleum ether=1:1v/V) to give the product in 35% yield, identified as (2-hydroxyethoxy) ethyl p-toluenesulfonate .1H NMR(400MHz,CDCl3)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 and substituting an equimolar amount of (2-hydroxyethoxy) ethyl-p-toluenesulfonate for 6- (3, 5-dimethyladamantan-1-yl) amino-1-hexanol to give 2- (2- (3, 5-dimethyladamantan-1-yl) amino) -ethoxyethanol in yield 61.0％.1H NMR(400MHz,CDCl3)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
Starting from the product of example 3.2, 6- (3, 5-dimethyladamantan-1-yl) amino-1-hexanol was replaced with an equimolar amount of 2- (2- (3, 5-dimethyladamantan-1-yl) amino) -ethoxyethanol, prepared as in example 1.3 to give 2- (2- (N-Boc-N- (3, 5-dimethyladamantan-1-yl)) amino) ethoxyethanol in yield 43％.1H NMR(400MHz,CDCl3)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
Starting from the product of example 3.3, 6- (N-Boc-N- (3, 5-dimethyladamantan-1-yl)) amino-1-hexanol was replaced with an equimolar amount of 2- (2- (N-Boc-N- (3, 5-dimethyladamantan-1-yl)) amino) ethoxy) ethanol, prepared as in example 1.4 to give 2- (2- (N-Boc-N- (3, 5-dimethyladamantan-1-yl)) amino) ethoxy) ethyl p-toluenesulfonate in yield 68.3％.1H NMR(400MHz,CDCl3)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) (prop-yl) amino) ethoxy) ethyl) (3, 5-dimethyladamantan-1-yl) carbamate (Pu-3O-Me (Boc))
From the product of example 3.4, 6- (N-Boc-N- (3, 5-dimethyladamantan-1-yl) amino) hexyl p-toluenesulfonate was replaced with an equimolar amount of 2- (2- (N-Boc-N- (3, 5-dimethyladamantan-1-yl) amino) ethoxy) ethyl p-toluenesulfonate, prepared as in example 1.5 to give 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 (designated Pu-3O-Me (Boc)) in the yield 31.6％.1H NMR(400MHz,CDCl3)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)
Starting from the product of example 3.5, pu-C6-Me (Boc) was replaced with an equimolar amount of Pu-3O-Me (Boc), prepared as in example 1.6 to give (6S) -N6- (2- (2- ((3, 5-dimethyladamantan-1-yl) amino) ethoxy) ethyl) -N6-propyl-4, 5,6, 7-tetrahydrobenzo [ d ] thiazole-2, 6-diamine (Pu-3O-Me, compound ML-B), yield 90％.1H NMR(400MHz,DMSO-d6)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 4 D2 receptor agonism assay
1. Reagent preparation
1.1 Sucrose-enriched slicing solution: 40mmol/L NaCl,4.5mmol/L KCl,1.25mmol/L NaH 2PO4,25mmol/L NaHCO3, 10mmol/L glucose, 148.5mmol/L sucrose, 1mmol/L ascorbate, 3mmol/L sodium pyruvate, 3mmol/L inositol, 0.5mmol/L CaCl 2,7mmol/L MgCl2.
1.2 Artificial cerebrospinal fluid ：125mmol/L NaCl,4.5mmol/L KCl,1.25mmol/L NaH2PO4,2.5mmol/L CaCl2,1.3mmol/L MgSO4,25mmol/L NaHCO3,15mmol/L glucose, 15mmol/L sucrose.
1.3 Recording action potential electrode inner liquid: 123mmol/L potassium methanesulfonate ,8mmol/L NaCl,15mmol/L HEPES,0.2mmol/L EGTA,1mmol/L MgCl2·H2O,2mmol/L MgATP,0.3mmol/L Na3GTP,7mmol/L Na2CrPi. osmotically at 290mOsm, pH was adjusted to 7.2 with KOH.
The slice fluid and the artificial cerebrospinal fluid are respectively used for rat brain slice and brain slice incubation, and the intra-electrode fluid is used for recording cell response.
2. Recording of cholinergic neuron current-induced action potentials
Chat-eGFP mice were selected, as shown in FIG. 1A, and the whole cell mode was used with patch clamp current clamp, and at least 5min was observed before recording was started, ensuring that the cells were in a more stable state. GABA current blocking agent (BIC, 10 μm) was added to the perfusate to clamp cholinergic neurons at-70 mV, current was injected, and the number of current-induced action potentials was recorded within 300 ms. Compound ML-B, pramipexole, compound ML-a, and compound ML-C prepared from physiological saline (0.9% NaCl solution) were perfused separately, and the effect on the number of action potentials was observed.
3. Experimental results
The results are shown in FIG. 1, and show that the compound ML-B does not have D2 receptor agonism at a concentration of 50. Mu.M, and has D2 receptor agonism at a concentration of 100. Mu.M, and also has D2 receptor agonism at a concentration of 200. Mu.M, and that the compound ML-B shows better dose dependence. Pramipexole (Prami) has D2 receptor agonism when the concentration reaches 200 μm; the concentrations of the compounds ML-A and ML-C, even if reaching 200. Mu.M, do not have D2 receptor agonism.
EXAMPLE 5 NMDA receptor antagonism assay
1. Reagent preparation
1.1 Sucrose-enriched slicing solution: 40mmol/L NaCl,4.5mmol/L KCl,1.25mmol/L NaH 2PO4,25mmol/L NaHCO3, 10mmol/L glucose, 148.5mmol/L sucrose, 1mmol/L ascorbate, 3mmol/L sodium pyruvate, 3mmol/L inositol, 0.5mmol/L CaCl 2,7mmol/L MgCl2.
1.2 Magnesium free artificial cerebrospinal fluid ：125mmol/L NaCl,4.5mmol/L KCl,1.25mmol/L NaH2PO4,2.5mmol/L CaCl2,25mmol/L NaHCO3,15mmol/L glucose, 15mmol/L sucrose.
1.3 Recording of excitatory postsynaptic currents the intra-electrode fluid ：119mmol/L CsMeSO4,8mmol/L TEACl,15mmol/L HEPES,0.6mmol/L EGTA,0.3mmol/L Na3GTP,4mmol/L MgATP,5mmol/L QX-314.CL,7mmol/L Na2CrPO4. osmotic pressure was 290mOsm, and the pH was adjusted to 7.2 with CsOH.
Recording of nmda receptor mediated excitatory postsynaptic currents
And a patch clamp voltage clamp whole cell recording mode is adopted, and before recording is started, at least 5 minutes are observed, so that the cells are ensured to be in a relatively stable state. GABA current blocking agent (BIC, 10 mu M) and AMPA receptor blocking agent (CNQX, 10 mu M) are added into the perfusion liquid, a stimulating electrode is placed near the stimulating electrode, cells are clamped at-70 mV, NMDA-mediated excitatory postsynaptic currents are recorded, different concentrations of compounds ML-B, pramipexole, ML-A and ML-C prepared from physiological saline are respectively perfused, and the influence of the compounds on the NMDA-mediated excitatory postsynaptic current amplitude is observed.
3 Results of experiments
As a result, as shown in FIG. 2, it was revealed that the compound ML-B had no NMDA receptor antagonism at 50. Mu.M, and had NMDA receptor antagonism at a concentration of 100. Mu.M, and also showed NMDA receptor antagonism at a concentration of 200. Mu.M, and that the compound ML-B showed a good dose-dependence. Pramipexole, ML-a, showed no significant NMDA receptor antagonism at a concentration of 200 μm, whereas compound ML-C, at a concentration of 200 μm, showed complete NMDA receptor antagonism.
As can be seen from the results of the D2 receptor agonism experiments in combination with example 4, the compound ML-B has both NMDA receptor antagonism and D2 receptor agonism.
EXAMPLE 6 experiments on the concentration measurement of Compound ML-B in plasma and brain homogenates
1. Solution preparation
1.1 Preparation of standard solutions of compounds: accurately weighing compound ML-B2.0 mg in a 10mL volumetric flask, dissolving with methanol to constant volume, preparing into 0.2mg/mL standard solution, and preserving at 4deg.C.
1.2 Preparation of internal standard: accurately weighing 4.15mg fluoxetine in a 10mL volumetric flask, dissolving with methanol to constant volume, and preparing into a standard solution with the concentration of 415 g/mL. Taking 0.1mL of standard solution in a 10mL volumetric flask, and fixing the volume by methanol to prepare the standard solution with the concentration of 4.15g/mL as an internal standard.
1.3 Preparation of dosing solution: accurately weighing compound ML-B4.8 mg, and adding 19.2mL of physiological saline for dissolution.
2. Detection conditions:
agilent 1200 series liquid phase system, using a Sunfire pretreatment C 18 column (4.6 mm. Times.250 mm), mobile phase 0.1% formic acid water (mobile phase A): methanol (mobile phase B) =80:20, V/V) flow rate 0.25mL/min.
3. Plasma pretreatment
100 Mu L of blank plasma is placed in a 1.5mL centrifuge tube, 10 mu L of internal standard is added, 300 mu L of precipitated protein of mixed solvent of methanol and acetonitrile=1:1V/V is added, the mixture is mixed and spun for 1min on a vortex machine, the mixture is centrifuged for 10min in a centrifuge at 12000r/min, and 10 mu L of supernatant is taken for sample injection.
4. Drawing of a Standard Curve
Blank plasma was prepared from standard compound solutions to give standard blank plasma series samples corresponding to compound ML-B plasma concentrations of 0.05, 0.1, 0.2, 1, 2.5, 5 μg/ML, and were treated under the "plasma pretreatment" term and measured under the "detection conditions" term. The chromatographic peak areas of the plasma sample and the internal standard are recorded, the concentration of the compound ML-B is taken as an abscissa, the peak area ratio (the ratio of the compound to the internal standard peak area) is taken as an ordinate, linear regression operation is carried out, the linear regression equation is obtained to be y= 1.2182x-0.1107, the r 2 value is 0.9943, and the result shows that the compound ML-B has good linear relation with the peak area ratio in the range of 0.05-5 mug/mL in the plasma.
5. Brain tissue pretreatment
The mice were rapidly sacrificed, brain tissues were dissected and removed, blood stains on the tissue surface were rinsed clean with normal saline, the tissue surface was blotted dry with filter paper, weighed, a fixed amount of normal saline (1 g of tissue was added with 3mL of normal saline) was added, and the tissue was homogenized with an electric homogenizer, and air bubbles were removed by ultrasound for 10 min.
100 Μl of blank brain tissue is placed in a 1.5mL centrifuge tube, 10 μl of internal standard is added, 300 μl of precipitated protein of mixed solvent of acetonitrile=1:1v/V is added, vortex is carried out on a vortex instrument for 1min, centrifuge is carried out for 10min at 12000r/min, and 10 μl of supernatant is sampled.
6. Drawing of brain homogenate standard curve
Taking 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 under the item of brain tissue pretreatment, measuring according to the item of detection conditions, recording chromatographic peak areas of a brain homogenate sample and an internal standard, carrying out linear regression operation by taking the compound concentration as an abscissa and the peak area ratio as an ordinate, obtaining a linear regression equation of y=0.0035x+0.0191 and r 2 value of 0.9931, and showing that the compound ML-B has good linear relation with the peak area ratio in the range of 10-600 ng/g in brain homogenate.
7. Abdominal administration experiment of mice
Mice were taken at 8-10 weeks of age, weighing 20-30g, randomly allocated to each time point, 2 per time point, and injected intraperitoneally with compound ML-B (formulated with physiological saline) at a dose of 2.5mg/kg. The mice are fasted and not water-forbidden for 12 hours, water is freely drunk during the test period, blood is taken from the orbital venous plexus respectively at 5min, 10min, 30min, 60min, 90min, 180min and 240min after administration, a plasma sample is placed in a centrifuge tube which is treated by heparin in advance, then the mice are sacrificed, and brain tissues are dissected and taken out; centrifuging the plasma sample in a centrifuge at 12000r/min for 10min, treating the plasma sample according to the 'plasma pretreatment' item, treating brain tissue according to the 'brain tissue pretreatment' item, analyzing the treated sample according to the analysis condition under the 'detection condition' item, substituting the ratio of the areas of the compound ML-B and the internal standard peak into a corresponding standard curve to obtain the plasma drug concentration and the brain tissue drug concentration of each time point of the compound ML-B, drawing a line graph, and calculating corresponding pharmacokinetic parameters.
TABLE 1 pharmacokinetic parameters of Compound ML-B in plasma
t1/2(min) 521.78
Tmax(min) 5
Cmax(ng/mL) 3268.57
The results are shown in Table 1 and FIG. 3, which demonstrate that the compound ML-B can exert therapeutic effects across the blood brain barrier.
EXAMPLE 7HE staining experiments
The experimental steps are as follows: C57/B6 mice (8-10 weeks old, 20-30 g) are randomly divided into a control group and an administration group; the compound ML-B (prepared into a solution with the concentration of 0.025% by adopting normal saline) is continuously injected into the abdominal cavity of the administration group for one month, the dosage is 2.5mg/kg, and the dosage is converted into 0.1mL/10 g; the control group was continuously intraperitoneally injected with physiological saline for one month, and the amount of physiological saline was converted according to the weight of the mice. After one month, the heart, liver, spleen, lung and kidney of the mice are taken after being perfused, respectively soaked in 10% neutral formalin solution, and then paraffin sections and HE staining are carried out, and then shooting is carried out.
The HE staining results of 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 important organs of mice.
Example 8
Establishment of mouse MPTP model
1. Animals: C57/B6 mice (8-10 weeks old, 20-30 g). Maintaining indoor temperature 22+ -2deg.C and illumination (12 h light and shade period), and optionally obtaining food and water.
2. Preparing a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine, MPTP) solution: the saline is used as solvent to prepare MPTP solution with concentration of 0.6%, and the administration volume is 0.05mL/10g, namely, the administration dosage is 30mg/kg.
3. Evaluation results: after MPTP injection, mice developed symptoms such as salivation, bow back, tremor of extremities.
4. The administration mode is as follows: mice were randomly divided into model group (MPTP), dosing group, positive drug group (Prami) and normal group (Ctrl); the mice in the model group are intraperitoneally injected with MPTP solution with concentration of 0.6 percent, the administration volume is 0.05mL/10g, namely, the administration dose is 30mg/kg, physiological saline is intraperitoneally injected after one hour, the administration volume is 0.05mL/10g, and the same operation is carried out for five continuous days; the mice in the administration group are intraperitoneally injected with MPTP solution with concentration of 0.6 percent, the administration volume is 0.05mL/10g, namely, the administration dosage is 30mg/kg, the same volume of ML-B solution with different concentrations (prepared by physiological saline, the concentration of the compound ML-B is 0.05 percent, the administration dosage is 2.5mg/kg, the concentration of the compound ML-B is 0.02 percent, the administration dosage is 1.0mg/kg, the concentration of the compound ML-B is 0.008 percent, the administration dosage is 0.4 mg/kg) is intraperitoneally injected after one hour, and the same operation is carried out continuously for five days; the positive drug group mice are intraperitoneally injected with MPTP solution with concentration of 0.6%, the administration volume is 0.05mL/10g, namely, the administration dosage is 30mg/kg, and the same volume of pramipexole solution (prepared by physiological saline, and the pramipexole concentration is 0.02% and the administration dosage is 1 mg/kg) is intraperitoneally injected after one hour, and the same operation is carried out for five consecutive days; normal mice were injected twice daily with normal saline, the two dosing times differing by one hour, each dosing volume being 0.05mL/10g, for five consecutive days with the same procedure.
Rotating rod experiment
The experimental steps are as follows: the mice were placed on a rotarod apparatus as shown in fig. 5A after molding and administration. The rod rotating instrument is set to increase the rotating speed from 5rpm/min to 40rpm/min at a constant speed, the rod time of the mice is recorded, and the rod residence time of each group of mice is compared.
The experimental results are shown in fig. 5B, which shows that: compound ML-B can prolong the on-stick residence time of MPTP model mice, and compound ML-B shows better dose correlation; compared with pramipexole, which is a positive drug, the compound ML-B has no obvious difference in effect on mice in the improvement model group.
Open field experiment
The experimental steps are as follows: the mice after the mould making and the drug administration are taken and placed in an open field box (40 cm multiplied by 40 cm), and the total movement distance, the movement speed, the percentage of the immobility time (speed less than 0.2 cm/s) and the movement track of each group of mice within 5min are recorded by using the behavioural experiment software.
The experimental results are shown in fig. 6, and fig. 6A, 6B, 6C and 6D are respectively a track diagram, a total movement distance, a movement speed and a percentage of immobility time of each group of mice, which indicate that: the compound ML-B can increase the total movement distance and movement speed of the MPTP model mice, and reduce the immobility time percentage of the mice; compared with pramipexole, which is a positive drug, the compound ML-B has no obvious difference in effect on mice in the improvement model group.
Immunofluorescence staining experiment of dopamine neurons in substantia nigra compact part
The experimental steps are as follows: after the behavioural investigation is finished, the mouse heart is perfused with 20mL of PBS solution and 40mL of 4% paraformaldehyde solution, then the head is broken, the brain is taken, the mouse brain is placed in the 4% paraformaldehyde solution for fixation overnight, the next day is changed into 30% sucrose solution for sugar precipitation for two days, a black compact part with the thickness of 20 mu m is frozen and sectioned after embedding by embedding agents, a patch is attached, the subsequent immunofluorescence staining of Tyrosine Hydroxylase (TH) is carried out, after the sealing of a glycerograp tablet containing dapi for resisting fluorescence quenching is adopted, the tablet is taken under a 10-time fluorescence microscope, and the number of TH staining positive cells is recorded.
The experimental results are shown in fig. 7, the dark place in the fluorescent chart is a background, the bright round dot is a TH positive cell, namely a dopamine neuron, and the area outlined by the dotted line is a mouse substantia nigra compact part. The more bright dots within the fluorescence framed area, i.e. the greater the number of dopamine neurons. The results show that: when the administration dosage of the compound ML-B is 2.5mg/kg, the damage of the nigra compact dopamine neurons of the MPTP model mice can be relieved, the reduction speed of the number of the dopamine neurons is reduced, and the neuroprotection effect is achieved; compound ML-B was not statistically different from pramipexole.
Example 9
New object identification experiment
The experimental steps are as follows: mice were randomly divided into model group (MPTP), dosing group, positive drug group (Prami) and normal group (Ctrl). The mice in the model group are intraperitoneally injected with MPTP solution with concentration of 0.6 percent, the administration volume is 0.05mL/10g, namely, the administration dose is 30mg/kg, physiological saline is intraperitoneally injected after one hour, the administration volume is 0.05mL/10g, and the same operation is carried out for five continuous days; the mice in the administration group are intraperitoneally injected with MPTP solution with concentration of 0.6%, the administration volume is 0.05mL/10g, namely, the administration dose is 30mg/kg, the same volume of ML-B solution (prepared by physiological saline, the concentration of the compound ML-B is 0.05 percent, the administration dose is 2.5 mg/kg) is intraperitoneally injected after one hour, and the same operation is carried out for five consecutive days; the positive drug group mice are intraperitoneally injected with MPTP solution with concentration of 0.6%, the administration volume is 0.05mL/10g, namely, the administration dosage is 30mg/kg, the equal volume of positive drug pramipexole solution (prepared by physiological saline, the pramipexole concentration is 0.02%, the administration dosage is 1 mg/kg) is intraperitoneally injected after one hour, and the same operation is carried out for five consecutive days; normal mice were injected twice daily with normal saline, the two dosing times differing by one hour, each dosing volume being 0.05mL/10g, for five consecutive days with the same procedure.
New object recognition experiments are generally divided into adaptation phase, familiarity phase and testing phase. At the time of adaptation period, the mice were placed in an experimental box (40X 40 cm), the mice were familiar with the environment. The following day of the adaptation phase, familiarity phase and test phase experiments were performed on the mice. As shown in fig. 8A, two identical objects are placed in the experiment box during the familiarity period, and the exploratory behaviors of the mice on the two objects within 10min are recorded; after 60min, a test period experiment is carried out, one of the two identical objects is replaced by an object with different colors and shapes, the position is unchanged, the exploration time of the mice for a familiar object (Familiar) and a Novel object (Novel) within 5min is recorded, and the learning and memory capacity of the mice is evaluated by using a discrimination index (discrimination index, DI).
The experimental results are shown in fig. 8B and 8C, which show that: compared with the mice in the model group, the time for exploring a new object by the mice in the compound ML-B group is obviously increased, which shows that the compound ML-B improves the cognitive dysfunction of the mice in the MPTP model, and the positive drug pramipexole has no obvious improvement effect on the cognitive dysfunction of the mice in the MPTP model.
Overhead maze test
The experimental steps are as follows: as shown in fig. 9A, the elevated plus maze generally comprises two opposing open arms (open arms) and two opposing closed arms (closed arms), the maze being 50cm high from the ground. And installing a camera right above the maze, placing the mouse at the end of the open arm far away from the central area and facing away from the central area, recording the time from the open arm to the first time the mouse enters the closed arm, and continuously measuring twice, wherein the interval time is 24h each time. The index for evaluating learning memory in mice is the ratio of the time to two passes from open arm to closed arm (TRANSFER LATENCY, TL).
The experimental results are shown in fig. 9B, which shows that: compared with the mice in the model group, the time for the compound ML-B group mice 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 positive drug pramipexole has no obvious improvement effect on the cognitive dysfunction of the mice in the MPTP model.
Example 10
Striatal cholinergic neuron spontaneous discharge and current evoked action potential recording experiment
1. Reagent preparation
1.1 Sucrose-enriched slicing solution: 40mmol/L NaCl,4.5mmol/L KCl,1.25mmol/L NaH 2PO4,25mmol/L NaHCO3, 10mmol/L glucose, 148.5mmol/L sucrose, 1mmol/L ascorbate, 3mmol/L sodium pyruvate, 3mmol/L inositol, 0.5mmol/L CaCl 2,7mmol/L MgCl2.
1.2 Artificial cerebrospinal fluid ：125mmol/L NaCl,4.5mmol/L KCl,1.25mmol/L NaH2PO4,2.5mmol/L CaCl2,1.3mmol/L MgSO4,25mmol/L NaHCO3,15mmol/L glucose, 15mmol/L sucrose.
1.3 Recording action potential and self-discharge electrode inner liquid: 123mmol/L potassium methanesulfonate ,8mmol/L NaCl,15mmol/L HEPES,0.2mmol/L EGTA,1mmol/L MgCl2·H2O,2mmol/L MgATP,0.3mmol/L Na3GTP,7mmol/L Na2CrPi. osmotically at 290mOsm, pH was adjusted to 7.2 with KOH.
2. Recording of cholinergic neuron spontaneous discharge and current-induced action potential
Mice after the end of the rod-rotating experiment and the open field experiment were used and divided into: control group (two intraperitoneal injections of saline), model group (one intraperitoneal injection of MPTP followed by one hour of intraperitoneal injection of equal volume of saline), and administration group (one intraperitoneal injection of MPTP followed by one hour of intraperitoneal injection of 2.5mg/kg of Compound ML-B).
As shown in fig. 10A, before starting recording in patch clamp voltage clamp cell attachment mode, at least 5min was observed to ensure that the cells were in a more stable state. GABA receptor blocker (BIC, 10 mu M) is added into the perfusion liquid, cells are clamped at-65 mV, current is gradually injected in a current clamp mode, and the number of current-induced action potentials under different injection currents is recorded.
3. Experimental results
The results are shown in fig. 10B and 10C, which show that: the compound ML-B can reduce the number of action potentials induced by current of the striatal cholinergic neurons of a PD model mouse and reduce the excitability of cholinergic neurons.
Example 11
Test for detecting NMDA receptor Activity of acanthus neurons in striatum
1. Reagent preparation
1.1 Sucrose-enriched slicing solution: 40mmol/L NaCl,4.5mmol/L KCl,1.25mmol/L NaH 2PO4,25mmol/L NaHCO3, 10mmol/L glucose, 148.5mmol/L sucrose, 1mmol/L ascorbate, 3mmol/L sodium pyruvate, 3mmol/L inositol, 0.5mmol/L CaCl 2,7mmol/L MgCl2.
1.2 Magnesium free artificial cerebrospinal fluid ：125mmol/L NaCl,4.5mmol/L KCl,1.25mmol/L NaH2PO4,2.5mmol/L CaCl2,25mmol/L NaHCO3,15mmol/L glucose, 15mmol/L sucrose.
1.3 Recording of excitatory postsynaptic currents the intra-electrode fluid ：119mmol/L CsMeSO4,8mmol/L TEACl,15mmol/L HEPES,0.6mmol/L EGTA,0.3mmol/L Na3GTP,4mmol/L MgATP,5mmol/L QX-314.CL,7mmol/L Na2CrPO4. osmotic pressure was 290mOsm, and the pH was adjusted to 7.2 with CsOH.
Recording of NMDA receptor mediated excitatory postsynaptic current and NMDA evoked current
The mice are randomly divided into a model group (MPTP), a dosing group, a positive drug group (Prami) and a normal group (Ctrl), wherein the model group mice are subjected to intraperitoneal injection of MPTP solution with the concentration of 0.6%, the dosing volume is 0.05mL/10g, namely 30mg/kg of dosing agent, physiological saline is injected into the abdominal cavity after one hour, the dosing volume is 0.05mL/10g, and the same operation is carried out for five continuous days; the mice in the administration group are intraperitoneally injected with MPTP solution with concentration of 0.6%, the administration volume is 0.05mL/10g, namely, the administration dose is 30mg/kg, the same volume of ML-B solution (prepared by physiological saline, the concentration of the compound ML-B is 0.05 percent, the administration dose is 2.5 mg/kg) is intraperitoneally injected after one hour, and the same operation is carried out for five consecutive days; the positive drug group mice are intraperitoneally injected with MPTP solution with concentration of 0.6%, the administration volume is 0.05mL/10g, namely, the administration dosage is 30mg/kg, and the same volume of pramipexole solution (prepared by physiological saline, and the pramipexole concentration is 0.02% and the administration dosage is 1 mg/kg) is intraperitoneally injected after one hour, and the same operation is carried out for five consecutive days; normal mice were injected twice daily with normal saline, the two dosing times differing by one hour, each dosing volume being 0.05mL/10g, for five consecutive days with the same procedure. After the rod rotating experiment and the open field experiment are finished, the mouse brain sections of each group are respectively taken, the section liquid and the artificial cerebrospinal fluid are used for incubating the mouse brain sections and the brain slices, and the electrode internal fluid is used for recording the cell reaction. And a patch clamp voltage clamp whole cell recording mode is adopted, and before recording is started, at least 5 minutes are observed, so that the cells are ensured to be in a relatively stable state. GABA current blocking agent (BIC, 10 μm) was added to the perfusate, AMPA receptor blocking agent (CNQX, 10 μm), stimulating electrode was placed, cells were clamped at-70 mV near the stimulating electrode, five stimulus intensities of 1V, 2V, 3V, 4V, 5V were sequentially given, and NMDAR-mediated excitatory postsynaptic currents were recorded in control, model, dosing, positive drug.
3. Experimental results
The results are shown in fig. 11, which shows that: the magnitude of NMDAR-mediated excitatory postsynaptic current change recorded in compound ML-B at the same stimulus intensity was less than that recorded in PD model mice at the same stimulus intensity, reducing aberrant NMDAR excitatory synaptic transmission in PD model mice.
Example 12
Striatal field potential recording experiments
1. Reagent preparation
1.1 Sucrose-enriched slicing solution: 40mmol/L NaCl,4.5mmol/L KCl,1.25mmol/L NaH 2PO4,25mmol/L NaHCO3, 10mmol/L glucose, 148.5mmol/L sucrose, 1mmol/L ascorbate, 3mmol/L sodium pyruvate, 3mmol/L inositol, 0.5mmol/L CaCl 2,7mmol/L MgCl2.
1.2 Artificial cerebrospinal fluid ：125mmol/L NaCl,4.5mmol/L KCl,1.25mmol/L NaH2PO4,2.5mmol/L CaCl2,1.3mmol/L MgSO4,25mmol/L NaHCO3,15mmol/L glucose, 15mmol/L sucrose.
1.3 Recording intraelectrode fluids for striatal field potentials: 1mol/L NaCl.
2. Recording of postsynaptic potential of striatal excitability
Mice were randomly divided into model group (MPTP), dosing group and normal group (Ctrl); the mice in the model group are intraperitoneally injected with MPTP solution (prepared by normal saline) with concentration of 0.6 percent, the administration volume is 0.05mL/10g, namely, the administration dose is 30mg/kg, the normal saline is intraperitoneally injected after one hour, the administration volume is 0.05mL/10g, and the same operation is carried out for five continuous days; the mice in the administration group are intraperitoneally injected with MPTP solution with concentration of 0.6%, the administration volume is 0.05mL/10g, namely, the administration dose is 30mg/kg, the same volume of ML-B solution (prepared by physiological saline, the concentration of the compound ML-B is 0.05 percent, the administration dose is 2.5 mg/kg) is intraperitoneally injected after one hour, and the same operation is carried out for five consecutive days; normal mice were injected twice daily with normal saline, the two dosing times differing by one hour, each dosing volume being 0.05mL/10g, for five consecutive days with the same procedure. After the rod rotating experiment and the open field experiment are finished, the mouse brain sections of each group are respectively taken, the section liquid and the artificial cerebrospinal fluid are used for incubating the mouse brain sections and the brain slices, and the electrode internal fluid is used for recording the cell reaction. Brain patch clamp experiments were performed, GABA receptor antagonists (BIC, 10. Mu.M) were added to the perfusate, and recorded in patch clamp current clamp mode, with the stimulating electrode placed in the cortex, the glass recording electrode placed in the striatum, and the field excitatory postsynaptic potential of the cortex-striatal pathway (field excitatory postsynaptic potential, fEPSP) recorded. The stimulus intensity is adjusted first so that fEPSP is within a proper range. Long-term inhibition (LTD) of fEPSP was induced by high frequency stimulation after 10min baseline was recorded steadily, and recording was continued for 45min after the end of the induction stimulation. The magnitude of fEPSP after high frequency stimulation is compared to the baseline magnitude.
3. Experimental results
The results are shown in fig. 12, which shows that: in MPTP model mice, the magnitude of fEPSP in the cortical-striatal pathway was not significantly changed by high frequency stimulation, and long-term inhibition (LTD) could not be induced. The compound ML-B can restore the synaptic plasticity damage condition in MPTP model mice and improve the overall output function of striatum.

Claims (4)

1. A compound of the formula:
2. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein: pharmaceutically acceptable salts are hydrochloride, hydrobromide, nitrate, perchlorate, phosphate, sulfate, formate, acetate, aconate, ascorbate, benzenesulfonate, benzoate, cinnamate, citrate, heptanoate, fumarate, glutamate, glycolate, lactate, maleate, malonate, mandelate, methanesulfonate, naphthalene-2-sulfonate, phthalate, salicylate, sorbate, stearate, succinate, tartrate or p-toluenesulfonate.
3. The use of a compound of claim 1, or a pharmaceutically acceptable salt thereof, for the preparation of a D2 receptor agonist and an NMDA receptor antagonist.
4. The use of a compound of claim 1 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the prevention and treatment of parkinson's disease.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009056805A1 (en) * 2007-10-31 2009-05-07 Motac Neuroscience Limited Medicaments

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Publication number Priority date Publication date Assignee Title
WO2009056805A1 (en) * 2007-10-31 2009-05-07 Motac Neuroscience Limited Medicaments

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Title
Novel multifunctional dopamine D2/D3 receptors agonists with potential neuroprotection and anti-alpha synuclein protein aggregation properties;Dan Luo et al.;《Bioorganic & Medicinal Chemistry》;1-46 *
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;张丽云 等;《Guizhou Medicinal Journal》;第44卷(第12期);1960-1961 *

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