CN112341404B - Thiazole derivative or salt, isomer, preparation method and application thereof - Google Patents

Thiazole derivative or salt, isomer, preparation method and application thereof Download PDF

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CN112341404B
CN112341404B CN202010772179.8A CN202010772179A CN112341404B CN 112341404 B CN112341404 B CN 112341404B CN 202010772179 A CN202010772179 A CN 202010772179A CN 112341404 B CN112341404 B CN 112341404B
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ethyl
morpholine
oxy
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thiazol
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CN112341404A (en
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曾燕群
鄢胜勇
马云龙
张涛
王颖
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Chengdu Easton Biopharmaceuticals Co Ltd
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    • C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members 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 to ring carbon atoms
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Abstract

Novel thiazole derivatives or salts, isomers, preparation methods and applications thereof. A compound of formula (I) having Sigma1 receptor antagonism and strong affinity for Sigma1R in vitro in terms of in vitro receptor affinity. Meanwhile, in the analysis of D2L radioligand binding and cAMP test of 5HT-1A receptor, CB1 receptor and CB2 receptor, the affinity of the ligand is poor for the receptors, and the ligand shows higher receptor selectivity. Through a mouse acetic acid torsion test, the compound has an analgesic effect. The compound has long half-life, low clearance rate and large AUClast through a rat oral medicine pharmacokinetic experiment; can reach Cmax after oral administration, has very fast absorption, and is suitable for medicinal use.

Description

Thiazole derivative or salt, isomer, preparation method and application thereof
Technical Field
The invention relates to the field of pharmaceutical chemistry, in particular to application of thiazole derivatives or salts and isomers thereof, a preparation method thereof and a pharmaceutical composition thereof in preparation of treatment and prevention, especially application in treatment of pain, drug addiction, opioid synergism, psychosis and the like.
Background
At present, the incidence rate of the world pain is about 35-45%, the incidence rate of the elderly is higher, about 75-90%, the pain is the third treatment field in global ranking, and the market is huge. Opioids are the strongest and most commonly used central analgesics at present, and past studies have shown that opioids (e.g., codeine, morphine, oxycodone, fentanyl) can effectively relieve pain. They bind to opioid receptors in the brain, spinal cord and gastrointestinal tract to reduce pain. Just because of the remarkable analgesic effect of opioids, abuse thereof has become a major public health problem in the united states; 78 people die each day due to opioid overdose. Therefore, there is a great clinical need for potent analgesics with new targets.
Sigma receptors are unique non-opioid receptors and are widely distributed in the central nervous system, and Sigma1 receptors are associated with the pathophysiology of all major central nervous system diseases, including mood disorders (anxiety and depression), psychosis and schizophrenia, as well as drug addiction and pain. Sigma1 receptor antagonists may also enhance the analgesic activity of opioid analgesics. The invention provides a Sigma1 receptor antagonist with a novel structure.
Disclosure of Invention
The present invention provides a novel compound which has Sigma1 receptor antagonism and which has strong affinity for Sigma1R in vitro in terms of in vitro receptor affinity. Meanwhile, in the analysis of D2L radioligand binding and cAMP test of 5HT-1A receptor, CB1 receptor and CB2 receptor, the affinity of the ligand is poor for the receptors, and the ligand shows higher receptor selectivity. Through a mouse acetic acid torsion test, the compound has an analgesic effect. And through the experiment of oral pharmacokinetics of rats, the compound of the invention has long half-life period, low clearance rate and AUC last Large; can reach C after oral administration max The absorption is extremely fast, and the medicine is suitable for medicines.
The invention provides a compound shown as a formula I or stereoisomers, geometric isomers, tautomers, oxynitride compounds, hydrates, solvent compounds, metabolites, pharmaceutically acceptable salts or prodrugs of the compound shown as the formula I,
Figure SMS_1
R 1 and R is 4 Independently selected from hydrogen, substituted or unsubstituted C 1-12 Alkyl, substituted or unsubstituted C 3-12 Cycloalkyl, substituted or unsubstitutedC 2-12 Alkenyl, substituted or unsubstituted C 5-12 Aryl, substituted or unsubstituted C 5-12 Heterocyclyl, substituted or unsubstituted alkoxy, substituted or unsubstituted alkylthio, substituted or unsubstituted amino, or halogen;
R 2 and R is 3 Independently selected from hydrogen, deuterium, methyl, ethyl, propyl, butyl, substituted or unsubstituted arylmethyl, substituted or unsubstituted arylethyl, substituted or unsubstituted arylheteromethyl, substituted or unsubstituted arylheteroethyl, or R 2 And R is 3 Taken together with the nitrogen atom to which they are attached form a substituted or unsubstituted morpholinyl, substituted or unsubstituted piperazinyl, substituted or unsubstituted pyrrolidinyl, substituted or unsubstituted piperidinyl, substituted or unsubstituted hexahydropyrimidinyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted thiomorpholinyl, substituted or unsubstituted azacyclooctyl, substituted or unsubstituted azepanyl;
wherein the substituents are selected from one or more of deuterium, halogen, hydroxy, C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, C 1 -C 6 Alkylthio, amino, C 1 -C 6 Alkylamino, C 1 -C 6 Alkanoylamino, C 5 -C 10 Aryl, C 5 -C 10 Aralkyl, C 5 -C 10 Heterocyclyl, NO 2 、CN、CF 3 Or they may be taken together to form a fused ring system.
Further, the compounds of the present invention have the structure shown in formula I':
Figure SMS_2
R 1 and R is 4 Independently selected from the group consisting of hydrogen, methyl, substituted or unsubstituted cyclopropyl, substituted or unsubstituted cyclohexyl, substituted or unsubstituted cyclopentyl, substituted or unsubstituted phenyl, substituted or unsubstituted pyridyl, substituted or unsubstituted pyrimidinyl, substituted or unsubstituted thienyl, substituted or unsubstituted thiazolyl, substituted or unsubstituted pyridinyl, substituted or unsubstituted pyrimidinyl, substituted or unsubstituted thiophenyl, substituted or unsubstituted thiazolyl, substituted or unsubstituted thiophenyl, and substituted or unsubstituted thiophenyl,Substituted or unsubstituted pyrrolyl, substituted or unsubstituted furan ring, substituted or unsubstituted naphthyl, substituted or unsubstituted quinolinyl, substituted or unsubstituted dibenzothiophenyl, substituted or unsubstituted propyl, substituted or unsubstituted allyl, substituted or unsubstituted benzothiophenyl, substituted or unsubstituted furanyl, substituted or unsubstituted benzofuranyl;
wherein the substituents are selected from one or more of deuterium, halogen, hydroxy, methyl, ethyl, cyclopropyl, t-butyl, methoxy, ethoxy, cyclopropyloxy, t-butoxy, methylthio, ethylthio, cyclopropylthio, t-butylthio, amino, methylamino, ethylamino, cyclopropylamino, t-butylamino, carboxamido, acetamido, cyclopropylamido, t-butyrylamino, NO 2 、CN、CF 3 。
Further, R 1 Selected from the group consisting of hydrogen, substituted or unsubstituted phenyl, substituted or unsubstituted naphthyl, substituted or unsubstituted biphenyl, substituted or unsubstituted benzothienyl, substituted or unsubstituted thienyl, substituted or unsubstituted furyl, substituted or unsubstituted benzofuranyl, and substituted or unsubstituted pyrazolyl;
R 4 selected from hydrogen, methyl, substituted or unsubstituted phenyl;
wherein the substituents are selected from one or more of deuterium, halogen, hydroxy, methyl, ethyl, cyclopropyl, t-butyl, methoxy, ethoxy, cyclopropyloxy, t-butoxy, methylthio, ethylthio, cyclopropylthio, t-butylthio, amino, methylamino, ethylamino, cyclopropylamino, t-butylamino, carboxamido, acetamido, cyclopropylamido, t-butyrylamino, NO 2 、CN、CF 3 。
Further, R 1 Selected from the group consisting of hydrogen, 4-trifluoromethoxyphenyl, 2-methoxyphenyl, 3, 4-dichlorophenyl, 2-chlorophenyl, 4-fluorophenyl, 2-methyl-4-fluorophenyl, 3-methoxyphenyl, biphenyl-4-yl, 4-cyanophenyl, 2-methylthiophenyl, 3, 4-difluorophenyl, 3, 5-difluorophenyl, 4-methoxyphenyl, 3-trifluoromethoxyphenyl, 4-fluoro-2-chlorophenyl, 2-methoxy-4-chlorophenyl, 2-tritolylFluoromethyl-4-chlorophenyl, 2-methoxy-4-fluorophenyl, 2, 4-dichlorophenyl, 2, 4-difluorophenyl, 2-trifluoromethyl-4-fluorophenyl, 3, 4-bis (trifluoromethyl) phenyl, furyl, benzofuryl, benzothienyl, thienyl, 1, 3-dimethyl-1H-pyrazol-4-yl;
R 4 selected from the group consisting of hydrogen, methyl, 2-naphthyl, 3, 4-difluorophenyl, phenyl, 3, 4-dichlorophenyl, 3, 5-difluorophenyl, 4-fluorophenyl, 2-methyl-4-fluorophenyl, 4-methoxyphenyl.
Further, preferred are the following compounds or stereoisomers, geometric isomers, tautomers, oxynitride compounds, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs thereof:
Figure SMS_3
Figure SMS_4
further, preferred are the following compounds or stereoisomers, geometric isomers, tautomers, oxynitrides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs thereof:
4- {2- { [5- (naphthalen-2-yl) thiazol-2-yl ] oxy } ethyl } morpholine;
4- {2- { [ 5-phenylthiazol-2-yl ] oxy } ethyl } morpholine;
4- {2- { [5- (3, 4-dichlorophenyl) thiazol-2-yl ] oxy } ethyl } morpholine;
4- {2- { [5- (3, 5-difluorophenyl) thiazol-2-yl ] oxy } ethyl } morpholine;
4- {2- { [5- (4-methoxyphenyl) thiazol-2-yl ] oxy } ethyl } morpholine;
4- {2- { {4- [4- (trifluoromethoxy) phenyl ] thiazol-2-yl } oxy } ethyl } morpholine;
4- {2- { [4- (2-methoxyphenyl) thiazol-2-yl ] oxy } ethyl } morpholine;
4- {2- { [4- (3, 4-dichlorophenyl) thiazol-2-yl ] oxy } ethyl } morpholine;
4- {2- { [4- (4-fluorophenyl) thiazol-2-yl ] oxy } ethyl } morpholine;
4- {2- { [4- (4-fluoro-2-methylphenyl) thiazol-2-yl ] oxy } ethyl } morpholine;
4- {2- { [4- ([ 1,1' -biphenyl ] -4-yl) thiazol-2-yl ] oxy } ethyl } morpholine;
4- [2- (2-morpholino) thiazol-4-yl ] benzonitrile;
4- {2- { [4- (2- (methylsulfanyl) phenyl) thiazol-2-yl ] oxy } ethyl } morpholine;
4- {2- { [ 5-methyl-4- (2- (methylsulfanyl) phenyl) thiazol-2-yl ] oxy } ethyl } morpholine;
4- {2- { { 5-methyl-4- [4- (trifluoromethoxy) phenyl ] thiazol-2-yl } oxy } ethyl } morpholine;
4- {2- { [4- (4-chloro-2-methoxyphenyl) thiazol-2-yl ] oxy } ethyl } morpholine;
4- {2- { {4- [2, 4-bis (trifluoromethyl) phenyl ] thiazol-2-yl } oxy } ethyl } morpholine;
4- {2- { [4- (furan-3-yl) thiazol-2-yl ] oxy } ethyl } morpholine;
4- {2- { [4- (benzofuran-2-yl) thiazol-2-yl ] oxy } ethyl } morpholine;
4- {2- { [4- (benzo [ b ] thiophen-2-yl) thiazol-2-yl ] oxy } ethyl } morpholine;
4- {2- { [4- (thiophen-3-yl) thiazol-2-yl ] oxy } ethyl } morpholine.
Further, the compounds of the invention or stereoisomers, geometric isomers, tautomers, oxynitride compounds, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs thereof, wherein hydrogen in said compounds may be substituted by one or more deuterium.
The invention also provides a preparation method of the compound, which comprises the following steps:
allowing the compound of formula IIa and the compound of formula VI to react at DMF, DMSO, CH 3 In CN and THF solvent, in K 2 CO 3 、Cs 2 CO 3 Carrying out substitution reaction at 0-120 ℃ under the alkaline condition of CsF or NaH for 4-16 hours to obtain a compound of a formula V;
continuing the compound of formula va at Na 2 CO 3 、K 2 CO 3 、Cs 2 CO 3 Or CsF and the like under alkaline conditions, and the organic boric acid shown in the formula III or the organic boric acid ester shown in the formula IV is prepared in a way of 1,4-dioxane, DMF, DMSO, DME or 1,4-dioxane/H 2 In a solvent such as O, pd (PPh) 3 ) 4 、Pd(dppf)Cl 2 、Pd(PPh 3 ) 2 Cl 2 、Pd(Cy 3 ) 2 Cl 2 Or Pd (OAc) 2 Reacting for 8-16 hours at 80-160 ℃ under the catalysis of an equal catalyst to prepare a compound shown in a formula I;
Figure SMS_5
the invention also provides another preparation method of the compound, which comprises the following steps:
the compound of formula IIb and the compound of formula VI are dissolved in DMF, DMSO, CH CN or THF in K 2 CO 3 、Cs 2 CO 3 Carrying out substitution reaction at 0-120 ℃ under the alkaline condition of CsF or NaH for 4-16 hours to obtain a compound of a formula VIII;
continuing the compound of formula vb at Na 2 CO 3 、K 2 CO 3 、Cs 2 CO 3 Or CsF and other alkaline conditions, and organic boric acid described in formula III or organic boric acid ester described in formula IV in 1,4-dioxane, DMF, DMSO, DME or 1,4-dioxane/H2O and other solvents, in Pd (PPh) 3 ) 4 、Pd(dppf)Cl 2 、Pd(PPh 3 ) 2 Cl 2 、Pd(Cy 3 ) 2 Cl 2 Or Pd (OAc) 2 Reacting for 8-16 hours at 80-160 ℃ under the catalysis of an equal catalyst to prepare a compound shown in a formula I;
Figure SMS_6
the invention also provides the use of a compound of formula I, I' in the manufacture of a medicament for the treatment or prophylaxis of a sigma receptor mediated disease or condition.
Further, the present invention relates to the use of compounds of formula I, I', in particular for the preparation of a medicament for the treatment or prophylaxis of diarrhea, lipoprotein disorders, migraine, obesity, arthritis, hypertension, cardiac arrhythmias, ulcers, cognitive disorders, chemical addiction, delayed spinal discs, ischemic stroke, epilepsy, stroke, depression, stress, psychosis, pain sensitization, cancer. In particular neuropathic pain, inflammatory pain, allodynia, cancer pain, postoperative pain and/or hyperalgesia.
Detailed Description
The present invention will be described in further detail with reference to the following examples and test examples, which are only for the purpose of illustrating the technical aspects of the present invention, but are not to be construed as limiting the present invention, and any equivalent substitution in the art according to the present disclosure is intended to be within the scope of the present invention.
The structure of the compound was determined by nuclear magnetic resonance (1H NMR) or liquid chromatography-mass spectrometry (LC-MS).
The liquid chromatography-mass spectrometer (LC-MS) is Agilent G6120B (matched with liquid phase Agilent 1260); the nuclear magnetic resonance (1H NMR) is Bruker AVANCE-400 or Bruker AVANCE-800, nuclear magnetic resonance 1 H NMR) shift (δ) given in parts per million (ppm) with solvent CDCl 3 The internal standard is Tetramethylsilane (TMS), and the chemical shift is 10 -6 (ppm) is given as a unit.
The term "room temperature" according to the invention means a temperature between 10 and 25 ℃.
Example 1: preparation of 4- {2- { [5- (naphthalen-2-yl) thiazol-2-yl ] oxy } ethyl } morpholine
Figure SMS_7
Step one, compound IIa (2.43 g,10 mmol), compound VI (1.97 g,15 mmol), cs are reacted at room temperature 2 CO 3 (9.75 g,30 mmol) was added to DMF (30 mL), heated to 100deg.C, and reacted for 6h, with the starting material reacted well. The reaction solution was cooled toAt room temperature, add H 2 O (30 mL), extracted three times with ethyl acetate (50 mL x 3), the organic phase washed successively with water, saturated brine, the organic phase dried over anhydrous sodium sulfate, filtered, the filtrate dried by spin, the crude product purified by eluent petroleum ether ethyl acetate=3:1, dried by spin, and dried under vacuum to give 2.50g of product. Yield: 85.2%.
Step two, compound Va (2.50 g,8.5 mmol), compound naphthalen-2-ylboronic acid (1.76 g,10.2 mmol), csF (1.55 g,10.2 mmol) was added to 1,4-dioxane/H at room temperature 2 O (30 mL, V/V=5/1), after nitrogen substitution, pd (dppf) Cl was added 2 (0.62 g,0.85 mmol), nitrogen was again replaced three times, heated to 100deg.C under nitrogen protection, and reacted for 16h, the starting material was completely reacted. The reaction mixture was cooled to room temperature, and H was added 2 O (30 mL), extracted three times with ethyl acetate (50 mL x 3), the organic phase washed successively with water, saturated brine, the organic phase dried over anhydrous sodium sulfate, filtered, the filtrate dried by spin-drying, the crude product using eluent petroleum ether: ethyl acetate = 2:1 column, spin-dry, vacuum drying to give 2.38g of product: 4- {2- { [5- (naphthalen-2-yl) thiazol-2-yl]Oxy } ethyl } morpholine. Yield: 82.2% purity 99.29%.
ESI-MS:m/z＝341.2(M+H) + 。
1 H NMR(400MHz,CDCl 3 )δ7.83–7.79(m,4H),7.60(dd,J＝8.7,1.5Hz,1H),7.53–7.44(m,3H),4.60(t,J＝5.6Hz,2H),3.77–3.73(m,4H),2.85(t,J＝5.6Hz,2H),2.63–2.53(m,4H)。
Example 2: preparation of 4- {2- { [ 5-phenylthiazol-2-yl ] oxy } ethyl } morpholine
Figure SMS_8
The procedure for the preparation of example 1 was followed by substituting equimolar amounts of phenylboronic acid for 2-naphthaleneboronic acid in step two to give the title compound in yield: 80.4% and purity of 99.46%.
ESI-MS:m/z＝291.2(M+H) + 。
1 H NMR(400MHz,CDCl 3 )δ7.46–7.42(m,2H),7.39–7.34(m,2H),7.31–7.27(m,2H),4.57(t,J＝5.6Hz,2H),3.76–3.73(m,4H),2.83(t,J＝5.6Hz,2H),2.61–2.54(m,4H)。
Example 3: preparation of 4- {2- { [5- (3, 4-dichlorophenyl) thiazol-2-yl ] oxy } ethyl } morpholine
Figure SMS_9
The procedure for the preparation of example 1 was followed by substituting equimolar amounts of 3, 4-dichlorobenzoboric acid for 2-naphthaleneboric acid in step two to give the title compound in yield: 82.5% and a purity of 99.77%.
ESI-MS:m/z＝359.2(M+H) + 。
1 H NMR(400MHz,CDCl 3 )δ7.51(d,J＝2.1Hz,1H),7.42(d,J＝8.4Hz,1H),7.30(s,1H),7.24(d,J＝2.1Hz,1H),4.58(t,J＝5.6Hz,2H),3.76–3.72(m,4H),2.82(t,J＝5.6Hz,2H),2.61–2.53(m,4H)。
Example 4: preparation of 4- {2- { [5- (3, 5-difluorophenyl) thiazol-2-yl ] oxy } ethyl } morpholine
Figure SMS_10
The procedure for the preparation of example 1 was followed by substituting equimolar amounts of 3, 5-difluorophenylboronic acid for 2-naphthaleneboronic acid in step two to give the title compound in yield: 81.2% and a purity of 99.91%.
ESI-MS:m/z＝327.2(M+H) + 。
1 H NMR(400MHz,CDCl 3 )δ7.33(s,1H),6.95(dd,J＝8.2,1.9Hz,2H),6.76–6.68(m,1H),4.58(t,J＝5.6Hz,2H),3.76–3.73(m,4H),2.83(t,J＝5.6Hz,2H),2.60–2.53(m,4H)。
Example 5: preparation of 4- {2- { [5- (4-methoxyphenyl) thiazol-2-yl ] oxy } ethyl } morpholine
Figure SMS_11
The procedure for the preparation of example 1 was followed by substituting equimolar amounts of 4-methoxyphenylboronic acid for 2-naphthaleneboronic acid in step two to give the title compound in yield: 79.8% of purity 99.62%.
ESI-MS:m/z＝321.2(M+H) + 。
1 H NMR(400MHz,CDCl 3 )δ7.39–7.33(m,2H),7.18(s,1H),6.93–6.87(m,2H),4.55(t,J＝5.6Hz,2H),3.83(s,3H),3.77–3.73(m,4H),2.82(t,J＝5.6Hz,2H),2.61–2.52(m,4H)。
Example 6:4- {2- { [4- {2- {4- [4- (trifluoromethoxy) phenyl }]Preparation of thiazol-2-yl } oxy } ethyl } morpholine
Figure SMS_12
Step one, compound IIb (2.43 g,10 mmol), compound VI (1.97 g,15 mmol), cs are reacted at room temperature 2 CO 3 (9.75 g,30 mmol) was added to DMF (30 mL), heated to 100deg.C, and reacted for 6h, with the starting material reacted well. The reaction mixture was cooled to room temperature, and H was added 2 O (30 mL), three extractions with ethyl acetate (50 mL x 3), successive washes with water, saturated brine, drying the organic phase over anhydrous sodium sulfate, filtration, spin-drying the filtrate, passing the crude product over column with eluent petroleum ether ethyl acetate=3:1, spin-drying, vacuum drying to give 2.56g of product. Yield: 87.3%.
Step two, compound vb (2.87 g,8.7 mmol), 4- (trifluoromethoxy) phenylboronic acid (2.15 g,10.44 mmol) and CsF (1.58 g,10.44 mmol) were added to 1,4-dioxane/H at room temperature 2 O (30 mL, V/V=5/1), after nitrogen substitution, pd (dppf) Cl was added 2 (0.62 g,0.85 mmol), nitrogen was again replaced three times, heated to 100deg.C under nitrogen protection, and reacted for 16h, the starting material was completely reacted. The reaction mixture was cooled to room temperature, and H was added 2 O (30 mL), extracted three times with ethyl acetate (50 mL x 3), the organic phase washed successively with water, saturated brine, the organic phase dried over anhydrous sodium sulfate, filtered, the filtrate dried by spin-drying, the crude product using eluent petroleum ether: ethyl acetate = 2:1 column, spin-dry, vacuum drying to give 2.64g of product: 4- {2- { [5- (naphthalen-2-yl) thiazol-2-yl]Oxy } ethyl } morpholine. Yield: 81.1% and a purity of 99.77%％。
ESI-MS:m/z＝375.2(M+H) + 。
1 H NMR(400MHz,CDCl 3 )δ7.83(d,J＝8.8Hz,2H),7.23(d,J＝8.4Hz,2H),6.87(s,1H),4.64(t,J＝5.6Hz,2H),3.76–3.73(m,4H),2.85(t,J＝5.6Hz,2H),2.62–2.55(m,4H)。
Example 7: preparation of 4- {2- { [4- (2-methoxyphenyl) thiazol-2-yl ] oxy } ethyl } morpholine
Figure SMS_13
The procedure of example 6 was followed, substituting equimolar amounts of 2-methoxyphenylboronic acid for 4- (trifluoromethoxy) phenylboronic acid in step two, to give the title compound in yield: 80.2% and purity of 99.12%.
ESI-MS:m/z＝321.2(M+H) + 。
1 H NMR(400MHz,CDCl 3 )δ8.13(dd,J＝7.7,1.7Hz,1H),7.36(s,1H),7.30–7.22(m,1H),7.02(t,J＝7.6Hz,1H),6.97(d,J＝8.2Hz,1H),4.65(t,J＝5.6Hz,2H),3.94(s,3H),3.78–3.74(m,4H),2.86(t,J＝5.3Hz,2H),2.67–2.54(m,4H)。
Example 8: preparation of 4- {2- { [4- (3, 4-dichlorophenyl) thiazol-2-yl ] oxy } ethyl } morpholine
Figure SMS_14
The procedure of example 6 was followed, substituting equimolar amounts of 3, 4-dichlorobenzoic acid for 4- (trifluoromethoxy) phenylboronic acid in step two, to give the title compound in yield: 82.5% and a purity of 99.23%.
ESI-MS:m/z＝360.2(M+H) + 。
1 H NMR(400MHz,CDCl 3 )δ7.88(dd,J＝7.8,1.7Hz,1H),7.43(dd,J＝7.9,1.0Hz,1H),7.33–7.28(m,1H),7.23(s,1H),4.62(t,J＝5.6Hz,2H),3.77–3.73(m,4H),2.84(t,J＝5.6Hz,2H),2.61–2.54(m,4H)。
Example 9: preparation of 4- {2- { [4- (4-fluorophenyl) thiazol-2-yl ] oxy } ethyl } morpholine
Figure SMS_15
The procedure of example 6 was followed, substituting equimolar amounts of 4- (trifluoromethoxy) phenylboronic acid in step two to 4-fluorobenzeneboronic acid, to give the title compound in yield: 84.2% and purity 99.31%.
ESI-MS:m/z＝309.2(M+H) + 。
1 H NMR(400MHz,CDCl 3 )δ7.81–7.74(m,2H),7.07(t,J＝8.7Hz,2H),6.79(s,1H),4.63(t,J＝5.6Hz,2H),3.76–3.73(m,4H),2.85(t,J＝5.6Hz,2H),2.62–2.54(m,4H)。
Example 10: preparation of 4- {2- { [4- (4-fluoro-2-methylphenyl) thiazol-2-yl ] oxy } ethyl } morpholine
Figure SMS_16
The procedure for the preparation of example 6 was followed by substituting equimolar amounts of 4- (trifluoromethoxy) phenylboronic acid in step two into 4-fluoro-2-methylphenylboronic acid to give the title compound in yield: 81.6% and a purity of 99.77%.
ESI-MS:m/z＝323.2(M+H) + 。
1 H NMR(400MHz,CDCl 3 )δ7.49(dd,J＝8.4,6.1Hz,1H),6.97–6.87(m,2H),6.58(s,1H),4.59(t,J＝5.6Hz,2H),3.77–3.72(m,4H),2.83(t,J＝5.6Hz,2H),2.63–2.51(m,4H),2.44(s,3H)。
Example 11: preparation of 4- {2- { [4- ([ 1,1' -biphenyl ] -4-yl) thiazol-2-yl ] oxy } ethyl } morpholine
Figure SMS_17
The procedure of example 6 was followed, substituting equimolar amounts of 4- (trifluoromethoxy) phenylboronic acid in step two into 4-methyl [1,1' -biphenyl ] boronic acid to give the title compound in yield: 80.9% and purity of 99.80%.
ESI-MS:m/z＝367.2(M+H) + 。
1 H NMR(400MHz,CDCl 3 )δ7.88(d,J＝8.3Hz,2H),7.63(d,J＝8.2Hz,4H),7.45(t,J＝7.6Hz,2H),7.39–7.32(m,1H),6.91(s,1H),4.66(t,J＝5.6Hz,2H),3.80–3.73(m,4H),2.86(t,J＝5.6Hz,2H),2.65–2.54(m,4H)。
Example 12: preparation of 4- {2- { [4- [2- (2-morpholino) thiazol-4-yl ] benzonitrile
Figure SMS_18
The procedure for the preparation of example 6 was followed by substituting equimolar amounts of 4- (trifluoromethoxy) phenylboronic acid in step two to 4-cyanophenylboronic acid to give the title compound in yield: 80.4% and the purity is 99.87%.
ESI-MS:m/z＝316.2(M+H) + 。
1 H NMR(400MHz,CDCl 3 )δ7.91(d,J＝8.4Hz,2H),7.67(d,J＝8.4Hz,2H),7.05(s,1H),4.65(t,J＝5.6Hz,2H),3.77–3.70(m,4H),2.85(t,J＝5.6Hz,2H),2.62–2.55(m,4H)。
Example 13: preparation of 4- {2- { [4- (2- (methylthio) phenyl) thiazol-2-yl ] oxy } ethyl } morpholine
Figure SMS_19
The procedure of example 6 was followed, substituting equimolar amounts of 2- (methylthio) phenylboronic acid for 4- (trifluoromethoxy) phenylboronic acid in step two, to give the title compound in yield: 81.6% and a purity of 99.75%.
ESI-MS:m/z＝337.2(M+H) + 。
1 H NMR(400MHz,CDCl 3 )δ7.62–7.58(m,1H),7.33–7.29(m,1H),7.28–7.24(m,1H),7.21–7.15(m,1H),6.96(s,1H),4.63(t,J＝5.6Hz,2H),3.76–3.73(m,4H),2.85(t,J＝5.6Hz,2H),2.62–2.52(m,5H),2.45(s,3H)。
Example 14: preparation of 4- {2- { [ 5-methyl-4- (2- (methylsulfanyl) phenyl) thiazol-2-yl ] oxy } ethyl } morpholine
Figure SMS_20
Step one: synthesis of 2, 4-dibromo-5-methyl-thiazole
Diisopropylamine (1.38 g,1.90mL,13.58 mmol) in THF (50 mL) was cooled to-78deg.C, then BuLi (5.43 mL,2.5M in hexane, 13.58 mmol) was added dropwise, and the temperature was maintained below-70deg.C. After complete addition, the mixture was removed from the ice-bath to 0 ℃ and the RBF was placed in an ice-water bath. The mixture was then cooled back to-78 ℃, followed by dropwise addition of 2, 4-dibromothiazole (3 g,12.35 mmol) in THF (10 mL) at-78 ℃ (temperature not exceeding-70 ℃). After all additions, stirring was continued for a further 30 minutes at-78 ℃. Methyl iodide (1.93 g, 845.6. Mu.L, 13.58 mmol) was added dropwise and the mixture was stirred at-78℃for 30 minutes and then allowed to warm to room temperature over 18 h. Quench by addition of water, dilute with Etoac, and collect the organic layer. The organics were then washed with salt and dried (MgSO 2 ) Filtration and vacuum concentration. Purification of the crude product by flash chromatography gave 2.22g of colorless solid in 70% yield.
Step two: the procedure of example 6 was followed, substituting 2, 4-dibromothiazole in step one with equimolar amounts of 2, 4-dibromo-5-methyl.
Step three: the procedure of example 6 was followed, substituting 4- (trifluoromethoxy) phenylboronic acid in step two with equimolar amounts of 2- (methylthio) phenylboronic acid and substituting compound vb with equimolar amounts of vc, to give the title compound in yield: 82.2% and a purity of 99.37%.
ESI-MS:m/z＝351.2(M+H) + 。
1 H NMR(400MHz,CDCl 3 )δ7.36–7.30(m,1H),7.26–7.22(m,2H),7.19–7.13(m,1H),4.53(t,J＝5.5Hz,2H),3.74–3.71(m,4H),2.80(t,J＝5.5Hz,2H),2.59–2.49(m,4H),2.41(s,3H),2.18(s,3H)。
Example 15: preparation of 4- {2- { [4- {2- { { 5-methyl-4- [4- (trifluoromethoxy) phenyl ] thiazol-2-yl } oxy } ethyl } morpholine
Figure SMS_21
The procedure of example 14 was followed, substituting equimolar amounts of 4- (trifluoromethoxy) phenylboronic acid for 2- (methylthio) phenylboronic acid in step two, to give the title compound in yield: 82.3% and a purity of 97.90%.
ESI-MS:m/z＝389.2(M+H) + 。
1 H NMR(400MHz,CDCl 3 )δ7.64–7.60(m,2H),7.25(d,J＝8.3Hz,2H),4.55(t,J＝5.6Hz,2H),3.77–3.71(m,4H),2.81(t,J＝5.6Hz,2H),2.60–2.52(m,4H),2.42(s,3H)。
Example 16: preparation of 4- {2- { [4- {2- { [4- (4-chloro-2-methoxyphenyl) thiazol-2-yl ] oxy } ethyl } morpholine
Figure SMS_22
The procedure for the preparation of example 6 was followed by substituting equimolar amounts of 4- (trifluoromethoxy) phenylboronic acid in step two into 4-chloro-2-methoxyphenylboronic acid to give the title compound in yield: 82.0% and a purity of 99.99%.
ESI-MS:m/z＝355.2(M+H) + 。
1 H NMR(400MHz,CDCl 3 )δ8.07(d,J＝8.4Hz,1H),7.33(s,1H),7.00(dd,J＝8.4,1.9Hz,1H),6.94(d,J＝1.8Hz,1H),4.62(t,J＝5.6Hz,2H),3.94(s,3H),3.77–3.72(m,4H),2.84(t,J＝5.6Hz,2H),2.62–2.53(m,4H)。
Example 17: preparation of 4- {2- { [4- {2- { {4- [2, 4-bis (trifluoromethyl) phenyl ] thiazol-2-yl } oxy } ethyl } morpholine
Figure SMS_23
The procedure of example 6 was followed, substituting equimolar amounts of 2, 4-bis (trifluoromethyl) phenylboronic acid for 4- (trifluoromethoxy) phenylboronic acid in step two, to give the title compound in yield: 79.8% of purity 99.21%.
ESI-MS:m/z＝427.2(M+H) + 。
1 H NMR(400MHz,CDCl 3 )δ7.99(s,1H),7.86–7.77(m,2H),6.85(s,1H),4.59(t,J＝5.6Hz,2H),3.78–3.68(m,4H),2.83(t,J＝5.6Hz,2H),2.60–2.51(m,4H)。
Example 18: preparation of 4- {2- { [4- {2- { [4- (furan-3-yl) thiazol-2-yl ] oxy } ethyl } morpholine
Figure SMS_24
The procedure of example 6 was followed, substituting equimolar amounts of 3-furanboronic acid for 4- (trifluoromethoxy) phenylboronic acid in step two, to give the title compound in yield: 82.4% and a purity of 99.14%.
ESI-MS:m/z＝281.2(M+H) + 。
1 H NMR(400MHz,CDCl 3 )δ7.40(d,J＝1.1Hz,1H),6.81(s,1H),6.65(d,J＝3.3Hz,1H),6.45(dd,J＝3.2,1.8Hz,1H),4.60(t,J＝5.6Hz,2H),3.77–3.70(m,4H),2.82(t,J＝5.6Hz,2H),2.61–2.50(m,4H)。
Example 19: preparation of 4- {2- { [4- {2- { [4- (benzofuran-2-yl) thiazol-2-yl ] oxy } ethyl } morpholine
Figure SMS_25
The procedure is as in example 6, substituting the 4- (trifluoromethoxy) phenylboronic acid in step two with equimolar amounts of 2-benzofuranboronic acid to give the title compound in yield: 80.9% and a purity of 99.94%.
ESI-MS:m/z＝331.2(M+H) + 。
1 H NMR(400MHz,CDCl 3 )δ7.58(d,J＝7.4Hz,1H),7.48(d,J＝8.1Hz,1H),7.32–7.28(m,1H),7.25–7.20(m,1H),7.09(s,1H),7.03(s,1H),4.65(t,J＝5.6Hz,2H),3.77–3.73(m,4H),2.85(t,J＝5.6Hz,2H),2.63–2.55(m,4H)。
Example 20: preparation of 4- {2- { [4- {2- { [4- (benzo [ b ] thiophen-2-yl) thiazol-2-yl ] oxy } ethyl } morpholine
Figure SMS_26
The procedure of example 6 was followed, substituting equimolar amounts of 2-benzo [ b ] thiopheneboronic acid for 4- (trifluoromethoxy) phenylboronic acid in step two, to give the title compound in yield: 83.2% and the purity is 99.85%.
ESI-MS:m/z＝347.2(M+H) + 。
1 H NMR(400MHz,CDCl 3 )δ7.77(dd,J＝20.0,7.4Hz,2H),7.61(s,1H),7.37–7.29(m,2H),6.88(s,1H),4.66(t,J＝5.6Hz,2H),3.77–3.72(m,4H),2.85(t,J＝5.6Hz,2H),2.64–2.54(m,4H)。
Example 21: preparation of 4- {2- { [4- {2- { [4- (thiophen-3-yl) thiazol-2-yl ] oxy } ethyl } morpholine
Figure SMS_27
The procedure of example 6 was followed, substituting the 4- (trifluoromethoxy) phenylboronic acid in step two with equimolar amounts of 3-thiopheneboronic acid, to give the title compound in yield: 81.5% and a purity of 99.74%.
ESI-MS:m/z＝297.2(M+H) + 。
1 H NMR(400MHz,CDCl 3 )δ7.35(d,J＝3.5Hz,1H),7.23(d,J＝4.8Hz,1H),7.03(dd,J＝4.9,3.7Hz,1H),6.73(s,1H),4.62(t,J＝5.6Hz,2H),3.77–3.72(m,4H),2.83(t,J＝5.6Hz,2H),2.62–2.54(m,4H)。
Test example 1: sigma1 in vitro affinity assay
1. Material and apparatus
Figure SMS_28
Figure SMS_29
2. Test procedure
2.1 preparation of sigma-1 receptor films
The guinea pig is broken, the brain cortex is quickly taken out by an operation on ice, and tissues are combined into a centrifuge tube. An appropriate amount of extract (0.05M Tris-HCl, w/0.32M sucrose) was added to the centrifuge tube, and the tissue was homogenized with the 4 th gear of the homogenizer for 3-4 seconds each time, 4 times. Adding the extract after homogenizing, adjusting the extract to 10mL/g, adjusting the weight of a centrifuge tube by a balance, and centrifuging at 1000rpm for 10 minutes by a high-speed refrigerated centrifuge; centrifuging, adding solution B into the supernatant to adjust the solution to 2mL/g, and centrifuging at 1000rpm and 4 ℃ for 10 minutes; taking supernatant, centrifuging at 11500rpm and 4 ℃ for 25 minutes; the precipitate was taken and adjusted to 3mL/g with the extract and incubated at 25℃for 15 minutes in a constant temperature water incubator. After incubation, the pellet was centrifuged at 11500rpm at 4℃for 25 minutes and stored at-80℃for further use.
2.2 test methods
1) Adding a proper amount of homogenate into the prepared membrane, and dispersing into suspension by a refiner; and the protein concentration (4 mg/ml) was measured.
2) 100. Mu.g of protein was added to each 96-well plate in a volume of 90. Mu.L;
3) 1. Mu.L of compound (final concentration 1. Mu.M, four-fold dilution, 8 concentrations) was added to the test wells, 1. Mu.L buffer was added to the ZPE wells, and 1. Mu.L of haloperidol (final concentration 1. Mu.M) was added to the HPE wells;
4) Add 10. Mu.L of [ to each well ] 3 H]- (+) -pentazocine (final concentration 10 nM);
5) Incubating the 96-well plate in a constant temperature water bath (25 ℃ C., 180 min); after incubation, the 96-well suspension was rapidly filtered under reduced pressure using GF/C plates prepared in advance in 0.25% pei solution, and after filtration GF/C was rinsed three times with assay buffer; washing and then drying in a baking oven at 37 ℃;
6) 50. Mu.L/well of scintillation fluid was added to GF/C plates;
7) And putting GF/C into a liquid flash counter, and reading the test value by program operation.
3. Test results
Table 1:
Figure SMS_30
Figure SMS_31
the results are shown in Table 1, and examples 3,4, 6, 9, 10 and 13 show strong in vitro affinities for sigma1R in terms of in vitro receptor affinities.
Test example 2: in vitro affinity assay for other targets
To assess the target selectivity of the example compounds, we determined their in vitro activity by D2L radioligand binding assay and 5HT-1A receptor, CB1 receptor, CB2 receptor cAMP assay.
D2L radioligand binding assay
1.1 test reagents
Figure SMS_32
1.2 test instruments
Figure SMS_33
1.3 preparation of reagents
Assay buffer: 50mM Tris-HCl pH 7.4,10mM MgCl 2 ,1mM EDTA,120mM NaCl
Washing buffer: 50mM Tris-HCl pH 7.4, stored at 4 ℃
0.5% PEI solution: 0.5mL PEI was dissolved in 100mL ddH 2 O, 4℃storage.
Extraction of 1.4D2L membrane proteins
1) D2L cells were digested with trypsin and then collected.
2) To LDL cells, 10 volumes of pre-chilled 0.32M sucrose (1 tablet of protease inhibitor pellet per 100 mL) were added and broken down with a high speed disperser.
3) The homogenate was centrifuged at 900g at 4℃for 10min. The supernatant was collected.
4) The supernatant was centrifuged at 40000g at 4℃for 60min, and the supernatant was discarded.
5) The pellet was resuspended in pre-chilled PBS (1 pellet per 100 mL). The resuspended liquid was transferred to a 50mL homogenizer for homogenization.
6) The homogenate was centrifuged at 40000g at 4℃for 10min and the supernatant was discarded.
7) The washing procedure was repeated three times.
8) The final pellet was resuspended in 4 volumes of PBS and stored at-80℃under liquid nitrogen treatment.
1.5 test methods
1) mu.L cpds (1% DMSO) and 100. Mu.L assay buffer were added to 96 well deep plates.
2) mu.L of membrane and 300. Mu.L of assay buffer were added to each well.
3) [3H ] -methylppirpirfenone (fianl concentration 0.5 nM) was added to 100. Mu.L assay buffer and incubated at 27℃for 30 min.
4) The UNIFILTER-96GF/B filter plates were pre-incubated with 0.5% PEI for 1 hour.
5) The UNIFILTER-96GF/B filter plate was washed twice with 1mL of wash buffer/well and the membrane mixture was transferred to the UNIFILTER-96GF/B filter plate and washed 4 times.
6) Plates were incubated at 55℃for 10min.
7) 40. Mu.L of ULTIMA GOLD was added to each well and the CPM of TopCount was read.
2. Test of the Activity of example Compounds in the 5-HT1A receptor cAMP assay
2.1 test reagent consumable
Figure SMS_34
2.2 test apparatus
Figure SMS_35
2.3 test methods
2.3.1 cell culture and reagent preparation
1) Cell lines: flp-In-CHO-5HT-1A
2) Complete medium Ham's F K+10% foetal calf serum+1 penicillin streptomycin+800 μg/mL hygromycin
3) Cell inoculation Medium Ham's F K+10% DFBS
4) Assay buffer 1*HBSS+20mM HEPES+0.1%BSA+500. Mu.M IBMX
2.3.2 determination of 5HT-1A receptor agonist Activity of test Compounds
(1) Cell culture and seed plates
1) Flp-In-CHO-5HT-1A cell lines were cultured at 37℃with 5% CO 2 In complete medium under environment;
2) After TrypLE digestion, the cells were resuspended in seeding medium, seeded in 60mm dishes at a density of 1 x 10≡6, at 37℃with 5% CO 2 Culturing overnight;
(2) Transfecting cells
1) The transfection reagent was equilibrated at room temperature.
2) Gi3 and transfection reagent were mixed and left at room temperature for 10 minutes.
3) The prepared plasmid was added to a cell culture dish (see step 2.3.1)
4) Cells at 37℃with 5% CO 2 Culturing for 5-6 hr.
(3) Cell plating
1) The transfected cells were resuspended in seeding medium and seeded into 384 cell culture plates at a seeding density of 8000 wells per well.
2) Cells at 37℃with 5% CO 2 Culturing overnight.
(4) Detection of
1) Test buffer 1*HBSS,0.1%BSA,5mM HEPES and 500. Mu.M IBMX were prepared.
2) The compound was diluted with buffer.
3) The medium in 384 plates was removed and 15. Mu.L buffer was added to each well.
4) 2.5. Mu.L of compound was added to each well and incubated at 37℃for 5 minutes.
5) Forskolin was diluted to 8 μm (8 x) with assay buffer.
6) 2.5 μl of 8 x forskolin diluted in step 5 was added and incubated at 37 ℃ for 10min.
7) Freeze thawing of cAMP-d2 and Anti-cAMP-Eu 3+ It was diluted 20-fold with test buffer.
8) Add 10. Mu.L of cAMP-d2 to the assay well, followed by 10. Mu.L of Anti-cAMP-Eu 3+ Into the test wells.
9) After the reaction plate was centrifuged at 200g at room temperature for 30s and left to stand at 25℃for 1 hour, data were collected by Envision.
2.4 data analysis
1)Z’factor＝1-3*(SDMax+SDMin)/(MeanMax-MeanMin)；
2)CVMax＝(SDMax/MeanMax)*100％；
3)CVMin＝(SDMin/MeanMin)*100％；
4)S/B＝Singal/Background；
5) Calculation of Compound EC using GraphPad nonlinear fitting equation 50 /IC 50 :
Y＝Bottom+(Top-Bottom)/(1+10^((LogEC 50 -X)*HillSlope))
Y＝Bottom+(Top-Bottom)/(1+10^((LogIC 50 -X)*HillSlope))
X is the log value of the compound concentration; y: action% or Inhibition%
3. Activity assay in CB1 receptor and CB2 receptor cAMP assay
3.1 reagent consumable for test
Figure SMS_36
3.2 test apparatus
Figure SMS_37
3.3 test methods
3.3.1 cell culture and reagent preparation
1) Cell lines: flp-In-CHO-CB1 and Flp-In-CHO-CB2;
2) Complete medium, ham's F K+10% fetal bovine serum+1% penicillin streptomycin+800 μg/mL hygromycin;
3) Assay buffer 1*HBSS+20mM HEPES+0.1%BSA+500. Mu.M IBMX.
3.3.2 determination of the Activity of test Compounds on CB1 and CB2 receptors
(1) Cell culture and seed plates
1) Flp-In-CHO-CB1 and Flp-In-CHO-CB2 cell lines were cultured at 37℃with 5% CO 2 In complete medium in the environment.
2) After the TrypLE digestion treatment, the cells were resuspended in complete medium, seeded into 384 cell culture plates at a density of 8000 per well.
3) Cells at 37℃with 5% CO 2 Culturing overnight.
(2) Detection of agonistic Activity
1) Test buffer 1*HBSS,0.1%BSA,20mM HEPES and 500. Mu.M IBMX were prepared.
2) The compound was diluted with buffer.
3) The medium in 384 plates was removed and 15. Mu.L buffer was added to each well.
4) 2.5. Mu.L of compound was added to each well and incubated at 37℃for 10 minutes.
5) Forskolin was diluted to 8 μm (8 x) with assay buffer.
6) 2.5 μl of diluted 8 x forskolin was added and incubated for 30min at 37 ℃.
7) Freeze thawing Eu-cAMP tracker and Ulight-anti-cAMP, diluted with cAMP detection buffer.
8) mu.L of Eu-cAMP tracker was added to the wells, followed by 10. Mu.L of Ulight-anti-cAMP.
9) After the reaction plate was centrifuged at 200g at room temperature for 30s and left to stand at 25℃for 1 hour, data were collected by Envision.
3.4 data analysis
1)Z’factor＝1-3*(SDMax+SDMin)/(MeanMax-MeanMin)；
2)CVMax＝(SDMax/MeanMax)*100％；
3)CVMin＝(SDMin/MeanMin)*100％；
4)S/B＝Singal/Background；
5) Calculation of Compound EC using GraphPad nonlinear fitting equation 50 /IC 50 :
Y＝Bottom+(Top-Bottom)/(1+10^((LogEC 50 -X)*HillSlope))
Y＝Bottom+(Top-Bottom)/(1+10^((LogIC 50 -X)*HillSlope))
X is the log value of the compound concentration; y: action% or Inhibition%
4. Radioligand binding assay to D2L assay results and Activity assay to 5HT-1A, CB1, CB2 receptor in CAMP assay
In vitro Activity of example Compounds against other targets in vitro affinity IC as measured by radioligand binding assay for D2L in example 10 is shown in Table 2 below 50 And in vitro agonistic activity EC in CAMP assays on 5HT-1A, CB, CB2 receptors 50 All greater than 1. Mu.M, indicating that example 10 has better sigma receptor selectivity.
Table 2:
Figure SMS_38
Figure SMS_39
test example 3: acetic acid torsion test for mice
1. Test materials
1.1 test reagents
Figure SMS_40
1.2 test instruments
1mL syringe, stopwatch, micropipette, electronic balance, 15mL centrifuge tube, 50mL centrifuge tube, and the like.
2. Test animals
Kunming mice, male and female halves, weigh 22-25g and are offered by Chengdu Biotechnology Inc.
3. Test method
3.1 preparation of test sample
HCl was added to normal saline to adjust pH to 4.5 for later use.
Each compound was weighed, added with DMSO (100 μl) and dissolved by shaking, and then added with the above physiological saline hydrochloric acid solution to prepare a test solution of 2 mg/mL. If still insoluble, it may be heated to dissolve.
3.2 Preparation of 0.8% acetic acid solution
And taking 0.2mL of glacial acetic acid in a 50mL centrifuge tube, adding 1.8mL of physiological saline, fully shaking and uniformly mixing, adding 23mL of physiological saline, and shaking and uniformly mixing to obtain the acetic acid solution with the required concentration. The solution is prepared on site, and the cover of the centrifuge tube is screwed after each use, so that acetic acid volatilization is prevented, and the test result is influenced.
3.3 administration and observations of the number of writhing
The test was carried out in a quiet environment at room temperature of 23-25 ℃. Mice were numbered prior to dosing and placed in corresponding numbered cages for 15min. After the adaptation, the mice were given 10mL/kg by intraperitoneal injection, and the doses were finally 20mg/kg, 10mg/kg, and 2.5mg/kg, respectively, and the negative control group was given the same volume of physiological saline. 10min after administration, the mice were intraperitoneally injected with 10mL/kg of the acetic acid solution, immediately placed in a cage, and observed for the number of twists within 15min. The indexes of the torsion body are as follows: a torsion is considered to occur when a mouse develops a typical abdominal indent accompanied by a characteristic reaction of trunk twisting, buttock lifting, and hindlimb extension.
4. Analysis of results
The average torsion number of 6 mice within 15min is used as a criterion for the analgesic effect of the compound of the example, and the analgesic percentage of the drug is calculated according to the following formula:
analgesic rate = (number of negative control torsion-number of compound torsion)/(number of negative control torsion) ×100%
The analgesic rate results of the compounds of the examples on mice pain are shown in table 3 below:
TABLE 3 analgesic Rate in mice when test example Compounds were intraperitoneally injected
Figure SMS_41
Test example 4: rat pharmacokinetic study
1. Test materials
Figure SMS_42
2. Test animals
SD rats, all male, 6, 220-300g, offered by Chengdu Biotechnology Inc.
3. Test method
Accurately weighing 30mg of each sample, respectively adding a proper amount of Kolliphor HS-15, uniformly mixing, then adding 0.9% sodium chloride injection, fixing the volume to 7.5mL, fully dissolving and uniformly mixing to obtain the sample solution.
SD rats were randomly divided into 2 groups (example 5 group, example 6 group) with 3 animals each at a dose of 20mg/kg and a dose volume of 5mL/kg.
Single tail vein injection is carried out, blood is taken from the jugular vein 5min, 10min, 15min, 30min, 1h, 2h, 4h, 6h, 8h and 24h before and after the administration, 0.2mL of whole blood is taken at each time point, the whole blood is placed in an anticoagulation tube containing EDTA-K2, and after centrifugation at the low temperature of 2000g, the blood plasma is separated and transferred into a microcentrifuge tube and stored in a refrigerator at the temperature of-80 ℃ for standby.
After the blood collection is completed, the concentration of the test substance in each blood sample is determined by autonomous analysis, and the drug metabolism parameter of each test substance is calculated.
4. Test results
The pharmacokinetic parameters of the rat oral preparation of example 5 and example 6 are shown in Table 4 below, and it can be seen that the compounds of the present invention have long half-life, low clearance and AUC last Large, can reach C after oral administration max The absorption is extremely fast.
Table 4:
subject T 1/2 (h) T max (h) C max (ng/mL) AUC last (h*ng/mL) Cl_F_obs(mL/h/kg)
example 5 2.1 0.2 2070.0 1477.9 13699.533
Example 6 3.8 1.1 869.3 6329.7 3174.399

Claims (6)

1. A compound of formula (I') or a pharmaceutically acceptable salt thereof,
Figure QLYQS_1
R 1 and R is 4 Independently selected from hydrogen, substituted or unsubstituted phenyl, unsubstituted naphthyl;
wherein the substituents in the substituted or unsubstituted phenyl groups are selected from one or more of halogen, methyl, methoxy, trifluoromethoxy or methylthio.
2. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein:
R 1 selected from hydrogen, substituted or unsubstituted phenyl;
R 4 selected from hydrogen, substituted or unsubstituted phenyl or unsubstituted naphthyl;
wherein the substituents in the substituted or unsubstituted phenyl groups are selected from one or more of halogen, methyl, methoxy, trifluoromethoxy or methylthio.
3. A compound according to claim 1 or claim 2, or a pharmaceutically acceptable salt thereof, wherein:
R 1 selected from hydrogen, 4-trifluoromethoxyphenyl, 4-fluorophenyl, 2-methyl-4-fluorophenyl, 2-methylthiophenyl;
R 4 selected from the group consisting of hydrogen, 2-naphthyl, phenyl, 3, 4-dichlorophenyl, 3, 5-difluorophenyl, 4-methoxyphenyl.
4. A compound as shown below, or a pharmaceutically acceptable salt thereof, selected from:
Figure QLYQS_2
。
5. the compound of claim 1, or a pharmaceutically acceptable salt thereof, selected from the following:
4- {2- { [5- (naphthalen-2-yl) thiazol-2-yl ] oxy } ethyl } morpholine;
4- {2- { [ 5-phenylthiazol-2-yl ] oxy } ethyl } morpholine;
4- {2- { [5- (3, 4-dichlorophenyl) thiazol-2-yl ] oxy } ethyl } morpholine;
4- {2- { [5- (3, 5-difluorophenyl) thiazol-2-yl ] oxy } ethyl } morpholine;
4- {2- { [5- (4-methoxyphenyl) thiazol-2-yl ] oxy } ethyl } morpholine;
4- {2- { {4- [4- (trifluoromethoxy) phenyl ] thiazol-2-yl } oxy } ethyl } morpholine;
4- {2- { [4- (4-fluorophenyl) thiazol-2-yl ] oxy } ethyl } morpholine;
4- {2- { [4- (4-fluoro-2-methylphenyl) thiazol-2-yl ] oxy } ethyl } morpholine;
4- {2- { [4- (2- (methylthio) phenyl) thiazol-2-yl ] oxy } ethyl } morpholine.
6. Use of a compound of any one of claims 1,4 or 5, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment or prophylaxis of a sigma receptor mediated disease or condition.
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