Substituted Heterocyclic Derivative, Preparation Method And Use Thereof
20170334896 · 2017-11-23
Inventors
- Zuguo LIU (Xiamen, Fujian Province, CN)
- Yan QIU (Xiamen, Fujian Province, CN)
- Jie REN (Xiamen, Fujian Province, CN)
- Yuhang LI (Xiamen, Fujian Province, CN)
- Longhe YANG (Xiamen, Fujian Province, CN)
- Chenggang ZHU (Xiamen, Fujian Province, CN)
Cpc classification
A61P29/00
HUMAN NECESSITIES
C07D207/273
CHEMISTRY; METALLURGY
C07D263/26
CHEMISTRY; METALLURGY
C07D413/06
CHEMISTRY; METALLURGY
International classification
C07D207/273
CHEMISTRY; METALLURGY
C07D207/28
CHEMISTRY; METALLURGY
C07D263/26
CHEMISTRY; METALLURGY
Abstract
The invention provides a compound as shown by Formula I having an enzyme activity which can inhibit endocannabinoid hydrolases NAAA and/or FAAH, or a pharmaceutically acceptable salt, hydrate or solvate thereof, and a preparation method and a use of the compound.
##STR00001##
Claims
1. A compound of Formula I, or a pharmaceutically acceptable salt, a hydrate or a solvate thereof, ##STR00118## wherein X is selected from the group consisting of C, N, and O; Y is selected from the group consisting of C and N; A is selected from the group consisting of R.sub.12, OR.sub.6, N R.sub.7R.sub.8 and CR.sub.9 R.sub.10R.sub.11; R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, R.sub.10, R.sub.11 are independently selected from the group consisting of —H, ═O, ═NOR′, halogen, halogenated group, polyhalogenated group, —(CH.sub.2).sub.nCN, —(CH.sub.2).sub.nNO.sub.2, —(CH.sub.2).sub.nR′, —(CH.sub.2).sub.nCOOR′, —(CH.sub.2).sub.nCONR′.sub.2, —(CH.sub.2).sub.nOR′, —(CH.sub.2).sub.nSR′, —(CH.sub.2).sub.nSOR′, —(CH.sub.2).sub.nSO.sub.2R′, —(CH.sub.2).sub.nNR′.sub.2, —(CH.sub.2).sub.nNR′COR′ and —(CH.sub.2).sub.nNR′SO.sub.2R′; R.sub.12 is —(CH.sub.2).sub.nR.sub.13; R.sub.13 is selected from the group consisting of H, a linear or branched C.sub.1-10alkyl, phenyl, thienyl, furyl, C.sub.4-6cycloalkyl, biphenylyl, phenoxyphenyl, benzyloxyphenyl, phenylethoxylphenyl, piperidyl, N-benzylpiperidyl, naphthyl, indolyl and uracil group, R.sub.13 is optionally substituted by one or two substituents selected from the group consisting of halogen, methyl, hydroxyl, C.sub.1-6alkyl, C.sub.1-6alkoxy, hydroxyl, nitro, trifluoromethyl, cyano, C.sub.1-6alkyl monosubstituted or polysubstituted by halogen, or C.sub.1-6alkoxyl monosubstituted or polysubstituted by halogen. n is an integer selected from 0-10; each R′ is independently H or is a group with no more than 20 carbon atoms and selected from a substituted or unsubstituted linear alkyl, branched alkyl, cycloalkyl, cycloheteroalkyl, alkenyl, alkynyl, a linear heteroalkyl, a branched heteroalkyl, heterocycloalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl or aryl.
2. A compound of Formula Ia, or a pharmaceutically acceptable salt, a hydrate or a solvate thereof, ##STR00119## wherein X is O or N; Y is N; n is an integer selected from 0-7; R.sub.4 is H, methyl, halogen, C.sub.1-6alkyl, C.sub.1-6alkoxy, hydroxyl, nitro, trifluoromethyl, cyano, CH.sub.3C(═O)CH.sub.2— or (CH.sub.3).sub.2NCH.sub.2—; R.sub.13 is selected from the group consisting of H, a linear or branched C.sub.1-10alkyl, phenyl, thienyl, furyl, C.sub.4-6cycloalkyl, biphenylyl, phenoxyphenyl, benzyloxyphenyl, phenylethoxylphenyl, piperidyl, N-benzylpiperidyl, naphthyl, indolyl and uracil group, R.sub.13 is optionally substituted by one or two substituents selected from the group consisting of halogen, methyl, hydroxyl, C.sub.1-6alkyl, C.sub.1-6alkoxy, hydroxyl, nitro, trifluoromethyl, cyano, C.sub.1-6alkyl monosubstituted or polysubstituted by halogen, or C.sub.1-6alkoxyl monosubstituted or polysubstituted by halogen.
3. The compound of Formula Ia, or a pharmaceutically acceptable salt, a hydrate or a solvate thereof according to claim 2, wherein: X is O or N; Y is N; n is an integer selected from 0-7; R.sub.4 is H, methyl, halogen, C.sub.1-4alkyl, C.sub.1-4alkoxy, hydroxyl, nitro, trifluoromethyl, cyano, CH.sub.3C(═O)CH.sub.2— or (CH.sub.3).sub.2NCH.sub.2—; R.sub.13 is selected from the group consisting of H, a linear or branched C.sub.1-8alkyl, phenyl, thienyl, furyl, cyclohexyl, biphenylyl, phenoxyphenyl, benzyloxyphenyl, phenylethoxylphenyl, piperidyl, N-benzylpiperidyl, naphthyl, indolyl and uracil group, R.sub.13 is optionally substituted by one or two substituents selected from the group consisting of halogen, methyl, hydroxyl, C.sub.1-4alkyl, C.sub.1-4alkoxy, hydroxyl, nitro, trifluoromethyl, cyano, C.sub.1-4alkyl monosubstituted or polysubstituted by halogen, or C.sub.1-4alkoxyl monosubstituted or polysubstituted by halogen.
4. The compound of Formula Ia, or a pharmaceutically acceptable salt, a hydrate or a solvate thereof according to claim 2, wherein: X is O or N; Y is N; n is 0, 1, 2, 3, 4, 5, 6 or 7; R.sub.4 is H, methyl, halogen, C.sub.1-4alkyl, C.sub.1-4alkoxy, hydroxyl, nitro, trifluoromethyl, cyano, CH.sub.3C(═O)CH.sub.2— or (CH.sub.3).sub.2NCH.sub.2—; R.sub.13 is selected from the group consisting of H, a linear or branched alkyl containing 8 carbon atoms, ##STR00120## ##STR00121##
5. The compound of Formula Ia, or a pharmaceutically acceptable salt, a hydrate or a solvate thereof according to claim 2, wherein: X is O or N; Y is N; n is 4, 5, 6 or 7; R.sub.4 is H, methyl, halogen, C.sub.1-4alkyl, C.sub.1-4alkoxy, hydroxyl, nitro, trifluoromethyl, cyano or CH.sub.3C(═O)CH.sub.2—; R.sub.13 is selected from the group consisting of H, a linear alkyl containing 8 carbon atoms, ##STR00122##
6. The compound of Formula Ia, or a pharmaceutically acceptable salt, a hydrate or a solvate thereof according to claim 2, wherein: X is O or N; Y is N; n is 4, 5, 6 or 7; R.sub.4 is H, methyl or CH.sub.3C(═O)CH.sub.2—; R.sub.13 is selected from the group consisting of H, a linear alkyl containing 8 carbon atoms, ##STR00123##
7. The compound of Formula Ia, or a pharmaceutically acceptable salt, a hydrate or a solvate thereof according to claim 2, wherein: X is O; Y is N; n is 4, 5, 6 or 7; R.sub.4 is H, methyl, halogen, C.sub.1-4alkyl, C.sub.1-4alkoxy, hydroxyl, nitro, trifluoromethyl, cyano or CH.sub.3C(═O)CH.sub.2—; R.sub.13 is selected from the group consisting of H, a linear alkyl containing 8 carbon atoms, ##STR00124##
8. The compound of Formula Ia, or a pharmaceutically acceptable salt, a hydrate or a solvate thereof according to claim 2, wherein: X is O; Y is N; n is 4, 5, 6 or 7; R.sub.4 is H, methyl or CH.sub.3C(═O)CH.sub.2—; R.sub.13 is selected from the group consisting of H, a linear alkyl containing 8 carbon atoms, ##STR00125##
9. The compound of Formula Ia, or a pharmaceutically acceptable salt, a hydrate or a solvate thereof according to claim 2, wherein: X is O; Y is N; n is 0 or 1; R.sub.4 is H, methyl, halogen, C.sub.1-4alkyl, C.sub.1-4alkoxy, hydroxyl, nitro, trifluoromethyl, cyano or (CH.sub.3).sub.2NCH.sub.2—; R.sub.13 is selected from the group consisting of H, a linear or branched alkyl containing 8 carbon atoms, ##STR00126##
10. The compound of Formula Ia, or a pharmaceutically acceptable salt, a hydrate or a solvate thereof according to claim 2, wherein: X is O; Y is N; n is 0 or 1; R.sub.4 is H or (CH.sub.3).sub.2NCH.sub.2—; R.sub.13 is selected from the group consisting of H, a linear or branched alkyl containing 8 carbon atoms, ##STR00127##
11. The compound, or a pharmaceutically acceptable salt, a hydrate or a solvate thereof according to claim 2, selected from the group consisting of: ##STR00128## ##STR00129## ##STR00130## ##STR00131## ##STR00132## ##STR00133## ##STR00134##
12. A method for preparing the compound of Formula Ia, or a pharmaceutically acceptable salt, a hydrate or a solvate thereof according to claim 2, comprising: in a solution, reacting a compound of Formula II with a compound of Formula III to obtain a compound of Formula Ia, ##STR00135## wherein X, Y, R.sub.4, R.sub.13 and n are as defined in claim 2.
13. The method according to claim 12, wherein the solution is a solution containing n-butyllithium, and the reaction temperature is −78° C.˜0° C.
14. A pharmaceutical composition, comprising at least the compound, or a pharmaceutically acceptable salt, a hydrate or a solvate thereof according to claim 2, and a pharmaceutically acceptable adjuvant.
15. A method for treating a disease or disorder, or alleviating severity of said disease or disorder, comprising administering to a patient in need of the treatment a therapeutically effective amount of at least one compound, or a pharmaceutically acceptable salt, a hydrate or a solvate thereof according to claim 2.
16. (canceled)
17. (canceled)
18. The method according to claim 15, wherein said disease or disorder is pain.
19. The method according to claim 18, wherein said pain is selected from the group consisting of: inflammatory pain.
20. The method according to claim 23, wherein said peripheral neuropathic pain is selected from the group consisting of: postherpetic neuralgia, pain caused by diabetic perineuropathy, neurothlipsis and exudation caused by tumor, lumbar surgery failure syndrome, neuropathic pain caused by lumbar disc protrusion, postpartum neuralgia, trigeminal neuralgia, chemotherapy-induced multiple neuropathic pain, post-radiotherapy plexopathy, and radicular neuralgia.
21. The method according to claim 23, wherein said central neuropathic pain is selected from the group consisting of: compression pain caused by spinal sclerosis, multiple sclerosis related pain, Parkinsonism related pain, dementia related pain, post-stroke pain, and pain following spinal cord injury.
22. The method according to claim 23, wherein said visceral inflammatory pain is selected from the group consisting of: appendicitis, gastritis, pancreatitis, prostatitis, myocarditis, interstitial cystitis, pain caused by hepatic, gall or kidney stone, irritable bowel syndrome, and chronic pelvic pain syndrome.
23. The method according to claim 19, wherein said neuropathic pain is selected from the group consisting of central neuropathic pain, and peripheral neuropathic pain, and/or wherein said inflammatory pain is selected from the group consisting of: osteoarthritis pain, fibromyalgia syndrome, inflammatory pain of rheumatic and rheumatoid arthritis, inflammatory pain of endometriosis, inflammatory toothache, ankylosing spondylitis pain, gouty arthritis pain, and visceral inflammatory pain, and/or wherein said mixed pain is selected from the group consisting of lumbodynia, shoulder pain, burning mouth syndrome, complex regional pain syndrome, migraine, cluster headache, and tension headache syndrome, and prosopodynia.
Description
DESCRIPTION OF THE DRAWINGS
[0144]
[0145]
[0146]
[0147]
[0148]
[0149]
[0150]
SPECIFIC MODES FOR CARRYING OUT THE INVENTION
[0151] The embodiments of the invention are described in detail by combining the following examples. However, a person skilled in the art understands that the following examples are only intender to illustrate the invention, and shall not be regarded as defining the scope of the invention. When the particular techniques or conditions are not indicated in Examples, the invention is carried out according to the techniques or conditions described in the prior art documents or according to the product instruction. The reagents or apparatuses, the manufacturers of which are not indicated, are the conventional products that are commercially available.
EXAMPLES
Preparation of Compounds 1-46 by the Following General Synthetic Method
[0152] At −78° C., to a tetrahydrofuran (THF) solution containing oxazolidinone (44 mg, 0.5 mmol), a hexane solution containing n-butyl lithium (0.55 mmol, 0.2 mL) (0.55 mmol, 0.2 mL) was slowly added dropwisely. After stirring for 10 min at the temperature, a tetrahydrofuran solution containing acyl chloride (0.55 mmol, 1 mL) was added dropwisely to the reaction solution. The reaction solution was stirred at −78° C. for 0.5 h, the temperature was increased slowly to room temperature within 4 h, and the reaction solution was stirred at room temperature for 1 h. After the reaction, saturated NH.sub.4Cl solution (5 mL) was added dropwisely to the reaction solution, ethyl acetate was used to extract the water phase for three times, the ethyl acetate phases were combined, and dried with anhydrous sodium sulfate, ethyl acetate was removed by evaporation under reduced pressure, and the residue was purified by silica gel chromatography to obtain the corresponding compound, wherein the solvent for purification was a mixture of ethyl acetate and petroleum ether, the ratio of which by volume was ethyl acetate:petroleum ether=1:5˜1:1.
Palmitoyl oxazolidinone (1)
[0153] ##STR00029##
[0154] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:5, the acyl chloride was palmitoyl chloride), the reaction was carried out to obtain product 1 (83 mg; yield: 51%, amorphous white powder).
[0155] IR (film) vmax: 2914, 2846, 1765, 1699, 1386 cm-1; 1H NMR (400 MHz, CDCl3) δ 0.88 (t, J=7.2 Hz, 3H), 1.26-1.32 (m, 24H), 1.62-1.69 (m, 2H), 2.91 (t, J=7.6 Hz, 2H), 4.02 (t, J=8.0 Hz, 2H), 4.41 (t, J=8.0 Hz, 2H) ppm; 13C NMR (100 MHz, CDCl3) δ 14.0, 22.6, 24.2, 29.1, 29.3, 29.4, 29.5, 29.6, 29.6, 31.8, 35.0, 42.5, 61.9, 153.5, 173.5 ppm; MS (ESI, m/z): 326 (M+H)+; Anal. calcd for C19H35NO3:C, 70.11; H, 10.84; N, 4.30. Found: C, 70.13; H, 10.86; N, 4.32.
Phenylhexanoyl oxazolidinone (2)
[0156] ##STR00030##
[0157] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:4, the acyl chloride was phenylhexanoyl chloride), the reaction was carried out to obtain product 2 (41 mg; yield: 31%, amorphous white powder).
[0158] IR (film) vmax: 2920, 2858, 1780, 1700, 1388, 1225, 1111, 1039 cm-1; 1H NMR (400 MHz, CDCl3) δ 1.36-1.44 (m, 2H), 1.61-1.73 (m, 4H), 2.61 (t, J=7.6 Hz, 2H), 2.90 (t, J=7.6 Hz, 2H), 3.97 (t, J=8.4 Hz, 2H), 4.36 (t, J=8.4 Hz, 2H), 7.16-7.18 (m, 3H), 7.24-7.28 (m, 2H) ppm; 13C NMR (100 MHz, CDCl3) δ 24.0, 28.6, 31.1, 34.9, 35.6, 42.4, 61.9, 125.5, 128.2, 128.3, 142.4, 153.4, 173.3 ppm; MS (ESI, m/z): 262 (M+H)+; HRMS (ESI) calcd for [C15H20NO3]+(M+H+): 262.1438; found:262.1451; Anal. calcd for C15H19NO3:C, 68.94; H, 7.33; N, 5.36. Found: C, 68.70; H, 7.31; N, 5.38.
Phenylheptanoyl oxazolidinone (3)
[0159] ##STR00031##
[0160] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:4, the acyl chloride was phenylheptanoyl chloride), the reaction was carried out to obtain product 3 (61 mg; yield: 44%, amorphous white powder).
[0161] IR (film) vmax: 2918, 2850, 1781, 1703, 1386, 1225, 1039 cm-1; 1H NMR (400 MHz, CDCl3) δ 1.35-1.42 (m, 4H), 1.58-1.69 (m, 4H), 2.60 (t, J=7.6 Hz, 2H), 2.90 (t, J=7.6 Hz, 2H), 3.98 (t, J=8.4 Hz, 2H), 4.36 (t, J=8.4 Hz, 2H), 7.16-7.17 (m, 3H), 7.24-7.28 (m, 2H) ppm; 113C NMR (100 MHz, CDCl3) δ 24.1, 28.9, 28.9, 31.2, 35.0, 35.8, 42.4, 61.9, 125.5, 128.1, 128.3, 142.6, 153.5, 173.4 ppm; MS (ESI, m/z): 276 (M+H)+; Anal. calcd for C16H21NO3:C, 69.79; H, 7.69; N, 5.09. Found: C, 69.83; H, 7.68; N, 5.10.
Phenyloctanoyl oxazolidinone (4)
[0162] ##STR00032##
[0163] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:4, the acyl chloride was phenyloctanoyl chloride), the reaction was carried out to obtain product 4 (61 mg; yield: 44%, amorphous white powder).
[0164] IR (film) vmax: 2920, 2848, 1780, 1703, 1386, 1225, 1020 cm-1; 1H NMR (400 MHz, CDCl3) δ 1.35-1.42 (m, 4H), 1.58-1.69 (m, 4H), 2.60 (t, J=7.6 Hz, 2H), 2.90 (t, J=7.6 Hz, 2H), 3.98 (t, J=8.4 Hz, 2H), 4.36 (t, J=8.4 Hz, 2H), 7.16-7.17 (m, 3H), 7.24-7.28 (m, 2H) ppm; 113C NMR (100 MHz, CDCl3) δ 24.1, 28.9, 28.9, 31.2, 35.0, 35.8, 42.4, 61.9, 125.5, 128.1, 128.3, 142.6, 153.5, 173.4 ppm; MS (ESI, m/z): 290 (M+H)+; Anal. calcd for C17H23NO3:C, 70.56; H, 8.01; N, 4.84. Found: C, 70.61; H, 7.99; N, 4.85.
3-Thienylhexanoyl oxazolidinone (5)
[0165] ##STR00033##
[0166] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:3, the acyl chloride was 3-thienylhexanoyl chloride); the reaction was carried out to obtain product 5 (57 mg; yield: 43%, amorphous white powder).
[0167] IR (film) vmax: 2917, 2849, 1777, 1697, 1386, 1222 cm-1; 1H NMR (400 MHz, CDCl3) δ 1.36-1.44 (m, 2H), 1.62-1.73 (m, 4H), 2.63 (t, J=7.6 Hz, 2H), 2.90 (t, J=7.6 Hz, 2H), 3.98 (t, J=8.0 Hz, 2H), 3.37 (t, J=8.0 Hz, 2H), 6.91-6.92 (m, 2H), 7.21-7.23 (m, 1H) ppm; 13C NMR (100 MHz, CDCl3) δ 23.9, 28.6, 29.9, 30.1, 34.9, 42.4, 61.9, 119.8, 125.0, 128.1, 142.7, 153.4, 173.3 ppm; MS (ESI, m/z): 268 (M+H)+; Anal. calcd for C13H17NO3S:C, 58.40; H, 6.41. Found: C, 58.57; H, 6.40.
Cyclohexylhexanoyl oxazolidinone (6)
[0168] ##STR00034##
[0169] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:5, the acyl chloride was cyclohexylhexanoyl chloride), the reaction was carried out to obtain product 6 (57 mg; yield: 43%, amorphous white powder).
[0170] IR (film) vmax: 2917, 2849, 1577, 1541, 1468, 1384, 1068, 1023 cm-1; 1H NMR (400 MHz, CDCl3) δ 0.82-0.90 (m, 2H), 1.15-1.23 (m, 6H), 1.32-1.34 (m, 4H), 1.63-1.70 (m, 7H), 2.92 (t, J=7.6 Hz, 2H), 4.03 (t, J=8.0 Hz, 2H), 4.42 (t, J=8.0 Hz, 2H) ppm; 13C NMR (100 MHz, CDCl3) δ 24.3, 26.4, 26.5, 26.7, 29.4, 33.4, 25.0, 37.3, 37.6, 42.5, 61.9, 153.5, 173.6 ppm; MS (ESI, m/z): 268 (M+H)+; Anal. calcd for C15H25NO3:C, 67.38; H, 9.42; N, 5.24. Found: C, 67.15; H, 9.41; N, 5.25.
3-Furylhexanoyl oxazolidinone (7)
[0171] ##STR00035##
[0172] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:3, the acyl chloride was 3-furylhexanoyl chloride), the reaction was carried out to obtain product 7 (6.3 mg; yield: 5%, white oily substance).
[0173] 1H NMR (400 MHz, CDCl3) δ 1.34-1.43 (m, 2H), 1.52-1.73 (m, 4H), 2.61 (t, J=7.6 Hz, 2H), 2.90 (t, J=7.6 Hz, 2H), 3.98 (t, J=8.0 Hz, 2H), 3.37 (t, J=8.0 Hz, 2H), 6.25(br, 1H), 7.19 (br, 1H), 7.33 (t, J=1.4 Hz, 1H) ppm; 13C NMR (100 MHz, CDCl3) δ 24.3, 24.0, 28.6, 31.1, 34.9, 42.4, 61.9, 110.9, 124.8, 138.7, 142.6, 153.4, 173.3 ppm; MS (ESI, m/z): 252 (M+H)+
3-Fluorophenylhexanoyl oxazolidinone (8)
[0174] ##STR00036##
[0175] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:5, the acyl chloride was 3-fluorophenylhexanoyl chloride), the reaction was carried out to obtain product 8 (57 mg; yield: 51%, amorphous white powder).
[0176] IR (film) vmax: 2918, 2850, 1779, 1699, 1617, 1585, 1387, 1223, 1044 cm-1; 1H NMR (400 MHz, CDCl3) δ 1.36-1.44 (m, 2H), 1.61-1.74 (m, 4H), 2.61 (t, J=7.6 Hz, 2H), 2.92 (t, J=7.6 Hz, 2H), 4.00 (t, J=8.4 Hz, 2H), 4.40 (t, J=8.4 Hz, 2H), 6.84-6.88 (m, 2H), 6.94 (d, J=7.5 Hz, 1H), 7.19-7.24 (m, 1H) ppm; 13C NMR (100 MHz, CDCl3) δ 24.0, 28.6, 30.8, 34.9, 35.4, 42.5, 61.9, 112.4,112.6,115.1,115.3, 124.0,124.1,145.0,145.1, 153.5, 161.7, 164.1, 173.4 ppm; MS (ESI, m/z): 280 (M+H)+; Anal. calcd for C15H18FNO3:C, 64.50; H, 6.50; N, 5.01. Found: C, 64.37; H, 6.49; N, 5.00.
3-Chlorophenylhexanoyl oxazolidinone (9)
[0177] ##STR00037##
[0178] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:3, the acyl chloride was 3-chlorophenylhexanoyl chloride), the reaction was carried out to obtain product 9 (57 mg; yield: 51%, amorphous white powder).
[0179] IR (film) vmax: 2917, 2849, 1777, 1692, 1573, 1537, 1468, 1383, 1202, 1066, 1036 cm-1; 1H NMR (400 MHz, CDCl3) δ 1.35-1.43 (m, 2H), 1.60-1.73 (m, 4H), 2.59 (t, J=7.6 Hz, 2H), 2.90 (t, J=7.6 Hz, 2H), 3.99 (t,=8.3 Hz, 2H), 4.39 (t, J=8.3 Hz, 2H), 7.04 (d, J=7.3 Hz, 1H), 7.13-7.20 (m, 3H) ppm; 13C NMR (100 MHz, CDCl3) δ 23.9, 28.5, 30.8, 34.9, 35.3, 43.4, 61.9, 125.7, 126.6, 128.4, 129.4, 133.8, 144.5, 153.5, 173.3 ppm; MS (ESI, m/z): 296 (M+H)+; Anal. calcd for C15H18ClNO3:C, 60.91; H, 6.13; N, 4.74. Found: C, 60.77; H, 6.15; N, 4.73.
4-Chlorophenylhexanovl oxazolidinone (10)
[0180] ##STR00038##
[0181] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:3, the acyl chloride was 4-chlorophenylhexanoyl chloride), the reaction was carried out to obtain product 10 (55 mg; yield: 49%, amorphous white powder).
[0182] IR (film) vmax: 2917, 2849, 1782, 1709, 1579, 1495, 1384, 1222, 1091, 1040 cm-1; 1H NMR (400 MHz, CDCl3) δ 1.35-1.42 (m, 2H), 1.59-1.71 (m, 4H), 2.58 (t, J=7.6 Hz, 2H), 2.90 (t, J=7.6 Hz, 2H), 4.00 (t, J=8.0 Hz, 2H), 4.39 (t, J=8.0 Hz, 2H), 7.09 (d, J=8.2 Hz, 2H), 7.23 (d, J=8.2 Hz, 2H) ppm; 13C NMR (100 MHz, CDCl3) δ 224.0, 24.4, 28.5, 31.0, 34.9, 42.4, 61.9, 128.3, 129.7, 140.9, 153.5, 173.4 ppm; MS (ESI, m/z): 296 (M+H)+; Anal. calcd for C15H18ClNO3:C, 60.91; H, 6.13; N, 4.74. Found: C, 60.83; H, 6.14; N, 4.75.
5-Acetonyl-3′-methylphenylhexanoyl oxazolidinone (11)
[0183] ##STR00039##
[0184] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:3, the acyl chloride was 3-methylphenylhexanoyl chloride, the reaction material oxazolidinone was replaced by 5-acetonyloxazolidinone), the reaction was carried out to obtain product 11 (72 mg; yield: 52%, amorphous white powder).
[0185] IR (film) vmax: 2955, 2917, 2849, 1776, 1701, 1571, 1541, 1465, 1384, 1069 cm-1; 1H NMR (400 MHz, CDCl3) δ 1.36-1.44 (m, 2H), 1.60-1.73 (m, 4H), 2.32 (s, 1H), 2.57 (t, J=7.6 Hz, 2H), 2.91 (t, J=7.6 Hz, 2H), 4.00 (t, J=8.2 Hz, 2H), 4.38 (t, J=8.2 Hz, 2H), 6.95-6.98 (m, 3H), 7.16 (dd, J=7.3, 7.3 Hz, 1H) ppm; 13C NMR (100 MHz, CDCl3) δ 21.4, 24.0, 28.7, 31.2, 34.9, 35.6, 42.5, 61.9, 125.4, 126.3, 128.1, 129.2, 137.7, 142.5, 153.5, 173.4 ppm; MS (ESI, m/z): 332 (M+H)+; Anal. calcd for C19H25NO4.
[0186] 5-Acetonyl-4′-methylphenylvaleryl oxazolidinone (12)
##STR00040##
[0187] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:3, the acyl chloride was 3-methylphenylhexanoyl chloride, the reaction material oxazolidinone was replaced by 5-acetonyloxazolidinone), the reaction was carried out to obtain product 12 (76 mg; yield: 55%, amorphous white powder).
[0188] IR (film) vmax: 2953, 2917, 2849, 1776, 1701, 1579, 1537, 1468, 1384, 1069, 1024 cm-1; 1H NMR (400 MHz, CDCl3) δ 1.35-1.43 (m, 2H), 1.59-1.73 (m, 4H), 2.31 (s, 1H), 2.57 (t, J=7.7 Hz, 2H), 2.90 (t, J=7.7 Hz, 2H), 4.00 (t, J=7.8 Hz, 2H), 4.38 (t, J=7.8 Hz, 2H), 7.04-7.09 (m, 4H) ppm; 13C NMR (100 MHz, CDCl3) δ 20.9, 24.0, 28.7, 31.2, 34.9, 35.2, 42.4, 61.9, 128.2, 128.9, 134.9, 139.4, 153.5, 173.4 ppm; MS (ESI, m/z): 332 (M+H)+; Anal. calcd for C19H25NO4.
3-Bibenzoyl oxazolidinone (13)
[0189] ##STR00041##
[0190] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1: 2, the acyl chloride was 3-bibenzoyl chloride), the reaction was carried out to obtain product 13 (81 mg; yield: 61%, amorphous white powder).
[0191] IR (film) vmax: 2916, 2848, 1633, 1565, 1406, 1107, 1037 cm-1; 1H NMR (400 MHz, CDCl3) δ 4.19-4.20 (m, 2H), 4.52 (br, 2H), 7.36-7.38 (m, 1H), 7.44 (br, 2H), 7.50-7.52 (m, 1H), 7.59-7.61 (m, 3H), 7.77-7.78 (br, 1H), 7.87 (s, 1H) ppm; 13C NMR (100 MHz, CDCl3) δ 43.7, 48.0, 48.2, 48.4, 48.6, 48.9, 62.6, 127.0, 127.6, 128.3, 128.7, 130.8, 140.0, 140.9, 153.9, 170.0 ppm; MS (ESI, m/z): 268 (M+H)+; Anal. calcd for C16H13NO3:C, 71.90; H, 4.90; N, 5.24. Found: C, 71.74; H, 4.89; N, 5.25.
3-Biphenylacetyl oxazolidinone (14)
[0192] ##STR00042##
[0193] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:3, the acyl chloride was 3-biphenylacetyl chloride), the reaction was carried out to obtain product 14 (63 mg; yield: 45%, amorphous white powder).
[0194] IR (film) vmax: 2916, 2848, 1776, 1697, 1598, 1478, 1387, 1366, 1223, 1180, 1109 cm-1; 1H NMR (400 MHz, CDCl3) δ 4.02 (t, J=8.0 Hz, 2H), 4.35 (s, 2H), 4.39 (t, J=8.0 Hz, 2H), 7.31 (d, J=7.2 Hz, 1H), 7.34 (d, J=7.2 Hz, 1H), 7.38-7.44 (m, 3H), 7.50 (d, J=7.6 Hz, 1H), 7.55-7.60 (m, 1H) ppm; 13C NMR (100 MHz, CDCl3) δ 41.1, 42.7, 61.9, 126.0, 127.2, 127.3, 128.6, 128.6, 128.7, 128.9, 134.0, 140.8, 141.5, 153.5, 171.2 ppm; MS (ESI, m/z): 282 (M+H)+; Anal. calcd for C17H15NO3:C, 72.58; H, 5.37; N, 4.98. Found: C, 72.71; H, 5.39; N, 4.96.
4-Biphenylacetyl oxazolidinone (15)
[0195] ##STR00043##
[0196] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:3, the acyl chloride was 4-biphenylacetyl chloride), the reaction was carried out to obtain product 15 (65 mg; yield: 46%, amorphous white powder).
[0197] IR (film) vmax: 2914, 2844, 1776, 1697, 1578, 1386, 1366, 1025 cm-1; 1H NMR (400 MHz, CDCl3) δ 4.00 (t, J=8.4 Hz, 2H), 4.31 (s, 2H), 4.36 (t, J=8.4 Hz, 2H), 7.33 (t, J=7.6 Hz, 1H), 7.37-7.44 (m, 4H), 7.54-7.58 (m, 4H) ppm; 13C NMR (100 MHz, CDCl3) δ 40.7, 42.6, 61.9, 127.0, 127.2, 128.7, 130.1, 132.5, 140.0, 140.7, 153.4, 171.1 ppm; MS (ESI, m/z): 282 (M+H)+; Anal. calcd for C17H15NO3:C, 72.58; H, 5.37; N, 4.98. Found: C, 72.67; H, 5.38; N, 4.97.
3-Phenoxybenzoyl oxazolidinone (16)
[0198] ##STR00044##
[0199] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1: 2, the acyl chloride was 3-phenoxybenzoyl chloride), the reaction was carried out to obtain product 16 (65 mg; yield: 48%, amorphous white powder).
[0200] IR (film) vmax: 2917, 2845, 1783, 1682, 1577, 1483, 1436, 1322, 1200 cm-1; 1H NMR (400 MHz, CDCl3) δ 4.14 (t, J=8.0 Hz, 2H), 4.46 (t, J=8.0 Hz, 2H), 7.04 (dd, J=8.6, 1.0 Hz, 2H), 7.12 (dd, J=7.4, 0.9 Hz, 1H), 7.18 (dddd, J=7.2, 7.2, 2.0, 2.0 Hz, 1H), 7.26 (br, 1H), 7.33-7.39 (m, 4H) ppm; 13C NMR (100 MHz, CDCl3) δ 43.6, 62.2, 119.1, 119.1, 122.6, 123.6, 123.7, 129.4, 129.9, 134.3, 153.0, 156.6, 156.8, 169.1 ppm; MS (ESI, m/z): 284 (M+H)+; Anal. calcd for C16H13NO4:C, 67.84; H, 4.63; N, 4.94. Found: C, 68.01; H, 4.64; N, 4.93.
3-Phenoxyphenylacetyl oxazolidinone (17)
[0201] ##STR00045##
[0202] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1: 2, the acyl chloride was 3-phenoxyphenylacetyl chloride), the reaction was carried out to obtain product 17(86 mg; yield: 53%, amorphous white powder).
[0203] IR (film) vmax: 2914, 2844, 1777, 1701, 1583, 1486, 1387, 1366, 1269, 1246, 1211 cm-1; 1H NMR (400 MHz, CDCl3) δ 3.99 (t, J=8.0 Hz, 2H), 4.24 (s, 1H), 4.37 (t, J=8.0 Hz, 2H), 6.89 (d, J=7.8 Hz, 1H), 6.96 (s, 1H), 7.01 (dd, J=8.4, 8.4 Hz, 3H), 7.09 (dd, J=7.8 Hz, 1H), 7.25 (d, J=7.8 Hz, 1H), 7.31 (dd, J=8.4, 8.4 Hz, 2H) ppm; 13C NMR (100 MHz, CDCl3) δ 40.9, 42.6, 61.9, 117.4, 118.9, 120.1, 123.2, 124.5, 129.7, 135.3, 153.4, 156.9, 157.2, 170.8 ppm; MS (ESI, m/z): 298 (M+H)+; Anal. calcd for C17H15NO4:C, 68.68; H, 5.09; N, 4.71. Found: C, 68.51; H, 5.08; N, 4.72.
4-Phenoxyphenylacetyl oxazolidinone (18)
[0204] ##STR00046##
[0205] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1: 2, the acyl chloride was 4-phenoxyphenylacetyl chloride), the reaction was carried out to obtain product 18 (88 mg; yield: 59%, amorphous white powder).
[0206] IR (film) vmax: 2916, 2848, 1777, 1577, 1537, 1486, 1467, 1385, 1237, 1108, 1040 cm-1; 1H NMR (400 MHz, CDCl3) δ 4.02 (t, J=8.0 Hz, 2H), 4.25 (s, 2H), 4.39 (t, J=8.0 Hz, 2H), 6.95 (d, J=8.4 Hz, 2H), 7.00 (d, J=7.6 Hz, 2H), 7.09 (t, J=7.6 Hz, 1H), 7.27 (d, J=8.4 Hz, 2H), 7.32 (t, J=7.6 Hz, 2H) ppm; 13C NMR (100 MHz, CDCl3) δ 40.3, 42.6, 62.0, 118.8, 118.9, 123.3, 128.2, 129.7, 131.0, 153.4, 156.4, 157.0, 171.3 ppm; MS (ESI, m/z): 298 (M+H)+; Anal. calcd for C17H15NO4:C, 68.68; H, 5.09; N, 4.71. Found: C, 68.73; H, 5.10; N, 4.72.
3-Benzyloxybenzoyl oxazolidinone (19)
[0207] ##STR00047##
[0208] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1: 2, the acyl chloride was 3-benzyloxybenzoyl chloride), the reaction was carried out to obtain product 19(82 mg; yield: 55%, amorphous white powder).
[0209] IR (film) vmax: 2916, 2849, 1785, 1679, 1634, 1579, 1436, 1383, 1325, 1244, 1217, 1196, 1145, 1098, 1037 cm-1; 1H NMR (400 MHz, CDCl3) δ 4.16 (t, J=7.6 Hz, 2H), 4.48 (t, J=7.6 Hz, 2H), 5.08 (s, 2H), 7.14-7.44 (m, 9H) ppm; 13C NMR (100 MHz, CDCl3) δ 43.7, 62.2, 70.3, 115.1, 119.3, 121.7, 127.6, 128.1, 128.6, 129.0, 133.9, 136.5, 153.1, 158.3, 169.5 ppm; MS (ESI, m/z): 298 (M+H)+; Anal. calcd for C17H15NO4:C, 68.68; H, 5.09; N, 4.71. Found: C, 68.81; H, 5.11; N, 4.71.
3-Benzyloxyphenylacetyl oxazolidinone (20)
[0210] ##STR00048##
[0211] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1: 2, the acyl chloride was 3-benzyloxyphenylacetyl chloride), the reaction was carried out to obtain product 20 (76 mg; yield: 49%, amorphous white powder).
[0212] IR (film) vmax: 2916, 2849, 1777, 1698, 1583, 1489, 1449, 1387, 1365, 1272, 1224, 1158, 1109, 1039 cm-1; 1H NMR (400 MHz, CDCl3) δ 3.97 (t, J=8.1 Hz, 2H), 4.25 (s, 2H), 4.35 (t, J=8.1 Hz, 2H), 5.04 (s, 2H), 6.88 (d, J=7.9 Hz, 1H), 6.91 (d, J=7.9 Hz, 1H), 6.96 (s, 1H), 7.22 (dd, J=7.9, 7.9 Hz, 1H), 7.30 (dd, J=7.2, 7.2 Hz, 1H), 7.37 (dd, J=7.2, 7.2 Hz, 2H), 7.42 (d, J=7.2 Hz, 2H) ppm; 13C NMR (100 MHz, CDCl3) δ 41.0, 42.6, 61.9, 69.9, 113.7, 116.2, 122.3, 127.5, 127.9, 128.5, 129.5, 135.0, 137.0, 153.4, 158.9, 171.0 ppm; MS (ESI, m/z): 312 (M+H)+; Anal. calcd for C18H17NO4:C, 69.44; H, 5.50; N, 4.50. Found: C, 69.20; H, 5.49; N, 4.51.
4-Benzyloxybenzoyl oxazolidinone (21)
[0213] ##STR00049##
[0214] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1: 2, the acyl chloride was 4-benzyloxybenzoyl chloride), the reaction was carried out to obtain product 21 (108 mg; yield: 73%, amorphous white powder).
[0215] IR (film) vmax: 2916, 2849, 1774, 1670, 1607, 1512, 1378, 1338, 1249, 1194, 1108, 1024 cm-1; 1H NMR (400 MHz, CDCl3) δ 4.14 (t, J=8.0 Hz, 2H), 4.47 (t, J=8.0 Hz, 2H), 5.11 (s, 2H), 6.99 (d, J=8.8 Hz, 2H), 7.34-7.43 (m, 5H), 7.69 (d, J=8.8 Hz, 2H) ppm; 13C NMR (100 MHz, CDCl3) δ 43.9, 62.2, 70.1, 114.0, 124.7, 127.5, 128.2, 128.6, 131.8, 136.2, 153.5, 162.4, 169.0 ppm; MS (ESI, m/z): 298 (M+H)+; Anal. calcd for C17H15NO4:C, 68.68; H, 5.09; N, 4.71. Found: C, 68.90; H, 5.11; N, 4.69.
4-Benzyloxyphenylacetyl oxazolidinone (22)
[0216] ##STR00050##
[0217] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1: 2, the acyl chloride was 4-benzyloxyphenylacetyl chloride), the reaction was carried out to obtain product 22 (77 mg; yield: 50%, amorphous white powder).
[0218] IR (film) vmax: 2916, 2849, 1777, 1698, 1607, 1511, 1387, 1365, 1240, 1177, 1111, 1040, 1016 cm-1; 1H NMR (400 MHz, CDCl3) δ 3.96 (t, J=8.0 Hz, 2H), 4.20 (s, 2H), 4.33 (t, J=8.0 Hz, 2H), 5.03 (s, 2H), 6.92 (d, J=8.4 Hz, 2H), 7.22 (d, J=8.4 Hz, 2H), 7.30 (t, J=7.2 Hz, 1H), 7.35-7.42 (m, 4H) ppm; 13C NMR (100 MHz, CDCl3) δ 40.1, 42.6, 61.9, 69.9, 114.8, 125.8, 127.4, 127.9, 128.5, 130.7, 136.9, 153.4, 157.9, 171.5 ppm; MS (ESI, m/z): 312 (M+H)+; Anal. calcd for C18H17NO4:C, 69.44; H, 5.50; N, 4.50. Found: C, 69.65; H, 5.51; N, 4.50.
5-N,N-dimethyl methylamino-4′-phenylethoxylbenzoyl oxazolidinone (23)
[0219] ##STR00051##
[0220] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1: 1, the acyl chloride was 4-phenylethoxylbenzoyl chloride, the reaction material oxazolidinone was replaced by 5-N,N-dimethyl methylaminooxazolidinone), the reaction was carried out to obtain product 23 (84 mg; yield: 54%, amorphous white powder).
[0221] IR (film) vmax: 2916, 2849, 1777, 1672, 1605, 1508, 1384, 1324, 1307, 1254, 1168, 1094, 1029 cm-1; 1H NMR (400 MHz, CDCl3) δ 3.11 (t, J=7.2 Hz, 2H), 4.14 (t, J=8.0 Hz, 2H), 4.22 (t, J=7.2 Hz, 2H), 4.46 (t, J=8.0 Hz, 2H), 6.90 (d, J=8.4 Hz, 2H), 7.23-7.34 (m, 5H), 7.67 (d, J=8.4 Hz, 2H) ppm; 13C NMR (100 MHz, CDCl3) δ 35.6, 43.9, 62.2, 68.8, 113.7, 124.4, 126.6, 128.5, 129.0, 131.8, 137.8, 153.5, 162.5, 169.0 ppm; MS (ESI, m/z): 312 (M+H)+; Anal. calcd for C21H24N2O4:C, 68.46; H, 6.57; N, 7.60; O, 17.37; Found: C, 68.23; H, 6.44; N, 7.78; O, 17.45.
4-Benzylcyclohexanoyl oxazolidinone (24)
[0222] ##STR00052##
[0223] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:5, the acyl chloride was 4-benzylcyclohexanoyl chloride), the reaction was carried out to obtain product 24 (84 mg; yield: 54%, amorphous white powder).
[0224] 1H NMR (400 MHz, CDCl3) δ 1.53-1.68 (m, 6H), 1.77-1.90 (m, 3H), 2.61-2.63 (m,2H), 3.60-3.66 (t, 1H), 3.99-4.03 (t, J=8.0 Hz, 2H), 4.36-4.40(t, J=8.0 Hz, 2H), 7.11-7.19(m,3H), 7.25-7.28(m,2H) ppm; 13C NMR (100 MHz, CDCl3) δ 25.2, 28.6, 36.4, 39.8, 40.1, 42.8, 61.8, 125.6, 128.1, 129.0, 141.3, 153.1, 176.6 ppm.
N-benzylpiperidine-4-carbonyl oxazolidinone (25)
[0225] ##STR00053##
[0226] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:3, the acyl chloride was N-benzylpiperidine-4-carbonyl chloride), the reaction was carried out to obtain product 25 (27 mg; yield: 46%, amorphous white powder).
[0227] 1H NMR (400 MHz, CDCl3) δ 1.72-1.83 (m, 4H), 2.64 (m, 1H), 3.92-4.05 (m, 8H), 4.39 (t, J=8.0 Hz, 2H), 7.21-7.25 (m, 3H), 7.28-7.30 (m,2H) ppm; 13C NMR (100 MHz, CDCl3) δ 27.9, 28.3, 34.7, 38.6, 42.6, 42.8, 60.3, 125.7, 128.4, 130.6, 140.7, 153.1, 176.6 ppm; MS (ESI, m/z): 289 (M+H)+; Anal. calcd for C16H20N2O3:C, 66.65; H, 6.99; N, 9.72; O, 16.65.
Phenylhexanoyl -5-methyl-oxazolidinone (27)
[0228] ##STR00054##
[0229] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:4, the acyl chloride was phenylhexanoyl chloride, the reaction material oxazolidinone was replaced by 5-methyloxazolidinone), the reaction was carried out to obtain product 27 (57 mg; yield: 42%, amorphous white powder).
[0230] IR (film) vmax: 2937, 2872, 1764, 1725, 1683, 1363, 1231, 1058 cm-1; 1H NMR (400 MHz, CDCl3) δ 0.97 (d, J=5.6 Hz, 3H), 1.32-1.40 (m, 2H), 1.58-1.62 (m, 4H), 2.63 (t, J=7.5 Hz, 2H), 2.87 (t, J=7.5 Hz, 2H), 4.05 (m, 1H), 4.42 (dd, J=10.6, 8.4 Hz, 2H), 7.15-7.18 (m, 3H), 7.24-7.29 (m, 2H) ppm; MS (ESI, m/z): 276 (M+H)+; C16H21NO3, C, 69.79; H, 7.69; N, 5.09; O, 17.43.
4-Hydroxyphenylhexanoyl oxazolidinone (31)
[0231] ##STR00055##
[0232] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1: 2, the acyl chloride was p-hydroxyphenylhexanoyl chloride), the reaction was carried out to obtain product 31 (53 mg; yield: 68%, amorphous white powder).
[0233] IR (film) vmax: 3362, 3130, 2837, 1764, 1717, 1538, 1519, 1267, 1243, 1075 cm-1; 1H NMR (400 MHz, CDCl3) δ 1.38 (m, 2H), 1.62-1.68 (m, 4H), 2.27 (s, 1H), 2.54 (t, J=7.0 Hz, 2H), 2.96 (t, J=7.0 Hz, 2H), 4.07 (t, J=7.2 Hz, 2H), 4.29 (t, J=7.2 Hz, 2H), 6.82 (d, J=6.9 Hz, 2H), 7.43 (d, J=6.9 Hz, 2H) ppm; 13C NMR (100 MHz, CDCl3) δ 21.7, 23.8, 26.7, 30.5, 31.3, 35.6, 41.7, 60.8, 114.7, 134.9, 136.4, 162.8, 172.4 ppm; MS (ESI, m/z): 278 (M+H)+; Anal. calcd for C15H19NO4:C, 64.97; H, 6.91; N, 5.05; O, 23.08.
3-Methylphenylhexanoyl oxazolidinone (32)
[0234] ##STR00056##
[0235] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:5, the acyl chloride was 3-methylphenylhexanoyl chloride), the reaction was carried out to obtain product 32 (72 mg; yield: 52%, amorphous white powder).
[0236] IR (film) vmax: 2955, 2917, 2849, 1776, 1701, 1571, 1541, 1465, 1384, 1069 cm-1; 1H NMR (400 MHz, CDCl3) δ 1.36-1.44 (m, 2H), 1.60-1.73 (m, 4H), 2.32 (s, 3H), 2.57 (t, J=7.6 Hz, 2H), 2.91 (t, J=7.6 Hz, 2H), 4.00 (t, J=8.2 Hz, 2H), 4.38 (t, J=8.2 Hz, 2H), 6.95-6.98 (m, 3H), 7.16 (dd, J=7.3, 7.3 Hz, 1H) ppm; 13C NMR (100 MHz, CDCl3) δ 21.4, 24.0, 28.7, 31.2, 34.9, 35.6, 42.5, 61.9, 125.4, 126.3, 128.1, 129.2, 137.7, 142.5, 153.5, 173.4 ppm; MS (ESI, m/z): 276 (M+H)+; Anal. calcd for C16H21NO3:C, 69.79; H, 7.69; N, 5.09. Found: C, 69.61; H, 7.67; N, 5.10.
4-Methylphenylhexanoyl oxazolidinone (33)
[0237] ##STR00057##
[0238] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:5, the acyl chloride was p-methylphenylhexanoyl chloride), the reaction was carried out to obtain product 33 (148 mg; yield: 72%, amorphous white powder).
[0239] IR (film) vmax: 3034, 2921, 2850, 1700, 1458, 1405 cm-1; 1H NMR (400 MHz, CDCl3) δ 1.34-1.41 (m, 2H), 1.58-1.70 (m, 4H), 2.31 (s, 3H), 2.34 (t, J=7.6 Hz, 2H), 2.56 (t, J=7.6 Hz, 2H), 7.05 (d, J=8.4 Hz, 2H), 7.08 (d, J=8.4 Hz, 2H) ppm; 13C NMR (100 MHz, CDCl3) δ 20.9, 24.5, 28.6, 31.1, 34.0, 35.2, 128.2, 128.9, 135.0, 139.3, 153.5, 180.4 ppm; MS (ESI, m/z): 207 (M+H)+; Anal. calcd for C13H18O2:C, 75.69; H, 8.80. Found: C, 75.78; H, 8.82.
Piperidin-3-yl-hexanoyl oxazolidinone (34)
[0240] ##STR00058##
[0241] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:3, the acyl chloride was piperidin-3-yl-hexanoyl chloride), the reaction was carried out to obtain product 34 (26 mg; yield: 36%, amorphous white powder).
[0242] IR (film) vmax: 3314, 3017, 1577, 1541, 1468, 1023 cm-1; 1H NMR (400 MHz, CDCl3) δ 0.82-0.90 (m, 2H), 1.15-1.23 (m, 6H), 1.63-1.70 (m, 5H), 2.92 (t, J=7.6 Hz, 2H), 3.39 (t, J=7.2 Hz, 2H), 3.54 (t, J=7.2 Hz, 2H), 4.03 (t, J=8.0 Hz, 2H), 4.42 (t, J=8.0 Hz, 2H) ppm; 13C NMR (100 MHz, CDCl3) δ 24.3, 26.4, 29.4, 33.4, 25.0, 37.3, 37.6, 42.5, 42.7, 46.6, 61.9, 153.5, 173.6 ppm; MS (ESI, m/z): 269 (M+H)+; Anal. calcd for C14H24N2O3:C, 62.66; H, 9.01; N, 10.44, O, 17.89.
Phenylvaleryl oxazolidinone (35)
[0243] ##STR00059##
[0244] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:5, the acyl chloride was phenylvaleryl chloride), the reaction was carried out to obtain product 35 (69 mg; yield: 56%, amorphous white powder).
[0245] IR (film) vmax: 2924, 2858, 1780, 1700, 1497, 1478, 1453, 1388, 1225, 1111, 1040 cm-1; 1H NMR (400 MHz, CDCl3) δ 1.69-1.71 (m, 4H), 2.64 (t, J=7.2 Hz, 2H), 2.94 (t, J=7.2 Hz, 2H), 3.98 (t, J=8.4 Hz, 2H), 4.37 (t, J=8.4 Hz, 2H), 7.17-7.19 (m, 3H), 7.25-7.29 (m, 2H) ppm; 13C NMR (100 MHz, CDCl3) δ 23.8, 30.7, 34.8, 35.5, 42.4, 61.9,125.7, 128.2, 128.3, 142.1, 153.5, 173.3 ppm; MS (ESI, m/z): 248 (M+H)+; Anal. calcd for C14H17NO3:C, 68.00; H, 6.93; N, 5.66. Found: C, 68.20; H, 6.91; N, 5.67.
3-Chlorophenylvaleryl oxazolidinone (36)
[0246] ##STR00060##
[0247] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:4, the acyl chloride was 3-chlorophenylvaleryl chloride), the reaction was carried out to obtain product 36 (32 mg; yield: 43%, amorphous white powder).
[0248] IR (film) vmax: 2917, 2849, 1777, 1692, 1573, 1537, 1468, 1383, 1202, 1066, 1036 cm-1; 1H NMR (400 MHz, CDCl3) δ 1.35-1.43 (m, 2H), 1.60-1.73 (m, 2H), 2.59 (t, J=7.6 Hz, 2H), 2.90 (t, J=7.6 Hz, 2H), 3.99 (t, J=8.3 Hz, 2H), 4.39 (t, J=8.3 Hz, 2H), 7.04 (d, J=7.3 Hz, 1H), 7.13-7.20 (m, 3H) ppm; 13C NMR (100 MHz, CDCl3) δ 23.9, 30.8, 34.9, 35.3, 43.4, 61.9, 125.7, 126.6, 128.4, 129.4, 133.8, 144.5, 153.5, 173.3 ppm; MS (ESI, m/z): 282 (M+H)+; Anal. calcd for C14H16ClNO3:C, 59.68; H, 5.72; Cl, 12.58; N, 4.97; O, 17.04.
4-Fluorophenylvaleryl oxazolidinone (37)
[0249] ##STR00061##
[0250] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:4, the acyl chloride was 4-fluorophenylvaleryl chloride), the reaction was carried out to obtain product 37 (73 mg; yield: 62%, amorphous white powder).
[0251] IR (film) vmax: 2926, 2852, 1774, 1709, 1371, 1045 cm-1; 1H NMR (400 MHz, CDCl3) δ 1.35-1.42 (m, 2H), 1.64 (m, 2H), 2.58 (t, J=7.6 Hz, 2H), 2.90 (t, J=7.6 Hz, 2H), 4.04 (t, J=8.0 Hz, 2H), 4.35 (t, J=8.0 Hz, 2H), 7.07 (d, J=8.2 Hz, 2H), 7.23 (d, J=8.2 Hz, 2H) ppm; 13C NMR (100 MHz, CDCl3) δ 23.8, 24.3, 31.4, 34.6, 42.5, 62.6, 128.3, 128.7, 140.9, 153.5, 173.4 ppm; MS (ESI, m/z): 266 (M+H)+; Anal. calcd for C14H16FNO3:C, 63.39; H, 6.08; F, 7.16; N, 5.28; O, 18.09.
2,4-Dichlorophenylheptanoyl oxazolidinone (38)
[0252] ##STR00062##
[0253] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:3, the acyl chloride was 2,4-dichlorophenylheptanoyl chloride), the reaction was carried out to obtain product 38 (7.6 mg; yield: 14%, amorphous white powder).
[0254] IR (film) vmax: 2928, 2860, 1784, 1706, 1617, 1223, 1067, 986 cm-1; 1H NMR (400 MHz, CDCl3) δ 1.36-1.48 (m, 2H), 1.65-1.78 (m, 6H), 2.67 (t, J=7.6 Hz, 2H), 2.91 (t, J=7.6 Hz, 2H), 4.04 (t, J=8.4 Hz, 2H), 4.42 (t, J=8.4 Hz, 2H), 6.88-6.92 (m, 3H) ppm; 13C NMR (100 MHz, CDCl3) δ 24.2, 24.6, 28.6, 30.5, 35.1, 35.4, 42.5, 61.9, 112.4, 112.6, 116.7, 124.3, 124.6, 145.1, 153.5, 173.4 ppm; MS (ESI, m/z): 344:346 (3:2, M+H)+; Anal. calcd for C16H19Cl2NO3:C, 55.83; H, 5.56; Cl, 20.60; N, 4.07; O, 13.94.
3,4-Dihydroxyphenylhexanoyl oxazolidinone (39)
[0255] ##STR00063##
[0256] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:1, the acyl chloride was 3,4-dihydroxyphenylhexanoyl chloride), the reaction was carried out to obtain product 39 (47 mg; yield: 36%, amorphous white powder).
[0257] IR (film) vmax: 3130, 3017, 2939, 1785, 1724, 1242, 1085 cm-1; 1H NMR (400 MHz, CDCl3) δ 1.35 (m, 2H), 1.68-1.74 (m, 4H), 2.76 (t, J=7.6 Hz, 2H), 2.87 (t, J=7.6 Hz, 2H), 4.03 (t, J=8.4 Hz, 2H), 4.39 (t, J=8.4 Hz, 2H), 6.90 (s, 1H), 7.07 (d, 1H), 7.46 (d, 1H) ppm; 13C NMR (100 MHz, CDCl3) δ 24.2, 28.3, 31.4, 33.7, 36.4, 42.5, 61.3, 105.3, 114.8, 124.7, 148.4, 153.5, 158.5, 159.7, 173.4 ppm; MS (ESI, m/z): 294 (M+H)+; Anal. calcd for C15H19NO5: C, 61.42; H, 6.53; N, 4.78; O, 27.27.
Palmitoyl imidazolidinone (40)
[0258] ##STR00064##
[0259] By a method similar to the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:5, the acyl chloride was palmitoyl chloride, the reaction material oxazolidinone was replaced by imidazolidinone), the reaction was carried out to obtain product 40 (37 mg; yield: 54%, amorphous white powder).
[0260] IR (film) vmax: 3312, 3017, 1768, 1715, 1388 cm-1; 1H NMR (400 MHz, CDCl3) δ 0.88 (t, J=7.2 Hz, 3H), 1.26-1.32 (m, 24H), 1.62-1.69 (m, 2H), 2.93 (t, J=7.6 Hz, 2H), 4.05 (t, J=8.0 Hz, 2H), 4.38 (t, J=8.0 Hz, 2H) ppm; 13C NMR (100 MHz, CDCl3) δ 14.0, 22.7, 24.1, 28.6, 29.3, 29.4, 29.5, 29.6, 29.6, 31.8, 35.0, 40.3, 45.2, 153.2, 174.8 ppm; MS (ESI, m/z): 325 (M+H)+; Anal. calcd for C19H36N2O2:C, 70.32; H, 11.18; N, 8.63; O, 9.86.
4-Phenoxybenzoyl oxazolidinone (41)
[0261] ##STR00065##
[0262] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:3, the acyl chloride was 4-phenoxybenzoyl chloride), the reaction was carried out to obtain product 41 (78 mg; yield: 55%, amorphous white powder).
[0263] IR (film) vmax: 2915, 2844, 1779, 1681, 1575, 1482, 1445, 1382, 1322 cm-1; 1H NMR (400 MHz, CDCl3) δ 4.17 (t, J=8.0 Hz, 2H), 4.50 (t,=8.0 Hz, 2H), 6.97 (ddd, J=8.4, 2.8, 2.0 Hz, 2H), 7.08 (dd, J=8.4, 0.8 Hz, 2H), 7.19 (dd, J=7.6 Hz, 1H), 7.37-7.42 (ddd, J=8.4, 7.6, 2.0 Hz 2H), 7.67 (ddd, J=8.4, 2.8, 2.0 Hz, 2H) ppm; 13C NMR (100 MHz, CDCl3) δ 43.9, 62.2, 116.7, 120.3, 124.6, 126.4, 130.0, 131.7, 153.4, 155.5, 161.7, 168.9 ppm; MS (ESI, m/z): 284 (M+H)+; Anal. calcd for C16H13NO4:C, 67.84; H, 4.63; N, 4.94. Found: C, 67.75; H, 4.63; N, 4.95.
3′Chloro-4-phenoxybenzoyl oxazolidinone (42)
[0264] ##STR00066##
[0265] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:3, the acyl chloride was 3′chloro-4-phenoxybenzoyl chloride), the reaction was carried out to obtain product 42 (36 mg; yield: 42%, amorphous white powder).
[0266] IR (film) vmax: 2935, 2768, 1765, 1673, 1571 cm-1; 1H NMR (400 MHz, CDCl3) δ 4.11 (t, J=8.0 Hz, 2H), 4.43 (t, J=8.0 Hz, 2H), 6.89 (d, J=72.0 Hz, 1H), 6.97 (ddd, J=8.4, 2.8, 2.0 Hz, 2H), 7.08 (dd, J=8.4, 0.8 Hz, 2H), 7.11 (dd, J=7.6, 2.0 Hz 1H), 7.47-7.50 (m, 2H) ppm; 13C NMR (100 MHz, CDCl3) δ 42.5, 60.4, 116.7, 119.7, 124.6, 126.4, 127.6, 130.3, 132.7, 136.4, 145.3, 153.4, 157.6, 161.7, 168.9 ppm; MS (ESI, m/z): 318:320 (3:1, M+H)+; Anal. calcd for C16H12ClNO4:C, 60.48; H, 3.81; Cl, 11.16; N, 4.41; O, 20.14.
3-Indolyl-n-hexanoyl oxazolidinone (43)
[0267] ##STR00067##
[0268] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1: 2, the acyl chloride was 3-indolyl-n-hexanoyl chloride), the reaction was carried out to obtain product 43 (14 mg; yield: 27%, amorphous white powder).
[0269] 1H NMR (400 MHz, CDCl3) δ 1.35 (m, 2H), 1.62-1.65 (m, 4H), 2.47 (t, J=6.8 Hz, 2H), 2.92 (t, J=7.0 Hz, 2H), 4.73 (m, 2H), 7.26 (m, 2H), 7.56 (d, J=6.6 Hz, 1H), 8.30 (d, J=6.6 Hz, 1H), 8.33 (s, 1H) ppm; MS (ESI, m/z): 301 (M+H)+, C17H20N2O3.
Uracil-1-yl-n-hexanoyl oxazolidinone (44)
[0270] ##STR00068##
[0271] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1: 1, the acyl chloride was uracil-1-yl-n-hexanoyl chloride), the reaction was carried out to obtain product 44 (18 mg; yield: 38%, amorphous white powder).
[0272] 1H NMR (400 MHz, CDCl3) δ 1.37 (m, 2H), 1.61-1.65 (m, 4H), 2.47 (t, J=6.8 Hz, 2H), 2.90 (t, J=7.0 Hz, 2H), 5.80 (d, J=8.4 Hz, 1H), 8.21 (d, J=8.4 Hz, 1H), 11.72 (br, 1H) ppm; MS (ESI, m/z): 296 (M+H)+, C13H17N3O5.
2-Naphthylhexanoyl oxazolidinone (45)
[0273] ##STR00069##
[0274] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:5, the acyl chloride was 2-naphthylhexanoyl chloride), the reaction was carried out to obtain product 45 (27 mg; yield: 32%, amorphous white powder).
[0275] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 1.36 (m, 2H), 1.60-1.64 (m, 4H), 2.45 (t, J=6.8 Hz, 2H), 2.91 (t, J=7.0 Hz, 2H), 7.18-7.29 (m, 7H) ppm; MS (ESI, m/z): 312 (M+H).sup.+, C19H21NO3.
7-fluoro-2-naphthylhexanoyl oxazolidinone (46)
[0276] ##STR00070##
[0277] By the general synthetic method 1 (solvent for purification: ethyl acetate:petroleum ether=1:4, the acyl chloride was 7-fluoro-2-naphthylhexanoyl chloride), the reaction was carried out to obtain product 46 (15 mg; yield: 26%, amorphous white powder).
[0278] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 1.36 (m, 2H), 1.60-1.64 (m, 4H), 2.47 (t, J=6.8 Hz, 2H), 2.87 (t, J=7.0 Hz, 2H), 7.20-7.31 (m, 6H) ppm; MS (ESI, m/z): 330 (M+H).sup.+, C19H20FNO3.
[0279] In Experimental examples according to the invention, compounds to be evaluated or compounds to be tested or test compounds used are the Compounds 1-46 prepared in the Examples according to the invention.
Experimental Example 1
Method for Determining Endocannabinoid Hydrolase Activity
[0280] In the Experimental example, endocannabinoid hydrolases used were Fatty Acid Amide Hydrolase (FAAH) and N-acylethanolamide hydrolyzing acid amidase (NAAA), which were prepared by the method described in the document (PMCID: PMC3423427, PMC3723234, PMC2692831, PMC3382457). The preparation method was as followed: a plasmid (pCDNA3.1/NAAA or pCDNA3.1/FAAH) carrying a whole NAAA/FAAH gene was constructed, wherein the plasmid carried Cytomegalovirus (CMV) promoter and Neomycin selectable gene; the plasmid was transformed into HEK-293 cell via lipid medium, stable cell lines expressing NAAA/FAAH at a high level were obtained by G418 screening and Western-blot method. HEK-293 recombinant cells were cultured and collected, washed with PBS for 2˜3 times, and ultrasonically treated in 20 mM Tris-HCl containing 0.32 M sucrose, then repeatedly frozen and thawed twice, and then centrifuged at 4° C., 800 g for 15 min. The supernatant (i.e., the desired protein) was collected, the protein concentration was determined by BCA method, and the protein was diluted to a concentration of 1 mg/mL, and sub-packaged and stored in a refrigerator at −80° C. for further use.
[0281] In the Experimental example, PBS solution used was prepared as followed: 8 g NaCl, 0.2 g KCl, 1.44 g Na.sub.2HPO.sub.4, and 0.24 g KH.sub.2PO.sub.4 were dissolved in 1 L ultrapure water, and the resultant solution was subjected to moist heat sterilization and stored at 4° C.
[0282] 30 μL (1 mg/mL) endocannabinoid hydrolase was added to a sample vial, and 2 μL DMSO (Blank control group) or a different concentration of a test compound (Compounds 1-46 prepared in the Examples according to the invention) was further added. The reaction was carried out at 37° C. for 10 min. 170 μL buffer (the buffer consisted of 50 mM disodium Hydrogen Phosphate, 0.1% Triton X-100, 3 mM DTT, 150 μL) containing enzymatic hydrolysis substrate (the substrate was a heptadecenoyl ethanolamine containing a double bond and 17 carbon atoms, abbreviated as 17:1 FAE) was further added, wherein the concentration of 17:1 FAE was 5 μM. The reaction was carried out at 37° C. for 30 min, and 200 μL methanol solution containing internal standard (the internal standard was margaric acid, at a concentration of 1 nmol) was then added to stop the reaction. LC-MS was used to determine the yield of the hydrolysate 17:1 FA (i.e., a heptadecenoic acid containing a double bond) of 17:1 FAE, then a graph was plotted with Graphpad Prism 5. Thereby, the IC.sub.50 of the test compound on endocannabinoid hydrolase was determined.
[0283] By the method above, the inhibitory effects of the Compounds 1-46 prepared in the invention on NAAA and FAAH were determined. The results were shown in Table 1, wherein IC.sub.50 (NAAA) represents a concentration that inhibits NAAA activity to 50% of the activity prior to inhibition, IC.sub.50 (FAAH) represents a concentration that inhibits FAAH activity to 50% of the activity prior to inhibition, and “>100 μM” represents that IC.sub.50 of a compound on a corresponding enzyme is above 100 μM, indicating that the compound has no inhibitory effect on the enzyme.
TABLE-US-00001 TABLE 1 Compound No. Compound structure IC.sub.50 (NAAA) IC.sub.50 (FAAH) 1
Experimental Example 2
Pain Assays
[0284] In the Experimental example, compounds to be evaluated or compounds to be tested or test compounds used are the Compounds 1-46 prepared in the Examples according to the invention. In the Experimental example, all the agents for injection, including compounds to be evaluated or compounds to be tested or test compounds, and positive control drug, were dissolved in a mixed solvent of PEG400:Tween 80:normal saline (at a volume ratio of 5:5:90), at a concentration of 5 mg/mL.
[0285] 1. Experimental Example of Neuropathic Pain
[0286] Neuropathic pain mouse model induced by spared nerve injury (SNI) is a common animal model for studying neuropathic pain medically. The invention uses the model to evaluate the therapeutic effects of the compounds on neuropathic pain, wherein the model can represent peripheral neuropathic pain including, but not limited to post-herpetic neuralgia, pain caused by diabetic perineuropathy, neurothlipsis and exudation caused by tumor, lumbar surgery failure syndrome, neuropathic pain caused by lumbar disc protrusion, postpartum neuralgia, trigeminal neuralgia, chemotherapy-induced multiple neuropathic pain, post-radiotherapy plexopathy, and radicular neuralgia.
[0287] C57BL/6 mice (purchased from Shanghai SLAC Laboratory Animal Co., Ltd) were anesthetized by intraperitoneal injection with pentobarbital sodium (40 mg/kg), and skin from knee joint to gluteal region was subjected to skin preparation and disinfection. After the mice were fixed, the knee joint part was cut about 1 cm longitudinally with bistoury, and muscle tissues were separated by blunt dissection to expose sciatic nerve. The tibial and common peroneal nerve branches of sciatic nerve stem were cut off carefully, and the cut was then stitched up. The mice was placed in a clean observation cage. In the sham-operation group, only sciatic nerve was exposed without cutting the tibial and common peroneal nerve branches. From the day before the experiment to 24 h after the operation, the plantar edges of the mice were stimulated everyday by Plantar Analgesia Meter for Thermal Paw (Ugo Basile Company (Italy), Product ID: 37450), and data was collected automatically to obtain the threshold value of mice to mechanical stimulation. The compound to be evaluated (10 mg/kg) was administered via intraperitoneal injection at 1 h before the test. The positive control drug gabapentin was administered at a dose of 100 mg/kg. All the agents to be injected were dissolved in a mixed solvent of PEG400:Tween 80:normal saline (at a volume ratio of 5:5:90), at a concentration of 5 mg/mL. By comparing the threshold values (the acting force of when the mouse retracting the paw) at different administration time in different administration groups, the compound was evaluated for its analgesic effect on neuropathic pain.
[0288] By the method above, the compounds to be evaluated were determined for their analgesic effects in SNI models, and the results were shown in
[0289] 2. Experimental Example of Abdominal Inflammatory Pain
[0290] Typical model for studying inflammation-induced visceral pain medically was used to evaluate the therapeutic effects of compounds on inflammatory visceral pain. The model can represent general visceral pain including, but not only limited to appendicitis, gastritis, pancreatitis, prostatitis, myocarditis, interstitial cystitis, pain caused by hepatic, gall or kidney stone, irritable bowel syndrome, and chronic pelvic pain syndrome.
[0291] Experimental Kunming mice (purchased by Xiamen University Laboratory Animal Center from Shanghai SLAC Laboratory Animal Co., Ltd) were in an experimental environment with free access to food and water for 1 week. The mice were fasted for 24 h before administration. The compound to be evaluated (10 mg/kg) was administered to the mice by intraperitoneal injection at 1 h before administration of acetic acid, wherein the positive control drug indometacin was administered at a dose of 20 mg/kg, and the blank control was normal saline. Acetic acid (50 μL, acetic acid dissolved in normal saline, at a concentration of 5%) was administered to the mice by intraperitoneal injection, and the mice were then placed in the observation cage. A video camera was used to record the behavior of the mice in the following 20 min. Pain-related behavior was analyzed statistically based on torsion times of the mice in said period.
[0292] As seen in
[0293] 3. Experimental Example of Headache
[0294] Nitroglycerin-induced headache rat model was a common model for studying inflammatory pain medically. The model was used in the invention to evaluate the therapeutic effects of the compounds on headache. The model can represent mixed pain including, but not only limited to: migraine, cluster headache, tension headache syndrome, prosopodynia, lumbodynia, shoulder pain, burning mouth syndrome, and complex regional pain syndrome.
[0295] Experimental Wistar rats with a body weight of about 250 g for each (purchased from Shanghai SLAC Laboratory Animal Co., Ltd) were in an experimental environment with free access to food and water for 1 week. The rats were fasted for 24 h before administration. The compound to be evaluated (10 mg/kg) was intraperitoneally injected to the rat at 1 h before the administration of nitroglycerin, wherein the positive control drug was indomethacin and the blank control was normal saline. Nitroglycerin (10 mg/kg, nitroglycerin dissolved in normal saline, at a concentration of 5%) was injected at epidermis of the rat neck, and the rats were then placed in the observation cage. A video camera was used to record the behavior of the rats in the following 60 min. Pain-related behavior was analyzed statistically based on the times that the rat scratched its head in said period.
[0296] By the method above, the tested Compound 2, 9 and 14 exhibited significant effects of inhibiting nitroglycerin-induced headache in rats, while Compounds 24 and 30 did not exhibit any effect of inhibiting headache in rats (see
[0297] 4. Experimental Example of Adjuvant-Induced Osteoarthritis Pain
[0298] Typical model for studying inflammatory pain medically was used to evaluate the therapeutic effects of compounds on inflammatory pain. 0.1 mL Freund's complete adjuvant was intradermally injected to toe of the left hind foot of the rats in each group to obtain adjuvant-induced arthritis rat models. Foot volume method was used to measure primary and secondary foot swelling degree (swelling degree=volume after inflammation−volume before inflammation), and polyarticular arthritis were scored (5 scoring grades: 0 represents no red swelling; 4 represents red swelling at all the paws including anklebone. The scores are accumulated according to the lesion degree of the rest three limbs not injected with adjuvant, and the highest score is 12). Before inflammation and at 6 h, 12 h, 18 h and 24 h after inflammation, Paw Volume Meter was used to measure the foot volume at the inflammatory side, and the swelling degree was calculated. The model can represent inflammatory pain including, but not only limited to: osteoarthritis pain and fibromyalgia syndrome, inflammatory pain of rheumatic and rheumatoid arthritis, inflammatory pain of endometriosis, inflammatory toothache, ankylosing spondylitis pain, and gouty arthritis pain.
[0299] By the method above, the tested Compound 2, 9, and 14 (10 mg/Kg) exhibited significant effects of inhibiting osteoarthritis pain, and the effective compounds of the invention had better effects than the positive control drug indometacin (10 mg/Kg).
[0300] Comparison of primary foot swelling degree of rats in each of the groups were shown in Table 2.
TABLE-US-00002 TABLE 2 Foot swelling degree after inflammation (X ± S, n = 10, mL) Group 6 h 12 h 18 h 24 h Normal control Group 0.08 ± 0.01 0.07 ± 0.03 0.08 ± 0.02.sup. 0.08 ± 0.03 Adjuvant-induced arthritis 0.98 ± 0.14.sup.a 1.78 ± 0.21.sup.a 1.95 ± 0.13.sup.a 1.87 ± 0.16.sup.a model group indometacin 0.57 ± 0.16.sup.c 0.96 ± 0.21.sup.c 1.32 ± 0.22.sup.b 1.08 ± 0.26.sup.c Compound 2 0.35 ± 0.07.sup.c 0.65 ± 0.13.sup.c 1.06 ± 0.14.sup.c 0.89 ± 0.21.sup.c Compound 9 0.44 ± 0.09.sup.c 0.75 ± 0.24.sup.c 1.24 ± 0.24.sup.b 1.06 ± 0.18.sup.c Compound 14 0.75 ± 0.11.sup.c 1.06 ± 0.19.sup.c 1.43 ± 0.25.sup.b 1.36 ± 0.18.sup.b Note: Compared to control group, .sup.aP < 0.01; compared to adjuvant-induced arthritis model group, .sup.bP < 0.05; .sup.cP < 0.01.
Experimental Example 3
Stability Assay
[0301] In the Experimental example, PBS solution used was prepared by the following method: 8 g NaCl, 0.2 g KCl, 1.44 g Na.sub.2HPO.sub.4, and 0.24 g KH.sub.2PO.sub.4 were dissolved in 1 L ultrapure water, subjected to moist heat sterilization, and stored at 4° C.
[0302] 1. Stability Assay in Rat Plasma
[0303] To each of several tubes sub-packaged with rat plasma, PBS solution was added in a volume equal to a quarter of plasma volume, and the resultant mixture was mixed well. To 1.5 mL diluted rat plasma, the compound to be tested (Compound 2, 8, 9, 11, 31, and 41) was added until the compound was at a final concentration of 2 μM, and the plasma sample containing the compound was incubated in a 37° C. incubator. At different time points (0, 10, 30, 60, 120, 240, 360, 720, 1440 min), 50 μL plasma sample was taken and added to 150 μL precooled methanol (containing the internal standard compound E8, with a structure of
##STR00117##
and a name of 1-(2-Biphenyl-4-yl)ethyl-carbonyl pyrrolidine, 1 nmol/sample), wherein three parallel samples were prepared for each time point. The samples were then vertically mixed well, and centrifuged at 4° C., 12000 g for 10 min. The supernatant was transferred to a sample vial. LC-MS/MS was used to measure the peak area at different time points, Analyst® version 1.4.1. software was used to analyze chromatographic peak, and the content of the target compound was calculated by virtue of the standard curve.
[0304] 2. Stability Assays at Acidic and Basic Conditions
[0305] To the prepared PBS, adjusted with dilute hydrochloric acid to pH 5.0, or adjusted with saturated NaOH to pH=7.4, the compound to be tested (Compound 2, 8, 9, 11, 31, and 41) was added until the compound to be tested was at a final concentration of 1 μM. The sample containing the compound to be tested was then incubated in a constant temperature shaker at 37° C. At different time points (0 min, 10 min, 30 min, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h), 50 μL sample was taken and added to 150 μL precooled methanol (containing the internal standard E8, 1 nmol/sample), wherein three parallel samples were prepared for each time point. The samples were then vertically mixed well, and transferred to a sample vial. LC-MS/MS was used to measure the peak area at different time points, Analyst® version 1.4.1. software was used to analyze chromatographic peak, and the content of the target compound was calculated based on the standard curve.
[0306] As seen from Table 3, the effective compounds of the invention had a half-life of more than 24 h at acidic or basic conditions, and a half-life of more than 120 min in rat plasma, i.e., their stability was much higher than the reported β-lactam type NAAA inhibitor (β-lactam type NAAA inhibitor had a half-life of less than 15 min in rat plasma).
[0307] 3. Assay on Drug Metabolism in Rat
[0308] 50 SD rats (purchased from Shanghai SLAC Laboratory Animal Co., Ltd) were divided into 5 groups randomly, 10 rats per group. After anesthesia with ether, rat was fixed on the operating table on its back, and was shaved at the operative site of neck. After disinfection with 75% alcohol, the neck was cut about 1.5 cm longitudinally, muscle tissues and nervi vascularis surrounding trachea were separated by blunt dissection, and carotid artery was separated by blunt dissection. Ligation was performed at distal end, an artery clamp was used at proximal part, and the distance between them was about 1 cm. A small cut was made with a pair of ophthalmic scissors at proximal part close to the ligated site, and arterial duct was introduced from the small cut to the proximal part, the artery clamp was loosened, the arterial ostium was clamped with smooth forceps, whilst the arterial duct was delivered to heart ventricle. Upon the introduction of the arterial duct into heart ventricle, the blood vessel was ligated. By using the same intubation method for artery, intubation was applied to jugular veins. After intubation, a tubule filled with heparin sodium was drawn out from artery and vein, which passed through the skin under the ear of the rat and drawn out from the dorsal part skin of the neck of the rat, and the tubule was fixed well. The muscles and skin were sutured at the surgical site of the rat, and normal saline containing heparin sodium was injected from time to time via the tubule connecting the artery and the vein to prevent tubule obstruction due to blood coagulation. The rat was placed in a warm and clean cage provided with water and food, waiting for the rat to wake up naturally.
[0309] After the rat regained consciousness and acted normally, assay on metabolic stability of compounds was performed in the rat. All the agents to be injected (including compounds to be tested and control drug) were dissolved in a mixed solvent of PEG400:Tween 80:normal saline (at a volume ratio of 5:5:90), at a concentration of 5 mg/mL. The agent was injected slowly at a dose of 30 mg/Kg via the tubule drawn out from the jugular vein. After administration, the time was recorded, and blood was collected from the tubule drawn out from carotid artery at 2, 5, 10, 20, 30, 60, 240, 360, and 720 min, in an amount of 200 μL for each time. The whole blood was placed in commercially available heparin sodium blood-collecting tube (Jiangxi Jingzhi Medical Equipment Co., Ltd.), centrifuged at 1800 g for 15 min. To the supernatant plasma, 150 μL pre-cooled methanol stopping buffer (containing internal standard E8, 1 nmol/sample) was added, vertically mixed well, and then centrifuged at 4° C., 12000 g for 10 min. The supernatant was transferred to a sample vial, the target compound was detected by LC-MS/MS, and the content of the compound was calculated by virtue of standard curve.
[0310] As seen from Table 3, the effective compounds of the invention had a half-life of 60-300 min in rat, i.e., their stability was much higher than the reported 13-lactam type NAAA inhibitor (β-lactam type NAAA inhibitor had a half-life of less than 5 min in rat).
TABLE-US-00003 TABLE 3 Half-life (min) Rat Metabolism Compound pH 5.0 pH 7.4 plasma in rat 2 >1440(100) >1440(100) 188 144 8 >1440(100) >1440(100) 165 93 9 >1440(80) >1440(100) 163 117 11 >1440(100) >1440(100) 267 184 31 >1440(92) >1440(100) 132 74 41 >1440(100) >1440(100) 320 175
Experimental Example 4
Assay on Cytotoxicity
[0311] In the Experimental example, PBS solution used was prepared by the following method: 8 g NaCl, 0.2 g KCl, 1.44 g Na.sub.2HPO.sub.4, 0.24 g KH.sub.2PO.sub.4 were dissolved in 1 L ultrapure water, subjected to moist heat sterilization, and stored at 4° C.
[0312] RAW264.7 cells in good growing state (purchased from American Type Culture Collection, ATCC, Beijing), at a density of about 80%-90%, were scraped off with a cell scraper to prepare a cell suspension. After counting, the cells were accurately seeded in a 96-well plate, at 100 μL per well, wherein the total number of cells retained the same, at about 20000 cells/well, and the same amount of culture medium (which was DMEM (Gibco, Shanghai) medium containing 10% fetal bovine serum (PAA), 1% penicillin-streptomycin (PAA)) was added. The 96-well plate was placed in an incubator to preincubate for 24 h (37° C., 5% CO.sub.2). The marginal wells of the plate were filled with an equal amount of PBS, but were not seeded with cells, so as to prevent evaporation of the culture medium in the experimental group. The old medium was removed with a pipette at 12 h after plating cells, 100 μL compound 2, 9 at a different concentration (at a concentration of 3, 10, 30, 100 μM, respectively) diluted with culture medium was added to the plate, and an equal amount of DMSO was added as control in control group, and three wells in parallel were used for each concentration. After incubating the plate in the incubator for a period of time (24 or 48 h), 10 μL CCK8 solution was added to each well, and then the plate was incubated in incubator for 1.5 h. the absorbance OD value was determined at 450 nm by microplate reader, and the OD value was proportional to the number of living cells. The cell survival rate was obtained by comparing the OD value in each group with the OD value in blank group. Cell viability×100%=[OD value (Administration group)−OD value (Blank group)]/[OD value (Control group)−OD value (Blank group)]×100%.
[0313] The experimental results showed that except for compound 9 at a high concentration of 100 μM, compound 2, 9 at other different concentrations had no effect on cell viability after incubation with cells for 24 h (see
Experimental Example 5
Assay on Toxicity to Early Development of Zebrafish Embryos
[0314] Fertilized eggs (donated by Zebrafish Research Group, School of Life Sciences, Xiamen University) were incubated in freshly prepared zebrafish embryo culture medium, which was replaced by fresh culture medium every day, and cultured in 28.5° C. incubator. Pure water was placed in the incubator to ensure a certain humidity in the culture environment. The formulation for zebrafish embryo culture medium was following: 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl.sub.2, 0.33 mM MgSO.sub.4, 10-5% methylene blue. The culture medium was prepared with pure water. For example, NaCl: 292.2 mg, KCl: 12.68 mg, CaCl.sub.2: 36.63 mg, MgSO.sub.4: 39.72 mg, and methylene blue: 0.1 μL were needed for the preparation of 1 L culture medium.
[0315] 7 Groups were set for each test compound, including 5 experimental groups relating to the test compound at different concentrations (solutions containing the test compound at different concentrations were prepared by using zebrafish embryo culture medium, wherein the concentrations were 10 mg/L, 3 mg/L, 1 mg/L, 0.3 mg/L, 0.1 mg/L), 1 solvent control group (DMSO) and 1 blank control group. The positive control drug was OTC analgesic ibuprofen at concentrations as described above. A 96-well cell culture plate was used to perform the experiment, three wells in parallel were used for each experimental group, and 10 fish eggs were contained in each well.
[0316] Culture medium containing a test compound was prepared, and the culture medium was added to a 96-well plate in advance, and each fertilized egg (by reference to the experimental method in OECD No. 212 document, fertilized eggs, which developed normally within 2 h after fertilization, were selected and subjected to exposure. Transparent fish eggs visible to naked eye were normally fertilized, while white fish eggs were dead and discarded. After the selection, the normally fertilized eggs had begun to divide as observed under microscope) was then independently placed in a different well, and cultured in the culture medium containing a test compound at a different concentration. Embryo development status at different stages was observed and photographed with invert microscope, including:
[0317] I. Morphological observation: the developmental states of fertilized eggs (yolk being coagulated or not, eye spot, pericardial sac being edematous or not, bloodstream being existing or not, tail development status, and the like) were observed and photographed at 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, and 96 h;
[0318] II. Hatching number and death number were counted at 72 h after fertilization, and hatching rate and mortality were calculated;
[0319] III. Body length of the hatched young fishes at 72 h was measured (measured after photographing with invert microscope);
[0320] IV. Embryonic abnormities after 72 h (manifestations of abnormalities: no formation of melanin, yolk sac disease, campylorrhachia, eye defect, tail defect, reduced heart rate, reduced hatching rate, etc.)
[0321] V. Heartbeat of young fishes (times/min) was recorded at 72 h and 96 h after fertilization (Recording method: heartbeat of young fishes could be clearly observed under microscope, and the number of heartbeats was recorded for 30 s).
[0322] Experimental results showed that at a concentration of 1 mg/L, the hatching rate for the group of Compound 2 and the group of Compound 9 reached up to 96.67% and 90% at 72 h, respectively, while the hatching rate for the ibuprofen group was only 56.67%; at concentrations of 0.3 mg/L and 0.1 mg/L, the hatching rate for the groups of Compound 2 and Compound 9 was significantly higher than that of the ibuprofen group at 72 h, while the mortality was much lower than that of the ibuprofen group (see Table 4). The research results above showed that at a dose lower than 3 mg/L, the toxicity of Compound 2 and Compound 9 on zebrafish embryo development was significantly lower than that of ibuprofen.
TABLE-US-00004 TABLE 4 The effects of compound 2, 9 and ibuprofen on zebrafish with respect to parameters such as hatch, heartbeat, and body length Death number (72 h) Unhatching number (72 h) Hatching number (72 h) Group Compound Compound Compound (n = 30) 2 9 ibuprofen 2 9 ibuprofen 2 9 ibuprofen Blank 1 0 4 0 0 0 29 30 26 Control 0 0 4 0 0 0 30 30 26 0.1 mg/L 3 2 8 0 0 0 27 28 22 0.3 mg/L 2 2 8 0 0 2 28 28 20 1 mg/L 1 3 13 0 0 0 29 27 17 3 mg/L 30 30 28 0 0 2 0 0 0 10 mg/L 30 30 30 0 0 0 0 0 0 Body length (mm, 72 h) Heartbeat (per min, 72 h) Group Compound Compound (n = 30) 2 9 ibuprofen 2 9 ibuprofen Blank 3.83 ± 0.02 3.87 ± 0.03 3.90 ± 0.06 124 ± 2.61 125 ± 1.23 124 ± 2.61 Control 3.90 ± 0.08 3.86 ± 0.02 3.98 ± 0.06 125 ± 1.10 126 ± 2.54 125 ± 2.74 0.1 mg/L 3.86 ± 0.05 3.90 ± 0.05 3.94 ± 0.14 130 ± 3.00 129 ± 2.73 125 ± 2.40 0.3 mg/L 3.87 ± 0.05 3.86 ± 0.03 3.95 ± 0.09 130 ± 3.58 130 ± 3.22 125 ± 2.48 1 mg/L 3.85 ± 0.06 3.88 ± 0.04 3.77 ± 0.18 128 ± 3.29 130 ± 2.76 126 ± 2.56 3 mg/L — — — — — — 10 mg/L — — — — — —
Experimental Example 6
Assay on Short-Term Acute Toxicity to Development of Zebrafish Embryos
[0323] By the same method described above, zebrafish fertilized eggs were cultured. 7 Groups were set for each test compound, including 5 experimental groups relating to the test compound at different concentrations (solutions containing the test compound at different concentrations were prepared by using zebrafish embryo culture medium, wherein the concentrations were 100 mg/L, 10 mg/L, 1 mg/L, 0.1 mg/L, 0.01 mg/L), 1 solvent control group (DMSO) and 1 blank control group. The fertilized eggs were placed and cultured in a 28.5° C. incubator, a certain humidity was ensured in the culture environment. Culture medium containing a test compound was prepared, and the culture medium was added to a 96-well plate in advance, and each fertilized egg (by reference to the experimental method in OECD No. 212 document, fertilized eggs, which developed normally within 24 h after fertilization, were selected and subjected to exposure. Transparent fish eggs visible to naked eye were normally fertilized, while white fish eggs were dead and discarded. After the selection, the normally fertilized eggs had begun to divide as observed under microscope) was then independently placed in a different well, and cultured in the culture medium containing a test compound at a different concentration. Embryo development status at different stages were observed and photographed with invert microscope, including:
[0324] I. Morphological observation: the developmental states of fertilized eggs (yolk being coagulated or not, eye spot, pericardial sac being edematous or not, bloodstream being existing or not, tail development status, and the like) were observed and photographed at 48 h, 72 h, and 96 h;
[0325] II. Hatching number and death number were counted at 72 h after fertilization, and hatching rate and mortality were calculated;
[0326] III. Body length of the hatched young fishes at 72 h was measured (measured after photographing with invert microscope);
[0327] IV. Embryonic abnormities after 72 h (manifestations of abnormalities: no formation of melanin, Yolk Sac disease, campylorrhachia, eye defect, tail defect, reduced heart rate, reduced hatching rate, etc.)
[0328] V. Heartbeat of young fishes (times/min) was recorded at 72 h and 96 h after fertilization (Recording method: heartbeat of young fishes could be clearly observed under microscope, and the number of heartbeats was recorded for 30 s).
[0329] The experimental results showed that in the solvent control group and the blank group, zebrafish developed normally. Zebrafish embryos exposed to 10 mg/L ibuprofen had a hatching rate of 0 and a mortality of 100% at 72 h, and in the ibuprofen group, the hatching rate decreased with the increase of dose at 72 h; at the same dose, the hatching rate of zebrafish embryos for compound 2 and compound 9 was 93.3%, and compound 2 and compound 9 had little effect on the hatching rate at a dose lower than 10 mg/L (See Table 5). Only when the F96 concentration was above 10 mg/L, it had a significant effect on hatching rate and mortality of zebrafish embryos at 72 h; while ibuprofen had an effect on hatching rate of zebrafish at 72 h even at a low concentration of 0.01 mg/L, and reduced the hatching rate of zebrafish by about half at 1 mg/L, and the effect was more significant with the increase of the concentration of ibuprofen.
TABLE-US-00005 TABLE 5 The effects of compound 2, 9 and ibuprofen on zebrafish with respect to parameters such as hatch, heartbeat, and body length Death number (72 h) Unhatching number (72 h) Hatching number (72 h) Group Compound Compound Compound (n = 30) 2 9 ibuprofen 2 9 ibuprofen 2 9 ibuprofen Blank 0 0 0 0 0 0 30 30 30 Control 0 0 0 0 0 0 30 30 30 0.01 mg/L 0 0 0 0 0 5 30 30 25 0.1 mg/L 0 0 0 0 0 8 30 30 22 1 mg/L 0 0 2 0 0 11 30 30 17 10 mg/L 0 0 30 2 2 0 28 28 0 100 mg/L 30 30 30 30 30 0 0 0 0 Body length (mm, 72 h) Heartbeat (per min, 72 h) Group Compound Compound (n = 30) 2 9 ibuprofen 2 9 ibuprofen Blank 4.01 ± 0.51 4.02 ± 0.42 3.94 ± 0.15 126 ± 1.41 125 ± 0.76 125 ± 1.60 Control 4.02 ± 0.01 4.02 ± 0.17 3.94 ± 0.08 124 ± 0.89 124 ± 1.42 125 ± 1.97 0.01 mg/L 3.90 ± 0.93 4.01 ± 0.24 3.97 ± 0.06 124 ± 1.67 124 ± 0.27 125 ± 2.16 0.1 mg/L 4.08 ± 0.09 4.03 ± 0.62 3.88 ± 0.15 123 ± 2.45 124 ± 1.17 124 ± 2.25 1 mg/L 3.92 ± 0.10 4.01 ± 0.26 3.97 ± 0.08 123 ± 3.03 124 ± 1.08 124 ± 1.67 10 mg/L 3.78 ± 0.05 3.82 ± 0.44 — 117 ± 3.90 117 ± 2.24 — 100 mg/L — — — — — —
[0330] It was found by further observation of the whole process of zebrafish embryo development that at 100 mg/L, 48 h, in the groups of Compound 2 and Compound 9, no zebrafish embryos died, but phenomena such as pericardial cyst, no heartbeat, no bloodstream, no tail development and the like occurred in embryos; in the ibuprofen group at the same dose, all the zebrafish embryos died. At 10 mg/L, 48 h, in the groups of Compound 2 and 9, the development of zebrafish embryos was basically normal; while in the ibuprofen group, phenomena such as pericardial cyst, no heartbeat, no bloodstream, and no tail development occurred in zebrafish. In the ibuprofen group at 1 mg/L, 72 h, embryos development delayed, with a hatching rate of only 56.7%, and mild edema occurred in pericardial sac of some zebrafishes, while Compound 2 and 9 at the same dose had no impact.
Experimental Example 7
Assay on Effects of Compounds 2, 9, 34 and COX2 Selective Inhibitor Celecoxib on hERG Potassium Channel
[0331] CHO-hERG cells (purchased from ATCC) were incubated in a culture flask. When the cell density reached 60-80%, the culture medium (Glutamax DMEM/F12 medium (Gibco, Shanghai) containing 10% fetal bovine serum) was removed. The cells were washed with 7 mL PBS once, and then digested by adding 3 mL Detachin cell dissociation buffer. After complete digestion, the resultant mixture was neutralized by adding 7 mL culture medium, and centrifuged. The supernatant was removed with a pipette, 5 mL culture medium was added to re-suspend the cells to ensure a cell density of 2˜5×10.sup.6/mL.
[0332] Intracellular fluid and extracellular fluid were prepared as described in the following table.
TABLE-US-00006 Reagent Extracellular fluid (mM) Intracellular fluid (mM) CaCl.sub.2 2 5.374 MgCl.sub.2 1 1.75 KCl 4 120 NaCl 145 — Glucose 10 — HEPES 10 10 EGTA — 5 Na-ATP — 4 pH 7.40 7.25
[0333] A certain amount of compound was dissolved in extracellular fluid at the following concentrations, to prepare the stock solution of compound. 2 μL stock solution of compound was added to 998 μL extracellular fluid, and was subjected to 5-fold serial dilution in extracellular fluid containing 0.2% DMSO to get the final concentrates to be tested. The highest test concentration for a test compound was 40 μM, followed by six concentrations, i.e., 40 μM, 8 μM, 1.6 μM, 0.32 μM, 0.064 μM, and 0.0128 μM. The highest test concentration of the positive compound, cisapride was 3 μM, followed by six concentrations, i.e., 3 μM, 0.6 μM, 0.12 μM, 0.024 μM, 0.0048 μM, and 0.00096 μM. In the final test concentration, the amount of DMSO was not more than 0.2%, as DMSO at the concentration had no effect on hERG potassium channel.
[0334] Single cell giga-seal and formation of whole-cell mode were automatically performed by Sophion Qpatch HT fully automated cell patch clamp systems. After obtaining the whole-cell recording mode, the cell was clamped at −80 mV. The cell first underwent pre-voltage of −50 mV for 50 msec, then underwent depolarization stimulation at 20 mV for 5 sec, and then underwent repolarization at −50 mV for 5 sec, and then the voltage return to −80mv. The cell underwent the stimulation at the voltage every 15 sec, and the data were recorded for 2 min, then extracellular fluid was administered, and then the data were recorded for 2 min. Then, the administration process begun. The concentration of a test compound started from the lowest concentration, each test concentration was administered for 2 min. After continuous administration of the test compound at all the concentrations, the positive control compound Cisapride was administered. At least three cells were tested for each concentration (n≧3). The experimental data was analyzed by XLFit software.
[0335] The experimental results showed that the median inhibitory concentration of Cisapride is 0.1 μM to hERG potassium channel, and the median inhibitory concentration of celecoxib is 6.4 μM to hERG potassium channel, while Compound 2, 9 and 34, at the highest concentration of 40 μM for evaluation, had maximal inhibition rate of 38.30%, 31.42%, 44.67% (See Table 6) for hERG potassium channel, respectively. These results showed that Compound 2, 9 and 34 had low inhibition rate for hERG potassium channel at high concentration, and had better cardiac safety than the commercially available analgesics COX.sub.2 selective inhibitor celecoxib.
TABLE-US-00007 TABLE 6 Inhibitory effects of Compounds 2, 9, 34 and celecoxib, Cisapride on hERG potassium channel. Maximum concentration- median inhibitory Compound inhibition rate (%) concentration (μM) 2 38.30 >40 9 31.42 >40 34 44.67 >40 celecoxib 74.81 6.4 Cisapride 98.46 0.10
[0336] Although the embodiments of the invention are described in detail, a person skilled in the art would understand that according to all the already disclosed teachings, these details can be modified and replaced, and these alterations all fall in the protection scope of the invention. The scope of the invention is defined by the claims and any equivalent thereof.