526
Vol. 62, No. 6
otherwise indicated δ; δ in ppm rel. to Me4Si, J in Hz. High 1H), 10.92 (s, 1H). HR-TOF-MS m/z 353.1436 ([M+H]+,
resolution-time-of-flight (HR-TOF)-MS were measured on a C19H21N4OS+; Calcd 353.1431).
BioTOFTM-Q mass spectrometer (Bruker); in m/z.
General Procedure for Preparing Compounds 3a–k
1-(4-Methoxyphenyl)-3-(5-(pyridine-3-yl)-1,3,4-thiadiazol-2-
A
yl)urea (6g): Yield 50.8%; mp 321.6–324.4°C. 1H-NMR
mixture of substituted benzoic acid (40.61mmol) and thios- (300MHz, DMSO-d6) δ: 3.75 (s, 3H), 6.92–7.94 (d, J=6Hz,
emicarbazide (48.73mmol) in POCl3 (45mL) was stirring for 2H), 7.42–7.44 (d, J=6Hz, 2H), 7.56–7.59 (m, 1H), 8.29–8.31
2h at 80°C. After cooling to room temperature, water (60mL) (d, J=6Hz, 1H), 8.70–8.71 (d, J=3Hz, 1H), 8.92 (s, 1H), 9.11
was added in 30min. Ice-water was added, and the pH was (s, 1H), 11.13 (s, 1H). HR-TOF-MS m/z 328.0868 ([M+H]+,
adjusted to 8 with 1M sodium hydroxide solution. The mixture C15H14N5O2S+; Calcd 328.0863).
was filtered. The obtained solid was crystallized from DMF–
H2O to give 3a–k.
1-(3,4-Dimethylphenyl)-3-(5-(pyridine-3-yl)-1,3,4-
thiadiazol-2-yl)urea (6h): Yield 58.9%; mp 318.2–321.6°C.
1H-NMR (300MHz, DMSO-d6) δ: 2.05–2.31 (m, 6H),
General Procedure for Preparing Compounds 6a–k
A
mixture of substituted benzoic acid (3mmol) and triethyl- 7.08–7.29 (m, 3H), 7.56 (s, 1H), 8.29–8.30 (m, 1H), 8.70 (s,
amine (3.3mmol) in anhydrous toluene 40mL was stirring for 1H), 8.90 (s, 1H), 9.10 (s, 1H), 11.16 (s, 1H). HR-TOF-MS m/z
45min at room temperature. To the above reaction mixture 326.1076 ([M+H]+, C16H16N5OS+; Calcd 326.1070).
DPPA (3.6mmol) was added and heated to 40°C for 1h. Then
1-(Pyridine-3-yl)-3-(5-(pyridine-3-yl)-1,3,4-thiadiazol-2-
the reaction mixture was refluxed for 3h. After cooling to yl)urea (6i): Yield 36.0%; mp 319.4–322.4°C. 1H-NMR
room temperature, intermediate 3a–k (3mmol) was added and (300MHz, DMSO-d6) δ: 7.34 (m, 1H), 7.57–7.58 (m, 1H),
refluxed for 10h. Cooled to room temperature and concentrat- 7.99–8.00 (d, J=3Hz, 1H), 8.29–8.31 (d, J=6Hz, 2H), 8.70 (s,
ed to give the crude product. The crude product was refluxed 2H), 9.10 (s, 1H), 9.34 (s, 1H), 11.78 (s, 1H). HR-TOF-MS m/z
in 50mL methanol for 0.5h. After cooling, the mixture was 299.0714 ([M+H]+, C13H11N6OS+; Calcd 299.0710).
filtered and the solid was collected as target product.
1-(5-Phenyl-1,3,4-thiadiazol-2-yl)-3-phenyl Urea (6j): Yield
1-(3,4-Dimethylphenyl)-3-(5-(4-methoxyphenyl)-1,3,4- 75.8%; mp 309.5–311.9°C. 1H-NMR (300MHz, DMSO-d6)
thiadiazol-2-yl)urea (6a): Yield 75.3%; mp 317.1–319.4°C. δ: 7.04–7.09 (m, 1H), 7.31–7.36 (m, 2H), 7.89–7.91 (m, 2H),
1H-NMR (300MHz, DMSO-d6) δ: 2.17–2.20 (d, 6H), 3.82 (s, 7.50–7.52 (m, 5H), 9.05 (s, 1H), 11.04 (s, 1H). HR-TOF-MS
3H), 7.06–7.84 (m, 7H), 8.81 (s, 1H), 10.88 (s, 1H). HR-TOF- m/z 295.0660 ([M−H]−, C15H11N4OS−; Calcd 295.0654).
MS m/z 355.1227 ([M+H]+, C18H19N4O2S+; Calcd 355.1223).
1-(5-(4-Fluorophenyl)-1,3,4-thiadiazol-2-yl)-3-(4-fluoro-
1
1-(5-(4-Methoxyphenyl)-1,3,4-thiadiazol-2-yl)-3-(pyridine-3- phenyl)urea (6k): Yield 81.2%; mp 335.7–336.9°C. H-NMR
yl)urea (6b): Yield 53.0%; mp 317.6–320.1°C. 1H-NMR (300MHz, DMSO-d6) δ: 7.14–7.20 (t, J=8.7Hz, 2H), 7.34–7.40
(300MHz, DMSO-d6) δ: 3.72 (s, 3H), 7.07–7.10 (d, J=9Hz, (t, J=8.7Hz, 2H), 7.51–7.55 (t, J=8.1Hz, 2H), 7.94–7.99 (t,
2H), 7.83–7.86 (d, J=9Hz, 2H), 7.34–7.38 (m, 1H), 7.97–8.00 J=8.4Hz, 2H), 9.13 (s, 1H), 11.14 (s, 1H). HR-TOF-MS m/z
(d, J=9Hz, 1H), 8.26–8.27 (d, J=3Hz, 1H), 8.69 (s, 1H), 9.291 331.0472 ([M−H]−, C15H9N4OSF2−; Calcd 331.0465).
(s, 1H). HR-TOF-MS m/z 328.0866 ([M+H]+, C15H14N2O2S+;
Calcd 328.0863).
In Vitro AChE Inhibition Assay AChE inhibitory activ-
ity was detected by a microtitre plate assay based on Ellman’s
1-(5-(4-Methoxyphenyl)-1,3,4-thiadiazol-2-yl)-3-(4- method26) (Rhee et al., 2001), using the Amplite™ Fluorimet-
methoxyphenyl)urea (6c): Yield 85.7%; mp 314.0–316.9°C. ric Acetylcholinesterase Assay Kit. In 96-well plates, 25µL
1H-NMR (300MHz, DMSO-d6) δ: 3.03 (s, 3H), 3.82 (s, Acetylcholine, 2µL Thiolite™ Green, 62.2µL of buffer and
3H), 6.89–6.92 (d, J=9Hz, 2H), 7.06–7.09 (d, J=9Hz, 2H), 10 µL of the test compound at concentration of 0.01, 0.1, 1, 10,
7.39–7.42 (d, J=9Hz, 2H), 7.82–7.85 (d, J=9Hz, 2H), 8.86 100, 1000µM, 10mM were added and the fluorescence intensity
(s, 1H), 10.9 (s, 1H). HR-TOF-MS m/z 357.1020 ([M+H]+, was measured at 485/528nm every 13s for five times. After
C17H17N4O3S+; Calcd 357.1016).
adding 25µL of 0.125U/mL AChE, the fluorescence intensity
1-(3,4-Dimethylphenyl)-3-(5-(4-methoxyphenyl)-1,3,4- was read again every 13s for five times. The fluorescence
thiadiazol-2-yl)urea (6d): Yield 76.4%; mp 329.4–331.7°C. intensity was measured using a Multi-Plate Reader (Synergy
1H-NMR (300MHz, DMSO-d6) δ: 2.28–2.31 (d, 6H), 3.74 (s, HT, BioTek Ltd.). Percentage of inhibition was calculated by
3H), 6.89–6.93 (d, J=9Hz, 2H), 7.39–7.42 (d, J=9Hz, 2H), comparing the rates for the sample to the blank (DMSO),
7.27–7.29 (d, J=6Hz, 1H), 7.60–7.62 (d, J=6Hz, 1H), 7.68 control contained all components except the tested extract.
(s, 1H), 8.86 (s, 1H), 10.90 (s, 1H). HR-TOF-MS m/z 355.1230 Galanthamine was used as positive control. The mean of four
([M+H]+, C18H19N4O2S+; Calcd 355.1223).
measurements for each concentration was determined (n=4).
1-(5-(3,4-Dimethylphenyl)-1,3,4-thiadiazol-2-yl)-3- The 50% inhibitory concentration (IC50) was calculated from a
(pyridine-3-yl)urea (6e): Yield 60.7%; mp 327.4–330.6°C. doseeresponse curve obtained by plotting the percentage of in-
1H-NMR (300MHz, DMSO-d6) δ: 2.28 (s, 3H), 2.30 (s, 3H), hibition versus the log concentration with the use of GraphPad
7.27–7.29 (d, J=6Hz, 1H), 7.61–7.63 (d, J=6Hz, 1H), 7.68 (s, Prism 5.0 software.
1H), 7.34–7.38 (m, 1H), 7.97–8.00 (d, J=9Hz, 1H), 8.26–8.27
(d, J=3Hz, 1H), 8.70 (s, 1H), 9.30 (s, 1H), 11.28 (s, 1H).
HR-TOF-MS m/z 326.1074 ([M+H]+, C16H16N5OS+; Calcd Natural Science Foundation for Colleges and Universities in
326.1070). Jiangsu Province (No. 11KJB350002), the Natural Science
Acknowledgments The work was supported by the
1-(3,4-Dimethylphenyl)-3-(5-(3,4-dimethylphenyl)-1,3,4- Foundation of Jiangsu Province (No. BK2012422) and the
thiadiazol-2-yl)urea (6f): Yield 81.9%; mp 341.2–344.6°C. National Natural Science Foundation of China (No. 81202393).
1H-NMR (300MHz, DMSO-d6) δ: 2.17–2.20 (d, 6H),
2.27–2.29 (d, 6H), 7.06–7.08 (d, J=6Hz, 1H), 7.20–7.29
(m, 3H), 7.60–7.62 (d, J=6Hz, 1H), 7.68 (s, 1H), 8.86 (s,