July 2003
NЈ-{1-[3-(4-Bromophenyl)-1-(4-nitrophenyl)-1H-pyrazol-4-yl]methyl-
843
3H, CH3), 7.35 (s, 1H, oxadiazoline C-2-H), 7.60—8.75 (m, 13 H,
aromaticϩpyrazole C-5 H); Anal. (C24H17BrN6O4) C, H and N.
ene}-2-chloronicotinohydrazide (28): Yield 80%; mp 347—348 °C; DMF;
IR (KBr, cmϪ1): 3300 (NH), 1670 (CϭO), 1600 (CϭN); 1H-NMR (DMSO-
d6): 7.20—8.80 (m, 12H, aromaticϩpyrazole C-5 H), 9.15 (s, 1H, CHϭN),
11.80 (br s, 1H, NH); Anal. (C22H14BrClN6O3) C, H, N and total halide.
1-(4-Nitrophenyl)-3-phenyl-1H-pyrazole-4-carbaldehyde Thiosemicar-
bazone (29): Yield 60%; mp 232—233 °C; alcohol; IR (KBr, cmϪ1): 3450,
3-Acetyl-2-(1-(4-nitrophenyl)-3-phenylpyrazol-4-yl)-5-(3-pyridyl)1,3,4-
oxadiazoline (41): Yield 65%; mp 246—247 °C; DMF-H2O; IR (KBr,
cmϪ1): 1660 (CϭO), 1600 (CϭN); 1H-NMR (DMSO-d6): 2.28 (s, 3H, CH3),
7.25 (s, 1H, oxadiazoline C-2-H), 7.25—8.76 (m, 14 H, aromaticϩpyrazole
C-5 H); Anal. (C24H18N6O4) C, H and N.
1
3-Acetyl-2-(3-(4-bromophenyl)-1-(4-nitrophenyl)pyrazol-4-yl)-5-(3-
pyridyl)-1,3,4-oxadiazoline (42): Yield 90%; mp 305—307 °C; DMF-H2O;
3400 (NH2), 3250(NH), 1600 (CϭN); H-NMR (DMSO-d6): 6.90 (s, 1H,
pyrazole C-5 H), 7.40—8.70 (m, 11H, aromaticϩNH2), 9.36 (s, 1H,
CHϭN), 11.40 (br s, 1H, NH); Anal. (C17H14N6O2S) C, H and N.
IR (KBr, cmϪ1): 1690 (CϭO), 1600 (CϭN); H-NMR (DMSO-d6): 2.20 (s,
1
3H, CH3), 7.15 (s, 1H, oxadiazoline C-2-H), 7.52—8.80 (m, 13 H,
aromaticϩpyrazole C-5 H); Anal. (C24H17BrN6O4) C, H, N and Br.
3-Acetyl-5-(2-chloro(3-pyridyl))-2-(1-(4-nitrophenyl)-3-phenylpyrazol-4-
yl)-1,3,4-oxadiazoline (43): Yield 85%; 201—202 °C; DMF-H2O; IR (KBr,
cmϪ1): 1700 (CϭO), 1600 (CϭN); 1H-NMR (DMSO-d6): 2.25 (s, 3H, CH3),
7.10 (s, 1H, oxadiazoline C-2-H), 7.20—8.70 (m, 13 H, aromaticϩpyrazole
C-5 H); Anal. (C24H17ClN6O4) C, H and N.
3-(4-Bromophenyl)-1-(4-nitrophenyl)-1H-pyrazole-4-carbaldehyde
Thiosemicarbazone (30): Yield 92%; mp 288—289 °C; DMF; IR (KBr,
1
cmϪ1): 3500, 3400 (NH2), 3200 (NH), 1610 (CϭN); H-NMR (DMSO-d6):
7.00 (s, 1H, pyrazole C-5 H), 7.40—8.70 (m, 10H, aromaticϩNH2), 9.36 (s,
1H, CHϭN), 11.40 (br s, 1H, NH); Anal. (C17H13BrN6O2S) C, H, N and Br.
1-(4-Nitrophenyl)-3-phenyl-1H-pyrazole-4-carbaldehyde N-Methylthio-
semicarbazone (31): Yield 60%; mp 230—231 °C; alcohol; IR (KBr, cmϪ1):
1
3-Acetyl-2-(3-(4-bromophenyl)-1-(4-nitrophenyl)pyrazol-4-yl)-5-(2-
chloro(3-pyridyl))-1,3,4-oxadiazoline (44): Yield 85%; mp 341—342 °C;
3300 (NH), 1600 (CϭN); H-NMR (DMSO-d6): 3.05 (s, 3H, CH3), 7.00 (s,
1H, pyrazole C-5 H), 7.35—8.48 (m, 9H, aromatic), 9.20 (s, 1H, CHϭN),
11.45 (br s, 1H, NH), 11.70 (br s, 1H, NH); Anal. (C18H16N6O2S) C, H and
N.
1
DMF-H2O; IR (KBr, cmϪ1): 1670 (CϭO), 1600 (CϭN); H-NMR (DMSO-
d6): 2.18 (s, 3H, CH3), 7.10 (s, 1H, oxadiazoline C-2-H), 7.40—8.80 (m, 12
H, aromaticϩpyrazole C-5 H); Anal. (C24H16BrClN6O4) C, H, N and total
halide.
N-{4-Acetyl-5-[1-(4-nitrophenyl)-3-phenyl-1H-pyrazol-4-yl]-4,5-dihydro-
[1,3,4]thiadiazol-2-yl}acetamide (45): Yield 95%; mp 252—253 °C; DMF-
H2O; IR (KBr, cmϪ1): 3200 (NH), 1700 (CϭO), 1680 (CϭO), 1600 (CϭN);
1H-NMR (DMSO-d6): 2.02 (s, 3H, NHCOCH3), 2.23 (s, 3H, COCH3), 7.06
(s, 1H, thiadiazoline C-2 H), 7.49—8.60 (m, 10H, aromaticϩpyrazole C-5
H), 9.57 (br s, 1H, NH); Anal. (C21H18N6O4S) C, H and N.
3-(4-Bromophenyl)-1-(4-nitrophenyl)-1H-pyrazole-4-carbaldehyde N-
Methylthiosemicarbazone (32): Yield 60%; mp 266—267 °C; alcohol; IR
1
(KBr, cmϪ1): 3300 (NH), 1590 (CϭN); H-NMR (DMSO-d6): 3.03 (s, 3H,
CH3), 7.00 (s, 1H, pyrazole C-5 H), 7.35—8.48 (m, 8H, aromatic), 9.24 (s,
1H, CHϭN), 11.20 (br s, 1H, NH), 11.45 (br s, 1H, NH); Anal.
(C18H15BrN6O2S) H and N, C: calcd. 47.07, found 47.80; EI-MS: m/zϭ458
(Mϩ) and 460 (Mϩϩ2).
General Procedure for Preparation of Compounds (33—48) A mix-
ture of the appropriate hydrazone (17—28), thiosemicarbazone (29—32)
(3 mmol) and 20 ml of acetic anhydride was refluxed for 4 h, then cooled and
added onto crushed ice. The precipitated product was filtered, dried and
crystallized.
N-{4-Acetyl-5-[3-(4-bromophenyl)-1-(4-nitrophenyl)-1H-pyrazol-4-yl]-
4,5-dihydro-[1,3,4]thiadiazol-2-yl}acetamide (46): Yield 91%; mp 331—
332 °C; DMF-H2O; IR (KBr, cmϪ1): 3300 (NH), 1700 (CϭO), 1680 (CϭO),
1
1600 (CϭN); H-NMR (DMSO-d6): 2.10 (s, 3H, NHCOCH3), 2.20 (s, 3H,
COCH3), 7.06 (s, 1H, thiadiazoline C-2 H), 7.65—8.41 (m, 9H, aromaticϩ
pyrazole C-5 H), 9.39 (br s, 1H, NH); Anal. (C21H17BrN6O4S) C, H and N.
N-{4-Acetyl-5-[1-(4-nitrophenyl)-3-phenyl-1H-pyrazol-4-yl]-4,5-dihydro-
[1,3,4]thiadiazol-2-yl}-N-methylacetamide (47): Yield 90%; mp 242—
244 °C; DMF-H2O; IR (KBr, cmϪ1): 1700 (CϭO), 1680 (CϭO), 1600
(CϭN); 1H-NMR (DMSO-d6): 1.96 (s, 3H, NHCOCH3), 2.25 (s, 3H,
COCH3), 3.03 (s, 3H, N–CH3), 7.02 (s, 1H, thiadiazoline C-2 H), 7.44—
8.60 (m, 10H, aromaticϩpyrazole C-5 H); Anal. (C22H20N6O4S) C, H and N.
N-{4-Acetyl-5-[3-(4-bromophenyl)-1-(4-nitrophenyl)-1H-pyrazol-4-yl]-
4,5-dihydro-[1,3,4]thiadiazol-2-yl}-N-methylacetamide (48): Yield 87%; mp
207—208 °C; DMF-H2O; IR (KBr, cmϪ1): 1700 (CϭO), 1680 (CϭO), 1600
(CϭN); 1H-NMR (DMSO-d6): 2.08 (s, 3H, NHCOCH3), 2.38 (s, 3H,
COCH3), 3.00 (s, 3H, N–CH3), 7.10 (s, 1H, thiadiazoline C-2 H), 7.44—
8.4060 (m, 9H, aromaticϩpyrazole C-5 H); Anal. (C22H19BrN6O4S) C, H
and N; EI-MS: m/zϭ542 (Mϩ) and 544 (Mϩϩ2).
3-Acetyl-2-(1-(4-nitrophenyl)-3-phenylpyrazol-4-yl)-5-phenyl-1,3,4-oxa-
diazoline (33): Yield 65%; mp 280—281 °C; DMF-H2O; IR (KBr, cmϪ1):
1
1670 (CϭO), 1600 (CϭN); H-NMR (DMSO-d6): 2.30 (s, 3H, CH3), 7.10
(s, 1H, oxadiazoline C-2-H), 7.40—8.30 (m, 15H, aromaticϩpyrazole C-5
H); Anal. (C25H19N5O4) C, H and N.
3-Acetyl-2-(3-(4-bromophenyl)-1-(4-nitrophenyl)pyrazol-4-yl)-5-phenyl-
1,3,4-oxadiazoline (34): Yield 68%; mp 364—366 °C; DMF-H2O; IR (KBr,
cmϪ1): 1660 (CϭO), 1600 (CϭN); 1H-NMR (DMSO-d6): 2.26 (s, 3H, CH3),
7.13 (s, 1H, oxadiazoline C-2-H), 7.59—8.60 (m, 14H, aromaticϩpyrazole
C-5 H); Anal. (C25H18BrN5O4) C, H, N and Br.
3-Acetyl-5-(2-chlorophenyl)-2-(1-(4-nitrophenyl)-3-phenylpyrazol-4-yl)-
1,3,4-oxadiazoline (35): Yield 85%; mp 228—229 °C; DMF-H2O; IR (KBr,
cmϪ1): 1670 (CϭO), 1600 (CϭN); 1H-NMR (DMSO-d6): 2.26 (s, 3H, CH3),
7.10 (s, 1H, oxadiazoline C-2-H), 7.27—8.33 (m, 14H, aromaticϩpyrazole
C-5 H); Anal. (C25H18ClN5O4) C, H and N.
In Vitro Cytotoxicity Assay20—22) The human tumor cell lines of the
cancer-screening panel were grown in RPMI 1640 medium containing 5%
fetal bovine serum and 2 mM L-glutamine. For a typical screening experi-
ment, cells were inoculated into 96 well microtiter plates in 100 ml at plating
densities ranging from 5000 to 40000 cells/well depending on the doubling
time of individual cell lines. After cell inoculation, the microtiter plates were
incubated at 37 °C, 5 % CO2, 95% air and 100% relative humidity for 24 h
prior to addition of experimental drugs.
After 24 h, two plates of each cell line were fixed in situ with
trichloroacetic acid (TCA), to represent a measurement of the cell popula-
tion for each cell line at the time of drug addition (Tz). Experimental drugs
were solubilized in dimethyl sulfoxide at 400-fold the desired final maxi-
mum test concentration and stored frozen prior to use. At the time of drug
addition, an aliquot of frozen concentrate was thawed and diluted to twice
the desired final maximum test concentration with complete medium con-
taining 50 mg/ml gentamicin. Additional four, 10-fold or 1/2 log serial dilu-
tions were made to provide a total of five drug concentrations plus control.
Aliquots of 100 ml of these different drug dilutions were added to the appro-
priate microtiter wells already containing 100 ml of medium, resulting in the
required final drug concentrations. Following drug addition, the plates were
incubated for an additional 48 h at 37 °C, 5% CO2, 95% air, and 100% rela-
tive humidity. For adherent cells, the assay was terminated by the addition of
cold TCA. Cells were fixed in situ by the gentle addition of 50 ml of cold
50% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 min at
4 °C. The supernatant was discarded, and the plates were washed five times
with tap water and air dried. Sulforhodamine B (SRB) solution (100 ml) at
3-Acetyl-2-(3-(4-bromophenyl)-1-4-nitrophenyl)pyrazol-4-yl)-5-(2-
chlorophenyl)-1,3,4-oxadiazoline (36): Yield 85%; mp 216—217 °C; DMF-
H2O; IR (KBr, cmϪ1): 1690 (CϭO), 1600 (CϭN); 1H-NMR (DMSO-d6):
2.28 (s, 3H, CH3), 7.10 (s, 1H, oxadiazoline C-2-H), 7.60—8.40 (m, 13H,
aromaticϩpyrazole C-5 H); Anal. (C25H17BrClN5O4) C, H, N and total
halide.
3-Acetyl-5-(4-hydroxyphenyl)-2-(1-(4-nitrophenyl)-3-phenylpyrazol-4-
yl)-1,3,4-oxadiazoline (37): Yield 90%; mp 199—200 °C; DMF-H2O; IR
(KBr, cmϪ1): 3500 (OH), 1670 (CϭO), 1600 (CϭN); 1H-NMR (DMSO-d6):
2.28 (s, 3H, CH3), 3.9 (br s, 1H, OH), 7.20 (s, 1H, oxadiazoline C-2-H),
7.25—8.35 (m, 14H, aromaticϩpyrazole C-5 H); Anal. (C25H19N5O5) C, H
and N.
3-Acetyl-2-(3-(4-bromophenyl)-1-(4-nitrophenyl)pyrazol-4-yl)-5-(4-hy-
droxyphenyl)-1,3,4-oxadiazoline (38): Yield 80%; mp 247—248 °C; DMF-
H2O; IR (KBr, cmϪ1): 3500 (OH), 1680 (CϭO), 1600 (CϭN); 1H-NMR
(DMSO-d6): 2.20 (s, 3H, CH3), 4.10 (br s, 1H, OH), 7.10 (s, 1H, oxadiazo-
line C-2-H), 7.60—8.40 (m, 13H, aromaticϩpyrazole C-5 H); Anal.
(C25H18BrN5O5) C, H, N and Br.
3-Acetyl-2-(1-(4-nitrophenyl)-3-phenylpyrazol-4-yl)-5-(4-pyridyl)-1,3,4-
oxadiazoline (39): Yield 60%; mp 180—181 °C; DMF-H2O; IR (KBr,
cmϪ1): 1700 (CϭO), 1620 (CϭN); 1H-NMR (DMSO-d6): 2.20 (s, 3H, CH3),
7.15 (s, 1H, oxadiazoline C-2-H), 7.25—8.76 (m, 14 H, aromaticϩpyrazole
C-5 H); Anal. (C24H18N6O4) C, H and N.
3-Acetyl-2-(3-(4-bromophenyl)-1-(4-nitrophenyl)pyrazol-4-yl)-5-(4-
pyridyl)-1,3,4-oxadiazoline (40): Yield 80%; mp 261—262 °C; DMF-H2O;
1
IR (KBr, cmϪ1): 1700 (CϭO), 1620 (CϭN); H-NMR (DMSO-d6): 2.28 (s,