Synthesis and anticancer evaluation of some new hydrazone derivatives of 2,6-dimethylimidazo[2,1-b][1,3,4]thiadiazole-5-carbohydrazide

Article abstract of DOI:10.1016/S0223-5234(03)00138-7

In this study, some novel 2,6-dimethyl-N′-substituted phenylmethylene-imidazo[2,1-b][1,3,4]thiadiazole-5-carbohydrazides (3a-3h) were synthesized from 2,6-dimethylimidazo-[2,1-b][1,3,4]thiadiazole-5-carbohydrazide (2). The newly synthesized compounds (3a-3h) were evaluated in the National Cancer Institute's 3-cell line, one dose in vitro primary cytotoxicity assay. Compounds 3c and 3h which passed the criteria for activity in this assay (20-29% growth percentages) were scheduled automatically for evaluation against the full panel of 60 human tumor cell lines at a minimum of five concentrations at 10-fold dilutions. Sulforhodamine B (SRB) protein assay was used to estimate cell stability or growth. 2,6-Dimethyl-N′-(2-hydroxyphenylmethylidene)imidazo[2,1-b][1,3,4] thiadiazole-5-carbohydrazide (3c) showed the most favorable cytotoxicity. This compound demonstrated the most marked effects in the National Cancer Institute's 60 human tumor cell line in vitro screen on an ovarian cancer cell line (OVCAR log₁₀GI₅₀ value -5.51).

Full text of DOI:10.1016/S0223-5234(03)00138-7

European Journal of Medicinal Chemistry 38 (2003) 781ꢀ786
/
www.elsevier.com/locate/ejmech
Short communication
Synthesis and anticancer evaluation of some new hydrazone
derivatives of 2,6-dimethylimidazo[2,1-b]-
[
1,3,4]thiadiazole-5-carbohydrazide
Nalan Terzioglu *, Aysel G u¨ rsoy
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Istanbul University, 34452 Istanbul, Turkey
Received 17 January 2003; received in revised form 2 June 2003; accepted 6 June 2003
Abstract
In this study, some novel 2,6-dimethyl-N?-substituted phenylmethylene-imidazo[2,1-b][1,3,4]thiadiazole-5-carbohydrazides (3aꢀ
h) were synthesized from 2,6-dimethylimidazo-[2,1-b][1,3,4]thiadiazole-5-carbohydrazide (2). The newly synthesized compounds
3aꢀ3h) were evaluated in the National Cancer Institute’s 3-cell line, one dose in vitro primary cytotoxicity assay. Compounds 3c
and 3h which passed the criteria for activity in this assay (20ꢀ29% growth percentages) were scheduled automatically for evaluation
against the full panel of 60 human tumor cell lines at a minimum of five concentrations at 10-fold dilutions. Sulforhodamine B
SRB) protein assay was used to estimate cell stability or growth. 2,6-Dimethyl-N?-(2-hydroxyphenylmethylidene)imidazo[2,1-
/
3
(
/
/
(
b][1,3,4]thiadiazole-5-carbohydrazide (3c) showed the most favorable cytotoxicity. This compound demonstrated the most marked
effects in the National Cancer Institute’s 60 human tumor cell line in vitro screen on an ovarian cancer cell line (OVCAR log10 GI50
value ꢁ5.51).
/
´
2003 Editions scientifiques et m e´ dicales Elsevier SAS. All rights reserved.
#
Keywords: Imidazo[2,1-b][1,3,4]thiadiazoles; Hyrazide-hydrazones; Anticancer activity
1
. Introduction
cytotoxic agents on the immune system also appears to
be very challenging (Fig. 1).
The search for anticancer drugs led to the discovery of
In an attempt to achieve new compounds with
possible anticancer properties, we designed and synthe-
sized some novel 2,6-dimethyl-N?-substituted phenyl-
methylene-imidazo[2,1-b][1,3,4]thiadiazole-5-carbohy-
several imidazo-fused heterocycles having anticancer
activity [1ꢀ5]. An early report on 2-amino-1,3,4-thia-
diazole derivatives deals with the activity of these
/
compounds against several transplanted animal tumors
[
drazide hydrazones (3aꢀ
properties.
/3h) to evaluate their anticancer
6]. Recently Gadad et al. [7] reported on the cytotoxic
effects of imidazo[2,1-b][1,3,4]thiadiazoles (I). Andreani
et al. [8] studied on some imidazo[2,1-b]thiazole guanyl
hydrazones (II) which were active against various cancer
cell lines. Much interest has also been focused on the
chemistry and anticonvulsant, analgesic [9], antibacter-
ial [10] and antisecretory [11] activities displayed by
compounds incorporating this heterocyclic system. Since
the imidazo[2,1-b][1,3,4]thiadiazole system is similar in
part to Levamisole, a well-known immunomodulator
2
. Chemistry
The synthetic pathway used in the preparation of the
compounds is outlined in Fig. 2. Thus the reaction of 2-
amino-5-methyl-1,3,4-thiadiazole and ethyl 2-chloroace-
toacetate
afforded
ethyl-2,6-dimethylimidazo[2,1-
[
12], the possibility of reducing the harmful effects of the
b][1,3,4]thiadiazole-5-carboxylate (1) which in turn was
refluxed with hydrazine hydrate to obtain 2,6-dimethy-
limidazo[2,1-b][1,3,4]thiadiazole-5-carbohydrazide (2)
[13].
* Corresponding author.
E-mail address: nalant@yahoo.com (N. Terzioglu).
´
0
223-5234/03/$ - see front matter # 2003 Editions scientifiques et m e´ dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0223-5234(03)00138-7

782
N. Terzioglu, A. G ¨u rsoy / European Journal of Medicinal Chemistry 38 (2003) 781ꢀ786
/
molecular weights. The major fragmentation pathway in
a, 3f and 3g involved the cleavage of the NÃN bond
3
/
and migration of the CH proton to the nitrogen atom,
giving the fragment at m/z 196 (Fig. 3). Additional
spectral characteristics are presented in Section 4.
3. Anticancer evaluation and discussion
The 2,6-dimethyl-N?-substituted phenylmethylidene-
Fig. 1.
imidazo[2,1-b][1,3,4]thiadiazole-5-carbohydrazides (3aꢀ
/
3h) were evaluated in the 3-cell line panel consisting of
NCI-H460 (Lung), MCF7 (Breast), and SF-268 (CNS).
Primary anticancer assay was performed in accordance
with the protocol of the Drug Evaluation Branch,
National Cancer Institute, Bethesda [14ꢀ
pounds were added at a single concentration (10
/
16]. The com-
4
ꢁ
M)
and the culture was incubated for 48 h. End point
determinations were made with a protein binding dye,
sulforhodamine B (SRB). Results for each compound
were reported as the percent of growth of the treated
cells when compared to the untreated control cells. The
2
-hydroxy derivative 3c and 4-nitro derivative 3h which
reduced the growth of the cell lines to 32% or less
negative numbers indicate cell kill) were passed on for
(
evaluation in the full panel of 60 human tumor cell lines.
Primary results are shown in Table 2. The cytotoxic and/
or growth inhibitory effects of the compounds were
tested in vitro against the full panel of 60 human tumor
cell lines derived from nine neoplastic diseases at 10-fold
ꢁ
4
dilutions of five concentrations ranging from 10
ꢁ
to
M. The percentage growth was evaluated spectro-
8
10
photometrically versus controls not treated with test
agents. A 48-h continuous drug exposure protocol was
followed and an SRB protein assay was used to estimate
cell viability or growth. For each compound, the 50%
growth inhibition (GI ) and total growth inhibition
5
0
(TGI) were obtained for all the cell lines. The log10 GI50
and log10 TGI were then determined, defined as the
Fig. 2. Synthesis of 3aꢀ3h.
/
mean of the log ’s of the individual GI and TGI
1
0
50
Condensation of 2 with appropriate aldehydes yielded
the corresponding hydrazide hydrazones (3aꢀ3h). All
the synthesized compounds are listed in Table 1 and
values. The lowest values are obtained with the most
sensitive cell lines. Compounds having values ꢁ4 and
less than ꢁ4 were declared to be active. The cell lines
/
/
/
their structures are determined by elemental analyses
1
and spectral data (IR, H-NMR, EI-MS).
used in the NCI screen were Leukemia (L) lines CCRF-
CEM, HL-60 (TB), K-562, MOLT-4, RPMI-8226, SR;
non-small cell lung cancer (NSCLC) lines A549/ATCC,
EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-
H322M, NCI-H460, NCI-H522; colon cancer (CL) lines
COLO 205, HCC-2998, HCT-116, HCT-15, HT29,
KM12, SW-620; central nervous system cancer
(CNSC) lines SF-268, SF-295, SF-539, SNB-19, SNB-
75, U251; melanoma (M) lines LOX IMVI, MALME-
3M, M14, SK-MEL-2, SK-MEL-28, SK-MEL-5,
UACC-257, UACC-62; ovarian cancer (OC) lines
IGROV1, OVCAR-3, OVCAR-4, OVCAR-5, OV-
CAR-8, SK-OV-3; renal cancer (RC) lines 786-O,
In the IR spectra, the NH and CÄ
/
O bands were
ꢁ
1
ꢁ1
observed in the 3460ꢀ
regions, respectively. In the H-NMR spectra of hydra-
zide hydrazones (3aꢀ
absorptions of the hydrazide 2 (dꢂ
presence of new resonances assigned to the Ã
proton of 3 provided evidence for hydrazone formation.
These protons resonated in 8.84ꢀ9.21 ppm region as
/
3446 cm
and 1695ꢀ
/
1690 cm
1
/
3h), the absence of the NH2
/5.25 ppm) and the
/
CHÄ
/
/
singlets. EI mass spectra of three representative exam-
ples (3a, 3f and 3g) provided molecular ions at m/z 299,
3
33 and 317 with different intensities confirming their

N. Terzioglu, A. G ¨u rsoy / European Journal of Medicinal Chemistry 38 (2003) 781ꢀ
/786
783
Table 1
Some characteristics of 3aꢀ3h
/
Compound Ar
Yield (%) Melting point (8C)
Formula (M
w
)
Analysis calc./found
C
H
N
3
3
3
3
3
3
3
3
a
b
c
d
e
f
C
6
H
5
40
51
43
68
87
63
83
62
288ꢀ
298
295ꢀ
/
289
C
14
C
15
C
14
C
15
C
14
C
14
C
14
C
14
H
H
H
H
H
H
H
H
13
15
13
15
N
N
N
N
5
5
5
5
OS (299.34)
OS (313.37)
56.17, 55.78 4.37, 4.19 23.39, 23.49
57.48, 57.37 4.82, 4.73 22.35, 22.52
53.32, 53.09 4.15, 4.05 22.21, 22.28
54.69, 54.47 4.59, 4.67 21.26, 21.91
43.41, 42.97 3.38, 2.74 18.08, 18.29
50.49, 51.06 4.23, 3.57 19.62, 19.82
52.98, 53.16 3.81, 3.91 22.07, 22.20
47.58, 48.14 3.70, 3.23 23.78, 23.81
4-CH
2-HOC
4-CH OC
4-BrC
4-CIC
4-FC
4-NO
3 6 4
C H
6
H
4
/297
O
O
2
S (315.35)
S (329.37)
3
6
H
4
290ꢀ
/
292
2
1
6
H
4
270
300
12BrN
12CIN
12FN OS (317.34)
12
5
OS×/ /
2
H
2
O (387.26)
1
2
6
H
4
5
OS×/ /
C
2
H
5
OH (356.82)
g
h
6
H
4
270
5
1
2
2
C
6
H
4
274ꢀ
/
276
N
6
O
3
S×/ /
H
2
O (353.36)
A498, ACHN, CAKI-1, RXF 393, SN12C, TK-10, UO-
1; prostate cancer (PC) lines PC-3, DU-145 and breast
cancer (BC) lines MCF7, NCI/ADR-RES, MDA-MB
cell line (SK-OV-3) and a BC cell line (MDA-MD-231/
ATCC) are ꢁ3.96, ꢁ3.90, ꢁ4.47 and ꢁ3.83, ꢁ3.49,
4.31, respectively. When these data are examined, it is
3
/
/
/
/
/
ꢁ
/
2
5
31/ATCC, HS 578T, MDA-MB-435, MDA-N, BT-
49, T-47D (Table 3).
The hydroxy derivative 3c was highly active in the in
observed that both the compounds (3c and 3h) are much
more active than chlorambucil, 5-fluorouracil and
melfalan against an OC cell line, OVCAR-3 and SK-
OV-3, respectively. In conclusion, these preliminary
results are promising and some of these compounds
may be potential candidates for new anticancer agents.
vitro screen on an OC cell line (OVCAR-3, log10 GI50
value ꢁ5.51), a BC cell line (MCF7, log10 GI50 value
ꢁ
/
/4.51) and an L cell line (HL-60(TB), log10 GI50 value
ꢁ
/
4.50). On the OC cell line (OVCAR-3), the log10 GI50
values of chlorambucil, 5-fluorouracil and melfalan used
as anticancer agents are ꢁ3.96, ꢁ4.53 and ꢁ4.42,
/
/
/
4. Experimental
respectively. In addition, the nitro derivative 3h demon-
strated the most marked effects on two CNSC cell lines
4.1. Chemistry
(
SF-539, log10 GI50 value ꢁ
value ꢁ4.63), an OC cell line (SK-OV-3, log10 GI50
value ꢁ4.55) and a BC cell line (MDA-MD-231/ATCC,
log10 GI50 value ꢁ4.62). The log10 GI50 values of
chlorambucil, 5-fluorouracil and melfalan on an OC
/
4.50 and U251, log10 GI50
/
Melting points were estimated with a B u¨ chi 530
melting point apparatus in open capillaries and are
uncorrected. Elemental analyses were performed on a
Carlo Erba 1106 elemental analyzer. IR spectra were
/
/
Fig. 3. The proposed fragmentation pattern of hydrazide hydrazones (3aꢀ3h).
/

784
N. Terzioglu, A. G ¨u rsoy / European Journal of Medicinal Chemistry 38 (2003) 781ꢀ786
/
Table 2
Three-cell panel (growth percentages after inoculation with com-
400 (400 MHz) spectrophotometer using DMSO-d (E.
6
Merck, Darmstadt, Germany). EI-MS were determined
on a VG Zab Spec (70 eV) mass spectrometer. Starting
materials were purchased from E. Merck (Darmstadt,
Germany).
pounds 3aꢀ/3h)
Compound
(
NCI-H460
(lung)
MCF7
(breast)
SF-268
(CNS)
ꢁ
4
10 M)
3
3
3
3
3
3
3
a
b
c
94
96
64
62
25
73
63
65
72
98
101
54
4.1.1. Synthesis of ethyl-2,6-dimethylimidazo[2,1-b]-
[1,3,4]thiadiazole-5-carboxylate (1)
29
d
e
106
86
103
49
0
.1 mol of 2-amino-5-methyl-1,3,4-thiadiazole was
refluxed with 0.1 mol of ethyl-2-chloroacetoacetate in
0 ml ethanol (70%) in the presence of 2 g pyridine for 5
g
h
104
85
93
20
5
h. The solid that separated was recrystallized with water;
m.p., 120 8C [13].
Table 3
In vitro tumor cell growth inhibition of 3c and 3h
4
[
.1.2. Synthesis of 2,6-dimethylimidazo[2,1-b]-
1,3,4]thiadiazole-5-carbohydrazide (2)
Panel/cell line
3c
3h
0.01 mol of 1 was refluxed with 0.04 mol of the
hydrazine hydrate (80%) in 20 ml ethanol for 2 h. The
solid that separated was recrystallized with ethanol
log10 GI50 log10 TGI Log10 GI50 log10 TGI
Leukemia
(
60%); m.p. 310 8C (decomp.) [13].
CCRF-CEM
HL-60 (TB)
MOLT-4
ꢁ
/
4.39
4.50
4.28
ꢀ/
ꢁ
/
4.00
4.00
4.00
ꢀ/
ꢁ
/
4.00
4.00
4.39
ꢀ/
ꢁ
/
4.00
4.00
4.00
ꢁ1
2
O). H-NMR [200 MHz, d, ppm, DMSO-d ]: 2.35 (3H,
: IR [KBr, n (cm )]: 3329, 3226 (NÃ
/
H), 1699 (CÄ
/
ꢁ/
ꢀ/
ꢁ/
ꢀ/
ꢁ
/
ꢀ/
ꢁ
/
1
ꢁ/
ꢀ/
ꢁ/
ꢁ/
ꢀ/
ꢁ
/
6
s, 6-CH ), 2.41 (3H, s, 2-CH ), 5.25 (2H, s, NH ), 13.10
3
3
2
Non-small cell lung cancer
(
1H, s, NH).
HOP-62
NCI-H460
NCI-H522
ꢀ
/
ꢁ
/
/
4.00
4.30
4.00
ꢀ
/
ꢁ
/
4.00
4.00
4.00
ꢁ
/
4.33
4.00
4.29
ꢀ
/
ꢁ
/
/
4.00
4.00
4.00
ꢁ
ꢁ
ꢀ
/
ꢁ
/
ꢀ
/
ꢁ
/
ꢀ
/
ꢁ
ꢁ
ꢀ/
/
ꢀ/
ꢁ/
ꢁ/
ꢀ/
/
4
.1.3. Synthesis of 2,6-dimethyl-N?-substituted
Colon cancer
COLO 205
HCT-116
phenylmethylidene-imidazo[2,1-b][1,3,4]thiadiazole-5-
carbohydrazides (3aꢀ3h): general procedure
.0025 mol of 2 was refluxed with 0.0025 mol of the
appropriate aldehyde in 50 ml ethanol (60%) for 5 h.
The solid that separated was washed with ethanol (60%).
ꢁ
ꢁ
ꢁ
ꢁ
/
4.23
4.29
4.38
4.26
ꢀ
/
ꢁ
/
4.00
4.00
4.00
4.00
ꢀ
ꢀ
ꢀ
ꢀ
/
ꢁ
/
/
4.00
4.00
4.00
4.00
ꢀ
/
ꢁ
ꢁ
ꢁ
ꢁ
/
4.00
4.00
4.00
4.00
/
/
ꢀ
/
ꢁ
/
/ꢁ
ꢀ
/
/
0
HCT-15
HCC-2998
/
ꢀ
/
ꢁ
/
/ꢁ
/
ꢀ
/
/
/
ꢀ/
ꢁ/
/ꢁ
/
ꢀ/
/
CNS cancer
SF-539
ꢁ
1
ꢀ
/
ꢁ
ꢁ
ꢁ
ꢁ
/
4.00
4.00
4.00
4.42
ꢀ
/
ꢁ
/
4.00
4.00
4.00
4.00
ꢁ
/
4.50
4.34
4.30
4.63
ꢀ
/
ꢁ
ꢁ
ꢁ
ꢁ
/
4.00
4.00
4.00
4.27
3a: IR [KBr, n (cm )]: 3446 (NÃ
/
H), 3014 (Ä
/
CH),
1
O). H-NMR [400 MHz, d, ppm, DMSO-d ]:
SNB-19
SNB-75
U251
ꢀ
/
/
ꢀ
/
ꢁ
/
ꢁ
/
ꢀ
/
/
1693 (CÄ
2
/
6
ꢀ/
/
ꢀ
/
ꢁ
/
ꢁ
/
ꢀ/
/
.37 (3H, s, 6-CH ), 2.44 (3H, s, 2-CH ), 7.51ꢀ7.62 (3H,
/
3
3
/
ꢀ/
ꢁ/
ꢁ/
/
m, arylidene C3,4,5
arylidene C2,6
NH). EI-MS [m/z (%)]: 299 [M ,100], 196 (94), 195
Ã
/
H), 7.90 (2H, d, Jꢂ
/7.59 Hz,
Melanoma
M14
Ã
/
H), 9.21 (1H, s, CHÄ
/), 13.29 (1H, s,
ꢁ
ꢁ
/
4.28
ꢀ
/
ꢁ
/
4.00
ꢀ
ꢀ
/
ꢁ
/
4.00
ꢀ
/
ꢁ
ꢁ
/4.00
ꢃ
SK-MEL-5
/
4.22
ꢀ
/-4.00
/
ꢁ
/
4.00
ꢀ/
/4.00
(38), 180 (1), 119 (2), 104 (30), 103 (50).
ꢁ1
Ovarian cancer
OVCAR-3
SK-OV-3
3
b: IR [KBr, n (cm )]: 3452 (NÃ
/
H), 3013 (Ä
/
CH),
1
O). H-NMR [200 MHz, d, ppm, DMSO-d ]:
ꢁ
/
5.51
ꢁ
/
4.04
ꢀ
/
ꢁ
/
4.00
ꢀ/
ꢁ
ꢁ
/4.00
1
2
690 (CÄ
/
ꢀ
/
ꢀ/
ꢁ
/
4.00
ꢁ
/
4.55
/4.06
6
.35 (3H, s, arylidene-CH ), 2.39 (3H, s, 6-CH ), 2.43
3
3
Renal cancer
(3H, s, 2-CH ), 7.36 (2H, d, Jꢂ
/
7.98 Hz, arylidene C3,5
Ã
/
786-O
A498
ꢁ
ꢁ
ꢁ
ꢁ
/
4.32
4.21
4.25
4.00
4.00
ꢀ
/
ꢁ
/
4.00
4.00
4.00
4.00
4.00
ꢀ
ꢀ
/
ꢁ
/
/
4.00
4.00
4.30
4.43
4.46
ꢀ
/
ꢁ
ꢁ
ꢁ
ꢁ
/
4.00
4.00
4.00
4.00
4.00
3
/
ꢀ
/
ꢁ
/
/ꢁ
ꢀ
/
/
H), 7.78 (2H, d, Jꢂ
(1H, s, CHÄ), 13.21 (1H, s, NH).
c: IR [KBr, n (cm )]: 3446 (NÃ
1
2
7
/
8.06 Hz, arylidene C2,6 H), 8.84
Ã
/
ACHN
TK-10
UO-31
/
ꢀ
/
ꢁ
/
ꢁ
ꢁ
ꢁ
/
ꢀ
/
/
/
ꢀ
/
/
ꢀ
/
ꢁ
/
/
ꢀ
/
/
ꢁ1
3
/
H), 3014 (Ä
/
CH),
ꢀ/
ꢁ
/
ꢀ/
ꢁ/
/
ꢀ/
ꢁ
/
1
693 (CÄ
/
O). H-NMR [400 MHz, d, ppm, DMSO-d ]:
6
Breast cancer
MCF7
.35 (3H, s, 6-CH ), 2.44 (3H, s, 2-CH ), 6.96 (1H, t, Jꢂ
/
3
3
ꢁ
ꢁ
ꢁ
ꢁ
/
4.51
4.21
4.00
4.21
ꢀ
/
ꢁ
/
4.00
4.00
4.00
4.00
ꢁ
/
4.04
4.00
4.62
4.00
ꢀ
/
ꢁ
ꢁ
ꢁ
ꢁ
/
4.00
4.00
4.31
4.00
.44 Hz, arylidene C Ã
/
H), 7.00 (1H, d, Jꢂ
/
8.30 Hz,
5
NCI/ADR-RES
MDA-MB 231/ATCC
T-47D
/
ꢀ
/
ꢁ
/
ꢀ
/
ꢁ
/
ꢀ/
/
arylidene C3Ã
/
H), 7.44 (1H, t, Jꢂ
H), 7.84 (1H, d, Jꢂ7.80 Hz, arylidene C6Ã
s, CHÄ), 10.40 (1H, s, ar-OH), 13.20 (1H, s, NH).
d: IR [KBr, n (cm )]: 3450 (NÃ
/
7.38 Hz, arylidene C Ã
4
/
ꢀ/
/
ꢀ
/
ꢁ
/
ꢁ
/
/
/
ꢀ/
ꢁ/
ꢀ
/
ꢁ/
ꢀ/
/
/
/
H), 9.22 (1H,
/
ꢁ
1
3
/
H), 3011 (Ä
/
CH),
1
recorded as KBr discs, using a Perkinꢀ
/
Elmer Model
600 FT-IR spectrometer. H-NMR spectra were ob-
tained on a Bruker AC 200 (200 MHz), a Bruker DPX
1694 (CÄ
/O). H-NMR [400 MHz, d, ppm, DMSO-d ]:
6
1
1
2.34 (3H, s, 6-CH ), 2.43 (3H, s, 2-CH ), 3.86 (3H, s, ar-
3
3
OCH ), 7.11 (2H, d, Jꢂ
/
8.14 Hz, arylidene C3,5 H),
Ã
/
3

N. Terzioglu, A. G ¨u rsoy / European Journal of Medicinal Chemistry 38 (2003) 781ꢀ
/786
785
7
.86 (2H, d, Jꢂ
CHÄ), 13.26 (1H, s, NH).
e: IR [KBr, n (cmꢁ1)]: 3011 (Ä
/
8.18 Hz, arylidene C2,6
Ã
/
H), 8.77 (1H, s,
ml of medium, resulting in the required final drug
concentrations.
Following drug addition, the plates were incubated
/
1
O). H-
3
/
CH), 1693 (CÄ
/
NMR [400 MHz, d, ppm, DMSO-d ]: 2.37 (3H, s, 6-
for an additional 48 h at 37 8C, 5% CO , 95% air and
6
2
CH ), 2.43 (3H, s, 2-CH ), 7.76 (2H, d, Jꢂ
/
8.48 Hz,
100% relative 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 incu-
bated for 60 min at 4 8C. The supernatant was dis-
carded, and the plates were washed five times with tap
water and air-dried. Sulforhodamine B (SRB) solution
(100 ml) at 0.4% (w/v) in 1% acetic acid was added to
each well, and plates were incubated for 10 min at room
temperature. After staining, unbound dye was removed
by washing five times with 1% acetic acid and the plates
were air-dried. Bound stain was subsequently solubilized
with 10 mM trizma base, and the absorbance was read
on an automated plate reader at a wavelength of 515
nm. For suspension cells, the methodology was the same
except that the assay was terminated by fixing settled
cells at the bottom of the wells by gently adding 50 ml of
80% TCA (final concentration, 16% TCA). Using the
seven absorbance measurements [time zero (Tz), control
growth (C), and test growth in the presence of drug at
the five concentration levels (Ti)], the percentage growth
was calculated at each of the drug concentrations levels.
Percentage growth inhibition was calculated as:
3
3
arylidene C3,5
C2,6
Ã
/
H), 7.85 (2H, d, Jꢂ
/
8.48 Hz, arylidene
Ã
/
H), 8.96 (1H, s, CHÄ
/
).
ꢁ
1
3
f: IR [KBr, n (cm )]: 3447 (NÃ
/
H), 3019 (Ä
/
CH),
O). H-NMR [200 MHz, d, ppm, DMSO-d ]:
1
1
2
695 (CÄ
/
6
.37 (3H, s, 6-CH ), 2.43 (3H, s, 2-CH ), 7.63 (2H, d,
3
3
Jꢂ
/
8.39 Hz, arylidene C3,5
Ã
/
H), 7.93 (2H, d, Jꢂ
/
8.55
), 13.30 (1H, s,
Hz, arylidene C2,6
Ã
/
H), 8.96 (1H, s, CHÄ
/
ꢃ
NH). EI-MS [m/z (%)]: 333 [M ,5], 211 (100), 196 (7),
95 (15), 180 (1), 155 (2), 153 (4), 140 (4), 139 (2), 138
3), 137 (3).
g: IR [KBr, n (cmꢁ1)]: 3460 (NÃ
1
(
3
/
H), 3013 (Ä
/
CH),
1
1
2
8
693 (CÄ
/
O). H-NMR [400 MHz, d, ppm, DMSO-d ]:
6
.37 (3H, s, 6-CH ), 2.44 (3H, s, 2-CH ), 7.40 (2H, t, Jꢂ
/
3
3
.84 Hz, arylidene C3,5
Ã
/
H), 7.98 (2H, dd, Jꢂ
/
7.96, 7.75
), 13.15 (1H, s,
Hz, arylidene C2,6
NH). EI-MS [m/z (%)]: 317 [M ,100], 196 (75), 195
Ã
/
H), 8.93 (1H, s, CHÄ
/
ꢃ
(
59), 180 (2), 137 (19), 122 (28), 121 (29).
ꢁ
1
3
h: IR [KBr, n (cm )]: 3446 (NÃ
/
H), 3015 (Ä
/
CH),
O). H-NMR [200 MHz, d, ppm, DMSO-d ]:
1
1
2
693 (CÄ
/
6
.42 (3H, s, 6-CH ), 2.45 (3H, s, 2-CH ), 8.15 (2H, d,
3
3
Jꢂ
/
8.75 Hz, arylidene C2,6
Ã
/
H), 8.35 (2H, d, Jꢂ
/
8.72
), 13.27 (1H, s,
Hz, arylidene C3,5
NH).
Ã
/
H), 9.23 (1H, s, CHÄ
/
(
Ti ꢁ Tz)
ꢄ100 for concentrations for which Ti]Tz
ꢄ100 for concentrations for which TiBTz
(
C ꢁ Tz)
4
.2. In vitro anticancer screening
(Ti ꢁ Tz)
Tz
The human tumor cell lines of the cancer screening
panel were grown in RPMI 1640 medium containing 5%
fetal bovine serum and 2 mM -glutamine. For a typical
Three doseꢀ
each experimental agent. Growth inhibition of 50%
(GI ) was calculated from [(TiꢁTz)/(CꢁTz)]ꢄ100ꢂ
/response parameters were calculated for
L
screening experiment, cells were inoculated into 96-well
microtiter plates in 100 ml at plating densities ranging
from 5000 to 40,000 cells per well depending on the
doubling time of individual cell lines. After cell inocula-
tion, the microtiter plates were incubated at 37 8C, 5%
/
/
/
/
5
0
50, which was the drug concentration resulting in a 50%
reduction in the net protein increase (as measured by
SRB staining) in control cells during the drug incuba-
tion. The drug concentration resulting in total growth
CO , 95% air and 100% relative humidity for 24 h prior
2
inhibition (TGI) was calculated from TiꢂTz. Values
/
to addition of experimental drugs.
were calculated for each of these parameters if the level
of activity was reached; however, if the effect was not
reached or was exceeded, the value for that parameter
was expressed as greater or less than the maximum or
minimum concentration tested.
After 24 h, two plates of each cell line were fixed in
situ with TCA, to represent a measurement of the cell
population for each cell line at the time of drug addition
(
Tz). Experimental drugs were solubilized in dimethyl
sulfoxide at 400-fold the desired final maximum test
concentration and stored frozen prior to use. At the time
of the drug addition, an aliquot of frozen concentrate
was thawed and diluted to twice the desired final
maximum test concentration with complete medium
Acknowledgements
ꢁ
1
containing 50 mg ml
gentamicin. Additional four,
0-fold or 1/2 log serial dilutions were made to provide a
We thank V.L. Narayanan from Drug Synthesis and
Chemistry Branch, National Cancer Institute, Bathesda,
MD, USA, for the evaluation of anticancer activity.
This work was partly supported by Istanbul University
1
total of five drug concentrations plus control. Aliquots
of 100 ml of these different drug dilutions were added to
the appropriate microtiter wells already containing 100
¨
Research Fund Project Number: O-194/221196.

786
N. Terzioglu, A. G ¨u rsoy / European Journal of Medicinal Chemistry 38 (2003) 781ꢀ786
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